bioactivity of nonedible parts ofpunica granatuml

Hindawi Publishing CorporationInternational Journal of Food ScienceVolume 2013 Article ID 602312 12 pageshttpdxdoiorg1011552013602312

Research ArticleBioactivity of Nonedible Parts of Punica granatum LA Potential Source of Functional Ingredients

Nawraj Rummun1 Jhoti Somanah2 Srishti Ramsaha3

Theeshan Bahorun4 and Vidushi S Neergheen-Bhujun3

1 Department of Health Sciences Faculty of Science University of Mauritius Reduit Mauritius2 Department of Biosciences and ANDI Centre of Excellence for Biomedical and Biomaterials ResearchUniversity of Mauritius Reduit Mauritius

3 Department of Health Sciences Faculty of Science and ANDI Centre of Excellence for Biomedical and Biomaterials ResearchUniversity of Mauritius Reduit Mauritius

4ANDI Centre of Excellence for Biomedical and Biomaterials Research University of Mauritius Reduit Mauritius

Correspondence should be addressed to Vidushi S Neergheen-Bhujun vneergheenuomacmu

Received 30 March 2013 Accepted 10 June 2013

Academic Editor Fabienne Remize

Copyright copy 2013 Nawraj Rummun et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Punica granatum L has a long standing culinary and medicinal traditional use in Mauritius This prompted a comparative studyto determine the bioefficacy of the flower peel leaf stem and seed extracts of the Mauritian P granatum The flower and peelextracts resulting fromorganic solvent extraction exhibited strong antioxidant activitieswhich correlatedwith the high levels of totalphenolics flavonoids and proanthocyanidins The peel extract had the most potent scavenging capacity reflected by high Troloxequivalent antioxidant capacity value (520601plusmn57848 120583molg air dry weight) very low IC

50values for hypochlorous acid (0004plusmn

0001mg air dry weightmL) and hydroxyl radicals scavenging (0111plusmn0001mg air dry weightmL) Peel extracts also significantlyinhibited S mutans (119875 lt 0001) S mitis (119875 lt 0001) and L acidophilus (119875 lt 005) growth compared to ciprofloxacin The flowerextract exhibited high ferric reducing nitric oxide scavenging and iron (II) ions chelation and significantly inhibited microsomallipid peroxidation Furthermore it showed a dose-dependent inhibition of xanthine oxidase with an IC

50value of 0058plusmn 0011mg

air dry weightmL This study showed that nonedible parts of cultivated pomegranates that are generally discarded are bioactivein multiassay systems thereby suggesting their potential use as natural prophylactics and in food applications

1 Introduction

Punica granatum L fruit or fruit juice has for the pastdecade been advocated as an interesting functional foodthat can confer health benefits beyond basic nutrition [1 2]P granatum L belongs to the family of Punicaceae and isindigenous to the Himalayas in northern India and to Iran[3] but has grown and been naturalized in a number of Asianand African countries including Mauritius The edible andnonedible parts (Figure 1) have been reported to treat differ-ent pathological conditions in different folklore medicine [4ndash6] Documented use of pomegranate in Mauritian folkloremedicine includes ingestion of macerated bark extracts to

treat asthma chronic diarrhea chronic dysentery relaxationof the larynx and intestinal worms [7]

Pomegranate extracts are known for their antidiabeticantibacterial anticarcinogenic antiatherogenic and antihy-pertensive potential amongst others [3] Pomegranate juiceis also used as mouthwash in oral hygiene [8] Consumptionof pomegranate juice has been linked with a decrease ininflammatory biomarkers levels and oxidation of both pro-teins and lipids in a randomized placebo-controlled trial [9]In the same vein the beneficial effect of pomegranate juicewas reported in an initial phase II clinical trial in patientswith prostate cancer [10]The health benefits of pomegranatehave been ascribed to the pluripharmacological effects of

2 International Journal of Food Science

(a) (b)

(c)

(d) (e) (f)

(g) (h) (i)

Figure 1 Different anatomical parts of P granatum tree and fruit (a) Unripe fruit (b) flower (c) stem (d) flower and tubular calyx (e) youngleaves at the branch endings (f) the fruitrsquos rind with membranous extensions forming compartments which contain the juicy arils (g) seeds(h) arils (juicy pulp coating the seed) (i) pomegranate inner membrane

the secondary metabolites more specifically its polyphenoliccompounds present in relatively high concentrations [11ndash13]

The phytophenolic compositions vary differently in theedible and nonedible parts of the plants and have been widelyinvestigated Pomegranate fruit (peel aril seeds and juice)has been reported to be rich in phenolic acids flavanolsflavones flavonones anthocyanidins and anthocyanin [3]Literature data reported glycated anthocyanins (pelargoni-din 35-diglucoside pelargonidin 3-glucoside) apart from

phenolic compounds common to the edible parts like gal-lic acid in the flowers [14] while the nonedible parts ofpomegranate comprising leaves roots and stem containedapigenin punicalin punicalagin and luteolin [3 15 16] Thisrich polyphenolic composition has been intrinsically linkedto the pluripharmacological effects of pomegranate extractsHowever it should be noted that sources of variation in thelevel of phytochemicals and nutrients arising from geneticvariability of a naturalized plant in addition to geographical

International Journal of Food Science 3

and environmental factors can result in diverse polyphenoliccompositions that modulate bioactivity level

Whilst recent years havewitnessed a surge in the scientificevaluation of the ethnopharmacological uses of pomegranatelimited works have been reported on the assessment of thenonedible discarded parts as a source of bioactive ingredientsfor the functional food industry Thus this study aimedat determining the antibacterial anti-inflammatory andantioxidant potential of the nonedible parts of the Mauritiancultivar of pomegranate with the view of promoting theirutilization in functional health and in potential food appli-cations

2 Methodology

21 Chemicals and Microorganisms Streptococcus mutans(ATCCR 5175) Streptococcus mitis (ATCCR 6249) and Lac-tobacillus acidophilus (ATCCR 4356) were purchased fromATCC Brain heart infusion agar (BHI) and de Man Rogosaand Sharpe agar (MRS) were purchased from Sigma-AldrichGermany BHI was used for growth of S mutans and S mitiswhile MRS agar was utilized for L acidophilus All otherchemicals used were of analytical grade

22 Plant Materials Pomegranate plant and fruit parts werecollected from a domesticated plant growing in a backyardin ldquoTrioletrdquo village situated in the Pamplemousses districtin the northern part of Mauritius Island during the monthof September 2011 and authenticated by the herbarium ofMauritius Sugar Industry Research Institute Reduit Punicagranatum leaves stems flowers and fruits were collectedfrom the same plantThe latter were air dried and the sampleshomogenized to a fine powder prior to extraction

23 Preparation of Extracts The plant material was extractedthrice with 70methanol (1 3 wv) and allowed tomacerateeach time at 4∘C for 24 hours The filtrates were pooledtogether and concentrated in vacuo at 37∘C The concen-trated aqueous extract was partitioned in dichloromethane toremove fats and chlorophyll and the aqueous phase was thencollected and lyophilized The lyophilized powders thereofderived were dissolved in deionized water and in 100methanol to a concentration of 1 g of air dried mass to 5mLfor the subsequent tests

24 Total Phenolic Content (TPC) The total phenolic con-tent was estimated using the Folin-Ciocalteu assay adaptedfrom Neergheen et al [17] The reaction mixture in a finalvolume of 5mL contained 025mL of the extracts 350mL ofdistilled water and 025mL of Folin-Ciocalteu reagent After3 minutes 075mL of 20 sodium carbonate solution wasadded The tubes were mixed thoroughly and heated for 40minutes in a water-bath set at 40∘C and then allowed to coolThe absorbance of the blue coloration was read at 685 nmagainst a blank Total phenolics were calculated with respectto a gallic acid standard curve (stock solution 250120583gmL) andresults expressed in mg of gallic acid equivalent (GAE)g airdry weight (ADW) of plant material

25 Total Proanthocyanidin Content (TPrC) A modifiedHClButan-1-ol assay adapted from Porter et al [18] was usedfor the quantification of total proanthocyanidin content ofthe methanolic plant extracts The reaction mixture in eachtube contained in a final volume of 335mL the followingin order of addition 025mL extract 3mL of n-BuOHHCl(95 5 vv) and 01mL of 2 NH

4Fe (SO

4)2sdot12 H

2O in

2M HCl and the tubes were incubated for 40 minutes at95∘C A red coloration was developed and the absorbancewas read at 550 nm against a blank standard containing025mL n-BuOHHCl (95 5 vv) instead of extract Theamount of proanthocyanidins in the extracts was calculatedin triplicates with respect to a cyanidin chloride standardcurve (stock solution 01mgmL) Results were expressed inmg of cyanidin chloride equivalent (CCE)g ADW of plantmaterial

26 Total Flavonoid Content (TFC) Total flavonoids weremeasured using a colorimetric assay adapted fromZhishen etal [19] A total of 150120583L of 5 aqueous NaNO

