Total Phenolic Contents and Antioxidant Capacities of Herbal and Tea Infusions

In order to supply new information on the antioxidant function of selected beverages for nutritionists and the general public, total phenolic contents of 51 kinds of herbal and tea infusions made in China were measured by the Folin-Ciocalteu method, and their antioxidant capacities were evaluated using ferric reducing antioxidant power (FRAP) and Trolox equivalent antioxidant capacity (TEAC) assays. A significant correlation between FRAP and TEAC values suggested that antioxidant components in these beverages were capable of reducing oxidants and scavenging free radicals. The high correlation between antioxidant capacities and total phenolic contents indicated that phenolic compounds could be one of the main components responsible for antioxidant activities of these beverages. Generally, these beverages had high antioxidant capacities and total phenolic contents, and could be important dietary sources of antioxidant phenolics for prevention of diseases caused by oxidative stress.


Introduction
Reactive oxygen species may cause a broad spectrum of damage to biological systems, and oxidative stress plays an important role in many chronic and degenerative diseases, such as cardiovascular diseases, cancer, diabetes mellitus and ageing [1][2][3]. Dietary supplements of antioxidants have become popular to enhance the body"s antioxidant defenses. Natural antioxidants may come from vegetables, fruits and beverages [4][5][6][7][8]. As an important category of phytochemicals, phenolic compounds universally exist in plants. They have attracted increasing attention as potential agents for preventing and treating many oxidative stress-related diseases. At present, there is considerable interest in determining the total phenolic contents and antioxidant capacities of diets. Many herbs and tea have been used to make infusions, and the term "rich in antioxidants" is often used to describe such infusions. However, it usually lacks scientific evidence.
A special kind of herbal infusion is called cool tea (Liang cha in Chinese), which originated from South China. The cool tea is made from some kinds of herbs, and has been drunk as a beverage for hundreds of years. The vendition of cool tea has been from South China to the whole of China, and from China to about 20 countries around the world, such as the United States of America, Canada, United Kingdom, France and Germany. The cool tea has the efficacies of clearing away heat, detoxification, dewetting, moistening lung and stopping thirsty. Similarly, tea has been widely drunk in China for thousands of years. Tea consumption is associated with reduced risks of cardiovascular disease and cancer, and health effects of tea come from its high content of phytochemicals with antioxidant activity [9]. Traditionally, tea is infused only before drinking. Nowadays, a variety of tea infusions have been produced and sold. However, total phenolic contents and antioxidant capacities of herbal and tea infusions made in China have not been evaluated.
The aim of this study was to systematically evaluate total phenolic contents and antioxidant capacities of 51 kinds of herbal and tea infusions made in China, to investigate the relationship between antioxidant capacity and total phenolic content, and to supply new information on the antioxidant function of these beverages for nutritionists and the general public.

Total Phenolic Content of 51 Infusions
Fifty-one kinds of commercial herbal and tea infusions were obtained from markets in Guangzhou, which represent main categories of the infusions made in China (Table 1). The total phenolic contents of 51 infusions were estimated using the Folin-Ciocalteu method, which relies on the transfer of electrons from phenolic compounds to the Folin-Ciocalteu reagent in alkaline medium, and is a simple and rapid method [10][11][12][13]. As shown in Table 2, the total phenolic contents varied from 0.032 ± 0.001 to 1.395 ± 0.068 g gallic acid equivalent (g GAE)/L with the difference of 44-fold, and the mean value was 0.480 g GAE/L for 51 infusions. Ping An Tang li yan cha had the highest total phenolic content (1.395 ± 0.068 g GAE/L), but Nian ci an run (chun cui hong se ting zhuang) showed the lowest total phenolic content (0.032 ± 0.001 g GAE/L) among the tested infusions.  For the herbal infusions, the total phenolic contents varied from 0.032 ± 0.001 to 1.395 ± 0.068 g GAE/L with the difference of 44-fold, and the mean value was 0.410 g GAE/L for the 28 herbal infusions ( Table 2). Ping An Tang li yan cha (1.395 ± 0.068 g GAE/L) had the highest total phenolic content, followed by Ping An Tang shi gan cha (1.192 ± 0.011 g GAE/L), Qing Xin Tang jiang huo wang (1.028 ± 0.055 g GAE/L), Qing Xin Tang er shi si wei (1.007 ± 0.039 g GAE/L), Qing Xin Tang zhi ke hua tan tang (0.909 ± 0.037 g GAE/L), Qing Xin Tang hou zheng tang (0.875 ± 0.019 g GAE/L) and Qing Xin Tang gan mao cha (0.844 ± 0.013 g GAE/L). Nian ci an run (chun cui hong se ting zhuang) had the lowest total phenolic content (0.032 ± 0.001 g GAE/L) among the tested herbal infusions.
When the total phenolic contents of these infusions were compared with those of Serbian white wines and Korean wines reported in the literature [8,14], there was no statistical difference (p  0.05), which indicated that these infusions could contribute the same health benefit as those wines in terms of polyphenols. Taking the negative health effect of alcohol in those wines into account, these infusions would have better health benefits for people [15].

