International Journal of Pharma and Bio Sciences

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2 International Journal of Pharma and Bio Sciences RESEARCH ARTICLE TOXICOLOGY PROTECTIVE EFFECTS OF PHYLLANTHUS FRUIT EXTRACT IN ADRIAMYCIN INDUCED GENOTOXICITY IN BONE MARROW CELLS OF MICE K. RUDRAMA DEVI* AND KUSUM LATA CHAMYAL Human Genetics and Toxicology, Department of Zoology, Osmania University,Hyderabad , A.P. India K. RUDRAMA DEVI Human Genetics and Toxicology, Department of Zoology, Osmania University, Hyderabad , A.P. India ABSTRACT Adriamycin (ADR) is the most commonly used anthracyclin effective in malignant lymphomas and used for various cancers. The aim of the present investigation was to assess the protective effects of curcumin on adriamycin induced genotoxicity on the bone marrow chromosomes of Swiss albino mice. In the present experiment four group of Swiss albino animals were maintained. The experimental group was administered with 4 mg/kg of ADR in a single dose. The subsequent experimental group was administered with Phyllanthus Fruit Extract PFE 340 mg/kg/bw orally for two weeks and on sixteenth day ADR (4 mg/kg/bw) was given intraperitonially as a single dose. For each experimental group control, animals were maintained. Two days after the administration of the last dose, the animals were sacrificed and air dried metaphase preparations were made and processed for identification of chromosomal aberrations in somatic cells of mice. In animals treated with single dose of ADR, an increase was observed when compared with the values of control group. But when animals primed with PFE + ADR group, there was a decrease in the frequency of chromosomal aberrations. Thus the results clearly indicated the protective role of PFE on adriamycin induced genotoxic damage in somatic cells of mice P - 133

3 KEYWORDS ISSN Vol 3/Issue 1/Jan Mar 2012 Somatic cells, Phyllanthus Fruit Extract, Adriamycin, Chromosomal aberrations. 1. INTRODUCTION Adriamycin(Doxorubicin) is an anthracycline antibiotic used as an antitumor agent against human malignancies such as leukemia, lymphomas and many solid tumors but which also has a wide variety of toxic sideeffects, including cardio toxicity, cytotoxicity and the induction of chromosomal aberrations 1-4. The majority of antineoplastic drugs, besides their generic growth property, display genotoxic effects which in turn contribute to growth inhibition 5. These genotoxic effects may lead to initiation of unrelated tumours years after cessation of chemotherapy 6. Free radicalmediated reactions are responsible for a wide range of chemotherapy-induced side effects and antioxidants are able to protect non-malignant cells and organs against damage caused by cytostatic agents 7. Most cancers can be controlled by adopting appropriate conventional treatments such as surgery, radiation and chemotherapy. However these treatments cause side effects. Hence the important of conventional therapies may decline. Alternative treatments founded in a back to nature approach might yield improved treatment avenues with fewer or no undesirable side effects. In the search of these new treatment, natural products are carving a path as prospective anticancer agents. According to believe in ancient Indian mythology, Phyllanthus emblica is the first tree to be created in the universe. It belongs to family Euphorbiaceae. It is also named as Amla, phyllanthus Emblica or Indian gooseberry. The species is native to India and also grows in tropical and subtropical regions including Pakistan, Uzbekistan, Srilanka, South East Asia, China and Malaysia. The fruits of PF are widely used in the Aryuveda and are believed to increase defense against diseases. It has its beneficial role in cancer, diabetes, liver treated, heart trouble, ulcer, anemia and various other diseases. Diet can modify the pathological processes, because certain naturally occurring substances known as antioxidants are present in plants and other sources have shown to be protective against mutagens or carcinogens or endogenous mutagens 8. Among the various phytonutrients, phyllanthus emblica posses good antioxidants. Phyllanthus was described in Indian Ayurvedic literature more than 200years ago. It has been widely used by traditional medical practitioners for the treatment of various diseases. It exhibits many properties like antiviral, antimutagenic, hepato protective activity, hypoglycemic activity etc In the present investigation, the studies were carried out on protective role of PFE on adriamycin induced genetic damage in somatic cells of mice. 2. MATERIALS AND METHODS 2.1 Drugs and Chemicals: Adriomycin(DOXORUBICIN)was given as a gift from MNJ Institute Of Oncology, Hyderabad. Trisodium citrate (Merck), NaCl(Loba Chemie), Methanol(s.d fine chemie), acetic acid(qualingens), Giemsa (s.d fine chemie). All other chemicals used in the experiments were of analytical grade. 2.2 PFE Extract preparation: Cameron and Puling 13 suggested the daily intake of vitamin C is 1-10g/day for human being. Data based on maximum ascorbate P - 134

