Stability of Tea Catechins and Antioxidant Properties of Green Tea Extracts as Affected by Boiling-treatment

De-Song Tang，Hui-Ling Liang

1.Tea Research Institute，Zhejiang University，Hangzhou 310058，China.

2.Hangzhou Tea Research Institute，All-China Federation of Supply and Marketing.

1.Introduction

Tea is a pleasant，popular，socially accepted，economical and safe drink that is enjoyed every day by hundreds of millions of people，and it has been suggested to be beneficial in the prevention of many human chronic diseases，such as cancer and cardiovascular diseases［1-3］.The benefits of tea are partly ascribed to its antioxidant components，such as tea polyphenols［4］.Tea polyphenols traditionally have been known to be mainly composed of four catechins including(-)-epicatechin(EC)，(-)-epigallocatechin-3-gallate(EGCG)，epicatechin-3-gallate(ECG)，and epigallocatechin(EGC)［5］.

In the production of green tea，the enzymic activity in freshly picked tea leaves by firing or steaming followed by thermal drying(microwave-，oven-，and sun-drying)［6］.And almost all the subjects reported preparing tea by brewing tea in hot water［7-8］.It is also well known that most conventional methods for preparing tea polypenols and catechins was carried out by extraction of tea leaves in hot water.Tea catechins undergo many chemical changes under thermal conditions，such as epimerization from epi-catechins into noepi-catechins during the manufacture of green tea［9］and when tea catechins were heat treated［10-11］.Besides of erpimerization，degradation of catechins also observed in an aqueous system heated by a microwave reactor［12］and when Longjing Green tea catechins were brewed at 98℃ for 7 h［13］.

It has been thought that the chemical changes during heat processing might cause changes in their bioactivity.Chengelis et al［14］investigated the potential toxicity of heat-treated tea catechins;Xu et al［15］isolated and purified each epi-catechin and epimer by various column chromatographic and semi-preparative HPLC techniques and examined the antioxidant activity and bioavailability of epimer of tea catechins.But until now the changes of catechins with the thermal treat duration and therefore the changes of bioactivities were few reported.The objectives of this work were to study the stabilities and antioxidant properties of green tea extracts during the prolonged boilingtreatment.Green tea extracts were prepared from hot brewed tea and then subjected to boiling-treatment for 1 to 5 h;The antioxidant properties of the boiling-treated samples were assayed in terms of antioxidant capacity by ferric reducing antioxidant power(FRAP)assay，scavenging abilities on 1，1-diphenyl-2-picrylhydrazyl(DPPH)，hydroxyl radicals(OH·)，and superoxide radical(O2·-).The stability of tea catechins and the antioxidant properties of green tea extracts under boiling-treatment were discussed.

2.Materials and methods

2.1.Catechins standards

(-)-EC，(-)-EGC，(-)-EGCG，(-)-ECG，(-)-gallocatechin((-)-GC)and(-)-catechin gallate((-)-CG)，(-)-Catechin((-)-C)，(-)-Gallocatechin gallate((-)-GCG)were purchased from Sigma Chemical Co.All other reagents were standard items from reputable commercial sources.

2.2.Preparation of green tea extracts

Green tea(Haiyong brand)was purchased from a local market.Tea extracts were obtained by solid-liquid extraction using deionized water at 100℃ tea leaf-water ratio was 1∶20(g/mL).The extraction time was 5 min.The extract solution was rapidly filtered through filter paper.The samples were immediately cooled down using cold running water.The extraction solution was fractionalized into 6 portions.One portion was used as raw control and the other five portions were subjected to boiling-treatment from 1 to 5 h，respectively.During the boiling-treatment，distilled water was added to avoid drying.After the treatment，the extract solutions were lyophilized using a freezedryer(3K30，Sigma Centrifuge，Germany).The effect of prolonged boiling-treatment on the stability of tea catechins and antioxidant activity of green tea extract were investigated.

