Medicinal composition extracted from cynomorium songaricum and preparation method and health food thereof

Abstract

The invention discloses a cynomorium songaricum extract pharmaceutical composition as well as a preparation method and health-care food thereof. A cynomorium songaricum plant material is extracted with an ethanol solution to obtain an extracting solution, the extracting solution comprises multiple cynomorium songaricum triterpenoids, and the multiple cynomorium songaricum triterpenoids jointly act to have remarkable effects on reducing fasting blood-glucose, improving glucose tolerance, improving insulin sensitivity and reducing white fat accumulation; insulin resistance can be improved by inhibiting inflammatory gene expression and reducing inflammatory reaction. The pharmaceutical composition can be used for preventing and treating type 2 diabetes mellitus.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine, and more specifically, to a Cynomorium songaricum extraction pharmaceutical composition, a preparation method thereof, and a health food. Background Art

[0002] Type 2 diabetes mellitus (T2DM) is a chronic metabolic disease. Insulin resistance is the fundamental pathophysiological mechanism of T2DM, and the imbalance of glucose metabolism homeostasis caused by abnormal increase in fat is one of the pathological bases of T2DM. The incidence of T2DM is increasing continuously and has become a serious public health problem.

[0003] Although chemical drug treatment can control the condition of T2DM to a certain extent, it is often accompanied by toxic and side effects and has a high production cost. Therefore, the development of natural compounds with hypoglycemic activity for intervening in T2DM has become a research hotspot. As one of the representatives of natural active compounds, food-derived triterpenoids have shown improvement effects on chronic metabolic diseases in a large number of past studies.

[0004] Cynomorium songaricum is a rare and endangered medicinal plant, which has the effects of tonifying the kidney and assisting yang, benefiting essence and blood, moistening the intestines and relieving constipation, etc., and is commonly used in traditional Chinese, Mongolian, and Tibetan medicines. Cynomorium songaricum triterpenoids are richly distributed in Cynomorium songaricum. In recent years, the research on Cynomorium songaricum extracts has focused on product preparation, component identification, and bioavailability, but the preventive and therapeutic effects of Cynomorium songaricum triterpenoids in preparing drugs on type 2 diabetic animal models and their effects are not clear. Summary of the Invention

[0005] The purpose of the present invention is to overcome the above-mentioned defects existing in the prior art, and to provide a Cynomorium songaricum extraction pharmaceutical composition, a preparation method thereof, and a health food.

[0006] In the first aspect, the present invention provides a Cynomorium songaricum extraction pharmaceutical composition for preparing a drug for preventing and treating type 2 diabetes, or for preparing a drug for preventing and treating diseases of abnormal glucose and lipid metabolism, or for preparing a drug for preventing and treating diseases mediated by oxidative stress and / or free radicals;

[0007] The pharmaceutical composition includes Cynomorium songaricum triterpenoids; the extraction method of the Cynomorium songaricum triterpenoids includes: extracting Cynomorium songaricum plant materials with an ethanol solution to obtain an extract, the extract includes the Cynomorium songaricum triterpenoids, and the pharmaceutical composition includes the extract.

[0008] In the second aspect, the present invention provides an extraction method of Cynomorium songaricum triterpenoids, including: extracting Cynomorium songaricum plant materials with an ethanol solution to obtain an extract, the extract includes the Cynomorium songaricum triterpenoids.

[0009] In a third aspect, the present invention provides a health food for preventing type 2 diabetes, or for preventing disorders of glucose and lipid metabolism, or for preventing diseases mediated by oxidative stress and / or free radicals, including cynomorium triterpenoids;

[0010] The extraction method of the cynomorium triterpenoids includes: extracting cynomorium plant materials with an ethanol solution to obtain an extract, and the extract includes the cynomorium triterpenoids.

[0011] Implementing the embodiments of the present invention will have the following beneficial effects:

[0012] In the embodiments of the present invention, cynomorium plant materials are extracted with an ethanol solution to obtain an extract, and the extract includes various cynomorium triterpenoids. The combined action of various cynomorium triterpenoids has significant effects in reducing fasting blood glucose, improving glucose tolerance, enhancing insulin sensitivity, and reducing white fat accumulation; insulin resistance can be improved by inhibiting the expression of inflammatory genes and reducing the inflammatory response. The pharmaceutical composition can be used for preventing and treating type 2 diabetes. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0014] Among them:

[0015] Figure 1 is a structural formula diagram of cynomorium triterpenoids in the present invention.

