Traditional Chinese medicine composition for treating metabolic-related fatty liver disease and fermentation method thereof

Abstract

The present invention relates to the technical field of traditional Chinese medicine, and in particular to a Chinese medicine composition and a fermentation method thereof for treating metabolic-related fatty liver disease. A Chinese medicine composition for treating metabolic-related fatty liver disease provided by the present invention is obtained by a fermentation method by the following components in parts by weight, wherein the components include 5-15 parts of hawthorn, 5-10 parts of roselle, 5-15 parts of capillaris, 5-15 parts of dandelion, or, the components include 5-15 parts of hawthorn, 5-10 parts of roselle, 5-15 parts of black-faced general, and 5-15 parts of fire tube leaves. The present invention is based on the understanding of the pathogenesis and treatment principles of metabolic-related fatty liver disease in traditional Chinese medicine, with reference to the research results of modern pharmacology, and according to the principle of treating different diseases with the same method under dialectical guidance, a scientific combination of heat-clearing and dampness-clearing, liver-soothing and gallbladder-promoting medicines is formed to improve and treat a prescription for metabolic-related fatty liver disease.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicines, and in particular to a traditional Chinese medicine composition for treating metabolism-related fatty liver disease and a fermentation method thereof. Background Art

[0002] Metabolic Dysfunction-Associated Fatty Liver Disease (MAFLD) is a stress-induced liver injury associated with lipid metabolism disorders based on insulin resistance and genetic susceptibility. In recent years, the global incidence of MAFLD has reached 38%. The disease is insidious and the early symptoms are often not significant. It requires the use of imaging technology for screening. If it is not prevented and treated in time, medium- and long-term pathological changes, such as liver fibrosis, cirrhosis, and hepatocellular carcinoma, will bring greater disease and economic burdens to patients, families, and society.

[0003] The onset of MAFLD is closely related to overnutrition caused by an unhealthy lifestyle based on family or specific genetic gene mutations. Currently, the important measures for managing MAFLD are mainly lifestyle intervention and drug therapy. However, lifestyle intervention is affected by patient compliance and there are great individual differences. Therefore, there is an urgent need to strengthen the research and development of safe and effective MAFLD drugs.

[0004] The prior art discloses a traditional Chinese medicine composition for treating metabolic-related fatty liver disease, as well as a preparation method and application thereof. The traditional Chinese medicine composition is formed by using components such as fried Citrus aurantium with bran, and alleviates MAFLD by weight loss, but does not improve the problem of liver inflammation damage. Summary of the invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the problem that the traditional Chinese medicine composition in the prior art is difficult to achieve in terms of sustained therapeutic effect after drug withdrawal, thereby providing a traditional Chinese medicine composition for treating metabolic-related fatty liver disease and a fermentation method thereof.

[0006] Another technical problem to be solved by the present invention is to overcome the defect that the Chinese medicine composition in the prior art cannot take into account the systemic pathogenesis of MAFLD, thereby providing a Chinese medicine composition for treating metabolic-related fatty liver disease and a fermentation method thereof, which can improve the problems of systemic insulin resistance and abnormal lipid metabolism.

[0007] In one aspect, the present invention provides a Chinese medicine composition for treating metabolic-related fatty liver disease.

[0008] The composition is obtained by fermentation of the following components in parts by weight, wherein the components include 5-15 parts of hawthorn, 5-10 parts of roselle, 5-15 parts of capillaris, and 5-15 parts of dandelion.

[0009] or,

[0010] The components include 5-15 parts of hawthorn, 5-10 parts of roselle, 5-15 parts of black-faced general, and 5-15 parts of fireweed leaves.

[0011] In some embodiments, the components include 8-12 parts of hawthorn, 6-9 parts of roselle, 8-12 parts of capillaris, and 8-12 parts of dandelion.

[0012] or,

[0013] The components include 8-12 parts of hawthorn, 6-9 parts of roselle, 8-12 parts of black-faced general, and 8-12 parts of fireweed leaves.

[0014] On the other hand, the present invention provides a method for fermenting a traditional Chinese medicine composition, wherein each component is weighed according to a formula, and fermented to obtain the traditional Chinese medicine composition.

[0015] In some of the embodiments, before fermenting the components, the steps of sterilizing and / or processing the components are also included, the sterilization temperature is 28°C-32°C, the sterilization time is 30 h-40 h, the processing temperature is 75°C-85°C, and the processing time is 15 min-25 min.

[0016] In some embodiments, the culture medium used for the fermentation includes MRS medium.

[0017] In some embodiments, the mass ratio of the mixture to the culture medium is (110-120):1, expressed in mg:mL.

[0018] In some embodiments, the fermentation comprises at least one of liquid fermentation and microbial fermentation.

[0019] Preferably, the fermentation temperature of the liquid fermentation is 30°C-35°C, and the fermentation time is 40 h-56 h.

[0020] In some of the embodiments, before liquid fermentation, each component is processed by water bath method, the water bath treatment temperature is 25° C.-35° C., and the water bath treatment time of each component is 30 h-40 h.

[0021] In some embodiments, the fermentation temperature of the microbial fermentation is 35° C.-42° C., and the fermentation time is 12 h-72 h.

[0022] In some of the embodiments, before fermentation with microorganisms, the processing temperature of each component is 75° C.-85° C., and the processing time is 15 min-25 min.

[0023] In some embodiments, the culture medium used in the microbial fermentation further includes bacterial agents and / or enzymes.

