Application of golden larch bark acetic acid in preparation of medicine for preventing or treating non-alcoholic fatty liver disease
By using Tujingpiacetic acid as an active ingredient, combined with pharmaceutical dosage form and administration methods, the problems of lipid accumulation and blood biochemical indicators in non-alcoholic fatty liver were solved, and effective protection and treatment of non-alcoholic fatty liver was achieved. Effect.
Patent Information
- Application Number
- CN202411235190.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-04
- Publication Date
- 2024-12-13
AI Technical Summary
The application of tujingpiacetic acid has not been studied in the prior art.
As the only active ingredient, the tujingpiacetic acid combines pharmaceutically acceptable amounts (10-300mg) through different dosage forms (such as tablets, granules, oral liquid preparations, etc.) and administration methods (such as oral administration). Salt/vector, used to prevent or treat non-alcoholic fatty liver, inhibit lipid deposition in AML12 hepatocytes, and improve biochemical indicators of triglycerides, total cholesterol, glutamate aminotransferase and alanine aminotransferase.
Tujingpiacetic acid significantly reduced lipid accumulation in non-alcoholic fatty liver and improved inflammatory invasive lesions in liver cells. Through cell oil red O staining and biochemical indicator detection, it proved that it has a protective effect on non-alcoholic fatty liver. It effectively reduced the liver lipid accumulation and serum biochemical index in mice induced by a high-fat diet.
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Figure CN119112876A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biomedicine and relates to a new use of an effective component of a traditional Chinese medicine, in particular to the use of high-purity tricholoma acetic acid in the preparation of a drug for preventing or treating non-alcoholic fatty liver disease. Background Art
[0002] At present, there have been many reports that Chinese herbal compound prescriptions such as Xiao Chaihu Decoction, Shenling Baizhu Powder, Yinchen Wuling Powder and monomer compounds such as hawthorn acid, white peony total glucosides, and fraxin can significantly improve non-alcoholic fatty liver disease, indicating that Chinese medicine has a good effect in the treatment of non-alcoholic fatty liver disease. Therefore, it is of great significance to deeply study the mechanism of action of the active ingredients of Chinese medicine and clarify their targets. Pseudolaric acid B (PAB) exists in plants of the genus Pseudolarix in the Pinaceae family. The content of Pseudolarix amabilis root bark is relatively high. It belongs to the diterpenoids. Pseudolaric acid B has a variety of pharmacological activities such as anti-tumor, anti-fertility, and anti-fungal effects. Pseudolaric acid B inhibits NF-κB signaling by activating peroxisome proliferator-activated receptor γ (PPARγ), thereby alleviating articular cartilage degeneration and synovitis. Other studies have shown that it can induce apoptosis of triple-negative breast cancer cells by inhibiting the PI3K / AKT / mTOR pathway. However, up to now, there has been no report on the use of oleacetic acid in the preparation of non-alcoholic fatty liver protection drugs. Summary of the invention
[0003] In view of this, the purpose of the present invention is to provide the use of rutin acetic acid in the preparation of a drug for preventing or treating non-alcoholic fatty liver disease. The present invention finds that rutin acetic acid has a protective effect on non-alcoholic fatty liver disease, especially on the improvement of lipid accumulation in liver tissue and blood biochemical indicators in non-alcoholic fatty liver disease.
[0004] The object of the present invention is to achieve the following:
[0005] The invention provides application of arbutin acetate in preparing a medicine for preventing or treating non-alcoholic fatty liver disease.
[0006] Based on the above technical solution, further, tricholoma acetic acid is used as the only active ingredient.
[0007] Based on the above technical solution, further, the drug comprises an effective amount of chasteberry acetic acid and a pharmaceutically acceptable salt / carrier.
[0008] Based on the above technical solution, further, the pharmaceutically acceptable salt includes sodium salt and potassium salt.
[0009] Based on the above technical solution, further, the pharmaceutically acceptable carrier includes a filler, a diluent, a binder, a disintegrant, an emulsifier and a drug-carrying carrier without toxic side effects.
[0010] Based on the above technical solution, further, the drug is prepared into pharmaceutically acceptable dosage forms, including tablets, granules, oral liquid preparations, drops, injection preparations and capsule preparations.
[0011] Based on the above technical solution, further, the drug is prepared in the form of a single-dose drug.