[0016] Figure 2 is the test result of mice in different treatment groups in the oral glucose tolerance test of the present invention. Among them, Figure A is a graph of blood glucose level changing with time, and Figure B is a graph for estimating the degree of impaired glucose tolerance by calculating the area under the curve of the blood glucose concentration-time graph.

[0017] Figure 3 is the test result of mice in different treatment groups in the insulin tolerance test of the present invention. Among them, Figure A is a graph of blood glucose level changing with time, and Figure B is a graph for estimating the degree of impaired insulin sensitivity by calculating the area under the curve of the blood glucose concentration-time graph.

[0018] Figure 4 is the measurement result of the body weight of mice in different treatment groups of the present invention over time.

[0019] Figure 5 is the measurement result of the fat weight of different parts of mice in different treatment groups of the present invention.

[0020] Figure 6 Test results of mice in different treatment groups in the blood lipid level test of the present invention. Among them, Figure A is the change graph of total triglyceride level, Figure B is the change graph of total cholesterol level over time, and Figure C is the change graph of high-density lipoprotein cholesterol / low-density lipoprotein cholesterol level.

[0021] Figure 7 It is the change graph of different treatment groups of the present invention at the levels of multiple biomarkers. Among them, Figure A is the change graph of adiponectin level, Figure B is the change graph of tumor necrosis factor α level, Figure C is the change graph of resistin level, and Figure D is the change graph of insulin level.

[0022] Figure 8 It is the influence graph of different treatment groups of the present invention on the expression levels of multiple mitochondrial genes. Among them, Figure A is the nrf1 mitochondrial gene, Figure B is the ppargc1a mitochondrial gene, Figure C is the tfam mitochondrial gene, Figure D is the twnk mitochondrial gene, and Figure E is the ucp1 mitochondrial gene.

[0023] Figure 9 It is the influence graph of different treatment groups of the present invention on the expression levels of multiple inflammation-related genes. Figure A is the CCR-5 inflammation gene, Figure B is the CXCL-10 inflammation gene, Figure C is the CCL-2 inflammation gene, Figure D is the CCR-2 inflammation gene, and Figure E is the tnf-α inflammation gene.

[0024] Figure 10 It is the histological change graph of different treatment groups of the present invention.

[0025] Figure 11 It is another influence graph of different treatment groups of the present invention on the expression levels of multiple mitochondrial genes. Among them, Figure A is the ucp1 mitochondrial gene, Figure B is the ppargc1a mitochondrial gene, Figure C is the tfam mitochondrial gene, Figure D is the twnk mitochondrial gene, and Figure E is the nrf1 mitochondrial gene. Detailed implementation manners

[0026] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts belong to the protection scope of the present invention.

[0027] The embodiments of the present invention provide a Cynomorium songaricum extraction pharmaceutical composition for preparing a drug for preventing and treating type 2 diabetes, or for preparing a drug for preventing and treating diseases of abnormal glycolipid metabolism, or for preparing a drug for preventing and treating diseases mediated by oxidative stress and / or free radicals.

[0028] The pharmaceutical composition comprises cynomorium triterpenoids; the extraction method of cynomorium triterpenoids includes: extracting cynomorium plant materials with an ethanol solution to obtain an extract, the extract includes cynomorium triterpenoids, and the pharmaceutical composition includes the extract.

[0029] Furthermore, the extract is chromatographically separated through a macroporous resin column to obtain cynomorium triterpenoids.

