Application of formosan lattuce herb extract for regulating insulin secretion function of pancreatic beta cells in preparation of hypoglycemic drugs

Abstract

The invention provides application of a lobular formosan lattuce herb extract with a function of regulating insulin secretion of pancreatic beta cells in preparation of a hypoglycemic drug, and relates to the technical field of natural product extracts. A preparation method of the lobular broadleaf holly leaf extract comprises the following steps: taking high-quality and dry lobular broadleaf holly leaves, crushing, carrying out microwave treatment, and carrying out enzyme treatment; and carrying out water extraction and alcohol extraction, and separating the product. Compared with the prior art, the preparation method has the advantages that the dual advantages of microwave and enzyme treatment are ingeniously fused, the efficiency of the extract is greatly improved, the extract has a remarkable effect in the aspect of improving type 2 diabetes symptoms of model mice, the broadleaf holly leaf component is highly utilized, the loss of precious components is effectively reduced, and the preparation method is suitable for industrial production. The yield is obviously improved. Besides, the preparation method disclosed by the invention is comprehensively optimized in process, is simple and convenient to operate, is stable and reliable, and brings unprecedented development potential and broad prospects for the market.

Description

Technical Field

[0001] This invention provides the use of a small-leaf bitter tea extract that regulates the function of pancreatic β-cells in secreting insulin in the preparation of hypoglycemic drugs, and relates to the field of natural product extract technology. Background Technology

[0002] Diabetes is a global chronic metabolic disease with an increasing incidence rate, posing a serious threat to human health. According to the World Health Organization (WHO), the number of people with diabetes is growing at an unprecedented rate, especially in developing countries. Long-term hyperglycemia can damage multiple organs and systems throughout the body, leading to a series of serious complications such as cardiovascular disease, retinopathy, and diabetic nephropathy, significantly reducing patients' quality of life and imposing a heavy economic burden on families and society. Diabetes is mainly divided into two types: type 1 diabetes and type 2 diabetes. Type 1 diabetes usually occurs in childhood and adolescence and is caused by the immune system mistakenly attacking and destroying the beta cells in the pancreas that produce insulin. Patients must inject insulin daily to survive. Type 2 diabetes is more common, usually occurring in adults, and is related to lifestyle and genetic factors. The body cannot effectively use insulin, leading to elevated blood sugar levels. Hyperglycemia refers to an excessively high concentration of glucose in the blood, usually exceeding the normal range. Hyperglycemia can be a symptom of diabetes or a result of other health problems, such as stress, the use of certain medications, or complications of certain diseases. Prolonged high blood sugar can damage multiple organs and systems in the body, making blood sugar control crucial for people with diabetes. Treatment for diabetes typically includes medication, dietary control, exercise, and lifestyle modifications. Medication includes oral hypoglycemic agents and insulin injections. Dietary control aims to help patients maintain a healthy weight, control carbohydrate intake, and avoid high-sugar and high-fat foods. Exercise can improve the body's insulin sensitivity, helping cells utilize blood sugar more effectively. Lifestyle modifications include quitting smoking, limiting alcohol consumption, maintaining a healthy weight, and reducing stress.

[0003] Small-leaf bitter tea is a common traditional Chinese medicine, also known as bitter tea, belonging to the genus *Kudingcha* in the family Theaceae. It is mainly distributed in southern my country, such as Guangdong, Guangxi, and Fujian. Small-leaf bitter tea has a slightly bitter, fragrant, and sweet taste, possessing a unique flavor. In addition to its unique taste, small-leaf bitter tea is rich in nutritional value and offers various health benefits. However, many questions remain to be addressed in the research and application of small-leaf bitter tea in treating diabetes and hyperglycemia. Summary of the Invention

[0004] To address the above problems, this invention provides the use of a small-leaf bitter tea extract that regulates the insulin secretion function of pancreatic β-cells in the preparation of hypoglycemic drugs, the specific details and scheme of which are as follows:

[0005] First, this invention provides a small-leaf bitter tea extract that regulates the function of insulin secretion by pancreatic β cells, the preparation method of which includes the following steps:

[0006] (1) Raw material pretreatment: Weigh high-quality, dry small-leaf bitter tea leaves, crush and sieve them to obtain tea powder; mix the obtained tea powder with water thoroughly and microwave it, then perform enzyme treatment.

[0007] (2) Extraction: The product obtained in step 1 is further extracted by adding water and then subjected to a first heating extraction. The product is filtered to obtain filtrate 1 and residue 1. The residue 1 is then extracted by adding an ethanol aqueous solution and then subjected to a second heating extraction. The product is filtered to obtain filtrate 2. Filtrate 1 and filtrate 2 are mixed to obtain an extract.

[0008] (3) Concentration: The extract was concentrated under reduced pressure using a rotary evaporator until it reached a viscous state;

[0009] (4) Organic solvent extraction: The concentrated extract was extracted and separated by petroleum ether, ethyl acetate and n-butanol in sequence, and each was repeated three times to obtain petroleum ether component, ethyl acetate component, n-butanol component and water-soluble component in sequence.

[0010] (5) Drying: The extracts of each component were vacuum dried to obtain petroleum ether component, ethyl acetate component, n-butanol component, water-soluble component and water-soluble component lyophilized powder, which, when mixed, became the small-leaf bitter tea extract that regulates the function of insulin secretion by pancreatic β cells.

[0011] Preferably, the sieving in step 1 is done through a 50-100 mesh sieve.

[0012] Preferably, the mass ratio of the tea powder to water in step 1 is 1:(2-3), the microwave processing power is 500-700W, the temperature is controlled at 60-80℃, the total processing time is 30-60s, and intermittent processing is adopted, with a 10s interval after each 15s processing before the next processing.

[0013] Preferably, the enzyme treatment in step 1 includes: adding a mixed enzyme solution of cellulase, hemicellulase and pectinase to the microwave-treated product, performing enzymatic hydrolysis, and then inactivating the enzymes.

[0014] Preferably, the enzyme activity ratio of cellulase, hemicellulase, and pectinase in the mixed enzyme solution is 1:1:0.5.

[0015] Preferably, the concentration of each enzyme in the mixed enzyme solution is 0.09-0.40 IU / ml; the mass ratio of the microwave-treated product to the mixed enzyme solution is 1:(1-1.2).

[0016] Preferably, in step 2, water is added, wherein the mass ratio of the product from step 1 to water is 1:(10-20), and the first heating extraction is performed at a temperature of 80-90°C for 30-60 minutes.

[0017] Preferably, in step 2, the ethanol in the aqueous ethanol solution accounts for 70-80 vol%, and the mass ratio of filter residue 1 to the aqueous ethanol solution is 1:(5-10). The second heating extraction is carried out at a temperature of 50-70°C for 30-60 minutes.

