ISSN 0976-111X

INTERNATIONAL JOURNAL OF PHARMA WORLD RESEARCH

(An International Quarterly Published Online Research Journal )

www.ijpwr.com Title: A STUDY ABOUT ANTIOXIDANT & ANTICANCER ACTIVITY OF ACTIVITY OF METHANOLIC EXTRACT OF SQUID, SEPIA BREVIMANA AND SEPIELLA INERMIS
C. Ilamparithi1, Anto Shering. M1, Brito Raj. S2, S.Kavimani1 1 Mother Therasa Post Graduate & Research Institute of Health Sciences, Puducherry. 2 Sri Venkateswara Colleges of Pharmacy, R.V.S.Nagar, Chittoor.

E-mail:editorijpwr@gmail.com

Correspondence to the author: C.Ilamparithi M.Pharm, Mother Therasa Post Graduate & Research Institute of Health Sciences, Puducherry E.mail: parithipharma@gmail.com

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ABSTRACT: Marine life is a vast resource, providing food, medicine and raw material all over the world. Methanolic extract obtained from flesh of fishes Squid, Sepia brevimana and Sepiella inermis contains fish oil, and in turn fish oil contains -3 unsaturated fatty acid is already been proved for its anti-cancer activity particularly breast, colon and prostate cancer. The fishes we selected for testing have -3 unsaturated fatty acid. In the present study was carried out comparative invitro anti oxidant activity using DPPH method. The main characteristic of an antioxidant is its ability to trap free radicals. We found that the Methanolic extract have the anti oxidant activity while comparing with the reference Ascorbic acid. The anti cancer study is carried out by Dye exclusion method using Daltons ascites lymphoma (DAL) cells. The methanolic extract of flesh of fishes shows significant free radical scavenging activity. It is also demonstrated the significant cytotoxicity activity at the tested dose level & the order potency is Sepia brevimana > Sepiella innermis > Squid.

KEY WORDS: Squid, Sepia brevimana, Sepiella inermis, Daltons ascites lymphoma method, DPPH method.

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I.

INTRODUCTION: Marine life is a vast resource, providing food, medicine and raw material, in addition to

helping to support recreation and tourism all over the world. Marine invertebrates and micro organisms have yielded substantially more bioactive natural products than seaweeds have, unlike the terrestrial environments, where plants are considerably richer in secondary metabolites 1, among the first bio-active compounds from marine sources, spongouridine and spongothymidine from the Caribbean sponge (Cryptotheca crypta) were isolated serendipitously in early 1950s. They were approved as an anticancer drug (cytosine, arabinoside, Ara-C) and an antiviral drug (Adenine arabinoside, Ara-C) respectively, 15 years later2. The secondary metabolites of marine organisms have been studied extensively over the past of 30 years, since a small number of academic chemists began to isolate and elucidate novel compounds from marine source in 1970s. Depth range from 10 to 100 m. Sepia brevimana is a shelf species, restricted to coastal waters. Around India, spawning occurs throughout the year. Several spawning peaks have been observed between July and February in eastern Indian waters. Sepiella inermis is a demersal, shallow water species found to 40 m depth. Squid are marine cephalopods of the order Teuthida, which comprises around 300 species. I.a. Antioxidant Activity: The main characteristic of an antioxidant is its ability to trap free radicals. Highly reactive free radicals and oxygen species are present in biological systems from a wide variety of sources. These free radicals may oxidize nucleic acids, proteins, lipids or DNA and can initiate degenerative disease. Antioxidant compounds like phenolic acids, polyphenols and flavanoids svavenge free
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radicals such as peroxide, hyperoxide or lipid peroxyl and thus inhibits the oxidative mechanisms that lead to degenerative diseases. Reactive oxygen species (ROS) such as superoxide anions, hydrogen peroxide hydroxyl and nitric oxide radicals, play an important role in oxidative stress related to the pathogenesis of various important diseases. Antioxidants act as a major defense against radical mediated toxicity by protecting the damages caused by free radicals. Antioxidant agents are effective in the prevention and treatment of complex diseases, like atherosclerosis, stroke, diabetes, Alzheimers disease and cancer 3. The various analytical methods used to measure the radical scavenging activity of antioxidants against free radicals are 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, the superoxide anion radical (O2), the hydroxyl radical (OH), peroxyl radical (ROO), the malondialdehyde (MDA) or thiobarbituric acid reactive substances (TBARS) assay. I.b. Determination of Antioxidant by DPPH Method:

A simple method that has been developed to determine the antioxidant activity utilizes the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. The structure of DPPH and its reduction by an antioxidant are shown above. The odd electron in the DPPH free radical gives a strong absorption maximum at 517 nm and is purple in color. The color turns from purple to yellow as the molar
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absorptivity of the DPPH radical at 517 nm reduces from 9660 to 1640 when the odd electron of DPPH radical becomes paired with hydrogen from a free radical scavenging antioxidant to form the reduced DPPH-H. The resulting decolorization is stoichiometric with respect to number of electrons captured. I.c. Evaluation of Cytotoxic Activity: The Cytotoxic activity of the Methanolic extract obtained from flesh of fishes Squid, Sepia brevimana and Sepiella inermis is carried out by Dye exclusion method using Daltons ascites Lymphoma cells bearing mice. This is an invitro method. The cells obtained from the mice were aspirated from the peritoneal cavity of tumor bearing mice. The cells were washed and diluted Phosphate buffer saline. The viability of the cells was checked using trypan blue. The number of cells in the 10-3 dilution was counted using a haemocytometer and the numbers of cells were adjusted to 1X106 cells/ml. The percentage cytotoxicity was calculated5. II. MATERIALS & METHODS:

II.a. Invitro Antioxidant Activity of the Methanolic Extraction: Test compound stock solution was diluted to final concentrations of 20, 40, 60, 80 and 100 g ml-1 in water, standard (Ascorbic acid) was diluted to final concentrations of 2, 4, 6, 8 and 10 g ml-1 in water. DPPH methanol solution (1ml, 0.1mmol) was added to 2.5 ml of drug solutions of different concentrations and allowed to react at room temperature. After 30 min the absorbance values were measured at 517 nm and converted into the percentage antioxidant activity. Methanol was used as the solvent and ascorbic acid as the standard 4.

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II.b. Evaluation of Cytotoxic Activity: Daltons ascites lymphoma cells bearing mice were obtained from Amala Cancer Research centre, Thrissur, Kerala. The cells were aspirated from the peritoneal cavity of tumor bearing mice. The cells were washed three times with Phosphate buffer saline [sodium chloride 8g, potassium chloride 0.2g, disodium hydrogen phosphate 1.44g, potassium dihydrogen phosphate 0.2g, distilled water up to 1000 ml]. The viability of the cells were checked using trypan blue (cell viability should above 98%) and different dilutions of 10 -1, 10-2 and 10-3 were made. The number of cells in the 10 -3 dilution was counted using a haemocytometer and the numbers of cells were adjusted to 1X10 6 cells/ml. The experiment was set up by incubating different concentrations of alcoholic extract (10, 20, 50, 100 and 200 g/ml) with 1X106 cells/ml. The final volume of the assay mixture was made up to 1 ml using Phosphate buffer saline and was incubated at 37C for 3 hours. 0.1 ml of Trypan blue solution was added after incubation period and the number of dead cells was counted using a haemocytometer5. Counting of non- viable cells randomly in every 100 cells is an indicative of anti-tumor activity. The percentage cytotoxicity was calculated by % Cytotoxicity = Number of dead cells Number of viable cells + Number of dead cells x 100

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III. RESULT & DISCUSSION

III.a. Anti Oxidant Activity: DPPH is a stable free radical available commensally and having characteristic absorption at 517 nm due to unpaired electron. The DPPH radical reacts with compounds having antioxidant activity and gets reduced, the reduced form is colorless. Hence the decrease in the absorbance of Ascorbic acid has been reported to be an effective antioxidant and free radical scavenger. Both in invitro and invivo conditions, it reduces oxidative and free radical induced damages6. The result were comparable to standard drug ascorbic acid which shows 98%, Squid and Sepia brevimana shows 79% where on Sepiella inermis shows 68% of free radical scavenging activity.

