Application of saffron crocus pigment composition in preparation of preparation for treating or improving metabolic syndrome

Abstract

The invention relates to the technical field of traditional Chinese medicine extract medicines, and discloses an application of a saffron crocus pigment composition in preparation of a preparation for treating or improving metabolic syndrome, and the saffron crocus pigment composition is extracted from traditional Chinese medicine gardenia. Research finds that the saffron pigment composition extracted from plants shows good blood glucose and lipid lowering activity in a metabolic syndrome mouse model induced by combination of 60% high-fat feed and intraperitoneal injection streptozotocin, and meanwhile, the saffron pigment composition can remarkably improve hepatic and nephropathy damage of the mouse; therefore, the composition has certain advantages in treatment or prevention of the metabolic syndrome.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine extract drugs, and particularly relates to the application of a saffron pigment composition in the preparation of a preparation for treating or improving metabolic syndrome. Background Art

[0002] As a pathological state in which multiple metabolic components abnormally aggregate, metabolic syndrome (MS) covers various metabolic disorders such as visceral obesity, dyslipidemia, hyperglycemia, and hypertension. Under the influence of economic development and lifestyle changes, its prevalence shows an upward trend globally. Moreover, the multiple risk factors brought about by MS greatly increase the risk of a series of diseases such as cardiovascular atherosclerosis and type 2 diabetes, seriously interfering with the normal life of patients.

[0003] Currently, the therapeutic drugs for MS clinically mainly deal with the symptoms. For example, orlistat is used to inhibit nutrient absorption, sulfonylurea drugs promote insulin secretion, liraglutide is used as an islet β-cell protector, rosiglitazone acts as an insulin sensitizer, metformin promotes glucose metabolism, acarbose delays glucose absorption, statins and fibrates are used for lipid-lowering, angiotensin II receptor antagonists lower blood pressure, and aspirin is used for anti-thrombosis, etc. In terms of treatment strategies, generally based on changing bad lifestyles, single drugs are used for a single major risk factor, or combination drugs are used for multiple risk factors.

[0004] However, these existing therapeutic drugs have many disadvantages and deficiencies. They are extremely likely to cause serious adverse reactions, such as hypoglycemia, gastrointestinal reactions, abnormal liver function, heart failure, lactic acidosis, rhabdomyolysis, etc., among which hypoglycemic reactions and hepatocyte damage are relatively common. Moreover, considering that MS patients often have liver and kidney function damage and are in a pro-thrombotic and pro-inflammatory state, whether it is single drug use or combination drug use, the toxic and side effects of drugs in the processes of combined application, metabolism, and excretion are becoming more and more significant, resulting in a great reduction in the therapeutic effect. Therefore, the research and development of anti-MS drugs with no tolerance effect and small toxic and side effects have broad market prospects and profound social significance. Summary of the Invention

[0005] The present invention aims to provide the application of a saffron pigment composition in the preparation of a preparation for treating or improving metabolic syndrome, so as to solve the technical problem of poor curative effect in the treatment of metabolic syndrome in the prior art.

[0006] To achieve the above object, the present invention adopts the following technical solution: An application of a saffron pigment composition in the preparation of a preparation for treating or improving metabolic syndrome, wherein the saffron pigment composition is obtained by extracting from the traditional Chinese medicine Gardenia jasminoides Ellis; it comprises the following raw materials in parts by mass: 30-40 parts of crocetin bis-β-D-gentiobioside, 8-12 parts of crocetin-β-D-glucopyranosyl-β-D-gentiobioside, 2-3 parts of crocetin bis-β-D-glucopyranoside, 12-18 parts of 13Z-crocetin bis-β-D-gentiobioside, 2-3 parts of neocrocin B, 15-20 parts of crocetin mono-β-D-gentiobioside, 2-3 parts of crocetin mono-β-D-glucopyranoside, 1-3 parts of 13Z-crocetin-8-O-β-D-gentiobioside, and 1-2 parts of 13Z-crocetin-8'-O-β-D-gentiobioside.

[0007] Preferably, as an improvement, the present solution also provides a preparation method of a saffron pigment composition, comprising the following steps:

[0008] Step 1: Extract Gardenia jasminoides Ellis fruits by solvent extraction method to obtain the total Gardenia jasminoides Ellis extract;

[0009] Step 2: Separate the above-mentioned total Gardenia jasminoides Ellis extract by macroporous adsorption resin chromatography, the eluent is water and 30-95 Vol% ethanol, collect the ethanol elution part of 65-75 Vol%, concentrate to obtain each component in the saffron pigment composition;

[0010] Step 3: Mix each component according to the above-mentioned mass parts to obtain the saffron pigment composition.

[0011] Preferably, as an improvement, in Step 1, the solvent extraction method includes any one or more of decoction method, heating reflux extraction method, ultrasonic oscillation extraction method, percolation method and impregnation method.

[0012] Preferably, as an improvement, when the heating reflux extraction method is used for extraction, it comprises the following steps: using 50-80% ethanol as the extraction solvent, the material-liquid ratio of Gardenia jasminoides Ellis and 50-80% ethanol (i.e., the mass ratio of raw material to extraction solvent) is 1:5-20, soak for 1-3 h and then reflux extract for 1-3 h, extract for 1-3 times. After extraction, filter the extract and remove the solvent to obtain the total Gardenia jasminoides Ellis extract.

[0013] Preferably, as an improvement, in step two, the steps of separating the total gardenia extract by macroporous resin chromatography are as follows: taking the above-mentioned total gardenia extract as the macroporous resin sample loading solution, after the sample loading is completed, collecting the liquid, repeating the sample loading, with a flow rate of 0.5 - 1.5 BV / h. After the sample loading is finished, successively elute with deionized water for 1 - 12 BV, 30% ethanol for 1 - 12 BV, and 70% ethanol for 1 - 12 BV, with an elution flow rate of 0.5 - 1.5 BV / h. Collect the 70% ethanol eluate, concentrate and dry it to obtain each component in the saffron pigment composition.

