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Chemico-Biological Interactions 173 (2008) 97–104

Contents lists available at ScienceDirect

Chemico-Biological Interactions

journa l homepage: www.e lsev ier .com/ locate /chembio int

ntioxidant-mediated protective effect of potato peel extract inrythrocytes against oxidative damage

andita Singh, P.S. Rajini ∗

ood Protectants and Infestation Control Department, Central Food Technological Research Institute, Mysore 570013, India

r t i c l e i n f o

rticle history:eceived 20 November 2007eceived in revised form 12 March 2008ccepted 17 March 2008vailable online 22 March 2008

eywords:otato peel extractat erythrocyteuman erythrocyte membranecanning electron microscopyxidative damagehenolic antioxidant

a b s t r a c t

Potato peels are waste by-product of the potato processing industry. They are reportedlyrich in polyphenols. Our earlier studies have shown that extracts derived from potato peel(PPE) possess strong antioxidant activity in chemical and biological model systems in vitro,attributable to its polyphenolic content. The main objective of this study was to investigatethe ability of PPE to protect erythrocytes against oxidative damage, in vitro. The protectionrendered by PPE in erythrocytes was studied in terms of resistance to oxidative damage,morphological alterations as well as membrane structural alterations. The total polyphe-nolic content in PPE was found to be 3.93 mg/g powder. The major phenolic acids presentin PPE were predominantly: gallic acid, caffeic acid, chlorogenic acid and protocatechuicacid. We chose the experimental prooxidant system: FeSO4 and ascorbic acid to inducelipid peroxidation in rat RBCs and human RBC membranes. PPE was found to inhibit lipidperoxidation with similar effectiveness in both the systems (about 80–85% inhibition byPPE at 2.5 mg/ml). While PPE per se did not cause any morphological alteration in the ery-

throcytes, under the experimental conditions, PPE significantly inhibited the H2O2-inducedmorphological alterations in rat RBCs as revealed by scanning electron microscopy. Further,PPE was found to offer significant protection to human erythrocyte membrane proteinsfrom oxidative damage induced by ferrous–ascorbate. In conclusion, our results indicatethat PPE is capable of protecting erythrocytes against oxidative damage probably by acting

ant.

as a strong antioxid

. Introduction

Increasing evidence suggests that oxidative damage toell components may play an important pathophysiologicalole in many types of human diseases [1]. The plasma mem-rane is a dynamic organelle system tightly controlling cel-

ular structure and function. Its structure and functions areusceptible to alterations as a consequence of interactions

ith xenobiotics and damage to any of its components may

n turn influence the integrity of cell structure and function.s is well known, lipid peroxidation (LPO) is the outcomef free radical mediated chain oxidation reaction of mem-

∗ Corresponding author. Tel.: +91 821 2513210; fax: +91 821 2517233.E-mail address: rajini29@yahoo.com (P.S. Rajini).

009-2797/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.cbi.2008.03.008

© 2008 Elsevier Ireland Ltd. All rights reserved.

brane polyunsaturated fatty acids (PUFAs), which disruptsthe structural and protective functions of cell membranes,and as a consequence, various pathological events areimplicated as a result of this oxidation [2]. The oxidativeprocess yields various lipid hydroperoxides and secondaryproducts, which ultimately result in structural disruptionof membrane lipid bilayer and produce deleterious effectson the activities of membrane-bound proteins [3].

Erythrocytes, potentially powerful promoters of oxida-tive processes, are extremely susceptible to oxidativedamage as a result of the high polyunsaturated fatty acid

content of their membranes and high cellular oxygen andhemoglobin (Hb) concentrations [4,5]. Malondialdehyde(MDA), the well-characterized product of the LPO of ery-throcytes, is a highly reactive and bifunctional molecule,which is shown to cross-link erythrocyte phospholipids

ological

98 N. Singh, P.S. Rajini / Chemico-Bi

and proteins to impair a variety of the membrane-relatedfunctions, which ultimately lead to diminished erythro-cyte survival [6–8]. Further, erythrocyte LPO is reportedlyinvolved in normal cell aging, and it has also been associ-ated with a variety of pathological events. Oxidants alsoproduce alterations in erythrocyte membranes as mani-fested by a decreased cytoskeletal protein content (lowmolecular weight, LMW), and production of high molecularweight (HMW) proteins [9], which can lead to abnor-malities in erythrocyte shape and disturbances in themicrocirculation [10].

