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Antioxidant and Antiproliferative Activities ofAnthocyanin/Ellagitannin-Enriched Extracts FromSyzygium cumini L. (Jamun, the Indian Blackberry)Farrukh Aqil a , Akash Gupta a , Radha Munagala a , Jeyaprakash Jeyabalan a , Hina Kausar a ,Ram Jee Sharma b , Inder Pal Singh b & Ramesh C. Gupta ca James Graham Brown Cancer Center, University of Louisville, Louisville, Kentucky, USAb National Institute of Pharmaceutical Education and Research, S.A.S. Nagar, Indiac James Graham Brown Cancer Center and Department of Pharmacology and Toxicology,University of Louisville, Louisville, Kentucky, USA

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To cite this article: Farrukh Aqil, Akash Gupta, Radha Munagala, Jeyaprakash Jeyabalan, Hina Kausar, Ram Jee Sharma,Inder Pal Singh & Ramesh C. Gupta (2012): Antioxidant and Antiproliferative Activities of Anthocyanin/Ellagitannin-EnrichedExtracts From Syzygium cumini L. (Jamun, the Indian Blackberry), Nutrition and Cancer, 64:3, 428-438

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Nutrition and Cancer, 64(3), 428–438Copyright C© 2012, Taylor & Francis Group, LLCISSN: 0163-5581 print / 1532-7914 onlineDOI: 10.1080/01635581.2012.657766

Antioxidant and Antiproliferative Activities ofAnthocyanin/Ellagitannin-Enriched Extracts From Syzygiumcumini L. (Jamun, the Indian Blackberry)

Farrukh Aqil, Akash Gupta, Radha Munagala, Jeyaprakash Jeyabalan, andHina KausarJames Graham Brown Cancer Center, University of Louisville, Louisville, Kentucky, USA

Ram Jee Sharma and Inder Pal SinghNational Institute of Pharmaceutical Education and Research, S.A.S. Nagar, India

Ramesh C. GuptaJames Graham Brown Cancer Center and Department of Pharmacology and Toxicology, Universityof Louisville, Louisville, Kentucky, USA

Colored fruits, particularly berries, are highly chemoprotectivebecause of their antioxidant, antiproliferative, and antiinflamma-tory activities. We report the cancer chemoprotective potential ofSyzygium cumini L., commonly known as jamun or Indian black-berry. Anthocyanins and other polyphenolics were extracted withacidic ethanol and enriched by amberlite XAD7/HP20 (1:1). Thepulp powder was found to contain 0.54% anthocyanins, 0.17% el-lagic acid/ellagitannins, and 1.15% total polyphenolics. Jamun seedcontained no detectable anthocyanins but had higher amounts of el-lagic acid/ellagitannins (0.5%) and total polyphenolics (2.7%) thanthe pulp powder. Upon acid hydrolysis, the pulp extract yielded 5anthocyanidins by HPLC: malvidin (44.4%), petunidin (24.2%),delphinidin (20.3%), cyanidin (6.6%), and peonidin (2.2%). Ex-tracts of both jamun pulp (1,445 ± 64 µmol of trolox equivalent(TE)/g) and seeds (3,379 ± 151 µM of TE/g) showed high oxygenradical absorbance capacity. Their high antioxidant potential wasalso reflected by 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulphonicacid)- and 2,2-diphenyl-1-picrylhydrazyl-scavenging and ferrousion-chelating activities. We also analyzed antiproliferative activ-ity of jamun extracts against human lung cancer A549 cells. Thehydrolyzed pulp and seed extracts showed significant antiprolifer-ative activity. However, unhydrolyzed extracts showed much lessactivity. These data showed that in addition to 5 anthocyanidins,jamun contains appreciable amounts of ellagic acid/ellagitannins,with high antioxidant and antiproliferative activities.

Submitted 7 April 2011; accepted in final form 11 January 2012.Address correspondence to Ramesh Gupta, Delia Baxter II, Room

304E, University of Louisville, 580 S. Preston Street, Louisville, KY40202. Phone: 502-852-6980. E-mail: rcgupta@louisville.edu

INTRODUCTIONReactive oxygen species (ROS) such as superoxide radicals

(O−2 ), hydroxyl radicals (•OH), and peroxyl radicals (ROO•)

have been associated with carcinogenesis, coronary heart dis-ease, and many other health issues related to advanced age (1).There are endogenous as well as exogenous systems to protectcells in animals. However, in many conditions, such as withaging, the endogenous antioxidants are depleted, and thereforean exogenous supply of antioxidants is essential. Antioxidantsplay an important role in biological systems (2) by suppressingthe formation of reactive oxygen species by reducing hydroper-oxides (ROO•) and H2O2 and scavenging free radicals.

Since antioxidants terminate ROS-mediated oxidative reac-tions, they may be used in prevention of aging-associated dis-eases, including cancer. This has led to an accelerated search forpure compounds and extracts of natural origin with antioxidantprinciples. Antioxidants have been detected in many agriculturaland food products, including fruits, vegetables, and oil seeds(3,4). However, many studies have conclusively shown that themajority of the antioxidant activity may be due to compoundssuch as flavonoids, isoflavones, flavones, and anthocyaninsrather than the traditionally considered vitamins C, E, and β-carotene (5,6). There is also a direct relationship between antiox-idant activity and polyphenolic content of plant extracts (6,7).Antioxidant activity of phenolics is mainly due to their redoxpotential, which allows them to act as reducing agents, hydrogendonors, singlet oxygen quenchers, and metal chelators (8,9).

Anthocyanins are a group of water-soluble flavonoids thatare responsible for the bright red, blue, and purple colorsin fruits, vegetables, and ornamental crops. The anthocyaninsfound in foods are glycosides of 6 main aglycon anthocyanidins:

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ANTIOXIDANT AND ANTIPROLIFERATIVE ACTIVITIES 429

FIG. 1. A: Structures of various anthocyanidins in jamun fruit. B: The Indian blackberry.

cyanidin, delphinidin, pelargonidin, peonidin, malvidin, andpetunidin (Fig. 1A). Health benefits associated with the useof anthocyanin-enriched (colored) foods include reduced riskof coronary heart disease (10), protection against obesity andhypoglycemia (11), memory enhancement (12), and preventionof cancer (13–15).

