Application of ketoisophorone and benzyl benzoate in preparation of medicine for treating diabetes

Abstract

The invention discloses application of ketoisophorone and benzyl benzoate in preparation of a medicine for treating diabetes, and belongs to the technical field of biological medicine. In-vivo and in-vitro studies find that after a T2DM animal model and a GlcN cell model are treated through KSP independent administration, BZT independent administration and KSP and BZT 1: 3 combined administration, compared with a model group, the blood glucose regulation effect of a combined administration group is most remarkable: the area under an fasting blood glucose and oral glucose tolerance test (OGTT) curve is in dose-dependent reduction, and the area under the curve is in dose-dependent reduction; an insulin tolerance test (ITT) result shows that the insulin sensitivity is obviously improved; in animal experiments, glycogen generation in liver tissues is increased, and balloon-like deformation and fatty degeneration accumulation conditions are relieved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of ketoisophorone and benzyl benzoate in the preparation of drugs for treating diabetes. Background Technology

[0002] Diabetes mellitus is a group of diseases characterized by hyperglycemia and related to impaired insulin secretion or its physiological function. Type 2 diabetes mellitus (T2DM) is the most common, accounting for approximately 90-95%. Exploring effective drugs for the prevention and treatment of diabetes from traditional Chinese medicine resources is of great significance.

[0003] Insulin resistance (IR) is a pathological process in which decreased sensitivity of insulin target organs and cells to insulin leads to impaired insulin regulation of glucose and lipid metabolism. It encompasses the entire process from the initial stage of compensatory hyperinsulinemia due to excessive insulin secretion, to pancreatic decompensation and the onset of type 2 diabetes mellitus (T2DM) symptoms. Therefore, effectively improving IR is crucial for treating T2DM. The development of IR is related to metabolic inflammatory responses, and numerous studies have shown that the NLRP3 inflammasome is a key mediator of this inflammatory response. The NLRP3 inflammasome is a protein complex composed of NLRP3, ASC, and caspase 1. Upon activation of the NLRP3 inflammasome, caspase 1 generates enzymatic activity through self-cleavage and cleaves proIL 1β to produce a large amount of active IL 1β. IL 1β, acting on cells themselves or other cells, triggers an amplified inflammatory cascade, interfering with insulin signaling and leading to the occurrence and development of IR.

[0004] The liver is one of the main target organs of insulin. Under the action of insulin, the liver maintains homeostasis of glucose and lipid metabolism by regulating activities such as hepatic glycogen synthesis and hepatic glucose output. However, in insulin resistance (IR), hepatic insulin signaling regulation is impaired, the ability of hepatic parenchymal cells to take up and utilize glucose decreases, gluconeogenesis levels increase, and lipid deposition and hepatic glucose output increase. Among these, increased hepatic glucose output is the main reason for the high blood glucose levels (especially fasting blood glucose) in patients with type 2 diabetes mellitus (T2DM). Therefore, effectively improving hepatic IR is key to the treatment of T2DM.

[0005] Ketoisophorone (KSP), a naturally derived terpene derivative, and benzyl benzoate (BZT), a common aromatic ester compound, have previously demonstrated potential activity in anti-inflammatory, antioxidant, and metabolic regulation fields. However, their application and mechanism of action in the treatment of type 2 diabetes mellitus (T2DM) have not been clearly reported. Given the complexity of the pathological mechanisms of T2DM, the liver, as the main organ for gluconeogenesis, experiences increased endogenous glucose production due to the overexpression of key enzymes such as PEPCK and G6Pase. Simultaneously, chronic inflammation induced by TXNIP / NLRP3 inflammasome activation further exacerbates insulin resistance. Furthermore, the AMPK signaling pathway, a core pathway for metabolic regulation, shows reduced activity, which is a significant contributing factor to glucose and lipid metabolism disorders. Based on this, this study hypothesizes that KSP and BZT may exert therapeutic effects on T2DM by regulating the aforementioned key pathways and molecules, thereby improving hepatic insulin resistance, inhibiting gluconeogenesis, and alleviating inflammatory responses. Summary of the Invention

[0006] The purpose of this invention is to provide the application of ketoisophorone (KSP) and benzyl benzoate (BZT) in the preparation of drugs for treating diabetes, thereby addressing the problems existing in the prior art. This invention has found that the combined administration of KSP and BZT in a 1:3 ratio inhibits overactivated gluconeogenesis and improves fasting hyperglycemia by downregulating the expression of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase).

