Changes in the Polyphenolic Profile, Carotenoids and Antioxidant Potential of Apricot

12 Pages·2017·1.86 MB·English

agriculture
Article
Changes in the Polyphenolic Profile, Carotenoids and
Antioxidant Potential of Apricot (Prunus armeniaca L.)
Leaves during Maturation
AlamZeb*,NadiaKhadimandWaqarAli
BiochemistryLaboratory,DepartmentofBiotechnology,UniversityofMalakand,Chakdara18800,Pakistan;
[email protected](N.K.);[email protected](W.A.)
* Correspondence:[email protected]@gmail.com;Tel.:[phone]
AcademicEditor:TakayukiShibamoto
Received:29December2016;Accepted:23January2017;Published:8February2017
Abstract: Apricot (Prunus armeniaca L.) leaves were studied to assess the potential of apricot
leavesforfuturestudiesandtheirapplicationsinnutraceuticalandbioactivefunctionalingredients.
Thechangesinthephenolicprofile,carotenoids,pigmentsandantioxidantpotentialwerestudiedat
fourmaturationstages. Polyphenolsandcarotenoidswerestudiedusingreversed-phaseHPLC-DAD.
Pigments, total phenolic contents and radical scavenging activity were also measured. Results
revealedtwelvephenoliccompoundsintheapricotleaves. Themajorphenoliccompoundswere
3-O-caffeoylquinicacid(14.6–49.6mg/g),4-O-caffeoylquinicacid(0.56–7.5mg/g),5-O-caffeoylquinic
acid (5.6–25.7 mg/g) and quercetin-3-O-glucosides (8.6–19.9 mg/g), while others include caffeic
acid and derivatives of coumaric acid and kaempferol. Significant changes were observed in
polyphenoliccompoundsduringmaturation. Lutein(56.7–65.7µg/g),neoxanthin(0.66–4.79µg/g),
5,6-epoxy-α-carotene(5.89–7.9µg/g),andβ-carotene(12.3–26.9µg/g)werethemajorcarotenoids.
Thereweresignificantvariationsinthecarotenoids,pigmentcontents,totalphenoliccontentsand
radicalscavengingactivityduringmaturation. Inconclusion,significantvariationoccurredinthe
polyphenolic profile, carotenoids contents and antioxidant potential of apricot leaves under the
studiedconditions.
Keywords: HPLC-DAD; phenolic compounds; carotenoids; apricot leaves; pigments contents;
antioxidantactivity;totalphenoliccontents;leavesmaturation
1. Introduction
Apricot(PrunusarmeniacaL.)isoneofthemostimportantplantswidelygrownandpopularfor
itscharacteristicfruit. Pakistanisthethirdmajorproducerintheworldwithannualproductionnow
morethan0.5millionMT[1]. NorthernareasandKhyberPakhtunkhwaarethemaincultivatingareas
forapricotwithanannualproductioncontributionofmorethan60%[1,2]. Thefruitfromtheseareasis
ofhighqualityandtasteandservesasasourceofeconomyandagriculture. Ali,MasudandAbbasi[1]
studiedthephysico-chemicalcharacteristicsofseveralapricotvarietiesfromPakistan. Theseauthors
reportedthatapricotfruitcontainsfat(2.1%–3%),proteins(6.18%–8.7%),fibre(11.85%–13.6%),ash
(9.45%–12.1%)andtotalsugars(56.8%–64.9%)onafreshweightbasis. Thereportedamountofascorbic
acid was 67.39–90.94 mg/100 g, total phenolic compounds were 4590–7310 mg GAE/100 g, total
carotenoids10.09–18.13mg/100gβ-caroteneandantioxidantactivityof56.84%–82.33%. Theauthors
concludedthatPakistaniapricotsareofhighnutritionalquality.
Apricotisagoodsourceofvariousphytochemicals,whichisoneofthereasonsthatitpossess
anti-microbial, anti-mutagenic, cardio-protective, hepato-protective and antioxidant activities [3].
Apricot is rich in carotenoids [4], fatty acids and sterols, volatile compounds, glycosides and
Agriculture2017,7,9;doi:10.3390/agriculture7020009 www.mdpi.com/journal/agriculture
Agriculture2017,7,9 2of12
polyphenols[3]. Polyphenolsincludescatechin,chlorogenicacid,cynidin-3-glucosides,epicatechin,
epigallocatechin,kaempferol-3-rutinoside,neochlorogenicacid,quercetin-3-glucosides,andrutin[5].
Thesephenoliccompoundsshowedvariationswithrespecttodifferentvarietyoffruitsandpeeling
(skin). Dragovic-Uzelac, et al. [6] evaluated different apricot varieties depending on the stage of
maturity and geographical regions. The authors concluded that the region of cultivation and the
cultivarweretwoimportantfactorsthatinfluencedthecarotenoidsandphenoliccompoundsofapricot
fruits. Differentphytochemicalsinapricotwerethusstudied, whichareaffectedbydifferentfruit
load[7]. Theapricotleavesplayasignificantroleinthedevelopmentoffruit[8]. Thereis,however,a
lackofinformationonthevariationsinthepolyphenolicprofile,carotenoidsandantioxidantpotential
ofapricotleavesduringmaturationunderspecificconditions. Thisstudy,therefore,hadtwoprincipal
objectives: (i) to determine the important polyphenolic composition, carotenoids and antioxidant
potential of the apricot leaves for potential biological applications and (ii) their variations during
maturationthatmayhavepotentialimportanceforfruitdevelopment.
2. MaterialsandMethods
2.1. Materials
Caftaric acid, quercetin-3-glucoside, 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid,
5-O-caffeoylquinic acid coumaric acid, lutein and β-carotene, DPPH & methanol were purchased
fromSigma-Aldrich(Steinheim,Germany),whilecaffeicacidwaspurchasedfromTCI(Tokyo,Japan).
GlacialaceticacidwaspurchasedfromDaejungChemicals(Namyangju,Korea). Ultrapuredeionized
double distilled water with less than 5 mΩ was used. All other chemicals and reagents were of
analyticalstandardofhighpurity.