Different models have been employed to detect andunderstand both the effects of reactive oxidizing speciesand the activity of natural and synthetic scavengers. Dueto their ready accessibility, ease of preparation, abundanceof polyunsaturated fatty acids and membrane proteins, andwealth of available information, erythrocyte membrane isan excellent model for the study of biomembrane toxicityin vitro [11,12] and have been extensively adopted [13,14].The key role of oxidative stress and iron release in a reac-tive form causing membrane protein damage via the Fentonreaction and hydroxyl radical production is well demon-strated.

There is an increasing interest in the protective bio-chemical function of naturally occurring antioxidants inbiological systems and on the mechanism of their action.Several plant constituents have been proven to possessconsiderable free radical scavenging or antioxidant activity[15–17]. Flavonoids and other phenolic compounds of plantorigin have been reported as scavengers and inhibitors oflipid peroxidation [18]. The primary protective mechanismof a phenolic antioxidant is to trap and stabilize free rad-ical species [19]. Recent studies have shown that differentflavonoids can interact with synthetic membranes (lipo-somes), protecting the bilayer from both the disruptioninduced by a detergent and free radical mediated lipid oxi-dation [20–22]. The molecular mechanisms underlying theantioxidant action of polyphenols have not yet been fullyelucidated and are still a matter of considerable debate.However, it has been speculated that the ability of thesecompounds to partition in cell membranes and the result-ing restriction on membrane fluidity could sterically hinderdiffusion of free radicals and thereby decrease the kineticsof free radical reactions [23].

In a previous study, we evaluated the antioxidant activ-ity of an extract of potato peel in several in vitro assaysystems [24] and also found that aqueous extract (PPE) ofpotato peel (a waste by-product of potato processing) isrich in various phenolic acids [25]. The different extracts ofpotato peel displayed phenolic contents ranging from 2.9to 4.2 mg/g potato peel powder. There was a strong corre-lation between the total phenolic content and antioxidantactivity. The objective of the present study was to exam-ine the protective effect of PPE against biochemical andmorphological alterations induced in RBC by exogenousoxidants. Further, intact rat erythrocytes exposed to proox-

idants and exposed to potato peel extract were observed byscanning electron microscope (SEM) while isolated humanerythrocyte membranes were studied for protein alterationby SDS–PAGE. The extent of lipid peroxidation was alsoassayed in both the systems.

Interactions 173 (2008) 97–104

2. Materials and methods

2.1. Chemicals

Hydrogen peroxide (H2O2), thiobarbituric acid (TBA),bovine serum albumin (BSA), N,N,N′,N′-tetramethylethyl-enediamine (TEMED), acrylamide, �-mercaptoethanol,bromophenol blue and ethylenediamine tetracetic acid(EDTA) and standard phenolic acids (Gallic acid, chloro-genic acid, caffeic acid and protocatechuic acid) werepurchased from M/s Sigma Chemicals Co. (St. Louis, MO,USA). Trichloroacetic acid (TCA), sodium dodecyl sulphate(SDS), methylene-bis acrylamide (Bis), glutaraldehyde, fer-rous sulphate, ascorbic acid, Coomassie Brilliant Blue,ammonium persulphate (APS), glycerol, Trizma base, andFolin’s reagent were purchased from M/s Sisco ResearchLaboratory (Mumbai, India). All other chemicals used wereof analytical grade.

