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Chemico-Biological Interactions 167 (2007) 125–134

Free radical scavenging reactions and antioxidant activity ofembelin: Biochemical and pulse radiolytic studies

Ravi Joshi a,∗, J.P. Kamat b, Tulsi Mukherjee a

a Radiation & Photochemistry Division, Chemistry Group, Bhabha Atomic Research Center,Mumbai 400085, India

b Radiation Biology & Health Sciences Division, Bhabha Atomic Research Center,Mumbai 400085, India

Received 27 November 2006; received in revised form 6 February 2007; accepted 6 February 2007Available online 15 February 2007

bstract

Embelin (from Embelia ribes) is a component of herbal drugs and possess wide range of medicinal properties. These propertiesay be, in part, due to scavenging of oxidizing free radicals. In this context, free radical scavenging reactions and antioxidant

ctivity of embelin (2,5-dihydroxy-3-undecyl-1,4-benzoquinone) have been studied. It has been found to scavenge DPPH radicalnd inhibit hydroxyl radical induced deoxyribose degradation. It has been also found to inhibit lipid peroxidation and restore impairedn-superoxide dismutase in rat liver mitochondria. Further, kinetics and mechanism of the reactions of embelin with hydroxyl, one-

lectron oxidizing, organo-haloperoxyl and thiyl radicals have been studied using nanosecond pulse radiolysis technique. Its redoxotential has been also evaluated with cyclic voltammetry. These studies suggest that embelin can act as a competitive antioxidantn physiological conditions.

2007 Elsevier Ireland Ltd. All rights reserved.

n vitro

eywords: Embelin; Free radicals; Antioxidant; Lipid peroxidation; I

. Introduction

In the last few decades, substantial increase in theesearch pertaining to natural antioxidants has beenbserved due to their non-toxicity and interaction witharious endogenous and exogenous free radicals in theiving cells [1–5]. In spite of the comprehensive network

f antioxidant defenses, many reactive oxygen speciesROS) still escape this surveillance and inflict variousytotoxic effects causing loss in structural/functional

∗ Corresponding author. Tel.: +91 22 25590301;ax: +91 22 25505151.

E-mail address: rjudrin@yahoo.com (R. Joshi).

009-2797/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reservdoi:10.1016/j.cbi.2007.02.004

studies; Pulse radiolysis

architecture of the cell. This oxidative damage leadsdirectly to cytotoxicity and/or indirectly to genotoxicitywith numerous serious anomalies favoring disharmonyand diseases [1,4]. Synthetic antioxidants such as buty-lated hydroxytoluene (BHT), butylated hydroxyanisole(BHA), etc. are widely used but they elicit adverse effectson human health in view of their inherent cytotoxicity[6].

The interest in antioxidants related to food and herbalmedicine is getting popularized due to their natural ori-gin, cost effectiveness, no side effects, and multifaceted

activities. These provide enormous scope in correctingthe oxidative imbalance, serving humans as food com-ponents, seasonings, beverages, cosmetics as well asmedicine. The World Health Organization has estimated

ed.

126 R. Joshi et al. / Chemico-Biological In

Scheme 1.

that about 80% of the earth’s inhabitants rely on tradi-tional medicine for their primary health care needs andmost of these therapies involve the use of preparationsfrom plant extracts [7]. Some of the popular Ayurvedicformulations used as drugs for anthelmintic, analgesicand antifertility properties [8–10], consist of parts of ashrub, Embelia ribes.

Embelin (2,5-dihydroxy-3-undecyl-1,4-benzoqui-none) is a constituent of E. ribes and has been alsoshown to possess antitumour properties [11]. Antitu-mour activity of such quinonic compounds is known tobe, in part, due to their redox reactions and formationof semiquinone radical [12]. Embelin is an interestingmolecule because of the presence of both quinone andphenolic groups on the same ring (Scheme 1). Embelinalso has a long alkyl chain (undecyl) as a substituent,which confers solubility in the non-polar phase and cellpermeability. Interestingly, the adjacent quinone andhydroquinone groups on the embelin form intramolecu-lar hydrogen bonds, which disappear on complexationwith inorganic metal ions [13]. The involvement ofquinonic groups in the antitumour activity of the drugsand intramolecular hydrogen bonding in embelin makeit an interesting molecule to study its reactions with thefree radicals.

