Application of corilagin in preparation of product for preventing or treating metabolism-related fatty liver disease

Abstract

The invention relates to the field of biological medicine, and particularly discloses application of natural plant component corilagin in prevention or treatment of metabolism-related fatty liver disease (MAFLD). Experiments prove that corilagin can significantly reduce the body weight and liver weight of MAFLD model mice, improve liver fatty degeneration and lipid deposition, reduce the levels of cholesterol, triglyceride, glutamic-pyruvic transaminase and glutamic oxalacetic transaminase in serum and liver tissues, and improve fasting blood glucose control and glucose metabolism capabilities. Mechanism research shows that the expression of fat synthesis related genes (Srebp1c, FASN and ACC1) is inhibited in a dose-dependent manner, and the expression of fat oxidation related genes (PPAR alpha, CPT1a and AMPK) is up-regulated, so that the regulation of lipid metabolism is realized. In-vitro experiments further prove that corilagin can inhibit lipid droplet accumulation in liver cells and reduce the content of triglyceride. The invention provides powerful technical support for development and application of corilagin in clinical treatment of MAFLD.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of corilagin in preparation of a product for preventing or treating metabolic associated fatty liver disease. BACKGROUND

[0002] Metabolic associated fatty liver disease (MAFLD) is a chronic liver disease characterized by liver fat accumulation, often closely related to obesity, type 2 diabetes and metabolic syndrome. The incidence of MAFLD is increasing year by year, and has become one of the important causes of chronic liver disease and its complications (such as liver fibrosis, liver cirrhosis, hepatocellular carcinoma) worldwide. At present, the clinical management of MAFLD mainly relies on lifestyle intervention (such as diet control, exercise) and symptomatic treatment of complications, but the patient compliance is low and the curative effect is limited. There is a lack of safe and effective drug treatment, and it is urgent to develop new prevention and treatment methods to meet the clinical needs.

[0003] In recent years, traditional Chinese medicine has shown unique advantages in the treatment of metabolic diseases. Its multi-target and multi-pathway characteristics are highly consistent with the complex pathological mechanism of MAFLD. According to traditional Chinese medicine theory, MAFLD is mainly caused by "internal retention of phlegm and dampness, liver stagnation and spleen deficiency". Traditional Chinese medicine compounds or single components can play a comprehensive effect by regulating lipid metabolism, inhibiting inflammatory response and improving insulin resistance. A large number of studies and clinical practices have shown that many traditional Chinese medicines and compounds have significant effects on MAFLD, providing important drug resources and theoretical basis for the prevention and treatment of the disease. Compared with single-target chemical drugs, traditional Chinese medicine treatment pays more attention to the restoration of overall metabolic balance, and has relatively high long-term drug safety, providing a new strategy for the intervention of MAFLD.

[0004] Corilagin is a natural tannin compound with good water solubility, stable chemical properties and high bioavailability. It has various pharmacological effects and can be isolated and extracted from various medicinal plants such as Phyllanthus emblica and Emblica officinalis. Studies have shown that corilagin has anti-inflammatory, antioxidant, antitumor and liver protection effects. In terms of liver protection, corilagin can protect against hepatitis, liver fibrosis and liver cancer caused by various factors through different mechanisms, and no obvious toxic reactions have been observed, indicating that corilagin has great application potential in the treatment of liver-related diseases. However, the mechanism of corilagin in the occurrence and development of liver-related diseases is still lacking in systematic understanding. Therefore, developing new drugs for preventing and treating MAFLD based on corilagin has important application prospects and clinical value. SUMMARY

[0005] In view of the deficiencies of the prior art, the purpose of the present application is to provide a new prevention or treatment method for metabolic associated fatty liver, and particularly relates to the application of corilagin in preventing or treating metabolic associated fatty liver.

[0006] To achieve the purpose of the present application, the following technical solutions are adopted:

[0007] The application of corilagin with a structural formula as shown in Formula I in preparing a product for preventing or treating metabolic associated fatty liver, wherein the corilagin has a molecular formula of C 27 H 22 O 18 , a molecular weight of 634.45,

[0008] Formula I:

[0009] Further, the metabolic associated fatty liver disease includes metabolic associated simple fatty liver, metabolic associated fatty liver hepatitis, liver fibrosis and liver cirrhosis.

