Rev. bras. farmacogn. vol.25 número2

(1)

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

HPLC-DAD-MS/MS

profiling

of

phenolics

from

Securigera

securidaca

flowers

and

its

anti-hyperglycemic

and

anti-hyperlipidemic

activities

Rana

M. Ibrahim

a

_,

_Ali

_M.

_El-Halawany

a,b,∗

_,

_Dalia

_O.

_Saleh

c

_,

_El

_Moataz

_Bellah

_El

_Naggar

d

_,

Abd

El-Rahman

O. El-Shabrawy

a

_,

_Seham

_S.

_El-Hawary

a

a_{Pharmacognosy}_Department,_Faculty_of_Pharmacy,_Cairo_University,_Kasr-El-Ainy_Street,_Cairo_11562,_Egypt

b_Department_of_Natural_Products,_Faculty_of_Pharmacy,_King_Abdulaziz_University,_Jeddah_21589,_Saudi_Arabia

c_Pharmacology_Department,_National_Research_Centre,_Dokki,₁₂₆₂₂_Cairo,_Egypt

d_Department_of_{Pharmacognosy,}_Faculty_of_Pharmacy,_Damanhour_University,_Damanhour_City,_Egypt

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received22December2014 Accepted21February2015 Availableonline23March2015

Keywords:

Securigerasecuridaca

Flowers HPLC-DAD-MS/MS Antidiabetic Anti-hyperlipidemic

a

b

s

t

r

a

c

t

Securigerasecuridaca(L.)Degen&Döefl.,Fabaceae,hasbeenwidelyusedintheIranian,Indianand Egyp-tianfolkmedicineasantidiabeticandanti-hyperlipidemicremedy.Phenolicprofilingoftheethanolic extract(90%)oftheflowersofS.securidacawasperformedviaHPLC-DAD-MS/MSanalysisinthepositive andnegativeionmodes.Thetotalpolyphenolsandflavonoidsintheflowersweredetermined colorimet-rically,andthequantificationoftheircomponentswascarriedoutusingHPLC-UV.Totalphenolicsand flavonoidsestimatedasgallicacidandrutinequivalentswere82.39±2.79mg/gand48.82±1.95mg/g

ofthedriedpowderedflowers,respectively.HPLC-DAD-MS/MS analysisoftheextractallowedthe identificationof39flavonoidsandeightphenolicacids.Quantitativeanalysisofsomeflavonoidsand phenolics(mg/100gpowderedflowers)revealedthepresenceofisoquercetrin(3340±2.1),hesperidin

(32.09±2.28),naringin(197.3±30.16),luteolin(10.247±0.594),chlorogenicacid(84.22±2.08),

cat-echin(3.94±0.57)andprotocatechuicacid(34.4±0.15),intheextract.Moreover,theacutetoxicity,

hypoglycemicandhypolipidemiceffectsoftheextractwereinvestigatedusingalloxaninduceddiabetes inratsinadoseof100,200,and400mg/kgbwt.Theethanolicextractwassafeuptoadoseof2000mg/kg. Alltesteddosesoftheflowerextractshowedmarkeddecreaseinbloodglucoselevelby31.78%,66.41% and63.8%at100,200and400mg/kgbwt,respectively,atp<0.05.Regardingtheanti-hyperlipidemic effect,adoseof400mg/kgoftheflowerextractshowedthehighestreductioninserumtriacylglycerides andtotalcholesterollevels(68.46%and51.50%,respectivelyatp<0.05).Thecurrentstudyprovedthe folkuseoftheflowersofS.securidacaasanti-diabeticandanti-hyperlipidemicagentwhichcouldbe attributedtoitshighphenoliccontent.

Introduction

Diabetes mellitus is a complex disorder that characterized

by chronic hyperglycemia and disturbances of fat and protein

metabolismassociatedwithmalfunctionininsulinsecretionand/or

insulinaction.Theutilizationofimpairedcarbohydrateleadsto

acceleratedlipolysis,resultedinhyperlipidemia(Kimetal.,2006).

TheMiddleEastandNorthernAfricahasthehighestprevalenceof

diabetesasaworldregionwith34milliondiabeticpersons

accord-ingtointernationaldiabetesfederation(IDFDiabetesAtlas,2011).

∗ Correspondingauthorat:PharmacognosyDepartment,FacultyofPharmacy, CairoUniversity,Kasr-El-AinyStreet,Cairo11562,Egypt.

E-mail:[email protected](A.M.El-Halawany).

Inrecentyears,thereisgrowingevidencethatplantpolyphenols

includingflavonoidsareuniquenutraceuticalsandsupplementary

treatmentsforvariousaspectsoftype 2diabetesmellitus.Plant

polyphenols can modulate carbohydrate and lipid metabolism,

attenuatehyperglycemia,dyslipidemia,insulinresistance,

allevi-ateoxidative stressand preventthedevelopment oflong-term

diabeticcomplications(Bahadoranetal.,2013).Nowadays,there

isagrowinginterestintheanalysisandidentificationof

medici-nalplants’phenolicconstituentsaimingatfindingnewsourcesfor

thesecompoundsandtoestablishtheirstructureactivity

relation-ship.

Securigera securidaca(L.) Degen &Dörfl.,Fabaceae,hasbeen

widely used in the Iranian, Indian and Egyptian folk medicine

asantidiabeticandanti-hyperlipidemicremedy(Alietal.,1998;

Azarmiyetal.,2009;PorchezhianandAnsari,2001).Chloroformic

http://dx.doi.org/10.1016/j.bjp.2015.02.008

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R.M.Ibrahimetal./RevistaBrasileiradeFarmacognosia25(2015)134–141

extract of S. securidaca decreased fasting serum glucose level,

increasedfoodconsumption,bodyweightandglycogencontent

oftheliverinrats(Zahedi-Asletal.,2005).Totalaqueousextractof

theseedsshowedsignificantdecreaseinbloodglucoselevel(−35%)

inglucoseloadedmice(Alietal.,1998).Inaddition,S.securidaca

seedsuspensionhasa protective effectagainstalloxan-induced

hyperglycemiaand oxidativestressin rats(Mahdiet al.,2011).

