ANTICANCER ACTIVITIY OF Cynodon dactylon L. EXTRACT ON EHRLICH ASCITES CARCINOMA

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1 ANTICANCER ACTIVITIY OF Cynodon dactylon L. EXTRACT ON EHRLICH ASCITES CARCINOMA Krishnamoorthy M.* and Ashwini Prabhu Department of Post graduate Studies and Research in Biosciences, Mangalore University, Mangalagangothri, Karnataka, (INDIA) Received November 14, 2010 Accepted January 4, 2011 ABSTRACT Cancer is the second leading cause of death worldwide next to cardiovascular diseases. Conventional therapies for treatment of cancer including chemotherapy and radiation therapy usually pose serious side effects. Use of herbal medicine for cancer treatment is gaining importance in this regard. India is a rich source of medicinal plants and a number of plant extracts are used against diseases in various medicinal systems. Cynodon dactylon L., (Family: Poaceae) commonly known as 'durva' grass is employed in India as a rejuvenator and for wound healing. In the present study, anticancer efficacy of Cynodon dactylon L. was assessed in Swiss albino mice Mus musculus. Mice inoculated with Ehrlich Ascites Carcinoma (EAC) cells were administered with three doses of Cynodon dactylon extract viz., 100, 200 and 400mg/kg body weight per oral for ten consecutive days. Anticancer activity of the extract was assessed based on Mean Survival Time (MST) and increase in life span (ILS) of experimental animals. In addition, hematological parameters like hemoglobin content, RBC count, WBC count and differential count of extract treated mice were also studied. Extract treatment enhanced the life span and restored the hematological parameters in EAC inoculated mice in comparison with tumor control group. Thus, the present study revealed that Cynodon dactylon L. extract showed significant anticancer activities in the tested animal models. Key Words : Cynodon dactylon, Ehrlich Ascites Carcinoma, Swiss albino mice, Anticancer, Meon Survival Time (MST) INTRODUCTION Cancer is the second leading cause of death worldwide next to cardiovascular diseases. The failure of conventional treatment modules including chemotherapy and radiation therapy pose a magnitude of severe side effects and in this regard, phytotherapy is emerging as a promising field in cancer therapy. Medicinal plants play a key role in the human healthcare. About 80% of the world s population rely on the use of traditional medicine, which is predominantly based on plant materials 1. A large number of medicinal plants and their constituents have been known to possess beneficial therapeutic potential 2. India is a rich source of medicinal plants and a number of plant extracts are used against diseases in various *Author for correspondence systems of medicine such as Ayurveda, Unani and Siddha. Only a few of them have been scientifically explored 3. The World Health Organization has recommended the evaluation of the plants effectiveness in conditions where we lack safe modern drugs 4. This has lead to an increasing demand of research on anticancer natural products which produces minimal or no side effects 5. Cynodon dactylon L (family: Poaceae) is commonly known as durva grass in India, has been regarded to possess various medicinal properties. It is a resilient perennial grass distributed all over the world and is especially native to the warm temperate and tropical regions. The plant possesses antimicrobial and antihypertensive activity and has also been used to treat urinary tract infection, calculi and prostatitis. The aqueous plant extract is used as anti inflammatory, antidiabetic, diuretic, antiemetic 551

