Application of schizophyllumcommuneh in preparation of medicine for treating type 2 diabetes mellitus

Abstract

The invention discloses an application of schizophyllumcommuneh in preparation of a medicine for treating type 2 diabetes mellitus, and belongs to the technical field of pharmacy. The active ingredient of the medicine for treating the type II diabetes is a schizophyllumcommuneh extract, and the preparation method of the schizophyllumcommuneh extract comprises the following steps: taking schizophyllumcommuneh as a raw material, extracting through an enzymolysis and microwave-assisted method, and then performing ethanol precipitation and spray drying to obtain the schizophyllumcommuneh extract. Based on a diabetic mouse model, in the aspect of monitoring the blood sugar content of a mouse, the Schizophyllumcommuneh is found to have prevention and treatment effects on the type 2 diabetes mellitus, and the Schizophyllumcommuneh is proved to have an exact curative effect on the type 2 diabetes mellitus. The Schizophyllumcommuneh extract prepared by the invention has the characteristics of safety and high efficiency, has potential development value for type 2 diabetes mellitus drugs, is a natural preparation, and is worthy of popularization.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the application of a type of white ginseng fungus in the preparation of a drug for treating type 2 diabetes. Background Technology

[0002] Diabetes is mainly classified into four categories: type 1 diabetes mellitus (T1DM), type 2 diabetes mellitus (T2DM), gestational diabetes mellitus (GDM), and other special types of diabetes. Type 2 diabetes mellitus (T2DM) is the most common type of diabetes and was once called "adult-onset diabetes," but in recent years, due to rising obesity rates, its incidence in adolescents and children has also increased significantly. Although type 2 diabetes is the most common type of diabetes, it differs significantly from other types of diabetes, such as type 1 diabetes mellitus (T1DM), gestational diabetes mellitus (GDM), and other special types of diabetes, in terms of etiology, pathogenesis, population distribution, treatment strategies, and prognosis. The difference between T2DM and T1DM is that T2DM is mainly characterized by insulin resistance, with normal or elevated insulin levels in the early stages, while T1DM is caused by autoimmune destruction of β cells, resulting in absolute insulin deficiency. Furthermore, T2DM can initially be treated primarily with lifestyle changes and oral medications, with insulin required only later, whereas T1DM requires lifelong insulin therapy once diagnosed. The difference between type 2 diabetes mellitus (T2DM) and gestational diabetes mellitus (GDM) lies in the fact that GDM is triggered by placental hormone-induced insulin resistance, resulting in relative β-cell insufficiency and occurring only during pregnancy. T2DM, on the other hand, is a combination of long-term insulin resistance and gradual β-cell dysfunction, and can occur at any time. GDM has treatment limitations, with oral hypoglycemic agents being contraindicated or used with caution, and insulin being the first-line treatment. In contrast, oral medications are the first-line treatment for T2DM. The difference between T2DM and other types of diabetes mellitus lies in the fact that other types can be caused by single-gene mutations (MODY, mitochondrial mutations), medications (glucocorticoids), pancreatic diseases (chronic pancreatitis), or endocrine disorders (Cushing's syndrome), while the etiology of T2DM involves multiple genes and lifestyle factors. In terms of treatment, MODY2 (GCK mutation) often only requires dietary control, and blood sugar levels can recover after discontinuing medication in drug-induced diabetes. However, T2DM typically requires long-term medication intervention.

[0003] The core pathology of type 2 diabetes is insulin resistance and the decline of pancreatic β-cell function, often accompanied by obesity, chronic low-grade inflammation, and gut microbiota dysbiosis. Unlike type 1 diabetes and gestational diabetes, type 2 diabetes is characterized by its "gradual" and "interventional" nature. Early intervention through lifestyle changes such as weight loss, exercise, and dietary control can even reverse or significantly slow disease progression. Because dietary intervention directly targets the key pathological mechanisms of type 2 diabetes, improving metabolic status and repairing some damaged functions by adjusting nutrient intake, it is not only a fundamental management tool for type 2 diabetes but also a crucial strategy for early "treatment."

[0004] Schizophyllum commune, a rare edible and medicinal fungus, is the fruiting body of the fungus Schizophyllum commune Fr. (tree flower). Studies have shown that the main component of Schizophyllum commune, polysaccharide, can significantly inhibit the survival of glioma cell lines U251 and U-87MG, achieving an anti-tumor effect. Simultaneously, its polysaccharide has strong antioxidant activity. In terms of immunomodulation, it can induce interleukin-10 to enhance anti-inflammatory activity, significantly delaying carbohydrate digestion and absorption, thereby improving postprandial blood glucose. Furthermore, Schizophyllum commune can suppress fluctuations in α-amylase and α-glucosidase levels. In addition, Schizophyllum commune can increase the abundance of beneficial intestinal bacteria (such as Lactobacillus and Bifidobacterium) and reduce the Firmicutes / Bacteroidetes ratio, thereby relieving constipation, improving intestinal barrier function, and inhibiting systemic inflammation. However, existing studies have mostly focused on single pathological models or in vitro mechanisms, and have not yet clarified the comprehensive effects of *Scutellaria baicalensis* as a dietary supplement in the early prevention of type 2 diabetes, nor have they conducted in-depth research on the application of *Scutellaria baicalensis* in the preparation of drugs for the treatment of type 2 diabetes. Summary of the Invention

[0005] In view of the aforementioned deficiencies in the prior art, this invention provides an application of *Gynostemma pentaphyllum* in the preparation of a drug for treating type 2 diabetes, solving the technical problem that the prior art has not yet used *Gynostemma pentaphyllum* as a main active ingredient in the treatment of type 2 diabetes. The drug for treating type 2 diabetes provided by this invention has good application prospects and market potential, providing a safe and effective drug for the prevention and treatment of type 2 diabetes.

