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Research highlights

FOCH 10667 No. of Pages 1, Model 5G

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" Subcritical water extraction of antioxidant compounds. " Evaluation of antioxidant activity in chemical and biological systems. " A simpleRP-HPLC method for the quantitative analysis of antioxidant compounds.

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Food Chemistry xxx (2011) xxx–xxx

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FOCH 10667 No. of Pages 9, Model 5G

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Analytical Methods

Subcritical water extraction of antioxid(Hippophae rhamnoides) leaves for the cactivity

M.S. Yogendra Kumar ⇑, Ruma Dutta, Dipti Prasad,Defence Institute of Physiology and Allied Sciences, Defence Research and Developme

a r t i c l e i n f o

Article history:Received 21 July 2010Received in revised form 2 January 2011Accepted 22 January 2011Available online xxxx

Keywords:SeabuckthornAntioxidant activitySubcritical water extractionRP-HPLC

a b s t r a c t

A novel environmentallextraction of antioxidanwas evaluated using comand antioxidant properstress in murine macropicity, reactive oxygen swhich is responsible foinhibited cytotoxicity, R

The chemical composGAE) and total flavonoidQuercetin-3-galactoside

1. Introduction

In recent years, there has been a wide interest in finding naturacompounds that could replace synthetic antioxidants commonlused in foods such as butylated hydroxytoluene (BHT) and butyated hydroxyanisole (BHA), because of its possible toxicity andue to a suspected action as promoters of carcinogenesis (Rodrguez-Meizoso et al., 2006). In this context, some studies have re

Contents lists av

Food

journal homepage: www

ported the use of herbs and spices, commonly employed as foodelerde-egs,t--–d

69s,70i,71)72

73n74-75e76a77e78

79e,80-81n82a83s84n85s86is87e

ingredients to flavour different types of food preparations, sincthey contain a wide variety of compounds that can have beneficiahealth effects. Also, several studies have revealed that plants havpotent antioxidants in the form of vitamins, flavonoids, and othephenolic compounds that act as scavengers of free radicals aninhibitors of lipid peroxidation (Upendra et al., 2008). Among thvarious plants reported for antioxidant activity, seabuckthorn (Hippophae rhamnoides L., Elaeagnaceae) has gained much importancas a versatile nutraceutical crop with diverse uses, from controllinsoil erosion to being a source of horse fodder, nutritious fooddrugs, and skin-care products (Fan, Ding, & Gu, 2007). This planis a native of Eurasia and has been domesticated in several countries (India, China, Nepal, Pakistan, Myanmar, Russia, Britain, Germany, Finland, Romania, France, etc.) at an altitude of 25004300 m (Negi et al., 2005). Different parts of this plant are use

0308-8146/$ - see front matter � 2011 Elsevier Ltd. All rights reserved.doi:10.1016/j.foodchem.2011.01.088

⇑ Corresponding author. Tel.: +91 986 889 4752; fax: +91 011 23914790.E-mail address: msyogendra@gmail.com (M.S.Y. Kumar).

1

Please cite this article in press as: Kumar, M. S. Y., et al. Subcritical water extleaves for the comparative evaluation of antioxidant activity. Food Chemistr

t compounds from Seabuckthornparative evaluation of antioxidant

shipra Misrarganisation, Lucknow Road, Timarpur, Delhi 110 054, India

iendly technique, subcritical water extraction (SWE) was employed for thmpounds from Seabuckthorn leaves (SBT). Antioxidant activity of the extraconly accepted chemical assays. Also, present study reports the cytoprotectivof SBT against tertiary-butyl hydroperoxide (tert-BOOH) induced oxidatives (Raw 264.7). Exposure of cells to tert-BOOH resulted, increase in cytotoxies (ROS) production and decrease in mitochondrial membrane potentiall in intracellular antioxidant levels. Pretreatment of cells with SBT extracproduction and maintained antioxidants levels similar to that of control cell

of the SWE extracts studied showed total phenol content (76.07–93.72 mg/ntent (47.06–66.03 mg/g rutin). Further, some of its phenolic constituents; (1) Kaempferol and (3) Isorhamnetin were quantified by RP-HPLC.

� 2011 Elsevier Ltd. All rights reserve

in traditional medicine for the treatment of diseases, such as flucardiovascular diseases, mucosal injuries, and skin disorders (Beveridge, Li, Oomah, & Smith, 1999; Upendra et al., 2008). Varioustudies of alcoholic and hydroalcoholic extracts of fruits, seedand leaves of seabuckthorn have confirmed its medicinal annutritional value (Geetha, Ram, Singh, Ilavazhagan, & Sawhne2002; Upendra et al., 2008). All parts of this wonder plant are considered to be a good source of a large number of bioactive compounds, including carotenoids, tocopherols, sterols, flavonoidlipids, vitamins, tannins, minerals, etc. (Hakkinen, KarenlampHeinonen, Mykkanen, & Torronen, 1999; Upendra et al., 2008which contribute to its wide usage as a natural antioxidant.

