Rev. bras. farmacogn. vol.27 número6

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w ww.e l s e v i e r . c o m / l o c a t e / b j p

Original

Article

Phytochemical

composition

and

chronic

hypoglycemic

effect

of

Rhizophora

mangle

cortex

on

STZ-NA-induced

diabetic

rats

Adolfo

Andrade-Cetto

a,∗

_,

_Sonia

_M.

_{Escandón-Rivera}

a

_,

_Gerado

_Mata

_Torres-Valle

a

_,

_Leovigildo

_Quijano

b

a_Laboratorio_de_{Etnofarmacología,}_Facultad_de_Ciencias,_Universidad_Nacional_Autónoma_de_México,_México,_DF,_Mexico

b_Instituto_de_Química,_Universidad_Nacional_Autónoma_de_México,_México,_DF,_Mexico

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received5May2017 Accepted25September2017 Availableonline2November2017

Keywords:

Type2diabetes Redmangrove Hypoglycemic Hypolipidemic

a

b

s

t

r

a

c

t

Type2diabetesisamajorhealthprobleminMexico,asitisinothercountries,isachroniccondition thatdevelopswhenthebodycannotproduceenoughinsulinorcannotuseitappropriately.Bothinsulin deficiencyandinsulinresistanceleadtohighbloodglucoselevels.InMexico,peoplewithdiabetesare knowntousethedecoctionofredmangrove(RhizophoramangleL.,Rhizophoraceae)barktocontrolblood glucoselevels.Therefore,inthisstudy,wesoughttoinvestigatethechronichypoglycemicand hypolipi-demiceffectsofR.mangle;wealsoelucidatesomeofthemajorphytochemicalcompoundsofR.mangle. Toanalyzethehypoglycemicandhypolipidemiceffects,weusedratswith streptozotocin–nicotinamide-inducedhyperglycemia;theratswereclassifiedintofourgroups(sixratseach),basedonthetreatment given,asfollows:group1,non-hyperglycemiccontrol;group2,hyperglycemiccontrol;group3, gliben-clamide(5mg/kgbodyweight);andgroup4,Rhizophoraethanol–waterextract(90mg/kg).Theextract orglibenclamidewasorallyadministered,dissolvedin1.5mlofphysiologicalNaCl-solution,twiceaday (inthemorningandintheevening)overaperiodof42days.Themethanolicextractwasusedtoelucidate themaincompoundspresentinR.mangleviaconventionalphytochemicalmethods,suchasTLC,HLPC, UPLC–DAD–MS,andNMR.Thefollowingcompoundsweredetected:cinchonainsIaandIb, catechin-3-O-rhamnopyranoside,epicatechin,lyoniside,andnudiposide.ThedailyadministrationofRhizophora

ethanol–waterextract,similartothetraditionalusagetocontroltype2diabetes,wasshowntoexert chronichypoglycemicandhypolipidemiceffects.Thiseffectmaybeassociatedwhittheconstituentsin theextract.ThesefindingssuggestthatR.mangleanditsconstituentscouldbepotentiallyusedtotreat type2diabetes.

Introduction

Diabetesisachronicconditionthatoccurswhenthebody can-notproduceenoughinsulinorcannotuseitappropriately.Intype2 diabetes(T2D),thebodycanproduceinsulinbutbecomesresistant toit,causingineffectivenessofinsulin.Consequently,insulin lev-elsmaybecomeinsufficient,andthusinsulinresistanceandinsulin deficiencyresultinhighbloodglucoselevels(IDF,2015)and(ADA, 2015).IndividualswithT2Dsufferfrominsulinresistanceand usu-allyrelative,ratherthanabsolute,insulindeficiency.However,at leastinitially,andoftenthroughouttheirlifetime,theseindividuals maynotrequireinsulintreatmenttosurvive.

∗ Correspondingauthor.

E-mail:aac@ciencias.unam.mx(A.Andrade-Cetto).

In2015,theIDFestimatedthat415millionpeopleareliving withdiabetesworldwide;Mexicostandssixthamongthetopten countries,with11.5millionpeople(IDF,2015).Diabetes-associated complications,suchascardiovascular disease,blindness,kidney failure,andlower-limbamputation,areamajorcauseofdisability, lowqualityoflife,andprematuredeath.

AmongtheWorldHealthOrganizationlistofessentialdrugs usedforthetreatmentofdiabetes,metformin(abiguanide)and gli-clazide(asulfonylurea)arewell-establishedmedicationsandthey shouldbeavailableandeasilyaccessible(accordingtoneed),toall patientswithT2D(IDF,2015).Itisofsignificancethatmetformin wasoriginally isolated from theFrench lilac(Galega officinalis)

(Witters,2001).

