Rev. bras. farmacogn. vol.26 número4

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

Original

Article

Evaluation

of

the

effects

of

passion

fruit

peel

flour

(

Passiflora

edulis

fo.

flavicarpa

)

on

metabolic

changes

in

HIV

patients

with

lipodystrophy

syndrome

secondary

to

antiretroviral

therapy

Simone

do

Socorro

Fernandes

Marques

_,

_Rosana

_Maria

_Feio

_Libonati

_,

Armando

Ubirajara

Oliveira

Sabaa-Srur

_,

_Rensheng

_Luo

_,

_Pushkar

_Shejwalkar

_,

Kenji

Hara

_,

_Thomas

_Dobbs

_,

_Robert

_E.

_Smith

a_{CentroUniversitáriodoPará,Belém,PA,Brazil}

b_{NúcleodeMedicinaTropical,UniversidadeFederaldoPará,Belém,PA,Brazil}

c_{InstitutodeNutric¸ão,CentrodeCiênciasdaSaúde,UniversidadeFederaldoRiodeJaneiro,RiodeJaneiro,RJ,Brazil} d_{DepartmentofChemistryandBiochemistry,UniversityofMissouri,St.Louis,USA}

e_{SchoolofEngineering,TokyoUniversityofTechnology,Hachioji,Tokyo,Japan} f_{U.S.FoodandDrugAdministration,Lenexa,KS,USA}

g_{ParkUniversity,Parkville,MO,USA}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received11May2015 Accepted21March2016 Availableonline2April2016

Keywords:

Passionfruitpeelflour HIVpatients

Lipodystrophysyndrome Dyslipidemia

a

b

s

t

r

a

c

t

Thisstudyevaluatedtheeffectsofusingpassionfruitpeelflourtogetherwithdiettherapyandcounseling in36patientswithHIVlipodystrophywhowereinanambulatoryclinicinauniversityhospital.The patientsweredividedintotwogroups.Onereceived30gofpassionfruitpeelflourdailyfor90daysand diettherapycounseling.Theothergroupreceivedonlydiettherapycounseling.Themetabolicchanges wereanalyzedbeforeandaftertheintervention,withasignificancelevelpredeterminedatp≤0.05. Theuseofpassionfruitpeelflourwaseffectiveinreducingtotalcholesterolandtriacylglyceridesafter 30days.TheconcentrationsofLDL-Cdecreased,whileHDL-Cincreasedinthebloodoflipodystrophy patientsafter90dayspassionfruitpeelflourtreatment.Nosignificantdifferencesinfoodconsumption wereseenbetweengroups.Theuseof30gofpassionfruitpeelflourfor90daystogetherwithdiet therapycounselingwaseffectiveinimprovingplasmaconcentrationsoftotalcholesterol,LDL-C,HDL-C andtriacylglycerides.

PublishedbyElsevierEditoraLtda.onbehalfofSociedadeBrasileiradeFarmacognosia.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Thetreatmentofacquiredimmunodeficiencysyndrome(AIDS) withreversetranscriptaseandproteaseinhibitorsrepresenteda positive impactonthesurvival rate ofindividuals carrying the humanimmunodeficiencyvirus,orHIV(Smith,2014).However, whenHAARTorhighlyactiveantiretroviraltherapy(Smith,2015) became a routine treatment, changes in body fat distribution anddyslipidemiawereobserved(Guimarãesetal.,2007).These changesarecalledHIVLipodystrophysyndrome(Tienetal.,2006;

Guimarãesetal.,2007;Araújoetal.,2007),whichcanincreasethe

riskofcardiovasculardiseases(Friis-Mølleretal.,2003).

∗ _{Correspondingauthor.}

E-mail:robert.smith@fda.hhs.gov(R.E.Smith).

Soluble fiber may increase short-chain fatty acid synthesis (Wongetal.,2006),therebyreducingendogenouscholesterol pro-ductionandassociatedwithadietlowincholesterolandsaturated fatcanreducebloodlevelsofLDL-Candtriacylglycerides(Liuetal.,

2000;Schneeman,2002; Solàetal.,2007).TheAmericanHeart

Association(2006)recommendedtheuseof5–10g/dayof

solu-blefiberforthereductionofdyslipidemia.Afiber-richdietmay bebeneficialinpreventingthedevelopmentoffatdepositionin peoplewithHIV(Hendricksetal.,2003).