[0018] Preferably, the temperature of the rotary evaporator in step 3 is 50-70℃ and the rotation speed is 40-60rpm.

[0019] Second, the present invention also provides a composition comprising the above-mentioned small-leaf bitter tea extract that regulates the function of pancreatic β-cells in secreting insulin, including small-leaf bitter tea extract that regulates the function of pancreatic β-cells in secreting insulin, oat β-glucan, vitamin D3, flaxseed extract, and mulberry leaf extract.

[0020] Preferably, the components in the composition are in the following proportions by mass:

[0021] The formula contains 20-40 parts of small-leaf bitter tea extract, 10-20 parts of oat β-glucan, 1-5 parts of vitamin D3, 5-15 parts of flaxseed extract, and 5-10 parts of mulberry leaf extract, which regulate the function of pancreatic β-cells in secreting insulin.

[0022] Third, the present invention also provides the use of the above-mentioned small-leaf bitter tea extract that regulates the function of pancreatic β-cells in secreting insulin in the preparation of pancreatic β-cell regulating foods, health products, compositions or drugs.

[0023] The beneficial effects of this invention are:

[0024] Compared with existing technologies, this invention innovatively combines microwave processing technology and enzyme processing technology, which enables the extracted components to effectively improve the symptoms of T2DM in model mice, efficiently utilize the components of bitter tea, reduce component loss, effectively improve yield, optimize the preparation process, and is reliable and simple, with broad market prospects.

[0025] The composition provided by this invention can further enhance the hypoglycemic effect through synergistic action. Among them, the small-leaf bitter tea extract, rich in various flavonoids and alkaloids, has significant hypoglycemic activity and is the core component of the composition. Oat β-glucan, as a high-quality dietary fiber, although it does not directly lower blood sugar, can improve the intestinal environment and delay carbohydrate absorption, indirectly contributing to blood sugar control. Vitamin D3 plays a key role in calcium metabolism; although it does not directly lower blood sugar, appropriate supplementation may help improve insulin sensitivity. Omega-3 fatty acids in flaxseed extract are beneficial to cardiovascular health and may indirectly affect glucose metabolism by improving blood lipids and reducing inflammation. Mulberry leaf extract contains various bioactive substances, such as flavonoids and polysaccharides, which help improve insulin resistance and promote insulin secretion. Although its hypoglycemic effect is relatively mild, its synergistic effect with other components can enhance the overall effect. The above components, through synergistic action, improve the overall hypoglycemic effect of the composition. Attached Figure Description

[0026] Figure 1 Effects of different extracts on typical symptoms of T2DM. (A) Schematic diagram created by BioRender.com to evaluate the effects of different SLK extract components on T2DM model mice. (B) Mouse weight records during high-fat diet feeding (n≥10). (C) Fasting blood glucose in mice 72 h after STZ injection. (D) Fasting blood glucose, (E) Body weight, (F) Metabolic capacity, (G) Water intake, (H) Food intake, (I) Body weight of each example and comparative example, (J) Fasting blood glucose of each example and comparative example. Control and model (T2DM) mice were given 0.2 mL / 10 g of physiological saline daily; the positive control group (metformin, 50 mg / kg / d) (Met), the petroleum ether group (SLK-PE), the n-butanol group (SLK-NA), and each example and comparative example group were all administered the component or product at 500 mg / kg / d (n=8). Data represent the mean ± SD. Compared with the control group, *P<0.05, **P<0.01. Compared with the model group, #P<0.05, ##P<0.01. Compared with the positive control group, ^P<0.05, ^^P<0.01.

[0027] Figure 2SLK-PE and SLK-NA improved oral glucose tolerance (OGTT), organ indices, serum biochemical indicators, and pathological conditions in T2DM mice. (A) OGTT and (B) AUC (n=8 mice / group). (C) Organ indices. (D) Mouse serum indicators: TG, CHO, HDL-C, GSP, LDL-C. (E) Mouse serum indicators: SOD, FINS, NEFA. (F) Calculation of pancreatic islet number using ImageJ software. Islet area (%) = (islet pixel area / tissue pixel area) × 100%. (G) Representative images of H&E-stained pancreatic, liver, spleen, lung, and kidney tissues. Compared with the control group, *P<0.05, **P<0.01. Compared with the model group, #P<0.05, ##P<0.01. Compared with the positive control group, ^P<0.05, ^^P<0.01.

[0028] Figure 3 Prediction and validation of pathway enrichment after SLK-PE and SLK-NA treatment in T2DM mice. (A) Intersection of target proteins of SLK-PE and SLK-NA active ingredients in T2DM treatment. (B) Pathway enrichment map of SLK-PE and SLK-NA active ingredients in T2DM treatment. (C) Protein-protein interaction network. (D) Western blot showing the regulatory effects of SLK-PE and SLK-NA on the expression of proteins related to the PI3K / AKT signaling pathway. (E) Quantification of D protein expression using ImageJ software. Compared with the control group, *P<0.05, **P<0.01. Compared with the model group, #P<0.05, ##P<0.01. Compared with the positive control group, ^P<0.05, ^^P<0.01.

[0029] Figure 4Effects of SLK-PE and SLK-NA on the expression of Nrf2, Keap-1, SIRT1, PGC-1α, INS, and GCG in pancreatic tissue of T2DM mice. (A) Immunohistochemical staining of pancreatic tissue (400x magnification, scale bar = 50 μm). (B) Quantitative calculation of the expression levels of Nrf2, Keap-1, and INS proteins in pancreatic tissue of group C using ImageJ. (C) Western blot showing the regulatory effects of SLK-PE and SLK-NA on the expression of Nrf2, Keap-1, and INS proteins. (D) Quantitative analysis of E protein expression using ImageJ software. (E) Immunofluorescence staining of SIRT1-positive pancreatic cells (red). PGC-1α-positive cells (green) and DAPI (blue) (600x magnification, scale bar = 50 μm). (F) Analysis and calculation of the positive area ratio of SIRT1 and PGC-1α using Aipathwell software. (G) Immunofluorescence staining of insulin-positive pancreatic cells (red). Glucagon-positive cells (green) and DAPI-positive cells (blue). (H) The positive area ratio of INS and GCG was analyzed and calculated using ImageJ software. Compared with the control group, *P<0.05, **P<0.01. Compared with the model group, #P<0.05, ##P<0.01. Compared with the positive drug group, ^P<0.05, ^^P<0.01.

[0030] Figure 5 Toxicity of SLK-PE and SLK-NA in mice. (A) Organ index of mice after administration of 32000 mg / kg extract. (B) Appearance of organs in mice during toxicity testing. (C) Representative H&E images of heart, liver, spleen, lung, kidney, and thymus tissues (400x magnification, scale bar = 50 μm). Compared with the control group, *P<0.05, **P<0.01. Compared with the model group, #P<0.05, ##P<0.01. Compared with the positive control group, ^P<0.05, ^^P<0.01.

