Food Chemistry 141 (2013) 3443–3450

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Food Chemistry

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Analytical Methods

Antioxidant and antimicrobial properties of phenolic rich fraction ofSeabuckthorn (Hippophae rhamnoides L.) leaves in vitro

0308-8146/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.foodchem.2013.06.057

⇑ Corresponding author. Tel.: +91 986 888 94752; fax: +91 011 23914790.E-mail address: msyogendra@gmail.com (M.S. Yogendra Kumar).

M.S. Yogendra Kumar a,⇑, R.J. Tirpude a, D.T. Maheshwari b, Anju Bansal a, Ksipra Misra a

a Defence Institute of Physiology and Allied Sciences, Lucknow Road, Timarpur, Delhi 110 054, Indiab Defence Bio-Engineering and Electromedical Laboratory, C V Raman Nagar, Bengaluru 560 093, India

a r t i c l e i n f o a b s t r a c t

Article history:Received 28 April 2011Received in revised form 5 June 2013Accepted 13 June 2013Available online 22 June 2013

Keywords:Antimicrobial activityAntioxidantPhenolHPLCSeabuckthorn

Phenolic rich fraction (PRF) from Seabuckthorn leaves was prepared by sequential fractionation. Totalphenolic content of PRF estimated as gallic acid equivalent was found to be 319.33 ± 7.02 mg/g of PRF.Its major constituents gallic acid, rutin, quercetin-3-galactoside, quercetin-3-glucoside, myricetin, quer-cetin, kaempferol and isorhamnetin, were found in the range of 1.551–196.89 mg/g of PRF as determinedby RP-HPLC. Antioxidant activity of PRF evaluated using 2,2-diphenyl-2-picrylhydrazyl, superoxide andnitric oxide scavenging assays. Reducing power of PRF increased with increasing amount of PRF; theequation of reducing power (y) and amount of PRF (x) was y = 8.004x (r2 = 0.99), indicating that reducingability correlated well with amount of PRF.

Antibacterial activity of PRF, tested against certain medically important bacterial species showedgrowth inhibiting effect against Escherichia coli, Salmonella typhi, Shigella dysenteriae, Streptococcus pneu-moniae and Staphylococcus aureus. In conclusion, PRF has potent antioxidant and broad spectrum antibac-terial properties.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

In healthy individuals, there is a dynamic balance between theamount of free radicals generated in the body and endogenousantioxidant defense system which scavenge them and protect thebody against the deleterious effects of free radicals. Free radicalseither formed by cellular metabolism, exogenous chemicals orstress are capable of oxidising biomolecules which may causemany diseases, including cancer, coronary heart disease, diabetes,and neurodegeneration (Moskovitz, Yim, & Choke, 2002; Sasaki,Ohta, & Decber, 1996). On the other hand, pathogenic bacteriacan cause food borne illnesses and a wide variety of infectious dis-eases (Begnami, Duarte, Furletti, & Rehder, 2010).

Seabuckthorn (Hippophae rhamnoides L., Elaeagnaceae) is a na-tive of Eurasia and has been domesticated in several countries (In-dia, China, Nepal, Pakistan, Myanmar, Russia, Britain, Germany,Finland, Romania, France, etc.) at an altitude of 2500–4300 m(Negi, Chauhan, Sadia, Rohinishree, & Ramteke, 2005). It has beenrecognised as a versatile nutraceutical crop with diverse uses, fromcontrolling soil erosion to being a source of horse fodder, nutritiousfoods, drugs and skin-care products (Fan, Ding, & Gu, 2007). Differ-ent parts of this plant are used in traditional medicine for the treat-ment of diseases, such as flu, cardiovascular diseases, mucosal

injuries and skin disorders (Beveridge, Li, & Oomah, 1999; Eccle-ston et al., 2002).

All parts of this plant are considered to be a good source of alarge number of bioactive compounds, including carotenoids, toc-opherols, sterols, flavonoids, lipids, vitamins, tannins, mineralsetc. (Chawla et al., 2007; Upendra et al., 2008) which contributeto its wide usage as a natural antioxidant. Phenol and flavonoidcontent is rich in fruits, leaves and bark of Seabuckthorn. High per-formance liquid chromatography (HPLC) is an indispensable toolfor the provisional identification of the main phenolic structurespresent in foods (Chirinos et al., 2009).