PlantshavebeenusedformedicinalpurposesinMexicosince pre-Hispanictimes.ThehighprevalenceofT2DamongMexicans, associatedtovulnerableeconomicstability,andthefactthatpeople trusttheeffectivenessofmedicinalplantshaveledtotheincreased useofplantstotreatT2D.Thesefactorshavemadeitessentialto

https://doi.org/10.1016/j.bjp.2017.09.007

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studythepharmacologicalandphytochemicalpropertiesofplants withhypoglycemicpropertiesinMexico.

RhizophoramangleL.,Rhizophoraceae(Andrade-Cettoand

Hein-rich,2005),traditionallyknownas“mangrove”or“redmangrove,”

iswidelyusedforthetreatmentofdiabetesinMexico.Itisa 25-m-talltreethatgrowsinmangrovesanddistributedalongthePacific andGulfCoastsofMexico.Ithasatall,straighttrunkwithabundant roots,aroundtreetopwithsympodialbranching,bitter-redwood, andcortex(PenningtonandSarukhán,1998).

The anti-hyperglycemic effect of the plant was previously reportedbyAlarcon-Aguilaraetal.(1998),theytesttheeffectof 28 plants in rabbits under a glucose tolerancetest, the results obtainedfromthevarianceanalysisshowedthatR.mangle signifi-cantlydecreasedthehyperglycemicpeakby16.1%.

Inapreviousstudyperformedbyourgroup,the ethnobotan-ical relevance and the acute hypoglycemic effect of R. mangle

werereported(Andrade-CettoandMares,2012),inthatstudywe confirmthat thedoseof 90mg/kg hasthebetterhypoglycemic effect, this dose is thetraditional used dose multiplied by 10. In the present study, we aimed to examine thechronic hypo-glycemiceffectoftheethanolicextractof thebarkofR.mangle

in streptozotocin–nicotinamide (STZ-NA)-induced diabetic rats; we also evaluated the lipid profile and glycated hemoglobin after chronicadministration. In addition, we sought to charac-terizethemajorphytochemicalcompoundspresentintheplant cortex.

Materialsandmethods

Plantextracts

Basedontheresultsofthepreviousstudyinwhichthewater and ethanol–water extracts (EW) were tested (Andrade-Cetto

andMares,2012),weselectedtheethanol–waterextractwhich

is similartothe traditional usedinfusion and presented better activity(Fig.1).New botanicalsamplesofRhizophoramangleL., Rhizophoraceae, werecollected withthe help of informants in Manialtepec,OaxacaMexico,theoriginalplantwasdepositedat theIMSS, Herbarium in MexicoCity withthe voucher number IMMSM15816.Theextracttobeusedinpharmacologicaltestswas preparedaspreviouslydescribed;inbrief;a 50gsampleofthe plantmaterialwasaddedto500mlofanethanol–watermixture (1:1),itwasthenheatedat40◦_C_for₄_h_before_being_filtered_for threetimes.Thiswasfollowedbyeliminationofthesolventunder reducedpressure ina Büchi rotaryevaporator.The yieldofthe extractethanol–water(1:1)was14.75g.

Forthephytochemicalidentificationofthemaincompoundsof thecortex;themethanolicextract(ME)waspreparedusing200g ofplantmaterialthroughSoxhletextraction.Defattingwith hex-ane(24h)followed bymethanol (MeOH)extraction(48h), and theresultingextractevaporatedunderreducedpressureuntilit reacheddrynessproducing15gofME.

Compoundsisolation

HPLC–DAD–MSanalysis was performed to confirm that the ethanol–water extract used in thepharmacological testing has a similarphytochemical profilethanthe water and methanolic extracts(Fig.1),butthelastonewasmoreaccessibleforthe isola-tionprocess.

AsampleofME(3g)wasdissolvedinMeOHandpartitioned withhexane toyield a hexane soluble fraction(HSF; 50mg), a MeOH-solublefraction(MSF, 2.90g), and a red precipitate(RP; 28mg).