This paper deals with the reactions of embelin withoxidizing free radicals and its antioxidant activity inin vitro systems. One of the physiologically importantfree radical, peroxyl radical, has been shown to induceoxidative damage to several biological components, likelipids, proteins and DNA [14–16]. The protective effectof embelin against the damage produced by these radi-cals has been elucidated in vitro in rat liver mitochondria.Effect of embelin on hydroxyl radical induced deoxyri-bose degradation and peroxyl radical induced loss ofmitochondrial Mn-SOD activity has also been studied.Further, its redox capacity has been studied using totalphenolic content assay, reduction of Fe(III) and mea-

surement of redox potential using cyclic voltammetry.Attempts have been made to understand the molecularmechanism of free radical scavenging reactions usingnanosecond pulse radiolysis technique.

teractions 167 (2007) 125–134

2. Materials and methods

2.1. Chemicals

Embelin was prepared by the method of Fieser andChamberlin [8]. Its purity has been checked as a sin-gle spot in TLC and a single peak in HPLC. 2-Thiobar-bituric acid, ascorbic acid, tris, 2,2′-azobis(2′-amid-inopropane) hydrochloride (AAPH), 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2-deoxyribose, gallic acid,quercetin, epinephrine, �-tocopherol and tetraethoxy-propane were purchased from Sigma Chemical Co. (St.Louis, MO, USA). All other chemicals were of analyt-ical reagent grade and solvents were of spectroscopygrade. De-ionised water (conductivity < 0.06 �S cm−1)obtained from a Barnstead nano-pure cartridge filtrationsystem (USA) was used for preparing solutions.

2.2. Methods

2.2.1. AnimalsWistar rats (male, 8–10 weeks) were bred in the

Animal House Facility and procured after obtainingclearance from Animal Ethics Committee. All the exper-iments were conducted with strict adherence to theethical guidelines laid down by the Committee for thePurpose of Control and Supervision of Experiments onAnimals (CPCSEA) constituted by the Animal WelfareDivision of Government of India on the use of animalsin scientific research. Animals were maintained undercontrolled laboratory conditions and fed standard animalfood and tap water ad libitum.

2.2.2. Isolation of rat liver mitochondriaThe isolation of mitochondria from rat liver was

carried out as described previously [17]. Briefly, malerats weighing 200 ± 20 g were fasted overnight, sac-rificed by cervical dislocation. Livers were removed,homogenized in 0.25 mol dm−3 sucrose containing1 × 10−3 mol dm−3 EDTA. The homogenate was cen-trifuged at 3000 × g for 10 min to remove cell debrisand the nuclear fraction. The resulting supernatantwas centrifuged at 10,000 × g for 10 min to sedimentmitochondria in a Sorvall RC5C centrifuge. The mito-chondrial pellets thus obtained were washed thrice with50 × 10−3 mol dm−3 potassium phosphate buffer (pH7.4) to remove sucrose and were suspended in the samebuffer at a concentration of 10 mg protein/mL.

2.2.3. Generation of ROS in rat liver mitochondriaRat liver mitochondria (1 mg protein/mL) were

exposed to AAPH that decomposes at 37 ◦C and gen-

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R. Joshi et al. / Chemico-Biolog

rates peroxyl radical [15]. Oxidative damage wasonitored after exposing mitochondria with and without

mbelin to AAPH.

.2.4. Measurement of oxidative damage initochondriaOxidative damage in liver mitochondria was mea-

ured using thiobarbituric acid reactive substancesTBARS) and superoxide dismutase (Mn-SOD) assays.fter exposing to AAPH, TBARS were isolated by boil-

ng mitochondrial samples for 15 min at 100 ◦C withhiobarbituric acid (TBA) reagent (0.5% 2-thiobarbituriccid/10% trichloroacetic acid (TCA)/0.63 mol dm−3

ydrochloric acid) and measuring the absorbance at32 nm against a blank unexposed to AAPH. Thealonaldehyde equivalents of the sample were calcu-

ated using tetraethoxypropane as standard. The resultsere expressed as nmol TBARS/mg protein, using= 1.56 × l05 dm3 mol−1 cm−1 [17].

