A traditional Chinese medicine composition and a microbial fermentation method for treating metabolic-related fatty liver

Abstract

The present application relates to the technical field of traditional Chinese medicine, and in particular to a traditional Chinese medicine composition for treating metabolic-related fatty hepatitis and a microbial fermentation method, The present application provides a traditional Chinese medicine composition for treating metabolic-related fatty hepatitis, which comprises the following components by weight: 10-30 parts of Rhodobryum gigantum, 5-15 parts of Smilax riparia, 5-15 parts of Sarcandra glabra, and 3-10 parts of dragon's blood; the traditional Chinese medicine composition is prepared by microbial fermentation of the above components. The present application combines Rhodobryum gigantum, Smilax riparia, Sarcandra glabra and dragon's blood to enhance the therapeutic effect of the traditional Chinese medicine composition on metabolic-related fatty hepatitis, and can significantly enhance the therapeutic effect on metabolic-related fatty hepatitis through multi-target and multi-pathway synergistic action. Furthermore, the present application uses microbial fermentation of the above components to obtain a traditional Chinese medicine composition fermentation product, which increases the content of effective components and enhances the efficacy, and has synergistic effects such as improving metabolic abnormalities, reducing adverse reactions, and improving taste.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine technology, specifically to a traditional Chinese medicine composition and microbial fermentation method for treating metabolic-related fatty liver disease. Background Technology

[0002] Metabolic steatohepatitis (MASH) is a critical stage in the progression of fatty liver disease, referred to as "liver stagnation" in Traditional Chinese Medicine (TCM). Its core pathological features are intrahepatic inflammation and fibrosis. With changes in modern lifestyles and the Westernization of dietary patterns, the incidence of MASH is rapidly increasing globally. Modern medical research shows that the livers of MASH patients exhibit characteristic pathological changes such as lipid deposition, inflammatory damage, and even partial fibrosis. TCM theory attributes its pathogenesis to "liver stagnation and spleen deficiency, with phlegm and blood stasis," closely related to emotional imbalances and improper diet. Epidemiological surveys show that the prevalence of MASH in Asian populations has reached 24.7%, and the age of onset is increasingly younger, with the incidence rate in the 30-40 age group nearly doubling compared to ten years ago, making it a significant public health problem threatening national health.

[0003] The clinical treatment of MASH currently faces severe challenges. Early symptoms of the disease are often insidious, and most patients are diagnosed with significant liver pathological changes. Modern medical treatments offer limited improvement in key pathological aspects of MASH, particularly in effectively reversing existing fibrosis. Developing treatments that can effectively intervene in the progression of MASH has become a crucial issue urgently needing to be addressed by the medical community. Currently, clinical management of MASH primarily relies on lifestyle interventions, but poor long-term patient adherence leads to significant individual differences in treatment outcomes. Rezdiffra, approved by the FDA in 2024 as the first targeted therapy for MASH, still faces numerous limitations in its clinical application: firstly, safety and efficacy data in different ethnic groups are insufficient; secondly, its efficacy in patients with mid-to-late-stage fibrosis is limited, and it is not suitable for pediatric patients. Notably, no drugs are currently recommended by guidelines for the clinical treatment of MASH in my country. The core dilemma of existing treatments lies in the fact that while early-stage MASH is reversible, treatment becomes significantly more difficult once it progresses to a stage with significant fibrosis. Against this backdrop, traditional Chinese medicine, with its multi-target effects, has demonstrated unique advantages. Its prevention and treatment experience based on the physical characteristics of the Chinese population provides important insights for developing MASH treatment plans that are more suitable for Chinese people. Summary of the Invention

[0004] Therefore, in order to improve the inflammatory damage and liver fibrosis caused by MASH, the present invention provides a traditional Chinese medicine composition and a microbial fermentation method for treating metabolic-associated fatty liver disease.

[0005] Traditional Chinese medicine (TCM) is characterized by its multi-target, multi-level, disease-symptom-combination approach and low toxicity in disease diagnosis and treatment, offering unique advantages over single-target chemotherapy for diseases with complex pathogenesis. Preliminary clinical observations have shown that this invention effectively improves inflammatory damage and fibrotic changes in MASH (muscular atrophy-associated inflammatory response). Animal experiments further suggest that the fermented TCM composition is superior to the unfermented TCM composition in reversing MASH and inhibiting liver inflammatory factors and fibrosis.

[0006] On the one hand, the present invention provides a traditional Chinese medicine composition for treating metabolic-associated fatty liver disease.

[0007] The traditional Chinese medicine composition comprises the following raw materials in parts by weight: 10-30 parts of Jiubiying, 5-15 parts of Chuanposhi, 5-15 parts of Zhongjiefeng, and 3-10 parts of Xuejie.

[0008] In some embodiments, the traditional Chinese medicine composition is prepared by microbial fermentation of 10-30 parts of *Smilax china*, 5-15 parts of *Smilax china*, 5-15 parts of *Smilax china*, and 3-10 parts of *Dracaena cochinchinensis*; the microorganisms include *Bacillus subtilis* (…). Bacillus subtilis ), Actinomycetes ( Actinomycetes ) and Streptomyces ( Streptomycetaceae At least one of the following.

