CN113577057A - Application of atractylenolide III in preparation of drug for treating non-alcoholic fatty liver disease - Google Patents

Application of atractylenolide III in preparation of drug for treating non-alcoholic fatty liver disease Download PDF

Info

Publication number
CN113577057A
CN113577057A CN202111088737.XA CN202111088737A CN113577057A CN 113577057 A CN113577057 A CN 113577057A CN 202111088737 A CN202111088737 A CN 202111088737A CN 113577057 A CN113577057 A CN 113577057A
Authority
CN
China
Prior art keywords
iii
fatty liver
liver
alcoholic fatty
liver disease
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202111088737.XA
Other languages
Chinese (zh)
Inventor
李曼
高月求
孙学华
张鑫
周振华
纪龙珊
高亚婷
方淼
江云
李茜
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shuguang Hospital Affiliated to Shanghai University of TCM
Original Assignee
Shuguang Hospital Affiliated to Shanghai University of TCM
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shuguang Hospital Affiliated to Shanghai University of TCM filed Critical Shuguang Hospital Affiliated to Shanghai University of TCM
Priority to CN202111088737.XA priority Critical patent/CN113577057A/en
Publication of CN113577057A publication Critical patent/CN113577057A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5067—Liver cells
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Hematology (AREA)
  • Cell Biology (AREA)
  • Public Health (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Food Science & Technology (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Epidemiology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Toxicology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Botany (AREA)
  • Mycology (AREA)
  • Nutrition Science (AREA)
  • Polymers & Plastics (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The invention relates to application of atractylenolide III in preparation of a medicament for treating non-alcoholic fatty liver disease. The invention discovers that atractylenolide III can reduce liver lipid deposition, reduce liver index, remarkably reduce serum blood lipid level and liver tissue TG, TC level and activate related signal molecules of AMPK/SIRT1 signal path; can also reduce MDA level, increase SOD and GSH-Px level, and improve oxidative stress. Atractylodes macrocephala lactone III can inhibit lipid deposition by activating an AMPK/SIRT1 signal channel, reduce oxidative stress reaction of liver cells, simultaneously improve CPT1A expression level, improve mitochondrial oxidative stress, and play a role in treating non-alcoholic fatty liver disease through the above mechanism. The atractylenolide III is a chemical substance derived from plants, has simple preparation steps and little environmental pollution, and is suitable for industrial production. Therefore, the atractylenolide III can be used for preparing medicines or health-care foods for treating the non-alcoholic fatty liver disease.

Description

Application of atractylenolide III in preparation of drug for treating non-alcoholic fatty liver disease
Technical Field
The invention relates to the technical field of traditional Chinese medicine extracts, in particular to application of atractylenolide III in preparation of a medicine for treating non-alcoholic fatty liver disease.
Background
Nonalcoholic fatty liver disease (NAFLD) refers to a clinical pathological syndrome characterized by diffuse hepatocellular steatosis, in addition to alcohol and other well-defined liver damage factors. The pathological process of the liver cirrhosis disease is from simple liver cell steatosis to nonalcoholic steatohepatitis (NASH) and further develops into hepatic fibrosis, liver cirrhosis and even hepatocellular carcinoma. Because the pathogenesis of NAFLD is not clear up to now, no exact effective therapy for NAFLD exists, and the treatment mainly aims at reducing the risk factors coexisting with cardiovascular diseases and metabolic syndrome.
Lifestyle intervention is a treatment prior to or in addition to drug therapy, and dietary therapy and exercise therapy are advocated. The Mediterranean diet model has been recommended by EASL-ESD-EASO clinical practice guidelines for the treatment of NAFLD. Exercise therapy recommends a combination of aerobic exercise, resistance exercise and reduced sedentary. After 3-6 months of life style intervention, NAFLD patients whose metabolic indexes (such as blood pressure, blood sugar, etc.) do not reach the expected range need to use related medicines.