2.2. Preparation of potato peel extract (PPE)

Fresh potato peel obtained from local potato chips mak-ing units were washed three times with tap water and thendried at 70 ◦C for 5 h in a ‘cross-flow air drier’. The dried peelwas ground in a multimill and passed through a 0.5 mmsieve to obtain a fine powder. 0.5 g of potato peel powderwas homogenized with 10 ml of distilled water for 5 minand the homogenate was centrifuged at 10,000 rpm for10 min. The supernatant was filtered through Whatman No.1 filter paper, and the resultant extract was lyophilized todryness in vacuo. The lyophilized powder (PPE) was storedin dark bottle at 4 ◦C until use.

2.3. Estimation of total phenolic content in PPE

Total phenolic content of the extract was quantifiedusing Folin–Ciocalteu reagent according to the modifiedmethod of Singleton and Rossi [26]. Briefly, aliquots of theextract with volume adjusted to 3 ml with distilled waterwere incubated with 0.5 ml of 95% ethanol and 0.25 ml ofFolin’s reagent (1:1 diluted with distilled water) for 5 minat room temperature. To this 0.5 ml of Na2CO3 (5%) solu-tion was added, mixed and the mixture was incubated for60 min at room temperature. The absorbance of the solu-tion was then read at 720 nm against a reagent blank. Gallicacid (0.1 mg/ml) was used as the standard and the phenoliccontent in the extract was expressed as milligram equiva-lent of gallic acid (GAE) per gram peel powder.

2.4. HPLC analysis of extract

Chromatographic analysis was performed on Shi-madzu LC-6AV HPLC (Tokyo, Japan), equipped with aternary pump delivery system. Chromatographic sepa-ration was performed using a Bond-pak C-18 column(250 mm × 4.6 mm i.d., particle size 10 �m; Shimpak,

Japan). Aliquots (15 �l) of extract or standard phenolicacid solutions (1 mg/ml) were injected for analysis. Themobile phase, water:methanol:acetic acid (64:35:1, v/v/v)was delivered isocratically at 0.7 ml/min, and the effluentwas monitored to identify and quantify the phenolic acid in

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3.93 mg/g powder. The major phenolic acids present in PPEwere: gallic acid, caffeic acid, chlorogenic acid and proto-catechuic acid (Fig. 1). The relative composition of the fourphenolic acids in PPE is presented in Table 1.

N. Singh, P.S. Rajini / Chemico-Bi

he extract and expressed as % phenolic acid in the extract27].

.5. Preparation of rat blood cells

Heparinised rat peripheral blood was collected fromFT-Wistar, male albino rats (150–200 g). Plasma and buffyoat were carefully removed after 10 min centrifugationt 500 × g and 4 ◦C. RBCs were harvested after repeatedlyashing in 10 vol. of isotonic buffered saline (PBS: 0.137 MaCl, 3 mM KCl, 1.9 mM NaH2PO4, 8.1 mM Na2HPO4, pH.4). Packed cells were suspended finally in known volumef isotonic buffer.

.6. Preparation of human erythrocyte ghosts (HEG)

Outdated acid-citrate-dextrose (ACD)-treated humanlood was used. Plasma and buffy coat were carefullyemoved after 10 min centrifugation at 500 × g and 4 ◦C.BCs were harvested after repeated washing in PBS.he packed cells were hemolysed by diluting 1:10 withypotonic sodium phosphate buffer (5 mM, pH 8) [28].BC ghosts were obtained after 30 min centrifugation at0,000 × g. Pelletted membranes were washed three timesr until they were pink, by repeated centrifugation at0,000 × g for 30 min. Ghosts were suspended in PBS.

.7. Lipid peroxidation assay

Rat RBCs and human erythrocyte ghosts (equivalento 1 mg protein) were incubated with ferrous sulphate10 �M) and ascorbic acid (100 �M) with or without.5 mg/ml of PPE for 60 min at 37 ◦C. Following incubation,he RBCs/HEG were washed and mixed with SDS (1%) andistilled water. Thiobarbituric acid (1% in 0.05 M NaOH) wasdded and the mixture was incubated in boiling water bathor 1 h. Following incubation, tubes were cooled and thebsorbance of the supernatant was read at 532 nm againstater blank [29].

