Application of calamint extract in preparation of medicine for preventing or treating non-alcoholic fatty liver disease

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of a clinopodium chinense extract in preparation of a medicine for preventing or treating a non-alcoholic fatty liver disease. Wherein the calamint is subjected to crushing, alcohol extraction and reduced pressure recovery to obtain a concentrate, the concentrate is extracted by petroleum ether and ethyl acetate to obtain an ethyl acetate extract, and the ethyl acetate extract is purified by macroporous resin to obtain the calamint extract. The extract obviously inhibits the accumulation of HepG2 cell lipid induced by palmitic acid, increases PPARalpha and CPT1A, reduces the protein expression of SREBP-1c, FASN and CD36, and inhibits the oxidative stress of liver cells. The extract can also obviously relieve lipid accumulation of NAFLD mice induced by high fat diet, improve oxidation resistance and relieve liver injury. The extract inhibits the activity of alpha-glucosidase and increases the expression of an adiponectin receptor 2. It is shown that the clinopodium megalanthum extract can effectively reduce blood fat, relieve liver lipid accumulation and inhibit oxidative stress, has the effects of inhibiting sugar absorption and protecting liver cells, and can be used for relieving the development of NAFLD.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and particularly relates to application of a Cyperus rotundus extract in preparing a drug for preventing or treating non-alcoholic fatty liver disease. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) is a clinical pathological syndrome caused by multiple other factors without a history of excessive drinking. The main feature of NAFLD is fatty degeneration of hepatocytes, which is often accompanied by obesity, hyperlipidemia, etc. According to the course of the disease, it can be divided into simple fatty liver, fatty liver hepatitis, liver fibrosis, liver cirrhosis and hepatocellular carcinoma. NAFLD can be caused by a variety of factors and diseases. The key to its occurrence lies in the imbalance of lipid metabolism such as the liver's uptake, oxidation, synthesis, esterification and transport of triglycerides. If one or more of these links are abnormal, it can induce triglyceride (TG) to accumulate in the liver to form fatty liver. In recent years, the widely recognized pathogenesis of NAFLD is the "multiple hit theory", which points out that multiple factors (insulin resistance, hormones secreted by adipose tissue, oxidative stress, endoplasmic reticulum stress, nutritional factors, intestinal microbiota, and genetic and epigenetic factors) induce NAFLD. Oxidative stress is still considered to be the main factor in liver damage and disease progression in the case of steatosis, and is the starting point of liver and extrahepatic damage. The incidence of NAFLD in the general population is as high as 20-30%, and 2-3% of NAFLD patients will develop non-alcoholic steatohepatitis (NASH). Therefore, the prevention and treatment of NAFLD is particularly important.

[0003] At present, in addition to changes in diet and lifestyle, the commonly used drugs for the treatment of NAFLD are mainly divided into three categories: insulin sensitizers (biguanides), lipid-lowering drugs (statins, etc.), and liver-protecting drugs (antioxidants). Although they have the effect of improving metabolic syndrome in animal experiments and clinical practice, they also have many side effects, such as myopathy, liver damage, and gastrointestinal reactions. In recent years, Rezdiffra, approved by the FDA, can be used in combination with diet and exercise to improve liver fibrosis and NASH symptoms, but its long-term clinical benefits are still under further study. Chinese herbal medicines have the pharmacological characteristics of multi-components, multi-targets, multi-pathway synergistic effects, low toxicity and side effects, and long-term medication. Therefore, it is very important to find Chinese herbal medicines that can prevent and treat NAFLD.

[0004] Clinopodium gracile (Benth.) Matsum, also known as thin wind wheel vegetable, belongs to the genus Clinopodium of the Lamiaceae family. It is distributed in various places south of the Yangtze River Basin. It has rich medicinal resources and has the effects of dispelling wind and heat, promoting qi and blood circulation, and detoxifying and reducing swelling. Clinopodium medicinal plants contain flavonoids and their glycosides, triterpenoid saponins, iridoid ether terpenoids, diterpenoids, free triterpenoids and other chemical components, which have hemostatic, antibacterial, anti-inflammatory, endothelial cell protection, immunomodulatory, antioxidant, and anti-tumor effects. Clinopodium gracile is used by the people in Fujian to treat diabetes and can improve the glucose and lipid metabolism disorders of diabetic patients.

[0005] The present invention aims to explore the improving effect of C. gracile extract (CGE) on NAFLD. Summary of the invention

[0006] Purpose of the invention: The present invention provides a method for preparing CGE, and its application in drugs for preventing and treating NAFLD.

[0007] Technical solution:

[0008] The invention provides an extract of Cyperus rotundus and application of the extract of Cyperus rotundus in preparing a medicine for preventing or treating non-alcoholic fatty liver disease.