[0024] In some embodiments, the concentration of the bacterial agent in the culture medium is 3 Vol%-7 Vol%, and the concentration of the bacterial species in the bacterial agent is 2.5×10 7 CFU / mL-4.5×10 8 CFU / mL.

[0025] Preferably, the bacterial agent is a probiotic, and the probiotic is Akkermansia muciniphila ( Akkermansia muciniphila ), Lactobacillus ( Lactobacillus ),

[0026] In some embodiments, the ratio of the enzyme to the culture medium is (1150-1350):1, expressed in U:g.

[0027] In some embodiments, the enzyme comprises a neutral protease. Preferably, the neutral protease is dispase.

[0028] In some embodiments, the MRS medium includes a carbon source, a nitrogen source and a metal salt, the pH value of the medium is 6-8, the concentration of the carbon source is less than or equal to 20 g / L, the concentration of the nitrogen source is 4 g / L-10 g / L, and the concentration of the metal salt is 0.2 g / L-0.5 g / L. The concentration of the carbon source is preferably 15 g / L-20 g / L.

[0029] More preferably, the concentration of the carbon source is 20 g / L, the concentration of the nitrogen source is 6.6 g / L, the concentration of the metal salt is 6 μg / mL, the concentration of the bacterial agent in the culture medium is 5 Vol%, and the ratio of the enzyme to the culture medium is 1250:1, in units of U:g.

[0030] In some of the embodiments, the carbon source is at least one of glucose and sucrose; the nitrogen source is at least one of yeast powder, ammonia water and peptone; the metal salt is at least one of ferrous salt, magnesium salt, calcium salt, potassium salt or zinc salt; and the pH value of the culture medium is 6.5-7.5.

[0031] Use of the above-mentioned Chinese medicine composition for treating metabolism-related fatty liver disease or the Chinese medicine composition prepared by the fermentation method of the above-mentioned Chinese medicine composition in the preparation of drugs for treating or assisting in the treatment of liver damage.

[0032] In some of the embodiments, the liver damage includes damage to the liver caused by metabolism-related fatty liver disease, liver lipid deposition and lipotoxicity, that is, the above-mentioned Chinese medicine composition for treating metabolism-related fatty liver disease or the Chinese medicine composition prepared by the fermentation method of the above-mentioned Chinese medicine composition is used in medicines for treating metabolism-related fatty liver disease, alleviating liver lipid deposition and lipotoxicity.

[0033] The technical solution of the present invention has the following advantages:

[0034] 1. The present invention provides a traditional Chinese medicine composition for treating metabolism-related fatty liver disease, which is obtained by a fermentation method from the following components in parts by weight, wherein the components include 5-15 parts of hawthorn, 5-10 parts of roselle, 5-15 parts of capillaris, and 5-15 parts of dandelion, or the components include 5-15 parts of hawthorn, 5-10 parts of roselle, 5-15 parts of black-faced general, and 5-15 parts of pyrophora leaves.

[0035] The present invention is based on the understanding and treatment principles of traditional Chinese medicine on the pathogenesis of metabolism-related fatty liver disease, refers to the research results of modern pharmacology, and according to the principle of treating different diseases with the same method under the guidance of dialectics, scientifically combines drugs for clearing away heat and dampness, soothing the liver and promoting bile secretion, to form a prescription that can improve and alleviate metabolism-related fatty liver disease.

[0036] Among them, hawthorn contains biologically active ingredients such as citric acid, chlorogenic acid and quercetin, which can promote digestion and increase gastrointestinal motility. At the same time, it can also reduce blood viscosity, improve blood circulation, lower cholesterol and resist atherosclerosis.

[0037] Roselle contains anthocyanins, polyphenols, organic acids and other biologically active ingredients, which have antioxidant, antibacterial and lipid-lowering activities, and have potential roles in improving abnormal glucose and lipid metabolism and oxidative stress and inflammatory damage.

[0038] The active ingredients of Artemisia capillaris mainly include flavonoids, phenols, organic acids, volatile oils, terpenes, etc., which have the functions of protecting liver function, inhibiting inflammatory response, lowering blood pressure, lowering blood lipids, resisting atherosclerosis, and promoting bile excretion. Among them, the main components of flavonoids are quercetin, naringenin, isorhamnetin, etc., which can regulate lipid synthesis, decomposition, transport and metabolism to achieve the effect of improving lipid metabolism and lowering blood lipids.

[0039] Dandelion contains quercetin, chlorogenic acid, β-sitosterol, para-hydroxyphenylacetic acid and other ingredients. It has good antioxidant, antibacterial, antiviral, lung function improvement, liver protection, immunity enhancement, blood circulation improvement and other effects. It can lower blood sugar, blood lipids, and relieve liver cell inflammatory damage.

[0040] Black-faced General is also known as wrinkled-leaf Amaranthus and crisp-leaf Amaranthus. It tastes spicy and cool in nature. It enters the lung, liver, stomach, and large intestine meridians. The whole plant or root is used as medicine. It has the effects of cooling blood and stopping bleeding, clearing away heat and dampness, as well as detoxifying and reducing swelling. Its active ingredients include tannic acid, saponins and steroid compounds.

[0041] The leaves of the fire tube plant, also known as red blowing wind, are pungent and cool in nature, and have the effects of clearing away heat and detoxifying. They belong to the spleen, stomach, and liver meridians. Black-faced general and fire tube leaves are commonly used anti-tumor and anti-inflammatory medicines.

