Traditional Chinese medicine composition for treating non-alcoholic steatohepatitis and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a traditional Chinese medicine composition for treating non-alcoholic steatohepatitis and application of the traditional Chinese medicine composition. The technical problem to be solved by the invention is to provide a new choice for patients with non-alcoholic steatohepatitis. According to the technical scheme, the traditional Chinese medicine composition for treating the non-alcoholic steatohepatitis is prepared from the following components: clove, herba cepbalanoplosis segeti, fructus gardeniae, peach kernels, semen hoveniae, stigma croci, pericarpium citri reticulatae, radix puerariae and herba taraxaci. The main purpose of the invention is to provide a traditional Chinese medicine composition for treating non-alcoholic steatohepatitis.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to a Chinese medicine composition for treating non-alcoholic fatty hepatitis and an application thereof. Background Art

[0002] Nonalcoholic Steatohepatitis (NASH) is a growing global epidemic and a severe form of nonalcoholic fatty liver disease (NAFLD). Pathological manifestations include fatty degeneration of more than 5% of hepatocytes, combined with intralobular inflammation and ballooning degeneration of hepatocytes. NASH significantly increases the risk of advanced liver disease (such as cirrhosis). In 2019, NASH has become the second most common indication for liver transplantation in the United States and the fastest growing indication. According to statistics, as of May 2021, the global prevalence of NAFLD has risen to 32.4%. Among NAFLD patients with biopsy indications, the estimated prevalence of NASH in Asia is 63.45%. Among NAFLD patients without biopsy indications, the estimated prevalence of NASH in Asia and North America is 6.67% and 29.85%, respectively. Among NASH patients, the liver-specific mortality rate and the total mortality rate are 11.77 / 1000 person / year and 25.56 / 1000 person / year, respectively. NASH has become an increasingly serious public health problem that requires widespread attention. However, to date, the clinical treatment of NASH still faces many problems, including the lack of effective preventive and therapeutic drugs and obvious toxic and side effects. Therefore, finding a suitable drug to treat NASH is an urgent problem to be solved clinically. Summary of the invention

[0003] The technical problem to be solved by the present invention is to provide a new option for patients with non-alcoholic fatty hepatitis.

[0004] The technical solution of the present invention is a traditional Chinese medicine composition for treating non-alcoholic fatty hepatitis, comprising the following ingredients: clove, thistle, gardenia, peach kernel, Hovenia dulcis, saffron, tangerine peel, kudzu root and dandelion.

[0005] Furthermore, the dosage of each ingredient is as follows: 1-9 parts by weight of cloves, 6-24 parts by weight of thistle, 5-20 parts by weight of gardenia, 5-20 parts by weight of peach kernel, 5-20 parts by weight of Hovenia dulcis, 1-6 parts by weight of saffron, 5-20 parts by weight of tangerine peel, 3-30 parts by weight of kudzu root and 5-20 parts by weight of dandelion.

[0006] Preferably, the dosage of each ingredient is as follows: 3 parts by weight of cloves, 12 parts by weight of thistle, 10 parts by weight of gardenia, 10 parts by weight of peach kernel, 10 parts by weight of Hovenia dulcis, 3 parts by weight of saffron, 10 parts by weight of tangerine peel, 15 parts by weight of kudzu root and 10 parts by weight of dandelion.

[0007] The present invention also provides a method for preparing the Chinese medicine composition, comprising the following steps: a. Mix clove, thistle, gardenia, peach kernel, Hovenia dulcis, saffron, dried orange peel, kudzu root and dandelion, add water to extract, extract twice, the first extraction obtains filtrate 1, the second extraction obtains filtrate 2; b. Combine filtrate 1 and filtrate 2, concentrate, dry and crush.

[0008] Furthermore, in step a, the extraction is carried out by a heating, condensation and reflux method.

[0009] Specifically, in step a, the liquid-to-solid volume-to-mass ratio (mL / g) during the first extraction is 1:6-1:20; and the liquid-to-solid volume-to-mass ratio (mL / g) during the second extraction is 1:6-1:10.