2.2. SampleCollectionandPreparation
Indigenous apricot leaves were collected from the apricot fields in Thana, District Malakand,
KhyberPakhtunkhwairrigatedwithfreshwater. Thesampleswerecollectedfourtimesfor2months
withanintervaloffifteendays(7Marchto21April). Thetemperatureduringeachcollectionwas
14.9,18.4,21.3and25.7◦C.Thetotalsunshinewas11hand40min,11hand47min,12hand46min
and 13 h and 45 min during the 1st, 2nd, 3rd and 4th collections, respectively. The samples were
collectedfromthestageofsprouting(20February)tomatureleaves. Sinceleavesmaturewithrespect
totimefromsproutingtofullsizedleavesbeforefruitdevelopment,thespecificinterval(oneweek)
wasconsideredasthematurationstage. Apricotleaveswereshadedriedaftercollection. Whenthe
sampleswerecompletelydried,theywerecrushedandgroundtoafinepowder. Theleafextracts
werepreparedfromthepowderedsamples.
2.3. SamplePreparation
Extraction of phenolic compounds was carried out using methanol and water (5:5, v/v) as a
solvent. For this purpose, a powdered sample of 1 g was mixed with a methanol-water mixture
(25mL).Theresultantmixturewaskeptat70◦Cforonehourinawaterbathandshakenvigorously.
Afilteredextractwasobtainedafter30minofcentrifugationat4000rpm. Thefilteredextract(2mL)
wasfilteredusingAgilentPFTEfilers(0.45µm)andtransferredinto2mLHPLCvials. Carotenoids
wereextractedusingcoldacetone,followedbysaponification,partitioningintoaqueousandhexane
layersandevaporationwithnitrogenunderambientconditionsanddaylight[9].
2.4. ChromatographyofPhenolicCompounds
The phenolic profile was determined using an Agilent 1260 HPLC system, consisting of a
quaternary pump, degasser, auto-sampler and diode array detector (DAD). The separation was
carried out using an Agilent rapid resolution column (Zorbax Eclipse C18, 406 × 250 mm, 5 µm).
Thegradientsystemusedinthisstudyconsistedofmethanol-aceticacid-deionizedwater(10:2:88,
Agriculture2017,7,9 3of12
v/v)assolventA,whilethesolventBcompositionwasmethanol-aceticacid-deionizedwater(90:2:8,
v/v)ataflowrateof1mL/min. Thebinarygradientprogramwasstartedwith100%Aat0min,85%
Aat5min,50%Aat20min,30%Aat25min[10]. Theseparationwasachievedwithin30minof
elution. TheDADdetectorwassetto320nmforanalysesofphenoliccompounds,whilethespectra
wererecordedintherangeof190to600nm. Theidentificationofphenoliccompoundswasbasedon
eithertheavailablestandards(caftaricacid,quercetin-3-glucoside,threecaffeoylquinicacids,caffeic
acid,coumaricacid),theirretentiontimesandabsorptionspectra,orbycomparingwithabsorption
spectrareportedinthescientificliterature. Thequantificationofidentifiedcompoundswasbasedon
thestandardcalibrationcurves(fivepointsinduplicates)ofthestandardcompoundsandisexpressed
asmg/gofdryweight(DW)ofthesample.
2.5. ChromatographyofCarotenoids
Carotenoidsweredeterminedusingareversed-phaseHPLCsystem. Theseparationwascarried
outusinganAgilentrapidresolutioncolumn(ZorbaxEclipseC18,4.6×100mm,3.5µm).Thegradient
systemconsistedofsolventA(methanol: deionizedwater,92:8,v/v,with10mMAmmoniumacetate),
solvent B (deionized water containing 0.01 mM) and solvent C as MTBE (100%) with a flow rate
of1mL/min. Thetertiarygradientprogramwasstartedwith80%:18%:2%ofA:B:Cat0minand
reached60%:0%:40%at40min[11]. Thespectrawererecordedintherangeof190to600nmandthe
chromatogramswereobtainedat450nm. Theidentificationofcarotenoidswasbasedoneitherthe
availablestandards(β-carotene&lutein),theirretentiontimes,orabsorptionspectrareportedinthe
literature[12]. Thequantificationofidentifiedcompoundswasbasedonpeakareaandisexpressedas
µg/gdryweight(DW)ofthesample.
2.6. DeterminationofPigmentsContents
Chlorophyll a & b, and total chlorophyll contents were determined using a method reported
previouslyusingthePharmaspec1700spectrophotometer(Shimadzu,Kyoto,Japan)[13]. Theextracts
ofpowderedsamplesweredissolvedinmethanol-waterandtheabsorbancewasmeasuredat645and
663nm. Thevaluesareexpressedasmg/mL.
2.7. DeterminationofTotalPhenolicContents
Totalphenoliccontents(TPC)ofapricotleavesextractsweredeterminedusingFolin-Ciocalteu
reagentasrecentlydescribedindetail[14]. TheFCreagentwaspreparedfreshinthelaboratory. Gallic
acidwasusedtoprepareastandardcalibrationcurve(sixpointsintriplicate). Theconcentrationrange
ofthecalibrationwasfrom5to100mg. TPCwasmeasuredusingaspectrophotometerandexpressed
asmgofgallicacidequivalents(GAE)per100gofthesample.
2.8. RadicalScavengingActivity
Theradicalscavengingactivity(RSA)ofeachsampleintriplicatewasdeterminedusingtheDPPH
method[15]. FreshDPPHsolution(0.1mM)waspreparedinmethanoleveryday. Formeasurement,
DPPHsolution(1.90mL)wasmixedwithmethanolicextract(10µL)ataconcentrationof1.01mg/mL
withtheleaves. Theresultantmixturewasplacedinadarkchamberfor30min. Thenabsorbanceof
thesamplewasmeasuredusingaPharmaspec1700spectrophotometer(Shimadzu,Japan)at515nm.
TheRSAofthesampleswascalculatedusingthegivenequation: %RSA=(Ac−As)/Ac,where,Acis
theabsorbanceofthecontrolandAsistheabsorbanceoftheapricotleafextract.
2.9. DataAnalyses
Allsamplesweremeasuredinreplicate(n=3). Datawereanalysedforvariationusingone-way
analysisofvariance(ANOVA)andTukey’smethodusingGraphPadPrism5forWindowsversion
5.03(GraphPadSoftwareInc.,LaJolla,CA,USA,2009).
