A traditional Chinese medicine composition for treating non-alcoholic fatty liver disease and a preparation method and application thereof

Abstract

The present application relates to a kind of traditional Chinese medicine compositions, including the following weight parts of preparation raw materials: Huanglian 15~20 parts, Fashi 15~18 parts, Gualoupi 30~45 parts, Juhong 10~15 parts, Danshen 15~20 parts, Baishu 15~20 parts.The traditional Chinese medicine composition prepared in the present application has obvious effect of resisting non-alcoholic fatty liver disease (NAFLD), compared with Rosuvastatin, can further improve the lipid accumulation and balloon-like change of liver, improve the level of triglyceride (TG), cholesterol (TC), low-density lipoprotein (LDL-C) and high-density lipoprotein (HDL-C) in blood fat, reduce liver damage index glutamic-pyruvic transaminase (ALT), inhibit the expression of inflammatory factors (TNF-alpha and IL-1beta).At the same time, the body weight of NAFLD mouse can be significantly reduced, glucose tolerance can be improved, insulin sensitivity can be improved, insulin resistance can be reduced, and the efficacy of regulating intestinal flora and increasing the abundance of intestinal Akkermansia bacteria can be achieved.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine technology, specifically to a traditional Chinese medicine composition for treating non-alcoholic fatty liver disease, its preparation method, and its application. Background Technology

[0002] Non-alcoholic fatty liver disease (NAFLD) is a metabolic disorder caused by the exclusion of alcohol and other liver-damaging factors. Its spectrum includes non-alcoholic steatosis and non-alcoholic steatohepatitis, and in severe cases, it can progress to cirrhosis and even liver cancer. NAFLD is often characterized by non-alcoholic lipid accumulation in the liver, ballooning degeneration of hepatocytes, and liver inflammation, and is frequently accompanied by abnormal glucose and lipid metabolism, obesity, and insulin resistance. Abnormal glucose and lipid metabolism are primarily associated with the development of NAFLD, increasing the risk of complications and mortality related to liver and vascular diseases. Abnormal glucose and lipid metabolism are not only the most typical clinical features of NAFLD but also significantly linked to metabolic syndromes such as diabetes, obesity, and hypertension. Its core pathology involves neuroendocrine disorders, chronic inflammatory responses, and gut microbiota dysbiosis.

[0003] Scholars who proposed the "multiple parallel attack hypothesis" believe that disruptions to the body's circadian rhythms, gut microbiota dysbiosis, and poor nutritional status collectively promote the occurrence and progression of NAFLD. NAFLD patients exhibit varying degrees of gut microbiota dysbiosis, and these changes show individual differences as the disease progresses. In particular, the gut microbiota can promote NAFLD development through multiple pathways, providing new insights into its prevention and treatment. Studies have confirmed that regulating the gut microbiota in patients can improve NAFLD-related laboratory indicators and slow its progression.

[0004] Modern medicine primarily treats NAFLD by changing unhealthy lifestyles, focusing on weight loss, lipid reduction, and liver protection. Traditional Chinese medicine (TCM) categorizes NAFLD under terms like "fat qi" and "accumulation." The *Nan Jing* (Classic of Difficult Issues), in its 56th chapter, states, "The accumulation in the liver is called fat qi." The *Danxi Medical Collection* states, "The spleen qi is the healthy yang qi of the body… phlegm and turbidity naturally disperse." Li Zhongzi's *Essential Readings for Physicians* further states, "Once this body exists, it relies on the qi of grains. Grains enter the stomach, are distributed throughout the six fu organs, and qi arrives, harmonizing with the five viscera to generate blood. Humans depend on this for life. Therefore, it is said that the foundation of acquired constitution lies in the spleen." The spleen is the foundation of acquired constitution, the source of qi and blood production. The essential nutrients from food and water required for the normal physiological activities of the five viscera and six fu organs all depend on the spleen's function of transportation and transformation. While transporting these nutrients, the spleen also distributes body fluids to nourish the whole body. It is also the pivot of the body's qi mechanism, playing a crucial role in fluid metabolism. When the spleen functions properly, qi and blood flow smoothly, body fluids are nourished, and water is distributed effectively. The spleen's function of governing transformation and transportation is similar to that of the gut microbiota, which is responsible for nutrient metabolism and regulating the body's immunity. The gut microbiota participates in the digestion and absorption of food and drink, the synthesis of nutrients, the maintenance of the intestinal barrier, and the regulation of the immune system. Zhang Zhicong stated in *Ling Shu Ji Zhu* that "when the essence overflows outward, the skin and flesh become plump and fatty; when it remains inward, the body becomes full of fat and grease." *Wei Sheng Bao Jian* states that "when a person's spleen and stomach are weak, and their diet is irregular or excessively cold or raw, they cannot digest and transform food, leading to accumulation and lumps." *Su Wen* states that "all dampness and swelling belong to the spleen." Physicians throughout history have believed that this disease is closely related to improper diet and damage to the spleen and stomach. When the spleen fails to function properly, water and dampness cannot be transformed, accumulating into phlegm. If this stagnation persists, it leads to qi stagnation, blood stasis, and the internal generation of damp-heat.

[0005] This invention posits that dampness, being a yin evil, is heavy, sticky, and persistent, obstructing the flow of qi. Lingnan (southern China) is particularly humid, with external dampness triggering internal dampness. In the early stages, dampness obstructs the flow of qi; in prolonged illness, it obstructs blood vessels, leading to blood stasis. This obstruction of blood vessels further hinders the circulation of qi, blood, and body fluids, thus generating internal phlegm and dampness. Spleen qi deficiency, internal generation of phlegm and dampness, and blood stasis transforming into heat are mutually causal and transformative, subsequently altering the intestinal microecological environment, leading to intestinal flora imbalance. Intestinal flora imbalance can further cause nutrient absorption disorders, abnormal glucose and lipid metabolism, damage to the intestinal barrier, and induce inflammatory responses, weakening the body's immunity and exacerbating spleen deficiency. Therefore, the "function of the spleen" in traditional Chinese medicine is closely related to the intestinal flora.