[0010] Preferably, in step a, the liquid-to-solid volume-to-mass ratio (mL / g) during the first extraction is 1:10; and the liquid-to-solid volume-to-mass ratio (mL / g) during the second extraction is 1:8.

[0011] Specifically, in step a, each extraction time is 1 to 3 hours.

[0012] Preferably, in step a, each extraction time is 1 hour.

[0013] The present invention also provides the use of the above-mentioned Chinese medicine composition or the Chinese medicine composition prepared by the above-mentioned method in preparing a medicine for treating or preventing non-alcoholic fatty liver disease.

[0014] The present invention also provides the use of the above-mentioned Chinese medicine composition or the Chinese medicine composition prepared by the above-mentioned method in the preparation of drugs for controlling body weight and food intake, reducing body fat percentage, improving glucose intolerance and insulin resistance, improving fatty liver, improving blood lipids, lowering blood sugar, reducing liver damage, improving inflammatory response, improving liver tissue oxidative stress level and / or improving liver steatosis, inflammation, fibrosis or liver damage caused by a high-fat and high-sugar diet.

[0015] The present invention also provides a medicine, food or health product for preventing or treating obesity, glucose and lipid metabolism disorders, and non-alcoholic fatty liver disease, including the above-mentioned Chinese medicine composition or the Chinese medicine composition prepared by the above-mentioned method.

[0016] Beneficial effects of the present invention: The present invention provides a Chinese medicine composition, the formula of which is as follows: clove, thistle, gardenia, peach kernel, Hovenia dulcis, saffron, tangerine peel, kudzu root and dandelion, which has the efficacy of treating non-alcoholic fatty hepatitis. The present invention provides an application of a Chinese medicine composition for treating non-alcoholic fatty hepatitis in the preparation of liver disease drugs. Based on the efficacy of the Chinese medicine composition in clinical treatment of NASH, a mouse NASH model was induced by a high-fat and high-cholesterol diet. After treatment with the Chinese medicine composition of the present invention, the changes in the weight, liver appearance, liver index, liver ultrasound, serum biochemical indexes, liver tissue pathology of the NASH mouse model and the effect of the Chinese medicine composition on insulin resistance of the NASH model animal were analyzed, and it was found that the Chinese medicine composition had a significant therapeutic effect on the NASH model of mice established by a high-fat and high-cholesterol diet. The present invention provides a new option for the treatment of non-alcoholic fatty hepatitis and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Effects of Chinese herbal compound on insulin resistance in NASH model mice. A: OGTT blood glucose curve; B: area under the OGTT curve, n=7-8; C: ITT blood glucose curve; D: area under the ITT curve, n=7-8; E: HOMA-IR, n=8-10. #P<0.05 VS.CON group, ###P<0.001 VS. CON group, **P<0.01 VS. HFHC group, ***P<0.001 VS. HFHC group.

[0018] Figure 2 Effect of Chinese herbal compound on NASH mouse model established by high-fat and high-cholesterol diet. A: Appearance of mice; B: Change curve of mouse body weight; C: Body fat ratio of mice; D: Change of blood glucose in mice. #P<0.05 VS. CON group, ##P<0.01 VS. CON group, ###P<0.001 VS. CON group, *P<0.05 VS. HFHC group, ***P<0.001 VS. HFHC group, n=8-10.

[0019] Figure 3 Effects of Chinese herbal compound on liver appearance, liver weight, liver index and liver ultrasound in NASH mouse model. A: liver appearance; B: liver weight, liver index, n=10; C: liver ultrasound image; D: gray value of liver ultrasound image, n=3. #P<0.05 VS. CON group, ##P<0.01 VS. CON group, *P<0.05 VS. HFHC group.

[0020] Figure 4The Chinese herbal compound can improve the levels of TG and TC in NASH mouse model. A: serum TG; B: serum TC. #P<0.05VS. CON group, ###P<0.001 VS. CON group, **P<0.01 VS. HFHC group, ***P<0.001 VS. HFHC group, n=8.

