Research Article Effects of Water Solutions on Extracting...

Hindawi Publishing CorporationThe Scientific World JournalVolume 2013 Article ID 368350 6 pageshttpdxdoiorg1011552013368350

Research ArticleEffects of Water Solutions on Extracting Green Tea Leaves

Wen-Ying Huang Yu-Ru Lin Ruei-Fen Ho Ho-Yen Liu and Yung-Sheng Lin

Department of Applied Cosmetology and Master Program of Cosmetic Science Hungkuang University Taichung Taiwan

Correspondence should be addressed to Yung-Sheng Lin linyssunrisehkedutw

Received 5 May 2013 Accepted 27 June 2013

Academic Editors A Garcıa-Vela D Kopelevich and I E Pavel

Copyright copy 2013 Wen-Ying Huang 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

This study investigates the effects ofwater solutions on the antioxidant content of green tea leaf extracts Green teas preparedwith tapwater and distilled water were comparedwith respect to four antioxidant assays total phenol content reducing power DMPD assayand trolox equivalent antioxidant capacity assay The results indicate that green tea prepared with distilled water exhibits higherantioxidant activity than that made with tap water The high performance liquid chromatography showed that major constituentsof green tea were found in higher concentrations in tea made with distilled water than in that made with tap water This could bedue to less calcium fixation in leaves and small water clusters Water solutions composed of less mineralisation are more effectivein promoting the quality of green tea leaf extracts

1 Introduction

Green tea is an infusion of the leaves of the Camellia sinensisplant a member of the Theaceae family [1 2] As a populardrink favoured by Asians in particular Chinese green teahas received much global attention for its promotion ofhuman health A number of studies have shown that green teapossesses a wide range of biological activities [3] includingantioxidant activity [4ndash9] anti-inflammatory activity [10]antimutagenic as well as anticarcinogenic activities [11ndash14] and neuroprotective effects [15] The major functionalconstituents of green tea catechins account for 8ndash15 of thedry leaf weight [16]

A variety of catechins which are members of thepolyphenol family are present in green tea leaves Thefour most abundant green tea epicatechins are (ndash)-epigallocatechin gallate (EGCG) (ndash)-epicatechin gallate(ECG) (ndash)-epigallocatechin (EGC) and (ndash)-epicatechin(EC) In addition four minor catechins are found in greentea leaves including catechin (C) catechin gallate (CG)gallocatechin (GC) and gallocatechin gallate (GCG) [16 17]Catechins have been reported to possess strong antioxidantactivity and are widely accepted as important antioxidantswhich eliminate free radicals [6]

It is presumed that different tea-processing proceduresand water components will result in different qualities of

green tea [18ndash21] Previous studies have demonstrated thatthe temperature [22ndash24] pH [25] organic solvent [26]pressure [27] soaking time [28] and elements of water [2930] affect the antioxidant activity of green tea However howthe physics-basedmechanism of the water solution affects teaextracts has rarely been discussed

The aim of this study is to compare the effects of makinggreen tea extracts with tap water versus distilled water onthe antioxidant activities of the extracts Comparisons aremade using the results of four antioxidant activity assaysand high performance liquid chromatography componentanalyses Following the literature review a possible physics-based mechanism to describe our observations is discussed

2 Materials and Methods

21 Chemicals and Reagents The green tea leaves were fromTenRen Tea Co Ltd Taipei Taiwan Gallic acid (GA) 221015840-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS)GC EC EGC and EGCG were obtained from Sigma(USA)NN-Dimethyl-p-phenylenediamine dihydrochloride(DMPD) and iron (III) chloride were purchased fromRiedel-de Haen (Germany) Folin-Ciocalteu reagent was from Fluka(Germany) Methanol acetonitrile and acetic acid werepurchased fromMerck (Darmstadt Germany)

