Composition capable of improving diabetes induced by obesity and application

Abstract

The invention relates to the field of microorganisms and the technical field of medicines, in particular to a composition capable of effectively improving diabetes induced by obesity and application. The invention relates to a composition capable of improving diabetes induced by obesity, the composition comprises a component A and a component B, the component A is bifidobacterium longum BKR-011, and the component B is puerarin; wherein the usage amount of the bifidobacterium longum BKR-011 is 0.5 * 10 < 8 > CFU / kg (B.W.)-2 * 10 < 10 > CFU / kg (B.W.), and the usage amount of the Pueralin is 2.5 mg / kg (B.W.)-25 mg / kg (B.W.). According to the invention, two probiotics and plant extracts which both have the effects of improving obesity and blood sugar are combined for the first time, on one hand, the strain BKR-011 can promote the absorption of Puranin by inhibiting the activity of UGTs and improve the utilization rate of the Puranin by an organism, and on the other hand, the strain BKR-011 and the Puranin can generate cross and complementary synergistic effects on a plurality of targets in treatment of obesity-diabetes mellitus.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbiology and medical technology, in particular to a composition and application for effectively improving diabetes induced by obesity. BACKGROUND

[0002] Diabetes mellitus is a well-defined disease characterized by elevated blood glucose, and is divided into type 1 diabetes mellitus (T1DM), type 2 diabetes mellitus (T2DM) and other specific types of diabetes mellitus, such as gestational diabetes. Unlike T1DM, T2DM is a non-autoimmune metabolic disease characterized by insulin resistance (IR). Before the onset of the disease, compensatory increase in insulin is often observed, during which the body's sensitivity to insulin decreases, and further, accompanied by dysfunction of pancreatic beta cells, leading to insufficient insulin secretion to maintain blood glucose homeostasis, eventually developing into T2DM.

[0003] More and more studies have shown that obesity can cause insulin resistance (IR), dysfunction of pancreatic beta cells, and ultimately lead to T2DM. The development of T2DM is usually the result of the combined effects of IR and beta cell dysfunction. Over time, T2DM can lead to a variety of long-term complications, including microvascular disease (retinopathy, nephropathy and neuropathy), macrovascular disease (stroke, myocardial infarction and peripheral arterial disease), heart failure and non-alcoholic fatty liver disease (NAFLD). In addition, it can also promote inflammation and cause the development of other diseases, such as Alzheimer's disease, Parkinson's disease, gout and rheumatoid arthritis.

[0004] Currently, T2DM is usually treated by reducing endogenous sugar production (biguanides, metformin), reducing small intestinal sugar absorption (AGI, acarbose), increasing sugar excretion (SGLT-is, dapagliflozin), increasing sugar conversion (TZD, rosiglitazone), promoting insulin secretion (SUs, glibenclamide) and reducing insulin degradation (DPP-4i, sitagliptin). However, these drugs often cannot delay the progression of T2DM complications, and often have side effects, such as metformin, which manifests as gastrointestinal irritation.

[0005] Pueraria lobata Pueraria lobata ( Willd. ) Ohwi is a medicinal and edible homologous plant with a long history. Puerarin (7,4'-dihydroxy-8-C-glucosylisoflavone) is the main bioactive component extracted from the roots of Pueraria lobata, which was first isolated in the 1950s [1]Puerarin has a wide range of pharmacological activities and is widely used in the treatment of cardiovascular and cerebrovascular diseases, diabetes and its complications, osteonecrosis, Parkinson's disease, Alzheimer's disease, endometriosis and cancer [2] However, despite its wide range of pharmacological activities, the clinical application of Puerarin is relatively limited, and only injection and eye drops are the main dosage forms of Puerarin [3] .

[0006] The main reason for this problem is that Puerarin belongs to isoflavone structure, and its structural characteristics result in low water solubility and low fat solubility, which in turn result in low oral absorption and low bioavailability. Based on its low solubility and low intestinal permeability characteristics, Puerarin can be classified as a class IV drug according to the biopharmaceutics classification system, and in order to improve its solubility, propylene glycol, ethylene glycol and polyvinylpyrrolidone and other solubilizers are often added in clinical injection preparations, but adverse reactions caused by solubilizers, including: vascular irritation, fever, allergy and hemolysis, often occur. In addition, due to the short elimination half-life of Puerarin, frequent or high-dose injection is required for administration, so it is of great significance to develop oral preparations that can improve the bioavailability of Puerarin.

