RevistaBrasileiradeFarmacognosia25(2015)142–144
w w w. s b f g n o s i a . o r g . b r / r e v i s t a
Original
Article
Study
of
hypocholesterolemic
activity
of
Algerian
Pistacia
lentiscus
leaves
extracts
in
vivo
Mohammed
Cheurfa
∗,
Rachida
Allem
LaboratoryofNaturalBioresources,DepartmentofBiology,FacultyofScience,UniversityofHassibaBenBoualiChlef,Box151,02000Chlef,Algeria
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received15September2014 Accepted1February2015 Availableonline31March2015
Keywords: Aqueousextract Ethanolicextract
Hypocholesterolemicactivity Pistacialentiscusleaves
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s
t
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t
Plantsarealargesourceofnewbioactivemoleculeswiththerapeuticpotentials.However,onlyasmall amountofworldwideplantshavebeenphytochemicallyinvestigated.Theaqueousandethanolicextracts
ofPistacialentiscusL.,Anacardiaceae,leaveswereevaluatedforhypocholesterolemicactivityinvivo.In
thisstudy,hypercholesterolemiawasinducedinanimalsbyfeedingthemhighcholesterol(1%)food.The extractsofP.lentiscuswereorallyadministeredatadoseof200mg/kgbodyweightalongwithahigh cholesteroldietforthirtysuccessivedays.Lipidparameterssuchastotalcholesterol,triacylglyceride, lowdensitylipoprotein,verylowdensitylipoproteinandhighdensitylipoproteinweremeasuredin theplasma.Totalphenolandflavonoidcontentswerealsoevaluated.Flavonoidcontentwasfoundto bemorepresentintheethanolicextract(8.218±0.009mgofQE/g)comparedtotheaqueousextract (3.107±0.014mgofQE/g).TheadministrationofP.lentiscusextractsproducedasignificantdecreasein totalcholesterol,triacylglycerideandlowdensitylipoprotein-cholesterol(154.6±18.10,71.2±4.38and 99.36±18.77mg/dlrespectively)intheethanolicextract,whiletheaqueousextractshowedasignificant decreaseintotalcholesterolandtriacylglyceride(203.6±9.18and97.6±3.57mg/dlrespectively).The resultsoftheinvestigationdemonstratedthatP.lentiscusleafextracthashypocholesterolemicproperties andmightbeusedforthepreventionofhypercholesterolemiaassociateddisorders.
©2015SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Allrightsreserved.
Introduction
Increases in cholesterol levels (hypercholesterolemia) have becomea significanthealth concernin recentyears. Hypercho-lesterolemia is known tobe a risk factor for thedevelopment of cardiovasculardiseases includingatherosclerosis, myocardial infarctionandcerebralparalysis(Avcıetal.,2006).This dysfunc-tionalsoenhancesfreeradicalgenerationinvariousways(Prasad andKalra,1993),aswellastheformationofoxygenfreeradicals, suchassuperoxideanionradicalorperoxynitrite,whichplaya sig-nificantroleinthepathogenesisofmanyotherdiseasesincluding, cancerandinflammatorydisorders(Dasetal.,2000).
Curinghypercholesterolemiawithoutanysideeffectsremains achallengeformodernmedicine.Plant-derivedproductsare fre-quentlyconsideredtobelesstoxic,withfewornosideeffects,than theirsyntheticequivalents.Plantsplayamajorroleinthe introduc-tionofnewtherapeuticagents,andhavereceivedmuchattention as sources of biologically active substances. Mastic (Pistacia lentiscus)isonesuchexample.
∗ Correspondingauthor.
E-mail:[email protected](M.Cheurfa).
P.lentiscusisverycommonintheMediterraneanregion.In Alge-ria,masticisfoundinboththeTellregionandinforestedareas.The aerialportionhastraditionallybeenusedasbothastimulantand adiureticinthetreatmentofhypertension,coughs,sorethroats andstomachaches.P.lentiscusleavescontainbioactivemolecules suchasphenoliccompounds(proanthocyanidintanninsandgallic), flavonoidglycosidesandanthocyanins.
Theaimofthisstudyishencetostudythehypocholesterolemic activityinvivoof aqueousandethanolic extractsofAlgerian P. lentiscusleaves.
Materialsandmethods
Plantmaterial
PistacialentiscusL.,Anacardiaceae,leaveswerecollectedlocally fromtheChlefregionofAlgeriain2012.Voucherspecimenswere collectedundertheregistrationnumberFSDB12.189,atthe herbar-iumofDepartmentofBiology,FacultyofSciences,Universityof HassibaBenBouali-Chlef.TheywereidentifiedbyMs.MedjahedK, abotanistattheInstituteofAgronomy,UniversityofHassibaBen Bouali-Chlef.Afterdryinginashadowatroomtemperature,the plantmaterialswerefragmented.
http://dx.doi.org/10.1016/j.bjp.2015.02.011
M.Cheurfa,R.Allem/RevistaBrasileiradeFarmacognosia25(2015)142–144 143
Preparationofaqueousextract
Theaqueousextractwaspreparedbydecoction.Fragmented leaves(100g)wereimmergedinoneliterofwaterat100◦Cfor 15min.TheaqueousextractwasfilteredthroughWhatmanpaper N◦1.Thefiltratewasconcentratedinarotaryvacuumevaporator at55◦C.Theextractionyieldwas11.88%.
