Effect of reverse osmosis concentration coupled with drying processes on polyphenols and antioxidant activity obtained from Tectona grandis leaf aqueous extracts

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Effect of reverse osmosis concentration coupled with

drying processes on polyphenols and antioxidant activity

obtained from Tectona grandis leaf aqueous extracts

Emmanuel N. Koﬀi, Emmanuelle Meudec, Félix A. Adjé, Paul R. Lozano,

Yves F. Lozano, Yves-Alain Bekro

To cite this version:

Emmanuel N. Koﬀi, Emmanuelle Meudec, Félix A. Adjé, Paul R. Lozano, Yves F. Lozano, et al.. Effect

of reverse osmosis concentration coupled with drying processes on polyphenols and antioxidant activity

obtained from Tectona grandis leaf aqueous extracts. Journal of Applied Research on Medicinal and

Aromatic Plants, Elsevier, 2015, 2 (2), pp.54-59. �10.1016/j.jarmap.2015.03.001�. �hal-01982717�

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ContentslistsavailableatScienceDirect

Journal

of

Applied

Research

on

Medicinal

and

Aromatic

Plants

jo u r n al h om ep age :w w w . e l s e v i e r . c o m / l o c a t e / j a r m a p

Effect

of

reverse

osmosis

concentration

coupled

with

drying

processes

on

polyphenols

and

antioxidant

activity

obtained

from

Tectona

grandis

leaf

aqueous

extracts

Emmanuel

N. Kofﬁ

a,∗

,

Emmanuelle

Meudec

b

_,

_Félix

_A.

_Adjé

c

_,

_Paul

_R.

_Lozano

d

_,

Yves

F. Lozano

d

_,

_Yves-Alain

_Bekro

a

a_Université_Nangui_Abrogoua,_Laboratoire_de_Chimie_Bio_Organique_et_de_Substances_Naturelles_(LCBOSN),₀₂_BP₈₀₁_Abidjan_02,_Cote

d’Ivoire

b_INRA,_UMR_SPO_(Sciences_pour_{l’œnologie),}_Plateforme_Polyphenols,₃₄₀₆₀_Montpellier_cedex_1,_France

c_INP-HB,_Laboratoire_de_procédés_Industriels,_de_Synthèse,_de_{l’Environnement}_et_des_Energies_Nouvelles_(LAPISEN),_{Yamoussoukro,}_Cote

d’Ivoire

d_CIRAD,_UMR-110_INTREPID_{(INTensiﬁcation}_Raisonnée_et_Ecologique_pour_une_Pisculture_Durable),_TA_B_110-16,₇₃_rue_J._F._Breton,

34098Montpelliercedex5,France

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received19October2014 Accepted2March2015 Availableonline25April2015 Keywords:

Tectonagrandisleafextracts Reverseosmosisconcentration Freeze-drying

Spray-drying Polyphenols Antioxidantcapacity

a

b

s

t

r

a

c

t

Tectona grandis leaf extracts obtained at pilot scale processes (ultrasound-assisted extraction,cross-flowmicrofiltration,reverseosmosisconcentration)containsphenolic compoundsthatexhibit antioxidantproperties.Thefinalreverseosmosis concentrate extractpresentedhighercontentofpolyphenol(21,080±117␮molg−1GAE)and antioxi-dantcapacity(8490±29␮molg−1_TE)_{comparatively}_to_crude_extract₍₁₃₀₀_±₁₂_␮mol_g−1 GAEforpolyphenoland430±2␮molg−1_TE_for_antioxidant_activity)_or_cross_flow micro-filtrationextract(1170±10␮molg−1GAEforpolyphenoland400±10␮molg−1TEfor antioxidant).Theconcentrationfactorsofpolyphenolandantioxidantcapacitywere18 and21,respectively.High-performanceliquidchromatography(HPLC)coupledto electro-sprayionizationmassspectrometry(ESI-MS)detectionnegativeionmodehasbeenused toidentifyandcharacterizedpolyphenolsintheconcentrateextractofT.grandisleaves. Sevenphenolicacidsandflavonoidswerecharacterized.Verbascoside(phenolicacid)was describedasthemostabundantphenoliccompoundsinthisconcentrateextract.Two dry-ingtechnologies(freeze-dryingandspray-drying)wereusedtoobtainedstablepowder fromconcentrateextract.Theeffectofthesedryingtechnologiesonphenoliccompounds andantioxidantactivitywerestudied.Freeze-dryingpresentedagoodrecoveryofphenolic compoundsandantioxidantcapacity.Thisdryingtechnologycouldbeusedforpreservation ofT.grandisextract.