[0007] The metabolism of Puerarin in vivo involves phase I (reduction and hydrolysis) and phase II (glucuronidation and sulfation) catalytic reactions [4,5] , but mainly phase II reaction. In vivo, both liver and intestine can metabolize Puerarin, and have similar metabolic profiles [6] . In human body, the metabolism (glucuronidation) of Puerarin is mainly realized by UGT1A1 and UGT1A9 in UGTs (uridine diphosphate glucuronosyltransferase) family [7] , while in rats it is believed to be realized by Ugt1a1 and Ugt1a7 [8] .

[0008] Studies have shown that certain pathological conditions can change the degree of oral absorption of Puerarin by the body, for example, in bacterial diarrhea rats, the absorption concentration of Puerarin is significantly higher than that in normal rats [9] ; while in diabetic rats, the AUC, C max and half-life (t 1 / 2 ) of Puerarin are significantly reduced

[10] . In addition, some traditional Chinese medicine prescriptions also show their influence on the absorption of Puerarin. Compared with Puerarin extract alone, Puerarin in Puerariae and Coptidis Decoction has higher blood absorption concentration and longer half-life

[11] . Although this mechanism has not been thoroughly revealed, it still indicates from another side the complexity of the metabolism and absorption of natural products in vivo.

[0009] References [1] Zhang B, Li M, Wang Q, et al. Exploring adverse effects of puerarin on catalase by multiple spectroscopic investigations and docking studies in vitro[J]. Journal of Biochemical and Molecular Toxicology, 2019, 33(5): e22296. [2] Zhou Y X, Zhang H, Peng C. Puerarin: a review of pharmacological effects[J]. Phytotherapy Research, 2014, 28(7): 961-975. [3] Luo C F, Cai B, Hou N, et al. UDP-glucuronosyltransferase 1A1 is the principal enzyme responsible for puerarin metabolism in human liver microsomes[J]. Archives of toxicology, 2012, 86: 1681-1690. [4] https: / / www.nmpa.gov.cn / datasearch / home-index.html#category=yp [5] Emi Y, Ikushiro S, Iyanagi T. Drug-responsive and tissue-specific alternative expression of multiple first exons in rat UDP-glucuronosyltransferase family 1 (UGT1) gene complex[J]. The journal of biochemistry, 1995, 117(2): 392-399. [6] Luo C F, Yuan M, Chen M S, et al. Metabolites of puerarin identified by liquid chromatography tandem mass spectrometry: similar metabolic profiles in liver and intestine of rats[J]. Journal of Chromatography B, 2010, 878(3-4): 363-370. [7] Srinivasan K, Viswanad B, Asrat L, et al. Combination of high-fat diet-fed and low-dose streptozotocin-treated rat: a model for type 2 diabetes and pharmacological screening[J]. Pharmacological research, 2005, 52(4): 313-320. [8] He M Y, Deng Y X, Shi Q Z, et al. Comparative pharmacokinetic investigation on baicalin and wogonoside in type 2 diabetic and normal rats after oral administration of traditional Chinese medicine Huanglian Jiedu decoction[J]. Journal of Ethnopharmacology, 2014, 155(1): 334-342. [9] Ling X, Xiang Y, Tang Q, et al. Comparative pharmacokinetics of eight major bioactive components in normal and bacterial diarrhea mini-pigs after oral administration of Gegen Qinlian Decoction[J]. Journal of Chromatography B, 2017, 1044: 132-141.

[10] Dong S, Zhang M, Niu H, et al. Upregulation of UDP-glucuronosyltransferases 1a1 and 1a7 are involved in altered puerarin pharmacokinetics in type II diabetic rats[J]. Molecules, 2018, 23(6): 1487.

[11] Zhang Y, Yuan J, Wang Y, et al. LC-MS / MS determination and pharmacokinetics study of puerarin and daidzein in rat plasma after oral administration of Gegenqinlian decoction and Radix Puerariae extract[J]. Pharmacognosy magazine, 2014, 10(39): 241. SUMMARY

[0010] The present application aims to provide a composition for improving obesity-induced diabetes and application.

