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LWT - Food Science and Technology 59 (2014) 1152e1158

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LWT - Food Science and Technology

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Value of off-season fresh Camellia sinensis leaves. Antiradical activity,total phenolics content and catechin profiles

Jos�e Baptista a, b, *, Elisabete Lima a, b, Lisete Paiva a, b, Ana R. Castro c

a Investigation Center on Natural Resources (CIRN), University of Azores, 9501-801 Ponta Delgada, S. Miguel, Açores, Portugalb Research Center for Agricultural Technology (CITA-A), University of Azores, 9700-042 Angra do Heroísmo, Terceira, Açores, Portugalc Innovation Technological Institute of the Azores (INOVA), 9504-540 Ponta Delgada, S. Miguel, Açores, Portugal

a r t i c l e i n f o

Article history:Received 27 December 2012Received in revised form3 February 2014Accepted 2 June 2014Available online 20 June 2014

Keywords:Tea plantation wasteRadical scavenging activityTotal phenolicsCatechins separationRP-HPLC

* Corresponding author. Research Center for AgricUniversity of Azores, 9700-042 Angra do HeroísmoTel.: þ351 296 650 182/296 650 172; fax: þ351 296 6

E-mail address: baptista@uac.pt (J. Baptista).

http://dx.doi.org/10.1016/j.lwt.2014.06.0040023-6438/© 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

“Normal-season” (bud plus first two leaves processed as commercial tea between ApreSep), “unused”(remaining leaves collected in Sep and Apr) and “off-season” (all leaves collected between SepeApr)green tea leaves samples from Camellia sinensis were extracted by water infusion followed by solventesolvent partition to recover catechins, that were separated and quantified by HPLC methods. Totalcatechins content ranged between 23.72 and 73.61 mg/g of the dry weight (DW) leaves for the off-seasonsamples and was 97.51, 115.12 and 184.62 mg/g DW for Apr, Sep and normal-season samples, respec-tively. The free radical scavenging activity of the off-season samples ranged between 45 and 80%, 79e90%and 90e92% for the 25, 50 and 100 ppm concentrations, respectively, after 20-min reaction time. Theother samples presented values of 87%, 91% and 94% (Sep), 88%, 92% and 93% (Apr) and 89%, 93% and 95%(normal-season), using the same conditions. Total phenolics content ranged between 43.21 and139.02 mg of gallic acid equivalents (GAE)/g DW for the off-season samples and was 182.23, 216.05 and221.32 mg of GAE/g DW for Apr, Sep and normal-season samples, respectively. Results revealed that theunused and off-season Azorean green tea leaves (catechins-rich waste products) have potential anti-radical activity that can be used for food and cosmetics preservation.

© 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Tea obtained from Camellia sinensis (L.) O. Kuntze is one of themost ancient and most widely consumed non-alcoholic beveragesworldwide. Its popularity is attributed to its sensory properties,relatively low retail price, stimulating effects and potential healthbenefits. The tea plant, originally from Southeast China, graduallyexpanded to India, Sri Lanka, and further into many tropical andsubtropical countries. Since the last decade of 19th century, tea hasalso been produced in one unique place in Europe e S. Miguel,Azores Islands (Baptista, Lima, Paiva, Andrade, & Alves, 2012).

In recent years, there has been a growing interest in identifyingthe pharmacological and physiological effects of green tea, whichinclude antioxidative (Vison, Dabbagh, Serry, & Jang, 1995; Yen &Chen, 1995), antimutagenic (Mukhtar, Wang, Katiyar, & Agarwal,

ultural Technology (CITA-A),, Terceira, Açores, Portugal.50 171.

1992; Okuda, Mori, & Hayatsu, 1984; Shiraki et al., 1994; Wanget al., 1989), anticlastogenic (Gupta, Saha, & Giri, 2002; Kuroda &Hara, 1999), anticarcinogenic (Fujiki et al., 1998; Imai andNakachi, 1997; Jankun, Selman, Swiercz, & Skrzypczak-Jankun,1997; Miyagawa et al., 1997; Stammler & Volm, 1997; Yang, Yang,Landau, Kim, & Liao, 1998) and other clinically relevant activities.About 30% of dry weight (DW) of tea leaves are comprised ofpolyphenols, that exit principally as flavonols (mostly catechins),flavonols usually as o-glycosides and phenolic acids that are themajor soluble components in tea liquor. The green tea polyphenols(GTP) are the compounds responsible for producing the referredhealth benefits, and their content in tea samples can vary with teaplant variety, age of the leaf, season, climate, type of soil, agricul-tural practices and particularly the processing and handling process(Graham, 1992).