Syzygium cumini L. (family, Myrtaceae), commonly knownas jamun or Indian blackberry, is native to India; it thrives ina tropical climate. It is also found in some parts of Americaas well as Asian and East African countries. The fruits are ob-long berries, deep purple or bluish in color with pinkish pulp(Fig. 1B), and are widely consumed as a fruit and also used forthe treatment of various diseases as an astringent, antiscorbutic,diuretic, antidiabetic, and in chronic diarrhea and enlargementof the spleen (16–18). Besides antidiabetic properties (19), ja-mun fruit has also been reported for strong antioxidant (20)and antigenotoxic potential (21). Though different parts of thisplant are used in herbal formulations, chemical components ofthe fruit and seed are poorly defined. There have been 3 reports(18,22,23) on the anthocyanin composition of jamun fruit. Thepulp of the fruit contains diglucoside of 5 anthocyanidins (22).The other recent report revealed the high content of phenolics inthe seeds (24). Anthocyanins exhibit anticarcinogenic propertiessuch as induction of cell-cycle arrest and apoptosis, as well asthe inhibition of tumor formation and growth in animals (15).The present study focused on extraction strategies and evaluat-ing the chemical profile of both the jamun pulp and seeds andassociated antioxidant and antiproliferative properties.

MATERIAL AND METHODS

ChemicalsXAD-761 (Amberlite polymeric adsorbent of 20–50 mesh)

and Diaion HP-20 were purchased from Supelco (Bellefonte,PA). Ascorbic acid, 1,1-diphenyl-2-picryl hydrazyl (DPPH),AAPH, fluorescein, and ABTS were procured from Sigma-Aldrich (St. Louis, MO). Anthocyanin and anthocyanidin stan-dards were obtained from ChromaDex, Inc. (Santa Ana, CA).All other chemicals used were of analytical grade.

Plant Material and Preparation of the ExtractSyzygium cumini (jamun) fruits were collected from the gar-

den of the National Institute of Pharmaceutical Education andResearch, S.A.S. Nagar, India. The pulp with skin (dark purplecolor) of the fruits were manually separated from the seeds andimmediately dried under vacuum at 40◦C for 2 days. Dried pulpwas powdered and stored at −20◦C in vacuum-sealed packsuntil use. Seeds were also powdered and stored likewise.

Jamun pulp and seed powders were extracted as describedschematically (Fig. 2). Briefly, 8 g each of pulp and seed powderwere extracted with 3 vol (w/v; 24 ml) of 75% aqueous ethanolcontaining 10 mM HCl. To increase the extraction efficiency,samples were sonicated for 15 min in a bath-type sonicator. Sam-ples were then centrifuged at 1,000 × g for 10 min and decanted.Residues were extracted 2 more times in each case. The pooledsupernatants were concentrated to about 12 ml volume undervacuum at 40◦C using a rotary evaporator (Model R 215, Buchi,Switzerland). Enrichment of the anthocyanins, ellagitannins,and other polyphenolics was achieved by loading the concen-trated extracts (6 ml) on a XAD-761 and diaion HP-20 (1:1; 24g total) column. XAD-761 has a high affinity for anthocyanins(25), whereas diaion HP-20 has an affinity for ellagitannins andother polyphenolics (26). Free sugars were eluted using 5 void-volumes of water. The polyphenolics, including anthocyaninsand ellagitannins, were eluted under gravity using methanol (34ml) containing 10 mM HCl. Pooled eluates were concentratedunder vacuum at 40◦C, and one half of the concentrated eluateswere hydrolyzed as described below.

Acid Hydrolysis of the Enriched Jamun Pulpand Seed Extracts

The enriched extracts of jamun pulp and seeds (100 ml)were hydrolyzed with 2N HCl (∼5 vol). Samples were refluxedat 110◦C for 1 h and then immediately cooled in an ice-bath.Hydrolysates were purified on a XAD-761 and diaion HP-20column as described above. Polyphenolics, including antho-cyanidins, were eluted with acidified (10 mM HCl) methanol.The enriched extracts were dried under vacuum using a SavantSpeed-Vac (Thermo-Savant, Holbrook, NY).

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430 F. AQIL ET AL.

Jamun fruits (Pulp or seeds)

(8.0 g dry powder)

Extraction(75% Aq. ethanol containing 10 mM HCl)

Sonication, centrifugation (1,000xg)and concentrated at reduced pressure

Pulp or seeds extract (50% extract)

Enriched on XAD7/ HP20 (1:1; 24 g) column

Washed 5x (15ml) with 10 mM HCl to remove sugars

Elute with 34 ml of methanol containing 0.1% HCl

Dried under vacuum, and stored at -20°C

Enriched on XAD7/ HP20 (1:1; 24 g) column

Hydrolyzed w/ 2 N HCl (5 vol) @ 110ºC and enriched

Pulp or seeds extract (50% extract)

x 3

Enriched on XAD7/ HP20 (1:1; 24 g) column

FIG. 2. Scheme for the isolation of enriched anthocyanins/anthocyanidins and phenolics.

HPLC AnalysisHPLC analysis was carried out according to Li et al. (22)

with minor modifications on a Shimadzu HPLC system (Kyoto,Japan) equipped with 2 LC-10ADvp pumps, autosampler, fluo-rescent, and diode array detectors and operated by Class VP (v.7.4 SP3). Jamun pulp and seed (hydrolyzed and unhydrolyzed)extracts (20 µL injection volume; 1 mg/ml) were analyzed ona ShimPack reverse phase column (Shimadzu; 250 × 4.6 mm,2.2 µm). Two separate experiments were carried out using dif-ferent gradients of 3.5% aqueous phosphoric acid (solvent A)and acetonitrile (solvent B). The first experiment was used toseparate all anthocyanidins, and the second was used for theseparation of anthocyanins, anthocyanidins, and other polyphe-nolics including ellagic acid and ellagitannins.

