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Analytical Methods

Subcritical water extraction of antioxidant compounds from Seabuckthorn

(Hippophae rhamnoides) leaves for the comparative evaluation of antioxidant

activity

M.S. Yogendra Kumar , Ruma Dutta, Dipti Prasad, Kshipra Misra

Defence Institute of Physiology and Allied Sciences, Defence Research and Development Organisation, Lucknow Road, Timarpur, Delhi 110 054, India

a r t i c l e i n f o

Article history:

Received 21 July 2010

Received in revised form 2 January 2011

Accepted 22 January 2011

Available online 2 February 2011

Keywords:

Seabuckthorn

Antioxidant activity

Subcritical water extraction

RP-HPLC

a b s t r a c t

A novel environmentally friendly technique, subcritical water extraction (SWE) was employed for the

extraction of antioxidant compounds from Seabuckthorn leaves (SBT). Antioxidant activity of the extracts

was evaluated using commonly accepted chemical assays. Also, present study reports the cytoprotective

and antioxidant properties of SBT against tertiary-butyl hydroperoxide (tert-BOOH) induced oxidative

stress in murine macrophages (Raw 264.7). Exposure of cells to tert-BOOH resulted, increase in cytotox-

icity, reactive oxygen species (ROS) production and decrease in mitochondrial membrane potential,

which is responsible for fall in intracellular antioxidant levels. Pretreatment of cells with SBT extracts

inhibited cytotoxicity, ROS production and maintained antioxidants levels similar to that of control cells.

The chemical composition of the SWE extracts studied showed total phenol content (76.0793.72 mg/g

GAE) and total flavonoid content (47.0666.03 mg/g rutin). Further, some of its phenolic constituents; (1)

Quercetin-3-galactoside, (2) Kaempferol and (3) Isorhamnetin were quantified by RP-HPLC.

2011 Elsevier Ltd. All rights reserved.

1. Introduction

In recent years, there has been a wide interest in finding natural

compounds that could replace synthetic antioxidants commonly

used in foods such as butylated hydroxytoluene (BHT) and butyl-

ated hydroxyanisole (BHA), because of its possible toxicity and

due to a suspected action as promoters of carcinogenesis

(Rodriguez-Meizoso et al., 2006). In this context, some studies have

reported the use of herbs and spices, commonly employed as food

ingredients to flavour different types of food preparations, since

they contain a wide variety of compounds that can have beneficial

health effects. Also, several studies have revealed that plants have

potent antioxidants in the form of vitamins, flavonoids, and other

phenolic compounds that act as scavengers of free radicals and

inhibitors of lipid peroxidation (Upendra et al., 2008). Among the

various plants reported for antioxidant activity, seabuckthorn (Hip-

pophae rhamnoidesL., Elaeagnaceae) has gained much importance

as a versatile nutraceutical crop with diverse uses, from controlling

soil erosion to being a source of horse fodder, nutritious foods,

drugs, and skin-care products (Fan, Ding, & Gu, 2007). This plant

is a native of Eurasia and has been domesticated in several coun-

tries (India, China, Nepal, Pakistan, Myanmar, Russia, Britain, Ger-

many, Finland, Romania, France, etc.) at an altitude of

25004300 m. Different parts of this plant are used in traditional

medicine for the treatment of diseases, such as flu, cardiovascular

diseases, mucosal injuries, and skin disorders (Beveridge, Li, Oo-

mah, & Smith, 1999; Upendra et al., 2008). Various studies of alco-

holic and hydroalcoholic extracts of fruits, seeds and leaves of

seabuckthorn have confirmed its medicinal and nutritional value

(Geetha, Ram, Singh, Ilavazhagan, & Sawhney, 2002; 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, flavonoids, lipids, vitamins, tan-

nins, minerals, etc. (Hakkinen, Karenlampi, Heinonen, Mykkanen,

& Torronen, 1999; Upendra et al., 2008) which contribute to its

wide usage as a natural antioxidant.

