A composition for the prevention or treatment of diabetes and its application

Abstract

This invention discloses a composition for the prevention or treatment of diabetes and its application, relating to the field of pharmaceutical technology. The composition is a combination of any of the following components: a mitochondrial protectant, a vanadium compound, and a hormone / pheromone. It can improve pancreatic β-cell activity and control fasting blood glucose, satiety blood glucose, glucose tolerance, and insulin levels within dietary dosage ranges.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more specifically to a composition for the prevention or treatment of diabetes and its application. Background Technology

[0002] Diabetes mellitus (DB) is a metabolic disease characterized by hyperglycemia, caused by insulin secretion defects and / or its biological dysfunction. It is divided into two main categories: insulin-dependent type I and non-insulin-dependent type II. Type I diabetes is mainly caused by the death of pancreatic β cells due to genetic and / or autoimmune damage, leading to insufficient insulin secretion. Type II diabetes is caused by various factors (life stress, obesity, unhealthy lifestyle, drug side effects, etc.) leading to tissue insulin resistance (IR, i.e., a reduced ability to utilize glucose per unit of insulin stimulation), resulting in insufficient insulin sensitivity in tissues, making normal insulin levels insufficient to lower blood glucose. However, overt diabetes generally involves both insulin resistance and insulin secretion defects.

[0003] The danger of diabetes lies in a series of metabolic disorders and functional impairments marked by high blood sugar. Although high blood sugar does not directly cause tissue damage, diabetes-related disorders of glucose and lipid metabolism and their associated signal transduction can lead to a series of serious complications that severely endanger health, mainly including: cardiovascular and cerebrovascular diseases, neuropathy, eye diseases, diabetic nephropathy, and diabetic foot. Furthermore, if insulin resistance occurs in the central nervous system, it may affect amyloid protein (Aβ) metabolism, a significant cause of Alzheimer's disease (AD); therefore, AD is also known as type III diabetes.

[0004] Statistics show that approximately 10% of the world's population currently suffers from diabetes, with 90-95% of these cases being type 2 diabetes. This number is continuously increasing. Diabetes treatment is currently extremely expensive; diabetic patients use medical services 3-4 times more frequently than non-diabetic individuals (including significantly increased hospitalizations and outpatient visits), and their medical expenses are 9 times higher than those of their age and sex without diabetes. Data shows that since 1993, the economic burden of diabetes has increased year by year, with direct medical expenses due to diabetes accounting for 13% of total national medical expenditures in 2010, and projected to reach 360 billion yuan by 2030. Therefore, research on the pathology and treatment of diabetes has always been a key area of ​​drug development.

[0005] Currently, the widely used drugs for treating diabetes mainly fall into the following categories: (1) Insulin preparations. (2) Carbohydrate absorption inhibitors: mainly α-glucosidase inhibitors. Representative drugs include acarbose (Glucobay) and voglibose. (3) Glucose excretion inhibitors: mainly sodium-glucose cotransporter 2 (SGLT2) inhibitors, such as canagliflozin, dapagliflozin, and empagliflozin. (4) Glycogenolysis inhibitors: these drugs mainly include glycogen phosphorylase inhibitors and glucokinase agonists. (5) Insulin secretion stimulants: these drugs mainly fall into the following three categories: (a) Sulfonylureas, such as glimepiride; (b) Glucagon-like peptide-1 (GLP-1) receptor agonists, such as byetda, exenatide, and liraglutide; (c) Dipeptidyl peptidase-4 (DDP-IV) inhibitors, such as sitagliptin, saxagliptin, vildagliptin, allogliptin, and linagliptin. (6) Insulin sensitizing drugs: These drugs mainly fall into two categories: biguanide drugs, such as metformin and phenformin; and thiazolidinedione drugs, such as rosiglitazone and pioglitazone.

[0006] Although a large number of insulin preparations and other hypoglycemic drugs are available on the market, the situation regarding the progression of diabetes is indeed becoming increasingly serious. This is due to several factors: the hypoglycemic mechanisms of these drugs are insufficient; simply controlling blood sugar is not enough to effectively reduce diabetic complications. Most hypoglycemic drugs are short-acting and must be taken for life, making side effects a significant concern. Currently, most drugs are not simple or convenient to use. Treatment cost is also a major issue; newly developed antidiabetic drugs are mostly expensive biotechnology drugs, limiting their accessibility to the general public. Therefore, developing combinations for the prevention and treatment of diabetes has become an important research direction. Summary of the Invention

[0007] In view of the above-mentioned technical problems existing in the prior art, the present invention provides a composition for the prevention or treatment of diabetes and its application, which improves the activity of pancreatic β cells and controls fasting blood glucose and satiety blood glucose within the range of dietary dosage.

