Traditional Chinese medicine composition for treating fatty liver, preparation and preparation process

Abstract

The invention relates to the technical field of traditional Chinese medicines, in particular to a traditional Chinese medicine composition for treating fatty liver and a preparation. The traditional Chinese medicine composition is prepared from raw radix astragali, radix salviae miltiorrhizae, stewed radix puerariae, rhizoma coptidis, rhizoma alismatis, semen cassiae, raw fructus crataegi, raw malt and lotus leaves. The compatibility of the traditional Chinese medicine composition accords with the principle of monarch, minister, assistant and guide, syndrome differentiation and disease differentiation are combined, raw radix astragali and radix salviae miltiorrhizae serve as monarch drugs, stewed radix puerariae and coptis chinensis serve as minister drugs, rhizoma alismatis, semen cassiae, hawthorn and raw malt serve as adjuvant drugs, lotus leaves serve as guide drugs, and the traditional Chinese medicine composition has the effects of replenishing qi, invigorating the spleen, soothing the liver, promoting diuresis, removing turbidity and dispersing blood stasis. Clinical research and animal experiments prove that the traditional Chinese medicine composition is wide in adaptation disease range and particularly has the remarkable curative effects of promoting GLP-1 secretion, relieving insulin resistance, protecting the liver, regulating and controlling the blood fat level and the like, an object using the traditional Chinese medicine composition does not have obvious adverse reaction, and safety is high.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine (IPC classification number: A61K36), and in particular to a traditional Chinese medicine composition and preparation for treating fatty liver. Background Art

[0002] Fatty liver mainly includes alcoholic fatty liver (AFL) and non-alcoholic fatty liver disease (NAFLD). In 2020, the international academic community recommended that NAFLD be renamed metabolic dysfunction-associated fatty liver disease (MAFLD). In 2024, the Chinese Medical Association's Hepatology Branch updated and republished the "Guidelines for the Prevention and Treatment of Metabolic-Associated (Non-Alcoholic) Fatty Liver Disease (2024 Edition)".

[0003] The global incidence of metabolic (non-alcoholic) fatty liver disease (MAFLD) is as high as 20%-30%. There is increasing evidence that MAFLD is a multi-system disease associated with metabolic disorders. It is more common in patients with obesity and type 2 diabetes (T2DM) and has similar adverse outcomes to these two comorbidities. MAFLD patients are twice as likely to die from cardiovascular disease as from liver disease. In my country, MAFLD has become the number one chronic liver disease, with the prevalence increasing from 15% to more than 31% in 10 years, becoming a key diagnosis and treatment topic for fatty liver.

[0004] For MAFLD, dietary management and exercise therapy are the main intervention measures, but due to poor patient compliance and inability to adhere to it for a long time, it is often difficult to achieve the desired therapeutic effect. At present, there is no specific drug approved for the treatment of the disease at home and abroad. Clinically commonly used hepatoprotective drugs and drugs for correcting metabolic disorders (lipid-lowering drugs, insulin sensitizers, hepatoprotective anti-inflammatory drugs) and other symptomatic treatments, but there are still technical problems such as limited efficacy and adverse reactions to varying degrees. Chinese patent application document CN104352863A discloses a drug for the treatment of insulin-dependent diabetes and its preparation method, with Pueraria root as the monarch, Radix Rehmanniae, Radix Astragali, Radix Trichosanthis, Stigma Corni, Fructus Schisandrae Chinensis, and Rhizoma Dioscoreae as auxiliary, to achieve the effect of quenching thirst, replenishing qi, promoting body fluid and nourishing yin without hurting the vital energy. Chinese patent CN102652826B discloses a Chinese medicine granule for treating diarrhea in children and its preparation method, selecting cloves, Poria, Fructus Schisandrae Chinensis, Flour-fried Pueraria root, Fructus Evodiae, Salvia miltiorrhiza, Rhizoma Coptis, and Sautéed Aucklandiae to effectively solve diarrhea in children. However, these existing technologies have not yet solved the above-mentioned technical problems and cannot exert a good therapeutic effect on MAFLD.

[0005] Traditional Chinese medicine compound has the clinical advantages of multiple pathways and multiple targets, which can not only effectively improve its clinical symptoms, but also reduce the adverse reactions caused by drugs. Therefore, finding a safe and effective traditional Chinese medicine composition and developing it into a suitable dosage form is of great clinical significance for the prevention and treatment of MAFLD. Summary of the invention

[0006] In order to solve the above problems, the first aspect of the present invention provides a traditional Chinese medicine composition for treating fatty liver. The traditional Chinese medicine composition has the effects of invigorating qi, strengthening the spleen and soothing the liver, removing dampness, removing turbidity and removing blood stasis, and can effectively treat metabolism-related fatty liver disease.

[0007] The Chinese medicine composition comprises a main drug and an auxiliary drug; the main drug comprises raw astragalus and purple salvia miltiorrhiza; the auxiliary drugs comprise stewed kudzu root and coptis root.

[0008] Furthermore, the Chinese medicine composition also includes adjuvants and guiding drugs, the adjuvants include at least one of oriental rhizome, cassia seed, raw hawthorn, and raw malt; the guiding drug includes lotus leaf.