[0009] The extract of Herba Cynanchifoliae is prepared according to the following steps:

[0010] The whole dried herb of Herba Cyperi is ground into powder, and refluxed or immersed in ethanol and its hydrate, methanol and its hydrate or propanol and its hydrate is used for extraction. The extract is concentrated under reduced pressure, and the concentrated extract is dispersed in water, and extracted with petroleum ether and ethyl acetate for 4 times in sequence. The ethyl acetate extract is subjected to column chromatography using a macroporous resin, and after impurities are eluted with water, ethanol is gradiently eluted (in the range of 10 to 90%), and the ethanol eluate (30-70%) is collected, concentrated and dried under reduced pressure to obtain Herba Cyperi extract CGE.

[0011] The CGE of the present invention can be mixed with conventional adjuvants and / or excipients to prepare various dosage forms for prevention or treatment. Injections, capsules, tablets, granules, dragees, solutions, etc. are all available pharmaceutical dosage forms. Dosage forms such as capsules, tablets, granules, dragees, etc. may contain one or more commonly used adjuvants - carriers or excipients: such as starch, microcrystalline fiber, etc.; binders: such as carboxymethyl cellulose, polyvinyl pyrrolidone, etc.; humectants: such as glycerol; disintegrants: such as calcium carbonate, etc.; adsorbents: such as kaolin, etc.; lubricants: such as talc, etc. Pharmaceutical excipients may be in various forms such as solid, semi-solid, liquid, etc., and formulation adjuvants may be of various types. Solutions may contain common solvents, solubilizers, emulsifiers, preservatives, etc., such as water, ethanol, glycerol, polyethylene glycol, benzyl benzoate, etc. Dosage forms such as capsules, tablets, granules, dragees, solutions, etc. can be prepared by known conventional methods. The extract of the effective part of Cyperus rotundus can be mixed with one or more excipients to prepare the dosage form.

[0012] The NAFLD cell model described in the present invention is HepG2 cells induced by palmitic acid (PA) and treated with CGE.

[0013] The CGE of the present invention has the effect of reducing lipid accumulation in HepG2 cells.

[0014] The CGE of the present invention has the effects of increasing the expression of PPARα, CPT1A and AdipoR2 and decreasing the expression of SREBP-1c, FASN and CD36 proteins.

[0015] The CGE of the present invention can alleviate the oxidative stress state in HepG2 cells induced by PA, and has the effects of increasing the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and reducing the contents of malondialdehyde (MDA) and reactive oxygen species (ROS).

[0016] The NAFLD model mice described in the present invention are induced by a high-fat diet and treated with CGE.

[0017] The CGE of the present invention can improve lipid accumulation and pathological damage in the liver of NAFLD mice.

[0018] The CGE of the present invention can alleviate the oxidative stress of the liver of NAFLD mice, and has the functions of reducing the serum MDA content and increasing the activities of SOD and GSH-Px.

[0019] The CGE of the present invention has a liver-protecting effect and can reduce the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum.

[0020] The CGE active ingredient of the present invention has affinity with PPARα protein.

[0021] The CGE of the present invention has the ability to significantly inhibit the activity of α-glucosidase.

[0022] Peroxisome proliferator-activated receptors (PPARs) regulate fatty acid metabolic homeostasis in multiple tissues, including the liver, adipose tissue, muscle, and other organs. The main expression of PPARα occurs in liver tissue, where it controls the regulation of fatty acid synthesis, translocation, and β-oxidation. First, carnitine palmitoyltransferase 1A (CPT1A) is an important regulator of fatty acid oxidation (FAO), and its regulation is regulated by PPARα. Mitochondria are the main organelles for fatty acid oxidation. In the cytoplasm, fatty acids are activated to form acyl-CoA, which is subsequently transported to the mitochondrial matrix through CAT1A-mediated mitochondrial membrane translocation for FAO. Second, activation of PPARα can effectively attenuate hepatic fatty acid synthesis and TG accumulation by reducing the expression of cholesterol-regulatory element binding protein (SREBP-1c). PPARα regulates the expression of fatty acid synthesis genes such as fatty acid synthase (FASN) and acetyl-CoA carboxylase 1 (ACC1), which are closely associated with SREBP-1c. Cluster of differentiation 36 (CD36) is a highly glycosylated single-chain transmembrane protein involved in free fatty acid uptake. PPARα specifically regulates CD36 to maintain the intracellular homeostasis of fatty acids. Therefore, PPARα is expected to become a potential target for the treatment of NAFLD. ROS are mainly produced in intracellular mitochondria to control the oxidative stress state. Lipid metabolism disorders lead to the interruption of the mitochondrial electron transport chain, resulting in excessive ROS production. Under normal circumstances, oxygen free radicals generated in the mitochondrial electron transport chain are cleared by GSH-Px and SOD. However, in NAFLD mice, excessive ROS produced by hepatocytes exceeds the clearance capacity of antioxidant enzymes, resulting in persistent oxidative stress. Excessive ROS generation reduces mitochondrial membrane potential, increases mitochondrial membrane permeability, and increases cytochrome C release, leading to cellular oxidative damage. Therefore, reducing the generation of ROS and inhibiting oxidative damage to hepatocytes may be an effective means to alleviate NAFLD.

