WWOORRLLDD JJOOUURRNNAALL OOFF PPHHAARRMMAACCYY AANNDD PPHHAARRMMAACCEEUUTTIICCAALL SSCCIIEENNCCEESS Padmaa World Journal of Pharmacy and Pharmaceutical Sciences SSJJIIFF IImmppaacctt FFaaccttoorr 55..221100 VVoolluummee 44,, IIssssuuee 0055,, 777788--778899.. RReesseeaarrcchh AArrttiiccllee IISSSSNN 2278 – 4357 COMPARISON OF IN VITRO ANTIOXIDANT ACTIVITY OF ARECA CATECHU LINN NUT BY MICROWAVE EXTRACTION AND SOXHLATION TECHNIQUE Dr. Padmaa M Paarakh* Department of Pharmacognosy, the Oxford College of Pharmacy, Bangalore 560 068, Karnataka, India. ABSTRACT Article Received on 27 Feb 2015, The aim of the present study is to compare the in vitro antioxidant activity of Areca catechu nut by two different techniques of extraction Revised on 21 March 2015, Accepted on 11 April 2015 viz., Microwave extraction and Soxhlation and correlate the antioxidant activity with the amount of flavonoids and phenol content *Correspondence for present in the extract. Areca catechu nuts [unroasted, roasted and Author commercial sample from shop] were extracted separately in microwave Dr. Padmaa M Paarakh extractor and soxhlet with methanol and distilled water. The study Department of were carried out with 2 extracts prepared by both methods using Pharmacognosy, the different in vitro antioxidant model viz., Phosphomolybdenum Oxford College of Pharmacy, Bangalore 560 antioxidant assay, Reducing power assay and DPPH radical 068, Karnataka, India. scavenging assay. Total flavonoid content and phenol content were also determined. The study exhibited strong antioxidant activity in different in vitro systems. The methanol and water extracts of commercial sample showed better results than other 2 extracts on evaluation with the different in vitro antioxidant methods. Flavonoid content and phenol content also correlated well the in vitro antioxidant activity. There was minute variation in microwave extraction and soxhlet extraction technique. This experiment has concluded the strong in vitro antioxidant properties of A.catechu. Further, investigation on in vivo antioxidant activity has to be carried out to understand its mode of action and to discover the main constituent of A.catechu nuts responsible for this antioxidant effect. KEYWORDS: Areca catechu, antioxidant activity, soxhlet extraction, microwave extraction, phosphomolybednum antioxidant assay, reducing power assay, DPPH assay. www.wjpps.com Vol 4, Issue 05, 2015. 778 Padmaa World Journal of Pharmacy and Pharmaceutical Sciences 1. INTRODUCTION Many diseases are caused due to oxidative stress. It has been implicated in the pathology of diseases for inflammatory conditions, cancer, atherosclerosis, Parkinsonism, diabetes and aging. Cardiovascular diseases, tumor growth, wrinkled skin, cancer, Alzheimer’s disease are contributed by accelerated cell oxidation rendering a decline in energy and endurance.[1] Oxygen is normally used in regular body processes like respiration and some cell mediated immune functions which tend to produce free radicals. The oxidation of cellular oxidizable substrates can be prevented by substances called antioxidants. The antioxidants act by scavenging reactive oxygen species, activating a number of detoxifying proteins or by preventing the generation of reactive oxygen species.[2,3] Many a times the body is unprotected from deleterious effects of free radicals as the antioxidation activity normally carried out by the body is unable to sufficiently quench or scavenge the free radicals. In order, to protect the human body from free radicals and retard the progress of many chronic diseases there has been a recent increase in interest in finding natural antioxidants.[4] α-tocopherol and ascorbic acid are the widely used natural antioxidants because they are regarded as safer and cause fewer adverse reactions. Therefore, there is a considerable interest to explore new natural sources that are safe thereby replacing the synthetic antioxidants.[5] A number of studies have been carried out on various plants, vegetables and fruits because they are rich sources of antioxidants, such as vitamin A, vitamin C, Vitamin E, carotenoids, polyphenolic compounds and flavonoids which prevent free radical damage thereby reducing risk of chronic diseases.