A ganoderma lucidum polysaccharide fraction and triterpene fraction pharmaceutical composition for preventing and treating type 2 diabetes and application thereof

Abstract

The application discloses a ganoderma lucidum polysaccharide part and triterpene part pharmaceutical composition for preventing and treating type 2 diabetes and application thereof. The pharmaceutical composition comprises the ganoderma lucidum polysaccharide part and the ganoderma lucidum triterpene part. The mass ratio of the ganoderma lucidum polysaccharide part to the ganoderma lucidum triterpene part is (0.5-2):1. The in-vitro alpha-glucosidase enzyme inhibition experiment, animal experiment and cell experiment prove that the composition composed of the ganoderma lucidum polysaccharide part and the ganoderma lucidum triterpene part can play a role in relieving diabetes symptoms and treating diabetes through the pathways of reducing blood sugar, resisting inflammation and inhibiting alpha-glucosidase, and the effect of the polysaccharide part and triterpene part composition is obviously better than that of a single part, has a multi-component synergistic effect and has a good clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology and relates to a combination drug with hypoglycemic function, specifically to a combination of Ganoderma lucidum polysaccharide and triterpenoid fractions for the prevention and treatment of type 2 diabetes and its application. Background Technology

[0002] Diabetes mellitus (DM) is a group of endocrine and metabolic syndromes characterized by elevated plasma glucose levels, caused by a variety of genetic and environmental factors. The disease is mainly classified into three types: type 1, type 2, and gestational diabetes. The etiology is primarily attributed to the combined effects of genetic and environmental factors. Insufficient insulin secretion or defective insulin action leads to disorders in the metabolism of carbohydrates, fats, and proteins, causing polydipsia, polyuria, polyphagia, and weight loss. In severe cases, acute complications such as ketoacidosis, hyperosmolar nonketotic diabetic coma, and various infections may occur, as well as chronic complications such as nephropathy, retinopathy, atherosclerosis, and diabetic neuropathy.

[0003] Reishi mushroom is the dried fruiting body of the fungus *Ganoderma lucidum* (Leyss. ex Fr.) Karst. or *Ganoderma sinense* Zhao, Xu et Zhang, belonging to the Polyporaceae family. The growth cycle of Reishi mushroom can be roughly divided into three stages: sporophyte, mycelium, and fruiting body. Reishi mushroom is beneficial to the "qi of the five internal organs: heart, liver, spleen, lungs, and kidneys," possessing the effects of invigorating heart qi, strengthening the body's resistance, and prolonging life. Furthermore, it is known for being non-toxic. In folk medicine, Reishi mushroom is considered an "immortal herb" and a "miracle drug." Therefore, its application in traditional Chinese medicine has a long history, spanning over 2000 years. Modern research has identified over 200 compounds isolated from Reishi mushroom, with the main active components being polysaccharides, triterpenes, sterols, proteins, and adenosine. Among these, Reishi polysaccharides are one of the most crucial active components, exhibiting good pharmacological effects in immune regulation, anti-tumor activity, antiviral activity, antioxidant activity, hypoglycemia, and lipid regulation.

[0004] Ganoderma lucidum polysaccharides are a class of highly active and functionally diverse natural polysaccharide macromolecules, composed of multiple monosaccharide molecules condensed together, and may also contain proteins or peptides bound to the sugar chains. Ganoderma lucidum polysaccharides have complex structures and are diverse in type, mainly composed of α-glucan and β-glucan, with β-glucan being the most prevalent. There are more than a dozen monosaccharides and their derivatives constituting Ganoderma lucidum polysaccharides, the most common being glucose, fructose, mannose, and arabinose. Extracellular polysaccharides secreted by Ganoderma lucidum mycelia are found in both liquid fermentation broth and solid culture media. Both intracellular and extracellular polysaccharides are effective polysaccharides. The pharmacological activity of Ganoderma lucidum polysaccharides is related to the glycosidic bonds, the three-dimensional structure of the polysaccharide molecule, and its molecular weight, which distinguishes them from other polysaccharides such as starch and cellulose. Studies have shown that Ganoderma lucidum polysaccharides possess pharmacological effects such as hypoglycemic, antitumor, antioxidant, immunomodulatory, and lipid-lowering properties.

