Rev. bras. farmacogn. vol.27 número4

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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

Comparative

study

of

the

hypocholesterolemic,

antidiabetic

effects

of

four

agro-waste

Citrus

peels

cultivars

and

their

HPLC

standardization

Nesrin

M. Fayek

a,∗

_,

_Amani

_H.

_El-Shazly

b

_,

_Azza

_R.

_Abdel-Monem

a

_,

_Mohamed

_Y.

_Moussa

a

_,

Samia

M. Abd-Elwahab

a

_,

_Nebal

_D.

_El-Tanbouly

a

a_Department_of_{Pharmacognosy,}_Faculty_of_Pharmacy,_Cairo_University,_Cairo,_Egypt

b_Department_of_{Pharmacology,}_Research_Institute_of_{Ophthalmology,}_Cairo,_Egypt

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received20July2016 Accepted2January2017 Availableonline29April2017

Keywords:

Agro-wasteCitruspeels Anti-diabetic HPLCquantification Hypocholesterolemic Pectin

Polymethoxyflavone

a

b

s

t

r

a

c

t

CitrusisaneconomicallyimportantfruitforEgypt,butitspeelalsoisoneofthemajorsourcesof

agri-culturalwaste.Duetoitsfermentation,thiswastecausesmanyeconomicandenvironmentalproblems. Thereforeitisworthwhiletoinvestigatewaystomakeuseofthiscitruswastegeneratedbythejuice industry.Thisstudywasaimedtoexplorethehypocholesterolemic,antidiabeticactivitiesoffour vari-etiesofcitruspeelsagrowastes,toisolatethemainflavonoidsintheactivefractionsandtoquantifythem byHPLCmethodfornutraceuticalpurposes.Allthetestedsamplesoftheagro-wasteCitrusfruitspeels showedsignificantdecreaseincholesterol,triacylglycerideandglucose.Themostdecreaseincholesterol levelwasobservedbymandarinpeelsaqueoushomogenateanditshexanefraction(59.3%and56.8%, respectively)reachingthesameeffectasthereferencedrugused(54.7%).Mostly,allsamplesdecrease tri-acylglyceride(by36%–80.6%)betterthanthereferencedrugused(by35%),while,glucosewasdecreased (by71.1%–82.8and68.6%–79.6%,respectively)mostlybytheaqueoushomogenates(exceptlime)and alcoholicextracts(exceptmandarin)ofCitrusfruitspeelsbetterthanthereferencedrugused(by68.3%). Alltheisolatedpectin,fromthefourcultivars,hassignificanteffectonthethreeparameters.The compar-ativeHPLCrapidquantificationofnobiletininthedifferentby-productcitrusvarietieshexanefractions revealedthatnobiletinispresentinhigherconcentrationinmandarin(10.14%)thantheotherspecies. Nobiletinand4′_{,5,7,8-tetramethoxy}_flavone_were_isolated_from_mandarin_peels_hexane_fraction_by chro-matographicfractionation.ThisisthefirstreportofthecomparativeHPLCquantificationofnobiletinand biologicalstudiesofdifferentcitruspeelsspeciesasagro-wasteproducts.Basedontheseresults,we sug-gestthepossibilitythatCitrusfruitspeelsmaybeconsideredasanantidiabeticandhypocholesterolemic nutraceuticalproduct.

Introduction

Oneofthemostimportantpopularthreatfactorsforcoronary

heartdisease,heartattackandstrokeisthehighcholesterol(AHA,

2014). Large amount of fruit peels are pitched as waste from

fruitprocessingindustryinspiteofthewelldeclaredbiological

activitiesof these peels compared to other discardedportions.

Among thefruits, Citrus fruits which its yield is approximated

as 80 million tons per year are regarded as precious healthy

dietsinceitsnutrientsboosthalenessandguardagainstchronic

disease(Gnanasaraswathietal.,2014).Hugeamountsofscrapsare

∗ Correspondingauthor.