[0009] Furthermore, the Chinese medicine composition includes raw astragalus, purple salvia miltiorrhiza, stewed kudzu root, coptis root, oriental water plantain, cassia seed, raw hawthorn, raw malt and lotus leaf.

[0010] Preferably, in parts by weight, the Chinese medicine composition comprises: 8-25 parts of raw astragalus, 5-20 parts of purple salvia miltiorrhiza, 8-30 parts of stewed kudzu root, 2-10 parts of coptis chinensis, 5-16 parts of oriental alisma, 5-20 parts of cassia seed, 4-22 parts of raw hawthorn, 7-28 parts of raw malt and 2-24 parts of lotus leaf.

[0011] More preferably, based on weight, the Chinese medicine composition includes: 12-21 parts of raw astragalus, 7-15 parts of salvia miltiorrhiza, 11-22 parts of stewed kudzu root, 4-7 parts of coptis chinensis, 8-14 parts of oriental water plantain, 7-15 parts of cassia seed, 8-15 parts of raw hawthorn, 10-23 parts of raw malt and 8-15 parts of lotus leaf.

[0012] The medicinal materials involved in the present invention are all recorded in the 2020 edition of the Chinese Pharmacopoeia, and the quality meets the requirements of the Pharmacopoeia.

[0013] Astragalus is sweet and slightly warm, and enters the spleen and lung meridians. It can replenish qi and strengthen the spleen, and replenish deficiency and strengthen the body.

[0014] Purple Salvia miltiorrhiza is bitter and slightly cold, and enters the heart and liver meridians. It is good at treating various diseases and symptoms caused by blood stasis, relieving blood stasis in the liver meridian, and dispersing the knots in the liver.

[0015] Stewed Pueraria root can raise the clear yang, and has a sweet and cool taste that clears the spleen.

[0016] Coptis chinensis is bitter and cold, and can clear away dampness and heat to lower turbid Yin. When combined with Pueraria root, the two can rise and fall respectively, thus bringing the clear and turbid into order.

[0017] Alisma orientalis is diuretic and eliminates dampness; raw hawthorn is sour and sweet, nourishes yin, promotes blood circulation and removes blood stasis; malt is sweet and warm, enters the stomach meridian, and can eliminate food stagnation and relieve swelling of the heart and abdomen.

[0018] Lotus leaves taste bitter and astringent, enter the liver and spleen meridians, clear phlegm, disperse blood stasis, resolve turbidity, and promote the production of clear qi.

[0019] By designing the formula of the Chinese medicine composition, raw astragalus and purple salvia miltiorrhiza are used as the main medicines, stewed kudzu root and coptis chinensis are used as the auxiliary medicines, oriental water chestnuts, cassia seed, raw hawthorn and raw malt are used as adjuvants, and lotus leaf is used as the guiding medicine. The raw materials are compounded synergistically to maximize the effects of invigorating qi and strengthening the spleen, tonifying deficiency and strengthening the body, soothing the liver and regulating qi, and resolving blood stasis and dispersing nodules.

[0020] By designing the formula of the traditional Chinese medicine composition, raw astragalus (Latin name: Astragalusmembranaceus(Fisch.)Bunge) and red salvia (Latin name: Radix et Rhizoma Salviayunnanensis) are used as the main medicines, stewed kudzu root (Latin name: Pueraria peduncularis Grah.) and coptis (Latin name: Coptis chinensis Franch.) are used as the assistant medicines, oriental water plantain (Latin name: Alisma plantago-aquaticaL.), cassia seed (Latin name: Semen Cassiae), hawthorn (Latin name: Crataegus pinnatifidaBunge) and raw malt (Latin name: Hordeum vulgare L.) are used as the adjuvant medicines, and lotus leaf (Latin name: Nelumbonucifera Gaertn.) is used as the guiding medicine. The raw materials are compounded to work synergistically, so as to maximize the effects of invigorating qi and strengthening the spleen, tonifying deficiency and strengthening the body, soothing the liver and regulating qi, and removing blood stasis and dispersing nodules. Clinical studies and animal experiments have shown that the Chinese medicine composition of the present invention can improve insulin resistance, regulate blood lipid levels, and intervene in the occurrence and development of MAFLD by promoting the production of glucagon-like peptide-1 (GLP-1), with significant therapeutic effects.

[0021] The second aspect of the present invention provides the use of the above-mentioned Chinese medicine composition in the preparation of a preparation for treating fatty liver (especially MAFLD); the preparation includes at least one of granules, decoctions, tablets, capsules, pills, oral liquids, injections, extracts, decoctions, fluid extracts, and syrups.

[0022] Furthermore, the raw materials for preparing the preparation include the above-mentioned Chinese medicine composition and excipients.

[0023] The excipients used in the present invention refer to additives (i.e., excipients) in the pharmaceutical preparation other than the main drug ingredients. The choice of excipients can be flexibly adjusted according to the usage requirements of different dosage forms; for example, diluents, fillers, wetting agents, adhesives, disintegrants, lubricants, cosolvents, emulsifiers, solubilizers, osmotic pressure regulators, etc.

