Chemical composition and in vitro antioxidant studies on Syzygium cumini fruit

Chemical composition and in vitro antioxidant studies on Syzygium cumini fruit Journal of the Science of Food and Agriculture J Sci Food Agric 87 2560–2569 (2007) Chemical composition and in vitro ant[.]

Journal of the Science of Food and Agriculture J Sci Food Agric 87:2560–2569 (2007) Chemical composition and in vitro antioxidant studies on Syzygium cumini fruit Palayyan Saraswathy Benherlal and Chami Arumughan∗ Agro-Processing and Natural Products Division, Regional Research Laboratory, CSIR, Trivandrum-695 019, India Abstract: Syzygium cumini, widely known as Jamun, is a tropical tree that yields purple ovoid fleshy fruit Its seed has traditionally been used in India for the treatment of diabetes Based on the available ethno-pharmacological knowledge, further studies were extended to understand the chemical composition and antioxidant activities of three anatomically distinct parts of fruit: the pulp, kernel and seed coat Fruit parts, their corresponding ethanol extracts and residues were evaluated for chemical composition The alcoholic extract was evaluated for its antioxidant potential against DPPH• , OH• , O2 •− and lipid peroxidation The whole fruit consisted of 666.0 ± 111.0 g kg−1 pulp, 290.0 ± 40.0 g kg−1 kernel and 50.0 ± 15.0 g kg−1 seed coat Fresh pulp was rich in carbohydrates, protein and minerals Total fatty matter was not significant in all three parts of fruit Detailed mineral analysis showed calcium was abundant in all fruit parts and extracts Total phenolics, anthocyanins and flavonoid contents of pulp were 3.9 ± 0.5, 1.34 ± 0.2 and 0.07 ± 0.04 g kg−1 , respectively Kernel and seed coat contained 9.0 ± 0.7 and 8.1 ± 0.8 g kg−1 total phenolics respectively Jamun pulp ethanol extract (PEE), kernel ethanol extract (KEE) and seed coat ethanol extract (SCEE) showed a high degree of phenolic enrichment DPPH radical scavenging activity of the samples and standards in descending order was: gallic acid > quercetin > Trolox > KEE > BHT > SCEE > PEE Superoxide radical scavenging activity (IC50 ) of KEE was six times higher (85.0 ± 5.0 µg mL−1 ) compared to Trolox (540.0 ± 5.0 µg mL−1 ) and three times compared to catechin (296.0 ± 11.0 µg mL−1 ) Hydroxyl radical scavenging activity (IC50 ) of KEE was 151.0 ± 5.0 µg mL−1 which was comparable with catechin (188.0 ± 6.0 µg mL−1 ) Inhibition of lipid peroxidation of the extracts was also studied and their activity against peroxide radicals were lower than that of standard compounds (BHT, 79.0 ± 4.0 µg mL−1 ; quercetin, 166.0 ± 13.0 µg mL−1 ; Trolox, 175.0 ± 4.0 µg mL−1 ; PEE, 342.0 ± 17.0 µg mL−1 ; KEE, 202.0 ± 13.0 µg mL−1 and SCEE, 268.0 ± 13.0 µg mL−1  2007 Society of Chemical Industry Keywords: Syzygium cumini; Jamun; chemical composition; antioxidant activity INTRODUCTION Free radicals are constantly generated in all living organisms as a result of metabolic activities that are presumed to trigger degenerative diseases: arthritis, coronary heart disease (CHD), diabetes, cataract, cancer, for example.1 – Apart from a range of harmful effects, they are also involved in numerous cellular processes such as vasodilation, signal transduction, gene expression, cell differentiation and development.6 – When the free radical production in a system exceeds its clearance, the sites of radical production undergo severe oxidative stress and damage various micro and macro molecules in the vicinity Antioxidants of endogenous and exogenous sources function as defence against oxidative stress by scavenging the excess free radicals and maintain the redox status Apart from the endogenous antioxidants, there is an array of non-nutrient exogenous antioxidants of plant origin; some of them are powerful free radical scavengers (e.g gallates, catechins) The exogenous antioxidants in biological systems should be a chemical substance(s) which when present at lower concentration, in relation to reactive oxygen species (ROS), significantly inhibit or delay tissue damage, while often being oxidised themselves The antioxidants can function either as chain breaking agents or in mechanisms involved in removal of ROS initiator Antioxidants are also reported to regulate expression of certain genes in response to cellular redox status.9 It has also been shown that polyphenols with relatively high antioxidant potential are able to induce translation of some mRNA.10 Consequent to the evidence on the ability of some antioxidants for their chemopreventive and therapeutic properties, the search for such functional antioxidants has actively been pursued Plants are rich in different antioxidants and many of them act together by different mechanisms to provide defense against free radical attack.11,12 Detailed understanding of these natural sources in terms of chemical composition, active molecules and ethno medicine would provide some information on their potential therapeutic uses For the present study Syzygium cumini (Skeels) popularly known as Jamun has been selected based on its use as antidiabetic agent in Indian traditional healthcare system, viz Ayurveda, folk medicine and ∗ Correspondence to: Chami Arumughan, Agro-Processing and Natural Products Division, Regional Research Laboratory, CSIR, Trivandrum-695 019, India E-mail: carumughan@yahoo.com Contract/grant sponsor: CSIR, India (Received April 2006; revised version received 26 January 2007; accepted February 2007) Published online 11 September 2007; DOI: 10.1002/jsfa.2957  2007 Society of Chemical Industry J Sci Food Agric 0022–5142/2007/$30.00 Composition and antioxidant studies on S cumini fruit tribal medicine S cumini is an evergreen tree distributed in the Indian sub-continent and southeast Asian countries The oval shaped fruit is about 2–3 cm long and has deep purple coloured fleshy pulp with a hard seed inside The fruit has delicate astringent taste and resembles blueberry in shape and colour Traditionally, this fruit has been used as an astringent, carminative, stomachic, antiscorbutic and diuretic Apart from the traditional knowledge