Traditional Chinese medicine composition for treating non-alcoholic fatty liver disease and application thereof

Abstract

The invention relates to a traditional Chinese medicine composition for treating non-alcoholic fatty liver disease. The traditional Chinese medicine composition is prepared from the following raw material medicines in parts by weight: 1-10 parts of oriental wormwood and 1-10 parts of liquorice. The invention further provides preparation of the medicine and application of the medicine to treatment of the non-alcoholic fatty liver disease. The traditional Chinese medicine composition has the advantages that a mouse non-alcoholic fatty liver disease model induced by high-fat and high-sugar feeding shows that the traditional Chinese medicine composition has an obvious effect of improving the non-alcoholic fatty liver disease, and the liver weight and the liver index of a non-alcoholic fatty liver disease model mouse can be obviously reduced; liver fatty degeneration and lipid droplet density can be remarkably improved in histopathology, and NAS integral of model mice is reduced. The TG and TC levels in serum and liver of the model mouse, the liver injury index ALT and AST levels and the liver lipid peroxidation index MDA level are reduced.

Description

[Technical field]

[0001] The present invention relates to the technical field of traditional Chinese medicine, and in particular to a traditional Chinese medicine composition having a therapeutic effect on non-alcoholic fatty liver disease and an application thereof. [Background technology]

[0002] Non-alcoholic fatty liver disease (NAFLD) is a metabolic stress-induced liver injury closely related to insulin resistance and genetic susceptibility. It is currently the most common chronic liver disease in the world, affecting about 30% of the world's population. NAFLD can be further divided into non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH); the latter can progress to liver fibrosis, cirrhosis and even hepatocellular carcinoma. In recent years, with the changes in living habits and dietary structure, the number of obese people has continued to increase; and NAFLD is strongly related to obesity, and its incidence rate increases in proportion to the increase in BMI. At the beginning of the 21st century, the prevalence of NAFLD in my country was estimated to be 23.8%. By 2018, the prevalence had increased to 32.9%, and the incidence rate had increased significantly. With the prevalence of obesity and metabolic syndrome, NAFLD has become the number one chronic liver disease in my country and the primary cause of abnormal liver biochemical indicators in health examinations, seriously threatening people's lives and health.

[0003] The pathogenesis of NAFLD is complex. The early "two-hit theory" believed that insulin resistance led to the accumulation of triglyceride lipids in the liver and the accumulated fatty acids caused oxidative stress damage in hepatocytes, leading to inflammation of hepatocytes as the main pathogenesis of NAFLD. The currently widely accepted "multiple-hit model" believes that multiple factors act together on genetically susceptible people to cause NAFLD. Although NAFLD has a high incidence rate and poses a great threat to health, the National Medical Products Administration (NMPA) has not approved any drugs for the treatment of NAFLD so far. Due to the lack of available drugs, the current intervention measures for NAFLD are mainly healthy diet and regular exercise; but patients have poor compliance with the above measures.

[0004] Traditional Chinese medicine is widely used in the clinical treatment of NAFLD in my country; Chinese patent medicines, in-hospital preparations, empirical prescriptions, syndrome differentiation and treatment, and effective ingredients of traditional Chinese medicines provide a variety of treatment options for patients with NAFLD. The multi-component and multi-target characteristics of traditional Chinese medicine compound have advantages in the treatment of NAFLD, a disease with a complex pathogenesis. Although there are some patents for traditional Chinese medicine compound prescriptions that can treat NAFLD, most of these compound prescriptions contain a large number of traditional Chinese medicines, some of which are expensive or difficult to obtain. Therefore, it is urgent to develop a traditional Chinese medicine composition that is relatively simple in composition, easy to prepare, and has good efficacy on NAFLD. [Summary of the invention]

[0005] The object of the present invention is to provide a Chinese medicine composition which can effectively treat non-alcoholic fatty liver disease. The Chinese medicine composition has the advantages of few components, simple preparation, controllable quality, significant efficacy and low cost.

[0006] The technical solution adopted by the present invention is as follows:

[0007] A traditional Chinese medicine composition for treating non-alcoholic fatty liver disease is prepared from the following traditional Chinese medicines in parts by weight: 1-10 parts of Artemisia capillaris and 1-10 parts of liquorice.

[0008] Preferably, the Chinese medicine composition is made of the following Chinese medicinal materials in parts by weight: 5-10 parts of Artemisia capillaris and 1-5 parts of Licorice.