It is well known that the use of natural antioxidants for scav-enging free radicals and consequently improve the oxidative sta-bility of food has recently received attention by the scientificcommunity due to the health awareness of more informed popu-lation and the worldwide trend to avoid the use of synthetic foodadditives. However, the radical scavenging activity of unused and

J. Baptista et al. / LWT - Food Science and Technology 59 (2014) 1152e1158 1153

off-season Azorean green tea leaves, particularly from autumn andwinter seasons, has not been investigated and is considered plan-tation waste generally being used as compost. Therefore, testing ofthe radical scavenging activities of these leaves that are available inhuge amount and determining the total phenolics content is ofinterest in order to find new sources of natural antioxidants thatcan be used to minimize oxidative damage to living cells and toprevent oxidative deterioration in products such as food andcosmetics.

The purpose of the present study is to optimize the methodol-ogy for the extraction of the biological active material, particularlycatechins, from unused and off-season Azorean green tea leaves(waste products), to evaluate its radical scavenging activity and thetotal phenolics, to quantify the total catechins and present the re-sults along with HPLC profile and also to compare the results withthose from spring/summer tea leaves used to produce commercialtea.

2. Material and methods

2.1. Chemicals and reagents

Tea catechins, namely (þ)-catechin (C, 98% e C1251), (þ)-epi-catechin (EC, 98% e E4018), (�)-epigallocatechin, (EGC, 98% e

E3768), (�)-epicatechin-3-gallate (ECG, 98% e E3893), (�)-epi-gallocatechin-3-gallate (EGCG, 95% e E4143) and (�)-galloca-techin-3-gallate (GCG, 98% e G6782), alkaloids, namely caffeine(CAF, 99% e C0750), theophylline (TP, 98% e T1633) and theobro-mine (TB, 98% e T4500), gallic acid (G7384), butylated hydrox-ytoluene (BHT), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and Folin-Ciocalteu reagent were obtained from SigmaeAldrich Co (St.Louis, MO, USA). Methanol, acetonitrile, hexane, ethyl acetate andchloroform (HPLC grade), used for liquid chromatography andextraction purposes, were purchased from Riedel-de Ha€en (Seelze-Aktiengesellschaft, Germany). Sodium carbonate and orthophos-phoric acid were obtained from E. Merck (Darmstadt, Germany)and other chemicals were analytical grade. Ultrapure glass distilledwater that was deionizedwithMilliporeMilli-Q purification system(Millipore Corp., Bedford, MA, USA) was used throughout all theexperiments.

2.2. Tea samples

Normal-season green tea leaves sample (used as a control),plucked as one bud and the first and second leaves from Azorean C.sinensis var. sinensis, between ends of Apr to middle of Sep/2010,was kindly donated by the Gorreana Tea Plant (S. Miguel Island,Azores, Portugal) in the form of commercial tea bag (dry powder).Unused (remaining leaves of the shoots, numbers 3e8) and off-season green tea leaves samples were collected directly from theplant during the period of Sep/2010 to Apr/2011 and were kindlydonated by Eng. Hermano Mota, the owner of the Gorreana teaplantation.

The fresh tea leaf enzymes, particularly polyphenol oxidase,were inactivated immediately after collecting at 70 �C during3e4min and prior the drying stepwhichwas performed in a dryingchamber at 70 �C until reached amoisture content of 4e5 g/100 g ofleaves DW (industrially, green tea leaves are rolled into variousshapes in continuous rolling as well as steaming or pan frying).However, the ideal and the most efficient method for the enzymesinactivation will be the use of a drying chamber associated to avacuum device that reduce the drying time and preserve thepolyphenols biological activity. Generally, the various types ofgreen tea are accomplished by changing how the tea is dried.

2.3. Extraction and preparation of polyphenols fractions from greentea leaves samples

Our protocol for the GTP extraction from the green tea leavessamples, shown in Fig. 1, was a slightly modified methodologydescribed by Baptista, Tavares, and Carvalho (1999). The driedsamples were grounded in a mortar to a particle size of 20e30 mm,stored under nitrogen in an airtight plastic container and keptunder refrigerated conditions until further experimental assays.The samples (0.1 g) were steeped at 70 �C for 30 min in 20 mL ofdistilled water, under nitrogen to prevent oxidation by the atmo-spheric oxygen, using a thermal flask. This extraction step wasrepeated three more times and the solution of the combinedaqueous extracts, after cooling for 10 min, was filtered under vac-uum through a 0.45 mm (pore size) cellulose acetate membrane toremove particulate matter, concentrated in a rotaevaporator at40 �C and reconstituted with distilled water in a 25 mL volumetricflask. Alternatively, to storage the samples, the material fromaqueous extracts can be freeze-dried and the green tea powderstored under refrigerated conditions for further assays. A volume of10 mL from the 25 mL (volumetric flask) or from the freeze-driedresidue dissolved in 25 mL (volumetric flask) of distilled waterwas used for the next extraction process by solventesolventpartition. The methylxanthines (caffeine, theobromine andtheophylline) and pigments were extracted with the same volumeof chloroform and the organic phase was rejected. Then, 10 mL ofethyl acetate was added to the same volume of aqueous solution forthe polyphenols extraction. This step was also repeated three moretimes and the extracts were combined. The ethyl acetate layer afterseparation was evaporated in a vacuum rotaevaporator and thelight brown residue (crude catechins) was dissolved in 500 mL ofwater and then subjected to HPLC analysis (10 mL of inj. vol.).

