Research Article Application of an Optimized HPLC Method for …downloads.hindawi.com/journals/jchem/2014/542121.pdf · 2019. 7. 31. · Research Article Application of an Optimized

Research ArticleApplication of an Optimized HPLC Method forthe Detection of Various Phenolic Compounds inApples from Lithuanian Cultivars

Mindaugas Liaudanskas1 Pranas Viškelis2 Valdas Jakštas1 Raimondas Raudonis1

Darius Kviklys2 Arvydas Milašius1 and Valdimaras Janulis1

1 Department of Pharmacognosy Faculty of Pharmacy Lithuanian University of Health Sciences A Mickeviciaus Street 9LT-44307 Kaunas Lithuania

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

Correspondence should be addressed to Mindaugas Liaudanskas mliaudanskasyahoocom

Received 23 January 2014 Revised 19 March 2014 Accepted 2 April 2014 Published 24 April 2014

Academic Editor Maria Luisa Serralheiro

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

A specific analytical procedure including sample preparation and HPLC analysis was developed and validated for the detection ofphenolic compounds in the samples of different apples from popular Lithuanian cultivars ldquoAldasrdquo ldquoAuksisrdquo ldquoLigolrdquo and ldquoSampionrdquoThe conditions for phenol extraction were optimized the solvent of the extraction was 70 (vv) ethanol and the extractionwas performed in an ultrasound bath for 20min at the temperature of 40∘C The HPLC mobile phase consisted of 2 (vv)acetic acid in water and 100 (vv) acetonitrile Using the HPLC technique 11 analytes were identified and their specificitywas confirmed procyanidin B1 (+)-catechin chlorogenic acid procyanidin B2 (minus)-epicatechin rutin hyperoside isoquercitrinavicularin quercitrin and phloridzin Chlorogenic acid was themajor component in ldquoAldasrdquo ldquoAuksisrdquo and ldquoLigolrdquo and procyanidinB2 in ldquoSampionrdquo Hyperoside and avicularin were the dominant compounds of all the identified quercetin derivatives in ldquoAldasrdquoand ldquoAuksisrdquo hyperoside in ldquoSampionrdquo and quercitrin in ldquoLigolrdquo The total content of phenolic compounds varied from 16410 plusmn479120583gg (cv ldquoLigolrdquo) to 42913 plusmn 1542 120583gg (cv ldquoAldasrdquo)

1 Introduction

In Lithuaniamdashlike in the whole Eastern European regionmdashapples are among the main vegetal food sources rich inphenolic compounds The Lithuanian Health Program indi-cates that the nutrition of school-age children should besupplemented with such local vegetal products as applescarrots and pears [1] Using more variable cultivars of fruitin the nutrition of adult and children population is advisedA greater variety of biologically active compounds in fruit isassociated with lower mortality [2]

Numerous studies have confirmed the benefit of phenoliccompounds for human health Studies have shown that dif-ferent groups of phenolic compounds as well as their in-dividual components have a specific effect on different phys-iological systems Phenolic compounds in apples have an

antiviral effect againstHerpes simplex strains and the flu virus[3 4] and they suppress cell proliferation [5] reduce bloodlevels of triglycerides and low density lipoproteins [6] andthus are useful in the prevention of cancer and cardiovasculardiseases Polyphenolic compounds protect the gastrointesti-nal mucosa and may be used as a preventive measure againstadverse effects of anti-inflammatory drugs [7] Phenoliccompounds in apples may be potentially useful in protectingthe lungs against damage from cigarette smoke [8] as wellas for the prevention of Alzheimerrsquos disease [9] Based onthese results of scientific studies it is expedient to concretizethe available knowledge about the distribution of phenoliccompounds in apples from popular Lithuanian cultivars

A reliable analytical instrument is required in order toobtain detailed results in the analysis of the phytochemicalcomposition of the apples The techniques of the qualitative

Hindawi Publishing CorporationJournal of ChemistryVolume 2014 Article ID 542121 10 pageshttpdxdoiorg1011552014542121

2 Journal of Chemistry

and quantitative analyses of the content of phenolic com-pounds in apples described in literature sources may notalways be suitable for implementation in other laboratoriesBecause of possible differences in the phytochemical compo-sition of the apples these techniques may be unsuitable forthe analysis of local cultivars Because of this a detailed sci-entific analysis requires the redevelopment of sample prepa-ration andmobile phase gradients of the techniques designedin other laboratories The need for the development of anovel technique was confirmed by our preliminary attemptsto adapt the techniques found in scientific literature sourcesthese attempts did not yield the desired results For thisreason the aim was set to develop optimize and validatethe suitable HPLC technique for the detection of phenoliccompounds in the lyophilized apple samples of the cultivarsldquoAldasrdquo ldquoAuksisrdquo ldquoLigolrdquo and ldquoSampionrdquo grown in Lithuania

2 Materials and Methods

21 Apples The following apple cultivars were included in thestudy ldquoAldasrdquo ldquoAuksisrdquo ldquoLigolrdquo and ldquoSampionrdquo the appleswere harvested in 2011The apples were supplied by the Insti-tute of Horticulture Lithuanian Research Centre for Agri-culture and Forestry Babtai Lithuania (55∘601015840N 23∘481015840E)The altitude of Babtai town is 57m above sea level Theyear 2011 was warmer than usual higher the mean monthlytemperatures were higher than the long-term average Thesummer of 2011 was distinguished by more rainfall in Julyand especially in August when double the normal monthlyrainfall was recorded 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 conditionsof the experimental orchard were the following clay loampH 73 humus 28 P

2O5 255mgkg and K

2O 230mgkg

The apple cultivars ldquoAuksisrdquo (early winter cv bred in Lithua-nia) ldquoSampionrdquo (winter cv bred in Czech Republic) andldquoLigolrdquo (winter cv bred in Poland) are the main cultivarsin Lithuanian commercial orchards The cv ldquoAldasrdquo (earlywinter cv bred in Lithuania) is recommended for ecologicalorchards Apples were harvested at the optimal harvest timeindividually established for every cultivar ldquoAldasrdquo 128DAFBldquoAuksisrdquo 114 DAFB ldquoLigolrdquo 168 DAFB and ldquoSampionrdquo 132DAFB The sample size comprised 20 ripe apples of eachcultivar

Each apple was cut into the slices of equal size (up to 1 cmin thickness) and the stalks and the seeds were removedThe apple slices were immediately frozen in a freezer (at ndash35∘C)with air circulation and then lyophilizedwith aZIRBUSsublimator 3 times 4 times 520 (ZIRBUS technology Bad GrundGermany) at the pressure of 001mbar (condenser tempera-ture minus85∘C)The lyophilized apple slices were ground to finepowder by using a Retsch 200 mill (Haan Germany)

Loss on drying before analysis was determined by dryingthe apple lyophilizate in a laboratory drying oven to complete

evaporation of water and volatile compounds (temperature105∘C the difference in weight between measurements upto 001 g) and by calculating the difference in raw materialweight before and after drying [10] The data were recalcu-lated for absolute dry lyophilizate weight

22 Chemicals All the solvents reagents and standards usedwere of analytical grade Aluminum trichloride hexahydratehexamethylentetramine acetonitrile acetone methanol andacetic and trifluoroacetic acids were obtained from Sigma-AldrichGmbH (Buchs Switzerland) and ethanol fromStum-bras AB (Kaunas Lithuania) Hyperoside rutin quercitrinphloridzin procyanidinB1 and procyanidin B2 and chloro-genic acid standards were purchased from Extrasynthese(Genay France) (+)-catechin and (minus)-epicatechin fromFluka (Buchs Switzerland) and avicularin and isoquercitrinfrom Chromadex (Santa Ana USA) In the study we useddeionized water produced by the Crystal E HPLC (AdronaSIA Riga Latvia) water purification system

