Alpha-glucosidase inhibitor and application thereof

Abstract

The invention relates to an alpha-glucosidase inhibitor and application thereof. The alpha-glucosidase inhibitor is vitamin C, dehydrogenated vitamin C, and 2, 3-diketone-L-gulonic acid. The influence of vitamin C, dehydrovitamin C and 2, 3-diketone-L-gulonic acid on alpha-glucosidase and HepG2 cell glucose consumption is researched through an in-vitro model, and the result shows that the vitamin C, dehydrovitamin C and 2, 3-diketone-L-gulonic acid can remarkably inhibit the activity of alpha-glucosidase and promote uptake and utilization of glucose by cells, so that the effect of improving insulin resistance is achieved.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an α-glucosidase inhibitor and its application. Background Technology

[0002] Diabetes mellitus is a metabolic disease characterized by chronic hyperglycemia, caused by insulin secretion defects or dysfunction. It can be classified into type 1 diabetes, type 2 diabetes, gestational diabetes, and other special types of diabetes. Type 2 diabetes is the most common, accounting for approximately 90% of all diabetes cases. It is a non-insulin-dependent type of diabetes. The causes of type 2 diabetes include genetic and environmental factors, primarily due to long-term unhealthy lifestyles and dietary habits that lead to partial damage to pancreatic β-cells or insulin resistance, resulting in relative insulin insufficiency, impaired glucose tolerance, and excessively high fasting blood glucose levels. The preference for high-sugar, high-fat, and high-salt foods, coupled with reduced physical activity, has led to a year-on-year increase in the incidence of type 2 diabetes. Treatment for type 2 diabetes primarily involves traditional hypoglycemic drugs, supplemented by dietary and exercise interventions. Drug therapy, based on different mechanisms of action, is mainly divided into five categories: insulin and its analogues, insulin sensitizers, insulin secretagogues, enzyme inhibitors, and sodium-glucose cotransporter 2 inhibitors.

[0003] Tyrosinase (EC. 1.14.18.1), also known as polyphenol oxidase or catechol oxidase, is a copper-containing metallooxidase widely found in microorganisms, plants, animals, and humans. It is a key rate-limiting enzyme regulating melanin biosynthesis. Tyrosinase first converts L-tyrosine hydroxyl groups to L-DOPA, then oxidizes L-DOPA to dopaquinone, ultimately leading to melanin biosynthesis. Therefore, its expression level and activity affect the rate and yield of melanin synthesis. In humans, melanin protects the skin from ultraviolet radiation; however, excessive melanin can lead to abnormal pigmentation disorders such as melasma, freckles, post-inflammatory melanosis, and pigmented acne scars, and even malignant melanoma. In addition, tyrosinase-catalyzed melanin biosynthesis also affects insect molting and wound healing, as well as the enzymatic browning process in fruits and vegetables. Therefore, tyrosinase inhibitors can inhibit melanin biosynthesis by reducing the catalytic efficiency of tyrosinase, and have a very broad application prospect in fields such as medical aesthetics, food preservatives, and novel pesticides.

[0004] Vitamin C (Ascorbic Acid), also known as L-ascorbic acid, is a water-soluble vitamin. Under normal circumstances, most of it is metabolized in the body into oxalic acid or combines with sulfuric acid to form ascorbic acid-2-sulfuric acid, which is excreted in the urine. A portion is also excreted directly in the urine. It has numerous physiological activities and functions, including: 1. Collagen synthesis; 2. Treatment of scurvy; 3. Prevention of gingival recession and bleeding; 4. Prevention of arteriosclerosis; 5. Antioxidant; 6. Treatment of anemia; 7. Cancer prevention; 8. Enhancement of the body's immunity; 9. Improvement of the body's stress response. The ability of vitamin C and its oxidation products to effectively inhibit the activity of α-glucosidase has not yet been discovered and utilized. Summary of the Invention

[0005] The purpose of this invention is to provide an α-glucosidase inhibitor and its application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: Application of vitamin C, dehydrovitamin C or 2,3-diketo-L-gulonic acid in the preparation of α-glucosidase inhibitors.