The first linear gradient contained solvent A initially 95% for10 min; 90% at 20 min; 87% at 30 min; 82% at 35 min; 75% at40 min; 40% for 41–61 min; and finally 95% in acetonitrile ata flow rate of 0.75 ml/min. The total run time was 61 min with10-min intervals between the injections. Anthocyanidins weremonitored at 520 nm. The second gradient contained solvent Ainitially: 90% for 0–5 min; 85% at 10 min; 80% at 15 min; 70%

at 24 min; 62% at 35 min; 94% for 40–43 min; and finally 90% at45 min. Ellagic acid/ellagitannins and other polyphenolics weremonitored at 366 and 280 nm, respectively. Anthocyanidins andellagic acid peaks were confirmed by cochromatography withthe reference materials.

Determination of Total Anthocyaninsby pH-Differential Method

Anthocyanin content was determined using the pH-differential method (27,28) and expressed as cyanidin-3-glucoside equivalent using molar extinction coefficient of26,900 L cm−1 mol−1 and molecular mass of 449.2 g mol−1.Briefly, 0.2-ml samples containing different concentrations ofextracts were mixed separately with 0.8 ml each of 0.025 Mpotassium chloride, pH 1.0 (adjusted with HCl), and 0.4 Msodium acetate, pH 4.5. The reaction mixtures were allowedto equilibrate at room temperature for 15 min and absorbancewas measured at 510 and 700 nm. The difference in absorbancebetween the 2 samples was then calculated using the formula:

Absorbance (A) = (A 510−A 700)pH1.0−(A 510−A 700)pH4.5.

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ANTIOXIDANT AND ANTIPROLIFERATIVE ACTIVITIES 431

Finally, total anthocyanins/anthocyanidins in the samples werecalculated by the following formula:

Total anthocyanins = [Absorbance (A) × 449 g mol−1

× dil factor]/26, 900 L cm−1mol−1.

Determination of Total Soluble SugarsTotal soluble sugars in the jamun pulp and seed extracts were

determined according to the method of Yemm and Willis (29).

Determination of Total PhenolicsTotal phenolic content in the jamun pulp and seed extracts

was determined with the Folin-Ciocalteau reagent as describedby Lister and Wilson (30) and expressed in mg of polyphenolsas gallic acid equivalent per gram of dry weight (mg GAE/gdw).

Antioxidant Assays

Total Antioxidant Potential by Oxygen Radical AbsorbanceCapacity Assay

Oxygen radical absorbance capacity (ORAC) capacity of thejamun pulp and seed extracts was determined at different con-centrations (31) using fluorescein as the “fluorescent probe.”Briefly, 25 µl of phosphate buffer (75 mM, pH 7.0), trolox stan-dard or jamun extracts were incubated with 150 µl of fluorescein(20 nM) at 37◦C for 1 h. The reaction was started by thermaldecomposition of AAPH (19 mM in 75 mM phosphate buffer,pH 7.0) and performed at 37◦C in 96-well flat-bottom plates.Fluorescence was measured immediately after the addition ofAAPH at excitation of 485 nm and an emission wavelength of535 nm for 2 h at 37◦C. The ORAC values, expressed as µMtrolox equivalents (µM TE), were calculated by applying thefollowing formula:

ORAC (µM TE) = (CTrolox ∗ (AUCSample

− AUCBlank) ∗ k)/(AUCTrolox − AUCBlank)

where CTrolox is the concentration (µM) of Trolox, k is the sam-ple dilution factor, and AUC is the area under the fluorescencedecay curve of the sample, blank, and trolox, respectively, cal-culated by Spectramax M2 software.

DPPH Radical-Scavenging AssayFree radical scavenging activity of jamun pulp and seed ex-

tracts against stable DPPH was determined spectrophotomet-rically (32). Fifty µl of the pulp and seed extracts, yielding12.5–100 µg/ml concentrations, were mixed with 0.5 ml of0.1 mM DPPH in methanol and 450 µl of 50 mM Tris-HCl,pH 7.4. Methanol (50 µl) was used as a vehicle control in theexperiment. After 30 min of incubation at room temperature, thereduction of the DPPH free radical was measured at 517 nm.

Inhibition percent was calculated from the following equation:

% Inhibition = [Absorbance of control

− Absorbance of test sample/Absorbance of control] × 100

ABTS Radical Cation Decolorization AssayThe spectrophotometric analysis of ABTS+ scavenging ac-

tivity was determined according to the method of Re et al. (33).ABTS+ was produced by reacting 2 mM ABTS in H2O with2.45 mM potassium persulfate, stored in the dark at room tem-perature overnight. The ABTS+ solution was diluted to give anabsorbance of 0.750 ± 0.025 at 734 nm in water. Then, 250µl of ABTS+ solution was added to 0.75 ml of jamun extractscontaining different concentrations (1.56–50 µg/ml). The ab-sorbance was recorded at 734 nm, 30 min after mixing, and thepercentage of radical scavenging was calculated for each con-centration against the blank (34). The scavenging capability oftest compounds was calculated using the following equation:

% ABTS+ scavenging activity = [Absorbance of control

− Absorbance of test sample/Absorbance of control] × 100

Ferric Reducing Antioxidant Power Assay(Fe3+ Reducing Power Assay)

The ferric reducing antioxidant power (FRAP) method (35)was used with minor modification. Briefly, different concentra-tions of extracts (10–80 µg/ml) in 0.75 ml of distilled waterwere mixed with 1.25 ml of 0.2 M sodium phosphate, pH 6.6,and 1.25 ml of 1% potassium ferricyanide. After incubation at50◦C for 20 min, the reaction mixture was acidified with 1.25 mltrichloroacetic acid (10%). Finally, 0.5 ml of FeCl3 (0.1%) wasadded to the reaction mixture and the absorbance was measuredat 700 nm. The increased absorbance of the reaction mixtureindicates greater reduction capability.

Cell Proliferation Assay and Measurements of CellViability

Inhibition of cell proliferation by jamun extracts was mea-sured in the human non-small-cell lung carcinoma cell lineA549. Cells were maintained in DMEM supplemented with10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/mlstreptomycin. Cells were plated in 96-well culture plates at 5× 103 cells/well. After 24-h incubation, cells were treated withextracts for 72 h, after which cell density was measured usingan MTT assay (36).

