Objective: The aim of this study was to compare the antioxidant activity of fruit of water apple and fragrant mango against 1,1-diphenyl-2- picrylhydrazyl as a free radical.Methods: This study was conducted by visible spectrophotometry.Results: The water apple and fragrant mango contain reducing compounds including vitamin C. The vitamin C content of water apple and fragrant mango fruit was 0.087% (w/w) and 0.050% (w/w), respectively. The inhibitory concentration 50% value of fresh fruit of water apple and fragrant mango was 4.857 Î¼g/ml and 4.379 Î¼g/ml, respectively.Conclusion: Antioxidant activity of water apple fruit was higher than fragrant mango fruit

Herawati, Irma Erika

Saptarini, Nyi Mekar

English

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Special Issue (May)Online - 2455-3891 Print - 0974-2441II-Indonesian Conference on Clinical PharmacyANTIOXIDANT ACTIVITY OF WATER APPLE (SYZYGIUM AQUEUM) FRUIT AND FRAGRANT MANGO (MANGIFERA ODORATA) FRUITNYI MEKAR SAPTARINI1*, IRMA ERIKA HERAWATI21Department of Pharmaceutical Analysis and Medicinal Chemistry, Faculty of Pharmacy, Universitas Padjadjaran, Indonesia. 2Department of Pharmacy, FMIPA, University of Al Ghifari, Indonesia. Email: nyi.mekar@unpad.ac.idReceived: 16 October 2016, Revised and Accepted: 24 January 2017ABSTRACTObjective: The aim of this study was to compare the antioxidant activity of fruit of water apple and fragrant mango against 1,1-diphenyl-2- picrylhydrazyl as a free radical.Methods: This study was conducted by visible spectrophotometry.Results: The water apple and fragrant mango contain reducing compounds including vitamin C. The vitamin C content of water apple and fragrant mango fruit was 0.087% (w/w) and 0.050% (w/w), respectively. The inhibitory concentration 50% value of fresh fruit of water apple and fragrant mango was 4.857 µg/ml and 4.379 µg/ml, respectively.Conclusion: Antioxidant activity of water apple fruit was higher than fragrant mango fruit.Keywords: 1,1-diphenyl-2-picrylhydrazyl, Free radical, Visible spectrophotometry, Vitamin C.INTRODUCTIONFree radicals are responsible for the damage of lipids, proteins, and nucleic acid in cells [1] which lead to cardiovascular diseases, cancers, and other age-related degenerative diseases [2]. Fruits and vegetables can reduce the risk of stroke and cancer because of the antioxidant contained in the plant parts. Different fruits have different capacities due to different dietary antioxidants such as vitamin C, flavonoids, and other phenolic compounds [3].Fruit of water apple (Syzygium aqueum (Burm.f.) Alston) and fragrant mango (Mangifera odorata Griffith) contains vitamin C. All physiological and biochemical actions of vitamin C are due to its action as an electron donor from a double bond. Vitamin C is an antioxidant, because of donating its electrons, which prevents other compounds from being oxidized. When vitamin C donates one electron, they are formed a free radical, semi-dehydroascorbic acid, or ascorbyl radical, which relatively stable (10−5 seconds of half-life) and fairly unreactive [4]. The aim of this study was to compare the antioxidant activity of fruit of water apple and fragrant mango against 1,1-diphenyl-2-picrylhydrazyl (DPPH) as a free radical.MATERIALS AND METHODSMaterialsFruit of water apple and fragrant mango was collected from Subang subdistrict, Indonesia, in July 2016. Fruits were identified by Laboratory of Plants Taxonomy, Department of Biology, Universitas Padjadjaran, Indonesia, with No. 450/HB/08/2016. All chemical reagents are analytical grade (Merck) including DPPH (Sigma-Aldrich).MethodsExtractionA total of 10 g of fruit and 15 ml of citrate buffer pH 4.2 were mashed and filtered into a 25 ml volumetric flask. The extract was centrifuged at 5000 rpm for 15 minutes to obtain supernatant. Citrate buffer pH 4.2 was added to the supernatant (1.0, 1.5, 2.0, 2.5, and 3 ml) in a 10 ml volumetric flask for antioxidant activity determination.The moisture content determinationA total of 5 g of fruit was dried on 105°C at atmospheric pressure for 5 hrs, then weighed. Drying and weighing continued, every 1 hr, until a constant weight [5].Qualitative analysis of reducing compoundsA total of 0.2 ml of 1% iodine solution was added to 2 