Agriculture2017,7,9 4of12
3. Results
Agriculture 2017, 7, 9  4 of 12 
3.1. PhenolicCompoundsinApricotLeaves
3. Results  
Figure 1A–D shows the HPLC-DAD chromatograms of the apricot leaves at the different
3.1. Phenolic Compounds in Apricot Leaves 
maturationstages. Atotaloftenpolyphenoliccompoundswasidentified. Theidentificationdetailsof
eachcompFoiguunrde r1eAp–rDes esnhtoewdsb tyhae pHePaLkCn‐uDmAbDe rchinrotmheatcohgrroammas toogf rtahme saaprreicsoht olweanveisn aTta bthlee 1d.iCffoermenpto und
1wasmidateunratitfiioend satasg3e-sO. A-c taoftfaelo oyfl qteuni npioclyapchiden(oclhicl ocroomgpeonuicndasc iwda)sw iditehntλified.o Tfh2e3 i4d,e2n9ti8fsichataionnd d3e2t5ailnsm  by
max
compoafr ienagchw ciothmaposutanndd raerpdrecsoemntpeodu bnyd .a Cpoemakp nouumndbe2r, iwn htihceh cwhraosmealutotgedramats5 a.2rem sihno,wwna sinc oTuamblea r1ic. acid
derivCatoimvep,owunitdh 1λ was iodfe2n8ti3fi,e2d8 a8s a3n‐Od‐c3a1f4fenomylq.uCinoimc apcoidu n(cdhl3orwogaesniidc eancitdifi) weditha sλ5m-axO o-fc 2a3ff4e, o29y8lqshu ainnidc acid
max
325 nm by comparing with a standard compound. Compound 2, which was eluted at 5.2 min, was 
(neo-chlorogenicacid)withλ of236,298shand326nm. Thiscompoundwaselutedat5.9min.
max
coumaric acid derivative, with λmax of 283, 288 and 314 nm. Compound 3 was identified as 
At6.1,6.3and6.6minofelution,coumaroylhexose(4),caffeicacid(5)and4-O-caffeoylquinicacid
5‐O‐caffeoylquinic acid (neo‐chlorogenic acid) with λmax of 236, 298 sh and 326 nm. This compound 
(6)wereidentified. Hydroxyferuloylglucosewasidentifiedattheretentiontimeof7.1minwithλ
was eluted at 5.9 min. At 6.1, 6.3 and 6.6 min of elution, coumaroyl hexose (4), caffeic acid (5) and  max
of 240, 305 and 326 nm as compound 7. No other compounds were identified until 14 min, when
4‐O‐caffeoylquinic acid (6) were identified. Hydroxyferuloylglucose was identified at the retention 
quercteimtine -3o-fg 7lu.1c omsiind ewwitahs λemluaxt eodf w24i0th, 3c0h5a raancdte 3ri2s6t icnmλm aasx ocofm25p6oaunndd 375. 4Nnom oathnedr mcoamrkpeodunadssc owmerpeo und
8 basieddenotinfiead cuonmtilp 1a4r imsoinn, wwihtehn tqhueerscteatnind‐a3r‐gdluccoomsidpeo uwnads .eluAtetdr ewteitnht icohnartaicmteerisstoicf λ1m6a.x7 oaf n2d561 a7n.2d min,
kaem3p5f4e rnoml haenxdo smidaerk(ecdo masp ocoumnpdo9u)nwd i8th bcahseadra ocnte rai sctoicmλparisoonf 2w8i5t,h3 t2h5e asntadn3d4a6rdn mcomanpdouknade. mApt ferol
max
derivraettievnetio(cno tmimpeosu onf d161.70 )anwde 1re7.2e lmutined, k.aCemompfperooul nhdex1o0sidhea d(coλmmpaxouonfd2 895) ,w3i2th4 cahnadra3ct4e5risntmic .λmHaxo owf ever,
theH2P8L5,C 3-2D5 AanDd m34e6t hnomd awnda skaneomtpsufefrfiocl ideenrtivtoatcivoer r(ceocmtlypoiduenndt 1if0y) twheered eelruivteedd. Ccoommppoounnedn t10o fhkadae λmmapx ferol.
of 285, 324 and 345 nm. However, the HPLC‐DAD method was not sufficient to correctly identify the 
Twomorecompounds,i.e.,coumaricacidandcaftaricacid,wereidentifiedatthestageof45daysof
derived component of kaempferol. Two more compounds, i.e., coumaric acid and caftaric acid, were 
maturationandwerelabelledascompound11and12,respectively. However,thesecompoundswere
identified at the stage of 45 days of maturation and were labelled as compound 11 and 12, 
notidentifiedinanyothermaturationstages.
respectively. However, these compounds were not identified in any other maturation stages.  

Figure 1. Representative HPLC‐DAD chromatograms of the apricot leaves during maturation at 320 
Figure 1. Representative HPLC-DAD chromatograms of the apricot leaves during maturation at
nm. (A) 15 days; (B) 30 days; (C) 45 days; and (D) 60 days. The identification of the peaks are given in 
320nm.(A)15days;(B)30days;(C)45days;and(D)60days.Theidentificationofthepeaksaregiven
Table 1. 
inTable1.
Agriculture2017,7,9 5of12
Table1.Identificationofpolyphenoliccompoundsinapricotleafsamples.
HPLC-DAD
Peak Rt(min) TentativeIdentification Identification
Agriculture 2017, 7, 9  λmax(nm) 5 of 12 
1 3.6 3-O-Caffeoylquinicacid 234,298sh,325 Standard
Table 1. Identification of polyphenolic compounds in apricot leaf samples.  