Extraction of antioxidants from plant tissues has usually beeaccomplished by conventional extraction processes such as solid–liquid extraction employing methanol, ethanol and acetonand also through steam distillation. Nowadays, there has beenhuge upsurge for developing rapid, reliable, and reproduciblmethods for the efficient extraction of bioactive compounds fromplants to increase their therapeutic functionality. In the literaturdifferent extraction techniques, such as maceration, Soxhlet, ultrasound-assisted extraction (UAE), microwave-assisted extractio(MAE) and subcritical extraction (SCE), are reported (Upendret al., 2008; Rodriguez-Meizoso et al., 2006). Recently, there habeen an increasing interest in the use of environmentally cleatechnologies able to provide high quality and high activity extractwhile precluding any toxicity associated to the solvents. In thsense, both, supercritical fluid extraction (SFE) with carbon dioxid

able at ScienceDirect

emistry

sevier .com/locate / foodchem

raction of antioxidant compounds from Seabuckthorn (Hippophae rhamnoides)y (2011), doi:10.1016/j.foodchem.2011.01.088

88 (CO2) as a solvent and sub critical water extraction (SWE) meet the89 requirements to be considered clean and safe processes (King,90 2000; Rodriguez-Meizoso et al., 2006).91 SWE, i.e., extraction using hot water under pressure sufficient to92 maintain water in the liquid state, has demonstrated its ability to93 selectively extract different classes of compounds depending on94 the temperature used, with the more polar extracted at lower tem-95 peratures while the less polar compounds were extracted at higher96 temperatures. The selectivity of SWE allows for manipulation of97 the composition of the extracts by changing the operating param-98 eters and it has been used for essential oil isolation (Ammann et al.,99 1999; Basile, Jimenez Carmona, & Clifford, 1998) as well as for anti-

100 oxidant extraction (Ibanez, Kubatova, Senorans, Cavero, & Reglero,101 2003; Rodriguez-Meizoso et al., 2006). The goal of the present102 investigation was to study the selectivity of SWE at several tem-103 peratures to extract antioxidant compounds from Seabuckthorn104 leaves (Hippophae Rhamnoides). SWE proposed in this work as a105 feasible process to concentrate and isolate antioxidant compounds106 to be used as nutraceuticals. In recent years, many papers have107 been published on the applicability of SWE for the extraction of108 bioactive compounds from plants. Nevertheless, to the best of109 our knowledge, there is no report available that could illustrate110 the feasibility of SWE as a rapid and efficient extraction tool for111 the determination of antioxidant activity of SBT leaves.112 Since the various bioactive properties of SBT, including antiox-113 idant, are attributed to the presence of phenolic compounds in it;114 hence, evidently, the other objective of this work was to investi-115 gate the feasibility of SWE for the rapid and efficient extraction116 of bioactive phenolics from the plant and comparing it to other117 extraction techniques (maceration and Soxhlet). Keeping this in118 mind, the antioxidant activity of SBT leaves extracts was evaluated119 in chemical and biological systems. Because, the antioxidant capac-120 ity of drugs can be evaluated using chemical methods, which are121 easy to execute and have high reproducibility. Nevertheless, such122 methods do not represent what happens in vivo (Soares, Andreaz-123 za124 ve125 se126 te127 (S128 ue129 Fig130 fo131 ex

132 2.

133 2.1

134

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139 2.2

140

141 US142 vic

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144

145 ac

1462,5,7,8-Tetramethylchroman-2-Carboxylic acid [Trolox], Rutin147(Sigma Aldrich Chemicals, USA), Folin–Ciocalteu reagent & Ascor-148bic Acid [Vitamin-C] (Sisco Research Laboratories, India).

1492.4. In vitro cell culture model

150Raw 264.7, a murine macrophage cell line was obtained from151National Centre of Cell Sciences (NCCS) Pune, India. Cells were152propagated in DMEM supplemented with 10% FBS, 100 lg/mL153ampicillin and 100 lg/mL streptomycin. They were maintained at15437 �C in a humidified CO2 incubator. The cells were grown to a den-155sity of 1 � 104 cells/well in 96 well plates (Greiner, Germany) for156determination of cytotoxicity, mitochondrial membrane potential157and ROS levels. The cells were grown in 24 well plates (Falcon158make) to a density of about 1 � 105/well for determination of159anti-oxidants levels.

1602.5. Extraction Procedure

1612.5.1. Maceration162100 g of powdered Seabuckthorn leaf sample was soaked in163500 mL of distilled water at room temperature. After 24 h, super-164natant was decanted, filtered through muslin cloth and stored in165amber coloured bottle. The solution was centrifuged at 8000 rpm166for 10 min. Finally, the supernatant solution was lyophilised and167the dried extract was stored at 5 �C for the further studies.

1682.5.2. Soxhlet extraction169100 g of powdered Seabuckthorn leaf sample was extracted170with 350 mL of 70% ethanol for 6–10 h in a Soxhlet apparatus.171The extract was filtered and Alcoholic content in extract was evap-172orated using Rota vapour at 40 �C. Finally, the supernatant solution173was lyophilised and the dried extract was stored at 5 �C for the fur-174ther studies.

