BIOLOGICAL TECHNOLOGY

INTERNATIONAL JOURNAL OF BIOLOGICAL TECHNOLOGY EDITOR –IN -CHIEF Dr. M. Maridass Director Gayathri Pharma Teknological Research Centre 58/9 Annanagar,Palyamkottai-627 011,Tamil Nadu, biotekeditor@yahoo.com ASSOCIATE EDITORS Dr.G.Raju Assistant Professor Department of Adv.Zoo.& Biotech, Pioneer Kumaraswamy College, Nagercoil-629003,Tamil Nadu Dr.R.Mahesh Postdoctoral Fellow Centre for Biodiversity and Forest Studies, Madurai Kamaraj University, Madurai - 21, Tamil Nadu Dr. D.Ganesh, Assistant Prof. Dept of Biotech, SPKCES Manonmaniam Sundaranar University, Alwarkurichi, Tamil Nadu. EXECUTIVE EDITORS Dr.A.Benniamin, Scientist "C" Botanical Survey of India Itanagar-791111 fernbenni@gmail.com Dr. A.Manimegalan Associate Professor Dept. of Envi. Sciences Bharathiar University Coimbatore Dr.U.Ramesh, Assistant Professor Dept of Molecular Biology Madurai Kamaraj University Madurai-21 EDITORIAL BOARD Dr. M.I. Zahir Hussain,Asst.Prof. Dept of Adv Zoo.& Biotechnology Sadakathullah Appa College, Palayamkottai - 627011-mizahirhussain@rediffmail.com Dr. Deepak Ganjewala Senior Assistant Professor, School of Biosciences and Technology, Vellore Institute of Technology University, Velllore-632 014, Tamil Nadu, India deepakganjawala73@yahoo.com Adnan J. AL Rehaily, Ph.D., Associate Professor Department of Pharmacognosy, College of Pharmacy, King Saud University, Dr. S. Gokul Shankar Faculty of Medicine, AIMST University Semeling, Kedah, Malaysia 08100. subushika@yahoo.com Dr. C. Rajasekaran Assistant Professor (SG) School of Bio Sciences and Technology VIT University Vellore - 632 104 drcrs70@gmail.com Dr. Pronobesh Chattopadhyay Professor College of Pharmacy IFTM, Lodhipur Rajput Moradabad -244001 (U.P), India chatto_pronobesh@rediffmail.com Dr. R. L. Khosa Professor & Director (Planning & Development), Pharmacy, BIT, Meerut. Prof. Shakti Prasad Pattanayak Division of Pharmacology Department of Pharmaceutical Sciences Birla Institute of Technology (BIT) Mesra, Ranchi – 835 215 sakthi_pattanayak@yahoo.co.in Dr.N.Siva, Assistant Professor Dept of Botany PTMTM College Kamuthi-623604 Annual Subscription Individual Rs.250/- ($ 50) Institution Rs.500/- ($ 100) Life Membership Rs.5000/- ($ 750) Dr.C.Vijayakumar, Assistant Prof (SG) Dept of Zoology St.Andrew's College, Goraghpur,UP Dr. M. Muralidharan Assistant Professor SPKCES, Manonmaniam Sundaranar University, Alwarkurichi, Tamil Nadu. Dr. Dilipkumar Pal, Professor, Seemanta Institute of Pharmaceutical Sciences, Jharpokharia, Mayurbhanj- 757 086, Orissa E-mail:drdilip2003@yahoo.co.in. Dr. A.P. Singh Associate Professor Dept of Dravyaguna, Shri Dhanwantry Ayurvedic College, Sector 46-B, Chandigarh Dr. Bharath Kumar Ravuru Associate Professor, Dept. of Biotechnology Vignan’s Engineering College Vadlamudi,Guntur-522224.Andhra Pradesh. bharathrkumar@yahoo.com, Prof. T. Kalaivani Assistant Professor School of Bio Sciences and Technology VIT University Vellore – 632 104 tkvani72@rediffmail.com raja_sekaran123@rediffmail.com Prof. A.Krishnaveni Asst Professor Dept of Pharmacognosy Madurai Medical College Madurai 625020, Tamilnadu. akrishnaveni72@rediffmail.com. Prof. Saumya Das, Asst. Professor (SG) Institute of Pharmaceutical Technology, NIET, Greater Noida. Dr.S.Vijayaragavan, Assistant Prof. Department of Zoology Presidency College Chennai, Tamil Nadu Dr.P.Ravichandran, Assistant Professor Department of Biotechnology JJ College of Arts and Science Pudukottai, Tamil Nadu Dr. Nachiket S. Dighe Assist.Prof. and HOD, Dept. of Medicinal Chemistry, Pravara Rural College of Pharmacy, Pravaranagar, Loni BK. India-413736. nachiket1111@rediffmail.com Prof. Sanjay Balasaheb Bhawar Asst.Prof. & Head Dept. Of