2was added to

250mL of extract After 5min 150120583L of 10 aqueous AlCl3

was added A total of 1mL of 1MNaOHwas added 1min afterthe addition of aluminum chloride The absorbance of thesolution was measured at 510 nm Flavonoid contents wereexpressed in 120583g querceting of ADW of plant material

27 Ferric Reducing Antioxidant Power Assay (FRAP) Thereducing power of the extracts was assessed using themethodof Benzie and Strain [20] A total of 100 120583L of sample wasadded to 300 120583L of distilled water followed by 3mL of FRAPreagent (40mM HCl and 20mL of 20mM ferric chloridein 200mL of 025M sodium acetate buffer at pH 36) Theabsorbance was read at 593 nm after 4min of incubation at37∘C Results were expressed in 120583mol Fe2+ equivalentg ofADW of plant material

28 Trolox Equivalent Antioxidant Capacity (TEAC) AssayThe free radical scavenging capacity of the extracts wasmeasured by the TEAC assay according to the method ofCampos and Lissi [21] A total of 050mL of diluted plantextract was added to 3mL of the ABTS∙+ solution generatedby a reaction between 22-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) diammonium salt (ABTS 050mM) andactivated MnO

2(1mM) in phosphate buffer (010M pH 7)

Decay in absorbance was monitored at 734 nm for 15minTEAC values are expressed in 120583mol Trolox equivalentg ofADW

29 Iron (II) Chelating Activity Themethod of Neergheen etal [17] was adapted to assess the iron (II) chelating activityof the extracts The reaction mixture contained in order ofaddition 200120583L of plant extract (varied concentrations) and50120583L of FeCl

2sdot4H2O (05mM) The reaction volume was

made up to 1mLwith distilled deionised water and incubatedfor 5 minutes at room temperature After incubation 50 120583Lof FerroZine (25mM) was added and the purple colorationformed was read at 562 nm The absorbance of the reactionmixture was read both before and after the addition of


FerroZine to account for possible interferences caused bythe plant extract The controls contained all the reactionreagents and water instead of the extract or the positivecontrol substance EDTA was used as a positive control Thepercentage chelating activity was calculated and results wereexpressed as mean IC

50(mg ADWmL)

210 Scavenging of Hypochlorous Acid (HOCl) The abilityof the extracts to scavenge HOCl was assessed essentiallyas described by Neergheen et al [17] Briefly the reactionmixture contained 100120583L taurine (10mM) 100 120583L HOCl(1mM) 100 120583L plant extract (variable concentrations) and700120583L phosphate saline buffer (pH 74) in a final volumeof 1mL The solution was mixed thoroughly and incubatedfor 10 minutes at ambient temperature The sample was thenassayed for taurine chloramine by adding 10 120583L potassiumiodide to the reacting mixture The I

2released was deter-

mined spectrophotometrically at 350 nm in the presence ofexcess Iminus as I

3

minus The absorbance of the reaction mixture wasread both before and after the addition of potassium iodide asto cater for possible interferences caused by the plant extractThe analyses were made in triplicates and the results wereexpressed as IC

50mg ADWmL for the plant extracts

211 Inhibition of Deoxyribose Degradation The hydroxylradical scavenging potential of the extracts was determinedusing the deoxyribose assay [22] The reacting mixturecontained in a final volume of 1mL the following reagentsorder of addition indicated 200120583L of 100mM KH

2PO4-

KOH 200120583L of 05mM FeCl3 100 120583L of 1mMEDTA 100 120583L

sample 200120583L of 15mM deoxyribose 100 120583L of 10mMH2O2 and 100 120583L of 1mM ascorbic acid Reaction mixtures

were incubated at 37∘C for 1 hourAt the end of the incubation period 1mL 1 (wv) thio-

barbituric acid (TBA) was added to each mixture followed bythe addition of 1mL 28 (wv) trichloroacetic acid (TCA)The solutionswere heated in awater bath at 80∘C for 20min todevelop the pink coloured MDA-(TBA)

2adduct The MDA-

(TBA)2chromogenwas extracted into 3mL butan-1-ol and its

absorbance measured at 532 nm The analyses were made intriplicates and the results were expressed as IC

50g ADWmL

for the plant extracts

212 Inhibition of Microsomal Lipid Peroxidation Beef livermicrosomes were prepared by tissue homogenization asdescribed by Neergheen et al [17] The formation of malon-dialdehyde measured as thiobarbituric reactive substances(TBARS) was used to monitor microsomal lipid peroxida-tionThe reactionmixture contained in a final volume of 1mLthe following 200120583L of 34mM phosphate buffered saline(pH 74) 200120583L of 05mgmLmicrosomal protein 400 120583L ofsample (variable concentrations) 100120583L of 1mM FeCl

3 and

100 120583L of 1mM ascorbate The mixture was incubated for 1hour at 37∘C

At the end of the incubation period 100 120583L 2 (wv) BHTwas added followed by 1mL 1 (wv) thiobarbituric acid and28 (wv) trichloroacetic acid The solutions were heated ina water bath at 80∘C for 20min to develop the pink coloured

MDA-(TBA)2adduct As turbidity was encountered the

MDA-(TBA)2chromogen was extracted into 2mL butan-1-

ol and its absorbance measured at 532 nm The inhibition ofmicrosomal lipid peroxidation was calculated and results areexpressed as mean IC

50(mg ADWmL)

213 Superoxide Anion Radical Scavenging Assay The super-oxide anion scavenging activity of the pomegranate extractswas measured according to the modified method of Kumaret al [23] One mL of 156120583M of nitroblue tetrazolium (NBT)aqueous solution and 1mL of 200 120583M beta-nicotinamideadenine dinucleotide reduced disodium salt hydrate (NADH)aqueous solution were mixed together followed by theaddition of 1mL of aqueous pomegranate extract (variedconcentration) The reaction was started by adding 100120583Lof 60120583Mphenazine methosulphate (PMS) aqueous solutionThe reaction mixture was incubated at 25∘C for 20 min-utes and the absorbance was measured at 560 nm againstcontrol sample Ascorbic acid was used as a positive con-trol IC

50value was calculated from the dose-dependent

curve obtained by plotting antioxidant activity () againsta concentration range for each pomegranate extract Theantioxidant activity was calculated as follows

antioxidant activity = [(1198600minus 1198601)

1198600

] times 100 (1)

where 1198600is the absorbance of the control (reaction mixture

without test sample) and 1198601is the absorbance of the test

sample

214 Nitric Oxide Radical Inhibition Assay Nitric oxideradical inhibition was evaluated according to the modi-fied method of Sunil et al [24] Griess Illosvoyrsquo reagentwas modified by using 01 (wv) naphthylethylenediaminedihydrochloride The reaction mixture contained 05mL ofextracts (variable concentrations) 2mL of 10mM aqueoussodium nitroprusside and 05mL phosphate saline bufferThe mixture was incubated at 25∘C for 180 minutes 05mLof the reaction mixture was pipetted out and 2mL of GriessIllosvoyrsquos reagent (033 sulphanilic acid in 20 glacialacetic acid and 01 naphthylethylenediamine dichloride)was added mixed and allowed to stand for 30 minutes Theabsorbance of the pink chromophore formed was measuredat 546 nm Ascorbic acid was used as positive control andthe percentage antioxidant activity was calculated IC

50value

was calculated from the dose-dependent curve obtainedby plotting antioxidant activity () against a concentrationrange for each pomegranate extract

215 Xanthine Oxidase (XO) Inhibition Assay Spectropho-tometric determination of XO inhibitory activity measuringuric acid production from xanthine substrate was used Themethod was adapted from Havlik et al [25] with some mod-ifications The mixture consisted of 250 120583L extract (variedconcentrations) 400120583L 012M phosphate buffer (pH 75)and 330 120583L xanthine (8mM in same buffer) The reactionwas initiated by adding 20120583L of xanthine oxidase (05UmL


Table 1 Total phenolics total flavonoids and total proanthocyani-din content of pomegranate parts extracts

TPC mg GAEgADW plusmn SD

TFC mg QEgADW plusmn SD

TPrC mg CCEgADW plusmn SD

Flower 33651 plusmn 070a21354 plusmn 314

a146 plusmn 006

ab

Peel 19027 plusmn 054b18010 plusmn 131

b248 plusmn 008

a

Leaf 8781 plusmn 047c6389 plusmn 062

c021 plusmn 001

b

Stem 5292 plusmn 062d4136 plusmn 052

d032 plusmn 001

b

Seed 065 plusmn 000e033 plusmn 000

e013 plusmn 000

b

LSD value at5 significance 096 285 129

ADW air dry weight CCE cyanidin chloride equivalent GAE gallic acidequivalent QE quercetin equivalent TPC total phenolic content TFCTotal Flavonoid content TPrC Total proanthocyanidin content Differentsuperscripts between rows in individual columns represent significantdifference between extracts Data expressed as mean plusmn standard deviation(119899 = 3)

in same buffer) which was prepared immediately before useThe tubes were incubated at room temperature for 5 minutesand the reaction stopped by the addition of 200120583L 1M HClAbsorbance for formation of uric acid was read at 295 nmAllopurinol was used as the positive control (concentrationrange 25ndash350 120583M) The inhibition was calculated andactivity of extract presented as calculated IC

