A synergistically acting auxiliary blood glucose lowering composition and use thereof

Abstract

The application discloses a kind of auxiliary hypoglycemic compositions with synergistic effect and application thereof, and belongs to the field of biological medicine.The auxiliary hypoglycemic composition includes 1-deoxynojirimycin and theaflavin.According to Chou-Talalay model, the combined action index is calculated, and it is proved that 1-deoxynojirimycin and theaflavin have synergistic effect in auxiliary hypoglycemic effect.The auxiliary hypoglycemic pharmaceutical composition provided by the application is derived from Chinese medicine mulberry leaf and black tea, and the components are safe with little side effect, which lays a theoretical foundation for developing a new way of combined hypoglycemic components of traditional Chinese medicine.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to an adjunctive hypoglycemic composition with synergistic effects and its application. Background Technology

[0002] Type 2 diabetes mellitus (T2DM) is a prevalent endocrine disorder that seriously endangers human health. Conventional drug treatment for T2DM primarily involves chemotherapy; however, these drugs cannot maintain blood glucose levels for extended periods, and even patients using them may experience secondary T2DM symptoms and drug side effects. Traditional Chinese medicine (TCM) monomers have been reported to have anti-T2DM effects, exerting their therapeutic effects by stimulating insulin secretion, protecting and repairing pancreatic β-cells, promoting glucose utilization, and improving oxidative stress levels. Therefore, exploring the active ingredients in TCM that may possess hypoglycemic capabilities and studying the effects of compound formulations is of great significance for the prevention and treatment of T2DM.

[0003] Mulberry leaves, a traditional Chinese medicine, are the dried leaves of the mulberry tree (Morus alba), a plant in the Moraceae family. They possess properties such as dispersing wind-heat, clearing the lungs and moistening dryness, and clearing the liver and improving eyesight. Mulberry leaves are rich in various active ingredients, including alkaloids, flavonoids, polysaccharides, and anthocyanins. Current scientific research indicates that 1-deoxynojirimycin (DNJ) is a major component of mulberry leaves, exhibiting antibacterial, antioxidant, anti-inflammatory, neuroprotective, antitumor, anti-HIV, anti-atherosclerotic, and blood sugar-lowering functions. DNJ indirectly affects blood sugar levels by interfering with humoral regulation. DNJ promotes the secretion of peptides and serotonin through the sodium-glucose cotransporter 3 (NGT3), thereby synergistically promoting glucose reabsorption in the kidneys and lowering blood sugar levels along with other family members.

[0004] Black tea, a plant belonging to the Theaceae family of the order Theales, possesses properties that promote blood circulation, reduce edema, fight inflammation, kill bacteria, and dilate blood vessels. Current research suggests that black tea is beneficial for diabetic patients, stimulating insulin secretion and lowering postprandial blood glucose peaks. During processing, black tea undergoes a chemical reaction centered on the enzymatic oxidation of tea polyphenols, resulting in a reduction of over 90% of polyphenols and the production of new components such as theaflavins and thearubigins. Theaflavins (TFs) are the main flavonoid components in black tea. Flavonoids can improve metabolic disorders by scavenging free radicals and increasing the activity of antioxidant enzymes in the liver; they may also lower blood glucose by protecting and repairing pancreatic islet cells and reducing insulin resistance. Tu Youying published a study in *Molecules* entitled "Study on the Improvement of Hyperlipidemia-Induced Insulin Resistance by Theaflavins through the Mitochondrial Pathway," discovering the ameliorative effect of high-purity theaflavins on a palmitic acid-induced insulin-resistant HepG2 cell model and its underlying mechanism, providing new theoretical basis for developing theaflavins for the treatment of diabetes.