[0009] Preferably, the Chinese medicine composition is made of the following Chinese medicinal materials in parts by weight: 1-5 parts of Artemisia capillaris and 5-10 parts of Licorice.

[0010] More preferably, the Chinese medicine composition is made of the following Chinese medicinal materials in parts by weight: 3 parts of Artemisia capillaris and 1 part of Licorice.

[0011] Wash Artemisia capillaris and Licorice, add 10 times the amount of distilled water, soak for 30 minutes, heat and boil for 30 minutes, filter and collect the filtrate, add 8 times the amount of distilled water to the residue, heat and boil for 30 minutes, filter and collect the filtrate, combine the two filtrates, heat and concentrate to a specified volume, which is the Chinese medicine composition of the present invention. The Chinese medicine composition of the present invention is an oral preparation, such as granules, capsules, and tablets.

[0012] The beneficial effects of the present invention are as follows: the Chinese medicine composition (Yingan mixture) described in the present invention has a significant therapeutic effect on non-alcoholic fatty liver disease induced by high-fat and high-sugar feed in C57BL / 6J mice. It can significantly reduce the liver weight and liver index of non-alcoholic fatty liver disease model mice, reduce ballooning degeneration and lipid droplet density in the liver of model mice, and reduce NAS scores. It can reduce triglycerides (TG) and total cholesterol (TC) in the liver and serum of model mice, reduce serum alanine aminotransferase (GPT / ALT) and aspartate aminotransferase (GOT / AST), liver malondialdehyde (MDA), and reduce the expression of inflammatory factors (TNF-α and IL-1β) in the liver of model mice.

Brief Description of the Drawings

[0013] Figure 1 :Gross morphology, liver weight and liver index of mice in each group (n=8, Mean ± SD). ## P <0.01, vsControl; * P <0.05,** P <0.01, vs HFHC.

[0014] Figure 2 :HE staining and NAS score of the liver of mice in each group. Control: blank control group, HFHC: high-fat and high-sugar model group, YGL: low-dose group of Yingan mixture, YGM: low-dose group of Yingan mixture, YGH: high-dose group of Yingan mixture, OCA: obeticholic acid group, YC: Artemisia capillaris group, GC: Licorice group. ## P <0.01, vs Control; * P <0.05,** P <0.01, vs HFHC.

[0015] Figure 3 : Oil red O staining of liver of mice in each group. Control: blank control group, HFHC: high-fat and high-sugar model group, YGL: low-dose group of Yingan mixture, YGM: low-dose group of Yingan mixture, YGH: high-dose group of Yingan mixture, OCA: obeticholic acid group, YC: Artemisia capillaris group, GC: Licorice group.

[0016] Figure 4 : TG and TC levels in serum and liver of mice in each group (n=8, Mean ± SD). # P <0.05,## P <0.01, vs Control; * P <0.05,** P <0.01, vs HFHC.

[0017] Figure 5 :Serum ALT and AST levels of mice in each group (n=8, Mean ± SD). ## P <0.01, vs Control; * P <0.05,** P <0.01, vs HFHC.

[0018] Figure 6 :The mRNA expression levels of inflammatory factors in the liver of mice in each group (n=8, Mean ± SD). # P <0.05,## P <0.01, vs Control; * P <0.05,** P <0.01, vs HFHC.

[0019] Figure 7 : MDA levels in the liver of mice in each group (n=8, Mean ± SD). ## P <0.01, vs Control; ** P <0.01, vs HFHC. [Specific implementation method]

[0020] The specific implementation modes provided by the present invention are described in detail below with reference to the accompanying drawings.

[0021] Example 1 Preparation of a Chinese medicine composition for treating non-alcoholic fatty liver disease

[0022] 5 parts of Artemisia capillaris and 5 parts of liquorice. Add 10 times the amount of water to the first decoction and 8 times the amount of water to the second decoction. Combine the two decoctions, heat and concentrate to the target volume.

[0023] Example 2 Preparation of a Chinese medicine composition for treating non-alcoholic fatty liver disease

[0024] 5 parts of Artemisia capillaris and 10 parts of liquorice. Add 10 times the amount of water to the first decoction and 8 times the amount of water to the second decoction. Combine the two decoctions, heat and concentrate to the target volume.

[0025] Example 3 Preparation of a Chinese medicine composition for treating non-alcoholic fatty liver disease

[0026] 3 parts of Artemisia capillaris and 10 parts of liquorice. Add 10 times the amount of water to the first decoction and 8 times the amount of water to the second decoction. Combine the two decoctions, heat and concentrate to the target volume.

