Use of the composition in preparing a drug for treating metabolic-related fatty liver disease

Abstract

The present invention relates to the technical field of traditional Chinese medicine, and in particular to the use of a composition in the preparation of a drug for treating metabolic-related fatty liver disease. The composition includes: coptis root, pinellia tuber, tuckahoe, immature bitter orange, safflower, lotus seed heart, sophora flavescens, cyperus rotundus, ginseng, ophiopogon japonicus and liquorice. The composition of the present invention is used to reduce the weight gain and liver index of rats with metabolic-related fatty liver disease, reduce the damage of metabolic-related fatty liver disease to rat liver, reduce the expression levels of TG, TC, LDL-C, ALT and AST in serum of metabolic-related fatty liver disease, increase the expression of HDL-C in serum, significantly upregulate the expression of PPARα, ACOX1, CPT-1, and LCAD mRNA in liver tissue, regulate the expression of key enzymes in the β-oxidation pathway of long-chain fatty acids, promote β-oxidation of long-chain fatty acids, reduce fat accumulation and the accumulation of triglycerides, thereby delaying liver fatty degeneration, inhibiting liver fat deposition, and playing a role in liver protection, and can be used for the treatment of 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 use of a composition in preparing a drug for treating metabolism-related fatty liver disease. Background Art

[0002] Metabolic-associated fatty liver disease (MAFLD), formerly known as non-alcoholic fatty liver disease (NAFLD), is currently the most common liver and metabolic disease in the world, with a global prevalence of 25%. An epidemiological study on China showed that the overall prevalence in China increased from 25.4% in 2008-2010 to 32.3% in 2015-2018, and the prevalence growth rate was twice that of Western countries. MAFLD has become the number one cause of chronic liver disease and liver biochemical abnormalities in health examinations in China, seriously endangering people's lives and health and causing a huge economic burden on society. Unhealthy lifestyle habits such as sitting for a long time and moving less, and unhealthy eating habits such as excessive dietary calories and unreasonable dietary structure are closely related to the increasing incidence of MAFLD. With the in-depth study of MAFLD, it was found that MAFLD is closely related to a variety of extrahepatic complications, including cardiovascular and cerebrovascular diseases, tumors, metabolic nephropathy, obstructive sleep apnea syndrome, osteoporosis, psoriasis, colorectal cancer, iron overload and various endocrine diseases (such as type 2 diabetes, thyroid dysfunction and polycystic ovary syndrome). MAFLD is a multisystem disease that affects multiple extrahepatic organ systems.

[0003] In terms of the treatment of MAFLD, it currently mainly includes lifestyle changes and personalized drug treatment. Among them, lifestyle changes include diet management, exercise intervention, biological clock adjustment and psychological intervention; personalized Western medicine treatment includes insulin sensitizers, vitamin E, weight loss and lipid-lowering drugs, liver protection and enzyme-lowering drugs, etc. Western medicine mainly focuses on protecting the liver, reducing enzymes, and regulating lipids, such as statins and fibrates. However, while lowering blood lipids, lipid-regulating drugs can also transport lipids to the liver, aggravate liver lipid metabolism, and may further aggravate liver fatty degeneration. Therefore, Western medicine treatment of MAFLD is still in the early exploration stage of clinical practice, lacks effective prevention and treatment methods, and existing Western medicine treatments have obvious side effects. In terms of traditional Chinese medicine treatment, there is no disease name for MAFLD in traditional Chinese medicine. According to the characteristics of the disease and clinical manifestations, it can be classified into the categories of "liver addiction", "flank pain", "accumulation", "liver attachment", "phlegm", and "phlegm turbidity". At present, most traditional Chinese medicine practitioners believe that NAFLD is located in the liver, closely related to the spleen, and related to the kidneys, and is a syndrome of deficiency and excess. The etiology and pathogenesis are mostly due to long-term addiction to fat and sweet food, obesity, laziness to work, or internal injuries due to seven emotions, improper regulation, or careless exposure to dampness and epidemic toxins, spleen failure, liver failure to dredge, and finally the transformation of water and grain into fine and fine is unfavorable, condensed into dampness and phlegm, blocking the liver network and causing disease. At present, the common treatment of TCM syndrome type includes phlegm-resolving and blood stasis method, liver-soothing and purging turbidity method, spleen-strengthening and kidney-tonifying method, liver-soothing and heat-clearing and dampness-removing method, etc. Traditional Chinese medicine treatment of MAFLD has obvious advantages in improving clinical symptoms and improving liver function, and has the characteristics of few adverse reactions and low side effects. At the same time, the existing Chinese patent medicines also have problems such as unclear syndrome differentiation and treatment in the treatment of MAFLD. Based on this, the technical problem solved by the present invention is to develop a Chinese medicine composition that can effectively prevent and treat metabolic-related fatty liver disease and has clear syndrome types. Summary of the invention

[0004] In view of this, the present invention aims to solve one of the technical problems in the related art at least to a certain extent.

[0005] The present invention proposes the use of a composition in the preparation of a drug for treating metabolic-related fatty liver disease. According to an embodiment of the present invention, the composition is used to treat metabolic-related fatty liver disease, laying a scientific research and application foundation for the treatment of metabolic-related fatty liver disease. The composition comprises coptis root, pinellia tuber, tuckahoe, immature bitter orange, radix changshan, lotus seed core, sophora flavescens, artemisia annua, ginseng, ophiopogon japonicus and liquorice.

