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A CONTRIBUTION TO THE MORPHOLOGICAL AND ETHNOPHARMACOLOGICAL STUDIES OF THE POLLEN GRAINS OF WETLAND PLANTS OF PUNJAB, PAKISTAN

ANDLEEB ANWAR SARDAR 12-GCU-PhD-BOT-08

DEPARTMENT OF BOTANY GC UNIVERSITY, LAHORE

A CONTRIBUTION TO THE MORPHOLOGICAL AND ETHNOPHARMACOLOGICAL STUDIES OF THE POLLEN GRAINS OF WETLAND PLANTS OF PUNJAB, PAKISTAN

Submitted to GC University, Lahore in partial fulfillment of the requirements for the award of degree of

Doctor of Philosophy

Botany

ANDLEEB ANWAR SARDAR 12-GCU-PhD-BOT-08

DEPARTMENT OF BOTANY GC UNIVERSITY, LAHORE

IN THE NAME OF ALLAH ALMIGHTY THE MOST BENEFICENT THE MOST MERCIFUL

DECLARATION

I, Ms. Andleeb Anwar Sardar Reg. No. 12-GCU-PhD-BOT- 08 student of Doctor of Philosophy in the subject of Botany, hereby declare that the matter printed in the thesis titled “A CONTRIBUTION TO THE MORPHOLOGICAL AND ETHNOPHARMACOLOGICAL STUDIES OF THE POLLEN GRAINS OF WETLAND PLANTS OF PUNJAB, PAKISTAN” is my own work and has not been printed, published and submitted as research work, thesis or publication in any form in any University, Research Institution, etc. in Pakistan or abroad.

______

Dated: ______Signature of Deponent

RESEARCH COMPLETION CERTIFICATE

Certified that the research work contained in this thesis titled “A CONTRIBUTION TO THE MORPHOLOGICAL AND ETHNOPHARMACOLOGICAL STUDIES OF THE POLLEN GRAINS OF WETLAND PLANTS OF PUNJAB, PAKISTAN” has been carried out and completed by Ms. Andleeb Anwar Sardar, Reg. No. 12-GCU-Ph.D-BOT-08 under my supervision.

______Supervisor Prof. Dr. Zaheer-ud-din Khan Department of Botany GC University, Lahore

______Co-Supervisor Prof. Dr. Anjum Perveen Director, Centre for Plant Conservation Department of Botany University of Karachi, Karachi

Submitted Through

______Chairperson Controller of Examinations Dr. Ghazala Yasmeen Butt GC University, Lahore Department of Botany GC University, Lahore

DEDICATION

This dissertation is dedicated to: o My parents, who have supported me all the way since the beginning of my studies o My respected teachers, friends and colleagues for their kind help and guidance o My husband for his practical and emotional support o My son Muhammad Abdullah Malkana, who has been my inspiration throughout this write- up.

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ACKNOWLEDGEMENTS I entreat before, Almighty Omnipotent, Who has conferred upon me His unending blessings and has inculcated sufficient courage in me, to accomplish this difficult endeavour. I summon tranquilty for the

Holy Prophet Hazrat Muhammad (PBUH) who is forever a light of knowledge , guidance and courage for the entire human race.

I am highly obliged in paying deepest gratitude to my most respected and dignified research supervisor Prof. Dr. Zaheer-ud-din

Khan, whose enthusiastic inspiration and fatherly affection enabled me to attain the objectives of this research endeavor without any difficulty.

I am greatly obliged to my honoured and meritorious research co- supervisior Prof. Dr. Anjum Perveen, Director, Centre for Plant

Conservation/Professor, Department of Botany, University of Karachi for her upright ittellect, discreet support and sage guidance during the morphological study of the pollen grains.

I would like to pay my regards and gratitude to Dr. Ghazala

Yasmeen Butt, Chairperson, Department of Botany for providing facilities to carry out and complete the present research work.

My present work was compounded only due to the virtuous prayers of my inviolable late grand father , Chaudhary Sardar Ahmed.

My deepest gratitude is a must, for the never ending impeccable love of my father, Muhammad Anwar Summan who sustained me in

difficulties and especially my mother, Mrs. Razia Anwar. For her the quote serves well: “Not only is a mother’s job never done, the definition keeps on changing.

I have to thanks my father in law, Ch. Nazir Ahmad and mother in law, Mrs Mumtaz Nazir for their love and support to achieve success.

Thank you both for giving me strength and prayers.

I am thankful to Ms. Sofia Qaiser for the help and support during my stay in Karachi to carry out the Scanning Electron Microscopic studies. I would like to express my very sincere gratitude to Ms. Rabia

Nazir for her absolute support in the compilation of the thesis.

Now, I wish to pay my special thanks to my senior colleagues Mr.

Saboor A. Khan and Mr. Babar Ali Khan and my friends, Ms. Uzma

Rafique, Ms. Mobina Ulfat, Ms. Uzma Hanif, Mr. Muhammad Sohaib and Mr. Nadeem Ullah for their sincere cooperation, constant encouragement and honourable support during the compilation of this research work.

Last but not the least, my final thanks and regards to my sisters, brothers and friends, who supported me to achieve success in every sphere of life and have always raised their hands in prayers for me.

ANDLEEB ANWAR SARDAR

ABSTRACT

The present study was carried out to explore the palynomorph of the wetland plants of

Punjab, Pakistan using light and scanning electron microscopy as well as to investigate the antioxidant activity of the crude extracts of pollen of some locally used, important medicinal wetland plants using different types of In vitro assays.

In first part of this study, morphological characteristics of pollen of 34 wetland plant species, belonging to 20 angiospermic families, including 13 dicotyledonous and

7 monocotyledonous, were accomplished using light and scanning electron microscopy. Takhtajan’s system (1980) of classification was rendered to arrange families. The pollen characteristics were found quite distinct for the identification of all the plant species investigated. The pollens were found generally free in most of the plant species and rarely united in tetrads, such as in Juncaceae and Typhaceae. Most of the pollen were radially symmetrical, isopolar-apolar, often heteropolar, as in

Trapa bispinosa Roxb., Nymphaea alba Linn., Eichhornia crassipes (Mart.) Roxb., while oblate-prolate spheroidal, infrequently prolate-subprolate as in Nelumbo nucifera Gaertn., Ranunculus muricatus Linn., Spergularia marina (Linn.) Criscb.,

Alternanthera sessilis (Linn.) DC., Persicaria species, Nasturtium officinale R. Br.,

Centella asiatica (Linn.) Urban etc. Non aperturate, poroid (false apertures), both simple (porate and colpate) and compound (colporate) apertures were observed in the pollen, whereas variations were recorded in tectum types, ranging from scabrate, reticulate to regulate, verrucate, echinate, striate, sub-psilate punctuate, finely reticulate with muri patterns, areolate and scabrate-areolate punctate. Four distinct types of pollen were recognized on the basis of tectum and apertural types, i.e. non- aperturate, porate, colpate and colporate.

The second part of this study comprised of the ethnopharmacological uses of the medicinal wetland plants of Punjab (Pakistan). Various visits of the study area were made during the years 2008-2011 to interview the local elderly, knowledgeable people and herbal healers to document the ethnobotanical data on wetland plants, which included local name, habit and habitat and traditional uses of these plants, with special emphasis on their therapeutic uses against different human diseases, ailments or disorders. A total of eighteen medicinally important aquatic and semi-aquatic plants belonging to three monocotyledonous and fourteen dicotyledonous families were reported and the pollen of some of these plants, viz; Typha domingensis Pers.,

Centella asiatica (Linn.) Urban and Nelumbo nucifera Gaertn. were explored for their antioxidant potential using Ferric Reducing Power, Metal Chelating Activity and

Trolox Equivalent Antioxidant Capacity assays (TEAC). The antioxidant components in the crude extracts of pollen were initially extracted in methanol and further fractionated in solvents of different polarity, such as n-hexane, chloroform, ethyl acetate and water exhibited reasonable antioxidant activity. Trolox Equivalent

Antioxidant Capacity (TEAC) ranged from 3.94 to 106.26 mM of Trolox equivalents and FRAP values ranged from 1.71 to 87.5 mM of FeSO4 equivalents. Using total phenolic and flavonoid content assays ranged from 143 to 8150 mg/ml of gallic acid and 110 to 5800 mg/ml of quercetin respectively. The highest Trolox Equivalent

Antioxidant Capacity (TEAC) value was found in the crude extract of Centella asiatica, whereas the total phenolic and flavonoid contents were significant in

Nelumbo nucifera.

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CONTENTS

Pg. No. Part I: Morphological Studies of Pollen Grains of Wetland Plants of Punjab, Pakistan 1. Introduction …………………………………………….…..………………….……. 01

Study Area…………………………………………………………………….…... 05

2. Review of Literature…………………………………………………………….…... 07

3. Materials and Methods ………………………………………………………….…… 10

4. Results and Discussion ………………………………………………………….…... 12 4.1 Results: Key to the Families……………………………………………………………….. 12 Morphology and General Description of the Pollen Grains………………….. 14 Dicotyledons……………………………………………………………..….……. 14 Monocotyledons……………………………………………………….....….…… 20 4.2 Discussion:…………………………..…………………………………………..…. 26 Part II: A Contribution to the Ethnopharmacological Studies of the Pollen Grains of Medicinal Wetland Plants of Punjab, Pakistan 1. Introduction……………………………………….…..…………………………….. 40

2. Review of Literature……………………………………………………………....… 44

3. Materials and Methods……………………………………………………………... 48

4. Results and Discussion…………………………………………………………...... 52 4.1 Results: Evaluation of Antioxidation Potential of Pollen………………………………... 52

4.2 Discussion:…………………………………………………………………………… 76

References ……………………………………………………….……………………….. 83

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LIST OF PLATES

Pg. No. Plate 1. Nymphaea alba: A. Pollen. B. Exine Pattern. Nelumbo nucifera: 32 C. Pollen D. Exine pattern. Spergularia marina: E. Pollen grain Phyla nodiflora: F. Pollen grain Plate 2. Persicaria glabra: A. Pollen. B. Exine Pattern. Polygonum 33 amphibium: C. Pollen D. Exine pattern. Trapa bispinosa: E. Pollen. F. Exine Pattern. Plate 3. Bacopa monnieri: A. Pollen B. Exine pattern. Ipomoea aquatica: 34 C. Pollen D. Exine pattern. Ipomoea carnea: E. Pollen F. Exine Pattern Plate 4 . Eclipta alba Linn: A. Pollen B. Exine Pattern . 35 Plate 5. Potamogeton nodosus: A. Pollen. B. Exine Pattern. 36 Schoenoplectus mucronatus: C. Pollen D. Exine pattern. Typha domigensis: E. Pollen F. Exine pattern Plate 6 . Cyperus rotundus: A. Pollen. B. Exine Pattern. Schoenoplectus 37 litoralis: C. Pollen D. Exine pattern. Cyperus conglomeratus: E. Pollen F. Exine Pattern Plate 7. A. Eclipta alba B. Nelumbo nucifera C. Typha domigensis D. 38 Persicaria glabra E. Dichanthium annulatum F. Potamogeton nodosus. Trapa bispinosa G. Typha domigensis H. Lemna aequinoctialis Plate 8. A. Lemna gibba B and C. Cyperus conglomeratus D. Cyperus 39 rotundus E. Eleocharis palustris F. Schoenoplectus lacustris G. Desmostachya bipinnata H. Echinochloa crus-galli Plate 9: Bundles of Typha plants collected to make mats and baskets 82 Plate 10: A person making mat from Typha plants 82

LIST OF TABLES

PART-I Pg. No.

Table I: Aquatic and Semi-Aquatic Plants of Punjab, Pakistan 03

Table II: Major Wetlands and Lakes of Punjab, Pakistan 05

Part-II

Table 1: Medicinal uses of Wetland Plants of Punjab, Pakistan for 55 treatment of Different Human Diseases

Table 2: Antioxidant activity of pollen of Typha domigensis 58

Table 3: Antioxidant activity of pollen of Centella asiatica 58

Table 4: Antioxidant activity of pollen of Nelumbo nucifera. 59

LIST OF FIGURES

Pg. No.

Figure 1: ABTS.+ assay of the pollen of Typha domingensis 60

Figure 2: Total phenolic contents of the pollen of T. domingensis 61

Figure 3: FRAP Assay of the pollen of T. domingensis 62

Figure 4: Metal Chelating Activity of the pollen of T. domingensis 63

Figure 5: Total Flavonoid Contents of the pollen of T. domingensis 64

Figure 6: ABTS.+ assay of the pollen of Centella asiatica 65

Figure 7: Total phenolic contents of the pollen of C. asiatica 66

Figure 8: FRAP Assay of the pollen of C. asiatica 67

Figure 9: Metal Chelating Activity of the pollen of C. asiatica 68

Figure 10: Total Flavonoid Contents of the pollen of C. asiatica 69

Figure 11: Superoxide Radical Anion Scavenging Activity of the 70 pollen of C. asiatica

Figure 12: ABTS.+ assay of the pollen of Nelumbo nucifera 71

Figure 13: Total Phenolic Contents of the pollen of N. nucifera 72

Figure 14: Total Flavonoid Contents of the pollen of N. nucifera 73

Figure 15: Metal Chelating Activity of the pollen of N. nucifera 74

Figure 16: FRAP Assay of the pollen of N. nucifera 75

PART I

A Contribution to the Morphological

Studies of Pollen Grains of Wetland Plants

of Punjab, Pakistan

1. INTRODUCTION

Palynology is the study of spores and pollen of seed plants. The examination of pollen, both recent and ancient, can be of value in a range of scientific studies including Taxonomy, Genetics, Melissopalynology, Forensic sciences, Allergic reaction studies, Atmospheric studies and retraction of individual and community vegetation. The pollen is a container which houses the male gametophytic generation in Angiosperms or Gymnosperms. Pollen contains phytochemicals, such as carotenoids, flavonoids and phytosterols. These are various features possessed by pollen grains which make them useful in a range of disciplines (Moore et al., 1991). Lindley (1830) was probably the first person to make use of pollen characters in the classification of Orchidaceae, and later the significance of pollen morphology in plant taxonomy has been stressed by several workers, notably by Mohl (1835), Fritzsche (1832), Fischer (1890), Selling (1946-47), Cranwell (1952), Erdtman (1952, 1957) and Qaiser & Ali (1978). Realizing its importance, palynology was recognized for the first time as a separate section of the International Botanical Congress in Stockholm Sweden in 1950 (Nair, 1964). Electron Microscopic studies have unveiled new details in the surface of pollen, even in monocotyledons where none had been found before (Bradley, 1958). Kuprianova (1948) has studied the pollen characteristics of whole of the monocotyledons and some of her conclusions are very suggestive. It is now unanimously accepted that pollen and spore morphology plays an important role in identification and tracing relationships of plants at various taxonomic levels (Nair, 1964). Alwadie (2008) studied the pollen morphology of six aquatic angiosperms from Saudi Arabia. In Pakistan the first contribution was made on the pollen characteristics of some medicinal plants by Malik et al. (1963). The importance of pollen morphology was greatly emphasized by several other workers. Palynology is a new upcoming science which has provided new dimensions to the complication of taxonomy. The classification of pollen is based on number, position nd character analysis known as ‘NPC’ system. Palynology was divided into Basic and applied palynology (Erdtman, 1963). Basic Palynology deals with pollen and spore morphology, the physical and chemical nature of sporoderm as well as the correlation between palynology and taxonomy. Recent palynological data are finding increasing application in the construction of diagnosis of new species. Applied

2 palynology comprises geopalynology, aeropalynology, mellitopalynology, pharmacopalynology, copropalynology and forensic palynology (Saxena, 1993). The tremendous contribution of palynology to the systematics and phylogeny of angiosperms is because of the evolutionary trends in pollen wall architecture, which is important source of phylogenetic information of major significance. Aristolochiaceae is one of the well known examples in which palynological data supports its isolated position in a monotypic order. Similarly the great diversity of pollen types in various genera of Nymphaeales supports its position as an old order (Walker and Doyle, 1975). The constant features and the sculpturing of the exine make pollen a highly recognizable object by which parent genus or even species may be recognized (Hariss, 1955; Moore and Webb, 1978). Palynology has established its rights by its various applications which are directly responsible to the service of mankind (Butt et al., 1987). Aquatic angiosperms are a significant part of the World’s flora. Water plants play an important role in healthy ecosystems while providing food, medicines and building materials. They are the primary oxygen producers, substrate of algae and cover of numerous invertebrates and help in cycling of nutrients (Kaufman, 1989). In addition to the role of palylnology in plant systematics, it also plays an important role in Food quality control, as it was narrated by local people during the survey of the study area for the documentation of local ethnobotanical knowledge on wetland or semi-wetland plants. They believe that the honey available in the local market is if without pollen grains in it, then it is a false honey and is a man made honey having concentrated sucrose solution. They believe that the pure honey is the one that contains pollen grains that are collected by honey bees. This view has already been supported by Butt et al. (1987). Pollen Flora of Aquatic Plants of Karachi has been studied by Perveen (1999). However, at present there is no separate documentation on the palynology of aquatic and semi-aquatic plants of Pakistan. Therefore, the present work was especially designed to enlist and provide detailed account on the pollen morphology and structure of exine patterns of aquatic and semi-aquatic plants of Punjab by Light and Scanning Microscopy. Moreover, the antioxidant activity of the inorganic/aqueous and organic crude extracts of the pollen of some medicinally important aquatic and semi-aquatic plants was also evaluated using different types of assays.