Statistical AnalysisAll calculations were performed with GraphPad Prizm sta-

tistical software (v. 4.03; La Jolla, CA). Data represent average± SD of 3 replicates. For cell proliferation assay, IC50 valueswere calculated using Microsoft Excel, XL Fit v. 5 software.

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432 F. AQIL ET AL.

Differences were considered a priori to be statistically signifi-cant if the P value was less than 0.05.

RESULTS

Extraction and Analysis of Jamun Pulp and SeedsJamun fruit has been reported to contain a variety of phyto-

chemicals. In this study, we analyzed anthocyanins and ellagicacid/ellagitannins in the jamun pulp and seeds and comparedtheir relative antioxidant and antiproliferative potency. The ja-mun pulp and seeds were extracted separately in acidified aque-ous ethanol (75% containing 10 mM HCl) and analyzed for 1)total phenolics, 2) anthocyanins, and 3) free sugars. Jamun pulppowder yielded at least 33% extract after 3 extractions, whereasseed powder yielded only 11%–12% (Table 1). Based on multi-ple extractions, we first determined that 3 repeat extractions canprovide at least 80% of extract (data not shown).

Jamun pulp and seed extracts were applied onto the XAD-761/HP-20 column (1:1) (24 g) for the enrichment of antho-cyanins and other polyphenolics, and one half of the enrichedextracts was hydrolyzed to convert the glycones to aglycones(e.g., anthocyanins to anthocyanidins). The hydrolyzed pulp andseed extracts yielded nearly 5% and 15% extracts, respectively(Table 2). The yields for unhydrolyzed pulp and seed extractswere higher.

Soluble SugarsAnthocyanins as well as some other phenolics (e.g., ellagitan-

nins) contain sugar moieties. We determined the sugar contentin both pulp and seed extracts. As expected, the pulp powderhad higher soluble sugars (9.0 ± 0.5%) than the seed powder(3.4 ± 0.4%); the enriched extracts were essentially free ofsoluble sugars (Tables 1 and 2).

Total PhenolicsJamun pulp powder contained 1.15% total polyphenolics

(w/w). Upon enrichment, the polyphenolic content in the pulpwas 1.77% that decreased to 1.31% upon hydrolysis and enrich-ment (Tables 1 and 2). Jamun seed powder contained twice asmuch polyphenolics (2.69%). Polyphenol content in the seedswas increased to 14.13% after enrichment, which decreased byhalf upon hydrolysis.

Anthocyanin ContentJamun pulp and seeds were analyzed for total anthocyanin

content using pH-differential method and expressed as cyanidin-3-glucoside equivalents. The jamun pulp powder contained0.54% anthocyanins. Upon enrichment, anthocyanins contentin the pulp was not changed. After hydrolysis, the enriched pulpextract yielded 0.23% of anthocyanidins. No anthocyanin wasdetected in the seed extracts (Tables 1 and 2).

HPLC AnalysisExtracts of the jamun pulp and seeds were analyzed by

HPLC monitoring the chromatogram at 366 nm for ellagicacid/ellagitannins, 280 nm for other polyphenolics, and 520 nmfor anthocyanins/anthocyanidins. Anthocyanins, the glycones ofdelphinidin, cyanidin, petunidin, peonidin, and malvidin, weredetected in unhydrolyzed pulp extracts. No peaks correspondingto anthocyanins were detected in the jamun seed extracts (Fig. 3).However, various peaks were detected at 366 and/or 280 nm,suggesting the presence of ellagitannins and other phenolic com-pounds. The presence of free ellagic acid and ellagitannins wasalso evident in jamun seeds. The identity of ellagic acid was es-tablished by cochromatography with reference compound. Theother peaks detected at 366 nm were presumably related to var-ious ellagitannins as the hydrolyzed extracts of both jamun pulpand seeds showed reduction in most of these peaks (10%–25%of relative area under curve) and enhancement in the ellagic acidpeak (Fig. 3), suggesting that the hydrolysis of the jamun pulpand seed extracts resulted in the conversion of some of the el-lagitannins (ellagic acid conjugates) to ellagic acid. Because ofcommercial unavailability of standards, the ellagitannin peakscould not be confirmed directly.

Further, upon hydrolysis, the jamun pulp extract revealed thepresence of 5 anthocyanidins, identified by cochromatographywith standards, as delphinidin, cyanidin, petunidin, peonidin,and malvidin (Fig. 4).

Antioxidant Assays

ORAC CapacityThe pulp and seed extracts exhibited concentration-

dependent total ORAC with respect to trolox equivalents.Fig. 5 shows the results of AAPH-mediated fluorescein oxi-dation in the presence and absence of the extracts in phosphate

TABLE 1Composition and characterization of Syzygium cumini (Jamun) extracts

Dry extract Soluble sugar Total phenolics Total anthocyaninsDry powder (g) (% yield) (g) (% yield) (g) (GAE) (% yield) (g) (% yield)

Pulp # 1 (8 g) 2.85 (35.6) 0.76 (9.5) 0.094 (1.18) 0.039 (0.49)Pulp # 2 (8 g) 2.63 (32.9) 0.68 (8.5) 0.089 (1.13) 0.046 (0.58)Seeds # 1 (8 g) 0.98 (12.3) 0.30 (3.8) 0.203 (2.54) Not detectedSeeds # 2 (8 g) 0.87 (10.9) 0.24 (3.0) 0.227 (2.84) Not detected

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TABLE 2Enrichment and analysis of anthocyanins and total phenolics in Syzygium cumini (Jamun) extracts by column chromatography

Enriched Soluble Total Total phenolics Free ellagicDry extracta extract (g) sugar (g) anthocyanins (g GAE) acid (mg)(weight) Treatment (% yield) (% yield) (g) (% yield) (% yield) (% yield)

Pulp extract 1 (2.63 g) Unhydrolyzed 0.33 (12.4) 0.003 (0.11) 0.015 (0.58) 0.05 (1.77) 5.40 (0.19)Pulp extract 2 (2.85 g) Hydrolyzed 0.14 (4.9) 0.001 (0.04) 0.007 (0.23) 0.04 (1.31) 3.94 (0.15)Seed extract 1 (0.87 g) Unhydrolyzed 0.26 (24.1) 0.001 (0.10) Not detected 0.12 (14.13) 36.34 (4.17)Seed extract 2 (0.98 g) Hydrolyzed 0.15 (14.8) 0.001 (0.10) Not detected 0.07 (7.14) 44.25 (4.51)

aExtracts were derived from 8 g material as in Table 1.