1. Introduction

Tectona grandis L. (Verbenaceae), commonly named

teak is used in folk medicine for a wide variety of

∗ Correspondingauthor.Tel.:+22507077180. E-mailaddress:emmanuelkofﬁ@ymail.com(E.N.Kofﬁ).

remedies(Aradhanaetal.,2010;TraBietal.,2008).

Sev-eralpharmacologicalactivitieshavebeenattributedtoT.

grandisextracts,mainlyantidiabeticactivity(Ghaisasetal.,

2010;PoojaandSamanta,2011)andantioxidantactivity

(NairaandKarvekar,2011;Raoetal.,2011).Previous

stud-iesrevealedthepresenceofphenoliccompoundsonthe

extractsofT. grandisleavessuchﬂavonoidsand

pheno-licacids(NairaandKarvekar,2010;Shuklaetal.,2010).

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Polyphenols are ofa great interest due totheir

beneﬁ-cialeffectforhumanhealth:preventionandtreatmentof

certaincancers,inﬂammatorydiseases,cardiovascularand

neurodegenerativediseases(PandeyandRizvi,2009).They

haveoftenbeenidentiﬁedasactiveprinciplesofnumerous

folkherbalmedicines(Apaketal.,2007).

Localpopulationsof Côted’Ivoire hasbeenextracted

thesebioactivecompoundsfromplantmaterialsby

decoc-tionorinfusionwithwaterassolvent.Manystudieshave

showedthatpolyphenolcontentsinaqueousextractswere

unstableduringstorage(Chedeaetal.,2011;Malickand

Bradford,2008).Inthisstudy,apilotplantscaleprocessing

ofdriedplantleaveswassettoproducestabilizedextracts

in apowder formtoincrease theshelf-lifeof theready

to use medicinal product. The process includes several

steps such as ultrasound-assisted water-maceration of

dried leaves, membrane ﬁltration and concentration of

theextractandstabilizationoftheconcentrateextractby

spray-dryingorfreeze-drying. Theeffectofstabilization

processesonpolyphenolcontentsandantioxidantcapacity

wasstudied.Polyphenolcontentswerealsoidentiﬁedand

characterizedbyHPLCcoupledtoUV–visdiodearray

detec-tionandmassspectrometrywithelectrosprayionization

(LC/DAD/ESI-MS2_).

2. Materialandmethods

2.1. Plantmaterial

LeavesofT. grandiswerecollectedfromteak

planta-tionsinthecentreofCôted’IvoirearoundYamoussoukro

area.Afterharvesting,theleaveswerebroughttoLAPISEN

laboratory(Yamoussoukro,Côted’Ivoire)fordryingatan

averagetemperatureof30◦Cduringdaytime,andkept

awayfromdirectsunexposureunderanopen-sidedshed.

Thedriedleaveswerepackedinplasticbagsandshipped

toCIRADlaboratory(Montpellier,France),wheretheywere

storedat4◦Cuntilprocessedandanalyzed.

2.2. Chemicals

Allreagentswereofanalyticalgrade.Sodiumhydroxide

(NaOH),sodiumcarbonatesalt (Na2CO3),monohydrated

citricacid,dihydratedmonosodiumphosphate(NaH2PO4,

2H2O),disodiumhydrogenphosphate(Na2HPO4),

Folin-Ciocalteu’sphenolreagentwerepurchasedfromCarloErba

(Spain).

Gallic acid, quercetin, trolox

(6-hydroxy-2,5,7,8-tetramethylchroman-1-carboxylic acid), ﬂuorescein,

AAPH (2,2-azobis (2-methylpropanimidamide)

dihy-drochloride),luteolin,caffeicandchlorogenicacidswere

purchasedfromSigma-Aldrich(Germany).