But so far to the best of our knowledge this is the first report onpolyphenol content and antioxidant as well as antimicrobial prop-erties of the Seabuckthon fraction. Hence, the aim of this study wasto evaluate the antioxidant and antimicrobial properties of pheno-lic rich fraction (PRF) of Seabuckthorn leaves by various in vitroassays.

2. Materials and methods

2.1. Apparatus

Waters high performance liquid chromatography (HPLC) sys-tem equipped with Waters 515 HPLC pump, Waters 717 plusauto-sampler and Waters 2487 UV detector, Mass spectrometermodel Q-TOF Ultima, Micro mass, Manchester, UK, fitted Alliance

3444 M.S. Yogendra Kumar et al. / Food Chemistry 141 (2013) 3443–3450

Waters 2695 DAD detector and with electron spray ionisation (ESI)source and Mass Lynx 4.0 SP4 data acquisition system, MilliporeWater Purification System (Elix-3 LWS Century & Milli-Q), Bio-Rad Spectrum 3000 Spectrophotometer, Buchi Rotavapor R-124,Allied Frost Lyophilizer FD-5, Afcoset Electronic balance FX-300,REMI Cooling centrifuge CPR-24.

2.2. Reagents

1,10-Diphenyl-2-picrylhydrazl (DPPH), 3,4,5-trihydroxybenzoicacid (gallic acid), phenazine methosulphate, nicotinamide adeninedinucleotide, nitroblue tetrazolium, butylated hydroxyl toluene(BHT), 2,4,6-tripyridy-s-triazine (TPTZ), rutin (Sigma AldrichChemicals, USA), ascorbic acid (Vitamin-C) (Sisco Research Labora-tories, India).

2.3. Plant material

Seabuckthorn leaves were collected in the month of Septemberfrom the plants in the hilly region of western Himalayas where theplant grows widely in natural condition. Voucher specimen is pre-served in Defence Institute of High Altitude Research, Leh after eth-no-botanical identification of species.

2.4. Extraction procedure

2.4.1. Preparation of Seabuckthorn leaves crude extract (SLE)Fresh leaves were cleaned thoroughly with ultrapure water,

dried under shade in a clean and dust-free environment. Macera-tion method was used to prepare the 70% ethanol extract of SBTleaves by soaking the powdered dry leaves in 70% ethanol (1:5w/v) at room temperature (25 ± 1 �C). After 24 h, the supernatantwas decanted and the residue was re-soaked in fresh solvent.The process was repeated three times for complete extraction.The supernatant was pooled, filtered through muslin cloth, andcentrifuged at 5000�g for 10 min at 4 �C. Ethanol content wasevaporated using Buchi Rotavapor R-124 at 40 �C. Finally, superna-tant solution was lyophilised in a Heto lyophilizer and the driedcrude extract was stored in airtight dark bottle at 4 �C.

2.4.2. Preparation of phenolic rich fraction (PRF)5 g of prepared SLE was dissolved in 100 ml water and sequen-

tially fractionated in separatory funnel using 100 ml hexane andethyl acetate. The SLE and its fractions (hexane fraction, HF; ethylacetate fraction, EAF; water fraction, WF) solvents were removedby using Rotavapor to dryness. Extract and fractions were dis-solved in 70% ethanol and appropriately diluted to obtain the de-sired concentrations for antioxidant activity evaluation.

2.5. Determination of total phenol content

Total phenol content of extract/extract fractions were deter-mined by the Folin–Ciocalteu method. 150 lL of extract, 2400 lLof ultrapure water and 150 lL of 0.25 N Folin–Ciocalteu reagent(Zhou & Yu, 2006) were combined and then mixed well. The mix-ture was allowed to react for 3 min then 300 lL of 1 N Na2CO3

solution was added and mixed well. The solution was incubatedat room temperature in the dark for 2 h. The absorbance was mea-sured at 725 nm using a spectrophotometer and the results wereexpressed in milligram of gallic acid equivalents (GAE) per gramextract/extract fractions.

