Research Article Phenolic Composition and Antioxidant...

Research ArticlePhenolic Composition and Antioxidant Activity ofMalus domestica Leaves

Mindaugas Liaudanskas1 Pranas Viškelis2 Raimondas Raudonis1

Darius Kviklys2 Norbertas Uselis2 and Valdimaras Janulis1

1 Department of Pharmacognosy Faculty of Pharmacy Lithuanian University of Health Sciences Eiveniu Street 450161 Kaunas Lithuania

2 Institute of Horticulture Lithuanian Research Centre for Agriculture and Forestry Kauno Street 30 Babtai 54333 Kaunas Lithuania

Correspondence should be addressed to Mindaugas Liaudanskas mliaudanskasyahoocom

Received 2 June 2014 Revised 11 August 2014 Accepted 25 August 2014 Published 11 September 2014

Academic Editor Wanchai De-Eknamkul

Copyright copy 2014 Mindaugas Liaudanskas et alThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in anymedium provided the originalwork is properly cited

The aim of this study was to determine the composition and content of phenolic compounds in the ethanol extracts of apple leavesand to evaluate the antioxidant activity of these extractsThe total phenolic content was determined spectrophotometrically as wellas the total flavonoid content in the ethanol extracts of apple leaves and the antioxidant activity of these extracts by the ABTSDPPH and FRAP assays The highest amount of phenolic compounds and flavonoids as well as the highest antioxidant activitywas determined in the ethanol extracts obtained from the apple leaves of the cv Aldas The analysis by the HPLC method revealedthat phloridzin was a predominant component in the ethanol extracts of the apple leaves of all cultivars investigatedThe followingquercetin glycosides were identified and quantified in the ethanol extracts of apple leaves hyperoside isoquercitrin avicularinrutin and quercitrin Quercitrin was the major compound among quercetin glycosides

1 Introduction

Studies on the chemical biodiversity of plants are recognizedas being relevant and are carried out with the aim ofenriching the assortment of raw medicinal plant materialsand to evaluate their potential application to the demandsof practical medicine A search for plants accumulatingphenolic compounds which have recently been consideredas an object ofmany scientific studies is especially promisingIt is of importance to assess the composition and content ofphenolic compounds in plant vegetative organs to determinethe patterns of their accumulation and identify new promis-ing sources of plant phenolic compounds

The domestic apple (Malus domestica Borkh) is one ofthe most widely cultivated fruit trees Although the chemicalcomposition of apples has been extensively investigated [1 2]we have failed to find any data on the composition andcontent of phenolic compounds in the leaves of differentapple cultivars grown in Lithuania Comprehensive data ofscientific research on the variation in the composition and

content of phenolic compounds would allow conducting pur-poseful studies leading to the usage of the raw materialobtained from apple leaves as a potential source of phenoliccompounds in practical medicine Biologically active com-pounds could lead to production of dietary supplementsand cosmetic preparations enriched in phenolic compoundsfound in apple leaves Small-scale studies on the chemicalcomposition of leaves have been published where phloretinglycosides phenolic acids catechins and some quercetin gly-cosides were identified as the main phenolic compounds [3ndash5] Other studies on the composition and content of phenoliccompounds in apple leaves were conducted in relation toVenturia inaequalis-caused infections in the vegetative organsof apple trees Phenolic compounds accumulated in fruitplants play an important role in the plant defense mechanismagainst different fungal diseases and different stresses [6ndash9]

Phenolic compounds acting as natural antioxidantsscavenge free radicals and inhibit their production stimulatethe synthesis of antioxidant enzymes and thus prevent oxida-tive stress resulting in damage to the structural molecules

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 306217 10 pageshttpdxdoiorg1011552014306217

2 The Scientific World Journal

of the body [10ndash12] The antioxidant activity of phenoliccompounds is associated with other biological propertiesof these compounds such as anti-inflammatory antimicro-bial anticancer cardiovascular system-improving and otheractivities [13ndash15] Apple leaves accumulate high amounts ofphloridzin [16 17] which exhibit antidiabetic activity [18]Therefore plant raw materials accumulating this compoundcan be potentially useful for the prevention of diabetesmellitus

Therefore the aimof this studywas to determine the com-position and content of phenolic compounds in the ethanolextracts of apple leaves harvested from the cultivars AldasAuksis Ligol and Lodel grown under Lithuanian climaticconditions to assess the variation of phenolic compoundsand to compare their antioxidant activity in the apple leafsamples obtained from different cultivars

2 Materials and Methods

21 Plant Material The following apple cultivars wereincluded into this study Auksis (early winter cv bred inLithuania) and Ligol (winter cv bred in Poland) that aretwo main cultivars in commercial apple orchards as wellas Aldas (early winter cv bred in Lithuania) and Lodel(winter cv bred in Poland) two cultivars resistant to applescab (Venturia inaequalis) Cultivars Aldas and Lodel arerecommended for organic orchards The apple trees weregrown in the experimental orchard of the Institute of Hor-ticulture Lithuanian Research Centre for Agriculture andForestry Babtai Lithuania (55∘601015840N 23∘481015840E) Aldas block4 row 16 trees 5ndash8 Auksis block 2 row 3 trees 25ndash28Ligol block 2 row 4 trees 21ndash24 Lodel block 4 row 5trees 12ndash15 The altitude of Babtai town is 57m above sealevel Average annual precipitation is 630mm and averagesum of active temperatures is (gt10∘C)ndash2300∘ Temperaturesduring the experiment year were close to long term averageThe summer of 2012 was characterised by more rainfall inApril June and July Trees were trained as slender spindlePest and disease management was carried out according tothe rules of the integrated plant protectionThe experimentalorchard was not irrigated Tree fertilization was performedaccording to soil and leaf analysis Nitrogen was appliedbefore flowering at the rate of 80 kgha and potassium wasapplied after harvest at the rate of 90 kgha Soil conditions ofthe experimental orchard were as follows clay loam pH 73organic matter 28 P

2O5 255mgkg and K

2O 230mgkg

Apple leaves were harvested from 10-year-old apple trees in2012The study sample comprised 20 healthy fully developedleaves collected from different places of each apple tree ofeach cultivar Apple leaves were lyophilized with a ZIRBUSsublimator 3 times 4 times 520 (ZIRBUS technology Bad GrundGermany) at a pressure of 001mbar (condenser temperaturendash85∘C) The lyophilized apple leaves were ground to a finepowder by using a Retsch 200 mill (Haan Germany) Losson drying before analysis was determined by the method ofthe European Pharmacopoeia [19] Data were recalculated forabsolute dry lyophilizate weight

22 Chemicals All the solvents reagents and standardsused were of analytical grade Acetonitrile and acetic acidwere obtained from Sigma-Aldrich GmbH (Buchs Switzer-land) and ethanol from Stumbras AB (Kaunas Lithuania)Hyperoside rutin quercitrin phloridzin phloretin caf-feic acid and chlorogenic acid standards were purchasedfrom Extrasynthese (Genay France) (+)-catechin and (ndash)-epicatechin from Fluka (Buchs Switzerland) and avicu-larin and isoquercitrin from Chromadex (Santa Ana USA)11-Diphenyl-2-picrylhydrazyl (DPPH∙) radical 221015840-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) potas-sium persulfate sodium acetate trihydrate iron (III) chloridehexahydrate and 246-tripyridyl-s-triazine (TPTZ) wereobtained from Sigma-Aldrich (Steinheim Germany) Folin-Ciocalteu reagent gallic acid monohydrate sodium carbon-ate aluminum chloride hexahydrate and hexamethylenete-tramine were purchased from Sigma-Aldrich GmbH (BuchsSwitzerland) Deionized water produced by the Crystal Ehigh-performance liquid chromatography (HPLC AdronaSIA Riga Latvia) water purification system was used