[0042] The present invention combines Artemisia capillaris and Taraxacum officinale with Hawthorn and Roselle to achieve synergistic effects, and has a significant effect on inhibiting liver lipid deposition and improving insulin sensitivity of insulin target organs (e.g., hepatocytes, adipocytes). Alternatively, the present invention combines Herba Artemisiae Argyi and Herba Pyriferae leaves with Hawthorn and Roselle, mainly utilizing the fact that Herba Artemisiae Argyi and Herba Pyriferae leaves belong to the liver meridian and have the effects of clearing away heat, detoxicating and anti-inflammatory. Because hepatocyte inflammatory damage is a pathological mechanism throughout the pathogenesis of MAFLD, and is closely related to the progression of late liver fibrosis and cirrhosis, improving hepatocyte inflammatory damage is the key to preventing further progression of hepatic fatty degeneration to MASH fibrosis. At the same time, it is observed that the Herba Artemisiae Argyi and Herba Pyriferae leaves are combined with Hawthorn and Roselle, and on the basis of improving MAFLD lipid deposition, they show better relief of hepatocyte inflammatory damage.

[0043] 2. The present invention provides a fermentation method of a Chinese medicine composition for treating metabolic-related fatty liver disease, wherein each component is weighed according to the formula, fermented, and a Chinese medicine composition is obtained. The present invention effectively changes the structure and content of some active ingredients in Chinese medicine by fermenting the components of certain components, improves the efficacy and curative effect of the Chinese medicine composition, improves the quality stability of Chinese medicine, and improves the sustained effect of Chinese medicine. We observed that oral non-fermented Chinese medicine composition, intervention for 3-6 months, ultrasound showed no fatty liver and then stopped the drug, but 42% of the patients re-examined the ultrasound after 6 months of discontinuation of the drug and showed mild fatty liver. 14% of the patients re-examined the ultrasound after 6 months of discontinuation of the drug and showed moderate fatty liver. Another group of patients matched with the condition, age and intervention time were given the same formula dose of fermented Chinese medicine composition, intervention for 3-6 months, ultrasound showed no fatty liver and then stopped the drug, 6 months of follow-up, compared with the patients intervened with non-fermented Chinese medicine composition, the above data decreased (42% vs. 31%, 14% vs. 8%, using fermented Chinese medicine composition vs using non-fermented Chinese medicine composition).

[0044] 3. The present invention provides a fermentation method of a Chinese medicine composition for treating metabolic-related fatty liver disease, wherein the fermentation includes at least one of liquid fermentation or microbial fermentation. The present invention can improve the taste through liquid fermentation on the one hand, and on the other hand, can increase the content of triterpenes, bacterial intracellular polysaccharides, microbial biomass, and bacterial extracellular crude polysaccharides in the Chinese medicine composition, and the fermentation product can better inhibit lipid precipitation and promote lipid metabolism, thereby improving the effect of improving MAFLD, and can also reduce adverse reactions of Chinese medicine, reduce the burden of liver metabolism and detoxification; the present invention can improve the content of small peptides, the types of organic acids, and the content of organic acids in the Chinese medicine composition by using microbial fermentation, which can not only improve MAFLD, but also reshape the diversity of intestinal microorganisms, increase the abundance of probiotics, and enrich the action pathway of the Chinese medicine composition to improve MAFLD from the perspective of intestinal microorganisms and their metabolites.

[0045] 4. The present invention provides a fermentation method for a traditional Chinese medicine composition for treating metabolic-related fatty liver disease. In the microbial fermentation of the present invention, a water bath heating treatment method is adopted to prepare each component, which improves the direct water boiling method, thereby increasing the yield and being able to more effectively remove impurities.

[0046] 5. The present invention provides a fermentation method for a traditional Chinese medicine composition for treating metabolic-related fatty liver disease. The bacterial agent in the microbial fermentation is probiotics. As a strain of microbial fermentation, the probiotics can utilize sugar metabolism to generate a variety of organic acids, reduce the pH value, and also play an antibacterial role, inhibit the growth of intestinal pathogens, and improve the body's immunity.

[0047] At the same time, the culture medium for microbial fermentation also includes enzyme substances. Enzyme substances can not only increase the number of live microorganisms and improve the acid production capacity during the fermentation process, but also produce short-chain fatty acids (acetic acid and propionic acid) by degrading mucin after entering the body, and interact with butyrate-producing bacteria to promote the production of butyrate, thereby improving the body's metabolism. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0049] Figure 1 The body composition results of each group of mice in Experiment 2 of the present invention are Figure 1 ;

[0050] Figure 2 The body composition results of each group of mice in Experiment 2 of the present invention are Figure 2 ;

[0051] Figure 3 The body composition results of each group of mice in Experiment 3 of the present invention are Figure 1 ;

[0052] Figure 4 The body composition results of each group of mice in Experiment 3 of the present invention are Figure 2 ;

[0053] Figure 5 The results of serum IL6 in mice of each treatment group in Experiment 3 of the present invention are Figure 1 ;

[0054] Figure 6 The results of serum IL6 in mice of each treatment group in Experiment 3 of the present invention are Figure 2 ;

[0055] Figure 7 The results of triglyceride content in the liver of mice in each treatment group in Experiment 3 of the present invention are Figure 1 ;

[0056] Figure 8 The results of triglyceride content in the liver of mice in each treatment group in Experiment 3 of the present invention are Figure 2 ;