Pharmacology, Pravara Rural College Of Pharmacy, Pravaranagar, Dr. Kailash Choudhary Assistant Professor Department of Biotechnology Lachoo Memorial College of Science and Technology JNVU, Jodhpur, Rajasthan India -342001 kchoudharylmc@gmail.com Prof. A. Saravana Kumar Assistant Professor Nandha College of Pharmacy, Erode-52. Ph: 04294-221174, e-mail: saravncp@gmail.com Dr.Nilesh Kumar, Assistant Professor Department of Pharmacognosy & Phytochemistry, Dept. of Pharmaceutical Sciences, Lovely Professional University, Jalandhar-Delhi G.T. Road, Phagwara, Punjab – 144402, INDIA mpharmnilesh@gmail.com Dr.S. Maneemegalai Vice-Principal,Department of Biochemistry, Bharathidasan University College for Women, Orathanadu-614 625. maneedevi@yahoo.co.in Advertisement Tariff Half P age : Rs.500/- Inner Page: Rs.1000/- Back Page : Rs.2000/- Subscription and Cost of reprints should be sent by Demand draft drawn in "GAYATHRI TEKNOLOGICAL PUBLICATION" Payable at Palayamkottai. © Gayathri Teknological Publication

International Journal of Biological Technology (2010) 1(1):1-123. Volume No: 1(1) i 1 2 Contents April, 2010 i-ii 1-7 8-11 3 4 5 6 7 8 Instruction to Authors Original Article Investigation of Phytochemical constituents from Eulophia epidendraea M. Maridass and U. Ramesh Pharmacognostical and phytochemical investigation studies on Gymnema sylvestre R.Br. C. Kalidass and V.R. Mohan Phytochemical, Pharmacognostical, Antimicrobial activity of Indigofera aspalathoids Vahl. (Fabaceae) Bojaxa A. Rosy, Henry Joseph, and Rosalie Effect of Plant Growth Regulator on In vitro Multiplication of Turmeric (Curcuma longa L. cv.Ranga ) Kambaska Kumar Behera, Debashrita Pani and Santilata Sahoo In vitro multiple shoot induction through axillary bud of Ocimum basilicum L. an important medicinal plant A. Daniel, C. Kalidass and V. R. Mohan Micropropagation in vitro flowering in Solanum nigrum linn. A medicinal plant M.S. Sundari, A. Benniamin and V.S. Manickam Clonal propagation of Adiantum capillus - veneris M. Maridass, R. Mahesh, G.Raju, and K. Muthuchezhian Estimation of culturable microbes present in heavy metal contaminated and non contaminated Agricultural soil R. Ajazhaja mohideen, R. Jeyaprakash, V. Thirumalai Arasu and T.Sivakumar Fluoride Removal from water by Sorbing on Plant and Fungal Biomass Rajneesh Prajapat, Ashish Bhatnagar, Rajarshi Kumar Gaur, and Vivek Bajpai Metal concentration in Manakudy estuarine sediments South West Coast of India A.Jeena Pearl Intra specific hybridization between Amhiprion sebae and A. polymnus under captive conditions A. Pushparaj Effect of Hesperidin on serum Heart Marker, Myocardial Tissues Parameter and Histopathological of Heart in isoproterenol Induced Myocardial infarction in Diabetic Rats Jagdish Kakadiya, Mehul Shah and Nehal Shah Azotobacter population in Rhizosphere and Non-Rhizosphere sediments of Tondi Coast V.Kalaigandhi, E.Kannapiran, Harimuraleedharan, A.Michael,T.Sivakumar and V.Thirumalai Arasu Application of statistical methods to optimize medium for increased yield of Oyster Mushroom (Pleurotus ostreatus) P. Prakash, S.Anuradha, P.K. Dhanalakshmi and N. Niveditha Biotechnological synthesis of gold nanoparticles of Azadirachta indica leaf extract A.Thirumurugan, Jiflin,G.J, Rajagomathi.G, Neethu Anns Tomy, S.Ramachandran, and R.Jaiganesh 12-15 16-23 24-28 29-32 33-37 38-42 43-46 47-51 9 10 11 52-56 12 57-62 13 63-65 14 66-74 15 75-77 Inauguration issue © Gayathri Teknological Publication