[0009] On the other hand, the present invention provides a method for preparing a traditional Chinese medicine composition for treating metabolic-related fatty liver disease, wherein the components are crudely extracted according to the proportions to obtain an extract.

[0010] In some embodiments, the crude extraction method is a water extraction method, which includes crushing the raw materials weighed according to the formula to form a mixture, mixing the mixture with water and boiling, filtering, and concentrating to obtain an extract.

[0011] In some embodiments, the method further includes a step of microbial fermentation of the traditional Chinese medicine composition, wherein the microbial culture is inoculated into a culture medium containing the extract, fermented in a fermenter, and the traditional Chinese medicine composition is obtained by solid-liquid separation; the microbial strains in the microbial culture include Bacillus subtilis (Bacillus subtilis). Bacillus subtilis ), Actinomycetes ( Actinomycetes ) and Streptomyces ( Streptomycetaceae At least one of the following.

[0012] In some embodiments, the mass ratio of the mixture to water is 1:8 to 1:12;

[0013] In some embodiments, the boiling time is 0.5-2 hours;

[0014] In some embodiments, the volume ratio of the concentrated extract to the mass ratio of the mixture is 1:1 to 1:2, in mL:g.

[0015] In some embodiments, during microbial fermentation, the microbial broth is inoculated into a culture medium containing the extract, with the volume ratio of the microbial broth to the culture medium containing the extract being 1:8 to 1:12. The fermentation temperature is 28-38°C, the humidity is 60%-70%, and the fermentation time is 24-72 hours at a rotation speed of 180-220 rpm and an aeration rate of 0.8-1.2 VVM.

[0016] In some embodiments, the bacterial strain includes Bacillus subtilis (B. subtilis). Bacillus subtilis ), Actinomycetes ( Actinomycetes ) and Streptomyces ( Streptomycetaceae );

[0017] In some embodiments, Bacillus subtilis (Bt) is prepared separately before microbial fermentation. Bacillus subtilis ), Actinomycetes ( Actinomycetes ) and Streptomyces ( Streptomycetaceae Seed solutions, their respective OD values 600 The values ​​are 0.8-1.0, 0.6-0.8, and 0.5-0.7 respectively;

[0018] In some embodiments, during microbial fermentation, Bacillus subtilis (… Bacillus subtilis ), Actinomycetes ( Actinomycetes ) and Streptomyces ( Streptomycetaceae The seed liquid of the microorganism was mixed in a volume ratio of 1-2:1-2:1-2 to prepare a microbial inoculum for fermentation;

[0019] In some embodiments, the total OD of the mixed microbial culture 600 The value is 1.9-2.5.

[0020] In some embodiments, the method for preparing the seed solution includes:

[0021] Bacillus subtilis ( Bacillus subtilis ), Actinomycetes ( Actinomycetes ) and Streptomyces ( Streptomycetaceae The first seed culture of each strain was obtained by inoculating the strains into culture medium for enrichment culture; the first seed culture was then inoculated into culture medium containing the extract for expansion culture to obtain the second seed culture of each strain.

[0022] OD of the first seed culture of Bacillus subtilis for expanded culture 600 The value was 2.5-3.5, and the OD value of the first seed culture of actinomycetes was 2.5-3.5. 600The value was 1.8-2.5, and the OD value of the first seed culture of Streptomyces was... 600 The value is 1.5-2.2;

[0023] In the preparation of the second seed solution, each of the obtained first seed solutions was inoculated into the culture medium containing the extract at a volume ratio of 1:10 to 1:20, and cultured at 35-38℃ and 200 rpm for 12 h.

[0024] In preparing the first seed culture, the volume ratio of the initial inoculum to the culture medium was 1:50-1:100, and the enrichment culture conditions were 32-37℃, 150-180rpm, and cultured for 12-16h.

[0025] In some embodiments, the culture medium is a seed culture medium and / or a fermentation culture medium; wherein, when the seed culture medium is used for Bacillus subtilis, it includes 0.5-2 g / L peptone, 0.3-1 g / L yeast extract, 0.5-1.5 g / L NaCl, and 0.5-2.0 g / L glucose; when used for actinomycetes and Streptomyces, it further includes 5-20 g / L cellulose and 1-3 g / L ammonium sulfate in addition to the seed culture medium; the fermentation culture medium is an extract containing the above-mentioned extract; the concentration of the extract in the culture medium containing the extract is 8-12 vol%.

[0026] In some embodiments, the solid-liquid separation step includes centrifugation and filtration, and the filtered filtrate is a traditional Chinese medicine composition, wherein the centrifugation speed is 8000 rpm and the centrifugation time is 8-12 min;

[0027] In some of these implementations, during microbial fermentation, the microorganisms are cultured at 28-31°C for 10-24 hours in the early stage and at 35-38°C for 14-48 hours in the later stage.