2.6. HPLC analysis

The HPLC system equipped with HPLC pump, auto-sampler andUV detector, interfaced with an IBM Pentium 4 personal computer.

The separation was performed on a Symmetry C18 (250 � 4.6 mm,ID; 5 lm) column (Waters, USA).The elution was performed withgradient flow rate programe at 1 ml/min for 16 min. The mobilephase consisted of Solution A (Water: Formic acid 99.9:0.1) andSolution B (acetonitrile:methanol 75:25) and components were de-tected at 370 nm with the following gradient; 80% A – 20% B in0 min, 60% A – 40% B in 5 min, 30% A – 70% B in 10 min and 80%A – 20% B in 15 min. Identification of compounds was performedusing direct injection to mass head of LC/MS system of all sepa-rately collected fractions obtained by HPLC, was based on theoccurrence of mass ions and matching the retention times of pur-chased analytical standards. For the preparation of the calibrationcurve, standard stock solutions of compounds gallic acid, rutin,quercetin-3-galactoside, quercetin-3-glucoside, myricetin, querce-tin, kaempferol and isorhamnetin (1 mg/2 ml) were prepared inabsolute ethanol, filtered through 0.22 lm filters (Millipore), andappropriately diluted (0.01–100 lg/ml) to obtain the desired con-centrations in the quantification range. The calibration graphswere plotted after linear regression of the peak areas versus con-centrations for each of the compound identified.

2.6.1. Direct infusion electrospray–mass spectrometry (ESI–MS)The extract and fraction were dissolved in HPLC grade methanol

and 1–2 lL of samples were directly infused on mass spectrometerfor mass ion analysis (Zarena & Udaya Sankar, 2011). The ESI-MS ofthe extract and fraction was obtained with mass spectrometeroperating at ESI negative mode. The interface and MSD parameterswere as follows: capillary voltage: 3.00 kV; cone: 100 �C; sourcetemperature: 120 �C, desolvation temperature: 300 �C; cone gasflow, 50 L/h; and de-solvation gas: 500 L/h. The mass range wasfrom 200 to 900 m/z, scan speed 1000 amu/s.

2.7. Antioxidant activity evaluation

2.7.1. DPPH radical scavenging activityThe DPPH radical scavenging activities of the test samples were

evaluated by the method of Blois (1958) with minor modifications.Initially, 0.1 ml of the samples at a concentration of 0.01, 0.025,0.05, 0.10 and 0.20 mg/ml was mixed with 1 ml of 0.2 mM DPPH(dissolved in methanol). The reaction mixture was incubated for20 min at 28 �C under dark. The control contained all reagents with-out the sample while methanol was used as blank. The DPPH radicalscavenging activity was determined by measuring the absorbanceat 517 nm using a spectrophotometer. The scavenging DPPH radicalactivity (%) of the tested sample was calculated as (1 � absorbanceof sample/absorbance of control) � 100. The DPPH radical scaveng-ing activity of BHT was also assayed for comparison.

2.7.2. Superoxide anion scavenging activityThe determination of superoxide anion scavenging activity of

extract, fraction and standard antioxidant was measured accordingto the slightly modified method of Nishimiki, Rao, and Yagi (1972).Superoxide radicals are generated in phenazine methosulphate(PMS)–nicotinamide adenine dinucleotide (NADH) systems bythe oxidation of NADH and are assayed by the reduction of nitro-blue tetrazolium (NBT). One milliliter of all the samples (0.01–0.10 mg/ml), 1.0 ml NBT solution (156 lM NBT in 100 mM phos-phate buffer, pH 7.4) and 1.0 ml NADH solution (468 lM in100 mM phosphate buffer, pH 7.4) were mixed. The reaction wasstarted by adding 100 lL of PMS solution (60 lM PMS in 100 mMphosphate buffer, pH 7.4) to the mixture. The mixture was incu-bated at 25 �C for 5 min, and its absorbance was measured at560 nm wavelength against blank samples. The decline of theabsorbance for the reaction mixture indicates an increasing super-oxide anion scavenging activity. The percentage inhibition ofsuperoxide anion generation was calculated using the following

