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BloodcompatibilitystudiesofSwarnabhasma(goldbhasma),anAyurvedicdrug

IntJAyurvedaRes.2011JanMar2(1):1422.

PMCID:PMC3157103

doi:10.4103/09747788.83183

BloodcompatibilitystudiesofSwarnabhasma(goldbhasma),anAyurvedicdrug
WilliPaulandChandraPrakashSharma
DivisionofBiosurfaceTechnology,BiomedicalTechnologyWing,SreeChitraTirunalInstituteforMedicalSciencesandTechnology,Thiruvananthapuram,
India
Addressforcorrespondence:Dr.ChandraPrakashSharma,DivisionofBiosurfaceTechnology,BiomedicalTechnologyWing,SreeChitraTirunal
InstituteforMedicalSciencesandTechnology,Thiruvananthapuram695012,India.Email:sharmacp@sctimst.ac.in
Received2010Jul22Accepted2011May7.
CopyrightInternationalJournalofAyurvedaResearch
ThisisanopenaccessarticledistributedunderthetermsoftheCreativeCommonsAttributionNoncommercialShareAlike3.0Unported,whichpermits
unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.

ThisarticlehasbeencitedbyotherarticlesinPMC.

Abstract
Swarnabhasma(goldbhasma)preparationsarewidelyutilizedastherapeuticagents.However,invitrobiological
evaluationsofbhasmapreparationsareneededalongwiththephysicochemicalcharacterizationforpresentday
standardizationofmetallicbhasmapreparationstomeetthecriteriathatsupportsitsuse.Therefore,anattempthas
beenmadetoevaluatetheproteinadsorption,bloodcompatibilityandcomplementactivationpotentialoftwo
batchesofSwarnabhasmapreparation,alongwithitsphysicochemicalcharacterization.Theparticlesize,
morphology,elementalanalysis,andinvitrocytotoxicitywereevaluatedinitially.Redbloodcellhemolysis,
aggregationstudieswithbloodcells,proteinadsorption,complementC3adsorption,plateletactivationandtight
junctionpermeabilityinCaco2celllinewereinvestigated.TheSwarnabhasmapreparationswithacrystallitesize
of2835nmdidnotinduceanybloodcellaggregationorproteinadsorption.Activationpotentialofthese
preparationstowardscomplementsystemorplateletswasnegligible.Theseparticleswerealsononcytotoxic.
SwarnabhasmaparticlesopenedthetightjunctionsinCaco2cellexperiments.Theresultssuggesttheapplication
ofSwarnabhasmapreparationsasatherapeuticagentinclinicalmedicinefromthebiologicalsafetypointofview.
Keywords:Bloodcompatibility,proteinadsorption,Swarnabhasma
INTRODUCTION
Fromasearlyas2500BC,thetherapeuticbenefitsofgoldpreparationshavebeenreportedinIndian,Arabicand
Chineseliterature.[1]Swarna(gold)bhasmahasbeenutilizedasatherapeuticagentinthetraditionalIndian
Ayurvedicmedicineforseveralclinicaldisordersincludingbronchialasthma,rheumatoidarthritis,diabetesmellitus,
andnervoussystemdiseases.[27]Swarnabhasmaisusuallygivenorallymixedwithhoney,gheeormilk.
Inrecentyears,therehasbeenarenewedinterestindrugdiscoverystrategieswherenaturalproductsandtraditional
medicinesarereemergingasattractiveoptions[8]andhencerenewedinterestsinagentslikeSwarnabhasma.
Recentresearchhasrevealedthatgoldnanoparticlesexhibitsizedependentabsorptionthroughratskinand
intestine,withsmallerparticles(~15nm)absorbedmorethanlargerparticles(>100nm).[9]Nanoparticlescanalso
beabsorbedthroughsublingualroutedirectlyintothebloodstream.[10]Therefore,itcanbepresumedthatsome
Swarnabhasmaparticlesmaygetabsorbedthroughthesublingualroutedirectlyintothebloodstream.Thishasnot