[0005] Ganoderma triterpenes are roostane-type triterpenoid compounds isolated from Ganoderma lucidum. Most Ganoderma triterpenes are tetracyclic triterpenoids, with a relative molecular weight generally between 400 and 600. They are mostly polar molecules and not easily volatile. Based on differences in structure and functional groups, Ganoderma triterpenes are further classified into ganoderic acids, ganoderic spore acids, ganoderic acid lactones, ganoderic acid methyl esters, ganoderic ketones, and ganoderic aldehydes. Most of these compounds in Ganoderma lucidum mycelium are triterpenic acids, with a carboxyl group at C-26 and a double bond at C-24 and C-25. Only a few compounds have a hydroxyl group at C-26, belonging to the triterpene alcohol class. Ganoderma triterpenes, as another important active ingredient in Ganoderma lucidum, exert anti-diabetic effects. Studies have shown that ganoderic acid A can significantly inhibit the activity of aldose reductase, which can convert glucose into sorbitol. The accumulation of sorbitol can lead to diabetic complications. Ganoderic acids can improve streptozotocin-induced type 2 diabetes by inhibiting α-glucosidase and α-amylase, thereby regulating gut microbiota and inflammatory responses. Other studies have shown that ganoderic acid B also inhibits α-glucosidase activity, and its effect is higher than that of acarbose, an α-glucosidase inhibitor that inhibits postprandial blood glucose levels.

[0006] According to the "Guidelines for the Prevention and Treatment of Type 2 Diabetes in China (2020 Edition)," the medications currently used in the clinical treatment of diabetes are mainly divided into oral hypoglycemic agents and insulin. Drug treatment for hyperglycemia is primarily based on correcting the two main pathophysiological changes that lead to elevated blood glucose levels in humans: insulin resistance and impaired insulin secretion. Based on their effects, oral hypoglycemic agents can be divided into those that primarily promote insulin secretion and those that lower blood glucose through other mechanisms. The former mainly includes sulfonylureas, meglitinides, and dipeptidyl peptidase IV inhibitors, while those that lower blood glucose through other mechanisms mainly include biguanides, thiazolidinediones, α-glucosidase inhibitors, and sodium-glucose cotransporter 2 inhibitors.

[0007] Medical nutrition therapy and exercise therapy are fundamental measures for controlling hyperglycemia in type 2 diabetes mellitus (T2DM). When diet and exercise fail to achieve target blood glucose control, drug therapy, including oral medications, should be initiated promptly. T2DM is a progressive disease. In the natural course of T2DM, pancreatic β-cell function gradually declines with disease progression, while the degree of insulin resistance remains relatively stable. Therefore, as T2DM progresses, the reliance on exogenous blood glucose control methods gradually increases. Clinically, oral hypoglycemic agents and combination therapies with oral and injectable hypoglycemic agents (insulin, glucagon-like peptide-1 receptor agonists) are often required. Currently, many monoclonal drugs for treating diabetes have numerous adverse reactions and toxic side effects, and some drugs may be unsuitable for patients with hepatic or renal insufficiency due to related metabolic reasons. For example, metformin, a first-line drug, is not suitable for patients with hepatic or renal insufficiency, and long-term use of metformin can lead to a decrease in vitamin B12 levels. Sulfonylureas and meglitinides carry the risk of hypoglycemia. Furthermore, various single-agent drugs target relatively single targets, often requiring combination therapy with multiple drugs in the later stages of type 2 diabetes mellitus (T2DM). The combined use of different drugs can further increase the metabolic burden on organs such as the liver and kidneys, and poses potential toxic side effects and adverse reactions. Therefore, there is an urgent need to develop drugs derived from natural sources with fewer toxic side effects. Summary of the Invention

[0008] The purpose of this invention is to provide a pharmaceutical composition of Ganoderma lucidum polysaccharide and triterpenoid fractions for the prevention and treatment of type 2 diabetes mellitus (T2DM) and its application. Addressing the disadvantages of clinically used drugs for T2DM, such as varying degrees of toxic side effects or adverse reactions, and the need for later combination therapy, this invention combines Ganoderma lucidum polysaccharide and triterpenoid fractions. Ganoderma lucidum polysaccharide excels at lowering blood sugar and reducing inflammation, while Ganoderma lucidum triterpenoids excel at inhibiting α-glucosidase, thereby achieving a synergistic effect of multiple components in the treatment of T2DM by lowering blood sugar and alleviating inflammation.

[0009] The objective of this invention can be achieved through the following technical solutions:

[0010] In a first aspect, the present invention claims protection for a pharmaceutical composition for the prevention and treatment of diabetes, the composition comprising a Ganoderma lucidum polysaccharide fraction and a Ganoderma lucidum triterpenoid fraction; wherein the mass ratio of the Ganoderma lucidum polysaccharide fraction to the Ganoderma lucidum triterpenoid fraction is (0.5~2):1; preferably (1~2):1.

