Original article

1 Div. Mol. Med., Bose Inst.,P-1/12 C.I.T. Scheme VII-M,Kolkata-700054, India,mandaln@rediffmail.com,nripen@jcbose.ac.in2 Lab. Pharm. Chem., Inst.Nat.Prod. Chem., Vietnam Acad.Sci. Technol., 18 Hoang QuocViet Street, Hanoi, Vietnam

In vitro assessment of phytochemicals, antioxidant and DNA protective potential of wild edible fruit of Elaeagnus latifolia Linn.Sourav PANJA1, Dipankar CHAUDHURI1, Nikhil BABAN GHATE1, Ha LE MINH2, Nripendranath MANDAL1*

* Correspondence and reprints

Received 30 September 2013Accepted 17 January 2014

Fruits, 2014, vol. 69, p. 303–314© 2014 Cirad/EDP SciencesAll rights reservedDOI: 10.1051/fruits/2014019www.fruits-journal.org

RESUMEN ESPAÑOL, p. 314

In vitro assessment of phytochemicals, antioxidant and DNA protectivepotential of wild edible fruit of Elaeagnus latifolia Linn.Abstract – Introduction. Elaeagnus latifolia Linn. is a type of wild edible fruit found in nor-theast India, Thailand and also in Vietnam. Although the fruit is reported to be a source of vita-mins, minerals, essential fatty acids and other bioactive compounds, only a few studies have beenconcerned with the antioxidant activity of this plant. Materials and methods. Our study revea-led in vitro antioxidant and free radical scavenging activity of 70% methanolic extract ofElaeagnus latifolia Linn. (ELME). Various tests including identification and quantification of phy-tochemicals, total antioxidant activity, hydroxyl radical, superoxide radical, singlet oxygen, hypo-chlorous acid scavenging, reducing power and DNA protection assays were performed. Resultsand discussion. Among the tests, ELME scavenged superoxide radical [IC50 = (150.78 ±4.2) µg⋅mL–1], hydroxyl radical [IC50 = (238.09 ± 11.63) µg⋅mL–1] and protected pUC18 DNA[P50 = (695.91 ± 15.84) µg⋅mL–1]; P50 signifies the concentration for 50% protection . The fruitis found to be a source of minute amounts of carbohydrates, ascorbic acid, tannins, phenolicsand flavonoids. HPLC data showed that purpurin, tannic acid, quercetin, catechin, reserpine andrutin are present in ELME. Conclusion. Our results provide evidence that 70% methanol extractof E. latifolia Linn. acts as a promising antioxidant as well as DNA protector, which is partly dueto the phenolic and flavonoid compounds present in it.

India / Elaeagnus latifolia / fruits / medicinal properties / free radicals /antioxidants

Évaluation in vitro des composés phytochimiques, des antioxydants et dupotentiel protecteur de l’ADN des fruits sauvages comestibles d’Elaeagnuslatifolia Linn.Résumé – Introduction. Elaeagnus latifolia Linn. est un type de fruit sauvage comestible trouvédans le nord de l’Inde, en Thaïlande et également au Vietnam.Bien que le fruit soit signalé commeune source de vitamines, minéraux, acides gras essentiels et autres composés bioactifs, seulesquelques recherches ont porté sur l’activité antioxydante de cette plante. Matériel et méthodes.Notre étude a révélé l’activité antioxydante et le potentiel de piégeage des radicaux in vitro depulpe d’Elaeagnus latifolia Linn. extraite à l’aide d’une solution aqueuse de méthanol à 70 %(ELME). Divers tests, dont l’identification et la quantification de composés phytochimiques, del’activité totale antioxydante, du radical hydroxyle, du radical superoxyde et de l’oxygène sin-gulet, et des essais du pouvoir réducteur et de piégeage de l’acide hypochloreux et de la pro-tection de l’ADN ont été effectués. Résultats et discussion. Parmi les tests réalisés, le radicalsuperoxyde piégé de ELME [IC50 = (150.78 ± 4.2) µg⋅mL–1], le radical hydroxyle [IC50 = (238.09 ±11.63) µg⋅mL–1] et l’ADN pUC18 protégé [P50 = (695.91 ± 15.84) µg⋅mL–1] ont présenté une pro-tection efficace. Le fruit se trouve être une source d’infimes quantités d’hydrates de carbone,d’acide ascorbique, de tanins, de composés phénoliques et de flavonoïdes. Les données HPLCont montré que la purpurine, l’acide tannique, la quercétine, la catéchine, la réserpine et la rutineétaient présents dans les extraits (ELME) présents. Conclusion. Nos résultats fournissent lapreuve que l’extrait d’E. latifolia Linn. agit comme un antioxydant prometteur et comme unprotecteur de l’ADN, ce qui est dû en partie aux composés phénoliques et flavonoïdes qui lecomposent.