Extraction of antioxidants from plant tissues has usually been

accomplished by conventional extraction processes such as so-

lidliquid extraction employing methanol, ethanol and acetone

and also through steam distillation. Nowadays, there has been a

huge upsurge for developing rapid, reliable, and reproducible

methods for the efficient extraction of bioactive compounds from

plants to increase their therapeutic functionality. In the literature,

different extraction techniques, such as maceration, Soxhlet, ultra-

sound-assisted extraction (UAE), microwave-assisted extraction

(MAE) and subcritical extraction (SCE), are reported (Upendra

et al., 2008; Rodriguez-Meizoso et al., 2006). Recently, there has

been an increasing interest in the use of environmentally clean

technologies able to provide high quality and high activity extracts

while precluding any toxicity associated to the solvents. In this

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: [email protected](M.S.Y. Kumar).

Food Chemistry 127 (2011) 13091316

Contents lists available at ScienceDirect

Food Chemistry

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / f o o d c h e m
http://dx.doi.org/10.1016/j.foodchem.2011.01.088mailto:[email protected]://dx.doi.org/10.1016/j.foodchem.2011.01.088http://www.sciencedirect.com/science/journal/03088146http://www.elsevier.com/locate/foodchemhttp://www.elsevier.com/locate/foodchemhttp://www.sciencedirect.com/science/journal/03088146http://dx.doi.org/10.1016/j.foodchem.2011.01.088mailto:[email protected]://dx.doi.org/10.1016/j.foodchem.2011.01.088

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sense, both, supercritical fluid extraction (SFE) with carbon dioxide

(CO2) as a solvent and sub critical water extraction (SWE) meet the

requirements to be considered clean and safe processes (King,

2000; Rodriguez-Meizoso et al., 2006).

SWE, i.e., extraction using hot water under pressure sufficient to

maintain water in the liquid state, has demonstrated its ability to

selectively extract different classes of compounds depending on

the temperature used, with the more polar extracted at lower tem-peratures while the less polar compounds were extracted at higher

temperatures. The selectivity of SWE allowsfor manipulation of the

composition of the extracts by changing the operating parameters

and it has been used for essential oil isolation (Basile, Jimenez Car-

mona, & Clifford, 1998) as well as forantioxidant extraction (Ibanez,

Kubatova, Senorans, Cavero, & Reglero, 2003; Rodriguez-Meizoso

et al., 2006). The goal of the present investigation was to study

the selectivity of SWE at several temperatures to extract antioxi-

dant compounds from Seabuckthorn leaves (HippophaeRhamno-

ides). SWE proposed in this work as a feasible process to

concentrate and isolate antioxidantcompounds to be used as nutra-

ceuticals. In recent years, many papers have been published on the

applicability of SWEfor theextraction of bioactivecompounds from

plants. Nevertheless, to thebest of our knowledge, there is no report

available that could illustrate the feasibility of SWE as a rapid and

efficient extraction tool for the determination of antioxidant activ-

ity of SBT leaves.

Since the various bioactive properties of SBT, including antiox-

idant, are attributed to the presence of phenolic compounds in it;

hence, evidently, the other objective of this work was to investi-

gate the feasibility of SWE for the rapid and efficient extraction

of bioactive phenolics from the plant and comparing it to other

extraction techniques (maceration and Soxhlet). Keeping this in

mind, the antioxidant activity of SBT leaves extracts was evaluated

in chemical and biological systems. Because, the antioxidant capac-

ity of drugs can be evaluated using chemical methods, which are

easy to execute and have high reproducibility. Nevertheless, such

methods do not represent what happens in vivo (Soares, Andreaz-

za, & Salvador, 2003). Assays using living cells have proven to bevery useful in the routine testing of various products, being fast,

sensitive, reproducible, as well as producing reliable results in

terms of the identification of biological and antioxidant activity

(Soares et al., 2003). Simultaneously, some of its phenolic constit-

uents (quercetin-3-O-galactoside, kaempferol, and isorhamnetin;

Fig. 1A) were determined with the help of reverse-phase high-

performance liquid chromatography (RP-HPLC) to demonstrate

the extraction efficiency and hence, antioxidant activity.

2. Materials and methods

2.1. Plant material

Seabuckthorn leaves were collected from hilly region of westernHimalyas, India in the month of September, in which the plant

grows widely under natural condition. Voucher specimen is pre-

served in Defence Institute of High Altitude Research, Leh after

ethanobotanical identification of species.

