A Chinese medicine composition and a product containing the same, a preparation method and use thereof

Abstract

The present invention discloses a Chinese medicine composition having the effect of alleviating NASH, and a product comprising the Chinese medicine composition, a preparation method and its use. The Chinese medicine composition is prepared with Artemisia capillaris, Hedyotis diffusa, Gardenia root, Morus alba, Salvia miltiorrhiza, Licorice and Rhubarb in a mass ratio of 1:1:0.5:0.5:1:0.5:0.5. Experiments have shown that the Chinese medicine compound of the present invention can effectively reduce liver swelling, improve liver pathological changes, reduce serum and liver transaminase, reduce liver lipid accumulation, inhibit the transcription of liver inflammatory factors, and protect the heart, effectively alleviating NASH. The Chinese medicine compound of the present invention has outstanding efficacy, simple dosage form, significant effect, and good application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicines, and specifically relates to a traditional Chinese medicine composition having the effect of alleviating NASH, and a product comprising the traditional Chinese medicine composition, a preparation method and uses thereof. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) is a metabolic liver disease characterized by lipid deposition in hepatocytes and excluding other causes of liver damage. Its disease spectrum includes non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis and hepatocellular carcinoma. With the changes in people's lifestyle and dietary structure, the global incidence of NAFLD has reached about 25.24%. Data show that about 59% of NAFLD patients who underwent liver biopsy have developed NASH. NASH is a subtype of NAFLD, which is a coexistence of hepatic fatty degeneration with liver damage and inflammation. It is worth noting that NASH has become the second most common indication for liver transplantation, a necessary stage for the development of simple fatty liver to cirrhosis, and the second largest cause of hepatocellular carcinoma. NAFLD is considered a multi-system disease. At the same time, NASH is also closely related to extrahepatic diseases such as type 2 diabetes, hypertension and cardiovascular disease.

[0003] The pathogenesis of NASH is complex, the course of the disease is long, and the pathogenesis is still unclear. At this stage, the intervention methods for NASH are still mainly diet control and physical exercise, but the difficulty in changing lifestyle and eating habits and the lack of persistence in exercise have become serious obstacles to treatment. At present, some clinical drugs, such as farnesoid X receptor agonists, PPARα / δ agonists and other targeted drugs are still in clinical trials, and their long-term efficacy and safety remain to be evaluated. Saroglitazar is the first therapeutic drug in the field of NASH in the world, but because of its safety and efficacy issues, it has only been approved for use in India (March 2020). To be precise, there is currently no specific drug for NASH in clinical practice. The market size of NAFLD-related drugs in China is expected to reach 3.2 billion yuan in 2025 and 35.5 billion yuan in 2030. Therefore, the market potential is huge.

[0004] Traditional Chinese medicine has a long history in my country. It has unique advantages in treating NASH, which may be related to the fact that traditional Chinese medicine compound contains multiple effective ingredients and has multi-level and multi-target treatment characteristics. Therefore, traditional Chinese medicine is an important intervention and treatment method to improve and reverse NAFLD and prevent NASH, cirrhosis and end-stage liver disease. Traditional Chinese medicine does not have a clear name for NAFLD / NASH, but according to its clinical manifestations, most of them belong to the categories of diseases and syndromes such as "liver disease", "flank pain" and "accumulation" in traditional Chinese medicine. As early as in the "Inner Canon of Medicine", there were descriptions of fatty liver, such as "Slightly urgent is fat gas under the ribs, like a cup". "Ancient and Modern Medical Mirror" discusses flank pain "If it is caused by violent anger, injury, sadness, qi stagnation, excessive diet... or phlegm accumulation flows into the blood, fighting with the blood, all of which can cause pain", pointing out that emotional disorders and unhealthy diet are important causes of this disease, and are closely related to pathological factors such as phlegm and dampness. Improper diet and overeating of fatty and sweet foods can lead to spleen deficiency, or emotional disorders, liver qi stagnation, wood overcoming spleen earth, which can also cause spleen dysfunction, and then dampness and turbidity stagnate inside. The pathogenesis of this stage is mainly liver depression and spleen deficiency, which is the stage of simple fatty liver; dampness evil accumulates for a long time, and turns into heat, and dampness and heat are accumulated inside. This stage is the stage from simple fatty liver to NASH; phlegm and turbidity cannot be transformed, blocking the qi, qi stagnation and blood stasis, blood stasis, phlegm and blood stasis in the liver, and develop into cirrhosis or even end-stage liver disease. It can be seen that the pathogenesis of NASH stage is mainly dampness and heat accumulation, and the treatment should be mainly heat-clearing and damp-removing. Clinically, heat-clearing and damp-removing prescriptions such as Yinchenhao Decoction and Yinchen Wuling Powder are used to treat NASH, and good results have been achieved. Summary of the invention

