Application of picria fel-terrae extract in treatment of metabolic dysfunction fatty liver disease

Abstract

The invention provides application of picria fel-terrae extract in treatment of metabolic dysfunction fatty liver disease. 75% ethanol is used as a solvent, and the picria fel-terrae coarse powder is subjected to hot reflux extraction to obtain the picria fel-terrae extract which is used for treating a hepatocyte lipid accumulation model and a metabolic dysfunction fatty liver disease mouse model. Experimental results show that the picria fel-terrae extract can effectively inhibit lipid accumulation in AML12 hepatocytes and remarkably relieve the liver tissue lipid deposition phenomenon in a metabolic dysfunction fatty liver disease model mouse, so that the TC content, the TG content, the AST content and the ALT content in serum of the metabolic dysfunction fatty liver disease mouse are reduced. Therefore, the crude extract of picria fel-terrae relieves liver lipid accumulation, improves liver cell functions and inhibits liver injury caused by fat deposition, picria fel-terrae, as a natural medicine, has multiple treatment advantages, is clear in curative effect and high in safety, and provides a scientific basis for developing a novel natural-source medicine for treating the metabolic dysfunction fatty liver disease.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to the application of Picria felterrae extract in the treatment of metabolic dysfunction-associated steatotic liver disease. Background Art

[0002] Metabolic Dysfunction-associated Steatotic Liver Disease (MASLD), formerly known as Nonalcoholic Fatty Liver Disease (NAFLD), includes a wide range of liver injuries and is one of the main causes of liver diseases globally. In 2023, a multi-society statement proposed using MASLD to replace NAFLD, which is defined as the presence of hepatic steatosis accompanied by at least one cardiometabolic risk factor, including evidence of overweight / obesity, type 2 diabetes, or metabolic dysregulation. MASLD begins with hepatic steatosis, which is characterized by the accumulation of excessive triglycerides (≥5% hepatocytes) in the liver. In a recent meta-analysis, 38% of adults globally had MASLD (2016 - 2019), an increase of 50% since 1990 - 2006. It is estimated that by 2040, the global prevalence of MASLD will reach 55.4%. Studies reported that in 2020, the overall estimated prevalence of MASLD in the general population was 39.43%.

[0003] Current treatment methods for MASLD emphasize intervention for metabolic abnormalities, including dietary adjustments, exercise, and possibly drug treatment, aiming to improve overall metabolic health. Among them, changing lifestyle through diet modification and regular exercise is the mainstay, but the treatment effect is often poor, and there are many adverse reaction reports. Although there are promising treatment options targeting the mechanisms for MASLD patients, they have not been approved by the international medical community. Currently, a large number of studies have shown that traditional Chinese medicine has unique advantages in improving the clinical symptoms of MASLD. Compared with Western medicine, traditional Chinese medicine has fewer side effects and has the characteristics of multi-system, multi-pathway, and multi-target effects.

[0004] In the concept of traditional Chinese medicine, the pathogenesis of MASLD can be summarized as spleen deficiency, damp-heat, phlegm stasis, cold coagulation, and qi stagnation. There have been many studies showing that Chinese herbal medicines can treat the symptoms. Among the 737 studies on the treatment of MASLD listed by Yan et al., 25 are traditional herbs, and silymarin and berberine have both been registered for phase 4 clinical trials for the treatment of MASLD or MASH.

[0005] The syndromes of NASLD patients can be classified into the following types: (1) spleen deficiency and phlegm turbidity; (2) liver qi stagnation; (3) accumulated damp-heat; (4) stasis blocking the channels and (5) liver-kidney deficiency. According to these traditional Chinese medicine syndromes, the treatment principles and related classical prescriptions for treating MASLD are as follows: (1) prescriptions for strengthening the spleen and resolving phlegm: Shenling Baizhu San, Simiao Powder, Sanzi Yangqin Decoction; (2) prescriptions for tonifying the liver and replenishing qi: Minor Bupleurum Decoction, Chaihu Shugan Powder; (3) prescriptions for clearing heat, moistening dampness and dissipating nodules: Major Bupleurum Decoction, Yinchenhao Decoction; (4) prescriptions for promoting blood circulation and removing stasis: Taohong Siwu Decoction; (5) prescriptions for warming and invigorating the spleen: Chaihu Lizhong Decoction, Linggui Zhugan Decoction, Aconite Lizhong Decoction, Sini Powder, Ganjianlingzhu Decoction. Classical prescriptions treat MASLD through various regulatory effects and different mechanisms.

[0006] Picria felterrae Lour. is a plant of the family Linderniaceae and the genus Picria. The medicinal material of Picria felterrae Lour. is a variety included in the 2010 edition of the Pharmacopoeia of the People's Republic of China. The dried whole herb can be used as medicine, which has the effects of clearing heat and detoxifying, reducing swelling and alleviating pain, and is used for symptoms such as wind-heat cold, sore throat, throat obstruction, abdominal pain, dysentery, traumatic injury, snake bite, etc. Picria felterrae Lour. is a large-scale and genuine medicinal material in Guangxi and is the raw material medicinal material for Chinese patent medicines such as Qingfei Sanjie Pills, Wantong Yankang Tablets, Fuyanjing Capsules, Xiaoyanling Tablets, Yanjianning Tablets, etc. However, there has been no report on the therapeutic effect of Picria felterrae Lour. extract on MASLD, so the present invention aims to explore its possible potential application value. Summary of the Invention

[0007] The present invention discovers the application of Picria felterrae Lour. extract in the preparation of drugs for treating metabolic dysfunctional fatty liver disease.