2.2. Apparatus

HPLC Waters, ASE 350 Dionex Corporation (Sunnyvale, CA,

USA), Spectrophotometer Bio-Rad. ELISA reader (Molecular De-

vices, USA), Spectrofluorimeter (Varian, USA).

2.3. Reagents

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

2,5,7,8-Tetramethylchroman-2-Carboxylic acid [Trolox], Rutin

(Sigma Aldrich Chemicals, USA), FolinCiocalteu reagent & Ascor-

bic Acid [Vitamin-C] (Sisco Research Laboratories, India).

2.4. In vitro cell culture model

Raw 264.7, a murine macrophage cell line was obtained from

National Centre of Cell Sciences (NCCS) Pune, India. Cells werepropagated in DMEM supplemented with 10% FBS, 100lg/mL

ampicillin and 100lg/mL streptomycin. They were maintained at

37C in a humidified CO2incubator. The cells were grown to a den-

sity of 1 104 cells/well in 96 well plates (Greiner, Germany) for

determination of cytotoxicity, mitochondrial membrane potential

and ROS levels. The cells were grown in 24 well plates (Falcon

make) to a density of about 1 105/well for determination of

anti-oxidants levels.

2.5. Extraction Procedure

2.5.1. Maceration

100 g of powdered Seabuckthorn leaf sample was soaked in

500 mL of distilled water at room temperature. After 24 h, super-

natant was decanted, filtered through muslin cloth and stored in

amber coloured bottle. The solution was centrifuged at 8000 rpm

for 10 min. Finally, the supernatant solution was lyophilised and

the dried extract was stored at 5C for the further studies.

2.5.2. Soxhlet extraction

100 g of powdered Seabuckthorn leaf sample was extracted

with 350 mL of 70% ethanol for 610 h in a Soxhlet apparatus.

The extract was filtered and Alcoholic content in extract was evap-

orated using Rota vapour at 40 C. Finally, the supernatant solution

was lyophilised and the dried extract was stored at 5C for the fur-

ther studies.

2.5.3. Subcritical water extraction

To perform the extractions, an Accelerated Solvent Extractionsystem ASE 350 equipped with a solvent controller unit from Dio-

nex Corporation (Sunnyvale, CA, USA) was used. Extractions were

carried out in triplicate using water. Individual extractions were

performed using the sample considering the following tempera-

tures: 25, 50, 100, 150 and 200C for 15 min extraction time at

each temperature. Previous to each experiment an extraction cell

heat-up was carried out for a given time, which changed according

to extraction temperature (the heat-up time is automatically fixed

by the equipment). Namely 5 min heat-up was used when extrac-

tion temperature was set at 50C and 100 C, 7 min at 150 C and

9 min at 200 C. Likewise, all extractions were performed in 33 mL

extraction cells, containing 2 g of sample.

The extraction procedure was as follows: (i) sample is loaded

into cell, (ii) cell is filled with solvent up to a pressure of1500 psi, (iii) initial heat-up time is applied, (iv) static extraction

with all system valves closed is performed for 15 min, (v) cell is

rinsed (with 60% cell volume using extraction solvent), (vi) solvent

is purged from cell with N2 gas and (vii) depressurisation takes

place. Between extractions, a rinse of the complete system was

made in order to overcome any extract carry-over. For solvent

evaporation a freeze dryer (Allied frost, India) was employed. The

collected extracts were kept protected from light, at 4C until use.

2.6. Determination of total phenol content (Thaipong, Boonprakob,

Crosby, Zevallos, & Byrne, 2006)

Total phenol content of extracts was determined by the Folin

Ciocalteu method. 150lL of extract, 2400lL of nanopure waterand 150 lL of 0.25 N FolinCiocalteu reagent were combined and

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then mixed well. The mixture was allowed to react for 3 min then

300lL of 1 N Na2CO3 solution was added and mixed well. The

solution was incubated at room temperature in the dark for 2 h.

The absorbance was measured at 725 nm using a spectrophotom-

eter and the results were expressed in milligram of gallic acid

equivalents (GAE) per 1 gram of extract.