The MSF was subjected tocolumn chromatography (CC) on 360gofsilicagel(70–230mesh,Merck)startingwithhexane100% (400ml),increasingthepolaritywithEtOAcusingamixtureof hex-ane/EtOAcaseluent,until100%(500ml),andsubsequentlywith MeOHuntil100%(500ml).Thisprocessledtofourteenprimary fractions(MSF1–MSF14).FractionMSF8(400mg)wassubjected tosilicagelCCelutedwithEtOAc/MeOH(10:0–0:10),thisprocess ledtofivesubfractions(MSF8.1–MSF8.5).Preparativethinlayer chromatography (TLC)(Macherey&Nagel,0.25mm)of fraction MSF8.2(20mg)usingEtOAc/MeOH/H2O,7:2:1,aseluentresulted intheisolationofamixtureof1and2(10mg).PreparativeTLC (EtOAc/MeOH/H2O,7:2:1)offractionMSF8.3(80mg)resultedin theisolationof3(23.7mg)and4(13mg).FSM8.4wasresolvedby HPLC(Nucleosil250×10mmi.d.,5␮m,C18,Macherey&Nagel); usingagradientofMeCN/H2Ostartingwith20/80to70/30during 17min(3ml/min;250and280nmUV-det.)toobtain10mgofa mixtureof5and6withanRt10.5min.

AnefficientmethodbasedonHPLC–DAD–MStechnique was usedforidentifyingtheisolatedcompoundsfromthe methano-licextractcorrespondingto(1–6)inthewaterandethanol–water extract.ThecomponentswereseparatedonaKinetexHPLC/UPLC XB-C18 column (50×2.1mm i.d., 2.6␮m) at 25◦_C. _The _mobile phaseconsistedofawatergradient(containing0.1%FA)(A)and acetonitrile(B).Thefollowinggradientelutionprogramwasused: 1%Bduring0.5min,1–35%B0.5–15min,35–100%B15–18min, 100–1%B18–20minataflowrateof0.2mlmin−1_,_the_injection volumenwas3␮l.Majoritycompoundsofthetradicional decoc-tionandtheethanol–waterextractwereidentifiedandareshown inFig.1.

Generalexperimentalprocedures

NMR spectraincludingHSQC,HMBC,COSY,andTOCSYwere recordedinaVarianInovaspectrometerat500(1_H)_and₁₂₅_MHz (13_C) _or _a _JEOL-ECA _at ₃₀₀ ₍1_H) _and ₇₅_MHz ₍13_C); chemi-cal shifts were recorded as ı values. HRESIMS were recorded on a Thermo Scientific LTQ Orbitrap XL hybrid FTMS (Fourier transform mass spectrometer). Data were collected in both positive and negative ionization modes via a liquid chromato-graphic/autosampler system that consisted of an Acquity UPLC system.ProfileHPLC–DAD–MSwereperformedusinganAgilent 1200InfinitysystemequippedwithaG1312-95006Binarypump, G1329-90012 Autosampler, controlled by Agilent ChemStation software,coupledtoaWatersdiodearraydetector(DAD)anda Squire6000BrukerESI-MSinnegativemodeionpolarity.

Analyticaland preparativeHPLCanalyseswereperformedin anAgilent1260InfinitysystemequippedwithaG1311B Quater-narypump,G1367EAutosampler,G1315CDADVL+andcontrolled byAgilentChemStationsoftware.Foranalyticaland semiprepara-tiveHPLC,Macherey-Nagel(NucleosilC18,250×4.6mmi.d.,5␮m), Macherey-Nagel(NucleosilC18,250×10mmi.d.,5␮m)columns, respectively,wereused.Columnchromatography(CC)wascarried outonsilicagel(70–230mesh,Merck).Thin-layer chromatogra-phyanalysiswascarriedoutonsilicagel60F254plates(Macherey &Nagel)usingcericsulfate(10%)solutioninH2SO4ascolorreagent.

Experimentalanimals

Eight-week-oldWistarratsweighing200–250gwereobtained fromtheBioteriumoftheScienceSchool,UNAM,andwere accli-matedwithfreeaccesstofoodandwaterforatleastoneweek inanair-conditionedroom(25◦_C_with_55%_humidity)_on_a₁₂_h light–dark cycle prior to performing theexperiments. The ani-malswerehandledaccordingtotheNationalInstituteofHealth,

USA(CommitteefortheUpdateoftheGuidefortheCareandUse

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A

B

C

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2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

AU

Minutes

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

Minutes

Fig.1.HPLC–DADprofilecomparisonofthemethanolic,ethanol–waterandwaterextracts.(A)Methanolicextract,(B)waterextract,(C)ethanol–waterextract.

as described by Masiello et al. (1998). In brief, the rats were fastedovernight and injected intraperitoneallywith 150mg/kg nicotinamide(NA)(Sigma,N3376)15minbeforeanintravenous injectionof65mg/kgstreptozotocin(STZ)incitratebuffer(Sigma, S0130).Diabetes was identified by polydipsia, polyuria and by measuringnon-fastingplasmaglucoselevels48hafterinjection. Animalswhichdidnotdevelopmorethan250mg/dlglucoselevels wererejected.

