Indian Journal of Natural Products and Resources Vol. 2(2), June 2011, pp. 174-178

Antioxidant activity of selected lesser known edible fruits from Western Ghats of India

S Karuppusamy*, G Muthuraja and K M Rajasekaran Department of Botany, Centre for Botanical Research, The Madura College (Autonomous), Madurai-625 011, Tamil Nadu, India

Received 21 May 2010; Accepted 8 February 2011

Six species of lesser known edible fruits from Western Ghats of India were analysed for their anthocyanin, ascorbic acid, total phenolics and flavonoid levels and their antioxidant activities using the DPPH (2,2-diphenyl-1-picrylhydrazyl) method. The data obtained shown the anthocyanin, ascorbic acid, total phenolics and flavonoid levels were significantly higher in the methanol extract of Mahonia leschenaultii (Wall. ex Wight & Arn.) Takeda fruits. Gaultheria fragrantissima Wall. and Rubus ellipticus Sm. fruits were also found to have significant amount of phytochemicals analyzed. M. leschenaultii fruits had highest antioxidant activity when compared to all other fruit extracts. There was a strong correlation between the content of phytochemicals and antioxidant activities in all fruit extracts in the following decreasing order: M. leschenaultii > G. fragrantissima > R. ellipticus > Grewia tiliaefolia Vahl > Ziziphus rugosa Lam.> Flueggea leucopyrus Willd.

Keywords: Anthocyanin, Antioxidant, Ascorbic acid, Flavonoids, Edible fruits, Phenolics, Western Ghats. IPC code; Int. cl. (2011.01)  A23L 1/00, A61K 36/00, A61P 39/06

Introduction of lesser known fruit species could lead to a better Wild edible plants contribute significantly to the understanding of the beneficial effects and an nutrition of inhabitants of rural areas of Western increased consumption of these fruits, including their Ghats1. Although these foods are consumed by people utilization in functional foods and as ingredients in throughout the year in fresh and dried forms, reliance nutraceuticals, medicine and pharmaceuticals5. on these foods increases during periods of cereal Research on wild fruits and other wild edible plants shortages. Now-a-days, however, a nutritional is also intended to promote the preservation of these transition is occurring in the poorest countries of the species, presently under threat by human activities. In world resulting in the replacement of traditional plant- addition to their nutritional value, the preservation of based diets that are rich in fruits and vegetables with these fruits also has economical advantages, as there diets that are rich in calories provided by animal fats is a significant trade with some of these wild edible and sugars, and low in complex carbohydrates2. Wild and medicinal fruits. Any scientific evidence for the edible foods include fruits, leaves, flowers and seeds health benefits of such wild fruits in addition to their from spontaneous trees and shrubs. Of these, fruits are nutritional value would be a value addition to the receiving increased interest from researchers working plants producing such fruits. Concerning their on wild edible plant species because of their medicinal properties, the most commonly studied nutritional value, vitamin and mineral contents. These benefit is their antioxidant effects. Antioxidants play a fruits are being investigated as potential food crucial role in the prevention of chronic ailments such supplements in the Western Ghats region so as to as heart disease, cancer, diabetes, hypertension, stroke increase quality of daily food for the rural and Alzheimer’s disease by combating oxidative 6 population3. stress . Currently, increasing attention has been paid by Numerous studies have shown that fruit and consumers to the lesser known fruits which have vegetables are sources of diverse nutrient and non- unusual flavours and qualities, and many of which are nutrient molecules, many of which display antioxidant rich in antioxidants and anthocyanins4. Detailed properties. In addition to vitamin C, a great number of information about the health promoting components other phenolics (especially the flavonoids) have —————— strong antioxidant activity in vitro. In fact, the vast *Correspondent author: E-mail: [email protected] majority of the activity seen in various fruit juice KARUPPUSAMY et al: ANTIOXIDANT ACTIVITY OF LESSER KNOWN EDIBLE FRUITS 175