[0012] Based on the above technical solution, further, the single-dose medicine contains 10 to 300 mg of chasteberry acetic acid.
[0013] Based on the above technical solution, further, the specific administration method is oral administration, wherein, oral administration of rutinaceous acetic acid (5, 10 and 20 mg / kg) to C57BL / 6 mice has a significant protective effect on non-alcoholic fatty liver disease; in clinical application, it is estimated that an adult (60 kg) takes about 60 to 240 mg (i.e. 1 to 4 mg / kg body weight) orally per day.
[0014] Based on the above technical scheme, further, the drug can inhibit lipid deposition in AML12 hepatocytes, improve the biochemical indicators of triglycerides (TG), total cholesterol (TC), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in non-alcoholic fatty liver disease; and improve lipid deposition and inflammatory infiltration lesions in hepatocytes in vivo.
[0015] The beneficial effects of the present invention compared with the prior art are as follows:
[0016] The present invention found that acetic acid has a protective effect on non-alcoholic fatty liver and can be used to prepare drugs for preventing or treating non-alcoholic fatty liver; the results of cell oil red O staining showed that compared with the control group, the sodium palmitate (PA) induction in the model group led to increased lipid accumulation in AML12 (normal mouse liver cells), while the acetic acid administration group had a significant decrease in lipid accumulation compared with the model group. The results of cell TC and TG determination showed that compared with the control group, the model group had significantly increased intracellular TC and TG contents after PA induction, while the acetic acid (0.025, 0.05 and 0.1 μmol / L) was administered in advance, indicating that acetic acid can inhibit PA-induced lipid accumulation in vitro. For high-fat diet (HFD)-induced non-alcoholic fatty liver disease in mice, compared with the model group, the lipid accumulation in the liver tissue was significantly reduced after treatment with tamarind acetic acid (5, 10 and 20 mg / kg), and the serum biochemical indexes ALT, AST, TC and TG levels in the treatment group were also significantly reduced compared with the model group, indicating that tamarind acetic acid can effectively improve HFD-induced non-alcoholic fatty liver disease in mice. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] In order to more clearly illustrate the embodiments of the present invention, the drawings related to the embodiments are briefly introduced below.
[0018] Figure 1 This is the inhibitory effect of tricholoma acetic acid on PA-induced AML12 cell proliferation in Example 1 of the present invention; compared with the model group, *p<0.05; **p less than 0.01 (n=6).
[0019] Figure 2 This is the Oil Red O staining result of lipid accumulation in cells induced by PA using tricholoma acetic acid in Example 1 of the present invention.
[0020] Figure 3 This is the test result of Example 1 of the present invention on the effect of acetic acid on the TC and TG contents in PA-induced AML12 cells.
[0021] Figure 4 This is the H&E test result of tricholoma acetic acid on HFD-induced non-alcoholic fatty liver liver tissue in mice according to Example 2 of the present invention.
[0022] Figure 5 This is the Oil Red O detection result of tricholoma acetic acid on HFD-induced non-alcoholic fatty liver liver tissue in mice according to Example 2 of the present invention.
[0023] Figure 6 These are the test results of Example 2 of the present invention on the effects of acetic acid on the serum biochemical indicators ALT, AST, TC and TG levels of non-alcoholic fatty liver disease in HFD-induced mice. DETAILED DESCRIPTION
[0024] The present invention is described in detail below with reference to the embodiments, but the embodiments of the present invention are not limited thereto. Obviously, the embodiments described below are only partial embodiments of the present invention. For those skilled in the art, other similar embodiments obtained without creative labor all fall within the protection scope of the present invention.
[0025] The experimental results in the examples are expressed as mean ± standard deviation (mean ± SD), and the data are analyzed using GraphPad 8.0 statistical analysis software; One-Way ANOVA analysis of variance was used for comparison of mean samples among multiple groups, and p < 0.05 was considered to be significantly different.
