ANTIOXIDANT ACTIVITIES OF SOME TROPICAL FRUITS

Tan Tze Guan1, Matthew Whiteman2 ABSTRACT There has been growing interest in the beneficial health effects of edible fruits and vegetables, as well as certain beverages such as wines and teas. Studies have shown that increased consumption of fresh fruits and vegetables is associated with a lower incidence of disease, particularly of degenerative ailments linked to the ageing process, such as cancer, cardiovascular disease, and Alzheimers disease. Their protective mechanisms are thought to be attributed to the presence of natural antioxidants, such as ascorbates, catechins, and flavanoids, which help to scavenge free radicals and reactive oxidants in the body. The antioxidant activities of three local fruits durian (durios zibethinus), rambutan (nephelium lappaceum), and mangosteen (garcinia mangostana) were investigated by means of the ABTS (2,2-azinobis-[3-ethylbenzthiazoline-6-sulphonic acid]) and DPPH (1,1-diphenyl-2picrylhydrazyl) assays. The TEAC (Trolox Equivalent Antioxidant Capacity) and IC50 values of the fruit extracts were calculated and compared. It was found that the mangosteen extract displayed the greatest antioxidant activity, followed by rambutan and durian. INTRODUCTION Radicals are chemical species with one or more unpaired electrons, and free radicals are radicals that have moved out of the immediate molecular environment of their generation. There are several endogenous sources of oxidants in the body: reduction of molecular oxygen in mitochondria during cellular respiration takes place in sequential steps, yielding the radical by-products superoxide O2-, hydroxyl HO, and hydrogen peroxide H2O2; degradation of fatty acids and other molecules in peroxisomes produce H2O2; phagocytosis results in an oxidative burst of nitric oxide (NO), which also reacts with superoxide to produce the oxidizing and nitrating species peroxynitrite (ONOO-) [1]. Free radicals and oxidants can trigger lipid peroxidation, as well as the oxidation of proteins and DNA, causing extensive damage to body cells. Oxidative stress resulted from an imbalance of oxidising species and natural antioxidants in the body has been thought to have contributed to aging, cell apoptosis, and severe diseases such as cancer, Parkinsons disease, Alzheimers disease, and even cardiovascular disorders [2]. Epidemiological studies and intervention trials on prevention of cancer and cardiovascular disease in people taking antioxidant supplements are suggestive that dietary intake of antioxidants can help scavenge free radicals and oxidants and protect the body against diseases [1]. It is also clear that most of the dietary antioxidants have low or minimal toxicity, and that intakes can be increased without adverse effects [3]. Many fresh fruits and vegetables have been found to contain natural antioxidants, mainly phenolic compounds such as ferulic acid, catechins, as well as ascorbic acid (vitamin C) [4]. The three fruit extracts used in this study are derived from tropical fruits durian (durios zibethinus), rambutan (nephelium lappaceum), and mangosteen (garcinia mangostana), all of which can be easily cultivated and obtained locally. These fruits are also consumed by a fairly large sector of the population in the tropics (Southeast Asia), especially the durian which is commonly referred to as the King of fruits. As such, these fruits have great potential to serve as alternative dietary antioxidants for many people should they be found to show considerable radical and oxidant scavenging abilities.
1 2

SRP Student, Raffles Junior College Department of Biochemistry, Faculty of Medicine, National University of Singapore

The TAA (total antioxidant activity) of the fruits was determined using the ABTS and DPPH methods. The ABTS assay involves the oxidation of ABTS (2,2-azinobis[3-ethylbenzothiazoline-6sulphonate]) to an intensely-coloured nitrogen-centred radical cation, ABTS+ (Fig. 1). It is thus useful for testing food extracts because ABTS+ has an absorption maxima at 734 nm and most food extracts are highly-coloured but do not absorb light at 734 nm [5]. It is also viable for both aqueous and lipophilic systems. The DPPH assay made use of a stable free radical DPPH (1,1-diphenyl-2picrylhydrazyl). The reaction involves a colour change from violet to yellow that can be easily monitored using a spectrophotomer at 520 nm [6].

Fig. 1: Oxidation of ABTS to ABTS+ radical MATERIALS AND METHODOLOGY All chemicals were obtained from Sigma-Aldrich Pte-Ltd, Singapore, except for ethanol. Fruit extracts were obtained by the following method: 50 +1 g of flesh of fruits mashed using a blender, then dissolved in 80% methanol and repeatedly stirred and filtered for at least three times; the fruit extracts (in solution) were concentrated to standard stocks of 2.5 g ml-1. The extracts were sonicated and vortexed to enhance their solubility in pure methanol. ABTS Assay The ABTS assay (Re et al) [5] was employed to measure the antioxidant activity of the fruit extracts. ABTS was dissolved in deionised water to 7 mM concentration, and potassium persulphate added to a concentration of 2.45 mM. The reaction mixture was left to stand at room temperature overnight (12 to 16 h) in the dark before usage. The resultant intensely-coloured ABTS+ radical cation was diluted with 0.01 M PBS (phosphate buffered saline), pH 7.4, to give an absorbance value of ~0.70 at 734 nm. The test compound (fruit extract) was diluted 100x with the ABTS+ solution to a total volume of 1 ml. Absorbance was measured spectrophotometrically at time intervals of 1, 2, 5, 10, and 15 min after addition for a range of four to eight concentrations for each extract. The assay was performed at least in triplicates. Controls without ABTS+ were used to allow for any absorbance of the extracts themselves, and 990 l of PBS was added to these control samples instead [3]. Fresh stocks of ABTS+ solution were prepared every five days due to self-degradation of the radical. The assay was first carried out on Trolox, the water-soluble -tocopherol (vitamin E) analogue, which served as a standard. The results of the assay were expressed relative to Trolox in terms of TEAC (Trolox equivalent antioxidant capacity) [5].