[0010] Further, the application includes a medicine or a health product or a food.

[0011] Preferably, the corilagin is used for reducing body weight, liver weight and fat weight.

[0012] Preferably, the corilagin is used for significantly reducing serum total cholesterol (TC), triglyceride (TG) and reducing liver alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, and improving liver lipid accumulation and liver damage state.

[0013] Preferably, the corilagin is used for significantly improving insulin resistance and regulating liver sugar and lipid metabolism disorder.

[0014] Preferably, the corilagin is used for inhibiting the expression of lipid synthesis genes SREBP-1c, FAS and ACC, and activating fatty acid oxidation gene CPT1a.

[0015] Preferably, the effective amount of the corilagin in cells is 1-50 μM, and the effective amount in animals is 1-100 mg / kg / d b.w.

[0016] The present application further discloses a product for preventing or treating metabolic associated fatty liver, which comprises the active ingredient of corilagin as claimed in claim 1, is derived from natural plant extracts, includes emblica, phyllanthus or longan, or is obtained through chemical synthesis.

[0017] Preferably, it further comprises a pharmaceutically acceptable excipient.

[0018] Preferably, the pharmaceutically acceptable adjuvant comprises at least one of a filler, a binder, a wetting agent, a disintegrant, an emulsifier, a co-solvent, a solubilizer, an osmotic pressure regulator, a surfactant, a coating material, a colorant, a pH regulator, an antioxidant, a bacteriostatic agent, or a buffer

[0019] Based on the above technical solutions, the advantages and beneficial effects of the present application are:

[0020] The present application found through animal experiments that corilagin can significantly reduce the body weight and liver weight of mice, significantly reduce the fatty degeneration of liver tissue, and effectively reduce the fat deposition in the liver. In addition, corilagin treatment also significantly reduces the levels of cholesterol, triglyceride, glutamic-pyruvic transaminase and glutamic-oxalacetic transaminase in the serum and liver of mice with metabolic-associated fatty liver disease, and promotes the recovery of liver function. Further studies have shown that corilagin can significantly improve the fasting blood glucose level of MAFLD mice and significantly enhance their glucose metabolism. In in vitro cell experiments, corilagin significantly reduces the accumulation of lipid droplets in hepatocytes and effectively reduces the triglyceride content, further verifying its regulation of lipid metabolism. Corilagin is derived from natural plants, has high safety and reliability, and has low acquisition cost, and has good clinical application potential. Therefore, the present application provides strong experimental support for corilagin as a potential drug for treating MAFLD, and opens up a new direction for developing new drugs or functional products for preventing or treating MAFLD, with broad market prospects and application value. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Effect of corilagin on the content of triglyceride in hepatocytes;

[0022] Figure 2 Effect of corilagin on lipid accumulation in hepatocytes (oil red O staining);

[0023] Figure 3 Effect of corilagin on the body weight of MAFLD mice;

[0024] Figure 4 Effect of corilagin on the liver weight of MAFLD mice;

[0025] Figure 5 Effect of corilagin on the fasting blood glucose of MAFLD mice;

[0026] Figure 6 Effect of corilagin on the glucose tolerance of MAFLD mice;

[0027] Figure 7 Effect of corilagin on the pathological changes (H&E staining) of the liver of MAFLD mice;

[0028] Figure 8 Effect of corilagin on pathological changes (oil red O staining) of the liver of MAFLD mice;

[0029] Figure 9 Effect of corilagin on the cholesterol and triglyceride contents of the serum and liver of MAFLD mice;

[0030] Figure 10 Effect of corilagin on the contents of serum glutamic-oxalacetic transaminase and glutamic-pyruvic transaminase of MAFLD mice;

[0031] Figure 11 Effect of corilagin on the expression of lipid metabolism genes in the liver of MAFLD mice. DETAILED DESCRIPTION

[0032] To make the technical problems, technical solutions and advantages of the present application clearer, the present application will be described in detail below in conjunction with the drawings and specific examples. Those skilled in the art should understand that the examples are only to help understand the present application and should not be regarded as specific limitations on the present application. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used, if not specifically stated, can be obtained from commercial channels.