.8. Scanning electron microscopy

Packed erythrocytes (50 �l) were incubated with H2O20.3%) with or without PPE (2.5 mg/ml) for 60 min at7 ◦C. After incubation, the suspension was centrifugednd the cell pellets were processed for SEM according toethod described earlier by Agrawal and Sultana [30] withinor modifications. RBCs were washed twice with saline

olution and finally pelletted. Then they were fixed withlutaraldehyde by adding one drop of each sample to plasticubes containing 1 ml of 2.5% glutaraldehyde in saline solu-ion. After incubation overnight at 4 ◦C, the fixed samplesere washed two times with saline, smeared on grease-

ree glass slides and air-dried. The slides were dehydrated in

scending series of acetone (30–100%) and then transferredo aluminium stubs with double-sided adhesive tape. Theells were gold coated for 3 min at 10–1 torr in a Polaron5000 sputter device. The observations and photographic

ecords were performed in an electron microscope (Model:EO 435 VP; LEO Electron Microscopy Ltd., Cambridge, UK).

Interactions 173 (2008) 97–104 99

2.9. Electrophoresis of membrane proteins

Polyacrylamide gel electrophoresis with dodecyl sul-phate (SDS–PAGE) was carried out employing the dis-continuous buffer system of Laemmli [31]. The monomerconcentration in the slab mini gels was 4% in the stackingand 10% in the running gel. Samples were dissociated bymixing them in SDS sample buffer (62.5 mM Tris–HCl, pH6.8, containing 2% SDS, 5% 2-mercaptoethanol, 10% glyceroland bromophenol blue as tracking dye) and then boiled for1 min. Amounts of 15 �g of protein were applied in eachlane. Protein concentrations had been previously deter-mined by the method of Lowry et al. [32]. A molecularweight protein standard was included in the slab gel. Atthe end of electrophoresis, the gels were stained with 0.2%Coomassie Brilliant Blue in 30% methanol/10% acetic acid.

2.10. Statistical analysis

Results are expressed as mean + S.E. Statistical analy-ses were performed using one- or two-way ANOVA whereapplicable using Statistica, Version 5 (Statsoft, USA).

3. Results

The total polyphenolic content in PPE was found to be

Fig. 1. HPLC profile of potato peel polyphenols: peak identification (GA,gallic acid; PCA, protocatechuic acid; CHA, chlorogenic acid; CA, caffeicacid). This experiment was repeated three times with similar resultsachieved (n = 3).

100 N. Singh, P.S. Rajini / Chemico-Biological Interactions 173 (2008) 97–104

Table 1Composition of phenolic acids in potato peel extract

Phenolic acid Relative %

Gallic acid (GA) 26.5 ± 0.2Caffeic acid (CA) 38.6 ± 0.1Protocatechuic acid (PCA) 18.8 ± 0.1

Table 2Lipid peroxidation in RBCs/membranes and its attenuation by PPE

System MDA (nmol/mg protein) % Control

Rat RBCsRBC 0.048 ± 0.08 100RBC + Fe/AA 0.157 ± 0.02 327RBC + Fe/AA + PPE 0.063 ± 0.07 131

Human erythrocyte ghost (HEG)HEG 1.23 ± 0.02 100HEG + Fe/AA 2.48 ± 0.21 202

since stacking gel is not included in the picture). Treatment

Chlorogenic acid (CHA) 16.0 ± 0.2

Data are expressed as mean ± S.E. of three replicates from two determina-tions.

To gain insight into the mechanism of protection by PPE,we determined LPO in RBCs and RBC membranes. We chosethe experimental prooxidant system: FeSO4 and ascorbicacid to induce lipid peroxidation, as a result of hydroxyl rad-icals (•OH) initiated by the Fenton reaction. PPE inhibitedLPO with similar effectiveness in both the systems, about80–85% inhibition by PPE at 2.5 mg/ml (Table 2).