[0005] However, current domestic and international research has not yet combined these two traditional Chinese medicine components with potential hypoglycemic effects to achieve the effect of assisting in hypoglycemia. Therefore, selecting appropriate doses and ratios of DNJ and TFs for synergistic use and studying the effects of synergistic use is of great significance for developing new approaches to combine traditional Chinese medicine components for hypoglycemia. Summary of the Invention

[0006] The purpose of this invention is to provide an auxiliary hypoglycemic composition with synergistic effects and its application, thereby solving the problems existing in the prior art. The auxiliary hypoglycemic composition provided by this invention is derived from the traditional Chinese medicines mulberry leaves and black tea. Its components are safe and have few side effects, laying a theoretical foundation for developing new approaches to combined hypoglycemic effects using traditional Chinese medicine components.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] The present invention provides an auxiliary hypoglycemic composition with synergistic effect, the auxiliary hypoglycemic composition comprising 1-deoxynojirimycin and theaflavins.

[0009] Furthermore, the mass ratio of 1-deoxynojirimycin to the theaflavins is (1-3):(1-3).

[0010] Preferably, the mass ratio of 1-deoxynojirimycin to the theaflavins is 2:1.

[0011] The present invention also provides the use of the aforementioned adjunctive hypoglycemic composition in the preparation of a medicament for treating diabetes.

[0012] Furthermore, the diabetes includes type 2 diabetes.

[0013] The present invention also provides a medicament for treating diabetes, the medicament comprising the aforementioned adjunctive hypoglycemic composition.

[0014] Furthermore, the mass ratio of 1-deoxynojirimycin to the theaflavins is (1-3):(1-3).

[0015] Preferably, the mass ratio of 1-deoxynojirimycin to the theaflavins is 2:1.

[0016] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0017] Furthermore, the diabetes includes type 2 diabetes.

[0018] The present invention discloses the following technical effects:

[0019] This invention employs network pharmacology bioinformatics to predict key targets and pathways of 1-deoxynojirimycin and theaflavins in synergistic hypoglycemic effects. The synergistic hypoglycemic ability of 1-deoxynojirimycin and theaflavins is evaluated based on their α-glucosidase inhibition capacity. The combined effect index is calculated using the Chou-Talalay model, confirming that 1-deoxynojirimycin and theaflavins have a synergistic effect in assisting hypoglycemia.

[0020] The hypoglycemic adjuvant composition provided by this invention is derived from the traditional Chinese medicine mulberry leaves and black tea. It has safe ingredients and few side effects, laying a theoretical foundation for developing new approaches to hypoglycemic adjuvants. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The diagrams show the molecular docking of TFs-T2DM-target, where A represents the molecular docking of TFs-PTGS2 and B represents the molecular docking of TFs-MMP9.

[0023] Figure 2 The α-glucosidase inhibition rates of DNJ, TFs, and the complex are expressed as mean ± standard deviation, n = 3; * represents P < 0.05, ** represents P < 0.01, relative to the TFs group; # represents P < 0.05, ## represents P < 0.01, relative to the DNJ group;

[0024] Figure 3The inhibition rates of α-salivary amylase by DNJ, TFs, and the complex are expressed as mean ± standard deviation, n = 3; * represents P < 0.05, ** represents P < 0.01, relative to the TFs group; # represents P < 0.05, ## represents P < 0.01, relative to the DNJ group;

[0025] Figure 4 The α-glucosidase inhibition rates of DNJ-TFs at different concentration ratios are shown in Figure A, where A represents the α-glucosidase inhibition rates obtained at each ratio when the final concentration of the mixed drug is 10 μg / mL; B represents the α-glucosidase inhibition rates obtained at each ratio when the final concentration of the mixed drug is 20 μg / mL; and C represents the α-glucosidase inhibition rates obtained at each ratio when the final concentration of the mixed drug is 30 μg / mL.

[0026] Figure 5 The effects of different concentrations of DNJ (A) and TFs (B) on the proliferation of HepG2 cells were detected using the CCK-8 assay.

[0027] Figure 6 The glucose content in the supernatant of HepG2 cells;

[0028] Figure 7 This refers to the uptake capacity of HepG2 cells for 2-NBDG.

