Wei Xu, Yang Zhou, Ling Lin, Dongyin Yuan, Yingqi Peng,Li Li, Wenjun Xiao,*, Zhihua Gong,*

a Key Laboratory of Tea Science of Ministry of Education, Hunan Agricultural University, Changsha 410128, China

b National Research Center of Engineering and Technology for Utilization of Botanical Functional Ingredients, Hunan Agricultural University, Changsha 410128, China

c Co-Innovation Center of Education Ministry for Utilization of Botanical Functional Ingredients, Changsha 410128, China

d Hunan Baojiachong Tea Farm Co.Ltd., Yiyang 413500, China

Keywords:

Black tea

Black brick tea (with fungal growth)

High blood glucose

Hypoglycemic effect

A B S T R A C T

Dark tea (containing Eurotium cristatum) and black tea have hypoglycemic effects.The black brick tea with fungal growth is obtained from black tea by adding E.cristatum, followed by steaming, pressing,fungal growth, and drying.However, the hypoglycemic effects of black brick tea are still unexplored.Here,we used black brick tea with fungal growth and black tea as raw materials to study their hypoglycemic effects in a hyperglycemic mice model.Both these types of black tea could lower the content of blood glucose and increase the content of hepatic glycogen by upregulating the proteins and mRNA expression of phosphatidylinositol-4,5-bisphosphate3-kinase, glycogen synthase, protein kinase B and phosphoinositidedependent protein kinase-1 and downregulating the protein and mRNA expression of glycogen synthase kinase 3β.These regulatory steps were followed by high activities of glutathione peroxidase and superoxide dismutase and low content of malondialdehyde.These teas can promote blood glucose transport and absorption by upregulating the protein and mRNA expression of insulin receptor substrate-1 and glucose transporter-2 and stimulate glycolysis by upregulating the protein and mRNA expression of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2.Our study suggests that the black brick tea was more effective than the black tea in terms of hypoglycemic.

1.Introduction

Hyperglycemia occurs when blood glucose levels are higher than 5.6 mmol/L in limosis, or higher than 7.8 mmol/L 2 h post meals, and is one of the main symptoms that characterize diabetes [1,2].Both genetic and environmental factors that lead to high blood glucose levels can cause disorders in the regulation of nerves and hormones.Several studies have shown that hyperglycemia is often accompanied by changes in the serum levels of superoxide dismutase (SOD),malondialdehyde (MDA), and glutathione peroxidase (GPx) [3,4].Oxidative stress is an important causing factor of hyperglycemia, and patients with hyperglycemia often show higher MDA activity and lower SOD and GPx activity [5].

Insulin plays an important role in hyperglycemia.Streptozotocin(STZ) can cause insufficient insulin secretion and high blood glucose levels by destroying islet β-cells [6].When insulin is insufficient,glucose transporters in the insulin-sensitive tissues cannot transport blood glucose to the appropriate organs and tissues for metabolism [7].Moreover, insufficient insulin levels cannot stimulate insulin receptors to combine with them.On the one hand, the phosphatidylinositol-4,5-bisphosphate3-kinase (PI3K)/AKT pathway is required for systemic metabolism insulin-dependent regulation; on the other hand, cellular metabolism is considered a major effector of insulin action [8,9].PI3K is not only an important type of lipid kinase, but also the first regulator of AKT activation.After PI3K activation, AKT is transported to the cell membrane and phosphorylated.Then, AKT phosphorylates glycogen synthase kinase 3β (GSK3β) and inhibits its activity.Insulin binding to the insulin receptor (INSR) leads to the phosphorylation of insulin receptor substrate-1 (IRS1) at the tyrosinebearing sites when the extracellular insulin signal is delivered to the cell via the insulin receptor [10].Phosphoinositide-dependent protein kinase-1 (PDK1) plays an important role in the AKT/PI3K signaling pathway by phosphorylating AKT [11].Moreover, the expression of glucose transporter-2 (GLUT2) can be regulated by various kinds of transcription factors, including activating protein-1, which can be phosphorylated and negatively regulated by GSK3β.