23 Extraction An amount of 25 g of lyophilized apple pow-der (exact weight) was weighed added to 30mL of ethanol(70 vv) and extracted in a Sonorex Digital 10 P ultrasonicbath (Bandelin Electronic GmbH amp Co KG Berlin Ger-many) for 20 minutes at 40∘C The conditions of extraction(the type of the extraction its duration temperature andthe solvent and its concentration) were chosen consider-ing the results of the optimization of the extraction Theextract obtained was filtered through a paper filter the applelyophilizate on the filter was washed twice with 10mL ofethanol (70 vv) in a 50mL flask The extract was filteredthrough a membrane filter with a pore size of 022120583m (CarlRoth GmbH Karlsruhe Germany)

24 Determination of the Total Flavonoid Content The totalflavonoid content in the extracts of lyophilized apple sampleswas determined by applying the technique described byUrbonaviciute et al [11] was calculated using the rutincalibration curve and was expressed as its equivalent (mgREg) for absolute dry lyophilizate weight

25 Instrumentation and Chromatographic Conditions AWaters 2695 chromatograph equipped with a Waters 2998PDA detector (Waters Milford USA) was used for HPLCanalysis Chromatographic separation was managed chro-matograms were recorded and data were processed with theEmpower v20 software (Waters Milford USA) Chromato-graphic separations were carried out by using a YMC-PackODS-A (5 120583m C

18 250 times 46mm id) column equipped

with a YMC-Triart (5 120583m C18 10 times 30mm id) precolumn

(YMC Europe GmbH Dinslaken Germany) The columnwas operated at a constant temperature of 25∘C The volumeof the extract being investigated was 10 120583L The flow ratewas 1mLmin and gradient elution was used The mobilephase consisted of 2 (vv) acetic acid in water (solventA) and 100 (vv) acetonitrile (solvent B) The followingconditions of elution were applied 0ndash30 minutes 3ndash15 B30ndash45 minutes 15ndash25 B 45ndash50 minutes 25ndash50 B and

Journal of Chemistry 3

50ndash55 minutes 50ndash95 B The total duration of the analysisincluding washing and reconditioning of the column was70 minutes The identification of the chromatographic peakswas achieved by comparing the retention times and spectralcharacteristics (120582 = 200ndash600 nm) of the eluting peakswith those of the reference compounds The compoundsidentified were confirmed by spiking the sample with thestandard compound and monitoring the changes in thepeak shape and spectral characteristics For quantitativeanalysis a calibration curve was obtained by the injectionof known concentrations of different standard compoundsThe concentrations of phenolic compounds identified inthe apple extracts were within the limits of the calibrationcurves Dihydrochalcones and flavan-3-ols were quantified at280 nm phenolic acids at 320 nm and flavonols at 360 nm

26 Statistical Data Analysis All the experiments were car-ried out in triplicate Means and standard deviations werecalculated with SPSS 200 software (Chicago USA) A singlefactor analysis of variance (ANOVA) along with the post-hoc Tukeyrsquos test was employed for statistical analysis TheKolmogorov-Smirnov test was applied to examine the nor-mality of the distribution To verify the hypothesis about theequality of variances Levenersquos test was employed Differenceswere considered to be significant at 119875 lt 005

3 Results and Discussion

31 Optimization of the Extraction Method The selection ofthe solvent and the conditions of extraction is an importantanalytical step in the development of the technique forthe qualitative and quantitative analysis of the biologicallyactive compounds in raw plant material The solvent of theextraction is themain factor in the prognosis of the qualitativeand quantitative composition of the isolated phenolic com-poundsThe extractants most commonly used for the extrac-tion of flavonoid glycosides their more polar aglycones andpolar phenolic acids aremethanol ethanol acetone and theirvarious aqueous mixtures of various concentrations [12 13]

To evaluate the effect of the extractant on the extractionof flavonoids the lyophilized apple samples were maceratedfor 4 hours in the dark and the total content of flavonoids wasdetermined by using spectrophotometry [11]The extractantsselected for the study were methanol ethanol and acetoneused at various concentrations The study showed that 70(vv) ethanol ensured the highest flavonoid output (27 plusmn01mg REg) (Figure 1) This extractant was selected forfurther analysis

Two techniques ensuring selective extraction of the phe-nolic compounds from solid-phase samples were chosen forcomparative studies of the effectiveness of the extractiontechniques maceration and ultrasound extraction (sonifica-tion) During the maceration of the samples at room temper-ature in dark environment at different time intervals (05ndash24hours) the highest total flavonoid content (32plusmn01mgREg)was registered after 24-hour extraction (Figure 2) To evaluatethe effectiveness of sonification apple samples were extractedin an ultrasound bath for 5ndash90min at different temperatures

0051015202530

20 30 40 50 60 70 80 90 95Tota

l flav

onoi

d co

nten

t(m

g RE

g D

W)

Solvent concentration (vv)

AcetoneEthanolMethanol

Figure 1 The total content of flavonoids in apple samples usingextractants at various concentrations

005101520253035

0 5 10 15 20 25

Tota

l flav

onoi

d co

nten

t (m

g RE

g D

W)

Time (h)

Figure 2 The total extracted flavonoid content when maceratingapple samples at different time intervals

0051015202530354045

0 10 20 30 40 50 60 70 80 90

Tota

l flav

onoi

d co

nten

t(m

g RE

g D

W)

Time (min)

20∘C

30∘C

40∘C

Figure 3 The total extracted flavonoid content when extractingapple samples in an ultrasound bath at different time intervals at thetemperature of 20ndash40∘C

(20ndash40∘C)The greatest extraction output (41plusmn02mgREg)was observed after extraction of the apple samples for 20minat the temperature of 40∘C (Figure 3)

The analysis showed that the greatest amount of flavon-oids (41 plusmn 02mg REg) from lyophilized apple samples wasextracted with 70 (vv) ethanol in an ultrasound bath after20 minutes of extraction at the temperature of 40∘C Theseconditions were selected for further analysis the extractionfrom lyophilized samples of different apple cultivars


Table 1 Characteristics of the quantitative evaluation of phenolic compounds

Compound Retention time(min)a

Wavelength(nm)

Confirmed linearityrange (120583gmL) Calibration equationb

Determinedquantitation limit

(120583gmL)R2

Procyanidin B1 156 280 625ndash100 y = 733 times 103x + 549 times 103 264 09998(+)-Catechin 197 280 5ndash80 y = 700 times 103x minus 187 times 103 220 09999Chlorogenic acid 207 320 625ndash100 y = 268 times 104x minus 302 times 104 293 09999Procyanidin B2 229 280 625ndash100 y = 558 times 103x + 715 times 103 286 09998(minus)-Epicatechin 273 280 5ndash80 y = 753 times 103x + 119 times 102 222 09999Hyperoside 400 360 5ndash80 y = 223 times 104x minus 101 times 104 242 09999Isoquercitrin 401 360 3125ndash50 y = 266 times 104x minus 210 times 104 154 09999Rutin 395 360 3125ndash50 y = 147 times 104x minus 766 times 103 279 09998Quercitrin 445 360 25ndash40 y = 177 times 104x minus 557 times 103 219 09998Avicularin 439 360 3125ndash50 y = 232 times 104x minus 112 times 104 256 09998Phloridzin 476 280 3125ndash50 y = 185 times 104x minus 709 times 103 240 09998aThe mean was calculated from 6 replicatesbx in the calibration equation indicates the concentration of a compound and y indicates a peak area