[6] This beneficial role of plants to provide natural antioxidants has led to increase in the search for newer plant based sources. Areca catechu Linn. an annual of the Palmaceae family is one of valuable medicinal plant. In many Asian cultures (such as India, Taiwan, and Southeast Asia), betel nut, Areca catechu L., is traditionally masticated either alone or as a quid along with a large variety of ingredients, such as betel leaf (Piper betel; family Piperaceae), slaked lime, catechu, different types of tobacco, and various additives, perfumes, and stimulants.[7] It is estimated that over 600 million individuals all over the world consume betel nut (also called areca nut) in one form or another.[8] In herbal medicine, the areca nut has been used medicinally as a drug against www.wjpps.com Vol 4, Issue 05, 2015. 779 Padmaa World Journal of Pharmacy and Pharmaceutical Sciences parasitic worms.[9] In old Indian scripts, such as Vagbhata (4th century), and Bhavamista (13th century), betel nut has been described as a therapeutic agent. Its use was recommended in many diseases, such as leucoderma, leprosy, anaemia, and obesity. Areca catechu L plant contains a diverse group of phenolic compounds with antioxidant activity, including flavonoids, lignans and stilbenes, and simple phenolic acids, such as hydroxybenzoic acids and hydroxycinnamic acids.[10,11] Most of the methods used in these studies are based on classical Soxhlet extraction. Nevertheless, this traditional sample extraction technique often uses large quantities of organic solvents and is usually time-consuming. In the last few years, established methods, such as supercritical fluid extraction (SFE), pressurized liquid extraction (PLE) and microwave-assisted extraction (MAE), were used to reduce the volume of solvents required, to improve the precision of analyte recoveries and to reduce extraction time. Of these techniques, SFE is the most selective extraction method, but its use has been limited by the strong matrix dependence of the extraction process. Most of the time, extraction conditions need to be optimized for each new matrix MAE technique offer advantages over the SFE and PLE methods as the extraction can be completed within minutes with less solvent.[12] Hence, the present study has been undertaken to investigate the two different technique of extraction viz., traditional and microwave assisted extraction technique and compare in vitro antioxidant properties of different extracts of the nuts[unroasted, roasted and commercial sample from shop] of A.catechu by various methods including Phosphomolybednum antioxidant assay, reducing power and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay. Antioxidant activity was correlated with the content of flavonoid and phenol content present in the extract. 2. MATERIALS AND METHODS 2.1 Plant material The Areca catechu nuts and commercial samples were collected from local market in Bangalore, Karnataka, India and it was identified and authenticated by Botanist, Natural Remedies Pvt Ltd., Bangalore. A voucher specimen was deposited in The Oxford College of Pharmacy, Bangalore. The nuts as such, roasted at 500C for 30 minutes and commercial samples were powdered coarsely respectively, passed through sieve no. 40 and stored in air tight container for further use. www.wjpps.com Vol 4, Issue 05, 2015. 780 Padmaa World Journal of Pharmacy and Pharmaceutical Sciences 2.2.1 Preparation of extract by soxhlet technique Coarsely powdered nuts [unroasted, roasted and commercial sample] of A.catechu 20 g, each were subjected to extraction in soxhlet extractor with methanol and boiled with distilled water [150 ml] respectively. The two extracts of three types of nuts were concentrated by rotary vacuum evaporator and evaporated to dryness. The yield was found to be 7.35, 9.4; 8.7, 9.8 and 34.75, 29.2 % w/w respectively with reference to the air dried unroasted, roasted and commercial samples. 