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Original
Article
Activity
of
Corylus
avellana
seed
oil
in
letrozole-induced
polycystic
ovary
syndrome
model
in
rats
Murside
Ayse
Demirel
a,
Mert
Ilhan
b,
Ipek
Suntar
b,
Hikmet
Keles
c,
Esra
Kupeli
Akkol
b,∗aLaboratoryAnimalsBreedingandExperimentalResearchesCenter,FacultyofPharmacy,GaziUniversity,Ankara,Turkey
bDepartmentofPharmacognosy,FacultyofPharmacy,GaziUniversity,Ankara,Turkey
cDepartmentofPathology,FacultyofVeterinaryMedicine,AfyonKocatepeUniversity,Afyonkarahisar,Turkey
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received5May2015 Accepted4September2015 Availableonline10November2015
Keywords:
Betulaceae
Corylusavellana
Phytosterol Letrozole
Polycysticovarysyndrome Antioxidant
a
b
s
t
r
a
c
t
Theaimofthepresentstudywastoassesstheactivityofthehazelnutoilinthetreatmentof
poly-cystic ovarysyndromein rats. Serum follicle-stimulatinghormone, luteinizinghormone, estradiol,
progesterone,testosterone,serumlipidparameters,leptinandglucoselevelswereevaluated.Moreover,
antioxidantactivitywastestedusingsuperoxidedismutase,malondialdehyde,catalase,glutathione
per-oxidaselevels.ThephytosterolcontentoftheoilwasdeterminedbyHPLC.Theplasmahighdensity
lipoprotein-cholesterollevelwasfoundtobesignificantlyhighandleptinandglucoseconcentrations
werefoundtobesignificantlylowinthetreatmentgroup.Accordingtothephytochemicalanalysis,the
maincomponentsoftheoilweredetectedas␣-tocopherol,␥-tocopherol,squalene,-sitosterol,
campes-terolandstigmasterol.Corylusavellanaoilwasfoundtobeeffectiveinthetreatmentofpolycysticovary
syndromeviaregulatinggonadotropins,steroidsandserumlipidparametersandpossessesantioxidant
activity.
©2015SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Allrightsreserved.
Introduction
Polycysticovarysyndrome(PCOS)isacommonendocrine disor-derwhichcausesanovulationinanimalandwomenofreproductive age(Dehghanetal.,2012;Zhaietal.,2012;Arikaweetal.,2013). Itshyperandrogenicmanifestationsincludemenstrualirregularity, acne,hirsutismandoligo-ovulation/anovulation.Metabolic abnor-malitiessuchasdyslipidemia,insulinresistance,therefore,diseases includingdiabetes,obesity,cancerandinfertilityaswellas coro-naryheartdiseasescouldbeseenalongwithPCOS(Maharjanetal., 2010;Zhaietal.,2012;Ghasemzadehetal.,2013).
PCOSischaracterizedbysmallarrestedantralfollicleformation inthedevelopmentprocess.InPCOSestrogenleveldecreases, how-ever,theprogesteronelevelincreasesandLH/FSHratiobecomes threetimesofthenormallevel.Androgensaresynthesizedbythe thecacells(Waltersetal.,2012).
CorylusavellanaL.,Betulaceae,isgrowingwildinEuropa, West-ernAsiaandNorthernAfricaaslargeshrubsorsmalltreesabout 3.5–4.5mhigh.Theleavesaredeciduous,rounded,6–12cmlong, softlyhairyonbothsurfaces,withadouble-serratemargin.The flowersare monoecious, withsingle-sex catkins,themale pale
∗ Correspondingauthor.
E-mail:esrak@gazi.edu.tr(E.KupeliAkkol).
yellowand 5–12cm long.Theediblepartofthehazelnutisthe roughlysphericalseed,whichiscoveredbyadarkbrownperisperm andprotectedbyahard,woodyshell.Theripeningnutisenclosed in agreen fringedtube(Könemann,1999; Continietal.,2011). ThisplantwasreportedtocontainvitaminsincludingvitaminE, B6andB9,unsaturatedfattyacids,plantsterolsandpolyphenols (Jakopic etal., 2011).Oneof themostimportant features ofC. avellanaistohavethehighestratioofunsaturated/saturatedfatty acids. Moreover,C. avellana wasshown toreduce plasma total andLDL cholesterolconcentrations,byitspolyunsaturatedfatty acid,phytosterolandsolubledietaryfibercontent(Weststrateand Meijer, 1998; Brownet al., 1999; Feldman, 2002).A highfiber diet canbebeneficialfor thehealthof heartand digestive sys-temandcanhelptoregulatethebloodglucoselevel(Anderson etal., 2009).C.avellana isa poorsourceofisoflavones,indeed, seedoilcontainsonlytraceamountsofphytoestrogens(Mazur, 2000).
