ISSN 2226-3063 e-ISSN 2227-9555 Modern Phytomorphology 12: 15–32, 2018 https://doi.org/10.5281/zenodo.1195691 RESEARCH ARTICLE Distribution, morphology, anatomy and histochemistry of Crepidium acuminatum Sebastian John Adams 1*, Thiruppathi Senthil Kumar 2, Gnanamani Muthuraman 1, Anju Majeed 1 1 Department of Phyto-Pharmacognosy, Research and Development, Sami Labs Ltd., 19/1 & 19/2, 1st main, 2nd phase, Peenya Industrial Area, 560058 Bangalore, India; * [email protected] 2 Department of Botany, Bharathidasan University, 620024 Tiruchirappalli, India Received: 11.11.2017 | Accepted: 06.03.2018 | Published: 11.03.2018 Abstract Crepidium acuminatum is largely confine to the Himalayan region although it has been reported in South India, where its presence distribution needs to be critically studied. This study describes the morphological, anatomical and histochemical aspects of the materials of the authenticated species collected from Himalayas. This study also highlights the features of histochemistry and anatomy that should be used for the correct identification and authentication of C. acuminatum, especially because of the therapeutic importance of the species and its possible adulteration by other orchids. Keywords: Malaxis acuminata, Microstylis wallichii, orchid mycorrhizae, pelotons, pseudobulb Introduction general debility, bleeding diathesis and phthisis (Pushpa et al. 2011; Balkrishna Crepidium acuminatum (D. Don) Szlach. 2012; Subedi et al. 2013; Adams et al. 2017). belongs to the family Orchidaceae. The main part of the plant applied in folk Commonly known as Jivaka, this Ashtavarga and Ayurvedic medicine is the pseudobulb, plant is mainly used for its cooling effect, which in fact is the aerial stem of the plant. febrifuge, spermopiotic and refrigerant Its Ayurvedic dynamics are: sweet in taste, activity; it acts against haematemesis, cold in potency, pacifies vata and aggravates fever, semen-related weakness, burning kapha (Singh 2006). It is also proven that the sensation, dipsia, emaciation, tuberculosis, ethanolic extract of pseudobulbs exhibits © The Author(s) 2018. Published by Andriy Novikov, State Natural History Museum NAS of Ukraine on behalf of Modern Phytomorphology. This is an open access article under the Creative Commons BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/) freely available on https://phytomorphology.org/ . 16 Adams S.J. et al. antiproliferative activity (Singh et al. 2017). (Foundation for Revitalization of Local It also forms a useful therapy in combination Health Traditions FRLHT, Bangalore) and with drugs from other ashtavarga plants. Prof. K.V. Krishnamurthy (Research and Astavarg Churna, Jevaniyo Dashko Development Consultant, Sami Labs Ltd, Mahakshay and Chyawanprash Linctus are Bangalore). The vouchers were deposited in some of the compound drugs from orchids, FRLHT herbarium, Bangalore. The voucher including this plant that are well-known raw drug material is also deposited there. in Ayurveda (Kaushik 1983; Bose et al. 1999; Some pseudobulbs are being cultivated in Chinmay et al. 2011). There are market the medicinal plant garden of that institute. problems in meeting the demands for this Fresh samples of leaf, pseudobulb, plant, as it is in very short supply giving protocorm and root were used for taking scope for possible adulterations / substitute transverse sections (about 10–20 µm of other orchids like Malaxis cylindrostachya in thickness), using razor blade and Kuntze and M. mackinnoni Duthie cryomicrotome Medi Meas MCM-ST. The (Balkrishna et al. 2012). The trade value of sections were stained using various staining this material is around 120 Rs/Kg in North- procedures (Tab. 1) and observed under a West Indian markets (Sultan & Singh 2006). microscope Nikon Eclipse Ci. Photographic Unsustainable harvest of wild populations images were captured using Nikon DS of this orchid for pharmaceutical and Ri2 attached to a microscope. The images nutraceuticals purpose has been a major were processed on Nikon Basic Essential threat to causing the rarity of this species software. (Hinsley et al. 2017). Hence, there is an Autofluorescence and induced urgent need for proper authentication fluorescence images were captured in the of this species, as well as defining its above mentioned microscope fitted with distribution in India so as to enable the epifluorescence unit and these results collection of genuine plants. This article were correlated with data obtained under describes the distribution, morphology and normal light. Calcium oxalate crystals were anatomy of C. acuminatum, all of which are localized using polarizing optics. very important in the proper authentication of this species. Results and discussion Material and methods Distribution and taxonomic