[0006] The specific technical solution is as follows: This invention provides the application of *Scutellaria baicalensis* in the preparation of a drug for treating type 2 diabetes.

[0007] Preferably, the active ingredient of the drug for treating type 2 diabetes is a white ginseng extract.

[0008] Preferably, the preparation method of the white ginseng extract includes the following steps: Step 1: Wash and dry the fruiting bodies of *Gynostemma pentaphyllum*, then pulverize them and pass them through a 100-120 mesh sieve to obtain *Gynostemma pentaphyllum* powder. Mix the *Gynostemma pentaphyllum* powder with 8-15 times the amount of water, adjust the pH to 4.5-5 with 0.1-0.3 mol / L citrate-sodium citrate buffer, add 0.8-1% w / v of a complex enzyme to the total solution system, and enzymatically hydrolyze at 30-50℃ for 2-5 hours. Step 2: Microwave extract the enzyme hydrolysate at 300-500W, 45-55℃, and 200-500rpm for 10-15 minutes; cool to room temperature, vacuum filter, and collect the filtrate. Alternatively, mix the enzymatic hydrolysate with a 0.5-2% w / v aqueous solution of cyclodextrin inclusion complex and stir for 8-12 min, then add a sodium citrate-EDTA complex solution and mix. The volume ratio of the enzymatic hydrolysate to the aqueous solution of cyclodextrin inclusion complex and the sodium citrate-EDTA complex solution is 100:1-2:0.5-1. Then, microwave extract for 10-15 min at 300-500 W, 45-55 °C, and 200-500 rpm. Cool to room temperature, vacuum filter, and collect the filtrate. Step 3: Mix the filtrate with anhydrous ethanol at a volume ratio of 1:3-5 and stir at 200-500 rpm for 20-40 min; then place it in a refrigerated environment at 1-5℃ for 10-15 h, and then centrifuge at 3000-5000 rpm at 1-5℃ for 8-15 min, collect the precipitate, and wash it; then add the precipitate to water to redissolve it, filter it, and obtain the filtrate; spray dry the filtrate to obtain the powder, which is the extract of white ginseng fungus.

[0009] Preferably, the composite enzyme is composed of cellulase and pectinase mixed in a mass ratio of 1-3:1.

[0010] Preferably, the preparation method of the cyclodextrin inclusion complex in step 2 includes the following steps: The functional agent and hydroxypropyl-β-cyclodextrin were mixed at a mass ratio of 1:8-12, added to water at 50-60℃, and then mixed and stirred at 400-800 rpm and 50-60℃ for 1-3 hours. After cooling, the mixture was freeze-dried to obtain the cyclodextrin inclusion complex.

[0011] Preferably, the functional agent is selected from one of epigallocatechin gallate and astragaloside I.

[0012] Preferably, the sodium citrate-EDTA composite solution in step 2 is prepared by mixing equal volumes of 0.1-0.2 mol / L sodium citrate aqueous solution and 0.01-0.2 mol / L EDTA aqueous solution, and then adjusting the pH to 5.8-6.2 with 0.1-0.2 mol / L HCl.

[0013] Preferably, in step 3, the inlet air temperature for spray drying is set to 185-190℃, the outlet air temperature is set to 75-80℃, the feed rate is set to 5-6mL / min, and the atomizing air pressure is set to 0.3-0.4MPa.

[0014] The present invention also provides a method for preparing a white ginseng extract, which is prepared by the above method.

[0015] Preferably, the drug for treating type 2 diabetes also includes pharmaceutical excipients.

[0016] Pharmaceutical excipients refer to the excipients and additives used in the production of drugs and the preparation of prescriptions; they are substances, other than the active ingredient, that have undergone reasonable safety assessments and are included in pharmaceutical formulations. Besides acting as excipients, carriers, and improving stability, pharmaceutical excipients also have important functions such as solubilization, co-solubilization, and sustained-release, and are important components that may affect the quality, safety, and efficacy of drugs.

[0017] More preferably, the above-mentioned medication for treating type 2 diabetes can be formulated into the following preparations: injection, tablet, pill, capsule, suspension, emulsion, granule, drop pill, powder, oral liquid, syrup, or tincture.

[0018] Regarding further explanation of the present invention, this application demonstrates a good preventive and therapeutic effect on type 2 diabetes mellitus (T2DM) through intervention with *Scutellaria baicalensis*. Considering that insulin resistance and pancreatic β-cell damage are the core pathological features of T2DM, this application hypothesizes that the intervention effect of *Scutellaria baicalensis* may be achieved through the following mechanisms: activating the AMPK signaling pathway to improve obesity-related insulin resistance; protecting pancreatic β-cells from oxidative stress-induced apoptosis by upregulating the expression of antioxidant enzymes; and regulating the gut microbiota to increase short-chain fatty acid levels, promote the secretion of glucagon-like peptide-1, and affect the IGF1 / PI3K / AKT signaling pathway to reduce blood glucose levels.