Ferric Reducing Antioxidant Power of 51 Infusions
The ferric reducing antioxidant power (FRAP) assay was used to evaluate antioxidant capacities of the 51 infusions. The FRAP assay is based on the capacity of antioxidants to reduce ferric(III) ions to ferrous(II) ions [16,17], which is a simple and widely used method for the evaluation of antioxidant capacity [18][19][20]. The FRAP values of 51 infusions are shown in Table 2. In general, these infusions had very high antioxidant capacities. As indicated in Table 2  The correlation between antioxidant capacities and the total phenolic content of the 51 infusions is shown in Figure 1. The result showed a positive linear correlation between the antioxidant capacities and total phenolic content (R 2 = 0.7929), which indicated that phenolic compounds could be one of the main components responsible for antioxidant activities of these beverages.

ABTS •+ Radical Scavenging Activity of 51 Infusions
The antioxidant capacities of samples may be influenced by lots of factors, such as test system, and cannot be fully described by one single method. Most natural antioxidants are multifunctional. A reliable antioxidant evaluation protocol requires different antioxidant activity assessments to be performed to take into account various mechanisms of antioxidant action [21]. Therefore, the Trolox equivalent antioxidant capacity (TEAC) assay was used to evaluate free radical scavenging capacities of 51 infusions. The TEAC assay is based on the ability of antioxidants to scavenge ABTS •+ radicals. It can measure antioxidant capacities of lipophilic and hydrophilic components in a sample, and is a method usually used for the evaluation of antioxidant capacity [22]. The TEAC values of 51 infusions are given in Table 2. Generally, these infusions had very strong free radical scavenging ability. As seen from Table 2, the TEAC values varied from 0.250 ± 0.006 to 19.296 ± 0.692 mol Trolox/L with the difference of 77-fold, and the mean value was 5.074 mol Trolox/L for the 51 infusions. Ping An Tang li yan cha had the highest free radical scavenging capacity (19.296 ± 0.692 mol Trolox/L), and Nian ci an run (chun cui hong se ting zhuang) showed the lowest free radical scavenging capacity (0.250 ± 0.006 mol Trolox/L) among the tested infusions.
The correlation between antioxidant capacities and the total phenolic content of the 51 infusions is shown in Figure 2. The result showed a positive linear correlation between the antioxidant capacities and total phenolic content (R 2 = 0.8043), which indicated that phenolic compounds could be one of the main components responsible for antioxidant activities of these infusions. In addition, the correlation between total antioxidant capacities obtained from FRAP and TEAC assays are shown in Figure 3. The results show a positive linear correlation (R 2 = 0.865) between them, which suggested that antioxidant components in these infusions could reduce oxidants (such as ferric ions) and scavenge free radicals. This result is in agreement with those of medicinal plants and wild fruits [23,24]. Maybe, this was because FRAP and TEAC assays are all electron transfer-based methods [25].  [26], and are shown in Table 3.
Because of their high antioxidant activities, it could be speculated that these infusions will be beneficial for the diseases caused by oxidative stress. For total phenolic content, FRAP value and TEAC value, the differences between herbal infusions and tea infusions, between bitter herbal infusions and sweet herbal infusions as well as between green tea infusions and black tea infusions were significant, but the difference between herbal infusions produced by Ping An Tang and those by Qing Xin Tang was not significant (Table 4). In addition, antioxidant capacities of tea infusions, bitter herbal infusions and green tea infusion were higher than those of herbal infusions, sweet herbal infusions and black tea infusion, respectively. Polyphenols are the most important antioxidants in the tea, and catechins are the major phenolic compounds in green tea. Black tea belongs to fermented tea, and its content of catechins was reduced to 20% of that in green tea [27]. Therefore, green tea usually had higher antioxidant capacity than black tea, which resulted in that green tea infusion might have higher antioxidant capacity than black tea infusion. The bitter herbal infusions are usually made from the medicinal plants under the "heat-clearing" category according to the classification of Chinese medicinal plants [12], or those used for prevention and treatment of cold, flu and cough [20], most of which showed the high antioxidant capacities [12,20], while sweet herbal infusions often contain fewer medicinal plants compared with bitter herbal infusions, resulting in lower antioxidant capacities.