about the therapeutic properties of Jamun fruit, investigation on the chemical and biochemical studies has been reported recently Acute, sub-acute and chronic antiinflammatory activities for the ethanolic extracts of S cumini bark have been investigated using rat models.13 A hypoglycaemic effect of S cumini leaves and antipyretic and antioxidant activities for Jamun seed have also been reported recently.14 – 18 Perusal of the previous reports revealed that a comprehensive approach to the chemical analysis and antioxidant studies is lacking for anatomically distinct parts of Jamun fruit The present investigation has therefore been designed to establish the chemical composition and antioxidant activities of different parts of the fruit and the result presented here is the first of the series MATERIALS AND METHODS Chemicals Xanthine, xanthine oxidase, thiobarbituric acid, 1,1diphenyl-2-picrylhydrazyl (DPPH), nitro-blue tetrazolium (NBT), tert-butyl hydroperoxide (t-BHP), quercetin, catechin, gallic acid and vitamin C were purchased from Sigma-Aldrich (St Louis, MO, USA) All other common chemicals and solvents were analytical grade and obtained from Merck (Mumbai, India) Sample preparation Fresh and fully ripened Jamun fruits were collected from three different locations of Thiruvananthapuram district of Kerala (a province in southern India) The fruits were washed and stored at −20 ◦ C in sealed polypropylene bags for future use The sample preparation scheme for composition analysis and antioxidant studies is depicted in Fig In order to obtain data on the anatomical parts, 500 g of fruits collected from each location were separated in to pulp (JP), kernel (JK) and seed coat (JSC) and yield of the parts was recorded separately for each location The data was subjected to one-way ANOVA to obtain the variations in the ratio of anatomical parts in the fruit For all the subsequent analysis, the corresponding anatomical parts from the three locations were pooled separately The pooled samples were used for ethanol extraction, composition analysis and antioxidant studies in triplicates Five hundred grams of the fresh samples (JP, JK and JSC) were extracted with ethanol with materialto-solvent ratio of 1:2 (w/v) The extraction was conducted at ambient temperature (25–30 ◦ C) under stirring for h At the end of the extraction, the J Sci Food Agric 87:2560–2569 (2007) DOI: 10.1002/jsfa Figure Scheme for separation and extraction of anatomical parts of Jamun fruit, their composition analysis and antioxidant studies slurry was filtered through muslin cloth to separate the ethanol fraction from the solid debris The extraction was repeated five times and the ethanol fractions were pooled The pooled extract was then centrifuged at 7500 × g for 10 and the supernatant was passed through Whatman 41 (pore size 20–25 µm) The clear extract thus obtained was concentrated under vacuum at 55–65 ◦ C using a rotary evaporator to dryness The sample from each anatomical part was prepared separately as mentioned above for chemical analysis and antioxidant activity studies The dried samples were reconstituted in ethanol (10 mg mL−1 ) and the samples thus prepared of pulp (PEE), kernel (KEE) and seed coat (SCEE) for evaluating antioxidant activity The residues from pulp (RP), kernel (RK) and seed coat (RSC) obtained after extraction were dried in shade and subjected to composition analysis Composition analysis Moisture, crude protein, crude fibre, starch and minerals (ash, Na, K, Ca and P) were estimated by the standard procedure of the AOAC.19 Ethanol soluble carbohydrate was determined by the anthrone method.20 Estimation of total phenolic compounds Total phenolic composition was determined using Folin–Ciocalteu reagent and expressed as gallic acid equivalent (GAE).21 The samples and standard gallic acid were diluted to 2–20 µg in 2.0 mL distilled water and 2.0 mL of commercial Folin–Ciocalteu reagent was added The content was mixed well and kept for at room temperature followed by addition of 2.0 mL of 10% aqueous sodium carbonate and incubated at room temperature for h Absorbance of the developed blue colour was read at 760 nm (Shimadzu UV-2450, Shimadzu Corporation, Kyoto, Japan) against a reagent blank Estimation of anthocyanins Anthocyanins of the whole fruit pulp were extracted with acidic methanol (0.1% HCl).22 Total monomeric 2561 P S Benherlal, C Arumughan anthocyanins in the extract were estimated by the pH differential method and expressed in cyanidin-3glucoside equivalency, where the MW of cyanidin-3glucoside is 449.2 and molar absorptivity is 26 900.23 Ten millilitres of extracted anthocyanin was made up to 50.0 mL using 0.025 mol L−1 potassium chloride buffer, pH 1.0 and 0.4 mol L−1 sodium acetate buffer, pH 4.5, separately The buffered anthocyanin extract was allowed to equilibrate for 15 at room temperature The absorbance of each buffered sample was measured at 520 nm (Shimadzu UV-2450) against a blank cell with distilled water The concentration of monomeric anthocyanin pigment (mg L−1 ) in the final solution was calculated using the formula A × MW × DF × 1000 ε×1 where A is absorbance, MW is molecular weight, DF is dilution factor, and ε is molar absorptivity Estimation of flavonoids Quantitative determination of flavonoids was performed by two complementary colorimetric methods: the aluminium chloride method and the 2,4-dinitrophenyl hydrazine method (2,4-DNPH) For the quantitative estimation of total flavonoids in the whole Jamun fruit, the extraction procedure described by Chang et al.24 was performed Aluminium chloride method Ten to 100 µg mL−1 of quercetin standard and appropriately diluted samples in 80% ethanol were taken in different test tubes (0.5 mL) and made up to mL with 95% ethanol followed by the addition of 0.1 mL of 10% aluminium chloride, 0.1 mL of mol L−1 potassium acetate and 2.8 mL of distilled water and incubated at room temperature (30–34 ◦ C) for 30 The intensity of colour developed was read at 415 nm (Shimadzu UV-2450) against a reagent blank Dinitrophenyl hydrazine method The reference standard used