The hyperglycemic animals wereclassified into four groups (1–4) each of them with six rats. Group 1 as normal control received1.5ml ofphysiologicalNaCl-solution(vehicle),group2

as hyperglycemic control received also 1.5ml of physiological NaCl-solution,group3wasgivena standard oralhypoglycemic agent, glibenclamide (5mg/kg bodyweight (bw)), in the same vehicle, while group 4 received Rm-EW (90mg/kg bw) dis-solved in 1.5ml of physiological NaCl-solution. The extract or the hypoglycemic agent was orally administered twice a day (in the morning and in the evening) over a period of 42 days. All groups were fed Purina Rodent Laboratory Chow 5001.

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fortheUpdateoftheGuidefortheCareandUseofLaboratory

Ani-mals(2015).Allmethodsusedinthisstudywereapprovedbythe

InternalCouncilofthe“FacultaddeCiencias”oftheUniversidad NacionalAutónomadeMéxico.Glucosemonitoringwasperformed weeklyand analyzedwithglucose teststripsand a glucometer Accutrend® plus.Glycatedhemoglobin(HbA1c)wasanalyzedin aDCAVantage®Siemens,equipment.Thelipidprofile(HDLTGand cholesterol)weremeasuredwithCardioCheck®andstripsPanels® PTS.VLDLwascalculatedusingthefollowingVLDL=0.2×TG.Both HbA1candlipidprofilesweremeasuredondays0,14,28and42 aftertheinitiationofadministrationoftreatments.

Statisticalmethods

Thedatawerestatisticallyanalyzedbyunpairedt-testwiththe helpofthesoftwareGraphPadPrism.Theplasmaglucoselevels wereexpressedasthemean(S.E.M.).

Resultsanddiscussion

Ethnobotany

TraditionaluseofR.mangle cortexforthetreatmentoftype 2diabeteswasconfirmedbythetraditionalhealerswhoassisted duringtheplantcollectioninManialtepec,Oaxaca,Mexico.Forthis purpose,aroughapproximationof20gofplantcortexareboiled in500mlofwaterfor15min,oncethedecoctioniscold,itis con-sumedthroughoutthedayasthesocalled“aguadeuso”.

Compoundidentification

CinchonainsIaandIb(1and2)

Compounds 1 and 2 were identified by their 1_H _and 13_C NMRspectraldata,included2Dexperiments(COSY,HSQC,HMBC, NOESY),andmassspectraldata,whichallowedtheidentification ofcompounds1and2asamixture.

Themassspectrum(ESI-MSnegativeionmode),showedonly onepseudomolecularionatm/z451.66[M−H]−_indicating_a molec-ularformula C24H20O9 for both compounds 1and 2, whilethe 1_H_and13_C_NMR_spectra_showed_duplicated_signals._The1_H_NMR

spectrum (CD3OD, 300MHz) showed pairs of signals at ıH(1/2)

4.81/4.87(brs,H-2),ıH(1/2)4.25/4.19(m,H-3)andıH(1/2)2.90/2.86

(m,H-4)suggestingthepresenceofa flavan-3-olmoietyinthe molecule.ThetypicalABXspinsystemduetothearomatic pro-tonsoftheB-ring,werealsopresentaspairsofsignals,atıH(1/2)

6.97/6.84(d,J=1.7/1.9Hz,H-2′_),_ı

H(1/2)6.75/6.69(d,J=8.2/8.1Hz,

H-5′₎_and_ı

H(1/2)6.80(dd,J(1)=1.7,7.9)/6.62(dd,J(2)=2.1,8.1Hz) (H-6′_). _Two _singlet _signals _at _ı_H(₁_/₂₎ _6.20/6.21 _suggested _a tri-substituted A-ring. The presence of a phenylpropanoid moiety relatedwithcaffeicacidwasindicatedbyanextraaromaticABX systemwithsignalsatıH6.64(d,J=7.6Hz, 1H,H-5′′),6.44(dd, J=8.0,2.2Hz,1H,H-6′′_),_6.55_(d,_J₌_2.2_Hz,_1H,_H-2′′_),_in_addition toan aliphaticABX systemdue to themethine CH-7′′ _and _the methyleneCH2-8′′_,_at_ı_H_4.55_(dd,_J₌_6.8,_1.3_Hz,_1H,_H-7′′_),_and_3.0 (m,2H,H-8′′_).

