Chemical composition and α-glucosidase inhibition As shown in Table S1, the contents of tea polysaccharides, catechin, and EC were higher in Se- BT than in BT, and BT had higher contents of tea polyphenols and total nitrogen, with significant differences. These results suggest that both Se- BT and BT have high nutrient contents and may play a role in regulating BG [1, 10]. In the α-glucosidase inhibition assay, Se-BT exhibited higher inhibition activity, which may be related to the high content of tea polysaccharides in Se-BT .
Effects of BT and Se-BT on hypoglycemic activity in vivo To evaluate the effects of Se-BT and BT on hyperglycemia, we established the hyperglycemic mouse model induced by a high-fat diet together with STZ. The BW of mouse was significantly decreased after five weeks of Se-BT intervention compared MC group, whereas BT did not play a notably role (Fig. 1A). Additionally, BT and Se-BT had similar effects in reducing BG (Fig. 1B). Oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) show that the induction of high-fat diet and STZ led to the notably decrease of insulin sensitivity and glucose tolerance. As expected, BT and Se-BT could markedly change this trend (Fig. 1C-F). Further histological analysis (Figure S1) show that hyperglycemia led to a large number of vacuoles in the liver tissue, and both hepatocytes and adipocytes appeared enlarged and swollen. Accordingly, BT and Se-BT significantly ameliorated these damages. Likewise, we also found that Se-BT showed a better improvement effect than BT. Previous research  show a significant improvement in OGTT in diabetic patients after ingestion of sucrose drinks containing BT polyphenols, but no significant difference in insulin tolerance. It may not be the tea polyphenols but other components that improve IR. Therefore, we speculate that the difference in the content of tea polyphenols and polysaccharides may be one of the factors for the distinction in hypoglycemic activity of BT and Se-BT. Additionally, our previous study shows that the binding of Se to polysaccharides led to the change in the structure of polysaccharides, which would affect their hypoglycemic activity . Previous research  reveals that Pluchea indica (L.) Less tea could inhibit lipid and carbohydrate accumulation in adipocytes, decrease perigonadal fat pad weight and adipocyte size, indicating that the hypoglycemic effect of BT and Se-BT may be related to lipid metabolism.
Dyslipidemia and metabolic disorders often occur in patients with hyperglycemia . Accordingly, we evaluated serum factor levels of lipid metabolism and oxidative stress-related. Figure 2A-F shows that hyperglycemia significantly induced the decreased levels of high density liptein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and the activity of superoxide dismutase (SOD), and considerably enhanced the levels of total cholesterol (TC) and triglycerides (TG) and glycosylated serum protein (GSP). As expected, Se-BT and BT treatments noticeably decreased the TG level and significantly increased HDL-C level. Differently, Se-BT notably decreased TC and LDL-C levels, while BT significantly reduced GSP levels. However, the effect on SOD activity was limited. Furthermore, IR can cause hepatic damage and inflammation . To reflect the degree of liver damage caused by hyperglycemia, we measured the levels of AST and ALT in serum. The results showed that AST was increased in the MC group, but not as significant as ALT (Fig. 2G-H), indicating the formed hepatic damage and inflammation . As expected, Se-BT and BT treatments reversed this trend. Excessive sugar and fat diets can cause disruptions in lipid and glucose metabolism, finally the hyperglycemia . TC and TG reflect the degree of lipid metabolism, and HDL-C and LDL-C are involved in the mediation of cholesterol transport . The level of GSP indicates the average BG concentration over a period of time and is an important indicator of BG level . Our previous study showed that Liubao tea intervention regulated TC, TG, HDL-C, LDL-C, AST and ALT levels, and then alleviated metabolic disorders caused by hyperglycemia . Gao et al.  found that BT decreased TG and ALT levels but had little effects on TC, HDL-C, and LDL-C levels. This may be related to the different sources, processing methods and distribution of active compounds in tea. Accordingly, BT and Se-BT could alleviate hyperglycemia by improving the pro-inflammation and regulating glucose and lipid metabolism.