Inde / Elaeagnus latifolia / fruit / propriété pharmacologique / radical libre /antioxydant

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1. Introduction

A number of reactive oxygen species andreactive nitrogen species are essential partsof aerobic life and metabolism [1]. Reactiveoxygen species (ROS) directly bring aboutdamage of cellular macromolecules, espe-cially DNA, which undergo strand breakage,and change and release of bases as well asmodification of sugar moieties [2, 3], thuspromoting a series of pathological events,viz. cancer and ageing [4]. On the otherhand, ROS along with pro-oxidants give riseto emphysema, cirrhosis, arteriosclerosis,inflammation, genotoxicity and other dis-ease conditions [5]. Various food products aswell as beverages containing antioxidantsact through different mechanisms, such asscavenging and chelating metals that cat-alyse the formation of free radicals, thusbeing important for the human body dueto their ability to counteract oxidativedamage [6, 7]. Synthetic antioxidants suchas butylated hydroxyanisole (BHA) andbutylated hydroxyltoluene (BHT) havebeen restricted for use due to their possi-ble toxicological effects [8]. Therefore, thesearch for natural antioxidants hasreceived much attention in order to iden-tify and develop more potent natural anti-oxidants to replace synthetic ones.

Elaeagnus latifolia Linn. is a type of edi-ble fruit; it belongs to the Elaeagnaceae fam-ily. Elaeagnus latifolia is a deciduous shrubgrowing to a maximum height of 3 m (9 ft)and expands maximally to 3 m (9 ft) with agrowing speed of medium rate. It is hardyto zone 9 (can tolerate a minimum temper-ature of −7 °C) and not frost-tender. It isfound in northeast India, Thailand and alsoin Vietnam. It is commonly known as Soh-Shang in Meghalya, Sibsagar (Dikho valleyof Assam), in India. The flowers are her-maphrodite (have both male and femaleorgans) and are pollinated by bees.It can fixnitrogen. The fruit is oblong in shape witha dark pink colour at the time of ripening.The fruits are eaten raw as well as used formaking chutney, jam, jelly and refreshingdrinks [9]. The fruit is a plentiful source ofvitamins, minerals and other bioactive com-pounds. It is also a source of essential fattyacids, which is fairly unusual for a fruit [9].

The objective of our study was to evalu-ate the phytochemical constituents, antioxi-dant potential, free radical scavengingactivity and DNA protection activity of 70%methanolic extract of Elaeagnus latifoliafruit (ELME).

2. Materials and methods

2.1. Chemicals

2,2′-azinobis-(3-ethylbenzothiazoline-6-sul-phonic acid) (ABTS) was procured fromRoche Diagnostics, Mannheim, Germany,and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) was obtainedfrom Fluka, Buchs, Switzerland. Potassiumpersulphate (K2S2O8), 2-deoxy-2-ribose,ethylene diamine tetra acetic acid (EDTA),ascorbic acid, trichloroacetic acid (TCA),mannitol, nitro blue tetrazolium (NBT), re-duced nicotinamide adenine dinucleotide(NADH), phenazine methosulphate (PMS),sodium nitroprusside (SNP), 1,10-phenan-throline, sulphanilamide, naphthyl ethylen-ediamine dihydrochloride (NED), L-histi-dine, lipoic acid, sodium pyruvate, quercetinand ferrozine were obtained from Sisco Re-search Laboratories Pvt. Ltd., Mumbai, India.HPLC-grade acetonitrile, ammonium ace-tate, hydrogen peroxide, potassium hexacy-anoferate, Folin-Ciocalteu reagent, sodiumcarbonate, mercuric chloride, potassium io-dide, anthrone, vanillin, thiourea, 2,4-dinitrophenylhydrazine, sodium hypochlorite, alu-minium chloride, xylenol orange, butylatedhydroxytoluene (BHT) and N,N- dimethyl-4-nitrosoaniline were obtained from Merck,Mumbai, India. 1,1-diphenyl-2-picrylhydra-zyl (DPPH), gallic acid, (+) catechin and cur-cumin were obtained from MP Biomedicals,France. Catalase, reserpine and sodium bi-carbonate were obtained from HiMedia Lab-oratories Pvt. Ltd., Mumbai, India. Evansblue was purchased from BDH, England. D-glucose was procured from Qualigens FineChemicals, Mumbai. Diethylene triaminepentaacetic acid (DTPA) was obtained fromSpectrochem Pvt. Ltd., Mumbai, India. Thio-barbituric acid (TBA) was obtained fromLoba Chemie, Mumbai, India.