[0024] The third aspect of the present invention provides a granule, wherein the raw materials for preparing the granule include the above-mentioned traditional Chinese medicine composition and an excipient.

[0025] Furthermore, the excipient includes at least one of dextrin, powdered sugar and starch.

[0026] The fourth aspect of the present invention provides a preparation process for the above-mentioned granules, which comprises: mixing the raw materials of the Chinese medicine composition according to the formula amount, adding a solvent for soaking, refluxing extraction, and concentrating under reduced pressure to obtain an extract; adding an excipient, mixing evenly, wet granulating, and drying to obtain the extract.

[0027] Preferably, the preparation process of the granules includes: mixing the raw materials of the Chinese medicine composition according to the formula amount, adding a solvent for soaking, then refluxing and extracting for multiple times, combining the extracts, concentrating under reduced pressure to obtain an extract; adding an excipient, mixing evenly, wet granulating, and drying.

[0028] Preferably, the preparation process of the granules comprises: mixing the raw materials of the Chinese medicine composition according to the formula amount, adding a solvent to soak for 0.5-1.5 hours, reflux extraction for multiple times, each time for 1-2 hours, combining the extracts, and concentrating under reduced pressure at 70-90°C and a vacuum degree of 0.08-0.09MPa to obtain an extract; adding an excipient, mixing evenly, wet granulating, and drying.

[0029] Furthermore, the relative density of the extract is 1.10-1.50.

[0030] Preferably, the relative density of the extract is 1.20-1.40.

[0031] Furthermore, the weight ratio of the extract to the excipient is 1:(1-4).

[0032] Preferably, the weight ratio of the extract to the excipient is 1:(1.5-2.5).

[0033] The present invention is directed to granules, and the weight ratio of the extract to the excipient is 1:(1-4), which can greatly improve the physical and chemical stability of the granules. The obtained granules are uniformly granulated, have a low water content, can be quickly dissolved, and are very convenient to use.

[0034] Furthermore, the mass ratio of the Chinese medicine composition to the solvent is 1:(5-15).

[0035] Preferably, the mass ratio of the traditional Chinese medicine composition to the solvent is 1:(8-12).

[0036] The solvent is not particularly limited, and any common solvent in the art may be used as long as it can achieve the purpose of dissolution, such as water or alcohol aqueous solution.

[0037] Beneficial effects:

[0038] (1) The Chinese medicine composition of the present invention complies with the principle of "monarch, minister, assistant and envoy", combines syndrome differentiation with disease differentiation, takes raw astragalus and purple salvia miltiorrhiza as monarch medicines, stewed kudzu root and coptis chinensis as minister medicines, takes oriental rhizome, cassia seed, raw hawthorn and raw malt as adjuvant medicines, and takes lotus leaf as envoy medicine. The Chinese medicine composition has the effects of invigorating qi, strengthening spleen and soothing liver, promoting dampness, removing turbidity and removing blood stasis.

[0039] (2) Clinical studies and animal experiments have shown that the Chinese medicine composition of the present invention has a wide range of indications, and in particular has significant therapeutic effects such as promoting the secretion of glucagon-like peptide-1 (GLP-1), alleviating insulin resistance, protecting the liver, and regulating blood lipid levels. In addition, the subjects did not experience obvious adverse reactions, and the Chinese medicine composition has a strong safety profile.

[0040] (3) The Chinese medicine preparation of the present invention has the advantages of simple preparation process, stable and controllable preparation quality, low price, and convenient carrying, which is convenient for commercial production and can be better promoted and applied in MAFLD patients.

[0041] (4) The Chinese medicine composition of the present invention can be made into granules with stable and reliable physical and chemical properties. The obtained granules comply with the provisions of the four general rules of the "Chinese Pharmacopoeia" (2020 edition) for granules, have good solubility, low water content, small difference in filling volume, and microbial indicators meet the requirements, and can be used with confidence. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The finished granule product obtained in Example 1;

[0043] Figure 2 The results of the pharmacodynamic effect of the Chinese medicine composition of Example 1 on anti-metabolism-associated fatty liver disease (MAFLD) in a mouse model; specifically (A) weight gain results, n=8; (BC) blood glucose curve and area under the curve (AUC) in the OGTT experiment, n=5; (D) liver tissue of each group of mice; (E) HE staining results of liver tissue, n=4; Oil red O staining of liver tissue, n=4; scale bar is 200μm; (F) daily energy intake results of each mouse; (GJ) serum total triglyceride, total cholesterol, high-density lipoprotein cholesterol and low-density lipoprotein cholesterol results, n=8; (KL) serum alanine aminotransferase and aspartate aminotransferase, n=8. (MN) liver total triglyceride and total cholesterol results, n=8.

[0044] Figure 3 , Effect of Chinese herbal medicine composition on small intestinal GLP-1 release in mice with metabolic-associated fatty liver disease (MAFLD) (A) PYY concentration in mouse serum, n = 6. (B) GLP-1 concentration in mouse serum, n = 4. (C) GLP-1 concentration in NCI-H716 cell supernatant, n = 5.

[0045] Figure 4 , Pueraria thin layer chromatogram (from left to right: puerarin reference, test sample, test sample, test sample, negative control).