The following aquatic and semi-aquatic plants were collected from different localities of Punjab: Table-I: Aquatic and Semi-Aquatic Plants of Punjab, Pakistan Sr. Name Family Place of

No. Collection

1. Alternanthera sessilis (Linn.) DC. Amaranthaceae Shakargarh

2. Bacopa monnieri (Linn.) Pennell Scrophulariaceae Rawalpindi

3. Centella asiatica (Linn.) Urban Umbelliferae Lahore

4. Cyperus arenarius Retz. Cyperaceae Gujranwala

5. Cyperus conglomerates Rottbl. Cyperaceae Gujranwala

6. Cyperus leavigatus Linn. Cyperaceae Sheikhupura

7. Cyperus rotundus Linn. Cyperaceae Sheikhupura

8. Desmostachya bipinnata (Linn.) Poaceae Gujrat

Stapf.

9. Dichanthium annulatum (Forssk.) Poaceae Gujrat

Stapf.

10. Echinochloa crus-galli (Linn.) P. Poaceae Gujranwala

Beauv.

11. Eclipta alba Linn. Compositae Lahore

12. Eichhornia crassipes (Mart.) Pontederiaceae Lahore

Sloms

13. Eleocharis palustris (Linn.) Roem. Cyperaceae Gujranwala

& Schult.

14. Ipomea aquatica Forsk. Convolvulaceae Burewala

15. Ipomea carnea Jacq. Convolvulaceae Burewala

16. Juncus articulatus Linn. Juncaceae Gujrat

17. Juncus maritimus Lam. Juncaceae Gujrat

18. Lemna aequinoctialis Welw. Lemnaceae Lahore

Sr. Name Family Place of

No. Collection

19. Lemna gibba Linn. Lemnaceae Lahore

20. Nasturtium officinale R. Br. Brassicaceae Jhelum

21. Nelumbo nucifera Gaertn. Nelumbonaceae Gujranwala

22. Nymphaea alba Linn. Nymphaceae Lahore

23. Persicaria amphibian Polygonaceae Hafizabad

(Linn.) A.Gray

24. Persicaria glabra (Willd.) Gomes Polygonaceae Bahawalpur

25. Phyla nodiflora (Linn.) Greene Verbenaceae Vehari

26. Potamogeton nodisis Poir. Potamogetonaceae Jhelum

27. Ranunculus muricatus Linn. Ranunculaceae Rawalpindi

28. Schoenoplectus lacustris (Linn.) Cyperaceae Kasoor

Palla

29. Schoenoplectus litoralis (Schard.) Cyperaceae Sialkot

Palla

30. Schoenoplectus mucronatus Cyperaceae Sialkot

(Linn.) Palla

31. Setaria pumila (Poir.) Roem. & Poaceae Narowal

Schult.

32. Spergularia marina (Linn.) Criscb. Caryophyllaceae Rawalpindi

33. Trapa bispinosa Roxb. Trapaceae Gujranwala

34. Typha domigensis Pers. Typhaceae Lahore

Study Area

The most populated province of Pakistan ‘Punjab’ with an area of 205,344 km2 (79,284 sq mi) at northwestern border between Latitude 30° 54' 58" N and Longitude 71° 51' 01" E of indo-Australian tectonic plate. Almost 62% population of the total population of Pakistan belongs to the province Punjab. This province is bordered by Azad and Jammu Kashmir to the northeast, Indian Punjab and Rajasthan to the east, Sindh to the south and Khayber Pakhtunkhwa to its western side. The capital city “Islamabad” of Pakistan is situated at its north.

The land of Punjab is extremely suitable for agricultural purposes due to canals and five main rivers flowing throughout the province. Some deserts are also a part of Punjab, e.g. Thar and Cholistan bordered by Rajasthan and Sulaiman Range. The temperature ranges from -2° to 47°C approximately. The maximum rainfall recorded in this season ranges from 200 to 1000 mm. Punjab is also beautifully landscaped with hot and barren hills to its south and cool hills of Himalaya to its extreme north edge. During summer season (July to September), land of this province is irrigated with heavy rains known as Moonsoon. Agricultural year begins with the advent of this season.

Artificial or naturally developed marshy areas with stagnant or running water are known as wetlands. These wetlands can exist permanently or temporarily having fresh or salty water. Major wetlands of Punjab are as follows:

Table-II: Major Wetlands and Lakes of Punjab, Pakistan Name of Wetland Status District/Area Bajwat Game Reserve Sialkot Ghamaghar Lake Not Protected Kasur Head Islam Game Reserve Multan Jahlar Lake Not Protected Khushab Kalar Kahar Lake Wildlife Sanctuary Chakwal Khabbaki Lake Wildlife Sanctuary Khushab Kharrar (Kharal) Lake Wildlife Sanctuary Okara

Name of Wetland Status District/Area Lal Suhanra (Patisar) Lake National Park Bahawalpur Mangla Dam Not Protected Jhelum Marala Headworks Not Protected Sailkot Nammal Lake Wildlife Sanctuary Khushab Qadirabad Barrage Not Protected Gujrawala Qadirabad Link Canal Game Reserve Gujrat Rasool (Rasul) Barrage Wildlife Sanctuary Gujrat Soan River Not Known Chakwal Taunsa Barrage Wildlife Sanctuary Muzaffargarh/DG Khan Ucchali Lake Ramsar Site Khushab Source: Ali and Akhtar (2006). The present study was carried out with the following objectives:  To document the plant inventory of aquatic and semi-aquatic plants of Punjab, Pakistan.  To explore the palynomorph of these plants  To provide the palynological data to the students/workers, involved in the studies of Paleobotany (Evolution of plants), Ecology, Plant conservation, Agriculture (Fisheries), Honey industry, Biodiversity, etc.

2. Review of Literature

The aquatic flowering plants draw a particular attention of Biologists due to their reproductive and physiological peculiarities, structural variations in morphology and anatomy (Perveen, 1999). Many authors have studied the pollen morphology of some species of aquatic families. Malik et al. (1963) studied the pollen characteristics of some medicinal plants. In a review paper by Bhutta (1968), the importance of pollen morphology was greatly emphasized. Soon after, Khan and Memon (1970) studied some pollens of the family Leguminosae of Sindh University, Jamshoro. Perveen and Qaiser (1995-2008) are already working on the Pollen Flora of Pakistan. Pollen Flora of Aquatic Plants of Karachi was studied by Perveen (1999).

Aiken (1978) reported the palynological data of nine Myriophyllum species from North America. Out of these nine Myriophyllum species, seven species were studied on scanning electron microscope with reference to wall sculpturing. Microrugulate and microverrucate type sculpturing was observed, that helped to make an identification key to the species of Myriophyllum. Guan et al. (1992) studied the pollen characters of twenty two aquatic plants representing eighteen genera of thirteen families. Takahashi (1994) studied development and structure of exine of a sub-merged plant, Ottellia alismoides. Verrucate type protrusions were observed on microspore plasma membrane at early tetrad stage. Cooper et al. (2000) studied and compared pollen ultrastructure of 13 Callitriche species, representing an aquatic dicots family Callitrichaceae, including three terrestrial, nine amphibious and one obligately submerged species by scanning and transmission electron microscope. Spheroidal, small and intectate pollens with poorly developed apertures were observed in all the species. Ornamentation of surface, aperture like structures, presence or absence and thickness of exine varied in all three types of species. However, the exine was absent in obligatory submerged species. Tanaka et al. (2004) studied morphology of pollen of twelve species representing the families Hydrocharitaceae and Najadaceae. They also studied structure and sculpturing of exine with reference to pollination mechanisms and the molecular phylogeny using scanning and transmission electron microscope.

Zetter and Ferguson (2001) reported the pollen of plants of family Trapaceae being unique and helpful in tracing fossil record. They examined pollen from different vacinities of four fossil species and explained two species first time viz; (i). Sporotrapoidites carlesii Zetter & Ferguson sp. nov. and S. cesarei Zetter & Ferguson sp. nov. The alterations in pollens with the passage of time were confered and the palaeoecology of the collection bodies in which these grow was also evaluated.

Meo and Khan (2004) studied palynology of a number of weeds of Asteraceae from Pakistan using light microscope. Pollen in the weeds of Asteraceae were found isopolar, symmetrical, trionocolporate, non-lacunate and echinat. Polar and equatorial shape, ratio, view, diameter; apertures class and type; spine length and their rows between colpi; shape of pollen were the main palynological features that were utilized to distinguish the taxa of weeds. However, the exine thickness which varies significantly among the weeds was used as cheif taxonomic character for the classification of weeds.

Samuli (2006) examined the palynological characters of a genus, i.e. Echinodorus (aquatic and semi-aquatic herbs) belonging to the family Alismataceae found in Argentina to USA naturally and used as an ornamental plants in aquariums. Ninety six morphological characters were taken to describe the phylogeny of Echinodorus species. Perveen and Qaiser (2006) examined the pollen characteristics and structure of fifty species in lieu of twenty seven genera belonging to family Umbelliferae/Apiaceae using light and scanning electron microscope from Pakistan. Tricolporate, radially symmetrical pollen, with striate-rugulate or rugulate-striate rarely simply striate tectum was found in umbelliferous species. On the basis of exine pattern three distinctive pollen types were recognized i.e., Pleurospermum hookeri- type, Bupleurum gilessii-type and Trachyspermum ammi-type. Zafar et al. (2007) studied pollen morphology and fertility estimation as a part of taxonomic description of 7 species of family Asteraceae from flora of Rawalpindi. The Pollen characters of these species varied greatly. Palynomorph catalog consisting of family, botanical, local and family names, species distribution, flower color and season and pollen description was developed. Shape, P/E ratio, surface of exine and pollen morphology were the chief characters studied. The fertility ratio (90-98.11%) showed the pollen flora well recognized. Moreover, the pollen flora was declared

9 extremely significant and useful not only for taxonomists but also for other scientists of pure and applied sciences. Perveen and Qaiser (2007) presented detailed palynological data on 13 species of 9 genera of family Verbenaceae from Pakistan by light and scanning electron microscope. Oblate-spheroidal, radially symmetrical, isopolar, tricolporate or tricolpate pollen with sexine much thicker or thinner than nexine was observed. Tectum type differed from subpsilate to reticulate, rugulate-reticulate often spinulose- reticulate. Eight distinct pollen types were renowned on the basis of apertures and exine ornamentation viz., Caryopteris grata-type, Caryopteris odrata-type, Chascanum marrubifolium-type, Clerodendrum phlomoides - type, Lantana indica– type, Phyla nodiflora–type and Verbena officinalis–type. Alwadie (2008) examined the pollen morphology of 6 species of 5 genera belonging to 5 families of aquatic angiosperms using both light and scanning electron microscopes from Saudi Arabia. Three distinct pollen types were recognized on the basis of apertural type. The correlation between pollen morphology and pollination was also discussed. Mazari et al. (2012) described pollen morphology of 7 species of family Asteraceae from Kaghan Valley viz., Achillea millefolium Linn., Chrysanthemum leucanthemum Linn., Gerbera gossypina (Royle) Beauv., Senecio chrysanthemoides DC. Prodr., Sonchus asper Linn., Tagetes erecta Linn. and Xanthium strumarium Linn. They illustrated substantial disparity in pollen shape, size, length and number of spines, colpi and sculpturing under both the light and electron microscopes.

3. MATERIALS AND METHODS

The plan of work used in the present investigation included:

1. Collection of Pollen The pollen of various aquatic and semi-aquatic plants was collected from their freshly collected plants or preserved plants in Herbaria. The wetland bodies of Punjab were visited, usually between July to October of each 2010, 2011 and 2012, to collect the flowers along with the plants. The voucher plant specimens were deposited in Dr. Sultan Ahmad Herbarium, GC University, Lahore. In total 34 species of 20 Angiospermic families belonging to 13 Dicotyledonous and 07 Monocotyledonous were collected. The anthers of the flowers of these plants were crushed to get the powdery pollen for microscopic studies. 2. Microscopic Study of the Pollen The polleniferous material was acetolyzed according to the methods of Erdtman (1952) and soaked either fresh or dry in glacial acetic acid for 12 to 18 hour. It was then crushed by a glass rod. The crushed material was centrifuged for 20 minutes at 5000 r.p.m and then the acid was decanted. A mixture, i.e. 1 part conc. Sulphuric acid added drop by the drop to 9 parts of Acetic anhydride, was added into 5ml of acetolysed polleniferous material and then heated in Water Bath upto boiling point. After cooling, the mixture was centrifuged for 20 minutes and the liquid part i.e. acetic acid was decanted. The material was washed with distilled water and sieved. The sieved material was again centrifuged and divided into two parts for scanning and light microscopic examination. (i) Scanning Electron Microscopy The first part of polleniferous material was mixed in water and shifted to a metallic stub by a fine pipette with double-sided adhesive tape. The stub with pollen material was placed overnight to dry at room temperature and then covered with gold coating in a sputtering chamber using Ion-sputter JFC-1500. Afterwards, the pollen material on stub was observed by Scanning Electron Microscope, Jeol (JSM-6380 LV) at the Centre for Plant Conservation, University of Karachi, Karachi.