[0007] The use of vitamin C, dehydrovitamin C, or 2,3-diketo-L-gulonic acid in the preparation of drugs to improve insulin resistance is characterized in that: vitamin C, dehydrovitamin C, or 2,3-diketo-L-gulonic acid, as α-glucosidase inhibitors, can inhibit α-glucosidase activity and promote the uptake and utilization of glucose by cells, thereby achieving the effect of improving insulin resistance. Attached Figure Description

[0008] Figure 1A : VC liquid phase retention time. Figure 1B VCJ liquid phase retention time. Figure 1C VCO liquid phase retention time.

[0009] Figure 2 The inhibitory mechanism of VC on α-glucosidase.

[0010] Figure 3 The inhibitory mechanism of VCJ on α-glucosidase.

[0011] Figure 4 The inhibitory mechanism of VCO on α-glucosidase.

[0012] Figure 5 : Types of inhibition of α-glucosidase by vitamin C.

[0013] Figure 6 : The type of inhibition of α-glucosidase by VCJ.

[0014] Figure 7: The type of inhibition of α-glucosidase by VCO.

[0015] Figure 8 Effects of different concentrations of VC, VCJ, and CVO on glucose consumption in IR-HepG2 cells. Detailed Implementation

[0016] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0017] The following abbreviations are used in the embodiments: Example 1 Vitamin C (VC), chemically known as ascorbic acid, is a water-soluble vitamin essential for humans and many animals. Dehydrovitamin C (VCJ) is an oxidation product of vitamin C, which can be reduced back to vitamin C. 2,3-Diketo-L-gulonic acid (VCO) is a further oxidation product of vitamin C and dehydrovitamin C.

[0018] Take a standard solution of vitamin C, or a standard solution of dehydrovitamin C, or a standard solution of 2,3-diketone-L-gulonic acid, and determine it by high performance liquid chromatography. Detection conditions: Column: Waters T3 column (4.6 mm × 250 mm, 5.0 μm); Mobile phase: methanol:water = 5:95; Flow rate: 1.0 mL / min.

[0019] As shown in Figure 1, the retention time of VC is 2.0 min ( Figure 1A The retention time of VCJ is 2.3 min. Figure 1B The retention time of VCO was 2.7 min. Figure 1C ).

[0020] Example 2 1. The inhibitory effects of VC, VCJ, and VCO on α-glucosidase. Alpha-glucosidase inhibitors are a class of oral hypoglycemic agents that treat diabetes by delaying the absorption of carbohydrates in the intestine. Their mechanism of action involves competitively inhibiting various alpha-glucosidases located in the small intestine, slowing the breakdown of starches into glucose, thereby reducing glucose absorption and lowering postprandial hyperglycemia. The principle of alpha-glucosidase inhibitory activity screening is as follows: p-Nitrophenol-α-D-glucosidase (PNPG) is a colorless substrate that, upon hydrolysis by alpha-glucosidase, releases p-nitrophenol (PNP). PNP is yellow under alkaline conditions; therefore, the concentration of PNP can be indirectly reflected by measuring the absorbance at 410 nm, thus indicating alpha-glucosidase activity. When the test compound competitively binds to alpha-glucosidase, the compound inhibits enzyme activity, leading to a decrease in PNP production and consequently a lower absorbance value. IC50 is typically used. 50 The IC50 value (half-maximal inhibitory concentration) is used to measure the inhibitory effect of a compound on α-glucosidase. 50 The smaller the value, the better the inhibitory effect of the compound on the enzyme.

[0021] Using PNPG as a substrate and a 96-well microplate as the reaction medium, with a final reaction volume of 130 μL, the inhibitory activity of α-glucosidase was determined. 30 μL of VC, VCJ, or VCO solutions at concentrations of 2, 4, 8, 16, and 32 μg / mL were added to each well of the microplate, followed by 30 μL of α-glucosidase solution at a concentration of 1.0 U / mL. The reaction was incubated at 37°C for 10 min, then 70 μL of 5 mmol / L PNPG solution was added, and the reaction was incubated at 37°C for 20 min. The reaction was terminated with 70 μL of 0.2 mol / L Na₂CO₃ solution, and the absorbance was measured at 405 nm. The IC₀ values ​​of VC, VCJ, and VCO for α-glucosidase inhibition were determined. 50 The values ​​are shown in the table below.