2.4. Analyses of crude catechins extracts by HPLC methods

Since the aromatic structural similarity of the green tea cat-echins made the separation difficult, an HPLC method could beadvantageous for the individual catechins separation, as alsorecommended by Goto, Yoshida, Kiso, and Nagashima (1996) andDalluge, Nelson, Thomas, and Sander (1998). In order to optimizethe methodology for the catechins separation, two different typesof reversed phase columns were tested in this study, Nova Pak C18e 4 mm column (3.9 mm � 15 cm) from Waters (Milford, MA,USA) and Ultropac Spherisorb ODS 2e3 mm column(4.6 mm � 10 cm) from LKB (Bromma, Sweden), under the sameanalytical conditions using the following solvent system: phase(A) 0.12% ammonium acetate (pH 5.5) and phase (B) methanol,using a linear gradient of 0e50% phase (B) over 30 min, at a flowrate of 1 mL/min. The Ultropac Spherisorb ODS 2 shows theability to separate the individual catechins with superior reso-lution, particularly between EC and EGCG, due to its differenthydrophobicity, carbon number (12%) and smaller particle size.The fine-tuning resolution was achieved with Spherisorb columnusing several acetonitrile aqueous-based mobile phases in com-bination with ethyl acetate and orthophosphoric acid. Finally, itwas found that acetonitrile:ethyl acetate:0.1% orthophophoricacid (8.5:2:89.5, v/v/v) e phase (A), during 10 min, followed by alinear gradient between the phase (A) and acetonitrile:water (1:1,v/v) e phase (B), reaching 20% (B) in 10 min, and held at thiscomposition until the end of the run, successfully separated allthe seven catechins (GC, EGC, C, EC, EGCG, GCG and ECG) within30 min. Before the HPLC analysis, the extracts were filteredthrough a polytetrafuoroethylene (PTFE) membrane cartridge.The column was maintained at 35 �C, attached to an AgilentTechnologies model 1200 HPLC system (Avondale, PA, USA),

Dried green tea leaves (100 mg)

Extracted three times with 20 mL of distilled water during 30 min (70 ºC under N2)

Filtration (cellulose acetate filter 0.45 μm) and evaporation in rotaevaporator

Green tea residue was reconstituted with distilled water in a 25 mL volumetric flask

Extracted with equal volume (10 mL) of chloroform (remove methylxantines and pigments)

Organic layer Aqueous layer(methylxantines and pigments)

Extracted three times with equal vol. of ethyl acetate (under N2)

Ethyl acetate layer Aqueous layer(polar components)

Evaporated in rotaevaporator

Crude catechin-residuedissolved in 500 μL of water

HPLC analysis (10 μL – inj. vol.)

Fig. 1. Protocol for the preparation of crude catechins from green tea leaves.

J. Baptista et al. / LWT - Food Science and Technology 59 (2014) 1152e11581154

equipped with a diode array detector (DAD). The chromatogramswere recorded according to the retention time, and the quanti-tative analysis was achieved by the external standard methodusing the ChemStation Chromatography Software from AgilentTechnologies. The average of triplicate measurements was usedto calculate the tea catechins content and the results wereexpressed in mg per g of DW. The sample concentration waslimited to the linearity range in order to avoid peak tailing andretention time (RT) shifting, which may occur when the sampleamount approaches the column sample load capacity. Peakidentity of the individual tea catechins were performed by RTbased on comparison with the authentic standards and/or byspiking the sample with same standards. The individual catechinswere also confirmed by chromatographic pattern (DAD) andsuperimposing the spectrum of the peak with the correspondingstandard spectrum.

The multiple level calibration curves for the C, EGC, EC, EGCGand ECG, components of green tea leaves, at five different con-centrations were constructed from the peak-area versus catechinsconcentrations. The linear regression analysis provided the equa-tions were y is the peak area and x is the concentration of catechinsin mg/g DW. Re-calibration was performed regularly.

