The Galloyl Catechins Contributing to Main Antioxidant Capacity of Tea Made from Camellia sinensis in China

Total polyphenol content, catechins content, and antioxidant capacities of green, dark, oolong, and black teas made from Camellia sinensis in China were evaluated. The total polyphenol content of 20 samples of tea was in the range of 7.82–32.36%. Total catechins content was in the range of 4.34–24.27%. The antioxidant capacity of tea extract was determined by the oxygen radical absorbance capacity (ORAC) test and the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging test. Total polyphenol content, catechins content, and antioxidant capacity decreased in the following order: green > oolong > black > dark tea. A positive correlation existed between the antioxidant capacity and total polyphenol content or catechins content (R 2 = 0.67–0.87). The antioxidant capacities of five major catechins (epigallocatechin gallate (EGCG), epicatechin gallate (ECG), epicatechin, epigallocatechin, and catechin) were determined by online HPLC DPPH radical-scavenging; the antioxidant activity of tea was mainly attributed to the esterified catechins (EGCG or ECG).


Introduction
Tea consumption has a long history of more than 2000 years. Originating in China and then spreading to Japan, Europe, and other areas, tea has become one of the most popular and frequently consumed beverages in the world [1]. All kinds of tea originate from Camellia sinensis, which has the two subspecies var. sinensis (China tea) and var. assamica (Assam tea) [2].
In China, six types of tea made from the leaves of Camellia sinensis including green, black, oolong, dark, white, and yellow tea have been classified based on the degree of fermentation and the color of the tea product [1]. Green and yellow teas are nonfermented tea, and oolong and white teas are called semifermented tea, while dark and black teas are fully fermented [3,4].
Of the six types of tea, four types (green, oolong, black, and dark teas) are widely consumed throughout the world. The manufacturing processes used to prepare the four common types of tea are as follows.
(1) Green tea: after the fresh tea is picked, it is allowed to wither for several hours. The tea leaves are then heated to remove moisture and to denature the enzymes that cause oxidation. The leaves are then kneaded and rolled while being dried. (2) Oolong tea: after the fresh leaves are picked, they are left out in the sun to wither for a few hours. Then the leaves are brought indoors and are laid out on large bamboo trays in a temperature-controlled room to oxidize. When the tea has reached the desired level of oxidation, the tea is exposed to heat to stop the oxidization. The tea is then shaped and roasted to dry. (3) Black tea: the fresh leaves are picked and allowed to wither in the sun. Once they have withered enough, the leaves are then bruised to cause oxidation. The leaves are then put into boxes to oxidize. When the leaves have turned to a dark red-copper color, they are fried and then rolled and shaped. (4) Dark tea: when the leaves are picked, they are fried, kneaded, and twisted, but instead of being dried as green tea is, the leaves are sprinkled with water and placed in huge piles under cloth to ferment before being dried. The resulting leaves have a dark black color [5]. Green tea is popular in China, Japan, India, and in Middle Eastern countries; oolong tea is mainly consumed in China, Japan, and Southeast Asia; dark tea is consumed in the southwestern regions of China; black tea is preferred in many countries throughout the world.
The radical-scavenging and antioxidant properties of tea polyphenols are frequently cited as important contributors to the above-mentioned health-protection benefits [22]. Nowadays, the content of tea polyphenols is regarded as a quality indicator of tea [23]. Therefore, it is desirable to investigate the total polyphenol contents and antioxidant capacities of different teas.
Many researchers have already begun to pay attention to the correlation between antioxidant capacity and the tea polyphenol content. Anesini et al. investigated the total polyphenol content and antioxidant capacity of commercially available tea (Camellia sinensis) in Argentina [25]. In their work, a high correlation was demonstrated between the two scrutinized properties. The antioxidant activity and phenolic profile in tea and herbal infusions were studied by Atoui et al. and their results suggested that black Ceylon tea and Chinese green tea infusions can be major sources of polyphenols that exhibit important antioxidant behavior [26]. Rusak et al. investigated the phenolic content and antioxidative capacity of green and white tea extracts [24]. The results showed that the antioxidative capacity of the investigated tea extracts correlated with the phenolic content. However, in the abovementioned studies, the profiles of catechins in tea were not elucidated and it remains unclear which compounds are responsible for their antioxidant property.
In the present study, total polyphenol content and antioxidant capacity of 20 samples from four types of tea (green, dark, oolong, and black teas) prepared from Camellia sinensis in China were determined and the correlation between the antioxidant capacity and total polyphenol content or catechins was investigated. Furthermore, the content of individual catechins (EGCG, EGC, ECG, EC, and catechin) and their contribution to the antioxidant capacity of tea were elucidated.
The Scientific World Journal 3

Preparation of Tea Extract.
The preparation of the tea extract was performed according to the method published by the International Organization for Standardization (ISO) [23]. Each tea sample was ground to a fine powder and 1.0 g of the powder was extracted with 25 mL of methanol/water (7 : 3, v/v) in a water bath at 70 ∘ C for 10 min. The extraction was repeated twice and the extracts were combined. After filtration through a filter paper and a 0.45 m membrane filter (Millipore), the extract volume was adjusted to 100 mL.

