LWT 39 (2006) 1059

ro&

. H

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in v

extracts showed high antioxidant activity measured as scavenging of DPPH, superoxide and hydroxyl radicals. Both the aqueous

and methanolic extracts inhibited microsomal lipid peroxidation and exhibited strong reducing power and metal chelating activity.

The antioxidant activity did not correlate with the phenolic content of the extracts. These results demonstrate the antioxidant

erative disorders and aging (Halliwell & Gutteridge,1999; Yu, 1994). The value of nutraceuticals in food has

decreased risk of degenerative diseases particularly

oxidant capacity (Wang, Cao, & Prior, 1997). Severalstudies have shown that plant derived antioxidant

are consumed as pickles and as a juice for its allegedhealth promoting properties. However, the roots of Dhhave not been investigated for their health promoting

ARTICLE IN PRESS

Corresponding author. Tel.: +918212513210;

potential. Earlier studies have shown that the rootscontain aldelydes, inositols, saponins, amyrins and lupeols

0023-6438/$30.00 r 2005 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.

doi:10.1016/j.lwt.2005.07.005

fax: +918212517233.

E-mail address: tshivanandappa@yahoo.com (T. Shivanandappa).long been recognized for their health benets (Klein,Sato, Meguid, & Miyata, 2000). Green tea (Cao, Soc,& Prior, 1996; Chen & Ho, 1995), red wine (Kinsella,Frankel, German, & Kanner, 1993) and ginseng (Fu &Ji, 2003; Kitts, Wiejewickreme, & Hu, 2000) are knownto have benecial effects on the prevention or progres-sion of diseases related to oxidative stress on account oftheir high antioxidant activity. It is believed that higherintake of antioxidant rich food is associated with

nutraceuticals scavenge free radicals and modulateoxidative stress-related degenerative effects (Thatte,Bagadey, & Dahanukar, 2000). Nutraceuticals arebecoming widely incorporated in functional food owingto their therapeutic effects in enhancing the well-being.There is a great deal of interest in newer naturalbioactive molecules with health promoting potential.Decalepis hamiltonii (Dh) (family: Asclepediaceae)

grows wild in the forests of peninsular India. Its tubersa new source of natural antioxidants or nutraceuticals with potential applications to reducing the level of oxidative stress and related

health benets.

r 2005 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.

Keywords: Decalepis hamiltonii; Lipid peroxidation; Free radical scavenging; Reducing power; Metal ion chelation; Phenolic content

1. Introduction

Free radicals have been implicated in many diseasessuch as cancer, atherosclerosis, diabetes, neurodegen-

cardiovascular diseases and cancer (Ames, Shigenaga,& Hagen, 1993; Joseph et al., 1999). Vegetables containseveral antioxidant nutrients in addition to vitamin C, Eand carotenoids which contribute to their total anti-potency of the root extracts which could be the basis for its alleged health promoting potential of Dh. The roots of Dh could serve asAntioxidant activity of the(Wight

Anup Srivastava, Shereen R

Department of Food Protectants and Infestation Control, Cen

Received 19 May 2005; received in rev

Abstract

Tuberous roots of Decalepis hamiltonii (Dh) are consumed in

antioxidant potential of the roots was measured using various1065

ots of Decalepis hamiltoniiArn.)

arish, T. Shivanandappa

ood Technological Research Institute, Mysore 570020, India

orm 8 July 2005; accepted 8 July 2005

ern India as pickles and beverage for their health benets. The

itro assays. Among the extracts, the methanolic and aqueous

www.elsevier.com/locate/lwt

Sequential extraction of the root powder was done

ARTICLE IN PRESSWTwith different solvents with increasing polarity i.e.hexane, chloroform, ethyl acetate, acetone, methanoland water. A total of 50 g of root powder was extractedin 0.5 l of the solvent in glass conical ask on a shakerfor 24 h at room temperature. The extract was lteredwith Whatman paper no. 1 and dried by ashevaporation/lyophilization.The methanolic extract was prepared by soxhlet

extraction in methanol. A 100 g of root powder wasextracted (1 l) at 50 1C for 24 h. Methanol was ashevaporated and the extract weighed (28 g). The aqueousextract was prepared by homogenizing the root powder(100 g) in 1 l of warm water (50 1C) and allowed to standfor 24 h, ltered with Whatman paper no. 1 and themine tetra-acetic acid (EDTA) were purchased fromM/sSigma Chemicals Co. (St. Louis, MO). Nicotinamideadenine dinucleotide-reduced (NADH), trichloroaceticacid (TCA), deoxyribose, ascorbic acid and otherchemicals were purchased from M/s Sisco ResearchLaboratories, Mumbai, India. All reagents were analy-tical grade.

