K. Sivasankara Prasad et al. / Journal of Pharmacy Research 2011,4(4),1041-1043 Research Article ISSN: 0974-6943 Available online through http://jprsolutions.info Evaluation of antimutagenic activity of Cucurbita pepo linn by bone marrow micro nucleus test in swiss albino mice against mitomycin-c induced mutations

K. Sivasankara Prasad*1, R.Anandan2, K.Mahesh Kumar1, K. Ravindrareddy 1, Sudhakar.Y 3 1Department of Pharmacology, P. Rami Reddy Memorial College of Pharmacy, Kadapa, Andhra Pradesh, India. 2Department of Pharmacology, Vinayaka Missions College of Pharmacy, Salem, Tamilnadu, India. 3 Department of Pharmaceutics, Govt.Polytechnic College, Kadapa (DT), Andhra Pradesh, India. Received on: 05-12-2010; Revised on: 14-01-2011; Accepted on:09-03-2011

ABSTRACT Toxicological studies have undergone a significant evolution during the past decade, with inclusion and great emphasis on chronic toxicity, carcinogenicity, teratoge- nicity and mutagenicity. Present study was taken up to evaluate antimutagenic activity of aqueous extract of Cucurbito pepo by bone marrow micronucleus test (MNT) in mice. Mitomycin C (4 mg/kg, i.p) was used as a geno toxic challenge and bone marrow of control and Mitomycin C treated mice was collected. In MNT, the bone marrow smears were stained with May-Grunwald’s followed by Giemsa stain. Polychromatic and Normochromatic erythrocytes were counted and P/N ratio was calculated. Hence, Cucurbito pepo has significant antimutagenic activity.

Key words: Antimutagenic activity, Macronuclei test, Mitomycin C, Cucurbito pepo Linn

INTRODUCTION Traditional medicine is very valuable resource because of the long of its use and thus being 27 – hexano cucurbit -5-en -11,20- dione. 2 –O-ß-D- Glycopyranoside and 16- hydroxyl -22, 23, evidence based. It is employed extensively in developing countries for primary health care, but 24, 25, 26, 27 hexanocucurbit -5 – en-11, 20- dione 3 – O – a - 1- Rhamnopyranosyl – (1,2) – of the late has aroused increasing interest in developed countries. One it is an alternative to high ß-D- 11, Glycopyranosiderespectively[16],ß–carotene[17],ProvitaminA caroteniods[18], VitaminA[19] cost medicines for promotive and preventive health care. Second one disease conditions with ,vitamin E [20], vitamin C[21] and alkaloid such as tannin [22] . Some of the compounds have been inadequate modern drugs and also for non life threatening diseases for lower incidences side reported to have role in the protection against gastric mucosal damage. effects reported than with modern drugs. WHO defined traditional medicine as the total knowledge ,skills and practices based on the theories , beliefs and experiences indigenous to MATERIALS AND METHODS different cultures , whether explicable or not used in the maintaince of the health and in the prevention ,diagnosis, improvement or treatment of physical and mental illness. The major Chemicals and Reagents resource base of the traditional medicine is medicinal plants with the introduction of modern Mitomycin C (Biochem pharmaceutical industries Ltd, Daman), Melatonin (Aristo pharmaceu- medicine. The increasing popularity in plant based drugs is now felt all over the world leading ticals, Mumbai), Giemsa stain (Himeida labs Pvt Ltd, Mumbai), May Grunewald’s stain to a fast growing market for plant based drugs, pharmaceuticals, neutraceticals, functional foods (Himeida labs Pvt Ltd, Mumbai), Dispovan syringes (Hindustan syringes and Medical devices and even cosmaceticals[1]. Ltd, Faridabad. Carboxy Methyl cellulose (Himeida labs Pvt Ltd, Mumbai).