[0006] The prescription of the composition of the present invention is based on the theory of phlegm and fire, and aims at the pathogenesis of the disease, which is the syndrome of phlegm and blood stasis. It has the effects of clearing away heat and dampness, regulating qi and resolving phlegm, and calming the heart and relieving palpitations. The coptis chinensis, pinellia ternata, poria, and immature bitter orange in the prescription can clear away heart heat, calm the heart and relieve palpitations; the radix scutellariae, sophora flavescens, lotus seed heart, and artemisia annua can clear heat and resolve phlegm; the ginseng and ophiopogon japonicus can invigorate qi and nourish yin; and the liquorice is sweet and mild, and harmonizes the various medicines.

[0007] According to an embodiment of the present invention, the use of the composition in preparing a drug for treating metabolism-related fatty liver disease may also have the following additional technical features:

[0008] According to an embodiment of the present invention, the composition is used in the preparation of a drug for reducing the expression levels of TG, TC and LDL-C in serum.

[0009] According to an embodiment of the present invention, the composition is used in the preparation of a drug for increasing the expression level of HDL-C in serum.

[0010] According to an embodiment of the present invention, the composition is used in the preparation of a drug for inhibiting the ALT content and AST content in serum.

[0011] It can be understood that blood lipids and liver function are two important indicators for evaluating lipid changes in MAFLD rat models. The composition of the present invention can reduce the expression of TG (triglycerides), TC (total cholesterol), LDL-C (low-density lipoprotein cholesterol), ALT (alanine aminotransferase), AST (aspartate aminotransferase) in serum and increase the expression of HDL-C (high-density lipoprotein cholesterol), which has the effect of reducing fatty degeneration and inhibiting liver fat deposition.

[0012] According to an embodiment of the present invention, the composition is used in the preparation of a drug for up-regulating the expression levels of PPARα, ACOX1, CPT-1, and LCAD mRNA in liver tissue.

[0013] It can be understood that the composition of the present invention intervenes in metabolism-related fatty liver disease through the PPAR signaling pathway, significantly upregulates the expression levels of PPARα, ACOX1, CPT-1, and LCAD mRNA in liver tissue, regulates the expression of key enzymes in the long-chain fatty acid β-oxidation pathway, promotes long-chain fatty acid β-oxidation, reduces fat accumulation and triglyceride accumulation, thereby delaying liver fatty degeneration and improving metabolism-related fatty liver disease.

[0014] According to an embodiment of the present invention, metabolism-related fatty liver disease causes fat vacuoles in liver cells and inflammatory cell infiltration in lobules, and the composition is used to reduce fat vacuoles and alleviate inflammatory cell infiltration, thereby alleviating the damage of metabolism-related fatty liver disease to rat liver.

[0015] According to an embodiment of the present invention, metabolic-related fatty liver disease increases the liver index, and the composition is used to reduce the liver index. It can be understood that the larger the liver index, the more serious the enlargement of the liver in the body. The composition of the present invention can be used to reduce the liver index and play a role in protecting the liver.

[0016] According to an embodiment of the present invention, the composition is prepared by the following raw materials in mass proportions: the mass ratio of coptis root, pinellia tuber, tuckahoe, immature bitter orange, radix changshan, lotus seed core, sophora flavescens, artemisia annua, ginseng, ophiopogon japonicus, and liquorice is 1:0.745:0.745:0.496:0.745:0.135:0.745:0.745:0.496:0.745:0.496. The composition with the above proportion can achieve a better therapeutic purpose.

[0017] According to an embodiment of the present invention, the composition is prepared by the following steps:

[0018] (1) cleaning, slicing, and drying the raw materials for later use;

[0019] (2) Take coptis root, immature bitter orange, radix chebulae, lotus seed core, bitter ginseng and 60% ethanol, reflux extract twice, add 8 times the amount of ethanol of the raw material for the first time, reflux extract for 2 hours, add 6 times the amount of ethanol of the raw material for the second time, reflux extract for 1.5 hours, combine the extracts, filter, and recover ethanol from the filtrate under reduced pressure at 0.04 MPa and 75° C., continue to concentrate to a thick paste with a relative density of 1.36 to 1.38 at 60° C., dry at 80° C. to form a dry paste, and discard the residue;

[0020] (3) Add 70% ethanol to ginseng, Poria cocos, and Pinellia tuber, and reflux extract twice. For the first time, add 10 times the amount of ethanol to extract the raw material, and reflux extract for 2.5 hours. For the second time, add 8 times the amount of ethanol to extract the raw material, and reflux extract for 2 hours. Combine the extracts, filter, and collect the filtrate in another container for later use.

[0021] (4) The residue obtained in step (3) is combined with Ophiopogon japonicus, Artemisia annua and Glycyrrhiza uralensis, and decocted twice with water, with 10 times the amount of the raw material added in the first decoction and 8 times the amount of the raw material added in the second decoction, each time for 1 hour, the decoction is combined, filtered, and the filtrate is concentrated to a clear paste with a relative density of 1.05 to 1.06 at 80°C, 95% ethanol is added to make the alcohol concentration reach 70%, mixed, and allowed to stand for 24 hours to precipitate, the supernatant is collected, filtered, and the filtrate is combined with the filtrate collected in another device in step (3), and ethanol is recovered under reduced pressure at 0.04 MPa and 75°C, and the mixture is further concentrated to a thick paste with a relative density of 1.36 to 1.38 at 60°C, and dried at 80°C to obtain a dry paste;

[0022] (5) The two dry pastes obtained in step (2) and step (4) are combined and crushed into fine powder to obtain the composition of the present invention.