[0031] Figure 6Effects of different concentrations of SLK-PE and SLK-NA on cell proliferation, cell morphology and glucose-stimulated insulin secretion in MIN6 cell model (A) Determination of safe concentrations of SLK-PE and SLK-NA by MTT assay. (B,C) Effects of SLK-PE and SLK-NA on proliferation of HFS-induced MIN6 cell injury model. Low, medium and high doses of SLK-PE and SLK-NA were 100, 200 and 400 μg / mL, respectively. (D,E) Effects of SLK-PE and SLK-NA on proliferation of STZ-induced MIN6 cell injury model. (F,G) Effects of SLK-PE and SL-NA on proliferation of HFS and STZ-induced MIN6 cell injury model. (H) Effects of SLK-PE and SLK-NA on cell morphology of HFS and STZ-induced MIN6 cell injury model. (H) Effects of different concentrations of SLK-PE on insulin release in MIN6 cell injury model under glucose stimulation. (1) Effects of different concentrations of SLK-PE on insulin release in MIN6 cell injury model under glucose stimulation. (J) Effects of different concentrations of SLK-NA on insulin release in a glucose-stimulated MIN6 cell injury model compared with the control group. *P<0.05, **P<0.01. Compared with the model group, #P<0.05, ##P<0.01.

[0032] Figure 7 Effects of SLK-PE and SLK-NA on PI3K / AKT signaling pathway and MafA / PCNA protein expression in MIN6 cells. (A) Western blot shows the regulatory effects of SLK-PE and SLK-NA on the expression of proteins related to the PI3K / AKT signaling pathway. (B, C, D, E) Quantitative analysis of protein A expression was performed using ImageJ software. (F) Western blot shows the regulatory effects of SLK-PE and SLK-NA on the expression of MafA and PCNA proteins. (G, H, I, J) Quantitative analysis of protein A expression was performed using ImageJ software. Compared with the control group, *P<0.05, **P<0.01. Compared with the model group, #P<0.05, ##P<0.01. Detailed Implementation

[0033] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.

[0035] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the experimental materials used in the following examples are all purchased from commercial channels.

[0036] Example 1: Preparation of a small-leaf bitter tea extract for regulating insulin secretion by pancreatic β-cells:

[0037] (1) Raw material pretreatment: Weigh high-quality, dry small-leaf bitter tea leaves, crush and sieve them to obtain tea powder; mix the obtained tea powder with water thoroughly and microwave it, then perform enzyme treatment.

[0038] (2) Extraction: The product obtained in step 1 is further extracted by adding water and then subjected to a first heating extraction. The product is filtered to obtain filtrate 1 and residue 1. The residue 1 is then extracted by adding an ethanol aqueous solution and then subjected to a second heating extraction. The product is filtered to obtain filtrate 2. Filtrate 1 and filtrate 2 are mixed to obtain an extract.

[0039] (3) Concentration: The extract was concentrated under reduced pressure using a rotary evaporator until it reached a viscous state;

[0040] (4) Organic solvent extraction: The concentrated extract was extracted and separated by petroleum ether, ethyl acetate and n-butanol in sequence, each repeated three times, to obtain petroleum ether component, ethyl acetate component, n-butanol component and water-soluble component (the water-soluble component comes from the extracted extract).

[0041] (5) Drying: The extracts of each component were vacuum dried to obtain petroleum ether component, ethyl acetate component, n-butanol component, water-soluble component and water-soluble component lyophilized powder, which, when mixed, became the small-leaf bitter tea extract that regulates the function of insulin secretion by pancreatic β cells.

[0042] The obtained petroleum ether component lyophilized powder is designated as SLK-PE component, the ethyl acetate component lyophilized powder as SLK-EA component, the n-butanol component lyophilized powder as SLK-NA component, and the water-soluble component lyophilized powder as SLK-WA component.

[0043] The sieving process described in step 1 involves passing the material through a 50-mesh sieve.

[0044] The mass ratio of tea powder to water in step 1 is 1:2. The microwave processing power is 500W, the temperature is controlled at 60℃, the total processing time is 30s, and intermittent processing is adopted, with a 10s interval after each 15s processing before the next processing.

[0045] Step 1, the enzyme treatment, includes: adding a mixed enzyme solution of cellulase, hemicellulase, and pectinase to the microwave-treated product, followed by enzymatic hydrolysis and enzyme inactivation. The enzyme activity ratio of cellulase, hemicellulase, and pectinase in the mixed enzyme solution is 1:1:0.5. The concentration of each enzyme in the mixed enzyme solution is 0.09 IU / ml; the mass ratio of the microwave-treated product to the mixed enzyme solution is 1:1.2.

[0046] In step 2, water is added, wherein the mass ratio of the product from step 1 to water is 1:10, and the first heating extraction is carried out at a temperature of 80°C for 60 minutes.

[0047] In step 2, the ethanol-water solution contains 70 vol% ethanol, and the mass ratio of filter residue 1 to the ethanol-water solution is 1:5. The second heating extraction is carried out at a temperature of 50°C for 60 min.

[0048] The rotary evaporator described in step 3 is set at a temperature of 50°C and a rotation speed of 60 rpm.

[0049] Example 2: Preparation of a small-leaf bitter tea extract for regulating insulin secretion by pancreatic β-cells:

[0050] (1) Raw material pretreatment: Weigh high-quality, dry small-leaf bitter tea leaves, crush and sieve them to obtain tea powder; mix the obtained tea powder with water thoroughly and microwave it, then perform enzyme treatment.

[0051] (2) Extraction: The product obtained in step 1 is further extracted by adding water and then subjected to a first heating extraction. The product is filtered to obtain filtrate 1 and residue 1. The residue 1 is then extracted by adding an ethanol aqueous solution and then subjected to a second heating extraction. The product is filtered to obtain filtrate 2. Filtrate 1 and filtrate 2 are mixed to obtain an extract.

[0052] (3) Concentration: The extract was concentrated under reduced pressure using a rotary evaporator until it reached a viscous state;

[0053] (4) Organic solvent extraction: The concentrated extract was extracted and separated by petroleum ether, ethyl acetate and n-butanol in sequence, each repeated three times, to obtain petroleum ether component, ethyl acetate component, n-butanol component and water-soluble component (the water-soluble component comes from the extracted extract).

[0054] (5) Drying: The extracts of each component were vacuum dried to obtain petroleum ether component, ethyl acetate component, n-butanol component, water-soluble component and water-soluble component lyophilized powder, which, when mixed, became the small-leaf bitter tea extract that regulates the function of insulin secretion by pancreatic β cells.

[0055] The sieving process described in step 1 involves passing the material through a 100-mesh sieve.