[0029] Figure 8 This refers to the glycogen synthesis capacity of HepG2 cells.

[0030] Figure 9 Effect of DNJ-TFs on PA-induced PTGS2 expression in HepG2 cells. Scale bar: 20 μm;

[0031] Figure 10 Effect of DNJ-TFs on PA-induced MMP9 expression in HepG2 cells. Scale bar: 20 μm.

[0032] Figure 11 The effects of DNJ-TFs intervention on the MMP9 / TNF-α / Akt / GLUT2 pathway in PA-induced insulin-resistant HepG2 cells were investigated. A: MMP9 protein expression level; B: RANKL protein expression level; C: TNF-α protein expression level; D: Akt and p-Akt protein expression levels; E: GLUT2 protein expression level.

[0033] Figure 12The effects of DNJ-TFs intervention on the PTGS2 / cAMP / GSK 3α / GSK 3β pathway in PA-induced insulin-resistant HepG2 cells were investigated. A: PTGS2 protein expression level; B: cAMP protein expression level; C: GSK 3α and p-GSK 3α protein expression levels; D: GSK 3β and p-GSK 3β protein expression levels.

[0034] Figure 13 Changes in TNF-α levels in HepG2 cells after the addition of an MMP9 inhibitor;

[0035] Figure 14 Changes in GSK 3α and GSK 3β levels in HepG2 cells after the addition of PTGS2 inhibitor. Detailed Implementation

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0038] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0039] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0040] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0041] Example 1: Network pharmacology analysis of the mechanism of action of DNJ-TFs against type 2 diabetes.

[0042] Network pharmacology was used to elucidate the potential molecular and target mechanisms of 1-deoxynojirimycin (DNJ) and theaflavins in combating type 2 diabetes mellitus (T2DM). Based on data extracted from the Swiss Target Prediction and PharmMapper databases, 127 potential targets for DNJ and TFs were identified. Data extracted from the DisGenet, DrugBank, OMIM, and GeneCards databases identified 5365 T2DM-related disease targets. Mapping these two datasets to construct a "DNJ, TFs-T2DM-target" dataset revealed 90 common targets. Network topology parameter analysis using NetworkAnalyzer, with the average "Degree" as a threshold, identified 36 core targets, including PTGS2, VEGFA, MMP9, DRD2, ACE, and GLB1, as shown in Table 1.

[0043] Table 1. Topological parameters of 36 key targets of DNJ-TFs in anti-type 2 diabetes.

[0044]

[0045]

[0046] The main biological processes by which 1-deoxynojirimycin and theaflavins combat T2DM include cellular carbohydrate metabolism, polysaccharide digestion, sugar alcohol and lipid metabolism, and angiotensin maturation. The signaling pathways they act upon mainly include the TNF signaling pathway, cAMP signaling pathway, and galactose metabolism pathway. Molecular docking results are shown in Table 2 and... Figure 1 As shown.

[0047] Table 2. Results of DNJ-TFs docking with key target molecules

[0048]

[0049] Table 2 shows that 1-deoxynojirimycin and theaflavins have high binding affinity to potential targets cyclooxygenase 2 (PTGS2) and matrix metalloproteinase 9 (MMP9). Figure 1 It was demonstrated that the binding of both TFs-PTGS2 and TFs-MMP9 groups can generate hydrogen bonds. Network pharmacology methods verified that 1-deoxynojirimycin and theaflavins can intervene in T2DM by binding with PTGS2 and MMP9.