2.2. Preparation of root powder and extraction

Tuberous roots (10 kg) of Dh were purchased from thelocal suppliers. They were washed with water and thencrushed with a roller to separate the inner woody coreand the outer eshy layer. The eshy portion wascollected, dried at 40 1C in a hot air oven and then nelypowdered. The powder (1.9 kg) was used for extraction.(Murti & Sheshadri, 1940, 1941a, b) as well as volatilecompounds such as 2-hydroxy-4methoxybenzaldehyde,vanillin, 2-phenyl ethyl alcohol, benzaldehyde, andothers (Nagarajan, Rao, & Gurudutt, 2001). The rootshave also been used as a substitute for Hemidesmusindicus in ayurvedic preparations of ancient Indianmedicine (Nayar, Shetty, Mary, & Yoganarshimhan,1978). This paper reports the antioxidant potential ofthe aqueous and methanolic extracts of the Dh rootemploying various in vitro assay systems, such asinhibition of lipid peroxidation (LPO), DPPH/super-oxide/hydroxyl radical scavenging, reducing power andmetal chelating activity.

2. Materials and methods

2.1. Chemicals

Butylated hydroxylanisole (BHA), nitroblue tetrazo-lium (NBT), 1,1-diphenyl-2-picrylhydrazyl (DPPH),phenazine methosulphate (PMS), thiobarbituric acid(TBA), bovine serum albumin (BSA) and ethylenedia-

A. Srivastava et al. / L1060ltrate was lyophilized and weighed (22 g).2.3. DPPH radical scavenging assay

DPPH radical scavenging activity was done accordingto Yamaguchi, Takamura, Matoba, and Terao (1998).Briey, 1ml of DPPH solution (0.1mmol/l, in 95%ethanol (v/v)) was incubated with different concentra-tions of the extract. The reaction mixture was shakenand incubated for 20min at room temperature and theabsorbance was read at 517 nm against a blank. Theradical scavenging activity was measured as a decreasein the absorbance of DPPH and calculated using thefollowing equation:

Scavenging effect % 1 ASample517 nm=AControl 517 nm 100.

2.4. Superoxide radical scavenging assay

The superoxide radical scavenging ability of theextracts was measured by the method of Nishikimi,Rao, and Yagi (1972). The reaction mixture containeddifferent concentrations of the extract, PMS (0.1mmol/l), NADH (1mmol/l) and NBT (1mmol/l) in phosphatebuffer (0.1mol/l, pH 7.4), was incubated at roomtemperature for 5min and the color was read at560 nm against a blank. The scavenging effect wascalculated using the equation described as in the case ofDPPH.

2.5. Hydroxyl radical scavenging assay

The reaction containing different concentrations ofthe extract was incubated with deoxyribose (10mmol/l),H2O2 (10mmol/l), FeCl3 (5mmol/l), EDTA (1mmol/l)and ascorbic acid (5mmol/l) in potassium phosphatebuffer (50mmol/l, pH 7.4) for 60min at 37 1C (Halliwell,Gutteridge, & Cross, 1987). The reaction was terminatedby adding TCA (5 g/100ml water) followed by theaddition of TBA (0.2 g/100ml water) and boiled in waterbath for 15min. The absorbance of the color wasmeasured at 535 nm against the reagent blank and theinhibition of the oxidation of deoxyribose was calcu-lated with respect to the control.