The number of diabetes cases is increasing due to lifestyle and diet changes [12,13].Currently, the drugs available for diabetes treatment are mainly western medicines (such as rosiglitazone,metformin, and acarbose) [14,15].However, their use is accompanied by many adverse effects such as liver and kidney damage [16].These medicines also cause an abrupt decrease in blood glucose levels.Therefore, it is necessary to search for natural, safe, and effective substitutes for such medicines.

Tea is a traditional healthy drink, and the activity of its functional ingredients in diabetes has been studied [17].A previous study has shown that epigallocatechin-3-gallate (EGCG) acts against insulin resistance in a Bmal1-dependent way by strengthening insulin signaling and eliminating oxidative stress, suggesting that EGCG may serve as a promising natural nutraceutical for the regulation of metabolic disorders related to circadian clocks [18].Meanwhile,Wu et al.[19]indicated that theabrownin in combination with swinging was found to significantly improve the serum lipid status and prevent obesity and insulin resistance in rats through the activation of circadian rhythms, protein kinase A, the adenosine monophosphateactivated protein kinase, and insulin signaling pathways.Tea has numerous active substances, such as polysaccharides, polyphenols,and pigments, which help to regulate the metabolism [20].Black tea(BT) belongs to the fermented tea class.One of the key processes for ensuring its quality is fermentation.During this process, the oxidation of polyphenols into theaflavins, thearubigins, and theabrownins form the foundation for BT quality characteristics.Moreover,theabrownins have high biological activity levels.Black brick tea with fungal growth (FBT) is a new kind of black tea product that is obtained by steaming, pressing, allowing fungal growth, and drying of BT.FBT not only has several biologically active substances, but is also better preserved.FBT uses BT as a raw material and increases the fungal growth process.Several reports have demonstrated the good hypoglycemic effects of BT andEurotium cristatum[21,22].Therefore, we speculated that FBT can lower blood glucose levels.To confirm this speculation, in this study, we established a hyperglycemic mouse model and explored the differences in hypoglycemic effects between FBT and BT.

2.Materials and methods

2.1 Experimental materials

Hunan Wuling Xiufeng Tea Co., Ltd.(Zhangjiajie, China)provided FBT and BT.BT was processed from one bud and three leaves of BiXiangZao via withering, simmering, fermenting, and drying.FBT was processed by steaming, pressing, allowing fungal growth, and drying of BT.After processing, FBT and BT samples weighed 600 g each.FBT and BT were extracted twice using water with a tea-to-water ratio of 1:15 during 30 min.Next, these materials were filtered and concentrated using a rotary evaporator at 55 °C to obtain a solid content of about 30%.FBT (147.70 g) and BT (143.50 g)freeze-dried powders were then obtained.

2.2 Chemicals and reagents

Metformin hydrochloride tablets were purchased from Squibb Pharmaceutical Co., Ltd.Shanghai, China.The primer was purchased from Sangon Biotech, Shanghai, China.STZ, EDTA, Tris, DEPC,APS, SDS, TEMED, Tween-20, acrylamide, glycine, methyl bisacrylamide, and ponceau were purchased from Sigma-Aldrich,Shanghai, China.The Reverse Transcription kit, Ultra SYBR Mixture,and DNA Marker were purchased from Kangwei Century, Beijing,China.SYBRGREEN I nucleic acid dye was purchased from Puboxin Bio, Beijing, China.TRIzol and SuperECL Plus hypersensitive luminescent liquid were purchased from Thermo Fisher, China.The EZNA Total RNA Kit I was purchased from Omega, GA, USA.The PrimeScript? RT Reagent Kit with gDNA Eraser (perfect real time)kit and TB GreenTM Premix Ex Taq II (Tli RNaseH Plus) kit were purchased from TaKaRa, Japan.Antibodies against β-actin (60008-1-Ig),PI3K (21890-1-AP), AKT (17609-1-AP), GSK-3β (22104-1-AP),PDK1 (20433-1-AP), GLUT2 (20436-1-AP), and IRS1 (16754-1-AP)were purchased from Proteintech (Rosemont, IL, USA).Antibodies against GS (ab134101) and PFKFB2 (ab181064) were purchased from Abcam (Cambridge, UK).

