K. Thirupathi et al. /BioMedRx 2013,1(4),392-396 Research Article Available online through http://jprsolutions.info

Anticancer Activity of collinus

K. Thirupathi1*, D.R. Krishna2, G. Krishna Mohan3, G.K.Mallaiah4 1Department of Pharmacy, University college, Satavahana university, Karimnagar, India 2Department of Pharmacology, University college, Kakatiya University, Warangal, India 3Center for pharmaceutical sciences, JNTU Hyderabad, India 4Vaagdevi Pharmacy College , Warangal, India

Received on:11-01-2013; Revised on: 07-02-2013; Accepted on:25-03-2013

ABSTRACT To evaluate the anticancer activity of methanolic extract and its fractions from the bark of Cleistanthus collinus against Earlisch Aceites Carcinoma (EAC) cells, HT 29 (colon cancer), H a549 (lung cancer) and MDA 231 (breast cancer) cells. Traditionally, the whole is used as poison and anticancer. Anticancer activity was studied using the methods viz. trypan blue and MTT assay. The ethyl acetate and its chromatographic fraction, and aqueous residue-I showed promising activity against cell lines used in the study. The observations suggest that the chromatographic fraction possesses potential anticancer activity and dose dependent.

KEYWORDS: Cleistanthus collinus; anticancer activity; Trypan blue method and MTT assay.

1. INTRODUCTION Cleistanthus collinus (Roxb.) Benth. belongs to family Euphorbiaceae, 2. MATERIALS AND METHODS is native to peninsular India and Srilanka. Traditionally, the whole plant is used as poison, anticancer and astringent. [1] Many parts of Cell lines the plant are reported to be toxic; the fruit and bark are employed to Earlisch Aceites Carcinoma (EAC) cells were obtained from Central poison fish. The extract of crushed leaves is used as cattle and fish Animal House H .S. K. College of Pharmacy, Bagalkot, Karnataka, poison, abortifacient, and in suicide and homicide attempts. [2] The India (IAEC/Clearance/2007/1-8) and used in Trypan blue method. wood is used as house post. For the severe headache, the head and HT 29-colrectal adenoma, H a549–Lung carcinoma and MDA 231- upper part of the body are bathed in water in which the leaves have Adenocarcinoma breast cancer cells were obtained from same source been steeped. An extract of the leaves and fruit acts as a violent and were used in MTT assay. gastro intestinal irritant. The bark is beneficial in skin diseases. [3, 4] Preparation of extracts There are very few published reports on the clinical and metabolic The methanolic extract of bark was bifurcated in to acetone soluble effects of this toxic compound in human beings. [5] Extracts of leaves and acetone insoluble fractions. Concentrated acetone fraction was reported to have anticancer, [6] neuromuscular junction blocking, [7] dispersed in 1 L of distilled water and extracted with ethyl acetate antifertility, [8] antifeedant [9] activities and root extract as anti-micro- (EAF-CBME) to get its corresponding fraction and aqueous residue bial activity. [10] (AQR-I). Acetone insoluble fraction was fractionated with butanone (BOT-CBME) and n-butyl alcohol (BOH-CBME) in succession to get Chemically lignans and its derivatives such as leucoanthocyanidins its corresponding fraction and aqueous residue (AQR-II). The ob- and arylnaphthalide lignans have been isolated from the different tained fractions and the aqueous residues were concentrated under parts of the plant. [11, 12] Therefore, in view of traditional use in cancer reduced pressure to yield corresponding extracts. and anticancer reports on leaves, it was aimed to carry out the anti- cancer activity of bark against EAC cells, HT 29, H a549 and MDA 231 Phytochemicals (mixture of lignans) were isolated from the EAF-CBME cancer cells in ordered to detect new sources of anticancer agents. using column chromatographic technique and given the code as C- EAF-CBME. The methanol wash was given the code as C1-EAF- [13] *Corresponding author. CBME. It was done according to standard procedures using silica K. Thirupathi gel (Kemphasol, Mumbai) of various grades like silica gel 100-200 Department of Pharmacy, mesh. University college, Satavahana university, Karimnagar, India