[0005] Based on the above situation, the present invention provides a Chinese medicine composition, which can effectively reduce liver swelling, improve liver pathological changes, reduce serum and liver transaminases, reduce liver lipid accumulation, inhibit the transcription of liver inflammatory factors, protect the heart, and effectively relieve NASH.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a Chinese medicine composition having the efficacy of alleviating, preventing and / or treating non-alcoholic fatty liver disease, wherein the Chinese medicine composition is prepared from the following seven medicinal materials: Artemisia capillaris, Hedyotis diffusa, Gardenia jasminoides, Morus alba, Salvia miltiorrhiza, Licorice and Rhubarb;

[0008] The mass ratios of the Artemisia capillaris, Hedyotis diffusa, Gardenia jasminoides, Morus alba, Salvia miltiorrhiza, Licorice and Rhubarb are 1:1:0.5:0.5:1:0.5:0.5, respectively.

[0009] Preferably, the mass ratios of Artemisia capillaris, Oldenlandia diffusa, Gardenia jasminoides, Morus alba, Salvia miltiorrhiza, Licorice and Rhubarb are 1:1:0.5:0.5:1:0.5:0.5, respectively.

[0010] The raw materials used to make the Chinese medicine composition of the present invention are proportioned according to parts by mass, and can be increased or decreased according to the corresponding proportion during production. For example, large-scale production can be measured in kilograms or tons, and small-scale production can also be measured in grams. The weight can be increased or decreased, but the mass ratio of the raw materials between the components remains unchanged.

[0011] Furthermore, the Chinese medicine composition of the present invention is prepared according to a method comprising the following steps:

[0012] (1) mixing the Artemisia capillaris, Hedyotis diffusa, Gardenia jasminoides, Morus alba, Salvia miltiorrhiza, and Licorice in proportion, adding water for boiling extraction, adding rhubarb in a proportion after 1-2 hours, boiling for another 10-20 minutes, filtering to remove residues, and obtaining an extract;

[0013] (2) The extract obtained in step (1) is concentrated under reduced pressure at a temperature of 45-55° C. to remove water, and then freeze-dried to obtain the product.

[0014] In step (1) of the above method, the amount of water added is 10-20 times the total mass of the Artemisia capillaris, Hedyotis diffusa, Gardenia jasminoides, Morus alba, Salvia miltiorrhiza, and Licorice.

[0015] In step (2) of the above method, the freeze-drying conditions are that the sample is pre-frozen at a temperature of -80°C and then vacuum freeze-dried at a temperature of -20°C for 10 hours.

[0016] The present invention also protects the use of the above-mentioned traditional Chinese medicine composition in the preparation of a product having the efficacy of alleviating, preventing and / or treating non-alcoholic fatty hepatitis.

[0017] At the same time, products having the efficacy of alleviating, preventing and / or treating non-alcoholic fatty hepatitis and prepared using the Chinese medicine composition provided by the present invention as an active ingredient also fall within the protection scope of the present invention.

[0018] The present invention has the effect of alleviating, preventing and / or treating non-alcoholic fatty liver disease in at least one of the following aspects: 1) reducing liver swelling (lowering liver index); 2) improving liver pathological changes and reducing fatty degeneration; 3) reducing liver lipid accumulation; 4) reducing serum and liver transaminases; 5) inhibiting the expression of liver inflammatory factors (such as reducing the levels of IL-6 and iNOS).