International Journal of Biological Technology (2010) 1(1):1-123. 16 Influence of media on protease production by Beauveria bassiana ( Bals.) Vuil. and stability towards commercially available detergents, surfactants and enzyme inhibitors S. Karthick Raja Namasivayam, S. Sivasubramanian and Ganesh Kumar Evaluation of amino acids profiles in human saliva using Reverse phase high performance liquid chromatography V. Nithya, S.Alagendran, G.Archunan, Anusha Bhaskar, X. Baskaran, Miller Samson, R.Anusha, E.A. Orozco-Bonilla, and Guevara Guzmán Rosalinda In vitro direct regeneration of nodal explant of Justicia prostrata Gamble R. Jeyachandran, X. Baskaran and L. Cindrella Genetic analysis of somoclonal variation among Jasminum auriculatum (Vohl.) and it’s callus Priya joy and D. Patric Raja Wound healing effect of chitosan in fresh water fish Cyprinus carpio L. U. Ramesh and M. Maridass In vitro Antibacterial activity of three Indian medicinal plants R.Jeyachandran, X. Baskaran and L.Cindrella Chemosystematics evaluation of Eugenia species based on molecular marker tools of flavonoids constituents M. Maridass and U. Ramesh In vitro propagation of wild yam, Dioscorea wightii through nodal cultures R. Mahesh, K.Muthuchelian, M. Maridass, and G.Raju Review An inside preview of Ethnopharmacology of Cissampelos pareira Linn. Amritpal Singh, Sanjiv Duggal, Jaswinder Singh and Shankar Katekhaye Newsletter Food for Space Charu Gupta and Sneh Gupta 78-83 17 84-89 90-93 18 19 94-98 99-102 103-106 20 21 22 107-110 111-113 23 24 114-120 121-123 25 Inauguration issue © Gayathri Teknological Publication

International Journal of Biological Technology Instructions to Authors International Journal of Biological Technology (IJBT) is published in the month of April, August and December. International Journal of Biological Technology (IJBT) dedicated to multidisciplinary field of Conservation biotechnology, Plant biotechnology, Medical biotechnology, Aquaculture biotechnology, and Environmental biotechnology. It publishes original contributions into several major areas such as Plant and animal tissue culture, Drug and vaccine development of natural product, Histopathology, toxicology and genotoxicology. Guidelines for preparation of manuscript should be following: The following types of papers are considered by the Editors: Research Articles, Review Articles, and Short Reports. Papers are preferable to be written in English. Papers are accepted on the understanding that they have not been published or being submitted for publication elsewhere. Manuscript Submission Three copies of the manuscript must be submitted to the Dr. M. Maridass, Editor, International Journal of Biological Technology (IJBT), 58, AnnaNagar, Palayamkottai-627011, Tamil Nadu, India. Submission may also be made electronically to biotekeditor@yahoo.com. If the article is accepted for publication, an electronic copy of the final version of the manuscript will be requested. If excerpts from other copyrighted works are included, the author must obtain written permission from the copyright owners and credit the source(s) in the article. The manuscript must be typed, double- spaced on one side of A4 paper (212 x 297 mm or 8.5 x 11 inches) using 1 inch margins. All pages in the manuscript should be numbered consecutively. The tables and figure should be grouped at the end of the manuscript. The manuscript should be in the following order: (1) Title, (2) Abstract, (3) Keywords, (4) Text, (5) Acknowledgements, (6) References, (7) Tables, (8) Figures. The text for research articles should contain the following sections: Introduction, Materials and Methods, Results, Discussion (or Results and Discussion) and Conclusions. Review of Manuscripts Upon receipt, a manuscript will be peer-reviewed. The reviewers' relevant comments and the editor's decision will be communicated to the authors. The qualified reviewer can be recommended by the authors; however