[0006] Based on the above, this unique theoretical feature of combining traditional Chinese medicine with modern medicine also provides a theoretical basis for the multi-target and all-round prevention and treatment of non-alcoholic fatty liver disease using traditional Chinese medicine. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a traditional Chinese medicine composition for treating non-alcoholic fatty liver disease, its preparation method, and its application. This traditional Chinese medicine composition can improve lipid accumulation and ballooning degeneration in the liver, improve the levels of triglycerides (TG), cholesterol (TC), low-density lipoprotein (LDL-C), and high-density lipoprotein (HDL-C) in blood lipids, reduce the liver damage marker alanine aminotransferase (ALT), inhibit inflammatory factors TNF-α and IL-1β, significantly reduce the body weight of NAFLD mice, improve glucose tolerance, increase insulin sensitivity, reduce insulin resistance, and simultaneously regulate the intestinal flora and increase the abundance of Akkermansia bacteria in the intestine.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] In a first aspect, the present invention provides a traditional Chinese medicine composition comprising the following raw materials in parts by weight: 15-20 parts of Coptis chinensis, 15-18 parts of Pinellia ternata, 30-45 parts of Trichosanthes kirilowii peel, 10-15 parts of Citrus reticulata peel, 15-20 parts of Salvia miltiorrhiza, and 15-20 parts of Atractylodes macrocephala.

[0010] This invention targets the key pathogenesis of spleen deficiency and dampness accumulation. It uses Trichosanthes peel as the chief ingredient to clear heat, resolve phlegm, and promote qi circulation and chest tightness. Coptis chinensis, bitter and cold, is used as the assistant ingredient to purge stomach heat and dry dampness, opening up heat stagnation in the middle jiao. Pinellia ternata, pungent and warm, dries spleen dampness and resolves phlegm, opening up dampness stagnation in the middle jiao. The combination of these two ingredients, one bitter and one pungent, balances the ascending and descending functions, harmonizing yin and yang, with the pungent opening and the bitter descending complementing each other. Salvia miltiorrhiza is added as an adjuvant to invigorate blood, remove blood stasis, and relieve pain. Citrus reticulata peel, one of the four major southern Chinese medicinal herbs and one of the ten major Guangdong medicinal herbs, has been known as "Southern Ginseng" since ancient times, with particularly significant effects in regulating qi, relieving chest tightness, and drying dampness and resolving phlegm. Atractylodes macrocephala strengthens the spleen, replenishes qi, dries dampness, and promotes diuresis. The combination of these herbs is synergistic, achieving the effects of strengthening the spleen, removing dampness, invigorating blood, and removing blood stasis.

[0011] Within the scope of this invention, the weight parts of the traditional Chinese medicine composition can regulate intestinal flora, repair the intestinal mucosal barrier, regulate lipid metabolism disorders, and have a regulatory effect on lipid accumulation, ballooning degeneration, liver damage indicators such as alanine aminotransferase and glucose tolerance, improve insulin sensitivity, and regulate insulin resistance inflammatory factors.

[0012] Preferably, the traditional Chinese medicine composition comprises the following raw materials in parts by weight: 14-16 parts of Coptis chinensis, 12-15 parts of Pinellia ternata, 30-33 parts of Trichosanthes kirilowii peel, 10-14 parts of Citrus reticulata peel, 15-18 parts of Salvia miltiorrhiza, and 15-18 parts of Atractylodes macrocephala.

[0013] Within the specified weight range, the traditional Chinese medicine composition slightly improves the structure and abundance of intestinal flora; has a weak repair effect on intestinal mucosal barrier damage; slightly reduces weight changes; and slightly decreases total cholesterol, triglycerides, and low-density lipoprotein cholesterol, while slightly increasing high-density lipoprotein cholesterol. It also improves lipid accumulation, ballooning degeneration, liver damage indicators such as alanine aminotransferase and glucose tolerance, and enhances insulin sensitivity, but has a weak regulatory effect on insulin resistance inflammatory factors.

[0014] More preferably, the traditional Chinese medicine composition comprises the following raw materials in parts by weight: 15 parts Coptis chinensis, 15 parts Pinellia ternata, 30 parts Trichosanthes kirilowii peel, 10 parts Citrus reticulata peel, 15 parts Salvia miltiorrhiza, and 15 parts Atractylodes macrocephala.

[0015] The traditional Chinese medicine composition described in this invention, when used in specific weight proportions, can improve lipid accumulation and ballooning degeneration in the liver, improve the levels of triglycerides (TG), cholesterol (TC), low-density lipoprotein (LDL-C), and high-density lipoprotein (HDL-C) in blood lipids, reduce the liver damage marker alanine aminotransferase (ALT), inhibit inflammatory factors TNF-α and IL-1β, significantly reduce the body weight of NAFLD mice, improve glucose tolerance, increase insulin sensitivity, reduce insulin resistance, and simultaneously regulate the intestinal flora and increase the abundance of Akkermansia bacteria in the gut.

[0016] Specifically, it improves the levels of triglycerides (TG), total cholesterol (TC), low-density lipoprotein (LDL-C), and high-density lipoprotein (HDL-C) in blood lipids, reduces the liver damage marker alanine aminotransferase (ALT), inhibits inflammatory factors TNF-α and IL-1β, significantly reduces the body weight of NAFLD mice, improves glucose tolerance, increases insulin sensitivity, reduces insulin resistance, and simultaneously regulates the gut microbiota, increasing the abundance of Akkermansia bacteria in the gut.