[0050] Example 2: Analysis of the synergistic hypoglycemic effect based on α-glucosidase and α-salivary amylase experiments

[0051] I. Experimental Methods

[0052] 1. Detection of the synergistic hypoglycemic ability of DNJ-TFs based on α-glucosidase inhibition ability

[0053] α-glucosidase and D-glucopyranoside were prepared into solutions of 0.26 U / mL and 1.8 mg / mL, respectively, using 0.1 mol / L sodium phosphate buffer (pH 6.8). 50 μL of phosphate buffer, 50 μL of α-glucosidase solution, and 50 μL of sample solution were transferred to a 96-well plate and incubated at 37 °C for 10 min. Then, 50 μL of D-glucopyranoside was added to initiate the reaction, and the reaction was carried out at 37 °C for 15 min. The reaction was terminated with 80 μL of 1 mol / L Na₂CO₃, and the absorbance was measured at 405 nm using a microplate reader. The blank group used buffer instead of α-glucosidase, and the control group used buffer instead of the sample. The percentage of α-glucosidase inhibition was calculated according to the manufacturer's instructions.

[0054] 2. Testing the synergistic hypoglycemic ability of DNJ-TFs based on α-salivary amylase inhibition.

[0055] Following the instructions of the α-salivary amylase assay kit, pre-incubate DNJ or TFs with 0.1 U / mL α-salivary amylase in sodium phosphate buffer at 37°C for 15 min. Add 50 μL of 0.4% (w / v) starch substrate to the reaction mixture, bringing the total volume to 200 μL, and incubate the reaction mixture at 37°C for 10 min. After incubation, stop the reaction by adding 1 mL of dinitrosalicylic acid (DNS) reagent to the reaction mixture. Then heat the mixture at 100°C for 10 min. After cooling the mixture to room temperature, read the absorbance at 540 nm using a microplate reader. Calculate the percentage of α-salivary amylase inhibition according to the manufacturer's instructions.

[0056] 3. Evaluation methods for synergistic hypoglycemic effects

[0057] Chou-Talalay model method: The calculation method of the combined effect index is shown in formula (1).

[0058]

[0059] In the formula:

[0060] (D)1---The effective concentration of test substance 1 when the combined effect of test substance 1 and test substance 2 is X;

[0061] (D)2---The effective concentration of test substance 2 when the combined effect of test substance 1 and test substance 2 is X;

[0062] (D X)1---The effective concentration when test substance 1 alone has an effect of X;

[0063] (D X )2---The effective concentration when test substance 2 alone has an effect of X;

[0064] (CI) X ---Combined effect index.

[0065] When CI > 1, the two have an antagonistic effect; when CI = 1, the two have an additive effect; when CI < 1, the two have a synergistic effect.

[0066] II. Results

[0067] 1. Evaluation of the synergistic hypoglycemic effect of DNJ-TFs based on α-glucosidase

[0068] The DNJ and TFs stock solutions were diluted to prepare test solutions of 30, 20, 10, 5, and 1 μg / mL. Simultaneously, DNJ and TFs solutions of the same concentration were mixed at a 1:1 volume ratio for a synergistic in vitro α-glucosidase experiment. The experimental results are as follows: Figure 2 As shown.

[0069] Depend on Figure 2 It can be seen that DNJ has a higher α-glucosidase inhibition rate when combined with TFs than when administered alone, with a significant difference starting from 5 μg / mL, indicating that the two have a certain synergistic effect on the α-glucosidase inhibition ability.

[0070] 2. Evaluation of the synergistic hypoglycemic effect of DNJ-TFs based on α-salivary amylase

[0071] Solutions of DNJ and TFs with a final concentration of 1 mg / mL were prepared separately. These solutions were then serially diluted to prepare test solutions of 30, 20, 10, 5, and 1 μg / mL. Simultaneously, DNJ and TFs solutions of the same concentration were mixed at a 1:1 volume ratio and subjected to a synergistic in vitro α-salivary amylase assay according to the instructions of the α-salivary amylase assay kit. The experimental results are as follows: Figure 3 As shown.

[0072] Depend on Figure 3 It can be seen that the inhibition rate of α-salivary amylase by DNJ in combination with TFs is higher than that of DNJ or TFs alone. The difference is significant starting from 1 μg / mL, indicating that the two have a certain synergistic effect on the inhibitory ability of α-salivary amylase.