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2.2. Animals

The Animal Ethical Clearance Committee ofthe Institute (registration number: 95/1999/CPCSEA) approved the use of adult maleSwiss Albino mice (Mus musculus), weigh-ing 20–25 g, for experimentation. Eachpolypropylene cage contained four mice ata time, supplied with an ad libitum labora-tory diet and water at (25 ± 2) °C and(60 ± 5)% humidity and a normal photocy-cle (12 h dark / 12 h light).

2.3. Plant material and extractpreparation

The fruits of E. latifolia were collected fromthe Garo Hills market in Assam (India). Theflesh pulps of fruit (100 g) were stirred usinga magnetic stirrer with 70% of methanol inwater (1000 mL) for 15 h; the mixture wascentrifuged at 2850 × g and the supernatantdecanted. The process was repeated by add-ing the solvent to the precipitated pellet. Thesupernatants were concentrated in a rotaryevaporator and lyophilised. The yield offruit in methanolic extract was 7.89%.Lyophilised E. latifolia 70% methanolicextract was stored at –20 °C until use.

2.4. Phytochemical analysis

Standard qualitative methods as describedpreviously were carried out in order to in-vestigate the resident phytochemicals suchas phenols, flavonoids, alkaloids, carbohy-drates, ascorbic acid, saponins, tannins, ter-penoids, triterpenoids, anthraquinones andglycosides in the extract [10,11].

2.4.1. Determination of total phenoliccontent

According to a previously described protocol[12], the total phenolic content of ELME wasdetermined by Folin-Ciocalteu (FC) reagent.All tests were performed six times. The phe-nolic contentwas calculated fromagallic acidstandard curve containing eight points.

2.4.2. Determination of total flavonoids

Total flavonoid content was determined ac-cording to a previously discussed method [12].The flavonoid content was calculated from

a quercetin standard curve containing sixpoints.

2.4.3. Quantification of alkaloid content

Quantification of alkaloid content for ELMEwas carried out using a method describedearlier [13]. All tests were performed sixtimes. The alkaloid content was evaluatedfrom the reserpine standard graph contain-ing nine points.

2.4.4. Quantification of carbohydratecontent

The carbohydrate content of the extract wasquantified using a previously describedmethod [13]. All tests were performed sixtimes. The carbohydrate content was eval-uated from a glucose standard curve con-taining six points.

2.4.5. Quantification of ascorbic acidcontent

This quantification was carried out accord-ing to a previously described method [13].All tests were performed six times. Theascorbic acid content was evaluated from anL-ascorbic acid standard curve containingeight points.

2.4.6. Quantification of tannin content

This was assayed and performed as per apreviously described method [14]. All testswere performed six times. The tannin con-tent was evaluated from a catechin standardgraph containing six points.