E-mail:[email protected](N.M.Fayek).

producedyearly,followingjuice manufacturing,fromaboutone

thirdofCitrusfruits(Lietal.,2006).Citruspeels,whichconstitute

the main residue, contain more bioactive compounds than do

juices(Boccoetal.,1998;Gorinsteinetal.,2001)andareasuitable

provenanceof pectin(Sakaiand Okushima,1980).While citrus

peelsexhibitpotentantioxidant,antimicrobial,anti-inflammatory

activities(Murakamietal.,2000;Linetal.,2011;Dhanavadeetal.,

2011)and have a reverse relationship withthecoronary heart

diseaseincidencebyitspotencyindecreasingplasmacholesterol

level(Boketal.,1999;Wilcoxetal.,2001;Whitmanetal.,2005;

Leeet al.,2011; Assiniet al.,2013),pectin isusefulin medical

purpose, inwhich itaids indecreasingserumcholesterol level,

dislodgingheavymetalions fromthebody, equilibratingblood

pressureandassistinginweightreduction(Tangetal.,2011).

A plentiful source of polyhydroxyl flavonoids, such as

hes-peridin,neohesperidinandnaringin,arethecitruspeelswhichare

http://dx.doi.org/10.1016/j.bjp.2017.01.010

(2)

alsotheuniquesourceofpolymethoxyflavoneswithhighcontent

suchasnobiletin,tangeretinandsinesetin(Lietal.,2006;Londo ˜no

etal.,2010).Inpreviouslyinvivostudies,lowerdosesof citrus

polymethoxylatedflavones(PMF)candecreaseplasmacholesterol

levelmorethanthatofflavanones(Morinetal.,2008)and

modu-latedlipidmetabolismincellsandanimals(Leeetal.,2011).Two

polymethoxylatedcitrusflavonoids,tangeretinandnobiletin,

mod-eratelyinhibitedbothcholesterol(CH)andtriacylglyceride(TG)

synthesis,whileweakereffectswerereportedbyotherPMF(e.g.,

sinensetin)andnon-PMF(e.g.,hesperetinandnaringenin).Hence,

attentionshouldbepaidfortheirproperextractionaspotential

compoundsandchecktheirsuitabilityastherapeutics. Thiswill

increasetheaggregatevalueoftheindustrialwaste.

Ourstudywascarriedoutonfouragro-wasteCitruspeelsspecies

cultivatedinEgypt,afterpreviouslyexploringtheirpeelsoil

bene-fits(Abd-Elwahabetal.,2016),[mandarin(CitrusreticulataBlanco

cv.Egyptian),sweetorange(Citrussinensis(L.)Osbeckcv.Olinda

Valencia),whitegrapefruit (C.paradisiMacfad.cv.Duncan) and

lime (C. paradisi aurantiifolia (Christm.) Swingle cv. Mexican)],

Rutaceae,toevaluateandcomparetheirhypocholesterolemicand

anti-diabeticactivitiesasagro-wasteproducts,toisolatethemain

flavonoidsintheactivefractionsandtoquantifythembyHPLC

methodfornutraceuticalpurposestofacilitatetheconversionof

thiswasteintohighvalue-addedproducts,thusallowingittobea

recycledcomponentoffunctionalfoodmaterial.

Materialandmethods

Plantmaterial

Samplesofthefreshfullymatureripecitrusfruitpeels

[man-darin (Citrus reticulata Blanco cv. Egyptian), sweet orange (C.

sinensis(L.)Osbeckcv.OlindaValencia),whitegrapefruit(C.

par-adisiMacfad. cv.Duncan) and lime(C. aurantiifoliaSwingle cv.

Mexican)],Rutaceae,wereidentifiedbyCitrusdepartment,

Horti-culturalInstitute,MinistryofAgriculture,Giza,Egyptandvoucher

specimensnumbers14-7-2016-I,14-7-2016-II,14-7-2016-IVand

14-7-2016-III,respectively,weredepositedattheMuseumofthe

PharmacognosyDepartment,FacultyofPharmacy,Cairo

Univer-sity,Egypt.ThematerialwerecollectedinFebruary2011(forsweet

orange),September2011(forlime),2ndhalfofDecember2011(for

grapefruit)and2ndhalfofJanuary2012(formandarin),fromthe

privateorchardofEl-Mazloomcompanyforhorticulture

produc-tionat78kmCairo-Ismailiaroad.

Preparationofextractsandisolationofpectin

Preparationofacitruspeelsaqueoushomogenates:theaqueous

homogenateswerepreparedbymixing1.5goffreshpeelofeach

specieswith100mldistilledwaterinablender,storedinbottle

andkeptinrefrigerator(2–5◦_C)_until_used.