samples is associated with molecules other than centrifugation were combined and filtered through a vitamin C7. Anthocyanins are a group of widespread Whatman no.1 filter paper, after which the filtrate was natural phenol compounds in plants. As a major sub- evaporated to dryness at 40°C under vacuum. The group, flavonoids and anthocyanins are water-soluble extracted phenolics were dissolved in methanol water plant pigments responsible for the blue, purple and (4:1, 10 ml). red colour of many plant tissues8. Anthocyanins have Determination of anthocyanin content been reported to be strong antioxidants and may exert Total anthocyanins were estimated by a pH- a wide range of health benefits through antioxidant or differential method. Two dilutions of fruit extracts other mechanisms9. The potential for these were prepared, one with potassium chloride buffer compounds to act as antioxidants in vivo is dependent (pH 1.0) (1.86g KCl in 1 litre of distilled water, upon their bioavailability, an area currently receiving pH value adjusted to 1.0 with concentrated HCl), and much attention10. the other with sodium acetate buffer (pH 4.5) (54.43 g Dietary supplements such as vitamin C have CH CO Na·3H O in 1 litre of distilled water, pH become popular for their perceived ability to enhance 3 2 2 value adjusted to 4.5 with concentrated HCl), diluting the body’s antioxidant defenses. Reactive oxygen each by the previously determined dilution factor species (ROS) have been shown to cause a broad [fruits extracts 1:5 (v/v)]. Absorbance was measured spectrum of damage to biological systems. simultaneously at 510 and 700 nm after 15 min Scavenging of ROS is part of a healthy, well- 11 incubation at room temperature. The content of total balanced, antioxidant defense system . A high anthocyanins was expressed in mg of cyanidin-3- scavenging activity of berry extracts towards glucoside equivalents (CGE) per 100g of berries using chemically generated ROS has been described in 12 a molar extinction coefficient of cyanidin-3-glucoside several studies . In this study, the content of and molar weight (MW) (449.2 g/mol). anthocyanins, ascorbic acid, phenolics and flavoniods and the antiradical activity of selected lesser known Determination of ascorbic acid edible fruit species were evaluated. The reduction properties of ascorbic acid are used in the measurement. A basic solution is made from Materials and Methods the chopped fruit sample with phosphoric acid, which is filtered. Then Fe3+ ions are added, which are Fruit samples reduced to Fe2+ ions by ascorbic acid. Thereinafter Fresh fruits samples of Flueggea leucopyrus Willd. 2+ (Euphorbiaceae), Grewia tiliaefolia Vahl (Tiliaceae), dipridyl reagent is added to the Fe ions, which Mahonia leschenaultii (Wall. ex Wight & Arn.) results a red complex. Commercial ascorbic acid was Takeda (Berberidaceae), Gaultheria fragrantissima used as standard for the comparison. The content Wall. (Ericaceae), Ziziphus rugosa Lam. of this complex is measured by spectrophotometer (a method by Paulovicsova et al)14. (Rhamnaceae) and Rubus ellipticus Sm. (Rosaceae) were collected (Plate 1) from Palni hills of Western Determination of total phenolics Ghats during the year 2008-2009. Fruits were The total phenolics of each fruit extract were botanically identified with the help of local Flora13 determined by the Folin-Ciocalteu method. The and the herbarium specimens were compared with diluted aqueous solution of each extract (0.5 ml) was authentic sheets deposited in the Botanical Survey of mixed with Folin Ciocalteu reagent (0.2 N, 2.5 ml). India, Southern Circle, Coimbatore, India. The fresh This mixture was allowed to stand at room fruits were washed with running tap water and kept temperature for 5 min and then sodium carbonate at 20 ± 2°C until extraction. solution (75 g/l in water, 2 ml) was added. After 2 h of incubation, the absorbance was measured at Sample extraction For each fruit sample, extraction was performed 760 nm against water blank. A standard calibration using 70% aqueous methanol. Crushed fruits were curve was plotted using gallic acid (0-200 mg/l). The homogenized and 1 g of the homogenate was results were expressed as mg of gallic acid equivalents (GAE)/100 g of fruit weight. extracted three times with 10 ml of solvent by shaking vigorously for 10 min. The samples with solvent were Determination of total flavonoids centrifuged (4000 rpm for 15 min in a bench The total flavonoids were estimated according to centrifuge). The three supernatants collected after Arvouet-Grand et al15. A diluted methanolic solution 176 INDIAN J NAT PROD RESOUR, JUNE 2011

Plate 1 : a. Flueggea leucopyrus; b. Grewia tiliaefolia; c. Mahonia leschenaultia; d. Gaultheria fragrantissima; e. Ziziphus rugosa; f. Rubus ellipticus (2 ml) of each fruit extract was mixed with a solution curve, and the results were expressed as mg of (2 ml) of aluminium trichloride (AlCl3) in methanol quercetin equivalents (QE)/100 g of fruit weight. (2 %). The absorbance was read at 415 nm after 10 min against a blank sample consisting of a methanol (2 ml) DPPH radical scavenging activity and plant extract (2 ml) without AlCl3. Quercetin was The hydrogen donating ability of extracts was used as reference compound to produce the standard examined in the presence of DPPH (2,2-diphenyl-1- KARUPPUSAMY et al: ANTIOXIDANT ACTIVITY OF LESSER KNOWN EDIBLE FRUITS 177