.2.5. Superoxide dismutase (SOD) assayThe assay of mitochondrial Mn-SOD activity was

ased on the spectrophotometric detection (absorbancet 320 nm) of adrenochrome formed by auto-oxidationf epinephrine in 50 × 10−3 mol dm−3 sodium carbonateuffer at pH 10. Appropriate sample blanks were takeno ensure that embelin did not interfere with the assay18].

.2.6. Inhibition of hydroxyl radical inducedeoxyribose degradation

The hydroxyl radical scavenging activity of embelinas determined as reported previously [19]. Briefly, the

eaction mixture (1 mL) contained 28 × 10−3 mol dm−3

-deoxyribose, 10 × 10−3 mol dm−3 Tris–HCl bufferpH 7.4), 1 × 10−4 mol dm−3 FeCl3, 1 × 10−4 molm−3 EDTA, 1 × 10−3 mol dm−3 H2O2 and embelin.he reaction was initiated by adding ascorbic acid

3 × 10−4 mol dm−3). After incubating the mixture forh at 37 ◦C, it was treated with 30% ice-cold HCl con-

aining 0.75% TBA. The mixture was boiled for 30 min,ooled and the absorbance of the supernatant was readt 532 nm. Mannitol was used as a positive control.

.2.7. DPPH assayDPPH is a stable free radical having an absorp-

ion maximum at 517 nm with ε = 9660 dm3 mol−1 cm−1

20]. In the presence of the compounds capable of

onating H-atom or an electron there is a decrease inbsorbance at 517 nm. We have studied the ability ofmbelin to neutralize DPPH radicals. One milliliter of00 × 10−3 mol dm−3 DPPH in methanol was mixed

eractions 167 (2007) 125–134 127

with equal volume of the embelin solution in phosphatebuffer (pH 7.4). The mixture was shaken and kept in darkfor 20 min. The absorbance at 517 nm was monitored inthe presence and absence of different concentrations ofthe embelin.

2.2.8. Reducing powerThe reducing power of embelin was measured using

chemical reduction of Fe(III) to Fe(II). The absorbanceof the reaction mixture was read at 700 nm. Theestimation was carried out by treating embelin atvarious concentrations (0.51–10.2 × 10−4 mol dm−3 inmethanol) with 1% potassium ferricyanide at 50 ◦Cfor 20 min. After centrifugation, clear supernatant wastreated with 0.1% FeCl3 and absorbance was measuredat 700 nm [2].

2.2.9. Measurement of total phenolic contentsThe measurement of total phenolic contents is

based on the reduction of Folin-Ciocalteu reagentby reducing equivalents from phenols. Briefly, Folin-Ciocalteu reagent was mixed equally with embelin(0.51–10.2 × 10−4 mol dm−3), mixture was treated with80% Na2CO3 and absorbance was measured at 730 nm[2].

2.2.10. Pulse radiolysisThe pulse radiolysis system giving pulses of 7 MeV

electrons from a linear electron accelerator for in situgeneration and study of free radicals has been used [21].High-energy electrons deposit energy in chemical sys-tem to generate its free radicals and molecular species(Eq. (1)):

(1)

The yields of these species depend on the radiationdose deposited by ionising radiation in the medium,which in turn depends on linear energy transfer ofhigh-energy electrons. The absorbed dose was mea-sured using an air-saturated aqueous solution containing5 × 10−2 mol dm−3 KSCN (Gε = 2.6 × 10−4 m2 J−1 at475 nm) [22]. The kinetic spectrophotometric detec-tion system covered the wavelength range from 250 to800 nm. The optical path length of the cell was 1.0 cm.The width of the electron pulse was 50 ns and the doseused was 16 Gy (1 Gy = 1 J energy deposited in 1 kg ofmaterial). Alkaline pH values were adjusted by adding

KOH only. High purity (>99.9%) N2O was used forpurging solutions as per requirement.