2.5. High-Performance LiquidChromatography (HPLC)standardisation of the extract

For HPLC analysis, stock solutions(10 µg⋅mL–1) were prepared in the mobilephase for the sample (ELME) [15] and pur-purin, tannic acid, quercetin, catechin, re-serpine and rutin as standards. Sampleswere then filtered through a 0.45-µm poly-tetrafluoroethylene (PTFE) filter (Millipore)to remove any particulate matter. Analysiswas performed using a HPLC ProminenceSystem RF10AXL (Shimadzu Corp., Japan)equipped with a degasser (DGU-20A5),quaternary pump (LC-20AT), auto-sampler

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(SIL-20A) and reflective index (RID-10A),fluorescence (RF-10AXL) and diode array(SPD-M20A) detectors. Twenty µL of sampleand standards were injected and analysed intriplicate. Gradient elution consecutive mo-bile phases of acetonitrile and 0.5 mM am-monium acetate in water, at a flow rate of1 mL⋅min–1 for 65 min through the column(Z1C-HILIC), were maintained at 25 °C. Thedetection was carried out at 254 nm.

2.6. In vitro antioxidant and freeradical scavenging assays

2.6.1. Total antioxidant activity

The antioxidant capacity of the ELME (0.05–10 mg⋅mL–1) was evaluated by ABTS⋅+ rad-ical cation decolourisation assay in compar-ison with the Trolox standard [16]. Theirabsorbance was measured at 734 nm.

2.6.2. DPPH radical scavenging assay

Complementary data supporting the studyof the antioxidant potential of ELME wereprovided by the DPPH radical scavengingassay [12]. The sample sets were then spec-trophotometrically measured at 517 nmafter 2 min. All the above tests were per-formed six times and in each case the per-centage of scavenging of the respectiveradical was calculated using the followingequation: % scavenging = [(A0 – A1) / A0] ×100, where A0 is the absorbance of the con-trol, and A1 is the absorbance in the pres-ence of the sample of fruit extracts and thestandard.

2.6.3. Hydroxyl radical scavengingassay

The hydroxyl radical scavenging assay wasperformed as described previously; it isbased on the Fenton reaction [17]. Mannitol,a classical OH⋅ scavenger, was used as a pos-itive control.

2.6.4. Superoxide radical scavengingassay

The scavenging activity of the superoxideradical for the ELME (0–120 µg⋅mL–1) wasmeasured spectrophotometrically at 562 nmas described previously [17]. Quercetin wasused as a standard.

2.6.5. Nitric oxide radical scavengingassay

Nitric oxide (NO⋅) generated from the SNPaqueous solution at physiological pH interactswith oxygen to produce nitrite ions, whichwere measured by Griess-Illosvoy reaction[12]. Various doses of ELME (0–70 µg⋅mL–1)along with the standard curcumin wereassessed for their respective activities.

2.6.6. Peroxynitrite radical scavengingassay

Peroxynitrite (ONOO–) was synthesised12 h before the experiment, as described byBeckman et al. [18]. The scavenging activityof the extract (0–200 µg⋅mL–1) was com-pared with that of the reference compoundgallic acid.

2.6.7. Singlet oxygen radicalscavenging assay

The production of singlet oxygen (1O2) wasdetermined by monitoring N,N-dimethyl-4-nitrosoaniline (RNO) bleaching using a pre-viously reported method [14]. The scaveng-ing activity of the extract (0–200 µg⋅mL–1)was compared with that of the standardlipoic acid.

2.6.8. Hypochlorous acid scavengingassay

The hypochlorous acid scavenging assaywas carried out as described previously [12].The scavenging activity of the extract (0–100 µg⋅mL–1) and the standard ascorbic acidwas evaluated by measuring the decrease inabsorbance of catalase at 404 nm.

2.6.9. Fe2+ chelation activity

The iron-chelating capacity of ELME (0–120 µg⋅mL–1) was spectrophotometricallyevaluated at 562 nm as described earlier[12]. EDTA was used as a positive control.

2.6.10.Lipid peroxidation assay

The ability of ELME to inhibit lipid peroxi-dation was assayed by a previouslydescribed method [17]. Various concentra-tions of the extract (2.5–25 µg⋅mL–1) wereassessed along with the standard Trolox.

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The absorbance of the reaction mixtureswas taken at 532 nm.

2.6.11. Measurement of reducing power

The Fe3+ reducing power of the extract wasdetermined by a method performed earlier[17]. Various concentrations (0–1.0 mg⋅mL–1)of the extract were tested and their absorb-ance was measured at 700 nm against anappropriate blank.