beenexperimentallyprovedforswarnabhasma.However,theantioxidant/restorativeeffectsofSwarnabhasma
againstglobalandfocalanimalmodelsofischemiahavebeenreported.[6]Acuteoraladministration(continuous
for8weeksonalbinomice10mg/20gb.w./day)ofSwarnabhasmahadnotreportedanytoxiceffectsasassessed
byliverfunctiontestsandhistologicalinvestigations.[11]
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Inmodernmedicine,goldnanoparticlesfindsignificantapplicationsindrugdeliveryastheyarecapableof
encapsulatingactivedrugsandtargeting.[12]Colloidalgoldnanoparticlesrepresentacompletelynoveltechnology
inthefieldofparticlebasedtumortargeteddrugdelivery.Themonolayerofpolyethyleneglycol(PEG)overgold
nanoparticleshasbeenfoundtoimprovethecellularinternalizationproperties.[13]Surfacemodificationofgold
nanoparticulatecarrierswithpoly(ethyleneglycol)hasemergedasastrategytoenhancesolubilityofhydrophobic
drugs,prolongcirculationtime,minimizenonspecificuptake,andallowforspecifictumortargeting.[13]Swarna
bhasmahasbeenwellcharacterizedphysicochemicallyandsinceitcontainsmorethan90%ofgoldparticles[14]it
mayalsobetherapeuticallyappliedinsimilarlineslikegoldnanoparticles.CellularinternalizationofSwarna
bhasmaand/oritsuptakeviaparacellularpathwayhavenotbeenestablishedyet.[15]Uptakeofnanoparticlescan
occurnotonlyviamicrofold(M)cells,thehighlyspecializedepithelialcellsinthePeyer'spatchesandisolated
folliclesofthegutassociatedlymphoidtissue(GALT),butalsoacrosstheapicalmembraneofenterocytes.[15]It
hasbeendemonstratedthatuptakeofgoldnanoparticlesoccurredinthesmallintestinebyabsorptionthrough
single,degradingenterocytesintheprocessofbeingextrudedfromavillusandgoldnanoparticlestypicallyless
than58nminsizeultimatelyreachesbloodandvariousorgansthroughblood.[16]Therefore,compatibilitywith
bloodisanextremelyimportantfactorwhentheseparticlesareabsorbedintothebloodstream.Bloodcompatible
materialscanbedefinedasthosematerialswhichdonotdamagebloodcomponentswhentheycomeincontact
withblood.[17]Invitrobiologicalevaluationsofbhasmapreparationsarealsoneededalongwiththe
physicochemicalcharacterizationandclinicalevaluationforpresentdaystandardizationofmetallicbhasma
preparationstomeetthecriteriathatsupportsitsuseworldwide.
Therefore,anattempthasbeenmadetostudythephysicochemicalcharacterizationandbloodcompatibilityoftwo
batchesofSwarnabhasma.
MATERIALSANDMETHODS
TwobottlesofSwarnabhasmawerepurchasedfromTheIndianMedicalPractitionersCoOperativePharmacy
andStoresLimited,Chennai,India(SwarnaBhasmaED)anddesignatedasSB1andSB2.Complementprotein
C3kitwasfromOrionDiagnostica,Finland.Plateletfactor(PF4)kit,AsserachromPF4,wasfromDiagnostica
Stago,France.Allotherchemicalsandotherreagentsusedwereofanalyticalreagentgrade.
Particlesizeandzetapotentialdeterminationbydynamiclightscattering

TheparticlesizesandthezetapotentialsofSwarnabhasmasampleswereanalyzedbyphotoncorrelation
spectroscopyandlaserDoppleranemometry,respectively,withaZetasizer,NanoZS(MalvernInstruments
Limited,UK)at25C.[18]
XRaydiffractionanalysis

TheXRaydiffraction(XRD)powderdiffractionpatternofSwarnabhasmawasrecordedonXraydiffractometer
(SiemensD5005Diffractometer)usingCuKradiation,l=1.5406overtherange30.080.0.
Scanningelectronmicroscopyandenergydispersivespectroscopy