2 5.2 p-Coumaricacidderivative 283,288,314 [16]
3 Peak  5.R9t  Tentative Id5e-nOti-fCicaatfifoeno ylquiHnPicLCa‐cDidAD  236,298sIhd,en3t2if6ication   Standard
4 6(m.1in)  p-Coumaroylhλemxaox s(nem)  285,316 [16]
1  3.6  3‐O‐Caffeoylquinic acid  234, 298sh, 325  Standard  
5 6.3 Caffeicacid 232,302,326 Standard
2  5.2  p‐Coumaric acid derivative 283, 288, 314  [16] 
6 6.6 4-Caffeoylquinicacid 232,303,326 Standard
3  5.9  5‐O‐Caffeoylquinic acid  236, 298sh, 326  Standard 
7 4  7.61.1  p‐CoumaHroyydl hreoxxoysef eruloylg28lu5,c 3o1s6e  240,305,326[16]  [15]
8 5  146..83  CaffeicQ auciedr c etin-3-glu23c2o, s3i0d2,e 326  256,354Standard  Standard
9 6  166..76  4‐CaffeoylquKinaiecm acpidf erolhe2x3o2,s 3id03e, 326  285,325,3S4ta6ndard  [17]
10 7  177..21  Hydroxy feruKloayel mglupcfoesreo lder2i4v0,a 3t0iv5,e 326  285,324,345[15]  [17]
11 8  81.41.8  Quercetin‐3‐glucCosoiudem  aricaci2d56, 354  230,312Standard  Standard
9  16.7  Kaempferol hexoside  285, 325, 346  [17] 
12 9.2 Caftaricacid 245,298sh,328 Standard
10  17.2  Kaempferol derivative   285, 324, 345  [17] 
11  8.1  Coumaric acid  230, 312  Standard 
12  9.2  Caftaric acid  245, 298sh, 328  Standard 
3.2. ChangesinthePhenolicCompounds
Figu3r.e2.2 Cshahnogwes sint thhee Pchhenaonligc eCsominpotuhnedsp  henoliccompoundsduringthematurationofapricotleaves.
ChlorogenicaFicgiudre( 32 -sOho-wcas ftfheeo cyhalqngueisn iinc thaec ipdh)enwolaics cpomrepsoeunntdas tduthriengh tihgeh meastturleatvioenl o(f1 a5p.6ri7comt lega/vegs). at15days
ofmaturaCthiolonro,gaenndic arceiadc (h3‐eOd‐csaiffgenoyifilqcuainnitcl ayci(dp) w<a0s .p0r1e)sehnitg aht tehre vhaiglhueests le(4ve8l. 7(165.a67n mdg4/g9). 6atm 15g d/agys) oafs  shownin
maturation, and reached significantly (p < 0.01) higher values (48.76 and 49.6 mg/g) as shown in 
Figure2A.Quercetin-3-glucoside(19.69mg/g)waspresentinthesecondhighestamount. Therewere
Figure 2A. Quercetin‐3‐glucoside (19.69 mg/g) was present in the second highest amount. There 
nosignificwaenret (npo >sig0n.0if1ic)acnht a(pn g> e0s.0d1)u crhinangg1es5 daunrding3 015d aanyds o30f mdaaytsu orfa tmioatnu,rawtihonil,e wahsilieg an isfiigcnainfictadnet creasewas
observeddaetcr4ea5sed wayass .obNseorvseidg anti 4fi5c daanyts.c Nhao nsiggensifiwcanetr echoanbgseesr wveerde aobts6er0vedda ayts 60o fdamysa otuf mraattiuornat.ioTnh. us,ahigh
correlatioTnh(uRs,2 a= h0ig.9h 1c1o4rr)eolaftitohne (sRe2 t=w 0o.91c1o4m) opf othuensde stwwo acsomobpsoeurnvdes dwdasu orbinsegrvtehde dsuerliencgt ethde msealetcuterda tionstages.
maturation stages. 

Figure 2. Changes in the polyphenolic compounds of apricot leaves during maturation.

(A)3-O-Caffeoylquinicacid,quercetin-3-glucoside;(B)5-O-Caffeoylquinicacid;(C)4-O-Caffeoylquinic
acid,hydroxyferuloylglucose,p-coumaricacidderivative;and(D)caffeicacid,kaempferolhexoside,
andlaempferolderivative.Significantlevelsarementionedinthetext.
Agriculture 2017, 7, 9  6 of 12 
Figure  2.  Changes  in  the  polyphenolic  compounds  of  apricot  leaves  during  maturation.  (A) 
3‐O‐Caffeoylquinic acid, quercetin‐3‐glucoside; (B) 5‐O‐Caffeoylquinic acid; (C) 4‐O‐Caffeoylquinic 
acid, hydroxyferuloyl glucose, p‐coumaric acid derivative; and (D) caffeic acid, kaempferol hexoside, 
and laempferol derivative. Significant levels are mentioned in the text. 
Agriculture2017,7,9 6of12
Compound 3 was tentatively identified as 5‐O‐caffeoylquinic acid (neo‐chlorogenic acid), 
whicCh owmaps othuen dth3irwd amsotestn atabtuivnedlyanidt econmtifipeoduansd5 i-nO t-ecramffse ooyf ltqhuei npiecrcaecnidt a(nmeoou-cnhtl oprroegseenntic ina ctihde) ,awprhiiccoht 
wleaasveths eatth diridffemreonstt asbtaugnedsa (nFtigcoumrep 2oBu)n. dSiignntiefricmasnto f(pth <e 0p.e0r5c)e inntcarmeaosuesn topccreusrernedti nduthrienagp trhiceo ftilresat vtehsreaet 
dstiaffgeerse notf smtaagtuesra(tFioignu, rweh2iBle) .aS sigignnifiifcicaanntt( dpe<cr0e.a0s5e) ioncccruerarseeds ionc cthuer raemdoduunrti nogf 5th‐Oe‐ficrasftfetohyrelqeusitnaigce ascoidf 
mata 6t0u rdaatyiosn o,fw mhailteuraastiigonn.i fiTchaen atmdeocurenat soef o5c‐cOu‐rcraefdfeionytlhqeuianmico aucnidt oaft 56-0O d-acayfsf ewoaysl qthuein siacmacei dasa tth6a0t data y30s 
odfaymsa toufr amtioatnu.raTthioena. mFoiguunrteo f2C5- Os-hcoawffeso ythlqaut inthice aacmidoautn6t 0odfa y4s‐Ow‐caasfftehoeyslqamuieniacs  athciadt adte3c0redaaseyds 
osifgmniafitcuarnattliyo nw.iFthig muraetu2rCatsihono.w As tshimatiltahre taremnodu wntaso fo4b-sOer-vceadff eiony tlhqeu cinasice aocfi dhyddercorxeyafseerdulsoigynl igfilcuacnotsley, 
wthieth ammaotuunrta toiof nw.hAicshim wilaasr htraelnf dofw thase oabmseoruvnetd oifn 4t‐hOe‐ccaasffeeoofyhlqyudirnoicx yafceirdu.l oTyhleg dlueccoresaes,eth ien atmheosuen ttwoof 
wcohmicphowunadssh awlfaso fsitghneiafimcaonutnlyt coofr4r-eOla-tceadff e(Roy2 l=q 0u.i9n9i9c)a wciidth. T ehaechd eocthreears. eTihnet dheesceretawseo icno mthpe oaumnodusnwt aosf 
s4i‐gOn‐icfiacfafenotlyylqcuorinreicla taecdid( Rw2a=s0 .h9i9g9h)lwy icthoreraeclahteodth e(Rr.2T =h e0d.6e7c9r4e)a sweiintht htheea minocurneatsoef 4in-O t-hcaef faemoyoluqnutin oicf 
a5c‐Oid‐cwafafseohliygqhulyincioc rarceilda.t eAd s(iRg2n=ifi0c.a6n7t9 (4p) <w 0it.h05t)h iencinrecaresae sweains tohbesearmveodu nint othfe5 -aOm-coaufnfet oolfy qpu‐cionuicmaacridic. 