2 The Scientific World Journal

22 Processing of Green Tea Instead of a brewing process[7 31] green tea solutions were prepared using tap waterand distilled water at 25∘C The former was taken from thesuburban area of the City of Greater Taichung Taiwan Dis-tilled water (lt1 120583Scm) was purified using a Millipore Alpha-Q Luton ultrapure water system such that both the organicand inorganic impurities were removed Two water sampleswere divided into 25mL aliquots in 50mL polypropylenecentrifuge tubes Aqueous extracts were prepared by soakinga 3125mg infusion of ground up and homogenized drygreen tea leaves in 25mL water at 25∘C for 20min Aftercentrifugation (1250 rpm) the green tea supernatant wascollected for further examination five replicates were madefor statistical analysis

23 Total Phenol Content The total phenol content of greentea was measured according to Gutfingerrsquos method [32] Eachgreen tea solution was mixed with 250120583L Folin-Ciocalteureagent for 1min and 50 120583L 2 Na

2CO3for 5min The

absorbance at 655 nm (Sunrise ELISA Plate Reader TecanAustria) denoted the total phenol content and it increasedwith the content of phenols

24 Reducing Power As described in a previous report [33]the reducing power of green tea was determined using amixture of 250120583Lphosphate buffer (200mM pH66) 250 120583Lpotassium ferricyanide (1 by weight) and 500120583L greentea The mixture was then incubated at 50∘C for 20minTrichloroacetic acid (250120583L 10 by weight) was mixed withdistilled water (250120583L) and FeCl

3(750 120583L 01 by weight)

allowed to react for 30min and the absorbance at 700 nmwasmeasured using a spectrophotometer Higher absorbanceindicated greater reducing power

25 DMPD Assay Antioxidant activities of green tea weremeasured according to Schlesierrsquos method [34] In principlecolorless DMPD will form a stable purple free radical cationDMPD+∙ in the presence of a suitable oxidant such as FeCl

3

Purple DMPD+∙ is scavenged and decolorized upon additionof an antioxidant Equal volumes of 24mM DMPD 24mMFeCl3 and 06M acetic acid were mixed together for 5min

to generate DMPD+∙ and the absorbance at 495 nm wasmeasured The antioxidant activity was inferred as the abilityto scavenge free radicals and a decrease in absorbance wasinferred as increasing scavenging power defined as follows

DMPD+ ∙ scavenging activity ()

= [1 minus (

A2A1)] times 100

(1)

where A1 denotes the absorbance of total free DMPD+∙radicals and A2 denotes the absorbance following additionof the antioxidant

26 Trolox Equivalent Antioxidant Capacity Assay The troloxequivalent antioxidant capacity (TEAC) assay was carried outusing the procedure described by Erkanrsquos method [35] Inbrief ABTS+∙ was produced by reacting 7mM ABTS with

245mM potassium persulfate in the dark at 4∘C for 12 h A30 120583L antioxidant aliquot was added to 2mL ABTS+∙ radicalsolution allowed to react for 10min and the absorbancemeasured at 734 nm

As stated previously the antioxidant capacitywas inferredas the ability to scavenge free radicals expressed as follows

ABTS+ ∙ scavenging activity ()

= [1 minus (

A4A3)] times 100

(2)

where A3 represents the absorbance of total free ABTS+∙radicals andA4 represents the absorbance following additionof the antioxidant

27 High Performance Liquid Chromatography The extractof green tea was mixed with an internal standard solution(05mg gallic acid diluted to 25mL with 70 methanol)at a ratio of 1 1 The samples were spiked with variousconcentrations of stock solutions and then analyzed

A stock solution was prepared by dissolving four markersubstances (GC 10mg EGC 50mg EGCG 25mg and EC100mg) in 1mL of 70 methanol the solution was thenstored in a refrigerator Stock solutions were diluted with70 methanol into a series of standard solutions (GC 078104 125 156 208 and 313 120583gmL EGC 250 625 7811041 1563 and 3125 120583gmL EGCG 260 313 391 521 and781 120583gmL EC 208 417 463 5 625 and 833 120583gmL) A20120583L aliquot of each solution was injected and analyzed twotimes using HPLC and the standard curves were plotted aspeak areas versus concentrations Recovery was determinedby comparing the amount of marker substances added to themarker substances found The limits of detection were basedon a signal to noise (SN) ratio of 3 1 as a minimum