[0007] To achieve the above objectives, the present invention provides the following solution: This invention provides the use of ketoisophorone (KSP) and benzyl benzoate (BZT) in the preparation of medicaments for treating diabetes.

[0008] Furthermore, the diabetes is type II diabetes.

[0009] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0010] Furthermore, the dosage form of the drug includes granules, tablets, powders, pills, capsules, or oral liquids.

[0011] The present invention provides a medicament for treating diabetes and / or relieving symptoms of diabetes, wherein the medicament is any pharmaceutically acceptable dosage form made of isophorone (KSP) and / or benzyl benzoate (BZT) as active ingredients and pharmaceutically acceptable excipients.

[0012] In one embodiment of the present invention, the dosage form of the drug includes, but is not limited to, tablets, dispersible tablets, lozenges, orally disintegrating tablets, sustained-release tablets, capsules, soft capsules, pills, granules, injections, powder injections, or aerosols. In one embodiment of the present invention, the diabetes is type II diabetes.

[0013] In one embodiment of the present invention, the drug further contains pharmaceutically acceptable pharmaceutical excipients; the pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field.

[0014] In one embodiment of the present invention, the excipients include one or more of the following: binders: cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone; diluents: pregelatinized starch, dextrin, sucrose, lactose, and mannitol; fillers: starch and sucrose; humectants: glycerol; disintegrants: sodium carboxymethyl starch, croscarmellose, and dry starch; absorption promoters: quaternary ammonium compounds; surfactants: polysorbates, fatty acid sorbitan, and fatty acid glycerides; colorants: titanium dioxide, sunset yellow, methylene blue, and pharmaceutical grade iron oxide red; lubricants: hydrogenated vegetable oil, talc, and polyethylene glycol; coating materials: acrylic resin, hydroxypropyl methylcellulose, povidone, and cellulose acetate; and other excipients may also be added to the composition: flavoring agents and sweeteners.

[0015] In one embodiment of the present invention, the isophorone (KSP) has the following structural formula: ; The structural formula of benzyl benzoate (BZT) is as follows: .

[0016] The present invention also provides the use of the above-mentioned isophorone (KSP) and / or benzyl benzoate (BZT) in the preparation of medicaments for treating diabetes and / or relieving symptoms of diabetes.

[0017] In one embodiment of the present invention, the diabetes is type II diabetes.

[0018] In one embodiment of the present invention, when isophorone (KSP) and benzyl benzoate (BZT) are used in combination, the ratio of KSP to BZT is 1:1 to 1:3.

[0019] In one embodiment of the present invention, the dosage form of the drug includes, but is not limited to, tablets, dispersible tablets, lozenges, orally disintegrating tablets, sustained-release tablets, capsules, soft capsules, pellets, granules, injections, powder injections, or aerosols.

[0020] In one embodiment of the present invention, the drug further contains pharmaceutically acceptable pharmaceutical excipients; the pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field.

[0021] In one embodiment of the present invention, the excipients include one or more of the following: binders: cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone; diluents: pregelatinized starch, dextrin, sucrose, lactose, and mannitol; fillers: starch and sucrose; humectants: glycerol; disintegrants: sodium carboxymethyl starch, croscarmellose, and dry starch; absorption promoters: quaternary ammonium compounds; surfactants: polysorbates, fatty acid sorbitan, and fatty acid glycerides; colorants: titanium dioxide, sunset yellow, methylene blue, and pharmaceutical grade iron oxide red; lubricants: hydrogenated vegetable oil, talc, and polyethylene glycol; coating materials: acrylic resin, hydroxypropyl methylcellulose, povidone, and cellulose acetate; and other excipients may also be added to the composition: flavoring agents and sweeteners.

[0022] In one embodiment of the present invention, the isophorone (KSP) has the following structural formula: ; The structural formula of benzyl benzoate (BZT) is as follows: .

[0023] The present invention also provides a method for preparing a medicament for treating diabetes and / or relieving symptoms of diabetes, the method comprising preparing any pharmaceutically acceptable dosage form with isophorone (KSP) and / or benzyl benzoate (BZT) as active ingredients and pharmaceutically acceptable excipients.

[0024] In one embodiment of the present invention, the diabetes is type II diabetes.

