Protective Effects of Boldine Against FreeRadical-induced Erythrocyte Lysis

Ines Jimenez1, Argelia Garrido 1, Roxana Bannach1, Martin Gotteland 2 and Hernan Speisky1*1Laboratory of Lipids and Antioxidants, Nutrition and Food Technology Institute, University of Chile, Santiago, Chile2Gastroenterology Unit, Nutrition and Food Technology Institute, University of Chile, Santiago, Chile

Boldine, an aporphine alkaloid extracted from the leaves and bark of boldo (Peumus boldusMol.), hasbeen shown to exhibit strong free-radical scavenger and antioxidant properties. Here, we report theinvitro ability of boldine to protect intact red cells against the haemolytic damage induced by the free radi-cal initiator 2,2 '-azobis-(2-amidinopropane) (AAPH). Boldine concentration-dependently prevented theAAPH-induced leakage of haemoglobin into the extracellular medium. Substantial and similar cyto-protective effects of boldine were observed whether the antioxidant was added 1 h prior to, or simulta-neously with, the azo-compound. The delayed addition of boldine, by 1 h relative to AAPH, diminishedbut did not abolish its cytoprotective effect. However, negligible effects of boldine were observed after itsaddition to erythrocytes previously incubated with AAPH for 2 h. The data presented demonstrate that,in addition to its well-established antioxidant effects, boldine also displays time-dependently strong cyto-protective properties against chemically induced haemolytic damage. Copyright# 2000 John Wiley &Sons, Ltd.

Keywords:boldine; free radicals; antioxidant; erythrocyte; cytoprotection.

INTRODUCTION

Exposure of erythrocytes to stringent oxidative condi-tions results in a myriad of free radical-mediatedreactions that ultimately lead to cell lysis (Orrenius,1993; Aruoma, 1998). Experimentally, oxidative haemo-lysis can be inducedin vitro by incubating intact isolatederythrocytes in the presence of synthetic organicperoxides such astert-butylhydroperoxide, or by theirexposure to conditions which favour an enhancedendogenous production of lipid and/or hydrogen peroxide(Corry et al., 1980; Trottaet al., 1981; Thornalleyet al.,1983). When the intracellular uptake and/or generation ofperoxides surpasses the capacity of the cell to reductivelyremove these species, part of the peroxide moleculesundergo decomposition, via a metal- or a P-450-catalysedreaction, into radical species capable of damagingproteins and polyunsaturated fatty acids present withinthe erythrocyte membrane. An additional experimentalmode to induce free radical-mediated oxidative damageto erythrocytes involves the use of so-called azo initiators(Niki, 1990). The latter compounds spontaneouslydecompose, at a temperature-controlled rate, yielding aknown flux of carbon-centred radicals that swiftly reactwith oxygen to yield peroxyl radicals. The rate ofdecomposition and the solubility of an azo-compound arefully determined by its chemical structure. 2,2'-azobis-(2-amidinopropane) is a highly hydrophilic radical initiator

previously employed to inducein vitro chain oxidation inpreparations such as lipid micelles, phospholipid lipo-somes, erythrocyte membranes and red cell suspensions(Yamamotoet al., 1985; Miki and Mino, 1985; Nikietal., 1988; Satoet al., 1995). In intact isolated erythro-cytes, AAPH has been shown to induce extensiveoxidation of membrane components (e.g. lipids andproteins) and to promote structural alterations which leadto the rupture of the erythrocyte membrane and tohaemoglobin leakage (Mikiet al., 1987; Satoet al., 1995;Celedonet al., 1997). It has been shown that, underinvitro conditions, the exogenous addition of some freeradical scavengers and/or chain-breaking antioxidantscan effectively prevent the azo-induced lysis of erythro-cytes, and that underin vivo conditions, the administra-tion of the former agents is useful in cytoprotectingagainst tissue damage induced by AAPH (Mikiet al.,1987; Terao and Niki, 1986; Nikiet al., 1988). Therefore,the combined use of intact erythrocytes and azo initiatorscan serve as a suitable experimental approach to quicklyassessin vitro the ability of new molecules to act,throughout their free radical scavenger properties, aspotentially beneficial cytoprotective agents.