1752.5176

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2 M.S.Y. Kumar et al. / Food Chemistry xxx (2011) xxx–xxx

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, & Salvador, 2003). Assays using living cells have proven to bery useful in the routine testing of various products, being fast,nsitive, reproducible, as well as producing reliable results inrms of the identification of biological and antioxidant activityoares et al., 2003). Simultaneously, some of its phenolic constit-nts (quercetin-3-O-galactoside, kaempferol, and isorhamnetin;. 1A) were determined with the help of reverse-phase high-per-

rmance liquid chromatography (RP-HPLC) to demonstrate thetraction efficiency and hence, antioxidant activity.

Materials and methods

. Plant material

Seabuckthorn leaves were collected from hilly region of westernmalyas, India in the month of September, in which the plantows widely under natural condition. Voucher specimen is pre-rved in Defence Institute of High Altitude Research, Leh afterhanobotanical identification of species.

. Apparatus

HPLC Waters, ASE 350 Dionex Corporation (Sunnyvale, CA,A), Spectrophotometer Bio-Rad. ELISA reader (Molecular De-es, USA), Spectrofluorimeter (Varian, USA).

. Reagents

1,1’-diphenyl-2-picrylhydrazl [DPPH], 3,4,5-trihydroxybenzoicid [Gallic Acid], TPTZ [2,4,6-tripyridy-s-triazine], 6 Hydroxy-

ease cite this article in press as: Kumar, M. S. Y., et al. Subcritical water extractionaves for the comparative evaluation of antioxidant activity. Food Chemistry (201

.3. Subcritical water extractionTo perform the extractions, an Accelerated Solvent Extraction

stem ASE 350 equipped with a solvent controller unit from Dio-x Corporation (Sunnyvale, CA, USA) was used. Extractions wererried out in triplicate using water. Individual extractions wererformed using the sample considering the following tempera-res: 25, 50, 100, 150 and 200 �C for 15 min extraction time atch temperature. Previous to each experiment an extraction cellat-up was carried out for a given time, which changed accordingextraction temperature (the heat-up time is automatically fixedthe equipment). Namely 5 min heat-up was used when extrac-n temperature was set at 50 �C and 100 �C, 7 min at 150 �C and

in at 200 �C. Likewise, all extractions were performed in 33 mLtraction cells, containing 2 g of sample.The extraction procedure was as follows: (i) sample is loaded

to cell, (ii) cell is filled with solvent up to a pressure of00 psi, (iii) initial heat-up time is applied, (iv) static extractionth all system valves closed is performed for 15 min, (v) cell issed (with 60% cell volume using extraction solvent), (vi) solventpurged from cell with N2 gas and (vii) depressurisation takes

ace. Between extractions, a rinse of the complete system wasade in order to overcome any extract carry-over. For solventaporation a freeze dryer (Allied frost, India) was employed. Thellected extracts were kept protected from light, at 4 �C until use.

. Determination of total phenol content (Thaipong, Boonprakob,osby, Zevallos, & Byrne, 2006)

Total phenol content of extracts was determined by the Folin–ocalteu method. 150 lL of extract, 2400 lL of nanopure waterd 150 lL of 0.25 N Folin–Ciocalteu reagent were combined and

of antioxidant compounds from Seabuckthorn (Hippophae rhamnoides)1), doi:10.1016/j.foodchem.2011.01.088

204 n205 e206 .207 -208 d209

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PLC

M.S.Y. Kumar et al. / Food Chemistry xxx (2011) xxx–xxx 3

FOCH 10667 No. of Pages 9, Model 5G

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then mixed well. The mixture was allowed to react for 3 min the300 lL of 1 N Na2CO3 solution was added and mixed well. Thsolution was incubated at room temperature in the dark for 2 hThe absorbance was measured at 725 nm using a spectrophotometer and the results were expressed in milligram of gallic aciequivalents (GAE) per 1 gram of extract.

2.7. Determination of Totalflavonoid content (Yanping Zou, Yanhua,Wei, 2004)

1.0 mL aliquot of appropriately diluted sample solution wamixed with 2 mL of distilled water and subsequently wit0.15 mL of a 5% NaNO2 solution. After 6 min, 0.15 mL of a 10% AlCsolution was added and allowed to stand for 6 min, then 2 mL of 4NaOH solution was added to the mixture. Then the mixture wathoroughly mixed and allowed to stand for another 15 min. Absorbance of the mixture was determined at 510 nm versus blank. Rutin was used as standard compound for the quantification of tota

Fig. 1. Structure of the quantified phenolic compounds (A) and H

flavonoid. All values were expressed as milligram of rutin equiva-lents per gram of dry raw material.

2.8. HPLC analysis

The HPLC system consisted of a Waters HPLC system (WatersCorporation, USA) equipped with Waters 515 HPLC pump, Waters717 plus autosampler and Waters 2487 UV detector, interfacedwith an IBM Pentium 4 personal computer. The separation wasperformed on a Symmetry C18 250 � 4.6 mm ID; 5 lm column(Waters, USA) by maintaining the gradient flow rate 0.75 mL/minof the mobile phase (Solution A; Water:O-Phosphoric acid

Please cite this article in press as: Kumar, M. S. Y., et al. Subcritical water extleaves for the comparative evaluation of antioxidant activity. Food Chemistr

99.7:0.3 and Solution B; Acetonitrile:Methanol 75:25) and peakwere detected at 370 nm. Identification of compounds was performed on the basis of the retention time, coinjections, and spectral matching with standards. For the preparation of thcalibration curve, standard stock solutions of compounds 1–(1 mg/2 mL) were prepared in methanol, filtered throug0.22 lm filters (Millipore), and appropriately diluted (0.01100 lg/mL) to obtain the desired concentrations in the quantification range. The calibration graphs were plotted after linear regression of the peak areas versus concentrations.