[0025] Beneficial effects This invention, using in vivo and in vitro studies, found that treatment with KSP alone, BZT alone, and a combination of KSP and BZT in animal and GlcN cell models of type 2 diabetes mellitus (T2DM) showed the most significant glycemic regulation compared to the model group, with the optimal KSP:BZT ratio of 1:3. Fasting blood glucose and the area under the curve (AUC) of the oral glucose tolerance test (OGTT) decreased in a dose-dependent manner, and the insulin tolerance test (ITT) results showed a significant improvement in insulin sensitivity. Animal experiments revealed increased glycogen production in liver tissue, alleviating ballooning degeneration and fatty degeneration accumulation. qPCR results showed that the 1:3 combination of KSP and BZT inhibited overactive gluconeogenesis and improved fasting hyperglycemia by downregulating the expression of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase). These results indicate that KSP and BZT, alone and in combination, have therapeutic effects on T2DM, and the combination therapy exhibits a synergistic effect. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 Quantification of glucose content, PEPCK activity, G6pase activity, PEPCK mRNA and G6pasemRNA in each experimental group showed that in HepG2 cells, GlcN (18 mM) induced enhanced gluconeogenesis and increased glucose synthesis in HepG2 hepatocytes. Figure 1 A), and upregulated the mRNA and enzyme activity of PEPCK / G6pase ( Figure 1 KSP and BZT, alone or in combination, can inhibit the transcription and activity of PEPCK / G6pase, suppress gluconeogenesis, and thus inhibit HepG2 glucose production. The combination of KSP (2.5 μM) and BZT (7.5 μM) showed the best effect.

[0028] Figure 2 Glycogen content and PAS staining of HepG2 cells in each experimental group: In the GlcN-induced glucose metabolism disorder cell model, KSP / BZT intervention alone or in combination increased cellular glycogen content and decreased gluconeogenesis. Figure 2 A), and can improve pathological damage in the model group ( Figure 2 B), and the combination of KSP (2.5μM) + BZT (7.5μM) showed better results.

[0029] Figure 3 This is a flowchart of animal experiments.

[0030] Figure 4 The glucose tolerance test (GTT) and insulin tolerance test (ITT) of each experimental mouse group were measured: In the T2DM animal model, KSP and BZT alone or in combination can reduce fasting blood glucose and improve insulin levels. At the same time, the GTT and ITT experiments showed that the combination of KSP (2.5 mg / kg) + BZT (7.5 mg / kg) had the best effect on improving glucose metabolism.

[0031] Figure 5 Liver glucose levels and PAS staining in mice of each group: In the type 2 diabetes mellitus (T2DM) model, T2DM reduces liver glucose levels ( Figure 5 A) and damage to tissue morphology ( Figure 5 B), while KSP and BZT, administered alone or in combination, can restore glycogen production and improve histopathological changes to varying degrees.

[0032] Figure 6 For safety assessment experiments. Detailed Implementation

[0033] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0034] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0035] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art; unless otherwise specified, the reagents used in the embodiments are all commercially available.

[0036] The CAS number of the isophorone (KSP) is 78-59-1, and its structural formula is as follows: ; The CAS number of the benzyl benzoate (BZT) is 120-51-4, and its structural formula is as follows: .

[0037] The mice used in the following examples were C57BL / 6 mice, purchased from Spifort Biotechnology Co., Ltd. The HepG2 cells used were purchased from Pronosei.

[0038] The detection methods involved in the following embodiments are as follows: PAS staining: Liver tissue was fixed, embedded, and sectioned into 5µm sections. These sections were mounted on glass slides, dewaxed, and stained using a glycogen PAS staining kit (Solarbio, Beijing, China). Glycogen PAS staining was performed according to the manufacturer's instructions. PAS staining of paraffin-embedded liver sections was used to detect changes in liver glycogen content and damage. The stained sections were analyzed using an optical microscope (Zeiss, Germany). HepG2 cells were plated to create a cell model. The treated cells were fixed with 4% paraformaldehyde (PFA) and permeabilized. Glycogen PAS staining was performed according to the manufacturer's instructions.