[0023] According to an embodiment of the present invention, the dosage form of the composition includes tablets, capsules, granules and pills. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solution of the present invention, the drawings required for use in the implementation mode will be briefly introduced below. Obviously, the drawings described below are only some implementation modes of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 HE staining results of rat liver tissue pathological sections (HE×400), where (1a) is group C, (1b) is group M, (1c) is group XSN-L, (1d) is group XSN-M, and (1e) is group XSN-H;

[0026] Figure 2 It is a network diagram of active ingredients and targets of the composition;

[0027] Figure 3 It is a Venn diagram of composition components-metabolism-related fatty liver disease targets;

[0028] Figure 4 It is a network diagram of composition-metabolism-related fatty liver disease PPI;

[0029] Figure 5 This is a GO functional enrichment analysis diagram;

[0030] Figure 6 This is the result of KEGG enrichment analysis;

[0031] Figure 7 It is a “component-target-pathway” network diagram;

[0032] Figure 8 This is a comparison of PPARα, ACOX1, CPT-1, and LCAD mRNA levels in the liver of rats in each group. DETAILED DESCRIPTION

[0033] The present invention is described in detail below with reference to the accompanying drawings and embodiments.

[0034] The terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0035] It should be noted that the term "composition" as used in the present invention refers to a product containing a specified amount of a specified component, and any product directly or indirectly produced by a combination of specified amounts of specified components. The meaning of this term related to a pharmaceutical composition includes a product containing an active ingredient (single or multiple) and an inert ingredient (single or multiple) constituting a carrier, as well as any product directly or indirectly produced by mixing, compounding or aggregating any two or more components, or by decomposing one or more components, or by other types of reactions or interactions of one or more components. Therefore, the composition of the present invention includes any composition prepared by mixing a compound of the present invention with a pharmaceutically acceptable carrier.

[0036] It should be noted that the term "treatment" of any disease or condition as used in the present invention refers to all that can slow down, interrupt, prevent, control or stop the progress of a disease or condition, but does not necessarily mean that all symptoms of all diseases or conditions disappear, and it also includes preventive treatment of the symptoms, especially in patients who are prone to such diseases or disorders. In some embodiments, it refers to improving a disease or condition (i.e., slowing down or preventing or alleviating the development of a disease or at least one clinical symptom thereof). In other embodiments, "treatment" refers to alleviating or improving at least one physical parameter, including physical parameters that may not be perceived by the patient. In other embodiments, "treatment" refers to regulating a disease or condition physically (e.g., stabilizing perceptible symptoms) or physiologically (e.g., stabilizing parameters of the body) or both of the above. In other embodiments, "treatment" refers to preventing or delaying the onset, occurrence or deterioration of a disease or condition.

[0037] The present invention proposes the use of a composition in the preparation of a drug for treating metabolic-related fatty liver disease. According to an embodiment of the present invention, the composition comprises coptis root, pinellia tuber, tuckahoe, immature bitter orange, radix changshan, lotus seed heart, sophora flavescens, artemisia annua, ginseng, ophiopogon japonicus and licorice. The mass ratio of the raw materials of the composition, coptis root, pinellia tuber, tuckahoe, immature bitter orange, radix changshan, lotus seed heart, sophora flavescens, artemisia annua, ginseng, ophiopogon japonicus and licorice is 1:0.745:0.745:0.496:0.745:0.135:0.745:0.745:0.496:0.745:0.496.

[0038] The prescription of the composition of the present invention is based on the theory of phlegm and fire, and has the effects of clearing away heat and dampness, regulating qi and resolving phlegm, and calming the heart and relieving palpitations, and the Rhizoma Coptidis, Rhizoma Pinelliae, Rhizoma Poriae, and Rhizoma Citrus Aurantii Immaturus in the prescription can clear away heart heat, calm the heart and relieve palpitations; Radix Dichroae, Radix Sophorae Flavescentis, Pyrus Nelumbinis, and Artemisia Annua can clear away heat and resolve phlegm; Ginseng and Radix Ophiopogonis can invigorate qi and nourish yin; Licorice can be sweet and mild, and harmonize the various medicines. At the same time, the dosage and compatibility of the components in the composition of the present invention will have a great influence on the medicinal properties and efficacy. Even if the same components are used in different dosage ratios, the efficacy will be different, and even the diseases treated will be different.

[0039] The composition of the present invention is used to reduce the weight gain and liver index of rats with metabolism-related fatty liver disease, reduce damage to rat livers, reduce liver fat vacuoles, reduce inflammatory infiltration of liver cells, reduce TG, TC, LDL-C, ALT and AST expression levels in serum of rats with metabolism-related fatty liver disease, increase HDL-C expression in serum, significantly upregulate PPARα, ACOX1, CPT-1, LCAD mRNA expression levels in liver tissue, regulate the expression of key enzymes in the long-chain fatty acid β-oxidation pathway, promote long-chain fatty acid β-oxidation, reduce fat accumulation and triglyceride accumulation, thereby delaying liver fatty degeneration, inhibiting liver fat deposition, and protecting the liver, and can be used for the treatment of metabolism-related fatty liver disease.