[0073] 3. Evaluation of the synergistic hypoglycemic capacity of DNJ-TFs based on the Chou-Talalay model

[0074] First, the inhibition rates of DNJ and TFs at different concentrations on α-glucosidase and α-salivary amylase were calculated, and the IC50 was calculated. 50 Value, based on Figure 2 and Figure 3 Calculate IC50 after combined administration 50 The value was substituted into the Chou-Talalay model calculation software "CompuSyn" according to formula (1) to analyze the combined effect index (CI) of the two. The CI calculation results are shown in Table 3 and Table 4.

[0075] Table 3. α-glucosidase assay: DNJ, TFs, and IC50 after synergistic reaction. 50 and CI value

[0076]

[0077] Table 4. DNJ, TFs, and IC50 after synergistic effect in α-salivary amylase assay. 50 and CI value

[0078]

[0079] Table 3 shows that after drug administration, the IC50 inhibition rate of DNJ on α-glucosidase was [value missing]. 50 The IC50 value of TFs for α-glucosidase inhibition was approximately 27.01 μg / mL. 50 The concentration was approximately 16.13 μg / mL, and the IC50 after synergy between the two was [value missing]. 50 The concentration was approximately 10.93 μg / mL. After substituting the values ​​into the Chou-Talalay model calculation software "CompuSyn", the CI index was calculated to be 0.85, which is less than 1. This indicates that DNJ, in conjunction with TFs, has a synergistic effect on the inhibitory capacity of α-glucosidase.

[0080] Table 4 shows that after drug administration, the IC50 inhibition rate of DNJ on α-salivary amylase was [value missing]. 50 The IC50 value of TFs for inhibiting α-salivary amylase was approximately 22.69 μg / mL. 50 The concentration was approximately 12.46 μg / mL, and the IC50 after synergy between the two was [missing information]. 50 The concentration was approximately 11.01 μg / mL. After substituting the values ​​into the Chou-Talalay model calculation software "CompuSyn", the CI index was calculated to be 0.76, which is less than 1. This indicates that DNJ, in conjunction with TFs, has a synergistic effect on the inhibitory capacity of α-salivary amylase.

[0081] Example 3

[0082] 1.1 Optimization of the optimal concentration ratio of deoxynojirimycin and theaflavins in synergistic use

[0083] To investigate the optimal dosing ratio of DNJ to TFs at a constant final concentration, and to provide a reference for subsequent cell and animal experiments, α-glucosidase inhibition experiments were conducted with DNJ to TFs ratios of 1:1, 1:2, 1:3, 3:1, and 2:1 at final concentrations of 10, 20, and 30 μg / mL. DNJ or TFs were pre-cultured with 0.1 U / mL α-salivary amylase in sodium phosphate buffer at 37°C for 15 min. 50 μL of 0.4% (w / v) starch substrate was added to the reaction mixture, bringing the total volume to 200 μL, and the reaction mixture was incubated at 37°C for 10 min. After incubation, the reaction was stopped by adding 1 mL of dinitrosalicylic acid (DNS) reagent to the reaction mixture. The mixture was heated at 100°C for 10 min. After cooling to room temperature, the absorbance was read at 540 nm using a microplate reader. The percentage of α-salivary amylase inhibition was calculated according to the instructions, and the combined effect index was calculated using the Chou-Talalay formula. Ultimately, at a fixed final concentration, a DNJ to TFs concentration ratio of 2:1 resulted in the optimal α-glucosidase inhibition rate, with a combined effect index of 0.894, higher than other groups. Figure 4 Therefore, a DNJ:TFs mass concentration ratio of 2:1 was selected for subsequent experiments, meaning the optimal mass ratio when the two are used together is DNJ:TFs = 2:1.