Accordingtothedatareportedinethnobotanicalstudies,C. avel-lanahasbeenusedforthetreatmentofvaricoseveins,hemorrhoids, diabetesmellitusandgynecologicaldisorders(Seziketal.,1997; Ramalhosai etal., 2011;Abeerand Amr,2013).Theaimof the presentstudywastoevaluatetheactivitypotentialofC.avellana oilinthetreatmentofPCOSandanalyzethephytochemical con-stituentsbyusingchromatographicmethodsinordertofindout thecompoundsresponsiblefromtheefficacy.
http://dx.doi.org/10.1016/j.bjp.2015.09.009
Materialsandmethods
Plantmaterial
SeedsofCorylusavellanaL.,Betulaceae,werecollectedfrom Tra-bzon,Akc¸abatvillage,TurkeyinAugust2013.Avoucherspecimen wasdepositedintheHerbariumofFacultyofPharmacy,Gazi Uni-versity,Ankara,Turkey.
Preparationoftheoil
Theplantmaterial(50g) wasair driedand grinded. Ground hazelnut was weighed (10g) and the oil was extracted with hexane in a Soxhlet apparatus during 6h (James, 1995). The extract was evaporated at 40◦C. The oil content of the hazel-nut was determined to be 51.23%. The oil was kept in a dark bottle in a freezer at −25◦C until the time of analy-sis.
Saponificationofsterol,tocopherolandsqualeneforHPLCanalysis
Theoil (30mg)was mixedthoroughly with50% KOH (w/v) (200l) and 1% ethanolic pyrogallol (w/v) (1.5ml). The tubes were keptat 70◦C in a water bath for 40min. Water (0.5ml) andhexane(3ml)wereaddedtothetubesaftercoolingonice. The tubes were shaken and centrifuged at 400×g for 10min. Thehexanephasewasremovedandtheextractionrepeatedwith hexane (1ml). The combined hexane extracts were dried and wasredissolvedinethanol(150l)andtransferredintoanHPLC vial.
Analysesofphytosterols,squaleneandtocopherolsbyHPLC
HPLCconditionsonVarianaProStarHPLCsysteminclude Var-ianProStar240Pump, VarianProStar410AutoSampler,Varian ProStar510ColumnOven,VarianProStar335UV/VIS Detector. Sample(10l)wasinjectedontoaPhenomenexlunaC85u Sil-ica100A(150mm×4.6mmLCColumn)forphytosterolanalysis. The chromatographic separation of the sample was performed usingamobilephaseconsistingof80%acetonitrileand20%water ataflow rateof 1.5ml/min.For theanalysesoftocopherol and squalene,SupelcosilLC-18Column,3m,(4.6mm×150mmi.d.; Supelco,Bellefonte,PA,USA)andmobilephaseconsistingof99% methanoland 1% water wasused ata flow rate of1.3ml/min. PeakareaswererecordedusingGalaxieChromatography Worksta-tionsoftwareversion1.9.3.2(Varian,Inc.MarketingDepartment 2700Mitchell Drive Walnut Creek, CA94598). Chromatograms wereextracted at 205nm, 292nm and 215nm for phytosterol, tocopherol and squalene analysis respectively (Maguire et al., 2004).
Animals
Eleven-week-old female, non-pregnant, eighteen mature Sprague Dawley rats (200–250g) with4–5 days regular estrus cycleswereusedinthestudy.AllratswerepurchasedfromKobay ExperimentalAnimalsLaboratory,Ankara,Turkeyandquarantined foratleasttwoweeks.Theanimalswerehousedinpolysulfone cagesat21–24◦C,40–45%humidity,andlightanddarkcyclesof 12hconditionsatLaboratoryAnimalsBreedingandExperimental ResearchesCenter,FacultyofPharmacy,GaziUniversity,Ankara, Turkey.Theexperimentswereconductedinaccordancewiththe directionsofGuidefortheCareandUseofLaboratoryAnimals. TheexperimentwasapprovedbytheExperimentalAnimalEthics CommitteeofGaziUniversity(G.Ü.E.T-14-022).