32 Development and Optimization of Chromatographic Sep-aration A broad variety of analytical methods have beenemployed for the detection of phenolics in rawplantmaterialsand their products Thin-layer and high-performance thin-layer chromatography mass infrared and ultraviolet spec-troscopy and other analytical methods have been applied forthe qualitative analysis of phenolics [14 15] For the quanti-tative evaluation of phenolic compounds in plant matricesHPLC and UV-visible absorption spectrophotometry are themost widely used methods capillary zone electrophoresis isapplied less frequently [16]

Experiments during the development stage showed thata better chromatographic separation was achieved by usingthe YMC-Pack ODS-A column (5 120583m C

18 250times46mm id)

and selecting acetonitrile rather than methanol as an organiccomponent For acidification (solvent A) two organic acidswere selected 2 acetic acid and 01 trifluoroacetic acid Abetter separation of the analytes was achieved by using 2acetic acid rather than 01 trifluoroacetic acid therefore 2acetic acid was used for further experiments The applicationof acetic acid to acidify themobile phase has been reported inother studies that investigated phenolic compounds in apples[3 17 18]

The method of gradient elution for the separation ofphenolic compounds (quercetin derivatives flavan-3-olsphenolic acids and dihydrochalcones) in ethanol extracts oflyophilized apple samples was optimizedThismethod allowsfor an effective separation of phenolic compounds present inlyophilized apple extracts The elution order of quercetin-3-O-glycosidesmdashalso reported in other studies [17 19 20]mdashwasas follows quercetin-3-O-rutinoside (rutin) quercetin-3-O-galactoside (hyperoside) quercetin-3-O-glucoside (iso-quercitrin) quercetin-3-O-arabinoside (avicularin) andquercetin-3-O-rhamnoside (quercitrin) The values of res-olution (Rs gt 2) were achieved in all the samples of standardsolutions and extracts Optimization of the HPLC methodallowed for an effective separation of quercetin glycosides

the separation of rutin and hyperoside in ethanol extractsof lyophilized apple samples with the resolution of 259the separation of hyperoside and isoquercitrin with theresolution of 312 and the separation of avicularin andquercitrin with the resolution of 283

The chromatograms of ethanol extracts of apple samplesat different wavelengths (280 320 and 360 nm) are depictedin Figure 4

33 Validation of the Method The validation of the methodwas performed on the basis of the ICH recommendations[21] The following characteristics of validation were evalu-ated the selectivity of the method (specificity) the repeata-bility of the results (precision) the quantitation limit of theanalytes and linearity

The evaluation of the selectivity of the method for peakidentification and purity was based on the comparison ofthe retention times and UV spectra of the analytes withthose of the standard compoundsThe compounds identifiedwere confirmed by adding the standard compound into theanalyzed extract and monitoring the changes in the peakshape and spectral characteristics

The quantitation limit of the analytes was assessed bycomparing the peak height to the baseline noise The ratio ofthe peak height to baseline noise (signal-to-noise ratio) usedfor the estimation of the quantitation limit was 10 1 Thedetermined quantitation limit varied from 154 120583gmL to293 120583gmL The obtained results confirm that this methodcan be used for the quantitative analysis of phenolic com-pounds The estimated determination coefficients (1198772)ofcalibration curves were greater than 0999 and this proves thelinearity of the quantitative determination method (Table 1)

The precision of the HPLC method was assessed onthe basis of two parameters repeatability and intermediateprecision The repeatability of the results was evaluated onthe basis of the results of consecutive analyses performed


Table 2 Precision of the HPLC method for the quantitative evaluation of phenolic compounds

Compound Relative standard deviation () for the peak areaIntradaya Interdayb Extraction after extraction

Procyanidin B1 132 239 377(+)-Catechin 149 226 356Chlorogenic acid 122 145 347(minus)-Epicatechin 152 235 392Procyanidin B2 140 251 242Hyperoside 138 261 277Isoquercitrin 126 207 236Rutin 137 244 271Quercitrin 146 254 327Avicularin 142 260 252Phloridzin 148 276 361aRepeatabilitybIntermediate precision

(AU

)

0002004006008

01012

(min)

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50

1 2

3

4 5 11

(a)

(min)

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50

(AU

)

0004008012016

02 3

(b)

(min)

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50

3

6

7

8

910

(AU

)

0001002003004

(c)

Figure 4 Chromatograms of ethanol extracts of the investigatedapple samples (cv ldquoAldasrdquo) (a) 120582 = 280 nm (b) 120582 = 320 nm (c)120582 = 360 nm Numbers indicate the peaks of analytes 1 procyanidinB1 2 (+)-catechin 3 chlorogenic acid 4 procyanidin B2 5 (minus)-epicatechin 6 rutin 7 hyperoside 8 isoquercitrin 9 avicularin10 quercitrin and 11 phloridzin

within the same day (6 consecutive analyses of a mixture ofstandards at 3 different concentrations performed within thesame day) The percent relative standard deviation (RSD)of the repeatability of the method determined according to

the peak area varied from 122 (chlorogenic acid) to 152((minus)-epicatechin) The intermediate precision of the resultswas assessed based on the results of analyses performedwithin 3 different days (6 consecutive analyses of a mixtureof standards at the mean concentration performed within thesame day 18 analyses in total) and its RSD for the peak areavaried from 145 (chlorogenic acid) to 276 (phloridzin)Extraction after the extraction of the lyophilized apple samplewas performed A set of 6 ethanol extracts was producedfrom the same lyophilized apple sample and was analyzed byapplying the developed and optimized HPLC method Theestimated RSD for the peak area did not exceed 392Theobtained results describing the precision of developed HPLCmethod are summarized in Table 2

34 Identification of Phenolic Compounds in Lyophilized AppleSamples Apples are among the most popular fruits con-sumed worldwide [22] They is a source of various bio-logically active substances especially vitamins and pheno-lic compounds [23] Apples contain phenolic compoundsof different classes flavonols (quercetin and isorhamnetinglycosides) dihydrochalcones (phloretin glycosides) flavan-3-ols (catechins [monomeric flavan-3-ols] and procyanidins[polymeric flavan-3-ols]) anthocyanins (cyanidin glycosidesespecially cyanidin galactoside) and phenolic acids (mostlychlorogenic acid) [24ndash26]

Considering the dietary habits of apple consumptionit is relevant to analyze apples without separating theirpeel from the flesh As the chemical composition of theapples of different cultivars can vary considerably [26 27]it is very important to compare and assess the chemicalcomposition of the unpeeled apples of different cultivars andto evaluate their quality as apples are used in diet preventionof various diseases preparation of safe and healthy foodsanddevelopment andproduction of dietary supplementsTheabove-mentioned circumstances allow for the developmentof analytical methods and the analysis of apples


Table 3 Content of phenolic compounds in ethanol extracts obtained from the apples of cultivars grown in Lithuania (expressed for absolutedry lyophilizate weight)

Compound Amount of phenolic compounds in different cultivarsa 120583ggldquoAldasrdquo ldquoAuksisrdquo ldquoLigolrdquo ldquoSampionrdquo

Procyanidin B1 957a 1081b 396c 832d

(+)-Catechin 1457a 1482b 501c 1297d

Chlorogenic acid 22284a 16188b 6872c 3413d


(minus)-Epicatechin 4476a 2991b 2365c 6840d

Rutin 164a 126b 148c 128d

Hyperoside 1912a 524b 683c 1373d

Isoquercitrin 403a 262b 257c 341d

Avicularin 1942a 588b 527c 812d

Quercitrin 1306a 446b 767c 700d

Phloridzin 1424a 907b 685c 638d

Total 42913 29140 16410 25686aValues are mean (n = 3) The different letters indicate significant differences between the values (P lt 005)