[0030] The cynomorium triterpenoids in this example include: 15α-hydroxy-3-oxo-urs-1,12-diene-28-acid, 3β-hydroxy-D_C-isolup-7-ene-28-acid, 3β-methylmalonyl urs-12-ene-28-acid, 3β-methylmalonyloxy olean-12-ene-28-acid, inophyllolide, ursolic acid, 3β-hydroxy olean-11,13(18)-diene-28-acid, oleanolic acid, 3β-hydroxy urs-20-ene-28-acid, δ-oleanolic acid, euscaphic acid, ferulol, cynomorium terpene, maslinic acid, isocoumaric acid, 2α-O-trans-p-hydroxycinnamoyl maslinic acid, 3β-O-trans-p-coumaroyl maslinic acid, p-coumaric acid, and 2α-hydroxy-3β-O-cis-p-coumaroyl urs-12-ene-28-acid.

[0031] Furthermore, the extraction is reflux extraction. Specifically, the dried stems of cynomorium are reflux extracted with an ethanol solution to obtain an extract; the extract is filtered and concentrated to obtain a concentrated extract; the concentrated extract includes cynomorium triterpenoids, and the concentrated extract is chromatographically separated through a macroporous resin column to obtain cynomorium triterpenoids.

[0032] Furthermore, the extraction time is 0.5h to 1.5h. When the extraction time is too short, the extraction is incomplete; while when the extraction time is too long, more impurities will be extracted. Preferably, the extraction time is 1h.

[0033] Furthermore, the concentration of the ethanol solution is 50% to 95%. When the concentration of the ethanol solution is too high, the polarity of the solvent may be reduced, thus affecting the solubility of triterpenoids and resulting in a decrease in the extraction rate. While when the concentration of the ethanol solution is too low, it affects the sufficient dissolution of triterpenoids and also affects the extraction effect.

[0034] Furthermore, the effective dose in the pharmaceutical composition is 40mg / kg to 120mg / kg.

[0035] In the embodiments of the present invention, Cynomorium songaricum plant materials are extracted with an ethanol solution to obtain an extract. The extract contains various Cynomorium songaricum triterpenoids. The combined action of these various Cynomorium songaricum triterpenoids has a significant effect on reducing fasting blood glucose, improving glucose tolerance, enhancing insulin sensitivity, and reducing white fat accumulation; it can improve insulin resistance by inhibiting the expression of inflammatory genes and reducing the inflammatory response. The pharmaceutical composition can be used for preventing and treating type 2 diabetes.

[0036] In a specific embodiment, the pharmaceutical composition further comprises excipients. The Cynomorium songaricum triterpenoids in the pharmaceutical composition are used as active ingredients for preventing or treating diseases, and the excipients are used to improve the physical, chemical, or biological properties of the drug. Exemplarily, the excipients may include fillers, binders, disintegrants, lubricants, preservatives, or flavoring agents.

[0037] The embodiments of the present invention also provide a health food for preventing type 2 diabetes, or for preventing diseases of abnormal glycolipid metabolism, or for preventing diseases mediated by oxidative stress and / or free radicals, which is characterized by comprising Cynomorium songaricum triterpenoids;

[0038] The extraction method of Cynomorium songaricum triterpenoids comprises: extracting Cynomorium songaricum plant materials with an ethanol solution to obtain an extract, and the extract contains Cynomorium songaricum triterpenoids.

[0039] The following are specific embodiments.

[0040] Example 1

[0041] (1) Grind the dry stems of Cynomorium songaricum to increase the extraction efficiency.

[0042] (2) According to the ratio of the weight to volume of Cynomorium songaricum and the ethanol solution being 1:8, use a 50% ethanol solution to carry out reflux extraction at 70°C for 1 hour. The extraction process is repeated twice to ensure the full extraction of triterpenoids in Cynomorium songaricum. After combining the extracts obtained from the two extractions, filter and concentrate under reduced pressure through a rotary evaporator to remove the solvent and concentrate the target compound to obtain a concentrated extract.

[0043] (3) Chromatographically separate the concentrated extract through an AB-8 macroporous resin column. Use a 70% ethanol solution aqueous solution as the eluent, and separate Cynomorium songaricum total triterpenes (CST) through the chromatographic column. CST contains various Cynomorium songaricum triterpenoids.

[0044] Comparative Example 1

[0045] Oleanolic acid is used as Comparative Example 1.

[0046] Comparative Example 2

[0047] Ursolic acid was used as Comparative Example 2.