Aacsidig ndiefircivaanttiv(pes< a0t.0 350) idnacryesa soef wmaastuorbasteirovne, dwinhitlhee naom soigunnitfoicfapn-tc ovuamriaatriiocna cwidasd eorbivseartviveeds fautr3t0hedra yass 
oshfomwantu irna tFioignu,rweh 2iCle. nTohesi gamnifioucannt tovfa croiautmioanrwoyals hoebxsoesrev esdigfnuirfitchaenrtalys s(ph o<w 0n.0i5n) iFnigcrueraes2eCd .aTt h3e0 admayosu notf 
omfactouuramtiaorno,y wl hheilxeo aset 4s5ig dnaifiycsa, nat lsyig(npif<ic0a.n0t5 )deinccrereaasese wdaast o3b0sderavyesdo. fAm fautrutrhaetri osnig,nwifhicilaenta t(p4 5< d0a.0y5s), 
ainscirgenaisfiec awnatsd eocbrseearsveewd aast o6b0s edravyesd .oAf mfuarttuhreartisoignn. iSfiicmanilta(rp t<re0n.d05s )winecrree aosbesweravsedob isne rtvheed aamt6o0undta yosf 
ocaffmfeaictu arcaitdi,o kna.eSmimpiflearrotlr henexdosswideer eanodb skearevmedpfinertohl edaemrivoautnivteosf acsa fsfheoicwanc iidn, tkhaee mFigpuferreo 2lDhe. xosideand
kaempferolderivativesasshownintheFigure2D.
3.3. Carotenoid Contents  
3.3. CarotenoidContents
Figure 3 shows the typical chromatogram of carotenoid contents in the apricot leaves. Using the 
availFaibgleu rseta3ndshaordwss atnhde atbyspoircpalticohnr sopmeacttroag irna mtheo frecpaorortteedn oliitdercaotunrteen, ttesni ncatrhoeteanporiidcos twleearev eids.enUtisfiinegd. 
tThheesaev caoilnatbalienesdta tnwdoa ridsosmaenrds oafb nseoorpxatinotnhisnp (eacltl‐rtarainns‐tnheeoxreapntohritne d& l9it‐ecrisa‐tnueroex,atnetnhicna)r, octies‐nγo‐icdasrowteenree, 
iadlle‐ntrtaifinse‐dv.ioTlahxeasnethcionn, taianlel‐dtratwnso‐luistoemine, rstohfrenee oxisaonmtheirns (alol-ft raαns‐c-naerootxeannet hin(5,&6‐e9p-coixs-yn‐eαo‐cxaarnottheinne),, 
c1i5s-‐γci-sc‐aαr‐octaernoet,eanlel- tarnadns 1-v3i‐ocilsa‐xαa‐nctahriont,eanlel-)t raannds- βlu‐tceairno,ttehnree eainsdom itesr 9s‐ocfisα i-scoamroetre naes (s5h,6o-wepno ixny t-hαe-c Taraobtleen 2e., 
1T5h-ecisse- αr-ecsaurlotste snheoawnedd1 3th-caits -αα‐-,c aβr‐ocaternotee)naen danβd- claurtoetienn weaenred pitrse9se-cnits iisno mhiegrh aasmshoouwntns iinn thalel Taapbrlieco2t. 
Tlehaevseesr. esultsshowedthatα-,β-caroteneandluteinwerepresentinhighamountsinallapricotleaves.

Figure 3. Representative HPLC‐DAD chromatograms at 450 nm of carotenoids of the apricot leaves. 
Figure3.RepresentativeHPLC-DADchromatogramsat450nmofcarotenoidsoftheapricotleaves.
Numbers above the peaks represent individual compounds with details given in Table 2. 
NumbersabovethepeaksrepresentindividualcompoundswithdetailsgiveninTable2.
Agriculture2017,7,9 7of12
Table2.IdentificationandchangesintheCarotenoidcontentsofApricotleafsamples.Identification
wasbasedoncomparisonwiththeabsorptionspectraofthestandardcompoundsanddatareportedin
theliterature[12].
Tentative HPLC-DAD Carotenoids(µg/g)
Peak Rt(min) Identification λmax(nm) 15 30 45 60
1 9.1 All-trans-neoxanthin 468,444,420 2.01±0.1a 4.79±0.3b 0.66±0.1c 4.01±0.3b
2 9.8 9-Cis-neoxanthin 464,436,418 2.46±0.2a 3.6±0.14b 1.01±0.2c 2.34±0.3a
3 10.1 Cis-γ-carotene 464,438,422 1.18±0.1a 1.55±0.1a 1.33±0.1a 2.34±0.1b
4 12.3 All-trans-violaxanthin 472,444,420,330 0.54±0.02a 0.42±0.01a 0.27±0.1b 0.26±0.1b
5 15.0 All-trans-lutein 474,446,422 65.7±1.4a 61.1±0.9b 58.2±0.4c 56.7±0.8d
6 17.2 5,6-epoxy-α-carotene 468,440,420 7.78±0.5a 7.9±0.3a 5.89±0.2b 6.41±0.7c
7 17.9 15-Cis-α-carotene 466,440,420,330 4.84±0.3a 4.69±0.2a 2.0±0.2b 4.14±0.1a
8 24.7 13-Cis-α-carotene 466,440,420 0.39±0.02a 0.32±0.02a 0.62±0.06b 0.21±0.03c
9 32.8 β-Carotene 476,450 12.3±0.1a 13.6±0.2a 26.9±1.1b 18.5±2.1c
10 33.2 9-Cis-β-carotene 468,446,335 0.81±0.04a 1.21±0.06b 2.2±0.1c 1.13±0.2b
Dataarepresentedasthemeanwithstandarddeviation(n=3). Differentletters(a–d)representsignificanceat
p<0.05(Tukey’stest).