Relative absorption of radicals in the UV–vis regionhas been observed against time and wavelength to get

ical Interactions 167 (2007) 125–134

128 R. Joshi et al. / Chemico-Biolog

kinetic and absorption characteristics, respectively. Thebimolecular rate constants were calculated by plottingpseudo-first order rate of formation of the transientagainst the concerned solute concentration. The timepoints in transient absorption spectra refer to the timeelapsed after the irradiation with the electron pulse. Theuncertainty in the measurement of wavelength, transientabsorption and least square fitting to get bimolecular rateconstants are ∼3 nm, ∼10% and <5%, respectively.

2.2.11. Cyclic voltammetryThe cyclic voltammetric experiment was carried out

using an Autolab Electrochemical System (Eco Chemie,The Netherlands) equipped with PGSTAT 20 and drivenby GPES software. The working electrode was a highlypolished glassy carbon electrode (GCE) with a diam-eter of 2 mm, and a platinum wire was used as acounter electrode. The reference electrode was saturatedcalomel electrode (SCE) with a salt bridge containing3 mol dm−3 KCl solution. The GCE surface was pol-ished with aqueous slurry of alumina powder before eachmeasurement. Solutions were de-aerated by bubblingN2 for 10 min prior to recording the cyclic voltammo-gram.

3. Results and discussion

3.1. Biochemical studies

ROS are known to cause deleterious oxidative dam-age to cell components and its biological consequenceson living cells are well known [1,5,23]. Among themthe hydroxyl radical (•OH) is one of the most dam-aging oxidizing radicals produced in the physiologicalsystems [24,25]. Generation of •OH is also reportedin AAPH solution [26,27]. Its reactions with variousbio-molecules (proteins, lipids, DNA bases, etc.) arediffusion-controlled and are well compiled [28]. Inter-estingly, lipid peroxidation process is associated with theformation of short-lived carbon-centered radical, whichis quenched by oxygen at diffusion-controlled rates toproduce alkylperoxyl radical [26,27]. Though cells pos-sess a comprehensive and integrated endogenous repairsystem, they are attacked by a wide range of oxidizingradicals [1,2,4,5,24,25]. Therefore, protective effect ofembelin was examined against these patho-physiologicalradicals.

Fig. 1 demonstrates the data on DPPH scav-

enging effect of embelin at various concentrations(0.51–10.2 × 10−4 mol dm−3). It can be seen thatembelin showed 25%, 32% and 50% scavenging ofDPPH radical, respectively, at 0.51 × 10−4, 1.02 ×

Fig. 1. Scavenging of DPPH radical by embelin.

10−4 and 2.55 × 10−4 mol dm−3 concentration. Furtherincrease in the embelin concentration above 2.55 ×10−4 mol dm−3 did not show significant change inthe absorbance of DPPH radical. The IC50 value forthe embelin, i.e. the concentration at which it scav-enges 50% of the DPPH radical, has been found to be2.55 × 10−4 mol dm−3. Therefore, further studies linkedto oxidative parameters have been carried out using0.85 × 10−4 and 1.7 × 10−4 mol dm−3 of embelin.

Fig. 2a demonstrates the data on rat liver mito-chondrial lipid peroxidation following exposure toperoxyl radical with and without embelin. A signif-icant increase in the formation of TBARS has beendemonstrated on AAPH exposure as compared to unex-posed control (control without AAPH exposure 1.2 ±0.098 nmol of TBARS/mg protein). The results showedsignificant inhibition in TBARS formation show-ing 42% and 58% decrease at 0.85 × 10−4 and1.7 × 10−4 mol dm−3 of embelin, respectively, as com-pared to the sample exposed to AAPH only. The effecthas been also compared with �-tocopherol, a well-known scavenger of peroxyl radical. �-Tocopherol hasbeen found to cause 41% reduction in TBARS forma-tion at 1.0 × 10−4 mol dm−3, which is comparable to thatproduced by embelin at 0.85 × 10−4 mol dm−3. Here, ithas to be noted that TBARS method measures the con-centration of total carbonyl present in the system, whichis formed by the oxidation of lipids, proteins and DNA[3].