A

(1) Gallic acid (2) Rutin

(3) Quercetin-3-galactoside (4) Quercetin-3-glucoside

(5) Myricetin (6) Quercetin

(7) Kaempferol (8) Isorhamnetin

Fig. 1. Structure of the quantified phenolic compounds (A), HPLC–UV chromatogram of phenolic compounds in Seabuckthorn SLE. Detection was at 370 nm. Peak: (1) gallicacid, (2) rutin, (3) quercetin-3-galactoside, (4) quercetin-3-glucoside, (5) myricetin, (6) quercetin, (7) kaempferol, (8) isorhamnetin (B), HPLC–UV chromatogram of phenoliccompounds in PRF. Detection was at 370 nm. Peak: (1) gallic acid, (2) rutin, (3) quercetin-3-galactoside, (4) quercetin-3-glucoside, (5) myricetin, (6) quercetin, (7) kaempferol,(8) isorhamnetin (C), infusion electrospray-mass spectrometry fingerprint of PRF (D).

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formula as (1 � absorbance of sample/absorbance of con-trol) � 100. The super oxide radical scavenging activity of BHTwas also assayed for comparison.

2.7.3. NO scavenging activityScavenging of NO was determined by incubating sodium nitro-

prusside (5 mM, in PBS) with different concentrations (0.01–0.20 mg/ml) of SLE and PRF at 25 �C. After 2 h, 0.5 ml of incubationsolution was withdrawn and mixed with 0.5 ml of Griess reagent(Green et al., 1981). The absorbance was measured at 550 nm.The percentage inhibition of nitric oxide was calculated using thefollowing formula as (1 � absorbance of sample/absorbance ofcontrol) � 100. The nitric oxide radical scavenging activity of BHTwas also assayed for comparison.

2.7.4. Determination of total reducing power1.0 ml of PRF solution (0.2–1.0 mg/ml) was mixed with 2.5 ml of

a 0.2 M phosphate buffer (pH 6.6) and 2.5 ml of a 1% (w/v) solution ofpotassium ferricyanide. The mixture was incubated in a water bathat 50 �C for 20 min. afterward, 2.5 ml of a 10% (w/v) trichloroaceticacid solution was added, and the mixture was then centrifuged at1000�g for 10 min. A 2.5-ml aliquot of the upper layer was com-bined with 2.5 ml of distilled water and 0.5 ml of a 0.1% (w/v) solu-tion of ferric chloride, and the absorbance was measuredspectrophotometrically (Yanping, Yanhua, & Dongzhi, 2004). Reduc-ing power of ascorbic acid was also determined for comparison.

2.8. Assay for antibacterial activity

Antibacterial activity of SLE and PRF using ampicillin (10 lg)(Hi Media Laboratory Pvt. Ltd., Mumbai, India) as positive control

Fig. 1 (continued)

3446 M.S. Yogendra Kumar et al. / Food Chemistry 141 (2013) 3443–3450

were evaluated against Escherichia coli (ATCC 9837), Salmonellatyphi (clinical isolate, AIIMS, Delhi), Shigella dysenteriae (clinicalisolate, AIIMS, Delhi), Staphylococcus aureus (ATCC 12600) andStreptococcus pneumoniae (clinical isolate, V. Patel Chest Institute,Delhi).

Sensitivity of different bacterial strains to various concentra-tions of SLE and PRF was measured in terms of zone of inhibitionusing agar diffusion assay (Bauer, Kirby, Sherris, & Turck, 1966).The plates containing Mueller–Hinton agar were spread with0.2 ml of the inoculum. Wells (8 mm diameter) were cut out from

M.S. Yogendra Kumar et al. / Food Chemistry 141 (2013) 3443–3450 3447

agar plates using a sterilised stainless steel borer. Stock solutions(1 mg/ml) of SLE and PRF prepared and each well was filled with0.1 ml of solution at the final concentration of 100, 200, 300,500 lg and 1 mg of SLE and PRF. The plates inoculated with differ-ent bacteria were incubated at 37 �C up to 24 h and diameter ofany resultant zone of inhibition was measured. For each combina-tion of SLE or PRF and the bacterial strain, the experiment was re-peated thrice. The bacteria with a clear zone of inhibition of morethan 8 mm were considered to be positive.

