Research Article New Sample Preparation Method for ...downloads.hindawi.com/journals/jamc/2015/964341.pdf · from the Sri Lankan tea germplasm by chemical analysis of tea leaves would

Research ArticleNew Sample Preparation Method for Quantification ofPhenolic Compounds of Tea (Camellia sinensis L Kuntze)A Polyphenol Rich Plant

P A Nimal Punyasiri1 Brasathe Jeganathan2 Jeevan Dananjaya Kottawa-Arachchi3

Mahasen A B Ranatunga3 I Sarath B Abeysinghe3

M T Kumudini Gunasekare4 and B M Rathnayake Bandara5

1 Institute of Biochemistry Molecular Biology and Biotechnology University of Colombo 00300 Colombo Sri Lanka2Department of Food Science and Technology Faculty of Agriculture University of Peradeniya 20400 Peradeniya Sri Lanka3Tea Research Institute of Sri Lanka 22100 Talawakelle Sri Lanka4Coordinating Secretariat for Science Technology amp Innovation 3rd Floor Standard Charted Building Janadhipathi Mawatha00100 Colombo Sri Lanka5Department of Chemistry Faculty of Science University of Peradeniya 20400 Peradeniya Sri Lanka

Correspondence should be addressed to P A Nimal Punyasiri nimalibmbbcmbaclk

Received 5 August 2015 Revised 24 August 2015 Accepted 28 September 2015

Academic Editor Krystyna Pyrzynska

Copyright copy 2015 P A Nimal Punyasiri et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Chemical analysis of the Sri Lankan tea (Camellia sinensis L) germplasm would immensely contribute to the success of thetea breeding programme However the polyphenols particularly catechins (flavan-3-ols) are readily prone to oxidation in theconventional method of sample preparation Therefore optimization of the present sample preparation methodology for theprofiling of metabolites is much important Two sample preparation methodologies were compared fresh leaves (as in theconventional procedures) and freeze-dried leaves (a new procedure) for quantification of major metabolites by employing twocultivars one is known to be high quality black tea and the other low quality black tea The amounts of major metabolites such ascatechins caffeine gallic acid and theobromine recorded in the new sampling procedure via freeze-dried leaves were significantlyhigher than those recorded in the conventional sample preparation procedure Additionally new method required less amount ofleaf sample for analysis of major metabolites and facilitates storage of samples until analysis The freeze-dried method would beuseful for high throughput analysis of large number of samples in shorter period without chemical deterioration starting from thepoint of harvest until usage Hence this method is more suitable for metabolite profiling of tea as well as other phenol rich plants

1 Introduction

Phenolic compounds are the most commonly studied ofall secondary metabolites because of their significant con-centration and their significant roles in plant tissues [1 2]This highly diverse group of secondary metabolites widely isdistributed in vegetable foods (legumes cereals and fruits)and beverages (tea cider and wine) which are importantconstituents of the human diet [3]

The rapid degradation of flavonoids during the extractionis a major drawback for the accurate quantification of thisubiquitous group of compounds Because of their importance

as potent antioxidants molecules quantification flavonoidsin fruits vegetable and other crops are important [4] Theybecome perishable mainly due to the enzymatic oxidationregulated by the enzyme polyphenol oxidase and light [5 6]Polyphenol oxidase is activated when the plant tissues aremacerated and the enzymes get mixed with the substrate andthe major parts of the native flavonoids are destroyed beforequantification

The sustainability of the tea industry mainly relies ondeveloping new tea (Camellia sinensis L) cultivars incorpo-rated with genetic resistance to biotic and abiotic stressesand capable of producing diverse tea products of desired

Hindawi Publishing CorporationJournal of Analytical Methods in ChemistryVolume 2015 Article ID 964341 6 pageshttpdxdoiorg1011552015964341

2 Journal of Analytical Methods in Chemistry

characterThe quality of tea product depends on the chemicalcomposition of fresh tea leaves [7] and the method ofmanufacture [8] The chemical composition of tea leaves isdetermined by the inherent genetic attributes of the accession[9] and geoclimatic factors [10]