(AU

)

0

0005

001

0015

002

0025

003

31 2 4 5 6

7

8

910 11

(min)0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50

(AU

)

00102030405060708

40 42446

7

8

9

10

(min)

times10minus2

(AU

)

minus002

0

002

004

006

008

18 19 20 21

3

2

(min)

times10minus2

(AU

)

minus002minus001

0001002003004005

13 14 15 16 17

1

(min)

times10minus2

Figure 5 Chromatogram of the ethanol extract of the investigated apple fruit sample (120582 = 320 nm cv ldquoSampionrdquo) Numbers indicate thepeaks of analytes 1 procyanidin B1 2 (+)-catechin 3 chlorogenic acid 4 procyanidin B2 5 (minus)-epicatechin 6 rutin 7 hyperoside 8isoquercitrin 9 avicularin 10 quercitrin and 11 phloridzin

In this study the developed optimized and validatedHPLC method was applied to investigate ethanol extracts oflyophilized apple samples The content of phenolic com-pounds in the apples of cultivars ldquoAldasrdquo ldquoAuksisrdquo ldquoLigolrdquoand ldquoSampionrdquo was qualitatively and quantitatively analyzedand determined An example of the chromatogram of theethanol extract of the apple fruit sample is shown in Figure 5and Table 3 summarizes the characteristics of the quantitativecontent

The following compounds of different phenolic groupswere identified in the investigated ethanol extracts pro-cyanidin B1 (+)-catechin chlorogenic acid procyanidin B2(minus)-epicatechin rutin hyperoside isoquercitrin avicularinquercitrin and phloridzin Neochlorogenic acid gallic acid5-hydroxymethyl furfural sinapic acid isorhamnetin-3-O-glucoside rosmarinic acid transcinnamic acid eupatorin-5-methyl ether sinensetin and protocatechuic acid in apples or

their products were detected by other authors [17 28ndash30] butthey were not found in the analyzed extracts

Chlorogenic acid was the major component in the applesof the cultivars ldquoAldasrdquo ldquoAuksisrdquo and ldquoLigolrdquo This acid hasproperties important for human health antioxidant activity[31] anti-inflammatory activity [32] the reduction of the riskof type 2 diabetes [33] the improvement of cardiovascularfunction [34] and the inhibition of the processes of carcino-genesis [35 36] The apples of the cv ldquoAldasrdquo contained thehighest concentration of chlorogenic acid and it was by 65-fold greater than that in the apples of the cv ldquoSampionrdquo (Ta-ble 3) The results confirm the findings of other studiesindicating that chlorogenic acid in apples is one of the mostcommon phenolic compounds [26 27]

In the studied extracts of apple samples we detectedand quantitatively evaluated monomeric flavan-3-ols (+)-catechin and (minus)-epicatechin (Table 3) which together with


procyanidins may be responsible for the cholesterol lowering[37] and vasodilating [38] effect of the apples they also inhibitsulfotransferases thus can regulate the biological activityof hydroxysteroids and can act as natural chemopreventiveagents [39]

The amount of the identified monomeric flavan-3-olsvaried from 2866plusmn114 120583gg (cv ldquoLigolrdquo) to 8137plusmn360 120583gg(cv ldquoSampionrdquo)Thehighest concentration of (minus)-epicatechinwas identified in the apple fruits of the cv ldquoSampionrdquo It wasby 29-fold greater than its lowest concentration in the applefruits of the cv ldquoLigolrdquo The concentration of (+)-catechin inthe apples of the cv ldquoAuksisrdquo was by 3-fold greater than itslowest concentration in the apples of the cv ldquoLigolrdquo Literaturereports that apples contain considerably greater amounts of(minus)-epicatechin than (+)-catechin [23 40] and this is inagreement with the results of our study The ratio of (+)-catechin to (minus)-epicatechin in the ethanol extracts of differentcultivar apple samples varied in the range from 1 20 (cvldquoAuksisrdquo) to 1 53 (cv ldquoSampionrdquo)

Duda-Chodak et al analyzed the lyophilized appleextracts of the cv ldquoSampionrdquo by HPLC The results of theirstudy showed that the analyzed extracts contained loweramounts of monomeric flavan-3-ols on the average2217120583gg of (minus)-epicatechin and 122 120583gg of (+)-catechin[41] Different apple cultivation and fertilization approachescould contribute to the differences in their quantitativecomposition the impact of meteorological and geographicalfactors cannot be excluded either

The compounds of the procyanidin (oligomeric flavan-3-ol) class have been identified in the plants of the familyRosaceae[42] and in various apple cultivars [24 40] Pro-cyanidins isolated from apples have an antineoplastic effect[43] a strong antioxidant effect [44] an anti-inflammatoryeffect [45] a cholesterol- and triglyceride-lowering effect [37]and a positive effect on the cardiovascular system [46 47]and therefore they are among the most promising phenoliccompounds in apples In the present study procyanidins B1and B2 were identified and quantified in the ethanol extractsof apple fruit samples Table 3 shows their quantitative con-tent

The amount of the identified procyanidins varied from3605 plusmn 126 120583gg (cv ldquoLigolrdquo) to 10144 plusmn 439 120583gg (cvldquoSampionrdquo)Thedata inTable 3 show that procyanidin B2wasthe major compound in the ethanol extracts of cv ldquoSampionrdquoapples and its concentrationwas by 29 times greater than thelowest concentration of procyanidin B2 found in the ethanolextracts of cv ldquoLigolrdquo apple samples Contrary to other 3cultivars procyanidin B2 rather than chlorogenic acid was adominant compound in the ethanol extracts of cv ldquoSampionrdquoapples

Ethanol extracts of lyophilized apple samples of all theanalyzed cultivars contained higher concentrations of pro-cyanidin B2 than those of procyanidin B1 (Table 3) Sim-ilar quantitative characteristics of these compounds in theextracts of apple samples have also been found by otherresearchers [26 41 48] The ratio of procyanidin B1 toprocyanidin B2 in the ethanol extracts of different cultivar

apple samples varied in the range from 1 42 (cv ldquoAuksisrdquo)to 1 112 (cv ldquoSampionrdquo) These data confirm the importanceof the cultivar as a factor for the quantitative composition ofapples

In the investigated ethanol extracts of apple samplesquercetin glycosides were the most abundant group of phe-nolic compounds Quercetin glycosides together with com-pounds of the flavan-3-ol group are most responsible forthe antioxidant effect of the complex of phenolic com-pounds in apples [44] The consumption of products richin quercetin glycosides reduces the risk of cancer and neu-rodegenerative diseases [49 50] Quercetin glycosides inhibitthe angiotensin-converting enzyme and therefore have anantihypertensive effect [46]

The following quercetin derivatives were identified anddetermined quantitatively rutin hyperoside isoquercitrinavicularin and quercitrin The total content of thesequercetin glycosides varied from 1685 plusmn 50 120583gg (cv ldquoAuk-sisrdquo) to 5323 plusmn 193 120583gg (cv ldquoAldasrdquo)

The triplet of quercetin glycosides namely rutin hypero-side and isoquercitrin where hyperoside was a predominantcomponent and the concentration of rutin was the lowestwas found to be characteristic of all apple cultivars analyzedin our study This is in line with the results of other studies[17 20 51] and therefore suggests that the rutin-hyperoside-isoquercitrin triplet (where hyperoside predominates and theconcentration of rutin is relatively low)mdashalong with the com-pounds of the dihydrochalcone classmdashcould be consideredas one of the characteristic features of the phytochemicalcomposition of apples allowing for the identification of appleproducts

