E ﬀ ects of Infusion and Storage on Antioxidant Activity and Total Phenolic Content of Black Tea

: This study determined antioxidant activity in terms of the 1,1-Diphenyl-2-picrylhydrazyl (DPPH) scavenging ability and total phenolic content of black tea under different infusion and storage conditions. High performance liquid chromatography analysis identified caffeine, ( − )-epigallocatechin, ( − )-epicatechin-3-gallate, ( − )-epigallocatechin-3-gallate and ( − )-gallocatechin-3-gallate in the tea sample. The water–tea leaves weight ratio did not affect the DPPH scavenging ability. However, infusion temperature affected the DPPH scavenging activity and the total phenolic content. In the present study, the 50% inhibitory concentrations (IC 50 ) for DPPH of black tea infused at 60 to 100 ◦ C ranged from 100.0 ± 13.7 to 28.4 ± 4.8 µ g / mL. The total phenolic content of black tea steeped at 60 to 100 ◦ C ranged from 50.4 ± 5.2 to 178.6 ± 16.4 mg gallic acid equivalent / g dry leaf. Black tea exhibited increased antioxidant activity when the infusion temperature was increased. Regarding short-term storage, the DPPH scavenging ability and total phenolic content of black tea did not significantly change within 15 days. This result was consistent for storage temperatures of 4, 9, and 25 ◦ C. ± 3.9 µ g / mL. The results indicate that no di ﬀ erence is observable among these IC 50 values with weight ratios from 25 to 500, as shown in Figure 2. The IC 50 for DPPH scavenging ability


Introduction
Tea, a popular beverage, is produced from the leaves of the plant Camellia sinensis (L.) O. Kuntze. Tea has been studied for its potential beneficial health effects and has been used for medical purposes in recent decades [1]. Tea may enhance physical health and physiological functions as well as reduce the risk of cardiovascular diseases and cancers [2]. Of the total tea production globally, black tea and green tea account for 78% and 20%, respectively. Black tea is mainly consumed in Western countries and in some Asian countries [3][4][5]. The biological activity of black tea is related to its chemical profile [3][4][5]. The typical compounds of black tea include catechins, theaflavins, amino acids and alkaloids [4]. The major polyphenolic components are catechins, theaflavins and thearubigins. Black tea extract products with an ultrahigh content of theaflavins have been marketed as dietary supplements [4].
The antioxidant capacity of black tea is associated with brewing conditions, including the infusion temperature, the amount of stirring and straining, and the particle size of the tea leaves [6,7]. Methods of infusing black tea vary worldwide. For example, infusion of black tea in China involves brewing black tea leaves with hot water at 100 • C for 20-40 s and repeating this infusion several times. In Ireland, 2 of 10 Canada, and the United Kingdom, black tea is mostly prepared using boiling water and consumed with milk and often sugar. A black tea infusion made with a cold steeping temperature (4 or 25 • C) for 2 h has become popular in Taiwan [8,9]. Determining the optimal method of infusing black tea is crucial to achieving high antioxidant activity. A previous paper reported that the total phenolic content of black tea leaves infused at 4 • C for 24 h was lower than those infused at 90 • C for 6 min, and this result correlated with the 1,1-Diphenyl-2-picrylhydrazyl (DPPH) scavenging activity [10]. Black tea produced using a short-duration, hot water extraction method exhibited the highest antioxidant activity. This result concurred with reports by Khokhar and Magnusdottir [6], Venditti et al. [8], and Hajiaghaalipour et al. [9]. However, few reports have discussed the effects of the water-tea leaves ratio on the antioxidant activity of black tea.
A low storage temperature of green tea could considerably extend catechin half-life, and catechins did not undergo significant degradation during a 6-month storage period at 4 • C [11,12]. The result of optimum storage of green tea at 4 • C concurred with another report in which no significant degradation of catechin or caffeine was observed during an 8-week storage period [13]. Moreover, no differences in concentrations were observed for the predominant catechins of green tea over the first 6 weeks of storage at 3 • C in different packages [14]. However, at 25 • C, the (−)-epi-catechin gallate and gallocatechin gallate of green tea decreased rapidly to zero or close to zero after 30 days; (−)-epigallocatechin decreased by 43% after 60 days of storage, and (−)-epigallocatechin gallate decreased by 68% after 90 days [11]. Both the DPPH scavenging ability and total phenolic content of green tea decreased considerably at 25 and 50 • C after a 3-month storage period, and they declined further at 50 • C [15].
Jiménez-Zamora et al. [15] also reported a decrease in the DPPH scavenging activity of black tea after 3 months, but the total phenolic content remained almost constant at 25 • C, even after 6 months of storage. Besides this example, few studies have reported the antioxidant properties of black tea following short-term storage. To understand the optimal infusion conditions and storage time of black tea before use, this study investigated the effects of the water-tea leaves weight ratio, infusion temperature, short-term storage, and storage temperature on the antioxidant activity of black tea infusion.