Passionfruitisthecommonnameforvariousspeciesofplants inthegenusPassiflora(Zeriaketal.,2010).Thetwospecieswith themostcommercialvalueareP.edulisfo.edulis(redpassionfruit) andP.edulisfo.flavicarpaO.Deg.(yellow),withtheyellowspecies beingthemostwidelycultivated(Zeriaketal.,2010).Ithasseveral healtheffects,includingloweringtheconcentrationsofLDL-Cand cholesterolintheblood(Ramosetal.,2007).Thepeelscanbedried

http://dx.doi.org/10.1016/j.bjp.2016.03.002

andmadeintoafoodproductcalledpassionfruitpeelflour(PFPF) thathasseveralhealtheffectsandissoldinBrazilasanadjuvant treatmentfordiabetesduetoitshypoglycemiceffect(Smithetal., 2012).Flourpreparedfromyellowpassionfruitpeelshasalsobeen showntoreducebloodglucoseindiabeticpeople.InaphaseI clin-icalstudy,passionfruitpeelflourwaswelltoleratedin36people betweenages20and60,ofbothsexes.Theyreceived10gofflour threetimesadayandweretoldtoputitintheirchoiceofjuice, soup,oranyotherfoodorbeverage.Therewasanaverage reduc-tionofbloodglucose,triacylglycerides,totalcholesterolandLDLof 5.2,15.0,18.2and19.0%,respectively.

InphaseIIstudies,flourpreparedfromyellowpassionfruitpeels reducedbloodglucose,cholesterol,LDL,bloodpressureandbody weightindiabeticpatients.

Thepeels,orrinds,ofHawaiianyellowpassionfruithavebeen fedtocattleandfoundanincreaseinmilkproduction(Otagakiand

Matsumoto,1958).Therearemanyanecdotalaccountsofit

increas-ingmilkproductionandpreventingbacterialinfection,butthese effectshavenotbeenquantified.

PFPFcontainsabout10%moisture,7.5%ash,4%protein,19% solublefiber,38%insolublefiberand21%solublecarbohydrates

(Córdovaetal.,2005;Pinheiroetal.,2008).Themajorcompound

inPFPFispectin,adietaryfiberthatisrichinpolygalacturonicacid anditsmethylester(Smithetal.,2012).However,tothebestof ourknowledge,thesolublecarbohydratesandothersoluble com-poundshaveneverbeenidentifiedoranalyzedbyeitherHPLCor NMR.

Thus,consideringthatPFPFis agoodsourceofsolublefiber, this studyevaluatedtheeffectivenessof PFPFin improvingthe bloodlipidprofileofindividualscarryingHIVandreceivingHAART who developed lipodystrophy. In addition,the hot, pressurized methanolicextract(100◦_{Cand10MPapressure)wasanalyzedby} HPLCandNMR.

Materialsandmethods

Chemicals

Chloroform(CHCl3),deuteratedchloroform(CDCl3),methanol (CH3OH) and deuteratedmethanol (CD3OD) were fromSigma– Aldrich,St.Louis,MO.

PreparationandcharacterizationofthePFPF

ThePFPFwasproducedfromsanitizedripeyellowpassionfruits (Passifloraedulisfo.flavicarpaO.Deg),thatwereidentifiedby AUOS-SandstoredattheFederalUniversityofRiodeJaneiroforfurther reference.Thepeelwasseparatedfromthepulp,dehydrated,and transformedintoflourusing aknifemill.Inorder to character-ize thePFPF,10gofit wasmixedwithenoughHydroMatrixTM (Sigma–Aldrich,St.Louis,MO)tofillthe100mlstainlesssteel sam-plecellusedinanAcceleratedSolventExtractor(ASE,ThermoFisher Scientific,Sunnyvale,CA).Then,CH3OHwasaddedwhilethe tem-perature and pressure were increased to 100◦_{C and 10.3MPa} (1500psi,100atm)overa3mintime(statictime).Next,the sol-ventwaspurgedintoacollectionvessel.Atotaloffourcycleswere runtostaticallyextractthesample,resultinginatotalvolumeof about160ml.Thesolventwasevaporatedoffandtheoilyresidues remainingwereweighed.Aportionoftheresidueremainingafter evaporatingtheCH3OHfromthemethanolicextractofeachsample wasredissolvedinCD3ODforNMRanalysis.Anotherportionwas dissolvedinmethanolforHPLCanalysis.

NMRanalysesweredoneusinganAgilentDD2600MHzNMR (SantaClara,CA).A30◦_{pulsewidthand1spulsedelaywereused} forthe1_{HNMR,whilea30}◦_{pulsewidthand2spulsedelaywere}

usedforthe1_H-coupled13_{C-NMRspectra,alsoknownas}13_C{1_H} -NMR.ChemicalshiftswerereferencedtotheCD3ODsignalsat3.35 and4.78ppm(for1_{H)and49.30ppm(for}13_{C)forthespectraofthe} methanolicextractsandtotheCDCl3signalsat7.27and77.23ppm, for1_Hand13_C{1_{H}-NMR,respectively.}

TheFTIRspectrumoftheresidueremainingfromthemethanolic extract,afterevaporatingoffthemethanolwasacquiredusingATR attachmentonShimadzuIRAffinity-1S.TheUV–Visspectrumofa 5␮g/mlsolutionoftheextractinmethanolwasobtainedusing ShimadzuUV-2600.Plainmethanolwasusedasblank.Thesource oflightwasshiftedtodeuteriumbulbat300nmandthespectrum windowwasopenedbetween900nmand185nm.

