The sensory evaluation of samples was evaluated according to the Chinese national standard procedure (GB/T 23776, 2018) (15): briefly, 3 g of tea leaves was steeped in 110 mL freshly boiled water for 5 min (of note, green tea was brewed for 4 min and dark tea for 2 min, after which the tea soup was poured out and brewed again for 5 min). Then, we can get the tea soup, and the tea residue was filtered out after brewing the infused leaves.
Biochemical components and sensory evaluation of the black tea with different extents of the fermentation. (a) Biochemical components in black tea fermented for 0, 2, 5, and 8 h. (b) 1) Heat map showing changes in biochemical components and sensory evaluation. The length of the rectangle and color intensities indicate the content. 2) The results of sensory evaluation in black tea with different fermentation times. As the degree of fermentation deepens, the color of the dry tea, the brewed tea soup, and the brewed tea leaves were gradually deepen.
Biochemical components and sensory evaluation of dark tea with different degrees of pile fermentation. (a) The content of biochemical components in dark tea fermented for 0, 10, 20, 30, 40, 50, and 60 days. (b) 1) Heat map showing changes in biochemical components and sensory evaluation. The length of the rectangle and color intensities indicate the content. 2) The results of sensory evaluation in dark tea with different fermentation times. As the degree of fermentation deepens, the color of the dry tea, the brewed tea soup, and the brewed tea leaves were gradually deepen.
Generally, the researches on uric acid-lowering effects of tea had focused on either a specific type of tea (mainly green tea) or a specific type of compound (mainly tea polyphenols and catechins). For example, Chen et al. (10) found that the polyphenols in green tea significantly reduced uric acid levels, and Jung et al. (31) demonstrated that green tea extract reversed hyperuricemia. Nevertheless, the uric acid-lowering effects of various tea made with different processing, and its components had not been systematically compared yet (32, 33). Therefore, in our study, we conducted a more comprehensive analysis of the effect of tea and its main bioactive compounds on reducing uric acid by inhibiting the production of uric acid at the biochemical and cellular levels.
For tea water extracts, using traditional six types of tea made from a single variety of Yinghong No. 9, we analyzed its effect on inhibiting the production of uric acid from the biochemical and cellular levels by targeting XOD. The results showed that all the six types of tea had a significant impact on it. Even the weakest dark tea also was similar to that of the XOD inhibitor allopurinol (34). Furthermore, we came to the conclusion that the lower the degree of fermentation, the better the effect of inhibiting the production of uric acid. In other words, the effect of green tea on inhibiting XOD activity was significantly higher than that of black tea also the other kinds of tea. Inconsistent with our results, Chuang et al. (3) reported that green tea extract had a slight uric-acid-lowering effect, whereas black tea significantly inhibited uric acid production (at the dose of 2 g/kg) in the kunming hyperuricemic mice. This may be caused by the following factors. First, the raw materials of black tea and green tea used in their research may come from different varieties, which leads to inconsistencies with our results. In our research, our samples are all made from Yinghong No. 9 tea plant varieties, and the same raw materials were used to make our conclusions more comparable. Second, in order to further verify the relationship between the effect of tea extract in inhibiting the production of uric acid and fermentation, we analyzed the effect of black tea and dark tea with different fermentation times on inhibiting uric acid production. In general, from the abundance of samples and the selection of single raw materials, our results may be more reliable.
For bioactive compounds in tea, accumulating evidence reported that the content of caffeine, theabrownins, and flavonoids was higher in the heavily fermented versus unfermented and/or lightly fermented teas from the same species, whereas total catechins are present at higher levels in the latter (4, 18). This was basically consistent with the law that the bioactive compounds reduced the production of uric acid in this paper. Then, we analyzed the effect of bioactive compounds on inhibiting the production of uric acid from the biochemical and cellular levels by targeting XOD. The results showed that gallic acid, tea polyphenols, and theaflavins had a significant effect on inhibiting the XOD activity, whereas tea polysaccharides, L-theanine, and caffeine had almost no effect on it. In particular, although theabrownins had no effect on inhibiting the XOD activity in the tube experiment, it had a significant effect on inhibiting XOD at the cellular level. According to the current research, tea polyphenols and theaflavins showed a significant effect on reducing uric acid production, which was consistent with previous studies (10, 13, 35). Regarding amino acids, the correlation analysis between the XOD activity and the component content of samples, free amino acids showed a significant correlation. However, L-theanine had almost no inhibitory effect at the cellular level. Free amino acids were a determinant of tea quality. Deb et al. reported that the content of free amino acids in a cup of green tea or black tea is around 6% (36), and L-theanine accounts for almost 50% of the total free amino acids (37). Therefore, we suspected that L-theanine may not be the key amino acid that inhibits the XOD activity. In addition, whether caffeine can lower uric acid was still controversial. Towiwat et al. (38) had shown that decaffeinated coffee can reduce serum uric acid levels in participants with hyperuricemia, while caffeinated coffee cannot. In addition, Park et al. (39) advocated that moderate coffee intake may have a primary preventive effect on hyperuricemia and gout in men and women. In this study, caffeine seemed almost no effect on inhibiting the XOD activity. Regarding theabrownins, as mentioned above, it showed a significant effect on reducing uric acid at the cellular level. Here, we put forward the hypothesis that theabrownins may be one of the important components for the remarkable effect of dark tea. Moreover, our research revealed the uric acid-lowering effect of L-theanine, theabrownins, and tea polysaccharide originally.