The limit of detection and the limit of quantification (LOD andLOQ; inj. vol. ¼ 10 mL) were defined as the amount injected samplewhich gave a signal to noise ratio of 3 and 10, respectively.

2.5. HPLC method validation

The catechins concentrations from the green tea leaves areexpressed as the means of three independent measurements andthe relative standard deviation (RSD) is reported. Linear regressionwas applied to develop equations to predict the epicatechins de-rivatives (ECDs) concentrations. The accuracy of the methodology

determination was evaluated by determining the recovery of twodifferent catechins: EC (medium RT) and ECG (longer RT) in thegreen tea leaves extract of a known level of 8.52 mg and 28.54 mg per10 mL of inj. volume. Three different amounts of standards wereadded to each sample which was subjected to the HPLC chroma-tography analysis. The recovery was calculated based on the dif-ference between the total amount determined in the spiked sampleand the amount observed in the non-spiked samples. All the ana-lyses were carried out in triplicate.

2.6. Determination of free radical scavenging activity (FRSA) inwater green tea leaves extracts

The FRSA potential of green tea leaves extracts and BHT (used asa reference sample) were tested in a methanolic solution of DPPHaccording to the method described by Molyneux (2004) andRainha, Lima, Baptista, and Rodrigues (2011). The antioxidants reactwith DPPH, used as a stable radical, that is a nitrogen-centredradical and converts it to 1,1-diphenyl-2-picryl hydrazine, due to itshydrogen-donating ability at a very rapid rate. A mixture withoutgreen tea leaves extract or BHT was used as the control sample. Theabsorbance was measured at 517 nm after 10 min and 20 min usinga Heyios aUV/VIS spectrophotometer (Unicam Ltd, Cambridge, UK).All determinations were performed in triplicate and averaged. Thedegree of discolouration indicates the scavenging potentials of theantioxidant. The activity of the extracts is attributed to theirhydrogen-donating ability. The antiradical activity (FRSA) of theantioxidant was calculated as a percentage of DPPH decolourationusing the following equation:

%FRSA ¼ ð1� Absorbance of sample=Absorbance of controlÞ� 100

Fig. 2. Reverse-phase HPLC of Azorean (Gorreana) green tea catechins on a 3 mmUltropac-Spherisorb ODS 2 column (100 � 4.6 mm i.d.). Mobile phase: phase (A) e

acetonitrile:ethyl acetate:0.1% orthophosphoric acid (8.5:2:89.5, v/v/v), during 10 min,followed by a linear gradient between (A) and phase (B) e acetonitrile:water (1:1, v/v),reaching 20% (B) in 10 min and held at this composition until the end of the run.Detection by UV (280 nm). Peaks: 1, gallic acid (GA); 2, (�)-gallocatechin (GC); 3,(�)-epigallocatechin (EGC); 4, (þ)-catechin (C); 5, caffeine (CAF); 6, (�)-epicatechin(EC); 7, (�)-epigallocatechin-3-gallate (EGCG); 8, p-coumaroylquinic acid (CA); 9,(�)-gallocatechin-3-gallate (GCG); 10, (�)-epicatechin-3-gallate (ECG).

J. Baptista et al. / LWT - Food Science and Technology 59 (2014) 1152e1158 1155

2.7. Determination of total phenolics content (TPC) in water greentea leaves extracts

TPC in green tea leaves extracts was determined by using Folin-Ciocalteu colorimetric methodology based on the oxidation/reduction reaction as described by Waterhouse (2002) and Rainha,Lima and Baptista (2011). Determinations were carried out intriplicate averaged and calculated from a calibration standard curveof gallic acid. The absorbance was measured at 765 nm usingmethanol as blank in a Heyios a UV/VIS spectrophotometer (Uni-cam Ltd, Cambridge, UK) and was plotted vs concentration. The TPCwas expressed as mg of gallic acid equivalents per gram of DW (mgGAE/g DW) of tea leaves material and was calculated using thefollowing equation:

C ¼ cV=m

where C is the total content of phenolic compounds, mg GAE/g DW;c the concentration of gallic acid obtained from the calibrationcurve, mg/mL; V the volume of extract, mL and m is the weight ofextract, g.