50(120583M)

216 Determination of Minimum Inhibition Concentration ofPunica granatum Sterilized molten agar was dispensed intosterile Petri dishes and allowed to solidify Microbial suspen-sion (150120583L) containing approximately 15 times 108 CFUmLwas spread evenly over the surface of the solidified mediumand left to air dry Meanwhile 20120583L of sample extracts andciprofloxacin (2mgmL) were loaded separately onto sterileoven-dried paper discs and placed firmly ontomedium usingforceps Each plate consisted of four impregnated discs twoextracts one positive control and one extract + positivecontrol The experiment was performed in quadruplicate

The Petri dishes were inverted and incubated at 37∘C for24 hours After the incubation period the diameter of thezone of inhibition defined as the area which was devoid ofor had minimal cell growth was measured to the nearestmillimeter The antimicrobial activity of the extract wasdetermined by the zone of inhibition of the extracts a higherinhibition zone indicated a more potent antimicrobial effectof the extract

217 Statistical Analysis All the antioxidant assays were car-ried out in triplicate and the results recorded were expressedas mean plusmn standard deviation All charts including standardcurves dose response curves and bar charts were generatedusing Microsoft Excel software (Version 2010) and GraphPadPrism version 601 fromGraphPad Software (SanDiego CAUSA) Correlation between phytoconstituent and antioxidantactivity was carried out using the Pearson correlation onSPSS (version 170) ANOVA (single factor) was performedin Microsoft Excel software (Version 2010) to test for signif-icant difference in mean values of the different extracts for

a

b

cd

e

b

a

cd

e0

1000

2000

3000

4000

5000

6000

7000

Flower Peel Leaf Stem Seed

120583m

olg

AD

W

120583mol Fe2+g ADW120583mol Troloxg ADW

Figure 2 TEAC and FRAP values of different pomegranate extractsADW air dry weight Different superscripts between columnsrepresent significant difference between extracts Data expressed asmean plusmn standard deviation 120583mol Trolox equivalentg air dry weightfor TEAC (119899 = 3) LSD = 92044 at 5 significance Data expressedas mean plusmn standard deviation 120583mol Fe2+g ADW for FRAP (119899 = 3)LSD = 10134 at 5 significance

each assay To test for null hypothesis the least significantdifference between extracts for each independent assay wascalculated

3 Results

31 Polyphenolic Content Total phenolics of the extractsranged between 065 plusmn 0004mg GAEg ADW and 33651 plusmn070mg GAEg ADW with the highest content measured inthe flower (Table 1) The amount of total phenolics differedsignificantly between the extracts (119875 lt 005) Total flavonoidswere between 0332 plusmn 0003mg QEg ADW and 21354 plusmn314mg QEg ADW Pomegranate flower extract had themost prominent flavonoid level followed by peel leaf andstem with only negligible amount measured in the seedextract Significant differences were observed in flavonoidcontent among the extracts (119875 lt 005) Relatively loweramount of total proanthocyanidins was present in the sam-ples compared to the amount of total phenolics and flavonoidcontents (Table 1)

32 Antioxidant Activities TEAC value ranged from 1404 plusmn240 120583mol Troloxg ADW to 520601 plusmn 57848 120583mol TroloxgADW with the peel exhibiting the highest TEAC value Theferric reducing potential ranged between 629 plusmn 038 120583molFe2+g ADW and 593300 plusmn 5406 120583mol Fe2+g ADW (119875 lt005) (Figure 2) The flowers had the highest ferric reducingpotential which was statistically different from the activity ofthe other extracts (119875 lt 005) (Figure 2)

All extracts showed dose-dependent iron (II) chelatingactivity However pomegranate flower exhibited the highestiron (II) cation chelating activity with the lowest calculatedIC50

value (Table 2) Statistically significant differences wereobserved between the IC

50values (119875 lt 005) with pome-

granate flower peel and stem being more potent than theleaf and the seed Similarly most of the extracts except


Table 2 Antioxidant activities of different pomegranate parts extract

Extract Fe(II) chelatingactivity

HOCl scavengingactivity

Inhibition of deoxyribosedegradation

Inhibition of lipidperoxidation

Nitric oxideinhibition

Superoxidescavenging

Flower 0113 plusmn 0006c0012 plusmn 0001

b0220 plusmn 0041

c0047 plusmn 0006

b0396 plusmn 0002

e0175 plusmn 0001

b

Peel 0157 plusmn 0006c0004 plusmn 0001

b0111 plusmn 0001

c0333 plusmn 0058

b0668 plusmn 0001

d0089 plusmn 0001

c

Leaf 0713 plusmn 0006b0017 plusmn 0002

b3752 plusmn 0091

b0601 plusmn 0100

b18155 plusmn 0005

b0072 plusmn 0001

d

Stem 0397 plusmn 0030c NA 0480 plusmn 0031

c1700 plusmn 0173

b4831 plusmn 0001

c0040 plusmn 0001

e

Seed 15400 plusmn 0310a5200 plusmn 0400

a14300 plusmn 0760

a35000 plusmn 2646

a48641 plusmn 0001

a2523 plusmn 0001

a

LSD value at 5significance 0300 0371 0630 2162 3231 1490

NA not available Data expressed as mean IC50 plusmn standard deviation (mgmL) (119899 = 3) different superscripts between rows in individual columns representsignificant difference between extracts

the stem extract showed dose-dependent hypochlorous acidscavenging activity with the peel extract being the mostpotent HOCl scavenger The calculated IC

50value ranged

between 0004 plusmn 0001mg ADWmL and 5200 plusmn 0400mgADWmL (Table 2)The calculated IC

50value of flower peel

and leaf extracts differed significantly from that of the seed(119875 lt 005) However the flower peel and leaf were observedto scavenge hypochlorous acid more efficiently than ascorbicacid used as a positive control (IC

50= 563 plusmn 021mgmL)

The samples analysed were also strong hydroxyl rad-ical scavengers The results were regarded as indicationsof hydroxyl radical scavenging propensity by virtue oftheir ability to inhibit deoxyribose degradation (Figure 3)Pomegranate peel afforded the highest protection followedby flower stem leaf and seed extracts (Table 2) Statisticallysignificant differences were observed in IC

50values among

the extracts (119875 lt 005)The degree of microsomal lipid peroxidation inhibition

induced by Fe3+ascorbate was evaluated by measuring theformation of MDA-(TBA)

2adduct spectrophotometrically

All the extracts protected microsome against lipid perox-idation in a dose-dependent manner Pomegranate floweroffered the most prominent protection followed by peel leafand stem extracts No significant difference was observedamong flower peel leaf and stem extracts as compared to theseed extract (119875 lt 005) Gallic acid used as positive control(IC50

value of 0014 plusmn 0002mgmL) was more potent thanthe flower extract

A similar trend was observed for nitric oxide radicalinhibitionThe flower and peel extracts were the most potentscavenger of NO∙ with the lowest calculated IC

50value

(Table 2) and were more effective than ascorbic acid used aspositive control (IC

501253141 plusmn 0002mgmL) (119875 lt 005)

All the extracts exhibited a dose-dependent effect againstsuperoxide radical However a different trend of activityfor the extracts under study was observed the stem extractbeing the most potent followed by leaf peel flower and seedextract The stem extract was a very powerful scavenger ofsuperoxide (Table 2) 100 folds more powerful than ascorbicacid (IC

5014191 plusmn 0001mgmL)

33 Anti-Inflammatory Effect of P granatum Extracts Thedegree of inhibition of xanthine oxidase by the extracts

0

10

20

30

40

50

60

70

80

0 02 04 06 08 1 12 14

Mea

n

inhi

bitio

n of

deo

xyrib

ose d

egra

datio

n

(mg ADWmL)extractflowerConcentration P granatumof

(a)

0102030405060708090

100

0 05 1 15 2 25 3 35 4

Mea

n

inhi

bitio

n of

deo

xyrib

ose d

egra

datio

n

0peel extract (mg ADWmL)Concentration P granatumof

(b)

Figure 3 Dose-dependent hydroxyl radical induced deoxyribosedegradation inhibition by pomegranate flower and peel extractsADW air dry weight data are representative of mean plusmn standarddeviation of three replicates IC

50values were extrapolated from the

graphs

was evaluated by measuring the formation of uric acidspectrophotometrically Only the flower extract showedxanthine oxidase inhibitory activity at the concentrationrange tested Pomegranate flower extract showed a dose-dependent inhibition of xanthine oxidase with an IC