[0057] Fig. 9 The results of free fatty acid content in the liver of mice in each treatment group in Experiment 3 of the present invention are Figure 1 ;

[0058] Fig.10 The results of free fatty acid content in the liver of mice in each treatment group in Experiment 3 of the present invention are Figure 2 ;

[0059] Fig.11 The results of serum alanine aminotransferase levels in mice in each treatment group in Experiment 3 of the present invention are Figure 1 ;

[0060] Fig.12 The results of serum alanine aminotransferase levels in mice in each treatment group in Experiment 3 of the present invention are Figure 2 ;

[0061] Fig.13 The results of serum aspartate aminotransferase levels of mice in each treatment group in Experiment 3 of the present invention are Figure 1 ;

[0062] Fig.14 The results of serum aspartate aminotransferase levels of mice in each treatment group in Experiment 3 of the present invention are Figure 2 ;

[0063] Fig.15 The anatomical morphology of the liver of mice in each treatment group in Experimental Example 3 of the present invention Figure 1 ;

[0064] Fig.16 The anatomical morphology of the liver of mice in each treatment group in Experimental Example 3 of the present invention Figure 2 ;

[0065] Fig.17 is a diagram showing changes in fat distribution and BMI before and after treatment in Experimental Example 4 of the present invention;

[0066] Figure 1-Figure 16 a represents the model group, b represents the Chinese medicine composition prepared by intervention comparison example 1, c represents the Chinese medicine composition prepared by intervention example 1, d represents the Chinese medicine composition prepared by intervention example 2, e represents the Chinese medicine composition prepared by intervention comparison example 2, f represents the Chinese medicine composition prepared by intervention comparison example 3, g represents the Chinese medicine composition prepared by intervention comparison example 4, and h represents the Chinese medicine composition prepared by intervention comparison example 5. DETAILED DESCRIPTION

[0067] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0068] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0069] Metabolic associated fatty liver disease (MAFLD) has no exact name in ancient Chinese medical books. According to its clinical manifestations, it can be classified into the categories of "flank pain", "accumulation", "jaundice", "liver disease", etc. It is mostly caused by addiction to fatty and sweet foods, improper work and rest, emotional imbalance, and transmission of other diseases, which lead to liver failure to release and spleen failure to function properly, and phlegm, heat, dampness, blood stasis and toxins accumulate in the liver, causing symptoms such as flank pain as the main symptom.

[0070] A large number of literatures have shown that damp-heat constitution is a common constitution type for MAFLD. Dampness (23.75%), heat (12.13%) or both dampness and heat (22.3%) are common pathogenic factors of MAFLD. Damp-heat syndrome is one of the most common clinical syndromes of MAFLD. Therefore, clearing away heat, detoxifying and removing dampness are the main treatment methods for MAFLD. The Western medical pathogenesis of MAFLD is still unclear. Currently, the "two-hit" theory and the "multiple parallel hits theory" are widely accepted. The first hit of the "two-hit theory" refers to the fatty liver caused by insulin resistance induced by overnutrition in the body, and the second hit refers to the release of a large amount of reactive oxygen species causing oxidative stress response as the cell metabolic pressure increases, further promoting lipid deposition and insulin resistance in liver cells, causing lipotoxicity and inflammatory response, aggravating disease progression such as liver tissue damage and fibrosis, and progressing from MAFL to MASH, etc. The "multiple parallel hits theory" is that the special metabolic microenvironment caused by the first and second hits, such as obesity, abnormal glucose and lipid metabolism, oxidative stress and inflammatory response, can hit or interact with liver tissue cells in parallel, and jointly promote the progression of MAFLD.

[0071] The combination and preparation methods of Chinese herbal medicines and prescriptions for improving metabolism in traditional Chinese medicine have a history of thousands of years. The advantage of traditional Chinese medicine lies in the comprehensive management of MAFLD from the perspective of holistic view and dialectical treatment. The treatment mainly focuses on the liver and spleen, while taking into account pathological products such as phlegm, heat, dampness, blood stasis and toxins.

[0072] The inventors found in their long-term research and practice that effective prescriptions and synergistic preparation methods were developed from the perspective of medicine and food homology, and a Chinese medicine composition with good improvement and therapeutic effects on MAFLD was obtained. The Chinese medicine composition provided by the present invention can improve the lipid deposition and inflammatory damage of MAFLD to a certain extent, but the disease relapses after drug withdrawal. The research team combined literature review and after 2 years of exploration, concluded that the Chinese medicine composition prepared by the special fermentation method for the treatment of mild MAFLD showed better improvement in lipid metabolism and relief of inflammatory damage, and after drug withdrawal, it still showed a sustained therapeutic effect.

[0073] The Akkermansia muciniphila mentioned in the present invention ( Akkermansia muciniphila , Akkermansia ) and Lactobacillus ( Lactobacillus ) are all commercially available, and the above-mentioned strains in the examples and comparative examples of the present invention were purchased from Shanghai Collection of Microorganisms Center.

[0074] Example 1

[0075] This embodiment provides a method for preparing a Chinese medicine composition for treating metabolic-related fatty liver disease, and the specific steps and parameters are as follows:

[0076] (1) Take 10 g of hawthorn, 8 g of roselle, 10 g of black-faced general and 10 g of fireweed leaves, wash, drain, air-dry, cut into sections, dry, and pass through a 500-mesh sieve to obtain a powder.