3. Results and discussion

3.1. Extraction of catechins from green tea leaves samples and HPLCseparation of the crude catechins extracts

The fresh green tea leaves are unusually rich in catechins whichremain practically unchanged in commercial green tea with a cor-rect manufacturing process, except a few enzymatically catalysedchanges which occur rapidly following plucking. The Azores Islands(Portugal) tea plantation produces and exports about 60 ton of drygreen tea leaves per year. The commercial Azorean green tea (budplus first two leaves) is processed between ends of Apr to middle ofSep and the amount of polyphenols are generally 1.4-fold higher insummer than in spring, presumably because of the higher growthrate andmetabolic activities of the young leaves during that season.The climate in the Azores Islands is mild and generally the leavesfell from the plant in the coldest windy months (Dec, Jan and Feb).In this Region there is no utilization, neither for the off-seasongreen tea leaves, neither for the unused leaves (remaining leavesfrom the shoots, numbers 3e8), and this study represent the firstcontribution to the tea plantation waste valorization as source offunctional ingredients.

According to Baptista, Tavares, and Carvalho (1998), the effect oftemperature on the extraction yield of the catechins gave rise todifferent types of behaviour: the concentration of EGCG increasesas the temperature increases from 50 �C, reaching the maximum at70 �C, and decreases after that temperature (dropping by 60%, w/w,from the maximumvalue at 100 �C); the concentrations of ECG andEC also increase as the temperature increases from 50 �C, reachingthe maximum in the range of 70e90 �C and decrease (dropping by50e60%, w/w from the maximum value at 100 �C) after that tem-perature; the concentration of EGC is maximum at 50 �C anddecrease rapidly from 50 �C to 70 �C and then slowly as the tem-perature increases, and the effect of temperature, on that range, forthe extraction of C is practically negligible. The influence ofextraction time on the extraction yield shows that the yield in-crease as the temperature increases reaching the maximum after30 min. The concentration of EGCG increases rapidly at 30e40 minand decreases slowly afterwards and the concentrations of ECG,EGC and EC increase slowly with increasing extraction time,reaching also the maximum at 30e40 min, and then decreaseslightly after that time, the effect of extraction time on the C con-centration is practically negligible. Based in these results we

adopted in our study the extraction temperature of 70 �C and30 min of extraction time.

The aqueous extracts of green tea leaves samples were sub-mitted to solventesolvent partition in order to obtain the crudecatechins extracts. The liquideliquid partitioning, methodology toseparate compounds based on their solubilities in two differentimmiscible liquids, was used as a pre-purification procedure(cleaning step) in order to improve the separation of the green teacatechins and consequently the precision and reproducibility of thequantitative determinations.

The developed HPLC method successfully separated all sevencatechins (GC, EGC, C, EC, EGCG, GCG and ECG) with a good reso-lution as compared to some reported results in literature. Our re-sults show that acid is always necessary in the mobile phase,whether a column has been equilibrated with acid-containingbuffers or not. This was different from the result given by Dallugeet al. (1998). Acetic acid has been found to have the same effecton the separation, but it is not as effective as orthophosphoric acid.Different quantities of orthophosphoric acid in the mobile phasewere tested in the present study. Experiments showed that theeffective amount of orthophosphoric acid in the mobile phase mustbe 0.04e0.1%. During the HPLC method development it became

J. Baptista et al. / LWT - Food Science and Technology 59 (2014) 1152e11581156

evident that temperature of the column oven affected the catechinsresolution. As the column temperature increased from room tem-perature to 50 �C, the retention times slightly decreased. Thetemperature of 35 �C was adopted during this study as completeseparation of all catechins was achieved at this temperature. Fig. 2illustrates the HPLC separation of Azorean green tea catechin resi-dues. The chromatograms were monitored at 280 nm and thecalibration curves of the catechins at this wavelength were linear inthe ranges referred in Table 1. However, ultraviolet spectroscopy isvery dependent on the sample environment (solvent, pH, temper-ature, etc.). Because of this, for data comparison, the sol-vochromatic shifts of catechins for different mobile phases must beconsidered. The sensitivity of the detection could be enhancedthree-fold by using a wavelength of 210 nm, however, the limit oflinearity was four fold lower.

The Table 1 shows the linear range (mmol/L) of multiple levelcalibration curves for the C, EGC, EC, EGCG and ECG, components ofgreen tea leaves, at five different concentrations. The linearregression analysis provided the equations and the correspondingcorrelation coefficient (R2) were y is the peak area and x is theconcentration of catechins in mg/g DW. The Table 1 also shows theLOD that ranged from 0.70 to 1.10 mmol/L for EGCG (componentwith longer RT) and EGC (component with medium RT), respec-tively, and the LOQ that ranged from 2.33 to 3.67 mmol/L for EGCGand EGC, respectively, in the experimental condition used.