FIG. 3. HPLC profile of jamun pulp and seeds (hydrolyzed and unhydrolyzed) extracts. Twenty µl of enriched extracts (1 mg/ml) were injected and analyzedusing photo diodarray detector on a C18 (250 × 4.6 mm ID × 5 µm) reverse phase column in a gradient of 3.5% aqueous phosphoric acid and acetonitrile.Chromatograms in the first column represent various polyphenolics detected at 280 nm. Chromatograms in the second column represent ellagitannins (ETs) andfree ellagic acid (EA) monitored at 366 nm. Chromatograms in the last column show anthocyanins and anthocyanidins. mAu, milliabsorbance units. For clarity ofdata, y-axis scales are different.

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434 F. AQIL ET AL.

FIG. 4. HPLC chromatogram of jamun anthocyanidins at 520 nm extracted from fruit pulp. Twenty µl of hydrolyzed pulp extract (1 mg/ml) was injected andanalyzed using photo diodarray detector on a C18 reverse phase column in a gradient of 3.5% aqueous phosphoric acid and acetonitrile for 61 min. Inset representsrelative (%) anthocyanidins content. Please note that solvent gradient used here was different than the gradient used in Fig. 3 (see text).

buffer. In the absence of extracts or trolox, a progressive de-crease of fluorescein fluorescence was observed, with almosttotal consumption after 30 min (Fig. 5A). The decay curve ofthe fluorescein was concentration-dependent when tested withtrolox and the jamun pulp and seed (Fig. 5B) extracts. The areaunder the curve of the kinetic profiles was linearly related to theamount of the extracts (Fig. 5A and 5B). This behavior was ob-served for both pulp and seed extracts. At equal concentrationsof hydrolyzed extracts, the jamun seeds showed a higher ORACvalue (3,379 ± 151 µM of TE/g) than the jamun pulp (1,445 ±64 µmol of TE/g) (Fig. 5C).

DPPH AssayThe free radical-scavenging activity of the jamun pulp and

seed extracts was measured as decolorizing activity follow-ing the trapping of the unpaired electron of DPPH as shownin Fig. 6A. A concentration-dependent response was observedwith both the jamun pulp and seed extracts at the concentra-tions tested (10–80 µg/ml). The seed extracts almost com-pletely inhibited DPPH absorption. Further, the unhydrolyzedseeds showed somewhat better inhibition (∼90% at 50 µg/ml)compared with hydrolyzed seeds (76% inhibition at 50 µg/ml).

Ascorbic acid, included as a positive control, inhibited DPPHabsorption by 91% at 50 µg/ml. The activity was markedlylower in the unhydrolyzed (25%) and hydrolyzed jamun pulpextracts (46%). The concentrations of extracts that scavenged50% of DPPH varied with, respectively, unhydrolyzed seeds,16.3 µg/ml; hydrolyzed seeds, 19.8 µg/ml; hydrolyzed pulp,79.0 µg/ml; and unhydrolyzed pulp, 90.0 µg/ml.

ABTS AssayFig. 6B shows the scavenging of ABTS radicals by the jamun

extracts. Both the seed and pulp extracts in unhydrolyzed andhydrolyzed forms at 50 µg/ml scavenged the ABTS radicalsby greater than 99%. Even 20 µg/ml of the extracts containedat least 50% ABTS radical scavenging activity. The activity ofthe seed extract was comparable to that of the ascorbic acidat all concentrations. However, pulp extracts showed somewhatless activity. The concentrations of extracts that scavenged 50%of ABTS radicals during the 30-min incubation, obtained byinterpolation of plotted data were, respectively, unhydrolyzedor hydrolyzed seed extracts, 4.6 µg/ml each; hydrolyzed pulpextract, 11.8 µg/ml; and unhydrolyzed pulp extract, 17.2 µg/ml(Fig. 6B).

FIG. 5. Oxygen radical absorbance capacity (ORAC) of jamun fruit pulp and seeds extracts. A: Fluorescence decay curve of fluorescein in the presence of eitherblank or different concentrations of trolox (from left to right, respectively, 0, 2.5, 5, 10, 20, and 25 µM) and AAPH. B: Inset represents the linear plot of area undercurve (AUC) versus different concentrations of trolox. C: ORAC values are equivalent to trolox (µM/ g of extracts). ∗∗P < 0.005; ∗∗∗P < 0.001.

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FIG. 6. Antioxidant potential of Syzygium cumini fruit extracts (pulp and seed, hydrolyzed and unhydrolyzed in 3 different assays: A: 2,2-diphenyl-1-picrylhydrazylfree radical scavenging activity; B: 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) scavenging activity; and C: ferric ion reducing power. Eachdata point represents the mean of 3 experiments with standard deviation.

FRAP AssayThe FRAP assay has been widely used in the evaluation of

antioxidant component in dietary polyphenols. The reducingability of the unhydrolyzed and hydrolyzed jamun pulp extractswere concentration-dependent (Fig. 6C). The FRAP values forthe seed extracts of the jamun fruits were significantly higher(P < .001) than those of pulp extracts. When compared withascorbic acid as a reference, the seed extract showed reduc-ing ability either similar or higher. However, the jamun pulpdemonstrated less activity than the ascorbic acid control. Theconcentrations of extracts that reduced 50% of ferric iron duringthe 20-min incubation, obtained by interpolation of plotted data,were, respectively, unhydrolyzed seed extracts, 23.6 µg/ml; hy-drolyzed seed extracts, 24.7 µg/ml; hydrolyzed pulp extract, 52µg/ml; and unhydrolyzed pulp extract, 80 µg/ml (Fig. 6C).