[0008] This invention discloses a composition for the prevention or treatment of diabetes, comprising a combination of any of the following components: a mitochondrial protectant, a vanadium compound, and a hormone / pheromone. These components can be combined in pairs or in all three.

[0009] The vanadium compounds are selected from inorganic vanadium compounds and organic vanadium compounds; the organic vanadium compounds include vanadium oxyoxide complexes, vanadium peroxide complexes, hydroxylamine vanadium complexes, and vanadium heteropolyacid complexes; wherein, the vanadium heteropolyacid complexes include vanadium benzoate, vanadium aspirin, vanadium acetylacetonate, bis(2-methyl-3-hydroxy-4-pyranone)vanadium oxyoxide (BMOV), 3-hydroxy-2-ethyl-4-pyranone (BEOV), and 3-hydroxy-2-isopropyl-4-pyranone (BI). Vanadium oxide (BOV), 3-hydroxy-2-n-butyl-4-pyranone (BnBOV), bis(pyridine-2-carboxylic acid)vanadium oxide (VO(Pa)2), vanadium pyridinedicarboxylate (VO-DPA), vanadium pyridinecarboxylate phthalamide oxyvanadium (VO-PAM), vanadium methylpyridinecarboxylate (VO-MPA), vanadium pyridinecarboxylate (VO-PA), vanadium malonic acid hydroxylamine oxyvanadium, vanadium oxalate hydroxylamine oxyvanadium, vanadium valine hydroxylamine oxyvanadium, vanadium leucine hydroxylamine oxyvanadium, and vanadium oxide complexes (VOL1-VOL6). Among them, the vanadium oxide complexes are prepared by introducing an antioxidant group at the 2-position of kojic acid pyranone as the parent core, such as the BSOV series, and by introducing antioxidant functional ligands with hydroxyaniline or hydroxyphenylethylamine derivative structures with aminotriacetic acid as the carbon skeleton.

[0010] The mitochondrial protectant is selected from exogenous antioxidants and endogenous antioxidants.

[0011] Exogenous antioxidants include coenzyme Q10, alpha-lipoic acid, idebenone, vitamin antioxidants, glutathione, tea polyphenols, and flavonoid antioxidants.

[0012] The vitamin antioxidants include B vitamins, vitamin C, vitamin E, and vitamin K.

[0013] Endogenous antioxidants include compounds that intervene in the following endogenous targets to enhance endogenous antioxidant stress capacity: nuclear factor NFE2-related factor (Nrf2), peroxisome proliferator-activated receptors (PPARs), adenosine monophosphate-activated protein kinase (AMPK), and uncoupling proteins (UCPs).

[0014] Among them, compounds that intervene in the endogenous target Nrf2 include α,β-unsaturated carbonyl compounds, which are Nrf2 agonists.

[0015] Nrf2 agonists include cinnamaldehyde, vanillin, syringaldehyde, asaraldehyde, coniferaldehyde, 5-hydroxymethylfurfural, 3,4-dimethoxybenzaldehyde, trans-3,5-dimethoxy-4-hydroxycinnamaldehyde, chalcone, 2,3-dihydro-3,5-dihydroxy-6-methyl-4(H)-pyran-4-one, 4-methoxy-6-methyl-2H-pyranone, genistein, 4-methoxy-6-[(E)-2-(4-methoxyphenyl)vinyl]-2H-pyran-2-one, kavanone, geraniol, 4-hydroxy-4'-methoxychalcone, licorice chalcone, phenethyl cinnamate, cinnamic acid, p-methoxycinnamic acid, o-methoxycinnamic acid, dimethyl fumarate, methyl 2-methoxy-5-sulfonamide benzoate, methyl 5-acetylsalicylate, and nicotinamide compounds.

[0016] The hormones are selected from adrenocortical hormones, sex hormones, thyroid hormones, insulin, and anterior pituitary hormones; among them, adrenocortical hormones include adrenocorticotropic hormone (ACTH), glucocorticoids, and mineralocorticoids; sex hormones include estrogens, progestins, androgens, anabolic steroids, and gonadotropins; thyroid hormones include thyroid-stimulating hormone (TSH) and thyroid hormones; insulin includes long-acting insulin, intermediate-acting insulin, and short-acting insulin; and anterior pituitary hormones include growth hormone, somatostatin, growth hormone-releasing hormone (GHR), and adrenocorticotropic hormone-releasing hormone (ACTH).

