Phenolic Profiles and Antioxidant Activities of 30 Tea Infusions from Green, Black, Oolong, White, Yellow and Dark Teas

Tea is among the most consumed drink worldwide, and its strong antioxidant activity is considered as the main contributor to several health benefits, such as cardiovascular protection and anticancer effect. In this study, the antioxidant activities of 30 tea infusions, which were obtained by the mimic of drinking tea of the public, from green, black, oolong, white, yellow and dark teas, were evaluated using ferric-reducing antioxidant power (FRAP) and Trolox equivalent antioxidant capacity (TEAC) assays, ranging from 504.80 ± 17.44 to 4647.47 ± 57.87 µmol Fe2+/g dry weight (DW) and 166.29 ± 24.48 to 2532.41 ± 50.18 µmol Trolox/g DW, respectively. Moreover, their total phenolic contents (TPC) were detected by Folin-Ciocalteu assay and were in the range of 24.77 ± 2.02 to 252.65 ± 4.74 mg gallic acid equivalent (GAE)/g DW. Generally, Dianqing Tea, Lushan Yunwu Tea, and Xihu Longjing Tea showed the strongest antioxidant activities among 30 teas. Furthermore, the phenolic compounds in tea infusions were identified and quantified, with catechins most commonly detected, especially in green tea infusions, which were main contributors to their antioxidant activities. Besides tea polyphenols, considerable content of caffeine also presented in 30 tea infusions.


Introduction
Tea (Camellia sinensis) is the most consumed drink in the world except for water, owing to its pleasant sensory properties, broad health benefits and unique sociocultural characteristics [1]. Usually, tea could be classified into six categories, i.e., green tea (unfermented), yellow tea (slight-fermented), white tea (mild-fermented), oolong tea (semi-fermented), black tea (fermented) and dark tea (post-fermented) according to the varying fermentation extent [2]. Accumulating clinical research has indicated the preventive and therapeutic effects of tea on oxidative stress-related diseases, like cardiovascular disease, cancer, liver disease, type 2 diabetes and cognitive dysfunction [3][4][5][6][7][8]. Moreover, several studies have shown that polyphenols and caffeine are the main ingredients responsible for the various bioactivities and distinctive sensory properties of teas [2,[9][10][11][12][13]. The most favorable antioxidant activities of tea infusions are attributed to polyphenols greatly [14][15][16]. The in vivo antioxidant activities of tea polyphenols cannot be simply extrapolated from their in vitro antioxidant effects due to their differences in bioavailability [17]. On the one hand, some polyphenols with strong in vitro antioxidant activities cannot pass the small intestinal barrier due to their polarity, solubility, or high molecular weight, and will possess weak in vivo antioxidant activities. On the other hand, some polyphenols with weak in vitro antioxidant activities can contribute to strong in vivo antioxidant activities after being transformed by intestinal enzymes or metabolized by gut microbiota [18]. In addition, the in vitro and in vivo antioxidant activities of some natural products could be consistent in certain conditions. Overall, in vitro antioxidant activity and phenolic profiles of tea can provide important references for the further study of in vivo antioxidant effects.
In a previous paper, the antioxidant polyphenols in different fractions of 30 teas, including fat-soluble, water-soluble, and insoluble-bound fractions, were evaluated after extraction by different organic solvents [19]. Using this method, it was expected that the antioxidants in tea could be completely extracted, and the total antioxidant capacity of tea could be obtained. However, this extraction method is very different compared to the habit of the public for tea drinking, where only water is used.
In this paper, therefore, we aimed to simulate the way of tea drinking to extract antioxidants in teas. The antioxidant activities of aqueous extracts (infusions) of 30 well-known teas from China, including 4 black teas, 5 dark teas, 9 green teas, 4 oolong teas, 3 white teas, and 5 yellow teas were evaluated using ferric-reducing antioxidant power (FRAP) and Trolox equivalent antioxidant capacity (TEAC) assays, and their total phenolic contents (TPC) were determined by the Folin-Ciocalteu method. Moreover, polyphenols and caffeine in 30 tea infusions were detected using high-performance liquid chromatography coupled with photodiode array detector (HPLC-PDAD). Tea infusions could have different types and contents of antioxidants compared to the fat-soluble, water-soluble, and insoluble-bound fractions of tea. The results obtained can provide guidance for the public to select tea with strong antioxidant capacity. The results are also very helpful for the nutritionist and epidemic experts to calculate/assess the intake amounts of antioxidants from tea.