4 concentrations in human body suggest a maximum body pool of around 5000mg, which is approximately 70mg/kg body weight in man 14. In the present study, a corresponding amount of an aqueous extract of PFE containing the same amount of vitamin C was used for mice, as calculated from daily 1 g intake for a 60kg person. The fruits were procured in bulk, cut into pieces and dried in sunlight. Known quantities weighed and kept in distilled water for 24hr. The AA content of the decoction was estimated b the 2, 6-dichlorophenol indophenol method 15 and it amounted to 685mg/kg body weight. 2.3 Animal treatment: The study was conducted after taking the approval of Institutional Ethical Committee on twenty adult male swiss albino mice 30 to 50 days old and weighing around to 30 to 40 g were maintained in plastic cages under controlled lighting conditions (12:12 light and dark cycle) relative humidity (50±5%) and temperature (37±2 o C) fed with mice feed and were given ad libitium access to water. A group of 5 mice per experiment were taken and treated with ADR and PEF. The doses were prepared daily in distilled water and were administered by gastric gavage method for PEF and 26G needle intraperitoneal injection for ADR treatment. The dose protocols were as follows: Group I control group were treated with 5 ml of physiological saline.. Group II the animals were treated with ADR 4 mg/animal/day intraperitoneally. Group III control were treated with PFE 340 mg/animal/day for two weeks daily. Group IV Experimental batch were pretreated with 340 mg/kg BW PFE for 15 days on the 16 th day single intraperitoneal dose of adriamycin 4 mg/kg/bw, were administered. 2.4 Analysis of chromosomal aberrations in somatic cells of mice: The animals were killed two days after administration of the last dose. The bone marrow was flushed into clean glass Petri dishes with hypertonic solution (0.56% KCl) were used to get a homogeneous cell suspension. It was then collected in clean centrifuge tubes and incubated at 37 o C for 45 minutes. Four slides for each were prepared from control and experimental animals. The staining was done within 24 h of preparation according to the method Preston et al., 16. The slides were screened for 50 well spread metaphases per animal for the presence of various types of chromosomal aberrations like gaps breaks, fragment, chromatid separations and polyploids in control and treated group of animals. The differences in the frequencies of chromosomal aberrations between control and treated groups were analyzed using Chi-Square test. For calculating mitotic index (MI) a minimum of 1000 cells were counted for each animal. 3. RESULTS AND DISCUSSION In the present investigation, the results on the assessment of genotoxicity in adriamycin showed a significant increase in the frequency of CAs. However the data on the incidence of chromosomal aberrations in somatic cells of mice showed non-mutagenic nature of PFE. But when the animals pre-treated with PFE and ADR administered showed a decrease in the percentage of chromosomal aberrations in somatic cells of mice. In adriamycin treated animals the mitotic index showed a decline, but when the animals pretreated with PEF for two weeks and co administration of ADR in single dose the mitotic index showed no change when compared with the controls. (Table1) P - 135