50value


01020304050607080

0 005 01 015 02 025 03 035 04Mea

n

xan

thin

e oxi

dase

inhi

bitio

n

flower extract (mg ADWmL)Concentration P granatumof

(a)

0

10

20

30

40

50

60

70

0 50 100 150 200 250 300 350 400

Xant

hine

oxi

dase

inhi

bitio

n (

)

Allopurinol concentration (120583M)

(b)

Figure 4 Dose-dependent xanthine oxidase inhibitory activity ofpomegranate flower extract and allopurinol ADW air dry weightData are representative of mean plusmn standard deviation of threereplicates IC

50value was extrapolated from the graph

of 0058 plusmn 0011mg ADWmL (Figure 4) Allopurinol usedas a positive control was however a more potent inhibitor(IC50value 00055 plusmn 00002mgmL)

34 Antibacterial Activity The antimicrobial activity of theextracts from P granatum on some indigenous oralmicrobio-tas known to rapidly colonize smooth surfaces and crevicesof the teeth and gums causing dental plaque and tooth carieswas evaluated Using the disc diffusion method it was notedthat bacterial growth was minimal in the presence of all con-centrated extracts Peel extract showed greater antibacterialactivity and produced the highest inhibition zones against Smutans S mitis and L acidophilus (1975mm 25mm and1475mm resp) (Table 3)This inhibitory effect was observedto be significantly greater than that of the positive controlciprofloxacin (119875 lt 0001) Leaf extract produced the secondhighest inhibition zones of 16 1825 and 875mm againstS mutans S mitis and L acidophilus respectively followedclosely by the stem extract The antibacterial activity effect ofthe flower extract was much less pronounced compared to itsplant counterparts Although bacterial cell proliferation wasminimal it was still significant compared to the activity ofciprofloxacin (119875 lt 0001)

Table 3 Zone of inhibition or minimum growth (mm) by variousparts of Punica granatum on S mutans S mitis and L acidophilus

Mean zone of inhibition plusmn SD (mm)Streptococcus

mutansStreptococcus

mitisLactobacillusacidophilus

Extract onlyPeel 1975 plusmn 050

lowastlowastlowast2500 plusmn 000


lowast

Flower 1225 plusmn 050lowast1475 plusmn 050


Leaf 1600 plusmn 000lowastlowastlowast1825 plusmn 050


Stem 1475 plusmn 050lowastlowastlowast1900 plusmn 115


Extract +ciprofloxacin

Peel 1075 plusmn 096 2241 plusmn 1411150 plusmn 100

Flower 1125 plusmn 096 1300 plusmn 000813 plusmn 103

Leaf 1250 plusmn 0581882 plusmn 082

825 plusmn 050

Stem 100 plusmn 00 1208 plusmn 058925 plusmn 126

Ciprofloxacin 1025 plusmn 050 875 plusmn 126 875 plusmn 050

Values are expressed as mean plusmn standard deviation Significance comparedto ciprofloxacin (positive control) 119875 lt 005lowast 119875 lt 001 119875 lt 0001lowastlowastlowast

0

5

10

15

20

25

30

Peel Flower Leaf Stem CiprofloxacinZone

of m

inim

alin

hibi

ted

grow

th (m

m)

Extract only

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

Extract + ciprofloxacin

Figure 5 Zone of inhibition or minimum growth (mm) byvarious parts of Punica granatum on Streptococcus mitis Values areexpressed as mean where error bars represent standard deviationSignificance compared to ciprofloxacin (positive control)119875 lt 005119875 lt 001

119875 lt 0001lowastlowastlowast

In addition to the study of sole plant extracts bacterialstrains were exposed to a combinational treatment (extract +ciprofloxacin) Such treatment produced inhibition zonesthat ranged between 1225 and 1975mm against S mutans1475 and 250mm against S mitis and 813 and 1150mmagainst L acidophilus The following trends were observedleaf gt flower gt peel gt stem peel gt leaf gt flower gt stem(Figure 5) and peel lt stem lt leaf lt flower respectively Forthe majority of the plant extracts tested the combinationaltreatment proved to exhibit a more efficient antibacterialeffect that significantly exceeded that of ciprofloxacin but notthat of individual plant extracts


4 Discussion and Conclusion

In the recent years P granatum L has received considerableattention for its pluripharmacological effects and its potentcontribution in the maintenance of human health The effi-cacy of pomegranate juice has been validated in clinical trialswherein its ability to decrease inflammatory biomarkers oxi-dation of lipids and proteins [9] and to prolong the doublingtime of prostate specific antigen in patients with prostatecancer [10] was reported Phytoconstituents encompassingseveral phenolic classes [26ndash28] fatty acids [29] sugarsand organic acids [27 30 31] have been characterized inpomegranate fruits and have been ascribed for the diversepharmacological effects The edible parts of pomegranatehave long been used as food while the nonedible parts likethe roots rinds and leaves have a number of applicationsin ethnomedicine [4 6] Thus it can be envisaged thatthe nonedible parts of pomegranate represent a beneficialsource of functional ingredients a statement warranting in-depth investigations The present study therefore aimed atdetermining the phytophenolic content and bioefficacy of thepomegranate plant that has been naturalized in the island ofMauritius since 1639 [7] Different plant parts namely theflower leaf stem peel and seeds were investigated to assessthe in vitro prophylactic potential

A significant variation in total phenolics concentrationwas found among the different parts of pomegranate studiedThe flower extract contained the highest phenolic level fol-lowed by the peel leaf and stem while the seed was relativelypoor (Table 1) However Zhang et al [32] reported that theethanolic extracts of pomegranate peel extract from Chinacontained higher levels of total phenolics (50898 plusmn 2419mggallic acid equivalentg DW) compared to the flower recep-tacles (45496 plusmn 1834mg gallic acid equivalentg DW) andleaf extracts (28976plusmn1482mg gallic acid equivalentg DW)Tehranifar et al [33] also reported higher total phenolicsin the methanolic extracts of peel (4235 plusmn 318mg gallicacid equivalentg DW) followed by seed (3847 plusmn 242mggallic acid equivalentg DW) while the lowest amounts weremeasured in the leaf extract (1333 plusmn 87mg gallic acidequivalentg DW)

Using the method of Zhishen et al [19] the follow-ing trend was established for the total flavonoids in thepomegranate extracts flower gt peel gt leaf gt stem gt seedextracts The HClButan-1-ol assay on the other hand indi-cated low levels of proanthocyanidins in the following orderpeel gt flower gt stem gt leaf gt seed extracts

The TPCmeasured in this study varied considerably withregard to data from the literature [32 33] Factors generallycontributing to these variations can include treatment modeof samples prior to extraction in this study plant parts wereair dried extraction methods and solvents [34] and cultivarsused [26] In addition phenolic and flavonoid contents havebeen reported to vary due to seasonal changes and thedegree of maturation of the plant parts For instance thebiosynthesis of flavonols has been documented to be lightdependent and can also be affected by temperature variation[35 36] Plants growing inMauritius are tolerant of high levelof environment stress induced by varying level of sunlight

ultraviolet radiation and temperature change throughoutthe year This may explain the interesting levels of phenoliccompounds in the parts studied

A multimethod approach was used to determine theantioxidant effect of the extracts since no one method canpredict the total antioxidant efficiency of an extract [17 37]Thus several independent methods differing in biologicalaction mechanisms were used to provide a thorough mech-anistic insight of the antioxidant actions of the extracts understudy Nevertheless a very strong correlation was observedbetween results of each antioxidant test For instance HOClscavenging activity was highly correlated with deoxyriboseassay results (119903 = 0968 119875 lt 0050) A similar relationshipwas observed between deoxyribose assay and lipid peroxi-dation assay while the superoxide anion radical scavengingactivity was significantly and highly correlated with the ironchelating activity (119903 = 0997 119875 lt 0001) the antioxidantactivity fromHOCl assay (119903 = 0999119875 lt 0001) deoxyriboseassay (119903 = 0964 119875 lt 001) lipid peroxidation assay (119903 =0997 119875 lt 0001) and nitric oxide assay (119903 = 0927 119875 lt0050) This is further supported by the very high correlationbetween the calculated IC

50values from iron chelation and

the inhibition of microsomal lipid peroxidation assay (119903 =09991 119875 lt 00001)

The TEAC value provided a ranking order of the antiox-idant capacity of the extracts mainly peel gt flower gt leaf gtstem gt seed extractsThe TEAC value measured in this studywas higher than that reported in the literature for instancethe TEAC value for peel extract was higher than that reportedby Shan et al [38] A very strong positive correlation betweenTEAC and proanthocyanidin content (119903 = 0921 119875 lt 005)and TFC was observed (119903 = 0936 119875 lt 005)

The extracts under study were also potent scavengersof a number of biologically relevant radicals The HOClscavenging assay indicated the peel extract as themost potentscavenger of hypochlorous acid The antioxidant capacityhierarchy based on the HOCl assay of the extracts was in thefollowing order of activity peelgt flowergt leafgt seed extractsthe seed extract showing similar efficacy to ascorbic acid (IC