[0022] Example 3 1. The inhibitory mechanism of VC, VCJ, and VCO on α-glucosidase was investigated using PNPG as a substrate and a 96-well microplate as the reaction carrier. The final reaction volume was 130 μL. The effect of different concentrations of the effector on the α-glucosidase-catalyzed L-DOPA oxidation activity was determined. As shown in the table below, 30 μL of LVC, VCJ, or VCO solution was added to the 96-well microplate, followed by 30 μL of α-glucosidase solution. The reaction was carried out at 37℃ for 10 min, followed by 70 μL of PNPG solution. The reaction was then carried out at 37℃ for 20 min, and finally terminated with 70 μL of 0.2 mol / L Na2CO3 solution. 1 2 3 4 substrate mmol / L 5.0 5.0 5.0 5.0 α-glucosidase U / mL 0.5 1.0 1.5 2.0 Effector μg / mL 0 5 20 40

[0023] Figures 2-4 The relationship between the residual enzyme activity of α-glucosidase after the action of the effector and the amount of enzyme added was expressed, resulting in a set of straight lines passing through the origin. As the content of the added effector increases, the slope of the line gradually decreases, indicating that the inhibitory effect of the effector on α-glucosidase is a reversible process. The decrease in α-glucosidase activity due to the increase in the effector content is due to the inhibition of enzyme activity and the reduction of catalytic efficiency, rather than the decrease in enzyme activity caused by the reduction of the effective amount of enzyme.

[0024] 2. Determination of the inhibition type and inhibition constant of VC, VCJ, and VCO on α-glucosidase: PNPG was used as the substrate, and a 96-well microplate was used as the reaction carrier. The final reaction volume was 130 μL. The effect of different inhibitor concentrations on enzyme activity was determined by Lineweaver Burk double reciprocal plot to determine the inhibition type. As shown in the table below, 30 μL of VC solution, VCJ solution, or VCO solution was added to a 96-well microplate, followed by 30 μL of α-glucosidase solution. The reaction was carried out at 37℃ for 10 min, followed by 70 μL of PNPG solution. The reaction was carried out at 37℃ for 20 min, and then the reaction was terminated with 70 μL of 0.2 mol / L Na2CO3 solution. 1 2 3 4 α-glucosidase U / mL 1.0 1.0 1.0 1.0 substrate mmol / L 0.5 1.0 2.0 4.0 Effector μg / mL 0 5 20 40

[0025] Depend on Figures 5-7 The experimental results show that VC, VCJ, and CVO form a set of straight lines intersecting on the ordinate, indicating that as α-glucosidase inhibitors, they can alter the maximum reaction rate (Vm) and Michaelis constant (Km) of the enzymatic reaction. The inhibition type is competitive. The Km value increases with increasing effector content, while Vm decreases with increasing effector content. This indicates that the effector affects both the affinity of α-glucosidase for the substrate and the catalytic activity of α-glucosidase. The effector can bind to both the free enzyme (E) and the enzyme-substrate complex (ES), but the inhibitory intensity differs. By plotting the slope and ordinate of the double reciprocal straight line against the effector content, and plotting the slope and ordinate against the extract content, the inhibition constants KI of the effector on the free enzyme can be calculated: KI = 18.37 for VC, KI = 10.78 for VCJ, and KI = 24.65 for CVO.