5 Table 1 Protective effect of PFE against adriamycin induce decline in mitotic index. Group Number Number of division of cells Number of non dividing cells Total number of cells Mitotic index Control group Group II ADR 4mg/kg//animal (single dose) * Group III PFE 340mg/kg/day/animal Group IV ADR+PFE 340mg/kg/animal/day for 15 days+4mg/kg/animal/day single dose *P< The actively proliferating cells from bone marrow provide maximum information on the effect of any test compound 16. The transition from proerythroblast to erythrocytes takes about seven cell division cycles. Each cell cycle takes hrs and the terminal mitosis is completed in about 10hrs before the transition of orthochromatid erythroblast to polychromatic erythrocytes. In view of the above to see the long and short term effect of test compound on cells, the sampling time ranged was from 6-72 hrs has taken in present observation There are different type of chromosomal aberrations observed in present analysis These aberration are classified into structural, numerical and other abnormalities. Structural aberration includes gaps, breaks, fragments, terminal deletion and centric fusion these end points serve as indicators for assessing the mutagenic effect of test substance. Phyllanthus emblica enjoyed a hallow position in Ayurveda an Indian system o medicine. It is a first tree to be created in the universe. Its fruit juice contains highest vitamin C contains as mg/100ml. It is used in the preparation of Indian pickles. The fruit when blended with other fruits boosted their nutritional quality in terms of vitamin C content 17. It is often used as Triphala which is a herbal formulation containing fruit of Terminalia chebula and Terminlia belerica in equal proportions. It has important medicinal value against various diseases. Invitro and invivo animal studies suggested wide range of potential therapeutic or preventive effects has been reported. Such effects in humans have not conformed so far. PFE when prepare in the Triphala delayed the development of fore stomach Papillomagness, breast cancer, skin tumors, liver fibrosis, diabetic cataract, Alzheimer s diseases Hence in our study we aimed to access the protective effects of PFE against the Adramycin induced genotoxicty. Chromosomal aberrations and a decrease with mitotic index are the most sensitive indicators of bone marrow damage Earlier we reported a decrease in mitotic index on the incidence of micronuclei in polychromatic erythrocytes of male mice when treated with adriamycin 26. In the present study an effort has been made to observe whether such toxic effects induced any adriamycin or neutralized or counter balanced by the treatment of PF fruit extract, primarily contains tannins alkoliods, phenolic compound, amino acids, carbohydrets and vitamin C. the PFE is prepared in formulations as P - 136

6 Triphala, kalpaamrutha and chyavana prash were showed therapeutic beneficial for infected wounds, coronary artery disease, arthritis, an opthcare in number of inflammatory and degenerative opthalamic disorders It has been exhibited antipyretic, anti tussive, dyslipidemia, snake venom neutralizer, anti microbial immunosuppression, anti mutagenic and anti carcinogenic properties However the geoprotective effect of PFE has not been evaluated against anticancer drug adriamycin. Hence, it is of interest to assess the genotoxicity of adriamycin and also the protection rendered my PFE against such genetic damage. Earlier we reported a significant increase in the frequency of micronuclei in bone marrow erythrocytes and sperm head abnormalities in male mice 26,37. Such cytotoxicity is the consequence of oxidative stress and generation of free radicals originate from exposed to the mutagens, which results in distributed homeostasis and also induction of such biological stress as shown by a sharp decline in mitotic index and elevation of chromosomal aberrations as observed in the present investigation (Table 1). Two different pathways of free radical formation of doxorubicin (adriamycin) have been described. First implicates the formation of semiquinone free radical. The semiquinone can be transferred to a C7 radical that can also mediates cellular damage. The reduction of doxorubicin by 2 electrons generates a secondary alcohol metabolite doxorubicinol 37. The second pathway doxorubicin free radicals come from a nonenzymatic mechanism that involves reactions with iron. For example Fe3+ reacts with doxorubin in a red-ox reaction after which the iron atom accepts an electron and a Fe2+ deoxyribicin free radical complex is produced. This iron doxorubicin complex can reduce oxygen to hydrogen peroxide and other active species Table2 Protective effects of PFE against adriamycin induced cytotoxicity in somatic cells of mice details of various types of aberrations observed in experiment group of animals Group Structural Numerical Total no. of G B F Ex P CS aberrations (8.00) (2.00) (0.00) (0.00) (0.00) (0.00) (2.00) (12.0) (2.00) (6.00) (0.00) (0.00) (2.00) (10.00)* Group - I Control Group - II ADR Group III PFE (1.20) (0.80) (1.20) (0.80) (0.00) (0.40) Group IV PFE+ADR (2.00) (0.80) (0.80) (0.00) (0.00) (0.00) G = Gaps, B = Breaks, F = Fragments, E = Exchanges, P = Polyploid, CS = Chromatid Separation Gaps and polyploids are not included the values in parenthesis are percentage. *P<0.05 In experimental animals, the mitotic index with PFE for two weeks and subsequently treated with a single dose of adriamycin (4mg/kg) did not show any significant decline and the value obtained was equivalent to the control group, there by indicating that adriamycin is rendered (2.80) 4 (4.00)* ineffective in inducing a decline in mitotic index as compared to the animals administered only adriamycin (Table 1). From the above data, it can be concluded that antioxidants such as PFE protects the damage from free radicals. A number of medicinal plants, traditionally used for P - 137