2.9. Statistical analysis

Each analysis was done three times from the same extract in or-der to determine their reproducibility. Results are expressed asmean ± SD. Statistical comparisons were made by one-way analy-sis of variance (ANOVA). Differences were considered to be signif-icant when the p values were <0.05.

3. Results and discussion

3.1. Extract yields and total phenol content

Seabuckthorn contain natural antioxidant constituents such asphenolic compounds, which have attracted a great deal of publicand scientific interest because of their health promoting effectsas antioxidants. Total phenolic content of SBT leaves, fruit, berriesand pulp are reported in the range of 1.9–10.8 mg/g of dry rawmaterial. Leaves found contain maximum phenolic contentfollowed by fruit, pulp and berries (Upendra et al., 2008;Zadernowski, Naczk, Czaplicki, Rubinsekiene, & Szakiewicz,2005). In our previous study, relatively higher phenolic compoundsand antioxidant activity was observed from 70% ethanolic extractin comparison with water extract (Upadhyay, Yogendra Kumar, &Gupta, 2010) of SBT leaves. We therefore used 70% ethanol asextraction solvent and obtained a 23.27% yield of extracted mate-rial from SBT leaves. From this SBT leaves crude extract (SLE),phenolic-rich fraction (PRF) was prepared by sequential extractionusing hexane and ethyl acetate. Total phenolic compounds of SLE,HF, EAF and WF was determined using Folin–Ciocalteau method isfound to be 245.65, 197.33, 319.33 and 206.00 (mg GAE/g extractor extract fraction) respectively. Results indicate that EAF con-tained the highest amount of total phenolic compounds followedby SLE, WF and HF. Therefore, ethyl acetate fraction is named asphenolic rich fraction (PRF) and it is used for further antioxidantand antimicrobial studies.

A simple and gradient elution-based reverse phase high perfor-mance liquid chromatography (RP-HPLC) method was developedfor the analysis and quantification of its major constituents gallicacid, rutin, quercetin-3-galactoside, quercetin-3-glucoside, myrice-tin, quercetin, kaempferol and isorhamnetin. For the developmentof an effective mobile phase, various solvent systems, including

Table 1Analysis of phenolic compounds by RP-HPLC–ESI-MS.

Phenolic compound RRT (min) [M�H

Gallic acid* 3.35 169Rutin* 7.11 609Quercetin-3-galactoside* 7.63 463Quercetin-3-glucoside* 7.97 463Myricetin* 9.45 317Quercetin* 11.21 301Kaempferol* 12.77 285Isorhamnetin* 13.01 315

RRT, relative retention time; SLE, seabuckthorn leaves extract; PRF, phenolic rich fractio* Identification based on authentic standard and mass spectrometry spectra.

# Values expressed as mean ± Standard deviation of three determinations.

different combinations of acetonitrile, methanol and water withformic acid were tried. Finally, a solvent system consisting of0.1% formic acid in water and acetonitrile:methanol (75:25) wasproved to be successful as it allows the separation of maximumcompounds with good resolution. Gallic acid, rutin, quercetin-3-galactoside, quercetin-3-glucoside, myricetin, quercetin, kaempf-erol and isorhamnetin (Fig. 1A) that might contribute to the antiox-idant behaviour were determined in SLE and PRF, as shown inFig. 1B and C. Quantitation was carried out by integration of thepeak using an external standard method, and results are presentedin mg/g of the PRF or SLE, for three replicate injections. The resultsindicate that the PRF was found to be rich in rutin, quercetin-3-galactoside, quercetin-3-glucoside, myricetin, quercetin, kaempf-erol and isorhamnetin contents (Table 1) in comparison to SLE,which further corroborates the observed trends in total phenoliccontent and antioxidant activity. Further, direct ESI ionisation(negative mode) was used to identify the phenolic compounds inthe SLE and PRF. The full scan negative mass spectra (Fig. 1D) al-lowed the identification of molecular masses of the Seabuckthornextract and fraction. Each compound gave reasonably intense[M�H]� ions. The ESI-MS fingerprint of SLE and PRF fractionshowed the presence of phenolic compounds such as gallic acid(m/z 169), rutin (m/z 609), quercetin-3-galactoside (m/z 463), quer-cetin-3-glucoside (m/z 463), myricetin (m/z 317), quercetin (m/z301), kaempferol (m/z 285) and isorhamnetin (m/z 315).