[0021] Figure 5 H&E staining and NAS score of mouse liver. A: H&E staining of liver tissue (200×); B: Hepatocyte fatty degeneration, intralobular inflammation, and ballooning degeneration score; C: Total NAS score. ##P<0.01 VS. CON group, ###P<0.001 VS. CON group, *P<0.05 VS. HFHC group, **P<0.01 VS. HFHC group, ***P<0.001 VS. HFHC group, n=6. DETAILED DESCRIPTION

[0022] Traditional Chinese medicine compound N (the traditional Chinese medicine composition of the present invention) is a clinical experience prescription. Based on its clinical efficacy in treating NASH, its improvement effect on hepatocyte fatty degeneration in the development of NASH was comprehensively evaluated through animal experiments.

[0023] The following examples are preferred embodiments of the present invention and further illustrate the present invention in detail, but do not limit the embodiments of the present invention in any way. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principle of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

[0024] The experimental animals used in the following examples are: Male C57BL / 6 mice, 8 weeks old and weighing 20-25 g, were housed in an SPF-grade animal room (20-25°C, 65-70%, 12 h light-dark alternation) at the Experimental Animal Center of Guangdong Pharmaceutical University. The animals were given free access to water and standard feed.

[0025] The experimental materials used in the following examples are: A high-fat, high-cholesterol diet was used to construct a non-alcoholic fatty liver disease mouse model: after 1 week of adaptive feeding, the control group (CON) was fed with ordinary feed, and the modeling group (50 mice) was fed with high-fat, high-cholesterol feed. At the 8th week of modeling, the mice were divided into the model group (HFHC), the low-dose group of Chinese medicine compound (NL), the medium-dose group (NM), the high-dose group (NH), and the atorvastatin group (ATV), with 10 mice in each group. The drug was administered for 8 weeks. The body weight of the mice was recorded every week, and the blood glucose of the mice was recorded every four weeks.

[0026] Example 1 Extraction and drying of medicine ① Prepare the raw materials: 3g of cloves, 12g of thistle, 10g of gardenia, 10g of peach kernel, 10g of Hovenia dulcis, 3g of saffron, 10g of dried tangerine peel, 15g of Pueraria lobata and 10g of dandelion (1-9g of cloves, 6-24g of thistle, 5-20g of gardenia, 5-20g of peach kernel, 5-20g of Hovenia dulcis, 1-6g of saffron, 5-20g of dried tangerine peel, 3-30g of Pueraria lobata and 5-20g of dandelion are also within the effective range and can also exert their medicinal effects).

[0027] ② Mix clove, thistle, gardenia, peach kernel, Hovenia dulcis, saffron, tangerine peel, kudzu root and dandelion, add water to extract, heat condensation reflux extraction twice, each time for 1 hour; during the first extraction, the liquid-to-solid volume-to-mass ratio (mL / g) is 1:10, and filtrate 1 is obtained; during the second extraction, the liquid-to-solid volume-to-mass ratio (mL / g) is 1:8, and filtrate 2 is obtained.

[0028] ③ Combine filtrate 1 and filtrate 2, concentrate, dry and crush to obtain.

[0029] Example 2 Effect of Chinese medicine compound N on insulin resistance in NASH mice The mice were given the drug for 8 weeks, and their body weight was recorded every week, and their blood glucose was recorded every four weeks. The glucose tolerance of mice was tested in the 7th week of drug administration: fasting for 6 hours, gavage of 1g / kg glucose, and the blood glucose level of mice was tested at 0 min, 15 min, 30 min, 60 min, 90 min, and 120 min after gavage. The insulin tolerance of mice was tested in the 8th week of drug administration: fasting for 4 hours, free drinking water, intraperitoneal injection of 0.5U / kg rapid-acting insulin, measuring blood glucose levels at 0, 15, 30, 45, and 60 min, drawing blood glucose change curves and calculating AUC. The blood glucose of mice was tested after fasting for 6 hours before sampling, and the fasting serum insulin content of mice was tested by taking blood from the tail vein to calculate the insulin resistance index.