The drugs currently being tried for the treatment of NAFLD/NASH are mainly of the following classes: the insulin sensitizer (including thiazolidinedione medicine and metformin medicine). The thiazolidone drugs such as pioglitazone and rosiglitazone have an improvement effect on transaminase and liver histology of patients suffering from NASH, but the use of rosiglitazone which increases the risk of coronary artery disease is strictly limited in the United states, and the long-term efficacy of pioglitazone is still to be evaluated. Metformin is still controversial for the treatment of NAFLD/NASH. ② antioxidant vitamin E, and the research has proved that the vitamin E can improve serum biochemical index and liver histopathological change of NASH patients. The long-lasting efficacy of vitamin E has not yet been determined, and its effects on end-point events, such as cirrhosis and long-term survival problems, have yet to be assessed. ③ lipid-lowering drugs (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, such as statins). The study demonstrated that atorvastatin was clinically effective but not histologically improved. At present, statins are not advocated to be used for treating fatty liver alone, and can be used safely if hyperlipidemia is combined. And fourthly, angiotensin II receptor Antagonist (ARB). ARB drugs are widely used to treat hypertension. Randomized controlled studies have shown that both telmisartan and valsartan are effective in NASH, but without placebo control it is difficult to assess whether ARB has a specific effect on NASH. The liver-protecting medicine is widely applied clinically, is used as an auxiliary treatment and is mainly used for assisting patients with simple fatty liver for basic treatment for NAFLD which is invalid for 6 months and has unknown disease cause.
At present, western medicine has the priority of curative effect in the aspect of treating NAFLD, and traditional Chinese medicine has unique clinical curative effect in the aspect of treating chronic liver diseases, thereby providing an important alternative method for treating NAFLD. Researches find that single traditional Chinese medicines such as coptis chinensis, salvia miltiorrhiza, rheum officinale, gardenia, cassia seeds, polygonum cuspidatum, radix paeoniae alba, pericarpium citri reticulatae, pseudo-ginseng, astragalus membranaceus and the like have the effect of improving NAFLD. The conventional prescriptions for clinically treating NAFLD are reported to be pueraria root qinlian decoction, modified xiaoyao powder, bupleurum root liver soothing powder, stagnation-eliminating and liver-softening granules, poria cocos powder, turbidity-eliminating granules, diaphragma-expelling and stasis-removing decoction, dampness-eliminating and stasis-removing prescription, ginseng and pueraria lobata prescription and the like. The traditional Chinese medicine compound has the advantages of treatment based on syndrome differentiation of patients, reliable and exact clinical curative effect, but the action target and related signal pathways are not clear.
Chinese patent document CN103933411A discloses a traditional Chinese medicine composition for treating fatty liver and a preparation method and application thereof. The traditional Chinese medicine composition for treating fatty liver comprises the following raw material medicines: 5-50 parts of radix bupleuri, 5-50 parts of fructus aurantii, 15-150 parts of oriental wormwood, 4-50 parts of cape jasmine fruit, 2-30 parts of rheum officinale, 15-150 parts of rhizoma alismatis, 8-100 parts of bighead atractylodes rhizome, 4-50 parts of pinellia ternate, 4-50 parts of mangnolia officinalis, 10-120 parts of coix seed, 2-30 parts of fructus amomi rotundus, 8-100 parts of talc, 6-80 parts of dried orange peel, 4-50 parts of ligusticum wallichii, 6-80 parts of rhizoma cyperi, 10-120 parts of white paeony root and 2-30 parts of liquorice. The composition can obviously improve the symptoms of the non-alcoholic fatty liver; the effect of improving the nonalcoholic fatty liver is achieved by improving the expression of adiponectin receptor AdipoR2mRNA and AdipoR1 mRNA; and has certain effect of improving liver fibrosis. Chinese patent document CN102423442A discloses a double-formula medicine for treating fatty liver and a preparation method thereof. The double-composition medicine is prepared by matching a composition A consisting of a lindera aggregate extract, a rhizoma corydalis extract, pseudo-ginseng powder, a clematis root extract, a black aconite root, an eucommia bark extract and the like with a composition B consisting of a prepared rehmannia root extract, a safflower extract, a radix bupleuri extract, an asparagus cochinchinensis extract, an ophiopogon root extract, a radix cyathulae extract, a bighead atractylodes rhizome extract and the like. However, no report is found about the application of atractylenolide III in preparing a medicament for treating non-alcoholic fatty liver disease at present. The structural formula of atractylenolide III is as follows:
Figure BDA0003266491430000021
disclosure of Invention
The invention aims to provide application of atractylenolide III in preparation of a medicament for treating non-alcoholic fatty liver disease aiming at the defects in the prior art.