Experimentaldesign
Letrozole (Letrasan®, DevaHolding A.S., Tekirdag, Turkey)
(concentrationof1mg/kg)wasdissolvedin0.5% carboxymethyl-cellulose(CMC)wasadministeredtotheratsbygavageoncedaily throughout21days.Thisdosewaschosenaccordingtothe pre-viousstudiesinwhichthecysticfollicleformationwasinduced (Rezvanfaretal.,2012a).Duringtheexperiment,theestruscycle was microscopicallyevaluated by the analyses of relative pro-portionofleukocytes,epithelialandcornifiedcells.Bodyweight changeduetotheadministrationofletrozolewasweeklyobserved. Allratswererandomlydividedintofollowingthreegroups consist-ingofsixratsineachgroup:(i)controlgroup(CMC;2ml/rat/day, p.o.),(ii)referencegroup(buserelinacetate;20mg/rat/week,s.c.) and(iii)treatmentgroup(C.avellana;2ml/rat/day,p.o.).Thetest materialswereadministeredthroughout45days.
Terminationoftheprocedure
Theanimalswereeuthanized24hafterthelastdoseofthe treat-ment.Bloodsampleswerecollectedbycardiacpuncture.Serum wasseparatedandkeptinafreezerat−20◦CfordeterminationFSH, LH,estradiol,progesterone,testosterone,TC,HDL-C,LDL-C,TGs, leptinandglucose,SOD,MDA,catalase,GPxlevels.Uteriandovaries weredissected,andweighedfortheevaluationoftheendocrine function.
Measurementofcirculatinglevelsofserumgonadotropinsand steroids
Serum gonadotropinlevels were determinedusing radioim-munoassay (RIA). All RIA kits were obtained according to the manufacturer’sinstructionfromBeckmanCoulterCo.,Marseille, France.Serum FSHwasassayedby sandwichRIAusing a com-merciallyavailableRIAkit.SerumLH,estradiol,progesterone,and testosterone weremeasuredby competitionradioimmunoassay withcommerciallyavailableRIAkits.
Evaluationofthebloodlipid,leptinandglucoselevels
Plasmaleptin(Cat.EZRL-83K)concentrationwasdeterminedby ELISAusingaratkit(LincoResearch,Inc.,St.Charles,USA).Plasma triacylglyceride,totalcholesterol,HDL-CLDL-Cvalueswere mea-suredwithcommerciallyavailableassaykits(HumanDiagnostica. GmbH,Germany).Bloodglucoselevelwasdeterminedusing Glu-coseRocheDiagnosticglucometerstrip.
Determinationofantioxidantactivity
SOD,GPx,MDA,catalaselevelswereanalyzedforthe determi-nationoftheantioxidantactivitypotential.SODandGPxactivities weremeasuredasdescribedinRezvanfaretal.(2012b).SODassay was evaluated by the rate of increase at 560nm. GPxactivity wasreadbythedetermination ofthedecreaseinabsorbanceat 365nm.MDAlevelandcatalaseactivitywerestudied biochemi-callyinerythrocytelysatesamples.Catalaseactivitywasmeasured andcalculatedaccordingtoAebi’smethodbyrecordingthe hydro-genperoxidedegradationspectrophotometricallyat240nm(Aebi, 1984).MDAwasmeasuredbythemodifiedYagi(1984).
Histology
Fig.1.Wievofthevaginalcytologyincontrol(A)andtreatment(B)groups.
sectionswithalight microscopeand Kameram® imageanalysis
system(Microsystem,Istanbul,Turkey).
Statisticalanalysis
StatisticalanalysiswasperformedusingSPSSversion16.The resultswereexpressedastheMean±S.D.Dunnett’stestwasused todeterminethesignificanceof differencesbetweengroups. In ordertocomparetwogroupsStudent’sttestwasused.Aprobability value<0.05wasconsideredstatisticallysignificant.