Currently about 600 germplasm accessions are beingmaintained in the field gene bankof theTeaResearch Instituteof Sri Lanka (TRI) and these accessions are not adequatelyevaluated for biochemical agronomic and molecular traits[11 12] Most of the earlier attempts concentrated on singlemetabolite variation of tea cultivars and work on measure-ment of a broad range of metabolites is relatively limited [13]

Thus metabolite profiling of representative accessionsfrom the Sri Lankan tea germplasm by chemical analysisof tea leaves would immensely contribute to the success ofa tea breeding programme The polyphenols particularlycatechins (flavan-3-ols) and their oxidation products whichare eventually responsible for the quality of tea [14] arereadily oxidized by polyphenol oxidase enzymes The collec-tion handling and storage of a large number of leaf samplesfor metabolite profiling thus require a sample preparationprocedure that would minimize the oxidation of polyphenolsand formation of artifacts In this backdrop this researchhas been carried out with the objective to optimize thesample preparation procedure to minimize the chemicaldeterioration that takes place in tea flush due to the inevitabledelay in analysis For this purpose a comparative studyhas been conducted between the fresh leaves (conventionalprocedures) and freeze-dried leaves (new sampling method)for quantification of some critical metabolitesmdashgallic acidcatechins and methylxanthines by employing two cultivarsone (DT1) of which is known to yield high quality black teaand the other (TRI2025) poor quality black tea

2 Materials and Methods

21 Standards and Solvents Caffeine gallic acid (minus)-epicatechin (minus)-epigallocatechin (minus)-epigallocatechin gal-late and (minus)-epicatechin gallate were purchased from Sigmachemical Co (St Louis MO USA) Methanol acetonitrileacetic acid and Folin-Ciocalteursquos phenol reagent were pur-chased from BDH UK Standard reference material (SRM3254) for green tea was obtained from the National Instituteof Standards and Testing (NIST) USA All reagents wereHPLC grade or higher AMilliporeMilli-Qwater purificationsystem was used to obtain high purity of water

22 Plant Materials and Experimental Location Two culti-vars (DT1 and TRI2025) were selected for this study fromex situ field gene bank at the Tea Research Institute of SriLanka Talawakelle (latitude 6∘541015840N longitude 80∘421015840E) Theplants were of same age and have been maintained undersimilar growth conditions recommended by the Tea ResearchInstitute of Sri Lanka

23 Extraction of Fresh Tea Leaves (Conventional Procedure)Five grams of fresh two leaves and a bud was placed in70 aqueous methanol (40mL) at boiling temperature for

10min The mixture was cooled to room temperature andhomogenized for 3min using a top-drive macerator (Ultra-Turrax USA) The homogenate was centrifuged (2415timesgfor 15min) and the supernatant was decanted into a 50mLvolumetric flask The residue was reextracted following theabove extraction procedure with 70 aqueous methanol(10mL) at boiling temperature The volume of the pooledextract was made up to 50mL with 70 aqueous methanolAn aliquot (10mL) of the above methanol extract wastransferred into a 200mL volumetric flask and topped upwith Milli-Q water

24 Preparation of Freeze-Dried Tea Sample (New Procedure)Approximately 10 g of fresh two leaves and an active bud fromeach cultivar was harvested and brought to the laboratoryin ice box at 4∘C The plant material was immediatelydeep frozen at minus80∘C for 6 h and then freeze-dried for24 h (Labconco-Freezone USA) The freeze-dried flush wasground to a fine powder (passed through 500120583m mesh) ina laboratory mill sealed in triple laminated aluminum foilpackages and stored until use at room temperature

25 Extraction of Freeze-Dried Tea Flush 02000 plusmn 0001 gfreeze-dried tea sample was weighed into a centrifugationtube and extracted with 50mL of 70 methanolwaterextraction mixture preheated to 70∘C for half an hourHeating of the test portions in the water bath was continuedfor 10min with mixing on the vortex mixer at the beginningafter 5min and at the end of 10minThen the extraction tubewas allowed to cool to room temperature and placed in a cen-trifuge at 1095timesg for 10min The supernatant was carefullydecanted into 10mL volumetric flask The extraction stepswere repeated on the residue the extractswere combined andthe volume was made up to 10mL with cold methanolwaterextraction mixture and mixed 10mL of the sample extractwas topped up to 5mL with freshly prepared stabilizingsolution containing 500 120583gmLminus1 of EDTA and 500 120583gmLminus1ascorbic acid in 10 volume fraction of acetonitrile Analiquot was filtered through 045 120583m regenerated cellulosesample filter 100 120583L of the sample was used for the HPLCanalysis All samples were analysed in triplicate