3.4. ChangesinCarotenoidContents
Table2showsthatasignificantincrease(p<0.05)occurredintheamountsofall-trans-neoxanthin
and its isomer with increasing maturation time. However, a significant loss in these carotenoids
was observed at 45 days. No significant changes were observed in the amounts of cis-γ-carotene
andall-trans-violaxanthin,exceptat60days,theamountofthesecarotenoidswerehigherorlower.
Asignificant(p<0.05)decreaseoccurredintheamountofall-trans-luteinfrom65.7to56.6µg/g. There
wasnosignificantchangesintheamountsofα-caroteneanditsisomersat15and30daysofmaturation,
whilesignificantchangeswereobservedat45and60daysofmaturation. Similarly,β-carotenewas
presentinthesecondhighestamount(12.3µg/g). Therewasnosignificantchangeduring15–30days,
whileasignificantincrease(p<0.05)occurredat45daysofmaturation. Theamountwaslessthan
at45daysandhigherthanat15and30daysofmaturation. Therewasasignificantincreaseinthe
amount of 9-cis-β-carotene with an increase in maturation; however, the amount decreased to the
original value measured at 30 days. These results showed that significant changes occurred with
maturationoftheapricotleaves.
3.5. ChangesinPigmentContents
Figure 4 shows the changes in the chlorophyll contents of the apricot leaves. Chlorophyll a
washigherthanchlorophyllb. Itwasobservedthatchlorophylladecreasedsignificantly(p<0.01)
to 5.40 mg/mL with maturation until 45 days from the 23.37 mg/mL level measured at 15 days.
Thevaluesincreasedto19.90mg/mLat60daysofmaturation. Similarobservationswasalsoreported
forchlorophyllb,wherevaluesof13.18,8.78,6.22mg/mLwereobtainedfor15,30and45daysof
maturation, respectively. This shows a significant (p < 0.01) decrease in chlorophyll b. The value
increased by 60 days of maturation. Total chlorophyll showed a similar trend of variation as that
describedaboveforchlorophylla&b.
Agriculture2017,7,9 8of12
Agriculture 2017, 7, 9  8 of 12 

Figure 4. Changes in the pigment contents of the apricot leaves during maturation. Different letters 
Figure4.Changesinthepigmentcontentsoftheapricotleavesduringmaturation.Differentletters
(a–d) in each column represent significant differences at p < 0.01 (Tukey’s test). 
(a–d)ineachcolumnrepresentsignificantdifferencesatp<0.01(Tukey’stest).
3.6. Changes in TPC & Radical Scavenging Activity 
3.6. ChangesinTPCandRadicalScavengingActivity
Figure 5A shows the TPC values of the apricot leaves during maturation. There was a 
Fsiiggnuirfieca5nAt (sph <o w0.0s5t)h iencTrPeaCsev ianl uthees ToPfCth feroampr 1ic5o ttol e45a vdeasysd uofr imngatumraattiuorna. tTiohne.reT whearse nwo asisganisfiicgannitfi cant
(p<0v.a0r5i)atiinocnr ebaetsweeiennt h30e aTnPdC 4f5r odmays1 5oft om4a5tudraatyiosno. fTmheasetu rreastuilotsn .shTohwe rtehawt atostnalo pshigennoifilicc acnotntveanrtsia tion
betweinecnre3a0seadn wdi4th5 mdaaytusroatfiomna. tFuigruatrieo 5nB. sThhoewses trhees uRlStsAs vhaoluwest hoaf tthtoe taaplrpichoet nleoalvicesc odnurteinngt smiantcurreaatsioend. with
There was a significant (p < 0.05) decrease in the RSA at 15 and 30 days of maturation. A significant 
maturation. Figure 5B shows the RSA values of the apricot leaves during maturation. There was
increase was then observed at 45 days and 60 days. The % RSA values at 30 days were similar to 
asignificant(p<0.05)decreaseintheRSAat15and30daysofmaturation. Asignificantincrease
those of 0.5 mg/mL of BHT, while the amounts at 15, 45 and 60 days were in the range of 0.5 to 1.5 
was then observed at 45 days and 60 days. The % RSA values at 30 days were similar to those of
mg/mL BHT equivalent % RSA. There was no significant variation in the RSA values at 45 and 60 
0.5mg/mLofBHT,whiletheamountsat15,45and60dayswereintherangeof0.5to1.5mg/mL
days of maturation. The results showed that the RSA values increased with maturation. The HPLC 
BHTequivalent%RSA.TherewasnosignificantvariationintheRSAvaluesat45and60daysof
data of the phenolic compounds were compared with RSA values during each maturation stage. It 
matuwraatsio nfo.uTnhde  rthesaut ltosnslyh otwhreede thcoamttphoeuRndSsA  hvaadlu eas  sintrcornega secodrrweliathtiomn awtuirthat itohne. TRhSeAH  vPaLluCesd, ait.ae.o, fthe
phen3o‐lOic‐ccaoffmeopyolquunidnisc waecride, cokmaepmaprfeedrowl idtherRivSaAtivve alauneds dquuerrincegtiena‐3c‐hglmucaotsuidrea.t ioAn  ssttraogneg. Iptowsiatisvfeo und
thatocnolryretlhatrieoen ccoomefpfiociuenntd s(Rh2 a=d 0a.8s2t2ro4)n gwcaos rfroeulantdio bnewtwiethent hReSRAS aAndv a3l‐uOe‐sc,aif.fee.o,y3l-qOu-icnaicf feaociydl,q wuihniilec acid,
kaemsptrfoenrogl dneegriavtiavteiv ceoarrnedlaqtiuonersc eotfi nR-23 -=g l0u.9co52s6id aen.dA Rst2r o=n 0g.8p4o0s6i twiveerec oorbreselarvtieodn fcoore ftfihcei eknate(mRp2fe=ro0l. 8224)
wasfdoeurnivdatbivetew aneedn qRueSrAceatinnd‐3‐3g-lOuc-coasfidfeeo. y lquinicacid,whilestrongnegativecorrelationsofR2=0.9526
andR2=0.8406wereobservedforthekaempferolderivativeandquercetin-3-glucoside.