[0023] Adiponectin is a fat factor secreted by adipocytes that is negatively correlated with obesity symptoms. It participates in the body's glucose metabolism and maintains energy balance. It has antioxidant, anti-inflammatory, anti-atherosclerotic and insulin antagonist effects. Increased adiponectin content in mammals can promote the oxidation of fatty acids. Existing studies have shown that adiponectin content decreases in patients with NAFLD. The effects of adiponectin are mainly mediated by adiponectin receptors (AdipoR). AdipoR1 is mainly expressed in skeletal muscle, while AdipoR2 is mainly expressed in the liver. AdipoR2 can also activate PPARα. Regulate lipid metabolism. By activating the AdipoR2 receptor, glucose and lipid metabolism can be effectively regulated, including promoting glucose uptake and utilization, thereby lowering blood sugar levels.

[0024] α-Glucosidase is a glycoside metabolic enzyme present in the brush border of small intestinal mucosal cells. This enzyme hydrolyzes disaccharides, allowing carbohydrates entering the small intestine to eventually form monosaccharides and be absorbed by the body. Inhibiting the activity of α-glucosidase can slow down the rate of carbohydrate hydrolysis and glucose production and delay the absorption of glucose, thereby effectively inhibiting the time and process of postprandial blood sugar increase. It can lower blood sugar in diabetic patients when used alone or in combination with other hypoglycemic drugs. Type 2 diabetes is a common complication of NAFLD, so inhibiting the activity of α-glucosidase can lower blood sugar in NAFLD patients with abnormal glucose metabolism.

[0025] Beneficial Effects

[0026] Fenofibrate (FNB) is used to treat hyperlipidemia in adults with unsatisfactory effects of dietary control therapy. Its effect on lowering triglycerides and mixed hyperlipidemia is more obvious than that on cholesterol. FNB can lower serum cholesterol and triglycerides. The main adverse reactions of fenofibrate include gastrointestinal irritation, such as diarrhea, nausea, constipation, etc., and may also cause myositis, myopathy, and rhabdomyolysis syndrome. Therefore, the present invention uses FNB as a positive control drug.

[0027] The present invention provides a new use of CGE in drugs for treating NAFLD. The results of the present invention show that CGE has good effects of reducing lipid accumulation, reducing oxidative damage, protecting the liver and inhibiting α-glucosidase activity. The results of in vitro experiments show that CGE significantly reduces the TG and TC content in HepG2 cells induced by PA, and reduces the oil red O staining area; significantly promotes the expression of PPARα, CPT1A and AdipoR2 proteins and inhibits the expression of SREBP-1c, FASN and CD36 proteins; significantly enhances the activity of SOD and GSH-Px and inhibits the generation of MDA and ROS. The results of in vivo experiments show that CGE can significantly reduce the oil red O staining area of ​​the liver of NAFLD mice and reduce liver pathological damage; significantly reduce serum TG, TC, NEFA, ALT and AST levels, reduce liver TG and TC content; significantly increase serum SOD, GSH-Px activity and reduce MDA generation. It is suggested that CGE has the effects of reducing fatty degeneration, inhibiting oxidative stress and protecting the liver, and can be used to prevent and treat NAFLD. Moreover, CGE has the same effect of inhibiting lipid accumulation as FNB, and its effect of reducing oxidative damage and protecting the liver is better than that of the positive drug FNB. FNB has no hepatoprotective effect. In addition, its side effects are large, such as long-term use can cause abnormal liver function. CGE regulates PPARα, and compared with FNB, the active ingredients of CGE (naringin, rosmarinic acid, apigenin, hesperidin or melissa glycosides) have a stronger affinity with PPARα protein, thereby alleviating NAFLD; CGE inhibits α-glucosidase with an IC50 of 74.1 μg / mL. This shows that CGE, as an extract of Sedum serrata, has a better therapeutic effect than FNB, and has the characteristics of hepatoprotective effect, small side effects, and a mechanism of action that inhibits α-glucosidase activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 , CGE on the lipid accumulation of HepG2 cells induced by PA; A. TG content of HepG2 cells. B. TC content of HepG2 cells. C. Representative pictures of Oil Red O staining. The results are expressed as mean ± SEM. ## P<0.01vs. normal group, *P<0.05, **P<0.01vs. model group.

[0029] Figure 2 Effects of CGE on the expression of PPARα, CPT1A, SREBP-1c, FASN, CD36 and AdipoR2 proteins related to glucose and lipid metabolism in HepG2 cells induced by PA; AB. Protein expression and statistical graphs of PPARα, CPT1A, SREBP-1c, FASN and CD36. CD. Protein expression and statistical graphs of AdipoR2. Results are expressed as mean±SEM. # P<0.05, ##P<0.01vs. normal group, *P<0.05, **P<0.01vs. model group.