2.6.12. DNA protection assay

The protection of the pUC18 plasmid DNA,damaged by Fenton reaction-generated OHradicals, was studied by quantifying thedecrease in supercoiled DNA after oxidativeattack using a previously described methodwith minor modifications [19]. Briefly,FeSO4 solution (15 µM), extract of varyingdoses, DNA (1 µg) and water were addedto Hepes buffer (pH 7.2, 13 mM) to makean initial reaction mixture. H2O2 solution(0.0125 mM) was then added to start thereaction. After 10 min, the reaction wasstopped by adding desferal (0.2 mM) as astopping reagent. Twenty µL of each reac-tion mixture was loaded in 1% agarose gel.After electrophoresis, the gel was stainedwith ethidium bromide (1 µg⋅mL–1) and vis-ualised in a UV transilluminator. The DNAbands were quantified through densitome-try and the following formula was used tocalculate the percentage of protection: % SC ={1.4 × SC/[OC + (1.4 × SC)] × 100}, whereSC = supercoiled; OC = open circular; 1.4= correction factor % protection = 100 ×[(control SC – chelator SC)/(control SC – nochelator SC) – 1].

The ability of the fruit extract to protectthe DNA supercoil can be expressed by theconcentration of sample required for 50%protection, designated as the P50 value.

2.6.13. Statistical analysis

All data were reported as the mean ± stand-ard deviation of six measurements. The sta-tistical analysis was performed by KyPlotversion 2.0 beta 13 (32 bit) and was plottedgraphically by Origin professional 8.0,wherever necessary. The IC50 values werecalculated by the formula: Y = 100 × [A1/(X +A1)], where A1 = IC50, Y = response

(Y = 100% when X = 0) and X = inhibitoryconcentration. The IC50 values were com-pared by paired t-test (two-sided) and one-sided ANOVA; wherever required, P < 0.05was considered significant.

3. Results and discussion

In physiological systems, the oxidationchain reaction produces many free radicalswhich disrupt biomolecules and can disruptthe immune system [20]. Antioxidants termi-nate these chain reactions by removing freeradical intermediates and inhibit other oxi-dation reactions. Previously, it was reportedthat the various types of edible fruits haveantioxidant activity [21, 22]. It would be bet-ter if dietary food supplements were apotent source of antioxidants. Our studyfocused on the evaluation of the antioxidantand free radical scavenging activity of ELME.

3.1. Phytochemical analysis

Our study showed that all phytochemicalsare moderately present in ELME except alka-loids, saponins and glycosides. The totalphenolic, flavonoid, ascorbate and tannincontents of ELME were calculated (table I).Phenolic compounds are very importantplant constituents due to their ability to con-fer free radical scavenging [23]. Moreover,flavonoids show their antioxidant proper-ties through scavenging or chelating proc-esses [24]. Different types of carbohydratesgive rise to different glycemic responses andthey are also able to stimulate lipogenesis[25]. In addition, ascorbic acid, terpenoidsand triterpenoids are good antioxidant phy-toconstituents. Tannins show internal anti-septic, anti-diarrhoea, antimicrobial andantifungal effects and also have astringentproperties [26]. Anthraquinones protectfrom Alzheimer’s disease [27]. The resultsstrongly suggest that phenolics are impor-tant components of these plants. The otherphenolic compounds such as flavonoids,which contain hydroxyls, are responsiblefor the radical scavenging effect in theplants. According to our study, the moderatecontent of carbohydrate, ascorbate, tannin,

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terpenoids and triterpenoids also in E. lati-folia can explain their good radical scaveng-ing activity. Our study carried out on ELMErevealed the presence of medically activeconstituents.

3.2. HPLC standardisation of ELME

Moreover, HPLC analysis was performed toidentify the presence of bioactive com-pounds in ELME by comparing with therespective retention time of the referencestandards. The chromatogram obtainedfrom HPLC analysis of the extract and sixmain peaks with retention times at (2.17,

3.35, 11.86, 18.00, 21.05 and 66.91) min cor-responded to those of purpurin, tannic acid,quercetin, catechin, reserpine and rutin,respectively (figure 1). It has been previ-ously reported that these six compoundshave antioxidant properties [28–32].