[0056] The mass ratio of tea powder to water in step 1 is 1:3. The microwave processing power is 700W, the temperature is controlled at 80℃, the total processing time is 30s, and intermittent processing is adopted, with a 10s interval after each 15s processing before the next processing.

[0057] Step 1, the enzyme treatment, includes: adding a mixed enzyme solution of cellulase, hemicellulase, and pectinase to the microwave-treated product, followed by enzymatic hydrolysis and enzyme inactivation. The enzyme activity ratio of cellulase, hemicellulase, and pectinase in the mixed enzyme solution is 1:1:0.5. The concentration of each enzyme in the mixed enzyme solution is 0.40 IU / ml; the mass ratio of the microwave-treated product to the mixed enzyme solution is 1:1.

[0058] In step 2, water is added, wherein the mass ratio of the product from step 1 to water is 1:20, and the first heating extraction is performed at a temperature of 90°C for 30 minutes.

[0059] In step 2, the ethanol-water solution contains 80 vol% ethanol, and the mass ratio of filter residue 1 to the ethanol-water solution is 1:10. The second heating extraction is carried out at a temperature of 70°C for 30 minutes.

[0060] The rotary evaporator described in step 3 is set at a temperature of 70°C and a rotation speed of 40 rpm.

[0061] Comparative Example 1: Preparation of a small-leaf bitter tea extract:

[0062] (1) Raw material pretreatment: Weigh high-quality, dry small-leaf bitter tea leaves, crush and sieve them to obtain tea powder; mix the obtained tea powder with water and then microwave it.

[0063] (2) Extraction: The product obtained in step 1 is further extracted by adding water and then subjected to a first heating extraction. The product is filtered to obtain filtrate 1 and residue 1. The residue 1 is then extracted by adding an ethanol aqueous solution and then subjected to a second heating extraction. The product is filtered to obtain filtrate 2. Filtrate 1 and filtrate 2 are mixed to obtain an extract.

[0064] (3) Concentration: The extract was concentrated under reduced pressure using a rotary evaporator until it reached a viscous state;

[0065] (4) Organic solvent extraction: The concentrated extract was extracted and separated by petroleum ether, ethyl acetate and n-butanol in sequence, each repeated three times, to obtain petroleum ether component, ethyl acetate component, n-butanol component and water-soluble component (the water-soluble component comes from the extracted extract).

[0066] (5) Drying: The extracts of each component are vacuum dried to obtain petroleum ether component, ethyl acetate component, n-butanol component, water-soluble component and water-soluble component lyophilized powder, which are then mixed to obtain small-leaf bitter tea extract.

[0067] The sieving process described in step 1 involves passing the material through a 100-mesh sieve.

[0068] The mass ratio of tea powder to water in step 1 is 1:3. The microwave processing power is 700W, the temperature is controlled at 80℃, the total processing time is 30s, and intermittent processing is adopted, with a 10s interval after each 15s processing before the next processing.

[0069] In step 2, water is added, wherein the mass ratio of the product from step 1 to water is 1:20, and the first heating extraction is performed at a temperature of 90°C for 30 minutes.

[0070] In step 2, the ethanol-water solution contains 80 vol% ethanol, and the mass ratio of filter residue 1 to the ethanol-water solution is 1:10. The second heating extraction is carried out at a temperature of 70°C for 30 minutes.

[0071] The rotary evaporator described in step 3 is set at a temperature of 70°C and a rotation speed of 40 rpm.

[0072] Example 3: A composition containing a small-leaf bitter tea extract that regulates the function of pancreatic β-cells in secreting insulin.

[0073] The composition includes *Kudzu chinensis* extract (which regulates insulin secretion from pancreatic β-cells), oat β-glucan, vitamin D3, flaxseed extract, and mulberry leaf extract. The components in the composition, by mass parts, are as follows:

[0074] Example 2 prepared 20 parts of small-leaf bitter tea extract, 20 parts of oat β-glucan, 5 parts of vitamin D3, 15 parts of flaxseed extract, and 10 parts of mulberry leaf extract to regulate the function of insulin secretion by pancreatic β cells.

[0075] Example 4: A composition containing a small-leaf bitter tea extract that regulates the function of pancreatic β-cells in secreting insulin.

[0076] The composition includes *Kudzu chinensis* extract (which regulates insulin secretion from pancreatic β-cells), oat β-glucan, vitamin D3, flaxseed extract, and mulberry leaf extract. The components in the composition, by mass parts, are as follows:

[0077] Example 2 prepared 40 parts of small-leaf bitter tea extract, 10 parts of oat β-glucan, 1 part of vitamin D3, 5 parts of flaxseed extract, and 5 parts of mulberry leaf extract to regulate the function of insulin secretion by pancreatic β cells.

[0078] Comparative Example 2: A composition:

[0079] The composition includes oat beta-glucan, vitamin D3, flaxseed extract, and mulberry leaf extract. The components in the composition, by mass parts, are as follows:

[0080] 10 parts oat beta-glucan, 1 part vitamin D3, 5 parts flaxseed extract, and 5 parts mulberry leaf extract.

[0081] Comparative Example 3: A composition:

[0082] The composition includes extracts of small-leaf bitter tea (which regulates insulin secretion from pancreatic β-cells), vitamin D3, flaxseed extract, and mulberry leaf extract. The components in the composition, by mass parts, are as follows:

[0083] Example 2 prepared 40 parts of small-leaf bitter tea extract, 1 part of vitamin D3, 5 parts of flaxseed extract, and 5 parts of mulberry leaf extract to regulate the function of insulin secretion by pancreatic β cells.

[0084] Comparative Example 4: A composition:

[0085] The composition includes extracts of small-leaf bitter tea (which regulates insulin secretion from pancreatic β-cells), oat β-glucan, flaxseed extract, and mulberry leaf extract. The components in the composition, by mass parts, are as follows:

[0086] Example 2 prepared 40 parts of small-leaf bitter tea extract, 10 parts of oat β-glucan, 5 parts of flaxseed extract, and 5 parts of mulberry leaf extract to regulate the function of pancreatic β-cells in secreting insulin.

[0087] Comparative Example 5: A composition:

[0088] The composition includes *Kudzu chinensis* extract (which regulates insulin secretion from pancreatic β-cells), oat β-glucan, vitamin D3, and mulberry leaf extract. The components in the composition, by mass parts, are as follows:

[0089] Example 2 prepared 40 parts of small-leaf bitter tea extract, 10 parts of oat β-glucan, 1 part of vitamin D3, and 5 parts of mulberry leaf extract to regulate the function of insulin secretion by pancreatic β cells.

[0090] Comparative Example 6: A composition:

[0091] The composition includes *Kudzu chinensis* extract, which regulates insulin secretion from pancreatic β-cells; oat β-glucan; vitamin D3; and flaxseed extract. The components in the composition, by mass parts, are as follows:

[0092] Example 2 prepared 40 parts of small-leaf bitter tea extract, 10 parts of oat β-glucan, 1 part of vitamin D3, and 5 parts of flaxseed extract to regulate the function of pancreatic β-cells in secreting insulin.