[0033] Example 1: Corilagin exerts a protective effect in a MAFLD hepatocyte model

[0034] The statistical analysis provided in the examples of the present application includes: all experimental data are statistically analyzed using Graph pad Prism 9.0 software, and are displayed using mean ± standard deviation (Mean ± SD). The difference between groups is analyzed by one-way analysis of variance. Compared with the model group (OA / PA group), "*" in the figure indicates p<0.05, "**" indicates p<0.01, "***" indicates p<0.001, and "****" indicates p<0.0001.

[0035] (1) Effect of corilagin on the triglyceride content of a MAFLD hepatocyte model

[0036] Experimental method: Take mouse normal hepatocytes (AML12) cells in the logarithmic growth phase, and inoculate 1×10 6The cells were seeded in 6-well plates at a density of 1.5 x 105cells / mL, and after the cells adhered, they were grouped and treated with drugs. The experiment was divided into 3 groups. The control group (Control group) was treated with DMEM medium. The model group (OA / PA group) was induced by using a medium containing oleic acid and palmitic acid to induce AML12 cells to establish a MAFLD cell model. The induction condition was to treat AML12 cells with 0.1 mM of oleic acid and palmitic acid (molar ratio of 2:1) for 24 hours. The corilagin treatment group (OA / PA+Cori group) was treated with a medium containing oleic acid and palmitic acid and 10 μM corilagin for 24 hours. After 24 hours of treatment, the original medium in the hole was discarded, and the cells were washed twice with PBS. Then, the cells were collected by RIPA lysis buffer and further broken by ultrasonic. The content of triglyceride in the cells was detected according to the operation steps of the triglyceride detection kit (Nanjing Jiancheng Biological Engineering Institute).

[0037] The experimental results are shown in Figure 1 Compared with the blank control group, OA / PA treatment significantly induced an increase in the TG content in the liver cells (p<0.001), and corilagin treatment could inhibit the increase in the TG content in the liver cells induced by OA / PA (p<0.01).

[0038] (2) Effect of corilagin on lipid accumulation in MAFLD hepatocyte model

[0039] Experimental method: The cell plating and grouping steps were consistent with those described in Example 1(1) above. After 24 hours of treatment, the original cell culture medium was removed, and the cells were gently washed twice with PBS buffer. Then, 4% (w / v) paraformaldehyde was added to fix the cells, and the fixation was performed for 20 minutes to ensure the integrity of the cell structure and lipid droplets. After fixation, the cells were washed twice with PBS. Next, oil red O staining was performed. The cells were pretreated with 60% isopropanol for 5 minutes to promote lipid staining. Then, the prepared oil red O staining working solution was added to each well, and the cells were stained at room temperature for 20 minutes to allow the lipid droplets to be fully stained. After staining, the cells were washed twice with PBS to remove excess staining solution and avoid excessive background staining. Hematoxylin staining was then performed to develop the cell nucleus. The cells were added with an appropriate amount of hematoxylin solution, and the staining was performed for 1 minute to make the cell nucleus blue. Finally, an inverted microscope was used to observe and photograph the formation of lipid droplets in the cells.

[0040] The experimental results are shown in Figure 2: It can be seen that the number of cells containing red lipid droplets in the model group (OA / PA group) significantly increased, indicating that lipid accumulation was obvious. However, in the cells treated with corilagin, the accumulation of lipid droplets was significantly reduced, indicating that corilagin had an inhibitory effect on lipid accumulation. In summary, the above results show that corilagin can effectively improve the lipid metabolism disorder in hepatocytes, reduce lipid droplet accumulation, and thus has a certain protective effect.

[0041] Example 2 Evaluation of the therapeutic effect of corilagin on MAFLD mice

[0042] The statistical analysis provided by the embodiments of the present application includes: all experimental data are statistically analyzed by Graph pad Prism 9.0 software, and are displayed as mean ± standard deviation (Mean ± SD). The difference between groups is analyzed by one-way analysis of variance. Compared with the model group (HFD), "*" in the figure represents p<0.05, "**" represents p<0.01, "***" represents p<0.001, and "****" represents p<0.0001.