3.3. Total antioxidant activity

The total antioxidant activity of theextract was determined based on the de-colourisation of the ABTS⋅+ produced by re-action of ABTS with potassium persulphateand measured spectrophotometrically at734 nm. The addition of ELME and Troloxconvert this preformed radical cation intoABTS in a concentration-dependent man-ner. The interaction of the ABTS⋅+ with theextract or the standard Trolox results in sup-pression of this cation and are plotted aspercentage inhibition of absorbance (fig-ure 2). The TEAC value of the extract wasmeasured as 0.070 ± 0.003 (table II). TheTEAC value obtained reflects the antioxi-dant potency of the extract.

3.4. DPPH scavenging assay

The DPPH stable free radical method is aneasy, rapid and sensitive way to survey theantioxidant activity of a specific compoundor plant extracts [33]. According to ourresults, it is evident that ELME scavengesDPPH radical moderately as compared withthe ascorbic acid (figure 3, table II). The IC50values of ELME and the standard were found

tive phytochemical analysis of 70% methanolic extract of Elaeagnus latifolia Linn.

are given for 100 mg extract

tal flavonoidsg quercetin Eq)

Total ascorbate(mg ascorbic acid Eq)

Carbohydrates(mg glucose Eq)

Tannins(mg catechin Eq)

5.44 ± 0.16 0.23 ± 0.09 10.96 ± 0.08 0.20 ± 0.03

Terpenoids Triterpenoids Anthraquinones Saponins Glycosides

Presence Presence Presence Absence Absence

Table I.Qualitative and quantita(ELME).

Quantitative analyses: results

Total phenolics(mg gallic acid Eq)

To(m

7.04 ± 0.27

Qualitative analyses

Alkaloids

Absence

Figure 1.HPLC chromatogram of 70%methanolic extract ofElaeagnus latifolia Linn (ELME).Inset shows expanded region ofthe chromatogram with theretention time of 8–24 min.

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to be (134.31 ± 8.39) µg⋅mL–1 and (5.29 ±0.28) µg⋅mL–1, respectively, which showthat ELME possesses a higher scavengingactivity than water methanolic extract ofanother species of Elaeagnus (E. pyri-formis) [34].

The effect of the plant extract in the scav-enging assay of DPPH radical furthermoreconfirmed the fact that the extract acts as anantioxidant, since the studies of TEAC andDPPH scavenging can be observed as com-plementary to each other.

3.5. Hydroxyl radical scavengingassay

The most detrimental of the free radicalsformed in biological systems is the hydroxylradical that causes enormous damage to bio-molecules of the living cells [35]. This com-pound forms a pink chromogen uponheating with TBA at low pH. As ELME orstandard mannitol was added to the reactionmixture the hydroxyl radicals were scav-enged and thereby sugar damage can beblocked (figure 4). The IC50 values (table II)of ELME and the standard in this assay were(238.09 ± 11.63) µg⋅mL–1 and (571.45 ±20.12) µg⋅mL–1, respectively. The resultsindicate that ELME is an excellent hydroxylradical scavenger; it is better than the stand-ard mannitol.

3.6. Superoxide scavenging assay

Superoxide anion is a harmful reactive oxy-gen species (ROS) with a detrimental effecton the cellular components in biologicalsystems [36]. Plant-derived antioxidantsexert their effects by enhancing the level ofantioxidant enzymes such as superoxidedismutase or by lowering the levels of lipidperoxides in the blood or liver [37]. Theextract and the reference compound quer-cetin have the ability to quench superoxideradicals in the PMS-NADH reaction mixture(figure 5). The IC50 values (table II) of thefruit extract and quercetin were (150.78 ±4.2) µg⋅mL–1 and (42.06 ± 1.35) µg⋅mL–1,respectively. The extract is not more potentthan the standard, yet a scavenger of super-oxide radical.

3.7. Hypochlorous acid scavengingassay

At the sites of inflammation, the neutrophilenzyme myeloperoxidase oxidises Cl- ionsand produces another harmful ROS,hypochlorous acid, which reacts with vari-ous types of biomolecules including nucleicacid, lipid and protein [36, 38, 39]. The dose-dependent hypochlorous acid scavengingactivity of ELME was compared with that ofascorbic acid (figure 6). The IC50 value was(667.11 ± 6.56) µg⋅mL–1; it was higher thanthat of ascorbic acid [(235.95 ± 5.75) µg⋅mL–1](table II). The inhibition of catalase deacti-vation in the presence of ELME indicates itsHOCl scavenging activity. So, from ourresults, it could be anticipated that ELME isa moderate HOCl scavenger but not as goodas ascorbic acid.