Preparationofa citrus peelsalcoholicextracts:thealcoholic

extractswere preparedby percolating200gof fresh peel with

80%methanol(600ml),filteredoffandtheresultingextractswere

evaporatedunderreducedpressure.

PreparationofaCitruspeelshexaneextracts:partofthe

previ-ouslypreparedalcoholicextractresidue(10g)foreachCitrusfruits

peelsweresuspendedseparatelyindistilledwater(30ml),then

extractedwithn-hexaneandtheresultinghexaneextractswere

evaporatedunderreducedpressure.

Isolation of pectin from citrus peels: each species of fresh

citrus peels (50g) was boiled with known volume of distilled

water (100ml), decanted, cooled to room temperature (25◦_C)

andthenfourvolumesofabsoluteethanol(400ml)wasaddedto

precipitatethepectin,keptinrefrigerator(2–5◦_C)_for _two_days

andthenfilteredtoobtainpectinwhichisfreezedriedandstored

indesiccatorsuntilused.

Citruspeelsalcoholicandhexaneextractsresiduesandthe

iso-latedpectins(1.5gofeach)weremixedwith100mldistilledwater

(1.5%),storedinbottleandkeptinrefrigerator(2–5◦_C)_until_used.

Materials

Forbiologicalstudy

Pure cholesterol powder analytical grade (C75209) and bile

saltspowder(48305)werepurchasedfromSigmaChemicalCo.,St.

Louis,USA.Metformin(Cidophage®):ChemicalIndustries

Devel-opmentCo. (CIDCo.), Giza,Egypt,as ananti-diabeticreference

drug.Atorvastatin5mg(Lipitor®):PfizerCompany,Cairo,Egypt,

asahypolipidemicreferencedrug.

Forchromatographicstudy

Silica gel 60 (89943Fluka) for column chromatography and

precoated silicagelplates 60 F254for TLCwereobtainedfrom

Sigma-AldrichCo.LLCandMerckMilliporeCorporation,Germany,

respectively. Acetonitrile (34860), methanol (34860) and

phos-phoricacid(79606)of HPLCgrade werepurchasedfromSigma

Chemical Co., St. Louis, USA. 1_H-(300_MHz) _NMR _spectra _were

recordedonVarianMercuryapparatusat25◦_C_using_TMS_as_an

internalstandardandchemicalshiftsweregiveninıvalues.

Isolationofpolymethoxyflavones

Theairdriedpowderedmandarinpeels(850g)were

macera-tedin75%alcohol(2×3l)atroomtemperatureandthealcohol

extractwasevaporatedundervacuum,toobtainayellowishbrown

residue (233g). This residue was suspended in distilled water

(500ml)andthenfractionatedwithhexane(250×3ml),toyield

5gresidueafterevaporationunderreducedpressure.Thelatter

waschromatographedonsilicagelcolumn;gradientelutionwas

carriedoutusinghexanecontaining10%stepwiseincrementsof

acetonetill100%acetone.Fractions,100mleach,werecollected

toyield40fractionsandsubjectedtoTLConpre-coatedsilicagel

plates,usingsolventsystemschloroform:methanol(98:2).

Frac-tions(17–20)elutedwithhexane:acetone(6:4)showedamajor

spotthatappearsyellowinUVat365nmandgaveayellowcolor

withp-anisaldehydesprayreagent,ammoniaandaluminum

chlo-ride.Thesefractions werepooledtogetherand thesolventwas

evaporatedunderreducedpressuretoyieldcompound1asa

yel-lowpowder(1.5g).Fractions(29–32)elutedwithhexane:acetone

(3:7)showeda majorspotthatappearsyellowinUVat365nm

andgaveayellowcolorwithp-anisaldehydesprayreagent,

ammo-niaandaluminumchloride.Thesefractionswerepooledtogether

andthesolventwasevaporatedunderreducedpressuretoyield

compound2asayellowpowder(0.75g).

HPLCanalysis

HPLCanalyseswereperformedusingAgientTechnologies1200

series; consisting of G1322A Degasser (serial No. JP94172767),

G1311AQuatpump(serialNo.DE62972789),G1314BVWD(serial

No.DE71365992)andG1328BMan.inj.(serialNo.DE60561522).