The term ‘antimutagen’ is used to describe the agents that reduce the frequency or rate of Plant collection and Authentication spontaneous and induced mutations by diverse mechanisms[2] It is well established that The whole fruits of Cucurbita pepo Linn belonging to the family Cucurbitaceae were collected, mutagenic and potentially Carcinogenic agents are omnipresent in the human environment and Kadapa district, Andhra Pradesh, India, in the month of may 2009. The plant was identified and it seems to be impossible to eliminate all of them. Moreover, several well-known mutagenic authenticated by the Botanist K.Madhava Shetty, Tirupathi, Andhra Pradesh. The plant scien- risk factors are closely connected with a modern lifestyle and their entire eradication appears to tific profile was mentioned in the table No.1 be very burdensome, even unattainable [3]. Therefore, there exists a need to reduce genotoxic effects of mutagenic and carcinogenic factors by the regular intake of antimutagenic agents. For Table ..1. The Cucurbita pepo plant scientific profile mentioned in the below table: this reason the antimutagenic agents should be taken commonly and continuously. The best Kingdom Plantae candidates appear to be natural diet components, taken in a sufficient antimutagenic concentra- Division Magnoliophyta Class Magnoliopsida tion during daily meals. Recent research has confirmed that many food articles contain Order Caryophyllales components that possess antimutagenic and anticarcinogenic properties [2, 4-7]. Previously we Family Cucurbitaceae described how alkylresorcinnls, natural amphiphilic compounds, commonly found in cereal Genus Cucurbita Specific epithet Pepo grains, markedly decreased mutagenic activity of several standard genotoxic agents in the Botanical name Cucurbita pepo Linn Ames test as well as in the Sister Chromatid Exchanges (SCEs) test with human lymphocytes in vitro [8]. The bran milling fraction of rye and wheat is a relatively rich source of alkylresorcinols Preparation methanolic extract from the pulp of Cucurbita pepo Linn [9]. The pulps were collected, air dried and powdered. The powdered pulp was exhaustively extracted with 95% methanol at room temperature for three days by maceration. The methanolic [10] Cucurbita Pepo Linn belonging to family cucurbitaceae commonly known as pumpkin is solution was filtered and concentrated under reduced pressure at 40° c to dryness. The semi available throughout India and consumed as vegetable in various parts of the plant of the world solid methanolic extract of Cucurbita pepo Linn was dispersed in 1%CMC suspension until .Different parts of the plant have been used as medicine ayurveda. The pulp of ripe fruit the moment of use. cucurbita pepo is used to relive intestinal inflammation or enteritis, dyspepsia [11]. And stomach disorders [12]. Its pulp used as dietary supplements for vitamin A [13] and is also used Preparation mitomycin C solution to treat liver disorders such as jaundice [14]. Extensive pharmacological investigations to Mitomycin C Solution was dissolved in distill water & administered intraperitoneally at a isolation of several active compounds syringicacid [15] cucurbitine and hexanocucurbitne glyco dose of 4 mg/kg according to the body weight of the animal. side such as cucurbitacin L 2 –O-ß-D- Glycopyranoside, 2, 6- dihydroxy -22, 23, 24, 25, 26, Preparation of Melatonin solution (Reference standard) *Corresponding author. Melatonin was dissolved in distill water and administered intreperitonially at a dose of 20 mg/ K.Sivasankar Prasad kg .Melatonin treatment started 24 hours prior to MMC administration. Normally (control) Asst.Professor, and MMC groups received the dose of an equal volume of 1 ml 1% CMC suspension Department of pharmacology, intrgastric administration (orally). P.Rami Reddy Memorial College of Pharmacy, Kadapa, Andhra Pradesh, India,

Journal of Pharmacy Research Vol.4.Issue 4. April 2011 1041-1043 K. Sivasankara Prasad et al. / Journal of Pharmacy Research 2011,4(4),1041-1043

Test procedure with a starting dose of 5mg/kg body weight and 1000 Normo chromatic erythrocytes (NCE) and the no.of micronuclei present in the serum (slides) were counted. Normal value =PCE/NCE=1:1 ratio. The slides were analyzed under binocular Olympus BH-2 microscope (10x100).

Stastical Analysis In order to analyze the antimutagenic activity of the cucurbita pepo extract, the frequency of the MNPCE from the treated groups was the treated was compared to the results obtained from the positive control goup by student’s T test, with p values lower than 0.05(P<0.05).