50

value 563 plusmn 021mgmL) Similarly the peel extract was thestrongest inhibitor of deoxyribose sugar degradation againsthydroxyl radicals generated via the Fenton reaction (Table 2)Only a moderate correlation between polyphenolic contentand IC

50values was found

The superoxide anion radical scavenging assay showeda different trend in activities compared to other antioxidantsystems Interestingly the stem extract was found to be morepotent than the leaf flower and seed extracts This findingis in line with data reported by Kaneria et al [34] wherebypomegranate stem extract exhibited higher antioxidant activ-ity than leaf extract in both DPPH antiradical assay and thesuperoxide anion radical assay On the other hand the flowerextract also exhibited interesting antioxidant potential Thelatter was the most potent inhibitor of nitric oxide followedby peel stem leaf and seed extracts

All the pomegranate extracts significantly inhibitedFe3+ascorbate-induced microsomal lipid peroxidation witha calculated IC

50of less than 17mg ADWmL except for


the seed extract The ability of the pomegranate peel extractto inhibit lipid peroxidation of beef liver microsome wasconsistent with the findings of Althunibat et al [39] whoreported decreased lipid peroxidation in liver and kidneyhomogenate of STZ-induced diabetic rat models The lipidprotective ability of the extracts may be partly attributed toits flavonoid content (119903 = 0630 119875 lt 005) For instance theO-dihydroxyl groups in the flavonol ring structure have beenreported to be a potent inhibitor of lipid peroxidation in cells[40]

Complex formation with reduced form of transitionmetals particularly those that can enhance metal-inducedfree radical generation has been proposed as an alternativeantioxidant mechanism of action ascribed to plant phytophe-nolic Flavonoids can act by chelating metal ions therebyinhibiting free radical production [41] In this line theiron (II) ions chelating ability of the different extracts wasinvestigated The hierarchy of metal iron chelation of theplant parts was flower gt peel gt stem gt leaf gt seed andparalleled the nitric oxide inhibition (Table 2) (119903 = 0947119875 lt 005) The similarity in activity trend in both assays maybe attributed to the involvement of the catechol moiety ofthe flavonoids as part of the mechanism employed in bothassays The structural requirements and the mechanism ofnitric oxide production inhibition by flavonoids have beenreported [42] The metal chelating activity correlated withTFC (119903 = 0626)whichmay be partly assigned to the chemicalstructure of flavonoids The catechol moiety in the ring Bthe 3-hydroxyl and 4- oxo groups in the heterocyclic ring Cand the 4- oxo and 5-hydroxyl groups between the C and Arings has been identified as binding sites for metal ions inthe flavonoid molecules [41 43] In addition the vital roleof Fe2+ in inducing and propagating lipid peroxidation hasbeen well documented in the literature [44 45] and thusiron chelation can be proposed as amechanism for the potentinhibition ofmicrosomal lipid peroxidation by the flower andpeel extracts

Similarly the FRAP assay based on the redox reactioninvolving electron transfer showed the following hierarchyof activity flower gt peel gt leaf gt stem gt seed The FRAPassay does not detect antioxidant compounds that act byhydrogen atom transfer The FRAP values of this studywere consistent with data from Ardekani et al [26] whoreported FRAP value of peel extract to vary between 3401 and4788120583mol Fe2+g DW among different cultivars Statisticallysignificant positive correlation was obtained between theFRAP and TPC (119903 = 0996 119875 lt 001) as well as with TFC(119903 = 0983 119875 lt 001) Numerous reports showed similartypes of linear relationship between antioxidant activities andphytophenolic contents of fruits [37 46]

Excess of uric acid in joints has been associated withinflammation [47] leading to pathological conditions Xan-thine oxidase an important enzyme involved in the con-version of hypoxanthine to xanthine and to uric acid hasbeen reported as an interesting target against inflamma-tion In this vein xanthine oxidase inhibitory activitiesof the pomegranate plant parts were thus assessed Thepomegranate flower extract exhibited a dose-dependent

enzyme inhibition propensity However the inhibitorypotential of the flower extract as measured by the calculatedIC50

(0058 plusmn 0011mg ADWmL) was weaker than allop-urinol (IC

50 00055 plusmn 00002mgmL) used as control Other

parts extracts showed no inhibitory activity at the testedconcentration

The extracts tested behaved differently in the variousexperimental systems showing varying hierarchy of activi-ties which were independent of the phenolic content mea-sured The general trend in bioefficacy demonstrated thatpomegranate flower and peel extracts were very potentfollowed by leafgt stemgt seed extractsThe findings indicatedthat polyphenolic compounds may act synergistically topotentiate the antioxidant activity of extracts It should alsobe noted that the pomegranate flower and peel extracts were100 times more potent than the red and yellow Psidiumcattleianum Sabine ldquoChinese guavardquo Mauritian exotic fruitsevaluated using the TEAC and FRAP assays [48] In additionpomegranate flower and peel extracts were found to bemuch more potent than the citrus extracts assayed usingsimilar methodology For instance the total phenolic contentof pomegranate flower extract was 200 folds higher thanFortunella margarita pulp extract (1694 plusmn 19 120583gg FW)[50] and 44 folds higher than C reticulata X C sinensisflavendo extract (7667 plusmn 93 120583gg FW) [38] Similarly thetotal flavonoid content were 200 folds higher than Citrusmaxima pulp extract (965 plusmn 7 120583gg FW) [50] and 35 foldshigher than C reticulata X C paradisi flavendo extract(5615 plusmn 93 120583gg FW) [38] Likewise the antioxidant propen-sities of pomegranate flower and peel extracts were signifi-cantly more important than the Mauritian citrus fruits pulpof flavendo extract thereby highlighting the prophylacticpotential of the extracts under study

Furthermore the growing prevalence of dental cariesgingivitis periodontitis and oral microbial infections casesamongst adults prompted the evaluation of the antibac-terial effects of the extracts against oral bacterial growthPathophysiological mechanisms including deficient nutri-tional intake alterations in host response to oral microfloracompromised neutrophil function and decreased phago-cytosis and leukotaxis have been increasingly suggested toaccount for these disorders A realistic management planincluding regular oral hygiene practice and basic dentaltreatment can be envisaged for managing dental caries andits associated oral complications Nowadays active con-stituents extracted from plants have been included in thepreparation of toothpaste mouth rinses dental floss andchewing gum to ensure a stronger antimicrobial activity [8]Ongoing studies focusing on the anticariogenic properties ofpolyphenols isolated from green tea [50] cranberry juice [51]and shiitake mushrooms [52] seemed promising Howeverdespite the numerous studies conducted on such functionalfoods only a handful of them can be clinically used tocontrol dental plaque caries formation andmouth infectionsdue to their effectiveness stability taste and economicfeasibility [53] George and Sumathy [54] reported effectiveantibacterial activity of aqueous and ethanolic extracts ofpomegranate against Streptococcus mutans Staphylococcussp Escherichia coli Lactobacillus sps and Candida albicans


isolated from the mouth In this study pomegranate wasreported to negatively influence the proliferation of GramPositive bacteria and concurrently to demonstrate potentiron-chelating capabilities Recently Kulkarni et al [55]found that punicalagin an ellagitannin isolated from thepomegranate peel could completely suppress iron catalyzingoxidant reactions in vitro It can therefore be speculated thatpotent pomegranate extracts under study in particular thepeel extract may be liable to remove iron from the brothmedium and deprive bacteria of the iron they need fornormal growth through chelation in view of the high iron(II) ions chelating efficiency The presence of both tanninsand alkaloids isolated from pomegranate pericarp and seedshas been extensively reviewed for their outstanding ability toblock bacterial surface adhesions and inhibit glycosyltrans-ferases thus deterring bacterial attachment to dental surfaceshence its colonization [56] Data from this study providebasic supplementary evidence of the antimicrobial activity ofpomegranate and support the imperative need to find neweffective bioagents that can avoid a negative impact upon thefuture oral health of communities affected by dental cariesand expenditure on dental services

While pomegranatersquo edible parts have common applica-tions in the food and food processing industries due to theirexcellent nutritional and health values [57] this study showedthe prospect of the nonedible parts Mauritian cultivar ofpomegranate An in-depth comparison of the broad classesof phytophenolic and the bioefficacies of the latter indicatedthe effectiveness of the flower and peel extracts The availableevidence indicates that these extracts might be of therapeuticbenefit in bacterial infections and be an ideal candidate forfunctional food health products Further investigations needto be directed towards determining the potential toxicity thephytochemistry of the nonedible parts and applicability ofthe extracts in various food matrices The use of functionalfoods enriched with pomegranate flower and peel extractshowever needs technologies for incorporating these health-promoting ingredients into food without reducing theirbioavailability or functionality