ThemorphologyandelementalcompositionofthebhasmasamplesweredeterminedbyEnvironmentalScanning
electronmicroscopy(SEM)(FEIQuanta)withenergydispersivespectroscopy(EDAX).Arepresentativeportion
ofeachsamplewassprinkledontoadoublesidecarbontapeandmountedonaluminumstubs,inordertogeta
higherqualitysecondaryelectronimageforSEMandEDAXexamination.
Invitrocytotoxicitystudies

TheL929fibroblastcellswereseededin24wellplatesatadensityof5105cells/well,culturedfor24hin
incubatorat37Cunder5%CO2.ThemediumwasreplacedwithSB1andSB2particlesuspensioninthemedium
ataconcentrationof5mg/ml/wellandincubatedfor20h.Mediumalonewasusedascontrol.Theparticleswere
removedand3(4,5dimethythiazol2yl)2,5diphenyltetrazoliumbromide(MTT)assaywasdone.
Bloodcellaggregationandhemolysisstudies

RBCswereseparatedbycentrifugingfreshbloodat700rpm.Thiswaswashedwithsalineanddilutedinsalinein
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aratioof1:4.WBCswereisolatedbycentrifugingthefreshbloodafterlayeringwithhistopaquefor20minat700
rpm.Plateletrichplasma(PRP)wascollectedbycentrifugingthefreshbloodat1000rpmfor20minlayeredon
histopaquesolution.To1mgeachofSB1andSB2particles,100lofthedilutedRBC,WBCsuspensionorPRP
wereaddedandincubatedfor30minat37C.Polyethyleneimine(PEI)andsalineweretakenaspositiveand
negativecontrols,respectively,forallstudies.Aggregationsifanywereobservedthroughaphasecontrast
microscope(LeicaDMIRB,Germany)atamagnificationof40.Hemolysisassaywasdoneontheparticlesas
reportedelsewhere.[19]Normalsalinewasusedasnegativecontrol(0%lysis)anddistilledwateraspositive
control(100%lysis).Theabsorbancewasmeasuredat541nmbyUVVisspectrophotometer(Varian).
Proteinadsorptionstudies

Theplasmawasseparatedbycentrifugationoffreshbloodat700rpm.TenmilligrameachofSB1andSB2
particlesweredispersedin200lofsaline.Tothis200lofplasmawasaddedandincubatedfor1h.After
incubationtheplasmawasseparatedbycentrifugationat10,000rpmanddilutedwithsaline.Theproteinsinthe
plasmasamplesbeforeandafterincubationwereseparatedbypolyacrylamidegelelectrophoresis(PAGE)using
discontinuousnativePAGEmethodofLaemmli.[20]Aresolvinggelof12%andastackinggelof4%wereused.
Electrophoresiswascarriedoutat100Vfor90minusingMiniPROTEANIIelectrophoresiscell(BioRad,CA,
USA).Thegelwasdigitalizedusinganimageanalyzer(LAS4000,Fuji)andthedensitometryscansweredone
withthesoftwareMultiGaugeV3.
Complementactivation

ComplementactivationbySwarnabhasmawasdeterminedbytheturbidimetricmethod,assessingthedepletionof
complementproteinC3onincubationwiththenanoparticles.Theparticlesuspensions(100l)wereincubatedfor
1hat37Cwith100lofcitratedblood.Thefinalconcentrationofthegoldbhasmaparticlesintheassaysystem
wasmaintainedat10g/mlofblood.Theassaywasdoneaspertheprotocolprovidedbythekitmanufacturer.
Plateletactivation

Humanblood(5ml)wascollectedfromconsentedvoluntarydonorinthemorninghoursafter25minrestwith
slightornostasis.Itwasimmediatelyplacedintheice/waterbathfor20min.Plateletrichplasma(PRP)was
collectedbycentrifugationat700rpmfor20min.Tenmilligrameachofbhasmasampleswereincubatedwiththe
freshPRPfor15min.Thiswascentrifugedat2500gfor20min.Plasmasupernatantwascollectedbyaspiration
andPF4wasassayedbyenzymelinkedimmunosorbentassay(ELISA)kit(DiagnosticaStago,France)according
tomanufacturer'sinstructions.Sampleswereassayedinduplicates.PF4levelswereexpressedinIU/ml.Precision
oftheassaywas0.7UI/mlinreplicatedeterminations.
Visualizationoftightjunctionsactinandzonaoccludens1(ZO1)

