Contents

1. Kalpana Godar and Shiva Kumar Rai Freshwater Green Algae from Raja-Rani Wetland, Bhogateni-Letang, Morang, 1

2. M. K. Adhikari New Records of Two Powdery Mildews (Erysiphales: Fungi) from Nepal 18 3. Nirmala Pradhan Records of Bryophytes from Godawari-Phulchoki Mountain Forest of Lalitpur District, Central Nepal 22 4. Chandrakala Thakur and Sangeeta Rajbhandary Fern and Fern Allies of Panchase Protected Forest, Central Nepal 39 5. Krishna Ram Bhattarai of Mai Pokhari Botanical Garden and Adjoining Areas, East Nepal 46 6. Usha Tandukar, Pramesh Bahadur Lakhey, Nishanta Shrestha, Amit Dhungana and Tara Datt Bhatt Antimicrobial Activity and Chemical Composition of Essential Oil of Fruits of Amomum subulatum Roxb. 57 7. Madan Raj Bhatta, Rajeswar Ranjitkar, Bhabani Prasad Adhikari, Parasmani Yadav and Laxman Bhandari Isolation of Curcumin and GC-MS Analysis of Oil in Curcuma longa L. 64 8. Parasmani Yadav, Bhabani Prasad Adhikari, Sarita Yadav, Sunita Khadaka, Devi Prasad Bhandari, Chetana Khanal Microscopic, UV- Fluorescence and FT-IR Analysis of the Powder of Five Medicinal Plants of Nepal 70 9. Laxman Bhandari, Madan Raj Bhatta and Rajeswar Ranjitkar Phytochemicals, Polyphenols and Antioxidant Activity of Camellia sinensis (L.) Kuntze (Tea ) from Ilam District, Nepal 78 10. Devi Prasad Bhandari, Rajeswar Ranjitkar and Laxman Bhandari Quantitative Determination of Antioxidant Potential of Five Selected Essential Oils of Nepalese Origin 84 11. Parasmani Yadav, Jyoti Joshi, Devi Prasad Bhandari and Sangita Swar Phytochemical and Physiochemical Properties of Sapindus mukorossi Gaertn. (Soapnut) of Far Western of Nepal 90 12. Tara Datt Bhatt, Parasmani Yadav, Amit Dhungana, Jyoti Joshi and Chintamani Basyal Study of the Variation of Major Chemical Constituents of Essential Oil Extracted from Rhizome and Leaf of Acorus calamus L. (Bojho) from Nepal 96 13. Tara Datt Bhatt, Amit Dhungana, Jyoti Joshi, Parasmani Yadav and Chintamani Basyal, Variation in Chemical Composition of Essential Oil Extracted from the Fruits of DC. (Timur) of Nepal 100 14. Pramesh Bahadur Lakhey, Usha Tandukar, Nishanta Shrestha and Rashmi Pradhan Some Nepalese Medicinal Plants Showing Potential Antimicrobial Activity Against Salmonella enterica subsp. enterica serovar Typhi 106 15. Nabin Rana, Saraswoti Khadka and Sabari Rajbahak In-Vitro Propagation of Lavender ( angustifolia Mill.) 112 Volume 16 Number 1 16. Sajita Dhakal, Bharat Babu Shrestha and Mohan Siwakoti Comparisons of Invasive Alien Richness between Tarai and Siwalik Regions of Central Nepal 119 17. Kamal Bahadur Nepali, Dipak Lamichhane and Srijana Shah Diversity of Butterfly and its Relationship with Plants in National Botanical Garden, Godawari, Lalitpur, Nepal 124 18. Mohan Prasad Devkota Government of Nepal religiosa G.Forst.: A Mythological Sacred from Nepal and its Medicinal Values 130 Ministry of Forest and Environment 19. Srijana Shah, Dipak Lamichhane, Ram Panthi and Kamal Bahadur Nepali Traditional Knowledge of Tamang Community on Utilization of Plant Resources in Dhading District, Central Nepal 134 Department of Plant Resources 20. Munesh Ratna Gubhaju and Anjana Bhattarai Thapathali, Kathmandu, Nepal Utilization Pattern of Bamboo in , Tansen-8, Palpa 141 2018 21. Pratiksha Shrestha Documentation of Ethnobotanical Knowledge of Magar Community of , Western Nepal 150 ISSN 1995 - 8579 Journal of Plant Resources, Vol. 16, No. 1

JOURNAL OF PLANT RESOURCES

Government of Nepal Ministry of Forest and Environment Department of Plant Resources Thapathali, Kathmandu, Nepal 2018 Advisory Board Mr. Sanjeev Kumar Rai Ms. Jyoti Joshi Bhatt Managing Editor Mr. Sishir Panthi Editorial Board Prof. Dr. Dharma Raj Dangol Mr. Deepak Lamichhane Ms. Usha Tandukar Mr. Tara Datt Bhatt Ms. Pratiksha Shrestha No. of Copies: 500

Date of Publication: 2018 April

Cover Photo: From top left in clock wise direction. Antimicrobial Activity of Amomum subulatum Roxb. oil against Enterococcus faecalis (Usha Tandukar) Pota nodosus (Sanjeev Kumar Rai) Rhododendron arboreum (Sanjeev Kumar Rai) Taxus wallichiana (Sanjeev Kumar Rai) Bryoerythrophyllum gymnostopum (Nirmala Pradhan) HPLC Instrument (Tara Datt Bhatt) © All rights reserved Department of Plant Resources (DPR) Thapathali, Kathmandu, Nepal Tel: 977-1-4251160, 4251161, 4268246, E-mail: [email protected]

Citation: Name of the author, year of publication. Title of the paper, J. Pl. Res. vol. 16, Issue 1 pages, Department of Plant Resources, Thapathali, Kathmandu, Nepal.

ISSN: 1995-8579

Published By: Publicity and Documentation Section Department of Plant Resources (DPR), Thapathali, Kathmandu, Nepal. Reviewers: Prof. Dr. Anjana Singh Dr. Bhaskar Adhikari Dr. Netra Lal Bhandari Prof. Dr. Bijaya Pant Prof. Dr. Bhaiya Khanal Dr. Bishnu Pandey Ms. Jyoti Joshi Bhatt Prof. Dr. Sangeeta Rajbhandari Prof. Dr. Ila Shrestha Dr. Keshab Raj Rajbhandari Dr. Kanti Shrestha Dr. Shiva Devkota Dr. Nirmala Joshi Prof. Dr. Amar Prasad Yadhav Dr. Susan Joshi Ms. Rose Shrestha Dr. E.N. Paudel Dr. Shandesh Bhattarai Prof. Dr. Rajani Malla Dr. Sitaram Adhikary Mr. Dhan Raj Kandel

The issue can be retrieved from http://www.dpr.gov.np Editorial

It is our Pleasure to bring out the current issue of Journal of Plant Resources, Volume 16, Number 1, a continuation of research publication by Department of Plant Resources. In this issue, 21 peer reviewed articles based on original research have been incorporated. The articles have been categorized as , phytochemisty, microbiology , biotechnology, ecology, ethnobotany. Two book reviews are also included. This issue intends to cover the research activities of the department as well as other research organization. We encourage the young researcher to pursue quality research and contribute to build scientific knowledge on Plant Resources.

The link between the result of scientific research and societies is dissemination of knowledge and information through publication. We believe that the research finding will be helpful to the scientific community as well as general public to enrich their knowledge and information on Plant Resources.

We would like to thank our valuable reviewers whose critical comments help to improve the quality of the journal. We acknowledge the contribution of the contributors for their interest in publishing their valued work in this publication and looking forward to further cooperation and collaboration with scientific institution. 2018J. Pl. Res. Vol. 16, No. 1, pp 1-17, 2018 Journal of Plant Resources Vol.16, No. 1

Freshwater Green Algae from Raja-Rani Wetland, Bhogateni-Letang, Morang, Nepal

Kalpana Godar* and Shiva Kumar Rai Phycology Research Lab, Department of , Post Graduate Campus, Tribhuvan University, Biratnagar, Nepal *E-mail: [email protected]

Abstract

A total 72 freshwater chlorophycean algae belonging to 33 genera, 12 families and 7 orders have been reported from Raja-Rani wetland. Desmids were found dominant by 64%. Cosmarium had maximum number of species (24%), followed by Staurastrum (10%), Euastrum, Scenedesmus and Micrasterias (each 6%).

Key words: Bambusina brebissonii, Chlorophyceae, Cosmarium, Rare species, Seasons

Introduction most studied algae contributed by Bando et al., 1989; Habib & Chaturvedi 1997; Rai, 2014. The Green algae (Chlorophyceae) are aquatic, chlorococcales from various localities of Nepal have photosynthetic organism which lack true roots, been studied by Nakano & Watanabe (1988) and Rai stems, , vascular tissue and have simple & Misra (2012). Algal flora of Raja-Rani wetland reproductive structure. It is the largest and most has not been studied hitherto. Hence, an endeavour diverse group of algae representing by more than is made to explore the green algae of this wetland as 8,000 species in the world (Guiry, 2012). They are a preliminary work. cosmopolitan in distribution and able to grow in wide range of habitats. They are common in moist tropical regions. Their size ranges from microscopic unicell Materials and Methods to large pseudo-parenchymatous structure exceeding Study area 50 m in length. Raja-Rani wetland (Dhimal Pokhari) (26°44.9’22'’N, Algae are being the topic of global interest 87°28.9’10'’E, altitude 470 m msl) is situated in nowadays. As world is facing a problem of energy Churia hill (Dhimal Danda), west from Chisang river crisis, scientists are working for the production of at Letang-Bhogateni municipality 4, Morang district, biofuels from algae as an alternative source of Nepal (figure 1). The wetland lies within the Raja- energy. Algae are widely using in the field of Rani community forest (1700 ha.) and covers about agriculture, medical science, industries and as 133 ha. The wetland has 3 ponds viz., Raja, Rani laboratory tools in genomics, proteomics and other and Rajkumari which altogether covers only about researches (Graham et al., 2016). Algae are also 20 ha. Based on the area covered, Rani Pond is large, using as biomonitors, both to assess the pollution of situated in the east and Raja Pond is quite small, river streams and to decide the environmental situated in the west side; both approximately 7 m conditions of the past. On the other hands, some depth in rainy season. South to the Rani Pond, there produce nuisance blooms causing a great threat to are two small Rajkumari ponds with 2 m depth in the aquatic ecosystem of the world. rainy season. Literature revealed that the freshwater green algae This wetland is important religious and historical of Nepal have been studied by various authors place particularly for Dhimal community. The area (Sahay et al., 1992; Kargupta & Jha, 1997; Rai, has dense mixed-forest dominated by Shorea robusta 2009). Among the green algae, the desmids are the and is rich in biodiversity (MoFSC, 2012). The

1 2018 Journal of Plant Resources Vol.16, No. 1 aquatic macrophytes are: floating species- of the lakes. Three seasonal trips were made at an Eichhornia cracipes, Pistia stratiotes, Spirodella interval of 3 months i.e., Falgun (winter), Jestha polyrhiza etc. and submerged species- (summer) and Bhadra (rainy), 2072/073 BS. Ceratophyllum demersum, Ottelia alismoides, Epiphytic algae were collected by squeezing Hydrilla verticillata etc. submerged leaves, stems and roots of macrophytes; free floating planktonic microalgae were collected The climate of Raja-Rani wetland is sub-tropical using plankton net (mesh size 0.5 mm) and benthic type with hot and humid summers, intense monsoon forms were collected by scrubbing the substratum rain and cold dry winters. The average annual like stone, pebbles etc. Finally, the collected samples minimum and maximum temperature ranges from were preserved in 4% formaldehyde solution in air 12°-19°C, and 22°-30°C, respectively. The total tight polythene bottles with proper tagging and annual rainfall in this region varies from 1,138 mm labeling (sample no, sampling site, date of collection to 2,671 mm (FRA/DFRS, 2014). etc.) Plant collection and identification The collected samples were brought to the Phycology A total 36 algae samples were collected from 12 sites Research Lab, Department of Botany, Post Graduate (9 from Raja and 3 from Rani lake) on the periphery Campus, Biratnagar for further investigation. All the

Figure 1: Map of Raja-Rani (Dhimal pokhari) wetland showing Raja, Rani and Rajkumari ponds and algal sampling sites

2 2018 Journal of Plant Resources Vol.16, No. 1 samples were screened under light microscope. Lab, Department of Botany, Post Graduate Campus Iodine and 1% methylene blue stains were used to Biratnagar, Nepal. confirm green and blue-green algae. Number and dominance of each alga were calculated. Algal Results and Discussion dimension (length, breadth etc.) was measured with the help of calibrated occular micrometer. In the present study, a total 72 spp. freshwater green Photomicrography was done for each taxa under 40X algae belonging to 33 genera, 12 families and 7 and 100X objectives using the Olympus Ch20i orders have been enumerated from 12 sites of Raja- microscope attached with camera. Identification was Rani wetland, Bhogateni, Morang, Nepal (table 1). carried out following Prescott, 1951; Scott & In total, desmidiales were represented by 64%. Prescott, 1961; Philipose, 1967; Nurul Islam, 1970; Chetophorales and Zygnematales have single Croasdale & Flint, 1986; Kouwets, 1987; Prasad & representatives i.e. Stigeoclonium fasciculare and Misra 1992 etc. by cross checking with illustrations Netrium digitus, respectively. Among the genera, and description given. Classification and current Cosmarium had maximum spp. (24%) followed by accepted name were confirmed from online algae Staurastrum (10%) and Euastrum, Scenedesmus, site www.algaebase.org. All the samples have been Micrasterias (each 6%). Algae classification is based deposited in the repository of Phycology Research on Guiry & Guiry (2018) and arranged alphabetically according to Silva (1980).

Table 1: Hierarchial listing of chlorophycean algae of Raja-Rani wetland, Morang Order Family Latin Name Chlamydomonadales Volvocaceae 1. Eudorina elegans 2. Pandorina morum Sphaerocystidaceae 3. Sphaerocystis schroeteri Chlorellales Chlorellaceae 4. Dictyosphaerium pulchellum Oocystaceae 5. Gloeotaenium loitlesbergerianum 6. Nephrocytium agardhianum Sphaeropleales Hydrodictyaceae 7. Pediastrum tetras var. tetraodon 8. Sorastrum spinulosum 9. Tetraedron minimum Scenedesmaceae 10. Dimorphococcus lunatus 11. Coelastrum cambricum var. intermedium 12. Coelastrum microsporum 13. Crucigenia crucifera 14. Scenedesmus acutiformis 15. Scenedesmus arcuatus var. platydiscus 16. Scenedesmus bijugatus var. gravenitzii 17. Scenedesmus hystrix Selenastraceae 18. Ankistrodesmus falcatus 19. Ankistrodesmus spiralis 20. Ankistrodesmus spiralis var. fasciculatus 21. Selenastrum bibraianum 22. Kirchneriella lunaris Chaetophorales Chaetophoraceae 23. Stigeoclonium fasciculare Oedogoniales Oedogoniaceae 24. Bulbochaete intermedia 25. Oedogonium undulatum Zygnematales Mesotaeniaceae 26. Netrium digitus Desmidiales Closteriaceae 27. Closterium dianae 28. Closterium ehrenbergii 29. Closterium gracile Desmidiaceae 30. Pleurotaenium trabecula 31. Euastrum elegans

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32. Euastrum insulare 33. Euastrum spinulosum 34. Euastrum turneri 35. Micrasterias mahabuleshwarensis var. surculifera 36. Micrasterias pinnatifida 37. Micrasterias radians 38. Micrasterias zeylanica 39. Actinotaenium cucurbita 40. Actinotaenium subglobosum 41. Cosmarium burkilli var. depressum 42. Cosmarium connatum 43. Cosmarium contractum 44. Cosmarium javanicum 45. Cosmarium lundellii 46. Cosmarium lundellii var. circulare 47. Cosmarium lundellii var. ellipticum 48. Cosmarium meneghinii 49. Cosmarium obsoletum 50. Cosmarium obtusatum 51. Cosmarium portianum 52. Cosmarium pseudoretusum 53. Cosmarium punctulatum 54. Cosmarium pyramidatum 55. Cosmarium quadrum 56. Cosmarium striolatum 57. Cosmarium subspeciosum var. validius 58. Xanthidium hastiferum var. javanicum 59. Staurodesmus convergens 60. Staurodesmus dejectus 61. Staurodesmus dickiei var. circularis 62. Staurastrum avicula 63. Staurastrum cf. ehrenbergianum 64. Staurastrum manfeldtii 65. Staurastrum cf. margaritaceum 66. Staurastrum sexangulare var. bidentatum 67. Staurastrum cf. tetracerum 68. Staurastrum tohopekaligense var. tohopokeligense f. minus 69. Teilingia granulata 70. Onychonema leave 71. Hyalotheca dissiliens 72. Bambusina brebissonii

Taxonomic description Tiffany and Britton 1952, P. 16, Pl. 1, Figure Each taxa is appended with author/s name, figure 13. number (in parenthesis), reference used for Colony 88 µm long, 62 µm broad with 16 motile identification followed by brief morphological cells; cells 14-18 µm long, 9-14 µm broad. characters including dimensions. 3. Sphaerocystis schroeteri Chodat (Figure 3) Prasad and Misra 1992, P. 7, Pl. 5, Figure 12. 1. Eudorina elegans Ehr. (Figure 1) Prescott Colony spherical, up to 500 µm in diameter, 4- 1951, P. 76, Pl. 1, Figures 24-26. 8 celled; cells spherical, 6-20 µm in diameter. Colony up to 200 ìm in diameter, 16-32 ovoid 4. Dictyosphaerium pulchellum Wood celled; cells 10-20 ìm in diameter. [Mucidosphaerium pulchellum (Wood) Bock, 2. Pandorina morum (Müller) Bory (Figure 2) Proschold and Krienitz] (Figure 4) Philipose

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1967, P. 199, Figure 110. with slightly thick projections. Colony 32 celled, arranged in series of 4 on 12. Coelastrum microsporum Nägeli (Figure 12) dichotomously branched threads; cells 3-10 µm Prescott 1951, P. 230, Pl. 53, Figure 3. in diameter. Colony spherical, 43-47.5 µm in diameter, 8- 5. Gloeotaenium loitlesbergereanum Hansg. 64 celled; cells spherical to ovoid, 7-10 µm in (Figure 5) Komarek 1983, P. 116, Pl. 17, Figure diameter, interconnected by short, stout 36. gelatinous processes, inter cellular spaces small. Colony 52-72 µm long, 38-52 µm broad, 2-8 13. Crucigenia crucifera (Wolle) Kuntze [Willea celled; cells 23-26 µm long, 17-19 µm broad, crucifera (Wolle) John, Wynne and Tsarenko] cells separated by dark-colored calcium (Figure 13) Philipose 1967, P. 240, Figure 149. carbonate, appears as 2 X-shaped bands. Colony rhomboidal, 24-27 µm in diameter, 4 6. Nephrocytium agardhianum Nägeli (Figure 6) celled, often 2-3 colonies together; cells Prescott 1951, P. 248, Pl. 54, Figures 15-16 elongate, 7.5 µm long, 4-5 µm broad, outer Colony ovate, 33-44 µm in diameter, 4-8 celled; margin concave, inner straight or slightly cells cylindrical or reniform, 10-16 µm long, convex. 3-6 µm broad, twisted, spiral arrangement 14. Scenedesmus acutiformis Schroder within the old mother cell wall. [Acutodesmus acutiformis (Schroder) Tsarenko 7. Pediastrum tetras var. tetraodon (Corda) and John] (Figures 14-15) Nakano and Hansgirg (Figure 7) Tiffany and Britton 1952, Watanabe 1988, P. 61, Figures 38-40. P. 112, Pl. 30, Figure 294. Colony flat, 4 celled, arranged linearly; cells Colony circular, 30 µm in diameter, 4-8-16 fusiform-elliptic, 17.5-21.5 µm long, 6-7.5 µm celled; marginal cell 10 µm long, 9-10 µm broad, cylindrical with acute ends and lateral broad, deeply incised,; inner cell 8.5 µm long, longitudinal ridge. 9.5 µm broad, 4-6 sided, single incision. 15. Scenedesmus arcuatus var. platydiscus Smith 8. Sorastrum spinulosum Nägeli (Figure 8) [Comasiella arcuata var. platydisca (Smith) Philipose 1967, P. 132, Figure 47. Hegewald et Wolf] (Figure 16) Prescott 1951, Colony spherical, 16 celled colony 35 µm in P. 275, Pl. 62, Figure 11. diameter, 4-8-16-32-128 celled; cells reniform Colony flat, 8 celled, arranged in 2 rows; cells to cuneate, 6-18 µm long, 8-20 µm broad, 3 oblong-ellipsoidal with rounded ends, 8-14 µm angled with 1-4 spines; spines 4-8 µm long. long, 4-5.5 µm broad, cells between 2 rows 9. Tetraedron minimum (Braun) Hansg. (Figure alternate. 9) Prescott 1951, P. 267, Pl. 60, Figures 12-15. 16. Scenedesmus bijugatus var. graevenitzii Cells small and flat, 6-20 µm in diameter, (Bernard) Philipose (Figure 17) Philipose 1967, tetragonal with rounded angles, without spines. P. 254, Figures 164 a-b. 10. Dimorphococcus lunatus Braun (Figure 10) Colony 4 to 8 celled; cells fusiform, ellipsoid Komarek 1983, P. 162, Pl. 34, Figure 94. to ovoid with obtuse ends, 12-16 µm long, 4.5- Colony 45-65 µm in diameter; group of 4 cells 6.5 µm broad, arrange alternately in contact only on the ends of fine-branched threads; cells 9- along a short portion of their length. 22 µm long, 7-15 µm broad, 2 inner cells ovate 17. Scenedesmus hystrix Lagerheim or subcylindric, 2 outer cells cordate. [Desmodesmus hystrix (Lagerheim) Hegewald] 11. Coelastrum cambricum var. intermedium (Figure 18) Prescott 1951, P. 278, Pl. 63, Figure (Bohlin) West (Figure 11) Prasad and Misra 12. 1992, P. 30, Pl. 4, Figure 5. Colony flat, 2-4-8 celled, arranged in a linear Colony spherical, 62-67.5 µm in diameter, 32 series; cell oblong-cylindrical with obtuse ends, celled; cells 15-17.5 µm in diameter, middle 13-19 µm long, 6-7 µm broad; cell wall covered cells spherical, peripheral cells sub-spherical with minute sharp spines.

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18. Ankistrodesmus falcatus (Corda) Ralfs (Figure 24, Figure 229. 19) Komarek 1983, P. 138, Pl. 25, Figure 64b. Vegetative cell capitellate, 66 µm long, 18.7 µm Cells needle to spindle shaped, 25-100 µm long, broad, 4-5 undulation per cell. 2-6 µm broad, solitary or in clustures of 2-32 26. Netrium digitus (Ehr.) Itzigs. et Rothe (Figure individuals, not inclosed in a colonial sheath. 27) Nurul Islam 1970, P. 908, Pl. 1, Figure 9; 19. Ankistrodesmus spiralis (Turn.) Lemm. (Figure Pl. 19, Figure 2. 20) Prescott 1951, P. 254, Pl. 56, Figures 11-12 Cells oblong-elliptic, 140 µm long, 40.5 µm Cells curved or sigmoid, 25-35 µm long, 2-3 broad, margins convex, not constricted, µm broad, attenuated from middle towards gradually attenuated from middle to truncate acute apices, middle portion coiled each other ends; apices 18-19 µm broad. in the colony, apical portions free. 27. Closterium dianae Ehr. ex Ralfs (Figure 28) 20. Ankistrodesmus spiralis var. fasciculatus Opute 2000, P. 136, Pl. 2, Figure 13. Smith [A. densus Korshikov] (Figure 21) Prasad Cells 162-320 µm long, 12.5-26.5 µm broad, and Misra 1992, P. 27, Pl. 4, Figure 8. tapering to obtusely rounded ends, outer margin Colony 50-180 µm in diameter, 34-50 celled; 108° arc; apices 2-3 µm broad, 140-315 µm cells curved or sigmoid, 52-110 µm long, 3-4.5 distant. µm broad, middle portion twisted around one 28. Closterium ehrenbergii Meneg. ex Ralfs another, apical portion of cell free. (Figure 29) Kouwets 1987, P. 203, Pl. 2, Figure 21. Selenastrum bibraianum Reinsch (Figure 22) 22. Tiffany and Britton 1952, P. 117, Pl. 32, Figure Cells stout, 246-380 µm long, 44-59 µm broad, 325. tapering to obtusely rounded ends; outer margin Cells generally curved, arcuate or lunate, 38- 100-125° arc, slightly inflated middle; apices 40 µm long, 5-7.5 µm broad, elongate, with 135-340 µm distant. markedly attenuated to obtuse, truncate, rostrate 29. Closterium gracile Ralfs (Figures 30-31) Flint or needles-like poles. and Williamson 1998, P. 77, Pl. 1, Figure 3. 22. Kirchneriella lunaris (Kirch.) Mçb. (Figure 23) Cells elongate, slender, linear, 120-260 µm Prasad and Misra 1992, P. 28, Pl. 4, Figure 3. long, 3-9 µm broad, straight 2/3 of length, Colony 50-200 µm in diameter, numerous cells margin parallel, gradually narrowed to the in groups of 4-16 within an envelope; cells obtuse apices; apices 2.5-3.5 µm wide. strongly curved, crescents with rather obtuse 30. Pleurotaenium trabecula NÓg. (Figures 32-33) points, 6-14 µm long, 3-7 µm broad. Nurul Islam and Yusuf Haroon 1980, P. 564, 23. Stigeoclonium fasciculare Küetz. (Figure 24) Pl. 4, Figure 56. Prasad and Misra 1992, P. 60, Pl. 9, Figure 4-5 Cells more or less straight, 350-520 µm long, (as var. glomeratum). 27-40 µm broad, margins slight inflation above, Thallus small, prostrate filaments profusely attenulated to rotundo-truncate apices; apices branched, branching usually alternate, rarely 20-22 µm broad, without tubercules. opposite; cells cylindrical, 30.5-60 µm long, 31. Euastrum elegans Ralfs (Figure 34) Kouwets 5.1-10.2 µm broad. 1987, P. 215, Pl. 8, Figures 7-8. 24. Bulbochaete intermedia De Bary ex Hirn Cells 26-37 µm long, 17-22 µm broad; (Figure 25) Tiffany and Britton 1952, P. 54, Pl. semicells with small rounded basal lobe, with 14, Figure 96. marginal teeth, flattened apex; apical incision Diocious, nannandrous, gunandrosporous; cells deep, narrow; isthmus 8 µm wide. 27-28 µm long, 18-20 µm broad; constriction 32. Euastrum insulare (Wittr.) Roy (Figure 35) at septa, 12.5-14 µm wide; stipe 5 µm long. Kouwets 1987, P. 216, Pl. 7, Figure 21. 25. Oedogonium undulatum Braun ex Hirn Cells 21-22 µm long, 14.8 µm broad, deeply (Figure 26) Tiffany and Britton 1952, P. 84, Pl. constricted; semicells truncate, 3 lobed; polar

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lobe truncate with shallow median notch, lateral 40. Actinotaenium subglobosum (Nordst.) (Figure lobes rounded; isthmus 4.5-5 µm wide. 43) Croasdale and Flint 1988, P. 38, Pl. 28, 33. Euastrum spinulosum Delponte (Figure 36) Figures 20-21. Nurul Islam 1970, P. 917, Pl. 17, Figure 3. Cells broadly ellipsoid, 32-48 µm long, 24-30 Cells 51-55 µm long, 47-48.5 µm broad; µm broad, slightly constricted. semicells 5 lobed, polar lobe truncate, 17-18 41. Cosmarium burkilli West et West var. µm broad, with 3 spines; lateral lobes rounded depressum Scott et Prescott (Figure 44) Scott with 5-6 spines; isthmus 11.5-13 µm wide. and Prescott 1961, P. 56, Pl. 30, Figures 8-9. 34. Euastrum turneri West (Figure 37) Croasdale Cells 51-52 µm long, 43.5-47 µm broad; and Flint 1986, P. 101, Pl. 21, Figure 21. semicells truncately pyramidal, convex sides, flat Cells 33-36 ìm long, 23-26 ìm broad; semicells apex; isthmus 12-12.5 µm wide. This specimen relatively broader, with a deeper incision is larger with 2 short spines at apical angles. between the basal and polar lobes; isthmus 6-8 42. Cosmarium connatum Breb. ex Ralfs (Figure ìm wide. 45) Croasdale and Flint 1988, P. 60, Pl. 37, 35. Micrasterias mahabuleshwarensis Hobs var. Figure 30. surculifera Lagerh. (Figure 38) Nurul Islam and Cells 60-105 µm long, 42-90 µm broad, Yusoof Haroon 1980, P. 572, Pl. 3, Figure 51. moderately constricted; sinus widely open; Cells 145 µm long, 125 µm broad; sinus open semicells transversely sub-elliptic with broad with acuminate extremity; semicells with 3 and flat apex; isthmus broad, 31-70 µm wide. large lobes, margins with small, acute spines; 43. Cosmarium contractum Kirch. (Figure 46) isthmus 22.5 µm wide. Croasdale and Flint 1988, P. 61, Pl. 33, 36. Micrasterias pinnatifida Ralfs (Figure 39) Figures1-2. Prasad and Misra 1992, P. 143, Pl. 20, Figure 4. Cells 30-54 µm long, 17-34 µm broad; sinus Cells 56-60 µm long, 55-60 µm broad; deep, opening widely from a sharp-angled semicells 3 lobed, all ends to bifid spines; lateral interior; semicells broadly elliptic; apex lobes horizontal; polar lobe triangular, 13-15 somewhat flattened; isthmus 8 µm wide. µm long, 35-38 µm broad; isthmus 10-11 µm 44. Cosmarium javanicum Nordstedt (Figure 47) wide. Bando et al. 1989, P. 16, Figure 7f. 37. Micrasterias radians Turn. (Figure 40) Scott Cells oblong-elliptic, 90-187 µm long, 45-90 and Prescott 1961, P. 51, Pl. 23, Figure 1. µm broad; sinus narrow, dilated at the apex; Cells subcircular, 103-110 µm long, 95-100 µm semicells truncate-pyramidate, truncately broad; semicells 5 lobed, furcate acuminate rounded ends; isthmus 35-53 µm wide. extremity; lateral lobes 2 lobuled; polar lobe 45. Cosmarium lundellii Delponte (Figure 48) retusely emarginate; isthmus 15-17 µm wide. Bharati and Hegde 1982, P. 742, Pl. 1, Figure 38. Micrasterias zeylanica Fritsch. (Figure 41) 3. Nurul Islam 1970, P. 922, Pl. 8, Figure 3. Cells 50.5-52 µm long, 50.5-53 µm broad, Cells 55 µm long, 56 µm broad; semicell scarcely deeply constricted; sinus linear and open; 5 lobed; polar lobe cuneate, faintly retuse, 12- semicells sub-semicircular to sub-pyramidate, 14 µm long, 39-41 µm broad, angles acuminate, basal angles rounded; isthmus 22.5 µm wide. inclined spine; isthmus 13.5 µm wide. 46. Cosmarium lundellii var. circulare (Reinsch) 39. Actinotaenium cucurbita (Bréb. ex Ralfs) Teil. Krieg. [C. circulare Reinsch] (Figure 49) Nurul (Figure 42) Croasdale and Flint 1988, P. 35, Pl. Islam and Irfanullah 1999, P. 93, Pl. 1, Figures 28, Figures 15-19. 4-5. Cells cylindrical to ellipsoid, 20-55 µm long, Cells 46-57 µm long, 37-44 µm broad, circular 12-30 µm broad; sinus a notch; sides and apex in outline, deeply constricted; sinus linear, straight, rounded or flat with rounded angles. somewhat dilated at the apex; semicells semi-

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circular, isthmus narrow, 13-18.5 µm wide. oblong-trapeziform, rounded angles, convex 47. Cosmarium lundellii var. ellipticum West et sides, truncate apex; isthmus 7-l4 µm wide. West (Figure 50) Scott and Prescott 1961, P. 54. Cosmarium pyramidatum Bréb. in Ralfs 61, Pl. 25, Figure 8. (Figure 57) Croasdale and Flint 1988, P. 91, Pl. Cells ellipsoid, 57-60 µm long, 42-43 µm broad, 35, Figures 8-9. deeply constricted; sinus linear; semicells sub- Cells 51-110 µm long, 32-70 µm broad; sinus semicircular, basal angles rounded, broadly deep, closed; semicells pyramidal with rounded rounded ends; isthmus 16-17 µm wide. angles, moderately convex sides and flattened 48. Cosmarium meneghinii Bréb. ex Ralfs (Figure apex; isthmus 9-25 µm wide. 51) Croasdale and Flint 1988, P. 75, Pl. 41, 55. Cosmarium quadrum Lund. (Figure 58) Figures 12-14. Croasdale and Flint 1988, P. 95, Pl. 54, Figures Cells 13-30 µm long, 10-22 µm broad; sinus 1-3. deep, closed; semicells transversely rectangular Cells 60-90 µm long, 54-85 µm broad; sinus below, pyramidal truncate above, margin retuse, deep, linear, dilated interior; semicells sub- angles rounded; isthmus 3-7 µm wide. rectangular, angles rounded, sides convex, apex 49. Cosmarium obsoletum (Hantz.) Reinsch retuse or straight; isthmus 18-30 µm wide. (Figure 52) Croasdale and Flint 1988, P. 80, Pl. 56. Cosmarium striolatum NÓg. ex Archer (Figure 29, Figure 1. 59) Bharati and Hedge 1982, P. 752, Pl. 10, Cells 23-56 µm long, 42-60 µm broad; sinus Figure 4. deep, closed; semicells depressed-globose, Cells 94-125 µm long, 52-68 µm broad, lower angles with conical thickening; side view constriction shallow; semicells broadly obovate- depressed-globose; isthmus 10-24 µm wide. elliptic, convex sides and apex; cell wall finely 50. Cosmarium obtusatum (Schm.) Schmidle granulate; isthmus broad, 42-52 µm wide. (Figure 53) Kouwets 1987, P. 228, Pl. 14, Fig 9. 57. Cosmarium subspeciosum var. validius Cells 48-60 µm long, 41-50 µm broad; sinus Nordst. (Figure 60) Bharati and Hegde 1982, deep, closed, dilated within; semicells semi- P. 752, Pl. 9, Figure 1. circular to truncate-pyramidal, sides with 8 Cells 45-50 µm long, 32.5-36.5 µm broad; sinus undulations; isthmus 15-18 µm wide. narrow, linear; semicells sub-rectangular, 51. Cosmarium portianum Arch. (Figure 54) Scott margin with 4 apical and 7 lateral crenations, and Prescott 1961, P. 65, Pl. 28, Figure 8. broadly truncate ends; isthmus 11-12 µm wide. Cells small, 20 µm long, 18 µm broad, deeply 58. Xanthidium hastiferum var. javanicum constricted; sinus gradually opening from a (Nordst.) Turn. [X. antilopaeum f. javanicum rounded extremity; semicells sub-reniform and (Nordstedt) Coesel] (Figure 61) Nurul Islam granulate; isthmus elongated, 16 µm wide and Irfanullah 1999, P. 103, Pl. 3, Figure 31. 52. Cosmarium pseudoretusum Ducellier [C. Cells 67-77 µm long, 77-87 µm broad (without pseudoretusum Ducellier] (Figure 55) Croasdale spines); sinus widely open; semicells and Flint 1988, P. 90, Pl. 42, Figures 3-5. subelliptic, paired apical spines from elevated Cells 25-37 µm long, 20-29 µm broad; sinus portion of apex; isthmus 12-16 µm wide. deep, closed; semicells 3 lobed, trapeziform, 59. Staurodesmus convergens (Ehr. ex Ralfs) basal lobe flat at bottom, retuse above, converging Lillieroth (Figure 62) Croasdale et al. 1994, P. to truncate apex; isthmus 6-10 µm wide. 41, Pl. 75, Figures 1-8. 53. Cosmarium punctulatum Bréb. (Figure 56) Cells 30-42 µm long, 32-46 µm broad; sinus Croasdale and Flint 1988, P. 90, Pl. 46, Figures open widely; semicells transversely elliptic, 8-10. lateral angles rounded-conical with stout, Cells small, 22-40 µm long, 20-38 µm broad; slightly incurved spine; isthmus 7.5-10 µm sinus linear, slightly dilated within; semicells wide.

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60. Staurodesmus dejectus (Breb. ex Ralfs) Teil. 1961, P. 107, Pl. 45, Figures 4-5. (Figures 63-64) Croasdale et al. 1994, P. 45, Cells 90 µm long, 102 µm broad with processes; Pl. 66, Figures 3-9. semicells depressed globose; long paired Cells 23-27 µm long, 25-31 µm broad; processes in 2 whorls, upper whorl divergent semicells cup-shaped, apex straight; isthmus and lower horizontal, not superimposed each slightly elongated, 5-10 µm wide; spines other, all end in 3-4 spines; margin of processes divergent, short, 2-4 µm long. with 3-4 denticulation; isthmus 27 µm wide. 61. Staurodesmus dickiei (Ralfs) Lill var. circularis 67. Staurastrum cf. tetracerum Ralfs ex Ralfs (Turn.) Croasdale (Figure 65) Croasdale et al. (Figures 75-76) Croasdale et al. 1994, P. 141, 1994, P. 46, Pl. 76, Figures 6-7. Pl. 101, Figures 1-7. Cells 21-50 µm long, 23-49 µm broad; Cells biradiate, 18-28 µm long, 18-30 µm broad semicells sem-icircular; spines small and with processes; semicell triangular; processes downwardly directed, arise from the base of divergent, denticulate, ends to 3-4 teeth; apex semicell, 3.5-6 µm long; isthmus 7-12 µm wide. concave; isthmus 4-6 µm wide. 62. Staurastrum avicula Bréb. (Figures 66-67) 68. Staurastrum tohopekaligense var. tohopekaligense f. Kouwets 1987, P. 242, Pl. 18, Figure 7. minus (Turn.) Scott et Prescott (Figure 77) Flint Cells 29-35 µm long, 35-42 µm broad with and Williamson 1998, P. 95, Pl. 9, Figure 2. spines; semicells sub-elliptical or sub- Cells 35 µm long, 33.5-35 µm broad with triangular, margins convex, angles ending in 2 processes; sinus acute, widely open; semicells small spines or 1 bifurcate spine; isthmus 9-11 obsemicircular base, straight apex; processes µm wide. in 2 whorls, 6 apical divergent, 3 lateral 63. Staurastrum cf ehrenbergianum Nägeli ex horizontal, all with 2 divergent spines; isthmus Archer (Figure 68) Croasdale et al. 1994, P. 96, 10 µm wide; processes 10 µm long. Pl. 90, Figures 6-7. 69. Teilingia granulata (Roy et Bisset) Bourrelly Cells 30-32 µm long, 28-34 µm broad, (Figure 78) Croasdale et al. 1994, P. 169, Pl. moderately constricted; semicells oval to 130, Figures 5-8. trapeziform, upper and lower angles projected Filaments long, straight; cells 6-15 µm long, 7- into bifurcate or emarginate verrucae. 17 µm broad; sinus deep rounded; apex flat with 64. Staurastrum manfeldtii Delp. (Figures 69-71) 2 granuales at each side; sides rounded, with Flint and Williamson 1998, P. 93, Pl. 9, Figure 5. clusture of granules; isthmus 3-7 µm wide. Cells 37-58 µm long, 33-100 µm broad with 70. Onychonema laeve Nordstedt (Figure 79) processes; semicells with swollen base, apex Croasdale et al. 1994, P.159, pl. 132, Figures 6-7. convex with row of emarginate verrucae; Filaments long, sheath present; cells 12-23 µm processes tapered, slightly convergent, long, 14-30 µm broad without spine; semicells tuberculate or spinulose, end to 3-4 spines; elliptic, 2 rods arise below apex; spine stout, isthmus 13-15 µm wide. convergent; isthmus 3-8 µm wide. 65. Staurastrum cf margaritaceum Meneghini ex 71. Hyalotheca dissiliens Bréb. ex Ralfs (Figure Ralfs (Figures 72-73) Croasdale et al. 1994, P. 80) Kouwets 1987, P. 256, Pl. 21, Figures 4-5 114, Pl. 104, Figures 1-7. Filaments long, straight, cylindrical with sheath; Cells 23-30 µm long, 15-48 µm broad with cells cylindric-discoidal, 12.5-15 µm long, 22.5- processes, constriction shallow; semicell 25 µm broad, width greater at middle, slightly fusiform; processes 5, short, convergent, with constricted, lens shaped space between cells; 3-4 circles of granules; isthmus 6-11 µm wide. sinus very shallow; isthmus 20-24 µm wide. 66. Staurastrum sexangulare Bulnheim ex 72. Bambusina brebissonii Kutz. ex. Kutz. [B. Rabenhorst var. bidentatum Gutwinski [S. borreli (Ralfs) Cleve] (Figure 81) Croasdale et elegans Borge] (Figure 74) Scott and Prescott al. 1994, P. 148, Pl. 144, Figures 1-8.

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Filaments long, twisted, thin sheath present; cells were dominant there but in Dhimal pokhari, barrel-shaped, 20-50 µm long, 10-30 µm broad; dominant genera were Cosmarium and Staurastrum. semicells with supra-isthmial swelling; apex 10-24 Similarly, Gonium, Cylindrocapsa, Glaucocystis, µm broad; isthmus 13-20 µm wide. Oocystis and Spirogyra reported in Bagh Jhoda wetland, Morang (Rajopadhyaya, 2016) were not In the present study, the maximum algae were reported from Raja-Rani wetland but rests of the reported in summer (38 spp.) followed by winter genera were common in both. The occurrence of (36 spp.) and rainy seasons (29 spp.). It indicated maximum number of algae in winter and least that, the summer and winter are the favourable number in rainy season at Bagh Jhoda wetland is season for algal growth. The change in season, quite differ from present study. ending of cold and starting of warm climate may influence their number by rapid growth and The morphological features of Staurastrum cf reproduction of algae. The appropriate sunlight, ehrenbergianum, S. cf. margaritaceum and S. cf optimum temperature, abundant nutritients and tetracerum reported from Raja-Rani wetland agree suitable pH were the essential factors for the well with the description given by Croasdale et al. existence of maximum algae in that particular (1994) except their dimensons. season. In rainy season, excessive flooding results the pond water muddier with less transparency and Conclusion thus disturb total aquatic ecosystem causing many algae to the death. The study showed that the algal flora of Raja-Rani wetland is rich and interesting. Out of 72 green algae Except the genera viz., Eudorina, Pandorina, reported from this wetland, desmids were dominant Tetraedron, Crucigenia, Netrium, Actinotaenium, with the following genera: Cosmarium (24%); Teilingia and Bambusina, all other algal genera were Staurastrum (10%); Euastrum, Scenedesmus, found in all three seasons. Netrium was not reported Micrasterias (each 6%) etc. The rare but interesting in winter whereas Eudorina, Pandorina, Tetraedron, species reported here were Tetraedron minimum, Crucigenia, Actinotaenium, Teilingia and Euastrum elegans, Micrasterias zeylanica, Bambusina were not reported in rainy seasons.The Cosmarium gracile, C. burkilli var. depressum, C. dominant genera in winter season were Cosmarium, connatum, C. meneghinii, C. obtusatum, C. Staurastrum, Euastrum, Closterium and pyramidatum and Bambusina brebissonii. It was Ankistrodesmus where as scarcely reported were observed that the number and types of algal species Netrium, Onychonema, Sorastrum etc. Similarly, in the lake have changed according to the seasons. dominant genera in summer were Cosmarium, Further extensive explorations are essential to Staurastrum, Closterium, Xanthidium, explore more interesting and new algae for their Staurodesmus, Scenedesmus and Hyalotheca and proper documentation and conservation. rare were Netrium, Bambusina etc. While observing the previous reports from Acknowledgements surrounding areas; genera viz., Oocystis, We would like to thank the Chairman, Department Dichotomosiphon, Spirogyra, Chara, Zoochlorella of Botany, Post Graduate Campus, Biratnagar for and Westella reported from Haseena wetland, laboratory facilities. First author is grateful to the Sundardulari (Rai, 2017) were not observed here but Department of Plant Resources, Thapathali and rest of the algae were common. Similarly, Nepal Academy of Science and Technology (NAST) Desmodesmus, Acutodesmus, Micractinium, for the financial assistance. Finally, we would like Ulothrix and Mougeotia reported from Raja-Rani to give special thank to the local people of Raja- lake, Dhankuta (Shrestha, 2015) were not found in Rani for their kind help and co-operation during our the present study. Scenedesmus and Selenastrum field trips.

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Figures: 1. Eudorina elegans 2. Pandorina morum 3. Sphaerocystis schroeteri 4. Dictyosphaerium pulchellum 5. Gloeotaenium loitelsbergerianum 6. Nephrocytium agardhianum 7. Pediastrum tetras var. tetraodon 8. Sorastrum spinulosum 9. Tetraedron minimum 10. Dimorphococcus lunatus 11. Coelastrum cambricum var. intermedium 12. C. microsporum 13. Crucigenia crucifera 14-15. Scenedesmus acutiformis 16. S. arcuatus var. platydiscus

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Figures: 17. Scenedesmus bijugatus var. gravenitzii 18. S. hystrix 19. Ankistrodesmus falcatus 20. A. spiralis 21. A. spiralis var. fasciculatus 22. Selenastrum bibraianum 23. Kirchneriella lunaris 24. Stigeoclonium fasciculare 25. Bulbochaete intermedia 26. Oedogonium undulatum 27. Netrium digitus 28. Closterium dianae 29. C. ehrenbergii 30-31. C. gracile 32-33. Pleurotaenium trabecula

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Figures: 34. Euastrum elegans 35. E. insulare 36. E. spinulosum 37. E. turneri 38. Micrasterias mahabuleshwarensis var. surculifera 39. M. pinnatifida 40. M. radians 41. M. zeylanica 42. Actinotaenium cucurbita 43. A. subglobosum 44. Cosmarium burkilli var. depressum 45. C. connatum 46. C. contractum 47. C. javanicum 48. C. lundellii 49. C. lundellii var. circulare

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Figures: 50. Cosmarium lundellii var. ellipticum 51. C. meneghinii 52. C. obsoletum 53. C. obtusatum 54. C. portianum 55. C. pseudoretusum 56. C. punctulatum 57. C. pyramidatum 58. C. quadrum 59. C. striolatum 60. C. subspeciosum var. validius 61. Xanthidium hastiferum var. javanicum 62. Staurodesmus convergens 63-64. S. dejectus 65. S. dickiei var. circularis

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Figures: 66-67. Staurastrum avicula 68. S. cf ehrenbergianum 69-71. S. manfeldtii 72-73. S. cf margaritaceum 74. S. sexangulare var. bidentatum 75-76. S. cf. tetracerum 77. S. tohopekaligense var. tohopekaligense f. minus 78. Teilingia granulata 79. Onychonema laeve 80. Hyalotheca dissiliens 81. Bambusina brebissonii

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Morang, Nepal. (M.Sc. Thesis), Department of Sahay, A.P., Das, P.K., & Verma, B.N. (1992). Botany, Post Graduate Campus, Biratnagar, Studies on the algal flora of Nepal-I: Chloro- Tribhuvan University, Nepal. phyceae. Geophytology, 20(2), 155-158. Rai, S.K. (2009). Some chlorophycean algae from Scott, A.M., & Prescott, G.W. (1961). Indonesian Maipokhari lake, Ilam, east Nepal. J. Nat. Hist. desmids. Hydrobiologia, 17(1-2), 1-132. Mus., Nepal, 24, 1-8. Shrestha, G. (2015). Studies on algal flora of Raja Rai, S.K. (2014). Additional desmids Rani lake, Dhankuta and its relationship with (Chlorophyceae) to eastern flora of Nepal. physic-chemical parameters of water. (M.Sc. Nepalese J. of Biosciences, 4, 40-46. Thesis), Department of Botany, Post Graduate Campus, Biratnagar, Tribhuvan University, Rai, S.K., & Misra, P.K. (2012). Taxonomy and Nepal. diversity of genus Pediastrum Meyen (Chlorophyceae, algae) in east Nepal. Our Nature, Silva, P.C. (1980). Names of classes and families of 10, 167-175. living algae. Regnum Vegetabile, 103, 1-156. Rajopadhyaya, R. (2016). Algae of Bagh Jhoda Tiffany, L.H., & Britton, M.E. (1952). The algae of wetland, Morang, Nepal. (M.Sc. Thesis), Illinois. New York: Hafner Publ. Co. Department of Botany, Post Graduate Campus, Biratnagar, Tribhuvan University, Nepal.

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New Records of Two Powdery Mildews (Erysiphales: Fungi) from Nepal

M. K. Adhikari GPO Box no. 21758, Kathmandu, Nepal E-mail: [email protected]

Abstract

The powdery mildews, Golovinomyces biocellatus (Ehrenb.) V.P. Heluta, parasitic on leaves of Ocimum tenuiflorum L. (Syn. Ocimum sanctum L.) and Pleochaeta indica N. Ahmad, A.K. Sarbhoy & Kamal, parasitic on leaves of Celtis australis L. are recorded as new to Nepal. The genus Pleochaeta is reported for the first time from Nepal. The description and distribution of both the species are provided here with.

Key words – Erysiphales, Golovinomyces, Pleochaeta

Introduction the identification of the species were based on the published monographs of Braun (2009) and Braun Very few authors have contributed their findings on & Cook (2012). Moreover the specimens were sent powdery mildews from various places of Nepal to Prof. U. Braun, Germany for authentic (Adhikari, 2009, 2017). Notably they are Adhikari identification. After identification the fungi were et al., 1997, 2001, 2006; Bhatt, 1966; Khadka & noted as new records for Nepal. The genus Shah, 1967; Khadka et al., 1968; Lama, 1976, 1977; Pleochaeta is entirely new to Nepal. The specimens Manandhar & Shah, 1975; Manandhar & Shah, gathered are housed in National Herbarium & Plant 1975; Pandey & Adhikari, 2005; Parajuli et al., 1999, Lab (KATH), Godavary, Martin-Luther-Universität, 2000; Pawsey, 1989; Singh, 1968; Singh & Nisha, Germany and Natural History Museum (NHM), TU, 1976 and Barun & Cook, 2012. The check reference Swayambhu, Kathmandu. The microscopic list of the previous documents, reports and additions description and distribution fungi in the globe have can be found in ‘Researches on the Nepalese been provided below. mycoflora-3: Erysiphales from Nepal’ (Adhikari, 2017). The recent approach on taxonomy and Enumeration of species nomenclature of this mycobiota group has tremendously changed. Golovinomyces biocellatus (Ehrenb.) Heluta, Ukrayins’k. Bot. Zhurn. 45(5): 62, 1988. Figure 325; The present study has been based on recent U. Braun & R. T. A. Cook’s (2012) Taxonomic collections. The macro and micro-photographs were Manual of the Erysiphales (Powdery Mildews).304 taken. The specimens were examined in the lab and - 305 pg. Figure 325. Figure no. 1 ABC

Figure 1: Golovinomyces biocellatus: A: Host Ocimum tenuiflorum infected with Golvinomyces biocellatus; B. Hyphae with nipple shaped appresoria C. Conidia of pathogen

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[Syn. Erysiphe biocellata Ehrenb., Nova Acta Phys.- (= Ocimum ocimi) and Oidium ocimi-sancti. Oidium Med. Acad. Caes. Leop.- Carol. Nat. Cur. 10: 211, ocimi is characterized by having nipple-shaped 1821; Erysiphe communis f. biocellata (Ehrenb.) Fr., hyphal appressoria, which undoubtedly belong to Syst. Mycol. 3: 239, 1829; Erysiphe biocellata var. Golovinomyces (Euoidium) (Braun & Cook, 2012, monardae (Nagy) U. Braun, Zentralbl. Mikrobiol. 306 pg.). Braun (1987) stated this species as parasitic 137:316, 1982; Golovinomyces simplex (Heluta) on leaves and stem of Ocimum, while Heluta, Ukrayins’k. Bot. Zhurn. 45(5): 63, 1988] Narayanswamy & Ramakrishana reported the [Anamorph: Oidium erysiphoides Fr., Syst.Mycol. species to be confined to upper surface of the leaf. 3: 432, 1832; Oidium ocimi S. Naray. & K. Ramakr., So, Narayanaswamy & Ramakrishnana proposed a Madras. Univ. Jour. 37–38: 87, 1967] new species as Ocimum ocimi, on the basis of epiphyllus whhte mycelium, hyphae hyaline, Specimen examined – Parasitic on leaves of Ocimum branched; conidiophores erect, cylindric, in chains, tenuiflorum L. (= Ocimum sanctum L.). 27 – 42 X 16- 22 µm U, Braun (1987, pg. 245). U. Bhanimandal, Lalitpur, 2067/3/24, no 201104.02 Braun & R. T. A. Cook (2012, 304 pg.) in their KATH, MK Adhikari, Germany, (HAL no. monograph treated Erysiphe biocellata Ehrenb [in unknown). Very commonly found in Kathmandu U. Braun, A monograph of the Erysiphales (powdery valley on Ocimum. Many of other collection mildews).Nova Hedwigia. 89, 245, 1989] and numbers are not provided here. Ocimum ocimi as . Mycelium parasitic on leaves stems an, According to Bruan & Cook (2012) another species amphigenous, covering partially or entire surface of Oidium ocimi-sancti Puzari, Sarbhoy, Ahmad & leaf, effused, thin, white irregular, Hyphae 4 – 5.4 Argawal [Indian Phytopathol. 59(1): 75 (2006)] µm broad, thin walled, hyaline, cylindrical, straight parasitic on Ocimum tenuiflorum L. (= Ocimum to curved. Appresoria nipple shaped. Conidiophores sanctum L.) is unclear. The combination of nipple 40.5 – 100 X 10.8 – 13.5 µm, thin walled, hyaline, shaped appressoria, conidia formed singly and some erect, cylindrical, with 2 – 3 shorter cells. Conidia lateral germ tubes is very unusual and doubtful. It 18 - 37.8 X 10.8 – 24 µm, ellipsoid-ovoid, to does not fit to any of the known anamorphic genera. dolliform, thin walled, hyaline, Cleistothecia not (Braun & Cook, 2012, 629 pg.). found. This species was recorded as Erysiphe biocellata Comment - Ocimum tenuiflorum L. (= Ocimum Ehrenb and Oidium ocimi-sancti Puzari, Sarbhoy, sanctum L.) is parasitized by Erysiphe biocellata Ahmad & Agrawal, Indian Phytopathol. 59(1): 75 Ehrenb (in Braun, A monograph of the Erysiphales (2006) (in Adhikari, 2014, 2017 and Adhikari & (powdery mildews). Nova Hedwigia. 89, 245, 1989) Bhattarai, 2014.

Figure 2: Pleochaeta indica: A: Host Celtis australis infected with Pleochaeta indica; B: Conidiophore; C: Hyphae with branched appresoria; D: Conidia of pathogen; E: Germinating conidia

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Not yet reported from Nepal. (americana, crassifolia, laevigata, lindheimeri, occidentalis, pumila, sp.) from North America Distribution – Caucasus, all Europe. USSR, , (USA), The species of Podosphaera and Erysiphe , India, North and South Africa, Africa, North differ from the present specimen in their structure America, South America (Braun, 1987; Braun & hyphal appresoria and conidia. Cook, 2012). The present specimen is more allied to Leveillula Pleochaeta indica N. Ahmad, A.K. Sarbhoy & species and resembles more with the description of Kamal, Mycol. Res. 99: 375, 1995, Figure 298; Braun Pleochaeta in it’s hyphal foot cells, nature, structure & Cook (2012) Taxonomic Manual of the and formation of conidia on conidiophores. Erysiphales (Powdery Mildews). 282pg, Figure 298 Pleochaeta shiraiana (Henn.) Kimbr & Korf, b. Figure no. 2 ABCDE parasitic on Celtis [Africana (= kraussiana), biondii, Mycelium superficial, hypophyllous, colonies white, bungeana, eriocarpa, rhamnifolia, sinensis var. thin walled. cylindrical, hyaline dense, effuse or in sinensis and var. japonica, tetrandra (= patches, often confluent or covering the entire yunnanensis), vandervoetiana] are reported from surface of the leaves, persistent. Hyphal appressoria Japan. lobed, 15 µm. solitary. Conidiophores arising from According to U. Braun, this species seems to be superficial hyphae, erect, slender, about up to 200 × Pleochaeta indica Ahmad et al., only known from 5–7 µm. Foot-cells twisted, followed by 2–3 shorter northern India (Himachal and Uttar Pradesh), which cells, forming conidia singly. Conidia dimorphic. is confined to Celtis australis (Personal communication Primary conidium oblong-cylindrical, ± lanceolate, in 25th July 2017). It is morphologically barely up to 56 x 10 - 15 ìm . Secondary conidia ellipsoid, distinguishable from Pleochaeta shiraiana but fusiform to clavate, pointed apically, 35– 66 × 15– genetically clearly distinct (Braun & Cook (2012:285). 25µm. Germ tubes apical, short to moderately long, 40 µm. straight, simple . Not yet reported from Nepal. Specimen examined – Parasitic on Celtis australis Distribution – India and Nepal. L., Harihar bhawan, Pulchowk, Lalitpur, 2074. 3 14 B. S. / 2017.6. 28 AD, no. 2074.3, (KATH), M. K. Acknowledgements Adhikari, HAL no. 3214 F, Germany; Pulchowk, Lalitpur, no. 2074.5.1. (16.8.2017), no, 2074.5 The author is obliged to Prof. Dr. Uwe Braun, KATH.Adhikari. Martin-Luther-Universität, Germany for his help in identification of the materials. Comment - Singh (1968) reported Oidium sp. on Celtis australis, collected from Tri Chandra College, References Kathmandu, which was mentioned as needed for recollection and study (Adhikari, 2017). Adhikari, M. K. (2009). Researches on the Nepalese mycoflora: Revised account on the history of According to Barun & Cook (2012), monograph, the mycological explorations (p. 90). Kathmandu, genus Celtis is parasitized by Podosphaera, Erysiphe Nepal. and Pleochaeta species. Podosphaera phytoptophila (Kellerm & Swingle) Braun & Takam, was reported Adhikari, M. K. (2012). Erysiphe cichoracearum on Celtis occidentalis, which is confined to USA, DC: the powdery mildew (Erysiphales) from Kansas. Erysiphe celtidis (Shvartsman & Nepal. Bull. Dept. Pl. Res., 34, 18 - 21. Kusnezowa) U. Braun & Takam. has been reported Adhikari, M. K. (2012). The Oidium species: parasitic on Celtis (australis, caucasica, glabrata) powdery mildews (Erysiphales) from Nepal. Bull. from Asia. Erysiphe parvula (Cooke & Peck) U. Dept. Pl. Res., 34, 26- 30. Braun & Takam., is reported parasitic on Celtis

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Adhikari, M. K. (2017). Researches on the Nepalese Khadka, B.B., Shah, S.M., & Lawat, K. (1968). Plant mycoflora-3: Erysiphales from Nepal. (p. 35). diseases in Nepal: A supplementary list. Tech. Kathmandu, GPO Box no. 21758, Nepal: K.S. Doc. 66 FAO Pl. Prot. Comm. South - Adhikari. and Pacific Region, Bangkok, . Adhikari, M. K., & Bhattarai, K. R. (2014). Lama, T.K. (1976). Some parasitic fungi from Catalogue of fungi preserved in National Pokhara (west Nepal). Jour. Sc., 6, 49-52. Herbarium and Plant Laboratories. (p. 107). Kathmandu, Nepal: Ministry of Forests and Soil Lama, T.K. (1977). Some parasitic fungi from Conservation, Department of Plant Resources, Pokhara. Jour. Nat. Hist. Mus., 1, 63-66. Lalitpur, Godavari, Nepal: National Herbarium Manandhar, K.L. & Shah, S.M. (1975). List of plant and Plant Laboratories. diseases in Nepal: second supplement. Tech. Doc. Adhikari, M.K., Manandhar, V., Joshi, L., & Kurmi, 97. FAO Pl. Prot. Comm. South - East Asia and P.P. (2006). Die back of sissoo in Pacific region, Bangkok, Thailand. western tarai belt of Nepal. Bull. Dept. Pl. Res., Pandey, B., & Adhikari, M.K. (2005). Odium citri: 27, 30 – 38. the Citrus disease in Nepal. Bull. Dept. Pl. Res., Bhatt, D.D. (1966). Preliminary list of plant diseases 26, 6 – 7 recorded in Kathmandu valley. Jour. Sc., 2(1), 13- Pawsey, R.G. (1989). A check reference list of plant 20. pathogens in Nepal. FRIC Occasional paper. no. 1/89, Kathmandu. Nepal. Braun, U. (1987). A monograph of of the Erysiphales (powerdry mildews). Beihefte Zur Nova Singh, S.C. (1968). Some parasitic fungi collected Hedwigia, 89, 1-700. from Kathmandu valley (Nepal). Ind. Phytopath., Braun, U., & Cook, R.T.A. (2012). Taxonomic 21, 23-30. manual of the Erysiphales (powdery mildews). Singh, S.C., & Nisha (1976). A contribution to the CBS Biodiversity Series, 11, 1-707 parasitic mycoflora of Nepal. Jour. Sc., 6, 11-14. Khadka, B.B. & Shah, S.M. (1967). Preliminary list of plant diseases recorded in Nepal. Nep. Jour. Agri., 2, 47-76.

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Records of Bryophytes from Godawari-Phulchoki Mountain Forest of Lalitpur District, Central Nepal

Nirmala Pradhan Natural History Museum, Swayambhu, Kathmandu, Nepal Email: [email protected]

Abstract

The species record of bryophytes made in different seasons of the year 2014-2017 has been presented in this paper. This revealed a list of 105 species of three classes of this plant which comprised two species of Anthocerotae, 25 species of Hepaticae including Jungermanniales, Marchantiales and Metzgeriales and 76 species of Musci. Two new records made in this study include Caduciella mariei (Besch.) Enroth (Leptodontaceae) and Bryoerythrophyllum gymnostomum (Broth.) P.C. Chen of Pottiaceae. Total diversity of 205 species of bryophytes has been counted in the Phulchoki mountain which also includes the diversity record made in the year 2000. This comes to be about 16.90% of the total species record of the country (1,213 species).

Key Words: Anthocerotac, Bryoerythrophyllum gymnostomum, Caduciella mariei, Hepaticae, Musci, New record

Introduction & Higuchi, 1995; Higuchi & Takaki, 1988, 1990). Takaki (1988), made a noteworthy study of Phulchoki, the southern ridge of Lalitpur district still bryophytes of the Godawari- Phulchoki, though a retains pristine forest at its altitudinal range of 1500- brief study, he made records of 6 spp. of mosses at 2747 m where varying ecosystems displaying diverse habitats for many species of bryophytes of different 1,530 to 2,500 of elevations. Higuchi & Takaki status categories are found. (1990), during their study in Central Nepal, they brought a list of 58 spp. of mosses of different status The distribution and species richness of bryophyte categories from Godawari Phulchoki area. Pradhan show a relationship with the physical gradients that (2000b) made a brief survey of the bryoflora in this differs with every changing altitude. Optimum level mountain which came up with a list of 30 spp. of of gradients favors the peak diversity of this flora at Hepaticae and 111 spp. of Musci including a new certain altitudinal habitat where temperature, humidity, record. Kattel & Adhikari (1992) have also soil pH, moisture and canopy are perfectly acting. mentioned 34 spp. of mosses of the Phulchoki The distribution of bryoflora is associated primarily mountain in their publication. with the depth of litter, the air temperature and the precipitation (Sun et al., 2013). According to Pharo Materials and Methods & Beattie (2002), substrate plays an important role in bryophyte diversity and composition both. Porley This study made at different altitudinal habitats in (2005) mentioned that the macroclimatic factors like the Phulchoki mountain forest covering different rainfall and temperature play important role for the seasons of the year 2014 to 2017. The species which distribution and diversity richness of bryophytes in were not readily identifiable in the field were a certain area. Similarly, the effect of shade, habitat collected and confirmed their identification humidity and temperature act significantly in the consulting books of Gangulee, 1969-1980, 1985; distribution of bryophytes specifically in the Eddy, 1988, 1990, 1996; Kashyap, 1972; Pradhan, mountain habitats (Pentecost, 1988). 2000a, 2000b and Smith, 1996. Book of Brummitt Notable works on Nepalese bryophytes especially & Powell (1992) was consulted for author’s citation. of the Godavari- Phulchoki mountain were done by Vegetation components associated to bryophyte the Japanese Bryologists in different periods (Furuki habitats were identified consulting the Flora of

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Phulchoki and Godawari (DPR, 1997). Collected The total record has been classified into 205 spp., specimens have been deposited in the Natural 106 genera, 48 families of the three classes of History Museum, Kathmandu. bryophytes. The class Anthocerotae included 2 genera, and 2 spp. under one family Study area Anthocerotaceae. Hepaticae came up with 27 genera, Phulchoki Mountain is located at its geographical 45 spp. under 17 families and the class Musci position of 27°33-27° 36’ N and 85°22’ – 85°36’ E represented 77 genera, 158 spp. of 30 families (figure with area coverage of 50 sq. km at southwest corner 2). of the Lalitpur district (figure 1). Temperature in this This study brought a list of diverse species of mountain normally varies from 10°C to 14°C in Marchantiales which represented 12 genera and 22 winter and 15°C to 30°C in summer. Snow fall is a spp. categorized into six families. Jungermanniales usual phenomenon in cold winter and heavy rainfall revealed 19 spp. of 12 genera and 8 families. occurs during monsoon. This mountain represents mixed broad leaved evergreen forest below 1,850 The largest record of 62 spp. of Hypnobryales was m, Oak (1,850-2,300 m) and dry oak made in the class Musci categorized into 32 genera forests above 2,300 m and dry Oak forest above and 11 families. Hookeriales and Leucodontales 2,300 m (Acharya et al., 1992). represented 1 sp. each of one genera and one family respectively (Appendix 1).

Figure 1: Black mark indicates study site area Figure 2: Species diversity of bryophytes at Godawari- Results and Discussion Phulchoki mountain forest This study recorded, 64 spp. of bryophytes which Caduciella mariei (Besch.) Enroth, Journ. Bryol. 16: now totals 205 species including the previous list of 612, 1991. 141 species (Pradhan, 2000b). This comprises 2 spp. of Anthocerotae, 15 spp. of Hepaticae and 47 spp. Pinnatella microptera M. Fleisch., Musci Fl. of Musci. Hepaticae represented 9 spp. of Buitenzorg 3: 915, 1908; Gangulee, Mosses E. India & Jungermanniales, 5 spp. of Marchantiales and 1 spp. Adj. Reg. 5: 1434, 1976. of Metzgeriales. Recorded 47 spp. of Musci comprised 1 spp. of Dicranales, 9 spp. of Eubryales, Leptodontaceae 2 spp. of Fissidentales, Funariales 1 spp., Plants robust, dark green, 4-5 cm long and 1 mm Hypnobryales 14 species, Isobryales 9 spp., wide with expanded leaves, irregularly branched Leucodontales 1spp., Polytrichales 2 spp., Pottiales growing intermingle with Piper longum L. on the 8 species including two new records of Caduciella bark of a tree. Stem yellowish brown and stiffy. mariei (Besch.) Enroth of Leptodontaceae and Leaves spathulate, small, 0.7 x 0.2 mm in size with Bryoerythrophyllum gymnostomum (Broth.) P.C. Chen round apex, percurrent costa ends far below apex of Pottiaceae. and entire margin, appressed to the stem on drying.

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Laminal cells small hexagonal, 6x9 µm in diameter. Philippines, Singapore, Sumatra, Thailand and Sporophytes not known (figure 3, 4). (MOBOT Result) Remarks: New records for Nepal. Bryoerythrophyllum gymnostomum P.C. Chen., Hedwigia, 80: 255, 1941; Gangulee, Mosses E. Ind. & Adj. Reg. 3: 745-747, 1972; Jiang Li-Xing et al., Moss Fl. China 2: 139, 2001. Didymodon gymnostomus Broth., Symbolae Sinicae 4: 39, 1929.

Pottiaceae This is a small, yellowish green acrocarpous moss with erect branched stem up to 12-20 mm tall; Leaves curved and contorted on drying and erectopatent when moist, ovate-lanceolate, 1.3 x 0.3 mm in size with acute apex and entire margin, costa Fig.3: Caduciella mariei (Besch.) Enroth.tif percurrent ends few cells below apex, apical cells elongated rectangular, 17 x 5 µm in diameter, middle laminal cells more or less quadrate, papillous, 9x8 µm and basal laminal cells quadrate, larger towards the costal region, 1 5 x 12 µm in diameter. Seta erect, light brown, 6-8 mm long, erect with cuculate long beaked operculum, peristome indistinct; spores spherical, yellowish-green, thin walled, 13-15 µm in diameter (figure 5).

Fig.4: Caduciella mariei, a leaf.tif

Habitats: Epiphytes on woody tree trunk. Specimens Examined: C. Nepal, Lalitpur, Godawari Bus Park, 1515 m, 27°35’22"N, 85°22’44"E, 19.09.2016, Pradhan & Shrestha, H.S., NP969 (NHM). Distribution: , Borneo, China, India, Macaronesia, , Papua New Guinea, Fig.5: Bryoerythrophyllum gymnostopum Broth

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Habitat: Dense tufts on soil.. Godawari-Phulchoki area. I am equally thankful to the Senior Botanists of the Department of Plant Specimens Examined: C. Nepal, Lalitpur, Phulchoki Resources Dr. Nirmala Joshi, Ms. Rose Shrestha, Marble factory site, 1644 m, 27°36’36" N, 85°22’2"E, 19.09.2016, Pradhan, NP1079 (NHM). Mr. Ramesh Basnet, Head of the Herbarium Section and Mr. Deepak Lamichhane, Head of the Garden Distribution: China and India (Chen, 1941). Section for their significant cooperation during field programs. I wish my sincere thanks to Mr. Dhana Remarks: New records for Nepal. Raj Kandel and other staffs of DPR who made Caduciella mariei is closely related to the genus significant cooperation in this work. My thanks also Leucodon due in having pinnate mode of branching, go to Mr. Devendra Maharjan, Ms. Ranju Shrestha narrowly imbricated leaves with broad obtuse apices and Sijan Shrestha of Natural History Museum for and entire margins (Enroth, 1991). This species is their kind help in the field. Last but not least I am known from the Tanzania as Pinnatella mariei thankful to Ms. Hira Shova Shrestha, Botanist and (Mattila & Enroth, 1990) and India as Pinnatella Mr. Madan K. Shrestha, Zoologist who also microptera (Robinson, 1964). With extensive study accompanied me in a separate field study program for its taxonomic relationships with Pinnatella in the Godawari-Phulchoki area. mariei now its name has been revised by Enroth (1991) as Caduciella mariei under the family References Leptodontaceae. Acharya, S.K., Shrestha, K.K., & Shah, J.P. (1992). Quantitative study of the family on Conclusion Phulchoki hill, Nepal. In Islam AKMN. QA Many preferable habitats for bryophytes were noted Fattah, IA Muttaqi and AZIZ (Eds.), Plant Science at Phulchoki mountain forest which included and Man; Problems and Prospects. Proc. Intl. substrates, tree barks, tree canopy, exposed roots, Bot. Conf., 10-12 January, 1991. Dhaka, moist ground, sandy soil, shade, exposed rocks, brick Bangladesh: Bangladesh Botanical Society. and concrete walls etc. The epiphytic species were Brummitt, R.K., & Powell, C.E. (1992). Authors of noticed mainly on the trunks of Alnus nepalensis, Plant names. UK: Royal Botanic Garden, Kew. axillaris, Schima wallichii, Ilex excelsa etc. Total diversity record of 205 spp. of Chen, P.C. (1941). Didymodon gymnostomus Broth. bryophytes has been made in the Phulchoki forest in Handel-Mazzetti. Symb. Sin., 4(39), 80, 255, that also combined the previous record of 141 spp. 1929 (Pradhan, 2000b). This included 2 spp. of DPR (1997). Flora of Phulchoki and Godawari. (pp. Anthocerotae, 15 spp. of Hepaticae and 47 spp. of 1-144). Thapathali, Kathmandu: Department of Musci including two new records of Caduciella Plant Resources, Ministry of Forests and Soil mariei of the family Leptodontaceae and Conservation. Bryoerythrophyllum gymnostomum of Pottiaceae Eddy, A. (1988). A Handbook of Malesian Mosses, Acknowledgements I. London: Nat. Hist. Mus. (BM). I am thankful to former Dean of the Institute of Eddy, A. (1990). A Handbook of Malesian Mosses, Science and Technology, Tribhuvan University, Mrs. II. London: Nat. Hist. Mus. (BM). Chirika Shova Tamrakar for providing funds for field Eddy, A. (1996). A Handbook of Malesian Mosses, work. Prof. Dr. Dharma Raj Dangol, now Chief of III. London: Nat. Hist. Mus. (BM). the Natural History Museum is acknowledged for planning and implementing this work in the Enroth, J. (1991). Notes on the Neckeraceae (Musci), 10. The Taxonomic Relationships of Pinnatella 25 2018 Journal of Plant Resources Vol.16, No. 1

mariei with the description of Caduciella Mattila, P., & Enroth, J. (1990). Pinnatella mariei (Leptodontaceae). Journ. Bryol., 16, 611-618. (Besch.) Broth: Newly recorded for the continental Africa and Tanzania. Journ. Bryol., Furuki, T., & Higuchi, M. (1995). Cryptogams of 16, 117-118. the Central and Eastern Nepal. Vol. III. In Watanabe and Hagiwara (Eds.), Hepaticae Pentecost, A. (1998). Some observations on the from Nepal collected by the botanical expedition biomass and distribution of cryptogamic of the National Science Museum, Tokyo in 1988. epiphytes in the upper montane forest of the 2. Metzgeriales and Marchantiales (pp. 143-149). Rwenzori Mountains, Uganda. Global Ecol. Tokyo, Japan: Department of Botany, National Biogeogr. Lett., 7, 273–284. Science Museum. Pharo, E.J., & Beattie, A.J. (2002). The association Gangulee, H. C. (1969-1980). Mosses of eastern between substrate variability and bryophyte and India and adjacent regions. Fasc. 1(8), 1-2145. lichen diversity in eastern Australian forests. Bryologist, 105, 11–26. Gangulee, H. C. (1985). Hand book of Indian Mosses. New Delhi, Bombay, Kolkata, New York: Porley, R.H.N (2005). Mosses and liverworts. Amerind Pubs. Co. Pvt. Ltd. London, UK: Harper Collins Publishers. Higuchi, M., & Takaki, N. (1988). Cryptogams of Pradhan, N. (2000a). Materials for a checklist of the Himalayas Central and Eastern Nepal. Vol. I. bryophytes of Nepal. (pp. 1-89). London: The In M. Watanabe & S. Malla (Eds.), A Collection Natural History, Museum. of the Hypnaceous mosses from the Kathmandu area, central Nepal (pp. 171-175). Tokyo, Japan: Pradhan, N. (2000b). Bryophytes of Phulchowki, Department of Botany, National Science central Nepal. Journ. Nat. Hist. Mus.,19, 57-81. Museum. Robinson, H. (1964). A small collection of Higuchi, M., & Takaki, N. (1990). Mosses from bryophytes from upper Assam, India. Journ. Nepal collected by botanical expedition of Hattori Bot. Lab., 27, 124-130. National Science Museum, Tokyo. In M. Smith, A.J.E. (1996). The Liverworts of Britain and Watanabe & S.B. Malla (Eds.), Cryptogams of Ireland. (pp. 1-362). Cambridge, UK: Cambridge the Himalayas, 2, 121-161. University Press. Jiang, Li-Xing, Crosby, M., & He, S. (2001). Moss Sun, S.Q., Wu, Y.H., Wang, G.X., Zhou, J., Yu, D., . Vol. II. (pp. 283). St. Louis, Bing, H.J., & Luo, J. (2013). Bryophyte species America: Missouri Bot. Garden Press. richness and composition along an altitudinal Kashyap, S. R. (1972). Liverworts of the western gradient in Gongga Mountain China. PLoS Himalayas and the Punjab plane. Delhi, India: ONE 8:e58131.doi:10.1371/ Research Co. journal.pone.0058131/(https://doi.org/10.1371/ journal.pone.0058131) Kattel, L.P., & Adhikari, M.K. (1992). Mosses of Nepal. (pp. 107) Nepal: Natural History Society Takaki, N. (1988). Some noteworthy mosses of the of Nepal. Kathmandu valley. In M. Watanabe & Malla, S.B (Eds.), Cryptogams. Himalayas, 1, 161-165. MBG Research. An annotated checklist and atlas of mosses of Thailand. Retrived from http:// www.mobot.org/MOBOT/moss/Thailand/thai- c.shtml.