2.3. Pilotplantextractionandextractconcentration

Ultrasound-assistedextractionofdriedleaves(1.65kg)

was performed with100L of acidiﬁed tap water (H2O,

0.01Ncitricacid)during40minusingultrasonic(US)pilot

plantunitequippedwithanchor-shapeandslow-motion

stirrer (40kHz US frequency, 200–500W US variable

power,REUS,Contes,France).Thewaterextractobtained

wasﬁltered using a nyloncloth to givea crude extrat

(CE),whichwasthenclariﬁedbyCross-ﬂow

microﬁltra-tion(CFM)togiveaCFMpermeatevolume(VCFM)ofabout

92L,whichwasthenconcentratebyreverseosmosis(RO)

ataconstanttrans-membranepressureof40bar.Theﬁnal

volumeoftheROconcentrateextractobtained(VRO)was

generally3L,whichwasclosetothatofthedeadvolumeof

theROpilotplantunit.TheperformanceofthisRO

concen-trationstepwascharacterizedbycalculatingconcentration

factor:

CF₌ TSPRO

TSPCFM

where CF, concentration factor; TSPRO, total soluble

polyphenolcontentintheROconcentrateandTSCFM,total

solublepolyphenolcontentintheTSPCFMpermeates.

2.4. Freezedryingprocess

Reverseosmosis(RO)concentrateextractwerefrozen

at−30◦_C_in_cold_room_and_dried_using_freeze_dryer_(type

cryonext,France)for48h.Sampletemperaturewassetat

−80◦_C_and_the_pressure_was_set_less_than_0.2_bar.

2.5. Spraydryingprocess

ROconcentrateextractsweredriedusingspraydryer

(MinisprayDryer,B-290MinisprayDryer)with120◦Cofan

inletairtemperatureand60◦Cofanoutletairtemperature.

2.6. Totalpolyphenolcontent

Totalpolyphenolcontentwasdeterminedby

colorime-try,usingtheFolin-Ciocalteu(F-C)method(Singletonand

Rossi,1965;Woodetal.,2002).To30␮Lsampleextract,

2.5mLofdilutedFolin-Ciocalteu’sphenolreagent(1/10)

wereadded.After2minofincubationinthedarkatroom

temperature,2mLofaqueoussodiumcarbonate(75gL−1)

wereadded.Afterslightstirring,themixturewasputin

awater bathat 50◦C for15minthencooleddown.The

absorbancewasmeasuredat=760nmusinga UV–vis

spectrophotometer (Jenway 6705, Barloworld Scientiﬁc

SAS,France).Totalpolyphenolcontentwasexpressedas

␮molGAE(GallicAcidEquivalent)pergramofdriedleaves

water-extracted.Sampleswereanalyzedintriplicate.

2.7. Antioxidantcapacity

Antioxidant capacity was carried by oxygen radical

absorbancecapacity(ORAC)assay.TheORACmethodused

wasdescribedbyOuetal.(2001).TheautomatedORAC

assaywascarriedoutonaVICTORTM_X3_Multilabel_Plate

Reader(Perkin–Elmer,USA)withﬂuorescenceﬁltersforan

excitationwavelengthat485nmandanemission

wave-lengthat535nm(Zuluetaetal.,2009).Thereactionwas

performedat37◦Casthereactionwasstartedbythermal

decompositionofAAPHin75mmolL−1 phosphatebuffer

(pH7.4).Astocksolutionofﬂuorescein(FL)wasprepared

byweighing22mgofFL,dissolvingitin100mLof

phos-phatebuffer(PBS)(75mmolL−1,pH7.4),andthenstoring

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workingsolution(78nmolL−1)wasprepareddailyby

dilu-tionof0.334mLofthestocksolutionin25mLofphosphate

buffer. The AAPH radical (221mmolL−1) was prepared

dailybytaking0.6gofAAPHandmakingitupto10mL

withPBS.100␮LofFLand100␮Lofdilutedsample,PBSor

standard(Trolox5–50␮molL−1)wereplacedineachwell

ofa96well-plateandpre-incubatedduring15min.After,

50␮LofAAPHwereaddedintothewells.Theﬂuorescence

wasmeasuredeveryminuteduring60minwithemission

andexcitationwavelengthof485and535nm,respectively,

whichwasmaintainedat37◦C.TheORACvalueswere

cal-culatedasareaunderthecurve(AUC)andwereexpressed

as␮molTE(TroloxEquivalent)pergramofdriedleaves

water-extracted.Sampleswereanalyzedintriplicate.

