Review Article Recent Updates in Neuroprotective and

www.mjms.usm.my © Penerbit Universiti Sains Malaysia, 2015 For permission, please email:[email protected]

Abstract Centella asiatica, locallywellknowninMalaysiaaspegaga, isa traditionalherb thathasbeen used widely in Ayurvedic medicine, traditional Chinese medicine, and in the traditionalmedicineofotherSoutheastAsiancountriesincludingMalaysia.Althoughconsumptionoftheplantisindicatedforvariousillnesses,itspotentialneuroprotectivepropertieshavebeenwellstudiedanddocumented.Inadditiontopaststudies,recentstudiesalsodiscoveredand/orreconfirmedthatC. asiaticaactsasanantioxidant,reducingtheeffectofoxidativestress in vitro and in vivo.Attheinvitrolevel,C. asiaticapromotesdendritearborisationandelongation,andalsoprotectstheneuronsfromapoptosis.InvivostudieshaveshownthatthewholeextractandalsoindividualcompoundsofC. asiaticahaveaprotectiveeffectagainstvariousneurologicaldiseases.MostoftheinvivostudiesonneuroprotectiveeffectshavefocusedonAlzheimer’sdisease,Parkinson’sdisease,learningandmemoryenhancement,neurotoxicityandothermentalillnessessuchasdepressionandanxiety,andepilepsy.Recentstudieshaveembarkedonfinding themolecularmechanismofneuroprotectionbyC. asiaticaextract.However,thecapabilityofC. asiatica inenhancingneuroregenerationhasnotbeenstudiedmuchandislimitedtotheregenerationofcrushedsciaticnervesandprotectionfromneuronalinjuryinhypoxiaconditions.Morestudiesarestillneededtoidentifythecompoundsand the mechanism of action of C. asiatica that are particularly involved in neuroprotectionand neuroregeneration. Furthermore, the extraction method, biochemical profile and dosageinformationoftheC. asiaticaextractneedtobestandardisedtoenhancetheeconomicvalueofthistraditionalherbandtoacceleratetheentryofC. asiaticaextractsintomodernmedicine.

Keywords: antioxidant, neuroprotective, neurological disease, neuronal injury, pegaga

Introduction

Thenervoussystem,consistingofthebrain,spinal cord, and peripheral nerves, is made ofcomplex and specialised structures which arevulnerable to various diseases and injury thatreducesensorimotorandcognitivefunctions,andmayalsobethecauseoflife-threateningproblemsin acute cases. Unfortunately, spontaneousregeneration and healing processes occur veryminimally in damaged tissues due to their highcomplexity.OurteamofresearchersattheTissueEngineering Centre, Universiti KebangsaanMalaysia has been conducting research on theregeneration of various tissues, and studies onnerve regeneration for development of cell and

tissue therapies have been going on for almosteightyears (1,2).Wehave identifiedvariouscellsources, ranging from stem cells to adult nervecells, and developed various scaffolds, rangingfrombiological tissue to synthetic hollow tubes,toenhanceinvitroandinvivonerveregeneration(3–6). Realising the historical nature ofmedicinalherbs,wewere attracted to scrutinisethe pharmacological effects of herb extracts insynergywith the provided cells and scaffolds tofurther enhance nerve regeneration, and also toimprovetheutilisationoftissue-engineerednervegraftsandcelltherapyinclinicalapplicationsfornervedegenerationandinjury. Ithasbeenreportedthatthereare250000plant species on the earth, and approximately 5

Review Article Recent Updates in Neuroprotectiveand Neuroregenerative Potential of Centella asiatica

Yogeswaran Lokanathan1, Norazzila omar1, Nur Nabilah ahmad Puzi1, Aminuddin Saim2, Ruszymah hj idruS1,3