A double-blind, placebo-controlled, cross-over design study showed that consumption of a beverage containing green tea catechins, caffeine, and calcium increases 24-h energy expenditure by 4.6%, but the contribution of the individual ingredients could not be distinguished. It was suggested that such modifications were sufficient to prevent weight gain. It has been reported that the body weights of rats and their plasma triglyceride, cholesterol, and low-density lipoprotein cholesterol were significantly reduced by feedings of Oolong, black, and green tea leaves to the animals. In addition, the inhibition of growth and suppression of lipogenesis in MCF-7 breast cancer cells may be through down-regulation of fatty acid synthase gene expression in the nucleus and stimulation of cell energy expenditure in the mitochondria [107,108]. When fed to mice, EGCG purified from green tea decreased diet-induced obesity in mice by decreasing energy absorption and increasing fat oxidation . The increased and prolonged sympathetic stimulation of thermogenesis by the interaction between polyphenols and caffeine could be of value in assisting the management of obesity .
Total RNA was extracted from three samples in accordance with the instructions included with the Quick RNA isolation Kit (Aidlab, Beijing, China). RNA extraction was performed with RNase-free DNaseI (TaKaRa, Dalian, China) to eliminate genomic DNA contamination. The extracted RNA concentration of each sample was calculated using a Nanodrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). The first-strand cDNA of each sample was synthesized in accordance with the instructions included with the PrimeScript RT reagent kit (TaKaRa, Dalian, China). The synthetized cDNA of each sample was diluted 18 times and used as template for quantitative real-time PCR (qRT-PCR) amplification. The experiments were repeated as three independent biological samples.
The expression levels of genes involved in AsA biosynthetic and recycling pathways in tea leaves under different temperature treatments were detected to explore the effects of temperature on the expression profiles of the genes of interest (Fig. 5). Genes involved in the AsA biosynthetic and recycling pathways were identified by referring to the tea plant transcriptome database.
High or low temperatures have various effects on plant morphology. For example, low temperature helps prevent the postharvest weight loss of fruits during storage50. By contrast, high temperature accelerates the postharvest weight loss of sugarcane cultivars during storage51. In this study, we found that different temperature treatments resulted in the postharvest weight loss of tea leaves. Postharvest weight loss often accompanies postharvest physiological deterioration in sweet cherry52. Temperature elevations during storage might promote the physiological deterioration of broccoli buds53. High temperature could cause cellular dehydration in plants54. The cell membrane mediates the selective transport of ions and organic molecules from the external environment55 and receives temperature stress signals56. It also maintains the shape of the cell by anchoring the cytoskeleton. Cell membrane damage causes cellular dehydration, which might affect weight loss in tea leaves at high temperatures.
External factors like sunlight, temperature, relative humidity, oxidative stress, and pollution influence AsA distribution and levels in plants57,58. In this study, the effects of different temperature conditions on the AsA level and distribution in tea leaves were detected and analyzed. AsA levels in tea leaves under low-temperature treatment are higher than those in tea leaves under room- or high-temperature treatment. AsA levels in sea buckthorn leaves and stems decreased with increasing temperature59. Temperature stress could affect the contents of antioxidant and lipid peroxidation enzymes60. Because AsA is an antioxidant, its levels could also be influenced by temperature stress9. Cold stress increases the AsA content of pepper (Capsicum annuum L.)61. A similar finding was observed for Cistus clusii under field conditions and a Mediterranean climate62. 2b1af7f3a8