BioMedRx Vol.1 Issue 4 .April 2013 392-396 K. Thirupathi et al. /BioMedRx 2013,1(4),392-396 Anticancer activity RESULTS: Medium with DMSO concentration less than one percentage is taken Trypan blue Method as control and the cell viabilities in the test drug treated are expressed The viability of cells to exclude trypan blue was determined by incu- as percentage inhibition with respect to DMSO. The concentration bating the cell suspension (0.1 ml) with 0.4% trypan blue (0.1 versus percentage inhibition graphs were given in the following pages. ml).Viability of the isolated cells was determined by trypan blue exclu- sion assay by counting the number of stained and unstained cells The CBME and its fractions were screened for cytotoxic activity

(viable cells). The concentration of the viable cells was adjusted to against Earlisch Aceites Carcinoma (EAC) cells. The IC50 of CBME 1x106 cells per ml. To 0.3 ml of cell suspension, 0.7 ml of drug solution found to be 36.58µg/ml. The maximum percentage inhibition (69.88) (20, 40 and 60 µg/ ml of CBME fractions) was added and incubating showed at the concentration of 60µg/ml. It showed significant dose for 72 hours. The media with drug solution was removed and the cells dependant inhibition on EAC cells but the results were not compa- were trypsinzed. To 0.1 ml of above resuspended cells (previously rable to the standard. When compared to cyclophosphamide CBME treated with drug), 0.1 ml of trypan blue dye (0.5% in PBS) was added was moderately active. The results of the present study are given in and allowed to stand for 2-3 min. Cells were then loaded on to a Table 1 and Fig 1. haemocytometer or on a glass slide, covered with cover slip and scored for dye uptake. A minimum of 100 cells were counted in three Table 1.Effect of CBME, cyclophosphamide on tumor (EAC cell line) separate fields using inverted microscope. [14] Percentage viability living cell number in ascetic fluid of tumor induced mice. was calculated as follows: Treatment Mean percentage growth inhibition 20µg/ml 40 µg/ml 60µg/ml Number of cells excluding dye % of viable cells = ——————————————— x 100 CBME 38.45 49.72 69.88 Total Number of cells counted Cyclophosphamide 52.24 96.87 98.43

MTT assay Cytotoxic activity on EAC cell line In vitro growth inhibition effect of the fractions of CBME was as- sessed by colorimetric determination of the conversion of MTT into 120 ‘formazan blue’ by the living cells. HT 29 (colon cancer), H a 549 100 (Lung cancer) and MDA 231(Breast cancer) cell lines were obtained. 80 All the cells were grown in plastic T-25 culture flask in Dulbecco’s CBME 60 Modified Eagle’s Medium (DMEM) expect MDA 231 cells grown in Cyclophosphamide Minimum assential media which were supplemented with 10% fetal 40 bovine serum (Gibco/BRL), 100 µg/ml pencillin, 200 µg/ml streptomy- 20 cin, 2 mM L-glutamine, 1% antibiotic/antimycotic solution (Gibco/ % growth inhibition 0 0 BRL) under 5% Co2 with 85% relative humidity at 37 C. After 2-3 days 20 40 60 cells were removed from the culture flask by trypsinizing the cells with trypsin-EDTA solution. Cell counts and viability were performed Concentration (µg/ml) using a standard trypan blue dye exclusion technique. The cell con- Fig 1. Effect of CBME on tumor (EAC cell line) living cell number in 4 centration was adjusted to 1x10 cells in each well with the same ascetic fluid of tumor induced mice, using Trypan blue method media in the 96 well plates. Afterb 24 h incubation the media was removed from each well and replaced with a fresh one containing The IC50 of EAF-CBME found to be 38.81µg/ml. The maximum per- series of concentrations of test compounds (10, 20 and 50 µg/ml of centage inhibition (72.12) showed at the concentration of 60µg/ml. CBME fractions), where as in control wells 1% DMSO (Vehicle) was Where as, the IC50 of AQR-I-CBME found to be 41.77µg/ml. The added. After 48 h incubation, media was replaced with 100 µl of fresh maximum percentage inhibition (69.21) showed at the concentration media without FBS. To each well 10 µl (5 mg/ml stock in PBS) of MTT of 60µg/ml. While C-EAF-CBME showed IC at 48.00µg/ml. The maxi- solution was added and incubated for 4 h at 370C. After incubation 50 mum percentage inhibition (58.32) showed at the concentration of supernatant was removed and the formazan crystals so formed was solublised in 200 µl of DMSO. Then the plate was read at 570 nm 60µg/ml. using UV-Visible spectrophotometer. Effect of test compounds on cell viability was calculated using cells treated with DMSO as con- BOT-CBME, BOH-CBME, AQR-II-CBME and C1-EAF-CBME were found to be inactive at 10µg/ml and they did not produce IC up to trol. IC50 values were determined from plot: % inhibition versus con- 50 centration. [15] 60µg/ml. The maximum percentage inhibition (29.88, 37.31, 39.32 and 27.58 respectively) showed at the concentration of 60µg/ml. Mean OD of control-Mean OD of test % of Growth inhibition = —————————————— x 100 Cyclophosphamide showed a dose dependant inhibition on EAC Mean OD of control cells. The IC50 of this standard found to be 11.84µg/ml. The maximum percentage inhibition (98.43) showed at the concentration of 60µg/ ml.