Theabovedataagreewiththosepublishedforthemixtureof cinchonainsIa(1)andIb(2),isolatedfromTrichiliacatigua, Meli-aceae (Pizzolatti et al.,2002). The13_C _NMR _data_and _2D _NMR experiments(COSY,HSQC,HMBC,NOESY;300MHz,acetone-D6) confirmedtheaboveassignments.HMBCcorrelationofH-8′′_and H-7′′ _with_the_tertiary_carbon_C-8₍_ı_C₁_/₂ _{105.7/105.9),}_indicating thatthephenylpropanoidunit wasattachedtotheC-8position oftheepicatechinmoiety.TherelativeconfigurationattheC-7′′ stereogeniccenterofcompounds1and2wasestablishedbased onthecorrelationsobservedintheNOESYexperiment.Thus,for compound1acorrelationbetweenH-2andH-2′′_/6′′_was_observed;

whilefor compound2NOESY correlationbetweenH-7′′ _and H-2′_/6′_was_observed._The_same_NOESY_interactions_were_previously reportedbyResendeetal.(2011)forcinchonainsIaandIb.

Catechin-3-O-rhamnopyranoside(3)

Compound 3 wasobtained as a yellowish amorphous solid, the molecular formula C21H24O10 was deduced from the ESI-MS[M+H]+_ion _at_m/z_437.314_and _435.73_[M₋_H]−_._The1_H_and 13_C_NMR_spectra_and_2D_NMR_experiments_(COSY,_HSQC,_HMBC,

NOESY,TOCSY;500MHz,CD3OD)indicatingthepresenceof cate-chinandrhamnosemoietiesinthemolecule.The1_H_NMR_spectrum showedtheABXaromaticsystemwithsignalsatıH/C6.84/115.1

(d,J=2.0Hz,1H,H-2′_/C-2′_),_6.76/116.1_(d,_J₌_8.0_Hz,_1H,_H-5′_/C-5′_), 6.71/119.8(dd,J=8.0,1.8Hz,1H,H-6′_/C-6′₎_due_to_the_B-ring pro-tons.AnAXsystemassociatedwithtwo-metacoupledaromatic protonswithsignalsatıH/C 5.94/96.4(d,J=2.5Hz,1H,H-6/C-6)

and5.86/95.5 (d,J=2.5Hz,1H, H-8/C-8)andanAMX2 spin sys-temwithresonancesatıH/C4.62/81.1(d,J=8.0Hz,1H,H-2/C-2),

3.93/75.9(m,1H, H-3/C-3),andat2.64/27.9 (dd,J=16.5, 8.5Hz, 1H,H-4␣/C-4),and2.88/27.9(dd,J=16.5,5.5Hz,1H,H-4␤/C-4), indicatedthepresenceofaflavan3-olskeleton.TheJ2,3coupling of8.0HztogetherwiththechemicalshiftofC-2atıH/C4.62/81.1

indicatea 2,3-transstereochemistry ofthecatechin. The HMBC spectrumshows correlationof H-3(ıH3.93)withtheanomeric carbon signalatıC 102.2(C-1′′)indicating a3-O-linkageof the

rhamnosetotheflavan.Alltheabovedataagreedwiththosefor catechin-3-O-rhamnopyranoside (Ishimaru et al., 1987). ESI-MS (positiveionmode):[M+H]+_m_/_z_437.314.

Epicatechin(4)

Compound4wasobtainedasaredamorphouspowder. ESI-MSpositiveionmode:[M+H]+_291.152_and_289.58_[M

−H]−_._The

1_H_NMR_revealed_an_ABX-type_aromatic_spin_system_with_proton

signalsatıH/C 7.01/115.3(d,J=2.0Hz,1H, H-2′/C-2′),6.78/115.9

(d,J=8.0Hz, 1H,H-5′_/C-5′_),_6.83/119.4_(dd,_J₌_8.0,_1.8_Hz,_1H, H-6′_/C-6′_),_and_an_AB_meta_coupled_aromatic_system_with_signals_at ıH/C5.94/96.4(d,J=2.5Hz,1H,H-6/C-6)and5.91/95.8(d,J=2.5Hz,

1H,H-8/C-8).ThepresenceofanAMX2 systemwithresonances at lower frequencies ıH/C 4.90/79.8 (d, J=3.5Hz, 1H, H-2/C-2),

4.18/67.5(ddd,J=1.54,3.10,4.64,1H,H-3/C-3),and2.74/29.2(dd,

J=16.8,2.8Hz,1H, H-4␣)and 2.88/29.2(dd, J=16.6, 4.3Hz, 1H, H-4␤),indicatethepresenceofaflavan3-olskeleton.TheJ2,3 cou-plingof3.5HztogetherwiththechemicalshiftofthemethineC-2 atıH/C4.90/79.8,indicatinga2,3-cisstereochemistry.Thus,

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Lyonisideandnudiposide(5and6)