[0019] The product can be a medicine, a health product or a food.

[0020] The pharmaceutical composition can be prepared directly or by adding pharmaceutically acceptable excipients to any pharmaceutically acceptable dosage form. For example, oral preparations include capsules, tablets, powders, granules, teas or oral liquids. The drugs in the above dosage forms can be prepared according to conventional methods in the pharmaceutical field.

[0021] The research group of the inventor of the present invention has previously discovered that a Chinese medicine compound can effectively relieve NASH, but it is not effective in reducing transaminase. The Chinese medicine compound of the present invention has been adjusted on the basis of the Chinese medicine compound, striving to increase the efficacy of reducing transaminase without changing its effect of relieving NASH.

[0022] The present invention is based on the concept of traditional Chinese medicine and is verified by modern pharmacological research methods. It is found that the Chinese medicine composition can effectively reduce liver swelling, improve liver pathological changes, reduce serum and liver transaminases, reduce liver lipid accumulation, inhibit the transcription of liver inflammatory factors, and effectively relieve NASH. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The body weights of rats in each group on the 1st and 28th days of the experiment (symbols represent significant differences: ###p<0.001 compared with the control group).

[0024] Figure 2 This is the heart, liver, spleen, lung and kidney index of rats in each group (heart, liver, spleen, lung and kidney index = heart, liver, spleen, lung and kidney weight / body weight*100%; symbols represent significant differences: compared with the control group #p<0.05, ##p<0.01, ###p<0.001; compared with the model group *p<0.05, ***p<0.001).

[0025] Figure 3 These are the results of macroscopic observation of the liver and HE staining of the heart, liver, spleen, lung and kidney tissues of rats in each group.

[0026] Figure 4 These are the biochemical indicators of serum and liver tissues of rats in each group (symbols represent significant differences: compared with the control group #p<0.05, ##p<0.01, ###p<0.001; compared with the model group *p<0.05, **p<0.01, ***p<0.001).

[0027] Figure 5 The transcription levels of inflammatory factors in rats in each group (symbols represent significant differences: compared with the control group, #p<0.05, ###p<0.001; compared with the model group, *p<0.05, ***p<0.001). DETAILED DESCRIPTION

[0028] The present invention is further described below in conjunction with specific examples, but the present invention is not limited to the following examples. The methods described are conventional methods unless otherwise specified. The raw materials described can be obtained from public commercial channels unless otherwise specified.

[0029] The Artemisia capillaris, Hedyotis diffusa, Gardenia jasminoides, Morus alba, Salvia miltiorrhiza, Licorice and Rhubarb used in the following examples all comply with the relevant provisions of the Chinese Pharmacopoeia (2020 edition) under the main text of each medicinal material. Before feeding, it was identified that the actual medicinal materials were consistent with the names and the quality met the standards.

[0030] Example 1: Preparation of a Chinese medicine composition for alleviating, preventing and / or treating non-alcoholic fatty hepatitis

[0031] Seven medicinal materials (Artemisia capillaris, Hedyotis diffusa, Gardenia jasminoides, Morus alba, Salvia miltiorrhiza, Licorice and Rhubarb) were prepared in a mass ratio of 1:1:0.5:0.5:1:0.5:0.5. First, the first six medicinal materials were mixed, and the extraction solvent-water was added at a solid-liquid ratio of 1:10 by mass, and the mixture was boiled and extracted for 1 hour, and then Rhubarb was added and boiled for another 10 minutes. The residue was filtered to remove the residue, and the extract was collected, and the water was removed by reduced pressure concentration at a temperature of 45-55°C using a rotary evaporator, pre-frozen at -80°C, and then vacuum freeze-dried at a temperature of -20°C for 10 hours.