HydroalcoholicextractofS.securidacaseedsproducedasignificant

reductionintheleveloftriglyceride,LDLaswellasdecreasein

lipidperoxidation(Fathietal.,2010).Totalseedextractimproved

endotheliumdependentvasodilationinhighfatfedratsby

lower-inglipidformationaroundtheaortainhypercholesterolemicrats

anddecreasingatheroscleroticlesions(Azarmiyetal.,2009).The

hypoglycemiceffectoftheseedwasestimatedtoberelatedtoits

flavonoidcontent(Hosseinzadehetal.,2002).Concerningthe

flow-ersofS.securidaca,twoflavonoids,kaempferolandastragalinwere

isolatedfromitsaqueousextract(Alietal.,1998)butnoreports

werefoundregardingtheeffectoftheflowersonplasmaglucose

andlipidsindiabeticrats.

In the present study, the acute toxicity, anti-diabetic,

anti-hyperlipidemiceffectofthealcoholicextractofS.securidacaflowers

wereevaluatedinalloxaninduceddiabetesmodelinrats.Thetotal

polyphenolsandflavonoidsintheflowersweredeterminedandthe

phenoliccompositionoftheextractwasdescribedusingHPLC/DAD

(high-performanceliquidchromatographic/diodearraydetector)

coupledwithESI-MS(electrosprayionization/massspectrometry)

toidentifythemajorphenoliccompoundspresentintheextract.

Inaddition,HPLC-DADquantificationofmajorphenolicacidsand

flavonoidswascarriedout.

Materialandmethods

Chemicals

Reagents for spectrophotometric determination of

pheno-lic compounds, Folin–Ciocalteu’s reagent: was obtained from

Loba-Chemie (Mumbai, India), sodium carbonate and t-butyl

hydroquinonewereobtainedfromSigma,USA.

RegardingHPLCanalysisofphenolic compounds;acetonitrile

and methanol used were of HPLC grade, and were purchased

fromSigma–Aldrich(Steinheim,Germany).o-Phosphoricacidused

wasofanalyticalgradefromSdfineChemlimited(Mumbai,India)

and formic acidwaspurchased from E-Merck(Darmstadt,

Ger-many).DistilledwaterwasfurtherpurifiedusingaMilli-Qsystem

(Millipore, MA, USA). Acetonitrile and acidulated water were

filteredthrough a 0.45pmmembrane filter (PallGelman

Labo-ratory, USA) and degassed in anultrasonic bathprior toHPLC

analysis.Isoquercetrin,rutin,gallicacid,trans-cinnamicacid,

sal-icylic acid, naringin, protocatechuic acid, ellagic acid, luteolin,

quercetin,caffeic acid, hesperetin, p-coumaric acid, kaempferol

and hesperidin were purchased fromsigma Co. (St.Louis, MO,

USA).

Plantmaterial

SamplesofSecurigerasecuridaca(L.)Degen&Dörfl,Fabaceae,

werecollected duringthe years (2010–2013)from The

Experi-mentalStationofMedicinalandAromaticPlants,Pharmacognosy

Department,FacultyofPharmacy,CairoUniversity,Giza.Plantwas

kindlyauthenticatedbyBotanyspecialist,Dr.MohamedEl-Gebaly,

DepartmentofBotany,NationalResearchCenter(NRC),Giza,Egypt

andavoucherspecimenwaskeptattheHerbariumofthe

Depart-mentofPharmacognosy,FacultyofPharmacy,CairoUniversity(no.

11-6-2013-2).

Determinationoftotalphenolicsandflavonoidscontents

Quantificationof phenoliccompounds wascarried outusing

Folin–Ciocalteau’smethodasreportedbySigeretal.(2008).Briefly;

1gofdriedpowderedflowerswashomogenizedwith40mlof80%

methanolusinga pestle andmortar,filteredthroughWatmann

No.1filterpaperandtransferredintoavolumetricflask(100ml)

with80%methanol.0.2mlofthemethanolicextractwasplaced

in avolumetric flask(10ml)and0.5mlFolin–Ciocalteureagent

(2N)wasadded.After3min,saturatedsodiumcarbonate(1ml)

(20%indistilledwater)wasaddedandthevolumewascompleted

withdistilledwater.After1h,absorbanceofbluecolorwas

mea-suredat_max725nmagainstablank(distilledwater)usingUnicam

UV–visible Spectrometer.Gallic acidwas used to compute the

standardcalibrationcurve(20,40,60,80, 100mg/ml).

Determi-nationswerecarriedoutintriplicates;resultswererepresentedas

themeanvalues±standarddeviationsandexpressedasmggallic

acidequivalentspergramdryweight(mgGAE/gD.W.).

Total flavonoids wereextracted according tothe methodof

Hertogetal.(1992),1gofdriedpowderedflowerswas

homoge-nizedwith40mlof62.5%methanolwith0.1gt-butylhydroquinone

(w/v)and10mlof6Nhydrochloricacidwasaddedcarefullyand

the mixture refluxedat 90◦_C _for ₂_h. _After _cooling, _the

super-natantwasfilteredandtransferredtoavolumetricflask(100ml)

with62.5% methanol.Sample (1ml), Folin–Ciocalteau’s reagent

(2N)(0.5ml) and Na2CO3 (200mg/ml) (3ml)were added,

vor-texedandthenallowedtostandfor15minatroomtemperature

ina darkplaceandabsorbancewasmeasuredat725nm.Rutin

wasusedasstandardandtheequivalents(w/w)weredetermined

froma standardconcentrationcurve(20,40,60,80,100mg/ml)

(Meenakshietal.,2009).Determinationswerecarriedoutin

trip-licates;resultswererepresented asthemeanvalues±standard

deviationsandexpressedasmgrutinequivalents(RE)pergramdry

weight.