[0030] like Figure 1 Compared with the CON group, the glucose tolerance, area under the insulin tolerance curve and insulin resistance index of the HFHC group were significantly increased; compared with the HFHC group, the area under the glucose tolerance curve of each group of Chinese herbal compound N showed a downward trend, the area under the curve of the high-dose group of Chinese herbal compound N was significantly reduced, and the insulin resistance index of each group of Chinese herbal compound N was significantly decreased.

[0031] Example 3 Effects of Chinese medicine compound N on appearance, body weight, body fat ratio and blood sugar in NASH model animals The weight of the mice was recorded using a weight scale, the fasting blood glucose value at the tip of the tail of the mice was detected using a Roche blood glucose meter and the data was recorded, and the body fat ratio of the mice was detected using a live mouse component analyzer and the data was recorded.

[0032] like Figure 2Compared with the CON group, the body weight, body fat ratio and blood glucose of mice in the HFHC group were significantly increased. Compared with the HFHC group, after the treatment of NASH mice with the Chinese medicine compound N, the body weight of mice showed a downward trend, and the body fat ratio and blood glucose of mice were significantly decreased; the effect of the Chinese medicine compound N was similar to that of the atorvastatin group (ATV), indicating that the Chinese medicine compound N has a significant therapeutic effect on the NASH model.

[0033] Example 4 Effects of Chinese medicine compound N on liver morphology, liver weight, liver index, and liver ultrasound in NASH model animals After 8 weeks of intervention of the NASH mouse model with the Chinese herbal compound N, the animals were anesthetized by isoflurane inhalation, and the abdominal fatty liver of each mouse was observed using the Vevo2100 small animal ultrasound imaging system.

[0034] like Figure 3 Compared with the CON group, the liver weight of mice in the HFHC group was significantly increased, and the ultrasonic grayscale value of the liver of mice in the HFHC group was significantly increased. Compared with the HFHC group, there was no statistically significant difference in the liver weight and liver index of mice after the Chinese medicine compound N treated NASH model mice; however, the liver ultrasound results showed that the Chinese medicine compound N could significantly inhibit the formation of fatty liver.

[0035] Example 5 Effect of Chinese medicine compound N on serum TG and TC levels in NASH mice According to the instructions of Nanjing Jiancheng triglyceride (TG) and total cholesterol (TC) kit, the TG and TC levels in animal serum were detected.

[0036] like Figure 4 Compared with the CON group, the levels of serum TG and TC in the HFHC group were significantly increased. Compared with the HFHC group, the Chinese medicine compound N could significantly reduce the serum TG level; meanwhile, the low and medium dose groups of Chinese medicine compound N could significantly inhibit the serum TC level.

[0037] Example 6 Effect of Chinese medicine compound N on liver pathology in NASH mice Fresh liver tissue was placed in an embedding box and immersed in tissue fixative. Paraffin sections were dehydrated by gradient ethanol dehydration method, then immersed in wax, embedded in paraffin, and placed on a paraffin slicer for sectioning. Frozen sections were dehydrated by sucrose dehydration method, embedded in OTC embedding agent, and placed on a freezing slicer for sectioning. Paraffin sections were placed in a 65°C oven for 1 hour, dewaxed in xylene three times, 10 minutes each time, and washed with various levels of ethanol to water: 100% ethanol 2 times for 5 minutes each time, 95% ethanol 2 times for 5 minutes each time, 80% ethanol 2 times for 5 minutes each time, and washed with water 2 times for 5 minutes. Hematoxylin staining for 5 minutes, distilled water washing for 5 minutes. 1% hydrochloric acid alcohol solution was differentiated for 5 seconds, and running water was rinsed to blue for 15 minutes. After staining in 0.5% eosin aqueous solution for 1 minute, it was rehydrated with various levels of ethanol: 80% ethanol for 2 minutes, 95% ethanol for 5 minutes each time, and 100% ethanol for 5 minutes each time. After clearing in xylene twice for 5 min each time, the slides were mounted with neutral gum.