26 2018 Journal of Plant Resources Vol.16, No. 1 , 2 , 7 9 m ) 1 s I . 0 d 7 2 T r ( 0 5 o i , , 1 c r , , m e o m i t n r h t 0 e 0 c a s 5 d 0 u u r 6 H 5 g a o 1 , i 1 i g , 2 . v H i i i , 7 e r i r k , 9 r a a n o 8 1 P a h 8 . w w t c 9 7 a a u l 1 0 d d z u . . i o o h 8 5 0 M G P G 1 . , ) e n ; , , ) p ; a M E E ) , o h ” ” l M . , H d - s 6 6 5 M s 7 i a H , N , 5 5 r e 7 r 9 , n H ( t ’ ’ . a i E 7 N , P 7 y E E 2 2 , ( a S ” N 8 P w , . t P ” ” h ( N 2 2 , , 4 E 0 a e 3 n ° ° 3 3 p N t H N 4 1 ’ 3 4 d N N 8 u 5 5 2 2 ’ o y , , E 1 ” 3 n 5 ’ ’ 3 o 7 o 8 8 y ” 2 h a ’ ” n a 5 2 7 5 5 P . r P , , 6 2 G . p h ’ a 5 1 ) h 9 M 2 2 . i ’ t P ) ° B N . ; h ’ 5 3 N 2 ) N N ° ° d s , p 5 ) ) 5 ’ ° , M N r d 5 8 ” ” a e 5 5 . M 3 n E . 8 E 4 r ° 7 r e a M 3 9 9 8 8 ) ° . H M M a n S ” r , , 2 ° 5 H h h 2 P . ) 4 4 , , 7 a H h t 8 ° P ’ 5 ’ N d 7 8 H H , , S M N N h 2 H d ( o 1 5 N N e , 5 1 5 M N e 2 , ( 2 ’ N N a d , t m * , ( H ” ” 8 e 3 0 3 n , ( ( . 1 r 2 t 2 H a N a i y n h ° ° 0 0 , 2 m r 5 N 5 1 1 t P 3 y ” 2 . h h a m 3 3 7 7 4 4 i ( N ’ t 2 0 2 m 7 ° N P , h 5 ’ ’ 1 h ( p 8 1 6 2 2 s l 5 0 i ” 4 8 8 w 5 a , P p 8 9 3 3 i 1 d 0 1 7 e , , i . 3 9 ’ 2 p 6 1 e x 8 P P 1 a r 3 3 o 6 e t N ° P P p 8 e 8 5 5 r e 8 , t s , ° ° m m 0 h 1 y i N N e 7 1 , 0 3 1 2 y , P 7 7 N N n . 1 r s S , h N , , ’ , 7 7 2 ° , 6 ) 1 , a h , 6 2 2 a P a n ” p i n n n n l 6 4 8 8 , 7 1 h p P i 1 , , i r e e m 2 & a a a a w i 1 M 3 4 4 2 N 0 r d p a 0 o m 4 a ° h h h h N p 0 m m 1 1 , a 2 a 0 m n s e H ’ 2 n . e d 7 i d d d d w , r . 2 , , 6 a 5 , , . 5 . a 5 8 i y i m c a 1 a a a a , o 2 N 4 P 1 h 6 r r 6 6 r r r r r 3 S 5 h 2 2 ( e 0 d 7 , 0 . , 5 5 d a a . ° G 1 1 1 o . 0 d P P P P 5 5 l p o 1 t - . 2 a 1 2 i 8 7 , 0 0 m a H 4 , , , , 1 1 w w r c a S 0 i 5 2 r , 5 o G 0 2 2 2 , e e e e 2 a a , , a i e 2 . . k P - 2 s t t t t 0 6 1 P , , a . r r , d d F 9 4 o , 2 y y y y D D , , , P a a h 9 n y o o e m m h 0 0 t 6 h h h h m I e n k e 0 l . . . d O O m w s N l n c . 2 G G p p p p ) e c d l 0 0 o 9 4 . n 7 i i i i e a - - , b o 0 , r x ) d 8 o . r M M u t a 0 0 o 1 2 r i 0 p p p p d n n i r M a 2 I I 2 h h a h 0 , 6 e e S S e e R n R e e 5 o a M , , 5 d C C c H G S 2 1 e , , , , P , , , , d d 1 E I I G E E M G 1 r r H . h S , , a 6 6 7 6 6 6 6 n , - - - - N t i i ” ” ” a a i , i i i i i g ( & h 1 1 1 1 1 1 1 r a e N r r r r r r o k k 4 t 0 3 a r ( e 0 0 0 0 0 0 0 . G G D a a a a a a s n 5 o o 4 i 4 2 p ) a B 2 2 2 2 2 2 2 B ’ e ’ ’ a 5 7 h h e e ...... v O w w w w w w h - r p 2 2 5 o h c c a 6 0 M n n 4 4 9 1 5 5 9 a a a a a a d h h l l i i 2 2 8 n d M 9 1 0 0 0 0 0 0 0 d d d d d d u g X A H u u S ° . . ° . ° . . . . I p p a h o P P o a o o o o l l a t r h h t 5 4 4 5 0 5 8 5 5 9 N C P 8 S ( G I P N 2 & N 2 8 2 8 P 0 0 G 0 1 A E N G A G B G G ) . s n n i f e f k t i a r . o h n & a t . d i . . c r r t h t l b S r o M . o n a c d t s o l . ) o r C n m . s s i u o G k m u ) a . e M . ( L s . . h u e h m D ) t s L o ) M h h i u s . P ( r D N m e s . p p . e ( h P u ) s n D e e - r e W . t C m L t t s i e u ) ( ) h a . a L r r . S . S u N s a h i ( t d l E ( t n a b L a u L i s v ( a i u i ( t u n i ( n s l r o c n s g m a n a w a s a e o i r t s F a r i i i e n y u t t i t d c a l c l s . v a i e i d r p a a l a o l e n o e d c l t o f a s e a L a u i s a a p a e c a o u i i l i l v n p m p u u s m i h r n n e a a i s G s u a d e m t p n n c n M h i o o h y a a f r c e a a a a r ) a a c p . i i i i s e g o e t a m m s e e y c n n n n i o r c r r o t o t c n n s o a a a a o e e o s p r l l l l n o u u e e l l l l g g e l y e h e e m o t i l t t a r u j u u u j n n u d e a h a n r h r r r e e y u u D C A C O F L C P F F F H J L J h s e p s l e o a l t e y a o i a r e r e n e b a e e c e n a e a s c a f a e e c a e e y i o d e l c a a o c a n i r h t c m a e a e t n i a i o o r c c y m a r e n c z c e t a a a e g i i y o e e g m A l l c F n o s r r n n a a / o r a p e u c l e u h h y e l g e w t o a J l p j u n e t v n e a e / e r i u o e N A C C F G J L r d a - e c s c i * t o e . a i h : N t p c e . e n e t 0 1 2 3 4 S o p 8 9 1 1 1 1 1 1 2 H 3 4 5 6 7 A S N

27 2018 Journal of Plant Resources Vol.16, No. 1 , , , , , , , ) ) ) ) , a 2 2 8 8 8 a 0 m h 7 7 M M M M 8 8 8 , y t 5 t 9 9 0 s 9 9 9 3 6 H H H H a e 1 1 5 1 1 1 8 2 . . . . r . . . N N N N 6 9 m , , 7 5 5 7 ( ( ( ( h 8 8 8 i 1 1 a 0 1 1 0 0 A S 0 0 . n , n n n n e , i , , , , i , , , , , , , , , 8 a r a a a a r t k 0 a h h h h m m m m m o m m m m m m m m m u o , t t d d d d i i ) z t i 0 0 0 0 0 h r r 6 0 0 0 0 0 4 0 0 0 a a a a i a r r r r c 0 0 5 0 0 o o 3 0 5 0 0 2 3 8 2 2 m M 2 l t t 4 4 5 4 4 M H P P P P 5 5 5 5 5 5 5 5 5 5 t t u u 7 H 2 2 2 2 2 , , , , , , a a 1 1 1 1 1 1 1 1 1 1 h S 9 , , , , , 6 6 6 6 7 7 2 2 , , , , , , , , . . N 1 H i H i i i i i P i i i i i i i i i i . ( 8 9 8 8 7 7 7 7 r r r r r r r r r r i i k k k k k k , , ; ; 7 9 9 9 9 9 9 9 9 i i a a a a a a a a a a n n n o o o o o o 2 2 0 1 1 1 1 1 1 1 1 h h i a a a . h h h h h h . . . . 7 . . . 7 . w w w w w w w w w w t t c c k h c c c c c c 6 9 6 9 9 9 6 6 7 9 7 a a a a a a a a a a u u l l l l l l u u u d 1 0 0 0 0 1 0 0 0 1 0 d d d d d d d d d d z z u u u u u u r ...... g g a i i , o o o o o o i i o o o o r h h h h h h u 5 4 2 2 7 0 2 5 7 5 P G 1 1 0 G G G 0 G F m P G H H P P M 0 2 0 P G P G G P 1 0 G M 1 k . c 3 ) i , , , 2 r e e e M 6 b n n n , P H . o o o , 9 ) , t t t N l N 2 E n l S S S , ( M 5 ” a a R , , , 1 m 6 h P H 6 6 6 w a 9 3 d 1 1 1 0 N N ’ h e 6 a t ( 0 0 0 5 t r 3 n s P e 2 2 2 6 2 P 3 . . . a e r 1 ° N r , 1 9 9 9 h c l , 5 h 7 i 0 0 0 n d n a . . . 8 S a o a o P e 9 9 9 r , . s , h r C . ) l N 1 1 1 P , l d ) N , A , , , , 6 a a n M n ” r 5 1 M a y m m m a w 9 H r P 1 0 h h H 4 0 0 0 o , 0 k 2 d N ’ t l d . 5 5 5 c N ( 2 i a c a 5 , i 3 ( r 6 6 6 o r r a 3 6 3 0 s P 1 1 1 . 6 b P ° F 1 0 , , , , , . 9 6 , , 7 1 e a a a 6 6 9 2 9 5 6 2 l 1 e e e 1 1 2 , 1 1 , P b r r r P 0 0 r , 0 0 m A A A N m 2 2 a N 2 2 . . m . . . . . 0 y y y n 0 1 1 n ) ) ) M 1 1 r r r 0 a 0 7 0 0 a 1 0 i . . o o o 5 h 5 . . 5 M M M r h t t t 8 8 5 d 1 8 8 a 1 c c c d H H H 0 0 a 1 , 1 0 a a a a , r w , , r , N N N , , F F F a D i D P ( ( ( P r m m d m m e e e , O , a O l l l l 7 5 6 . o l i 7 5 . i 0 0 b b b 6 6 6 w ) M o 4 4 M r r r G o 0 0 0 0 0 I a I a a a 7 5 S s ; 6 6 1 1 1 M d ) C 2 2 C , , 1 1 I P o P P I M M M H , , 6 6 - , , i i M i i i i i N G N N 1 1 a a N r r r r r k k r r ( 0 H 0 ; a a a a a n n n o o ) a a 2 2 a a a N 3 h h . . w w w w w h h ( h h h c c 2 M 9 5 a a a a a d d l l l d d d 6 0 0 d d d d d u u l H u u . . a a a o o P a o o o a a r r r h h 9 5 N 1 w 0 ( P P P G P G N N G P G G N e . . . t r . . b m u e l w m n z u i r l e , h C e i ) e d e B . . M e d d d n l , L . g & h i n l . ) d r . n . o . p t & C & o a e L a o t t U w m z h e r t & l . y . t r a P R n u s p ) a l r y . m m i t i e . G & S e m ) ) a h H s u F e h . ( t . a H ) L m h c H l . . e n p T h ( ) n S . R L m m s h e f u e a ( s S i ( a m p L a r f S h e a c L c i e y a e h m l e u ) i s e i B ( t s r ) a t L l m . e i h c i . u p d t v ( a r c L i s K y s S s t a s a G c i i h N n d i ( n a i f n n h s ( e r s n ( o n i n p e n i a e n p o o u c e r e a a e K g p r M a s t r l a . n t i t o o d e p a h r ( t s i e c l p t a n o a r c h e e a u i i S p l a a u m e h u l l j a p a c p f ( r r c s e a s s l i i z i t t c u i i n e a S m s i t m e e s t l s i o a a a o s s l l e l n p s m p r e u n m a a e n l s u m l m m m a M u & e . u e h p a a a a . a a G N s s s a u t u u i i i i h l l e ) h b k m w i l u v t t t t t i i a a a j ) ) c h h e i r n . . . i t n p n n n n e n h h h h h a a a a r p t h e l i i . b b l b l l n c c c c c a a a a a s r e a h l r l l l l a c a d o i n n n a l l e e c o o o o o h h h h a s a a e e e u S l l n i i i i i e e e h t n h e s s c r r r c c c b o i . t o . e e c i g g g g g d d d s s r n r r r c e e e n r r o b z L N P t t t P o a a n n a n a a a a y a a a a a . o o l s l o s s l l l e t i i i r u G & & M M P L C L ( K M P A L P P A A M P P P R M M M P T T s e e l a e a e a i a c t e e a e c n c l e a a a a a i l a e h t i h e c n p c h c a a e r c i a c l h a o n l i c o o e r g t n M r a a y o / l o e C M P P A a c i t a p e 6 7 8 9 0 1 2 3 4 8 9 0 1 2 3 4 5 5 6 7 2 2 2 2 3 3 3 3 3 1 1 2 2 H 2 2 2 2 1 1 1

28 2018 Journal of Plant Resources Vol.16, No. 1 , , , , , , ) 6 a , 7 7 6 8 , i h 8 j M , 9 9 9 8 , t r 9 m h s 9 9 9 9 8 H e 1 e g 1 1 1 1 9 . 6 n r . . . . N n 9 9 3 a ( h i 8 6 8 8 1 0 5 0 0 0 0 B S S . . ; n ; ; ; ; a 1 i , , i , , i i i i , , , , , , , , 8 2 a , , r r y , k k k k 0 0 t h m m m m m o o m m m m m m m m n a m m a a a . . a , , t t d ) ) ) ) ) e t t k k k k 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 a a m a d a a a a r 0 0 0 0 0 0 0 5 2 3 0 3 2 8 0 3 8 r M M M M M 4 0 0 0 8 4 8 H A H T P T T T 5 5 5 5 5 5 5 8 5 5 a H H H H H 2 2 2 2 1 2 1 , , , , , , , , 1 1 1 1 1 1 1 1 1 1 G , , , , , , , 2 7 6 6 7 6 7 6 2 6 , , , , , , , , , , N N N N N i i i i i i i i i i i i i i i i i i ( ( ( ( ( 7 7 8 8 7 8 7 8 7 8 r r r r r r r r r r r k k k k k k k 9 9 2 9 9 9 9 9 9 9 9 a a a a a a a a a a a n n n n n o o o o o o o 1 1 5 1 1 1 1 1 1 1 1 i a a a a a h h h h h h h ...... w w w w w w w w w w w 9 k h h h h h c c c c c c c 7 6 9 9 6 9 6 9 7 9 a a a a a a a a a a a l l l l l l l 1 u d d d d d 0 0 0 0 0 0 0 0 0 0 d d d d d d d d d d d u u u u u u u r ...... a a a a a o o o o o o o o o o o r r r r r h h h h h h h u 5 0 5 7 2 0 7 0 7 5 7 P P G P G P P G G G P 1 F 2 P 0 1 G 0 G P 2 1 P G P 2 G G 2 P 1 P G 1 . , ) , n n n l , i a a a M o h h h m H s d d d 3 , N a 5 a a , , 1 ( r r r 6 4 , 7 E R 1 P P P 5 6 E ” P , 0 , , , 2 1 ” 6 l l l a 2 l l l N , 8 0 . 3 h i a a a ’ t 2 9 P n k s ’ 3 w w w 0 a o e N . 3 2 h r h e e e . 5 ° 2 d h c n n n ° 1 5 l R a S . o o o 5 , 8 r u t t t , . 8 , s s s h E P D ) , , ” , , , , , l P N l l i 3 6 6 6 M N i ; o o ” ) 4 1 1 1 ” o s H ’ g 9 s 0 0 0 6 , 2 n M 4 N , 2 2 2 3 ’ 5 . . . a ( 2 ’ 6 H 5 ° 1 9 5 9 5 1 D 0 3 5 0 0 0 0 N 3 0 . . . 1 ° ( 8 2 ° . 2 9 5 9 & 1 7 , . 7 9 1 1 0 1 1 2 ” 1 n 2 4 1 , , , , P 9 a 0 . , 6 . 4 h . 8 m m m N m P 8 ’ m ) d 1 0 4 3 4 0 4 n 0 N a , . M r , 3 a 2 4 7 4 7 , ) ° m h P 5 6 5 6 E 5 9 H 7 ” d , 1 1 1 1 1 3 M 0 N 2 a e 3 , , , , 6 , ( 5 t r H , 6 P y 6 D D D D D P 2 8 N m h 1 , ’ N ( . 9 O O O O O - l p ) ) 3 i , 5 . 7 i 0 7 ) 2 M M M M o 1 3 M p P 0 M M 9 I I I I ° I 1 7 e S 5 M 7 N 5 C C C C H H 6 1 C , , 1 I I I I 8 I H , a 6 6 P P - - - - N N , i , i i i i i ( ( h 1 1 a N r r r r r N N k t r ( 0 0 . N a a a a a s 8 8 , , o ) a 2 2 e ’ 6 4 6 2 7 h . . w w w w w h r 9 c 0 5 0 0 2 M 5 5 a a a a a d h l 3 1 9 1 6 5 0 0 d d d d d u H u S . ° . P P P P o o o o o a P h 5 7 5 N ( 0 N & N N 2 0 N G G G P G G N N ) . r l . l e t t e g & a u e ) a d H . l M J . p t . . e l s t u t . a . l z s S e . a o o y e C e s A n e b n ) n ( . h u e . e N u t r n e ) . s N t . . s e h e i a t N n e M K A R ) u t L p d . ( . N n i . ) K e D s n i . . M n b t i a ) m s i C a l p s L L r W d M i h S u u l a m L ( ) o t i n ( o n r S r r s p . a l i u c ( i c e l a e l r a g a n s i l a l y l r L t a S g l o e n i r a a o h u a y ( r t t a t r e n v r o a g h m i S g n T e l a g t u b e a o l i ( ( f u y p t b l s v L n r u a i l b b t o m j r o a u o a a a y n o i s u s u m r a g s u z n l p c t n e n p s h n u u y i d o a s s s s s c a a o c c h h a a a a p m m a e t u u u u u i i p l y i r u u u t t p p p p p l L a a r a a i i e u i i q i i n n i a o o o o o a l a d d r r t l l l l l f r n d a a c r c & a r t e e o o y y y y y r e o i h h a a o o o i g g a h h p p p p p s h a h i i c c h i t t z z t t d s g i c l c c r r t t m m m m m m a r a l r r o o e c c c e e a a u r y a y a a a a a r i e i r i a a P B D M B R C P R C P R C T M C C C C C M M s e e a l e e e a a a c i e e e a r e s a c l e c e e l a e e a a a a l i e g e i c e r e r z a c a n c e c e t e a n o a a e g n i i n i i a r s z l r g c a t l e u M r l c i c r e i n i a / e c i e M P D R T W A a D c / i i t c a s p u e 5 6 7 8 9 0 1 2 3 7 8 9 0 1 2 3 4 5 6 4 4 4 M 4 4 5 5 5 5 3 3 3 4 4 4 4 H 4 3 3

29 2018 Journal of Plant Resources Vol.16, No. 1 . , ) n , i i ) a 2 k h M 7 h M c o 9 d H u h H 1 a c g . , N r l N 7 o ( 0 P u ( 0 3 . N , h 5 n ; ; ; ; n 5 , , , , , , 7 i i i i i i i i i i P , , , , , , 1 a a , 1 , 9 k k k k k k k k k k ; . , h h m m m m m m , m m m m m m i ) a a a a a a a a m a a 9 n m d d h m a k k k k k k k k k k 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 a a M . c a a a a a a a a a a h r r 0 0 0 5 0 0 0 3 3 0 3 3 3 3 0 7 H d 4 5 7 8 0 0 6 O T P T T T T T T T P T T 0 5 5 5 5 5 5 5 0 a N 2 2 1 1 2 2 2 . 5 , , , , , , , , , , , , , r 1 1 1 1 1 1 1 ( 1 , , , , , , , 5 6 7 6 2 6 6 6 6 6 6 7 6 6 5 P , , , , , , , i i i i i i i i i i i i i i , 6 , 1 8 9 8 7 8 8 8 8 8 8 9 8 8 7 i r r r r r r r i k k k k k k k 8 , 9 9 9 9 9 9 9 9 9 9 9 9 9 9 k a a a a a a a h o o o o o o o 9 ) 1 1 1 1 1 1 1 1 1 1 1 1 1 1 o c h h h h h h h m ...... 1 w w w w w w w h , . u c c c c c c c M 9 7 9 7 9 9 9 9 9 9 6 8 9 . a a a a a a a c 9 l l l l l l l h 0 l l 0 0 0 0 0 0 0 0 0 0 0 0 0 d d d d d d d 0 g u H u u u u u u . . . 0 ...... a u . o o o o o o o o h h h h h h h 2 7 2 4 5 6 7 2 7 7 7 8 7 7 h t 2 N 0 P 1 G ( P 0 2 1 G P 1 1 0 2 2 G G P G G P 1 P 2 2 1 P G e P N 0 , , . ) n 6 a 1 , M l , n h , 0 l a d H 2 a N E . n h a ” N a r w ” 1 d ( 2 h 9 0 a P ’ e . r d 3 5 2 n 8 , ’ , a P 1 l 2 o r 0 5 , t ° 1 o N . , P s 3 e 5 1 o ) t ° ” , , E 8 l h P y 7 2 ” i 6 M c , h 2 2 N 3 o 1 s ’ p H s N , 1 , 0 i r . 5 , 4 2 p N e ” m . 3 S ’ i 6 ( e . 6 ° 1 4 v 1 7 , 3 2 7 H 0 a 2 0 8 ’ 7 . 6 2 ° , 2 4 6 1 8 X . 0 , a 5 1 3 0 0 . 9 1 h 8 t ° m , t 2 , 0 i , P . 7 S s . r 5 5 m e 2 8 f N N a r 2 0 , 2 o . 6 ” h n 4 w . , l 5 4 m a 5 a ) 1 S l 0 5 h 2 d m a , 4 ’ i , 2 M d o & r 4 w 0 4 , a a m H i 6 G , r 5 n 3 . - k ° 1 7 a 4 w P ) N 0 n o ( 7 , 1 h a 1 , 2 e h e M 2 0 d d e , 5 t 2 d t i c a i 2 o r r 1 l H 8 , . y r s a a u , 7 G 9 h P N m e n h ( 0 . ; . G P p w l , . i e ) ) ) l P 7 a l b i N 6 9 - p d r a 4 d i o r M M M 3 e 1 a c n 7 o s n a i 7 , , a H H H 2 e , g n G M P E E h h , 6 - a i N N N i i ” ” t d r g ( ( ( N 1 n k k 4 6 a o a a a 0 r 9 4 3 n o o 4 5 b b 2 B w ’ ’ a 0 6 6 P h h i . - t a 2 2 o h c c 1 0 0 , 9 h l l n d 2 2 n d 1 1 k 1 0 g u u a ° ° . o a h c P P P a t r h h h 5 5 9 o G N 8 8 1 N N r P P P S E B r . . e . l k l g ) j E . e i e . ) k e u a . ) l v . e J . W d r l t o & v M i . t . ) a r u . r i & . . . k a M A g g k H B y o r C w n M ) ( o H . z ( a o d a ) & t . m o . o e t t m H m u i G t y k M m H i D u H u z ( o u e ) . t m ( t M . t m o s o M s a o ( f a w m a u t n f . l m u d D n H y s i s d u t e n i ( i ( r m u u u e l w e o d t a a c c i u n m i i s e r a m d G i t n l H a e r e b c ( u p p h i e i d l a i e d c e c d L a x g r f n a i H a m a c e i m n o a u s n e o u m v & y r a o o c m e u m m m m m m m e t s a c u i u a h l t u u u u u u u c i n a x r m l i i i i i i i c r n a i n e e h a u s n n n n n n n l a o p o g L y f p u h d e e e e e e e r h d a r d a p B a a a c m m m m m m m p o b o & e g t . . e e l y y y y y y y e ) r n o n l . a . k k h b b h h h h m m h h h a o a k b m c c c t k c c c c c c c d o u u r l m t r r r s t o M i n a a a a y y a a a c m e o i c o i a a o r n r r r r r r r r r r y M K B & B O N A B B B B B B S T B B B D G G e a e c a y e s r a e b l e o c a c a y u y r r e b L B u E / i c s u 4 5 6 7 8 9 7 8 9 0 1 2 3 4 5 6 6 6 6 6 6 6 5 5 5 M 6 6 6 6 5 5 5

30 2018 Journal of Plant Resources Vol.16, No. 1 , , 6 6 0 8 8 0 9 9 9 1 1 . . , n i a 7 7 k . i h 1 1 o ; h d , , , , , , , i i i i i i , , N h c , , a k k k k k k , c r m m m m m m m u m m i l a a a a a a m m P g h u k k k k k k 0 0 0 0 0 0 0 0 0 0 0 , c o a a a a a a h 0 0 0 0 0 0 0 3 3 3 3 7 2 4 7 4 4 8 9 P O N T T T T T T 5 5 5 5 9 2 2 2 2 2 1 1 ; , , , , , , , , 1 1 1 1 9 , , , , , , , 6 6 6 6 6 6 2 5 6 , , , , 1 i i i i i i i i i i i . 8 8 8 8 8 8 7 7 8 r r r r k k k k k k k 6 9 9 9 9 9 9 9 9 9 . a a a a o o o o o o o ) 0 1 1 1 1 1 1 1 1 1 i i h . h h h h h h ...... w w w w k k c c c c c c c M 8 9 9 9 9 9 8 7 6 8 a a a a a a l l l l l l l 2 0 0 0 0 0 0 0 0 0 d d d d k k u H u u u u u u ...... , o o o o a a h h h h h h h 7 2 2 2 1 7 5 5 7 N P 1 m ( 0 P T G P 0 0 P P T 1 2 P G P G 1 G 1 2 , , , , l , e N i , 6 l 0 n i o 1 6 ” 1 o o s 4 0 1 t 3 6 , s 8 , s 1 2 0 2 , 8 , , . d P N , 8 E 2 7 2 1 . n N E E ’ ” P N ” E P a ” 0 ” 1 4 8 ” ” . , , 6 . N l 6 0 0 N ) 3 1 1 0 8 . i 6 8 3 , 3 2 ’ ° , ’ . 9 1 2 0 8 o 0 ’ ’ 1 2 ) M 8 ’ 7 5 s 3 , 0 0 8 3 3 1 2 ’ 0 3 2 3 , H , 2 P 2 2 E M ° 8 . ° 5 , 7 2 5 ° ° ” E 5 N ° 1 7 N P 3 6 H 1 P 5 5 3 ( ” 8 ° . 5 0 2 1 0 8 8 . N , 1 3 ) N N , 7 8 , 6 0 2 ( , , 8 , 3 1 4 . 2 , . 3 2 n N ’ M 0 9 m 4 . 1 3 N N , a 6 ’ 4 R N , 7 ” 1 1 H 9 0 h . P 2 4 ” ” . , m 2 0 P ” . ) 7 m ° 7 d . a 2 9 N 6 8 ) N 4 6 ( 0 a P 5 ° 5 N 8 h 4 3 ’ 1 2 M r , t 3 ’ ’ 7 8 5 1 , 0 M 5 , N 7 ’ 2 s 0 - P 5 5 5 H , 8 3 , 3 4 e 5 6 3 H 5 , m 3 3 1 , ° 2 r l n 7 3 N N 2 6 m 2 ° ° i , h 7 7 N ( - ° a 0 N i P 5 7 7 P ( o ” 2 r S 6 6 P 7 h 0 4 s 1 2 2 1 ” N a , 4 N 4 d 2 2 8 2 , N , , , 5 5 a 5 , 5 & , 8 1 w , m 8 . r E 2 2 - n D 2 2 m m 6 a n ’ ’ n ” P m P 0 R a 0 , , a d P 4 4 a O 6 i i 0 3 5 . . , 5 h , 0 N h . o ) 3 3 h 0 k k 7 2 2 N 6 a 5 d ) 5 , ° ° d M d 2 o o 5 5 5 G a 1 h 1 I 1 a M n 7 7 ’ t a R r h h 1 1 1 M , r 0 , , r a s 2 2 C 4 , i c c P H , , , 2 2 P I e l l h H P , , 2 . a k D , 9 r , i ° u u N d D D D , h 5 o e l N r h 6 ( m m t O a i e 5 h h ( 0 h n O a O O s r . S P o n 8 P P 7 6 7 c o e M 1 P 5 s , - l - w t o , r 0 N 4 4 M M l M I i i t 9 s , , 0 u a l I I I h 8 7 7 s n n N C 7 n k , , a h d E S e 2 2 e C C C , I P a c P o ” ” P E E I I I w h h , , 6 - - h o N i i ” ” 6 6 i i i i g g N G & r 1 a d r r d r r , k k 0 6 0 8 a a r 0 . ; a a a e a a n n n d o o 2 1 2 2 . ) ) a t r 2 B B ’ ’ ’ ’ a a a ) e h h . w w w w h - - n P s 3 3 4 4 h h h c c M M 5 a a a a e d h h l l M o 2 2 2 3 d d d k 0 d d d d u g g H H u u ° ° ° . ° p a a a m c H o o a o o a a r r r h h 5 5 5 5 7 x e N N o N 8 P 8 P ( G ( 8 0 P e r B 2 P G c P N G B G . i i ) M l d l i ) e y m . n . & e r k h i k . . l c l c y h A h o a s B . o c e . c . . L c r ) u w M r O E & . r M a . r g a r d . l a ) & e a L l . e v g B & b l h & a e l h m e . h . d ) . c H t y u y . & a u w t c y s d t n . b . o w z h d s i t z h o a r o o w i K d h t m M d o r r p o c i o L r d c h n e u B . B a ( h o i r S s D B ( c e B D & r c M s ( C a H t C s ( u L S s c s S n a n ( m n s m C i a e a u B a r m e a e m u s e m u c v c m r p e m u m s i g d m r r u i n d u e l s m u i i o i x u s o n e e v c t o h t u e t n u a g h i l o t o r o a g t n o i s r v e a i i y y n y e d r v r n C n r r o g k f t r v i e m o h t e a a m a u c e ) a f e n a b c j g s u c r . t p r l c g i u l o a o y o i l e a a s s s s n a l a e e r c c i j r . h l g s u g p p r e . n n n n b r p h m e e e e u h . a o a m m m m m c a s m m m m m m m c d d d d i i m r i i i i h l d u u u u u s l M d i t u u u u u u u i i i i i i i s i s s s r h o r n . h y y y y y y y s s s s e n e n n n n i a r i r r o h i i i r r r r a c l C P K ( S B F B M F B B M P R M M M M F F F B B B B e a e s c e a l e t a a n t e e n c d e a i i s d n i s i s s F M i F / i c s u 2 3 4 5 6 7 4 5 6 7 8 9 0 1 3 0 1 2 8 8 M 8 8 8 8 7 7 7 7 7 7 7 8 8 7 7 7

31 2018 Journal of Plant Resources Vol.16, No. 1 i i i i i 2 ; k k , , , , , , , i i i i i i i i , , , , , , h h h 9 , , u u k k k k k k k k c c c m m m m m m m 5 s s m m m m m m a a a a a a a a u u u t t m m B k k k k k k k k 0 0 0 0 0 0 0 a a g g g 0 0 0 0 0 0 0 0 i i i a a a a a a a a 5 0 0 0 0 0 0 3 3 2 3 2 2 3 3 w w M 6 1 4 2 3 5 1 H H H T T T I T T T T T I 5 5 5 5 5 5 5 5 1 2 2 2 2 2 2 , , , , , . , , , , , , , , H 1 1 1 1 1 1 1 1 , , , , , , , 8 6 6 8 8 8 2 8 6 6 6 6 6 5 , , , , , , , , N i i i i i i i i i i i i i i i 8 8 8 8 8 8 7 8 8 8 8 8 8 7 r r r r r r r r , k k k k k k k 9 9 9 9 9 9 9 9 9 9 9 9 9 9 . a a a a a a a a n o o o o o o o ) 1 1 1 1 1 1 1 1 1 1 1 1 1 1 a h h h h h h h ...... w w w w w w w w h c c c c c c c M 0 9 9 9 9 9 7 9 8 9 9 9 9 7 a a a a a a a a l l l l l l l d 1 0 0 0 0 0 0 0 0 0 0 0 0 0 d d d d d d d d H u u u u u u u ...... a o o o o o o o o h r h h h h h h 4 2 7 7 4 4 5 7 7 1 7 7 7 5 N ( P 0 P 0 G P G 1 P 2 0 0 1 G G P P G 2 2 1 1 1 P G G G 1 P 0 4 5 . 6 & R P , , , n n , N 6 a i , 6 a , 1 a h 1 E m h t t 0 0 R ” s d n 5 2 2 , e 0 a . u . , 2 r r a 2 9 o 9 e h ’ 5 h P 0 0 t p 3 . 1 S m . , s o 2 9 , , , e 9 l e i l t ° 1 r s 6 i r 1 y 5 , h a 1 o , n h 8 0 S i . E S E w p , ) a ” 2 , i , ” . a t . l ) 0 N p l 5 2 d n M 1 ’ 4 e a 1 o ” u 0 ’ M 2 H , . . 0 w 6 o 2 ) G 2 6 8 H 2 N 3 2 ° . ; k 1 m 0 ( ’ ° ) M N 5 1 c 0 , , 5 ( i 5 1 1 8 H 2 r 3 . M 8 . 1 m m , 4 ° b 8 N , 5 8 H 6 7 ( , 5 2 N 1 0 . 9 N 2 . 5 4 3 N P , ) ” 8 ( P , 5 1 5 5 . ” 6 5 . N m M 0 0 2 2 ) 5 6 N m 3 9 ’ , 2 , , n ’ 0 1 , H . i i P . 5 a 3 M 6 E 5 7 1 k k N 3 S h 2 ” N 8 6 P . H ( 1 o o ° ° d 5 . 3 1 n h h 7 N a H N 7 1 2 8 2 , a c c r ( 2 ’ i , 2 , 3 1 l l n h P , 5 k a , 7 u u , 6 a D d , 2 h o h h 8 P h m t a m ° m k h O r 7 s P P d c 5 N c 8 . 4 e a 0 P P l - - ) o 8 r M r 0 i i 4 n 5 , u I , h N l 6 R P n n a 6 6 h M l C , S e e 1 , , h N 1 1 a P I H h h , 6 6 d R - - , , i ” i i i w g g & a 1 1 , a N r r r k 0 r a a r ( 0 0 . a e a a a n o 4 ) t a P 2 2 h B B ’ a 4 h . . e t w w w h - , - 3 r h s c 4 M 5 1 a a a e d h h l c 3 e d 6 0 0 d d d u p g g H u r ° . . n a P o o a o a a o r h h 7 5 8 o l N S ( 2 0 N c G P 0 B G N B s P G . . e h c . A t u k i o ) r l ) r . k . . ) p o . ) k a h B t . . w S o k c t t M h ) i t d s . ) a o t . i i . e t . x o T t . e M i & H r i h l w M ( H ( w c ( d D F y . B m ) i s M . a d . ( e i z . . h i ) i u ( f t . e f m n o e f w t d n e H f l i o t u M i a ( H d e a a m i r r i D . r F n e s r f r e u m a B . s G f t a m G o u e M . a i r ( H c t u i ( a h u r r h i a t e i s M m r s n i c t r a c n u s h i r n u t G i i i r u a u n h n i p c o e i o i c M m e b s i b f w l s c i a s n l i t i l a m a l o a v a i e n a l a i f l c f i r o m m m m w c i l p v g b r i m b u l u u u u x e p n b i n i i i o y m g d c r i o y a r o c c c u o y t z r n n s u e n o t r l i s e e e i d h d l h s s s s s s u h h h o y a a t p t t m n n n n n n e a a i i r h i h y y y i e e e e e e o o n . t r r r r a p h h h n c d d d d d d s h o . h e o o i i i i i i i h a t s t t c l c c l h o i r g d s s s s s s t n y a y y a i o r a a a y r s s s s s s e r r y i r r n r r r i i u h i i i i a a a a J B B P B E C T B B B C C F F F P T F F F F e e a a e e c c e a s e a i a e e i a l c e a s g e e c a e e c e s l a h t y c a e t s n i a l r a i y i y h n a b l r r h i c o o a e b c t r a l n l n a k a m a r u p o p n A B F S D o y u H H F / / / i i i c c c s s s 0 1 2 3 4 5 u u u 9 0 0 0 0 0 0 2 3 4 5 6 7 8 1 8 9 0 M 9 M 1 1 1 1 1 1 9 9 9 M 9 9 9 9 9 8 8 9

32 2018 Journal of Plant Resources Vol.16, No. 1 , 8 8 , 9 m 1 . 7 9 & 4 0 i i 7 , , i i i i i i i i i i , , h h . , , , , , , , , 1 k k k k k k k k k k c c i m m m m , a a a a a a a a a a u u k m m m m m m m m t i k k k k k k k k k k 0 0 a g g 0 0 0 0 0 0 0 0 0 0 i i a a a a a a a a a a 0 k 0 3 3 3 3 0 3 0 3 3 2 m a 9 9 H H T T T T T T T T T T 5 5 5 5 9 5 6 5 5 5 u 1 1 , , , , , , , , , , , , T 1 1 1 1 1 1 2 1 1 1 S , , 6 6 6 6 6 8 6 6 6 6 8 6 , , , , , , , , , , i i i i i i i i i i i i i 8 8 8 8 8 8 8 8 8 8 8 8 & r r r r r r r r r r k k k 9 9 9 9 9 9 9 9 9 9 9 9 i a a a a a a a a a a o o . o 1 1 1 1 1 1 1 1 1 1 1 1 i h h h h ...... w w w w w w w w w w k c c c c 9 9 9 9 9 9 9 9 9 9 9 9 a a a a a a a a a a a l l l u 0 0 0 0 0 0 0 0 0 0 0 0 d d d d d d d d d d k u u u ...... g o o i o o o o o o o o a h h h 2 7 2 2 2 5 2 7 2 2 4 7 P 0 1 G 0 G 0 P H G 0 0 T 0 1 G G 0 0 0 G G P G G 1 G . a ) . h 1 ) t M , 1 0 s 5 4 & M , e 1 H ; 1 1 r 0 ) 1 , , , , n H , m 9 N 0 h 1 . , P ( a E E E E 0 ) E 2 N M S P 8 . h ” ” ” ” E ( 1 ” N 2 0 & 1 d 4 ” H 0 0 M 2 N 2 , 1 & ’ 0 6 , a P ’ 1 4 4 2 2 , n 0 . N 8 H r ’ ’ ’ 0 2 1 2 4 ( n n a m N 6 8 P 2 ’ P 3 3 1 2 , 2 N a a 9 h , 9 1 ° ( 2 2 , e 2 2 ° 1 6 . h h N 0 d . t , ° ° ° e 5 2 5 i 1 P ) S P d d , a 2 t 8 . . 5 ° 5 5 . 8 s E 8 r 5 ) a a y 9 P N 8 5 N 8 8 ” , r r M , 1 R H P y h 0 , , 8 , , , 3 M r N P P . , , , , N p H 1 N i , 2 S i o , , a a e N S N N r H . ’ . P t t ” . N ” e e p h h 2 N a c t t 5 ( y ” ” t t 6 N H R 6 N E H 2 2 a s s y y ( 2 h 6 6 w 3 , , 3 , 2 , , 3 2 e e ° ’ h h F . ’ a p 3 3 a a ’ ’ r r 5 4 a i 6 ’ ’ 5 6 p p 6 d S h h e 5 5 h h 5 i i 6 h . 1 p t t 5 5 8 3 l 3 o t 3 3 6 p p s s S S ° 0 3 3 ° , E s b H P ° ° e e e e G r ° ° 7 2 e 7 , r r P . 7 7 N , , a r a ; N 7 7 & & 2 2 5 h h 9 2 2 ) 5 6 h h N 2 2 ” , 1 t n n , 0 , , , S S M 1 1 S , , , 0 . s 0 M a a . ; m 0 0 R 4 9 e m m m 2 ) h h m m & & ’ r 2 2 . H R , & 1 . . . 0 d d 4 6 6 3 h a 1 , , 3 3 M n n N a a 1 6 5 n 4 1 1 a 3 h 1 S ( r r a a 2 2 E t 1 0 . 6 a ° H 6 5 5 h . . h s h P P 5 5 ” t 8 1 h 3 7 1 1 1 8 0 & e N s d d , , 1 1 0 1 , d 6 2 r , ( , , 1 2 i a a e e e , , 4 i i i a n 9 t t r h r r , , , ’ k r r r r , . 0 a y y D D P h S P P ) a a a 2 o P 0 h m m m h h m S , , , 2 h O O N , w w w 6 l l l d p p M l ° , c i i i 0 0 0 i i 5 a a a a i l P , e 5 M M r o o o 5 0 5 H . p p 2 d d d o u I I p 8 N 6 6 6 S e S s S e P 5 o o o S h N o . , C C . 1 1 1 , , , , , , , ( 1 l I P I G G G , , , s 7 7 6 5 7 6 7 H N - - R - , - - - i i i 3 i i i i 1 1 1 1 1 1 1 , a , a a r r r r n k k k ” 0 r r r 0 0 0 0 0 0 0 . . . a i a a a a a o o o 6 6 ) ) ) a a a a 2 2 2 2 2 2 2 h h ’ h h h t ...... t t w w w w h h h P 5 s s c c c M M M n 9 9 5 1 9 5 9 a a a a d d d l l l N 3 e e u 0 0 0 1 0 0 0 d d d d u u u H H H u u u r r . . . . ° . . . , o a o o a a o o . h h h h h 0 0 5 8 7 0 5 0 N N N ( 2 S 2 N 0 1 2 ( 2 0 2 ( G S m R G N P N P P G G . . . ) w g . ) r . r n W . o ) l r e i . t . t g t e g e P N k y e n o g e r m R a e o . ) ) o e a r e . ( u B e c o t J a e w C g w t a p p M ) J . i g e h H d r ( m ) r L . . ( e a c m e e a u A s J M a ) a S i s c A w n p . a ( ) H J . . ( s r t . . r n o d ( l ) H t l . w l e n u . r e a t e A l m t s s ) i e m e d c . c c t A e . T i ) i e u i H . u e s u ( . a h r o u r k r ) ( d M f e e h . r c i b i e f o ( H u m M f i M v c s c s c i l M ( a o . f h ( i o z i b s r s . a a u i i a t . c i c e g l s l s . r s r u u r d l l H f s e a C G p s u m n h i C ( n a l d ( o a j F t G L s a a n e i p ( n F p s m i . o l i s w i m m m u p l r e u o u u m m ) s t s h r d u u u u e . i e r r c l d M i i u u i a u t i B d r i d i i t c u a i n c m m m n n n ) n g t r o b ) c i r e r . a y a . u e u u l r i a o n u e m m m r a u h i t i i S p p r g l l m m d o s h C c g a a a h h h t s o d s o e c c c h h h n n n n n n n n . l & o t t t d o n a h n l n n & o o o o o o o o y b . r r r o o o l c h y y y a . d d d d d d d d p o e w l r r r o h c n h h h n t o o o o o o o o h e . h c c c m R s m t t t r y t t t t t r r H r e y c c u a a a o . o i a n n n r u h n n n n n u u a R S B P ( & ( R M C E E E E M M E R E E E E E M H H E E e e a a e e c c a i a t m n o o c d o o l t y n H E 7 8 9 0 1 2 3 8 9 0 1 2 3 4 5 6 6 7 1 1 1 2 2 2 2 0 0 1 1 1 1 1 1 1 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

33 2018 Journal of Plant Resources Vol.16, No. 1 , , 6 6 0 9 . 8 5 9 l , , 9 8 a 1 g . 1 1 ; m t n . 0 . n , e l 0 o 9 i & & & n 0 a i 1 5 k L a i i i i 0 n i h , , , , , i i o , , , , , h h h h 1 a k c & ; , , , B k k h c c c c 2 h m m m m m a u o m m m m m i a a - c u u u u m m m d k l M g d h k k 0 0 0 0 0 0 g g g g 0 0 0 0 0 0 0 0 a a u c i i i o i n a a r 0 0 0 0 0 0 H 3 5 0 2 3 3 3 5 h T u 3 1 3 7 0 7 H H H N H A P T T 5 5 5 5 5 5 5 5 N , g P 2 2 2 1 2 2 , , , , , , , , , i 1 1 1 1 1 1 1 1 8 ; n , , , , , , 6 8 6 6 8 6 2 6 2 , , , , , , , , i i i H i i i 8 6 i i i i i i i i a 8 8 8 8 8 8 7 8 7 r r r r r r r r k k k k k k 9 , 9 h 9 9 9 9 9 9 9 9 9 i a a a a a a a a o o o o o o 1 6 9 ) d . 1 1 1 1 1 1 1 1 1 i i i h h h h h h h ...... 8 . 1 w w w w w w w w a k k k c r c c c c c c M 8 0 9 9 9 9 9 7 9 9 7 a a a a a a a a . g a a a l l l l l l u P 0 1 0 0 0 0 0 0 0 1 0 d d d d d d d d n k k k H u u u u u u ...... g G , . o o o o o o o o i a a a o h h h h h h 1 2 7 2 2 5 5 5 5 0 5 N ( m 3 D 0 G G G 2 G 0 0 T 0 T G P G G 0 P L 1 P T 0 G H P 1 P P 1 , . E ) , ” . , , . 0 6 5 S M , ) . . 2 1 1 H ’ S , 0 0 H M . 9 3 , 2 2 N , E 6 . . H 2 ( H a E , ” ° 9 4 1 , ” h N 0 1 , 5 t 0 1 N P a ( 4 . . 2 1 s 8 h N ’ 4 ” 4 8 N e t 1 1 , ’ 3 r 6 s 2 1 ” , 7 1 2 . 2 h e N 3 , , 6 0 P 2 r ° ’ S ’ S . 1 E E ” ° h 5 6 5 N ” ” 6 5 P 8 S 3 H 3 & 2 2 3 8 , ° ° , , N . ’ 5 5 , 7 n a 7 & , 5 N R 1 1 . a 2 h 2 N ’ ’ 3 t , n h ” , , R ” ° 3 3 s a a d 6 , 2 e 7 2 2 m h h m a r 3 a t ° ° 2 2 r d ’ ’ s h 0 h 8 5 5 , . a P t 5 e 5 4 S ) 0 r 8 8 s . r , 3 m 3 6 ) 6 , , P e ° h e ° M r 1 t & 1 , 0 7 S N N 7 M h , y , e H 5 2 n 2 e ” ” h e S n H 6 t , a t & , N 5 5 p i o i 1 ( h i N t s 9 m - 9 n & S m ( d p ’ ’ S 5 a a y 3 5 y n 5 5 5 E , 5 0 h r r r a 2 2 3 3 1 . , 6 7 5 d o P o ) ° ° h 5 5 t 5 6 1 t a 6 P , 7 7 d 1 1 c r 1 c 1 0 P e M a a N 2 2 , , t a P , 0 2 r . N , , F y H k F 2 , D D , P . 9 r . h e n m . m e N e , 9 a 0 . O O l p l l ( . a ) n i i 0 R P b 0 0 b . 9 h o r p M M 2 o , r 2 2 t s M 9 1 d I I a a a 6 E s S 5 5 u a 1 , h C C H 9 , , , r 1 1 t , M I I B E M 6 6 P 6 P s , - - , N i ” E i i i i i i ( 1 1 1 e , r r r r r r N k ” 0 r 0 0 0 . e a a a a a a 5 , o 2 4 t ) h 2 2 2 ’ ’ 6 5 h . . . y w w w w w w S 2 2 c 0 5 M 9 5 5 h a a a a a a , l 2 2 l 1 6 0 0 0 p d d d d d d H i u . ° . . ° i P P o o o o o o h o 9 5 5 5 5 p N ( N 1 e 8 0 N S G 0 G 8 G G G P G . . d a i M e r r . . ) h . ) p b . D t . B r r o M C d o ) m ) . g . g h r . n ) ) u i e . . e n k t C w e f M a . ) a t o K f E . c . d i o ) i ) i o w w . s . k w e a ) r ) d t h l t . . h d t o M i h t h H n c i H i G h ( c e ( c c w o ( ( u i c A . S W d S t H s M g s M s H ( m ( i s e ( i ( e ( u m e e i M e u t s s a a u H a H u a e a e a B a t n n e m ( c r ( r r d u r e e a r n m a e m e e i i u r n r r u t g n o l l m h a t g t e f m e i t o a i p i n r c a s s i u t f s l i i i u r o s e s o a l u l e s a x p u s l r a h r d c i o e m a t n a e y - c k e m a g t r u i m e r n . j l h a m i m d a u l o t s r h p o i p m u r a n t r u s m e c i a l l u d i i a e a . w l s u x a l a c d p r m r o l d l e m l g t w l m a e c l i g o a a e t I u d . . . . t y y a i l a e c m m m e i s u o e t h h h h . r ) h c h n m a u u u u d l m . o a d h c c c c a i l u t p o o a u d n n n t g s s s s t w . i t t e l . n i u o n e c i i i i i t l a i l p p p y e r x x h s p p e y n u i e e e e i o y y y o h i h t h o s r l l c l e a e e l C M F ( P F D M S N F * L C R P T R F H H H L L P F G B e a e e c a a e e l c e l a a a y e e t e a h c n c e a p o a i c o r d d t a i u o a n t t c p y p s m y h e e e L L R S H 2 3 4 5 6 7 8 9 6 7 8 9 0 1 5 4 3 3 3 3 3 3 3 3 2 2 2 2 3 3 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

34 2018 Journal of Plant Resources Vol.16, No. 1 , . l , a . l a & i i i i & k h , , , , , , , i i i i i i i i i i i , , , , , , , , , h h i c u k k k k k k k k k k k c c h m m m m m m m s u m m m m m m m m m a a a a a a a a a a a u u t c g k k k k k k k k k k k 0 0 0 0 0 0 0 a g g 0 0 0 0 0 0 0 0 0 u i i o a a a a a a a a a a a 0 0 0 0 5 0 0 3 3 3 3 2 3 3 3 2 g w 0 0 2 3 6 1 3 H H N T T T T T T T T T T I T 5 5 5 5 5 5 5 5 5 o 2 2 2 2 2 2 2 , , , , , , , , , , , , , , , 1 1 1 1 1 1 1 1 1 N , , , , , , , 6 6 6 6 6 8 6 6 6 8 6 6 2 6 2 , , , , , , , , , i i i i i i i , i i i i i i i i i 8 8 8 8 8 8 8 8 8 8 8 8 7 8 7 r r r r r r r r r k k k k k k k 5 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 a a a a a a a a a o o o o o o o 7 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 h h h h h h h ...... w w w w w w w w w 9 c c c c c c c 9 9 9 9 9 9 9 9 9 9 9 9 7 9 7 a a a a a a a a a 1 l l l l l l l . 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 d d d d d d d d d u u u u u u u ...... n o o o o o o o o o h h h h h h h 1 1 3 7 2 4 2 2 1 4 7 7 5 7 5 n 1 G 1 G 1 P G 1 0 0 0 0 P 1 0 G P G G G 1 P 1 P G G 1 P a P 1 1 . . ) . 0 ) a ) 6 . , h M ; . . t M 7 ) ) M s S H P . H e H M M , r N , , , , N , , H N ( h N ( 8 H H N E E E E , , ( N N S 2 ” ” ” ” 2 , 6 a 4 ” N N 1 4 0 0 0 0 8 ” ” ( ( h 5 0 9 N t & 1 9 2 2 2 2 9 5 P 7 8 3 s 1 9 ’ ’ ’ ’ n ” 1 7 ’ 3 3 P e N P 6 5 3 3 3 3 a ’ ’ 6 r P P 5 , 7 7 2 2 2 2 h N 5 5 3 N h . 3 ° ° ° ° N N ’ d , 3 3 P P ° , S R 5 5 5 5 . . . a ° ° 6 n 7 r N N . 8 8 8 8 7 7 3 a R R R 2 & P ° , , , , 2 2 h n n , D , , , , 7 , , a a d n a a a , N N N N 5 2 l m a a h h h h h m m r ” ” ” ” t t t 1 , o h d d 7 s s s P 6 6 6 6 0 g a a d 7 0 m e e e 8 r r , 3 3 3 3 2 n a 8 2 r r r . ’ ’ ’ ’ 4 r e P P a 4 4 5 h h h t 1 5 5 5 5 1 P , , 4 1 1 y S S S D 1 3 3 3 3 , e e , . 6 i , , h t t ° ° ° ° e i i r 8 1 y y t p 7 7 7 7 & & & & r r a i 1 , y h h a a 2 2 2 2 p n n n n e , w h p p , , , , t e a a a a w w i i a i p m a a , h h h h i s p p m m m m d d d e e 6 d d d d p 0 o y 3 8 0 3 a a a a o o e , , 1 r 5 r r r r 2 2 7 2 G , 6 6 0 o 6 G G P P P P 5 5 5 5 t - 6 1 1 2 1 . c , , , , 1 1 1 1 n n n 1 0 0 , 4 a e e e e , , , , e e e i 0 1 1 t t t t . . 0 r F d d d 2 . y y y y D D D D . a r r r . 5 5 ) 4 e h h h h a a a 9 0 0 O O O O l w . . 2 p p p p 0 . M b i i i i a G G G . 5 5 , ) r M M M M p p p p d 9 0 0 I I I I H c c c a E e e e e i i o i M 1 , , ” C C C C N , , , , n n n , M I I I I 2 G ( E E H 6 6 6 6 a a a - - - - - i ” ” 5 t t t E i i i i 1 1 1 1 4 a N r r r r k ” 6 6 1 o o o r ( 0 0 0 0 . . 3 a a a a o 2 1 1 2 b b b ) ) a 2 2 2 2 6 ’ ’ ’ ’ - - - 5 h . . . . w w w w h 2 3 3 3 P o o o c 6 M M 5 5 5 5 a a a a d l 2 2 2 2 n n n 9 0 0 0 0 d d d d N u u H H ° ° . . ° . . ° h h h P o o o o a . t t t h 5 5 5 5 5 5 5 5 N N N 8 P ( ( 8 0 0 8 0 0 8 G E G E G G R N E . r r ) ) . & ) p ) e e & l . . k s . y . l g . ) g l . & l q k s . & z e k e l e s s o p . A . o a u a o s w o e r k h . i J ) J m o M d e c . D c L e . M . a . M e s n u v M H h ) . r K r . A o & H A . c & H ) B ) e a ( & . C p ) ( . s p ) ) B . . y ( i u ) . . v s l l H . t y t w z m l e ) r l s . F t m t ( t m . l z t t i a . a i o u . d e m t u t i ) u e t F s o i i e . h u u S l i . m s t H l H D l M . i M ( c ( ( ( M l v a D u u w ( H o ( s M M ( i t r ( f M ( y e m d i M i e w ( a . n i s m m m h t a u e m s I m p i ) e s m i B u u t u u p C . o s h H u u ) i m i r H l ( l s u u n l h . l c o e l m b l ( i t u a g u m o i a c u g n y o a t e m o l u t n l . i i r f n i v f n u c l c s o o h u r i u i u i t a h r n t i s o t e a w s t c o c o b e r a p e M . l u i n t u g m m M o i o n u ( s t i c y n p . k c p n i r n r m i m v u u u o s a l e i e s s l l r a l a m r v C o u a u r l l B u o i m m b u ( p e c f g h k m m c u y y n n n n ) a d u u i o n e . m i i h h M l t o o o o n l a i . i m m m m m m m m n l l d d p p n . d d d d a r i k m u u u u u u u u t e c t t r o o o o & l m o h o i i i i i i i o o o i r d . r u t t a u e e o y o p p c o u d n d d d d d d d d o h e l . y n a a m m m m i i i i i i i i l s p t g e d t o o h a o g i z p W M p t r o l o o o t r y e u u u u u u u u c M o m m a a i b e e t o y a e r e . . a l l a n n n n n a l h h h h h h h h e e a u r n J R C D L F B & T C H M i A s T T T T T T T A A A C C H H T I E S S e a e c a e l l a y e c h a p i o d i e r u e h t T S 2 3 4 5 6 7 8 3 4 5 6 7 8 9 0 1 0 1 2 5 5 5 5 5 5 5 4 4 4 4 4 4 4 5 5 4 4 4 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

35 2018 Journal of Plant Resources Vol.16, No. 1 , 2 7 9 1 . 5 i i i i i i i i 0 h h h h h h h h , , , , , , , , i i i i , , , , , c c c c c c c c k k k k m m m m m m m m u u u u u u u u m m m m a a m a a g g g g g g g g k k k k 0 0 0 0 0 0 7 0 0 0 0 0 0 o o o o o o o o a a a a 0 5 0 0 0 0 4 0 0 3 0 3 3 . l 4 6 4 1 1 0 7 1 1 N N N N N N N N T T T T 5 7 5 5 a 2 1 2 2 2 2 2 2 2 , , , , , , , , , , , , 1 1 1 1 , , , , , , , , , 6 2 6 2 2 2 2 2 6 6 2 2 , , , , i i i i i i i i i & i i i i 8 7 8 7 7 7 7 7 8 8 7 7 r r r r k k k k k k k k k i 9 9 9 9 9 9 9 9 9 9 9 9 a a a a o o o o o o o o o h 1 1 1 1 1 1 1 1 1 1 1 1 h h h h h h h h h ...... c w w w w c c c c c c c c c 9 7 9 7 7 7 7 7 9 9 7 7 a a a a u l l l l l l l l l 0 0 0 0 0 0 0 0 0 0 0 0 d d g d d u u u u u u u u u ...... o o o o o h h h h h h h h h 1 5 7 5 5 5 5 5 2 1 5 5 1 P G 1 1 G P 1 P P P 1 P 1 1 P 1 N 0 P 1 G 1 P G 1 4 ; ; 2 . ) . 6 ) ) & , M P M - , 6 n M H . h a , , , 1 N E H , H g S h 0 ” N , 4 E E E . N ( a N , , d 2 6 N ” ” ” 8 ( . ( a e a R 0 B 3 3 0 7 , ” 1 t 3 4 r h , 2 2 2 2 ; 9 P 1 2 t y 4 0 N P ’ ’ ’ ’ a ) . s 4 8 7 h 0 , , N 4 3 5 3 h ’ ” e 4 6 p t P 1 e M r , 2 2 2 2 . 5 i 5 2 t N s . P ) ° P ° ° ° h N 3 p 2 , e H y ” 5 R 5 5 5 ° r S e N 6 N h E M n N 8 8 . 8 8 8 ’ . h 7 , , . p ” ( a ) n 2 , , , , i e 5 2 H 5 S n D 6 ’ h t a p 3 2 , a 1 , N , N N N M 1 N y 4 d h ° e l 3 h e ’ 0 ( a h 3 m t ” d ” ” ” 7 , o H 6 d r 2 3 ° p y a . 3 8 0 0 6 2 6 g a i 7 2 P P r 7 N h r 1 0 2 4 4 3 , 1 n ° ( 8 p 2 ’ P ’ ’ ’ p N 1 7 P . a 0 5 e 4 i . m , 4 , 5 3 5 5 , e , P 2 8 , 1 . p 8 t D e . 3 3 3 3 1 e 5 m t , 3 e , y 1 ° t ° ° ° N 9 R 8 i . y 4 1 , y h , 7 7 7 7 , & r ) 0 2 N , P h . 5 0 5 . h p 2 a n 2 2 2 a 2 i m n ” p ) 9 1 2 1 N a M p , , , , h i 6 . . a w i p 9 t 1 , 0 ) , h 0 p 2 2 a H e M h s p m 3 m m m , d 2 n d 5 , ’ 0 . e d e E , d M n i a . a N H 6 r 2 5 3 3 m 5 a 1 o , , 6 r ( u k h 8 r h 1 2 H 2 2 2 3 N 1 6 6 1 P o 5 d o 1 G . ( 5 , ° 6 S P 5 5 5 1 1 0 N i a p 2 h , 8 1 7 2 . , , 1 1 1 ( n r 0 0 2 9 k c 5 1 . 0 e e e 2 , , , , e l m m 2 1 P 8 . t t t o i 1 1 , . . 1 1 , ) u d o , , 7 y y y D D h D k 4 , r 1 4 0 P h e m . c h h h C m P o n 6 a m 0 0 M O O l O n P . . 8 p p p N h e P 0 0 u N i 0 i i G o H 8 4 0 R - c , d M M M t 0 0 h i . l . p 7 p p r N , 0 2 I I I P c N 0 6 e e e S n n u 5 P i S a , , , ( E . C C C . 2 e 1 a , , , D , h 1 n I I I ” E E G – h h , , 6 6 6 2 6 a , H - - R - - P i i ” ” 6 t i i i i i i i g d 1 1 1 2 1 , , r - r r r r r r k k 5 6 0 o a a i 0 0 0 0 . 5 . a a a a a a a a a o o r 9 5 2 b ) ) n 2 2 2 2 h h B ’ ’ ’ - B h h . . . . t t P w w w w w w w - e 2 2 4 o s s c c M M 1 1 5 5 a a a a a a a h h l l , M 2 2 2 e e n 0 1 0 0 d d d d d d d l g g H H u u r r . . . ° ° ° . i h o H o o o o o o a a t h h h h 8 8 5 5 5 5 5 o N N s 0 B G ( S 1 0 8 8 N G ( 8 S G P G 0 P G G B E G . . r ) ) . . h e r . t . k M g . g . o o e ) A ) r e h M . . o a . C . a l ) c k . ) ( B J . l s h . . & H . A l o g i w t l . e ) ( l l . e ) o o m y h o u e M r M . l e A n r a c z l u u . H N g ) u l o ) ) p M G o . n S ( B . . h r e e t ) r ) ( t . M a . . t v c ) a M t d D x u a t i . r i e s . s m k ( t C t e . n c w a i t e ( n n u o M e C . i M o a C M h s l e g ( o ( H B . d r ( ( l t M c d ( o m a i e ( M o e t t a H C n a S a u ( n r a m ( ( m a u ( n s t l n d u - a t c l h r u e u . a a s i o a b a a a e i a a s . l M p i a r e h t l n t i . e u m c m s m r c o i h r r . c p r r a r o h s l g a c o n s s o f u i o u b p i l c u n a n C c l i i i t s s r i s l s f a y s i i s b e a e f o o a g l y m i e a x l e l i r r e r r i r e a a a l e m l m e f a c i F b a i i i l d d m m m s h e e e a a F r c r r i i s s u p n F n c f s a u u . . i i s e . r s t a a a i i L d e e . a s s h p h a a a i d d d d e d . d d t M i i i c p p i l i M k o m m m n n n h M & . r r r s o o . j . i . o y c y o o t ) l i i i ) u u u i u u u . r . r r a a a r r r ) h l h h l y . i i i . e o t e r r l l l . l b b b e h o e e l b w b r c c c l l l a a h d d d l z h i e e i i n k i i i c i i i s s s e k k t a o o o c F r l r r e n g g i i i t B o s m m p p p c c r r u u u o h y a . o u e o o o e e e e e e e e r o e e l e r a a a e l l l l l r h h h p a a M B ( M F J F A M M F H B M F J S B P P P T H N N A F F M T T T e e a e a e e a e c a e c a e c s a i c i a e g r r l a i e o h a w e k p y t c d r y e e e r b N C H M o s I / i c s 9 0 1 2 3 4 5 6 2 3 4 5 6 7 8 9 0 1 u 6 7 7 7 7 7 7 7 6 6 6 6 6 6 6 5 6 6 1 1 1 1 1 1 1 1 M 1 1 1 1 1 1 1 1 1 1

36 2018 Journal of Plant Resources Vol.16, No. 1 ) . n M l a a H ; h . d N L L & . , . ( a r e i i i i G t G 9 n n . h h h h P , , , , , , , , , , ) y , , a a 8 c c c c , h h h h h m m m m m m m m m m 5 u u u u k t t M p d d i i g g g g c i B 0 0 0 0 0 0 0 0 0 0 a a H o o o o o p m r r m 0 0 0 5 0 0 0 0 0 0 M N 7 8 7 8 1 7 4 1 7 4 S R E N N N S N P P ( ) 1 1 1 1 2 1 2 2 1 2 , , , , , , , , , , H , , , , , , , , , , 2 7 7 7 7 2 7 2 2 2 2 M N i i i i i i i i i i 7 8 9 9 8 7 9 7 7 7 7 k k , k k k k k k k k H 9 9 9 9 9 9 9 9 9 9 5 n o o o o o o o o o o 1 1 1 1 1 1 1 1 1 1 N a B h h h h h h h h h h ...... ( h c c c c c c c c c c 7 6 6 7 7 7 7 7 7 7 l l l l l l l l l l M d 4 0 0 0 0 0 0 0 0 0 0 u u u u u u u u u u ...... a P H h h h r h h h h h h h 5 2 0 8 5 8 5 5 5 5 . P P N 1 0 P P P 2 P 2 1 N P P 2 P 1 P 1 1 P 1 , . . a ) n ) ; h a ) . t M , ) h M s M P d E H e 3 H , M , r a ” 2 H N , , N r 5 h E 0 N H N 5 ( , , ( ” P E E N 7 . S 5 P ’ ( ” N ” ” ’ , 6 7 N S ) ) 8 , ( 4 . 9 e 3 3 2 8 N P 4 & t 1 ” 2 4 N 2 2 2 7 4 , M M H 6 y ° 7 ’ ’ 9 ’ N ° n n 6 P , 7 ” h 5 5 5 4 5 P a H H , 5 a 7 a ’ 6 P p 8 3 2 2 h N 8 h i N N N h P 5 3 ° ° ° R , d , t ( ( , N d p ’ . , . 3 7 5 5 s a N , a N 6 ° n r N E 6 2 e 2 8 8 D R r , n a r 3 7 , 1 1 P ” , , , , ” P n a ° h , h ’ 2 6 7 , 5 a l a 4 , h 7 N N m d 4 , S 1 5 h P P o h e 5 d a 2 t 3 t 0 ” ” ’ 1 7 r g d a m s ° , N N y & 2 0 0 r 5 0 8 a n e P . 7 . h r 5 r 4 4 3 2 n m P 4 a , n 5 . 2 n ’ ’ p ° 2 l a h P 1 a i 0 i a 1 , 4 D 5 , 3 7 5 h . , S , h p h o 1 e 4 3 3 i . 2 e 1 d 5 . m t s e d r t ) ° ° d - 6 & a , 0 8 y & a , , a a y 7 7 r 5 1 . r , 7 r 1 h n M 6 4 m h 2 2 4 , n P w P a p 8 , P 1 1 p , , m e a i a 5 H 7 i , e h 4 t , . 0 0 l t h p d 2 i 0 p m d m e e 4 i 1 N 2 2 s y d o , ( . E e 5 a p p 4 o , i , a h 5 3 r i 1 , , s y 1 G o o 6 r k 1 5 r p 2 2 r l l P 0 6 6 , i 1 a o P 8 . 0 s s 5 n 5 - o . 1 1 6 p , t h , , 0 8 e 1 1 n w 7 0 0 1 n n c y c e e 1 0 E d i i e a , , l t t 1 2 2 0 a a r , , . . a a P d d f u y y , 2 t t D a D . r 4 4 . w o h h h ) m m N n n e a m 0 0 9 O G O l p p P e . . , u u G i i 5 0 0 0 G M b - h 4 4 e . o o - R r p p M M 2 0 0 t i n n 2 2 9 c I I . H a , 5 6 0 E i m m E i n e ) , , n 1 a C C e 2 1 2 N n , p , , , d o , M I I l ( E E h r a h , , , M 6 6 6 6 - - t t i i i i i ” ” a E i i g A s 1 1 1 1 4 r r H o k k k k k 6 6 ” a e 0 0 0 0 ; . G 1 a a o o o o o 5 5 r 2 ) ) B N 2 2 2 2 6 B ’ ’ ’ h h h h h e . . . ( . h w w - 2 2 2 P o c c c c c M n M 5 5 1 4 a a S h l l l l l 4 i 2 2 2 n 0 0 0 0 d d N g H u H u u u u . ° ° . . 1 . ° p d h o o l a , t h h h h h 5 5 5 5 8 1 4 5 n N N 0 G 8 8 ( 0 0 a 1 2 A P ( B P G 8 R P E P P . . . d d i ) ) i M & r . . r & & ) v v B y . B ) x & l u u . n . y z ) l e ) . . a a e . z . k P . o e r t e e k r n t h o o R u ) i D t o g . B B B o o . ) D o e o ) s , . . . . M ( w r . l M x H t a l v i i . t R P P H ( d l ) r B i ( ( ( ( w . m e e D . C a a ) W t v h ( u s u . l a a M t H i r m a t t H c i n ( v u s t s ( a m u t i a a r S M a m a l l l n y i ( u a M s a l H . m m e e u a u u n ( p u p l l u t u o r r H c a C r s m h t d c a ( s r u n r r ( a i n i a s u u e n c e e e p r u i r t a L . c a i o i s h s s e c a g d r i u a r y r l o s i d g n . s s r n i g d c a o a e b h n & i i r n r i l c t u e e r u s s i a s s u r m c a c a n h i i c j m n n u p p v o e u a s c s o m o o w i l . . s s i p p f d r m m m m m m d d h h h o t m h o o o n u u u u u u c i o o c c a u t t t t t t y y n B l o s s . l p p S a . r r l i i m a a a a a a i . a t d r ) h y y e n ) b e e b . . o n n n n n n p v h i . e o o l l c h h e r . o o k k o o o o o o u o t s k p c v h t d c c l l F F r r c i t t a g c g g g g g r c n e a u a a i e o o . . e e a i r u e a e t o a t o o o o o a l e r r M C B P Z D M F M P L R M M G P D T T P P P P P N M e e e a a a e s e e e e a e e c a c c e l a s a e a a c a e t e c d t l a h c n a o c h a n a o y i l p c r o h r i s d i y t c b r d e p o i h t o l o o o c r c y h r a c t c l u t i a e r t a r y e o t u l t e P P R T L O o o L P P / / / i i i c c c s s s 7 8 9 0 1 2 9 0 1 2 3 4 5 6 7 8 u u u 8 8 8 9 9 9 7 8 8 8 8 8 8 8 7 7 M 1 1 1 1 1 1 M 1 1 1 1 1 1 1 M 1 1 1

37 2018 Journal of Plant Resources Vol.16, No. 1 ) B 6 M 7 M P H H N N ; N . ( . s n n K , , K , 2 e , , a a , i , , , 4 h h m m m c m m , o o m o 7 l d d e t t t i 0 0 0 0 i i i 0 0 a a B p r r a a o a 5 5 0 5 0 3 s 5 6 8 6 S S S S P P M 6 5 1 2 2 1 2 , , , , , , 1 1 ) H 4 , , , , 6 6 2 2 7 2 , , i i i i i i 1 M N 8 8 7 7 9 7 r r k k , k k 9 9 9 9 9 9 , a a H n o o o o ) 1 1 1 1 1 1 a h h h h ...... w w N h c c c c M ( 9 9 7 7 6 7 a a l l l l d 0 0 0 0 0 0 d d H u u u u 0 ...... a o o h h r h h 2 2 2 8 5 8 5 N 1 0 G ( 0 G P 2 P 1 P P P 2 1 , 8 1 . 7 2 E ) 6 4 1 8 ” 6 9 0 9 7 0 9 3 9 M P 7 3 , 2 P 8 5 1 5 0 H 5 N , E , , P P 4 9 N 1 . ’ 4 P p ” N , E E P l . ) N N , 4 ( 3 o 1 P , ” ” 6 a , N t S , , , N 2 7 2 . . . 0 0 M 1 N E , 3 N i ’ ° N . P N 2 2 ” 0 & n 8 H R R k 2 ” 5 H ’ ’ S n ” a 2 3 . . 6 , , , N ” o 2 8 6 . 3 3 a N 6 h 1 a a a ° , P h 6 R , 3 1 ( H 2 2 h 3 d 4 h ’ h h e 5 c 3 ° ° , 0 ’ , d t t t ’ N l N a ’ 9 6 p 8 a 5 5 s s s a 2 a r 6 4 , u 6 7 3 , o r ” h e e e 8 8 h . 3 P 2 l 6 h t ° r r r 3 t 0 1 P ° , , ° s . 8 N s , 1 ° s P 7 h h h 1 ) ) l 5 , 7 5 e 0 e 0 l 7 N N ; 2 ” n S S S r P e 2 . . r 2 8 , i a 2 t 2 , ’ M M 6 ) ) h . ” ” h , a , N y E , 4 3 t w & & & 1 6 6 S H H ’ m S ’ , h M M N m 3 n . . 0 3 3 , m s 4 k n n 6 . p ° N N ’ ’ 4 u n n H e H 5 i ” 2 4 c ( ( a a & 3 8 7 i 4 3 3 4 o a a i ° 1 p 1 4 ° h h , N c r N 0 2 4 3 3 6 h h 7 8 5 0 ( 5 ( 6 7 m E d d n e b ° ° , , 6 1 8 8 d d 8 2 2 a a a 1 2 , , p , 7 7 . 7 1 7 a a , E ’ 6 6 r r , m s h , , 6 6 r r 5 2 2 1 e 1 ” , 4 4 i P P P P d t 1 1 N P P 1 e , , 7 1 7 3 2 6 i m 3 k , , . a t 0 0 N N 0 l s l 4 ° 1 i 0 P r o P ” , , m m i i 8 2 2 0 , , s ’ 2 7 7 r r 4 1 . . h P e . o o 1 N N ’ 0 n n l 4 a a 0 8 2 2 c 1 1 s , s y 3 l l , , a a l . 4 6 b 3 , 7 2 r , l l l 0 0 , 6 , 0 r i i n i h h u 2 ° . . 1 . p R 5 5 o 6 1 6 a a ° m t p p o d d h 7 8 8 , 9 o , 1 1 1 0 1 i h c a 5 a 2 S 0 0 P a 2 e e , , 7 r r M T 0 2 0 k a d 8 t t , , , , . h - , f 4 2 i 2 i o P P a e e t D D i , . 9 . r 7 r r r s m m m m k k h , , e m 9 0 9 O O N l c c 2 a e P c . o k k o l r 5 0 0 0 0 0 b n n 0 , , c c . 9 h . h ” w r M M u h 2 5 0 0 o o 5 p k c c o o 9 1 9 1 I I a a l h l 5 6 5 2 c c S r r 5 c o 1 , 1 5 d C C u u 2 1 2 2 , , , , , P 1 o , , M T 2 I I o E l h h , , , , 6 6 6 5 6 ’ , - - - i i i i i i ” E E i i i i b G P P 1 1 1 1 1 4 r r r r k k k k k k ” 1 ” 0 0 0 0 0 . ; . ; ; . . e 2 a a a a o o o o o o 2 2 2 ) ) ) ) ) t ) ) ) ° 2 2 2 2 2 ’ ’ ’ h h h h h h . . . . . e w w w w 7 2 2 2 r c c c c c c M M M M M M M M 5 1 5 1 1 a a a a l l l l l l 2 c 2 2 2 0 0 0 1 0 d d d d H H H H H u H u H u u H u u ° ° ° . . . . . n , o o o o h h h h h h 5 5 5 5 8 5 8 8 o N N N N N N N N ( ( 8 8 ( ( ( 8 0 0 0 m 1 c 0 P G G P G ( ( G P P ( P P r . e k ) r j . g . i t e e m t . g C i a u . r W e J n r . p P t t a ) M . e t . s ( J ) i f h m m . A o . i f i . f i i u r C t ) M f i r L r A t . h i . t i t v r ) c a ) t G s . r . i s . C i i ( l . l o G M d u e k l . ( G i i r o P t i c . M i o r a f t m i c ( e i i ) a v S o e u n . B r w . x r t u a p o h m H e M i u s c B a t s l s ( l i i u m m ( c n u o l r p a o p u u e l m t r a t f c e l l a 5 s t r i s y l l r m l o c h B 0 n i x u o h y y a a a h ( l r 2 t e C r o m p h h t l i o m c m . . u s o p p m m v p u i o n m u x r n l o o C u u i i n n . . n i i u m h r r y i o m e i t t d n a n n P D t a a c h h o s g y i a t t a e t o o ) ) l r s o a a n a a y y . . g s i h g g l l l l r e r r r n a l o h o o e w w u t u u C o e e a e e n r r p n c i d d b y b b . o o m d d m o e r r e r r e e i M y y m m C y y y y h a r r e n a a . i y B H H P ( & g ( C B * B B H H H H R T A B B s e e i l a i e c m a a i t F t o 8 4 P 3 4 5 6 7 8 9 0 1 2 3 4 5 0 0 0 9 9 9 9 0 0 0 9 9 9 2 2 2 1 1 1 1 2 2 2 1 1 1

38 2018J. Pl. Res. Vol. 16, No. 1, pp 39-45, 2018 Journal of Plant Resources Vol.16, No. 1

Fern and Fern Allies of Panchase Protected Forest, Central Nepal

Chandrakala Thakur1* and Sangeeta Rajbhandary2 1District Plant Resources Office, Makwanpur, Nepal 2Central Department of Botany, Tribhuvan University, Kirtipur, Kathmandu, Nepal *E-mail: [email protected]

Abstract

This floristic study of fern and fern allies was carried out in Panchase protected forest of central Nepal within the year 2014 and 2015. From the present study, all together 92 species of fern and fern allies belonging to 50 genera and 22 families were documented. The study showed that Polypodiaceae was the largest family containing 11 genera and 21 species. The major habitats of the collected species were terrestrial with 47 species followed by epiphytes with 28 species and five species as lithophytes While some species are found in different habitats as seven species as terrestrial and lithophytes, three species as lithophytes and epiphytes and two species as terrestrial and epiphytes.

Keywords: Altitude, Distribution, Epiphytic, Habitat

Introduction Fern and fern allies make an important contribution to earth’s plant diversity. A present study shows that, Fern and fern allies are groups of seedless but there is the existence of all together 300 genera of spore producing plants, developed within two fern and fern allies including approximately 9600 different lineages i.e. Lycophylls and Euphylls ferns and 1400 Lycophytes worldwide (Smith et al., (Pryer et al., 2001; Smith et al., 2006). In the past, 2006). In Nepal, according to the recent publication fern species were believed to be mystical plants a checklist of 580 species of fern and fern allies because of their capacity to reproduce without (pteridophytes) is listed (Fraser-Jenkins et al., 2015). bearing a , a fruit or seed (Moran, 2004). Ferns An extensive field survey of Panchase fern flora has are considered to be the first vascular land plant that had a flourishing past in dominating the vegetation not been carried out and information has not been of the earth about 280-230 million years ago (Khare, published yet. The effective conservation of 1996). Although, in the existing flora today, ferns Panchase fern flora will depend largely on how are now largely replaced by the seed bearing vascular effective the conservation of natural forests is done plants (angiosperms/gymnosperms) but still in the area. For this, minimizing of fragmentation constitute a fairly high-flying part of the present day and habitat loss through effective land use planning vegetation of the world (Borthakur et al., 2001). and a sound policy framework is a must. But in Ferns show a range of variations in size, form and addition, to conservation the fern flora enumeration habitat. Most of the fern are herbaceous (annual or of the fern flora is equally necessary. To understand perennial), but some are also creepers or climbers; their distribution, collection and identification is in terms of size some ferns can attain the size of a necessary to know the rare and threatened species, tree (called the tree fern, Cyathea spinulosa), edible, medicinal, ornamental ferns from the study whereas some ferns like Hymenophyllum, area. This will help in the management and Salvinia and Azolla are very minute (not more than protection of the fern and fern allies of this protected 2-3 cm in size) (Gurung, 1991). It is the largest group forest. The main focus of this paper is only to of living primitive vascular plants with more than highlights the overview of fern and fern allies of 10,000 species in the world (Dixit & Vohra, 1984; Panchase protected forest, central Nepal and present WCMC, 1992), of which about 97% are ferns and about 16.20% of the total species of the fern and 3% the fern allies or Lycophytes (Rajbhandary, fern allies of Nepal. Even though there are many 2016). medicinal, edible and ornamental species, we

39 2018 Journal of Plant Resources Vol.16, No. 1 focused only to identify the species and give its recorded in notebook. The collected samples were overview in this paper. pressed and dried. The dried specimen were mounted and labelled with field note and deposited at Materials and Methods Tribhuvan University Central Herbarium (TUCH). The Panchase protected forest is situated in between The collected specimen were identified with the help the latitudes of 28º12" and 28º18"N and longitudes relevant taxonomic literature like Smith, 1879; 83º45" and 83º57" E. The area lies at the junction of Beddome, 1865-70, 1883, 1892; Schneider, 1894; three districts, Kaski, Parbat and Syanja (IUCN, Blatter & d’Almeida, 1922; Iwatsuki, 1988; Gurung, 2013; Maren et al., 2013). The forest is bordered by 1991; Gurung, 1984; Gurung, 1986; Khullar, 1994; 3 districts covering an area of 45.93 sq. Km (figure Khullar, 2000; Borthakur et al., 2001; Bista et al., 1). The altitude of this forest area ranges from 1405 2002; Fraser-Jenkins, 2008; Fraser-Jenkins et al., m to 2,500 m at the peak region (IUCN, 2013). 2010, Fraser-Jenkins et al., 2015; Bhattarai & Rajbhandary, 2017. Some specimens were also identified with their comparison to the specimen deposited at KATH and TUCH.