Thewavelengthusedforthequantificationoftheflavanones

gly-cosideswiththeUVdetectorwas325nm.Thechromatographic

separationwascarriedoutonLiChrosher(R)100RP-18endcapped

(5␮m) column. LiChroCART(R) 250-4 HPLC-Cartridge, Agilent

Technologies,PartNo.79925ODE-584,CartridgeNo.031399and

(3)

Table1

Ingredientcompositionofthedietfedtorats.

Standardlaboratorydiet Percentage(%)

Vitaminmixture 1

Mineralmixture 4

Cornoil 10

Sucrose 20

Cellulose 0.2

Starch 54.3

Casein(95%pure) 10.5

Theend-cappedcolumnwasheldat37◦_C_and_the_flow_rate_was

setat1ml/min.ThechosenmobilephasewasasthatusedbySun etal.(2010)butwithlittlemodificationasfollowing:themobile

phaseconsistedoftwosolvents;90%phosphoricacid(0.3%)(A)and

10%acetonitrile(B).Thesolventgradientinvolumeratioswasas

follows:10–40%Bover1min.Thesolventgradientwasmaintained

at40%Bfor1min,increasedto45%Bover18min,andto100%

Bover2min,thenmaintainedat100%Bfor7min.Theinjection

volumewas20␮l.Analyseswereperformedatleastthreetimes

andonlymeanvalueswerereported.

ThefourtestedCitruspeelshexaneextractswerepreparedat

aconcentrationof2mg/mlandfilteredonEkicrodisc®3.AcroLC

(3mmdiameter,0.45␮m,HPLCcertified,LOTNo.A10422144P/N

E031)beforeuse.Theisolatedstandardnobiletinwaspreparedat

astockconcentrationof500␮g/ml.Calibrationstandardsample

waspreparedbyappropriate dilutionswithmethanolfromthe

stocksolutions andfilteredonEkicrodisc®3.Acro LCbeforeuse.

Calibrationcurveswereobtainedbyplottingthepeakareaofthe

standardversusitsconcentrations(0.92<R2_<_0.99)._{Concentrations}

ofthenobiletininsamplesweredeterminedbyapplicationofthe

obtainedstandardcurve.

Experimentalanimalsandprotocols

MaleWistarstrainratsweighing150–160g,agedthreemonths,

utilizedfor assessment ofthedifferentpharmacological effects,

weresuppliedfromtheResearchInstituteofOphthalmology.They

wereindividuallyhousedsixpercage(320cm×180cm×160cm)

understandardized temperature (25–28◦_C), _humidity_(50–60%)

andlight(12hlight/darkcycleswiththelightonat7a.m.andhad

freeaccesstotapwaterandfoodpelletsconditions.Theywerefed

thestandardlaboratorydietasshowninTable1.

HypercholesterolemiawasinducedbyfeedingthemaleWistar

ratswithstandardlaboratorydietmixedwith1%cholesteroland

0.25%bilesaltspowdersfromthedietweight(Berrouguietal.,

2003;Owens,2006).Bloodsamplesweretakenattheeighthweek

ofexperimentandthencentrifugedat715×gforcefor10minfor

biochemicalanalysesofplasmaparameters.Theclearplasmawas

separatedanddividedintothreeportionstomeasuretheplasma

glucose,triacylglycerideand cholesterollevelsby usingspecific

kits.The separated plasmawas stored at−80◦_C _until _analysis.

ThiswascarriedoutaccordingtoOdetolaetal.(2006)andOwens

(2006).Untreatedmodel(highfatdiet)andthenegativecontrol:

receiveddaily0.1mlofdistilledwaterorallybesidestandard

labo-ratorydiet.

Treatedmodelgroups(16groups):administered0.1mlofthe

correspondingextract or pectin or aqueous homogenateorally

besidestandardlaboratorydiet.

Biochemicalplasmaanalyses

Plasmaglucose,TGandtotalcholesterollevelsweredetermined

byenzymaticmethodsusingcommercialassaykits(glucosekits,

triacylglyceridekits andtotal cholesterol kits)according tothe

manufacturer’sprotocols,purchasedfromBiodiagnosticCompany

(Egypt).SpectoUV–visDoubleBeamPC,8scanningautocell,

UVD-3200(LABOMED,Inc.)wasusedforevaluationofanti-diabeticand

hypocholesterolemicactivitiesbymeasurementofthecolor

inten-sityat515–520nm.