[0032] Comparative Example 1

[0033] Four medicinal materials (Artemisia capillaris, Hedyotis diffusa, Gardenia jasminoides, and Morus alba) were prepared in a mass ratio of 1:1:0.5:0.5. First, the four medicinal materials were mixed, and an extraction solvent - water was added at a solid-liquid ratio of 1:10 by mass. The extract was boiled and extracted for 40 minutes. The residue was removed by filtration, and the extract was collected. The extract was concentrated under reduced pressure using a rotary evaporator at a temperature of 45-55°C to remove water, and then pre-frozen at -80°C, and then vacuum freeze-dried at -20°C for 10 hours.

[0034] Example 2: Pharmacodynamics study of Chinese medicine composition

[0035] 1. Purpose of the test:

[0036] The aim of the present invention is to study the improving effect of this Chinese medicine composition on non-alcoholic fatty liver disease.

[0037] 2. Test Materials

[0038] 2.1 Test samples:

[0039] The Chinese medicine composition prepared in Example 1. Samples used in animal experiments: The Chinese medicine composition in Example 1 was reconstituted with pure water into a solution with a crude drug concentration of 5 g / mL and filtered with a 0.22 μm microporous membrane.

[0040] The Chinese medicine composition prepared in Comparative Example 1. Samples used in animal experiments: The Chinese medicine composition of Comparative Example 1 was reconstituted with pure water into a solution with a crude drug concentration of 3 g / mL and filtered with a 0.22 μm microporous membrane.

[0041] 2.2 Experimental animals

[0042] Male SD rats (190±10 g).

[0043] 3 Experimental methods

[0044] 3.1 Animal modeling and drug administration

[0045] 7-week-old male SD rats were kept in an SPF animal room (temperature 21±2℃, light and dark cycle 12 hours), with free access to food and water, and all animal experimental operations were carried out in accordance with the guidelines for experimental animal welfare and trial issued by NIH. In this experiment, a rat model of non-alcoholic fatty hepatitis was established by feeding with methionine and choline deficient diet (MCD diet). MCD diet is one of the internationally recognized methods for establishing NASH models, which can replicate liver pathological conditions close to human NASH. The rats were randomly divided into 6 groups, namely control group, model group, positive drug group, low-dose group, high-dose group, and original group (Comparative Example 1). The control group was fed with MCS feed (control feed of MCD feed, containing methionine and choline), and the model group, positive drug group, low- and high-dose group, high-dose group, and original group were fed with MCD feed; the positive drug group was given 120 mg / kg / d of polyene phosphatidylcholine capsules (PPC, Yishanfu) at the same time as modeling, the low- and high-dose groups and the high-dose groups were given 1.67 g / kg / d and 5 g / kg / d of the above compound by gavage at the same time as modeling, and the original group was given 3 g / kg / d (raw drug concentration) of the comparative compound by gavage. The control group and the model group were given an equal amount of pure water by gavage for 4 weeks. After 4 weeks, the animal weight was measured and the animal was anesthetized by intraperitoneal injection of 10% chloral hydrate (dose 3 mL / kg), the liver was collected and weighed, and the liver tissue samples were prepared according to the relevant methods and preserved for testing.

[0046] 3.2 Pathological observation (HE staining)

[0047] Small pieces of heart, liver, spleen, lung, and kidney tissues from the same position of rats were taken, rinsed with PBS, fixed in 4% paraformaldehyde for 12 hours, dehydrated overnight, and routinely embedded in paraffin. The sections were cut into 5 μm slices, dewaxed, counterstained with hematoxylin for 5 minutes, differentiated with hydrochloric acid alcohol, rinsed with PBS to turn blue, counterstained with eosin for 3 minutes, and routinely dehydrated to xylene; the sections were sealed with neutral gum and examined under a microscope. Observed under a microscope.

[0048] 3.3 Detection of serum and liver tissue biochemical indicators

[0049] The rat abdominal aorta blood was collected and centrifuged at 4°C and 2000 rpm for 10 min. The supernatant was taken and the levels of triglyceride (TG), cholesterol (TC), low-density lipoprotein (LDL-c), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum were detected.

[0050] Take an appropriate amount of liver tissue, add physiological saline at a ratio of 1:9 and perform tissue homogenization. Centrifuge at 4°C and 3500 rpm for 10 min, take the supernatant, and detect the ALT and AST concentrations in the liver tissue.