in this assay was naringenin Five hundred, 1000, 1500 and 2000 µg of naringenin and 100–1000 µg of extracts were made up to 1.0 mL with methanol in separate test tubes Then, 2.0 mL of 1% 2,4-DNPH reagent and 2.0 mL of methanol were added to the reaction system and the constituents were mixed thoroughly The tubes were stoppered and incubated at 50 ◦ C for 50 in a constant temperature water bath After incubation the tubes were cooled and 5.0 mL of 1.0% potassium hydroxide (1.0 g potassium hydroxide in 100 mL 70% methanol) was added Finally, 1.0 mL of the reaction mixture was taken from each tube and mixed with 5.0 mL methanol The precipitates formed were removed by centrifugation at 7500 × g for 10 The supernatant was collected and adjusted to 25.0 mL and the absorbance of the final solution was measured at 415 nm (Shimadzu UV-2450) against the blank Total 2562 flavonoid was expressed as the sum of % flavonoid obtained in each method DPPH radical scavenging activity DPPH radical scavenging activity was estimated according to the method of Brand-Williams et al.25 The assay contained 2.9 mL of 0.1 mmol L−1 DPPH in ethanol and 0.1 mL of various concentrations of extracts and standards in the same solvent and were taken in a glass cuvette The contents were mixed well immediately and the degree of reduction of absorbance was recorded for 30 in an UV–visible spectrophotometer at 517 nm (Shimadzu UV-2450) Optical densities at time zero (OD t0 ) and at 30 (OD t30 ) were used for calculating percentage radical scavenging activity Percentage radical scavenging activity was plotted against the corresponding antioxidant substance concentration to obtain the IC50 value, which is defined as the amount of antioxidant material required to scavenge 50% of the free radicals in the assay system IC50 values are inversely proportional to the antioxidant potency Superoxide radical scavenging activity Superoxide radical scavenging activity study was performed according to the method of Parejo et al.26 using the xanthine–xanthine oxidase system Fifty to 250 micrograms of appropriately diluted samples and standards (catechin, Trolox and gallic acid) were taken in a 1.0 mL cuvette and xanthine was added to obtain a final concentration of 0.2 mmol L−1 Sixtythree microlitres of 1.0 mmol L−1 NBT was added to the reaction system and the final volume was made up to 1.0 mL with phosphate buffer (50 mmol L−1 , pH 7.5) excluding the volume of enzyme Sixtythree microlitres of xanthine oxidase (1.2 U µL−1 ) was added to the system and mixed well to start the reaction The blue colour developed by the reduction of NBT by superoxide radicals was measured at 560 nm for 15 (Shimadzu UV-2450) A blank was prepared without sample and standards are considered as 100% radicals A decrease in NBT reduction in the presence of added antioxidant extract and standard compounds was monitored and % radical scavenging activity (RSA) was calculated by the formula 
Asample × 100 RSA = − Ablank where the RSA is in %, Asample is the OD of the sample or standard, and Ablank is the OD of the blank Hydroxyl radical scavenging activity Hydroxyl radical scavenging activity was studied according to the method of Klein et al.27 Different concentrations of appropriately diluted extracts and standards (vitamin C, BHT, gallic acid, Trolox, catechin and quercetin) were taken in a series of test tubes and the following reagents were added: 1.0 mL iron EDTA solution (0.13% ferrous ammonium J Sci Food Agric 87:2560–2569 (2007) DOI: 10.1002/jsfa Composition and antioxidant studies on S cumini fruit sulfate and 0.2% EDTA), 0.5 mL EDTA (0.018%) and 1.0 mL phosphate buffered dimethyl sulfoxide (DMSO) (0.855% DMSO in 0.1 mmol L−1 phosphate buffer, pH 7.4, v/v) The contents were mixed well and the reaction was started by adding 0.5 mL 0.22% ascorbic acid All tubes were closed and heated in a constant temperature water bath at 90 ◦ C for 15 The reaction was stopped by adding 1.0 mL 17.5% ice cold trichloroacetic acid (TCA) Finally 3.0 mL of Nash reagent (75.0 g ammonium acetate, 3.0 mL glacial acetic acid and 2.0 mL acetyl acetone were mixed and made up to 1.0 L with distilled water) was added and kept at room temperature (30–34 ◦ C) for 15 to develop colour The yellow colour developed was read at 412 nm (Shimadzu UV-2450) against a reagent blank Percentage of radical scavenging activity was calculated by measuring decrease in optical density in the presence of added radical scavenger with reference to blank Inhibition of lipid peroxidation Inhibition of lipid peroxidation was assessed using the red blood cell model system as described by Manna et al.28 Heparinised whole blood was collected from healthy volunteers The blood was centrifuged for 10 at 1000 × g to separate plasma and red blood cells (RBCs) After removing plasma and buffy coat, the packed RBCs were resuspended in isotonic saline and washed several times to remove plasma protein Finally, the RBCs were resuspended to a final concentration of 5% (v/v) in isotonic saline The assay system contained a final strength of 2.0% RBC suspension, appropriately diluted extract and 500 µmol L−1 t-BHP The final volume was made up to 5.0 mL with isotonic saline and incubated at 37 ◦ C in a water bath for h After oxidative treatment, the tubes were centrifuged at 1000 × g for 10 to separate RBCs Two millilitres of the cell-free supernatant was collected and mixed with 1.0 mL of 30% (w/v) trichloroacetic acid The tubes were gently mixed and further centrifuged for 15 at 5000 × g Two millilitres of the supernatant was collected and added 0.5 mL 1% (w/v) thiobarbituric acid (TBA) in 0.05 mol L−1 NaOH The mixture was heated in boiling water bath for 10 to develop colour The absorbance of pink chromogen developed was read at 532 nm (Shimadzu UV-2450) against a reagent blank Percentage reduction of pink colour (inhibition of lipid peroxidation) in the presence of added standard antioxidants and samples with reference to blank was plotted against the concentration to get IC50 values Total reducing power Total reducing power was estimated