3.8. Fe2+ chelation activity

The results demonstrated that the formationof the Fe2+-ferrozine complex is inhibited inthe presence of test and reference com-pounds (figure 7). The IC50 values of theELME and EDTA were (993.68 ±50.74) µg⋅mL–1 and (1.27 ± 0.05) µg⋅mL–1,respectively (table II). According to the

FTeraa

FTeraa

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igure 2.otal antioxidant activity of fruitxtract and Trolox in the ABTSadical cation decolourisationssay. All data are expresseds mean ± S.D. (n = 6).

igure 2.otal antioxidant activity of fruitxtract and Trolox in the ABTSadical cation decolourisationssay. All data are expresseds mean ± S.D. (n = 6).

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results, the extract was not as efficient as thestandard EDTA, but the decrease in the con-centration-dependent colour formation inthe presence of the extract indicates that ithas iron-chelating properties.

3.9. DNA protection assay

The Fe2+-H2O2-mediated DNA breakdownis protected by ELME (figure 8). pUC18supercoiled DNA was used as a control(lane 1). The addition of extract to the reac-tion mixture substantially decreased theDNA strand scission in a dose-dependentmanner, leading to the protection of DNAunder oxidative stress. The treatment ofsupercoiled DNA with Fenton’s reagent ledto the conversion of DNA to the open cir-cular form (lane 2). The addition of different

oxidant, free radical scavenging and iron-chelating activities of 70% methanolic extract. (ELME) and reference compounds.

Values for ELME1 Standard Values of standard compounds

0.070 ± 0.003 – –

695.91 ± 15.84 – –

f ELME in

134.31 ± 8.39 Ascorbic acid 5.29 ± 0.28***

enging 238.09 ± 11.63 Mannitol 571.45 ± 20.12***

venging 150.78 ± 4.20 Quercetin 42.06 ± 1.35***

cavenging 667.11 ± 6.56 Ascorbic acid 235.95 ± 5.75***

993.68 ± 50.74 EDTA 1.27 ± 0.05***

ivities is µg⋅mL–1, except the TEAC value, which is expressed in mg⋅mL–1.n of the extract (in µg⋅mL–1) required for 50% protection of pUC18 DNA.S.D, (n = 6), where ***: P < 0.001 vs. ELME.

Figure 5.Scavenging effect of 70% methanolic extract ofElaeagnus latifolia Linn. (ELME) and the standardquercetin on superoxide radical. All data areexpressed as mean ± S.D. (n = 6). ***: P < 0.001vs. 0 µg⋅mL–1.

Table II.Quantification of the antiof Elaeagnus latifolia Linn

Activity

TEAC value

DNA protection, [P]50

IC50 values for the activities o

DPPH scavenging

Hydroxyl radical (OH⋅) scav

Superoxide anion (O2⋅−) sca

Hypochlorous acid (HOCl) s

Fe2+ chelation1 Unit for IC50 values of all act[P]50 signifies the concentratioData are expressed as mean ±

Figure 3.Effect of the 70% methanolicextract of Elaeagnus latifoliaLinn. (ELME) and standardascorbic acid in the DPPHradical scavenging study. Theresults are mean ± S.D. (n = 6).***: P < 0.001 vs. 0 µg⋅mL–1.

Figure 4.Hydroxyl radical scavengingactivity of 70% methanolicextract of Elaeagnus latifoliaLinn. (ELME) and the referencecompound mannitol. Theresults are mean ± S.D. (n = 6).**: P < 0.01 and ***: P < 0.001vs. 0 µg⋅mL–1.

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concentrations of an extract resulted in therestoration of DNA in the supercoiled form(lane 3 to 9). The significant reduction in theformation of nicked DNA and increase in

supercoiled DNA in the presence of theextract determined its excellent DNA protec-tive effect; also reflected by the [P]50 value(concentrationof the extract required for 50%protection of the supercoiled DNA), whichwas found to be (695.91 ± 15.84) µg⋅mL–1

(table II). DNA protection can be achievedby two pathways, either Fe+ chelation orhydroxyl radical scavenging. In our studyELME did not have good Fe+ chelation prop-erties. So, ELME protects DNA throughhydroxyl radical scavenging.