[0019] The beneficial effects of this invention are: 1. Compared with existing technologies, this invention provides an application of *Gynostemma pentaphyllum* in the preparation of drugs for treating type 2 diabetes. *Gynostemma pentaphyllum*, as a rare edible and medicinal fungus, possesses various physiological activities and beneficial pharmacological effects, including anti-tumor, immune-regulating, antioxidant, and anti-inflammatory properties, which are beneficial to health. This invention, through optimized extraction processes and controlled dosage, fully leverages the therapeutic effects of *Gynostemma pentaphyllum* on type 2 diabetes. At therapeutic doses for type 2 diabetes, it is safe, reliable, and highly effective, demonstrating potential development value for drugs for type 2 diabetes.

[0020] 2. Compared with the existing technology, the present invention introduces cyclodextrin inclusion complex and sodium citrate-EDTA complex solution for synergistic use in the preparation of white ginseng extract, which significantly reduces the polysaccharide oxidation loss rate during the extraction process and achieves a significant improvement in polysaccharide extraction rate. At the same time, through dual protection of active ingredients and synergistic regulation of hypoglycemic pathway, it enhances pancreatic islet protection and achieves a significant reduction in fasting blood glucose level. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the fasting blood glucose levels in mice in Example 1; Figure 2Schematic diagram of the blood glucose change and glucose area curve of mice by oral glucose tolerance in Example 1. Detailed implementation mode

[0022] Parameters and sources of specific chemical substances are used.

[0023] Schizophyllum commune, a commercially available product, produced in Dafang County, Guizhou Province; Cellulase, enzyme activity ≥ 5000 U / g, commercially available; Pectinase, enzyme activity ≥ 3000 U / g, commercially available Epigallocatechin gallate, pharmaceutical grade, commercially available; Astragaloside I, pharmaceutical grade, commercially available; Hydroxypropyl-β-cyclodextrin, pharmaceutical grade, commercially available.

[0024] In this study, 6-week-old male C57BL / 6J mice (weighing 16 - 20 g) provided by the Guangdong Provincial Medical Experimental Animal Center were used. The experimental animal production license number is SCXK (Yue) 2022 - 0002. All experimental operations were strictly carried out in accordance with the guiding principles formulated in the national standard GB / T35892 - 2018 "Guidelines for the Ethical Review of Laboratory Animal Welfare", and were submitted and approved by the Laboratory Animal Ethics Committee of Guangdong Pharmaceutical University (Laboratory animal use license number: SYXK (Yue) 2022 - 0125).

[0025] Example 1 Establishment, grouping and administration of mouse models Take 50 6-week-old male C57BL / 6J mice (weighing 16 - 20 g) and adaptively feed them in a SPF-level environment for 1 week. The mice are raised in a constant temperature environment of 25 °C, with 12 hours of light / 12 hours of darkness. During the feeding period, the mice can freely obtain ordinary feed and water; after 1 week, 12 mice are selected as the normal group and drink normal water and eat ordinary feed every day; the remaining mice are in the high-fat diet group, drink normal water every day and eat 60% high-fat diet (product number: D12492, brand: ReadyDietech); after 6 weeks, 24 mice with a 20% higher weight gain ratio than the normal group mice are selected from the high-fat diet group for injection with low-dose streptozotocin (STZ, brand: Sigma) to make diabetic mice.

[0026] Mouse diabetes research Preparation of streptozotocin injection: Dissolve 2.1g of citric acid in 100mL of ultrapure water to prepare solution A; dissolve 2.94g of sodium citrate in 100mL of ultrapure water to prepare solution B; before injection, mix solutions A and B in a 1:1 ratio and adjust the pH to 4.4 to prepare the AB mixture, which is then stored at 4℃ for later use; calculate the streptozotocin dosage based on a dose of 100mg / kg, weigh the streptozotocin powder using an electronic balance, and mix it with the AB mixture to prepare the streptozotocin injection; the entire process should be conducted in the dark, and the resulting liquid should be stored at 4℃. The injection should be completed within 15 minutes after the liquid is prepared.

[0027] Preparation of sodium citrate buffer: Dissolve 2.1g of citric acid in 100mL of ultrapure water to prepare solution A; dissolve 2.94g of sodium citrate in 100mL of ultrapure water to prepare solution B; before injection, mix solution A and solution B in a 1:1 ratio and adjust the pH to 4.4 to prepare sodium citrate buffer, store at 4℃ for later use.

[0028] The specific procedure for inducing diabetic mice with low-dose streptozotocin injection was as follows: 24 mice were randomly divided into an experimental group (20 mice) and a normal group (4 mice). The mice were then fasted for 12 hours. After fasting, the experimental group mice were injected with streptozotocin via the tail vein, while the normal group mice were injected with sodium citrate buffer via the tail vein. 72 hours later, the fasting blood glucose (FBG) concentration was measured using a glucometer. A FBG concentration of not less than 11.1 mmol / L on days 3 and 7 after modeling was considered a successful model. Mice that did not meet the blood glucose standard or died during modeling were excluded, resulting in a model success rate of 50%. The successfully modeled mice were randomly divided into a diabetic model group and a *Gynostemma pentaphyllum* powder diabetic group, with 4 mice in each group. The diabetic model group was recorded as the DM group and administered 10 ml / kg of physiological saline by gavage. The *Gynostemma pentaphyllum* powder diabetic group was recorded as the DMS group and administered 0.625 g / kg of *Gynostemma pentaphyllum* powder (obtained by crushing the fruiting body of *Gynostemma pentaphyllum* and passing it through a 120-mesh sieve) by gavage. The administration lasted for 28 days.