Caco2cellswereseeded(at20,000cells/well)ontofourwellcellcultureplates(Nunc).Thecellsweremaintained
inincubatorat37Cunder5%CO2andusedfortransportexperiments6dayspostseeding.[21]Mediumwas
replacedwithHank'sbufferedsaltsolution(HBSS)transportmedium,andcellswereequilibratedatleastfor2h
beforeuptakeexperiments.Cellsweretreatedwith500lSBparticlesataconcentrationof10mg/mlfor1h.The
particleswereremovedbywashingthecellsthreetimeswithphosphatebufferedsaline(PBS).Thecellswerefixed
with250lof4%paraformaldehydefor20minatroomtemperature.Thenthecellswerepermeabilizedusing
0.2%TritonX100inblockingsolution,madeof1%(w/v)bovineserumalbumin(BSA)inPBS,for20min,soas
tomakethecellwallpermeabletothestain.ThepermeabilizedcellswerethenwashedtwicewithPBSand
incubatedwith250lof1%BSAfor30min.
Foractinfilamentvisualization,theblockingsolutionwasremovedandcellswereincubatedwith200l
rhodaminephalloidinsolution(0.2g/ml)for20minatroomtemperature.Afterremovalofrhodaminephalloidin,
thecellsweretreatedwith1%BSAasbefore.ThecellswerewashedwithPBS,anddriedovernightat4C.
ImageswereobtainedusingCarlZeissLSMMeta510invertedconfocallaserscanningmicroscope(CarlZeiss,
Germany),equippedwithHe/Nelaser543.Thevisualizationofrhodaminephalloidinwasdoneusingexcitation
andemissionwavelengthsof543and605nm,respectively.ForZO1stainingtheblockingsolutionwasremoved
andcellswereincubatedwith200lofZO1antibody(0.1g/ml)overnightat4C.AfterremovalofZO1
antibodythecellsweretreatedwith1%BSAasbefore.Theblockingsolutionwasremovedandthecellswere
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incubatedwith250lFITCantirabbitIgGforonehouratroomtemperature.ThecellswerewashedwithPBS,
anddriedovernightat4C.ImageswereobtainedusingCarlZeissLSMMeta510invertedconfocallaserscanning
microscope(CarlZeiss,Germany),equippedwithArgon2laser.ThevisualizationofFITCwasdoneusing
excitationandemissionwavelengthsof488and505530nm,respectively.
RESULTS
TheparticlesizedistributionsofthetwobatchesofSwarnabhasmaparticlesevaluatedbydynamiclightscattering
areshowninFigure1.SB1hadameanparticlediameterof717nmandSB2hadameanparticlediameterof669
nm.ThezetapotentialsofnanoparticlesatneutralpH(pH7.4)werefoundtobe17.40.55mVand16.30.37
mV,respectively,forSB1andSB2preparations.
Figure1
ParticlesizedistributionsofSwarnabhasmapreparationsSB1andSB2

TheXRDpatternsofSwarnabhasmaareshowninFigure2.ThesizeofgoldcrystallitesinSwarnabhasmawas
calculatedfromtheXRDpatternusingtheScherrerformulaanddeterminedtobethesame(28nm)forbothSB1
andSB2.MorphologiesofSB1andSB2byscanningelectronmicroscopyareshowninFigures3aand3b.The
elementalcompositionoftheSwarnabhasmasampleswereanalyzedbyEDAXasshowninTable1.
Figure2
XraydiffractionpatternsofSwarnabhasmapreparationsSB1andSB2