[0046] Figure 5 , thin layer chromatogram of Coptis chinensis (from left to right: berberine hydrochloride reference substance, Coptis chinensis reference medicinal material, test sample, test sample, test sample, negative control).

[0047] Figure 6 , thin layer chromatogram of Astragalus, including A. view under sunlight, B. view under ultraviolet light (from left to right: Astragaloside IV reference substance, test substance, test substance, test substance, negative control). DETAILED DESCRIPTION

[0048] In order to make the above-mentioned purpose, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below.

[0049] Except as shown in the operating examples or otherwise indicated, all numbers used in the specification and claims to indicate the amount of ingredients, physicochemical properties, etc. are understood to be adjusted by the term "about" in all cases. Therefore, unless otherwise indicated, the numerical parameters listed in the above specification and the attached claims are approximate values, and those skilled in the art can use the teachings disclosed herein to seek to obtain the desired properties and appropriately change these approximate values. The use of numerical ranges expressed as endpoints includes all numbers within the range and any range within the range, for example, 1 to 5 includes 1, 1.1, 1.3, 1.5, 2, 2.75, 3, 3.80, 4 and 5, etc.

[0050] Example 1

[0051] This embodiment provides a traditional Chinese medicine composition for treating fatty liver. The raw materials are composed of 15g raw astragalus, 10g purple salvia miltiorrhiza, 15g stewed kudzu root, 5g coptis root, 10g oriental rhizome, 10g cassia seed, 10g raw hawthorn, 15g raw malt and 10g lotus leaf by weight.

[0052] The preparation method of the decoction containing the Chinese medicine composition is: the Chinese medicine decoction is made according to the traditional decoction method, that is, the raw materials of the Chinese medicine composition are mixed according to the formula amount, 10 times the amount of water is added to mix, and then decocted for 1 hour, and the decoction is collected.

[0053] Example 2

[0054] This embodiment provides a traditional Chinese medicine composition for treating fatty liver. The raw materials are composed of 12g raw astragalus, 8g purple salvia miltiorrhiza, 12g stewed kudzu root, 4g coptis root, 8g oriental rhizome, 8g cassia seed, 8g raw hawthorn, 12g raw malt and 8g lotus leaf by weight.

[0055] The preparation method of the decoction containing the Chinese medicine composition is: the Chinese medicine decoction is made according to the traditional decoction method, that is, the raw materials of the Chinese medicine composition are mixed according to the formula amount, 10 times the amount of water is added to mix, and then decocted for 1 hour, and the decoction is collected.

[0056] Example 3

[0057] This embodiment provides a traditional Chinese medicine composition for treating fatty liver. The raw materials are composed of 21g raw astragalus, 14g purple salvia miltiorrhiza, 21g stewed kudzu root, 7g coptis root, 14g oriental rhizome, 14g cassia seed, 14g raw hawthorn, 21g raw malt and 14g lotus leaf by weight.

[0058] The preparation method of the decoction containing the Chinese medicine composition is: the Chinese medicine decoction is made according to the traditional decoction method, that is, the raw materials of the Chinese medicine composition are mixed according to the formula amount, 10 times the amount of water is added to mix, and then decocted for 1 hour, and the decoction is collected.

[0059] Comparative Example 1

[0060] The Jiangzhimai'an decoction is composed of the following raw materials by weight: 15g of Astragalus, 15g of Salvia miltiorrhiza, 13g of malt, 13g of Crataegus and 14g of Cassia seed. It is prepared into a Chinese medicine decoction according to the traditional decoction method. The specific preparation method is the same as that in Example 1.

[0061] Application Example 1

[0062] The preparation process of the granules containing the Chinese medicine composition is as follows: according to the raw material composition of the Chinese medicine composition in Example 1, the raw materials are mixed, 10 times the amount of water is added to soak for 1 hour, reflux extraction is performed twice, each time for 1.5 hours, the extracts are combined, and reduced pressure concentration is performed at 80°C and a vacuum degree of 0.09 MPa to obtain an extract, and the relative density of the extract is 1.3; dextrin is added in an amount of 2 times the weight of the extract, mixed evenly, wet granulated, dried, granulated, and packaged into 15g / bag, and the obtained granule product is shown in FIG. Figure 1 .

[0063] Performance Testing

[0064] Note: Unless otherwise specified, all solutions involved in the test methods are aqueous solutions.

[0065] 1. Clinical trials

[0066] The Chinese herbal decoctions prepared in Example 1 and Comparative Example 1 were subjected to clinical trials, and the test methods were as follows.

[0067] (1) Treatment samples

[0068] A total of 138 patients with metabolic associated fatty liver disease (MAFLD) who met the case observation requirements were collected from Kunshan Traditional Chinese Medicine Hospital, including 77 cases in the treatment group (28 cases of liver depression and spleen deficiency type, 24 cases of dampness and turbidity retention, 20 cases of damp-heat accumulation, and 5 cases of phlegm and blood stasis); and 61 cases in the control group (22 cases of liver depression and spleen deficiency type, 19 cases of dampness and turbidity retention, 17 cases of damp-heat accumulation, and 3 cases of phlegm and blood stasis).