Scanning electron micrographs of erythrocytes treatedin vitro with H2O2 and PPE are shown in Fig. 2. Untreatederythrocytes appear as typical discocytes, while exposureto H2O2 resulted in a significant change in the cell shapeand distinct echinocyte formation. Upon treatment of cellswith H2O2, formation of multiple blebs on cell membranewas observed. PPE per se did not cause any morphologicalalterations in the cells. However, under the experimental

conditions, PPE significantly inhibited the H2O2-inducedmorphological alterations.

Results in Fig. 3 present the extent of protectionoffered by PPE to membrane cytoskeletal proteins against

Fig. 2. Scanning electron micrographs of rat RBCs before and after treatment wit(I) Untreated RBC: typical biconcave appearance(II) RBC treated with PPE: normal morphology(III) RBC treated with H2O2: echinocytes(IV) RBC treated with H2O2 + PPE: most of the cells showing biconcave shape, a

This experiment was repeated three times with similar results achieved; n = 3.

HEG + Fe/AA + PPE 1.48 ± 0.07 120

Fe, FeSO4; AA, Ascorbic acid.Data are expressed as mean ± S.E. of three replicates from three determi-nations.

ferrous–ascorbate induced oxidative damage. The majorproteins of erythrocyte cytoskeleton could be identified inthe lane (lane 1) containing untreated erythrocyte ghosts.The bands were numbered according to the electrophoreticmobility following Fairbanks et al. [33]. Treatment withferrous–ascorbate led to decreased intensities of bands 1–3and total disappearance of bands 4.1, 4.2, 5 and 6 (lane2) while a few new bands appeared in the low molecu-lar weight region and high molecular weight aggregatesappeared on the stacking gel (not visible in Fig. 3, lane 2,

with PPE alone did not affect any of the band intensi-ties (lane 3). However, in membranes co-incubated withferrous–ascorbate and PPE (varying concentrations), mostbands were clearly visible (lanes 4–6).

h H2O2 (0.3%) in the absence or presence of PPE (2.5 mg) [×2700].

lbeit some echinocytes.

N. Singh, P.S. Rajini / Chemico-Biological

Fig. 3. Protective effect of PPE on ferrous–ascorbate induced changes inhuman erythrocyte membrane proteins (HEG) analyzed using SDS–PAGE.Lane 1: untreated, intact erythrocyte membranes; lane 2: membranesincubated with ferrous–ascorbate; lane 3: membrane incubated with PPE(1.88 mg/ml); lane 4: membrane incubated with ferrous–ascorbate + PPE(0.9 mg/ml); lane 5: membrane incubated with ferrous–ascorbate + PPE(1.39 mg/ml); lane 6: membrane incubated with ferrous–ascorbate + PPE(Tmt

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1.88 mg/ml). The amount of layered protein was 15 �g in each lane.he protein bands have been numbered according to the electrophoreticobility following Fairbanks et al. [33]. This experiment was repeated

hree times with similar results achieved (n = 3).

. Discussion

Erythrocytes are particularly sensitive to oxidative dam-ge due to the presence of high polyunsaturated fattycid content in their membranes and high cellular con-entrations of oxygen and hemoglobin [34] and hencehey represent a convenient model to study the effectf oxidative stress on plasma membrane [35]. Erythro-yte damage includes changes in membrane protein andipid structure, which in turn induce alterations on exter-al surface of the cell. In the present study, we choserythrocytes and its membrane as the in vitro mod-ls to study the efficacy of polyphenol-rich potato peelxtract as inhibitor of ferrous/ascorbate and H2O2-inducedipid/protein peroxidation. Our results clearly demonstratehat ferrous/ascorbate and H2O2-induced lipid/protein per-xidation in erythrocytes was significantly suppressed byPE, which could be attributed to its antioxidant properties.