identity Crepidium acuminatum is reported to be Fresh plants were collected from Raisaar mainly distributed in the temperate to Devta, Tehri Garhwal District, Uttarakhand, subtropical Himalayas (Clarke 1885) at an India (30.34465, 78.415371) and identified altitude of 1200–2100 m a.s.l. It is found in by Dr. Amit Singh (G.B. Pant National Himachal Pradesh, Uttarakhand, Arunachal Institute of Himalayan Environment and Pradesh, Assam, Nagaland, Manipur, Sustainable Development, Mohal-Kullu, Mizoram and Tripura states of India Himachal Pradesh), Dr. T.N. Manohara (Samant et al. 1998; Chauhan 1999; Singh (Scientist, Rain Forest Research 2005, 2006; Dhyani et al. 2010; Balkrishna et Institution, Jorhat, Assam), and verified al. 2012). Ved et al. (2003) and Lohani et al. and authenticated by Dr. K. Ravikumar (2013) have reported that it is a rare and Modern Phytomorphology 12, 2018 Distribution, morphology and histochemistry of Crepidium acuminatum 17 Table 1. Localization of chemicals components and applied procedures. Chemical components Method References Alkaloid Dragendroff’s Reagent Yoder & Mahlberg 1976; Ferreira et al. 1998 Acidic polysaccharide Toluidine Blue O (TBO) Krishnamurthy 1988 Anthocyanin Vanillin-Perchloric Acid Abraham et al. 1988; Narayana et al. 2002 Lignin Phloroglucinol–HCl; TBO Krishnamurthy 1988 Polyphenols TBO Krishnamurthy 1988 Lipids Oil Red O, Sudan Black Krishnamurthy 1988 Cutin, Suberin Auramin O, Azure B Krishnamurthy 1988 Starch Iodine-Potassium Iodide (Lugol’s Iodine) Krishnamurthy 1988 vulnerable species of the Himalayan region. flower. According to the checklist prepared C. acuminatum has also been reported from based on the earlier reports and literature, South India especially in the South Western Nayar et al. (2014) concluded that only Ghats and recently in Andhra Pradesh two species of Crepidium are distributed (Gamble & Fischer 1915–1936; Abraham & in Western Ghats, namely C. mackinnonii Vatsala 1981; Henry et al. 1989; Reddy et al. (Duthie) Szlach. [syn. Microstylis mackinnonii 2001, 2006; Murugesan & Balasubramaniam Duthie, Malaxis mackinnonii (Duthie) Ames] 2008; Karuppusamy et al. 2009; Cheruvathur and C. purpureum (Lindl.) Szlach. [syn. et al. 2010; Dutt et al. 2010; Aravindhan Microstylis purpurea Lindl, Microstylis wallichii et al. 2011; Chinmay et al. 2011; Manikandan auct. non. Lindl., Malaxis purpurea (Lindl.) & Lakshminarasimhan 2012; Gupta 2016; Kuntze, Malaxis acuminata auct. non. Ved et al. 2016). According to Matthew D. Don]. Thus, according to these authors (1983), this species (recorded as Malaxis Crepidium acuminatum (D. Don) Szlach. acuminata D. Don) is found in Servarayans [syn. Malaxis acuminata D. Don, Microstylis Hills (Yercaud) of Tamil Nadu Eastern Ghats wallichii Lindl.] does not occur in Western at an altitude of 1500 m, on the mountain Ghats. We also consulted Dr. C. Sathish slopes. He distinguished this species from Kumar, former Scientist of Jawaharlal Malaxis rheedii B. Heyne ex Wallace on the Nehru Tropical Botanic Garden & Research basis of that there are large auricles and a Institute, Trivandrum, and a leading mid lobe with a split at apex of the perianth orchidologist of India. He has categorically lip in M. acuminata, while the lip is without stated that C. acuminatum is restricted to the distinct auricles in M. rheedii. However, he Himalaya region and that all reports of this mentioned that M. acuminata, although species in South India are based on wrong is a different species from M. rheedii in identification. Indian species of Malaxis Yercaud hills, he still was not satisfied Sol. ex Sw. were merged with Crepidium with his identification and requested for Blume, Liparis Rich., Oberonia Lindl. and some more work in order to confirm the Seidenfia Szlach., and it was suggested that presence of this species in this region. the genus Malaxis s.l. (including the species Ridley (1887) distinguished the species M. acuminata) probably is not represented Microstylis rheedii Wt. from Crepidium Blume in South India. It was also suggested that by the colour and structural characters of further studies may be made to verify Modern Phytomorphology 12, 2018 18 Adams S.J. et al. whether the South India samples reported smooth, shinning outside and fleshy inside. as M. acuminata represent natural hybrid Mature stem is purple in colour at least on between M. acuminata and M. rheedii (both of the side bearing the leafy sheath. Stem is them now are known under the new names). 2 to 12 cm long and 1 to 4 cm in diameter, Such possibilities for natural hybridization mucilaginous when in contact with water. in Crepidium have been reported by The dry stem is covered with the shiny Nuammee et al. (2016). However, we feel translucent grayish pink coloured