Embelin was examined for its modulating effect onantioxidant defense enzyme, mitochondrial superoxide

dismutase (Mn-SOD) with AAPH exposure. The Mn-SOD activity has been found to reduce significantly to23% on AAPH exposure. Embelin showed significanteffect resulting in extensive restoration (Fig. 2b) to 91%

R. Joshi et al. / Chemico-Biological Interactions 167 (2007) 125–134 129

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ig. 2. Antioxidant effect of embelin on rat liver mitochondria exposed1) unexposed, (2) exposed, (3) exposed + embelin 0.85 × 10−4 mol dm.58 × 10−4 mol dm−3 and (6) exposed + �-tocopherol 1.0 × 10−4 mo

nd 96% at 0.85 × 10−4 and 1.70 × 10−4 mol dm−3,espectively, as compared to the control. On the contrary,-tocopherol has not shown such protective effect at.58 × 10−4 mol dm−3 but almost complete restorationas observed at 1.0 × 10−4 mol dm−3. These studies

how that embelin is better than �-tocopherol in inhibit-ng lipid peroxidation and preventing Mn-SOD fromeroxyl radical attack.

Fig. 3a demonstrates concentration dependent scav-nging of hydroxyl radical by embelin using deoxyriboseegradation assay. The inhibition observed with embelint 0.51 × 10−4, 1.02 × 10−4, 2.55 × 10−4, 5.1 × 10−4,0.2 × 10−4 mol dm−3 was 20%, 50%, 62%, 69% and9%, respectively. This suggests high reactivity of embe-in with hydroxyl radical, which has been also studied

sing nanosecond pulse radiolysis technique.

The phenolic compounds from food and herbs arenown to be responsible for antioxidant activity and phe-olic groups also serve as index of antioxidant function

Fig. 3. (a) Hydroxyl radical scavenging activity and (b) total availa

PH for TBARS formation (a), and superoxide dismutase activity (b) atxposed + embelin 1.70 × 10−4 mol dm−3, (5) exposed + �-tocopherol

[2]. Therefore, the estimation of total available phenoliccontents of embelin has been carried out (Fig. 3b) to findH-donating capacity of the two hydroxy groups presenton the benzoquinone ring of embelin. Gallic acid hasbeen used as a positive control. Embelin showed phenoliccontents as 7.2, 11.8, 25.2, 42.4 and 63.0 �g equivalentgallic acid at 0.51 × 10−4, 1.02 × 10−4, 2.55 × 10−4,5.1 × 10−4, 10.2 × 10−4 mol dm−3, respectively. Thissuggests that on an average less than one phenolic groupper embelin molecule is available for redox reactions,which could be due to strong intramolecular hydrogenbonding of phenolic groups with the adjacent quinonicgroups.

The ferric reducing ability (Fe(III) → Fe(II)) ofembelin was measured in the concentration range

0.51–10.2 × 10−4 mol dm−3 (Fig. 4). In this assay,increased absorbance indicated increased reducingpower. The reducing power of embelin was detectedeven at 0.51 × 10−4 mol dm−3. However, it was found to

ble phenolic content of embelin at various concentrations.

130 R. Joshi et al. / Chemico-Biological Interactions 167 (2007) 125–134

Fig. 4. Fe(III) reducing ability of embelin.

be four times higher at 1.02 × 10−4 mol dm−3 than thatat 0.51 × 10−4 mol dm−3. At 2.55 × 10−4 mol dm−3, itwas further increased by 1.5 times. A steep increasewas observed above 2.55 × 10−4 mol dm−3. The highFe(III) reducing ability of embelin shows its hydrogenatom donating capacity resulting in antioxidant action.The Fe(III) reducing ability of embelin can also result inprooxidant effect by increasing the concentration of theFe(II) for Fenton reaction. However, embelin has beenfound to be an efficient hydroxyl radical scavenger evenat 1.02 × 10−4 mol dm−3 (Fig. 3a).