26 Determination of Dry Matter Content The dry mattercontent was determined by loss in mass at 103∘C determinedon a portion of the test sample in accordance with ISO1573[15] method for both fresh flush leaves and freeze-dried flush

27 Determination of Total Polyphenols (TPP) in Fresh Flushand Freeze-Dried Flush Extracts A series of gallic acidstandard solutions was prepared with concentrations rangingfrom 10 to 50120583gmLminus1 The TPP in the standards andthe extracts were determined in quadruplicate using Folin-Ciocalteursquos phenol reagent according to ISO 14502-1 [16]method using a spectrophotometer (CARY 50Bio VarianUSA) set at 765 nm

Journal of Analytical Methods in Chemistry 3

28 Analysis of Metabolites in Fresh Leaf and Freeze-DriedExtracts Using High Performance Liquid Chromatography(HPLC) Caffeine EC ECg EGC EGCg gallic acid andtheobromine were quantified in duplicate in two trials ofsamples using ISO 14502-2 [17] method by HPLC (Agilent1260 HPLC Infinity System) on a phenyl-bonded columnusing elution with UV detection at 278 nm

29 HPLC Analysis

291 Instrumentation The HPLC instrument consisted ofAgilent 1260 Infinity HPLC system with solvent deliverysystem temperature-controlled column oven autosamplerdegasser unit and a photodiode array detector and OpenLabChemstation software system

292 Adjustment of the Apparatus Luna-5120583m Phenyl-Hexyl-46 times 250mm column (Phenomenex Inc USA) witha guard column made of the same material (Security-GuardPhenomenex Inc USA) was used Mobile phase A was 9acetonitrile containing 2acetic acid (BDHHipersolv-HPLCgrade) Mobile phase B was 80 aqueous acetonitrile (BDHHipersolv-HPLC grade) Solvent gradient was reset to 100mobile phase A and allowed to equilibrate for 10mins beforethe next injectionThe elutionwas reset to 100mobile phaseA and allowed to equilibrate for 10min before next injection

Flow rate was set to 100mLmin and column oventemperature was kept at 35 plusmn 05∘C The injection volumewas 10 120583LThemetabolites were detected at 278 nm Once theflow rate of the mobile phase and temperature were stablethe column was conditioned with a blank gradient run Aftereach batch of analysis the chromatographic system and col-umn were thoroughly flushed with 50 aqueous acetonitrileand column sealing plugs were replaced if disconnected forstorage

210 Identification and Quantification of Gallic Acid Cate-chins and Methylxanthines The individual catechins (ECECg EGC and EGCg) and gallic acid were identified bycomparing retention times from sample chromatogramswiththose recorded for a variety of standards obtained under thesame chromatographic conditions Calibration curves wereconstructed for the individual compounds The use of diodearray detection allowed the UV profile of the catechin peaksto be scrutinized and peak purity to be assessed which wasparticularly useful for the determination of the low levels ofcatechins in tea cultivars

Alternatively caffeine was used as a standard in con-junction with individual catechins considering the RelativeResponse Factors (RRFs) established by an ISO internationalinterlaboratory test Data was collected using the data collec-tionintegration system for all peaks in the extracts

211 Validation Procedure Standard referencematerial (SRM3254) for green tea certified by NIST USA was used for thevalidation of the analytical methodology

Table 1 Comparison of dry matter and the sample required foranalysis of cultivars DT1 and TRI2025

Cultivars Sample Dry matter () Sample requiredfor analysis (g)

DT1 Fresh 2038 500Freeze-dried 9118 020

TRI2025 Fresh 2141 500Freeze-dried 9349 020

212 Statistical Analysis Completely Randomized Design(CRD) was used as the experimental model Significant dif-ferences (119875 lt 001) were tested using two tailed paired 119905-testat 95 confidence level Least Significant Difference (LSD)was used as themean separation technique All these analyseswere carried out with the statistical analysis software package(SAS Version 91 for Windows SAS Institute Inc USA)