(ii) Light Microscopy Glycerine (50%) was added to the second part of polleniferous material and then centrifuged for 20 minutes. Glycerine was decanted and rest of the material containing pollen was transferred onto a glass slide having unstained glycerine jelly (made after Kisser’s Method, 1937). The slide was allowed to dry and then observed under light microscope, Nikon (Type-2). For thin walled pollen The delicate pollen of some plants (especially of Gramineae and Cyperaceae) are usually shriveled and wrinkled. To avoid shrinkage, the pollen was suspended in distilled water directly instead of doing acetolysis. On the other hand, the anthers of grasses taken from plants preserved on herbarium sheets were soaked in Potassium hydroxide (45%) for one to two minutes and then washed with distilled water for 3-5 times. The washed polleniferous material was crushed with glass rod and 50% glycerine was added into this material. It was centrifuged for 2-3 minutes and after which the glycerine was decanted. However, the remaining onwards procedure was the same, i.e. preparing glass slided for light microscopy. Photomicrographs were taken and the results were presented as Plates 01-08. 3. Statistical Analysis of the Data For each species 2-3 specimens were studied and the measurements were based on 10-15 pollen per specimen. SPSS 12.0 version was used to carry out statistical analysis. Pollen size including polar axis (P), and equatorial axes or diameter (E) as well as aperture size, apocolipium, mesocolpium, exine thickness (excluding spinules), colpi length and spine length were computed. Values in the text were mean ± SD. Significant differences were evaluated using One Way ANOVA. The terms in the present study were used according to Wodehouse (1928); Erdtman (1952); Faegri and Iversen (1964); Kremp (1965) and Walker and Doyle (1976). Artificial keys to the families, genera and species were designed on the basis of pollen characters except in some cases, where family and generic keys were based on quantitative characters.

4. RESULTS AND DISCUSSION 4.1 RESULTS

On the basis of the pollen morphology recorded in the present study, the following key was framed to delimit families of the plants investigated: Key to the Families: 1. + Pollen single 3 - Pollen united in groups 2 2. + Pollen monoporate 20. Typhaceae - Pollen non-aperturate 16. Juncaceae 3. + Pollen aperturate 4 - Pollen non-aperturate 14. Potamogetonaceae 4. + Pollen colpate and colporate 5 - Pollen Porate 15 5. + Pollen colpate 6 - Pollen colporate 11 6. + Pollen mono-bicolpate 7 - Pollen tricolpate 8 7. + Tectum reticulate 01. Nymphaeaceae - Tectum areolate 15. Pontederiaceae 8. + Tectum reticulate-rugulate 9 - Tectum Scabrate 10 9. + Tectum reticulate 07. Brassicaceae/Cruciferae - Tectum rugulate 02. Nelumbonaceae 10. + Pantocolpate, isopolar, sub-prolate to oblate spheroidal, colpi narrow 13.17-18.45 µm long 04. Caryophyllaceae - Tricolpate, isopolar rarely apolar to sub-prolate, colpi narrow with acute ends 03. Ranunculaceae 11. + Tectum echinate with sparsely punctate base 13. Compositae/Asteraceae - Tectum not as above 12 12. + Pollen oblate-spheroidal 13 - Pollen subprolate to prolate 14 13. + Tectum finely scabrate 06. Polygonaceae (P.P)

- Tectum coarsely reticulate with irregular muri pattern 11. Scrophulariaceae 14. + Pollen subprolate, Tectum sub-psilate-punctate 12. Verbenaceae - Pollen prolate, Tectum striate 10. Umbelliferae/Apiaceae 15. + Pollen mono-diporate 16 - Pollen pantoporate 17 16. + Tectum areolate or scabrate 18. Poaceae/Gramineae (P.P) - Tectum spinulose or spinulose punctate. 19. Lemnaceae 17. + Finely perforate echinate tectum with granules 09. Convolvulaceae - Tectum not as above 18 18. + Tectum reticulate 06. Polygonaceae (P.P) - Tectum scabrate, Exine (3.43-) 4.21±1.6 (-5.25) µm thick 05. Amaranthaceae 19. + Pollen triangular 20 - Pollen spheroidal 18. Poaceae/Gramineae (P.P) 20. + Tectum areolate-punctuate or scabrate-punctuate 17. Cyperaceae - Tectum twisted, smooth regulate, perforated with germinal structures 08. Trapaceae

Morphology and General Description of the Pollen Grains Pollen grains are generally free, united in tetrads as in Juncaceae. Exine patterns also varied in immense extent, such as scabrate, reticulate to regulate, verrucate, echinate, striate, sub-psilate punctuate, finely reticulate with muri patterns. Areolate and scabrate-areolate punctat. Sexine is thicker or thinner or equally thick to nexine. Both simple and compound apertures are found. However non-aperturate/poroid pollen are also observed as in Potamogetonaceae, Juncaceae and Cyperaceae, the Monocots. DICOTYLEDONS 1. Family Nymphaceae Pollen grains heteropolar, bilateral, monocolpate, boat shaped, sexine thinner than nexine, tectum rough reticulate. Represented by a single aquatic species in the study area. Nymphaea alba Linn. Pollen grains elliptic, bilateral, heteropolar, boat shaped, monocolpate, size: Length (26.35-) 31.20±1.05 (-34.26) and breadth (31.38-) 32.75±0.62 (35.57) µm. P/E ratio: 0.95. Colpus (17.13-) 20.45±0.83 (-24.40) µm long, nexine thicker than sexine, tectum rough reticulate.

2. Family Nelumbonaceae Nelumbonaceae is characterized by having tricolpate, subprolate pollen grains that are radially symmetrical and isopolar. Tectum rugulate. This is a monotypic family represented by two species of a single genus (Willis, 1973). Only one species found in study area. Nelumbo nucifera Gaertn. Prolate spheroidal, isopolar, radially symmetrical pollen grains. Size: Length (50.07-) 58.50±2.39 (-65.87) and breadth E (47.43) 56.94±2.30 (-63.25) µm. Ratio (P/E): 1.09. Tricolpate, rounded trilobed, length of colpi (39.53-) 45.33±1.64 (-50.07) µm, Colpal membrane densely fossulate, Mesocolpium (34.26-) 41.11± 2.07 (-47.43) µm, Apocolpium (5.25-) 8.44± 0.84 (-10.50) µm. Exine (2.64-) 4.85± 0.63 (-7.11) µm thick. Nexine thinner than sexine, tectum rugulate.

3. Family Ranunculaceae

Frequently radially symmetrical pollen grains that are isopolar rarely apolar to sub- prolate, tricolpate, often pantoporate, nexine thicker or thinner than sexine. Tectum usually spinulose, often striate, verrucate or scabrate. Ranunculus muricatus Linn. Prolate-spheroidal and tricolpate pollen grains. Size: P (23.32-) 26.01±0.98 (-28.99) and E (21.08-) 22.08± 0.78 (-27.35) µm. Ratio (P/E): 1.13. Ectocolpus long narrow with acute ends, colpi (13.17-) 15.52± 0.83 (-18.45) µm long, mesocolpium (11.54-) 13.85±0.50 (-15.17) µm and apocolpium (5.36-) 6.95±0.40 (-7.90) µm. Exine (2.64-) 3.48±0.22 (-3.95) µm thick. Nexine thicker than sexine, tectum verrucate to scabrate.

4. Family Caryophyllaceae Tricolpate-pantocolpate, isopolar, sub-prolate to oblate spheroidal, radially symmetrical, fossaperturate pollen grains with small colpi, colpal membrane sub- psilate to granulated, nexine thinner or thicker than sexine. Tectum densely scabrate to spinulated-punctate. Following aquatic species is found in the study area: Spergularia marina (Linn.) Criscb. Pollen grains pantocolpate, oblate-spheroidal, size: Length (21.08-) 27.83±2.21 (- 36.85) µm and breadth (18.45-) 24.77±2.30 (-34.26). P/E ratio: 1.12. Colpi narrow, (13.17-) 16.55±0.82 (-18.45) µm long, colpal membrane minutely granulated. Exine (2.64-) 3.43±0.37 (-5.01) µm thick. Tectum finely scabrate, sexine thicker than nexine.

5. Family Amaranthaceae Pollen grains usually radially symmetrical, polyforate, spheroidal, polyporate with small-large pores, non-operculate or operculate with rounded or star like operculum, exine commonly very thick, baculate, undulated. Tectum lightly to compactly scabrate. Represented by one aquatic specie in the study area. Alternanthera sessilis (Linn.) DC. Pollen grains oblate-spheroidal, panto-porate (occasionally hexa-porate), non- operculated, hexagonal in shape, P/E ratio: 1.10. Size: Polar axis (21.08-) 27.83±2.21 (-36.85) and diameter (18.45-) 21.87±1.20 (-26.35) µm. Apertures circular (5-9.7) µm in diameter, covered with muroid ridges. Exine (3.43-) 4.21±1.6 (-5.25) µm thick. Scabrate spinulose tectum.

6. Family Polygonaceae Pollen grains generally tri-tetra colporate, rarely porate, oblate speroidal-prolate, polyrugate, radially symmetrical, colpal membrane narrow granulated to scabrate, sexine slightly thicker, thicker or thinner than nexine. Tectum varies from finely scabrate to punctate or medium-coarsely reticulate. Two aquatic species are found in the present study area. Key to the species + Tectum scabrate with even forms of muri Persicaria glabra - Tectum finely scabrate Persicaria amphibia Persicaria glabra (Willd.) Gomes Pollen grains apolar, polypantoporate, oblate-spheroidal, radially symmetrical, size: (44.79-) 58.21±3.68 (-79.05) µm in length and (50.07-) 61.18±3.17 (77.73) µm in diameter. P/E ratio: 0.95. Pore±oblong, (7.91-15.8) µm. Exine (3.64-) 5.32±0.34 (- 7.25) µm, sexine thicker than nexine. Tectum scabrate with even forms of muri. Persicaria amphibian (Linn.) A.Gray Polypantoporate , isopolar, oblate-spheroidal pollen grains with fossapertures. Size: length (44.80-) 55.60±3.29 (-68.50) and diameter (42.18-) 54.75±3.00 (-63.25) µm. P/E ratio: 1.01. Colpi (25.62-) 34.97±2.53 (-46.11) µm thick, mesocolpium (15.55-) 21.13±2.01 (-27.67) µm, apocolpium (12.12-) 15.44±1.02 (-18.45) µm. Exine thickness (1.64-) 3.22±0.38 (-4.25) µm, finely scabrate tecum, nexine and sexine are equally thick.

7. Family Brassicaceae/Cruciferae Pollen grains usually tricolpate, oblate-spheroidal to sub-prolate, tectum generally reticulate, nexine thinner than sexine,. Single aquatic species of this family found in the study area. Nasturtium officinale R. Br. Radially symmetrical, tricolpate, sub-prolate pollen grains are found in this species. P/E ratio: 1.18. Size: Length (21.08-) 24.24±1.30 (-28.99 and breadth (18.45-) 20.55±0.84 (-23.71) µm. Long sunken colpi with acute ends (13.18-) 14.97±0.43 (- 15.81) µm, mesocolpium (8.43-) 9.59±0.33 (-10.54) µm, apocolpium (5.25-) 6.72±0.36 (-7.64) µm. Tectum coarsely reticulate, exine thickness (2.37-) 2.95±0.24 (- 3.95) µm, nexine thinner than sexine.

8. Family Trapaceae Polllen grains in polar view triangular obtuse and in equatorial view rhombic, tricolporate or porate, protruding having a collar of sexine, angulaperturate, nexine thicker than sexine, tectum perforate, verrucate-rugulate at poles. Trapa bispinosa Roxb. Pollen grains triangular obtuse, heteropolar, wedge shaped with combined ridges, tricolporate, size: length (36.89-) 47.64±2.54 (-55.33) µm and breadth (28.91-) 46.11±3.94 (-57.97) µm. P/E ratio: 1.03. colpi (18.45-) 30.49±3.34 (-39.53) µm long, exine thickness (1.94-) 3.06±0.40 (-4.89) µm, nexine thicker than sexine. Tectum twisted, smooth-rugulate, perforate with germinal structures at poles.

9. Family Convolvulaceae Pollen grains usually isopolar, seldom apolar, radially symmetrical, pantoporate, subprolate-prolate or ± spheroidal-oblate spheroidal, echinate, sexine and nexine are equally thick, sexine frequently slightly thinner or thicker than nexine. Uniformly echinate, coarsely perforated tectum. Spines sharp or blunt, few spines with slightly recurved apices. Two species of a single genus were found in the study area. Key to the species + Tectum finely perforated Ipomoea aquatica - Tectum medium-finely perforated Ipomoea carnea Ipomoea aquatica Forsk. Pollen grains spheroidal, echinate, apolar, size: Polar axis P (60.60-) 69.56±2.06 (- 81.69) µm and equatorial diameter E (63.24-) 73.52±2.19 (-84.32) µm. P/E ratio: 0.95. pantoporate, pores ± circular, diameter (5.25-) 8.17±0.44 (-9.90) µm. Exine thickness (3.43-) 4.80±0.37 (-6.06) µm, nexine slightly thicker than sexine. Finely perforate echinate tectum with granules. Spines with blunt apices, length (4.74-) 6.58±0.95 (-7.91) µm. Ipomoea carnea Jacq. Pollen grains apolar, spheroidal, size: Polar axis P (65.88-) 77.47±2.70 (-84.32) µm and equatorial diameter E (68.51-) 74.83±2.07 (-81.69) µm. P/E ratio: 1.03. pantoporate, pores ± circular, diameter (7.90-) 9.75±0.45 (-10.45) µm. Exine thickness (3.95-) 3.48±0.33 (-6.06) µm. Sexine and nexine are equally thick. Tectum echinate moderate to coarsely perforate having granules. Spines are distinctly

18 perforated at base with blunt and recovered apices, uniformly distributed, length (4.47-) 6.06±0.52 (7.64) µm.

10. Family Umbelliferae/Apiaceae Pollen grains radially symmetrical, isopolar, tricolporate rarely tetra-colporate or porate, prolate to sub-prolate, colpi with costae, sexine is equally thick to nexine, or slightly thinner than nexine. Tectum is striate-rugulate or simple striate. A single species is present in the study area. Centella asiatica (Linn.) Urban Pollen grains tricolporate, subprolate, angul-aperturate, isopolar. P/E ratio: 1.58. size: Length (16.01-) 20.55±0.25 (-23.71) and diameter (11.86-) 12.98±1.42 (-13.49) µm. Colpi in length (7.90-) 11.33±0.77 (-13.17) µm, mesocolpium (6.59-) 7.11±0.23 (- 7.90) µm, apocolpium (5.25-) 6.01±0.26 (-6.59) µm. Exine thickness (2.64-) 5.25±0.46 (-3.90) µm, nexine thinner than sexine. Tectum striate.

11. Family Scrophulariaceae Isopolar, radially symmetrical pollen grains having oblate-spheroidal, prolate- spheroidal or sub-prolate shape. Tricolporate, angulaperturate, colpal membrane granulated, sexine slightly thicker or equal in thickness to nexine. Tectum reticulate. Only one aquatic species found in the study area. Bacopa monnieri (Linn.) Pennell Pollen grains oblate-spheroida, tricolporate P/E ratio: 0.88. Size: Length (13.18-) 21.52±1.94 (-26.39) and diameter (15.80-) 24.51±2.58 (-34.26) µm. Colpal length (13.18-) 16.68±1.03 (-21.08) µm, mesocolpium (9.01-) 10.54±1.10 (-12.18) and apocolpium (5.36-) 8.35±1.75 (-10.55) µm, exine (3.95-) 5.06± 0.56 (-7.91) µm, sexine as thick as nexine, finely reticulate with muri. Tectum coarsely reticulate with irregular muri pattern.

12. Family Verbenaceae Pollen grains tricolporate to colpate, isopolar, radially symmetrical, prolate-spheroidal to sub-oblate, angulaperturate, colpal membrane sub-psilate to granulated. Sexine is equally thick, or much thicker or thinner to nexine. Sub-psilate, superbly regulate or reticulate tectum. Only one species is seen in the present work.