Extractionprocedures

The air-dried powdered S. securidaca flowers (1.6kg) were

exhaustively extracted with 90% ethanol by cold maceration.

Thetotalalcoholicextract wascombinedandevaporatedunder

reducedpressureatatemperaturenotexceeding50◦_C,_yielding

250gofdryresidue.Forbiologicalstudytheextractdissolvedin

bi-distilledwaterbytheaidofanultrasonicbathjustpriortothe

investigation.

PreparationoftheextractforHPLC-DAD-MS/MSanalysis

Thepreviouslypreparedethanolicextractoftheflowers(20mg)

was dissolved in HPLC grade methanol (2ml). The methanolic

extractwasplacedinultrasonicbathfor5minandfilteredthrough

0.4␮mmembrane filter.Aliquot of 10␮lwasinjected into the

LC/DAD/MSanalysissystem.

HPLC-DAD-ESI–MSapparatus

The analysis was performed using a Hewlett-Packard 1100

(Waldbronn,Germany)composedofaquaternarypumpwithanon

linedegasser,athermostatedcolumncompartment,aphotodiode

arraydetector(DAD),anautosampler,and1100ChemStation

soft-ware,coupledwithelectrosprayionization(ESI)interfacedBruker

Daltonik Esquire-LC ion trapmass spectrometer (Bremen,

Ger-many) and an Agilent HP1100 HPLC system equipped withan

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ConditionsforHPLC-DAD-MS/MSanalysisofflavonoids

TheHPLCseparationwasperformedonEclipseXDBC18column

(50mm×2.1mm,1.8␮m,AgilentCompany,USA).Mobilephase

consistedoftwosolvents,(A)methanoland(B)0.2%formicacid.

Separationofcompoundswascarriedout withgradientelution

profile:0min,A:B10:90;36min,A:B70:30;50min,A:B100:0;

60min.Chromatographywasperformedat30◦_C_with_a_flow-rate

of0.8ml/min.UVtracesweremeasuredat254,360andUVspectra

(DAD)wererecordedbetween190and900nm.

Massspectrophotometricconditions

Theionizationparameterswere asfollows:capillary voltage

4000V, end platevoltage−500V;nebulizinggasof nitrogenat

35.0p.s.i.;dryinggasof10l/minnitrogenat350◦_C._Mass_analyzer

scannedfrom50to1300␮.TheMS–MSspectrawererecordedin

auto-MS–MSmode.Thefragmentationamplitudewassetto1.0V.

Massspectraweresimultaneouslyacquiredusingelectrospray

ion-izationinthepositiveandnegativeionizationmodes.

QuantitativedeterminationofphenoliccompoundsbyHPLC

Quantitativedetermination ofphenolic compoundswas

per-formed using HPLC apparatus, Agilent Series 1200 apparatus

(Agilent, USA) equipped with autosampling injector, solvent

degasser, quaternary HP pump (series 1200) and ultraviolet

(UV)detector(set at280nmfor phenolic acidsand 330nm for

flavonoids).TheanalysiswasachievedonazobraxODSC18column

(particlesize5␮m,250mm×4.6mmØ).Columntemperaturewas

maintainedat35◦_C._Flavonoid_separation_was_done_adopting_the

methodofMattilaetal.(2000),usingamobilephaseconsisting

of50mMH3PO4,pH2.5(solutionA)andacetonitrile(solutionB)

aceticacid(40:60,v/v)inthefollowinggradient:isocraticelution

95%A:5%B,0–5min;lineargradientfrom95%A:5%Bto50%A:50%B,

5–55min;isocraticelution50%A:50%B,55–65min;lineargradient

from50%A:50%Bto95%A:5%B,65–67min.Theflowrateofthe

mobilephasewas0.7ml/min.Phenolicacidsseparationwasdone

adoptingthemethodofGoupyetal.(1999)withasolvent

sys-temconsistingofA(aqueousaceticacid2.5%),B(aqueousacetic

acid8%)andC(acetonitrile)inthefollowinggradient:at0min,5%

B;at20min,10%B;at50min,30%B;at55min,50%Bat60min,

100%B;at 100min,50%B and50% C; at110min,100% C until

120min.Thesolventflowratewas1ml/min.Theinjectionvolumes

were5␮l.Standardflavonoidsandphenolicacidswerepreparedas

10mg/50mlsolutionsinmethanolandtheyweredilutedtomake

concentrations(20–40␮g/ml)andinjectedintoHPLC.

Quantifica-tionofcompoundswasperformedbasedonpeakareacomputation

(externalstandardmethod).Theanalysiswasrunintriplicatesand

theconcentrationsoftheidentifiedcompoundswereexpressedas

(mg±SD/100gdryweight)andlistedinTable2.

Experimentalanimals

Adult Wister male albino rats, weighing 180–250g, were

obtainedfromtheAnimalHouseColonyoftheNationalResearch

Center(Dokki,Giza,Egypt),andwerehousedunderconventional

laboratoryconditionsthroughouttheperiodofexperimentation.

Theanimalswerefedastandardratpelletdietandallowedfree

accesstowater.

Drugsandkits

Alloxanmonohydratepowder(Sigma–Aldrich, St.Louis, MO,

USA),Gliclazide(Servier,Egypt)wereusedinthepresent

inves-tigation.Thebiochemicalkitsusedinthestudywereglucosekits

(Biodiagnostic,Egypt),cholesterolkits(Biodiagnostic,Egypt)and

triacylglycerideskits(Biodiagnostic,Egypt).