according to Zhu et al.29 The reaction system consist of appropriately diluted (100–500 µg) extracts in 1.0 mL of water, 2.5 mL of phosphate buffer (0.2 mmol L−1 pH 6.6) and 2.5 mL of 1% potassium ferricyanide The reaction system was closed and incubated at 50 ◦ C in a water bath for 30 After the incubation J Sci Food Agric 87:2560–2569 (2007) DOI: 10.1002/jsfa period 2.5 mL 10% TCA was added to the assay system and the contents were mixed well The mixture was centrifuged at 3000 × g for 30 to remove precipitate Supernatant (2.5 mL) was collected and mixed with 2.5 mL of distilled water and 0.5 mL 0.1% ferric chloride The colour developed was read at 700nm (Shimadzu UV-2450) against a reagent blank Statistical analysis Sample collection was performed as described above and subjected to one-way ANOVA to understand the variation in the content of anatomical parts in the fruit collected from different locations For subsequent composition analysis and antioxidant studies representative fruit samples were taken from each location and pooled From the pooled fruits, samples were taken for composition analysis and activity studies and the results were analysed for standard error All experiments were repeated three times and the results were reported as mean ± SEE (standard error of estimate) Statistical analysis was performed using Microsoft Excel RESULTS AND DISCUSSION Composition analysis The mean yield of pulp, seed coat and kernel of fully ripened Jamun fruits with an average weight of 6.0 ± g is shown in Table The mean anatomical constituents observed in Jamun fruit collected from three different locations were significantly different (P < 0.05) The fully ripened Jamun fruit studied here had 666.0 ± 111 g kg−1 pulp, 290.0 ± 40 g kg−1 kernel and 50.0 ± 15 g kg−1 seed coat on fresh weight JP, JK and JSC were subjected to ethanol extraction and further analysis was performed on the anatomical parts, extracts and residue and the results are expressed on dry weight Composition of anatomical parts Chemical composition of anatomical parts of fresh Jamun fruit is shown in Table While the fresh pulp (JP) had the highest moisture content, (850.0 ± 40.0 g kg−1 ) JSC recorded the lowest (100.0 ± 20.0 g kg−1 ) In terms of quantity, bulk of the fruit parts was composed of starch, soluble sugars, fibre and protein with negligible amount of fat Alcohol extracted most of the soluble sugars and minerals Table Yield of anatomical parts of fresh Jamun fruit Anatomical part Yield (g kg−1 ) Pulp Kernel Seed coat 666.0 ± 111.0 290.0 ± 40.0 50.0 ± 15.0 The samples were collected from three different locations and subjected to one-way ANOVA The mean values for anatomical parts (pulp, kernel and seed coat) were significantly different (P < 0.05) All values are expressed in mean ± SEE 2563 P S Benherlal, C Arumughan Table Chemical composition of fruit parts, their ethanol extract and residue Sample JP PEE RP JK KEE RK JSC SCEE RSC Moisturea TFMb Proteinb TESCb Crude fibreb Starchb Ashb 850.0 ± 40 – – 470.0 ± 30 – – 100.0 ± 20 – – 16.0 ± 2.0 Tr 36.0 ± 4.0 3.5 ± 0.1 Tr 4.5 ± 0.4 5.0 ± 0.7 Tr 4.7 ± 0.6 66.0 ± 6.0 13.0 ± 0.25 100.0 ± 7.5 68.0 ± 0.4 8.0 ± 0.5 78.0 ± 2.2 160.0 ± 5.0 9.0 ± 0.3 138.0 ± 0.4 400.0 ± 33.0 520.0 ± 9.0 139.0 ± 7.0 120.0 ± 8.0 600.0 ± 10.0 201.0 ± 6.0 210.0 ± 7.0 710.0 ± 13.0 69.0 ± 1.3 7.0 ± 13.0 ND 20.0 ± 2.0 29.0 ± 2.0 ND 27.1 ± 1.1 120.0 ± 6.0 ND 146.0 ± 5.0 350.0 ± 20.0 – 672.0 ± 23.0 600.0 ± 33.0 – 616.0 ± 15.5 430.0 ± 15.0 – 603.0 ± 65.0 45.0 ± 0.6 40.0 ± 3.5 26.0 ± 1.8 20.0 ± 1.0 17.0 ± 0.3 18.0 ± 1.0 25.0 ± 0.7 16.0 ± 0.8 24.0 ± 0.4 All results are given as g kg−1 a Fresh basis; b dry basis JP, Jamun pulp; PEE, pulp ethanol extract; RP, residual pulp; JK, Jamun kernel; KEE, kernel ethanol extract; RK, residual kernel; JSC, Jamun seed coat; SCEE, seed coat ethanol extract; RSC, residual seed coat; TFM, total fatty matter; TESC, total 80% ethanol soluble carbohydrate Tr, trace; ND, not detected (n = ± SEE) present in the fruit parts, the alcohol extract therefore was enriched with these constituents in terms of quantity The residue obtained after alcohol extraction contained mostly fibre and fatty matter Results of mineral composition (Fig 2) indicated that JP was rich in total minerals (45.0 ± 0.6 g kg−1 ) on dry weight followed by JSC (25.0 ± 0.7 g kg−1 ) and JK (20.0 ± 1.0 g kg−1 ) Among the minerals the most abundant were Ca and K in all the fruit parts and that indicates that the edible part of fruit (JP) is a rich source for these essential minerals Alcohol extracts of these fruit parts showed a similar trend in their mineral content as those of the whole fruit parts Composition of the bioactive compounds in Jamun fruit parts is shown in Table Cyanidin-3-glucoside equivalent anthocyanin content in JP was 1.34 ± 0.2 g kg−1 and the PEE contained 3.20 ± 0.27 g kg−1 The cyanidin-3-glucoside equivalent anthocyanins were not detected in KEE and SCEE Total flavonoids estimated by the two complementary methods: the aluminium chloride method (specific for flavones flavonols and isoflavones) and the 2,4-DNPH method (specific for flavonones) JP and JK were found to contain flavonols, flavones and isoflavones and they also contained flavonones as estimated by the above methods While the JSC contained a comparable amount of aluminium chloride reactive flavonoids as observed in JK, the 2,4-DNPH reactive flavonoids were not found in detectable amount in JSC Total phenolic content in JK and JSC were 9.0 ± 0.7 g kg−1 and 8.1 ± 0.8 g kg−1 , respectively, which were almost two-fold higher than that of JP (3.9 ± 0.5 g kg−1 ) The presence of anthocyanins in fully ripened Jamun pulp has previously been reported, namely cyanidin, petunidin and malvidin.30 In another study by the analysis of anthocyanin was limited to the skin of Jamun fruit The results of these studies, obviously, are not comparable with those of the present study.18 Total flavonoid contents of edible part of various fruit have been reported previously, The results of the present study indicate that Jamun fruit contained one to three times more flavonoid than