the selection of reviewers will be at the discretion of the Editor. Papers accepted become the copyright of the Journal. Title The title should be on the first page and provide precise information about the contents. Title should not exceed 120 characters (space included). The title should be followed by the list of author(s) and affiliation(s). Corresponding author should be indicated with the postal address, phone and e- mail address. Abstract and Keywords Papers must begin with an abstract not exceeding 250 words. It should contain a brief description of the aim, principal results and major conclusions. 5-10 informative English keywords are required. Text For research article, It should contain Introduction, Materials and Methods, Results, Discussion (or Results and Discussion) and Conclusion sections The chemical terms must conform to IUPAC rules and the SI units must be used throughout. The specification of materials and equipment must be given. For experiments involving human or animal subjects, authors must state in the manuscript the approval of the protocol by an institutional or local ethics committee. The i © Gayathri Teknological Publication

International Journal of Biological Technology Declarations of Helsinki, Tokyo for humans, the European Community and Indian Council of Medical Research guidelines as accepted principles for the use of experimental animals, must be adhered to. Equations must be consecutively numbered on the right hand side using numbers in parentheses. Tables Each table should be titled, typed on separate sheets in consecutive order using Arabic numerals. Indicate SI units of measure in parentheses. Figures Figures must be presented on separate sheets in consecutive order using Arabic numerals. The legend should be typed separately from the figures. Indicate SI units of measure in parentheses. The original photograph, chromatogram or drawings are required with the submission. Due to the technical complications which can arise, please submit in addition black and white prints corresponding to the color figures. References Literature citations in the text must be presented in numerical order in parentheses. The abbreviations of journals should follow the system used by Index Medicus or Chemical Abstracts. References must be arranged as follows: Yamada, H., Yoshino, M., and Matsumoto, T., 1987. Effects of phytosterols on anti- complementary activity. Chem. Pharm. Bull.,35: 4851- 4855. Edwards, S. A.,2006. The Nanotech Pioneers, Wiley-VCH Verlag, Weinheim, pp. 1-14. Meltzer, P.S. Kallioniemi, A. and Trent, J.M.,2002. Chromosome alterations in human solid tumors. In: B. Vogelstein, and K.W. Kinzler (eds.), The Genetic Basis of Human Cancer, McGraw-Hill, New York, pp. 93-113. Aviv, H. Friedman, D. Bar-Ilan, A. and Vered, M.,1996. Submicron emulsions as ocular drug delivery vehicles, U.S. Patent US 5492811. Short Reports The short report section is open to interesting results worthy of publication without requiring extensive introduction and discussion. This section should contain the same heading and subheadings as research articles. The length of the article should not more than 8 pages (A4) or 2500 words. Tables, figures and references are to be arranged according to research papers. Review Articles The organization of review articles can be arranged at the author's discretion. Abstract and keywords are requested. Tables, figures and references are to be arranged according to research papers. Referees Three Experts should be evaluation of the Manuscript. Correcting Proofs The editorial office will send the proofs to authors for correction. Only typesetting errors may be corrected; changes in, or additions to, the accepted