Acutetoxicity

AcuteoraltoxicityoftheethanolicextractoftheflowersofS.

securidacaL.wasperformedfollowingthemethodofLorke(1983).

Anti-hyperglycemicactivityandanti-hyperlipidemicactivity

Ratswereweighedandinjectedintraperitoneallywithalloxan

(150mg/kg)dissolvedindistilledwater.After48hbloodsamples

werewithdrawnfromtheretro-orbitalvenousplexusunderlight

etheranesthesiaandtheserumwasseparatedbycentrifugation

forthedeterminationofglucoselevel.Onlyratswithserumglucose

levelsmorethan250mg/dlwereselectedandconsideredas

hyper-glycemicanimalsaccordingtomethodofNeshwarietal.(2012).

Thehyperglycemicratswerethendividedintofivegroups(10rats

each).Thefirstgroupofdiabeticratsservedascontrol;thesecond

tofourthgroupsreceivedalcoholicextractoftheflowersatdoses

100,200and400mg/kgorallyfor10consecutivedays;andthefifth

groupofdiabeticratsreceivedGliclazide(antidiabeticstandard)at

doseof5mg/kgbwtorallyfor10consecutivedays.Theextractand

Gliclazidewerestarted48hafteralloxaninjectionatwhichtime

hyperglycemiawasconfirmed.Twenty-four hoursafterthelast

doseofeitherdrugtreatment,abloodsamplewaswithdrawnfrom

theretro-orbitalvenousplexusfrom18hfood-deprivedratsand

wascentrifugedat3000rpmfor10min.Theserumwasobtained

fordeterminationoftheserumglucoselevel,triacylglyceridesand

totalcholesterollevel.

Determinationofserumglucoselevel

Glucose level wasdetermined as quinineamine usinga test

reagent kit (Biodiagnostic, Egypt) according to the method of

Trinder(1969).Theabsorbancewasmeasuredat510nmandthe

resultswereexpressedasmg/dl.

Determinationofserumtriacylglyceridelevel

Triacylglycerideswereestimatedbyenzymaticmethodsusing

diagnostickit(Biodiagnostic,Egypt)accordingtothemethodof

Fossati and Prencipe (1982). The absorbance was measured at

510nmandtheresultswereexpressedasmg/dl.

Determinationofserumtotalcholesterollevel

Totalcholesterolwasestimatedbyenzymaticmethodsusing

diagnostickit(Biodiagnostic,Egypt)accordingtothemethodof

Allainetal.(1974).Theabsorbancewasmeasuredat500nmand

theresultswereexpressedasmg/dl.

Statisticalanalysis

Statisticalanalysiswascarriedoutbyonewayanalysisof

vari-ance (ANOVA)followedbyTukeytest. Resultsareexpressedas

means±SEM(n=10).

Resultsanddiscussion

TotalphenolsandflavonoidscontentsofS.securidacaflowers

wereinvestigated.Thevalueoftotalphenolicswas82.39±2.79mg

gallicacidequivalent(GAE)/g(D.W.)andthatofthetotalflavonoids

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Table1

PeakassignmenetofmetabolitesinethanolicextractofS.securidacausingLC-DAD/MSinpositiveandnegativeionizationmodes.

No. RT [M−H]−_m/z _[M+H]+_m/z _Fragment_ions _Identified_compounds

1 0.9 147 – 147,62 trans-Cinnamicacid

2 1.2 – 139 137,93 Salicylicacid

3 1.5 – 216 170[(M+H)-HCOOH],125,97 Gallicacid

4 1.8 – 152 153,109 Protocatechuicacid

5 2.8 – 292 289,271,245,227,205,179,125 Catechin

6 11.1 447 – 327[(M−H)-120],285[(M−H)-162],284,255,227 Kaempferol-7-O-glucoside

7 11.7 448 – 447,357[(M−H)-90],327[(M−H)-120],

285[(M−H)-162]

Orientin

8 12.4 625 – 505[(M−H)-120],463[(M−H)-162],300,

301[(M−H)-162]

Quercetin-3,7-di-O-glucoside

9 12.7 447 – 327[(M−H)-120],284[(M−H)-H-162],255

[aglycone-2H-CO],243,241,217,213,199,175,149

Luteolin-3′-O-glucoside

10 12.8 447 – 327[(M−H)-120],285[(M−H)-162],284,

257[aglycone-CO],243,241,217,213,199,175,149

Luteolin-7-O-glucoside

11 13.2 593 – 503[(M−H)-90],473[(M−H)-120],431[(M−H)-162],

311[(M−H)-120–162],283

Isovitexin-4′-O-glucoside

12 13.6 609 – 489[(M−H)-120],447[(M−H)-162],

357[(M−H)-90–162],327[(M−H)-120–162]

Isoorientin-4′-O-glucoside

13 14.2 593 – 575[(M−H)-18],503[(M−H)-90],473[(M−H)-120],

383[(M−H)-90–120],353[(M−H)-120–120]

Apigenin6,8-di-C-glucoside (vicenin-2)

14 14.9 593 – 503[(M−H)-90],473[(M−H)-120],431,341,311

[(M−H)-120–162]

Isovitexin-7-O-glucoside (saponarin)

15 15 – 595 449,433,284

Kaempferol-O-neohesperidosideee

16 15.4 623 – 503[(M−H)-120],461[(M−H)-162],

341[(M−H)-120–162],315[(M−H)-146–162],297,

195,179,161,153,135

Isorhamnetin-3-O-glucoside-7-O-rhamnoside

17 15.9 448 – 447,429,357[(M−H)-90],327[(M−H)-120],

285[(M−H)-162]