7 thousands of years are present in a group of herbal preparations of the Indian traditional health care system (Ayurveda) named rasayana identified for anti oxidant properties PFE have been supported for its anti oxidant activity. It contains tannoid principles of embliconin A, embliconin B, punigluconin and peunculagin, have been reported to poses antioxidant activity invivo and invitro Further the PFE accounts for approximately 45-70% of the antioxidant activity. Further the phenolic compounds derived from plant exhibit a number of beneficial effects and potentially inhibited several stages of carcinogenesis. Phenolic compounds are the major components from the fruit juice of PFE and from the branches, leaves and roots showed stronger inhibition against P16F10 cell growth than against Hela and MK1 cell growth 44. PFE and its medicinal preparations may prove beneficial as a component of combination therapy in phase I clinical trial in cancer patients under the cyclophamide treatment 45. Ethanolic extract of PFE was experimentally evaluated for protection against genotoxicity induced by DMBA. PFE administrated orally at different concentrations (100, 250, 500mg/kg body weight) for 7 consecutive days in swiss albino mice prior to a single intro peritonial injection of DMBA decreased the frequency of bone marrow micronuclei 46. These observations are in accord with our results where PFE has been showed to render protection against adriamycin induced toxicity (Table 2). When animals pre-treated with PFE and subsequently treated with adraimycin, it was observed that M1 was equivalent to that of control group. Such observations indicate that when organisms. administered with fruit extract for a longer period of time than perhaps toxic agents such as anticancer drugs might not be effective in induction of chromosomal aberrations. CONCLUSION From the above studies, it is concluded that phyllanthus emblica was a potential candidate as protective agent to adriamycin induced genotoxic effect in somatic cells of mice. The combined treatment of adriamycin and PFE holds a promise as a safe and effective chemotherapeutic strategy. REFERENCES 1. Young RC, Ozols RF and Myers CE. The antharacycline antineoplastic drugs. New Engl J Med 305: (1981) 2. Booser DJ and Hortobagyi GN. Anthracycline antibiotics in cancer therapy: Focus on drug resistance. Drugs 47: (1994) 3. Singal PK, Li T, Kumar D, Danelisen I and Iliskovic N. Adriamycin- induced heart failure:mechanism and modulation. Mol and Cell Biochem 207: (2000) 4. Jung K and Reszka R. Mitochondria as subcellular targets for clinically useful anthracyclines. Adv Drug Deliv Rev 49: (2001) 5. Buschini,A., Poli,P. and Rossi,C. Saccharomyces cerevisiae as an eukaryotic cell model to assess cytotoxicity and genotoxicity of three anticancer anthraquinones. Mutagenesis, 18, (2003) 6. Beretta G., Cancer Treatment Medical Guide, 10th ed., Farmitalia Carlo Erba-Erbamont, Milan. (1991). 7. Weijil, N.I., Cleton, F.J. and Osanto, S. Free radicals and antioxidants in chemotherapyinduced toxicity. Cancer Treat. Rev: 23: (1997). 8. Khan KH, Roles of Emblica officinalis in Medicine A review (2009) 9. Ferguson L.R., Antimutagens as cancer chemopreventive agents in the diet. Mutat Res. May 1:207 (1) (1994) P - 138

8 ISSN Vol 3/Issue 1/Jan Mar 2012 Ramakrishnan P.N., Murugasen R, Palamichamy S and Murugesh N. Indian Journal of Pharmaeuti. Sci. 44, 1: 10. (1982). Syamasunder, K.V., Singh,B., Thakur, R.S., Hussain, A., Kiso,Y. and Hikino, H.J. Ethanopharmacol. Sp., 14(1):41-4, (1985). Venkateswaran, P.S., Millman, I. and Blumberg, B.S. Effects of an extract from Phyllanthus niuri on hepatitis B and wood chuck hepatitis viruses: Invitro and Invivo studies. Proc. Natl. Acad. Sci. U.S.A., 84(1); ,(1987). Cameron, E, and L Pauling. Cancer and Vitamin C. Linus Pauling Institute of Science and Medicine, California, (1979). Counsell, J, N and D. H Horning. Vitamin C and Ascorbic acid. Applied Science Publishers London (1981) Pearson. D., The chemical analysis of food 7th edition churchchill, living stone, London (1952) Preston R J., BJ Dean, S Galloway, H, Holden, AF Mcfee and M Shelby Mammalian invivo cytogenetic assay: analysis of chromosomal abberations boicmmeh53l07-7mm6z3o0cz-56mu5u3e07-7j583 0CjnC56-z; P - 139