[0011] In specific embodiments of the present invention, the mass ratio of Ganoderma lucidum polysaccharide fraction to Ganoderma lucidum triterpenoid fraction can be 1:2, 1:1, or 2:1. The most preferred ratios are 1:1 and 2:1.

[0012] Furthermore, the Ganoderma lucidum polysaccharide fraction is prepared by the following method: crude polysaccharide is extracted from Ganoderma lucidum mycelium by water extraction and alcohol precipitation, and purified by removing proteins using the Sevag method.

[0013] In a specific embodiment of the present invention, dried Ganoderma lucidum mycelium is pulverized, extracted with boiling water for 1-3 hours, and the extraction is repeated 2-4 times. The extracts are combined, concentrated under reduced pressure, and anhydrous ethanol is added to the concentrated extract. After standing, the precipitate is collected and freeze-dried to obtain crude Ganoderma lucidum mycelium polysaccharide. The protein in the polysaccharide is removed using the Sevag method to obtain the purified Ganoderma lucidum polysaccharide fraction. More specifically, 8-15 times the volume of water is added to the Ganoderma lucidum mycelium, and the extract is extracted with boiling water for 1-3 hours, repeated 2-4 times. The extracts are combined and concentrated under reduced pressure. 3-5 times the volume of anhydrous ethanol is added to the concentrated extract, stirred evenly, and placed at 4°C overnight. The next day, it is centrifuged (3,000 rpm, 15 min) and the precipitate is collected. After freeze-drying, crude Ganoderma lucidum mycelium polysaccharide is obtained.

[0014] Furthermore, the Ganoderma triterpenoid fraction is prepared by the following method: obtained from Ganoderma mycelium by hot reflux extraction with 80%-95% ethanol.

[0015] In a specific embodiment of the present invention, dried Ganoderma lucidum mycelium is taken, pulverized, and 80%-95% ethanol is added at a material-to-liquid ratio of 1:10-20 (g / mL). The mixture is then refluxed for 0.5-2 hours, and the extraction is repeated 2-4 times. The extracts are combined, the supernatant is collected by centrifugation and concentrated, and then freeze-dried to obtain the Ganoderma lucidum triterpenoid fraction.

[0016] Furthermore, the pharmaceutical composition is prepared into a pharmaceutical formulation with pharmaceutically acceptable excipients; the pharmaceutical formulation is selected from ointments, liniments, patches, lotions, solutions, powders, sprays, syrups, suppositories, tablets, pills, granules, capsules, injections, nano-encapsulations, liposomes, or enteric-coated sustained-release formulations.

[0017] Secondly, the present invention claims protection for the use of the above-described composition in the preparation of a medicament for treating type 2 diabetes.

[0018] Thirdly, the present invention seeks protection for the use of the above-described composition in the preparation of a medicament for lowering blood sugar.

[0019] Fourthly, the present invention claims protection for the use of the above-described composition in the preparation of a medicament for reducing inflammatory responses in diabetic patients.

[0020] Fifthly, the present invention claims protection for the use of the above-described composition in the preparation of a medicament for inhibiting α-glucosidase.

[0021] Sixthly, the present invention claims protection for a medicament comprising the aforementioned composition as an active ingredient, and pharmaceutically acceptable excipients.

[0022] Traditional Chinese medicine (TCM) is characterized by its multi-component, multi-pathway, and multi-target nature. Ganoderma lucidum polysaccharides and triterpenes, as two of the most studied active ingredients for treating diabetes, regulate different pathways and target different substances. The combined use of these two components aligns with the synergistic effects of TCM's multi-component and multi-target approach. Ganoderma lucidum polysaccharides primarily improve diabetes symptoms through mechanisms such as improving insulin resistance, anti-inflammation, anti-oxidation, and regulating gut microbiota. Ganoderma lucidum triterpenes, on the other hand, show good efficacy against key enzymes regulating glucose metabolism, such as α-glucosidase and α-amylase. The combined use of Ganoderma lucidum polysaccharides and triterpenes can target different substances to lower blood sugar, reduce inflammation, and inhibit α-glucosidase, synergistically treating type 2 diabetes mellitus (T2DM) and constructing a novel combination therapy regimen for T2DM.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) All components of this invention are natural medicines that are classified as superior and have the function of strengthening the body and consolidating the foundation in the "Shennong's Classic of Materia Medica". The two parts are used in combination to treat diabetes through multiple pathways such as lowering blood sugar, anti-inflammation and inhibiting α-glucosidase.

