Research Article ISSN: 0974-6943

M.Meenakshi Sundaram et al. / Journal of Pharmacy Research 2010, 3(9),2332-2333

Available online through www.jpronline.info Antimicrobial and Anticancer Studies on Euphorbia heterophylla
M.Meenakshi Sundaram*, K.Karthikeyan @ D.Sudarsanam # and P.Brindha* *School of Chemical & Biotechnology, SASTRA University, Thanjavur 613401. TamilNadu, INDIA. @ Department of Biotechnology, Srimad Andavan Arts and Science College, Tiruchirapalli-620005, TamilNadu, INDIA. # Dept of Adv. Zoology & Biotechnology, Loyola College, Nungambakkam, Chennai-34. TamilNadu, INDIA.

Received on: 15-04-2010; Revised on: 18-05-2010; Accepted on:13-07-2010 ABSTRACT

Euphorbia heterophylla is a local medicinal plant used in ethno medicine for the treatment of constipation, bronchitis and asthma. E. heterophylla is medicinal plant with the common name spurge weed. In the present study selected plant drug was screened for its anticancer activity by Invitro and Insilco methods. Preliminary phytochemical screening revealed the presence of Terpenoids, Quinones, Alkaloids, Sterol, Coumarin, Starch, and Protein. Ethanol extract was tested for their antibacterial property against various organisms. The extract showed significant antimicrobial activity particularly in Proteus vulgaris and Staphylococcus aureus. Alcoholic extract of plant material showed significant in-vitro cytotoxic activity on EAC (Ehrlich ascites carcinoma) cell lines. GC-MS was carried out in ethanol extract of Euphorbia heterophylla.31 compounds were identified. Bioinformatics tools were employed in understanding the mechanism of action. 14 of the compounds were docked with the Bcl-2 protein, among them Allantoic acid was considered as best candidate as it is having least energy in docking.

Key words: Ehrlich ascites carcinoma, Docking studies, Bcl-2

INTRODUCTION Herbs are now very popular in developing countries on account of improved knowledge about the safety, efficacy and quality assurance of ethno- medicine. In recent years, secondary plant metabolites (phytochemicals) have been extensively investigated as a source of medicinal agents.Euphorbia has been used for many years as a homeopathic remedy and has been reported to treat successfully some cancers. In homeopathy many varieties of the herb are made into homeopathic preparation and each one has a specific action. The use of spurges (Euphorbia spp.) to treat tumors is well documented in Greek and Roman medical literature (J.L. Hartwell., 1969), and modern studies have shown that these plants are still employed to treat cancerous conditions in traditional medicine in many areas of the world. Traditionally, Euphorbia species have been used internally as laxatives and externally for rheumatism and skin conditions. Euphorbia heterophylla leaf is used in traditional medical practices as laxative, anti gonorrheal, migraine and wart cures. The plant lattices have been used as fish poison, insecticide and ordeal poisons (Rodriguez et al., 1976, Falodun et al, 2003). The leaves of E. heterophylla have been reported to contain quercetin (Falodun and Agbakwuru, 2004). Diterpenoids have also been reported in the root of E. heterophylla (Rowan and Onwukaeme, 2001). The skin irritant, tumor-promoting and anti-tumor/anticancer and recently anti-HIV activities of Euphorbia species have also been reported in E.heterophylla leaf Linn (Williams et al., 1995). Euphorbia is stated to possess antitussive, antifungal and antitumor properties. There is mixed evidence showing Euphorias effectiveness for chronic bronchitis, eczema, epilepsy and oral inflammation. In the present study selected plant drug was screened for its anticancer activity by Invitro and Insilco methods. MATERIALS AND METHODS Plant material The fresh plants of E. heterophylla were obtained from the in and around Trichy, in December, 2009, Collected plant material were identified and authenticated by Dr.P.Brindha., Dean, CARISM, SASTRA university, Thanjavur. Phytochemical screening The crude plant powdered sample was subjected to phytochemical, screening, testing for the presence of Alkaloids, Tannins, Flavonoids and Saponins using standard experimental procedure.

Extraction The powdered leaves (600 g) were extracted first with n-hexane, chloroform, and ethanol at room temperature. The extracts were evaporated to dryness using an evaporator. Assay of Antimicrobial Activity The ethanolic extract of Euphorbia heterophylla was tested against various organisms such as Escherichia coli, Proteus vulgaris, Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeuroginosa, Bacillus subtilis. Assay of antimicrobial activity was performed by disc diffusion method. (Bauer.,et al, 1966) Cells
Ehrlich ascites carcinoma is a spontaneous murine mammary adenocarcinoma adapted to ascites form. EAC cells were obtained through the courtesy of AMALA cancer research center, Trichur, Kerala. They were maintained by intraperitoneal inoculation of 106 cells/mouse.