Determination of Total Polyphenols by Folin-Ciocalteu
Method. The total polyphenol content in 20 samples from four types of tea (green, dark, oolong, and black) was determined according to the Folin-Ciocalteu method as described by ISO 14502-1 [23] with some minor modifications. One milliliter of the tea extract was diluted with water to 100 mL. The diluted extract solution (1.0 mL) was then mixed with 5.0 mL of 50% Folin-Ciocalteu reagent. After 5 min, 4.0 mL of 7.5% Na 2 CO 3 solution was added and mixed. Then the mixture was incubated at room temperature for 60 min. The absorbance of the solution was measured at 765 nm with a Shimadzu UV-2550 UV-Vis spectrophotometer against a reagent blank. The determination was three times. The total polyphenol content was expressed as gallic acid equivalents (GAE, units: g/100 g sample).

Determination of Catechins by HPLC.
The content of catechins in 20 samples from four types of tea (green, dark, oolong, and black teas) was determined according to the ISO 14502-2 method [27]. Quantification of the major catechins (EC, EGC, ECG, EGCG, and catechin) was performed using an HPLC system (Jasco, Japan) equipped with a 1580 pump and a 975 UV-Vis detector. Chromatographic separation was achieved with a HiQ Sil C 18 V reversed phase column (250 mm × 4.6 mm i.d.) packed with 5 m particles and the column temperature was maintained at 35 ∘ C. Mobile phase A was a mixture of 9% acetonitrile and 2% acetic acid with 20 g/mL EDTA. Mobile phase B was a mixture of 80% acetonitrile and 2% acetic acid with 20 g/mL EDTA. Baseline separation of EC, EGC, ECG, EGCG, catechin, gallic acid, and caffeine was achieved with a binary gradient elution program as follows: 100% mobile phase A for 10 min, then over 15 min a linear gradient to 68% mobile phase A, 32% mobile phase B and hold at this composition for 10 min. The detection wavelength was 278 nm and the flow rate was 0.8 mL/min. One milliliter of tea extract was diluted with water to 100 mL, the diluted extract was filtered through a 0.45 m membrane filter (Millipore), and the filtrate was injected into the HPLC apparatus. Standard solutions of the major catechins (EC, EGC, ECG, EGCG, and catechin), gallic acid, and caffeine were prepared by accurately weighing standard powders and dissolving them in 10% acetonitrile with 500 g/mL of EDTA. The stock solutions were stored at 4 ∘ C. All standard solutions were filtered through a 0.45 m membrane filter (Millipore) and the filtrate was injected. The total catechins content was expressed as a percentage by mass of the sample on a dry 4 The Scientific World Journal matter basis and was obtained by summation of the EGC, catechin, EC, EGCG, and ECG content.

Oxygen Radical Absorbance Capacity (ORAC) Assay.
The oxygen radical absorbance capacity (ORAC) method is used to measure the antioxidant capacity of biological samples in vitro. The ORAC assay was first developed by Cao et al. in 1993 [28]. This approach has been recognized and is now frequently used in analysis of polyphenol antioxidant capacity. 2,2 -Azobis(2-amidinopropane) dihydrochloride (AAPH) is thought to produce the peroxyl radical upon heating, which damages the fluorescent molecule and results in a loss of fluorescence [29,30]. The fluorescence decay is monitored as fluorescein in the solution decomposes, a process that should be slower in the presence of antioxidants. Decay curves are recorded and the area between the two decay curves (with or without antioxidant) is calculated. Calculating the area under the fluorescence decay curve (AUC) of trolox, which is used as a standard antioxidant, a standard curve is prepared that can be used to evaluate the radical-scavenging activity of other antioxidants in terms of trolox equivalents (TE).
The ORAC assay was performed using a fluorescence spectrophotometer (F-7000, Hitachi, Japan), following a previously described procedure [31] with minor modification. Samples were diluted with sodium phosphate buffer (75 mM, pH 7.4). Sodium fluorescein solution (600 L, 40 nM) was added to 100 L of phosphate buffer (blank), to 100 L aliquots of trolox solution (1.25, 2.5, 5, 10, 20, 40, and 80 M), or to 100 L of diluted sample. The respective mixtures were incubated for 15 min at 37 ∘ C. Immediately before initiating the measurements, 100 L of AAPH (150 mM) was added into the test solution. With the excitation filter set to 485 nm and the emission recorded at 535 nm, readings were collected every minute for 60 min. The results were calculated based on the differences in AUC between the blank and the sample and were expressed as in units of micromoles TE per 1 g of dry tea. The AUC was calculated as follows: where 0 is the fluorescence reading at initiation of the reaction, is the fluorescence reading at minutes, and 60 is the final measurement. Net AUC was calculated as follows: 2.7. DPPH Radical-Scavenging Activity. The antioxidant capacity in 20 samples from four types of tea (green, dark, oolong, and black teas) was determined by reduction of the 2,2diphenyl-1-picryhydrazyl (DPPH) radical according to the published method [32]. A stock solution was prepared by stirring 75 mg of DPPH in 1 L of methanol overnight. A 0.75 mL aliquot of sample extract was mixed with 1.5 mL of DPPH solution. For each extract, a blank of 1.5 mL of methanol was used without DPPH reagent to correct for any sample absorbance at 521 nm. Sample and blank absorbance were recorded at 521 nm after 6 min. The percentage inhibition of DPPH was calculated according to the equation below [33]: where 0 is the absorbance of the DPPH solution, 1 is the absorbance of the tea extract in the presence of DPPH solution, and is the absorbance of the tea extract solution without DPPH. Each sample was analyzed in triplicate.
The EC50 value was calculated by a graphical method as the concentration that caused 50% inhibition of DPPH. The anti-DPPH efficiency (AE) was also calculated as log ⋅ (1/EC50) [34].