Sample Preparation
The 30 teas (Table 1) were purchased from China. The sample (1 g) was extracted by 10 mL boiling distilled water in a 98 • C water bath shaker for 5 min, and the extraction procedures were repeated six times. All infusions were combined, and their antioxidant activities were evaluated immediately.

Detection of Antioxidant Capacity and Total Phenolic Content (TPC)
The FRAP assay was utilized to determine the reducing ability of tea infusions based on literature previously published with minor modification [20]. In brief, the properly diluted sample of 100 µL was mixed with 3 mL of FRAP reagent (a mixture of 300 mmol/L sodium acetate-acetic acid buffer, 10 mmol/L TPTZ solution, and 20 mmol/L ferric chloride solution at a volume ratio of 10:1:1). The reaction was conducted at room temperature for 4 min, and the absorbance was recorded at 593 nm. FeSO 4 was used as a standard, and the results were expressed as µmol Fe 2+ /g dry weight (DW). The TEAC assay was used to measure the free radical-scavenging ability of tea infusions based on previously published literature that was slightly modified [21]. Briefly, 0.1 mL of appropriately diluted sample was mixed with 3.8 mL of ABTS• + working solution with absorbance controlled at 0.710 ± 0.050. After the reaction at room temperature for 6 min, the absorbance was measured at the wavelength of 734 nm. Trolox was used as a standard, and the results were presented as µmol Trolox/g DW.
The TPC was measured by the Folin-Ciocalteu method [22]. The 0.5 mL of properly diluted sample was mixed with 2.5 mL of 0.2 mol/L Folin-Ciocalteu reagent. After 4 min of reaction in dark at room temperature, 2 mL of saturated sodium carbonate solution (75 g/L) was added, and the reaction continued for 2 h in dark at room temperature. The absorbance was measured at a wavelength of 760 nm. Gallic acid was used as a standard, and the results were expressed as mg gallic acid equivalent (GAE)/g DW.

High-Performance Liquid Chromatography (HPLC) Analysis
Tea infusions were subjected to HPLC coupled with a PDAD (Waters, Milford, MA, USA) and an Agilent Zorbax Extend-C18 column (4.6 × 250 mm, 5 µm) for determination of phenolic compounds and caffeine. The HPLC method referred to previous literature with minor modification [23]. In brief, the mobile phase A and B were methanol and 0.1% formic (v/v), respectively, and the temperature was 35 • C with the flow rate of 1.0 mL/min. The elution gradient was set as follows: 5% A (0 min), 20% A (10 min), 22% A (15 min), 25% A (20 min), 40% A (40 min), 42% A (50 min), 50% A (60 min), 95% A (70 min), 5% A (70.1 min) and 5% A (75 min). Phenolic compounds and caffeine in tea infusions were identified via comparing their retention time and ultraviolet-visible (UV-vis) spectra with those of standard compounds. Quantitative analysis was performed according to the peak area under the maximal absorbance wavelength, and the contents were expressed as mg/g DW. The limit of detection (LOD) and limit of quantity (LOQ) of this method were 2 and 5 µg/mL, respectively.

Statistical Analysis
All data obtained after 3 repetitions were analyzed using SPSS 20.0. (IBM SPSS Statistics, IBM Corp, Somers, NY, USA) and expressed as mean ± standard deviation (SD). Significant differences among groups were manifested using one-way analysis of variance (ANOVA) followed by an LSD post hoc test, with a significant level set at 0.05. For potential correlation among measurements, the linear regression analysis was performed. Statistical significance was defined at p < 0.05.