Experimentaldesign

A clinical therapeutic randomized trial was performed with individuals carrying HIV, presenting Lipodystrophy Syn-drome,receivingHAART,andshowingdyslipidemia (hypercholes-terolemia,hypertriglyceridemia,orboth)asdefinedbytheNational

Cholesterol Education Program (2002). These individuals were

monitoredat theLipodystrophyAmbulatory Centerin theJoão deBarrosBarretoUniversityHospital(HUJBB),duringtheperiod of JanuarytoDecember 2009,and fulfilled thecriteria for par-ticipationin the study.Thesample wascalculated through the calculationofProportion:twosamples.Forthefirstsample(from thegroupusingPFPF),theestimatedproportionofplasma choles-terolimprovementwas50%;forthesecondsample,thisestimate wasof10%.Theratiobetweenthesesampleswasstipulatedas1:1. Thepowerofthetestwasfixedas0.8,andtheunilateralalphalevel in0.05.Thesamplewascalculatedfromsixteenindividualsineach group.

The36individualsparticipatinginthestudywere systemati-callydividedintotwogroups.Group1(n=18)receiveddiettherapy counselingand30g/dayofPFPF,consumedasadilutesolutionin water,juices,and/orfruitsmoothies.Group2(n=18)receiveddiet therapycounselingonly.Therewere12menand6womenineach group.

Informationaboutpathology,weight,height,andfoodintake wasassessedintheinitialevaluationbythe24hrecordingmethod (R24)atthetimeoftheinitial(beginningoftheintervention)and lastassessments(endofthestudy).

Individualsfromthetwogroupswereinstructedtohave bio-chemicaltestsperformedmonthly(totalcholesterol–TC,LDL-C, HDL-C,andtriacylglycerides)attheHUJBB’slaboratory.Thesetests wereperformedinthemorning,after12hfastingandat4time pointsinthestudy:beforetheintervention(T0),at30(T30),60 (T60),and90days(T90)aftertheintervention.

Thefollowingparameterswerefollowedasinclusioncriteria inthestudy:beanadultpatient(anygender)receivingHAART, attending treatmentattheLipodystrophy ambulatory centerat theHUJBB,presentingchangesintheTCand/orLDL-Cand/or tri-acylglycerides,presentingundetectableHIVviralloadandLTCD4+ above300cells/mm3 _{andagreeingtoparticipateinthestudyby} voluntarilysigninganinformedconsentform.

Thefollowingcriteriawerefollowedforstudyexclusion: indi-viduals presenting triacylglycerides above 700mg/dl, or using lipid-loweringdrugs,whocouldnotcompletethe90daysof mon-itoring, didnot tolerate theuseof PFPF,missed appointments, displayedmentalillness,wereunderage18(children,and ado-lescents),anddidnotaccepttoparticipateinthestudy.

Thepairedt-test wasusedtoassessthevariation intheresults fromlaboratorytests.

Thestudywasconductedaccordingtotheguidelineslaiddown intheDeclarationofHelsinkiandNurembergCoderespectingthe standardsofresearchinvolvinghumansubjects(resolution196/96) oftheNationalHealthCouncil.Thisstudywasauthorizedbythe EthicsCommitteeforResearchwithHumanBeingsoftheHUJBB fromtheFederalUniversityofParáunderProtocolNo.1945/08. Writteninformedconsentwasobtainedfromallpatients.

Thisresearchreceivednospecificgrantfromanyfundingagency inthepublic,commercialornot-for-profitsectors.

Results

Drymethanol at 100◦_{C and 10MPa (100atm) pressure in a} sealedcontainersolubilized31%ofthePFPF.TheFTIRspectrumof theresidueremainingafterevaporatingoffthemethanolisshown in Fig.1. Thepeaks in theregion from1400to 1800cm−1 _are listedinTable1.TheUV–Visspectrumofa5␮g/mlsolutionofthe extractinmethanolisshowninFig.2.The1_Hand13_C{1_H}-NMR spectraoftheextractareshowninFigs.3and4.The13_Cchemical shiftsaretabulatedintheSupplementaryMaterial.Signalsdueto theCHOandCH2Oofcarbohydratesaccountedfor75.9%ofthe totalpeakarea(Fig.1).TherewerealsosignalsduetoCH3(CH2)n

groupsinfattyacylsthatarecovalentlyboundtocarbohydrates asfattyacidglycosidesinboththe1_Hand13_C{1_{H}-NMRspectra}

(Figs. 1 and 2).On the other hand,several of thesignals that

areproducedbyd-fructosewerenotseen,includingtheonesat 105.93,99.30,84.24,83.31,77.60,76.84,71.93,66.00,65.19and 64.28ppm. So, even though d-fructose is soluble in methanol,

100

98

96

94

92

90

88

86

3700 3200 2700 2200 Wavenumber (cm–1₎

% tr

ansmission (a.u.)