Statisticalanalysis

Allthedatawereexpressedasthemean±standarderrorofthe

mean(SEM).Thestatisticalsignificanceofdifferencesbetweenthe

meanvaluesforthetreatmentgroupswasanalyzedbyone-way

analysisofvariance(ANOVA)followedbyPostHoctestand

Bonfer-roni(SnedecorandCochran,1982)usingtheSPSSsoftware(SPSS

Inc.,Chicago,USA).Avalueofp<0.05wasconsideredstatistically

significantfor analysis.Correlationanalysiswascarriedoutand

R-squarevalueswerereported.

Resultsanddiscussion

Thepercentageyieldofpeelsandpectinobtainedfromdifferent

agro-wastepeels

Bothwhitegrapefruitandmandarinhavethehighest

percent-ageyieldoffreshpeels,recording25.81%and23.31%respectively,

whileloweryieldwasobservedfortheothertworemainingtested

Citrusfruits,sweetorangeandlime,recordingthesameresult18%.

Upondryingthepeels,alltheCitrus fruitsshownearbyresults;

5.80%inwhitegrapefruitto7.19%inlimefruits.Alltheisolated

pectinfromthetestedagro-wastepeelsoccursasawhitepowder.

Sweetorangehavethehighestcontentofpectin(21.33%)followed

bylime(19.7%)andgrapefruit(11.66%),whilemandarinhavethe

lowestpectincontents(9.14%).Ourresultsshowedavariationfrom

thepreviouslyreportedones,RouseandCrandall(1976)reported

8.15%pectinyieldinfreshgroundValenciaorangepeelsand6.35%

inDuncangrapefruitpeelsextractedwithnitricacid.BothLiuetal.

(2001)andSakaiandOkushima(1980)recorded3.68%and0.4%for

MarrsandNavelorangepeel,respectively,and2.99%and0.2%for

MarshandNavelgrapefruitpeel,respectively,preparedbyclassical

hotacidprocedureandwaterextraction.Whilethepectinyields

ofsweetorangeandmandarinextractedbytheadditionof

sul-phuricacidwas43.7%and37.3%,respectively(Wangetal.,2008).

Therefore,thepectinyielddependsnotonlyonthespeciesand/or

cultivarsbutalsoonthemethodusedforpreparation.

IdentificationoftheisolatedCitrusflavonoid

The structure of the isolated compound (1) was identified

asnobiletin [2-(3,4-dimethoxyphenyl)-5,6,7,8-tetramethoxy-4H

-1-benzopyran-4-one]bycomparingitsphysicalandspectraldata

withthereportedones(Dandanetal.,2007;Johannetal.,2007).

Compound1:colorlessneedles;C21H22O8;EI/MSm/z(70ev)402

[M]+_,₃₈₇_[M-CH

3]+;mp.137–138◦C;IRmax(KBrcm−1):2943.7,

2839.7, 1645.9, 1592.2, 1520.1, 1462.5, 1370.7,1277.7, 1016.2,

840.7,803.4;1_H_NMR₍₃₀₀_Hz,_DMSO)_ı_:_7.63_(1H,_dd_,_J₌_8.7_and

2.1Hz,H-6′_),_7.53_(1H,_d_,_J₌_2.1_Hz,_H-2′_),_7.14_(1H,_d_,_J₌_8.7_Hz,

H-5′_),_6.83_(1H,_s_,_H-3),_4.02,_3.97_(each_3H,_s_,_OMe),_3.88,_3.85,_3.84,

3.78(12H,overlapped,4×OMe).

Thestructureoftheisolatedcompound(2)wasidentifiedas

4′_{,5,7,8-tetramethoxyflavone}_by_comparing_its_physical_and

spec-traldatawiththereportedone(Uckooetal.,2012).Compound2:

colorlessneedles;C19H18O6;EI/MSm/z(70ev)342[M]+,327

[M-CH3]+,311[M-OCH3]+;mp.141–142◦C;1HNMR(300Hz,CDCl3)

ı:7.88(1H,d,J=8.7Hz,H-2′ _&₆′_), _7.02_(1H,_d_,_J₌_8.4_Hz,_H–3′ _&

5′_),_6.62_(1H,_s_,_H-3),_6.44_(1H,_s_,_H-8),_6.62_(1H,_s_,_H-3),_4.02,_3.99,

3.96,3.89(each3H,s,OMe).13_C_NMR₍₇₅_Hz,_CDCl

3)ı:176.37(C-4),

162.37(C-2),160.28(C-4′_),_156.86_(C-9),_156.25_(C-7),_151.69

(4)

Table2

Meanconcentraionandpercentageoftheisolatedstandardnobelitininthefour testedhexanecitruspeelsextracts.