[0029] Monitoring of mouse body weight and fasting blood glucose Mouse body weight and FBG concentration were measured weekly before and during drug administration. When monitoring FBG concentration in each group, mice in each group were fasted for 12 hours on the day of blood collection (but water was allowed). Blood was collected by puncturing the capillaries at the tip of the mouse tail with a lancet, and the FBG concentration in the mouse blood was measured using a blood glucose meter.

[0030] Effects on oral glucose tolerance in diabetic mice Mice were fasted (but allowed water) for 12 hours. The amount of glucose solution to be administered was calculated based on the mice's body weight, with a dose of 1 g / kg. Before gavage, the blood glucose level at 0 min was measured using a blood glucose meter. Based on the calculated glucose volume, the mice were given the corresponding dose of glucose solution via gavage. Blood glucose levels were monitored at 15, 30, 60, 90, and 120 min.

[0031] Data processing and analysis Experimental data are expressed as mean values. SPSS 11.5 software was used for statistical processing. ANOVA was used for one-way ANOVA. P < 0.05 was considered statistically significant, and P < 0.01 was considered highly statistically significant. GraphPad Prism 6.0 software was used for plotting.

[0032] Test result data as follows Figure 1 (Compared with the NC group, #### is P<0.0001; compared with the DM group, **** is P<0.0001) and Figure 2 (Compared with the NC group, #### is P<0.0001; compared with the DM group, **** is P<0.0001) as shown.

[0033] Depend on Figure 1 It can be seen that, compared with the NC group, the FBG of diabetic mice in the DM group was significantly increased, which is statistically significant; compared with the DM group, the FBG of mice in the DMS group after intervention with *Bletilla striata* was significantly decreased.

[0034] Depend on Figure 2 It was found that the dynamic changes in blood glucose levels in the DMS group and the DM group showed significant differences. The DMS group reached its peak blood glucose level 15 minutes after gavage, while the DM group reached its peak after 30 minutes. Significant differences were observed between the DMS and DM groups at 30, 60, and 120 minutes. After reaching their peak, blood glucose levels in both groups gradually decreased over time, but the decrease was more significant in the DMS group. At 120 minutes, the blood glucose level in the DM group was still significantly higher than that in the DMS group. AUC, representing the area under the glucose curve, allows for a more intuitive, effective, and reliable assessment of the overall glucose tolerance level in the organism. Calculations showed that the AUC of the DMS group was significantly lower than that of the DM group, indicating that *Pleurotus ostreatus* intervention prevented the increase in oral glucose tolerance in type 2 diabetic mice.

[0035] Experiments have shown that the intervention of *Scutellaria baicalensis* can reduce blood sugar levels and improve glucose tolerance, indicating that *Scutellaria baicalensis* has a certain preventive and therapeutic effect on type 2 diabetes.

[0036] Example 2 A method for preparing an extract of *Gynostemma pentaphyllum* includes the following steps: Step 1: Wash and dry the fruiting bodies of *Gynostemma pentaphyllum*, then pulverize them and pass them through a 120-mesh sieve to obtain *Gynostemma pentaphyllum* powder. Mix the *Gynostemma pentaphyllum* powder with 10 times the amount of water, adjust the pH to 4.6 with 0.1 mol / L citrate-sodium citrate buffer, add 0.8% w / v of the total solution system of the complex enzyme, and enzymatically hydrolyze at 45℃ for 3 hours. Step 2: Microwave extract the enzyme hydrolysate at 400W, 50℃, and 200rpm for 12 minutes; cool to room temperature, vacuum filter, and collect the filtrate; Step 3: Mix the filtrate with anhydrous ethanol at a volume ratio of 1:3.5 and stir at 200 rpm for 30 min. Then, place the mixture at 4℃ and refrigerate for 12 h. After that, centrifuge at 4000 rpm at 4℃ for 10 min, collect the precipitate, and wash the precipitate twice with 75 wt% ethanol. Then, dissolve the precipitate in water to a concentration of 4% w / v, filter it using a 0.45 μm microporous membrane, and obtain the filtrate. Spray dry the filtrate with the following settings: inlet air temperature of 186℃, outlet air temperature of 78℃, feed rate of 5 mL / min, and atomizing air pressure of 0.3 MPa, to obtain the powder, which is the extract of *Gynostemma pentaphyllum*.

[0037] The complex enzyme is composed of cellulase and pectinase mixed in a mass ratio of 2:1.

[0038] Example 3 A method for preparing an extract of *Gynostemma pentaphyllum* includes the following steps: Step 1: Wash and dry the fruiting bodies of *Gynostemma pentaphyllum*, then pulverize them and pass them through a 120-mesh sieve to obtain *Gynostemma pentaphyllum* powder. Mix the *Gynostemma pentaphyllum* powder with 10 times the amount of water, adjust the pH to 4.6 with 0.1 mol / L citrate-sodium citrate buffer, add 0.8% w / v of the total solution system of the complex enzyme, and enzymatically hydrolyze at 45℃ for 3 hours. Step 2: Mix the enzymatic hydrolysate with a 1% w / v cyclodextrin inclusion complex aqueous solution and stir for 10 min. Then add the sodium citrate-EDTA complex solution and mix. The volume ratio of the enzymatic hydrolysate to the cyclodextrin inclusion complex aqueous solution and the sodium citrate-EDTA complex solution is 100:1:0.5. After that, microwave extract for 12 min at 400 W, 50 °C and 200 rpm. Cool to room temperature, vacuum filter and collect the filtrate. Step 3: Mix the filtrate with anhydrous ethanol at a volume ratio of 1:3.5 and stir at 200 rpm for 30 min. Then, place the mixture at 4℃ and refrigerate for 12 h. After that, centrifuge at 4000 rpm at 4℃ for 10 min, collect the precipitate, and wash the precipitate twice with 75 wt% ethanol. Then, dissolve the precipitate in water to a concentration of 4% w / v, filter it using a 0.45 μm microporous membrane, and obtain the filtrate. Spray dry the filtrate with the following settings: inlet air temperature of 186℃, outlet air temperature of 78℃, feed rate of 5 mL / min, and atomizing air pressure of 0.3 MPa, to obtain the powder, which is the extract of *Gynostemma pentaphyllum*.