1700 1200 700

100

98

96

94

92

90

88

86

1800 1750 1700 1650 1600 1550 1500 1450 1400 Wavenumber (cm–1)

% tr

ansmission (a.u.)

Fig.1.FTIRspectrumofthehot,pressurizedmethanolicextract(100◦_Cand10MPa

pressure)ofpassionfruitpeelflour,afterevaporatingoffthemethanol.Thepeaks canbeusedasatypeoffingerprintthatcanbeusedtoidentifyit.

185 1

0.8

0.6

0.4

0.2

0

285 385 485 585

Wavelength (nm) 270 nm

Nor

maliz

ed absorbance units

685 785 885

Fig.2.UV–Visspectrumofthehot,pressurizedmethanolicextract(100◦

Cand 10MPapressure)ofpassionfruitpeelflour,inmethanol.Themaximumat270nm canbeusedtohelpidentifythepassionfruitpeelflour.

8 7

HC = of aromatic compounds

(CH₂)_n

CD3OD CD3OD

CHO and CH2O of

carbohydrates

CH3

6 5 4 3 2 1 ppm

Fig.3.1_{HNMRofthehot,pressurizedmethanolicextract(100}◦

Cand10MPa pres-sure)ofpassionfruitpeelflour.Thesignalscanbeusedasatypeoffingerprintthat canbeusedtoidentifyit.

ppm 20 40 60 80 100 120 140 160 180

C=O

HC = and C=C of aromatic compounds

(CH₂)_n CH₃ CD3OD

CHO and CH₂O of carbohydrates

Fig.4.13_C{1_{H}-NMRofthehot,pressurizedmethanolicextract(100}◦_Cand10MPa

Table1

PeaksintheFTIRspectrumofthehot, pres-surizedmethanolicextract(100◦_Cand10MPa

pressure)ofpassionfruitpeelflour.

cm−1 _cm−1

1796 1610

1792 1609

1785 1603

1781 1595

1723 1591

1767 1577

1762 1570

1757 1565

1751 1559

1749 1555

1744 1549

1740 1540

1734 1533

1730 1528

1723 1521

1719 1517

1716 1507

1707 1499

1700 1497

1696 1490

1684 1481

1681 1477

1676 1473

1669 1465

1663 1458

1654 1448

1647 1437

1636 1430

1628 1424

1624 1420

1617 1412

itis notpresent ata sufficientconcentrationtobedetectedby 13_C{1_{H}-NMR.Glucoseandsucrosearenotsolubleinmethanol,}

butarenotknowntobeinPFPF,whichdoesnothaveasweettaste. Still,there isa significantconcentrationof fattyacidglycosides inthemethanolicextractofPFPF.Theyproducednotonlysignals duetoCH3(CH2)n,butalsothecarbonylcarbonduetoestersat

170.397and172.635ppm.Thereisalsoatleastonefreefattyacid, asindicatedbythecarbonylcarbonat181.573ppm.Finally,there arealsosignalsduetoHC CRcarbonsandprotonsduetoaromatic compoundsinthe1_Hand13_C{1_{H}-NMRspectra.}

AsshownintheSupplementaryMaterial,analysisbyHPLC sup-portsthehypothesisthattherearearomaticcompounds.

Outofthe41individualsselectedtoparticipateinthestudy, three wereeliminated fromGroup 1 (tworeported unpleasant tastewhenusingPFPF,andoneinitiatedtheuseoflipid-lowering drugsduringthestudy).InGroup2,twopatientswereeliminated (one initiated the useof lipid-lowering drugs, and one missed subsequent appointments).Thirty-six patientsremained in the studyandwereevaluated.Table2describesthecharacteristicsof thepatients.Thestatisticalanalysisdemonstratednodifference betweenthegroupsaccordingtotheevaluatedvariables.The aver-agevaluesforTC,LDL-C,HDL-C,andtriacylglyceridesarepresented

inTables3and4forpatientsinGroup1and2,respectively.

TheincreaseinHDL-C,inthegroupusingPFPFduring90days averaged5.9±10.7mg/dl,whichcorrespondedto13.4%ofthe ini-tialvalue.On theotherhandinGroup2,theincreaseaveraged 1±10.4mg/dlandcorrespondedto2.3%oftheinitialvalue.