Agriculture 2017, 7, 9  9 of 12 
(A)

Figure5.Cont.

Figure 5. Changes in the total phenolic contents and radical scavenging activity of the apricot leaves 
during maturation. Different letters (a–e) in each column represent significant differences at p < 0.05 
(Tukey’s test).  
4. Discussion 
Apricot leaves are rich in various phenolic compounds as shown in Figure 1. The present results 
showed high amounts of caffeoyl quinic acids such as 3‐O‐caffeoylquinic acid, 4‐O‐caffeoylquinic 
acid (crypto‐chlorogenic acid) and 5‐O‐caffeoylquinic acid (neo‐chlorogenic acid). These compounds 
were identified from authentic standards and previous reports [16–18]. Neo‐chlorogenic acid and 
chlorogenic acid were previously identified in apricot peel extract [19], and unpeeled and peeled 
canned apricot fruit [20]. Similarly, Dragovic‐Uzelac, et al. [21] showed that caffeoyl quinic acids 
were the dominant polyphenols during apricot fruit ripening. This suggests that caffeoyl quinic 
acids are a class of compounds that are present in various products of Apricot and can thus be used 
as a marker for variations or identification of food products. The increase in the chlorogenic acid in 
the apricot leaves may be due to the heat stress, which increased during maturation [22]. Coumaric 
acid  and  coumaroyl  hexose  were  identified  in  relatively  small  amounts.  Coumaric  acid  was 
identified in apricot fruit [21] and has strong antioxidant potential. Similarly, caffeic acid, which was 
determined using a standard compound, was previously identified in raw and puree apricot 
cultivars [21]. 
Kaempferol derivatives were identified in the apricot leaves in this study and have been 
identified in apricot fruit by several authors [6,20,21]. Quercetin‐3‐O‐glucoside is an important 
glycoside  formed  from  quercetin  and  is  an  important  component  of  apricot  fruit  [23].  This 
compound  was  identified  in  apricot  fruit  by  several  authors  [5,6].  Campbell,  Merwin  and 
Padilla‐Zakour [5] showed that maturity had no effect on the quercetin‐3‐glucoside contents of 
different  varieties  of  apricot  fruits.  The  present  results  show  a  significant  decrease  in 
quercetin‐3‐glucoside in the apricot leaves during maturation. This decrease may be due to the heat
Agriculture 2017, 7, 9  9 of 12 
Agriculture2017,7,9 9of12

(B)
Figure 5. Changes in the total phenolic contents and radical scavenging activity of the apricot leaves 
during maturation. Different letters (a–e) in each column represent significant differences at p < 0.05 
Figure5. (A)TotalPhenolicContents(mg/100g);(B)RadicalScavengingActivity(%). Changesin
(Tukey’s test).  
thetotalphenoliccontentsandradicalscavengingactivityoftheapricotleavesduringmaturation.
Differentletters(a–e)ineachcolumnrepresentsignificantdifferencesatp<0.05(Tukey’stest).
4. Discussion 
4. DiAscpursisciootn leaves are rich in various phenolic compounds as shown in Figure 1. The present results 
showed high amounts of caffeoyl quinic acids such as 3‐O‐caffeoylquinic acid, 4‐O‐caffeoylquinic 
ApricotleavesarerichinvariousphenoliccompoundsasshowninFigure1. Thepresentresults
acid (crypto‐chlorogenic acid) and 5‐O‐caffeoylquinic acid (neo‐chlorogenic acid). These compounds 
showed high amounts of caffeoyl quinic acids such as 3-O-caffeoylquinic acid, 4-O-caffeoylquinic
were identified from authentic standards and previous reports [16–18]. Neo‐chlorogenic acid and 
acid(crypto-chlorogenicacid)and5-O-caffeoylquinicacid(neo-chlorogenicacid). Thesecompounds
chlorogenic acid were previously identified in apricot peel extract [19], and unpeeled and peeled 
were identified from authentic standards and previous reports [16–18]. Neo-chlorogenic acid and
canned apricot fruit [20]. Similarly, Dragovic‐Uzelac, et al. [21] showed that caffeoyl quinic acids 
chlorogenic acid were previously identified in apricot peel extract [19], and unpeeled and peeled
were the dominant polyphenols during apricot fruit ripening. This suggests that caffeoyl quinic 
cannedapricotfruit[20]. Similarly,Dragovic-Uzelac,etal.[21]showedthatcaffeoylquinicacidswere
acids are a class of compounds that are present in various products of Apricot and can thus be used 
thedominantpolyphenolsduringapricotfruitripening. Thissuggeststhatcaffeoylquinicacidsarea
as a marker for variations or identification of food products. The increase in the chlorogenic acid in 
classofcompoundsthatarepresentinvariousproductsofApricotandcanthusbeusedasamarker
the apricot leaves may be due to the heat stress, which increased during maturation [22]. Coumaric 
forvariationsoridentificationoffoodproducts. Theincreaseinthechlorogenicacidintheapricot
acid  and  coumaroyl  hexose  were  identified  in  relatively  small  amounts.  Coumaric  acid  was 
leavesmaybeduetotheheatstress, whichincreasedduringmaturation[22]. Coumaricacidand
identified in apricot fruit [21] and has strong antioxidant potential. Similarly, caffeic acid, which was 
coumaroylhexosewereidentifiedinrelativelysmallamounts. Coumaricacidwasidentifiedinapricot
determined using a standard compound, was previously identified in raw and puree apricot 
fruit[21]andhasstrongantioxidantpotential. Similarly,caffeicacid,whichwasdeterminedusinga
cultivars [21]. 
standardcompound,waspreviouslyidentifiedinrawandpureeapricotcultivars[21].