HPLC was performed on an Agilent 1220 series systemSatisfactory separation of the markers was obtained using areversed-phase column (Cosmosil 5C18-AR II 5120583m 25 cmtimes46mm ID Nacalai Tesque Kyoto Japan) at 280 nm anelution flow rate of 08mLmin and a linear solvent gradientof A-B (A 10mM KH

2PO4(pH 40) B CH

3CN CH

3OH

and H2O at a ratio of 15 25 1 respectively [vvv]) as

follows 0min 20 B 5min 20 B 15min 30 B 45min60B

28 Statistical Analysis The paired t-test method was usedto evaluate differences between groups A value of 119875 lt 005was considered statistically significant (lowast) and 119875 lt 001 washighly significant (lowastlowast)

3 Results and Discussion

31 Total Phenol Content Figure 1 shows the differences inA655

between the tea solutions Indicating the total amountof phenol the A

655for the control (no green tea extract)

green tea made with distilled water and green tea made withtap water were 006 plusmn 002 081 plusmn 003 and 069 plusmn 003respectively Phenols are regarded as significant constituentsof green tea because of their free-radical scavenging ability

The Scientific World Journal 3

0010203040506070809

Tap water Distilled water Blank

Abso

rban

ce

lowastlowast

Figure 1 Effects of water solution on the total phenol content ofgreen tea

0

01

02

03

04

05

06


Abso

rban

ce

lowast

Figure 2 Effects of water solution on the reducing power of greentea

32 Reducing Power The reducing power of a compoundserves as an indicator of its antioxidant activity [33] Figure 2shows the reducing power (absorbance at 700 nm) of the3 groups tested The A

700for the control green tea made

with distilled water and green tea made with tap water were011 plusmn 002 053 plusmn 003 and 047 plusmn 006 respectively

33 DMPD Assay Figure 3 shows that the inhibition ofDMPD+∙ radical cations by green tea was 8404plusmn 310whenmade with distilled water and 8215 plusmn 246 whenmade withtap water Compared to tea made with tap water there was a189 improvement inDMPD+∙ scavenging activity in the teamade with distilled waterThus the distilled water was able toextract more antioxidant substances from the green tea leavesas determined by DMPD+∙ scavenging activity

34 Trolox Equivalent Antioxidant Capacity Assay Figure 4indicates that ABTS+∙ radicals were inhibited by 7875 plusmn181 and 7101 plusmn 141 in green tea made using distilledwater and tap water respectively Results of the TEACassay demonstrate that green tea made with distilled waterhas superior ABTS+∙ scavenging activity (774 increase)compared to green tea made with tap water These resultssuggest that distilled water has a clear advantage over tapwater in terms of its ability to extract more antioxidants intothe tea solution

35 High Performance Liquid Chromatography Calibrationcurves were prepared by plotting the peak-area ratios (using

60

65

70

75

80

85

90

Tap water Distilled water

lowast

DM

PD+∙

scav

engi

ng ac

tivity

()

Figure 3 Effects of water solution on DMPD+∙ scavenging activityin green tea

60

65

70

75

80

85

90


lowast

ABT

S+∙

scav

engi

ng ac

tivity

()

Figure 4 Effects of water solutions on ABTS+∙ scavenging activityin green tea

gallic acid as an internal standard) against the correspondingconcentrations Using linear regression to analyze data inthe concentration range of interest the detection limits werebetween 0078 and 125 120583gmL (SN = 3) for the components

By substituting the peak-area ratios of individual peaksacquired usingHPLC the contents of individual componentsin the green tea were determined The average amounts of 4constituents in teas made using tap water and distilled waterrespectively were (mgg plusmn SD) GC 443 plusmn 094 and 335 plusmn053 EGC 586 plusmn 178 and 2103 plusmn 047 EGCG 143 plusmn 007and 1236 plusmn 003 EC 552 plusmn 021 and 1026 plusmn 018 Of these 4constituents EGC EGCG and EC show higher amounts inthe distilled water sample than its tap water counterpart asshown in Figure 5