[0027] Example 4 Preparation of a Chinese medicine composition for treating non-alcoholic fatty liver disease

[0028] 10 parts of Artemisia capillaris and 5 parts of liquorice. Add 10 times the amount of water to the first decoction and 8 times the amount of water to the second decoction. Combine the two decoctions, heat and concentrate to the target volume.

[0029] Example 5 Preparation of a Chinese medicine composition for treating non-alcoholic fatty liver disease

[0030] 10 parts of Artemisia capillaris and 2 parts of liquorice. Add 10 times the amount of water to the first decoction and 8 times the amount of water to the second decoction. Combine the two decoctions, heat and concentrate to the target volume.

[0031] Example 6 Preparation of a Chinese medicine composition for treating non-alcoholic fatty liver disease

[0032] 3 parts of Artemisia capillaris and 1 part of Licorice root. Add 10 times the amount of water to the first decoction and 8 times the amount of water to the second decoction. Combine the two decoctions, heat and concentrate to the target volume.

[0033] Example 7 Preparation of granules, tablets and capsules of Chinese medicine composition for treating non-alcoholic fatty liver disease

[0034] Take any of the drugs described in Examples 1-6, and boil them in water according to the above method. Add 3 times the volume of alcohol to the obtained decoction concentrate and stir overnight. Take the supernatant and concentrate it to a thick extract, add pharmaceutical excipients, vacuum dry, crush and granulate; granulate into granules, press into tablets or fill capsules.

[0035] Example 8 Animal efficacy experiment

[0036] 1 Experimental Materials

[0037] 1.1 Drugs

[0038] Artemisia capillaris was purchased from Shanghai Yanghetang Chinese Medicine Pieces Co., Ltd. Licorice was purchased from Shanghai Kangqiao Chinese Medicine Pieces Co., Ltd. Obeticholic acid was purchased from Panso Pharmaceutical Co., Ltd.

[0039] 1.2 Experimental animals

[0040] Male C57BL / 6J mice, SPF grade, male, weighing 18-22 g; purchased from Shanghai Slake Experimental Animal Co., Ltd., raised and used for subsequent experiments in the Animal Experimental Center of Shanghai University of Traditional Chinese Medicine.

[0041] 1.3 Reagents and instruments

[0042] Oil red dye kit, ALT detection kit, AST detection kit, TG detection kit, TC detection kit, MDA detection kit, RNA extraction reagent, reverse transcription kit, PCR amplification kit.

[0043] Electronic balance, pure water system, high-speed refrigerated centrifuge, multifunctional microplate reader, reverse transcription PCR instrument, StepOnePlus real-time fluorescence quantitative PCR instrument.

[0044] 2 Methods

[0045] 2.1 Preparation of Yingan mixture

[0046] Weigh 900g of Artemisia capillaris and 300g of Licorice (Artemisia capillaris: Licorice = 3:1) in a beaker, add 12L (10 times the amount) of distilled water, soak for 30min, heat on an induction cooker, filter after boiling for 30min, collect the filtrate, add 9.6L (8 times the amount) of distilled water to the residue, heat on an induction cooker, filter after boiling for 30min, collect the filtrate, combine the two filtrates, heat and concentrate to 800mL, which is a high-dose Artemisia capillaris mixture (raw drug amount 1.5g / mL); cool and store at -80℃. The medium and low-dose Artemisia capillaris mixtures were prepared by the same method, and heated and concentrated to 1200mL and 2400mL respectively (raw drug amounts were 1.0g / mL and 0.5g / mL respectively).

[0047] 2.2 Mouse modeling and grouping and drug administration

[0048] After one week of adaptive feeding, C57BL / 6J mice were randomly divided into a control group (n=8) and a model group (n=56) according to their body weight. The mice in the control group were fed with conventional ordinary feed and double distilled water, while the mice in the model group were fed with high-fat feed and high-sugar drinking water. The mice were allowed to drink water freely, and the feed and drinking water were changed every 2 days for 12 weeks. After 12 weeks of modeling, the mice in the model group were divided into 7 groups according to their body weight, namely, the model group (HFHC, n=8), the low-dose Yingan mixture group (YG-L, n=8), the medium-dose Yingan mixture group (YG-M, n=8), the high-dose Yingan mixture group (YG-H, n=8), the obeticholic acid group (OCA, n=8), the Yinchen decoction group (YC, n=8) and the licorice decoction group (GC, n=8). The corresponding drug solution was given by gavage at 10 mL / kg daily, and the high-fat and high-sugar feeding was continued for 8 weeks. The control group (Control, n=8) was given double distilled water by gavage and fed with regular feed for 8 weeks.