2.7. Determination of Total flavonoid content (Yanping Zou, Yanhua, &

Wei, 2004)

1.0 mL aliquot of appropriately diluted sample solution was

mixed with 2 mL of distilled water and subsequently with

0.15 mL of a 5% NaNO2solution. After 6 min, 0.15 mL of a 10% AlCl3solution was added and allowed to stand for 6 min, then 2 mL of 4%

NaOH solution was added to the mixture. Then the mixture wasthoroughly 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 total flavonoid. All values were expressed as milligram of rutin

equivalents per gram of dry raw material.

2.8. HPLC analysis

The HPLC system consisted of a Waters HPLC system (Waters

Corporation, USA) equipped with Waters 515 HPLC pump, Waters

717 plus autosampler and Waters 2487 UV detector, interfaced

with an IBM Pentium 4 personal computer. The separation was

performed on a Symmetry C18 250 4.6 mm ID; 5lm column

(Waters, USA) by maintaining the gradient flow rate 0.75 mL/minof the mobile phase (Solution A; Water:O-Phosphoric acid

99.7:0.3 and Solution B; Acetonitrile:Methanol 75:25) and peaks

were 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 the cali-

bration curve, standard stock solutions of compounds 13 (1 mg/

2 mL) were prepared in methanol, filtered through 0.22lm filters

(Millipore), and appropriately diluted (0.01100lg/mL) to obtain

the desired concentrations in the quantification range. The calibra-

tion 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 with100 mL methanol and then stored at 20C until needed. The

working solution was obtained by mixing 10 mL stock solution

with 45 mL methanol to obtain an absorbance of 1.10 0.02 units

at 515 nm using the spectrophotometer. 150lL of root extract

solution was allowed to react with 2850 lL of the DPPH solution

for 2 h in the dark. Then the absorbance was taken at 515 nm.

The standard curve was linear between 25 and 200 ppm Trolox. Re-

sults are expressed in mg TE/g of dry raw material.

2.9.2. FRAP assay (Thaipong et al., 2006)

The stock solutions included 300 mM acetate buffer (3.1 g

C2H3NaO23H2O and 16 mL C2H4O2), pH 3.6, 10 mM TPTZ (2,4,6-tri-

pyridyl-s-triazine) solution in 40 mM HCl, and 20 mM FeCl36H2O

solution. The fresh working solution was prepared by mixing25 mL acetate buffer, 2.5 mL TPTZ solution, and 2.5 mL FeCl36H2O

Fig. 1. Structure of the quantified phenolic compounds (A) and HPLC chromatogram of subcritical water extract of Seabuckthorn leaves (B).

M.S.Y. Kumar et al. / Food Chemistry 127 (2011) 13091316 1311

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solution and then warmed at 37C before using. 150 lL of leaves

extract solution was allowed to react with 2850lL of the FRAP

solution for 30 min in the dark condition. Readings of the coloured

product [ferrous tripyridyltriazine complex] were then taken at

593 nm. The standard curve was linear between 25 and 150 ppm

Trolox. Results are expressed in mg TE/g of dry raw material.

2.9.3. Determination of total reducing power (Zou et al., 2004)1.0 mL of leaves extract solution (0.21.0 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 of potassium ferricyanide. The mixture was incu-

bated in a water bath at 50C for 20 min. afterward, 2.5 mL of a

10% (w/v) trichloroacetic acid solution was added and the mixture

was then centrifuged at 3000 rpm for 10 min. A 2.5 mL aliquot of

the upper layer was combined with 2.5 mL of distilled water and

0.5 mL of a 0.1% (w/v) solution of ferric chloride, and the absor-

bance was measured spectrophotometrically at 700 nm. Vitamin

C is used for the standard graph.

2.10. Determination of cytotoxicity and antioxidant activity in cell

culture model

2.10.1. Determination of cytotoxicity

The optimal incubation time and concentration of tert-BOOH

required to produce cytotoxicity on Raw cells were determined.

To determine the efficacy of various antioxidants the cells were

supplemented with antioxidants before tert-BOOH addition. The

lowest concentration of antioxidant that provided the maximum

protection against tert-BOOH induced cytotoxicity was used in

the present study. Raw cells were incubated with 100lM tert-

BOOH for 1 h in the presence and absence of different antioxidant

for determination of cytotoxicity and ROS. For measuring GSH lev-

els the cells were exposed to tert-BOOH for 2 h.