[0030] Figure 3 , Effects of CGE on oxidative stress of HepG2 cells induced by PA; A. SOD activity of HepG2 cells. B. GSH-Px activity of HepG2 cells. C. MDA content of HepG2 cells. D. Representative images of intracellular ROS (DCFH-DA staining). E. Relative fluorescence intensity of DCFH-DA staining. Results are expressed as mean±SEM. ## P<0.01 vs. normal group, *P<0.05, **P<0.01 vs. model group, & P<0.05vs.FNB group.

[0031] Figure 4 , CGE effects on body weight and liver weight of NAFLD mice induced by high-fat diet; A. Mouse body weight. B. Mouse liver weight. C. Mouse liver weight coefficient. Results are expressed as mean ± SEM. ## P<0.01 vs. normal group, *P<0.05, **P<0.01 vs. model group, & P<0.05vs.FNB group.

[0032] Figure 5 , Effects of CGE on liver histopathology of NAFLD mice induced by high-fat diet; A. Appearance of liver. B. Representative images of hematoxylin and eosin (H&E) staining.

[0033] Figure 6 , Effects of CGE on lipid accumulation in NAFLD mice induced by high-fat diet; A. Serum TC content in mice. B. Serum TG content in mice. C. TC content in mouse liver. D. TG content in mouse liver. E. Serum NEFA content in mice. F. Representative pictures of Oil Red O staining in mouse liver. Results are expressed as mean ± SEM. ## P<0.01vs. normal group, *P<0.05, **P<0.01vs. model group.

[0034] Figure 7 , CGE on the activity of serum ALT and AST in NAFLD mice induced by high-fat diet; A. ALT activity in serum of mice. B. AST activity in serum of mice. The results are expressed as mean±SEM. ## P<0.01 vs. normal group, *P<0.05, **P<0.01 vs. model group, & P<0.05, && P<0.01vs.FNB group.

[0035] Figure 8 , Effects of CGE on oxidative stress in NAFLD mice induced by high-fat diet; A. Activity of SOD in mouse serum. B. Activity of GSH-Px in mouse serum. C. Content of MDA in mouse serum. Results are expressed as mean±SEM. ## P<0.01 vs. normal group, **P<0.01 vs. model group, && P<0.01vs.FNB group.

[0036] Fig. 9 , the affinity of the active ingredients of CGE to PPARα protein. DETAILED DESCRIPTION

[0037] The present invention describes the above contents of the present invention in detail through the following embodiments, but the present invention is not limited thereto, and all technologies realized based on the above contents of the present invention belong to the scope of the present invention.

[0038] Experimental materials: Human hepatoma cell HepG2 cells were purchased from Type Culture Collection Center, USA. PA was purchased from China Pharmaceutical Chemical Reagent Co., Ltd. Fenofibrate was purchased from Solebao Biotechnology Co., Ltd. PPARα antibody was purchased from Shenyang Wanlei Biological Co., Ltd., China. SREBP-1c antibody was purchased from Santa Cruz Biotechnology. Anti-GAPDH antibody was purchased from Bioworld Technology. CPT1A and FASN antibodies were purchased from Wuhan Proteintech, China. Total cholesterol (TC) and TG biochemical indicators were purchased from Zhejiang Dongou Diagnostic Products Co., Ltd. Free fatty acids (NEFA) SOD, GSH-Px, MDA, ALT and AST detection kits were purchased from Nanjing Jiancheng Bioengineering Institute. Reactive oxygen detection kits were purchased from Biyuntian Biotechnology Co., Ltd.

[0039] Experimental animals: SPF male C57BL / 6 mice (6-8 weeks old, weighing 18-22 g) were provided by the Animal Medical Center of Yangzhou University. High-fat feed was purchased from Nantong Trophy Feed Technology Co., Ltd.

[0040] Example 1 Preparation of extract from effective parts of Herba Cynanchifoliae

[0041] The extraction steps are as follows: the whole herb of dried Herba Cynoglossi (1.5 kg) was crushed, extracted with 80% ethanol by reflux, and the concentrated extract was extracted 4 times with petroleum ether and ethyl acetate respectively. The ethyl acetate part was separated with macroporous resin D101, and eluted with 10%, 30%, 50%, 70%, and 90% ethanol solutions in sequence. Finally, the 30% to 70% ethanol eluate was collected, concentrated and dried under reduced pressure to finally obtain CGE (14.1 g). A total of 6 components were identified in the Herba Cynoglossi extract, namely, naringin, hesperidin, rosmarinic acid, melissa glycoside, apigenin and isosakura glycoside. Among them, the contents of naringin, hesperidin, melissa glycoside and isosakura glycoside in CGE were 48.35, 80.36, 115.67 and 11.58 mg / g, respectively.

[0042] Example 2: Improvement of PA-induced hepatocyte lipid accumulation by CGE

[0043] 1. Detect the content of TG and TC in HepG2 cells.