[0040] The present invention is described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and do not limit the present invention in any way.

[0041] Example 1 Preparation of the composition

[0042] The formula of the composition: the mass ratio of coptis chinensis, pinellia ternata, poria, immature bitter orange, radix changshan, lotus seed core, sophora flavescens, artemisia annua, ginseng, ophiopogon japonicus and licorice is 1:0.745:0.745:0.496:0.745:0.135:0.745:0.745:0.496:0.745:0.496.

[0043] The process steps are as follows:

[0044] (1) cleaning, slicing, and drying the raw materials for later use;

[0045] (2) Take coptis root, immature bitter orange, radix chebulae, lotus seed core, bitter ginseng and 60% ethanol, reflux extract twice, add 8 times the amount of ethanol of the raw material for the first time, reflux extract for 2 hours, add 6 times the amount of ethanol of the raw material for the second time, reflux extract for 1.5 hours, combine the extracts, filter, and recover ethanol from the filtrate under reduced pressure at 0.04 MPa and 75° C., continue to concentrate to a thick paste with a relative density of 1.36 to 1.38 at 60° C., dry at 80° C. to form a dry paste, and discard the residue;

[0046] (3) Add 70% ethanol to ginseng, Poria cocos, and Pinellia tuber, and reflux extract twice. For the first time, add 10 times the amount of ethanol to extract the raw material, and reflux extract for 2.5 hours. For the second time, add 8 times the amount of ethanol to extract the raw material, and reflux extract for 2 hours. Combine the extracts, filter, and collect the filtrate in another container for later use.

[0047] (4) The residue obtained in step (3) is combined with Ophiopogon japonicus, Artemisia annua and Glycyrrhiza uralensis, and decocted twice with water, with 10 times the amount of the raw material added in the first decoction and 8 times the amount of the raw material added in the second decoction, each time for 1 hour, the decoction is combined, filtered, and the filtrate is concentrated to a clear paste with a relative density of 1.05 to 1.06 at 80°C, 95% ethanol is added to make the alcohol concentration reach 70%, mixed, and allowed to stand for 24 hours to precipitate, the supernatant is collected, filtered, and the filtrate is combined with the filtrate collected in another device in step (3), and ethanol is recovered under reduced pressure at 0.04 MPa and 75°C, and the mixture is further concentrated to a thick paste with a relative density of 1.36 to 1.38 at 60°C, and dried at 80°C to obtain a dry paste;

[0048] (5) The two dry pastes obtained in step (2) and step (4) are combined and crushed into fine powder to obtain the composition of the present invention.

[0049] The dosage forms of the composition include tablets, capsules, granules and pills. The formulations and process steps of different dosage forms are the same as those in Example 1, and different dosage forms are prepared by adding different excipients and following the conventional preparation methods of each dosage form.

[0050] In the following examples, the composition prepared in Example 1 was used to carry out relevant research.

[0051] Example 2

[0052] In this example, a high-fat diet was used to construct a MAFLD rat model. After the administration of the composition of the present invention, the changes in rat body weight, liver index, liver tissue pathological morphology and serum biochemical index detection experiments showed that the composition of the present invention can be used for intervention treatment of metabolism-related fatty liver disease.

[0053] 2.1 Experimental Materials

[0054] (1) Experimental drugs

[0055] The composition liquid of the present invention (accurately weigh 3 parts of the composition powder of the required mass according to the ratio of 1:2:4, add a certain amount of 0.5% CMC-Na solution to prepare the drug suspension with concentrations of 0.052 g / mL, 0.104 g / mL and 0.208 g / mL, respectively, to obtain the low-, medium- and high-dose liquids of the composition of the present invention); distilled water (Guangzhou Watsons Food and Beverage Company); high-fat feed D12451 (Xiaoshu Youtai Biotechnology Co., Ltd.).

[0056] Table 1 List of drugs used in the experiment

[0057]

[0058] (2) Experimental instruments

[0059] Table 2 List of experimental instruments

[0060]

[0061]

[0062] (3) Experimental animals

[0063] The experimental animals were 50 healthy male Wistar rats, provided by Beijing Weitonglihua Experimental Animal Technology Co., Ltd. The breeding conditions were artificial light 12h / d; temperature 24±2℃; ventilation rate 15 times / h and humidity 50±10%, and all rats were free to eat and drink. The animal experiment ethics number is: TCM-LAEC2022125.

[0064] 2.2 Construction of animal model

[0065] After 7 days of adaptive feeding, the experimental animals were divided into 5 groups, each with 10 animals, namely: blank group (group C), model group (group M), low-dose liquid group of the composition of the present invention (group XSN-L, 0.52g / kg, clinical equivalent dose), medium-dose liquid group of the composition of the present invention (group XSN-M, 1.04g / kg, 2 times the clinical equivalent dose), and high-dose liquid group of the composition of the present invention (group XSN-H, 2.08g / kg, 4 times the clinical equivalent dose). Except for group C, which was fed with ordinary feed, the other 4 groups were all fed with high-fat feed (D12451) for a total of 8 weeks. Starting from the 3rd week, each group was gavaged at a ratio of 10ml / kg / d, and groups C and M were given an equal amount of normal saline for 6 consecutive weeks. The standard for successful model construction is that when observed under a microscope, hepatocyte fatty degeneration with a degree of fatty degeneration of liver tissue of more than 30% is regarded as metabolic-related fatty liver disease.