We have previously reported the antioxidative proper-ties of boldine, an aporphine alkaloid (Fig. 1) abundantlypresent in the leaves and bark of the Chilean boldo tree(Peumus boldusMol.) (Speisky and Cassels, 1994). Lowconcentrations of boldine prevent ( IC50 = 15–20mM) theauto-oxidation of brain homogenates, inhibit the AAPH-induced peroxidative damage to erythrocyte ghostmembranes, protect lysozyme against AAPH-inducedinactivation (Speiskyet al., 1991) and prevent enzyma-tically catalysed peroxidative damage to microsomalmembranes without inhibiting cytochrome P-450 or itscorresponding reductase activity (Cederbaumet al.,

PHYTOTHERAPY RESEARCHPhytother. Res.14, 339–343 (2000)

Copyright# 2000 John Wiley & Sons, Ltd.

* Correspondence to: Dr H. Speisky, Laboratory of Lipids and Antioxidants,Nutrition and Food Technology Institute, University of Chile, POB 138-11,Santiago, Chile.E-mail: hspeisky@uec.inta.uchile.clContract/grant sponsor: FONDECYT; Contract/grant number: 1950258.Contract/grant sponsor: PHO/WHO.

Received 30 April 1999Accepted 7 September 1999

1992).Althoughboldineis relativelyunreactivetowardssuperoxideanions,it behavesasan excellentscavengerof hydroxyl radicals (Cederbaumet al., 1992). Inaddition, boldine effectively protects non-biotic sub-stratessuchasfishoil againstperoxidation,behavingasachain-breakingantioxidant (Valenzuela et al., 1991;Ganga et al., 1998). Prompted by its free radicalscavengerand chain-breakingantioxidantproperties,inthe presentwork we haveaddresseda possiblecytopro-tective effect of boldine against the AAPH-inducedhaemolysisof intacterythrocytes.Wehavecharacterizedthe time-courseand concentration-dependence of itscytoprotectiveeffects. We have also studied the im-portance of the time of addition of this antioxidantrelative to the azo initiator as a determinantof itscytoprotectiveaction.

MATERIALS AND METHODS

2,2'-azobis-(2-amidinopropane) dihydrochloride waspurchasedfrom Polysciences,Inc. (Warrington, PA,USA). Boldine was extractedfrom the bark of Peumusboldus with methanol. Following evaporationof thesolvent,theresiduewaspartitionedbetweentheaqueousacid and chloroform phaseto remove non-alkaloidalconstituents.The total alkaloidswere re-extractedwithchloroformafteradjustingthepH of theaqueousphaseto8–9. Boldine was finally isolated from the crudealkaloidal mixture by several recrystallizationsfromchloroform.The alkaloid waschromatographicallypureand its identity was establishedby IR and NMRspectrometry(Speiskyet al., 1993).All otherchemicalswere of analytical grade and obtained from SigmaChemicalCo (St. Louis, MO).

Blood was obtainedfrom lightly ether-anaesthetizedrats and quickly centrifuged(1500� g, 10min, 22°C).The erythrocyteswere washedthree times with saline(0.9% NaCl, 4°C) and centrifuged(1500� g, 10min,22°C) to obtain a constantlypackedcell volume. Thelatter was subsequentlyused to prepare a 10% v/verythrocyte suspensionwith incubation buffer (salinebrought to pH 7.4 with sodium phosphate).Aliquots(2.5mL each)of thecell suspensionwereaddedto 25mLErlenmeyerflasksand incubatedat 37°C with constantand gentle shaking.Each suspensionwas preincubatedfor 5 min before the addition of any further chemical.Haemolysiswasinducedby thedirect additionof 50mLof anAAPH stocksolution(preparedin saline).Boldine,when tested,was addedto the incubation medium as25mL of a freshstocksolution(preparedin saline).

Samplesof thesuspensionswereremovedat different

times and the degree of haemolysis was assessedspectrophotometricallyby readingthe OD of 1500� gsupernatantsat540nm.Totalhaemolysiswasinducedbyvigorously vortexing a mixture of 0.1mL of theerythrocyte suspensionwith 4.9mL of cold distilledwater.The haemolysedsamplewascentrifugedandthesupernatantused to estimatethe 100% of haemolysisvalue. Partial haemolysiswas assessedin supernatantsobtained by centrifuging a mixture of 0.1mL ofincubatedsuspensionsplus 0.9mL of buffered saline.Percentageof haemolysiswascalculatedby multiplyingby 100 the ratio of the readingsfor partial and totalhaemolysis.