[0017] The pheromones are selected from androstenone, estradiol, and animal hormones.

[0018] The concentration range of the mitochondrial protectant is 0.1 μM-100 μM, the concentration range of the vanadium compound is 0.1 μM-20 μM, and the concentration range of the hormone / pheromone is 0.1 nM-1 μM. Preferably, the concentration range of the mitochondrial protectant is 50 μM-100 μM, the concentration range of the vanadium compound is 0.5 μM-2 μM, and the concentration range of the hormone / pheromone is 1 nM-100 nM.

[0019] Preferably, the composition comprises a combination of two or three of the following components: 50 μM-100 μM coniferaldehyde, 1 μM acetylacetonate, and 10 nM β-estradiol. A concentration of 50 μM for coniferaldehyde is optimal. The composition shows the best efficacy in diabetic cell models and animal models when combined in pairs or in all three combinations.

[0020] The above composition is used for the prevention or treatment of diabetes. More specifically, the composition is used to: improve pancreatic β-cell activity; and control fasting blood glucose, satiety blood glucose, glucose tolerance, or insulin levels. The composition can be prepared as a medicine or health food for the prevention and treatment of diabetes.

[0021] Among them, mitochondrial protectants are used to maintain the energy and reduction balance of pancreatic cells and resist oxidative stress in the early stages of diabetes, vanadium compounds are used to activate diabetes-related cellular stress responses, and hormones / pheromones are used to link with cellular stress signals and enhance the effects of vanadium compounds.

[0022] Studies have shown that some important mechanisms leading to insulin resistance include: energy metabolism disorders; endoplasmic reticulum stress; oxidative stress; impaired mitochondrial function; downregulation of silencing signaling pathways; inflammatory response; and central nervous system regulatory disorders.

[0023] Studies have revealed the dynamics and temporal nature of molecular events involved in pancreatic islet tissue damage during the pathogenesis of diabetes: In the early stages (4–6 weeks), the metabolic type of islet cells gradually shifts from aerobic oxidative phosphorylation (OXPHOS) to anaerobic glycolysis, accompanied by a low-level inflammatory response in the islet tissue and a compensatory increase in insulin synthesis. As the disease progresses, in the later stages (8–24 weeks), the inflammatory response in islet tissue intensifies dramatically, accompanied by the loss of functional compensatory mechanisms of islet β cells. Furthermore, early relief of oxidative stress can effectively improve the secretory function of islet β cells and reduce peripheral blood glucose levels; therefore, oxidative stress may be a crucial molecular event in the early stages of islet function damage in diabetic animal models. In addition, oxidative stress can act on mitochondria, exacerbating their functional abnormalities. Large amounts of free radicals can damage mitochondrial DNA, membrane lipids, and proteins, leading to oxidative damage and mitochondrial functional defects. Therefore, based on the factors that may cause mitochondrial damage in the early stages of diabetes, ingredients such as increasing mitochondrial enzyme activity, stabilizing the mitochondrial membrane, or supplementing with exogenous antioxidants and promoting endogenous antioxidant effects can protect mitochondria and their functions, and play a role in assisting in the intervention of diabetes.

[0024] Based on long-term research on the regulation of cellular life processes and their molecular mechanisms by metal ions / trace elements, it has been found that metal ions can regulate cellular stress response systems to exert pharmacological effects. In studies on vanadium compounds for combating diabetes, vanadium complexes can induce the interaction between the heat shock protein Hsp60 and PPARγ, thereby activating PPARs-AMPK signaling, eliminating insulin resistance, and enhancing glucose / lipid metabolism. Vanadium compounds can also induce the expression of the heat shock protein family members Grp78 and Grp75, thereby regulating the unfolded protein response (UCP) of cells and playing a protective role for pancreatic islet cells and nerve cells; and UCP is one of the pathways regulating aging.

[0025] Furthermore, reproduction and energy metabolism are closely related, with gonadal steroids playing a crucial role in specific energy metabolism processes under various physiological conditions. For a long time, the gonads were considered endocrine glands, producing sex steroids such as estrogen, androgens, and progesterone solely for the purposes of sex differentiation, puberty, and reproduction. Estrogens, androgens, and progesterone also regulate insulin secretion in a sex-specific manner via different receptors in pancreatic β-cells.