[0026] Example 1 Study on the protective effect of acetic acid on lipid accumulation induced by PA in vitro
[0027] 1. CCK8 assay to determine cell viability
[0028] Normal mouse liver cells (AML12) that have grown well were digested with trypsin and centrifuged to prepare a cell suspension. 5 Cells were inoculated into 96-well plates at 100 μL / mL, with 100 μL of cell suspension in each well. The cells were grown under normal incubation conditions, and the experiment was performed when the cells grew to about 60% of the bottom area of the well. The groups were as follows: control group (Control), model group (sodium palmitate PA 150 μM), low-dose administration group (PA 150 μM + 0.025 μM acetic acid), medium-dose administration group (PA 150 μM + 0.05 μM acetic acid) and high-dose administration group (PA 150 μM + 0.1 μM acetic acid). Different concentrations of acetic acid solution were first added to each group according to the different groups, and PA solution was added to each group after 24 hours, and CCK8 solution was added after 24 hours, and the absorbance value of each well at 450 nm was detected, and the cell viability was calculated.
[0029] This example investigates the effect of acetic acid on PA-induced cell viability. Figure 1 It can be seen that compared with the control group, the cell viability of the model group was significantly decreased, while the cell viability of the group treated with acetic acid was significantly increased compared with the model group.
[0030] 2. Cell Oil Red O Staining
[0031] Normal mouse liver cells (AML12) that have grown well were obtained, digested with trypsin, centrifuged, and prepared into a cell suspension at a concentration of 1×10 6100 μM / mL was inoculated into a six-well plate, 2 mL per well. After culturing under normal conditions until the cells covered 60% of the bottom surface, different concentrations of acetic acid solution were given, and 150 μM PA solution was given after 24 hours. After 24 hours, the culture medium was discarded, washed twice with PBS, fixed with cell fixative for 30 minutes, discarded the fixative, washed twice with distilled water, added 60% isopropanol to soak for 30 seconds, discarded the isopropanol, added the newly prepared Oil Red O staining solution, soaked for 20 minutes, discarded the staining solution, and rinsed with 60% isopropanol for 30 seconds until the stroma was clear. Wash with water 2-5 times until there is no excess staining solution. Add distilled water to cover the cells and observe under a microscope.
[0032] In this study, Oil Red O staining was used to investigate the effect of acetic acid on lipid accumulation in PA-affected AML12 cells. Figure 2 As can be seen from the figure, compared with the control group, the red area of the cells in the model group increased significantly, indicating that the intracellular lipid accumulation was serious. After 24 hours of advance administration of scutellaria baicalensis acetic acid, the red area in the cells decreased significantly and the lipid accumulation decreased, indicating that scutellaria baicalensis acetic acid can effectively reduce the lipid accumulation in AML12 cells.
[0033] 3. Detection of intracellular TC and TG content
[0034] Normal mouse liver cells (AML12) that have grown well were obtained, digested with trypsin, centrifuged, and prepared into a cell suspension at a concentration of 1×10 6 100 μM / mL was inoculated into a six-well plate, 2 mL per well. After culturing under normal conditions until the cells covered 60% of the bottom surface, different concentrations of acetic acid solution were given, and 150 μM PA solution was given after 24 hours. After 24 hours, the culture medium was discarded, washed twice with PBS, and the cells were scraped off with a cell scraper, centrifuged at 1000 rpm for 10 minutes, the supernatant was discarded, the cell pellet was retained, 0.2-0.3 mL of homogenization medium was added, and ultrasonic disruption was performed under ice water bath. The prepared homogenate was added to a 96-well plate without centrifugation, and then the working solution in the kit was added (the working solution of the TC kit was Good's buffer containing cholesterol esterase, cholesterol oxidase, peroxidase, 4-AAP, phenol, bovine serum albumin and sodium azide; the working solution of the TG kit was Tris-HCl buffer containing lipase, glycerol kinase, 3-phosphoglycerol oxidase, peroxidase, ATP, 4-aminoantipyrine and parachlorophenol), the absorbance value of each well at 500 nm was measured, and the TC and TG contents were calculated.
[0035] In this study, the effects of acetic acid on PA-induced AML12 cells were evaluated by measuring the TC and TG contents in the cells. Figure 3It can be seen that compared with the blank group, the intracellular TC and TG contents in the model group were significantly increased, while after administration of acetic acid, the intracellular TC and TG levels were significantly decreased, indicating that acetic acid can effectively reduce the increase of TC and TG contents in AML12 cells induced by PA.