[0039] Real-time quantitative PCR (RT-PCR): RNA was extracted using the Freezon Reagent kit (Vazyme, Nanjing, China), and RNA integrity and concentration were assessed using a NanoDrop spectrophotometer. Subsequently, an equal volume of RNA (1 μg) was reverse transcribed using the Hifair® III 1st Strand cDNA Synthesis SuperMix (yeasen Biotechnology, Shanghai, China). Real-time quantitative PCR was performed using the SYBR Green PCR Master Mixture (yeasen Biotechnology, Shanghai, China) on an Applied Biosystems QuantStudio 3 (ThermoFisher, USA). A 2- The Ct method was used to analyze relative gene expression levels, where Ct represents the cycle threshold number normalized to β-actin. Primers are shown in Table 1 below: Table 1: HepG2 primer sequences

[0040] Enzyme-linked immunosorbent assay (ELISA): Fasting insulin levels were measured using an Elabscience ELISA kit according to the manufacturer's instructions.

[0041] Measurement of intracellular glucose and glycogen content: Glucose and glycogen levels were determined using commercial kits and according to the manufacturer's instructions. The simplified procedure is as follows: First, the cell culture medium was replaced with DMEM medium free of phenol red and supplemented with sodium pyruvate (2 mM) and sodium lactate (20 mM). After 3 hours of incubation, the buffer used for glucose production detection was collected, and the glucose oxidase-peroxidase assay kit (Nanjing Jiancheng Biotechnology Institute, China) was used for measurement. Glycogen detection is based on the principle that glycogen can be dehydrated in concentrated sulfuric acid to form aldehyde derivatives, which react with anthrone to form a blue compound. Based on this principle, glycogen levels were determined using a glycogen assay kit (Nanjing Jiancheng Biotechnology Institute, China) and following the operating procedures. Finally, all data were standardized to the total protein content of each sample. To observe glycogen storage in HepG2 cells, the collected cells were fixed with 4% paraformaldehyde, followed by PAS staining (periodic acid-Schiff staining), and observed under an Olympus BX50 optical microscope. The red-stained areas were considered glycogen.

[0042] PEPCK and G6pase activity assays: The activities of G6Pase and PEPCK were detected using a commercially available kit (Nanjing Jiancheng Biotechnology Institute, China). In brief: PEPCK activity was determined based on its reaction with oxaloacetate to produce phosphoenolpyruvate and carbon dioxide. The latter, in a cascade catalysis of pyruvate kinase and lactate dehydrogenase, promotes the conversion of NADH to NAD+. This process can be reflected by monitoring the decrease in NADH absorbance at 340 nm, indicating enzyme activity. G6Pase activity was quantified by detecting the amount of NADPH generated at 340 nm (derived from NADP). Finally, the activities of both enzymes were standardized according to the total protein content of each sample.

[0043] Blood glucose measurement: Using a blood glucose meter (Yuwell) and test strips (Yuwell), fasting blood glucose levels in mice were assessed, and glucose tolerance and insulin tolerance were tested.

[0044] Data Analysis: All experiments and analyses were performed at least three times. Quantitative data are expressed as mean ± standard deviation (SD). Data analysis was performed using GraphPad Prism 8. Unpaired t-tests and one-way ANOVA were used to assess differences between groups, followed by Dunnett's multiple comparison test. A p-value <0.05 was considered statistically significant.

[0045] Example 1: Cell Experiment The specific steps are as follows: 1. Cell experiments: (1) Culture of HepG2 cells: HepG2 cells were cultured in DMEM complete medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin in a humidified incubator at 37°C and 5% CO2. Cells were rapidly thawed at 37°C, centrifuged at 1000 rpm for 5 min to remove the cryopreservation solution, resuspended, and seeded. The medium was changed after 24 h to remove dead cells. Cells were passaged when the confluence reached 80%–90%. Wash the cells with DPBS first, then add 0.25% trypsin-EDTA and digest at 37°C for 1-2 min. Once the intercellular spaces are observed to increase and the cells become rounded under a microscope, immediately add complete culture medium to stop the digestion. Gently pipette to form a single-cell suspension and inoculate into a new culture flask at a ratio of 1:3 to 1:5. Subculture every 2-3 days. Replace the culture medium with fresh medium when the pH value decreases (the color turns yellow).

[0046] For long-term cryopreservation, select cells in the logarithmic growth phase, resuspend them in cryopreservation solution containing 10% DMSO, program the temperature, and transfer them to liquid nitrogen for storage. Strict aseptic operation must be followed throughout the process to avoid contamination and to precisely control the digestion time to prevent cell damage.