2.6. Inhibition of microsomal lipid peroxidation

Liver excised from adult male Wistar rats, washomogenized (20 g/100ml Tris buffer) in 0.02mol/l, trisbuffer (pH 7.4). Microsomes were isolated by thecalcium aggregation method (Kamath & Rubin, 1972).100 ml of liver microsomal suspension (0.5mg protein)was incubated with 1mmol/l each of FeSO4 andascorbic acid with or without extract in a total volumeof 1ml in 0.1mol/l phosphate buffer (pH 7.4). Afterincubation at 37 1C for 60min, the reaction mixture was

39 (2006) 10591065boiled with TBA (0.67 g/100ml water) for 15min.

Formation of TBA reactive substances (TBARS) wascalculated from the absorbance at 535 nm (Buege &Aust, 1978). BHA was used as the positive control.

2.7. Measurement of reducing power

The reducing power of the extracts was measured byincubating the reaction mixture (1ml) containing theextract in phosphate buffer (0.2mol/l, pH 6.6) withpotassium ferricyanide (1 g/100ml water) at 50 1C for

Microsomal protein estimation was done by themethod of Lowry, Rosenbrough, Farr, and Randall(1951) using BSA as the standard.

2.10. Statistical analysis

Data were expressed as mean7S.E. of three separateexperiments and subjected to the analysis of variance(Po0:05) using the computer programme Excel andStatistica software (1999).

by both aqueous and methanolic extracts and was

ARTICLE IN PRESS

(%)

A. Srivastava et al. / LWT 39 (2006) 10591065 106120min. The reaction was terminated by adding TCAsolution (10 g/100ml water), centrifuged at 3000 rpm for10min and the supernatant was mixed with ferricchloride (0.1 g/100ml water), the absorbance measuredat 700 nm (Yen & Chen, 1995). Increased absorbance ofthe reaction mixture indicated increased reducingpower.

2.8. Metal ion chelating assay

The Fe2+-chelating ability of the extract was mea-sured by the ferrous iron-ferrozine complex at 562 nm(Decker & Welch, 1990). The reaction mixture contain-ing FeCl2 (2mmol/l) and ferrozine (5mmol/l) along withextracts was adjusted to a total volume of 0.8ml withmethanol, mixed and incubated for 10min at roomtemperature. The absorbance of the mixture was read at562 nm against a blank. EDTA was used as positivecontrol. The ability of the extract to chelate ferrous ionwas calculated using the equation described for DPPH.

2.9. Total phenolic content

Total phenolic content was estimated by FolinCio-calteau method (Singleton & Rossi, 1965). To 6.0mldouble distilled water, a 0.1ml sample and 0.5mlFolinCiocalteau reagent was mixed followed by theaddition of 1.5ml Na2CO3 (20 g/100ml water) and thevolume was made up to 10.0ml with distilled water.After incubation for 30min at 25 1C, the absorbance wasmeasured at 760 nm and the phenolic content wascalculated with a guaicol standard and expressed asguaicol equivalents.

Table 1

Antioxidant activitya of the sequential extracts of D. hamiltonii

Solvent Extract yield (g) DPPH radical scavenging

Hexane 3.78 3.670.4Chloroform 4.76 36.0672.6Ethyl acetate 1 47.5173.1Acetone 1.19 55.4773.6Methanol 12.95 69.7774.2Water 8.36 73.5474.7aAntioxidant activity was assayed at a concentration of 1mg/ml for all thSuperoxide radical scavenging (%) LPO (% inhibition)

0 43.372.80 64.773.613.772.1 71.474.227.273.4 76.575.149.974.6 68.873.986.274.8 6073.33. Results and discussion

3.1. Antioxidant activity of the extracts

Antioxidant activity of the sequential extracts arepresented in Table 1. Among the extracts, maximumantioxidant activity was shown by the methanolic andaqueous extracts and therefore, they were chosen forfurther study. The results indicate the choice of thesolvent for obtaining the extract with high antioxidantactivity.

3.2. DPPH radical scavenging activity

A high radical scavenging activity was observed inboth the aqueous and methanolic extracts in aconcentration dependent manner. The aqueous extractwas slightly more active than the methanolic extract(IC50 0.29 and 0.36mg/ml, respectively) (Table 2 andFig. 1). Proton-radical scavenging action is an impor-tant attribute of antioxidants, which is measured byDPPH radical scavenging assay. DPPH, a protonatedradical, has characteristic absorbance maxima at 517 nmwhich decreases with the scavenging of the protonradical (Yamaguchi et al., 1998). Hydrogen-donatingability of the antioxidant molecule contributes to its freeradical scavenging nature (Chen & Ho, 1995).