Figure3
MorphologyofSwarnabhasmapreparations(a)SB1and(b)SB2by
scanningelectronmicroscopy
Table1
ElementalanalysisofswarnabhasmabyEDAX
InvitrocytotoxicityoftheseparticleshasbeendonewithL929fibroblastcellsasperISOstandard.[22]Ithasbeen
confirmedbytheinvitrocytotoxicitystudiesthatthebhasmaparticlesarenontoxic.Ascomparedtocontrol
(medium)theparticlesshowed100%cellviability.
TheaggregationsofthebloodcellsoninteractionwiththenanoparticlesareshowninFigures4,5and6,
respectively,forRBC,WBCandplatelets.ItrevealednoaggregationofbloodcellsonincubationofSwarna
bhasmaatahigherinteractionratioof10mg/ml.Polyethyeleneimine(PEI)whichwasusedaspositivecontrol
showedaggregationwhereassalineusedasnegativecontroldidnotshowanyaggregation.Thesamewasvisible
withthehemolyticpropertyofthenanoparticles.ThehemolysisinducedbySB1was0.05%andthatforSB2was
0.3%whichwaswellwithintheacceptablelimitsof1%.[23]
Figure4
AggregationofRBCbyincubationof(a)SB1,(b)SB2,(c)normalsaline
(negativecontrol)and(d)polyethyleneimine(positivecontrol)
Figure5
AggregationofWBCbyincubationof(a)SB1,(b)SB2,(c)normalsaline
(negativecontrol)and(d)polyethyleneimine(positivecontrol)

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Figure6
Aggregationofplateletsbyincubationof(a)SB1,(b)SB2,(c)normalsaline(negativecontrol)
and(d)polyethyleneimine(positivecontrol)
TheproteinadsorptionstudiesevaluatedusingnativePAGEelectrophoresisdemonstratednosignificantadsorption
ofproteinsoccurringontoSB1orSB2asshowninthedensitometryscanofthetreatedplasma[Figure7].The
figureshowsthepeaksofalbumin,globulinregionandfibrinogen.Comparedtothedensitometryscanofcontrol
plasma,thepeakheightsofalbumin,fibrinogenorglobulinsofplasmatreatedwithbhasmadidnotshowany
changeindicatingnosignificantadsorptionofbloodproteins.
Figure7
DensitometryscanofnativePAGEofplasmaproteinsbeforeandafter
incubationwithSB1andSB2

MeasuringC3aorC5ainbloodorserumaftercontactwithamaterialhasbeenthemostusualwayofassessing
complementactivation.Ithasbeenclaimedthatasurfaceisbiocompatibleifthesemarkersarenotincreasedinthe
fluidphase.[24]SinceC3iscleavedtoC3aandC3bbythecontactofthesurfacewithblood,irrespectiveof
whethertheactivationoccursviaclassicaloralternativepathways,andalsoC3acouldbeadsorbedontothe
materialsurfacejustlikeanyotherproteins,C3depletioninthemediumcanbetakenasanindirectmeasureof
complementactivation.TheamountofC3inblood(preincubation)was127mg%.AfterincubationwithSB1and
SB2,itwas126mg%and131mg%,respectively,indicatingnosignificantchangesinthecomplementprotein
level.
Plateletfactor4(PF4)isaplateletspecificproteinsecretedwhenaplateletisactivatedandbelongstotheCXC
chemokinefamily.MeasurementsofplasmalevelsofPF4havebeenshowntobethemarkerofplatelet
degranulation,andincreasedlevelofPF4isusedtodetectplateletactivationofthecirculatingpoolofplatelets.[25]
OnincubationwithSwarnabhasmathelevelofplateletfactor4inplasmadidnotchangeappreciablycomparedto
controlplasma.ThePF4levelincontrolplasmawas5.430.10IU/mlandafterincubationwithSB1for15minit
was4.820.4IU/mlandforSB2itwas5.07+0.3IU/ml.Theplateletadheredontothebhasmaparticleswere
observedthroughscanningelectronmicroscopyafterincubatingtheSBparticleswithplateletrichplasma.There
werefewcellsobservedadheringontotheparticles.OnlyonecellwasfoundadheredontotheSB2samplewithno
activationordeformationoftheplateletsasshowninFigure8.
Figure8
MorphologyofplateletadheredontoSwarnabhasmapreparationby
scanningelectronmicroscope