Phyla nodiflora (Linn.) Greene Pollen grains tricolporate, prolate-spheroidal, triangular. P/E ratio: 1.17. Size: length (23.72-) 27.14±1.04 (-30.30) and breadth (18.45-) 23.19±1.44 (-27.67) µm. Colpi long narrow with acute ends, (15.81-) 17.65±21.08 (-21.08) µm, mesocolpium (11.03- ) 12.40±1.16 (-14.10) and apocolpium (5.27-) 8.4±1.03 (-11.86) µm, exine thickness (1.64-) 1.90±1.14 (-2.43) µm, nexine thinner than sexine. Tectum sub-psilate- punctate.

13. Family Compositae/Asteraceae Usually radially symmetrical, oblate spheroidal-prolate spheroidal, isopolar, hardly apolar, echinate-echinolophate, often non echinate, lophate pollen grains, in polar view hexagonal to octagonal, tricolporate, often tetracolporate or porate, colpal membrane sub-psilate, sexine equally thick to nexine or slightly thinner or thicker. Tectum echinate or echinolophate, spines with sharp or acute ends and punctate base, echinolophate pollen grains are perforated in the depressions and on the ridges. In the study area only a single species found. Eclipta alba Linn. Pollen grains tricolporate, oblate spheroidal, echinate. P/E ratio: 1.31. Size: length (26.35-) 32.36±1.05 (-39.56) µm and breadth (18.45-) 24.72±1.30 (-34.26) µm. Fossaperturate, colpi with acute ends (18.18-) 23.27±0.43 (-28.98) µm long. Exine thickness (2.64-) 4.11±0.73 (-5.25) µm, nexine thinner than sexine. Tectum echinate, spines pointed at ends with sparsely punctate base, (7.91-) 10.65±1.40 (-13.18) µm long.

MONOCOTYLEDONS 14. Family Potamogetonaceae Pollen grains radially symmetrical, apolar, non-aperturate, spheroidal, sexine equally thick or thicker to nexine. Reticulate tectum having irregular muri. In study area is represented by only one species aquatic in nature. Potamogeton nodosus Poir. Pollen grains spheroidal, non-aperturate, apolar, radially symmetrical. P/E ratio: 1.06. Size: length (17.45-) 21.61±1.04 (-23.72) and breadth (17.13-) 19.92±1.97 (-23.72) µm. Exine (1.64-) 2.16±1.16 (-2.96) µm thick, nexine thinner than sexine. Reticulate tectum havin irregular pattern of muri.

15. Family Pontederiaceae Pollen grains heteropolar, oblong, mono-bicolpate, sexine is equally thick to nexine or thinner. Tectum areolate. Only one aquatic species found in the present study area. Eichhornia crassipes (Mart.) Sloms Heteropolar, oblong pollen grains. Size: Length (28.99-) 32.41±0.84 (-34.26) µm and Breadth (28.99) 35.31±2.30 (-44.86) µm. P/E ratio: 0.92. Mono-bicolpate, colpi (18.45-) 25.30±2.70 (-34.26). Exine (2.64-) 4.01±0.43 (-5.25), sexine is thinner than nexine or equally thick. Tectum areolate.

16. Family Juncaceae Pollen grains united in tetrahedral tetrads, non-aperturate, nexine thinner than sexine. Tectum lophate to reticulate. A genus Juncus represented with the following two species is present in the study area: Key to the Species + Lophate to reticulate with fine scabrae tectum Juncus articulatus - Tectum stratification obscure Juncus maritimus Juncus articulatus Linn. Pollen grains non aperturate, united in tetrads, size: Length (25.82-) 28.62±0.95 (- 31.62) µm and breadth (14.49-) 18.66±1.03 (-21.08) µm. Exine thickness (1.43-) 2.32±1.27 (-3.25) µm, nexine thinner than sexine. Tectum lophate-reticulate, with fine scabrae.

Juncus maritimus Lam. Pollen grains tetrahedral united in tetrads, non aperturate, size: Length (36.85-) 41.71±1.12 (-44.79) µm and breadth (36.85-) 39.29±0.66 (-42.18) µm. Exine pattern obscure, (1.95-) 2.48±0.49 (-3.91) µm thick, sexine thinner than nexine.

17. Family Cyperaceae Pollen grains triangular to pear shaped, heteropolar, 1-4 faintly marked aperturates, in pear shaped grains one of aperture is always situated on the proximal face and is considered to be the germ pore (Dunbar, 1973), often tenui-exinous, sexine is equally thick to nexine or usually thicker. Tectum scabrate-punctate, or areolate-punctate. Key to the Pollen Types + Tectum scabrate-punctate Type I - Tectum areolate-punctate Type II Type I: Scabrate-punctate Size: 23.71-52.70 µm in length and 21.03-38.20 µm in breadth, Triangular, 4- aperturate one end is broad with pore like aperture, other end is rounded or tapering with colpus like apertures. Exine thickness 1.64-2.69 µm, nexine thicker or thinner than sexine. Tectum scabrate-punctate. The following two species are found in the present study area falling in above mentioned pollen type: Cyperus leavigatus Linn. Cyperus arenarius Retz. Type II: Areolate-punctate Size: 21.08-31.62 µm in length and 16.31-37.08 µm in breadth, Triangular, 4- aperturate one end is broad with pore like aperture, other end is rounded or tapering with colpus like apertures. Exine thickness 3.28-5.38 µm, nexine thinner or thicker than sexine. Tectum areolate-punctate. List of 03 genus representing 06 species in the present study area falling in above mentioned pollen type is given below: Cyperus conglomeratus Rottbl. Cyperus rotundus Linn. Eleocharis palustris (Linn.) Roem. & Schult. Schoenoplectus mucronatus (Linn.) Palla Schoenoplectus lacustris (Linn.) Palla

Schoenoplectus litoralis (Schard.) Palla Cyperus Linn. Pollen grains triangular, heteropolar, 4-aperturate, nexine thicker or thinner than sexine. Tectum scabrate-areolate. A genus with 600 species represented by following four species in the study area: C. arenarius Retz. Pollen grains triangular, size: Length (23.71-) 31.62±2.37 (-39.53) and Breadth (21.08-) 25.98±1.38 (-29.78) µm. 4-aperturate, one pore like aperture at the broader pole, and three elongated colpus like apertures towards the tapering pole. Exine thickness (2.64-) 4.64±0.56 (-6.58) µm, nexine thinner than sexine. Scabrate, sparsely punctate tectum. C. conglomeratus Rottbl. Pollen grains triangular, ± with tapering ends, size: Length (21.08-) 25.48±1.18 (- 28.72) µm and Breadth (17.12-) 20.03±0.96 (-23.71) µm. 4-aperturate, one pore like aperture at the broader pole and three elongated colpus like apertures towards the tapering pole. Exine thickness (4.21-) 5.06±0.26 (6.06) µm, nexine thinner than sexine. Areolate, sparsely punctate tectum. C. laevigatus Linn. Pollen grains triangular, ± with rounded ends, size: Length (34.26-) 42.16±2.91 (- 52.70) µm and Breadth (26.35-) 32.15±1.75 (-38.20) µm. 4-aperturate, one pore like aperture at the broader pole and three elongated colpus like apertures towards the tapering pole. Exine thickness (2.64-) 3.11±0.18 (-3.69) µm, nexine thicker than sexine. Scabrate, sparsely punctate tectum. C. rotundus Linn. Pollen grains triangular, ± with rounded ends, size: Length (28.99-) 34.26±2.92 (- 47.43) µm and Breadth (21.08-) 26.24±1.58 (-31.62) µm. 4-aperturate, one pore like aperture at the broader pole and three elongated colpus like apertures towards the tapering pole. Exine thickness (5.01-) 6.27±0.41 (7.38) µm, nexine thinner than sexine. Areolate, sparsely punctate tectum. Eleocharis R. Br. Pollen grains triangular, heteropolar, 4-aperturate, nexine thicker or thinner than sexine Tectum areolate-scabrate. A genus of almost 150 species, represented by one following species in the study area:

Eleocharis palustris (Linn.) Roem. & Schult. Pollen grains triangular, ± with rounded ends, size: Length (23.71-) 30.99±2.43 (- 39.53) µm and Breadth (18.18-) 23.08±1.32 (-26.35) µm. 4-aperturate, one pore like aperture at the broader pole and three elongated colpus like apertures towards the tapering pole. Exine thickness (2.64-) 2.90±1.41 (-3.43) µm, nexine thinner than sexine. Tectum areolate. Schoenoplectus Linn. Pollen grains triangular, heteropolar, 4-aperturate, nexine thicker or thinner than sexine. Tectum areolate with scabrae. A genus of almost 200 species, represented by three following species in the study area: S. lacustris (Linn.) Palla Pollen grains triangular, ± with tapering ends, size: Length (26.35-) 33.00±2.41 (- 42.16) µm and Breadth (15.81-) 23.08±1.87 (-28.46) µm. P/E ratio: 1.43. 4-aperturate, one pore like aperture at the broader pole and three elongated colpus like apertures towards the tapering pole. Exine thickness (2.48-) 3.98±1.34 (-4.25) µm, nexine thinner than sexine. Tectum areolate-punctate. S. litoralis (Schard.) Palla Pollen grains triangular, ± with rounded ends, size: Length (31.62-) 35.84±1.14 (- 39.53) µm and Breadth (21.08-) 23.50±0.73 (-26.35) µm. P/E ratio: 1.55. 4-aperturate, one pore like aperture at the broader pole and three elongated colpus like apertures towards the tapering pole. Exine thickness (1.96-) 3.79±0.19 (-4.48) µm, nexine thicker than sexine. Tectum areolate having scabrae. S. mucronatus (Linn.) Palla Pollen grains triangular, ± with rounded ends, size: Length (27.76-) 31.41±1.16 (- 35.57) µm and Breadth (36.89-) 42.61±1.37 (-47.43) µm. P/E ratio: 0.74. 4-aperturate, one pore like aperture at the broader pole and three elongated colpus like apertures towards the tapering pole. Exine thickness (2.95-) 3.88±0.49 (-4.38) µm, nexine thinner than sexine. Tectum areolate with scabrae.

18. Family Poaceae/Gramineae Pollen grains monoporate to diporate, rarely triporate, apolar, spheroidal, operculate to non-operculate, annulate to non-annulate or reduced annulate, generally sexine is

24 equally thick to nexine or often thicker, sometimes thinner. Tectum usually areolate- scabrate and rarely areolate cum scabrate. Poaceae is one of the biggest families of angiosperms. Four species aquatic and semi- aquatic in nature are found in the study area. The pollen morphology of various taxa at generic level even at tribal level is outstandingly same. Most of the pollen characters e.g. size, aperture, shape, exine etc play a little role in the classification and delimitation of various taxa of the family. Therefore, this is not easy to construct a workable identification key. Key to the species 1. + Tectum areolate 2 - Tectum areolate cum scabrate Dichanthium annulatum 2. + Operculate Desmostachya bipinnata - Non-operculate 3 3. + complete annulus Echinochloa crus-galli - reduced annulus Setaria pumila Desmostachya bipinnata (Linn.) Stapf. Pollen grains spheroidal, (18.44-) 27.14±2.99 (-39.53) µm in diameter, mono- diporate, operculate, annulate, annulus (5.25-) 8.06±0.94 (-10.54) µm in diameter, (1.85-) 2.77±0.23 (-3.43) µm thick, pore (2.11-) 2.95±0.34 (-4.21) µm in diameter, Exine (0.79-) 1.63±0.27 (-2.64) µm thick, tectum areolate. Dichanthium annulatum (Forssk.) Stapf. Pollen grains spheroidal, (18.45-) 26.35±2.92 (-36.89) µm in diameter, monoporate, operculate, annulate, annulus (8.17-) 10.33±0.83 (-13.17) µm in diameter, (2.90-) 3.74±0.31 (-4.74) µm thick, pore (1.32-) 2.27±0.26 (-2.90) µm in diameter, Exine (0.79-) 1.37±0.33 (-2.90) µm thick, tectum areolate cum scabrate. Echinochloa crus-galli (Linn.) P. Beauv. Pollen grains spheroidal, (21.08-) 24.51±2.11 (-34.26) µm in diameter, monoporate, non-operculate, annulate, annulus (6.59-) 7.64±0.37 (-8.96) µm in diameter, (1.05-) 2.27±0.32 (-3.16) µm thick, pore (2.64-) 3.06±0.23 (-3.95) µm in diameter, Exine (2.64-) 2.90±0.14 (-3.43) µm thick, tectum areolate. Setaria pumila (Poir.) Roem. & Schult. Pollen grains spheroidal, (26.35-) 35.31±2.61 (-44.80) µm in diameter, monoporate, non-operculate, annulus reduce, pore diameter (2.64-) 2.97±0.14 (-3.43) µm, Exine (2.64-) 2.79±1.09 (-3.16) µm thick, tectum areolate.

19. Family Lemnaceae Pollen grains apolar, monoporate, spheroidal, Sexine is equally thick to nexine or sometimes thicker. Tectum spinulose to spinulose punctate. In the study area two species are present. Key to the species + Spinulose punctate tectum Lemna aequinoctialis - Spinulose tectum Lemna gibba Lemna aequinoctialis Welw. Pollen grains spheroidal, monoporate very small, Exine thickness (1.85-) 2.01±1.16 (- 2.45) µm, sexine and nexine are equally thick. Tectum spinulose punctate. Lemna gibba Linn. Pollen grains spheroidal, monoporate very small, Exine thickness (1.95-) 2.15±1.11 (- 3.02) µm, sexine and nexine are eqully thick. Tectum spinulose.

20. Family Typhaceae Pollen grains monads or tetrads, monoporate, nexine thinner than sexine, Tectum reticulat-rugulate or thickly foveolate. In study area is represented by a species only. Typha domigensis Pers. Radially symmetrical, monads, spheroidal to apolar pollen grains. Size: length (18.45- ) 21.24±1.97 (-24.03) and breadth (18.45-) 23.08±1.17 (-25.35) µm. Exine thickness (2.64-) 3.48±0.23 (-4.22) µm, nexine thinner than sexine. Reticulate tectum with irregular murri. Monoporate, pore ± circular, diameter (5.25-) 6.17±0.32 (-7.38) µm.