Materials
Pali (referring to origin) black tea leaves were obtained from tea trees planted on the campus of the National United University, Miaoli, Taiwan. DPPH, caffeine, epigallocatechin, epicatechin gallate, epigallocatechin gallate, and gallocatechin gallate were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Folin-Denis reagent and gallic acid were obtained from Fluka (Neu-Ulm, Germany). Sodium carbonate was obtained from Riedel-de Haën Chemical Co. (Seelze, Germany). Methanol was obtained from Macron Chemical Co. (Washington, DC, USA).

Infusion Temperature
A total of 0.4 g of black tea leaves was mixed with 40 mL of water at varying temperatures (60, 70, 80, 90, and 100 • C) for 1 min. Then, the black tea infusions were filtered through 0.47-µm filter paper and cooled to room temperature rapidly on ice. After that, the black tea infusion was diluted to different concentrations as test samples.

Storage Time and Temperature
A total of 1.2 g of black tea leaves was added to 120 mL of boiling water and infused for 1 min. Next, the black tea infusions were filtered through 0.47-µm filter paper and cooled to room temperature rapidly on ice, and then placed in a dark incubator at different temperatures (4, 9, and 25 • C) for 15 days. Each sample's DPPH scavenging activity and total phenolic content was calculated every 3 days.

Determination of Total Phenolic Content
The total phenolic content of the tea sample was determined using the Folin-Denis method devised in previous reports [16][17][18]. Every black tea sample was mixed with 0.8 mL of Folin-Denis reagent for 3 min. Then, 0.8 mL of 10% sodium carbonate and 1.6 mL of water were added to induce a reaction. The mixture was shaken and kept in the dark at room temperature for 1 h. The absorbance was measured at 700 nm. Gallic acid was used as a reference standard, and the results were expressed as milligrams of gallic acid equivalent (GAE) per gram of black tea leaf. The measured calibration curve of gallic acid was y = 0.0171 x + 0.0999 (R 2 = 0.9978) in the range 0-65 µg/mL, where x and y were concentration and absorbance, respectively.

High Performance Liquid Chromatography (HPLC) Analysis
The Waters 600 HPLC system (Waters, Milford, MA, USA) with Empower 2 Pro Software (Waters, Milford, MA, USA) had an integrated controller, a quaternary pump, a column temperature controller, an autoinjector, and a photodiode array detector. The mobile phase consisted of a mixture of A (0.085% phosphoric acid), B (acetonitrile), and C (water) [18]. The percentage of the mobile phase was as follows

Determination of DPPH Scavenging Activity
The DPPH scavenging activity of the tea sample on a DPPH radical was estimated according to the procedure developed in previous studies [16,17,19]. The stock solution was prepared by dissolving 80-mg DPPH with 100 mL of methanol. A 2-mL aliquot tea sample was mixed with 2 mL of DPPH solution. The mixture was shaken and incubated for 30 min in the dark at room temperature. The absorbance was measured spectrophotometrically at 517 nm. The DPPH scavenging activity was calculated using the following equation, and the antioxidant activity was evaluated based on IC 50 (µg/mL) value, which was determined from the calibration curve inhibition of DPPH free radical (%) vs. infusion concentration (µg/mL).

Statistical Analysis
To ensure statistical representation, all of the experiments were performed at least in triplicate, and means between the treatment levels were compared using the SAS software (version 9.4, SAS Institute, Cary, NC, USA). Statistical analyses were based on the analysis of variance (ANOVA). If the treatment was significant (p < 0.05), the treatment mean was compared with the Tukey's test. The correlation among the variables was measured using the CORR procedure in SAS.

Statistical Analysis
To ensure statistical representation, all of the experiments were performed at least in triplicate, and means between the treatment levels were compared using the SAS software (version 9.4, SAS Institute, Cary, NC, USA). Statistical analyses were based on the analysis of variance (ANOVA). If the treatment was significant (p < 0.05), the treatment mean was compared with the Tukey's test. The correlation among the variables was measured using the CORR procedure in SAS.

Effect of the Water-Tea Leaves Weight Ratio
Appl. Sci. 2020, 10, x 5 of 11 with weight ratios from 25 to 500, as shown in Figure 2. The IC50 for DPPH scavenging ability ranged from 30.4 ± 3.3 to 26.6 ± 3.9 μg/mL. Therefore, the black tea infusion was fixed with a 100:1 weight ratio for determining the antioxidant capacity in this study to allow comparison with previous reports [6,8,[20][21][22].