The Table 2 shows the content of ECDs in the studied tea plantleaves samples. The results revealed a superior value of total ECDsin unused leaves collected in the end of summer (Sepe 115.12mg/gDW) and beginning of spring (Apr e 97.51 mg/g DW) and lowercontent of ECDs during autumn and winter seasons that rangedfrom 23.72 to 73.6 mg/g DW. As expected, the highest value of totalECDs (184.62 mg/g DW) was obtained for normal-season sample(tea bag). Considering the quantitative analysis of the individualcatechins (Table 2), the results revealed also a decreasing contentduring autumn and winter seasons reaching the lowest valuesduring the cold months (Dec and Jan). The ECDs (in mg/g of DW)decreased 83.47% (24.98e4.13), 53.57% (1.68e0.78), 85.63%(10.65e1.53), 75.21% (42.08e10.43) and 80.83% (35.73e6.85) forEGC, C, EC, EGCG and ECG, respectively, from the Sep (end of thenormal-season) to Dec (more cold month of the off-season period),presumably because of the lower growth rate and the lowermetabolic activities of the leaves during off-season period.Furthermore, in this ECDs mixture, EC was the most affected and C(the minor constituent) was the least affected by the lower tem-perature during the off-season period.

3.2. HPLC method validation

The repeatability of HPLC analysis was evaluated in intraday of aknown level of EC and ECG, tea components that present mediumand higher coefficient of diffusion in the HPLC column. The resultspresented in Table 3 show that the intraday repeatability (%RSD)were 1.78% and 2.91% for EC and ECG, respectively, whereas theinterday precision (data acquired over a period of 5 days) was for

Table 1The linear range, regression equations, correlation coefficient (R2) and the limits of dete

Analytes Linear range (mmol/L) Linear equation

C 11.7e187.5 y ¼ 436935x þ 13554EGC 0.125e2.0 y ¼ 115784x � 85881EC 23.4e375 y ¼ 555366x � 23354EGCG 0.312e5.0 y ¼ 963349x þ 929779ECG 0.093e3.0 y ¼ 1E þ 06x þ 322547

a Chromatographic conditions and identity of compounds as in Fig. 2. Inj. vol. ¼ 10 mL

both better than 4.97% (data not shown in Table 3) indicating a highdegree of the repeatability for the determination of green tea cat-echins content under the analytical conditions used. The accuracyof the EC and ECG determinationwas evaluated by determining therecovery of EC and ECG in a sample of a known level of thesecomponents as referred in methods. Results with the RSD areshown in Table 4. The RSD was better than 3.15% for EC and themean recovery ranged from 96.0% to 97.7% and better than 2.70%for ECG and the mean recovery ranged from 99.67% to 100% indi-cating a high degree of the accuracy for the determination of cat-echins under the analytical conditions used.

3.3. FRSA activity of water green tea leaves extracts

The FRSA measure the hydrogen atom or electron donor ca-pacity of an extract to the stable radical DPPH formed in alcoholicsolution. The Table 5 shows the results of all samples that weremeasured in triplicate using three different concentrations (25, 50and 100 ppm) and during the time period of 10min and 20min. Thecommercial tea bag, Sep and Apr samples were the most activeshowing 89%, 87% and 88%, 93%, 91% and 92%, and 95%, 94% and 93%for the three concentrations of 25, 50 and 100 ppm, respectively,after 20 min reaction time. The autumn and winter samples pre-sented lower FRSA activity as compared with spring and summersamples ranging between 45 and 80%, 79 and 90% and 90 and 92%for the same concentrations and the same reaction time period.Above 90% can be considered as a full absorption inhibition ofDPPH, because after completing the reaction the final solution al-ways possesses some yellowish colour and therefore its absorptioninhibition compared to colourless methanol solution not reach100% (Miliauskas, Venskutonis, & van Beek, 2004). In all samplesthe activity increased accordingly to the concentration and to thereaction time. The BHT, used in the same conditions, as a positivecontrol, presents a FRSA values of 82%, 91% and 93% for 25, 50 and100 ppm concentrations, respectively, during the time period of20 min.

3.4. Determination of TPC in water green tea leaves extracts

The TPC of the green tea leaves extracts was calculated fromregression equation of calibration curve (y ¼ 1.0091x � 0.0141;R2 ¼ 0.998) and was expressed as mg of GAE per g of DW. The TPCvalues ranged from 43.21 to 221.32 mg GAE/g DW (Table 5). Theresults shows that the greatest amounts of TPC (221.32 mg GAE/gDW)were found for green tea leaves from commercial tea bag, usedas a control, followed by Sep and Apr samples with 216.05 and182.23 mg GAE/g DW, respectively. The lowest amounts of TPCwere found from the autumn and winter samples with valuesranging from 43.21 to 139.02 mg GAE/g DW, being Dec the monththat presents the lowest content (about 31% of the Apr value). Theresults also show that there is a direct correlation between TPC andantioxidant activity that also shows a lower value of FRSA in thewinter months particularly in Dec.

ction (LOD) and quantification (LOD) of green tea leaves catechinsa.