1 Tissue Engineering Center, UKM Medical Center, 56000 Cheras, Kuala Lumpur, Malaysia

2 Ear, Nose and Throat Consultant Clinic, Ampang Puteri Specialist Hospital, 68000 Ampang, Selangor, Malaysia

3 Department of Physiology, Faculty of Medicine, Universiti Kebangsaan Malaysia, 56000 Cheras, Kuala Lumpur, Malaysia

Submitted:15Sept2015Accepted:25Nov2015

4Malays J Med Sci. Jan-Feb 2016; 23(1): 4-14

Review Article |RecentUpdatesinPotentialofCentella asiatica

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000 species have specific therapeutic value (7).Generally,C. asiatica is a traditional medicinalherb that belongs to the Apiaceae family and isknown as pegaga in Malaysia. It has also beenidentified by different names such as Indianpennywort in English, gotu kola in Sri Lanka,brahmi in Hindi and mandukaparni in theAyurvedicmedicineofIndia,buakbokinThailand,kakikudainIndonesiaandyuhong-yuhonginthePhilippines (8,9). This review focuses on recentdiscoveries,preferablywithinthepastfiveyears,onthepropertiesofC. asiatica(L.)inprotectionagainstneural-relateddiseasesandregenerationofinjurednerves,frominvitrostudiestoinvivoandclinicalstudies. Geographically,thisparticularherbisnativetoIndia,China,SriLanka,Madagascar,Indonesiaand Malaysia, and grows well in swampy anddampareasoffields(10).Pertainingtoitsmedicalbenefits, it has been found broadly across itsboundaries in Turkey, South America, and theWest Indies (11,12). Over thousands of years, ithadbeenusedbypeopleallaroundtheworldasaremedyformanytypesofdisease.IthadbeenusedwidelyinAyurvedicmedicinetorejuvenatethe body, improve the intellect and combatcognitive disorders (13). It is known as a braintonic and has also been applied in traditionalChinese medicine. In addition, it has also beenincorporatedasadruginothermedicalpracticessuchastheGermanHomeopathicPharmacopoeia(GHP)andtheEuropeanPharmacopoeia(14). C. asiatica is getting more popular in themodern world due to its versatility and efficacythathavebeenprovenbymanyscientificstudies.C. asiatica is well known as a wound-healingagent, due to its ability to heal small wounds,scratches, burns, and skin irritation (15,16). Aswell as skin re-epithelisation, Ruszymah andher colleagues (17) remarked on the capacity ofthis herb to treat cornea epithelialwounds.Thefew innovations using C. asiatica extracts inhealthcare applications by Ruszymah and hercolleagues won awards from the InternationalExposition of Research and Inventions ofInstitutions of Higher Learning (PECIPTA)as early as 2007. As technology develops, theundiscovered potential of this herb is furtherscrutinisedand ithasbeendocumented tohaveanti-inflammatory,antimicrobialandantifungal,antidepressant,antioxidantandanticancereffects(18–21). C. asiatica was also found to have anantiproliferativeeffectagainsthumanrespiratoryepithelialcellsinvitroanditalsoreducedspermcount,motility,andviabilityinmalerats,besideshaving other antifertility effects (12,22). Even

though many preclinical investigations havebeenperformed, furtherdetailedclinical studiesare highly needed to scientifically evaluatepharmacological values and standardise thebiochemical profile of the extract before usein various therapeutic applications. Since thetoxicity effects of C. asiatica vary according toits geographical distribution, standardising itsbioactivecomponentsduringextractpreparationwould be appear to be momentous. This isbecause such fluctuations can lead to variouscomplicationswhichwouldconsequentlylimititstherapeuticvalue.

Chemical Composition and Preparations of C. asiatica

Brinkhaus et al. (10) adequately summedup the chemical composition of C. asiatica,discovered as early as 1956 (23). About 0.1% isessential oils,while 1–8% is saponin-containingtriterpene acids and their sugar esters. Furtherstudies on chemical constituents were done onhydroponically grown C. asiatica by Othman(24).Theauthorstudiedthenutritionalcontent,the total polyphenol content between air-driedsamplesandfreshsamples,salicylicacidcontentand flavonoid content, and found that thenutritionalcontentisaboutthesamethroughouttheplant fromthe leaf to theroot;however, theleaf contains higher P, Fe,Na,Mg and ascorbicacid,whiletherootscontainhigherCaandK.Thepolyphenolandsalicylicacidcontentwashigherinfreshsamplescomparedtoair-driedsamples.Analysisof sampleswithHPLCshowed that themajorflavonoidcompoundswerecatechin,rutin,quercetin,andnaringin. Next,OyedejiandAfolayan(25)investigatedthe content of C. asiatica essential oils fromplantsgrowninSouthAfrica,andfoundthatthemajor essential oils are a-humulene (21.06%),b-caryophyllene (19.08%), bicyclogermacrene(11.22%), germacrene B (6.29%) and myrcene(6.55%). Siddiqui et al. (26) isolated andcharacterised the structure of three novelcompounds in C. asiatica: centellin, asiaticinand centellicin with the structure of 6-acetoxy-trideca-1,7-dien-4-yn-3-ol, p-benzoyloxymethyl-butyl benzoate, and 1-(20,30-dihydroxypropyl)-2-en-3-methyl-6-hydroxy-9-yn-undecanoate,respectively. Zainol et al. (27) profiled twoaccessions ofC. asiatica grown in Johor Bharuandfoundthatitshoweddifferentphytochemicalcontentindifferentparts,withleavescontainingthe most phytochemicals. Zhang et al. (28)evaluated content of the herb from different