Results and Discussion From this study, all together 92 spp. of fern and fern allies were documented belonging to 50 genera and 22 families (table 1). The diagram below (figure 2, table 1) shows the species and genus composition of the families reported from the study site. This reveals that Polypodiaceae is the Figure 1: Study sites (Source: Department of Survey, Kathmandu, Nepal) largest family which contains Overall two field trips were performed in two highest 11 genus and 21 species. Followed by different seasons i.e. month of June (Pre-monsoon) Dryopteridaceae with six genera and 14 spp. and and October (Post-monsoon) in 2014 and 2015. The Pteridaceae and Woodsiaceae with 4 genera each collection of sample was done mainly in Bhadaure, including 11 and 9 spp. respectively. The families Bhadare, Bansingh, Arukharka, Panchase peak and like Selaginellaceae contains single genus with 6 spp. many other parts of the forest. Plants were collected Other families like Asplenicaceae, Davalliaceae, at all the possible habitat such as plains, streams, Hymenophyllaceae and Lycopodiaceae contains 4 rivers, dense forest, meadows and peak region and spp. each, while Dennstaedtiaceae have 3 spp. The vegetation zone covering the altitude range from families Equisetaceae, Glecheniaceae, Oleandraceae 1,405 m-2,500 m. The samples were collected with and Vittariaceae all contains 2 spp. and remaining the help of digging tools and clean cutter and also all other families viz. Blechnaceae, Cyatheaceae, by hands. The collected plants were kept in polythene Lygodiaceae, Lindsaeaceae, Ophioglossaceae are bags and some were pressed in field with the help monotypic. of newspaper. Each collected specimens were photographed, numbered and their detail notes were

40 2018 Journal of Plant Resources Vol.16, No. 1

Table 1: Name list of pteridophytes found in Panchase protected area, Nepal. * Epi=Epiphyte, Lit=Lithophyte, Ter= Terrestrial S.N. CN Scientific name Family Altitude(m) Habit* 1 236 Aleuritopteris formosana (Hayata) Pteridaceae 1690 Ter 2 185 Arachniodes spectabilis (Ching) Ching Dryopteridaceae 1600 Ter 3 296 Arthromeris lehmanni (Mett.) Ching Polypodiaceae 2285 Epi 4 208 Arthromeris wallichiana (Spreng.) Ching Polypodiaceae 2250 Lit 5 300 Asplenium ensiforme Wall. ex Hook. & Grev. Aspleniaceae 2330 Epi 6 188 Asplenium normale D.Don Aspleniaceae 1800 Lit 7 190 Asplenium tenuifolium D. Don Aspleniaceae 1600 Ter 8 152 Asplenium yoshinagae subsp. indicum (Sledge) Ching & S.K. Wu Aspleniaceae 1890 Epi 9 191 Athyrium distans T.Moore Woodsiaceae 1600 Ter 10 491 Athyrium fangii Ching Woodsiaceae 2400 Ter 11 310 Athyrium fimbriatum (Wall. ex Hook.) T. Woodsiaceae 2490 Ter 12 216 Athyrium foliolosum T. Moore ex R. Sim Woodsiaceae 1584 Ter 13 237 Athyrium pectinatum (Wall. ex Mett.) T. Moore Woodsiaceae 1700 Ter 14 316 Athyrium setiferum C. Chr. Woodsiaceae 2475 Ter 15 259 Blechinum orientale L. Blechnaceae 1660 Ter 16 305 Botrychium lanuginosum Wall. ex Hook. & Grev. Ophioglossaceae 2345 Ter 17 205 Coniogramme fraxinea (D. Don) Fée ex Diels Pteridaceae 1980 Lit 18 204 Coniogramme pubescens Hieron. Pteridaceae 2340 Ter 19 320 Ctenitis apiciflora (Wall. ex Mett.) Ching Dryopteridaceae 2220 Trer 20 289 Cythea spinulosa Wall. ex Hook. Cytheaceae 1600 Ter 21 266 Davallia bullata Wall.ex Hook. Davalliacae 1650 Epi 22 249 Davallodes membranulosum (Wall. ex Hook.) Copel. Davalliacae 2300 Epi 23 242 Dennstaedtia zeylanica(Sw.) Zink ex Fraser-Jenk. & Kandel Dennstaedtiaceae 1751 Ter 24 345 Dicranopteris taiwanensis Ching & P.S.Chiu Glecheniaceae 1400 Ter 25 254 Diplazium esculentum (Retz.) Sw. Woodsiaceae 1670 Ter 26 244 Diplopterygium giganteum (Wall. ex Hook. & Bauer) Nakai. Glecheniaceae 1700 Ter 27 169 Drynaria propinqua (Wall. ex Mett.) Polypodiaceae 1910 Epi 28 273 Dryopteris cochleata (D. Don) C. Chr. Dryopteridaceae 1520 Ter 29 317 Dryopteris lepidopoda Hayata Dryopteridaceae 2480 Ter 30 235 Dryopteris sparsa (D. Don) Kuntze Dryopteridaceae 1690 Ter 31 312 Dryopteris woodsiisora Hayata Dryopteridaceae 2360 Ter, Lit 32 294 Elaphoglossum marginatum (Wall. ex Fee) T. Moore Elaphoglossaceae 2220 Epi, Lit 33 295 Equisetum arvens L. Equisetaceae 2250 Epi 34 263 Equisetum ramosissimum Desf. Equisetaceae 1647 Ter 35 291 Gonophebium argutum (Wall. ex Hook.) J. Sm. ex Hook. Polypodiaceae 2177 Epi 36 246 Huperzia hamiltonii (Spreng.) Trevis Lycopodiaceae 1874 Epi 37 245 Huperzia pulcherima (Wall. ex Hook. & Grev.) T. Sen & U. Sen Lycopodiaceae 1874 Epi 38 200 Hymenophyllum badium Hook. & Grev. Hymenophyllaceae 1980 Epi 39 197 Hymenophyllum exsertum Wall. ex Hook. Hymenophyllaceae 1980 Epi 40 299 Hypolepis polypodioides (Blume) Hook. Dennstaedtiaceae 1650 Ter 41 314 Katoella pulchra (D.Don) Fraser-Jenk, Kandel & Pariyar Davalliacae 2480 Ter, Epi 42 473 Lepisorous clathratus (Clarke)Ching Polypodiaceae 2400 Ter 43 233 Lepisorous morrisonensis (Hayata)H.Ito Polypodiaceae 2250 Epi 44 186 Lepisorus contortus (Christ) Ching Polypodiaceae 1980 Epi 45 402 Lepisorus henryi (Hierin. Ex C. Chr.) L. Wang Polypodiaceae 1410 Epi 46 209 Lepisorus loriformis (Wallich ex Mettenius) Ching Polypodiaceae 2400 Epi 47 213 Lepisorus scolopendrium (Ching) Mehra & Bir Polypodiaceae 1920 Epi 48 159 Lepisorus sublinearis (Baker ex Takeda) Ching Polypodiaceae 1910 Epi 49 169 Leptichilus insignus (Blume) Fraser-Jenk. Polypodiaceae 1665 Ter 50 253 Leucostegia truncata (D.Don) Fraser-Jenk. Davalliacae 2360 Ter 51 299 Loxogramme involuta (D.Don) C.Presl Polypodiaceae 2000 Epi 52 227 Loxogramme porcata M.G. Price Polypodiaceae 1905 Epi  41 2018 Journal of Plant Resources Vol.16, No. 1

53 264 Lycopodiella cernua (L.) Pic. Serm. Lycopodiaceae 1610 Ter, Lit 54 151 Lycopodium japonicum Thunb. Lycopodiaceae 1810 Ter 55 265 Lygodium japonicum (Thumb.) Sw. Lygodiaceae 1570 Ter 56 274 Microsorum membranaceum (D. Don) Ching Polypodiaceae 1420 Lit, Ter 57 279 Nephrolepis cordifolia (L.) K. Presl Nephrolepidaceae 1450 Ter 58 256 Odontosoria chinesis (L.)J.Sm. Subsp.chinensis Lindsaeaceae 1910 Epi 59 183 Oleandra pistillaris (Sw.) C. Chr. Oleandraceae 1550 Lit, Epi 60 231 Oleandra wallichii (Hook.) C.Presl Oleandraceae 1980 Lit, Epi 61 223 Onychium siliculosum (Desv.) C.Chr. Pteridaceae 1650 Ter 62 202 Peranema aspididiodies (Blume)Mett. Dryopteridaceae 1980 Ter 63 192 Peranema cyatheoides D.Don Dryopteridaceae 1900 Ter 64 163 Peranema paleolata (Pic.Serm.) Fraser-Jenk. Dryopteridaceae 1920 Ter 65 161 Polypodiodes amoena (Wall. ex Mett.)Ching Polypodiaceae 1920 Ter 66 214 Polypodiodes lachnopus (Wall. ex Hook.) Ching Polypodiaceae 1594 Epi 67 192 Polystichum lentum (D. Don) T. Moore Dryopteridaceae 1600 Lit, Ter 68 232 Polystichum nepalense (Spreng.)C.Chr Dryopteridaceae 2400 Ter 69 345 Polystichum semifertile (C. B. Cl.) Ching Dryopteridaceae 1520 Ter 70 238 Polystichum squarrosum (D. Don) Fee D76ryopteridaceae 1730 Ter 71 309 Pteridium revolutum (Bl.) Nakai, Dennstaedtiaceae 2445 Ter 72 247 Pteris aspericaulis Wallich ex J. Agardh Pteridaceae 1878 Epi 73 262 Pteris biaurtia L.subsp. biaurita Pteridaceae 1584 Ter 74 243 Pteris pellucens J.Agardh Pteridaceae 1841 Ter 75 218 Pteris puberula Ching Pteridaceae 1634 Ter 76 421 Pteris subquinata Wall. ex J. Agardh Pteridaceae 1500 Ter 77 422 Pteris vittata L. Pteridaceae 1420 Ter 78 209 Pyrrosia mannii (Giesenh.) Ching Polypodiaceae 2250 Epi 79 410 Pyrrosia pyrosa (C. Presl) Hovenkamp, Blumea Polypodiaceae 1700 Epi 80 314 Selaginella bisulcata Spring Selaginellaceae 2480 Ter 81 276 Selaginella bryopteris (L.) Baker Selaginellaceae 1420 Lit 82 239 Selaginella chrysocaulos (Hook & Grev.) Spring, Selaginellaceae 1730 Ter 83 283 Selaginella pennata (D. Don) Spring Selaginellaceae 1470 Ter 84 278 Selaginella subdiaphana (Wall.ex Hook & Griv.)Spring Selaginellaceae 1450 Ter, Lit 85 292 Selliguea ebenipes (Hook.) S.Lindsay Polypodiaceae 1920 Lit 86 305 Tomophyllum donianum (Spreng)Fraser-Jenk. & Paris Grammitidaceae 1647 Epi, Ter 87 189 Tricholepidium normale (D. Don) Ching Polypodiaceae 1600 Epi 88 194 Trichomanes auriculatum Blume Hymenophyllaceae 1600 Lit, Ter 89 193 Trichomanes striatum D. Don Hymenophyllaceae 1600 Lit, Ter 90 299 Vittaria flexuosa Fee Vittariaceae 2330 Epi 91 170 Vittaria linearifolia Ching Vittariaceae 1420 Epi 92 259 Woodsia elongata R. Br. Ex Richardson Woodsiaceae 2480 Ter

On thethe basis basis of of species species number, number, the thelargest largest family family is iscomposition Polypodiaceae of contains forest. 21Rakotondrainibe species and most &of Polypodiaceaethem are epiphytes contains (Figure 21 spp. 2, Table and most 1). Asof themQuercusRaharimalada, semecarpifolia 1998; and Prajapati,Rhododendron 2013, arboreumRajbhandary, are are epiphytes (figure 2, table 1). As Quercus 2013 & Shrestha, 2017 also reported similar results. major species at the upper part of the area which seems to be suitable for epiphytic growth of the fern Figure semecarpifoliaspecies.Even though and Rhododendron the number of arboreumepiphytes variesare greatly according to the altitude than the nature of 2: Nephrolepidaceae Famili majorforests, species in case at of the the upper study part area, of duethe areato the which dominance ofLindsaeaceae Quercus semicarpifolia quite a number of es with Elaphoglossaceae numbe seems to be suitable for epiphytic growth of the fern Blechnaceae rof

species were found to be epiphytic at the altitudinal ranges from 2200-2400 m. The higher number of

e species

i Oleandraceae l i

species.epiphytes Even may though be due the to number altitude of epiphytesand composition varies of forest.m Equisetaceae Rakotondrainibe & Raharimalada, 1998; a

F Lycopodiaceae Amo greatlyPrajapati, according 2013, Rajbhandary, to the altitude 2013 than & the Shrestha, nature 2017of also reportedDavalliacae similar results. ng the Selaginellaceae gener forests, in case of the study area, due to the Pteridaceae a, dominance of Quercus semicarpifolia quite a Polypodiaceae Lepis 0 5 10 15 20 25 orus number of species were found to be epiphytic at the is the Number of species larges altitudinal range from 2,200-2,400 m. The higher t with sevenFigure species 2: followed Families by Athyrium with with number six species of (Figure species 3, Table 1) followed by Selaginella which number of epiphytes may be due to altitude and contains five species. Similarly Asplenium, Polystichum, Dryopteris contain four species each. The genera like Microsorum, Blechinum, Cythea, Paradavallodes, Davallia, Katoella, Leucostegia, 42 Dennstaedtia, Pteridium, Hypolepis, Arachniodes, Ctenitis, Diplopterygium, Dicranopteris, Diplazium, Onychium, Cyathea, Tricholepidium etc. are monotypic genera.

s e

i c e

5 p s f

o  r e  b m

u  N  

Figure 3: Number of species in different genera

Habitat wise distribution of species Three distinct types of habitat namely terrestrial, lithophytes and epiphytic were identified for fern and fern allies collected from the present study. Besides these three habitats some species were also found on both i.e. epiphtytes or terrestrial and epiphytes or lithophytes habitats. These major three habitats from different areas of Nepal for fern and fern allies have also been reported by Iwatsuki, 1988; Gurung, 1991; Bhattarai, 2013; Prajapati, 2013; Rajbhandary, 2013 and Rajbhandary, 2016.

From the present study among the 92 species collected, the highest number of species were terrestrial 47, followed by 28 epiphytic and five lithophytes (Figure 4, Table 1). Apart from this there were some 6 2018 Journal of PlantFigure Resources Vol.16, No. 1 2: Famili es with Among the genera, Lepisorus is the largest with pulchra,numbe Tomophyllum donianum) in terrestrial and r of sevens species followed by Athyrium with 6 spp. epiphytic habitat. Lycopodiella cernua and Microsorum

e species i l i

m membranaceum (figurea 3, table 1) followed by Selaginella which have also reported to be foundSeven (in ,

F Amo contains 5 spp. Similarly Asplenium, Polystichum, ndifferentg the habitat by Rajbhandary (2016) and Katoella omanes ageneruriculatum, Trichomanes striatum) , followed by Dryopteris contain 4 spp. each. The genera like threepulchraa, species found ( both as terrestrial and epiphytic habitat) lithophyticLepis and wo species ( ) in terrestrial and Microsorum, Blechinum, Cythea, Paradavallodes, epiphyticby Shrestha habitat. (2017) whichand Microsorum supports membranaceum the present 0 5 10 15 20 25 orus isdiff the 2016 and Katoella pulchra terrestrial and epiphytic Davallia, Katoella, Leucostegia, Dennstaedtia, habitatfindings. by Shrestha, 2017 Number of species larges Pteridium, Hypolepis, Arachniodes, Ctenitis, t with seven species followed by Athyrium with six species (Figure 3, Table 1) followed by Selaginella which containsDiplopterygium five species. Similarly ,Asplenium Dicranopteris, Polystichum, Dryopteris, Diplazium contain four species, each. The  generaOnychium like Microsorum, Cyathea,, Blechinum, Cythea Tricholepidium, Paradavallodes, Davallia etc., Katoella are, Leucostegia,  Dennstaedtia, Pteridium, Hypolepis, Arachniodes, Ctenitis, Diplopterygium, Dicranopteris, Diplazium, 

Onychium, Cyathea, Tricholepidium etc. are monotypic genera. s

monotypic genera. e

i  c e

p  s

f o  r

e

b  s e m i c u  e N

p s  f

o 

r e  b  m

u  N  

Figure 4: 4: Number of species in different habitat

Figure 3: Number 3: Numberof species in different of species genera in different genera Distribution of species in different habitat along

Habitat wise distribution of species thataltitudinal require different gradient micro- ThreeHabitat distinct typeswise of habitatdistribution namely terrestrial, of lithophytes species and epiphytic were identified for fernaltitudinal and range features the fern allies collected from the present study. Besides these three habitats some species were also foundLandscape on diversity, climatic andl species edaphic of fern conditionsand fern alli . In the present study bothThree i.e. epiphtytes distinct or terrestrial types and epiphytesof habitat or lithophytes namely habitats. These terrestrial, major three habitats from rom 1400-1700 different areas of Nepal for fern and fern allies have also been reported by Iwatsuki, 1988; Gurung, of1991; the study area seems to be- 2000favourable m holds 9 for the 14 epiphytes and Bhattarai,lithophytes 2013; Prajapati, and 2013; epiphytic Rajbhandary, 2013 were and Rajbhandary, identified 2016. for fern luxuriant growth of ferns as -2500it provides different and fern allies collected from the present study. two lithophytes (Figure 4, Table 1 On the altitude ranging from 2000 m From the present study among the 92 species collected, the highest number of species were terrestrialQuercusmicrohabitats 47, and Rhododendron suitable for differentepiphytic types fern diversity of fern thick bark followedBesides by 28 these epiphytic three and five habitats lithophytes (Figure some 4, Table species 1). Apart were from this also there were thussome supporting the Similar fi y other studies done in Manangspecies (Shrestha, that 2017require different micro-environmental20013). found on both i.e. epiphtytes6 or terrestrial and conditions. Due to occurrence of varying topography epiphytes or lithophytes habitats. These major three and altitudinal range features, the Panchase protected habitats from different areas of Nepal for fern and forest has developed immense habitat variation and fern allies have also been reported by Iwatsuki, 1988; that has facilitated the luxurious growth of several Gurung, 1991; Bhattarai, 2013; Prajapati, 2013; species of fern and fern allies. In the present study Rajbhandary, 2013 and Rajbhandary, 2016. according to the collection, elevation ranges from 7 From the present study among the 92 spp. collected, 1400-1700 m holds 28 terrestrial spp. eight epiphytes the highest number of species were terrestrial 47 spp. and eight lithophytes. Likewise, 1,700- 2,000 m followed by 28 spp. epiphytic and 5 spp. lithophytes holds 9 terrestrial spp., 14 epiphytes and four (figure 4, table 1). Apart from this, there were some lithophytes. And at an elevation from 2,000-2,500 common species which were found in more than one m holds 16 terrestrial spp., 11 epiphytes and 2 habitat. Seven (Dryopteris woodsiisora, Lycopodiella lithophytes (figure 4, table 1). On the altitude ranging cernua, Microsorum membranaceum, Polystichum from 2,000 m above it is mostly covered by Quercus lentum, Selaginella subdiaphana, Trichomanes and Rhododendron forest which support the auriculatum, Trichomanes striatum) spp. belonging to epiphytic fern diversity as the have thick bark terrestrial and lithophytic habitat, followed by 3 spp. thus supporting the epiphytic species. Similar (Elaphoglossum marginatum, Oleandra pistillaris, findings have been reported by other studies done Oleandra wallichii) which were found in both in Manang (Shrestha, 2017), in Daman (Prajapati, lithophytic and as epiphytes while 2 spp. (Katoella 2013; Rajbhandary, 20013). 43 2018 Journal of Plant Resources Vol.16, No. 1

 References  

s Beddome, R. H. (1865-70). The ferns of British

e  i c e

p 

S India. Vol. 1 & 2. Madras, India. f

o  Lithophytes r e

b  Epiphytic

m Beddome, R. H. (1883). Handbook to the ferns of

u  Terrestrial N  British India, Ceylon and the Malay Peninsula. Calcutta, India: Thacker Spink & Co.  1400-1700 1700-2000 2000-2500 Beddome, R. H. (1892). Supplement to the ferns of Altitude (m) Figure 4: Distribution of species in different habitat along altitudinal gradient British India, Ceylon and the Malay Peninsula. Figure 4: Distribution of species in different habitat along Conclusion Calcutta, India: Thacker Spink & Co. Fromaltitudinal the inventory gradient work conducted in the Panchase protected forest area, all together 92 species of fern and fern allies belonging to 22 families and 50 genera were recorded. Most of the species collected wereBhattarai , S. (2013). Pteridophytes of Nubri valley, found to be growing along terrestrial habit followed by epiphytic species and lithophytes. On the basis of species number, the largest family is Polypodiaceae contains 21 species and most of them are epiphytes. Manaslu conservation area, central Nepal. (M.Sc. AsConclusion Quercus semecarpifolia and Rhododendron arboreum are major species at the upper part of the area which seems to be suitable for epiphytic growth of the fern species. Some species like Lycopodiella Thesis), Central Department of Botany, Tribhuvan cernua, Oleandra pistillaris, Oleandra wallichii, Katoella pulchra, Microsorum membranaceum and ElaphoglossumFrom the marginatum inventory etc. shown work to have growingconducted in more than in one the habitat. Panchase University, Kathmandu, Nepal. Acknowledgementsprotected forest area, all together 92 spp. of fern and We are grateful to Prof. Dr. Mohan Siwakoti, Head, and all other faculties and administrative staffs ofBhattarai, S., & Rajbhandary, S. (2017). Pteridophyte Centralfern Departmentallies belongingof Botany, TU for providingto 22 necessaryfamilies facilities and to accomplish 50 genera the research. We would also like to thank Mr. Dhana Raj Kandel for the identification and providing the relevant flora of Manaslu conservation area, central Nepal. literatureswere recorded.for the study. We Most would also of like the to express species our sincere collected gratitude and werethank Narahari Chapagain and the entire family of District Plant Resources Office, Makwanpur Mr. Bishnu Rijal, Mr. American Journal of Plant Sciences, 8, 680-687. Pravinfound Bhandari, to be Mr. growing Roshan Gaudel along Chhetri, Ms.terrestrial Santa Budha habitMagar, Mr. followed Rajesh Tamang and Shreehari Bhattarai for their continuous support and encouragement. by epiphytic species and lithophytes. On the basis Bista, M.S., Adhikari, M.K., & Rajbhandari, K.R. References Beddome,of species R. H. (1865-70). number, The ferns of British India.the Vol. largest 1 & 2. Madras, India.family is (2002). Pteridophytes of Nepal. Bull. Dept. Plant Beddome, R. H. (1883). Handbook to the ferns of British India,Ceylon and the Malay Peninsula. PolypodiaceaeCalcutta, India: Thacker contains Spink & Co. 21 spp. and most of them Resources, 19, 175. Beddome, R. H. (1892). Supplement to the ferns of British India, Ceylon and the Malay Peninsula. are Calcutta,epiphytes. India: Thacker As Spink &Quercus Co. semecarpifolia and Bhattarai, S. (2013). Pteridophytes of Nubri valley, Manaslu conservation area, central Nepal. (M.Sc.Blatter, E. & d’Almeida, J.F. (1922). The ferns of RhododendronThesis), Central Department arboreum of Botany, Tribhuvan are University, major Kathmandu, species Nepal. at the Bhattarai, S., & Rajbhandary, S. (2017). Pteridophyte flora of Manaslu conservation area, central Nepal. Bombay. Bombay, India: D. B. Taraporevala Sons upperAmerican part Journal of the of Plant area Sciences which, 8, 680-687. seems to be suitable for Bista, M.S., Adhikari, M.K., & Rajbhandari, K.R. (2002). Pteridophytes of Nepal. Bull. Dept. Plant & Co. epiphyticResources, 19growth, 175. of the fern species. Some species Blatter, E. & d'Almeida, J.F. (1922). The ferns of Bombay. Bombay, India: D. B. Taraporevala Sons &Borthakur, S.K., Deka, P., & Nath, K.K. (2001). likeCo. Lycopodiella cernua, Oleandra pistillaris, 8 Illustrated manual of ferns of Assam. Dehra Dun, Oleandra wallichii, Katoella pulchra, Microsorum India: Bishen Singh Mahendra Pal Singh. membranaceum and Elaphoglossum marginatum etc. shown to have growing in more than one habitat. Dixit, R.D., & Vohra, J.N. (1984). A dictionary of the pteridophytes: Flora of India series- 4. Acknowledgements Howrah: Botanical Survey of India. Fraser-Jenkins, C.R., Pangtey, Y.P.S., & Khullar, S.P. We are grateful to Prof. Dr. Mohan Siwakoti, Head, (2010). Asplenium Laciniatum D. Don and all other faculties and administrative staffs of (Aspleniaceae): A critical complex and confused Central Department of Botany, TU for providing species in the Indian subcontinent. Indian Fern necessary facilities to accomplish the research. We J., 27, 178-215. would also like to thank Mr. Dhana Raj Kandel for the identification and providing the relevant Fraser-Jenkins, C.R. (2008). Taxonomic revision of literatures for the study. We would also like to three hundred Indian subcontinental express our sincere gratitude and thank Narahari pteridophytes with a revised census-list. Dehra Chapagain and the entire family of District Plant Dun, India: Bishen Singh Mahendra Pal Singh. Resources Office, Makwanpur Mr. Bishnu Rijal, Mr. Fraser-Jenkins, C.R., Kandel, D.R., & Pariyar, S. Pravin Bhandari, Mr. Roshan Gaudel Chhetri, Ms. (2015). Fern and fern alliles of Nepal, Vol I. Santa Budha Magar, Mr. Rajesh Tamang and Kathmandu, Nepal: National Herbarium and Plant Shreehari Bhattarai for their continuous support and Laboratories, Department of Plant Resources. encouragement.

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Gurung, V.L. (1984). Ferns of Nepal. I. Majupuria, Pryer, K.M., Schieder, H., Smith, A.R., Cranell, R., T.C. (Ed.), Nepal Natures Paradise (pp. 198-211). Wolf, P.G., Hunt, J.S., & Stipes, S.D. (2001). Gurung, V.L. (1986). Pteridophytes. In S.B. Malla, Horsetails & fens are a evidence on the ancient S.B. Rajbhandari, T.B. Shrestha, P.M. Adhikari, evolutionary split in vascular lands plants Science S.R. Adhikari & P.R. Shakya (Eds.) Flora of 1697-1699: A classification for extant ferns. Kathmandu valley. Bull. Dept. Med. Plants. Taxon, 55, 705-731. Thapathali, Kathmandu: Department of Plant Rajbhandary, S. (2013). Inventory of pteridophytes Resources. of Daman VDC, Makwanpur district, Central Gurung, V.L. (1991). Ferns the beauty of Nepalese Nepal with application of GIS. A report submitted flora. Kathmandu, Nepal, Sahayogi Press Pvt. to University Grants Commission (UGC) Nepal Ltd. (2012-2013) for Faculty Research Grant. IUCN (2013). Biodiversity resource inventory Rajbhandary, S. (2016.) Fern and fern allies of Nepal. ecosystem assessment of Bhadaure Tamagi VDC, In P.K. Jha, M. Siwakoti, & S. Rajbhandary Kaski. Nepal: International Union for (Eds.), Frontiers of Botany (pp 124-150). Conservation of Nature. Kathmandu, Nepal, Central Department of Botany, Tribhuvan University. Iwatsuki, K. (1988). An enumeration of the pteridophytes of Nepal. In H. Ohba & S.B. Malla Rakotondrainibe, F., & Raharimalada, F. (1998). The (Eds.), The Himalaya plant Bull. 31, (pp. 231- pteridophytes of the reserve speciale Anjanahari 339). Tokyo, Japan: Univeresity of Tokyo. Besud, Madagascar: Floristic analysis and altitudinal distribution. In S.M. Goodman, & N.S. Khare, P. B. (1996). Ferns and fern-allies- their Fieldiana, Zoology (Eds.). A floral and faunal significance and fantasies. Applied Botany inventory of the Réserve Spéciale Abstracts, Lucknow, India: NBRI. d’Anjanaharibe-Sud, Madagascar: With Khullar, S.P. (1994). An illustrated fern flora of west reference to elevational variation, 90, 1-246. himalaya. Vol. I. Dehra Dun, India: Intrernationl Schneider, G. (1894). The book of choice ferns. Vol. Book Distributors. III. London, England: L. Upcott Gill, London and Khullar, S.P. (2000). An illustrated fern flora of west Strand, W.C. himalaya. Vol. II. Dehra Dun, India: Internationl Shrestha, H.S. (2017). Floristic study of fern and Book Distributors. fern allies in altitudinal gradient from Besishahar Maren, I.E., Bhattarai, K.R., & Chaudhary, R.P. to lower Manang, western Nepal. (M.Sc.Thesis), (2013). Forest ecosystem services and Central Department of Botany, Tribhuvan biodiversity in contrasting himalayas forest University Kathmandu, Nepal. management services. Environmental Smith, J. (1879). Ferns: British & foreign. London: Conservation, 41(1), 73-83. David Bogue. Moran, R.C. (2004). A natural history of ferns. Smith, A.R., Pryer, K.M., Schuetpelz, E., Korall, P., Portland : Timber Press. Schneider, H., & Wolf, P.G. (2006). A Prajapati, S. (2013). Pteridophytes of Daman and classification for extant ferns. Taxon, 55, 705-731. adjoining areas, Makwanpur district, central WCMC (1992). Global biodiversity: Status of the Nepal. (M.Sc. Thesis), Central Department of earth’s living resources. World Conservation Botany, Tribhuvan University, Kathmandu, Monitoring Centre, London: Champman and Nepal. Hall.

45 2018J. Pl. Res. Vol. 16, No. 1, pp 46-56, 2018 Journal of Plant Resources Vol.16, No. 1

Plants of Mai Pokhari Botanical Garden and Adjoining Areas, East Nepal

Krishna Ram Bhattarai Department of Plant Resources, Thapathali, Kathmandu, Nepal E-mail: [email protected]

Abstract

Mai Pokhari area is a habitat of some endangered , rare orchids and an endemic bryophyte: Sphagnum nepalense. A preliminary survey was carried out in Mai Pokhari botanical garden and adjoining area of Ilam district, east Nepal to document flowering plants and some non-flowering plants (pteridophytes and bryophytes). This study documented, 238 plant species belonging to 195 genera and 98 families. Among them, 135 species were conserved naturally (in- situ), 51 species were domesticated (ex-situ) and 52 species were conserved in-situ as well as ex-situ in the field of Mai Pokhari botanical garden. This area is facing the impact of climate change and anthropogenic disturbances.

Key words: Biodiversity, Endangered, Ex-situ, Orchid, Wetland

Introduction There are some studies related to the documentation of flora of Mai Pokhari and its adjoining area such Nepal is rich in biological and cultural resources as as Rai, 1999; Basnet, 2003; DFO, 2012; Parajuli, the diverse climate and extreme altitudinal gradient 2012. These studies related with documentation of favors the occurrence of wide variety of flora, fauna phanerogams and vascular cryptogams. Rai (2005, and ethno-linguistic diversity (Martin et al., 2015). 2009) studied diatoms and Chlorophyceae from Mai Besides the local factors like temperature, rainfall, Pokhari lake. snowfall and aspects, the country is standing at the central position in the Himalayas and act as merging Nepal being one of the signatory countries of place of eastern and western Himalayan floristic Convention on Biological Diversity (CBD) has elements (MoFSC, 2014). The country has 35 forest committed to document the biological resources of types (Stainton, 1972), 75 vegetation types and 118 Nepal, and publish the Flora of Nepal up to 2020. ecosystems (BPP, 1995) providing habitat to over The Aichi targets of the CBD’s “Strategic Plan for 11,614 species of flora ranging from fungi to Biodiversity 2011-2020” set 20 targets to value, angiosperms (Shrestha, 2015). Since, more than two conserve, restore and wisely use biodiversity by hundred years, it has been the centre of attraction 2050, maintaining ecosystem functions and services, for the naturalists, plant explorers, expedition teams take effective and urgent action to halt the loss of and tourists. biodiversity in order to ensure resilient ecosystem (Chaudhary et al., 2015). Realizing the importance Several works have been done in the past on different of flora, this study aims to document plants of Mai aspects of biodiversity of Nepal. Some notable Pokhari and adjoining areas. This document will also works done in the field of plant diversity by Hara & provide valuable baseline information regarding the Williams, 1979; Hara et al., 1982; Malla et al., 1982; conserved plants in Mai Pokhari Botanical garden Polunin & Stainton, 1984; Manandhar, 1991; and its adjoining areas and for the implementation Shrestha & Ghimire, 1996; Maden & Dhakal, 1998; of conservation and research activities in Mai Siwakoti & Varma, 1999; Shrestha et al., 2004, 2008; Pokhari Ramsar site. Pandey, 2009; Bhuju, 2010; Limbu et al., 2012 are conducted in the eastern part of Nepal.

46 2018 Journal of Plant Resources Vol.16, No. 1

Materials and Methods Field survey

Study area The field survey was conducted in two seasons, first on September 23-29, 2015 and second on June 7- Mai Pokhari area lies in the middle of Kanchenjunga- 12, 2016. The plants were collected along with the Singhalila landscape complex, one of the route with the help of local guide. The photographs biodiversity hotspots as identified by the Critical of all the specimens were taken in their natural Ecosystem Partership Fund. The landscape was habitat before or after their collection. The plant recognized as the Kanchenjunga biodiversity specimens were collected and pressed in between hotspot, endemic birds areas, amphibian diversity newspapers. They were then dried in using a natural rich site and centre for plant species dispersal and drying technique in sunlight (Forman & Bridson, diversity (NCDC, 2005). 1989). Scientific names were determined by using Mai Pokhari wetland was declared as wetlands of different books (Polunin & Stainton, 1984; Stainton, international significance (Ramsar site) on 28 1988; Baral & Kurmi, 2006; Raskoti, 2009). For October 2008. The Ramsar site located at Sandakpur nomenclature of plant Press et al., (2000) was rural municipality of Ilam district, east Nepal (figure followed. Voucher specimens were deposited at the 1). It lies between 2,080 m to 2,164 m altitude and District Plant Resources Office (DPRO), Ilam. its total area of 90 hectare (ha.) includes 1.85 ha. of adjoiningwater areas body and for (Mai the Pokhari).implementation Over of conservationall climate and of Mairesearch activitiesResults in Maiand Discussion Pokhari Ramsar site. Pokhari area is temperate with cold and humid The study recorded, 238 plant species which belong Materialsconditions and Methods almost throughout the year. The average Study area to 195 genera and 98 families (table 1). Among Mai Pokhariannual area rainfall lies in the is middle 3,000 of mm. Kanchenjunga-Singhalila The average minimum landscape complex,them, one 197 of thespp. were angiosperms with 152 dicots biodiversitytemperature hotspots as atidentified winter by is the 2°C Critical and Ecosystem average Partershipmaximum Fund. The landscape was recognized as the Kanchenjunga biodiversity hotspot, endemic birds areas, amphibian(64%), diversity 45 monocots (19%), 8 gymnosperms (3%), rich sitetemperature and centre for plant at summer species dispersal is 18°C and diversity(Pradhan (NCDC, & Joshi, 2005). 30 pteridophytes (13%) and 3 bryophytes (1%) 2017). It has been identified as a biodiversity corridor (figure 2). In this study, the most dominant family Mai Pokhari(NCDC, wetland 2005; was declared DFO, 2012).as wetlands Cloud of international forest ecosystem significance (Ramsar site) on 28 October 2008. The Ramsar site located at Sandakpur rural municipality of Ilamwas district, east (27 spp.) followed by Rosaceae Nepal (Figurewith east-Himalayan1). It lies between 2,080 Oak-Laurel m to 2,164 m forest altitude is and the its maintotal area of(11 90 spp.)hectare and (ha.) (10 spp.). Rest of the families includescomponent 1.85 ha. of water of body the (Mai vegetation. Pokhari). Over The all climatevegetation of Mai Pokhariis area is temperate with cold and humid conditions almost throughout the year. The average annualis rainfall represented is 3,000 by less than 10 spp. Herbs were the mm. Thecapable average minimumof condensing temperature fogat winter water. is 20 CMai and averagePokhari maximum temperature at 0 most dominant species (111 spp.) followed by trees summerecosystem is 18 C (Pradhan offers & uniqueJoshi, 2017). opportunity It has been identifiedto observe as a biodiversity(60 spp.), corridor (42 spp.), climbers (15 spp.), woody (NCDC,impacts 2005; DFO, of 2012).climate Cloud change forest ecosystemon fauna with and east-Himalayan flora and Oak-Laurel forest is the main component of the vegetation. The vegetation is capable of condensingclimbers fog water. (5 Mai spp.) and creepers (5 spp.). Pokharisubsequent ecosystem offers mitigation unique opportunity (Pradhan to observe & Joshi, impacts 2017). of climate change on fauna and flora, and subsequent mitigation (Pradhan & Joshi, 2017). Among 152 species of dicots, trees were dominant (52 spp.) followed by herbs (44 spp.), shrubs (34 spp.), climbers (13 spp.), woody climbers (5 spp.) and creepers (4 spp.). Among them, 98 spp. were found naturally, 25 spp. were conserved naturally as well as domesticated and 29 spp. were domesticated (ex-situ) in the field. Among 45 spp. of monocots, herbs were dominant (37 spp.) followed by shrubs (6 spp.) and climbers (2 spp.). Among them, 18 spp. were found naturally, 11 spp. were found naturally as well as planted and 16 spp. were domesticated in the field. Among gymnosperms, 7 spp. were trees and 1 spp. was Figure 1: 1 Map: Map of Maipokhari of Maipokhari Ramsar site, Ramsar Ilam, Nepal site, Ilam, Nepal of which 2 spp. were found naturally and 6 spp. were

Field survey The field survey was conducted in two seasons, first on September 23-29, 2015 and second on June 7-12, 2016. The plants were collected along with the route with the help of local guide. The 47 photographs of all the specimens were taken in their natural habitat before or after their collection. The plant specimens were collected and pressed in between newspapers. They were then dried in using a natural drying technique in sunlight (Forman & Bridson, 1989). Scientific names were determined by using different books ( Polunin and Stainton, 1984; Stainton, 1988;

2 Baral and Kurmi, 2006; Raskoti, 2009). For nomenclature of plant Press et al., 2000 was followed. Voucher2018 specimens were deposited at the District Plant Journal Resources of OfficePlant (DPRO),Resources Vol.16, No. 1 Ilam.

Results and Discussionconserved as ex-situ. Out of 30 spp. of pteridophytes, lake but becoming threatened due to pollution and The study recorded,15 spp. 238 plantwere species conserved which belongin the to fern 195 housegenera andand 98 fern families anthropogenic(Table 1). activities like boating and peat Among them, garden197 spp. and were rest angiosperms species were with found152 dicots naturally. (64%), Among45 monocotsremoving (19%), 8 activities in the pond (WWF, 2007). For gymnosperms (3%), 30 pteridophytes (13%) and 3 bryophytes (1%) (Figure 2). In this study, the most dominant familybryophytes, was Orchidaceae Sphagnum (27 spp.) nepalense followed by isRosaceae endemic (11 spp.) to andits Ericaceae protection, District Plant Resources Office, Ilam (10 spp.). Rest Nepalof the andfamilies conserved is represented in-situ by sinceless than its population10 species. Herbs was werewith the mostthe Mai Pokhari Religious forest user group dominant speciesfound (111 spp.)decreasing followed in by alarming trees (60 spp.), rate. shrubs (42 spp.), climbersdeclared (15 spp.), a small area where the species still found woody climbers (5 spp.) and creepers (5 spp.). along the periphery of pond as “The Sphagnum Among 152 speciesIn this of dicots, study trees area, were Basnet dominant (2003), (52 spp.) documented followed by herbs 350 (44 spp.),Conservation shrubs Site”. This area was fenced with (34 spp.), climbersspp. (13 of spp.), vascular woody plantsclimbers where(5 spp.) asand Parajulicreepers (4 (2012), spp.). Amongbarbed them, 98 wire and an information board was kept there spp. were foundrecorded naturally, 25296 spp. spp. were including conserved algae,naturally fungi, as well lichens as domesticated and and 29 spp. were domesticated (ex-situ) in the field. Among 45 spp. of monocots, herbs were dominantto protect (37 from visitor’s disturbance (DPRO, 2016). spp.) followed cropby shrubs plants. (6 spp.) DFO and climbers (2012), (2 spp.).recorded Among 231them, spp.18 spp. werePradhan found & Joshi (2016) documented the experiences naturally, 11 spp.belonging were found to naturally 185 genera as well and as planted78 families and 16 with spp. wereorchids domesticatedof about in the 90 respondents in Maipokhari area about field. Among gymnosperms,(17 spp.), magnolia 7 spp. were (3trees spp.), and 1 rhododendron spp. was shrub of (5which spp.) 2 spp. were found naturally and 6 spp. were conserved as ex-situ. Out of 30 spp. of pteridophytes, 15climate spp. were change in terms of temperature, conserved in theand fern medicinal house and andfern aromaticgarden and plantsrest spp. (62 were spp.) found from naturally.precipitation, Among fog and water level in Mai Pokhari bryophytes, SphagnumMai Pokhari nepalense area.Thisis endemic to studyNepal and documented conserved in-situ more since its population was found decreasing in alarming rate. lake. To analyze the effect of climate change in Mai orchid spp. than DFO (2012) but out of 27 spp. of Pokhari area, regular monitoring of the floristic In this study area,orchids Basnet 10 (2003) spp. weredocumented not found 350 spp.in the of wild.vascular Similarly, plants where compositionas Parajuli should also be conducted. (2012) recordedthis 296 spp.study including documented algae, fungi, more lichens pteridophytes and crop plants. DFOthan (2012) recorded 231 spp. belonging to 185 genera and 78 families with orchids (17 spp.), magnolia (3 spp.), previous study of Basnet (2003) and DFO (2012). rhododendron (5 spp.) and medicinal and aromatic plants (62 spp.) from Mai PokhariConclusion area.This study documentedMany more speciesorchid spp. of than the DFO plants (2012) reported but out of 27earlier spp. of were orchids 10 spp. were not found in therarely wild. Similarly, found this in study this documented study more due pteridophytes to human than previous study of Basnet, 2003 and DFO, 2012. Many species of the plants reported earlier were rarelyMai found Pokhari in area is an important religious and this study due toencroachment human encroachment and and probably probably byby climateclimate change. change. aesthetic site of Nepal. It has national and international significance as it is in Ramsar list. The Bryophytes present investigation in Mai Pokhari area Pteridophytes 1% 13% documented, 238 plant spp. with 152 spp. of dicots, Gymnosperms 3% 45 spp. of monocots, 8 spp. of gymnosperms, 30 spp. of pteridophytes and 3 spp. of bryophytes.

Dicots Among the total documented plant spp., 136 spp. Monocots 64% 19% were found naturally (in-situ), 51 spp. were domesticated (ex-situ) and 51 spp. were found naturally as well as conserved in the field of Mai Figure 2: CategoryFigure of documented 2: Category plants of documented plants Pokhari botanical garden. This study did not cover all the category of flora so further exploration should Among the total documented3 plant species, 136 spp. be conducted in this area at different seasons. To were found naturally (in-situ), 51 spp. were study the effect of climate change, emphasis should domesticated (ex-situ) and 51 spp. were found be given to the documentation of epiphytic plants, naturally as well as conserved in the field of Mai orchids, lichens and mosses. Pokhari botanical garden. Of the domesticated 51 spp., most of them were rare or not found naturally Acknowledgements in this study area. Analyzing the local of naturally available plants, 57 spp. were I am grateful to Mr. Sanjeev Kumar Rai, Director common, 59 spp. were not so common and 66 spp. General and Ms. Jyoti Joshi Bhatta, Deputy Director were rare in this area. Basnet, 2003; DFO, 2012 and General of DPR for providing support to this study. Parajuli, 2012 did not mention their local I would like to thank Ms. Jeny Niraula, Mr. Dipen conservation status as well as whether they were Bam, Mr. Tula Ghimire, Mr. Ram Rai, Mr. Lakpa conserved in-situ or ex-situ. An endemic moss Sherpa and Mr. Padam Acharya of DPRO Ilam for Sphagnum nepalense is found in the Mai Pokhari their help and support during the field work and

48 2018 Journal of Plant Resources Vol.16, No. 1 herbarium management. I would like to give special Hara, H., & Williams, L.H.J. (1979). An enumeration thanks to Prof. Dr. Devendra Man Bajracharya and of the flowering plants of Nepal. Vol. II. London: Prof. Dr. Mukesh Kumar Kshetri for their presence British Natural History Museum. in orchid identification at the study site. Hara, H., Charter, A.H., & Williams, L.J.H. (1982). An enumeration of the flowering plants of Nepal. References Vol. III. London: British Natural History Museum. Baral, S.R., & Kurmi, P.P. (2006). A Compendium Maden, K., & Dhakal, M.R. (1998). General survey of medicinal plants in Nepal. Maiju Bahal, of edible wild fruits from Koshi zone, eastern Chabahil, Kathmandu, Nepal: Mrs. Rachana Nepal. T.U. Journal, 21(1), 77–84. Sharma. Malla, S.B., Rajbhandari, S.B., Shrestha, T.B., Basnet, B.K. (2003). Floral diversity of Mai Pokhari, Adhikari, P.M., & Adhikari, S.R. (Eds.) (1982). Ilam. Bulletin of Department of Plant Resources, Wild edible plants of Nepal. Bull. Dept. Med. 22, 60-69. Plants, Nepal, 9, Thapathali, Kathmandu: Bhuju, D.R. (2010). The churia hills of Nepal: Land Department of Plant Resources. use change and biodiversity. Proceedings of Manandhar, N.P. (1991). Some additional note on National seminar on Churia region environmental wild food plants of Nepal. J. Nat. Hist. Mus., Study, Department of Soil Conservation and 12(1– 4), 19–32. Watershed Management, Kathmandu, Nepal. Martin, G., Turin, M., Tuladhar-Douglas, W., & BPP (1995). Biodiversity profile of the midhills Chhdtri, R.B. (2015). People. In G. Miehe, C.A. physiographic zone. Technical Publication No. Pendry & R. Chaudhary ( Eds.), Nepal : An 13. Nepal: Government of Nepal, Ministry of introduction to the natural history, ecology and Forest and Soil Conservation and Netherland: human environment of the Himalayas (pp. 251- Government of the Netherlands, Ministry of 269). UK: Royal Botanic Garden Edinburgh. Foreign Affairs. MoFSC (2014). Nepal biodiversity strategy and Chaudhary, R.P., Uprety, Y., Joshi, S.P., Shrestha, action plan: 2014-2020. Kathmandu, Nepal: K.K., Basnet, K.B., Basnet, G., Shrestha, K.R., Government of Nepal, Ministry of Forests and Bhatta, K.P., Acharya, K.P., & Chettri, N. (2015). Soil Conservation. KL Nepal: From conservation and development perspectives. Kathmandu, Nepal: Ministry of NCDC (2005). Participatory conservation corridor Forests and Soil Conservation (MoFSC), development strategy and action plan for Government of Nepal; Research Centre for transborder areas along the Kangchenjunga Applied Science and Technology (RECAST), landscape in eastern Nepal. Ilam: Namsaling Tribhuvan University; and International Centre Community Development Center (NCDC). for Integrated Mountain Development (ICIMOD). Pandey, J. (2009). Plant diversity in lower Kangchenjungha- Singhalila ridge, Panchthar DFO (2012). Management plan of Mai Pokhari district, eastern Nepal. (M.Sc. Thesis), Central Ramsar site, Ilam, Nepal. Ilam, Nepal: District Department of Botany, Tribhuvan University, Forest Office. Kathmandu, Nepal. DPRO (2016). Annual Report (Text: in Nepali). Ilam, Parajuli, R.R. (2012). Preliminary enumeration of Nepal: District Plant Resources Office (DPRO). major flora of Mai Pokhari Ramsar site. Ilam: Ilam, Nepal: District Plant Resources Office. Forman, L., & Bridson, D. (1989). The herbarium handbook. UK: Royal Botanic Gardens, Kew. 49 2018 Journal of Plant Resources Vol.16, No. 1

Polunin, O., & Stainton, A. (1984). of the Shrestha, K.K., & Ghimire, S.K. (1996). Plant Himalaya. New Delhi, India. Oxford University diversity inventory of the proposed Kanchenjunga Press. conservation area (Ghunsa and Simbua khola valleys). A Report submitted to World Wildlife Pradhan, S.M., & Joshi, R.M. (2017). Wetland, Fund (WWF) Nepal. cloud-forest and climate change: In the context of Mai Pokhari Ramsar site. Nepal: The East Shrestha, K.K., Kunwar, R.M., Dhamala, M.K., Foundation. Humagain, K., Pandey, J., & Khatri, N.B. (2008). Conservation of plant resources in Press, J.R., Shrestha, K.K., & Sutton, D.A. (2000). Kangchenjungha - Singhalila ridge, eastern Annotated checklist of the flowering plants of Nepal. Journal of Plant Science, 2, 62-68. Nepal. London: The Natural history Museum. Shrestha, K.K., Rajbhandary, S., Tiwari, N.N., Rai, L.R. (1999). Flora of Mai Pokhari and adjoining Poudel, R.C., & Uprety, Y. (2004). Ethnobotany area, Ilam district, east Nepal. (M.Sc. Thesis), in Nepal: Review and perspectives. Kathmandu: Central Department of Botany, Tribhuvan World Wildlife Fund (WWF) Nepal Program. University, Kathmandu, Nepal. Siwakoti, M., & Varma, S.K. (1999). Plant diversity Rai, S.K. (2005). Preliminary report of diatoms from of eastern Nepal: Flora of plains of eastern Mai Pokhari lake, Ilam, Nepal. Our Nature, 3, Nepal. Dehara Dun, India: Bishen Singh 26-30. Mahendra Pal. Rai, S.K. (2009). Some chlorophycean algae from Stainton, A. (1988). Flowers of the Himalaya: A Mai Pokhari lake, Ilam, east Nepal. Journal of supplement. New Delhi, India: Oxford University Natural History Museum, 24, 1-8. Press. Raskoti, B.B. (2009). The orchids of Nepal. Nepal: Stainton, J.D.A. (1972). Forests of Nepal. London: Bhakta Bahadur Raskoti and Rita Ale. John Murray. Shrestha, K.K. (2015). Flora of Nepal: Issues, WWF (2007). Mai Pokhari Ilam-Importance of progress and way forward. In M. Siwakoti & S. religion, culture and tradition in conservation. Rajbhandari (Eds.), Taxonomic tools and flora Nepal: World Wildlife Fund. writing (pp. 25-52). Kathmandu, Nepal: Department of Plant Resources & Central Department of Botany, Tribhuvan University.

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Table 1. Plant list of Mai Pokhari area DD=icotyleodons Domesticated; N= Natural; C= Common; Nc= Not so common; R= Rare; H= Herb; Sh= Shrub; T= Tree; Cl= Climber, TWablec= Woody 1. Plant climber; list of Mai Cr.= Pokhari Creeper area Plant category; Local DicotyleodonsS.N. Scientific name Family Local name habit, collection status Plantnumber category; Local S.N.1 Acer oblongumScientific Wall. ex DC.name Aceraceae Family Putli Local name N; R Dicot;habit, T; 9collection status 2 Achyranthes aspera L. Amaranthaceae Datiwan/ankhle N; C Dicot; H;number 4 13 AcerAconitum oblongum ferox Wall. ex DC.Ser. AceraceaeRanunculaceae PutliBish N;D R Dicot; T;H; 951 24 AchyranthesAconitum heterophyllum aspera L. Wall. ex Royle RanunculaceaeAmaranthaceae BikhmaDatiwan/ankhle DN; C Dicot; H; 84 35 AconitumActinidia callosaferox Wall. Lindl. ex Ser. Actinidiaceae Ranunculaceae ThekiBish Phal N;D R Dicot; Wc;H; 51 64 46 AconitumAeschynanthus heterophyllum hookeri C. Wall. B. Clarke ex Royle GesneriaceaeRanunculaceae Bikhma N;D R Dicot; H; 1358 57 ActinidiaAeschynanthus callosa sikkimensis Lindl. (C. B. Clarke) ActinidiaceaeGesneriaceae Theki Phal N; R Dicot; Wc;H; 139 64 6 AeschynanthusStapf hookeri C. B. Clarke Gesneriaceae N; R Dicot; H; 135 78 AeschynanthusAgapetes serpens sikkimensis (Wight) Sleumer (C. B. Clarke) Gesneriaceae Ericaceae Khursane N; RNc Dicot; H;Sh; 139 201 9 StapfAnaphalis adnata Wall. ex DC. Buki Phool N; C Dicot; H; 5 108 ArtemisiaAgapetes serpensindica Willd. (Wight) Sleumer AsteraceaeEricaceae TitepatiKhursane N; CNc Dicot; H;Sh; 36 201 119 AstilbeAnaphalis rivularis adnata Buch.–Ham. Wall. ex DC. ex D.Don AsteraceaeSaxifragaceae BukiBudho Phool okhati N;N/D; C R Dicot; H; 578 1210 AzaleaArtemisia alabamensis indica Willd. (Rehder) Ashe Ericaceae Asteraceae AzaliaTitepati DN; C Dicot; Sh;H; 36 204 1311 BaliospermumAstilbe rivularis corymbiferum Buch.–Ham. Hook.ex D.Don f EuphorbiaceaeSaxifragaceae ChaulaneBudho okhati N;N/D; C R Dicot; Sh;H; 78 30 1412 BegoniaAzalea alabamensis picta Sm. (Rehder) Ashe BegoniaceaeEricaceae MagarkancheAzalia N/D;D Nc Dicot; Sh;H; 113 204 1513 BerberisBaliospermum aristata corymbiferum var. aristata Hook.DC. f EuphorbiaceaeBerberidaceae ChutroChaulane N/D;N; C C Dicot; Sh; 330 1614 BergeniaBegonia picta ciliata Sm. (Haw.) Sternb. BegoniaceaeSaxifragaceae MagarkanchePakhanbed N/D;D/N; NcR Dicot; H; 113202 1715 BetulaBerberis alnoides aristata Buch.-Ham. var. aristata ex DC. D. Don Betulaceae Berberidaceae SaurChutro N;N/D; R C Dicot; T;Sh; 101 3 1816 BistortaBergenia amplexicaulis ciliata (Haw.) (D. Sternb. Don) Greene PolygonaceaeSaxifragaceae PakhanbedBhale D D/N; R Dicot; H; 117202 17 Betula alnoides Buch.-Ham. ex D. Don Betulaceae Saurpakhenved N; R Dicot; T; 101 1918 BrassaiopsisBistorta amplexicaulis mitis C. B. (D. Clarke Don) Greene PolygonaceaeAraliaceae BhaleDhapre N;D Nc Dicot; H;T; 203117 20 Camellia sinensis (L.) Kuntze Theaceae Chiyapatepakhenved D Dicot; Sh; 211 2119 CastanopsisBrassaiopsis hystrixmitis C. Miq. B. Clarke AraliaceaeFagaceae PataleDhapre katus N; NcC Dicot; T; 203209 2220 CastanopsisCamellia sinensis indica (L.) (Roxb.) Kuntze Miq. TheaceaeFagaceae ChiyapateKatus DN; C Dicot; Sh;T; 210 211 2321 Castanopsis tribuloideshystrix Miq. (Sm.) A. DC. Fagaceae PataleMusure katus katush N; C Dicot; T; 100209 2422 CentellaCastanopsis asiatica indica (L.) (Roxb.) Urb. Miq. UmbelliferaeFagaceae KatusGhodtapre N; C Dicot; T;H; 21055 2523 CereusCastanopsis peruvianus tribuloides (L.) Mill.(Sm.) A. DC. CactaceaeFagaceae Musure katush DN; C Dicot; T;Sh; 100 149 2624 CeropegiaCentella asiatica pubescens (L.) Urb.Wall. UmbelliferaeAsclepiadaceae Ghodtapre N; CR Dicot; Cl;H; 55123 2725 ChoerospondiasCereus peruvianus axillaris (L.) Mill. (Roxb.) B. L. Burtt CactaceaeAnacardiaceae Lapsi D Dicot; T;Sh; 208 149 26 &Ceropegia A. W. Hill, pubescens Wall. Asclepiadaceae N; R Dicot; Cl; 123 2827 CinnamomumChoerospondias bejolghota axillaris (Roxb.)(Buch.-Ham.)Sweet B. L. Burtt AnacardiaceaeLauraceae LapsiSinkauli DN/D; R Dicot; T; 20898 29 & A. W. Hill, camphora (L.) J. Presl Kapur D Dicot; T; 109 3028 Cinnamomum glanduliferumbejolghota (Buch.-Ham.)Sweet (Wall.) Meisn. Lauraceae SinkauliMalagiri DN/D; R Dicot; T; 10498 3129 CleistocactusCinnamomum sp.camphora (L.) J. Presl Cactaceae Lauraceae Kapur D Dicot; Sh;T; 109 181 3230 ClematisCinnamomum buchananiana glanduliferum DC. (Wall.) Meisn. RanunculaceaeLauraceae JungeMalagiri lahara N;D R Dicot; Cl;T; 104 121 3331 CupheaCleistocactus procumbens sp. Cav. LythraceaeCactaceae D Dicot; Sh; 189181 3432 CyathulaClematis buchananianacapitata Moq. DC. RanunculaceaeAmaranthaceae ChuireJunge lahara kuro N; RC Dicot; H;Cl; 59121 3533 DaphneCuphea procumbensbholua Buch.-Ham. Cav. ex D.Don LythraceaeThymelaeaceae Lokta N/D;D Nc Dicot; Sh; 34189 3634 DichroaCyathula febrifuga capitata Moq.Lour. HydrangeaceaeAmaranthaceae ChuireBashak kuro N;N/D; C Nc Dicot; Sh;H; 59 65 3735 DigitalisDaphne bholua purpurea Buch.-Ham. L. ex D.Don ThymelaeaceaeScrophulariaceae LoktaDigitalis N/D;D Nc Dicot; Sh;H; 132 34 3836 DioscoreaDichroa febrifuga kamoonensis Lour. Kunth HydrangeaceaeDioscoreaceae RaniBashak vyakur N;N/D; R Nc Dicot; Cl;Sh; 4665 3937 DrymariaDigitalis purpurea cordata LL. CaryophyllaceaeScrophulariaceae AbijaloDigitalis N;D C Dicot; H; 118132 4038 EchinocactusDioscorea kamoonensis sp. Kunth DioscoreaceaeCactaceae Rani vyakur N;D R Dicot; Cl;Sh; 46187 39 Drymaria cordata L Caryophyllaceae Abijalo N; C Dicot; H; 118 41 gardneri (Wall.) Meisn. Arghali N/D; C Dicot; Sh; 106 40 Echinocactus sp. Cactaceae D Dicot; Sh; 187 42 Elaeocarpus lanceifolius Roxb. Elaeocarpaceae Bhadrakcha N/D; R Dicot; T; 107 41 Edgeworthia gardneri (Wall.) Meisn. Thymelaeaceae Arghali N/D; C Dicot; Sh; 106 43 Elatostema sessile var. sessile J. R. & G. Gagleto N; C Dicot; H; 240 42 Elaeocarpus lanceifolius Roxb. Elaeocarpaceae Bhadrakcha N/D; R Dicot; T; 107 Forst. 43 Elatostema sessile var. sessile J. R. & G. Urticaceae Gagleto N; C Dicot; H; 240 Forst.

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44 Elsholtzia blanda (Benth.) Benth. Ban silam N; Nc Dicot; H; 238 45 Elsholtzia ciliata (Thunb.) Hyland Lamiaceae Bhotepaati N; R Dicot; Sh; 35 46 Elsholtzia strobilifera Benth. Lamiaceae Phurke jhar/Ban N; C Dicot; H; 41 bawari 47 Engelhardia spicata Lesch. ex Blume Mauwa N; R Dicot; T; 207 48 Erythrina arborescens Roxb. Phaledo/Theki N; R Dicot; T; 88 kath 49 Euodia fraxinifolia (D. Don) Hook. f. Rutaceae Khanakpa N; R Dicot; T; 76 50 Eupatorium adenophorum Spreng. Asteraceae Kalijhar N; C Dicot; H; 77 51 Euphorbia milii Des Moul. Simari D Dicot; Sh; 182 52 Euphorbia royleana Boiss. Euphorbiaceae Siundi D Dicot; T; 180 53 Eurya acuminata DC. Theaceae Jhigane/phalam N; Nc Dicot; T; 15 e 54 Exbucklandia populnae (R.Br.ex Griff)R.W. Hamamelidaceae Pipli N/D; Nc Dicot; T; 99 Br 55 Ferocactus sp. Cactaceae D Dicot; Sh; 183 56 neriifolia Sm. Dudhilo N; C Dicot; T; 142 57 Fragaria nubicola Lindl. ex Lacaita, Rosaceae Bhuin ainselu N; C Dicot; Cr; 146 58 pinnata Roxb. Dabdabe N; C Dicot; T; 141 59 Gentiana sp. Gentianaceae Tite N; Nc Dicot; H; 161 60 diversifolia (Link) Friis Urticaceae Allo N; Nc Dicot; H; 152 61 Gnaphalium affine D. Don Asteraceae Buki phool- N; C Dicot; H; 162 yellow 62 Gonostegia hirta (Blume) Miq. Urticaceae Bhuinchiple N; C Dicot; Cr; 163 63 Hatiora sp. Cactaceae D Dicot; Sh; 188 64 Hemiphragma heterophyllum Wall. Scrophulariaceae Lalgedi/Nasjhar N; Nc Dicot; Cr; 148 65 Heracleum nepalense D.Don Apiaceae Chimphing N/D; Nc Dicot; H; 95 66 Herpetospermum pedunculosum (Ser.) Baill Ban karela N; Nc Dicot; Cl; 26 67 Holboellia latifolia var. latifolia Wall. Lardizabalaceae Gulpha/gufla N; R. Dicot; Cl; 40 68 aspera Buch.-Ham. ex D. Don Nilkamal N/D; R Dicot; Sh; 147 69 Hymenopogon parasiticus Wall. Rubiaceae Phirphire N; R Dicot; Sh; 19 70 cordifolium Choisy Clusiaceae Kamile jhar/ N; Nc Dicot; Sh; 70 Urilo 71 Ilex dipyrena Wall. Aquifoliaceae Kande liso N; R Dicot; T; 67 72 Impatiens edgeworthii Hook. f. Balsaminaceae Ban tiuri N; C Dicot; H; 24 (Bhale) 73 regia L Juglandaceae Okhar D Dicot; T; 145 74 Laportea terminalis Wight Urticaceae Patale Sisnu N; Nc Dicot; H; 25 75 Leucosceptrum canum Sm. Lamiaceae Ghurpis/Bhusur N; C Dicot; T; 14 e 76 Lindera neesiana (Wall. ex Nees) Kurz Lauraceae Siltimur N/D; Nc Dicot; T; 75 77 Lindera pulcherrima (Nees)Hook. f. Lauraceae Sishi N; Nc Dicot; T; 165 78 Lithocarpus elegans (Blume)Soepadmo Fagaceae Arkhaule N; Nc Dicot; T; 144 79 Lithocarpus pachyphylla (Kurz) Rehder Fagaceae Bante, Gante N; R Dicot; T; 150 80 ovalifolia (Wall.)Drude Ericaceae Angeri N/D; Nc Dicot; Sh; 22 81 Macaranga pustulata King ex Hook. f. Euphorbiaceae Maleto N; C Dicot; T; 164 82 Machilus odoratissima Nees Lauraceae Kaulo N/D; R Dicot; T; 175 83 Magnolia campbellii Hook.f.& Thomson Ghoge champ N; R Dicot; T, 122 84 Magnolia doltsopa (Buch.-Ham. ex DC.) Magnoliaceae Rani champ N; R Dicot; T; 116 Figlar 85 Magnolia languinosa (Wall.)Figlar & Noot. Magnoliaceae Phusre champ N; R Dicot; T; 115 86 Mahonia acanthifolia G. Don Berberidaceae Keshari N/D; Nc Dicot; Sh; 87 87 Mammillaria sp. Cactaceae D Dicot; Sh; 185 88 Mucuna macrocarpa Wall. Fabaceae Pangra/baldangr N; R Dicot; Wc; 50 a 89 Nelumbo nucifera Gaertn. Nymphaceae Kamal N; Nc Dicot; H; 124

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90 Nepata erecta (Royle ex Benth.) Benth. Lamiaceae N; C Dicot; H; 191 91 Nicotiana tabacum L. Solanaceae Kancho pat N; R Dicot; H; 90 92 Opuntia monacantha (Willd.) Haw. Cactaceae D Dicot; Sh; 186 93 Osbeckia stellata Buch.-Ham. ex D. Don Melastomataceae Lote phool N; C Dicot; Sh; 190 94 Oxalis corniculata L. Oxalidaceae N; C Dicot; H; 205 95 Oxalis latifolia Humb. Oxalidaceae N; C Dicot; H; 237 96 Oxyspora paniculata (D. Don) DC Melastomataceae Lote Phool N; C Dicot; Sh; 7 97 Panax pseudo-ginseng Wall. Araliaceae Ginsing N/D; R Dicot; H; 119 98 Persea odoratissima (Nees) Kosterm. Lauraceae Seto Kaulo N/D; Nc Dicot; T; 120 99 Persicaria capitata (Buch.-Ham. ex D. Don) Ratnaulo N; C Dicot; H; 58 H. Gross 100 Phytolacca acinosa Roxb. Phytolaccaceae Jaringo rato N; Nc Dicot; Sh; 131 101 Pieris formosa (Wall.) D. Don Ericaceae Balu/Lekh D Dicot; Sh; 108 angeri 102 Piper mullesua Buch.-Ham. ex D. Don Piperaceae Chabo N; R Dicot; Cl; 31 103 Plantago erosa Wall. Kane jhar N; C Dicot; H; 56 104 Potentilla fulgens Wall. ex. Hook. Rosaceae Bajradanti/ N/D; R Dicot; H; 60 Banmula 105 Primula sp. Primulaceae N/D; C Dicot; H; 155 106 Prunus cerasoides D. Don Rosaceae Painu N; R Dicot; T; 110 107 Pyracantha crenulata (D.Don) Roem. Rosaceae Ghangaru D Dicot; Sh; 105 108 Pyrularia edulis (Wall. ex Roxb.) DC. Santalaceae Amphi N; R Dicot; T; 71 109 Pyrus pashia Buch.-Ham. ex D. Don Rosaceae Mel N; R Dicot; T; 83 110 Quercus lamellosa Sm. Fagaceae Bajrath N; R Dicot; T; 84 111 Quercus semecarpifolia Sm. Fagaceae Khashru N; R Dicot; T; 97 112 Rheum australe D. Don Polygonaceae Padamchal D Dicot; H; 154 113 Rhododendron arboreum Sm Ericaceae Lali gurans N/D; Nc Dicot; T; 125 114 Rhododendron dalhousieae Hook.f. Ericaceae Chimal N/D; R Dicot; T; 129 115 Rhododendron grande Wight Ericaceae Potlingo D Dicot; T; 94 116 Rhododendron griffithianum Wight Ericaceae Chimal N/D; R Dicot; T;33 117 Rhus javanica Miller Bhakimlo D Dicot; T; 54 118 Rhus succedanea var. succedanea L. Anacardiaceae Rani bhalayo N; R Dicot; T; 49 119 Rosa sp. Rosaceae D Dicot; Sh; 160 120 Rubia manjith Roxb. ex Fleming Rubiaceae Majitho N; C Dicot; Cl; 2 121 Rubus acuminatus Sm. Rosaceae Lahare ainselu, N; C Dicot; Cl; 48 darme 122 Rubus ellipticus Sm. Rosaceae Ainselu N; C Dicot; Sh; 159 123 Rubus foliolosus D. Don Rosaceae N; C Dicot; Wc; 158 124 Rubus hoffmeisterianus Kunth & Bouche Roaceae Darme/ainselu N; C Dicot; Wc; 39 125 Rubus sp.sp. Rosaceae Darme-Thulo N; C Dicot; Wc; 27 126 Rumex nepalensis Spreng. Polygonaceae Halhale N; C Dicot; Cr; 37 127 Sambucus canadensis L. Sambucaceae Kanike phool N; Nc Dicot; T; 80 128 Saurauia napaulensis DC. Saurauiaceae Gogan N; C Dicot; T; 57 129 Schefflera impressa (C. B. Clarke) Harms Araliaceae Bhale chinde N; R Dicot; T; 96 130 Schisandra propinqua (Wall.) Baill. Schisandraceae Nyalchu N; Nc Dicot; Cl; 157 131 Selinum tenuifolium Wall.ex C.B.Clarke Apiaceae Bhutkesh N/D; R Dicot; H; 62 132 Stenocerus sp. Cactaceae D Dicot; Sh; 184 133 Swertia angustifolia Buch.-Ham. ex D. Don Gentianaceae Bhale chiraito N; R Dicot; H; 20 134 Swertia bimaculata (Sieb. & Zucc.) C. B. Gentianaceae Bhale chiraito N; R Dicot; H; 17 Clarke 135 Swertia chirayita (Roxb. ex Fleming) Karsten Gentianaceae Chiraito N/D; R Dicot; H; 10 136 Symplocos pyrifolia Wall. ex G. Don Symplocaceae Kholme N; R Dicot; T; 11 137 Symplocos ramosissima Wall. ex G. Don Symplocaceae Kharane N; Nc Dicot; T; 9 138 Tetrastigma bracteolatum (Wall.) Planch. Vitaceae Charchare N; Nc Dicot; Cl; 12 139 Tetrastigma serrulatum (Roxb.) Planch., Vitaceae Charchare N; Nc Dicot; Cl; 156 140 Thunbergia coccinea Wall. ex D. Don Acanthaceae Kanase lahara N; Nc Dicot; Cl; 47

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141 Trichosanthes wallichiana (Ser.) Wight Cucurbitaceae Indreni N/D; Nc Dicot; Cl; 28 142 Urtica ardens Link Urticaceae Sisno-ghariya N; C Dicot; H; 82 143 Urtica dioca L. Urtucaceae Sisno N; C Dicot; H; 166 144 Vaccinium nummularia Hook. f. & Thomson Ericaceae N; Nc Dicot; Sh; 86 ex C. B. Clarke 145 Vaccinium vacciniaceum (Roxb.) Sleumer Ericaceae N; Nc Dicot; Sh; 153 146 Valeriana jatamansii Jones Valerianaceae Sugandhawal D Dicot; H; 127 147 Viburnum cylindricum Buch.-Ham. ex D. Sambucaceae Ghode khari N; R Dicot; T; 114 Don 148 Viburnum erubescens Wall. ex DC Sambucaceae Asare N; Nc Dicot; T; 126 149 Viola sp. Violaceae N; Nc Dicot; H; 128 150 Zanthoxylum acanthopodium DC. Rutaceae Boke timur N; Nc Dicot; Sh; 13 151 Zanthoxylum armatum DC. Rutaceae Parpare timur D Dicot; T;151 152 Zanthoxylum oxyphyllum Edgew. Rutaceae Boketimur N; Nc Dicot; T; 79

Monocotyledons Plant category; Local S.N. Scientific name Family Local name habit, collection status number 1 Acorus calamus L. Araceae Bojho N/D; R Monocot; H; 93 2 Agave americana L Agavaceae Ketuke D Monocot; Sh; 130 3 Aloe vera (L.) Burm. f. Asphodelaceae Ghyukumari D Monocot; H; 192 4 Arisaema nepenthoides (Wall.) Mart. ex Araceae Laduwa/sarpa N; C Monocot; H; 42 Schott makai 5 Arundina graminifolia (D.Don) Hochr. Orchidaceae D Monocot; H; 193 6 Arundinaria maling Gamble Poaceae Malingo N/D; C Monocot; Sh; 195 7 curillus Buch.-Ham.ex D.Don Kurilo D; R Monocot; H; 136 8 guttulatum (Hook.f.)Balakr. Orchidaceae N; R Monocot; H; 167 9 Bulbophyllum leopardinum(Wall.)Lindl. Orchidaceae N; R Monocot; H; 215 10 Calanthe chloroleuca Lindl. Orchidaceae D Monocot; H; 226 11 Calanthe mannii Hook.f. Orchidaceae N/D; Nc Monocot; H; 222 12 Cardiocrinum giganteum (wall.) Makino Liliaceae Chameli/Ghiu N/D; R Monocot; H; 223 pat 13 Chlorophytum nepalense (Lindl.) Baker Liliaceae Ban alu N; Nc Monocot; H; 85 14 Coelogyne cristata Lindl. Orchidaceae N; R Monocot; H; 134 15 Coelogyne nitida Lindl. Orchidaceae D Monocot; H; 194 16 Curculigo crassifolia (Baker) Hook. f. Hypoxidaceae Dhotisaro N/D; R Monocot; Sh; 44 17 iridioides D. Don Orchidaceae D Monocot; H; 232 18 Cymbidium devonianium Lindl. ex Paxton Orchidaceae N; R Monocot; H; 236 19 Cymbidium erythraeum Lindl. Orchidaceae D Monocot; H; 230 20 Cymbidium hookerianum Rchb.f. Orchidaceae D Monocot; H; 237 21 Cymbidium longifolium D.Don Orchidaceae D Monocot; H; 224 22 Cymbopogon citratus (DC.)Trin. Poaceae lemongrass D Monocot; H; 233 23 Dactylorhiza hatagirea (D. Don) Soo Orchidaceae Panchaunle D Monocot; H; 238 24 Dendrobium eriaeflorum Griff Orchidaceae N; R Monocot; H; 234 25 Dendrobium longicornu Lindl. Orchidaceae N; Nc Monocot; H; 229 26 Drepanostachyum falcatum (Nees) Keng f. Poaceae Diu nigalo D Monocot; Sh; 225 27 Drepanostachyum intermedium (Munro) Poaceae Paryang, nigalo N; C Monocot; Sh; 231 Keng f. 28 Cautleya spicata (Sm.) Baker Zingiberaceae Sarato N; R Monocot; H; 45 29 Epigenium rotundatum (Lindl.) Summerh. Orchidaceae N/D; R Monocot; H; 228 30 Eria coronaria (Lindl.) Rchb.f. Orchidaceae N/D; R Monocot; H; 239 31 Euluphia sp. Orchidaceae N; R Monocot; H; 242 32 Habenaria sp. Orchidaceae N; R Monocot; H; 227 33 Lilium nepalense D. Don Liliaceae Ban lasun/okhe D Monocot; H; 248 alu

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34 Liparis sp. Orchidaceae N; R Monocot; H; 243 35 Otochilus albus Lindl. Orchidaceae D Monocot; H; 241 36 Paris polyphylla Sm Trilliaceae Satuwa N/D; R Monocot; H; 246 37 Phaius tankervilliae (Banks) Blume Orchidaceae D Monocot; H; 240 38 Pleione hookeriana (Lindl.) Kuntze Orchidaceae N/D; R Monocot; H; 235 39 Pleione praecax (Sm.) D. Don Orchidaceae N; R Monocot; H; 168 40 Rhaphidophora decursiva (Roxb.) Schott Araceae kanchirno D Monocot; Cl; 102 41 Satyrium nepalense D. Don Orchidaceae N; Nc Monocot; H; 219 42 Smilax aspera L. Smilaceae Kukurdiano N; R Monocot; Cl; 244 43 Spiranthes sinensis (Pers.) Ames Orchidaceae N; R Monocot; H; 43 44 Thamnocalamus spathiflorus var. crassinodus Poaceae Ghunre Nigalo N/D; C Monocot; Sh; 245 (Yi) Stapleton 45 Vandopsis undulata Sm. Orchidaceae N; R Monocot; H; 16

Gymnosperms Plant category; Local S.N. Scientific name Family Local name habit, collection status number 1 Abies spectabilis (D.Don) Mirb. Pinaceae Ghoge sallo D Gymnosperm; T; 61 2 Cryptomeria japonica (L. f.) D. Don Taxodiaceae Japanese sallo N; C Gymnosperm; T; 140 3 Juniperus horizontalis Moench Cupressaceae Dhupi D Gymnosperm; Sh; 138 4 Pinus roxburghii Sarg. Pinaceae Khote sallo D Gymnosperm; T; 169 5 Pinus wallichiana A. B. Jacks Pinaceae Gobre sallo N;Nc Gymnosperm; T; 170 6 Taxus wallichiana Zucc. Taxaceae Lauth salla D Gymnosperm; T; 89 7 Thuja orientalis (L.)Franco Cupressaceae Dhupi D Gymnosperm; T; 137 8 Tsuga dumosa (D. Don) Eichler Pinaceae Thingre salla D Gymnosperm; T; 66

Pteridophytes Plant category; Laocal S.N. Scientific name Family Local name habit, collection status number 1 Adiantum sp. Pteridaceae N; C Pteridophytes; H; 68 2 Aleuritopteris albomarginata(C.B.Clarke) Aleuritopteris N; C Pteridophytes; H; 103 Ching 3 Arthromeris wallichiana (spreng.) ching Polypodiaceae N/D; C Pteridophytes; H; 21 4 Athyrium sp. Athyriaceae N/D; Nc Pteridophytes; H; 38 5 Botrychium multifidum (Gmel.) Rupr. Ophioglossaceae N/D; R Pteridophytes; H; 112 6 Cythea chinensis Copel. Cyatheaceae Rukh uneu N/D; R Pteridophytes; T; 111 7 Dennstaedtia sp. Dennstaedtiaceae N/D; Nc Pteridophytes; H; 172 8 Dicranopteris splendida (Hand.-Mazz.) Tagawa Gleicheniaceae N/D; Nc Pteridophytes; H; 197 9 Diplazium maximum (D.Don) C.Chr. Woodsiaceae N/D; C Pteridophytes; H; 176 10 Doryopteris chrysocoma (Christ)C.Chr. Dryopteridaceae N; Nc Pteridophytes; H; 133 11 Drynaria propinqua (Wall.ex Mett.) J.sm. ex Polypodiaceae N; Nc Pteridophytes; H; 171 Bedd. 12 Dryopteris chrysocoma (christ) C.chr. Dryopteridaceae N/D; Nc Pteridophytes; H; 217 13 Dryopteris wallichiana (Spreng.)Hyl. Dryopteridaceae N; C Pteridophytes; H; 177 14 Equisetum sp. Equisetaceae Kurkure jhar N; C Pteridophytes; H; 212 15 Glechenia gigantea Wall. ex Hook. Gleicheniaceae N; C Pteridophytes; Sh; 174 16 Goniophlebium sp. Polypodiaceae N; Nc Pteridophytes; H; 178 17 Hymenophyllum sp. Hymenophyllaceae N; Nc Pteridophytes; H; 214 18 Lepisorus loriformis (wall. ex Mett.) ching Polypodiaceae N; Nc Pteridophytes; H; 179 19 Leucostegia truncata (D.Don) Fraser-Jenk. Davalliaceae Chamsure uneu N/D; C Pteridophytes; H; 175 20 Lycopodium japonicum Thunb. Lycopodiaceae Nagbeli N; R Pteridophytes; H; 18 21 Nephrolepis cordifolia (L.) Presl Davalliaceae Pani amala N/D; C Pteridophytes; H; 213 22 Oleandra wallichii (Hook.) C. Presl Oleandraceae N; C Pteridophytes; H; 74 23 Peranema cyatheoides D.Don Dryopteridaceae N/D; Nc Pteridophytes; H; 218

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24 Polypodiodes24 lachnopusPolypodiodes (Wall. lachnopus Ex Hook.) (Wall. Polypodiaceae Ex Hook.) Polypodiaceae N; Nc Pteridophytes; N; Nc H; Pteridophytes;221 H; 221 Ching Ching 25 Polypodiodes25 microrhizomaPolypodiodes (C.B. microrhizoma Clarke ex (C.B.Polypodiaceae Clarke ex Polypodiaceae N; Nc Pteridophytes; N; Nc H; Pteridophytes;196 H; 196 Baker) Ching Baker) Ching 26 Pteridium revolutum26 Pteridium (Blume) revolutum Nakai (Blume) Nakai Dennstaedtiaceae Dennstaedtiaceae N/D; Nc Pteridophytes; N/D; Nc H;Pteridophytes; 216 H; 216 27 Pteris vittata27 L. Pteris vittata L. Pteridaceae Pteridaceae N/D; Nc Pteridophytes; N/D; Nc H;Pteridophytes; 173 H; 173 28 Pteris wallichiana28 Pteris J.Agardh wallichiana J.Agardh Pteridaceae Pteridaceae N/D; Nc Pteridophytes; N/D; Nc H;P 199teridophytes; H; 199 29 Selaginella 29sp. Selaginella sp. Selaginellaceae Selaginellaceae N; C Pteridophytes; N; C H; Pteridophytes; 220 H; 220 30 Thelypteris 30sp. Thelypteris sp. Thelypteridaceae Thelypteridaceae N/D; Nc Pteridophytes; N/D; Nc H;Pteridophytes; 99 H; 99

Bryophytes Bryophytes Plant category; PlantLocas category; S.N. Scientific name Family Locas Local name habit, collection S.N. Scientific name Family Local name habit,status collection status number number 1 Marchantia sp. Marchantiaceae N, C Bryphyte; H; 239 1 Marchantia sp. Marchantiaceae N, C Bryphyte; H; 239 2 Sphagnum nepalense H. Suzuki Sphagnaceae Galainche jhyau N; R Bryophyte; H; 198 2 Sphagnum nepalense3 Funaria H. Suzukisp. Sphagnaceae FunariaceaeGalainche jhyau N; R Bryophyte; N; C H; 198 Bryophyte; H; 200 3 Funaria sp. Funariaceae N; C Bryophyte; H; 200 D= Domesticated; N= Natural; C= Common; Nc= Not so common; R= Rare; H= Herb; Sh= Shrub; T= Tree; Cl= Climber, D= Domesticated;Wc= N= Woody Natural; climber; C= Common; Cr.= Creeper Nc= Not so common; R= Rare; H= Herb; Sh= Shrub; T= Tree; Cl= Climber, Wc= Woody climber; Cr.= Creeper

12  56 12  2018J. Pl. Res. Vol. 16, No. 1, pp 57-63, 2018 Journal of Plant Resources Vol.16, No. 1

Antimicrobial Activity and Chemical Composition of Essential Oil of Fruits of Amomum subulatum Roxb.

Usha Tandukar*, Pramesh Bahadur Lakhey, Nishanta Shrestha, Amit Dhungana and Tara Datt Bhatt Department of Plant Resources, Thapathali, Kathmandu, Nepal *E-mail: [email protected]

Abstract

Essential oil from fruits of Amomum subulatum was hydrodistilled and antimicrobial activity was studied in seven species of bacteria. Chemical composition of oil was studied through Gas Chromatography and Mass Spectrometry (GC-MS). Major components of oil were eucalyptol (62.48%), β pinene (8.81%), α-terpineol (8.30%), α-pinene (5.30%), terpinen-4-ol (3.06%), sabinene (2.29%), myrcene (1.38%), δ-terpineol (1.31%) and γ-terpinene (1.24%). The oil showed antimicrobial activities against Bacillus subtilis ATCC 6501, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 8739, Proteus vulgaris ATCC 6380, Salmonella enterica subsp enterica serovar Typhi clinical isolate, Shigella dysenteriae ATCC 13313 and Staphylococcus aureus ATCC 6538P among which the least MBC was observed for Escherichia coli, Enterococcus faecalis and Shigella dysenteriae.

Key words: Antibacterial, Cardamom oil, Chemical components, Hydrodistillation

Introduction occurs in July to September. Seeds are aromatic, stomachic, appetizer, carminative and useful in Amomum is a genus of family Zigiberaceae which neuralgia. Seeds are also used as aphrodisiac, is one of the essential-oil-bearing plant families. antidote to scropion-sting and snake bite. Oil from Amomum subulatum Roxb. is a terrestrial, seeds is aromatic, stimulant, stomachic and applied rhizomatous, perennial herb, naturally distributed in to eye lids to allay inflammation (DPR, 2016). Nepal, India, tropical Asia, Africa and is cultivated in Nepal, North-west Bengal, and Assam Now a days, utilization of essential oil as an hills (Nadkarni, 1976). Cardamom is valuable spices alternative to synthetic antimicrobials is an yielding tradable fruits and essential oil. There are increasing trend. Plant essential oils are aromatic oily two main types of cardamom: (a) small green liquids which can be obtained by expression, cardamom (Elettaria cardamom (L.) Maton) and (b) fermentation, enfleurage, extraction, steam or large red/ black cardamom (A. subulatum). Large hydrodistillation from different parts of plants (Burt, (big) cardamom is mainly grown in Nepal, India and 2004). Chemically, essential oils are derived from (Arora & Kaur, 2007). A. subulatum bush terpenes and their oxygenated compounds. They are grows to a height of about 90-100 cm. Leaves are made up of isoprene units (C6H8) and are usually simple, large, oblong, lanceolate, bright green and mono-, sesqui- and diterpenes with emperical glaborous on both surfaces. Flowers are yellowish formulae as C5H8, C10H16, C15H24 and C20H32 white with reddish brown born in globose, respectively (Kokate et al., 2008). It has been shortly peduncled spikes growing at the base of the reported that the antimicrobial activity of essential plant. Fruits are reddish brown, densely echinate, oils is generally due to phenolic and terpenoid globose capsules (DPR, 2016). It grows best in warm compounds (Hinneburg et al., 2006; Hoque et al., humid place where there is plenty of rain and rich 2008; Dixit et al., 2015) as well as aliphatic soil. It can grow in altitudes upto 1370 m. The bush compounds (Anusuya et al., 2012). Therefore, the requires shade and usually grows under natural forest objectives of the present study were to investigate cover. The plant produces flowers after it is 2-3 years antimicrobial activities and to determine chemical old (Badei et al., 2002). Flowering and fruiting compositions of essential oils of A. subulatum.