2.9. Determination of antioxidant activity in chemical system

2.9.1. DPPH assay (Thaipong et al., 2006)The stock solution was prepared by dissolving 24 mg DPPH wit

100 mL methanol and then stored at �20 �C until needed. Thworking solution was obtained by mixing 10 mL stock solutiowith 45 mL methanol to obtain an absorbance of 1.10 ± 0.02 unit

chromatogram of subcritical water extract of Seabuckthorn leaves (B).

246at 515 nm using the spectrophotometer. 150 lL of root extract247solution was allowed to react with 2850 lL of the DPPH solution248for 2 h in the dark. Then the absorbance was taken at 515 nm.249The standard curve was linear between 25 and 200 ppm Trolox. Re-250sults are expressed in mg TE/g of dry raw material.

2512.9.2. FRAP assay (Thaipong et al., 2006)252The stock solutions included 300 mM acetate buffer (3.1 g253C2H3NaO2�3H2O and 16 mL C2H4O2), pH 3.6, 10 mM TPTZ (2,4,6-tri-254pyridyl-s-triazine) solution in 40 mM HCl, and 20 mM FeCl3�6H2O255solution. The fresh working solution was prepared by mixing25625 mL acetate buffer, 2.5 mL TPTZ solution, and 2.5 mL FeCl3�6H2O

raction of antioxidant compounds from Seabuckthorn (Hippophae rhamnoides)y (2011), doi:10.1016/j.foodchem.2011.01.088

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4 M.S.Y. Kumar et al. / Food Chemistry xxx (2011) xxx–xxx

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lution and then warmed at 37 �C before using. 150 lL of leavestract solution was allowed to react with 2850 lL of the FRAPlution for 30 min in the dark condition. Readings of the colouredoduct [ferrous tripyridyltriazine complex] were then taken at3 nm. The standard curve was linear between 25 and 150 ppmolox. Results are expressed in mg TE/g of dry raw material.

.3. Determination of total reducing power (Zou et al., 2004)1.0 mL of leaves extract solution (0.2–1.0 mg/mL) was mixed

th 2.5 mL of a 0.2 M phosphate buffer (pH 6.6) and 2.5 mL of a(w/v) solution of potassium ferricyanide. The mixture was incu-

ted in a water bath at 50 �C for 20 min. afterward, 2.5 mL of a% (w/v) trichloroacetic acid solution was added and the mixtures then centrifuged at 3000 rpm for 10 min. A 2.5 mL aliquot of

e upper layer was combined with 2.5 mL of distilled water andmL of a 0.1% (w/v) solution of ferric chloride, and the absor-

nce was measured spectrophotometrically at 700 nm. Vitaminis used for the standard graph.

0. Determination of cytotoxicity and antioxidant activity in celllture model

0.1. Determination of cytotoxicityThe optimal incubation time and concentration of tert-BOOH

quired to produce cytotoxicity on Raw cells were determined.determine the efficacy of various antioxidants the cells were

pplemented with antioxidants before tert-BOOH addition. Theest concentration of antioxidant that provided the maximum

otection against tert-BOOH induced cytotoxicity was used ine present study. Raw cells were incubated with 100 lM tert-OH for 1 h in the presence and absence of different antioxidant

r determination of cytotoxicity and ROS. For measuring GSH lev-the cells were exposed to tert-BOOH for 2 h.Cytotoxicity was studied by using neutral red uptake (Sairamal., 2000), a supra-vital dye, that is selectively taken by the livells. Briefly, 10 ll of neutral red dye (0.1%) was added to the cellsd incubated at 37 �C in CO2 incubator for 45 min. Later the cellsre washed with PBS followed by the addition of 200 ll ethanol-

etic acid (50:1) solution. The OD was measured at 570 nm usingsa reader (Molecular Devices).

0.2. Measurement of reactive oxygen species (ROS)It was measured (Catchart, Schwiers, & Ames, 1983) using fluo-

scent probe 2,7 dichlorofluorescein (DCFHDA). Briefly after incu-tion, 10 ll of DCFHDA stock solution (200 lM in DMSO) wasded to 190 ll of medium in 96 well plate to get final concentra-n of 10 lM. The cells were incubated at 37 �C for 30 min in CO2

cubator. The cells were washed thrice with PBS and fluorescences measured by multiwell spectrofluorimeter (Molecular De-es) with excitation at 485 nm and emission at 530 nm. Alter-tely radical scavenging activity of the SBT extracts weretermined in cultured cells using fluorescent probe DCFHDA.iefly, after incubation, 10 ll of DCFHDA stock solution (200 lM

le 1raction of Sea buckthorn leaves, determination of total phenol and flavonoid content.