2.3 Animals and experimental design

All experimental procedures involving animals were performed in strict compliance with the Animal Care and Use Guidelines and Regulations of the Ethics Committee of Hunan Agricultural University (registry number: 015063506).Male SPF Kunming mice(8 weeks old) were purchased from Hunan SJA Laboratory Animal Co., Ltd.(Changsha, China) and housed in the Tea Institution of Hunan Agricultural University under controlled temperature ((25 ± 2) °C)and humidity (50%–70%) conditions and maintained under a 12 h light/dark cycle.All mice had free access to food and water.They were acclimatized for one week prior to the commencement of the experiments.The required amount of STZ was determined according to mouse weight.STZ was dissolved in physiological saline to prepare a solution and intraperitoneally injected (50 mg/kg) for 5 consecutive days, once a day.After the last injection, mice were fasted for 16 h and blood glucose levels were measured.If blood glucose levels were higher than 11.1 mmoL/L, the model was considered successful.

Ninety healthy male SPF Kunming mice were categorized into 9 groups according to their body weight (n= 10/group): normal group(NC), model group (MC), black tea high-weight group (BTH), black tea middle-weight group (BTM), black tea low-weight group (BTL),black brick tea with fungal growth high-weight group (FBTH),black brick tea with fungal growth middle-weight group (FBTM),black brick tea with fungal growth low-weight group (FBTL), and metformin hydrochloride group (MHT).The mice in the BTH, BTM,and BTL groups were administered 1 000, 500, and 300 mg/kg BT,respectively.The mice in the FBTH, FBTM, and FBTL groups were administered 1 000, 500, and 300 mg/kg FBT, respectively.The mice in the NC group were administered physiological saline according to their weight.The administration volume was fixed at 0.1 mL/10 g of the body weight of each mouse.All mice were gavaged continuously for 28 days, fasted for 12 h after the last gavage, and then euthanized using sodium pentobarbital.Mouse livers were dissected and stored at–80 °C until use.

2.4 Determination of biochemical composition

The contents of caffeine, simple catechins, ester catechins, and total catechins in the dry FBT and BT powders were determined by HPLC according to the method reported by Li et al.[23].The contents of tea polyphenols in the dry FBT and BT powders were determined by UV according to the method reported by Nanjegowda et al.[24].The contents of thea flavins, thearubigins, and theabrownins in the dry FBT and BT powders were determined by HPLC according to the method reported by Kumar et al.[25].

2.5 Determination of blood glucose and MDA levels, and GPx and SOD activity

Blood glucose levels were determined according to the kit instructions.Liver tissues were sampled from each mouse and homogenized in ice-cold 0.9% sterile saline on ice at a concentration of 2 g/10 mL.The homogenate was centrifuged at 8 000 r/min for 10 min at 4 °C, and the supernatant was collected and stored at –20 °C.SOD and GPx activity and the MDA content were detected according to the kit instructions.

2.6 Real-time quantitative PCR (qRT-PCR)

The total RNA from the liver tissues was isolated using the E.Z.N.A.? Total RNA Kit I (Omega, GA, USA; R6834-01) according to the manufacturer’s instructions.cDNA was synthesized using the TaKaRa RNA PCR kit according to the manufacturer’s instructions and stored at –20 °C for qRT-PCR.