2 and purifying agent 6. In India, the plant is reputed Toxicological evaluation of the plant extract for the treatment of anorexia, burning sensation Before selecting the doses for anticancer study, of the body, pruritis, miscarriage and the acute toxicity of the extract was carried out erycephalis 7,8. In Iranian folk medicine, the roots in Swiss albino mice using up and down and rhizomes of the plant have been used for the procedure 12. The acute toxicity test was carried treatment of depression, epilepsy and out to investigate the acute dose lethal to 50% of hemorrhage 9. However, there was no proof till the animals thereby establishing the therapeutic date about the anticancer activity of this plant for index. The animals were fasted overnight and then which the present study was designed. they were administered with different doses of Cynodon dactylon hydroalcoholic extract in a AIMS AND OBJECTIVES single dose viz., 1.75mg, 55 mg, 175 mg, 550 mg The present study was carried out to investigate and 2000 mg/kg body wt. Animals were kept under the anticancer efficacy of Cynodon dactylon observation up to 72 hours. Parameters such as hydroalcoholic extract on Ehrlich Ascites behavioural changes, autonomic nervous system Carcinoma (EAC) cells in in vivo Mus musculus profiles and central nervous system profiles were system. analyzed. The observations were tabulated according to Irwin s table. MATERIAL AND METHODS Preparation of the plant extract and Preliminary phytochemical screening: The leaves of Cynodon dactylon L. were collected from surroundings of Mangalore, Kranataka, India. The plant material was identified as per database on medicinal plants used in Ayurveda- Volume The leaves were washed; shade dried and powdered using a kitchen blender. Leaf powder was subjected to hot continuous percolation using ethanol in a Soxhlet apparatus. The extract was further concentrated to obtain a thick paste. It was stored at -20 C until use. Preliminary phytochemical screening of the extract was carried out 11. Animals Line bred Swiss albino mice, 8 weeks old, weighing 25± 2 g were used for the present study. They were maintained under aseptic conditions in the animal house of Biosciences department, Mangalore University. The animals were housed in polypropylene cages and provided with standard feed pellets and tap water ad libitum, under 12 hrs of light/dark cycle. The study was conducted after obtaining institutional animal ethical committee clearance (No: CPCSEA 232). Tumor cells Ehrlich Ascites Carcinoma (EAC) cells were initially procured through the courtesy of Radiobiology laboratory, KMC, Manipal, Karnataka, India. They were maintained by weekly intraperitoneal (i.p.) inoculation of 10 6 cells/mouse. 552 Effect of Cynodon dactylon leaf extract on mean survival time (MST) and average body weights of EAC bearing mice Ascitic fluid was aspirated from the peritoneal cavity of full grown tumor model and viable cell count was determined using trypan blue dye exclusion test. Animals were injected intrapertoneally with tumor cells in 0.1 ml of physiological saline. They divided into six groups. Group I served as tumor control wherein the animals received injection of tumor cells alone. Group II was administered with 0.1 ml of 10% ethanol for ten consecutive days which served as solvent control. Group III was treated with standard anticancer drug 5- fluorouracil (20 mg/ kg body wt. i.p.) 24 hrs after EAC inoculation. Groups IV, V and VI were treated respectively with 100mg/kg, 200mg/kg and 400mg/kg of the Cynodon dactylon leaf extract for ten consecutive days, the first administration starting from 24 hrs after EAC injection. Mean survival time (MST) of each group, consisting of five mice was noted. Increase in the life span of Cynodon dactylon extract treated group in comparison with the tumor control group was calculated using the formula: Increase in life span ILS (%) = T-C/C x 100 Where, T = Number of days the treated animals survived C = Number of days the control animals survived. 13

3 Increase in the average body weights of the animals was also recorded in these groups. Effect of Cynodon dactylon extract on hematological parameters In order to assess the effect of the extract on hematological parameters of EAC bearing mice, a comparison was made among six groups (n = 5) of mice on the 14th day after EAC transplantation. Group I comprised of normal mice. Group II and III served as tumor control and solvent control respectively. Group IV, V and VI were treated with Cynodon dactylon leaf extract viz., 100mg/kg, 200mg/kg and 400mg/kg p.o. consecutively for ten days after EAC injection. Animals were sacrificed, blood was drawn through cardiac puncture and RBC count, hemoglobin content, total WBC count and Differential counts were made 14. Statistical analysis All values were expressed as mean ± SEM. The data was statistically analyzed by student s t test. P values < 0.05 were considered significant. RESULTS AND DISCUSSION Results of Acute toxicity studies of Cynodon dactylon leaf extract are summarized in Table 1.The animals did not show any changes in the behavioral, neurological and autonomic responses It was evident from this study that the extract administration did not pose any toxic effects to experimental animals and hence was safe for consumption. Table 1 : Irwin s table showing the response of swiss albino mice Mus musculus after administration of Cynodon dactylon extract 553

4 The effect of Cynodon dactylon extract on mean survival time of tumor bearing mice is tabulated in Table 2. The Mean Survival Time of the control group was 22 ± 0.31, whereas it was found to be 22 ± 0.44, 40 ± 0.63, 23 ± 0.31, 31 ± 0.44 and 33 ± 0.31 for the groups treated with 10% alcohol, 5-Fluorouracil(20mg/kg), Cynodon dactylon alcoholic extract 100mg/kg, 200mg/kg and 400mg/ kg respectively. Increase in life span of animals treated with 5-FU and extract at different doses was shown to be 81.81%, 4.54%, 40.9% and 50% respectively. The average weight gain due to ascetic fluid accumulation in tumor control group was found to be 13.1 ± 0.04 whereas it was 13.0 ± 0.04, 5.78 ± 0.06, ± 0.12, 9.52 ± 0.17 and 7.46 ± 0.12 for the groups administered with 10% alcohol, 5-FU(20mg/kg), Cynodon dactylon extract 100mg/kg, 200mg/kg and 400mg/kg respectively. Table 2 : Effect of Cynodon dactylon leaf extract on Mean survival time (MST) and body weights of EAC bearing mice Treatment Schedule MST(days) ILS(%) Average increase in body weights(g) Tumor control 22 ± ± % alcohol 22 ± ± Fluorouracil 40 ± 0.63* ± 0.06* ETC (100 mg/kg) 23 ± ± 0.12** ETC (200 mg/kg) 31 ± 0.44* ± 0.17* ETC (400 mg/kg) 33 ± 0.31* ± 0.12* MST = Mean Survival Time, ILS = Increase in Life Span, ETC = Ethanolic extract of Cynodon dactylon n=5 animals in each group, *p<0.01,**p<0.05 when compared with tumor control Values are expressed as Mean ± SEM Evaluation of haematological parameters is summarized in Table 3. Hemoglobin level and RBC count was found to be decreased whereas WBC count was found to be increased in tumor control group. Groups treated with Cynodon dactylon extract showed restoration of these haematological parameters in a dose dependent manner. The future of cancer chemoprevention remains open to innovation, with a specific need for emphasizing cancer prevention in public health policy 15. Fundamental treatments for cancer include radiation therapy and chemotherapy which usually pose drawback of side effects such as myelosuppression, nausea, vomiting, alopecia, impotence and immunosuppression 16,17. It seems probable to overcome the above shortcomings with the use of drugs of natural origin. Cancer prevention using natural products that inhibit cell transformation and prevent metastasis would be less painful, more economical and rational approach for cancer control. Use of natural and dietary agents is being increasingly utilized as an effective way for the treatment of many types of cancers 18,19. In this study, an attempt has been made to evaluate the anticancer efficacy of 554

