Application of nortriterpenoids in preparation of hypoglycemic drugs or functional food

Abstract

The invention provides application of a reduced triterpenoid compound demethylzeylasteral shown in a formula I or pharmaceutically acceptable salt, ester and solvate thereof in preparation of drugs or functional food for reducing blood sugar and preventing and / or treating diabetes. The invention further provides a pharmaceutical composition which comprises the compound shown in the formula I and pharmaceutically acceptable pharmaceutic adjuvants, the pharmaceutical composition is used as a pharmaceutical preparation, and the medicine is a hypoglycemic medicine and a medicine for preventing and / or treating diabetes mellitus: # imgabs0 # formula I.

Description

Technical Field

[0001] The present invention relates to the application field of medicine or health care products, and specifically to the application of a triterpenoid compound in the preparation of medicines or functional foods, and especially to the application of a triterpenoid compound in the preparation of medicines or functional foods for lowering blood sugar, preventing and / or treating diabetes. Background Art

[0002] Diabetes is a chronic metabolic disease that continues to be prevalent worldwide, especially type 2 diabetes, which accounts for about 90% of all diabetic patients. According to the International Diabetes Federation (IDF), diabetes will become the seventh leading cause of death by 2030. Long-term uncontrolled high blood sugar in diabetics can cause serious damage to multiple systems of the human body, especially the nervous and cardiovascular systems. It is the main cause of cardiovascular and cerebrovascular diseases, stroke, blindness, renal failure, and lower limb amputation. It has become the third most serious non-communicable disease threatening people's health and life after cardiovascular and cerebrovascular diseases and malignant tumors.

[0003] The core of diabetes treatment is to stabilize blood sugar, and insulin is the key factor in regulating blood sugar levels in the human body. When the body's insulin production is insufficient or cannot be effectively utilized, abnormal blood sugar or hyperglycemia will occur, resulting in diabetes. The existing clinical diabetes treatment drugs mainly include insulin, DPP4 inhibitors, GLP-1 receptor agonists, insulin secretagogues, insulin sensitizers, α -Glucosidase inhibitors, metformin and SGLT-2 inhibitors, most of which are related to the level and function of insulin. The research and development of new hypoglycemic drugs or functional foods is still a hot topic in the field of anti-diabetic treatment.

[0004] Insulin acts on peripheral tissues (such as adipose tissue, skeletal muscle, etc.), activates their intracellular signal transduction pathways, and then initiates glucose uptake. This series of reactions plays a key role in the body's regulation of glucose levels. Different insulin stimulation concentrations are linearly related to the amount of glucose uptake by adipose tissue / skeletal muscle caused by insulin stimulation within the effective range. By detecting the amount of glucose uptake by adipocytes, their sensitivity to insulin and their hypoglycemic activity can be reflected.

[0005] Insulin sensitizers are a type of hypoglycemic drugs used in clinical anti-diabetic treatments, mainly thiazolidinediones represented by rosiglitazone, which have excellent hypoglycemic effects, but their clinical application is limited by side effects such as weight gain and cardiovascular disease risks. At the same time, increasing the synthesis of glycogen by liver cells and muscle cells is also an effective way to lower blood sugar. Therefore, the market has an urgent need for safer and more effective new hypoglycemic drug molecules with multiple effects such as insulin sensitization and increased glycogen synthesis. Summary of the invention

[0006] In view of the problems of the prior art, the object of the present invention is to provide a new type of blood sugar lowering drug, in particular to provide a use of triterpenoid compounds in the preparation of blood sugar lowering, prevention and / or treatment of diabetes drugs or functional foods.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In the first aspect, the present invention provides the use of a triterpenoid compound of formula I or a pharmaceutically acceptable salt, ester, or solvate thereof in the preparation of a drug or functional food for lowering blood sugar, preventing and / or treating diabetes:

[0009]

[0010] Formula Ⅰ.

[0011] Preferably, the blood sugar lowering is to reduce high blood sugar in type 1 diabetes or type 2 diabetes.

