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Evaluation of Antioxidant and Chemopreventive Effect of Cocos Nucifera L.

Shrabanti Dev*
Pharmacy Discipline, Khulna University, Khulna-9208, Bangladesh

Anha Afrin Shefa
Pharmacy Discipline, Khulna University, Khulna-9208, Bangladesh

Archana Mandal
Pharmacy Discipline, Khulna University, Khulna-9208, Bangladesh

Projit Roy Gayen
Pharmacy Discipline, Khulna University, Khulna-9208, Bangladesh

Kaniz Asma
Pharmacy Discipline, Khulna University, Khulna-9208, Bangladesh

Md. Abdullah Al Bari
Pharmacy Discipline, Khulna University, Khulna-9208, Bangladesh

Masum Shahriar
Department of Pharmacy, Jahangirnagar University, Savar, Dhaka-1342, Bangladesh

Asish Kumar Das
Pharmacy Discipline, Khulna University, Khulna-9208, Bangladesh

Abstract/ Executive Summary:

This study revealed the antioxidant and anticancer activities of the ethanolic extract of the tender fruits of Cocos nucifera. In antioxidant screening, IC50 value was found to be 7.71µg/mlfor ascorbic acid and44.67 µg/ml for C. nucifera fruits. The phenolic content, total flavonoid and total tannin content were 537.89 mg GAE/100 gm, 40.69mg of QE/100 gm and 44.61 mg of GAE/100 gm of dry powder of C. nucifera respectively. In DMBA and croton oil-induced skin cancer in model mice,the extract significantly decreased the number, size, yield and burden of tumor when compared with carcinogenic control. The extract increased the level of natural antioxidants like GSH, SOD and Catalase. Moreover, substantial decrease in SGPT, SGOT (liver activity marker) was observed at different doses of the crude extract. Lipid profile of mice treated with C. nucifera was brought back to normal level to some extent when compared to carcinogenic control. In conclusion,C. nucifera fruits have antioxidant activity as well as preventive role on cancer initiation and propagation without major toxicity.

Keywords: Cocos nucifera, DMBA, Croton oil, GSH, SOD, Catalase.

Location: Pharmacy Discipline, Khulna University, Khulna-9208, Bangladesh

Start Date:

End Date:

Program Area: Resource Development and Management

Commodity: Coconut

Objectives:

The aim of the present study is to elucidate the antioxidant and chemopreventive activity of C. nucifera tender fruit extract.

Methodology:

Sample Collection: The fruits of Cocos nucifera were collected from surrounding area of Khulna university. Drying & Grinding: The collected fruits were air dried for three weeks and were ground into a fine powder with the help of a suitable grinder. The powder was stored in an airtight container and kept in a cool, dark and dry place until analysis commenced. Extraction: Dried fruit powder sample of 400g was taken in 1500 ml of 99% ethanol and kept in air tight bottle. After 7 days ethanol was filtered by What man No. 1 filter paper. The filtrate wasdried with rotary evaporator at 40⁰C and kept in the refrigerator for further use. The yield was 11.2% of the dry weight of powder. Test Animals: Male Swiss albino mice (6-7 weeks) were used for this study. Mice were maintained in animal house at temperature 22 ± 3⁰ C and 12 hour light and dark cycle. Mice were house in polypropylene cage. Standard food and water were provided ad libitum. All experiments were performed following the standard guidelines developed by the ethical committee of Khulna University, Bangladesh. Chemicals: Ascorbic acid, Folin-Ciocalteu’s phenol reagent, (+)-Catechin, Gallic acid, Super oxisde dismutase (SOD), Catalase, 7,12-Dimethyl Benzanthracene (DMBA) and croton oil were purchased from Sigma-Aldrich Co. (St. Louis,MO). DPPH was purchased from Wako Pure Chemical Industry Ltd., Osaka, Japan. All other chemicals and reagents were of analytical grade. Evaluation of antioxidant activity i) DPPH radical scavenging activity: The anti-oxidant potential of the ethanol extract was determined on the basis of their scavenging activity of the stable 1, 1-diphenyl-2picryl hydrazyl (DPPH) free radical. DPPH is a stable free radical containing an odd electron in its structure and usually utilized for detection of the radical scavenging activity in chemical analysis. The aliquots of the different concentrations (5-100 μg/mL) of the extract were added to 6 mL of a 0.004% w/v solution of DPPH. Absorbance at 517 nm was determined after 30 min, and IC50 (Inhibitory conc. 50%) was determined. IC50 value denotes the concentration of sample required to scavenge 50% of the DPPH free radicals (Gupta et. al., 1971). The percent inhibition is calculated using the formula: % inhibition = (Blank Absorbance - Sample Absorbance/ Blank Absorbance) × 100. ii) Determination of Total Phenolic Content (TPC): Total Phenolic Content was determined by using Folin-Ciocalteu (FC) reagent with analytical grade gallic acid as the standard (Marinova et al., 2005). Extract or standard solution (15.62-500 mg/l) of 1 ml was added to distilled water (9 ml). Then 1 ml FC reagent (10 times diluted with distilled water) was added. After 5 minutes; 10 ml 7% Na₂CO₃ was added to the mixture and kept for 30 minutes at room temperature. Then absorbance was measured against blank at 750 nm using UV spectrophotometer. Total phenolic content of the extract was determined from the standard curve and expressed as mg gallic acid equivalent (GAE)/100 g dried plant material. iii) Determination of total flavonoid content (TFC): Total flavonoid content was determined in the sample extract by reaction with sodium nitrite, followed by the development of coloured flavonoid-aluminum complex formation using aluminum chloride in alkaline condition which can be monitored spectrophotometrically at maximum wavelength of 369 nm (Bakar et al., 2009). Evaluation of Chemopreventive Activity i) Induction of tumor: The hairs on the dorsal region of mice were shaven 3 days before the commencement of the experiment. For the induction of the tumours, a two stage protocol consisting of initiation with a single topical application of carcinogen DMBA followed two weeks later by a promoter, croton oil, three times a week, were employed as per our previous modified method of Berenblum,1941 (Prashar & Kumar, 1994). Four groups (10 animals per group) of Swiss albino mice were used for the study. Animals were dorsally shaved with hair clipper. Group-I was served as negative control which was given2% Tween 80 in water (10 ml/kg bodyweight) orally and 50 µL of acetone topically on their shaven back. Group-II was treated as carcinogenic control and received a single topical application of DMBA (100 µg in 50 µL of acetone per animal) on their shaven back. Two week after the DMBA application, 0.1 ml of 1% croton oil in acetone was topically applied three times per a week until the termination of the experiment at 16 weeks. Group-III and IV were served as test groups and treated orally with the ethanolic extract of fruits of C. nucifera at dose of 250 mg/kg body weight and 500 mg/kg body weight respectively from 15 days before initiation of carcinogenesis by DMBA and continued throughout the experimental protocol on daily basis. ii) Tumor study: Tumour incidence, tumour yield, and tumour burden were calculated after the termination of the experiment. Tumor incidence: The number of mice carrying at least onetumor, expressed as a percentage incidence. Tumor yield: The average number of papilloma per mouse. Tumor burden: The average number of tumors per tumorbearing mouse. Biochemical study of tumor and liver sample: Tissue homogenate was prepared as described previously by Sharma et al., 2010. Determination of total protein concentration was performed following the method described by Lowry et al., 1951. Reduced glutathione (GSH) was estimated using the method of Ellman et al., 1959. Misra & Fridorich (1972) described the basis for the assay of Superoxide Dismutase (SOD) enzyme, which involved auto-oxidation of epinephrine to adrenochrome by superoxide radicals. This method was followed for SOD analysis. Catalase (CAT) activity was evaluated following the method developed by Bergmeyer, H. U. (1983), which is based of H₂O₂ degradation by the action of CAT. Statistical analysis: Values are presented as mean ± SE. The data obtained from different groups was analyzed by Student’s t-test. The value P ?0.05 was considered statistically significant for all conducted experiments.

Source of Information: Jahangirnagar University J. Biol. Sci. 6(2): 47-58, 2017 (December)

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Achievement/Findings:

The present study suggests that the ethanol extract of C. nucifera tender fruit has potential antioxidant, and chemo preventive activity. The results are quite promising and demand for further investigations.

Knowledge Management: Journal