[0077] (2) Mix 20 g of glucose, 4 g of yeast powder and 0.2 g of ferrous sulfate, dilute to 1 L with deionized water, adjust the pH value to 7, and sterilize at 121°C for 20 min to prepare a basal culture medium;

[0078] The activated Akkermansia muciniphila ( Akkermansia muciniphila The Akk bacteria) were washed with sterile 8.5 mg / mL saline, centrifuged, and then the aforementioned saline was added to the original volume and mixed. The process was repeated 3 times to obtain a bacterial suspension of AKK probiotics. The bacterial concentration of the bacterial suspension was 6×10 7 CFU / mL;

[0079] The bacterial suspension was added to the basal medium at a concentration of 5 Vol% to form a fermentation medium.

[0080] (3) The powder was heated in a water bath at 80°C for 20 min. After cooling to room temperature, the powder was added to the fermentation medium at a ratio of 110:1 (mg:mL) and neutral protease was added to the fermentation medium at a rate of 1250 U / g. The medium was incubated at 37°C for 48 h to obtain a Chinese medicine composition. The neutral protease was a commercially available product.

[0081] The centrifuge tube containing the fermentation liquid was balanced and placed in a centrifuge, and centrifuged at 2500 rpm for 10 minutes. The supernatant was taken out and made into dry powder using a spray dryer.

[0082] Example 2

[0083] This embodiment provides a method for preparing a Chinese medicine composition for treating metabolic-related fatty liver disease. The specific steps and parameters are the same as those in Example 1, except that the formula of step (1) is different, namely, (1) 10 g of hawthorn, 8 g of roselle, 10 g of capillaris, and 10 g of dandelion are washed, drained, air-dried, cut into sections, dried, and passed through a 500-mesh sieve to obtain a powder.

[0084] Example 3

[0085] This embodiment provides a method for preparing a Chinese medicine composition for treating metabolic-related fatty liver disease. The specific steps and parameters are the same as those in Example 1, except that the fermentation temperature in step (3) is 35°C.

[0086] Example 4

[0087] This embodiment provides a method for preparing a Chinese medicine composition for treating metabolic-related fatty liver disease. The specific steps and parameters are the same as those in Example 1, except that the fermentation temperature in step (3) is 40°C.

[0088] Example 5

[0089] This embodiment provides a method for preparing a Chinese medicine composition for treating metabolic-related fatty liver disease. The specific steps and parameters are the same as those in Example 1, except that the fermentation time in step (2) is 36 hours.

[0090] Example 6

[0091] This embodiment provides a method for preparing a Chinese medicine composition for treating metabolic-related fatty liver disease. The specific steps and parameters are the same as those in Example 1, except that in step (2), the amount of bacterial suspension added to the basal culture medium is 3 Vol%.

[0092] Example 7

[0093] This embodiment provides a method for preparing a Chinese medicine composition for treating metabolic-related fatty liver disease. The specific steps and parameters are the same as those in Example 1, except that in step (2), the amount of bacterial suspension added to the basal culture medium is 4 Vol%.

[0094] Example 8

[0095] This embodiment provides a method for preparing a traditional Chinese medicine composition for treating metabolic-related fatty liver disease. The specific steps and parameters are the same as those in Example 1, except that in step (3), the amount of neutral protease added to the fermentation medium is 1100 U / g.

[0096] Example 9

[0097] This embodiment provides a method for preparing a Chinese medicine composition for treating metabolic-related fatty liver disease, and the specific steps and parameters are as follows:

[0098] (1) Wash 10 g of hawthorn, 8 g of roselle, 10 g of black-faced general, and 10 g of fireweed leaves, drain, air-dry, cut into sections, dry, and pass through a 500-mesh sieve to obtain a powder.

[0099] (2) Steam the powder at 30°C for 35 hours, where the steam pressure is 0.1 MPa. After steaming, cool and mix.

[0100] (3) Mix 20 g of glucose, 4 g of yeast powder and 0.2 g of ferrous sulfate, dilute to 1 L with deionized water, adjust the pH value to 7, and sterilize at 121 °C for 20 min to prepare a basal culture medium;

[0101] The powder steamed and cooled in step (2) was injected into the basic culture medium at a ratio of 110 mg:1 mL, and fermented at 35° C. for 50 h to obtain a Chinese medicine composition.

[0102] The fermented product was centrifuged at a rotation speed of 2500 rpm for 10 minutes to obtain a supernatant, and the supernatant was taken out and made into dry powder using a spray dryer.

[0103] Example 10

[0104] This embodiment provides a method for preparing a Chinese medicine composition for treating metabolic-related fatty liver disease. The specific steps and parameters are the same as those in Embodiment 10, except that an equal mass of ammonia water is used to replace the yeast powder in step (3).

[0105] Embodiment 11

[0106] This embodiment provides a method for preparing a Chinese medicine composition for treating metabolic-related fatty liver disease. The specific steps and parameters are the same as those of Example 10, except that an equal mass of peptone is used to replace the yeast powder in step (3).

[0107] Example 12

[0108] This embodiment provides a method for preparing a traditional Chinese medicine composition for treating metabolic-related fatty liver disease. The specific steps and parameters are the same as those in Example 10, except that the content of glucose in step (3) is 15 g.