4.2 DISCUSSION

Size, symmetry, shape, polarity, exine sculpturing and apertural types are the major significant characteristics for identification and cataloging of pollen grains (Wodehouse, 1935; Walker & Doyle, 1975). In the present study, morphology and characteristics of pollen grains of 34 species belonging to 20 Angiospermic families of 13 dicotyledonous and 7 monocotyledonous of aquatic plants of Punjab has been investigated. Takhtajan’s system (1980) of classification has been rendered to arrange families. Pollen grains of dicotyledons are much particular and show a large diversity in their morphology and characteristics e.g. in size, shape, polarity, apertures, exine and tectum patterns. However, pollen of monocots are least specialized due to their porate, monocolpate pollen with illdefined apertures than dicotyledons. But the pollen grains of monocotyledons have also large distinctions in their exine patterns. Some dicotyledons with ancient monocolpate pollen type are considered as ancestors of monocots (Takhtajan, 1969). Following four distinctive pollen types are documented on the basis of apertures: Type-I is recognized by non-aperturate pollen grains. The species of family Juncaceae and Potamogetonaceae have non-aperturate pollen. But the pollen of Potamogeton is in monads (Pettitt and Jermy, 1975; Cook, 1988; Sorsa, 1988) and in Juncaceae are united in tetrads. Type-II is readily recognized by having colpate pollen in six different species of monocot and dicots. The colpi number ranges from mono-pantocolpate. Only a single species of monocots, the Eichhornia crassipes has mono-bicolpate pollen (Horn, 1987; Barrett, 1988). Amongst dicots Nymphaea is totally different from other species by having monocolpate pollen. However, tricolpate pollen was observed in three species such as Nelumbo nucifera, Nasturtium officinale, Ranunculus muricatus. In the present study panto-colpate pollen are only found in single species of the family Caryophyllaceae. Type-III is easily delimited by having porate/poroid pollen. In the present study twenty species belonging to the families Amaranthaceae, Convolvulaceae, Cyperaceae, Poaceae, Lemnaceae and Typhaceae have been observed by having this type of pollen. In three species of dicots, such as Alternanthera sessilis, Ipomoea aquatica and Ipomoea carnea pantoporate pollen grains were studied (Sengupta,

1972). Persicaria glabra and Persicaria amphibia are found with polypanto-porate pollen (Van-Leeuwan et. al., 1988). Monoporate pollen was observed mostly in the monocots as in Poaceae, Lemnaceae and Typhaceae (Cook, 1988; Landolt, 1986; Perveen and Qaiser, 2012). The species of the family Cyperaceae have been observed with 1-4 faintly marked poroid or elongate apertures, one at the thick end and three at lateral ends. Type-IV is readily characterized by having tricolporate pollen grains. Five species of aquatic dicots such as Trapa bispinosa, Centella asiatica, Bacopa monnieri, Phyla nodiflora and Eclipta alba have been observed with this type of pollen grains. Dicotyledons: From the family Nymphaceae only one genus Nymphaea has been examined. The features of pollen grains of the family Nymphaeaceae are totally different from other families of dicotyledons by having heteropolar, bilateral symmetric and monocolpate pollen. However, 2-3 sulcate pollen had been reported in Nymphaea by Erdtman (1952) and Walker (1974). In family Nelumbonaceae belonging to a monotypic order Nelumbonales, pollen of Nelumbo nucifera has been examined. Pollen grains were usually tricolpate, isopolar, sub-prolate, sexine thicker than nexine. Erdtman (1952) and Walker (1974) also made similar observations on the other species of Nelumbo. Previously, families Nelumbonaceae and Nymphaeaceae were considered as a single entity, but presently treated as two distint families and even placed under two separate orders i.e. Nelumbonales and Nymphaeales (Cronquist, 1968; Takhtajan, 1969). Pollen morphology also confirms them as two distinct entities. Both families are extremely different due to characteristics of pollen, such as shape, symmetry, number, position of apertures and exine sculpturing. Within family Ranunculaceae only a single species Ranunculus muricatus has been examined. Pollen grains were characterized by having pollen tricolpate, prolate- spheroidal, verrucate-scabrate tectum. Erdtman (1952) considered that Ranunculaceae has more or less similar pollen grains as in Alismataceae. Caryphyllaceae with a single aquatic species Spergularia marina has been observed. Pollen morphology of Spergularia was quite distinct such as pantocolpate, oblate-spheroidal grains with scabrate-punctate tectum are found. The monotypic order Polygonales (Polygonaceae) one of the most palynologically diverse order in dicotyledons, as indicated by Nowicke and Skvarla (1977, 1979) has been examined. It is represented by two species in the study area. Polygonaceae

28 depicts considerable variations in apertures, surface sculpturing, and size of the grains. Polypantoporate, heteropolar types of grains occur within the two species of the genus Persicaria having scabrate, finley scabrate tectum with even forms of muri in both species. Similar types of grains have also been reported in the other species of Polygonaceae (Fageri & Iversen, 1964; Moore & Webb, 1978; Van-Leeuwen et al. 1988). Size of the grains was also highly variable ranging from 11-15 um Polar axis in Polygonum amphibium R. Br. and 20-25 um in Persicaria glabra (Willd.) Gomes. Similarly, tectum also showed continous variations within the family. In Persicaria coarsely reticulate with baculate lumina, in Polygonum finely scabrate was found. It was suggested by Takhtajan (1969), that Polygonaceae and Caryophyllaceae are probably derived from the same ancestory. However, this is not supported by our palynological data because Polygonaceae is an eurypalynous and impartially large family. Pollen morphology of only a single aquatic species Nasturtium officinale R. Br. of the family Brassicaceae has been examined in the study area. Tricolpate, sub- prolate, coarsely reticulate tectum grains are found. In the family Brassicaceae reticulate and tricolpate pollen has also been reported by Appel & Al-Shehbaz (2003). However, they described pollen with lightly spinulose punctate tectum in Heliophila and 10-colpate pollen in some genera of Brassicaceae. This family was also classified by having reticulate and tricolpate pollen by Moore and Webb (1978). On the foundation of lumina size, the Brassicaceae was described with three pollen types by Khalik (2002). Perveen et al. (2004) divided Brassicaceae into four distinct pollen types on the basis of tectal surface, i.e. Erysimum melicentae - type, Arabis bijuga – type, Draba lanceolata - type and Farsetia ramosissima - type. Nasturtium officinale R. Br. is recognized in Draba lanceolate - type by having coarsely reticulate tectum. Pollen of Trapa bispinosa Roxb. has been studied. Trapa is a genus of aquatic plants, which because of its edible fruits has been the focus of human interest for thousand of years. It is surprising that there is still no consensus regarding its botanical affinities, while some botanists considered it belonging to family Lythraceae (Graham et al., 1998). On the other hand, it is considered to represent a separate family Trapaceae (Hutchinson, 1969; Cronquist, 1981; Cook, 1996; Takhtajan, 1997). Pollen grains of Trapa are spheroidal, heteropolar, wedge shaped with combined

29 ridges, tricolporate, tectum twisted, smooth to rugulate, perforate at poles with germinal structures. Convolvulaceae a eurypalynous family has been examined represented by genus Ipomoea with two aquatic species in the study area. In this family pollen characters are extremely variable such as polarity, shape, apertural types and exine sculpturing. Most striking feature is found in exine pattern and apertural types. The family Convolvulaceae is divided into two groups, based on spinulose and non- spinulose pollen and are further classified on the basis of types of apertures (Gamble, 1923). Similarly, Erdtman (1952) distinguished two well-marked types i.e. Ipomoea- type and Convolvulus-type. Ipomoea-type has been recognized in the present study having apolar, pantoporate grains with echinate tectum. A single genus Ipomoea is included in this type. Within Umbelliferae/Apiaceae, pollen grains of Centella asiatica Linn. has been examined. Pollen are tricolporate, subprolate, angul-aperturate, isopolar with striate tectum. Perveen & Qaiser (2006) have also reported tricolporate pollen grains with striate tectum in the family Umbelliferae. Cerceau-Larrival (1962) divided the pollen of Umbelliferae into 4 distinct types on the basis of P/E ratio. Punt (1984) supposed that this family is very typical by having slit like ectocolpi, broad-band like costae and bone shaped pollen. He recognized a number of pollen types in the family. On the source of tectum three pollen types are made by Perveen & Qaiser (2006). Within family Scrophulariaceae, pollen grains of Bacopa monnieri (Linn.) Pennell grows in wetlands has been examined. Pollen are mostly tricolporate with reticulate tectum. However, sufficient variations are observed within the shape and exine thickness by which genera of this family can be separated. In the genus Bacopa pollen are oblate-spheroidal. Pollen grains of Scrophulariaceae show taxonomic relationship with the Salvadoraceae, which belongs to subclass Rosidae. From the order Lamiales, Verbenaceae family with a genus Phyla representing a single species has been investigated. In this family both colpate and colporate grains occur. Colpate, colporate and rugate grains have already been reported in this family particularly in the genus Verbena by Erdtman (1952); Punt and Langewis (1988). However, in the present studied species Phyla, pollen grains are tricolporate in shape. In addition to aperture, shape, size and exine pattern is also extremely varied. Asterales is a monotypic order with a single family Compositae which is well defined, easily field recognized and largest family of dicotyledons (Cronquist, 1981).

In the present work, compositae with a single genus Eclipta grows in wetlands or near banks of canals has been examined. The pollen morphology of Asteraceae is also distinctive as of floral morphology. It is also a eurypalynous family (Erdtman, 1952). On the foundation of exine patterns three distinct pollen types have been recognized. Type-I is branded due to non-echinate pollen. Type II is characterizes by having spinulose tectum, a large number of genera belongs to this type e.g. Eclipta also lies in this pollen type. The pollen type III is also classified by having echinolophate tectum. Monocotyledons: Pollen of monocots are least specialized due to their porate, monocolpate pollen with illdefined apertures than dicotyledons. But the pollen grains of monocotyledons have large distinctions in exine. As of Magnoliidae, monocots were suggested as monosulcate group by Walker & Doyle (1975). Some dicotyledons with ancient monocolpate pollen type are considered as ancestors of monocots (Takhtajan, 1969). In the current study, pollen morphology of whole four sub classes belonging to 07 orders has been described. Each order is represented by a single family. From sub class Alismidae, Potamogetonales has been examined, representing a single family Potamogetonaceae. This family is also defined as a stenopalynous by Sorsa (1988). Pollen grains are generally apolar, non-aperturate, with coarsely reticulate tectum. Within this family, a single genus Potamogeton has been studied. The genus has apolar, non-aperturate pollen grains that are comparatively innovative. However, all other monocotyledons have monocolpate pollen grains (Walker & Doyle, 1975). From Liliidae, the family Pontederiaceae with Eichhornia has been studied. Pollen grains of this genus are less peculiar due to boatshaped and bilateral symmetric pollen than other species of monocotyledons, which are considered to be primitive pollen type in the angiosperms. Takhtajan (1969) suggested that Pontederiaceae evidentally is related to Liliaceae. Within Commelinidae, Juncales, Cyperales and Poales have been studied. From Juncales only a single family Juncaceae has been examined representing two species of Juncus. This family is unique in their pollen type by having non-aperturate tetrads with lopho-reticulate tectum. Cyperales is a monotypic order, fairly uniform in their pollen morphology, pollen grains are generally heteropolar, 1-4 colpate with scabrate or areolate tectum. Walker & Doyle (1975) reported asymmetrical, ovoid grains with

31 one or four blotchy apertures. Takhtajan (1969) considered that Cyperales is a fairly advanced taxon and probably originated from primitive Juncaceae. Poales is a monotypic order, with a stenopalynous family Poaceae. The present study is based on 04 species aquatic in nature. Pollen grains are generally apolar, normally monoporate. However, diporate or triporate grains are also found, within the species Desmostachya bipinnata. In the family Gramineae diporate pollen has been studied (Siddique & Qaiser, 1988; Perveen & Qaiser, 2012). Two types of grains (mono and diporate) within the same species are the results of polyploidy (Stebbins, 1949). Tectum of the family is generally areolate, areolate cum scabrate, or simply scabrate. Similarly type of tectum has also been observed in the other members of this family by Faegri & Iversen (1964) and Andersen & Bertelsin (1972). Poales with porate and apolar pollen grains is much distinctive than other monocots. The sedges and grasses are placed in Cyperales by Cronquist (1968). However, these are separated in orders Poales and Cyperales respectively by Takhtajan (1969). This interpretation of Takhtajan is supported by palynology, as Poaceae is definitely diverse from Cyperaceae due to porate (2-3 porate) and apolar pollen. While in Cyperaceae heteropolar, 1-4 aperturate grains are present. Arales and Typhales of Arecidae was examined in the present study, e.g. two Lemna species of Lemnaceae. Family Lemnaceae was found characterized by apolar, monoporate grains with spinulose tectum. Landolt (1986) reported spiny often less than 20 µm size grains in Lemnaceae. Walker & Doyle (1975) have also reported spinulose grains with reduced apertures in this family. Typhaceae with a single species of Typha has been examined within the order Typhales. Inspite of the fact that, Typhaceae is a monotypic family yet it is quite variable in their pollen type. For example monoporate pollen with reticulate or reticulate-rugulate tectum are studies in Typha domigensis (Cook 1988). Palynologically, Typhaceae is more specialized than Lemnaceae.

Scanning Electron Micographs of Pollen Grains of Dicots

A B

D C

E F

Plate 1. Nymphaea alba: A. Pollen. B. Exine Pattern. Nelumbo nucifera: C. Pollen D. Exine pattern. Spergularia marina: E. Pollen grain Phyla nodiflora: F. Pollen grain

A B

C D

E F

Plate 2. Persicaria glabra: A. Pollen. B. Exine Pattern. Persicaria amphibia: C. Pollen D. Exine pattern. Trapa bispinosa: E. Pollen. F. Exine Pattern.

A B

C D

E F

Plate 3. Bacopa monnieri: A. Pollen B. Exine pattern. Ipomoea aquatica: C. Pollen D. Exine pattern. Ipomoea carnea: E. Pollen F. Exine Pattern

A B

Plate 4 . Eclipta alba Linn: A. Pollen B. Exine Pattern

Scanning Electron Micographs of Pollen Grains of Monocots

A B

C D

E F

Plate 5. Potamogeton nodosus: A. Pollen. B. Exine Pattern. Schoenoplectus mucronatus: C. Pollen D. Exine pattern. Typha domigensis: E. Pollen F. Exine pattern.

A B

C D

E F

Plate 6. Cyperus rotundus: A. Pollen. B. Exine Pattern. Schoenoplectus litoralis: C. Pollen D. Exine pattern. Cyperus conglomeratus: E. Pollen F. Exine Pattern

Light Microscopy Photomicographs of Pollen Grains

A B

C D

E F

H G

Plate 7. A. Eclipta alba B. Nelumbo nucifera C. Typha domigensis D. Persicaria glabra E. Dichanthium annulatum F. Potamogeton nodosus . Trapa bispinosa G . Typha domigensis H. Lemna aequinoctialis

A B

C D

E F

G H

Plate 8. A. Lemna gibba B and C. Cyperus conglomeratus D. Cyperus rotundus E. Eleocharis palustris F. Schoenoplectus lacustris G. Desmostachya bipinnata H. Echinochloa crus-galli

PART II

A Contribution to the Ethnopharmacological Studies of the Pollen Grains of Medicinal Wetland Plants of Punjab, Pakistan

1. INTRODUCTION

Ethnopharmacology, is the systematic study of substances in plants used to cure common diseases and their relationship with the ethnic/local people. Holmstedt and Bruhn (1983) consider it as "the interdisciplinary scientific investigation of biologically dynamic agents used or observed by men conventionally ". It is a joint effort of botanists, chemists, anthropologists and pharmacologists. Sickness, curing and human physiology is considered as a cycle of interrelationships among nature, supernature, society and the individual in Traditional Medical Systems (Fabrega, 1975). Ethnobotany is providing a novel tool for pharmaceutical research. Public and private Pharmaceutical Industries began to invest money to assemble traditional information of medicinal plants and gather samples for laboratory testing especially in tropical regions, e.g. America and Africa (Chadwick and Marsh, 1994).

The knowledge of plants used by Humans is thousands of years old, gained by “trial and error” methods. Medicinal plants are commonly used for survival, home therapies, trade and helpful in lessen human suffering/poverty (Kunwar et al., 2006). In the developing countries of the world which do not have modern health facilities, medicinal plants play a vital role in the existence of rural peoples (Ahmad, 2003). Conventional medicines obtained from plants meets daily primary health requirements of about 70-80 % indigenous population in the developing countries Worldwide (Eddouks et al., 2002). Ayurvedic and Unani are the two most consistent systems in the field of medicine that present basic health facilities to the rural peoples.

The use of traditional medicine is widespread, and plants still present a large foundation of natural antioxidants that can be a good source of new drugs (Perry et al., 1999). Medicinal plant research is considered as a productive approach towards the investigation of new medicines/drugs (Svendsen and Scheffer 1982; Samuelson 1989). Plants are now well known due to the presence of medically functional compounds active on biological systems. The bioactivity of such compounds involves therapeutic value. Pathological studies of numerous chronic disorders e.g cancer, cardiac and degenerative brain diseases revealed that such disorders entails oxidative disorder to cellular components (Ebrahimzadeh et al., 2009). The chemically unstable free radicals can harm cells to a great extent due to the imbalance

41 between the generation of reactive oxygen species (ROS) and the antioxidant enzymes. .The destructive effects of ROS cause per-oxidation of lipids and aggression of tissue proteins in membrane, spoil of DNA and enzymes (Husain et al., 1987). Antioxidants are most important in the cure of chronic diseases and health troubles by ceasing radical-mediated oxidative reactions and nonstop ROS attacks (Kalpana et al., 2011). Due to the existence of antioxidant compounds e.g. phenolics, proanthocyanidins and flavonoids, plants reveal wonderful antioxidant activity (Rice- Evans, 1995).