[0039] The complex enzyme is composed of cellulase and pectinase mixed in a mass ratio of 2:1.

[0040] The preparation method of the cyclodextrin inclusion complex includes the following steps: Epigallocatechin gallate and hydroxypropyl-β-cyclodextrin were mixed at a mass ratio of 1:10, added to water at 60°C, and then stirred at 500 rpm and 60°C for 2 hours. After cooling, the mixture was freeze-dried to obtain the cyclodextrin inclusion complex.

[0041] The sodium citrate-EDTA composite solution was prepared by mixing equal volumes of 0.1 mol / L sodium citrate aqueous solution and 0.01 mol / L EDTA aqueous solution, and then adjusting the pH to 6 with 0.1 mol / L HCl.

[0042] Example 4 A method for preparing *Gynostemma pentaphyllum* extract differs from Example 3 only in that the method for preparing the cyclodextrin inclusion complex includes the following steps: Astragalus saponin I and hydroxypropyl-β-cyclodextrin were mixed at a mass ratio of 1:10, added to water at 60℃, and then mixed and stirred at 500 rpm and 60℃ for 2 hours. After cooling, the mixture was freeze-dried to obtain the cyclodextrin inclusion complex.

[0043] Example 5 A method for preparing *Gynostemma pentaphyllum* extract differs from Example 3 only in that the method for preparing the cyclodextrin inclusion complex includes the following steps: The functional agent was mixed with hydroxypropyl-β-cyclodextrin at a mass ratio of 1:10, added to water at 60°C, and then mixed and stirred at 500 rpm and 60°C for 2 hours. After cooling, the mixture was freeze-dried to obtain the cyclodextrin inclusion complex.

[0044] The functional agent is composed of epigallocatechin gallate and astragaloside I in a mass ratio of 1:1.

[0045] Comparative Example 1 A method for preparing an extract of *Gynostemma pentaphyllum* includes the following steps: Step 1: Wash and dry the fruiting bodies of *Gynostemma pentaphyllum*, then pulverize them and pass them through a 120-mesh sieve to obtain *Gynostemma pentaphyllum* powder. Mix the *Gynostemma pentaphyllum* powder with 10 times the amount of water, adjust the pH to 4.6 with 0.1 mol / L citrate-sodium citrate buffer, add 0.8% w / v of the total solution system of the complex enzyme, and enzymatically hydrolyze at 45℃ for 3 hours. Step 2: Mix the enzymatic hydrolysate with a 1% w / v aqueous solution of cyclodextrin inclusion complex and stir for 10 min, wherein the volume ratio of the enzymatic hydrolysate to the aqueous solution of cyclodextrin inclusion complex is 100:1; then microwave extract for 12 min at 400 W, 50 °C and 200 rpm; cool to room temperature, vacuum filter and collect the filtrate. Step 3: Mix the filtrate with anhydrous ethanol at a volume ratio of 1:3.5 and stir at 200 rpm for 30 min. Then, place the mixture at 4℃ and refrigerate for 12 h. After that, centrifuge at 4000 rpm at 4℃ for 10 min, collect the precipitate, and wash the precipitate twice with 75 wt% ethanol. Then, dissolve the precipitate in water to a concentration of 4% w / v, filter it using a 0.45 μm microporous membrane, and obtain the filtrate. Spray dry the filtrate with the following settings: inlet air temperature of 186℃, outlet air temperature of 78℃, feed rate of 5 mL / min, and atomizing air pressure of 0.3 MPa, to obtain the powder, which is the extract of *Gynostemma pentaphyllum*.

[0046] The complex enzyme is composed of cellulase and pectinase mixed in a mass ratio of 2:1.

[0047] The preparation method of the cyclodextrin inclusion complex is the same as that in Example 3.

[0048] Comparative Example 2 A method for preparing an extract of *Gynostemma pentaphyllum* includes the following steps: Step 1: Wash and dry the fruiting bodies of *Gynostemma pentaphyllum*, then pulverize them and pass them through a 120-mesh sieve to obtain *Gynostemma pentaphyllum* powder. Mix the *Gynostemma pentaphyllum* powder with 10 times the amount of water, adjust the pH to 4.6 with 0.1 mol / L citrate-sodium citrate buffer, add 0.8% w / v of the total solution system of the complex enzyme, and enzymatically hydrolyze at 45℃ for 3 hours. Step 2: Mix the enzymatic hydrolysate with a 1% w / v aqueous solution of cyclodextrin inclusion complex and stir for 10 min, wherein the volume ratio of the enzymatic hydrolysate to the aqueous solution of cyclodextrin inclusion complex is 100:1; then microwave extract for 12 min at 400 W, 50 °C and 200 rpm; cool to room temperature, vacuum filter and collect the filtrate. Step 3: Mix the filtrate with anhydrous ethanol at a volume ratio of 1:3.5 and stir at 200 rpm for 30 min. Then, place the mixture at 4℃ and refrigerate for 12 h. After that, centrifuge at 4000 rpm at 4℃ for 10 min, collect the precipitate, and wash the precipitate twice with 75 wt% ethanol. Then, dissolve the precipitate in water to a concentration of 4% w / v, filter it using a 0.45 μm microporous membrane, and obtain the filtrate. Spray dry the filtrate with the following settings: inlet air temperature of 186℃, outlet air temperature of 78℃, feed rate of 5 mL / min, and atomizing air pressure of 0.3 MPa, to obtain the powder, which is the extract of *Gynostemma pentaphyllum*.