Iso-orientin

18 15.9 564 – 545,503[(M−H)-60],473[(M−H)-90],

443[(M−H)-120],425,413,383,353[(M−H)-120–90],

Apigenin-6-C-pentoside–8-C-hexoside

19 16.1 639 – 639(M−H)-,519[(M−H)-120],477[(M−H)-162],459

[(M−H)-180],357[(M−H)-120–162],314,

315[(M−H)-162–162]

Isorhamnetin-O-sophoroside

20 16.3 593 – 503[(M−H)-90],473[(M−H)-120],447[(M−H)-146],

429[(M−H)-146-H2O],413,395,383,353,329,299

Isoorientin-2′′_{-O-rhamnoside}

21 17.3 431 – 431,353,341[(M−H)-90],311[(M−H)-120],269 Isovitexin

22 17.6 577 – 577,413[(M−H)-146–18],457[(M−H)-120],341,311,

293[aglycone+41–18]-,173

Isovitexin-2′′-O-rhamnoside

23 17.7 – 580 579,459,271,235 Naringin

24 17.9 463 – 301[(M−H)-162],300,271,255,179,151 Isoquercetrin

25 18.1 – 303 301,258,143 Hesperetin

26 18.1 – 464 343,301,179,151 Hyperoside

27 18.2 – 303 257,229,185 Ellagicacid

28 18.9 578 – 431[(M−H)-146],308,285,269[(M−H)-162–146] Apigenin-7-O-rutinoside

29 19.2 608 – 301,281,237,326 Hesperidin

30 19.3 577 – 431[(M−H)-146],285[(M−H)-146–146] Kaempferol-3,7-dirhamnoside

(kaempferitrin)

31 19.5 – 609 301,464,179,151 Rutin

32 19.5 448 – 285[(M−H)-162],284,255,267,257,256,241,229,

213,163

Kaempferol-3-O-glucoside (astragalin)

33 19.5 607 – 608[(M−H)-H],463[(M−H)-146],447[(M−H)-162],

299[(M−H)-162–146]

Quercetin-3-O-glucoside-7-O-rhamnoside

34 19.7 – 287 287,285,217,241,175 Luteolin

35 19.7 593 – 593(M−H),447[(M−H)-146],285[(M−H)-146–162]

Kaempferol-3-O-glucoside-7-rhamnoside 36 19.9 477 – 357[(M−H)-120],315[(M−H)-162],314,286,285,271,

243,299

Isorhamnetin-3-O-glucoside

37 20.1 479 – 357[(M−H)-120],315[(M−H)-162],314,286,285,271,

299

Isorhamnetin-7-O-glucoside

38 23.3 479 – 477,301[(M−H)-176],273,257,179,193,151 Quercetin-3-glucuronide

39 24.9 625 – 449,461[(M−H)-162],447[(M−H)-176],337,287,

285[(M−H)-162–176]

Luteolin-7-O-glucuronide-3-O-glucoside

40 25.4 609 – 449[(M−H)-162],431[(M−H)-180],301,

287[(M−H)-162–162]

Luteolindi-O-glucoside

41 26.4 563 – 440[(M−H)-120],323,269[(M−H)-132–162] Apigenin-O-pentosyl-hexoside

42 33 – 301 301,151,179 Quercetin

43 34.3 – 286 285,257,151,169,241 Kaempferol

44 38.5 – 179 179,135,107 Caffeicacid

45 38.5 593 – 473[(M−H)-120],447[(M−H)-146],

301[(M−H)-146–146],299

Quercetin-3,7-dirhamnoside

46 38.8 – 353 353,191,190 Chlorogenicacid

(5)

0

0.0

6

4

2

0 0.5 1.0 2.0 1.5 2.5

0 5

A

= (–) BPC at 254

B

= (–) BPC at 360 nm

C

= (+) BPC at 230 nm 10

1 29

25

27 23

3134

42

43 44

46

47 5

4 3 2

24

67 10 12

13

16

18

19

15 26

303533 17 11 14

21

20

37 32

36

38 3939

41 45

28 22

9 8

BPC 50-1300 –AII MS

15 20 25 30 35 40 45 Time [min]

2 4 6 8 Intens x105

Intens x105

0 5 10

BPC 50-1300 –AII MS

15 20 25 30 35 40 45 Time [min]

0 5 10

BPC 300-400 +AII

15 20 25 30 35 40 45 Time [min]

Fig.1.HPLC-ESI-MS/MSbasepeakchromatograms(BPC)ofthecrudeethanolicextractoftheflowersofS.securidacaL.recordedat254nm(A)and360nm(B),negativeion mode,andat230nm(C),positiveionmode.PeaknumbersfollowthoselistedinTable1.

HPLC-DAD-MS/MSanalysisofphenoliccompounds

TheHPLC-DAD-MS/MSanalysiswascarriedoutin both

neg-ative and positive ionizationmodes. The HPLC-DAD base peak

chromatograms(BPC)recordedat254nm(A),360nm(B)inthe

negativemodeandBPCinthepositivemodeobtainedat230nm

(C)areshowninFig.1.Theidentities,retentiontimesandobserved

molecularandfragmentionsforindividualcompoundsare

pre-sentedinTable1.

Atotalof47phenoliccompoundshavebeententatively

iden-tifiedbycomparingretentiontimesandMSdataofthedetected

peakswiththatreportedintheliteratureandbysearching

phyto-chemicaldictionaryofnaturalproductsdatabase(CRC).Identified

compoundsbelongedtovariousclasses(Table1)includingeight

phenolicacidsand39flavonoids.Thephenolicacidsincludedthree

hydroxybenzoicacids(salicylicacid,gallicacidandprotocatechuic

acid)andfourhydroxycinnamicacidderivatives(trans-cinnamic

acid,caffeicacid,chlorogenicacidandp-coumaricacid),inaddition

toellagicacid.Flavonoidswerepresentmostlyasflavones

(includ-ingbothO-andC-glycosideswithapigeninorluteolinasaglycone)

andflavonols(derivedfromtheaglycones;quercetin,kaempferol

andisorhamnetin),flavanones(naringeninandhesperitinandtheir

glycosides). While, only one flavan-3-ol (catechin) was

identi-fied.Sugar moieties consists of hexosides,deoxyhexosides and

pentosidesasdeducedfromthelossof162␮,146␮and132␮,

respectively.