23 Extraction An amount of 025 g of lyophilized appleleaf powder (exact weight) was weighed added to 10mLof ethanol (70 vv) and extracted in a Sonorex Digital10 P ultrasonic bath (Bandelin Electronic GmbH amp CoKG Berlin Germany) for 40 minutes at 60∘C The extractobtained was centrifuged for 7 minutes at 6000 rpm with aHermle Z206A centrifuge (Denville Scientific Inc USA)Theextract was collected and filtered through a membrane filterwith a pore size of 022120583m (Carl Roth GmbH KarlsruheGermany)

24 Spectrophotometric Studies

241 Determination of Total Phenolic and Flavonoid Con-tent All the spectrophotometric measurements were carriedout with a Genesys-10UVVis spectrophotometer (ThermoSpectronic Rochester USA) The total phenolic content(mg GAEg DW) in the ethanol extracts of apple leaveswas determined by the Folin-Ciocalteu method [20] Thetotal amount of flavonoids in the ethanol extracts of appleleaves was determined using the describedmethodology [21]calculated from a rutin calibration curve and expressed asmgg rutin equivalent (RE) per gram of absolutely dry weight(DW) (mg REg DW)

242 Determination of Antioxidant Activity

(1) DPPH∙ Free Radical Scavenging Assay The DPPH∙ freeradical scavenging activity was determined using the methodproposed by Brand-Williams et al [22] DPPH∙ solution in963 vv ethanol (3mL 6times10minus5M)was mixed with 10120583L ofthe ethanol extract of apple leaves A decrease in absorbancewas determined at a wavelength of 515 nm after keeping thesamples for 30 minutes in the dark

(2) ABTS∙+ Radical Cation Decolorization Assay An ABTS∙+radical cation decolorization assay was applied according tothemethodology described byRe et al [23] A volumeof 3mL

The Scientific World Journal 3

of ABTS∙+ solution (absorbance 0800plusmn 002) wasmixedwith10 120583L of the ethanol extract of apple leaves A decrease inabsorbance was measured at a wavelength of 734 nm afterkeeping the samples for 30 minutes in the dark

(3) FRAP Assay The ferric reducing antioxidant power(FRAP) assay was carried out as described by Benzie andStrain [24] The working FRAP solution included TPTZ(001M dissolved in 004MHCl) FeCl

3sdot6H2O (002M in

water) and acetate buffer (03M pH 36) at the ratio of1 1 10 A volume of 3mL of a freshly prepared FRAP reagentwas mixed with 10120583L of the apple leaf extract An increase inabsorbance was recorded after 30 minutes at a wavelength of593 nm

(4) Calculation of Antioxidant Activity of the Ethanol Extractof Apple Leaves The antioxidant activity of extracts wascalculated from Trolox calibration curve and expressed as120583mol Trolox equivalent (TE) per gram of absolutely dryweight (DW) TE was calculated according to the formulaTE = 119888 times 119881119898 (120583molg) 119888 the concentration of Troloxestablished from the calibration curve (in 120583M)119881 the volumeof leaf extract (in L)119898 the weight (precise) of lyophilized leafpowder (in g)

25 High-Performance Liquid Chromatography Qualitativeand quantitative analysis of phenolic compounds was per-formed according to the previously validated and describedhigh-performance liquid chromatography (HPLC) method[25] A Waters 2695 chromatograph equipped with a Waters2998 photodiode array (PDA) detector (Waters MilfordUSA) was used for HPLC analysis Chromatographic separa-tions were carried out by using a YMC-Pack ODS-A (5 120583mC18 250 times 46mm id) column equipped with a YMC-Triart (5 120583m C18 10 times 30mm id) precolumn (YMC EuropeGmbH Dinslaken Germany) The column was operated ata constant temperature of 25∘C The volume of the extractbeing investigated was 10 120583L The flow rate was 1mLminand gradient elution was used The mobile phase consistedof 2 (vv) acetic acid in water (solvent A) and 100 (vv)acetonitrile (solvent B) The following conditions of elutionwere applied 0ndash30 minutes 3ndash15 B 30ndash45 minutes 15ndash25B 45ndash50minutes 25ndash50B and 50ndash55minutes 50ndash95 B

The identification of the chromatographic peaks wasachieved by comparing the retention times and spectralcharacteristics (120582 = 200ndash600 nm) of the eluting peaks withthose of reference compounds The compounds identifiedwere confirmed by spiking the sample with the standardcompound andmonitoring the changes in the peak shape andspectral characteristics For quantitative analysis a calibra-tion curvewas obtained by injection of known concentrationsof different standard compounds Dihydrochalcones and cat-echins were quantified at 280 nm phenolic acids at 320 nmand flavonols at 360 nm

26 Statistical Data Processing Methods All the experimentswere carried out in triplicate Means and standard deviationswere calculated with the SPSS 200 software (Chicago USA)

Table 1 The total amounts of phenolic compounds (TP) andflavonoids (TFd) in the ethanol extracts of apple leaves of differentcultivars

cv TP (mgGAEgDW)a TFd (mgREgDW)a

Aldas 16335 plusmn 436 4502 plusmn 090

Auksis 9881 plusmn 151 2159 plusmn 052

Ligol 10793 plusmn 294 2697 plusmn 063

Lodel 15986 plusmn 402 3964 plusmn 131

119875 lt 005b

119875 lt 005b

aValues are means plusmn standard deviations (119899 = 3)bBy ANOVA test

A single factor analysis of variance (ANOVA) along withthe post hoc Tukey test was employed for statistical analysisThe Kolmogorov-Smirnov test was applied to examine thenormality of distribution To verify the hypothesis aboutthe equality of variances Levenersquos test was employed Thecorrelation was evaluated by Pearson analysis Differences at119875 lt 005 were considered to be significant

3 Results and Discussion

31 Determination of Total Phenolic and Flavonoid ContentsIn order to determine the patterns of the accumulation ofbiologically active compounds in plants it is important toidentify their composition and content in separate plantorgans The secondary metabolites of plant metabolismphenolic compounds have been detected in apple leaves[3 16] therefore this study aimed at determining the com-position and content of phenolic compounds in plants and atdetermining the patterns of their variation and accumulation

In this study the total amount of phenolic compoundsin the ethanol extracts of apple leaves varied from 9881 plusmn151mg GAEg DW (cv Auksis) to 16335 plusmn 436mg GAEgDW (cv Aldas) (Table 1) In a study by Iqbal et al [26]the total phenolic content in the ethanol extracts of appleleaves was 15706mgGAEg DW meanwhile one Slovenianstudy reported a lower total phenolic content in apple leavesranging from 80mgGAEg DW to 115mgGAEg DW [16]Both scab-resistant cultivars Aldas and Lodel accumulatedsignificantly higher amounts of total phenolics and totalflavonoids Such differences among the cultivars susceptibleand resistant to apple scab have been established in otherstudies as well [27]

Studies investigating the total phenolic content in the leafsamples of other plants belonging to the Rosaceae family havebeen conducted as well Methanol extracts from the leavesof various Sorbus species were found to have lower totalamounts of phenolics ranging from 606 to 909mgGAEgDW [28] Hua et al [6] who investigated the samples ofpear leaves reported a lower total phenolic content (681ndash833mgGAEg DW)

The biological effects of many plant rawmaterials dependon flavonoids therefore studies on the variation in theircontent are important and relevant This study determinedthe total flavonoids content in the ethanol extracts of appleleaves which ranged from 2159 plusmn 052mgREg DW (cv


Auksis) to 4502 plusmn 090mgREg DW (cv Aldas) (Table 1)Iqbal et al who investigated the ethanol extracts of appleleaves reported a higher total flavonoids content reaching12186mgREg DW [26] In contrast to that the ethanolextracts of hawthorn leaves were found to have a lower totalflavonoids content (95ndash130mgREg DW) [21]