Data plotted representthe meanof 3–4 independentexperiments,eachconductedin triplicate. Sinceresultswere reproducible within 10% of the correspondingstandarddeviations,only the meanof the valueswereconsideredwhenplotting of eachdatapoint.Student’st-test was applied to determinethe significanceof thedifferencesbetweenthe haemolysisobservedfor theAAPH groupandanothergroupundercomparison,at theearliesttime at which thehaemolysisinducedby AAPHalone becomessignificantly different from that of asuspensionincubatedin the absenceof any of theseagents.The sametestwasappliedto determinewhetherstatistical significance exists between the maximalhaemolysis attained at the end of each incubationexperiment.Employing those data points (�3) whichdefinethelinearpartof eachhaemolysiscurve(r � 0.94),linear regressionanalysiswasapplied,andthe time (t50)correspondingto the theoretical50% haemolysisesti-

Figure 1. Chemical structure of boldine [S]-2,9-dihydroxy-1,10-dimethoxyaporphine.

Figure 2. Effect of a ®xed concentration of boldine on theAAPH-induced erythrocyte lysis. AAPH was added (25 mM

closed circles; 50 mM closed squares) either alone orsimultaneously with 50 mM boldine (corresponding opensymbols). Controls were incubated in the sole presence ofboldine (open pentagons), or in the absence of both addedagents (asterisks). Boldine and AAPH were added simulta-neously at time 0. Signi®cant differences were found after 2 hbetween suspensions incubated with 50 mM AAPH in theabsence versus the presence of boldine, and after 3 h,between suspensions incubated with 25 mM AAPH alone orplus boldine. Signi®cant differences were also observed after6 h of incubation between the groups 25 and 50 mM AAPH,and between each AAPH group and its correspondingboldine-added group.

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matedandexpressedasmean� SD.Student’st-testwasappliedto establishthesignificanceof thedifferencesint50 betweencurves. Differences were consideredsig-nificantat the level of p< 0.05.

RESULTS

As shownin Fig.2, raterythrocytessuspendedin isotonicphosphate-salinebuffer,pH 7.4,andincubatedat37°C inthe absenceof the radical initiator AAPH, remainedvirtually intact for up to 6 h of incubation.By theendofthe incubationperiod,only minor haemolysis(lessthan6%) wasdetectedwhich, most likely, resultedfrom theshaking-relatedmechanical stress.Erythrocytes incu-batedin the absenceof AAPH, but in the presenceofboldine, showeda slightly but not significantly greaterviability comparedwith cellsincubatedin theabsenceofboth agents(Fig. 2). The addition of AAPH (25 and50mM) to the erythrocytesuspensionwascharacterizedby an induction phase—duringwhich no significanthaemolysistook place—andfollowed by a sharp andsigmoid-like haemolysiscurve. AAPH concentration-dependentlyincreasedthe extentof the haemolysis(atanygiventime)andloweredtheestimatedt50 haemolysisvalues.However,in thepresenceof afixedconcentrationof boldine(50mM), thetime neededfor AAPH to initiatethe haemolysis was always increased.Boldine alsoappearedto decreasethe rateof AAPH-inducedhaemo-lysisasevidencedby theoccurrenceof lowerhaemolysisvaluesthroughoutthewholeincubationperiod.A similarpatternof protectionwas observedwhen the effectsofincreasingconcentrations(12.5–100mM) of boldinewere

studiedon thehaemolysisinducedby a fixed concentra-tion (50mM) of AAPH (Fig. 3). Boldinedelayedthestartof haemolysisand,at eachstudiedtime, it decreasedtheextentof lysis in a concentration-dependent manner.Atthe highest testedboldine concentration(100mM), thehaemolysisinduced by AAPH amounted,after 6 h ofincubation,to lessthan50%of that inducedby AAPH.