[0014] Preferably, as an improvement, this solution also provides a pharmaceutical preparation for treating metabolic syndrome, including a saffron pigment composition and pharmaceutically acceptable excipients.

[0015] Preferably, as an improvement, the pharmaceutical preparation is any one of powder, granule, tablet, emulsion, syrup, aerosol, soft capsule, hard capsule, sterile injection, and sterile powder.

[0016] Preferably, as an improvement, the administration route of the pharmaceutical preparation is any one of oral administration, nasal inhalation, or parenteral administration.

[0017] Preferably, as an improvement, this solution also provides a method for constructing a type II diabetic animal experimental model, which is used to verify the effect of the above-mentioned pharmaceutical preparation in treating metabolic syndrome, including constructing by using 60% high-fat diet combined with intraperitoneal injection of streptozotocin.

[0018] Preferably, as an improvement, this solution also provides a type II diabetic animal experimental model, which is the MS mouse model constructed by the above method.

[0019] The principle and advantages of this solution are:

[0020] 1. In this solution, the saffron pigment composition can effectively improve the glycolipid metabolism disorder and liver and kidney damage in mice with metabolic syndrome, indicating that it can be used as an anti-metabolic syndrome drug, providing a new solution for the research and treatment of metabolic syndrome. Specifically, in this solution, intragastric administration of the saffron pigment composition significantly improved the glycolipid metabolism disorder and liver and kidney damage in the MS mouse model induced by 60% high-fat diet combined with STZ. The specific results are as follows: In terms of glucose metabolism, compared with the model group, continuous administration of the saffron pigment composition for 5 weeks significantly reduced the fasting blood glucose level in MS mice, significantly increased the oral glucose tolerance and serum insulin content in MS mice, and at the same time alleviated the pancreatic pathological damage in MS mice; in terms of lipid metabolism, the saffron pigment composition significantly reduced the blood lipid level in MS mice and significantly reduced the deposition of triglyceride (TG), total cholesterol (TC) and lipid droplets in the liver of MS mice; in terms of liver and kidney damage induced by 60% high-fat diet combined with STZ, the saffron pigment composition reduced the liver index in MS mice and significantly reduced the levels of liver and kidney damage-related biochemical indexes such as alanine amioTransferase (ALT), aspartate transaminase (AST) and blood urea nitrogen (BUN) in the serum of MS mice, and at the same time alleviated the liver and kidney pathological damage in MS mice. In the present invention, the hypoglycemic effect of the saffron pigment composition is weaker than that of the positive drug rosiglitazone, but its lipid-lowering effect is significantly better than that of rosiglitazone, and the saffron pigment composition significantly improves the liver and kidney damage in MS mice, while rosiglitazone shows a certain degree of nephrotoxicity. In summary, the saffron pigment composition is safe and effective for the treatment of MS.

[0021] 2. In this solution, a type II diabetes animal experimental model was constructed by combining 60% high-fat diet with intraperitoneal injection of streptozotocin. Among them, STZ can damage pancreatic islet β cells through the nitrosourea in its structure and is a commonly used drug to induce MS and diabetes. Combining with 60% high-fat diet induction can better simulate the development state of MS. And the constructed model is relatively stable and can better simulate the relevant characteristics of MS in terms of pathology, biochemistry, pathogenic mechanism, etc. The present invention successfully evaluated the activity of the saffron pigment composition in the treatment of MS using the above model.

[0022] 3. In this solution, each component of the saffron pigment composition was obtained by extraction from Gardenia jasminoides Ellis, and the formula of each component was optimized to effectively relieve related symptoms when used in the treatment of MS and have low kidney damage. It not only broadens the application scope of the saffron pigment composition extracted from Gardenia jasminoides Ellis, but also provides a direction for the research and development of new drugs for the treatment of MS. Description of the Drawings

[0023] Figure 1 Effect of the saffron pigment composition in Example 2 of the present invention on the fasting blood glucose level of MS mice( *** P < 0.001 vs. control group mice; # P < 0.05, ### P < 0.001 vs. model group mice).

[0024] Figure 2 Effect of the saffron pigment composition in Example 2 of the present invention on the oral glucose tolerance of MS mice( *** P < 0.001 vs. control group mice; # P < 0.05, ## P < 0.01, ### P < 0.001 vs. model group mice).

[0025] Figure 3 Effect of the saffron pigment composition in Example 2 of the present invention on the serum insulin level of MS mice( ** P < 0.01, *** P < 0.001 vs. control group mice; # P < 0.05, ## P < 0.01, ### P < 0.001 vs. model group mice).

[0026] Figure 4 Effect of the saffron pigment composition in Example 2 of the present invention on the blood lipid level of MS mice( * P < 0.05, ** P < 0.01, *** P < 0.001 vs. control group mice; # P < 0.05, ## P < 0.01 vs. model group mice).

[0027] Figure 5 Effect of the saffron pigment composition in Example 2 of the present invention on the liver lipid accumulation of MS mice( *** P < 0.001 vs. control group mice; # P < 0.05, ## P < 0.01 vs. model group mice).

[0028] Figure 6 Effect of the saffron pigment composition in Example 2 of the present invention on the organ index of MS mice( * P < 0.05, *** P < 0.001 vs. control group mice; # P < 0.05, ###P < 0.001 vs. the model group mice).

[0029] Figure 7 This is the effect of the saffron pigment composition in Example 2 of the present invention on the serum biochemical levels of MS mice ( ** P < 0.01, *** P < 0.001 vs. the control group mice; # P < 0.05, ### P < 0.001 vs. the model group mice).