Interest of ethnopharmacologists and chemists in Phytomedicines has been increased in the last decade due to their inhibitory action to propagate free radical reactions, to defend the human body from ailments and hinder lipid oxidative rancidity in food. Flavonoids and other phenolic compounds seem to be most valuable phytochemicals obtained from raw materials of herbs, seeds and fruits (Abbasi et al., 2010). Phenolic compounds obtained from raw materials of plants are strong lipid peroxidation inhibitors and efficient free radical scavengers (Chang et al., 2007). A variety of herbal medicines have got tremendous repute for the treatment of several diseases. Therefore, now a days various folk medicines in single and or in combination are used to treat diverse types of inflammatory and arthritic ailments (Paula et al., 2003). Medicines derived from raw materials of plants are also getting popularity due to the belief that “Green medicine” is secure with fewer side effects than the synthetic drugs (Parekh & Chanda, 2006). Like other raw materials of plants, pollen has also been reported by various researchers to house effective medicinal compounds/substances. Bee pollen has been utilized for many years in traditional medicines and supplementary nutrition mainly due to its health benefits. Its nutritional composition consists of sugars, lipids, proteins, mineral salts, fibers, vitamins and amino acids (Serra & Escola , 1997; Isla et al., 2001; Kroyer & Hegedus, 2001). Moreover, pollen also contains polyphenolic substances, mainly flavonoids which are antioxidant and antimicrobial in action (Basim &Ozcan, 2006). Apart from the above mentioned scope and applications of palynological data, it also plays an important role in the determination of the quality of honey, as it was depicted by local herbalists/hakims, during the ethnobotanical surveys of the study

42 area. They believe that the honey having a reasonable number of pollen grains in it is much better in quality and action against various human ailments as compared to the one having less or no pollen grains. They also believe that the source of pollen if is a plant that has a significant medicinal value, then this honey is much superior in its quality as far as its action against human ailments is concerned. In herbal medicine community of Turkey, female flowers of Typha species are utilized outwardly to stop blood loss (Sezik et al., 1997). Other than this, Typha pollen are also used for curing wounds and burns (Yesilada, 2002). Typha pollen recognized for removing stasis and haematemesis, are frequently recommended to cure nose and uterine bleedings (Qin & Sun, 2005). In Pakistan, Typha pollen are being eaten orally to increase flow of urine, lessen fever, prevent bleedings as well as cure injuries. Market of the herbal medicines is going up annually at the rate of 20% in India due to their increasing global demand (Srivastava, 2000; Subrat, 2002). The world market was valued of U.S. $ 19.4 billion in 1999 for herbal medicines (Laird and Pierce 2002). Various drugs have come into the International Market through study of ethnopharmacology and conventional herbal medicines (Bussmann, 2002). Tiwari and Joshi (1990) estimated that 25 % of the medicines prescribed by medical practitioners hold active principle obtained from higher plants. About 7,000 natural compounds present in plants and used in modern medicines, had also been utilized centuries ago by Asiatic, Amerindian and European healers. Now a days, 25 % prescriptions of US medical practitioners contain medicines having more than one ingredients derived from plants (Farnsworth and Morris 1976). Plants are award of nature to mankind to fight against microbes and diseases. Therefore, it is crucial to promote proper use of plants and to determine their potential for new medicines (Parekh and Chanda 2007). Ethnopharmacology has got much consideration and a lot of research is being carried out in this field Worldwide. Because of their widespread occurrence terrestrial angiosperms have got much attention for antimicrobial activities and other properties. However, aquatic angiosperms from rivers, lakes, etc. get less consideration. Although submerged aquatic angiosperms contain promising antimicrobial agents (Morales et al., 2006; Bushmann & Ailstok, 2006). Unfortunately, in Pakistan no significant work has been done on the Ethnopharmacology of wetland Plants. Some preliminary studies on antioxidant and antibacterial potential of the pollen of some

43 plants have been carried out, but no comprehensive study on the radical scavenging and antioxidant capacity has been endeavored as yet. Moreover, most of the studies have been accomplished on the pollen collected by Bees and not on the freshly/directly collected pollen from the plants. Therefore, the present study was especially designed with the following objectives:  To document the plant inventory of locally used medicinal aquatic and semi- aquatic plants of Punjab, Pakistan  To explore the medicinal potential of the pollen of some of the wetland plants of Punjab for their possible use as antioxidant agents  To provide the ethnopharmacological data thus obtained to the people/workers of local Honey industry, ethnopharmacology for new drug development etc.

2. REVIEW OF LITERATURE

The uses of aquatic plants for food, medicine, economic and other purposes have been studied by many authors (Sculthorpe, 1967; Pirie, 1968; Gupta & Lamba, 1976). This shows that herbal plants are being utilized over centuries ago for curing a variety of diseases especially under Unani systems. Therefore, World Health Organization

(WHO) is emphasizing the wider use of medicinal plants for different ailments.

Benefits of herbal drugs have also been proven by modern day scientific system

(Alam et al., 1999). Moreover, allopathic medication has side effects from mild to harsh (Said, 1996; Chan, 1993).

The current review is a citation of traditional and ethnopharmacological uses of aquatic and semiaquatic plants. Many of them have a multiplicity of uses ranging from medicinal, famine food to fodder and others. Yang-Me et al. (2007) evaluated antioxidant activity of different extracts of Nelumbo nucifera Gaertn. Various extracts of different polarities of N. nucifera rhizome were subjected to β-carotene bleaching and DPPH (2, 2’- diphenylpicrylhydrazyl) assays. The values were compared with ascorbic acid and BHA (butylated hydroxyanisole). Plant extract of all polarities showed highest antioxidant activity than that of standards. Ghosh et al. (2008) studied In vivo antioxidant and antidiabetic effects of ethanolic extracts of aerial parts of Bacopa monnieri Linn. on glycosylation of alloxan induced hyperglycemic rats. The plant extracts showed considerable decrease in blood glucose level of rats in contrast of control and compared with the standard medicine glibenclamide. The rats medicated with ethanolic extracts gained weight and reversed to normal. Ozbay and Alim (2009) assessed antimicrobial activity of some aquatic plants by disc diffusion method from Northeastern Anatolian region of Turkey against five gram- negative, three gram-positive and one fungus species. No inhibitory zone was examined with any microorganism of methanolic extracts of plants, whereas, the acetone extract of all the studied plants exhibited strong antimicrobial activity.

Jagtap et al. (2009) studied aqueous, ethanol and petroleum ether extracts of Centella asiatica to determine antimicrobial activity by agar diffusion method. The zone of inhibition produced against different strains was compared with that of the ciprofloxacin, the standard antibiotic (10 µg/ml). Small zone of inhibitions was found in water and petroleum ether extracts as compared to that of ethanol. Abbasi et al. (2010) determined comparative antioxidant prospective of methanolic and water extracts of Ipomoea carnea Jacq. separated in n-butanol, ethyl acetate, n-hexane and chloroform. The total antioxidant potential assessed by DPPH (2, 2’-diphenyl-1-picrylhydrazyl) activity, FRAP and ferric thiocyanate assays demonstrated antioxidant potential. However, n.butanol fraction gave highest value of DPPH free radical activity and the chloroform fraction showed uppermost values of total phenolic contents and antioxidant activity. Kaya et al. (2010) evaluated In vitro antioxidant and antibacterial activities of various extracts in increasing polarity of Ranunculus marginatus d’Urv and R. sprunerianus Boiss. In trolox equivalent antioxidant capacity and DPPH radical scavenging assays, maximum antioxidant activity was examined in methanolic extracts. The total phenolic contents were estimated by Folin-Ciocalteau reagent colorimetrically and total flavonoid contens by spectrophotometric methods. The antibacterial activity was evaluated by micro-well dilution and paper disk diffusion methods. The antibacterial activity was displayed in paper disk diffusion method against all the tested bacteria. Dodoala et al. (2010) studied anti-urolithiatic activity of ethanolic extracts of whole plant of Phyla nodiflora (Linn.) Greene against renal stones of calcium oxalate type induced in rats by administering Gentamycin and Calculi producing food was prevented and the pre-formed stones were also dissolved by ethanolic extracts of P. nodilfora. The plants extracts also showed momentous effects on In vivo and In vitro antioxidant parameters. Madhusudhanan et al. (2011) evaluated antioxidant activity of extracts of aqueous and ethanolic flowers of Nymphaea alba Linn. by In vitro DPPH, hydrogen peroxide and nitric oxide assays. They reported that flowers of N. alba contained dynamic alkaloids, nymphaeine, nupharine and are sedative. Both extracts showed magnificent activity, however, the ethanolic extract revealed more antioxidant activity than aqueous activity.

Venkatesh and Dorai (2011) explored In vitro antioxidant and antibacterial potential of both pink and white flowers of Nelumbo nucifera Gaertn. against different bacterial strains. Minimum Inhibitory Concentration (MIC) and zone of inhibition were compared with the standard antibiotics and their potential was suggested as alternative antibiotics to cure the ailments caused by these bacteria. Both white and pink flowers exhibited highest antioxidant activity in FRAP (Ferric reducing antioxidant power) assay. They suggested that maximum antioxidant activity in both the flower extracts showed was because of alkaloids, phenols and flavonoids in them. Kalpana et al. (2011) studied methnolic extracts of Desmostachya bipinnata (Linn.) Stapf. roots to find out antioxidant activity and the compounds responsible for antioxidant activity in its’ roots. Total phenolic contents were calculated by Folin- ciocalteu method while total flavonoids by aluminium colorimetric assay and tannins by Folin-denis method. Free radical scavenging activity was evaluated by FRAP and ABTS assays. By their findings of antioxidant phytochemicals, they suggested that D. bipinnata could serve as a future drug. Choudhary et al. (2011) highlighted the important utilities of Polygonum species (Knotweeds) of India, to make an inventory of those species that require re- cultivation and accessibility of increasing natural medicinal products. They reported 72 species of Polygonum, out of which 34 were found in use excessively as medicines, famine food, ornamentals and others. Borah et al. (2011) evaluated In vitro antioxidant activity of various extracts of Alternanthera sessilis (Linn.) DC. using DPPH and Phophomolybdate methods. The maximum antioxidant activity was examined by methanolic extracts in both the assays. Nitric oxide radical scavenging, superoxide radical scavenging and ferrous chelating activities showed highest values in acetone and methanol extracts. Increase in the concentration elevated antioxidant potential. This study indicated that A. sessilis is a good source of antioxidants. Prabu et al. (2011) suggested E. alba as an excellent antimicrobial and antioxidant, after studying the antibacterial, antifungal and antioxidant properties of methanolic extracts of Eclipta alba Linn. using 2, 2’-diphenyl-1-picrylhydrazyl (DPPH) assay and disc diffusion methods. The plant extracts showed strong antioxidant effects as compared to the standard ascorbic acid. Esra et al. (2011) studied external use of female flowers of Typha domingensis Pers. for healing burns and wounds in Turkish folk medicine. Both (male and female)

47 flowers were evaluated In vivo by wound healing (circular excision and linear incision) models. Among n-hexane, chloroform, ethanol and water extracts, the significant wound healing activity was observed in methanolic and water extracts, as compared to standard ointment. Shanab and Shalaby (2012) determined anticorrosion efficiency and biological activity of separated fractions of Eicchhornia crassipes (Mart.) Sloms. by spectroscopic methods. Although E. crassipes is an persistent aquatic weed causing serious problems in irrigation canals and drainage ponds worldwide, yet is exposed as a prosperous source of bioactive compounds having antioxidant, antiviral, antimicrobial and antitumoral activities. Bukhari (2000) has carried out a research work to investigate the study of palynology, skin irritancy and antimicrobial activity of the pollen grains of Abutilon indicum, Gossypium hirsutum and Malvastrum coromandelianum species of the family Malvaceae. The results showed that the extracts of pollen grains of Malvastrum coromandelianum indicated a stronger irritant response and antimicrobial activity against four bacteria and two fungi.

3. MATERIALS AND METHODS

The present study was carried out under the following plan of work: 1. Ethnobotanical study 2. Collection of pollen 3. Ethnopharmacological Study 4. Statistical analysis of the data 1. Ethnobotanical Study: This study was conceded by various visits of Wetlands of Punjab, Pakistan during the years 2008-2011. Local senior knowledgeable people and herbal healers were interviewed to get the data i.e. local name of plants, habit and habitat, traditional uses of plants, with emphasis on therapeutic uses. The data was confirmed by frequent queries. The fresh plants especially with flowers and fruits were collected, dried, compressed, mounted and afterwards recognized/identified by Flora of Pakistan (Nasir & Ali, 1970-1995; Ali & Qaiser, 1995-2005). Names of plants identified were inveterated in Herbarium, Centre of Plant Conservation, University of Karachi, Karachi. The voucher specimens are placed in Herbarium, Botany Department, GC University, Lahore. The data thus obtained was verified with the existing literature on these plants. 2. Collection of Pollen: Pollen of Typha domigensis were collected directly by shedding its, inflorescence on a filter paper. However, the inflorescence of Nelumbo nucifera and Centella asiatica were spreaded out on filter paper and allowed to dehiscence. Afterwards the pollen was gathered from the paper in powdery form. 3. Ethnopharmacological Study: This study included investigation of antioxidant activity of pollen as follows: Antioxidant Activity Chemicals The reagents used to explore the antioxidant activity in all experimental assays were of purified grade, labeled with Fluka and Merck Trade Marks.