[0049] The complex enzyme is composed of cellulase and pectinase mixed in a mass ratio of 2:1.

[0050] The preparation method of the cyclodextrin inclusion complex is the same as that in Example 4.

[0051] Comparative Example 3 A method for preparing an extract of *Gynostemma pentaphyllum* includes the following steps: Step 1: Wash and dry the fruiting bodies of *Gynostemma pentaphyllum*, then pulverize them and pass them through a 120-mesh sieve to obtain *Gynostemma pentaphyllum* powder. Mix the *Gynostemma pentaphyllum* powder with 10 times the amount of water, adjust the pH to 4.6 with 0.1 mol / L citrate-sodium citrate buffer, add 0.8% w / v of the total solution system of the complex enzyme, and enzymatically hydrolyze at 45℃ for 3 hours. Step 2: Mix the enzymatic hydrolysate with the sodium citrate-EDTA composite solution at a volume ratio of 100:0.5, stir well, and then microwave extract for 12 min at 400W, 50℃, and 200rpm; cool to room temperature, vacuum filter, and collect the filtrate. Step 3: Mix the filtrate with anhydrous ethanol at a volume ratio of 1:3.5 and stir at 200 rpm for 30 min. Then, place the mixture at 4℃ and refrigerate for 12 h. After that, centrifuge at 4000 rpm at 4℃ for 10 min, collect the precipitate, and wash the precipitate twice with 75 wt% ethanol. Then, dissolve the precipitate in water to a concentration of 4% w / v, filter it using a 0.45 μm microporous membrane, and obtain the filtrate. Spray dry the filtrate with the following settings: inlet air temperature of 186℃, outlet air temperature of 78℃, feed rate of 5 mL / min, and atomizing air pressure of 0.3 MPa, to obtain the powder, which is the extract of *Gynostemma pentaphyllum*.

[0052] The complex enzyme is composed of cellulase and pectinase mixed in a mass ratio of 2:1.

[0053] The sodium citrate-EDTA composite solution was prepared by mixing equal volumes of 0.1 mol / L sodium citrate aqueous solution and 0.01 mol / L EDTA aqueous solution, and then adjusting the pH to 6 with 0.1 mol / L HCl.

[0054] Test Example 1 Polysaccharide extraction rate determination The polysaccharide extraction rate of the *Gynostemma pentaphyllum* extracts provided in Examples 2-5 and Comparative Examples 1-3 of this invention was determined. The polysaccharide content in the *Gynostemma pentaphyllum* extract was determined using the sulfuric acid-anthrone method, with dextran as the standard. The total sugar content in the *Gynostemma pentaphyllum* raw material (the *Gynostemma pentaphyllum* powder obtained in step 1) and the *Gynostemma pentaphyllum* extract (the *Gynostemma pentaphyllum* extract obtained in step 3) was determined. The test method is as follows: Take 0.1, 0.2, 0.3, 0.4, and 0.5 mL of a 100 μg / mL dextran solution, respectively, and bring the volume to 1.0 mL with distilled water. Add 4 mL of anthrone sulfate solution to each tube, and cool them in ice water. After all the solutions have been added, heat them in a water bath for 10 min, remove them, and cool them in tap water for 10 min. Using the corresponding reagents as blanks, measure the absorbance at a wavelength of 620 nm. Plot a standard curve with absorbance on the ordinate and dextran concentration on the abscissa.

[0055] Accurately weigh 1.0 mL of a 1 mg / mL white ginseng extract solution, measure the absorbance using the method described above, and calculate the mass fraction W (%) of the polysaccharide using the following formula.

[0056] W=C1V1 / M1×10 -4 ×100% In the formula: C1 is the concentration in the test solution obtained from the standard curve (ug / mL); V1 is the sample volume (mL); M1 is the mass of the white ginseng extract or white ginseng raw material (g).

[0057] Extraction rate (%) = W1m1 / W2m2 × 100% In the formula: W1 is the polysaccharide content (%) in the white ginseng extract; W2 is the polysaccharide content (%) in the white ginseng raw material; m1 is the mass (g) of the white ginseng extract used; m2 is the mass (g) of the white ginseng raw material used.

[0058] The specific test results are shown in Table 1 below.