[0044] HepG2 cells with good growth in the logarithmic growth phase were selected and, after digestion, the HepG2 cell suspension was inoculated into a 6-well cell culture plate (1×10 5 / well). When the cells grew to 80% confluence, the experimental groups were as follows: the normal group was natural culture of HepG2 cells, the model group was co-cultured with 250μM PA, and the other groups were co-cultured with CGE (7.5, 15, 30μg / mL) and FNB (100μM) and HepG2 cells and PA. After 24h of culture, the culture medium was discarded, and the cells were washed 3 times with PBS. 100μL of lysis buffer was added to each well, lysed on ice for 15min, and the cells were scraped off. 4℃, 12000g / min, centrifuged for 10min, and the supernatant was collected. The cell protein content was detected according to the bovine serum albumin (BCA) kit, and the intracellular TG and TC content were detected according to the instructions of the TG and TC kits. The experiment was repeated 3 times in parallel.

[0045] The results are as follows Figure 1 As shown in A, compared with the normal group, the TG content in HepG2 cells caused by PA was significantly increased. Compared with the model group, the CGE (15, 30 μg / mL) group and the FNB (100 μM) (i.e., FNB 36 μg / mL) group could significantly reduce the TG content in HepG2 cells, with inhibition rates of 73.6%, 76.6%, and 65.8%, respectively. Figure 1 As shown in B, compared with the normal group, the TC content in HepG2 cells induced by PA was extremely significantly increased. Compared with the model group, the CGE (15, 30 μg / mL) group and the FNB (100 μM) group could significantly reduce the TC content in HepG2 cells.

[0046] 2. Oil Red O staining to observe lipid droplets in HepG2 cells

[0047] Cells were grouped and cultured according to the steps in Example 2.1. The cells were removed from the incubator, washed three times with phosphate buffer solution (PBS), fixed with 4% paraformaldehyde for 15 min, then stained with Oil Red O for 1 h, and counterstained with hematoxylin for 15 s. Finally, the Oil Red O-stained cells were observed under an Olympus microscope.

[0048] The results are as follows Figure 1 As shown in C, the number of lipid droplets in HepG2 cells caused by PA increased significantly; compared with the model group, the CGE (15, 30 μg / mL) group significantly reduced the Oil Red O staining area of ​​HepG2 cells. It can be seen that CGE can effectively inhibit lipid accumulation in the NAFLD cell model.

[0049] Example 3 Regulatory effect of CGE on PA-induced PPARα, CPT1A, SREBP-1c, FASN, CD36 and AdipoR2 proteins in hepatocytes

[0050] Cells were grouped and cultured according to step 1 of the example. After the cells were treated, proteins were extracted, quantified by the BCA method, and the protein expression of PPARα, CPT1A, SREBP-1c, FASN, CD36 and AdipoR2 was detected by western blot. The steps are as follows: HepG2 cell samples were lysed with lysis buffer, and the protein samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to PVDF membranes. Then, TBST buffer containing 6% skim milk or 5% BSA was used to block at room temperature for 2 hours. Primary antibody (1:1000 dilution) was added and incubated at 4°C for 12 hours. Then, TBST was washed 3 times, and secondary antibody (1:5000 dilution) was added and incubated at room temperature for 2 hours. Finally, the electrochemiluminescence color development system was used for fixing and color development, and then the gel imaging system was used for photography, and Image J software was used for relative quantitative analysis.

[0051] PPARα plays an important role in maintaining lipid metabolism balance. It is involved in multiple links such as fatty acid synthesis, uptake and β-oxidation. PPARα can inhibit CD36 expression to reduce intracellular fatty acid uptake. PPARα can also increase CPT1A gene expression to promote fatty acid oxidation. In addition, FASN and ACC1 are downstream target genes of SREBP-1c. Activating PPARα can reduce SREBP-1c expression and inhibit FASN and ACC1 target gene transcription, thereby weakening liver fatty acid synthesis. Results Figure 2As shown in AB, compared with the normal group, the expression levels of PPARα and CPT1A proteins in HepG2 cells induced by PA were extremely significantly reduced, and the expression levels of SREBP-1c, FASN and CD36 proteins were significantly increased. Compared with the model group, the CGE (15, 30 μg / mL) group could significantly upregulate the expression levels of PPARα and CPT1A proteins, and significantly downregulate the expression levels of SREBP-1c, FASN and CD36. Figure 2 As shown in CD, compared with the normal group, the protein expression level of AdipoR2 in HepG2 cells induced by PA was significantly reduced. Compared with the model group, the protein expression level of AdipoR2 in the CGE (30 μg / mL) group was significantly upregulated. It can be seen that CGE can improve the glucose and lipid metabolism disorders in the NAFLD cell model by inhibiting fatty acid synthesis and uptake and promoting fatty acid β-oxidation. Its mechanism of action is related to the upregulation of PPARα protein expression.