Theaveragevalue(Table3)forbasaltotalcholesteroldetected inGroup1(227.3±42.9mg/dl)isconsideredaborderlinevalue accordingtotheNCEPIII(2003),however,after90daysof inter-vention with PFPF, this value decreased to 192.1±45.2mg/dl, whichisclassifiedasoptimal.Thisreductionwashighlysignificant (p=0.001)andalready noticedasasignificantdecrease30days

Table2

EffectsofPFPFontheplasmaconcentrationsofcholesterol,LDL-C,HDL-CandTGin HIVpatientswithlipodystrophysyndromesecondary.

Variable Group1 Group2 pvalue

Average SD Average SD

Age(years) 46.0 8.4 46.6 7.0 0.83

Timesincediagnosis(years) 9.1 4.9 11.0 5.4 0.28 DurationofHAART(years) 8.2 4.5 10.0 5.0 0.25

Weight(kg) 59.7 10.1 57.5 10.4 0.52

BMI(kg/m2_{) 23.3 2.8 22.1 3.5 0.28}

Therewere12menand6womenineachgroup.Thedataobtainedwere tabu-latedandanalyzedusingtheBiostat5.0program(2007)withasignificancelevelof

p≤0.05.Thedietproprogram(EstevesandMonteiro,2002)wasusedforthe eval-uationofR24h.Thebilateralt-testwasusedfortheverificationofhomogeneityin theobservedvaluesbetweenthegroups.Thepairedt-testwasusedtoassessthe variationintheresultsfromlaboratorytests.

aftertheintervention(p=0.007).TheLDLconcentrationsin indi-vidualswhousedPFPFduring30and60daysshoweddiscreteand non-significantreduction.AsignificantLDL-Creductionwasonly observedafter90daysofintervention(p=0.0082).

AdiscreteandnotsignificantelevationintheHDL-C concentra-tionswereobservedinindividualswhousedPFPFduring30days; however,asignificantincreasewasobservedafter90daysofits use(p=0.0294).TherewerenosignificantchangesintheHDL-C concentrationsinGroup2throughouttheinterventionperiod.

Theplasmatriacylglyceridesconcentrationsdecreased signifi-cantly(p=0.034)after30daysofPFPFconsumptiontogetherwith diettherapycounseling.However,therewasnofurthersignificant reductionafterwards,showingthatPFPFassistedinreducingthe levelsoftriacylglyceridesinjustthefirst30daysandmaintaining thislowerlevelthroughoutthestudy.

Table3

Averagesandstandarddeviationsanddifferencesbetweentheaveragevaluesof theevaluatedparametersduringtheinterventioninGroup1(whichreceiveddiet therapycounselingand30g/dayofPFPF).Comparisonbetweenthebasalvaluesand valuesobservedinotherperiodsthroughoutthestudy.

Evaluation Differencebetweenaverages(SD)

Average SD T0/T30 T0/T60 T0/T90

TC

T0 227.3 42.9 26.8(40.8) 34.5(27.4) 35.2(39.5) T30 200.6 51.2 p=0.007a _p<0.0001a _p=0.001a

T60 192.8 45.5 T90 192.1 45.2

LDL-C

T0 125.8 33.1 13.2(36.4) 14.5(32.6) 32.3(34.72) T30 112.6 47.7 p=0.14 p=0.1 p=0.0082a

T60 111.3 42.6 T90 93.5 45.8

HDL-C

T0 43.9 12.2 −1.1(9.2) 0.6(9.7) −5.9(10.7) T30 44.8 16.9 p=0.33 p=0.4 p=0.0294a

T60 43.1 13.9 T90 49.7 19.1

TG

T0 323.9 114.4 68.6(140.3) 97.7(149.1) 96.47(164.6) T30 255.2 115.2 p=0.034a _p=0.0078a _p=0.0140a

T60 226.1 118.4 T90 227.4 139.3

TC,totalcholesterol;TG,triacylglycerides.

a_{p-value<0.05.Thedataobtainedweretabulatedandanalyzedusingthe}

Bio-stat5.0program(2007)withasignificancelevelofp≤0.05.Thedietproprogram (EstevesandMonteiro,2002)wasusedfortheevaluationofR24h.Thebilateral

Table4

Averagesandstandarddeviationanddifferencesbetweentheaveragevaluesof theevaluatedparametersduringtheinterventioninGroup2(whichreceiveddiet therapycounselingonly).Comparisonbetweenthebasalvaluesandvaluesobserved inotherperiodsthroughoutthestudy.