[0048] Test Example

[0049] The total triterpenoids of Cynomorium songaricum extracted in Example 1 were subjected to component identification. First, in the dissolution step, the total triterpenoid extract of Cynomorium songaricum was dissolved in methanol to prepare a sample solution to be tested. Subsequently, in the separation step, a Thermo Scientific Hypersil GOLD chromatographic column (size: 2.1×100 mm, particle size: 1.9 μm) was used for the separation of compounds. The analytical system used was the UHPLC-Q-Exactive Orbitrap MS / MS system, which is an integration of the DIONEX Ultimate 3000 HPLC system and the Q-Exactive Focus Orbitrap MS mass spectrometer of Thermo Fisher Scientific to achieve high-precision compound analysis.

[0050] Furthermore, in the mass spectrometry coupling step, the SCIEX ExionLC HPLC system was combined with the Triple Quad TM 5500 QTRAP Ready mass spectrometer, which was equipped with a Turbo Spray ion source for quantitative analysis of the identified compounds. In the HPLC method, a flow rate of 0.1 mL / min was used, and the mobile phase consisted of Solvent A (aqueous solution containing 0.1% formic acid) and Solvent B (methanol). The gradient elution program was set to optimize the separation effect of the compounds. Specifically, from 0 minute to 4 minutes, the proportion of Solvent B increased from 10% to 35%, then from 5 minutes to 10 minutes it increased to 50%, and then from 10 minutes to 12 minutes it increased to 95%, and the proportion of Solvent B was maintained at 95% until 13.5 minutes, and finally it was restored to the initial 10% Solvent B condition from 13.5 minutes to 14 minutes. Through this gradient elution program, a comprehensive analysis and identification of the compounds in the CST extract can be achieved, providing an efficient and accurate analytical method for the chemical characteristics of Cynomorium songaricum triterpenoids.

[0051] Table 1 shows the compounds contained in the total triterpenoids of Cynomorium songaricum detected in the negative ion mode by liquid chromatography-mass spectrometry (LC-MS / MS), including retention time (RT), compound name, molecular formula, calculated molecular weight (Calc.MW), mass-to-charge ratio (m / z), maximum peak area (Area(Max.)), and reference ions.

[0052] Table 1 CST Compounds Detected by LC-MS / MS

[0053]

[0054]

[0055]

[0056] Animal experiment 1

[0057] In this experiment, 8-week-old male C57BL / 6 mice were randomly divided into a control group (Control), a model group (Model), a metformin group (Metformin), a high-dose CST group (CSTH group), and a low-dose CST group (CSTL group), with 10-15 mice in each group.

[0058] The mice in the control group were only fed a normal diet (ND), while the mice in the model group, metformin group, CSTH group, and CSTL group were fed a high-fat diet (HFD). In addition, the mice in the metformin group, CSTH group, and CSTL group were respectively given the corresponding drugs by oral gavage every day: metformin 10 mg / kg, CST 120 mg / kg, 40 mg / kg, for 8 weeks.

[0059] Test results of animal experiment 1

[0060] 1) Oral glucose tolerance test (OGTT)

[0061] After 8 weeks of the experiment, the mice were fasted for 6 hours and then given glucose (2 g / kg) by oral gavage. Blood glucose was measured using a blood glucose meter, and the results are shown in Figure 2 A. Blood was collected from the tip of the tail vein at time points of 0, 15, 30, 60, and 120 minutes after glucose administration. The degree of impaired glucose tolerance was estimated by calculating the area under the curve (AUC) of the blood glucose concentration (Blood glucose level) versus time (Time) graph, and the results are shown in Figure 2 B. The asterisks in the figure indicate statistically significant differences, and ** indicates p < 0.01.

[0062] As can be seen from Figure 2 A: Compared with the control group, the mice in the model group, metformin group, CSTH group, and CSTL group had a weakened ability to tolerate glucose and an increase in blood glucose. After 30 min, the blood glucose of the mice in the CSTH group and CSTL group was lower than that of the model group, indicating that CST can improve the glucose tolerance of mice and play a role in lowering blood glucose.