Cytotoxicity was studied by using neutral red uptake (Sairam

et al., 2000), a supra-vital dye, that is selectively taken by the live

cells. Briefly, 10 ll of neutral red dye (0.1%) was added to the cells

and incubated at 37C in CO2incubator for 45 min. Later the cells

were washed with PBS followed by the addition of 200ll ethanol-

acetic acid (50:1) solution. The OD was measured at 570 nm using

Elisa reader (Molecular Devices).

2.10.2. Measurement of reactive oxygen species (ROS)

It was measured (Catchart, Schwiers, & Ames, 1983) using fluo-

rescent probe 2,7 dichlorofluorescein (DCFHDA). Briefly after incu-

bation, 10ll of DCFHDA stock solution (200lM in DMSO) was

added to 190ll of medium in 96 well plate to get final concentra-

tion of 10lM. The cells were incubated at 37 C for 30 min in CO2incubator. The cells were washed thrice with PBS and fluorescence

was measured by multiwell spectrofluorimeter (Molecular De-

vices) with excitation at 485 nm and emission at 530 nm. Alter-

nately radical scavenging activity of the SBT extracts were

determined in cultured cells using fluorescent probe DCFHDA.

Briefly, after incubation, 10 ll of DCFHDA stock solution (200lM

in DMSO) was added to cells (1 106 cells). The cells were incu-

bated at 37C for 30 min in CO2incubator. The cells were washed

twice with PBS. The ROS production was monitored by Flowcytom-

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

2.10.3. Determination of mitochondrial transmembrane potential

(mitochondrial integrity)

Mitochondrial membrane potential (MMP) was determinedusing fluorescent probe Rhodamine123 (Jiang & Acosta, 1993).

After the cells were exposed to tert-BOOH, 10ll Rhodamine 123

(10 lg/mL) was added to cells and incubated for 30 min. The cells

were washed three times with PBS and fluorescence was measured

using spectrofluorimeter (Spectra Max M2 Molecular Devices) with

an excitation of 485 nm and emission at 531 nm.

2.10.4. Determination of reduced glutathione levels

After incubation, the cells were lysed by adding 200 ll of lysis

buffer (100lM Tris, 20 mM EDTA, 0.25% Triton X-100 pH 8.0).

The reduced and oxidised glutathione (GSSG) levels in the cells

were determined fluorimetrically (Hissin & Hilf, 1976).

2.11. Statistical analysis

Each analysis was done three times from the same extract in or-

der to determine their reproducibility. Results are expressed as

mean SD. Statistical comparisons were made by one-way analy-

sis of variance (ANOVA). Differences were considered to be signif-

icant when thep values were

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In this study, the effect of sub critical water extraction method,

for the efficient extraction of antioxidative compounds from Sea-

buckthorn leaf was investigated. Until now, to the best of our

knowledge, there is no such report available that could highlight

the feasibility of sub critical water extraction procedure as an effi-

cient method for the extraction of antioxidant compounds from

Seabuckthorn leaf. For comparison, Seabuckthorn leaf was also ex-

tracted by maceration and Soxhlet extraction methods. Duringextraction, it was seen that maximum extraction yield (49.20%)

was achieved with sub critical water extraction followed by

Soxhlet (31.65%), and maceration (15.41%). However, taking into

consideration the solvent consumption and time needed for

extraction, SWE was found to be the best feasible approach for

the rapid and efficient extraction of antioxidant compounds.

3.2. Total phenol and flavonoid contents

As a part of chemical composition analysis, total flavonoid and

total phenol content of Sea buckthorn leaf extracts were deter-

mined by colorimetric method. The results as given in theTable 1

indicate the presence of higher total phenolic and flavonoid con-

tent in the sub critical water extract obtained at 150

C followedby the extracts obtained by Soxhlet and maceration methods.

Phenolics are the major plant compounds with antioxidant activ-

ity. This activity is believed to be mainly due to redox properties,

which play important role in adsorbing and neutralising free radi-

cals, quenching singlet and triplet oxygen, or decomposing perox-

ides (Costantino, Albasini, Rastelli, & Benvenuti, 1992). Results

obtained in the present study revealed that the level of these phe-

nolic compounds in the SBT leaf extracts were considerable. The

results strongly suggest that phenolics are important components

of this plant, and some of its pharmacological effects could be

attributed to the presence of these valuable constituents.