Preparationofethanolicextract
Theethanolicextractwaspreparedbyhomogenizing100gof fragmentedleavesin 900mlof ethanolsolution(50%)for three days,withfrequent agitation.Theethanolicextractwasfiltered throughWhatmanpaperN◦1.Thesolventwasremovedfromthe sampleusingarotaryvacuumevaporatorat48◦C(Peixotoetal.,
2011).Theextractionyieldwas15.88%.Alloftheabovedryextracts werestoredat4◦Cinsterileglassjarsand,sealedforuseinfurther studies.
Phytochemicalsscreeningoftheleavesextracts
Determinationoftotalflavonoidcontent
Thetotalflavonoidcontentintheextractswasdetermined spec-trophotometricallyusinganaluminumchloridemethodinvolving theformationofaflavonoid–aluminumcomplexat420nm(Zhou etal.,2005).Theconcentrationofthetotalflavonoidcontentwas calculatedbycomparison withtheabsorbanceof different con-centrationsof quercetin(QE), and theresult was expressedas milligramsofQEequivalentspergramofplantpowder.Samples werepreparedintriplicateforeachanalysis,andthemeanvalue ofabsorbancewasobtained.
Assayofhypocholesterolemicactivityinvivo
Animals
Twenty-five male Swiss albino mice weighing between 25 and 30g were used in the present study. They were housed in polypropylene cages with controlled levels of temperature (24±2◦C)andlight(a12hlight,12hdarkphotoperiod),andfed withstandardlaboratorypellets.Foodandwater weregivenad libitum.
Theprotocolemployedwasapprovedbythelaboratoryof tox-icologyandpharmacology(Antibioticalgroup–Medea,Algeria), followingtherecommendationsoftheEuropeanPharmacopoeia 8.0underreferencenumber215/2013.
Experimentaldesign
Hypercholesterolemiawasinducedinthemicevia administer-ingof foodcontaining highcholesterol (1%)for thirty days. All micehad freeaccesstofoodand water fortheduration ofthe experiment.Thetestgroupreceived200mg/kgbodyweightplant extractsat10ml/kgeverymorning,administeredorally.Themice weredividedintofivegroupsoffiveanimalseachasfollows:
Group1:Receivednormaldiet(controlgroup).
Group2:Fedwithahighcholesteroldietandreceived0.5mlof sterilewater,administeredorally.
Group3:Fedwithahighcholesteroldietandreceivedaqueous extractofP.lentiscus,200mg/kgbodyweightfor30days, admin-isteredorally.
Group4:Fedwithahighcholesteroldietandreceivedethanolic extractofP.lentiscus,200mg/kgbodyweightfor30days, admin-isteredorally.
Group5:Fedwithahighcholesteroldietandreceivedreference drugAtorvastatin,10mg/kgbodyweight(KheraandBhatia,2012) forthirtydays,administeredorally.
Attheendoftheexperiment,bloodsampleswerecollectedfrom themiceinallgroupsinvialswithoutanti-coagulant.Biochemical parametersmeasuredinthestudywereTC(totalcholesterol),TG (triacylglyceride),LDL(lowdensitylipoprotein),HDL(highdensity lipoprotein)andVLDL(verylowdensitylipoprotein),usingassay kits(BeckmanCoulter,Ref:467825,445850,969706,and650207 –USA.).
Statisticalanalysis
The experimental results were expressed as mean SEM (standarderroroftheman).DatawereassessedbyANOVA.Tukey’s testwasthenappliedusingXLStatPro7.5software.Apvalueof <0.05wasconsideredtobestatisticallysignificant.
Results
Flavonoidcontent
Flavonoid levelsin the aqueousand ethanolicextracts of P. lentiscusleaveswere3.107±0.014and8.218±0.009mgofQE/g respectively.Ebrahimzadehetal.(2008)testedthegumofP. lentis-cus.Theyfoundtheflavonoidlevelstobe30.52±1.10mgofQE/g. Aflavonoidcontentof38.7±0.02mgofQE/gwasfoundbyCherbal etal.(2012)forthemethanolextractofP.lentiscusleaves.
Hypocholesterolemicactivityinvivo
Table1showsthevaluesofserumlipidprofileinthenormal, hypercholesterolemic,controlandextracttreatedgroups.
Cholesterolandtriacylglycerideareimportantbuildingblocks in the structure of biological membranes. They are also used in thebiosynthesisof steroidhormones.However,high choles-terolconcentrationinthebloodincreasestheriskofdeveloping atherosclerosisandrelatedcardiovasculardiseases.
In the present study, mice fed with a high cholesterol diet had higher (p<0.05) levels of total cholesterol in the serum (253±31.60mg/dl) than mice fed with a normal diet (116±0.89mg/dl).
The treatment of group 3 with aqueous extract of P. lentiscus at 200mg/kg body weight, significantly decreased TC (203.6±9.18mg/dl)andTG(97.6±3.57mg/dl)comparedtothe hypercholesterolemiccontrol(group 2).However,there wasno significantdecreaseinLDL-cholesterol.