The mouseβ-actin endogenous reference gene primer and oligonucleotide primers were synthesized by Sangon Bioengineering Co., Ltd.(Shanghai, China).Details of oligonucleotide primers are listed in Table 1.To quantify gene expression, the synthesized cDNA was subjected to qRT-PCR in a total reaction volume of 20 μL, according to the manufacturer’s instructions (Dalian Bao Bioengineering Co., Ltd., Dalian, China).Briefly, the qRT-PCR reaction mixture comprised of 10 μL of TB Green Premix ExTaqII(Tli RNaseH Plus), 2 μL of cDNA template, 0.4 μL of each forward and reverse primers, 0.4 μL of ROX Reference Dye II (50 ×), and 6.8 μL of sterilized water.The PCRs were performed on the ABI 7500 Fast real-time PCR system (Applied Biosystems Inc., CA,USA).The protocol included denaturation at 95 °C for 30 s, followed by 40 cycles at 95 °C for 5 s, and amplification at 60 °C for 30 s.To determine the melting curve, the temperature was increased from 60 °C to 95 °C.β-Actin was used as the housekeeping gene for normalizing the transcript levels of target genes.The threshold cycle (CT) value of each reaction was determined at the end of the qRT-PCR run.The change in the transcription level of each target gene, normalized to the expression level ofβ-actin, was calculated using the following formula: relative mRNA level of the target gene = 2-ΔΔCT; where, ΔΔCT = (CT Target - CTβ-actin) treatment - (CT Target - CTβ-actin) control.

Table 1Primers of genes for RT-PCR.

2.7 Western blotting

Total proteins from the liver were isolated using an enzyme inhibitor lysate; western blotting was performed according to the method reported by Gong et al.[26].

2.8 Statistical analysis

All statistical analyses were performed using IBM SPSS software(version 22.0).The inter-group differences were assessed by multiple comparisons using the DUNCAN test.The results are expressed as mean ± SE.A value ofP＜ 0.05 was considered to be statistically significant.

3.Results

3.1 Body weight changes during and after STZ, BT, and FBT supplementation

As shown in Table 2, the body weight of hyperglycemic mice was significantly lower after intraperitoneal injection of STZ (P＜ 0.05).Additionally, with the continuation of BT or FBT treatment, the body weight of hyperglycemic mice gradually returned to normal.

Table 2Body weight (g) of hyperglycemia mice by injection of STZ and treatment with BT and FBT.

3.2 FBT and BT chemical compositions

As shown in Table 3, there was no significant difference in the amounts of polyphenols, simple catechins, ester catechins, total catechins, caffeine, thea flavins, and thearubigins in FBT versus BT.However, the theabrownin content was significantly higher in FBT than in BT (P< 0.01) due to fungal growth.

Table 3Biochemical composition (%) of FBT and BT.

3.3 Effects of FBT and BT on fasting blood glucose and fasting insulin level

As shown in Table 4, the fasting blood glucose levels of the MC group were significantly higher (P< 0.01) than those of the NC group after STZ administration, whereas the insulin levels of the MC group were significantly lower (P< 0.01) than those of the NC group.The fasting blood glucose levels of mice in the MC group were higher than 5.6 mmol/L, which is a characteristic of limosis.

Table 4Fasting blood glucose level and insulin level of hyperglycemic mice (n = 10).

3.4 Effects of FBT and BT on SOD and GPx activities, and MDA and hepatic glycogen contents

After STZ administration, SOD and GPx activities were lower in the MC group than in the NC group, while the MDA content was higher in the MC group than in the NC group.After BT treatment,SOD and GPx activities were significantly higher in the BT group than in the MC group (P＜ 0.05) (Figs.1A, 1B), especially in the BTH group.On the other hand, the MDA content was significantly lower in the BT group than in the MC group (P< 0.01) (Fig.1C), especially in the BTH group.After FBT treatment, SOD and GPx activities were significantly higher in the FBT group than in the MC group (P< 0.01),especially in the FBTM and FBTH groups, whereas the MDA content was significantly lower in the FBT group than in the MC group(P< 0.01), especially in the FBTM and FBTH groups.Therefore,FBT was more effective than BT in inducing SOD and GPx activities,thereby decreasing the MDA content.