[0030] Figure 8 This is the effect of the saffron pigment composition in Example 2 of the present invention on the pathological damage of the liver, kidney and pancreas of MS mice ( *** P < 0.001 vs. the control group mice; # P < 0.05, ## P < 0.01, ### P < 0.001 vs. the model group mice).

[0031] Figure 9 This is the effect of the saffron pigment composition in Example 3 of the present invention on the survival rate of IR-HepG2 cells.

[0032] Figure 10 This is the effect of the saffron pigment monomer compound in Example 3 of the present invention on the survival rate of IR-HepG2 cells. Detailed implementation manners

[0033] The present invention will be further described in detail below with reference to the embodiments, but the implementation manners of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used can all be obtained from commercial channels.

[0034] General description of the solution

[0035] This solution provides an application of a saffron pigment composition in the preparation of a preparation for treating or improving metabolic syndrome. The saffron pigment composition is obtained by extracting from the traditional Chinese medicine Gardenia jasminoides Ellis, and includes the raw material components and mass parts as shown in Table 1.

[0036] Table 1 Raw material components and mass parts of the saffron pigment composition

[0037] Serial number Compound name Parts by weight / part 1 Crocin bis-β-D-gentiobioside 30～40 2 Crocin-β-D-glucopyranosyl-β-D-gentiobioside 8～12 3 Crocin bis-β-D-glucopyranoside 2～3 4 13Z-Crocin bis-β-D-gentiobioside 12～18 5 neocrocin B 2～3 6 Crocin mono-β-D-gentiobioside 15～20 7 Crocin mono-β-D-glucopyranoside 2～3 8 13Z-Crocin-8-O-β-D-gentiobioside 1～3 9 13Z-Crocin-8'-O-β-D-gentiobioside 1～2

[0038] The preparation method of the saffron pigment composition in this solution includes the following steps:

[0039] Step 1: Extract Gardenia jasminoides Ellis fruits by solvent extraction method to obtain the total Gardenia jasminoides Ellis extract;

[0040] Among them, the solvent extraction method includes any one or more of decoction method, heating reflux extraction method, ultrasonic oscillation extraction method, percolation method and maceration method. When the heating reflux extraction method is used for extraction, 50-80% ethanol is used as the extraction solvent, and the material-liquid ratio of gardenia and 50-80% ethanol (i.e., the mass ratio of raw material to extraction solvent) is 1:5-20. After soaking for 1-3 h, reflux extraction is carried out for 1-3 h, and extraction is carried out 1-3 times. After extraction, the extract is filtered and the solvent is removed to obtain the total gardenia extract.

[0041] Specifically, the extraction steps of the total gardenia extract in this solution are as follows: Weigh 50 g of gardenia medicinal materials, add 5-20 times the amount of 50-80% ethanol, soak for 1-3 h, reflux extract for 1-3 h, and extract 1-3 times. Combine the extracts and concentrate until there is no alcohol smell. Dilute the concentrated solution with water, centrifuge at 5000 rpm for 5 min, and take the supernatant as the total gardenia extract.

[0042] Step 2: Separate the above-mentioned total gardenia extract by macroporous adsorption resin chromatography. The eluent is water and 30-95 Vol% ethanol. Collect the ethanol elution part of 65-75 Vol%, concentrate it to obtain each component in the saffron pigment composition.

[0043] As an improvement, during the separation process, gradient elution is carried out successively with 3-5 times the column bed volume of water, 25-35 Vo1%, 45-55 Vol%, 65-75 Vol%, and 90-100 Vol% ethanol.

[0044] Specifically, the steps of separating the total gardenia extract by macroporous adsorption resin chromatography in this solution are as follows: Take the above-mentioned total gardenia extract as the macroporous resin sample solution. After sampling, collect the liquid, repeat sampling, and the flow rate is 0.5-1.5 BV / h. After sampling, successively use deionized water for elution for 1-12 BV, 30% ethanol for elution for 1-12 BV, and 70% ethanol for elution for 1-12 BV. The elution flow rate is 0.5-1.5 BV / h. Collect the 70% ethanol eluate, concentrate and dry it to obtain each component in the saffron pigment composition.

[0045] Step 3: Mix each component according to the parts by mass in Table 1 to obtain the saffron pigment composition. Among them, the structures of each component in the saffron pigment composition prepared in this solution are as follows: 1. Crocin bis-β-D-gentiobioside

[0046]

[0047] 2. Crocin-β-D-glucopyranosyl-β-D-gentiobioside

[0048] 3. Crocin Di-β-D-glucopyranoside

[0049]

[0050] 4. 13Z-Crocin Di-β-D-gentiobioside

[0051]

[0052] 5. neocrocin B

[0053]

[0054] 6. Crocin Mono-β-D-gentiobioside

[0055]

[0056] 7. 13Z-Crocin-8-O-β-D-gentiobioside

[0057]

[0058] 8. 13Z-Crocin-8'-O-β-D-gentiobioside

[0059]

[0060] 9. Crocin Mono-β-D-glucopyranoside

[0061]

[0062] This solution also provides a pharmaceutical preparation for treating metabolic syndrome, including a crocin pigment composition and pharmaceutically acceptable excipients. Among them, the pharmaceutical preparation is any one of powder, granule, tablet, emulsion, syrup, aerosol, soft capsule, hard capsule, sterile injection and sterile powder. The administration route of the pharmaceutical preparation is any one of oral administration, nasal inhalation, or parenteral administration.

[0063] Specifically, the components of the crocin pigment composition used in the pharmaceutical preparation and their preparation methods are as described above. In order to enable the pharmaceutical preparation to release the active ingredients rapidly, continuously and for a long time, the pharmaceutical preparation of the present invention can be manufactured according to the conventional methods disclosed in those in this technical field, which will not be elaborated here.