2.2.2 Preparation of extract by Microwave assisted extraction technique Coarsely powdered nuts [unroasted, roasted and commercial sample] of A.catechu 20 g, each were subjected to extraction in Microwave extractor [Ragatech Company Microwave extractor with Power 3, set at temperature 500C for 30 minutes with continuous stirring] with methanol and water [150 ml] respectively. The two extracts of three types of nuts were concentrated by rotary vacuum evaporator and evaporated to dryness. The yield was found to be 4.65, 4.8; 8, 4.8 and 11.95, 4.7 % w/w respectively with reference to the air dried unroasted, roasted and commercial samples. 2.3 Drugs and Chemicals The compounds, 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 100% ethanol, ascorbic acid, potassium hydrogen phosphate, potassium di-hydrogen phosphate, sodium phosphate, ammonium molybedate, sulphuric acid were purchased from SD Fine chemicals ltd, Mumbai. Potassium ferricyanide, trichloroacetic acid (TCA), ferric chloride, ascorbic acid, rutin, gallic acid and other reagents were procured from Loba chemicals, Mumbai. All chemicals and reagents used in this study were at least of analytical grade. 2.4. Determination of total flavonoid content The total flavonoid content in the extracts were determined by method modified by Zhishen’s method.[13] Different concentration of extracts in methanol[ 3 ml] was mixed with 0.1 ml of 10 % aluminum chloride followed by 0.1 ml of 1 M potassium acetate solution. Add 2.8 ml of water and kept for incubation at room temperature for 30 min. The absorbance was measured at 415 nm. The total flavonoid content is expressed as Rutin equivalent [mg/100 g] of the dried weight. www.wjpps.com Vol 4, Issue 05, 2015. 781 Padmaa World Journal of Pharmacy and Pharmaceutical Sciences 2.5 Determination of Total Phenol Content The total phenol content was determined by Folin-Ciocalteu assay.[14] Different concentration of extracts were made up to 3.5 ml, then 0.5 ml of Folin-Ciocalteu reagent followed by 2 ml of 7.5 % sodium carbonate solution. The above solution is incubated at room temperature for 10 min and absorbance was measured at 650 nm. Total phenolic content are expressed as gallic acid equivalent [mg/g] of the dried weight. 2.6 In vitro Antioxidant Activity 2.6.1 Determination of total antioxidant capacity (phosphomolybednum antioxidant assay) The total antioxidant capacity of the extracts were evaluated by the phosphomolybednum assay method of Prieto et al.[15] which is based on the reduction of Mo (VI) to Mo (V) by the compounds and subsequent formation of a green phosphate - Mo (V) complex in acidic condition. 0.3ml (25,50,100 and 200 μg/ml) of different extracts were combined with 3ml of reagent solution (600 mM sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate) and the reaction mixture in the tubes were capped and incubated in a boiling water bath at 95°C for 90 min. After the samples had cooled to room temperature, the absorbance of the aqueous solution of each was measured at 695 nm against a blank. A typical blank solution contained 3 ml of reagent solution and the appropriate volume of the same solvent used for the sample and it was incubated under the same conditions. The antioxidant capacity was expressed as the number of gram equivalents of ascorbic acid (25- 100 μg/ml) which was also processed and incubated under the same conditions. 2.6.2 Reducing power assay The reducing power was determined by the Fe3+ and Fe2+ transformation in the presence of fractions described by Oyaizu.[16] The Fe2+can be monitored by measuring the formation of Perl’s Prussian blue at 700 nm. One ml of the different extracts (25,50,100 and 200 μg/ml), 2.5ml of phosphate buffer (pH 6.6) and 2.5 ml of 1% potassium ferricyanide was incubated at 500C for 30 min and 2.5 ml of 10% trichloroacetic acid was added to the mixture and centrifuged at 3000 rpm for 10 min. About 2.5 ml of supernatant was diluted with 2.5 ml of water and shaken with 0.5 ml of freshly prepared 0.1% ferric chloride solution. The absorbance was measured at 700 nm. Ascorbic acid (100-500 μg/ml) was used as standard. 2.6.3 DPPH radical scavenging assay The free radical scavenging activity of the different extracts were measured in vitro by 1,1- diphenyl-2-picrylhydrazyl (DPPH) assay.[17] About 0.3 mM solution of DPPH in 100% www.wjpps.com Vol 4, Issue 05, 2015. 