In a first aspect, an application of atractylenolide III in preparation of a medicament for treating non-alcoholic fatty liver disease is provided.
Preferably, the non-alcoholic fatty liver disease is Simple Fatty Liver (SFL), non-alcoholic steatohepatitis (NASH) or cirrhosis associated therewith.
In another aspect of the invention, the application of atractylenolide III in preparing a medicament for inhibiting and inducing non-alcoholic fatty liver disease cells is provided.
Preferably, the non-alcoholic fatty liver disease-inducing cell is a HepG2 cell.
In another aspect of the present invention, a pharmaceutical composition for treating non-alcoholic fatty liver disease is provided, wherein the pharmaceutical composition comprises atractylenolide iii as an active ingredient, and further comprises a pharmaceutically acceptable carrier.
Preferably, the dosage form of the pharmaceutical composition is an external dosage form or an internal dosage form.
More preferably, the dosage form of the pharmaceutical composition is a patch, a paste, an ointment, a gel, a film coating agent, a cataplasm, a tablet, an oral liquid, an injection, a capsule or a granule.
In another aspect of the invention, the application of atractylenolide III as a target point in screening and preparing a medicament for treating non-alcoholic fatty liver disease is provided.
The invention has the advantages that:
1. the invention is applied to a simple high fat induced fatty liver animal model, finds that the atractylenolide III can reduce the weight and liver lipid deposition of a model mouse, reduce liver index, and remarkably reduce ALT, AST, TG, TC, HDL and LDL levels of serum and TG and TC levels of liver tissues; atractylenolide III can reduce the contents of MDA, SOD and GSH-Px in serum and liver tissues of a model mouse, and improve the oxidative stress level; atractylodes macrocephala lactone III can activate related signal molecules of AMPK/SIRT1 signal pathway.
2. The invention is applied to a non-alcoholic fatty liver cell model, finds that the atractylenolide III has no influence on cell activity, can obviously reduce lipid deposition and TG and TC expression levels in cells, reduces MDA level, increases SOD and GSH-Px levels, and improves oxidative stress.
3. The invention is applied to inhibitor experiments, and proves that atractylenolide III can inhibit lipid deposition, reduce oxidative stress reaction by activating an AMPK/SIRT1 signal channel, simultaneously improve CPT1A enzyme activity and improve mitochondrial oxidative stress, and plays a role in treating non-alcoholic fatty liver disease through the mechanisms. Therefore, the atractylenolide III has the prospect of being developed into a medicament or health-care food for treating the non-alcoholic fatty liver disease. The atractylenolide III is a chemical substance derived from plants, has simple preparation steps and small environmental pollution in the preparation process, and is suitable for industrial production.
Drawings
FIG. 1. A: the chemical structural formula of atractylenolide III; b: mouse model making and administration time specification; c: weight change in mice between 0-16 weeks; d: weight gain in mice during dosing; e: average daily food intake in mice.
FIG. 2. A: mouse liver index; b: gross mouse liver, HE staining and oil red staining; c: balloon-like variation score and NAS score; d: serum ALT, AST levels; e: serum TG, TC levels; f: serum LDL, HDL levels; g: hepatic tissue TG, TC levels.
FIGS. 3. A-C: mouse serum MDA, SOD, GSH-Px levels; D-F: mouse liver tissue MDA, SOD, GSH-Px levels.
FIG. 4. A-B: WB detects the protein expression difference of mice of different groups and carries out quantitative analysis.
FIG. 5. A: the influence of different concentrations of FFAs on HepG2 cells on cell viability at different times; b: the effect of different concentrations of ATL III on the activity of HepG2 cells at different times; c: effect of ATL III 24h after 24h of FFAs on cell viability.
FIG. 6. A: HepG2 cell oil red staining; b: oil red staining semi-quantitative analysis; C-F: ALT, AST, TG, TC levels in HepG2 cells.