2.8. HPLC–ESI-SManalyses

Aniontrapmassspectrometer(BrukerDaltonics

Ama-zon,Bremen,Germany)wasconnectedviaanelectrospray

ionization (ESI) interface for high performance liquid

chromatography–tandemmassspectrometry(HPLC-SM2₎

to UPLC-DAD (Waters Acquity, Milford, MA) equipped

withaRP18column(AcquityBEHcolumn,10mm×1mm,

1.7␮mparticlesize,Waters,Milford,MA)placedina

con-trolledtemperatureovensetat35◦C.Theinjectionvolume

was0.5␮L.Themobilephasewasabinarysolventsystem

ofA(water:formicacid,99:1,v/v)andB(methanol:formic

acid,99:1,v/v).Themulti-lineargradientproﬁlewas:2%B

fromstartto1min,2–30%B,from1to10min,30%Bfrom10

to12min,30–75%Bfrom12to25min,75–90%Bfrom25to

30min,and90%Bfrom30to35min.Theelutionﬂowrate

wassetat0.08mLmin−1.Themassspectrometeroperated

innegativeionmode(capillaryvoltage:2.5kV;endplate

offset:−500V;temperature:200◦C;nebulizergas:10psi

anddrygas:5Lmin−1;collisionenergyforfragmentation

inMS/MSsetat1).Polyphenolsweredetectedat280nm.

UV–visspectrawererecordedfrom210nmto600nm.The

dataanalysissoftwarewasusedfordataacquisitionand

processing.

2.9. Statisticalanalysis

Resultswereexpressedasmean±standarddeviationof

threereplicate.Datawereevaluatedbyone-wayanalysis

ofvariance(ANOVA)usingStatistica7.1(StatSoft,Inc.,USA)

solfware.Newman-keulstestwasperformedtodetermine

signiﬁcantdifferencesatp<0.05.

3. Resultsanddiscussion

3.1. Extractionandconcentrationprocess

The ultrasound-assisted extract of T. grandis leaves

obtainedinpilotscalewasclariﬁedbycrossﬂow

micro-ﬁltration(CFM)andthenconcentratedbyreverseosmosis

(RO).Table1presents totalpolyphenol andantioxidant

content from the three co-products (crude

extract-CFM extract-concentrate ROretentate). Theamounts of

polyphenols and antioxidants were higher in RO

con-centrateextract than crude extractor CFM extract.The

Table1

TotalpolyphenolcontentsandantioxidantactivityofT.grandisL.leaf extract.

Processco-products Totalpolyphenols (␮molg−1_GAE) Antioxidant capacity (␮molg−1TE) Crudeextract 1300±12a ₄₃₀_±₂a CFMpermeate 1170±10a ₄₀₀_±₁₀a ROconcentrate 21,080±117b ₈₄₉₀_±₂₉b CF 18 21

GAE,gallicacidequivalent;TE,troloxequivalent;CFM,crossﬂow mem-brane;RO,reverseosmosis;CF,concentrationfactor.Foreachcolumn, lettersequalsindicatethatthemeansdifferenceisnotsigniﬁcantat p<0.05.

concentrationfactors(CF)oftotalpolyphenoland antiox-idantcapacitywere18and21;respectively.Theamount of polyphenols and antioxidant capacity obtained in CFM extract was lower than those obtained in crude extract. Statisticalanalysis didnotshow signiﬁcant dif-ferencesatp<0.05betweentheconsideredvalues.These resultsindicatethatconcentrationprocessdidnotdegrade polyphenolsandantioxidantactivityfromT.grandisleaves, asfoundbyAdjéetal.(2012).