In the cholesterol-fed mice (group 4), the treatment with 200mg/kgbodyweightofP.lentiscusethanolicextractalso signif-icantlydecreasedTC(154.6±18.10mg/dl),TG(71.2±4.38mg/dl) andLDL-cholesterol(99.36±18.77mg/dl).
TC,TGandLDL-cholesterolalsoshowedasignificantdecrease ascomparedtothehighcholesterol-fedmicereceiving10mg/kg bodyweightdosesofatorvastatin(group5).
P.lentiscus leafextractsshowedanon-significantincreaseof HDL-cholesterollevelincomparisontothehypercholesterolemic control(group2).
Discussion
144 M.Cheurfa,R.Allem/RevistaBrasileiradeFarmacognosia25(2015)142–144
Table1
EffectofaqueousandethanolicextractofP.lentiscusonserumlipidprofileofmicefedwithhighcholesteroldietafter30days.
Groups Dose(mg/kg) TC(mg/dl) TG(mg/dl) HDL-c(mg/dl) LDL-c(mg/dl) VLDL-c(mg/dl)
Controlgroup 116±0.89 146.4±10.89 48.2±6.40 38.52±8.13 29.28±2.17 Hypercholesterolaemicgroup 253±31.60# 200±12.32# 31.6±1.74# 160±31.60# 40±2.46#
AqP.lentiscus 200 203.6±9.18* 97.6±3.57*** 38.2±1.48 145.88±9.76 19.52±0.71**
EtP.lentiscus 200 154.6±18.10*** 71.2±4.38*** 41±1.87 99.36±18.77* 14.24±0.87**
Atorvastatin 10 130.6±8.14*** 121.8
±42.65*** 62.8
±3.49*** 43.44
±9.35*** 24.36
±8.53
Aq:aqueousextract. Et:ethanolicextract.
* p<0.05significantfromhypercholesterolaemiccontrol.
** p<0.01significantfromhypercholesterolaemiccontrol.
***p<0.001significantfromhypercholesterolaemiccontrol.
#p<0.05significantfromcontrolanimals.
(suchasgeographicandclimaticfactors),geneticfactors,degreesof maturationoftheplant,andstoragetimeallhaveastronginfluence onthecontentofphenoliccompounds(Fallehetal.,2008).
Highlevelsofcholesterolinthediethavebeenshownto ele-vatetotalcholesterol,andmayincreasetheriskofcardiovascular complications.
Regarding thecholesterol-lowering effect of P. lentiscus leaf extractsinmicefedwithahigh-cholesteroldiet,theP.lentiscus
extractsshowedhypocholesterolemicproperties.Thismaybedue toeithertheindividualorthesynergisticactionofthephenolic components. A possible mechanism here may bethe hydroly-siscontrolofcertainlipoproteinsandtheirselectiveuptakeand metabolismbydifferenttissues.
Flavonoidsmayenhancethelecithinacyltransferase(LCAT)by increasingtheiractivity,whichregulatesbloodlipids(Yooetal., 2008).LCATplaysakeyroleintheincorporationoffreecholesterol intoHDL(thismayincreaseHDL)anditstransferbacktoVLDLand LDL,whicharelaterreturnedinlivercells(DobiásováandFrohlich, 1999).
Flavonoidsmight representanother beneficial groupof nat-urallyoccurringhypolipidemiccompounds (Cookand Samman, 1996), and they appear tohave intensivebiological properties thatpromotehumanhealthandhelpreducetheriskofdisease. Flavonoidsactasantioxidants,protectLDLcholesterolfrom oxida-tion,inhibitplateletaggregation,andactasanti-inflammatoryand anti-tumoragents(CookandSamman,1996;Manachetal.,1996). Inadditiontodirectoxidantscavenging,flavonoidsmayinhibit theenzymesinvolved ingeneratingpro-oxidant molecules.For example,flavonoidshavebeenshowntoinhibitthegenerationor releaseoffreeradicalsderivedfromlipoxygenase(LOX).Ithasbeen suggestedthatLOXisinvolvedintheearlyeventsof atherosclero-sisbyinducingplasmaLDLoxidationinthesubendothelialspace ofthearterialwall(Huangetal.,1997).
Inconclusion,thepresentstudyclearly demonstratesthatP. lentiscusleafextractisrichinphenoliccompounds.Furthermore, intermsofhypercholesterolemicactivity,P.lentiscusextracts sig-nificantlydecreaseTClevelsinplasma.Futurestudiesareproposed inordertoconfirmtheseresults.
Authors’contributions
MCdidtheanalysis,interpretationandacquisitionofthedata anddraftedthepaper.RAdesignedthestudy,supervisedthe labo-ratoryworkandcontributedtocriticalreadingofthemanuscript.
Boththeauthorshavereadthefinalmanuscriptandapprovedfor submission.
Conflictsofinterest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgements
TheauthorswouldliketothankMr.BenonaM,BoukhelkhalK andMs.NegabI,fromAntibioticalgroup,Medea,Algeria.
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