[0051] 3.4 Real-time quantitative polymerase chain reaction (PCR) detection of liver inflammatory factor levels

[0052] Total RNA was extracted using an RNA extraction kit, reverse transcribed using the kit, and analyzed using quantitative PCR.

[0053] 3.5 Statistical analysis

[0054] SPSS for Windows 19.0 was used for statistical analysis, and the data were expressed as mean ± standard deviation (SD). The differences between the groups were analyzed by one-way ANOVA, and the test level p < 0.05 was considered statistically significant. GraphPad Prism 6.0 was used for graphics.

[0055] 4 Experimental results

[0056] 4.1 Results of rat body weight and organ indexes

[0057] We recorded the weight changes of rats during the experiment, such as Figure 1 As shown in the figure, on the first day of the experiment, there was no significant difference in the body weight of rats in each group (p>0.05). After the end of the experiment (day 28), the body weight of rats in the model group was significantly lower than that of the control group (p<0.001), but the positive control drug and the Chinese medicine composition of the present invention could not reverse the weight loss. This is mainly because the MCD feed has a poor taste, the appetite of rats has decreased significantly, and the food intake of rats cannot be intervened by drugs.

[0058] Figure 2 As shown, the cardiac index of the model group was significantly lower than that of the control group (p<0.01), and the cardiac index of rats in the low-dose group and the high-dose group was significantly higher than that of the model group (p<0.05). The liver index of the model group was significantly higher than that of the control group (p<0.001), and the liver index of rats in the low-dose group and the high-dose group was lower than that of the model group (p<0.05, p<0.05). There was no significant difference in the spleen index, lung index and kidney index of rats in each group. The results show that this Chinese medicine composition can reduce the liver index of NASH rats and increase the cardiac index, and has the effect of protecting the liver and heart.

[0059] 4.2 Conditions of the rat heart, liver, spleen, lungs and kidneys

[0060] like Figure 3 As shown in the figure, from the perspective of liver tissue morphology, the liver of the rats in the control group was dark red, with normal capsule, clear edges and elastic touch. The liver of the rats in the model group was swollen, yellowish in color, tense in capsule, blunt edges and soft to the touch. The low-dose group and the high-dose group were superior to the model group in terms of liver size, color and touch. The results of HE staining showed that the cell boundaries of the liver tissue of the rats in the control group were obvious, the blue staining of the cell nucleus was located in the center of the cell, the hepatic cord structure was clear, and there was no obvious lesion. In the liver tissue of the model group, there were a large number of fat vacuoles, and even adjacent cells were fused into one piece, the cell boundaries were unclear, the cell nucleus was squeezed to one side, and the hepatic cord structure was unclear. Compared with the model group, the fat vacuoles in the liver tissue of the rats in the low-dose group and the high-dose group were reduced, and the hepatic cord structure was restored to a certain extent. There were no significant changes in heart tissue, spleen tissue, lung tissue and kidney tissue. The results show that the Chinese medicine composition can effectively improve the pathological changes of liver tissue in NASH rats, reduce fatty degeneration, and is non-toxic to other organs.

[0061] 4.3 Effects on biochemical indicators of rats

[0062] We first tested the serum lipid levels, such as Figure 4 As shown in the results, the levels of serum total cholesterol (TC), total triglyceride (TG) and low-density lipoprotein (LDL-c) in the model group were significantly lower than those in the control group (p<0.001, p<0.05, p<0.001), and the serum TC levels of rats in the low-dose group and high-dose group were significantly higher than those in the model group (p<0.05, p<0.001), and the effect was dose-dependent. However, although the levels of TG and LDL-c showed an increasing trend, the difference was not significant compared with the model group.

[0063] The serum alanine aminotransferase (ALT) level of rats in the model group was significantly higher than that in the control group (p<0.01), but the aspartate aminotransferase (AST) level was not significantly increased. The ALT and AST levels in the liver tissue of rats in the model group were significantly higher than those in the control group (p<0.001, p<0.001). The low-dose group and the high-dose group could significantly reduce the ALT and AST levels in a dose-dependent manner. The original prescription group could not reduce the serum transaminase level, but could slightly reduce the transaminase level in the liver tissue.