[0017] Secondly, the present invention provides a method for preparing the above-mentioned traditional Chinese medicine composition, comprising the following steps:

[0018] (1) Weigh the raw materials according to the weight ratio, soak them in water for 30-40 minutes, decoct them, filter the decoction to obtain the Chinese medicine liquid;

[0019] (2) The Chinese medicine liquid obtained in step (1) is evaporated, and the liquid after rotary evaporation is collected; then dried and concentrated to obtain concentrated extract.

[0020] (3) Freeze-dry the concentrated extract obtained in step (2) to obtain the traditional Chinese medicine composition.

[0021] Preferably, in step (1), the volume ratio of water to the raw materials is (8:10):1; the decoction is performed more than twice; and the total decoction time is 1.5h-2h.

[0022] Preferably, in step (2), the rotary evaporation temperature is 80°C and the rotation speed is 60 rpm.

[0023] Preferably, in step (3), the freezing temperature is -80℃ and the freezing time is 24h.

[0024] Freeze-drying yields lyophilized powder for subsequent animal experiments.

[0025] Thirdly, the present invention provides a medicament for treating non-alcoholic fatty liver disease, comprising the above-mentioned traditional Chinese medicine composition.

[0026] Preferably, the medicament for treating non-alcoholic fatty liver disease further includes pharmaceutically acceptable excipients.

[0027] Preferably, the dosage form of the drug for treating non-alcoholic fatty liver disease is granules, tablets, pills, or capsules.

[0028] Preferably, the drug is used to reduce lipid accumulation and ballooning degeneration in the liver of NAFLD; reduce serum ALT levels in NAFLD patients; reduce serum TG, TC, LDL-C, and HDL-C in NAFLD patients; inhibit inflammatory factors TNF-α and IL-1β in NAFLD patients; reduce fasting blood glucose levels in NAFLD patients; improve glucose tolerance; increase insulin sensitivity; and regulate gut microbiota.

[0029] The beneficial effects of this invention are as follows:

[0030] The traditional Chinese medicine composition of this invention (Shenju Qushi Formula) has a significant effect against non-alcoholic fatty liver disease (NAFLD). It can improve lipid accumulation and ballooning degeneration in the liver, improve the levels of triglycerides (TG), cholesterol (TC), low-density lipoprotein (LDL-C), and high-density lipoprotein (HDL-C) in blood lipids, reduce the liver damage marker alanine aminotransferase (ALT), inhibit inflammatory factors TNF-α and IL-1β, significantly reduce the body weight of NAFLD mice, improve glucose tolerance, increase insulin sensitivity, reduce insulin resistance, and regulate intestinal flora, increasing the abundance of Akkermansia bacteria in the intestine. Attached Figure Description

[0031] Figure 1 The results of Oil Red O staining and HE staining of the livers of mice in each group are shown.

[0032] Figure 2 A bar chart showing the percentage of lipid droplet area in the liver of mice in each group after Oil Red O staining; * indicates p<0.05.

[0033] Figure 3The effect of the traditional Chinese medicine composition on the body weight of mice; * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, **** indicates p<0.0001.

[0034] Figure 4 The effect of the traditional Chinese medicine composition on triglycerides (TG), cholesterol (TC), low-density lipoprotein (LDL-C) and high-density lipoprotein (HDL-C) in mice; * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, **** indicates p<0.0001.

[0035] Figure 5 The effects of a traditional Chinese medicine composition on the liver inflammatory factors TNF-α and IL-1β and the liver injury marker alanine aminotransferase (ALT) in mice were investigated. A represents the changes in IL-1β expression in mouse serum of different groups, B represents the changes in TNF-α expression in mouse serum of different groups, and C represents the changes in ALT expression in mouse serum of different groups. * indicates p<0.05; ** indicates p<0.01, *** indicates p<0.001, and **** indicates p<0.0001.

[0036] Figure 6 The effects of the traditional Chinese medicine composition on fasting blood glucose, glucose tolerance, and insulin sensitivity in mice are shown in Figure 1. A represents the results of glucose tolerance and insulin tolerance tests, B represents the fasting blood glucose in mice, and C represents the area under the glucose tolerance curve and the area under the insulin tolerance curve. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001.

[0037] Figure 7 The effect of a traditional Chinese medicine composition on the triglyceride-glucose index (TYP), a substitute indicator for insulin resistance in mice; * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, **** indicates p<0.0001.

[0038] Figure 8 Partial least squares regression analysis of gut microbiota in each group of mice.

[0039] Figure 9 This is a graph showing the abundance of gut microbiota in mice.

[0040] Figure 10 Heatmap of gut microbiota distribution in each group of mice.

[0041] Figure 11 Comparison of differences among the top 10 bacterial strains. Detailed Implementation

[0042] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0043] The raw materials for this invention are sourced as follows:

[0044] Trichosanthes peel: The dried, mature pericarp of *Trichosanthes kirilowii* Maxim., a plant in the Cucurbitaceae family. Sweet and cold in nature. It enters the Lung and Stomach meridians. Atractylodes macrocephala: The dried rhizome of *Atractylodes macrocephala* Koidz., a plant in the Asteraceae family. Bitter, sweet, and warm in nature. It enters the Spleen and Stomach meridians. Coptis chinensis: The dried rhizome of *Coptis chinensis* Franch., a plant in the Ranunculaceae family. Bitter and cold in nature. It enters the Heart, Spleen, Stomach, Liver, Gallbladder, and Large Intestine meridians. Pinellia ternata: The dried tuber of *Pinellia ternata* (Thunb.) Breit., a plant in the Araceae family. Pungent, warm, and toxic. It enters the Spleen, Stomach, and Lung meridians. Salvia miltiorrhiza: The root of *Salvia miltiorrhiza*, a plant in the Lamiaceae family. Bitter and slightly cold in nature. It enters the Heart and Liver meridians. Orange peel: The dried, mature peel of Citrus reticulata Blanco and its cultivated varieties, belonging to the Rutaceae family. It is bitter, pungent, and warm in nature. It enters the lung and spleen meridians.