Phenolic compounds exist in most plant tissues as secondarymetabolites, i.e. they are not essential for growth, developmentor reproduction but may play roles as antioxidants and in interac-tions between the plant and its biological environment. Phenolicsare also important components of the human diet due to their po-tential antioxidant activity, their capacity to diminish oxidativestress induced tissue damage resulted from chronic diseases andtheir potentially important properties such as anticancer activities(Khadem & Marles, 2010). Some of these properties derive from thefree radical-scavenging activities of phenols. Therefore there aremany reports relating to the reactivities of phenols with activeoxygen species. Recent interest in these substances has been stim-ulated by the potential health benefits arising from their antioxi-dant activity. The antioxidant activity of plant extract cannot beevaluated by only a single method due to the complex nature ofphytochemicals. It has recently been recommended to employ atleast two different in vitro models because of the differencesbetween various free-radical scavenging assay systems (Schlesieret al., 2002). Thus, the SLE and PRF were subjected to three differ-ent antioxidant bioassays.

3.2. Antioxidant activity evaluation

3.2.1. DPPH radical scavenging activityThe PRF fraction showed stronger DPPH free radical scavenging

activity than SLE at all the concentrations tested (Fig. 2A). At0.2 mg/ml, the highest percentage of DPPH radical scavenging

]� (m/z) SLE# (mg/g) PRF# (mg/g)

314.08 ± 4.71 191.89 ± 9.010.41 ± 0.03 0.52 ± 0.072.41 ± 0.12 8.26 ± 0.730.73 ± 0.06 1.55 ± 0.040.19 ± 0.02 1.93 ± 0.220.26 ± 0.02 2.912 ± 0.260.20 ± 0.01 2.85 ± 0.450.86 ± 0.01 9.53 ± 0.71

n.

3448 M.S. Yogendra Kumar et al. / Food Chemistry 141 (2013) 3443–3450

activity of 79.56% was observed in the PRF fraction, significantlyhigher (p < 0.05) than that of SLE (47.25%). From 0.01 to 0.20 mg/ml, the DPPH radical scavenging activity of SLE and PRF increasedbut increased slowly when the concentration exceeded 0.20 mg/mlprobably because the reaction of the scavenging radical activitygradually tended to stabilise at 0.20–0.50 mg/ml. A similar obser-vation was noticed by Sun, Shi, Jiang, Xue, and Wei (2007) in lon-gan pericarp. Flavonoids with free hydroxyl groups have a potentantioxidant activity. The results revealed that the DPPH radical

A

0

10

20

30

40

50

60

70

80

90

100

0 0.05 0.1

Concentration

DPP

H s

cave

ngin

g ac

tivity

(%)

BHTPRFSLE

B

0

10

20

30

40

50

60

70

80

90

100

0 0.02 0.04

Concentra

Supe

roxi

de o

xide

sca

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ing

activ

ity (%

)

BHTPRFSLE

C

0

10

20

30

40

50

60

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80

0 0.02 0.04 0.06 0.08

Concentr

Nitr

ic o

xide

sca

veng

ing

activ

ity (%

) BHTPRFSLE

Fig. 2. DPPH radical scavenging activity of (A), superoxide radical scavenging activ

scavenging activity of SLE and PRF might be attributed to the elec-tron donating ability.