[0012] Preferably, the medicine or functional food has the effects of promoting cellular glucose uptake and lowering blood sugar.

[0013] The Chinese name of Formula I is demethylzeylasteral, and its English name is demethylzeylasteral. It can be isolated from the Celastraceae plant Tripterygium wilfordii and has high safety.

[0014] Preferably, the medicine or functional food further comprises pharmaceutically or food acceptable excipients, wherein the excipients include any one or a combination of at least two of carriers, diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, antibacterial agents or buffers;

[0015] Preferably, the pharmaceutical dosage form is a tablet, capsule, injection, granule or oral liquid;

[0016] Preferably, the daily dosage of the nortriterpenoid compound is 0.01-100 mg / kg calculated based on body weight.

[0017] The present invention unexpectedly found that the triterpenoid compound shown in formula I or its pharmaceutically acceptable salt, ester, solvate can promote the cell's uptake of glucose, promote cell glycogen synthesis, increase insulin sensitivity and directly reduce the blood sugar of diabetic mice, and can be used to reduce blood sugar or prevent and treat diabetes. The present invention found that the compound demethylzeylasteral has a significant effect of promoting the uptake of glucose by 3T3-L1 adipocytes ( Figure 1 ), significantly promote hepatocyte glycogen synthesis ( Figure 2 ), significantly promote insulin sensitivity of adipocytes ( Figure 3 ) and significantly reduce blood sugar in db / db diabetic mice (Table 1), which can lower blood sugar and play a therapeutic and preventive role in type 1 or type 2 diabetes.

[0018] In a second aspect, the present invention provides the use of a triterpenoid compound represented by formula I or a pharmaceutically acceptable salt, ester, solvate or a combination thereof in the preparation of a cellular glucose uptake promoter and / or a blood glucose lowering preparation in vivo:

[0019]

[0020] Formula Ⅰ.

[0021] In a third aspect, the present invention provides a pharmaceutical composition comprising a triterpenoid compound of formula I or a pharmaceutically acceptable salt, ester, solvate thereof and a pharmaceutically acceptable excipient thereof, for use as a pharmaceutical preparation, wherein the drug is a blood sugar lowering drug, a drug for treating or preventing diabetes:

[0022]

[0023] Formula Ⅰ.

[0024] Preferably, the composition contains 0.001-99.9wt% of the nortriterpenoid compound or a pharmaceutically acceptable salt, ester, or solvate thereof, based on the total weight of the composition;

[0025] Preferably, the daily dosage of the triterpenoid compound is 0.01-100 mg / kg calculated based on body weight.

[0026] Preferably, the pharmaceutically acceptable excipients include any one or a combination of at least two of carriers, diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, antibacterial agents or buffers, such as a combination of binders and diluents, a combination of disintegrants and fillers, a combination of disintegrants, fillers and lubricants, etc. Other arbitrary combinations are not described here one by one.

[0027] Preferably, the pharmaceutical preparation is a tablet, a capsule, an injection, a granule or an oral liquid.

[0028] The triterpenoid compounds or pharmaceutically acceptable salts, esters and solvates thereof of the present invention can also be used in combination with other drugs to achieve better hypoglycemic effects.

[0029] In the present invention, the administration route of the drug can be selected according to actual needs, such as oral administration, sublingual administration, intravenous injection, intramuscular injection or subcutaneous injection. Oral administration is preferred.