[0045] Laboratory animals: C57BL / 6 mice (male, 6–8 weeks old, weighing 20–22 g) were purchased from Guangdong Provincial Medical Laboratory Animal Center and housed in an SPF environment (temperature 22±2℃, humidity 50±10%), provided with food and water. ApoE- / - mice (male, 6–8 weeks old, weighing 20–22 g) were purchased from Jiangsu Yaokang Jicui Biotechnology Co., Ltd. and housed in an SPF environment (temperature 22±2℃, humidity 50±10%), provided with food and water.

[0046] Experimental reagents: 4% paraformaldehyde (Beyotime, China); Total cholesterol (TC) assay kit (Nanjing Jiancheng, China); Triglyceride (TG) assay kit (Nanjing Jiancheng, China); High-density lipoprotein (HDL-C) assay kit (Nanjing Jiancheng, China); Low-density lipoprotein (LDL-C) assay kit (Nanjing Jiancheng, China); Alanine aminotransferase (ALT / GPT) test kit (Nanjing Jiancheng, China); Hematoxylin staining solution (Beyotime, China); Oil Red O stock solution (Sigma-Aldrich); Isopropanol (Guangzhou Chemical Plant); 4% paraformaldehyde fixative; PBS; Glycerol (Sigma-Aldrich).

[0047] Experimental instruments and consumables: Disposable syringe (1mL) (Kangli Medical Technology Co., Ltd., China); blood glucose meter (Roche, Switzerland); blood glucose test strips (Roche, Switzerland); electronic analytical balance (Mettler-Toledo, Switzerland); inverted microscope (Olympus, USA); ELISA reader (BioTek, USA); pipette (Eppendorf, Germany); refrigerated high-speed centrifuge (Eppendorf, Germany); tissue embedding machine (Thermo Scientific, USA); paraffin microtome (Leica, Germany); fully automated staining and mounting workstation (Leica, Germany).

[0048] Example 1:

[0049] An embodiment of the traditional Chinese medicine composition of the present invention includes the following raw materials in parts by weight: 15g of Coptis chinensis, 15g of Pinellia ternata, 30g of Trichosanthes kirilowii peel, 10g of Citrus reticulata peel, 15g of Salvia miltiorrhiza, and 15g of Atractylodes macrocephala.

[0050] The preparation method of the traditional Chinese medicine composition described in this embodiment includes the following steps:

[0051] (1) Weigh the raw materials according to the weight ratio, soak them in water for 30-40 minutes, and then decoct them twice. Each time, decoct them with 10 times the weight of the medicine in water for 1.5 hours each time to obtain the Chinese medicine liquid.

[0052] (2) The Chinese herbal medicine liquid was rotary evaporated at a temperature of 80°C and a rotation speed of 60 rpm. The evaporated liquid was collected and stored in a refrigerator at 4°C for drying to obtain a concentrated extract.

[0053] (3) The concentrated extract is frozen at -80°C for 24 hours to obtain a frozen extract, and then the frozen extract is freeze-dried to obtain a traditional Chinese medicine composition.

[0054] Example 2:

[0055] An embodiment of the traditional Chinese medicine composition of the present invention includes the following raw materials in parts by weight: 16g of Coptis chinensis, 12g of Pinellia ternata, 35g of Trichosanthes kirilowii peel, 13g of Citrus reticulata peel, 17g of Salvia miltiorrhiza, and 18g of Atractylodes macrocephala.

[0056] The preparation method of the traditional Chinese medicine composition described in this embodiment is the same as that in Embodiment 1.

[0057] Example 3:

[0058] An embodiment of the traditional Chinese medicine composition of the present invention includes the following raw materials in parts by weight: 14g of Coptis chinensis, 12g of Pinellia ternata, 33g of Trichosanthes kirilowii peel, 14g of Citrus reticulata peel, 18g of Salvia miltiorrhiza, and 16g of Atractylodes macrocephala.

[0059] The preparation method of the traditional Chinese medicine composition described in this embodiment is the same as that in Embodiment 1.

[0060] Example 4:

[0061] An embodiment of the traditional Chinese medicine composition of the present invention includes the following raw materials in parts by weight: 20g of Coptis chinensis, 18g of Pinellia ternata, 45g of Trichosanthes kirilowii peel, 15g of Citrus reticulata peel, 20g of Salvia miltiorrhiza, and 20g of Atractylodes macrocephala.

[0062] The preparation method of the traditional Chinese medicine composition described in this embodiment is the same as that in Embodiment 1.

[0063] Comparative Example 1:

[0064] A comparative example of the traditional Chinese medicine composition of the present invention includes the following raw materials in parts by weight: 30g of Coptis chinensis, 30g of Pinellia ternata, 50g of Trichosanthes kirilowii peel, 20g of Citrus reticulata peel, 25g of Salvia miltiorrhiza, and 25g of Atractylodes macrocephala.

[0065] The preparation method of the traditional Chinese medicine composition described in this comparative example is the same as that in Example 1.

[0066] Comparative Example 2:

[0067] A comparative example of the traditional Chinese medicine composition of the present invention includes the following raw materials in parts by weight: 3g of Coptis chinensis, 3g of Pinellia ternata, 5g of Trichosanthes kirilowii peel, 4g of Citrus reticulata peel, 3g of Salvia miltiorrhiza, and 3g of Atractylodes macrocephala.