3.2.2. Superoxide anion scavenging activitySuperoxide anion scavenging activities of SLE, PRF and BHT

were analysed, and were concentration dependent (Fig. 2B). ThePRF fraction exhibited an excellent superoxide anion scavengingactivity, higher than SLE in a dose-dependent manner. At 0.1 mg/ml, the superoxide anion scavenging activity of the BHT, PRF and

0.15 0.2

(mg/ml)

0.06 0.08 0.1

tion (mg/ml)

0.1 0.12 0.14 0.16 0.18 0.2

ation (mg/ml)

ity of (B) and nitric oxide radical scavenging activity (C) of SLE, PRF and BHT.

0

0.2

0.4

0.6

0.8

1

1.2

0 0.02 0.04 0.06 0.08 0.1

Concentration (mg/ml)

Abs

orba

nce

at 7

00 n

m

Vit-CPRFSLE

Fig. 3. Reducing power of SLE, PRF and Vit-C. Higher absorbance values indicate higher reducing activity.

M.S. Yogendra Kumar et al. / Food Chemistry 141 (2013) 3443–3450 3449

SLE were 92.73 ± 0.53%, 57.43 ± 1.72% and 46.66 ± 1.14%, respec-tively. Therefore, the superoxide anion scavenging activity wasfound in this increasing order, the BHT > PRF > SLE. It was reportedthat the superoxide anion scavenging activity could be due to theaction of a free hydroxyl group (Siddhuraju, Mohan, & Becker,2002).

3.2.3. Nitric oxide scavenging activityNitric oxide scavenging activities of SLE, PRF and BHT were ana-

lysed, and were concentration dependent (Fig. 2C). The PRF frac-tion exhibited an excellent nitric oxide scavenging activity,higher than SLE in a dose-dependent manner. At 0.1 mg/ml, thesuperoxide anion scavenging activity of the BHT, PRF and SLE were60.74 ± 0.78%, 40.38 ± 0.86% and 30.39 ± 1.59%, respectively.

3.2.4. Reducing powerThe reducing power of PRF, which may serve as a significant

reflection of the antioxidant activity, was determined using a mod-ified iron (III) to iron (II) reduction assay. In this assay, the yellowcolour of the test solution changes to various shades of green andblue depending on the reducing power of extracts or compounds.The presence of reductants in the solution causes the reductionof the Fe3+/ferricyanide complex to the ferrous form. Therefore,the Fe2+ can be monitored by measurement of the formation ofPerl’s Prussian blue at 700 nm (Yanping et al., 2004).

Table 2Antibacterial property of sea buckthorn extracts (SLE and PRF) using agar diffusion assay.

Concentration Zone of inhibition (mm)

E. coli S. typhi S. dysente

SLE PRF SLE PRF SLE

100 lg – 8.0 ± 0.67* – 8.0 ± 0.47* –200 lg – 9.33 ± 0.41* – 10.0 ± 1.63* 9.28 ± 0.5300 lg – 11.33 ± 1.70 11.00 ± 0.82 15.0 ± 0.76 11.45 ± 0500 lg 10.54 ± 0.26 14.0 ± 2.16 12.26 ± 1.43 16.0 ± 2.04 13.48 ± 11 mg 11.63 ± 0.52 15.38 ± 0.38 13.69 ± 0.63 18.38 ± 0.73 15.23 ± 0Ampicillin

(10 lg)18.43 ± 1.06 22.33 ± 1.69 22.66 ± 1

SLE, Seabuckthorn leaf extract; PRF, Phenolic rich fraction.(–) Indicates zone of inhibition less than 8 mm.Values are expressed as mean ± SD (n = 3) for each observation.Values bearing the same superscripts are significantly different (p < 0.05) from each otherpneumoniae; (b) SLE and PRF values are significantly different from each other as tested* Significantly different (p < 0.05) from control (ampicillin) in the respective group.