57 2018 Journal of Plant Resources Vol.16, No. 1

Materials and Methods using 24 hours cultures of the organisms. The prepared cell suspensions were uniformly spread Plant material collection over the dry surface of Muller-Hinton Agar (MHA) The fruits of A. subulatum were collected from the plates. The inoculated plates were left for maximum Pilot Plant Section of Department of Plant Resources 15 minutes to allow absorption of excess surface at Godavari, Lalitpur in October, 2017 and were sun moisture. Using sterilized cork bores, 5 agar wells dried for 3 weeks. The sun-dried fruits were then of 6 mm diameter were made in the inoculated plates. hydro-distilled in Clevenger apparatus for 5 hrs in Test solution of the essential oil of 5%, 10% and triplicate run. The percentage of oil obtained was 20% concentrations (w/v) were prepared by noted. dissolving it in 10% (v/v) aqueous dimethyl sulphoxide with 5% (v/v) polysorbate 80. Gas chromatography–mass spectrometry (GC- Micropipettes were used to place 50 µL of the MS) analysis solution of three different concentrations, pure Chemical constituents of the essential oil was essential oil and 10% (v/v) aqueous dimethyl analyzed by Gas Chromatography (Shimadzu sulphoxide with 5% (v/v) polysorbate 80 as negative GC 2010) having an RTx-5MS column control into each of the five wells. The plates were (25m×0.25mm×0.25µm) and using Helium as place in upright condition with lids closed for 3 hours carrier gas. The sample (1 µL) diluted with so that the solutions diffused into the media. The spectroscopic grade hexane in a ratio 1:10 was plates were then incubated in inverted position at injected into the GC inlet maintaining constant 35±2°C for 18 to 24 hrs. After the incubation period, column flow 0.68 mLmin-1 and purge flow 3 mLmin-1 the diameter of zone of inhibition was measured in split mode. The initial column oven temperature using a digital caliper to the nearest whole millimeter. was set at 40ºC and the injection temperature was The organisms in which the essential oil showed 250ºC. zone of inhibition were selected for the The constituents separated were detected and determination of Minimum Inhibitory Concentration identified by a Mass Spectrometer (Shimadzu (MIC) and Minimum Bactericidal Concentrations GCMS-QP 2010 plus). During the analysis, the ion (MBC) by two-fold serial dilution method (Forbes source and the interface temperature was set at 250ºC et al., 2007). Stock solution of the essential oil of -1 and 200ºC respectively. The detector gain mode was 750 mg.mL concentration was prepared in 10% relative, scanning time was from 4.00 min to 68.00 (v/v) aqueous dimethyl sulphoxide with 5% (v/v) min and scan speed was 666 with m/z range of 40.00- polysorbate 80. Twelve vials, each containing 1 mL 350.00. The MS library used for comparison was of Muller Hinton Broth were prepared and labelled FFNSC 1.3 and NIST 11. from 0 to 11. In no. 0 vial, the media was removed and only 1 mL stock solution was placed. In no. 1 Antimicrobial activity vial, 1 mL of the stock solution was added to 1 mL of the medium and mixed evenly by vortexing to The oil sample was screened against seven bacterial obtain a solution of concentration 375 mgmL-1; 1 species (Bacillus subtilis, Enterococcus faecalis, mL of the 375 mgmL-1 solution in no. 1 vial was Escherichia coli, Proteus vulgaris, Salmonella transferred to no. 2 vial and was mixed evenly to enterica subsp enterica serovar Typhi, Shigella obtain a solution of concentration 187.5 mgmL-1. dysenteriae, Staphylococcus aureus) using agar well This process was repeated upto vial no 10 to obtain diffusion method as described by Perez et al. (1990). solutions of concentrations 93.75 mgmL-1, 46.875 Cell suspension of the organisms with turbidity mgmL-1, 23.4375 mg.mL-1, 11.71875 mgmL-1, equivalent to 0.5 McFarland Nephelometric 5.859375 mg.mL-1, 2.929688 mgmL-1, 1.464844 Standard and containing microbial load of 1.5×108 mgmL-1 and 0.732422 mgmL-1. Vial no. 11 was left cfu.mL-1 was prepared in sterilized normal saline with media only. Each vial was inoculated with 20

58 2018 Journal of Plant Resources Vol.16, No. 1 inoculated organisms at different concentrations of the essential oils and were incubated at 35±2°C for µL18 of to the 24 cell hrs. suspension MBC was ofdetermined the organisms as the containing minimum concentrationreported 1.1% in whichessential no growthoil yield was in observed. A. subulatum microbial load of 1.5×108 cfu.mL-1 and were fruit sample collected from local market of Lahore, incubatedResult and at 35±2°CDiscussion for 18 to 24 hrs. The MIC of Pakistan while Agnihotri et al. (2012) have reported theThe oil percentage sample was yield visually of essential determined oil after as the hydrodistillation lowest 0.8% of yield fruits from of A.local subulatum market ofrun Himachal for five hours Pradesh, in concentrationClevenger apparatus that did notin showtriplicate bacterial run was growth 2.28% as v/w.India. However, Essential Gilani oils et are al. very(2006) complex reported natural 1.1% indicatedessential oilby yield turbidity in A. subulatumin comparison fruit sample to the collectedmixtures from localwhich market can of containLahore, Pakistanabout 20–60while uninoculatedAgnihotri et al.medium. (2012) haveFor thereported determination 0.8% yield offrom componentslocal market atof quiteHimachal different Pradesh, concentrations. India. Essential They MBC,oils are the very vials complex were sub-cultured natural mixtures on NA whichplates canto containare characterized about 20–60 by componentstwo or three majorat quite components different determineconcentrations. the viability They areof the characterized inoculated byorganisms two or threeat fairlymajor highcomponents concentrations at fairly (20–70%) high concentrations compared to at(20–70%) different concentrationscompared to others of the components essential oils present and in otherstrace amounts. components Detailed present compositional in trace analysis amounts. is achieved by gas chromatography and mass spectrometry of essential oils from different plant parts like were incubated at 35±2°C for 18 to 24 hrs. MBC Detailed compositional analysis is achieved by gas rhizome, leaves, stem, fruits and flowers (Dorman & Deans, 2000). The data of essential oil composition was determined as the minimum concentration in chromatography and mass spectrometry of essential of fruits of A. subulatum is given in table 1. which no growth was observed. oils from different plant parts like rhizome, leaves, Hydro-distilled essential oils of A. subulatum when analyzed by Gas Chromatography –Mass Spectroscopy (GC-MS) showed presence of 26 chemicalstem, fruits components, and flowers among (Dorman which & Deans, eucalyptol 2000). Results(62.48%), and β pinene Discussion (8,81%), α-terpineol (8.30%), α-pineneThe data (5.30%), of essential terpinen-4-ol oil composition (3.06%), of fruitssabinene of A. (2.29%), myrcene (1.38%), δ-terpineol (1.31%) and γ-terpinenesubulatum (1.24%) is given werein table major 1. components (Table The percentage yield of essential oil after 1). Gilani et al. (2006) also analyzed A. subulatum essential oil using GC-MS and found 26 compounds hydrodistillation of fruits of A. subulatum run for Hydro-distilled essential oils of A. subulatum when among which 1,8-cineol was the major component (55.37%). Similarly, Agnihotri et al. (2012) have five hours in Clevenger apparatus in triplicate run analyzed by Gas Chromatography–Mass identified 26 components in the essential oil sample using GC with FID detector. was 2.28% v/w. However, Gilani et al. (2006) Spectroscopy (GC-MS) showed presence of 26 Table 1: Chemical composition of essential oil of A. subulatum Peak S.N. Retention time Area Aera (%) Name of compound 1. 11.472 1526882 0.46 α- Thujene 2. 11.768 17425212 5.30 α- Pinene 3. 13.615 7515731 2.29 Sabinene 4. 13.753 28954640 8.81 β- Pinene 5. 14.452 4552817 1.38 Myrcene 6. 15.661 2240922 0.68 α-Terpinene 7. 16.074 523635 0.16 o-Cymene 8. 16.479 205400149 62.48 Eucalyptol 9. 17.731 4062817 1.24 γ-Terpinolene 10. 18.149 2300768 0.70 (E)-β Ocimene 11. 19.176 1048057 0.32 Terpinolene 12. 19.674 1452048 0.44 trans-Sabinene hydrate 13. 20.799 633580 0.19 3,5-Methanocyclopentapyrazole,3,3a,4,5,6,6a- hexahydro-3a,4,4-trimethyl 14. 21.663 1108041 0.34 Bicyclo[3.1.1] heptan-3-ol,6,6-dimethyl-2-methylene- ,[1S-(1alpha.,3alpha.,5.alpha 15. 22.835 519083 0.16 Pinocarvone 16. 23.016 4295159 1.31 δ-Terpineol 17. 23.511 10074975 3.06 Terpinen-4-ol 18. 24.163 27300859 8.30 α-Terpineol 19. 24.459 1590780 0.48 Myrtenol 20. 31.085 1106457 0.34 Epoxy-alpha-terpenyl acetate 21. 37.044 705940 0.21 Germacrene D 22. 37.265 637921 0.19 β-Selinene 23. 37.691 513444 0.16 Bicyclogermacrene 24. 40.203 16411388 0.50 (E)-Nerolidol 25. 40.926 694129 0.21 Spathulenol 26. 41.474 927324 0.28 Diethyl-phthalate 328752813 100.00

 59 2018 Journal of Plant Resources Vol.16, No. 1 chemical components, among which eucalyptol ZOIs of 18.26 mm and 19.96 mm in Lactobacillus (62.48%), β pinene (8,81%), α-terpineol (8.30%), acidophillus and Escherichia coli respectively. α-pinene (5.30%), terpinen-4-ol (3.06%), sabinene Agnihotri et al. (2012), screened antimicrobial (2.29%),A. subulatum myrcene essential (1.38%), oil, δ in-terpineol 5%, 10% (1.31%) and 20% and solution,activity showed of the no essential actimicrobial oil against activity. 10 organisms However, with in pure form, the oil showed antimicrobial activities against all seven bacterial species. Maximum ZOI (23 ã-terpinene (1.24%) were major components (table maximum ZOI in Escherichia coli (20 mm) and mm) was observed against Enterococcus faecalis while minimum ZOI (14 mm) was observed against 1).Bacillus Gilani etsubtilis al. (2006), (Table also 2; analyzedPlate 1, 2A. & subulatum 3). MIC of Staphylococcusthe essential oil aureus sample (20 could mm). not The be antimicrobial determined essentialvisually oil due using to the GC-MS turbid and nature found of 26 the compounds sample. MBCactivity determination of plant essentialby subculturing oils is due of to the their MIC chemical vials amongshowed which that 1,8-cineolamong the wasseven the microorganisms major component used forstructure, screening, in particular minimum to theMBC presence (2.92969 of hydrophilic mg.mL-1) (55.37%).was observed Similarly, for Escherichia.Agnihotri et colial. (2012),, second have minimumfunctional MBC (5.85938groups suchmg.mL as-1 hydroxyl) were observed groups inof identifiedEnterococcus 26 components faecalis and in the Shigella essential dysenteriae oil sample. Maximumphenolic components MBC (more and/or than lipophilicity375 mg.mL -1of)was some usingobserved GC with for FIDBacillus detector. subtilis) (Table 2, Plate 4, 5 essential& 6). Bhatt oil components et al (2013) (Dorman have reported & Deans, that 2000). A. subulatum oil showed antimicrobial activities againstBecause Bacillus ofsubtilis the great (ZOI number16 mm), ofStaphylococcus constituents, A.aureus subulatum (14 mm), essential Escherichia oil, in 5%,coli (1810% mm) and and 20% Pseudomonasessential aeruginosaoils seem (13to havemm). noGilani specific et al. cellular(2006) solutionfound thatshowed the noessential actimicrobial oil gave activity. ZOIs ofHowever, 18.26 mm targets.and 19.96 As typicalmm in lipophiles,Lactobacillus they acidophillus pass through and the inEscherichia pure form, thecoli oil respectively. showed antimicrobial Agnihotri etactivities al. (2012) cellscreened wall antimicrobialand cytoplasmic activity membrane, of the essential disrupt oilthe againstagainst all 10 seven organisms bacterial with species. maximum Maximum ZOI in ZOIEscherichiastructure coli of(20 their mm) different and Staphylococcus layers of polysaccharides, aureus (20 (23mm). mm) The was antimicrobial observed against activity Enterococcus of plant essential faecalis oilsfatty is due acids to andtheir phospholipids chemical structure, and permeabilize in particular them. to the presence of hydrophilic functional groups, such as hydroxyl groups of phenolic components and/or while minimum ZOI (14 mm) was observed against Cytotoxicity appears to include such membrane Bacilluslipophilicity subtilis of (tablesome 2;essential Plate 1, oil 2 &components 3). MIC of (Dorman the & Deans, 2000). Because of the great number of constituents, essential oils seem to have no specificdamage cellular (Carsontargets. As et typicalal., 2002). lipophiles, In bacteria, they pass the essential oil sample could not be determined visually permeabilization of the membranes is associated duethrough to the the turbid cell wallnature and of cytoplasmic the sample. membrane, MBC disrupt the structure of their different layers of polysaccharides, fatty acids and phospholipids and permeabilizewith loss of them. ions Cytotoxicity and reduction appears of membraneto include determination by subculturing of the MIC vials potential, collapse of the proton pump and depletion showedsuch membrane that among damage the seven (Carson microorganisms et al., 2002). used In bacteria, the permeabilization of the membranes is associated with loss of ions and reduction of membraneof the potential,ATP pool collapse(Sikkema of et the al., proton1994; Helandorpump and et for screening, minimum MBC (2.92969 mgmL-1) depletion of the ATP pool (Sikkema et al., 1994; Helandoral., 1998; et al., Ultee 1998; et Ultee al., 2002; et al., Turina2002; Turinaet al., 2006).et al., was2006). observed Essential for Escherichia oils can coagulate coli, second the cytoplasm minimum (Gustafson et al., 1998) and damage lipids and proteins -1 Essential oils can coagulate the cytoplasm MBC(Burt (5.85938et al., 2004). mg.mL Damage) towere the cellobserved wall and in membrane(Gustafson can lead et al.,to the1998) leakage and ofdamage macromolecules lipids and Enterococcusand to lysis (Juven faecalis et al.,and 1994; Shigella Cox etdysenteriae al., 2000; Oussalah. et al., 2006) Similarly in the case of essential -1 proteins (Burt et al., 2004). Damage to the cell wall Maximumoil of A. subulatumMBC (more, major than components 375 mgmL like) waseucalyptoland (ether),membrane β pinene can (), lead to the leakageα-terpineol of observed(monocyclic for Bacillus alcohol), subtilis α-pinene) (table 2,(), Plate 4, 5 & terpinen-4-olmacromolecules (monoterpene and to lysis (Juven alcohol), et al., 1994;sabinene Cox 6).(monoterpene), Bhatt et al. myrcene(2013), have(monoterpene), reported δthat-terpineol A. et(monocyclic al., 2000; Oussalah alcohol), etγ -terpineneal., 2006) Similarly(monoterpene) in the may be responsible for the antimicrobial activity. Sonboli et al. (2006) reported 1,8- cineole (eucalyptol) subulatum oil showed antimicrobial activities case of essential oil of A. subulatum, major as the major component responsible for antimicrobial activity in essential oil from Salvia sps. Likewise, against Bacillus subtilis (ZOI 16 mm), components like eucalyptol (ether), β pinene StaphylococcusYousefzadi et al.aureus (2007) (14 demonstrated mm), Escherichia components coli of essential oil 1,8 cineole and α-pinene of Salvia sclarea as having highest antimicrobial activity. Similarly,(monoterpenes), Subulal et al. α -terpineol(2006) conducted (monocyclic studies alcohol), on the (18 mm) and Pseudomonas aeruginosa (13 mm). α-pinene (monoterpene), terpinen-4-ol Gilaniessential et al. oil (2006), of A. cannicarpum found that the having essential promising oil gave antimicrobial activity which showed major component was β-pinene (14%) and trace α-terpineol (1.51%). (monoterpene alcohol), sabinene (monoterpene),

Table 2: Zone of inhibition, minimum inhibitory concentration and minimum bactericidal concentration of A. subulatum essential oil ZOI (mm) MBC S.N. Organisms MIC 5% 10% 20% Pure oil (mg.mL-1) 1. Bacillus subtilis ATCC 6051 0 0 0 14 Could not >375 2. Enterococcus faecalis ATCC 29212 0 0 0 23 be 5.85938 3. Escherichia coli ATCC 8739 0 0 0 16 determined 2.92969 4. Proteus vulgaris ATCC 6380 0 0 0 15 due visually 11.7188 Salmonella enterica subsp.enterica due to the 5. 0 0 0 16 23.4375 serovar Typhi Clinical Isolates turbid 6. Shigella dysenteriae ATCC 13313 0 0 0 16 nature of the 5.85938 7. Staphylococcus aureus ATCC 6538P 0 0 0 17 sample 46.875

60  2018 Journal of Plant Resources Vol.16, No. 1 myrcene (monoterpene), δ-terpineol (monocyclic General of Department of Plant Resources for alcohol), γ-terpinene (monoterpene) may be inspiring to carry out the study. The authors are also responsible for the antimicrobial activity. Sonboli grateful to all the assistants of lab who helped in et al. (2006) reported 1,8- cineole (eucalyptol) as carrying out the experiments. the major component responsible for antimicrobial activity in essential oil from Salvia sps. Likewise, References Yousefzadi et al. (2007) demonstrated components of essential oil 1,8 cineole and α-pinene of Salvia Agnihotri, S.A., Wakode, S.R., & Ali, M. (2012). sclarea as having highest antimicrobial activity. Chemical composition, antimicrobial and topical Similarly, Subulal et al. (2006) conducted studies anti-inflammatory activity of essential oil of on the essential oil of A. cannicarpum having Amomum subulatum fruits. Acta Poloniae promising antimicrobial activity which showed Pharmaceutica. Drug Research, 69(6), 1177-1181. major component was β-pinene (14%) and trace α- Anusuya, N., & Manian, S. (2012). Antioxidant and terpineol (1.51%). free radical scavenging potential of different solvent extracts of Indigofera tinctoria L. Leaves. J. Pharm. Conclusion Pharm. Sci., 5(1), 142-147. The study demonstrated the volatile oil of A. Arora, D.S., & Kaur, G.J. (2007). Antibacterial activity subulatum was composed of 26 components. The of some Indian medicinal plants. J. Nat. Med., 61(3), major components were eucalyptol followed by â 313-317. pinene, α-terpineol, α-pinene, terpinen-4-ol, Badei, A.Z, El-Akel, A.T., Faheid, S.M., & Mahmound, sabinene, myrcene, δ-terpineol and γ-terpinene. The B.S. (2002). Application of some spices in flavoring study suggested the antimicrobial activity of fruits and preservation of cookies: 1-Antioxidant of A. subulatum of essential oil is resultant effect of properties of cardamom, cinnamon and clove. Dtsch major and minor components in essential oil Lebensmitt Rundsch, 98, 176-183. composition showing significant antimicrobial Bhatt, M.R., Juyal, V., & Singh, A. (2013). activity against Escherichia coli followed by Antimicrobial activity of essential oil from the fruits Shigella dysenteriae and Enterococcus faecalis as of Ammomum subulatum. International Journal of evidenced by comparative MBC values. Fruther Pharmacy and Life Sciences, 4(12), 3190-3192. research is required to separate components of essential oils and study of antimicrobial activity of Burt, S. (2004). Essential oils: their antibacterial each component to identify which component of properties and potential applications in foods - a essential oil is actually resposible for the review. Int. J. Food Microbiol., 94, 223–253. antimicrobial activity Carson, C.F., Mee, B.J., & Riley, T.V. (2002). Mechanism of action of Melaleuca alternifolia (tea Acknowledgements tree) oil on Staphylococcus aureus determined by time-kill, lysis, leakage and salt tolerance assays and The authors are grateful to Mr. Jay Govind electron microscopy. Antimicrobial Agents and Chaudhary (Assistant Botanist) for collecting the Chemotherapy, 46(6), 1914-1920. samples of A. subulatum from Pilot Plant Section, Cox, S.D., Mann, C.M., Markham, J.L., Bell H.C., Godavari, Lalitpur of Department of Plant Resources Gustafson, J.E., Warmington, J.R., & Wyllie, S.G. and to Mr. Chintamani Bashyal, Instrument Section, (2000). The mode of antimicrobial action of essential Department of Plant Resources for helping in sample oil of Melaleuca alternifolia (tea tree oil). J. Appl. preparation in GC-MS analysis of the sample. Micr., 88(1), 170–175. Heartfelt gratitude is also due to Mr. Sanjeev Kumar Rai, Director General of Department of Plant Dixit, A., Singh, H., Sharma, R.A., & Sharma, A. Resources and Ms. Jyoti Joshi Bhatta, Deputy (2015). Estimation of antioxidant and antibacterial

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activity of crude extracts of Thevetia peruviana constituents. J. App. Bacteriol., 76(6), 626–631. (Pers.) K. Schum. Int. J. Pharm. Pharm. Sci., 7(2), Kokate, C.K., Purohit, A.P., & Gokhale, S.B. (2008). 55-59. Pharmacognosy. 42nd edition. Pune, India: Nirali Dorman, H.J.D., & Deans, S.G. (2000). Antimicrobial Prakashan. agents from plants: Antibacterial activity of plant Nadkarni, K,M. (1976). Indian materica medica. 3rd volatile oils. J. Appl. Microbiol., 88(2), 308–316. edition, (p. 1260), Bombay, India: Popuplar Frobes, B.A., Sahm, D.F., & Weissfeld, A.S. (2007). Prakashan. th Bailey and Scott’s Diagnostic Microbiology. 12 Oussalah, M., Caillet, S., & Lacroix, M. (2006). edition. Missouri, USA: Mosby Elsevier. Mechanism of action of Spanish oregano, Chinese Gilani, S.R., Shahid, I., Javed, M., Mehmud, S., & cinnamon, and savory essential oils against cell Ahme, R. (2006). Antimicrobial activities and membranes and walls of Escherichia coli O157:H7 physico-chemical properties of the essential oil from and Listeria monocytogenes. J. Food Protect., 69(5), Amomum subulatum. International Journal of 1046–1055. Applied Chemistry, 2(2), 81-86. Perez, C., Paul, M., & Bazerque, P. (1990). Antibiotic DPR (2016). Medicinal Plants of Nepal. 2nd edition. assay by agar well diffusion method. Acta Biol. Med. Kathmandu, Nepal: Government of Nepal, Exp., 15, 113-115. Department of Plant Resources. Sabulal, B., Dan, M., Pradeep, N.S., Valsamma, R.K., Gustafson, J.E., Liew, Y.C., Chew, S., Markham, J.L., & George, V. (2006). Composition and antimicrobial Bell, H.C., Wyllie, S.G., & Warmington, J.R. (1998). activity of essential oil from the fruits of Amomum Effects of tea tree oil on Escherichia coli. Letters in cannicarpum. Acta Pharm., 56, 473-480. Applied Microbiology, 26, 194–198. Sikkema, J., De Bont, J.A.M., & Poolman, B. (1994). Helander, L.M., Alakomi, H.L., Latva-Kala, K., Interactions of cyclic hydrocarbons with biological Mattila-Sandholm, T., Pol, I., Smid, E.J., Gorris, membranes. J. Biol.Chem., 269(11), 8022-8028. L.G.M., & Wright, A.V. (1998). Characterization of Sonboli, A., Babakhani, B., & Mehrabian, A.R. (2006). the action of selected essential oil components on Antimicrobial activity of six constituents of essential gram negative bacteria. Journal of Agricultural oil from Salvia. Z. Naturforsch., 61, 160–164. Chemistry, 46, 3590-3595. Turina, A.V., Nolan, M.V., Zygadlo, J.A., & Perillo, Hinneburg, I., Dorman, D.H., & Hilunen, R. (2006). M.A. (2006). Natural terpenes: self-assembly and Antioxidant activities of extracts from selected membrane partitioning. Biophys. Chem., 122(2), culinary herbs and spices. Food Chem., 97(1), 122- 101–113. 129. Ultee A., Bennik, M.H., & Moezelaar R. (2002). The Hoque, M.M., Bari, M.L., Juneja, V.K., & Kawamoto, phenolic hydroxyl group of carvacrol is essential S. (2008). Antimicrobial activity of clove and for action against the food-borne-pathogen Bacillus cinnamon extracts, against food borne pathogens and cereus. Appl. EnViron. Microbiol., 68(4), 1561– spoilage bacteria, and inactivation of Listeria 1568. monocytogenes in ground meat with their essential oils. Rep., Natl., Food Res., Inst., 2, 9-13. Yousefzadia, M., Sonboli, A., Farah, K., Samad N.E., Behvar A., & Amineh, Z. (2007). Antimicrobial Juven, B.J., Kanner, J., Schved, F., & Weisslowicz, H. activity of some salvia species essential oils from (1994). Factors that interact with the antibacterial Iran. Z. Naturforsch., 62, 514-518. action of thyme essential oil and its active

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Plate 1: ZOI shown by different concentrations of A. subulatum Plate 2: ZOI shown by different concentrations of A. subulatum oil against Enterococcus faecalis oil against Escherichia coli

Plate 3: ZOI shown by different concentrations of A. subulatum Plate 4: MBC of A. subulatum oil against Enterococcus oil against Shigella dysenteriae faecalis

Plate 5: MBC of A. subulatum oil against Escherichia coli Plate 6: MBC of A. subulatum oil against Shigella dysenteriae

63 2018J. Pl. Res. Vol. 16, No. 1, pp 64-69, 2018 Journal of Plant Resources Vol.16, No. 1

Isolation of Curcumin and GC-MS Analysis of Oil in Curcuma longa L.

Madan Raj Bhatta*, Rajeswar Ranjitkar, Bhabani Prasad Adhikari, Parasmani Yadav and Laxman Bhandari Natural Products Research Laboratory, Thapathali, Kathmandu, Nepal *E-mail:[email protected]

Abstract

Hydrodistilled oil of moist crude rhizome of Turmeric (Curcuma longa) was found to be 2.1% and analyzed by GC-MS. Oil content tumerone 19.01%, curcumene <α-> 5.93%, zingiberene <α- > 8.18%, sesquiphellandrene <β_> 9.27%, caryophylleneoxide 9.11%, terpinolene 9.54%, eucalyptol 2.30%, benzenesulfonamide 8.73% as a major compound. Curcumin was isolated from rhizome by column chromatography and the isolated curcumin was characterized by comparing melting points, Retardation factor (Rf), Infrared spectroscopy (IR) spectra with authentic curcumin

Key words: Column chromatography, Essential oil, Solvent extraction, TLC

Introduction damage to tissues, diabetic cataracts, diabetic damage to pancreatic insulin-producing cells, Curcuma longa (Turmeric) is rhizotomous rheumatoid arthritis, inflammatory bowel disease, herbaceous perennial, native to nearly crohn’s disease, psoriasis, inherited peripheral 4000 years to the vedic culture in India and neuropathies. (Rukkumani et al., 2003, Nishiyama subcontinent, where used as a culinary, spice and et al., 2005). had religious significance. Since 1280, that exhibited qualities so similar to that of saffron recognized as Previous study shows that the major constituents in pharmaceutical crop (Devkota & Rajbhandari, 2015) fresh rhizome were ar-turmerone (24.4%), α- belongs to family zingiberaceae has long been used turmerone (20.5%), β-turmerone (11.1%), α- as a powerful anti-inflammatory in Chinese and santalene (7.2%), ar-curcumene(6.6%), β- Indian systems of medicine (Ching et al., 2014). In caryophyllene (9.8%) and β-sesquiphellendrene ayurvedic system, turmeric is used for flavor, color, (7.1%) by GC-MS analysis (Singh et al., 2010). preservative, coloring agent, as well as for various Isolation of curcumin by column chromatography purposes like dye, food spice, source of industrial from 95% ethanol extract provide information about starch (Cousines et al., 2007). the yield and quality of turmeric rhizome from The oil content in rhizomes range to (0.16-1.94) % Kailali district. on fresh weigh basis (Garg et al., 1999) and the active The main objectives of this research were ingredient in it is curcumin, which is about 2% by investigation of chemical constituents of turmeric weigh (Andrew et al., 2000). Curcumin is a yellow oil of Nepalese origin from Kailali district and to color compound used in food and textile industries isolate curcumin with 95% ethanol by using simple combined with demethoxy curcumin, bisdemethoxy cold percolation method. curcumin and water soluble protein turmerin (Susana, 2012). According to Kulkarni et al. (2012) Materials and Methods and Revathyl, et al. (2011), curcumin yield is maximum in acetone extract and better TLC Plant material resolution in solvent system CHCl3:MeOH (95:5). Uses of curcumin have reported as suppressive The fresh home cultivated turmeric rhizomes were effects, high serum cholesterol levels, free radical purchased from a vendor of Dhangadhi, Kailali

64 Na2SO4 is used as a moisture free agent for hydro distillated oil from turmeric rhizome. Silica gel 60-120 mesh size and column 29/32 size were used for isolation and all the solvent and chemical are of analytical grade. 2018 Journal of PlantVolatile Resources oil extraction Vol.16, No. 1 100 g sample was taken into 500 ml R.B. flask. Distilled water was poured into the flask up to the mark district in Poush 2073. The rhizomes were washed, Solventof turmeric extraction which wetmethod it. The Clevenger apparatus was fitted on the mouth of the flask and it was heated to boil. The heat was continued for 6 hours (hrs.). The oil was collected in the trap of the Clevenger chopped into small pieces, air dried and stored in a Step 1 plastic air tight container for hydro distillation of apparatus. The oil and water layer was separated completely and drawn off slowly and collected in the Extractiontest tube and of 40dried g turmeric over Na powderSO and with store 95% it alcohol at 40 C for GC-MS analysis & further use. percentage essential oil, GC-MS analysis and 2 4 0 (150Quantitative mL). Left analysis out 24 of hrs. essential and sonication oil by GC-MS at 50 C isolation of curcumin from column chromatography. forThe 15 chemical minutes thenconstituents filter and inremain the essential residue X oil 3 timeswere separated using a Shimadzu gas chromatograph (GC- Step 2 Chemicals and reagent 2010) with Rtx-5 MS column (25 m × 0.25 mm× 0.25 µm). 1 µl of the essential oil was diluted with Separationspectroscopic of Oleoresin:grade hexane extract (10:1) was was washed injected with into the GC inlet maintaining column flow rate of 1 hexane then 0.1 % HCl. Na2SO4 is used as a moisture free agent for hydro mL/min and purge flow 3 mL/min after fixing the split ratio at 120. The initial column oven temperature distillated oil from turmeric rhizome. Silica gel 60- Stepwas set3 at 40° C and the injection temperature was 250 °C. The qualitative analysis of the essential oil 120 mesh size and column 29/32 size were used for Forwas purificationfurther continued total extractin a Shimadzu was dissolved GCMS-QP-2010 in plus. During the analysis, the ion source isolation and all the solvent and chemicals are of mixturetemperature of chloroform: and the interface acetone temperature(60:40, v/v) and was kept set at 200° C and 250° C respectively. Detector scanning analytical grade. overnightstart time wasat -4°C 4 minutes, temperature end time for was precipitation. 68 minutes and scan speed was 666 with scanning range of m/z Yellow-orange40.00-350.00. crystals solid was obtained with high Volatile oil extraction puritySolvent and extraction conformed method by thin layer chromatography (TLC).Step 1 100 g sample was taken into 500 mL R.B. flask. Extraction of 40 g turmeric powder with 95% alcohol (150 mL). Left out 24 hrs. and sonication at 500C Distilled water was poured into the flask up to the Columnfor 15 minutes. chromatography then filter and remain residue X 3 times mark of turmeric which wet it. The Clevenger TheStep purified2 extract coming from chloroform: apparatus was fitted on the mouth of the flask and it Separation of Oleoresin: extract was washed with hexane then 0.1 % HCl. acetone (60:40) was chromatographed on a 100 g was heated to boil. The heat was continued for 6 Step 3 silica gel 60-120 mesh size, 29/32 column size eluted hours (hrs.). The oil was collected in the trap of the For purification total extract was dissolved in mixture of chloroform: acetone (60:40, v/v) and kept with chloroform: methanol (95:5). The fractions Clevenger apparatus. The oil and water layer was overnight at -4°C temperature for precipitation. Yellow-orange crystals solid was obtained with high werepurity collected and conformed consisting by ofthin 10-12 layer mL chromatography in 20 mL test (TLC). separated completely and drawn off slowly and tube. Each fraction was monitored by TLC using collected in the test tube and dried over Na SO and Column chromatography 2 4 solvent system chloroform: methanol (95:5). The sub store it at 4°C for GC-MS analysis & further use. The purified extract coming from chloroform: acetone (60:40) was chromatographed on a 100 g silica fractionsgel 60-120 were mesh pulled size, into 29/32 a, b,column c, d, esize and eluted f. The with chloroform: methanol (95:5). The fractions were fractions “d” where found to be single spot which Quantitative analysis of essential oil by GC-MS collected consisting of 10-12 mL in 20 mL test tube. Each fraction was monitored by TLC using solvent wassystem later chloroform: concentrated methanol and (95:5).purified. The Thesub fractionssub were pulled into a, b, c, d, e and f. The fractions The chemical constituents in the essential oil were fractions“d” where ‘c’ found & ‘e’ to alsobe single contains spot some which curcumin was later concentrated and purified. The sub fractions c & e separated using a Shimadzu gas chromatograph (GC- whichalso contains was separated some curcumin by further which TLC wasMethod. separated by further TLC Method. 2010) with Rtx-5 MS column (25 m × 0.25 mm× Table 11:: The The TLC TLC sub Sub fractions fractions from from Column Column Chromatography Chromatography 0.25 µm). 1 µl of the essential oil was diluted with S.N. Test tube Sub fractions spectroscopic grade hexane (10:1) was injected into 1 Below 15 Discarded the GC inlet maintaining column flow rate of 1 mL/ 2 15,16,17 mixed a-subfraction min and purge flow 3 mL/min after fixing the split 3 18,19,20 mix b-subfraction 4 21,22 mix c-subfraction ratio at 120. The initial column oven temperature 5 23 single d-subfraction was set at 40°C and the injection temperature was 6 24 single e-subfraction 250°C. The qualitative analysis of the essential oil 7 25,26 mix f-subfraction was further continued in a Shimadzu GCMS-QP- 8 Above 27 Discarded. 2010 plus. During the analysis, the ion source Purification, crystallization and melting point determination temperature and the interface temperature was set Purification, crystallization and melting point The obtained sub-fraction “d” isolated curcumin was recrystallizing in warm water. The melting point determination at 200°C and 250°C respectively. Detector scanning was determined by using digital auto melting point apparatus. start time was 4 minutes, end time was 68 minutes TheMeasurement obtained sub-fraction of spectra “d”by Infra isolated red curcumin (IR) Analysis was and scan speed was 666 with scanning range of m/z recrystallizing in warm water. The melting point was 40.00-350.00. determined by using digital auto melting point 2 apparatus.

65 IR spectrum was measured by using shimadzu and authenticated by certified reference materials (CRM) curcumin. IR spectrum was measured by using shimadzu and authenticated by certified reference materials (CRM) Result & Discussion curcumin. Oil percentage from turmeric rhizome Result & Discussion The essential oil percentage obtainedOil percentage by hydro from turmericdistillation rhizome method was 2.1% having water slight yellowish color with characteristic turmericThe essential odor. oil Whichpercentage is obtainedslightly byviscous hydro thandistillation water. method was 2.1% having water slight Isolation of curcumin yellowish color with characteristic turmeric odor. Which is slightly viscous than water. Isolation of curcumin Extractive value for column chromatographyExtractive value obtained for column from chromatography solvent extraction obtained from method solvent wasextraction 9 g. method The was 9 g. The isolated yield of curcumin by columnisolated chromatography yield of curcumin chloroform: by column chromatography methanol (95:5) chloroform: was methanol1.9 g. (95:5) was 1.9 g. TLC profile and Melting Point of IsolatedTLC profile Curcumin and Melting Point of Isolated Curcumin The TLC profile of isolated curcumin of main fraction is shown in table 2 & figure 1: The TLC profile of isolated curcuminTable of 2: mainThe TLC fraction profile of fraction is shown along with in tableCo-TLC 2 with & thefigure authentic 1: curcumin chromatogram. Table 2: The TLC profile of fraction along with Co-TLC with the authentic curcumin chromatogram. Rf value ChromatographicRf observation value of various fractions Solvent system & Rf value of CHCl :MeOH (95:5) TLC Isolated 0.75 Chromatographic observation of variousStandard fractions Solvent system & Rf value 0.75of CHCl :MeOH (95:5) TLC Isolated 0.75 Mode of visualization Standard Naked eye 0.75 Yellow-orange 2018 JournalShort UV of (254nm) Plant Resources Light yellow Vol.16, brown No. 1 Long (366nm)Mode of visualization Intense bright yellow black Naked eye Yellow-orange Measurement of spectra by Infrared (IR) Analysis TLC profile and Melting PointMelting of Point Isolated Curcumin Short UV (254nm) Isolated curcumin Light yellow brown 183 IR Longspectrum (366nm) was measured by using shimadzuAuthentic and curcuminThe IntenseTLC profilebright yellow of isolated black 182curcumin of main authenticated by certified reference materials (CRM) Meltingfraction Point is shown in table 2 & figure 1: curcumin.Isolated curcumin 183 Figure 1: Authentic curcumin 182 TLC of authentic and sub Results & Discussion fractions ‘d’ Figure 1: curcumin in Oil percentage from turmeric rhizome chloroform: TLC of methanol The essential oil percentage obtained by hydro authentic (95:5) distillation method was 2.1% having water slight and sub fractions ‘d’ GC-MS yellowish color with characteristic turmeric odor. curcumin in data Figure 1: TLC of authentic and sub fractions ‘d’ curcumin in analysis Which is slightly viscous than water. chloroform: chloroform: methanol (95:5) Chemical methanol Isolation of curcumin composition of the essential oil turmeric sample along with identification and tentative percentage composition of theGC-MS constituents data analysispresent in the oil sample. The following table(95:5) 3 and figure 2 shows the Extractive value for column chromatographynumber of constituents identified and their relative percentage in the essential oil. obtained from solvent extraction method was 9 g. Chemical composition of the essential oil turmeric GC-MS The isolated yield of curcumin by column sample along with identification and tentative data chromatography chloroform: methanol (95:5) was percentage composition of the constituents present analysis 1.9 g. in the oil sample. The following table 3 and figure 2 Chemical composition of the essential oil turmeric sampleshows along the number with ofidentification constituents identified and tentative and their percentage composition of the constituents present in the oilrelative sample. percentage The following in the essential table 3oil. and figure 2 shows the number of constituents identified and their relative percentage in the essential oil. IR spectrum was measured by using shimadzu and authenticated by certified reference materials (CRM) Figure 2: curcumin. Turmeric chromatogram essential oil 3 Result & Discussion Oil percentage from turmeric rhizome The essential oil percentage obtained by hydro distillation method was 2.1% having water slight yellowish color with characteristic turmeric odor. Which is slightly viscous than water. Isolation of curcumin Extractive value for column chromatography obtained from solvent extraction method was 9 g. The isolated yield of curcumin by column chromatography chloroform: methanol (95:5) was 1.9 g. Figure 2: TLCTurmeric profile chromatogram and Melting essential Point ofoil Isolated Curcumin FigureThe TLC 2: Turmeric profile chromatogram of isolated curcuminessential oil of main fraction is shown in table 2 & figure 1: Table 2: The TLC profile of fraction along with Co-TLC with the authentic3 curcumin chromatogram. Table 2: The TLC profile of fraction along with Co-TLC with the authentic curcumin chromatogram. Rf value

Chromatographic observation of various fractions Solvent system & Rf value of CHCl3:MeOH (95:5) TLC Isolated 0.75 Standard 0.75 Mode of visualization Naked eye Yellow-orange Short UV (254 nm) Light yellow brown Long (366 nm) Intense bright yellow black Melting point Isolated curcumin 183ŀ Authentic curcumin 182ŀ

Figure 1: 66 TLC of authentic and sub fractions ‘d’ curcumin in chloroform: methanol (95:5) GC-MS data analysis Chemical composition of the essential oil turmeric sample along with identification and tentative percentage composition of the constituents present in the oil sample. The following table 3 and figure 2 shows the number of constituents identified and their relative percentage in the essential oil.

Figure 2: Turmeric chromatogram essential oil

3 2018 Journal of Plant Resources Vol.16, No. 1 Table 3: Chemical compounds present in the turmeric oil sample identified by GC-MS. Table 3: Chemical compounds present in the turmeric oil sample identified by GC-MS. Retention S.N. Name of chemical constituents Area%(MS) time(min) 1. Terpinene 15.707 0.49 2. Cymene 16.098 0.55 3. Eucalyptol 16.432 2.30 4. Terpinolene 19.236 9.54 5. Cymen-8-ol 23.936 0.85 6. Caryphyllene<(E)_> 34.544 9.11 7. Humulene 35.956 2.02 8. Curcumene 37.073 5.93 9. Zingiberene 37.593 8.18 10. Bisabolene 38.121 2.04 11. Sesquiphellandrene 38.753 9.27 12. Nerolidol<(E)_> 40.244 0.45 13. Tumerol 40.935 0.56 14. Caryophyllene oxide 41.206 1.32 15. 3,4-Dimethylbenzyl isothiocyanate 41.912 1.38 16. Fokienol 42.044 0.45 17. .alpha.-Bisabolol 42.236 0.56 18. Cedren-13-ol,8_ 43.042 0.57 19. Tumerone 44.229 15.48 20. Tumerone 44.391 19.01 21. Benzenesulfonamide,N-(2,6-dimethylphenyl)-4-methoxy-3-tetrazol-1-yl- 45.567 8.73 22. Zinc,bis[2-(1;1-dimethyl-2-propenyl)-3,3-dimethylcyclopropyl]-,[1.alpha.(1R*,2 47.157 0.61 23. 6.beta.Bicyclo[4.3.0]nonane,5.beta.-ioamethyl-1.beta.-isopropenyl-4.alpha.,5.alpha 49.259 0.62 Total 100

IR analysisAnalysis An infrared spectrum of reference curcumin and isolated curcumin was taken using FTIR An infrared spectrum of reference curcumin and -1 spectrophotometer (IR Prestige-21, Shimadzu, Japan).Japan). The scaning The scaning range was range 250-5000 was 250-5000 cm -1 and cm the and IR isolated curcumin was taken using FTIR spectra of samples were obtained using ATR Diamond.the IR spectra of samples were obtained using ATR spectrophotometer (IR Prestige-21, Shimadzu, Diamond.

Figure 3: IR spectrum of isolated curcumin Figure 3: IR spectrum of isolated curcumin

67 4

Figure 4: IR spectrum of authentic curcumin Conclusion The result of this investigation shows that isolated curcumin from this method is very convenient in lab scale for better yield. This research will be helpful for qualitative and quantative analysis of essential oil of cultivated turmeric rhizomes of Kailali District. This work is also commercially beneficial to all

5 2018 Journal of Plant Resources Vol.16, No. 1 Figure 3: IR spectrum of isolated curcumin

FigureFigure 4: 4: IR IR spectrum spectrum of of authentic authentic curcumin curcumin Conclusion Conclusion Refrences: The result of this investigation shows that isolated curcumin from this method is very convenient in lab The scaleresult for of betterthis investigation yield. This researchshows that, will isolated be helpfulAndrew, for qualitative M.A., Matthew, and quantative S.M., & analysis Ram, S.M. of essential (2000). oil curcuminof cultivated from this turmeric method rhizomes is very convenientof Kailali inDistrict. IsolationThis work of curcuminis also commercially from turmeric. beneficial J. Chem. to all lab scale for better yield. This research will be helpful Educ., 77(3), 359. for qualitative and quantitative analysis of essential 5 oil of cultivated turmeric rhizomes of Kailali district. Ching, W.Y., Yusoff, Y.S., & Wan-Amarina, W.B. This work is also commercially beneficial to all (2014). Extraction of essential oil from Curcuma public level. Though for further structure illucidation longa. Jounal of Food, Chemistry and Nutrition, and to develop less hazardous green extraction 2(01), 1-10. technique HPLC, NMR and further research is Cousins, M., Adelberg, J., Chenb, F., & Rieck, I. required. (2007). Antioxidant capacity of fresh and dried rhizomes from four clones of turmeric (Curcuma Acknowledgements longa L.) grown in-vitro. Ind.crop prod., 25, 129- 135. The authors are like to acknowledge the Director General Mr. Sanjeev kumar Rai and Deputy Director Devkota, L., & Rajbhandari, M. (2015). General Mrs. Jyoti Joshi Bhatta and Chief of Natural Composition of essential oils in turmeric rhizome. Product Research Laboratory Devi Prasad Bhandari Nepal Journal of Science and Technology, 16(1), for his great motivation and encouragement for this 87-94. work. We are obliged to Mr. Rajendra Sharma, Mrs. Bipasana Shakya, Mr. Tara Datta Bhatta, Mr. Krishna Garg, S.N., Bansal, R.P., Gupta, M.M., & Sushil, K. Kumar Shah for their participation and team work. (1999). Variation in the rhizome essential oil and

68 2018 Journal of Plant Resources Vol.16, No. 1

curcumin contents and oil quality in the land races identification of curcuminoids from turmeric of turmeric Curcuma longa of North Indian (curcuma longa L.) by column chromatography. plains. Flavour and Fragrance Journal, 14(5), Journal of Experimental Sciences, 2(7), 21-25. 1099-1026. Rukkumani, R., Sri Balasubashini, M., & Menon, Kulkarni, S.J., Maske, K.N., Budre, M.P., & V.P. (2003). Protective effects of curcumin and Mahajan, R.P. (2012). Extraction and purification photo irradiated curcumin on circulatory lipids of curcuminoids from turmeric curcuma longa and lipid proxidation products in alcohol and L. International Journal of Pharmacology and polyunsaturated fatty acid-induced toxicity. Pharmaceutical Technology, 1(2), 2277-3436. Phytotherapy Research, 17, 925-929. Nishiyama, T., Mae, T., Kishida, H., Tsukagawa, M., Singh, G., Kapoor, S., Singh, P., Carola, S., Minaki, Y., Kuroda, M., Sashida, Y., Takahashi, Lampasona, C., & Catalan, A.N. (2010). K., Kawada, T., Nakagawa, K., & Kitahara, M. Comparative study of chemical composition and (2005). Curminoids and sesquiterpenoids in antioxidant activity of fresh and dry rhizomes of turmeric (Curcuma longa L.) suppress an increase turmeric (Curcuma longa L.) Food and Chemical in blood glucose level in type 2 diabetic KK-Ay Toxicology, 48(4), 1026-1031. Mice. Journal of Agriculture and Food Chemistry, 53, 959-963. Susana, Z. (2012). Isolation and synthesis of Curcumin. (Bachelors Thesis), Department of Revathyl, S., Elumalanil, S., Antony, S., & Benny, Physics, Chemistry & Biology, Linkoping M. (2011). Isolation, purification, and University.

69 2018J. Pl. Res. Vol. 16, No. 1, pp 70-77, 2018 Journal of Plant Resources Vol.16, No. 1

Microscopic, UV- Fluorescence and FT-IR Analysis of the Powder of Five Medicinal Plants of Nepal

Parasmani Yadav1*, Bhabani Prasad Adhikari1, Sarita Yadav1, Sunita Khadaka2, Devi Prasad Bhandari1 and Chetana Khanal1 1Natural Products Research Laboratory, Kathmandu, Nepal 2Trichandra Multiple Campus, Tribhuvan University, Kathmandu, Nepal *E-mail: [email protected]

Abstract

The present research was conducted to evaluate the pharmacognostic, UV-fluorescence and FT- IR analysis of medicinal plants which will be useful in standardization for quality, purity and sample identification. The pharmacognostic characters observed in this study will be helpful in correct identification and characterization of medicinal plants. The fluorescence analysis showed that, the crude drugs exhibited clear fluorescence behaviors at different radiation due to the presence of various phytochemical constituents after treated with different chemical reagents. Whereas FT-IR analysis showed different functional groups based on its peak ratio in the powder medicinal plants. This study helps in the authenticity due to change in morphological form, assay for detection and composition of plant molecules between two species, intentional and unintentional adulteration, procurement of raw herb, process development for production and quality assurance, heavy metal contamination and post harvest surveillance of herbals.

Keywords: Identification, Powder microscopy, Standardization

Introduction also to a lack of adequate or accepted research methodology for evaluating traditional medicine Traditional herbal medicine and their preparations (WHO/EDM/TRM, 2000). have been widely used for the thousands of years in developing and developed countries owing to its This study is focused on microscopic, UV- natural origin and lesser side effects or dissatisfaction fluorescence and FT-IR spectroscopy of five with the results of synthetic drugs. However, one of medicinal plants that are i. Sapindus mukorossi the characteristics of oriental herbal medicine Gaertn. (powder of pericarp) ii. Justicia adhatoda preparations is that all the herbal medicines, either L. (rhizome powder) iii. Percea odoratissima (bark presenting as single herbs or as collections of herbs powder) iv. Butea monosperma (Lam.) Taub. (Leaf in composite formulae is extracted with boiling water powder) and v. Pogostemon bengalensis (Burm. f.) during the decoction process (Hamburger & Kuntze (leaf powder). It will help in the authenticity Hostettman, 1991). This may be the main reason why due to change in morphological form, assay for quality control of oriental herbal drugs is more detection and composition of plant molecules difficult than that of western drug (WHO/EDM/ between 2 species, intentional and unintentional TRM, 2000). Despite its existence and continued adulteration, procurement of raw herb, process use over many centuries, its popularity and extensive development for production and quality assurance, use during the last decade, traditional medicine has heavy metal contamination and postharvest not been officially recognized in most countries. surveillance of herbals. Consequently, education, training and research in this area have not been accorded due attention and Materials and Methods support. The quantity and quality of the safety and efficacy data on traditional medicine are far from The plant material was collected in an appropriate sufficient to meet the criteria needed to support its stage of its growth from different places of Nepal. use world-wide. The reasons for the lack of research Sapindus mukorossi was collected from Baitadi data are due to not only to health care policies but district and Percea odoratissima was collected from

70 2018 Journal of Plant Resources Vol.16, No. 1

Salyan distrct, a temperate zone. Similarly, Justicia and every analysis was repeated twice for the adhatoda,Dried plant Butea material monosperma was finely and powdered Pogostemon in blenderspectrum and subjected confirmation in chemicals (Janakiraman for analysis. et al., At 2011). first bengalensissufficient amount were collected of powder from was the taken tropical in chloral-hydrate zone solution on a slide, covered with cover slip Chitwanand was districtleft for and18 hours were andauthenticated finally observe through under microscopeResults and model Discussion number MBL 2100. Starch test detailwas also taxonomical done with iodine study solution and (Ansari,air dried 2006). for The pharmacognostic characters are helpful in correct pharmacognosticalidentification and characterizationstudy. of medicinal plants The(Gupta, different 2006) plants were powdered then subjected to the microscopic study. The microscopic pictures Fluorescence analysis in dried powder of the powder of the pericarp of Sapindus mukorossi PowderThe fluorescence microscopy analysis of dried powdered of leaf and rhizome was carried out by treating with are in figure 1. The microscopic study of the powder Driedvarious plant chemicals. material wasIf the finely substances powdered themselves in blender are not fluorescent, they may often be converted into of Justicia adhatoda are shown in figure 2. The andfluorescent subjected derivatives in chemicals by applyingfor analysis. chemical At first, reagents. A small quantity (1 gm) of dried and finely microscopic pictures of the bark of Percea sufficientpowdered amount leaf and of rhizome,powder wasfruits taken of medicinal in chloral- plants was treated with freshly prepared acids, alkaline odoratissima are shown in figure 3. Similarly, hydratesolutions solution and different on a slide, solvents. covered They with were cover subjected slip to fluorescence analysis in daylight, in short UV- microscopic study pictures of the powder of leaves andlight was (254 left nm) for and18 hours long UV-and finallylight (365 observe nm) (Kavitha,under 2014) of Butea monosperma are shown in figure 4. The microscopeFT-IR spectroscopy model number analysis MBL 2100. Starch test microscopic pictures of leaves of Pogostemon wasFourier also donetransform with infrarediodine solution spectroscopy (Ansari, analysis 2006). was performed using SHIMADZU spectrophotometer bengalensis are shown in figures 5. The(FT-IR, pharmacognostic model number charactersIR Prestige-21). are helpfulThe powders in were scanned in the wavelength ranging from 300- correct1100 nm identification and the characteristic and characterization peaks were detected. of ThePowder peak valuesanalysis of the plays FT-IR a weresignificant recorded. role Each in medicinaland every plants analysis (Gupta, was repeated 2006). twice for the spectrumidentification confirmation of(Janakiraman crude drug. et These al, 2011). characters will help in the identification of right variety and search FluorescenceResults and Discussionanalysis in dried powder for adulterants. Powder microscopy is one of the TheThe fluorescence different plants analysis were ofpowdered dried powdered then subjected of leaf to thesimplest microscopic and cheapest study. The methods microscopic to start pictures with offor andthe rhizome powder wasof the carried pericarp out byof treatingSapindus with mukorossi various areestablishing in figure i. The the microscopic correct identity study ofof thethe powder source chemicals.of Justicia Ifadhatoda the substances are shown themselves in figure ii. are The not microscopicmaterials. pictures It is ofuseful the bark for furtherof Percea pharmacological odoratissima fluorescent,are shown inthey figure may iii. often Similarly, be converted microscopic into studyand picturestherapeutic of the evaluationpowder of leavesalong ofwith Butea the fluorescentmonosperma derivatives are shown by applying in figure chemical iv. The reagents.microscopicstandardization pictures of leaves of plantof Pogostemon material. Powdered bengalensis drug are of Ashown small quantityin figures (1 v. gm) of dried and finely powdered different parts of plant gave different fluorescence leaf and rhizome, fruits of medicinal plants was under day light and ultraviolet radiation of both long treatedPowder with analysis freshly plays prepared a significant acids, alkaline role in solutions identificationand of short crude wave drug. lengths.These characters Therefore, will florescencehelp in the andidentification different solvents.of right variety They andwere search subjected for adulterants. to evaluation Powder ismicroscopy used for identificationis one of the simplestof plant and and fluorescencecheapest methods analysis to instart daylight, with for in establishing short UV- light the correctpowdered identity drug. of the Some source crude materials. drugs are It often is useful assessed for (254further nm) pharmacological and long UV- lightand (365therapeutic nm) (Kavitha, evaluation alongqualitatively with the in standardization this way and ofit isplant an importantmaterial. 2014).Powdered drug of different parts of plant gave differentparameter fluorescence of pharmacognostical under day light evaluation. and ultraviolet radiation of both long and short wave lengths. Therefore florescence evaluation is used for identification IR and its advanced forms FTIR spectroscopy are of plant and powdered drug. Some crude drugs are often assessed qualitatively in this way and it is an FT-IR spectroscopy analysis used to gather the functional properties and structural important parameter of pharmacognostical evaluation. Fourier transform infrared spectroscopy analysis was information of a compound. Plants and plant performed using SHIMADZU spectrophotometer products have been characterized by characteristic IR and its advanced forms FTIR spectroscopy are used to gather the functional properties and structural (FT-IR, model number IR Prestige-21). The powders peaks in FTIR spectrum, indicating the presence or information of a compound. Plants and plant products have been characterized by characteristic peaks in were scanned in the wavelength ranging from 300- absence of functional groups and molecular skeleton, FTIR spectrum, indicating the presence or absence of functional groups and molecular skeleton, even 1100 nm and the characteristic peaks were detected. even from different geographical locations. The types from different geographical locations. The types of bond need to be recognized are given in table 1. The peak values of the FT-IR were recorded. Each of bond need to be recognized are given in table 1. Table 1: Types of bond need to be recognized *Broad due to hydrogen bonding between O-H groups Bond Functional group Absorbance (cm-1) O – H Alcohol 3200 – 3600 / strong and broad* O – H Carboxylic acid 2500 – 3200 / medium and very broad* C=O Aldehydes / ketones / carboxylic acids/ esters 1680 – 1750 / strong and sharp C-O Alcohols / esters / ethers 1050 – 1300 / medium C-H Alkanes / alkenes etc 2850 – 3100 / medium

1. Powder microscopy 71 2018 Journal of Plant Resources Vol.16, No. 1

1. Powder microscopy

ABCD E Figure 1: Sapindus mukorossi (pericarp of fruits): A. Fibers, B. Stone cell, C. Starch grains, D. Rossete crystals, E. Pitted vessel

ABCD Figure 2: Justicia adhatoda (leaf): A. Vessel, B. Starch grains, C. trichome, D. Fiber

AB CDEF Figure 3: Percea odoratissima (bark): A. Stone cell, B. Oil globule, C. Fiber, D. Cork cell, E. Brown content, F. Trichome

AB C D Figure 4: Butea monosperma (leaf): A. Epidermal cells, B. Epidermis and pallisade, C. Fiber, D. Trichome

AB C D E Figure 5: Pogostemon bengalensis (leaf): A. Cells, B. Vessel, C. Fiber, D. Stomata, E. Trichome

72 2018AB JournalC of Plant ResourcesDE Vol.16, No. 1 Figure 5: Pogostemon bengalensis (leaf): A. Cells, B. Vessel, C. Fiber, D. Stomata, E. Trichome 2. Fluorescence analysis of crude powder with different chemical reagents 2. Fluorescence analysis of crude powder with different chemical reagents DifferentDifferent chemical chemical reagents reagents are addedare added with with the powderedthe powdered materials materials then then different different colors colors are observedare observed with differentwith differenttypes of lighttypes and of lightthey andare tabulatedthey are tabulated as follows. as follows.

Table 2: TableSapindus 2: Sapindusmukorossi mukorossi (pericarp (pericarpof fruits) of fruits) S. N. Powdered drug + chemical reagent Day light UV short UV long 1. Sapindus mukorossi + Conc. HCl Brown Light green Black 2. Sapindus mukorossi + Dil.HCl Brown Green Black 3. Sapindus mukorossi + Iodine solution (2%) Brown Green Black 4. Sapindus mukorossi + Glacial acetic acid Brown Green Dark brown

5. Sapindus mukorossi + Conc. HNO3 Brown Black Black 6. Sapindus mukorossi + Dil. HNO3 Brown Dark green Grey 7. Sapindus mukorossi + Glacial acetic acid +HNO3 Brown Light green Green 8. Sapindus mukorossi + Dil. H2So4 Brown Green Black 9. Sapindus mukorossi +Conc. H2So4 Brown Green Black 10. Sapindus mukorossi + Fehling’s reagent Brown Dark brown Dark brown 11. Sapindus mukorossi + Picric acid solution (Hager’s reagent) Brown Green 12. Sapindus mukorossi + NaOH (10%) Brown Green Black 13. Sapindus mukorossi + KOH (10%) Brown Green Black 14. Sapindus mukorossi + Wagner’s reagent Brown Green Brown

15. Sapindus mukorossi + CuSo4 (5%) Brown Green Black 16. Sapindus mukorossi + Lead acetate Brown Bluish green Black 17. Sapindus mukorossi + Methanol Brown Green Brown 18. Sapindus mukorossi + Ethanol Brown Light green Purple

Table 3: Justicia adhatoda (leaf) Table 3: Justicia adhatoda (leaf) S. N. Powdered drug + chemical reagent Day light UV short UV long 1. Justicia adhatoda + Conc. HCl Yellow Light green Black 2. Justicia adhatoda + Dil.HCl Light green Light green Black 3. Justicia adhatoda + Iodine solution (2%) Yellow Green Black 4. Justicia adhatoda + Glacial acetic acid Yellowish green Light green Brown

5. Justicia adhatoda + Conc. HNO3 Red Green Black 6. Justicia adhatoda + Dil. HNO3 Yellow Green Black 7. Justicia adhatoda + Glacial acetic acid +HNO3 Yellow Green Brown 8. Justicia adhatoda + Dil. H2So4 Yellowish green Green Black 9. Justicia adhatoda +Conc. H2So4 Brown Green Black 10. Justicia adhatoda + Fehling’s reagent Dark green Light green Black

11. Justicia adhatoda +2% FeCl3 Brown Green Black 12. Justicia adhatoda +NaOH (10%) Yellow Green Brown 13. Justicia adhatoda + KOH (10%) Green Light green Black 14. Justicia adhatoda + Wagner’s reagent Yellow Green Black

15. Justicia adhatoda +CuSo4 (5%) Green Light green Black 16. Justicia adhatoda + Mayer’s reagent Light yellow Green Black 17. Justicia adhatoda + Diethyl ether Green Light green Brown 18. Justicia adhatoda + Ethanol Greenish yellow Light green Brown 19. Justicia adhatoda +Acetone Light green Light green Brown 20. Justicia adhatoda + Ethyl acetate Yellowish green Light green Brown

Table 4: Percea odoratissima (Kaulo ) bark S. N. Powdered drug + chemical reagent Day light UV short UV long 1. odoratissima + Conc. HCl Brown Green Brown 2. odoratissima + Dil.HCl Brown Light green Black 3. odoratissima + Iodine solution (2%) Yellow Green Black 4. odoratissima + Glacial acetic acid Brown Light brown Black 73 5. odoratissima + Conc. HNO3 Red Green Black

6. odoratissima + Dil. HNO3 Brown Green Black

7. odoratissima + Glacial acetic acid +HNO3 Brown Dirty green Black

8. odoratissima + Dil. H2So4 Brown Light green Black 9. odoratissima +Conc. HSo Black Brown Black 10. odoratissima + Fehling’s reagent Blue Black Black

11. odoratissima +2% FeCl3 Brown Green Black 12. odoratissima + NaOH (10%) Brown Green Black 13. odoratissima + KOH (10%) Dark brown Green Black 14. odoratissima + Wagner’s reagent Yellow Green Black

15. odoratissima +CuSo4 (5%) Brown Dark brown Black 16. odoratissima + Mayer’s reagent Brown Dark brown Black 17. odoratissima + Diethyl ether Brown Dirty green Black 18. odoratissima + Ethanol Brown Creamy Black 19. odoratissima +Acetone Brown Dark green Reddish brown 20. odoratissima + Ethyl acetate Brown Light green Black Table 3: Justicia adhatoda (leaf)

S. N. Powdered drug + chemical reagent Day light UV short UV long 1. adhatoda + Conc. HCl Yellow Light green Black 2. adhatoda + Dil.HCl Light green Light green Black 3. adhatoda + Iodine solution (2%) Yellow Green Black 4. adhatoda + Glacial acetic acid Yellowish green Light green Brown 5. adhatoda + Conc. HNO Red Green Black 6. adhatoda + Dil. HNO Yellow Green Black 7. adhatoda + Glacial acetic acid +HNO Yellow Green Brown 8. adhatoda + Dil. H So Yellowish green Green Black 9. adhatoda +Conc. HSo Brown Green Black 10. adhatoda + Fehling’s reagent Dark green Light green Black 11. adhatoda +2% FeCl Brown Green Black 12. adhatoda +NaOH (10%) Yellow Green Brown 13. adhatoda + KOH (10%) Green Light green Black 14. adhatoda + Wagner’s reagent Yellow Green Black 15. adhatoda +CuSo (5%) Green Light green Black 16. adhatoda + Mayer’s reagent Light yellow Green Black 17. adhatoda + Diethyl ether Green Light green Brown 18. adhatoda + Ethanol Greenish yellow Light green Brown 19. adhatoda +Acetone Light green Light green Brown 2018 Journal of Plant Resources Vol.16, No. 1 20. adhatoda + Ethyl acetate Yellowish green Light green Brown

Table 4:Table Percea 4: Perceaodoratissima odoratissima (Kaulo (Kaulo) bark ) bark S. N. Powdered drug + chemical reagent Day light UV short UV long 1. Percea odoratissima + Conc. HCl Brown Green Brown 2. Percea odoratissima + Dil.HCl Brown Light green Black 3. Percea odoratissima + Iodine solution (2%) Yellow Green Black 4. Percea odoratissima + Glacial acetic acid Brown Light brown Black

5. Percea odoratissima + Conc. HNO3 Red Green Black

6. Percea odoratissima + Dil. HNO3 Brown Green Black

7. Percea odoratissima + Glacial acetic acid +HNO3 Brown Dirty green Black

8. Percea odoratissima + Dil. H2So4 Brown Light green Black

9. Percea odoratissima +Conc. H2So4 Black Brown Black 10. Percea odoratissima + Fehling’s reagent Blue Black Black

11. Percea odoratissima +2% FeCl3 Brown Green Black 12. Percea odoratissima + NaOH (10%) Brown Green Black 13. Percea odoratissima + KOH (10%) Dark brown Green Black 14. Percea odoratissima + Wagner’s reagent Yellow Green Black

15. Percea odoratissima +CuSo4 (5%) Brown Dark brown Black 16. Percea odoratissima + Mayer’s reagent Brown Dark brown Black 17. Percea odoratissima + Diethyl ether Brown Dirty green Black 18. Percea odoratissima + Ethanol Brown Creamy Black 19. Percea odoratissima +Acetone Brown Dark green Reddish brown 20. Percea odoratissima + Ethyl acetate Brown Light green Black

Table 5: Butea monosperma (Palas) leaf Table 5: Butea monosperma (Palas) leaf S. N. Powdered drug + chemical reagent Day light UV short UV long 1. Butea monosperma + Conc. HCl Light green Light green Black 2. Butea monosperma Dil.HCl Green Brown Black 3. Butea monosperma + Iodine solution (2%) Green Green Black 4. Butea monosperma + Glacial acetic acid Light green Light green Black

5. Butea monosperma + Conc. HNO3 Red Light green Black 6. Butea monosperma + Dil. HNO3 Dirty green Green Black 7. Butea monosperma + Glacial acetic acid +HNO3 Yellow Green Black 8. Butea monosperma + Dil. H2So4 Green Green Brown 9. Butea monosperma Conc. H2So4 Brown Green Black 10. Butea monosperma + Fehling’s reagent Blue Greenish black Black

11. Butea monosperma + 2% FeCl3 Brown Dirty green Black 12. Butea monosperma + NaOH (10%) Dirty green Green Black 13. Butea monosperma + KOH (10%) Dirty green Light green Black 14. Butea monosperma Wagner’s reagent Yellow Green Black

15. Butea monosperma +CuSo4 (5%) Green Light green Black 16. Butea monosperma +Mayer’s reagent Light green Light green Black 17. Butea monosperma + Ethanol Light green Light green Black 18. Butea monosperma + Diethyl ether Dirty green Green Black 19. Butea monosperma + Acetone Green Green Black 20. Butea monosperma + Ethyl acetate Yellowish green Light green Black

Table 6: Pogostemon bengalensis (Rudilo) leaf

S. N. Powdered drug + chemical reagent Day light UV short UV long 1. bengalensis + Conc. HCl Dirty green Orange Black 2. bengalensis + Dil.HCl Green Light green Black 74 3. bengalensis + Iodine solution (2%) Dirty green Light green Black 4. bengalensis + Glacial acetic acid Yellowish green Light green Black 5. bengalensis + Conc. HNO Red Green Black 6. bengalensis + Dil. HNO Dirty green Green Black 7. bengalensis + Glacial acetic acid +HNO Brown Green Black 8. bengalensis +Dil. HSo Dirty green Green Black 9. bengalensis +Conc. HSo Light yellow Light green Brown 10. bengalensis + Fehling’s reagent Green Light green Black 12. bengalensis + NaOH (10%) Geenish yellow Green Black 13. bengalensis + KOH (10%) Yellow Green Black 14. bengalensis + Wagner’s reagent Yellow Green Black 15. bengalensis +CuSo (5%) Green Dirty green Black 16. bengalensis + Mayer’s reagent Dirty green Green Black 17. bengalensis + Diethyl ether Dirty green Green Black 18. bengalensis + Ethanol Green Light green Brown 19. bengalensis +Acetone Green Light green Brown 20. bengalensis + Ethyl acetate

3. FT-IR spectroscopy analysis of Powder Powder of plant materials were subjected to the FT-IR spectroscopy for the analysis of presence of various functional groups which are shown below in the figures. Table 5: Butea monosperma (Palas) leaf

S. N. Powdered drug + chemical reagent Day light UV short UV long 1. monosperma + Conc. HCl Light green Light green Black 2. monosperma Dil.HCl Green Brown Black 3. monosperma + Iodine solution (2%) Green Green Black 4. monosperma + Glacial acetic acid Light green Light green Black 5. monosperma + Conc. HNO Red Light green Black 6. monosperma + Dil. HNO Dirty green Green Black 7. monosperma + Glacial acetic acid +HNO Yellow Green Black 8. monosperma + Dil. H So Green Green Brown 9. monosperma Conc. H So Brown Green Black 10. monosperma + Fehling’s reagent Blue Greenish black Black 11. monosperma + 2% FeCl Brown Dirty green Black 12. monosperma + NaOH (10%) Dirty green Green Black 13. monosperma + KOH (10%) Dirty green Light green Black 14. monosperma Wagner’s reagent Yellow Green Black 15. monosperma +CuSo (5%) Green Light green Black 16. monosperma +Mayer’s reagent Light green Light green Black 17. monosperma + Ethanol Light green Light green Black 18. monosperma + Diethyl ether Dirty green Green Black 19. monosperma + Acetone Green Green Black 201820. monosperma + Ethyl acetate Journal of Plant ResourcesYellowish green Light green Black Vol.16, No. 1

Table 6: Pogostemon bengalensis (Rudilo) leaf Table 6: Pogostemon bengalensis (Rudilo) leaf S. N. Powdered drug + chemical reagent Day light UV short UV long 1. Pogostemon bengalensis + Conc. HCl Dirty green Orange Black 2. Pogostemon bengalensis + Dil.HCl Green Light green Black 3. Pogostemon bengalensis + Iodine solution (2%) Dirty green Light green Black 4. Pogostemon bengalensis + Glacial acetic acid Yellowish green Light green Black

5. Pogostemon bengalensis + Conc. HNO3 Red Green Black 6. Pogostemon bengalensis + Dil. HNO3 Dirty green Green Black 7. Pogostemon bengalensis + Glacial acetic acid +HNO3 Brown Green Black 8. Pogostemon bengalensis +Dil. H2So4 Dirty green Green Black 9. Pogostemon bengalensis +Conc. H2So4 Light yellow Light green Brown 10. Pogostemon bengalensis + Fehling’s reagent Green Light green Black 12. Pogostemon bengalensis + NaOH (10%) Geenish yellow Green Black 13. Pogostemon bengalensis + KOH (10%) Yellow Green Black 14. Pogostemon bengalensis + Wagner’s reagent Yellow Green Black

15. Pogostemon bengalensis +CuSo4 (5%) Green Dirty green Black 16. Pogostemon bengalensis + Mayer’s reagent Dirty green Green Black 17. Pogostemon bengalensis + Diethyl ether Dirty green Green Black 18. Pogostemon bengalensis + Ethanol Green Light green Brown 19. Pogostemon bengalensis +Acetone Green Light green Brown 20. Pogostemon bengalensis + Ethyl acetate

3. 3.FT-IR FT-IR spectroscopy spectroscopy analysis analysis of ofpowder Powder PowderPowder of ofplant plant materials materials were were subjected subjected to the to FT-IR the FT-IR spectroscopy spectroscopy for the for analysis the analysis of presence of presence of various of functionalvarious functionalgroups which groups are shownwhich arebelow shown in the below figures. in the figures.

Figure 6:Sapindus mukorrossi (pericarp of fruits)

Figure: 7 Justicia adhatoda (Asuro) leaf

75 2018 Journal of Plant Resources Vol.16, No. 1

Figure: 8 Percea odoratissima (bark)

Figure: 9 Butea monosperma (leaf)

Figure: 10 Pogostemon bengalensis (Rudilo) leaf

Conclusion Based on the results obtained in the present and composition of plant molecules between two investigation, it may be concluded that microscopic, species, intentional and unintentional adulteration, UV- fluorescence and FT-IR spectroscopy are procurement of raw herb, process development for different for crude powder of different plants. This production and quality assurance, heavy metal research may help in the study in authenticity due to contamination and postharvest surveillance of change in morphological form, assay for detection herbals.

76 2018 Journal of Plant Resources Vol.16, No. 1

Acknowledgments Hamburger, M., & Hostettman, K. (1991). Bioactivity in plants: The link between The authors would like to thank Mr. Devi Prasad phytochemistry and medicine. Phytochem., Bhandari, Chief of Natural Products Research 30(12), 3864-3874. Laboratory for giving his co-operation and support for managing the research work. The authors would Janakiraman, N., Sahaya Sathish, S., & Johnson, M. also like to thank Department of Plant Resources (2011). UV-VIS and FTIR spectroscopic studies and Natural Products Research Laboratory for giving on Peristrophe bicalyculata (Retz.) Nees. Asian an opportunity for research work in a very much J. Pharmaceu. and Clin. Res., 4(4), 125-29. friendly and sound environment. Kavitha, R. (2014) Fluorescence and ft-IR analysis of leaf and fruit of Trichosanthes dioica Roxb. References World Journal of Pharmacy and Pharmaceutical Ansari, M.M., Ahmad, J., & Ansari, S.H. (2006). Sciences, 3(10), 563-572. Pharmacognostic evaluation of the stem bark of WHO/EDM/TRM (2000). General guidelines for Balanitesa egyptica Delile “Hingot”. methodologies on research and evaluation of Hamdardmedicus, 50, 82-94. traditional medicines. Geneva: World Health Gupta, M.K., Sharma, P.K., Ansari, S.H., & Organization. Lagarkha, R. (2006). Pharmacognostical evaluation of Grewia asiatica fruits. Int. J. Plant Sci., 1(2), 249-51.

77 2018J. Pl. Res. Vol. 16, No. 1, pp 78-83, 2018 Journal of Plant Resources Vol.16, No. 1

Phytochemicals, Polyphenols and Antioxidant Activity of Camellia sinensis (L.) Kuntze (Tea Leaf) from Ilam District, Nepal

Laxman Bhandari*, Madan Raj Bhatta and Rajeswar Ranjitkar Natural Products Research Laboratory, Department of Plant Resources, Thapathali, Kathmandu, Nepal *E-mail: [email protected]

Abstract

Preliminary phytochemical screening of dried tea leaves has been carried out in different solvent system. Quantitative determination of Total phenolic content (TPC) and Total flavonoid content (TFC) was carried out using Folin-Ciocalteau reagent and aluminium chloride reagent by spectrophotometric technique. Gallic acid and quercetin is used as standard for calibration for phenols and flavonoid. The standard calibration curve for phenolic is Y= 0.01X+0.001 and R2= 0.99 and flavonoid is Y= 0.004X and R2 = 0.98. The maxiumum phenolic and flavonoid content in methanolic extracts was found to be 321.25± 2.1651 mg GAE/ g and 126.88 3.2476 mg QE/ g. Antioxidant activity of extracts were measured using 2, 2 -Diphenyl-1-picrylhydrazyl (DPPH)

method. The Half maximal inhibitory concentration (IC50 ) value in methanolic extract is 25.15.

This indicates that the higher is TPC, the lower is IC50.

Keywords: DPPH method, Phytochemical screening, Total flavonoid content, Total phenolic content

Introduction such as cancer, inflammatory disease, asthma, cardiovascular diseases, neurodegenerative disease Camellia sinensis (L.) Kuntze, family Theaceae is and premature aging (Ghimire et al., 2011). an evergreen shrub, cultivated and flowering on Medicinal plants containing large amounts of March-April (Parmer et al., 2012). Leaves of this antioxidants polyphenols which play important role plant are bitter and use as astringent, stimulant, in absorbing and neutralizing free radicals, gently excitant, diuretic, appetizer and digestive quenching singlet and triplet oxygen or decomposing (Tarqq et al., 2010). Leaves yield caffeine, peroxides (Siddique et al., 2010). theobromine, theophylline, xanthine, ascorbic acid, nicotinic acid, riboflavin, pantothenic acid, Polyphenols are commonly found edible and inedible quercetin, kaempferol and inositol (DPR, 2007). plants with medicinal and aromatic values. They are secondary metabolites present in fruits, seeds, herbs The antioxidants obtained from plants are of and vegetables. They are reported to have multiple greater benefit in comparison to synthetic ones. biological effects antitumor, antimutagenic and Synthetic antioxidants used frequently in food industry are butylatedhydroxy toluene (BHT) and antibacterial including antioxidant activity (Bhandari butylatedhydroxy anisole (BHA) proven to cause et al., 2003). Therefore, consumers should increase highly carcinogenic and hemorrhaging. Therefore, their intake of food rich in antioxidant compounds synthetic antioxidants need to be replaced by natural that lower the risk of chronic health problems and oxidants and it is of great important to find new compounds responsible for such antioxidant activity sources of safe and economic natural antioxidants can be isolated and used for prevention and treatment (Tipoe et al., 2007). of free-radical related disorders (Ghimeray et al., 2009). So, the recent attention is to find naturally

Reactive oxygen species (ROS) such as H2O2, HOCl occurring phenolic antioxidants for use in food and and free radicals such as (OH-) and superoxide anion medicine (Salluca et al., 2008). - (O2 ) are produced as normal products of cellular metabolism (Adedapo et al., 2009). Rapid Nepal is natural storehouse of medicinal plants. production of free radicals can lead to oxidative Approximately, 70-80% of the population of Nepal damage to biomolecules and may cause disorders depends on traditional medicines (Shrestha, 2002).