Figure 4 depictsthe effects of the early addition ofboldine(75mM) relativeto thatof AAPH (50mM) to thesuspension.In thissetof experiments,boldinewasaddedeitheruponstartingtheincubation(time0) but1 h beforeAAPH, or simultaneouslywith AAPH but 1 h after theincubationwas initiated. In the absenceof boldine, theadditionof AAPH 1 h afterinitiating theincubationledtoa haemolysiscurve similar in shapebut shifted to theright comparedwith that resultingfrom the addition ofAAPH at time 0. The estimatedt50 haemolysisvalueswere 3.8� 0.3h and 2.8� 0.2h, respectively.Boldineadditionat time0, 1 h prior to thatof AAPH, resultedin avirtually identicalandoverlappingpatternof cytoprotec-tion (e.g. induction time andshapeof the curve) to thatseen when both AAPH and boldine were addedsimultaneously1 h after initiating the incubation.

The effects of the delayed addition of boldine onAAPH-inducederythrocytelysisareshownin Fig.5.Theaddition of boldine 1 h after incubation with AAPH,slightly but significantly affected the course of thehaemolysisby shifting the curve towardsthe right; t50values were 2.9� 0.2h and 3.6� 0.2h, respectively.However,after 2 h of incubationwith the azo-initiator,

Figure 3. The effect of increasing concentrations of boldineon the AAPH-induced erythrocyte lysis. AAPH (50 mM) wasadded in the absence (closed squares) or presence (opensymbols) of boldine as follows: squares, 12.5 mM; pentagons,25 mM; circles, 50 mM; crosses, 75 mM; asterisks, 100 mM.Boldine and AAPH were added simultaneously at time 0.Signi®cant differences were found, after 2 h, between thesuspensions incubated with AAPH in the absence versus thepresence of all concentrations of boldine, and after 6 h,between suspensions incubated with AAPH in the absenceversus the presence of either 50, 75 or 100 mM boldine.

Figure 4. The effect of the early addition of boldine on AAPH-induced erythrocyte lysis. The closed symbols, circles andtriangles, represent cell suspensions added solely AAPH(50 mM), either at time 0 or 1 h after initiated the incubation,respectively. The open pentagons represent suspensionsadded boldine (75 mM) at time zero and AAPH 1 h later. Theopen squares represent suspensions added boldine andAAPH simultaneously, 1 h after initiated the incubation.Signi®cant differences were observed from 4 h of incubationon, between suspensions incubated with AAPH alone(whether added at time 0 or 1 h later) and suspensionsincubated with AAPH plus boldine (whether added at time 0or 1 h later).

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the addition of boldine had no significanteffect on thetime-courseof cell lysis.

DISCUSSION

Thefreeradicalscavengerandchain-breakingpropertiesof boldine are well established(Speiskyet al., 1991;Cederbaumet al., 1992;Casselset al., 1995;Bannachetal., 1996). Of several systems used to assessitsantioxidantproperties,boldinewasshownto beparticu-larly effective in preventing the peroxidativedamageinduced by AAPH to erythrocyte ghost membranes(Speiskyet al., 1991). Data presentedhere show that,in addition to its demonstratedantioxidative effects,boldine also displays strong cytoprotectivepropertiesagainst the lytic damageinduced by AAPH to intacterythrocytes.

Our resultsshow that the time neededby AAPH toinitiate the haemolysisand the extent of the cell lysisinduced by this peroxyl radical generator are bothinversely and directly proportional,respectively,to itsextracellular concentration.Under such experimentalconditions,boldine was shownto concentration-depen-dently lengthen the haemolysis induction time anddecreaseits maximalextent.Giventheability of boldineto scavengeAAPH-derivedperoxyl radicals(Speiskyetal., 1991), its cytoprotectiveeffectsare likely to resultfrom it diminishing the net concentrationof AAPH-derivedperoxyl radicalsgeneratedalongthe incubation.

It is possiblethat during the induction period, boldinemoleculesareprogressivelyconsumedby AAPH-derivedperoxyl radicals, and that, only after their completeconsumptionhas taken place,erythrocytelysis ensues.Such scavengingaction of boldine would lower theextracellularconcentrationof peroxyl radicalsto a levelthataccountedfor thereductionin themaximalextentofthe haemolysisseenwhen boldine is addedto the cellsuspension.