[0025] (2) All compositions of the present invention are composed of active parts of Chinese medicine derived from natural drugs, with little toxic side effects, relatively clear components, relatively clear mechanisms, and similar efficacy to positive control drugs, and the quality is controllable.

[0026] (3) Both parts and the composition of the present invention have no obvious toxicity. Both mouse experiments and cell experiments show that they have therapeutic effects of reducing blood sugar and alleviating inflammation.

[0027] (4) The results of the in vitro inhibition experiment of α-glucosidase showed that the Ganoderma triterpenoid fraction of the present invention can effectively inhibit the action of α-glucosidase.

[0028] (5) The various parts of the present invention can reduce blood glucose in T2DM mice, and the Ganoderma lucidum polysaccharide part can also reduce the level of inflammatory factors in the serum of T2DM mice, which has a significant therapeutic effect on T2DM.

[0029] (6) The composition in this invention can significantly reverse the reduction in glucose consumption in the insulin-resistant HepG-2 cell model. Attached Figure Description

[0030] Figure 1The changes in fasting blood glucose (FBG) over time were shown in different treatment groups; where Con: blank group, Mod: model group, GLP: Ganoderma lucidum polysaccharide group, GLT: Ganoderma lucidum triterpenoid group, and Ipr: positive control group (iogglitazone).

[0031] Figure 2 The levels of inflammatory factors TNF-α, IL-1β, and IL-10 in the serum of mice in different treatment groups were calculated. Among them, Con: blank group, Mod: model group, GLP: Ganoderma lucidum polysaccharide group, GLT: Ganoderma lucidum triterpenoid group, and Ipr: positive control group (iogglitazone). Compared with the blank group, ### P < 0.001; compared with the model group, * P<0.05, ** P<0.01, *** P<0.001.

[0032] Figure 3 Dose-response curves of glucose consumption in insulin-resistant HepG-2 cells after administration of three Ganoderma lucidum extracts. Detailed Implementation

[0033] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0034] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0035] In this embodiment of the invention, the mycelium of Ganoderma lucidum strain SS30 is used as one of the raw material sources; this strain information is only an example and does not constitute a limitation of the invention. Those skilled in the art can use Ganoderma lucidum sources with similar active ingredients, such as Ganoderma lucidum var. rubrum, Ganoderma lucidum var. aurantiacus, or their artificially cultivated mycelium, to prepare Ganoderma lucidum polysaccharide fractions and Ganoderma lucidum triterpenoid fractions according to the method described in this invention and implement this invention.

[0036] In this embodiment of the invention, the mycelium of Ganoderma lucidum strain SS30 is used as one of the raw material sources. This strain information is merely illustrative and does not constitute a limitation of the invention. Related strain information can be found in published document CN114350525A. Those skilled in the art can use Ganoderma lucidum sources with similar active ingredients, such as Ganoderma lucidum var. rubrum, Ganoderma sinense, or their artificially cultivated mycelium, to prepare Ganoderma lucidum polysaccharide fractions and Ganoderma lucidum triterpenoid fractions according to the method described in this invention and implement this invention.

[0037] Example 1: Preparation of Ganoderma lucidum polysaccharides

[0038] 1. Experimental Methods

[0039] Dried Ganoderma lucidum mycelium (SS 30 grade) was pulverized and mixed with 10 times its volume of water. The mixture was extracted with boiling water for 2 hours, repeated three times. The extracts were combined and concentrated under reduced pressure. Four times its volume of anhydrous ethanol was added to the concentrated extract, and the mixture was stirred thoroughly and incubated overnight at 4°C. The next day, the extract was centrifuged (3,000 rpm, 15 min) and the precipitate was collected. After freeze-drying, crude polysaccharide from the Ganoderma lucidum mycelium was obtained. Protein was removed from the polysaccharide using the Sevag method to obtain purified Ganoderma lucidum polysaccharide (GLP). The protein content of the Ganoderma lucidum polysaccharide was determined using a BCA kit, and the polysaccharide content was determined using the sulfuric acid-anthrone method with anhydrous glucose as a control.

[0040] 2. Experimental Results

[0041] The protein content of the prepared Ganoderma lucidum polysaccharide, as determined by the BCA kit, was below the detection limit, indicating that the prepared Ganoderma lucidum polysaccharide was free of protein impurities. The polysaccharide content of the prepared GLP, determined by the sulfuric acid-anthrone method, was 66.81% ± 2.38%.