[0043] (1) Effect of corilagin on food intake, body weight and organ weight of MAFLD mice

[0044] Experimental method: In this embodiment, 24 8-week-old male C57BL / 6 mice were adaptively fed for 1 week, and 6 of them were randomly selected as a blank group (Chow) and fed with ordinary feed. The remaining mice were fed with 60% high-fat diet for 12 weeks to establish a MAFLD model. After modeling, 18 MAFLD mice were divided into a model group (HFD), a low-dose corilagin treatment group (Cori-L, 15 mg / kg / d b.w.), and a high-dose corilagin treatment group (Cori-H, 30 mg / kg / d b.w.). Each treatment group continued to be fed with 60% high-fat diet, and was given intragastrically with the corresponding dose every two days. The normal group and the model group were given intragastrically with the same volume of normal saline. Drug intervention lasted for 4 weeks, during which the body weight and food intake of mice in each group were monitored and recorded every week. After the experiment, the liver, epididymal fat (eWAT), subcutaneous fat (sWAT), and brown adipose tissue (BAT) of the mice were collected and weighed. Part of the tissue was fixed in 4% (w / v) paraformaldehyde for subsequent preparation of pathological sections; the remaining tissue was quickly transferred to a -80℃ ultra-low temperature refrigerator for storage for subsequent analysis.

[0045] Experimental results: The body weight changes of mice in each group are shown in Figure 3 Compared with the blank group mice, the body weight of the model group mice was significantly increased (p<0.0001) compared with the normal group. Compared with the model group, corilagin can significantly inhibit the increase in body weight of mice caused by high-fat diet in a dose-dependent manner (p<0.01, p<0.001) after four weeks of administration. Figure 4The liver weights of the mice in each group were shown. Compared with the normal group, the liver weights of the mice in the model group were significantly increased (p<0.0001). However, compared with the model group, the liver weights of the mice in the low-dose and high-dose corilagin treatment groups were significantly decreased (p<0.05, p<0.0001), indicating that corilagin can effectively inhibit the increase in body weight and liver induced by high-fat diet.

[0046] (2) Effect of corilagin on fasting blood glucose level and glucose metabolism ability of MAFLD mice

[0047] Experimental method: In this embodiment, the glucose metabolism ability of mice in each group was evaluated by glucose tolerance test (GTT). The glucose used in the experiment was diluted with normal saline, and the intraperitoneal injection dose was 2 g / kg. Before the experiment, the mice were subjected to overnight fasting and body weight measurement. After calculating the required injection amount according to the body weight, intraperitoneal glucose injection was performed. At 0, 30, 60, 90 and 120 minutes after injection, blood samples were collected for blood glucose level determination and recording. The blood glucose value at 0 minute was fasting blood glucose. Finally, the blood glucose change curve was plotted according to the blood glucose change data, and the area under the curve was calculated to evaluate the glucose metabolism status of the mice.

[0048] Experimental results: The results of fasting blood glucose and glucose tolerance test are shown in Figure 5 and Figure 6 . The experimental results show that the fasting blood glucose of the mice in the model group is significantly higher than that in the normal group, indicating that high-fat diet leads to the increase in fasting blood glucose. However, after low-dose and high-dose corilagin treatment, the increase in fasting blood glucose caused by high-fat diet can be significantly inhibited (p<0.005, p<0.01). In addition, after intraperitoneal injection of glucose, the blood glucose change curve of the mice in the model group at each time point is significantly higher than that in the normal group, showing the characteristics of abnormal glucose metabolism. However, after high-dose corilagin treatment, the area under the blood glucose curve is significantly lower than that in the model group (p<0.01), indicating that corilagin can improve the glucose metabolism ability of MAFLD mice.

[0049] (3) Effect of corilagin on liver pathological changes (H&E and oil red O staining) of MAFLD mice

[0050] Experimental method: In this embodiment, H&E staining and oil red O staining techniques were used to evaluate the liver pathological changes of mice in each group.