Method of extraction Extraction temperature (�C) % of Yield Total phenol (mg/g

Maceration 25 ± 5 15.41 28.35 ± 1.31Soxhlet 80 31.65 77.85 ± 2.36

30 24.00 43.77 ± 1.7250 27.25 60.22 ± 1.93

Subcritical water 100 30.50 76.07 ± 2.41150 46.45 93.72 ± 2.83200 49.20 86.70 ± 2.53

ata expressed as mean ± standard deviation (SD) of three replicates.

ease cite this article in press as: Kumar, M. S. Y., et al. Subcritical water extractionaves for the comparative evaluation of antioxidant activity. Food Chemistry (201

DMSO) was added to cells (1 � 106 cells). The cells were incu-ted at 37 �C for 30 min in CO2 incubator. The cells were washedice with PBS. The ROS production was monitored by Flowcytom-

er equipped with cell quest software (Beckton Dickinton, USA).

0.3. Determination of mitochondrial transmembrane potentialitochondrial integrity)Mitochondrial membrane potential (MMP) was determined

ing fluorescent probe Rhodamine123 (Jiang & Acosta, 1993).ter the cells were exposed to tert-BOOH, 10 ll Rhodamine 1230 lg/mL) was added to cells and incubated for 30 min. The cellsre washed three times with PBS and fluorescence was measured

ing spectrofluorimeter (Spectra Max M2 Molecular Devices) withexcitation of 485 nm and emission at 531 nm.

0.4. Determination of reduced glutathione levelsAfter incubation, the cells were lysed by adding 200 ll of lysisffer (100 lM Tris, 20 mM EDTA, 0.25% Triton X-100 pH 8.0).e reduced and oxidised glutathione (GSSG) levels in the cellsre determined fluorimetrically (Hissin & Hilf, 1976).

1. Statistical analysis

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

Results and discussion

. Subcritical water extraction

Sub critical water extraction (SWE), i.e., extraction using hotter under pressure, has recently emerged as a useful tool to re-

ace the traditional extraction methods. SWE is an environmen-lly-clean technique that, in addition, provides higher extractionlds to extract solid samples (Rodrıguez-Meizoso et al., 2006).SWE is carried out using hot water (from 100 �C until 374 �C,

e latter being the water critical temperature) under high pres-re (usually up to 100 bar) enough to maintain water in the liquidte. Table 1 provides the extraction yield of SBT leaves in differ-t extraction conditions. The most important factor to take intocount in this type of extraction procedures is the dielectric con-nt (e). This parameter can be modulated easily, within a wide

nge of values, by only tuning the extraction temperature. Waterroom temperature is a very polar solvent, with a dielectric con-nt close to 80. However, this level can be significantly decreasedvalues close to 27 when water is heated up to 250 �C while

aintaining its liquid state applying pressure. This dielectric con-nt value is similar to that of ethanol being therefore appropriatesolubilise less-polar compounds (Miller & Hawthorne, 2000;

driguez-Meizoso et al., 2006).

) ± SD*of dry raw material Total flavonoid (mg/g) ± SD*of dry raw material

14.14 ± 1.1242.93 ± 1.9329.09 ± 1.6339.81 ± 1.7947.06 ± 2.1566.03 ± 2.4166.40 ± 2.36

of antioxidant compounds from Seabuckthorn (Hippophae rhamnoides)1), doi:10.1016/j.foodchem.2011.01.088

352 In this study, the effect of sub critical water extraction method,353 for the efficient extraction of antioxidative compounds from Sea-354 buckthorn leaf was investigated. Until now, to the best of our355 knowledge, there is no such report available that could highlight356 the feasibility of sub critical water extraction procedure as an effi-357 cient method for the extraction of antioxidant compounds from358 Seabuckthorn leaf. For comparison, Seabuckthorn leaf was also ex-359 tracted by maceration and Soxhlet extraction methods. During360 extraction, it was seen that maximum extraction yield (49.20%)361 was achieved with sub critical water extraction followed by Soxh-362 let (31.65%), and maceration (15.41%). However, taking into con-363 sideration the solvent consumption and time needed for364 extraction, SWE was found to be the best feasible approach for365 the rapid and efficient extraction of antioxidant compounds.

366 3.2. Total phenol and flavonoid contents

367 As a part of chemical composition analysis, total flavonoid and368 -369 e370 -371 y372 -373 is374 h375 s,376 s377 d378 -379

380

381

382

383

384

385

386

387

388

389

390

391

392

393

394

395

396

397

398

399

400

401d402-403-404y405-406n407-408t409g410

411

412s413e414-415a416t417o,418

419.420d421f422n423t424r425-426s427

428-

M.S.Y. Kumar et al. / Food Chemistry xxx (2011) xxx–xxx 5

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total phenol content of Sea buckthorn leaf extracts were determined by colorimetric method. The results as given in the Tabl1 indicate the presence of higher total phenolic and flavonoid content in the sub critical water extract obtained at 150 �C followed bthe extracts obtained by Soxhlet and maceration methods. Phenolics are the major plant compounds with antioxidant activity. Thactivity is believed to be mainly due to redox properties, whicplay important role in adsorbing and neutralising free radicalquenching singlet and triplet oxygen, or decomposing peroxide(Costantino, Albasini, Rastelli, & Benvenuti, 1992). Results obtainein the present study revealed that the level of these phenolic com

pounds in the SBT leaf extracts were considerable. The resultsstrongly suggest that phenolics are important components of thisplant, and some of its pharmacological effects could be attributedto the presence of these valuable constituents.