[0049] 2.3 Collection of materials

[0050] Before treatment, mice were fasted overnight, anesthetized with sodium pentobarbital, and blood was collected from the retroorbital venous plexus. After standing, the blood was placed in a precooled centrifuge and centrifuged at 4000rpm for 10 minutes at 4°C. The upper serum was aspirated and transferred to a clean 1.5mL EP tube and stored in a -80°C refrigerator for later use. The mouse abdominal cavity was opened, and the liver tissue was peeled off with a blunt surface of surgical scissors. The liver was rinsed with saline until the surface was clean. After naked eye observation and photographing, the liver was divided into the following parts: two undamaged liver lobe tissues were carefully cut, one was placed in an embedding box and immersed in 4% paraformaldehyde for subsequent preparation of liver pathological sections, and the other was embedded in OCT glue and quickly frozen in liquid nitrogen for preparation of frozen sections; the remaining liver was placed in a 1.5mL centrifuge tube with Trizol added in advance for qPCR to determine the mRNA expression of the target gene.

[0051] 2.4 HE staining and Oil Red O staining of liver tissue sections

[0052] Dewax the paraffin sections, soak them in xylene for 10 minutes, soak them in anhydrous ethanol for 5 minutes, then soak them in different concentration gradients of ethanol (95%, 80%, 70%) for 3 minutes, wash the ethanol with tap water, and wash them with distilled water. Soak them in hematoxylin stain for 5-10 minutes, wash the floating color with tap water, wash them with distilled water, differentiate them with 75% hydrochloric acid ethanol, rinse them with tap water until they turn blue, wash them with distilled water, soak them in eosin stain for 1-2 minutes, rinse them with tap water, and wash them with distilled water. Soak them in 95% ethanol for 1 minute, repeat the operation once, soak them in 100% ethanol for 1 minute, soak them in xylene for 1 minute, and seal them with neutral resin.

[0053] Prepare Oil Red O staining solution, filter it through filter paper and set aside. Take out the frozen sections and warm them up to room temperature for 10 minutes. Soak them in Oil Red O staining solution for 10-15 minutes, wash them with 37℃ double distilled water for 1 minute, soak them in re-staining solution for 5 minutes, wash them with double distilled water for 1 minute, and seal them with aqueous sealing medium.

[0054] 2.5 Evaluation of lipid-lowering effects

[0055] Weigh an appropriate amount of liver tissue and add physiological saline to prepare liver tissue homogenate. Follow the steps in the Nanjing Jiancheng TG (triglyceride) and TC (total cholesterol) kit instructions, use an enzyme reader to read the absorbance, and determine the TG and TC content in the liver. Take a serum sample, thaw it at 4°C, vortex mix, take an appropriate amount of serum sample, follow the steps in the Nanjing Jiancheng TG and TC kit instructions, use an enzyme reader to read the absorbance, and determine the levels of TG and TC in the serum.

[0056] 2.6 Evaluation of anti-inflammatory effects

[0057] Serum samples were taken, thawed at 4°C, vortexed and mixed, and appropriate amount of serum samples were taken. The steps in the Nanjing Jiancheng ALT / GPT and AST / GOT instructions were followed, and the absorbance was read using a microplate reader to determine the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum. RNA was extracted from liver tissue, reverse transcribed into cDNA, and qPCR was used to detect the mRNA expression levels of inflammatory factors (TNF-α and IL-1β).

[0058] 2.7 Evaluation of anti-lipid peroxidation effect

[0059] Weigh an appropriate amount of liver tissue, add physiological saline to prepare liver tissue homogenate, follow the steps in the Nanjing Jiancheng MDA kit instructions, use an ELISA reader to read the absorbance, and determine the MDA content in the liver.

[0060] 2.7 Statistical analysis

[0061] The experimental data were statistically analyzed using GraphPad Prism 9.0 software, and the quantitative data were expressed in the form of "mean ± standard deviation". One-way analysis of variance was used, and Dunnett's t test was used for inter-group comparison. When p < 0.05, it was considered to be significantly different, and when p < 0.01, it was considered to be very significantly different.