[0011] To achieve the above-mentioned object, the technical scheme adopted by the present application is as follows: The composition for improving obesity-induced diabetes comprises two components A and B, wherein A is Bifidobacterium longum BKR-011, and B is puerarin; the amount of Bifidobacterium longum BKR-011 is 0.5×10 8 CFU / kg (B.W.)~2×10 10CFU / kg (BW), Puerarin dosage is 2.5 mg / kg (BW) ~ 25 mg / kg (BW).

[0012] The amount of Bifidobacterium longum BKR-011 used in the composition is 0.2 × 10⁻⁶. 9 CFU / kg (BW) ~ 1×10 9 CFU / kg (BW), Puerarin dosage is 5 mg / kg (BW) ~ 20 mg / kg (BW).

[0013] The Bifidobacterium longum ( Bifidobacterium longum BKR-011 was deposited on July 26, 2021, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 22949. The depository address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0014] The puerarin is either elemental puerarin or an extract containing puerarin obtained from natural extraction.

[0015] The extract containing Puerarin is from the legume kudzu (Pueraria lobata). Pueraria lobata ( Willd .) Ohwi Leguminosae plant, kudzu vine ( Pueraria thunbergiana Benth. Puerarin-containing extracts are obtained by extracting one or more of the following plants: puerarin and others.

[0016] The use of the composition described herein for improving obesity-induced diabetes, the composition being used in the preparation of medicaments, health foods and other foods for treating or improving obesity-induced diabetes and its complications.

[0017] An agent for improving obesity-induced diabetes, the agent comprising the composition thereof, wherein the composition comprises Bifidobacterium longum BKR-011 and puerarin.

[0018] The dosage form of the preparation includes, but is not limited to, tablets, powders, capsules, and suspensions.

[0019] The formulation consists of Bifidobacterium longum and puerarin, which can be packaged together or separately.

[0020] The strain BKR-011 in the composition has the functions of reducing the metabolic rate of Puerarin by inhibiting the expression level of glucuronidation enzyme UGTs in the intestinal tract and liver, enhancing the bioavailability, increasing the secretion of intestinal hormone GLP-1, and then increasing the secretion of insulin, thereby effectively improving blood sugar, increasing the secretion of adiponectin and leptin, and realizing the comprehensive treatment of obesity-induced diabetes.

[0021] The Puerarin in the composition can increase the expression level of GLP-1 receptor and enhance the sensitivity, and can also increase the expression of the key regulator TCF7L2 for maintaining the survival and regeneration of beta cells, protect the function of islet beta cells, thereby producing a synergistic effect on the hypoglycemic effect of the strain BKR-011, effectively reducing the dosage of the two, improving the overall treatment effect, and providing a safer and more efficient intervention scheme for obesity-induced diabetes patients.

[0022] Compared with the prior art, the technical advantages of the present application are embodied as follows: The present application firstly combines two kinds of probiotics and plant extracts which can improve obesity and blood sugar, on the one hand, the strain BKR-011 can promote the absorption of Puerarin by inhibiting the activity of UGTs, and on the other hand, the strain BKR-011 and Puerarin can produce cross and complementary synergistic effects on multiple targets in the treatment of obesity-induced diabetes, including GLP-1, GLP-1 receptor, TCF7L2, adiponectin and leptin, etc., thereby enhancing the curative effect, greatly reducing the dosage of single use, and reducing the use cost.

[0023] The composition of the present application can improve the bioavailability of Puerarin by adjusting the UGTs in the intestinal tract. Bifidobacterium longum The strain BKR-011 can reduce the degradation of Puerarin in the body by adjusting the UGTs in the intestinal tract, thereby improving the bioavailability of oral Puerarin. Bifidobacterium longum The present application provides a rat plasma Puerari concentration-time curve. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The present application provides a rat plasma Puerari concentration-time curve.

[0025] Figure 2The mRNA level effect graph of Ugt1a1 and Ugt1a7 in rat liver and small intestine provided by the embodiment of the present application; wherein, A is Ugt1a1, and B is Ugt1a7.

[0026] Figure 3 The effect graph of Puerarin and strain BKR-011 on the body weight of mice provided by the embodiment of the present application.

[0027] Figure 4 The effect of Puerarin and strain BKR-011 on the fasting blood glucose of mice.

[0028] Figure 5 The intraperitoneal glucose tolerance test (IPGTT) results of mice in each group after 5 weeks of treatment provided by the embodiment of the present application.