those in blueberry, strawberry, apple, grape, for example Flavonoid content of cranberry is almost similar to that of Jamun 2564 Figure Mineral profile of (A) Jamun pulp (JP), kernel (JK), seed coat (JSC); (B) Jamun pulp ethanol extract (PEE), kernel ethanol extract (KEE), seed coat ethanol extract (SCEE); (C) residual pulp (RP), residual kernel (RK) and residual seed coat (RSC) All values are expressed in dry weight basis (n = ± SEE) J Sci Food Agric 87:2560–2569 (2007) DOI: 10.1002/jsfa Composition and antioxidant studies on S cumini fruit Table Free polyphenols and anthocyanin content of fresh Jamun fruit parts and their corresponding ethanol extract Flavonoids (g kg−1 ) Sample JPa PEEb JKa KEEb JSCa SCEEb Total free phenol (g kg−1 ) 3.9 ± 0.5 340.0 ± 1.7 9.0 ± 0.7 370.0 ± 7.8 8.1 ± 0.8 270.0 ± 3.4 Anthocyanins (g kg−1 ) 1.34 ± 0.20 3.20 ± 0.27 – – – – AlCl3 method 2,4-DNPH method Total 0.05 ± 0.03 8.60 ± 0.50 0.44 ± 0.17 30.00 ± 0.43 0.41 ± 0.08 25.30 ± 0.37 0.02 ± 0.01 1.40 ± 0.32 0.08 ± 0.03 2.00 ± 0.09 ND ND 0.07 ± 0.04 10.000 ± 0.82 0.52 ± 0.20 32.00 ± 0.52 0.41 ± 0.08 25.30 ± 0.37 a Fresh weight basis; b dry weight basis JP, Jamun pulp; PEE, pulp ethanol extract; JK, Jamun kernel; KEE, kernel ethanol extract; JSC, Jamun seed coat; SCEE, seed coat ethanol extract ND, not detected (n = ± SEE) The total anthocyanin content was also substantially high in the edible part of Jamun fruit (JP) as compared to that in blueberry.31 Antioxidant activities Antioxidant activities of PEE, KEE and SCEE against DPPH radical, superoxide radical, hydroxyl radical and peroxyl radicals were evaluated using various assay methods DPPH radical scavenging activity DPPH radical scavenging activities of extracts and standard compounds were evaluated and the results are shown in Fig DPPH is a stable free radical (purple colour) and it transforms to non-radical form (yellow colour) by abstracting one electron and hence it is widely used as measure for the electron donation capacity of the antioxidant under the assay conditions.32 A linear relation was observed up to a certain level between percentage radical scavenging activity and sample concentrations; but in different rate with respect to the chemical composition of samples and nature of standard compounds tested Antioxidant power of KEE was extremely high with an IC50 of 8.6 ± 3.0 µg mL−1 However, SCEE (IC50 , 48.0 ± 9.0 µg mL−1 ) and PEE (IC50 , 158.0 ± 5.0 µg mL−1 ) also showed a reasonable antioxidant activity with that of standard compounds tested here (vitamin C, 7.0 ± 0.76 µg mL−1 ; Trolox, 4.3 ± 1.0 µg mL−1 ; and catechin 6.0 ± 0.2 µg mL−1 ) (Table 4) Although the TPC contents between the samples tested varied in close range, the antioxidant power of KEE with 370.0 ± 7.8 g kg−1 TPC was 17 times more than that of PEE with 340.0 ± 1.7 g kg−1 TPC in terms of DPPH radical scavenging activity Superoxide radical scavenging capacity The major source of free radical production in vivo is through superoxides, which are produced by the leakage of a free electron during its transport in mitochondria.33 A dose dependent superoxide radical scavenging activity was observed in all samples and standard molecules (Fig 4) KEE with IC50 value of 85.0 ± 5.0 µg mL−1 was found to be a very strong J Sci Food Agric 87:2560–2569 (2007) DOI: 10.1002/jsfa Figure DPPH radical scavenging activity of Jamun pulp ethanol extract (PEE), kernel ethanol extract (KEE), seed coat ethanol extract (SCEE) and standard BHT, vitamin C, quercetin, gallic acid, catechin and Trolox (n = ± SEE) superoxide radical scavenger and the activity was significantly higher than those of standard compounds such as gallic acid (225.0 ± 6.0 µg mL−1 ) and catechin (296.0 ± 11.0 µg mL−1 ) The IC50 values for PEE and SCEE were far higher with 18 and times lower activity, respectively, than that for KEE The polyphenol content of the samples did correlate with their superoxide radical scavenging activity, suggesting that the chemical structure of polyphenols may have bearing on their superoxide radical scavenging activity 2565 P S Benherlal, C Arumughan Table IC50 values of the ethanol extracts of Jamun fruit parts and standard compounds using different radical scavenging assay methods IC50 value (µg ml−1 ) Sample DPPH Superoxide Hydroxyl Inhibition of radical radical radical lipid scavenging scavenging scavenging peroxidation PEE 158 ± KEE 8.6 ± SCEE 48 ± Gallic acid 1.3 ± 0.05 Vitamin C ± 0.76 BHT 40.6 ± Quercetin 2.4 ± 2.3 Trolox 4.3 ± Catechin ± 0.2 1703 ± 85 ± 759 ± 14 225 ± – – – 540 ± 296 ± 11 310 ± 10 151 ± 261 ± – 44 ± 7.2 21 ± 102 ± 190 ± 38 188 ± 342 ± 17 202 ± 13 268 ± 13 – – 79 ± 166 ± 13 175 ± – PEE, pulp ethanol extract; KEE, kernel ethanol extract; SCEE, seed coat ethanol extract (dry weight basis) (n = ± SEE) Figure Hydroxyl radical scavenging activity of Jamun pulp ethanol extract (PEE), kernel ethanol extract (KEE), seed coat ethanol extract (SCEE), vitamin C, BHT, gallic acid, Trolox, catechin and quercetin (n = ± SEE) (DMSO) and form formaldehyde The hydroxyl radical scavenging activity of samples is related to the reduction in formaldehyde production and it is quantified using Nash reagent A dose dependent hydroxyl radical scavenging activity was observed in all three extracts (Fig 5) The hydroxyl radical scavenging activity (IC50 ) of KEE, SCEE and PEE were 151.0 ± 5.0 µg mL−1 , 261.0 ± 4.0 µg mL−1 and 310 ± 10.0 µg mL−1 , respectively suggesting that KEE was more active than SCEE and PEE Further, activity of KEE was comparable with those of standard quercetin (IC50 , 102.0 ± 8.0 µg mL−1 ) Trolox (IC50 , 190.0 ± 38.0 µg mL−1 ) and catechin (IC50 , 188.0 ± 6.0 µg mL−1 ) Activity of BHT and vitamin C was found to be substantially lower than that of the samples tested here (Table 4) Figure Superoxide radical scavenging activity of Jamun kernel ethanol extract (KEE), seed coat ethanol extract (SCEE), pulp ethanol extract (PEE), catechin, Trolox and gallic acid (n = ± SEE) Hydroxyl radical scavenging activity In the present investigation hydroxyl radical scavenging activity of different samples and standard