[0064] Here, the term "pharmaceutically acceptable" means that the compound is physiologically acceptable when administered to humans and does not cause gastrointestinal disorders, dizziness and other allergic reactions or systemic allergic reactions similar to these allergic reactions.

[0065] In the present invention, "pharmaceutically acceptable excipients" include but are not limited to: binders (such as microcrystalline cellulose, alginate, gelatin, and polyvinylpyrrolidone), fillers (such as starch, sucrose, glucose, and anhydrous lactic acid), disintegrants (such as cross-linked PVP, sodium croscarmellose, sodium carboxymethylcellulose cross-linked, and low-substituted hydroxypropyl cellulose), lubricants (magnesium stearate, aluminum stearate, talc, polyethylene glycol, sodium benzoate), wetting agents (such as glycerol), surfactants (such as cetyl alcohol), as well as absorption promoters, flavoring agents, sweeteners, diluents, coating agents, etc.

[0066] The features and properties of the present invention will be further described in detail with reference to the following examples:

[0067] Example 1 Preparation of crocin pigment composition from Gardenia jasminoides

[0068] The crocin pigment composition was prepared by the following steps:

[0069] Step 1: The fruits of Gardenia jasminoides were extracted by solvent extraction to obtain the total extract of Gardenia jasminoides. The steps are as follows:

[0070] Weigh 50 g of Gardenia jasminoides medicinal materials, add 5 - 20 times the amount of 50 - 80% ethanol, soak for 1 - 3 h, reflux and extract for 1 - 3 h, and extract 1 - 3 times. Combine the extraction solutions and concentrate until the alcohol smell disappears. Dilute the concentrated solution with water, centrifuge at 5000 rpm for 5 min, and take the supernatant as the total extract of Gardenia jasminoides.

[0071] Step 2: The above-mentioned total extract of Gardenia jasminoides was separated by macroporous adsorption resin chromatography. The steps are as follows:

[0072] Take the above-mentioned total extract of Gardenia jasminoides as the macroporous resin sample loading solution. After sample loading, collect the liquid, repeat sample loading, with a flow rate of 0.5 - 1.5 BV / h. After sample loading, successively elute with deionized water for 1 - 12 BV, 30% ethanol for 1 - 12 BV, and 70% ethanol for 1 - 12 BV, with an elution flow rate of 0.5 - 1.5 BV / h. Collect the 70% ethanol eluate, concentrate and dry it to obtain each component in the crocin pigment composition.

[0073] Step 3: Mix each component according to the parts by mass in Table 1 to obtain the crocin pigment composition.

[0074] Example 2 Pharmacodynamic evaluation of crocin pigment composition using an MS mouse model induced by 60% high-fat diet combined with intraperitoneal injection of STZ

[0075] 1 Establishment of MS mouse model and administration method

[0076] 1.1 Establishment of MS mouse model:

[0077] Mice were randomly divided into a control group and a model group according to their body weight, and were fed with normal diet and 60% high-fat diet for 12 weeks respectively. Then all mice were fasted but allowed to drink water for 12 h. The mice in the model group were intraperitoneally injected with STZ (50 mg / kg) for three consecutive days, and the blank control group was injected with an equal dose of citrate buffer solution (pH 4.4). Roche blood glucose meter was used to measure the fasting blood glucose every week (mice were fasted but allowed to drink water for 5 h). At week 0, the average fasting blood glucose was 12.976 ± 3.425 mmol / L.

[0078] 1.2 Administration method:

[0079] According to the fasting blood glucose values, the mice in the model group were randomly divided into a model group, a composition group (i.e., saffron pigment composition, 50 mg / kg, the same below) and a rosiglitazone group (10 mg / kg). After 24 h, the mice in the control group and the model group were intragastrically administered with the corresponding dose of 0.05% CMC-Na, and the remaining groups were administered with the corresponding dose of drugs once a day for 5 consecutive weeks. During the administration period, the control group mice were continuously fed with normal diet, and the remaining groups of mice were fed with 60% high-fat diet. Roche blood glucose meter was used to measure the fasting blood glucose of each group of mice every week (fasted but allowed to drink water for 5 h). One day before sacrificing the mice, an oral glucose tolerance test was performed. After the last administration, the mice were fasted but allowed to drink water for 4 h, then the eyeballs were removed to collect blood. The blood samples were allowed to stand at room temperature for 2 h, centrifuged at 3500 rmp for 15 min, and the separated serum was stored frozen at -80°C. The pancreas, liver, kidney and abdominal fat of the mice were taken and stored frozen at -80°C or stored at room temperature in 4% paraformaldehyde for subsequent research.

[0080] 60% high-fat diet (D12492) was purchased from SPF (Beijing) Biotechnology Co., Ltd. The positive control drug rosiglitazone sodium tablets were purchased from Taizhou Group Chongqing Fuling Pharmaceutical Factory Co., Ltd. (National Drug Approval Number: H20041399); trisodium citrate (batch number: 20200401), citric acid (batch number: 20210515) were all purchased from Tianjin Damao Chemical Reagent Factory; streptozotocin (STZ) was purchased from Sigma (product number: S0130-500MG). Preparation: STZ was dissolved in 4°C citrate buffer solution (PH = 4.4) to prepare a solution, which was prepared immediately before use, refrigerated in the dark on ice bath, and used up within 15 min; the saffron pigment composition was prepared into a suspension of 5 mg / ml with 0.5% CMC-Na solution, and rosiglitazone was prepared into a uniform solution of 1 mg / ml with 0.5% CMC-Na solution.