2.3.Total phenols contents determination

Total phenolic compounds was determined by the Folin-Ciocalteu method［16］:2.5mL of Folin-Ciocalteu reagent，previously diluted with distilled water(1∶10，v/v)，and 2mL of 75 g/L aqueous solution of sodium carbonate were added to 0.5mL of an aqueous solution of the extract.The mixture was kept for 5 min at 50℃ and，after cooling，the absorbance at 760 nm was measured by a UV-Vis spectrophotometer(Cary50，Varian，USA).The total phenolic compounds was calculated as gallic acid equivalent from the calibration curve of gallic acid(GA)standard solutions(2-40 μg/mL).

2.4.Green tea catechins determination

The lyophilized samples were dissolved in deionized water and filtered through a 0.22 μm filter for HPLC analysis according to the reported method［17］.The Shimadzu HPLC system(Shimadzu SCL-10A，Shimadzu Cooperation，Tokyo，Japan)with auto sampler was employed in the present study.A Diamonsil TM C18 column(250 mm ×4.6 mm，i.d.)was used at temperature of 40℃ and the injection volume was 10 μl.A binary solvent A:acetonitrile/acetic acid/water(6∶1∶193，v/v/v)and solvent B:acetonitrile/acetic acid/water(60∶1∶139，v/v/v)was used as mobile phase;Gradient 100%of solvent A to100%of solvent B by linear gradient during first 45 min and then 100%of solvent B until to 60 min;Measurements were performed at a flow rate of 1.0ml/min，Detection was done by a Shimadzu SPD ultraviolet detector at 280 nm.

2.5.Antioxidant capacity

The antioxidant capacity of the extracts was determined by the ferric reducing antioxidant power(FRAP)assay［16］，in which the antioxidants present in the sample reduce the Fe(Ⅲ)/tripyridyltriazine(TPTZ)complex to the blue ferrous form with an increase in absorbance at 593 nm.The FRAP assay was carried out as following:0.1 mL of an aqueous solution of the extracts was transferred to a test tube and 3.0mL of freshly prepared FRAP reagent(25 mL of 300 mmol/L acetate buffer，pH 3.6;2.5 mL of 10mmol TPTZ in 40 mmol/L HCl;2.5 mL of 20 mmol/L FeCl3·6H2O)was added.The tubes were vortexed and left at 37℃ for exactly 40 min.The absorbance of the mixture was measured at 593 nm.The relative activities of samples were calculated from the calibration curve of L-ascorbic acid(2-50μg/mL)and the results were expressed as μg ascorbic acid equivalent(AAE)/mg of extract(on a dry basis).

2.6.Free radicals scavenging ability

2.6.1.Assay of DPPH(1，1-diphenyl-2-picrylhydrazyl)radical scavenging activity

The scavenging activity of pigment extract on 1，1-diphenyl-2-picrylhydrazyl(DPPH)radicals(Sigma，analysis grade)was measured according to the method of Wang，Li，Zeng and Liu［18］.DPPH solutions(2 mL)in ethanol(2×10-4mol/l)and 2 mL of tested samples with different concentrations were mixed in the tubes.The mixture was incubated for 60 min in the dark at 25℃，and the decrease in absorbance at 517 nm was measured against ethanol using a UV/Vis spectrophotometer.Methanol was used as the blank.The scavenging activity of pigment on DPPH was calculated according to the following equation:where Sa is the scavenging activity of tested sample(%)，Aiis the absorbance of 2 mL DPPH solution and 2 mL sample solution，Ajis the absorbance of 2 mL sample solution and 2 mL ethanol，A0is the absorbance of 2 mL DPPH solution and 2 mL sample solvent.

2.6.2.Assay of Superoxide radical O-·2scavenging activity

Superoxide radical scavenging activity was measured by the pyrogallol autoxidation［18］.Tris-HCl buffer(5.6 ml of 50 mM)(pH 8.2)and 0.2 ml tested samples were mixed in tubes with lids and then the mixture was incubated for 10 min in the water bath at 25℃.Meanwhile，0.2 ml of 0.95 mM pyrogallol preheated at 25℃ was added immediately.The absorbance of sample and control were determined by UV/Vis spectrophotometer(Varian，USA)at 325 nm every 30 s.Scavenging activity was calculated using the following equation:

where Sa is the inhibition rate of tested sample(%)，Asis the absorbance of the tested sample，Abis the absorbance of the control.