Identificationofflavonoids

SeventeenflavoneswereidentifiedonthebasisoftheirMS/MS

fragmentation.Threecompoundsweremono-C-glycosylflavones

(peaks7,17and21)producingMSfragmentationpatterns

charac-teristictoC-glycosidesflavonoidsincludingdehydrationandcross

ringcleavageoftheglucosemoietythatproducing0,2crossring

cleavage [(M−H)-120]and 0, 3 cross ring cleavage[(M−H)-90]

(Figueirinhaetal.,2008).Compounds7,17and21showed

pseu-domolecularionsatm/z448,m/z448andm/z431,respectively,

andexhibitedtypicalfragmentationpatternsofC-glycosides,hence

theywereassignedasorientin,isoorientinandisovitexin,

respec-tively.

Peaks 13 and 18 were di-C-glycosyl flavones and showed

fragmentationpatternof[(M−H)-18],[(M−H)-90],[(M−H)-120],

[aglycone+113], and [aglycone+83] found in negative mode

(6)

pseudomolecularionatm/z593andcharacteristicfragmentions

at 575 [(M−H)-18], 503[(M−H)-90], 473[(M−H)-120], and 383

[(M−H)-90–120].

Compound18wasidentifiedasisochaftoside(6-C

-pentosyl-8-C-hexosylapigenin)(Figueirinhaetal.,2008;Zhangetal.,2011).

It showed a pseudomolecular ion at m/z 564 and a

fragmen-tation pattern typical of the asymmetric di-C-glycosides. MS2

datashowedfragmentsatm/z473[(M−H)-90]and443[(M−

H)-120], indicating the presence of a C-hexosyl unit. In the same

spectrumafragmentwasobservedatm/z503[(M−H)-60],

cor-respondingtothefragmentationofapentose.Thebasepeakat

m/z 473 [(M−H)-90] and the high abundance of the fragment

atm/z503[(M−H)-60]revealedthepresenceof a6-Cpentosyl

unit.Theionsatm/z353(aglycone+83)and383(aglycone+113)

supportedtheconclusionthatapigenin(MW270)wasthe

agly-cone.

SixcompoundswereidentifiedasO-glycosylflavones(peaks9,

10,28,39,40and41)andshowedfragmentationpatternbeginning

withthecleavageoftheO-sugarbond(Zhangetal.,2011).Five

com-poundswereidentifiedasO-,C-glycosylflavones(compounds11,

12,14,20and22)producingacharacteristicfragmentionsofO-,

C-glycosylflavonesat[(M−H)-120],[(M−H)-90]and[(M−H)-162]

or[(M−H)-146](Figueirinhaetal.,2008;Zhangetal.,2011).For

example,isoorientin-2′′_-_O_-rhamnoside₍₂₀₎_had_a

pseudomolecu-larionatm/z593thatrevealsaluteolinglycosidewithahexoseand

deoxyhexose.Fragmentsatm/z447[(M−H)-146],corresponding

tolossofonedeoxyhexoseandanotheratm/z429[(M−

H)-146-H2O]correspondingtothelossofrhamnose+H2Owereobserved.

In addition,the absenceof the aglyconeion is consistent with

anO-,C-diglycosidestructure(Figueirinhaetal.,2008).MS2_data

alsoexhibitedfragmentsatm/z473[(M−H)-120](basepeak)and

a minor ion atm/z 503[(M−H)-90],which indicated the

pres-enceofaC-glucosylunit.Compound11(isovitexin-4′_-_O_-glucoside)

exhibited apseudomolecular ion atm/z 593witha

fragmenta-tionpattern ofapigenindihexoside and characteristic fragment

ionsofO-,C-glycosylflavoneswiththelossofaO-hexosylmoiety

(−162␮).

Eighteen flavonols were identified in the ethanolic extract

ofthe flowersofS. securidaca.The identificationofthese

com-poundswasfacilitatedbytheanalysisoffragmentationpathways

of(M−H)−/(M+H)+ ionsin thenegativeandpositive ionmodes

and the observation of glycosidic residues (rhamnosyl (146␮)

and glucosyl (162␮)) werecleaved sequentiallyand generated

characteristicaglyconefragmentscompared totheavailable

lit-erature.Amongthesecompoundsfivecompoundswereidentified

askaempferolglycosides(6,15,30,32and35)and sevenwere

identifiedasglycosidesofquercetin(8,24,26,31,33,38and45).

Inaddition,fourisorhamnetinglycosides(16,19,36and37)were

identified.

Flavanonesusually occuras O-glycosyl derivatives,withthe

sugarmoietyboundtotheaglyconehydroxylgroupateitherC-7or

C-3.Amongthesecompounds,theO-diglycosidesareadominant

categoryandtheirstructuresareusuallycharacterizedbythe

link-ageofeitherneohesperidoseorrutinosetotheflavonoidskeleton.

Compound23wasfoundtobetheneohesperidosidenaringin.The

precursorandproductionsofthiscompoundwerem/z579and

271, respectively,indicating thelossofO-diglycoside(m/z308)

(Zhangetal.,2011)andcompound29wasfoundtobethe

ruti-nosidehesperidinwithprecursorandproductionsofm/z609and

299,respectively.