R2 LOD (mmol/L) LOQ (mmol/L)

0.9983 1.04 3.060.9951 1.10 3.670.9999 1.10 3.620.9926 0.70 2.330.9990 0.72 2.40

.

Table 2HPLC quantitative determination of ECDs content (in mg/g of DW) in Azorean green tea leaves samples (Camellia sinensis) from different batches collected during 2010/2011.a

ECDs Green tea samples from normal- and off-season leaves and from unused leaves of Sep and Apr months

Name RT (min) Tea bagb Sep/10c Oct/10d Nov/10(fst wk)d

Nov/10(last wk)d

Dec/10d Jan/11d Feb/11d Apr/11(fst wk)d

Apr/11(last wk)c

EGC 9.24 31.34 ± 1.10 24.98 ± 1.00 8.55 ± 0.32 8.75 ± 0.35 13.53 ± 0.54 4.13 ± 0.17 7.80 ± 0.31 13.53 ± 0.54 15.95 ± 0.80 19.63 ± 0.79C 10.72 2.51 ± 0.09 1.68 ± 0.07 0.73 ± 0.43 0.68 ± 0.03 0.48 ± 0.02 0.78 ± 0.03 0.58 ± 0.02 2.48 ± 0.02 2.40 ± 0.10 1.70 ± 0.07EC 15.18 14.56 ± 0.51 10.65 ± 0.43 3.80 ± 0.14 3.75 ± 0.15 1.92 ± 0.08 1.53 ± 0.06 1.78 ± 0.07 3.98 ± 0.08 9.88 ± 0.40 8.35 ± 0.33EGCG 17.55 97.69 ± 3.45 42.08 ± 1.68 14.73 ± 0.58 14.23 ± 0.56 16.08 ± 0.64 10.43 ± 0.42 13.45 ± 0.54 18.08 ± 0.64 23.70 ± 0.91 36.43 ± 1.43ECG 28.09 38.52 ± 1.35 35.73 ± 1.43 13.85 ± 0.63 13.60 ± 0.54 6.98 ± 0.28 6.85 ± 0.27 6.73 ± 0.23 13.98 ± 0.28 21.68 ± 0.79 31.40 ± 1.26Total ECDs e 184.62 ± 6.52e 115.12 ± 4.60 41.66 ± 1.65 41.01 ± 1.84 39.20 ± 1.56 23.72 ± 0.95 30.34 ± 1.21 42.05 ± 1.56 73.61 ± 2.94 97.51 ± 3.90

a Chromatographic conditions and identity of compounds as in Fig. 2. Values are mean ± SD (n ¼ 3). DW, dry weight. RT, retention time. ECDs, epicatechin derivatives. Fst,first. Wk, week.

b Commercial tea bag sample from normal-season (bud plus two first tea leaves, processed between ends of Apr to middle of Sep).c Unused leaves samples (remaining leaves of the shoots, numbers 3e8) collected directly from the tea plant.d Off-season leaves samples collected directly from the tea plant (all leaves).e Similar to the published ECDs value of 186.8 mg/g DW (Baptista et al., 1999).

Table 3Intra-day precision data for retention time (RT), standard deviation (SD), relativestandard deviation (RSD) and coefficient of variation (CV) of EC and ECG.a

Analytes Intra-day precision (n ¼ 10)

RT mean (min) SD (min) RSD (%)b CV (%)c

EC 15.183 0.118 1.78 0.78ECG 28.098 0.214 2.91 0.76

a Chromatographic conditions as in Fig. 2. EC, (�)-epicatechin. ECG, (�)-epi-catechin-3-gallate.

b RSD (%) ¼ (Mean deviation)2/SD � 100.c CV (%) ¼ (SD/Mean) � 100.

Table 4Recovery of EC (medium RT) and ECG (longer RT) from green tea samples (n ¼ 3).a

Analytes Concentration (inj. vol 10 mL) Recovery (%) RSD (%)

Catechins (mg) Spiked (mg) Measured (mg)

EC 10.65 ± 1.71 10 20.18 97.7 2.6220 29.41 96.0 2.8630 39.43 97.0 3.15

ECG 35.73 ± 5.61 20 35.69 99.89 2.1030 65.51 99.67 2.7050 85.71 100.00 2.51

a Chromatographic conditions as in Fig. 2. EC, (�)-epicatechin. ECG, (�)-epi-catechin-3-gallate.