The rutin-hyperoside-isoquercitrin ratio varied depend-ing on the apple cultivar It was 1 116 25 in the ethanolextracts of cv ldquoAldasrdquo apples 1 42 21mdashin cv ldquoAuksisrdquoapples 1 46 17mdashin cv ldquoLigolrdquo apples and 1 107 27mdashin cvldquoSampionrdquo apples

The data in Table 3 show that the apple samples of thecv ldquoAldasrdquo contained the highest levels of all the identifiedflavonols The concentration of hyperoside in this cultivarwas by 37-fold greater than that in the cv ldquoAuksisrdquo Theconcentration of isoquercitrin in the apples of the cv ldquoAldasrdquowas by 16-fold greater than that in the apples of the cvldquoLigolrdquo The concentration of avicularin in the apples of thecv ldquoAldasrdquo was by a 37-fold higher compared to the lowestconcentration of avicularin found in the apples of the cvldquoLigolrdquo The concentration of quercitrin found in the applesof the cv ldquoAldasrdquo was by 29 times higher than the lowestconcentration of quercitrin in the apples of the cv ldquoAuksisrdquo(Table 3) In the apples of the cv ldquoSampionrdquo hyperoside wasthe major compound of all quercetin derivatives identifiedand this is in line with the results of other studies [41 48]Rutin was a minor component among all the identifiedquercetin derivatives and these results are consistent withthose reported by other authors [20 23] The highest con-centration of rutin (164 plusmn 07 120583gg) was detected in ethanolextracts of the cv ldquoAldasrdquo apple samples This concentrationwas by 13 times greater than the lowest concentration of rutindetected in the ethanol extracts of cv ldquoAuksisrdquo apple samples


Qualitative and quantitative analysis of the compoundsof the dihydrochalcone class is of great importance as thesecompounds can be regarded as chemotaxonomic markersfor the identification of apple cultivars as well as productsmade from apples [17 52 53] Phloridzin exhibits antidiabeticactivity [54 55] and therefore apples as plant raw materialsaccumulating this compound can be potentially useful for theprevention of diabetesmellitus [9]The highest concentrationof dihydrochalcone phloridzin was in the cv ldquoAldasrdquo (1424 plusmn63 120583gg) and it was by 23 times higher than the lowestconcentration of phloridzin detected in the cv ldquoSampionrdquo(638 plusmn 27 120583gg)

Our analysis showed a wide variation in the phenoliccontent of different apple cultivars grown in Lithuania Thetotal content of phenolic compounds identified in the ethanolextracts of lyophilized apple samples ranged from 16410 plusmn479 120583gg (cv ldquoLigolrdquo) to 42913plusmn1542 120583gg (cv ldquoAldasrdquo)Thecultivar itself as a factor has an impact on the parameters ofqualitative and quantitative content of the apples [20] anddepending on the apple cultivar these parameters can varyconsiderably [26]Other factors thatmay affect the qualitativeand quantitative content of apples include the geochemicalcomposition of the soil the geographic region [41 56ndash58] and the climatic-meteorological [59] and cultivation orstorage conditions [60 61] Some authors found increasedlevels of phenolic compounds in apple peel due to sunlightexposure [62 63] Orchard fertigation and foliar fertilizationlead to increased phenol contents [64]However these factorswere not considered in this study and we are planning toinclude them in our future studies

4 Conclusions

The HPLC-based analytical procedure for the determinationof phenolic compounds in the apple samples from 4 pop-ular cultivars was optimized and validated The specificityprecision and assaying range confirm the suitability of thismethod

The examination of the apple samples revealed thedistribution of 11 different groups of phenolic compoundsimportant for human health Chlorogenic acid was the majorcomponent in cultivars ldquoAldasrdquo ldquoAuksisrdquo and ldquoLigolrdquo andprocyanidin B2 in cv ldquoSampionrdquo Hyperoside and avicularinwere the dominant compounds among all the identifiedquercetin derivatives in the apples of the cultivars ldquoAldasrdquo andldquoAuksisrdquo hyperoside in the cv ldquoSampionrdquo and quercitrin inthe cv ldquoLigolrdquo

Abbreviations

cv CultivarDAFB Days after full bloomDW Dry weightHPLC High-performance liquid chromatographyPDA Photodiode arrayRE Rutin equivalentRSD Percent relative standard deviationvv Volumevolume

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] Nacionalines sveikatos tarybos metinis pranesimas Lietuvossveikatos programa rezultatai ir isvados = Annual report of theNational Health Board Lithuanian health program results andconclusions Vilnius Lithuania 2011

[2] Food and Heath in Europe A New Basis for Action WHORegional Publications European Series no 96 WHO 2004

[3] B Suarez A L Alvarez Y D Garcıa G D Barrio A P Loboand F Parra ldquoPhenolic profiles antioxidant activity and in vitroantiviral properties of apple pomacerdquo Food Chemistry vol 120no 1 pp 339ndash342 2010

[4] R-R He MWang C-ZWang et al ldquoProtective effect of applepolyphenols against stress-provoked influenza viral infection inrestraint micerdquo Journal of Agricultural and Food Chemistry vol59 no 8 pp 3730ndash3737 2011

[5] S Reagan-Shaw D Eggert H Mukhtar and N AhmadldquoAntiproliferative effects of apple peel extract against cancercellsrdquo Nutrition and Cancer vol 62 no 4 pp 517ndash524 2010

[6] S H Thilakarathna H P V Rupasinghe and P W NeedsldquoApple peel bioactive rich extracts effectively inhibit in vitrohuman LDL cholesterol oxidationrdquo FoodChemistry vol 138 no1 pp 463ndash470 2013

[7] C Carrasco-Pozo H Speisky O Brunser E Pastene and MGotteland ldquoApple peel polyphenols protect against gastroin-testinal mucosa alterations induced by indomethacin in ratsrdquoJournal of Agricultural and Food Chemistry vol 59 no 12 pp6459ndash6466 2011

[8] M J Bao J Shen Y L Jia et al ldquoApple polyphenol protectsagainst cigarette smoke-induced acute lung injuryrdquo Nutritionvol 29 no 1 pp 235ndash243 2013

[9] A Francini and L Sebastiani ldquoPhenolic compounds in apple(Malus x domestica Borkh) compounds characterization andstability during postharvest and after processingrdquoAntioxidantsvol 2 no 3 pp 181ndash193 2013

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

[11] 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

[12] C Santos-Buelga S Gonzalez-Manzano M Duenas and AM Gonzalez-Paramas ldquoExtraction and isolation of phenoliccompoundsrdquo Methods in Molecular Biology vol 864 pp 427ndash464 2012

[13] A KhoddamiM AWilkes and T H Roberts ldquoTechniques foranalysis of plant phenolic compoundsrdquoMolecules vol 18 no 2pp 2328ndash2375 2013


[14] C D Stalikas ldquoExtraction separation and detection methodsfor phenolic acids and flavonoidsrdquo Journal of Separation Sciencevol 30 no 18 pp 3268ndash3295 2007

[15] M Naczk and F Shahidi ldquoExtraction and analysis of phenolicsin foodrdquo Journal of Chromatography A vol 1054 no 1-2 pp 95ndash111 2004

[16] R Tsao and Z Deng ldquoSeparation procedures for naturallyoccurring antioxidant phytochemicalsrdquo Journal of Chromatog-raphy B vol 812 no 1-2 pp 85ndash99 2004