[0052] Example 4 CGE inhibits PA-induced hepatocyte oxidative stress

[0053] 1. Effects of CGE on PA-induced SOD, GSH-Px and MDA in hepatocytes

[0054] Cells were grouped and cultured according to the steps in Example 2.1, and the cells were lysed. The content of cell protein was detected according to the BCA kit, and the activity of SOD, GSH-Px and MDA content in the cells were detected according to the kit instructions.

[0055] The results are as follows Figure 3 As shown in AB, compared with the normal group, the PA-induced SOD and GSH-Px activities of HepG2 cells were significantly decreased. Compared with the model group, the SOD activities of hepatocytes in the CGE (15, 30 μg / mL) group and the FNB (100 μM) group were significantly increased, with promotion rates of 87.8%, 152.7% and 93.9%, respectively. The GSH-Px activities of hepatocytes in the CGE (30 μg / mL) group and the FNB (100 μM) group were significantly upregulated, with promotion rates of 112.4% and 49.5%, respectively. Moreover, the upregulated GSH-Px activity of hepatocytes in the CGE (30 μg / mL) group caused by PA was significantly stronger than that in the FNB (100 μM) group. Figure 3 As shown in C, compared with the normal group, the MDA content in HepG2 cells induced by PA was significantly increased. Compared with the model group, the MDA content in hepatocytes in the CGE (15, 30 μg / mL) group and the FNB (100 μM) group was significantly reduced, and the effect of the CGE (15, 30 μg / mL) group on reducing the MDA content in hepatocytes induced by PA was significantly stronger than that in the FNB (100 μM) group. This indicates that the antioxidant effect of CGE is better than that of FNB.

[0056] 2. Effect of CGE on PA-induced ROS generation in hepatocytes

[0057] Cells were grouped according to the steps in Example 2.1. HepG2 cells (1×10 5 Cells / well) were seeded in 6-well plates and placed at 37°C with 5% CO 2 Culture in an incubator. According to the kit instructions, add 1 mL of serum-free medium containing DCFH-DA probe (2 μM) to each well of cells and incubate in a 37°C constant temperature incubator for 30 minutes. Wash three times with serum-free medium to remove excess probes, and observe the cells with an inverted fluorescence microscope.

[0058] The results are as follows Figure 3 As shown in DE, compared with the normal group, the ROS generation in HepG2 cells caused by PA increased significantly. Compared with the model group, the CGE (15, 30 μg / mL) group could significantly reduce the ROS content. It can be seen that CGE can effectively inhibit the oxidative stress state in the NAFLD cell model.

[0059] Example 5: The preventive and therapeutic effects of CGE on NAFLD mice induced by a high-fat diet

[0060] Establishment and administration of NAFLD animal model: C57BL / 6 mice were adaptively raised in an environment (23±2℃, 12h / 12h light-dark cycle) for 3 days. Fifty mice were divided into five groups, namely normal diet group (ND), high-fat diet group (HFD group), HFD+CGE 40mg / kg group, HFD+CGE 80mg / kg group, and HFD+FNB 80mg / kg group, with 10 mice in each group. The normal control group was fed with ordinary feed, and the HFD group was fed with 60% high-fat feed (60% fat, 19.4% protein, 20.6% carbohydrate) + 1% cholesterol + 0.25% bile salt (TP23300-X, purchased from Nantong Trophy Company), and free drinking water. The mice were fed with a high-fat diet for 10 weeks, and CGE and FNB were given treatment from the 3rd to the 10th week. Animals were treated at the end of 10 weeks. They were fasted but not watered for 12 h before treatment. Blood was collected from the abdominal aorta and centrifuged at 4°C, 12,000 g / min for 10 min to collect serum.

[0061] 1. Effects of CGE on body weight and liver weight of NAFLD mice induced by high-fat diet

[0062] The mouse liver was removed and weighed. Liver weight coefficient = liver (g) / body weight (100g)

[0063] NAFLD is often accompanied by obesity and increased liver weight. Figure 4As shown in the figure, compared with the normal group, the body weight, liver weight and liver index of NAFLD mice induced by high-fat diet increased significantly. Compared with the model group, the body weight of mice in the CGE (40, 80 mg / kg) group and the FNB (80 mg / kg) group was significantly reduced, with inhibition rates of 63.2%, 75.4% and 108.8%, respectively; the liver weight decreased significantly, with inhibition rates of 71.4%, 85.7% and 57.1%, respectively. Compared with the model group, the liver index of the CGE (40, 80 mg / kg) group decreased significantly, with inhibition rates of 100% and 128.57%, respectively. Although the body weight and liver weight of the FNB (80 mg / kg) group decreased, the liver index increased to a certain extent. Compared with the positive drug FNB group, the liver index of the CGE 80 mg / kg group was significantly reduced. The above results show that CGE can effectively inhibit the increase of body weight and liver weight of NAFLD mice induced by HFD, and has a liver protective effect, while FNB has no liver protective effect.