[0109] Example 13

[0110] This embodiment provides a method for preparing a Chinese medicine composition for treating metabolic-related fatty liver disease. The specific steps and parameters are the same as those in Embodiment 10, except that equal masses of magnesium sulfate, potassium nitrate, zinc sulfate, and calcium sulfate are used to replace ferrous sulfate in step (3).

[0111] Embodiment 14

[0112] This embodiment provides a method for preparing a Chinese medicine composition for treating metabolic-related fatty liver disease. The specific steps and parameters are the same as those in Example 10, except that the pH value in step (3) is 6.

[0113] Embodiment 15

[0114] This embodiment provides a method for preparing a Chinese medicine composition for treating metabolic-related fatty liver disease. The specific steps and parameters are the same as those in Example 10, except that the pH value in step (3) is 8.

[0115] Comparative Example 1

[0116] This comparative example provides a method for preparing a Chinese medicine composition for treating metabolic-related fatty liver disease. The specific steps and parameters are the same as those in Example 1, except that the formula in step (1) contains only hawthorn and roselle, i.e., (1) 10 g of hawthorn and 8 g of roselle are taken, washed, drained, air-dried, cut into sections, dried, and passed through a 500-mesh sieve to obtain a powder.

[0117] Comparative Example 2

[0118] This comparative example provides a method for preparing a traditional Chinese medicine composition for treating metabolic-related fatty liver disease. The specific steps and parameters are the same as those in Example 1, except that the formula of step (1) is different, namely, (1) (1) take 10 g of hawthorn, 8 g of roselle, and 10 g of black-faced general, wash, drain, dry, cut into sections, dry, and pass through a 500-mesh sieve to obtain a powder.

[0119] Comparative Example 3

[0120] This comparative example provides a method for preparing a traditional Chinese medicine composition for treating metabolic-related fatty liver disease. The specific steps and parameters are the same as those in Example 1, except that the formula of step (1) is different, namely, (1) (1) take 10 g of hawthorn, 8 g of roselle, and 10 g of pyrocyrrhiza uralensis leaves, wash, drain, air-dry, cut into sections, dry, and pass through a 500-mesh sieve to obtain a powder.

[0121] Comparative Example 4

[0122] This embodiment provides a method for preparing a Chinese medicine composition for treating metabolic-related fatty liver disease. The specific steps and parameters are the same as those in Example 1, except that the formula of step (1) is different, namely, (1) 10 g of hawthorn, 8 g of roselle, and 10 g of dandelion are washed, drained, air-dried, cut into sections, dried, and passed through a 500-mesh sieve to obtain a powder.

[0123] Comparative Example 5

[0124] This embodiment provides a method for preparing a Chinese medicine composition for treating metabolic-related fatty liver disease. The specific steps and parameters are the same as those in Example 1, except that the formula of step (1) is different, namely, (1) taking 10 g of hawthorn, 8 g of roselle, and 10 g of capillaris, draining, air-drying, cutting into sections, drying, and passing through a 500-mesh sieve to obtain a powder.

[0125] Experimental Example 1

[0126] The contents of triterpenes, intracellular polysaccharides, microbial biomass, citric acid, acetic acid, propionic acid, and small peptides in the purified traditional Chinese medicine composition obtained in Examples 1-15 were tested.

[0127] Method for determining triterpene content: A Phenomenex Luna C18 chromatographic column was used, the detection wavelength was 281 nm, the mobile phase was methanol-water-tetrahydrofuran (25:70:12), the flow rate was 1.0 mL / min, the drift tube temperature was 110°C, the carrier gas flow rate was 2.8 L / min, the drug sample was injected into the high performance liquid chromatograph, the chromatogram was drawn, and compared with the standard curve, and finally the triterpene content was calculated.

[0128] Method for measuring polysaccharide content: Take a drug sample and perform infrared detection. Press the sample directly on a sample table coated with conductive silver glue, spray gold atoms (Au) using a DEC-A2 surface treatment instrument, and observe using a scanning electron microscope (Hitachis-570, Hitachi, Japan). Record the wavelength absorption, and finally determine the polysaccharide content based on the characteristic absorption peaks of specific functional groups in the polysaccharide molecules.

[0129] Method for determining microbial biomass: Add the drug fermentation broth and the diluted horseshoe crab reagent to the microwells and mix thoroughly. Incubate the mixture at 37°C for 60 minutes, and then immediately read the optical density using a spectrophotometer. Use the microbial biomass in the serial dilution standard as a positive control to draw a standard curve. Compare the optical density values ​​of the experimental tube and the control tube to calculate the microbial biomass.

[0130] Method for determining citric acid content: filter to remove turbidity, dilute the sample, mix with polyphthalamide or polyvinylpolypyrrolidone, stir for 1 minute, filter, and use a UV-visible spectrophotometer to directly determine the absorbance of the filtered liquid with the reagent blank as the control. Calculate the citric acid content based on the regression line equation in the standard curve.

[0131] Method for determination of acetic acid, propionic acid, and small peptide content: German Bruker Esquire3000 high performance liquid chromatography-electrospray ionization-ion trap multistage mass spectrometer (HPLC-ESI-MS / MS) was selected, the chromatographic column was Microsorb ODS column (4∙6mm i∙d∙×250 mm 5μm), the mobile phase was acetonitrile-water (containing 0∙05% TFA1mmol / L SDS) solution (volume ratio of 35:65), the flow rate was 0.4mL / min, and the wavelength was 210nm. Using a known concentration of small peptide standard, HPLC analysis was performed to establish a relationship curve between the concentration of small peptides and their peak area or peak height. The drug sample was injected into the liquid chromatograph through a syringe, and the chromatographic peak in the sample was compared with the standard curve, and finally the content of acetic acid, propionic acid, and small peptides in the sample was determined.