compounds were evaluated using the ascorbic acid–iron–EDTA system Hydroxyl radicals generated in the system react with dimethyl sulfoxide 2566 Inhibition of lipid peroxidation Oxidation of membrane phospholipids causes the loss of membrane integrity and hence diminishes normal cellular function in terms of transport and signalling Oxidised low-density lipoproteins (LDLs) is reported to trigger plaque formation in the inner lining of the artery and ultimately cause atherosclerosis Events such as initiation, propagation and termination are the major steps in the progression of lipid J Sci Food Agric 87:2560–2569 (2007) DOI: 10.1002/jsfa Composition and antioxidant studies on S cumini fruit Figure Total reducing power of Jamun kernel ethanol extract (KEE), seed coat ethanol extract (SCEE), pulp ethanol extract (PEE) and vitamin C (n = ± SEE) Total reducing power Evaluation of total reducing power showed that KEE had reducing activity greater than SCEE and PEE However vitamin C was found to be more active than the test samples A linear relation was observed between the phenolic content and reducing activity within each samples (Fig 7) Figure Inhibition of lipid peroxidation in RBC membrane by Jamun kernel ethanol extract (KEE), seed coat ethanol extract (SCEE), pulp ethanol extract (PEE), BHT, quercetin and Trolox (n = ± SEE) oxidation.34 Polyunsaturated fatty acid containing bis allylic positions are more vulnerable to free radical attack by hydrogen abstraction There are two mechanisms involved in the inhibition or prevention of lipid peroxidation One is the chain-breaking action of antioxidants which donate one electron to the free radical formed and further progression is terminated Second is the inhibition of chain initiation by scavenging reactive oxygen and nitrogen species.35 In the present investigation, lipid peroxidation was induced in RBCs by t-BHP The inhibition of lipid peroxidation was found to be dose dependent as observed in the case of other radical scavenging assays Percentage antiperoxidative activity of different samples (KEE, PEE and SCEE) and standard compounds are shown in Fig Among different samples, KEE (IC50 , 202.0 ± 13.0 µg mL−1 ) was more effective than SCEE (IC50 , 268.0 ± 13.0 µg mL−1 ) and PEE (IC50 , 342.0 ± 17.0 µg mL−1 ) BHT showed a high degree of antiperoxidative activity (IC50 , 79.0 ± 4.0 µg mL−1 ) than other standard compounds Activity (IC50 ) of Trolox and quercetin was 175.0 ± 4.0 µg mL−1 and 166.0 ± 13.0 µg mL−1 , respectively (Table 4) The extracts evaluated here thus showed lower activity than that of standards in the case of their ability to inhibit peroxidation of membrane lipids J Sci Food Agric 87:2560–2569 (2007) DOI: 10.1002/jsfa Nutraceutical significance of Jamun The composition analysis of Jamun fruit parts brought out its nutritional and nutraceutical importance The fresh pulp of Jamun fruit has slight astringency with highly acceptable taste and flavor The anthocyanins rich edible part (JP) of Jamun was comparable with that of blueberry, blackberry and blackcurrent, whose nutraceutical properties are well documented, suggesting the potential nutraceutical value of Jamun fruit Anthocyanins in these fruits are reported to be powerful antioxidant and stability studies showed that they are stable up to months in dry pulps.18 Anthocyanins (cyanidin glucosides) have been shown to protect cell membrane lipids from oxidation.36 According to Rice-Evans, some cyanidins are many times more powerful antioxidants than tocopherols.37 Bertuglia et al.38 showed that anthocyanin supplements effectively inhibited inflammation and subsequent blood vessel damage and maintained the integrity of vascular micro capillaries in animal model Chemopreventive action and the molecular mechanism of anthocyanidins have been recently reviewed by Hou et al.39 Recent reports on the ability of anthocyanins to modulate insulin secretion have generated interest in fruits with deep colours such as blueberry, blackberry and raspberry.40 However, a variety of richly coloured tropical fruits is available but have not been investigated for their therapeutic properties though these fruits have been consumed for centuries Jamun fruit is one of such fruits with a deep purple colour and is rich in anthocyanins, as shown in the present studies It is grown widely in the Indian sub-continent There are very limited studies conducted on Jamun fruit 2567 P S Benherlal, C Arumughan for its chemical composition and biological activities, though the fruit parts are used in Indian traditional medicine for management of hyperglycaemia In a recent study Anandharajan et al.41 reported the ability of Jamun seed extract to modulate glucose transport mediated through expression of specific receptors using myocytes This finding support the health claim of Jamun seed as antidiabetic agent by practioners of Indian traditional medicine However, numerous studies have been reported concerning the health benefits of anthocyanin-bearing fruits such as cranberry and raspberry.42,43 The potential of black raspberry methanol extract to inhibit tumour development in mouse epithelial cells mediated by impairing signal transduction pathways leading to activation of transcription factors like activator protein (AP1) and nuclear factor kappa B (NF-κB) leading to down regulation of vascular endothelial growth factors (VEGF) and COX-2 expressions.44 In another study a specific anthocyanin (cyanidin-3-glucoside) isolated from blueberry has been shown to inhibit UVB radiation and 12-O-tetradecanolyphorbol-13-acetate (TPA) induced transactivation of NF-kB, AP1 and expression COX-2 and tumor necrosis factor-alpha (TNF-α) and attributed these effects to the inhibition of mitogen-activated protein kinase (MAPK) activity in the cultured JB6 cell line.45 Cyanidin-3-glucoside from blackberry is further reported to suppress nitric oxide production, indicating anti-inflammatory properties of this anthocyanin.46 The results of the in vitro models not necessarily mean that the anthocyanins are biologically active under in vivo conditions