Antiproliferative ActivityTo evaluate the effect of jamun extracts on cell viability of

human non-small-lung cancer cells (A549), an MTT assay wasperformed. A concentration-dependent response was observedwith both the jamun pulp and seed extracts at the concentra-tions tested (12.5–200 µg/ml) (Fig. 7). Hydrolyzed jamun pulpshowed strong antiproliferative activity with an IC50 value of59 ± 4 µg/ml. However, unhydrolyzed pulp had a very weakeffect at the tested concentrations. The hydrolyzed seed extractshowed the highest antiproliferative activity (IC50 = 38 ± 3 µg/ml; P < 0.05). However, the activity of unhydrolyzed seed ex-tract (IC50 = 64 ± 5 µg/ml) was similar to that of hydrolyzedpulp.

DISCUSSIONBerries are an excellent source of various bioactive polyphe-

nolics, including anthocyanins, proanthocyanidins, ellagitan-nins, flavonols, and phenolic acids. In this study, Syzygiumcumini (jamun, the Indian blackberry) fruit pulp and seed pow-der were extracted using 75% aqueous ethanol and tested forcontents of anthocyanins, ellagic acid, and other polyphenolics,as well as their activities. Thirty percent of jamun pulp powderwas extractable, which decreased to 2%–4% upon enrichment.Similarly, jamun seed powder yielded 2%–3% enriched extract.This approach of using a mixture of XAD-761/diaion HP-20

FIG. 7. Antiproliferative activity of Syzygium cumini pulp and seed extractsby MTT assay. Cells were treated with different jamun extracts at 12.5–200µg/ml concentrations for 72 h. Data are expressed as percentage of untreatedcells, mean ± SD (n = 3). Each data point represents the mean of 3 experimentswith standard deviation.

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served as an effective means to enrich both anthocyanins andellagitannins along with other polyphenolics. The column wasreusable multiple times after regeneration and provided repro-ducible results.

Anthocyanins content in the jamun pulp powder used in thisstudy (0.54) was more than the reported anthocyanins (0.23%)(18) where the pigments were extracted from the fresh pulp.The 3.5% anthocyanins reported in jamun extract is twice theamount found in our pulp extracts (1.5–1.8%) (22). Differentgeographical regions and climatic conditions could potentiallyexplain these differences, although we cannot rule out if thelower content found in our preparation was due to the extractionprocedure and drying method of the pulp. No anthocyanins weredetected in the seed extracts either by pH differential method orHPLC analysis, consistent with the findings of others (22).

The conversion of essentially all anthocyanins into antho-cyanidins confirms the complete hydrolysis. Irrespective of sam-ples, various peaks were detected at 280 nm, indicating the pres-ence of unidentified polyphenolics (Fig. 3). Jamun pulp extractscontained 0.17% free ellagic acid in our preparation. This EAwas “free” because it was detected in addition to several peaksassociated with ellagitannins without hydrolysis of the extract;upon hydrolysis, ellagitannins were converted to ellagic acid, asdiscussed below. Ellagic acid/ellagitannin content in seed extract(4.17%) is in agreement with the reported level (4.2%) (24).

In this study, quantification of ellagitannins was not possibledue to unavailability of authentic standards. Ellagic acid in theseed extracts was almost 20-fold higher than that in pulp ex-tracts (Table 2). In addition to ellagic acid, various compoundsdetected at 366 nm in the unhydrolyzed pulp and seeds are pre-sumably related to ellagitannins. It is noteworthy that the levelof ellagic acid increased upon hydrolysis of the extract. Sinceellagitannins are complex derivatives of ellagic acid and yieldedellagic acid upon hydrolysis, these unidentified peaks detectedat 366 nm may be related to ellagitannins. Jamun pulp powderused in this study contained 1.15% polyphenolics (inclusive ofanthocyanins), and it is double the amount (0.56%) reported(18). Enriched pulp contains 1.77% polyphenols that reduced to1.31% upon hydrolysis. This decrease can be attributed to lossof sugar during the conversion of anthocyanins to anthocyani-dins. Phenolic content in seed powder was twice or high (2.7%)than pulp and increased to 14.13% upon enrichment.

Jamun appears to be the only berry that contains the 5 an-thocyanidins present in blueberry/bilberry along with significantamounts of ellagic acid/ellagitannins. In view of the known anti-carcinogenicity potential of these 5 anthocyanidins in blueberryand cyanidin together with ellagic acid/ellagitannins in blackraspberry against estrogen-mediated breast cancer (13,37), thiscomposition of anthocyanidins and ellagic acid/ellagitanninsin jamun could potentially provide additive, if not synergistic,effects.

Antioxidants have attracted much interest with respect totheir protective effects against free radical damage involved inmany degenerative diseases, including cancer. The multiplicity

of antioxidants present in whole fruits and vegetables is con-sidered to elicit higher activity than any individual constituents(8). Both pulp and seed of jamun had high antioxidant capacitiesrelative to trolox. The higher ORAC value of the jamun seedextract (double that of pulp) may be attributed to the higher totalpolyphenolics compared to those in the pulp extract, given thelinear relationship between ORAC and total phenolic content inthe fruit of the Vaccinium species (38).

Jamun unhydrolyzed extract had maximum antioxidant po-tential both in DPPH and ABTS assays. The concentrations ofextracts that scavenged 50% of DPPH varied with, respectively,unhydrolyzed seeds, 16.3 µg/ml; hydrolyzed seeds, 19.8 µg/ml; hydrolyzed pulp, 79.0 µg/ml; and unhydrolyzed pulp, 90.0µg/ml. The cyanidin- and delphinidin-derived pigments havea catechol (ortho-dihydroxyl) group and a pyrogallol (vicinaltrihydroxyl) group, respectively. These structural features arelikely to contribute a significant radical scavenging capacityof these compounds (8). Likewise, the presence of cis-diols inellagic acid/ellagitannins is known to scavenge free radicals in-duced by redox cycling of estrogen catechols (13,39). It is alsosuggested that aglycones have higher antioxidant activity overtheir glycone counterparts (15).