[0068] The preparation method of the traditional Chinese medicine composition described in this comparative example is the same as that in Example 1.

[0069] Based on relevant animal experiments and analyses, the traditional Chinese medicine compositions prepared in Examples 1-7 above have the following advantages and disadvantages compared to those prepared in the examples:

[0070] Example 1 demonstrates a significant anti-nonalcoholic fatty liver disease (NAFLD) effect, improving lipid accumulation and ballooning degeneration in the liver, reducing triglyceride (TG), total cholesterol (TC), low-density lipoprotein (LDL-C), and high-density lipoprotein (HDL-C) levels in the blood, lowering the liver damage marker alanine aminotransferase (ALT), inhibiting inflammatory factors TNF-α and IL-1β, significantly reducing the body weight of NAFLD mice, improving glucose tolerance, increasing insulin sensitivity, reducing insulin resistance, and simultaneously regulating the intestinal flora and increasing the abundance of Akkermansia bacteria in the gut.

[0071] Compared with Example 1, Example 2 showed weaker effects in regulating gut microbiota, repairing the intestinal mucosal barrier, and regulating lipid metabolism disorders. This was manifested in the following ways: slight improvement in gut microbiota structure and abundance; a weak repair effect on damaged intestinal mucosal barrier; slightly reduced weight changes; a slight decrease in total cholesterol, triglycerides, and low-density lipoprotein cholesterol, and a slight increase in high-density lipoprotein cholesterol; and weaker effects on lipid accumulation, ballooning degeneration, liver damage indicators such as alanine aminotransferase and glucose tolerance, improved insulin sensitivity, and regulation of inflammatory factors related to insulin resistance.

[0072] Compared with Example 1, Example 3 showed weaker effects in regulating gut microbiota, repairing the intestinal mucosal barrier, and regulating lipid metabolism disorders than Formula 2. It improved lipid accumulation and ballooning degeneration in the liver and had a certain regulatory effect on inflammatory factors, but the effect was weaker than that of Example 2. This was reflected in the following: differences in gut microbiota structure and abundance; a reduction in pathogenic bacteria after intervention in Example 3, but no significant regulatory effect on Akkermansia; a decrease in triglycerides and low-density lipoprotein cholesterol among blood lipid indicators, while high-density lipoprotein cholesterol and total cholesterol showed no significant difference; it could reduce liver damage indicator alanine aminotransferase, improve glucose tolerance, increase insulin sensitivity, reduce insulin resistance, and inhibit inflammatory factors TNF-α and IL-1β, but without statistical significance.

[0073] Compared with Example 1, Example 4 showed no significant effect in regulating gut microbiota, repairing the intestinal mucosal barrier, regulating lipid metabolism disorders, improving hepatic lipid accumulation, and reducing ballooning degeneration. These effects were weaker than in Example 1, manifested in the following ways: no significant changes in gut microbiota structure and abundance; persistent damage to the intestinal mucosal barrier; no significant changes in body weight; and no significant changes in blood lipid indicators such as total cholesterol, triglycerides, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol. It also had no regulatory effect on improving glucose tolerance, increasing insulin sensitivity, reducing insulin resistance, or regulating inflammatory factors.

[0074] Compared with Example 1, Comparative Example 1 showed weaker effects in regulating gut microbiota, repairing the intestinal mucosal barrier, and regulating lipid metabolism disorders. This was reflected in the following: slight improvement in gut microbiota structure and abundance; weak repair effect on damaged intestinal mucosal barrier; slight reduction in weight change; slight decrease in total cholesterol, triglycerides, and low-density lipoprotein cholesterol, and slight increase in high-density lipoprotein cholesterol among blood lipid indicators; and weaker effects on lipid accumulation, ballooning degeneration, liver damage indicators such as alanine aminotransferase and glucose tolerance, improved insulin sensitivity, and regulation of inflammatory factors related to insulin resistance.

[0075] Compared with Example 1, Comparative Example 2 showed weaker effects in regulating gut microbiota, repairing the intestinal mucosal barrier, and regulating lipid metabolism disorders. This was reflected in the following: slight improvement in gut microbiota structure and abundance; weak repair effect on damaged intestinal mucosal barrier; slight reduction in weight change; slight decrease in total cholesterol, triglycerides, and low-density lipoprotein cholesterol, and slight increase in high-density lipoprotein cholesterol among blood lipid indicators; and weaker effects on lipid accumulation, ballooning degeneration, liver damage indicators such as alanine aminotransferase and glucose tolerance, improved insulin sensitivity, and regulation of inflammatory factors related to insulin resistance.

[0076] In summary, Example 1 is the optimal solution. Preparing the traditional Chinese medicine composition described in the above examples into oral formulations such as capsules, granules, tablets, powders, pills, or oral liquids using conventional molding processes does not affect the effect of the traditional Chinese medicine composition in regulating lipid metabolism disorders.

[0077] The pharmacological effects of the traditional Chinese medicine composition prepared in Example 1 of this invention are described in detail below. To verify the therapeutic effect of the traditional Chinese medicine composition of this invention on non-alcoholic fatty liver disease and the improvement of intestinal flora imbalance, the following animal experiments were conducted.

[0078] Experimental Example 1

[0079] I. Experimental Methods (I) Construction of Glucose and Lipid Metabolism Phenotype in Mice with Nonalcoholic Fatty Liver Disease

[0080] ApoE- / - mice were acclimatized in an SPF environment for one week, followed by a high-fat diet for four weeks to establish a non-alcoholic fatty liver disease (NAFLD) animal model. Subsequently, the low-dose group, high-dose group, and positive control group of the traditional Chinese medicine (TCM) composition were administered the high-dose, low-dose, and positive control drug rosuvastatin via gavage, respectively, on top of the high-fat diet. The administration period was 16 weeks, and the general condition of the mice was recorded. At the end of the experiment, the mice were euthanized by enucleation after pentobarbital anesthesia and blood collection. Blood and liver tissues were collected on the day the experiment ended for subsequent testing.