Trolox made in ethanol was used as standard antioxidant. Ultra-Violet 1700 Pharma Spectrophotometer, Ultra-Violet Visible Spectrophotometer (Shimadzu, Japan operational through CPS controller) were used throughtout the experimental work. Preparation of Plant Extracts The crude extracts of pollen of Typha domigensis, Nelumbo nucifera and Centella asiatica were obtained in methanol using soxhelt apparatus. This methanol extract was evaporated in rotary evaporator at 300C to get residue, which was dissolved in suitable quantity of distilled water and partitioned with n-hexane, chloroform and ethyl acetate respectively. These extracts of varying polarity and water fraction were concentrated further on rotary evaporator to get stock solution for use in the following antioxidant assays: ABTS•+ Decolorization Assay ABTS (2,2’-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) radical cation protocol was used after Re et al. (1999). 2,2’-azinobis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) was mixed with water to get a concentration of 7mM. ABTS stock solution was dissolved in potassium persulfate (2.45 mM) and set aside (12-16 hours) without light to obtain desired ABTS.+. To analyze antioxidant activity of each pollen, the extract was diluted in relevant organic solvent with which they were extracted. Now 10 μL of this solution was mixed with diluted ABTS•+ solution (2.99 mL) for measurement of absorbance (30 °C) after one minute interval using Spectrophotometer at 517nm for a total duration of eight minutes. Solvent without pollen extract were also run parallel for accurate readings. The %age of decoloration was computed with the help of following prescription:

In above mentioned equation, Ao = absorbance of radical cation

Af = absorbance of the addition of sample/standard antioxidants in stock soultion Results were obtained by the comparison of antioxidants concentration and Trolox. Total Phenolic Contents Assay Total phenolic contents were measured by a stated method of Slinkard & Singleton (1977). To obtain a stock solution, 0.5 grams of gallic acid was dissolved in 10 mL of ethanol and final volume was raised to 100mL by adding distilled water. Anhydrous

50 sodium carbonate (200 grams) was mixed with double distilled water (800 mL) in order to get Sodium carbonate solution. Few crystals of sodium carbonate were also added after boiling and cooling. The solution was set aside for a day and night. It was then filtered and final volume was made upto one litre by adding double distilled water. The different volumes (0, 1, 2, 3, 5 and 10 mL) of stock solution of phenol were diluted with double distilled water up to 100 mL. A volume of 40 μL was taken into separate cuvettes from each dilution for further dilution with double distilled water up to 3.16 mL. A blank was also run in parallel. Then Folin- Ciocalteu's reagent (200 μL) was mixed and subsequently sodium carbonate solution (600 μL) was also added and placed for half an hour at 40ºC. Each solution and blank was measured at 765 nm for absorbance. FRAP (Ferric Reducing Antioxidant Power) Assay Ferric Reducing capacity was analyzed by a reported method of Benzie & Strain, (1999). Freshly prepared FRAP solution was containing 25ml (pH 3.6) of acetate buffer (300 mM), TPTZ solution (2.5 mL of 10 mM) in 40 mM of HCl solution and ferric chloride solution (2.5 mL of 20 mM). 100 μL of each of sample solution was taken in separate test tubes and then FRAP solution was added in each to make a volume of 3mL. Absorbance was measured after one minute for a total duration of six minutes at 593 nm. The readings were contrasted with ferrous sulphate standard curve. Metal Chelating Activity The method of Dinis et al. (1994) was utilized to determine chelation of ferrous ions and standards. Aliquots (1 ml) of the plant extracts and control were taken separately in test tubes, followed by mixing with 50 μl of 2mM FeSO4.7H2O and 150 μl of 5 mM ferrozine. The mixtures were shaken well and placed for 10min at room temperature. Each solution was measured in a spectrophotometer at 562 nm for absorbance. The %age inhibition of antioxidants was measured with the help of following equation:

%age Inhibition = [(A0 – A1)/A0] x 100 Here,

A0 = absorbance of control

A1 = absorbance in presence of the plant extracts or standards. EDTA was utilized as a reference compound. Total Flavonoid Content

Total Flavonoid contents were determined by using colorimetric method after Jia et al. (1999). Distilled water (1.25 ml) was mixed in sample extract and then Sodium nitrite solution (0.075 ml, 5%) was also added and incubated for five minutes. 0.15 ml of 10% aluminum chloride was added after incubation and then one Molar sodium hydroxide (0.5 ml) was finally mixed after an interval of six minutes. This was diluted with distilled water of 0.275 ml and measured at 510 nm instantly for absorbance by comparing with standard curve of quercetin. Superoxide Anion Radical Scavenging Activity This activity of plant extracts was evaluated by the method devised by Nikishimi et al. (1972). In this method, NADH-PMS system was employed for the In vitro generation of superoxide radical anions. The reaction mixture was prepared by mixing 100μl of sample, 624μM of NADH, 200μM NBT and 80 μM PMS in phosphate buffer (0.1M) of pH 7.4. Then absorbance of reaction mixture was measured at 560 nm. The %age scavenging of each sample was calculated from the formula: %age scavenging = [1– AS /AB X 100] Where AB and AS were the absorbance of blank and sample solutions respectively. 4. Statistical Analysis All the experiments were carried out by using 3 replica of each sample and values were computed from final results. The values were articulated by taking mean at ±SD (n = 3) using One Way ANOVA (SPSS version 12.0).

4. RESULTS AND DISCUSSION

4.1 RESULTS Ethnopharmacological study Ethnopharmacological information of eighteen (18) wetland or semi-wetland plants belonging to seventeen (17) genera of 17 families, collected from conventional and local people is presented in Table 1. Botanical names mentioned in alphabetic order are chased by their family name, part used and ethnopharmacolgical uses. Evaluation of Antioxidation Potential of Pollen The antioxidation potential of Pollen grains of Typha domingensis Pers., Centella asiatica (Linn.) Urban and Nelumbo nucifera Gaertn. Pollen was explored by using three basic working mechanisms of antioxidants. For this purpose, ABTS.+ assay was employed to determine free radical scavenging ability, FRAP assay to identify ferric reducing ability and metal chelating ability for its chelating potential. Super oxide anion radical scavenging activity was employed for free radicals of Centella asiatica pollen. Moreover, to correlate antioxidant ability with its phytochemical composition, the total phenolic and flavonoid contents were also determined. The results of antioxidation activity of T. domigensis were summarized in Table 2 and Figs. 1-5, while those of C. asiatica in Table 3 and Figs. 6-11 and N. nucifera in Table 4 and Figs. 12-16.  ABTS•+ Decolorization Assay Protocol Trolox Equivalent Antioxidant Capacity (TEAC) values were obtained by measuring the percentage inhibition values of pollen extracts with Trolox curve. TEAC values of T. domingensis ranged from 3.94-8.96 mM, while that of C. asiatica 12.34 to 106.26 mM and N. nucifera 6.0-9.1 mM of trolox equivalents. Amongst different fractions of T. domingensis, n-hexane, choloroform and ethyl acetate showed higher TEAC values, whereas in C. asiatica water and ethyl acetate showed highest TEAC readings and in N. nucifera, n-Hexane, methanol and water showed highest values. However, the remaining polar fractions showed small TEAC values indicating relatively low free radical scavenging ability. The graphical representation of results of ABTS.+ of T. domingensis, , C. asiatica and N. nucifera are shown in Fig. 1, Fig. 6 and Fig. 12 respectively.

 Total Phenolic Contents (TPC) Phenolic compounds having hydroxyl groups in their structures are very powerful antioxidants (Robbins, 2003). Total Phenolic Content values of T. domingensis ranged from 262-354 mg/l, while that of C. asiatica from 143-1155 mg/l and of N. nucifera from 5410-8150 mg/l of Gallic Acid. The graphical representation of total phenolic contents of T. domingensis, C. asiatica and N. nucifera are shown in Fig. 2, Fig. 7 and Fig. 13 respectivley. All the fractions of T. domigensis extracts showed high values of total Phenolic Content with pollen. However, in case of H. asiatica methanol, chloroform and water extracts showed highest TPC values.  Ferric reducing antioxidant power (FRAP) The Ferric Reducing Antioxidant Power assay of Benzie and Strain (1999) engrosses a single electron reduction of the Fe (TPTZ)2 (III) complex (pale yellow) to the Fe

(TPTZ)2 (II) complex (blue) by sample antioxidants at acidic pH. Any antioxidant species with lower reduction potential than that of Fe (III) TPTZ salt (0.7 V) may be 3+ 2+ able to reduce Fe -TPTZ to Fe -TPTZ contributing to FRAP value (Dejian et al., 2005). This reduction was observed spectrophotometrically at 593 nm. Appearance of deep blue coloration was a sign of presence of reducing components in the sample. The FRAP values of the fractions of Pollen extracts were calculated by way of comparison with a calibration curve obtained using iron (II) sulfate as the standard reductant. Ferric reducing antioxidant power values for different fractions varied from

1.312-5.944 mg/L of FeSO4 equivalents of T. domingensis, 4.58-13.87 mg/L of FeSO4 equivalents of C. asiatica and 49.6-87.5 of FeSO4 of N. nucifera. The graphical representation of results of FRAP assay of T. domingensis, C. asiatica and N. nucifera are shown in Fig. 3, Fig. 8 and Fig. 16 respectively.  Metal chelating activity An important mechanism of antioxidant activity is their ability to chelate/deactivate transition metals, which have hydroperoxide decomposition catalyzing capacity and Fenton-type effects (Quang-Vinh, 2011). Chelating agents may also act as secondary antioxidants because they reduce redox potential, as a result stabilizing the oxidized forms of metal species. So, the %age bound iron capabilities of the extracts were monitored. In the existence of chelating agents, complex formation between ferrozine and Fe2+ is interrupted, resulting in reduction in the red colour of the complex. The graphical representation of metal chelating capacities of the n-Hexane, Chloroform,

Ethyl Acetate, Methanol and water extracts of pollen grains of T. domingensis, C. asiatica and N. nucifera are shown in Fig. 4, Fig. 9 and Fig. 15 respectively.  Total Flavonoid Contents Flavonoids, as one of the most varied and general groups of natural compounds, are the most vital natural phenolic constituents (Agrawal, 1989). Therefore, the contents of flavonoids in the extracts were also evaluated. The graphical representation of results of total flavonoid contents of T. domingensis, C. asiatica and are shown in Fig. 5, Fig. 10 and Fig. 14 respectively.  Superoxide Radical Anion Scavenging Activity This activity as evolved by Nikishimi et al. (1972) is hereby used to find out superoxide radical anions in the pollen extracts of C. asiatica only. These superoxide radical anions reduce Nitroblue tetrazolium (NBT) to formazan which lessens the yellow color of the NBT to blue. The blue color shows the production of superoxide radicals. These superoxide radicals are scavenged by sample having antioxidant potential by donating electrons. The absorbance of the samples was taken at 560 nm and decrease in absorbance showed the scavenging of superoxide radicals. The results of this assay were expressed in terms of %age scavenging of superoxide radical anion. The decrease in absorbance at 560 nm with the sample and the standard compound, quercetein indicates their abilities to quench superoxide radicals in the reaction mixture. All the fractions of C. asiatica showed higher values of superoxide radical anion. The highest values with ethyl acetate and n-Hexane were also showing similar scavenging levels, as shown in Fig. 11.

Table 1: Medicinal uses of Wetland Plants of Punjab, Pakistan for treatment of Different Diseases Sr. Botanical Name Family Part Used Ethnopharmacological Uses No. 1. Alternanthera sessilis Amaranthaceae Whole plant It is used to treat cough and fever, (Linn.) DC. wounds, eye infections and acne. 2. Bacopa monnieri (Linn.) Scrophulariaceae Whole plant It is used to cure a number of ailments Pennell in India and Pakistan e.g. epilepsy, anxiety, depression, asthma, bronchitis, cardiac and digestive disorders. 3. Centella asiatica (Linn.) Umbelliferae/Apiaceae Whole Plant It is employed to treat dysentery, skin diseases, brain disorders, tuberculosis Urban and ulcer. 4. Desmostachya bipinnata Poaceae/Gramineae Whole plant It is used in diarrhea, dysentery, (Linn.) Stapf. jaundice and skin diseases. 5. Eclipta alba Linn. Compositae/Asteraceae Leaves, Roots and Commonly prescribed by herbal flowers healers for liver disorders, also used to reduce hepatic and spleen ailments. Fresh juice of leaves is used to improve digestion and increase hunger. 6. Eichhornia crassipes (Mart.) Pontederiaceae Whole plant It is used for cardiovascular diseases Sloms and disorders of brain. Leaves are diuretic that may also be useful to cure cough and asthma.

Sr. Botanical Name Family Part Used Ethnopharmacological Uses No. 7. Ipomoea carnea Jacq. Convolvulaceae Roots, Juice and Boiled roots are used to provoke Leaves menstruation while fresh juice and leaves by the traditional healers for skin diseases 8. I. aquatica Forsk. Convolvulaceae Leaves Generally used for the treatment of liver disorders. 9. Nasturtium officinale R. Br. Brassicaceae/Cruciferae Whole plant It has been used in earliest time to treat liver disorders and skin ailments. 10. Nelumbo nucifera Gaertn. Nelumbonaceae Flowers Whole flower is used to cure liver diseases and a range of other ailments like fever, hypertension, diarrhea, weakness etc 11. Nymphaea alba Linn. Nymphaceae Flowers Useful for the treatment of anxiety and inhibition of renal stress. 12. Phyla nodiflora (Linn.) Verbenaceae Whole plant It is used for curing ulcer, burning and Greene asthma. 13. Persicaria amphibia (Linn.) Polygonaceae Leaves, Stems and A combination of leaves and stems is A. Gray Leaves used to cure stomach pains and diarrhoea. Roots are used to treat cough, cold and fever.

Sr. Botanical Name Family Part Used Ethnopharmacological Uses No. 14. Potamogeton nodosus Poir. Potamogetonaceae Leaves A pain balm is prepared by the maceration of leaves. 15. Ranunculus muricatus Linn. Ranunculaceae Roots and Flowers Commonly used to treat jaundice, constipation, dropsy and swellings of joints. 16. Spergularia marina (Linn.) Caryophyllaceae Whole plant It is commonly used as an antidiabetic. Criscb. 17. Trapa bispinosa Roxb. Trapaceae Fruits Fruits are edible, sweet, nutritive, appetizer and diuretic with cooling effect, mainly used to treat leucorrhoea, seminal weakness, leprosy, fever and diarrhea. 18. Typha domingensis Pers. Typhaceae Inflorescence Flowers are used to cease bleeding. Pollens are recognized to increase flow of urine, lessen fever, prevent bleedings as well as cure injuries.

Table 2: Antioxidant activity of pollen of Typha domigensis Sample TEAC Value FRAP %age TPC (mg/L TFC (mg/L of mMol Value bound iron of GAE) QE) (mMol) n-Hexane 8.96±0.02 1.80±0.76 50.40±0.98 289.99±0.32 409.18±0.91 Chloroform 6.82±0.04 2.80±0.86 58.80±0.15 271.49±0.86 258.27±0.05 Ethyl Acetate 5.70±0.10 1.71±0.93 45.20±0.06 262.49±0.92 401.90±0.88 Methanol 3.94±0.25 5.94±0.48 41.40±0.05 354.99±0.75 372.82±0.16 Water 4.00±0.98 3.98±0.72 47.90±0.76 272.94±0.66 110.09±0.17 The data shown is mean ±SD (n=3).

Table 3: Antioxidant activity of pollen of Centella asiatica Sample TEAC FRAP %age %age TPC TFC Value Value bound scavenging of mg/mL of mg/mL of (mM) (mM) Iron Superoxide GAE QE radicals 13.93±0.7 4.58±0.5 27.5±0. 1301.81±0. n-Hexane 8 5 18 89.1±0.36 250±0.986 68 17.64±0.0 12.27±0. 28.36± 1281.81±0. Chloroform 6 49 0.49 88.6±0.74 1070±0.654 26 85.09±0.0 7.84±0.2 45.63± 911.81±0.3 Ethyl acetate 4 0 0.40 89.1±0.26 143±0.169 6 12.34±0.1 12.54±0. 55.63± 2488.18±0. Methanol 6 24 0.70 84.5±0.48 1155±0.467 67 106.26±0. 13.87±0. 45.63± 1488.18±0. Water 56 89 0.84 84.33±0.43 1106±0.556 06

The data shown is mean ±SD (n=3).

Table 4: Antioxidant activity of pollen of Nelumbo nucifera TEAC FRAP %age TPC mg/mL TFC mg/mL Sample Value Value bound Iron of GAE of QE (mM) (mM) n-Hexane 7.8±0.002 49.6±0.068 47.8±0.07 6970±0.046 4412.5±0.93

Chloroform 6±0.002 68.6±0.05 50.3±0.02 5800±0.43 4600±0.35 Ethyl Acetate 6.6±0.35 63.6±0.09 57.5±0.50 5410±0.098 4985±0.023

Methanol 9.1±0.07 87.5±0.07 79.4±0.05 8150±0.25 5800±0.13 Water 8.8±0.57 74.5±0.08 71.5±0.78 7910±0.098 5665±0.94

The data shown is mean ±SD (n=3).