[0066] 2.3 Sample collection

[0067] Blood was collected from the abdominal aorta, left to stand at room temperature for 2 h, and centrifuged at 4°C, 3500 r / min for 10 min. The upper serum was collected and stored in a -80°C ultra-low temperature refrigerator for later use.

[0068] The animals were killed, and the rat liver tissue was removed. The residual blood was washed with pre-cooled physiological saline, and the residual water was dried with filter paper. The liver index was weighed and recorded, and the liver index was calculated (liver index = liver weight / body weight × 100%). The liver lobule was fixed in 10% formalin fixative for HE staining. The remaining tissue was placed in a cryopreservation tube, quenched with liquid nitrogen, and stored in a -80°C ultra-low temperature refrigerator for use.

[0069] 2.4 Statistical methods

[0070] The experimental results were statistically analyzed using GraphPad Prism 8 software. The statistical differences among the groups were compared using one-way-ANOVA. The data were expressed as mean±SEM. P<0.05 indicated statistically significant differences.

[0071] 2.5 Experimental Results

[0072] (1) The composition of the present invention is used to resist the increase in body weight gain in MAFLD model rats.

[0073] The rats were weighed and recorded at the end of each week of the experiment, and the weekly weight changes of rats in each group during the modeling process and the weight gain (%) before and after modeling were analyzed.

[0074] The formula for calculating weight gain is as follows:

[0075]

[0076] Table 3 Comparison of weight gain of rats in each group

[0077]

[0078] Note: Compared with group C, #### P<0.0001; compared with group M, *p<0.05, **p<0.01

[0079] As shown in Table 3, before the experiment, there was no significant difference in the body weight of rats in each group. After 8 weeks, the body weight gain of rats in group M increased significantly compared with group C. Compared with group M, the body weight gain of groups XSN-L, XSN-M and XSN-H decreased significantly. This indicates that the composition of the present invention can resist the increase in body weight gain of rats in the MAFLD model.

[0080] (2) The composition of the present invention significantly reduces the liver index of MAFLD model rats.

[0081] The statistical results of liver index in each group are shown in Table 4. Compared with group C, the liver index of group M was significantly increased (P<0.01), and the difference was statistically significant; compared with group M, the liver index of group XSN-L and group XSN-M showed a downward trend compared with group M, and the liver index of group XSN-H was significantly reduced (P<0.05), and the difference was statistically significant.

[0082] Table 4 Results of liver index determination in rats of each group

[0083]

[0084] Note: Compared with group C, ## P<0.01; *p<0.05 compared with group M

[0085] (3) The composition of the present invention is used to reduce fat vacuoles and alleviate inflammatory cell infiltration in MAFLD model rats.

[0086] The rat liver lobules were selected and stained with HE. The results of liver tissue pathology sections are as follows Figure 1 As shown, the liver cells of rats in group C were normal in size, arranged neatly, and had no obvious abnormalities; the liver cells of rats in group M and the drug solution group of the present invention had a certain degree of damage, the liver cells of rats in group M were of different sizes and arranged disorderly, there were fat vacuoles of different sizes in the liver cells, and inflammatory cells infiltrated in the lobules; compared with group M, the damage was alleviated in groups XSN-L, XSN-M, and XSN-H after administration, with fewer fat vacuoles and less inflammatory cell infiltration, and the damage was alleviated more significantly in group XSN-H.

[0087] (4) The composition of the present invention reduces the expression levels of TG, TC and LDL-C in the serum of MAFLD model rats, increases the level of HDL-C, and inhibits the levels of ALT and AST in the serum.

[0088] Take an appropriate amount of serum and measure the levels of TG (triglyceride), TC (total cholesterol), LDL-C (low-density lipoprotein cholesterol), HDL-C (high-density lipoprotein cholesterol), ALT (alanine aminotransferase), and AST (aspartate aminotransferase) in the serum according to the relevant instructions and operations of the kit.

[0089] Blood lipids are an important indicator for evaluating lipid changes in the MAFLD model. As shown in Table 5: Serum TG levels. Compared with group C, the TG levels of rats in group M were significantly increased, and the difference was extremely statistically significant (P<0.0001). Compared with group M, the TG levels of groups XSN-L, XSN-M, and XSN-H were significantly decreased, and the difference was statistically significant (P<0.01, P<0.001); serum TC levels. Compared with group C, the TC levels of rats in group M were increased. Compared with group M, the XSN-L and XSN-M groups showed a downward trend, and the TC level of group XSN-H was significantly decreased, and the difference was statistically significant (P<0.001); serum LDL-C levels. Compared with group C, the LDL-C levels of rats in group M were increased. Compared with the M group, the levels of XSN-L group, XSN-M group, and XSN-H group showed a downward trend; compared with the C group, the HDL-C level in the serum of rats in the M group was significantly lower, and the difference was statistically significant (P<0.001). Compared with the M group, the levels of XSN-L group and XSN-H group showed an upward trend, and the level of XSN-M group was significantly increased, and the difference was statistically significant (P<0.05).