[0081] (II) Experimental Grouping and Drug Administration: The experimental mice were divided into the following 5 groups: control group (C57BL / 6J mice + normal diet); NAFLD group (ApoE- / - mice + high-fat diet); NAFLD+SJQS_L group (ApoE- / - mice + high-fat diet + low-dose traditional Chinese medicine combination); NAFLD+SJQS_H group (ApoE- / - mice + high-fat diet + high-dose traditional Chinese medicine combination); NAFLD+rosuvastatin group (ApoE- / - mice + high-fat diet + rosuvastatin). Low-dose traditional Chinese medicine group: dosage 50mg / g / day (medicinal material weight / mouse weight / day, the same below);

[0082] High-dose traditional Chinese medicine group: dosage 100mg / g / day;

[0083] Rosuvastatin group: dose 0.15 mg / g / day.

[0084] Seven animals were in each group, and the administration details were as follows: NAFLD+SJQS_L group: the traditional Chinese medicine composition prepared in Example 1, dose 50 mg / g / day. NAFLD+SJQS_H group: the traditional Chinese medicine composition prepared in Example 1, dose 100 mg / g / day. NAFLD+rosuvastatin group: rosuvastatin, the positive control drug, dose 0.15 mg / g / day. All three groups (NAFLD+SJQS_L, NAFLD+SJQS_H, and NAFLD+rosuvastatin) were administered the medicine by gavage for 16 weeks.

[0085] (III) Measurement of relevant indicators of non-alcoholic fatty liver disease

[0086] 1. Fasting blood glucose (FBG) measurement

[0087] During the experimental period, the mice's weight was measured weekly and their fasting blood glucose was measured every two weeks. The fasting blood glucose measurement method was as follows: all mice were fasted for 8 hours but allowed free access to water. The scissors were sterilized by high temperature and autoclaving the day before the experiment. After calming the mice, a small incision was made in their tails, the first drop of blood was discarded, and a drop of fresh blood was placed directly onto a blood glucose test strip (using a Roche blood glucose meter and its matching test strips; the test strips were for single use only). The blood glucose value was read and recorded. Then, a suitable amount of alcohol was used to treat the mouse's tail wound.

[0088] 2. Oral glucose tolerance test (OGTT)

[0089] In week 16 of the experiment, the mice underwent an oral glucose tolerance test (OGTT): all mice were fasted for 12 hours but allowed free access to water. Then, each group of mice was administered glucose solution at 10 ml / kg by gavage. Blood glucose levels at 0 h, 0.5 h, 1.0 h, 1.5 h, and 2 h were measured and recorded at the time of gavage (0 h). The blood glucose measurement method was the same as that for fasting blood glucose (FBG).

[0090] 3. Insulin Tolerance Test (IGITT)

[0091] In week 16 of the experiment, IGITT was measured in mice: all mice were fasted but allowed free access to water for 12 hours. Insulin was diluted with physiological saline to prepare an insulin concentration of 0.5 U / 10 mL. The weight of the mice was measured, and the insulin injection dose (0.1 ml / 10 g) was calculated based on the weight. Blood glucose was measured before insulin injection. After intraperitoneal injection of insulin, blood glucose was measured at 15 min, 30 min, 45 min, and 60 min. The blood glucose measurement method was the same as that for FBG measurement. After the experiment, each cage was replenished with feed.

[0092] 4. Calculation of the Triglyceride-Glucose Index (TYP), a substitute indicator for insulin resistance.

[0093] TYP = Triglycerides * Fasting Glucose / 2

[0094] 5. Blood lipid panel test

[0095] After the 16-week experiment, serum samples were collected from mice to measure four lipid parameters: total cholesterol (TC), triglycerides (TG), high-density lipoprotein (HDL-C), and low-density lipoprotein (LDL-C). All four parameters were measured using the corresponding kits, and the procedures were performed according to the kit instructions.

[0096] 6. HE staining method

[0097] Liver tissues were harvested from seven mice in each group. After routine fixation, dehydration, embedding, and sectioning, 4 μm paraffin sections were prepared. The sections were dewaxed and baked using xylene, graded ethanol, and distilled water. Hematoxylin and eosin (HE) staining was performed according to the instructions of the HE staining kit. After staining, the sections were dehydrated, cleared, and mounted. Finally, liver tissue sections were photographed using an optical microscope to observe the pathological changes in the mouse liver.

[0098] 7. Oil Red O staining of tissue

[0099] Remove frozen sections from the -20°C freezer and allow to recover to room temperature for 10 min. Pre-incubate with 60% isopropanol at room temperature for 8 min. Stain with Oil Red O solution and incubate at 37°C in the dark for 25 min. Differentiate with 60% isopropanol for 10 s. Wash three times with PBS for 30 s each time, gently shaking. Stain with hematoxylin for 20 s. Wash three times with PBS for 30 s each time, gently shaking. Allow the slides to air dry. Mount the slides with glycerol gelatin.

[0100] 8. Inflammatory factor detection

[0101] Total RNA was extracted from mouse liver tissue and cDNA was obtained by reverse transcription. Primers for mouse TNF and IL-1β were designed, and inflammatory factors were measured by real-time quantitative PCR.

[0102] 9. Measurement of alanine aminotransferase (ALT), a marker of liver damage

[0103] After the 16-week experiment, the serum of mice was collected to measure their alanine aminotransferase (ALT). The index was measured using the corresponding kit, and the operation was performed according to the kit instructions.