[17] A Schieber P Keller and R Carle ldquoDetermination of phenolicacids and flavonoids of apple and pear by high-performanceliquid chromatographyrdquo Journal of Chromatography A vol 910no 2 pp 265ndash273 2001

[18] N-N Chen S-C Zhao L-G Deng et al ldquoDetermination offive polyphenols by HPLCDAD and discrimination of applevarietiesrdquo Chromatographia vol 73 no 5-6 pp 595ndash598 2011

[19] R Tsao R Yang J C Young andH Zhu ldquoPolyphenolic profilesin eight apple cultivars using high-performance liquid chro-matography (HPLC)rdquo Journal of Agricultural and Food Chem-istry vol 51 no 21 pp 6347ndash6353 2003

[20] S C Marks W Mullen and A Crozier ldquoFlavonoid andchlorogenic acid profiles of English cider applesrdquo Journal of theScience of Food and Agriculture vol 87 no 4 pp 719ndash728 2007

[21] N Baber ldquoInternational conference on harmonisation of tech-nical requirements for registration of pharmaceuticals forhuman use (ICH)rdquoBritish Journal of Clinical Pharmacology vol37 no 5 pp 401ndash404 1994

[22] A Mari I Tedesco A Nappo G L Russo A Malorni andV Carbone ldquoPhenolic compound characterisation and antipro-liferative activity of ldquoAnnurcardquo apple a southern Italian cultivarrdquoFood Chemistry vol 123 no 1 pp 157ndash164 2010

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

[24] L Montero M Herrero E Ibanez and A Cifuentes ldquoProfilingof phenolic compounds from different apple varieties usingcomprehensive two-dimensional liquid chromatographyrdquo Jour-nal of Chromatography A vol 1313 pp 275ndash283 2013

[25] D De Paepe K Servaes B Noten et al ldquoAn improved massspectrometric method for identification and quantification ofphenolic compounds in apple fruitsrdquo Food Chemistry vol 136no 2 pp 368ndash375 2013

[26] 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

[27] L Jakobek R Garcıa-Villalba and F A Tomas-BarberanldquoPolyphenolic characterisation of old local apple varieties fromSoutheastern European regionrdquo Journal of Food Compositionand Analysis vol 31 no 2 pp 199ndash211 2013

[28] M C Soares E T Ribeiro E M Kuskoski et al ldquoCompositionof phenolic acids content in apple (Malus sp) pomacerdquo Sem-inaCiencias Agrarias vol 29 no 2 pp 339ndash348 2008

[29] A Schieber P Keller P Streker I Klaiber and R CarleldquoDetection of isorhamnetin glycosides in extracts of apples(Malus domestica cv ldquoBrettacherrdquo) by HPLC-PDA and HPLC-APCI-MSMSrdquo Phytochemical Analysis vol 13 no 2 pp 87ndash942002

[30] M Amzad Hossain S M Salehuddin M J Kabir S MM Rahman and H P V Rupasinghe ldquoSinensetin rutin 31015840-hydroxy-5 6 7 41015840-tetramethoxyflavone and rosmarinic acidcontents and antioxidative effect of the skin of apple fruitrdquo FoodChemistry vol 113 no 1 pp 185ndash190 2009

[31] Y Sato S Itagaki T Kurokawa et al ldquoIn vitro and in vivoantioxidant properties of chlorogenic acid and caffeic acidrdquoInternational Journal of Pharmaceutics vol 403 no 1-2 pp 136ndash138 2011

[32] X Zhang H Huang T Yang et al ldquoChlorogenic acid protectsmice against lipopolysaccharide-induced acute lung injuryrdquoInjury vol 41 no 7 pp 746ndash752 2010

[33] T Thomas and A F H Pfeiffer ldquoFoods for the prevention ofdiabetes how do they workrdquo DiabetesMetabolism Researchand Reviews vol 28 no 1 pp 25ndash49 2012

[34] Y Kanno R Watanabe H Zempo M Ogawa J Suzuki andM Isobe ldquoChlorogenic acid attenuates ventricular remodelingafter myocardial infarction in micerdquo International Heart Jour-nal vol 54 no 3 pp 176ndash180 2013

[35] N Cinkilic S K Cetintas T Zorlu et al ldquoRadioprotectionby two phenolic compounds chlorogenic and quinic acid onX-ray induced DNA damage in human blood lymphocytes invitrordquo Food and Chemical Toxicology vol 53 pp 359ndash363 2013

[36] B Stefanska H Karlic F Varga K Fabianowska-Majewskaand A G Haslberger ldquoEpigenetic mechanisms in anti-canceractions of bioactive food componentsmdashthe implications incancer preventionrdquoBritish Journal of Pharmacology vol 167 no2 pp 279ndash297 2012

[37] A T Serra J Rocha B Sepodes et al ldquoEvaluation of cardiovas-cular protective effect of different apple varietiesmdashcorrelationof response with compositionrdquo Food Chemistry vol 135 no 4pp 2378ndash2386 2012

[38] B A R Lina A A Reus O Hasselwander Q Bui and PP Tenning ldquoSafety evaluation of EvesseTM EPC an applepolyphenol extract rich in flavan-3-olsrdquo Food and ChemicalToxicology vol 50 no 8 pp 2845ndash2853 2012

[39] C Huang Y Chen T Zhou and G Chen ldquoSulfation of dietaryflavonoids by human sulfotransferasesrdquoXenobiotica vol 39 no4 pp 312ndash322 2009

[40] L Panzella M Petriccione P Rega M Scortichini and ANapolitano ldquoA reappraisal of traditional apple cultivars fromSouthern Italy as a rich source of phenols with superiorantioxidant activityrdquo Food Chemistry vol 140 no 4 pp 672ndash679 2013

[41] A Duda-Chodak T Tarko P Satora P Sroka and T TuszynskildquoThe profile of polyphenols and antioxidant properties ofselected apple cultivars grown in Polandrdquo Journal of Fruit andOrnamental Plant Research vol 18 no 2 pp 39ndash50 2010

[42] M Braunlich R Slimestad H Wangensteen et al ldquoExtractsanthocyanins and procyanidins from Aronia melanocarpa asradical scavengers and enzyme inhibitorsrdquo Nutrients vol 5 no3 pp 663ndash678 2013

[43] M E Maldonado-Celis S Bousserouel F Gosse A Lobsteinand F Raul ldquoApple procyanidins activate apoptotic signalingpathway in human colon adenocarcinoma cells by a lipid-raftindependentmechanismrdquoBiochemical andBiophysical ResearchCommunications vol 388 no 2 pp 372ndash376 2009

[44] X Bai H Zhang and S Ren ldquoAntioxidant activity and HPLCanalysis of polyphenol-enriched extracts from industrial applepomacerdquo Journal of the Science of Food and Agriculture vol 93no 10 pp 2502ndash2506 2013


[45] C M Andre J M Greenwood E G Walker et al ldquoAnti-inflammatory procyanidins and triterpenes in 109 apple vari-etiesrdquo Journal of Agricultural and Food Chemistry vol 60 no42 pp 10546ndash10554 2012

[46] N Balasuriya and H P V Rupasinghe ldquoAntihypertensiveproperties of flavonoid-rich apple peel extractrdquo Food Chemistryvol 135 no 4 pp 2320ndash2325 2012

[47] E-B Byun S Korematsu T Ishikawa et al ldquoApple procyanidinsinduce hyperpolarization of rat aorta endothelial cells viaactivation of K+ channelsrdquo Journal of Nutritional Biochemistryvol 23 no 3 pp 278ndash286 2012

