Optimizing synchronous extraction and antioxidant activity evaluation of polyphenols and polysaccharides from Ya'an Tibetan tea (Camellia sinensis)

Abstract The optimal synchronous conditions to extract tea polysaccharides (TPS) and tea polyphenols (TPP) from Ya'an Tibetan tea were investigated, and the antioxidative capacity of TPS and TPP was measured, and the tea was analyzed to identify the polyphenol compounds it contained. On the basis of single‐factor experiments, a Box–Behnken design and response surface methodology were applied to optimize the hot water extraction conditions. The optimal extraction technology was determined as extraction temperature of 83°C, time of 104 min, and liquid‐to‐material ratio of 41 ml/g, yielding TPP and TPS at 42.70 ± 2.38 mg/g and 53.86 ± 3.79 mg/g, respectively. The TPS and TPP in Ya'an Tibetan tea have high eliminating activities on DPPH and strong reducing power, with TPP showing a higher antioxidant activity than TPS. UHPLC‐QqQ‐MS/MS analysis identified EGCG, GCG, and ECG as major polyphenol components in Ya'an Tibetan tea. These findings might promote the application of Ya'an Tibetan tea in the food industry.


| INTRODUC TI ON
Tea (Camellia sinensis) is one of the most popular beverages consumed worldwide. Tea is classified into green tea, oolong tea, black tea, and dark tea according to the fermentation level (Weerawatanakorn et al., 2015). Chinese dark tea is postfermented tea, the history of which dates back to the Ming Dynasty at 1,500 A.D. Tea was initially produced in Southwestern China and then carried via the mountains to Tibet and Xinjiang. In contrast to black tea, the process used for the fermentation of dark tea is known as "postfermentation" (or "wodui" in Chinese, meaning "pile-fermentation" as a type of "controlled composting"), whereby these wet teas are compressed and formed into brick, mushroom, square, bowl, and cake shapes so as to be convenient for transportation. The bricks of tea then undergo controlled fermentation with a mixture of bacteria and mold that excludes disease-causing microbes, and the polyphenols in tea also limit the growth of bacteria that can cause disease and spoilage while allowing the correct microbes to break down the material.
With increasing fermentation, the color of the tea becomes darker, the tea softens in texture, and the flavor becomes concentrated and rich but is no longer astringent (Zhang, Zhang, Zhou, Ling, & Wan, 2013
Another important bioactive component in tea is TPS, although there has been a lack of studies on this substance. Recently, there have been studies showing that TPS has many health benefits including antioxidant, antitumor, antibacteria, anti inflammation, and detoxification Li, Wang, Yuan, Pan, & Chen, 2018;Mao et al., 2018;Ren, Hu, Luo, & Yang, 2015).
Kangzhuan brick tea, also known as Tibetan tea, is a typical dark tea with a dark brown color. Tibetan tea is mainly produced in Ya'an, Sichuan, China and is the most popular drink for the Tibetan people.
Tibetan tea has a history of thousands of years and contains multiple beneficial elements and bioactive substances. Tibetan tea is often consumed with high-fat foods, such as beef and cheese, to promote better digestion of fat and cholesterol. However, there have been a few studies on the active ingredients of Tibetan tea, and to guide the effective use of Tibetan tea, further study is required.
There are many studies describing how to extract either TPP or TPS alone. However, to the best of our knowledge, combinative extraction of TPP together with TPS from Tibetan tea has not been reported, which limits its commercial development. TPP and TPS extractions are affected by many factors. In many different research fields, factor analysis and experimental design are used to determine effective parameters and improve performance (Bilgin, Elhussein, Özyürek, Güçlü, & Şahin, 2018). And response surface methodology (RSM) is often used to determine the optimal parameters in the extraction research.
The objective of present study was to optimize the conditions of simultaneous extraction of TPP and TPS in Tibetan tea and to evaluate the antioxidant activities of TPP and TPS and analyzed the TPP compounds using ultra-high-performance liquid chromatography-triple quadrupole-mass spectrometry (UHPLC-QqQ-MS).
Ammonium formate and methanol are HPLC grade. Trichloroacetic acid and potassium ferricyanide were purchased from Solarbio Science & Technology Co., Ltd.