Conflict of Interests

The authors declare that there is no conflict of interests

References

[1] C Guo J Wei J Yang J Xu W Pang and Y Jiang ldquoPome-granate juice is potentially better than apple juice in improvingantioxidant function in elderly subjectsrdquo Nutrition Researchvol 28 no 2 pp 72ndash77 2008

[2] E Gonzalez-Molina D A Moreno and C Garcıa-Viguera ldquoAnew drink rich in healthy bioactives combining lemon andpomegranate juicesrdquo Food Chemistry vol 115 no 4 pp 1364ndash1372 2009

[3] J Jurenka ldquoTherapeutic applications of pomegranate (Punicagranatum L) a reviewrdquoAlternativeMedicine Review vol 13 no2 pp 128ndash144 2008

[4] M C Mathabe R V Nikolova N Lall and N Z NyazemaldquoAntibacterial activities of medicinal plants used for

the treatment of diarrhoea in Limpopo Province SouthAfricardquo Journal of Ethnopharmacology vol 105 no 1-2 pp286ndash293 2006

[5] M-J Song and H Kim ldquoEthnomedicinal application of plantsin the western plain region of North Jeolla Province in KoreardquoJournal of Ethnopharmacology vol 137 no 1 pp 167ndash175 2011

[6] P Tetali C Waghchaure P G Daswani N H Antia andT J Birdi ldquoEthnobotanical survey of antidiarrhoeal plants ofParinche valley Pune district Maharashtra Indiardquo Journal ofEthnopharmacology vol 123 no 2 pp 229ndash236 2009

[7] G Rouillard and J Gueho Les PLantes Et Leur Histoire A LrsquoILeMaurice 1999

[8] I E Alsaimary ldquoEfficacy of some antibacterial agents againstStreptococcus mutans associated with tooth decayrdquo InternetJournal of Microbiology vol 7 no 2 2010

[9] L Shema-Didi S Sela L Ore et al ldquoOne year of pomegranatejuice intake decreases oxidative stress inflammation and inci-dence of infections in hemodialysis patients a randomizedplacebo-controlled trialrdquo Free Radical Biology amp Medicine vol53 pp 297ndash304 2012

[10] A J Pantuck J T Leppert N Zomorodian et al ldquoPhase IIstudy of pomegranate juice formenwith rising prostate-specificantigen following surgery or radiation for prostate cancerrdquoClinical Cancer Research vol 12 no 13 pp 4018ndash4026 2006

[11] L S Adams Y Zhang N P Seeram D Heber and SChen ldquoPomegranate ellagitannin-derived compounds exhibitantiproferative and antiaromatase activity in breast cancer cellsin vitrordquo Cancer Prevention Research vol 3 no 1 pp 108ndash1132010

[12] E H Endo D A Garcia Cortez T Ueda-Nakamura C VNakamura and B P Dias Filho ldquoPotent antifungal activityof extracts and pure compound isolated from pomegranatepeels and synergismwith fluconazole againstCandida albicansrdquoResearch in Microbiology vol 161 no 7 pp 534ndash540 2010

[13] M Haidari M Ali S Ward Casscells III and M MadjidldquoPomegranate (Punica granatum) purified polyphenol extractinhibits influenza virus and has a synergistic effect withoseltamivirrdquo Phytomedicine vol 16 no 12 pp 1127ndash1136 2009

[14] L Zhang Q Fu and Y Zhang ldquoComposition of anthocyaninsin pomegranate flowers and their antioxidant activityrdquo FoodChemistry vol 127 no 4 pp 1444ndash1449 2011

[15] M I Gil F A Tomas-Barberan B Hess-Pierce D M Holcroftand A A Kader ldquoAntioxidant activity of pomegranate juiceand its relationship with phenolic composition and processingrdquoJournal of Agricultural and Food Chemistry vol 48 no 10 pp4581ndash4589 2000

[16] T Tanaka G-I Nonaka and I Nishioka ldquoTannins andrelated compounds XL Revision of the structures of punicalinand punicalagin and isolation and characterization of 2-O-galloylpunicalin from the bark ofPunica granatumLrdquoChemicaland Pharmaceutical Bulletin vol 34 no 2 pp 650ndash655 1986

[17] V S Neergheen M A Soobrattee T Bahorun and O IAruoma ldquoCharacterization of the phenolic constituents inMauritian endemic plants as determinants of their antioxidantactivities in vitrordquo Journal of Plant Physiology vol 163 no 8 pp787ndash799 2006

[18] L J Porter L N Hrstich and B G Chan ldquoThe conversionof procyanidins and prodelphinidins to cyanidin and delphini-dinrdquo Phytochemistry vol 25 no 1 pp 223ndash230 1985

[19] J Zhishen TMengcheng andW Jianming ldquoThedeterminationof flavonoid contents in mulberry and their scavenging effects


on superoxide radicalsrdquo Food Chemistry vol 64 no 4 pp 555ndash559 1999

[20] I F F Benzie and J J Strain ldquoThe ferric reducing ability ofplasma (FRAP) as a measure of ldquoantioxidant powerrdquo the FRAPassayrdquo Analytical Biochemistry vol 239 no 1 pp 70ndash76 1996

[21] AM Campos and E A Lissi ldquoKinetics of the reaction between221015840-azinobis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS)derived radical cations and phenolsrdquo International Journal ofChemical Kinetics vol 29 no 3 pp 219ndash224 1997

[22] O I Aruoma ldquoDeoxyribose assay for detecting hydroxylradicalsrdquoMethods in Enzymology vol 233 pp 57ndash66 1994

[23] M Kumar M Chandel S Kumar and S Kaur ldquoStudies on theantioxidant genoprotective activity of extracts of Koelreuteriapaniculata laxmrdquo American Journal of Biomedical Sciences vol1 pp 177ndash189 2011

[24] C Sunil P Agastian C Kumarappan and S IgnacimuthuldquoIn vitro antioxidant antidiabetic and antilipidemic activitiesof Symplocos cochinchinensis (Lour) S Moore barkrdquo Food andChemical Toxicology vol 50 no 5 pp 1547ndash1553 2012

[25] J Havlik R G de la Huebra K Hejtmankova et al ldquoXanthineoxidase inhibitory properties of Czech medicinal plantsrdquo Jour-nal of Ethnopharmacology vol 132 no 2 pp 461ndash465 2010

[26] M R S Ardekani M Hajimahmoodi M R Oveisi et alldquoComparative antioxidant activity and total flavonoid contentof Persian pomegranate (Punica granatum l) cultivarsrdquo IranianJournal of Pharmaceutical Research vol 10 no 3 pp 519ndash5242011

[27] P Mena C Garcıa-Viguera J Navarro-Rico et al ldquoPhytochem-ical characterisation for industrial use of pomegranate (Punicagranatum L) cultivars grown in Spainrdquo Journal of the Science ofFood and Agriculture vol 91 no 10 pp 1893ndash1906 2011

[28] A Tehranifar M Zarei Z Nemati B Esfandiyari and MR Vazifeshenas ldquoInvestigation of physico-chemical propertiesand antioxidant activity of twenty Iranian pomegranate (PunicagranatumL) cultivarsrdquo ScientiaHorticulturae vol 126 no 2 pp180ndash185 2010

[29] A Parashar N Sinha and P Singh ldquoLipid contents and fattyacids composition of seed oil from twenty five pomegranatesvarieties grown in Indiardquo Advance Journal of Food Science andTechnology vol 2 no 1 pp 12ndash15 2010

[30] P Melgarejo D M Salazar and F Artes ldquoOrganic acids andsugars composition of harvested pomegranate fruitsrdquo EuropeanFood Research and Technology vol 211 no 3 pp 185ndash190 2000

[31] N Hasnaoui R Jbir M Mars et al ldquoOrganic acids sugarsand anthocyanins contents in juices of Tunisian pomegranatefruitsrdquo International Journal of Food Properties vol 14 no 4pp 741ndash757 2011

[32] L Zhang X Yang Y Zhang L Wang and R Zhang ldquoInvitro antioxidant properties of different parts of pomegranateflowersrdquo Food and Bioproducts Processing vol 89 no 3 pp 234ndash240 2011

[33] A Tehranifar Y Selahvarzi M Kharrazi and V J BakhshldquoHigh potential of agro-industrial by-products of pomegranate(Punica granatum L) as the powerful antifungal and antioxi-dant substancesrdquo Industrial Crops and Products vol 34 no 3pp 1523ndash1527 2011

[34] M J Kaneria M B Bapodara and S V Chanda ldquoEffectof extraction techniques and solvents on antioxidant activityof pomegranate (Punica granatum L) leaf and stemrdquo FoodAnalytical Methods vol 5 no 3 pp 396ndash404 2012

[35] E Braidot M Zancani E Petrussa et al ldquoTransport and accu-mulation of flavonoids in grapevine (Vitis vinifera L)rdquo PlantSignaling and Behavior vol 3 no 9 pp 626ndash632 2008