FIG. 7. A: detecting intracellular ROS level by flow cytometry; b: quantitatively analyzing the ROS level; C-E: HepG2 cells MDA, SOD and GSH-Px levels.
FIG. 8. A-B: WB detection of HepG2 cell protein expression difference and quantitative analysis.
FIG. 9. A: AMPK and SIRT1 inhibitors stain oil red after acting on HepG2 cells; b: oil red staining semi-quantitative analysis; C-D: the levels of TG, TC after AMPK and SIRT1 inhibitors act on HepG2 cells.
FIG. 10. A: detecting intracellular ROS levels of the AMPK and SIRT1 inhibitors after acting on HepG2 cells by flow cytometry; b: quantitatively analyzing the ROS level; C-E: MDA, SOD and GSH-Px levels after action of AMPK and SIRT1 inhibitors on HepG2 cells.
FIGS. 11. A-C: WB detects the protein expression difference of the AMPK and the SIRT1 inhibitor after acting on HepG2 cells and carries out quantitative analysis.
FIG. 12. A-C: WB detects the protein expression difference of the AMPK and the SIRT1 inhibitor after acting on HepG2 cells and carries out quantitative analysis.
Detailed Description
The following detailed description of the present invention will be made with reference to the accompanying drawings.
Example 1 animal experiments with Atractylodes macrocephala lactone III for the treatment of non-alcoholic fatty liver disease
1 materials of the experiment
1.1 Experimental animals and cells
30 male C57BL/6J mice with the age of 5 weeks are 14-18g in weight, and are sourced from Shanghai BiKai laboratory animal Limited liability company, and the license numbers of the laboratory animals are as follows: SYXK (Shanghai) 2020-. The mice are raised in an SPF grade barrier system of the experimental animal center of Shanghai medical university, the temperature is 24 +/-2 ℃, the humidity is 55%, and the mice freely take feed and drinking water. Animal ethical IACUC No. PZSHUTTCM 210320005.
1.2 Experimental drugs and Primary reagents
Atractylodes macrocephala lactone III (ATL III) was purchased from Shanghai Hotan biotechnology, 60 kcal% fat calorie high fat mouse food (HF60) was purchased from Toez biotechnology, Dulbecco's Modified Eagle's Medium (DMEM), Fetal Bovine Serum (FBS), penicillin and streptomycin were purchased from Gibco, CCK-8 kit, Compound C, EX 527 was purchased from AbMole, ALT/AST/TG/HDL/LDL/MDA/GSH-Px/SOD/ROS test kit, oil red O dye kit, HE dye kit was purchased from Nanfang biotechnology research institute, RIPA lysate, PAGE protein quantification kit, SDS-gel preparation kit, Cell mitochondria isolation kit is purchased from Biyuntian biotechnology, Inc., CPT1A activity detection kit is purchased from Genmed sciences, glycine, sodium chloride, Tris base, Sodium Dodecyl Sulfate (SDS), TWEEN20, isopropanol, methanol, ethanol, etc. are purchased from the national drug Group, AMPK, p-AMPK, LKB1, p-LKB1 antibody, Protease/Phosphatase Inhibitor Cocktail is purchased from Cell Signaling Technology, SIRT1, Nrf2, CPT1A, SIRT3, PGC1 alpha, GAPDH antibody are purchased from Proteinch Group.
2 method of experiment
2.1 grouping
After 30 male C57BL/6J mice were fed adaptively for one week, the mice were divided into a normal group, a model group and a drug group by block according to body weight, wherein 8 mice were used in the normal group, and 11 mice were used in the model group and the drug group, respectively.
2.2 Molding and identification
Normal ordinary feed is given to the normal group, high-fat feed is given to the other groups, 3 mice are taken from the normal group and the model group after 12 weeks, and model identification is carried out through gross observation, detection of blood fat, liver fat and serum transaminase and pathological staining.
2.3 administration of drugs
After 12 weeks, the normal group and the model group were given tail vein injection of physiological saline, and the drug group was given tail vein injection of ATL iii. The dose was 10mg/kg, and the weight was measured every 3 days and every 2 weeks, and the dose was adjusted according to the weight. The administration time was 4 weeks.