2.6.3.Assay of hydroxyl radical-scavenging activity

The scavenging activity for hydroxyl radicals was measured，with the Fenton reaction［19］.Reaction mixture contained 0.5 mL of 7.5 mM FeSO4，0.5 mL of 7.5 mM 1，10-phenanthroline，2.5 mL of 0.2 M phosphate buffer(pH 7.8)，0.5 mL of 0.1%H2O2and 0.5 mL of sample solution.The reaction was started by adding H2O2.After incubation at room temperature for 45 min，the absorbance of the mixture at 536 nm was measured.Hydroxyl scavenging activity was calculated using the following equation:

where，Asis the absorbance in the presence of sample and H2O2;Acis the absorbance in the presence of H2O2without sample;Abis the absorbance without sample and H2O2.

2.7.Statistical analysis

Each experiment was carried out in triplicate.Data analyses were performed on the Statistical Analysis System(SAS，version 8.0，SAS Institute Inc.，USA).T-test in One-way ANOVA was used to determine the significant difference of the means at P ＜5%level.

3.Results and discussion

3.1.Variation of catechins during the treatment

The changes of the catechins under the boiling-treatment were shown in Fig.1.It was evident that the epi-form catechins，(-)-EGCG，(-)-EGC，(-)-ECG and(-)-EC decreased and their corresponding epimers，(-)-GCG，(-)-GC，(-)-CG and(-)-C continuously increased during the boiling-treatment in the range of experiment.The results indicated that the predominant change of catechins appeared to be epimerized from the epistructure to the nonepistructure.Table1 suggested that the reduction of total catechins were not significant(P ＞0.05)when the green tea extract was boiling-treated for 1h，and it became significant(P ＜0.05)when boilingtreated for more than 2 h comparing with the untreated sample.There were significant(P ＜ 0.05)changes observed for the total of(-)-EC and(-)-C and the total of(-)-EGC and(-)-GC when the samples were treated for 1h while the reduction of other total of epi-and nonepi-form catechins remained at the same level with the untreated samples，indicating that(-)-EC and(-)-C，(-)-EGC and(-)-GC were less stable than other catechins under thermal condition.The reduction of total catechins content indicated that not only the epimerization but also the degradation of catechins occurred during the boiling-treatment.

The results were consistent with the reported that epigallocatechin gallate(EGCG)and epicatechin gallate(ECG)were partially epimerized into gallocatechin gallate(GCG)and catechin gallate(CG)respectively when tea catechins extract was heated in water or dry tea was extracted in water at 100℃［11］，the epimerization of EGCG to(-)gallocatechin gallate(GCG)was observed when Longjing green tea catechins and pure EGCG were autoclaved at 120℃ for 20 min［13］， and the tea epi-catechinsepimerized to nonepi-catechinsduring pasteurization［20］.Epimerisation of the catechins is thought to be one of the most important reactions in the manufacture of green tea［12］.This is thought to be due to the difference in stereochemistry:trans-forms，in general，being more stable than cis-forms.However，these results do not support the reported that was inferred when temperature increased to 98℃ and above，the epimerization from(-)-GCG to(-)-EGCG became prominent［21］and there was an increase of catechins in processed green tea beverage resulted from the"isomerization"of epicatechins［22］.

It was reported that the epimerisation takes place more easily in tap water than in purified water at temperatures above 80℃［9］.The epimerization rates of authentic tea catechins in distilled water are much lower than those in tea infusion or in pH 6.0 buffer solution.Suzuki et al［23］found that the metal ions in tea infusion might affect the rate of epimerization by the addition of ethylene diamine tetra acetic acid，disodium salt(Na2EDTA)to decreased the epimerization of catechins in the pH 6.0 buffer solution.Chen et al［24］reported that the addition of ascorbic acid significantly increased the stability of all four derivatives of catechins，particularly EGC and EGCG.Liang et al［11］reported that the epimerization of the catechins could be inhibited if the the dry tea was extracted in 50%(v/v)ethanol or in water at 80℃ or below.This provided an idea for preparation of tea catechins with hight content of epi-catehins.