[0026] Coniferalderhyde (CFA) is a phenolic compound found in some foods and traditional Chinese medicines. It is widely available and has a high safety profile. CFA can activate the Nrf2 pathway to exert its antioxidant effect. CFA can inhibit the damage to mitochondrial structure and energy metabolism caused by Aβ load, including restoring normal mitochondrial dynamics and regulating energy metabolism towards a higher propensity for oxidative phosphorylation. Therefore, it is a good mitochondrial protectant.

[0027] Furthermore, vanadium compounds can synergistically protect nerve cells with mitochondrial protectants and cinnamaldehyde; and in type II diabetic animals, vanadium compounds not only effectively control blood sugar but also significantly improve hyperinsulinemia and reduce basal insulin levels. This invention selects dietary doses of the trace element vanadium (non-hypoglycemic dose) as a representative element of metal ions / biological trace elements.

[0028] Studies have shown that estrogen deficiency can lead to a decrease in the body's metabolic rate, resulting in a series of symptoms such as central obesity and dyslipidemia. In a high-fat diet-induced diabetic animal model, chronic estrogen administration can protect mice from adverse reactions such as glucose tolerance caused by a high-fat diet. Furthermore, estrogen also plays an important role in improving insulin resistance, inhibiting glucose-lipid toxicity, and preventing oxidative stress. Therefore, this invention selects β-estradiol as a representative compound for hormone / pheromone research.

[0029] Studies have shown that, within the designed concentration range (dietary dose), neither coniferaldehyde alone, nor vanadium alone at normal physiological concentrations, nor β-estradiol alone have the ability or effect to lower blood sugar. However, the combined administration of vanadium at dietary doses and β-estradiol at normal physiological concentrations, or the combined administration of coniferaldehyde, vanadium at dietary doses, and β-estradiol at normal physiological concentrations, showed significant synergistic effects.

[0030] Studies have shown that combinations for the prevention or treatment of diabetes, especially triple therapy, can effectively protect the vitality of pancreatic β cells; under dietary doses of vanadium, they can control fasting blood glucose and random blood glucose (satiety blood glucose) in diabetic animals, improve the characteristics of polydipsia in diabetes, improve insulin resistance, and reduce the occurrence of diabetic complications.

[0031] Compared with the prior art, the beneficial effects of the present invention are: improving pancreatic β-cell activity; and controlling fasting blood glucose, satiety blood glucose, glucose tolerance, and insulin levels within the range of dietary dosage. Attached Figure Description

[0032] Figure 1 This is a graph showing the detection results of the effect of the composition of the present invention on the activity of mouse pancreatic β cells;

[0033] Figure 2 This is a graph showing the detection results of the effect of the composition in Example 1 on the body weight of the animal model;

[0034] Figure 3 This is a graph showing the detection results of the effect of the composition in Example 1 on the food intake of an animal model;

[0035] Figure 4 This is a graph showing the detection results of the effect of the composition in Example 1 on the water intake of an animal model;

[0036] Figure 5 This is a graph showing the detection results of the effect of the composition in Example 1 on fasting blood glucose in an animal model;

[0037] Figure 6 This is a graph showing the detection results of the effect of the composition in Example 1 on satiety blood glucose in an animal model;

[0038] Figure 7 This is a graph showing the detection results of the effect of the composition in Example 1 on glucose tolerance in an animal model;

[0039] Figure 8 This is a graph showing the detection results of the effect of the composition in Example 1 on serum insulin levels in an animal model;

[0040] Figure 9 The graph shows the results of detecting the effect of the composition in Example 2 on fasting blood glucose and saturated blood glucose in animal models.

[0041] Figure 10 This is a graph showing the detection results of the effect of the composition in Example 2 on glucose tolerance in an animal model. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0043] The present invention will now be described in further detail with reference to the accompanying drawings:

[0044] Preparation of compositions: preparation of coniferaldehyde (CFA), dietary doses of vanadium (VAC or V) and β-estradiol (E2), and their combinations in pairs and in triplet formulations.

[0045] Weigh 17.18 mg (0.1 mmol) of coniferaldehyde (CFA), dissolve it in 10 mL of DMSO to prepare a 10 mM coniferaldehyde (CFA) stock solution; take 100 μL of the 10 mM coniferaldehyde (CFA) stock solution and dilute it in 0.9 mL of DMEM (containing 10% serum) to prepare a 1 mM coniferaldehyde (CFA) solution; then dilute the 1 mM coniferaldehyde (CFA) solution to 240 μM, 150 μM and 100 μM respectively to prepare coniferaldehyde (CFA) solutions of different concentrations.