2.4 sample Collection
(1) At 12 weeks, 3 mice were taken from each of the normal group and the model group, and blood and liver were collected.
(2) After 4 weeks of administration, fasting for more than 12h overnight before sacrifice, weighing and recording the weight of each group of mice, after anaesthetizing with a proper amount of 1% sodium pentobarbital, disinfecting the abdominal skin with an alcohol cotton ball, cutting a small opening on the lower abdomen of the mice, cutting the peritoneum with a U-shaped cutter, taking blood, placing the blood in a 1.5ml EP tube (coated with heparin sodium anticoagulation), standing for 2h at room temperature, centrifuging at 3000rpm for 10min, collecting serum and subpackaging, and storing at-80 ℃ for later use. Carefully taking down the liver and spleen with forceps and scissors, draining blood in normal saline, drying with filter paper, weighing the weight of liver, and cutting 3 pieces of liver tissue at 5mm away from the maximum leaf distance edge of liver(about 0.5X 0.5cm3)2 blocks are fixed in 4 percent neutral formaldehyde solution, 1 block is immediately put in liquid nitrogen and left for ice cutting for subsequent pathological staining, and the rest liver tissue is stored at minus 80 ℃ for later use.
2.5 detection of various indexes
2.5.1 general conditions of animals
The weight, food intake, mental state, hair color, activity and the like of each group of mice were observed and recorded.
2.5.2 HE staining of liver tissue of mice in each group
Tissue dehydration and embedding
The liver tissue is fixed in 4% neutral formaldehyde for 1 week and then taken out, washed with tap water for 5-6h, dehydrated with different concentrations of alcohol step by step (i.e., 50% ethanol, 45min → 70% ethanol, 1.5h → 80% ethanol 1.5h → 90% ethanol 1h → 95% ethanol, 1h → 100% ethanol I, 1h → 100% ethanol II, 1h), xylene transparent (xylene I, 20min → xylene II, 20min), and wax impregnated (paraffin I, 1h → paraffin II, 1 h). After dehydration, paraffin embedding is carried out, and after cooling, the product is stored at normal temperature.
② paraffin tissue section and HE staining
Continuously slicing with a rotary slicer at a thickness of 5 μm in a 50 deg.C water bath, taking out the slide, and heating to 60 deg.C
Baking the slices for 2h and then carrying out HE dyeing, wherein the specific steps are as follows:
1) xylene 2X 10min
2) 100% alcohol 2X 5min
3) 95% alcohol 2X 5min
4) Flushing with running water for 10min
5) The content of hematoxylin is 2min,
6) flushing with running water for 5min
7) 1% hydrochloric alcohol (concentrated hydrochloric acid: 75% alcohol) 4s
8) Flushing with running water for 10min
9) Eosin 100s
10) Flushing with running water for 10min
11) 95% alcohol 2X 10s
12) 100% alcohol 2X 1min
13) Xylene 2X 1min
14) Neutral gum sealing sheet: adding a drop of neutral gum, sealing with a cover slip, drying at room temperature, observing under a microscope, taking a picture, and storing in a slide box.
2.5.3 oil Red O staining of liver tissue of mice in each group
(1) Frozen tissue section
a. Material taking:
cutting the largest liver leaf with uniform edge of 0.5 × 0.5 × 0.5cm3The tissue block is put into a container with the bottom layer filled with OCT glue, then the OCT glue is covered on the tissue and quickly stretches into liquid nitrogen, and after the tissue becomes white, the tissue block is taken out and is wrapped by tinfoil paper and stored at minus 80 ℃.
b. Slicing:
melting tissue, covering with iron cover for ice cutting, covering tissue surface with OCT glue, freezing at low temperature in microtome, cutting into 10 μm thick slices, adsorbing on slide glass, and placing in slice box, and embedding the rest liver tissue with OCT glue at-80 deg.C.
(2) Dyeing with oil red O:
a. preparing an oil red O application liquid according to the proportion of a storage liquid to a diluent to 5: 2;
b. temperature return: taking out the ice-cut white slices from-80 ℃, balancing the room temperature for 5-10min, and taking the sample out of water;
c. placing into a dye vat containing oil red O application liquid for 10-15 min;
ddH at d.37 deg.C2O washing for 5-20 s;
e. dyeing for 3-5min with a counterdyeing solution;
f. washing with water for 30-60s (the water flow speed is low);
g. after the surface water is completely dried, a water-based sealing agent (heated to be liquid in 60 ℃ warm water) can be dripped on the surface of the glass slide and the glass slide is covered with a cover glass.