The data on the patterns of variation in the total phenolicand flavonoid contents of apple leaves are scarce Thereforethis study provides new knowledge about total phenolics andflavonoids content in the apple leaves of the cultivars grownunder Lithuanian climatic conditions allows the comparisonof the results obtained with those of other studies and isvaluable to carrying out a search for promising biologicallyactive substance-accumulating plant raw materials

32 Measurements of Antioxidant Activity in Extracts Afterstudying the total phenolics and flavonoids content of appleleaves harvested fromdifferent cultivars grown under Lithua-nian climatic conditions it is important to examine and assessthe antioxidant activity in the extracts of apple leaves Theresults obtained during studies will be useful for the selectionof apple cultivars in order to provide a consumer with prod-ucts rich in antioxidants will be useful for the assessment andstandardization of quality of plant raw materials and theirproducts and will allow predicting an antioxidant effect ofapple leaves in vivo

Herbal extracts are multicomponent matrices withantioxidant activity determined by the set of different mech-anism reactions so antioxidant effect cannot be adequatelytested using only one method [29 30] For these reasons itis recommended to use at least two different methods fordetermination of antioxidant activity in herbal extracts [31]In order to thoroughly evaluate the antioxidant activity ofthe ethanol extracts of apple leaves different antioxidantcapacity assays (DPPH ABTS and FRAP) were employedThe in vitro antioxidant effect of the investigated extracts wasevaluated by the DPPH and ABTS assays as a capability ofDPPH∙ and ABTS∙+ compounds possessing an antiradicalactivity to scavenge free radicals [22 23] and by the FRAPassay as a capability of antioxidants to reduce Fe(III) to Fe(II)[24]The results of in vitro antioxidant activity determined inthe ethanol extracts of apple leaves (cultivars Aldas AuksisLigol and Lodel) are summarized in Figure 1

It has been previously reported that antioxidant capacitydetermined by in vitro assays differs [32ndash34] TE valuesobtained from samples of apple leaf extracts antioxidantactivity studies according to the used methods can bearranged in the following order DPPH lt ABTS lt FRAP(Figure 1) This pattern could be explained by differences ofapplied antioxidant activity determination methods FRAPassay is a method for measuring total reducing power of elec-tron donating substances whilst ABTS and DPPH assays aremethods formeasuring the ability of antioxidantmolecules toscavenge ABTS∙+ and DPPH∙ free radicals respectively [3536] ABTS and DPPHmethods have several differences fromone another which causes their TE values to be differentSome authors indicate that TE or VCE values determined byDPPH method are lower than those determined by ABTS

0

50

100

150

200

250

300

350

400

Aldas Auksis Ligol LodelApple cultivar

DPPH

ABTS

FRAP

TE (120583

mol

g D

W)

Figure 1The antioxidant activity of ethanol extracts obtained fromapple leaves and determined by using DPPH ABTS and FRAPassays Values aremeans and errors bars indicate standard deviations(119899 = 3)

method [37ndash40] probably because the DPPH method hasmore limitations ABTS radical cation (ABTS∙+) is solublein water and organic solvents which allows determiningantiradical activity of the hydrophilic and lipophilic com-pounds [30 41] DPPH∙ radicals are only soluble in organicsolvents which limits the evaluation of antioxidant activity ofhydrophilic antioxidants [36 42]Wang et al found that someof the compounds which have ABTS∙+ scavenging activitymay not show DPPH∙ scavenging activity [43] Arts et alreported that some products of ABTS∙+ scavenging reactionmay have a considerable contribution to the antioxidantcapacity and can continually react with ABTS∙+ [44]

The analysis of the antioxidant activity in apple leavesrevealed differences in the antioxidant activity among theethanol extracts of the apple leaves of the cultivars inves-tigated (Figure 1) The ethanol extracts obtained from theapple leaves of the cv Aldas showed the highest TE valuesThey reached 14195 28023 and 35554 120583moLg DW forthe DPPH ABTS and FRAP assays respectively To ourknowledge no studies on the antioxidant activity of appleleaves have been carried out so far Therefore the findingsfrom this study on antioxidant activity were compared withthose obtained in studies on other plants belonging to theRosaceae family The methanol extracts obtained from theleaves of different Sorbus species showed a higher antioxidantactivity that is the TE values were higher than thosedetermined in the present study 3440ndash6283 120583moLg DWby the DPPH assay 2625ndash4673120583moLg DW by the ABTSassay and 8616ndash16504 120583moLg DW by the FRAP assay[28] These results could be explained by the fact that appleleaves accumulate high amounts of phloridzin belonging tothe dihydrochalcone class [3 16] which possesses a lowerantioxidant activity than the phenolic compounds belongingto other classes [4 45]

In order to determine the relationship between the anti-oxidant activities of the ethanol extracts of apple leaves


(AU

)

000005010015020025030035040

(min)2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 4600 4800 5000 5200

31

24 5

6

78

9

10 11

Figure 2 Chromatogramof ethanol extract of apple leaf sample (cv Aldas) investigated (120582= 320 nm) Numbers indicate the peaks of analytes1 (+)-catechin 2 chlorogenic acid 3 caffeic acid 4 (ndash)-epicatechin 5 rutin 6 hyperoside 7 isoquercitrin 8 avicularin 9 quercitrin 10phloridzin and 11 phloretin

Table 2 Content of quercetin glycosides in ethanol extracts obtained from the apple leaves of cultivars grown in Lithuania

Compound Content of quercetin glycosides mgg (expressed for absolute dry weight)a

Aldas Auksis Ligol LodelHyperoside 895 plusmn 035a 567 plusmn 020b 459 plusmn 017c 703 plusmn 020d

Isoquercitrin 348 plusmn 012a 279 plusmn 011b 184 plusmn 007c 240 plusmn 008d

Rutin 033 plusmn 001a 075 plusmn 003b 067 plusmn 002c 054 plusmn 002d

Avicularin 248 plusmn 010a 282 plusmn 010b 209 plusmn 007c 251 plusmn 008d

Quercitrin 1336 plusmn 051a 1029 plusmn 048b 777 plusmn 027c 1231 plusmn 059daValues are means plusmn standard deviations (119899 = 3) The different letters indicate significant differences between the values (119875 lt 005)

assessed by the DPPH ABTS and FRAP assays and totalphenolic as well as flavonoid contents in these extracts thecorrelation analysis was carried out There was a strong pos-itive correlation between total phenolic as well as flavonoidcontents and the antioxidant activity assessed by all themethods (119903 = 084minus098119875 lt 005)The strongest correlationwas found between the antioxidant activity of the ethanolextracts of apple leaves determined by the FRAPmethod andtotal flavonoid content (119903 = 096 119875 lt 005) as well as totalphenolic content (119903 = 098 119875 lt 005) in these extracts Otherstudies published earlier also reported strong correlativerelationships between phenolic as well as flavonoid contentsand the antioxidant activity of plant extracts evaluated bydifferent assays [46ndash48]

33 Identification and Quantification of Phenolic Compoundsby HPLC The phenolic compounds of various groups (+)-catechin chlorogenic acid caffeic acid (ndash)-epicatechin rutinhyperoside isoquercitrin avicularin quercitrin phloretinand phloridzin in the analyzed ethanol extracts obtainedfrom the apple leaves of the cultivars Aldas Auksis Ligoland Lodel were identified by the HPLC method The valuesof resolution (Rs gt 2) were achieved in all the samples ofextracts The applied HPLC method allowed for an effectiveseparation of quercetin glycosides the separation of rutinand hyperoside in ethanol extracts of lyophilized appleleaf samples with the resolution of 263 the separation ofhyperoside and isoquercitrin with the resolution of 328 and

the separation of avicularin and quercitrinwith the resolutionof 294 The chromatograms of ethanol extracts of appleleaf samples obtained from all the cultivars investigated areidentical regarding the number of analytes and retentiontimeThe example of chromatogram of the ethanol extract ofthe apple leaf sample (cv Aldas) is shown in Figure 2