78 2018 Journal of Plant Resources Vol.16, No. 1

Despite widespread use of wild plants as medicine prepared by dissolving 1 mg gallic acid in 1 mL of in Nepal, little is known about the antioxidant methanol (1 mg/mL). Various concentrations of properties of phenolic compounds and their gallic acid such as 200, 100, 50 and 25 µg/mL were contribution to human health (Sharma et al., 2011). prepared by serial dilution of stock solution. An aliquot of 1 mL gallic acid of each concentration in Materials and Methods methanol was added to 20 mL test tube. To that 5 mL of Folin-Ciocalteu reagent (10%) and 4 mL of Plant materials 7% Na2CO3 were added to get a total of 10 mL. The Tea leaves were collected from tea garden of near to blue colored mixture was shaken well and incubated Ilam bazaar factory side southern part in the month for 30 minutes at 40ºC in a water bath. Then the of Bhadra 2073. Herbariums of plants were absorbance was measured at 760 nm against blank. authenticated by Pharmacognosy section of NPRL. The absorbance values obtained at different The plant sample was air dried and the leaves were concentrations of gallic acid were used to plot the crushed into powder and stored in polythene bags calibration curve. for further use Similarly, TFC was determined by AlCl3 colorimetric Chemicals and reagents assay (Acharya, 2013). Concentration of standard quercetin viz, 2 mg/mL, 1 mg/mL, 0.5 mg/mL and Folin–Ciocalteu’s phenol reagent, Gallic acid and 0.25 mg/mL were prepared by serial dilution of stock DPPH was purchased from sigma chemical solution of concentration of 4 mg/mL. An aliquot of company, USA. All the solutions are prepared in 1 mL quercetin of each concentration in MeOH was distilled water. Chemicals and reagents used were added to 10 mL v.f. containing 4 mL of double of analytical grade. distilled water. At the zero time, 0.3 mL, 5% sodium nitrite was added to the flask. After 5 min, 0.3 mL Soxhlet extraction method of 10% AlCl3 was added to the flask. At 6 min, 2 mL 50 gm dried powdered tea leaves were extracted with of 1 M NaOH was added to the mixture. 500 mL petroleum ether extract on Soxhlet apparatus Immediately, the total volume of the mixture was for 8 hours on 2073/08/01. The residue were made up to 10 mL by the addition of 2.4 mL double extracted successively with methanol, 50% ethanol distilled water and mixed thoroughly. Absorbance and water extract respectively. The extracts were of the pink colored mixture was determined at 510 filtered and solvents were evaporated in rotatary nm versus a blank containing all reagents except evaporator under reduced pressure. quercetin. Absorbance values obtained at different concentrations of quercetin were used to plot the Preliminary phytochemical screening calibration curve. The extracts were used for the preliminary Preparation of samples for phenolic content and phytochemical analysis. All the tests were performed flavonoid content: Stock solutions of all extracts in triplicate mode. The standard operating procedures were prepared by dissolving 1 mg in 1 mL of MeOH. taken for analysis (Harborne, 1969; Sofowora, Serial dilutions were carried out to get the 1993). concentration of 200, 100, 50 and 25 µg/mL. To these diluted solution FCR and Na CO were added and Determination of total phenolic content and total 2 3 flavonoid content incubated for 30 minutes as in the case of standard gallic acid preparation and absorbance was measured Preparation of standard for phenolic content and at 760 nm. flavonoid content: The TPC of extract was estimated by Folin-Ciocalteau reagent described by Singleton Similarly, various concentrations of the extracts viz & Rossi, (1965). Gallic acid stock solution was 2 mg/mL, 1 mg/mL, 0.5 mg/mL and 0.25 mg/mL

79 2018 Journal of Plant Resources Vol.16, No. 1 were prepared. Following the procedure described mL distilled water and 2.5 mL 0.1 mM methanolic above in flavonoid, absorbance for each DPPH solution. These samples were well shaken and concentration of extract was recorded. TFC of the kept in dark for 30 minutes at a room temperature. extracts was expressed as mg quercetin equivalents The absorbance of the mixture was measured (QE) per gram of extract in dry weight (mg/g). spectrophotometrically at 517 nm against the blank solution consisting 2.5 mL MeOH and 0.5 mL Calculation for TPC and TFC and statistical distilled water. analysis The radical scavenging activity was expressed as the The total phenolic content and flavonoid content was radical scavenging percentage using the following calculated using the following formulas: equation: cV C = ……….... (1) (Ac - As ) m DPPH % scavenging activity =   ×100  Ac  where C= total contents of compounds in mg/g, in Where, A =absorbance of control and methanol, A = mg GAE/ g or total flavonoid content mg QE/ g dry c s absorbance of sample solution and DPPH radical. extract, c= concentration of gallic acid established from the calibration curve in mg/ml or concentration IC50 value is the concentration of sample required to of quercetin obtain from calibration curve, mg/ml, scavenge 50% of DPPH free radical and was V= the volume of extract in ml, m= the weight of calculated from the plotted graph of radical plant extract in g. scavenging activity against concentration of extracts. The antioxidant activity was determined by DPPH Calculation of linear correlation coefficient R2 and assay and the free radical scavenging activity (IC ) correlation analysis were carried out using Microsoft 50 value was calculated. The highest the phenolic Office Excel 2007. The linear regression equation content the lowest the IC value. is given as, 50 y = mx + C… (2) Results and Discussion y = absorbance of extract, m= slope of the calibration curve, x= concentration of the extract, C=intercept The research work was carried out on the medicinal plant which shows the potent phytochemical Determination of antioxidant activity using constituent’s summarized in table 1. DPPH free radical method Calibration curve for Total phenolic content DPPH radical scavenging activity of extracts was (TPC) and Total flavonoid content (TFC) carried out according to Brands et al. Method (Brand- Williams, 2012). The total phenolic content in plant extract was determined by using Folin-Ciocalteu colourimetric DPPH solution (0.1 mM) in MeOH was prepared method. The absorbance values obtained at different by dissolving 3.9 mg of DPPH in 100 mL methanol concentrations of gallic acid was used for the and stirred overnight at 4°C. Thus prepared purple construction of calibration curve. Absorbance values colored DPPH free radical solution was stored at - for gallic acid measured at 760 nm using Folin- 20°C for further use. Ciocalteu reagent. Similarly the total flavonoid Three different concentrations (5, 10 and 15 µg/mL) contents were determined by a colorimetric assay of methanolic solutions of each extracts were using aluminum chloride. Absorbance values for prepared by the serial dilution of the stock solution quercetin measured at 430 nm using aluminum of the respective extract. To each 0.5 ml extract chloride coulometric assay were shown in table 2 solution, 2.5 mL, 0.1 mM methanolic DPPH solution and calibration curve in figure 1 and figure 2. was added. A control was prepared by mixing 0.5

80 TableTable 1: Preliminary 1: Preliminary phytochemical phytochemical screening screening of Camellia of Camellia sinensis sinensis (tea leaves) (tea leaves) Note: Note:Result Result + means + means presence presence – means – means absence absence of phytochemicals of phytochemicals PetroleumPetroleum MeOH MeOH 50% ethanol50% ethanol S.N. S.N. ExperimentExperiment Test Test H O extractH O extract EtherEther Extract Extract extract extract extractextract 2 2 1. 1. Volatile Volatile oils oils Spot Te/Residue Spot Te/Residue test test ------Table2. 1: Preliminary2. Alkaloids Alkaloids phytochemical Mayers screening Mayers Regent of Camellia Regent Test Testsinensis (tea leaves)+ + + + + + + + Note:3. Result3.Flavonoid + meansFlavonoid presence – means Shinoda absence Shinoda test of tphytochemicalsest + + + + + + + + Petroleum MeOH 50% ethanol S.N.4. 4.Steroids ExperimentSteroids Steroid Steroid t Testest test - - + + H- O extract- + + 5. 5.Terpenoids Terpenoids Terpenoids Terpenoids test test Ether Extract - extract- -extract - - 2 - + + 1.6. Volatile6.Tannins Tannins oils Spot 0.1% Te/Residue F 0.1%eCl FtestTesteCl Test - + -+ + - + + - + + + 2. Alkaloids Mayers Regent Test + + + + 20187. 7.Reducing Reducing sugar sugarFehlings Fehlings Test Journal Test of Plant Resources+ + + + Vol.16,+ +No. 1 + + 3. Flavonoid Shinoda test + + + + 4.8. Steroids8.Glycosides Glycosides Steroid Salkowski’s test Salkowski’s test test - - +- + - + + + + - - Table5.9. 1: PreliminaryTerpenoids9.Phenols Phenols phytochemical Terpenoids Phenolic screening Phenolic test test of Camellia test sinensis (tea- leaves) - - - + - + + + + + + Note:10.6. Result 10.TanninsSaponins + meansSaponins presence 0.1%– means Froth/ FeCl absence Froth/FTestoam of FToam estphytochemicals Test + - +- - + - - + - - - 11.7. 11.ReducingProtein Protein sugar Fehlings Ninhydrin T Ninhydrinest Test Test Petroleum+ + MeOH++ 50%+ ethanol+ + + + + + + S.N. Experiment Test H O extract 12.8. 12.GlycosidesCarbohydrate Carbohydrate Salkowski’s Molish Molish Ttestest Test ether extract- - extract+- +extract + + +2 - + + + 13.9.1. 13. VolatilePhenols Phytosterols Phytosterolsoils SpotPhenolic Liebermann Te/Residue test Liebermann test Burchard Burchard test test - - + -+ + + -+ + + -+ + - - 10.2. AlkaloidsSaponins MayersFroth/ F regentoam T esttest +- +- +- +- 11.3. FlavonoidProtein ShinodaNinhydrin test T est ++ ++ ++ ++ Calibration12.4. CalibrationSteroidsCarbohydrate curve curve for Total for SteroidMolish Totalphenolic tTestest phenolic content content (TPC) (TPC) and --Total and Totalflavonoid ++flavonoid content content-+ (TFC) (TFC) ++ The13.5. ThetotalTerpenoids Phytosterols totalphenolic phenolic content TerpenoidsLiebermann content in t plantestBurchard in plantextract test extract was wasdetermined- + determined - + by using by using - + Folin-Ciocalteu Folin-Ciocalteu+ - colourimetric colourimetric method.6. method.Tannins The absorbanceThe absorbance 0.1% values FeCl 3valuestest obtained obtained at different at different+ concentrations concentrations+ of+ gallic of gallic acid +acidwas usedwas usedfor thefor the constructionCalibration7. constructionReducing curve of sforugarcalibration ofTotal calibration Fehlingsphenolic curve. test content curve. Absorbance (TPC) Absorbance and Totalvalues flavonoid+values for gallic forcontent+ gallic acid (TFC) acidmeasured + measured at 760 at+ nm760 usingnm using Folin- Folin- 8. Glycosides Salkowski’s test - + + - CiocalteuThe totalCiocalteu phenolicreagent reagent .Similarlycontent .Similarly in plantthe total theextract totalflavonoid was flavonoid determined contents contents bywere using weredetermined Folin-Ciocalteu determined by a bycolorimetric colourimetric a colorimetric assay assay using using method.9. PhenolsThe absorbance Phenolicvalues tobtainedest at different concentrations- + of gallic acid+ was used +for the aluminumconstruction10. aluminumSaponins chloride.of calibration chloride. Absorbance Froth/ curve. Absorbance Foam Absorbance t estvalues values for values quercetinfor for quercetin -gallic measured acid measured measured- at 430 atat -760 nm430 nmusingnm using using aluminum- Folin- aluminum chloride chloride 11. Protein Ninhydrin test + + + + coulometricCiocalteucoulometric reagent assay .Similarly assay were wereshown the showntotal in Tableflavonoid in Table 2 and contents 2 calibration and calibrationwere determined curve curve in Figure by in a Figurecolorimetric 1 and 1 Figure and assay Figure 2. using 2. Table12. Table 2: AbsorbanceCarbohydrate 2: Absorbance value Molishvaluefor gallic for test gallicacid and acid quercetin and quercetin measured measured- for calibration for+ calibration curve curve+ + aluminum13. Phytosterols chloride. Absorbance Liebermann values Burchard for testquercetin measured + at 430 + nm using + aluminum chloride - GallicGallic acid usedacid asused standard as standard for calibration for calibration of of coulometric assay were shown in Table 2 and calibration curveQuercetin Quercetinin Figure is used 1 is and asused standard Figure as standard 2. for calibration for calibration of flavonoid of flavonoid TableCalibration 2: AbsorbanceAbsorbance curve for valuevalue Total forforphenols phenolicgallicgallicphenols acid content and quercetin (TPC) measured and Total for flavonoid calibrationcalibration content curvecurve (TFC) TheConcentration GallictotalConcentration acidphenolic used as content standard in for plant calibration extract of was determined by using Folin-Ciocalteu colourimetric AbsorbanceAbsorbance QuercetinConcentration is Concentrationused as standard (µg/mL) ( µforg/mL) calibration of Absorbance flavonoidAbsorbance method.(µ g/mL)The(µ absorbanceg/mL) phenols values obtained at different concentrations of gallic acid was used for the Concentration for gallicfor gallicacid measured acid measured valuesvalues for quercetin for quercetin measured measured construction of calibration curve.Absorbance Absorbance valuesConcentration for gallic ( µacidg/mL) measured at 760Absorbance nm using Folin- (µg/mL) Ciocalteu10 reagent10 .Similarlyfor gallic the acid total 0.11measured flavonoid 0.11 contents were determined10 10 by values a colorimetric for quercetin assay measured 0.04 using 0.04 25 25 0.25 0.25 25 25 0.11 0.11 aluminum10 chloride. Absorbance0.11 values for quercetin measured10 at 430 nm using aluminum 0.04 chloride coulometric2550 assay50 were shown in0.25 Table 0.49 2 and0.49 calibration curve25 in Figure50 501 and Figure 2. 0.11 0.23 0.23 Table 2: Absorbance5075 75 value for gallic acid0.49 and0.74 quercetin 0.74 measured for calibration50 75 curve 75 0.23 0.30 0.30 Gallic75100 acid used100 as standard for0.74 calibration 1.01 1.01 of 75 100 100 0.30 0.39 0.39 Quercetin is used as standard for calibration of flavonoid 100 phenols 1.01 100 0.39 Concentration Absorbance Concentration (µg/mL) Absorbance (µg/mL) 8 8 for gallic acid measured 0.5 0.5 values for quercetin measured 8 0.5   10 0.11 0.410 0.4 0.04

0.4 e e e 25 e 0.25 25 0.11 c c e c c e 8 8 c n n c

n 8 n

50 0.49 n 50 0.23

a 0.3 a 0.3 n a a

a 0.3 b b a b b b

75 0.74 r 75r 0.30 b 8 8 y = 0.004xy = 0.004x r r 8 r y = 0.004x r o o o o o 7 Y 86U87 Y 86U8 0.2 0.2 s s o 7 Y 86U8 0.2 100 1.01 s 100 0.39 s s s R² = 0.98 R² = 0.98R² = 0.98 b b b b b b 88 8 Y8 Y8 Y8 A A A A A A 0.1 0.1 0.1 88 8 8 0.50 0 0    0 50 100 150      0.4 00 50 50 100 100 150 150 e

e          c

c 8 Concentration µg/mL n n

Concentration µg/mL a 0.3 ConcentrationConcentration µg/mL µg/mL a b

b ConcentrationConcentration µg/mL µg/mL

8 r y = 0.004x r o

o 7 Y 86U8 0.2 s

Figures 1: Calibration curve for authentic gallic acid Figure 2: Calibration curve for authenticR² = 0.98 quercetin b Figureb 1:8 Calibration curve for Y8 authentic gallic acid Figure 2: Calibration curve for authentic quercetin A FigureA Figure 1: Calibration 1: Calibration curve curvefor authentic for authentic gallic gallicacid 0.1acid Figure Figure 2: Calibration 2: Calibration curve curvefor authentic for authentic quercetin quercetin Calculation8 of total phenolic and total flavonoid contents in extracts Calculation of total phenolic and total flavonoid 0 in (mg/g). The concentrations of flavonoid in each CalculationThe concentrationCalculation of total ofof totalphenolicphenolic phenolic inand extract totaland totalflavonoidwas calculatedflavonoid contents fromcontents inthe extracts calibrationin extracts curve by regression Thecontentsequation Theconcentration inY= concentrationextracts 0.01x+0.001, of phenolic Rof2=0.999. phenolic inThe extract TFCin extract was was calculatedtest0 wascalculated samples calculated using 50 were fromthe formula 100calculated fromthe calibrationC=cV/mthe 150 from calibration theand calibrationcurve expressed curve by regressionby regression      2 The concentration of phenolic2 in 2extract was curve by regression equation Y=0.004x, R = 0.99. equationequation Y= 0.01x+0.001, Y= 0.01x+0.001, R =0.999. R =0.999. The TFCThe4 TFCwas calculatedwasConcentration calculated using µg/mL using the formula the formula C=cV/m C=cV/m and expressed and expressed calculated from Concentrationthe calibration µg/mL curve by regression TFC of the extracts were calculated using the 2 formula, C=cV/m and expressed as mg quercetin equationFigure 1: CalibrationY= 0.01x+0.001, curve for authenticR =0.999. gallic The acid TFC Figure was 2: Calibration4 4curve for authentic quercetin calculated using the formula C=cV/m and expressed equivalents (QE) per gram extract in (mg/g).The asCalculation mg gallic acidof total equivalents phenolic (GAE) and total per gflavonoid of extract contentsTPC in& extractsTFC was calculated given in table 3. The concentration of phenolic in extract was calculated from the calibration curve by regression 81 equation Y= 0.01x+0.001, R2=0.999. The TFC was calculated using the formula C=cV/m and expressed 4 as mg gallic acid equivalents (GAE) per g of extract in (mg/g). The concentrations of flavonoid in each test samples were calculated from the calibration curve by regression equation Y=0.004x, R2= 0.99. TFC of the extracts were calculated using the formula, C=cV/m and expressed as mg quercetin equivalents (QE) per gram extract in (mg/g).The TPC & TFC was calculated given in Table 3. asTable mg 3 :gallic Total acidphenolic equivalents content (TPC) (GAE) and Total per gflavonoid of extract content in (mg/g).(TFC) in differentThe concentrations extract of tea leaves of flavonoid in each test samples were calculated from the calibration curve by regression equation Y=0.004x, R2= 0.99. TFC TPC and TFC content (Mean) MeOH extract 50% EtOH extract H O extract of the extracts were calculated using the formula, C=cV/m and expressed as mg quercetin2 equivalents (QE)2018mg GAE/ per gramg (Mean extract TPC inS.D) (mg/g).The TPC Journal &321.25 TFC of wasPlant2.1651 calculated Resources given103.75 in Table2.1651 3. 53.44 Vol.16,2.0492 No. 1 QE mg/ g (Mean TFC S.D) 126.88 3.2476 67.50 2.5000 17.19 4.1810 Table 3: Total phenolic content (TPC) and Total flavonoid content (TFC) in different extract of tea leaves

DPPHTPC assay and for TFC antioxidant content (mean) activities MeOH extract 50% EtOH extract H O extract The DPPH assay was carried out and absorbance values measured at wavelength 517 nm2 for different concentrationsmg GAE/ g (Mean and TPC theƏ controlS.D) are given in 321.25TableƏ 4.2.1651 The calculated103.75 percentageƏ2.1651 of inhibition53.44 showedƏ 2.0492 that extractQE mg/ antioxidant g (Mean TFC activity.Ə S.D) 126.88 Ə 3.2476 67.50 Ə2.5000 17.19 Ə4.1810

DPPHTable 4: assay Absorbance for antioxidant and a control activities measured at wavelength 517 nm in the DPPH assay, % inhibition and IC50 The DPPH assay was carried out and absorbance values% measured at wavelength 517 nm for different Plant extract Conc. µg/ml Absorbance IC TPC (mgGAE/g) TFC (mg QE/g) concentrations and the control are given in Table 4.inhibition The calculated 50 percentage of inhibition showed that extract antioxidant activity.5 0.70 24.73 25.15 321.25Ə 2.1651 126.88 Ə 3.2476 Methanolic 10 0.66 29.03 Table 4: Absorbance and a control15 measured at0.58 wavelength 51737.63 nm in the DPPH assay, % inhibition and IC 5 0.79 15.05 46.02 103.75 Ə 2.1651 67.5050 Ə 2.5000 % 50Plant % ethanolic extract Conc.10 µ g/ml Absorbance0.76 18.27 IC TPC (mgGAE/g) TFC (mg QE/g) 15 0.71 inhibition23.66 50 5 0.700.91 24.732.14 265.157.83 321.2553.44Ə 2.04922.1651 126.8817.19 Ə 4.18103.2476 AqueousMethanolic Extract 10 0.660.86 279.03.54 15 0.580.84 397.63.68 Control 5 0.790.93 15.05 4 6.02 103.75 2.1651 67.50 2.5000 50 % ethanolic 10 0.76 18.27 15 0.71 23.66 DPPHFolin-Ciocalteu assay for (FC) antioxidant method activitieswas applied for the determinationradical disorders. of total Thephenolic phytochemical using gallic screening, acid as a standard based on the transfer5 of electrons 0.91 in alkaline2.14 medium67.83 from53.44 the2.0492 phenolic 17.19compounds4.1810 to Aqueous Extract 10 0.86 7.54quantification of phenolic compounds and The DPPH assay was carried out and absorbance 4- phosphomolybedic/phosphotungstic 15 acid0.84 complexes to9.68antioxidant form blue coloredactivity complexes,in plant extracts (PMoW is 11influencedO40) that valuesareControl determined measured spectrophotometrically at wavelength 517 nm forat0.93 760nm. different by the chemical nature of the analyte, assay method, concentrationsThe DPPH assay and is the based control on the are capability given in table of an 4. antioxidantselection to of donate standards hydrogen and presenceradical which of interfering is stable Folin-CiocalteuThefree calculatedradical with percentage (FC) deep method violet of inhibitioncolor. was applied When showed anfor odd thatthe electrondeterminationsubstances. become ofMany paired total plant phenolicin the of presenceNepal using has gallic of not free acidyet radical giveas a standardextractscavenger antioxidant based of antioxidant on activity. the transferagent, DPPHof electrons radicals inget alkaline reducedattention medium to ascorresponding a source from of the antioxidant hydrazine,phenolic polyphenols compoundsDPPH-H form due to phosphomolybedic/phosphotungsticand the solution gets decolorized from acid its complexesinitial deep to violet form to blue light colored yellow complexes, color. (PMoW O )4-that Folin-Ciocalteu (FC) method was applied for the to lack of commercial application. The result11 40showed are determined spectrophotometrically at 760nm. determination of total phenolic using gallic acid as that this plant is potent antioxidant property and TheConclusions DPPH assay is based on the capability of an antioxidant to donate hydrogen radical which is stable aThis standard research based helps on to the analyze transfer the ofphenol electrons and flavonoid in maximum contents phenolic which havecontent potential in methanolic effect in extracts. human free radical with deep violet color. When an odd electron become paired in the presence of free radical health in prevention and treatment of free radical disorders.So they The could phytochemical be the richscreening, source quantification of natural scavengeralkaline medium of antioxidant from the agent, phenolic DPPH compounds radicals getto reduced to corresponding hydrazine, DPPH-H form of phenolic compounds and antioxidant activity in plantantioxidants. extracts is The influenced correlation by thebetween chemical IC valuenature and of andphosphomolybedic/phosphotungstic the solution gets decolorized from acid its complexes initial deep violet to light yellow color. 50 the analyte, assay method, selection of standards4- and phenolicpresence contentof interfering is: the substances.higher is phenolic Many plantcontent of to form blue colored complexes, (PMoW11O40) that ConclusionsareNepal determined has not spectrophotometricallyyet give attention as a at source 760 nm. of antioxidantthe lower polyphenolsis IC50 value. due to lack of commercial Thisapplication. research The helps result to analyze showed the that phenol this plantand flavonoid is potent contentsantioxidant which property have potential and maximum effect inphenolic human This research will certainly help to quantify the total healthThecontent DPPH in in prevention methanolic assay is andbased extracts treatment on theso theycapabilityof free could radical ofbe an thedisorders. rich source The phytochemicalof natural antioxidants. screening, The quantification correlation antioxidant to donate hydrogen radical which is phenolic and flavonoid content and antioxidant effect ofbetween phenolic IC 50compounds value and phenolicand antioxidant content activityis: the higher in plant is phenolicextracts is content influenced the lower by the is chemicalIC50 value. nature of of Nepalese Ilam leaf tea Camellia sinensis. The thestable analyte, free radical assay withmethod, deep selection violet color. of standards When an and presence of interfering substances. Many plant of This research will certainly help to quantify the totalPolyphenols phenolic and from flavonoid leaves used content as potent and biochemicalantioxidant Nepalodd electron has not become yet give paired attention in the presenceas a source of free of antioxidant polyphenols due to lack of commercial effect of Nepalese Ilam leaf tea Camellia sinensis. The Polyphenols from leaves used as potent application.radical scavenger The result of antioxidantshowed that agent, this plant DPPH is potentand showedantioxidant antioxidant property property and maximum so they couldphenolic be biochemical and showed antioxidant property so they could be rich source of natural antioxidants. The contentradicals inget methanolic reduced to extracts corresponding so they couldhydrazine, be the richrich source source ofof natural antioxidants.antioxidants. TheThe future correlation of tea industry in Nepal is very bright. Development of this betweenDPPH-H IC form50 value and andthe phenolicsolution contentgets decolorized is: the higher is phenolic content the lower is IC50 value. from its initial deep violet to light yellow color. 5 sector helps not only the farmers and the stakeholder, This research will certainly help to quantify the totalbut phenolic also helps and strengtheningflavonoid content the andwhole antioxidant national effect of Nepalese Ilam leaf tea Camellia sinensis. The Polyphenols from leaves used as potent Conclusions economy and health benefits. biochemical and showed antioxidant property so they could be rich source of natural antioxidants. The This research helps to analyze the phenol and Acknowledgements flavonoid contents which have potential effect in 5 We would like to thanks Mrs. Chetana Khanal for human health in prevention and treatment of free identification of the plants. The authors are grateful

82 2018 Journal of Plant Resources Vol.16, No. 1 and like to offer profound gratitude to Mr. Sanjeev African Journal of Biotechnology, 8(13), 3084- Kumar Rai, Director General and Mrs. Jyoti Joshi 3091. Bhatta, Deputy Directory General, Department of Harborne, J.B. (1969). Phytochemical method: A plant Resources. We offer sincere thanks to Mr. Devi guide to modern techniques of plant analysis. Prasad Bhandari, Chief of NPRL for his great Phytochem., 8, 419-423. motivation and encouragement and Mr. Rajendra Parmer, N., Rawat, M., & Kumar, J.V. (2012). Sharma, for his valuable suggestion. We are obliged Camellia sinensis (green tea): A review. Global to Ramesh K. Yadav, Bhawani Prasad Adhikari, Journal of Pharmacology, 6(2), 52-59. Parasmani Yadav and all the staff of NPRL as well as DPR for their co-operation and team work. Salluca, T.G., & Penarrieta, J.M. (2008). Determination of total phenolic content & antioxidant capacity of Andean tubers and roots. References Revista Boliviana de quimica, 58-61. Acharya, P. (2013). Isolation of catechin from Sharma, G.N., Dubey, S.K., Sati, N. &, Sanadya, J. catechu: Estimation of total flavonoid content in (2011). Phytochemical screening and estimation Camellia sinensis and Camellia assamica Kuntze of total phenolic content in Aegle marmelos seeds. collected from different geographical region and International Journal of Pharmaceutical and their antioxidant activities. (M.Sc.Thesis), Clinical Research, 3(2), 27-29. Central Department of Chemistry, Tribhuvan University, Kathmandu, Nepal. Shrestha, A.R. (2002). Biological and phytochemical Studies on root and rzhizome of Rheum emodi of Adedapo, A.A., Jimoh, F.O., Afolayan, A.J., & Nepalese origin. (M.Sc.Thesis), Central Masika, J. (2009). Antioxidant properties of the Department of Chemistry, Tribhuvan University methanol extracts of the leaves and stems of Celtis Kathmandu, Nepal. africana. Rec. Nat. Prod., 3(1), 23-31. Siddique, N.A., & Mohd, M. (2010). Antioxidant, DPR, (2007). Medicinal Plants of Nepal. total phenolic content, total flavonoid content of Kathmandu, Nepal: Government of Nepal, selected plant. African Journal of Plant Science, Ministry of Forest and Soil Conservation, 4(1), 1-5. Department of plant Resources. Singleton, V.L., & Rossi J.A. (1965). Colorimetry Bhandari, M.R., Kasai, T., & Kawabata, J. (2003). of total phenolics with phosphomolybdic- Nutritional evaluation of wild yam (Dioscorea phosphotungstic acid reagents. Am. J. Enol. Vitic., spp.) tubers of Nepal. Food Chemistry, 82(4), 16, 144-158. 619-623. Sofowora, A.O. (1993). Medicinal plants and Brand-Williams W., Cuvelier, M.E., & Berset, C. traditional medicine in Africa: , II edition. (pp. (2012). Use of free radical method to determine 320). UK: University of Ife Press. the antioxidant activity. LBT-Food Science and Tarqq, M., Naveed, A., & Barkat, Ali, K. (2010). Technology, 6(28), 4481-4488. The morphology, characteristics and medicinal Ghimire, B.K., Seong, E.S., & Kim, E.H. (2011). properties of camellia sinensis tea. J. Med. Plants Comparative evaluation of the antioxidant Res. 4 (19), 2028-33. activity of some medicinal plants used in Nepal. Tipoe, G.L., Lenung, T.M., Hung, M.W., & Fung, J. Med. Plant Res., 5(10), 1884-1891. M.L. (2007). Green tea polyphenols as an Ghimeray, A.K., Jin, C.W., & Ghimire, B.K. (2009). antioxidant and anti-inflammatory agent for Antioxidant activity and quantitative estimation cardiovascular protection. Drug targets, 7(2), of Azardirachta indica using methanolic extract. 135-144.

83 2018J. Pl. Res. Vol. 16, No. 1, pp 84-89, 2018 Journal of Plant Resources Vol.16, No. 1

Quantitative Determination of Antioxidant Potential of Five Selected Essential Oils of Nepalese Origin

Devi Prasad Bhandari*, Rajeswar Ranjitkar and Laxman Bhandari Natural Products Research Laboratory, Thapathali, Kathmandu, Nepal. *E-mail: dpbhandari_chem @yahoo.com

Abstract

Antioxidants are the substances which inhibit oxidation, which have the ability to remove the potentially damaging oxidizing agents in a living organism. Preliminary antioxidant activities of 21 common essential oils were analyzed. Among them five positive essential oils were selected for further quantitative analysis. In this study, antioxidant activity was performed by DPPH (1, 1- diphenyl-2-picryl hydrazyl) method for selected essential oils which is compared with reference standard ascorbic acid. Ocimum sanctum, Cinnamomum tamala, Acorus calamus, Curcuma aromatica and Aegle marmelous oil exhibited strong antioxidant DPPH (1,1-diphenyl-2-

pycrylhydrazyl) radical scavenging activity with half maximal inhibitory concentration (IC50) value 119.07, 131.61, 312.64 , 433.34 and 534.51 µg/mL respectively. The results obtained indicate that these essential oils have antioxidant activity that can be useful as natural antioxidant in medicine, food industry and cosmetics for scavenging free radicals.

Keywords: DPPH Method, Essential oil, Free radical, IC50 value, Scavenging activity

Introduction to deterioration producing toxic products like peroxides which alters taste, aroma, flavors, Since ancient times, essential oils are recognized for nutritional quality and safety in foods (David & their medicinal value and they are very interesting Choe, 2003). Existence of antioxidants in oils is and powerful natural plant products. They continue technically a simplest way of reducing oxidation and to be of paramount importance until the present day. delay the oxidation of other molecules by inhibiting Essential oils have been used as perfumes, flavors, the initiation of oxidizing chain reactions by free for foods and beverages or to heal both body and radicals (Li et al., 2009). The reactive oxygen species mind for thousands of years (Wei & Shibamoto, 2010). Besides that the utilization of essential oil is play an important role related to the degenerative or very extensive and covers a wide range of human pathological processes of various serious diseases activity and some of the important uses are as; such as aging (Burns et al., 2001), cancer, coronary ingredients in the manufacture of soaps, cosmetics, heart disease, alzheimer’s disease (Smith et al., 1996; perfumery, healthcare herbal products, confectionary, Diaz et al., 1997), neurodegenerative disorders, aerated water, syrups, disinfectants, insecticides, atherosclerosis, cataracts and inflammation fungicides etc. (Margaris et al., 1982; Moss et al., (Aruoma, 1998). 2003). Essential oil compounds are fat soluble thus Basil essential oil (Ocimum basilicum) has been used they have the ability to permeate the membranes of traditionally as a natural therapeutic agent such as the skin before being captured by the micro- asthma, headache, and cough. Several studies circulation and drained into the systemic circulation, demonstrated, this oil exhibited various biological which reaches all target organs (Adorjan & activities such as antioxidant, anticonvulsant along Buchbauer, 2010; Baser & Buchbauer, 2010). with hypnotic activities, antiplasmodial activity, Antioxidant, a molecule which neutralizes harmful antiviral and anti-inflammatory (Bayala et al., 2014; free radical compounds that damage living cells, Jayasinghe et al., 2003; Ismail et al., 2006; Akono added to foodstuffs and oils routinely to prevent the et al., 2014; Kubiça et al., 2014). Sweet flag (Acorus damage caused by free radical. Rancidity in oil leads calamus) rhizomes has been used in ayurvedic

84 2018 Journal of Plant Resources Vol.16, No. 1 practice for curing several diseases like fever, asthma Louis, MO, USA) was used. Solvents and all other and bronchitis and as a sedative. Native tribes used reagents were of analytical grade. it to treat a cough, made a decoction as a carminative and as an infusion for cholic (Venskutonis et al., Rapid screening of antioxidant by dot-blot and 2003). Tejpat (Cinnamomum tamala) has been used DPPH staining for flavoring food and antioxidant as well as in Antioxidant capacity of the essential oils was eye- medicine for antidiabetic, antidiarrhoeal, detected by a rapid DPPH staining TLC method. antihyperlipidemic, antioxygenic, anti-inflammatory, Each essential oil was applied as a dot on a TLC acaricidal, hepatoprotective, gastroprotective, layer that was then stained with DPPH solution. This antibacterial and immunomodulatory activities ( Kar method was typically based on the inhibition of the et al. 2003, Chakraborty, 2010; Rao et al., 2008; accumulation of oxidized products. The generation Dhulasavant et al., 2010; Semwal et al., 1999; of free radicals was inhibited by the addition of Gambhire et al., 2009 ). Bel (Aegle marmelos) has antioxidants and scavenging of the free radicals been used traditionally for the treatment of various shifted the end point. The reduced ascorbic acid was diseases such as dysentery, fever, diabetes, asthma, used as a positive control. Initial faint spots appeared heart problems, ophthalmia, haemorrhoids and and 1 hour later, weak spots could be observed in urinary problems (Bansal & Bansal, 2011, Pino et sample row. White spots with strong intensity al., 2005, Raju et al., 1999). Yellow zedoary oil appeared were considered positive for further (Curcuma zedoaria) has been used as a stomachic, quantitative analysis (Chang et al., 2007). The results it has been recognized by its anti tumor (Kim et al., is shown in table 1. 2000), hepatoprotective (Matsuda et al., 2001), anti inflammatory (Jang et al., 2001) and analgesic effects DPPH (1, 1-diphenyl-2-pycrylhydrazyl) free (Navarro et al., 2002, Atiqur et al., 2014). radical scavenging assay The main aim of this research is to find out the This method is based on the reduction of a methanol antioxidant activity of selected essential oils of solution of 0.1 mM DPPH in the presence of a hydrogen-donating antioxidant due to the formation Nepalese origin. The quantitative IC50 value of Ocimum sanctum, Cinnamomum tamala, Acorus of the non-radical form DPPH-H (Soler et al., 2000). calamus, Curcuma aromatica and Aegle marmelous The samples were prepared by dissolving oil to oil has been studied. Such study in Nepal is yet to methanol and taken (100, 200 and 300 ìg/mL, 2 mL give more attention so it will certainly help to of a methanol added to DPPH free radical (Brand et quantify the antioxidant activity of selected essential al., 1995). The reaction mixture was shaken by mixer oil thereby helping its use in pharmaceutical, food and then kept in the dark for 30 minutes under industry and cosmetics. ambient conditions. The change in colour from purple to yellow was measured spectrophotometrically by using UV-Spectrophotometer. Materials and Methods DPPH* + AH DPPH + A* Plant Materials Essential oils were purchased from different suppliers in Katmandu, Nepal. These were confirmed with standard oils by CO- TLC method.

Chemicals and Reagents Ascorbic acid was purchased from Merck, Germany. DPPH (1,1-diphenyl-2-pycrylhydrazyl) manufactured by Sigma Chemical Co. Ltd. (St. 1: Diphenylpicrylhydrazyl (free radical)

85 2018 Journal of Plant Resources Vol.16, No. 1

and their variance was analyzed using one-way ANOVA procedure as represented in table 2.

Results and Discussion Preliminary antioxidant screenings of 21 common essential oils were analyzed by a rapid DPPH staining TLC method. Among them Cinnamomum tamala, Ocimum sanctum, Aegle marmelous, Acorus 2: Diphenylpicrylhydrazine (nonradical) calamus and Curcuma aromatica shown white spots with strong intensity, considered as positive for The percentage of inhibition of antioxidant capacity further quantitative analysis. was computed by measuring the absorbance at 517 nm using the following formula, The positive DPPH free radical scavenging suggests that the compounds are electron donors. The Inhibition (%) = [(A Control –A Sample) /A Control] × 100 scavenging activity of positive control; Vitamin C Where, A Control is the absorbance of the control increased with increasing concentrations and the and A Sample is the absorbance of the sample at maximum inhibition of DPPH radical was more than 95 % at 300 ìg/mL. Similarly, the scavenging 517 nm IC50 value denotes the concentration of sample required to scavenge 50% DPPH radicals. activity of Ocimum sanctum, Cinnamomum tamala, Antioxidant activity has been calculated by plotting Acorus calamus, Curcuma aromatic and Aegle percentage inhibition against different marmelous was found 92%, 83%, 43%, 33% and concentrations of oils. All observation has been 26% at 300 µg/mL respectively. Among the above carried out in triplicate was recorded as mean ± SD essential oil, Ocimum sanctum has highest IC50 value Table 1: Rapid preliminary screening of antioxidant by Dot-Blot and DPPH staining of essential oils Table 1: Rapid preliminary screening of antioxidant by Dot-Blot and DPPH staining of essential oils Preliminary S.N. Local name Latin name Parts used screening by DPPH assay 1 Taalishpatra (Gobre sallo) Abeis spectabilis (D.Don) Mirb. Leaves Negative 2 Bojho Acorus calamus L. Rhizomes Positive√ 3Bel Aegle marmelous (L.) Correa Leaves Positive√ 4 Chamomile Matricaria chamomilla L. Flower Negative 5 Sugandhakokilaa Cinnamomum glaucescens (Nees) Hand.-Mazz Fruits Negative 6 Tejpat Cinnamomum tamala (Buch.-Ham.) T.Nees & Eberm. Leaves Positive√ 8 Kachur Curcuma zedoaria Rosc. Rhizomes Negative 9 Citronella Cymbopogon citratus (DC.) Stapf Leaves Negative 10 Lemon grass Cymbopogon flexuosus (Nees ex Steud.) W.Watson Leaves Negative 11 Palmarosa Cymbopogon martini (Roxb.) Watson Leaves Negative 12 Masalaa Eucalyptus citriodora Hook. Leaves Negative 13 Dhupee Juniperus spp. Leaves Negative 14 Pudinaa Mentha arvensis L. Leaves Negative 15 Jataamasi Nardostachys grandiflora DC. Rhizomes Negative 16 Tulasi Ocimum sanctum L. Areal Parts Positive√ 17 Patchouli Pogostemon cablin (Blanco) Benth. Leaves Negative 18 Apricot Prunus spp. Seeds Negative 19 Sunpaatee Rhododendron anthopogon D.Don Leaves Negative 20 Sugandhawaal Valeriana jatamansi Jones Rhizomes Negative 21 Timur Zanthoxylum armatum DC. Fruits Negative

86The positive DPPH free radical scavenging suggests that the compounds are electron donors. The scavenging activity of positive control; Vitamin C increased with increasing concentrations and the maximum inhibition of DPPH radical was more than 95 % at 300 µg/mL. Similarly, the scavenging activity of Ocimum sanctum, Cinnamomum tamala, Acorus calamus, Curcuma aromatic and Aegle marmelous, was found 92%, 83%, 43%, 33% and 26% at 300 µg/mL respectively. Among the above essential oil, Ocimum sanctum has highest IC50 value (119.07 µg/mL) and Aegle marmelous has lowest IC50 value (534.51 µg/mL). The result shows that, the scavenging activity of Ocimum sanctum is somewhat equivalent with Vitamin C at same concentration.

Table 2: DPPH scavenging activity of five selected essential oils

Concentration Antioxidant activity (%) of Ascorbic Ocimum Cinnamomum Acorus Curcuma Aegle essential oils acid sanctum tamala calamus aromatic (Yellow marmelous (Bel (µg/mL) (Vitamin C) (Tulasi oil) (Tejpat oil) (Bojho oil) zedoary oil oil) 100 85.72±1.16 63.52±1.32 59.43±1.23 28.08±1.12 15.12±1.16 4.74±1.21 200 91.4±1.19 80.32±1.33 76.61±1.24 37.38±1.21 25.11±1.18 15.89±1.32 300 95.54±1.21 92.64±1.12 83.72±1.22 43.54±1.12 33.72±1.14 26.36±1.21 IC (µg/mL) 86.57±1.23 119.07±1.22 131.61±1.34 312.64±1.14 433.34±1.19 534.51±1.23 Values represent means ± SD, n=3

4 Table 1: Rapid preliminary screening of antioxidant by Dot-Blot and DPPH staining of essential oils

Preliminary S.N. Local name Latin name Parts used screening by DPPH assay 1 Taalishpatra (Gobre sallo) Abeis spectabilis (D.Don) Mirb. Leaves Negative 2 Bojho Acorus calamus L. Rhizomes Positive 3 Bel Aegle marmelous (L.) Correa Leaves Positive 4 Chamomile Matricaria chamomilla L. Flower Negative 5 Sugandhakokilaa Cinnamomum glaucescens (Nees) Hand.-Mazz Fruits Negative 6 Tejpat Cinnamomum tamala (Buch.- Leaves Positive 8 Kachur Curcuma zedoaria Rosc. Rhizomes Negative 9 Citronella Cymbopogon citratus (DC.) Stapf Leaves Negative 10 Lemon grass Cymbopogon flexuosus (Nees ex Steud.) W.Watson Leaves Negative 11 Palmarosa Cymbopogon martini (Roxb.) Watson Leaves Negative 12 Masalaa Eucalyptus citriodora Hook. Leaves Negative 13 Dhupee Juniperus spp. Leaves Negative 14 Pudinaa Mentha arvensis L. Leaves Negative 15 Jataamasi Nardostachys grandiflora DC. Rhizomes Negative 16 Tulasi Ocimum sanctum L. Areal Parts Positive 17 Patchouli Pogostemon cablin (Blanco) Benth. Leaves Negative 18 Apricot Prunus Spp. Seeds Negative 19 Sunpaatee Rhododendron anthopogon D.Don Leaves Negative 20 Sugandhawaal Valeriana jatamansi Jones Rhizomes Negative 21 Timur Zanthoxylum armatum DC. Fruits Negative

The positive DPPH free radical scavenging suggests that the compounds are electron donors. The scavenging activity of positive control; Vitamin C increased with increasing concentrations and the maximum inhibition of DPPH radical was more than 95 % at 300 µg/mL. Similarly, the scavenging activity of Ocimum sanctum, Cinnamomum tamala, Acorus calamus, Curcuma aromatic and Aegle marmelous, was found 92%, 83%, 43%, 33% and 26% at 300 µg/mL respectively. Among the above essential oil, Ocimum sanctum has highest IC50 value (119.07 µg/mL) and Aegle marmelous has lowest IC50 value (534.51 µg/mL). The result shows that, the scavenging activity of Ocimum sanctum is somewhat equivalent with Vitamin C at same concentration. 2018 Journal of Plant Resources Vol.16, No. 1 Table 2: DPPH scavenging activity of five selected essential oils

Table 2: DPPH scavenging activity of five selected essential oils Concentration Antioxidant activity (%) of Ascorbic Ocimum Cinnamomum Acorus Curcuma Aegle essential oils acid sanctum tamala calamus aromatic (Yellow marmelous (Bel (µg/mL) (Vitamin C) (Tulasi oil) (Tejpat oil) (Bojho oil) zedoary oil oil) 100 85.72±1.16 63.52±1.32 59.43±1.23 28.08±1.12 15.12±1.16 4.74±1.21 200 91.4±1.19 80.32±1.33 76.61±1.24 37.38±1.21 25.11±1.18 15.89±1.32 300 95.54±1.21 92.64±1.12 83.72±1.22 43.54±1.12 33.72±1.14 26.36±1.21

IC50 (µg/mL) 86.57±1.23 119.07±1.22 131.61±1.34 312.64±1.14 433.34±1.19 534.51±1.23 Values represent means ± SD, n=3

(119.07 µg/mL) and Aegle marmelous has lowest Acknowledgements ). IC50 value (534.51 µg/mL The result shows that, the scavenging activity of Ocimum sanctum is The authors are thankful to Director General of somewhat equivalent with Vitamin C at same Department of Plant Resources Mr. Sanjeev Kumar concentration. Rai and Deputy Director General of Department of Plant Resources Ms. Jyoti Joshi Bhatta for  4 facilitating laboratory to conduct this research HQ`G1H H1R  successfully. We would also give thanks to Mr. H1I%I:JH %I Rajendra sharma, Mr. Paras Mani Yadav, Mr. Ram

n  o

i ^ %C:1_1C t Dev Mandal, Mr. Madan Raj Bhatta, Mr. Bhabani i b i  1JJ:IQI%I :I:C: h Adhikari and Mrs. Chetana Khanal for their n

I ^ V=]: _1C  % cooperation. HQ`%H:C:I%  ^Q=.Q_Q1C %`H%I::`QI: 1H:^  References :J.:CVRQ_Q1C  V$CVI:`IVCQ%^VC_ Adorjan, B., & Buchbauer, G. (2010). Biological Q1C QJHVJ `: 1QJ ^j$LIC_ properties of essential oils: An updated review. Flavour Fragr. J., 25, 407-426. Figure 1: DPPH scavenging activity of five essential oils Figure 1: DPPH scavenging activity of five essential oils Akono Ntonga, P., Baldovini, N., Mouray, E., Mambu, L., Belong, P., & Grellier, P. (2014). Conclusion Activity of Ocimum basilicum, Ocimum canum, and Cymbopogon citratus essential oils against The essential oil contain antioxidants of natural Plasmodium falciparum and mature-stage larvae origin that counter the deleterious free radicals which of Anopheles funestus s.s. Parasite, 21(33). abstract H+ from hydrogen donors and accept electron from electron rich species causing oxidative Aruoma, O.I. (1998). Free radicals, oxidative stress stress in aerobic mechanism. Although all the five and antioxidants in human health and disease. J. essential oil exhibit antioxidant property, Ocimum Am. Oil Chem. Soc., 75, 199-212. sanctum and Cinnamomum tamala possesses remarkable antioxidant property which is similar to Atiqur, R., Monira, A., Rafiquel, I., Kazi D., Amzad that of standard ascorbic acid. So, these can prove Hossain, M., & Minkyun, N. (2014). In-vitro highly beneficial as natural antioxidant in antioxidant potential of the essential oil and leaf pharmaceutical, food industry and cosmetics for extracts of Curcuma zedoaria Rosc. Journal of scavenging free radicals. Further studies are Applied Pharmaceutical Science, 4(02), 107-111. required to identify specific active constituents of Bansal, Y., & Bansal, G. (2011). Analytical methods these essential oils for the significant antioxidant for standardization of Aegle marmelos: A review. effect. J. Pharm. Educ. Res., 2, 37–44.

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Baser, K.H.C., & Buchbauer, G. (2010). Handbook (2009). Anti-inflammatory activity of aqueous of essential oils: Science, technology and extract of Cinnamomum tamala leaves by in-vivo Applications. London, New York: CRC Press. and in-vitro methods. Journal of Pharmacy Research, 2(9), 1521-1524. Bayala, B., Bassole, I.H., Gnoula, C., Nebie, R., Yonli, A., Morel, L., Figueredo, G., Nikiema, J.B., Jang, M.K., Sohn, D.H., & Ryu, J.H. (2001). A & Lobaccaro, J.M. (2014). Antioxidant, anti- curcuminoid and sesquiterpenes as inhibitors of Inflammatory and anti-proliferative activities of macrophage TNF-alpha release from Curcuma essential oils of plants from Burkina Faso. J. zedoaria. Planta Med., 67, 550-552. Chemical composition, 9(3), 92122. Jayasinghe, C., Gotoh, N., Aoki, T., & Wada, S. Brand William, W., Curvelier, M.E., & Berset, C. (2003). Phenolics composition and antioxidant (1995). Use of a free radical method to evaluate activity of sweet basil (Ocimum basilicum L.). J. antioxidant activity. LWT-Food Science and Agric. Food Chem., 51, 4442–9. Technology, 28(1), 25-30. Ismail, M. (2006). Central properties and chemical Burns, J., Gardner, P.T., Matthews, D., Duthie, G.G., composition of Ocimum basilicum L. essential Lean, M.E., & Crozier, A. (2001). Extraction of oil. Pharm. Biol., 44, 619–26. phenolics and changes in antioxidant activity of red wines during vinification. J. Agric. Food Kar, A., Choudhary, B.K., & Bandyopadhyay, N.G. Chem., 49, 5797-5808. (2003). Comparative evaluation of hypoglycaemic activity of some Indian medicinal Chakraborty, U., & Das, H. (2010). Antidiabetic and plants in alloxan diabetic rats. J. antioxidant activities of Cinnamomum tamala Ethnopharmacol., 84, 105- 108. leaf extracts in STZ-treated diabetic rats. Global Journal of Biotechnology & Biochemistry, 5(1), Kim, K.I., Kim, J.W., Hong, B.S., Shin, D.H., Cho, 12-18. Y., Kim, H.K., & Yang, H.C. (2000). Antitumor, genotoxicity and anticlastogenic activities of David Min, B., & Choe, E. (2003). Effects of singlet polysaccharide from Curcuma Zedoaria. J. Mol. oxygen oxidation on the flavor of foods and Cell, 10, 392-398. stability of vitamins. Journal of Food Science and Biotechnology, 38(1), 582-586. Kubiça, T.F., Alves, S.H., Weiblen, R., & Lovato, L.T., (2014). In-vitro inhibition of the bovine viral Dhulasavant, V., Shinde, S., Pawar, M., & Naikwade, diarrhoea virus by the essential oil of Ocimum N.S., (2010). Antihyperlipidemic activity of basilicum (basil) and monoterpenes. Braz. J. Cinnamomum tamala Nees, on high cholesterol Microbiol., 45(1), 209-14. diet induced hyperlipidemia. International Journal of Pharm. Tech., 2(4), 2517-2521. Li, X.Q., Ji, C., Sun, Y.Y., Yang, M.L., & Chu, X.G. (2009). Analysis of synthetic antioxidants and Diaz, M.N., Frei, B., Vita, J.A., & Keaney, J.F. preservatives in edible vegetable oil by HPLC/ (1997). Antioxidants and atherosclerotic heart TOF-MS. Food Chemistry, 113(2), 692-700. disease. N. Engl. J. Med., 337, 408-416. Margaris, N., Koedam, A., & Vokou, D. (1982). Chang, H.Y., Ho, Y.L., Sheu, M.J., Lin, Y.H., Tseng, Aromatic plants: Basic and applied aspects. The M.C., Wu, S.H., Huang, G.J., & Chang, Y.S. Hague, London, Boston: Martinus Nijhoff (2007). Antioxidant and free radical scavenging Publishers. activities of Phellinus merrillii extracts. Botanical Studies, 48, 407-417. Matsuda, H., Morikawa, T., Niinomiya, K., & Yoshikawa, M. (2001). Hepatoprotective Gambhire, M.N., Juvekar, A.R., & Wankhede, S.S. constituents from Zedoariae rhizoma: Absolute

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stereostructures of three new carabrane type Semwal, A.D., Sharma, G.K., & Arya, S.S. (1999). sesquiterpenes, curcumenolactones A, B And C. Pro-or antioxygenic activity of tejpat Bio.-Organ. Med. Chem., 9, 909-916. (Cinnamomum tamala) and red chilli (Capsicum annum) in sunflower oil. Journal of the Science Moss, M., Cook, J., Wesnes, K., & Duckett, P. of Food and Agriculture, 79(12), 1733-1736. (2003). Aromas of rosemary and lavender essential oils differentially affect cognition and Smith, M.A., Perry, G., Richey, P.L., Sayre, L.M., mood in healthy adults. International Journal of Anderson,V.E., & Beal, M.F. (1996). Oxidative Neuroscience, 113, 15-38. damage in alzheimer’s. Nature, 382, 120-121. Navarro, D.D., Souza, M.M., Neto, R.A., Golin, V., Soler-Rivas, C., Espýn, J.C., & Wichers, H.J. (2000). Niero, R.,Yunes, R.A., Delle Monache , F., & An easy and fast test to compare total free radical Cechinel, V. (2002). Phytochemical analysis and scavenger capacity of foodstuffs. Phytochemistry analgesic properties of Curcuma zedoaria grown Analysis, 11(5), 1-9. in Brazil. Phytomed., 9, 427-432. Venskutonis, R., & Dagilyte, A. (2003). Composition Raju, P.M., Agarwal, S.S., Ali, M., Velasco- of essential oil of sweet flag (Acorus calamus L.) Negueruela, A., & Pérez-Alonso, M.J. (1999). leaves at different growing phases. Journal of Chemical composition of the leaf oil of Aegle Essential Oil Research, 5, 10-19. marmelos (L.) Correa. J. Essen. Oil Res., 11, 311- 313. Wei, A., & Shibamoto, T. (2010). Antioxidant/ lipoxygenase inhibitory activities and chemical Rao, C.V., Vijayakumar, M., Sairam, K., & Kumar, compositions of selected essential oil. J. Agric. V. (2008). Antidiarrhoeal activity of the Food Chem., 58, 7218-7225. standardised extract of Cinnamomum tamala in experimental rats. Journal of Natural Medicines, 62(4), 396-402.

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Phytochemical and Physiochemical Properties of Sapindus mukorossi Gaertn. (Soapnut) of Far Western of Nepal

Parasmani Yadav*1, Jyoti Joshi2, Devi Prasad Bhandari1 and Sangita Swar2 1Natural Products Research Laboratory, Kathmandu, Nepal 2Department of Plant Resources, Kathmandu, Nepal E-mail: [email protected]

Abstract

The objective of this work was to investigate powder microscopy, physicochemical parameters like Loss on drying, Extractive values, Total ash values, Acid insoluble ash value, Water soluble ash value, pH value, Fluorescence analysis activity and Phytochemicals present in the methanolic extracts of the pericarp of the fruits of Sapindus mukorossi (Ritha).

Key words: Phytochemical screening, powder microscopy,

Introduction Sapindus mukorossi Gaertn. (l/¶f - ‘Rittha’ in Nepali) Soapnuts are versatile washing products that are is commonly known by several names such as natural, eco-friendly and bio degradable and organic soapnut, soapberry, washnut, reetha and aritha. It is generally used by ethnic group. Soapnuts contain a tree widely grown in the Mid hill region, ‘saponin’, which have the ability to clean and wash. Shiwalik and sub Himalayan tracts at altitudes from When in contact with water, it creates mild suds, 200 m to 1500 m (Bhattarai & Ghimire, 2006). The which was similar to soap. Soapnuts can be used for S. mukorossi is a fairly large, deciduous tree with a cleaning basically washing clothes, as a liquid soap, straight trunk up to 12 m in height, sometimes cleaning and shining ornaments, household cleaner attaining a height of 20 m and a girth of 1.8 m with etc. (Dutta, 2007). a globosely crown and rather fine leathery foliage. Bark is dark to pale yellow, fairly smooth with many Soapnuts are also highly-effective and gentle. It will vertical lines of lenticels and fine fissures exfoliating leave the laundry fresh and clean and compared to in irregular wood scales. The blaze is 0.8-1.3 cm, other detergents, its mildness will keep colors bright, hard, not fibrous, pale orange brown, brittle and maintaining fabric structure of clothes for longer granular. Leaves are 30-50 cm long, alternate, par periods. It can be used on all fabrics and at all pinnate; common very narrowly bordered, temperatures. Soapnuts are allergy free and hence glabrous; leaflets 5-10 pairs, opposite or alternate, are good for skin especially for babies, eczema and 5-18 by 2.5-5 cm, lanceolate, acuminate, entire, sensitive skin. This chemical free product is excellent glabrous, often slightly falcate or oblique. for washing children’s clothing. It is one of the is compound, terminal panicle, 30 cm prioritized medicinal plant for economic or more in length with pubescent branches. Flowers development of Nepal (Baral & Kurmi et al., 2006). are about 5 mm across, small, terminal, polygamous, It has also antibiotic (Bauer et al., 1996), antifungal green wash white, sub-sessile, numerous, mostly (Charpinella, 2007) and antibacterial (Devkota, bisexual, sepals 5, each with a woolly scale on either 2000) properties. Soapnuts are good both side above the claw. Fruits are globosely, fleshy, 1- ecologically and economically when compared to seeded drupe, sometimes 2 drupels together, about other forms of detergents. Soapnuts are found in 1.8-2.5 cm across. Seeds are 0.8-1.3 cm in diameter, Nepal, India and some other countries in south east globose, smooth, black and loosely placed in dry (Bhattarai & Ghimire, 2006). fruit (Bhattarai & Ghimire, 2006).

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Materials and Methods without delay. Acid-insoluble ash content was calculated as mg/g of air-dried material. Collection of plant material Determination of extractive values: Considering the The fruits of Soapnut (ritha) were collected from diversity and chemical nature of drug, different Gokuleshor municipality of Baitadi district and solvents viz. water, ethanol, ethyl acetate, diethyl identification was done in Natural Products Research ether and petroleum ether were used for Laboratory, Thapathali, Kathmandu, Nepal. determination of extractive values. About 5 g of Shade drying of the plant samples powdered leaf material was subjected for cold maceration extraction with 100 mL of above All the collected fruit samples of S. mukorossi were solvents. Determination of extractive values of a cleaned, chopped and shade dried in the blotting paper crude drug was beneficial in its evaluation process at room temperature. The drying place was maintained wherever evaluation of chemical components in dark in order to prevent the degradation of bioactive drugs was not possible by any other means. After components of by interfering through light. extraction, the extracts were concentrated in rota- vaporizer and dried in vacuum desicator. Then the Grinding of the plant samples extractive values were calculated as percentage w/ After drying, pericarp of S. mukorossi was ground w of solvent soluble extractive with reference to the into fine powder. air dried drug.

Physicochemical studies (Quality control Determination of water-soluble ash: 25 mL of water methods for herbal materials) was added to the crucible containing the total ash, covered with a watch-glass and boiled gently for 5 Determination of ash values: For determining ash minutes. Insoluble matter was collected on an ash content of drug, about 3 g powder was spread in a less filter paper. Washed with hot water and ignited pre-ignited and weighed silica crucible. Then the in a crucible for 15 minutes at a temperature not crucible was incinerated gradually to make the exceeding 450°C in a muffle furnace. Allowed the crucible free from carbon. After cooling, crucible residue to cool in a suitable desiccator for 30 minutes was weighed to get the total ash content and then and then weighed without delay. The weight of the the ash was subjected for determining the acid residue was subtracted in mg from the weight of total insoluble and water soluble ash. The percentage of ash. Water soluble ash content was calculated as total ash was calculated by taking the air dried mg/g of air-dried material. sample as standard. Determination of loss on drying: For this 5 g of drug Determination of acid-insoluble ash: 25 mL of (powdered leaf material) was taken and kept in an hydrochloric acid (~70g/L) TS was added to the oven at 105oC till a constant weight was obtained. crucible containing the total ash, covered with a Loss on drying in the sample was calculated as watch-glass and boiled gently for 5 minutes. The reference to the air dried material. watch-glass was rinsed with 5 mL of hot water and added to the crucible. The insoluble matter was Determination of pH range: The pH of different collected on an ash less filter-paper (Whatmann 41) formulations in 1% w/v (1 g:100 mL) and 10% w/v and washed with hot water until the filtrate was (10 g:100 mL) of water soluble portions of whole neutral. The filter-paper containing the insoluble powder of drugs was determined using standard matter was transferred to the original crucible, simple glass electrode. ignited by gradually increasing the heat to 550°C for 3 hours in a muffle furnace (Nabertherm) to Preliminary phytochemical screening constant weight. The residue was allowed to cool in The preliminary phytochemical screening of the a suitable desiccator for 30 minutes, and then weigh methanol (hot) extracts of plant powder of drugs

91 2018 Journal of Plant Resources Vol.16, No. 1 were carried out using standard laboratory Dragendroff s test: Extracts were dissolved procedures. To detect the presence of different individually in dilute hydrochloric acid and filtered. secondary metabolites (phytochemical constituents) Filtrates were treated with Dragendroff s reagent. such as alkaloids, flavonoids, saponins, tannins, steroid glycosides, phenols, coumarins, reducing Detection of reducing sugar: Formation of Fehling’s sugars, protein, anthraquinones, quinines, fixed oils test- To a test tube 1 mL each a Fehling’s A and B and fats. solutions weremixed. To this, ~2 mL of plant extract was added and heated. Formation of red precipitate Determination of phenolic compounds: 2-3 drops indicates the presence of presence of reducing sugar/ of 1% ferric chloride (FeCl3) solution were added in carbohydrates. to 2 mL portions (1%) of each extract. Phenolic compounds produce a deep violet color with ferric Xanthoproteic test: The extracts were treated with ions. few drops of conc. Nitric acid. Formation of yellow color indicates the presence of proteins. Determination of tannins: Ferric chloride test- A small quantity of the extract was boiled with water Determination saponins: Foam Test- 0.5 g of extract and filtered. Two drops of ferric chloride was added was shaken with 2 mL of water. If foam produces to the filtrate, formation of a blue-black or green persists for ten minutes it indicates the presence of blackwash color in the presence of ferric chloride saponins. precipitate was taken as evidence for the presence Fluorescence analysis: 1 g of crude drug was taken of tannins. in watch glass and subjected for fluorescent analysis Determination of flavonoids: Shinoda test- The as such and after treatment with different reagents. extract was dissolved in methanol (50%, 1-2 mL) Determination of foaming index: Many herbal by heating. To an alcoholic solution of each of the materials contain saponins that can cause persistent extract, 3 pieces of magnesium chips were added foam when an aqueous decoction was shaken. The followed by a few drops of concentrated foaming ability of an aqueous decoction of herbal hydrochloric acid. Appearance of an orange, pink materials and their extracts was measured in terms or red to purple color indicates the presence of of a foaming index. flavonoids. Foaming index =1000/a Determination of steroid glycosides: Liebermann Burchard’s test- Extract was dissolved in equal Where a = the volume in ml of the decoction used volumes of anhydrous acetic acid and chloroform for preparing the dilution in the tube where foaming to a height of 1 cm was observed. (CHCl3) and cooled to 0°C. The mixture was transferred to a dry test tube and concentrated Powder microscopy sulfuric acid (H2SO4) was introduced to the bottom of the tube. Formation of a reddish brown or violet- Dried plant material was finely powdered in blender brown ring at the interface of the 2 liquids indicates and subjected in chemicals for analysis. At first, the presence of steroids. sufficient amount of powder was taken in chloral- hydrate solution on a slide, covered with cover slip Determination of alkaloids: Meyer’s Test- 1 ml and was left for 18 hours and finally observe under portions of each extract was acidified with 2-3 drops microscope. Starch test was also done with iodine of 1M hydrochloric acid and treated with 4-5 drops solution. of Mayer’s regent (Potassium mercuric iodide) Formation of a yellow or white colored precipitate or turbidity indicates the presence of alkaloids.

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Results The Preliminary phytochemical screening for various functional groups is tabulated as table 3. The organoleptic characters of the dried pericarp of Fluorescence analysis of powdered drugs is the fruits of S. mukorossi powder are tabulated as presented in table 4. Powder microscopy of pericarp table 1. The Average physicochemical parameters of the fruits of S. mukorossi is shown in figure 1. of the S. mukorossi powder are tabulated as table 2.

Table 1: Organoleptic evaluation parameters S. N. Organoleptic parameters Results 1. Color Brown 2. Odor Characteristic 3. Taste Bitter 4. Nature Crystalline 5. Texture Rough 6. Solubility Acid Soluble in water, ethanol and methanol

Table 2: Physicochemical study of powder of soapnut S. N. Parameters Results 1 Ash Value (%) Total ash 2.35 Acid insoluble ash 0.08 Water soluble ash 0.70 2 Successive extractive in Successive extractive values (%) Petroleum ether 0.0 Diethyl ether 1.1 Ethyl acetate 9.3 Methanol 59.8 Ethanol 42.6 Aqueous 77.8 3 Loss on drying 10.74 pH value (1% water solution) 4.77 @ 17oC 4 5 Foaming index 5000

Table 3: Preliminary screening of major phytochemicals in methanolic extract of the pericarp of soapnut S. N. Chemical constituents Tests / reagent Results ( extract) 1. Alkaloids Dragendorff’s + Wagners + Mayer’s + 2. Carbohydrate Molisch’s + Fehling’s + Benedict’s + 3. Flavonoids Mg ribbon and dilHCl + 4. Glycosides NaOH test + 5. Tannins/Phenols Ferric chloride + 6. Saponin Foam Test + 7. Fixed oil and Fat Filter Paper Test + 8. Steroids Libermann + 9. Terpenoids + 10. Protein/amino acid Xanthoproteic Test -

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Table 4: Fluorescence analysis of crude powder with different chemical reagents S. N. Pericarp of fruits of soapnut + chemical reagents Day light UV short UV long 1. Powder + Conc. HCl Brown Light green Black 2. Powder+ Dil.HCl Brown Green Black 3. Powder+ Iodine solution (2%) Yellow Green Black 4. Powder+ Glacial acetic acid Yellow Green Dark Brown

5. Powder+ Conc. HNO3 Yellow Black Black 6. Powder+ Dil. HNO3 Yellow Dark Green Grey 7. Powder+ Glacial acetic acid +HNO3 Yellow Light Green Green 8. Powder+ Dil. H2SO4 Yellow Green Black 9. Powder+ Conc. H2SO4 Yellow Green Black 10. Powder+ Fehling’s reagent Yellow Dark Brown Dark Brown 11. Powder+ Picric acid solution Yellow Green 12. Powder+ NaOH (10%) Yellow Green Black 13. Powder+ KOH (10%) Yellow Green Black 14. Powder+ Wagner’s reagent Yellow Green Brown

15. Powder + CuSO4 (5%) Yellow Green Black 16. Powder+ Lead acetate Yellow Bluish green Black 17. Powder+ Methanol Yellow Green Brown 18. Powder+ Ethanol Yellow Light green Purple

Microscopic analysis Microscopic analysis

AB C D E Figure 1: Powder microscopic analysis of the pericarp of fruits of Sapindus mukorossi: A. Fibers, B. Pitted vessel, C. Starch grains, D.A Rossete crystals, E.B Stone cell C D E

PhysicochemicalFigure 1: Powder microscopic parameters analysis like of the ash pericarp values, of fruits ofpresence Sapindus of mukorossi glycosides,; A. Fibers, tannins, B. Pitted flavonoids, vessel, C. saponins Starch moisture content and fluorescencegrains, analysis D. Rossete are crystals,and triterpinoidsE. Stone cell in more quantity where as proteins helpful in determining the physiological and non- in less quantity. Presence of such phytochemical physiologicalPhysicochemical ash, parameters possibility likeof microbialash values, growth moistureconstituents content and fluorescencemay be responsible analysis are for helpful various in ordetermining contamination the physiological and presence and of impuritiesnon-physiologicalpharmacological ash, possibility activities of microbialof this elite growth medicinal or respectively.contamination The and relative presence low of impuritiesacid insoluble respectively. ash plant. The relativeMedicinal low plantsacid insoluble are considered ash value as 0.08% living valueand a 0.08%high ratio and of a highwater ratio soluble of water ash content soluble 0.7% ash of factoriesSapindus as mukorossi they produce indicates various that phytochemicalsthe crude drug contains more physiological ash or contents (i.e. related to plant tissue) than the non- physiological content 0.7% of Sapindus mukorossi indicates that viz., alkaloids, flavonoids, glycosides, phenols, content. The relative moisture content indicates that the drugs give equal possibility for microbial the crude drug contains more physiological ash or saponins, sterols etc in the form of secondary growth and contamination. The preliminary phytochemical investigations in Sapindus mukorossi contents (i.e. related to plant tissue) than the non- metabolites. These serve as life saving drugs. Due methanolic extract indicated the presence of glycosides, tannins, flavonoids, saponins and triterpinoids physiologicalin more quantity content. where The as relativeproteins moisture in less quantity. content Presenceto this, of day such by phytochemical day demand constituentsfor crude drugsmay be is indicatesresponsible that for the various drugs pharmacologicalgive equal possibility activities for ofincreasing this elite andmedicinal also adulteration plant. Medicinal of crude plants drugs isare in microbialconsidered asgrowth living andfactories contamination. as they produce The variousmalpractice. phytochemicals Hence, standardization viz., alkaloids, of aflavonoids, crude drug preliminaryglycosides, phenols, phytochemical saponins, sterolsinvestigations etc in the formin ofis secondaryessential to metabolites. avoid and identifyThese serve adulteration. as lifesaving Sapindusdrugs. Due mukorossi to this, day methanolic by day demandextract indicated for crude the drugs is increasing and also adulteration of crude drugs is in malpractice. Hence, standardization of a crude drug is essential to avoid and identify adulteration. 94 2018 Journal of Plant Resources Vol.16, No. 1

Conclusion Baral, S.R., & Kurmi, P.P. (2006). A compendium of medicinal plants in Nepal. Maiju Bahal, In the present investigation, a set of studies of Chabahil, Kathmandu, Nepal: Mrs. Rachana organoleptic properties, physiochemical properties, Sharma. phytochemistry and florescence analysis were conducted on S. mukorossi as WHO guidelines and Bauer, A.W., Kirby, W.M., Sherris, M., & Turck, J.C. other standard methods. These studies revealed the (1996). Antibiotic suspecttibility testing by a presence of various important bioactive compounds standardized single disk method. American and proved that these plant drugs are also Journal of clinical pathology, 45, 493-496. medicinally important. These results may help in standardization, identification and in carrying out Charpinella, M.C., Herrero, G.G., Alonso, R.A., & further research S. mukorossi based drugs which are Palacios, S.M. (1999). Antifungal activity of used in ayurveda and modern pharmacopoeia. Melia azedarch fruit extract. Fitterapia, 70, 296- 298. With the availability of primary information, further studies can be carried out like phytopharmacology Devkota, K.P., Acharya, R., Baral, M.P., & Pokherel, of different extracts, standardization of the extracts, R.P. (2000). Antimicrobial activity of some herbal plants used in traditional medicine in Nepal. identification and isolation of active principles and rd pharmacological studies of isolated compounds. Proceedings, 3 National Conference on Science & Technology, RONAST, II: 1311-1317. Acknowledgements Devkota, K.P., & Dutta, I.C. (2001). Antibacterial activities of commercially traded herbs used in The authors would like to thank Mr. Sanjeev Kumar traditional medicines in communities of Doti Rai, Director General of Department of Plant district, Nepal. Report submitted to IUCN, Resources for giving his co-operation and support Kathmandu, Nepal. for managing the research work .The authors would also like to thank KAILASH-CDI PROGRAM for Dutta, I.C. (2007). Non timber forest products of financial support and Natural Products Research Nepal: Identification, classification, Laboratory for giving an opportunity for research ethnic uses & cultivation. (p. 98). Kathmandu, work in a very much friendly and sound environment Nepal: Hill Side Press. without which the research work was not possible.

References Bhattarai, K.R., & Ghimire, M.D. (2006). Commercially important medicinal and aromatic plants of Nepal and their distribution pattern and conservation measure along the elevation gradient of the Himalayas. Banko Jankari, 16, 3-13.

95 2018J. Pl. Res. Vol. 16, No. 1, pp 96-99, 2018 Journal of Plant Resources Vol.16, No. 1

Study of the Variation of Major Chemical Constituents of Essential Oil Extracted from Rhizome and Leaf of Acorus calamus L. (Bojho) from Nepal

Tara Datt Bhatt*, Parasmani Yadav, Amit Dhungana, Jyoti Joshi and Chintamani Basyal Department of Plant Resources, Thapathali, Kathmandu, Nepal *E-mail: [email protected]

Abstract

The objective of this work was to study the variation of major chemical compositions of essential oil obtained by hydro-distillation of stem and leaf of Acorus calamus found in Nepal by GC-MS analysis. The essential oil contained asarone (Z) and asarone (E) as a major constituents ranging from 40.59-76.33% and 1.29–10.48% respectively.

Keywords: Asarone(E), Asarone(Z), GC-MS, Leaf, Rhizome

Introduction In the present study, major constituents of essential oil of the stem and leaf of Acorus calamus from Acorus calamus L. (English: Sweet flag; Nepali: different parts of Nepal were analyzed by GC-MS. af]emf]) belongs to the family Araceae. It is a known The purpose of this study was, to generate data for herbal drug commonly used in traditional medicine the essential oil and to create directly comparable (Parki et al., 2017). It grows as herbs with perennial chemical components of the essential oil of Acorus tuberous thick rhizomes in wetlands, particularly calamus due to geographical variation. marshes (Keddy et al., 2010). It is herb with aromatic rootstock, leaves broad, ensiform with distinct midrib. Spathe is cylindrical and slightly curved. Materials and methods Flower is bisexual each with a perianth of six Collection of plant material orbicular concave segments, 6, ovary conical, fruits are oblong berries (Joshi & Joshi, Rhizome and leaf of Acorus calamus were collected 2001). Monoterpene hydrocarbons, sequestrine from four different places of Nepal in the same ketones, asarone (2, 4, 5-trimethoxy-1- season. Five samples were collected from Salyan propenylbenzene) and β-asarone are the main and Banke district, mid-western region of Nepal. constituents of the essential oil distillates from rhizome . It is used as an analgesic for the relief of Extraction of essential oils toothache and headache, to cure/prevent a hangover, A clevenger apparatus were used for the extraction to treat a cough, as a carminative for colic, to treat of essential oil from the rhizome of Acorus calamus diabetes, to reduce bowel pains, to lessen swelling through hydro-steam distillation. The rhizomes were and for constipation, to cure fevers, asthma, thoroughly washed and placed in distillation flask bronchitis and as sedative (Bajracharya, 1979; and subjected to hydro-steam distillation for about Davidson & Parish, 1989). It exhibited a good anti- 8 hours. The steam and vaporized oil were condensed fungal activity as well. The rhizome roots essential into liquid by a vertical condenser and collected in oil extracted distilled from these plant parts have measuring tube. Being immiscible and lighter than been reported to several biological activity including water, the volatile oil separated out as an upper layer. antifungal, antibacterial, allelophatgice, anticellcullar The oil was then separated from water and collected and immunosuppressive (Grosvenor & Suprino, in small glass bottles, dried with anhydrous sodium 1995). Essential oil of the species possesses anti- sulphate, sealed, labelled and stored in light resistant gonadal activity in (Devkota et al., 1999). vials at 4–6ºC for further use.

96 Extraction of essential oils A clevenger apparatus were used for the extraction of essential oil from the rhizome of Acorus calamus through hydro- steam distillation. The rhizomes were thoroughly washed and placed in distillation flask and subjected to hydro-steam distillation for about 8 hours. The steam and vaporized oil were condensed into liquid by a vertical condenser and collected in measuring tube. Being immiscible and lighter than water, the volatile oil separated out as an upper layer. The oil was then separated from water and collected in small glass bottles, dried with anhydrous sodium sulphate, sealed, labelled and stored in light resistant vials at 4–6º C for further use. 2018 Journal of Plant Resources Vol.16, No. 1

µL of the essential oil diluted with spectroscopic grade hexane (10:1) was injected into the GC inlet maintaining column flow rate of 0.68 mL/min and purge flow 3 mL/min in the split mode. The initial column oven temperature was set at 40°C and the Extraction of essential oils injection temperature was 250°C. A clevenger apparatus were used for the extraction of essential oil from the rhizome of Acorus calamus through hydro- steam distillation. The rhizomes were thoroughly washed and placed in The qualitative analysis of the essential oil was distillation flask and subjected to hydro-steam distillation for about 8 hours. The steam and further continued in a Shimadzu GCMS-QP2010 vaporized oil were condensed into liquid by a vertical condenser and collected in measuring Plus. During the analysis, the ion source temperature tube. Being immiscible and lighter than water, the volatile oil separated out as an upper layer. and the interface temperature was set at 250°C and The oil was then separated from water and collected in small glass bottles, dried with anhydrous 200°C respectively. The detector scanning start time sodium sulphate, sealed, labelled and stored in light resistant vials at 4–6º C for further use. was 4 minutes and end time was 68 minutes; scan speed was 666 with scanning range of m/z 40.00- FigureFigure 1: 1:leaf leaf of Acorusof Acorus calamus calamus Figure350.00. 2: Rhizome of Acorus calamus (x10,000) 1.00 208Identification of compounds 0.75 Identification of compounds was done by comparing Gas chromatography- mass spectrometry (GC-MS)193 analysis 0.50The chemical constituents in the essential oils werethe separatedMass spectral using data a Shimadzupresent in thegas library. Along 165 with the Mass data relative index of compound are 0.25chromatograph Mass Spectrophotometer137 (GCMS QP 2010 Plus) with Rtx-5MS column 133 (25mX0.25mmX0.2569 79 91 µ105m). 1 µL of the essential oil dilutedused forwith identification.The spectroscopic grade MS hexane library used in the 53 177 0.00(10:1) was injected into the GC inlet maintaining columnanalysis215 flow 231 rate process 248 of 0.68 263 was mL/min 279 NIST 295 and11 312 and purge 327FFNSC 343 1.3. flow50.0 3 mL/min 75.0 in the 100.0 split 125.0mode. The 150.0 initial 175.0 column 200.0 oven temperature 225.0 250.0 was 275.0set at 40 300.00Cand 325.0the 350.0 Figureinjection 8: Mass temperature fragmentation was of 250 asarone0C. Z Relative quantification of components

The qualitative analysis of the essential oil was further continuedThe relative in a Shimadzupercentage GCMS-QP2010 of each constituents present Plus. During the analysis, the ion source temperature andin essentialthe interface oil hastemperature calculated was as set its atarea percentage 2500C and 2000C respectively. The detector scanning startpresent time was in the4 minutes chromatogram and end time of essential was oil. 68 minutes; scan speed was 666 with scanning range of m/z 40.00-350.00 (x10,000) 1.00 208Results and Discussion Figure 1: leaf of Acorus calamus 0.75 Figure 2:2: RhizomeRhizome of of Acorus Acorus calamus calamus 193 Altogether, five samples of the Acorus calamus plant 0.50 Gas Chromatography- Mass Spectrometry165 (GC- 137 including rhizome and leaf under study were 0.25 MS) analysis 91 133 69 79 105 collected from four different places of Nepal348 in 2074 53 177 Gas chromatography- mass spectrometry (GC-MS)0.00 analysis BS. The226 232 essential254264 oils281 were295 analyzed 312 by 341 GCMS and The chemical50.0 constituents 75.0 100.0 in 125.0the essential 150.0 oils 175.0 were 200.0 225.0 250.0 275.0 300.0 325.0 The chemical constituents in the essential separatedoils were using separated a Shimadzu using gasa Shimadzu Chromatograph gas it was found that chemical constituents varies from Figure 9: Mass fragmentation of asarone E 2 chromatograph Mass Spectrophotometer (GCMSMass SpectrophotometerQP 2010 Plus) with (GCMS Rtx-5MS QP 2010column Plus) 7 to 35 compounds in which some compounds were (25mX0.25mmX0.25µm). 1 µL of the essentialwith oil Rtx-5MS diluted columnwith spectroscopic (25mX0.25mmX0.25 grade hexaneµm). 1 unidentified. The major compounds found were (10:1) was injected into the GC inlet maintaining column flow rate of 0.68 mL/min and purge flow 3 mL/min in the split mode. The initial column oven temperature was set at 400Cand the Table 1: Major compounds found in the essential oil of Acorus calamus collected from four different places of 0 Table 1: Major compounds found in the essential oil of Acorus calamus collected from four different places of Nepal injection temperature was 250 C. Nepal analyzed by GCMS. analyzed by GCMS.

The qualitative analysis of the essential oil was further continued in a Shimadzu GCMS-QP2010Parts used in extraction of Essential Major constituents in Essential oil in % Places Plus. During the analysis, the ion source temperature and the interface temperature was set at oil Asarone (Z) Asarone (E) 2500C and 2000C respectively. The detector scanningDhakeri start Botanical time was Garden, 4 minutes Banke and end time was Rhizome 40.59 1.29 68 minutes; scan speed was 666 with scanning rangeMulpani of m/z Botanical 40.00-350.00 Garden, Salyan Rhizome 76.33 10.41 Mulpani (wild), Salyan Rhizome 73.92 10.48 Damarkhola, Salyan Rhizome 68.72 8.90 Luham, Salyan Leaf 72.21 6.95

97

2

5 c I are The MS library used in the analysis process was NIST 11 and FFNSC 1.3. qc

Results and Discussion Altogether, five samples of the Acorus calamus plant including rhizome and leaf under study were collected from four different places of Nepal in 2074 . The essential oils were analyzed by GCMS and it was found that chemical constituents varies from 7 to 35 compounds in which some compounds were unidentified. The major compounds found were asarone Z and asarone E in each essential oil. The chromatograms of the essential oil and mass fragmentation pattern of the major compounds asarone Z and asarone E are given below.

c 5.0(x1,000,000)

I TIC (1.00) 6 are The MS library 4.0 used in the analysis process was NIST 11 and FFNSC 1.3. qc 3.0

2.0

Results and Discussion 1.0 7 4 5

Altogether, five samples of the Acorus calamus plant including rhizome and leaf under study 2 1 3 were collected from four different places of Nepal in 2074 . The essential oils were analyzed 10 20 30 40 50 by GCMS and it was found that chemical constituents varies from 7 to 35 compounds in which some compounds2018 were unidentified. The major compounds found were Journal asaroneFigure Zof and 3: PlantGCMS asarone chromatogramResources E of essential oil of rhizome of Acorus calamus collected Vol.16, from No. Mulpani 1 botanical in each essential oil. The chromatograms of the essential oil and mass fragmentation pattern of garden, Salyan the major compounds asarone Z and asarone E are given below. asarone (Z) and asarone (E) in each essential oil are tabulated in table 1. The chromatograms of the essential (x1,000,000)oil and mass fragmentation pattern of the major compounds asarone (Z) and asarone (E) are given below. TIC (1.00) 1 7.5 1 (x1,000,000) 5.0(x1,000,000)

TIC (1.00) 7

TIC (1.00) 6 5.0 (x1,000,000)

TIC (1.00) 1 7.5 1 5.0 4.0 4.0

3.0 3.0 5.0

2.5 4 2.0 1 2.0 8 3 5 9 2.5 1.0 4 1 7 1.0 6 5 4 0 2 1 7 3 9 7 1 1 1 6 1 1 1 2 8 3 5 6 5 3 4 1 4 2 8 5 2 10 201 30 403 50 6 5 4 0 2 1 7 3 9 7 1 1 1 6 1 1 1 10 20 30 40 50 2 10 20 30 40 50 10 20 30 40 50 Figure 5: GCMS chromatogram of essential oil of leaf of Acorus calamus collected from Luham, Salyan Figure 3: GCMS chromatogram of essential oil of rhizomeFigure 4: of GCMS chromatogramFigure 4: GCMS of essential chromatogram oil of rhizome of Acorus of essential calamus collected oil of fromrhizome Mulpani of (wild), Salyan Figure 3:GCMS chromatogram of essential oil of rhizome of Acorus calamus collected from Mulpani botanical Figure 5: GCMS chromatogram of essential oil of leaf of Acorus calamus collected from Luham, Salyan Acorus calamus collectedgarden, from Salyan Mulpani botanical garden, Acorus calamus collected from Mulpani (wild), Salyan Salyan

(x1,000,000) (x1,000,000)

4 3

TIC (1.00) 1 (x1,000,000) 1 7.5 TIC (1.00) 1

TIC (1.00) 4 1 3.0(x1,000,000) TIC (1.00) 3.0 5.0 7 5.0 2.0 4.0 2.0

3.0 2.5 4 1.0 1.0 1 8 3 6 6 5 1 1 2 2 1 2.0 1 6 0 0 3 5 4 3 0 1 7 1 2 3 1 9 1 7 1 7 5 1 1 4 1 7 6 7 5 1 1 1 2 1 4 1 7 1 2 1 1 1 3 5 8 9 2 1 1 6 1 3 5 8 9 1 6 10 20 30 4010 5020 30 40 50 1.0 10 20 30 40 9 50 5 6 3 4 1 2 Figure 6: GCMS chromatogramFigure 6: ofGCMS essential chromatogram oilFigure of rhizome 6: GCMS of chromatogram Acorusof essential calamus of essentialoil collected of rhizomeoil8 offrom rhizome Damarkhola, Figureof of Acorus 5:Figure GCMS Salyancalamus chromatogram 5:collected GCMS from of essential Damarkhola,chromatogram oil of leafSalyan of Acorus of calamusessential collected oil from of Luham,leaf ofSalyan Acorus calamus10 collected 20 from Damarkhola, 30 Salyan 40 50 Acorus calamus collected from Luham, Salyan Figure 4: GCMS chromatogram of essential oil of rhizome of Acorus calamus collected from Mulpani (wild), Salyan (x1,000,000) 4

(x1,000,000) TIC (1.00) 1

TIC (1.00) 2 (x1,000,000) 7.5 3.0 1 2

TIC (1.00) 1 7.5 3 2.0 5.0 5.0 1.0 6 1 2.5 1 3 2 1 0 3 2 1 3 1 7 5 4 7 3 4 1 1 3 2 1 1 3 5 8 9 1 6 2 0 8 6 2 2 6 4 4 8 1 1 1 1 3 2 2 1 2 5 9

2.5 7 1 2 9 3 5 4 2 3 7 0

0 10 20 30 40 50 1 3 9 5 6 1 5 1 1 2 3 1 2 3 1 7 8 2 2 3 1 2 3 3 4

(x10,000) 3 0 2 8

1.00 6 2 2 4 6 8 4 1

10 201 30 40 50

1 208 2 2 3 2 1 5 9

7 Figure 6: GCMS chromatogram of essential oil of rhizome of Acorus calamus collected from Damarkhola, Salyan 1 2 9 5 7 0 2 4 3 0 1 3 9 6 5 1 5 1 1 2 2 1 3 3 1 7 2 8 2 3 1 10Figure0.75 7: GCMS 20Figure chromatogram 7: GCMS 30chromatogram of essential of essential 40 oil oil of ofrhizome rhizome 50 of Acorus of calamus Acorus collected calamus from Dhakeri, collected Banke from Dhakeri, Banke 193 (x10,000) 0.50 1.00 Figure 7: GCMS chromatogram of essential oil of rhizome of Acorus calamus collected165 from Dhakeri,208 Banke (x1,000,000) 2

137 TIC (1.00) 1 0.25 0.75 7.5 69 91 105 133 79 177 53 193 215 231 248 263 279 295 312 327 343 0.00 0.50 50.0 75.0 100.0 125.0 150.0165 175.0 200.05.0 225.0 250.0 275.0 300.0 325.0 350.0 0.25 137 Figure 8: Mass fragmentation of asarone133 Z 69 79 91 105 53 177 4 2152.5 231 248 263 279 295 312 327 343 1

0.00 3 2 3 3 50.0 75.0 100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 300.04 325.0 350.0 3 0 2 8 6 2 2 4 6 8 4 1 1 1 2 2 3 2 1 5 9 7 1 2 9 5 7 2 0 4 3 0 1 3 9 6 5 1 5 1 1 2 2 1 3 3 1 7 2 8 2 3 FigureFigure 8: Mass 8: Mass fragmentation fragmentation of asarone of Z asarone Z 1 4 10 20 30 40 50 (x10,000) 1.00 Figure 7: GCMS208 chromatogram of essential oil of rhizome of Acorus calamus collected from Dhakeri, Banke

0.75

(x10,000) 193 0.50 1.00 165 208 137 0.25 0.75 91 133 69 79 105 348 53 177 193 0.50 226 232 254264281 295 312 341 0.00 165 50.0 75.0 100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 300.0 325.0 137 0.25 91 133 4 FigureFigure 9: Mass 9: fragmentationMass69 79 fragmentation1 05of asarone of E asarone E 348 53 177 0.00 226 232 254264281 295 312 341 50.0 75.0 100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 300.0 325.0 98 Figure 9: Mass fragmentation of asarone E Table 1: Major compounds found in the essential oil of Acorus calamus collected from four different places of Nepal analyzed by GCMS.