Table 5FRSA (reaction time 10 min and 20 min) at different concentrations (25, 50 and 100 ppmsinensis) from different batches (normal-season, off-season and unused leaves).a

Tea samples and BHT Free radical scavenging activity (FRSA) (%)

25 ppm 50 ppm

10 min 20 min 10 min

Tea bagb 53 ± 0.4 89 ± 0.9 92 ± 0.5Sep/10c 52 ± 0.2 87 ± 0.7 90 ± 0.6Oct/10d 51 ± 0.4 51 ± 0.5 89 ± 0.6Nov/10 (fst wk)d 48 ± 0.2 48 ± 0.3 87 ± 0.5Nov/10 (last wk)d 47 ± 0.2 48 ± 0.3 80 ± 0.4Dec/10d 45 ± 0.1 46 ± 0.2 79 ± 0.4Jan/11d 44 ± 0.5 45 ± 0.3 79 ± 0.5Feb/11d 45 ± 0.3 45 ± 0.3 79 ± 0.4Apr/11 (fst wk)d 50 ± 0.3 80 ± 0.5 85 ± 0.3Apr/11 (last wk)c 52 ± 0.1 88 ± 0.3 91 ± 0.3BHT e 82 ± 0.5 e

b-d Legend as referred in Table 2.a Values are mean ± SD (n ¼ 3). BHT (butylated hydroxyltoluene) was used as positiv

J. Baptista et al. / LWT - Food Science and Technology 59 (2014) 1152e1158 1157

4. Conclusion

Tea from C. sinensis species of the Theaceas family is one of themost popular consumed beverages, at least in some parts of theworld. The composition of the Azorean green tea catechins reflectthe influence of geographic origin of the tea leaf, climate, season,variety, type of soil (volcanic) and the horticultural and processingtechniques (Baptista & Tavares, 1998; Baptista et al., 1998). TheHPLC methodology allowed the determination and the comparisonof the catechins content on green tea leaves from normal-seasonand waste products (unused and off-season leaves) with the helpof calibration curves prepared using authentic standards at knownconcentrations. The total ECDs content was found to be lower forthe off-season period (particularly during the coldest months) withcorresponding lower values of TPC, as compared to the unused andnormal-season samples. Results also revealed that the extractsfrom unused and off-season samples possess potential antiradicalactivity (evaluated using DPPH) that could be used for preservationpurposes in food and beverages formulations, scavenging freeradicals, or as a beneficial ingredient in the cosmetic products.

According to the information provided by the Gorreana teaplantation (with an annual production of 60 ton of dry tea leavesthat are exported to several countries), an amount of leaves on theorder of tens of tonnes eventually fall on the ground during the off-season period if they are not collected. With a weighted average oftotal ECDs of ca 60 mg/g of DW throughout the off-season period

) and total phenolics content (TPC) of Azorean green tea leaves samples (Camellia

TPC (mg GAE/g DW)

100 ppm

20 min 10 min 20 min

93 ± 0.7 95 ± 0.3 95 ± 0.7 221.32 ± 6.7991 ± 0.5 94 ± 0.8 94 ± 0.8 216.05 ± 6.7890 ± 0.6 92 ± 0.7 92 ± 0.4 77.95 ± 2.4587 ± 0.9 90 ± 0.6 91 ± 0.1 77.03 ± 2.4281 ± 0.5 89 ± 0.7 90 ± 0.6 73.25 ± 2.3179 ± 0.4 90 ± 0.2 90 ± 05 43.21 ± 1.3679 ± 0.5 90 ± 0.1 90 ± 0.3 56.36 ± 1.7280 ± 0.5 90 ± 0.3 90 ± 0.4 90.18 ± 2.8385 ± 0.3 91 ± 0.2 91 ± 0.5 139.02 ± 4.3692 ± 0.5 93 ± 0.4 93 ± 0.3 182.23 ± 5.7291 ± 0.2 e 93 ± 0.5 e

e control. GAE, gallic acid equivalents. DW, dry weight. - not applied.

J. Baptista et al. / LWT - Food Science and Technology 59 (2014) 1152e11581158

plus the unused leaves from Sep and Apr, ca 15 Kg of crude cate-chins can be extracted from each ton of fresh green tea leaves(250 kg of DW for each ton of fresh tea leaves) assuming 75 g ofmoisture/100 g of dry green tea leaves (Baptista& Tavares, 1998). Inother words, the polyphenols (catechins) from the unused and off-season leaves may have a surplus value that has the potential forthe large-scale extraction of natural antioxidants that can be aprofitable investment using methodologies without the use oforganic solvents (e.g. Chang, Chiu, Chen, & Chang, 2000).

Acknowledgements

This work was supported by funds of CIRN and Department ofTechnological Sciences and Development e University of Azores.

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