3.3. Scavenging of superoxide radical

Superoxide radical scavenging activity was showne extracts.

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75xy

l rad

ical

scav

engi

ng

50

25

WTTable 2

Antioxidant activity of the extracts of D. hamiltonii roots

Extracts IC50 (mg/ml)

DPPH Superoxide Hydroxyl

radical

Microsomal

LPO

Metal

chelation

Aqueous 0.29 0.53 1.84 0.51 5.49

Methanolic 0.36 2.12 3.95 0.46 32.49

100

75

DPP

H ra

dica

l sca

veng

ing

(%)

50

25

00 0.4

Concentration of extract (mg/ml)0.8 1.2

Fig. 1. DPPH scavenging effect of D. hamiltonii root extracts.

(Control absorbance 0.74) KAqueous extract, Methanolic

A. Srivastava et al. / L1062concentration dependent, with an IC50 value of 0.53 and2.12mg/ml, respectively (Table 2 and Fig. 2). Aqueousextract was markedly a more potent scavenger ofsuperoxide anion than the methanolic extract. Super-oxide radicals are generated during the normal physio-logical process mainly in mitochondria. Althoughsuperoxide anion is by itself a weak oxidant, it givesrise to the powerful and dangerous hydroxyl radicals aswell as singlet oxygen both of which contribute to theoxidative stress (Dahl & Richardson, 1978; Meyer &Isaksen, 1995). Therefore superoxide radical scavengingby antioxidants has physiological implications.

3.4. Hydroxyl radical scavenging activity

Both aqueous and methanolic extracts of D. hamilto-nii displayed hydroxyl radical scavenging activity. IC50values for the aqueous and methanolic extract were 1.84and 3.95mg/ml, respectively (Table 2 and Fig. 3). Thehydroxyl radical is an extremely reactive free radicalformed in biological systems and has been implicated asa highly damaging species in free radical pathology,capable of damaging biomolecules of the living cells(Gordon, 1990; Halliwell, 1991; Hochestein & Atallah,1988). Hydroxyl radical has the capacity to cause DNAstrand breakage, which contributes to carcinogenesis,mutagenesis and cytotoxicity (Aruoma, Halliwell, &

extract.100

75

Supe

roxi

de ra

dica

l sca

veng

ing

(%)

50

25

00 1.40.7

Concentration of extract (mg/ml)2.8 3.52.1

Fig. 2. Superoxide radical scavenging by D. hamiltonii root extracts.

(Control, DA340 nm/min 0.1022) K Aqueous extract, Metha-nolic extract.

100

(%)

39 (2006) 10591065Dizdaroglu, 1989). In addition, this radical species isconsidered as one of the quick initiators of the LPOprocess, abstracting hydrogen atoms from unsaturatedfatty acids (Kappus, 1991).

3.5. Inhibition of microsomal lipid peroxidation

As shown in Fig. 4, both aqueous and methanolicextracts were equipotent in inhibiting LPO of livermicrosomes. IC50 values for the aqueous and methanolicextract were 0.51 and 0.46mg/ml, respectively (Table 2and Fig. 4). LPO has been broadly dened as theoxidative deterioration of polyunsaturated lipids(Kappus, 1991). Initiation of a peroxidation sequencein a membrane or polyunsaturated fatty acid is due toabstraction of a hydrogen atom from the double bond inthe fatty acid. The free radical tends to stabilize by amolecular rearrangement to produce a conjugated diene,which then readily reacts with oxygen molecule to give aperoxy radical (Jadhav, Nimbalkar, Kulkarni & Mad-havi, 1996). Peroxy radicals can abstract a hydrogen

Hyd

ro

00 63

Concentration of extract (mg/ml)9

Fig. 3. Hydroxyl radical scavenging by D. hamiltonii extracts. (Control

absorbance 0.67) K Aqueous extract, Methanolic extract.

since they reduce the redox potential thereby stabilizingthe oxidized form of the metal ion (Gordon, 1990).