Table 1: Major compounds found in theParts essential used oil in ofextraction Acorus calamus of Essential collected fromMajor four constituents different places in Essential of oil in % Places Nepal analyzed by GCMS. oil Asarone (Z) Asarone (E) Dhakeri Botanical Garden, Banke Rhizome 40.59 1.29 Parts used in extraction of Essential Major constituents in Essential oil in % Mulpani BotanicalPlaces Garden, Salyan Rhizome 76.33 10.41 Mulpani (wild), Salyan Rhizomeoil Asarone 73.92 (Z) Asarone 10.48 (E) Damarkhola,Dhakeri Botanical Salyan Garden, Banke Rhizome Rhizome 40.59 68.72 1.29 8.90 Luham,Mulpani Salyan Botanical Garden, Salyan Rhizome Leaf 76.33 72.21 10.41 6.95 Mulpani (wild), Salyan Rhizome 73.92 10.48 Damarkhola, Salyan Rhizome 68.72 8.90 Luham, Salyan Leaf 72.21 6.95

5

5 2018 Journal of Plant Resources Vol.16, No. 1

90 References

80

70 Bajracharya, M.B., (1979). Ayurvedic medicinal

60 A Dhakeri Botanical Garden,Banke plants and general treatment. (p. 230) r 50 Mulpani Botanical Garden, Salyan e Kathmandu, Nepal: Piyusavarsi Ausadhalaya a 40 Mulpani (wild), Salyan % 30 Damarkhola,Salyan Mahaboudha. Luham,Salyan 20 10 Davidson, P.M., & Parish, M.E., (1989). Methods 0 for testing the efficacy of food antimicrobials. Asarone (Z) Asarone (E)

Figure 2: Bar diagram for the representation of major chemical constituents on the basis of area percentage of Food Technol., 43,148-155. GCMS chromatogram Figure 2: Bar diagram for the representation of major chemical Cconstituentsonclusion on the basis of area percentage of GCMS Devkota, K.P., Acharya, R., Baral, M.P., & Adhikari, By studying the GCMS analysis of essential oil of leaf and rhizome of Acormus calamus collectedchromatogram from different places of Nepal, essential oils are somewhat similar but the percentage R.P., (1999). Antimicrobial activities of some of main chemical constituent has varied from place to place. Nepalese calamus oil contained high amount of asarone (Z) 76.33% and asarone (E) 10.48% in leaf and rhizome. herbal plants used in traditional medicine in rd AcknowledgementsConclusion Nepal. In: Proceding of 3 National Conference The authors would like to thank Mr. Sanjeev Kumar Rai, Director General of Department of Plant Resources for giving his co-operation and support for managing the research work. The on Science and Technology. 2, 1311-1317. authorsBy studyingwould also like tothe thank GCMS Department ofanalysis Plant Resources of for essential giving an opportunity oil of for research work in a very much friendly and sound environment and for financial support without wleafhich the and research rhizome work was not of possible. Acormus calamus collected from Grosvenor, P.W., Suprino, A., & Gray, D.O., (1995). different places of Nepal, essential oils are somewhat Medicinal plants from Riau province, Sumatra, similar but the percentage of main chemical Indonesia: Part 2: Antibacterial and antifungal constituent has varied from place to place. Nepalese activity. J. Ethnopharmacol., 45(2), 97-111. calamus oil contained high amount of asarone (Z) Joshi, K.K, & Joshi, S.D., (2001). Genetic heritage 76.33% and asarone (E) 10.48% in leaf and rhizome. of medicinal and aromatic plants of Nepal Himalayas. (p. 239). Kathmandu, Nepal: Buddha Acknowledgements Academic Publishers and Distributors Pvt. Ltd. The authors would like to thank Mr. Sanjeev Kumar Keddy, P.A., (2010). Wetland Ecology: Principles Rai, Director General of Department of Plant nd 6 and Conservation. 2 edition. Cambridge, UK: Resources for giving his co-operation and support Cambridge University Press. for managing the research work. The authors would also like to thank Department of Plant Resources Parki, A., Chaubey, P., Om Prakash, Kumar, R., & for giving an opportunity for research work in a very Pant, A.K., (2017). Seasonal variation in essential much friendly and sound environment and for oil compositions and antioxidant properties of financial support without which the research work Acorus calamus L. Accessions. Medicines was not possible. (Basel)., 4(4). doi: 10.3390/medicines4040081.

99 2018J. Pl. Res. Vol. 16, No. 1, pp 100-105, 2018 Journal of Plant Resources Vol.16, No. 1

Variation in Chemical Composition of Essential Oil Extracted from the Fruits of Zanthoxylum armatum DC. (Timur) of Nepal

Tara Datt Bhatt*, Amit Dhungana, Jyoti Joshi, Parasmani Yadav and Chintamani Basyal, Department of Plant Resources, Thapathali, Kathmandu, Nepal E-mail: [email protected]

Abstract

The purpose of this study was to generate data and to create directly comparable major chemical components of the essential oil of timur due to geographical/topographical variation. Fruits of Zanthoxylum armatum were collected from different parts of Nepal. The extraction of essential oil was performed by hydro-distillation using Clevenger apparatus and then their chemical composition was identified by gas chromatography coupled with mass spectrometry (GC-MS). The results of chromatographic analysis have shown diverse chemical profiles of studied species. By GCMS analysis, 30 compounds were identified in which 6 compounds were taken as major compounds. The major compounds were (36.29-45.61%), beta- phellandrene (19.93- 38.38%), myrcene (4.13-8.73%), methyl cinnamate (3.12-22.14%) and sabinene (1.57-4.78%). These compounds are considered as interesting components that are needed in industrial and pharmaceutical goals.

Keywords: Beta-phellandrene, Clevenger, GC-MS, Linalool

Introduction In the present study, essential oil is extracted from the fruits of Zanthoxylum armatum collected from Zanthoxylum armatum DC. of the Rutaceace family different parts of Nepal and were analyzed by is an important medicinal plant of Nepal commonly Schimadzu GC-MS QP 2010 Plus. The purpose of known as Timur, Nepal pepper and toothache tree. this study was to generate data and to create directly It is a major indigenous spice of Nepal. The ripen comparable major chemical components of the fruit follicles are usually reddish in color and 4-5 essential oil of timur due to geographical/ mm in diameter. The fruits contain an aroma that is topographical variation. present in brown fruit wall (pericarp-shell). It may be able to develop numbing or anesthetic feeling on the tongue. Seeds are solitary, globose, shining and Materials and Methods have bitter taste (Bharati et al., 2015). Timur is not Collection of plant material only used as flavouring in cooking but also its seed oil and crushed seeds are added to cereal seeds and The fruits of Z. armatum were collected from legumes to protect them against damages caused by different places of Nepal. 3 samples were collected stored grain pests (Kala et al., 2005). It is also used from Dadeldhura district, 2 samples from Surkhet as larvicidal agent against mosquitoes (Tiwari et al., district and 2 samples were from Myagdi district. 2007). Timur is the main non-timber forest product of Nepal. This spice is mostly cultivated in the mid- Extraction of essential oil western region of Nepal. A Clevenger apparatus was used for the extraction The essential oil extracted from leaves, seeded leaves of essential oil from the fruits of Z. armatum through and pericarp of the Zanthoxylum armatum can be hydro distillation (Waheed et al., 2011). The fruits used as a commercial source of different chemical were thoroughly washed and placed in distillation compounds which are used as a valuable perfumery flask and subjected to hydro distillation for about 8 and flavoring ingredients in industries (Mohan et hours. The steam and vaporized oil were condensed al., 2012). into liquid by a vertical condenser and collected in

100 Gas chromatography- mass spectrometry (GC-MS) The chemical constituents in the essential oils were separated using a Shimadzu gas chromatograph Mass Spectrophotometer (GCMS QP 2010 Plus) with Rtx-5MS column (25mX0.25mmX0.25µm). 1 µL of the essential oil diluted with spectroscopic grade hexane (10:1) was injected into the GC inlet maintaining column flow rate of 0.68 mL/min and purge flow 3 mL/min in the split mode. The initial column oven temperature was set at 400C and the injection temperature was 2500C (Keshav et al., 2017).

The qualitative analysis of the essentialGas oil chromatography- was further mass continued spectrometry in (GC-MS)a Shimadzu GCMS-QP2010 Plus. During the analysis, the ion source temperatureThe chemical constituentsand the ininterface the essential temperature oils were separated was usingset at a Shimadzu2500C andgas chromatograph 2000C respectively. The detector scanningMass Spectrophotometerstart time was (GCMS 4 minutes QP 2010 and Plus) end with time Rtx-5MS was column 68 minutes; (25mX0.25mmX0.25 scan µm). 1 µL of the essential oil diluted with spectroscopic grade hexane (10:1) was injected into the GC inlet speed was 666 with scanning range ofmaintaining m/z 40.00-350.00. column flow rate Identificationof 0.68 mL/min and of purge compounds flow 3 mL/min was in thedone split by mode. The initial comparing the mass spectral data presentcolumn in the oven mass temperature spectral was setlibrary at 400C NIST and the 2011injection and temperature FFNSC was 1.3. 250 0C (Keshav et al., 2017).

2018 Journal of Plant Resources Vol.16, No. 1 The qualitative analysis of the essential oil was further continued in a Shimadzu GCMS-QP2010 Plus. During the analysis, the ion source temperature and the interface temperature was set at 2500C and measuring tube. Being immiscible and lighter than 2000C respectively. The detector scanning start time was 4 minutes and end time was 68 minutes; scan water, the volatile oil separated outspeed as anwas upper 666 layer.with scanning range of m/z 40.00-350.00. Identification of compounds was done by The oil was then separated fromcomparing water and the collected mass spectral data present in the mass spectral library NIST 2011 and FFNSC 1.3. in small glass bottles, dried with anhydrous sodium sulphate, sealed, labelled and stored in light resistant vials at 4–6ºC for further use. The oil percentage was found to be in the range 2.84 to 4.22 % (v/w).

Gas Chromatography- Mass Spectrometry (GC-MS) The chemical constituents in the essential oils were separated using a Shimadzu gas chromatograph Mass Spectrophotometer (GCMS QP 2010 Plus) with Rtx-5MS column (25mX0.25mmX0.25µm). 1 µL of the essential oil diluted with spectroscopic Figure 1: Collected fruits of timur grade hexane (10:1) was injected into the GC inlet Figure 2: 2: Plant Plant of timur of timur maintaining column flow rate of 0.68 mL/min and Figure 1: Collected fruits of timur purge flow 3 mL/min in the split mode. The initial Results and Discussion Figure 2: Plant of timur column oven temperature was set at 40°C and the Altogether seven samples of the plant under study Results and Discussioninjection temperature was 250°C (Keshav et al., were collected from four different places of Nepal 2017). Altogether seven samples of the plant underResults studyand Discussion were collectedin 2073 BS . fromThe oil fourpercentages different of Z. armatumplaces fruitsof Nepal in Altogether seven samples of the plant under study were collected from four different places of Nepal in The qualitative analysis of the essential oil was obtained during hydro-distillation are tabulated 2073 BS. The oil percentages of Z. armatum2073 BS .fruits The oil obtained percentages during of Z. armatum hydro-distillation fruits obtained during are hydro-distillation tabulated below are tabulated below further continued in a Shimadzu GCMS-QP2010 below in table 1. in Table 1. in Table 1. Plus. During the analysis, the ion source temperature Table 1: Oil % of timur fruit samples collected from four and the interface temperature was set at 250°C Tableand 1: Oildifferent % of Timur places fruit of Nepal. samples collected from four different places of Nepal. 200°CTable respectively. 1: Oil % Theof Timur detector fruit scanning samples start collected time from four different places of Nepal. S. N. Name of places Oil percentage (v/w) (%) was 4 minutes and end time was 68 minutes; scan Oil percentage (v/w) S. N. Name of places 1. Dadeldhura (Darkholi) 4.22 speed was 666 with scanning range of m/z 40.00- 2. Dadeldhura(%) (Chamsal) 3.97 350.00. Identification1. ofDadeldhura compounds (Darkholi) was done by 3. Dadeldhura 4.22 (Mada) 2.84 4. Myagdi (Toripani) 3.13 comparing the mass2. spectralDadeldhura data present (Chamsal) in the mass 3.97 spectral library NIST 2011 and FFNSC 1.3. 5. Myagdi (Neptachaur) 3.32 3. Dadeldhura (Mada) 6. Sukhet 2.84 (Birendranagar) 3.28 4. Myagdi (Toripani) 7. Surkhet 3.13 (Ratanagla) 2.27 5. Myagdi (Neptachaur) 3.32 6. Sukhet (Birendranagar) 3.28 7. Surkhet (Ratanagla) 2.27

Figure 1: Collected fruits of timur

101 2018 Journal of Plant Resources Vol.16, No. 1 1 0 (x10,000,000) (x10,000,000) 1 TIC (1.00) TIC (1.00) 1 1.75 1.50

188.10 (100.00) 6 7

1.50 1.25 4

1.25 4 1.00 9 5 1.00 1 0.75 0.75 0.50 3 0.50 3

0.25 2 0.25 2 8 1 5 7 1 1 6 8 3 0 2 4 1 1 6 1 1 1 1 5 0.00 9 10 20 30 40 10 20 30 40 Figure 3: GCMS chromatogram of essential oil of timur Figure 4: GCMS chromatogram of essential oil of timur collected from Dadeldhura^:`@.QC1_ collected from Dadeldhura (Chamsal)

(x10,000,000) 1.75 TIC (1.00) 8

(x10,000,000) 1.50 TIC (1.00) 9 1 1.50 1 1.25 1.25 1.00 1.00 0.75 0.75 4

0.50 0.50 2 0.25 4 0.25 1 0 1 9 2 6 1 5 3 1 3 5 3 7 1 1 1 1 1 7 1 8 1 4 7 6 2 10 20 30 40 9 1 1 5 Figure 5: GCMS chromatogram of essential3 oil of timur Figure 6: GCMS chromatogram of essential oil of timur collected from Dadeldhura (Mada) collected from Myagdi (Neptechaur) 2

(x10,000,000) 4 1.75 TIC (1.00)

9 (x10,000,000) TIC (1.00) 1.50 6 2 1.75 1

1.25

1.50 8 8 1.00 1 1.25 3 2 0.75 1.00 1 8

0.50 4 0.75 4 0.25 0.50 2 3 2 1 0 9 1 4 1 6 5 1 1 1 8 7 7 1 1 2 1 1 5 1 3 0.25

10 20 30 4 6 1 4 2 4 8 1 9 1 1 1 1 6 0 3 1 5 9 2 7 3 5 2 0 1 2 1 1 2 1 7 1 Figure 7: GCMS chromatogram of essential oil of timur 10 20 30 4 collected6 from Myagdi (Toripani) Figure 8: GCMS chromatogram of essential oil of timur  collected from Surkhet (Ratanagle)

102 1 2 1 1 3 1

2018 Journal of Plant Resources Vol.16, No. 1

(x10,000,000)

TIC (1.00) 1

(x10,000,000) 0 0

1.50TIC (1.00) 1 7 1.50 1.25 1.25 1.00 1.00 0 0.75 2 0.75

0.50 5 0.50 0.25 4 6 8 3 0.25 2 9 1 1 1 6 1 1 1 2 1 3 5 9 7 1 1 4 1 1 8 8 1 7 2 9 1 9 4 2 1 2 1 8 1 1 6 4 1 25.0 1

25.05 Figure 9: GCMS chromatogram of essential1 oil of timur collected 6 Figure 9: GCMS chromatogramfrom3 Surkhet of (Birendranagar)essential oil of timur collected from Surkhet (Birendranagar)

The abundance of each constituents present in essential oil analyzed by GCMS has calculated as its area TheThe abundance abundance of of each each constituents constituents present present5 inin essentialessential oiloil analyzedanalyzed by GCMS has calculatedcalculated asas itsits areaarea percentage present in the chromatogram and are listed in Table 2. percentagepercentage present present in in the the chromatogram chromatogram andand areare listedlisted in Table 2. Table 2: Chemical composition of essential oil of fruits of Z. armatum analyzed by GCMS TableTable 2: 2: Chemical Chemical composition composition of of essential essential oil oil of of fruits fruits of of Z. Z. armatum armatum analyzedanalyzed byby GCMSGCMS * A: Dadeldhura (Darkholi), B: Dadeldhura (Chamsal). C: Dadeldhura (Mada), D: Myagdi (Toripani), E: Myagdi * *A:A: Dadeldhura Dadeldhura (Darkholi), (Darkholi), B: Dadeldhura B: Dadeldhura (Chamsal). (Chamsal). C: Dadeldhura C: Dadeldhura (Mada), D:(Mada), Myagdi D: (Toripani), Myagdi E:(Toripani), Myagdi (Neptechaur),E: Myagdi (Neptechaur), F: Surkhet (Ratanagle), G: Surkhet (Birendranagar) (Neptechaur), F: Surkhet (Ratanagle), G: Surkhet (Birendranagar) 2 F: Surkhet (Ratanagle), G: Surkhet (Birendranagar) 0 SampleSample collected collected places* places* S.N.S.N. ChemicalChemical Constituents Constituents ABCDEFABCDEF G G 1. 1. 1,3,5,7-Cyclooctatetraene1,3,5,7-Cyclooctatetraene 0.56 0.56 0.51 0.51 2. 2. PinenePinene 0.310.31 0.390.39 0.460.46 0.5 0.5 0.54 0.54 0.4 0.4 3. 3. SabineneSabinene 1.57 1.57 3.123.12 3.463.46 1.7 1.7 4.78 4.78 3.65 3.65 2.24 2.24 4. 4. MyrceneMyrcene 4.13 4.13 6.106.10 6.186.18 4.884.88 8.73 8.73 4.86 4.86 6.32 6.32 5. 5. PinenePinene 0.35 0.35 0.250.25 0.51 0.51 0.36 0.36 0.26 0.26 6. 6. CareneCarene 0.310.31 7. 7. PhellandrenePhellandrene 0.54 0.54 8. 8. .gamma.-Terpinene.gamma.-Terpinene 7 0.4 0.4 9. 9. TerpineneTerpinene 0.33 0.33 0.58 0.58 0.29 0.29 10.10. CymeneCymene 0.5 0.5 0.22 0.22 0.35 0.35 11.11. PhellandrenePhellandrene 19.93 19.93 26.3926.39 25.0325.03 25.0925.09 38.3838.38 20.2120.21 26.9126.91 12.12. Ocimene Ocimene <(E)-, <(E)-, beta-> beta-> 0.76 0.76 13.13. TerpineneTerpinene 0.28 0.28 0.56 0.56 0.570.57 0.99 0.99 0.21 0.21 0.34 0.34 14.14. TerpinoleneTerpinolene 0.48 0.48 0.740.74 0.710.71 0.720.72 1.12 1.12 0.68 0.68 0.63 0.63 15.15. LinaloolLinalool 45.61 45.61 41.2941.29 36.2936.29 42.6542.65 39.0939.09 43.5143.51 39.6139.61 16.16. SabineneSabinene hydrate hydrate 2.142.14 1.43 1.43 1.471.47 1.76 1.76 17.17. Ocim-(4E,6Z)-eneOcim-(4E,6Z)-ene 0.330.33 0.330.33 0.530.53 0.61 0.61 0.81 0.81 18.18. TerpineolTerpineol 0.350.35 19.19. Non-(2Z)-enolNon-(2Z)-enol 0.45 0.45 0.3 0.3 0.32 0.32 0.36 0.36 20.20. Terpinen-4-olTerpinen-4-ol 0.72 0.72 0.600.60 0.990.99 1.581.58 0.81 0.81 1.67 1.67 1.08 1.08 21.21. CryptoneCryptone 0.42 0.42 0.26 0.26 0.84 0.84 0.38 0.38 1.08 1.08 22.22. TerpineolTerpineol 0.33 0.33 0.530.53 0.35 0.35 0.66 0.66 0.47 0.47 23.23. DecanalDecanal 0.27 0.27 24.24. PiperitonePiperitone 0.56 0.56 0.39 0.39 0.5 0.5 0.64 0.64 0.53 0.53 0.59 0.59 25.25. Undecan-2-oneUndecan-2-one 0.33 0.33 26.26. PhellandralPhellandral 0.41 0.41 27.27. Cinnamate Cinnamate (Z)-> 0.37 0.37 0.44 0.44 0.32 0.32 0.4 0.4 28.28. CinnamateCinnamate <(E)-,methyl-> <(E)-,methyl-> 22.14 22.14 20.9920.99 21.4821.48 16.2616.26 3.12 3.12 18.81 18.81 15.3215.32 29.29. CaryophylleneCaryophyllene <(E)-> <(E)-> 0.550.55 0.580.58 0.270.27 0.41 0.41 0.55 0.55 30. Caryophyllene oxide 0.38 30. Caryophyllene oxide 0.38 103 2018 Journal of Plant Resources Vol.16, No. 1

TableTable 3: Major 3: Major chemicals chemicals present present in the in essential the essential oils of oils Z. ofarmatum Z. armatum fruits fruits

Major constituents on the basis of area percentage of GC-MS chromatogram Places Linalool Beta-phellandrene Methyl- cinnamate Myrcene Sabinene Dadeldhura (Darkholi) 45.61 19.93 22.14 4.13 1.57 Dadeldhura (Chamsal) 41.29 26.39 20.99 6.10 3.12 Dadeldhura (Mada) 36.29 25.03 21.48 6.18 3.46 Myagdi (Toripani) 43.51 25.09 16.26 4.88 1.7 Myagdi (Neptachaur) 39.61 38.38 3.12 8.73 4.78 Sukhet (Ratanagle) 42.65 20.21 18.81 4.86 3.65 Surkhet (Birendranagar) 39.09 26.91 15.32 6.32 2.24

50 45 40 35 A Dadeldhura (Darkholi) 30 r Dadeldhura (Chamsal) e 25 Dadeldhura (Mada) a 20 15 Myagdi (Toripani) % 10 Myagdi (Neptachaur) 5 Surkhet (Ratanagle) 0 Surkhet (Birendranagar) Linalool Phellandrene Methyl Myrcene Sabinine cinnamate Major compounds

FigureFigure 10: 10: Bar Bar diagram diagram for for the the major major chemical chemical constituents constituents present present in in the the essential essential oil oil of of timur timur by by GCMS GCMS analysis analysis Conclusion ConclusionThe oil percentage in the fruits of Z. armatum collectedAcknowledgments from 7 places of Nepal were varied from 2.27- 4.22 % (v/w). By studying the chromatograme of essential oil of different places, they are somewhat Thesimilar oil butpercentage some compounds in the fruits were of presentZ. armatum only in oneThe authorsor two wouldplaces likeof essentialto thank Mr. oil. Sanjeev The results Kumar of collectedchromatographic from 7 places analysis of Nepalhave shown were varied diverse from chemicalRai, profiles Director of General studied and species. Dr. Akhileswar By GCMS Lal analysis Karna, 2.27-4.2230 compounds % (v/w). were By studyingidentified the in chromatograms which 6 compoundsformer were Director taken asGeneral major ofcompounds. Department The of Plantmajor ofcompounds essential oil were of different linalool places, (36.29-45.61%), they are somewhat beta-phellandreneResources (19.93-38.38%), for giving their co-operation myrcene (4.13-8.73%), and support similarmethyl butcinnamate some compounds (3.12-22.14%) were and present sabinene only (1.57-4.78%). in for managing the research work .The authors would one or two places of essential oil. The results of also like to thank Department of Plant Resources chromatographicAcknowledgments analysis have shown diverse for giving an opportunity for research work in a very chemicalThe authors profiles would of likestudied to thank species. Mr. BySanjeev GCMS Kumarmuch Rai, friendly Director and General sound and environment Dr. Akhileswar and forLal analysis,Karna, former 30 compounds Director were General identified of Department in which 6 of Plantfinancial Resources support forwithout giving which their the co-operation research work and compoundssupport for weremanaging taken theas majorresearch compounds. work .The The authorswas would not possible. also like to thank Department of Plant Resources for giving an opportunity for research work in a very much friendly and sound environment major compounds were linalool (36.29-45.61%), and for financial support without which the research work was not possible. beta-phellandrene (19.93-38.38%), myrcene (4.13- 8.73%), methyl cinnamate (3.12-22.14%) and References sabineneBharati, S.,(1.57-4.78%). & Bhusan, B., (2015). Phytochemical and pharmacological activities of Z. armatum DC.: An overview. Research Journal of Pharmaceutical, Biological and Chemical sciences, 1403- 1409. 104 2018 Journal of Plant Resources Vol.16, No. 1

References Paudel, K., Bhatt, T.D., Adhikari, A.K., & Basyal, C., (2017). Composition comparison of essential Bharati, S., & Bhusan, B., (2015). Phytochemical oils of Zanthoxylum armatum DC by GC-MS, and pharmacological activities of Z. armatum Journal of Plant Resource, 15, 81-85. DC.: An overview. Research Journal of Pharmaceutical, Biological and Chemical Tiwari M., Naik S.N., Tewary D.K., Mittal P.K., & sciences, 1403-1409. Yadav S. (2007), Chemical Composition and larvicidal activities of Essential oil of Kala, C.P., Farooquee N.A., & Dhar, U., (2005). Zanthoxylum armatum DC (Rutaceae) against the Traditional uses and conservation of Timur three mosquito vectors. J. Vect. Borne Dis., 44(3), (Zanthoxylum armatum DC.) through social 198-204. institutions in Uttaranchal Himalaya, India. Conservation and Society, 3, 224-30. Waheed, A., Mahmud, S., Akhtar, M., & Nazir, T. (2011) Studies on components of essential oil of Mohan, M., Seth, R., Singh P., Singh, R., & Gupta Zanthoxylum armatum by GC-MS, American S., (2012). Variation in chemical constituents Journal of Analytical Chemistry, 2, 258-261 from the different parts of Zanthoxylum armatum DC. essential oils from Uttarakhanda Himalaya, India. Medicinal Plants, 4. doi: 10.5958/j.0975- 4261.4.4.026.

105 2018J. Pl. Res. Vol. 16, No. 1, pp 106-111, 2018 Journal of Plant Resources Vol.16, No. 1

Some Nepalese Medicinal Plants Showing Potential Antimicrobial Activity Against Salmonella enterica subsp. enterica serovar Typhi

Pramesh Bahadur Lakhey*, Usha Tandukar, Nishanta Shrestha and Rashmi Pradhan Department of Plant Resources, Thapathali, Kathmandu, Nepal *E-mail: [email protected]

Abstract

Typhoid, known locally as “Myadhejoro”, is an infectious fever caused by the bacterium Salmonella enterica subsp. enterica serovar Typhi. Transmitted through faeco-oral route, it causes severe worldwide morbidity and mortality, particularly in developing countries like Nepal with poor hygienic condition and contaminated water supply. Development of multiple-disease-resistant strain of this obnoxious pathogen has induced the scientific community to constantly look for neo-antibiotics. This study was conducted to screen the extracts of some Nepalese plants for antimicrobial activity against this pathogen. During the study, 53 extracts of 37 plant species were screened for antimicrobial activity against S. Typhi. Among them, 9 extracts showed antimicrobial property against the bacterium out of which ethyl acetate extract of bark of Punica granatum showed largest Zone of inhibition (ZOI) 17 mm while methanolic extract of Asparagus racemosus showed smallest ZOI 9mm.

Keywords: Agar well diffusion, Antibacterial, Plant extracts, Typhoid

Introduction parasite growing in the intestines and blood (CDC, 2016). This disease is spread by eating or drinking Typhoid fever, colloquially called “Myadhejoro” in food or water contaminated with the feces of an Nepali, is an infectious enteric fever characterized infected person (WHO, 2008). The problem is by intestinal irritation and eruption of red spots on aggravated by the fact that between 1% and 6% of the chest and abdomen. Observant patients in the individuals infected with S. Typhi become chronic, pre-antibiotic era discovered that the fever caused asymptomatic carriers which can easily transmit the by typhoid usually lasts for about three weeks that disease to new hosts (Gopinath et al., 2012). People is the illness came with a certain time frame (myadh) (ENPHO, 2018). It occurs predominantly in living in poor sanitary and unhygienic conditions association with poor sanitation and lack of clean are at higher risk of being infected (Wain et al., 2015) drinking water, the conditions common in Since 1989, multidrug-resistant typhoid fever, developing countries such as Nepal. According to resistant to the first-line recommended drugs for the estimates made in 2014, 21 million episodes of treatment such as chloramphenicol, ampicillin and typhoid occur worldwide resulting in 222,000 deaths trimethoprim-sulfamethoxazole has caused (WHO, 2018). According to the annual report for outbreaks in several countries in the developing FY 2015/16 published by Department of Health world, resulting in increased morbidity and mortality, Services, in Nepal, typhoid was the second leading especially in affected children below five years of cause of waterborne disease after diarrhoea and age and those who are malnourished (Zaki & gastroenteritis (DoHS, 2016). In addition, it is one Karande, 2011).In Nepal, a massive, single–point of the leading diagnoses of fever in most of the source, multidrug-resistant outbreak, caused by hospitals in Nepal (ENPHO, 2018). The disease, multidrug-resistant isolates with a plasmid encoding along with other enteric fevers, was so prevalent in Kathmandu that it was previously coined as enteric for resistance, was recorded in the summer of 2002 fever capital of the world (Karkey et al., 2008). from Bharatpur affecting a total of 5,963 individuals. This was considered as the largest single-point Salmonella enterica subsp. enterica serovar Typhi, source outbreak of multidrug-resistant typhoid fever the causal agent of typhoid fever, is an obligate till date (Lewis et al., 2005).

106 2018 Journal of Plant Resources Vol.16, No. 1

In the wake of emergence of multiple drug resistance minutes to allow absorption of excess surface in S. Typhi, it is only highly pertinent that the moisture. Using sterilized cork borer, agar wells of scientific world constantly looks into different 6 mm diameter were made in the inoculated plates, potential sources of neo drugs which can be effective 4 to 5 wells per plate. Micropipettes were used to against this challenging malady. Plants have place 50 µL of the test solution into each well. traditionally been proven and are being used to cure Extraction solvent (50µL) was also added to one of several diseases from time immemorial. Further, the wells of each plate as negative control. The plates drugs such as morphine, aspirin, codeine, quinine, were placed in upright condition until the test paclitaxel, artemisinin which are being used in solution was absorbed into agar and the wells allopathic medicine have been extracted, derived or appeared dry. The plates were then incubated in fabricated from different plants (Veeresham, 2012). inverted position at 35±2°C for18 to 24 hrs. After This is a non-deniable evidence of plant biodiversity the incubation period, the diameters of zones of being the most important potential source of such inhibition (ZOIs), interpreted as the clear areas new antimicrobial drugs. Thus, screening of plant around the agar wells, were measured using a digital extracts and secondary metabolites may provide an caliper to the nearest whole millimeter. important lead to the discovery of highly efficient ideal drug against S. Typhi. Grading of the antimicrobial activities of the extracts for further study was then done on the basis of ZOIs following Khakurel et al. (2014). Extracts with ZOIs Materials and Methods above 18 mm were considered as highly The screening of the antimicrobial activities was encouraging, 14-18 mm as encouraging, 10-14 mm done at Department of Plant Resources, Biological as moderate and less than 10 as weak. Section between 2070 and 2073. The test plant materials were air dried and then powdered in a Results and Discussion grinder. Extraction was done using cold percolation method or soxhlet extraction method with hexane, From 2070 to 2073, 53 extracts of 37 plant spp. were ethyl acetate, methanol or 50% ethyl alcohol. The screened for antimicrobial activity against S. Typhi extract was concentrated in a rotary vacuum by agar well diffusion method. Out of them, 9 evaporator at 60°C under reduced pressure and extracts extracted from different parts of 6 plant finally dried in water bath. species, including methanol and 50% ethanol extracts of roots of Asparagus adscendens, methanol Agar well diffusion method was used to assess the extract of roots of A. racemosus, methanol extract antimicrobial activity of the obtained extracts (Perez of rhizome of Curcuma longa, ethyl acetate extracts et al., 1990). Solution of the extract was prepared in of bark, flower and leaf of Punica granatum, -1 extraction solvent at 0.1 mgmL concentration. The methanol extract of stem of Selinum wallichianum isolate of S. Typhi used for antimicrobial screening and methanol extract of Thymus linearis, showed was a clinical isolate obtained from Tribhuvan antimicrobial activity against the bacterium. When University Teaching Hospital, Maharajgunj, graded following Khakurel et al. (2010), out of the Kathmandu. 9 extracts, the antimicrobial activity of 1 extract Cell suspension of the organism with turbidity (methanol extract of A. racemosus) fell under weak equivalent to 0.5 McFarland Nephelometric category, 6 under moderate category (methanol and Standard and containing microbial load of 1.5×108 50% ethanol extracts of A. adscendens, methanol cfu.mL-1 was prepared in sterilized normal saline extract of C. longa, ethyl acetate extract of leaves of using 18 to 24 hours culture. The prepared cell P. granatum, methanol extract of stem of S. suspension was uniformly spread over the dry wallichianum, methanol extract of entire plants of surface of Muller-Hinton Agar (MHA) plates. The T. linearis), 2 under encouraging caterogy (ethyl inoculated plates were left ajar for maximum 15 acetate extracts of bark and flower of P. granatum)

107 2018 Journal of Plant Resources Vol.16, No. 1 and none fell under excellent activity. Ethyl acetate showed smallest ZOI (9 mm) among the tested extract of bark of P. granatum showed largest ZOI extracts (table 1). (17 mm) while methanolic extract of A. racemosus

Table 1: Antimicrobial activity of the plant extracts against Salmonella enterica subsp. enterica serovar Typhi Plant part Zone of ZOI Analysed S.N Names of plants` Solvent Process used inhibition (mm) grading year 1. Acorus calamus L. Rhizome Methanol Soxhlet - - 2071/72 2. Aloe vera (L.) Burm.f. Leaf Methanol Soxhlet - - 2071/72 3. Alstonia scholaris (L.) R. Br. Bark 50% ethanol Cold - - 2070/71 4. Asparagus adscendens Roxb. Root methanol Soxhlet 12 Moderate 2072/73 Asparagus adscendens Roxb. Root 50% ethanol Soxhlet 11 Moderate 2072/73 5. Asparagus racemosus Willd. Root Methanol Soxhlet 9 Weak 2072/73 Asparagus curillus Buch.-Ham. 50% ethyl 6. Rhizome Cold - - 2070/71 ex Roxb. alcohol 7. Asparagus penicillatus H.Hara Root 50% ethanol Soxhlet - - 2072/73 8. Azadirachta indica A. Juss. PlantLeaf part Methanol Soxhlet Zone- of ZOI - Analysed 2071/72 S.N Names of plants` Solvent Process 9. Catharanthus roseus (L.) G.Don used Leaf Methanol Cold inhibition - (mm) grading - 2070/71year 1. AcorusCinnamomum calamus tamala L. (Buch.- Rhizome Methanol Soxhlet - - 2071/72 10. Leaf Ethanol Soxhlet - - 2071/72 2. AloeHam.) vera T. (L.)Nees Burm.f. & Eberm. Leaf Methanol Soxhlet - - 2071/72 3. AlstoniaCinnamomum scholaris tamala (L.) (Buch.-R. Br. Bark 50% ethanol Cold - - 2070/71 Leaf Methanol Soxhlet - - 2071/72 4. AsparagusHam.) T. Nees adscendens & Eberm. Roxb. Root methanol Soxhlet 12 Moderate 2072/73 AsparagusCinnamomum adscendens tamala (Buch.- Roxb. Root 50% ethanol Soxhlet 11 Moderate 2072/73 Leaf Ethyl acetate Soxhlet - - 2071/72 5. AHam.)sparagus T. Nees racemosus & Eberm. Willd. Root Methanol Soxhlet 9 Weak 2072/73 AsparagusCinnamomum curillus tamala Buch.-Ham. (Buch.- 50% ethyl 6. RhizomeLeaf Pet. ether SColdoxhlet - - - 2071/722070/71 exHam.) Roxb. T. Nees & Eberm. alcohol 7.11. AsparagusCitrus maxima penicillatus (Burm.) H.Hara Merr. LeavesRoot 50% Hexane ethanol Soxhlet - - - 2072/73 2072/73 8. AzadirachtaCitrus maxima indica (Burm.) A. Juss. Merr. LeavesLeaf EthylMethanol acetate SSoxhletoxhlet - - - 2072/73 2071/72 9. Catharanthus roseus (L.) G.Don Leaf Methanol Cold - - 2070/71 Citrus maxima (Burm.) Merr. Leaves Methanol Soxhlet - - 2072/73 Cinnamomum tamala (Buch.- 10. Leaf 50%Ethanol ethyl Soxhlet - - 2071/72 12. Ham.)Corydalis T. Nees chaerophylla & Eberm. DC. Roots Cold - - 2071/72 alcohol Cinnamomum tamala (Buch.- Leaf Methanol Soxhlet - - 2071/72 13. Ham.)Curcuma T. Nees longa & L. Eberm. Rhizome Methanol Soxhlet 12 Moderate 2071/72 Cinnamomum tamala (Buch.- 50% ethyl 14. Dioscorea hamiltonii Hook. f. Leaf Bark Ethyl acetate SoxhletCold - - - 2070/71 2071/72 Ham.) T. Nees & Eberm. alcohol CinnamomumEupatorium adenophorum tamala (Buch.- 15. WholeLeaf plant Pet.Methanol ether SSoxhletoxhlet - - - 2072/73 2071/72 Ham.)Spreng. T. Nees & Eberm. 50% ethyl 11.16. CitrusGaultheria maxima fragrantissima (Burm.) Merr. Wall. LeavesLeaf Hexane SoxhletCold - - - 2070/71 2072/73 Citrus maxima (Burm.) Merr. Leaves Ethylalcohol acetate Soxhlet - - 2072/73 Gentiana capitate Buch.-Ham. 17. Citrus maxima (Burm.) Merr. Whole Leaves plant Methanol SoxhletCold - - - 2071/72 2072/73 ex D. Don 50% ethyl 12. Corydalis chaerophylla DC. Roots Cold - - 2071/72 Flowering 50%alcohol ethyl 18. Hypericum cordifolium Choisy Cold - - 2070/71 13. Curcuma longa L. Rhizomebranch Malcoholethanol Soxhlet 12 Moderate 2071/72 50% ethyl 14.19. DioscoreaIris clarkei hamiltonii Baker ex Hook.Hook. f.f. Roots Bark Cold - - - 2071/722070/71 alcohol Eupatorium adenophorum 80% 15.20. Lantana camara L. WholeSeed plant Methanol Soxhlet - - - 2072/73 2072/73 Spreng. methanol 50% ethyl 16.21. GaultheriaLyonia ovalifolia fragrantissima (Wall.) Drude Wall. Branch,Leaf twig Cold - - - 2070/712070/71 alcohol GentianaMaesa chisi capitatea Buch.-Ham. Buch.-Ham. ex D. 50% ethyl 17.22. WholeBark plant Methanol Cold - - - 2071/72 2071/72 ex D.Don Don alcohol Flowering 50% ethyl 18.23. HypericumMentha arvensis cordifolium L. Choisy Leaves Hexane SoxhletCold - - - 2072/732070/71 Mentha arvensis L. branchLeaves Ethylalcohol acetate Soxhlet - - 2072/73 50% ethyl 19. IrisMentha clarkei arvensis Baker L.ex Hook. f. LeavesRoots Methnol SoxhletCold - - - 2072/732071/72 alcohol 50% ethyl 24. Moringa oleifera Lam. Leaf 80% Cold - - 2070/71 20. Lantana camara L. Seed Soxhlet - - 2072/73 methanolalcohol 50% ethyl 21. Lyonia ovalifolia (Wall.) Drude Branch, twig Cold - - 2070/71 alcohol 108 Maesa chisia Buch.-Ham. ex D. 50% ethyl 22. Bark Cold - - 2071/72 Don alcohol 23. Mentha arvensis L. Leaves Hexane Soxhlet - - 2072/73 Mentha arvensis L. Leaves Ethyl acetate Soxhlet - - 2072/73 Mentha arvensis L. Leaves Methnol Soxhlet - - 2072/73 50% ethyl 24. Moringa oleifera Lam. Leaf Cold - - 2070/71 alcohol 2018 Journal of Plant Resources Vol.16, No. 1

Neanotis gracilis (Hook.f.) W. 25. Entire plant Methanol Soxhlet - - 2072/73 H.Lewis Neanotis gracilis (Hook.f.) W. 26. Entire plant Methanol Cold - - 2072/73 H. Lewis 27. Ocimum tenuiflorum L. Leaf Methanol Soxhlet - - 2071/72 28. Piper nigrum L. Fruit Essential oil - - 2071/72 29. Plantago major L. Entire plant Methanol Soxhlet - - 2072/73 30. Punica granatum L. Rind Methanol Cold - - 2070/71 Punica granatum L. Rind Hexane Cold - - 2070/71 Punica granatum L. Bark Ethyl acetate Cold 17 Encouraging 2071/72 Punica granatum L. Flower Ethyl acetate Cold 14 Encouraging 2071/72 Punica granatum L. Leaf Ethyl acetate Cold 12 Moderate 2071/72 31. Reinwardtia indica Dumort. Leaf Methanol Soxhlet - - 2072/73 Reinwardtia indica Dumort. Shoot Methanol Soxhlet - - 2072/73 Selinum wallichianum (DC.) Soxhlet 32. Stem Methanol 10 Moderate 2071/72 Raizada & H.O. Saxena Stellaria monosperma Buch.- 33. Entire plant Methanol Soxhlet - 2072/73 Ham. ex D. Don Tanacetum dolichophylum (L.) 50% ethyl 34. Root Cold - - 2071/72 Druce alcohol Aerial 50% ethyl 35. Terminalia alata Wall. Cold - - 2070/71 branch alcohol 36. Thalictrum foliolosum DC Root Ethanol Soxhlet - - 2072/73 Thalictrum foliolosum DC Shoot Ethanol Soxhlet - - 2072/73 Thalictrum foliolosum DC Root Methanol Soxhlet - - 2072/73 Thalictrum foliolosum DC Root Methanol Soxhlet - - 2072/73 37. Thymus linearis Benth. Entire plant Methanol Soxhlet 11 Moderate 2071/72

Choi et al. (2011) reported that P. granatum peel ethanolic extract had significantly reduced the Choi et al. (2011), reported that P. granatum peel Mandal et al. (2000), tested the antimicrobial activity mortality rate and the number of viable S. typhimurium in the faeces of affected mice. Prashanth et al. ethanolic(2001) tested extract the antimicrobial had significantly activities reduced of petroleum the ofether, methanol chloroform, extract methanolof Asparagus and racemosuswater extracts against of mortalityP. granatum rate peel and and thefound number that, against of viable S. Typhi, S. petroleum,ten bacterial chloroform species includingand methanolic S. Typhi extracts and reported were typhimuriumequally effective in whilethe faeces water extractof affected was least mice. effective.that theMostafa extract et had al. considerable (2007) have in reported vitro antibacterial that the Prashanthmethanolic et extract al. (2001), of P. granatum tested the was antimicrobial effective againstefficacy all tested against bacteria the including test organisms. S. Typhi. Chouhan Ahmadi et& activitiesal. (2015) of foundpetroleum that ether,the flower chloroform, extracts methanol of P. granatumPande flower(2014), showed found thata semi-strong methanolic antibacterialand aqueous andactivity water on extracts S. Typhi of P.with granatum ZOI 31.1 peel mm. and Similarly,found extractsaccording of rootsto Mahboubi of A. racemosus et al. (2015), exhibited methanol broad that,fraction against of ethanol S. Typhi, extract petroleum, of bark chloroformof P. granatum and var.spectrum pleniflora activity was more against effective bacteria against causing S. Typhi UTI methanolicthan water, extractsethyl acetate were and equally chloroform effective fractions while of theincluding ethanol extractSalmonella implying sp. thatin comparison the traditional to use the wateras antibacterial extract was agent least against effective. foodborne Mostafa disease et wasal. justified.ethanol and ethyl acetate extracts. In the tests done (2007), have reported that the methanolic extract of by Ganesan et al. (2015), S. typhimurium was found P.Mandal granatum et al.was (2000) effective tested against the allantimicrobial tested bacteria activityto ofbe methanolmost sensitive extract to ofmethanolic Asparagus extract racemosus of A. includingagainst ten S. bacterialTyphi. Ahmadi species et includingal. (2015), S. foundTyphi that and reportedracemosus that along the extract with Bacillus had considerable subtilis. Similarly, in vitro theantibacterial flower extracts efficacy of P.against granatum the testflower organisms. showed aChouhanfindings & Pandeof Shevale (2014), et foundal. (2015) that indicatemethanolic that and the semi-strongaqueous extracts antibacterial of roots activity of A. racemosuson S. Typhi exhibited with ethanolbroad spectrum extract ofactivity roots ofagainst A. racemosus bacteria causingshowed ZOIUTI 31.1including mm. Similarly,Salmonella according sp. in comparison to Mahboubi to theet ethanolstrong andantimicrobial ethyl acetate activity extracts. against In the S. testsTyphi done over al.by (2015),Ganesan methanol et al. (2015), fraction S. of typhimurium ethanol extract was foundof theto beacetone most sensitiveextract andto methanolic the standard extract antibiotic of A. barkracemosus of P. granatumalong with var.Bacillus pleniflora subtilis .was Similarly, more findingsciprofloxacin. of Shevale Tinrat et &al. Sila-asna(2015) indicate (2017), that showed the effectiveethanol extract against of S. roots Typhi of than A. racemosus water, ethyl showed acetate strongthat antimicrobial natural distilled activity water against extract S. ofTyphi A. racemosus over the acetone extract and the standard antibiotic ciprofloxacin. Tinrat & Sila-asna (2017) showed that natural and chloroform fractions of the ethanol extract roots had significant antimicrobial effect on S. distilled water extract of A. racemosus roots had significant antimicrobial effect on S. typhimurium with implying that the traditional use as antibacterial agent typhimurium with minimum inhibitory concentration minimum inhibitory concentration of 1600 mg.mL-1. against foodborne disease was justified. of 1600 mgmL-1.

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Naz et al. (2015) evaluated the activity of crude, n- Resources for encouraging us in our research hexane, chloroform, ethyl acetate and butanol activities. extracts of Thymus linearis against 9 bacterial strains including S. Typhi and found out that the crude References extract and chloroform extract showed ZOIs of 23 mm and 19 mm respectively. Zengin & Baysal Ahmadi, A., Soleimani, N., & Tajbakhsh, M. (2015). (2015) tested the antimicrobial properties of essential Antibacterial effects of Punica granatum flower oil of Thymus sp. against S. typhimurium in the extract on Salmonella typhi. https:// ground beef and reported that the essential oil www.researchgate.net/publication/319059476. treatment inhibited the growth of the bacterium. CDC (2016). Typhoid fever. Retrived from https:// Ishaq (2015), tested the antimicrobial properties of www.cdc.gov/typhoid-fever/index.html. methanolic extract of T. linearis against various Choi, J., Kang, O., Lee, U., Chae, H., Oh, Y., Brice, multiple-drug-resistant strains of pathogenic bacteria and found that the crude methanolic extract was most O., Kim, M., Sohn, D., Kim, H., Park, H., Shin, effective against S. Typhi. The extracts of different D., Rho, J., & Kwon, D. (2011). In-vitro and in- species of Thymus have also been reported to show vivo antibacterial activity of Punica granatum antimicrobial activities against S. Typhi (Karaman peel ethanol extract against Salmonella. Evid. et al., 2001; Maksimovic et al., 2008; Rota et al., Based Complement. Alternat. Med., 1-8. 2008; Hussain et al., 2011). doi:10.1093/ecam/nep105. Chouhan, D.A., & Pande, P.S. (2014). Studies on Conclusion antibacterial potential of Asparagus racemosus extract against bacteria causing UTI. From the current study, it could be concluded that International Journal of Pharmaceutical ethyl acetate extracts of P. granatum flower and bark Research, 6(2), 100-106. have significant antimicrobial property against S. Typhi. The findings suggest that, further researches ENPHO. (2018). Typhoid: The Neglected Urgent in to confirm the potential of these extracts against S. Nepal. Retrived from http://enpho.org/featured/ typhoid-the-neglected-urgent-in-nepal/ typhi, its multiple drug resistant strains, identification of active ingredient, toxicity testing to confirm their Ganesan, R., Latha, T., & Mangai, S. (2015). applicability in human subjects are justified. Other Phytochemical analysis and antibacterial activity plant extracts such as T. linearis methanol extract, of roots of Asparagus racemosus Willd. Asian P. granatum leaf ethyl acetate extract can also be Journal of Phytomedicine and Clinical Research, developed into effective drugs against the causal 3(4), 132-139. agent of typhoid after further confirmatory DoHS. (2016). Annual Report: Department of researches. These researches will not only help in Health Services 2072/73 (2015/16). Kathmandu, seeking out viable sources of antimicrobial agents Nepal: Government of Nepal, Department of against notorious typhoid but also valorize the local Health Services. plant resource of Nepal. Gopinath, S., Carden, S., & Monack, D. (2012). Shedding light on Salmonella carriers. Trends in Acknowledgements Microbiology, 20(7), 320-327. We would like to express our sincere gratitude to Hussain, A.I., Anwar, F., Nigam, P.S., Sarker, S.D., Mr. Sanjeev Kumar Rai, Director General, Moore, J.E., Rao, J.R., & Mazumdar, A. (2011). Department of Plant Resources for providing us an Antibacterial activity of some Lamiaceae essential excellent opportunity to conduct this research. We oils using resazurin as an indicator of cell growth. are also deeply indebted to Ms. Jyoti Joshi Bhatta, LWT-Food Sci Technol, 44, 1199-1206. Deputy Director General, Department of Plant Isaq, M.S. (2015). In vitro phytochemical and

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antimicrobial screening of Thymus linearis. screening of Thymus linearis. Bangladesh J. Bangladesh J. Pharmacol., 10, 21-26. Pharmacol., 10, 21-16. Karaman, S., Digrak, M., Ravid, U., & Ilcim, A. Parashanth, D., Asha, M.K., & Amit, A. (2001). (2001). Antibacterial and antifungal activity of Antibacterial activity of Punica garanatum. the essential oils of Thymus revolutus Celak from Fitoterapia, 72(2001), 171-173. Turkey. J. Ethnopharmacol., 76, 183-86. Perez, C., Pauli, M., & Bazerque, P. (1990). An Karkey, A., Aryjal, A., Basnyat, B., & Baker, S. antibiotic assay by agar-well diffusion method. (2008). Kathmandu, Nepal: Still an enteric fever Acta Biologiae et. Medecine Experimentalis, 15, capital of the world. J. Infect. Developing 113-115. Countires Countries, 2(6), 461-465. Rota, M.C., Herrera, A., Martínez, R.M., Sotomayor, Khakurel, B.K., Pradhan, R., & Lakhey, P.B. (2014). J.A., & , M.J. (2008). Antimicrobial A preliminary screening of some Nepalese activity and chemical composition of Thymus medicinal plants for antimicrobial activity. Plant vulgaris, Thymus zygis and Thymus hyemalis Resources, 36, 72-75. essential oils. Food Control, 19, 681-687. Lewis, M.D., Serichantalergs, O., Pitarangsi, C., Shevale, U.L., Mudrawale, A.S., Yadav, S.R., Chunak, N., Mason, C.J., Regmi, L.R., Pandey, Chavan, J.J., Jamdade, C.B., & Patil, D.B. (2015). P., Laskar, R., Shrestha, C.D., & Malla, S. (2005). Phytochemical and antimicrobial studies on Typhoid fever: a massive, single-point source, Asparagus racemosus. World Journal of multidrug-resistant outbreak in Nepal. Clinical Pharmaceutical Research, 4(9), 1805-1810. Infectious Disease, 40, 554-561. Tinrat, S., & Sila-asna, M. (2017). Phytochemical Mahboubi, A., Asgarpanah, J., Sadaghiyani, P.N., screening and influence of extraction solvents on & Faizi, M. (2015). Total phenolic and flavonoid antioxidant and antimicrobial activities of content and antibacterial activity of Punica Asparagus racemosus Willd. root. International granatum L. var. pleniflora flowers (Golnar) Journal of Phytomedicine, 9(2), 343-351. against bacterial strains causing foodborne Veeresham, C. (2012). Natural products derived from diseases. BMC Complementary and Alternative plants as a source of drugs. J. Adv. Pharma. Medicine, 15(1), 366. Technol. Res., 3(4), 200-201. Maksimovic, Z., Stojanovic, D., Sostaric, I., Dajic, Wain, J., Hendriksen, R.S., Mikoleit, M.L., Keddy, Z., & Ristic, M. (2008). Composition and radical- K.H., & Ochiai, R.L. (2015). Typhoid fever. scavenging activity of Thymus glabrescens Willd. Lancet., 385(9973), 1136–1145. (Lamiaceae) essential oil. J. Sci. Food Agric., 88, 2036-2041. WHO (2008). Typhoid vaccines: WHO position paper. Wkly Epidemiol Rec., 83(6), 49–59. Mandal, S.C., Nandy, A., Pal, M., &Saha, B.P. (2000). Evaluation of antibacterial activity of WHO (2018). Immunization, vaccines and Asparagus racemosus Willd. root. Phytother Res., biologicals: Typhoid. Retrived from http:// 14(2), 118-119. www.who.int/immunization/diseases/typhoid/en/ Mostafa, A.A., Al-Askar, A.A., Almaary, K.S., Zaki, S.A, & Karande, S. (2011). Mutidrug-resistant Dawoud, T.M., Sholkamy, E.N., & Bakri, M.M. typhoid fever: a review. J. Infect. Dev. Ctries., (2017). Antimicrobial activity of some plant 5(5), 324-327. extracts against bacterial strains causing food Zengin, H., & Baysal, A.H. (2015). Antioxidant and poisoning diseases. Saudi Journal of Biological antimicrobial activities of thyme and clove Sciences, 25, 361-366. essential oils and application in minced beef. Naz, A., Saeed, M., Hussain, M.M., & Ishaq, M.S. Journal of Food Processing and Preservation, (2015). In vitro phytochemical and antimicrobial 39, 1261-1271.

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In-Vitro Propagation of Lavender (Lavandula angustifolia Mill.)

Nabin Rana, Saraswoti Khadka and Sabari Rajbahak* Biotechnology Section, Department of Plant Resources, Thapathali, Kathmandu, Nepal *Email: [email protected]

Abstract

Lavender (Lavandula angustifolia) is a that is endemic to the Mediterranean region. Lavender is widely used in the food, perfume and pharmaceutical industries. To reach supply of demand and the large-scale production of lavender efficient biotechnology-based approaches such as tissue culture technique is required. To initiate the in-vitro culture, lavender seeds were disinfected at 0.1% concentration of NaOCl for 5 minutes. Appropriate medium to establish the culture was found to be Murashige & Skoog (1962) Medium. Seed explants, cotyledonary buds (0.4-0.6 cm in length) were used for micropropagation of Lavender by manipulating the cytokinin and auxin on MS media. In this study we found that, hormone combination with 1.0 mg/L BAP and 0.1 mg/L NAA gave optimum growth results. The acclimatization of bottles with mature plantlets were done in a greenhouse at 20±5°C for a week. Plant lets were established successfully ex-vitro in non-sterile sand. The rooted plants were then transferred in polybags containing garden soil, organic matter and sand (1:1:1). Mass propagation of lavender plants by tissue culture technique was established (95%) on MS medium enriched with BAP and NAA and adapted ex- vitro with 88% survival.

Keywords: Explants, Medium, Micropropagation, Murashige & Skoog (MS), Tissue culture

Introduction al., 1999). The trace amount of perillyl alcohol, a Lavandula angustifolia an annual herb is a member monoterpene in L. angustifolia has been an of the Lamiaceae family: rich in aromatic essential increasing interest (Perrucci et al., 1996) in its oils which is valuable for its pharmaceutical, aeromatic chemopreventative and chemotherapeutic properties and culinary properties (Hanamanthagouda et al., (Schulz et al., 1994; Hohl, 1996; Peffley & Gayen, 2010; Ebadollahi et al., 2014; Mendez-Tovar et al., 2003). 2015). It is relatively rich in phenolic constituents Alongside to its use as medicine, lavender is valued with flavons,phytosterols & anthocyanins for its strong and pleasant fragrance. In ancient time, (Horbourne & Williams, 2002; Lis-Balchin, 2012), lavender was known as an aphrodisiac, later it was main components includes linalool, 4-ol-terpinen, used for the storage of laundry and to disguise á-terpineol, linalyl anthranilate, gernanyl acetate, objectionable odours (Chu & Kemper, 2001). coumarin, herniarinborneol, lavandulol acetate and Nowadays, we commonly find lavender in a wide minerals (Cavanagh & Wilkinson, 2005; Costa et variety of perfumes and soaps. They are also used al., 2011; Georgiev et al., 2009; Spiridon et al., 2011). in the food industry because of their nutraceutical As a medicinal remedy Lavandula angustifolia is effects (Hsu et al., 2007). stated to be carminative, spasmolytic, anti- depressant, neuralgia, rheumatism, depression, Lavender has gained attention for their diverse insomnia, windy colic, fainting toothache, sprains, secondary metabolites. Realizing the fact of their sinusitis, stress, migraine which enhances the human usefulness, many countries over the world are health (Chu & Kemper, 2001).The sedative effect establishing many perfumes, scented bags, & antispasm property of Lavender oil are useful for pharmaceutical companies. On the flip side of high patients with respiratory distress syndrome & demand of lavender flowers its supply has been effective against fungal infection (Lis Balchin & scanty (Hassiotis et al., 2014). It is because of Hart, 1999; Behmanesh et al., 2015; Hohmann et difficulties in the planting and caring process (Stanev

112 2018 Journal of Plant Resources Vol.16, No. 1 et al., 2016). As a matter of fact plant tissue culture seedling of the lavender were transferred to sterile technique is important technique in the field of plant petri plates with the sterile forceps under asceptic propagation (Hartmann & Kester, 1975). Tissue culture conditions. The seed coat and excess leaves were technique helps to proliferate the plant in large removed from the cotyledonary bud. Those buds quantities in a short time, high productivity and disease- were transferred aceptically into shoot induction free plant, improving survival rate of plantlets (Badawy medium. et al., 2003). Growth regulators are used to enhance in-vitro plantlets behaviour, 6-benzylaminopurine Culture media and in-vitro growth conditions of (BAP), for example, promotes the growth of plantlets the explants shoots (Murashige & Skoog, 1962). Inorganic salts and vitamins of MS were used as a Establishment of the tissue culture of lavender can basal medium. Sucrose 30g/L was used as carbon be achieved from seedlings obtained by germinating source, plant growth regulator (BAP, NAA, IAA) seeds, from bud culture, shoot generation from tissue were added and pH was adjusted to 5.8 before or callus also from cells and protoplasts. In this study, autoclaving. The media was solidified using 0.8% we investigated propagation of lavender by agar and autoclaved at 121°C for 15 minutes. The germinating seeds to establish the best culture cultures were incubated at 16 h photoperiod with medium supplemented with growth regulator for i) light intensity of 3000 lux using florescent tube lights higher shoot proliferation of lavender ii) for the and temperature of 25±2°C for 2-3 weeks. production of high quality plantlets. (Rajbahak et al., 2014). In-vitro shoots proliferation Materials and Methods Cotyledonary buds were used as an explant source Plant material and were inoculated in shoot initiation medium which consisted of different medium as Murashige Lavender seeds were provided form Department of & Skoog (1962) medium, Modified Medium (MM) Plant resources. and Woody Plant medium (WPM). For each Surface sterilization treatment three explants in a single flask were used and each experiment was repeated 3 times. Explants Seeds might have bacteria and fungi on their outer were grown in 3 different initiation media for 2-3 coat. Some of these microorganism may be weeks. Among them, the best shoot initiation pathogenic and overwhelm the seedling to sterilize medium was selected. Explants were transferred into the lavender seeds 0.1% NaoCl and 0.05% HgCl2 shoot proliferation medium supplemented with were prepared. Seed was surface sterilized with 0.1% different concentration of BAP, NAA and IAA (0.5 NaoCl for 2, 3.5, 5, 6.5, 8 minutes respectively mg/mL, 1.0 mg/mL, 2.0 mg/L, 2.5 mg/L, 5.0 mg/ likewise seeds were treated with 0.05% of HgCl2 LBAP, 0.1 mg/L IAA and NAA). For each treatment, for 1, 1.5, 2, 2.5, 3 minutes to standardize the fifteen explants were used and each experiment was appropriate time for surface sterilization. After repeated three times. treatment of NaoCl and HgCl2 seeds were washed three times with sterile distilled water then Hardening and acclimatization of plantlets inoculated in murashige & Skoog (MS) Medium for After 2-3 successive sub culture into proliferation germination. media, the culture bottles were moved to green house Seed germination and explant isolation for a week for acclimatization. After a week, plantlets were removed from bottles and washed with distilled Lavender seeds were germinated after 10- 15 days. water to remove media from the plantlets. Then, the The explants consists of transferable half (0.4-0.6 plantlets were transferred into sand trays and covered cm in length) of cotyledonary bud. Those germinated with polythene hood to maintain moisture. The

113 2018 Journal of Plant Resources Vol.16, No. 1 temperature and humidity of the greenhouse was maintained at 20±5°C and 80% humidity respectively. PlantsConcentration were assessed and fortreatment rooting time at 3-4 of weeks.NaOCl After and HgCl2for the rate of sterile explants optimizingSurface sterilization the growth of of explants the rooted were plantlets, done using these 0.1% concentration of NaOCl & 0.05% concentration of HgCl . Where 0.1% concentration of NaOCl is proper at 5 minutes sterilization time (75.84% of were transferred2 to nursery polybags containing sterile seed and 41.20% of germinated seeds) while in case of 0.05% concentration HgCl is suitable at 2 garden soil organic matter and sand (1:1:1). 2 minutes sterilization time (75% of sterile seed and 32% of germinated seeds) as results showed in table 1.

ResultsTable 1: Results of sterilization of lavender seeds using NaOCl and HgCl2 * Rate of germinated seeds (%) = (number of germinated seeds/total number of seeds) x 100 Concentration* Rate of sterile seedsand (%)treatment = (number time of sterile of NaOCl seeds/total and number of original seeds) x 100

Concentration of Treatment time Rate of germinated seeds HgCl2for the rate of sterile explants Rate of asceptic seeds (%) NaOCl & HgCl (minutes) Figure 1: Germinated seeds of lavender on(%) MS medium Surface sterilization of explants were 2done using 46.00after 10-15 days of inoculation38.00 0.1% concentration of NaOCl 3.5& 0.05% 54.33 25.66 lavender shoots. The cultures were incubated at 16 concentration(NaOCl) of 0.1% HgCl . Where 0.1% concentration5 75.84 41.20 2 h photoperiod with light intensity of 3000 Lux using of NaOCl is proper at 5 minutes sterilization6.5 time 83.66 31.80 8 100.00 28.73 (75.84% of sterile seed and 41.20% of 1germinated 50.67 22.64 seeds) while in case of 0.05% concentration1.5 HgCl2 67.88 26.58 is suitable(HgCl at2 )2 0.05% minutes sterilization time2 (75% of 75.31 32.67 sterile seed and 32% of germinated seeds)2.5 as results 87 20.82 showed in table 1. 3 100 17.54

TheConcentration effects of shoot and treatment initiation time on ofthe NaOCl growth and of HgCl2for the rate of sterile explants lavenderSurface shootssterilization of explants were done using 0.1% concentration of NaOCl & 0.05% concentration of HgCl2. Where 0.1% concentration of NaOCl is proper at 5 minutes sterilization time (75.84% of Among three different shoot initiation media MS sterile seed and 41.20% of germinated seeds) while in case of 0.05% concentration HgCl2 is suitable at 2 medium was found to be suitable for the growth of minutes sterilization time (75% of sterile seed and 32%Figure of germinated 2: The growth seeds) of lavender as results shoots showed on MS inmedium table 1.

Table 1: Results of sterilization of lavender seeds using NaOCl and HgCl2 * Rate of germinated seeds (%) = (number of germinated seeds/total number of seeds) x 100 * Rate of sterile seeds (%) = (number of sterile seeds/total number of original seeds) x 100

Concentration of Treatment time Rate of germinated seeds Rate of asceptic seeds (%) NaOCl & HgCl2 (minutes) (%) 2 46.00 38.00 3.5 54.33 25.66 (NaOCl) 0.1%Figure 1: Germinated seeds5 of Lavender on MS medium 75.84after 10-15 days of inoculation 41.20 6.5 83.66 31.80 The effects of shoot initiation on the growth8 of lavender shoots 100.00 28.73 Among three different shoot initiation1 media MS medium was 50.67 found to be suitable for 22.64the growth of Lavender shoots.The cultures were incubated1.5 at 16 h photoperiod67.88 with light intensity of 300026.58 Lux using (HgCl ) 0.05% florescent tube2 lights and temperature 2of 25± 2°C. Time taken 75.31for the shoot initiation was32.67 2-3 weeks of 2.5 87 20.82 primary culture. 3 100 17.54 Table 2: The effect of shoot initiation media on the growth of lavender shoots *Average number of shoot and average height of shoots were given as mean±standard error Mineral Number of culture Number of weeks for Average number of shoot Average height of media bottles shoot initiation (Shoot/explants) shoots (cm) MM 5 2-3 1.50±0.05 2.56±0.2 WPM 5 2-3 2.66±0.04 3.23±0.1 MS 5 2-3 3.78±0.03 3.68±0.2

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Figure 1: Germinated seeds of Lavender on3 MS medium after 10-15 days of inoculation

The effects of shoot initiation on the growth of lavender shoots Among three different shoot initiation media MS medium was found to be suitable for the growth of Lavender shoots.The cultures were incubated at 16 h photoperiod with light intensity of 3000 Lux using florescent tube lights and temperature of 25± 2°C. Time taken for the shoot initiation was 2-3 weeks of primary culture.

Table 2: The effect of shoot initiation media on the growth of lavender shoots *Average number of shoot and average height of shoots were given as mean±standard error Mineral Number of culture Number of weeks for Average number of shoot Average height of media bottles shoot initiation (Shoot/explants) shoots (cm) MM 5 2-3 1.50±0.05 2.56±0.2 WPM 5 2-3 2.66±0.04 3.23±0.1 MS 5 2-3 3.78±0.03 3.68±0.2

3 2018 Journal of Plant Resources Vol.16, No. 1 florescent tube lights and temperature of 25±2°C. shoots within 2-3 weeks. Number of shoots/explants Time taken for the shoot initiation was 2-3 weeks of was 4.06±0.05 and maximum average height of primary culture. shoots was 3.46±0.04 cm. MS medium supplemented with other hormones gave lesser The effects of hormone concentrations on the number of shoots (table 3). shoot multiplication of lavender The effect of different concentration of BAP and Hardening and acclimatization of plantlets NAA is shown in table 3. The efficacy of MS medium with 2.5µM NAA was used for rooting combination of growth hormones was assessed according to Echeverrigaray et al. (2005). The based on number of shoots induced after inoculation. plantlets were then hardened in the greenhouse for Shoot formation on cotyledon explants cultured on a week. Up to 95% of those plantlets showed rooting MS medium supplemented with BAP 1.0 mg/ after two weeks on sand at 20±5°C greenhouse and L+NAA 0.1 mg/L gave rise to luxuriantly growing 80% humidity. About 88% of the plants were

Figure 2: The growth of lavender shoots on MS medium

The effects of hormone concentrations on the shoot multiplication of lavender The effect of different concentration of BAP and NAA is shown in table 3. The efficacy of combination of growth hormones was assessed based on number of shoots induced after inoculation. Shoot formation on cotyledon explants cultured on MS medium supplemented with BAP 1.0 mg/L+NAA 0.1 mg/L gave (A) (B) rise to luxuriantly growing shoots within 2-3 weeks. Number of shoots/explants was 4.06±0.05 and maximumFigure average 3: (A) & height (B) Induction of shoots of lavender was 3.46±0.04 shoot on MS cm. medium MS medium supplemented supplemented with 1.0 mg/L with concentration other hormones of BAP and 0.1 mg/L NAA gave lesser number of shoots (Table 3). Table 3: Effects of growth hormones (BAP+NAA) on shoot multiplication *Number of shoots/explants and average height of shoots were given as mean±standard error. Plant growth No. of culture No of weeks for shoot No. of shoots Average height of regulators bottles initiation (Shoots/explants) shoots (cm) BAP+NAA (mg/L) 0.5+0.1 5 2-3 3.70±0.07 2.87±0.00 1.0+0.1 5 2-3 4.06±0.05 3.46±0.04 1.5+0.1 5 2-3 3.19±0.06 3.31±0.01 2.0+0.1 5 2-3 3.45±0.05 3.29±0.02 2.5+0.1 5 2-3 3.86±0.05 3.27±0.06

Table 4: Effect of growth hormones (BAP+IAA) on shoot multiplication *Number of shoots/explants and Average height of shoots were given as mean±standard error. Plant growth No. of culture No of weeks for shoot No. of shoots/explants Height of shoots (cm) regulators bottles initiation BAP+IAA (mg/L) 0.5+0.1 5 2-3 2.87±0.06 1.80±0.02 1.0+0.1 5 2-3 2.91±0.07 2.23±0.05 1.5+0.1 5 2-3 3.55±0.05 2.56±0.01 2.0+0.1 5 2-3 3.68±0.02 2.21±0.07 2.5+0.1 5 2-3 3.97±0.04 2.90±0.05

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(A) (B) Figure 4: (A) The roots of lavender buds cultured on MS medium supplemented with NAA (1mg/L), (B) Lavender plantlets transferred to the Greenhouse

hardened successfully. Those hardened and case of HgCl2 but the germination rate decreased acclimatized plants were shifted to the soil bag significantly (38.00-28.73% in NaOCl and 22.64- containing garden soil organic matter and sand 17.54% in HgCl2). (1:1:1). Variation in shoot proliferation rate among different growth medium has been previously reported (Tien Discussion Vinh et al., 2017; Goncalves & Romano, 2013). Tien Tissue culture techniques are gaining increasing Vinh suggest that WPM medium supplemented with importance as a valuable supplement to the BAP (0.1mg/L) was appropriate for the growth of conventional methods (Bayliss, 1980). It has been L. angustifolia. Here, our results revealed that MS used to generate plantlets with better qualities, medium supplemented with 1.0 mg/L BAP and greater vigor, higher yield and disease resistance 0.1mg/L NAA gave rise to luxuriantly growing (Barba & Nitchell, 1969). shoots of Lavandula angustifolia. Number of shoots/ explants was 4.06±0.05 and maximum average The success of tissue culture lies in i) to develop height of shoots was 3.46±0.04 cm. MS medium contamination free culture ii) induction/ regeneration supplemented with other hormones gave lesser of plantlets and iii) finally to transfer the in vitro number of shoots regenerated plantlets to field conditions. Bacterial and fungal contamination continually threatens plant Data represented in table 3 & 4 showed that MS tissue cultures throughout the duration of the culture media with different concentration of BAP, NAA period. In this step it has to operate the proper and IAA (0.5 mg/L, 1.0 mg/L, 2.0 mg/L, 2.5 mg/L, inoculation process to overcome the contamination. 5.0 mg/L BAP, 0.1 mg/L IAA and NAA). This Concentration of 0.1% NaOCl was proper at 5 variation may have resulted from the different minutes sterilization time (75.84% of sterile seed requirements of shoot proliferation in lab such as and 41.20% of germinated seeds) while in case of light intensity and temperature given, pH measurement of media etc. Moreover, differences 0.05% concentration HgCl2 is suitable at 2 minutes sterilization time (75% of sterile seed and 32% of in stimulation of shoot proliferation may be related germinated seeds). While increasing the sterilization to differences in macronutrients (Matt & Jehle, 2005; Liu & Pijut, 2008; Ruzic & Vujovic, 2008). Increase time of NaOCl and HgCl2 8 minutes and 3 minutes respectively, the rate of sterile seeds increased and decrease in the concentration of BAP keeping markedly (83.66-100% in NaOCl and 87-100% in NAA and IAA adversely affect on the shoot

116 2018 Journal of Plant Resources Vol.16, No. 1 proliferation and growth. Culture medium and Behmanesh, F., Pasha, H., Sefidgar, A. A., Taghizadeh, cytokinins used, effect on proliferation rate of M., Moghadamnia, A. A., Adib Rad, H., & Shirkhani, micropropagation process (Dias et al., 2002). So L. (2015). Antifungal effect of lavender essential oil there is greater essence of research to observe the (Lavandula angustifolia) and clotrimazole on shoot proliferation of Lavandula angustifolia by : an in vitro study. Scientifica,2015, varying the cytokinin concentration along with auxin 261397. in MM and WPM media. Cavanagh, H. M., & Wilkinson, J. M. (2005). Lavender essential oil: A review. Australian infection Conclusion control, 10(1), 35-37. Lavender seeds sterilized at 0.1% concentration of Chu, C. J., & Kemper, K. J. (2001). Lavender (Lavandula spp.) Longwood Herbal Task Force, 1-32. NaOCl at 5 minutes sterilization time was best. MS medium supplemented with 1.0 mg/L BAP and 0.1 Costa, P., Gonçalves, S., Andrade, P. B., Valentão, P., & mg/L NAA, sucrose 30g/L and agar 8 g/L was Romano, A. (2011). Inhibitory effect of Lavandula suitable for maximum number of shoot proliferation. viridis on Fe2+-induced lipid peroxidation, Tissue culture technique of the lavender plant was antioxidant and anti-cholinesterase properties. Food established (95%) on MS medium enriched with chemistry, 126(4), 1779-1786. BAP and NAA and adapted ex vitro with surviving Dias, M. C., Almeida, R., & Romano, A. (2002). Rapid rate up to 88% of healthy plants. clonal multiplication of Lavandula viridis L’Her through in vitro axillary shoot proliferation. Plant Acknowledgement Cell, Tissue and Organ Culture, 68(1), 99-102. Ebadollahi, A., Jalali Sendi, J., Aliakbar, A., & Razmjou, It is our privilege to express our sincere thanks deep J. (2014). Chemical composition and acaricidal sense of gratitude to Director General Mr. Sanjeev effects of essential oils of Foeniculum vulgare Kumar Rai, Deputy Director General Mrs. Jyoti Mill.(Apiales: Apiaceae) and Lavandula angustifolia Joshi Bhatt for facilitating lab making it suitable to Miller (: Lamiaceae) against Tetranychus conduct this research successfully. We sincerely urticae Koch (Acari: Tetranychidae). Psyche: A thank former Deputy Director General (DDG), Rose Journal of Entomology, 2014, 1-6. Shrestha for providing us with the sampling materials for in-vitro propagation of lavender. We Echeverrigaray, S., Basso, R., & Andrade, L. B. (2005). acknowledge Aashis Shrestha for his kind supports Micropropagation of Lavandula dentata from in lab work and Lila Rai for her kind support for the axillary buds of field-grown adult plants. Biologia Plantarum, 49(3), 439-442. acclimatization of plantlets in greenhouse. Georgiev, M., Abrashev, R., Krumova, E., Demirevska, References K., Ilieva, M., & Angelova, M. (2009). Rosmarinic acid and antioxidant enzyme activities in Lavandula Badawy, E., Sakr, S., El-Sharnouby, M., Szoke, E., vera MM cell suspension culture: a comparative Mathe, I., Blunden, G., & Kery, A. (2003). Production study. Applied biochemistry and biotechnology, and composition of lavender plants through tissue 159(2), 415. culture as affected with gamma irradiation Gonçalves, S., & Romano, A. (2013). In vitro culture treatments. Horticuturae, 297, 325-328. of lavenders (Lavandula spp.) and the production of Barba R., & Nickell L. (1969) Nutrition and organ secondary metabolites. Biotechnology advances, differentiation in tissue cultures of sugarcane, a 31(2), 166-174. . Plant, 89, 299–302. Hanamanthagouda, M. S., Kakkalameli, S. B., Naik, P. Bayliss, M. W. (1980). Chromosomal variation in plant M., Nagella, P., Seetharamareddy, H. R., & Murthy, tissues in culture. Int. Rev. Cytol.(Suppl), 11, 113- H. N. (2010). Essential oils of Lavandula bipinnata 144. and their antimicrobial activities. Food Chemistry,

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118(3), 836-839. product of Lavandula latifolia essential oil Harborne, J. B., & Williams, C. A. (2002). distillation as source of antioxidants. Journal of Food Phytochemistry of the genus Lavandula (pp. 86-99). and Drug Analysis, 23(2), 225-233. London, UK : CRC Press. Murashige, T., and Skoog, F. (1962) A revised medium Hartmann, H. T., & Kester, D. E. (1975). Plant for rapid growth and bioassays with tobacco tissue propagation: principles and practices. Prentice-Hall. culture. Physiol. Plant, 15,473-497. doi: 10.1111/ j.1399- 3054.1962.tb08052.x Hassiotis, C. N., Ntana, F., Lazari, D. M., Poulios, S., & Vlachonasios, K. E. (2014). Environmental and Peffley, D. M., & Gayen, A. K. (2003). Plant-derived developmental factors affect essential oil production monoterpenes suppress hamster kidney cell 3- and quality of Lavandula angustifolia during hydroxy-3-methylglutaryl coenzyme a reductase flowering period. Industrial Crops and Products, 62, synthesis at the post-transcriptional level. The 359-366. Journal of Nutrition, 133(1), 38-44. Hohl, R. J. (1996). Monoterpenes as regulators of Perrucci, S., Macchioni, G., Cioni, P. C., Flamini, G., malignant cell proliferation. In Dietary Morelli, I., & Taccini, F. (1996). The activity of phytochemicals in cancer prevention and volatile compounds from Lavandula angustifolia treatment (pp. 137-146). Boston: Springer. against Psoroptes cuniculi. Phytotherapy Research, 10(1), 5-8. Hohmann, J., Zupkó, I., Rédei, D., Csányi, M., Falkay, G., Máthé, I., & Janicsák, G. (1999). Protective effects Rajbahak, S., Chapagain, N., Shrestha, J., & Basnet, P. of the aerial parts of Salvia officinalis, Melissa (2014). Clonal propagation of Paulownia tomentosa officinalis and Lavandula angustifolia and their Steud for commercial production. Bull. Dpr. Pl. Re., constituents against enzyme-dependent and enzyme- 36. independent lipid peroxidation. Planta Medica: Ru•iæ, D.V., & Vujoviæ, T.I. (2008). The effects of Natural Products and Medicinal Plant cytokinin types and their concentration on in vitro Research, 65(6), 576-578. multiplication of sweet cherry cv. Lapins (Prunus Hsu, C.K., Chang, C.T., Lu, H.Y., & Chung, Y.C. (2007). avium L.). Horticultural Science, 35(1), 12-21. Inhibitory effects of the water extracts of Lavendula Schulz, S., Bühling, F., & Ansorge, S. (1994). Prenylated sp. on mushroom tyrosinase activity. Food proteins and lymphocyte proliferation: Inhibition by Chemistry, 105(3), 1099-1105. d limonene and related monoterpenes. European Lis-Balchin, M. T. (2012). Lavender. In handbook of Journal of Immunology, 24(2), 301-307. herbs and spices (2nd edition), Vol. II. (pp. 329-347). Spiridon, I., Colceru, S., Anghel, N., Teaca, C.A., Bodirlau, R., & Armatu, A. (2011). Antioxidant Lis Balchin, M., & Hart, S. (1999). Studies on the mode capacity and total phenolic contents of oregano of action of the essential oil of Lavender, Lavandula (Origanum vulgare), lavender (Lavandula angustifolia P. Miller. Phytotherapy Research, 13(6), angustifolia) and lemon balm (Melissa officinalis) 540-542. from Romania. Natural Product Research, 25(17), Liu, X., & Pijut, P. M. (2008). Plant regeneration from 1657-1661. in vitro leaves of mature black cherry (Prunus serotina). Plant Cell, Tissue and Organ Stanev, S., Zagorcheva, T., & Atanassiov, I. (2016). Culture, 94(2), 113-123. Lavender cultivation in Bulgaria-21st century developments, breeding challenges and Matt, A., & Jehle, J. A. (2005). In vitro plant regeneration oppurtunities. Bulgarian Journal of Agricultural from leaves and internode sections of sweet cherry Science, 22(4), 584-590. (Prunus avium L.). Plant Cell Reports, 24(8), 468-476. Tien Vinh, D., Hoa, M.T., Khai, P.C., & Van Minh, T. (2017). Micropropagation of lavender (Lavandula Méndez-Tovar, I., Herrero, B., Pérez-Magariño, S., angustifolia). Seeds, 50(10), 50-100c. Pereira, J. A., & Manzanera, MCAS. (2015). By-

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Comparisons of Invasive Alien Plant Species Richness between Tarai and Siwalik Regions of Central Nepal

Sajita Dhakal1*, Bharat Babu Shrestha2 and Mohan Siwakoti2 1National Botanical Garden Godawari, Lalitpur, Kathmandu, Nepal 2Central Department of Botany, Tribhuvan University, Kirtipur, Kathmandu, Nepal *E-mail: [email protected]

Abstract

Invasive alien plant species (IAPS) are the sub-category of naturalized plant species and one of the major drivers of environmental changes and the leading cause of biodiversity loss after . High concentration of IAPS is found on the southern half of the country which includes which two physiographic regions i.e. Tarai and Siwalik regions with tropical to subtropical vegetation. A detailed survey of vascular plants in different vegetation types of Tarai and Siwalik regions in central Nepal was carried out following Modified Whittaker vegetation sampling method to analyze diversity of IAPS. Altogether, 38 plots of 0.1 ha. (20 m × 50 m) were sampled in 12 sampling sites six in each Siwalik and Tarai, including four vegetation types. In each vegetation type, three plots were sampled. Among the 16 species of IAPS recorded from the study area, Siwalik region have high IAPS richness than Tarai region. Chromolaena odorata was the most dominant species with highest cover and frequency in both physiographic regions.