[0090] Table 5 Comparison of blood lipid levels in rats in each group

[0091]

[0092] Note: Compared with group C, ### P < 0.001, #### P<0.0001; compared with group M, * P<0.05, ** P<0.01, *** P<0.001

[0093] Liver function is another important indicator for evaluating MAFLD. The expression levels of serum ALT and AST in each group are shown in Table 6: serum ALT level, compared with group C, the ALT expression level of rats in group M increased. Compared with group M, the ALT expression levels of rats in groups XSN-L, XSN-M, and XSN-H all tended to decrease; serum AST level, compared with group C, the AST expression level of rats in group M increased. Compared with group M, the AST expression levels of rats in groups XSN-L, XSN-M, and XSN-H all tended to decrease.

[0094] Table 6 Changes of ALT and AST levels in serum of rats in each group

[0095]

[0096] Example 3

[0097] This example uses a combination of network biology, cheminformatics and bioinformatics to reveal that the treatment of MAFLD by the composition of the present invention is regulated by multiple components, multiple targets and multiple pathways, and PPAR signaling pathway is the main pathway for the treatment of MAFLD by the composition of the present invention.

[0098] The specific steps are as follows:

[0099] Step 1: Prediction of the active ingredients and their targets of the composition of the present invention

[0100] The chemical components of the Chinese medicines that make up the composition of the present invention are retrieved and obtained through TCMSP (Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform), and preliminary screening is performed based on oral bioavailability OB ≥ 30% and drug-like property DL ≥ 0.18 to obtain active compounds and their targets, and supplement them according to existing literature reports. Finally, the protein targets are standardized according to the protein database Uniprot (Universal Protein Resource). A total of 474 active ingredients of the composition of the present invention are obtained, including 57 active ingredients from Pinellia ternata, 19 from Dichroa fescue, 16 from Poria cocos, 132 from Glycyrrhiza uralensis, 25 from Coptis chinensis, 44 from Sophora flavescens, 12 from Nelumbo nucifera, 1 from Ophiopogon japonicus, 75 from Artemisia annua, 66 from Ginseng, and 27 from Citrus aurantium. Figure 2 shown.

[0101] Step 2: Prediction of MAFLD disease-related targets

[0102] Using "non-alcoholic fatty liver" as the keyword, we searched the OMIM (Online Mendelian Inheritance in Man), TTD (Therapeutic Target Database), Drugbank and Genecard databases, and obtained a total of 1749 MAFLD disease-related targets. After deleting duplicate options, 1721 related targets were obtained.

[0103] Step 3: Common target screening of “composition of the present invention-MAFLD” and construction of PPI network

[0104] In order to further reveal the effect of the composition of the present invention on MAFLD, the screened active ingredient targets of the composition of the present invention and the disease targets of MAFLD were intersected, and a Venn diagram was drawn to obtain 192 common targets of the composition of the present invention and MAFLD, see Figure 3 In order to obtain the interaction relationship between targets, the intersection targets were submitted to the STRING11.0 platform to construct the interaction (protein-protein interaction, PPI) network between target proteins, and the minimum interaction threshold was set to "highest confidence" (>0.9), and the PPI network diagram of "the composition of the present invention-MAFLD" was obtained, see Figure 4The network contains 191 nodes and 477 edges. Each node represents a target, and each edge represents the interaction relationship between targets. The average node degree is 4.99, and the average local clustering coefficient is 0.402.

[0105] Step 4: GO and KEGG enrichment analysis of common targets of “composition of the present invention-MAFLD”

[0106] In order to reveal the functional distribution of the composition of the present invention on MAFLD, the Metascape data platform was used to perform GO and KEGG analysis and visualization on the targets related to the intervention of the composition of the present invention on MAFLD. In the results of GO enrichment analysis, the biological processes of the composition of the present invention in treating MAFLD include: response to exogenous stimuli, response to hormones, response to nutrient levels, cell response to organic cyclic compounds, response to inorganic substances, monocarboxylic acid metabolic process, response to oxygen levels, regulation of small molecule metabolic process, organic hydroxyl compound metabolic process, etc.; the cellular components include: vesicle cavity, membrane raft, lysis vacuole, receptor complex, cell apical part, transcriptional regulatory complex, peroxisome, membrane side, etc.; the molecular functions include: carboxylic acid binding, oxidoreductase, RNA polymerase II specific DNA binding transcription factor binding, protein homodimerization activity, protease binding, heme binding, NADP binding, protein domain specific binding, nitric oxide synthase regulatory factor activity, etc. Figure 5 shown.

[0107] The results obtained from the KEGG pathway enrichment analysis mainly include: lipid and atherosclerosis signaling pathway, chemical carcinogen-receptor activation signaling pathway, cytochrome P450 metabolic signaling pathway of exogenous substances, diabetic cardiomyopathy signaling pathway, PPAR signaling pathway, arginine biosynthesis signaling pathway, non-alcoholic fatty liver, MAPK signaling pathway, etc. Figure 6 As shown. At present, the medical community generally believes that the main cause of MAFLD is the excessive intake of high-fat, high-sugar, and high-protein by the human body, which leads to lipid accumulation in the body, affects lipid metabolism, and causes MAFLD. The PPAR signaling pathway can regulate the expression of a variety of key genes in triglyceride metabolism, affect peroxisomal β-oxidation, mitochondrial β-oxidation, lipoprotein assembly and transport, fatty acid uptake and binding, and many other processes, thus becoming a key regulatory point for liver triglyceride catabolism. Therefore, the PPARsignaling pathway was selected as the main signaling pathway for MAFLD for verification.