[0069] There were 41 males and 36 females in the treatment group, with an average age of 45.52±12.7 years; there were 32 males and 29 females in the control group, with an average age of 47.39±13.6 years. The two groups were comparable in age, sex ratio, liver function, and blood lipids before treatment (P>0.05).

[0070] (2) Treatment methods

[0071] Treatment group medication: patients took the Chinese medicine decoction obtained in Example 1 (prepared by the preparation room of Kunshan Traditional Chinese Medicine Hospital), the method of taking was oral, 2 times a day, 1 bag each time, each bag was 200 ml, and it was taken warm 1 hour after meal.

[0072] Control group medication: patients took the comparative example 1 Jiangzhimai An decoction, which was taken orally, twice a day, 1 bag each time, each bag was 200 ml, and it was taken warm 1 hour after meal.

[0073] The treatment course for both groups of patients was 3 months. No other lipid-lowering or enzyme-lowering drugs were taken during the period. In accordance with the requirements of the "Guidelines for the Prevention and Treatment of Non-alcoholic Fatty Liver Disease (2018 Update)" formulated by the Fatty Liver and Alcoholic Liver Disease Group of the Hepatology Branch of the Chinese Medical Association, all patients received health education before treatment, and during the treatment, they adjusted their energy intake and diet structure, exercised moderately, and corrected their unhealthy lifestyles and behaviors.

[0074] (3) Treatment outcomes

[0075] 1) Evaluation of liver elasticity imaging CAP value (liver stiffness value)

[0076] CAP value classification standard: normal <238dB / m; 238dB / m≤ mild <259dB / m; 259dB / m≤

[0077] Moderate <292dB / m; 292dB / m ≤ severe.

[0078] Recovery: CAP value indicates that the liver is normal;

[0079] Markedly effective: CAP value showed recovery from severe to mild, i.e. fatty liver grade was reduced by 2 grades compared with the previous one;

[0080] Effective: CAP value indicates recovery from severe to moderate or from moderate to mild, that is, the fatty liver grade is reduced by 1 grade compared with the previous one;

[0081] Ineffective: CAP value indicated that the grade of fatty liver did not change significantly or even worsened.

[0082] The CAP values ​​of the two groups of patients before and after treatment were compared with the rank sum test of two-sample one-way ordered classification. The results showed that there was a statistically significant difference between the two groups (Z=-2.422, P=0.015). The total effective rate of the two groups was compared with the two-sample chi-square test, and the results showed that there was also a statistically significant difference (chi-square=7.198, P=0.007). The effective rate of the treatment group (90.90%) was significantly higher than that of the control group (73.77%). See Table 1 for details.

[0083] Table 1 Comparison of CAP values ​​between the two groups before and after treatment

[0084]

[0085] Note: Compared with the control group after treatment, P<0.05.

[0086] 2) Comparison of liver function and blood lipid changes between the two groups of patients before and after treatment (see Table 2): After treatment, the levels of ALT (alanine aminotransferase), AST (fatty liver aspartate aminotransferase), TG (triglyceride), and TC (total cholesterol) in the two groups of patients were reduced, and the level of HDL-C (high-density lipoprotein cholesterol) was increased, which was significantly different from before treatment (P<0.05), and the treatment group was better than the control group (P<0.05).

[0087] Table 2 Comparison of liver function and blood lipid changes between the two groups of patients before and after treatment

[0088]

[0089] Note: Compared with the group before treatment﹡P<0.05; compared with the control group after treatment▲P<0.05.

[0090] 3) HOMA-IR index of the two groups of patients before and after treatment (see Table 3): After treatment, the HOMA-IR (insulin resistance index) levels of the two groups of patients decreased, which was significantly different from before treatment (P<0.05), and the treatment group was better than the control group (P<0.05).

[0091] Table 3 Comparison of HOMA-IR index and serum ADP level between the two groups before and after treatment

[0092]

[0093] Note: Compared with the group before treatment ﹡P<0.05; compared with the control group after treatment ▲P<0.05 (4) Conclusion

[0094] The Chinese medicine composition provided in Example 1 of the present invention has more advantages than the conventional Jiangzhi Mai'an granules (Comparative Example 1) in terms of the total effective rate of improving the CAP value of liver elastic imaging, the cure rate, liver function, and glucose and lipid metabolism.

[0095] 2. Animal Experiments

[0096] (1) Experimental animals and modeling and drug administration

[0097] Sixty 8-week-old SPF C57BL / 6J male mice were randomly divided into 6 groups: normal group, model group, Chinese herbal medicine decoction group (high, medium and low doses of 20g / kg, 10g / kg and 5g / kg of raw medicine, respectively), and positive control group (metformin). The metabolic-associated fatty liver disease (MAFLD) model was established by feeding with a high-fat diet for 8 weeks, and drug intervention was performed for 4 weeks. The body weight and food intake of mice were recorded daily.

[0098] (2) Specimen collection

[0099] OGTT and ITT assays: After fasting for 14 h, an oral glucose tolerance test (OGTT) was performed, and blood glucose levels were measured in the tail vein every 0, 15, 30, 60, 90 and 120 min using a Roche blood glucose meter after oral administration of 1 g / kg glucose. Insulin tolerance test (ITT) was performed in mice fasted for 5 h. Insulin (0.75 U / kg) was administered intraperitoneally and blood glucose concentrations were measured every 15, 30, 45, 60 and 90 min.