Initially, we demonstrated the ability of thee2+/ascorbate system to induce lipid peroxidation inormal rat RBCs as well as human erythrocyte membranes.

n Fe2+/ascorbate-dependent model of oxidative stress,ue to the hydroxyl radical production via the Fenton

eaction, the oxidative damage is reported to occur bothn protein and lipid components of the membrane [36].ome authors have reported that the increase in lipideroxides renders the RBC membrane rigid by altering

Interactions 173 (2008) 97–104 101

the amino-phospholipid organization [37]. Further, mal-ondialdehyde (MDA) generated through decomposition ofperoxidized lipids has been shown to affect the intrinsicmechanical properties of the RBCs membrane resulting indecreased deformability [35,38]. MDA can also cause anenhanced cross-linking of hemoglobin to the membraneskeletal proteins [39] leading ultimately to increased RBCsmembrane rigidity and deformability impairment. Ourresults are consistent with those of earlier findings inerythrocytes subjected to oxidative stress.

Oxidants are reported to induce alterations in theerythrocyte membrane as manifested by a decreasedcytoskeletal protein content and production of high molec-ular weight proteins which can lead to abnormalities inerythrocyte shape and rheological properties [40]. Forinstance, H2O2 and Fe2+/ascorbate induce an echinocytictype of shape alteration, i.e., develop a form character-ized by blebs or protuberances over the cell membrane,indicative of oxidative damage [41]. Our SEM observationsshowed that H2O2 induced morphological alterations inthe red cells from a discoid to an echinocytic form. H2O2,which crosses the RBC membrane and acts on the intra-cellular moiety, forms ferryl radical or hydroxyl radical byinteracting with hemoglobin and initiates a series of reac-tions ultimately resulting in RBC lysis [42]. The formationof multiple blebs on the plasma membrane is consid-ered to be an early sign of cell oxidative injury [43] andthese blebs enlarge, and eventually break releasing theintracellular components. While it has been observed thatblebbing often accompanies a loss of membrane asymme-try [44], peroxide-induced cytoskeletal re-organization andbleb formation are found to correlate with the degree ofGSH oxidation [45] and hence, membrane lipid peroxida-tion and damage to cytoskeleton proteins is considered tobe an important factor in bleb formation [46].

According to the bilayer couple hypothesis [47,48],the shape changes induced in erythrocytes by foreignmolecules are due to differential expansion of the twomonolayers of the red cell membrane and spiculated-shaped echinocytes are produced when it locates into theouter moiety. Our major observation that H2O2 inducedthe formation of echinocytes probably indicates that itwas inserted in the outer leaflet of the erythrocyte mem-brane. The generation of echinocytes after exposure toH2O2 appears also to be related to peroxidation of mem-brane protein, including spectrin–hemoglobin complex.The mechanism of echinocyte formation may also berelated to condensation of the inner monolayer lipids asa result of spectrin–hemoglobin complex formation. Thebilayer couple hypothesis entails that a decrease in innerlipid monolayer area will result in echinocyte formation[47,49].

In the present study, a significant protection was offeredby PPE in terms of retaining the morphological integrityof the erythrocytes as well as reducing the extent of lipidperoxidation when subjected to oxidative stress. Numerous

cals (polyphenols) to reduce markers of oxidative stress,including protection of RBC against free radical [13,50,51].Polyphenols show considerable antioxidant activity both invitro and in vivo [52], which is attributed mainly to their

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102 N. Singh, P.S. Rajini / Chemico-Bi

capacity to scavenge oxygen and nitrogen active species[53] and to chelate redox-active metals [54]. Our ear-lier studies had adequately demonstrated the free radicalscavenging activity of PPE in vitro [24]. The molecular mech-anisms of the antioxidant action of polyphenols have notyet been full elucidated and are still a matter of consid-erable debate. It has been suggested that polyphenols iflocalized within the cell membrane, may alter the rigidityof the membrane by mediating a fluidizing effect withinthe membrane [55]. Hence, another possible contribu-tory mechanism toward their antioxidant activities is theirability to stabilize membranes by decreasing membranefluidity [23]. The interaction of polyphenols with bilayerscould be a relevant mechanism in their protection againstmembrane oxidation. While Arora et al. [23] reported thatflavonoids and isoflavonoids partition into the hydrophobiccore of large unilamellar vesicles (LUV) decreased its fluid-ity, Nakagawa et al. [56] indicated that certain flavonoidsmight stabilize membranes by locating in the lipid andaqueous interphase. Accordingly, it is quite possible thatthe location of the extract components into the membranebilayer and the resulting restriction on its fluidity mighthinder the diffusion of H2O2 and its consequent damagingeffects. This conclusion can also imply that this restrictioncould apply to the diffusion of free radicals into the cellmembranes and the consequent decrease of the kinetics offree radical reactions [57].