Flavonoids form a class of benzo-c-pyrone derivatives include

flavones, flavanes, flavonols, anthocyanidines, and catechins. They

possess 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, Gker, Ertan, & Pindur, 1989; Jagtap

et 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 many

reports relating to the reactivities of flavonoids with active oxygen

species. Recent interest in these substances has been stimulated by

the potential health benefits arising from their antioxidant activity.

3.3. Antioxidant activity evaluation of SBT extracts in chemical system

3.3.1. DPPH assay

The 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

at 517 nm in visible spectroscopy. As the electron becomes paired

off in the presence of free radical scavenger, the absorption van-

ishes, and the resulting decolourisation is stoichiometric with re-

spect to the number of electrons taken up. From the DPPH assay

results it may be postulated that SBT reduces the radical to the cor-

responding hydrazine on reacting with the hydrogen donors in

SBT. The bleaching of DPPH represents the capacity of SBT to scav-

enge free radicals independent of enzymatic activity. The presentinvestigation shows that SBT is sufficiently effective in scavenging

DPPH radicals (Badami, Gupta, & Suresh, 2003).

3.3.2. FRAP assay

The antioxidant potentials of the aqueous and alcoholic extracts

of the leaves were estimated from their ability to reduce TPTZ-Fe

(III) complex to TPTZ-Fe (II). Antioxidant activity increased propor-

tionally with the phenol content. According to recent reports, a

highly positive relationship between total phenols and antioxidant

activity appears to be the trend in many plant species (Adedapo,

Jimosh, Koduru, Afolayan, & Masika, 2008).

The results of both FRAP and DPPHassay are listed inTable 2.

The trolox equivalents antioxidant capacity (TEAC) values obtained

for the extracts submitted to the FRAP assay were in the range of2.03182.13 mg/g, while the values for the DPPH assay were in

the range 6.97282.75 mg/g. In addition, the higher antioxidant

activity exhibited by the sub critical water extracts (Table 2) over

the other Soxhlet and maceration extraction methods clearly dem-

onstrates the relative advantage of SWE for obtaining formulations

with high antioxidant compounds.

It will be relevant to mention here that earlier papers

(Costantino et al., 1992) have demonstrated the correlation be-

tween the phenolic content of plants to their antioxidant power.

In this study also, a good correlation was indicated between the

phenolic content and the antioxidant power of extracts. For the

measurement of total phenolic content, the absorbance of Sea-

buckthorn leaf extracts was determined spectrometrically accord-

ing to the FolinCiocalteu method and calculated as GAE. It is clear

from the Table 1 that the maximum total phenolic content was

achieved with sub critical water extraction followed by Soxhlet

and maceration, which is evidently in accordance with the ob-

served antioxidant activity.

3.3.3. Reducing power

The reducing power of SBT extracts, which may serve as a sig-

nificant reflection of the antioxidant activity, was determined

using a modified iron (III) to iron (II) reduction assay. In this assay,

the yellow colour of the test solution changes to various shades of

green and blue depending on the reducing power of extracts or

compounds. The presence of reductants in the solution causes

the reduction of the Fe3+/Ferricyanide complex to the ferrous form.

Therefore, the Fe2+ can be monitored by measurement of the for-

mation of Perls Prussian blue at 700 nm.

Fig 2shows the reducing power of SBT extracts and compared

to ascorbic acid. All the extracts have showed some degree of

Table 2

Antioxidant activity evaluation in chemical system.

Method of extraction Extraction temperature (C) DPPH mg Trolox eqt./g SD* of dry raw material FRAP mg Trolox eqt./g SD* of dry raw material

Maceration 25 5 86.35 2.93 93.91 3.72

Soxhlet 80 255.87 5.51 217.77 4.29

30 133.31 5.72 144.99 4.53

50 164.03 6.28 179.62 5.72

Subcritical water 100 194.76 6.71 219.03 6.28

150 353.43 8.75 276.93 7.71

200 343.86 7.71 261.54 6.12

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


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duced glutathione (GSH) by about 23% and an increase in oxidised

glutathione (GSSG) by about 2.9-fold in Raw cell line as compared

to control cells (Fig. 3C). Further there was a considerable fall in

GSH/GSSG ratio from 3.6 in control cells to 0.96 in cells incubated

with tert-BOOH. Supplementation of SBT extract (25lg/mL) re-

sulted in significant increase in GSH and decrease in GSSG levels.