[0042] Example 2 Preparation of total triterpenes from Ganoderma lucidum

[0043] 1. Experimental Methods

[0044] Dried Ganoderma lucidum mycelium of SS 30 was pulverized and extracted with 85% ethanol at a material-to-liquid ratio of 1:15 (g / mL) by hot reflux for 1 h, repeated 3 times. The extracts from the three extractions were combined, centrifuged (3,000 rpm, 15 min), and the supernatant was concentrated to a certain volume. After freeze-drying, the Ganoderma lucidum triterpene (GLT) was obtained. The content of the prepared Ganoderma lucidum triterpene was determined by vanillin-glacial acetic acid method using oleanolic acid as a reference standard.

[0045] 2. Experimental Results

[0046] The total triterpenoid content of GLT prepared by vanillin-glacial acetic acid method was 1.09% ± 0.05%.

[0047] Example 3: Preparation of total Ganoderma lucidum polypeptides

[0048] 1. Test Methods

[0049] Dried Ganoderma lucidum mycelium (SS 30) was pulverized and extracted with 10% ethanol at a material-to-liquid ratio of 1:15 (g / mL) using ultrasonic-assisted extraction at 60℃ and 100% power for 1.5 h. The extraction was repeated twice. The two extracts were combined, centrifuged (3,000 rpm, 15 min), and the supernatant was collected and concentrated to a certain volume. Four volumes of 95% ethanol were added, mixed thoroughly, and incubated overnight at 4℃. The next day, the supernatant was collected by centrifugation (3,000 rpm, 15 min). The supernatant was concentrated under reduced pressure to a certain volume and freeze-dried to obtain the total Ganoderma lucidum polypeptide (GLPP). The total GLPP content was determined using a BCA kit.

[0050] 2. Experimental Results

[0051] The total polypeptide content of the prepared GLPP was determined to be 6.06% ± 0.36% using a BCA kit.

[0052] Example 4: In vitro α-glucosidase inhibition experiment

[0053] Alpha-glucosidase inhibitors lower postprandial blood glucose by inhibiting carbohydrate absorption in the upper small intestine, making them suitable for patients with elevated postprandial blood glucose levels whose main food source is carbohydrates. Systematic reviews of clinical studies in individuals with type 2 diabetes mellitus (T2DM) showed that alpha-glucosidase inhibitors reduced HbA1c by 0.50% and caused weight loss. Therefore, inhibiting alpha-glucosidase can effectively lower postprandial blood glucose and improve diabetic symptoms. This invention employs an in vitro alpha-glucosidase inhibition assay, using an enzyme-linked immunosorbent assay (ELISA) reader to detect the inhibitory effects of the two sites involved in this invention on alpha-glucosidase.

[0054] 1. Experimental Methods

[0055] A concentration gradient of 5 mg / mL, 2.5 mg / mL, 1.25 mg / mL, 0.625 mg / mL, and 0.3125 mg / mL was set for GLP and GLT samples. 24 μL of sample solution was added to each well of a 96-well plate, followed by 96 μL of 0.1 mol / L PBS solution and 10 μL of α-glucosidase solution. The mixture was thoroughly mixed and incubated at 37 °C for 15 min. Then, 30 μL of p-nitrobenzene-α-D-glucosidase solution was added, and the reaction was incubated at 37 °C for 20 min. The reaction was terminated by adding 80 μL of 0.2 mol / L Na₂CO₃ solution. The absorbance (OD₁) was measured at 405 nm using a microplate reader.

[0056] For the negative control, PBS buffer was used instead of the α-glucosidase solution, and all other tests were performed the same as the standard, measuring absorbance (OD2). For the negative control, pure water was used instead of the sample solution, and all other tests were performed the same as the standard, measuring absorbance (OD3). For the negative control blank, pure water was used instead of the sample solution, and PBS buffer was used instead of the α-glucosidase solution, and all other tests were performed the same as the standard, measuring absorbance (OD4). The positive control drug was acarbose, and the detection method was the same as the standard. The inhibition rate of the samples against α-glucosidase was calculated, and the half maximal inhibitory concentration (IC50) of each sample against α-glucosidase was calculated. 50 ).

[0057] The inhibition rate is calculated using the following formula: SR% = [1 - (OD1 - OD2) / (OD3 - OD4)] × 100%.

[0058] 2. Experimental Results

[0059] Table 1 shows that the total triterpenoid fraction of Ganoderma lucidum exhibited good inhibitory activity against α-glucosidase, while the polysaccharide fraction of Ganoderma lucidum did not inhibit α-glucosidase activity at any concentration.