The percentage antioxidant activity (% inhibition) is presented in Table No 1.

Table No: 1 Anti Oxidant of methanolic extract of Squid, Sepia brevimana and Sepiella inermis

SL.NO.

COMPOUND USED

% INHIBITION AT A CONCENTRATION (g/ml) 20 35.7 28.8 15.7 78.46 40 48 42.8 33.4 95.58 60 63 56.5 56 96.40 80 76.7 76.9 63.8 97.52 100 79.5 79.36 68.4 98.43

1 2 3 4

Squid Sepia brevimana Sepiella inermis Standard (Ascorbicacid)

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III.b. Cytotoxic Activity: The present study demonstrated the significant cytotoxicity activity at the tested dose level. The results of effect of methanolic extract of Squid, Sepia brevimana and Sepiella inermis on Daltons ascites lymphoma cells were shown in table no. 2 and figure no. 1 The order potency was found to be Sepia brevimana > Sepiella innermis > Squid at the end of sixth hour.

Table 2: Effect of methanolic extract of Squid, Sepia brevimana and Sepiella inermis on DAL cells

Sl.No

Time (Hours) Squid

% Cytotoxicity Sepia brevimana 0 2 6 18 36 66 82 Sepia inermis 0 1 4 16 28 51 70

1 2 3 4 5 6 7

0 1 2 3 4 5 6

0 8 15 20 29 48 55

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Fig 1: Effect of methanolic extract of Squid, Sepia brevimana and Sepiella inermis on DAL cells

90 80 70

60
50

40
30 20 10 0 0 1 2 3 4 5 6

Time (Hours)
Squid Sepia brevimana Sepia brevimana

IV.

CONCLUSION Many compound derived from various natural resources have been found to have anti-

angiogenesis and anti-tumor activities using both invitro and invivo models. These compounds include extracts and fraction from cartilage, fish oil etc7. Among n-3 fatty acids [omega-3], neither long-chain nor short-chain forms were consistently associated with breast cancer risk. High levels of docosahexaenoic acid, however, the most abundant n-3 PUFA [omega-3] in erythrocyte membranes, were associated with a reduced risk of breast cancer. The future studies include the separation of components which are responsible from cytotoxic activity and going for clinical trials to use this for human use.
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V. REFERENCE

1. Faulkner D J (2002). Marine natural products. Nat Prod Rep. 19, 1-48. 2. Newman D J and Cragg G M (2004). Marine natural products and related compounds in clinical
and advanced preclinical trials. J Nat Prod. 67, 1216-1238..

3. T P A Devasagayam, J C Tilak, K K Boloor, K S Sane, S S Ghaskadbi, R D Lele. J.

Assoc.Phys. India., 52, 794-804, (2004).

4. Rajasekaran S, GopalKrishna Rao, Sanjay Pai P N and Gurpreet Singh Sodhi, Synthesis,
Antibacterial and invitro Antioxidant Activity of 2,3-Substituted Quinazolin-4(3H)-ones, J. Chem. Pharm. Res., 2010, 2(1): 482-488

5. Gopakumar G, Viji MO, Nair CKK. Radiosensitization of murine tumor (Daltons

ascites lymphoma) cells with Iron oxide Sanazole (Fe3O4 - AK 2123) nanoparticle complex. Amala Research Bulletin 2008; 28: 105-10.

6. Dani Mathew M, Kagiya V.T and C.K.K. nair, Ascorbic acid monoglucoside as antioxidant and
Radio protector, Amala research Buletin, vol.26,168.(2006)

7. Lee, A., and Langer, R., Shark cartilage contains inhibitors of tumor angiogenesis.
Science 1993; 221: 1185

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