Due to its hydrophilic character,AAPH undergoesthermal decompositionextracellularly. Therefore,bol-dine moleculeswould prevent AAPH-derived peroxylradicals within the aqueousphasefrom initiating theoxidative modification of membrane-locatedmoleculartargetsthat are critical for defining the integrity of theerythrocyte.Theinterpretationthatboldinetrapsinitiatorradicalsis in line with theearlyobservationby Niki etal.(1988) that uric and ascorbicacids, two highly water-solublemolecules,promotetheir cytoprotectiveeffectsby scavengingAAPH-derivedperoxyl radicals.

Recentstudiesby Satoet al. (1995)haveshownthatthehaemolyticactionof AAPH couldbeassociatedwiththe oxidation of lipid components present in theerythrocytemembranes.Thus,thepossibilitythatboldineexertsits cytoprotectiveeffectsat themembranelevel,asa chain-breakingantioxidant,shouldalsobeconsidered.In fact, Miki and Mino (1985) establishedthat underexperimentalconditionssimilar to thoseemployedhere,thehaemolysisensuesonly afterAAPH hasloweredtheerythrocytemembranealpha-tocopherolconcentrationsto a critically low level. Therefore,boldine by directlyinteractingwith lipoperoxyl radicals(Valenzuelaet al.,1991; Gangaet al., 1998),may sparealpha-tocopherolmolecules,lengtheningthe inductionphase.

On the other hand,boldine may alternativelyprotectred cells from undergoing lysis by preventing theoxidative damageof membraneproteins.According toa recent report by Celedon et al. (1997), membraneproteindegradationconstituteda majoreventassociatedwith the AAPH-induced lysis of erythrocytes.Usinglysozyme inactivation by AAPH as an experimentalmodel of protein oxidation, we have establishedthatboldineprotectsagainsttheoxidativeinactivationof thisenzyme (Speisky et al., 1991; Casselset al., 1995).Further,ongoingwork revealsthat in the samemodel,carbonyl formation and tryptophan loss are bothpreventedby boldine to the sameextent to which theantioxidant protects against the loss of the enzymeactivity.

Ourstudyalsoshowsthatdelayingfor 2 h theadditionof boldine relative to that of AAPH resulted in thecomplete loss of the effectiveness of boldine tocytoprotect.This resultsuggestedthatwithin thiselapsedtime, AAPH has alreadytriggeredoxidative modifica-tions that canno longerbe preventedor reversedby thepresenceof boldine.In contrastto thelatter,theability ofboldineto lengthenthe inductionphaseandto decreasethe maximal extent of the haemolysisremainedcom-pletely unalteredwhenthe antioxidantwasaddedto thesuspension1 h beforeAAPH. On the basisof the aboveconsiderations,theresultsof theearlyadditionof boldinewould suggestthat its extracellular concentrationre-mainedessentiallyunmodifiedduring the time elapsedbefore AAPH addition. Alternatively, it may besuggestedthat duringsucha period,boldineis biotrans-formed into metaboliteswhich either fully retain their

Figure 5. The effect of the delayed addition of boldine on theAAPH-induced erythrocyte lysis. Boldine (75 mM) was addedat 0 h (open circles), 1 h (open pentagons) or 2 h (opensquares) after the addition of AAPH (50 mM, added at time 0).The closed circles represent suspensions incubated in thesole presence of AAPH (added at time 0). No signi®cantdifferences were found point-to-point along the incubationbetween suspensions added AAPH alone or AAPH plusboldine (added 2 h later). Signi®cant differences wereobserved from 3 h of incubation on, between suspensionsincubated in the presence of AAPH alone or AAPH plusboldine (added 1 h after initiated the incubation).

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original antioxidantproperties,or that, despitelackingsuchproperties,their sole formation is associatedwithintracellularchangeswhichendowtheerythrocytewith acytoprotective capacity similar to that afforded byboldinemolecules.

Sincetheleakageof haemoglobininto theextracellularmedium representsonly a final event to measuretheextentof cell lysis, it is notpossiblefrom thepresentdatato determinethemechanism(s)by which boldineexertedits cytoprotectiveeffects.However,giventhepossibilitythat the oxidation of membranelipids and/or proteins

representsa significant event underlying erythrocytelysis, future work will addresswhetherboldinepreventsthe oxidation of both membranecomponentsand howsucheffectswould relate to its reportedcytoprotectiveactions.

Acknowledgements

This researchwas partially supportedby funds from FONDECYT(Fondode Investigacion en Cienciay Tecnologı´a) grantno 1950258,andthePHO/WHO.

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