[0060] Table 1. Inhibitory effects of GLP and GLT on α-glucosidase (n=3, x̄±SD)

[0061] GLT 41.83±4.45 GLP / Acarbose 60.15±2.56

[0062] Example 5: Effects of Ganoderma lucidum polysaccharide and triterpenoid fractions on STZ-induced T2DM mouse model

[0063] 1. Experimental Methods

[0064] 1.1 T2DM mouse model

[0065] Five-week-old male SPF-grade healthy C57BL / 6J mice, weighing 21±1 g, were fed regularly, kept at an indoor temperature of 25±2℃ and a humidity of 60±5%. All mice had free access to food and were kept in an environment with alternating 12-hour day and night cycles, fed standard feed for one week to acclimatize to the environment.

[0066] One week later, the mice were randomly divided into two groups: a control group (n=10) and a high-fat group (n=40). The control group was fed a normal diet (10 kcal% fat diet), while the model group was fed a high-fat diet (60 kcal% fat diet).

[0067] During the rearing period, mice were housed in cages of 10, maintaining a temperature of 25±2℃ and a relative humidity of 60±5%. They had free access to food and water, with a 12 / 12-hour day / night cycle. Bedding and water were changed every two days, and mice were randomly swapped between groups to minimize intra-group variations. Cages were rotated weekly in one direction to avoid positional influence. After 4 weeks, the mice were weighed to ensure the model group mice maintained a weight of approximately 30 g.

[0068] A T2DM model was established in high-fat mice by inducing streptozotocin solution (STZ). The specific modeling method was as follows: high-fat mice were injected intraperitoneally with STZ citrate buffer solution at a dose of 40 mg / kg body weight, and normal mice were injected intraperitoneally with an equal volume of sodium citrate buffer. The injections were given every other day for a total of 3 times.

[0069] Solution preparation

[0070] Citric acid-sodium citrate buffer (pH=4.5): Solution A (0.1 mol / L citric acid): Weigh 2.10 g of citric acid and add 100 mL of pure water; Solution B (0.1 mol / L sodium citrate): Weigh 2.94 g of sodium citrate and add 100 mL of pure water; Store solutions A and B separately at 4°C. Before use, mix solutions A and B at a ratio of 1:1.32 (v / v) to prepare a 0.1 mol / L citric acid buffer and adjust the pH to 4.5.

[0071] Streptozotocin solution: Weigh 10 mg of STZ into a 1.5 mL centrifuge tube, dissolve in 1 mL of 0.1 M citrate buffer, shake well, and operate on ice protected from light. Prepare immediately before use and use within 30 min. Administer the prepared solution intraperitoneally at a dose of 40 mg / kg.

[0072] 1.2 Experimental Grouping

[0073] T2DM mice that successfully developed the model were randomly divided into four groups: model group (T2DM, n=10), positive control group (eoglitazone, 6.5 mg / kg, n=10), GLP group (300 mg / kg, n=10), and GLT group (300 mg / kg, n=10). All groups continued to be fed a high-fat diet. The drug administration groups received different drugs via gavage, while the blank control group and T2DM group received an equal volume of 0.5% (m / v, g / 100ml) CMC-Na solution daily.

[0074] 1.3 Detection of fasting blood glucose (FBG) in T2DM mice

[0075] Mice were grouped according to "1.2 Experimental Grouping". After 6 weeks of drug administration and culture, mice in the blank group, model group, positive control group and different drug administration groups were fasted for 6 hours. Blood glucose was then measured by taking blood from the tail tip using a blood glucose meter.

[0076] 1.4 ELISA method for detecting serum concentrations of TNF-α, IL-1β, and IL-10

[0077] Mice were grouped according to the experimental grouping in section 1.2. After 6 weeks of drug administration and culture, blood was collected by enucleation. Serum was collected after centrifugation at 3,000 rpm for 10 min. The levels of TNF-α, IL-1β, and IL-10 in the serum of mice in the blank group, model group, positive control group, and different drug administration groups were detected by ELISA.

[0078] 2. Experimental Results

[0079] During the modeling phase, blood was collected from the tail cuttings (tip) of the modeling mice 5 and 7 days after injection, and the FBG level of the mice after fasting for 6 hours was measured using a blood glucose meter / blood glucose test strip. If the FBG level was >11.1 mmol / L for two consecutive times, and accompanied by symptoms such as polydipsia, polyphagia, polyuria, and weight loss, the T2DM mouse model was considered to have been successfully established.