| Extraction technological process and singlefactor experiment
The synchronous extraction process that we employed to extract TPP and TPS is as Figure 1. Tibetan tea was ground until the particle size less than 355 μm. The single-factor experiment for synchronous extraction was conducted, and the effect of extraction temperature (50, 60, 70, 80, 90°C), extraction time (60, 80, 100, 120, 140 min), and liquid/material ratio (20:1, 30:1, 40:1, 50:1, 60:1) on the extraction yield has been studied. For each tea extraction, distilled water was added to a flask containing 1 g of tea powder, and the extractions were performed in the given parameters. After

F I G U R E 1
The synchronous extraction process of TPP and TPS the hot water extraction, the extracts were centrifuged at 3,000 g/ min for 10 min. The supernatant was blended with the equal volume of ethyl acetate, and then, the organic phase was removed for determination of TPP. The aqueous phase was retained, to which 95% EtOH in a 1:4 (v/v) ratio at room temperature was added.
The solution was left undisturbed for 12 hr and then collected the precipitate by 3,000 g/min centrifugation, and the precipitate was dried at 60°C to obtain TPS.

| Response surface methodology (RSM)
Based on the results of the single-factor experiments, a response surface methodology experiment was carried out to obtain the optimal synchronous extraction parameters, and the data were analyzed by Design-Expert (8.0.6.1, Stat-Ease Inc., USA). In the experiment, both extraction yield of TPS and TPP were used as the response value, and the liquid-to-material ratio (X 1 ), extraction temperature (X 2 ), and extraction time (X 3 ) were independent variables ( Table 2). The quadratic equation of the experiment is as follows: where Y denotes the response variables, X i and X j denote the independent variables, β 0 is the intercept, β i is the linear, β ii is the quadratic, and β ij is the interaction coefficients.

| Tea polyphenols content assays
The TPP content was analyzed by the Folin-Ciocalteu colorimetric method (Bilgin et al., 2018) with some modifications. First, 1.0 ml diluted sample was combined with 5.0 ml 10% Folin-Ciocalteu reagent. After 5 min, 4.0 ml of 7.5% Na 2 CO 3 was added. After 1 hr, the TPP content was determined at 765 nm using a PT-3502C microplate spectrophotometer (Potenov Co. Ltd.). The result was expressed as gallic acid equivalent per g of Tibetan tea (mg/g dry basis).

| Tea polysaccharides content assays
The amount of TPS was determined using the phenol-sulfuric acid method (Guo, Guo, Zhu, & Jiang, 2016), and the result was expressed as glucose equivalent per g of Tibetan tea (mg/g dry basis).
The mobile phase consisted of 10 mmol/L ammonium formate (A) and methanol (B) at a flow rate of 0.4 ml/min, and the injection volume was 5 μl at 30°C column temperature. A gradient elution was this as follows: 90%-10% B in 6 min, 10% B in 7 min, 90% B in 7.5 min, and 90%-90% B in 10 min.
ESI in negative ionization mode was performed with the following parameters: the drying gas flow rate was 10.0 ml/min, gas temperature was set at 350°C, and capillary voltage was 3,500 V.
A multiple reaction monitoring (MRM) mode was used, and all the transitions (Table 1) were monitored over the run time with dwell times of 20 ms.

| 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay
The DPPH assay was determined according to a previously published method with some modification (Guo et al., 2016). The samples were analyzed at 517 nm against a blank sample without DPPH. DPPH radical scavenging capacity was calculated as follows: where A i is the sample with DPPH-ethanol solution, A j is the absorbance of the sample with ethanol, and A c is the distilled water mix with DPPH-ethanol solution.

| Reducing power assay
The reducing power assay was determined following a reported method with slight modifications (Qiu, Wang, Song, Deng, & Zhao, 2018). The sample solution (100 μl) was mixed with 250 μl 0.2 mol/L phosphate buffer and 250 μl 1% potassium ferricyanide. The mixed solution was heated to 50°C for 20 min and then cooled rapidly. Then, 250 μl 10% TCA was added to the solution and centrifuged at 1580g for 10 min.
Next, 250 μl of the supernatant was mixed with 250 μl distilled water and 50 μl 0.1% FeCl 3 , and the mixtures were measured at 700 nm using a PT-3502C microplate spectrophotometer. The results were expressed as absorbance values.