[48] A Duda-Chodak T Tarko and T Tuszynski ldquoAntioxidantactivity of applesmdashan impact of maturity stage and fruit partrdquoActa Scientiarum Polonorum Technologia Alimentaria vol 10no 4 pp 443ndash454 2011

[49] F Dajas ldquoLife or death neuroprotective and anticancer effectsof quercetinrdquo Journal of Ethnopharmacology vol 143 no 2 pp383ndash396 2012

[50] M Gonzalez-Vallinas M Gonzalez-Castejon A Rodrıguez-Casado and A Ramırez de Molina ldquoDietary phytochemicalsin cancer prevention and therapy a complementary approachwith promising perspectivesrdquo Nutrition Reviews vol 71 no 9pp 585ndash599 2013

[51] A Alberti A A F Zelinski D M Zardo I M DemiateA Nogueira and L I Mafra ldquoOptimisation of the extractionof phenolic compounds from apples using response surfacemethodologyrdquo Food Chemistry vol 149 pp 151ndash158 2014

[52] R M Alonso-Salces K Ndjoko E F Queiroz et al ldquoOn-linecharacterisation of apple polyphenols by liquid chromatogra-phy coupledwithmass spectrometry and ultraviolet absorbancedetectionrdquo Journal of Chromatography A vol 1046 no 1-2 pp89ndash100 2004

[53] C Gosch HHalbwirth andK Stich ldquoPhloridzin biosynthesisdistribution and physiological relevance in plantsrdquo Phytochem-istry vol 71 no 8-9 pp 838ndash843 2010

[54] 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

[55] M Najafian M Z Jahromi M J Nowroznejhad et alldquoPhloridzin reduces blood glucose levels and improves lipidsmetabolism in streptozotocin-induced diabetic ratsrdquoMolecularBiology Reports vol 39 no 5 pp 5299ndash5306 2012

[56] E Delian V Petre I Burzo L Badulescu and D HozaldquoTotal phenols and nutrients composition aspects of some applecultivars and new studied breeding creations lines grown inVoinesti areamdashRomaniardquo Romanian Biotechnological Lettersvol 16 no 6 pp 6722ndash6729 2011

[57] M A Awad and A De Jager ldquoRelationships between fruitnutrients and concentrations of flavonoids and chlorogenic acidin ldquoElstarrdquo apple skinrdquo ScientiaHorticulturae vol 92 no 3-4 pp265ndash276 2002

[58] T KMcGhieMHunt and L E Barnett ldquoCultivar and growingregion determine the antioxidant polyphenolic concentrationand composition of apples grown in New Zealandrdquo Journal ofAgricultural and Food Chemistry vol 53 no 8 pp 3065ndash30702005

[59] L Mainla U Moor K Karp and T Pussa ldquoThe effect ofgenotype and rootstock on polyphenol composition of selectedapple cultivars in Estoniardquo Zemdirbyste vol 98 no 1 pp 63ndash702011

[60] N T T Hoang J B Golding and M A Wilkes ldquoThe effectof postharvest 1-MCP treatment and storage atmosphere onldquoCripps Pinkrdquo apple phenolics and antioxidant activityrdquo FoodChemistry vol 127 no 3 pp 1249ndash1256 2011

[61] A A Van der SluisMDekker A De Jager andWM F JongenldquoActivity and concentration of polyphenolic antioxidants inapple effect of cultivar harvest year and storage conditionsrdquoJournal of Agricultural and Food Chemistry vol 49 no 8 pp3606ndash3613 2001

[62] S F Hagen G I A Borge G B Bengtsson et al ldquoPhenolic con-tents and other health and sensory related properties of applefruit (Malus domestica Borkh cv Aroma) effect of postharvestUV-B irradiationrdquo Postharvest Biology and Technology vol 45no 1 pp 1ndash10 2007

[63] S Sun L Xin H Gao J Wang and P Li ldquoResponse of phenoliccompounds in ldquoGolden Deliciousrdquo and ldquoRed Deliciousrdquo applespeel to fruit bagging and subsequent sunlightre-exposurerdquoScientia Horticulturae vol 168 pp 161ndash167 2014

[64] S Murtic H Civic M ETHuric et al ldquoThe content of someantioxidants in apple depends on the type of fertilizationrdquoPolishJournal of Environmental Studies vol 22 no 2 pp 475ndash4802013

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


and quantitative analyses of the content of phenolic com-pounds in apples described in literature sources may notalways be suitable for implementation in other laboratoriesBecause of possible differences in the phytochemical compo-sition of the apples these techniques may be unsuitable forthe analysis of local cultivars Because of this a detailed sci-entific analysis requires the redevelopment of sample prepa-ration andmobile phase gradients of the techniques designedin other laboratories The need for the development of anovel technique was confirmed by our preliminary attemptsto adapt the techniques found in scientific literature sourcesthese attempts did not yield the desired results For thisreason the aim was set to develop optimize and validatethe suitable HPLC technique for the detection of phenoliccompounds in the lyophilized apple samples of the cultivarsldquoAldasrdquo ldquoAuksisrdquo ldquoLigolrdquo and ldquoSampionrdquo grown in Lithuania

2 Materials and Methods

21 Apples The following apple cultivars were included in thestudy ldquoAldasrdquo ldquoAuksisrdquo ldquoLigolrdquo and ldquoSampionrdquo the appleswere harvested in 2011The apples were supplied by the Insti-tute of Horticulture Lithuanian Research Centre for Agri-culture and Forestry Babtai Lithuania (55∘601015840N 23∘481015840E)The altitude of Babtai town is 57m above sea level Theyear 2011 was warmer than usual higher the mean monthlytemperatures were higher than the long-term average Thesummer of 2011 was distinguished by more rainfall in Julyand especially in August when double the normal monthlyrainfall was recorded 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 conditionsof the experimental orchard were the following clay loampH 73 humus 28 P

2O5 255mgkg and K

2O 230mgkg

The apple cultivars ldquoAuksisrdquo (early winter cv bred in Lithua-nia) ldquoSampionrdquo (winter cv bred in Czech Republic) andldquoLigolrdquo (winter cv bred in Poland) are the main cultivarsin Lithuanian commercial orchards The cv ldquoAldasrdquo (earlywinter cv bred in Lithuania) is recommended for ecologicalorchards Apples were harvested at the optimal harvest timeindividually established for every cultivar ldquoAldasrdquo 128DAFBldquoAuksisrdquo 114 DAFB ldquoLigolrdquo 168 DAFB and ldquoSampionrdquo 132DAFB The sample size comprised 20 ripe apples of eachcultivar

Each apple was cut into the slices of equal size (up to 1 cmin thickness) and the stalks and the seeds were removedThe apple slices were immediately frozen in a freezer (at ndash35∘C)with air circulation and then lyophilizedwith aZIRBUSsublimator 3 times 4 times 520 (ZIRBUS technology Bad GrundGermany) at the pressure of 001mbar (condenser tempera-ture minus85∘C)The lyophilized apple slices were ground to finepowder by using a Retsch 200 mill (Haan Germany)

Loss on drying before analysis was determined by dryingthe apple lyophilizate in a laboratory drying oven to complete

evaporation of water and volatile compounds (temperature105∘C the difference in weight between measurements upto 001 g) and by calculating the difference in raw materialweight before and after drying [10] The data were recalcu-lated for absolute dry lyophilizate weight