2.5.4 detection of Biochemical indicators of serum of mice in each group
Mouse serum 50 μ l was diluted 4-fold with physiological saline, and then sent to the department of examination of eosin hospital affiliated to medical university at Shanghai for detection using a fully automatic biochemical analyzer. The detection indexes are as follows: alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), Triglyceride (TG), Total Cholesterol (TC), low density cholesterol ester (LDL-c), and high density cholesterol ester (HDL-c).
2.5.5 detection of TG and TC content in liver tissue of each group of mice
50mg of liver tissue was placed in 300. mu.l of RIPA lysate and homogenized in 10% tissue under ice bath.
Homogenizing for 3 times at 10 s/10 r/min, and detecting the content of TG and TC in liver tissue by biochemical method. The specific operation steps are carried out according to the kit instructions.
2.5.6 detection of MDA, SOD and GSH-Px content in blood serum and liver tissue of each group of mice
Serum and tissue homogenates were taken and worked up according to the kit instructions.
2.5.7Western Blot for detecting the expression of AMPK/SIRT1 signal related protein in mouse liver tissue
30mg of liver tissue is put into cell lysate containing 1% PMSF, the cell lysate is placed on ice for 30min after being homogenized, 12000g of liver tissue is centrifuged for 15min, supernatant is taken, and the protein concentration is measured and sampled by a BCA method. The amount of each sample was 30. mu.g, and the volume of each sample was 10. mu.l. Separating protein by using 12% separation gel, transferring the protein to a PVDF membrane by a wet transfer method, sealing for 1h by using 5% skimmed milk powder, incubating overnight at 4 ℃ for the first time, washing the membrane for 4 times by using TBST, adding the second antibody, washing the membrane for 4 times by using TBST, developing, and counting gray values by using ImageJ software to calculate relative expression amount. Detecting the expression levels of mouse liver tissues Adiopor1, Adiopor2, p-LKB1/LKB1, p-AMPK/AMPK, SIRT1, SIRT3, CPT1A, Nrf2 and PGC1 alpha.
3 results of the experiment
3.1 general conditions and weight changes in mice
General conditions of mice: the weight of the normal group of mice is naturally increased, the mental state is good, the response is sensitive, the hair is tidy and smooth, the normal group of mice is tightly attached to the skin, and the diet and the defecation are normal; the mouse in the model group has disordered hair, no luster, adhesion and greasy feeling, slow reaction and weakened activity; mice in the drug group of ATL III have poor spirit, slightly dull reaction, tidy hair compared with a model group, no obvious greasy feeling and reduced activity in different degrees.
After successful model identification for 12 weeks, the groups were treated for 4 weeks and the weights of each group were recorded before and after dosing. The weight of the normal group and the model group is increased, and the weight of the model group is obviously increased compared with the normal group (P is less than 0.001); compared with the model group, the medicine group can obviously reduce the weight of the mouse, and the difference has statistical significance (P is less than 0.001), as shown in figure 1.
3.2 gross and index of liver in mice
Liver gross and liver index: the liver capsule of the normal group of mice is smooth, the edge is neat, and the normal group of mice is dark red, bright and glossy; compared with the normal group, the liver of the model group is obviously enlarged, the edge is dull, the color is light and slightly yellow, the texture is silt-like and greasy, and a large amount of fat tissue deposition can be seen in the viscera; the liver volume of the drug group ATL III is smaller than that of the model group, the capsule is smoother, and the greasy feeling of the section is not obvious.
Calculating by the formula: liver index (%) -, liver mass (g)/body mass (g) × 100%
Compared with a normal group, the liver index of the mouse in the model group is obviously increased (P is less than 0.001); compared with the model group, the drug group significantly reduced the liver index (P < 0.001).