Keywords: Community, Ecology, Invasive, Vegetation

Introduction species Chromolaena odorata, Eichhornia crassipes, Lantana camara and Mikania micrantha The introduction and establishment of IAPS is are included in the list of 100 of the world’s worst considered second largest threats to global invasive species (Lowe et al., 2000). The aim of this biodiversity, after the habitat destruction (Sala et al., research work is to address gaps to some extent by 2000). Invasion is a multi-step process comprised analyzing the frequency and coverage of different of three phases; introduction: which involves initial IAPS in both physiographic regions in southern dispersal (where an organism moves from its native lowland of central Nepal. habitat, often over long distances, to a new habitat outside of its home range), establishment: of self- Materials and methods sustaining populations within the new habitat and spread: to nearby habitats (Liebhold & Tobin, 2008). This study was carried out in Tarai (Bara and Frequent availability of unused resources of the Rupandehi) and Siwalik (Makwanpur and habitat increase vulnerability to invasion (Davis et Nawalpaarasi) regions of central Nepal. al., 2000) while human activities increase propagule pressure of IAS (Simberloff, 2009). Nepal lies in the transition zone between several floristic regions of Asia and floral elements of all regions are well represented within Nepal (Dobremez, 1976). In Nepal, there are all together 26 IAPS have been reported (Shrestha et al., 2017). Majority of IAPS species in Nepal are confined to low land particularly in central Nepal (Shrestha et al., 2016). As the land use system is more intensive in lower elevation of central Nepal, it has to lead to the exposure of terrestrial ecosystem to disturbance, making the land Figure 1: Map 1: ofMap the study of thearea. (Source:study Departmentarea (Source: of Survey, Department GoN, Arc Map version of Survey, 10.2.1) GoN, Arc Map version 10.2.1) more susceptible to IAPS (Poudel, 2011). Four The climate of the study area ranges tropical to subtropical types. Based on weather data recorded at Department of Hydrology and Meteorology of Government of Nepal (GoN) for the period of last 10 years (2006-2015), the mean maximum/minimum temperature recorded at Hetauda and Dumkauli of Siwalik region were 29.63ºC/17.23ºC and119 30.82ºC/18.95ºC respectively. Similarly, the mean maximum/minimum temperature recorded in Simara and Butwal are 27.51ºC /16.46ºC and 30.04ºC/18.69ºC respectively. Rainfall showed strong seasonal patterns with highest rainfall values recorded between June to September in both physiographic regions. The present study was based on the field survey conducted during October and November 2015 and May of 2016. Where in possible, different types of vegetations were sampled in the same locality. Otherwise, vegetation located at the relatively far distance was sampled. In each of vegetation, three sample plots (20 m×50 m) were located subjectively in the area where IAPS was relatively common. A detailed analysis of vegetation was made by Modified Whittaker Nested Vegetation Sampling Plot Design (Stohlgren et al., 1995).

0.5 m × 2m

measurement 5m× 20 m

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The climate of the study area ranges tropical to literature and expert consultations. Analysis was subtropical types. Based on weather data recorded performed only for IAPS. at Department of Hydrology and Meteorology of Government of Nepal (GoN) for the period of last Numerical Analysis 10 years (2006-2015), the mean maximum/minimum Frequency: From our sampling data, the frequency temperature recorded at Hetauda and Dumkauli of of each IAPS was calculated according to Zobel et Siwalik region were 29.63ºC/17.23ºC and 30.82ºC/ al. (1987) using the following relation; 18.95ºC respectively. Similarly, the mean maximum/ Number of sample plots in which species occurred minimum temperature recorded in Simara and Frequency (%) = ×100 Butwal are 27.51ºC /16.46ºC and 30.04ºC/18.69ºC Total number of sample plots respectively. Rainfall showed strong seasonal (Note: sample plot refers to 20 m×50 m plot) patterns with highest rainfall values recorded between June to September in both physiographic Frequency of IAPS was also calculated for each of regions. the two physiographic regions (Tarai and Siwalik) using the same formula. Figure 1: Map ofThe the study present area. (Source: study Department was of Survey, based GoN, on Arc Mapthe version field 10.2.1) survey

The climate conductedof the study area during ranges October tropical to andsubtropical November types. Based 2015 on andweather dataCover: Coverage (%) of IAPS was evaluated by recorded at Department of Hydrology and Meteorology of Government of Nepal (GoN) for visualthe estimation method, considering each plot as period of lastMay 10 years of 2016.(2006-2015), Where the mean in possible, maximum/minimum different temperature types ofrecorded at Hetauda andvegetations Dumkauli of Siwalikwere regionsampled were in29.63ºC/17.23ºC the same andlocality. 30.82ºC/18.95ºC100 percent. Two types of cover i.e. species wise by respectively. Similarly, the mean maximum/minimum temperature recorded in Simara eachand IAPS for all plots and plot wise by all IAPS for Butwal are 27.51ºCOtherwise, /16.46ºC andvegetation 30.04ºC/18.69ºC located respectively. at the Rainfall relatively showed strong far seasonal patterns with distancehighest rainfall was values sampled. recorded between In each June of to vegetation,September in both three physiographiceach plot were calculated by formula given below; regions. sample plots (20 m×50 m) were located subjectively The present study was based on the field survey conducted during October and November 2015 Total coverage of particular IAPS and May of 2016.in the Where area in possible,where differentIAPS typeswas ofrelatively vegetations werecommon. sampled inA the sameFor Individual species: Cover (%) = ×100 locality. Otherwise, vegetation located at the relatively far distance was sampled. In each of Total number of sample plots vegetation, threedetailed sample plots analysis (20 m×50 m)of were vegetation located subjectively was inmade the area bywhere IAPS was relativelyModified common. A Whittakerdetailed analysis Nested of vegetation Vegetation was made bySampling Modified Whittaker Nested Vegetation Sampling Plot Design (Stohlgren et al., 1995). Results and Discussion Plot Design (Stohlgren et al., 1995). A total of 16 invasive alien plant species (IAPS) were 0.5 m × 2m 2m× 5 m recorded in Tarai and Siwalik regions. Of total, 15 spp. of IAPS were found in Siwalik region while only 12 spp. were found in Tarai region. Ipomoea Canopy carnea subsp. fistulosa was observed only in Tarai measurement

m 5m× 20 m region. Three IAPS Mikania micrantha, Senna 0 2 occidentalis and Ageratina adenophora were found only in Siwalik region. 2 Frequency and cover of IAPS 50 m Among 16 spp. of IAPS, the frequency and coverage Figure 2: Outline of modified-Whittaker nested vegetation of Chromolaena odorata was the highest while sampling plot design (Stohlgren et al., 1995) frequency of Ipomoea carnea subsp. fistulosa was At different vegetation types, a 50 m×20 m plot was the least in most of the cases. Ageratina adenophora defined. In total 36 plots were sampled in 12 sites had the lowest cover of all IAPS. and four vegetation types were selected in Tarai as By analyzing separately the frequency and coverage well as Siwalik regions. Three replicate sampling in each physiographic region, both frequency and was performed for single vegetation type. coverage of Chromolaena odorata was again the Further, the identification of collected plant highest in both physiographic regions (figure 2). In specimens was done using relevant taxonomic Siwalik region, there was highest frequency of

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Chromolaena odorata followed by Bidens pilosa, in Chromolaena odorata is prolific with up to 87,000 Ageratum houstonianum, Lantana camara and so seeds per mature plant (McFadyen, 1989). Also the on while in Tarai region, highest frequency of high frequency and coverage of Chormolaena Chromolaena odorata was followed by Spermococe odorata among 16 IAPS in both physiographic alata, Ageratum houstonianum and so on. In Siwalik regions might be due to high efficiency of region the highest coverage of Chromolaena odorata reproduction. The coverage and frequency of was followed by Ageratum houstonianum, Lantana Chromolaena odorata is low under low light camara, Parthenium hysterophorus etc. while in intensities, increased to intermediate light intensities Tarai region highest coverage of Chromolaena (Norbu, 2004). Similarly, Chromolaena odorata is odorata was followed by Ipomoea carnea sub sp. better able to tolerate high temperature (≥40ºC) and fistulosa, Parthenium hysterophorus etc. mostly invaded hot and humid region (Joshi et al., 2006). This might be the reason for high frequency and coverage in Tarai and Siwalik regions. Sal forest of Tarai and Siwalik regions of Nepal is best for availability of such understory light intensity and supports the high reproduction, rapid growth and spread of IAPS species like Chromolaena odorata (Joshi, 2001). Good dispersal ability can be attributed to relatively high frequency of Bidens pilosa and Ageratum houstonianum. Production of high number of seeds and ability to thrive in any environment are the important survival strategy of Bidens pilosa. A single plant of Bidens pilosa can produce 3000-6000 seeds (Bartolome et al., 2013). The high coverage of Parthenium hysterophorus might be due to large quantities of viable seeds with seed production of Figure 2: 20,000 per plant and is easily spread by vehicles, FigureFigure 2: 2: Frequency and coverage of IAPS in Siwalik and and farm machinery equipments (Belgeri InfestationTarai regions of IAPS of central Nepal. A. Frequency of IAPS in two Infestationphysiographic of IAPS regions & B. Coverage of IAPS in two et al., 2012). Also this species is able to germinate, Chromolaena odorata physiographic regions Chromolaena odorata grow and flower over a wide range of temperature Chromolaena odorata and photoperiods in its introduced habitat (Williams Nepal (Tiwari et al., 2005). Chromolaena odorata NepalInfestation (Tiwari et al., of 2005). IAPS & Groves, 2006). Parthenium hysterophorus is The highest coverage of Theproduction,The highest highest rapid coverage g IAPS of richness of Siwalik region than , 1996).commonly The found in areas with poor ground cover production,seeds production rapid gin , 1996).such The as wastelands, grazed pasture and roadsides as seeds(McFadyen,Tarai production region in was may be related to life history traits among 16 (McFadyen,and bioclimatic origin of species. Chromolaena amongwell 16as in construction sites (Adkins & Shabbir, coverage and frequency of coverageodorata and wasfrequency first of dominant, 2004). species Similarly, in terms of bothrata is better2014). able to It has strong allelopathic effects which enable tolerate high temperature ( , 2004). Similarly, rata is betteret al., able 2006). to toleratecoverage high temperature and frequency ( mainly in forested area. This et al.,the 2006). plant to invade and persist in wide range of result was also supported by previous4 study i.e. the ecosystems (Kanchan et al., 1980). Mikania frequency of Chromolaena odorata4 was highest in micrantha was abundant in riverine Dalbergia forest forests of Tarai in Nepal (Tiwari et al., 2005). in Siwalik regions. This was also supported by Sapkota (2007). He reported high abundance of The highest coverage of Chromolaena odorata Mikania micrantha in natural stand of Bombax ceiba might be due to small seed mass, prolific production, and plantations of Dalbergia sissoo forest in Chitwan rapid growth and high efficiency of competition for National Park. nutrition (Rejmanek, 1996). The seeds production

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Conclusion Belgeri, A.M., Sheldon, C.N., & Adkins, S.W. (2012). Screening parthenium weed (Parthenium Siwalik region have high IAPS richness than Tarai hysterophorus L.) seedling for their allelopathic region. Among 16 species of IAPS recorded in Tarai potential. Weed Science Research, 18, 727-731. and Siwalik regions, Chromolaena odorata was the most dominant species in terms of both frequency Davis, M.A., Grime, J.P., & Thompson, K. (2000). and coverage. In Siwalik region, there was highest Fluctuating resources in plant communities: A frequency of Chromolaena odorata > Bidens pilosa general theory of invasibility. Journal of Ecology, > Ageratum houstonianum > Lantana camara and 88, 528-534. so on while in Tarai region highest frequency of Dobremez, J.F. (1976). Le Neìpal: eìcologie et Chromolaena odorata > Spermococe alata>Ageratum biogeìographie. Paris France: Eìditions du Centre houstonianum and so on. Similarly, the coverage of National de la Recherche Scientifique. Chromolaena odorata is highest in both physiographic regions. Joshi, C. (2001). Invasive banmara (Chromolaena odorata): Spatial detection and prediction. (MSc Acknowledgements Thesis), International Institute for Geo- information Science and Earth Observation. We are grateful to the Head of Department of Central Enschede, Netherland. Department of Botany, Tribhuvan University and to the research project ‘Assessment of the effect of Joshi, C., Leeuw, J.D., Andal, J., Skidmore, A.K., climate change on distribution of invasive alien plant Lekhak, H.D., Duren, I.C., & Norbu, N. (2006). Indirect remote sensing of a cryptic forest species in Nepal’ which is funded under Climate understorey invasive species. Forest Ecology and Change Research Grants Program implemented by Management, 225, 243-256. the Nepal Academy of Science and Technology (NAST). We would like to thanks to Mr. Krishna Kanchan, S.D., & Jayachandra (1980). Allelopathic Prasad Sharma, Mr.Yadu Nath Poudel, Sangita effects of Parthenium hysterophorus L. to Thapa, Samikshya Banjade, Kussum Shress, leaching of inhibitors from aerial vegetative parts. Gajendra Chataut and Sonia Pujara for their Plant and Soil, 55, 61-66. continuous support during the field survey. Moreover, this work could not have been carried out Liebhold, A.M., & Tobin, P.C. (2008). Population ecology of invasions and their without the help of the management committee of management. Annual Review of Entomology, 53, Mahila sirjansil community forest; Makwanpur, 387-408. Binayi community forest; Nawalparasi, Karahiya community forest, and Ramnagar community forest; Lowe, S., Browne, M., Boudjelas, S., & Poorter, Rupandehi so they are especially acknowledged. D.M. (2000). 100 of the world’s worst invasive alien species. A selection from the global invasive References species database. The Invasive Species Specialist Group (ISSG), New Zealand: Auckland. Adkins, S., & Shabbir, A. (2014). Biology, ecology and management of the invasive parthenium weed McFadyen, R.E.C. (1989). Siam weed: A new threat (Parthenium hysterophorus L.). Pest to Australia’s north, Queensland Department of Management Science, 70, 1023-1029. lands. Plant Protection Quarterly, 4, 3-7. Bartolome, A.P., Villasenor, I.M., & Yang, W.C. Norbu, N. (2004). Invasion success of Chromolaena (2013). Bidens pilosa L. (Asteraceae): Botanical odorata in the Tarai of Nepal. (M.Sc. thesis), properties, traditional uses, phytochemistry and International Institute for Geo-information pharmacology. Evidence-Based Complementary Science and Earth Observation, Enschede, and Alternative Medicine, 13, 1-51. Netherlands.

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Poudel, A. (2011). Germination response of fallow In B.K. Joshi, H.B. KC & A.K. Acharya (Eds.), land plant species of central Nepal to allelopathic Conservation and Utilization of Agricultural effect of Parthenium hysterophorus L. (M.Sc. Plant Genetic resources in Nepal. (pp.446-452). Thesis), Central Department of Botany, Proceedings of 2nd National Workshop, 22-23 Tribhuvan University, Kathmandu, Nepal. May 2017, Dhulikhel; NAGRC, FDD, DoA and MoAD; Kathmandu, Nepal. Rejmánek, M., & Richardson, D.M. (1996). What attributes make some plant species more Simberloff, D. (2009). The role of propagule pressure invasive? Ecology, 77, 1655-1661. in biological invasion. Annual Review of Ecology, Evolution and Systematics, 40, 81-102. Sala, O.E., Chapin, F.S. III., Armesto, J.J., Berlow, R., Bloomfield, J., Dirzo, R., Huber-Sanwald, E., Stohlgren, T.J., Falkner, M.B., & Schell, L.D. (1995). Huenneke, L.F., Jackson, R.B., Kinzig, A., A modified Whittaker Nested Vegetation Leemans, R., Lodge, D., Mooney, H.A., sampling method. Vegetatio., 117, 113-121. Oesterheld, M., Poff, N.L., Sykes, M.T., Walker, B.H., Walker, M., & Wall, D.H. (2000). Global Tiwari, S., Siwakoti, M., Adhikari, B., & Subedi, K. biodiversity scenarios for the year 2100. Science, (2005). An inventory and assessment of invasive 287, 1770-1774. alien plant species of Nepal. IUCN- The World Conservation Union, Nepal. Sapkota, L. (2007). Ecology and management issue of Mikania micrantha in Chitwan national park, Williams, J.D., & Groves, R.H. (2006). The Nepal. Banko Janakari, 17, 27-39. influence of temperature and photoperiod on growth and development of Parthenium Shrestha, B.B. (2016). Invasive alien plant species hysterophorus L. Weed Research, 20, 47-52. in Nepal. In P.K. Jha, M. Siwakoti & S. Zobel, D.B., Jha, P.K., Behan, M.J., & Yadav, U.KR. Rajbhandary (Eds.), Frontiers of Botany (pp-269- (1987). A practical manual for ecology. 284). Nepal: Central Department of Botany, Kathmandu, Nepal: Ratna Pustak Distributor. Tribhuvan University. Shrestha, B.B., Siwakoti, M., & Ranjit, J.D. (2017). Status of Invasive Alien Plant Species in Nepal.

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Diversity of Butterfly and its Relationship with Plants in National Botanical Garden, Godawari, Lalitpur, Nepal

Kamal Bahadur Nepali*, Dipak Lamichhane and Srijana Shah National Botanical Garden, Godawari, Lalitpur, Nepal *E-mail: [email protected]

Abstract

Present work document, 84 butterfly species belonging to 56 genera of 6 families have been reported with their relationship with different plant species in National Botanical Garden (NBG), Nepal, conducted in 2016-2017. Hence, this garden has good diversity of butterfly and they are interrelated with each other. Butterfly depends on different plant species for nectar, shelter and to complete their life cycle and also helps to plant for pollination.

Keywords: Benefits, Distribution, Flora, Inter-relationship

Introduction Although relatively little is known about the lifecycles and ecology of the majority of butterflies Butterflies include some of the most highly and moths, it seems likely that a large number of developed insects. Their diversity is astounding more species are involved in mutually beneficial relation- than 17,000 species in the world and 660 species in ships with other organisms (Hoskins, 2017). Nepal (Smith, 2011). In Nepal, the area above 3,000 m is occupied mostly by palearctic butterflies while Several works have been done (Devries et al., 1997; the temperate, subtropical and tropical species Kunte, 1997; Tiple & Khurad, 2009) and relationship are sequentially distributed below this altitude. with plants (Pierce, et al., 1985; Rodriguez et al., The temperate zone has many micro-habitats to 1994; Peterson, 1997; Nylin & Janz, 2009) in the offer different species of butterflies (Khanal et al., globe. In Nepal, few works concerning butterfly 2013). diversity (Prajapati et al., 2000; Khanal, 2008; A few species of butterfly are generalists, able to Khanal, et al., 2013; Khanal, et al., 2014; Khanal, et exist in a wide variety of habitats. The adults can al., 2015) have been done but its relationship with feed on nectar from a wide range of flowers and the plants has not been comprehensively studied. Thus, caterpillars are able to feed on the leaves of several the study was carried out in National Botanical different types of plant. Most butterflies however Garden (NBG), Godawari to fulfil certain gaps on are far more specialised, each species having its own relationship between butterflies and plants. particular requirements regarding habitats, temperature, humidity, larval food plants and adult Objective food sources (Hoskins, 2016). Often the relationship The objective of this study is to know the diversity between a butterfly and its larval food plants only of butterfly and its relationship with plant species in benefits the butterfly and many plants actively defend National Botanical Garden (NBG), Godawari, themselves against being eaten. Passiflora plants for Lalitpur, Nepal. example produce tiny growths which resemble butterfly eggs, deterring Heliconius butterflies from Materials and Methods laying real eggs on them. They will not lay further eggs if others are already present, presumably Study area because the larvae are cannibalistic. Many plants defend themselves by producing toxins that can kill This study, National Botanical Garden Godawari larvae, but the larvae are often able to evolve (NBG) is located at Lalitpur district, Nepal. It lies methods of combating this defense. in the upper midhill region of central Nepal with

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altitude 1515 m. NBG is the first botanical garden hours varied from 2-5 hr. per day being less during of Nepal which was established in 28th October 1962 monsoon and winter seasons (August, 1-3hr./day; under the Depatrment of Plant Resources (DPR), April-May-June, 4-5 hr./day; September-December, Ministry of Forests and Soil Conservation (MoFSC), 3-4 hr./day). Thus, a total 198 hrs. sampling was Government of Nepal and it lies between 83°55'56" carried out during the entire study period. Voucher to 83°58'50" E longitude and 28°11'40" to 28°12'25" specimens collected during field visit were preserved N latitude. The garden covers an area of 82 hectare as herbarium and were identified with the help of of varying topography and exposure. It is surrounded various literatures. They were identified using by natural forest of Alnus nepalensis, Schima standard literatures (Hara & Williams, 1979; Hara wallichii Castanopsis indica, Prunus cerasoides, et al., 1982) and comparing specimens at National Pyrus pashia, Rhododendron arboretum, Herbarium and Plant Laboratories (KATH), incurva, Ilex excelsa, Berberis asiatica, Rubus Godawari, Lalitpur, Nepal. This study, National Botanical Garden Godawari (NBG) is located at Lalitpur district, Nepal. It lies in the upperellipticus, midhill region ofAstilbe central Nepal rivularis with altitude 1515 and m. NBGlies is thein first the botanical sub- garden of Nepal which was established in 28th October 1962 under the Depatrment of Plant Resources (DPR), Ministrytropical of Forests and ecological soil Conservation zone (MoFSC), (Sharma Government et of al.,Nepal 2003). and it lies betweenThere 83˚55'56”Results and Discussion to 83˚58'50” E longitude and 28˚11 40” to 2812˚ 25” N latitude. The garden covers an area of 82 hector ofare varying many topography thematic and exposure. gardens It is surrounded where by differentnatural forest ofspecies Alnus nepalensis, Schima wallichii Castanopsis indica, Prunus cerasoides, Pyrus pashia, Rhododendron arboretumOur, study showed, a total of 84 species of butterfly Ziziphusof incurva, seasonal Ilex excelsa, flowers Berberis asiatica,are being Rubus ellipticus, planted Astilbe in rivularis different and lies in the sub tropicalseasons ecological throughout zone (Sharma etthe al., year.2003). There This are studymany thematic was gardens carried where differentbelonging to 56 genera of 6 families. Among them, species of seasonal flowers are being planted in different seasons throughout the year. This study was carried out in 2016-2017.in 2016-2017. 5 spp. belong to Hesperiidae, 10 spp. belong to , 54 spp. belong to , 10 spp. belong to Papilonidae and 5 spp. belong to Pieridaeae family (Annex). Species of Nymphalidae family has been found higher than other family (figure 2). Khanal et al. (2015), showed that total species record belong to Papilonidae and 5 spp. belong to Pieridaeae family (Annex). Species of Nymphalidae family has been found higherof all than the other families family (Figure in Godavari-Phulchoki 2). Khanal et al., 2015, showed included that total species record of all the families43 in Godavari-Phulchokispp. at 1500 m,included 62 43spp. spp. at at 15002000 m, 62m, spp. 21 at spp.2000 m,at 21 spp. at 2500 m and 7 spp. at 27002500 m. m and 7 spp. at 2700 m.

60 54

y 50 l f

Figure 1: Map of study area r e t Figure 1: Map of study area t 40 Sampling u B

Specimens of the butterflies were caught and photographed by using butterfly nets and camera during f o

. 30 regular Samplingmonitoring of the total butterfly fauna throughout NBG from April 2016 to October 2017. o During this period 84 species of butterflies were identified from NBG with the help of various literatures N (Smith, 1994, 2010, 2011; Smetacek, 2017) and the collected specimens were preserve in the museum of 20 NBG. CollectionSpecimens of specimens of were the made inbutterflies April to May (spring), were June tocaught July (pre-monsoon), and August 10 10 (monsoon), September to November (post-monsoon) and December (winter). Sample collection was 10 5 5 carried photographedout every day except holiday by using(eighteen month)butterfly throughout nets the dayand from camera11:00am.-04:00pm. (5hr/day) but the sampling hours varied from 2-5 hr. per day being less during monsoon and winter 0 seasons during(August, 1-3hr./day; regular April-May-June, monitoring 4-5 hr./day; of the September-December, total butterfly 3-4 faunahr./day). Thus, a total 198throughout hrs. sampling was NBG carried outfrom during April the entire 2016 study period.to October Voucher specimens 2017. collected Hesperiidae Lycaenidae Nymphalidae Papilionidae Pieridae during field visit were preserved as herbarium and were identified with the help of various literatures. Family They wereDuring identified this using period,standard literatures 84 species (Hara & Williams, of butterflies 1979; Hara et wereal., 1982) and comparing specimens at National Herbarium and Plant Laboratories (KATH), Godawari, Lalitpur, Figure 2: Number of species with family Nepal. identified from NBG with the help of various Figure 2: Number of species with family Resultsliteratures and Discussion (Smith, 1994, 2010, 2011; Smetacek, Maximum species of butterflies were seen in June Our study showed, a total of 84 species of butterfly belonging to 56 genera of 6 families.Maximum Among species them, 5 of butterflies were seen in June to July (pre-monsoon) due to the presence different spp. belong2017) to Hesperiidae, and the 10 collectedspp. belong to specimensLycaenidae, 54 spp. were belong preserve to typesNymphalidae, of flowerin 10 spp.into the July garden. (pre-monsoon) NBG is rich in biodiversity due to wherethe presence more than 850 different plant species have been the museum of NBG. Collection2 of specimensconserved were by collectingtypes from of different flower altitudinal in the range garden. of Nepal. WeNBG found is that rich all butterflies in depend on plants for their nectar, shelter and to complete their lifecycle likewise butterfly helped in pollination for made in April to May (spring), June to Julyplants. (pre- This studybiodiversity also showed that, where 84 different more butterfly than 850 spp. haveplant 34 species different spp.have of host plant and monsoon), August (monsoon), Septembertop 3 host to plant beenof butterflies conserved were Tagetes by erecta collecting (15 spp.), Petuniafrom pinnatadifferent (8 spp.), and hybrida (8 spp.). Gilbert, 1975 & Benson, 1978, also found that a female butterfly’s decision to lay an November (post-monsoon) and December (winter).egg is made in twoaltitudinal phase: during arange pre-alighting of Nepal.phase she mustWe searchfound for a thatfood plantall upon which to Sample collection was carried out every dayland except and during abutterflies post-alighting dependphase she onmust plants assess the for suitability their nectar, of that food shelter plant for the survival of her offspring. holiday (eighteen month) throughout the day from and to complete their lifecycle likewise butterfly 11:00 am.-04:00 pm. (5hr./day) but the samplingStudy by Khanal helpedet al., 2015 in also pollination showed that Godavari-Phulchoki for plants. This (1500-2734 study m) alsois influenced mainly with subtropical to temperate bio-climatic zones as this district showed a stepwise change in elevation followed with change in ecosystem where flora and fauna of different status categories are sheltered. The localities especially the inner area of the Bardia National Park is remarkably125 interesting where multitude species of various status categories are under the safe line of protection (Khanal, 2008). Hoskins, 2017, found that, in some cases the relationship is obvious and simple e.g. butterflies pollinating flowers in exchange for nectar but in many species it is very complex and can involve several species of organism at different stages of the butterfly's lifecycle. Conclusion There are more than 850 plant spp. conserved in NBG within 82 hector area and this study found that 84 spp. of butterfly, it may be due to the natural forest in Phulchoki hill, seasonal flowers in different thematic gardens and unpolluted environment. Plants and butterflies were found to be mutually benefitted as butterfly gets nectar, shelter as well as resting place from plants and plants gets medium for pollination. In this study, top three host plants of butterflies were Tagetes erecta (15 spp.), Petunia

3 2018 Journal of Plant Resources Vol.16, No. 1 showed that, 84 different butterfly spp. have 34 General of Department of Plant Resources for their different spp. of host plant and top 3 host plant of support and encouragement. Our sincere thanks go butterflies were Tagetes erecta (15 spp.), Petunia to all the staff of National Botanical Garden, pinnata (8 spp.), and Dahlia hybrida (8 spp.). Gilbert Godawari, Lalitpur for providing necessary support (1975) & Benson (1978), also found that a female during the field work. butterfly’s decision to lay an egg is made in two phase: during a pre-alighting phase she must search References for a food plant upon which to land and during a post-alighting phase she must assess the suitability Benson, W.W. (1978). Resource partitioning in of that food plant for the survival of her offspring. passion vina butterflies. An International Journal of Organic Evolution, 32(3), 493-518. Study by Khanal et al. (2015) also showed that Godavari-Phulchoki (1500-2734 m) is influenced Devries, P.S., Murray, D., & Lande, R. (1997). mainly with subtropical to temperate bio-climatic Species diversity in vertical, horizontal and zones as this district showed a stepwise change in temporal dimensions of a fruit feeding butterfly elevation followed with change in ecosystem where community in an Ecuadorian rainforest. flora and fauna of different status categories are Biological Journal of the Linnean Society, 62, sheltered. The localities especially the inner area of 343-364. the Bardia National Park is remarkably interesting Gilbert, L.E. (1975). Annual review of ecology & where multitude species of various status categories systematic. Butterfly Ecology, 6, 365-395. are under the safe line of protection (Khanal, 2008). Hoskins (2017), found that, in some cases the Hara, H., & Williams, L.H.J. (1979). An enumeration relationship is obvious and simple e.g. butterflies of the flowering plants of Nepal. Vol. II. London, pollinating flowers in exchange for nectar but in UK: British Museum (Natural History). many species it is very complex and can involve Hara, H., Chater, A.O., & Williams, L.H.J. (1982). several species of organism at different stages of the An enumeration of the flowering plants of Nepal. butterfly’s life cycle. Vol. III. London, UK: British Museum (Natural History). Conclusion Hoskins, A. (2016). Learn about butterflies: The There are more than 850 plant spp. conserved in complete guide to the world butterflies and moths. NBG within 82 hectare area and this study found Retrive from: www.learnaboutbutterfly.com. that 84 spp. of butterfly, it may be due to the natural forest in Phulchoki hill, seasonal flowers in different Hoskins, A. (2017). Learn about butterflies: the thematic gardens and unpolluted environment. Plants complete guide to the world butterflies and moths. and butterflies were found to be mutually benefitted Retrive from: www.learnaboutbutterfly.com. as butterfly gets nectar, shelter as well as resting place Khanal, B. (2008). Diversity and status of butterflies from plants and plants gets medium for pollination. in lowland districts of west Nepal. Journal of In this study, top three host plants of butterflies were Natural History Museum, 23, 92-97. Tagetes erecta (15 spp.), Petunia pinnata (8 spp.) and Dahlia hybrida (8 spp.). So, it can be concluded Khanal, B., Chalise, M.K., & Solanki, G.S. (2013). that NBG is good place for butterfly. Threatened butterflies of central Nepal. Journal of Threatened Taxa, 5(11), 4612-4615. Acknowledgements Khanal, B., Shresth, K., & Shrestha, M.K. (2014). We are grateful to Mr. Sanjeev Kumar Rai Director Status monitoring and conservation issues of General and Mrs. Jyoti Joshi Bhatta, Deputy Director Hope (,

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Papilionidae): An endangered butterfly of Nepal. Prajapati, B., Shrestha, U., & Tamrakar, A.S. (2000). J. Nat. Hist. Mus., 28, 49-56. Diversity of butterfly in Daman area of Makawanpur district, central Nepal. Nepal Khanal, B., Chalise, M.K., & Solanki, G.S. (2015). Journal of Science and Technology, 2, 71-76. Distribution of Nymphalid butterflies (Lepidoptera: Nymphalidae) at different Rodriguez, J., Jordane, D., & Heoger, J.F. (1994). altitudinal ranges in Godavari-Phulchpki Spatial heterogeneity in a butterfly-host plant mountain forest, central Nepal. Diversity, interaction. Journal of Animal Ecology, 63(1), 31- Natural History & Conservation, 5, 41-48. 38. Kunte, K.J. (1997). Seasonal patterns in butterfly Smith, C. (1994). Butterflies of Nepal. Bangkok, abundance and species diversity in four tropical Thailand: Tecpress Services L.P. habitats in Northern Western Ghats. J. Biosci., 22(5), 593-603. Smith, C. (2010). Lepidoptera of Nepal. Kathmandu, Nepal: Sigma General Offset Press. Nylin, S., & Janz, N. (2009). Butterfly host plant range: An example of plasticity as a promoter of Smith, C. (2011). A photographic pocket guide to: speciation? Evolutionary Ecology, 23(1), 137- Butterflies of Nepal. Basantapur, Kathmandu, 146. Nepal: Himalayan Map House (P.) Ltd. . Peterson, M.A. (1997). Host plant phenology and Smetacek, P. (2017). A naturalist guide to the butterfly dispersal: Causes and consequences of butterflies of India. Malaysia: Times Offset (M) uphill movement. Ecology, 78(1), 167-180. Sdn. Bhd. Pierce, N. E., & Elgar, M. A. (1985). The influence Tiple, A.D., & Khurad, A.M (2009). Butterfly species of ants on host plant selection by Jalmenus diversity, habitats & sesonal distribution in and evagoras, a Myrmecophilous lycaenid butterfly. around Nagpur city, central India. World Journal Behav. Ecol. Sociobiol., 16, 209-222. of Zoology, 4(3), 153-162.

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Annex

ListList of butterfliesButterflies and host plants S.N. Scientific Name Comman Name Family Host plants 1. fylla Doubl. Dark judy Lycaenidae Cinnamomum tamala (Buch.-Ham.) T. Nees & Eberm. 2. Abrota ganga Moore Serageant major Nymphalidae Narcissus tazetta L. 3. Acytolepis puspa Horsfield Common Hedge Lycaenidae Stevia rebaudiana (Bertoni) Bertoni BLUE 4. Aglais cashmirensis Fruh. Indian fortoiseshell Nymphalidae Petunia hybrida Vilm., Lantana camara L. 5. Argyreus hyperbius India fritillary Nymphalidae hyssopifolia Kunth. Linnaeus 6. Ariadne merione Cramer Common castor Nymphalidae Petunia hybrida Vilm. 7. Athyma cama Moore Orange staff seargant Nymphalidae Tagetes erecta L. 8. Athyma jina Moore Bhutan sergeant Nymphalidae Tagetes erecta L., Petunia hybrida Vilm. 9. Athyma opalina Kollar Himalayan sergeant Nymphalidae Narcissus tazetta L. 10. Athyma perius Linnaeus Athyma perius Nymphalidae Tagetes erecta L. linnaeus 11. Athyma selenophora Kollar Staff sergeant Nymphalidae Woodfordia fruticosa (L.) Kurz 12. Atrophaneura latreillei Rose windmill Papilionidae Petunia hybrida Vilm. Donavan 13. caleta Hewitson Angled pierrot Lycaenidae Pyrus pashia Buch.-Ham. ex D. Don 14. Callerebia hybrid Butler Hybrid argns Nymphalidae Dahlia pinnata Cav. 15. Celaenorrhinus dhanada Himalayan yellow Hesperiidae Murraya koenigii (L.) Spreng., Lantana camara Moore banded flat L. 16. Cethosia biblis Drury Red lacewing Nymphalidae Chrysanthemum morifolium Ramat. 17. Childrena childreni Gray Common jester Nymphalidae Tagetes erecta L. 18. Colias fieldii Menetries Dark clouded yellow Pieridae Cuphea hyssopifolia Kunth. 19. Cyrestis thyodamas Common mal Nymphalidae Narcissus tazetta L. Boisduval 20. Delias belladonna Fabricius Hill jezebel Pieridae Dahlia pinnata Cav. 21. Dilipa morgiana Westwood Dillipa morgiana Nymphalidae Pyrus pashia Buch.-Ham. ex D. Don moore 22. egeon West. Orange punch Lycaenidae Drymaria cordata subsp. diandra (Blume) J. A. Duke, Tagetes erecta L. 23. Dodona eugenes Bates Tailed punch Lycaenidae Woodfordia fruticosa (L.) Kurz 24. Euploea core Cramer Comman indian craw Nymphalidae Drepanostachyum falcatum (Nees.) Keng.f., Prunus cerasoides Buch.-Ham. ex D. Don 25. Euthalia duda Stanudinger Blue duchess Nymphalidae Zinnia elegans L. 26. Euthalia nara Moore Bronze duke Nymphalidae Melampodium paludosum Kunth 27. Euthalia patala Kollar Euthalia patala kollar Nymphalidae nepalense Sweet. 28. Euthalia sahadeva Moore Green duke Nymphalidae Drepanostachyum falcatum (Nees.) Keng.f., Prunus cerasoides Buch.-Ham. ex D. Don 29. Graphium Agamemnon Tailed jay Papilionidae Tagetes erecta L., Dipsacus atratus Hook. f. & Linnaeus Thomson ex C. B. Clarke, Lantana camara L. 30. Graphium Chironides Veined Jay Papilionidae Lantana camara L. Honrath 31. Graphium cloanthus Glassy blue bottle Papilionidae Zinnia elegans L. Westwd 32. nama Doubleday Circe Nymphalidae Dahlia pinnata Cav. 33. Hypolymnas bolina Drury Great eggfly Nymphalidae Dipsacus atratus 34. Iraota timoleon Stoll Silver streak bluw Lycaenidae Narcissus tazetta L. 35. Junonia iphita Cramer Chocolate pansy Nymphalidae Chrysanthemum morifolium Ramat. 36. Kaniska cance Linnaeus Blue admiral Nymphalidae Astilbe rivularis Buch.-Ham. ex D. Don 37. Lampides boeticus Linnaeus Pea blue Lycaenidae Petunia hybrida Vilm. Dahlia pinnata Cav. 38. Lethe rohria Fabricius Common tree brown Nymphalidae Drepanostachyum falcatum (Nees.) Keng. f. 39. Lethe kansa Moore Bamboo forester Nymphalidae Hypericum uralum Buch.-Ham.ex D. Don

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40. Melanitis leda Cramer Common evening Nymphalidae Melampodium paludosum Kunth brown 41. Aaporia agathon Gray Great blank vein Pieridae mutabilis L. 42. Mycalesis perseus Fab. Common bushbrown Nymphalidae Crinum amoenum Ker Gawl. ex Roxb. 43. Neptis ananta Moore Yellow sailer Nymphalidae Narcissus tazetta L. 44. Neptis cartica Moore Plain saiter Nymphalidae Prunus cerasoides Buch.-Ham. ex D. Don 45. Neptis hylas Moore Common soviler Nymphalidae Trifolium repens L. Petunia hybrida Vilm. 46. Neptis manasa Moore Pale hockeystick Nymphalidae Impatiens scabrida DC., Salvia coccinea Buc'hoz sailer ex Etl. 47. Neptis miah Moore Small yellow sailor Nymphalidae Prunus cerasoides Buch.-Ham. ex D. Don 48. Neptis nycteus Deniceville Hockeystick sailer Nymphalidae Dahlia pinnata Cav. 49. Notocrypta curvifascia Restricted demon Hesperiidae Petunia hybrida Vilm. Felder & Felder 50. Nymphalis xanthomelas Large tortoiseshell Nymphalidae Narcissus tazetta L., Lantana camara L. Stichel 51. Orinoma damaris Gray Tigerbrown Nymphalidae Trifolium repens L., Dahlia pinnata Cav. 52. Papilio paris Fruh. Paris peacock Papilionidae Trifolium repens L. 53. Papilio polytes Linnaeus Common mormon Papilionidae Tagetes erecta L. 54. Papilio helenus Linnaeus Red hellen Papilionidae Tagetes erecta L. 55. Papilio janaka Moore Tailed redbreast Papilionidae Cuphea hyssopifolia Kunth., Dahlia pinnata Cav. 56. Papilio protenor Fruh. Spangle Papilionidae Nephrolepis cordifolia (L.) C. Presl., Mahonia napaulensis DC. 57. Parantica aglea Stoll Glassy tiger Nymphalidae Tagetes erecta L. Petunia hybrida Vilm. 58. Peris canidia Linnaeus Indian cabbage white Pieridae Tagetes erecta L. 59. Phaedyma Aspasia Fujioka Great hockeystick Nymphalidae Pyrus pashia Buch.-Ham. ex D. Don sailer 60. Polytremis eltola Hewitson Yellow spot swift Hesperiidae Tagetes erecta L., Narcissus jonquilla L. 61. Polyura athamus Drury Common nawab Nymphalidae Trifolium repens L. 62. Precis almanac Linnaeus Large silver stripe Nymphalidae Drymaria cordata subsp. diandra (Blume) J. A. Duke 63. Precis atlites Linnaeus Gery pansy Nymphalidae Lantana camara L. 64. Precis hierta Fab. Yellow pansy Nymphalidae Wisteria sinensis (Sims) Sweet 65. Precis iphita Cramer Chocolate pansy Nymphalidae Prunus cerasoides Buch.-Ham. ex D. Don 66. Precis orithya Hubner Blue pansy Nymphalidae Melampodium paludosum Kunth 67. Pseudocolodenia dan Falvous pied flat Hesperiidae Tagetes erecta L. Aeschylus 68. Rapala nissa Kollar Common flash Lycaenidae Narcissus tazetta L. Tagetes erecta L. 69. Stibochiona nicea Gray Popinjay Nymphalidae Campsis radicans (L.) Seem. 70. Sumalia dudu Westwood Nymphalidae Prunus sp. 71. Symbrenthia hypselis Moore Spotted jester Nymphalidae Trifolium repens L. 72. Symbrenthia lilaea Moore Chocolate jester Nymphalidae Narcissus jonquilla L. Tagetes erecta L. 73. Tanaecia julii Menetr. Common earl Nymphalidae Bauhinia purpurea L. 74. Eurema blanda Boisduval Three spot grass Pieridae Calanthe tricarinata Lindl. yellow 75. Tirumala limniace Cramer Blue tiger Nymphalidae Tagetes erecta L. 76. Tirumala septentrionis dark blue tiger Nymphalidae Lonicera japonica Thunb., Tagetes erecta L., Butler 77. Troides Helena Felder Common birdwing Papilionidae Campsis radicans (L.) Seem. 78. Udara albocaerulea Moore Albocaerulean Lycaenidae Prunus cerasoides Buch.-Ham. ex D. Don, Trifolium repens L. 79. Udara dilecta Moore Pale hedge blue Lycaenidae Tagetes erecta L. 80. Udaspes folus Cramer Grass demon Hesperiidae Bougainvillea glabra Choisy. 81 Vagrans egista Cramer Vagrant Nymphalidae Prunus cerasoides Buch.-Ham. ex D. Don 82. Vanessa cardui Linnaeus Peacock pansy Nymphalidae Melampodium paludosum Kunth 83. Herbst Indian red admiral Nymphalidae Bougainvillea glabra Choisy., Melampodium paludosum Kunth 84. Ypthima baldus Fab. Common five ring Nymphalidae Dahlia imperialis Roezl ex Ortgies

7 129 2018J. Pl. Res. Vol. 16, No. 1, pp 130-133, 2018 Journal of Plant Resources Vol.16, No. 1

Crateva religiosa G.Forst.: A Mythological Sacred Tree from Nepal and its Medicinal Values

Mohan Prasad Devkota Botany Department, Amrit Science Campus, Tribhuvan University, Kathmandu, Nepal E-mail: [email protected]

Abstract

This article has investigated the occurrence of Crateva religiosa G.Forst. in Nepal and its religious values in Nepalese communities. This highly sacred tree has a very small population in Patan district of Kathmandu valley although, it has been reported to occur naturally in Chitwan and Jajarkot districts of Nepal. The uses and values of this tree have not been reported from outside the valley but it has been considered as a highly sacred tree and worshiped by the local people. Agnishala temple of Patan was visited to investigate the tree population and its religious values. Non-formal interviews were conducted with the secretary of the temple management committee and the priests to record various myths associated with tree and its sacredness. The tree has received little recognition in Nepal both botanically and religiously and is poorly known due to its limited geographical distribution within the country. This article has reported the tree from Agnishala temple of Patan area, ward number 19 of Lalitpur district, central Nepal for the first time and is locally called as Varuna Briksha (j?0f j[If) with immense religious value for the people of local community. The occurrence of the tree from Patan of Lalitpur district has not been listed in the recently published Flora of Nepal Vol. 3 and is also named as Siplikan in Nepali language. Therefore, it is recommended to correct its local name and include its occurrence in the list.

Key words: , Economic importance, Siplikan, Varuna Briksha

Introduction The tree is called as Varun Briksha in Nepali language and belongs to the family Capparaceae. In Like many other sacred trees in Hindu religion, English it is known as Sacred garlic pear and Three- Crataeva religiosa G. Forst. is one of the trees that leaved whereas Varun Briksha in Sanskrit. The has been given little recognition to its sacredness in tree is also known with the name of spider tree Nepalese societies all over the country. The tree is because the showy flowers produce many long considered as sacred to Lord Shiva and the leaves are stamens resembling a spider. used to worship him during Mahashivaratri as well as Rahu (Planet Neptune), one of the nine planets or General characteristics of the tree the Navagrahas (ecs.com.np). It has been believed that the fruits of C. religiosa were considered as a Crateva religiosais deciduous tree of 3 to 15 m. supplement of energy in ancient times. This was one height with many crooked branches. Leaves are tri- of the reasons sages used to eat its fruits before going foliate compound, leaflets ovate-orbicular, dull green into deep meditation for months (ecs.com.np). Since, above and grey below. It flowers profusely during the tree grows in places with plenty of water in its March to May, when it sheds all its leaves, natural habitat; it is also ‘believed that the presence are 10-20 flowered corymbose of tree is the indicator of plenty of water in the ground . The flowers are pale to yellow, large, and this is the reason it is mythologically related to hermaphrodite, actinomorphic and complete. the God Varuna (God of water). In both India and the Flowers open in the evening between 7 PM and 9 Pacific Islands the tree is credited with occult powers PM (personal communication with Mr. Sharma). and consequently was planted around temples and Fruit is ovoid to obovoid, grey to dust colored with because of this association the specific epithet was circular ash yellow flecks and ripe from July to chosen by the original author G. Forster. August.

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Distribution and ecology 2011), the tree is reported from Chitwan and Jajarkot The tree is native to Japan, Australia, much of Districts of Nepal from 200 to 1,200 m altitude in and several South Pacific islands. It these districts along the roadside and fields. The tree is grown elsewhere for its edible fruit, especially in has been doubtfully given local name as “Siplikan”, parts of the African continent (EOL). It has global in Flora of Nepal (Vol. 3, 2011) and has been unable distribution including India, , Sri Lanka, to report it from the ward number 19 of Patan Sub- Malaysia, Indonesia and China. In India, it is found Metropolitan of Lalitpur District of Central Nepal. in Peninsular India, Western India, Gangetic Plains, There are 16 medium sized trees of Crateva religiosa and Eastern India, up to Tripura and Manipur growing in the Agnishala Temple premises of Patan (Williamson, 2002). at an elevation of 1326 m., (27º 40' 20.88" N & 85º 19’16.45" E) and the temple has been a center of The tree enjoys humid river valleys and open religious site for the local communities. monsoon forests. It has been reported that the tree prefers to grow along the river bank, streams and Medicinal properties of the tree near to temple side. Warrier (1995) and Panda (2004) Despite its immense medicinal value and uses highlighted that the tree found in periodically inundated forests but usually below 100 m, altitude. worldwide, not much information is available on its In Philippines, it has been reported to grow in waste uses in Nepal. Almost whole tree has medicinal value places, along streams and in thickets near the sea. It but especially trunk bark, leaves and root bark are is also cultivated in the gardens for its ornamental mostly used to cure many medical disorders (Patil and medicinal value as well (Sharma et al., 2006). & Gaikwad, 2011). Its berry like fruit is edible and used as astringent while young shoots are used in curries. Fruits are also used as spice because of its Materials and Methods garlic taste C. religiosa is valuable in treating Vata A simple methodology was adapted to investigate (blood flow, waste elimination and breathing), Pitta the detail information on the occurrence and (fever and metabolic disorder) and Kapha (joint medicinal values of Crateva religiosa from Nepal. lubrication, skin moisture, wound healing, strength Agnishala Temple located at ward number 19 of and vigor, memory loss, heart and lung weakness Patan was visited to observe the trees. Non-formal and weak immune system CSIR (1987). Plant is used interview were conducted with the Member Secretary ethno-pharmacologically as diuretic, laxative, of the temple Mr. B. N. Sharma and priests to lithonotriptic, antirehumatic, antiperiodic, bitter, investigate the myth associated with the tree and its tonic, rubifacient and counterirritant (Bhattachargee, religious and medicinal values. Relevant literatures 2001; Nadkarni, 1979). The bark is used in the related to flora of Nepal were searched to find out urinary disorders including kidney and bladder its general ecology and distribution pattern and other stones, antiemetic and calculous affections and as related websites were also searched. Especially, the an antidote in snakebite (Bhattachargee, 2001).Roots 3rd volume of Flora of Nepal (2011) was reviewed and bark are laxative and lithontipic and increase to find out its distribution pattern in Nepal, similarly appetite and biliary secretion (Malini et al., 1995). the KATH at Godawari, Lalitpur was also visited to Leaves are used as externally rubifacient and used study the deposited specimens. in rheumatism. According to Gurrero [http/www.bpi.da.gov.ph., Results and Discussion 2009], in Philippines, leaves are useful in irregular menstruation and also in stomachic, whereas the bark Record of occurrence of Crataeva religiosa from is used to cure convulsions and tympanites. Sanyal Nepal & Ghose [http/www.bpi.da.gov.ph., 2009], After carefully searching in the most recent botanical speculated that the crushed leaves are applied in the records of Nepal, especially Flora of Nepal,(Vol. 3, form of paste for swelling of feet and also for a

131 2018 Journal of Plant Resources Vol.16, No. 1 burning sensation in the soles of feet. The bark and is also believed to be 4,000 years old temple and the leaves are pounded and applied in the form of a since that time fire has been kept burning as poultice in rheumatism. The fresh leaves bruised everlasting (personal communication with Mr. with little vinegar applied to skin. Traditionally, the Sharma). Present population of sixteen trees of C. plant is used as oxitosic, in rheumatic fever in kidney religiosa at the temple are believed to have been stones, bladder stone and as tonic (Ghani, 1998). It propagated from the same walking stick used by the is useful as antipyretic, antilithitic, antihelminthic, brothers. The tree has been reported to occur only in demulcent, in blood and chest diseases (Dury, 1978). Patan and nowhere else in Nepal according to the temple management committee authorities. Long Berry like globose fruits of Crataeva are edible and back the Agnishala Temple was believed to be used as astringent (Parker, 1999) and rind of the fruit situated at the confluence of Bagmati River and is used as mordant in dying (Dury, 1978). Fruits of Prabhawati River (Nakkhu Khola) of Kathmandu this tree are also used as spice because of its garlic Valley which are now far separated from each other. taste (Seidmann, 2005).The juice of fruit, leaves and bark is applied to cure snakebite, infected wounds Assumption on its historical occurrence and cuts. It increases appetite and controls other skin diseases (Sapkota, 2003). The decoction of the bark If we consider the past history of the temple which is useful in the treatment of urinary organs (Sapkota, was believed to be established at the confluence of 2003), leaves are used as vegetable and the dried two rivers in the valley suggest that the tree might leaves are smoked in caries of nasal bones, the smoke have once dominated the flood plains of Bagmati being exhaled through the nose in neurologic pains and Prabhawati rivers as its natural habitat. But if (Dastur, 1962). we consider the altitude of Kathmandu Valley, is too high for the natural altitudinal distribution for Legend of the tree the tree. Assuming that, if it has once occurred in the valley floor then why it is absent from the other The trees of Varuna Brikshaya, in Agnishala temple, parts of the valley? What were the environmental are associated with very old and interesting legend factors that made this tree to disappear from the and myth. According to one legend, believed to be valley? more than 1,000 years back, two brothers named Bidyadhar and Yashodhar, during their pilgrimage, arrived to the present location of Agnishala Temple Conclusion to stay overnight (personal communication with Mr. In Hinduism, the legends are so intricately related Sharma). Next morning when they woke up they with the religious practices and sentiments of the found that the walking stick which they had been local people and are deeply rooted in the societies. carrying during pilgrimage has propagated giving Therefore, if we look at the myth associated with out roots and shoots overnight. After seeing this the tree it is very hard to believe but it is a fact that miracle they decided to settle at Agnishala temple all Hindu myths are associated with a particular God for the rest of their lives. Yashodhar stayed at which cannot be ignored as well. But regarding the Agnishala and later Bidyadhar moved to Bu:Bahal, existence of trees definitely some investigation can also known as Yashodhar Mahavihara located in be carried out to disclose the age of the tree at least. ward no. 18 of Patan. Bidyadhar later started As an attempt, dendrochronological study can help practicing Buddhism and established Yashodhar in determining the age of these trees from the temple Mahavihara. He also planted many such trees in the area. But, how it will affect the local belief system premises of the Mahavihara (Monastery) but only and the values if the trees are not found from that one has survived. It is also believed that the brothers time period? These are some of the questions that established the Agnimath and maintained fire since need to be answered very carefully by a detail that time. According to another legend, Agnishala botanical research without extinguishing temple’s

132 2018 Journal of Plant Resources Vol.16, No. 1 religious values and local belief. An inventory can Malini, M.M., Baskar, R., & Varalakshmi, P. (1995). be also carried out in the similar habitats in the valley Effect of lupeol, a pentacyclictriterpene in urinary to find out the existence of the tree as well. enzyme in hyperoxaluric rats. Japanese Journal of Concerning to the medicinal values of the tree more Medical Science and Biology, 48, 211-220. research is needed especially the traditional practices adopted by the local people supplemented by modern Nadkarni, A.K.C. (1979). Indian materia medica. analytical research. Mumbai, India: Popular publication Pvt. Ltd. Panda, H. (2004). Handbook of medicinal plants. Acknowledgements Delhi, India: Asia Pacific Publication. I am thankful to Mr. Bhushan Nath Sharma, Parker, R. N. (1999).Common Indian trees and how Secretary of the Agnishala Management Committee, to know them. (pp. 32). Jaipur, India: Pointer for providing valuable information to prepare this publication. article. Patil, U.H., & D.K. Gaikwad.(2011). Medicinal profile of a scared drug in ayurveda: Crataeva References religiosa: A review. Journal of Pharmacological Bhatachargee, S.K. (2001). Handbook of Medicinal Science& Research, 3(1), 923-929. Plants. Jaipur, India: Aavishkar Publication and Retrived from ecs.com.np. Distributors. Retrived from http/www.bpi.da.gov.ph., 2009. CSIR (1987). Crataeva: Wealth of India. Vol. II. New Delhi: National Institute of Science Communication Sapkota, J. (2003). Biologically active and Information Resources. pentacyclictriterpeneslupeol and their current medicine significations. Journal of Applied Dastur, J.F. (1962). Medicinal plants of India and Biomedicine, 31, 7-12. Pakistan. 2nd ed., Bombay. Taraporvala Sons and Company Private Limited. Seidmann, J. (2005). World spice plants: Economic usages, botany, taxonomy.(e-book). Retrived from Dury, C.H. (1978). The useful plants of India. (pp. https://www.springer.com/us/book 353). Deharadun, India: International Book Publications. Sharma, S.B., Rana, A., & Chauhan,S.V.S. (2006). Reproductive biology of Crataeva religiosa Forst. Encyclopedia of Life. (May 28, 2016). Retrived Current Science, 90(5), 716-720. from: http://eol.org/pages/485000/details. Warrier, P.K. (1995). Indian medicinal plants: A Flora of Nepal (2011). Magnoliaceae to Rosaceae. compendium of 500 species. Arya V. S. (Ed.),. Vol. III. (pp. 96-97) Nepal Academy of Science and France: Orient Publication. Technology, Nepal, Royal Botanic Garden, Edinburgh, The society of Himalayan Botany, Williamson, M. (2002). Major herbs of ayurveda. University of Tokyo. (pp. 111-116). Scotland: Churchill Livingston Publication, Elsevier Science, Ltd. Ghani, A. (1998). Medicinal plants of Bangladesh with Chemical constituents and uses. 2nd edition. (pp - 184). Dhaka: Asiatic Society of Bangladesh.

133 2018J. Pl. Res. Vol. 16, No. 1, pp 134-140, 2018 Journal of Plant Resources Vol.16, No. 1

Traditional Knowledge of Tamang Community on Utilization of Plant Resources in Dhading District, Central Nepal

Srijana Shah*, Dipak Lamichhane, Ram Panthi and Kamal Bahadur Nepali National Botanical Garden, Godawari, Lalitpur, Nepal *E-mail: [email protected]

Abstract

The present work documents 81 plant species belonging to 78 genera of 50 families used by Tamang community of Dhunibesi municipality of Dhading district, Central Nepal conducted in 2016. Group discussion was done with 30 respondents including traditional healers and knowledgeable persons both male and female. The information collected includes local name, form of use, parts used and uses. 23 plant species were used for medicinal purpose, 30 used as fodder and fruits of 20 plant species were edible other uses included firewood, timber, veterinary use, etc. This study concludes that Tamang community of the study area have been using plant resources since the past and are still dependent on it for their livelihood. Hence, this community had good traditional knowledge on using the plants for various purposes.

Keywords: Conservation, Ethnic community, Ethnobotany, Livelihood

Introduction 2003; Malla & Chhetri, 2009; Luitel et al., 2014; Shah & Lamichhane, 2017). Due to changing life Nepal is rich in biodiversity (Chaudhary, 1998). Most style, extreme secrecy of traditional healers and of the rural people directly depend on forest negligence of youngsters, the practice and biodiversity for meeting their daily requirements as dependence of ethnic societies in folk medicines is well as economic well being (MoFSC, 2014). Local in rapid decline globally. Ethnobotanical studies help people have adapted their lives according to the for conservation of cultural tradition, sustainable use resources available around them (Rajbhandary & of plants as well as for socio-economic growth of Winkler, 2015). The practice of using plant resources ethnic communities (Malla & Chhetri, 2009; Mesfin vary according to location, tradition, climatic et al., 2013). Traditional knowledge studies help for conditions and vegetation type of the place (Kunwar conservation of cultural tradition, sustainable use of & Bussmann, 2008). The ethnic communities have plants as well as for socio-economic growth of the significant customary knowledge on utilization of ethnic communities. This knowledge seems to be plant and plant parts (Acharya & Acharya, 2009). decreasing in the younger generation because of Ethnobotanical research aims to document the modernization people search for quick efficacy rather indigenous knowledge of different ethnic groups that than following the traditional methods. Therefore, have been practiced through generations for the ethnobotanical exploitation and documentation of benefit of the society (Chaudhary, 1998; Malla & indigenous knowledge about the usefulness of such Chhetri, 2009). Tamang is one of the major ethnic a vast pool of genetic resources is needs to be groups consisting of 1,539,830 populations. They continued so as to preserve traditional knowledge, cover 5.8 percent of total population of Nepal (CBS, skill and practices (Kurmi & Baral, 2004; Singh et 2011). The important settlement districts are al., 2012). Kabhrepalanchok, Dhading, Sindhupalchok and Lalitpur in central Nepal (Manandhar, 2002). Major objective of this study is documentation of traditional knowledge and indigenous practices of Several studies regarding the use of plants by Tamang community and exhibit the plant made Tamang community in different parts of Nepal have materials used by them in ethnobotanical museum been conducted in the past (Shrestha, 1988; Tamang, of National Botanical Garden, Godawari, Lalitpur,

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Nepal and plantation of important indigenous plants The information collected included local name of in ethnobotanical garden for conservation and plants, uses, form of use and parts used. The graphs information sharing. Specific objectives are, were prepared using MS-Excel. Map of the study • to know about the various plants and plant parts area was prepared by using Arc GIS. used by people of Tamang community, • to understand the purpose of using various plants Voucher specimens of 50 plant species were • to document the indigenous knowledge, skill and collected during field visit for herbarium preparation. practices of the Tamang people for future record. They were identified using standard literatures (Hara et al., 1978, 1982; Hara & Williams, 1979; Press et • to know about the various plants and plant parts used by people of Tamang community,al., 2000) and comparing specimens at National • to understand the purposeMaterials of using various and plants, Methods and Herbarium and Plant Laboratories (KATH), • to document the indigenous knowledge, skill and practices of the Tamang people for future record. Study area Godawari, Lalitpur, Nepal. The herbarium specimens were deposited in KATH. Materials and Methods Dhading district lies between 80°17' to 84°35' N Study Area longitude and 27°40' to 28°17' E latitude. Its total Results and Discussion Dhading district lies between 80area˚17' tois about84˚35' 1926N longitude sq. km. andThis 27 study˚40' to was 28 ˚carried17 E latitude. Its total area is about 1926 sq.km. This study wasout oncarried Chhatredeurali, out on Chhatredeurali, Dhunibesi municipality- Dhunibesi municipality- 1 of Altogether 1 of Dhading 81 plant spp. belonging to 78 genera of district in 2016. The site consistedDhading of subtropical district typein 2016. of vegetation. The site Theconsisted total population of 50 families was 7,687 were with collected and their local name, uses, 3,665 male and 4,022 female. subtropical type of vegetation. The total population parts used and form of uses were noted down was 7,687 with 3,665 male and 4,022 female. (Appendix 1). Two spp. of pteridophytes and remaining 76 spp. were of dicots among which 11 herbs, 19 shrubs, 37 trees, 8 climber and 4 spp. belong to monocots. The family Compositae (5 spp.) represented the highest number of plants followed by Leguminosae (4 spp.), Rosaceae (4 spp.), Solanaceae (4 spp.) and Poaceae (3 spp.). Most of the plants 30 spp. were used for fodder, 23 spp. for medicinal purposes followed by miscellanous uses (22 spp.), fruit edible (20 spp.) and others as shown in figure 2. Some of the common Themedicinal family Compositae uses were(5 spp.) representedin fever, the toothache, highest number labour,of plants followed by Leguminosae (4 spp.), Rosaceae (4 spp.), Solanaceae (4 spp.) and Poaceae (3 spp.). Mostpressure, of the plantssugar, (30 increase spp.) were lactation,used for fodder, cut (23and spp.) wounds, for medicinal purposes followed by miscellanouseye problem, uses (22 etc. spp.), Miscellanous fruit edible (20 spp.) uses and include others as shownmaking in figure 2. Some of the common medicinal uses were in fever, toothache, labour, pressure, sugar, increase lactation, cut and wounds, eye problem,soap, shampoo, etc. Miscellanous plates uses include(tapari), making button, soap, shampoo, fermentation, plates (tapari), button, fermentation, etc. Four of the plants were used for curing animal diseases. Several species were found to be used for more thanetc. one Four purpose. of the plants were used for curing animal

   

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Figure 1: Map of study area l 

Figure 1: Map of study area p  f

o 

. o Plant species were collected fromPlant the studyspecies site. were The collected taxonomic from characters the study and site. other The necessaryN  information taxonomic characters and other necessary   were noted down. To obtain detail information, the plant specimens collected from the field were exhibited during group discussion with 30information respondents were mostly noted including down. Totraditional obtain detailhealers and knowledgeable persons both male and female. Theinformation, information the collected plant specimens included collected local name from of the plants, uses, form of use, and parts used. The graphs werefield prepared were usingexhibited MS-Excel. during Mapgroup of discussion the study areawith was prepared by using USES Figure 2: Number of plants used by Tamang people for various purposes Arc GIS. 30 respondents mostly including traditional healers Figure 2: Number of plants used by Tamang people for Amongvarious the purposes different parts, leaves of most of the plants (26 spp.) were used by Tamang people for various and knowledgeable persons both male and female. purposes followed by fruit (23 spp.) and others as shown in figure 3. The study revealed that whole plant Voucher specimens of 50 plant species were collected during field visit for herbariumparts preparation. like root, rhizome, They branches, leaves, fruit, flower, bark, stem, seed, tuber, flower and tender shoots were used for medicinal purpose. 135 were identified using standard literatures (Hara et al., 1978, 1982; Hara & Williams, 1979; Press et al., 2000) and comparing specimens at National Herbarium and Plant Laboratories (KATH), Godawari,   s t

Lalitpur, Nepal. The herbarium specimens were deposited in KATH. n  a l

p  f o

.  Result and Discussion o  N   Altogether 81 plant spp. belonging to 78 genera of 50 families were collected and their local name, uses, parts used and form of uses were noted down (Appendix 1). Two spp. of pteridophytes and remaining 76 spp. were of dicots among which 11 herbs, 19 shrubs, 37 trees, 8 climber and 4 spp. belong to monocots. Parts used

Figure 3: Number of plant parts used by Tamang people

Previous studies also showed that people of Tamang community fulfill their different requirements from plants (Manandhar, 2002; Malla & Chhetri, 2009; Luitel et al., 2014). Kunwar et al. (2006) recorded 106 plants used by people of Dhading district among which most of the plants were used as medicine (96 species), followed by food, fodder, fuel wood and religious, where as in this study apart from medicinal use The family Compositae (5 spp.) represented the highest number of plants followed by Leguminosae (4 spp.), Rosaceae (4 spp.), Solanaceae (4 spp.) and Poaceae (3 spp.). Most of the plants (30 spp.) were used for fodder, (23 spp.) for medicinal purposes followed by miscellanous uses (22 spp.), fruit edible (20 spp.) and others as shown in figure 2. Some of the common medicinal uses were in fever, toothache, labour, pressure, sugar, increase lactation, cut and wounds, eye problem, etc. Miscellanous uses include making soap, shampoo, plates (tapari), button, fermentation, etc. Four of the plants were used for curing animal diseases. Several species were found to be used for more than one purpose.

 2018 Journal of Plant Resources Vol.16, No. 1  

s   t  n a

diseases.l  Several species were found to be used for vegetables, pickles, juices, jams, beverages or in

p  f

o  more. than one purpose. fermentation of alcohol. Sales of these plant o N  resources are important source of income generation Among the different parts, leaves of most of the  for poor people (Rajbhandary & Winkler, 2015). plants (26 spp.) were used by Tamang people for various purposes followed by fruit (23 spp.) and others as shown in figureUSES 3. The study revealed that Conclusion Figure 2: Number of plants used by Tamang people for various purposes whole plant parts like root, rhizome, branches, Present study shows that people of Tamang Among the different parts, leaves of most of the plants (26 spp.) were used by Tamang people for various leaves,purposes followed fruit, by flower, fruit (23 spp.) bark, and othersstem, as shownseed, in tuber,figure 3. Theflower study revealedcommunity that whole plant still practice using wild plants for various andparts like tender root, rhizome, shoots branches, were leaves, used fruit, for flower, medicinal bark, stem, purpose. seed, tuber, flower and tender shoots were used for medicinal purpose. purposes most importantly as wild edible fruits and

  for medicinal value. Though the study area is very   s

t nearby the Kathmandu valley and people mostly

n  a l p  dependent on modern medicine but still they practice f o

.  o  N   the traditional healing methods as basic treatment  for prevalent diseases in the study area. Hence, it is necessary to properly document the indigenous knowledge for future record. Ex-situ and in-situ Parts used conservation of ethnobotanically important plants Figure 3: Number of plant parts used by Tamang people Figure 3: Number of plant parts used by Tamang people should be promoted. Further study of other places is Previous studies also showed that people of Tamang community fulfill their differentalso requirements recommended. from plants (Manandhar, 2002; Malla & Chhetri, 2009; Luitel et al., 2014). Kunwar et al. (2006) recorded 106 Previousplants used by studies people of alsoDhading showed district among that which people most ofof theTamang plants were used as medicine (96 communityspecies), followed byfulfill food, fodder, their fuel different wood and religious, requirements where as in this from study apart from medicinal use Acknowledgements plants (Manandhar, 2002; Malla & Chhetri, 2009; Luitel et al., 2014). Kunwar et al. (2006) recorded We are grateful to Mr. Sanjeev Kumar Rai, Director 106 plants used by people of Dhading district among General of Department of Plant Resources for his which most of the plants were used as medicine (96 support and encouragement. Our sincere thanks goes spp.), followed by food, fodder, fuel wood and to the local people of Chhatredeurali, Dhunibesi, religious, where as in this study apart from medicinal Dhading for sharing their valuable information and use plant species were found to be used mostly as kind cooperation during this study. We would also fodder, food, religious and other uses. Their study like to thank Mr. Roshan Tamang and Ram Prasad also showed that plants of Leguminosae family were Kuikel for their assistance during the field work. the most used ones which is similar to this work where Compositae followed by Leguminosae plants References are mostly used. Leguminosae being an economically important family and also the seeds Acharya, R., & Acharya, K.P. (2009). consist of high protein content. Study conducted in Ethnobotanical study of medicinal plants used by Kavrepalanchowk district by Shah & Lamichhane Tharu community of Parroha VDC, Rupandehi (2017) and Acharya (2012) in Gulmi district also district, Nepal. Scientific World, 7(7), 80-84. found that leaves and fruits were the most frequently Acharya, R. (2012). Ethnobotanical study of used parts for edible as well as medicinal purpose medicinal plants of Resunga hill used by Magar may be because they are easily available and contain community of Badagaun VDC, Gulmi district, high concentration of bioactive compounds. Another Nepal. Scientific World, 7, 54-65. study conducted in Dhading district Aryal (2007) also showed that, people of in this district regard Aryal, K.P. (2007). Uncultivated plants: An option uncultivated plants as primary source of food. for livelihood support of the people in mid- people Various plant parts are consumed as food and wild in mid-hills of Nepal hills of Nepal hills of Nepal. food plants are used in a variety of ways such as (Master Thesis), Uppasala University, Sweden.

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137 2018 Journal of Plant Resources Vol.16, No. 1 m t r t o o r o c f e c o b e o b b b b b n f b b b b b b b b u e e e u u e e e u u e e e n i m r r r r r r r r o r r r r r e e e e e e e e e i o e e e e e e e e l L r r r r r r r r r h h h h h M H H H M S T H T T C T S S T T S S T T H H H T H , s e g r o s i t n e f u i s s o n a g e i e r e n v g p r e i . i ) a t l c e i p d a e a r r e n a d e r e i a u L t h o r , p n , c d s F n a d a g l n i t t , i m a n ( a n m d i o o b s y i a t l p b e o l d e o a t r p n a n e h a e d i h i m l o r a h p s n h c t o p u t s a r y o e s y r t n u e p g e r s d r o m y a r d b s r e n n s d e o e n i i e d u t w e g d m n n g b d o f k s o i t n e n d u n n f a a d i d a a s n e i i o m o U o n s p e s f u k e e m w m e a a a F w s r , a t l o s m r e s e a e , , , u r a r i r u l o m s s s a c e o o n g d f c t t t k r i , s d h . n n e u n u u c c s n h e i u i d i e i o i d c c c t r e g l o o p c d s d u i v n b f n d o n n n t e i e u i t i i i a s g e a i e r s i e y t o e a s k n l e e e e e e i f a M s l e s r a i u e s s s s s l g b d , , , , g r r i u i u u u u u e e e e e e e m g f e l s u r l l l l l l m s l l l l l e e o i v n p r o b b b b b b o u t a d l r a a a a s e i i i i i i f t o s n t , n , o , , n n n n n n f d d d d d d i i i i i a i r r r r r r r r r r r o u o e e e e e e e i c c c c c e e e e e e e e e e e d f t c i i i i i n t t t t t t w n d d d d d d d d d e o a i i i i i i n w w e d d d d d i e a t i d d d d d d d d d t o l u u u u u u e e e e e o o r e c a o r r r r o o o o o r o o r o l i l e s l a F F F F F W F E F F M F F F F F F c F F M M F F l M M b a f n o o e i s s s t s m e e e c r u t v e v e v e o o o c c c a a a c i i i F o e e e e u u u L d R j L j L j t t s s s i t t o e e e d u n n o v v v e r a a a s a a h l l s F e e s e e u p p e , , , , L r r r L L s s s s s s s s s s s m h s e e , e e e t e e e e e e e e e e e d d , , c , l l o d t t t t t t t r d v v v v v v v v v v v o o z o o n i i i i i w w o o o i a a a a a a n a a a a a a a h o h o u u u u u o o o h o o r e e e e e e e e e e e e r r r r r l l 1 P W F L L F L W R F W L W L T L F R L F L L W R B F L F L x i d e n m a e a r o n l p o d m e a l n a a g p i h a h e k a i n c h w p p h r a r n a e A n c r a u a c a i i g a l p g i o e a n s a g t a a b r s a h n h o n i m e t j a k m m g p a n o t i d c a t a u k a e i a r m u n m m y r l l n a k n k p j b t l a n e h y a a a a a a a h o h T h i a a a e o e a a u l M P B A S C S K K T T T T G L P D P K B T D K C a p e a e w N i u s , t m h t u r r a t a l t a a c k a a a i n h h p r h h j j s i i a K t t n i l e r p r i a u r s e e e r o t a e k i a i r i h o l t s h h u g o a i s a n o r a a i p a a l d s d t d l o p m s n s k o s i k r r e h m p o i u a i r l r n u n m a m e j i n n n j p t u t g m t N h o u h o u a a t a a a o a h h h a e i a a e a n i B G D B K B C L G B K U T S D D S T G B D T K L d a h e a D e e e e c a e e e n a a e e y a i e a a e a l i e e a e e e e e a e i s s c s r a c c e e e a a a a a e e a e y a e o a o e o t t t t t a a a a c t c i a e a i m i i l i i e t a e c i n n n e e a d e s s s s a s i e c d e u i a e i i a e a a i c c n a b r o o c o a o b o l c n e r a h c e t F a a r a r a m m m u a a e u p p c e e p p p a c l l a p r e c n u u u u i r a c n b b a u u o a m m o m p m m c m i r r m n t r t g b a g g u g r i l n c e o o a e e o y e o u o o e a e e i a o a c r m C C L C L P A A B B F L C C S B P C V L C L C L C o c n . . ) . g o L ) h . n M . t ) D . l . a B n . d R e ) m n m . D ) i a B . a b ( L x . g H x T . e - l s n o t x . . m . . f e e h c e e d h S R r L a d l o g . ( c m i . . l p m ) i l a o a V b u z o . e s l i S i a i i n l x z ( a . B g H L . a r W d o a V o o r ( - l ( N i d . d f . r . a a l . . . s u l l i ) W l l p D R s r e a u y . l l h t l L b u o L ( l l i s i e a C e i . o a c h a s L x s r D e a c . x L . a d h u ( a a p p i D w o D l c t i o e o W a . L W l c p r e L m n o a s n B o a o i p i s i a m r R . r i N o a s . a a c z i t m s a m s a a g n p d e s t u i c H d c c n s t r a b L y s s v a a e u t u g n o e i c k i p a i n o e i t . e i l n o o n i n d a n t d d r r a a d m s r r m l e b l a i d a a o i m a i r s R B s o a l n u b c n u a r s a a W u a e h i r c l E o c i i s . o l . t a a p v o s g a l s a k n l p a c b m n a a p i s u a p u l A p p H o e m a m a o o a a o i a a & i j c s e e n s p r g i i i a s o c i j o i u s i r b b t s n o s a m a t t i s a e n n r r . n a u s n o & i & o a i a l r i i u i i e c x r e a a a s u b s b n a l s t m i d C o z n e d c h h e e n r r s r t g a t i b n e l u e i u e a n r s o c r l l d e e m u u a o r t n u d t p D n b n d N r M i . r a a a a h i o o o r u g g l l r r s a a a e e i c . . t K S A B C C C C C C C C C C A A A A A A A B B B B B B A B T f o t s N i . 5 6 7 8 9 0 1 2 3 4 5 0 1 2 3 4 S L 1 1 1 1 1 2 2 2 2 2 2 2 3 4 5 6 7 8 9 1 1 1 1 1 1

138 2018 Journal of Plant Resources Vol.16, No. 1 e t y h r r p e e o b b d b b b b s b s e i s s m e e u u e u e e e e u e e u e e e e e e e n r m i e a a r r r r r r e e e e e e e e e e e e e e e e i l r e r r l r r r r r r r r r r r r r r r r e h h h h h t C T T T F S T S T T S T G T C T T T T S T S T G P T T T e c , d i s . n u o e j e a l s s r t a a l e a , u g o d l a e e ) n n t n n o b e o r i s r i a i e r n o i r d k u t o i k d i e a d f t s l f a m f a e c s p a a o e i , m ) o n t e y t e a t i v r i m l a i r t e r n i r d c s e l e k ( t s g r n u u l e p a e a n s s d r r u n o i m s n d s p a f e i d f o d m a c e e a l d n n a f , r l t p o , i t n e d a s a n e f ( m y a r b m p a a e p a n a f s i l h r i j a i e o i s s s s r a , n e p r h u r s h u c e n e t a t e r a a i s i d i p p t d s g d k g n , r u g , d d , a T u n e s a n e n d l o l p g h k u e e r r s i i s u o e l s a o c n p n m e h t w k u i s n y o i a c k a t , M c a g a u d c l e o n n e d , w g g N J i n t g m i n i u s h e m e n i , d n n n s p o n o l r c i u i i n n d a e d i t e k r d h i i b n a u i s p s e k m g t e n m h a s o a d r s o d n a s k o u p e e f n u c u t l r u t e r i r i a o m a m s s u h , t e e e o n l o s o s e m a h e k r i o u u i p r u e h g b e w u d f s m d b a e h w c m t g y o e n b u a r s s v o o i d t d i t o f e n e f i d v l q o m n a u u n n n l i o o g e d c i i o i i g n e s e n d o o d f i b s i ) w n g n o n i e i i a e i r l e i i e e e e e a i n L R W f a e s s g g r s r e d v s s s s l s r r k a , , , , i i t a v u r d o e d e a u a l l e u u u u u a o e e e e o f e e y e d e e l r l l f l e d , n c y l l l l l e e o r s d m s r r m i r s b o b b b d e e L t d a d a a a a r s c c ( w h i i i i u i l f u t o n a i i u e n o n , o n n , , n n n n s o c d d d a d b r o e i i i i i n d f r r r r r r u s u i z o s e i e o i e p e e e e j e i j i a r i c c c c c t g e e e e e e s n , e r l r t w i i i i i t r d e b g t t t t t t c t w i a d d d d d y v d b d e e e i i i i i i i i w d d d t d d e l n o e d l e l t l r d d d d d a d e l r r g i m u u u u u u u e e e e e o s r i a o n l e u e i e r r e o o r o r r e o r o r l i a e l h n e n F T p F F M F F B S F F F L A V M M F F F F F F F t w M I F R i g c M v e e y e e e g t c n c b c c s n i i i i i o f , a r u t g i u u u s o j t p e a j j j n s e c i f e d e t e t t v d k i t i o n a m h v o r a w e r s c u u e e a o a e r e a r t u o e y e L F S b P l p L R B F d S b t r s b t t l e o e d , d n n u o o v k o w a a o r a h l l o B s o a s e l p p e , W r r r B L F W s s s s s s h , e e e e e s e e e e e e d , d c d , , , l l t e t t t t t t t t d v v v v v v o o o o n o d i i i i i i i i i w w m v a a a a a a n a e o h h o o u u u u u u u u u a o e o r e e e e e e e r r r r r r r r e r l t l e F L L W L L L F L L F F F W B F W F W F W S F F S T F o d o n d g g e n n n g e a m o o l n o e g t l m o a n a e e g i r d h r n o a e a r , a n p h n i a c e p n m p m n i k u a e o y r a s c o w a u a a e a e o u s n s l e r s m k h a m k n i u o t m a r y l a a m t u g u m r t u k c a n g m n s d h l p a o m p u y o s e a a a a r h a a i i i a e i a a a i i L B A R M P B M B D K N N K S R K P K T B T L D G S t a p u o r e h s c a a A o l l a l e o a n h , i o r u e t n a r i K k m n t r e u i m a n a r r i o k u i o o a o , r l a a a a b l k a o o e u r s g i e r j a s k t k m a i u i t e i y l a h y b g t v u n a a u f i l u k n n t r m n r l a i p r u k n u n a i a s a a a i a h h h a a a a u i i h P K B O U K B B L P K M K J D A K B B T S K S S A C e a e e e c a a e a e e e e e e e e e a a d a a e a e c a e a i a c e s e e e e a e a a e a e e e p e e a c c c o a a e c e e e e e i c e c e e c a e a a a a e e a e c e n a c a a g a l i a c d a a a a e r i e e e c e a a c a c a d e e e r r n o i a n e e l h c n a a c o c e e a a c c r r d n c t a i a a m c c c t a u o e e a c a c c i n a a s r i e a a i c h i n p a u a r r s o c c a a n l r r r r a l c d a p a b s p m p e e l u g a a o e e p g i e g o o y y i t y n y i c l l e u i o a h a e r o o a r o u P J H S A M T D L A M R M O O B H R L M M P N S P C E P . . . l J n s D . . o . e h r x . H D c m s e P e ) . . s r a . . . d l y e u l & b D u s H C e a z . m P . r - x o r s a . f x a ) . . ) o . r . h . . D u e . h l W R l . k l e H L c R l b L C C ) - K . . a ) o . ( a P ( a . u s x m . r l l D . ) i o l l h ) a m o . ) a m u t a B L . W . . . a y ) c W L i s a ( e H u o . L l R H . i s L p C L h i u a c ( ( - i . a r a l ( o H L L s a t W a L p t r a f r - B D h ( ( o d c L c a - i . a m r o s n f . a m e i a c i a l r r n i s d l r f h y a a u t a r u . o u h g u r i L a d t t t u r o i d n c l c u c p d h t c l c e u L b o u a r n i b y o b e t B c a i u . a f a l e n s t l o a i t i e r i c b n o i n i l o d f c a b u p r r r l L c a i B a m l r l i a i c i e a n i s a i l o o b u a u a m a y h b l i i n g r t s m a l a s r i n a c c n e d a d d e i f e p m a o m a b u e e s f d s v p u e a y o a h l r m e n v c e e u s c a e n h z h r n o l n c t a e r a t u a c o t a a u a n s m a o a e a o l i j l a C a a s n i o o n a v k n o n s n a i d a r r ( i y r i a c a r i c c t a e s s a o u b s u a s i s e o i x m h e i t t e n y s x l h l o n u u t r c s e o r r l n p n o o p s r c p a m o p c o r i a c g s c c o u u y y t a e o r i i y i r y e i y r u i i r h a e i y u u m M M M M M N N N O D D E E F F F H I J J L L M M C C D F T L D 6 7 5 6 7 8 9 0 1 2 3 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 8 4 4 4 4 4 5 5 5 5 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 2 2 2

139 2018 Journal of Plant Resources Vol.16, No. 1 r r r r r e e e e e b b b b b b b b b b b b b b u b b b u u e u e u e e e u u e e u e u e e e r m m m m m r r r r r r r r r r r e e e e e e e e e e e i i i i i h e e e l l l l l r r r r r r r r r r r h h h h h h h h S S S T S T C S T C T T C S T S S T H C H T S H T T T C , d e e r g n l s , e n a i u o m w s r p r a s o e g p e l n l u i t h n a f t o h n n a . n , i o i s e i r r y l g t r r e e i s t t c h n l o t l . e d a o i d k e a a g d d a w d b n r c i r n g d o m m , u o i n g o p i f n o t s i t h h i s f n d s n s t s s i p m e t a t r s a o n a i k r s y i a e a , p t a b o o u y w o s t , e e w r r n n d d , t s m e w l d i o e n l o h s e o o g u b n l t f e n n p b a i e o n d o u . i i b e m f y a v i i d p l n k r e a t i o i e h w i n h e e e g s t e c t o r e t g i c v e s s i o t e w t u l o d e e t r a g a e i u l u i g e d s c o d e k s b m h d u f r b r l r e i n r s o r . e v i c n r g n a o , f v r a a f r u s d i a e d a r o s n n o o e , , o d n s i c e B f i e d a a s f i s s s d c i m l a a a c e k , t t t n g r u g i r u p o e b r s n , a . i r n u a u n u e j u t o i e a d l i e b i e e n e e i c c s c s t t d e e m r o e w c d e t i r w g d s . l r a i o e s n i n n n n u r o c d p e e d o u n m l e k i i i i i , l d o t l n k i n e s s o y i h h e o c o , n i n F s a a a e e e e e i i R F k r f a u c c F u e t d o u s , s s s s r h s g p r , , , , l e e o t s f , o m e d j a i e e f u u u u u t a p i e e e e e e l o l j h a u e o g l l l l l l y y r , s o l l l l l t e b e g s d g h s b b n b b e i a o n c d d t a d r a a a a a g g i i i i i u s e i n f u h g d , n o , n n n n h i s n n d d k d d t g g g g r i i i i i t r r r r r o e e n n i i o k e u e a e e e i c n n n c c n c c i i l l e e e e e s m e f t i i i i i i i i i a l l g b t t t t i w n a d d d d i i o e k d m i i i i i p w d k k k d d k d d i a e e i u r l d d d l n k m k o l u u u u m e a a a e e a e e o r r r s s e a e r e i r u r r o o r o i l e i l o n n n n F F M F M P c T F B M f F R M F i M B F F M M i M i M F b F T d i e e e c e e e e t t i c c c c s s i i i u i r r a a j u u u u e e p p j j j j r t t t t e w w e e f k i i o o b r o o c c a u u l l i i o o a u e r r u u F F F R j T L F R B j t t s k i t t o r e u n n t a o v r a a o h p a l l B F l o s e p p , , , , , r l u r r r r r R s s s s s s s e e e , p s e r e e e e e e e e e e e d , , d l l t t t d t t t t t t t i i e w d d v v v v v v v o o k o o k i i i i i i o i w w w o u u b r r o n a a a n a a a a h o h o u u u u u u u o o o o l r r o a a e u e e e e e e e e r r r r r r r l l l h F F F F F B W W T T L L W F F F F F L w T s L F L B L W F F L g n a a n r r r a a a a l m i y o h h a e n h m k i e g a d g h s g w l r e s p e h n a c g n o l n g i e a a o t i i g e n r c i g i e y o n k r s h n s i r p y y l s h a o a n g n g h o a s a c f a l r i g a a r k k m i p h t a a i n l d i d a n y i d a i l o a u n b a a p b a g o j t o a a n a k g g m n a y e p l h m l i r d n a w a y y h m a a a a r a h a a e h u i o a a u a o h A K M L H P W B L N K S L P N P P T L T K P P K D N o r o i s a t n l i i i i h n a a r y i e i a k a o y a p a h i m n b u i r i r d o i k b i l i n l a a h r e u a n u h s l a l o a i b l e o l o a u i a u d o e e l t y g p a r l j h a s u l o l u r i t y y k a n n t r d u a n i l u t l a i n i t a d m s u u h u i m a m a a a a h i h a h y h a i i n K A L A H B I S G A P P C R T K S C P D K N M K e e e e a a a a e e e e e e c c a c c a a a e e e a a e e e t t e a e a c a a e e a e a a e c e e a m m e a e e e e a a e a a a i e r r e c a e e c e e e a c m m c t a c e e e e e e a e e a b a e c i a a a a a e e a a e o o a c a c c c a r e e p p c a b e t n e t e e c n a c c a e a d a d a e l o c s s r c s a s c e c c c a i e a a r c i i n n n n a c a i a l a c a l u h m a a a a r b c i t a h a a a i l n n a i l c a s c b x a s s r p e t l l t a l u p n i n e e e e n x e r o u u u h o o r i u h y a m o o a o o a i S E S V R M O L M C E S P M T U S R B P R S L M L S R R n . o r s n e D . . o m K d n C x D o u . . o e x x g r r . D e h . e . e t r z D n u . ) T i D . r . . e S m . o m n m h u r b m a x . t m L . o D M f & e t t S x . r & e e K a t n r l . e H l ) o f a D o t s x l . . . ) - t o e i r t H m F . . . e r . o R m - K s a e m . u k h . ( w u . L m . u x . D a M h d K ) a r . ( b c h r o b o i u S . ( e b c a e L g ) c x . u G o i x p H L . B x . r a r o m . C l D a a g - o u s ( i L o a s B a b o t . o H u o e e w n s H u D c m a o f R B s a x h i b R S n s s c a s i R ( s i c i l S . c c t r l c S o t . e a i a n a i o s i i a a n i K u a a t i i a r a a b t u a i t c e d r ( r s i L n d R a c t l m r l d a i t i v l h u h a i o o u e i t l B n m n g u r r o u l n r o s o l o g c h i n k a e c l n a o f e e c f e f o s c i v t u r e o i i a i d l i i i e u l r i a n e r r t i a c l a s l s j u t h h a m e v n n a e i u p H c t d p o t r i h s n m a u t a r n b r a u i s t c a i n a s e n n u ) a d l v x h d l a a a o s c . g r a i w l c a r t e a a d s r r o i o o p c l e m m m u o e o n s m n o d p r u a s k o m a n f c n a x u u u d s s a p o . a a o s e c h u p s i i a a s l e s d w o l a n n n n h m c h l x d i u s e n r l p s n l i u i s u i c o n o a a a c p i n i r e w a y y o b b b y t y r i r l l l p r u h i n t n z o e o o r s s x x a i h i r y e h u u e h i r i m o o o a a c t H S S S S T T T U V W Z P P P P R R R R S S S S O O O O P G D 4 5 6 7 1 2 3 4 5 6 7 8 9 0 1 9 0 1 2 3 4 5 6 7 8 9 0 8 7 7 7 7 7 7 7 7 7 8 8 5 6 6 6 6 6 6 6 6 6 6 7 5 5 5 5 5

140 2018J. Pl. Res. Vol. 16, No. 1, pp 141-149, 2018 Journal of Plant Resources Vol.16, No. 1

Utilization Pattern of Bamboo in Madanpokhara, Tansen-8, Palpa

Munesh Ratna Gubhaju* and Anjana Bhattarai Tribhuvan Multiple Campus, Tribhuvan University, Palpa, Nepal *Email: [email protected]

Abstract

Altogether, 10 species of bamboo plants were documented along with scientific name, use categories and parts used. 10 plants species were used for agricultural implements, construction, fuel, fodder, making household tools, 9 species were used for cultural aspect, 8 species were used for food and 3 species were used for medicinal purpose. The knowledge on the traditional use of bamboo particularly medicinal value has been limited only to traditional healers and elderly people of the study area. Bamboo and its product were mainly consumed on agriculture field to make supportive stalk for plants like tomato, bean and cucumber.