The apple leaves of the cv Aldas contained the highesttotal amount of the quercetin glycosides identified andquantified It was 17 times higher than their lowest amountfound in the apple leaves of the cv Ligol (Table 2) Quercitrinwas a predominant component among all the quercetinglycosides identified and quantified in the ethanol extractsof apple leaves The apple leaves of the cv Aldas had thehighest amount of quercitrin The results obtained are inagreement with literature data that quercitrin is one of themain flavonols found in apple leaves [5 49]The results of thisstudy are in line with those reported by the Slovenian studywhere the quercitrin content in the apple leaves collectedin the Maribor region ranged from 63mgg to 108mgg(cv Golden Delicious) and from 37mgg to 85mgg (cvJonagold) [49] The factors such as a geographical regionand climatic-meteorological and cultivation conditions couldinfluence these quantitative differences

Rutin was the minor component among all the quercetinglycosides quantified in all ethanol extracts of the apple leafsamples analyzed The results obtained are confirmed bypreviously published data on the variation in the compositionand content of quercetin glycosides in apple leaves [5]


The highest amount of rutin was detected in the apple leavesof the cv Auksis (Table 2) However the Slovenian studyreported contrary findings on the flavonol content in appleleaves The authors of that study demonstrated that rutinalong with quercitrin was one of the major compounds inapple leaves [49]

When comparing quercetin glycoside content and com-position variation in apple fruits and leaves grown in Lithua-nian climatic conditions it was found that apple leaves havehigher amounts of these compounds For example cv Aldascv Auksis and cv Ligol apple fruits had hyperoside amountsfrom 005mgg (cv Auksis) to 019mgg (cv Aldas) [25]which constitutes only 09ndash21 hyperoside content foundin leaves of the same apple cultivars Similar quantitativedifferences between apple fruit and leaf grown in Lithuaniaare also typical in other quercetin group compounds

All the quercetin glycosides identified and quantified inthe ethanol extracts of apple leaf samples can be rankedin the following ascending order by their content rutin ltisoquercitrinlt avicularin lt hyperoside lt quercitrin Thisorder was characteristic of the apple leaves of the cultivarsAuksis Ligol and Lodel In the apple leaves of the cvAldas the amount of isoquercitrin was higher than that ofavicularin

Phloridzin and phloretin which belong to the dihy-drochalcone class were identified in the ethanol extractsof apple leaf samples Phloridzin was the major phenoliccompound in the apple leaves It accounted for 769 to842 of all phenolic compounds identified and quantifiedin the extracts of apple leaves by the HPLC method Theresults obtained confirm literature data that phloridzin is apredominant component of phenolic compounds in appleleaves [16 17] Determined phloretin levels were significantlylower They make up 10ndash18 of all identified and quan-tified phenolic compounds This could be interpreted byphloretin and phloridzin molecules structural differencesPhloridzin is phloretin glycoside which has glucose linkedat hydroxy group in moleculersquos 21015840 position Gosch et alresearched dihydrochalcone synthesis processes in appleleaves and found that phloridzin is formed from phloretinby glycosylation reaction affected by enzyme dihydrochal-cone 21015840-O-glucosyltransferase [17] Sugar molecule link tothe aglycone influences the physical-chemical propertiesof flavonoid Commonly flavonoid glycosides have lowerantioxidant activity and are more hydrophilic than theirrespective aglycones [50 51] These characteristics relate tothe fact that the flavonoid aglycone has more free hydroxygroups than their corresponding glycosides which have oneor several hydroxy groups with bonded sugar [52 53] Rezket al indicates that formation of phloridzin by glycosylationof hydroxy group at phloretin moleculersquos 21015840 position reducesits antioxidant activity 18 times compared to phloretin[54] Other researchers who conducted comparative studiesof phloretin and phloridzin anti-inflammatory effects andtheir influence on lipid oxidation report similar patternsPhloretin has significantly stronger anti-inflammatory [55]and lipid oxidation inhibitory effect [56] than phloridzinThe flavonoid aglycone reactivity explains why glycosides arethe most common flavonoid in plants [57 58] This pattern

is also characteristic for dihydrochalcone group compoundsin apple leavesmdashglycoside phloridzin levels are significantlyhigher than its aglycone phloretin levels [3 59]

Phloridzin has a wide spectrum of biological effectsinhibits the growth of cancer cells [60] improves memory[61 62] and is useful in the bone fracture prevention [63 64]One of the most important and potentially valuable phlo-ridzin biological effects is antidiabetic activity [18 65] Mainphloridzin pharmacological mechanism of action leadingto its antidiabetic effect is to produce renal glycosuria andblock intestinal glucose absorption through inhibition ofsodium-glucose symporters in kidneys and small intestine[66] The principle uses of phloridzin are associated with itsability to reduce plasma glucose without changing insulinlevels [67 68] Phloridzin ability to reverse glucotoxicity andreduce blood glucose levels without increasing body weightdetermines its benefits in prophylaxis and treatment of type 2diabetes [69ndash71] Phloridzin reduces bodyweight by blockingthe absorption and resorption of glucose [72 73] and theweight loss is one of the most important type 2 diabetesprevention methods [74 75] Phloridzin consumption toreduce glucose concentration in blood plasma does not causebody fluid loss and hypoglycemia risk [66 76] For thereasons stated above it is purposeful to investigate plantmaterials and extracts that accumulate phloridzinWe believeit is a promising research in developing medicines and foodsupplements for body weight reduction in the prevention ofdiabetes Our assumption is supported by other scientistrsquosresearch which offers to use apple leaves to enrich phenoliccompounds composition with phloridzin in apple juice [77]

The apple leaves of the cv Ligol contained the highest con-centration of phloridzinThe identified amounts of phloretinwere lower than those of phloridzin (Table 3) Such trends inthe content of compounds belonging to this dihydrochalconeclass have been demonstrated by other studies as well [3] Incontrast to the leaves of apple cultivars grown in Lithuaniaphloridzin was not the dominant compound in apple fruitsDetermined phloridzin levels were 006ndash014mgg [25] andit was only 39ndash55 of identified and quantified phenoliccompounds tested in varieties cv Aldas cv Auksis and cvLigol There was also a distinction of composition of phe-nolic compounds between the apple fruit and the apple leafsamples Phloretin was identified only in apple leaf samplesbut not in apple fruit samples [25 78]

The highest total amount of phenolic acids identified andquantified by the HPLCmethod (161 plusmn 007mgg) was foundin the apple leaves of the cv Auksis (Table 3) The ethanolextracts of apple leaves contained caffeic acid its amount waslower than that of chlorogenic acid The levels of chlorogenicacid were similar or slightly higher than those reportedby the earlier studies [49 79] Fruit and leaf compositionand content of apples cultivated in Lithuanian climaticconditions differed In cv Aldas cv Auksis and cv Ligolapples chlorogenic acid was the predominant compounddetermined amounts (069ndash223mgg) were 548ndash696 ofall identified and quantified phenolic compounds [25] Inapple leaf extracts phloridzin was dominant and chlorogenicacid was only 03ndash10 of all identified phenolic compounds


Table 3 Content of dihydrochalcones phenolic acids and catechins in ethanol extracts obtained from the apple leaves of cultivars grown inLithuania

Compound Content of dihydrochalcones phenolic acids and catechins mgg (expressed for absolute dry weight)a

Aldas Auksis Ligol LodelPhloridzin 10601 plusmn 423a 1089 plusmn 432b 11443 plusmn 472c 10951 plusmn 462d

Phloretin 181 plusmn 007a 152 plusmn 006b 240 plusmn 009c 140 plusmn 006d

Chlorogenic acid 048 plusmn 002a 138 plusmn 006b 112 plusmn 005c 086 plusmn 003d

Caffeic acid 026 plusmn 002a 023 plusmn 003b 015 plusmn 001c 014 plusmn 001d

(+)-Catechin 005 plusmn 001a 027 plusmn 002b 009 plusmn 001c 017 plusmn 001d

(minus)-Epicatechin 072 plusmn 002a 038 plusmn 002b 076 plusmn 002c 039 plusmn 001daValues are means plusmn standard deviations (119899 = 3) The different letters indicate significant differences between the values (119875 lt 005)