Results
Thelevelsoftotal␣-tocopherolandgama-tocopherolwas calcu-latedas301.5g/gand52.8g/g,respectively.173.7g/gsqualene wasdetectedinthenut.Thelevelsofphytosterolsweredetected asfollows;-sitosterol875.4g/g;campesterol75.4g/gand stig-masterol44.9g/g.
Afterletrozoleadministrationtotherats,reproductivecycle wasfoundtobe irregular.Followingtheadministration of test materials,thecontrolgroupdisplayedirregularestrouscyclesand exhibitedconstantestrus(Fig.1A),whereasthetreatmentgroup andthereferencegroupexhibitedregularestrouscycles(Fig.1B).
Twenty one days after the letrozoleadministration, all rats exhibitedsignificantincreaseinthebodyweight(controlgroup: 384.5±12.5g, treatment group: 384.7±13.0g and reference group:386.8±12.7g).Afterthetreatmentwithhazelnutoiland thereferencedrugbuserelin,aremarkabledecreasewasobserved in the body weight which was detected as 304.7±11.0g and 308.2±12.1g,respectively.However,thebodyweightofthe con-trolgroupanimalsincreasedto424.6±10.6g.
cf cf
cf cl
cl
cl
cl
cl
A
B
C
Fig.2.Histopathologicalviewoftheexperimentalgroups.Theimagesarepresented werestainedwithHxE.Theoriginalmagnificationwas1.25×andthescalebars represent1000m.(A)Controlgroup;(B)treatmentgroup;(C)referencegroup. Abbreviationsasshownontheimages;cl:corpusluteum,cf:cysticfollicle.
In comparison withthe control group,hazelnut oiland the reference drug administration caused remission in the ovar-ian weight at the end of the experiment (25.6±1.2mg/100g, 21±1.0mg/100g and 22±1.2mg/100g, (p<0.05) respectively). However, after the administration of hazelnut oiland the ref-erence drugthe uterineweightwas increasedwhen compared to control group (88.2±1.2mg/100g, 92.0±1.4mg/100g and 51±1.1mg/100g,(p<0.05)respectively).
Follicular cysts, seconder follicles and atretic follicles were recordedassevereinthecontrolgroup,moderateinthetreatment group,andmildinthereferencegroup.Lumenoffollicularcysts wasborderedwithsingleormorelayeredgranulosacells. Degener-ationanddesquamationofthesecellsandthickenedthecainterna weresevereinthecontrolgroup.Comparedtotheothergroups regressionofcorpusluteumwasprominentinthereferencegroup, moderateinthetreatmentgroupandmildinthecontrolgroup (Fig.2).
Table1
Serumgonadotropinandsteroidlevelsincontrol,treatmentandreferencegroups.
Groups FSH(ng/ml) LH(ng/ml) Estradiol(pg/ml) Progesterone(ng/ml) Testosteron(ng/dl)
Control 108.3±5.8 8.7±1.6 2.6±0.3 9.85±1.7 267.46±23.5
Treatment 87.6±3.2 2.8±0.2*** 9.06±1.3** 26.8±1.5*** 92.4±7.3***
Reference 75.2±7.5* 3.6±0.4*** 10.8±1.9** 36.4±5.2** 72.64±6.2***
* p<0.05. ** p<0.01.
***p<0.001(treatmentandreferencegroupswerecomparedwiththecontrolgroup).
DatapresentedasStatisticalMean±StandardDeviation(SD).
Table2
EffectsofCorylusavellanaonplasmaconcentrationsofTC,TG,HDL-C,LDL-C,leptinandglucose.
Groups TC(mg/dl) TG(mg/dl) HDL-C(mg/dl) LDL-C(mg/dl) Leptin(g/l) Glucose(mg/dl)
Control 52.01±4.11 114.81±10.24 53.59±5.72 19.61±6.38 5.94±1.79 136.45±10.93 Treatment 41.58±3.18 102.55±11.93 78.36±3.15** 15.50±4.19 3.98±1.04* 107.74±3.41* Reference 31.46±1.09* 84.72±9.08* 85.32±2.10** 12.48±3.07* 2.46±1.12** 98.06±2.22**
* p<0.05.