Thecontrolcellsstainedwithrhodaminephalloidintovisualizeactinproteinshoweduniformstainingpattern[
Figure9a].CellstreatedwithSB1andSB2particlesshoweddisruptedstainingpattern,thoughthedisruptionwas
higherwithSB1asseenfromFigures9bandc.Actinfilamentswereobservedtobediscontinuousanddisrupted
asevidencedfromthestainingpatternandtheclumping.Tofurtherinvestigatetheeffectonthetightjunction
proteinsimmunofluorescencestudiesusingantiZO1wasdone.ZO1isatightjunctionassociatedprotein,which
playsanimportantroleintightjunctionfunctionalregulation.Tightjunctionsarecomposedoftransmembrane
proteinsoccludin,claudinsandjunctionaladhesionmoleculeswhichintercalatewithcorrespondingproteinsfrom
adjacentcellstoformtheintercellularbarrier.Theseproteinsassociatearewithperipheralmembraneproteins
includingthemembraneproteinszonulaoccludens(ZO1to3)whichjoinsthetransmembraneproteinstotheactin
cytoskeleton.ZO1andoccludingphosphorylationareassociatedwithstimulusinducedtightjunctiondisassembly
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andparacellularpermeabilitychanges.TheeffectofSB1andSB2particlesonZO1tightjunctionalproteinswas
evaluatedonCaco2cellmonolayersasshowninFigure10.IntheuntreatedcellsZO1isobservedassmooth
linesatcellcelljunctions[Figure10a].Theimmunofluorescentstainingintensityofbhasmaparticletreatedcells
wereobservedtobeweakercomparedtothecontrolwhichindicatedthelossofZO1fromsitesofcellcell
contact[Figure10aandb].
Figure9
Confocalimages(20)ofCaco2cellactin.(a)and(b)Cacocellsexposed
to5mgofSB1andSB2,respectively,for1h(c)Cacocellswithoutany
treatment(control)

Figure10
Confocalimages(20)ofCaco2celltightjunctionproteinZO1.(a)and(b)
Cacocellsexposedto5mgofSB1andSB2,respectively,for1h(c)Caco
cellswithoutanytreatment(control)