These biochemical studies demonstrated a significantprotection by embelin against various oxidative stres-sors. The formation of TBARS, an index of peroxidationreactions, is significantly reduced in the presence ofembelin. Peroxyl radical induced depletion of Mn-SODis also effectively prevented by embelin. In addition,its protective capacity is found to be better than �-tocopherol, an established chain breaking antioxidant.The available phenolic groups for H-donation have beenfound to be lesser than those present on each molecule(two phenolic groups per molecule). Embelin has beenfound to be highly reactive to hydroxyl radical. It alsoshows high potential for reduction of Fe(III) → Fe(II)justifying antioxidant function.

3.2. Pulse radiolytic studies

To understand the molecular mechanism for antiox-idant action of embelin, kinetics and mechanism of itsreaction with hydroxyl, one-electron oxidizing, peroxyl

and thiyl radicals have been studied using nanosecondelectron pulse radiolysis. Since embelin has poor solu-bility in neutral aqueous solutions due to intramolecularhydrogen bonding, experiments have been performed in

Fig. 5. Transient absorption spectrum observed from N2O-saturatedaqueous solution for embelin (1 × 10−4 mol dm−3) at pH 10. Inset:corrected spectrum at 90 �s. Dose = 16 Gy.

alkaline media in aqueous solutions. In aqueous-ethanolsolution pKa has been found to be 6.8.

3.2.1. Reaction with oxidantsReaction of embelin with hydroxyl radical (•OH) at

pH 10 has produced a broad transient absorption spec-trum in the wavelength region of 380–480 nm up to100 �s (Fig. 5). Transient absorption spectrum at 90 �s(inset of Fig. 5) corrected for the ground state absorp-tion of embelin showed absorption bands at 320 and∼450 nm.

Further, the reaction of embelin with one-electronoxidants namely, azidyl (N3

•) and trichloromethyl per-oxyl radicals (•CCl3O2) has been studied to distinguishthe absorption bands of phenoxyl type radical fromOH-adduct and transient due to H-abstraction. Thesereactions at pH > 10 produced transient absorption spec-tra different from that observed with •OH radical andhave absorption maximum at ∼410 nm in both the cases(Fig. 6). The 410 nm absorption has been found todecay by second order kinetics (2k/ε = 7 × 104 s−1) up to1 ms and later both 410 and 370 nm absorption decayedslowly.

Reaction of embelin (O2E(OH)2) with hydroxyl rad-ical and oxidants is expected to produce adducts andphenoxyl type radical, respectively (Scheme 2). A com-parison of transient absorption spectra observed in itsreaction with hydroxyl radical (Fig. 5) to those withone-electron oxidants (Fig. 6) suggests that the broadabsorption band in the 380–480 nm region can be

ascribed to transients produced by OH-addition (I inScheme 2) and/or H-abstraction. Similarly, the absorp-tion bands observed at 410 and 330 nm (with N3

•) canbe ascribed to phenoxyl type radical (II in Scheme 2)

R. Joshi et al. / Chemico-Biological Interactions 167 (2007) 125–134 131

F saturated aqueous solution of embelin (1 × 10−4 mol dm−3) and NaN3

( in the inset. (B) Aerated aqueous solution of embelin (1 × 10−4 mol dm−3),t and kinetic traces at (a) 370 nm and (b) 410 nm in the inset. Dose = 16 Gy.

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Table 1Rate constants (k, dm3 mol−1 s−1) and transient absorption maximumfor the reaction of embelin with various radicals

Radical pH λmax (nm) k

•OH 10.0 440 7.2 × 109

N3• 10.5 410, 370 3.5 × 109

CCl3O2• 11.4 410, 370 8.0 × 108

• 8

ig. 6. Transient absorption spectrum observed from (A) N2O-1 × 10−2 mol dm−3) at pH 10.5 and corrected absorption spectrumert-butanol (2 mol dm−3) and CCl4 (1 × 10−2 mol dm−3) at pH 11.4

Eqs. (2) and (3)) [29,30]:

•OH + O2E(OH)2 → O2E(OH)3• (I)

→ O2E(OH)O• (II) + H2O (2a)

2E(OH)3• (I) → products (2b)