** p<0.01(treatmentandreferencegroupswerecomparedwiththecontrolgroup).
DatapresentedasStatisticalMean±StandardDeviation(SD).
Table3
SerumlevelsofMDA,SOD,catalaseandGPxlevelsinallgroups.
Groups MDA(mmol/ml) SOD(u/mgprotein) Catalase(mmol/ml) GPx(u/mgprotein)
Control 3.41±0.58 846.12±20.03 7.46±2.32 112.42±9.52 Treatment 2.63±0.21* 994.05±17.22 9.24±1.62* 107.25±5.27
Reference 1.24±0.14** 1032.26±25.47* 8.43±1.89 104.71±6.13
* p<0.05.
** p<0.01(treatmentandreferencegroupswerecomparedwiththecontrolgroup).
DatapresentedasStatisticalMean±StandardDeviation(SD).
tothetreatmentandreferencegroups.Whileestradiol(p<0.01) andprogesterone(p<0.001)levelssignificantlyincreased,LHand testosteron(p<0.001) levels decreased in the treatmentgroup when compared to the control group (Table 1). However this differencewasnotsignificantbetweenthereferencegroupand treatmentgroup.
Serum TC, TG, HDL-C, LDL-C, leptin and glucose lev-els were respectively 41.58±3.18mg/dl, 102.55±11.93mg/dl, 78.36±3.15mg/dl, 15.50±4.19mg/dl, 3.98±1.04g/l, 107.74± 3.41mg/dlinthetreatmentgroup.TheplasmaHDL-C(p<0.01), leptinandglucose(p<0.001)concentrationswerefoundtobe sig-nificantin thetreatment groupwhen compared to thecontrol group(Table2).
Serum MDA, SOD, catalase and GPx levels were deter-mined as 2.63±0.21mmol/ml, 994.05±17.22u/mg protein, 9.24±1.62mmol/mland107.25±5.27u/mgprotein,respectively in thetreatmentgroup. Serum catalase(p<0.05) levels signifi-cantlyincreasedinthetreatmentgroupwhencomparedtothe control group. There was no statistically significant difference between the treatment and reference groups in terms of the antioxidantactivity(p>0.05)(Table3).
Fortheacutetoxicityevaluation,theoilwasadministeredorally totheratsat1,2and4ml/rat/daydoses.Theanimalswereobserved for48handneithertoxicitynormortalitywasrecorded.
Discussion
ThereareseveralhypothesesontheetiologyofPCOS,which isacommonendocrinedisorderinwomenandanimals,however, thereislimitedinformationregardingthemechanismsofits devel-opment(Ball and Peters, 2004;Padmanabhanand Veiga-Lopez, 2013).Treatmentof PCOSishighly importantfor the economi-callyvaluable animalssuchaslivestock(Ball andPeters, 2004). ThetherapeuticapproachestoPCOSaremainlysymptomaticsince
littleinformationisavailableontheetiopathogenesisofthe dis-ease.Somepharmacological agentssuchas oralcontraceptives, antiandrogens,GnRHagonists,steroids,insulinsensitizerssuchas metformin,troglitazonearepreferredbothforhumanandanimal inthetreatmentofPCOS(Dehghanetal.,2012).However,duetothe adverseeffectscausedbysyntheticdrugs,theplantbasedremedies aremuchpreferableinthetreatmentofPCOS.
Previous studies onthe family Betulaceaerevealed that the plantsarerichinthephenolicconstituentsincludingflavonoids, tannins,caffeicacidderivatives,diarylheptanoid-typecompounds (Jin etal.,2007; Martineauet al.,2010).Diarylheptanoidswere found topossess various pharmacological effects such as anti-inflammatory,antioxidant,anticancerandantiadipogenicactivities (Jinetal.,2007;Martineauetal.,2010).Moreover,diarylheptanoids arethemajorcomponents,whichexhibithighestrogenicactivity byASPP049mediatedtranscriptionalactivationthrougha ligand-dependentER␣-estrogen-responsiveelement-drivenpathwayand anon-genomicactiononvascularrelaxationthroughthe ER-Akt-endotheliumnitric oxidesynthase pathway(Suksamrarnet al., 2008;Winuthayanonetal.,2009a,b;Intapadetal.,2009).