3.8. Total phenolic content

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hamiltonii extracts. (Control value 6.5 n moles MDA/mg protein)

Met

al c

h40

20

00 60453015

Concentration of extract (mg/ml)75

Fig. 6. Metal ion chelating effect of D. hamiltonii extracts. (Control

absorbance 0.70) K Aqueous extract, Methanolic extract.

WT3.6. Reducing power

The reducing power of D. hamiltonii extracts wasconcentration-dependent (Table 2 and Fig. 5). Metha-nolic extract was being slightly more active than theaqueous extract. It is believed that antioxidant activityand reducing power are related (Duh, 1998; Duh, Tu, &Yen, 1999; Tanaka, Kuie, Nagashima, & Taguchi,1988). Reductones inhibit LPO by donating a hydrogenatom from another molecule to give lipid hydroper-oxide, R-OOH. A probable alternative fate of peroxyradicals is to form cyclic peroxides; these cyclicperoxides, lipid peroxides and cyclic endoperoxidesfragment to aldehydes such as malondialdehyde(MDA) and polymerization products. MDA and 4-hydroxy nonenal are the major break down products ofLPO. MDA is usually taken as a marker of LPO andoxidative stress (Janero, 1990).

K Aqueous extract, Methanolic extract.100

75

LPO

inhi

bitio

n (%

)

50

25

00 0.80.60.40.2

Concentration of extract (mg/ml)1

Fig. 4. Inhibition of liver microsomal lipid peroxidation by D.

A. Srivastava et al. / Latom and thereby terminating the free radical chainreaction (Yen & Chen, 1995).

3.7. Metal ion chelating activity

The ferrous ion-chelating effect was shown by both ofaqueous and methanolic extracts of Dh with IC50 valuesof 5.49 and 32.49mg/ml, respectively (Table 2 andFig. 6). Iron is known to generate free radicals throughthe Fenton and HaberWeiss reaction (Halliwell &Gutteridge, 1990). Metal ion chelating activity of anantioxidant molecule prevents oxyradical generationand the consequent oxidative damage. Metal ionchelating capacity plays a signicant role in antioxidantmechanism since it reduces the concentration of thecatalysing transition metal in LPO (Duh et al., 1999). Itis reported that chelating agents, which form s-bondswith a metal, are effective as secondary antioxidants0.6

0.4

Red

ucin

g po

wer

0.2

00 4321

Concentration of extract (mg/ml)5

Fig. 5. Reducing power of D. hamiltonii extracts. K Aqueous

extract, Methanolic extract.

100

80

60el

atio

n (%

)

39 (2006) 10591065 1063Phenolics content in the methanolic extract of Dh washigher than that of the aqueous extract (21.473.7 and13.872.4mg guaicol equivalents/g, respectively). Anti-oxidant activity of the plant extract is often associatedwith the phenolic compounds present in them. Hydro-gen donating property of the polyphenolic compounds isresponsible for the inhibition of free radical inducedLPO (Yen, Duh, & Tsai, 1993). In our study, thereseemed to be little correlation between the phenoliccontent and antioxidant activity of the extracts sinceaqueous extract with lower phenolic content showedhigher antioxidant activity. However, it is known thatnonphenolic antioxidants could also contribute to theantioxidant activity of an extract (Harish & Shivanan-dappa, 2005; Mariko, Hassimotto, Genovese, & Lajolo,2005).In order to characterize antioxidant activity of a plant

extract, it is desirable to subject it to a battery of tests

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Acknowledgements

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ARTICLE IN PRESSA. Srivastava et al. / LWT 39 (2006) 10591065 1065

Antioxidant activity of the roots of Decalepis hamiltonii (Wight & Arn.)IntroductionMaterials and methodsChemicalsPreparation of root powder and extractionDPPH radical scavenging assaySuperoxide radical scavenging assayHydroxyl radical scavenging assayInhibition of microsomal lipid peroxidationMeasurement of reducing powerMetal ion chelating assayTotal phenolic contentStatistical analysis

Results and discussionAntioxidant activity of the extractsDPPH radical scavenging activityScavenging of superoxide radicalHydroxyl radical scavenging activityInhibition of microsomal lipid peroxidationReducing powerMetal ion chelating activityTotal phenolic content

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