[0093] The hypoglycemic effects of the extracts or compositions prepared in the above examples and comparative examples were tested using mouse experiments, as detailed below:

[0094] I. Experimental Methods

[0095] Laboratory Animals: All animal experiments were approved by the Laboratory Animal Ethics Committee of Guizhou Medical University (No. 2101015, Animal Production License No.: SCXK(Beijing)2019-0010). Healthy male C57BL / 6J mice (6 weeks old, 18-24g) were purchased from Beijing Speford Biotechnology Co., Ltd. and housed at the Guizhou Provincial Natural Products Research Center in a specific pathogen-free facility with individual ventilation cages (IVC) barriers. The ambient temperature was (22±2)℃, the humidity was (50±10)%, and the normal light-dark cycle was maintained in an SPF-grade animal room with an IVC barrier environment. The housing environment temperature was (22±2)℃, the humidity was (50±10)%, and normal light-dark cycle was maintained. Mice had free access to food and water. The mouse feed and bedding were SPF-grade, and the drinking water was distilled water sterilized by high temperature and high pressure.

[0096] Animal model of type 2 diabetes mellitus (T2DM): After one week of acclimatization, the control group was fed a conventional diet, while the model mice were fed a high-fat diet (HFD: 32.5% maintenance basal diet, 28% lard, 8% sucrose, 10.8% whole milk powder, 13.5% casein, 3% experimental animal premix, 2% microcrystalline cellulose, 1.8% dicalcium phosphate, and 0.4% limestone) for 4 weeks. After a 12-hour fast, streptozotocin (STZ) was injected intraperitoneally (STZ 200 mg / kg, twice, 72 hours apart). The control group received the same volume (0.2 mL / 10 g) of sterile citrate buffer. On the third day after injection, all mice were fasted for 12 hours, and fasting blood glucose was measured from the tail vein. A blood glucose level ≥11.1 mmol / L was considered a successful establishment of the type 2 diabetes mouse model.

[0097] Animal treatment groups

[0098] Mice were randomly assigned to Metformin (50 mg / kg / day), the respective control and comparative treatment groups, and a control group. Following our previous study, all extracts were administered at a dose of 500 mg / kg / day. Control and model rats were given 0.2 mL / 10 g / day of physiological saline daily. Food intake, water intake, and urine output were recorded every 4 days. After administration, experimental group mice were fasted for 12 hours weekly, and a small amount of tail blood was collected to measure fasting blood glucose (FBG).

[0099] In week 8 of the experiment, an oral glucose tolerance test (OGTT) was performed. Mice were fasted for 8 hours but allowed free access to water, and then administered anhydrous glucose solution by gavage at a dose of 2 g / kg. Blood glucose concentrations were measured by collecting blood from the tail vein before administration and at 30, 60, 90, and 120 minutes after administration.

[0100] Biochemical index determination: Using kits and in accordance with the manufacturer's instructions, the levels of serum triglycerides (TG), cholesterol (CHO), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), glycated serum protein (GSP), fasting insulin (FINS), superoxide dismutase (SOD), and free fatty acids (NEFA) in mice were measured.

[0101] Histopathological examination: Pancreatic tissues from mice in each group were fixed with 4% paraformaldehyde for 48 h, embedded in conventional paraffin, and cut into 4 μm sections for H&E staining. Morphological changes of the pancreas were observed and photographed under a Leica inverted FL fluorescence microscope (400x magnification).

[0102] Immunohistochemistry: Pancreatic tissue was fixed in 4% paraformaldehyde solution. Antigen retrieval and dewaxing were performed. After blocking nonspecific binding, the tissue was incubated overnight at 4°C with bovine serum albumin and tested for Nrf2 (1:1000), Keap-1 (1:1000), and INS (1:2000) with primary antibodies. Sections were incubated with anti-rabbit IgG-HRP, followed by DAB staining and dehydration. Finally, sections were cleared in xylene and sealed with neutral adhesive. Images were obtained at 400x magnification using a microscope and analyzed using Image-Pro Plus 6.0 software.

[0103] Immunoblot: Total protein was extracted from 10% pancreatic tissue lysate using RIPA buffer (containing 1% PMSF). After freezing for 30 min, the mixture was centrifuged at 12000 rpm for 10 min at 4 °C. The supernatant was collected, and proteins were separated by SDS-PAGE and transferred to a PVDF membrane. Incubation with 5% BSA blocked nonspecific binding. After washing with TBST, the membrane was incubated overnight at 4 °C with primary antibody. The membrane was washed with TBST solution, and incubated with secondary antibody on a level shaker at room temperature for 60 min. After washing with TBST solution, protein bands were detected using a Bio-Rad ChemiDoc MP membrane scanner. The optical density of the protein bands was measured using ImageJ software, with β-actin as an internal control.

[0104] Optimization analysis: The extract (0.15 g) was ultrasonically extracted in a 25 mL colorimetric tube for 40 min. After filtration through a 0.22 μm microporous membrane, 1 mL of each extract was transferred to a 10 mL volumetric flask and diluted twice. Chromatographic analysis was then performed under the conditions listed in the table below. Table 1 Liquid chromatography-mass spectrometry conditions

[0105]

[0106]

[0107]

[0108] Enrichment pathway prediction: Simplified molecular input line input system (SMILES) structural formulas were obtained. The compound structures were imported into the Swiss Target Prediction database (http: / / www.swisstargetprediction.ch / ) to predict target compounds. The GeneCard database was used to search for diabetes-related genes, and the obtained target protein names were converted to official gene symbols using the UniProt database (https: / / www.uniprot.org / ) for subsequent analysis. Through Kyoto Encyclopedia of Genetics and Genomes (KEGG) pathway analysis, a protein-protein interaction (PPI) network was constructed in the String database (String: functional protein association networks: https: / / cn.string-db.org / ) to screen for core targets.

[0109] Toxicity in mice: In the preliminary experiment, healthy 6-week-old Kunming mice were randomly divided into the following experimental groups, with N=4 mice in each group. SLK-PE and SLK-NA were administered by gavage at low, medium, and high doses, respectively, at doses of 4000, 8000, and 16000 mg / kg (0.2 mL / 10 g). Control group mice were given 0.2 mL / 10 g of physiological saline. Mice were fasted for 16 hours before administration but had free access to water. Mice were observed for 7 days after oral administration. No deaths or toxic effects were observed in the highest dose group (16000 mg / kg). Due to limitations in extract solubility, the dose was doubled in the formal toxicity study. In the formal toxicity study, 60 healthy male Kunming mice aged 6 weeks were selected and divided into 3 groups (n=20 / group). SLK-PE and SLK-NA were administered by gavage at 32000 mg / kg (0.2 mL / 10 g), while the control group was administered physiological saline (0.2 mL / 10 g) by gavage. Mice were fasted for 16 hours before gavage but had free access to water. Mice were observed for 4 hours after oral administration, followed by once-daily observation for 7 days. Acute toxicity and mortality were recorded during this period. Mice were euthanized by dislocation at the end of the experiment, and their organs were harvested. The volume and color of each organ were observed.