[0008] Furthermore, the hepatocyte lipid accumulation model described in the present invention refers to being induced by palmitic acid and oleic acid and treated with Picria felterrae Lour. extract.

[0009] Furthermore, the Picria felterrae Lour. extract described in the present invention has the function of reducing the lipid level in AML12 cells.

[0010] Furthermore, the Picria felterrae Lour. extract described in the present invention can relieve the lipid accumulation in the liver of MASLD mice.

[0011] Furthermore, the Picria felterrae Lour. extract described in the present invention has the effect of reducing the levels of TC, TG, AST, and ALT in the serum of MASLD mice. Description of the Drawings

[0012] Figure 1 It is the Oil Red O staining map of lipid droplets in AML12 cells after the treatment in Example 3;

[0013] Figure 2 It is the measurement map of the TC content in AML12 cells after the treatment in Example 4;

[0014] Figure 3 It is a measurement chart of the TG content in AML12 cells after the treatment of Example 4;

[0015] Figure 4 It is an Oil Red O staining chart of lipid droplets in the liver of mice after the treatment of Example 6;

[0016] Figure 5 It is a measurement chart of the TC content in the serum of mice after the treatment of Example 7;

[0017] Figure 6 It is a measurement chart of the TG content in the serum of mice after the treatment of Example 7;

[0018] Figure 7 It is a measurement chart of the AST content in the serum of mice after the treatment of Example 7;

[0019] Figure 8 It is a measurement chart of the ALT content in the serum of mice after the treatment of Example 7;

[0020] * indicates compared with the blank group, * p < 0.05, ** p < 0.01; # indicates compared with the model group, # p < 0.05, ## p < 0.01; Abbreviations: TC: total cholesterol; TG: triglyceride; AST: aspartate aminotransferase; ALT: alanine aminotransferase. Detailed implementation manners

[0021] The present invention is further described by the following examples, which do not limit the present invention in any way. Any modification or change that is easily achieved by those of ordinary skill in the art to the present invention without departing from the technical solution of the present invention will fall within the scope of the claims of the present invention.

[0022] Materials:

[0023] Picria felterrae was purchased from Guangxi region; Oil Red O powder was purchased from Sigma - Aldrich, product catalog number: 215 - 295 - 3; SPF - grade male C57BL / 6 mice, 20 - 22 g, were provided by Liaoning Changsheng Biotechnology Co., Ltd., and the experimental animal production license number: SCXK(Liao)2020 - 0001; 60% high - fat diet D124926 was provided by Xiaoshu Youtai (Beijing) Biotechnology Co., Ltd., license number: SCXK(Jing)2023 - 0010; TC, TG, AST, and ALT measurement kits were purchased from Nanjing Jiancheng Bioengineering Institute.

[0024] Example 1: Preparation of Picria felterrae extract (PE)

[0025] ① Extract the active ingredients: Grind the Picria fel-terrae raw materials into coarse powder, add 75% ethanol solution according to the solid-liquid ratio of 1:10 (g / mL), and perform three extractions by the hot reflux extraction method for 1 hour each time. Combine the extracts, concentrate under reduced pressure to remove ethanol, dry at 60 °C after concentrating into an extract, and obtain the Picria fel-terrae extract.

[0026] ② Preparation of the preparation: Dissolve the extract in PBS to obtain PE at 80 mg / ml and dissolve it in physiological saline to obtain PE at 50 mg / kg.

[0027] Example 2: Treatment of AML12 cells

[0028] ① Cell culture: Seed AML12 cells in a 6-well plate at a density of 1×10 6 cells per well, and the culture medium is DMEM / F12 medium containing 10% FBS, 1% penicillin-streptomycin, 1% ITS, and 40 ng / L dexamethasone. Place it in a cell culture incubator at 37 °C containing 5% carbon dioxide for culture. After 12 hours, replace it with a medium containing only DMEM / F12 to starve AML12 cells for 3 hours;

[0029] ② Experimental grouping: Incubate AML12 cells with 80 mg / ml PE for 1 hour and then add OA (660 μM) + PA (330 μM) to incubate the cells for 24 hours; Divide into a normal group (DMEM / F12 medium), a model group (OA (660 μM) + PA (330 μM)), a drug control group (80 mg / ml PE), and a treatment group (80 mg / ml PE + OA (660 μM) + PA (330 μM)).