3 Results and Discussion

A major problem in the quantification of catechins is theirhighly perishable nature One of themost important fractionsin tea flush is the proteins because they include the enzymeswhich are required for the metabolism in the tea plant andfor black tea manufacture [7] Polyphenol oxidase is animportant enzyme available in tea which is activated by light[18] and is responsible for the oxidation of flavonols in thepresence of atmospheric oxygen In the conventional samplepreparation methodology there is an inevitable delay in theanalysis during which the tea flush is subjected to mechan-ical damage This results in the formation of characteristiccompounds of black tea [19 20] Though it is a positivereaction initiated in the rolling process of tea manufacturingit hinders accurate and precise measurement of metabolitesin the tea flush Further the content of catechins is dependentupon other factors including particularly light since some ofthe important enzymes of the flavonoid biosynthetic pathwayare activated by light [21] Since the analysis of the batch ofcultivars sampled has to be done on the same day it was notpossible to handle a large number of samples at a time

In contrast the new method suppressed chemical dete-rioration starting from the point of harvest until usage Asdescribed in Figure 1 the samples harvested from the fieldwere immediately collected into and ice box at 4∘C wherebythe chemical deterioration of themetabolites wasminimizedPrefreezing at minus80∘C for 6 h to two weeks is a convenientfirst step in sample preparation prior to freeze-drying Freeze-drying is a preservation technique by which moisture isremoved and the shelf-life is increased approximately upto one year The dry matter content of the freeze-driedsamples was eight times less than that of the fresh flushTherefore substantially less amount of sample was requiredfor analysis in the new method (Table 1) and convenienthandling of many samples was possible at a time Alsofreeze-dried leaf samples can be stored for a long time whenpacked in triple laminated aluminum sheets and it facilitatessampling at once and stored for metabolite analysis Thus


Leaf sampling

Transportation

Prefreezing

Freeze drying

Grinding

Storing

sample

Boiling(70 methanol)


Vortex

CentrifugationCentrifugation

leaves

Homogenizing

Dilution

HPLC

Catechins

Leaf sampling

UV-Vis

Total polyphenols

Conventionalprocedure

Proposedprocedure

50 g of fresh02 g of ground

for 24hr

(at 4∘C)

(at minus80∘C 6hr)

Figure 1 Steps of conventional and new sample preparation pro-cedure for metabolite analysis of tea

environmental influence on metabolite availability could beminimized specially when handling large number of samples

Maximum recovery of the metabolites required a mini-mum of 70 methanol in the extractant which was sufficientto inactivate polyphenol oxidase [22] Multiple extractionprocedures of short durations (10min) at moderate tem-perature (70∘C) in the new method were more favourablethan those involving boiling temperature and long extrac-tion times in a macerator as such conditions would leadto decomposition of catechins [23] Efficient separation ofcaffeine (minus)-epicatechin (EC) (minus)-epigallocatechin (EGC)(minus)-epigallocatechin gallate (EGCg) (minus)-epicatechin gallate(ECg) gallic acid and theobromine in the tea extracts(Figure 2) was achieved in less than 25min of a chromato-graphic run by the gradient elution with acetonitrile waterand acetic acid All calibration curves displayed excellentcorrelation coefficients (ge0998) Although the sensitivity ofthe analysis can be increased by threefold by using a shorterdetection wavelength the chance of interference by other

(min)0 5 10 15 20 25 30 35

(mAU

)

050

100150200250300350400450

250

82

671

308

63

304

363

0 513

36

671

974

2

124

9113

853

155

30

181

30

214

42

64

512

3

7

(1) Gallic acid(2) Theobromine(3) EGC(4) Caffeine

(5) Epicatechin(6) EGCg(7) ECg

Figure 2 HPLC chromatogram of representative tea accessionquantified using ISO 14502-2 method

components present in the tea extract is high [23] Hence278 nm was used as the detector wavelength

The Folin-Ciocalteursquos phenol reagent used for TPP anal-ysis contains phosphotungstic acid as oxidants which onreduction yield a blue colour with a broad maximum absorp-tion at 765 nm As this reagent reacts with a wide range ofpolyphenol compounds it is often used to estimate the ldquototalpolyphenolrdquo content in plant material rich in polyphenolsalthough the response can vary with the individual com-ponents depending on their chemical structures Howeverthe Folin-Ciocalteursquos reagent could simultaneously oxidizeseveral nonphenolic organic compounds as well as someinorganic substances to give elevated apparent phenoliccontent [24] As it is based on the reduction properties ofsample components it really measures the total reducingcapacity of samples