Compounds5and6wereisolatedasabrownamorphoussolid. TheUVspectrumshowedabsorptionsat236,and276nm(MeOH). Themolecularformulafor5and6C27H36O12wasdeducedfromthe

pseudo-molecularionpeaksatm/z575.446[M+Na]+_,_and_551.65 [M−H]−_, _obtained _by _ESI-MS _in _positive _and _negative _modes, respectively.Theidentificationof5and6waspossiblebycareful analysisof1_H_and13_C_NMR_spectra,_included_1D_and_2D exper-iments(COSY, HSQC,HMBC,TOCSY);at 500MHz,using D2Oas solvent.The13_C_NMR_spectrum_of_the_mixture_showed_some_dual signals,whilethe1_H_NMR_spectrum_showed_differences_only_in_the chemicalshiftsoftheanomericprotonH-1′′_,_and_some_alicylic pro-tonsofthexylosemoiety.The1_H_and13_C_NMR_spectra_displayed thetypicalsignalsofalignanskeleton,includinganaromatic pro-tonsingletatıH/C6.81/108.8(s,1H,H-2′/C-2′),andtwoequivalent

aromaticprotonssingletatıH/C6.53/106.9(s,2H,H-2,H-6/C-2,C-6)

for5;andatıH/C6.81/108.7(s,1H,H-2′/C-2′),and6.55/106.9(s,2H,

H-2,H-6/C-2,C-6)for6.The1_H_and13_C_NMR_spectra_also_showed foursingletsignalsduetofivemethoxylgroups,twoofthem sym-metricallyequivalents[ıH/C3.91/57.1(s,3H,OMe-3′),3.81/57.3(s,

6H,OMe-3,OMe-5),3.43/60.7(s,3H,OMe-5′₎_for₅_;_3.91/57.1_(s, 3H,OMe-3′_), _3.82/57.3_(s,_6H, _OMe-3, _OMe-5),_3.42/60.7_(s, _3H, OMe-5′₎_for₆_]._The_NMR_spectra_of₅_and₆_revealed_other_signals, consistentwiththepresenceofaxylosemoiety[ıH/C4.37/104.7(d, J=7.8Hz,H-1′′_,_anomeric_proton),_3.34/73.9_(dd,_J₌_8.5_Hz,_16.5_Hz, 1H,H-2′′_/C-2′′_),_3.47/76.5_(t,_J₌_9.2_Hz,_1H,_H-3′′_/C-3′′_),_3.63/70.2_(m, 1H,H-4′′_/C-4′′_),_3.59/66.0_(dd,_J₌_11.3_Hz,_4.3_Hz,_1H,_H-5_␣′′_/C-5′′_), 3.95/66.0(m,1H,H-5␤′′_/C-5′′₎_for₅_;_and_4.07/103.4_(d,_J₌_7.5_Hz, H-1′′_,_anomeric_proton),_3.28/73.7_(m,_1H,_H-2′′_/C-2′′_),_3.63/70.1_(m, 1H,H-3′′_/C-3′′_),_3.09/76.6_(t,_J₌_11.1_Hz,_1H,_H-4′′_/C-4′′_),_3.59/66.1 (dd,J=11.3Hz,4.3Hz,1H,H-5␣′′_/C-5′′_),_3.95/66.1_(m,_1H,_H-5_␤′′ /C-5′′₎_for₆_]._The_value_of_the_coupling_constant₍_J₌_7.5_Hz)_between theanomericprotonandC-2′′_position,_suggested_the_␤_-orientation oftheglycosidiclinkagein5and6.TheHMBCcorrelationfrom H-1′′₍_ıH_4.37_for₅_;_4.07_for₆₎_to_C-9₍_ı_C_71.6_for₅_and₆₎_indicated thatthexyloseunitwaslinkedtotheoxygenatC-9.Theabovedata

wereinagreementwiththoseforlyonisideandnudiposide(5and

6)(Sadhuetal.,2007).

Themajor metabolitesisolated(3–6)of ME,wereidentified throughtheelaborationofanHPLC–ESI-MSchromatographic pro-fileofWaterextractofR.mangle.

Efficacyindiabeticrats

WeconfirmedthattheStz-Namodelissuitableforachronic experimentinwhichglucosevaluesareevaluated.Theglucose val-uesforthegroup1(normal)remainstablearound125mg/dlover the42daysofexperimentation,whereasthevaluesforthegroup 2(hyperglycemic)werearound170mg/dlinthesameperiod;the group2 presentedstatisticallysignificanthighervalues as com-pared to the group 1 (Table 1). The glycated hemoglobin and triacylglycerides(Table2)arealsohigherinthegroup2in con-trasttothegroup1andtheincreaseinHb1Acandtriacylglycerides levelsissignificantafter14daysoftheinjection.