3.2.Total phenolic compounds(TPC)

Total phenolic compounds of boiling-treated green tea extracts were of a similar magnitude(P ＞0.05)under the boiling-treatment within 4 h(Table1).The TPC of sample boiling-treated for 5 h reduce significantly(P＜0.05)comparing with the untreated sample，but it was not significantly(P ＞0.05)changed when comparing with the samples treated for 1 to 4 h.The results indicated that effect of boiling-treatment on TPC of green tea extract was limited.

Many reports indicated that there was a significant reduction in total phenolic contents under the conditions of heat treatment［6，25-26］and the degree of polyphenols degradation depends very much on the processing time and the size of the vegetables［26］.Boiling could significantly reduced the total phenols contents，while steaming resulted in a greater retention of TPC［27-28］and microwaving，frying or further warm holding did not affect the levels of polyphenols although their levels were partly degraded during boiling［29］.TPC of the combined cooked tissues of peppers and cooking water were reduced after boiling was extended to 30 min，the decline was not appreciable in comparison with boiling for 5 min［29］.It suggested that the polyphenols leached into liquor during the boilingtreatment.

There are many reports related to the increasing of TPC after thermal treatment.The effects of post harvest treatment and heat stress(100℃)on wheat grain flour indicated an increase in phenolics［30］.The maximum TPC of whole grape seed extract(WGSE)were achieved when the grape seeds were heat-treated at 150℃ for 40 min，according to the GC-MS analysis，several low-molecular-weight phenolic compounds such as azelaic acid，3，4-dihydroxy benzoic acid，and o-cinnamic acid were newly formed［31］.The TLC analysis showed that during thermal processes of sauerkraut juice，some substances with reactivity towards Folin-Ciocalteu reagent were released［32］.Thermal processing at 115℃for 25 min significantly increased phytochemical content such as ferulic acid by 550%and total phenolics by 54%［33］.Most of the phenolics were bound or attached to cell wall structures［34］，the increase of TPC under thermal processing could be due to the release of bound phenolic acids from the breakdown of cellular constituents and cell walls.

It was also reported［35］that condensed tannins，such as proanthocyanidins，were present in low concentration in fresh must，showed an increasing trend with the increase of cooking due to polymerization of single polyphenols upon oxidation and further condensation reactions.In this experiment，the total catechins content reduced but only moderate reduction for TPC was observed.It is perhaps some substances with reactivity towards Folin-Ciocalteu reagent formed under the boiling-treatment，such as polymerization of single green tea catechins.

3.3.Antioxidant capacity(AC)

It was showed in Table2 that no significant(P ＞0.05)reduction of the AC was observed when the samples were boiling-treated for 1h comparing with the untreated sample.Only moderate decline occurred when the samples were boiling-treated for 1 to 4h.It indicated that AC of green tea extract was moderately adversely affected under the boiling-treatment within the range of experiment.