36 Ion Concentration The extraction efficiency of green tealeaves depends on the presence of electrolytes Thereforethe ions present in water were further determined by theinductively coupled plasma mass spectrometry (SCIEX Elan5000 Perkin Elmer Uberlingen Germany) Table 1 showsthe cation contents in two waters of this study There isa higher cation concentration in tap water than distilledwater Corresponding to previous reports [36] the higherthe mineralisation the lower the green tea leaves extractionyields of organic matter The reason may be also attributedto that calcium is taken up by leaves from highly mineralisedwater and assumed to be complexed with pectins in cell wallsThe formation of complex will retard extraction of greentea leaves thus explaining the decrease in extraction yield


IS

GC EGC EGCGEC

25 5 75(min)

201751512510

(mAU

) 3025201510

50

(a)

IS

GC EGC EGCGEC

25 5 75(min)

201751512510

(mAU

)

3035

25201510

50

(b)

Figure 5High performance liquid chromatography spectra of green tea preparedwith differentwater solutions (a) Tapwater and (b) distilledwater (IS = gallic acid GC = gallocatechin EGC = (ndash)-epigallocatechin EGCG = (ndash)-epigallocatechin gallate and EC = (ndash)-epicatechin)

Table 1 Cation compositions of tap water and distilled water

Water Cation concentration (ppb)Ca2+ Al3+ Na+ Mg2+ K+

Tap water 21690 1333 10120 6698 1361Distilled water 1152 0465 9035 2159 0941

Besides tea polyphenols which are the leading functionalcomponent may combine with ions such as Ca2+ and Mg2+to be partially retained in tea residue [37]

Except calcium fixation in leaves the water structuremay also affect extraction efficiency Water structures havebeen studied for over 100 years in various research fieldsincluding physics chemistry and physical chemistry [38]Many theoretical and experimental approaches for study-ing the structure of water have been well developed (egstatistical thermodynamics molecular dynamics simulationinfrared spectrum Raman spectrum X-ray diffraction neu-tron diffraction and NMR) [39ndash41] Electrolytes have beenshown to affect the median number of water clusters andthe cluster size [39 42] This observation supports theidea that water organized into smaller clusters can extractmore functional constituents of green tea leaves and henceenhances the antioxidant activity of green tea The rate-determining step in tea leaf infusion was determined to bethe diffusion of solutes through the leaf matrix to the surface[43] Smaller water clusters provide larger surface areas thusincreasing contact opportunities between water moleculesand green tea leaves and allowing the extraction of moresolutes from the leaves In addition the interaction betweenthe sample and the extracting solvent is enhanced due tothe increased number of water clusters resulting in a highlyefficient extraction In this study cations increase the medianwater cluster size [39] and decrease extraction of green tealeaves

4 Conclusions

This study investigated the effects of water conditions on theefficiency of constituent extraction from green tea leavesTheincrease in polyphenol extraction in green tea prepared with

distilled water increases its antioxidant activity Our resultsrevealed that distilled water may be a good choice for extract-ing compounds probably due to its less calcium fixation inleaves and smaller water cluster size thus enhancing theantioxidant activity of green tea made with distilled waterThese results provide an alternative application of distilledwater in the processing of certain foods

Conflict of Interests

The authors declare that they have no competing interestsAll authors have no financial relation with the commercialidentities mentioned in the paper

Acknowledgment

The authors feel deeply indebted to the National ScienceCouncil Taiwan for all the resources gained under Grant noNSC101-2622-E-241-002-CC3

References

[1] M D Brown ldquoGreen tea (Camellia sinensis) extract and itspossible role in the prevention of cancerrdquo Alternative MedicineReview vol 4 no 5 pp 360ndash370 1999

[2] J V Higdon and B Frei ldquoTea catechins and polyphenols healtheffectsmetabolism and antioxidant functionsrdquoCritical Reviewsin Food Science and Nutrition vol 43 pp 89ndash143 2003

[3] A B Sharangi ldquoMedicinal and therapeutic potentialities of tea(Camellia sinensis L)mdasha reviewrdquo Food Research Internationalvol 42 no 5-6 pp 529ndash535 2009