[0029] Figure 6 The concentration effect graph of insulin and GLP-1 in the fasting serum of mice in each group after the treatment provided by the embodiment of the present application; wherein, A is the insulin level in the serum of mice, and B is the GLP-1 level in the serum of mice.

[0030] Figure 7 The expression level effect graph of GLP-1R, TCF7L2 and insulin in the islets of mice provided by the embodiment of the present application.

[0031] Figure 8 The level effect graph of triglyceride (TG), total cholesterol (TC), adiponectin and leptin in the serum provided by the embodiment of the present application; wherein, A is the triglyceride level in the serum, B is the total cholesterol level in the serum ， C is the adiponectin level in the serum. DETAILED DESCRIPTION

[0032] The specific embodiments of the present application are further described in conjunction with examples, and it should be pointed out that the specific embodiments described herein are only for the purpose of illustrating and explaining the present application, and are not limited to the present application.

[0033] Bifidobacterium longum in the composition of the present application Bifidobacterium longum BKR-011 reduces the degradation of Puerarin in vivo by regulating UGTs in the intestinal tract, thereby achieving the improvement of the bioavailability of oral Puerarin; meanwhile, the two exhibit significant synergistic effect in the treatment of diabetes and related complications caused by obesity, and therefore have wide application prospects.

[0034] The Bifidobacterium longum involved in the following embodiments Bifidobacterium longumBKR-011 has been described in the patent document with application number 202211089417.0 and the invention name of "a strain of Bifidobacterium longum and its application", and the corresponding preservation certificate is provided. Its classification name is Bifidobacterium longum (BKR-011) Bifidobacterium longum ) was preserved in China General Microbiological Culture Collection Center on July 26, 2021, and the preservation number is CGMCC No. 22949.

[0035] The culture medium involved in the following examples is as follows: MRS culture medium (g / L): 10 g / L of proteose peptone, 10 g / L of beef extract, 15 g / L of glucose, 15 g / L of lactose, 5 g / L of yeast powder, 2 g / L of diammonium hydrogen citrate, 2.6 g / L of K2HPO4·3H2O, 0.1 g / L of MgSO4·7H2O, 0.05 g / L of MnSO4, 1 mL / L of Tween 80, 0.5 g / L of cysteine amino acid salt.

[0036] The method for preparing the bacterial suspension involved in the following examples is as follows: the strain BKR-011 preserved in the glycerol tube is inoculated in the MRS culture medium, and cultured at 37°C until the OD 600 >1.2, the fermentation broth after culture is loaded into a centrifuge tube, centrifuged at 4000 rpm for 10 min, and the supernatant is discarded to recover the bacterial cells. The bacterial cells are resuspended to 1.0×10 10 CFU / ml for standby.

[0037] Puerarin with a purity of >99% is purchased from Chengdu Longquan High-tech Natural Medicine Co., Ltd. (Chengdu, China).

[0038] The test result data is statistically analyzed by using GraphPad, # represents p<0.05 compared with the control group, which has a difference, * represents p<0.01 compared with the control group, which has a significant difference.

[0039] Example 1: Strain BKR-011 has the ability to improve the oral bioavailability of Puerarin The test animals are male Sprague / Dawley (SD) rats with a body weight of 180-220 g. The rats are raised under environmental control conditions, with a temperature of 22±2°C, a relative humidity of 50±5%, and a light / dark cycle of 12 hours. At least one week before the experiment, the rats can freely eat and drink water. The rats are fasted overnight before administration, but can freely drink water. All animal experiments are carried out in accordance with the "Guide for the Care and Use of Laboratory Animals" (8th edition, revised in 2011) issued by the National Institutes of Health (NIH).

[0040] Take 18 SD rats, divided into 3 groups, the first group is the control group (CON), the first 7 days free diet, the second group is the high dose of probiotics group (High), the first 7 days in addition to free diet, but also intragastric suspension of the above obtained 0.1 ml (1 x 10 9 CFU / each / day). The third group is the low dose of probiotics group (Low), the first 7 days in addition to free diet, but also intragastric suspension of the above obtained 0.02 ml (2 x 10 8 CFU / each / day), the above groups were cultured to the 8th day, respectively, to each group of Puerarin (250 mg / kg) was given, and then the mouse tail blood was collected at 0.17, 0.33, 0.50, 0.75, 1.0, 1.5, 2.0, 3.0, 4.0, 6.0 and 10 hours, respectively. The plasma was collected and analyzed.