[0063] As can be seen from Figure 2It can be seen that: compared with the control group, the AUC value of the model group increased significantly, indicating that the area under the blood glucose response curve of the model group was larger, meaning that the blood glucose level fluctuated more. After treatment with metformin and different doses of CST, the AUC values all decreased. In particular, the AUC value of the CSTL group decreased significantly compared with the model group and was close to the control group level. This indicates that CST has a significant effect on reducing the area under the blood glucose response curve at higher doses and helps to improve blood glucose control.

[0064] 2) Insulin tolerance test (ITT)

[0065] After 8 weeks of experiment, the mice were fasted for 5 hours and then intraperitoneally injected with insulin (0.5 IU / kg). Blood glucose was measured using a blood glucose meter, and the results are as Figure 3 shown in A. Blood was collected from the tip of the tail vein at time points of 0, 30, 60, 90, and 120 minutes after insulin administration. The degree of impaired insulin sensitivity was estimated by calculating the area under the curve of the blood glucose concentration-time graph, and the results are as Figure 3 shown in B. The asterisks in the figure indicate statistically significant differences, where ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001.

[0066] From Figure 3 A, it can be seen that: compared with the control group, the insulin sensitivity of the mice in the model group, metformin group, CSTH group, and CSTL group decreased and the blood glucose increased. The blood glucose of the mice in the CSTH group and CSTL group was lower than that of the model group. Combining Figure 3 with B, there were significant differences between the CSTH group, CSTL group and the model group, indicating that CST can improve the insulin sensitivity of mice and play a role in reducing blood glucose.

[0067] 3) Body weight measurement

[0068] During the 8-week experiment, the food intake and body weight of the mice were monitored weekly, and the percentage change in body weight of the mice was calculated. The results are as Figure 4 shown.

[0069] From Figure 4 it can be seen that: compared with the control group, the body weights of the mice in the model group, metformin group, CSTH group, and CSTL group increased. The body weights of the mice in the CSTH group and CSTL group were significantly lower than those of the model group, indicating that CST can play a role in reducing body weight.

[0070] 4) Body fat measurement

[0071] At the end of the 8-week experiment, blood samples were collected by cardiac excision after isoflurane anesthesia, and then the mice were sacrificed by cardiac excision. Different fat pads including subcutaneous fat, intestinal fat, and epididymal fat were dissected and weighed, and the results are as Figure 5 .

[0072] As can be seen from Figure 5 : Compared with the control group, the fat weight of the mice in the model group was significantly increased, while in the group treated with CST, especially the CSTH group, the fat weight of the mice was significantly lower than that in the model group, indicating that CST can play a role in reducing fat accumulation.

[0073] 5) Biochemical analysis

[0074] At the end of the 8-week experiment, blood samples were collected by cardiac excision after isoflurane anesthesia, and then the mice were sacrificed by cardiac excision. Muscle and liver samples were dissected and removed for biochemical analysis.

[0075] ① The triglycerides (TG) were measured using an enzyme kit (Nanjing Jiancheng, China), and the results are as Figure 6 shown in A; the total cholesterol (TC) level was measured, and the results are as Figure 6 shown in B; the high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) were measured, and the HDL / LDL ratio was calculated, and the results are as Figure 6 shown in C. The asterisks in the above figures indicate statistically significant differences, where ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001.

[0076] As can be seen from Figure 6 A: Compared with the control group, the total triglyceride level in the model group was significantly increased, while in the group treated with CST, especially the CSTH group, the total triglyceride level was significantly decreased, and there were significant differences between the CSTH group, CSTL group and the model group respectively.

[0077] As can be seen from Figure 6 B: Compared with the control group, the total cholesterol level in the model group was significantly increased, while in the group treated with CST, especially the CSTL group, the total cholesterol level was decreased, and there was a significant difference between the CSTL group and the model group.

[0078] As can be seen from Figure 6 C: Compared with the control group, the HDL-C / LDL-C level in the model group was significantly lower than that in the control group, while the LDL-C level in the CST treatment group increased, and the increase in the CSTH group was more significant.

[0079] These results indicate that CST has potential therapeutic effects in regulating blood lipid levels, especially in reducing harmful cholesterol (such as LDL-C) and increasing beneficial cholesterol (such as HDL-C).