Online HPLC DPPH Screening Analysis of Tea Samples.
The antioxidant activity of EC, EGC, ECG, EGCG, catechin, gallic acid, and caffeine was measured using the online HPLC DPPH screening method [35]. The online HPLC DPPH method was developed using a methanolic solution of DPPH stable free radical (50 mg/L). The instrumental setup is depicted in Figure 2. The flow of HPLC-separated analytes and DPPH solution was 0.8 mL/min and 0.2 mL/min, respectively. The HPLC-separated analytes reacted with the DPPH solution in post-column mode, which was photometrically detected as a negative peak at 521 nm. The length of the capillary (5.1 m × 0.5 mm i.d.) used for the postcolumn reaction was adjusted to achieve a reaction time of 1.0 min. The HPLC conditions were the same as described above.

Total Polyphenol and Catechins Content in Four Types
of Tea. Using the Folin-Ciocalteu method, total polyphenol contents in 20 samples from four types of tea were calculated from the standard curve for gallic acid, ranging from 10 to 50 g/mL ( = 0.183 − 0.025, 2 = 0.9987). The results are summarized in Table 2 Table 2. It can be seen from Table 2    polyphenols and catechins. Although there was a significant difference in the content of polyphenols and catechins in the same type of tea, as a whole, the contents of total polyphenols and catechins decreased in the following order: green tea > oolong tea > black tea > dark tea.
In addition, the correlation between catechins and total polyphenols in the 20 samples from the four types of tea was tested. The results, shown in Figure 3, reveal a strong positive correlation between catechins and total polyphenols ( 2 = 0.96). Table 2 lists the contents of individual catechins in the four types of tea. It is apparent that the content of esterified catechins (EGCG or ECG) is higher than that of nonesterified catechins (catechin, EC, or EGC). In comparison of the four types of tea, EGCG content was the highest in green tea   (5.25-14.39% on dry tea) and in oolong tea (4.57-13.08% on dry tea). With the increase in the degree of fermentation, the EGCG content gradually decreased and the range of EGCG content in dark tea is only 0.36-0.93% on dry tea. Among the four types of tea, the ECG content was the highest in black tea (3.38-5.63% on dry tea) and in dark tea (1.92-4.73% on dry tea).     ranged from 909.28 to 3092.51 mol TE/g. Of the selected teas, the antioxidant activity as assessed by the ORAC method decreased in the following order: green tea > oolong tea > black tea > dark tea. Huang shan mao feng (green tea) showed the strongest antioxidant activity. Figure 5 shows the relationships between the ORAC value and total polyphenols and catechins, respectively. A positive correlation was observed in each case, with 2 = 0.67 for total polyphenols and 2 = 0.77 for catechins. In addition, the correlations between individual catechin content and ORAC values in 20 samples were evaluated. The correlations between ORAC value and content of catechin, EGC, EC, EGCG, and ECG, respectively, are shown in Table 3, which shows a positive correlation for each case. In green tea, the EC content showed a relatively higher positive correlation ( 2 = 0.73) with ORAC values than catechin, EGC, EGCG, and ECG, and the values were significantly different ( < 0.01). For black tea, the EGCG content showed a relatively higher positive correlation ( 2 = 0.82) with ORAC values than catechin, EC, EGC, and ECG, and the values were significantly different ( < 0.01). In contrast, the correlation coefficients between ORAC value and content of individual catechin in oolong tea and in dark tea infusions were not significantly different ( < 0.01).