because of the biotransformation of these molecules.47 Studies using extracts from blackberry, blueberry and other anthocyanincontaining fruits have demonstrated their effects on inflammation, neuroprotection, oxidative stress, for example.48 – 50 However, in another study the consumption of cranberry juice was not found to be effective against heart disease and cancer in healthy human volunteers.47 Nevertheless, epidemiological data suggests that consumption of fruits and vegetables has been associated with lower incidence of cancer, CHD and inflammation through the chemopreventive and antioxidant properties of the phytochemicals present The non-edible part of many fruits, particularly the kernels, are rich in polyphenols and flavonoids with high antioxidant activity The biological properties of some of them have been validated scientifically, while many of them are yet to be studied Most of these seeds are not palatable and therefore not consumed as food Plant polyphenols comprise different classes of compounds, such as phenolic acids, flavonoids, anthocyanins and stilbene Many plantderived medicines are reported to contain substantial amounts of flavonoids and are proven to have antibacterial, anti-inflammatory, anti-allergic, antimutagenic, antiviral, anti-neoplastic, anti-thrombotic and vasodilatory activities.51 2568 Extracts of Jamun fruit parts evaluated in this study using four assay methods had antioxidant activity: KEE > SCEE > PEE Among these extracts, KEE was found possesses antioxidant activity comparable or better than that of standard antioxidants in terms of DPPH, superoxide and hydroxyl radical scavenging properties Comparison between the activity obtained in six different methods is not relevant because of the complex and diverse constituents of phytochemicals and their different mechanisms.in different assay systems The same level of phenolic content in different anatomical parts of the fruit, viz PEE and KEE, thus did not show a similar antioxidant response perhaps due to their constituent phytochemicals and this was supported by previous authors.26 The present study is the first in the series to establish the possible therapeutic and chemopreventive properties of Jamun fruit which is very rich in anthocyanins and antioxidant phytochemicals that may have similar biological effects as those demonstrated in the case of blueberry and blackberry fruits Detailed characterisation of the phytochemicals based on activity-guided fractionation of JP and JK is in progress, and is expected to lead to isolation of the active principle Jamun fruit thus has high potential to yield products of therapeutic or nutraceutical value The Jamun seed used in traditional medicine as a hypoglycaemic agent requires further investigation to establish the relation, if any, between its antioxidant property and reported hypoglycaemic effect ACKNOWLEDGEMENT The authors gratefully acknowledge the grant from CSIR, India REFERENCES Bauerova K and Bezek A, Role of reactive oxygen and nitrogen species in etiopathogenesis of rheumatoid arthritis Gen Physiol Biophys 18:15–20 (1999) Marzani B, Felzani G, Rosa Grazia B, Vecchiet J and Marzatico F, Human muscle aging: ROS-mediated alterations in rectus abdominis and vastus lateralis muscles Exp Geront 40:959–965 (2005) Dhalla NS, Temsha RM and Netticadan T, Role of oxidative stress in cardiovascular diseases J Hypertens 18:655–673 (2000) Hunt JV, Smith CCT and Wolff SP, Auto-oxidative glycosylation and possible involvement of peroxides and free radicals in LDL modification by glucose Diabetes 39:1420–1424 (1990) Truscott RJW, Age-related nuclear cataract – oxidation is the key Exp Eye Res 80:709–725 (2005) Finkel T and Holbrook NJ, Oxidants, oxidative stress and the biology of ageing Nature 408:239–247 (2000) Li YP, Chen Y, Li AS and Reid MB, Hydrogen peroxide stimulates ubiquitin-conjugating activity and expression of genes for specific E2 and E3 proteins in skeletal muscle myotubes Am J Physiol, Cell Physiol 285:C806–C812 (2003) Lima ES, Bonini MG, Augusto O, Barbeiro HV, Souza HP and Abdalla DSP, Nitrated lipids decompose to nitric oxide and lipid radicals and cause vasorelaxation Free Rad Biol Med 39:532–539 (2005) Allen RG and Tresini M, Oxidative stress and gene regulation Free Rad Biol Med 28:463–499 (2000) J Sci Food Agric 87:2560–2569 (2007) DOI: 10.1002/jsfa Composition and antioxidant studies on S cumini fruit 10 Choi HS and Moore DD, Induction of c-fos and c-jun gene expression by phenolic antioxidants Mol Endocrinol 7:1596–1602 (1993) 11 Caragay AB, Cancer preventive foods and ingredients Food Technol 46:65–68 (1992) 12 Shahidi F, Natural Antioxidants: Chemistry, Health Effects and Applications AOCS Press, Champaigen, IL, pp 4–5 (1997) 13 Muruganandan S, Pant S, Srinivasan K, Chandra S, Tandan SK, Lal J, et al, Inhibitory role of Syzyguim cumini on autocoid-induced inflammation in rats Ind J Physiol Pharmacol 46:482–486 (2002) 14 Coimbra TC, Danni FF, Blotta RM, Pereira CA, Guedes MD and Graf RG, Plants employed in the treatment of diabetes mellitus; results of an ethnopharmacological survey in Porto Alegre, Brazil Fitoterapia 63:320–322 (1992) 15 Gosh K, Chakraborty D, Chaterjee GK, Choudari AKN and Upal M, Studies on anti inflammatory and anti pyretic activities of Syzygium cumini Linn seed IRCS Med Sci Biochem 13:340–341 (1985) 16 Chakraborty D, Chaudari AKN and Maha Patra PK, Studies on psychopharmaceutical actions of Syzygium cumini Linn Seed extract IRCS Med Sci Biochem 13:746–747 (1985) 17 Ravi K, Ramachandran B and Subramanian S, Effect of Eugenia jambolana seed kernel on antioxidant defense system in streptozotocin-induced diabetes in rats Life Sci 75:2717–2731 (2004) 18 Banerjee A, Dasgupta N and De B, In vitro study of antioxidant activity of Syzygium cumini fruit Food Chem 90:727–733 (2005) 19 Horowitz W, Official Methods of Analysis of AOAC International, AOAC International, Gaithersburg, MD, USA (2000) 20 Pons A, Roca P, Aguilo C, Garcia FJ, Alemany M and Palou A, A method for the simultaneous determinations of total carbohydrate and glycerol in