5 Cynodon dactylon, which is traditionally used as an agent to control diabetes in India and also has many other pharmacological claims including anti inflammatory, anticystitis, antihypertensive and antiviral activities. In the present study, animals administered with different doses of Cynodon dactylon extract showed a dose dependent increase in Mean survival time and life span with respect to control group, thereby proving role of the extract in retardation of tumor development. Tumor growth is generally associated with remarkable changes in haematopoiesis, immune response, myelosuppression and anaemia 20.The reliable criteria for judging the value of anticancer drug is the prolongation of life span and decrease of WBCs from blood 21,22. Hematological studies have revealed that administration of Cynodon dactylon extract to EAC inoculated animals restored Hemoglobin level, RBC and WBC counts almost near to normal values, which indicated the protective effect of the extract on haemopoietic system. Table 3 : Effect of Cynodon dactylon leaf extract on hematological paramets of EAC bearing mice Parameter Normal Tumor control 10% alcohol ETC 100mg/kg ETC 200 mg/kg Hb (g%) 12.28± ± ± ± 0.12** 8.38 ± 0.14* RBC (10 6 cells/mm 3 ) WBC (10 3 cells/mm 3 ) 4.26 ± ± ± ± ± 0.10* 6.43 ± ± ± ± 0.05* ± 0.12* Lymphocytes (%) 70.4 ± ± ± 0.83** 52.2 ± ± 0.15* Monocytes(%) 1.8 ± ± 0 1 ± 0 1 ± 0 1 ± 0 Neutrophils(%) 24.2 ± ± ± ± 0.73** ± 0.33* Eosinophils(%) 1.6 ± ± 0 1 ± 0 1 ± 0 1 ± 0 Basophils(%) 2 ± 0 1 ± 0 1 ± 0 1 ± ± 0.20 ETC 400 mg/kg ± 0.11* 3.9 ± 0.07* 8.16 ± 0.09* ± 0.29* 1 ± ± 0.09* 1 ± 0 1 ± 0 n=5 animals in each group, *p<0.01, **p<0.05 when compared with control, values are expressed as mean ± SEM Preliminary phytochemical screening of Cynodon dactylon extract indicated the presence of phenolic compounds, alkaloids, flavonoids and sterols.phenolics are known to exhibit antioxidant capacity due to their ability to chelate metal ions involved in the production of free radicals 23. Phenolic compounds have been reported to display some relevant biological activities such as antibacterial, antiviral, immune stimulating and estrogenic effects 24 as well as antiproliferative and cytotoxic properties in several tumor cells 25,26. Several alkaloids are known to possess cytotoxic 555 properties on human cancer cell lines 27. Flavonids have a chemopreventive role in cancer through their effects on signal transduction in cell proliferation 28 and angiogenesis 29. CONCLUSION The presence of ceratin phytoconsttuents including phenolic compounds, alkaloids and sterols might have played a possible role in the mediation of anticancer activity of Cynodon dactylon extract. These results indicate the possibility of developing Cynodon dactylon extract into a potential and novel chemoptheraeutic agent. Further studies to