BioMedRx Vol.1 Issue 4 .April 2013 392-396 K. Thirupathi et al. /BioMedRx 2013,1(4),392-396 All fractions showed significant dose dependant inhibition on EAC cell lines HT-29, H a549 and MDA-231 respectively at the concentra- cells. The EAF-CBME, AQR-I-CBME and C-EAF-CBME were com- tion of 50µg/ml. When compared to cyclophosphamide, EAF-CBME parable to the standard and moderately potent, but BOT-CBME, BOH- showed moderate activity against H a549 cells and moderate activity CBME, AQR-II-CBME and C1-EAF-CBME not comparable to the stan- towards HT-29 and MDA-231. dard and less potent in nature. The results of the present study are given in Table 2 and Fig 2. Where as, the IC50 of C-EAF-CBME found to be 9.52, 14.00 and 7.12µg/ ml against cell lines HT-29, H a549 and MDA-231 respectively. The Table 2.Effect of CBME fractions on tumor (EAC cell line) living cell maximum percentage inhibitions showed at the concentration of 50µg/ number in ascetic fluid of tumor induced mice. ml were 78.42, 71.17 and 73.02. It showed significant dose dependant Treatment Mean percentage growth inhibition cytotoxic activity against all cell lines used and comparable to the 10 µg /ml 20µg/ml 40 µg/ml 60µg/ml cyclophosphamide. C-EAF-CBME exhibited more cytotoxic activity against HT-29 and H a549 cells and moderate activity towards MDA- EAF-CBME 11.12 37.32 51.21 72.12 231. While C1-EAF-CBME did not show significant and comparable C-EAF-CBME 11.22 35.43 43.21 58.32 C1-EAF-CBME 6.43 10.31 21.12 27.58 anticancer activity against all cell lines used. The maximum percent- AQR-I-CBME 9.21 38.32 44.32 69.21 age inhibitions observed were 17.01, 15.52 and 16.98 against cell lines BOT-CBME 5.72 28.21 29.32 29.88 HT-29, H a549 and MDA-231 respectively at the concentration of BOH-CBME 5.21 25.32 31.21 37.31 50µg/ml. AQR-II-CBME 6.21 23.31 32.21 39.32 Cyclophosphamide 14.31 52.24 96.87 98.43 AQR-I-CBME showed IC50 at 33.36 and 23.43µg/ml against H a549

Cytotoxic activity on EAC cell line and MDA- 231 respectively at the concentration of 50µg/ml. The

120 maximum percentage inhibitions were found to be 46.37, 56.11 and

100 66.79 at the concentration of 50µg/ml. It showed dose dependant

80 cytotoxic activity against all cell lines used. It showed moderate ac-

60 tivity against H a549 and MDA-231, and not comparable to the cyclo- 10 µg /ml 40 20µg/ml phosphamide. 40 µg/ml % growth inhibition 20 60µg/ml