Key words: Agriculture, Construction, Healers, Medicinal plants, Traditional knowledge

Introduction “Bans” and “Nigalo” in Terai, while in the high mountain it is “Bans”, “Nigalo” and “Malingo”. Bamboo plays an important role for the people, Bamboo species are found in different ecological particularly in the rural areas. It has been used for zones of Nepal. The distribution and abundance of various purposes both in the traditional as well as bamboo seem to be directly linked to the amount of modern sector. Bamboo is used in nearly every aspect rainfall (Bamboo, 2017). In previous work, bamboo of daily life. Its importance is better felt and distribution was found high in the eastern and central understood in areas where it abounds or where timber region of the country whereas western and mid and other traditional construction material are not western region had low distribution (Shrestha, 2018). readily available or are extremely expensive. It supports many major industries such as housing and Bamboos are grown in homesteads and unproductive construction, handicraft and furniture. land and are common in natural forests. From the utilization point of view, there is no other plant with The bamboos which are giant, woody, tree-like as much importance to the rural people as bamboo. grasses have a long history as exceptionally versatile In the hills, bamboos are used for making furniture, and widely-used resource. Especially in Asia, where agricultural implements, baskets, houses, bridges, it is known variously as the “poor man’s timber”, for scaffolding, fencing, fodder, fuel wood and even the “cradle-to coffin plant” and “green-gold” as water supply pipes. Besides the new shoots are bamboo has and still provides the materials needed used as vegetables. The use of bamboo varies from for existence. Bamboo is also an eminently place to place depending upon the choice of the local renewable resource under the right conditions they people as well as on the availability of a particular display prodigious rates of growth. species in that area. In the Terai, these are most commonly used in house construction, fencing, as Bamboo belongs to the family Poaceae and scaffolding for big buildings and in making baskets subfamily Bambusoideae. Globally, there are more etc. than 1573 spp. under 90 genera of bamboos, which are unevenly distributed in the various parts of the Bamboos are used in more than 180 ways in Nepal tropical, subtropical and temperate regions of the (Das, 2002). Because of its multiple uses world (Giri & Das, 2013). Das (2002) reported, 53 (construction, household, food, medicine etc.) spp. of 12 genera, which are growing naturally or in bamboo has been an important source of income, cultivated land. There are 2 types of bamboos: sustaining the livelihood of rural people. It is

141 2018 Journal of Plant Resources Vol.16, No. 1 estimated that about 3.3 million families are involved the potentially rich area for species abundance and with the bamboo sub-sector either as producers or bamboos occurring in the area were identified from user of bamboo based products (Pant, 2006). The secondary data and literature review. demand of the bamboo products is growing in the The primary data about information of preparation national and international markets due to their and utilization pattern were collected applying environment friendly nature. survey and inventory technique (Martin, 1995; In northern Indian state of Assam, the fermented Rastogi et al., 1998; Cunningham, 2001). Secondary bamboo paste known as khorisa is known locally as data or information was collected from books, a folk remedy for the treatment of impotence, articles, magazine, published or unpublished report, infertility, and menstrual pains (Bamboo, 2017). websites etc. The information about population From the ancient traditional knowledge, distribution pattern of the study area was obtained pharmaceutical preparations of bamboo shoots like from Central Bureau of Statistics, Kathmandu. bamboo salt, bamboo vinegar, bamboo extracts are The collected plant samples, prepared voucher being used to control diabetes and keep the specimens and herbarium specimens were identified cholesterol level within normal limit (Singhal et al., by consulting books on flora and other taxonomic 2013). It has been established that bamboo extract literature (Hooker, 1872-1897; Hara et al., 1978, has antioxidant activities and anti-inflammatory 1979, 1982; Polunin & Stainton, 1984; Stainton, effects (Hu et al., 2000; Jung et al., 2005). Bambusa 1988) and for further identification photographs were arundinacea is highly reputed ayurvedic medicinal used. The voucher herbarium specimens were plant (Rathod et al., 2011). submitted to Department of Botany, Tribhuvan Bamboo panels, especially floors are more and more Multiple Campus, Tansen, Palpa. in demand all over the world because they have the texture of marble and the elegance of wood; in Results addition, they are strong, durable, smooth, clean, There are 12 different ethnic/caste groups living in non-sliding and resistant to humidity. Bamboo the study area among which Magar and Sarki ethinic products which are used in household are many. groups have much knowledge about the utilization Furniture, decorative instrument, pulp and paper, pattern of bamboo product. Elder respondents and textile instruments (clothing towels, pillows, persons engaged in agriculture are found to possess blankets, etc.), cooking instruments (chopstick, sufficient knowledge on bamboo utilization. They cutting boards, etc.), in the purpose of post harvest have been using bamboo as food, traditional system as mandro, bhakari, dalo, supo, brush, chalno medicine, fuel, agriculture field, construction, etc. Weaving of bamboo strips is very popular, as household tools and cultural purpose etc. People 70% of the farmers grow bamboo on their farms and from the low economic status pay more interest on around the homesteads for the purpose (Karki et al., practices and utilization of bamboos as they are 1998). The price of bamboo culms and its products easily available and affordable. Some of the bamboo are increasing day by day but the demand for such species have been used by people for income traditionally used products is decreasing (Jha & generation, producing handicraft product and Yadava, 2015). directly selling them. The annual income of the local people ranges from NPR 10,000 to 45,000 Materials and Methods annually. The present research was carried out in Based on the uses, the bamboo species are grouped Madanpokhara, Tansen-8, Palpa, Nepal and includes into eight different categories, i.e. food, fuel, fodder, field surveys, evaluation, monitoring, formal agriculture, construction, household tools, medicine interviews and questionnaires. Before going field, and cultural aspect. Total number of bamboo species

142 species found with their use category is presented in table 1. Result showed that 10 bamboo species are 2018used for fodder, fuel, household tools, Journalagriculture of Plant tools Resources and construction purposes. Nine species Vol.16, haveNo. 1 cultural value, eight species are consumed as food and three species are used for medicinal purpose. Table 1: Plant use categories Note: Fdr = Fodder, Fl = Fuel, HT = Household tools, Ag = Agricultural tools, Cnst = Construction, Cult = Cultural aspect, Fd = Food and Med = Medicine Name of Bamboo Fdr Fl HT Ag Cnst Cult Fd Med Ampelocalamus patellaris (Gamble) Stapleton 1 1 1 1 1 1 1 Bambusa balcooa Roxb. 11 1 1 1 1 1 1 Bambusa multiplex (Lour.) Raeusch. ex Schult. 11 1 1 1 1 Bambusa nepalensis Stapleton 11 1 1 1 1 1 Bambusa tulda Roxb. 11 1 1 1 1 1 1 Dendrocalamus giganteus Munro 11 1 1 1 1 1 1 Dendrocalamus hamiltonii Neex & Arn. ex Munro 11 1 1 1 1 1 Dendrocalamus strictus (Roxb.) Nees 11 1 1 1 1 1 Himalayacalamus fimbriatus Stapleton 11 1 1 1 1 1 Melocanna baccifera (Roxb.) Kurz 11 1 1 1 Total 10 10 10 10 10 9 8 3 The result showed that all the bamboos have multiple uses, i.e. the species representing more than one founduse category. with their Melocanna use category baccifera is presented is used in for table five differentin agriculture categories practice, that 50 is houses fodder, use fuel, in construction, household 1.tools, Result agricultural showed that, tools 10 bambooand construction. spp. are used Bambusa for household multiplex tools, has differentfuel and food.six uses;46 HH fodder, use bamboo fuel, fodder,household fuel, tools, household agricultural tools, tools,agriculture construction tools and and culturalas fodder value. and Ampelocalamus 6 houses use bamboo patellaris as, medicinalBambusa constructionnepalensis, Dendrocalamuspurposes. 9 spp. hamitoniihave cultural, Dendrocalamus value, 8 purpose. strictus and Himalayacalamus fimbriatus have spp.seven are different consumed use as categories; food and 3fodder, spp. are fuel, used household for tools, agricultural tools, construction, cultural medicinalvalue and purpose.food. Bambusa balcooa, Bambusa tulda andDifferent Dendrocalamus parts of bamboosgiganteus are are used used forin alldifferent eight categories that are fodder, fuel, household tools, agriculturalpurposes. tools, Same construction, bamboo plant cultural parts value, may be food used and for Themedicine. result showed that, all the bamboos have a number of purposes and various parts of bamboos multiple uses i.e. the species representing more than may also be used for a single purpose. Among all Among the total 50 household (HH) surveyed it was found that the local people are using bamboos for one use category. Melocanna baccifera is used for 5 the collected bamboo species, maximum utilized different multiple purposes. 48 HH are use bamboo in agriculture practice, 50 houses use in differentconstruction, categories household i.e. fodder, tools, fuel, fuel household and food. tools,46 HH useplant bamboo parts are as fodderculm (stem), and 6 leafhouses and use root bamboo (rhizome) as agriculturalmedicinal purpose. tools and construction. Bambusa (10 spp.), shoot (8 spp.). multiplex has different six uses; fodder, fuel, householdDifferent partstools, of agricultural bamboos aretools, used construction for different and purposes.Plant Samesub-use bamboo categories plant parts may be used for a number of purposes and various parts of bamboos may also be used for a single purpose. Among all the cultural value. Ampelocalamus patellaris, Bambusa In each major bamboo use category different sub collected bamboo species, maximum utilized plant parts are culm (stem), leaf and root (rhizome) (10 nepalensis, Dendrocalamus hamitonii, Dendrocalamus use categories are studied. Following sections strictusspp.), shoot and Himalayacalamus (8 spp.). fimbriatus have seven Plant sub-use categories describe the number of bamboo species in different different use categories; fodder, fuel, household In each major bamboo use category different sub use categoriessub-use categories. are studied. Following sections describe tools, agricultural tools, construction, cultural value the number of bamboo species in different sub-use categories. and food. Bambusa balcooa, Bambusa tulda and Agriculture field: There are 10 bamboo spp. which DendrocalamusAgriculture field: giganteus There are are10 bambooused in species all eight whichare are used used inin agricultureagriculture field i.e. to makemake differentdifferent categoriesagricultural that equipment are fodder, and bamboofuel, household used for tools,other purposesagricultural are presented equipment in tableand bamboo2. The bamboo used for leaves other agriculturalare used in tools, providing construction, shades tocultural plant. value, The culms food arepurposes used to are make presented baskets in table(doko, 2. Theshayagu, bamboo piringo, leaves andnamlo, medicine. makhara, khondra, kholyas), vegetable stalks (forare usedtomato, in providing bean, cucumber shades toetc.), plant. greenhouse The culms and are fish traps etc. used to make baskets (doko, shayagu, piringo, Among the total 50 household (HH) surveyed, it was namlo, makhara, khondra, kholyas), vegetable stalks found that the local people are using bamboos for (for tomato, bean, cucumber etc.), greenhouse and different multiple purposes. 48 HH are use bamboo fish traps etc.

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143 2018 Journal of Plant Resources Vol.16, No. 1

Table 2: Use of Bamboo in agriculture field

Table 2: UseUses of Bamboo in agriculture field Plant name Plant part No. of species As shade Ampelocalamus patellaris Leaf 10 Uses Bambusa balcooa Plant name Plant part No. of species As shade AmpelocalamusBambusa multiplex patellaris Leaf 10 BambusaBambusa balcooa nepalensis BBambusaambusa multiplex tulda BambusaDendrocalams nepalensis hamitonii BambusaDendrocalamus tulda giganteus DendrocalamsDendrocalamus hamitonii strictus DendrocalamusHimalayacalamus giganteus fimbriatus DendrocalamusMelocanna baccifera strictus Baskets(doko, HimalayacalamusAmpelocalamus patellaris fimbriatus Culm 10 shayagu, piringo, MelocannaBambusa balcooa baccifera Baskets(doko,namlo, makhara, AmpelocalamusBambusa multiplex patellaris Culm 10 shayagu,khondra, piringo,kholyas) BambusaBambusa balcooa nepalensis namlo, makhara, BambusaBambusa multiplex tulda khondra, kholyas) BambusaDendrocalams nepalensis hamitonii BambusaDendrocalamus tulda giganteus DendrocalamsDendrocalamus hamitonii strictus DendrocalamusHimalayacalamus giganteus fimbriatus DendrocalamusMelocanna baccifera strictus Vegetable stalks HimalayacalamusAmpelocalamus patellaris fimbriatus Culm 8 (tomato, bean, MelocannaBambusa balcooa baccifera Vegetablecucumber poles)stalks AmpelocalamusBambusa nepalensis patellaris Culm 8 (tomato, bean, BambusaBambusa balcooa tulda cucumber poles) BambusaDendrocalams nepalensis hamitonii BambusaDendrocalamus tulda giganteus DendrocalamsDendrocalamus hamitonii strictus DendrocalamusMelocanna baccifera giganteus Dendrocalamus strictus Greenhouse Bambusa balcooa Culm 7 Melocanna baccifera Bambusa nepalensis Greenhouse Bambusa balcooa Culm 7 Bambusa tulda BambusaDendrocalams nepalensis hamitonii Bambusa tulda Dendrocalamus giganteus Dendrocalams hamitonii Dendrocalamus strictus Dendrocalamus giganteus Melocanna baccifera Dendrocalamus strictus Fish traps Bambusa multiplex Culm 2 Melocanna baccifera Himalayacalamus fimbriatus Fish traps Bambusa multiplex Culm 2 Culinary (food): BambooHimalayacalamus shoots are fimbriatus used as a vegetable, fermented pickle. Total eight species of Culinary (food): Bamboo shoots are used as a vegetable, fermented pickle. Total 8 spp. of bamboos were bamboos were used as food in this area. usedCulinary as food (food) in this: Bambooarea. shoots are used as a vegetable, fermented pickle. Total eight species of bamboosTable 3: Name were of used bamboo as foodspecies in which this area. are used as food Table 3: Name of bambooScientific species whichname are used as food Local name Ampelocalamus patellaris Nibha/Ghopi/Lyas Scientific name Local name Bambusa balcooa ban/bhalu/dhanu Ampelocalamus patellaris Nibha/Ghopi/Lyas Bambusa nepalensis Choya/phusre/ Tama Bambusa balcooa ban/bhalu/dhanu Bambusa tulda Taru bans Bambusa nepalensis Choya/phusre/ Tama Dendrocalamus giganteus Bhalu bans/ Dhunge bans Bambusa tulda Taru bans Dendrocalamus hamiltonii Choya/ Tama Dendrocalamus giganteus Bhalu bans/ Dhunge bans Dendrocalamus strictus Lathi bans/ Taru bans Dendrocalamus hamiltonii Choya/ Tama DendrocalamusHimalayacalamus strictus fimbriatus LathiTite bans/nigalo Taru bans HimalayacalamusConstruction: Sevenfimbriatus species are used in making house,Tite supportive nigalo poles, ladder etc., and for making ConstructionConstructionanimal case: tenSeven: Seven bamboo species species plants are are used are used used. in makingin making house, house, supportive supportive poles, poles, ladder ladder etc., andetc., for and making for making animal caseanimal ten bamboocase ten bambooplants are plants used. are used.

144 4 4 2018 Journal of Plant Resources Vol.16, No. 1

Table 4: Bamboo use as construction Table 4: Bamboo use as construction Table 4: BambooUses use as construction Bamboos name Part use No. of species use Uses Bamboos name Part use No. of species use Animal case Uses Ampelocalamus Bamboos patellaris name Culm Part use No. of species10 use Animal case Ampelocalamus patellaris Culm 10 Animal case MelocannaAmpelocalamus baccifera patellaris Culm 10 Melocanna baccifera HMelocannaimalayacalamus baccifera fimbriatus Himalayacalamus fimbriatus HBambusaaimalayacalamus balcooa fimbriatus Bambusaa balcooa BambusaaBambusa nepalensis balcooa Bambusa nepalensis Bambusa nepalensismultiplex Bambusa multiplex BambusaDendrocalamus multiplex hamiltonii Dendrocalamus hamiltonii Dendrocalamus hamiltoniigiganteus Dendrocalamus giganteus Dendrocalamus giganteusstrictus Dendrocalamus strictus DendrocalamusBambusa tulda strictus Bambusa tulda Building/ House/ BambusaDendrocalamus tulda hamiltonii Culm 7 Building/ House/ Dendrocalamus hamiltonii Culm 7 Building/Ladder/construction House/ Dendrocalamus hamiltoniigiganteus Culm 7 Ladder/construction Dendrocalamus giganteus supportiveLadder/construction poles Dendrocalamus strictusgiganteus supportive poles Dendrocalamus strictus supportive/ beam/ Electric poles pole BambusaDendrocalamus nepalensis strictus / beam/ Electric pole Bambusa tuldanepalensis Bambusa tuldabalcooa Bambusa balcooamultiplex Bambusa multiplex HouseholdHousehold tools tools: Ten: Ten bamboo bamboo plants plants are areused used as household as household tools tools for making for making basket, basket, furniture furniture and rack and and rack Householdand only 6 spp.tools are: Ten used bamboo for making plants bed. are used as household tools for making basket, furniture and rack onlyand six only species 6 spp. are are used used for for making making bed. bed. Tableand only 5: Bamboo 6 spp. usedare usedin making for makinghousehold bed. tools Table 5: Bamboo used in making household tools Table 5: BambooUses used in making household tools Bamboo name Use part No. of species use Basket(Mandro,Uses Ampelocalamus Bamboo patellaris name Culm Use part No. of species10 use Basket(Mandro,supo, dalo, chalni, AmpelocalamusBambusa multiplex patellaris Culm 10 supo,Bhakari) dalo, chalni, Bambusa multiplexnepalensis Bhakari)furniture (table) Bambusa nepalensistulda furnitureRack (table) BambusaBamusa balcooa tulda Rack DendrocalamusBamusa balcooa giganteus Dendrocalamus giganteushamiltonii Dendrocalamus hamiltoniistrictus DendrocalamusHimalayacalamus strictus fimbriatus HimalayacalamusMelocanna baccifera fimbriatus Bed MelocannaBambusa balcooa baccifera Culm 6 Bed Bambusa multiplexbalcooa Culm 6 Bambusa multiplextulda BambusaDendrocalamus tulda giganteus Dendrocalamus giganteushamiltonii Dendrocalamus hamiltoniistrictus Dendrocalamus strictus Medicine: As a medicine Dendrocalamus3 bamboo spp. strictus are used. For boils 2 bamboo plants (paste of root) are used; for Medicine:diabetesMedicine: Asand As a cholesterolamedicine medicine 3 33bamboo bamboobamboo spp. spp. plants are are used. shootused. For Forare boils boilsused 2 2bambooand bamboo for plantsinfertility plants (paste (paste and of of menstrualroot) root) are are used; painused; fortwo for diabetesbamboodiabetes and speciesand cholesterol cholesterol in fermented 3 bamboo3 bamboo form plants are plants used. shoot shoot are usedare usedand forand infertility for infertility and menstrual and menstrual pain 2 bamboopain two spp.bamboo in fermented species formin fermented are used. form are used. Table 6: Medicinal use of Bamboo Table 6: Medicinal use of Bamboo Table 6:Ailments Medicinal use of Bamboo Bamboo name Used part No. of species use CholesterolAilments Bambusa balcooa Bamboo name Shoot Used part No. of species3 use CholesterolDiabetes Bambusa balcooatulda Shoot 3 Diabetes BambusaDendrocalamus tulda giganteus Boils DendrocalamusBambusa balcooa giganteus Root 2 Boils DendrocalamusBambusa balcooa giganteus Root 2 Infertility and DendrocalamusBambusa balcooa giganteus Fermented 2 InfertilityMenstrual andpain BambusaDendrocalamus balcooa giganteus Fermentedshoot 2 Menstrual pain Dendrocalamus giganteus shoot Cultural: Bamboo is an integral part of forestry and the backbone of Nepal’s rural culture (Das, 2002). WhenCultural person: Bamboo dies, isHindu an integral people partmake of coffinforestry to andcarry the dead backbone body. Inof this Nepal’s purpose rural six culture bamboo (Das, plants 2002). are When person dies, Hindu people make coffin to carry dead body. In this purpose six bamboo plants are 145 5 5 2018 Journal of Plant Resources Vol.16, No. 1

Cultural: Bamboo is an integral part of forestry and the backbone of Nepal’s rural culture (Das, 2003). When person dies, Hindu people make coffin to carry dead body. In this purpose, 6 bamboo plants are used toused make to coffin. make Bamboocoffin. Bamboo leaves are leaves considered are considered to be pure to sitting be pure mat sittingin puja matand 9in bamboo puja, and plant’s nine leaf bamboo are usedplant's for leafthis arepurpose used andfor thisto stitch purpose leaf andplate to knownstitch leafas tapari plate andknown to makeas tapari fine and sticks to makeof bamboo fine sticks called of sinkaa,bamboo 2 bamboo called sinkaa, plants aretwo used. bamboo Whole plants plants are of used. 3 bamboo Whole spp. plants are ofused three to make bamboo Hindus species flag. are used to make Hindus flag. Table 7: Cultural use of bamboo Uses Bamboo name Used part No. of species use As a mat in cultural Ampelocalamus patellaris Leaf 8 program Bambusa balcooa Bambusa multiplex Bambusa tulda Bumbusa nepalensis Dendrocalamus giganteus Dendrocalamus hamiltonii Dendrocalamus strictus Coffin Bambusa balcooa Culm 6 Bambusa tulda Bumbusa nepalensis Dendrocalamus giganteus Dendrocalamus hamiltonii Dendrocalamus strictus Flag(Lingaa) Ampelocalamus patellaris Upper-ground 3 Bambusa multiplex part Himalayacalamus fimbriatus Sinkaa Ampelocalamus patellaris Culm 2 Himalayacalamus fimbriatus Fuel and fodder: All ten species of bamboo are used as fuel wood and fodder. Dry culms and rhizome of Fuelbamboo and fodder are used: All as 10 fire spp. wood of bamboo when it arewas used dry. asGreenBoth leaves elder are usedmainly as fodder male plant.respondents possess fuel wood and fodder. Dry culms and rhizome of sufficient knowledge on uses of bamboo because Discussion and Conclusion bamboo are used as fire wood when it was dry. Green they used them as traditional medicine, food, There are five different major ethnic groups in Madanpokhara area viz. Magar, Brahmin, Sarki, Chhetry leaves are used as fodder plant. household tools, agriculture tools, on construction, and Kami (CBS, 2014). Among these the most dominant group is Magar and Brahmin having their own cultural program and as fuel etc. People from young tradition and culture. There is close relationship between their culture and plants. Madanpokhara is Discussiondominated by and Hindu Conclusion religion. People use bamboo generationfor many purposes have little from knowledge birth to death on usesincluding of bamboos. Brahmin and Chhetry ethnic group have Thereseveral are religious five different activities major during ethnic lifespan. groups They in used sinka to stitch leaf plate (tapari) and bamboo leaf is use for sitting purpose. It is thought that bamboo leaflittle is knowledgepure for cultural about program how to and prepare bamboo bamboo plant is Madanpokhara area viz. Magar, Brahmin, Sarki, used as flag. They have been using it since time immemorial.products in comparison to Magar and Sarki ethnic Chhetry and Kami (CBS, 2014). Among these, the people. The declining of knowledge transfer to the mostBoth dominant elder mainly group male is Magar respondents and Brahmin possess having sufficientyoung knowledge generation on uses in of the bamboo study becausearea is theydue usedto theirthem own as traditional tradition medicine,and culture. food, There household is close tools, knowledgeableagriculture tools, persons on construction, feel hesitate culturalto transfer program their and as fuel, etc. People from young generation have little knowledge on uses of bamboos. Brahmin and relationship between their culture and plants. knowledge, lack of interest on traditional occupation, Chhetry ethnic group have little knowledge about how to prepare bamboo products in comparison to Madanpokhara is dominated by Hindu religion. due to lack of proper management, market price of Magar and Sarki ethnic people. The declining of knowledge, transfer to the young generation in the People use bamboo for many purposes from birth to bamboo product is not so satisfactory and the trend deathstudy including area is due several to knowledgeable religious activities persons during feel hesitate to transfer their knowledge, lack of interest on traditional occupation, due to lack of proper management,of job service market in priceurban ofarea bamboo to abroad. product People is fromnot so lifespan. They used sinka to stitch leaf plate (tapari) the low economic status and who are involved in andsatisfactory bamboo leafand theis use trend for of sitting job service purpose. in urbanIt is area to abroad. People from the low economic status and who are involved in agriculture paid more interestagriculture on practices paid and more utilization interest of onbamboo practices as they and do thought that, bamboo leaf is pure for cultural not have any alternative jobs. Elder females are utilizationmostly involved of bamboo in household as they dowork. not Theyhave rarelyany program and bamboo plant is used as flag. They have participate in outside work and economic developmentalternative job. It is jobs. concluded Elder femalesthat, due are to mostlydivision involved of labour been using it since time immemorial. between male and female, females less active in outsidein household work and work. in making They rarely bamboo participate products in outside so they have little knowledge about the bamboo as compare to elder male. In this area bamboo may one of the 146alternative sources of income and there should be initiation of awareness program to promote for bamboo production and commercialization.

6 2018 Journal of Plant Resources Vol.16, No. 1 work and economic development job. It is concluded medicinal value of bamboo plants. However majority that, due to division of labour between male and of the villagers are unfamiliar to it. Only a few old female, females less active in outside work and in people and traditional healers of Brahmin ethnic making bamboo products so they have little group are aware about the importance and use of knowledge about the bamboo as compare to elder bamboo for medicinal purpose. In Madanpokhara, male. In this area, bamboo may one of the alternative there is lack of knowledge about it, may be because sources of income and there should be initiation of of the easy access of fully facilitated hospitals in awareness program to promote for bamboo nearby the village and it is easy to take synthetic production and commercialization. medicine and it works fast as compare to traditional medicinal practice. In Madanpokhara, marginalized people with low economic status use to make house from bamboo. It Bamboo culm, rhizome can be used as fuel. In may be because it is one of the cheapest ways for Madanpokhara dry culm and rhizome are used as construction. Altogether 10 spp. of bamboo are used firewood while in other developed country, bamboo for construction purpose, e.g. in making building are used as bio-fuel, charcoal, gastification, house, as supportive beam, pole, roof, wall, ladder, briquettes, pyrolysis, pellets and biomass and many animal case etc. Similar experiences are found in other useful products which is lacking in study area other countries as well like China, Hongkong (Bamboo Import Europe, 2016). It may be because (Landler, 2002). lack of technological advancement and proper investment by related authorities. Eight bamboo spp. are used as the food. New shoots are used as vegetable, fermented bamboo, pickle as This study showed that, the local people are highly tama. Mainly in Teej festival, fry fresh tama curry is dependent on bamboo resources for various purposes the main dish for Hindus people in this area. of their daily needs. Their income from these However, the uses of bamboo for food are found to products is also almost similar with that of earlier be quite different as practiced in other communities reported from mid hills (Shakya et al., 2012). Their or country (Bamboo, 2017). income can be increased if the cultivations is properly managed. Following measures are Bamboo products are being popular in the world in recommended in order to document, preserve the form of Bamboo vinegar, bamboo wine, bamboo tea, traditional knowledge of the local people for the bamboo beer, charcoal coated peanuts etc. (Bamboo betterment of the life standards of the people. Import Europe, 2016; Bamboo, 2017) which are not Ø The bamboo species should be cultivated in a practiced in Madanpokhara area. It may be because larger scale in their personal lands as well as bare of lack of knowledge, low economic status, no part of the forest areas. This will help in market assurance, budget etc. upgrading socio-economic status. Either rural or urban area, bamboo products are used Ø Market is the main constraint for the in their houses. Furniture, muda, racks, bed, basket, development of the resources. So, the market brush, broom etc. are used in this study area. should be identified or created. The over- However in other developed countries, products like exploitation and early harvesting should be textile (cloth), paper and automobile products and discouraged. so on are prepared (Bamboo Import Europe, 2016). Ø Training should be provided to the weavers to They use them by the industrialization of bamboo produce newer products suitable for both the sources. In Madanpokhara, there is a lack of such rural as well as urban consumers. Modern project or industry. weaving accessories and techniques should be introduced at local level. However, such Some medicinal use of bamboos also found in intervention must be compatible to the local Madanpokhara. Only 6 family members know the socio-cultural conditions.

147 2018 Journal of Plant Resources Vol.16, No. 1

Acknowledgements Giri, A., & Das, A.N (2013). Bamboo. In Jha, P.K., Neupane, F.P., Shrestha, M.L., and Khanal, I.P. We are grateful to Lecturer Mr. Chandra Bahadur (Eds.), Bilogical Diversity and Conservation (pp. Thapa, Chairman, Department of Botany, T.M.C. 162-166). Khumaltar, Lalitpur: Nepal Academy Palpa, Lecturer Radhe Shyam Pathak, Pratiksha of Science and Technology (NAST). Shrestha, Toyanath Belbase and Technical Assistant Mr. Tek Bahadur Karki. We are thankful to the Hara, H., & Williams, L.H.J. (1979). An enumeration Library staff Mr. Bal Bahadur Birkatta and Mrs. of the flowering plants of Nepal. Vol II. London: Mina Neupane for providing book facilities for Trustees of British Museum (Natural History). reference work. We can’t forget the help of Binod Hara, H., Charter, A.O., & Williams, L.H.J. (1982). Bhattarai, Yub raj Gaha, Mira Khadka, Ankita An enumeration of the flowering plants of Nepal. Dhakal, Shristy K.C., Punam Ghimire, Nitu Somai Vol. III. London: Trustees of British Museum and Mrs. Bal Kumari Somai for their help during (Natural History). the compilation of this work. We are greatly indebted to local people of study area for providing valuable Hara, H., Stern, W.T., & Williams, L.H.J. (1978). information. An enumeration of the flowering plants of Nepal Vol. I. London: Trustees of British Museum References (Natural History). Bamboo Import Europe (2016). Retrived from https:/ Hooker, J.D. (1872-1897). The flora of British India. /www.bambooimport.com/en/blog/produt- made- Vol. I-VII. London: Reeve and Co. from- bamboo. Accessed on (28/09/2016). Hu, C., Zhang, Y., & Kitts, D.D. (2000). Evaluation of antioxidant and pro-oxidant activities of Bamboo (2017). Retrieved from https:// bamboo Phyllostachys nigra var. henonis leaf en.wikipedia.org/wiki/Bamboo. Accessed on (27/ extract in-vitro. J. Agril. Food Chem., 48, 3170– 07/2017). 3176. CBS (2014). National Population and Housing Jha, R.K., & Yadava, J.N. (2015). Economic Census, 2011 (Village Development Committee/ potential and marketing trend of bamboo in Municipality). Palpa. Kathmandu, Nepal: Nepal: A case study from Rautahat District. Banko Government of Nepal, National Planning Jankari, 25(1), 63-75. Commission Secretariat, Central Bureau of Statistics. Jung, H.J., Nam, J.H., Choi, J., Lee, K.T., & Park, H.J. (2005). Anti-inflammatory effects of Cunningham, A.B. (2001). Applied ethnobotany: chiisanoside and chiisanogenin obtained from the People, wild plant use and conservation. London leaves of Acanthopanax chiisanensis in the and sterling, VA: Earthscan publications Ltd. carrageenan and Freund’s complete adjuvant- Das, A.N. (2002). Bamboo growing and its market induced rats. J. Ethno. pharmacology, 97, 359- development potential for sustaining rural 367. livelihood and poverty reduction in eastern Nepal. Karki, M.B., Sherchan, G.R., & Karki, J.B.S. (1998). Banko Janakari, 12(1), 8-19. Extensive bamboo production to consumption Das, A.N. (2013). Bamboo: Socio-economic aspects. systems in Nepal: A case study. INBAR Working In P.K. Jha, F.P. Neupane, M.L. Shrestha & I.P. Paper No. 17. Beijing, China: International Khanal (Eds.), Environment and Natural Network for Bamboo and Rattan. Resources (pp. 115-120). Khumaltar, Lalitpur: Landler, M. (2002, March 27). Hong Kong Journal~ Nepal Academy of Science and Technology For Raising Skyscrapers, Bamboo Does Nicely. (NAST). New York Times.

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Martin, G.J. (1995). Ethnobotany: A methods properties of Bambusa arundinacea as a potential manual. London: Chapman and Hall. medicinal tree: An overview. J. Applied Pharmaceutical Sci., 01(10), 27-31. Pant, A. (2006). Accessing competitiveness of bamboo sub sector. A study report submitted to Shakya, M.B., Rajbhandary, S., & Das, A.N. (2012). GTZ-PSP/RUFIN, Kathmandu, Nepal. Socio-economic impacts of bamboo enterprises in the mid-hills of Nepal: A case study on Pahari Polunin, O. & Stainton, A. (1984). Flowers of the community at Badikhel Village, Lalitpur. Banko Himalayas. New Delhi, India: Oxford Press. Jankari, 22(2), 19-25. Press, J.R., Shrestha, K.K., & Sutton, D.A. (2000). Shrestha, K. (2018). Distribution and status of Annotated checklist of the flowering plants of bamboos in Nepal. Retrieved from https:// Nepal. London: Natural History Museum and www.bioversityinternational.org/fileadmin/ Kathmandu: Central Department of Botany, bioversity/publications/Web_version/572/ Tribhuvan University. ch29.htm. Accessed on (5/3/2018). Rastogi A., Godbole, A., & Shengji, P. (1998). Singhal, P., Ball, M., Satya, S., Sudhakar, P., & Naik, Applied Ethnobotany in Natural Resource S.N. (2013). Bamboo shoots: A novel source of Management-Traditional Home Gardens. nutrition and medicine, 53(5), 517-534. Kathmandu, Nepal: International Centre for Integrated Mountain Development (ICIMOD). Stainton, A. (1988). Flowers of the Himalaya- a supplement. New Delhi, India: Oxford Press. Rathod, J.D., Pathak, N.L., Patel, R.G., Jivani, N.P., & Bhatt N.M. (2011). Phyto-pharmacological

149 2018J. Pl. Res. Vol. 16, No. 1, pp 150-155, 2018 Journal of Plant Resources Vol.16, No. 1

Documentation of Ethnobotanical Knowledge of Magar Community of Palpa District, Western Nepal

Pratiksha Shrestha Department of Plant Resources, Thapathali, Kathmandu, Nepal E-mail: [email protected]

Abstract

The study has made to document the ethnobotanical knowledge of Magar community of Village Development Committee (VDC) of Palpa district, Western Nepal. Different field visits were made during 2013 to 2014 to collect the ethnobotanical knowledge of plants used for different purposes by interview, group discussion and observation. Altogether, 84 plant species belonging to 49 families and 73 genera which include 70 dicots, 13 monocots and 1 gymnosperm were recorded from study area. Out of 84 species recorded, 45 species are trees followed by 14 shrubs, 20 herbs and 5 climbers. Magar people use plants as medicine, fodder, religious, timber and other purposes. They use different plant parts for different purposes. Many plants are documented for multiple uses.

Key words: Medicinal use, Mujhung VDC, Plant parts, Traditional knowledge

Introduction in a traditional way to fulfill the requirements of their daily life. They use plants and plants products for The relationship between human beings and nature different purposes such as medicine, food, fodder, is the basis of life and the natural resources play a fuel wood, ornaments, etc. vital role. Peoples are dependent on the plants and its product not only for daily needs but also the In the census of 2011, population of Nepal was 26 essence of life. The history of human civilization million. The total caste ethnic groups of Nepal are and economic development are interwoven with 125 and spread throughout the 77 districts. There is biological resources (Ravi & Pushpangandan, 1998). no district with a single caste/ethnic group. The Nepal is multi-cast, multi-lingual, multi-religious, population of Chhetri is 4,398,053 (16.59%), Bahun, multi-cultural country. Indigenous knowledge is as 3,226,903 (12.18%), Magar, 1,321,933 (7.13%), old as the human civilization but the term Tharu, 1,737,470 (6.56%), Tamang, 1,539,830 “Ethnobotany” was first applied by an American (5.81%), Newar, 1,321,933 (4.99%) (CBS, 2012). botanist John W. Harshberger in 1896 to the study The indigenous people have traditional knowledge of plants used by primitive and aboriginal people. on use of plants which are easily available and Ethnobotany is a multi-disciplinary approach that inexpensively. contributes in analyzing how local people interact with plant resources. Thus, the main aim of Ethnobotany Several studies have been conducted to document is to document the knowledge about plants that has the ethnobotanical knowledge mainly focusing on come through generations and use the knowledge medicinal use in different parts of Nepal. In Palpa for the benefit of society (Chaudhary, 1998). district, only some work was done on ethnobotanical knowledge of Magar community (Ale et al., 2009; Ethnobotany can contribute to the conservation of Gubhaju, 2009; Joshi & Joshi, 2000; Mahato & natural resources and community development by Chaudhary, 2005; Pangeni, 2009; Singh et al., 2011). documentation and utilization of valuable But in my study area i.e. Mujhung VDC, Palpa, there indigenous knowledge on the traditional use and is no work done in knowledge of Magar community management of plant resources, among different in plant use pattern.The objective of this study is to ethnic communities. The indigenous peoples living document the ethnobotanical knowledge of the in remote rural areas use plants and plant products Magar people of Mujhung VDCs in Palpa district.

150 Materials and Methods The study was conducted in Palpa district, located in in the Western Development Region of Nepal. It lies between 27˚34'-27˚57' N and 83˚15'-83˚22' E in the central part of Nepal. This study was conducted in Mujhung VDC in 2013 to 2014. It is 16 km. in west from Tansen and bounded by Palung Mainadi in east, Chahara and in west, Somadi and Khyaha in north and Pheka and Chahara in south. The total population of VDC is 2,147 (male: 933 and female: 1,214) in 532 household. Among them, total population of Magar are 1,033 (male: 463, female: 570) (CBS, 2012). The information about ethnobotanical knowledge on plant resources was obtained by using PRA (Participatory Rural Appraisal) method. The use and values of medicinal plants, vegetables, food, fodder, fuel, edible fruits and making agricultural tools were collected with the help of interviews and focus group discussions. The interviews were conducted among the knowledgeable villagers, heads of household, elderly people, herder who had knowledge and experiences about medicinal plants and their uses. A total of 30 informants were interviewed between the age group of 20-85 years including both male and female. Plants specimens were collected from study area and identified the local name by local people and later identified scientific name with the help of standard floras (Malla et al., 1986; Polunin & Staintion, 1984; Shrestha, 2018 Journal of Plant1998; Resources Stainton, 1988). The collected plant specimens were Vol.16, deposited No. at Tribhuvan1 Multiple Campus, Tansen, Palpa.  Materials and Methods Resultpeoples and Discussionuse plants to cure different common diseases Altogether,like tooth 84 plant ache, spp. belonging common to 49 familiescold, andstomach 73 genera werepain, recorded from study The study was conducted in Palpa district, located area as ethnobotanical use values which include 70 dicots, 13 monocots and 1 spp. belongs to gymnosperm.diarrhoea, Out increase of 84 spp. belongslactation, to 45 trees, pressure, 14 shrubs, diabetes,20 herbs and 5 climbers were in Lumbini zone in the Western Development Region usedheadache as different etc. purposes they for alsodaily life. used many plants to cure of Nepal. It lies between 27°34'-27°57' N and 83°15'- Most of the plants were used as medicinal purposes followed by fodder, religious, miscellaneous,animal disease. wild edible Peoples fruit and othersused (Figure many 1). plantsMost of theas Magar dye, peoples use plants 83°22' E in the central part of Nepal. This study was tobroom, cure different vegetables, common diseases pickle, like tooth toothbrush, ache, common soap cold, stomachetc. pain, diarrhea, conducted in Mujhung VDC in 2013 to 2014. It is increase lactation, pressure, diabetes, headache etc. they also used many plants to cure animal disease.several Peoples plants used are many used plants as as multipledye, broom, purposes.vegetables, pickle, toothbrush, soap etc. 16 km. in west from Tansen and bounded by Palung several plants are used as multiple purposes. Mainadi in east, Chahara and Somadi in west, 37 Somadi and Khyaha in north and Pheka and Chahara   27  s in south. The total population of VDC is 2,147 (male: t  17 20 18 n  16 a 11 l  7 933 and female: 1,214) in 532 household. Among P  f

o

r  them, total population of Magar are 1,033 (male: e b m

463, female: 570) (CBS, 2012). The information u N about ethnobotanical knowledge on plant resources was obtained by using PRA (Participatory Rural Use catagory Appraisal) method. The use and values of medicinal Figure 1: Number of plants used by Magar community for various purposes Figure 1: Number of plants used by Magar community for plants, vegetables, food, fodder, fuel, edible fruits Amongvarious different purposes plants parts leaves (29spp.) of the most of plants are used in different and making agricultural tools were collected with purposes by Magar community followed by stems (27 spp.), fruits (18 spp.), flowers (11 spp.) the help of interviews and focus group discussions. andAmong other parts.different The whole plants plants parts were leaves also used (29 in spp.)many purposesof the (figure 2). The The interviews were conducted among the most of plants are used in different purposes by knowledgeable villagers, heads of household, elderly Magar community followed by stems (27 spp.), fruits people, herder who had knowledge and experiences (18 spp.), flowers (11 spp.) and other parts. The about medicinal plants and their uses. A total of 30 whole plants were also used in many purposes (figure informants were interviewed between the age group 2). The ethnobotanical knowledge of Magar people of 20-85 years including both male and female. by scientific name of plants, local name, nepali name, Plants specimens were collected from study area and life form, parts used and use values are given in ethnobotanical knowledge of Magar people by scientific name of plants, local name, Nepali identified the local name by local people and later tablename, life 1. form, parts used and use values are given in table 1. identified scientific name with the help of standard 35 29 30 27 s

floras (Malla et al., 1986; Polunin & Staintion, 1984; t n

a 25 l

Shrestha, 1998; Stainton, 1988). The collected plant P 18 f 20 o r specimens were deposited at Tribhuvan Multiple e 15

b 11

m 8 Campus, Tansen, Palpa. u 10

N 5 5 3 3 2 22 0 Results and Discussion Altogether, 84 plant spp. belonging to 49 families and 73 genera were recorded from study area as Plant parts use ethnobotanical use values which include 70 dicots, Figure 2: Number of plants parts used by Magar community for various purposes 13 monocots and 1 spp. belongs to gymnosperm. Figure 2: Number of plants parts used by Magar community forSingh various (2011) foundpurposes that the Magar people of Dovan VDC of Palpa had great knowledge of Out of 84 spp. belongs to 45 trees, 14 shrubs, 20 plant used in medicine purpose. They found 48 plants species to cure different diseases and medicinal plants are highly threatened due to human activities. Ale (2009) also found that, herbs and 5 climbers were used as different purposes SinghMagar people (2011) of found VDC that, has vast the knowledge Magar ofpeople using plant of resourcesDovan as medicinal, wild for daily life. VDCfruit, food, of religiousPalpa hadand other great various knowledge domestic purposes. of plant Pangeni used (2009) in showed that the medicineMagar peoples purpose. of Siluwa and They found VDCs 48 plantsof Palpa speciesdistrict used to plants for different Most of the plants were used as medicinal purposes daily purposes. She found that 63 plants species belonging to 42 families and 61 genera as curemedicinal, different fodder, spices, diseases edible and andmiscellaneous medicinal use. plants are followed by fodder, religious, miscellaneous, wild highly threatened due to human activities. Ale (2009) edible fruit and others (figure 1). Most of the Magar Conclusion alsoThe present found study that, concluded Magar that peoplethe local peopleof Siluwa of Mujhung VDC VDC has have vast knowledge about the use of plant resources for different purposes like medicinal, wild edible, fodder, fire wood, religious and others. They mainly used the plants to cure human and animal diseases. They also used many plants as religious purposes. 151

Acknowledgements I am thankfull to Jamuna Karki and Jayanti Karki for their support during field work. The author is also grateful to the people of Magar community of Mujhung VDC, Palpa for providing valuable information about ethnobotany.

References Ale R., Raskoti, B.B., & Shrestha, K. (2009). Ethnobotanical knowledge of Magar community in Siluwa VDC, Palpa district, Nepal. Journal of Natural History Museum, 24. CBS (2012). Statistical year book of Nepal. Kathmandu, Nepal: Government of Nepal, National Planning Commission Secretariate, Central Burea of Statistics. Chaudhary, R.P. (1998). Biodiversity in Nepal: Status and conservation. Uttar Pradesh, India: S. Devi,Saharanpur and Bangkok, Thailand: Tecpress Books,. 2018 Journal of Plant Resources Vol.16, No. 1 vast knowledge of using plant resources as utilization. (M.Sc. Thesis), Central Department medicinal, wild fruit, food, religious and other of Botany, Tribhuvan University, Kirtipur, various domestic purposes. Pangeni (2009) showed Kathmandu, Nepal. that the Magar peoples of Siluwa and Ringneraha Joshi, A.R., & Joshi, K. (2000). Indigenous VDCs of Palpa district used plants for different daily knowledge and uses of medicinal plants by local purposes. She found that 63 plants species belonging communities of the Kali-Gandaki Watershed to 42 families and 61 genera as medicinal, fodder, Area, Nepal. Journal of Ethnopharmacology, 73, spices, edible and miscellaneous use. 175-183. Conclusion Mahato, R.B., & Chaudhary, R.P. (2005). Ethnomedicinal study and antibacterial activities The present study concluded that the local people of selected plants of Palpa district, Nepal. of Mujhung VDC have vast knowledge about the Scientific World, 3(3), 26-30. use of plant resources for different purposes like medicinal, wild edible, fodder, fire wood, religious Malla, S.B., Rajbhandari, S.B., Shrestha, T.B., and others. They mainly used the plants to cure Adhikari, P.M., Adhikari, S.R., & Shakya, P.R. human and animal diseases. They also used many (1986). Flora of kathmandu valley. Kathmandu, plants as religious purposes. Nepal: Government of Nepal, Ministry of Forests and Soil Conservation. Acknowledgements Pangeni, B. (2009). Ethnobotanical study of Magar I am thankfull to Jamuna Karki and Jayanti Khadka community of Palpa district, western Nepal. for their support during field work. The author is (M.Sc. Thesis), Central Department of Botany, also grateful to the people of Magar community of Tribhuvan University, Kirtipur, Kathmandu, Mujhung VDC, Palpa for providing valuable Nepal. information about ethnobotany. Polunin, O. & Stainton, J.D.A. (1984). Flowers of the Himalaya. New Delhi: Oxford University References Press. Ale R., Raskoti, B.B., & Shrestha, K. (2009). Ravi, K. & Pushpangadan, P. (1998). Application of Ethnobotanical knowledge of Magar community environmental valuation techniques for economic in Siluwa VDC, Palpa district, Nepal. Journal of evaluation of biodiversity: A critical Natural History Museum, 24. investigation. New Delhi: Oxford & IBH CBS (2012). Statistical year book of Nepal. Publishers. Kathmandu, Nepal: Government of Nepal, Shrestha, K. (1998). Dictionary of Nepalese Plant National Planning Commission Secretariate, Names. Kathmandu, Nepal: Mandala Book Point. Central Burea of Statistics. Singh, A.G., Gautam, L.P., & Tewari, D.D. (2011). Chaudhary, R.P. (1998). Biodiversity in Nepal: Status Folk Use of Some Medicinal Plants of and conservation. Uttar Pradesh, India: S. VDC of Palpa District, Western Nepal. Journal Devi,Saharanpur and Bangkok, Thailand: of Phytology, 3(8),62-67. Tecpress Books,. Staiton, A. (1988). Flowers of the Himalaya: A Gubhaju, M.R. (2009). Non timber forest products supplement. India: Oxford University Press, (NTFPs) in community forests of Dovan , Palpa: Oxford. Diversity, population, status and patterns of

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Table 1: List of plants used by Magar people of Mujhung VDC, Palpa, West Nepal. *H=Herb, S=Shrub, T= Tree, C= Climber S. Nepali Life Parts Table 1: ListFamily of plants used by MagarLatin people name of Mujhung VDC, Palpa, WestMagar Nepal. name Uses **N. L= Leaves, S= Steam, F= Fruit, Fl= Flower, Rh= Rhizome, nameSe= Seed, W= Whole plant,form* R= used**Root, Br= Bark 1S. Amaranthaceae Achyranthes bidentata Blume DatiwanNepali Life H Parts W Religious, Tooth pain Family Latin name Magar name Uses 2N. Acoraceae Acorus calamus L. Bhojhoname Malabaru form* H used** Rh Medicinal 31 RutaceaeAmaranthaceae AegleAchyranthes marmelos bidentata (L.) Correa Blume BelDatiwan Bel H T W F FruitReligious, Tooth pain 2 Acoraceae Acorus calamus L. Bhojho Malabaru H Rh Medicinaledible,medicinal 34 RutaceaeAsparagaceae AegleAgave marmeloscantala (Haw.) (L.) Correa Roxb. ex BelKetuki Bel TS FS FruitFiber Salm-Dyck edible,medicinal 54 CompositaeAsparagaceae AgeratinaAgave cantala adenophora (Haw.) Roxb. (Spreng.) ex BanmaraKetuki Banmara HS LS MFiberedicinal R.M.KingSalm-Dyck & H.Rob. 65 Compositae AgeratumAgeratina conyzoidesadenophora (L.) (Spreng.) L. Banmara Gandhe Jhar GanauneBanmara Jhar H WL FodderMedicinal 7 Xanthorrhoeaceae AloeR.M.King vera (L.)& H.Rob. Burm.f. Gheukumari H L Medicinal 68 CompositaeAmaranthaceae AgeratumAmaranthus conyzoides caudatus (L.)L. L. LatteGandhe Jhar Ganaune Bhartu Jhar H Seed, W FodderMedicinal, leaves 7 Xanthorrhoeaceae Aloe vera (L.) Burm.f. Gheukumari Hleaves L Medicinalcooked as vegetable 89 AmaranthaceaeCompositae AmaranthusArtemisia indica caudatus Willd. L. LatteTitepati Bhartu Pati H S Seed, L Religious,Medicinal, medicinalleaves 10 Asparagaceae Asparagus racemosus Willd. Kurilo Bikh S leaves Youn Medicinal,cooked as vegetable young 9 Compositae Artemisia indica Willd. Titepati Pati S Lg Religious,shoot used medicinalas 10 Asparagaceae Asparagus racemosus Willd. Kurilo Bikh S Younshoot vegetableMedicinal, young 11 Leguminosae Bauhinia purpurea L. Tanki T Lg Fodder,shoot used firewood as 12 Leguminosae Bauhinia vahlii Wight & Arn. bhorla Bharala T shoot L/S vegetable Fodder, Agricultural 11 Leguminosae Bauhinia purpurea L. Tanki T L Fodder,tools, leaves firewood used as 12 Leguminosae Bauhinia vahlii Wight & Arn. bhorla Bharala T L/S Fodder,plate Agricultural 13 Leguminosae Bauhinia variegata L. Koiralo T Fl/L tools, Fodder, leaves flower used used as plateas vegetable and 13 Leguminosae Bauhinia variegata L. Koiralo T Fl/L medicine Fodder, flower used 14 Berberidaceae Berberis asiatica Roxb. ex DC. Chutro Chutara S F/R Fruitas vegetable edible, rootand medicine medicinal 14 Berberidaceae Berberis asiatica Roxb. ex DC. Chutro Chutara S F/R Fruit edible, root 15 Saxifragaceae Bergenia ciliata (Haw.) Sternb. Pakhane Pakhane H W Medicinal medicinal 16 javanica (L.) Merr. Bhakimlo S F Fruit edible, 15 Saxifragaceae Bergenia ciliata (Haw.) Sternb. Pakhane Pakhane H W Medicinal medicinal 16 Simaroubaceae Brucea javanica (L.) Merr. Bhakimlo S F Fruit edible, 17 Apocynaceae Calotropis gigantea (L.) Dryand. Aank Aank S W medicinalReligious 1718 ApocynaceaeFagaceae CalotropisCastanopsis gigantea acuminatissima (L.) Dryand. AankMusure PataleAank Jheru TS W S TimberReligious 18 Fagaceae Castanopsis(Blume) A.DC. acuminatissima MusureKatus Patale Jheru T S Timber 19 Fagaceae (Blume)Castanopsis A.DC. indica (Roxb. ex KatusDalne Katus Jheru T S/F Fruit edible, timber, 19 Fagaceae CastanopsisLindl.) indica (Roxb. ex Dalne Katus Jheru T S/F agricultural Fruit edible, tools timber, 20 Zingiberaceae Lindl.)Cautleya spicata (Sm.) Baker Jangali Gandhak H Rh Magriculturaledicinal tools 20 Zingiberaceae Cautleya spicata (Sm.) Baker Jangalibesar Gandhak H Rh Medicinal 21 Apiaceae Centella asiatica (L.) Urb. besar Ghodtapre Tapre H W Medicinal 2122 ApiaceaeAmaranthaceae CentellaChenopodium asiatica album (L.) L.Urb. GhodtapreLude Seto Tapre lude H S We M Medicinaledicinal 2223 AmaranthaceaeLauraceae ChenopodiumCinnamomum albumtamala L. (Buch.- TejpatLude Seto Tejpat lude H T S Le M Usededicinal as spices 23 Lauraceae CinnamomumHam.) T.Nees tamala& Eberm. (Buch.- Tejpat Tejpat T L Used as spices 24 Menispermaceae Ham.)Cissampelos T.Nees pareira & Eberm. L. Badalpate C R medicinal 2425 MenispermaceaeCapparaceae CissampelosCrateva unilocularis pareira L.Buch.- BadalpateSiplighan Sibli CT L/BrR Fmodder,edicinal m edicinal, 25 Capparaceae CratevaHam. unilocularis Buch.- Siplighan Sibli T L/Br usedFodder, as vegetablemedicinal, 26 Convolvulaceae Ham.Cuscuta reflexa Roxb. Aakas beli Aakas beli C S used Medicinal as vegetable 2627 ConvolvulaceaeSolanaceae CuscutaDatura metel reflexa L. Roxb. AakasDhaturo beli Aakas beli C S SF MedicinalReligious, medicinal 2728 SolanaceaePoaceae DaturaDendrocalamus metel L. hamiltonii Nees Tama Dhaturo Bas Churga S S/L/y F Fodder, Religious, religious, medicinal 28 Poaceae Dendrocalamus& Arn. ex Munro hamiltonii Nees Tama Bas Churga S S/L/young agriculturalFodder, religious, & Arn. ex Munro shootoung tools,youngagricultural shoot shoot usedtools,young as vegetable shoot 29 Dioscoreaceae Dioscorea alata L. Ban tarul Namya C T used Edible as vegetable 29 Dioscoreaceae Dioscorea alata L. Ban tarul Namya C T Edible

153 2018 Journal of Plant Resources Vol.16, No. 1

30 Dioscoreaceae Dioscorea bulbifera L. Gitthaa C T Edible 31 Ebenaceae Diospyros malabarica (Desr.) Khalluk Khalluk T F/S Fruit edible, timber Kostel. 32 butyracea (Roxb.) Chiuri Chiuri T F/L Religious, medicinal, H.J.Lam leaves used as plate 33 Poaceae Drepanostachyum intermedium Nigalo Nigalo S S Religious (Munro) Keng f. 34 Juglandaceae Engelhardia spicata var. integra mauwaa T S Timber, Agricultural (Kurz) Grierson & Long tools 35 Leguminosae Erythrina stricta Roxb. Phaledo T L Fodder 36 Pentaphylacaceae Eurya acuminata DC. Jhingane Jhingane T L/Fl Fodder, medicinal 37 Euphorbiaceae Falconeria insignis Royle Khirro T S Timber, agricultural tools 38 Moraceae Ficus benghalensis L. Bar Bar T W Religious 39 Moraceae Ficus benjamina L. Swami T W Religious 40 Moraceae Ficus glaberrima Blume Bedulno Pakhri T L Fodder 41 Moraceae Ficus hispida L.f. Thotne T L Fodder 42 Moraceae Ficus lacor Buch.-Ham Kavro Ghonsibar T Fl/L Fodder, medicinal, young flower used as vegetable 43 Moraceae Ficus religiosa L. Peepal T W Religious 44 Moraceae Ficus semicordata Buch.-Ham. Khaniyo T F/S Fruit edible, fodder, ex Sm. Religious 45 floribunda Wall. Lakuri T S/L Fodder, Timber, fire wood 46 Compositae Gamochaeta malvinensis Buki phool Bokisar H W Religious (H.Koyama) T.R.Dudley 47 Burseraceae Garuga pinnata Roxb. Dabdabe T L/S Fodder, Fire wood, medicinal 48 Grewia optiva J.R.Drumm. ex Phosro T L/S Fodder, Timber, Burret firewood 49 Poaceae Imperata cylindrica (L.) Shiru H R/W Fodder, medicinal Raeusch. 50 Acanthaceae Justicia adhatoda L. Asuro S Fl Medicinal 51 Lauraceae Litsea monopetala (Roxb.) Pers. Kutmero T L/S Fodder, timber, firewood 52 Ericaceae Lyonia ovalifolia (Wall.) Drude Angeri T Fl Medicinal 53 Anacardiaceae indica L. Aap Satak T F Fruit edible, bark used as dye 54 Meliaceae Melia azedarach L. Bakaino T S Timber, firewood 55 Lamiaceae Mentha spicata L. Pudina H W Medicinal, leaves used as pickle 56 Moraceae Morus serrata Roxb. Kimbu Khembe T L/F Fruit edible, fodder 57 Musaceae Musa paradisiaca L. Ban kera Dinla H Fl/F Fruit edible, flower medicinal 58 Myricaceae Myrica esculenta Buch.-Ham. ex Kaphal T F/S Fruit edible, timber, D. Don religious, firewood 59 Oleaceae Nyctanthes aculeata Craib Parijat T Fl Religious 60 Santalaceae Osyris lanceolata Hochst. & Nundhiki T L/S Fodder, firewood Steud. 61 Oxalidaceae Oxalis corniculata L. Chariamilo Jhyamruk H W Medicinal, leaves used as pickle 62 Phyllanthaceae Phyllanthus emblica L. Amala Ghormet T F Fruit edible, medicinal 63 Urticaceae Pilea scripta (Buch.-Ham. ex D. Chiple T L Fodder Don)

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64 Pinaceae Pinus roxburghii Sarg. Salla Dansin T S Timber 65 Rosaceae Prunus cerasoides Buch.-Ham. Paiyu T FL/L Fodder, religious ex D. Don 66 Rosaceae Prunus persica (L.) Batsch Aaru Ghurli T F/S Fruit edible, firewood 67 Rosaceae Pyrus pashia Buch.-Ham. ex D. Mayal Ghorpal T F/S Fruit edible, timber, Don religious 68 Ericaceae Rhododendron arboreum Sm. Laligurans Rogan T Fl Religious, medicinal 69 Rosaceae Rubus ellipticus Sm. Ainselu Cighwan S F Fruit edible 70 Poaceae Saccharum officinarum L. Uukhu Khum H S Religious 71 Sapindaceae Sapindus mukorossi Gaertn. Rittha Jharlyan T F/L Fodder, fruit used as soap 72 Theaceae Schima wallichii Choisy Chilaune Ghyansin T S Timber, agricultural tools 73 Shorea robusta Gaertn. Saal Phoksin T S/L Timber, religious, agricultural tools, leaves used as plate 74 Malvaceae Sida acuta Burm.f. Balu jhar Balu H R Medicinal 75 Menispermaceae Stephania elegans Hook. f. & Gane gurjo C R Medicinal Thomson 76 Syzygium cumini (L.) Skeels Phader Kyamuna T F/S Fruit edible, timber, medicinal 77 Combretaceae Terminalia alata Wall. Saaja Darsin T L/S Fodder, timber 78 Poaceae Themeda triandra Forssk. Khar Gijan H W Fodder 79 Poaceae Thysanolaena latifolia (Roxb. ex Amriso S L/Fl Fodder, flower used Hornem.) Honda as broom 80 Meliaceae Toona ciliata M.Roem. Tuni T S Timber, firewood 81 Urticaceae Urtica dioica L. Sisno Ghyo H L Medicinal, leaves used as vegetable 82 Woodfordia fruticosa (L.) Kurz Dhayero Jharek S L/Fl Fodder, medicinal 83 Rutaceae Zanthoxylum armatum DC. Timur S Se Medicinal, seed used in pickle 84 Zingiberaceae Zingiber officinale Roscoe Aduwa Chebuk H Rh Medicinal *H=Herb, S=Shrub, T= Tree, C= Climber ** L= Leaves, S= Steam, F= Fruit, Fl= Flower, Rh= Rhizome, Se= Seed, W= Whole plant, R= Root, Br= Bark

155 2018 Journal of Plant Resources Vol.16, No. 1

BOOK REVIEW: Flowering Plants of Nepal: An Introduction

Rajbhandari, K.R., Rai, S.K., Bhatt, G.D., Chhetri, R., In the next Chapter “Studies on the Flowering Plants of and Khatri, S. (2017). Flowering Plants of Nepal: An Nepal”, the authors have reviewed progress of flowering Introduction. Department of Plant Resources, Thapathali, plants of Nepal as well as neighbouring countries such Kathmandu, Nepal, XVI+432p. ISBN: 978-9937-0-3148- as India and Bhutan,and classified taxonomic studies on 6. the flowering plants under three periods: (i) Period I (between 1802 to 1947); Period II (between 1948 to 1982; Plant species occurring in Nepal Himalaya is a valuable and (iii) Period III (between 1983-2017). Brief mention heritage of the country and humankind. Botanists should has been made about some Masters and Ph.D. theses on be committed and responsible to undertake taxonomic taxonomic revision of Nepalese flowering plants carried study of plants, its conservation and sustainable use. For outparticularly during over 200 years, naturalists periods II and III. The “Flora and botanistsof the world – of Nepal, vol. 3”, is the only both professional and volume published out under amateur have focused to international collaboration study plant species of Nepal in 2011 so far out of 10 which is a global biodiversity volumes.Another important hotspot in the Himalayas. In book “Nepal: An this context, a book introduction to the natural “Flowering Plants of Nepal: history, ecology, and human An Introduction” has been environment of the authored by five scientists Himalayas” as a companion and published by the to the Flora of Nepal has Department of Plant been published in 2015 Resources (DPR). Since its under international establishment in 1961, the collaboration. In addition, DPR has been actively local floras, fascicles, and involved in taxonomic work catalogues have also been on Nepalese plants alone as published by the well as in collaboration with Department of Plant national and international Resources. The chapter is institutions. supported by a total of 26 In the book, “Foreword” appendices (Appendix 1 to written by Dr. Akhileshwar 26). Appendix 27 deals with Lal Karna (then Director revised list of families, General) has highlighted the number of genera and need to understand the species on the Nepalese diversity and status of plant species for the preparation flowering plants; and Appendix 28 provides important of Flora of Nepal. information on list of the endemic flowering plants of Nepal based on published literatures. The book begins with “Summary” (in Nepali language) and “Introduction” that provide a concise overview of The follow up chapter “Flowering Plant Diversity of historical sketch of taxonomic initiatives carried out by Nepal” presents information on the status of floral European, mainly British botanists followed by British, (flowering plants) diversity of Nepal. This chapter is Japanese and Indian explorerson the flowering plants of supported by a total of eight tables.The tables comprise Nepal. The present status of plant diversity of Nepal is information about characteristic taxonomic featuresof highlighted. Flora of Nepal,such as:big families of flowering plants

156 2018 Journal of Plant Resources Vol.16, No. 1 with more than 100 species;list of big genera having 10 • To make the book user friendly, a condensed or more than 10 species;list of scientific names of statement or outline of all “Appendices’, in particular Nepalese flowering plants with Nepal, names of Nepalese big Appendices (such as Appendix 1, 5, 7, 12, 28) persons and place names as species, subspecies or variety; would help users to quickly browse the content. names of places, mountains and rivers of Nepal used as • A gap has been observed in compilation of few specific epithets of the flowering plants& scientific related references. names of the Nepalese flowering plants with specific • A brief introduction about contributors would inspire epithets in the names of places, mountains, rivers, etc. of them to further bring out quality contribution. Nepal;list of scientific names in honour of Nepalese • Printing as well as binding qualities need to be persons; list of endemic flowering plants with families thoroughly improved. and genera with number of species; list of families having All in all, the book is a compilation of taxonomic 10 or more than 10 species of endemic flowering plants information on Flora of Nepal, and some sources include of Nepal; and list of genera having 5 or more than 5 archival documentation which were not previously easily species of endemic flowering plants of Nepal. available. The target groups would be basically students, researchers and professionals who wish to get quick The next chapter “Recent Activities (1993-2017) on the taxonomic information aboutan overview of flowering Publication of Flora of Nepal” deals with initiatives plants of Nepal. However, at the writing phase of Flora (workshops, seminars, meetings, orientation trainings, of Nepal, original taxonomic references on Flora, memorandum of understandings-MoUs, etc.) undertaken Monograph, Revision, Checklistfor specific taxonomic after 1993 for preparation of Flora of Nepal.In the information such as synonyms, distribution, etc. need to “References” chapter,530plus taxonomic literatures have be consultedby experts. Let us hope that preference and been provided. top priority would be given to forthcoming volumes of There are 152 photo plates taken by S.K. Rai with caption Flora of Nepal, and all volumes be accomplished on of their scientific name and local vernacular name, and time.Publication of complete account of the “Flora of these photographs are helpful to identify the plant species. Nepal” would benefit the countryper seas well as serve wide range of users. Authors could have considered the following in the book. Ram P. Chaudhary • Latin pronunciation should be followed while Professor Emeritus translating scientific names of taxa into Nepalese Tribhuvan University, Kirtipur, Nepal language. Email: [email protected] & [email protected]

157 2018 Journal of Plant Resources Vol.16, No. 1

BOOK REVIEW: A Handbook of the Flowering Plants of Nepal Volume I

Authors: Keshab Raj Rajbhnadari and Sanjeev Kumar Rai followed by Habit , habitat, altitudinal and phytogeographical distribution, collection information- district name, locality, date Published by Government of Nepal, Ministry of Forests and of collection, altitude, collector name and specimens where Soil Conservation, Department of Plant Resources. Date of preserved is given in herbarium acronym. Colour photo plates publication 2017. Hard bound cover, Copies of production: of 304 species are presented, two photo plates in one page. 1000. This book has updated nomenclature and classification is Photos mostly by Sanjeev Kumar Rai, cover photos: names arranged according to most recent system (Angiosperm not mentioned, may be Phylogeny Group APG IV 2016).It is a Uvariahamiltonii front and time demanding approach. It gives a Aeridesmultiflora (orchid) way to reshuffle the previous designed back.Page number 656 + viii. volumes of Flora of Nepal. The volume The book foreword by the Director covers from Angiosperm Basal Clade General of DPR with due to . The larger formal and acknowledgement to different informal clades such as superrosids, Herbaria and persons involved , Superasterids, are left while preparing themanuscript. A for remaining two volumes. Out of 58 single page summary of the book families listed in this volume, 2 families is given in Nepali language. A brief (Nymphaeaceae, Schisandraceae) from introduction of the book provides basal clade, 7 families ( Saururaceae, the institutional and publication Piperaceae, Aristolochiaceae, history of Department of Plant Myristicaceae, Magnoliaceae, Resources, including the Flora Annonaceae, Lauraceae) from Nepal Project with its collaboration clade, two families from and progress. There is mentioned independent clade (Chloranthaceae, that the book is an outcome of the Ceratophyllaceae), 19 families decision made by the 7th (Acoraceae, Araceae, Tofieldiaceae, International Editorial Board Alismataceae, Butamaceae, meeting held at Kathmandu on Hydrocharitaceae, Aponogetanaceae, 2015. The book initiates to Juncaginaceae, Potamogetonaceae, implement one of the activities of the National Biodiversity Nartheciaceae, Burmaniaceae, Dioscoraceae, Pandanaceae, Strategy and Action Plan (2014-2020) for publishing the Flora Orchidaceae, Hypoxidaceae, Iridiaceae, Xanthorroeaceae, of Nepal by 2020. It seems an updated version of the lists of Amarrophyllaceae, Asparaginaceae, Melanthioceae, flowering plants of Nepal started after the publications of an Colchicaceae, Smilaceae, Liliaceae) from Monocots clade, Enumeration of the Flowering Plants of Nepal by Hara et al. 13 monocot families (Arecaceae, Commelinaceae, (1978-1982).The Department of Plant Resources has been Pontederiaceae, Musaceae, Costaceae, Zingeberaceae, planned to enumerate all the flowering plants of Nepal in three Typhaceae, Xyriadaceae, Eriocaulaceae, Juncaceae, volumes by 2020. The first volume covers 58 families, 421 Cyperaceae, Poaceae) from Commelinids Clade. Similarly, 12 genera and 1715 species of flowering plants of Nepal. families (Papaveraceae, Circaesternceae, Lardizabalaceae, This volume is based on the herbarium specimens preserved Menispermaceae, Berberidaceae, Ranunculaceae, Sabiaceae, in the herbaria of Nepal, India, UK, Japan, Germany, France, Nelumbonaceae, Proteacese, Trochdendraceae, Buxaceae, Switzerland, Sweden, Russia, Belgium, Denmark, Netherlands, Dilleniaceae) from Eudicots clade.References are given with USA, Australia, etc. whenever possible. It listed the species selected literature. At last portion, index is given which makes based on specimens (only species with voucher specimens cited user friendly to find out species. in literature are considered).The number of species may be This book is useful to teachers, students and researchers in the different from previous publications (380 species of field of plant science, natural science, including policy makers, Orchidaceaethan previous 451 species). environmentalists, conservationists and general public Regarding to the format of species information, valid name is interested to plants. The wide circulation with affordable cost given in bold letter along with author citation and its original is expected for the proper utilization of this book. publication. It followed by synonyms, priority is given related Mohan Siwakoti to basionyms. Nepali name is also given, if available. This is

158 Contents

1. Kalpana Godar and Shiva Kumar Rai Freshwater Green Algae from Raja-Rani Wetland, Bhogateni-Letang, Morang, Nepal 1

2. M. K. Adhikari New Records of Two Powdery Mildews (Erysiphales: Fungi) from Nepal 18 3. Nirmala Pradhan Records of Bryophytes from Godawari-Phulchoki Mountain Forest of Lalitpur District, Central Nepal 22 4. Chandrakala Thakur and Sangeeta Rajbhandary Fern and Fern Allies of Panchase Protected Forest, Central Nepal 39 5. Krishna Ram Bhattarai Plants of Mai Pokhari Botanical Garden and Adjoining Areas, East Nepal 46 6. Usha Tandukar, Pramesh Bahadur Lakhey, Nishanta Shrestha, Amit Dhungana and Tara Datt Bhatt Antimicrobial Activity and Chemical Composition of Essential Oil of Fruits of Amomum subulatum Roxb. 57 7. Madan Raj Bhatta, Rajeswar Ranjitkar, Bhabani Prasad Adhikari, Parasmani Yadav and Laxman Bhandari Isolation of Curcumin and GC-MS Analysis of Oil in Curcuma longa L. 64 8. Parasmani Yadav, Bhabani Prasad Adhikari, Sarita Yadav, Sunita Khadaka, Devi Prasad Bhandari, Chetana Khanal Microscopic, UV- Fluorescence and FT-IR Analysis of the Powder of Five Medicinal Plants of Nepal 70 9. Laxman Bhandari, Madan Raj Bhatta and Rajeswar Ranjitkar Phytochemicals, Polyphenols and Antioxidant Activity of Camellia sinensis (L.) Kuntze (Tea Leaf) from Ilam District, Nepal 78 10. Devi Prasad Bhandari, Rajeswar Ranjitkar and Laxman Bhandari Quantitative Determination of Antioxidant Potential of Five Selected Essential Oils of Nepalese Origin 84 11. Parasmani Yadav, Jyoti Joshi, Devi Prasad Bhandari and Sangita Swar Phytochemical and Physiochemical Properties of Sapindus mukorossi Gaertn. (Soapnut) of Far Western of Nepal 90 12. Tara Datt Bhatt, Parasmani Yadav, Amit Dhungana, Jyoti Joshi and Chintamani Basyal Study of the Variation of Major Chemical Constituents of Essential Oil Extracted from Rhizome and Leaf of Acorus calamus L. (Bojho) from Nepal 96 13. Tara Datt Bhatt, Amit Dhungana, Jyoti Joshi, Parasmani Yadav and Chintamani Basyal, Variation in Chemical Composition of Essential Oil Extracted from the Fruits of Zanthoxylum armatum DC. (Timur) of Nepal 100 14. Pramesh Bahadur Lakhey, Usha Tandukar, Nishanta Shrestha and Rashmi Pradhan Some Nepalese Medicinal Plants Showing Potential Antimicrobial Activity Against Salmonella enterica subsp. enterica serovar Typhi 106 15. Nabin Rana, Saraswoti Khadka and Sabari Rajbahak In-Vitro Propagation of Lavender (Lavandula angustifolia Mill.) 112 Volume 16 Number 1 16. Sajita Dhakal, Bharat Babu Shrestha and Mohan Siwakoti Comparisons of Invasive Alien Plant Species Richness between Tarai and Siwalik Regions of Central Nepal 119 17. Kamal Bahadur Nepali, Dipak Lamichhane and Srijana Shah Diversity of Butterfly and its Relationship with Plants in National Botanical Garden, Godawari, Lalitpur, Nepal 124 18. Mohan Prasad Devkota Government of Nepal Crateva religiosa G.Forst.: A Mythological Sacred Tree from Nepal and its Medicinal Values 130 Ministry of Forest and Environment 19. Srijana Shah, Dipak Lamichhane, Ram Panthi and Kamal Bahadur Nepali Traditional Knowledge of Tamang Community on Utilization of Plant Resources in Dhading District, Central Nepal 134 Department of Plant Resources 20. Munesh Ratna Gubhaju and Anjana Bhattarai Thapathali, Kathmandu, Nepal Utilization Pattern of Bamboo in Madanpokhara, Tansen-8, Palpa 141 2018 21. Pratiksha Shrestha Documentation of Ethnobotanical Knowledge of Magar Community of Palpa District, Western Nepal 150 ISSN 1995 - 8579