3.10. Assays showing no significantresults

ELME extract did not showed any significantremarkable result in the nitric oxide, hydro-gen peroxide, peroxynitrite, singlet oxygenscavenging, reducing power or lipid perox-idation inhibition assays, so the correspond-ing results and figures are not provided.

4. Conclusions

From the present study, we can concludethat a 70% methanolic extract of Elaeagnuslatifolia contains small amounts of phenols,flavonoids, ascorbic acid, carbohydratesand tannins. Various in vitro assays indicatethat the extract exhibits moderate antioxi-dant and free radical scavenging activitiesand, hence, the fruit is considered to be asignificant source of natural antioxidants,which might be helpful in preventing theprogress of oxidative stress in the body sys-tem. Therefore, further studies should becarried out to isolate active compoundsfrom this fruit material with antioxidantproperties.

Acknowledgements

The authors would like to thank Mr.Rhitajit Sarkar and Dr. Bibhabasu Hazra forcritically reviewing the paper. The authorsalso extend their thanks to Mr. Ranjit KumarDas and Mr. Pradip Kumar Mallick for

Figure 6.Hypochlorous acid scavenging activity of 70%methanolic extract of Elaeagnus latifolia Linn.(ELME) and the standard ascorbic acid. All dataare expressed as mean ± S.D. (n = 6). *: P < 0.05and ***: P < 0.001 vs. 0 µg⋅mL–1.

Figure 7.Effect of 70% methanolic extract of Elaeagnuslatifolia Linn. (ELME) (above) and the standardEDTA (below) on ferrozine-Fe2+ complexformation. The results are mean ± S.D. (n = 6).***: P < 0.001 vs. 0 µg⋅mL–1.

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technical assistance in sample preparationand handling of lab equipment and animalsin experimental procedures.

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Evaluación in vitro de los compuestos fitoquímicos, de los antioxidantes ydel potencial protector del ADN de los frutos silvestres comestibles deElaeagnus latifolia Linn.

Resumen – Introducción. Elaeagnus latifolia Linn. es un tipo de fruto silvestre comestible,que se encuentra en el norte de la India, en Tailandia, así como en Vietnam. A pesar de queel fruto se considere una fuente de vitaminas, minerales, ácidos grasos esenciales y otroscompuestos bioactivos, sólo existen pocas investigaciones sobre la actividad antioxidante deesta planta. Material y métodos. Nuestro estudio reveló la actividad antioxidante y el poten-cial de captación de los radicales in vitro de pulpa de Elaeagnus latifolia Linn. extraída con laayuda de una solución acuosa de metanol al 70 % (ELME). Se realizaron diversos tests, entrelos cuales la identificación y la cuantificación de compuestos fitoquímicos, de la actividadtotal antioxidante, del radical hidroxilo, del radical superóxido y de oxígeno singlete, asícomo ensayos del poder reductor y de captación del ácido hipocloroso y de la protección delADN. Resultados y discusión. Entre los tests realizados, el radical superóxido captado deELME [IC50 = (150.78 ± 4.2) µg⋅mL–1], el radical hidroxilo [IC50 = (238.09 ± 11.63) µg⋅mL–1] yel ADN pUC18 protegido [P50 = (695.91 ± 15.84) µg⋅mL–1] presentaron una protección eficaz.El fruto resulta ser una fuente de ínfimas cantidades de hidratos de carbono, de ácido ascór-bico, de taninos, de compuestos fenólicos y de flavonoidos. Los datos HPLC mostraron que lapurpurina, el ácido tánico, la quercetina, la catequina, la reserpina y rutina se encontrabanpresentes en los extractos (ELME) presentes. Conclusión. Nuestros resultados aportan unaprueba de que el extracto de E. latifolia Linn. actúa como un antioxidante prometedor aligual que como protector del ADN, lo que se debe, en parte, a los compuestos fenólicos y fla-vonoides que le componen.

India / Elaeagnus latifolia / frutas / propiedades medicinales / radicales libres /antioxidantes

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