3.3HE staining and NAS scoring of mouse liver and oil Red staining
HE staining results show that liver cords in the normal group are arranged regularly and the cell structure is complete; compared with the normal group, the hepatic lobular structure of the model group is fuzzy, the hepatic cords are disorderly arranged, the hepatic cells have a small amount of ballooning change (the hepatic cells are swelled and have less cytoplasm), the fatty change is radially distributed around the central vein, the hepatic lobules have mixed fatty degeneration mainly with vesicularity, and necrotic foci can be seen in the hepatic lobules. The drug group exhibited a different degree of reduction in adiposity compared to the model group, as shown in FIG. 2.
The balloon-like variation scoring and NAS scoring results were: normal group values were 0.38 + -0.22 and 1.66 + -0.51; compared with the normal group, the model group is obviously increased (P is less than 0.001), and the numerical values are 2.13 +/-0.42 and 4.93 +/-0.47; the dose group was significantly lower (P < 0.01) than the model group, with values of 1.44. + -. 0.32 and 2.76. + -. 0.52, as shown in FIG. 2.
The result of oil red staining shows that the liver tissue of a normal mouse only has a small amount of small orange fat droplets; the model group is a large red lipid drop; the number of drops of medicinal lipid is significantly reduced, see fig. 2.
3.4 Biochemical indices of serum and liver tissue in mice
Compared with a normal group, the serum ALT, AST, TG, TC, HDL and LDL levels and the liver tissue TG and TC levels of the model group mice are all obviously increased (P is less than 0.001); compared with the model group, the medicine composition can obviously reduce the ALT, AST, TG, TC, HDL and LDL levels of serum and the TG and TC levels of liver tissues (P is less than 0.001), and the formula is shown in figure 2.
3.5 mouse serum and liver tissue MDA, SOD and GSH-Px levels
Mitochondria are an important energy pool, but NAFLD occurs, and is often associated with mitochondrial dysfunction, leading to reduced beta oxidation and concomitant increase in ROS, further leading to inflammation, necrosis and fibrosis of the liver.
Therefore, we examined indicators of oxidative stress. Therefore, the contents of MDA, SOD and GSH-Px in the serum liver tissue homogenate are detected by a biochemical method. The results show that compared with the normal group, the contents of MDA, SOD and GSH-Px in the model group are obviously increased (P is less than 0.001); the drug group significantly reduced the levels of MDA, SOD and GSH-Px (P < 0.001) compared to the model group, as shown in FIG. 3.
3.6 mouse liver tissue AMPK/SIRT1 pathway protein expression level
AMPK is a receptor of cellular energy, and under low-energy conditions, AMPK phosphorylates specific enzymes and growth control nodes to increase ATP production and reduce ATP consumption. After LKB1 is activated, Thr 172 of the α subunit of AMPK is further phosphorylated, which activates downstream signaling. Mitochondria are a producer of body energy, and the ingestion of a large amount of free fatty acids in hepatocytes, mitochondria are overloaded to operate, resulting in incomplete mitochondrial beta oxidation, severe impairment of mitochondrial structure and function, further accumulation of fat, resulting in massive steatosis in hepatocytes, and increased oxidative stress in the liver due to intermediary toxic metabolites such as DAG, ceramides, etc.
The results show that the AMPK/SIRT1 signals of the liver of the NAFLD model mouse are damaged, and the SIRT3, the PGC1 alpha, the Nrf2 and the CPT1A are used as downstream signal molecules and play important roles in resisting oxidative stress reaction and promoting fatty acid oxidation. To explore the potential mechanisms by which ATL iii inhibits the oxidative stress of the liver and promotes fatty acid oxidation in NAFLD model mice, the protein expression levels of these signaling molecules were examined. Compared with the normal group, the expression of the model group P-LKB1, P-AMPK, SIRT1, SIRT3, PGC1 alpha, Nrf2 and CPT1A is remarkably reduced (P is less than 0.001); the drug combination significantly increased the expression levels of these proteins compared to the model group (P < 0.001), as shown in FIG. 4.
EXAMPLE 2 cellular experiment of therapeutic Effect of Atractylodes macrocephala lactone III
1 materials of the experiment
1.1 test cells
Human hepatoma cell line HepG2 cells were purchased from Bai Biotech Co., Ltd, Kyoto, Japan.