manuscript will be allowed. The reprints in PDF will be supplied free of charge to the corresponding author. The authors are responsible for the contents appeared in their published manuscripts. Charges Printing & process charges National Rs.500/- and International US $100/- . Copyright and Photocopying All rights reserved for ©2010 Gayathri Teknological Publication Ltd. And with the exception of good dealing for the purposes of research or criticism or review, no part of this publication may be reproduced, stored or transmitted in any form or by any means without the prior permission in writing from the copyright holder. ****************** © Gayathri Teknological Publication ii

International Journal of Biological Technology (2010) 1(1):1-7. Original Article Investigation of Phytochemical constituents from Eulophia epidendraea M. Maridass and U. Ramesh Animal Health Research Unit, St. Xavier’s College (Autonomous), Palayamkottai - 627002, Tamil Nadu, India. Email: orchideyadass@yahoo.com Received :12.4.2009 Revised :25.8.2009 Accepted: 11.12.2009 Published: 15.4.2010 Abstract The tuber and leaf of Eulophia epidendraea was phytochemically examined. Chromatographic procedures led to the isolation of four phytochemicals in β-sitosterol (I), β-sitosterolglucoside (II), β – amyrin (III) and lupeol (IV) from the tuber and also four flavonoids of apigenin, luteolin, kaempferol, and quercetin were identified from the leaves of E. epidendraea. Keywords: Orchidaceae, Eulophia epidendraea, tuber, leaf, Phytochemicals Introduction Higher plants are major sources of natural products such as pharmaceuticals, agrochemicals ingredients of flavor and fragrance, food additives, and pesticides (Balandrin and Klocke,1988). The pharmaceutically well known phychemical of morphine was isolated from opium poppy, Papaver somniferum by Sertuner (Burger,1960) quinine from Chinchona officinalis (Cragg et al.,2002), reserpine from Rauvolfia serentina, ephedrine from Ephedra vulgaris and taxol from Taxus brevifolia (Wani et al.,1971). These phytochemicals constitute some of the most exiting chemotherapeutic agents currently available for use in a clinical medicine. Orchid Eulophia epidendraea (Retz.) Fischer belongs to the family Orchidaceae, Which has been traditionally used by the local people of Yadav community for the treatment of tumour, abscess and healing of wound (Maridass et al., 2008). Recently, pharmacological studies on the wound - healing activity of tuber extract of E. epidendraea were reported. The present study was, therefore, carried out to identify the chemical constituents of the tuber and leaf of Eulophia epidendraea (Retz.) Fischer. About 1.0 kg of this powder was extracted with petroleum ether (30-60 o C) benzene (80.3 o C), chloroform (61 o C), acetone (56 o C) and methanol (65 o C) in a Soxhlet apparatus. The extraction process was performed for 8h. The solvents were evaporated under reduced pressure. After determining the yields, sediment extracts were stored at 4 o C for further study. The methanolic extract (8.0g) was then fractionated by column chromatography on silica gel and eluted with ethyl acetate (EtOAc) followed by EtOAc - MeOH, and gradient 25 ml fractions were collected: fractions1- 4 (EtOAc), fractions 5-17 (10% MeOH), fractions18-21 (20% MeOH), fractons 22-33 (30% MeOH), fractions 34-38 (40% MeOH), fractions 39-56 [EtOAc-MeOH- H2O (10:5:1v/v/v)]. Fractions 5-17 contained compound (I). Fractions 18- 21 contained (II); fractions 34-38 contained (III); fractions 39-56 contained (IV) respectively. Identifications were made by comparison with the data from previous IR, UV, NMR and mass spectra (Pandey et al., 1996; Mučaji et al., 2000). Instrumentation UV spectra were obtained on a Shimadzu UV-160 spectrophotometer, and IR spectra were determined in KBr discs on a Perkin-Elmer 781 spectrophotometer. 