[0046] Weigh 26.51 mg (0.1 mmol) of acetylacetonate vanadyl oxyacetylene (VAC), dissolve it in 10 mL of distilled water to prepare a 10 mM acetylacetonate vanadyl oxyacetylene (VAC) stock solution; dilute 10 μL of the acetylacetonate vanadyl oxyacetylene (VAC) stock solution in 9.99 mL of DMEM (containing 10% serum) to prepare a 10 μM acetylacetonate vanadyl oxyacetylene solution; then dilute the 10 μM acetylacetonate vanadyl oxyacetylene (VAC) solution to 2 μM.

[0047] Weigh 27.24 mg (0.1 mmol) of β-estradiol (E2), dissolve it in 10 mL of DMSO to prepare a 10 mM β-estradiol (E2) stock solution; take 1 μL of the β-estradiol (E2) stock solution and dilute it in 9.999 mL of DMEM (containing 10% serum) to prepare a 1 μM β-estradiol (E2) solution; then dilute it with 1 μM acetylacetonate (V) solution to 20 nM.

[0048] A coniferaldehyde (CFA) and acetylacetonate (VAC) compound formulation was prepared by mixing a 100 μM coniferaldehyde (CFA) solution and a 2 μM acetylacetonate (VAC) solution in a 1:1 ratio. The final concentrations of coniferaldehyde (CFA) and acetylacetonate (VAC) were 50 μM and 1 μM, respectively.

[0049] A 1:1 mixture of 100 μM coniferaldehyde (CFA) solution and 20 nM β-estradiol (E2) solution was used to prepare a coniferaldehyde (CFA) combined with β-estradiol (E2) compound (CFA+E2) formulation with final concentrations of 50 μM and 10 nM, respectively.

[0050] A 2 μM solution of acetylacetonate vanadyl (VAC) and a 20 nM solution of β-estradiol (E2) were mixed in a 1:1 ratio to prepare a VAC+E2 compound formulation with final concentrations of 1 μM and 10 nM of acetylacetonate vanadyl (VAC) and β-estradiol (E2).

[0051] A trivalent (CFA+VAC+E2) compound formulation of coniferaldehyde (CFA), acetylacetonate vanadyl (VAC), and β-estradiol (E2) was prepared by mixing a 240 μM / 150 μM coniferaldehyde (CFA) solution, a 3 μM acetylacetonate vanadyl (VAC) solution, and a 30 nM β-estradiol (E2) solution in a 1:1:1 ratio. The final concentrations of coniferaldehyde (CFA), acetylacetonate vanadyl (VAC), and β-estradiol (E2) were 80 μM / 50 μM, 1 μM, and 10 nM, respectively.

[0052] Detection of the effect of the composition on the viability of mouse pancreatic β cells:

[0053] Pancreatic β-cells (NIT-1 cells) pre-culture and passage: Cells were cultured at 37°C and 5% CO2 in DMEM (containing 10% fetal bovine serum), 100 U / ml penicillin and 100 μg / ml streptomycin. (1) Preheat culture medium, trypsin and PBS in a 37°C water bath; (2) Discard the old culture medium in the culture flask, take 2ml of PBS and add it to a 25cm cell culture flask, wash the cell surface 3 times and discard the PBS; (3) Add 1mL of 0.05% trypsin to digest the cells, observe under a microscope until the cell morphology changes slightly, then pour out the trypsin in a clean bench and keep about 100μL, place the cells in a 37°C cell culture incubator and incubate for 3min until digestion is complete; (4) Stop digestion with 2mL of cell culture medium, and pipette the culture flask wall, transfer the cells to a centrifuge tube, and centrifuge at 1000rpm for 3min; (5) After centrifugation, discard the supernatant, add 1mL of fresh DMEM (containing 10% fetal bovine serum) to the culture flask and pipette to mix the cells, passage the cells in an appropriate ratio, label the cell name, passage number and passage date on the flask and place it in an incubator.