[0028] This invention provides the use of the above-mentioned traditional Chinese medicine composition for treating metabolic-associated steatohepatitis, or the traditional Chinese medicine composition prepared by the above-mentioned method, in the preparation of drugs for relieving or treating metabolic-associated steatohepatitis.

[0029] The above uses include use in the preparation of medicines for relieving or treating non-alcoholic steatohepatitis.

[0030] The technical solution of this invention has the following advantages:

[0031] 1. This invention provides a traditional Chinese medicine composition for treating metabolic-related fatty liver disease, comprising the following components in parts by weight: 10-30 parts of *Smilax glabra*; 5-15 parts of *Chuanposhi*; 5-15 parts of *Zhongjiefeng*; and 3-10 parts of *Xuejie*. The traditional Chinese medicine composition is obtained by microbial fermentation of the above components. *Smilax glabra* contains saponins, quercetin, kaempferol, etc., which have significant anti-inflammatory and antioxidant effects, can reduce liver inflammation, regulate lipid metabolism, reduce liver lipid deposition, inhibit fatty liver formation, enhance insulin sensitivity, and improve abnormal glucose metabolism. *Chuanposhi* contains bioactive components such as alkaloids, luteolin, and phenolic acids, which have significant hepatoprotective effects, can reduce hepatocyte damage, promote bile secretion, improve cholestasis, lower cholesterol levels, regulate blood lipid metabolism, reduce liver fat accumulation, and have antioxidant effects. The effects are significant, capable of scavenging free radicals and reducing oxidative stress damage to the liver. *Symplocos rubra* contains bioactive components such as eucalyptol, limonene, apigenin, and polysaccharides, which promote hepatocyte repair, improve liver function, enhance immunity, and improve metabolic disorders. *Dracaena cochinchinensis* contains bioactive components such as daphne, catechins, and polysaccharides, which can promote blood circulation, improve liver microcirculation, reduce liver congestion, regulate lipid metabolism, and inhibit the progression of liver fibrosis, significantly improving liver lipid metabolism, reducing fat deposition, and inhibiting the formation of fatty liver. This invention, through the compounding of *Symplocos rubra*, *Symplocos rubra*, *Symplocos rubra*, and *Dracaena cochinchinensis*, enhances the therapeutic efficacy of the traditional Chinese medicine composition on metabolic-related fatty liver disease. It can significantly enhance the therapeutic effect on metabolic-related fatty liver disease through multi-target and multi-pathway synergistic effects. Furthermore, this invention utilizes microbial fermentation of the above components to obtain a traditional Chinese medicine composition that increases the content of effective ingredients, enhances efficacy, and can significantly improve metabolic abnormalities, reduce adverse reactions, and improve taste, resulting in a synergistic effect.

[0032] 2. This invention provides a method for preparing a traditional Chinese medicine composition for treating metabolic-related fatty liver disease, comprising the following steps: crudely extracting each component according to a specified ratio to obtain an extract; inoculating a microbial culture solution into a culture medium containing the extract for enrichment and fermentation; and separating the solid and liquid components to obtain the traditional Chinese medicine composition. This invention utilizes microorganisms to ferment the crude extracts of each component, resulting in a simple preparation process and a high content of effective components in the obtained traditional Chinese medicine composition.

[0033] 3. This invention provides a method for preparing a traditional Chinese medicine composition for treating metabolic-related fatty liver disease, wherein the bacterial strain includes Bacillus subtilis (…). Bacillus subtilis ), Actinomycetes ( Actinomycetes ) and Streptomyces ( StreptomycetaceaeThis invention utilizes three microbial strains to ferment the extract, which comprehensively and thoroughly decomposes the complex components in the extract, significantly increasing the content of active ingredients. Experiments have shown that, compared to a single strain, the fermentation method using multiple strains in this invention can increase the content of flavonoids and phenolic compounds. The combination of different strains diversifies metabolites, enhances the multi-target effect of the drug, and can more effectively improve liver lipid metabolism, reduce cholesterol and triglyceride levels, alleviate liver inflammation and oxidative stress. It can also indirectly improve liver metabolic function and enhance insulin sensitivity by regulating the intestinal flora. Attached Figure Description

[0034] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0035] Figure 1 This is a comparison chart of liver stiffness, liver function, and blood lipids in MASH patients before and after treatment in Experimental Example 2 of this invention (Before: before treatment; After: after treatment).

[0036] \nFigure 2 This refers to HE / O staining of mouse livers in Experiment Example 3 of this invention; the two images on the left are HE-stained images of the livers of two mice; the two images on the right are O-stained images of the livers of two mice.

[0037] \nFigure 3 These are anatomical images of mouse livers in Experiment Example 3 of this invention (A. Model group; B. Traditional Chinese medicine fermentation group; C. Traditional Chinese medicine group).