[0041] The method for determining Puerarin in plasma is as follows: 100 μL of rat plasma is taken, 10 μL (200 μmol / L) of methyl-p-hydroxybenzoic acid (internal standard, IS) is added, mixed thoroughly, 500 μL of methanol is added, vortexed for 1 min, centrifuged at 15000 g for 20 min, the supernatant is collected, blown dry under nitrogen at 45°C, 100 μL of acetonitrile is added to remix, centrifuged at 15000 g for 20 min, and 20 μL of the supernatant is subjected to HPLC analysis. A reversed-phase C 18 Ecosil analysis column (250 mm x 4.6 mm, 5 μm) is used, the mobile phase is acetonitrile and ammonium acetate (2.5 mmol / L, pH 7.4), the column temperature is 35°C, and the absorbance is detected at 250 nm. The Puerarin concentration and time curve in the plasma of the three groups of rats is shown in Figure 1 .

[0042] As can be seen from Figure 1 , Puerarin is rapidly absorbed orally, and the highest blood drug concentration can be reached in each group at 0.75 h, which is 25.4 ± 4.1 μmol / L for the control group, 59.0 ± 4.2 μmol / L for the high dose of probiotics group, and 43.3 ± 3.9 μmol / L for the low dose of probiotics group.

[0043] Further analysis of the change of Puerarin concentration in the plasma of the three groups of rats at different times is carried out. The pharmacokinetic parameters are analyzed by non-compartment model, and the DAS 2.1.1 software (Mathematical Pharmacology Committee of Chinese Pharmacological Society, Shanghai, China) is used for processing. The data is expressed as mean ± standard deviation. Statistical differences are evaluated by one-way ANOVA (see Table 1).

[0044] The analysis results are shown in Table 1. For the pharmacokinetic parameters of Puerarin, including AUC 0-t , AUC0-∞ CON group was significantly different from high dose group (P<0.001) and low dose group (P<0.01) of BKR-011, which showed that after oral administration of BKR-011, the AUC value was significantly increased, and the CLz / F and Vz / F values were decreased. This indicated that BKR-011 could enhance the absorption of Puerarin in rats and delay its degradation, which played a good guarantee for the more effective pharmacological effect of Puerarin in vivo.

[0045] Table 1. Effects of different doses of BKR-011 on the pharmacokinetic parameters of oral Puerarin.

[0046] Then the above treated mice were further tested to determine the ability of BKR-011 to inhibit the mRNA expression levels of Ugt1a1 and Ugt1a7 genes in the liver and intestinal tract After determining the Puerarin blood concentration of the above groups of SD rats, the rats in each group were sacrificed, and liver and small intestine samples were extracted. The mRNA levels of Ugt1 a1 and Ugt1 a7 in the liver and small intestine of SD rats were detected by the following method: TRIzol® kit was used to treat liver and small intestine tissues, and total RNA was isolated based on the instructions. The purity of total RNA was measured using a spectrophotometer, and the wavelength absorption ratio (260 / 280 nm) of all preparations was between 1.8 and 2.0. GoTaq® 2-Step RT-qPCR System (Promega) and specific primers (Applied Biosystem ®) were used for reverse transcription of total RNA to cDNA and qPCR. The primer sequences used are as follows: Uridine diphosphate glucuronosyltransferase gene Ugt1a1 : Forward 5'-GCACGAAGTGGTGGTCAT-3' Reverse 5'-CGGAAGGAAAGGGTCTGT-3' Uridine diphosphate glucuronosyltransferase gene Ugt1a7 : Forward 5'-AGTGTCCGTTTGGTTGTT-3' Reverse 5'-TTCCATCGCTTTCTTCTC-3' Reference gene glyceraldehyde-3-phosphate dehydrogenase gene GAPDH : Positive 5'-GCCTTCCGTGTTCCTACC-3' Reverse 5'-GCCTGCTTCACCACCTTC-3' qRT-PCR was used to measure the mRNA levels of Ugt1a1 and Ugt1a7 in the liver and small intestine of rats. The results are as follows: Figure 2 As shown.