[0132] According to the detection results of the effective ingredients of the traditional Chinese medicine compositions prepared in Example 1 and Example 5, it can be seen that the organic acid content increases with the continuous increase of the fermentation time. After fermentation for 36 hours, the contents of citric acid, acetic acid and propionic acid tend to be stable. The acid content after fermentation for 48 hours is significantly increased compared with before, and the contents of various organic acids also tend to be stable after fermentation for 48 hours.

[0133] According to the test results of the effective components of the Chinese medicine composition prepared in Example 1 and Example 3-4, it can be seen that the content of small peptides in the Chinese medicine composition is the highest at a fermentation temperature of 40°C, which is 109.78 mg / g. The content of small peptides is not much different at 35°C and 37°C, which shows that 40°C is suitable for enzymatic hydrolysis by neutral proteases, indicating that relatively high temperatures can enhance the activity of neutral proteases. The organic acid content is the highest at 37°C, among which the acetic acid content is the highest, which is consistent with the fact that probiotics grow best at 37°C.

[0134] According to the effective component detection results of the traditional Chinese medicine compositions prepared in Example 1 and Examples 6-8, it can be seen that the content of small peptides increases with the increase of the inoculation amount of the bacterial solution, tends to be stable when the inoculation amount is 5%, and the content of propionic acid is the highest under the condition of 5% inoculation amount. When the enzyme addition amount is 1250U / g, the content of small peptides tends to be stable, and the highest content of propionic acid reaches 3.34mg / g.

[0135] According to the detection results of the effective ingredients of the Chinese medicine compositions prepared in Examples 9-11, it can be seen that the triterpene yield of the Chinese medicine compositions obtained by fermentation with yeast powder as the nitrogen source can reach 0.514 g / L after compatibility. Compared with Examples 10 and 11, the triterpene yield is increased by 74% and 30%.

[0136] According to the detection results of the effective ingredients of the traditional Chinese medicine compositions prepared in Examples 9 and 12, it can be seen that when the glucose concentration is controlled below 20 g / L, the bacterial intracellular polysaccharide content is relatively high. Among them, when the concentration of the carbon source is 20 g / L, the bacterial intracellular polysaccharide content is the highest, reaching 1.12 g / L.

[0137] Exogenous substances can enhance the body's resistance and increase the content of effective ingredients. According to the test results of the effective ingredients of the Chinese medicine compositions prepared in Examples 9 and 13, it can be seen that the influence of the culture medium containing different metal ions on the content of the liquid fermentation polysaccharide of the drug can be seen from the results. 2+ The microbial biomass and bacterial extracellular crude polysaccharide content obtained by fermentation with the culture medium were the highest, which were 7.299 g / L and 0.720 g / L, respectively.

[0138] According to the detection results of the effective components of the Chinese medicine compositions prepared in Examples 9 and 14-15, it can be seen that a higher initial pH value (7.0-8.0) is conducive to the synthesis of extracellular polysaccharides, and a lower pH value (6.0-7.0) is conducive to the growth of bacteria.

[0139] Experimental Example 2

[0140] 2.1 Construction of MAFLD model mice

[0141] The replication of the rat MAFLD model was carried out according to the previous modeling method of the research group: the MAFLD model was administrated by oral administration of high-fat emulsion to reduce the model differences caused by uneven eating. The formula of high-fat emulsion was: 50wt% lard, 10wt% cholesterol, 2wt% bile salt, 20wt% propylene glycol, and 12wt% purified water. The oral administration dose was 1mL / 50g (high-fat emulsion: mouse body weight), once a day.

[0142] The model was established for 8 consecutive weeks. The hepatic steatosis was observed dynamically during the modeling process. Two rats were randomly selected for HE staining at the 4th and 8th week of modeling to observe the hepatic steatosis. If macrovesicular or predominantly macrovesicular steatosis involving more than 5% of hepatocytes was observed, which could be accompanied by mild nonspecific inflammation, it indicated that the MAFLD model was successfully established. The mice with successful modeling were intervened by the following methods.

[0143] 2.2 Setting up treatment groups

[0144] 80 mice were randomly divided into 8 groups (n=10, n is the number of mice in each group), and the model was established according to the above-mentioned MAFLD model construction method. After the model was successfully established, 7 groups of model mice were infused with the aqueous solution of the Chinese medicine composition prepared by Examples 1-2 and Comparative Examples 1-5, 1 mL / day, for 60 days. The model group was not given drug intervention, but was infused with 1 mL / day of normal saline for 60 days.

[0145] The concentration of the aqueous solution of the traditional Chinese medicine composition is 34.5wt%.

[0146] 2.3 Detection

[0147] After 2 months of intervention, time-domain nuclear magnetic resonance (TD-NMR) technology was used to measure the body weight of mice, including liver weight, muscle weight, and visceral fat weight (inguinal adipose tissue IngWAT, epididymal adipose tissue EpiWAT).