Kaempferol derivatives were identified in the apricot leaves in this study and have been 
Kaempferolderivativeswereidentifiedintheapricotleavesinthisstudyandhavebeenidentified
identified in apricot fruit by several authors [6,20,21]. Quercetin‐3‐O‐glucoside is an important 
in apricot fruit by several authors [6,20,21]. Quercetin-3-O-glucoside is an important glycoside
glycoside  formed  from  quercetin  and  is  an  important  component  of  apricot  fruit  [23].  This 
formed from quercetin and is an important component of apricot fruit [23]. This compound was
compound  was  identified  in  apricot  fruit  by  several  authors  [5,6].  Campbell,  Merwin  and 
identifiedinapricotfruitbyseveralauthors[5,6]. Campbell,MerwinandPadilla-Zakour[5]showed
Padilla‐Zakour [5] showed that maturity had no effect on the quercetin‐3‐glucoside contents of 
that maturity had no effect on the quercetin-3-glucoside contents of different varieties of apricot
different  varieties  of  apricot  fruits.  The  present  results  show  a  significant  decrease  in 
fruits. Thepresentresultsshowasignificantdecreaseinquercetin-3-glucosideintheapricotleaves
quercetin‐3‐glucoside in the apricot leaves during maturation. This decrease may be due to the heat 
during maturation. This decrease may be due to the heat stress, which increased during the leaf
maturation [22]. Studies have shown that quercetin-3-O-glucoside possesses strong antioxidant

effects[24]. Theoralsupplementationofquercetin-3-O-glucoside&queretinshowedthatglucoside
waswellabsorbedbytheintestinalsystemoftheratscomparedwithquercetinalone[25].Theseresults
suggestthatapricotleavescontainimportantpolyphenoliccompounds,whichhavepotentialbiological
importance. Thechangesinthepolyphenoliccompoundsintheleavesduringmaturationmaybe
correlatedwithfruitdevelopmentandthusshouldbeconsideredinapricotagriculture. Variationin
thesecompoundsmayoccurduetothevariety,climate,soilandenvironmentalconditions. Theexact
structuresofeachderivativecompoundsinthisstudycanbedeterminedusingmassspectrometry.
Apricot leaves are rich in lutein, α- and β-carotenes. These carotenoids are among the major
carotenoidsintheapricotleavesandfruits[3,26]. Katayama,etal.[27]showedthatviolaxanthinand
Agriculture2017,7,9 10of12
β-caroteneincreasedduringthematurationofapricotfruits,whilethisstudyshowedthattheamount
ofthesecarotenoidsdecreasedinthefirsttwostagesofmaturation. Thismaybeduetothetransport
ortransformationsofthesecarotenoidstoothercarotenoidsinthefruit. Theseauthorsalsoreported
thatluteindecreasedwithmaturation,whichmightberelatedtoitslowerlevelintheleaves. These
resultsindicatethatimportantcarotenoidswerepresentintheapricotleavesandchangesinthese
compoundswereobservedduringmaturation,whichmightbeimportantforfruitdevelopment.
Pigmentssuchaschlorophyllsplayakeyroleinthephotosynthesisanddefenceoftheplant
againstexternalstress. Inthepresentstudy,chlorophyllsdecreasedsignificantlywithmaturationuntil
45days. Sabiretal.[28]showedthatthechlorophyllcontentofyoungleaveshadahighcorrelation
with iron concentration, which is an indicator of the vital role of iron in the chlorophyll content
ofleaves.
Thetotalphenoliccontent(TPC)oftheapricotleavesincreasedwithmaturationasshownin
Figure4A.Theincreasewashighlycorrelatedwiththeincreaseordecreaseinthepolyphenolicprofile
oftheleaves. Phenoliccompoundshavehighantioxidantactivityasindicatedbythehighcorrelation
coefficientandRSAvalues.ThepresentresultsshowedthatRSAvaluesincreasedwithmaturationand
eachmaturationstagewasknownforitsincreaseinheatstress. Theseresultsareinagreementwiththe
resultsofMadrau,Piscopo,Sanguinetti,DelCaro,Poiana,RomeoandPiga[21],whoshowedthatheat
stressincreasedtheantioxidantactivityoftheapricotfruits. Theseresultsindicatethatapricotleaves
havehighamountsofphenoliccompoundsandimportantcarotenoids,withhighlevelsofantioxidant
activityandthuscouldbeusedasapotentialbiologicalsourceofnaturalantioxidantsubstances.
5. Conclusions
In conclusion, apricot leaves were collected at four maturation stages. HPLC-DAD analyses
revealedtwelvepolyphenoliccompoundsintheapricotleaves. Themajorphenoliccompoundswere
3-O-caffeoylquinicacid,4-O-caffeoylquinicacid,5-O-caffeoylquinicacidandquercetin-3-O-glucosides.
Otherphenoliccompoundsincludedcoumaricacidoritsderivatives,kaempferoloritsderivatives,
andcaffeicacid. Significantchangeswereobservedinthepolyphenoliccompoundsduringmaturation.
Lutein,neoxanthin,α-,andβ-caroteneswerethemajorcarotenoids. Thereweresignificantvariations
inthecarotenoids,pigmentcontents,totalphenoliccontentsandradicalscavengingactivityduring
maturation. Inconclusion,apricotleavesareagoodsourceofpolyphenoliccompounds&carotenoids
andthechangesinthesecompoundsmayaffectfruitdevelopmentandthusshouldbeconsideredfor
thebestagriculturepracticeofthisplant.
Acknowledgments:TheworkwascarriedoutunderNRPUprojectNo.2344andtherefore,theHigherEducation
CommissionofPakistanisgreatlyacknowledged.
AuthorContributions:A.Z.andW.A.designedtheexperiments,N.K.&A.Z.carriedouttheexperiments,A.Z.
wrotethemanuscript.Allauthorscontributedequallytothisworkandagreeuponthisfinalversionofmanuscript.
ConflictsofInterest:Theauthorsdeclarenoconflictofinterest.
Abbreviations
Thefollowingabbreviationsareusedinthismanuscript:
HPLC-DAD Highperformanceliquidchromatographywithdiodearraydetection
TPC Totalphenoliccontents
RSA Radicalscavengingactivity
GAE Gallicacidequivalent
DPPH Diphenylpicrylhydrazineradical
ANOVA Analysisofvariance