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Malays J Med Sci. Jan-Feb 2016; 23(1): 4-14

locations in China and developed a chemicalfingerprinting method for quality evaluation.Chong and Aziz (29) reviewed the chemicalcomponentsofC. asiaticafrom1949to2011.Theyfoundthatthechemicalcomponentswerediverseandthattriterpeneswerethemajorconstituents.Hashim et al. (30) specifically studied thetriterpene constituents and their biologicalactivities in ethanolic-aqueous extract from C. asiatica grown in a forest reserve in Malaysia.Theyfoundthatthereweresignificantamountsofmadecassosideandasiaticoside,butlowamountsofasiaticandmadecassicacid.Finallyandmostrecently,Devkotaetal.(31)studiedthecontentofessentialoilsfromC. asiaticagrownindifferenthabitats inNepal. They found that the essentialoilsvaried fromtheones found inSouthAfrica.Thehabitatalsohadaninfluence,withC. asiaticagrown in shady grasslands containing 39 oils,open grassland 34, and open agricultural lands36. Traditional and modern preparations varybetweencountries.InMalaysia,herbalremediessuch as C. asiatica are prepared traditionallyby traditional healers and tailored to individualneeds.Mostareintheformofjuiceandoil,andmodernpreparations are in tablet form (32). InIndia,C. asiaticaisusedasapasteappliedtotheskin,coldpoulticeusedforrheumatism,leafjuicerubbedontheforeheadforheadache,orointmentorpowdermixedwithbathwater(33).Theleavesarealsocookedandeaten,whileinMalaysiaitiseatenrawasasalad(34).

Enhancement and Regeneration of Nerve Cells

Assyntheticdrugscanleadtolungandkidneytoxicity, many clinicians and scientists havesearchedvigorouslyforotheralternativestotreattheirpatients.Herbalplantswhicharenaturallyrich in therapeutic value,more eco-friendly andhave lesser side effects have been subsequentlystudied for utilisation in medical applications.ThishasbroughtC. asiatica,which isknown tohave memory and cognitive enhancement, intoscientific investigations for nerve regenerationand neurological functions before therapeuticuse. Researchon theneuroregenerativecapacityofC. asiaticaon thecentralnervoussystemhasbeen widely conducted, focusing on brain cells.Soumyanath et al. (35) revealed the ability ofasiatic acid (AA) to promote the elongation ofneurites using an in vitro experimental model.Theyalsoperceivedthatthisactioncouldbeco-