[0047] (2) Different treatments of GlcN-induced HepG2 cells: GlcN (glucosamine)-induced HepG2 cells (model group): GlcN was added to cultured HepG2 cells to establish the model (GlcN concentration was 30 mM). After 24 h of modeling (conditions: 37℃, 5% CO2), without drug intervention, the cells were cultured for another 24 h (conditions: 37℃, 5% CO2), and samples were collected. Ketoisophorone (KSP) group: Following the model group method, GlcN was first used to establish the model. After 24 h of modeling, 10 μM KSP was added and the intervention was carried out for 24 h (conditions: 37℃, 5% CO2). Samples were then collected. Benzyl benzoate (BZT) group: Following the model group method, GlcN was first used to establish the model. After 24 h of modeling, 10 μM BZT was added and the intervention was carried out for 24 h (conditions: 37℃, 5% CO2). Samples were then collected. KSP / BZT-1 group: Following the model group method, GlcN modeling was first performed. After 24 h of modeling, KSP was administered at a ratio of 5 μM KSP + 5 μM BZT, and intervention was carried out for 24 h (conditions: 37℃, 5% CO2). Samples were then collected. KSP / BZT-2 group: Following the model group method, GlcN modeling was first performed. 24 h after modeling, the drug was administered in a ratio of 3.33 μM KSP + 6.67 μM BZT and the intervention lasted for 24 h (conditions: 37℃, 5% CO2). Samples were then collected. KSP / BZT-3 group: Following the model group method, GlcN was first used to establish the model. After 24 h of modeling, the drug was administered in a ratio of 2.5 μM KSP + 7.5 μM BZT and the intervention lasted for 24 h (under conditions of 37℃ and 5% CO2). Samples were then collected.

[0048] Control group (Con group): No reagents were added, and the samples were collected after culturing at 37℃ and 5% CO2 for 48 hours.

[0049] (3) After the experiment, the glucose content of HepG2 cells, the activity of key gluconeogenesis enzymes PEPCK and G6Pase, the relative mRNA level of PEPCK, the relative mRNA level of G6Pase, and the intracellular glycogen content were detected in each sample.

[0050] 2. Experimental Results: Ketoisophorone (KSP) and benzyl benzoate (BZT) inhibited glucose synthesis and the expression and content of key gluconeogenesis enzymes in HepG2 cells; results are as follows. Figure 1 and Figure 2 As shown in Table 2: Table 2: Experimental Results

[0051] The results show: (1) Treatment of HepG2 cells with the same concentration of KSP / BZT (10 μM), the combined application of KSP (5 μM) and BZT (5 μM) (KSP / BZT-1 group), the combined application of KSP (3.33 μM) and BZT (6.67 μM) (KSP / BZT-2 group), or the combined application of KSP (2.5 μM) and BZT (7.5 μM) (KSP / BZT-3 group) showed that the administration significantly reduced gluconeogenesis in HepG2 cells and increased intracellular glycogen content. Among them, the combined application of KSP (2.5 μM) and BZT (7.5 μM) had the most significant therapeutic effect.

[0052] (2) Figure 2The tissue section results showed that: in the Con group, the abundance and uniform distribution of purplish-red glycogen granules in the cytoplasm were sufficient, and glycogen reserves were at a physiological homeostatic level; in the GlcN group, the number of purplish-red glycogen granules in the cytoplasm was significantly reduced, with only a few scattered light staining signals, indicating a severe deficiency of glycogen reserves; in the GlcN+KSP (10μM) group, the abundance of purplish-red glycogen granules in the cytoplasm was increased compared to the GlcN group, and glycogen reserves were effectively restored; in the GlcN+BZT (10μM) group, the number of purplish-red glycogen granules in the cytoplasm was increased compared to the GlcN group, and glycogen reserves were effectively restored; in the GlcN+KSP (5μM)+BZT (5μM) group, the abundance of purplish-red glycogen granules in the cytoplasm was close to that of the Con group, with uniform and dense distribution, and polysaccharide reserves were restored to near physiological levels; in the GlcN+KSP (3.33μM)+BZT (6.67μM) group, the abundance of purplish-red glycogen granules in the cytoplasm was higher than that of the GlcN group. The polysaccharide reserves in the group were significantly improved, and the polysaccharide reserves were in good condition. In the GlcN+KSP (2.5μM)+BZT (7.5μM) group, the abundance of purple-red glycogen granules in the cytoplasm was significantly increased compared with the GlcN group, and the polysaccharide reserves were effectively improved.