Flavonoids form a class of benzo-c-pyrone derivatives includeflavones, flavanes, flavonols, anthocyanidines, and catechins. Theypossess a wide spectrum of biological activities such as anticancer,antibacterial, antifungal, antiviral, spasmolytic, hypoglycaemic,antihistaminic and radioprotective potential (Amella et al., 1985;Cazarolli et al., 2008; Ertan, Göker, Ertan, & Pindur, 1989; Jagtapet al., 2009; Londhe, Devasagayam, Foo, & Ghaskadbi, 2009; Perry& Foster, 1994). Some of these properties derive from the free rad-ical-scavenging activities of flavonoids. Therefore there are manyreports relating to the reactivities of flavonoids with active oxygenspecies. Recent interest in these substances has been stimulated bythe potential health benefits arising from their antioxidant activity.

3.3. Antioxidant activity evaluation of SBT extracts in chemical system

3.3.1. DPPH assayThe free radical scavenging activity of SBT was studied by its

ability to bleach the stable radical DPPH. This assay provides infor-mation on the reactivity of compounds with a stable free radical.Because of the odd electron, DPPH shows a strong absorption band

Table 2Antioxidant activity evaluation in chemical system.

Method of extraction Extraction temperature (�C) DPPH mg Trolox eqt

Maceration 25 ± 5 86.35 ± 2.93Soxhlet 80 255.87 ± 5.51

30 133.31 ± 5.7250 164.03 ± 6.28

Subcritical water 100 194.76 ± 6.71150 353.43 ± 8.75200 343.86 ± 7.71

* Data expressed as mean ± standard deviation (SD) of three replicates.

Please cite this article in press as: Kumar, M. S. Y., et al. Subcritical water extleaves for the comparative evaluation of antioxidant activity. Food Chemistr

at 517 nm in visible spectroscopy. As the electron becomes paireoff in the presence of free radical scavenger, the absorption vanishes, and the resulting decolourisation is stoichiometric with respect to the number of electrons taken up. From the DPPH assaresults it may be postulated that SBT reduces the radical to the corresponding hydrazine on reacting with the hydrogen donors iSBT. The bleaching of DPPH represents the capacity of SBT to scavenge free radicals independent of enzymatic activity. The preseninvestigation shows that SBT is sufficiently effective in scavenginDPPH radicals (Badami, Gupta, & Suresh, 2003).

3.3.2. FRAP assayThe antioxidant potentials of the aqueous and alcoholic extract

of the leaves were estimated from their ability to reduce TPTZ-F(III) complex to TPTZ-Fe (II). Antioxidant activity increased proportionally with the phenol content. According to recent reports,highly positive relationship between total phenols and antioxidanactivity appears to be the trend in many plant species (AdedapJimosh, Koduru, Afolayan, & Masika, 2008).

The results of both FRAP and DPPH� assay are listed in Table 2The trolox equivalents antioxidant capacity (TEAC) values obtainefor the extracts submitted to the FRAP assay were in the range o2.03–182.13 mg/g, while the values for the DPPH� assay were ithe range 6.97–282.75 mg/g. In addition, the higher antioxidanactivity exhibited by the sub critical water extracts (Table 2) ovethe other Soxhlet and maceration extraction methods clearly demonstrates the relative advantage of SWE for obtaining formulationwith high antioxidant compounds.

It will be relevant to mention here that earlier papers (Costan

429tino et al., 1992; Awika, Rooney, Wu, Prior, & Cisneros-Zevallos,4302003) have demonstrated the correlation between the phenolic431content of plants to their antioxidant power. In this study also, a432good correlation was indicated between the phenolic content and433the antioxidant power of extracts. For the measurement of total434phenolic content, the absorbance of Seabuckthorn leaf extracts435was determined spectrometrically according to the Folin–Ciocalteu436method and calculated as GAE. It is clear from the Table 1 that the437maximum total phenolic content was achieved with sub critical438water extraction followed by Soxhlet and maceration, which is evi-439dently in accordance with the observed antioxidant activity.

4403.3.3. Reducing power441The reducing power of SBT extracts, which may serve as a sig-442nificant reflection of the antioxidant activity, was determined443using a modified iron (III) to iron (II) reduction assay (Awika444et al., 2003). In this assay, the yellow colour of the test solution445changes to various shades of green and blue depending on the446reducing power of extracts or compounds. The presence of reduc-447tants in the solution causes the reduction of the Fe3+/Ferricyanide448complex to the ferrous form. Therefore, the Fe2+ can be monitored449by measurement of the formation of Perl’s Prussian blue at 700 nm.450Fig. 2 shows the reducing power of SBT extracts and compared451to ascorbic acid. All the extracts have showed some degree of

./g ± SD* of dry raw material FRAP mg Trolox eqt./g ± SD* of dry raw material

93.91 ± 3.72217.77 ± 4.29144.99 ± 4.53179.62 ± 5.72219.03 ± 6.28276.93 ± 7.71261.54 ± 6.12

raction of antioxidant compounds from Seabuckthorn (Hippophae rhamnoides)y (2011), doi:10.1016/j.foodchem.2011.01.088

452 re453 wa454 in455 in456 am457 let458 3.7459 re460 am461 ea462 ph

463also, a good correlation has been indicated between the phenolic464content and the antioxidant power of extracts (Costantino et al.,4651992). The reducing power of ascorbic acid, and SBT extracts fol-466lowed the following order: ascorbic acid > SWE 150 > Soxhlet >467Maceration.