DPPH Assay DPPH was dissolved in 80% ethanol to 200 M and sonicated for 5 min to obtain the stable free radical DPPH. The test compound was diluted in the ratio 1:1 with the DPPH solution in a 96-well microplate. Appropriate controls were run in each series. Fresh DPPH solution was prepared daily. Each fruit extract was tested in triplicate at three concentrations, such that a 50% fall in absorbance of the DPPH can be calculated. The absorbance of the reaction mixture was measured after 25 min using a microplate spectrophotometer [6]. The IC50 (concentration causing 50% inhibition) value of each extract was determined and compared with the corresponding TEAC. RESULTS Trolox Equivalent Antioxidant Capacity The ABTS assay was calibrated with the water-soluble -tocopherol analogue, Trolox (Fig. 2). All three fruit extracts displayed antioxidant activities as they were able to scavenge the ABTS+ radical cation. All three extracts displayed a near-linear variance between the concentration of extract added and the corresponding fall in absorbance (Fig. 3). The TEAC of each extract at each time interval was determined and plotted (Fig. 4). Since TEAC is a measurement of the effective antioxidant activity of the extract, a higher TEAC would imply greater antioxidant activity of the sample. It was observed that at the 15 min point, garcinia mangostana had the highest TEAC of 24.84 (Table 1), followed by nephelium lappaceum (5.97), and durios zibethinus (3.39). In effect, the TAA of garcinia mangostana is nearly four times that of nephelium lappaceum and seven times that of durios zibethinus, both of which have relatively low TEAC. Moreover, the TEAC of each extract was calculated from a range where the fall in absorbance is proportional to the concentration of extract at 15 min. At this point, ABTS+ depletion for garcinia mangstana had ceased, and reaction with Trolox for the other two extracts was nearly complete. Thus the values of TEAC for the three extracts could be underestimates.
Graph of Fall in absorbance against Concentration of Trolox
0.8 0.7

Fall in absorbance

0.6 0.5 0.4 0.3 0.2 0.1 0 0 5 10 15 20 25 30

Trolox concentration/micromole

Fig. 2: Trolox standard curve. The results of the ABTS assay are expressed in comparison to Trolox.

Graph of Fall in absorbance against Concentration of Durio zibethinus extract (1 to 15 min)

0.8
0.7

Graph of Fall in absorbance against Concentration of Nephelium lappaceum extract (1 to 15 min)

Fall in absorbance

0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0 5 7.5 10 15 20 25

Fall in absorbance Concentration of durian extract/mg per ml

0.6 0.5 0.4 0.3 0.2 0.1 0 0 2.5 5 6.25 7.5 8.75 10 12.5

Concentration of rambutan extract/mg per mL

1 min 2 min 5 min 10 min 15 min 1 min 2 min 5 min 10 min 15 min

Graph of Fall in absorbance against Concentration of Garcinia mangostana extract (1 to 15 min)

0.8 0.7

Fall in absorbance

0.6 0.5 0.4 0.3 0.2 0.1 0 0 1 2 3 4 5 6 7 8

Concentration of mangosteen extract/mg per mL

1 min 2 min 5 min 10 min 15 min

Fig. 3: Concentration-response curves for inhibition of the absorbance of ABTS+ cation at 734 nm at five different time points (1, 2, 5, 10, 15 min) for the three fruit extracts.
Graph of TEAC against time
25

TEAC (mM Trolox)

20 15 10 5 0 0 5 10 15 20

Time (min)
Durios zibethinus Nephelium lappaceum Garcinia mangostana

Fig. 4: The variation of the Trolox Equivalent Antioxidant Capacity of each extract with time (up to 15 min) Inhibition (50 per cent) by fruit extracts The change in colourisation from violet to yellow and subsequent fall in absorbance of the stable radical DPPH was measured at 515 nm for three concentrations, and the results were plotted (Fig. 5). The IC50 value for each fruit extract, defined as the concentration of extract causing 50 per cent inhibition of absorbance, was determined from the curves plotted and tabulated (Table 1). Since IC50 is a measure of inhibitory concentration, a lower IC50 value would reflect greater antioxidant activity of the sample. Hence garcinia mangostana once again displayed the highest TAA with the lowest IC50 of 4.85. Extracts of durios zibethinus and nephelium lappaceum had comparatively high IC50 values,

with nephelium lappaceum exhibiting a slightly higher TAA than durios zibethinus. The IC50 of both durios zibethinus and nephelium lappaceum were nearly eight-fold that of garcinia mangostana.
Graph of Fall in absorbance against Concentration of extract
0.8