[4] S M Henning Y T Niu N H Lee et al ldquoBioavailability andantioxidant activity of tea flavanols after consumption of greentea black tea or a green tea extract supplementrdquo AmericanJournal of Clinical Nutrition vol 80 no 6 pp 1558ndash1564 2004

[5] L A Beltz D K Bayer A L Moss and I M Simet ldquoMecha-nisms of cancer prevention by green and black tea polyphenolsrdquoAnti-Cancer Agents in Medicinal Chemistry vol 6 no 5 pp389ndash406 2006

[6] N P Seeram S M Henning Y Niu R Lee H S Scheuller andD Heber ldquoCatechin and caffeine content of green tea dietarysupplements and correlation with antioxidant capacityrdquo Journal


of Agricultural and Food Chemistry vol 54 no 5 pp 1599ndash16032006

[7] S-D Lin E-H Liu and J-L Mau ldquoEffect of different brewingmethods on antioxidant properties of steaming green teardquoLWTmdashFood Science and Technology vol 41 no 9 pp 1616ndash16232008

[8] M Bancirova ldquoComparison of the antioxidant capacity and theantimicrobial activity of black and green teardquo Food ResearchInternational vol 43 no 5 pp 1379ndash1382 2010

[9] A S G Costa M A Nunes I M C Almeida et alldquoTeas dietary supplements and fruit juices a comparativestudy regarding antioxidant activity and bioactive compoundsrdquoLWTmdashFood Science and Technology vol 49 pp 324ndash328 2012

[10] H Cao M A Kelly F Kari et al ldquoGreen tea increases anti-inflammatory tristetraprolin and decreases pro-inflammatorytumor necrosis factor mRNA levels in ratsrdquo Journal of Inflam-mation vol 4 article 1 2007

[11] J Khatiwada M Verghese L T Walker L Shackelford C BChawan and R Sunkara ldquoCombination of green tea phyticacid and inositol reduced the incidence of azoxymethane-induced colon tumors in Fisher 344 male ratsrdquo LWTmdashFoodScience and Technology vol 39 no 10 pp 1080ndash1086 2006

[12] J D van der Merwe E Joubert E S Richards et al ldquoAcomparative study on the antimutagenic properties of aqueousextracts of Aspalathus linearis (rooibos) different Cyclopia spp(honeybush) andCamellia sinensis teasrdquoMutationResearch vol611 no 1-2 pp 42ndash53 2006

[13] C S Yang S Sang J D Lambert Z Hou J Ju and G LuldquoPossible mechanisms of the cancer-preventive activities ofgreen teardquo Molecular Nutrition amp Food Research vol 50 pp170ndash175 2006

[14] H-S Jeong S JangM-J Jang et al ldquoEffects of (ndash)-epigallocate-chin-3-gallate on the activity of substantia nigra dopaminergicneuronsrdquo Brain Research vol 1130 no 1 pp 114ndash118 2007

[15] S Mandel O Weinreb T Amit and M B H Youdim ldquoCellsignaling pathways in the neuroprotective actions of the greentea polyphenol (ndash)-epigallocatechin-3-gallate implications forneurodegenerative diseasesrdquo Journal of Neurochemistry vol 88no 6 pp 1555ndash1569 2004

[16] Y Yoshida M Kiso and T Goto ldquoEfficiency of the extractionof catechins from green teardquo Food Chemistry vol 67 no 4 pp429ndash433 1999

[17] S C Lee S Y Kim S M Jeong and J H Park ldquoEffect of far-infrared irradiation on catechins and nitrite scavenging activityof green teardquo Journal of Agricultural and Food Chemistry vol54 pp 399ndash403 2006

[18] Y Huang J Sheng F Yang and Q Hu ldquoEffect of enzymeinactivation by microwave and oven heating on preservationquality of green teardquo Journal of Food Engineering vol 78 no2 pp 687ndash692 2007

[19] V R Sinija H N Mishra and S Bal ldquoProcess technology forproduction of soluble tea powderrdquo Journal of Food Engineeringvol 82 no 3 pp 276ndash283 2007

[20] D Komes D Horzic A Belscak K K Ganic and I VulicldquoGreen tea preparation and its influence on the content ofbioactive compoundsrdquo Food Research International vol 43 pp167ndash176 2010