stimulatedwiththeotheractivecompoundsfoundintheherbthathavesynergisticeffectswithAA.Inparallelwiththis,Raoetal.(36)reportedthatfreshC. asiaticaleafextractsignificantlyincreasesthe dendritic arborisation of hippocampal CA3neuronsinvivo.InviewofC. asiaticaasanervetonic(37),itisbelievedtohavetherapeuticeffectson theperipheralnervous system too.However,furtherinvestigationsareneededtobringit intoevidence,astheliteratureonsuchnervoussystembenefitsisfartoolimitedtodate. Since variation of extract constituentscan lead to various complications, scientistsfrom Thailand have recently established astandardisedextractofC. asiaticaknownasECa233 (38). The extract was clearly demonstratedto have stimulatory effects on the elongation ofneuroblastomacellneuritesatamaximumdoseof100µg/mL(39).Inspiteofdendriticarborisation,thisparticularherbalsoexhibitsneuroprotectiveproperties. Zhang and his colleagues (40) haverecentlydisclosedthatAAamelioratestheactionofC2-ceramideininducingneuronalcelldeathinaconcentration-dependentmanner. Thoughtheunderlyingmechanismswherebytheherbexertsitseffectsarepoorlyunderstood,the aforementioned properties synchronisewiththemasteryofthisherbtophosphorylateseveralsignalling pathways to mediate its functions.Indeed, the mechanisms involved in neuritedevelopment are quite complex, but the MEK/ERK and PI3/Akt signalling pathways havegained significant attention (41). A previousreport elucidated that AA in C. asiatica elicitsits neuroregenerative effect via the MAPkinase pathway (35). In agreement with this,Wanakhachornkrai et al. (39) also revealed thatECa233isMEK/ERK-andPI3K/AKT-dependentinpromotingtheelongationofneurites. Besides that, Omar et al. (42) recentlydisclosedtheregulationofthecaspase-9pathwaybyC. asiaticainmodulatingneuroncellsurvivalagainst apoptosis. In this study, l-buthionine-(S,R)-sulfoximine(BSO)-inducedhumanneuroncelldeathwastreatedwithethanolicextractofC. asiaticaintherangeof5–500µg/mL.Theyfoundthat, at low concentrations, this extract is ableto protect neuron cells against oxidative stress.This may point to the capacity of this herb ininhibitingthecaspase-9pathwaybyhinderingtheproduction of proapoptotic proteins, promotingantiapoptotic proteins and imitating the latter(43,44). Though this mechanism is apparentlyfocused on neuroprotective effects againstapoptosis, such amechanism could be a subsetin the regeneration and enhancement of nerve


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cellinjuries.Alltheaforementionedmechanismsoverlap each other in maintaining nerve cellgrowth.

Effects of C. asiatica on Systemic Diseases

Among the many uses of C. asiatica intraditionalmedicineisasabraintonictoincreasememory performance (45,46). Also, C. asiaticahasbeenusedtotreatneurodegenerativediseases(47)suchasAlzheimer’sdisease,characterisedbyadeclineincognitivefunctions,andinParkinson’sdisease, characterised by loss of locomotorcontrolduetoadecreaseofneurotransmittersinthebrain.Thecommondenominatorinthesetwodiseasesisthepurportedmechanismunderlyingtheirpathologies,whichisoxidativestress.Inthisreview, the effect ofC. asiatica on amelioratingoxidative stress in vitro and in vivo will bedescribedfirst,followedbytheeffectofC. asiaticaon Alzheimer’s disease and Parkinson’s disease.Othermental illnessesof interestaredepressionandanxiety,andepilepsy. Oxidative stress occurswhenmore reactiveoxygen species (ROS) than antioxidants arepresent in thebody.ROSprotect thebody fromforeign insults such as from xenobiotics whichenterthebody.However,wheninexcessorwhenthe antioxidant level in the body is reduced,ROS cause damage to native nucleic acids,proteins and lipids andhavebeen implicated inneurodegeneration,cancerandageing(48). The effect of C. asiatica in improvingmental performance has been attributed to itspropertyasanantioxidantandasapromoterofantioxidant production. This has been validatedthrough many ROS scavenging assays (radicalscavengingassays)invitroandinvivo.Anandetal. (49)reported that,with the1, 1diphenyl–2–picrylhydrazyl (DPPH) free radical scavengingactivityassay,themethanolextractofC. asiaticahas the highest free radical scavenging activityfollowed by the hexane extract. This can beattributed to the higher levels of polyphenolsand flavonoids present in this fraction, whichimparttheantioxidantfunction.Anandetal.alsoreportedonthereducingcapabilityofC. asiaticaextracttoinactivatehydroxyls,themostreactiveROS.Again,methanolextractshowedthehighestreducingpower. Three assays were conducted by Chippadaand Vangalapati (50) on the methanol extractofC. asiatica:DPPH,reducingpowerandnitricoxide (NO) radical scavenging assays. Theyfound thatmethanolic extract ofC. asiatica has