Fall in absorbance

0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0 10 20 30 40 50 60

Concentration of extract/mg per ml

Durios zibethinus Nephelium lappaceum Garcinia mangostana

Fig. 5: Scavenging capacity of the three extracts on DPPH determined by measuring absorbance of the reaction mixture at 515 nm 25 min after addition Fruit Durios zibethinus Nephelium lappaceum Garcinia mangostana TEAC (mM Trolox) 3.39 5.97 24.84 IC50 (mg/ml) 41.6 35.6 4.85

Table 1: Antioxidant capacities as TEAC (in the ABTS+ decolourisation assay) and IC50 values (in the DPPH assay). The assay was performed at least in triplicate for at least three concentrations. Results expressed as TEAC were determined at 15 min. DISCUSSION The results of both assays are in agreement that the extract of garcinia mangostana displayed the highest antioxidant capacity, followed by nephelium lappaceum, then durios zibethinus. It was also noted that the difference between the TEAC and IC50 values of nephelium lappaceum and durios zibethinus is slight (Table 1), indicating approximate TAA for both fruits, whilst those of garcinia mangostana were significantly higher. In comparison with other fruits, vegetables, and beverages, the TEAC of durios zibethinus and nephelium lappaceum are fairly low. Berries, especially blackcurrants, and grapes have been reported to be extremely potent antioxidants, with TEAC values reaching 500 mM Trolox/kg, and red wines have shown ABTS+ scavenging capacities equivalent to 12 to 24 mM Trolox [4]. Extracts of garcinia mangostana, with a TEAC of 24.84 and IC50 of 4.85, thus prove that the fruit has considerable potential to serve as an effective dietary antioxidant. Both the ABTS and DPPH assays measure the TAA of the fruit extracts in an organic medium (since all extracts were prepared in methanol). It is important to determine the relative antioxidant capacity of the fruits in both aqueous and lipophilic mediums since free radicals and oxidants in the body commonly cause damage to aqueous-based cellular structures and organelles as well as lead to peroxidation of lipids. However, the species of free radicals and oxidants scavenged by the fruit extracts and the antioxidant compounds present cannot be elucidated using these two assays. Based on knowledge gathered from previous experiments, ascorbic acid (vitamin C) and tocopherols (vitamin E) are major sources of antioxidants in fruits and vegetables [1, 7]. Great focus has also been made on the powerful antioxidant activities of phenolic constituents such as catechins, carotenoids, flavanoids, as well as anthocyanins. Similarly, it can be deduced that the antioxidant activities of the three fruits

can also be largely attributed to these phenolic compounds. The findings of this study are also limited because the TAA of the extracts was only measured in vitro. Bioavailability of the antioxidants in these fruits cannot be ascertained, and thus the efficacy of their antioxidant activities in vivo cannot be accurately determined too. ACKNOWLEDGEMENTS I would like to extend my gratitude to my mentor, Dr Matthew Whiteman for his guidance and advice throughout the course of my work, my teacher-advisor Mr Tay Ming Chee, as well as those in the Antioxidants Research laboratory who have helped me in one way or another. REFERENCES [1] Frei, B. (1994) San Diego: Academic Press. Natural antioxidants in human health and disease. [2] Giasson, B. I., Ischiropoulos H., Lee V. M. Y., and Trojanowski J. Q. (2002) The relationship between oxidative/nitrosative stress and pathological inclusions in Alzheimers and Parkinsons diseases. Free Radical Biology and Medicine 32, 1264-1275. [3] Johnson I. T., and Fenwick G. R. UK: The Royal Society of Chemistry. Dietary anticarcinogens and antimutagens: chemical and biological aspects. [4] Long L. H., Chua D. T. K., and Halliwell B. The antioxidant activities of seasonings used in Asian cooking. Powerful antioxidant activity of dark soy sauce revealed using the ABTS assay. Free Radical Biology and Medicine 32, 181-186 [5] Re R., Pellegrini N., Proteggente A., Pannala A., Yang M., and Rice-Evans C. (1999) Antioxidant activity applying an improved ABTS radical cation decolourisation assay. Free Radical Biology and Medicine 26, 1231-1237. [6] Choi D. W., Leininger-Muller B., King Y. C., Leroy P., Siest G., and Wellman M. (2002) Differential role of CYP2E1 binders and isoniazid on CYP2E1 protein modification in NADPHdependent microsomal oxidative reactions: FR scavenging ability of isoniazid. Free Radical Research 36, 893-904 [7] Packer L., Hiramatsu M., and Yoshikawa T. (1999) San Diego: Academic Press. Antioxidant food supplements in human health