[0080] ② The levels of adiponectin were measured using an enzyme kit (Animaluni, Shanghai, China), and the results are as shown in Figure 7 Figure A; the levels of tumor necrosis factor α (TNF-α) were measured, and the results are as shown in Figure 7 Figure B; the levels of resistin were measured, and the results are as shown in Figure 7 Figure C; the levels of insulin were measured, and the results are as shown in Figure 7 Figure D. The asterisks in the figures indicate statistically significant differences, where * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001.

[0081] As can be seen from Figure 7 Figure A: Compared with the control group, the adiponectin level in the model group was significantly lower than that in the control group, while the adiponectin level in the CST treatment group increased, and the increase in the CSTL group was more significant. There were significant differences between the CSTH group, the CSTL group and the model group respectively. Adiponectin is an endogenous bioactive polypeptide or protein secreted by adipocytes, and has physiological functions such as enhancing insulin sensitivity, regulating glucose and lipid metabolism, anti-inflammatory, and anti-atherosclerotic effects.

[0082] As can be seen from Figure 7 Figure A: The TNF-α level in the model group was significantly higher than that in the control group, indicating an enhanced inflammatory response. The TNF-α level in the CSTL group decreased, showing the inhibitory effect of CST on inflammation. The TNF-α level in the CSTH group decreased further, showing a stronger inhibitory effect of high-dose CST. The TNF-α level in the CSTH group was close to the control group level, showing the effectiveness of high-dose CST. There were significant differences between the model group and the control group, as well as between the CSTL group and the high-dose group and the model group.

[0083] Figure 7 Figure C shows that the resistin level in the model group was significantly higher than that in the control group, indicating an increase in insulin resistance. The resistin level in the CSTL group decreased, showing the improvement effect of CST on insulin resistance. The resistin level in the CSTH group decreased further, showing a stronger improvement effect of high-dose CST. The resistin level in the CSTH group was close to the control group level, showing the effectiveness of high-dose CST. There were significant differences between the model group and the control group, as well as between the CSTL group and the high-dose group and the model group.

[0084] In Figure 7 D, the insulin level in the model group was significantly higher than that in the control group, indicating insulin resistance. The insulin level in the CSTL group decreased, showing the improvement effect of CST on insulin resistance. The insulin level in the CSTH group further decreased, showing a stronger improvement effect of high-dose CST. The insulin level in the CSTH group was close to that in the control group, showing the effectiveness of high-dose CST. There were significant differences between the model group and the control group, and between the CSTL group and the high-dose groups and the model group.

[0085] These results indicate that CST can improve the body's sensitivity to insulin. Among them, high-dose CST shows a more significant effect, suggesting that dose plays an important role in the treatment effect. These findings provide strong evidence for CST as a treatment strategy for improving insulin resistance and related metabolic disorders.

[0086] 6) RT-qPCR (Real-time fluorescence quantitative PCR) analysis

[0087] Quantification was performed using a NanoDrop spectrophotometer (Thermo Scientific, USA), and reverse transcription was performed using a cDNA kit. Primers for nrfl, ppargc1a, tfam, twnk, ucp1, ccr2, ccr5, ccl2, cxcl-10, and tnf-α were purchased from Biosyntech (Suzhou, China). The expression of target genes was measured by semi-quantitative q-PCR using iTaqTM Universal SYBR Green Supermix (Vazyme, China) on a Thermo real-time PCR system. Actin was used as the reference gene for the analysis. The relative expression of target genes was determined using the ddCt method. The results are as Figure 8 、 Figure 9 shown. The asterisks in the figure indicate statistically significant differences, where * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001.

[0088] Figure 8 showed the effects of different treatment groups on the expression levels of mitochondrial genes of nrfl, ppargc1a, tfam, twnk, and ucp1. From Figure 8 it can be seen that: compared with the control group, the gene expression in the model group was significantly decreased, while the CST treatment group could significantly increase the expression levels of these genes, and the gene expression levels were close to or exceeded those of the control group.

[0089] These results indicate that CST can increase the mRNA expression levels of mitochondrial biogenesis genes and effectively regulate mitochondrial biogenesis.