Antioxidant Capacities of Tea Infusions
The FRAP values of different tea infusions were quite different and varied from 504.80 ± 17.44 to 4647.47 ± 57.87 µmol Fe 2+ /g DW (

Total Phenolic Content of Tea Infusions
The total phenolic contents of 30 tea infusions were quite differed ranging from 24.77 ± 2.02 to 252.65 ± 4.74 mg GAE/g DW (Table 1 and Figure 3

Correlations between Values of Ferric-Reducing Antioxidant Power (FRAP), Trolox Equivalent Antioxidant Capacity (TEAC), and TPC
The correlations between values of FRAP, TEAC, and TPC of 30 teas are presented in Figure 4. There was a strong correlation between FRAP and TEAC values of 30 teas, with R 2 of 0.840 (p < 0.001), which indicated that components in tea infusions possessed both good reducing ability and free radicals cleaning capacity. Moreover, the antioxidant activities of 30 teas were strongly correlated with their total phenolic contents (FRAP vs. TPC, R 2 = 0.915, p < 0.001; TEAC vs. TPC, R 2 = 0.946, p < 0.001). Thus, the phenolic compounds could play key roles in the strong antioxidant activities of teas.

Correlations between Values of Ferric-Reducing Antioxidant Power (FRAP), Trolox Equivalent Antioxidant Capacity (TEAC), and TPC
The correlations between values of FRAP, TEAC, and TPC of 30 teas are presented in Figure 4. There was a strong correlation between FRAP and TEAC values of 30 teas, with R 2 of 0.840 (p < 0.001), which indicated that components in tea infusions possessed both good reducing ability and free radicals cleaning capacity. Moreover, the antioxidant activities of 30 teas were strongly correlated with their total phenolic contents (FRAP vs. TPC, R 2 = 0.915, p < 0.001; TEAC vs. TPC, R 2 = 0.946, p < 0.001). Thus, the phenolic compounds could play key roles in the strong antioxidant activities of teas.

Correlations between Values of Ferric-Reducing Antioxidant Power (FRAP), Trolox Equivalent Antioxidant Capacity (TEAC), and TPC
The correlations between values of FRAP, TEAC, and TPC of 30 teas are presented in Figure 4. There was a strong correlation between FRAP and TEAC values of 30 teas, with R 2 of 0.840 (p < 0.001), which indicated that components in tea infusions possessed both good reducing ability and free radicals cleaning capacity. Moreover, the antioxidant activities of 30 teas were strongly correlated with their total phenolic contents (FRAP vs. TPC, R 2 = 0.915, p < 0.001; TEAC vs. TPC, R 2 = 0.946, p < 0.001). Thus, the phenolic compounds could play key roles in the strong antioxidant activities of teas.
Aside from phenolic compounds, caffeine was also quantified in this study (

Discussion
It is difficult to fully evaluate the antioxidant activities of natural antioxidants just using a single determination method, so employing methods involved in different determination principles are essential [24][25][26]. In this study, 2 commonly used different assays for the determination of antioxidant activities, i.e., FRAP (reducing Fe 3+ to Fe 2+ ) and TEAC (scavenging ATBS radical), were combined to evaluate comprehensively the antioxidant activities of tea infusions [27,28].