1 H NMR spectra were recorded with a Varian Gemini NMR spectrometer at 200, 400 MHz or with a Bruker Avance NMR spectrometer at 500 MHz in CDCl 3 . 13 C NMR spectra were recorded with a Varian Gemini NMR spectrometer at 50, 100 MHz or with a Bruker Avance NMR spectrometer at 125 MHz in CDCl 3 . EI - MS were obtained with a JEOL JMS - HX110 Materials and Methods Plant materials The orchid Eulophia epidendraea (Retz.) Fischer was collected from Kambli Malaikovil Forest, (75 o 50’E and 9 o 20’N) near Tenkasi, Tirunelveli District, Tamil Nadu, India. Solvent extraction and isolation The tuber of Eulophia epidendraea (Retz.) Fischer was air - dried and powdered. © Gayathri Teknological Publication 1

International Journal of Biological Technology (2010) 1(1):1-7. spectrometer and HREI - MS with a Finnigan MAT 95S spectrometer. Isolation of leaf flavonoids The powdered leaf of Eulophia epidendraea was studied using hydrolyzed extract following the method of Harborne (1973). About 100g of dried leaves were cut into small pieces and extracted in 20 ml of 2M HCl, then boiled in a water bath at 100 o C for 1hr. The hydrolyzed extract was allowed to cool and filtered through a filter paper to remove debris from the extract. The filtrate was treated twice with ethyl acetate; the upper layer containing flavones and flavonol was then separated from lower aqueous layer by a separating funnel. Amyl alcohol was added to the latter layer to extract anthocynidins. These extracts were allowed to evaporate to dryness overnight in a dark fume chamber. Then five drops each of ethanol (95%) and methanol (100%) were added to dissolve flavones and flavonol which were ready for spotting into the plates. Thin layer chromatograpic (TLC) analysis The hydrolyzed extracts were run single dimensionally in solvent forestal, at room temperature of 21 - 28 o C. The concentrated extracts were spotted on the lower left corner of the TLC plate using 5µl micropipette. Fifteen loads of the extracts were applied and allowed to dry using a hair dryer before each subsequent load. The diameter of the spot in each chromatogram was normally about 5mm. Authentic markers of flavones (luteolin and apigenin) and flavonols (myricetin, quercetin and kaempferol) obtained commercially were co-chromatographed. Identification of the hydrolyzed compounds of these extracts was made by examination of the spots under UV light and by changes in colour under day light after application of ammonia. Rf values of these spots in comparison with the Rf values of authentic markers, coupled with those values given for each known compound in Harborne (1973), were of great help in identification of these spots. Nine chromatographic spots were identified in this study. They indicated four known compounds and five unidentified compounds (Table -2). of chromatographic separations over silica-gel, resulting in the isolation of β-sitosterol (I), β- sitosterolglucoside (II), β – amyrin (III) and lupeol (IV). The structures of these compounds were determined by comparing their spectral data with those reported or analyzing their various 13 C and 1 H- NMR spectral data and determined in comparison with the literature data. Compound (I) was obtained as colourless needle. The molecular formula C 29 H 50 O was assigned by HREI-MS spectrophotometer. On the basis of 1 H and 13 C-NMR spectral data from the previous literature compound (I) was established as β – sitosterol (Kovganko et al., 1999). H HO Fig.1. β- Sitosterol In the present study, β-sitosterol was isolated from tuber of