Evaluation Differencebetweenaverages(SD)

Avg SD T0/T30 T0/T60 T0/T90

TC

T0 234 59.7 8.8(36.2) 7.0(26.3) 4.83(35.4) T30 226 56.7 p=0.16 p=0.14 p=0.28 T60 228 55.3

T90 230 50.1

LDL-C

T0 143 52.6 16.9(49.5) 18.5(51.8) 12.7(34.0) T30 126 51.6 p=0.11 p=0.10 p=0.09 T60 125 45.8

T90 131 42.5

HDL-C

T0 43.6 16.5 −0.6(10.6) 2.77(7.8) −1(10.4) T30 44.2 17.3 p=0.40 p=0.09 p=0.35 T60 40.9 14.9

T90 44.6 17.7

TG

T0 294 120.4 −16.2(131.4) 5.33(72.3) −7.94(118.9) T30 310 137.4 p=0.30 p=0.38 p=0.39 T60 288 115.9

T90 302 153.6

TC,totalcholesterol;TG,triacylglycerides.

ThedataobtainedweretabulatedandanalyzedusingtheBiostat5.0program(2007) withasignificancelevelofp≤0.05.Thedietproprogram(EstevesandMonteiro, 2002)wasusedfortheevaluationofR24h.Thebilateralt-testwasusedforthe verificationofhomogeneityintheobservedvaluesbetweenthegroups.Thepaired

t-testwasusedtoassessthevariationintheresultsfromlaboratorytests.

Table5showstheevaluationofeffectsfromtheconsumption

ofmacronutrientsanddietaryfibers,constituentsofthedietused inthestudy,beforeandafterintervention.Therewasnodifference intheconstituentsofthedietbetweenthegroups,thisconfirming theeffectsofthePFPFintheobtainedresults.

Table5

Averageandstandarddeviationoftheconsumptionlevelsofthemainfood compo-nents,beforeandafterintervention,inGroups1and2.

Nutrients Group1 Group2 p-value

Average SD Average SD

Calories(kcal)

Before 2190 688 2265 830 0.77

After 1883 499 1804 670 0.69

Saturatedfat(g)

Before 16.9 7.9 16.0 6.7 0.70

After 13.3 7.0 14.5 13.7 0.74

Monounsaturatedfat(g)

Before 16.5 7.4 18.5 9.2 0.47

After 12.4 8.0 14.3 10.3 0.56

Polyunsaturatedfat(g)

Before 11.8 9.1 16.7 15.5 0.25

After 5.9 4.7 5.7 4.4 0.87

Cholesterol(mg)

Before 354.6 304 383 327 0.79

After 184.7 120 221 191 0.50

Fibers(g)

Before 32.7 55.5 29.6 20.7 0.48

After 36.6 56.4 32.7 7.1 0.62

ThedataobtainedweretabulatedandanalyzedusingtheBiostat5.0program(2007) withasignificancelevelofp≤0.05.Thedietproprogram(EstevesandMonteiro, 2002)wasusedfortheevaluationofR24h.Thebilateralt-testwasusedforthe verificationofhomogeneityintheobservedvaluesbetweenthegroups.Thepaired

t-testwasusedtoassessthevariationintheresultsfromlaboratorytests.

Discussion

To the best of our knowledge, this is the first report that describestheFTIR,UV–VisandNMRanalysisofPFPF.Hot, pres-surized,dry methanolwaschosenasthesolventtoextractthe PFPF,becausepreviousstudieshaveshownthatitisbetterthan ultrasound,Soxhletextractionor othermethods forsolubilizing solids in fruits(Richter et al., 1996; Richards et al.,2014).Dry methanolat100◦_{Cand10MPa(100atm)pressureinan} acceler-atedsolventextractorwasabletosolubilized-fructose,fattyacid glycosidesandothercompoundsfromavarietyoffruits(Richards etal.,2014).d-fructoseproducedmorethansixsignals,becauseit existsasamixtureofthreeisomersinmethanol:␤-fructopyranose, ␣-fructofuranoseand␤-fructofuranose(Richardsetal.,2014).Fatty acidglycosideshavebeenshowntohaveimportant neuromodula-toryeffects(Akihisaetal.,2007;Lietal.,2008;Smith,2015).

TheFTIRspectrumhadalarge,broadpeakfromabout3000to 3500cm−1_{.Thisindicatesthepresenceofhydroxylgroups(Smith,}

2014).Therewere alsopeaks from1650to 1780,and 1600to 1650cm−1_{duetoC OandC Obonds,respectively(Smith,2014).} TheUV spectrum hadmaximum absorbance(max)at 205and 270nm.Themaxat270nmindicatesthepresenceofoneormore aromaticcompounds.The1_Hand13_C{1_{H}-NMRspectracontained} signalsfromabout3.0to5.4ppmand64to110ppm,respectively, duetoCHOandCH2Ogroupsincarbohydrates.Fructoseistheonly simplesugarthatdissolvesinmethanol.However,PFPFdoesnot tastesweetandtherewereseveralsignalsthatfructoseisknown toproducein13_{C-NMRspectra(Richardsetal.,2014)thatwere} not seen inthe spectrum. Also, there weresignals from 0.6to 1.0ppmand14.5to34ppminthe1_Hand13_C{1_{H}-NMRspectra} duetotheCH3and(CH2)noffattyacylgroups.Thissupportsthe