•ox + O2E(OH)2 → [O2E(OH)2]•+

→ O2E(OH)O• (II) + H+ (3)

n the reaction of embelin with N3• and •CCl3O2 radicals

here is a simultaneous decay of absorption at 410 nm andelayed formation at ∼330 nm in the corrected absorp-ion spectrum (insets of Fig. 6A and B). This delayedormation of absorption band at ∼330 nm could be dueo loss of alkyl chain leading to another carbon-centeredransient. We have observed similar evolution of tran-ients earlier with folic acid on removal of alkyl chain

31]. Such time resolved evolution of absorption has noteen observed with •OH radical. This absence of timeesolved evolution with hydroxyl radical could be dueo formation of •OH-adduct with ring or H-abstraction

Scheme 2

GS 3.9 430, 320 7.9 × 10Emb• + AscH− 10.0 410, 360 5.8 × 107

from side alkyl chain of embelin (Eq. (2b)) [29,30]. Thishas been confirmed by scavenging of 440 nm absorption,observed in the reaction of embelin and •OH, with oxy-gen (k = 8.8 × 108 dm3 mol−1 s−1). Corrected transientabsorption spectrum for the reaction with azidyl radical(inset of Fig. 6A) also showed time resolved evolutionof absorption bands with maximum at 330 and 410 nm.

The reaction rate constants and absorption maxima ofthe transient/s observed in the reaction of embelin withvarious radicals are given in Table 1. The reaction withN3

• and •CCl3O2 radicals produce mainly one-electron

.

ical Interactions 167 (2007) 125–134

Fig. 7. Transient absorption spectrum observed from aeratedaqueous solution for embelin (1 × 10−4 mol dm−3), ascorbic

132 R. Joshi et al. / Chemico-Biolog

oxidized transient of aromatic compounds whereas•OH causes adduct formation and/or H-abstractionalso. Therefore, the absorption bands formed initiallyat ∼410 and ∼330 nm with N3

• and •CCl3O2 havebeen ascribed to the phenoxyl type radical of embe-lin, namely O2E(OH)O•. A broad absorption band inthe 380–480 nm region has been ascribed to OH-adductformation and/or H-abstraction. Radiolysis of embelinin non-polar solvents, CH2Cl2 and 1-chlorobutane, hasnot resulted in oxidation reaction probably due to strongintramolecular hydrogen bond. A comparison of tran-sient absorption data at 410 nm also suggests that only35% of the •OH radical cause one-electron oxidationand rest cause either formation of •OH adducts on thering and/or H-abstraction from alkyl side chain. Further,85% of •CCl3O2 radical causes one-electron oxidationas compared to that with N3

• radical reaction.

3.2.2. Reaction with RS• and scavenging byascorbate

The medicinal use of embelin makes it imperative tostudy reactions of embelin with other free radicals ofbiological interest and repair of embelin free radical bycommonly used antioxidant, ascorbic acid.

Thiyl radicals (RS•) are generated in the cellularredox processes and in the antioxidant action of glu-tathione. These are reactive oxidants and attack lipidsto cause their oxidative degradation or isomerization[32,33]. Therefore, the repair of thiyl radical is neces-sary for the storage of thiols (RSH) for their antioxidantactivity as well as to protect lipids. Thiyl radical isgenerally prepared by irradiation of N2O saturated 1:1water–ethanol solution at pH below 7 to avoid the for-mation of unwanted disulfide radical anion (RSSR•−)(Eqs. (4)–(6)):

H•/•OH + RCH2OH → H2/H2O + RC•HOH (4)

RC•HOH + RSH → RS• + RCH2OH (5)

RS− + RS• � RSSR•− (6)

Reaction of embelin with glutathiyl radical (GS•), at pH3.9, has shown an absorption spectrum similar to thatof phenoxyl radical with maxima at ∼430 and 320 nm(figure not shown here). However, no time resolutionhas been observed in the absorption spectrum with GS•.This suggests that GS• reacts with embelin to produceits phenoxyl type radical only.