[0036] Example 2 Protective effect of acetic acid on HFD-induced non-alcoholic fatty liver disease in mice
[0037] 1. Experimental animal grouping, modeling and treatment
[0038] Fifty healthy male SPF C57BL / 6 mice were randomly divided into five groups: blank control group (Control), model group (Model), and acetic acid (PAB) administration group: model group + different doses of acetic acid (5, 10 and 20 mg / kg) were administered by gavage, and the experiment began after one week of free drinking water and food intake. The model group and PAB administration group were given a high-fat diet, and the blank group was given a normal diet. During this period, the administration group was given drugs by gavage, once a day, and acetic acid was added to 0.5% sodium carboxymethyl cellulose (CMC-Na) solution and fully mixed to make a suspension. The blank group and the model group were given the same dose of 0.5% CMC-Na solution for eight weeks. After eight weeks, blood was collected and the mice were killed. The blood was centrifuged at 3500rpm for 10min, and the upper serum was taken and stored at -80℃; part of the liver tissue was fixed in 4% paraformaldehyde for pathological research; the remaining liver tissue was stored at -80℃ for later use.
[0039] 2. Histopathology
[0040] The liver tissues of the above groups were fixed with 4% paraformaldehyde, embedded and sectioned in a conventional manner, and fixed on glass slides. The pathological changes of the tissues were observed and analyzed by hematoxylin-eosin (H&E) staining.
[0041] Results Figure 4 The results showed that in the HFD-induced non-alcoholic fatty liver model in mice, obvious balloon-like changes appeared in the liver tissue of the model group, and the hepatocytes were loosely arranged, with certain inflammatory infiltration. Compared with the model group, the balloon-like changes in the acetic acid-treated group were reduced, and the hepatocytes were tightly arranged, indicating that acetic acid can significantly protect the HFD-induced lipid accumulation in the liver.
[0042] 3. Oil Red O staining of liver tissue
[0043] Part of the liver tissues of each group were embedded and frozen into sections, fixed on glass slides, and stained with Oil Red O staining solution to observe and analyze the pathological changes of the tissues.
[0044] Results Figure 5The results showed that in the HFD-induced non-alcoholic fatty liver model in mice, the area of red lipid droplets in the liver tissue of the model group increased significantly. Compared with the model group, the red lipid droplets in the liver tissue of the drug-treated group decreased significantly, indicating that tamarind acetic acid can significantly reduce HFD-induced hepatic lipid accumulation.
[0045] 4. Serum biochemical index detection
[0046] The serum after centrifugation was taken and the contents of various biochemical indices in the serum were determined according to the instructions of the TC, TG, ALT and AST kits.
[0047] Results Figure 6 The results showed that in the HFD-induced non-alcoholic fatty liver model in mice, the biochemical indicators of the model group were significantly increased compared with the control group, while after treatment with tricholoma acetic acid, the biochemical indicators were significantly decreased, indicating that tricholoma acetic acid can reverse the changes in serum biochemical indicators induced by HFD, thereby playing a protective role against non-alcoholic fatty liver.
[0048] 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 replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.
Claims
1. Application of acetic acid of scutellaria baicalensis in the preparation of drugs for preventing or treating non-alcoholic fatty liver disease.
2. The use according to claim 1, characterized in that: Arnica acetic acid as the only active ingredient.
3. The use according to claim 1, characterized in that: The medicine comprises an effective amount of chasteberry acetic acid and a pharmaceutically acceptable salt / carrier.
4. The use according to claim 3, characterized in that: The pharmaceutically acceptable salts include sodium salt and potassium salt.
5. The use according to claim 3, characterized in that: The pharmaceutically acceptable carriers include fillers, diluents, binders, disintegrants, emulsifiers and drug carriers without toxic or side effects.
6. The use according to claim 1, characterized in that: The drug is prepared into pharmaceutically acceptable dosage forms, including tablets, granules, oral liquid preparations, drops, injection preparations and capsule preparations.
7. The use according to claim 1, characterized in that: The medicine is prepared in the form of a single-dose medicine, and the single-dose medicine contains 10 to 300 mg of chasteberry acetic acid.
8. The use according to claim 1, characterized in that: The drug can inhibit lipid deposition in AML12 hepatocytes and improve biochemical indicators of triglyceride (TG), total cholesterol (TC), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in non-alcoholic fatty liver disease. Improve lipid deposition and inflammatory infiltration lesions in liver cells in vivo.