[0038] \nFigure 4 This refers to the biochemical detection of mice in Experiment Example 3 of this invention (A. Model group; B. Traditional Chinese medicine fermentation group; C. Traditional Chinese medicine group).

[0039] \nFigure 5 HE staining of mouse liver tissue in Experiment Example 3 of this invention (A. Model group; B. Traditional Chinese medicine fermentation group; C. Traditional Chinese medicine group). Detailed Implementation

[0040] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0041] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0042] The Bacillus subtilis mentioned in this invention ( \nBacillus subtilis ), Actinomycetes ( Actinomycetes ) and Streptomyces ( Streptomycetaceae All strains were commercially available. The strains used in this embodiment were purchased from Beina Biotechnology, including Bacillus subtilis (…). Bacillus subtilis The purchased item, with serial number BNCC195731, contains actinomycetes (…). Actinomycetes The product name is *Streptomyces* DNA Standard, product number BNCC371919, and it is made from *Streptomyces*. Streptomycetaceae The product name is Streptomyces farinaceus BNCC153298.

[0043] Sodium carboxymethyl cellulose and sodium cellulose mentioned in this invention are both commercially available and purchased from Beina Biotechnology. The product number of sodium carboxymethyl cellulose is D11895 and the product number of sodium cellulose is R033059.

[0044] Example 1

[0045] This embodiment provides a method for preparing a traditional Chinese medicine composition for treating metabolic-related steatohepatitis, with the specific steps and parameters as follows:

[0046] (1) Take 30g of Jiubiying, 10g of Chuanposhi, 10g of Zhongjiefeng and 10g of Xuejie, mix them, crush them and pass them through a 60-mesh sieve;

[0047] The powder was placed in distilled water at a mass ratio of 1:10, boiled, kept for 1 hour, and then filtered to obtain filtrate and residue.

[0048] Repeat the above extraction steps twice on the filter residue, combine the filtrates, concentrate them to a volume of 60 mL, and obtain the extract.

[0049] (2) Weigh 1.5g of peptone, 0.8g of yeast extract, 1.0g of NaCl and 1.5g of glucose, dissolve them in 1L of distilled water and adjust the pH to 7.0. Sterilize the culture medium at 121℃ for 20min to obtain the culture medium.

[0050] (3) Bacillus subtilis ( Bacillus subtilis The inoculum was added to the culture medium prepared in step (2) at a volume of 2% (v / v) of the culture medium. The culture was carried out at 37°C and 160 rpm for 12 hours to obtain the first seed culture. The OD of the first seed culture was... 600 The value is 2.5-3.5;

[0051] The first seed culture was inoculated into a culture medium containing 10% extract at a volume ratio of 1:15. The culture was then incubated at 37°C and 200 rpm for 12 hours to obtain the second seed culture. The culture medium was the same as that used in step (2), and contained 10 vol% of the extract from step (1). The OD of the second seed culture... 600 The value is 0.8-1.0.

[0052] (4) The seed liquid is inoculated into the fermenter for fermentation according to the volume ratio of the second seed liquid to the culture medium of 1:10. The fermentation conditions are 37°C, 60% humidity, 200 rpm rotation speed and 1 VVM aeration rate, and fermentation for 72 h. The culture medium in the fermenter is the culture medium of step (2), and the culture medium contains 10 vol% of the extract obtained in step (1). The pH value of the culture medium is 7.

[0053] (5) After fermentation, the fermentation broth was centrifuged at 8000 rpm for 10 min to remove the cells and insoluble matter, and the supernatant was collected. Then, the supernatant was concentrated to 1:1 (i.e., 1 g of raw medicinal material corresponds to 1 mL of concentrated liquid) using a rotary evaporator (60℃, -0.09 MPa vacuum) to obtain the fermented Chinese medicine composition, which is about 60 mL of viscous extract (density ≈ 1.2-1.4 g / mL, water content 30%-50%).

[0054] Example 2

[0055] 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 in Example 1, except that: the fermentation conditions in step (4) are shown in Table 1.

[0056] Table 1 Fermentation conditions

[0057]

[0058] The purpose of early fermentation is to promote the microbial cells to adapt to the environment and grow stably; the purpose of late fermentation is to enhance the synthesis of secondary metabolites (such as flavonoids and phenols).

[0059] Example 3

[0060] 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 in Example 2, except that the fermentation time in step (4) is 72 hours, the initial fermentation time is 24 hours, and the later fermentation time is 48 hours.

[0061] Example 4

[0062] 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 in Example 1, except that:

[0063] In step (3):

[0064] The preparation method of the first seed liquid is as follows: Bacillus subtilis (… Bacillus subtilis ), Actinomycetes ( Actinomycetes ) and Streptomyces ( Streptomycetaceae The inoculum was added to the culture medium from step (2), with an inoculum volume of 1% of the culture medium volume. The culture was carried out at 37°C and 180 rpm for 12 hours to obtain the first seed culture. The first seed culture of Bacillus subtilis (OD) was then obtained. 600 Value 2.5-3.5), Actinomycete first seed culture (OD) 600 Value 1.8-2.5) and Streptomyces first seed culture (OD) 600 Value 1.5-2.2).