The mean values of TPP and the amounts of fourcatechins (EC ECg EGC and EGCg) caffeine theobromineand gallic acid recorded for the two cultivars DT1 andTRI2025 corresponding to the two sampling procedures(fresh leaves and freeze-dried leaves) are given in Table 2The concentration of each metabolite varied with the cul-tivar For both cultivars DT1 and TRI2025 the contents ofall the metabolites (TPP EC ECg EGC EGCg caffeinetheobromine and gallic acid) determined by employing newmethod were found to be higher than in the conventionalmethod the difference was statistically significant (119875 lt 005)for each of the metabolites except for theobromine in cultivarDT1

National Institute of Standards and Testing (NIST) cer-tified SRM 3254 quantified via these two methods confirmsthat the new method is accurate and precise for the determi-nation of the major metabolites in tea

4 Conclusion

The freeze-dried sampling procedure recorded significantlyhigher amount of metabolites with small leaf quantityand facilitates storage of samples without chemical dete-rioration starting from the point of harvest until usage


Table 2 Metabolite composition of tea flush of cultivars DT1 and TRI 2025 determined using two methods of sampling with fresh tea flush(conventional procedure) and freeze-dried tea flush (new procedure)

Metabolites DT1 (mg gminus1) TRI 2025 (mg gminus1)Fresh Freeze-dried 119875 LSD CV Fresh Freeze-dried 119875 LSD CV

TPPdaggerdagger 26649 plusmn 279 28227 plusmn 377 lowastlowast 356 121 26633 plusmn 272 27807 plusmn 354 lowastlowast 339 116ECdagger 1588 plusmn 029 1927 plusmn 027 lowastlowast 048 159 627 plusmn 054 747 plusmn 041 lowast 082 69ECgdagger 3765 plusmn 122 4143 plusmn 053 lowast 163 238 2530 plusmn 145 3071 plusmn 049 lowastlowast 187 386EGCdagger 3399 plusmn 091 4221 plusmn 056 lowastlowast 131 199 1754 plusmn 106 2001 plusmn 092 lowast 172 528EGCgdagger 8400 plusmn 515 9361 plusmn 114 lowast 645 420 10417 plusmn 322 12895 plusmn 233 lowastlowast 487 241Caffeinedagger 3531 plusmn 136 3867 plusmn 033 lowast 171 267 3452 plusmn 013 4171 plusmn 059 lowastlowast 074 112Theobrominedagger 217 plusmn 015 225 plusmn 002 NS 018 490 462 plusmn 032 589 plusmn 017 lowastlowast 045 491Gallic aciddagger 029 plusmn 001 055 plusmn 003 lowastlowast 003 426 031 plusmn 001 083 plusmn 001 lowastlowast 002 159Means plusmn SD (119899 = 8) 119875 probability lowastsignificant at 5 level of probability lowastlowastsignificant at 1 level of probability NS not significantly different CV coefficientof variance LSD Least Significant Difference daggerdaggerdetermined by ISO-14052-1 Spectrophotometric method daggerdetermined by ISO-14052-2 HPLC method andTPP total polyphenols

Hence the method is more suitable for metabolite profilingof tea germplasm using high throughput analysis where largenumber of samples need to be handled

Conflict of Interests

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

Acknowledgments

This work was supported by the National Research Councilof Sri Lanka (Grant no NRC11023)

References

[1] P G Waterman and S Mole Analysis of Phenolic Plant Metabo-lites Blackwell Scientific Publications Oxford UK 1994

[2] J B Harborne ldquoGeneral procedures and measurement of totalphenolicsrdquo in Methods in Plant Biochemistry Plant Phenolicspp 1ndash29 Academic Press London UK 1989

[3] L Bravo R Abia M A Eastwood and F Saura-CalixtoldquoDegradation of polyphenols (catechin and tannic acid) in therat intestinal tract effect on colonic fermentation and faecaloutputrdquo British Journal of Nutrition vol 71 no 6 pp 933ndash9461994