9 30. Ganju, L., D. Karan, S. Chanda, K.K. Srivastava, R.C. Sawhney and W. Selvamurthy,. Immunomodulatory effects of agents of plant origin. Biomed Pharmacother., 57(7): ( 2003) 31. Perianayagam, J.B., S.K. Sharma, A. Joseph and A.J. Christina,. Evaluation of anti-pyretic and analgesic activity of Emblica officinalis Gaertn. J. Ethnopharmacol., 95(1): 83-5.( 2004) 32. Sai, R.M., D. Neetu, P. Deepti, M. Vandana, G. Ilavazhagan, D. Kumar and W. Selvamurthy,. Cytoprotective activity of Amla (Emblica officinalis) against chromium (VI) induced oxidative injury in murine macrophages. Phytother Res., 17(4): ( 2003) 33. Kim, H.J., T. Yokozawa, H.Y. Kim, C. Tohda, T.P. Rao and L.R. Juneja,. Influence of amla (Emblica officinalis Gaertn.) on hypercholesterolemia and lipid peroxidation in cholesterol-fed rats. J Nutr Sci Vitaminol (Tokyo)., 51(6): ( 2005) 34. Alam, M.I. and A. Gomes,. Snake venom neutralization by Indian medicinal plants (Vitex negundo and Emblica officinalis) root extracts. J. Ethnopharmacol., 86(1): ( 2003) 35. Kaur, S., S. Arora, K. Kaur and S. Kumar,. The invitro antimutagenic activity of Triphala-- an Indian herbal drug. Food Chem Toxicol., 40(4): ( 2002) 36. Madhavi Dand K. Rudrama Devi. Modulatory effects of Phylantus fruit extract against lead induced Genotoxicity in somatic cells of mice". Journal of Environmental Biology Vol. 28(1): , (2007). 37. Adriamycin induced sperm head abnormalities in swiss albino mice C. Kusum Lata and K. Rudrama Devi International journal of agricultural biological research..vol:27(2):91-97,(2011). 38. Granados- principal S, Quiles JL, Ramirez- Tortosa CL, Sanchez-Rovira P and Ramirez- Tortosa MC, New advances in molecular mechanism and prevention of Adriamycin toxicity by antioxidant nutrients. Food and chemicals toxicology, 48: , (2010). 39. Quiles JL, Huertas JR, Battino M, Matrix J and Ramirez-Tortosa MC, antioxidant nutrients and adriamycin toxicity. Txicology, 180: (2002). 40. Sanocka D and Kurpisz M, Reactive oxygen species and sperm cells. Repod. Bio. Endocrinol., 2: 1-7, (2004). 41. Xu X, Persson H L, and Richerdson D R, molecular pharmacology of interaction of anthracyclins with iron. Mol Pharmacol, 68: , (2005). 42. Scartezzini, P. and E. Speroni,. Review on some plants of Indian traditional medicine with antioxidant activity. J Ethnopharmacol., 71(1-2): ( 2000) 43. Bhattacharya, A., S. Ghosal and S.K. Bhattacharya,. Antioxidant activity of tannoid principles of Emblica officinalis (amla) in chronic stress induced changes in rat brain. Indian J Exp Biol., 38(9): ( 2000) 44. Zhang, Y.J., T. Nagao, T. Tanaka, C.R. Yang, H. Okabe and I. Kouno,. Antiproliferative activity of the main constituents from Phyllanthus emblica. Biol Pharm Bull., 27(2): ( 2004) 45. Haque R, B Bin-Hafeez, I. Ahmad, S. Parvez, S Pandey, and S Raisuddin. Protective effects of Emblica officinalis Gaertn. in cyclophosphamide-treated mice. Human and Experimental Toxicology, 20(12): (2001). 46. Banu, S.M., K. Selvendiran, J.P. Singh and D. Sakthisekaran,. Protective effect of Emblica officinalis ethanolic extract against 7, 12- dimethylbenz(a) anthracene (DMBA) induced genotoxicity in Swiss albino mice. Hum Exp Toxicol.,23(11): ( 2004) P - 140

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