[0064] 2. Effect of CGE on liver histopathology in NAFLD mice induced by high-fat diet

[0065] A portion of the liver of the experimental mouse was fixed with 10% formalin solution, embedded in OCT, and made into paraffin sections. The sections were stained with hematoxylin-eosin (H&E staining), and then the staining of the liver tissue was observed under a microscope.

[0066] The results are as follows Figure 5 As shown in the figure, compared with the ND group, the liver of NAFLD mice induced by high-fat diet was significantly enlarged and yellowish in color. Compared with the model group, this phenomenon was alleviated to varying degrees after administration of CGE 40 and 80 mg / kg. The results of H&E staining of liver tissue showed that the hepatocytes in the normal group were arranged in a cord-like structure with normal structure. In the model group, diffuse lipid vacuoles were observed, and there was fusion, and a certain amount of inflammatory infiltration appeared around the portal area. After treatment with CGE 40 and 80 mg / kg, the degree of fatty degeneration and inflammatory response of the liver were significantly improved.

[0067] 3. Effect of CGE on lipid accumulation in NAFLD mice induced by high-fat diet

[0068] The kit was used to detect the content of TC, TG and NEFA in mouse serum. 10 mg of liver was taken from each mouse and rinsed twice in normal saline, then a 10% liver homogenate was made with isopropanol, and the homogenate was centrifuged at 4°C, 12000g / min for 10 minutes, and the supernatant was collected. The TG and TC content in the liver tissue was detected according to the instructions of the TG and TC kit. Part of the liver of the experimental mouse was frozen and stained with Oil Red O, and then the staining of the liver tissue was observed under a microscope.

[0069] Serum lipids and liver lipid content can reflect the degree of lipid accumulation in mice, and Oil Red O staining of liver tissue can further observe the number and size distribution of lipid droplets in the mouse liver. Figure 6 As shown in AE, compared with the normal group, the TC and TG levels in the serum and liver tissues of NAFLD mice induced by a high-fat diet were significantly increased, and the serum NEFA content was significantly increased. Compared with the HFD group, the TC and TG levels in the serum of the CGE (40, 80 mg / kg) group and the FNB (80 mg / kg) group were significantly reduced (TC inhibition rates were 35.0%, 91.5% and 80.5%, respectively; TG inhibition rates were 68.0%, 84.0% and 96.0%, respectively), and the TC and TG levels in the liver tissues were significantly reduced (TC inhibition rates were 40.6%, 77.5% and 94.8%, respectively; TG inhibition rates were 59.4%, 88.9% and 104.0%, respectively), and the serum NEFA content was also significantly reduced (inhibition rates were 40.1%, 90.8% and 101.3%, respectively). Figure 6 As shown in F, a large number of lipid droplets accumulated in the liver tissue of mice in the model group, and the number of lipid droplets in the liver of mice significantly decreased after CGE treatment. The above results indicate that CGE can significantly reduce lipid accumulation in NAFLD mice induced by HFD.

[0070] 4. Effect of CGE on serum ALT and AST activities in NAFLD mice induced by high-fat diet

[0071] Abnormal increase in ALT and AST enzyme activities is an important marker of liver damage. Figure 7 As shown in AB, a high-fat diet led to a significant increase in the activity of serum ALT and AST in mice. Compared with the model group, the activity of serum ALT in mice in the CGE (80 mg / kg) group decreased significantly, with an inhibition rate of 101.8%, and the activity of serum AST in mice in the CGE (40, 80 mg / kg) group decreased significantly (inhibition rates were 92.4% and 100.0%, respectively); the serum ALT in mice in the FNB (80 mg / kg) group showed an increasing trend, and there was no significant difference in AST activity compared with the model group. Compared with the positive drug FNB group, the activity of serum ALT and AST enzymes in mice in the CGE 80 mg / kg group was significantly reduced. This shows that CGE alleviates liver damage induced by a high-fat diet in NAFLD mice and has a liver protective effect, while FNB has no protective effect on the liver.

[0072] 5. Effect of CGE on oxidative stress in NAFLD mice induced by high-fat diet

[0073] Oxidative stress damage is a key factor in the development of NAFLD to NASH. Together with lipid accumulation, it forms a "parallel attack" to accelerate the development of NAFLD. Antioxidant enzyme activity and lipid peroxidation products in mice can reflect the oxidative stress status in mice. Figure 8 AC shows that compared with the normal group, the activities of serum SOD and GSH-Px in NAFLD mice induced by high-fat diet were extremely significantly reduced, and the MDA content was significantly increased. Compared with the model group, CGE (40, 80 mg / kg) can significantly increase the activities of serum SOD and GSH-Px in mice (the SOD promotion rates were 80.3% and 91.6%, respectively; the GSH-Px promotion rates were 62.7% and 70.4%, respectively). At the same time, compared with the model group, the CGE (40, 80 mg / kg) group and the FNB (80 mg / kg) group significantly reduced the MDA content, with inhibition rates of 20.7%, 79.0% and 70.7%, respectively). Compared with the FNB (80 mg / kg) group, the activity of serum GSH-Px in mice in the CGE (40, 80 mg / kg) group was significantly upregulated. This shows that CGE can effectively improve the oxidative stress state of NAFLD mice, and its antioxidant effect in vivo is better than that of FNB.