[0090] Figure 9 showed the effects of different treatment groups on the expression levels of inflammation-related genes including ccr2, ccr5, ccl2, cxcl-10, and tnf-α. From Figure 9 it can be seen that: compared with the control group, the expression levels of CCR-5, CXCL-10, CCL-2, TNF-α, and CCR-2 genes in the model group were significantly increased. The metformin and CST treatment groups could significantly reduce the expression levels of CCR-5, CXCL-10, and CCR-2, but had no significant effect on the expression of CCL-2 (ns indicates no significant difference).

[0091] These results indicate that CST can inhibit the expression of inflammatory genes and reduce the inflammatory response to improve insulin resistance.

[0092] 7) Tissue observation

[0093] Mouse adipose paraffin sections were made, and the pathological changes of adipose tissue were observed under a microscope after staining. The results were as Figure 10 shown. Figure 10 The first row is hematoxylin-eosin (HE) staining of liver tissue (Liver), the second row is oil red O staining of white adipose tissue (WAT), and the third row is hematoxylin-eosin (HE) staining of white adipose tissue (WAT).

[0094] From Figure 10 it can be seen that: compared with the control group, the liver tissue of the model group showed obvious fatty degeneration, manifested as the accumulation of a large number of vacuoles and lipid droplets; in adipose tissue, the model group showed significant adipocyte enlargement and inflammatory cell infiltration. Oil red O staining showed an increase in lipid accumulation, and HE staining showed disordered tissue structure. However, after treatment with metformin and CST (120 mg / kg), these pathological changes were significantly improved, manifested as a decrease in the size of adipocytes, a reduction in lipid accumulation, a decrease in inflammatory cell infiltration, and the restoration of tissue structure.

[0095] The results of these histological changes indicate that CST has potential therapeutic effects on improving lipid accumulation, inflammatory response, and tissue structure disorder in the model group.

[0096] In summary, in this embodiment, the total triterpenoid extract of Cynomorium songaricum can not only significantly inhibit the biosynthesis of TG, improve the body's insulin sensitivity, but also increase the mRNA expression levels of mitochondrial biogenesis genes, effectively regulate mitochondrial biogenesis, and CST can be used as a therapeutic strategy to improve insulin resistance and related metabolic disorders, thereby inhibiting the occurrence of type 2 diabetes. Therefore, the Cynomorium songaricum triterpenoid extract prepared by the present invention can be applied to improve type 2 diabetes, prevent and treat glycolipid metabolism disorders, and prevent and treat diseases mediated by oxidative stress and / or free radicals.

[0097] Animal experiment 2

[0098] In this experiment, 8-week-old male C57BL / 6 mice were randomly divided into a control group (Control), a model group (Model), a metformin group (Metformin), a high-dose oleanolic acid group (OAH group), a low-dose oleanolic acid group (OAL group), a high-dose ursolic acid group (UAH group), a low-dose ursolic acid group (UAL group), a high-dose CST group (CSTH group), and a low-dose CST group (CSTL group), with 10 - 15 mice in each group.

[0099] The mice in the control group were only fed a normal diet (ND), while the mice in the model group, metformin group, OAH group, OAL group, UAH group, UAL group, CSTH group, and CSTL group were fed a high-fat diet (HFD). In addition, the mice in the metformin group, UAH group, UAL group, CSTH group, and CSTL group were respectively orally gavaged with the corresponding drugs daily for 8 weeks. The metformin concentration in the metformin group was 10 mg / kg, the UA concentration in the UAH group was 30 mg / kg, the UA concentration in the UAL group was 10 mg / kg, the OA concentration in the OAH group was 30 mg / kg, the OA concentration in the OAL group was 10 mg / kg, the CST concentration in the CSTH group was 120 mg / kg, and the CST concentration in the CSTL group was 40 mg / kg.

[0100] Test results of animal experiment 2

[0101] RT-qPCR analysis was performed. The specific experimental steps of RT-qPCR were the same as those in animal experiment 1 and will not be repeated here.