[0053] Therefore, the combined application of KSP (2.5 μM) and BZT (7.5 μM) is the most effective treatment for HepG2 cells.

[0054] Example 2: Establishment of T2DM mouse model and drug administration Six-week-old male C57BL / 6J mice were purchased from Spiefol Biotechnology Co., Ltd. (Nanjing, China). Mice were placed in a humidity- and temperature-controlled environment and provided with standard food and clean water free of charge during a 12-hour light-dark cycle. After one week of acclimatization, we evaluated the effects of ketoisophorone (KSP) and benzyl benzoate (BZT) on a type 2 diabetes mellitus (T2DM) mouse model. All animal experiments in this study were approved by Jiangnan University. Mice were fed a 60% fat diet for 12 weeks to induce glucose metabolism disorder and establish a T2DM model. Mice with T2DM+KSP or T2DM+BZT were administered KSP (10 mg / kg / day) or BZT (10 g / kg / day) by gavage for 6 consecutive weeks starting at week 6. Mice with T2DM+KSP+BZT were administered KSP (2.5 mg / kg / day) and BZT (7.5 g / kg / day) by gavage for 6 consecutive weeks. All mice were housed at Jiangnan University.

[0055] The specific steps are as follows: 1. Construction of T2DM mice (type II diabetes model) Model building such as Figure 3 As shown.

[0056] After a week of acclimatization in the quarantine room, C57BL / 6J mice were randomly divided into a control group and a model T2D group. The specific steps are as follows: Weeks 0-12: Feeding with high-fat or regular feed; Control group: The group was fed a normal diet (14.7 kJ / g, 13% of the energy was fat) for 12 weeks throughout the experiment. T2D group (model): fed a high-fat diet (21.8 kJ / g, 60% fat, D12492) for 12 weeks; At the end of 12 weeks, mice were fasted for 10 hours but allowed free water, and their fasting blood glucose (FBG) was measured.

[0057] 2. Animal experiments (12 weeks in total): Blank control group: No type II diabetes model was established. This group served as a blank control throughout the entire process and was fed only normal feed (regular feed). Starting from week 8 of feeding, 100 μL of 1% (v / v) DMSO saline was administered by gavage once daily for 4 consecutive weeks.

[0058] Model group: A T2DM model was established using a high-fat diet. No drugs were administered throughout the experiment, and the diet was maintained on a high-fat diet for 12 weeks. Starting from week 8 of the high-fat diet, 100 μL of 1% (v / v) DMSO saline was administered by gavage once daily for 4 consecutive weeks.

[0059] Ketoisophorone (KSP) group: A type 2 diabetes mellitus (T2DM) model was established using a high-fat diet, with the diet being fed a high-fat diet for 12 weeks. Starting from week 8 of the high-fat diet, KSP (10 mg / kg / d in physiological saline solution of 1% (v / v) DMSO, 100 μL) was administered by gavage once daily for 4 weeks.

[0060] Benzyl benzoate (BZT) group: A T2DM model was established using a high-fat diet, with the patient continuously fed a high-fat diet for 12 weeks. Starting from week 8 of the high-fat diet, BZT (solvent: 1% (v / v) DMSO in physiological saline, gavage dose: 100 μL) was administered by gavage once daily for 4 consecutive weeks.

[0061] Ketoisophorone (KSP) and benzyl benzoate (BZT) 1:3 combination therapy group: A type 2 diabetes mellitus (T2DM) model was established using a high-fat diet, with continuous high-fat feeding for 12 weeks. Starting from week 8 of high-fat feeding, 2.5 mg / kg KSP + 7.5 mg / kg BZT (total dose 10 mg / kg, both in 1% (v / v) DMSO saline solution, with gavage doses of 25 μL KSP and 75 μL BZT, respectively) were administered once daily for 4 consecutive weeks.

[0062] 3. Experimental Results (1) After 12 weeks, fasting blood glucose (FBG), insulin level, glucose tolerance test (AUC of GTT), and insulin tolerance test (AUC of ITT) were measured respectively. The results are as follows: Figure 4 As shown in Table 3.