[0081] 2 Detection indexes

[0082] (1) Determination of fasting blood glucose (FBG): At 9:00 am on the 0th, 1st, 2nd, 3rd, 4th, and 5th weeks after administration, the mice in each group were fasted but allowed to drink water. At 2:00 pm, the blood glucose concentration of the tail tip blood of the mice in each group was measured using a Roche blood glucose meter (vital type), which was the fasting blood glucose value.

[0083] (2) Oral glucose tolerance test (OGTT): One day before the end of the experiment, the OGTT experiment was conducted. At 6:00 am, the mice in each group were fasted but allowed to drink water. At 2:00 pm, the mice in each group were gavaged with glucose (2 g / kg). The blood glucose concentration of the mice in each group was measured using a Roche blood glucose meter at 0, 30, 60, 90, and 120 min.

[0084] (3) Determination of fasting serum insulin (FINS), insulin resistance index (Homeostatic Model Assessment of Insulin Resistance, HOMA-IR), and insulin sensitivity index (Insulin sensitivity index, ISI) in MS mice: The insulin content in the mouse serum was determined according to the instructions of the mouse insulin enzyme-linked immunosorbent assay kit (Nanjing Jiancheng Bioengineering Institute). According to the obtained FINS and FBG, the insulin resistance index and insulin sensitivity index of the mice in each group were calculated: HOMA-IR = FBG × FINS / 22.5; ISI = 1 / [FBG × FINS].

[0085] (4) Determination of mouse serum biochemical indexes: The levels of TG, TC, LDL-C, HDL-C, ALT, AST, BUN, and CRE in the mouse serum were measured using a Mindray BS-240 automatic biochemical analyzer and the corresponding assay kits (Shenzhen Mindray Bio-Medical Electronics Co., Ltd.).

[0086] (5) Determination of TG and TC contents in mouse liver: The determination was carried out according to the instructions of the TG and TC test kits (Nanjing Jiancheng Bioengineering Institute).

[0087] (6) Oil Red O staining of mouse liver: After the last administration, mice in each group were fasted but allowed to drink water for 4 h. The livers of the mice were removed and fixed in 4% paraformaldehyde. After 24 h, the fresh 4% paraformaldehyde was replaced and the samples were stored at room temperature for further use. The fixed mouse tissues were taken out, the surface liquid was blotted dry, and then embedded in OCT. Serial sections with a thickness of about 8 μm were made on the largest surface of the mouse liver using a cryostat. Fixation of fresh frozen sections: The frozen sections were allowed to warm to room temperature and air-dried, then fixed in the fixing solution for 15 min, washed with distilled water and air-dried. The sections were immersed in Oil Red staining solution for 10 min (covered to avoid light). The sections were taken out and left for 3 s, then placed successively in two cylinders of 60% isopropanol for differentiation for 3 s and 5 s respectively. The sections were then successively immersed in two cylinders of distilled water for 10 s each. The sections were taken out and left for 3 s, then counterstained with hematoxylin for 5 min, and then successively immersed in three cylinders of distilled water for 5 s, 10 s, and 30 s respectively. Differentiated with the differentiating solution (using 60% alcohol as the solvent) for 8 s, washed successively in two cylinders of distilled water for 10 s each, blued with the bluing solution for 1 s, and the sections were gently immersed in two cylinders of tap water for 5 s and 10 s respectively. The staining effect was examined under a microscope; sealed with glycerol gelatin mounting medium; observed under an upright optical microscope and images were collected for analysis.

[0088] (7) Determination of mouse organ index: After the last administration, mice in each group were fasted but allowed to drink water for 4 h. The body weights of the mice were recorded. The livers and kidneys of the mice were taken out, and after blotting the peritoneal fluid on the surface of the organs with filter paper, the weights of each organ were measured and the organ index was calculated (organ index = (organ weight ÷ mouse body weight) × 100%).

[0089] (8) Hematoxylin-eosin staining (HE staining) of mouse pancreas, liver, and kidney: After the last administration, the mice in each group were fasted for 4 h without water deprivation. The pancreas, liver, and kidney of the mice were taken and fixed in 4% paraformaldehyde. After 24 h, the fresh 4% paraformaldehyde was replaced and stored at room temperature for standby. The fixed mouse tissues were taken out, the surface liquid was blotted dry, and then paraffin embedding was carried out. A pathological slicer was used to make continuous sections on the largest surface of each mouse tissue, with a thickness of about 4 μm. The mouse sections were pasted, baked, dewaxed to water (the sections were successively placed in xylene I for 20 min - xylene II for 20 min - environmentally friendly dewaxing solution III for 10 min - absolute ethanol I for 5 min - absolute ethanol II for 5 min - 75% alcohol for 5 min - washed with distilled water); the sections were placed in hematoxylin staining solution for 5 min and washed with distilled water; differentiated with differentiating solution for 10 s and washed with distilled water; blued with bluing solution for 5 min and rinsed with running water for 30 min; the sections were successively dehydrated in gradient alcohols of 85% and 95% for 5 min each, and then stained in eosin staining solution for 5 min; the sections were successively placed in absolute ethanol I for 5 min - absolute ethanol II for 5 min - absolute ethanol III for 5 min - xylene I for 5 min - xylene II for 5 min for clearing, and sealed with neutral balsam; observed under an upright optical microscope and images were collected for analysis. Pathological scoring was performed on the HE staining results of the mouse pancreas, liver, and kidney. The scoring criteria were as follows: Scoring was performed on pancreatic edema, cell necrosis, hemorrhage, and inflammatory cell infiltration; according to the recognized NAS standard, scoring was performed on hepatocyte steatosis, intrahepatic lobular inflammation, and the degree of hepatocyte ballooning degeneration; the Paller standard was used to score renal tubular dilation, brush border injury, interstitial inflammation, and the situation of cell exfoliation and necrosis in the renal tubular lumen.