782 Padmaa World Journal of Pharmacy and Pharmaceutical Sciences Ethanol was prepared and 1ml of this solution was added to 3 ml of different extracts dissolved in ethanol at different concentrations (25,50,100 and 200 μg/ml). The mixture was shaken and allowed to stand at room temperature for 30 min and the absorbance of the solution was measured at 517 nm against a blank using spectrophotometer. The % scavenging activity at different concentrations was determined and the IC value of the fractions was 50 compared with that of ascorbic acid, which was used as the standard. Decreasing of the DPPH solution absorbance indicates an increase of the DPPH radical scavenging ability. DPPH radical scavenging activity was calculated according to the following equation: Scavenging effect (%) = [(A -A )/A x 100] 0 1 0 The results were expressed as mean values ± standard deviation. The extract concentration providing 50% inhibition (IC ) was calculated from the graph of scavenging effect 50 percentage against the extract concentration. Ascorbic acid (0.125-1 μg/ml) was used as standard. 3. RESULTS AND DISCUSSION 3.1 Total flavonoid content Fig.1 shows the flavonoid content of different extracts by soxhlation and microwave assisted extraction technique. It is very clear that the amount of flavonoid by microwave assisted extraction was found to highest in methanol roasted followed by methanol unroasted, water commercial sample, methanol commercial sample, water unroasted and water roasted. By soxhlet extraction, the content of flavanoid was found to be highest in methanol unroasted, methanol roasted, water commercial sample, water roasted, water unroasted and methanol commercial sample. 3.2 Total phenol content Fig.2 shows the phenol content of different extracts by soxhlation and microwave assisted extraction technique. It is very clear that the amount of phenol by microwave assisted extraction was found to highest in methanol commercial sample, water commercial sample, methanol roasted, methanol unroasted, water unroasted and water roasted. By soxhlet extraction, the content of phenol was found to be highest in methanol commercial sample, water commercial sample, methanol roasted, water roasted, methanol unroasted and water unroasted. www.wjpps.com Vol 4, Issue 05, 2015. 783 Padmaa World Journal of Pharmacy and Pharmaceutical Sciences 3.3 Total antioxidant capacity (phosphomolybednum antioxidant assay) The data for the antioxidant capacity by phosphomolybednum method was given in the Fig 3.The antioxidant capacity of the fractions was measured spectrophotometrically through Phosphomolybdenum method, which was based on the reduction of Mo (VI) to Mo (V) by the sample analyte and the subsequent formation of green phosphate/Mo (V) compounds with a maximum absorption at 695 nm. The antioxidant capacity of the extracts of A.catechu. was found to decrease in this order in microwave assisted extraction as methanol commercial sample, water commercial sample, methanol roasted, methanol unroasted, water unroasted and water roasted. The antioxidant capacity of the extracts of A.catechu was found to decrease in this order in soxhlet extraction as methanol commercial sample, water commercial sample, water roasted, methanol roasted, water unroasted and methanol unroasted. 3.4 Reducing power assay Fig 4 shows the reducing power of the A.catechu extract by both the method. In this assay, the yellow color of the test solution changes to various shades of green and blue, depending on the reducing power of each compound. The presence of reducers causes the reduction of the Fe 3+/ferricyanide complex to the ferrous form. Therefore, by measuring the formation of Perl's Prussian blue at 700 nm, we can monitor the Fe2+ concentration. The reducing properties are generally associated with the presence of reductones[18], which have been shown to exert antioxidant action by breaking the free radical chain by donating a hydrogen atom.