[0065] The preparation method of the second seed culture is as follows: The three first seed cultures mentioned above were inoculated into a culture medium containing 10 vol% extract at a volume ratio of 1:15 (seed culture: culture medium), and then independently cultured at 37℃ and 200 rpm for 12 h to obtain the second seed culture of Bacillus subtilis (OD). 600 Value 0.8-1.0), second seed culture of actinomycetes (OD) 600 Value 0.6-0.8) and Streptomyces second seed culture (OD) 600 (Value 0.5-0.7).

[0066] In step (4):

[0067] The three types of second seed liquids were mixed in a volume ratio of 1:1:1 to obtain the total OD. 600 A mixed seed culture with a value of 1.9-2.5 was used for inoculation in subsequent fermentation steps. The inoculation volume was the same as that of the second seed culture of Bacillus subtilis in Example 1, and the other fermentation conditions were the same as those in step (4) of Example 1.

[0068] Example 5

[0069] 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 in Example 4, except that the culture medium in step (2) also includes 15 g / L cellulose.

[0070] Example 6

[0071] 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 in Example 4, except that the culture medium in step (2) also includes 3 g / L ammonium sulfate.

[0072] Example 7

[0073] This embodiment provides a method for preparing a traditional Chinese medicine composition for treating metabolic-related steatohepatitis, with the specific steps and parameters as follows:

[0074] (1) Take 30g of Jiubiying, 10g of Chuanposhi, 10g of Zhongjiefeng and 10g of Xuejie, crush them and pass them through a 60-mesh sieve;

[0075] The powder was placed in distilled water at a mass ratio of 1:10, boiled, kept for 1 hour, and then filtered to obtain filtrate and residue.

[0076] Repeat the above extraction steps twice on the filter residue, combine the filtrates, concentrate them to a volume of 60 mL, and obtain the extract.

[0077] (2) Weigh 1.5g of peptone, 0.8g of yeast extract, 1.0g of NaCl, 1.5g of glucose, 15g of sodium carboxymethyl cellulose and 2g of ammonium sulfate, dissolve them in 1L of distilled water, adjust the pH to 7.0, sterilize at 121℃ for 20min to obtain the culture medium.

[0078] (3) Bacillus subtilis ( Bacillus subtilis ), Actinomycetes ( Actinomycetes ) and Streptomyces ( Streptomycetaceae The initial inoculum to culture medium was inoculated at a volume ratio of 1:100 into the culture medium prepared in step (2), and cultured at 37°C and 160 rpm for 12 h to obtain the first seed culture. The OD values ​​of Bacillus subtilis, Actinomycetes and Streptomyces in the first seed culture were... 600 The values ​​are 2.5-3.5, 1.8-2.5, and 1.5-2.2 respectively;

[0079] The first seed culture was inoculated into the culture medium at a volume ratio of 1:15, and cultured at 37°C and 200 rpm for 12 hours to obtain the second seed culture. The culture medium was the same as that in step (2), and contained 10 vol% of the extract from step (1). The second seed culture of Bacillus subtilis (OD) was then obtained. 600 Value 0.8-1.0), second seed culture of actinomycetes (OD) 600 Value 0.6-0.8) and Streptomyces second seed culture (OD) 600 (Value 0.5-0.7).

[0080] (4) Mix the above three types of second seed liquids in a volume ratio of 1:1:1 to obtain the total OD. 600 A mixed seed culture with a pH of 1.9-2.5 was inoculated into a fermenter at a volume ratio of 1:10 between the mixed seed culture and the culture medium. Fermentation was divided into an early stage and a late stage. The fermentation conditions are shown in Table 2. The culture medium in the fermenter was the culture medium from step (2), and the culture medium contained 10 vol% of the extract from step (1). The pH of the culture medium was 7.

[0081] Table 2 Fermentation conditions

[0082]

[0083] (5) After fermentation, centrifuge at 8000 rpm for 10 min to remove the cells, retain the supernatant, and concentrate the filtrate to 1:1 (i.e., 1g of raw medicinal material corresponds to 1mL of concentrated liquid) to obtain about 50-60mL of Chinese medicine composition (containing 30%-50% water).

[0084] Example 8

[0085] The traditional Chinese medicine composition provided in this embodiment is the extract obtained in step (1) of Example 1, which is concentrated to 1:1 (i.e., 1g of raw medicinal material corresponds to 1mL of concentrated liquid).

[0086] Experimental Example 1

[0087] The active ingredients and OD in the traditional Chinese medicine compositions prepared in each embodiment 600 The method for determining the value is shown below.

[0088] Method for determining quercetin content: A C18 column (250 mm × 4.6 mm, 5 μm) was used, with a detection wavelength of 370 nm. The mobile phase was methanol-0.1% phosphoric acid water (55:45, v / v), the flow rate was 1.0 mL / min, the column temperature was 30 ℃, and the injection volume was 10 μL. 0.5 mL of the concentrated solution was taken and diluted to 10 mL with 70% methanol. The solution was extracted ultrasonically for 30 min, filtered through a 0.22 μm filter membrane, and injected into a high-performance liquid chromatograph. A chromatogram was plotted, and the quercetin content was calculated by comparing it with a standard curve.