[0090] 3 Statistical analysis

[0091] The experimental results were statistically analyzed using Graphpad Prism 8.0 software. The experimental data were expressed as Mean±SEM. The difference between different groups was compared by one-way analysis of variance (One-way ANOVA), and then Turkey test was used. P<0.05 was considered to indicate a statistically significant difference.

[0092] 4 Analysis of experimental results

[0093] 4.1 Crocus pigment composition reduces fasting blood glucose in MS mice

[0094] The fasting blood glucose of the mice was measured at weeks 0, 1, 2, 3, 4, and 5 after administration, as Figure 1As shown, with the extension of the administration time, the fasting blood glucose value of the mice in the composition group gradually decreased. At the 5th week of administration, the fasting blood glucose value of the mice in the composition group was significantly lower than that of the mice in the model group. The hypoglycemic effect of the positive drug rosiglitazone was relatively significant, and the fasting blood glucose value of the mice was significantly decreased at the 1st week of administration. The above results indicate that the saffron pigment composition can decrease the fasting blood glucose value of MS mice, but its hypoglycemic effect is weaker than that of rosiglitazone.

[0095] 4.2 Saffron pigment composition increases the oral glucose tolerance of MS mice

[0096] OGTT is a classic method for judging whether there is abnormal glucose metabolism in the body. As Figure 2 shown in A, 30 min after intragastric administration of glucose, the blood glucose levels of the mice in each group except the control group mice increased significantly within 30 min and then began to decrease; the blood glucose level of the mice in the rosiglitazone group quickly dropped to the normal level, while the blood glucose level of the mice in the composition group decreased more slowly, but the blood glucose level was always lower than that of the mice in the model group; the area under the OGTT curve can be used to evaluate the glucose tolerance level of MS mice. The results show that the area under the curve of the model group mice increased significantly compared with that of the control group mice, while the area under the curve of the mice decreased significantly after administration of the saffron pigment composition, but the decrease amplitude was smaller than that of rosiglitazone ( Figure 2 B). The above results indicate that the saffron pigment composition can improve the abnormal glucose metabolism of MS mice, but its effect is weaker than that of rosiglitazone.

[0097] 4.3 Saffron pigment composition increases the serum insulin content of MS mice

[0098] In this experiment, the ELISA method was used to measure the serum insulin content of MS mice, and the insulin resistance index and insulin sensitivity index were calculated to judge the insulin resistance degree of MS mice. As Figure 3 shown, compared with the control group mice, the model group mice showed obvious insulin resistance. The saffron pigment composition and rosiglitazone could significantly increase the serum insulin content of the mice ( Figure 3 A), significantly decrease the insulin resistance index of the mice ( Figure 3 B), and significantly improve the serum insulin sensitivity of the mice ( Figure 3 C). The above results indicate that the saffron pigment composition can improve the insulin resistance state of MS mice, but its effect is weaker than that of rosiglitazone.

[0099] 4.4 Saffron pigment composition decreases the blood lipid level of MS mice

[0100] The blood lipid level of MS mice was evaluated by measuring the contents of total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) in the serum of the mice. As Figure 4As shown, it was found that the mice in the model group showed obvious dyslipidemia, that is, the contents of TC, TG, and LDL-C in the serum were significantly higher than those in the control group of mice. The saffron pigment composition could significantly reduce the levels of TC, TG, and LDL-C in the serum of mice, and rosiglitazone could significantly reduce the levels of TC and LDL-C in the serum of mice. The above results indicate that the saffron pigment composition can regulate the dyslipidemia in MS mice, and its effect is comparable to that of rosiglitazone.

[0101] 4.5 Saffron pigment composition reduces lipid accumulation in the liver of MS mice

[0102] In this experiment, the lipid accumulation in the liver of mice was evaluated by detecting the contents of TG and TC in the liver of mice and oil red O staining. The results showed that the contents of TG and TC in the liver of the model group of mice were significantly higher than those in the control group of mice. Administration of the saffron pigment composition by gavage could significantly reduce the contents of TG and TC in the liver of mice, and rosiglitazone could significantly reduce the content of TC in the liver of mice, but had no obvious effect on the content of TG ( Figure 5 A, B). The results of oil red O staining showed that compared with the control group, a large number of lipid droplets were formed in the liver tissue of the model group of mice, which were largely accumulated around the hepatic sinusoids, and the liver tissue was accompanied by obvious vacuolar degeneration. After treatment with the saffron pigment composition, the lipid droplet aggregation and vacuolar degeneration around the hepatic sinusoids of MS mice were significantly reduced, and rosiglitazone also reduced the area of lipid droplet aggregation and vacuolar degeneration to a certain extent ( Figure 5 C). The above results indicate that the saffron pigment composition can reduce the excessive lipid accumulation in the liver of MS mice, and its effect is stronger than that of rosiglitazone.

[0103] 4.6 Effect of saffron pigment composition on the organ indices of MS mice

[0104] The damage condition was preliminarily judged by calculating the liver and kidney indices of mice. As Figure 6 shown, compared with the control group of mice, the liver and kidney indices of the model group of mice were significantly increased. The saffron pigment composition could significantly reduce the liver index of mice ( Figure 6 A), and also had a certain alleviating effect on the enlargement of the kidneys of mice ( Figure 6 B), but there was no statistical significance. Rosiglitazone had no effect on the enlargement of the liver of mice, could significantly reduce the kidney index of mice, and the kidney index was lower than that of the control group of mice, suggesting that it may have certain nephrotoxicity.