[4] M Stobiecki and P Kachlicki ldquoIsolation and identification offlavonoidsrdquo inThe Science of Flavonoids E Grotewold Ed pp47ndash69 Springer New York NY USA 2006

[5] M Obanda P O Owuor and C K Njuguna ldquoThe impact ofclonal variation of total polyphenols content and polyphenoloxidase activity of fresh tea shoots on plain black tea qualityparametersrdquo Tea vol 13 no 2 pp 129ndash133 1992

[6] Y S Wang L P Gao Z R Wang et al ldquoLight-inducedexpression of genes involved in phenylpropanoid biosyntheticpathways in callus of tea (Camellia sinensis (L) O Kuntze)rdquoScientia Horticulturae vol 133 no 1 pp 72ndash83 2012

[7] A Robertson ldquoThe chemistry and biochemistry of black teaproductionmdashthe non-volatilesrdquo inTea Cultivation toConsump-tion K C Willson and M N Clifford Eds pp 555ndash601Chapman amp Hall London UK 1992

[8] M Obanda P O Owuor R Mangrsquooka and M M KavoildquoChanges in thearubigin fractions and theaflavin levels due tovariations in processing conditions and their influence on blacktea liquor brightness and total colourrdquo Food Chemistry vol 85no 2 pp 163ndash173 2004

[9] K Wei L Wang J Zhou et al ldquoCatechin contents in tea(Camellia sinensis) as affected by cultivar and environment andtheir relation to chlorophyll contentsrdquo Food Chemistry vol 125no 1 pp 44ndash48 2011

[10] Y Chen Y Jiang J Duan J Shi S Xue and Y KakudaldquoVariation in catechin contents in relation to quality of lsquoHuangZhi Xiangrsquo Oolong tea (Camellia sinensis) at various growingaltitudes and seasonsrdquo Food Chemistry vol 119 no 2 pp 648ndash652 2010

[11] M T K Gunasekare ldquoCurrent status and future directionsin breeding teardquo in Current Status and Future Directions ofPlant Breeding Research in Sri Lanka H P M Gunasena andP C Girihagama Eds pp 111ndash124 Sri Lanka Council forAgricultural Research Policy Colombo Sri Lanka 2007

[12] M A B Ranatunge and M T K Gunasekare ldquoAssemblingof preliminary core collection of tea (Camellia sinensis (L) OKuntze) genetic resources in Sri Lankardquo Plant Genetic ResourcesNewsletter vol 155 pp 41ndash45 2008

[13] J D Kottawa-Arachchi M T K Gunasekare M A BRanatunga L Jayasinghe and R P Karunagoda ldquoAnalysisof selected biochemical constituents in black tea (Camelliasinensis) for predicting the quality of tea germplasm in SriLankardquo Tropical Agricultural Research vol 23 no 1 pp 30ndash412012

[14] U H Engelhardt ldquoChemistry of teardquo in Comprehensive NaturalProducts II Chemistry and Biology L Mender and H W LiuEds pp 1000ndash1027 Elsevier London UK 2010

[15] International Organization for Standardization ldquoTea Deter-mination of loss in mass at 103∘Crdquo ISO 1573 InternationalOrganization for Standardization 1980

[16] ISO ldquoDetermination of substances characteristic of greenand black tea-part I content of total polyphenols in teamdashcolorimetricmethodusing Folin-Ciocalteu reagentrdquo ISO 14502-1 2005

[17] International Organization for Standardization ldquoDetermina-tion of substances characteristic of green and black teamdashpart2 content of catechins caffeine and gallic acid in teamdashmethod


using high performance liquid chromatographyrdquo ISO 14502-2International Organization for Standardization 2005

[18] Y S Wang L P Gao Y Shan Y L Liu Y W Tian and T XialdquoInfluence of shade on flavonoid biosynthesis in tea (Camelliasinensis (L) O Kuntze)rdquo Scientia Horticulturae vol 141 pp 7ndash16 2012

[19] A Robertson ldquoEffects of physical and chemical conditions onthe in vitro oxidation of tea leaf catechinsrdquo Phytochemistry vol22 no 4 pp 889ndash896 1983

[20] A Robertson ldquoEffects of catechin concentration on the for-mation of black tea polyphenols during in vitro oxidationrdquoPhytochemistry vol 22 no 4 pp 897ndash903 1983