[0062] 3 Results

[0063] 3.1 Yingan mixture reduces liver weight and liver index

[0064] like Figure 1As shown in the figure, compared with the control group, the gross morphology of the liver in the model group showed fatty degeneration. The gross morphology of the liver improved to a certain extent after treatment with a medium dose of Ingan mixture, and significantly improved after treatment with a high dose of Ingan mixture. Compared with the control group, the liver weight and liver index of mice in the model group increased significantly; and compared with the model group, different doses of Ingan mixture treatment could significantly reduce the liver weight and liver index of mice. This suggests that Ingan mixture has a therapeutic effect on non-alcoholic fatty liver disease in mice successfully induced by a high-fat and high-sugar diet.

[0065] 3.2 Improvement of liver histopathology by Yingan mixture

[0066] like Figure 2 As shown, Figure 2 As shown in the figure, compared with the control group, the liver in the model group had obvious lobular inflammation and ballooning degeneration, and the lobular inflammation and ballooning degeneration of the liver were significantly improved after treatment with medium and high doses of Ingan mixture. Compared with the control group, the NAS score of the model group reached the level of NASH, and the NAS score was significantly reduced after treatment with low doses of Ingan mixture, and the NAS score was very significantly reduced after treatment with medium and high doses of Ingan mixture. Figure 3 As shown in the figure, compared with the control group, there were a large number of red-stained lipid droplets in the liver of the model group. The red lipid droplets decreased after treatment with low-dose Ingan mixture, and the reduction of red lipid droplets was more obvious after treatment with medium and high-dose Ingan mixture. The above results suggest that Ingan mixture can alleviate liver lipid accumulation and inflammation, and its effect shows a certain degree of dose dependence.

[0067] 3.3 Yinggan mixture can alleviate liver lipid accumulation

[0068] like Figure 4 As shown in the figure, compared with the control group, the levels of TG and TC in the liver and serum of the model group were significantly increased, while after treatment with different doses of Yingan mixture, the levels of TG and TC in the liver and serum were significantly decreased; and its effect showed a certain dose dependence. This shows that Yingan mixture can alleviate liver lipid accumulation.

[0069] 3.4 Yingan mixture can inhibit liver inflammation

[0070] like Figure 5 As shown in the figure, compared with the control group, the ALT and AST levels in the serum of the model group were significantly increased, and after treatment with different doses of Yingan mixture, the levels of ALT and AST in the serum were significantly decreased. This suggests that Yingan mixture can improve liver function. Figure 6As shown in the results, compared with the control group, the mRNA expression levels of inflammatory factors (TNF-α and IL-1β) in the liver of the model group increased significantly, while the mRNA expression levels of inflammatory factors (TNF-α and IL-1β) in the liver decreased significantly after treatment with different doses of Yingan mixture, indicating that Yingan mixture can inhibit liver inflammation.

[0071] 3.5 Yingan mixture can alleviate liver lipid peroxidation

[0072] like Figure 7 As shown in the figure, compared with the control group, the MDA level in the liver of the model group mice was significantly increased, and the MDA level in the liver was significantly decreased after treatment with different doses of Yingan mixture, indicating that Yingan mixture can alleviate liver lipid peroxidation.

[0073] The above description is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. A Chinese medicine composition for treating non-alcoholic fatty liver disease, characterized in that: The invention is prepared from the following Chinese medicinal materials by weight: 1-10 parts of Artemisia capillaris and 1-10 parts of liquorice.

2. The Chinese medicine composition according to claim 1, characterized in that: The invention is prepared from the following Chinese medicinal materials by weight: 5-10 parts of Artemisia capillaris and 1-5 parts of liquorice.

3. The Chinese medicine composition according to claim 2, characterized in that: The invention is prepared from the following Chinese medicinal materials by weight: 1-5 parts of Artemisia capillaris and 5-10 parts of liquorice.

4. The Chinese medicine composition according to claim 3, characterized in that: It is made from the following Chinese medicinal materials by weight: 3 parts of Artemisia capillaris and 1 part of liquorice.

5. The Chinese medicine composition according to claims 1-4, characterized in that: The pharmaceutical dosage form of the drug is granules, capsules, and tablets.

6. Use of the Chinese medicine composition according to claims 1-4 in preparing a drug for treating non-alcoholic fatty liver disease.

7. The use according to claim 6, characterized in that: The non-alcoholic fatty liver disease is simple fatty degeneration and non-alcoholic fatty hepatitis.

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