Vitamin C (ascorbic acid or AscH ) is a well-known

2water-soluble physiological antioxidant [34]. It has pKaat 4.1 and 11.8 and exists as anion (AscH−) at pH 7.Its one-electron oxidized radical (Asc•−) has absorp-tion maximum at 360 nm with ε = 3300 mol−1 dm3 cm−1

acid (5 × 10−5 mol dm−3), tert-butanol (2 mol dm−3) and CCl4(3 × 10−2 mol dm−3) at pH 10. Inset: kinetic traces at (a) 360 nm and(b) 410 nm under identical conditions. Dose = 16 Gy.

[35]. Therefore, scavenging of embelin phenoxyl radi-cal (II in Scheme 2) by ascorbate has been studied. Ithas been observed that there is a simultaneous decay ofabsorption of II at 410 nm and growth of Asc•− at 360 nmat pH 10 (Fig. 7). The absorption–time profiles (insetof Fig. 7) show that embelin phenoxyl radical (410 nm)is oxidizing in nature and is scavenged by AscH− toproduce Asc•− (360 nm). Though, direct oxidation ofascorbate occurs in the first step but the later forma-tion of ascorbate radical (>20 �s) clearly confirms thepostulation.

3.3. Cyclic voltammetry of embelin

Cyclic voltammetry has been shown to determine theredox potential and the total antioxidant capacity of natu-ral products, biological samples, etc. [36]. The potentialat which the anodic current is maximum and the areaunder the current–voltage curve are indicators of theantioxidant potential and capacity, respectively. There-fore, oxidation of embelin has been studied using cyclicvoltammetry at pH 9.5 in water. The cyclic voltammo-gram of embelin shows (Fig. 8) a maximum at +0.495 Vversus SCE (+0.72 V versus NHE), which has beenassigned to O2E(OH)O•/O2E(OH)2 couple (Eq. (7)):

O2E(OH)O• + H+ + e− → O2E(OH)2

(E = +0.72 V versus NHE) (7)

•OH + e− → −OH (E = +1.9 V versus NHE) (8)

R. Joshi et al. / Chemico-Biological Int

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ig. 8. Cyclic voltammogram of oxidation of embelin at pH 9.5 inater with SCE as reference electrode.

⇒ •OH + O2E(OH)2 → O2E(OH)O• + H2O

(ΔE = +1.18 V) (9)

nd

O2E(OH)O• + AscH− → Asc•− + O2E(OH)2

(ΔE = +0.39 V) (10)

he measured reduction potential (+0.72 V versus NHE)f embelin couple (O2E(OH)O•/O2E(OH)2) is lesserhan those of physiologically present oxidizing radi-als namely, hydroxyl (•OH/−OH, +1.9 V), alkylperoxylRO2

•/RO2−, >1.0 V), and thiyl (RS•/RS−, ∼1.1 V)

adicals and more than that of ascorbic acid cou-le (Asc•−/AscH−, +0.33 V) [37]. This suggests thatmbelin can scavenge hydroxyl, peroxyl and glutathiyladicals, as reported in earlier sections, to produceess reactive embelin phenoxyl radical (Eqs. (8) and9)). Further, embelin phenoxyl radical produced inhese scavenging reactions has been found to be scav-nged by ascorbic acid (Asc•−/AscH−, +0.33 V) (Eq.10)). The reduction potential value of embelin coupleO2E(OH)O•/O2E(OH)2) measured with cyclic voltam-etry study agrees well with the free radical scavenging

ctivity of embelin and scavenging of embelin radical byscorbic acid.

. Conclusion

Embelin has been found to efficiently scavenge phys-ologically relevant oxidizing radicals. Interestingly,mbelin scavenged hydroxyl, glutathiyl and peroxyl rad-cal to produce embelin phenoxyl radical (λmax 410 nm),

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eractions 167 (2007) 125–134 133

which in turn is scavenged by ascorbate anion. Inhibitionof lipid peroxidation, reduction of Fe(III) and restorationof Mn-superoxide dismutase in in vitro studies suggestthat it can be an efficient antioxidant in physiologicalsystem. Further, its free radical scavenging activity hasbeen also found to be better than �-tocopherol. It can beinferred that in addition to its biochemical roles, antiox-idant action of embelin may also be attributed to itsmedicinal properties.

Acknowledgement

Authors wish to thank Dr. Shilpa Tawade for the assis-tance with cyclic voltammetric measurements.

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