[0054] MTS method for detecting cell viability: (1) Seeding cells: After preparing a cell suspension with DMEM (containing 10% fetal bovine serum) medium, seed 3000-4000 cells per well into a 96-well plate, with a volume of 100 μL per well; (2) After the cells reach about 60% confluence, add the drug and incubate for 36 h; (3) Prepare a 10% MTS solution with DMEM (containing 10% fetal bovine serum) medium. After incubating for 36 h, discard the original solution in the culture wells, add 100 μL of 10% MTS solution to each well, and continue incubation for 2 h; (3) After 2 h, measure the absorbance of each well with an ELISA reader (excitation wavelength 490 nm). Plot a bar chart of drug action with drug concentration as the x-axis and absorbance as the y-axis. Figure 1 The components of each composition are shown in Table 1.

[0055] Table 1

[0056]

[0057] Figure 1 The results showed that single-drug CFA at a concentration of 50 μM and single-drug E2 at a concentration of 10 nM significantly promoted NIT-1 cell viability. Single-drug V at a concentration of 1 μM also improved NIT cell viability, but the difference was not statistically significant. Compared with single-drug therapy, the cell viability-promoting effects of all two-drug combinations (CFA+V, CFA+E2, and V+E2) were further enhanced. Furthermore, the cell viability was further improved in the triple-drug combination group (CFA+V+E2), and the combined effect of CFA at a concentration of 50 μM was superior to that at a concentration of 80 μM.

[0058] Example 1

[0059] First batch of animal experiments:

[0060] Clean-grade BKS-Leprem2Cd479 / Gpt male diabetic mice (6 weeks old) were provided by the Nanjing Animal Model Center. Animal husbandry and handling were conducted in accordance with animal welfare and ethical guidelines. After one week of acclimatization, mice with blood glucose levels greater than 11 mM were randomly divided into groups of 8 mice per group, housed in cages of 2-3 mice per cage, under a 12 / 12h light / dark cycle, at an ambient temperature of 22-26℃. When the mice reached approximately 7 weeks of age, they were fed according to the methods described in Table 2.

[0061] Table 2

[0062]

[0063] During the experiment, the mice were weighed every 2-3 days. Figure 2 ), measuring and recording food intake ( Figure 3 ) and water intake ( Figure 4 Fasting blood glucose should be measured once a week. Figure 5 ) and satiety blood sugar ( Figure 6 Before measuring fasting blood glucose, one must fast overnight but not drink water; before measuring random blood glucose (full blood glucose), one must fast overnight but not drink water, and then measure blood glucose using a Roche blood glucose meter and blood glucose test strips after 12 hours by drawing blood from the tail vein.

[0064] Before the end of the experiment (day 37), a glucose tolerance test was conducted. BKS-DB diabetic mice were fasted for 12 hours with unlimited water intake, and their fasting blood glucose was measured and recorded as time T0. Immediately afterwards, a 20% glucose solution was administered at a dose of 1 g / kg. -1 Mice were administered the medication by gavage, and blood glucose levels were measured at 15, 30, 60, 120, 180, and 240 minutes post-gavage. Figure 7 The mice were fasted for 12 hours on the night before sacrifice (day 42), but water was allowed. After weighing the mice, they were anesthetized, their eyeballs were enucleated, and blood was collected in EP tubes. The tubes were incubated at room temperature for 2 hours, then centrifuged (4000 rpm, 20 min). Serum was collected, and fasting serum insulin levels were measured by ELISA. Figure 8 ).

[0065] Mouse weight changes as follows Figure 2 As shown, the body weight of BKS-DB mice gradually increased as the experiment progressed. Compared with the control group 21, there was no significant difference in the weight of the single-drug CFA group 23 and the dual-drug group 24 (CFA+VAC). However, the weight increase trend was more obvious in the CFA+VAC+E2 group 25, with its average weight being higher than that of the control group and other experimental groups. No significant weight loss was observed in the BKS-DB mice throughout the entire experimental period.

[0066] Changes in food intake in mice as follows Figure 3 As shown, during the monitoring period of the drug administration week, the food intake of mice fluctuated within the normal range, but there was no significant difference between the groups. However, the changes in water intake were more significant (e.g., Figure 4 Compared with the control group 21, the water consumption of the CFA+VAC+E2 group 25 was significantly reduced, with the average value lower than that of the control group and other experimental groups. Furthermore, during the rearing of the mice, the bedding in the cages was observed; the bedding in the CFA+VAC+E2 group 25 was drier, while the bedding in the control group 21 was relatively damp.