Thestandardhypoglycemicagentglibenclamidecouldcontrol theglucoselevelsfromday7andtheHb1Acafter28days.The Rm-EWextractcontrolstheglucosevalues fromday7,andthe Hb1Acafter28days,theHb1Acresultsarestatisticallydifferent fromtheirowntime0butnotfromgroup2,theRm-EWextract alsocontrolsthetriacylglyceridesandtheVLDLlevelsafter28days

(Tables1and2).

R.mangletraditionalusagetotreattype2diabetesisthrough drinkingthedecoctionthroughouttheday,thiswayof administra-tionisnoticeableanditisnotassociatedwithmeals,thismeans theplantisnotusedinthepostabsorptivestate.Thisfactcouldbe relatedtotheactionmechanism.

Someof themaincomponentsofR. manglecortexwere iso-latedfromthemethanolsolublefraction(MSF)byrepeatedcolumn chromatographyinsilicagelandpurified byHPLC.Thisprocess allowed the isolation and identification of two flavalignans: 1 and2(cinchonainsIaandIb),twoflavanols:3(catechin-3-O-␣ -rhamnopyranoside)and4(epicatechin),andtwolignanglycosides: 5and6(lyonisideandnudiposide).Thesecompoundshadnotbeen previouslyreportedforR.mangle.Thechromatographicprofilefor

Table1

ChronichypoglycemiceffectofRhizophoramanglecortexonSTZ-NAinduceddiabeticrats.Thevaluesrepresentthemean±SEM.Superscriptedlettersinthesamerow indicatestatisticallysignificantdifferencescomparedwithtime0.Superscriptednumbersinthesamecolumnindicatestatisticallysignificantdifferencescomparedwith therespectivecontrolgroup(a,1)(p<0.05).

Groups Glucose

T0(mg/dl) T7(mg/dl) T14(mg/dl) T21(mg/dl) T28(mg/dl) T35(mg/dl) T42(mg/dl)

1Norm. 123±3 129±1 124±2 127±1 125±4 118±6 131±3

2Hyperg. 175±01 ₁₇₁

±21 ₁₇₁

±101 ₁₅₄

±41 ₁₆₈

±71 ₁₆₂

±41 ₁₆₈

±91 3Glib.5mg/kg 179±2 129±5a1 ₁₄₈

±9a ₁₃₂

±6a1 ₁₅₀

±9a ₁₃₄

±11a1 ₁₅₃

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Table2

ChronichypoglycemiceffectsoftheRhizophoramanglecortexonSTZ-NAinduceddiabeticrats.Thevaluesrepresentthemean±SEM.Superscriptedlettersinthesamerow indicatestatisticallysignificantdifferencescomparedwithtime0.Superscriptednumbersinthesamecolumnindicatestatisticallysignificantdifferencescomparedwith therespectivecontrolgroup(a,1)(p<0.0).

Groups T0 T14 T28 T42

HbA1c(%) Tg (mg/dl)

vLDL (mg/dl)

HbA1c (%)

Tg (mg/dl)

vLDL (mg/dl)

HbA1c(%) Tg(mg/dl) vLDL (mg/dl)

1Norm. 3.6±0.1 71±4.0 14±1 3.6±0.1 79±16 16±3 3.54±0.1 63±4 12±1 3.62±0.1 61±10 12±2 2Hyperg. 3.7±0.1 52±11 ₁₀_±₁1 _4.1_±_0.21 _77.6_±₁₄ ₁₅_±₃ _4.2_±_0.11a ₁₁₉_±₁₅1a ₂₄_±₃1a _4.3_±_0.11a ₁₁₃_±₂₆a ₂₃_±₅a 3Glib.5mg/kg 3.8±0.2 69±11 14±2 4.2±0.2 89±13. 18±3 3.9±0.2 100±14 20±3 3.9±0.21 ₁₁₅

±19 23±4a 4Rm-EW90mg/kg 3.4±0.2 78±10 15±21 _4.1

±0.2a ₇₆

±13 15±3 3.9±0.1 65±41 ₁₃

±11 _4.0

±0.2 72±10 14±2

allextracts(Fig.1)indicatesthat3–6arepartofthemain com-poundsfoundinR.mangle;however,itisstillnecessarytocontinue thechemicalanalysisoftheothersubfractionsforabetter under-standingofthechemicalprofileofthisplant.

Amongplantmetabolites,phenolsarefoundtopossessawide range of biological effects. In recent years, plant polyphenols includingphenolicacids,flavonoids,stilbenesandlignans,based oninvitrostudies,animal modelsand someclinicaltrials,have beenproposedaseffectivesupplementsfordiabetesmanagement andpreventionofitslong-termcomplications(Bahadoranetal.,

2013).