[0089] Method for determining the content of quercetin: A C18 column (250 mm × 4.6 mm, 5 μm) was used, with a detection wavelength of 203 nm. The mobile phase was acetonitrile-water (35:65, v / v), the flow rate was 1.0 mL / min, the column temperature was 30 ℃, and the injection volume was 10 μL. 0.5 mL of the concentrated solution was taken and diluted to 10 mL with 70% methanol. The solution was extracted ultrasonically for 30 min, filtered through a 0.22 μm filter, and injected into a high-performance liquid chromatograph. A chromatogram was plotted, and the quercetin content was calculated by comparing it with a standard curve.

[0090] Preparation of standard curves for quercetin and saponins: A series of standard solutions of quercetin (0.01-0.5 mg / mL) and saponins (0.05-1.0 mg / mL) were prepared, and standard curves were constructed using peak area (Y) against concentration (X).

[0091] Method for determining kaempferol content: A C18 column (250 mm × 4.6 mm, 5 μm) was used, with a detection wavelength of 367 nm. The mobile phase was acetonitrile-0.1% phosphoric acid water (45:55, v / v), the flow rate was 1.0 mL / min, the column temperature was 30 ℃, and the injection volume was 10 μL. 0.5 mL of the concentrated solution was taken and diluted to 10 mL with 70% methanol. The solution was extracted ultrasonically for 30 min, filtered through a 0.22 μm filter membrane, and injected into a high-performance liquid chromatograph. A chromatogram was plotted, and the kaempferol content was calculated by comparing it with a standard curve.

[0092] Preparation of kaempferol standard curve: Prepare a series of kaempferol concentration standard solutions (0.005-0.2 mg / mL), and construct a standard curve using peak area (Y) against concentration (X).

[0093] Method for determining luteolin content: A C18 column (250 mm × 4.6 mm, 5 μm) was used, with a detection wavelength of 350 nm. The mobile phase was methanol-0.1% phosphoric acid water (50:50, v / v), the flow rate was 1.0 mL / min, the column temperature was 30 ℃, and the injection volume was 10 μL. 0.5 mL of the concentrated solution was taken and diluted to 10 mL with 70% methanol. The solution was ultrasonically extracted for 30 min, filtered through a 0.22 μm filter, and injected into a high-performance liquid chromatograph. A chromatogram was plotted, and the luteolin content was calculated by comparing it with a standard curve.

[0094] Preparation of luteolin standard curve: Prepare a series of luteolin concentration standard solutions (0.005-0.1 mg / mL), and construct a standard curve using peak area (Y) against concentration (X).

[0095] Method for determining the content of daphne: A C18 column (250 mm × 4.6 mm, 5 μm) was used, with a detection wavelength of 440 nm. The mobile phase was acetonitrile-0.1% phosphoric acid water (42:58, v / v), the flow rate was 1.0 mL / min, the column temperature was 30 ℃, and the injection volume was 10 μL. 0.5 mL of the concentrated solution was taken and diluted to 10 mL with 70% methanol. The solution was extracted by sonication for 30 min, filtered through a 0.22 μm filter membrane, and injected into a high-performance liquid chromatograph. A chromatogram was plotted, and the daphne content was calculated by comparing it with a standard curve.

[0096] Preparation of standard curve for strychnine: Prepare a series of standard solutions of strychnine at different concentrations (0.01-0.3 mg / mL), and construct a standard curve using peak area (Y) against concentration (X).

[0097] Method for determining polysaccharide content: The phenol-sulfuric acid method was used. Take 0.1 mL of the concentrated solution, add ultrapure water to 2.0 mL, add 1.0 mL of 6% phenol solution, mix well, and then quickly add 5.0 mL of concentrated sulfuric acid. Shake to mix, let stand for 10 min, and then react in a 40℃ water bath for 20 min. Cool to room temperature. Using glucose as a standard, measure the absorbance at a wavelength of 490 nm. Compare with the standard curve to calculate the polysaccharide content (calculated as glucose).

[0098] Preparation of polysaccharide standard curve: Prepare glucose standard solutions of various concentrations (0.01-0.1 mg / mL) and construct a standard curve using absorbance (Y) against concentration (X).

[0099] OD 600 Determination method of OD value: Take the fermentation broth sample, dilute it appropriately (usually 10-50 times), use sterile culture medium as a blank control, and measure the absorbance value at a wavelength of 600 nm using a UV-Vis spectrophotometer. Calculate the OD value of the original sample according to the dilution factor. 600 value.