4683.4. Antioxidant activity evaluation of SBT extracts in biological system

4693.4.1. Reactive oxygen species470The free radical quenching activity of SBT was evaluated in cul-471tured cells against tert-BOOH induced oxidative stress as measured472by fluorescent probe 2,7 dichlorofluorescein diacetate. There was473significant increase by about 2.1 times in cells exposed to tert-BOOH474re475(Fi476cia477su478fre

4793.4480

481ica4823.5483ce484tra

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0.1

0.2

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6 M.S.Y. Kumar et al. / Food Chemistry xxx (2011) xxx–xxx

FOCH 10667 No. of Pages 9, Model 5G

8 February 2011

Plle

ducing power; however, as anticipated, their reducing powers inferior to ascorbic acid, which is known to be a strong reduc-

g agent. Reducing power of the extracts increased with increas-g amount of extract. The equation of reducing power (y) and

ount of extract (x) for the extract prepared by maceration, Soxh-and SWE methods were y = 3.3266x + 0.007 (r2 = 0.9965), y =76x � 0.015 (r2 = 0.9993) and y = 3.958x � 0.0125 (r2 = 0.9987)

spectively. Indicating that reducing ability correlated well withount of the extracts. It will be relevant to mention here that

rlier reports have demonstrated the correlation between theenolic content of plants to their antioxidant power. In this study

00 0.05 0.1 0.15 0.2 0.25

Concentration mg/ml

Fig. 2. Reducing powers of SBT extracts and ascorbic acid.

0.000

20.000

40.000

60.000

80.000

100.000

120.000

140.000

160.000

180.000

200.000

Control tert-BOOH Soxhlet Subcritical Maceration Vit C

Fluo

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(au)

*#

* P < 0.01 # P < 0.05

0

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100

150

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Control tert-BOOH Soxhlet Subcritical Maceration Vit C0

0.5

1

1.5

2

2.5

3

3.5

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GSH GSSG GSH/GSSG

A

C

. 3. Determination of reactive oxygen species (A), Determination of mitochondrialotoxicity (D).

ease cite this article in press as: Kumar, M. S. Y., et al. Subcritical water extractionaves for the comparative evaluation of antioxidant activity. Food Chemistry (201

lative to control cells indicating the generation of intracellular ROSg. 3A). Pretreatment of cells with SBT extract (25 lg/mL) appre-bly inhibited the ROS generation induced by tert-BOOH. Howeverbcritical water extract was found to possess significantly highere radical quenching activity followed by Soxhlet and maceration.

.2. Mitochondrial transmembrane potentialAddition of tert-BOOH (100 lM) to Raw cells resulted in signif-nt decrease in mitochondrial transmembrane potential by abouttimes in cells exposed to tert-BOOH (Fig. 3B). Pretreatment of

lls exposed to SBT extract (25 lg/mL) restored the mitochondrialnsmembrane potential similar to that of normal cells.

.3. Antioxidant statusThe GSH level was determined both during 1 and 2 h of tert-OH treatment to understand the dynamics of GSH metabolism

0

50

100

150

200

250

Control tert-BOOH Soxhlet Subcritical Maceration Vit C

* P < 0.01 # P < 0.05

#*

0

20

40

60

80

100

120

Control tert-BOOH Soxhlet Subcritical Maceration Vit C

* P < 0.01 # P < 0.05

# *

brane potential (B) and determination of glutathione levels (C), evaluation of

of antioxidant compounds from Seabuckthorn (Hippophae rhamnoides)1), doi:10.1016/j.foodchem.2011.01.088

488 s489 ls490 -491 d492 d493 n494 d495 -496 s.497 -498 l499 n500

501

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M.S.Y. Kumar et al. / Food Chemistry xxx (2011) xxx–xxx 7

FOCH 10667 No. of Pages 9, Model 5G

8 February 2011

in the Raw cell line. Since no significant change in GSH level waobserved at 1 h, the data at 1hr was not shown. Exposure of celto tert-BOOH for 2 h resulted in considerable fall in intracellular reduced glutathione (GSH) by about 23% and an increase in oxidiseglutathione (GSSG) by about 2.9-fold in Raw cell line as compareto control cells (Fig. 3C). Further there was a considerable fall iGSH/GSSG ratio from 3.6 in control cells to 0.96 in cells incubatewith tert-BOOH. Supplementation of SBT extract (25 lg/mL) resulted in significant increase in GSH and decrease in GSSG levelThe GSH/GSSG ratio in SBT treated cells were increased significantly, when compared to control cells. However GSH levedropped to further 40% of the control cells after 3 h incubation ipresence of tert-BOOH.