concentration-dependent DPPH scavenging,reducing power and NO radical scavengingactivityupto2000µg/mL.TheethanolicextractofC. asiaticawasalsoconfirmed,usingtheDPPHandsuperoxideradicalscavengingactivityassays,to have significant antioxidant activity (51).Ariffin et al. (52) testedC. asiatica prepared asherbal teas with the DPPH and ferric-reducingantioxidantpotential(FRAP)assays.TheyfoundthatC. asiatica herbal teas possess significantlyhighantioxidantproperties,withnon-fermentedteas having a higher antioxidant activity thanfermentedones. A study on reproductivity in male rats bySainath et al. (53) found antioxidant activity ofC. asiaticaagainstlead-inducedoxidativestress.TheratsweregivenaccesstowatercontainingleadadlibitumandagroupwasalsogivenC. asiaticaextract.After70days,theratsweresacrificedandtheliver,brain,kidney,testis,epididymis,prostategland, vas deferens and seminal vesicles wereexamined.Theyfoundthat,inthegroupgiventheextract,therewasadecreaseinlipidperoxidationandan elevationof antioxidant enzymeactivity.The extract showed antioxidant activity in allthree assays. On comparison of the methanolextractwithascorbicacid,Singhetal.(54)foundthe extract to have concentration-dependentantioxidant activity. All of these studies clearlyindicatedtheantioxidantpotentialofC. asiaticaextract,specificallyofthemethanolicextract. There are two hypotheses for thedevelopment of Alzheimer’s disease: thecholinergic hypothesis and the amyloid cascadehypothesis (52). In the first, it is hypothesisedthat cognitive decline in Alzheimer’s patients isdue to degeneration of cholinergic neurons orcholinergic transmission. Therefore, an increaseintheavailabilityofacetylcholineisadvantageousand is the reason for currentmedical treatmentwith acetylcholinesterase inhibitors. The otherhypothesis is that neuronal damage is due todeposition of amyloid plaques formed frombeta-amyloids (Aβ). These plaques are thoughttobeformedduetooxidativeinsultswhichleadto inflammation, lipid peroxidation and plaqueformationanddepositioninthebrain. To investigate this theory, Ramesh et al.(55) studied the effect of C. asiatica aqueousextract on beta-amyloid fraction 42 (β42) invitro.TheyfoundthattheextractdidnotinhibitAβ aggregation anddidnot disintegrate alreadyformedfibrils.They thereforeproposed that thebeneficialeffectofC. asiaticaseeninAlzheimer’spatients could be due to antioxidative or anti-inflammatory properties of C. asiatica, or due

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to an enhanced secretase pathway, an enzymepathway that degrades the formed plaques.Defillipo et al. (56) found that water extract ofC. asiatica inhibited phospholipase A2 (PLA2)enzymes, particularly cPLA2 and sPLA2, whichare thekeyplayers inAβ-inducedneurotoxicity.Further studies on the water extract done byGray et al. (57,58) found caffeoylquinic acids asthe active substances preventing Aβ-inducedcell death, having antioxidative properties andenhancing mitochondrial biogenesis. Thus,C. asiatica was suggested as a prospectiveremedy for Aβ-induced neuroinflammationand oxidative stress. Then, Rahman et al. (51)provedthatC. asiaticahasantioxidantpropertiesby further testing the ethanolic extract on theactivity of acetylcholinesterase (AchE) andbutyrylcholinesterase (BchE). They found thattheextractinhibitedthesetwoenzymesandcouldtherefore increase availability of acetylcholinefor cholinergic transmission. Focusing at themolecularlevel,C. asiaticawasfoundtoincreasephosphorylation of cAMP response element-binding protein (CREB) in neuroblastoma cellsexpressing Aβ, possibly mediated by the ERK/RSKsignallingpathway(59). Soumyanathetal.(35)experimentedontheTg2576 transgenic mouse model of Alzheimer’sdisease.ThismodelcontainsAβplaques,astrocyticandmicroglialactivationanddystrophicchanges.On behavioural analysis with the Morris watermaze test, the mice showed impaired spatialmemory. On treatment with aqueous extract ofC. asiatica,therewasanimprovedlearningcurvecomparable to wild-type mice. On dissection,however,therewasnosignificantdifferenceofAβcontent.Inthispreliminarystudy,theyfoundthattheaqueousextractofC. asiaticadoesnotshowinhibitionofAchEandBchE,offersnoprotectionagainst hydrogen peroxide oxidative stress andhasnoeffectonglutamateneurotoxicity.AqueousextractofC. asiatica isknowntobevoidofAA,itsmain antioxidant.This couldbe the cause oftheabsenceofantioxidativefunctions.However,sincetheaqueousextractdoesshowbehaviouralimprovementscomparabletowild-typemice,theauthorsproposedtheabilityoftheaqueousextractto affect the pathology of Alzheimer’s diseasethrough a different mechanism or pathway,and also the presence of novel compounds yetundeterminedintheextract. Xuetal.(60)testedAAinvitroonglutamate-induced neurotoxicity in SH-SY5Y cells and invivoonamonosodiumglutamate(MSG)-induceddementiaanimalmodel.Oxidativestresscancause