[0064] The results showed that the Chinese medicine composition could effectively reduce the transaminase level of NASH rats and had a protective effect on the liver, which was superior to the original prescription group.

[0065] 4.4 Levels of inflammatory factors in rat liver tissue

[0066] like Figure 5As shown, compared with the control group, the IL-6 and iNOS gene levels in the liver tissue of the model group rats were significantly increased (p<0.05, p<0.001), and the IL-6 and iNOS levels in the liver tissue of the low-dose group and the high-dose group were significantly lower than those in the model group. The results show that the Chinese medicine composition can effectively reduce the IL-6 and iNOS levels in the liver tissue of NASH rats.

[0067] 4.5 Summary of drug efficacy

[0068] The above experiments prove that the Chinese medicine compound of the present invention can effectively reduce liver swelling, improve liver pathological changes, reduce serum and liver transaminases, reduce liver lipid accumulation, inhibit the transcription of liver inflammatory factors, protect the heart, and effectively relieve NASH.

[0069] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Chinese medicine composition having the efficacy of preventing and / or treating non-alcoholic fatty hepatitis, the Chinese medicine composition being prepared from the following seven medicinal materials: Artemisia capillaris, Hedyotis diffusa, Gardenia jasminoides, Morus alba, Salvia miltiorrhiza, Licorice and Rhubarb; The mass ratios of the Artemisia capillaris, Hedyotis diffusa, Gardenia jasminoides, Morus alba, Salvia miltiorrhiza, Licorice and Rhubarb are 1:1:0.5:0.5:1:0.5:0.5, respectively.

2. The method for preparing the Chinese medicine composition according to claim 1, comprising the following steps: (1) Mix the Artemisia capillaris, Hedyotis diffusa, Gardenia jasminoides, Morus alba, Salvia miltiorrhiza and Licorice in proportion, add water for boiling extraction, add rhubarb in a proportion after 1-2 hours, boil for another 10-20 minutes, filter to remove residues, and obtain an extract; (2) The extract obtained in step (1) is concentrated under reduced pressure to remove water, and then freeze-dried to obtain the product.

3. The preparation method according to claim 2, characterized in that: In the step (1), the amount of water added is 10-20 times the total mass of the Artemisia capillaris, Hedyotis diffusa, Gardenia jasminoides, Morus alba, Salvia miltiorrhiza, and Licorice.

4. The preparation method according to claim 2 or 3, characterized in that: In the step (2), the reduced pressure concentration is carried out at a temperature of 45-55°C, and the freeze-drying condition is that the sample is pre-frozen at a temperature of -80°C and then vacuum freeze-dried at a temperature of -20°C for 10 hours.

5. Use of the Chinese medicine composition according to claim 1 or the Chinese medicine composition prepared by the method according to any one of claims 2 to 4 in the preparation of a medicine having the efficacy of preventing and / or treating non-alcoholic fatty hepatitis.

6. The use according to claim 5, characterized in that: The effects of preventing and / or treating non-alcoholic fatty liver disease are embodied in the following aspects: 1) reducing liver swelling; 2) improving liver pathological changes and reducing fatty degeneration; 3) reducing liver lipid accumulation; 4) reducing serum and liver transaminases; 5) inhibiting the expression of liver inflammatory factors.

7. The use according to claim 5, characterized in that: The dosage form of the medicine is an oral preparation, including capsules, tablets, powders, granules, tea or oral liquid.

8. A medicine having the efficacy of preventing and / or treating non-alcoholic fatty hepatitis, wherein the active ingredient is the Chinese medicine composition according to claim 1 or the Chinese medicine composition prepared by the method according to any one of claims 2 to 4.

9. The drug according to claim 8, characterized in that: The dosage form of the medicine is an oral preparation, including capsules, tablets, powders, granules, tea or oral liquid.

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