As shown in Fig. 3, the PRF exhibited higher reducing powerthan SLE, suggesting that the PRF possessed a stronger electrondonating capacity. Reducing power of ascorbic acid was deter-mined for comparison as a standard reducing agent. Furthermore,a linear relationship existed between the concentration and reduc-ing power of the ascorbic acid, PRF and SLE with their correlationcoefficients of 0.9979 (y = 11.46x), 0.9867 (y = 8.0038x) and0.9593 (y = 4.3633x), respectively. It will be relevant to mentionhere that studies have reported the existence of linear relation-ships between antioxidant capacity (assessed as copper reducingpower) and total phenol content (Li, Wang, Li, Li, & Wang, 2009;Yogendra Kumar, Dutta, Prasad, & Misra, 2011). In this study also,a good correlation has been indicated between the flavonoid con-tent and the antioxidant power of SLE and PRF.

3.3. Antibacterial property

The results of antibacterial property of SLE and PRF in terms ofzone of inhibition (mm) are presented in Table 2. Antibacterialactivity of SLE and PRF was compared with ampicillin as a standardantibiotic. Both had shown marked antibacterial activity againstGram positive and Gram negative bacteria which included E. coli,S. typhi, S. dysenteriae, S.pneumoniae and S. aureus. The SLE hadmaximum zone of inhibition (15.23 ± 0.84 mm) against S. dysente-riae and minimum zone of inhibition (8.33 ± 1.12 mm) for S. pneu-moniae whereas, PRF had maximum zone of inhibition

riae S. pneumoniae S. aureus

PRF SLE PRF SLE PRF

8.33 ± 0.34* – 10.87 ± 0.58* – 9.35 ± 0.45*

3* 13.4 ± 0.82 8.33 ± 1.12* 12.67 ± 0.14 8.63 ± 0.35* 10.67 ± 1.26.26 17.0 ± 1.63 11.12 ± 0.66 15.66 ± 1.70 10.67 ± 0.58 16.76 ± 2.05.03 19.35 ± 0.94 13.36 ± 0.48 18.56 ± 0.88 11.76 ± 0.83b 18.66 ± 1.27b

.84 20.67 ± 1.54 13.84 ± 0.46a 19.0 ± 2.16a 13.64 ± 0.74 18.84 ± 0.78

.56 22.0 ± 1.16 21.33 ± 1.24

(a) SLE and PRF values are significantly different from each other as tested against S.against S. aureus).

3450 M.S. Yogendra Kumar et al. / Food Chemistry 141 (2013) 3443–3450

(20.67 ± 1.54 mm) for S. dysenteriae and minimum zone of inhibi-tion (8.0 ± 0.47 mm) for S. typhi.

Overall, PRF of SBT was found to be more active than the SLEagainst all bacterial species tested. Further, PRF was found effectiveat lower concentration (100 lg) than the SLE. Upadhyay et al.,(2010) studied the antibacterial properties of aqueous and hydro-alcoholic leaf extracts of Seabuckthorn and found efficacy of hydro-alcoholic leaf extract at higher concentration (>125 lg/ml). Thus,our study indicated that PRF has an edge over other extracts. Phe-nol constituents of the plant extracts have shown potent antimi-crobial properties (Klanckin, Guzy, Kolar, Abramovic, & Mozina,2009). The observed antibacterial activity could be attributed tothe phenol constituents of the SBT leaf extracts, especially flavo-noids. Further, antibacterial activity observed in the present studyjustified the traditional uses of SBT for wound healing, skin disor-ders and other infectious conditions.

4. Conclusion

The data of the present study suggests that PRF of Seabuckthornleaves has the potent antioxidant and antimicrobial activities.Eight individual phenolic compounds in the PRF sample identifiedby RP-HPLC may be responsible for antioxidant and antimicrobialactivities. Obtained results show that the PRF can be used as sourceof natural antioxidants and as a possible food supplement. Furtherstudies of PRF on antioxidant status in animal models are neededto evaluate their potential benefits.

Acknowledgement

Authors are thankful to Director, DIPAS, Delhi, Dr. G. Ilavazha-gan, DLS, New Delhi, Dr. K. Udaya Sankar, CFTRI, Mysore and Dr.T.M. Kotresh, DEBEL, Bangalore for their constant support andencouragement.

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