[0102] Figure 11 Shows the effects of different treatment groups on the expression levels of mitochondrial genes ucp1, ppargc1a, tfam, twnk, and nrfl. From Figure 11It can be seen that: compared with the control group, the gene expression in the model group was significantly decreased, and the CST treatment group could significantly increase the expression levels of these genes, and the gene expression levels were close to or exceeded those of the control group. The gene expression in the CST treatment group was higher than that in the OA treatment group and the UA treatment group, indicating that various compounds in total triterpenoids of Cynomorium songaricum Rupr. can play a synergistic role and obtain better drug effects than single triterpenoids.

[0103] As mentioned above, only the preferred specific embodiments of the present invention are described, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, makes equivalent substitutions or changes, and should be covered by the protection scope of the present invention.

[0104] The above embodiments only represent several embodiments of the present invention, and the description is relatively specific and detailed, but it cannot be understood as a limitation to the scope of the patent application. It should be noted that for those of ordinary skill in the art, without departing from the inventive concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A Cynomorium songaricum extraction pharmaceutical composition, characterized in that For preparing a drug for preventing and treating type 2 diabetes, or for preparing a drug for preventing and treating diseases of abnormal glucose and lipid metabolism, or for preparing a drug for preventing and treating diseases mediated by oxidative stress and / or free radicals; The pharmaceutical composition comprises cynomorium triterpenoids; the extraction method of the cynomorium triterpenoids comprises: extracting cynomorium plant materials with an ethanol solution to obtain an extract, and the extract comprises the cynomorium triterpenoids.

2. The Cynomorium songaricum Rupr. extract pharmaceutical composition according to claim 1, characterized in that The cynomorium triterpenoids comprise: 15α-hydroxy-3-oxo-urs-1,12-diene-28-oic acid, 3β-hydroxy-D_C-isolup-7-ene-28-oic acid, 3β-methylmalonylurs-12-ene-28-oic acid, 3β-methylmalonyloxyolean-12-ene-28-oic acid, obtusifoliol, ursolic acid, 3β-hydroxyolean-11,13(18)-diene-28-oic acid, oleanolic acid, 3β-hydroxyurs-20-ene-28-oic acid, δ-oleanolic acid, pomolic acid, uvaol, cynomorium terpene, maslinic acid, isocoumaric acid, 2α-O-trans-p-hydroxycinnamoylmaslinic acid, 3β-O-trans-p-coumaroylmaslinic acid, p-coumaric acid and 2α-hydroxy-3β-O-cis-p-coumaroylurs-12-ene-28-oic acid.

3. The Cynomorium songaricum Rupr. extract pharmaceutical composition according to claim 1, characterized in that The effective dose of the pharmaceutical composition is 40 mg / kg to 120 mg / kg.

4. The Cynomorium songaricum Rupr. extract pharmaceutical composition according to claim 1, characterized in that, It is characterized in that It further comprises excipients.

5. The Cynomorium songaricum Rupr. extract pharmaceutical composition according to claim 1, characterized in that It is characterized in that The extraction is reflux extraction.

6. The Cynomorium songaricum Rupr. extract pharmaceutical composition according to claim 1, characterized in that, The extraction time is 0.5 h to 1.5 h.

7. The Cynomorium songaricum Rupr. extract pharmaceutical composition according to claim 1, characterized in that, The concentration of the ethanol solution is 50% to 95%.

8. The Cynomorium songaricum Rupr. extract pharmaceutical composition according to claim 1, wherein The extract is chromatographically separated through a macroporous resin column to obtain the cynomorium triterpenoids.

9. A preparation method of the Cynomorium songaricum extraction pharmaceutical composition according to any one of claims 1 to 8, characterized in that, The extraction method of the cynomorium triterpenoids comprises: extracting cynomorium plant materials with an ethanol solution to obtain an extract, and the extract comprises the cynomorium triterpenoids.

10. A health food for preventing type 2 diabetes, or for preventing disorders of glucose and lipid metabolism, or for preventing diseases mediated by oxidative stress and / or free radicals, characterized in that Comprising cynomorium triterpenoids; The extraction method of the cynomorium triterpenoids comprises: extracting cynomorium plant materials with an ethanol solution to obtain an extract, and the extract comprises the cynomorium triterpenoids.

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