[0030] The present invention unexpectedly found that the demethylzeylasteral compound shown in formula I or its pharmaceutically acceptable salt, ester, solvate can significantly promote cellular glucose uptake, increase hepatocyte glycogen synthesis, improve adipocyte insulin sensitivity, and significantly reduce blood sugar in db / db diabetic mice, and can be used to prepare hypoglycemic drugs or functional foods, especially suitable for preparing drugs for preventing or treating diabetes, and can reduce high blood sugar in type 1 diabetes or type 2 diabetes. Therefore, it is expected that demethylzeylasteral can be used in clinical hypoglycemic, diabetes treatment drugs or functional foods, and can be suitable for type 1 and type 2 diabetes patients. The compound exists in large quantities in plants such as Tripterygium wilfordii, has multiple pharmacological activities such as anti-inflammatory, immunosuppressive and anti-cancer, and has low toxic and side effects and high safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 : is the chemical structure of formula Ⅰ

[0032] Figure 2: Effects of demethylzelaminaldehyde on glucose uptake rate and cell viability of 3T3-L1 adipocytes (***p<0.001)

[0033] Figure 3: Effect of norzelaminaldehyde on the relative content of glycogen in insulin-resistant HepG2 cells (***p<0.001)

[0034] Figure 4:Effects of norzelaminaldehyde on glucose uptake rate in insulin-resistant 3T3-L1 adipocytes (ac represents the difference between the data of each group, and the data of each group are arranged from large to small, the largest is marked as a, the data with significant difference from a is marked as b (p < 0.05), and the data with significant difference from b is marked as c (p < 0.05). The same letters indicate that there is no significant difference between the two (p> 0.05)) Specific embodiments

[0035] The technical scheme of the present invention is further illustrated by specific examples below. Those skilled in the art should understand that the examples are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0036] The process, conditions, reagents, experimental methods, etc. for implementing the present invention, except for the contents specifically mentioned below, are all common knowledge and common common sense in the art, and the present invention has no particular restrictions. The experimental methods without specifying specific conditions in the embodiments are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0037] Unless otherwise specified, all technical terms and scientific terms used in this specification have the same meaning as those generally understood by those skilled in the art to which the present invention belongs. However, in case of any conflict, the present specification including the definitions shall prevail.

[0038] Drugs and reagents such as norzelamidin, metformin, insulin, and glucose were purchased from Sigma and other companies.

[0039] Example 1

[0040] Effect of norzelaminaldehyde (Dem) on glucose uptake activity in adipocytes

[0041] Experimental principle:

[0042] Insulin regulates GLUT4 through specific signaling pathways such as the PI3K signaling pathway to transport glucose from outside the cell to the cell, allowing the cell to complete the uptake of glucose. Therefore, the cell's uptake of glucose is one of the indicators that directly reflects insulin sensitivity. We test the activity of the test substance on the cell at a certain concentration. If the glucose uptake is increased, we will further conduct a dose-effect relationship study.

[0043] Experimental methods:

[0044] The experiment was divided into three groups, namely blank control, positive drug group (insulin, Ins), and compound group (demethylzeylasteral, Dem). First, the differentiated 3T3-L1 cells were inoculated into a 96-well plate and cultured overnight. After washing the 3T3-L1 cells in the 96-well plate once with PBS buffer, 200 μL of low-glucose culture medium containing different samples were added respectively. The blank control group was incubated with the corresponding concentration of DMSO, the positive control was incubated with 100nmol / L insulin, and the compound group was incubated with a final concentration of 20 μmol / L demethylzeylasteral. Each sample was repeated three times. After incubation at 37°C for 24 hours, 10 μl of cell culture medium was aspirated respectively, and the glucose concentration in the culture medium was determined by the glucose oxidase-peroxidase method, and the absorbance was measured at 405 nm using a microplate reader. After the glucose uptake test, the CellTiter 96 ® Cell viability was determined by water cell proliferation assay. 15 μL CellTiter 96 ® Aqueous cell proliferation test reagent. After incubation at 37°C for 8 hours, the absorbance was measured at 490 nm using an enzyme reader to determine whether the compound was toxic to adipocytes. The glucose consumption rate (Formula 1) and relative cell viability (Formula 2) were calculated as follows:

[0045] Glucose consumption rate (%) = (A1− A2) / A1× 100 (Formula 1)

[0046] Where A1 = absorbance of initial glucose, A2 = absorbance of blank control, positive control or sample group.