Fig.1 Effects of FBT and BT on activity of SOD (A) and GPx (B), and content of MDA (C) and hepatic glycogen (D) in the liver in mice.Values are expressed as the mean ± SE.Different letters indicate significant differences (P < 0.05).

After STZ administration, the hepatic glycogen content was lower in the MC group than in the NC group.Meanwhile, after BT treatment, the hepatic glycogen content was higher in the BT group than in the MC group; this was significant in the BTM and BTH groups (P＜ 0.05) (Fig.1D).After FBT treatment, the hepatic glycogen content was higher in the FBT groups than in the MC group; this was significant in the FBTM and FBTH groups (P＜ 0.05).Compared with BT, FBT was thus more effective at increasing the content of hepatic glycogen at the same dosage.

3.5 Effects of FBT and BT on the mRNA expression of key genes and proteins involved in glucose metabolism

The effects of FBT and BT on the mRNA expression of key genes and proteins involved in glycogen synthesis in the liver of mice are described in Table 5 and Fig.2.After treatment with STZ,GSK3βmRNA expression was higher in the MC group than in the NC group(P< 0.01), whilePI3K,AKT, andPDK1mRNA expression was lower in the MC group than in the NC group (Table 5).Simultaneously,GSK3β protein expression was higher in the MC group than in the NC group (P< 0.01) (Fig.2).After BT and FBT treatment,GSK3βmRNA expression was lower in the BT and FBT groups than in the MC group, whereasPI3K,AKT, andPDK1mRNA expression was higher in the BT and FBT groups than in the MC group (Table 5).Meanwhile, GSK3β protein expression was lower in the BT and FBT groups than in the MC group and PI3K, AKT, and PDK1 protein expression was higher in the BT and FBT groups than in the MC group (Fig.2); this was significant (P< 0.05) in the BT, BTH, FBTM,and FBTH groups.FBT was, therefore, more effective than BT in upregulating PI3K, AKT, and PDK1 expression and downregulating GSK3β expression.

Fig.2 Effects of FBT and BT on activity of SOD (A) and GPx (B), and content of MDA (C) and hepatic glycogen (D) in the liver in mice.Values are expressed as the mean ± SE.Different letters indicate significant differences (P < 0.05).

Table 5Effect on mRNA expression of GSK3β, AKT, PI3K and PDK1 in the liver in hyperglycemic mice of FBT and BT (n = 10).

Table 6 describes the effects of FBT and BT on the mRNA expression of key genes involved in regulating glucose transport,absorption, and metabolism as well as regulating glycolysis in the liver of mice.After treatment with STZ,GLUT2,IRS1,GS, andPFKFB2mRNA expression was lower in the MC group than in the NC group (P< 0.01) (Table 6).Meanwhile, GLUT2, IRS1, GS, and PFKFB2 protein expression was lower in the MC group than in the NC group (P＜ 0.01) (Fig.3).After FBT or BT treatment,GLUT2,IRS1,GS, andPFKFB2mRNA expression was higher in the FBT and BT groups than in the MC group (Table 6).Simultaneously, GLUT2,IRS1, GS, and PFKFB2 protein expression was higher in the FBT and BT groups than in the MC group (Fig.3); this was significant(P＜ 0.05) in the BTH, FBTM, and FBTH groups.Therefore, FBT was more effective than BT in upregulating GLUT2, IRS1, GS, and PFKFB2 expression.