In addition to key cytokines in serum, we measured some BG-related mRNA levels. As can be seen in Fig. 2I-N, the mRNA level of PTEN of mice in MC group was significantly increased compared with NC group, while the mRNA levels of IRS-1, GLUT-2, Akt, PDX-1 and PI3K were considerably declined. As expected, the intervention of BT or Se-BT could significantly prevent this phenomenon. Notably, Se-BT is more effective in mRNA levels of PTEN, PI3K and Akt. The PI3K/Akt pathway is closely associated with lipid metabolism and glucose homeostasis, and further T2D-associated metabolic syndrome is also closely related to its imbalanced expression . IRS-1, located upstream of the PI3K-Akt pathway, is an insulin receptor substrate and shows a great significance in insulin signal transduction . However, IR can lead to a blocked binding of insulin to IRS-1 on cell membrane, thus inhibiting the failed insulin delivery and the decrease in IRS-1 expression . For PI3K-Akt transportation, PTEN and PDX-1, located in the core region of the pathway, can inhibit and activate insulin-signaling transportation, respectively [19, 21]. Notably, PTEN inhibits the activity of Akt . An experiment  in diabetic mice shows that tea polysaccharides could activate the PI3K/Akt signaling pathway, transport GLUT4 to the membrane surface and enhance glucose uptake. In addition, Liubao tea could activate the PI3K-Akt-PPARs-GLUT2 cascade signaling pathway to improve metabolic disorders and IR . Accordingly, we hypothesize that BT could regulate glucose metabolism and IR by activating PI3K/Akt signaling pathway.
Effects of BT and Se-BT on the gut microbial The diversity and stability of the intestinal flora can be disrupted by excessive ingestion of sugar and fat, resulting in intestinal inflammation, IR, and eventually, type 2 diabetes . The Chao index, which indicates the bacterial richness, was significantly increased by the interventions of BT and Se-BT (Fig. 3A). Besides, the Shannon index, which indicates species and community richness, was markedly lower in MC group than in the BT and Se-BT groups (Fig. 3B). In the PLS-DA analysis (beta-diversity), MC was obviously different compared to the NC, BT and Se-BT intervention groups. And BT group more closely resembling the NC group (Fig. 3C). This suggests the effectiveness of BT and Se-BT in preventing hyperglycemia and the strong link between the regulation of gut microbiota and symptom improvement.
To further confirm the importance of changing dietary habits in regulating hyperglycemia, we analyzed the abundance of dominant species at the genus and phylum levels. At the phylum level, the ratio of Firmicutes/Bacteroidetes was reduced after intervention with BT and Se-BT, and the effect of Se-BT was more pronounced (Figure S2). Many members of Firmicutes and Bacteroidetes can encode carbohydrate-active enzymes , thus we initially speculated that BT or Se-BT might enhance glucose metabolism by improving the abundance of Firmicutes and Bacteroidetes. At the genus level, the top 10 genera were Lactobacillus, Allobaculum, Unspecified Clostridiales, Unspecified_S24_7, Turicibacter, Unspecified Lachnospiraceae, Bacteroides, Ruminococcus, Ruminococcus_1, Oscillospira (Fig. 3D-M). Allobaculum produced short-chain fatty acids (SCFAs) and alleviated diabetes and obesity by reducing the migration of endotoxins into the bloodstream . SCFA can affect host glucose metabolism by activating intestinal gluconeogenesis through a complementary mechanism, including acetic acid, propionic acid, butyric acid, etc. In mice with diet-induced obesity, SCFA supplementation improved IR . Similarly, in a study of glucose metabolism in diabetic rats, Lachnospiraceae was significantly decreased in treated rats and was significantly positively correlated with 2 h-glucose, isobutyric acid and isovaleric acid, and negatively correlated with butyric acid . The main fermentation products of bacteria are acetic acid, butyric acid and succinic acid, while the main fermentation products of lactic acid bacteria are lactic acid, etc . Ruminococcus_1 was significantly increased in patients with T2DM and was a conditionally pathogenic bacterium . Turicibacter is a genus associated with disorders of glucolipid metabolism and also with intestinal butyric acid, which increases insulin secretion and sensitivity and has significant anti-obesity and anti-inflammatory effect . Recent evidence  suggests that the abundance of Roseburia, Blautia, Allobaculum, Alistipes and Turicibacter was increased in mice fed wih a high-fat diet after Berberine intervention, and these microbiota showed a positive anti-inflammatory effect.
Based on Spearman correlation analysis (Fig. 4), 15 of the top 50 OTUs correlated with at least one parameter associated with hyperglycemia. Among them, Enterococcus, Klebsiella, Akkermansia, Sutterella, Prevotella, Paraprevotella, Weissella showed significant negative correlations with hyperglycemia-related parameters. While Ruminococcus, Coprobacillus, Odoribacter, Turicibacter, Allobaculum, Adlercreutzia, Clostridium showed significant positive correlation with hyperglycemia-related parameters. Coprobacillus is an important butyric acid producer with high abundance in obese animals or humans . Odoribacter is a common, short-chain fatty acid producing member . Both of them can affect intestinal function and mediate related intestinal diseases . This further proves that black tea extract may play a crucial role in alleviating hyperglycemia and IR by affecting the secretion of SCFA and altering the composition of gut microbes. However, it remains unclear how SCFA regulates glucose and lipid metabolism and alleviates hyperglycemia. Further research is needed to explore in hypoglycemic mechanism of BT.