ml of supernatant. The discoloration of the brown color was indicated the positive reaction.Quantitative analysis of vitamin CA total of 2 ml of 25 µg/ml methylene blue and 2 ml of distilled water incubated for 10 minutes. The absorbance was measured at 400-900 nm with spectrophotometer against distilled water as blank. A total of 2 ml of 25 µg/ml methylene blue was added to 2 ml of standard vitamin C (2, 4, 6, 8, and 10 µg/ml) or supernatant in 10 ml volumetric flask, accomplished with citrate buffer pH 4.2. The mixture was incubated for 10 minutes, then the absorbance was measured at maximum wavelength [6].Antioxidant activity determinationThe modified method of Molyneux (2004) was conducted [7]. A total of 1.5 ml of 40 µg/ml DPPH was added to various concentrations of 1.5 ml of vitamin C or supernatant. The mixtures were incubated in a dark chamber for 30 minutes, then absorbance was measured at 517 nm using spectrophotometer (Ray Leigh). The blank was 96% ethanol. Percentage of antioxidant activity was calculated using this formula:% of DPPH inhibition =(Ab-Aa)(Ab)×100Aa and Ab are the absorbance values of the sample and the blank, respectively. A percent inhibition versus concentration was plotted, and the concentration of sample required for 50% inhibition was determined and expressed as inhibitory concentration 50% (IC50) value.Statistical analysisThe results were presented as the mean ± standard error of the mean. Data comparisons between groups were done by one-way ANOVA Research Article© 2017 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4. 0/) DOI: http://dx.doi.org/10.22159/ajpcr.2017.v10s2.1948755Special Issue (May) Saptarini and Herawati followed by t-test. Values were considered statistically significant at p<0.05.RESULTSA total of 10.127±0.005 g of water apple fruit was produced the turbid pink extract, whereas 10.036±0.232 g of fragrant mango was produced the turbid yellow extract. The turbid extract was centrifuged to obtain a clear supernatant, pink for water apple and yellow for fragrant mango. The moisture content for water apple and fragrant mango was 92.847±0.059% and 84.927±0.099%, respectively. Water apple and fragrant mango supernatant were given the positive results.There were statistically significant differences between groups on vitamin C content (p=3.65×10−4) and IC50 value (p=2.19×10−6).DISCUSSIONCitrate buffer pH 4.2 was used to extract the reducing compounds, especially vitamin C, which is present in the fruit. Citrate buffer pH 4.2 was selected because of vitamin C is stable at acidic pH. Aqueous solutions are most stable at pH 5-6 and very unstable at alkaline pH [8]. The water apple and fragrant mango extracts were turbid, so centrifugation was conducted to separate the supernatant which contains the secondary metabolites from the residues. The moisture content for both fruits was high (more than 80%), which shown the content of water and volatile compounds in fruits. These values were determined to calculate the vitamin C content in dried plant materials because the used samples were fresh fruits.Qualitative analysis of reducing compounds, including vitamin C in the supernatant, was showed the positive results. There was the oxidation-reduction reaction, the vitamin C is oxidized by the iodine into dehydroascorbic acid, and the iodine is reduced [9]. These results indicate the presence of a reducing compound in the supernatant which can react with DPPH as free radical oxidants.Quantitative analysis of vitamin C was based on oxidation-reduction reactions which observed from discoloration of methylene blue to leucomethylene blue, from blue to colorless [6]. The methylene blue solution was provided the maximum wavelength at 664.4 nm. This wavelength was consistent with the literature [6]. The instrument response was proportional to the vitamin C content, due to the correlation coefficient was 0.9993 (Fig. 1) which meets the criteria by ICH [10].The vitamin C content in both fruits was small (Table 1) because of the used sample was fresh fruit with high moisture content. If converted to a dried plant material, the vitamin C content for water apple and fragrant mango was 1.216±0.001% and 0.332±0.001% (w/w), respectively. High vitamin C content in water