[0026] Example 4 Digestive fluid treatment group: Take 1.0 mL of 100 μg / mL effector (selected from VC, VCJ, VCO, acarbose), adjust the pH to 3.0 with 1 mol / L HCl solution, then add 1.0 mL of simulated gastric juice and 5 μL of 0.3 mol / L CaCl2, then add pepsin to a final concentration of 170 U / mL, stir well, adjust the pH to 2.0, and place in a constant temperature shaker at 37℃ and 120 rpm for 2 h. After digestion is completed, take a sample quickly, centrifuge at 10000 rpm for 10 min, and collect the supernatant, which is the sample after simulated gastric digestion. Take 1.0 mL of a sample simulated by gastric digestion, adjust the pH to 6.0 with 1 mol / L sodium hydroxide, add 1.0 mL of simulated intestinal fluid, then add 20 μL of 0.3 mol / L CaCl2, followed by trypsin at a final concentration of 200 U / mL, and 10 mg of porcine bile salt. Mix well, then adjust the pH to 7.4 with 1 mol / L sodium hydroxide solution. Place in a constant temperature shaker at 37℃ and 120 rpm for 2 hours. After digestion, quickly take a sample and centrifuge at 10,000 rpm for 10 min, collect the supernatant, heat in an 85℃ water bath for 5 min, then stop the enzymatic reaction to obtain the sample to be tested. Using PNPG as the substrate and a 96-well microplate as the reaction carrier, the final reaction volume is 130 μL, and the α-glucosidase inhibitory activity is measured. Add 30 μL of the sample to be tested to a 96-well microplate, then add 30 μL of 1.0 U / mL α-glucosidase solution and react at 37℃ for 10 min. Add 70 μL of 5 mmol / L PNPG solution and react at 37℃ for 20 min. Finally, terminate the reaction with 70 μL of 0.2 mol / L Na2CO3 solution.

[0027] Water treatment group: The operating procedures are basically the same as those of the digestive fluid treatment group. The difference is that an equal amount of water is used instead of simulated gastric juice and simulated intestinal juice.

[0028] As shown in the table below, the digestive fluid has a certain effect on the enzyme inhibitory activities of VC, VCJ, and VCO. The control group (acarbose) has the least effect, the sample group (VCO) has the least effect, and the VC has the greatest effect. VCO+ digestive fluid VCO + Water Vitamin C + Digestive Fluid Vitamin C + Water Inhibition rate % 60.96±5.91 88.63±4.07 33.40±6.95 76.89±3.12 VCJ+ digestive juice VCJ + Water Acarbose + digestive juices Acarbose + Water Inhibition rate % 52.31±5.63 91.87±3.57 74.84±7.63 89.78±1.28

[0029] Example 5 IR-HepG2 cells were seeded at 5000 cells / well in 96-well plates and cultured for 24 h. The old culture medium was discarded, and 100 μL of a DMEM high-glucose medium containing 10% fetal bovine serum (FBS) was added to each well. The cells were cultured for 48 h to complete cell modeling. A culture medium group and a drug-free control group were also set up. The culture medium was discarded, and the cells were washed once with PBS. 100 μL of serum-free medium was added, and the cells were cultured for 48 h. Glucose content was measured using the GLU assay kit (GOD method). Cell viability was measured using the CCK-8 assay, and glucose consumption per cell was calculated.

[0030] IR-HepG2 cells were seeded at 5000 cells / well in 96-well plates and cultured for 24 h. The old culture medium was discarded, and 100 μL of a medium containing 0.01 μmol / L insulin and 5 μmol / L dexamethasone (prepared with 10% fetal bovine serum) was added to each well. The cells were cultured for 48 h to complete cell modeling. A culture medium group and a drug-free control group were also set up. The culture medium was discarded, and the cells were washed once with PBS. 100 μL of different concentrations of samples prepared with serum-free medium were added, and the cells were cultured for 48 h. Glucose content was measured using the GOD method of a GLU assay kit. Cell viability was measured using the CCK-8 assay, and the glucose consumption per cell was calculated.

[0031] Depend on Figure 8 It can be seen that VC, VCJ, and CVO significantly promoted glucose consumption in IR-HepG2 cells over 24 hours at concentrations ranging from 12.5 to 100 μg / mL, with highly significant differences, and the effect increased with increasing concentration. VC, VCJ, and CVO can significantly improve the glucose consumption capacity of insulin-resistant HepG2 cells and alleviate insulin resistance.

Claims

1. Application of vitamin C, dehydrovitamin C or 2,3-diketo-L-gulonic acid in the preparation of α-glucosidase inhibitors.

2. The use of vitamin C, dehydrovitamin C, or 2,3-diketo-L-gulonic acid in the preparation of drugs for improving insulin resistance, characterized in that: Vitamin C, dehydrovitamin C, or 2,3-diketo-L-gulonic acid, as α-glucosidase inhibitors, can inhibit α-glucosidase activity and promote the uptake and utilization of glucose by cells, thereby improving insulin resistance.

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