| Statistical analysis
All data are presented as the mean ± SD and subjected to LSD tests using the software SPSS 16.0 statistical software package (SPSS Inc., Chicago, IL, USA). p values < .05 were considered to be significant.

| Influence of extraction parameters on total tea polyphenol and tea polysaccharide yield
In the present study, the single-factor experiment was adopted to study the effect of three extraction parameters (temperature, time, liquid-to-material ratio) on the extraction yield.
F I G U R E 2 Influence of extraction temperature, extraction time, and liquid/ material ratio on the extraction of TPP and TPS from Ya'an Tibetan tea, and columns not sharing a common letter showed significant difference (p < .05)

| Extraction temperature
In the present study, the extraction yield of TPP and TPS was significantly affected by temperature ( Figure 2). Moreover, extraction content of TPP reached the maximum when the temperature increased to 80°C from 50°C (p < .05); then, as the temperature continued to increase, there was no significant difference in TPP yield (p > .05). Similar results were found for Yunwu tea (Guo et al., 2016).
The study indicated that high temperature benefited the dissolution and release of TPS and TPP in water by reducing the viscosity and improving the diffusion coefficient. The similar phenomenon was seen in TPS extraction. Therefore, 80°C was applied in RSM study.

| Extraction time
As shown in Figure 2

| Liquid-to-material ratio
In the present study, extraction content of TPP in the 40:1 group was significantly higher than that in other groups (p < .05), and the ratio of 20:1 showed significantly lower yield than other groups (p < .05) ( Figure 2). These results are in accordance with previous findings that a higher liquid-to-material ratio leads to a higher yield of polyphenols, and a higher liquid-material ratio generates a decrease in the consumption of tea material and decrease in the cost of extraction (Ćujić et al., 2016). However, when the ratio of liquid to material decreases to a certain extent, TPP are gradually saturated in the solution, and then the amount of dissolved TPP becomes low. As for TPS extraction, the extraction yield in the 40:1 group and 50:1 group was significantly higher than that in the other groups (p < .05). It is suggested that a high ratio of liquid to material accelerated the reaction between the surfaces of polysaccharides and the liquid, then resulted in the high yield of TPS (Cho, Getachew, Saravana, & Chun, 2019). Therefore, the liquid-to-material ratio of 40:1 was applied in the further study.

3
(2) TPS = 53.52 + 3.21X 1 + 6.93X 2 + 2.18X 3 − 0.16X 1 X 2 + 1.21X 1 X 3 − 2.13X 2 X 3 − 9.71X 2 1 − 14.16X 2 2 − 8.30X 2 3 No. and TPS (p = .0004) was significant, which suggested that the model was good fit with the experimental data. The p value for lack of fit was insignificant (p > .05). Moreover, the R 2 Adj of TPP model and TPS model was 0.9895 and 0.9862, respectively, the results showed that the experimental data of RSM had a high degree of accuracy and reliability and that the observed and predicted data correlated well. Content of TPP was significantly affected by extraction temperature (p < .0001) and extraction time (p < .01), and the yield of TPP increased by increasing the extraction time. Furthermore, for the extraction of TPP, the liquid-to-material ratio had no remarkable effect on TPP yield (p < .05). The results also showed that all three extraction factors significantly affected the extraction rate of TPS (p < .05).
Response surface plots of TPP yield and TPS yield are shown in Figure 3a-c. The effect of extraction time and liquid-to-material ratio on the yield of TPP and TPS is shown in Figure 2a. The result showed that the interaction between extraction time and liquid-to-material ratio (X 1 X 2 ) showed a significantly positive effect on the TPP yield (p < .05); however, the TPS yield was not significantly affected by the interaction (p > .05). Moreover, interaction between X 1 X 3 and X 2 X 3 also had significant effect on the TPP extraction yield. (1) and (2)