[0110] Cell Culture: Normal mouse pancreatic β cells (MIN6, Beijing Dingguo Biotechnology Co., Ltd., Beijing, China) were cultured in DMEM medium containing 10% fetal bovine serum (FBS) (Gibco Laboratories, Grand Island, NY, USA), 100 IU / mL penicillin, and 100 μg / mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA) at 37°C with 5% CO2. Cells were seeded at a density of 4000 cells / well in 96-well plates and cultured for 12 hours. Cells were then exposed to 30 mmol / L glucose, 0.4 mmol / L sodium palmitate, and 3 mM STZ (Sigma-Aldrich, St. Louis, MO, USA) for 24 hours.

[0111] Cell viability assay: MIN6 cells in logarithmic growth phase were seeded at a concentration of 4000 cells / well in 96 wells, with 100 μL of cell suspension added to each well. Five composite wells were set up per group. Blank wells were left at the edge, and PBS was added to prevent evaporation of the cell suspension. Cells were removed after the preset time, and the culture medium in the wells was discarded. 10% CCK-8 solution was added to each well (ensuring no air bubbles in the wells), and the cells were incubated in the culture plate for the specified time. Cell color was observed at the predetermined time, and enzyme labeling was used for detection. The absorbance was set at 450 nm, and the absorbance values ​​of different groups were measured.

[0112] Glucose-stimulated insulin secretion (GSIS) assay: MIN6 cells were seeded for 12 h, then incubated with HFS and STZ, and different concentrations of SLK-PE and SLK-NA for 24 h. They were then incubated with 0.1% BSA (Sigma-Aldrich; St. Louis, MO, USA) in KRB buffer (Sigma-Aldrich) for 1 h, followed by incubation with KRB buffer containing low or high glucose for 1 h. Insulin levels were measured from the cell supernatant.

[0113] PI3K / AKT signaling pathway and MafA / PCNA protein expression: Immunowestern blot sample collection: MIN6 cells in logarithmic growth phase, 2 × 10⁶ 5 Cells were seeded at a density of 100 cells / mL in 6-well plates, with 2 mL of cell suspension added to each well. After cell adhesion, cells were treated according to different experimental groups. Cells were further processed 24 hours after intervention. Western blot analysis was performed as described above.

[0114] Statistical analysis: Statistical analysis was performed using Excel and SPSS 26.0. Quantitative data are expressed as mean ± standard deviation (SD). One-way ANOVA was used for comparisons among multiple groups. A p-value < 0.05 was considered statistically significant, and a p-value < 0.01 was considered highly significant.

[0115] II. Experimental Results

[0116] 1. Determination of chemical composition of extracts:

[0117] The extraction rates of SLK-PE, SLK-EA, SLK-NA, and SLK-WA were 1.12%, 3.27%, 9.82%, and 4.30%, respectively. Qualitative analysis of the substances in the different extracts was performed using liquid chromatography-mass spectrometry (LC-MS). The compounds identified in SLK-PE and SLK-NA are shown in Table 2.

[0118] Table2.Chemical constituents ofdifferent SLK extracts

[0119]

[0120] 2. Effects of the extracts and compositions provided by this invention on physiological indicators of mice.

[0121] A type 2 diabetes mellitus (T2DM) mouse model was established using a high-fat diet combined with STZ, achieving a modeling success rate of 91.428%. This study aimed to evaluate the effects of different extracts of SLK on T2DM model mice, as illustrated in the diagram below. Figure 1 As shown in Figure A, during the high-fat diet feeding period, the weight gain rate of the model group rats was higher than that of the control group ( Figure 1 B). Furthermore, the fasting blood glucose level in the model mice after STZ injection was significantly higher than that in the control group ( Figure 1 C). After gavage administration of different SLK extract components, various physiological indicators of mice in the SLK-PE and SLK-NA groups improved, while SLK-EA and SLK-WA had no significant effect on mice. Therefore, our subsequent experiments mainly focused on SLK-PE and SLK-NA. FBG concentrations in all groups of mice continued to increase, reaching a peak 2–3 weeks after STZ injection. Figure 1 D). At week 8, compared with the T2DM group, the FBG concentrations in the Met, SLK-PE, and SLK-NA groups were significantly lower (P<0.01), and after 6 weeks, the effect of the SLK-PE group was significantly greater than that of the Met group (P<0.01). The T2DM group exhibited typical symptoms of diabetes, such as polydipsia, polyphagia, polyuria, and weight loss. The body weight of mice in the SLK-PE group gradually increased over time and was significantly higher than that in the T2DM group (P<0.01). The effect of SLK-PE was significantly greater than that of Met and SLK-NA (P<0.01). Figure 1 E). The T2DM group mice maintained high levels of food intake, water consumption, and metabolic capacity, while these symptoms in the Met group mice began to decline on day 24. Figure 1(FH). These symptoms in the SLK-PE group mice began to decrease on day 16 and gradually reached levels similar to the control group. The SLK-NA group was more effective than the Met group, but less effective than the SLK-PE group. Therefore, the results indicate that SLK-PE is more effective than the positive control drug Met in improving T2DM symptoms. From Figure 1 I, Figure 1 As can be seen, the composition provided by the present invention can improve the symptoms of T2DM more effectively than a single component.

[0122] 3. The extracts and compositions provided by this invention improve oral glucose tolerance (OGTT) and biochemical indicators.