Discussion
It is difficult to fully evaluate the antioxidant activities of natural antioxidants just using a single determination method, so employing methods involved in different determination principles are essential [24][25][26]. In this study, 2 commonly used different assays for the determination of antioxidant activities, i.e., FRAP (reducing Fe 3+ to Fe 2+ ) and TEAC (scavenging ATBS radical), were combined to evaluate comprehensively the antioxidant activities of tea infusions [27,28]. The  The antioxidant capacities and total phenolic contents of 30 teas differed greatly. Cultivar type, place of production, planting condition, harvesting time, leaf grade, and the manufactured process could affect antioxidant capacities and total phenolic contents of teas [11,31]. With respect to manufacturing, the antioxidant properties of teas (obtained from a single cultivar type and planted under the same conditions) could decrease as the fermentation degree increase [32]. Generally speaking, the antioxidant properties of the six categories of teas tested were in decreasing order of green tea, yellow tea, oolong tea, black tea, dark tea, and white tea.
Green tea possessed the highest antioxidant capacity and total phenolic content, which could be due to the minimized oxidation degree of young leaves and the bud due to inactivated enzymes during the steaming process [33]. The antioxidant properties of black and dark teas decreased during extended fermentation by polyphenol oxidases and microbes, respectively [13]. Additionally, the antioxidant activity of white tea, expected to be intermediate between yellow and oolong teas, was similar to fermented teas, which might be related to its special manufacturing process. In the production of white tea, the polyphenol oxidases and peroxidases remain active due to lack of steaming [34]. Furthermore, polyphenols in white tea are gradually lost due to enzymatic and non-enzymatic oxidation reactions in the process of withering.
Phenolic compounds refer to a large class of secondary metabolites in natural products involved in an extensive range of bioactivities ameliorating risks of oxidative stress-related diseases [35,36]. Some phenolic compounds (e.g., tannins) could reduce diet digestibility due to binding and precipitating dietary carbohydrates, proteins, and digestive enzymes, which were considered as antinutrients [37]. From the point of view, malnourished people should drink less tea, and overnourished/obese people could drink more tea. Additionally, some phenolic compounds have several health benefits, like cardiovascular protection and anticancer effects, which are associated with their strong antioxidant activities [3,8]. There were strong correlations between values of FRAP, TEAC and TPC of 30 teas, which indicated that rich phenolic compounds were the main sources responsible for the potent antioxidant activities regarding reducing and free radicals scavenging abilities.
In this study, polyphenols in 30 teas were determined with catechins being the dominant phenolic compounds. Moreover, green tea was a richer source of catechins among 6 categories of teas, in which catechins comprised 11.08%-16.21% of the dry weight. EGCG was the richest catechins. It has been reported that EGCG possessing the most number of phenolic hydroxyl groups manifests the strongest antioxidant activity in catechins, which is even stronger than vitamins C and E [38]. It has been reported that EGCG could exhibit multiple bioactivities, especially anticancer effects, through inhibiting cancer stem cells and modulating molecular events associated with cancer cell proliferation, apoptosis, immunity and so on [39]. Moreover, gallic acid, a well-known phenolic compound, widely presented in all 30 teas in low contents. A study reported that gallic acid could exhibit stronger DPPH radical scavenging capacity than vitamin C [40]. During the process of enzymatic fermentation, catechins in fresh tea leaves could partially convert into the complex condensation product, e.g., theaflavins, driven by polyphenol oxidase-catalyzed oxidative polymerization [41]. In this study, the theaflavin could be detected in two black teas, i.e., Dianhong Congou Black Tea and Yichang Congou Black Tea. A study suggested that theaflavins present in black tea could possess similar levels of antioxidant activities as catechins in green tea in terms of inhibiting Cu 2+ -mediated low-density lipoprotein (LDL) oxidation [42]. Therefore, wide spectra of phenolic compounds were included in the bioactive constituents of tea infusions.
Apart from phenolic compounds, habitual tea infusion intake could also introduce high contents of caffeine making up 1.24%-4.75% of the dry weight of tea leaves. A clinical trial has demonstrated that high caffeine intake could accelerate weight loss via relatively higher thermogenesis and fat oxidation [43,44]. However, the nervous system stimulating effects of caffeine should also be taken into consideration [45].

Conclusions
In this study, the infusions of 30 selected teas were prepared mimicking daily practices, and their antioxidant activities were evaluated using different determination methods. The results suggested that 30 tea infusions possessed strong antioxidant activities and rich phenolic compounds, which varied greatly in specific varieties. Additionally, phenolic compounds were the main contributors to the antioxidant capacities of teas. Thus, tea is a good dietary source of natural antioxidants, especially phenolic compounds, which have a good potential to be developed into a functional drink or dietary supplements. Furthermore, the antioxidant activities and the profiles of specific bioactive substances (such as EGCG, theaflavin and caffeine) in tea varieties were quite different. Generally speaking, green tea possessed the highest antioxidant capacity and total phenolic content, which was also a richer source of polyphenols, especially catechins. Among 30 teas, Dianqing Tea, Lushan Yunwu Tea, and Xihu Longjing Tea are good sources of natural antioxidants. Thus, consumers should take into account the characteristics of different teas comprehensively for the selection of teas that are best suited to their needs. Moreover, the results are also very helpful for the nutritionist and epidemic experts to calculate or assess the intake amounts of antioxidants from tea.