[0059] Table 1 As shown in Table 1, compared with Example 2, the polysaccharide extraction rates of Examples 3-5 and Comparative Examples 1-3 were significantly improved. A comparison of Examples 3-5 and Comparative Examples 1-3 revealed that the polysaccharide extraction rate of Examples 3-5 was higher than that of Comparative Examples 1-3, with Example 5 exhibiting the highest polysaccharide extraction rate at 66.2%. This indicates that the synergistic use of cyclodextrin inclusion complexes and sodium citrate-EDTA complex solution during the preparation of *Gynostemma pentaphyllum* extract is beneficial for improving the polysaccharide extraction rate. The reason for this may be that during the extraction process, the antioxidant activity of epigallocatechin gallate in the cyclodextrin inclusion complex can inhibit polysaccharide oxidative breakage, increasing the polysaccharide molecular weight retention rate and thus directly improving the polysaccharide yield. Furthermore, astragaloside I can activate the metabolic pathways of *Gynostemma pentaphyllum* cells to promote intracellular polysaccharide release, thereby improving polysaccharide dissolution efficiency. Simultaneously, β-cyclodextrin can disrupt the chitin-cellulose complex structure of the *Gynostemma pentaphyllum* cell wall, complementing cellulase and pectinase, increasing cell wall permeability, which is beneficial for improving the total polysaccharide yield. Furthermore, EDTA in the sodium citrate-EDTA composite solution can strongly chelate... / This reduces the polysaccharide oxidation loss rate, while sodium citrate can maintain a weakly acidic environment to prevent polysaccharide hydrolysis, thus contributing to an increase in the total polysaccharide yield.

[0060] Test Example 2 Treatment efficacy for type 2 diabetes The therapeutic effects of the *Panax notoginseng* extracts provided in Examples 2-5 and Comparative Examples 1-3 of this invention on type 2 diabetes were determined. The test methods are the same as in Example 1, and are as follows: Mouse model establishment, grouping and drug administration Fifty 6-week-old male C57BL / 6J mice (weighing 16-20g) were acclimatized in an SPF-grade environment for one week. The mice were housed in a constant temperature environment of 25℃ with 12 hours of light and 12 hours of darkness. During the feeding period, the mice had free access to normal food and water. After one week, 12 mice were selected as the normal group, which drank normal water and ate normal food daily. The remaining mice were in the high-fat diet group, which drank normal water daily and ate 60% high-fat food (product number: D12492, brand: ReadyDietech). After 6 weeks, 24 mice from the high-fat diet group that gained 20% more weight than the normal group were selected and injected with low-dose streptozotocin (STZ, brand: Sigma) to induce diabetic mice.

[0061] The streptozotocin injection solution was prepared according to Example 1; Sodium citrate buffer solution was prepared according to Example 1; The specific procedure for inducing diabetic mice with low-dose streptozotocin injection was as follows: 24 mice were randomly divided into an experimental group (20 mice) and a normal group (4 mice). The mice were then fasted for 12 hours. After fasting, the mice in the experimental group were injected with streptozotocin via the tail vein, while the mice in the normal group were injected with sodium citrate buffer via the tail vein. 72 hours later, the fasting blood glucose (FBG) concentration was measured using a glucometer. A FBG concentration of not less than 11.1 mmol / L on days 3 and 7 after modeling was considered a successful model. Mice that did not meet the blood glucose standard or died during modeling were excluded, resulting in a model success rate of 50%. The successfully modeled mice were randomly divided into a diabetic model group and a diabetic group containing *Gynostemma pentaphyllum* extract, with 4 mice in each group. The diabetic model group was administered 10 mL / kg of physiological saline by gavage, while the diabetic group containing *Gynostemma pentaphyllum* extract was recorded as Examples 2-4 and Comparative Examples 1-3, respectively, and administered 0.625 g / kg of *Gynostemma pentaphyllum* extract by gavage. The administration lasted for 28 days.

[0062] Monitoring of mouse body weight and fasting blood glucose Mouse body weight and FBG concentration were measured weekly before and during drug administration. When monitoring FBG concentration in each group, mice in each group were fasted for 12 hours on the day of blood collection (water was permitted). Blood was collected by puncturing the capillaries at the tip of the mouse tail with a lancet, and the FBG concentration in the mouse blood was measured using a blood glucose meter. The fasting blood glucose reduction rate (%) was calculated based on the fasting blood glucose values ​​measured in the diabetic model group and Examples 2-5 and Comparative Examples 1-3, using the following formula: Fasting blood glucose reduction rate (%) = (F1 - F n ) / F1×100% In the formula, F1 represents the fasting blood glucose level (mmol / L) of mice in the diabetic model group; F n The fasting blood glucose level (mmol / L) of mice in the diabetic group (i.e., Examples 2-5 and Comparative Examples 1-3) of the white ginseng extract group. The specific test results are shown in Table 2 below.

[0063] Table 2 Table 2 shows that, compared with the diabetic model group, the fasting blood glucose levels of Examples 2-5 and Comparative Examples 1-3 were reduced. Comparing Examples 2-5 and Comparative Examples 1-3, the reduction rate of fasting blood glucose in Examples 3-5 was higher than that in Examples 2 and Comparative Examples 1-3, with Example 5 showing the highest reduction rate, exhibiting a significant decrease in fasting blood glucose compared to the diabetic model group. This may be attributed to the synergistic use of the cyclodextrin inclusion complex and the sodium citrate-EDTA complex solution, which, through dual protection of the active ingredients and synergistic regulation of the hypoglycemic pathway, achieves a significant reduction in fasting blood glucose levels. Encapsulating epigallocatechin gallate and astragaloside I in cyclodextrin ensures sustained release of the functional agent after reaching the intestine. Epigallocatechin gallate can enhance antioxidant capacity by activating the Nrf2 pathway, compensating for the lack of direct protection of pancreatic β-cells by *Scutellaria baicalensis*. Simultaneously, astragaloside I can activate the AMPK signaling pathway, synergistically improving insulin resistance with *Scutellaria baicalensis*, thereby achieving the effect of reducing fasting blood glucose levels. Meanwhile, the sodium citrate-EDTA composite solution can improve the molecular weight retention rate of white ginseng polysaccharide and protect astragaloside I from glycosidic bond hydrolysis, thereby promoting AMPK activation efficiency.