[0123] In the OGTT, blood glucose levels decreased in all five groups at 60 min, 90 min, and 120 min after glucose administration. Figure 2 A). Blood glucose levels in both the SLK-PE and SLK-NA groups were lower than those in the T2DM group. AUC calculations of the OGTT results for each group showed that the control group had the smallest AUC area, while the AUCs of the T2DM and Met groups were significantly higher than those of the SLK-PE and SLK-NA groups (P<0.01), suggesting that SLK-PE and SLK-NA can improve glucose regulation in T2DM mice. Figure 2 B). There were no significant differences in cardiac indices among the groups of mice. T2DM mice developed hepatic steatosis, and the liver, spleen, and pancreas indices in the SLK-PE and SLK-NA groups were significantly different from those in the T2DM group (P<0.01). The lung indices in the T2DM, Met, and SLK-NA groups were significantly different from those in the control group (P<0.01), while there was no significant difference in the SLK-PE group (P<0.01). The kidney index in the SLK-PE group was significantly lower than that in the T2DM group (P<0.01). The results show that both SLK-PE and SLK-NA can improve the indices of the spleen, kidney, and pancreas in T2DM mice, reducing the risk of T2DM. Figure 2 C). In the T2DM group, the levels of triglycerides (TG), glycosylated serum protein (GSP), low-density lipoprotein cholesterol (LDL-C), cholesterol (CHO), and non-esterified fatty acids (NEFA) were higher than those in the control group, while the levels of high-density lipoprotein cholesterol (HDL-C), superoxide dismutase (SOD), and fasting insulin (FINS) were lower, and diabetic symptoms were more pronounced. After administration of SLK-PE and SLK-NA, the levels of TG, CHO, GSP, HDL-C, and NEFA were significantly decreased (P<0.05 or P<0.01), while the levels of HDL-C, SOD, and FINS were significantly increased (P<0.05 or P<0.01). Figure 2DE). The results showed that SLK-PE and SLK-NA improved the pathological state of organs such as liver, spleen, lung, kidney, and pancreas in T2DM mice and reduced the risk of T2DM complications, suggesting that pancreatic islet cells were damaged in T2DM mice. Compared with the control group, the SLK-PE group (P<0.01), SLK-NA group (P<0.01), and Met group (P<0.05) showed significantly better morphological damage and islet number of pancreatic islet cells than the T2DM group, and nuclear optical staining was significantly improved ( Figure 2 (FG), among which the SLK-PE group showed the best results.

[0124] 4. Prediction and verification of pathway enrichment

[0125] A total of 286 target proteins were found to intersect among the T2DM, SLK-PE, and SLK-NA groups. Figure 3 A). Among all the relevant pathways in the PPI network, the PI3K / AKT pathway is associated with the highest levels of proteins. Figure 3 (BC). Furthermore, KEGG enrichment analysis showed that the PI3K / AKT pathway was the most enriched. The PI3K / AKT signaling pathway is mainly involved in physiological activities such as cell proliferation, differentiation, apoptosis, and glucose and lipid metabolism. Next, we examined the expression levels of related genes. Compared with the T2DM group, SLK-PE and SLK-NA treatment significantly increased the expression levels of P-PI3K / PI3K, P-AKT / AKT, and Glut4, which are related to the PI3K / AKT signaling pathway, in mouse pancreatic tissue (P<0.01 or P<0.05), and SLK-PE showed a better ability to upregulate Glut4 protein expression than Met (P<0.01). Compared with the T2DM group, SLK-PE and SLK-NA treatment downregulated the expression of FoXO1 and GSK-3β in mouse pancreatic tissue (P<0.01 or P<0.05). Figure 3 DE). The SLK-PE group showed a significantly better ability to downregulate the expression of FoXO1 and GSK-3β proteins than the SLK-NA and Met groups (P<0.01 or P<0.05). These results suggest that SLK-PE and SLK-NA may affect blood glucose levels by interfering with the expression of the PI3K / AKT signaling pathway and its related proteins.

[0126] 5. SLK-PE and SLK-NA activate the expression of genes regulating β-cell function.

[0127] Activation of Nrf2 helps maintain β-cell quality by inhibiting β-cell death and promoting their proliferation. Insulin (INS) is mainly secreted by β-cells and is crucial for regulating blood glucose levels. Immunohistochemical analysis showed that, after 8 weeks of drug administration, compared with the model group, the expression of Nrf2 and INS proteins was upregulated in pancreatic tissue of the SLK-PE and SLK-NA groups, while the expression of Keap-1 protein was significantly downregulated (P<0.01). Figure 4 AB). Western blot analysis of the expression levels of Nrf2, Keap-1, and INS proteins yielded results consistent with immunohistochemical findings. Figure 4 CD). Considering that activation of SIRT1 and PGC-1α helps improve pancreatic β-cell function, the effects of SLK-PE and SLK-NA on SIRT1 and PGC-1α protein expression in pancreatic tissue of T2DM mice were detected by confocal microscopy after immunofluorescence staining. Figure 4 Compared with the T2DM group, the expression of SIRT1 and PGC-1α proteins in the SLK-PE and SLK-NA groups was significantly upregulated (P<0.01), and the effect was greater than that in the Met group. The T2DM group showed a significant reduction in insulin-positive area, a significant decrease in the proportion of β cells, and an increase in α cell infiltration into the islet center. Administration of SLK-PE and SLK-NA partially prevented β cell damage, maintained β cell quality, and reduced α cell infiltration (P<0.01 or P<0.05). Figure 4 The above results indicate that SLK extract can improve the function and quality of pancreatic β cells, which is beneficial for lowering blood glucose levels.

[0128] 6. Toxicity

[0129] No mice showed signs of death, abnormal appearance, abnormal stool, abnormal mental state, or abnormal behavior during the study period. Due to solubility limitations, there was no significant difference in mean daily food intake and water consumption between the control and treatment groups after oral gavage administration of the maximum dose of 3200 mg / kg of SLK-PE and SLK-NA. Figure 5 A). The liver weight of mice in the SLK-PE and SLK-NA groups was slightly increased compared to the control group, but the increase was not significant. Figure 5 B). This difference may be related to the increased hepatic metabolic load caused by high-dose SLK extract. Representative H&E staining images of the heart, liver, spleen, lung, kidney, and thymus of mice in each group are shown below. Figure 5 As shown in Figure C, H&E staining of the heart, liver, spleen, lungs, kidneys, and thymus in both the SLK-PE and SLK-NA groups showed no significant pathological changes compared to the control group. These experiments demonstrate that SLK-PE and SLK-NA have good safety profiles.

[0130] 7. Effects on MIN6 proliferation activity, cell morphology, and GSIS

[0131] The effects of SLK-PE and SLK-NA on MIN6 cell viability as detected by MTT assay are as follows: Figure 6 As shown in Figure A, the OD values ​​of cells treated with SLK-PE and SLK-NA for 24 h showed that the 400 μg / mL group had the highest MIN6 cell viability. The β-cell population is considered to be mainly maintained through proliferation; the CCK8 assay was used to detect the effects of SLK-PE and SLK-NA on the proliferation of the MIN6 cell injury model. Compared with the control group, the cell proliferation activity of the MIN6 cell injury model groups treated with high-fat / high-glucose and STZ alone, as well as those treated with high-fat / high-glucose + STZ, was significantly reduced (P<0.01). Compared with the model group, the cell viability of cells treated with different concentrations of SLK-PE and SLK-NA increased in a dose-dependent manner (P<0.01 or P<0.05). Figure 6 BG). At the same concentration, we found that SLK-PE had a better improvement effect than SLK-NA. Under light microscopy, the control group MIN6 cells were adherent cells with distinct angles. MIN6 cells established by HFS combined with STZ showed shrunken angles and reduced cell number. After intervention with SLK-PE and SLK-NA, cell morphology was improved (BG). Figure 6 H). To investigate the effects of SLK-PE and SLK-NA on pancreatic β-cell function, we performed a GSIS experiment ( Figure 6 The results showed that MIN6 cell function was impaired after treatment with HFS and STZ. There was no statistically significant difference in insulin secretion between the two groups under 2.8 mM hypoglycemic conditions, but insulin secretion was significantly increased under 16.7 mM glucose stimulation (P>0.05). Treatment with SLK-PE and SLK-NA significantly or extremely significantly increased insulin secretion in damaged MIN6 cells (P<0.05 or P<0.01). These results indicate that SLK-PE and SLK-NA can improve the function of MIN6 pancreatic islet cells and enhance the sensitivity of MIN6 cells to glucose-stimulated pancreatic hormone secretion.