[0105] 4.7 Effect of saffron pigment composition on biochemical indices related to liver and kidney injury in MS mice

[0106] Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are sensitive indicators of liver cell injury, and creatinine (CRE) and blood urea nitrogen (BUN) are sensitive indicators reflecting kidney function. In this experiment, the above indicators were detected to evaluate the effect of saffron pigment composition on liver and kidney injury in MS mice. As Figure 7 shown, the levels of serum ALT, AST, CRE, and BUN in the MS group mice were significantly increased, indicating liver and kidney injury in the mice. After treatment with saffron pigment composition, the levels of serum ALT, AST, and BUN in the mice were significantly decreased, and the CRE level showed no obvious change. While rosiglitazone could significantly increase the serum CRE level in MS mice, suggesting again that it may have certain nephrotoxicity. The above results indicate that saffron pigment composition has a certain improvement effect on MS-related liver and kidney injury, and its effect is stronger than that of rosiglitazone.

[0107] 4.8 Effect of saffron pigment composition on pathological injury of liver, kidney and pancreas in MS mice

[0108] The pathological injury conditions of the liver, kidney and pancreas in MS mice were evaluated by HE staining. As Figure 8 shown in A, treatment with saffron pigment composition could improve the inflammatory infiltration and vacuolar fatty degeneration in the liver of MS mice, and rosiglitazone had no improvement on liver pathological injury. The degrees of hepatocyte steatosis, intrahepatic lobular inflammation and hepatocyte ballooning degeneration were scored. After treatment with saffron pigment composition, the pathological score of the liver in MS mice was significantly decreased, while rosiglitazone had no obvious effect on it ( Figure 8 D). Figure 8 As shown in B, nodular sclerosis changes and unclear boundaries could be seen in the glomeruli of the MS group mice, the brush border structure inside the renal tubules disappeared, the renal tubular epithelial cells had vacuolar changes and necrosis and exfoliation, and some renal tubules were dilated, and obvious inflammatory cell infiltration was seen in the renal interstitium. After treatment with saffron pigment composition, the above-mentioned kidney pathological injury could be improved to a certain extent, the glomerular structure was clear but some renal tubules were significantly dilated, and rosiglitazone had no improvement effect on kidney injury. The degrees of renal tubular dilation, brush border injury, interstitial inflammation, and cell exfoliation and necrosis in the renal tubular lumen were scored. Saffron pigment composition significantly decreased the pathological score of kidney injury in MS mice ( Figure 8 E). Figure 8 As shown in C, the structure of the pancreatic tissue in the model group mice was disordered, the islet morphology was irregular and the boundaries were unclear, the number of cells in the islets was small and the arrangement was irregular, and the islet capillaries were significantly dilated. After treatment with saffron pigment composition and rosiglitazone, the islet tissue structure was clear and the capillary dilation was alleviated. The scores of pancreatic edema, cell necrosis, bleeding, and inflammatory cell infiltration in each group of mice were found. Both saffron pigment composition and rosiglitazone could reduce the pathological injury score of the pancreas in MS mice ( Figure 8F). The above results indicate that the crocinoid composition can improve the pathological damage of the liver, kidney and pancreas in MS mice, and its improvement effect on liver and kidney damage is significantly better than that of rosiglitazone.

[0109] 5. Summary of the experiment

[0110] In this experiment, an MS mouse model established by a 60% high-fat diet combined with intraperitoneal injection of streptozotocin was used to evaluate the therapeutic effect of the test substance, the crocinoid composition, on MS. The experimental results show that this composition can reduce the fasting blood glucose level of MS mice, improve glucose tolerance and insulin resistance, indicating that the crocinoid composition has good hypoglycemic activity, but its effect is not as good as that of rosiglitazone; the crocinoid composition can regulate dyslipidemia in mice and reduce lipid accumulation in the liver of mice, indicating that the crocinoid composition has obvious lipid-lowering activity, and its effect is better than that of rosiglitazone; in addition, the crocinoid composition has a particularly significant therapeutic effect on liver and kidney damage in MS mice, which is better than that of rosiglitazone. This shows that the crocinoid composition has a significant therapeutic effect on the MS mouse model induced by a high-fat diet combined with STZ, indicating that this composition has certain anti-MS activity, suggesting that the research and development of the crocinoid composition as an anti-MS drug has broad application prospects.

[0111] Example 3: Protective effects of the crocinoid composition and monomers in Gardenia jasminoides Ellis on insulin-induced insulin resistance in HepG2 cells

[0112] 1 Cell culture

[0113] HepG2 cells were cultured in MEM medium containing 10% FBS and 1% penicillin-streptomycin, and cultured and passaged in an incubator at 37 °C and 5% CO2. Cells in the logarithmic growth phase with good growth conditions were taken for subsequent experiments.

[0114] 2 Determination of the ability of the crocinoid composition and monomers in Gardenia jasminoides Ellis to improve insulin resistance in HepG2 cells

[0115] Take HepG2 cells in the logarithmic growth phase, dilute them with MEM to a cell suspension of 1x10 per milliliter, seed 100 μL per well into a 96-well plate, and perform starvation treatment for 12 h. The experiment was set up with a blank group, a model group, a positive group (1.5 mmol·L 5 metformin hydrochloride), and groups of crocinoid compositions / monomers at different concentrations. Discard the old culture medium, and add DMEM high-glucose medium (containing 10% FBS + 1% penicillin-streptomycin) containing 0.8 μmol·L -1 insulin to all groups except the blank group, and induce culture in an incubator at 37 °C and 5% CO2 for 24 h. The absorbance value was measured at 450 nm by the CCK-8 method and the cell survival rate was calculated ( -1 ), and Figure 9, Figure 10 )。In addition, after the same grouping and administration method, the cell supernatant was taken, and a kit was used to detect and calculate glucose uptake. The results are shown in Table 2.