[0080] like Figure 1 As shown, FBG in the model group mice increased significantly, and administration of positive control drugs (iogglitazone), GLP, and GLT could all reduce FBG in T2DM mice.

[0081] like Figure 2 As shown, serum TNF-α and IL-1β levels in the model group mice were significantly increased, while IL-10 levels were significantly decreased, and these effects were significantly reversed after GLP administration. GLP administration was more effective than GLT and the positive control in reversing inflammatory factor levels.

[0082] Example 6: Effects of Ganoderma lucidum polysaccharides, triterpenes, and polypeptide fractions on insulin-resistant HepG-2 cells

[0083] 1. Experimental Methods

[0084] 1.1 Establishment of an insulin-resistant HepG-2 cell model

[0085] HepG-2 cells in the logarithmic growth phase were digested with trypsin, then added to complete culture medium to prepare a single-cell suspension. After counting, the cell density was adjusted to 1 × 10⁻⁶ cells / cells. 5Add 3 mL of cell suspension to each well of a 6-well plate and incubate in a constant temperature incubator. When the cells reach 60% confluence, discard the culture medium, wash with PBS, and add 2% serum-containing medium (v / v) to prepare 200 μmol / L palmitic acid for further treatment for 24 h. Detect the glucose content in the culture supernatant using a glucose kit and calculate the glucose consumption (GC) of each group of cells.

[0086] 1.2 Experimental Grouping

[0087] After determining the concentration range that had no effect on cell growth using the CCK8 assay, model groups, GLP groups, GLT groups, GLPP groups, GLP + GLT groups, GLP + GLPP groups, GLT + GLPP groups, and a positive control group were established. The corresponding drug doses were administered according to Tables 2 and 3. The model group was incubated with 200 μmol / L palmitic acid in 2% serum-containing medium (v / v), while the other groups were incubated with 200 μmol / L palmitic acid in 2% serum-containing medium containing the corresponding dose of Ganoderma lucidum extract. After 24 h of incubation, the supernatant of the culture medium was used to determine GC.

[0088] 1.4 Calculation of the combination index of combined administration of GLP, GLT, and GLPP

[0089] GraphPad Prism was used to perform four-parameter logistic curve fitting on the dosage and glucose consumption of the three drug administration groups to obtain the fitted curves. Based on the fitted curves, the equivalent dose of the single-dose administration corresponding to the glucose consumption of the combined drug administration group was calculated, and the combination index (CI) was calculated. The dosage of the combined drug administration is shown in Table 3.

[0090] 2. Experimental Results

[0091] As shown in Table 2, the GC of HepG-2 cells in the model group was significantly decreased. The GC of the positive drug and other treatment groups was significantly lower than that of the model group, indicating that there was GC in the cells at all three sites. Furthermore, Ganoderma lucidum polysaccharide fraction (GLP), Ganoderma lucidum total triterpenoid fraction (GLT), and Ganoderma lucidum total polypeptide fraction (GLPP) showed a dose-dependent glycemic inhibitory effect in the cell model (e.g., Figure 3 As shown): The four-parameter fitting curve of GLPP for glucose consumption is y = 78.7 + 21.3 / [1 + (503.1 / x)]. 1.269 Rsquared = 0.9946; The four-parameter fitting curve of GLP for glucose consumption is y = 78.7+21.3 / [1 +(335.2 / x)]. 1.217R squared = 0.9975; the four-parameter fitting curve of GLT for glucose consumption is y = 78.7 + 19.72 / [1 + (251.7 / x)]. 1.242 R squared = 0.9984.

[0092] Table 2. GC of HepG-2 cells in different treatment groups (n=3, x̄ ± SD)

[0093] Con 100.00±10.72 Mod <![CDATA[78.69±2.46 ### ]]> Metformin (1 mM) <![CDATA[92.54±1.22 *** ]]> GLPP (50 μg / mL) 79.76±3.74 GLPP (100 μg / mL) 81.18±2.81 GLPP (200 μg / mL) 83.31±2.81 GLPP (300 μg / mL) 85.79±4.30 GLPP (400 μg / mL) <![CDATA[88.63±1.06 ** <!-- 7 -->]]> GLP (50 μg / mL) 80.82±5.03 GLP (100 μg / mL) 82.24±4.64 GLP (200 μg / mL) 85.08±4.03 GLP (300 μg / mL) <![CDATA[88.28±2.68 * ]]> GLP (400 μg / mL) <![CDATA[90.76±4.64 * ]]> GLT (50 μg / mL) 80.47±1.62 GLT (100 μg / mL) 83.66±3.25 GLT (200 μg / mL) <![CDATA[86.86±2.21 * ]]> GLT (300 μg / mL) <![CDATA[89.34±3.25 ** ]]> GLT (400 μg / mL) <![CDATA[91.12±2.68 *** ]]>

[0094] Note: Compared with the blank group, ### P < 0.001, compared with the model group, * P<0.05, ** P<0.01, *** P<0.001.