[0047] Depend on Figure 2 It was found that both Ugt1a1 and Ugt1a7 were expressed in both the liver and small intestine, and strain BKR-011 effectively reduced Ugt1a1 expression in both high-dose and low-dose groups. Figure 2 A) and Ugt1a7 ( Figure 2 B) expression levels, with P < 0.001 in the high-dose group and P < 0.05 in the low-dose group. Since Ugt1 is considered a key enzyme for the degradation of Puerarin in vivo, and the inhibition of this pathway by strain BKR-011 can improve the bioavailability of Puerarin.

[0048] Example 2. The effect of a combination of strain BKR-011 and Puerarin on the improvement of obesity-induced diabetes. The experimental animals were 4-week-old male C57BL / 6J mice. Mice were housed under controlled environmental conditions: temperature 22±2℃, relative humidity 50±5%, and a 12-hour light / dark cycle. The experiment consisted of 5 groups, with 12 mice in each group. Group 1 was the negative control group (ND), fed freely for 17 weeks (the first 12 weeks were the modeling period, and the last 5 weeks were the experimental period); Group 2 was the positive control group (HFD), fed a high-fat diet (Table 2) daily for 17 weeks (the first 12 weeks were the modeling period, and the last 5 weeks were the experimental period); Group 3 was the Puerarin group (HFD+Pue), fed a high-fat diet daily for 12 weeks, followed by daily gavage administration of Puerarin 50 mg / kg for 5 weeks; Group 4 was the strain BKR-011 group (HFD+BKR-011), fed a high-fat diet daily for 12 weeks, followed by daily gavage administration of strain BKR-011 1×10 9 The concentration of CFU / kg was maintained for 5 weeks; Group 5 was the Puerarin + strain BKR-011 combination group (HFD + Puerarin + BKR-011), fed a high-fat diet daily. After 12 weeks of feeding, Puerarin 25 mg / kg and strain BKR-011 5 × 10 CFU / kg were also administered by gavage daily. 8 The CFU / kg level was maintained for 5 weeks. An intraperitoneal glucose tolerance test (IPGTT) was performed at the end of the feeding period for each group, and blood samples and pancreatic tissue were collected simultaneously.

[0049] Table 2. High-fat diet formula (10 kg).

[0050] The test method of IPGTT is that the mice are intraperitoneally injected with glucose at a dose of 2 mg / g of body weight after fasting for 12 h. Blood samples are collected at 0, 30, 60, 90 and 120 minutes, and glucose measurement is performed using a blood glucose meter (Accu-Chek Active; Roche).

[0051] The measurement method of each physiological index in serum is that the collected blood samples are centrifuged at a speed of 3000 rpm for 10 minutes at 4°C, and the serum is stored frozen until analysis. DPP-4 inhibitor Diprotin A and aprotinin are added to each blood sample (final concentrations are 100 μmol and 85 μg / mL, respectively). Insulin is determined using a mouse insulin ELISA kit (Alpco). Total cholesterol and triglycerides are evaluated by a kit (Nanjing Jiancheng). Serum adiponectin and leptin are evaluated by a mouse ELISA kit (Boster). GLP-1 is measured using a mouse / rat specific GLP-1 (7-36) ELISA assay (Phoenix). All steps are performed according to the manufacturer's instructions.

[0052] The detection method of GLP-1 receptor and related target genes in mouse islets is as follows: refer to the determination method based on mRNA transcription level in Example 1, and the required target gene primers are as follows: GLP-1R Forward: 5'-CCTGAGGAACAGCTCCTGTC-3'; Reverse: 5'-GGATGCAAACAGGTTCAGGT-3'.

[0053] T-cell factor 7-like 2 (TCF7L2) TCF7L2 ) Forward: 5'-CAGGGAAGAACAGGCAAAAT-3'; Reverse: 5'-GGGGGAGGCGAGTCTAGTAA-3'.

[0054] Insulin (INS) Ins1 ) Forward: 5'-TTCTTCTACACACCCA-3'; Reverse: 5'-CTAGTTGCAGTAGTTCT-3'.

[0055] After 5 weeks of intervention with a high-fat diet in mice using Puerarin and strain BKR-011, the changes in mouse body weight were as follows: Figure 3 As shown.