[0100] Collection of other biological samples: mice were terminally blooded, and 50 mg / kg sodium pentobarbital solution (5 mg / mL) was intraperitoneally injected with a dosing volume of 0.1 mL / 10 g. Orbital blood was collected after anesthesia, and the mice were killed by spinal dislocation. The liver was removed, rinsed with saline, and dried with filter paper and weighed. A piece of liver lobe was taken and fixed in paraformaldehyde, and the remaining part was quickly frozen with liquid nitrogen and stored at -80°C.

[0101] (3) Experimental results

[0102] 1) Pharmacological effects of a Chinese herbal medicine combination on metabolic-associated fatty liver disease (MAFLD) in a mouse model

[0103] The results of drug efficacy are shown in Figure 2 .from Figure 2It can be seen that the low, medium and high doses of the Chinese medicine decoction in Example 1 have different degrees of improvement on the weight gain, liver steatosis, insulin resistance and dyslipidemia of mice with metabolic associated fatty liver disease (MAFLD), and alleviate the symptoms of MAFLD in mice fed a high-fat diet. In addition, it was found that the three doses of the Chinese medicine composition can significantly reduce the energy intake of mice with metabolic associated fatty liver disease (MAFLD). Figure 2 It can be seen that after administering low, medium and high doses of the Chinese medicine composition to mice with metabolic-associated fatty liver disease (MAFLD), the weight gain was significantly reduced ( Figure 2 Glucose tolerance improved in a dose-dependent manner ( Figure 2 Figure BC), indicating that the Chinese medicine composition improved the insulin sensitivity of mice with metabolic-associated fatty liver disease (MAFLD) mediated by a high-fat diet. After high-fat diet modeling, the model group mice had obvious small oil droplets in the liver tissue. After drug treatment, the small oil droplets were significantly reduced compared with the modeling group (Figure D). Liver oil red staining and hematoxylin staining showed that it effectively alleviated the liver fatty degeneration and abnormal deposition of lipids mediated by a high-fat diet ( Figure 2 Each dose can reduce the energy intake of mice ( Figure 2 Meanwhile, the high-dose Chinese medicine composition can significantly improve serum TG, TC, HDL-c, LDL-c, ALT and AST, as well as liver TC and TG levels ( Figure 2 The above results show that the Chinese medicine composition of the present invention alleviates the symptoms of metabolic-associated fatty liver disease (MAFLD) in mice fed a high-fat diet. Figure 2 All values ​​are expressed as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 compared with ND mice; #p < 0.05, ##p < 0.01, ###p < 0.001 compared with HFD mice.

[0104] 2) Effect of Chinese herbal medicine combination on GLP-1 secretion in mice with metabolic associated fatty liver disease (MAFLD)

[0105] The content of GLP-1 in mouse serum was determined by ELISA kit, and it was found that the content of GLP-1 in mouse serum increased significantly after administration. In the serum of mice with metabolic-associated fatty liver disease (MAFLD), administration of the Chinese medicine composition increased the content of GLP-1 in a dose-dependent manner ( Figure 3 B), while PYY (peptide tyrosine tyrosine) showed no significant difference ( Figure 3 Then, the Chinese medicine composition was diluted to a certain concentration and administered to the NCI-H716 cell line, and it was found that it significantly increased the secretion of GLP-1 in a dose-dependent manner ( Figure 3 (Figure C).

[0106] Note: Figure 3 Compared with ND mice, *p<0.05, **p<0.01, ***p<0.001; compared with HFD mice, #p<0.05, ##p<0.01, ###p<0.001.

[0107] (4) Conclusion

[0108] The Chinese medicine composition of Example 1 of the present invention has a significant pharmacodynamic effect on mice with non-alcoholic fatty liver disease and can limit the energy intake of mice; it is speculated that the Chinese medicine composition of the present invention can regulate the body's metabolism, thereby promoting the production of GLP-1, effectively alleviating insulin resistance, and achieving the therapeutic effect of non-alcoholic fatty liver disease.

[0109] 3. Physical and chemical identification

[0110] (1) Properties

[0111] The granules prepared in Application Example 1 are light yellow in color, slightly fragrant, and slightly bitter in taste.

[0112] (2) Identification

[0113] 1) Stewed Pueraria root

[0114] Take 10g of granules, grind them into powder, add 20mL of methanol and place in a stoppered conical bottle, ultrasonicate for 30min, filter, and concentrate the clear liquid to 2ml as the test solution. Take another negative granule (without pueraria root) and prepare a negative control solution in the same way. Take the puerarin reference substance and add methanol to prepare a solution containing 1mg per 1ml as the reference substance solution.

[0115] Take 2 μL of each of the three solutions and spot them on the same silica gel G thin layer plate, use chloroform-methanol-water (volume ratio 7:2.5:0.25) as the developing agent, develop, take out, dry, and examine under ultraviolet light (365nm). In the chromatogram of the test sample, at the corresponding position of the chromatogram of the reference sample, a fluorescent spot of the same color appears. The test results are shown in Figure 4 .