biological samples with the anthrone reagent J Biochem Biophys Methods 4:227–231 (1981) 21 Jayasinghe C, Gotoh N, Aoki T and Wada S, Phenolic composition and antioxidant activity of sweet basil J Agric Food Chem 51:4442–4449 (2003) 22 Giusti MM, Saona LE and Wrolstad RE, Spectral characteristic, molar absorbtivity and colour of pelargonidin derivaties J Agric Food Chem 47:4631–4637 (1999) 23 Wrolstad RE, Putnam TP and Varseveld GW, Color quality of frozen straw berries: Effect of anthocyanin, PH, total acidity and ascorbic acid variability J Food Sci 35:448–452 (1970) 24 Chang C-C, Yang M-H, Wen H-M and Chern J-C, Estimation of total flavonoid content in propolis by two complementary colorimetric methods J Food Drug Anal 10:178–182 (2002) 25 Brand-Williams W, Cuvelier ME and Berset C, Use of a free radical method to evaluate antioxidant activity Lebensm-Wiss Food Sci Technol 28:25–30 (1995) 26 Parejo I, Viladomat F, Bastida J, Rosas-Romero A, Flerlage N, Burillo J, et al, Comparison between the radical scavenging activity and antioxidant activity of six distilled and nondistilled Mediterranean herbs and aromatic plants J Agric Food Chem 50:6882–6890 (2002) 27 Klein SM, Cohen G and Cederbaum AI, Production of formaldehyde during metabolism of dimethyl sulfoxide by hydroxyl radical generating system Biochemistry 20:6006–6012 (1991) 28 Manna C, D’Angelo S, Migliardi V, Loffredi E, Mazzoni O, Marrica P, et al, Protective effect of phenolic fraction from virgin olive oils against oxidative stress in human cells J Agric Food Chem 50:6521–6526 (2002) 29 Zhu QV, Hackman RM, Jodilensunsa XX, Holt RR and Keen CL, Antioxidative activities of Oolong tea J Agric Food Chem 50:6229–6934 (2002) 30 Chadha YR, Wealth of India National Institute of Science Communication and Information Resources, Delhi, India, pp 100–102 (2003) J Sci Food Agric 87:2560–2569 (2007) DOI: 10.1002/jsfa 31 Franke AA, Custer LJ, Arakaki C and Murphy CP, Vitamin C and flavanoid levels of fruits and vegetables consumed in Hawai J Food Comp Anal 17:1–35 (2004) 32 Molyneux P, The use of the stable free radical diphenylpicryl hydroxyl (DPPH) for estimating antioxidant activity J Sci Technol 26:211–219 (2004) 33 Vaya1 J and Aviram M Nutritional antioxidants: Mechanisms of action, analyses of activities and medical applications Curr Med Chem: Immunol, Endocrinol Metab Agents 1:99–117 (2001) 34 Gardner HW, Oxygen radical chemistry of polyunsaturated fatty acids Free Rad Biol Med 7:65–86 (1989) 35 Ingold KU, Inhibition of the autoxidation of organic substances in the liqiud phase Chem Res 61:563–589 (1961) 36 Tsuda T, Diatery cyanidin 3-O-beta-D-glycoside increases in vivo oxidative, resistance of serum in rats Lipids 6:583–588 (1998) 37 Rice-Evans CA, The relative antioxidant activities of plant derived polyphenolic flavanoids Free Radicals 22:3785–3793 (1995) 38 Bertuglia S, Malandrino S and Colantuoni A, Effect of Vaccinium myrtillus anthocyansides on ischemia reperfusion injury in hamster cheek pouch microcirculation Pharmacol Res 31:183–187 (1995) 39 Hou D-X, Fujii M, Terahara N and Yoshimoto M, Molecular mechanisms behind the chemopreventive effects of anthocyanidins J Biomed Biotechnol 5:321–325 (2004) 40 Jayaprakasam B, Vareed SK, Olson LK and Nair MG, Insulin secretion by bioactive anthocyanins and anthocyanidins present in fruits J Agric Food Chem 53:23–38 (2005) 41 Anandharajan R, Jaiganesh S, Shankernarayanan NP, Viswakarma RA and Balakrishnan A, In vitro glucose uptake activity of Aegles marmelos and Syzygium cumini by activation of Glut-4, PI3 kinase and PPARγ in L6 myotubes Phytomedicine 13:434–441 (2006) 42 Dvaranauskaite A, Venskutonis PR and Labokas J, Radical scavenging activity of raspberry (Rubus idaeus L.) fruit extracts Acta Alimentaria 35:73–83 (2006) 43 He X and Rui HL, Cranberry phytochemicals: Isolation, structure elucidation, and their antiproliferative and antioxidant activities J Agric Food Chem 54:7069–7074 (2006) 44 Lu H, Li J, Zhang D, Stoner GD and Huang C, Molecular mechanisms involved in chemoprevention of black raspberry extracts: From transcription factors to their target genes Nutrition and Cancer 54:69–78 (2006) 45 Ding M, Feng R, Wang SY and Bowman L, Cyanidin-3glucoside, a natural product derived from blackberry, exhibits chemopreventive and chemotherapeutic activity J Biol Chem 281:17359–17368 (2006) 46 Pergola C, Rossi A, Dugo P, Cuzzocrea S and Sautebin L, Inhibition of nitric oxide biosynthesis by anthocyanin fraction of blackberry extract Nitric Oxide – Biology and Chemistry 15:30–39 (2006) 47 Duthie SJ, Jenkinson AMcE, Crozier A and Mullen W, The effects of cranberry juice consumption on antioxidant status and biomarkers relating to heart disease and cancer in healthy human volunteers Eur J Nutr 45:113–122 (2006) 48 Galli RL, Bielinski DF, Szprengiel A, Shukitt-Hale B and Joseph JA, Blueberry supplemented diet reverses age-related decline in hippocampal HSP70 neuroprotection Neurobiol Aging 27:344–350 (2006) 49 Rossi A, Serraino I, Dugo P, Di Paola R, Mondello L and Genovese T, Protective effects of anthocyanins from blackberry in a rat model of acute lung inflammation Free Rad Res 37:891–900 (2003) 50 Wang SY and Lin HS, Antioxidant activity in fruits and leaves of blackberry, raspberry, and strawberry varies with cultivar and developmental stage J Agric Food Chem 48:140–146 (2000) 51 Alan L and Miller ND, Antioxidant flavonoids: Structure, function and clinical usage Alternative Medicine Review 11:103–111 (1996) 2569 .. .Composition and antioxidant studies on S cumini fruit tribal medicine S cumini is an evergreen tree distributed in the Indian sub-continent and southeast Asian countries The oval shaped fruit. .. aluminium chloride method (specific for flavones flavonols and isoflavones) and the 2,4-DNPH method (specific for flavonones) JP and JK were found to contain flavonols, flavones and isoflavones and. .. mechanisms involved in the inhibition or prevention of lipid peroxidation One is the chain-breaking action of antioxidants which donate one electron to the free radical formed and further progression