[21] J Hu Y Chen and D Ni ldquoEffect of superfine grinding onquality and antioxidant property of fine green tea powdersrdquoLWTmdashFood Science and Technology vol 45 pp 8ndash12 2012

[22] J Xi D Shen S Zhao B Lu Y Li and R Zhang ldquoChar-acterization of polyphenols from green tea leaves using ahigh hydrostatic pressure extractionrdquo International Journal ofPharmaceutics vol 382 pp 139ndash143 2009

[23] M von Staszewski AM R Pilosof andR J Jagus ldquoAntioxidantand antimicrobial performance of different Argentinean greentea varieties as affected by whey proteinsrdquo Food Chemistry vol125 no 1 pp 186ndash192 2011

[24] X Jun S Deji L Ye and Z Rui ldquoComparison of in vitroantioxidant activities and bioactive components of green teaextracts by different extraction methodsrdquo International Journalof Pharmaceutics vol 408 pp 97ndash101 2011

[25] B F Zimmermann and M Gleichenhagen ldquoThe effect ofascorbic acid citric acid and low pH on the extraction of greentea how to get most out of itrdquo Food Chemistry vol 124 no 4pp 1543ndash1548 2011

[26] F Li F Wang F Yu et al ldquoIn vitro antioxidant and anticanceractivities of ethanolic extract of selenium-enriched green teardquoFood Chemistry vol 111 no 1 pp 165ndash170 2008

[27] J Xi D Shen Y Li and R Zhang ldquoUltrahigh pressureextraction as a tool to improve the antioxidant activities of greentea extractsrdquoFoodResearch International vol 44 pp 2783ndash27872011

[28] Q V Vuong C E Stathopoulos J B Golding M H Nguyenand P D Roach ldquoOptimum conditions for the water extractionof L -theanine fromgreen teardquo Journal of Separation Science vol34 no 18 pp 2468ndash2474 2011

[29] D R Zhou Y Q Chen and D J Ni ldquoEffect of water quality onthe nutritional components and antioxidant activity of green teaextractsrdquo Food Chemistry vol 113 pp 110ndash114 2009

[30] M S Bae and S C Lee ldquoEffect of deep sea water on theantioxidant activity and catechin content of green teardquo Journalof Medicinal Plants Research vol 4 no 16 pp 1662ndash1667 2010

[31] S-D Lin C-H Liang E-H Liu and J-L Mau ldquoAntioxidantproperties of water extracts from parching green teardquo Journal ofFood Biochemistry vol 34 no 3 pp 477ndash500 2010

[32] T Gutfinger ldquoPolyphenols in olive oilsrdquo Journal of the AmericanOil Chemists Society vol 58 no 11 pp 966ndash968 1981

[33] Y-S Shyu J-T Lin Y-T Chang C-J Chiang and D-J YangldquoEvaluation of antioxidant ability of ethanolic extract from dill(Anethum graveolens L) flowerrdquo Food Chemistry vol 115 no 2pp 515ndash521 2009

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

[35] N Erkan G Ayranci and E Ayranci ldquoAntioxidant activities ofrosemary (Rosmarinus Officinalis L) extract blackseed (Nigellasativa L) essential oil carnosic acid rosmarinic acid andsesamolrdquo Food Chemistry vol 110 no 1 pp 76ndash82 2008

[36] A Mossion M Potin-Gautier S Delerue I Le Hecho and PBehra ldquoEffect of water composition on aluminium calcium andorganic carbon extraction in tea infusionsrdquo Food Chemistry vol106 no 4 pp 1467ndash1475 2008

[37] D Zhou Y Chen and D Ni ldquoEffect of water quality on thenutritional components and antioxidant activity of green teaextractsrdquo Food Chemistry vol 113 pp 110ndash114 2009

[38] F H Stillinger ldquoWater revisitedrdquo Science vol 209 no 4455 pp451ndash457 1980

[39] R Li Z Jiang H Yang and Y J Guan ldquoEffects of ions innatural water on the 17ONMR chemical shift of water and theirrelationship to water clusterrdquo Journal of Molecular Liquids vol126 pp 14ndash18 2006