DISCUSSION
DifferentmethodsofpreparingSwarnabhasmahavebeenreportedinvariousAyurvedictexts.[2,3]Thishasbeen
donebytheincinerationofgoldwithvariouscompoundslikemercury,mercurysulfide,sulfur,orpiment(As2S3),
realgar(AsS),chalcopyrite,etc.outofwhichtheprocedurewithmercuryisconsideredtobetherapeutically
effective.[26]VariousattemptshavebeendoneonthestandardizationofSwarnabhasmapreparationsforclinical
applications.[27]However,thebloodcompatibilityaspectshavenotbeeninvestigatedtillnow.
Althoughtheparticlesizesofdifferentbatchesshowedsimilarity,itseemsthattheseparticlesareaggregatesof
muchsmallerparticles.Whendispersedinanaqueousmedium,goldcolloidsformanegativelycharged
hydrophobicparticlesuspension.Thishydrophobicitygivesthesegoldparticlesatendencytoaggregatetogetherto
formlargerparticles.[28]BothbatchesofSwarnabhasmaexhibitedlargersizesandagglomerationoftheparticles.
However,thecrystallitesizecalculatedfromXRDwasmuchsmaller.Therefore,thecomparativelylargersizemay
beduetotheagglomerationoftheparticlesbyrepeatedcyclesofcalcinationsinvolvedinpreparationasreported
earlier.[29]Zetapotentialhasbeensuggestedtoplayanimportantroleinparticleuptakebecausethesurfaceofthe
intestinalmucosaisnegativelychargedowingtothepresenceofglycocalix.Particleswithahighpositivesurface
chargelikechitosanareusuallyattractedbytheintestinalmucosa,whichhelpsinincreasingtheintestinal
absorptionoftheencapsulateddrug.However,thestrongelectrostaticinteractionbetweenthepositivelycharged
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particlesandthenegativelychargedglycocalixmayslowdowntheprogressionandpenetrationoftheseparticles
towardstheepithelialcellsurfacereducingtheiruptake.Alsoithasbeenshownthatnonionizedparticleshavea
greateraffinityforMcellsthanforionizedparticles[30]andpositivelychargedparticles.[31]Ithasbeensuggested
thatgoldbhasmaparticleswithlownegativezetapotentialandnanosizemaybeuptakenbyasimilarmanner.
TheXraydiffractionpeaksat2=38.2,44.4,64.6and77.6ofSwarnabhasmawereidenticalwiththose
reportedforthestandardgoldmetal(Au)(JCPDSFileNo.040784).Noothermajordiffractionpeakswere
observedconfirmingthattheSwarnabhasmaiscomposedofmainlygoldnanoparticles.Thehighintensityof
XRDlinesintheXRDpatternsuggestsitscrystallinenature.Ithasbeenreportedthatnanoparticlesexhibiteda
sizedependentuptakefromtheintestine,anditspassageviathemesenterylymphsupplyandlymphnodestothe
liver,[32,33]withsignificantabsorptionforparticleslessthan100nm.Therefore,uptakeofSwarnabhasma
particlesof28nmthroughtheintestinecanbeexpected.
FromtheEDAXresultsitwasconfirmedthat90%ofSwarnabhasmacontainspuregoldandisincorrelationwith
XRDdata.EDAXprovidegoodestimateoftheconcentrationofmainelementsinthesampleinasignificantly
fasterwayandprovidesusefulinformationonthedistributionoftheelementformingthesampleandtheirpossible
chemicalform.[34]
TheAyurvedicmultiingredientcompoundsareformulatedinawaythattheingredientsarecapableofcounter
balancingtoxiceffects,ifany,presentintheherbsormetals(bhasma).[35]Theseparticlespassthroughextensive
processingbeforetheyaredeclaredfitforinternaluse.TheprocessesconsistofShodhanandMarana.Cultureof
variouscelltypeswithcolloidalgoldshowednoevidenceofcytotoxicity.[3639]Noinvivocytotoxicityhasbeen
reportedwiththeuseofcolloidalgoldadministratedintravenouslytoponiesandpigsatdosesof400mgofgold.
[40]Theinitialeventwhenamaterialcomesincontactwithbloodistheadsorptionofproteins.Thenatureof
proteinandamountofproteinadsorbedwilldirectlyinfluencethecompatibilityoftheparticleswiththeblood.
Therewasacorrelationoftheadsorptionoftheglobularproteinswiththebloodcellaggregationshowingno
activationoraggregationofcellsonincubationwithSwarnabhasma.Activationofplateletsinitiatesthe
deformationofthecellswithpseudopodformationandendswithbloodcoagulationorthrombusformation.[41]In
thepresentstudyplateletsseemtobenotactivatingandadheringontothebhasmaparticlesandeventheveryfew
plateletsadheredarenotactivatedasseenfromtheirroundshape.Thisisanindicationoftheveryhighplatelet