apoptosisofcellsandcompromisemitochondrialfunction.AA reduced the incidenceof apoptosisby 5% compared to untreated SH-SY5Y cells.AA also prevented the decline inmitochondrialmembranepotential (MMP)which is ameasureof mitochondrial function. In gene expressionanalysis,AAtreatmentcausesupregulationofSirt1and PGC-1 genes responsible for mitochondrialbiogenesis and functions, and hence positivelyaffectstheirsurvivalandlongevity. Parkinson’sdisease,on theotherhand, isaneurodegenerative disease associated with thelossofdopaminergicneuronsinthebasalganglia.The exact mechanism of neurodegeneration iscurrentlyunknown,butoxidativestressisthoughttobeoneofthecauses.MitochondrialdysfunctionandROSgenerationisalsothoughttobeinvolved.Haleagrahara and Ponnusamy (61) evaluatedthe effect of aqueous extract of C. asiatica on1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)-inducedParkinsonisminagedSprague-Dawley rats. MPTP binds in mitochondrialcomplex 1 and causes dopamine depletionin the striatum. The rats received the extractorally for 21 days, and were then sacrificedand analysed for lipid hydroperoxides, proteincarbonyl content, xanthine oxidase, superoxidedismutase, glutathione peroxidase, catalaseand total antioxidants. In all the parameters,C. asiatica when given together with the MPTP-inducedoxidativestress,reversedtheneurotoxiceffect of MPTP towards values in the normalcondition. Focusing more on the bioactiveconstituentofC. asiatica,Xuetal.(62)evaluatedtheeffectofasiaticosideonanMPTP-inducedratmodel of Parkinson’s disease. They performedtwo behavioural tests: open-field testing andladder walking. Then, they sacrificed the ratsto measure dopamine (DA) and its metabolitemalonyldialdehyde (MDA), reduced glutathione(GSH) and gene expression. They found thattreatment with asiaticoside for 7 and 14 daysresulted in attenuation of the effects of MPTPon thebehavioural tests, reducedMDAwhich isamarkerofoxidative stress, andmodulated thereductionofDAwith increasedconcentrationofGSH.Xu et al. (63) repeated the same protocolwith madecassoside and found similar results.In addition, they evaluated expression of BDNF(brain-derived neurotrophic factor), which isusually reduced in Parkinson’s disease. Indeed,madecassosideupregulatesBDNFexpression. The role ofC. asiatica hasnot beenwidelyresearchedforitsapplicationinmentalstressorillness.However, it has been used in Ayurvedic


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medicine as a rasayana, a health tonic. In thisregard,C. asiatica is hypothesised to act as anadaptogen to the daily stresses impacting themind (64). Segen’s Medical Dictionary definesadaptogens as a family of natural substancesthatcompensateforfluctuationsinhomeostasis.Jana et al. (64) studied 33 individuals from theage of 18 to 60 with diagnosis of generalisedanxietydisorder(GAD);theywerewithheldfromantidepressantmedicationandgivenC. asiaticain500mgcapsulesfor60days.Scalesofstress,anxiety, depression, adjustment and attentionwereevaluated.Allthescalescoresimprovedafter60 days, with no side effects of vertigo, nauseaand dizziness as experienced with the commonmedications. Kalshetty et al. (65) evaluatedthe antidepressant effect of C. asiatica extractstandardised to asiaticoside (INDCA) on ananimalmodelofchronicbehaviouraldepression.Anolfactorybulbectomywasdoneontheratstoinduce the chronic depression. Treatment withINDCA showed dose-dependent reversal of thebody weight, body temperature and heart beatreduction seen in behavioural depression, withnormal activity in open-field and elevated plusmazetests.ItwasproposedthatINDCAexertsanantidepressanteffectintheserats. TheeffectofethanolicextractofC. asiaticaon an acute depression ratmodel (forced swimtestmodel)was investigatedbySelvi et al. (21).They found thatC. asiatica has a similar effectto the commercial antidepressants imipramineand diazepam, but at a lower level. Theycaused reduced immobility time and increasedexploratorybehaviour.Evaluatingparametersofbehaviour, Wanasuntronwong et al. (38) testedstandardised C. asiatica extract, ECa 233, onchronicallyimmobilisedmice,amodelofanxiety.Theytestedthemiceinelevatedplusmaze,light-dark box and open-field tests and measuredtheir body weight and serum corticosterone.Diazepam, a benzodiazepine used to treatanxiety, acts by binding to theGABAA receptorand inhibits its activity while downregulatingcorticotrophinreleasinghormone(CRH),leadingto a reduced corticosterone level in the blood.Based on the behavioural test, ECa 233 showedan anxiolytic effect, and blood analysis showeda reduced corticosterone level. Therefore, thereis a possibility that the anxiolytic effect of ECa233worksbythesamemechanismasdiazepam.SaravananandSarumati(66)furtherinvestigatedthe effect of aqueous extract of C. asiatica onimmobilisation-induced stress in rats. Theymeasured erythrocyte and leucocyte counts andAchEactivity.Theseparameterswerereducedon