6 characetrize the active principles and to elucidate the mechanism of Cynodon dactylon are in progress. REFERENCES 1. Surendra V. et al., Hepatoprotective activity of aerial parts of Cynodon dactylon against CCl4-induced in Rats, Phacog. Mag., 4 (16), , (2008). 2. Agbar Z et al., Comparative antioxidant activity of some edible plants, Turk. J. Biol., 32(3), , (2008). 3. Osawa T., Kawakishi S. and Namiki M., In Kuroda Y., Shankel D. M., Waters MD (Ed), Antimutagenesis and anticarcinogenesis mechanism II, New York, Plenum,, , (1990). 4. Day C., Traditional plant treatment for Diabetes Mellitus: pharmaceutical foods, Brit. J. Nut., 80(1), 5-6, (1998). 5. Rao B.K., Kesavulu M. M. and Giri R. A. C., Antidiabetic and hypolipidemic effects of Momordica cymbalaua hook fruit powder in Alloxan diabetic rats, J. Ethnopharmaco., 67(1), , (1999). 6. Ahmed S., Reza M.S. and Jebbar A., Antimicrobial activity of Cynodon dactylon, Fitoterapia, 65(1), , (1994). 7. Lepor atti M. L., and Corradi L., Ethnopharmacobotanical remarks on the province of Cheiti town, Abruzzo, Central Italy, J. Ethnopharmacol. 74(1), 17-40, (2001). 8. Shinwari M. I., and Khan M. A., Folk use of medicinal herbs of Margalla Hills National Park, Islamabad, J. Ethnopharmacol., 69(1), 45-56, (2000). 9. Miraldi E., Ferri S. and Mostaghimi V., Botanical drugs and preparations in the traditional medicine of West Azerbaijan (Iran), J. Ethnopharmacol., 75(2-3), 77-87, (2001). 10. Billore et al., Database on medicinal plants used in Ayurveda, Central Council for 556 Research in Ayurveda and Siddha, New Delhi, 38-53, (2004). 11. Wagner H., Bladt S. and Zgainski E.M., Plant drug analysis, Springer Verlag Publishers, New York, , (1984). 12. OECD (Organization of Economic Cooperation Development), The OECD guidelines for testing of chemical, 420, Acute Oral toxicity, OECD, Paris, 1-14, (2001). 13. Suffness M. and Douros J., Methods in Cancer Research, In : Devita VT(Ed.), Academic Press, New York, 73-75, (1978). 14. Verma P. S., Tyagi B. S. and Agarwal V. K., Animal Physiology, S. Chand and Co. Ltd., New Delhi, , (2002). 15. Tsao A. S., Sim E.S. and Hong W.K., Chemoprevention of Cancer, CA. Cancer J. Clin., 54(3), , (2004). 16. Marklund et al., Coppr and Zinc containing superoxide dismutase, catalase and glutathione peroxidase in normal and neoplastic human cell lines and normal human tissues, Cancer Res., 42(5), , (1982). 17. Price V. E. and Greenfield R. E., Anemia in cancer, Adv. Cancer Res., 5(1), , (1958). 18. Wenzel et al., Dietary flavone is a potent apoptosis inducer in human colon carcinoma cells, Cancer Res., 60(14), , (2000). 19. Miyoshi et al., Dietary ginger constituents, galanals A and B, are potent apoptosis inducers in human T lymphoma Jurkat Cells, Cancer Lett., 199(2), , (2003). 20. Kumarappan C.T. and Mandal S.C., Antitumor activity of polyphenolic extract of Ichnocarpus frutescens, Exp. Oncol., 29 (2), , (2007). 21. Clarkson B.D. and Bur chenal J.H., Preliminary screening of antineoplastic drugs, Prog. Clin. Cancer, 1(1), , (1965).

7 Journal of Environmental Research And Development Vol. 5 No. 3 January-March Obiling C. and Guerin M., Advances in Cancer Research 2, Academic Press, New York, , (1954). 23. Yang et al., Inhibition of carcinogenesis by dietary polyphenolic compounds, Ann. Rev. Nut., 21(1), , (2001). 24. Rice Evans C.A., Miller N.J. and Paganga C., Structure antioxidant activity relationships of flavonoids and phenolic acids, Free Rad. Biol. Med., 20(1), , (1996). 25. Rapta et al., Redox intermediates of flavonoids and Caffeic acid esters from Propolis: An EPR Spectroscopy and cyclic voltametry study, Free Rad. Biol. Med., 18(5), , (1995). 26. Agollo et al., Relationship between flavonoid structure and inhibition of phosphatidylinosiol 3- kinase and Protein Kinase C inhibition, Biochem. Pharmacol., 53(11), , (1997). 27. Shoeb M., Anticancer agents from medicinal plants, Bangla. J. Pharmacol., 1(2), 35-41, (2006). 28. Chen Y.C., Shen S.C. and Len H.Y., Rutinoside at C7 attenuates the apoptosisinducing activity of flavonoids, Biochem. Pharmacol., 66(7), , (2003). 29. Shen et al., Differential apoptosis inducing effect of quercetin and its glycosides in human promyelolukemic HL-60 cells by alternative activation of the caspase-3 cascade, J. Cell. Biochem., 89(5), , (2003). 557