[0084] 2. Verification of molecular docking of DNJ-TFs with specific sites of α-glucosidase and α-salivary amylase

[0085] To determine the binding affinity of DNJ-TFs to the binding sites of α-glucosidase and α-salivary amylase, molecular docking of DNJ-TFs with α-glucosidase and α-salivary amylase was performed using Schrodinger software. Three available crystal structures were used: 2QMJ (N-terminus of maltase-glucosidase), 3TOP (C-terminus of maltase-glucosidase), and 3LPP (N-terminus of sucrase-isomaltase). The results are shown in Table 5.

[0086] Table 5. Binding energies of DNJ, TFs with α-glucosidase (2QMJ, 3LPP) and α-salivary amylase (3TOP)

[0087]

[0088] Table 5 shows that for N-terminal maltase glucosylamylase (2QMJ), the binding energy of DNJ at the active site is -5.60 kcal / mol, and the binding energy of TFs is -9.50 kcal / mol. For C-terminal maltase glucosylamylase (3TOP), the binding energy of DNJ at the active site is -5.90 kcal / mol, and the binding energy of TFs is -10.20 kcal / mol. For N-terminal sucrose isomaltase (3LPP), the binding energy of DNJ at the active site is -5.40 kcal / mol, and the binding energy of TFs is -8.80 kcal / mol.

[0089] For the three subunits of α-glucosidase and α-salivary amylase, the binding energy of DNJ to TFs is less than -5, indicating a greater affinity. This may be the reason why they have a strong inhibitory rate of α-glucosidase and α-salivary amylase after combined administration.

[0090] Example 41 - Cellular Experiment on the Synergistic Use of Deoxynojirimycin and Theaflavins to Assist in Hypoglycemic Control

[0091] 1.1-Cytotoxicity assay of deoxynojirimycin and theaflavins

[0092] Human-derived hepatocellular carcinoma cells (HepG2) purchased from the Cell Bank of the Chinese Academy of Sciences, China, after culture and resuscitation, were treated with DNJ (concentrations of 50, 40, 30, 20, and 10 μg / mL) and TFs (concentrations of 40, 30, 20, 10, and 5 μg / mL) for 24 h, with three replicates per group. After treatment, the cells were incubated for 24 h. The effects of DNJ and TFs on the proliferation of HepG2 cells were determined. Cell proliferation was calculated using the following formula:

[0093]

[0094] Effects of DNJ and TFs on the proliferation of HepG2 cells, such as Figure 5 As shown, by Figure 5 As shown in A, low concentrations of DNJ (20 or 10 μg / mL) affected the proliferation of HepG2 cells, but not significantly (P>0.05). High concentrations of DNJ (50, 40, or 30 μg / mL) significantly inhibited cell proliferation. These results indicate that DNJ concentrations of 30 μg / mL and above are highly toxic and should not be used. Therefore, DNJ solutions with concentrations of 20 and 10 μg / mL were selected for subsequent experiments.

[0095] Depend on Figure 5As shown in B, low concentrations of TFs (10 or 5 μg / mL) affected the cell proliferation of HepG2 cells, but not significantly (P>0.05). High concentrations of TFs (40, 30 or 20 μg / mL) significantly inhibited cell proliferation. These results indicate that TFs at concentrations of 20 μg / mL and above are highly toxic and should not be used. Therefore, TF solutions with concentrations of 10 and 5 μg / mL were selected for subsequent experiments.

[0096] 2. Cell experiments

[0097] DNJ solutions were selected at concentrations of 20 and 10 μg / mL, and TFs solutions at concentrations of 10 and 5 μg / mL. Based on the conclusion from the α-glucosidase experiment that a concentration ratio of 2:1 yielded the best hypoglycemic effect, 10 μg / mL DNJ solution and 5 μg / mL TFs solution were designated as low-concentration intervention group A (hereinafter referred to as group A), and 20 μg / mL DNJ solution and 10 μg / mL TFs solution were designated as high-concentration intervention group B (hereinafter referred to as group B). Both groups were prepared using PA medium containing 300 μM and subjected to CCK-8 assays. It was found that neither group significantly affected cell proliferation (P>0.05), but both showed a certain proliferative effect. Therefore, groups A and B will be used as intervention groups for subsequent experiments.