[0063] Table 3: Experimental Results for Each Group

[0064] The results show: Both KSP and BZT can lower blood glucose in mice, and their combined use is even more effective.

[0065] (2) After the animal experiment, liver tissues of mice in each group were taken to detect liver glycogen content (μg / mg protein) and analyze liver tissue sections.

[0066] 1) Liver glycogen content The results are as follows Figure 5 As shown: Blank control group: The liver glycogen content of mice in the untreated control group was 48.40±2.98.

[0067] Model group: The liver glycogen content of mice on a high-fat diet for 12 weeks was 21.98±1.73.

[0068] Ketoisophorone (KSP) group: After treatment with ketoisophorone (KSP) in T2DM mice, the liver glycogen content was 32.25±1.77.

[0069] Benzyl benzoate (BZT) group: After treatment with benzyl benzoate (BZT) in T2DM mice, the liver glucose content was 33.07±1.82.

[0070] It is evident that ketophorone (KSP) and benzyl benzoate (BZT) increased glycogen content in the livers of T2DM mice.

[0071] Drug combination therapy: When ketoisophorone (KSP) and benzyl benzoate (BZT) were used in a 1:3 ratio, the effect of increasing liver glucose levels in patients with type 2 diabetes mellitus (T2DM) was better than that of using either drug alone, as shown in the results. Figure 5 As shown, the combined use of KSP (2.5 mg / kg / d) and BZT (7.5 mg / kg / d) to treat T2DM mice resulted in the best increase in liver glucose content, which was 45.20 ± 1.91.

[0072] 2) Tissue section analysis: The results are as follows Figure 5 As shown, ketophorone (KSP) and benzyl benzoate (BZT) restored the normal glycogen content in T2D liver tissue (Control). The abundance of purple-red glycogen granules in the cytoplasm was high and evenly distributed, showing a continuous and dense filling state, with no obvious glycogen deficiency areas, suggesting that the liver's glycogen synthesis and storage functions were in a physiological homeostasis. In the diabetes model group (T2D), the number of purplish-red glycogen granules in the cytoplasm was significantly reduced, with only scattered light-stained signals. The glycogen distribution showed a discrete characteristic, and the overall abundance was significantly lower than that of the control group, reflecting a severe impairment of liver glycogen reserve capacity. In the 10 mg / kg KSP intervention group (T2D+KSP), the abundance of purple-red glycogen granules in the cytoplasm significantly increased, the distribution uniformity improved, the discrete region decreased, and the glycogen reserve level was significantly restored compared with the T2D group. In the 10 mg / kg BZT intervention group (T2D+BZT), the abundance of purplish-red glycogen granules in the cytoplasm significantly increased, the uniformity of distribution improved, the discrete regions decreased, and the glycogen reserve level was significantly restored compared with the T2D group. In the combined intervention group (T2D + 2.5 mg / kg KSP + 7.5 mg / kg BZT), the abundance of purplish-red glycogen granules in the cytoplasm was close to that of the control group, and the distribution was uniform and dense. The glycogen reserve status was basically restored to the physiological level, suggesting that the combined intervention had the best effect on liver glycogen repair.

[0073] Example 3: Safety Assessment Experiment (1) Using cell viability as an indicator, we investigated the toxic effects of different concentrations of KSP on HepG2 cells.

[0074] The results are as follows Figure 6 As shown, with 0 μM (solvent control) cell viability as the baseline, there was no significant difference in cell viability between the control group and the control group after treatment with low concentrations of KSP ranging from 1 to 10 μM. Cell viability did not fluctuate abnormally in the 50-200 μM high concentration groups; even at a high concentration of 500 μM, cell viability remained at the control group level, and no toxicity-related decrease in viability was observed.

[0075] The above results indicate that KSP at concentrations ranging from 1 to 500 μM has no significant toxicity to cells.

[0076] (2) Using the same cell viability assay system, the cytotoxicity of different concentrations of BZT on HepG2 cells was evaluated.

[0077] The results are as follows Figure 6 As shown, with 0 μM (solvent control) cell viability as the baseline, the cell viability after treatment with 1~10 μM low concentration BZT remained consistent with that of the control group. No inhibitory changes in cell viability were observed in the 50-200 μM high concentration group; the cell viability in the 500 μM high concentration group remained similar to that of the control group, and no toxicity-induced decrease in viability was observed.