[0038] like Figure 5 NAS scores were calculated and counted for H&E staining of the livers of mice in each group. The results showed that compared with the CON group, the scores of hepatocyte fatty degeneration, intralobular inflammation, and ballooning lesions in the HFHC group were significantly increased. Compared with the HFHC group, the scores of hepatocyte fatty degeneration in the medium and high dose groups of Chinese herbal compound N were significantly decreased; the scores of intralobular inflammation in the low and high dose groups of Chinese herbal compound N were significantly decreased; and the scores of hepatocyte ballooning lesions in each drug administration group showed a downward trend. Compared with the CON group, the total NAS score in the HFHC group was significantly increased. Compared with the HFHC group, the total NAS scores in each group of Chinese herbal compound N were significantly decreased.

Claims

1. A Chinese medicine composition for treating non-alcoholic fatty hepatitis, characterized in that: Includes the following ingredients: clove, thistle, gardenia, peach kernel, hovenia dulcis, saffron, tangerine peel, kudzu root and dandelion.

2. The Chinese medicine composition according to claim 1, characterized in that: The dosage of each component is as follows: 1-9 parts by weight of cloves, 6-24 parts by weight of thistle, 5-20 parts by weight of gardenia, 5-20 parts by weight of peach kernel, 5-20 parts by weight of Hovenia dulcis, 1-6 parts by weight of saffron, 5-20 parts by weight of tangerine peel, 3-30 parts by weight of kudzu root and 5-20 parts by weight of dandelion.

3. The Chinese medicine composition according to claim 2, characterized in that: The dosage of each ingredient is as follows: 3 parts by weight of cloves, 12 parts by weight of thistle, 10 parts by weight of gardenia, 10 parts by weight of peach kernel, 10 parts by weight of Hovenia dulcis, 3 parts by weight of saffron, 10 parts by weight of tangerine peel, 15 parts by weight of kudzu root and 10 parts by weight of dandelion.

4. The method for preparing the Chinese medicine composition according to any one of claims 1 to 3, characterized in that: The steps include: a. Mix clove, thistle, gardenia, peach kernel, Hovenia dulcis, saffron, dried orange peel, kudzu root and dandelion, add water to extract, extract twice, the first extraction obtains filtrate 1, the second extraction obtains filtrate 2; b. Combine filtrate 1 and filtrate 2, concentrate, dry and crush.

5. The preparation method according to claim 4, characterized in that: In step a, the extraction is carried out by a heating, condensing and reflux method.

6. The preparation method according to claim 4, characterized in that: In step a, the liquid-to-solid volume-to-mass ratio (mL / g) is 1:6-1:20 during the first extraction; the liquid-to-solid volume-to-mass ratio (mL / g) is 1:6-1:10 during the second extraction; and the extraction time for each extraction is 1-3 hours.

7. The preparation method according to claim 6, characterized in that: In step a, the liquid-to-solid volume-to-mass ratio (mL / g) during the first extraction was 1:10; the liquid-to-solid volume-to-mass ratio (mL / g) during the second extraction was 1:8; and the extraction time for each extraction was 1 h.

8. Use of the Chinese medicine composition according to any one of claims 1 to 3 or the Chinese medicine composition prepared by the method according to any one of claims 4 to 7 in the preparation of a medicament for treating or preventing non-alcoholic fatty liver disease.

9. Use of the Chinese medicine composition according to any one of claims 1 to 3 or the Chinese medicine composition prepared by the method according to any one of claims 4 to 7 in the preparation of drugs for controlling body weight and food intake, reducing body fat percentage, improving glucose intolerance and insulin resistance, improving fatty liver, improving blood lipids, lowering blood sugar, reducing liver damage, improving inflammatory response, improving oxidative stress level in liver tissue and / or improving liver steatosis, inflammation, fibrosis or liver damage caused by a high-fat and high-sugar diet.

10. A medicine, food or health product for preventing or treating obesity, glucose and lipid metabolism disorders, and non-alcoholic fatty liver disease, characterized in that: The invention comprises the Chinese medicine composition according to any one of claims 1 to 3 or the Chinese medicine composition prepared by the method according to any one of claims 4 to 7.

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