Table: 2. Results of consolidated DATA of PCE, NCE, Micronucleus, Mitomycin –C, Melatonin

Cucurbita pepo linn Cucurbita pepo linn Cucurbita pepo linn extract 100mg/kg+ MMC extract 200mg/kg+ MMC extract 400mg/kg+ MMC

Slide : 1-3 Slide : 1-3 Slide : 1-3 PCE NCE MN N/P Ratio PCE NCE MN N/PRatio PCE NCE MN N/PRatio 1031 969 12 0.939 1031 969 8 0.939 1031 969 5 1.164 924 1076 9 1.164 901 1099 6 1.143 924 1076 7 0.939 1089 929 8 0.853 924 1076 9 1.164 1231 769 4 0.624

MELATONIN 20mg/kg MMC(4 mg/kg) Normal control (CMC suspension) + MMC (4 mg/kg)

Slide : 1-3 Slide : 1-3 Slide : 1-3 PCE NCE MN N/PRatio PCE NCE MN N/PRatio PCE NCE MN N/PRatio 1021 979 4 0.958 1039 969 17 1.072 1081 929 1 0.859 945 1051 6 1.112 924 1076 09 1.164 901 1099 2 1.219 1103 897 4 0.813 1139 961 10 0.843 1039 961 1 0.924 per step 3 animals of a single sex (normally females) are used a: Unclassified 0,1,2,3;Number of mentioned or dead animals at each step Testing at 5000mg/kg b.w.: see Annex.2. Micronucleus test photographs GHS: Globally Hamonised classification system (mg/kg b.w.) Figure: 1 OECD Guide line chart

Experimental animals 20 female swiss albino mice obtained from the facility, Sugen life sciences were used for the study. All mice were certified for good health at receiving time .Age of the start of the treatment was approximately 8 to 12 weeks and kept at air-controlled rooms. Animal experiments were conducted according to guidelines and following the approval of the Institutional Animal Ethical Committee. Four groups, consisting of five animals each were used for the study.

Acute toxicity study Acute toxicity study was done according to OECD (Organization for Economic Co-operation and Development) Guideline 423. Toxicity study was performed up to the dose level of 2000 mg/kg, No mouse died. So the LD 50 was 2000 mg/kg. And the ED 50 was 1/10 the of LD 50 was 2000/10 =200 mg/kg. OECD Guide line chart mentioned in the figure No: 1 Figure 2:CONTROL (CMC) Figure 3 :MMC (4mg/kg) Induced Micronuclei

ANTIMUTAGENIC ACTIVITY Animals were divided in to five groups as control (n=6), Mitomycin C inducing agent (n=6), Mitomycin C+ Melatonin (standard drug) (n=6), Mitomycin C + Methanolic extract of Cucurbita pepo 100 mg/kg bw p.o (n=6), Mitomycin C + Methanolic extract of Cucurbita pepo 200 mg/kg bw p.o (n=6), and Mitomycin C + Methanolic extract of Cucurbita pepo 400 mg/kg bw p.o (n=6), Where n was the number of animals included in the study.

Treatment Protocol GROUP 1: Normal control (1 ml of 1% CMC).p.o GROUP 2: Mitomycin-C 4 mg/kg (mutations inducing agent) i.p. GROUP 3: Melatonin 20 mg/kg (Standard reference as an antimutagenic) i.p. +Mitomycin-C (4mg/kg) i.p Figure 4 MMC (4mg/kg) + Melatonin (20 mg/kg) Figure 5Cucurbita pepo (100 mg/kg) + MMC (4mg/kg) GROUP 4: Methanolic extract of Cucurbita pepo Linn.(100 mg/kg). +Mitomycin-C (4 mg/kg). GROUP 5: Methanolic extract of Cucurbita pepo Linn. (200 mg/kg). +Mitomycin-C (4 mg/kg). GROUP 6: Methanolic extract of Cucurbita pepo Linn. (400 mg/kg). + Mitomycin-C (4 mg/kg).

Micronucleus Test (As per OECD guide line no: TG 473) The respective groups of mice were collected from the animal cage. The mice were sacrificed by cervical dislocation method. The femur bone was isolated after removing the muscles surrounding it. Both ends of the femur were cut, the femur bone was completely dislocated from mice. The excess muscles adhering to femur bone were grafted with grafting scissor. The upper end of the femur girdle was removed with scissor, to make a small orifice upon it. The foetal bovine serum was collected in the respective 1ml syringes. The foetal bovine serum was aspirated into the respective eppendroff tube through orifice of the femur bone. The tubes were centrifuged the with 2000 rpm for 10 minutes. The supernatant liquid was removed from the tubes; the remaining serum was mixed well with water pillar tube. Thin smears on slides Figure 6 Cucurbita pepo (200mg/kg) +MMC Figure 7Cucurbita pepo (400mg/kg) + MMC were prepared. The slides were kept aside for overnight. (4mg/kg) (4mg/kg)