Fig.3 Effects of FBT and BT on the proteins expression of GLUT2 (A), GS (B), p-IRS1 (C) and PFKFB2 (D) in the liver in mice.IRS1 was regulated through a complex mechanism involving phosphorylation of ＞ 50 serine/threonine residues within their long, unstructured tail regions.Insulin-stimulated kinases(including AKT and PI3K) mediate feedback serine/threonine residues phosphorylation of IRS, with positive effects on insulin sensitivity.Values are expressed as the mean ± SE.Different letters indicate significant differences (P ＜ 0.05).

Table 6Effect on mRNA expression of GLUT2, IRS1, GS and PFKFB2 in the liver in hyperglycemic mice of FBT and BT (n = 10).

4.Discussion

Fasting blood glucose and insulin levels are the most important indicators for determining hyperglycemia.Multiple studies have reported that treating mice with STZ significantly increases their fasting blood glucose and decreases their fasting insulin levels [27].Our study showed that the fasting blood glucose levels in NC mice were lower than those in MC mice after FBT or BT intake, whereas fasting insulin levels in NC mice were higher than those in MC mice after FBT or BT intake.After 28 days of treatment, the levels of fasting blood glucose in the FBT groups were lower than those in the BT groups, while the levels of fasting insulin in the FBT groups were higher than those in the BT groups.These results illustrate the success of this experimental model and show that FBT is more effective than BT at treating hyperglycemia in mice by lowering fasting blood glucose levels and promoting higher fasting insulin levels.

Numerous studies have found that oxidative stress is closely related to the occurrence and development of diabetes [28,29].Oxidative stress induces protein oxidative damage, which may cause diabetic complications.MDA is a product of lipid peroxide degradation that can indirectly reflect the degree of damage caused by oxygen free radicals to body cells, while SOD and GPx are important antioxidant enzymes that can reflect the oxidative stress in the body.In this study, we found that, during the development of diabetes,SOD and GPx activities gradually decreased, while the MDA content increased [30].Hyperglycemia can weaken the body’s ability to scavenge free radicals and cause many complications.In our study,SOD and GPx activities decreased and the MDA content increased in hyperglycemic mice treated with STZ; this partially agrees with the results of a previous study [31].Another previous study has confirmed that blood glucose can affect SOD activity and MDA content in patients with diabetes; however, the mechanism is not clear [32].Enhancing the free radical scavenging ability by increasing SOD activity and decreasing the MDA content can protect the islet nerve in hyperglycemic conditions, and thus regulate the blood glucose levels.In our study, FBT and BT treatments increased SOD and GPx activities and decreased the MDA content.This indicates that both FBT and BT have the ability to scavenge free radicals and suppress anti-oxidative stress in hyperglycemic mouse models.

Glycogen synthesis is an important pathway for glucose metabolism.The liver is the main site for the synthesis of glycogen,and it plays a crucial role in regulating glucose metabolism.Our results indicate that FBT and BT efficiently increase hepatic glycogen content in hyperglycemic mice.The process of insulin binding with the insulin receptor activates PI3K and AKT and promotes the glycogen synthesis process.GSK3β is not only a core enzyme in the process of hepatic glycogen synthesis, but it is also a negative regulator of the glycogen synthesis process.Glycogen synthesis is regulated by PI3K and AKT, which are the key protein kinases in this signaling pathway [33].In the PI3K/AKT signaling pathway,increasing PI3K expression enhances the expression of PDK1,which plays a part in promoting damaged islet β-cell recovery,increasing insulin secretion, and improving insulin resistance.AKT is phosphorylated in this process.In our study, the intake of BT and FBT increased PI3K expression.Furthermore, PI3K upregulated PDK1 expression and then stimulated AKT phosphorylation and downregulated GSK3β expression.This partially agrees with the results of previous studies [34].Moreover, FBT was more effective than BT in upregulating PI3K, AKT, and PDK1 and downregulating GSK3β.This could be a plausible reason for the glycogen content increase in the liver after FBT and BT intake.