hypothesisthatPFPFcontainsfattyacidglycosides.Therewere sig-nalsfromabout6.8to8.2ppmand128to136ppminthe1_Hand 13_C{1_{H}-NMRspectraduetoC CandHC Cbonds.Thisis}

consis-tentwiththeUVspectrumthatshowedthepresenceofaromatic compounds.Finally,thereweresignalsinthe13_C{1_H}-NMR spec-trumfromabout171to182ppmduetoC Obondsinestersand carboxylicacids.TheFTIRspectrumisagoodfingerprintofthePFPF, whiletheUVandNMRspectrawerealsoquiteinformative.

Aclinicaltrialwasconductedinwomenpresenting

dyslipide-mia(Ramosetal.,2007)usingthesame30g/daydoseofPFPFthat

wasusedinthepresentstudy.Similarreductionsinthe concen-trationsofcholesterolinthebloodwereseen(Ramosetal.,2007). Areductionof31.7±28.0mg/dlinthisvaluewasobservedafter 30daysofinterventionand47.0±29.5mg/dlafter60days(Ramos etal.,2007).Bothwerestatisticallysignificant,withp=0.0001and p=0.0000,respectively(Ramosetal.,2007).

Inanotheropenclinicaltrialwasconductedon36healthyadults ofbothgenders,whowereinstructedtoeat10gofPFPFthreetimes aday,dailyforduringeightweeks(Medeirosetal.,2009).A sig-nificantreductionintotalcholesterolconcentrationsintheblood (17.0%forwomenand19.5%formen)wasobserved(Medeirosetal.,

2009).

Elevatedtotalcholesterolisariskfactorfordeveloping cardio-vascular disease(Smith, 2015).Thishasbecomemore frequent amongHIV+ individualswho receiveHAART drugtherapy. Car-diovasculardiseasesaretheleadingcauseofdeathintheUS,but theirincidencecanbeloweredbytakingdrugslikestatinsthat reducebloodcholesterollevels(Smith,2015),andbyconsuming moredietaryfiber(Brownetal.,1999).TheNationalCholesterol EducationProgramintheUSA(2002)estimatedthatforevery1% reductionincholesterolconcentrationintheblood,adecreaseof 2%intheriskofcardiovasculardiseasescouldbeobserved.

compositionofhealthybacteriainthegut,withconcomitanthealth effects.Finally,thefattyacidglycosidesmaybeabletoboostthe immunesystem,reducesmolderinginflammation,whichcanhelp preventcardiovascularandneurodegenerativediseasesaswellas cancer(Smith,2015).

ThereductionofLDL-Cobservedinthisstudyissimilartothe resultsobservedbyothers(Ramosetal.,2007)whosawadecrease of33.6±33.3mg/dlintheLDL-C(p=0.0003)whenintroducing6g ofsolublefiberintheusualdietof52women. Onestudy eval-uatedtheintroductionof14goffiberper dayin womenliving inacommunityinFortCollinsandtherewasan8%reductionin theamountsofLDL-Cafter90daysoffollow-up,lowerthanthe presentstudythatthedecreasewasonaverage32.3±34.7mg/dl ingroup1whichequals25.6%(Davyetal.,2002).Ameta-analysis wasperformedontheeffectsoffiberintakeoncholesterollevels andconcluded,thattheincreaseinsolublefiberinthediet,produce significantresultsontheLDL-Cvalues(Brownetal.,1999).

LDL-Cistheprimarylipoproteinintheplasma,actsasa choles-terol transporter and seems to be the most atherogenic of all

(GoldsteinandBrown,1977).Thedepositionofcholesterolisa

con-sequenceofoxidationofcirculatingcholesterol-richlipoproteins, inparticular LDL-C(Steinber,1997;Cobbold etal.,2002).Thus, theNCEPhasidentifiedthislipoproteinasthemaintargetinthe treatmentofdyslipidemia(NCEP,2002).TheincreaseinLDL-C lev-elspromotesatherogenesishowever,therestorationoftheideal serumlevelsreducestheriskofatheroscleroticcardiovascular dis-easesignificantlyeveninpatientswhoalreadypresentadvanced pathology(Grundyetal.,2004;Tabasetal.,2007).Thereduction of39mg/dlinthislipoproteinlevelreducesin30%therelativerisk fordevelopingcoronaryarterydisease(Grundyetal.,2004).

It wasfoundthatadults of bothsexes who received30gof PFPFdailyforeightweekshadsignificantlyloweraverageHDL-C concentrationsattheendofthestudycomparedtothebeginning

(Janebroetal.,2008).Thispatternwasalsoobservedinthepresent

study,inbothfourthandeighthweek.Thevaluesreachedthe nor-malrangeforbothwomenandmeninthislatestevaluation,andthe respectiveaverageswere53.50±10.8mg/dland43.2±8.13mg/dl

(Janebroetal.,2008).

