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[.]

Chemical composition and in vitro

antioxidant studies on Syzygium cumini

fruit

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 • , O 2 •− 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 (IC 50 ) of KEE was six times higher (85.0 ± 5.0µg mL −1 ) compared to Trolox (540.0 ± 5.0µg mL −1 ) and three times compared to catechin (296.0 ± 11.0µg mL −1 ) Hydroxyl radical scavenging activity (IC 50 ) of KEE was 151.0 ± 5.0µg mL −1 which was comparable with catechin (188.0 ± 6.0µg 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 ± 4.0µg mL −1 ; quercetin, 166.0 ± 13.0µg mL −1 ; Trolox, 175.0 ± 4.0µg mL −1 ; PEE, 342.0 ± 17.0µg mL −1 ; KEE, 202.0 ± 13.0µg mL −1 and SCEE, 268.0 ± 13.0µg 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,

can-cer, for example.1 – 5 Apart from a range of harmful

effects, they are also involved in numerous cellular

pro-cesses such as vasodilation, signal transduction, gene

expression, cell differentiation and development.6 – 8

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

concentra-tion, in relation to reactive oxygen species (ROS),

significantly inhibit or delay tissue damage, while often being oxidised themselves The antioxidants can func-tion 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 antioxi-dant 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.11,12Detailed understanding

of these natural sources in terms of chemical compo-sition, active molecules and ethno medicine would provide some information on their potential therapeu-tic 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 7 April 2006; revised version received 26 January 2007; accepted 8 February 2007)

Published online 11 September 2007;DOI: 10.1002/jsfa.2957

tribal medicine S cumini is an evergreen tree

distributed in the Indian sub-continent and

south-east 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

anti-inflammatory 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,

1,1-diphenyl-2-picrylhydrazyl (DPPH), nitro-blue

tetra-zolium (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

analyt-ical 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 1 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

material-to-solvent ratio of 1:2 (w/v) The extraction was

conducted at ambient temperature (25 – 30◦C) under

stirring for 4 h At the end of the extraction, the

Figure 1 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 min and the supernatant was passed

through Whatman 41 (pore size 20 – 25µm) 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).21The samples and standard gallic acid were diluted to 2 – 20µg 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 5 min at room temperature followed by addition

of 2.0 mL of 10% aqueous sodium carbonate and incubated at room temperature for 1 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).22Total monomeric

anthocyanins in the extract were estimated by the

pH differential method and expressed in

glucoside equivalency, where the MW of

cyanidin-3-glucoside 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 min 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

per-formed by two complementary colorimetric

meth-ods: 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.24was performed

Aluminium chloride method

Ten to 100µg 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

2 mL with 95% ethanol followed by the addition of

0.1 mL of 10% aluminium chloride, 0.1 mL of 1 mol

L−1 potassium acetate and 2.8 mL of distilled water

and incubated at room temperature (30 – 34◦C) for

30 min 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 2000µg

of naringenin and 100 – 1000µg 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 min 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 min 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

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−1DPPH

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 min in an

UV – visible spectrophotometer at 517 nm (Shimadzu

UV-2450) Optical densities at time zero (OD t0)

and at 30 min (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 IC50value, 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

Sixty-three microlitres of 1.0 mmol L−1NBT 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 Sixty-three 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 min (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

RSA=

1−Asample

Ablank



× 100

where the RSA is in %, Asampleis the OD of the sample

or standard, and Ablankis 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

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−1phosphate

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 min.

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 min 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.28Heparinised whole blood was collected

from healthy volunteers The blood was centrifuged

for 10 min 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

500µmol L−1t-BHP The final volume was made up

to 5.0 mL with isotonic saline and incubated at 37◦C

in a water bath for 2 h After oxidative treatment,

the tubes were centrifuged at 1000× g for 10 min

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 min 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 min 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 IC50values

Total reducing power

Total reducing power was estimated according to Zhu

et al.29 The reaction system consist of appropriately

diluted (100 – 500µg) 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 min After the incubation

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 min 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± 3 g is shown in Table 1 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−1seed 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 2 While the fresh pulp (JP) had the highest moisture

con-tent, (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 Alco-hol extracted most of the soluble sugars and minerals

Table 1 Yield of anatomical parts of fresh Jamun fruit

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.

Table 2 Chemical composition of fruit parts, their ethanol extract and residue

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= 3 ± SEE).

present in the fruit parts, the alcohol extract

there-fore 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 3 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

Figure 2 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= 3 ± SEE).

Table 3 Free polyphenols and anthocyanin content of fresh Jamun fruit parts and their corresponding ethanol extract

Flavonoids (g kg −1)

Sample

Total free phenol (g kg −1)

Anthocyanins

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= 3 ± 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 3 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.32A 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 ± 3.0µg mL−1.