[36] D Treutter ldquoManaging phenol contents in crop plants byphytochemical farming and breeding-visions and constraintsrdquoInternational Journal of Molecular Sciences vol 11 no 3 pp807ndash857 2010

[37] D Ramful T Bahorun E Bourdon E Tarnus and O IAruoma ldquoBioactive phenolics and antioxidant propensity offlavedo extracts of Mauritian citrus fruits potential prophylac-tic ingredients for functional foods applicationrdquo Toxicology vol278 no 1 pp 75ndash87 2010

[38] B Shan Y-Z Cai J D Brooks and H Corke ldquoThe invitro antibacterial activity of dietary spice and medicinal herbextractsrdquo International Journal of FoodMicrobiology vol 117 no1 pp 112ndash119 2007

[39] O Y Althunibat A H Al-Mustafa K Tarawneh K MKhleifat B H Ridzwan and H N Qaralleh ldquoProtective roleof Punica granatum L peel extract against oxidative damage inexperimental diabetic ratsrdquo Process Biochemistry vol 45 no 4pp 581ndash585 2010

[40] I-W Peng and S-M Kuo ldquoFlavonoid structure affects theinhibition of lipid peroxidation in Caco-2 intestinal cells atphysiological concentrationsrdquo Journal of Nutrition vol 133 no7 pp 2184ndash2187 2003

[41] D Malesev and V Kuntic ldquoInvestigation of metal-flavonoidchelates and the determination of flavonoids via metal-flavonoid complexing reactionsrdquo Journal of the Serbian Chemi-cal Society vol 72 no 10 pp 921ndash939 2007

[42] H Matsuda T Morikawa S Ando I Toguchida and MYoshikawa ldquoStructural requirements of flavonoids for nitricoxide production inhibitory activity and mechanism of actionrdquoBioorganic and Medicinal Chemistry vol 11 no 9 pp 1995ndash2000 2003

[43] D Prochazkova I Bousova and N Wilhelmova ldquoAntioxidantand prooxidant properties of flavonoidsrdquo Fitoterapia vol 82 pp513ndash523 2011

[44] L Tang Y Zhang Z Qian and X Shen ldquoThe mechanism ofFe2+-initiated lipid peroxidation in liposomes the dual functionof ferrous ions the roles of the pre-existing lipid peroxides andthe lipid peroxyl radicalrdquo Biochemical Journal vol 352 no 1 pp27ndash36 2000

[45] M Valko C J Rhodes J Moncol M Izakovic and M MazurldquoFree radicals metals and antioxidants in oxidative stress-induced cancerrdquo Chemico-Biological Interactions vol 160 no 1pp 1ndash40 2006

[46] R L Prior G Cao A Martin et al ldquoAntioxidant capacity asinfluenced by total phenolic and anthocyanin content maturityand variety of Vaccinium speciesrdquo Journal of Agricultural andFood Chemistry vol 46 no 7 pp 2686ndash2693 1998

[47] A Chandrasekara and F Shahidi ldquoAntiproliferative potentialand DNA scission inhibitory activity of phenolics from wholemillet grainsrdquo Journal of Functional Foods vol 3 no 3 pp 159ndash170 2011

[48] A Luximon-Ramma T Bahorun and A Crozier ldquoAntioxidantactions and phenolic and vitamin C contents of commonMauritian exotic fruitsrdquo Journal of the Science of Food andAgriculture vol 83 no 5 pp 496ndash502 2003

[49] D Ramful E Tarnus O I Aruoma E Bourdon and T Baho-run ldquoPolyphenol composition vitamin C content and anti-oxidant capacity of Mauritian citrus fruit pulpsrdquo Food ResearchInternational vol 44 no 7 pp 2088ndash2099 2011


[50] S Otake M Makimura T Kuroki Y Nishihara and MHirasawa ldquoAnticaries effects of polyphenolic compounds fromJapanese green teardquo Caries Research vol 25 no 6 pp 438ndash4431991

[51] J Babu C Blair S Jacob and O Itzhak ldquoInhibition of Strepto-coccus gordoniimetabolic activity in biofilm by cranberry juicehigh-molecular-weight componentrdquo Journal of Biomedicine andBiotechnology vol 2012 Article ID 590384 7 pages 2012

[52] C Signoretto G Burlacchini A Marchi et al ldquoTesting a lowmolecular mass fraction of a mushroom (Lentinus edodes)extract formulated as an oral rinse in a cohort of volunteersrdquoJournal of Biomedicine and Biotechnology vol 2011 Article ID857987 2011

[53] R A Bagramian F Garcia-Godoy and A R Volpe ldquoTheglobal increase in dental caries A pending public health crisisrdquoAmerican Journal of Dentistry vol 22 no 1 pp 3ndash8 2009

[54] S George and V J H Sumathy ldquoAnti-bacterial and anti-fungalactivity of botanical extracts against microorganisms isolatedfrom mouth florardquo Biosciences International vol 1 pp 74ndash772012

[55] A P Kulkarni H S Mahal S Kapoor and S M Aradhya ldquoInvitro studies on the binding antioxidant and cytotoxic actionof punicalaginrdquo Journal of Agricultural and Food Chemistry vol55 no 4 pp 1491ndash1500 2007

[56] G K Jayaprakasha P S Negi and B S Jena ldquoAntimicrobialactivities of pomegranaterdquo in From Ancient Foods to ModernMedicine Chapter 11 pp 168ndash183 Taylor and Francis 2006

[57] L U Opara M R Al-Ani and Y S Al-Shuaibi ldquoPhysico-chemical properties vitamin C content and antimicrobial pro-perties of pomegranate fruit (Punica granatum L)rdquo Food andBioprocess Technology vol 2 no 3 pp 315ndash321 2009

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


(a) (b)

(c)

(d) (e) (f)

(g) (h) (i)

Figure 1 Different anatomical parts of P granatum tree and fruit (a) Unripe fruit (b) flower (c) stem (d) flower and tubular calyx (e) youngleaves at the branch endings (f) the fruitrsquos rind with membranous extensions forming compartments which contain the juicy arils (g) seeds(h) arils (juicy pulp coating the seed) (i) pomegranate inner membrane

the secondary metabolites more specifically its polyphenoliccompounds present in relatively high concentrations [11ndash13]

The phytophenolic compositions vary differently in theedible and nonedible parts of the plants and have been widelyinvestigated Pomegranate fruit (peel aril seeds and juice)has been reported to be rich in phenolic acids flavanolsflavones flavonones anthocyanidins and anthocyanin [3]Literature data reported glycated anthocyanins (pelargoni-din 35-diglucoside pelargonidin 3-glucoside) apart from

phenolic compounds common to the edible parts like gal-lic acid in the flowers [14] while the nonedible parts ofpomegranate comprising leaves roots and stem containedapigenin punicalin punicalagin and luteolin [3 15 16] Thisrich polyphenolic composition has been intrinsically linkedto the pluripharmacological effects of pomegranate extractsHowever it should be noted that sources of variation in thelevel of phytochemicals and nutrients arising from geneticvariability of a naturalized plant in addition to geographical




2 Methodology






4Fe (SO

4)2sdot12 H

2O in



2was added to












50(mg ADWmL)




3




2PO4-





2adduct The MDA-



50g ADWmL



3 and






50(mg ADWmL)





1198600

] times 100 (1)



sample


50value









a146 plusmn 006

ab


b248 plusmn 008

a


c021 plusmn 001

b


d032 plusmn 001

b


e013 plusmn 000

b




50(120583M)




a

b

cd

e

b

a

cd

e0

1000

2000

3000

4000

5000

6000

7000


120583m

olg

AD

W




3 Results
















b0220 plusmn 0041

c0047 plusmn 0006

b0396 plusmn 0002

e0175 plusmn 0001

b


b0111 plusmn 0001

c0333 plusmn 0058

b0668 plusmn 0001

d0089 plusmn 0001

c


b3752 plusmn 0091

b0601 plusmn 0100

b18155 plusmn 0005

b0072 plusmn 0001

d


c1700 plusmn 0173

b4831 plusmn 0001

c0040 plusmn 0001

e


a14300 plusmn 0760

a35000 plusmn 2646

a48641 plusmn 0001

a2523 plusmn 0001

a




50value ranged






50values among







50value


501253141 plusmn 0002mgmL) (119875 lt 005)




0

10

20

30

40

50

60

70

80

0 02 04 06 08 1 12 14

Mea

n

inhi

bitio

n of

deo

xyrib

ose d

egra

datio

n


(a)

0102030405060708090

100

0 05 1 15 2 25 3 35 4

Mea

n

inhi

bitio

n of

deo

xyrib

ose d

egra

datio

n


(b)



graphs


50value


01020304050607080

0 005 01 015 02 025 03 035 04Mea

n

xan

thin

e oxi

dase

inhi

bitio

n


(a)

0

10

20

30

40

50

60

70

0 50 100 150 200 250 300 350 400

Xant

hine

oxi

dase

inhi

bitio

n (

)