Composition differences were also determined apple leavescontained caffeic acid while apple fruits did not

The chemical composition of catechins (monomericflavan-3-ols) in ethanol extracts obtained from the appleleaves of the cultivars Aldas Auksis Ligol and Lodel wasstudied The highest and lowest total amounts of the cat-echins identified were found in the apple leaves of thecultivars Ligol and Lodel respectively (085 plusmn 004mggversus 056 plusmn 002mgg) (Table 3) The amounts of (ndash)-epicatechin in the extracts studied were higher than thoseof (+)-catechin Similar amounts of (ndash)-epicatechin werereported by other authors [3] (+)-Catechin (005ndash015mgg)and (ndash)-epicatechin (024ndash045mgg) levels determined inapple fruits of Lithuanian cultivated cv Aldas cv Auksisand cv Ligol were similar to those determined in appleleaves Composition differences were also determinedmdashprocyanidins (oligomeric flavan-3-ols) group compoundsprocyanidin B1 and procyanidin B2 were identified in applefruits [25 78] while in apple leaves they were not

The HPLC analysis of ethanol extracts obtained fromapple leaves revealed that phloridzin was the major com-pound in the samples investigated and its amounts wereconsiderably higher than those of other phenolic compoundsQuercitrin was a predominant component among quercetinglycosides The results of the HPLC analysis show that appleleaves are a valuable natural source of dihydrochalcones andquercetin glycosidesThis encourages further research on thisplant as a raw material for use in pharmacy

4 Conclusions

In conclusion the results of this study will provide newknowledge about the composition and content of phenoliccompounds in apple leaves and the antioxidant activity oftheir extracts which will give a wide range of possibilities toemploy these plants as the source of phenolic compoundsThe highest total amounts of phenolic compounds andflavonoids were determined in the apple leaves of the cvAldas (16335plusmn 436mgGAEgDWand4502plusmn 090mgREgDW resp) Phloridzin was the major compound in theethanol extracts of apple leaves of all the cultivars investi-gatedThe apple leaves of the cv Ligol had the highest amountof phloridzin (11443 plusmn 472mgg DW) Quercitrin was thepredominant component among the quercetin glycosides

identified and quantified in ethanol extracts and its amountin the apple leaves of different cultivars ranged from 777 to1336mgg DW

The preliminary in vitro experiments examining theantioxidant activity of apple leaf extracts by theABTSDPPHand FRAP assays have shown that these extracts possess astrong antioxidant activity which positively correlated withthe total phenolic and flavonoid contents (119903 = 084ndash098119875 lt 005)The ethanol extracts obtained from the apple leavesof the cv Aldas showed the highest TE values 14195 120583molgDW by the DPPH assay 28023 120583molg DW by the ABTSassay and 35554120583molg DW by the FRAP assay

The results reported in this study prompt further researchon the chemical composition and biological effect of appleleaves by evaluating the antioxidant activity of individual phe-nolic compounds in vitro and in vivo and confirm a potentialof these plants as a rawmaterial in medical practice as well asthe development and production of dietary supplements andcosmetic preparations rich in biologically active compounds

Abbreviations

ANOVA A single factor analysis of varianceABTS 221015840-azino-bis(3-ethylbenzothiazoline-6-

sulphonic acid)cv CultivarDPPH 22-diphenyl-1-picrylhydrazylDW Dry weightFRAP Ferric reducing antioxidant powerGAE Gallic acid equivalentHPLC High-performance liquid chromatographyRs ResolutionTE Trolox equivalentTFd Total amount of flavonoidsTP Total amount of phenolic compoundsTPTZ 246-tripyridyl-s-triazineVCE Vitamin C equivalent

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper


Acknowledgments

This study was supported by the Foundation of the Lithua-nian University of Health Sciences and a Grant from theResearch Council of Lithuania (no SVE-022011)

References

[1] J Wu H Gao L Zhao et al ldquoChemical compositional charac-terization of some apple cultivarsrdquo Food Chemistry vol 103 no1 pp 88ndash93 2007

[2] M Ceymann E Arrigoni H Scharer A Bozzi Nising and RF Hurrell ldquoIdentification of apples rich in health-promotingflavan-3-ols and phenolic acids by measuring the polyphenolprofilerdquo Journal of Food Composition and Analysis vol 26 no1-2 pp 128ndash135 2012

[3] A Picinelli E Dapena and J J Mangas ldquoPolyphenolic patternin apple tree leaves in relation to scab resistance a preliminarystudyrdquo Journal of Agricultural and Food Chemistry vol 43 no8 pp 2273ndash2278 1995

[4] D Bonarska-Kujawa S Cyboran J Oszmianski and HKleszczynska ldquoAntioxidant properties of apple leaves and fruitsextracts from apple leaves and fruits as effective antioxidantsrdquoJournal ofMedicinal Plants Research vol 5 no 11 pp 2339ndash23472011

[5] U Mayr S Michalek D Treutter and W Feucht ldquoPhenoliccompounds of apple and their relationship to scab resistancerdquoJournal of Phytopathology vol 145 no 2-3 pp 69ndash75 1997

[6] YHuaHGuo X-G Zhou et al ldquoCorrelations between solublesugar and phenol contents in leaves and pear scab resistancerdquoJournal of Plant Pathology amp Microbiology vol 5 no 1 pp 1ndash42014

[7] J Schovankova and H Opatova ldquoChanges in phenols composi-tion and activity of phenylalanine-ammonia lyase in apples afterfungal infectionsrdquo Horticultural Science vol 38 no 1 pp 1ndash102011

[8] R Veberic M Trobec K Herbinger M Hofer D Grill and FStampar ldquoPhenolic compounds in some apple (Malus domesticaBorkh) cultivars of organic and integrated productionrdquo Journalof the Science of Food and Agriculture vol 85 no 10 pp 1687ndash1694 2005

[9] U Mayr D Treutter C Santos-Buelga H Bauer and WFeucht ldquoDevelopmental changes in the phenol concentrationsof ldquogolden deliciousrdquo apple fruits and leavesrdquo Phytochemistryvol 38 no 5 pp 1151ndash1155 1995

[10] M S Fernandez-Panchon D Villano A M Troncoso and MC Garcia-Parrilla ldquoAntioxidant activity of phenolic com-pounds from in vitro results to in vivo evidencerdquo CriticalReviews in Food Science and Nutrition vol 48 no 7 pp 649ndash671 2008

[11] B Halliwell J Rafter and A Jenner ldquoHealth promotion byflavonoids tocopherols tocotrienols and other phenols director indirect effects Antioxidant or notrdquo The American Journalof Clinical Nutrition vol 81 no 1 pp 268Sndash276S 2005

[12] N Balasundram K Sundram and S Samman ldquoPhenolic com-pounds in plants and agri-industrial by-products antioxidantactivity occurrence and potential usesrdquo Food Chemistry vol99 no 1 pp 191ndash203 2006

[13] G G Duthie S J Duthie and J A M Kyle ldquoPlant polyphenolsin cancer and heart disease implications as nutritional antiox-idantsrdquo Nutrition Research Reviews vol 13 no 1 pp 79ndash1062000

[14] Y Curin and R Andriantsitohaina ldquoPolyphenols as potentialtherapeutical agents against cardiovascular diseasesrdquo Pharma-cological Reports vol 57 pp 97ndash107 2005

[15] E Middleton Jr C Kandaswami and T C Theoharides ldquoTheeffects of plant flavonoids on mammalian cells implicationsfor inflammation heart disease and cancerrdquo PharmacologicalReviews vol 52 no 4 pp 673ndash751 2000