TheresultsfromtheVeteransAffairsHigh-Density Lipoprotein Intervention Trial reinforced the idea that elevated HDL-C can reducetherisk ofcoronarydisease(Rubinsetal.,2000).In this study,the22%reductionindeathsfromcoronaryeventsor non-fatal myocardial infarctions was correlated to thereduction in cholesterolandtriacylglycerideslevels,by4%and31%,respectively, andintheincreaseofHDL-Cby6%,withoutsignificantchanges in LDL-C(Rubinset al., 2000).It shouldbe emphasizedthat in thepresent study,theHDL-CinthegroupusingPFPFincreased from 43.9±12.2mg/dl at dayzero to 49.7±19.1 after 90 days

(Table3).OntheotherhandHDL-Cchangedfrom43.6±16.5to

44.6±17.7mg/dlafter30days.

DespitethefibersnotinterferingdirectlyintheHDL-Clevels, thislipoproteinvariesinareversedwaywiththetriacylglycerides (Vegaetal.,2014).Thereductioninlipolysisofthelipoproteins richintriacylglycerideslowersthelevelsofavailablesubstratefor thematurationofHDL-C(deManetal.,2003).Inaddition,the enrichmentofHDL-Cwithtriacylglyceridesincreasesthecatabolic rateandtherefore,reducesitsplasmaconcentration.Also,thelipid exchangebetweenHDL-Candthelipoproteinsrichin triacylglyc-eridesarereducedleadingtoafasterdisappearanceoftheplasma HDL-C(Genestetal.,2006).Foreach1%increaseinHDL-Cthere wasa1%reductionincoronaryeventsregardlessofthevariation ofLDL-C,becauseHDL-Cpromotestheesterficationofcholesterol andprotectsthearteriesduetothereversetransportof choles-terol.Thispromotesthewithdrawalofcholesterolfromthetissues peripheraltotheliverwhereitisexcretedinthebile(Abbottetal.,

1988;Barter,2004;Daminellietal.,2008).

In agreement with the present study, 43 volunteers who received30g/dayPFPFexperiencedasignificantreductionin tria-cylglyceridesafter8weeksusingthisflour.Theaveragevaluesfell from212.0±119.3to161.2±91.1mg/dl(Janebroetal.,2008).

Hypertriglyceridemiaisanindependentriskfactorfor athero-genesis,stroke,andacutemyocardialinfarction.Itwasobservedin ameta-analysiswithseventeenprospectiveepidemiological stud-iesthateveryincreaseof89mg/dlinthetriglyceridemialedtoan increasein therisk ofcardiovasculardiseaseof 32%and76%in menandwomen,respectively(Abbottetal.,1988).Thehigherthe triglyceridemia,thegreatertheformationofLDL-CtypeB,which aresmalleranddenser.Thisisimportantbecausesmallanddense LDL-Cparticlesmaybemoresusceptibletooxidativemodification. Also,anincreasednumberofatherogenicparticlesmayadversely influencecardiovascularrisk(Passarelietal.,2007;Milleretal.,

2011).

Conclusion

Itwasconcludedthattheuseof30gofpassionfruitpeelflour during30daystogetherwithdiettherapycounselingwaseffective inreducingcholesterolandtriacylglycerideslevels.PFPFtreatment showedgoodeffectsagainstlipodystrophyinHIVpatientstreated for90days.Theuseofthisproductfor90dayswaseffectivein reducingtheLDL-C,andincreasingtheHDL-Cvaluesinindividuals presentingHIV Lipodystrophysyndromeanddyslipidemia.PFPF wasfoundtocontainmethanol-solublefattyacidglycosidesand aromaticcompounds whenanalyzed byNMR. FTIRandUV–Vis spectraarealsousefulinestablishinga“fingerprint”ofPFPF.

Ethicaldisclosures

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

Confidentialityofdata. Theauthorsdeclarethattheyhave fol-lowed theprotocolsof theirwork centeronthe publicationof patientdata

Righttoprivacyandinformedconsent.Theauthorsdeclarethat nopatientdataappearinthisarticle.

Authors’contributions

SSFM,RMFLandAUOSSdesignedtheexperimentsandcarried themout.SSFMwrotethefirstdraft.RMFLisheradvisor.She con-ceivedtheproject,anddidthestatisticalanalyses.RESaddedmore backgroundinformation,rewrotethepaperandsubmitteditand revisedit basedon reviewers’comments. TDdidthemethanol extractions.PSandSHacquiredtheFTIRandUV–Visspectraand analyzedthem.RLdidtheNMRanalyses.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

AppendixA. Supplementarydata

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