[0108] Step 5: Construction of the network of “ingredients of the composition of the present invention - target - signaling pathway”

[0109] The data of the components, targets and signal pathways of the composition of the present invention were respectively imported into the Cytoscape 3.7.0 software for integration to obtain a network diagram of "components of the composition of the present invention - targets - signal pathways", as shown in FIG. Figure 7 The network includes 278 nodes and 856 edges in total, including 121 component targets, 137 gene targets and 20 signal pathways.

[0110] Step 6: Confirmation of the main signaling pathways of MAFLD intervened by the composition of the present invention

[0111] Peroxisome proliferator-activated receptor α is a member of the nuclear receptor family of transcription factors activated by ligands. It is mainly expressed in the liver and heart and is related to sugar, lipid, and protein metabolism. It can regulate the expression of a variety of key genes in triglyceride metabolism, affect peroxisomal β-oxidation, mitochondrial β-oxidation, lipoprotein assembly and transport, fatty acid uptake and binding, and many other processes, thus becoming a key regulatory point for hepatic triglyceride catabolism. Therefore, PPAR signaling pathway is the main pathway of MAFLD.

[0112] Example 4

[0113] In this example, the liver tissue of rats was tested by real-time fluorescence quantitative PCR to illustrate the changes in the gene level of the composition of the present invention in intervening MAFLD. It was further verified that the composition of the present invention in Example 3 played a role in intervening MAFLD through the PPARsignaling pathway.

[0114] The experimental materials, animal model construction, sample collection, and statistical methods in this example are the same as 2.1, 2.2, 2.3, and 2.4 in Example 2. The animals were grouped into a blank group (Group C), a model group (Group M), and a medium-dose drug solution group of the composition (Group XSN-M, 1.04 g / kg, 2 times the clinical equivalent dose).

[0115] 4.1 Experimental methods

[0116] Step 1: Extract total RNA

[0117] Take 30mg of tissue and add it to a pre-cooled grinding tube containing 1mL of total RNA extraction reagent. Grind it thoroughly with a homogenizer until it is completely lysed. Centrifuge at 4℃ and 12000rpm for 10min and take the supernatant. Add 0.2mL of chloroform to the supernatant tube, mix it upside down and let it stand for 3min. Centrifuge it again for 10min and take the upper aqueous phase. Mix the upper aqueous phase with isopropanol in an equal volume ratio, mix it upside down, centrifuge it for 10min, discard the supernatant, leave the precipitate, and add 1mL of 75% ethanol (ethanol + EDPC water) to the precipitate to rinse the precipitate. Centrifuge, discard the supernatant, and air-dry at room temperature for 10min. Add 20μL of RNasefree water to dissolve the RNA. Determine the RNA concentration and dilute it appropriately to make its final concentration 100-500ng / μL.

[0118] Step 2: Reverse transcription of cDNA

[0119] Table 7 Reaction system

[0120]

[0121] The prepared reaction solution was placed on a PCR instrument for reverse transcription reaction. The reaction conditions were 50°C / 20min and 85°C / 5min. The obtained cDNA was frozen in a -20°C refrigerator for later use.

[0122] Step 3: PCR amplification reaction

[0123] The gene primer sequences are shown in Table 8, and the reaction system is shown in Table 9.

[0124] Table 8 Primer sequences

[0125]

[0126]

[0127] Table 9 Reaction system

[0128]

[0129] PCR amplification conditions:

[0130] Pre-denaturation: 95℃, 3 minutes

[0131] 45 cycles: 95°C, 30 seconds

[0132] 60℃,30seconds

[0133] Melting curve: 95℃, 10seconds→65℃, 1minutes→95℃

[0134] Cooling: 40℃, 10seconds.

[0135] Step 4: Calculation of relative gene expression

[0136] After the reaction, according to the Ct value of the target gene and the internal reference, 2 -ΔΔCt The ratio of the target gene to the internal reference gene was calculated as the relative expression level of the target gene mRNA.

[0137] 4.2 Experimental Results

[0138] As shown in Table 10, Figure 8 As shown in the figure: PPARα mRNA expression in liver. Compared with group C, the expression of PPARα mRNA in liver of rats in group M was significantly decreased, and the difference was statistically significant (P<0.001). Compared with group M, the expression of PPARα mRNA in group XSN-M was significantly increased, and the difference was statistically significant (P<0.01); ACOX1 mRNA expression in liver. Compared with group C, the expression of ACOX1 mRNA in liver of rats in group M was significantly decreased, and the difference was statistically significant (P<0.05). Compared with group M, the expression of ACOX1 mRNA in group XSN-M was significantly increased, and the difference was statistically significant (P<0.05); CPT-1 mRNA expression in liver. Compared with group C, the expression of CPT-1 mRNA in liver of rats in group M was significantly decreased, and the difference was statistically significant (P<0.01). Compared with the M group, the CPT-1 mRNA expression in the XSN-M group was significantly increased, and the difference was statistically significant (P<0.05); compared with the C group, the LCAD mRNA expression in the liver of the M group was significantly decreased, and the difference was statistically significant (P<0.01). Compared with the M group, the LCAD mRNA expression in the XSN-M group was significantly increased, and the difference was statistically significant (P<0.05).