[0056] Depend on Figure 3 It was observed that at week 0, there was a significant difference in body weight between the negative control group (group 1) and the positive control group (group 2) (P<0.001), demonstrating successful model establishment. By week 1 of intervention, the Puerarin and BKR-011 combination group (group 5) showed improvement in obesity (P<0.05). By week 2, the Puerarin single-component group (group 3) and the BKR-011 group (group 4) also showed improvement in obesity (significantly P<0.05 and P<0.01, respectively). By week 4, the Puerarin and BKR-011 combination group (group 5) showed a greater effect on improving body weight than the single-component groups (P<0.05) (groups 3 and 4), demonstrating that although the dosage of each component in the combination was reduced (1 / 2), the synergistic effect of both enhanced the weight loss effect.

[0057] During the 5-week intervention, the changes in fasting blood glucose in mice were as follows: Figure 4 As shown.

[0058] Depend on Figure 4 It was observed that in week 0, there was a significant difference in fasting blood glucose between the negative control group (group 1) and the positive control group (group 2) (P<0.001), proving the successful establishment of the model. By week 1, the BKR-011 group (group 4) showed a significant hypoglycemic effect (P<0.05), while the Puerarin group (group 3) showed a significant effect in week 2 (P<0.05). By week 3, the hypoglycemic effect of the Puerarin and BKR-011 combination group (group 5) was significantly higher than that of the single-component groups (P<0.01) (groups 3 and 4), further indicating that Puerarin and BKR-011 have a synergistic effect.

[0059] After 5 weeks of treatment, mice in each group underwent an intraperitoneal glucose tolerance test (IPGTT). Changes in blood glucose levels over time in each group were as follows: Figure 5 As shown.

[0060] Depend on Figure 5It was found that blood glucose levels in all groups of mice peaked at 30 minutes. The blood glucose levels, from highest to lowest, were: positive control group (Group 2), Puerarin-only group (Group 3), BKR-011-only group (Group 4), combined group (Group 5), and negative control group (Group 1). The positive control group (Group 2) showed a significantly lower rate of blood glucose decline after 60 minutes compared to the other groups, suggesting possible insulin deficiency or insulin resistance in these mice. The Puerarin group (Group 3) and the BKR-011-only group (Group 4) showed significant improvement. The Puerarin group (Group 3) exhibited a higher rate of blood glucose decline, while the BKR-011-only group (Group 4) showed a lower peak blood glucose concentration. The combined group (Group 5) showed good improvement in both peak blood glucose concentration and rate of blood glucose decline.

[0061] After 5 weeks of treatment, the fasting serum insulin and GLP-1 levels in mice of each group were as follows: Figure 6 As shown.

[0062] Depend on Figure 6 As shown in Figure A, the insulin levels in the positive control group (Group 2) were lower than those in the negative control group (Group 1), suggesting that pancreatic function may be impaired under a sustained high-fat diet, thus failing to provide sufficient compensatory insulin to control blood glucose. The Puerarin group (Group 3) did not show a significant increase in insulin levels, a result consistent with... Figure 5 The observation that no significant change in blood glucose levels occurred in the Puerarin group (Group 3) at 30 minutes during the IPGTT test in mice was corroborated by this finding. Unlike the Puerarin group (Group 3), the single-strain BKR-011 group (Group 4) significantly increased fasting insulin levels (P<0.05), suggesting that strain BKR-011 may protect pancreatic function or improve blood glucose levels by stimulating insulin secretion through other pathways. The combined group (Group 5) further increased serum insulin levels in mice (P<0.01), further suggesting a synergistic effect between Puerarin and strain BKR-011.

[0063] Depend on Figure 6As shown in Figure B, unlike the Puerarin group (Group 3), the single-strain BKR-011 group (Group 4) and the combined group (Group 5) significantly increased the level of GLP-1 in mice (P<0.01). GLP-1, as an enterogenic insulin secretagogue, stimulates insulin secretion from pancreatic β cells. Since there was no significant difference in GLP-1 secretion levels between the single-strain BKR-011 group (Group 4) and the combined group (Group 5), it is believed that the increased GLP-1 secretion was the result of the single action of strain BKR-011. This also confirms that... Figure 6 The phenomenon that strain BKR-011 in group A can increase insulin levels in mice.

[0064] At the end of the 5-week treatment period, the gene expression levels of GLP-1 receptor (GLP-1R), transcription factor TCF7L2, and insulin in the mouse islets were observed. Figure 7 .