Anumberofreportshavesuggestedthatphytosterolspossess estrogenic activity and therefore, have potentialeffects onthe reproductivesystem.Accordingtothepreviouspublishedarticles especially -sitosterolwasfound toact as anestrogenic agent (MaliniandVanithakumari,1992;Mellanenetal.,1996).Although theestrogenicpotentialof-sitosterolwasconfirmedbyseveral invitroandinvivoexperiments,amechanisticstudysuggestedthat
-sitosterolisnotestrogenicitselfbutneedstobemetabolizedto ahormonallyactivemetabolite(Mellanenetal.,1996).
Alterationintheoxidant-antioxidantprofileisknowntooccur in polycystic ovary syndrome (Verit and Erel, 2008). Reactive oxygenspeciescausetheformationoflipidhydroperoxides, con-jugateddienes,andMDA,aswelloxidizebiomoleculesleadingto extensivelipidperoxidation(LPO) in proteins, membranes,and genes(Kucukkurtetal.,2008).TheevaluationofMDAisanindicator offree-radicaldamagethroughmembraneLPO(Kelesetal.,2012). Inthepresentstudy,bloodMDAleveldecreasedinthetreatment groupwhen comparedtocontrolgroupwhichsuggesthazelnut includeactivecomponentsagainstLPO.Antioxidantsinvolve enzy-maticsystemssuchasSOD,glutathioneperoxidase,andCATwhich preventthe oxidative cell injury on tissues (Kyle et al., 1987). SODprotectsthetissuesfromtheharmfuleffectsofsuperoxide radicalsandCATenzymehydrolyzesH2O2 intoH2Oand1/2O2. Theseenzymeshaveanimportantroleintheresistanceofcellular lipids,proteins,andDNA(MatésandSánchez-Jiménez,1999).In thepresentstudy,thelevelsoferythrocyteSODandCATactivities increasedwhereasGPxleveldecreasedwhencomparedtocontrol group.
TheethanolicextractofC.avellanaseeddisplayedremarkable antioxidant activity in total antioxidant and radical scaveng-ing tests, which could be attributed to the relatively high total phenol content of the extract (Shahidi et al., 2007). Myricetin-3-O-rhamnoside,quercetin-3-O-rhamnoside,gallicacid, protocatechuic acid, phloretin-2′-O-glucoside, (+)-catechin, (− )-epicatechinandprocyanidinswerereportedasthemajorphenolic compoundsofC.avellanaseed(WeststrateandMeijer,1998).
Phytosterolssuchassqualene,campesterol,-sitosterol, stig-masterol, tocopherol have been determined as the potential antioxidantagentsinthepreviousstudies(Smith,2000;Kmiecik etal.,2011;deFranc¸aFerreiraetal.,2014).Therefore,itwas sug-gestedthat phytosterolsdeterminedherein,probablypromoted thetreatmentofPCOSbytheirantioxidanteffect.
InsulinplaysanimportantroleinthemodulationofbloodFSH levels(Sudhaetal.,2000).TheantioxidantactivityofC.avellana couldberesponsiblefromtheregulatoreffectofgonadalhormone levelsinratswithhighlevelglucoseduetoPCOSasa reproduc-tiveproblem.Therefore,theimprovementinthebloodglucoseand seruminsulinlevelscouldbeassociatedwiththeimprovementin theserumFSHandLHhormones.
Themaincomponentsoftheoilweredetectedas␣-tocopherol,
␥-tocopherol,squalene,-sitosterol,campesterolandstigmasterol. C.avellanaoilwasfoundtobeeffectiveinthetreatmentofPCOS viaregulatinggonadotropins,steroidsandserumlipidparameters andpossessantioxidantactivity.
Authors’contribution
MADcontributedtothelaboratoryworks,analysisofthedata. MI contributedtolaboratorywork,dataanalysis andcollection ofplantsamples.IScontributedtomanuscriptwriting,laboratory worksandcollectionofplantsamples.HKhelpedin histopatho-logicalstudies.EKAdesignedthestudy,supervisedthelaboratory works,manuscriptwriting,helpedincollectionofplantsamples.
Conflictsofinterest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgement
AuthorswouldliketothankFeratS¸AH˙INfromTurkishMedicines andMedicalDevicesAgency,Ankara,Turkey,forhisvaluablehelp duringHPLCanalysis.
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