[0139] Table 10 PPARα, ACOX1, CPT-1, LCAD mRNA levels in the liver of rats in each group

[0140]

[0141] Note: Compared with group C, # P<0.05, ## P<0.01, ### P<0.001; compared with group M, * P<0.05, ** P<0.01

[0142] A high-fat diet causes cells to take in too much long-chain fatty acids, and the amount taken in is greater than the amount that can be oxidized and decomposed, causing them to accumulate in the liver. Due to the disorder of fatty acid metabolism in the liver, the ability to oxidize and decompose fatty acids and clear lipids is insufficient, so lipids that have not been oxidized and decomposed accumulate in liver cells, causing fatty degeneration of liver cells and ultimately forming MAFLD.

[0143] The composition of the present invention significantly up-regulates the expression levels of PPARα, ACOX1, CPT-1, and LCAD mRNA in liver tissue, regulates the expression of key enzymes in the long-chain fatty acid β-oxidation pathway, promotes long-chain fatty acid β-oxidation, reduces fat accumulation and triglyceride accumulation, thereby delaying liver fatty degeneration and improving metabolism-related fatty liver disease. The composition of the present invention plays a role in intervening in metabolism-related fatty liver disease through the PPAR signaling pathway, which is consistent with the results of network pharmacology research.

[0144] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of the composition in preparing a drug for treating metabolic-related fatty liver disease, characterized in that: The composition is prepared by mixing the following raw materials in proportion by weight: the mass ratio of coptis root, pinellia tuber, tuckahoe, immature bitter orange, radix chebulae, lotus seed core, sophora flavescens, artemisia annua, ginseng, ophiopogon japonicus and liquorice is 1:0.745:0.745:0.496:0.745:0.135:0.745:0.745:0.496:0.745:0.

496.

2. The use according to claim 1, characterized in that The composition is used in preparing a drug for reducing the expression levels of TG, TC and LDL-C in serum.

3. The use according to claim 1, characterized in that The composition is used in preparing a drug for increasing the expression level of HDL-C in serum.

4. The use according to claim 1, characterized in that The composition is used in preparing a drug for inhibiting the ALT content and AST content in serum.

5. The use according to claim 1, characterized in that: The composition is used in preparing a drug for up-regulating the expression levels of PPARα, ACOX1, CPT-1 and LCAD mRNA in liver tissue.

6. The use according to claim 1, characterized in that: Metabolism-related fatty liver disease causes fat vacuoles in liver cells and inflammatory cell infiltration in lobules. The composition is used to reduce fat vacuoles and alleviate inflammatory cell infiltration.

7. The use according to claim 1, characterized in that Metabolism-related fatty liver disease increases the liver index, and the composition is used to reduce the liver index.

8. The use according to claim 1, wherein the composition is prepared by the following steps: (1) cleaning, slicing, and drying the raw materials for later use; (2) Take coptis root, immature bitter orange, radix chebulae, lotus seed core, bitter ginseng and 60% ethanol, reflux extract twice, add 8 times the amount of ethanol of the raw material for the first time, reflux extract for 2 hours, add 6 times the amount of ethanol of the raw material for the second time, reflux extract for 1.5 hours, combine the extracts, filter, and recover ethanol from the filtrate under reduced pressure at 0.04 MPa and 75° C., continue to concentrate to a thick paste with a relative density of 1.36 to 1.38 at 60° C., dry at 80° C. to form a dry paste, and discard the residue; (3) Add 70% ethanol to ginseng, Poria cocos, and Pinellia tuber, and reflux extract twice. For the first time, add 10 times the amount of ethanol to extract the raw material, and reflux extract for 2.5 hours. For the second time, add 8 times the amount of ethanol to extract the raw material, and reflux extract for 2 hours. Combine the extracts, filter, and collect the filtrate in another container for later use. (4) The residue obtained in step (3) is combined with Ophiopogon japonicus, Artemisia annua, and Glycyrrhiza uralensis, and decocted twice with water, with 10 times the amount of the raw material added in the first decoction and 8 times the amount of the raw material added in the second decoction, each time for 1 hour, the decoction is combined, filtered, and the filtrate is concentrated to a clear paste with a relative density of 1.05 to 1.06 at 80° C., 95% ethanol is added to make the alcohol concentration reach 70%, mixed, and allowed to stand for 24 hours to precipitate, the supernatant is collected, filtered, and the filtrate is combined with the filtrate collected in another device in step (3), and ethanol is recovered under reduced pressure at 0.04 MPa and 75° C., and the mixture is further concentrated to a thick paste with a relative density of 1.36 to 1.38 at 60° C., and dried at 80° C. to obtain a dry paste; (5) The two dry pastes obtained in step (2) and step (4) are combined and crushed into fine powder to obtain.

9. The use according to any one of claims 1 to 8, characterized in that The dosage forms of the composition include tablets, capsules, granules and pills.

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