Oneflavan-3-olwasidentified,compound5,whichproduceda

protonatedmolecularionpeakatm/z(292)andyieldedfragment

ionsatm/z245,205,and179characteristicfor(+)-catechin.The

fragmention atm/z245,correspondingto[M+H-44]+_,_was

pro-ducedbythelossofa(CH)₂OHgroupfromthecatechinmolecule

(Sunetal.,2007).

Identificationofphenolicacids

Eight phenolic acids belonged to various classes have been

identifiedbycomparingtheirretentiontimesandfragmentation

patternswiththatreported(Sánchez-Rabanedaetal.,2003;Sun

etal.,2007).Inthepositiveionmodehydroxybenzoicacids

pro-ducedaprotonated[M+H]+_molecule_and_a_[M+H-44]+_fragment_ion

vialossofaCO2groupfromthecarboxylicacidmoiety(Sunetal.,

2007).Threehydroxybenzoicacidshavebeenidentified;salicylic

acid,gallicacid,andprotocatechuicacid(2,3,and4).

Four hydroxycinnamic acids were identified; trans-cinnamic

acid,caffeicacid,chlorogenicacidandp-coumaricacid(1,44,46

and47).Caffeicandp-coumaricacidsproducedprotonated

molec-ularionsatm/z179and165,respectively,andMS2_spectra_due_to

lossofCO₂groupfromthecarboxylicacidfunction(fragmentions

atm/z135and119,respectively,[(M−H)-44])(Sunetal.,2007).

Chlorogenicacidshowedamolecularionpeakat(m/z353)anda

fragmentationionthatcorrespondingtothedeprotonatedquinic

acid(m/z191)(Sunetal.,2007).Compound27hadan[M+H]+_ion

atm/z303whichyieldedamajorionatm/z301andminorionsat

m/z284,257,and229characteristicofellagicacidfragmentation

(SandhuandGu,2010).

QuantitativedeterminationofsomephenoliccompoundsinS. securidacaflowers

Absolute quantification of phenolics using the available

standardswascarriedout(Table2).Eightphenolicacidsi.e.trans

-cinnamicacid(2.36±0.98mg/100g),salicylicacid(15.54±1.91),

protocatechuicacid(34.4±0.155),ellagicacid(13.47±3.95),

caf-feicacid(5.4±1.43),chlorogenicacid(84.22±2.08),p-coumaric

acid(7.58±1.51)andgallicacid(9.5±0.14)weredetermined.In

additiontosevenflavonoidsi.e.isoquercetrin(3340±2.1),naringin

(19.73±3.016), hesperidin (32.098±2.28), luteolin (10.247±

0.594),quercetin(1.16±0.022),kaempferol (0.62±0.129),

cate-chin(39.44±5.73)andhesperetin(0.109±0.013).

Acutetoxicity

TheethanolicextractoftheflowersofS.securidacawasfound

tobesafeuptoadoseof2g/kgbwtwithnomortalityorsignsof

behavioralchangesortoxicityobservedwhichsuggestsitssafety

(Osweileretal.,1985).

Table2

QuantificationsofsomephenoliccompoundsidentifiedinS.securidacausingHPLC analysis.

Compound *Concentration(mg/100g)

trans-cinnamicacid 2.36±0.98

Salicylicacid 15.54±1.91

Protocatechuicacid 34.4±0.15

Naringin 19.73±3.01

Ellagicacid 13.47±3.95

Luteolin 10.24±0.59

Isoquercetrin 3340±2.1

Quercetin 1.16±0.02

Kaempferol 0.62±0.12

Caffeicacid 5.4±1.43

Catechin 39.44±5.73

Hesperidin 32.09±2.28

p-Coumaricacid 7.58±1.51

Hesperetin 0.10±0.01

Gallicacid 0.95±0.014

Chlorogenicacid 8.42±2.08

(7)

350

A

B

Anti-hyperglycemic activity

Anti-hyperlipidemic activity @

@ @

* *

* * *

Normal Diabetic Gliclazide (5 mg/kg)

Dose I (100 mg/kg)

Dose II (200 mg/kg)

Dose III (400 mg/kg)

Serum triacylglycerides (mg/dl) Serum cholesterol (mg/dl) *

* *

250

150

50

0

Nor mal

Diabetic

Gliclazide (5 mg/kg)Dose I (100 mg/kg)

Dose II (20

0 mg/kg)

Dos e III

(400 m g/kg) 50

100 200 250

150 100

Ser

um glucose le

vel (mg/dl)

Concentr

ation (mg/dl)

200 300

Fig.2. BiologicalactivitiesoftheethanolicextractoftheflowersofS.securidacaL. (A)Effectonserumglucoselevelinalloxan-inducedhyperglycemicrats.(B)Serum triacylglyceridesandcholesterollevelsinalloxan-inducedhyperglycemicrats. @_Significant_difference_from_normal_rats_p_<_0.05,_*_Significant_difference_from hyper-glycemicratsp<0.05.

Anti-hyperglycemicactivity

The ethanolic extract of the flowers showed marked anti-diabeticactivityonbloodglucoselevelsinalloxan-induceddiabetic ratsatalltesteddoses100,200and400mg/kgbwtwith poten-ciesof 31.78, 66.41and 63.8% respectively (Fig. 2A).The most

potentreductioninserumglucoselevelwasrecordedwithadose

of200mg/kgbwtcomparedtoGliclazideatadoseof5mg/kgbwt

(64.85%).

Anti-hyperlipidemicactivity

Theethanolicextractoftheflowersexhibitedpotent

hypolipi-demic effect on the elevated serum triacylglycerides and

cholesterollevelsinalloxaninducedhyperglycemicrats(Fig.2B)

with39.68%and41.46%decreasesinserumtriacylglyceridesand

cholesterollevelsatdose200mg/kg,68.46%and51.50%fordose

400mg/kg,comparedtogliclazideatdoseof5mg/kgbwt

show-ingreductioninserumtriacylglyceridesandcholesterollevelswith

73.01%and62.20%,respectively.