3.2. Identiﬁcationofphenoliccompoundsfrom

concentrateROextract

HPLC-DADproﬁleofpolyphenolsfromROconcentrate

extractwasshowninFig.1.Phenolicacidsandﬂavonoids

wereidentiﬁed.

3.2.1. Phenolicacidsidentiﬁcation

ThemolecularstructuresofphenolicacidsinT.grandis

leavesextract,wereconﬁrmedonthebasisoftheirLC–MS

fragmentationMS,MS2_and_MS3_and_on_the_shape_of_their

UV–visspectraasshowninTable2,andwerecompared

withpreviouspublishedstudies.Sevenphenolicacidswere

identiﬁedinaqueousextractofT.grandisleaves.

Compound1gave[M−H]−_at_m/z₁₅₃_with_UV–vis_max

at 260 and 294nm spectra are typicalfor

protocatech-uic acid. Compounds2and 7had [M−H]− _at_m/z₌_353.

The MS2 _{fragmentation} _patterns _produced _ions _at _m/z

191, 179, 135, correspondingto3-O-caffeoylquinic acid

(Compound2).Thefragmentationatm/z191([M-H-162]),

179([M-H-174]),173([M-H-180])and 135([M-H-218])

werecharacteristicsof4-O-caffeoylquinicacid(Stalmach

et al., 2009). The product ions m/z 191for quinicacid

and179forcaffeicacidrevealedtheconstituentof3-CQA

prior tocondensation.Loss ofa caffeoylmoiety yielded

the other dominant fragment ion m/z 173. The

struc-tureof4-CQA(Compound7)wasconﬁrmedbyco-elution

with thestandard. Compound3 wasidentiﬁed as

2-O-caffeoylhydroxycitricacidwithm/z−at369andMS2_at₂₀₇

afterlossof162amu(caffeoylmoiety).Asimilar

fragmen-tationwasreportedbyParveenetal.(2008).Compound

4with[M−H]−_at_m/z₄₈₇_and_a_fragment_at₁₇₉_(caffeic

acid)obtainedafterloss308amu.InMS2 _{fragmentation,}

itobservedthatcompound3lostneutralmassionatm/z

44correspondingtolossofCO2.So,compoundshouldbe

identiﬁedascaffeoylacidderivative.Compound6,which

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Fig.1.HPLCchromatogramobtainedat280nmforaqueousextractofT.grandisleaves. Table2

LC–MSdataobtainedfromtheanalysisofT.grandisleavesextract.

Peak Compounds max MW [M−H+_] _Fragments _Neutral_loss _Percentage_(%)

1 Protocatechuicacid 296 154 153 0.9

2 3-O-caffeoylquinic(3-CQA) 324 354 353 191-179-(135) 1.3

3 2-O-caffeoylhydroxycitricacid 326 370 369 207 162 3.2

4 Caffeoylacidderivative 326 488 487 179-135 308-44 2.2

6 Caffeicacid 297sh-325 180 179 1.8

7 4-O-caffeoylquinic(4-CQA) 326 354 353 173-191-179-135 180-162-174–218 12.2

10 Apigenin7-O-diglucuronide 269–330 624 623 447(271) 176(2×176) 8.0 11 Luteolin7-O-diglucuronide 255–349 638 637 351-(285-193) 286-(352-444) 9.5 14 luteolin7-O-glucuronide 289sh-333 462 461 285 176 2.8 15 Verbascoside 289sh-333 624 623 461-315 162-308 31 16 Luteolindiglucuronide 269–340 637 461–285 176*2 2.3 17 Apigeninglucuronide 266–336 446 445 269 176 1.0 19 Luteolinglucuronide 268–340 462 461 285 176 0.9 20 Luteolin 254–349 286 285 175-239-241 0.4

typicalforcaffeicacid(Fernandesetal.,2011).Thenature

ofthiscompound wasconﬁrmedbyco-elutionwiththe

standard. Caffeicacidwasalready reportedinT.grandis

extract(NayeemandKarvekar,2010).Compound15gave

[M−H]− _at ₆₂₃_and_fragments _at₄₆₁_amu_and ₃₁₅_amu

afterlossof162amu(caffeoylmoiety)and308amu

(rham-nosidemoiety),respectively.ItsUV–visspectrumshowed

amaximalatmax=333withashoulderat289shtypical

forverbascoside(Petreskaetal.,2011).Verbascosidewas

identiﬁedasthemostabundantcompound(30%oftotal

peakareaat280nm)inextractofT.grandisleaves.