Claims

1. Use of white ginseng mushroom in the preparation of a drug for treating type 2 diabetes.

2. Use according to claim 1, characterized in that: The active ingredient of the drug for treating type 2 diabetes is a white ginseng mushroom extract.

3. Use according to claim 2, wherein: The preparation method of the white ginseng mushroom extract comprises the following steps: Step 1: After washing and drying the white ginseng mushroom fruiting body, it is crushed and sieved through a 100-120 mesh sieve to obtain white ginseng mushroom powder; the white ginseng mushroom powder is mixed with 8-15 times the amount of water, and the pH value is adjusted to 4.5-5 with a 0.1-0.3 mol / L citric acid-sodium citrate buffer solution; a total solution system of 0.8-1% w / v complex enzyme is added, and the enzyme is hydrolyzed at 30-50°C for 2-5 hours; Step 2: The enzyme hydrolysate is subjected to microwave extraction at 300-500W, 45-55°C, and 200-500rpm for 10-15 minutes; after cooling to room temperature, vacuum filtration is performed, and the filtrate is collected; or, the enzyme hydrolysate is mixed with a 0.5-2% w / v cyclodextrin inclusion compound aqueous solution and stirred for 8-12 minutes, then a sodium citrate-EDTA composite solution is added, wherein the volume ratio of the enzyme hydrolysate, the cyclodextrin inclusion compound aqueous solution, and the sodium citrate-EDTA composite solution is 100:1-2:0.5-1; then the mixture is subjected to microwave extraction at 300-500W, 45-55°C, and 200-500rpm for 10-15 minutes; after cooling to room temperature, vacuum filtration is performed, and the filtrate is collected; Step 3: The filtrate is mixed with anhydrous ethanol at a volume ratio of 1:3-5, and stirred at 200-500rpm for 20-40 minutes; then it is placed in a cold storage at 1-5°C for 10-15 hours, and then centrifuged at 3000-5000rpm and 1-5°C for 8-15 minutes; the precipitate is collected and washed; then the precipitate is dissolved in water, filtered, and the filtrate is obtained; the filtrate is spray dried to obtain a powder, which is the white ginseng mushroom extract.

4. Use according to claim 3, wherein: The complex enzyme in step 1 is a mixture of cellulase and pectinase at a mass ratio of 1-3:

1.

5. The use according to claim 3, wherein the compound is ###0002### The preparation method of the cyclodextrin inclusion compound in step 2 comprises the following steps: a functional agent is mixed with hydroxypropyl-β-cyclodextrin at a mass ratio of 1:8-12, and then added to 50-60°C water, and then stirred at 400-800rpm and 50-60°C for 1-3 hours; after cooling, freeze-drying is performed to obtain the cyclodextrin inclusion compound; the functional agent is selected from at least one of epigallocatechin gallate and astragaloside I.

6. The use according to claim 3, characterized in that: The sodium citrate-EDTA composite solution in step 2 is prepared by mixing equal volumes of 0.1-0.2 mol / L sodium citrate aqueous solution and 0.01-0.2 mol / L EDTA aqueous solution, and then adjusting the pH to 5.8-6.2 with 0.1-0.2 mol / L HCl.

7. The use according to claim 2, wherein: The drug for treating type 2 diabetes also includes pharmaceutical excipients.

8. A method for preparing a white ginseng mushroom extract, characterized by, The method comprises the following steps: Step 1, after the white mushroom fruiting body is washed, dried and crushed, it is passed through a 100-120 mesh sieve to obtain white mushroom powder; the white mushroom powder is mixed with 8-15 times the amount of water, the pH value is adjusted to 4.5-5 with 0.1-0.3 mol / L citric acid-sodium citrate buffer solution, 0.8-1% w / v complex enzyme is added to the total solution system, and the enzyme is hydrolyzed at 30-50°C for 2-5 h; Step 2, the enzyme solution is extracted by microwave at 300-500W, 45-55°C, 200-500rpm for 10-15 min; cooled to room temperature, vacuum filtration, collect the filtrate; or, the enzyme solution is mixed with 0.5-2% w / v cyclodextrin inclusion compound aqueous solution and stirred for 8-12 min, then add sodium citrate-EDTA complex solution, wherein the volume ratio of enzyme solution, cyclodextrin inclusion compound aqueous solution and sodium citrate-EDTA complex solution is 100:1-2:0.5-1; then microwave extraction at 300-500W, 45-55°C, 200-500rpm for 10-15 min; cooled to room temperature, vacuum filtration, collect the filtrate; Step 3, the filtrate is mixed with anhydrous ethanol at a volume ratio of 1:3-5, and stirred at 200-500rpm for 20-40 min; then placed in 1-5°C cold storage for 10-15h, then centrifuged at 3000-5000rpm, 1-5°C for 8-15 min, collect the precipitate, wash; then add water to the precipitate, dissolve, filter to obtain the filtrate; the filtrate is spray dried to obtain powder, which is the white mushroom extract.

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