[0067] The results of fasting blood glucose changes in mice during drug administration are as follows: Figure 5As shown. One week after administration, compared with the control group 21, the CFA group 23, CFA+VAC group 24, and CFA+VAC+E2 group 25 significantly reduced fasting blood glucose in diabetic mice. However, in the second week and subsequent weeks, the fasting blood glucose of diabetic model mice in the CFA group 23 and CFA+VAC group 24 showed an upward trend, which continued until the end of the monitoring period without any relief. In contrast, the CFA+VAC+E2 group 25 significantly reduced fasting blood glucose in diabetic mice one week after administration, and in the following weeks of testing, the triple experimental group maintained the fasting blood glucose of diabetic mice at a normal level (~10mM).

[0068] Random (satiety) blood glucose results as follows Figure 6 As shown, the blood glucose level of control diabetic mice reached a high value (~30 mM) at approximately 8–9 weeks of age. After administration of the drugs, compared with the DB group 21, the CFA group 23 and the CFA+VAC group 24 did not show a significant trend of improvement in random blood glucose in diabetic mice; however, after one week of administration, the random blood glucose level of the diabetic mice in the CFA+VAC+E2 group 25 began to decline steadily, and in the following weeks of testing, the triple therapy reduced the random blood glucose level of the diabetic mice to a normal level (~12 mM).

[0069] Results of oral glucose tolerance test ( Figure 7 It was found that after gavage administration of glucose, blood glucose levels in all groups reached their maximum value of approximately 32 mM at 15 minutes. After 30 minutes, the blood glucose levels in DB group 21, CFA group 23, and CFA+VAC group 24 showed no significant decrease. In contrast, the blood glucose level in CFA+VAC+E2 group 25 began to decrease rapidly, returning to normal levels by 2 hours. The fasting blood glucose levels in other groups remained high at 3 hours without significant decrease. The experiment indicates that CFA+VAC+E2 group 25 significantly improved fasting glucose tolerance and glucose loading capacity in mice, suggesting that the triple therapy may improve glucose metabolism in diabetic mice and promote their tissue utilization and absorption of glucose.

[0070] Fasting serum insulin was measured in mice before sacrifice. Figure 8 Compared with wild-type healthy mice group 26 (WT), the fasting serum insulin level in DB group 21 was significantly higher than that in WT group 26. Compared with DB group 21, the average fasting serum insulin level was reduced in CFA group 23, CFA+VAC group 24 and CFA+VAC+E2 group 25.

[0071] Among them, the CFA+VAC+E2 group 25 could still effectively control fasting blood glucose, satiety blood glucose and glucose tolerance in diabetic mice even with reduced insulin levels, indicating that it increased insulin sensitivity in diabetic mice.

[0072] Example 2

[0073] The second batch of animal experiments: Clean-grade BKS-Leprem2Cd479 / Gpt male diabetic model mice (6 weeks old) were provided by the Nanjing Animal Model Center. Animal husbandry and handling were carried out in accordance with animal welfare and ethical guidelines. After one week of acclimatization, mice with blood glucose levels greater than 11 mM were randomly divided into groups of 8 mice per group, housed in cages of 2-3 mice per cage, under a 12 / 12h light / dark cycle, at an ambient temperature of 22-26℃. After 7 weeks of age, the mice were fed according to the methods in Table 3.

[0074] Table 3

[0075]

[0076] During the experiment, fasting blood glucose and random blood glucose were measured weekly. Before fasting blood glucose measurement, patients needed to fast overnight but could drink water; before random blood glucose measurement, patients could fast overnight but could drink water. Blood was drawn from the tail vein 12 hours later and measured using a Roche blood glucose meter and test strips. (See [link to details]). Figure 9 .

[0077] Before the end of the experiment, a glucose concentration experiment was conducted. BKS-DB diabetic mice were fasted for 12 hours with unlimited water intake, and their fasting blood glucose was measured and recorded as time T0. Immediately afterwards, a 20% glucose solution was administered at a dose of 1 g / kg. -1 Blood glucose levels were measured at 15, 30, 60, 120, 180, and 240 minutes after gavage, following body weight administration. (See [reference needed]). Figure 10 .