It was previous thought that many food antioxidants can be significantly lost as a consequence of sterilisation，pasteurisation，dehydration，as well as during prolonged storage.But many reports indicate that processing and storage are not always responsible for depletion in the antioxidant properties of foods.The antioxidant activity of food has been linked to its phenolic compounds.Odriozola-Serrano，Soliva-Fortuny and Martín-Belloso［36］reported that no significant differences were observed in phenolic content and antioxidant capacity between treated and untreated tomato juices samples just after processing.It was reported that cooking of peppers by microwave heating and stir-frying was better to ensure a higher retention of the bioactive components in peppers，this was accompanied by the TP contents which did not affect by microwave heating and stir-frying while their levels were partly degraded during boiling［29］.Xu et al［28］reported that pressure steaming resulted in a greater retention of TPC，F(xiàn)RAP values in both yellow and black soybeans as compared to the boiling-treatments.And Francisco［37］reported that heat treatments(＜135℃，15 min)could further enhance the antioxidant capacity of peanut skins.Thermal treatment increased the antioxidant activity of grape seed extracts and the maximum TPC of whole grape seed extract(WGSE)were achieved when the seeds were heat-treated at 150℃ for 40 min［31］.Antioxidant activity in tomato and carrot purée was significantly higher than in untreated or thermally processed samples［38］.Thermal treatment could induce the formation of compounds with novel antioxidant properties，which could maintain or even enhance the overall antioxidant potential of foods［39］.Increase in AC following thermal treatment could be attributed to the release of bound phenolic compounds brought about by the breakdown of cellular constituents and the formation of new compounds with enhanced antioxidant properties.

Studies addressing the impact of thermal processing on total phenolics and antioxidant activity in foods are becoming more important due to its role in human health and disease management.Processed fruits and vegetables have been long considered to have lower nutritional value than the fresh produce due to the loss of vitamin C during processing.Dewanto et al［33］found that thermal processing at 115℃ for 25 min significantly elevated the total antioxidant activity of sweet corn by 44%and increased phytochemical content such as ferulic acid by 550%and total phenolics by 54%，although 25% vitamin C loss was observed.It suggested that processed fruits and vegetables might retain their antioxidant activity despite the loss of vitamin C.Ikeda et al［20］reported that heat-epimerized catechins are more effective than green tea catechins to prevent atherosclerosis by plasma-cholesterol lowering and antioxidative activities.In the present study，the reduction of antioxidant properties of green tea catechins accompanied the decrease of TPC and the loss in AC of boiling-treated green tea extracts may be attributed to thermal degradation of phenolic compounds.

3.4.Free radical-scavenging and antioxidant activities

The free radical scavenging abilities of the boiling-treated green tea extract against DPPH，O2·-，and OH·were tested，and the results were presented in Table2.It was found that all the boiling-treated samples were efficient in scavenging the DPPH radical，The IC50values were below 5.0 μg/mL.While the IC50values for O2·-and OH· free radicals were shown to be less active(IC50＞100 μg/mL).No difference(P ＞0.05)was observed on the free radicals-scavenging ability against DPPH and O2·-for the samples under boiling-treatment less than 2 h，and no difference(P ＞0.05)on the scavenging ability against OH· when the samples were treated even for 5 h.This indicated that the free radical scavenging ability of green tea extract was not greatly affected under boilingtreatment less than 2 h.

Free radical scavenging activity also accompanied with the phenolic content in food tissue.Thermal processing in general resulted changes of phenolic content and thus leaded to variation of antioxidant capacity and free radical scavenging activity.Chuah et al［29］reported that microwave heating and stir-frying did not affect the free radical scavenging abilities，Xu et al［28］reported that pressure steaming caused significant increases in DPPH scavenging activity in yellow soybeans as compared with the raw soybeans and Kim et al［31］reported that thermal treatment increased the radical scavenging activity of grape seed extracts.Studies in vegetables confirm that thermal processing significantly alters biochemical composition.

4.Conclusions

Pimerization from epi-catechins to the corresponding nonepi-catechins occurred within the range of experiment.The reduction of total catechins was not significant when the green tea extract was boiling-treated for 1h，and total phenolic content was of a similar magnitude under the boiling-treatment from 1 to 4 h.Boiling-treatment influenced the stability of green tea catehins，but had limited effect on the total phenolic compounds of green tea extract.There was no significant reduction of the antioxidant capacity when the samples were boiling-treated for 1 h.No difference was observed on the free radicals-scavenging ability of less than 2h boiling-treated samples against DPPH and O2·-，and no difference on the scavenging ability against OH· the samples were boiling-treated even for 5h.Green tea extracts still remained excellent in antioxidant properties and free radical scavenging activity after the boilingtreatment for 1h，and moderate loss of these bioactivities would occur when boiling-treated for more than 1 h.

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