0 BOH-CBME did not show significant and comparable cytotoxic ac- EAF-CBME C-EAF-CBME C1-EAF-CBME AQR-I-CBME BOT-CBME BOH-CBME AQR-II-CBME Cyclophosphamide tivity against all cell lines used. The maximum percentage inhibitions Groups observed were 14.25, 18.25 and 30.50 against cell lines HT-29, H a549 and MDA-231 respectively at the concentration of 50µg/ml. It exhib- Fig 2. Effect of CBME fractions on tumor (EAC cell line) living cell ited mild anticancer activity. number in ascetic fluid of tumor induced mice, using Trypan blue method The IC50 of BOT-CBME found to be 32.66µg/ml against HT-29 at the The CBME and its fractions were screened for cytotoxic activity concentration of 50µg/ml. The anticancer activity was moderate when against HT- 29 colorectal adenoma, H a549 –Lung carcinoma and compared to the standard. It did not exhibit significant cytotoxic MDA-231-Adenocarcinoma breast cancer cells by MTT method. The activity against H a549 and MDA-231 but exhibited dose dependant results of the evaluation have been viewed by taking Cyclophospha- cytotoxic activity towards all cell lines used. The maximum percent- mide (IC 20 mM) as the standard. But, many of the fractions were age inhibitions observed were 66.98, 30.46 and 22.11 against cell lines 50 HT-29, H a549 and MDA-231 respectively at the concentration of comparable in cytotoxicity with that of standard Cyclophosphamide 50µg/ml. employed. The IC of AQR-II-CBME found to be 43.43µg/ml against MDA-231 The IC of CBME found to be 58.52, 22.05 and 35.69µg/ml against cell 50 50 at the concentration of 50µg/ml. The anticancer activity was moder- lines HT-29, H a549 and MDA-231 respectively. The maximum per- ate against HT-29 and MDA-231 where as in H a549, it was mild. It centage inhibitions showed at the concentration of 50µg/ml were exhibited dose dependant cytotoxic activity towards all cell lines used 47.58, 73.19 and 58.77. It showed significant dose dependant cyto- but not comparable to the standard. The maximum percentage inhibi- toxic activity against three cell lines. The CBME exhibited significant tions observed were 44.25, 17.38 and 51.26 against cell lines HT-29, H cytotoxic activity against H a549 cells and moderate activity towards a549 and MDA-231 respectively at the concentration of 50µg/ml. HT-29 and MDA-231 when compared to the cyclophosphamide. It showed comparable results in the activity against H a549 cells but Cyclophosphamide showed a dose dependant cytotoxic activity not against HT-29 and MDA-231. against all cell lines used. The IC50 of standard found to be 20.92, 10.95 and 17.23µg/ml against cell lines HT-29, H a549 and MDA-231 The IC of EAF-CBME found to be 30.79µg/ml against H a549. The 50 respectively. The maximum percentage inhibitions were 81.36, 88.22 maximum percentage inhibitions were 38.68, 63.67 and 47.88 against and 93.50 at the concentration of 50µg/ml. It exhibited dose depen-

BioMedRx Vol.1 Issue 4 .April 2013 392-396 K. Thirupathi et al. /BioMedRx 2013,1(4),392-396 dant and potent anticancer activity against MDA-231 while moderate 4. DISCUSSION activity towards HT-29 and H a549. The results of the study are pre- are the invaluable source for searching potential anticancer sented in Table 3 and Fig 3. agents. In this study, there may be inhibitors and active ingredients in the Cleistanthus collinus extracts, which can induce the cytotoxic Table 3.Effect of CBME and its fractions on cancer cell lines Treatment HT 29-colrectal adenoma H a549 –Lung carcinoma MDA 231-adenocarcinoma breast cancer 10µg/ml 20µg/ml 50 µg/ml 10µg/ml 20µg/ml 50µg/ml 10µg/ml 20µg/ml 50µg/ml

EAF-CBME 3.21 15.66 38.68 36.19 41.50 63.67 20.90 39.81 47.88 C-EAF-CBME 58.22 65.95 78.42 42.78 60.04 71.17 54.48 64.94 73.02 C1-EAF-CBME 4.08 16.57 17.01 13.39 14.84 15.52 3.42 10.80 16.98 AQR-I-CBME 29.20 34.04 46.37 38.55 47.17 56.11 41.19 48.19 66.79 BOH-CBME 12.68 12.80 14.25 08.00 9.07 18.25 2.93 25.62 30.50 BOT-CBME 29.09 36.63 66.98 06.90 14.55 30.46 6.89 9.20 22.11 AQR-II-CBME 15.00 25.64 44.25 02.44 09.03 17.38 28.05 46.34 51.26 CBME 34.90 34.91 47.58 41.61 46.47 73.19 29.81 43.06 58.77 Cyclophosphamide 33.89 54.09 81.36 39.60 72.11 88.22 38.55 56.03 93.50