picrylhydrazyl) stable radical16. One milliliter of positive correlation between the phytochemcial 0.3 mM DPPH ethanol solution was added to 2.5 ml contents analyzed and the antioxidant activity was sample of different fruit extracts and allowed to react established (R = 0.962) in all fruit extracts. Highest at room temperature. After 30 min, the absorbance phytochemical contents corresponded to highest values were measured at 517 nm. Methanol extract of antioxidant activity. A correlation between the each sample (1.0 mL) with water (2.5 ml) was used phenolics and flavonoid content and the antioxidant as a blank for respective samples. DPPH solution activity has been established for many herbs17,18. A (1.0 ml, 0.3 mM) with methanol (2.5 ml) served as direct correlation with total polyphenol content has negative control. The antioxidant content was been established in surveys of commercial fruits determined using a standard curve of ascorbic acid analyzed for antioxidant potential19. (0-10 µg/ml). The results were expressed as mg of Different Rubus species were recognized as berries ascorbic acid equivalent antioxidant content (AEAC) with high antioxidant activity20-22, for that reason R. per 100 g of fruit weight. ellipticus was chosen as a species to which the antioxidant activity and the polyphenol content of the Statistical analysis All data presented are means of six determinations other fruits was compared. There are data about along with standard deviations. Statistical analysis a moderate antioxidant activity of R. ellipticus and used the MS Excel software (CORREL Statistical high antioxidant activity of M. leschenaultii fresh function) to calculate quercetin, ascorbic acid and fruits, while data about the antioxidant properties of gallic acid equivalents, to determine inhibition G. tiliaefolia, Z. rugosa and F. leucopyrus are limited. percentage and to establish linear regression Our study demonstrates highest antioxidant activity equations. The SigmaStat2.0 was used to determine for two fruit species of M. leschenaultii and correlation coefficients (R). G. fragrantissima, which are 2.5 times higher than the antioxidant activity of the Z. rugosa and Results and Discussion F. leucopyrus extracts. Medium antioxidant activity Present study established the determination of of fruits extracts was observed from R. ellipticus and anthocyanin, ascorbic acid, total phenolics and flavoid G. tiliaefolia. Different antioxidant and radical level of six lesser known wild edible fruits from scavenging activity may partly be due to wide variety Western Ghats (Table 1). M. leschenaultii fruits of antioxidant constituents such as anthocyanins, showed highest level of anthocyanin, ascorbic acid, vitamins, phenolics, flavonoids and carotenoids. total phenolics and flavonoids in methanol extracts. Also types of antioxidants, inhibitors of free radicals The reduction of all four phytochemicals analysed which initiate oxidation and inhibitors of free radical were in the order: M. leschenaultii > G. chain propagation reactions are known. Different fragrantissima > R. ellipticus > G. tiliaefolia > Z. mechanisms of action and kinetics of the inhibitory rugosa > F. leucopyrus. Highest antioxidant activity effect of these antioxidants using different procedures was established for M. leschenaultii fruits resulted in the discrepancy of these findings23. Owing (361.2±3.69 AAE/g). The antioxidant activities of to the complexity of the antioxidant materials and other fruit extracts decreased almost in the order of their mechanism of actions, it is obvious that no phytochemical contents (Table 1). A high degree of single testing method is capable of providing a

Table 1  Total content of anthocyanin, ascorbic acid, phenolics and flavonoids and antioxidant activity of selected lesser known fruit extracts Fruit species Anthocyanin Ascorbic acid Total phenolics Total flavonids Antioxidant activity (CGE/100g) (AAE/100g) (GAE/100g) (QE/100g) DPPH (µg/ml) Flueggea leucopyrus 0.34 ± 0.09 39.4 ± 4.93 31.7 ± 4.92 42.7 ± 0.46 76.8 ± 1.22e Gaultheria fragrantissima 2.47 ± 0.07 67.6 ± 8.41 80.4 ± 3.18 94.3 ± 1.35 240.2 ± 1.28b Grewia tiliaefolia 2.60 ± 0.09 70.5 ± 3.07 44.1 ± 1.81 47.1 ± 0.92 120.2 ± 1.86d Mahonia leschenaultii 8.58 ± 0.02 69.9 ± 9.76 86.8 ± 0.30 95.5 ± 1.76 361.2 ± 3.69a Rubus ellipticus 1.71 ± 0.08 44.0 ± 4.95 72.0 ± 1.25 86.4 ± 2.04 196.4 ± 1.84c Ziziphus rugosa 0.34 ± 0.02 35.0 ± 3.21 41.8 ± 0.20 41.8 ± 1.20 116.9 ± 1.02d (Results are mean ± SD; n=6). *Values with the same letter are not significantly different (P> 0.05). 178 INDIAN J NAT PROD RESOUR, JUNE 2011

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