[16] M M Petkovsek A Slatnar F Stampar and R Veberic ldquoTheinfluence of organicintegrated production on the content ofphenolic compounds in apple leaves and fruits in four differentvarieties over a 2-year periodrdquo Journal of the Science of Food andAgriculture vol 90 no 14 pp 2366ndash2378 2010

[17] C Gosch H Halbwirth J Kuhn S Miosic and K StichldquoBiosynthesis of phloridzin in apple (Malus domestica Borkh)rdquoPlant Science vol 176 no 2 pp 223ndash231 2009

[18] S Masumoto Y Akimoto H Oike and M Kobori ldquoDietaryphloridzin reduces blood glucose levels and reverses Sglt1expression in the small intestine in streptozotocin-induceddiabetic micerdquo Journal of Agricultural and Food Chemistry vol57 no 11 pp 4651ndash4656 2009

[19] European Pharmacopoeia vol 1 Council of Europe StrasbourgFrance 7th edition 2010

[20] R Bobinait P Viskelis and P R Venskutonis ldquoVariation of totalphenolics anthocyanins ellagic acid and radical scavengingcapacity in various raspberry (Rubus spp) cultivarsrdquo FoodChemistry vol 132 no 3 pp 1495ndash1501 2012

[21] A Urbonaviciute V Jakstas O Kornysova V Janulis and AMaruska ldquoCapillary electrophoretic analysis of flavonoids insingle-styled hawthorn (Crataegus monogyna Jacq) ethanolicextractsrdquo Journal of Chromatography A vol 1112 no 1-2 pp339ndash344 2006

[22] W Brand-Williams M E Cuvelier and C Berset ldquoUse of a freeradical method to evaluate antioxidant activityrdquo LWTmdashFoodScience and Technology vol 28 no 1 pp 25ndash30 1995

[23] R Re N Pellegrini A Proteggente A PannalaM Yang andCRice-Evans ldquoAntioxidant activity applying an improved ABTSradical cation decolorization assayrdquo Free Radical Biology ampMedicine vol 26 no 9-10 pp 1231ndash1237 1999

[24] I F F Benzie and J J Strain ldquoFerric reducingantioxidant powerassay direct measure of total antioxidant activity of biologicalfluids and modified version for simultaneous measurementof total antioxidant power and ascorbic acid concentrationrdquoMethods in Enzymology vol 299 pp 15ndash27 1998

[25] M Liaudanskas P Viskelis V Jakstas et al ldquoApplication of anoptimized HPLC method for the detection of various phenoliccompounds in apples from Lithuanian cultivarsrdquo Journal ofChemistry vol 2014 Article ID 542121 10 pages 2014

[26] M Iqbal M Sharma R F Ali M Yousuf and A HussainldquoIn vitro antioxidant activity and spectrophotometric quan-tification of total phenolic and flavonoid contents of MalusdomesticardquoWorld Journal of Pharmaceutical Research vol 3 no1 pp 452ndash471 2013

[27] V Usenik M Mikulic-Petkovsek A Solar and F StamparldquoFlavonols of leaves in relation to apple scab resistancerdquo Journalof Plant Diseases and Protection vol 111 no 2 pp 137ndash144 2004

[28] M A Olszewska and P Michel ldquoAntioxidant activity of inflo-rescences leaves and fruits of three sorbus species in relation totheir polyphenolic compositionrdquoNatural Product Research vol23 no 16 pp 1507ndash1521 2009

[29] W Li A W Hydamaka L Lowry and T Beta ldquoComparisonof antioxidant capacity and phenolic compounds of berries


chokecherry and seabuckthornrdquo Central European Journal ofBiology vol 4 no 4 pp 499ndash506 2009

[30] R L Prior X Wu and K Schaich ldquoStandardized methodsfor the determination of antioxidant capacity and phenolicsin foods and dietary supplementsrdquo Journal of Agricultural andFood Chemistry vol 53 no 10 pp 4290ndash4302 2005

[31] K Schlesier M Harwat V Bohm and R Bitsch ldquoAssessmentof antioxidant activity by using different in vitromethodsrdquo FreeRadical Research vol 36 no 2 pp 177ndash187 2002

[32] P C Wootton-Beard A Moran and L Ryan ldquoStability ofthe total antioxidant capacity and total polyphenol content of23 commercially available vegetable juices before and after invitro digestion measured by FRAP DPPH ABTS and Folin-Ciocalteu methodsrdquo Food Research International vol 44 no 1pp 217ndash224 2011

[33] S E Celik M Ozyurek K Guclu and R Apak ldquoSolventeffects on the antioxidant capacity of lipophilic and hydrophilicantioxidants measured by CUPRAC ABTSpersulphate andFRAP methodsrdquo Talanta vol 81 no 4-5 pp 1300ndash1309 2010

[34] G-L Chen S-G Chen Y-Y Zhao C-X Luo J Li and Y-QGao ldquoTotal phenolic contents of 33 fruits and their antioxidantcapacities before and after in vitro digestionrdquo Industrial Cropsand Products vol 57 pp 150ndash157 2014

[35] M N Alam N J Bristi and M Rafiquzzaman ldquoReview on invivo and in vitro methods evaluation of antioxidant activityrdquoSaudi Pharmaceutical Journal vol 21 no 2 pp 143ndash152 2013

[36] R Apak K Guclu B Demirata et al ldquoComparative evaluationof various total antioxidant capacity assays applied to phenoliccompounds with the CUPRAC assayrdquo Molecules vol 12 no 7pp 1496ndash1547 2007

[37] K SMinioti andC A Georgiou ldquoComparison of different testsused in mapping the Greek virgin olive oil production for thedetermination of its total antioxidant capacityrdquoGrasas YAceitesvol 61 no 1 pp 45ndash51 2010

[38] A Floegel D-O Kim S-J Chung S I Koo and O K ChunldquoComparison of ABTSDPPH assays to measure antioxidantcapacity in popular antioxidant-rich US foodsrdquo Journal of FoodComposition and Analysis vol 24 no 7 pp 1043ndash1048 2011

[39] M M B Almeida P H M de Sousa A M C Arriaga et alldquoBioactive compounds and antioxidant activity of fresh exoticfruits from northeastern Brazilrdquo Food Research Internationalvol 44 no 7 pp 2155ndash2159 2011

[40] J Piljac-Zegarac T Stipcevic and A Belscak ldquoAntioxidantproperties and phenolic content of different floral origin hon-eysrdquo Journal of ApiProduct and ApiMedical Science vol 1 no 2pp 43ndash50 2009

[41] A Wojdyło J Oszmianski and R Czemerys ldquoAntioxidantactivity and phenolic compounds in 32 selected herbsrdquo FoodChemistry vol 105 no 3 pp 940ndash949 2007

[42] P-Y Chao S-Y Lin K-H Lin et al ldquoAntioxidant activity inextracts of 27 indigenous Taiwanese vegetablesrdquo Nutrients vol6 no 5 pp 2115ndash2130 2014

[43] M Wang J Li M Rangarajan et al ldquoAntioxidative phenoliccompounds from sage (Salvia officinalis)rdquo Journal of Agricul-tural and Food Chemistry vol 46 no 12 pp 4869ndash4873 1998

[44] M J T J Arts G R M M Haenen H-P Voss and ABast ldquoAntioxidant capacity of reaction products limits theapplicability of the Trolox Equivalent Antioxidant Capacity(TEAC) assayrdquo Food and Chemical Toxicology vol 42 no 1 pp45ndash49 2004

[45] Y Lu and L Y Foo ldquoAntioxidant and radical scavengingactivities of polyphenols from apple pomacerdquo Food Chemistryvol 68 no 1 pp 81ndash85 2000

[46] W Zheng and S Y Wang ldquoAntioxidant activity and phenoliccompounds in selected herbsrdquo Journal of Agricultural and FoodChemistry vol 49 no 11 pp 5165ndash5170 2001