| Antioxidant activity of TPP and TPS from Tibetan tea
In the present study, the DPPH assay and reduction power assay were carried out to compare the antioxidant activity of TPP, TPS, and Vc in the range of 0.02-0.07 mg/ml. Both DPPH assay and reduction power assay are widely used to evaluate the antioxidant activity in active ingredients for food (Guo et al., 2014). As a very stable radical, DPPH appears purple when dissolved in ethanol and has a maximum absorption peak at 517 nm, and the stronger the scavenging ability of antioxidant, the lower the absorbance (Wang, Chen, Jia, Tang, & Ma, 2012;Wang, Yang, & Wei, 2012). Figure 4a shows that TPP possesses stronger scavenging of DPPH, and the effects depend on the dose in the range of experimental concentrations (p < .05). Moreover, there was no significant difference between TPP and Vc (p > .05), and the DPPH scavenging activity of TPS was lower than that of TPP and Vc for the concentrations of 0.02-0.07 mg/ml (p < .05). Other dark tea, such as Fuzhuan tea, Pu-erh tea and Liubao tea, also displayed high DPPH radical scavenging ability (Lv, Zhang, Shi, & Lin, 2017).
The antioxidant activity also could be evaluated by reducing power in several research. In the assay, the Fe 3+ of ferricyanide complex could be reduced to the Fe 2+ by the antioxidant, and then, the potassium ferrocyanide is further reacted with FeCl 3 to form Perl's Prussian blue with the maximum absorption peak at 700 nm (Guo et al., 2016). As shown in Figure 4b, the reducing power of TPP and TPS increased with concentration, and the activity of TPP was significantly stronger than that of TPS in the tested concentrations of our experiment (p < .05).
Furthermore, the reducing power of TPP and TPS was lower than that of Vc for all of the concentrations tested in the present study. TA B L E 3 ANOVA for the effect of temperature, time, and liquid-to-material ratio on the content of TPP and TPS using the response surface model Many studies have confirmed the strong antioxidant activity of TPP. High antioxidant activity has been observed for extracts of Puerh tea (Huang, Zhang, et al., 2013;Huang, Chen, et al., 2013). Fan et al. (2013 also reported that Zijuan Pu-erh tea reduced lipid-membrane oxidation by scavenging free radicals. Moreover, Li, Chen, Zhu, and Meng (2017) investigated the antioxidant activity of TPP from three dark tea and found that Ya'an Tibetan tea showed the highest antioxidant activity. TPS from brick tea also exhibited dose-dependent antioxidant activity (Yang et al., 2017), and it was reported that the DPPH radical scavenging activities of TPS could be attributed to the carboxyl group in hexuronic acid (Wang, Chen, et al., 2012;Wang, Yang, et al., 2012). Due to the difference of the tea processing technology and origin, there were also found significant differences in the composition of TPS, and affecting its antioxidant activity. In the fermentation process, antioxidant activity of deeply fermented oolong tea increased by the conjugation between its polysaccharide and protein (Wang, Chen, et al., 2012;Wang, Yang, et al., 2012).
However, Zhao, Huangfu, Dong, & Liu, (2014) reported that TPS extracted from nonfermented green tea had stronger antioxidant activity than that extracted from fully fermented black tea. The changes in the antioxidant activity of TPS in Tibetan tea during fermentation require further study.

F I G U R E 3
Response surface plots for the effects of temperature and time (a), temperature and liquid-to-material ratio (b), time and liquid-to-material ratio (c) on the yield of TPP and TPS