In the glucose absorption and transport process, IRS1 plays an important role in the PI3K/AKT signaling pathway.Insulin binding with the insulin receptor (INSR) leads to IRS phosphorylation at the tyrosine-bearing sites.Therefore, this process upregulates GLUT2 expression.PFKFB2 plays an important role in glycolysis, and phosphorylated PFKFB2 enhances insulin sensitivity [35].In our study, FBT and BT intake stimulated AKT and PI3K phosphorylation.Furthermore, AKT and PI3K expression was upregulated.Moreover,FBT was more effective than BT in upregulating GLUT2, GS, IRS1,and PFKFB2.This process laid the foundation for GS phosphorylation and GSK3β inhibition.This may be because the active ingredients in BT and FBT, such as polyphenols and pigments, play a regulatory role in this process, and this partially agrees with the results of a previous study, which showed that tea polyphenols and pigments have a positive effect in regulating genes and proteins at low glucose levels [36,37].Additionally, the phosphorylated GS stimulates GLUT2 expression, increasing insulin secretion and sensitivity.Additionally, the ability of the liver to intake glucose improves.Our results showed that BT and FBT may promote glucose absorption and insulin secretion by promoting GS, IRS1, and PFKFB2 expression.

Regarding hepatic glycogen and blood glucose, we have directly demonstrated that FBT is more effective than BT in treating hyperglycemic mice.Based on the results of MDA content and SOD and GPx activities, we infer that FBT is more effective than BT at scavenging free radicals.Meanwhile, FBT can regulate the expression of genes and proteins that are associated with glucose metabolism.FBT has a higher content of theabrownins than BT because the preparation of the former has an additional process of fungal growth.This results in a 7.03% higher content of theabrownins in FBT than that in BT, which may be responsible for the better hypoglycemic effects of FBT than those of BT [38].This finding partially agrees with that of a previous study, which showed that theabrownin supplementation prevents insulin resistance [39].To the best of our knowledge, this is the first study to provide a scientific basis for the hypoglycemic effect and mechanism of FBT theabrownins.Nevertheless, FBT and BT contain several active ingredients, and currently, we cannot confirm whether theabrownins are the only effective substances.Thus, it is imperative to investigate the other active substances in FBT and BT and verify whether they are also responsible for the hypoglycemic effects of these products [40].Furthermore, the specific pathways involved in these hypoglycemic processes remain undetermined.As the liver is not the only organ responsible for glucose metabolism, other organs also influence the hypoglycemic effect.These aspects were not evaluated in this study.As healthy functional drinks, FBT and BT cannot completely replace a drug, but they can play a supporting role in regulating human health.The functional ingredients in FBT and BT can be used as food additives for nutritional intervention.In the future, we will focus on the hypoglycemic effect of FBT using proteomic and transcriptomic approaches.

5.Conclusions

Overall, our results showed that FBT and BT intake decreased blood glucose levels and increased the hepatic glycogen content.Moreover, it promoted SOD and GPx activities and reduced the MDA content.In addition, our results also showed that FBT and BT regulated glycogen synthesis by upregulating PI3K, PDK1, GS, and AKT protein and mRNA expression and downregulating GSK3β protein and mRNA expression.FBT and BT can regulate glucose absorption and transport by upregulating IRS1 and GLUT2 protein and mRNA expression.In addition, they can stimulate glycolysis by upregulating GS and PFKFB2 protein and mRNA expression.Compared to BT, FBT significantly upregulated PI3K, AKT, PDK1,IRS1, and GLUT2 protein and mRNA expression and downregulated GSK3β protein and mRNA expression in higher levels.Therefore,FBT is more effective than BT in scavenging free radicals and inducing hypoglycemic effects.

Conflicts of interest

There is no conflicts to declare.

Acknowledgement

We would like to thank Editage for English language editing.This work was supported by The National Natural Science Foundation of China (grant number 31871804), Natural Science Foundation Project of Hunan Province, China (grant number 2020JJ4036), and the Key R&D Program of Hunan Province (2020NK2030).