ABTS radicals are more reactive than DPPH radicals and,unlike the reaction with DPPH radicals, which involve H-atomtransfer, the reactions with ABTS+ radicals involve an electron-transfer process. Jamun extracts are better scavengers of ABTScompared to DPPH, with much lower concentration’s that scav-enged 50% of tested radicals.

Metal ion chelation is another important antioxidant property,and few compounds (e.g., resveratrol) have been shown for theirability to compete with ferrozine for ferrous ion in solution(40). In this study, jamun showed a similar trend for antioxidantpotential. Hydrolyzed seeds had activity comparable to ascorbicacid.

Compounds containing 2 or more of the functional groupssuch as -OH, -SH, -COOH, -PO3H2, C O, -NR2, -S-, and -O-in a favorable structure-function configuration can show metalchelation activity (34). Jamun pulp contained anthocyaninsalong with ellagic acid/ellagitannins, whereas seeds contained ahigher content of ellagic acid/ellagitannins. These compoundshave catechol and many -OH groups that might contribute totheir antioxidant potential.

Endogenous oxidative DNA damage has been considered tobe a significant factor in the initiation of human cancer. Thecancer-protective effect of vegetables and fruits is attributed tothe ability of the antioxidants in them to scavenge free radi-cals, preventing DNA damage and subsequent mutation. In thepresent study, jamun pulp and seed extracts decreased the viablecell number of human lung cancer cell line. Hydrolyzed jamunpulp has strong antiproliferative activity, whereas unhydrolyzedextract is less effective. We have demonstrated that jamun pulpcontained all 5 major anthocyanidins in different proportions.The antiproliferative activity of individual anthocyanidins hasbeen shown in different cancer types (15). The activity in the

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jamun hydrolyzed and unhydrolyzed seed extracts may be at-tributed to the high content of polyphenolics, including variousellagitannins and EA. Ellagitannin-enriched fractions showed asimilar effect against a breast cancer cell line (41).

CONCLUSIONXAD-7 and Diaion HP-20 resins provide an effective method

for removing sugars as well as enriching a variety of polyphe-nolics, including anthocyanins and ellagic acid/ellagitannins.In addition to anthocyanins, jamun pulp is rich in ellagicacid/ellagitannins. On the other hand, jamun seeds are richin ellagic acid/ellagitannins in addition to other unidentifiedpolyphenolics. Jamun is the only berry that contains 5 antho-cyanins and appreciable amounts of ellagic acid/ellagitannins.Phytochemicals in both jamun pulp and seed extracts were aneffective antioxidant in different in vitro assays, including as-says of free radical-scavenging (DPPH and ABTS), reducingpower, and ORAC, when compared with standard antioxidantcompounds such as ascorbic acid and trolox. Together, our datashow high antioxidant and antiproliferative activities in jamun,and this berry may be a viable candidate for chemopreventionof lung cancer.

ACKNOWLEDGMENTSThis work was supported by Agnes Brown Duggan Endow-

ment and the USPHS grant CA-125152. Dr. Ramesh Guptaholds the Agnes Brown Duggan Chair in Oncological Research.Thanks to Technology Development Centre, NIPER, S.A.S. Na-gar, India for vacuum drying of jamun pulp. Ms. Ellen Snell ofthe University of Louisville Writing Center is acknowledged forgoing through the manuscript.

REFERENCES1. Steer P, Millgard J, Sarabi DM, Basu S, Vessby B, et al.: Cardiac and vascu-

lar structure and function are related to lipid peroxidation and metabolism.Lipids 37, 231–236, 2002.

2. Cheeseman KH and Slater TF: An introduction to free radical biochemistry.Br Med Bull 49, 481–493, 1993.

3. Yu L, Haley S, Perret J, Harris M, Wilson J, et al.: Free radical scavengingproperties of wheat extracts. J Agric Food Chem 50, 1619–1624, 2002.

4. Naczk M and Shahidi F: Phenolics in cereals, fruits and vegetables: oc-currence, extraction and analysis. J Pharm Biomed Anal 41, 1523–1542,2006.

5. Liu F and Ng TB: Antioxidative and free radical scavenging activities ofselected medicinal herbs. Life Sci 66, 725–735, 2000.

6. Savikin K, Zdunic G, Jankovic T, Tasic S, Menkovic N, et al.: Pheno-lic content and radical scavenging capacity of berries and related jamsfrom certificated area in Serbia. Plant Foods Hum Nutr 64, 212–217,2009.

7. Ivanova D, Gerova D, Chervenkov T, and Yankova T: Polyphenols andantioxidant capacity of Bulgarian medicinal plants. J Ethnopharmacol 96,145–150, 2005.

8. Rice-Evans CA and Miller NJ: Antioxidant activities of flavonoids as bioac-tive components of food. Biochem Soc Trans 24, 790–795, 1996.

9. Perron NR and Brumaghim JL: A review of the antioxidant mechanisms ofpolyphenol compounds related to iron binding. Cell Biochem Biophys 53,75–100, 2009.

10. Mazza GJ: Anthocyanins and heart health. Ann Ist Super Sanita 43,369–374, 2007.

11. Jayaprakasam B, Olson LK, Schutzki RE, Tai MH, and Nair MG: Amelio-ration of obesity and glucose intolerance in high-fat-fed C57BL/6 mice byanthocyanins and ursolic acid in Cornelian cherry (Cornus mas). J AgricFood Chem 54, 243–248, 2006.

12. Andres-Lacueva C, Shukitt-Hale B, Galli RL, Jauregui O, Lamuela-Raventos RM, et al.: Anthocyanins in aged blueberry-fed rats arefound centrally and may enhance memory. Nutr Neurosci 8, 111–120,2005.

13. Aiyer HS, Srinivasan C, and Gupta RC: Dietary berries and ellagic acid di-minish estrogen-mediated mammary tumorigenesis in ACI rats. Nutr Can-cer 60, 227–234, 2008.

14. Stoner GD, Wang LS, Zikri N, Chen T, Hecht SS, et al.: Cancer preventionwith freeze-dried berries and berry components. Semin Cancer Biol 17,403–410, 2007.