Staining procedure Three coupling jars were taken. 1st coupling jar: Containing conc, macgrownolds stain in which the slides were kept for RESULTS 3 minutes. The obtained results of the control group (Mentioned in table no: 2, figure 2), inducing group 2nd coupling jar: Macgrownolds stain (1:1 dilution), The slides were kept for 2 minutes. Mitomicyin -C (4mg/kg per day each) orally (Mentioned in the table no: 2, figure 3) ,standard 3rd coupling jar: Giemsa stain (1:6 dilution), The slides were kept for 10 minutes group (MMC (4mg/kg) + Melatonin (20 mg/kg) (Mentioned in the table no: 2, figure 4), extract After staining the slides, were dried for few minutes ,1000 polychromatic erythrocytes (PCE) of Cucurbita pepo Linn co-treated with 4mg.kg-1 of MMC the results shows that the lowest dose 1041-1043 Journal of Pharmacy Research Vol.4.Issue 4. April 2011 K. Sivasankara Prasad et al. / Journal of Pharmacy Research 2011,4(4),1041-1043 (100mg/kg + 4mg/kg MMC) (Mentioned in table no: 2, figure 5), the Cucurbita pepo did not REFERENCES modulate the mutagenic activity of MMC. However the antimutagenic effect was strongly 1. Akerele O., summary of WHO guide line for the assessment of the herbal medicines, herbal gram, observed for all others doses tested (200 & 400mg.kg of Cucurbita pepo extract + 4mg.kg of 1993, Vol 28 pages 13-19 2. M.D. Water, H.F. Stack, M.A. Jackson, H.E. Brockman, S. De Flora, Activity profiles of antimutagens: MMC) (Mentioned in table no: 2, figure 6, 7). in vitro and in vivo data, Mutat. Res. 350 1996, 109-129. 3. S. De Flora, Problems and prospects in antimutagenesis and anticarcinogenesis, Mutat. Res. 202 The inhibitory effect of Cucurbita pepo linn against mutagenicity induced by Mitomycin – C ,1988, 279-283. 4. L.W. Wattenberg, Inhibition of carcinogenesis by minor dietary constituents, Cancer Res. 52 is shown in Tables 3. In the present study, Cucurbita pepo showed time dependent inhibitory (Suppl.) (1992) 20855- 20915. effect on the frequency of MN in PCE as well as NCE. Decrease in P/N ratio due to Mitomycin 5. B. Stavric, Role of chemo preventers in human diet, Clin. Biochem. 27 (1994) 319-332. – C was also inhibited by Cucurbita pepo linn. 6. L.W. Wattenberg, Chemoprevention of cancer, Cancer Res. 1985, 45 , 1-7. 7. G. Block, B. Patterson, A. Subar, Fruit, vegetables and cancer prevention: a review of epidemio- logical evidence, Nutr. Cancer 1992, 18, l-29. Table 3: Mean Micro nucleated Polychromatic Erythrocytes (MNPCE) 8. K. Qsiorowski, K. Szyba, J.B. Brokos, A. Kozubek, Antimutagenic activity of alkylresorcinols from cereal grains, Cancer Lett. 1996, 106 109-I 15. GROUP DOSE LEVEL SEX MNPCE mean±SD (n=6) 9. Kozubek, J.H.P. Tyman, Cereal grain resorcinolic lipids: mono and dienoic homologues are present in rye grains, Chem. Phys. Lipids 1995,78 , 29-35. Control 0.0 mg/kg Male 1.33 ± 0.58 10. Paris HS , Yonash N, Portnoy V, Mozes- Daube N, Tzuri G & kaatzir N assessment of gentic Pulp. Extract +MMC 100mg/kg +4 mg/kg Male 9.67 ± 2.08 relationships in cucurbita pepo using in DNA markers , J Theroetical Appl Gen, 2003 106-971 Pulp. Extract + MMC 200mg/kg +4 mg/kg Male 7.67 ± 1.53 11. Orlandelli M, Further results of treatment of dyspepsia and enteritis with pumpkin pulp, clin pediatr Pulp. Extract + MMC 400mg/kg + 4mg/kg. Male 5.33 ± 1.52 , 1951, 33(2) ,69. MMC 4 mg/kg Male 12.00 ± 4.36 12. Lanc C, Comparision of plants used for skin and stomach problems in Trinidad and Tobago with Melatonin+ MMC 20 mg/kg + 4mg/kg Male 4.67 ± 1.15 asian ethnomedcine, J ethnobiol ethnomed, 2007, 33. 