[0104] 10. Weight measurement: Tested once a week during the experiment.

[0105] 11. Intestinal flora detection

[0106] (1) Sample collection: The entire intestine of the mouse was removed using sterile surgical forceps and scissors. The contents of the cecum to colon section were cut off and placed in sterile cryovials for preservation. The contents were then frozen in liquid nitrogen for more than 2 hours to ensure sufficient freezing before being transferred to a -80°C freezer for storage.

[0107] (2) Extraction of intestinal microbiota DNA: Intestinal microbiota DNA was extracted using a fecal extraction kit according to the instructions.

[0108] (3) 16S rDNA sequencing.

[0109] (iv) Statistical methods

[0110] SPSS 22.0 statistical software was used. Quantitative data were expressed as mean ± standard deviation (mean ± SD). One-way ANOVA was used for intergroup comparisons, and LSD test was used for pairwise comparisons. The significance level was set at α = 0.05.

[0111] II. Measurement results of indicators related to non-alcoholic fatty liver disease:

[0112] 1. Effects of traditional Chinese medicine composition on liver pathological changes induced by a high-fat diet in mice

[0113] The livers of experimental mice were stained with hematoxylin and eosin (HE), and cells were stained with Oil Red O to observe morphological changes and lipid deposition. Figure 1 HE staining results showed that the control group mice had clear hepatic cord structures, tightly packed hepatocytes, and no obvious fatty degeneration, necrosis, inflammation, or other pathological changes. The NAFLD group mice showed widespread hepatocyte fatty degeneration, cell swelling, and cytoplasm filled with round vacuoles. The high-dose group of the traditional Chinese medicine composition showed a significant reduction in the degree of ballooning degeneration and fatty degeneration of hepatocytes. Oil Red O staining results were consistent with HE staining of the liver.

[0114] The results showed that compared with the control group, a high-fat diet induced a significant increase in the positive area of ​​red lipid droplets in mouse hepatocytes and a significant increase in the percentage of lipid droplet area, indicating that lipid deposition in the liver of mice in the NAFLD group was significantly aggravated. Both the high-dose group of the traditional Chinese medicine combination (NAFLD+SJQS_H) and the rosuvastatin group could inhibit hepatic lipid accumulation. However, compared with the rosuvastatin group, the NAFLD+SJQS_H group showed a significant decrease in the positive area of ​​red lipid droplets in hepatocytes and a significant decrease in the percentage of lipid droplet area (see...). Figure 2 The results indicate that the traditional Chinese medicine composition significantly inhibits lipid accumulation in the liver. The pathological section results also show that the traditional Chinese medicine composition has a stronger protective effect against non-alcoholic fatty liver disease compared to rosuvastatin.

[0115] 2. Effects of traditional Chinese medicine composition on lipid metabolism and body weight in mice induced by a high-fat diet.

[0116] The results of the four tests on body weight and blood lipids of mice in each group are as follows ( Figure 3 , Figure 4 As shown in the figure, the effects of the traditional Chinese medicine composition on mouse body weight, triglycerides (TG), total cholesterol (TC), low-density lipoprotein (LDL-C), and high-density lipoprotein (HDL-C) are observed. The results showed that compared to the control group, the NAFLD group mice had significantly increased body weight, total cholesterol, triglyceride levels, and LDL-C levels. Both the high- and low-dose groups of the traditional Chinese medicine composition and the rosuvastatin group reduced TC and LDL-C. Compared to the rosuvastatin group, the high- and low-dose groups of the traditional Chinese medicine composition significantly reduced mouse body weight and TG. All three groups showed an increasing trend in HDL-C compared to the high- and low-dose groups of the traditional Chinese medicine composition and the rosuvastatin group.

[0117] 3. Effects of traditional Chinese medicine composition on liver damage and inflammation induced by a high-fat diet in mice.

[0118] One characteristic of NAFLD is liver damage. Alanine aminotransferase (ALT) is an enzyme that reflects hepatocyte damage, and ALT is mainly found in the cytoplasm of hepatocytes. When hepatocytes are damaged or necrotic, ALT levels rise significantly. We measured ALT levels in the serum of experimental mice. The results showed that compared with the control group, serum ALT activity was significantly increased in the NAFLD group, while high and low doses of the traditional Chinese medicine composition significantly inhibited the increase in ALT activity (see...). Figure 5 C), while the ALT level in the rosuvastatin group was not different from that in the NAFLD group, indicating that the traditional Chinese medicine composition can alleviate liver damage induced by a high-fat diet and has a significant liver-protective effect, with better efficacy than rosuvastatin.

[0119] Subsequently, we further evaluated the expression of inflammatory cytokines TNF-α and IL-1β in the liver. Notably, both the high- and low-dose groups of the traditional Chinese medicine composition and the rosuvastatin group significantly inhibited the expression of inflammatory cytokines (see...). Figure 5 A and 5B).

[0120] 4. Effects of traditional Chinese medicine composition on glucose metabolism induced by a high-fat diet in mice

[0121] Figure 6-7 This study investigates the effects of a traditional Chinese medicine composition on fasting blood glucose, glucose tolerance, insulin sensitivity, and triglyceride-glucose index in mice. A represents the results of glucose tolerance and insulin tolerance tests; B represents fasting blood glucose levels in mice; and C represents the area under the glucose tolerance curve and the area under the insulin tolerance curve. Fasting blood glucose data showed that the NAFLD group had a significantly higher blood glucose level than the control group (see...). Figure 6B) There was no significant difference in efficacy between the high- and low-dose groups of the traditional Chinese medicine composition and the rosuvastatin group. Glucose tolerance and insulin tolerance test results showed that both the high- and low-dose groups of the traditional Chinese medicine composition were effective, significantly improving glucose tolerance and increasing insulin sensitivity. The rosuvastatin group showed impaired glucose tolerance, which was related to the effects of statins on glucose metabolism. Figure 6 A, Figure 6 C). Figure 7 The triglyceride-glucose index calculation results showed that the NAFLD group had significant insulin resistance compared with the control group. The high and low dose groups of the traditional Chinese medicine composition had significant efficacy, while rosuvastatin had little effect. The efficacy of the high and low dose groups of the traditional Chinese medicine composition against insulin resistance was significantly better than that of the rosuvastatin group.