TestedhexaneCitruspeel (2mg/ml)

Meanconcentraionof nobelitina₍_␮_g/ml)

±SE

Percentageof nobelitin(%)

Mandarin 202.91±3.63 10.14 Sweetorange 73.15±3.08 3.6 Grapefruit 18.13±0.33 0.9

Lime 0.09±0.34 0.0045

a_Mean_concentraion_of_nobelitin_is_based_on_the_average_of_three

determina-tions±standarderror.

(C-5′_),_114.49_(C-3′_),_108.52_(C-10),_106.31_(C-3),_93.44_(C-6),_61.02

(CofOMeatC8),60.66(CofOMeatC7),55.90(CofOMeatC5),

55.05(CofOMeatC4′_).

O

R

1

H

₃

CO

H

₃

CO

CH

₃

O

R

₂

OCH

₃

1 R

₁

=R

₂

=OCH

₃

2 R

₁

=R

₂

=H

HPLCresults

In thecurrent study,the chromatographicprofiles of

differ-entagro-waste citruspeelsrevealedthatmadarainhexanepeel

extact has the highest concentration of the isolated nobiletin

(202.91␮g/ml±3.63; 10.14%), nearly more than twice that in

sweet orange (73.15␮g/ml±3.08; 3.6%) and ten times as that

ingrapefruit(18.13␮g/ml±0.33;0.9%).However,limeisnearly

depletedofit(Table2;Fig.1).ThisisthefirstHPLCstudy

compar-ingthepercentageofnobiletininthesefournewEgyptiancitrus

varieties.

Theanti-diabeticandhypocholesterolemiceffectsofthedifferent

agro-wastecitruspeels

Ingeneral,allthetestedsamples(thealcoholicextracts,hexane

extracts,isolatedpectinsandtheaqueoushomogenates)ofthefour

testedagro-wastecitruspeelshadshownsignificantdecreaseinall

thetested parameters(cholesterol,triacylglycerideand glucose)

(Table3;Fig.2).

Thealcoholicextract,hexaneextractandaqueoushomogenate

of mandarinpeels show thehighest decrease (by 48.9%, 56.8%

and 59.3%) in cholesterol level (77.5±13.3, 65.5±20.6 and

61.8±17.6mg/dl,respectively)equivalenttothatofatorvastatin

referencedrug(68.7±1.09mg/dl[54.7%]).Mandarinalcoholpeel

extractwaspreviouslyreportedtoimprovebloodcholesterol

pro-fileindosedependantmanner(Adelinaetal.,2008).Thepresence

ofthemainbioactivecomponentsasflavanones(hesperetin,

narin-genin),flavoneglycosides(hesperidin,naringin)andmethoxylated

flavones(PMF)inCitrusfruits(MantheyandGuthrie,2002)could

clarifythehypocholesterolemicimpactsofbothalcoholicextracts

and aqueoushomogenates of thefour tested agro-waste citrus

peelsastheexistenceofhesperidinandnaringin,andtheir

agly-coneshesperetinandnaringenin,havebeenaccountedforlowering

plasmaand hepaticcholesteroland triacylglycerolbyinhibiting

thesehepaticenzymesinexperimentalanimals(Kimetal.,2003)

mAU

mAU 700

600

500

400

300

200

100

0

300

250

200

150

100

50

0

120

100

80

60

40

20

200

175

150

125 100 75 50

25 0

120

100

80

60

40

20

0 0

0 5 10 15 20 25 min

A

B

C

D

E

(5)

Table3

Themeanvalues(M±SE)oftheplasmacholesterol,triacylglycerideandglucoselevels(mg/dl)ofthecontrolandmodelgroupstreatedwithalcoholicextracts,n-hexane extracts,isolatedpectinandaqueoushomogenatesofthetestedCitruspeels.