DPPH Radical-Scavenging Activity in Four Types of Tea.
The free radical-scavenging activity of the 20 samples of the four types of tea was assessed by DPPH assay; the results are shown in Figure 6. All sample extracts showed good inhibitory activity against the DPPH radical in a dosedependent manner. The EC50 and AE values of the tea sample extracts are summarized in Table 4, where lower EC50 values or higher AE values suggest higher antioxidant activity. For the 20 selected tea samples, EC50 was in the range 0.142-0.764 mg tea/mL and the AE values ranged from 0.117 to 0.848. Generally, DPPH radical-scavenging efficiency decreased in the following order: green tea > oolong tea > black tea > dark tea. The scavenging efficiency of Huang shan mao feng (green tea) against the DPPH radical was the strongest; its EC50 and AE values were 0.142 mg tea/mL and 0.848, respectively. Figure 7 shows the respective correlations between the AE values and the content of total catechins and total polyphenols. In each case, good correlation was observed ( 2 = 0.87 for total catechins; 2 = 0.77 for total polyphenols).
The correlations between AE values and the content of individual catechins in the 20 tea infusions are summarized in Table 5; significant positive correlation was observed in each case. Among the individual catechins in green tea, the EGCG content showed a relatively higher positive correlation ( 2 = 0.86) with AE values than those of catechin, EC, EGC, and ECG, and the values were significantly different ( < 0.01).

Online HPLC DPPH Radical-Scavenging Activity of Tea
Samples. In the tests of antioxidant capacity in the 20 samples of tea, as measured by the DPPH method, it was not clear which component played the important role in antioxidant activity. The recent development of the online HPLC DPPH method was used to our advantage in this study. This method, which is now used widely in the testing of food and agricultural products and in the pharmaceutical industry [35][36][37], allows the radical-scavenging activity of a single substance The Scientific World Journal   to be measured. This allows the calculation of a component's contribution to the overall radical-scavenging activity of a mixture of antioxidants [37]. Thus, the radical-scavenging activities of the major catechins (EGC, EC, EGCG, ECG, and catechin), gallic acid, and caffeine were determined by the online HPLC DPPH screening method. The chromatograms of the mixed catechin standards and that of a tea sample are shown in Figure 8, illustrating the excellent baseline separation of EGC, EC, EGCG, ECG, catechin, gallic acid, and caffeine. The areas of the positive peaks at 278 nm were used for quantitative analysis of EC, EGC, ECG, EGCG, gallic acid, catechin, and caffeine, while the areas of the negative peaks at 521 nm were used to evaluate the DPPH-scavenging activity. The results showed that while caffeine had almost no DPPH-scavenging activity, strong positive correlations were observed for the contents of the respective HPLC-separated catechins and for gallic acid (see Figure 9). For the same reaction time (1.0 min), the antioxidant activity as assessed by the online DPPH radicalscavenging method decreased in the following order: EGC > gallic acid > EGCG > EC > ECG > catechin. The contributions of gallic acid and the respective catechins in tea to the DPPH-scavenging activity were calculated based on the area ratios of corresponding negative peaks to the total area of all negative peaks at 521 nm; the results are shown in Figure 10. EGCG can be seen as the main contributor to DPPH-scavenging activity in green and oolong teas, while ECG was a significant contributor in black and dark teas. In general, the DPPH-scavenging activity can be mainly attributed to the esterified catechins (EGCG or ECG). In addition to gallic acid and catechins, other compound/s in tea appear to contribute to the DPPH-scavenging activity. Based on the results shown   in Figure 10, these minor contributions are in the range of 18.2-28.3%.

Conclusions
We conducted a systematic study on the total polyphenol and catechins content, as well as antioxidant activity, in four types of tea (green, dark, oolong, and black) prepared from Camellia sinensis in China. The highest content of polyphenols and total catechins was detected in green tea, followed by oolong, black, and dark tea. There was a positive correlation between the antioxidant capacity and total polyphenol content or catechins content ( 2 = 0.67-0.87, < 0.05). In general, it appears that the longer-fermented teas have lower total polyphenol content, lower catechins content, and lower antioxidant capacity. According to our results, green tea in China is of very high quality in terms of antioxidant capacity when compared with the other teas. Results of online HPLC DPPH radical-scavenging activity tests of tea samples showed that gallic acid, EGCG, ECG, EC, EGC, and catechin were responsible for the antioxidant capacity of the extracts from tea samples. Furthermore, the antioxidant capacity of tea extracts was mainly attributed to the esterified catechins (EGCG or ECG). The results also suggested that other compound/s in tea may also contribute to the antioxidant capacity to a minor degree.
While consumers will always rely on personal preferences relating to color, taste, and aroma when selecting tea, we believe that the antioxidant capacity should also be considered. The results of this study should prove useful to any consumers who wish to select tea based on antioxidant capacity.