[0030] Example 3: Oil Red O staining of AML12 cells

[0031] Discard the culture medium and wash the cells 2 times with PBS; Fix with 4% paraformaldehyde for 30 minutes, then discard the fixative and wash the cells 2 times with PBS; Moisten with 60% isopropanol for 2 s in advance and then stain with 0.5% Oil Red O staining solution prepared with 60% isopropanol for 30 minutes; Discard the staining solution and wash the cells multiple times with PBS until the remaining Oil Red residues are washed away; Stain the nuclei with hematoxylin for 1 - 2 minutes, then discard the staining solution and wash the cells 3 times with PBS. After sealing with glycerol, observe the lipid droplets with an optical microscope at a magnification of 200 times.

[0032] The experimental results are shown in the appendix Figure 1 . The results of Oil Red O staining showed that the model group could significantly increase the formation of lipid droplets in AML12 cells, and PE in the treatment group could effectively reduce the lipid droplet accumulation induced by OA (660 μM) + PA (330 μM).

[0033] Example 4: Determination of TC and TG Contents in AML12 Cells

[0034] Collect the cells, centrifuge at 3000 rpm for 5 min, discard the supernatant, and keep the cell pellet; add cell lysis buffer containing phosphatase inhibitor and protease inhibitor, mix well and let stand for 5 min to break the cells; measure according to the kit manufacturer's instructions, and use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance by colorimetry.

[0035] The measured TC and TG contents are shown in the appendix Figure 2 、 3 . The results showed that compared with the normal group, OA (660 μM) + PA (330 μM) could significantly increase the TC and TG levels in AML12 cells. Compared with the model group, the PE drug experimental group could reduce the TC and TG levels in AML12 cells (p < 0.01).

[0036] Example 5: Establishment of a MASLD Mouse Model

[0037] House the mice in an SPF environment at room temperature with a relative humidity of 40% - 80%. Randomly divide each group into 6 mice: normal group (normal diet, saline), model group (HFD, saline), drug control group (normal diet, 50 mg / kg PE), drug treatment group (HFD, 50 mg / kg PE), for a total of 12 weeks. In the last 4 weeks, the model group and the drug treatment group were gavaged with 50 mg / kg of the crude extract of Picria felterrae in saline, and the mice were sacrificed to collect blood and liver.

[0038] Example 6: Oil Red O Staining of Mouse Hepatocytes

[0039] Take the mouse liver, fix it in 4% paraformaldehyde solution for 4 h, then transfer it to 200 g / L sucrose-PB solution for dehydration. After 24 h, change to 300 g / L sucrose-PB solution. After 24 h, place the tissue in the embedding agent. Let the liver tissue sections air-dry at room temperature for 20 min. After the water vapor on the glass slide disappears, perform Oil Red O staining as in Example 3.

[0040] The experimental results are shown in the appendix Figure 4 , and a high-fat diet can significantly increase lipid accumulation in mouse hepatocytes. Compared with the model group, the treatment group can significantly relieve lipid accumulation in mouse hepatocytes induced by a high-fat diet.

[0041] Example 7: Determination of TC, TG, AST, and ALT Contents in Mouse Serum

[0042] A method of obtaining mouse blood by taking blood from the eyeball was used; the blood was allowed to stand at room temperature until serum was precipitated, centrifuged at 3000 rpm / min for 5 min, and the upper layer of serum was aspirated; according to the test kit instructions, the contents of TC, TG, AST, and ALT in the serum were measured, and the absorbance values of each well were measured with an enzyme-linked immunosorbent assay (ELISA) reader.

[0043] The experimental results are shown in Appendix Figure 5 、 6 、 7, 8. The results showed that compared with the blank group, the model group significantly increased the contents of TC, TG, AST, and ALT in the serum of mice (p < 0.01). Compared with the model group, the drug treatment groups could significantly reduce the contents of TC, TG, AST, and ALT in the liver of mice (p < 0.01).

[0044] One-way analysis of variance was used to perform multiple comparisons and statistical significance between different groups. Statistical analysis was performed using GraphPad Prism 9.0.

Claims

1. Application of Scrophularia ningpoensis extract in the preparation of drugs for the treatment of metabolic dysfunction fatty liver disease.

2. The use according to claim 1, characterized in that: The Scrophularia dahurica extract is extracted by ethanol.

3. The use according to claim 2, characterized in that: The bitter Scrophulariaceae was crushed into coarse powder, and 75% ethanol solution was added at a solid-liquid ratio of 1:10 (g / mL). The hot reflux extraction method was repeated three times, and the bitter Scrophulariaceae was extracted for 1 h each time; the extracts were combined, concentrated under reduced pressure to remove ethanol, and concentrated to an extract and dried at 60°C to obtain the bitter Scrophulariaceae extract.

4. The use according to claim 1, characterized in that: The extract of Scrophularia dahurica has the function of reducing the lipid level in AML12 cells.

5. The use according to claim 1, characterized in that: The extract of Scrophularia dahurica has the function of reducing lipid accumulation in the liver of metabolic dysfunction fatty liver disease.

6. The use according to claim 1, characterized in that: The bitter figwort extract can reduce the levels of TC, TG, AST and ALT in the serum of metabolic dysfunction fatty liver disease.

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