Effect of Infusion Temperature
The antioxidant capacity for DPPH scavenging activity and the total phenols of black tea are dependent on infusion temperature. Figure 3 confirms that the DPPH scavenging ability of black tea was affected by infusion temperature. A higher DPPH scavenging ability was noted at higher temperatures, revealing that the scavenging activity for the DPPH radical of black tea prepared with boiling water was better than the others at the same tea concentration. However, Figure 3 indicates that the DPPH scavenging activity increased with tea concentration regardless of infusion temperature. Furthermore, the effects of tea concentration on the DPPH scavenging activity were more prominent at a higher infusion temperature. According to Figure 3, the IC50 values were 100.0 ± 13.7, 63.0 ± 4.2, 46.2 ± 5.5, 35.3 ± 5.2, and 28.4 ± 4.8 μg/mL for temperatures from 60 to 100 °C (Figure 4). The total phenolic contents for 60 to 100 °C were 50.4 ± 5.2, 72.6 ± 7.9, 104.1 ± 7.0, 135.2 ± 8.5, and 178.6 ± 16.4 mg GAE/g dry leaf, respectively (Table 1). An increase in the DPPH scavenging ability and the total phenolic content associated with an increase in the infusion temperature concurred with the findings in previous reports [6,8,10,[23][24][25]. Thus, the infusion temperature of black tea is critical in the preparation process when aiming for high antioxidant capacity. Black tea with lower DPPH IC50 had higher total phenols, and the IC50 of DPPH scavenging ability showed a negative correlation with the value of the total phenolic content, as seen in Figure 5. This result concurred with previous reports [23,[26][27][28]. Theaflavins, a type of phenolic compound in black tea, may contribute to strong antioxidant properties that affect the DPPH scavenging activity and total phenols [23,[29][30][31][32]. However, the therapeutic activities of black tea are attributed not only to the antioxidant activity of its components but also to the ability of bioactive molecules to interact with several biological targets [33,34]. At a high temperature, chemical degradation can occur, changing the structure of temperature-sensitive molecules such as (-)-epigallocatechin-3-gallate.
Wide variations are noted among different kinds of black tea in terms of DPPH scavenging ability [9,22,[35][36][37][38][39] and total phenolic content [6,20,32,37,40]; these are highly dependent on the genetic background and growing conditions (such as temperature, nitrogen availability, and light conditions) of the plant material. The IC50 of black tea in this study was 28.4 ± 4.8 μg/mL, and it falls

Effect of Infusion Temperature
The antioxidant capacity for DPPH scavenging activity and the total phenols of black tea are dependent on infusion temperature. Figure 3 confirms that the DPPH scavenging ability of black tea was affected by infusion temperature. A higher DPPH scavenging ability was noted at higher temperatures, revealing that the scavenging activity for the DPPH radical of black tea prepared with boiling water was better than the others at the same tea concentration. However, Figure 3 indicates that the DPPH scavenging activity increased with tea concentration regardless of infusion temperature. Furthermore, the effects of tea concentration on the DPPH scavenging activity were more prominent at a higher infusion temperature.
Appl. Sci. 2020, 10, x 6 of 11 within the range of 12.7-118.7 μg/mL found by other authors [35,39]. The total phenolic content in this study was 178.6 ± 16.4 mg GAE/g dry leaf, and it was in the range from 50.9 ± 4.1 mg GAE/g dry leaf [32] to 274.3 ± 0.0 mg GAE/g dry leaf [20]. Different brewing conditions, including infusion temperature and time, may affect the DPPH IC50 and total phenolic content. Pastoriza et al. [7] reported that an increase in the infusion temperature increased antioxidant activity, and Ramalho et al. [20] demonstrated that the total phenolic content increased with infusion time. According to Figure 3, the IC 50 values were 100.0 ± 13.7, 63.0 ± 4.2, 46.2 ± 5.5, 35.3 ± 5.2, and 28.4 ± 4.8 µg/mL for temperatures from 60 to 100 • C (Figure 4). The total phenolic contents for 60 to 100 • C were 50.4 ± 5.2, 72.6 ± 7.9, 104.1 ± 7.0, 135.2 ± 8.5, and 178.6 ± 16.4 mg GAE/g dry leaf, respectively (Table 1). An increase in the DPPH scavenging ability and the total phenolic content associated with an increase in the infusion temperature concurred with the findings in previous reports [6,8,10,[23][24][25]. Thus, the infusion temperature of black tea is critical in the preparation process when aiming for high antioxidant capacity. Black tea with lower DPPH IC 50 had higher total phenols, and the IC 50 of DPPH scavenging ability showed a negative correlation with the value of the total phenolic content, as seen in Figure 5. This result concurred with previous reports [23,[26][27][28]. Theaflavins, a type of phenolic compound in black tea, may contribute to strong antioxidant properties that affect the DPPH scavenging activity and total phenols [23,[29][30][31][32]. However, the therapeutic activities of black tea are attributed not only to the antioxidant activity of its components but also to the ability of bioactive molecules to interact with several biological targets [5,33]. At a high temperature, chemical degradation can occur, changing the structure of temperature-sensitive molecules such as (−)-epigallocatechin-3-gallate. temperature and time, may affect the DPPH IC50 and total phenolic content. Pastoriza et al. [7] reported that an increase in the infusion temperature increased antioxidant activity, and Ramalho et al. [20] demonstrated that the total phenolic content increased with infusion time.