Groupsa _Mean

±SEof

Cholesterol(mg/dl)b _{Triacylglyceride}_(mg/dl)b _Glucose_(mg/dl)b

Negativecontrol 64.9±0.80 72.7±2.98 83.2±1.43

Modeltreatedwithreferencedrugc _68.7_±_1.09 _153.3_±_1.95 _86.7_±_1.34

Modelofhypercholesterolemicnontreatedrats 151.9±27.1 236±36.0 274.2±42.6

Modeltreatedwithpeelalcoholicextractof

Mandarin 77.5±13.3 69.9±24.8f _149.8

±24.8f

Sweetorange 125.9±20.7 83.3±18.5f _55.9_±_17.5f

Whitegrapefruit 153.1±14.5 75.5±28.0f _86.0_±_13.8f Lime 211.6±14.1d,e _45.7_±_16.3e,f _56.4_±_15.7f

Modeltreatedwithpeeln-hexaneextractof

Mandarin 65.5±20.6 53.9±7.3e,f _153.4_±_18.7f

Sweetorange 78.6±21.3 63.8±22.9f _111.6_±_17.6f

Whitegrapefruit 109.8±22.8 63.6±16.5f _91.0_±_13.2f

Lime 73.2±7.3 83.5±7.9f _124.0

±13.6f

Modeltreatedwithpeelpectinof

Mandarin 98.6±29.8 50.3±20.0e,f _189.0_±_24.8d

Sweetorange 82.6±24.1 61.6±16.8f _151.9

±7.8f

Whitegrapefruit 98.5±4.5 116.4±12.4f _168.4

±6.5f

Lime 75.4±18.2 73.3±18.2f _202.7

±11.4d,e

Modeltreatedwithpeelaqueoushomogenateof

Mandarin 61.8±17.6 150.9±8.6f _54.5

±7.9f

Sweetorange 80.6±29.8 165.9±141d _46.9

±9.5f Whitegrapefruit 80.4±17.6 117.4±8.8f _79.2_±_27.4f

Lime 81.4±11.0 107.2±5.6f _160.5_±_14.8f

a_Number_of_male_Wistar_strain_rats_(150–160_g,_aged_three_months)_in_each_group_is_six.

b _{Concentration}_of_each_parameter_is_based_on_average_of_four_{determinations}_±_standard_error_of_mean_for_group.

c _Reference_drug_for_glucose_is_Metformin₍₂₀_mg/kg_b.wt)._Reference_drug_for_cholesterol_and_{triacylglyceride}_is_atorvastatin₍₅_mg/kg_b.wt). d _Value_is_significant_difference_when_compared_to_the_negative_control_group_at_p_<_0.05.

e_Value_is_significant_difference_when_compared_to_the_model_treated_with_reference_drug_at_p_<_0.05.

f _Value_is_significant_difference_when_compared_to_the_untreated_{hypercholesterolemia}_model_(high_fat_diet)_at_p_<_0.05.

350

300

250

200

150

100

50

0

Negative

control Negativecontrol

Negative control

Negative control Reference

drug

Reference drug

Reference drug High fat

diet

High fat diet

Mandarin Mandarin

Aqueous homogenaate Hexane extract Sweet

orange

Sweet orange

Pectin

Grapefruit Grapefruit

Alcohol extract

Lime Lime

Mean le

vel

±

S

.E.

(mg/dl)

Mean le

vel

±

S

.E.

(mg/dl)

Mean le

vel

±

S

.E.

(mg/dl)

Mean le

vel

±

S

.E.

(mg/dl)

350

300

250

200

150

100

50

0

350

300

250

200

150

100

50

0 350

300

250

200

150

100

50

0

Cholesterol Triacylglyceride Glucose

(6)

andregulatingthefattyacidandcholesterolmetabolism(Jungetal., 2006).

Mostly, all the tested samples decrease TG (by 36.0–80.6%)

(ranging from 150.9±8.68 to 45.7±16.35mg/dl) better than

theatorvastatinreferencedrugused(153.3±1.95mg/dl[35.0%]),

while,glucosewasdecreasedmostlybytheaqueoushomogenates

(except lime) (by 71.1–82.8%) (ranging from 79.2±27.4 to

46.9±9.58mg/dl) and alcoholic extracts (except mandarin) by

(68.6–79.6%)(rangingfrom86.0±13.8to55.9±17.5mg/dl)of

Cit-rus fruitspeelsbetter thanthe metforminreference drugused

(86.7±1.34mg/dl[68.3%]).