[0148] Results Figure 1-Figure 2 It can be seen that the fermentation product of the Chinese medicine composition provided by the present invention can significantly improve the body's fat metabolism. It can reduce visceral fat, especially visceral fat in the liver, testicles, groin, etc., indicating that the weight loss mainly comes from the reduction of visceral fat, rather than the loss of water, muscle, etc. In particular, it does not affect the weight of the muscle (no muscle loss), overcoming the adverse reactions of many current lipid metabolism regulating drugs such as weight loss and muscle loss or muscle lysis risks, indicating that the composition provided by the present invention reduces visceral fat, especially improves liver lipid deposition, without the side effect of muscle loss.

[0149] Experimental Example 3

[0150] 3.1 Construction of MAFLD model mice

[0151] The model mice were constructed according to the construction method in 2.1 of Experimental Example 2.

[0152] 3.2 Setting up treatment groups

[0153] The fermented products of the traditional Chinese medicine compositions prepared in Examples 1-2 and Comparative Examples 1-5 (all with a concentration of 34.5 wt%) were respectively administered by intragastric administration at 1 mL / day for 60 days; the model group was administered with normal saline at 1 mL / day for 60 days, and the number of mice in each group was 10.

[0154] 3.3 Detection

[0155] After 2 months of intervention, the body weight, liver weight, liver lipid metabolism indexes and liver function indexes of the mice were measured. Among them, triglyceride (TG), free fatty acid (FFA), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected by biochemical enzymatic method.

[0156] Depend on Figure 3-16 It can be seen that the Chinese medicine composition provided by the present invention can effectively reduce the levels of visceral fat, liver weight, liver triglycerides, liver free fatty acids, serum IL6, serum alanine aminotransferase and serum aspartate aminotransferase in model mice, and the Chinese medicine compositions prepared in Examples 1-2 and Comparative Examples 1-5 are significantly different from those in the model group in the above indicators.

[0157] Experimental Example 4

[0158] Ten patients with MAFLD were recruited, and the granules of Example 1 (raw materials: 10 g of hawthorn, 8 g of roselle, 10 g of black-faced general, and 10 g of fire-tube leaves) were mixed evenly with 150 mL of warm water and taken orally once a day, 1 dose each time, for 60 days. The BMI, liver fat fraction, and pancreatic fat fraction of each patient before and after treatment (1.5T magnetic resonance imaging MRI-IDEAL-IQ technology) were measured, and the results are shown in Fig.17 It can be seen that the Chinese medicine composition provided by the present invention can significantly reduce the patient's BMI value, and at the same time, has a significant effect on reducing the fat content of the left liver, right liver, pancreatic head and pancreatic tail.

[0159] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A Chinese medicine composition for treating metabolic-related fatty liver disease, characterized in that: The following components in parts by weight are obtained by microbial fermentation: The components are 5-15 parts of hawthorn, 5-10 parts of roselle, 5-15 parts of black-faced general, and 5-15 parts of fire tube leaves. The culture medium used in the microbial fermentation includes a carbon source, a nitrogen source, a metal salt, a bacterial agent and an enzyme. The bacterial agent is Akkermansia muciniphila ( Akkermansia muciniphila ), The concentration of the bacterial agent is 3 Vol%-7 Vol%, and the concentration of the bacterial species in the bacterial agent is 2.5×10 7 CFU / mL-4.5×10 8 CFU / mL, The enzyme is a neutral protease, The ratio of the enzyme to the culture medium is 1150-1350:1, and the unit is U:g. The fermentation temperature of the microbial fermentation is 35° C.-42° C., and the fermentation time is 12 h-72 h.

2. The Chinese medicine composition according to claim 1, characterized in that: In parts by weight, the components include 8-12 parts of hawthorn, 6-9 parts of roselle, 8-12 parts of black-faced general, and 8-12 parts of fireweed leaves.

3. A fermentation method for the Chinese medicine composition according to any one of claims 1 to 2, characterized in that: The components are weighed according to weight, and fermented to obtain a Chinese medicine composition, wherein the fermentation is microbial fermentation.

4. The fermentation method according to claim 3, characterized in that The fermentation temperature of the microbial fermentation is 35° C.-42° C., and the fermentation time is 12 h-72 h.

5. The fermentation method according to claim 4, characterized in that The culture medium used in the microbial fermentation includes a carbon source, a nitrogen source and a metal salt. The pH value of the culture medium is 6-8, The concentration of the carbon source is less than or equal to 20 g / L, The concentration of nitrogen source is 4 g / L -10g / L, The concentration of the metal salt is 0.2 g / L-0.5 g / L.

6. The fermentation method according to claim 5, characterized in that The carbon source is at least one of glucose and sucrose; The nitrogen source is yeast powder; The metal salt is at least one of ferrous salt, magnesium salt, calcium salt, potassium salt or zinc salt; The pH value of the culture medium is 6.5-7.

5.

7. The fermentation method according to claim 4, characterized in that The culture medium used in the microbial fermentation also includes bacterial agents and enzymes. The bacterial agent is Akkermansia muciniphila ( Akkermansia muciniphila ), The concentration of the bacterial agent is 3 Vol%-7 Vol%, and the concentration of the bacterial species in the bacterial agent is 2.5×10 7 CFU / mL-4.5×10 8 CFU / mL, The enzyme is a neutral protease, The ratio of the enzyme to the culture medium is 1150-1350:1, and the unit is U:g.

8. Use of a Chinese medicine composition for treating metabolism-related fatty liver disease according to any one of claims 1-2 or a Chinese medicine composition obtained by the fermentation method of the Chinese medicine composition according to any one of claims 3-7 in the preparation of a drug for treating metabolism-related fatty liver disease.

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