[0098] HepG2 cells were divided into 5 groups, with 3 replicates per group: a blank control group (Con); a metformin (10 μg / mL) positive control group; a model group (Model) treated with PA (300 μM) for 24 h; a low-dose PA+DNJ-TFs group A (containing 300 μM PA, 10 μg / mL DNJ, and 5 μg / mL TFs in the culture medium); and a high-dose DNJ-TFs group B (containing 300 μM PA, 20 μg / mL DNJ, and 10 μg / mL TFs in the culture medium).

[0099] The glucose oxidase micro-method was used to detect the glucose content in the supernatant of HepG2 cells, such as... Figure 6 As shown, PA (300 μM) significantly increased the glucose content in the supernatant of HepG2 cells (P<0.01), while the drug-treated group significantly reversed this trend (P<0.01) and improved glucose consumption capacity.

[0100] Glucose uptake was measured using the 2-NBDG fluorescence method. The effects of DNJ-TFs on 2-NBDG uptake in insulin-resistant HepG2 cells were compared. The results are as follows: Figure 7As shown, PA (300 μM) significantly reduced the uptake of 2-NBDG by HepG2 cells (P<0.01), while the treatment group significantly reversed this trend (P<0.05, P<0.01). These results indicate that DNJ-TFs can improve the insulin sensitivity of PA-treated HepG2 cells.

[0101] The anthrone method was used to detect glycogen content in cells, and the effects of DNJ-TFs on glycogen synthesis in insulin-resistant HepG2 cells were compared. The results are as follows: Figure 8 As shown, PA (300 μM) caused a significant decrease in glycogen content in HepG2 cells (P<0.01), while the drug-treated group significantly reversed this trend (P<0.05, P<0.01), restoring glycogen synthesis homeostasis. These results indicate that DNJ-TFs can improve the glycogen synthesis disorder in PA-treated HepG2 cells and restore the body's glycogen synthesis capacity.

[0102] Immunofluorescence assays revealed that PA intervention increased the fluorescence intensity of PTGS2 and decreased the fluorescence intensity of MMP9, while intervention with positive control drugs and DNJ-TFs reversed this phenomenon. These results indicate that DNJ-TFs can downregulate PA-induced increases in PTGS2 expression, increase the fluorescence intensity of MMP9, and improve cellular insulin resistance. Figure 9-10 ).

[0103] Western blotting analysis revealed changes in downstream molecules at two key targets after drug binding. The results are as follows: Figure 11-12 As shown in the figure, DNJ-TFs upregulated GLUT2 protein levels and significantly reversed PA-induced changes in p-Akt levels (P<0.01), while increasing MMP9 protein levels and reducing the production of inflammatory factors, thus alleviating insulin resistance symptoms by reducing inflammation and improving glucose transport. These results indicate that DNJ-TFs may improve glucose uptake and insulin sensitivity through the MMP9 / TNF-α / Akt / GLUT2 pathway; DNJ-TFs can inhibit PTGS2 protein levels and reverse PA-induced changes in cAMP levels (P<0.05), reduce GSK 3α and GSK 3β phosphorylation, and alleviate insulin resistance symptoms by improving the body's glycogen synthesis capacity. These results suggest that DNJ-TFs may improve glucose uptake and insulin sensitivity. PA induces insulin resistance in HepG2 cells through the PTGS2 / cAMP / GSK 3α / GSK 3β pathway.