Graphical representation of results of antioxidant activity of Typha domingensis

ABTS Assay

8 7

6 5 4 3

TEAC Value(mMol) TEAC 1 0 n-Hexane Chloroform Ethyl Methanol Water

Acetate

Figure 1: ABTS.+ assay of the Pollen of T. domingensis

Total phenolic contents

450 400 350 300 250 200 150 100 TPC TPC (mg/l of GAE) 50 0 n-Hexane Chloroform Ethyl Methanol Water Acetate

Figure 2: Total phenolic contents of the pollen of T. domingensis

FRAP Assay

8 7 6 5 4 3

FRAP Value FRAP 2 1 0

e ate nol Water oroform etha n-Hexan hl l Acet M C Ethy

Figure 3: FRAP Assay of the pollen of T. domingensis

Metal chelating activity

70 60 50 40 30 20

%age bound iron bound %age 10 0 n-Hexane Chloroform Ethyl Methanol Water Acetate

Figure 4: Metal Chelating Activity of the pollen of T. domingensis

Total Flavonoid contents

500 450 400 350 300 250 200 150 TFC (mg/l QE) (mg/l TFC 100 50 0 n-Hexane Chloroform Ethyl Methanol Water Acetate

Figure 5: Total Flavonoid Contents of the pollen of T. domingensis

Graphical representation of results antioxidant activity of Centella asiatica

Figure 6: ABTS.+ assay of the pollen of C. asiatica

Figure 7: Total phenolic contents of the pollen of C. asiatica

Figure 8: FRAP Assay of the pollen of C. asiatica

Figure 9: Metal Chelating Activity of the pollen of C. asiatica

Total Flavonoid contents 3000 2500 2000 1500 1000

TPC (mg/L of QE) 500 0 Ethyl Methanol Chloroform Water n-Hexane Acetate

Figure 10: Total Flavonoid Contents of the pollen of C. asiatica

Figure 11: Superoxide Radical Anion Scavenging Activity of the pollen of C. asiatica

Graphical representation of results of antioxidant activity of Nelumbo nucifera

ABTS Assay 10 9 8 7 6 5 4

TEAC (mM) Value 3 2 1 0 n-Hexane Ethyl Water Chloroform Methanol Acetate

Figure 12: ABTS.+ assay of the pollen of N. nucifera

Total Phenolic contents 9000 8000

) 7000 6000 5000 4000 3000

TPC (GAE mg/mL (GAE TPC 2000 1000 0 n-Hexane Ethyl Water Chloroform Methanol Acetate

Figure 13: Total Phenolic Contents of the pollen of N. nucifera

Total Flavonoid contents 7000

6000

5000

4000

3000 TFC(QE mg/mL) 2000

1000

0 n-Hexane Ethyl Water Chloroform Methanol Acetate

Figure 14: Total Flavonoid Contents of the pollen of N. nucifera

Metal chelating activity 90 80 70 60 50 40 30 %age Bound Iron %age 20 10 0 n-Hexane Ethyl Water Chloroform Methanol Acetate

Figure 15: Metal Chelating Activity of the pollen of N. nucifera

FRAP ASSAY 100 90

) 80 70 60 50 40 30 FRAP (mM Value 20 10 0 n-Hexane Ethyl Water Chloroform Methanol Acetate

Figure 16: FRAP Assay of the pollen of N. nucifera

4.2 DISCUSSION

Depending upon the external and internal uses, only 18 (53%) wetland plants species belonging to seventeen families and having highly ranked medicinal values were explored for the documentation of their ethnopharmacological uses. These can be classified into following different categories according to their medicinal applications and therapeutic properties:  Aquatic plants used to treat cough, asthma and fever e.g. Alternanthera sessilis, Eichhornia crassipes, Nelumbo nucifera, Persicaria amphibian, Trapa bispinosa, Phyla nodiflora and Typha domingensis.  Aquatic plants used to reduce digestive, spleen, renal, hepatic and liver disorders e.g. Bacopa monnieri, Desmostachya bipinnata, Eclipta alba, Centella asiatica, Ipomoea aquatica, Nasturtium officinale, Nelumbo nucifera, Nymphaea alba, Phyla nodiflora, Persicaria amphibian, Ranunculus muricatus and Trapa bispinosa.  Aquatic plants useful for brain and cardio-vascular diseases e.g. Bacopa monnieri, Eichhornia crassipes, Centella asiatica, Nelumbo nucifera and Nymphaea alba.  Aquatic plants used against skin diseases e.g. Desmostachya bipinnata, Ipomoea carnea and Nasturtium officinale.

It has been determined from the above mentioned data that, Punjab is vastly diversified with wild aquatic medicinal plants that are being used in Gastrontology (66%), Neurology and Cardiovascular diseases (28%), Respiratory diseases (39%) and Dermatology (17%). This is because of the traditions living in Punjab, where most of the inhibitants are rural with an extensive experience in fold medicines as local herbalists and healers. It can be eagerly sought after the present research work that wetlands of Punjab comprise a significant medicinal flora. It is obvious from this ethnobotanical survey of aquatic plants of Punjab, that researchers should pay attention to evaluate their ethnopharmacological activities for the improvement of pharmaceutical industry by using natural drug resources for the synthesis of new drugs that can be very effective against resistant pathogens. This can also lead to

77 the protection of these plants especially from threatening and endangering factors. Most of the young people do not know about such ethnomedicinal uses of these plants. Awareness campaigns and projects regarding conservation and uses of wetland plants should be started by Government so that local people may know about ethnomedicinal effectiveness of the locally found aquatic and semi-aquatic plants, as per WHO demands. For example, inspite of frequent use of Spergularia marina as an antidiabetic in lowering blood glucose level, its antidiabetic and antioxidant activities have not been evaluated. Similarly Eichhornia crassipes, commonly used for cardiovascular diseases and disorders of brain, having leaves being diuretic and may even be useful to cure cough and asthma needs such ethnopharmacological investigations. This is because E. crassipes contains several biologically active and valuable medicinal compounds that may be useful for the cure of cardiac ailments, cancer and brain disorders. This finding has also been supported by Shanab & Shalaby (2012) after completing such studies on this plant. Moreover, harvesting of E. crassipes for extraction of natural antioxidants and pharmaceutical purposes may be helpful in controlling the wide spread growth of this plant in water bodies and canals, because it is one of the serious problems in the drainage of ponds and canals Worldwide.

As documented during the present ethnobotanical surveys, Eclipta alba is commonly prescribed by herbal healers for liver disorders and to reduce hepatic and spleen ailments. Fresh juice of leaves of this plant is used to improve digestion and increase hunger. The same has been suggested by Prabu et al. (2011) while evaluating the antimicrobial and antioxidant activity of E. alba for curing human diseases. Nymphaea alba is useful for the treatment of anxiety and renal stress. This is because of the presence of phenolic constituents in N. alba. Madhusudhanan et al. (2011) agree with the findings of the present work that the ethanolic and aqueous extracts of the flowers of Nymphaea alba Linn. contain phenolic compounds like gallic and ellagic acid. Ipomoea carnea growing along the canal banks is ethnomedicinally important and has been used in Punjab by herbal healers to cure various ailments. The important bioactive compounds and antioxidants have been reported in polar fractions of I. carnea by Abbasi et al. (2010). Similarly according to indigenous knowledge in Sri Lanka,

Ipomoea aquatica may have insulin like activity and is also used as a vegetable throughout the region of Southeast Asia (Huang et al., 2005) just like its’ use in Punjab, Pakistan.Whereas it has also been used to treat liver disorders, as recorded during the surveys in the study areas in the Punjab for the documentation of ethnomedicinal data on wetland or semi-wetland plants. Persicaria amphibia is used very often as a potent source of antioxidants due to the presence of flavonoids, stilbenoids, tannins and coumarins. This is in agreement with various authors that the different species of Persicaria contain several chemical compounds of such types (Sartor et al., 1999; Datta et al., 2002; Lopez et al., 2006). The flowers of Nelumbo nucifera are used against various diseases. This is because of the alkaloids present in it such as neferine, nuciferine, remrefidine, liensinine and flavonoids like quercetin, norcoclaurine present in Nelumbo flowers (Venkatesh and Dorai, 2011). Roots and flowers of Ranunculus muricatus are used to treat jaundice, constipation, dropsy and swellings of joints. In Turkish traditional medicine, Ranunculus species are utilized to treat wounds because of their wound healing properties, as reported by Kaya et al. (2010). Antioxidant, antiviral and antimicrobial studies by various authors have shown that Ranunculus species contain several biologically active constituents, responsible for such activities (Sener et. al., 1998; Li et. al., 2005; Barbour et. al., 2004; Noor et al., 2006). The entire plant of Nasturtium officinale R. Br. is medicinally useful. The antioxidant study of N. officinale extract has been carried out by Bahramikia and Yazdanparast (2010) and they declard it an effective remedy against free radical-related ailments. Fruits of Trapa bispinosa are edible and used to cure various diseases. Trapa bispinosa fruit contains sugars, starch, proteins, fats, manganese and nitrogenous substances (Ghani, 2003). The grass Desmostachya bipinnata is also used as a potential source of natural antioxidants, as confirmed by Kalpana et al. (2011). Other than these medicinal values, aquatic plants play an important role in water bodies. They are the main source of food for zooplanktons, e.g. Gheese and ducks that depend upon Lemna, Potamogeton, Polygonum species for food. Songbirds use fibres of Typha species to make their nests. Typha plants are also used to make baskets and mats

(Fig 17 and 18). Aquatic plants provide shelter to small animals such as aquatic insects, crustaceans and snails that are ultimately used by fishes and waterfowls for food. They are also a source of food for young frogs, salamanders and fishes. Young fishes and amphibians use aquatic plants as source of cover for prevention from predators. They protect shorelines from erosion and alleviate sediments for water clarity. Now a days, aquatic plants are getting much attention for being their use to saturate pollutants as phytoremediators and indicators of water quality, as it was recorded in some towns in Punjab during the ethnobotanical surveys of the study area. Antioxidant Activity: Typha domigensis In general, it is concluded that Typha domigensis pollen have a high antioxidant potential, as all the extracts showed a distinct antioxidant activity in all the In vitro working mechanisms of antioxidant activity. The results showed that methanol extract displayed the highest total phenolic contents and also possessed the strongest reducing power. Moreover, the highest metal chelating activity was found in the chloroform extract and total flavonoid content in n-hexane fraction. The fractions of increasing polarity extractive solvents showed higher value of TEAC indicating high solubility of phenolic and other antioxidant components. High Total Phenolic Content values are due to the presence of sterols, terpenoids, flavones, gallic acid, long chain hydrocarbons, caffeic acid and p-coumeric acid in pollen of Typha (Esra et al., 2011). The crown FRAP values were obtained in more polar solvents, e.g. methanol. It is apparent that the polarity of the extractive solvent has vast influence on the extraction of antioxidant compounds. In metal chelating activity, chloroform and n-Hexane fractions owned higher metal chelating activities in contrast to the other solvents. The metal chelating activity of the fractions was tremendously dependent on the solvent due to the different antioxidant potentials of the compounds. The compounds with high polarity were responsible for metal chelating activity. However results demonstrate that all extracts had useful capacity for iron binding and therefore, their action as peroxidation inhibitors may be correlated to their iron-binding capacity. The yield of flavonoids depended not only upon plant type but also upon the extraction solvent. The pollen of Typha extract in n-Hexane (a non-polar solvent) contained higher flavonoid contents as compared to the other solvents. This

80 means that the highest polar solvent (water) is not suitable for the extraction of flavonoids compounds from Typha pollen. Centella asiatica n-Hexane, Chloroform, Ethyl acetate, Methanol and water extracts of Centella asiatica pollen showed significant antioxidant activity in all the In vitro assays. C. asiatica growing as a creeper, is a very active medicinal plant. Several chemical compounds have been reported in C. asiatica such as asiatic acid, brahmic acid, asiaticin, glucose, terpenoids, rhamnose, stearic acid, ascorbic acid, calcium, iron, phosphate, etc. (Sahu et al., 1989; Williamson, 2002; Pan et al., 2007). As per the findings of the present work, aqueous extract showed highest total antioxidant activity with TEAC assay. Ethyl acetate fraction showed moderate activity. It may be suggested that C. asiatica extract reacts with the hydrogen donor in the polar aqueous medium and converts it into hydrazine. It also shows that reducing power is increased by increasing the extract. The antioxidant activity of pollen by FRAP assay showed similar results with TEAC values whereby the aqueous medium showed highest mean antioxidant activity. In metal chelating activity polar medium methanol extract have highest %age iron bond than other extracts. Pittella et al. (2009) has also proved while evaluating the antioxidant activity of C. asiatica that the potent antioxidant activity in polar extracts is due to the presence of hydroxyl groups in them. Scavenging activity based on superoxide radicals was also determined in pollen of C. asiatica. All extracts from non polar to polar scavenged superoxide radicals effectively. The polar fractions such as water and methanol extracts possessed the highest phenolic contents, which could be due to the presence of hydroxyl groups. In humans polyphenols shows inhibitory effects on mutagenesis and carcinogenesis (Chippada and Vangalapati, 2011). In the present study, one mg of methanolic extract contained highest phenolic contents, which show that pollen of C. asiatica contains gallic acid, tannins, hydroxyl and polyphenols. The total flavonoid contents were highest in methanol extract whereas, the non polar medium n-hexane extract also showed a potent amount of flavonoids. The findings of this section of the study propose that pollen grains of C. asiatica can be a good source of antioxidants that may be used to treat diseases like dysentery, skin diseases, brain disorders, tuberculosis,

81 ulcer, aging and age associated oxidative stress, as has been stated by a number of workers (Jagtap et al., 2009; Pittella et al., 2009; Kunwar et al., 2009). Nelumbo nucifera The data obtained from antioxidant activity showed that the pollen of N. nucifera possess strong antioxidants in all In vitro assays. Several phytochemical constituents e.g. flavonoids, alkaloids and phenols have been screened from both pink and white flowers of N. nucifera (Durairaj and Dorai, 2010). n-Hexane, Chloroform, Ethyl acetate, Methanol and water extracts were used to determine antioxidant activity in all the In vitro assays. The major objective of the present study was to carry out the antioxidant potential of pollen grains of both pink and white lotus flowers and as per our results, it is concluded that N. nucifera flowers have the highest antioxidant activity. It is evident from Figures 13 and 14 that all extracts of pollen of lotus flowers have highest phenolic and flavonoid contents. Sridhar and Rajeev (2007) have also concluded that the major constituents of lotus flowers are alkaloids and flavonoids. The present phytochemical analysis of lotus pollen also revealed the presence of alkaloids, flavonoids and phenolic compounds in them. In general, it is concluded that T. domigensis, C. asiatica and N. nucifera pollen have a high antioxidant potential, as all the extracts showed a distinct antioxidant potential in all In vitro working mechanisms of antioxidant activity. Therefore, it is suggested that pollen extracts of varying polarity of medicinally important aquatic flowers may be used for pharmaceutical uses. This study supports the belief of the local herbalists/hakims that the presence of pollen of plants having medicinal value in the honey declares it a valid/pure honey. The honey without or with less pollen in it is either a false honey or is not good in its’ quality. Some honey types are even considered to be superior in quality and very effective against serious human diseases. This may be because of the rich amount of plant secondary metabolites present in the pollen grains that are found in such honey samples. Such phytochemicals like carotenoids, flavonoids and phytosterols have already been reported in the pollen grains by Moore et al. (1991). During the field survey of the wetland and semi-wetlands of Punjab, a variety of bees, including honey bees and butterflies were found visiting and collecting pollen of the

82 flowers of aquatic plants. This means that the pollen grains of the aquatic plants are also added up into the honey hives in the study area.

Figure 17: Bundles of Typha plants collected to make mats and baskets

Figure 18: A person making mat from Typha plants

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