22 Chemicals All the solvents reagents and standards usedwere of analytical grade Aluminum trichloride hexahydratehexamethylentetramine acetonitrile acetone methanol andacetic and trifluoroacetic acids were obtained from Sigma-AldrichGmbH (Buchs Switzerland) and ethanol fromStum-bras AB (Kaunas Lithuania) Hyperoside rutin quercitrinphloridzin procyanidinB1 and procyanidin B2 and chloro-genic acid standards were purchased from Extrasynthese(Genay France) (+)-catechin and (minus)-epicatechin fromFluka (Buchs Switzerland) and avicularin and isoquercitrinfrom Chromadex (Santa Ana USA) In the study we useddeionized water produced by the Crystal E HPLC (AdronaSIA Riga Latvia) water purification system

23 Extraction An amount of 25 g of lyophilized apple pow-der (exact weight) was weighed added to 30mL of ethanol(70 vv) and extracted in a Sonorex Digital 10 P ultrasonicbath (Bandelin Electronic GmbH amp Co KG Berlin Ger-many) for 20 minutes at 40∘C The conditions of extraction(the type of the extraction its duration temperature andthe solvent and its concentration) were chosen consider-ing the results of the optimization of the extraction Theextract obtained was filtered through a paper filter the applelyophilizate on the filter was washed twice with 10mL ofethanol (70 vv) in a 50mL flask The extract was filteredthrough a membrane filter with a pore size of 022120583m (CarlRoth GmbH Karlsruhe Germany)

24 Determination of the Total Flavonoid Content The totalflavonoid content in the extracts of lyophilized apple sampleswas determined by applying the technique described byUrbonaviciute et al [11] was calculated using the rutincalibration curve and was expressed as its equivalent (mgREg) for absolute dry lyophilizate weight

25 Instrumentation and Chromatographic Conditions AWaters 2695 chromatograph equipped with a Waters 2998PDA detector (Waters Milford USA) was used for HPLCanalysis Chromatographic separation was managed chro-matograms were recorded and data were processed with theEmpower v20 software (Waters Milford USA) Chromato-graphic separations were carried out by using a YMC-PackODS-A (5 120583m C

18 250 times 46mm id) column equipped

with a YMC-Triart (5 120583m C18 10 times 30mm id) precolumn

(YMC Europe GmbH Dinslaken Germany) The columnwas operated at a constant temperature of 25∘C The volumeof the extract being investigated was 10 120583L The flow ratewas 1mLmin and gradient elution was used The mobilephase consisted of 2 (vv) acetic acid in water (solventA) and 100 (vv) acetonitrile (solvent B) The followingconditions of elution were applied 0ndash30 minutes 3ndash15 B30ndash45 minutes 15ndash25 B 45ndash50 minutes 25ndash50 B and








0051015202530

20 30 40 50 60 70 80 90 95Tota

l flav

onoi

d co

nten

t(m

g RE

g D

W)




005101520253035

0 5 10 15 20 25

Tota

l flav

onoi

d co

nten

t (m

g RE

g D

W)

Time (h)


0051015202530354045

0 10 20 30 40 50 60 70 80 90

Tota

l flav

onoi

d co

nten

t(m

g RE

g D

W)

Time (min)

20∘C

30∘C

40∘C







Wavelength(nm)



(120583gmL)R2




18 250times46mm id)













(AU

)

0002004006008

01012

(min)

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50

1 2

3

4 5 11

(a)

(min)

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50

(AU

)

0004008012016

02 3

(b)

(min)

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50

3

6

7

8

910

(AU

)

0001002003004

(c)










(+)-Catechin 1457a 1482b 501c 1297d




Rutin 164a 126b 148c 128d







(AU

)

0

0005

001

0015

002

0025

003

31 2 4 5 6

7

8

910 11

(min)0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50

(AU

)

00102030405060708

40 42446

7

8

9

10

(min)

times10minus2

(AU

)

minus002

0

002

004

006

008

18 19 20 21

3

2

(min)

times10minus2

(AU

)

minus002minus001

0001002003004005

13 14 15 16 17

1

(min)

times10minus2























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








0051015202530

20 30 40 50 60 70 80 90 95Tota

l flav

onoi

d co

nten

t(m

g RE

g D

W)




005101520253035

0 5 10 15 20 25

Tota

l flav

onoi

d co

nten

t (m

g RE

g D

W)

Time (h)


0051015202530354045

0 10 20 30 40 50 60 70 80 90

Tota

l flav

onoi

d co

nten

t(m

g RE

g D

W)

Time (min)

20∘C

30∘C

40∘C







Wavelength(nm)



(120583gmL)R2




18 250times46mm id)













(AU

)

0002004006008

01012

(min)

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50

1 2

3

4 5 11

(a)

(min)

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50

(AU

)

0004008012016

02 3

(b)

(min)

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50

3

6

7

8

910

(AU

)

0001002003004

(c)










(+)-Catechin 1457a 1482b 501c 1297d




Rutin 164a 126b 148c 128d







(AU

)

0

0005

001

0015

002

0025

003

31 2 4 5 6

7

8

910 11

(min)0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50

(AU

)

00102030405060708

40 42446

7

8

9

10

(min)

times10minus2

(AU

)

minus002

0

002

004

006

008

18 19 20 21

3

2

(min)

times10minus2

(AU

)

minus002minus001

0001002003004005

13 14 15 16 17

1

(min)

times10minus2























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




Wavelength(nm)



(120583gmL)R2




18 250times46mm id)













(AU

)

0002004006008

01012

(min)

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50

1 2

3

4 5 11

(a)

(min)

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50

(AU

)

0004008012016

02 3

(b)

(min)

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50

3

6

7

8

910

(AU

)

0001002003004

(c)










(+)-Catechin 1457a 1482b 501c 1297d




Rutin 164a 126b 148c 128d







(AU

)

0

0005

001

0015

002

0025

003

31 2 4 5 6

7

8

910 11

(min)0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50

(AU

)

00102030405060708

40 42446

7

8

9

10

(min)

times10minus2

(AU

)

minus002

0

002

004

006

008

18 19 20 21

3

2

(min)

times10minus2

(AU

)

minus002minus001

0001002003004005

13 14 15 16 17

1

(min)

times10minus2























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

)

0002004006008

01012

(min)

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50

1 2

3

4 5 11

(a)

(min)

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50

(AU

)

0004008012016

02 3

(b)

(min)

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50

3

6

7

8

910

(AU

)

0001002003004

(c)










(+)-Catechin 1457a 1482b 501c 1297d




Rutin 164a 126b 148c 128d







(AU

)

0

0005

001

0015

002

0025

003

31 2 4 5 6

7

8

910 11

(min)0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50

(AU

)

00102030405060708

40 42446

7

8

9

10

(min)

times10minus2

(AU

)

minus002

0

002

004

006

008

18 19 20 21

3

2

(min)

times10minus2

(AU

)

minus002minus001

0001002003004005

13 14 15 16 17

1

(min)

times10minus2























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





(+)-Catechin 1457a 1482b 501c 1297d




Rutin 164a 126b 148c 128d







(AU

)

0

0005

001

0015

002

0025

003

31 2 4 5 6

7

8

910 11

(min)0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50

(AU

)

00102030405060708

40 42446

7

8

9

10

(min)

times10minus2

(AU

)

minus002

0

002

004

006

008

18 19 20 21

3

2

(min)

times10minus2

(AU

)

minus002minus001

0001002003004005

13 14 15 16 17

1

(min)

times10minus2























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




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































































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










Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

Documents

Research Article Application of an Optimized HPLC Method for …downloads.hindawi.com/journals/jchem/2014/542121.pdf · 2019. 7. 31. · Research Article Application of an Optimized