Based on several in vitro, animal models and some human studies,dietaryplantpolyphenolsandpolyphenol-richproducts, modulatecarbohydrateandlipidmetabolismaswellas attenu-atehyperglycemia,dyslipidemiaandinsulinresistance(Bahadoran

etal.,2013).

Sietal.(2011)showsthatepicatechintreatmentcausedchanges

indiabeticmice.Thesechangesareassociatedwithahealthierand longerlifespan,includingimprovedskeletalmusclestressoutput, reducedsystematicinflammationmarkersandserumLDL choles-terol,increasedhepaticantioxidantglutathioneconcentrationand totalsuperoxidedismutaseactivity,decreasedcirculating insulin-likegrowthfactor-1,andimprovedAMP-activatedproteinkinase activityin theliverandskeletalmuscle.Recentstudies(Litterio etal.,2015)showedthatepicatechinpreventedhypertensionina

invivomodeldietwith10%(w/v)fructoseinthedrinkingwater (high fructose, HF) for eight weeks on rats, decreasing super-oxideanion production and elevatingNOSactivity,favoring an increase in NO bioavailability. Epicatechin and epicatechin-rich foodsimprovesinsulinsensitivityinhighfatdiet-fedinamouse modelofobesityandT2Dtriggeredbyhighfatconsumptioninmice

(Cremoninietal.,2016).

Cinchonain Ib, from Eriobotrya japonica (Thunb.) Lindl., Rosaceae,leavesenhancedinsulinsecretionfromINS-1cells(rat insulinomacell), aswellasreduced plasmainsulinlevelinrats after108mg/kgoraladministration,however,itdidnotinduceany changesinbloodlevels(Qa’danetal.,2009).

Indiabeticratstheethanol–waterextractexertsahypoglycemic effectaftersevendaysofadministration,theeffectwassustained untilday42,thedecreaseinglucoselevelswerereflectedinthe Hb1Aclevelsafter28days,despitebeingsignificantnotbefore42 daysafterthebeginningoftheexperiment.Thereasonforthisis; Hb1Acisformedinanon-enzymaticglycationwhenhemoglobin isexposedtoplasmaglucose,whentheaverageamountofplasma glucoseincreases,thefractionofglycatedhemoglobinincreases. Thisservesasamarkerforaveragebloodglucoselevelsoverthe previousthreemonths.

Insulindeficiencyandinsulinresistancecauseinconsequence anincreasein lipolysisconductedby adipocytes.Theactivation ofthis pathwaycontributes todyslipidaemia, conditionpresent in type 2 diabetics (De Fronzo et al., 2015). In the present study,decreaseoftriacylglyceridesandplasmaglucoselevelswas observed,suggestingthecontrolofinsulinresistanceasapossible mechanismofaction.Suchdecreasemaybeduetotheactivation

oftheAKTkinasepathwaywhichisresponsibleforinhibitingthe enzymesinvolvedin lipolysiswherebytheconcentrationof tri-glyceridesintheblooddecreases.Anotherimportantfactoristhat AKTalsoisresponsibleforinhibitingtheactivityofGSK3 increas-ingglycogensynthesiswhichdecreasesthereleaseofglucoseby theliver(Suniletal.,2012),forthis reasonwesuggestthatthe hypoglycemiceffectoftheplantmaybelinkedtotheliverglucose output.

Insummary, thedecoctionofR.mangle exertsachronic(42 daysof treatment)hypoglycemicandhypolipidemiceffect.This effects couldbeassociated withthe compoundspresent in the waterextract:catechin-3-O-rhamnopyranoside,lyoniside, nudipo-sideandespecially byepicatechin.However,furtherstudiesare neededtotacklethemechanismofactionresponsibleofitseffects.

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare thattheproceduresfollowedwereinaccordancewiththe regula-tionsoftherelevantclinicalresearchethicscommitteeandwith thoseoftheCodeofEthicsoftheWorldMedicalAssociation (Dec-larationofHelsinki).

Confidentialityofdata. Theauthorsdeclarethatnopatientdata appearinthisarticle.

Righttoprivacyandinformedconsent.Theauthorsdeclarethat nopatientdataappearinthisarticle.

Authors’contributions

AA-Cidealizedthestudy,writethemanuscriptandgetthe finan-cialsupport;GMT-Vperformthepharmacologicalexperiments; SME-Rperformsthephytochemicalexperiments;LQreviewedthe phytochemicalexperimentaldata.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

This project was partially sponsored by the DGAPA, PAPIIT IN28216andCONACyTCB-0151264.

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