[0078] These results suggest that BZT in the concentration range of 1–500 μM has no significant toxicity to cells, and the concentration used in the experiment (e.g., 10 μM) has good cell safety.

[0079] (3) Animal experiments to verify toxic effects The specific experimental method was the same as that of the model group and blank group in the animal experiment of Example 2. After 4 weeks of culture, there was no significant difference in the growth status of the two groups of mice (including core indicators such as weight gain trend, overall activity status, feeding and drinking behavior and general physiological signs). No growth retardation, abnormal activity or physiological imbalance caused by model construction was observed, which proves that the dosage of the present invention has good safety.

[0080] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A drug, characterized in that, The drug is used to treat diabetes and / or relieve symptoms of diabetes, and the drug is any pharmaceutically acceptable dosage form made with isophorone (KSP) and / or benzyl benzoate (BZT) as active ingredients and pharmaceutically acceptable excipients.

2. The drug according to claim 1, characterized in that, The dosage forms of the drug include, but are not limited to, tablets, dispersible tablets, lozenges, orally disintegrating tablets, sustained-release tablets, capsules, soft capsules, pellets, granules, injections, powder for injection, or aerosols. Preferably, the diabetes is type II diabetes.

3. The drug according to claim 2, characterized in that, The drug also contains pharmaceutically acceptable excipients; the excipients refer to conventional drug carriers in the pharmaceutical field.

4. The drug according to claim 3, characterized in that, The excipients include one or more of the following: binders: cellulose derivatives, alginate, gelatin and polyvinylpyrrolidone; diluents: pregelatinized starch, dextrin, sucrose, lactose, mannitol; Fillers: starch, sucrose; wetting agents: glycerin; disintegrants: sodium carboxymethyl starch, croscarmellose, and dry starch; absorption enhancers: quaternary ammonium compounds; surfactants: polysorbate, fatty acid sorbitan, and fatty acid glycerides; colorants: titanium dioxide, sunset yellow, methylene blue, and pharmaceutical grade iron oxide red. Lubricants: hydrogenated vegetable oil, talc, and polyethylene glycol; Coating materials: acrylic resin, hydroxypropyl methylcellulose, povidone, and cellulose acetate; Other excipients may also be added to the composition: flavoring agents and sweeteners; Preferably, the isophorone (KSP) has the following structural formula: ； The structural formula of benzyl benzoate (BZT) is as follows: 。 5. The use of isophorone (KSP) and / or benzyl benzoate (BZT) in the preparation of medicines for treating diabetes and / or relieving symptoms of diabetes.

6. The application according to claim 5, characterized in that, The dosage forms of the drug include, but are not limited to, tablets, dispersible tablets, lozenges, orally disintegrating tablets, sustained-release tablets, capsules, soft capsules, pellets, granules, injections, powder for injection, or aerosols.

7. The application according to claim 6, characterized in that, The drug also contains pharmaceutically acceptable excipients; the excipients refer to conventional drug carriers in the pharmaceutical field.

8. The application according to claim 7, characterized in that, The excipients include one or more of the following: binders: cellulose derivatives, alginate, gelatin and polyvinylpyrrolidone; diluents: pregelatinized starch, dextrin, sucrose, lactose, mannitol; Fillers: starch, sucrose; wetting agents: glycerin; disintegrants: sodium carboxymethyl starch, croscarmellose, and dry starch; absorption enhancers: quaternary ammonium compounds; surfactants: polysorbate, fatty acid sorbitan, and fatty acid glycerides; colorants: titanium dioxide, sunset yellow, methylene blue, and pharmaceutical grade iron oxide red. Lubricants: hydrogenated vegetable oil, talc, and polyethylene glycol; Coating materials: acrylic resin, hydroxypropyl methylcellulose, povidone, and cellulose acetate; Other excipients may also be added to the composition: flavoring agents and sweeteners.

9. The application according to claim 5, characterized in that, The structural formula of the isophorone (KSP) is as follows: ； The structural formula of benzyl benzoate (BZT) is as follows: 。 10. A method for preparing a medicament for treating diabetes and / or alleviating symptoms of diabetes, characterized in that, The method includes any pharmaceutically acceptable dosage form made with isophorone (KSP) and / or benzyl benzoate (BZT) as active ingredients and pharmaceutically acceptable excipients.

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