However, SCEE (IC50, 48.0 ± 9.0µg mL−1) and PEE

(IC50, 158.0 ± 5.0µg mL−1) also showed a reasonable

antioxidant activity with that of standard compounds

tested here (vitamin C, 7.0 ± 0.76µg mL−1; Trolox,

4.3 ± 1.0µg mL−1; and catechin 6.0 ± 0.2µg 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.33A dose dependent superoxide radical

scavenging activity was observed in all samples and

standard molecules (Fig 4) KEE with IC50 value of

85.0 ± 5.0µg mL−1 was found to be a very strong

Figure 3 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= 3 ± SEE).

superoxide radical scavenger and the activity was significantly higher than those of standard compounds

such as gallic acid (225.0 ± 6.0µg mL−1) and catechin

(296.0 ± 11.0µg mL−1) The IC

50 values for PEE and SCEE were far higher with 18 and 8 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

Table 4 IC50 values of the ethanol extracts of Jamun fruit parts and

standard compounds using different radical scavenging assay

methods

IC50value ( µg ml −1)

Sample

DPPH

radical

scavenging

Superoxide radical scavenging

Hydroxyl radical scavenging

Inhibition of lipid peroxidation

PEE, pulp ethanol extract; KEE, kernel ethanol extract; SCEE, seed

coat ethanol extract (dry weight basis) (n= 3 ± SEE).

Figure 4 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= 3 ± SEE).

Hydroxyl radical scavenging activity

In the present investigation hydroxyl radical

scav-enging activity of different samples and

stan-dard compounds were evaluated using the ascorbic

acid – iron – EDTA system Hydroxyl radicals

gener-ated in the system react with dimethyl sulfoxide

Figure 5 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= 3 ± 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 radi-cal scavenging activity (IC50) of KEE, SCEE and

PEE were 151.0 ± 5.0µg mL−1, 261.0 ± 4.0µg mL−1

and 310± 10.0µg mL−1, respectively suggesting that

KEE was more active than SCEE and PEE Fur-ther, activity of KEE was comparable with those

of standard quercetin (IC50, 102.0 ± 8.0µg mL−1)

Trolox (IC50, 190.0 ± 38.0µg mL−1) and catechin

(IC50, 188.0 ± 6.0µg mL−1) Activity of BHT and

vitamin C was found to be substantially lower than that of the samples tested here (Table 4)

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

Figure 6 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= 3 ± 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 6 Among different

samples, KEE (IC50, 202.0 ± 13.0µg mL−1) was more

effective than SCEE (IC50, 268.0 ± 13.0µg mL−1)

and PEE (IC50, 342.0 ± 17.0µg mL−1) BHT showed

a high degree of antiperoxidative activity (IC50,

79.0 ± 4.0µg mL−1) than other standard compounds.

Activity (IC50) of Trolox and quercetin was 175.0±

4.0µg mL−1 and 166.0 ± 13.0µg 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

Figure 7 Total reducing power of Jamun kernel ethanol extract

(KEE), seed coat ethanol extract (SCEE), pulp ethanol extract (PEE)

and vitamin C (n= 3 ± 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)

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, suggest-ing the potential nutraceutical value of Jamun fruit Anthocyanins in these fruits are reported to be power-ful antioxidant and stability studies showed that they are stable up to 6 months in dry pulps.18Anthocyanins (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 effec-tively 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

anthocyani-dins have been recently reviewed by Hou et al.39

Recent reports on the ability of anthocyanins to mod-ulate insulin secretion have generated interest in fruits with deep colours such as blueberry, blackberry and raspberry.40However, a variety of richly coloured trop-ical 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

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.41reported the ability

of Jamun seed extract to modulate glucose

trans-port 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

ben-efits 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

sig-nal transduction pathways leading to activation of

transcription factors like activator protein 1 (AP1)

and nuclear factor kappa B (NF-κB) leading to

down regulation of vascular endothelial growth

fac-tors (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

prop-erties of this anthocyanin.46

The results of the in vitro models do not

necessar-ily mean that the anthocyanins are biologically active

under in vivo conditions because of the

biotransfor-mation of these molecules.47 Studies using extracts

from blackberry, blueberry and other

anthocyanin-containing fruits have demonstrated their effects on

inflammation, neuroprotection, oxidative stress, for

example.48 – 50 However, in another study the

con-sumption of cranberry juice was not found to be

effec-tive against heart disease and cancer in healthy human

volunteers.47 Nevertheless, epidemiological data

sug-gests 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

dif-ferent classes of compounds, such as phenolic acids,

flavonoids, anthocyanins and stilbene Many

plant-derived medicines are reported to contain

substan-tial amounts of flavonoids and are proven to have

antibacterial, anti-inflammatory, anti-allergic,

antimu-tagenic, antiviral, anti-neoplastic, anti-thrombotic and

vasodilatory activities.51

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 proper-ties 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 characterisa-tion 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

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