[0116] 2) Coptis chinensis

[0117] Take 10g of granules, grind them into powder, add 20ml of methanol and place them in a stoppered conical flask, ultrasonically treat for 30min, filter, and concentrate the clear liquid to 2ml as the test solution. Take 0.25g of Coptis chinensis reference medicinal material, add 20ml of methanol and place them in a stoppered conical flask, ultrasonically treat for 30min, filter, and obtain the Coptis chinensis reference medicinal material solution. Take berberine hydrochloride reference substance, add methanol to make a solution containing 0.5mg per 1ml, as the reference substance solution. Take another 10g of negative granules (lacking Coptis chinensis) and prepare a negative control solution in the same way as the test solution.

[0118] Take 2 μL of each of the above solutions and spot them on the same high-efficiency silica gel G thin layer plate, use toluene-isopropanol-ethyl acetate-methanol-concentrated ammonia test solution (volume ratio 5:1.5:3:1.5:1.5) as the developing agent, place it in a developing cylinder pre-saturated with concentrated ammonia test solution for 20 minutes, develop, take out, dry, and examine under ultraviolet light (365nm). In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions of the chromatograms of the reference medicinal materials and reference sample. Test results are shown in Figure 5 .

[0119] 3) Astragalus

[0120] Take 30g of granules, grind them into powder, add 80mL of hot water to dissolve, cool and extract with ether (twice, 100mL each time), discard the ether solution, extract the water layer with water-saturated n-butanol (twice, 100mL each time), combine the n-butanol solution, wash with ammonia test solution (twice, 100mL each time), discard the ammonia test solution, evaporate the n-butanol solution, dissolve the residue in 1mL of methanol as the test solution. Take another reference substance of astragaloside IV and add methanol to prepare a solution containing 2mg per 1ml as the reference substance solution. Take another 30g of negative granules (lacking astragalus) and prepare a negative control solution in the same way.

[0121] Take 5 μL of each of the above solutions and spot them on the same high-efficiency silica gel G thin layer plate, use the lower layer solution placed at 10°C in chloroform-methanol-water (volume ratio 13:7:2) as the developing agent, and develop at 10°C, take out, dry, spray with 10% sulfuric acid ethanol solution, and heat at 105°C until the spots are clearly colored. Check under sunlight and ultraviolet light (365nm), and the same color fluorescent spots will appear at the corresponding positions in the chromatogram of the test sample and the chromatogram of the reference sample. Test results are shown in Figure 6 .

[0122] Figure 4 , 5 The results of 6 confirm that the granules of the present invention contain stewed Pueraria root, Coptis chinensis and Astragalus membranaceus.

[0123] (3) Inspection

[0124] 1) Particle size

[0125] Referring to the second double screening method of Part IV General Rules 0982 of the "Chinese Pharmacopoeia" (2020 edition), three batches of granule samples of Application Example 1 were inspected for particle size using No. 1 sieve and No. 5 sieve. The particles that could not pass through the No. 1 sieve and the fine powder that could pass through the No. 5 sieve were collected and accurately weighed, and the proportion of the sum of the two (i.e., particle size %) was calculated. The results are shown in Table 4.

[0126] Table 4 Particle size inspection results (n=6)

[0127]

[0128] 2)Moisture

[0129] Referring to the second method of drying method in Part IV of the Chinese Pharmacopoeia (2020 edition), three batches of granule samples of Application Example 1 were evenly spread in a flat weighing bottle with a constant weight, with a thickness of 5 to 10 mm, accurately weighed, and kept open at 100 to 105 ° C. Dry for 5 hours, take out, cover and place in a desiccator for 30 minutes until cold, accurately weighed, dried again for 1 hour, cooled, and accurately weighed. The water content (i.e., moisture %) of the sample was calculated until the difference between two consecutive times did not exceed 5 mg. The results are shown in Table 5.

[0130] Table 5 Moisture test results (n=6)

[0131]

[0132] 3) Solubility

[0133] According to the inspection requirements of "Soluble Particles Inspection Method" under Item 0104 of the Fourth General Rules of the Chinese Pharmacopoeia (2020 Edition), 15 g of each of the granule samples of three batches of Application Example 1 were taken, 200 mL of hot water was added, and stirred for 5 minutes. The solubility of the particles to be tested was observed, and the inspection results are shown in Table 6.

[0134] Table 6 Solubility test results (n=6)

[0135]

[0136] 4) Difference in loading volume

[0137] According to the requirements of Item 0104 of the General Rules of Part IV of the "Chinese Pharmacopoeia" (2020 edition), 10 bags of granule samples from three batches of Application Example 1 were taken, the packaging was removed, and the weight of the contents of each bag was accurately weighed. The filling amount and the average filling amount of each bag were calculated, and the filling amount difference limit was calculated. The inspection results are shown in Table 7.

[0138] Table 7 Results of filling quantity difference inspection (n=10)

[0139]

[0140] 5) Microbial limits

[0141] According to the relevant requirements of Items 1105, 1106, and 1107 of the General Rules of Part IV of the "Chinese Pharmacopoeia" (2020 Edition), three batches of granule samples of Application Example 1 were inspected for various microbial indicators, and the inspection results are shown in Table 8.