Short term in-vitro cytotoxicity (Sheeja KR et al., 1997) Short term in-vitro cytotoxicity was assessed using EAC cell lines by incubating the different concentration of drugs at 37c for 3 hours. The tumor cells were aspirated from peritoneal cavity of tumor bearing mice using a 10 ml syringe and transferred to a test tube containing isotonic saline. The cells were then washed in normal saline and cell number was determined using a haemocytometer and adjusted at 10 x 106 cells/ml. for the cytotoxicity assay , different concentrations of drug (50,75&100g/ml) were added to each and the final volume was adjusted to one ml with normal saline. Control tubes were kept with the solvent and without the solvent along with tumor cells. All the tubes were incubated at 37c for 3 hours. After incubation 0.1ml of 1% trypan blue dye in isotonic saline was added to each tube and the number of viable (unstained) and dead (stained) cells were counted using haemocytometer. The percent cytotoxicity (%dead cells) was calculated using the formula. The dead cells were calculated by the formula, Total cells counted total viable cells %Dead cells = Total cells counted Gas Chromatography and Mass Spectrometry This is technique used to separate the compounds present in the given sample (solvent extraction), and this is combination of gas chromatography as well as mass spectrometry technique .We can get result a chromatogram graph in this GC-MS. Docking Studies Bcl-2 protein, a anti apoptotic protein selected as target molecule. The 3D

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*Corresponding author.
M.Meenakshi Sundaram Asst.Professor School of Chemical & Biotechnology Sastra University Thanjavur 613401.Tamilnadu, INDIA. Email:msundar77@yahoo.com Mobile: +91-9894475375

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M.Meenakshi Sundaram et al. / Journal of Pharmacy Research 2010, 3(9),2332-2333

structure of the compounds present in the sample was drawn in chemsketch tool. The 3D structure of the compounds was converted into PDB file by using molecular converter and was taken as ligand molecule. The active site in the target protein was predicted by using the online tool Q-site finder. The PDB file of target protein, the PDB file of the ligand and text file of aminoacids in the active site were taken as input files and autodocking was carried out. The 3D structure of the docked molecules and the bonds, residues involved the docking was viewed by using Pymol viewer. RESULTS Phytochemical screening The Preliminary phytochemical tests answered positively for the presence of Terpenoids, Quinones, Alkaloids, Sterol, Coumarin, Starch, and Protein in case of raw drug powder. Phytochemical screening of Hexane extract shows positive result for Terpenoids, Sterol, Coumarin. Phytochemical screening of Chloroform extract shows positive result for Quinine, Sterol, Coumarin, starch. Phytochemical screening of Ethanol extract shows positive result for Alkaloid, Sterol, Coumarin, protein. Antimicrobial activity The ethanolic extract of Euphorbia heterophylla was tested against various organisms such as Escherichia coli, Proteus vulgaris, Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeuroginosa& Bacillus subtilis. . The extract showed significant antimicrobial activity particularly in Proteus vulgaris and Staphylococcus aureus. Invitrocytotoxic assay Ethanolic extract of Euphorbia heterophylla was subjected to the invitro cytotoxic assay. Euphorbia heterophylla showed significant invitro cytotoxic activity against Ehrlich Ascites carcinoma cell lines. At the dose level of 100g/ml 46.49% of cell death noticed. (Table1) Table 1: Invitro cytotoxic assay for ethanolic extract of Euphorbia hetrophylla
Conc.of Ethanol extract(g/ml) Control 50 75 100 No. of viable cells 294 336 291 229 Viable cells % 85.21 80 75 53.50 No. of dead cells 51 84 97 199 Dead cells % 14.78 20 25 46.49