Our data on the qualitative/quantitative analysis of PPEfor phenolic acids showed the presence of chlorogenic acid,gallic acid, caffeic acid and protocatechuic acids [25]. Thisis in accordance with the studies of Rodriguez de Sotilloet al. [27,58] who also showed the presence of chlorogenicacid, gallic acid, protocatechuic acid and caffeic acid as themajor phenolics in PPE, although their relative proportionwere slightly varied from that of ours. The observed protec-tive effect by PPE may hence be attributed to the combinedeffect of the major phenolic acids present by way of scav-enging peroxy radicals as well as by stabilizing the cellmembrane. Inhibitory effect of chlorogenic acids on hemol-ysis and peroxidation of mouse erythrocytes have beendemonstrated earlier [59]. However, the biological effectof these polyphenols will ultimately depend on both thepolyphenol and also the extent to which they associate withthe cell [60].

We also investigated the protective effect of PPEon oxidative denaturation of the erythrocyte membraneproteins in vitro employing human erythrocyte ghostmembranes. It is reported that an oxidative attack by ‘site-specifically’ generated reactive hydroxyl radical (•OH) frommetal catalyzed Haber–Weiss reaction could lead to cross-linking and/or degradation of RBC membrane proteins [61].In the Fe2+/ascorbate model of oxidative stress, anothermechanism namely, the oxidative fragmentation of pro-teins, appears to play a major role. It is likely that hydroxylradical generated via iron catalyzed Fenton reaction maybe responsible for oxidative attack on the polypeptide

chain and subsequent non-enzymatic protein fragmen-tation [62]. Further, erythrocyte membrane proteins areshown to be susceptible to degradation by membrane-bound serine protease activity after oxidative modificationof the membranes [63]. Changes in erythrocyte mem-

Interactions 173 (2008) 97–104

brane ion permeability, lipid peroxidation, formation ofdisulphide bonds and activation of proteolysis have beenreported following the challenge of erythrocytes by vari-ous oxygen radical generating systems [62,63]. In our study,treatment of HEG with Fe2+/ascorbate produced a decreasein HMW protein band content and an increase in new pro-teins with low molecular weights. This finding is consistentwith the earlier reports [36]. However, most of the pro-tein bands were clearly visible in the membranes exposedto antioxidant rich PPE as well those exposed to both PPEand Fe2+/ascorbate clearly establishing the effective protec-tion offered by PPE against oxidative damage to membraneproteins.

In conclusion, our studies have shown that while PPE perse did not alter the morphology of rat RBCs in vitro, its pres-ence attenuated markedly the morphological degenerationof the cells induced by H2O2. Further PPE offered signifi-cant protection to erythrocyte membrane against oxidativedamage to its membrane proteins. These results provideadditional support for our earlier findings [24] on the strongradical quenching activity of PPE. However, a comprehen-sive and systematic analysis will be required to furtherelucidate the mechanism of protective action and whetherthese findings can be extrapolated to in vivo conditions,require further study.

Acknowledgements

The authors wish to thank the Director, CFTRI, for hissupport in this study. The authors also are very grateful tothe technical assistance of Mr. K. Anbalagan in ScanningElectron Microscopy. The first author (NS) acknowledgesthe financial support from the Council of Scientific andIndustrial Research, India, in the form of senior researchfellowship.

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