Thus the observed anti-diabetic and hypocholesterolemic

effectsofthedifferentagro-wastecitruspeelsextractscouldbe

attributedtothepresenceofnobiletin(Tsutsumietal.,2014)with

theobviousdecreaseincholesterollevelincaseofmandarindue

toitshighestpercentageofnobiletinasrevealedfromHPLC

anal-ysis(Table2).Previous studiesonCitrus peelsreportedsimilar

effects (Kurowskaand Manthey, 2004;Nagata et al.,2010; Lee

etal.,2014;Tsutsumietal.,2014)andcontributed themtothe

presenceofPMFlikenobiletinwhichstimulateslipolysisin

differ-entiatedadipocytes,attenuateddyslipidemiathroughareduction

inVLDL-triacylglyceridesecretion,preventedhepatic

triacylglyc-eride accumulation, enhanced fatty acid B-oxidation (Mulvihill

etal.,2011).

All the isolated pectin, from the four cultivars, has

sig-nificant effect on the three tested parameters, with more

pronounceddecreaseontriacylglycerideby50.7–78.7%(ranging

from116.4±12.4to50.3±20.0mg/dl)andlittledecreaseonboth

cholesterol and glucose by 30–50.3% (ranging from 98.6±29.8

to75.4±18.2mg/dl)and26–44.6%(rangingfrom202.7±11.4to

151.9±7.85mg/dl),respectively.Thiswasexplainedbythe

previ-ouslyreporteddatathattheviscosity-enhancingandgelforming

properties of pectin coulddelay gastric emptying and possibly

reducetheabsorptionrateinsmallintestine(deEscaladaPlaetal.,

2007).Besides,pectinasakindofsolubledietaryfiber,lowersblood

cholesterollevelsandLDLcholesterolfractionwithoutchanging

HDLcholesterol(Liuetal.,2006;Shahaetal.,2013).

Sincetheaqueoushomogenateisrichofbothpectinand

fla-vanoids (PMF),this could explain itswell-pronouncedeffect in

loweringcholesterollevelsviamodulatinghepaticHMG-CoA

lev-els,possiblybybindingbileacidsandincreasingtheturnoverrate

ofbloodandlivercholesterol(Marouneketal.,2007).

Conclusion

Thisisthefirstreportaboutcomparingpositiveeffectoffour

newEgyptianvarietiesofagro-wastecitruspeelsextractsandtheir

pectin onhigh-fat diet rats.The observed hypocholesterolemic

andhypotriglycermiceffectofthetestedsamplesisdirectly

pro-portionaltotheircontentofnobiletin.Otherconstituentsrather

thanPMFalsoparticipateintheobservedbiologicalactivitiesas

limeexhibitedcertainactivitiesalthoughHPLCstudyrevealedits

depletionofthesecompounds.Theaqueoushomogenatesofthese

agro-waste citruspeels canbeusedas anticholesterolemic and

anti-diabeticdrugstosavetimeandchemicalconsumptionduring

extractionoftheseagro-wasteproducts.

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare

thattheproceduresfollowedwereinaccordancewiththe

regula-tionsoftherelevantclinicalresearchethicscommitteeandwith

thoseoftheCodeofEthicsoftheWorldMedicalAssociation

(Dec-larationofHelsinki).

Confidentialityofdata. Theauthorsdeclarethatnopatientdata

appearinthisarticle.

Righttoprivacyandinformedconsent.Theauthorsdeclarethat

nopatientdataappearinthisarticle.

Authors’contribution

NMF(PhDstudent)contributedincollectingtheplantmaterial,

preparationofallextracts,runningthelaboratorywork,helpin

biologicalstudies,performingtheHPLCanalysis,statistical

analy-sisofthedataandconstructingthemanuscript.AEcontributedin

performingthebiologicalstudy.ARAcontributedinrevisingthe

manuscript.SA,NEandMYMcontributedinsuggestingthepoint

oftheresearchandsupervisingthework.Alltheauthorshaveread

thefinalmanuscriptandapproveditssubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

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