compatibilityofSwarnabhasmapreparations.
Oneofthenegativeeffectsoftheclinicalapplicationofvariousbloodcontactingmaterialsistheactivationofthe
complementsysteminducedbytheforeignsurface.Theresponseofbloodincontactwiththematerialdependson
physicochemicalfeaturessuchassurfacearea,surfacecharge,hydrophobicity/hydrophilicityetc.Theresponse
dependsdirectlyonthesurfacearea.AdsorptionofC3triggerscomplementactivation.[41]Ithasbeen
demonstratedinthisstudythattheadsorptionsofC3onSwarnabhasmapreparationsareinsignificantindicating
thatthesepreparationsdonotinduceanycomplementactivationwhenitreachesthesystemiccirculation.
Pharmacologicaleffectsexertedbythetherapeuticagentsdependuponitsabilitytocrossthebiologicalmembranes
intothesystemiccirculationandreachthesiteofaction.Thisisusuallyoccurredbyoneofthetwopathways
paracellularortranscellular.Mostdrugsaretransportedtranscellularlydependingontheirphysicochemical
propertieshowever,theparacellularrouteisusuallythemainrouteofabsorptionfornanoparticles.Thisis
governedbythetightjunctions(TJs).TJsareamultipleunitstructurecomposedofmultiproteincomplexthat
affiliateswiththeunderlyingapicalactomyosinring.TJproteinsidentifiedincludetransmembraneproteins
occludinandclaudin,andcytoplasmicplaqueproteinsZO1,ZO2,ZO3,cingulin,and7H6.Althoughthe
adaptivemechanismsandspecificregulationofthesetightjunctionsareareasofactiveinvestigationandremain
incompletelyunderstood,itisknownthatsomepolymerscanpromotetheirwidening,facilitatingabsorptionofthe
particlesintothesystemiccirculation.Ithasbeenestablishedinthisstudybythetightjunctionvisualizationstudies
thattheSwarnabhasmaparticlesarecapableofopeningtightjunctions,thusfacilitatingthebhasmaparticlestobe
absorbedintothesystemiccirculationandcomesindirectcontactwithblood.ThustheSwarmabhasmaparticles
shouldbehighlycompatiblewithblood.
CONCLUSION
BhasmasareAyurvedicmetalbasedpreparationsmadebymanysystematicprocesseswithherbs,convertingraw
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metalintoitstherapeuticform.Swarnabhasma,atherapeuticformofgoldmetalofnanosizedparticlesfoundto
bewithacrystallitesizeof2835nmandwas90%puregoldasvisiblefromXraydiffractionandelemental
analysis.TheyhadalownegativezetapotentialinaphysiologicalpH.TheSwarnabhasmapreparationsdidnot
induceanybloodcellaggregationoranyproteinadsorption.Activationpotentialofthesepreparationstowards
complementsystemorplateletswasnegligible.Theseparticleswerealsononcytotoxic.Caco2cellexperiments
ontightjunctionintegrityinthepresenceofSwarnabhasmaparticlesdemonstratedtheirabilitytoopenthetight
junctions.Ithasbeendemonstratedthatuptakeofgoldnanoparticlesoccurredinthesmallintestinebyabsorption
throughsingle,degradingenterocytesintheprocessofbeingextrudedfromavillusandgoldnanoparticles
typicallylessthan58nminsizereachingvariousorgansthroughblood,[16]whichsuggeststheimportanceofthe
bloodcompatibilitystudiesforthestandardizationofbhasmapreparations.SincegoldintheSwarnabhasmais
approximately2835nminsize,itcanreachtheaffectedsiteonoraladministrationviaintestinalabsorptionand
possiblycanreleaseAu(I)ionsinasustainedmannerrequiredfortherapeuticaction.[42]Theseresultsreinforcethe
applicationofSwarnabhasmaasatherapeuticagentinclinicalmedicinefromthesafetypointofview.These
testingprotocolsmaybeadoptedasascreeningtestforallbhasmapreparationstomeetthecriteriathatsupportsits
useworldwide.
ACKNOWLEDGEMENT
WearegratefultotheDirectorandtheHeadBMTWingofSCTIMSTforprovidingfacilitiesforthecompletionof
thiswork.Authorshavefullaccesstoallofthedatainthestudyandtakeresponsibilityfortheintegrityofthedata
andtheaccuracyofthedataanalysis.ThisworkwassupportedbytheDepartmentofScienceandTechnology,
Govt.ofIndiathroughtheprojectFacilityfornano/microparticlebasedbiomaterialsadvanceddrugdelivery
systems#8013,undertheDrugsandPharmaceuticalsResearchProgramme.
Footnotes
SourceofSupport:Nil
ConflictofInterest:Nonedeclared.

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