[0100] Measurements showed that the fermentation process of this invention significantly increased the content of active ingredients (increase ≥20%) and the biomass of the microorganisms, which was significantly better than the unfermented traditional Chinese medicine group in Example 8. Specifically, the OD values ​​after fermentation in Examples 1, 3, 4, and 7 were... 600 The values ​​and the specific contents of active ingredients in Examples 1, 3, 4, 7, and 8 are shown in Table 3.

[0101] Table 3 OD in each embodiment 600 Values ​​and content of each active ingredient

[0102]

[0103] Experiment Example 2

[0104] Twenty-three MASH patients were recruited. A decoction of 30g *Jiu Bi Ying*, 10g *Chuan Po Shi*, 10g *Zhong Jie Feng*, and 10g *Xue Jie* was prepared and administered orally once daily. Preparation method: Soak the herbs in 400mL of purified water for half an hour, then decoct for 40 minutes each time to obtain 150mL of liquid. Mix the two decoctions thoroughly and divide into two portions of 150mL each. Administer 150mL orally twice daily for 60 days. CAP (liver stiffness index), ALT (alanine aminotransferase), AST (aspartate aminotransferase), TG (triglycerides), TC (total cholesterol), and FFA (fatty acids) were measured before and after treatment in each patient. Independent samples t-tests were used to compare the means among multiple groups.

[0105] See results Figure 1It can be seen that the unfermented traditional Chinese medicine composition provided by the present invention can significantly reduce the liver stiffness index of patients, and at the same time, serum ALT, AST, TG, TC and FFA are significantly reduced, which has a significant therapeutic effect.

[0106] Experimental Example 3

[0107] 3.1 Constructing the MASH model mouse

[0108] Eighteen 5-week-old SPF-grade male C57BL / 6J mice were purchased and housed in a constant temperature (22±2℃) and humidity (55%±5%) environment with a 12-hour light / 12-hour dark cycle, and free access to food and water. All mice underwent acclimatization for one week before the experiment. The rat MASH model was established according to the group's previous modeling method: the MASH model was fed a high-fat, high-cholesterol diet with free access to food. The commercially available high-fat, high-cholesterol diet formula (TP 26304-100) included protein (casein, L-cysteine), carbohydrates (starch, sucrose), fat (milk fat), dietary fiber (cellulose), a mineral / vitamin mixture, choline supplement, antioxidant (tert-butylhydroquinone), and cholesterol. The diet contained 4.5 kcal / g of energy; its composition included 42% fat, 14% protein, 44% carbohydrates, and 1% cholesterol.

[0109] The model was established for 19 consecutive weeks, and the changes in liver fat were dynamically observed during the modeling process. At week 19, liver tissue from two mice was randomly selected for HE / Red Oil O staining. If the liver tissue showed macrovesicular or predominantly macrovesicular fatty degeneration, involving more than 5% of hepatocytes, and may be accompanied by mild nonspecific inflammation and partial fibrosis, it indicated that the MASH model was successful. Figure 2 Mice that successfully developed the model were treated with the following methods.

[0110] 3.2 Setting up processing groups

[0111] Eighteen mice were used to construct the MASH model as described above. After successful modeling, they were randomly divided into a model group (n=6, where n is the number of mice in each group), an unfermented herbal medicine group (n=6) from Example 8, and a fermented herbal medicine group (n=6) from Example 7. These 18 mice were continued to be fed a high-fat, high-cholesterol diet. In addition, group A was perfused with 1 mL of physiological saline per day, group B was perfused with 1 mL of the concentrated extract after fermentation prepared in Example 7 per day, and group C was perfused with 1 mL of the ordinary concentrated extract prepared in Example 8 per day, for a total of 8 weeks.

[0112] 3.3 Detection

[0113] Eight weeks after the intervention ended, mouse liver tissue was collected to observe its external morphology, and liver sections were stained with hematoxylin and eosin (HE) to observe pathological changes. Serum was collected from mice to measure serum ALT and AST levels; liver tissue was collected to measure TG and TC levels. Independent samples t-tests were used to compare the means among multiple groups.

[0114] See results Figures 3 - 5 We observed that compared to the model group mice, both the traditional Chinese medicine (TCM) and fermented TCM groups significantly reduced liver volume and lipid content, suggesting that both the ordinary concentrated TCM extract and the fermented concentrated extract can improve hepatic lipid deposition and reduce serum transaminase levels, indicating that both have good hepatoprotective effects. However, compared to the unfermented TCM concentrate, the fermented concentrate significantly reduced hepatic macrovesicular steatosis, suggesting that the fermented concentrate is more effective in improving hepatic inflammation and fibrosis.

[0115] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A traditional Chinese medicine composition for treating metabolic-associated fatty liver disease, characterized in that: Made from the following parts by weight of raw materials, 10-30 parts of Jiubiying, 5-15 parts of Chuanposhi, 5-15 parts of Zhongjiefeng, and 3-10 parts of Xuejie; Alternatively, a traditional Chinese medicine composition consisting of 10-30 parts of Jiubiying, 5-15 parts of Chuanposhi, 5-15 parts of Zhongjiefeng, and 3-10 parts of Xuejie may be prepared by microbial fermentation; the microorganisms are composed of Bacillus subtilis, actinomycetes, and Streptomyces farinaceus.

