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Research Article ISSN: 0974-6943

K. Sivasankara Prasad et al. / Journal of Pharmacy Research 2011,4(4),

Available online through www.jpronline.info Evaluation of antimutagenic activity of Cucurbita pepo linn by bone marrow micro neclues 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 Department of Pharmacology, P. Rami Reddy Memorial College of Pharmacy, Kadapa, Andhra Pradesh, India. 2 Department of Pharmacology, Vinayaka Missions College of Pharmacy, Salem, Tamilnadu, India. 3 Department of Pharmaceutics, Govt.Polytechnic College, Kadapa (DT), Andhra Pradesh, India.
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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, teratogenicity 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-Grunwalds 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 evidence based. It is employed extensively in developing countries for primary health care, but of the late has aroused increasing interest in developed countries. One it is an alternative to high cost medicines for promotive and preventive health care. Second one disease conditions with inadequate modern drugs and also for non life threatening diseases for lower incidences side 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 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 resource base of the traditional medicine is medicinal plants with the introduction of modern medicine. The increasing popularity in plant based drugs is now felt all over the world leading to a fast growing market for plant based drugs, pharmaceuticals, neutraceticals, functional foods and even cosmaceticals[1]. The term antimutagen is used to describe the agents that reduce the frequency or rate of spontaneous and induced mutations by diverse mechanisms[2] It is well established that mutagenic and potentially Carcinogenic agents are omnipresent in the human environment and it seems to be impossible to eliminate all of them. Moreover, several well-known mutagenic risk factors are closely connected with a modern lifestyle and their entire eradication appears to 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 this reason the antimutagenic agents should be taken commonly and continuously. The best candidates appear to be natural diet components, taken in a sufficient antimutagenic concentration during daily meals. Recent research has confirmed that many food articles contain components that possess antimutagenic and anticarcinogenic properties [2, 4-7]. Previously we described how alkylresorcinnls, natural amphiphilic compounds, commonly found in cereal grains, markedly decreased mutagenic activity of several standard genotoxic agents in the 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 [9]. Cucurbita Pepo Linn belonging to family cucurbitaceae [10] commonly known as pumpkin is available throughout India and consumed as vegetable in various parts of the plant of the world .Different parts of the plant have been used as medicine ayurveda. The pulp of ripe fruit 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 to treat liver disorders such as jaundice [14]. Extensive pharmacological investigations to isolation of several active compounds syringicacid [15] cucurbitine and hexanocucurbitne glyco side such as cucurbitacin L 2 O--D- Glycopyranoside, 2, 6- dihydroxy -22, 23, 24, 25, 26, 27 hexano cucurbit -5-en -11,20- dione. 2 O--D- Glycopyranoside and 16- hydroxyl -22, 23, 24, 25, 26, 27 hexanocucurbit -5 en-11, 20- dione 3 O a - 1- Rhamnopyranosyl (1,2) -D- 11, Glycopyranosiderespectively[16],carotene[17],ProvitaminA caroteniods[18], VitaminA[19] ,vitamin E [20], vitamin C[21] and alkaloid such as tannin [22] . Some of the compounds have been reported to have role in the protection against gastric mucosal damage. MATERIALS AND METHODS Chemicals and Reagents Mitomycin C (Biochem pharmaceutical industries Ltd, Daman), Melatonin (Aristo pharmaceuticals, Mumbai), Giemsa stain (Himeida labs Pvt Ltd, Mumbai), May Grunewalds stain (Himeida labs Pvt Ltd, Mumbai), Dispovan syringes (Hindustan syringes and Medical devices Ltd, Faridabad. Carboxy Methyl cellulose (Himeida labs Pvt Ltd, Mumbai). Plant collection and Authentication The whole fruits of Cucurbita pepo Linn belonging to the family Cucurbitaceae were collected, Kadapa district, Andhra Pradesh, India, in the month of may 2009. The plant was identified and authenticated by the Botanist K.Madhava Shetty, Tirupathi, Andhra Pradesh. The plant scientific profile was mentioned in the table No.1
Table ..1. The Cucurbita pepo plant scientific profile mentioned in the below table:
Kingdom Division Class Order Family Genus Specific epithet Botanical name Plantae Magnoliophyta Magnoliopsida Caryophyllales Cucurbitaceae Cucurbita Pepo Cucurbita pepo Linn

Preparation methanolic extract from the pulp of Cucurbita pepo Linn 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 solution was filtered and concentrated under reduced pressure at 40 c to dryness. The semi solid methanolic extract of Cucurbita pepo Linn was dispersed in 1%CMC suspension until the moment of use. Preparation mitomycin C solution Mitomycin C Solution was dissolved in distill water & administered intraperitoneally at a dose of 4 mg/kg according to the body weight of the animal. Preparation of Melatonin solution (Reference standard) Melatonin was dissolved in distill water and administered intreperitonially at a dose of 20 mg/ kg .Melatonin treatment started 24 hours prior to MMC administration. Normally (control) and MMC groups received the dose of an equal volume of 1 ml 1% CMC suspension intrgastric administration (orally).