[0074] Example 6 CGE active ingredients have affinity for PPARα protein

[0075] The binding energy and interaction mode of CGE active compounds with PPARα target were analyzed using protein-ligand docking software AutoDockTools-1.5.7. The molecular structures of naringin, hesperidin, rosmarinic acid, melissin, apigenin, isosakuratin and FNB were retrieved from PubChem website (https: / / pubchem.ncbi.nlm.nih.gov / ). The protein structure of PPARα (PDB ID, 1I7G; resolution, ) were downloaded from the PDB website (http: / / www.rcsb.org / pdb / home / home.do). Molecular docking was performed using AutoDockTools-1.5.7 software.

[0076] Molecular docking analysis was used to evaluate the affinity of CGE active compounds to the PPARα target. Fig. 9 As shown in Table 1, each active compound binds to the PPARα protein through visible hydrogen bonds. The binding energy of PPARα with naringin, rosmarinic acid, apigenin, hesperidin, or melis glycosides is lower than that of FNB (-7.6 kcal / mol). It can be seen that the active ingredients of CGE (naringin, rosmarinic acid, apigenin, hesperidin, or melis glycosides) have a stronger affinity with the PPARα protein. This further supports that CGE alleviates NAFLD by regulating PPARα.

[0077] Table 1 Docking scores of CGE active ingredients and PPARα protein

[0078]

[0079] Example 7 Inhibitory effect of CGE on α-glucosidase

[0080] The activity of α-glucosidase was determined using 4-nitrophenol-α-D-furanoglucoside (PNP-G) as a substrate. 40 μl of CGE (final concentration of 7.5, 15, 30, 60, 120 μg / mL) and 30 μl of α-glucosidase (0.35 mg / mL) were added to 500 μl of KH 2 PO 4 Buffer (67mM, pH=6.8) was incubated in a 37°C water bath for 10 min, and then 30 μL of substrate PNP-G (0.01M) was added to start the reaction. After reacting at 37°C for 10 min, 0.1M Na 2 CO 3 1mL was added to terminate the reaction. The OD value of p-nitrobenzene (PNP) released from PNP-G under the action of the enzyme was measured at a wavelength of 405nm. The enzyme activity unit is defined as the amount of enzyme required to hydrolyze PNP-G to release 1μmol PNP within 1min at 37℃ and pH=6.8, and the inhibition rate was calculated. The enzyme inhibition test was repeated 3 times, with 3 replicates each time. The inhibition rate of α-glucosidase was calculated using the following formula: Inhibition rate = (OD value of the control group - OD value of the drug group) / OD value of the control group × 100%.

[0081] The results are shown in Table 2: Compared with the control group, CGE (7.5-120 μg / mL) had a very significant inhibitory effect on α-glucosidase in a dose-dependent manner. IC of CGE for inhibiting α-glucosidase 50 It is 74.1μg / mL.

[0082] Table 2 Inhibitory effect of CGE on α-glucosidase ( n=9)

[0083]

[0084] Note: ** P<0.01vs control group.

Claims

1. Application of the extract of Cyperus rotundus in the preparation of a drug for preventing or treating non-alcoholic fatty liver disease.

2. The use according to claim 1, characterized in that: The extract of Herba Cyperi is prepared according to the following steps: the whole dried Herba Cyperi is ground, extracted by reflux with ethanol and its hydrates, methanol and its hydrates or propanol and its hydrates, or extracted by immersion, the extract is concentrated under reduced pressure, the concentrated extract is dispersed in water, petroleum ether and ethyl acetate are used in sequence, the ethyl acetate extract is subjected to column chromatography with a macroporous resin, impurities are eluted with water, and then gradient eluted with ethanol, the ethanol eluate is collected, concentrated under reduced pressure and dried to obtain the Herba Cyperi extract.

3. The application according to claim 2, characterized in that: The ethanol gradient elution is 10-90% ethanol elution, and the ethanol eluate is collected as 30-70% ethanol eluate.

4. The use according to claim 2 or 3, characterized in that: The main components of the Herba Cyperi serratae extract are naringin, hesperidin, rosmarinic acid, melissa officinalis, apigenin and isosakurogen.

5. The use according to any one of claims 1 to 4, characterized in that The extract of Herba Cynanchifoliae is a pharmaceutical composition formed with pharmaceutically acceptable auxiliary materials.

6. The use according to claim 5, characterized in that The pharmaceutical composition is prepared into solution, capsule, tablet, granule or dragee.

Images