15. Wang LS and Stoner GD: Anthocyanins and their role in cancer prevention.Cancer Lett 269, 281–290, 2008.

16. Achrekar S, Kaklij GS, Pote MS, and Kelkar SM: Hypoglycemic activityof Eugenia jambolana and Ficus bengalensis: mechanism of action. In Vivo5, 143–147, 1991.

17. Chaturvedi A, Bhawani G, Agarwal PK, Goel S, Singh A, et al.: Antidiabeticand antiulcer effects of extract of Eugenia jambolana seed in mild diabeticrats: study on gastric mucosal offensive acid-pepsin secretion. Indian JPhysiol Pharmacol 53, 137–146, 2009.

18. Veigas JM, Narayan MS, Laxman PM, and Neelwarne B: Chemical na-ture, stability and bioefficacies of anthocyanins from fruit peel of syzygiumcumini Skeels. Food Chem 105, 619–627, 2007.

19. Prince PS, Menon VP, and Pari L: Hypoglycaemic activity of Syzigiumcumini seeds: effect on lipid peroxidation in alloxan diabetic rats.J Ethnopharmacol 61, 1–7, 1998.

20. Kaur C and Kapoor HC: Antioxidant activity of some fruits in Indian diet.Acta Hort (ISHS) 696, 593–565, 2005.

21. Arun R, Prakash MV, Abraham SK, and Premkumar K: Role of Syzygiumcumini seed extract in the chemoprevention of in vivo genomic damage andoxidative stress. J Ethnopharmacol 134, 329–333, 2011.

22. Li L, Adams LS, Chen S, Killian C, Ahmed A, et al.: Eugenia jambolanaLam. berry extract inhibits growth and induces apoptosis of human breastcancer but not nontumorigenic breast cells. J Agric Food Chem 57, 826–831,2009.

23. Benherlal PS and Arumughan C: Chemical composition and in vitro antiox-idant studies on Syzygium cumini fruit. J Sci Food Agric 87, 2560–2569,2007.

24. Sharma M, Li L, Celver J, Killian C, Kovoor A, et al.: Effects of fruitellagitannin extracts, ellagic acid, and their colonic metabolite, urolithin A,on Wnt signaling. J Agric Food Chem 58, 3965–3969, 2010.

25. Kahkonen MP, Heinamaki J, Ollilainen V, and Heinonen M: Berry an-thocyanins: isolation, identification, and antioxidant activities. J Sci FoodAgric 83, 1403–1411, 1984.

26. Seeram NP, Lee R, Hardy ML, and Heber D: Rapid large-scale purificationof ellagitannins from pomegranate husk, a by-product of the commercialjuice industry. Separ Purific Technol 41, 49–55, 2005.

27. Lee J, Durst RW, and Wrolstad RE: Determination of total monomericanthocyanin pigment content of fruit juices, beverages, natural colorants,and wines by the pH differential method: collaborative study. J AOAC Int88, 1269–1278, 2005.

28. Guisti MM and Wrolstad RE: Characterization and measurement of an-thocyanins by UV-visible spectroscopy. In: Currents Protocols in FoodAnalytical Chemistry, Wrolstad RE (ed.). New York, NY: John Wiley &Sons, 2001, pp. 1–13.

29. Yemm EW and Willis AJ: The estimation of carbohydrates in plant extractsby anthrone. Biochem J 57, 508–514, 1954.

Dow

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by [

Stan

ford

Uni

vers

ity L

ibra

ries

] at

22:

51 0

5 O

ctob

er 2

012

438 F. AQIL ET AL.

30. Lister E and Wilson P: Measurement of total phenolics and ABTS assay forantioxidant activity. Crop Research Institute, Lincoln, New Zealand, 2001.

31. Ou B, Hampsch-Woodill M, and Prior RL: Development and validation ofan improved oxygen radical absorbance capacity assay using fluorescein asthe fluorescent probe. J Agric Food Chem 49, 4619–4626, 2001.

32. Gyamfi MA, Yonamine M, and Aniya Y: Free-radical scavenging actionof medicinal herbs from Ghana: Thonningia sanguinea on experimentallyinduced liver injuries. Gen Pharmacol 32, 661–667, 1999.

33. Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, et al.: Antioxidantactivity applying an improved ABTS radical cation decolorization assay.Free Radic Biol Med 26, 1231–1237, 1999.

34. Ak T and Gulcin I: Antioxidant and radical scavenging properties of cur-cumin. Chemico-Biological Interac 174, 27–37, 2008.

35. Oyaizu M: Studies on product of browning reaction prepared from glucoseamine. Jpn J Nutr 44, 307–315, 1986.

36. Munagala R, Kausar H, Munjal C, and Gupta RC: Withaferin A inducesp53-dependent apoptosis by repression of HPV oncogenes and upregulation

of tumor suppressor proteins in human cervical cancer cells. Carcinogenesis32, 1697–1705, 2011.

37. Aiyer HS and Gupta RC: Berries and ellagic acid prevent estrogen-inducedmammary tumorigenesis by modulating enzymes of estrogen metabolism.Cancer Prev Res (Phila) 3, 727–737, 2010.

38. Prior RL, Cao GH, Martin A, Sofic E, McEwen J, et al.: Antioxidantcapacity as influenced by total phenolic and anthocyanin content, maturity,and variety of Vaccinium species. J Agric Food Chem 46, 2686–2693, 1998.

39. Aiyer HS, Vadhanam MV, Stoyanova R, Caprio GD, Clapper ML, et al.: Di-etary berries and ellagic acid prevent oxidative DNA damage and modulateexpression of DNA repair genes. Int J Mol Sci 9, 327–341, 2008.

40. Kehrer JP: The Haber-Weiss reaction and mechanisms of toxicity. Toxicol-ogy 149, 43–50, 2000.

41. Adams LS, Zhang Y, Seeram NP, Heber D, and Chen S: Pomegranateellagitannin-derived compounds exhibit antiproliferative and antiaromataseactivity in breast cancer cells in vitro. Cancer Prev Res (Phila) 3, 108–113,2010.

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