2. A method for preparing the traditional Chinese medicine composition as described in claim 1, characterized in that: Includes the following steps, The components were crudely extracted according to the ratio to obtain an extract; the traditional Chinese medicine composition consisting of 10-30 parts of Jiubiying, 5-15 parts of Chuanposhi, 5-15 parts of Zhongjiefeng, and 3-10 parts of Xuejie was prepared by microbial fermentation; the microorganisms consisted of Bacillus subtilis, actinomycetes, and Streptomyces farinaceus.

3. The method for preparing the traditional Chinese medicine composition according to claim 2, characterized in that: The crude extraction method is water extraction, which includes crushing the raw materials weighed according to the formula into a mixture, mixing the mixture with water and boiling, filtering, and concentrating to obtain an extract. And / or, it further includes the step of microbial fermentation of the traditional Chinese medicine composition, wherein the microbial liquid is inoculated into a culture medium containing the extract, fermented in a fermenter, and the traditional Chinese medicine composition is obtained by solid-liquid separation; the microbial strains in the microbial liquid are composed of Bacillus subtilis, actinomycetes, and Streptomyces farinaceus.

4. The method for preparing the traditional Chinese medicine composition according to claim 3, characterized in that: The mass ratio of the mixture to water is 1:8 to 1:12; And / or, the boiling time is 0.5-2 hours; And / or, the volume ratio of the concentrated extract to the mass ratio of the mixture is 1:1 to 1:2, in mL:g; And / or, during microbial fermentation, the microbial culture is inoculated into a culture medium containing the extract, with a volume ratio of microbial culture to culture medium containing the extract of 1:8-1:12, a fermentation temperature of 28-38℃, a humidity of 60%-70%, and a fermentation time of 24-72 h at a rotation speed of 180-220 rpm and an aeration rate of 0.8-1.2 VVM.

5. The method for preparing the traditional Chinese medicine composition according to claim 4, characterized in that: Before microbial fermentation, seed cultures of Bacillus subtilis, actinomycetes, and Streptomyces were prepared separately, and their respective OD values ​​were measured. 600 The values ​​are 0.8-1.0, 0.6-0.8, and 0.5-0.7 respectively; And / or, during microbial fermentation, the seed liquids of Bacillus subtilis, actinomycetes viscous and Streptomyces farinaceus are mixed in a volume ratio of 1-2:1-2:1-2 to prepare a microbial inoculum for fermentation; And / or, the total OD of the mixed microbial culture 600 The value is 1.9-2.

5.

6. The method for preparing the traditional Chinese medicine composition according to claim 5, characterized in that: The method for preparing the seed liquid includes: Bacillus subtilis, actinomycetes viscous, and Streptomyces farinaceus were inoculated into culture medium for enrichment culture to obtain the first seed culture of each strain; then the first seed culture was inoculated into culture medium containing extract for expansion culture to obtain the second seed culture of each strain. OD of the first seed culture of Bacillus subtilis for expanded culture 600 Value 2.5-3.5, OD of first seed culture of *Actinomyces viscous* 600 The value is 1.8-2.5, and the OD value of the first seed culture of Streptomyces farinaceus is... 600 The value is 1.5-2.2; In the preparation of the second seed solution, each of the obtained first seed solutions was inoculated into the culture medium containing the extract at a volume ratio of 1:10 to 1:20, and cultured at 35-38℃ and 200 rpm for 12 h. In preparing the first seed culture, the volume ratio of the initial inoculum to the culture medium was 1:50-1:100, and the enrichment culture conditions were 32-37℃, 150-180rpm, and cultured for 12-16h.

7. The method for preparing the traditional Chinese medicine composition according to any one of claims 3-6, characterized in that: The culture medium is a seed culture medium and / or a fermentation culture medium; wherein the seed culture medium comprises 0.5-2 g / L peptone, 0.3-1 g / L yeast extract, 0.5-1.5 g / L NaCl, 0.5-2.0 g / L glucose, 5-20 g / L cellulose, and 1-3 g / L ammonium sulfate; the fermentation culture medium is a culture medium containing the extract as described in claim 4 or 5; the concentration of the extract in the culture medium containing the extract is 8-12 vol%. And / or, the solid-liquid separation step includes centrifugation and filtration, the filtrate after filtration is a traditional Chinese medicine composition, wherein the centrifugation speed is 8000 rpm and the centrifugation time is 8-12 min; And / or, during microbial fermentation, the initial culture should be carried out at 28-31℃ for 10-24 hours, and the subsequent culture should be carried out at 35-38℃ for 14-48 hours.

8. The use of a traditional Chinese medicine composition for treating metabolic-associated steatohepatitis as described in claim 1, or a traditional Chinese medicine composition prepared by any one of claims 2-7, in the preparation of a drug for relieving or treating metabolic-associated steatohepatitis.

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