[0065] like Figure 7 As shown, although Puerarin failed to significantly increase the concentration of GLP-1 in plasma ( Figure 6 B), but it can significantly increase the level of GLP-1 receptor (GLP-1R) (P<0.001), while strain BKR-011 can increase GLP-1 ( Figure 6 The levels of GLP-1 (P<0.01) were increased, but the levels of GLP-1 receptor (GLP-1R) were not increased. This phenomenon suggests a synergistic mechanism between the two components: strain BKR-011 increased GLP-1 production, while Puerarin enhanced the expression intensity of GLP-1 receptor, thereby enhancing the promoting effect of GLP-1 on insulin.

[0066] TCF7L2 is an important transcription factor in the wingless-type MMTV integration site (Wnt) / β-catenin signaling pathway and a key regulator for maintaining β-cell survival and regeneration [12,13]. Figure 7 As shown, unlike strain BKR-011, Puerarin can increase the expression level of the TCF7L2 gene. Therefore, it is believed that Puerarin also has the ability to maintain pancreatic β-cell function. This also reflects the mechanism by which Puerarin and strain BKR-011 synergistically improve obesity-induced diabetes from different factors and different targets.

[0067] Effects of Puerarin and strain BKR-011 on other indicators in obesity-induced diabetic mice, such as Figure 8 As shown in the figure.

[0068] Depend on Figure 8AB It was found that both Puerarin and BKR-011 could significantly reduce the content of TG and TC in mice (P<0.01), and the combination of Puerarin and BKR-011 could more significantly reduce the content of TG and TC in mice. Figure 8 C It was found that BKR-011 could significantly increase the level of adiponectin, and the combination of Puerarin and BKR-011 could more significantly increase the level of adiponectin in mice. Figure 8 D It was found that the level of leptin was compensatorily increased in the positive control group (group 2), and significantly reduced in the Puerarin group (group 3), which indicated that Puerarin could reduce the increase of leptin caused by high-fat diet by improving fat metabolism. However, the combination group (group 5) and the single-strain BKR-011 group (group 4) did not significantly reduce the level of leptin, which indicated that BKR-011 had the ability to increase the level of leptin in the body, which further indicated that the synergistic mechanism between Puerarin and BKR-011 was extensive.

Claims

1. A composition for improving obesity-induced diabetes, characterized in that: The composition consists of two components, A and B. Component A is *Bifidobacterium longum* BKR-011, and component B is puerarin. The dosage of *Bifidobacterium longum* BKR-011 is 0.5 × 10⁻⁶. 8 CFU / kg (BW) ~2×10 10 CFU / kg (BW), Puerarin dosage is 2.5 mg / kg (BW) ~ 25 mg / kg (BW).

2. The composition according to claim 1 for improving obesity-induced diabetes, characterized in that: The amount of Bifidobacterium longum BKR-011 used in the composition is 0.2 × 10⁻⁶. 9 CFU / kg (BW) ~ 1×10 9 CFU / kg (BW), Puerarin dosage is 5 mg / kg (BW) ~ 20 mg / kg (BW).

3. The composition according to claim 1 or 2 for improving obesity-induced diabetes, characterized in that: The Bifidobacterium longum ( Bifidobacterium longum BKR-011 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 26, 2021, with accession number CGMCC No. 22949.

4. The composition according to claim 1 or 2 for improving obesity-induced diabetes, characterized in that: The puerarin is either elemental puerarin or an extract containing puerarin obtained from natural extraction.

5. The composition according to claim 4 for improving obesity-induced diabetes, characterized in that: The extract containing Puerarin is from the legume kudzu (Pueraria lobata). Pueraria lobata ( Willd .) Ohwi Leguminosae plant, kudzu vine ( Pueraria thunbergiana Benth. Puerarin-containing extracts are obtained by extracting one or more of the following plants: puerarin and others.

6. The application of the composition of claim 1 for improving obesity-induced diabetes, characterized in that: The composition is used in the preparation of medicaments, health foods and other foods for the treatment or improvement of diabetes and its complications induced by obesity.

7. A preparation for improving obesity-induced diabetes, characterized in that: The formulation comprises the composition of claim 1, wherein the composition is Bifidobacterium longum BKR-011 and puerarin.

8. The preparation according to claim 7 for improving obesity-induced diabetes, characterized in that: The dosage forms mentioned include, but are not limited to, tablets, powders, capsules, and suspensions.

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