| UHPLC-QqQ-MS analysis
Green tea exhibits strong bioactivity mainly because of its abundance of TPP, such as EGCG (Hamilton-Miller, 1995). It was pointed out the total amount of polyphenols in tea is inversely proportional to the degree of fermentation. Along with the fermentation, the constituents and content of TPP were altered significantly by oxidation and polymerization in dark tea. We determined that there were 12 compounds in both TPP and tea soup of Ya'an Tibetan tea, including protocatechuic acid, epicatechin (EC), epigallocatechin gallate (EGCG), epicatechin gallate (ECG), fumalic acid, gallic acid (GA), gallocatechin gallate (GCG), gallocatechin (GC), caffeic acid, kaempferol, quercetin, and rutin (Figure 5a-d). Consistent with other studies, EGCG, GCG, ECG, and GA were identified as the major components in both TPP and tea soup of Ya'an Tibetan tea. In Fuzhuan brick tea, the content of EGCG, EGC, and ECG was much higher as compared to other catechins (Zhu et al., 2015). EGCG and ECG were also noted to be the predominant active compounds in brick tea (Zheng et al., 2015). Furthermore, as the major components of TPP, EGCG, GCG, and ECG were mainly responsible for the antioxidant activity of tea (Guo et al., 2016). In tea, the significant positive correlation between the content of EGCG and the antioxidant activity has also been proven by the FRAP and ABTS experiments Xu, Hu, et al., 2015;Xu, Zhang, et al., 2015). These studies indicate that the antioxidant activity of Tibetan tea might be attributed to its polyphenolic components, such as EGCG, GCG, and ECG.
With the development of the fungal postfermentation, the content of galloyl catechins decreased gradually, especially EGCG and ECG (Zhu et al., 2015). The reduction of catechin in microbial fermented tea mainly due to polymerization and oxidative cleavage of aromatic rings, and then, the humic acids were produced (Zheng et al., 2015). Similarly, the level of EGCG was significantly higher in raw Pu-erh tea than that of fermented Pu-erh tea .
Interestingly, in the present study, the level of EGCG in Ya'an Tibetan tea was significantly higher than that in other brick teas, and it was suggested that the EGCG in Ya'an Tibetan tea was poorly degraded by microorganisms during the fermentation process when the raw dark tea turned into dark tea. For the postfermented tea, microorganisms play a key role in tea quality. Chen et al. (2018) found Aspergillus cristatellus present in Fuzhuan brick tea produced in Shanxi province. In Pu-erh tea, the main microbes were Rhizopus, Saccharomyces, Aspergillus, Penicillium, and Bacterium (Zhao et al., 2013). Therefore, we consider that during the fermentation stage, the main microbes in Ya'an Tibetan tea may be different from those in other brick tea. However, because the microbiological method of action is unknown during the pile-fermentation processing, the specific mechanism requires further study.
Moreover, the concentrations of these polyphenols in TPP were considerably higher than that in tea soup. It was suggested that TPP can be obtained more effectively through the RSM extraction process. Therefore, effective extraction technology is useful for the most efficient extraction of TPP from Ya'an Tibetan tea.

| CON CLUS ION
A Box-Behnken design based on RSM was demonstrated to be sufficient to optimize conditions for the hot water extraction of TPP and TPS from Ya'an Tibetan tea, and the liquid-to-material ratio, temperature, and time had significant effects on the extraction. The highest yields of TPP and TPS were obtained with a liquid-to-material ratio of 41 ml/g, extraction temperature of 83°C,

F I G U R E 4
The antioxidant activity of Tibetan tea. A. DPPH radical scavenging activities of TPP, TPS, and Vc (mean ± SD, n = 3). Different letters (a, b, c) indicate significant differences (p < .05) between TPP, TPS, and Vc as the same concentration. Compared with the previous column data of TPP, an asterisk (*) represents p < .05. B. Ferric reducing antioxidant power of TPP, TPS, and Vc (mean ± SD, n = 3). Compared with the previous column data of TPS, an asterisk (#) represents p < .05 F I G U R E 5 Concentrations of 12 compounds in TPP (a) and tea soup (b) and Chromatogram of 12 compounds in TPP (c) and tea soup (d) and extraction time of 104 min. Furthermore, the DPPH assay and reduction power assay indicated that both TPP and TPS of Tibetan tea possessed strong antioxidant capability, with TPP showing higher antioxidant activity than TPS. Moreover, EGCG, GCG, ECG, and GA were identified as major polyphenol components in both TPP and tea soup from Ya'an Tibetan tea. These findings are beneficial to the development and application of Ya'an Tibetan tea in the food industry.

ACK N OWLED G M ENT
Thank all those who have helped in carrying out research. This study was supported by Chongqing Science and Technology Committee (cstc2016jcyjA0414).

CO N FLI C T O F I NTE R E S T
The authors declare that there is no conflict of interests.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.

I N FO R M E D CO N S E NT
Written informed consent was obtained from all study participants.