[19] However, as anticipated, the reduction power of ascorbic acid was relatively more pronounced than that of A.catechu . The reducing powers for the different extracts in the microwave assisted extraction were in the following order: methanol commercial sample > water commercial sample > methanol roasted > methanol unroasted > water unroasted> water roasted. The reducing powers for the different extracts in the soxhlet extraction were in the following order: methanol commercial sample > water commercial sample > methanol roasted > methanol unroasted > water roasted> water unroasted. 3.5 DPPH radical scavenging activity (RSA) The RSA of the A.catechu extracts were evaluated using an ethanol solution of the stable free radical, DPPH. The model for scavenging the stable DPPH radical is widely used model to evaluate antioxidant activities in a relatively short time to compare with other methods. The effect of antioxidants on DPPH radical scavenging was thought to be due to their hydrogen www.wjpps.com Vol 4, Issue 05, 2015. 784 Padmaa World Journal of Pharmacy and Pharmaceutical Sciences donating ability. DPPH is a stable free radical and accept an electron or hydrogen radical to become a stable diamagnetic molecule and therefore inhibit the propagation phase of lipid peroxide.[20,21] 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 when the odd electron of DPPH radical becomes paired with a 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. The reduction capability of DPPH radicals was determined by decrease in its absorbance at 517 nm induced by antioxidants. Hence, DPPH is usually used as a substrate to evaluate antioxidative activity of antioxidants. The RSA values of unroasted, roasted and commercial sample are presented in Table 1. IC 50 values (concentration of sample required to scavenge 50% of free radicals) of A. catechu extracts, ascorbic acid are indicated in Table 1. The antioxidant capacity of the extracts of A.catechu was found to decrease in this order in microwave assisted extraction as methanol unroasted, methanol roasted, methanol commercial sample, water commercial sample , water roasted and water unroasted. The antioxidant capacity of the extracts of A.catechu was found to decrease in this order in soxhlet extraction as methanol unroasted, methanol commercial sample, water commercial sample, water roasted, water unroasted and methanol roasted. Table 1: DPPH Scavenging activity of nuts of A.catechu by microwave assisted and soxhlet extraction IC value 50 S no. Sample content Solvent used Microwave assisted Soxhlet extraction extraction [µg/ml] [µg/ml] 1 Unroasted nut Water 22.83 ± 0.28 20.72 ± 0.80 2 Unroasted nut Methanol 14.07 ± 0.05 13.98 ± 0.46 3 Roasted nut Water 17.9 ± 0.40 16.57 ± 0.55 4 Roasted nut Methanol 14.07 ± 0.25 32.43 ± 0.15 5 Commercial sample Water 16.82 ± 0.37 15.12 ± 0.63 6 Commercial sample Methanol 14.44 ± 0.68 14.63 ± 0.35 7 Standard Ascorbic acid 5.00 ± 0.13 www.wjpps.com Vol 4, Issue 05, 2015. 785 Padmaa World Journal of Pharmacy and Pharmaceutical Sciences www.wjpps.com Vol 4, Issue 05, 2015. 786 Padmaa World Journal of Pharmacy and Pharmaceutical Sciences 4. CONCLUSIONS This experiment has concluded the strong in vitro antioxidant properties of A.catechu. The methanol and water extracts of commercial sample showed better results than other 2 extracts on evaluation with the different in vitro antioxidant methods. Flavonoid content and phenol content also correlated well with the in vitro antioxidant activity. There was minute variation in Microwave extraction and soxhlet extraction technique. This experiment has concluded the strong in vitro antioxidant properties of A.catechu. Further, investigation on in vivo antioxidant activity has to be carried out to understand its mode of action and to discover the main constituent of A.catechu nuts responsible for this antioxidant effect. 5. ACKNOWLEDGEMENT The authors are grateful to Department of Pharmacognosy, The Oxford College of Pharmacy, Bangalore, for providing the facilities. 6. ETHICAL ISSUES There is none to be applied. 7. CONFLICT OF INTEREST None to be declared. 8. REFERENCES 1. Finkel T, Holbrook NJ. Oxidants, oxidative stress and biology of ageing. Nature, 2000; 408: 239-247. 2. Halliwell B. Antioxidants in human health and disease. Annu Rev Nutr, 1996; 16: 33-50. www.wjpps.com Vol 4, Issue 05, 2015. 787