Cytotoxic activity on HT- 29 colrectal adenoma cells action against cancer cells and initiate anti-proliferation pathway lead- 90.00 ing to cancer cell death. Cleistanthus collinus is a member of 80.00 70.00 Euphorbiaceae, the family which comprises more than 60.00 7,500 species. Some Euphorbiacea plants are found to have antitumor 50.00 [16] 40.00 activity, for example niruri and P. emblica . The com- 30.00 pounds isolated from stems and fruits of Ostodes paniculata have 20.00

% growth inhibition [17] 10.00 significant in vitro cytotoxic effect on cancer cell lines (p-388). 0.00 EAF-CBME C-EAF-CBME C1-EAF-CBME AQR-I-CBME BOH-CBME BOT-CBME AQR-II-CBME CBME Cyclophosphamide

Treatment 10µg /ml 20µg /ml 50µg /ml The CBME and its fractions were shown to have inhibitory effect on cancer cells. Mouse Earlisch Aceites Carcinoma (EAC) cells were Fig 3. Anticancer activity of CBME and its fractions against HT 29- sensitive to the CBME and its fractions at various doses in vitro colrectal adenoma cells, using MTT assay. condition. They were also effective in the suppression of prolifera- tion of the three human cancer cell lines such as HT- 29 colorectal Cytotoxic activity on H a549- Lung carcinoma cells adenoma, H a549 –Lung carcinoma and MDA-231-Adenocarcinoma 100.00 breast cancer in a dose-dependent pattern. Among the tested frac- 90.00 80.00 tions, EAF-CBME showed the strongest anticancer activity (EAC 70.00 cells), and was then subjected to further chromatographic fraction- 60.00 50.00 ation on silica gel to obtain two fractions. They were coded as C- 40.00 30.00 EAF- CBME and C1-EAF- CBME. 20.00 % growth inhibition 10.00 0.00 In this study, we found interesting results with C-EAF- CBME. Of the EAF-CBME C-EAF-CBME C1-EAF-CBME AQR-I-CBME BOH-CBME BOT-CBME AQR-II-CBME CBME Cyclophosphamide fractions, it was found to exhibit strongest anticancer activity against Treatment 10µg /ml 20µg /ml 50µg /ml all cell lines used in the study. It showed potent activity against HT- 29 and H a549, while moderate activity against MDA-231 and EAC Fig 4. Anticancer activity of CBME and its fractions on H a549 – cells, when compared to the standard. This may due to presence of Lung carcinoma cells, using MTT assay. lignans in the fraction. AQR-I-CBME exhibited moderate activity against all cell lines used. It may due to the presence of saponins. The Cytotoxic activity on MDA 231 adenocarcinoma breast cancer cells BOT-CBME exhibited potent cytotoxic activity against HT-29 cells 100.00 90.00 but did not show significant activity in other cell lines. 80.00 70.00 60.00 Chemical investigation of the resin of Podophylum species has re- 50.00 40.00 vealed the presence of several lignans, including podophyllotoxin, a- 30.00 peltatin and 5-peltatin which show antitumour activity in mice. [18]

% growth inhibition 20.00 10.00 Diphyllin is one of the arylnaphthalene derivatives which has cyto- 0.00 EAF-CBME C-EAF-CBME C1-EAF-CBME AQR-I-CBME BOH-CBME BOT-CBME AQR-II-CBME CBME Cyclophosphamide static activity. This powerful action may be related to its close struc- [19] Treatment 10µg /ml 20µg /ml 50µg /ml tural relationship with podophyllotoxin. Lignane compounds ob- tained from leaves and heart wood of same plant reported to possess Fig 5. Anticancer activity of CBME and its fractions on MDA 231- anticancer activity. [20] Saponins isolated from different plants and Adenocarcinoma breast cancer cells, using MTT assay.