[0078] During the first 3 weeks of the experiment, groups CFA (32), VAC (33), E2 (34), and CFA+VAC+E2 (38) were set up, as follows: Figure 9 a and Figure 9 As shown in b, compared to the control group (groups 31-34), both fasting blood glucose and satiety blood glucose showed no improvement and instead trended upward. After week 3, dual-drug therapy was administered to each of the single-drug groups. Figure 9 As shown in c, compared with DB group 31, CFA+VAC group 35 did not significantly reduce fasting blood glucose in diabetic mice; while VAC+E2 group 36, CFA+E2 group 37 and CFA+VAC+E2 group 38 all significantly reduced fasting blood glucose in diabetic mice. After 15 weeks of combined administration (total administration time 18 weeks), these three experimental groups reduced fasting blood glucose to a completely normal level (~7mM).

[0079] Random (satiety) blood glucose results as follows Figure 9As shown in Figure d, the blood glucose level in the control group 31 reached a plateau (~32 mM) in approximately 3 weeks. After combination therapy, except for the CFA+VAC group 35, the VAC+E2 group 36, CFA+E2 group 37, and CFA+VAC+E2 group 38 all significantly reduced the random blood glucose level in diabetic mice. After 15 weeks of combined administration (total administration time 18 weeks), the VAC+E2 group 36 and the CFA+VAC+E2 group 38 reduced the random blood glucose level in diabetic mice to normal levels (~12 mM), and the CFA+E2 group 37 also controlled the random blood glucose in diabetic mice. The VAC+E2 group 36 and the CFA+VAC+E2 group 38 performed better than the CFA+E2 group 37. Furthermore, it can be seen that during the 18-week testing period, both fasting blood glucose and satiety blood glucose showed an overall decreasing trend without significant rebound.

[0080] The results of the oral glucose tolerance test are as follows: Figure 10 As shown, after glucose gavage, blood glucose levels in all experimental groups reached their maximum values ​​(~32 mM) at 15 minutes. Slightly different from the first batch of experiments, the blood glucose levels in VAC+E2 group 36, CFA+E2 group 37, and CFA+VAC+E2 group 38 were lower than those in DB group 31 and CFA+VAC group 35, suggesting that longer-term drug treatment may significantly improve the glucose metabolism capacity of diabetic mice. After 30 minutes, blood glucose levels in VAC+E2 group 36, CFA+E2 group 37, and CFA+VAC+E2 group 38 began to gradually decrease, returning to normal levels by 3 hours, with no significant difference among these three groups. However, blood glucose levels in DB group 31 and CFA+VAC group 35 remained high at 4 hours, showing no significant decreasing trend. Therefore, the experimental results indicate that the VAC+E2, CFA+E2, and CFA+V+E2 groups significantly improved fasting glucose tolerance and glucose loading capacity in mice, demonstrating the potential to improve glucose metabolism and promote tissue utilization and absorption of glucose in mice.

[0081] This invention illustrates the detailed experimental procedure and the cell model and BKS-Lepr used in this invention through the above embodiments. em2Cd479 The invention relates to a Gpt transgenic mouse model, but is not limited to the detailed experimental procedures, diabetic cells, and animal models described above. That is, it does not mean that the invention must rely on the detailed experimental procedures, diabetic cells, and animal models described above to be implemented. Those skilled in the art should understand that any improvements to the invention, any structural changes to the product, mitochondrial protectants, vanadium compounds, and equivalent substitutions for hormones or information exchangers all fall within the scope of protection and disclosure of this invention.

Claims

1. A composition for the prevention or treatment of diabetes, characterized in that, The composition is selected from one of the following combinations: The combination of coniferaldehyde and β-estradiol; The combination of acetylacetone vanadium oxide and β-estradiol; A combination of coniferaldehyde, acetylacetone vanadium, and β-estradiol; The concentration range of coniferaldehyde is 0.1 μM - 100 μM, the concentration range of acetylacetone vanadium is 0.1 μM - 20 μM, and the concentration range of β-estradiol is 0.1 nM - 1 μM.

2. The composition according to claim 1, characterized in that, The concentration range of coniferaldehyde is 50 μM - 100 μM, the concentration range of acetylacetone vanadium is 0.5 μM - 2 μM, and the concentration range of β-estradiol is 1 nM - 100 nM.

3. The composition of claim 2, wherein, The concentration range of coniferaldehyde is 50 μM - 100 μM, the concentration range of acetylacetone vanadium is 1 μM, and the concentration range of β-estradiol is 10 nM.

4. Use of a composition for the manufacture of a medicament for the prevention or treatment of diabetes, characterized in that, The composition is any one of claims 1 to 3, and the composition is used to: improve pancreatic β-cell activity; and control fasting blood glucose, satiety blood glucose, glucose tolerance, or insulin levels.

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