[0047] Relative cell viability (%) = A2 / A1 × 100 (Formula 2)

[0048] Where A1 = absorbance of blank control, A2 = absorbance of sample or positive control.

[0049] The experimental results are shown in Figure 2 The results showed that compared with the blank control group, demethylzeylasteral at a concentration of 20 μmol / L for 24 hours had a significant activity in promoting glucose uptake in adipocytes (***p<0.001), and its activity was stronger than that of the positive control insulin group; at the same time, at this concentration, demethylzeylasteral had no significant effect on the viability of 3T3-L1 adipocytes.

[0050] Example 2

[0051] Effects of norzelaminaldehyde on glycogen synthesis in insulin-resistant HepG2 cells

[0052] Experimental methods:

[0053] Establishment of HepG2 cell insulin resistance model

[0054] HepG2 cells in the logarithmic growth phase were plated in a 6-well plate at a density of 3 × 10 5 cells / well, and when the cells were fused to 80%, HepG2 cells were intervened with MEM EAGLE medium (without serum) containing 55.5 mmol / L glucose for 48 h, which successfully established the HepG2 cell insulin resistance model.

[0055] Glycogen synthesis experiment of HepG2 cells in insulin resistance

[0056] HepG2 cells with insulin resistance model successfully established were randomly divided into model control group (Model control), positive control group (Met-1.0 mmol / L), demethylzeramunal administration group (Dem-20 μmol / L), blank control group was given 2.0 mL of MEM EAGLE medium (without serum) containing 5.5 mmol / L glucose per well, and the other groups were added with experimental samples of corresponding concentrations on the basis of blank control group. After 24 h of drug intervention, the cell supernatant was discarded, and the cells were collected after trypsin digestion. The relative glycogen content in HepG2 cells was detected (anthrone method) according to the instructions of glycogen detection kit. The relative glycogen content is calculated as shown in formula 3.

[0057] Relative glycogen content = (O s – O b1 ) / (O m – O b2 ) × 100% (Formula 3)

[0058] Note: O s Detect OD value for each group of samples. b1 is the OD value of blank detection corresponding to each group of samples, O m The OD value of the model control group samples was detected. b2 is the OD value of the model control group corresponding to the blank test.

[0059] The experimental results are shown in Figure 3 The results showed that compared with the blank control group, demethylzeylasteral at a concentration of 20 μmol / L for 24 hours had a significant increase in the relative content of glycogen in insulin-resistant HepG2 cells (***p<0.001), and its activity was stronger than that of the positive control metformin group, indicating that demethylzeylasteral has a significant promoting effect on glycogen synthesis in HepG2 cells.

[0060] Example 3

[0061] Effects of norzelaminaldehyde on glucose uptake in insulin-resistant 3T3-L1 adipocytes

[0062] Experimental methods:

[0063] Differentiation of 3T3-L1 preadipocytes

[0064] 3T3-L1 preadipocytes in logarithmic growth phase were inoculated into 75 cm 2 Cell culture flasks (culture system: 10% CS High Glucose DMEM + 1% P / S) were cultured in an incubator at 37 °C and 5% CO2 for 48 h, and cell differentiation began when the cells grew to 80%-90% confluence. The cells covering the bottom of the culture flask were first starved for 48 h, and then replaced with High Glucose DMEM containing 0.5mmol / L 3-isobutyl-1-methylxanthine, 100 nmol / L insulin, 1.0 μmol / L dexamethasone, 1.0 μmol / L rosiglitazone, 10% FBS, and 1% P / S for 72 h. After 72 h, the culture system was replaced with High Glucose DMEM containing 100nmol / L insulin, 10% FBS, and 1% P / S for 24 h.