[0132] 8. Expression of PI3K / AKT signaling pathway and MafA / PCNA proteins

[0133] To verify the effects of SLK-PE and SLK-NA on the expression levels of regulatory proteins in the PI3K / AKT signaling pathway and β-cell function, we performed Western blot experiments. Figure 7As shown, protein detection results indicated that, compared with the control group, the expression of PI3K / P-PI3K and AKT / P-AKT proteins in the model group was decreased (P<0.01). Compared with the model group, the expression of PI3K / P-PI3K and AKT / P-AKT proteins in the low, medium, and high dose groups of the SLK-PE and SLK-NA groups was increased (P<0.05 or P<0.01). Figure 7 (AE). The effects of SLK-PE and SLK-NA were weakened after the addition of the PI3K pathway inhibitor LY290042 (P<0.01). Compared with the control group, the expression levels of MafA and PCNA in cells of the HFS+STZ group were decreased (P<0.01). Compared with the HFS+STZ group, the expression levels of MafA and PCNA proteins in both the SLK-PE and SLK-NA groups were increased (P<0.05 or P<0.01). The effects of SLK-PE and SLK-NA were weakened in the LY290042 group (P<0.01). Figure 7 (FJ). The results showed that SLK-PE and SLK-NA can improve MIN6 function through the PI3K / AKT signaling pathway, increasing MIN6 proliferation and insulin secretion.

[0134] In this study, we extracted and isolated the natural components of SLK and preliminarily identified its active ingredients. Through a series of studies, the components in SLK-PE and SLK-NA can alleviate pancreatic islet cell damage, increase insulin secretion, and improve symptoms in T2DM mice by regulating proteins related to the PI3K / AKT signaling pathway involved in glucose and lipid metabolism and oxidative stress. Due to the large number of genes involved in T2DM, we only analyzed the expression levels of proteins related to the PI3K / AKT signaling pathway. Therefore, we cannot rule out the possibility that other signaling pathways play a role in the mechanism by which SLK alleviates T2DM. Further research is needed to clarify this issue. In conclusion, SLK extract may have significant potential value as a nutritional and health product.

[0135] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0136] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention. The actual application is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar methods and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A small-leaf bitter tea extract that regulates the function of insulin secretion by pancreatic β cells, characterized in that: Its preparation method includes the following steps: (1) Raw material pretreatment: Weigh high-quality, dry small-leaf bitter tea leaves, crush and sieve them to obtain tea powder; mix the obtained tea powder with water thoroughly and microwave it, then perform enzyme treatment. (2) Extraction: The product obtained in step 1 is further extracted by adding water and then subjected to a first heating extraction. The product is filtered to obtain filtrate 1 and residue 1. The residue 1 is then extracted by adding an ethanol aqueous solution and then subjected to a second heating extraction. The product is filtered to obtain filtrate 2. Filtrate 1 and filtrate 2 are mixed to obtain an extract. (3) Concentration: The extract was concentrated under reduced pressure using a rotary evaporator until it reached a viscous state; (4) Organic solvent extraction: The concentrated extract was extracted and separated by petroleum ether, ethyl acetate and n-butanol in sequence, and each was repeated three times to obtain petroleum ether component, ethyl acetate component, n-butanol component and water-soluble component in sequence. (5) Drying: The extracts of each component were vacuum dried to obtain petroleum ether component, ethyl acetate component, n-butanol component, water-soluble component and water-soluble component lyophilized powder, which, when mixed, became the small-leaf bitter tea extract that regulates the function of insulin secretion by pancreatic β cells.

2. The small-leaf bitter tea extract according to claim 1, characterized in that: Step 1, the enzyme treatment, includes adding a mixed enzyme solution of cellulase, hemicellulase, and pectinase to the microwave-treated product, followed by enzymatic hydrolysis and enzyme inactivation.

3. The small-leaf bitter tea extract according to claim 2, characterized in that: The enzyme activity ratio of cellulase, hemicellulase, and pectinase in the mixed enzyme solution is 1:1:0.

5.

4. The small-leaf bitter tea extract according to claim 3, characterized in that: The concentration of each enzyme in the mixed enzyme solution is 0.09-0.40 IU / ml; the mass ratio of microwave-treated product to mixed enzyme solution is 1:(1-1.2).

5. The small-leaf bitter tea extract according to claim 1, characterized in that: The mass ratio of tea powder to water in step 1 is 1:(2-3). The microwave processing power is 500-700W, the temperature is controlled at 60-80℃, the total processing time is 30-60s, and intermittent processing is adopted, with a 10s interval after each 15s processing before the next processing.

6. The small-leaf bitter tea extract according to claim 1, characterized in that: In step 2, water is added, wherein the mass ratio of the product from step 1 to water is 1:(10-20). The first heating extraction is carried out at a temperature of 80-90℃ for 30-60 minutes.

7. The small-leaf bitter tea extract according to claim 1, characterized in that: In step 2, the ethanol aqueous solution contains 70-80 vol% ethanol, and the mass ratio of filter residue 1 to the ethanol aqueous solution is 1:(5-10). The second heating extraction is carried out at a temperature of 50-70℃ for 30-60 min.

8. A composition comprising the extract of *Kuding jasmine* as described in any one of claims 1-7, which regulates the function of pancreatic β-cells in secreting insulin, characterized in that: It includes small-leaf bitter tea extract, coenzyme Q10, grape seed extract, soybean lecithin, and inulin, which regulate the function of pancreatic β-cells in secreting insulin.

9. The composition according to claim 8, characterized in that: The components in the composition are as follows by mass: 20-50 parts of small-leaf bitter tea extract, 5-15 parts of coenzyme Q10, 10-30 parts of grape seed extract, 5-20 parts of soybean lecithin, and 5-20 parts of inulin.

10. The use of the small-leaf bitter tea extract according to any one of claims 1-7 in the preparation of hypoglycemic foods, health products, compositions or drugs.

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