[47] A Luximon-Ramma T Bahorun M A Soobrattee and O IAruoma ldquoAntioxidant activities of phenolic proanthocyanidinand flavonoid components in extracts of Cassia fistulardquo Journalof Agricultural and Food Chemistry vol 50 no 18 pp 5042ndash5047 2002

[48] Y S Velioglu GMazza L Gao and B D Oomah ldquoAntioxidantactivity and total phenolics in selected fruits vegetables andgrain productsrdquo Journal of Agricultural and Food Chemistry vol46 no 10 pp 4113ndash4117 1998

[49] M Mikulic Petkovsek F Stampar and R Veberic ldquoIncreasedphenolic content in apple leaves infected with the apple scabpathogenrdquo Journal of Plant Pathology vol 90 no 1 pp 49ndash552008

[50] E Tripoli M L Guardia S Giammanco D D Majo and MGiammanco ldquoCitrus flavonoids molecular structure biologicalactivity and nutritional properties a reviewrdquo Food Chemistryvol 104 no 2 pp 466ndash479 2007

[51] G Kottra and H Daniel ldquoFlavonoid glycosides are not trans-ported by the human Na+glucose transporter when expressedin Xenopus laevis oocytes but effectively inhibit electrogenicglucose uptakerdquoThe Journal of Pharmacology and ExperimentalTherapeutics vol 322 no 2 pp 829ndash835 2007

[52] P Montoro A Braca C Pizza and N de Tommasi ldquoStructure-antioxidant activity relationships of flavonoids isolated fromdifferent plant speciesrdquo Food Chemistry vol 92 no 2 pp 349ndash355 2005

[53] A Hopia and M Heinonen ldquoAntioxidant activity of flavonolaglycones and their glycosides in methyl linoleaterdquo Journal ofthe American Oil Chemistsrsquo Society vol 76 no 1 pp 139ndash1441999

[54] B M Rezk G R M M Haenen W J F Van der Vijgh andA Bast ldquoThe antioxidant activity of phloretin the disclosure ofa new antioxidant pharmacophore in flavonoidsrdquo Biochemicaland Biophysical Research Communications vol 295 no 1 pp9ndash13 2002

[55] W-T Chang W-C Huang and C-J Liou ldquoEvaluationof the anti-inflammatory effects of phloretin and phlorizinin lipopolysaccharide-stimulated mouse macrophagesrdquo FoodChemistry vol 134 no 2 pp 972ndash979 2012

[56] H P V Rupasinghe and A Yasmin ldquoInhibition of oxidationof aqueous emulsions of Omega-3 fatty acids and fish oil byphloretin and phloridzinrdquo Molecules vol 15 no 1 pp 251ndash2572010

[57] M Pikulski and J S Brodbelt ldquoDifferentiation of flavonoid gly-coside isomers by using metal complexation and electrosprayionization mass spectrometryrdquo Journal of the American Societyfor Mass Spectrometry vol 14 no 12 pp 1437ndash1453 2003

[58] W Majak ldquoReview of toxic glycosides in rangeland and pastureforagesrdquo Journal of Range Management vol 54 no 4 pp 494ndash498 2001

[59] M M Petkovsek A Slatnar F Stampar and R VebericldquoPhenolic compounds in apple leaves after infection with applescabrdquo Biologia Plantarum vol 55 no 4 pp 725ndash730 2011

[60] S Veeriah T Kautenburger N Habermann et al ldquoAppleflavonoids inhibit growth of HT29 human colon cancer cells


and modulate expression of genes involved in the biotransfor-mation of xenobioticsrdquoMolecular Carcinogenesis vol 45 no 3pp 164ndash174 2006

[61] M M Boccia S R Kopf and C M Baratti ldquoPhlorizin acompetitive inhibitor of glucose transport facilitates memorystorage in micerdquo Neurobiology of Learning and Memory vol 71no 1 pp 104ndash112 1999

[62] S-Y Zhang B-Y Li X-L Li et al ldquoEffects of phlorizin on dia-betic retinopathy according to isobaric tags for relative andabsolute quantification-based proteomics in dbdb micerdquoMolecular Vision vol 19 pp 812ndash821 2013

[63] C Puel A Quintin J Mathey et al ldquoPrevention of bone loss byphloridzin an apple polyphenol in ovariectomized rats underinflammation conditionsrdquoCalcified Tissue International vol 77no 5 pp 311ndash318 2005

[64] C-L Shen V von BergenM-C Chyu et al ldquoFruits and dietaryphytochemicals in bone protectionrdquoNutrition Research vol 32no 12 pp 897ndash910 2012

[65] M Najafian M Z Jahromi M J Nowroznejhad et al ldquoPhlo-ridzin reduces blood glucose levels and improves lipids metab-olism in streptozotocin-induced diabetic ratsrdquo Molecular Biol-ogy Reports vol 39 no 5 pp 5299ndash5306 2012

[66] J R L Ehrenkranz N G Lewis C R Kahn and J Roth ldquoPhlo-rizin a reviewrdquoDiabetesMetabolism Research and Reviews vol21 no 1 pp 31ndash38 2005

[67] M Kobori S Masumoto Y Akimoto and H Oike ldquoPhloridzinreduces blood glucose levels and alters hepatic gene expressionin normal BALBcmicerdquo Food and Chemical Toxicology vol 50no 7 pp 2547ndash2553 2012

[68] H Zhao S Yakar O Gavrilova et al ldquoPhloridzin improveshyperglycemia but not hepatic insulin resistance in a transgenicmouse model of type 2 diabetesrdquo Diabetes vol 53 no 11 pp2901ndash2909 2004

[69] V Poitout and R P Robertson ldquoMinireview secondary 120573-cellfailure in type 2 diabetes a convergence of glucotoxicity andlipotoxicityrdquo Endocrinology vol 143 no 2 pp 339ndash342 2002

[70] T C Hardman and S W Dubrey ldquoDevelopment and potentialrole of type-2 sodium-glucose transporter inhibitors for man-agement of type 2 diabetesrdquo Diabetes Therapy vol 2 no 3 pp133ndash145 2011

[71] T Ohta H Morinaga T Yamamoto and T Yamada ldquoEffectof phlorizin on metabolic abnormalities in SpontaneouslyDiabetic Torii (SDT) ratsrdquoOpen Journal of Animal Sciences vol2 no 2 pp 113ndash118 2012

[72] Q Cai B Li F Yu et al ldquoInvestigation of the protective effectsof phlorizin on diabetic cardiomyopathy in dbdb mice byquantitative proteomicsrdquo Journal of Diabetes Research vol 2013Article ID 263845 9 pages 2013

[73] W-D Lu B-Y Li F Yu et al ldquoQuantitative proteomics studyon the protective mechanism of phlorizin on hepatic damage indiabetic dbdb micerdquo Molecular Medicine Reports vol 5 no 5pp 1285ndash1294 2012

[74] J A Tayek ldquoIs weight loss a cure for type 2 diabetesrdquo DiabetesCare vol 25 no 2 pp 397ndash398 2002

[75] American Diabetes Association ldquoStandards of medical care indiabetesmdash2014rdquo Diabetes Care vol 37 supplement 1 pp S14ndashS80 2014

[76] M Singhal S Rasania A Gaur V R M Vishnu S V R Rajuand Y Upadhyay ldquoOverview on sodium glucose transportinhibitors as a therapeutic tool against diabetesmellitusrdquoGlobalJournal of Pharmacology vol 6 no 2 pp 86ndash93 2012

[77] J Kolniak-Ostek J Oszmianski and A Wojdyło ldquoEffect ofapple leaves addition on physicochemical properties of cloudybeveragesrdquo Industrial Crops and Products vol 44 pp 413ndash4202013

[78] D Kviklys M Liaudanskas V Janulis et al ldquoRootstock geno-type determines phenol content in apple fruitsrdquo Plant Soil andEnvironment vol 60 no 5 pp 234ndash240 2014