[0116] 3 Experimental Results

[0117] In this experiment, the CCK8 method was used to detect the effects of saffron pigment compositions at different concentrations (1, 10, 25, 50, 100 μg / mL) and monomers (0.1, 1, 10 μM) on the viability of IR-HepG2 cells. As Figures 9 - 10 shown, compared with the CON group, after treatment with saffron pigment compositions / monomers at different concentrations for 24 h, no cytotoxicity was exhibited, and there was no significant effect on the proliferation and viability of IR-HepG2 cells. In addition, as shown in Table 2, the glucose uptake rate of the model group was 68.8 ± 4.4%, and compared with the CON group, the glucose uptake rate of the MOD group was significantly decreased (P < 0.001), indicating that the HepG2 cell IR model was successfully established. The glucose consumption of IR-HepG2 cells after treatment with saffron pigment compositions / monomers at different concentrations increased to varying degrees. Among them, the increase in the uptake rate of the high-dose composition was the most significant (P < 0.001), which was comparable to the effect of the positive drug. It shows that saffron pigment compositions can effectively promote the glucose consumption of insulin-resistant HepG2 cells and improve the disorder of glucose metabolism.

[0118] Table 2 Effects of saffron pigment compositions and monomers on glucose consumption of IR-HepG2 cells

[0119]

[0120] Note: Compared with the control group, ### P < 0.001; compared with the model group * P < 0.05, ** P < 0.01, *** P < 0.01

[0121] The above are only examples of the present invention. Specific technical solutions and / or common knowledge such as characteristics well known in the art are not described in detail here. It should be noted that for those skilled in the art, without departing from the technical solution of the present invention, several deformations and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the implementation effect of the present invention and the practicality of the patent. The protection scope required by this application should be based on the content of its claims, and the specific implementation manners described in the specification can be used to explain the content of the claims.

Claims

1. Use of a saffron pigment composition in the preparation of a preparation for treating or improving metabolic syndrome, characterized in that: The saffron pigment composition is obtained by extracting from the traditional Chinese medicine Gardenia jasminoides Ellis; it includes the following raw materials in parts by mass: 30-40 parts of crocetin bis-β-D-gentiobioside, 8-12 parts of crocetin-β-D-glucopyranosyl-β-D-gentiobioside, 2-3 parts of crocetin bis-β-D-glucopyranoside, 12-18 parts of 13Z-crocetin bis-β-D-gentiobioside, 2-3 parts of neocrocin B, 15-20 parts of crocetin mono-β-D-gentiobioside, 2-3 parts of crocetin mono-β-D-glucopyranoside, 1-3 parts of 13Z-crocetin-8-O-β-D-gentiobioside, 1-2 parts of 13Z-crocetin-8'-O-β-D-gentiobioside.

2. A preparation method of a saffron pigment composition, characterized in that: It includes the following steps: Step 1: Extract Gardenia jasminoides Ellis fruits by solvent extraction method to obtain the total Gardenia jasminoides Ellis extract; Step 2: Separate the above-mentioned total Gardenia jasminoides Ellis extract by macroporous adsorption resin chromatography, the eluent is water and 30-95Vol% ethanol, collect the ethanol elution part of 65-75Vol%, concentrate to obtain each component in the saffron pigment composition; Step 3: Refer to the mass parts of each component in the saffron pigment composition described in claim 1, and mix each component in step 2 to obtain the saffron pigment composition.

3. The preparation method according to claim 2, characterized in that: In step 1, the solvent extraction method includes: any one or more of decoction method, heating reflux extraction method, ultrasonic oscillation extraction method, percolation method and impregnation method.

4. The preparation method according to claim 3, characterized in that: When the heating reflux extraction method is used for extraction, it includes the following steps: using 50-80% ethanol as the extraction solvent, the material-liquid ratio of Gardenia jasminoides Ellis and 50-80% ethanol (that is, the mass ratio of raw material to extraction solvent) is 1:5-20, soak for 1-3h and then reflux extract for 1-3h, extract 1-3 times. After extraction, filter the extract and remove the solvent to obtain the total Gardenia jasminoides Ellis extract.

5. The preparation method according to claim 2, characterized in that: In step 2, the steps of separating the total Gardenia jasminoides Ellis extract by macroporous adsorption resin chromatography are as follows: use the above-mentioned total Gardenia jasminoides Ellis extract as the macroporous resin sample solution, after sample loading is completed, collect the liquid, repeat sample loading, the flow rate is 0.5-1.5BV / h, after sample loading is completed, sequentially use deionized water to elute 1-12BV, 30% ethanol to elute 1-12BV, 70% ethanol to elute 1-12BV, and the elution flow rate is 0.5-1.5BV / h. Collect the 70% ethanol eluate, concentrate and dry to obtain each component in the saffron pigment composition.

6. A pharmaceutical preparation for treating metabolic syndrome, characterized in that: It includes the saffron pigment composition prepared by the method described in any one of claims 2-5, and pharmaceutically acceptable excipients.

7. The pharmaceutical preparation according to claim 6, characterized in that: The pharmaceutical preparation is any one of powder, granule, tablet, emulsion, syrup, aerosol, soft capsule, hard capsule, sterile injection and sterile powder.

8. The pharmaceutical preparation according to claim 6, characterized in that: The administration route of the pharmaceutical preparation is any one of oral administration, nasal inhalation, or parenteral administration.

9. A method for constructing an animal experimental model of type II diabetes, characterized in that: The experimental model is used to verify the role of the above-mentioned pharmaceutical preparation in the treatment of metabolic syndrome, and it is constructed by using 60% high-fat diet combined with intraperitoneal injection of streptozotocin.

10. An animal experimental model for type II diabetes, characterized in that: The MS mouse model constructed by the construction method described in claim 9 is adopted.

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