[0065] Glucose uptake experiment in 3T3-L1 adipocytes with insulin resistance

[0066] Successfully differentiated 3T3-L1 adipocytes were cultured at 1.5 × 10 5 Each well was inoculated into a 24-well plate, and then each well was given 1µmol / L dexamethasone for 72 h to construct an insulin resistance cell model. After 72 h, a blank control group, drug treatment group 1, drug treatment group 2, and drug treatment group 3 were set up. Each well of the blank control group was given 0.5 mL of DMEM medium (complete medium) containing 10.5 mmol / L glucose, each well of drug treatment group 1 was added with 0.5 mL of complete medium containing 20 μmol / L demethylzelaminaldehyde, each well of drug treatment group 2 was added with 0.5 mL of complete medium containing 100 nmol / L insulin, and each well of drug treatment group 3 was added with 0.5 mL of complete medium containing 100 nmol / L insulin and 20 μmol / L demethylzelaminaldehyde. After 24 h of drug intervention, 10 μL of culture medium supernatant was precisely aspirated from each well, and 190 μL of glucose content detection reagent was added. The OD value of the samples in each experimental group at 505 nm was detected by glucose oxidase method, and the glucose uptake rate of each experimental group was calculated by formula 4 to evaluate the effect of demethylzelaminaldehyde on the glucose uptake ability of insulin-resistant 3T3-L1 adipocytes.

[0067] Glucose uptake rate (%) = (O a -O s ) / O a × 100% (Formula 4)

[0068] Note: Oa is the OD value of glucose concentration in each experimental well at the time of drug administration, which is the glucose concentration before ingestion; Os is the OD value of glucose concentration in each experimental well 24 hours after drug administration, which is the glucose concentration after ingestion.

[0069] The experimental results are shown in Figure 4 The results showed that compared with the blank control group, the glucose uptake rate of 3T3-L1 adipocytes increased by 8.5% when norzelaminaldehyde acted alone, and the glucose uptake rate of 3T3-L1 adipocytes increased by 13.6% when insulin acted alone. When the two were used together, the theoretical glucose uptake rate should be 58.1%, while the actual glucose uptake rate was 64.1%, which was 6% higher than the theoretical value. The combined effect 1+1>2, indicating that norzelaminaldehyde has a certain effect of increasing the sensitivity of insulin-resistant 3T3-L1 adipocytes to insulin.

[0070] Example 4

[0071] Evaluation of the hypoglycemic effect of norzelaminaldehyde on db / db diabetic mice

[0072] Experimental principles and methods:

[0073] Experimental principle: db / db spontaneous diabetic mice are a type 2 diabetes model. The animals begin to overeat and gain weight at one month old, followed by uncontrolled increases in blood sugar, high blood insulin, pancreatic β cells, late peripheral neuropathy, and slowed healing, and generally die within 10 months. It is an ideal model for studying new drugs for the treatment of human type 2 diabetes.

[0074] Experimental methods: 8-week-old db / db diabetic male mice were selected, and random blood glucose and random body weight were measured at 9:00 am 2 days before administration. After that, fasting blood glucose and fasting body weight were measured after fasting for 6 hours (water was not allowed). 18 db / db mice were selected, and their random blood glucose was greater than 15mmol / L. According to the random blood glucose, random body weight, fasting blood glucose and fasting body weight of mice, the mice were divided into 3 groups, 6 in each group, and there were no significant differences in body weight and blood glucose among the groups. They were model control group (same dose of CMC-Na+ saline), positive drug metformin hydrochloride (Met) control group and demethylzeylasteral (Dem) group. Oral gavage was administered once a day, and the dosage of demethylzeylasteral and positive drug metformin hydrochloride groups was 40mg / kg.bw and 250mg / kg.bw, respectively, for 12 days. At the end of the experiment, the random blood glucose, random body weight, fasting blood glucose and fasting body weight of each group of mice were measured before and after administration.

[0075] The blood sugar reduction rate was calculated according to the following formula:

[0076]

[0077] The experimental results are shown in Table 1. The results show that continuous oral administration of demethylzeylasteral for 12 days has a significant effect on reducing both random blood glucose and fasting blood glucose in db / db diabetic mice, and the effect of the demethylzeylasteral administration group on reducing fasting blood glucose is better than that of the positive drug metformin hydrochloride group.