The GSH/GSSG ratio in SBT treated cells were increased signifi-

cantly, when compared to control cells. However GSH level

dropped to further 40% of the control cells after 3 h incubation in

presence of tert-BOOH.

3.5. Evaluation of Cytotoxicity

We assessed the cytotoxicity of tert-BOOH by exposing Raw

264.7 murine macrophages to various concentration of tert-BOOH

(10500 lM). The LD50 was observed at 100 lM concentration of

tert-BOOH. Various concentration of SBT (10200lg) was also

tested to determine the optimum dose and cytotoxicity, if any to-

wards Raw cells. We found that upto100lg/mL of SBT drug had no

cytotoxicity. However, a concentration as low as 25lg/mL was

effective and showed viability > 95% that was maintained through

the test period. Addition of tert-BOOH (100lM) to Raw cells re-sulted in significant increase in cytotoxicity (55%) as revealed by

the fall in neutral red uptake as compared to control cells. Pretreat-

ment of cells with SBT extract (25lg/mL) significantly attenuated

Raw 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 was

developed for the analysis of Quercetin-3-galactoside, kaempferol

and isorhamnetin (Fig. 1A) in the extracts. For the development

of an effective mobile phase, various solvent systems, including

different combinations of acetonitrile, methanol and water withortho phosphoric acid were tried. Finally, a solvent system

consisting of 0.3% ortho phosphoric acid in water and acetonitrile:

methanol (75:25) was proved successful because it allows for the

separation of maximum compounds with good resolution

(Fig. 1B). Quercetin-3-galactoside, Kaempferol and Isorhamnetin,

these might contribute to the antioxidant behaviour of the plant

was identified in extracts, as shown in Figure 6. Identification of

compounds was performed on the basis of the retention time, co

injections, and spectral matching with standard. For quantification,

standard stock solutions of Quercetin-3-galactoside, Kaempferol

and Isorhamnetin was (1 mg/2 mL) was prepared in ethanol, fil-

tered through 0.22lm filters (Millipore), and appropriately diluted

(0.01100lg/mL) to obtain the desired concentrations for quanti-

fication. The results as given in theTable 3indicate the presence ofhigher Quercetin-3-galactoside, Kaempferol and Isorhamnetin con-

tent in the sub critical water extract obtained at 150C followed by

the extracts obtained by Soxhlet and maceration methods.

4. Conclusion

Subcritical water extraction of SBT leaves was found to be a bet-

ter approach than Soxhlet and maceration because the use of sub-

critical water imparted higher antioxidant and cytoprotectiveactivities to the extracts besides ensuring low solvent consump-

tion, ease, and rapidity of the overall method than Soxhlet and

maceration extraction methods. Antioxidant activity of the ex-

tracts was evaluated using commonly accepted chemical assays

(DPPH & FRAP). Also, present study report the cytoprotective and

antioxidant properties of SBT against tertiary-butyl hydroperoxide

(tert-BOOH) induced oxidative stress in murine macrophages (Raw

264.7). SBT extracts inhibited cytotoxicity, ROS production and

maintained antioxidants levels similar to that of control cells.

Simultaneously, a simple RP-HPLC method was developed for

the identification and quantification of three phenolic compounds

(Quercetin-3-galactoside, kaempferol and isorhamnetin) present in

the extracts of SBT, to demonstrate the increased antioxidant

power. The results are promising and demonstrate the practicalfeasibility of subcritical water extraction to substitute the tradi-

tional time-consuming techniques for efficient extraction of anti-

oxidative compounds to provide nutraceutical-rich formulations.

Acknowledgements

Authors are thankful to Dr. G. Ilavazhagan, Director, DIPAS,

Delhi for the constant support and encouragement. Dr. K Udaya

Sankar, CFTRI, Mysore and Dr. Ashoke Banerji, Amrita Vishwa Vid-

ya Peetham, Amritapuri.

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Determination of Quercetin-3-galactoside, kaempferol and isorhamnetin in seabuckthorn leaves by HPLC.

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