Diabetesmellitusisacomplexdisorderthatcharacterizedby

chronichyperglycemiaanddyslipidemia.Thediseasebecomesa

realproblemofpublichealthindevelopingcountries,whereits

prevalenceisincreasingsteadilyandadequatetreatmentisoften

expensiveorunavailable. Resultsof thepresent studyrevealed

thatalloxan-inducedhyperglycaemiaisassociatedwithmetabolic

changes.Alloxaninduceschemicaldiabetesinratsbydamaging

insulinsecretingpancreatic␤-cellsleadingtodecreaseininsulin

release(Kimetal.,2006).

Hyperlipidaemiaisamajorcharacteristicofdiabetes(Pushparaj

etal.,2000).DMinducedhyperlipidaemiaisattributabletoexcess

mobilizationoffatfromtheadiposetissueduetotheutilizationof

theglucose.Moreover,studiessuggestedthathyperlipidemiaisone

ofthemostcommonfeaturesinalloxan-inducedhyperglycaemia

inexperimentalrats(Krishnakumaretal.,2000).Inthisstudy,an

increaseinthelevelsoftotalcholesterolandtriglycerideshasbeen

observedinalloxan-inducedhyperglycaemicrats.Plantsusedin

traditionalmedicinetotreatdiabetesmellitusrepresenta

valu-ablealternativeforthecontrolofthisdisease(KumarandVerma,

2011).Inthepresentstudy,weevaluatetheacutetoxicity,

anti-hyperglycemicandhypolipidemiceffectsoftheethanolicextract

oftheflowersofS.securidacaL.Moreover,thephenoliccomposition

oftheextractwascharacterizedusingHPLC-DAD-ESI/MStechnique

tohelpinchemicalprofilingandstandardizationoftheextract.Oral

treatmentofhyperglycemicratswiththeethanolicextractofthe

flowers(100,200and400mg/kgbwt)significantlydecreasedthe

elevatedserumglucoselevelaswellasserumtotalcholesteroland

triglycerideslevelsindiabeticratswithpotenciescomparableto

gliclazide.Hence,wecouldsaythatextracthadbeneficialeffects

oncarbohydratemetabolisminhyperglycaemicrats.

HPLC-DAD-ESI/MSanalysisrevealedthattheethanolicextract

containscomplexmixtureofphenoliccompoundsincluding

differ-entclassesofphenolicacidsandflavonoids.Mostofthedetected

phenolic compoundswere reportedtohave anti-diabeticeffect

thoughdifferentmechanisms.Isovitexin,luteolin7-O-glucoside,

hyperoside and isorientin were reported to possess

antihyper-glycemicaction(Brahmachari,2011;delPilarNicasio-Torresetal.,

2012;Foladoretal.,2010).Vicenin-2wasreportedtobean

antiox-idantthatstronglyinhibited␣-glucosidaseandexhibitedpotent

anti-glycation properties (Islam et al.,2014). Isoquercetrin and

astragalinwerefoundtobeglycationinhibitorshavingcomparable

activitytothatofaminoguanidine(Brahmachari,2011).Rutinwas

reportedtopossesspotenthypoglycemicandhypolipidemic

activ-itiesbyenhancingperipheralglucoseutilizationbyskeletalmuscle

andstimulationof␤-cells(JadhavandPuchchakayala,2012).

The respective aglycones, quercetin and kaempferol were

found toimprove insulin-stimulated glucose uptake in mature

adipocytes(Figueirinhaetal.,2008).Isorhamnetin-3-O-glucoside

wasreportedtolowerserumglucoseconcentration,sorbitol

accu-mulationinthelenses,redbloodcellsbyexertingpotentinhibitory

activity against rat lens aldose reductase,leading to improved

diabeticcomplications(Brahmachari,2011).Rutinandhesperidin

were reportedto preventtheprogression of hyperglycemiaby

increasinghepaticglycolysis,glycogenconcentrationand

lower-inghepaticgluconeogenesis(Jungetal.,2004).Throughadocking

study,catechinshowedpotentialagonistcharacteristictoinsulin

receptor (insulin mimetic) (Pitchai and Manikkam, 2012).

Gal-licacidwasreportedtohavehypoglycemicandhypolipidaemic

effects against streptozotocin induced diabetic rats (Latha and

Daisy,2011),whilechlorogenicacidwasreportedtoexhibit

hypo-glycemic, hypolipidemic, and antioxidant properties (del Pilar

Nicasio-Torresetal.,2012).

Conclusion

In this workwe have evaluatedtheanti-hyperglycemic and

(8)

flower.Phenolicacidsandflavonoidsextractedwith90%ethanol

havebeenidentifiedandquantified.Theethanolicextractofthe

flowerswassafeuptodoseof2g/kg.Theextractshowedpotent

anti-diabeticandhypolipidemiceffectinalloxaninduced

hyper-glycemicinrats.Thecurrentresultsindicatethatthe

flavonoid-andphenolicacid-richextractofS.securidacaflowersisapromising

naturalpharmaceuticalforcombatingdiabetes.

Conflictsofinterest

Theauthorsdeclarethattheyhavenocompetinginterests.

Author’scontributions

RIwrotethemanuscript,carriedoutextractionproceduresand

analyzeddata.AMwrotethemanuscript,planedtheworkand

ana-lyzeddata.DScarriedoutbiologicalactivity.ENcarriedoutLCMS

analysisandinterpreteddata.AErevisedthemanuscriptand

super-visedwork.SEsuggestedthepointandrevisedthemanuscript.

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