13. Kune GA, Bannerman S , Field B, Watson LF, Cleland H, Merenstein D, & Vietta L diet,alcohol, n=6, Values are expressed in Mean ± SEM, One way ANOVA followed by student’s T test 0.05Vs smoking, serum beta-carotene , and vitamin A in male nonmelanocytic skin cancer patients and controls, Nutr cancer 1992, 18(3) 237. Control, *(P<0.05) Vs Mitomycin - C. 14. Sezik E, Yesilada E Shadidoyatov H, Kulivey Z, Nigmatullaev A M, Apriov HN, Takaishi Y,Takeda Y & Honda G , Folk mecine in Uzbekistan I Toshkent, Djizzax, and samarqand provinces, J DISCUSSION ethnopharmacol, 2004, 92,197. 15. Dragovic –Uzelac V, Deloonga K,Levaj B,Djakovic S & Popisil J ,Phenolic profilies of raw The aim of the work was to evaluate the mutagenic activity methanolic extract of Cucurbita apricots, Pumpikn, and their purees in the evaluation of apricots, Nectar and jam authenticity, J pepo linn by mice bone marrow micronucleus test. This is a short term assay which has been afric Food Chem , 2005,53,12,4836. widely employed to detect the antimutagenic activity of substances in vivo .Micro nuclei 16. Wang DC, Pan HY, Deng XM, XiangH, Gao HY, Cai H & Wu LJ cucurbitane and Hexanorcucurbitane glycosides from the fruits of cucurbito pepo, CV dayanguna, J asian Nat Prod Res, 2007,9(6) ,525. separated from in addition to the main nucleus a cell are the results of a centric chromosomes 17. Veda S, Kamath A, Platel K, Begum K & srinivasan K, Determination of bioaccessbility of beta- or lagging chromosomes that fail to incorporate into either of daughter nuclei during the caratone in vegetables by in vitro methods, Mol Nutr Food Res, 2006 ,50 (11) 1047. telophase of the miotic cells. The frequency of the MN in poly chromatic erythrocytes of mice 18. Azevedo-Melerio CH & Rodriguez-Amaya DB, Qualitative and quantitative in carotenoid com- bone marrow is a very sensitive index of damaging produced by ionizing radiation and by position cucurbito moschata, cucurbita maxima, and cucurbita pepo, J Agri Food Chem,55 (10) 4027. chemical mutagens. The test results some advantages compared to other kind of assays, in 19. Lans C ,Turner N, Khan T, Breuer G& Boepple W, Ethnoveterianary medicines used for ruminants which we mainly mention the low cost and reliabity. In addition this assay utilizies mamma- in british Columbia, Canada, J ethnobiol Ethnomed, 2007,3 ,11. lians, in which present capacity of metabolism similar to humans that hardly can reproduced 20. Tadmor Y, Paris Hs, Meir A, Schaffer AA, & lewinsohn E, Dual role of the pigmentation gene B in affecting carotenoid and vitamin E content in squash mesocarp (cucurbito pepo), J Agri Food in totally in vitro assays. Chem, 2005,53 (25) 9759. 21. Hancock RD, McRae D, Haupt S & viola R , Syntheis of L-ascorbic acid in the phloem ,Plant ACKNOWLEDGEMENT boil , 2003,37. 22. Silveria NA, Alvarez –Leite J I ,Siliva ME, Viera LQ, Bambirra EA & Vieira EC , Effect of high We are thankful to Management of vinayaka mission’s College of Pharmacy, Salem, Tamilnadu ingestion of dehydrated pumpikin ( cucurbito pepo) on the cholesterol metabolism and on the for the providing the all facilities for carried out this work. hepatic histology in mice , Arquivos de Biologiae Tecnologia , 1996 , 39 ,961.

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

Journal of Pharmacy Research Vol.4.Issue 4. April 2011 1041-1043