[0122] 5. The traditional Chinese medicine composition can regulate the intestinal flora of mice with non-alcoholic fatty liver disease.

[0123] Figure 7 Partial least squares regression analysis was performed on the gut microbiota of each group of mice. The scales on the horizontal and vertical axes represent relative distances and have no practical significance. plsda1 and plsda2 represent suspected influencing factors affecting the shift in microbial composition in each group. 16S rDNA sequencing results showed that the traditional Chinese medicine composition of this invention can affect the gut microbiota of NAFLD mice. Partial least squares regression analysis showed that the gut microbiota composition of the NAFLD group differed significantly from the control group, and the NAFLD+SJQS_H group exhibited significant changes in gut microbiota.

[0124] The gut microbiota is a vast and complex community, comprising 500-1000 species and a total bacterial count of 100 trillion, ten times the total number of human cells. 16S rDNA sequencing results showed that after intervention with the herbal composition of this invention in NAFLD mice, the Akkermansia genus in the NAFLD group showed a decreasing trend, while after adjustment with high and low doses of the herbal composition of this invention, it showed varying degrees of increasing trend. Rosuvastatin had little effect on regulating Akkermansia (e.g., ...). Figure 8-10 ).

[0125] Akk bacteria are currently recognized as beneficial bacteria and are a normal part of the human gut microbiota. The abundance of Akk bacteria in the gut microbiota of obese patients is significantly reduced. Akk bacteria are negatively correlated with obesity, diabetes, cardiovascular disease, and low-grade inflammation. Akk bacteria not only protect the integrity of intestinal epithelial cells and the mucus layer, playing a metabolic protective role, but also exert anti-inflammatory effects during inflammatory responses by regulating T cells, the endocannabinoid system, and non-classical Toll-like receptors.

[0126] In summary, the traditional Chinese medicine composition of the present invention has significant therapeutic effects and is safe to use. It can prevent and treat NAFLD by specifically increasing the abundance of Akk bacteria in the intestinal flora, regulating intestinal flora imbalance, repairing the intestinal mucosal barrier, and regulating abnormal glucose and lipid metabolism. Based on the above comprehensive evaluation, the efficacy of the traditional Chinese medicine composition is superior to rosuvastatin, especially in avoiding the hyperglycemia that occurs when using statin drugs.

[0127] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A traditional Chinese medicine composition for treating non-alcoholic fatty liver disease, characterized in that, It is prepared from the following raw materials in parts by weight: 15-20 parts of Coptis chinensis, 15-18 parts of Pinellia ternata, 30-45 parts of Trichosanthes kirilowii peel, 10-15 parts of Citrus reticulata peel, 15-20 parts of Salvia miltiorrhiza, and 15-20 parts of Atractylodes macrocephala.

2. A traditional Chinese medicine composition for treating non-alcoholic fatty liver disease, characterized in that, It is made from the following raw materials in parts by weight: 14-16 parts Coptis chinensis, 12-15 parts Pinellia ternata, 30-33 parts Trichosanthes kirilowii peel, 10-14 parts Citrus reticulata peel, 15-18 parts Salvia miltiorrhiza, and 15-18 parts Atractylodes macrocephala.

3. The traditional Chinese medicine composition for treating non-alcoholic fatty liver disease as described in claim 2, characterized in that, It is made from the following raw materials in parts by weight: 15 parts Coptis chinensis, 15 parts Pinellia ternata, 30 parts Trichosanthes kirilowii peel, 10 parts Citrus reticulata peel, 15 parts Salvia miltiorrhiza, and 15 parts Atractylodes macrocephala.

4. A method for preparing a traditional Chinese medicine composition for treating non-alcoholic fatty liver disease as described in any one of claims 1-3, characterized in that, Includes the following steps: (1) Weigh the raw materials according to the proportion of weight, soak them in water for 30-40 minutes, decoct them, and filter the decoction to obtain the Chinese medicine liquid; (2) Collect the Chinese herbal medicine liquid obtained in step (1) by rotary evaporation, and dry it to obtain concentrated extract; (3) Freeze-dry the concentrated extract obtained in step (2) to obtain the traditional Chinese medicine composition.

5. The method for preparing the traditional Chinese medicine composition for treating non-alcoholic fatty liver disease as described in claim 4, characterized in that, In step (1), the volume ratio of water to raw materials is (8-10):1; the decoction is performed more than twice; and the total decoction time is 1.5h-2h.

6. The method for preparing the traditional Chinese medicine composition for treating non-alcoholic fatty liver disease as described in claim 4, characterized in that, In step (2), the rotary evaporation temperature is 80℃ and the rotation speed is 60 rpm; in step (3), the freezing temperature is -80℃ and the freezing time is 24h.

7. A medicament for treating non-alcoholic fatty liver disease, characterized in that, It is made from the traditional Chinese medicine composition for treating non-alcoholic fatty liver disease as described in any one of claims 1 to 3.

8. The medicament for treating non-alcoholic fatty liver disease as described in claim 7, characterized in that, Pharmaceutically acceptable excipients are also added.

9. The medicament for treating non-alcoholic fatty liver disease as described in claim 7, characterized in that, The dosage form of the drug is granules, tablets, pills, or capsules.

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