(b)







mutansStreptococcus





lowast











825 plusmn 050




0

5

10

15

20

25

30


of m

inim

alin

hibi

ted

grow

th (m

m)

Extract only

lowastlowastlowast


lowastlowastlowast

















50


50values was found




















References





































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




2 Methodology






4Fe (SO

4)2sdot12 H

2O in



2was added to












50(mg ADWmL)




3




2PO4-





2adduct The MDA-



50g ADWmL



3 and






50(mg ADWmL)





1198600

] times 100 (1)



sample


50value









a146 plusmn 006

ab


b248 plusmn 008

a


c021 plusmn 001

b


d032 plusmn 001

b


e013 plusmn 000

b




50(120583M)




a

b

cd

e

b

a

cd

e0

1000

2000

3000

4000

5000

6000

7000


120583m

olg

AD

W




3 Results
















b0220 plusmn 0041

c0047 plusmn 0006

b0396 plusmn 0002

e0175 plusmn 0001

b


b0111 plusmn 0001

c0333 plusmn 0058

b0668 plusmn 0001

d0089 plusmn 0001

c


b3752 plusmn 0091

b0601 plusmn 0100

b18155 plusmn 0005

b0072 plusmn 0001

d


c1700 plusmn 0173

b4831 plusmn 0001

c0040 plusmn 0001

e


a14300 plusmn 0760

a35000 plusmn 2646

a48641 plusmn 0001

a2523 plusmn 0001

a




50value ranged






50values among







50value


501253141 plusmn 0002mgmL) (119875 lt 005)




0

10

20

30

40

50

60

70

80

0 02 04 06 08 1 12 14

Mea

n

inhi

bitio

n of

deo

xyrib

ose d

egra

datio

n


(a)

0102030405060708090

100

0 05 1 15 2 25 3 35 4

Mea

n

inhi

bitio

n of

deo

xyrib

ose d

egra

datio

n


(b)



graphs


50value


01020304050607080

0 005 01 015 02 025 03 035 04Mea

n

xan

thin

e oxi

dase

inhi

bitio

n


(a)

0

10

20

30

40

50

60

70

0 50 100 150 200 250 300 350 400

Xant

hine

oxi

dase

inhi

bitio

n (

)


(b)







mutansStreptococcus





lowast











825 plusmn 050




0

5

10

15

20

25

30


of m

inim

alin

hibi

ted

grow

th (m

m)

Extract only

lowastlowastlowast


lowastlowastlowast

















50


50values was found




















References





































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



50(mg ADWmL)




3




2PO4-





2adduct The MDA-



50g ADWmL



3 and






50(mg ADWmL)





1198600

] times 100 (1)



sample


50value









a146 plusmn 006

ab


b248 plusmn 008

a


c021 plusmn 001

b


d032 plusmn 001

b


e013 plusmn 000

b




50(120583M)




a

b

cd

e

b

a

cd

e0

1000

2000

3000

4000

5000

6000

7000


120583m

olg

AD

W




3 Results
















b0220 plusmn 0041

c0047 plusmn 0006

b0396 plusmn 0002

e0175 plusmn 0001

b


b0111 plusmn 0001

c0333 plusmn 0058

b0668 plusmn 0001

d0089 plusmn 0001

c


b3752 plusmn 0091

b0601 plusmn 0100

b18155 plusmn 0005

b0072 plusmn 0001

d


c1700 plusmn 0173

b4831 plusmn 0001

c0040 plusmn 0001

e


a14300 plusmn 0760

a35000 plusmn 2646

a48641 plusmn 0001

a2523 plusmn 0001

a




50value ranged






50values among







50value


501253141 plusmn 0002mgmL) (119875 lt 005)




0

10

20

30

40

50

60

70

80

0 02 04 06 08 1 12 14

Mea

n

inhi

bitio

n of

deo

xyrib

ose d

egra

datio

n


(a)

0102030405060708090

100

0 05 1 15 2 25 3 35 4

Mea

n

inhi

bitio

n of

deo

xyrib

ose d

egra

datio

n


(b)



graphs


50value


01020304050607080

0 005 01 015 02 025 03 035 04Mea

n

xan

thin

e oxi

dase

inhi

bitio

n


(a)

0

10

20

30

40

50

60

70

0 50 100 150 200 250 300 350 400

Xant

hine

oxi

dase

inhi

bitio

n (

)


(b)







mutansStreptococcus





lowast











825 plusmn 050




0

5

10

15

20

25

30


of m

inim

alin

hibi

ted

grow

th (m

m)

Extract only

lowastlowastlowast


lowastlowastlowast

















50


50values was found




















References





































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







a146 plusmn 006

ab


b248 plusmn 008

a


c021 plusmn 001

b


d032 plusmn 001

b


e013 plusmn 000

b




50(120583M)




a

b

cd

e

b

a

cd

e0

1000

2000

3000

4000

5000

6000

7000


120583m

olg

AD

W




3 Results
















b0220 plusmn 0041

c0047 plusmn 0006

b0396 plusmn 0002

e0175 plusmn 0001

b


b0111 plusmn 0001

c0333 plusmn 0058

b0668 plusmn 0001

d0089 plusmn 0001

c


b3752 plusmn 0091

b0601 plusmn 0100

b18155 plusmn 0005

b0072 plusmn 0001

d


c1700 plusmn 0173

b4831 plusmn 0001

c0040 plusmn 0001

e


a14300 plusmn 0760

a35000 plusmn 2646

a48641 plusmn 0001

a2523 plusmn 0001

a




50value ranged






50values among







50value


501253141 plusmn 0002mgmL) (119875 lt 005)




0

10

20

30

40

50

60

70

80

0 02 04 06 08 1 12 14

Mea

n

inhi

bitio

n of

deo

xyrib

ose d

egra

datio

n


(a)

0102030405060708090

100

0 05 1 15 2 25 3 35 4

Mea

n

inhi

bitio

n of

deo

xyrib

ose d

egra

datio

n


(b)



graphs


50value


01020304050607080

0 005 01 015 02 025 03 035 04Mea

n

xan

thin

e oxi

dase

inhi

bitio

n


(a)

0

10

20

30

40

50

60

70

0 50 100 150 200 250 300 350 400

Xant

hine

oxi

dase

inhi

bitio

n (

)


(b)







mutansStreptococcus





lowast











825 plusmn 050




0

5

10

15

20

25

30


of m

inim

alin

hibi

ted

grow

th (m

m)

Extract only

lowastlowastlowast


lowastlowastlowast

















50


50values was found




















References





































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










b0220 plusmn 0041

c0047 plusmn 0006

b0396 plusmn 0002

e0175 plusmn 0001

b


b0111 plusmn 0001

c0333 plusmn 0058

b0668 plusmn 0001

d0089 plusmn 0001

c


b3752 plusmn 0091

b0601 plusmn 0100

b18155 plusmn 0005

b0072 plusmn 0001

d


c1700 plusmn 0173

b4831 plusmn 0001

c0040 plusmn 0001

e


a14300 plusmn 0760

a35000 plusmn 2646

a48641 plusmn 0001

a2523 plusmn 0001

a




50value ranged






50values among







50value


501253141 plusmn 0002mgmL) (119875 lt 005)




0

10

20

30

40

50

60

70

80

0 02 04 06 08 1 12 14

Mea

n

inhi

bitio

n of

deo

xyrib

ose d

egra

datio

n


(a)

0102030405060708090

100

0 05 1 15 2 25 3 35 4

Mea

n

inhi

bitio

n of

deo

xyrib

ose d

egra

datio

n


(b)



graphs


50value


01020304050607080

0 005 01 015 02 025 03 035 04Mea

n

xan

thin

e oxi

dase

inhi

bitio

n


(a)

0

10

20

30

40

50

60

70

0 50 100 150 200 250 300 350 400

Xant

hine

oxi

dase

inhi

bitio

n (

)


(b)







mutansStreptococcus





lowast











825 plusmn 050




0

5

10

15

20

25

30


of m

inim

alin

hibi

ted

grow

th (m

m)

Extract only

lowastlowastlowast


lowastlowastlowast

















50


50values was found




















References





































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


01020304050607080

0 005 01 015 02 025 03 035 04Mea

n

xan

thin

e oxi

dase

inhi

bitio

n


(a)

0

10

20

30

40

50

60

70

0 50 100 150 200 250 300 350 400

Xant

hine

oxi

dase

inhi

bitio

n (

)


(b)







mutansStreptococcus





lowast











825 plusmn 050




0

5

10

15

20

25

30


of m

inim

alin

hibi

ted

grow

th (m

m)

Extract only

lowastlowastlowast


lowastlowastlowast

















50


50values was found




















References





































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













50


50values was found




















References





































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

















References





































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

bioactivity of nonedible parts ofpunica granatuml

Documents

university of mauritius

biomaterials research

potential use

introductionpunica granatum

mgair dry weightml

unrestricted use

molg air dry weight

faculty of science