K.Sivasankar Prasad Asst.Professor, Department of pharmacology, P.Rami Reddy Memorial College of Pharmacy, Kadapa, Andhra Pradesh, India, Tel.: 07702765184. E-mail: sivasankar_prasad@rediffmail.com

Journal of Pharmacy Research Vol.4.Issue 4. April 2011

K. Sivasankara Prasad et al. / Journal of Pharmacy Research 2011,4(4),


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 students 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 extract 100mg/kg+ MMC Slide : 1-3 PCE NCE 1031 969 924 1076 1089 929 MN 12 9 8 N/P Ratio 0.939 1.164 0.853 Cucurbita pepo linn extract 200mg/kg+ MMC Slide : 1-3 PCE NCE 1031 969 901 1099 924 1076 MMC(4 mg/kg) Slide : 1-3 PCE NCE 1039 969 924 1076 1139 961 MN 8 6 9 N/PRatio 0.939 1.143 1.164 Cucurbita pepo linn extract 400mg/kg+ MMC Slide : 1-3 PCE NCE 1031 969 924 1076 1231 769 MN 5 7 4 N/PRatio 1.164 0.939 0.624

MELATONIN 20mg/kg + MMC (4 mg/kg) Slide : 1-3 PCE NCE 1021 979 945 1051 1103 897 per step 3 animals of a single sex (normally females) are used 0,1,2,3;Number of mentioned or dead animals at each step GHS: Globally Hamonised classification system (mg/kg b.w.) : Unclassified Testing at 5000mg/kg b.w.: see Annex.2. MN 4 6 4 N/PRatio 0.958 1.112 0.813

Normal control (CMC suspension) Slide : 1-3 PCE NCE 1081 929 901 1099 1039 961

MN 17 09 10

N/PRatio 1.072 1.164 0.843

MN 1 2 1

N/PRatio 0.859 1.219 0.924

Micronucleus test photographs

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 Figure 2:CONTROL (CMC) 50 was 2000/10 =200 mg/kg. OECD Guide line chart mentioned in the figure No: 1 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).

Figure 3 :MMC (4mg/kg) Induced Micronuclei

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 were prepared. The slides were kept aside for overnight. (4mg/kg) Staining procedure Three coupling jars were taken. 1st coupling jar: Containing conc, macgrownolds stain in which the slides were kept for 3 minutes. 2nd coupling jar: Macgrownolds stain (1:1 dilution), The slides were kept for 2 minutes. 3rd coupling jar: Giemsa stain (1:6 dilution), The slides were kept for 10 minutes After staining the slides, were dried for few minutes ,1000 polychromatic erythrocytes (PCE)

Figure 7Cucurbita pepo (400mg/kg) + MMC (4mg/kg)

RESULTS The obtained results of the control group (Mentioned in table no: 2, figure 2), inducing group Mitomicyin -C (4mg/kg per day each) orally (Mentioned in the table no: 2, figure 3) ,standard group (MMC (4mg/kg) + Melatonin (20 mg/kg) (Mentioned in the table no: 2, figure 4), extract of Cucurbita pepo Linn co-treated with 4mg.kg-1 of MMC the results shows that the lowest dose

Journal of Pharmacy Research Vol.4.Issue 4. April 2011

K. Sivasankara Prasad et al. / Journal of Pharmacy Research 2011,4(4),


(100mg/kg + 4mg/kg MMC) (Mentioned in table no: 2, figure 5), the Cucurbita pepo did not modulate the mutagenic activity of MMC. However the antimutagenic effect was strongly observed for all others doses tested (200 & 400mg.kg of Cucurbita pepo extract + 4mg.kg of MMC) (Mentioned in table no: 2, figure 6, 7). The inhibitory effect of Cucurbita pepo linn against mutagenicity induced by Mitomycin C is shown in Tables 3. In the present study, Cucurbita pepo showed time dependent inhibitory effect on the frequency of MN in PCE as well as NCE. Decrease in P/N ratio due to Mitomycin C was also inhibited by Cucurbita pepo linn.
Table 3: Mean Micro nucleated Polychromatic Erythrocytes (MNPCE)
GROUP Control Pulp. Extract +MMC Pulp. Extract + MMC Pulp. Extract + MMC MMC Melatonin+ MMC DOSE LEVEL 0.0 mg/kg 100mg/kg +4 mg/kg 200mg/kg +4 mg/kg 400mg/kg + 4mg/kg. 4 mg/kg 20 mg/kg + 4mg/kg SEX Male Male Male Male Male Male MNPCE meanSD (n=6) 1.33 0.58 9.67 2.08 7.67 1.53 5.33 1.52 12.00 4.36 4.67 1.15

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n=6, Values are expressed in Mean SEM, One way ANOVA followed by students T test 0.05Vs Control, *(P<0.05) Vs Mitomycin - C.

14. 15. 16. 17. 18. 19. 20. 21.

DISCUSSION The aim of the work was to evaluate the mutagenic activity methanolic extract of Cucurbita pepo linn by mice bone marrow micronucleus test. This is a short term assay which has been widely employed to detect the antimutagenic activity of substances in vivo .Micro nuclei separated from in addition to the main nucleus a cell are the results of a centric chromosomes or lagging chromosomes that fail to incorporate into either of daughter nuclei during the telophase of the miotic cells. The frequency of the MN in poly chromatic erythrocytes of mice bone marrow is a very sensitive index of damaging produced by ionizing radiation and by chemical mutagens. The test results some advantages compared to other kind of assays, in which we mainly mention the low cost and reliabity. In addition this assay utilizies mammalians, in which present capacity of metabolism similar to humans that hardly can reproduced in totally in vitro assays. ACKNOWLEDGEMENT We are thankful to Management of vinayaka missions College of Pharmacy, Salem, Tamilnadu for the providing the all facilities for carried out this work.

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

Journal of Pharmacy Research Vol.4.Issue 4. April 2011

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