[79] M Mikulic Petkovsek V Usenik and F Stampar ldquoThe roleof chlorogenic acid in the resistance of apples to apple scab(Venturia inaequalis (Cooke) G Wind Aderh)rdquo ZbornikBiotechniske Fakultete Univerze v Ljubljani Kmetijstvo vol 81no 2 pp 233ndash242 2003

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy


Carbohydrate Chemistry

International Journal of


Journal of

Chemistry


Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


Journal of


Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


of the body [10ndash12] The antioxidant activity of phenoliccompounds is associated with other biological propertiesof these compounds such as anti-inflammatory antimicro-bial anticancer cardiovascular system-improving and otheractivities [13ndash15] Apple leaves accumulate high amounts ofphloridzin [16 17] which exhibit antidiabetic activity [18]Therefore plant raw materials accumulating this compoundcan be potentially useful for the prevention of diabetesmellitus

Therefore the aimof this studywas to determine the com-position and content of phenolic compounds in the ethanolextracts of apple leaves harvested from the cultivars AldasAuksis Ligol and Lodel grown under Lithuanian climaticconditions to assess the variation of phenolic compoundsand to compare their antioxidant activity in the apple leafsamples obtained from different cultivars

2 Materials and Methods

21 Plant Material The following apple cultivars wereincluded into this study Auksis (early winter cv bred inLithuania) and Ligol (winter cv bred in Poland) that aretwo main cultivars in commercial apple orchards as wellas Aldas (early winter cv bred in Lithuania) and Lodel(winter cv bred in Poland) two cultivars resistant to applescab (Venturia inaequalis) Cultivars Aldas and Lodel arerecommended for organic orchards The apple trees weregrown in the experimental orchard of the Institute of Hor-ticulture Lithuanian Research Centre for Agriculture andForestry Babtai Lithuania (55∘601015840N 23∘481015840E) Aldas block4 row 16 trees 5ndash8 Auksis block 2 row 3 trees 25ndash28Ligol block 2 row 4 trees 21ndash24 Lodel block 4 row 5trees 12ndash15 The altitude of Babtai town is 57m above sealevel Average annual precipitation is 630mm and averagesum of active temperatures is (gt10∘C)ndash2300∘ Temperaturesduring the experiment year were close to long term averageThe summer of 2012 was characterised by more rainfall inApril June and July Trees were trained as slender spindlePest and disease management was carried out according tothe rules of the integrated plant protectionThe experimentalorchard was not irrigated Tree fertilization was performedaccording to soil and leaf analysis Nitrogen was appliedbefore flowering at the rate of 80 kgha and potassium wasapplied after harvest at the rate of 90 kgha Soil conditions ofthe experimental orchard were as follows clay loam pH 73organic matter 28 P

2O5 255mgkg and K

2O 230mgkg

Apple leaves were harvested from 10-year-old apple trees in2012The study sample comprised 20 healthy fully developedleaves collected from different places of each apple tree ofeach cultivar Apple leaves were lyophilized with a ZIRBUSsublimator 3 times 4 times 520 (ZIRBUS technology Bad GrundGermany) at a pressure of 001mbar (condenser temperaturendash85∘C) The lyophilized apple leaves were ground to a finepowder by using a Retsch 200 mill (Haan Germany) Losson drying before analysis was determined by the method ofthe European Pharmacopoeia [19] Data were recalculated forabsolute dry lyophilizate weight

22 Chemicals All the solvents reagents and standardsused were of analytical grade Acetonitrile and acetic acidwere obtained from Sigma-Aldrich GmbH (Buchs Switzer-land) and ethanol from Stumbras AB (Kaunas Lithuania)Hyperoside rutin quercitrin phloridzin phloretin caf-feic acid and chlorogenic acid standards were purchasedfrom Extrasynthese (Genay France) (+)-catechin and (ndash)-epicatechin from Fluka (Buchs Switzerland) and avicu-larin and isoquercitrin from Chromadex (Santa Ana USA)11-Diphenyl-2-picrylhydrazyl (DPPH∙) radical 221015840-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) potas-sium persulfate sodium acetate trihydrate iron (III) chloridehexahydrate and 246-tripyridyl-s-triazine (TPTZ) wereobtained from Sigma-Aldrich (Steinheim Germany) Folin-Ciocalteu reagent gallic acid monohydrate sodium carbon-ate aluminum chloride hexahydrate and hexamethylenete-tramine were purchased from Sigma-Aldrich GmbH (BuchsSwitzerland) Deionized water produced by the Crystal Ehigh-performance liquid chromatography (HPLC AdronaSIA Riga Latvia) water purification system was used

23 Extraction An amount of 025 g of lyophilized appleleaf powder (exact weight) was weighed added to 10mLof ethanol (70 vv) and extracted in a Sonorex Digital10 P ultrasonic bath (Bandelin Electronic GmbH amp CoKG Berlin Germany) for 40 minutes at 60∘C The extractobtained was centrifuged for 7 minutes at 6000 rpm with aHermle Z206A centrifuge (Denville Scientific Inc USA)Theextract was collected and filtered through a membrane filterwith a pore size of 022120583m (Carl Roth GmbH KarlsruheGermany)

24 Spectrophotometric Studies

241 Determination of Total Phenolic and Flavonoid Con-tent All the spectrophotometric measurements were carriedout with a Genesys-10UVVis spectrophotometer (ThermoSpectronic Rochester USA) The total phenolic content(mg GAEg DW) in the ethanol extracts of apple leaveswas determined by the Folin-Ciocalteu method [20] Thetotal amount of flavonoids in the ethanol extracts of appleleaves was determined using the describedmethodology [21]calculated from a rutin calibration curve and expressed asmgg rutin equivalent (RE) per gram of absolutely dry weight(DW) (mg REg DW)

242 Determination of Antioxidant Activity

(1) DPPH∙ Free Radical Scavenging Assay The DPPH∙ freeradical scavenging activity was determined using the methodproposed by Brand-Williams et al [22] DPPH∙ solution in963 vv ethanol (3mL 6times10minus5M)was mixed with 10120583L ofthe ethanol extract of apple leaves A decrease in absorbancewas determined at a wavelength of 515 nm after keeping thesamples for 30 minutes in the dark

(2) ABTS∙+ Radical Cation Decolorization Assay An ABTS∙+radical cation decolorization assay was applied according tothemethodology described byRe et al [23] A volumeof 3mL




3sdot6H2O (002M in












119875 lt 005b

119875 lt 005b














0

50

100

150

200

250

300

350

400


DPPH

ABTS

FRAP

TE (120583

mol

g D

W)






(AU

)

000005010015020025030035040

(min)2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 4600 4800 5000 5200

31

24 5

6

78

9

10 11



































4 Conclusions





Abbreviations






Acknowledgments


References





























































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




3sdot6H2O (002M in












119875 lt 005b

119875 lt 005b














0

50

100

150

200

250

300

350

400


DPPH

ABTS

FRAP

TE (120583

mol

g D

W)






(AU

)

000005010015020025030035040

(min)2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 4600 4800 5000 5200

31

24 5

6

78

9

10 11



































4 Conclusions





Abbreviations






Acknowledgments


References





























































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of







0

50

100

150

200

250

300

350

400


DPPH

ABTS

FRAP

TE (120583

mol

g D

W)






(AU

)

000005010015020025030035040

(min)2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 4600 4800 5000 5200

31

24 5

6

78

9

10 11



































4 Conclusions





Abbreviations






Acknowledgments


References





























































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


(AU

)

000005010015020025030035040

(min)2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 4600 4800 5000 5200

31

24 5

6

78

9

10 11



































4 Conclusions





Abbreviations






Acknowledgments


References





























































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of






















4 Conclusions





Abbreviations






Acknowledgments


References





























































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


Acknowledgments


References





























































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of
































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

Research Article Phenolic Composition and Antioxidant...

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