BioMedRx Vol.1 Issue 4 .April 2013 392-396 K. Thirupathi et al. /BioMedRx 2013,1(4),392-396 animals have been shown to specifically inhibit the growth of cancer of Pharmaceutical science, 46, 1984, 95–96. cells in vitro. [21] The saponins or lignans contained in methanolic 11. Ganguly AK, Seshadri TR, Subramanian P, A study of extract and fractions may contribute to anticancer activity. The tradi- leucoanthocyanidins of plants-I, Isomers of leucodelphinidin tional utilization of the plant is thus validated. from karada bark and eucalyptus gum, Tetrahedron 3, 1958, 225-229. 5. CONCLUSION 12. Ramesh C, Nasi Ravindranath, Tejomoortula Siva Ram, It is required to purify the C-EAF- CBME to isolation of active com- Biswanath Das, Arylnaphthalide Lignans from Cleistanthus pounds for establish novel cytotoxic drugs as it showing strong collinus, Chemical and Pharmaceutical Bulletin, 51(11), 2003, anticancer activity. 1299—1300. 13. Kremmer T. Gel Chromatography, John-Wiley and Sons, REFERENCES Wiley-Interscience Publications, New York, 1979. 14. Rajender G, Benarjee B, Prasad MSK, Krishna DR, Studies 1. Joy PP, Thomas J, Samuel Mathew, Skaria Baby P, Medicinal on the cytotoxic effect of antiserum raised against riboflavin Plants, Kerala Agricultural University, Aromatic and Me- binding protein on in vitro cell cultures, Asian Journal of dicinal Plants Research Station, Kerala, 1998. Experimental Biological Sciences, 1(3), 2010, 524-530. 2. Chatterjee A, pakrash SC, The treatise on Indian medicinal 15. Jalil Tavakkol Afshari, Moosa-alreza Hajzadeh, Ahmed plants, National institute of science communication and in- Ghorbani, Heydar parsai, Ethanolic extract of Allium sativum formation resources, New delhi, 1994. has antiproliferative effect on Hep 2 and L929 cell line, Phar- 3. Kirtikar KR, Basu BD, Indian Medicinal Plants, 2nd edition, macognosy Magzine, 2, 2006, 31-34. International Book Distributors, Dehradun, 1995. 16. Joao B Calixto, Adair RS Santos, Valdir Cechinel Filho, 4. Modi NJ, Subrahmanyam BV, Modi’s Medical Jurisprudence Rosendo A, Yunes. A review of the plants of the genus and Toxicology, 22nd edition, Butterworths, New Delhi, 1999. phyllanthus: their chemistry, pharmacology, and therapeutic 5. Thomas K, Dayal AK, Narasimhan, Ganesh A, Sheshadri potential inc. Medicinal Research Review, 18 (4), 1998, 225– MS, Cherian AM, Bhanu M, Metabolic and cardiac effects 258. of Cleistanthus collinus poisoning, Journal of Association 17. Sukhdev S Handa, Douglas Kinghorn A, Geoffrey A. Cordell, of Physician India, 39(4),1991, 312-314. Norman R. Farnsworth, Plant Anticancer Agents. XXII. Iso- 6. Rajkumar S, Bhatia AL, Shanmugam G, Molecular Mecha- lation of a phorbol diester and its Ä5,6-7â-Hydroperoxide nisms Underlying the Inhibition of Cell Proliferation by derivative from Ostodes paniculata. Journal of Natural Cleistanthins, Asian Journal of Experimental Sciences, 15 Products 46 (1), 1983, 123–126. (1&2), 2001, 9-16. 18. Dymock W. Pharmacographia Indica, The Journal of the In- 7. Vijayalakshmi KM, Nanda Kumar NV, Isolation of a neuro- stitute of Health and Týbbi (Medical) Research, Pakistan, muscular junctional blocking agent from Cleistanthus Zain Packaging Industries Ltd, Karachi, 16, 1972. collinus plant leaf extract, Phytotherapy Research 8, 1994, 19. Skeria MG, Amer KM. Lignans from Haplophyllum 245-247. tuberculatum. Phytochemistry 23, 1984, 1151. 8. Choudhary DN, Singh JN, Singh BP, Effect of some medici- 20. Pradheep Kumar CP, Pande G, Shanmugam G, Cleistanthin B nal plants on fertility of albino rats. Indian Journal of Phar- causes G1 arrest and induces apoptosis in mammalian cells, macology, 23(4), 1991, 253-257. Apoptosis, 3, 1998, 413–419. 9. Anand, Jagadiswari Rao, Botanical pesticides in agriculture, 21. Kuznetzova TA, Anisimov MM, Popov AM, A comparative In behavioural and physiological approaches in pest man- in vitro study of physiological activity of triterpene glyco- agement, Coimbatore publication, 1996, 137. sides of marine invertebrates of echinoderm type, Compara- 10. Satyanarayana P, Subramanyam P, Koteswara RP. Chemical tive Biochemistry and Physiology, 73C, 1982, 41–43. constitutents of Cleistanthus collinus roots, Indian Journal

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BioMedRx Vol.1 Issue 4 .April 2013 392-396