Figure1: Allantoic acid docked with BCl2

DISCUSSION The plant was commonly used in treating constipation, bacterial and inflammatory disease conditions (arthritis and rheumatism). Traditionally the leaf of the plant is used to treat ear pain (otitis media or externa) while the poultice induces milk flow from breast. In the present study selected plant drug was screened for its anticancer activity by Invitro and Insilco methods. Phytochemical screening helps to reveal the chemical nature of the constituents of the plant extract and the one that predominates over the others. It may also be used to search for bioactive agents that could be used in the synthesis of very useful drugs (Yakubu et al., 2005). Phytochemical screening of the leaves of the selected plant revealed the presence of Terpenoids, Quinones, Alkaloids, Sterol, Coumarin, Starch, and Protein as the major phytochemical components. Bcl-2 (Codes for a protein that blocks cell suicide mechanism) Suppresses apoptosis in a variety of cell systems including factor-dependent lymphohematopoietic and neural cells. The compounds isolated from ethanolic extract of Euphorbia heterophylla were docked with the Bcl-2 protein. Their docking structure view in pymol. 14 of the compounds were docked with the Bcl-2 protein; only 8 compounds were produce hydrogen bond with Bcl-2. Allantoic acid seems to be best candidate because it had least energy among 8 compounds. CONCLUSION To conclude Preliminary phytochemical screening revealed the presence of Terpenoids, Quinones, Alkaloids, Sterol, Coumarin, Starch, and Protein. The extract showed significant antimicrobial activity particularly in Proteus vulgaris and Staphylococcus aureus. Ethanolic extract of plant material showed significant in-vitro cytotoxic activity on EAC cell lines. Insilico studies were employed in understanding the mechanism of action. Thus present study revealed that ethanolic extract of Euphorbia heterophylla possessed significant antitumour activity. REFERENCES
1. Abiodun Falodun, Sajjad Ali, Irfan Mohammed Quadir and Iqbal M. I Choudhary (2008). Phytochemical and biological investigation of chloroform and ethylacetate fractions of Euphorbia heterophylla leaf (Euphorbiaceae) Journal of Medicinal Plants Research Vol. 2(12), 365-369 Bauer, A. W., W. M. Kirby, J. C. Sherris, and M. Turck. 1966. Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol. 45:493-496. Falodun A, Agbakwuru EOP. (2004). Phytochemical Analysis and Laxative Activity of Euphorbia heterophylla Linn (Euphorbiaceae). Pak. J. Sci. Res. 47 (5): 345 - 348. Huey,R., Morris,G.M., Olson,A.J., and Goodsell,D.S. (2007). A semiempirical free energy force field with charge-based desolvation. J.L. Hartwell, Plants used against cancer. A survey, Lloydia 32 (1969), pp. 153205. Morris .G., P. Khodade., R. Prabhu., N. Chandra., S. Raha and R. Govindarajan.,(2005): Parallel implementation of AutoDock. J. Appl. Cryst. 40, 598-599 Rodriguez E., Twers GHN, Mitchell JC (1976). Biological activities of sesquiterpene Lactones. Phytochemistry 15: 1573. Rowan NG, Onwukaeme DN. (2001) Deterpnoid esters of Euphorbiaceae in Euphorbia hyles. Nig. J. Pharmacol.32; 60 - 64. Sheeja KR., Kuttan G., Kuttan R., (1997): cytotoxic and antitumour activity of berberin. Amala Res bull. 17:73-76 Williams CA, Hoult, JRS, Harborne JB, Greenham J (1995) Phytochemistry 38 (1): 267. Yakubu MT, Akanji MA, Oladiji AT (2005). Aphrodisiac potentials of the aqueous extract of Fadogia agrestis (Schweinf. Ex Hiern) stem in male albino rats. Asian J. Androl. 7(4): 399-404.

GC-MS analysis GC-MS was carried out in ethanol extract of Euphorbia heterophylla.31 compounds were identified in the analysis. among them 6 of the compounds had high percentage peak area that are Aceticacid,3-hydroxy-6-isopropenyl-4,8a-dimethyl-1,2,3,5,6,7,8,8a octahydronaphthalen-2-yl ester. Bicyclo[4.2.0]octa-1,3,5-triene. 1,2-Benzenedicarboxylic acid, diisooctyl ester. 2-Benzyloxyethylamine. Styrene. 3-Trifluoroacetoxydodecane Insilico studies Anticancer activity of Euphorbia hetrophylla was studied by insilico method Bioinformatics tools were employed in understanding the mechanism of action. 14 of the compounds were docked with the Bcl-2 protein, among them Allantoic acid was carried as best candidate as it is having least energy in docking. (Table 2&Figure1) Table 2: Dock score for the compounds
S.no 1 2 3 4 5 6 7 8 Compounds 2-Benzyloxyethylamine 2-Formylhistamine Allantoic acid Benzeneethanamine, 2-fluoro-,3-dihydroxy-N-methyl3-Trifluoroacetoxydodecane Pterin-6-carboxylic acid Imidazole, 2-amino-5-[(2-carboxy)vinyl]Acetic acid, 3-hydroxy-6-isopropenyl-4,8a-dimethyl1,2,3,5,6,7,8,8a-octahydronaphthalen-2-yl ester Docking Score -7.11 -7.90 -8.16 -6.77 -8.14 -7.51 -7.72 -7.78

2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Source of support: Nil, Conflict of interest: None Declared

Journal of Pharmacy Research Vol.3.Issue 9.September 2010

2332-2333