stress,butwithco-administrationofC. asiatica,thevaluesrevertedbacktonormal,suggestingitsanti-stressproperty. As for epilepsy, Visweswari et al. (67)evaluated the effect of pre-treatment for oneweek with n-hexane, chloroform, ethylacetate,n-butanol and water extracts of C. asiaticaon pentylenetetrazol (PTZ)-induced seizures.During the seizures, there is a high content ofacetylcholine and reduced AchE. However, intheratspre-treatedwithn-hexane,ethylacetateand n-butanol extract ofC. asiatica, therewerereduced Ach and increased AchE levels. This isconsidered as anti-seizure activity. In anotherpaper, Visweswari et al. (68) again evaluateddifferent extractions of C. asiatica on Na+/K+,Mg2+ and Ca2+ ATPases in rat brains duringPTZ-induced epilepsy, because these enzymesplay a crucial role in maintaining membranepotentialtopreventneuronhyperexcitabilityandareinhibitedduringseizures.Pre-treatmentwithC. asiaticawasfoundtoincreaseATPaseactivitylevelsthroughoutthebrain.

In Vivo Studies of C. asiatica on Nerve or Brain Tissue Regeneration

In a study by Soumyanath et al. (35),ethanolicextractofC. asiaticawassupplementedin the drinking water of Sprague-Dawley ratsat a concentration of 2mg/mL. The studydemonstrated that C. asiatica promotes morerapidfunctionalrecovery(toe-spreadandwalkingonset)andincreasedaxonalregeneration(largeraxondiameterandgreaternumberofmyelinatedaxons) in ratswith sciatic bilateral nerve crush.StudiesontheeffectofC. asiaticaascrudeextractoritscomponentsonperipherynerverepairandregenerationareverylimitedasnoinvivostudieson periphery nerve regeneration besides thisstudyhavebeenconductedtodate. A study on amygdaloid neurons andhippocampalCA3neuronsofratpups(7daysold)fedwith2,4and6mL/kgbodyweightoffreshofC. asiaticaleafjuicefor2,4and6weeksshoweda significant increase in dendritic length anddendriticbranchingpoints.Theeffectsweredose-and duration-dependent as only the neurons ofratstreatedwith4and6mLC. asiatica/kgbodyweight/day for longer periods of time (4 and 6weeks) showed significant increases (36,69).Enhancementofneuronaldendriticarborisationby fresh C. asiatica leaf juice supplementationinneonatalratshasbeenfoundtocorrelatewithimprovedlearningandmemory(70).

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Effects of C. asiatica on Neurotoxicity and Brain Injury

A study in ratswithmiddle cerebral arteryocclusion (MCAO) showed that C. asiaticaimproves neurobehavioral activity and reducestissuedeathduetolackofoxygen.Theratswereorally fedwithC. asiaticaextract(100,200and300mg/kgbodyweight)for21days.Subsequently,theratsunderwentMCAOfor2hfollowedby22hreperfusion.Neurobehavioralanalysisbyflexiontest, spontaneousmotoractivityevaluation,gripstrength and muscular coordination showedthattheratsadministratedwithC. asiatica(200and 300mg/kg body weight) were significantlyless affected byMCAO compared to the controlgroup. The infarction volume upon MCAO wasalso shown tobe significantlydecreased inboththe200and300mgC. asiatica/kgbodyweightgroups. Furthermore, rats in these groups alsohad significant protection fromneuronal injury,evidenced by more intact hippocampal andcorticalneurons(71).