[21] P J Hilton ldquoTeardquo in Encyclopedia of Industrial ChemicalAnalysis F J Snell and L S Eltre Eds vol 18 pp 455ndash518Inderscience Publishers New York NY USA 1973

[22] K Robards ldquoStrategies for the determination of bioactive phe-nols in plants fruit and vegetablesrdquo Journal of ChromatographyA vol 1000 no 1-2 pp 657ndash691 2003

[23] Y Zuo H Chen and Y Deng ldquoSimultaneous determinationof catechins caffeine and gallic acids in green oolong blackand pu-erh teas using HPLC with a photodiode array detectorrdquoTalanta vol 57 no 2 pp 307ndash316 2002

[24] A Pekal P Drozdz M Biesaga and K Pyrzynska ldquoEvaluationof the antioxidant properties of fruit and flavoured black teasrdquoEuropean Journal of Nutrition vol 50 no 8 pp 681ndash688 2011

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


characterThe quality of tea product depends on the chemicalcomposition of fresh tea leaves [7] and the method ofmanufacture [8] The chemical composition of tea leaves isdetermined by the inherent genetic attributes of the accession[9] and geoclimatic factors [10]

Currently about 600 germplasm accessions are beingmaintained in the field gene bankof theTeaResearch Instituteof Sri Lanka (TRI) and these accessions are not adequatelyevaluated for biochemical agronomic and molecular traits[11 12] Most of the earlier attempts concentrated on singlemetabolite variation of tea cultivars and work on measure-ment of a broad range of metabolites is relatively limited [13]

Thus metabolite profiling of representative accessionsfrom the Sri Lankan tea germplasm by chemical analysisof tea leaves would immensely contribute to the success ofa tea breeding programme The polyphenols particularlycatechins (flavan-3-ols) and their oxidation products whichare eventually responsible for the quality of tea [14] arereadily oxidized by polyphenol oxidase enzymes The collec-tion handling and storage of a large number of leaf samplesfor metabolite profiling thus require a sample preparationprocedure that would minimize the oxidation of polyphenolsand formation of artifacts In this backdrop this researchhas been carried out with the objective to optimize thesample preparation procedure to minimize the chemicaldeterioration that takes place in tea flush due to the inevitabledelay in analysis For this purpose a comparative studyhas been conducted between the fresh leaves (conventionalprocedures) and freeze-dried leaves (new sampling method)for quantification of some critical metabolitesmdashgallic acidcatechins and methylxanthines by employing two cultivarsone (DT1) of which is known to yield high quality black teaand the other (TRI2025) poor quality black tea

2 Materials and Methods

21 Standards and Solvents Caffeine gallic acid (minus)-epicatechin (minus)-epigallocatechin (minus)-epigallocatechin gal-late and (minus)-epicatechin gallate were purchased from Sigmachemical Co (St Louis MO USA) Methanol acetonitrileacetic acid and Folin-Ciocalteursquos phenol reagent were pur-chased from BDH UK Standard reference material (SRM3254) for green tea was obtained from the National Instituteof Standards and Testing (NIST) USA All reagents wereHPLC grade or higher AMilliporeMilli-Qwater purificationsystem was used to obtain high purity of water

22 Plant Materials and Experimental Location Two culti-vars (DT1 and TRI2025) were selected for this study fromex situ field gene bank at the Tea Research Institute of SriLanka Talawakelle (latitude 6∘541015840N longitude 80∘421015840E) Theplants were of same age and have been maintained undersimilar growth conditions recommended by the Tea ResearchInstitute of Sri Lanka

23 Extraction of Fresh Tea Leaves (Conventional Procedure)Five grams of fresh two leaves and a bud was placed in70 aqueous methanol (40mL) at boiling temperature for

10min The mixture was cooled to room temperature andhomogenized for 3min using a top-drive macerator (Ultra-Turrax USA) The homogenate was centrifuged (2415timesgfor 15min) and the supernatant was decanted into a 50mLvolumetric flask The residue was reextracted following theabove extraction procedure with 70 aqueous methanol(10mL) at boiling temperature The volume of the pooledextract was made up to 50mL with 70 aqueous methanolAn aliquot (10mL) of the above methanol extract wastransferred into a 200mL volumetric flask and topped upwith Milli-Q water