[0095] As shown in Table 3, in the case of combined administration, when the GLP:GLT ratio was 1:1 and 1:2, the combination index of GLP+GLT combined administration was less than 0.9, indicating that the combined use of GLP and GLT has a synergistic hypoglycemic effect. The combination index of the GLP+GLPP group and the GLT+GLPP group was greater than 0.9, indicating that the combined administration had no synergistic effect.

[0096] Table 3. GC and combined index of HepG-2 cells in different treatment groups (n=3, x̄±SD)

[0097] Con 100.00±10.72 / Mod <![CDATA[78.69±2.46 ### ]]> / GLPP+GLP (200 μg / mL+100 μg / mL) 85.44±1.84 1.37 GLPP+GLP (150 μg / mL+150 μg / mL) <![CDATA[86.86±2.17 * ]]> 1.16 GLPP+GLP (100 μg / mL+200 μg / mL) <![CDATA[86.86±1.62 * ]]> 1.20 GLPP+GLT (200 μg / mL+100 μg / mL) <![CDATA[87.21±1.23 * ]]> 1.05 GLPP+GLT (150 μg / mL+150 μg / mL) <![CDATA[87.57±1.06 * ]]> 1.10 GLPP+GLT (100 μg / mL+200 μg / mL) <![CDATA[88.64±1.07 ** ]]> 1.01 GLP+GLT (200 μg / mL+100 μg / mL) <![CDATA[88.28±1.62 ** ]]> 1.14 GLP+GLT (150 μg / mL+150 μg / mL) <![CDATA[90.41±1.23 *** ]]> 0.82 GLP+GLT (100 μg / mL+200 μg / mL) <![CDATA[91.12±1.22 *** ]]> 0.75

[0098] Note: Compared with the blank group, ### P < 0.001, compared with the model group, * P<0.05, ** P<0.01, *** P<0.001.

Claims

1. A pharmaceutical composition for the prevention and treatment of diabetes, characterized in that, The pharmaceutical composition comprises Ganoderma lucidum polysaccharide fraction and Ganoderma lucidum triterpenoid fraction; wherein the mass ratio of the Ganoderma lucidum polysaccharide fraction to the Ganoderma lucidum triterpenoid fraction is (0.5~2):

1.

2. The pharmaceutical composition according to claim 1, characterized in that, The preferred mass ratio of Ganoderma lucidum polysaccharide fraction to Ganoderma lucidum triterpenoid fraction is (1~2):

1.

3. The pharmaceutical composition according to claim 1, characterized in that, The Ganoderma lucidum polysaccharide fraction was prepared by the following method: crude polysaccharide was extracted from Ganoderma lucidum mycelium by water extraction and alcohol precipitation, and the protein was removed and purified by Sevag method.

4. The pharmaceutical composition according to claim 1, characterized in that, The Ganoderma triterpenoid fraction was prepared by the following method: it was obtained from Ganoderma mycelium by hot reflux extraction with 80%-95% ethanol.

5. The pharmaceutical composition according to any one of claims 1 to 4, characterized in that, The pharmaceutical composition is prepared together with pharmaceutically acceptable excipients to form a pharmaceutical preparation; the pharmaceutical preparation is selected from ointments, liniments, patches, lotions, solutions, powders, sprays, syrups, suppositories, tablets, pills, granules, capsules, injections, nano-encapsulations, liposomes, or enteric-coated sustained-release preparations.

6. Use of the pharmaceutical composition according to any one of claims 1 to 5 in the preparation of a medicament for treating type 2 diabetes.

7. Use of the pharmaceutical composition according to any one of claims 1 to 5 in the preparation of a medicament for lowering blood glucose.

8. Use of the pharmaceutical composition according to any one of claims 1 to 5 in the preparation of a medicament for reducing inflammatory response in diabetic patients.

9. Use of the pharmaceutical composition according to any one of claims 1 to 5 in the preparation of a medicament for inhibiting α-glucosidase.

10. A drug, characterized in that, It comprises the pharmaceutical composition of any one of claims 1 to 5 as the active ingredient, and pharmaceutically acceptable excipients.

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