Pre-viousstudyshowedsigniﬁcantantihyperglycemicactivity

of verbascoside (Shukla et al., 2010). Thisproperty can

bebeneﬁcialforthetreatmentofdiabetics.Otherstudies

assignedtothiscompoundantioxidant,anti-inﬂammatory,

photo-protective and anti-gastriculcer activities (Singh

etal., 2010;Vertuanietal., 2011).So, leaveswhichare

thedischargesofwoodindustriescouldbeinterestedby

pharmaceuticalorcosmeticindustries.

3.2.2. Flavonoididentiﬁcation

Sevenﬂavonoidswereidentiﬁedonbetweenbasisof

HPLCcoupledtoUV–visdiodearraydetectionandmass

spectrometry withelectrospray ionization

(LC/DAD/ESI-MS2_).

Themolecularionpeak[M−H]−_for_the_compound₁₁

wasm/z623withfragmentsionsatm/z447([M-H-176]−)

and271([M-H-176–176]−),typicalfragmentsofapigenin

7-O-diglucuronide (Meng et al., 2006). Compounds 12

and16wereassignedtoluteolindiglucuronide.HPLC-SM

Table3

Effectofdryingprocessesonpolyphenolcontentsandantioxidantactivity.

Co-product Polyphenol Antioxidant

Content(␮molg−1_GAE) _Recovery_(%) _Capacity_(␮mol_g−1_TE) _Recovery_(%)

ROconcentrate 21,080±117a _100.0 ₈₄₉₀_±₂₉a _100.0

Powder1 18,170±75b _86.19 ₆₉₈₀_±₁₂b _82.21

Powder2 13,960±38c _66.22 ₅₂₁₀_±₅c _61.36

RO,reverseosmosis;GAE,gallicacidequivalent;TE,troloxequivalent.Foreachcolumn,lettersequalsindicatethatthemeansdifferenceisnotsigniﬁcant atp<0.05.

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characteristicsindicatedinSM1_,_m/z_at₆₃₇_[M–H]−_with

fragmentsions at m/z461([M-H-176]− and 285 ([M-H-(2_×176)]−),respectivelyafterlossofoneglucuronidacid andtwoglucuronidacids.Compound14with[M−H]−_at

m/z461anda fragmentat285(luteolin)obtainedafter loss176(glucuronic acid)wasassignedtoluteolin 7-O-glucuronide. A similar fragmentation of the compound wasreportedbyJohnsonetal.(2011)inRusselia

equiseti-formisextract.Compound18 wasidentiﬁedas apigenin

glucuronide showed the loss of a glucuronic acid (m/z

176)andproducedthepredominantfragmentatm/z269

correspondingtodeprotonatedapigenin.Similar

fragmen-tationofthecompoundwasreportedbyZimmermannetal.

(2011)whenanalysingSalviaofﬁcinalisL.extracts.

Com-pound19wasluteolinglucuronidewithm/z−at461and

MS2_ion_at₂₈₅_(luteolin)_due_to_the_loss_of₁₇₆_amu

cor-respondingtoglucuronideacid.Thesimilarfragmentation

haspreviouslybeenbyPatoraandKlimek(2002)fromthe

leavesofMelissaofﬁcinalis.Compound20hada[M−H]−

ionatm/z285andwasassignedtoluteolinaglycone.The

co-elutionwithastandardconﬁrmedthepresenceof

lute-olin.