24 Preparation of Freeze-Dried Tea Sample (New Procedure)Approximately 10 g of fresh two leaves and an active bud fromeach cultivar was harvested and brought to the laboratoryin ice box at 4∘C The plant material was immediatelydeep frozen at minus80∘C for 6 h and then freeze-dried for24 h (Labconco-Freezone USA) The freeze-dried flush wasground to a fine powder (passed through 500120583m mesh) ina laboratory mill sealed in triple laminated aluminum foilpackages and stored until use at room temperature

25 Extraction of Freeze-Dried Tea Flush 02000 plusmn 0001 gfreeze-dried tea sample was weighed into a centrifugationtube and extracted with 50mL of 70 methanolwaterextraction mixture preheated to 70∘C for half an hourHeating of the test portions in the water bath was continuedfor 10min with mixing on the vortex mixer at the beginningafter 5min and at the end of 10minThen the extraction tubewas allowed to cool to room temperature and placed in a cen-trifuge at 1095timesg for 10min The supernatant was carefullydecanted into 10mL volumetric flask The extraction stepswere repeated on the residue the extractswere combined andthe volume was made up to 10mL with cold methanolwaterextraction mixture and mixed 10mL of the sample extractwas topped up to 5mL with freshly prepared stabilizingsolution containing 500 120583gmLminus1 of EDTA and 500 120583gmLminus1ascorbic acid in 10 volume fraction of acetonitrile Analiquot was filtered through 045 120583m regenerated cellulosesample filter 100 120583L of the sample was used for the HPLCanalysis All samples were analysed in triplicate

26 Determination of Dry Matter Content The dry mattercontent was determined by loss in mass at 103∘C determinedon a portion of the test sample in accordance with ISO1573[15] method for both fresh flush leaves and freeze-dried flush

27 Determination of Total Polyphenols (TPP) in Fresh Flushand Freeze-Dried Flush Extracts A series of gallic acidstandard solutions was prepared with concentrations rangingfrom 10 to 50120583gmLminus1 The TPP in the standards andthe extracts were determined in quadruplicate using Folin-Ciocalteursquos phenol reagent according to ISO 14502-1 [16]method using a spectrophotometer (CARY 50Bio VarianUSA) set at 765 nm



29 HPLC Analysis
















Leaf sampling

Transportation

Prefreezing

Freeze drying

Grinding

Storing

sample



Vortex


leaves

Homogenizing

Dilution

HPLC

Catechins

Leaf sampling

UV-Vis

Total polyphenols


Proposedprocedure


for 24hr

(at 4∘C)





(min)0 5 10 15 20 25 30 35

(mAU

)

050

100150200250300350400450

250

82

671

308

63

304

363

0 513

36

671

974

2

124

9113

853

155

30

181

30

214

42

64

512

3

7








4 Conclusion









Acknowledgments


References




































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



29 HPLC Analysis
















Leaf sampling

Transportation

Prefreezing

Freeze drying

Grinding

Storing

sample



Vortex


leaves

Homogenizing

Dilution

HPLC

Catechins

Leaf sampling

UV-Vis

Total polyphenols


Proposedprocedure


for 24hr

(at 4∘C)





(min)0 5 10 15 20 25 30 35

(mAU

)

050

100150200250300350400450

250

82

671

308

63

304

363

0 513

36

671

974

2

124

9113

853

155

30

181

30

214

42

64

512

3

7








4 Conclusion









Acknowledgments


References




































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


Leaf sampling

Transportation

Prefreezing

Freeze drying

Grinding

Storing

sample



Vortex


leaves

Homogenizing

Dilution

HPLC

Catechins

Leaf sampling

UV-Vis

Total polyphenols


Proposedprocedure


for 24hr

(at 4∘C)





(min)0 5 10 15 20 25 30 35

(mAU

)

050

100150200250300350400450

250

82

671

308

63

304

363

0 513

36

671

974

2

124

9113

853

155

30

181

30

214

42

64

512

3

7








4 Conclusion









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










Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

Documents

Research Article New Sample Preparation Method for ...downloads.hindawi.com/journals/jamc/2015/964341.pdf · from the Sri Lankan tea germplasm by chemical analysis of tea leaves would