[0078] Table 1. Hypoglycemic effect of demethylzeylasteral on db / db diabetic mice

[0079]

[0080] Example 5

[0081] Tablets: 4 g of the compound demethylzeylasteral is mixed with 2.5 g of lactose and 3.0 g of starch, and the mixture is evenly moistened with water, sieved and dried, and then sieved again, and 0.5 g of magnesium stearate is added, mixed, and tableted into 100 tablets, with a tablet weight of 100 mg and a content of 40 mg / tablet.

[0082] Example 6

[0083] Capsules: 4 g of the compound demethylzeylasteral is mixed evenly with 2.5 g of lactose and 0.5 g of magnesium stearate, sieved, and mixed evenly. The resulting mixture is filled into 100 hard gelatin capsules, capsule weight: 70 mg, content: 40 mg / capsule.

[0084] Example 7

[0085] Oral ampoule: Add 4g of the compound demethylzeylasteral to conventional additives and purified water used to prepare oral solution, dilute to 0.5L, and aseptically fill into 100 ampoules, 5ml each, content: 40mg / bottle.

[0086] Example 8

[0087] Granules: Mix 4g of the compound demethylzeylasteral with 4g of lactose and 2g of starch, moisten them evenly with water, sieve the moistened mixture, dry it, sieve it again, and then make the mixture into granules and pack them into 100 bags, each bag weighing 100mg, with a content of 40mg / bag.

[0088] The above are only several embodiments of the present application, and are not intended to limit the present application in any form, that is, it does not mean that the present invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of the raw materials of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention. Within the scope of the technical concept of the present invention, a variety of simple variations can be made to the technical solution of the present invention, and these simple variations all belong to the protection scope of the present invention.

Claims

1. Use of the triterpenoid compound represented by formula I or its pharmaceutically acceptable salt, ester, solvate in the preparation of drugs or functional foods for lowering blood sugar, preventing and / or treating diabetes:

2. The use according to claim 1, characterized in that: The blood sugar lowering refers to lowering high blood sugar in type 1 diabetes or type 2 diabetes.

3. The use according to claim 1, characterized in that: The medicine or functional food has the effects of promoting cell glucose uptake, promoting cell glycogen synthesis and increasing insulin sensitivity.

4. The use according to claims 1 to 3, characterized in that: The medicine or functional food further comprises pharmaceutically or food acceptable excipients; Preferably, the auxiliary material comprises any one or a combination of at least two of a carrier, a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an emulsifier, a cosolvent, a solubilizer, an osmotic pressure regulator, a surfactant, a coating material, a colorant, a pH regulator, an antioxidant, an antibacterial agent or a buffer; Preferably, the pharmaceutical dosage form is a tablet, capsule, injection, granule or oral liquid; Preferably, the daily dosage of the nortriterpenoid compound is 0.01-100 mg / kg calculated based on body weight.

5. Use of the triterpenoid compound of formula I or its pharmaceutically acceptable salt, ester, solvate in the preparation of a cellular glucose uptake promoter and / or a hypoglycemic pharmaceutical preparation:

6. A pharmaceutical composition comprising a triterpenoid compound of formula I or a pharmaceutically acceptable salt, ester, solvate thereof and a pharmaceutically acceptable excipient, for use as a pharmaceutical preparation, wherein the drug is a hypoglycemic drug, a drug for treating and / or preventing diabetes:

7. The composition according to claim 6, characterized in that Based on the total weight of the composition, it contains 0.001-99.99wt% of the nortriterpenoid compound or its pharmaceutically acceptable salt, ester, solvate; Preferably, the daily dosage of the nortriterpenoid compound is 0.01-100 mg / kg calculated based on body weight.

8. The composition according to claim 6, characterized in that The pharmaceutical preparation is a tablet, capsule, injection, granule or oral liquid.

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