[40] S J Suresh K Kapoor S Talwar and A Rastogi ldquoInternalstructure of water around cationsrdquo Journal of Molecular Liquidsvol 174 pp 135ndash142 2012

[41] S Shimokawa T Yokono T Mizuno H Tamura T Erata andT Araiso ldquoEffect of far-infrared light irradiation on water asobserved by X-ray diffraction measurementsrdquo Japanese Journalof Applied Physics 2 vol 43 no 4 pp L545ndashL547 2004

[42] G Markovich S Pollack R Giniger and O CheshnovskyldquoPhotoelectron spectroscopy of Cl- Br- and I mdashsolvated inwater clustersrdquo The Journal of Chemical Physics vol 101 no 11pp 9344ndash9353 1994

[43] C Astill M R Birch C Dacombe P G Humphrey and P TMartin ldquoFactors affecting the caffeine and polyphenol contentsof black and green tea infusionsrdquo Journal of Agricultural andFood Chemistry vol 49 no 11 pp 5340ndash5347 2001

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CatalystsJournal of


22 Processing of Green Tea Instead of a brewing process[7 31] green tea solutions were prepared using tap waterand distilled water at 25∘C The former was taken from thesuburban area of the City of Greater Taichung Taiwan Dis-tilled water (lt1 120583Scm) was purified using a Millipore Alpha-Q Luton ultrapure water system such that both the organicand inorganic impurities were removed Two water sampleswere divided into 25mL aliquots in 50mL polypropylenecentrifuge tubes Aqueous extracts were prepared by soakinga 3125mg infusion of ground up and homogenized drygreen tea leaves in 25mL water at 25∘C for 20min Aftercentrifugation (1250 rpm) the green tea supernatant wascollected for further examination five replicates were madefor statistical analysis

23 Total Phenol Content The total phenol content of greentea was measured according to Gutfingerrsquos method [32] Eachgreen tea solution was mixed with 250120583L Folin-Ciocalteureagent for 1min and 50 120583L 2 Na

2CO3for 5min The

absorbance at 655 nm (Sunrise ELISA Plate Reader TecanAustria) denoted the total phenol content and it increasedwith the content of phenols

24 Reducing Power As described in a previous report [33]the reducing power of green tea was determined using amixture of 250120583Lphosphate buffer (200mM pH66) 250 120583Lpotassium ferricyanide (1 by weight) and 500120583L greentea The mixture was then incubated at 50∘C for 20minTrichloroacetic acid (250120583L 10 by weight) was mixed withdistilled water (250120583L) and FeCl

3(750 120583L 01 by weight)

allowed to react for 30min and the absorbance at 700 nmwasmeasured using a spectrophotometer Higher absorbanceindicated greater reducing power

25 DMPD Assay Antioxidant activities of green tea weremeasured according to Schlesierrsquos method [34] In principlecolorless DMPD will form a stable purple free radical cationDMPD+∙ in the presence of a suitable oxidant such as FeCl

3

Purple DMPD+∙ is scavenged and decolorized upon additionof an antioxidant Equal volumes of 24mM DMPD 24mMFeCl3 and 06M acetic acid were mixed together for 5min

to generate DMPD+∙ and the absorbance at 495 nm wasmeasured The antioxidant activity was inferred as the abilityto scavenge free radicals and a decrease in absorbance wasinferred as increasing scavenging power defined as follows

DMPD+ ∙ scavenging activity ()

= [1 minus (

A2A1)] times 100

(1)

where A1 denotes the absorbance of total free DMPD+∙radicals and A2 denotes the absorbance following additionof the antioxidant

26 Trolox Equivalent Antioxidant Capacity Assay The troloxequivalent antioxidant capacity (TEAC) assay was carried outusing the procedure described by Erkanrsquos method [35] Inbrief ABTS+∙ was produced by reacting 7mM ABTS with

245mM potassium persulfate in the dark at 4∘C for 12 h A30 120583L antioxidant aliquot was added to 2mL ABTS+∙ radicalsolution allowed to react for 10min and the absorbancemeasured at 734 nm