[0051] H&E staining: First, the prepared liver paraffin sections were treated with deparaffinization, xylene immersion, gradient alcohol dehydration and water washing. Then, the sections were immersed in hematoxylin solution for 5 minutes, washed with running water, differentiated with 1% (v / v) hydrochloric acid, and then re-stained with eosin solution for 2 minutes. Next, the sections were dehydrated with alcohol gradient, and finally mounted with neutral resin. The sections were observed under a microscope and photographed for recording to evaluate the morphological changes of liver tissue.

[0052] Oil red O staining: Fresh liver tissue was made into frozen sections, and after section fixation, it was washed thoroughly with distilled water. Then, the sections were immersed in oil red O working solution for 10-15 minutes, washed with 60% (v / v) isopropanol, and then stained with a solution of hematoxylin for 2 minutes. The sections were again immersed in distilled water and mounted with glycerol gelatin. Finally, the lipid droplets in the liver were observed under a microscope, and photographed for recording to evaluate the accumulation of lipids.

[0053] Experimental results: The results of H&E staining are shown in Figure 7 As shown, the liver structure of normal group mice was complete, the liver lobules were arranged regularly, the hepatocyte morphology was normal, the liver sinusoids were clearly visible, and no lipid droplets or inflammatory cells were observed. In contrast, the liver of model group mice was swollen, the hepatocytes showed extensive fatty degeneration, a large amount of fat vacuoles accumulated in the liver, and was accompanied by local infiltration of inflammatory cells. After low-dose and high-dose corilagin treatment, the liver structure of mice was restored to some extent, the hepatocyte and liver lobule morphology tended to be normal, the fat vacuoles were significantly reduced, and only a small amount of lipid droplets and mild inflammatory reaction were observed in a few areas.

[0054] The results of oil red O staining are shown in Figure 8 Compared with the liver of blank group mice, a large number of orange lipid droplets accumulated in the hepatocytes of model group mice. After low-dose and high-dose corilagin treatment, the lipid droplets in the liver were significantly reduced, and some of the lipid droplets were reduced, indicating that corilagin effectively reduced fat accumulation. In summary, corilagin can significantly improve the fatty degeneration and damage of the liver of MAFLD mice, and has a certain protective effect.

[0055] (4) Effect of corilagin on biochemical indicators of liver of MAFLD mice

[0056] Experimental method: In this experiment, the reagent kit produced by Nanjing Jiancheng Biological Engineering Institute (cholesterol, triglyceride, glutamic transaminase, glutamic transaminase) was used to determine the related indicators of serum and liver homogenate of each group of mice. The specific operation steps were carried out according to the instructions in the reagent kit to ensure the accuracy and reliability of the detection.

[0057] Experimental results: As shown in Figure 9 and Figure 10As shown, compared with the normal group, the contents of cholesterol (p<0.0001), triglyceride (p<0.0001), glutamic-pyruvic transaminase (p<0.0001) and glutamic-oxalacetic transaminase (p<0.0001) in the serum and liver of the model group mice were significantly increased, suggesting that the model group mice had obvious abnormal lipid metabolism and liver damage. After treatment with different doses of corilagin, the contents of cholesterol, triglyceride, glutamic-oxalacetic transaminase and glutamic-pyruvic transaminase in the serum and liver of each dose group mice were significantly lower than those of the model group (p<0.05). These results showed that corilagin could effectively improve the lipid metabolism disorder of MAFLD mice, reduce liver damage, and had a significant liver protection effect.

[0058] (5) Effect of corilagin on the expression of liver lipid metabolism genes in MAFLD mice

[0059] Experimental method: About 50 mg of frozen liver tissue was taken, and total RNA was extracted according to the operation of the RNA Isolator total RNA extraction kit (R401-01) of Vazyme. The RNA concentration and purity were detected by NanoDrop microspectrophotometer. After confirmation, the first strand of cDNA was synthesized using the HiScript III first strand cDNA synthesis kit (Vazyme, R312-02). The synthesized cDNA was used as a template, and the fluorescence quantitative PCR (qPCR) was performed using the BIO-RAD real-time PCR system and Hieff qPCR SYBR Green premix (Yeasen, 11201ES08) to select lipid metabolism related genes (Srebp-1c, FASN, ACC1, PPARα, CPT1a and AMPK, etc.) and internal reference genes (β-actin). The reaction system and program were set according to the kit instructions, and three replicate holes were set for each sample. The relative expression amount of each target gene was calculated by 2 -ΔΔCt method.