[0142] Table 8 Microbial limit test results (n=3)

[0143]

[0144] (6) Content determination (HPLC)

[0145] A Shimadzu LC-20AD high performance liquid chromatograph was used, with an Agilent C18 column (4.6 mm × 250 mm, 5 μm); acetonitrile-0.1% phosphoric acid aqueous solution (22:78) as the mobile phase; the detection wavelength was 286 nm; the flow rate was 1.0 mL / min; the column temperature was 35°C, and the analysis time was 60 min. The theoretical plate number calculated based on the salvianolic acid B peak should not be less than 6000.

[0146] Reagents: Salvianolic acid B reference substance (National Institute for the Control of Pharmaceutical and Biological Products, batch number: 111562-201716), acetonitrile and phosphoric acid were all chromatographically pure, and other reagents were of analytical grade.

[0147] Preparation of reference solution: Take an appropriate amount of salvianolic acid B reference substance, accurately weigh it, and add methanol to make a solution containing 142.4 μg per 1 mL to obtain the reference solution.

[0148] Preparation of the test solution: Take the granule sample of Application Example 1, grind it into powder, weigh 5 g, add 20 mL of 50% methanol aqueous solution, place it in a stoppered Erlenmeyer flask, weigh it, shake it to mix, sonicate for 30 min, make up the weight with 50% methanol aqueous solution, centrifuge (10000 r / min, 10 min), take the supernatant, filter it, and obtain the test solution.

[0149] Determination: Accurately pipette 10 μL of reference solution and test solution respectively, inject into high performance liquid chromatography for qualitative and quantitative detection. The determination results show that this product contains salvianolic acid B, with a content of not less than 0.4 mg per gram of particles.

Claims

1. A Chinese medicine composition for treating fatty liver, characterized in that: The Chinese medicine composition comprises a main drug and an auxiliary drug; the main drug comprises raw astragalus and purple salvia miltiorrhiza; the auxiliary drugs comprise stewed kudzu root and coptis root.

2. The Chinese medicine composition for treating fatty liver according to claim 1, characterized in that: The Chinese medicine composition also includes adjuvants and guiding drugs, wherein the adjuvants include at least one of oriental rhizome, cassia seed, raw hawthorn and raw malt; and the guiding drug includes lotus leaf.

3. The Chinese medicine composition for treating fatty liver according to claim 1, characterized in that: The Chinese medicine composition comprises, by weight: 8-25 parts of raw astragalus, 5-20 parts of purple salvia miltiorrhiza, 8-30 parts of stewed kudzu root, 2-10 parts of coptis root, 5-16 parts of oriental arisaema, 5-20 parts of cassia seed, 4-22 parts of raw hawthorn, 7-28 parts of raw malt and 2-24 parts of lotus leaf; Preferably, in parts by weight, the Chinese medicine composition comprises: 12-21 parts of raw astragalus, 7-15 parts of purple salvia miltiorrhiza, 11-22 parts of stewed kudzu root, 4-7 parts of coptis chinensis, 8-14 parts of oriental alisma, 7-15 parts of cassia seed, 8-15 parts of raw hawthorn, 10-23 parts of raw malt and 8-15 parts of lotus leaf.

4. Use of the Chinese medicine composition according to claim 1 in preparing a preparation for treating fatty liver, characterized in that: The preparation comprises at least one of granules, decoctions, tablets, capsules, pills, oral solutions, injections, extracts, decoctions, fluid extracts and syrups.

5. A granule, comprising the Chinese medicine composition according to any one of claims 1 to 3 and an excipient; characterized in that: The excipient includes at least one of dextrin, powdered sugar and starch.

6. A process for preparing the granules according to claim 5, characterized in that: The preparation process of the granules comprises: mixing the raw materials of the Chinese medicine composition according to the formula amount, adding a solvent for soaking, refluxing extraction, and concentrating under reduced pressure to obtain an extract; adding an excipient, mixing evenly, granulating, and drying to obtain the granules.

7. The process for preparing the granules according to claim 6, characterized in that: The preparation process of the granules comprises: mixing the raw materials of the Chinese medicine composition according to the formula amount, adding a solvent for soaking, then refluxing and extracting for multiple times, combining the extracts, and concentrating under reduced pressure to obtain an extract; adding an excipient, mixing evenly, wet granulating, and drying to obtain the granules.

8. The process for preparing the granules according to claim 7, characterized in that: The soaking time of adding solvent is 0.5-2h; the reflux extraction time each time is 1-2h; the conditions of reduced pressure concentration are: 70-90°C, vacuum degree 0.08-0.09MPa.

9. The process for preparing the granules according to any one of claims 6 to 8, characterized in that: The relative density of the extract is 1.10-1.50; preferably 1.20-1.40; The weight ratio of the extract to the excipient is 1:(1-4); preferably 1:(1.5-2.5).

10. The process for preparing the granules according to any one of claims 6 to 8, characterized in that: The mass ratio of the traditional Chinese medicine composition to the solvent is 1:(5-15); preferably 1:(8-12).

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