apple due to the time harvest was the harvest season for water apple in July to August in Indonesia [11]. Low vitamin C content in fragrant mango due to the time harvest was not the harvest season, i.e., from September to December in Indonesia [12]. This study was conducted in July, so we suggested that the secondary metabolites content in fragrant mango were not maximally produced.The chemical composition of horticultural crops was influenced by climatic conditions including light and average temperature [13]. The intensity and amount of light during the growing season influence the vitamin C content because of vitamin C was synthesized from sugars through photosynthesis. The outside fruit which exposed to maximum sunlight contains a higher amount of vitamin C than inside and shaded fruit on the same plant [14]. Climatic conditions and cultural practices also influence the preharvest [8,15]. The plant maturity when harvested, harvesting method, postharvest handling conditions [16], Fig. 1: Calibration curve of standard vitamin C (n=3)Table 1: Vitamin C content in fruitSample Absorbance Vitamin C content (µg/ml)Vitamin C content  (% w/w)Water apple 0.886±0.001 4.047±0.115 0.087±0.002Fragrant mango 0.894±0.001 2.313±0.115 0.050±0.002Values are mean±SD (n=3), SD: Standard deviationTable 2: The antioxidant activitySample Concentration (µg/ml) Absorbance % inhibition Linear regression equation IC50 (µg/ml)Vitamin C standard 2 0.432±0.003 32.448±0.477 y=7.7318x+17.755R²=0.9984.1704 0.325±0.011 49.167±1.6856 0.221±0.005 65.521±0.7868 0.138±0.003 78.490±0.50210 0.031±0.005 95.104±0.705Water apple fruit 4 0.571±0.002 45.587±0.220 y=6.0857x+20.444R²=0.9954.8575 0.523±0.002 50.159±0.1986 0.461±0.002 56.127±0.1987 0.383±0.003 63.556±0.2408 0.318±0.004 69.714±0.343Fragrant mango fruit 2 0.766±0.014 16.571±1.746 y=9.2476x+9.8794R²=0.9904.3793 0.645±0.001 27.016±1.3614 0.537±0.001 38.540±0.1105 0.475±0.001 48.889±0.1106 0.366±0.004 54.762±0.095Values are mean±SD (n=3), SD: Standard deviation56Special Issue (May) Saptarini and Herawati storage duration and conditions, processing, and physical damage [17] also affect the vitamin C content.The antioxidant activity with the DPPH method was characterized by the color alteration, from purple to yellow after incubation for 20 minutes. It is because of the free radical DPPH was reduced to DPPH [18]. Table 2 showed that IC50 value for water apple and fragrant mango was similar as standard vitamin C. Both fruits were classified as a very strong antioxidant because the IC50 value is less than 50 µg/ml [18] due to high vitamin C content (Table 1). The reactive free radical (DPPH) was reduced by vitamin C (ascorbic acid), and the ascorbyl radical formed in its place is less reactive [19]. Then, the ascorbyl radical loss of a second electron to form the dehydroascorbic acid, which its stability (only a few minutes) depends on factors such as pH and temperature [20]. The vitamin C content and the IC50 value for both fruits were statistically significant, due to harvest time which also influenced by climate.CONCLUSIONThe vitamin C content and antioxidant activity of water apple fruit were higher than fragrant mango fruit.ACKNOWLEDGMENTThe authors thank Rubiyani Sumantri and Kiki Rizky Fitriyani for technical assistance.REFERENCES1. Alothman M, Bhat R, Karim AA. UV radiation-induced changes of antioxidant capacity of fresh-cut tropical fruits. Innov Food Sci Emerg Technol 2009;10:512-6.2. Scalzo J, Politi A, Pellegrini N, Mezzetti B, Battino M. Plant genotype affects total antioxidant capacity and phenolic contents in fruit. Nutrition 2005;21(2):207-13.3. Saura-Calixto F, Goni I. Antioxidant capacity of the Spanish Mediterranean diet. Food Chem 2006;94:442-7.4. Bielski BH, Richter HW, Chan PC. Some properties of the ascorbate free radical. Ann N Y Acad Sci 1975;258:231-7.5. USP, editor. US Pharmacopeia 32. 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ANTIOXIDANT ACTIVITY OF WATER APPLE (Syzygium aqueum) FRUIT ANDFRAGRANT MANGO (Mangifera odorata) FRUIT

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ANTIOXIDANT ACTIVITY OF WATER APPLE (Syzygium aqueum) FRUIT ANDFRAGRANT MANGO (Mangifera odorata) FRUIT

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