Sample Preparation
Twenty-eight kinds of herbal infusions and twenty-three kinds of tea infusions were bought from local markets (Table 1), which are commercial preparations and in the form of tin with aquatic solution. The samples were kept in the refrigerator at 4 °C until analysis. The various infusions were centrifuged at 3,500 rpm for 30 min, and the resulting supernatants were used for the determination of total phenolic contents and antioxidant capacities.

Determination of Total Phenolic Content
Total phenolic content of the infusion was determined according to the literature [10,28]. Briefly, 0.50 mL of the diluted infusion (a dilution factor of 10-times with water) was added into 2.5 mL of 1:10 diluted Folin-Ciocalteu reagent. After 4 min, 2 mL of saturated sodium carbonate solution (about 75 g/L) was added. The absorbance of the mixture was measured at 760 nm after incubation for 2 h at room temperature. Gallic acid was used as a reference standard and the results were expressed as gram gallic acid equivalent (g GAE)/L of infusion.

Ferric-Reducing Antioxidant Power (FRAP) Assay
The FRAP assay of the infusion was carried out according to the procedure described in the literature [16,17]. Briefly, the FRAP reagent was prepared from sodium acetate buffer (300 mM, pH 3.6), 10 mM TPTZ solution (40 mM HCl as solvent) and 20 mM iron (III) chloride solution in a volume ratio of 10:1:1, respectively. The FRAP reagent was prepared fresh daily and warmed to 37 °C in a water bath before use. One hundred microliters of the diluted infusion was added to 3 mL of the FRAP reagent. After 4 min, the absorbance of the mixture was measured at 593 nm using a Shimadzu UV-2450 ultraviolet-visible spectrophotometer (Japan). The standard curve was constructed using FeSO4 solution, and the results were expressed as mol Fe(II)/L of infusion.

Trolox Equivalent Antioxidant Capacity (TEAC) Assay
The TEAC assay of the infusion was carried out according to the method established in the literature [22]. Briefly, the ABTS •+ stock solution was prepared from 7 mM ABTS and 2.45 mM potassium persulfate in a volume ratio of 1:1, and then incubated in the dark for 16 h at room temperature, which should be used within 2 days. The ABTS + working solution was prepared by diluting the stock solution with ethanol to an absorbance of 0.70 ± 0.05 at 734 nm. All infusions were aptly diluted to provide 20-80% inhibition of the blank absorbance. One hundred microliters of the diluted infusion was mixed with 3.8 mL ABTS + working solution. After 6 min of incubation at room temperature, the absorbance of the mixture was measured at 734 nm, and the percent of inhibition of absorbance was calculated. Trolox solution was used as a reference standard, and the results were expressed as mol Trolox/L of infusion.

Statistical Analysis
All the experiments were carried out in triplicate, and the results were expressed as mean ± SD (standard deviation). Statistical analysis was performed using SPSS 13.0 and Excel 2003. The p value less than 0.05 was considered to be statistically significant.

Conclusions
The total phenolic contents and antioxidant capacities of 51 kinds of herbal and tea infusions made in China were evaluated. A high correlation between antioxidant capacity and total phenolic content indicated that phenolic compounds could be one of the main components responsible for antioxidant activities of these beverages. A significant correlation between the FRAP value and the TEAC value suggested that antioxidant components in these beverages were capable of reducing oxidants and scavenging free radicals. Generally, these beverages had high total phenolic contents and antioxidant capacities. These beverages could be important dietary sources of antioxidant phenolics for prevention of diseases caused by oxidative stress. This study supplied new information on the antioxidant function of these beverages for consumers, nutritionists and food policy makers. In the future, health effects of these beverages for the consumers should be explored by the epidemiologic method.