Manystudies wereinvestigated polyphenolcontents

of T.grandis leafextracts. Amongof polyphenol

identi-ﬁedonlychlorogenicacid(Ghareeb etal.,2013),caffeic

acid (Naira and Karvekar, 2010; Shalini and Rachana,

2009),verbascoside(Shuklaetal.,2010;Singhetal.,2010)

and luteolin (Shukla et al., 2010) were identiﬁed

dur-ing previous studies.Others phenolic compounds were

reportedfor theﬁrsttime (namely protocatechuic acid,

2-O-caffeoylhydroxycitricacid,Caffeoylacidderivative,

4-O-caffeoylquinicacid,apigenin7-O-diglucuronide,luteolin

7-O-diglucuronide, luteolin glucuronide, luteolin

diglu-curonide,apigeninglucuronide,luteolinglucuronide).The

compositioninpolyphenolofourextractswasforgreater

partdifferentfromthoseofpreviousstudies.Manahetal.

(2004)wasreportedthat polyphenolcontents ofplants

wereaffectedbynumerousfactors.Thesefactorsinclude

genetic,ripenessat time harvest,environmental factors

(soiltype,sunexposure,andrainfall),processing,and

stor-age.

3.3. Effectofdryingprocessesonreverseosmosis

concentrateextract

Thereverseosmosis concentrateextractsweredried

by freeze-drying and spray-drying to obtain powder 1

and2,respectively.Thepowdersobtainedareallbrown.

Asshown in Table3, the effect of drying processes on

polyphenolsandantioxidantcapacityfromreverse

osmo-sis(RO)concentrateextract.Theamountsofpolyphenols

obtained after drying process are lower than those of

theconcentrateextract:powder2(13,960±38␮molg−1

GAE)<powder1(18,170±75␮molg−1GAE)<RO

concen-trateextract(21,080±117␮molg−1GAE).Theamountof

antioxidantcapacityofpowder2(5210_±5_␮molg−1 TE)

wasalsolowerthanthoseofpowder1(6980±12␮molg−1

TE)andROconcentrateextract(8490±29␮molg−1GAE).

Recoveryofpolyphenolcontentsandantioxidantcapacity

inpowders,weregenerallybetterthan61%.Freeze-drying

gavebetteryields(>82%)thandidspray-drying(61–67%).

Similar result was reported by Da Silva et al. (2011)

whendryingpropolis.Phaechamudetal.(2012)alsowere

demonstratedthatthermaldryingprocessaffected

signif-icantlytheamountofphenoliccompoundsinextract.

Thisstudy showedthat freeze-drying is a good

pro-cesstostabilizephenolicantioxidantfromT.grandisleaves,

as indicated by Munin and Edwards-Lévy (2011). They

reportedthatfreeze-driedparticleswerestableoverlong

periodsandprovidedtopolyphenolsaneffective

protec-tionagainstoxidationphenomenonduringtheirstorage,

whereasantioxidantactivityremainedidentical.

4. Conclusion

T.grandisleavesextractsobtainedatpilotscalecontain

phenolic compounds that exhibit antioxidant

proper-ties. Reverse osmosis concentrate extract have higher

amountofphenoliccompoundsandantioxidantcapacity

comparatively tocrude extract.In this extract,fourteen

phenolic compounds as ﬂavonoids and phenolic acids

were identiﬁed and characterized. The most abundant

polyphenolinthisextractwasverbascoside.Others

com-pounds were reported for the ﬁrst time in T. grandis

(namely protocatechuic acid, 3-O-caffeoyl quinic acid,

2-O-caffeoylhydroxycitricacid,Caffeoylacidderivative,

4-O-caffeoylquinicacid,apigenin7-O-diglucuronide,luteolin

7-O-diglucuronide, luteolin glucuronide, luteolin

diglu-curonide, apigenin glucuronide, luteolin glucuronide).

Whenaconcentrateextractwasdriedbyfreeze-dryingand

spray-drying,thefreeze-driedextracthasbeenpresented

agoodrecoveryofpolyphenolsandantioxidantcapacity.

Thepowderformofleafwater-extractsobtainedby

freeze-dryingcouldbeapotentialadvantageforpreservationof

itsqualityduringstorageandmarketingofthistraditional

medicineatvillagelevelintropicalcountries.

Acknowledgement

WegratefullyacknowledgetheFrenchEmbassyinCôte

d’IvoireforthedoctoralscholarshipprovidedtoE.K.and

CIRAD(DRS) fortechnicalassistanceand otherﬁnancial

support.

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