Learning and Memory Enhancement by C. asiatica

Memoryenhancement effectsofC. asiaticahave been documented in Ayurvedic medicinesince ancient times (72). Few randomised,placebo-controlledand/orblindedclinicalstudieshaveproventheeffectofC. asiaticainimprovingcognitive function and enhancingmemory (73–76).ThestudiesfoundthatC. asiaticaintakeforaperiodrangingfrom2to6monthsreducedage-related decline in cognitive function in healthymiddle-aged adults and the elderly with mildcognitiveimpairment(MCI).Furthermore,otherMCI-related conditions such as hypertension,insomnia, lossofappetiteandconstipationwerealso found to be improved followingC. asiaticaintake (76). Recent animal studies have alsoconfirmed that C. asiatica supplementationimproves memory performance in rats withmemory dysfunction due to oxidative stress(77,78).However, a studybyJared (79) showedthat C. asiatica uptake by rats only enhancedretention of memory but did not improve thelearningprocess. Recent preclinical studies have startedto explore the compounds of C. asiatica thatare responsible for cognitive and memoryenhancement function. AA, a pentacyclictriterpeneinC. asiatica,wasfoundtosignificantly

reduceglutamate-inducedcognitivedeficits.TheadministrationoforalAA(100mg/kg)wasfoundto restore levels of lipid peroxidation andGSH,and the activity of superoxide dismutase in thehippocampusandcortextocontrollevels(55).ItwasalsofoundthatAAprotectsagainstglutamate-induced dementia and enhanced recovery fromlearning and memory deficit due to glutamateuptakeinamousemodel(48).AnotherstudybyNasiretal.(80)alsofoundthatsupplementationofAAinnormalratssignificantlyimprovedmemoryandlearninginadose-dependentmanner.

Future Direction

As can be observed from the studiesmentioned above,C. asiatica does have provenneuroprotectiveandneuroregenerativepropertiesinanimalmodelsandalsoinhumans.However,more studies are still needed to identify thecompounds of C. asiatica that are particularlyinvolvedinneuroprotectiveandneuroregenerativeproperties and also themechanism of action ofthese compounds. Besides that, the extractionmethod,biochemicalprofile,dosageinformationandinfluenceofgeographicalandotherfactorsindetermining thebiochemicalprofileof theplantneedtobeascertainedandstandardisedbymorescientificstudies.Furthermore,theeffectsoftheC. asiaticaextractoninvitromesenchymalstemcelldifferentiationintoneurallineagecellsandonthe invivomodelofnerve injury still remain tobeexplored.Thus,furtherstudiesonthoseareasofC. asiatica need to be undertaken to greatlyenhance the economic value of this traditionalherb and to accelerate the usage of this herb inclinicalapplications.

Acknowledgement

This work was supported by NRGS grant(NH1014D048)fromtheMinistryofAgricultureMalaysia.

Conflicts of Interest

The authors declare they have no conflict ofinterest.

Funds

This work was supported by NRGS grant(NH1014D048)fromtheMinistryofAgricultureMalaysia.


www.mjms.usm.my 11

Authors’ Contributions

Conceptionanddesign:YL,RHIAnalysisandinterpretationofthedata,collectionandassemblyofdata:YL,NO,NNAP,ASDraftingofthearticle:YL,NO,NNAP,AS,RHICritical revision of the article for importantintellectualcontent:YL,AS,RHIFinalapprovalofthearticle:YL,NO,NNAP,AS,RHIObtaining of funding and administrative,technical,orlogisticsupport:RHI

Correspondence

ProfessorDrRuszymahHajiIdrusMD(UKM),PhD(UKM)TissueEngineeringCentreUniversitiKebangsaanMalaysiaMedicalCentreJalanYaacobLatif,BandarTunRazak56000CherasKualaLumpur,MalaysiaTel:03-91457704Fax:03-91457678Email:[email protected]

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