Eulophia epidendraea, and the same was reported in many species including Tephrosia strigosa and Heliotropium indicum, Ajuga macrosperma aerial part of Brillantaisia palisatii, Elaphoglossum spathulatum, Parahancornia amapa, Conyza bonariensis, Lilium longiflorum and Tulipa gesneriana, Zhongguo zhongyao, Atractylodes chinenese (Sreenivasulu and Sarma, 1996; Pandey et al., 1996; Dinda et al.,1997; Carvalhoa et al.,2001, Kong et al.,2001; Berrondo et al.,2003; Socolsky et al.,2003; Endoh et al.,1981). The occurrence of β- sitosterolglucoside was reported from plants such as Lilium candidum, Olea europaea and Heliotropium indicum (Pandey et al., 1996; Mučaji et al., 2000; Kadowaki et al., 2003). The intake of β-sitosterolglucoside capsule in marathon runner provide less inflammatory and reduced immunosuppressed activity excessive of physical stress (Bauic et al.,1999). Compound (II) was obtained as colourless needle. The molecular formula C 35 H 60 O 6 was assigned by HREI-MS 2 Results and Discussion The methanolic-tuber extract of Eulophia epidendraea was subjected to a series © Gayathri Teknological Publication

International Journal of Biological Technology (2010) 1(1):1-7. spectrophotometer. On the basis of 1 H and 13 C- NMR spectral data from the previous literature compound (II) was established as β - sitosterolglucoside. (Swift, 1952). The 13 C-NMR spectrum of the β - sitosterol showed 35 carbon signal, including the signals corresponding to two olefinic carbon at δ121.9(C-6) and δ140.9 (C-5). Furthermore, the 1 H-NMR spectrum exhibited one olefinic proton signal at δ5.35 (H- 6), two angular methyl groups at δ0.89(s,H-18), δ0.93(s,H-19) an isopropyl (δ0.86(H- 26),0.89(H-27), 1.68 (H-25) and ethyl (δ0.66(H- 29),1.26(H-28) group (Fig.1. ) 29 28 22 20 17 13 16 15 immunomodulatory agents. Donald et al., (1997) reported that the β-sitosterol and β- sitosterolglucoside were used in the treatment of pulmonary tuberculosis. Compound (III) 29 30 H 12 25 1 3 27 26 28 21 18 12 19 1 2 3 GlcO 5 4 6 10 11 9 8 7 23 25 24 HO 23 24 27 Fig.2. β- Sitosterolglucoside Strong absorption due to many hydroxyl group (3400 cm -1 ) in the IR spectrum and the signal in the 1 H-NMR (δ3.97 (H- 5’),4.07(H-2’),4.30(H-3’,4’), 4.43(H- 13 6’α),4.58(H-6’β),5.06 (H-1’) and C-NMR (δ62.8 (C-6’), 71.7 (C-4’),75.4 (C-2’),78.6 (C- 3’),102.6 (C-1’) spectra suggested that the compound was a steroidal glycoside. This is the first report of steroidal glycoside from this orchid genus. Beta-sitosterol (β-sitosterol) and its glucoside (β-sitosterolglucoside) are the most abundant sterols found in plants. In common with other phytosterols they are not endogenously synthesised in the human body and are derived exclusively from the diet (Ling et al.,1995). Although they differ from cholesterol by only an extra ethyl group in the side chain, they show profound biological effects in a number of experimental animal models. These include, inter alia, reduction of carcinogen-induced colon cancer, anti- inflammatory (Yamamoto et al., 1991), and anti- complement activity (Yamada et al., 1987). Bouic et al., (1996), reported that the β-sitosterol and β -sitosterol glucoside stimulate the proliferation of human peripheral blood lymphocytes and they can be used as an Fig.3. β- Amyrin Compound β- Amyrin was obtained as colourless needle. The molecular formula was established as C 30 H 50 O on the basis EI-MS: m/z 426 (M + , 0.6%), 411(0.1%), 218 (100%), 272 (0.1%), 189 (30%), 135 (34%), 95(48%); UV spectrum showed the maxima at 240.2 nm (UV λ max (MeOH); IR spectrum suggested the presence of IR γ max (KBR) cm -1 : 3397, 2932, 1645, 1465, 1379, 1029, 900; 1 H (200MHZ, CDCl3): δ 5.12 (m, H-12), 3.23 (m, H-3), 1.13, 0.99, 0.97,0.94, 0.87(x2), 0.83, 0.79(CH 3 ). 