[0060] Table 1 Primer sequences for qPCR

[0061]

[0062] Experimental results: To explore the mechanism of corilagin in NAFLD mice, the mRNA expression levels of genes closely related to lipid metabolism in the liver of mice, such as Srebp1c, FASN, ACC1 (fatty synthesis related) and PPARα, CPT1a, AMPK (fatty oxidation related), were detected. Compared with the blank control group, the expressions of Srebp-1c, FASN and ACC1 in the liver of the model group mice were significantly up-regulated (p<0.01), while the expressions of PPARα, CPT1a and AMPK were significantly down-regulated (p<0.01), suggesting that the NAFLD model was successfully constructed and showed the characteristics of enhanced fatty synthesis and inhibited fatty oxidation.

[0063] After the intervention of corilagin, the expression levels of Srebp-1c, FASN and ACC1 in the liver of mice in the low-dose group and the high-dose group were all decreased in a dose-dependent manner, especially in the high-dose group (p<0.01). At the same time, the expression of PPARα, CPT1a and AMPK was significantly up-regulated (p<0.01), indicating that corilagin can effectively inhibit the expression of liver fatty synthesis genes and promote the activation of fatty oxidation-related genes, thereby playing a good lipid-lowering effect.

[0064] In summary, corilagin shows a significant improvement effect on the pathological changes of metabolic-related fatty liver disease in animal models and cell experiments. This study not only deepens the understanding of the target of corilagin from the pharmacological mechanism level, but also provides a solid scientific basis for its application in the prevention and treatment of metabolic liver disease.

[0065] The above is only the preferred specific embodiment of the present application, but the protection scope of the present application is not limited to this. Any person skilled in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. Use of corilagin of structural formula as Formula I in the preparation of a product for preventing or treating metabolic-related fatty liver disease, Formula I:

2. Use according to claim 1, characterized in that, The metabolic-related fatty liver disease includes metabolic-related simple fatty liver, metabolic-related fatty liver hepatitis, liver fibrosis and liver cirrhosis.

3. Use according to claim 1, characterized in that The use includes a medicine or health product or food.

4. Use according to claim 1, characterized in that The use prevents or treats metabolic-related fatty liver disease by inhibiting the expression of lipid synthesis genes Srebp1c, FASN and ACC1, and activating fatty acid oxidation genes PPARa, CPT1a and AMPK.

5. The use according to claim 1, characterized in that, The effective amount of the corilagin in cells is 1-50 μM, and the effective amount in animals is 1-100 mg / kg / d b.w.

6. Use according to claim 1, characterized in that The corilagin is derived from natural plant extracts, including emblica, phyllanthus or longan, or obtained by chemical synthesis.

7. Use according to claim 1, characterized in that, According to claim 1, the dosage form of the medicine or health product is oral preparation, including capsules, tablets, granules, powders, solutions or tea infusion.

8. The product for preventing or treating metabolic-related fatty liver disease according to claim 7, wherein the product is a composition for preventing or treating metabolic-related fatty liver disease, which comprises the extract of claim 1 as an active ingredient. It also includes pharmaceutically acceptable excipients.

9. The product for preventing or treating metabolic-related fatty liver disease according to claim 8, wherein the product is a composition for preventing or treating metabolic-related fatty liver disease, which comprises the extract of claim 1 as an active ingredient. The pharmaceutically acceptable excipients include at least one of a filler, a binder, a wetting agent, a disintegrant, an emulsifier, a cosolvent, a solubilizer, an osmotic pressure regulator, a surfactant, a coating material, a colorant, a pH regulator, an antioxidant, a bacteriostatic agent or a buffer.

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