3.5. Evaluation of Cytotoxicity

Table 3Determination of Quercetin-3-galactoside, kaempferol and isorhamnetin in seabuc

Method of extraction Extractiontemperature (�C)

Quercetin-3-galactoof dry raw material

Maceration 25 ± 5 41.21 ± 2.71Soxhlet 80 58.34 ± 3.72

30 176.26 ± 5.72Subcritical water 50 233.52 ± 6.28

100 263.17 ± 6.71150 423.39 ± 8.75200 169.86 ± 7.71

* Data expressed as mean ± standard deviation (SD) of three replicates.

We assessed the cytotoxicity of tert-BOOH by exposing Raw264.7 murine macrophages to various concentration of tert-BOOH(10–500 lM). The LD50 was observed at 100 lM concentration oftert-BOOH. Various concentration of SBT (10–200 lg) was alsotested to determine the optimum dose and cytotoxicity, if any to-wards Raw cells. We found that upto100 lg/mL of SBT drug had nocytotoxicity. However, a concentration as low as 25 lg/mL waseffective and showed viability > 95% that was maintained throughthe test period. Addition of tert-BOOH (100 lM) to Raw cells re-sulted in significant increase in cytotoxicity (55%) as revealed bythe fall in neutral red uptake as compared to control cells. Pretreat-ment of cells with SBT extract (25 lg/mL) significantly attenuatedRaw cell viability (P < 0.01) compared to cells treated with tert-BOOH alone (Fig. 3D).

3.6. Identification and quantification of marker compounds by RP-HPLC

A simple and gradient elution-based RP-HPLC method wasdeveloped for the analysis of Quercetin-3-galactoside, kaempferoland isorhamnetin (Fig. 1A) in the extracts. For the developmentof an effective mobile phase, various solvent systems, includingdifferent combinations of acetonitrile, methanol and water withortho phosphoric acid were tried. Finally, a solvent system consist-ing of 0.3% ortho phosphoric acid in water and acetonitrile: meth-anol (75:25) was proved successful because it allows for theseparation of maximum compounds with good resolution(Fig. 1B). Quercetin-3-galactoside, Kaempferol and Isorhamnetin,these might contribute to the antioxidant behaviour of the plantwas identified in extracts, as shown in Figure 6. Identification ofcompounds was performed on the basis of the retention time, coinjections, and spectral matching with standard. For quantification,standard stock solutions of Quercetin-3-galactoside, Kaempferoland Isorhamnetin was (1 mg/2 mL) was prepared in ethanol, fil-tered through 0.22 lm filters (Millipore), and appropriately diluted(0.01–100 lg/mL) to obtain the desired concentrations for quanti-

Please cite this article in press as: Kumar, M. S. Y., et al. Subcritical water extleaves for the comparative evaluation of antioxidant activity. Food Chemistr

fication. The results as given in the Table 3 indicate the presence ohigher Quercetin-3-galactoside, Kaempferol and Isorhamnetin content in the sub critical water extract obtained at 150 �C followed bthe extracts obtained by Soxhlet and maceration methods.

4. Conclusion

Subcritical water extraction of SBT leaves was found to be a beter approach than Soxhlet and maceration because the use of subcritical water imparted higher antioxidant and cytoprotectivactivities to the extracts besides ensuring low solvent consumption, ease, and rapidity of the overall method than Soxhlet anmaceration extraction methods. Antioxidant activity of the extracts was evaluated using commonly accepted chemical assay(DPPH & FRAP). Also, present study report the cytoprotective anantioxidant properties of SBT against tertiary-butyl hydroperoxid

rn leaves by HPLC.

(lg/g) ± SD* Kaempferol (lg/g) ± SD*

of dry raw materialIsorhamnetin (lg/g) ± SD*

of dry raw material

23.72 ± 1.32 27.91 ± 2.125.54 ± 1.72 65.69 ± 3.7310.74 ± 1.53 52.92 ± 3.7211.24 ± 2.71 83.31 ± 3.2119.62 ± 1.71 92.92 ± 4.2846.43 ± 2.28 112.65 ± 5.7520.44 ± 2.12 24.41 ± 1.71

550(tert-BOOH) induced oxidative stress in murine macrophages (Raw551264.7). SBT extracts inhibited cytotoxicity, ROS production and552maintained antioxidants levels similar to that of control cells.553Simultaneously, a simple RP-HPLC method was developed for554the identification and quantification of three phenolic compounds555(Quercetin-3-galactoside, kaempferol and isorhamnetin) present in556the extracts of SBT, to demonstrate the increased antioxidant557power. The results are promising and demonstrate the practical558feasibility of subcritical water extraction to substitute the tradi-559tional time-consuming techniques for efficient extraction of anti-560oxidative compounds to provide nutraceutical-rich formulations.

5615. Uncited references

562Andreas et al. (1999); Geetha et al. (2003).

563Acknowledgements

564Authors are thankful to Dr. G. Ilavazhagan, Director, DIPAS,565Delhi for the constant support and encouragement. Dr. K Udaya566Sankar, CFTRI, Mysore and Dr. Ashoke Banerji, Amrita Vishwa567Vidya Peetham, Amritapuri.

568References

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Please cite this article in press as: Kumar, M. S. Y., et al. Subcritical water extraction of antioxidant compounds from Seabuckthorn (Hippophae rhamnoides)leaves for the comparative evaluation of antioxidant activity. Food Chemistry (2011), doi:10.1016/j.foodchem.2011.01.088