13 C NMR (50.29 MHz,CDCl 3 ): δ 38.14 (C-1), 27.51(C-2), 79.12 (C-3), 38.88 (C-4), 55.27 (C- 5), 18.44 (C-6), 33.03 (C-7), 38.82 (C-8), 47.81 (C-9), 37.00 (C-10), 23.48 (C-11),121.82 (C-12), 145.28 (C-13), 42.18 (C-14), 26.12 (C-15), 2737 (C-16), 32.04 (C-17), 47.22 (C-18), 46.93 (C-19), 31.34 (C-20), 34.83 (C-21), 37.26 (C- 22), 28.22 (C-23), 15.47 (C-24), 15.72 (C-25), 16.97 (C-26), 25.26 (C-27),28.44 (C-28), 33.44 (C-29), 23.48 (C-30). Compound (IV) was obtained as colourless needle. The molecular formula C 30 H 60 O was assigned by HREI-MS 1 spectrophotometer. On the basis of H and 13 C- NMR spectral data from the previous literature compound (IV) was established as lupeol [Ref: Aratanechemuge et al., 2004]. In the present work, the compound β- amyrin was isolated from Eulophia epidendraea which was also found in several plants such as Tephrosia strigosa and Heliotropium indicum, Brillantaisia palisatii, Lychnophora pinaster, 3 © Gayathri Teknological Publication

International Journal of Biological Technology (2010) 1(1):1-7. Luxemburgia nobilis, Chiococca braquiata, Parahancornia amapa, Atractylodes chinenese, Atractylodes chinenese (Sreenivasulu and Sarma, 1996; Pandey et al., 1996, Carvalhoa et al.,2001; Ding et al.,2000; Berrondo et al.,2003, Silveira et al.,2005; Oliveira et al.,2002; Lopesa et al.,2004). 30 20 19 29 18 25 11 12 13 14 1 2 3 OH 24 4 10 5 6 23 9 8 7 27 15 17 16 22 28 21 OH HO O OH OH O OH Fig.7. Kaempferol HO O OH OH OH O Fig. 8. Quercetin Quercetin, a phytochemical belonging to the flavonoids, has antioxidant activities, inhibition of protein kinases (Davies et al., 2000) and DNA topoisomerases (Constantinou et al., 1995) regulate gene expression (Moon et al., 2003) and also modulate gene expression related to oxidative stress and in the antioxidant defence system (Moskaug et al.,2004). According to Van Wiel et al., (2001) and Tsanova- Savova and Ribarora (2002), the most common flavonoids in grape wine were flavonols (quercetin, kaempferol, and myricetin). Betes- Saura et al., (1996) detected quercetin, kaempferol in leaves and exocarps of grape Vitis labruscana cv. Kyoho and, Vitis vinifera L. fruits. Lilium auratum, L. henryi, L.martagon, L. myrciphyllum and L. willmottiae. L. henryi, L. martagon, L. myrciphyllum, L. willmottiae, L. leichtlinii.Apigenin and luteolin which were structurally elucidated from 13 C-NMR, where spectral data reported earlier (Loo et al., 1986; Wagner, 1976). These compounds were isolated from the roots of Glossostemon bruguieri, Conyza bonariensis (Meselhy, 2003; Kong et al., 2001). Luteolin was also isolated from the fruit of Terminalia chebula (Klika et al., 2004). So far anti-venom compounds isolated from plants include β-sitosterol, β-sitosterolglucoside, β - amyrin, kampferol and quercetin (Martz ,1992 ;Houghton et al.,1993; Abubakar et al.,2000; Reyes-Chilpa et al.,1994).t was concluded that the phytochemicals isolated from Eulophia epidendraea may appear to be a good resource of biologically active compounds. Fig.4. Lupeol Similarly the compound Leupol isolated from Eulophia epidendraea was also reported from plants such as Brillantaisia palisatii, Lychnophora pinaster, Parahancornia amapa, (Ding et al.,2000; Carvalhoa et al.,2001; Berrondo et al.,2003, Silveira et al.,2005). Aratanechemuge et al.,(2004), reported that the suppression of growth of the HL-60 cells by lupeol results from the induction of apoptosis by this compound. Badami et al.,(2003) reported that the lupeol isolated from the bark of Grewia tiliaefolia, had weak cytotoxic properties. OH HO O OH O OH OH Fig.5. Apigenin HO O OH O Fig.6. Luteolin © Gayathri Teknological Publication 4