[0104] MMP9 inhibitors or PTGS2 inhibitors were added to the original drug-treated groups. After 24 hours of culture, changes in the content of each component were observed using an ELISA kit to determine whether DNJ-TFs affect downstream components by intervening in MMP9 and PTGS2. Results are as follows: Figure 13-14 After the addition of an MMP9 inhibitor, TNF-α levels significantly increased compared to the group without the inhibitor (P<0.01), and DNJ-TFs still showed an inhibitory trend within the same group (P<0.05, P<0.01). This demonstrates that MMP9 inhibition exacerbates TNF-α levels, and the stable presence of MMP9 can inhibit TNF-α to some extent. After the addition of a PTGS2 inhibitor, GSK 3α and GSK 3β levels significantly decreased compared to the group without the inhibitor (P<0.01), and DNJ-TFs still showed an inhibitory trend within the same group (P<0.05, P<0.01). This demonstrates that PTGS2 inhibition also inhibits GSK 3α and GSK 3β levels, and PTGS2 may cause insulin resistance damage by interfering with the body's normal glycogen synthesis capacity.

[0105] This embodiment examined the effects of synergistic administration of 1-deoxynojirimycin and theaflavins on the glucose consumption, 2-NBDG uptake, and glycogen synthesis in HepG2 cells after PA intervention. Immunofluorescence was used to verify the expression of two key proteins predicted by network pharmacology during insulin resistance injury. Western blotting was used to analyze the changes in downstream molecules after the two key targets bound to the drugs. Finally, administration of MMP9 and PTGS2 inhibitors verified that the drugs affect insulin resistance injury through the two key proteins, thereby influencing the body's inflammatory damage and glycogen synthesis homeostasis, thus intervening in the condition of T2DM.

[0106] Example 4: Effect of combined administration of DNJ-TFs on the improvement of type 2 diabetes model mice induced by high-fat diet.

[0107] 1. Preparation of experimental animals and type 2 diabetic mouse models

[0108] Laboratory mice (4-week-old male C57BL / 6J mice, SPF grade) were fed with standard feed in an environment with a temperature of 20-25℃ and a relative humidity of 50-70%. After one week of acclimatization to the new environment, they were divided into groups. Forty mice were randomly divided into 5 groups and housed individually.

[0109] The control group (n=8) continued normal feeding; the model group (n=8) was fed a high-sugar, high-fat diet; the acarbose positive control group (Aca group) (n=8) was fed a high-fat diet, and acarbose was dissolved in drinking water at 10 mg / mL, with the dressing changed daily; the DNJ-TFs synergistic low-dose administration group A (group A) (n=8) was fed a high-fat diet, and DNJ was dissolved in drinking water at 10 mg / mL and TFs at 5 mg / mL, with the dressing changed daily; the DNJ-TFs synergistic high-dose administration group B (group B) (n=8) was fed a high-fat diet, and DNJ was dissolved in drinking water at 20 mg / mL and TFs at 10 mg / mL, with the dressing changed daily.

[0110] 2. Blood glucose measurement

[0111] Ten weeks after administration, mice were fasted for 12 hours but allowed free access to water. Blood was then collected from the tail vein to measure their fasting blood glucose levels.

[0112] 3. Results Analysis

[0113] The results of blood glucose measurement in each group of mice are shown in Table 6.

[0114] Table 61 - Effects of deoxynojirimycin and theaflavins on blood glucose levels in mice with type 2 diabetes induced by a high-sugar diet.

[0115]

[0116] Note: Compared with the control group, ##: P < 0.01. Compared with the model group, **: P < 0.01.

[0117] Table 6 shows that after 10 weeks of induction with a high-sugar, high-fat diet, blood glucose levels in the model group increased significantly, indicating that the T2DM model was successfully established. Blood glucose levels were significantly controlled after 10 weeks of treatment with the positive control drug group, the low-dose group receiving synergistic administration of 1-deoxynojirimycin and theaflavins, and the high-dose group receiving synergistic administration of 1-deoxynojirimycin and theaflavins.

[0118] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of an adjuvant hypoglycemic composition with synergistic effects in the preparation of a medicament for treating diabetes, characterized in that, The adjunctive hypoglycemic composition is 1-deoxynojirimycin and theaflavins; The mass ratio of 1-deoxynojirimycin to the theaflavins is 2:

1.

2. The application according to claim 1, characterized in that, The diabetes mentioned is type 2 diabetes.

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