As stated previously the antioxidant capacitywas inferredas the ability to scavenge free radicals expressed as follows

ABTS+ ∙ scavenging activity ()

= [1 minus (

A4A3)] times 100

(2)

where A3 represents the absorbance of total free ABTS+∙radicals andA4 represents the absorbance following additionof the antioxidant

27 High Performance Liquid Chromatography The extractof green tea was mixed with an internal standard solution(05mg gallic acid diluted to 25mL with 70 methanol)at a ratio of 1 1 The samples were spiked with variousconcentrations of stock solutions and then analyzed

A stock solution was prepared by dissolving four markersubstances (GC 10mg EGC 50mg EGCG 25mg and EC100mg) in 1mL of 70 methanol the solution was thenstored in a refrigerator Stock solutions were diluted with70 methanol into a series of standard solutions (GC 078104 125 156 208 and 313 120583gmL EGC 250 625 7811041 1563 and 3125 120583gmL EGCG 260 313 391 521 and781 120583gmL EC 208 417 463 5 625 and 833 120583gmL) A20120583L aliquot of each solution was injected and analyzed twotimes using HPLC and the standard curves were plotted aspeak areas versus concentrations Recovery was determinedby comparing the amount of marker substances added to themarker substances found The limits of detection were basedon a signal to noise (SN) ratio of 3 1 as a minimum

HPLC was performed on an Agilent 1220 series systemSatisfactory separation of the markers was obtained using areversed-phase column (Cosmosil 5C18-AR II 5120583m 25 cmtimes46mm ID Nacalai Tesque Kyoto Japan) at 280 nm anelution flow rate of 08mLmin and a linear solvent gradientof A-B (A 10mM KH

2PO4(pH 40) B CH

3CN CH

3OH

and H2O at a ratio of 15 25 1 respectively [vvv]) as

follows 0min 20 B 5min 20 B 15min 30 B 45min60B

28 Statistical Analysis The paired t-test method was usedto evaluate differences between groups A value of 119875 lt 005was considered statistically significant (lowast) and 119875 lt 001 washighly significant (lowastlowast)

3 Results and Discussion

31 Total Phenol Content Figure 1 shows the differences inA655

between the tea solutions Indicating the total amountof phenol the A

655for the control (no green tea extract)

green tea made with distilled water and green tea made withtap water were 006 plusmn 002 081 plusmn 003 and 069 plusmn 003respectively Phenols are regarded as significant constituentsof green tea because of their free-radical scavenging ability


0010203040506070809


Abso

rban

ce

lowastlowast


0

01

02

03

04

05

06


Abso

rban

ce

lowast








60

65

70

75

80

85

90


lowast

DM

PD+∙

scav

engi

ng ac

tivity

()


60

65

70

75

80

85

90


lowast

ABT

S+∙

scav

engi

ng ac

tivity

()






IS

GC EGC EGCGEC

25 5 75(min)

201751512510

(mAU

) 3025201510

50

(a)

IS

GC EGC EGCGEC

25 5 75(min)

201751512510

(mAU

)

3035

25201510

50

(b)







4 Conclusions





Acknowledgment


References
























































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


0010203040506070809


Abso

rban

ce

lowastlowast


0

01

02

03

04

05

06


Abso

rban

ce

lowast








60

65

70

75

80

85

90


lowast

DM

PD+∙

scav

engi

ng ac

tivity

()


60

65

70

75

80

85

90


lowast

ABT

S+∙

scav

engi

ng ac

tivity

()






IS

GC EGC EGCGEC

25 5 75(min)

201751512510

(mAU

) 3025201510

50

(a)

IS

GC EGC EGCGEC

25 5 75(min)

201751512510

(mAU

)

3035

25201510

50

(b)







4 Conclusions





Acknowledgment


References
























































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


IS

GC EGC EGCGEC

25 5 75(min)

201751512510

(mAU

) 3025201510

50

(a)

IS

GC EGC EGCGEC

25 5 75(min)

201751512510

(mAU

)

3035

25201510

50

(b)







4 Conclusions





Acknowledgment


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

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