Effect of Storage Time and Storage Temperature
The storage time and storage temperature have a great influence on determining the antioxidant activity and total phenolic content of black tea. Figures 6 and 7 show the DPPH scavenging activity and total phenolic content of black tea at different temperatures for different storage times, respectively. As shown in Figure 6, the difference between the two means of the DPPH scavenging ability is not statistically significant at 4 °C and 25 °C. In addition, at 9 °C, the trend was similar for 0-day to 15-day storage periods, except for the 9-day storage period, which was lower than the other storage times. These results indicated that the DPPH scavenging activity of black tea did not decline within 15 days even though it was kept at 25 °C. No notable difference was observed in the scavenging activity against the DPPH radical during 15-day storage.  Wide variations are noted among different kinds of black tea in terms of DPPH scavenging ability [9,22,[34][35][36][37][38] and total phenolic content [6,20,32,36,39]; these are highly dependent on the genetic background and growing conditions (such as temperature, nitrogen availability, and light conditions) of the plant material. The IC 50 of black tea in this study was 28.4 ± 4.8 µg/mL, and it falls within the range of 12.7-118.7 µg/mL found by other authors [34,38]. The total phenolic content in this study was 178.6 ± 16.4 mg GAE/g dry leaf, and it was in the range from 50.9 ± 4.1 mg GAE/g dry leaf [32] to 274.3 ± 0.0 mg GAE/g dry leaf [20]. Different brewing conditions, including infusion temperature and time, may affect the DPPH IC 50 and total phenolic content. Pastoriza et al. [7] reported that an increase in the infusion temperature increased antioxidant activity, and Ramalho et al. [20] demonstrated that the total phenolic content increased with infusion time.

Effect of Storage Time and Storage Temperature
The storage time and storage temperature have a great influence on determining the antioxidant activity and total phenolic content of black tea. Figures 6 and 7 show the DPPH scavenging activity and total phenolic content of black tea at different temperatures for different storage times, respectively. As shown in Figure 6, the difference between the two means of the DPPH scavenging ability is not statistically significant at 4 • C and 25 • C. In addition, at 9 • C, the trend was similar for 0-day to 15-day storage periods, except for the 9-day storage period, which was lower than the other storage times. These results indicated that the DPPH scavenging activity of black tea did not decline within 15 days even though it was kept at 25 • C. No notable difference was observed in the scavenging activity against the DPPH radical during 15-day storage. Jiménez-Zamora et al. [15] reported that the DPPH scavenging activity of black tea exhibited considerable decline at 25 • C after 3 months; however, no perceptible difference was observed in the DPPH scavenging activity among the three storage temperatures in this study because of the relatively short 15-day storage period.   In terms of the total phenolic content, no significant difference was observed from 0-day to 15-day storage periods at 4 • C and 25 • C, as shown in Figure 7. As with the result of the DPPH scavenging ability, there was a similar trend for the total phenolic content from 0-day to 15-day storage at 9 • C, except 12-day storage, which was lower than the other storage times.
In summary, the black tea exhibited similar scavenging ability and total phenolic content at 4, 9, and 25 • C regardless of storage time in this study. Although at 9 • C, the performance of DPPH scavenging ability and total phenolic content was lower than the other storage times at 9-day and 12-day, respectively. The situation might have resulted from sampling and the significant level of p < 0.05. Moreover, the performance of the total phenolic content in this study concurred with a previous report indicating that the total phenolic content of black tea remained almost constant after storage at 25 • C or 50 • C for 6 months [15].

Conclusions
This study investigated the effects of infusion conditions and short-term storage on the antioxidant activity and total phenolic content of black tea. The results indicated that the water-tea leaves weight ratio from 10 to 500 did not have a significant impact on DPPH scavenging ability. However, the DPPH scavenging ability and total phenolic content increased with infusion temperature. The IC 50 of DPPH scavenging activity and the total phenolic content had a negative correlation. Moreover, the results indicated that the antioxidant capacity and total phenolic content of black tea did not exhibit perceptible changes during 15-day storage. Both the DPPH scavenging ability and the total phenolic content of black tea had negligible variation at 4, 9, and 25 • C storage temperatures.