1.2 Experimental drugs and Primary reagents
Atractylodes macrocephala lactone III (ATL III) is purchased from Shanghai Hotan Biotechnology Limited, fatty acid-free Bovine Serum Albumin (BSA), Oleic Acid (OA), Palmitic Acid (PA) is purchased from Sigma-Aldrich, dimethyl sulfoxide (DMSO) is purchased from Tauto-Biotech, Dulbecco's Modified Eagle's Medium (DMEM), Fetal Bovine Serum (FBS), penicillin and streptomycin are purchased from Gibco, CCK-8 kit, Compound C, EX 527 is purchased from AbMole, ALT/AST/TG/TC/HDL/LDL/MDA/GSH-Px/SOD/ROS test kit, oil red O staining solution kit, HE staining solution is purchased from Nanjing Biotech institute, RIPA lysis solution, BCA protein quantification kit, SDS-PAGE gel preparation kit, cell mitochondrial separation kit is purchased from Biyunnan Biotechnology Limited, CPT1A activity detection kit is purchased from Genmed Sci, glycine, sodium chloride, Tris base, Sodium Dodecyl Sulfate (SDS), TWEEN20, isopropanol, methanol, ethanol, etc. were purchased from the national drug Group, AMPK, p-AMPK, LKB1, p-LKB1 antibody, Protease/Phosphatase Inhibitor Cocktail were purchased from Cell Signaling Technology, SIRT1, Nrf2, CPT1A, SIRT3, PGC1 alpha, GAPDH antibody were purchased from Proteitech Group.
2 method of experiment
2.1 preparation of chemical reagents
Stock solutions of 20mM OA and 40mM PA were prepared in DMEM medium with 1% BSA and diluted with DMEM medium with 1% BSA to obtain the desired final concentration. 3mM FFA is a mixture of 2mM OA and 1mM PA (OA: PA, 2: 1). 200ng/ml ATL III stock solutions were prepared in DMSO, and the final DMSO concentration was less than 0.1% in all experiments.
2.2 cell culture, NAFLD cell model and drug treatment
HepG2 cells were cultured in DMEM medium containing 10% FBS at 37 ℃ with a volume fraction of 5% CO2Culturing for 72h in a saturated humidity incubator, observing and recording the growth state of the cells, subculturing when the cells are 70-80% fused, and taking the cells in the logarithmic phase for experiment. HepG2 cells were incubated with 1Mm FFA for 24 hours to stimulate lipid accumulation; cells were then treated with ATL III (0-50. mu.g/ml) and their effect on lipid metabolism was observed. The control group was given an equal amount of 1% BSA solution. The control and FFA treated groups were added with the same volume of DMSO.
2.3 cell viability assay
To examine the effect of FFAs and ALT III on HepG2 cell viability, 5X 10 cells were taken3One/well was inoculated into 96-well dishes and incubated overnight. Different concentrations of FFA (0-1.5mM) or ATL III (0-200. mu.g/ml) were added for 24h and 48h of incubation. Next, 10. mu.l/well of CCK-8 solution was added to the medium and incubated at 37 ℃ for 2 hours. The OD of the absorbance at 450nm was measured by a microplate reader.
2.4 oil Red O staining
Oil red O staining detected lipid droplet levels in HepG2 cells. HepG2 cells were seeded in 6-well plates at a density of 5X 105Cells/well, cultured overnight. FFA (1mM) was added to the medium for 24 hours, ATL III (0-50. mu.g/ml), and the mixture was further cultured for 24 hours. Cells were first rinsed 3 times with PBS and incubated for 15min at room temperature with 4% paraformaldehyde. Washed 2 times with PBS and stained with oil red O working solution (oil red O dye to diluent at a ratio of 5: 2) for 30 min. Cells were then washed with 60% isopropanol to remove excess dye and washed with PBS. Finally, the stained cells were observed with a microscope. To further identify intracellular lipid content, images were analyzed using ImageJ software.
2.5 Biochemical analysis of cells
HepG2 cells were seeded in 6-well plates at a density of 5X 105Individual cells/well. Discarding the culture solution after 12h, treating with FFA for 24h, thenCells were then incubated for 24 hours with different concentrations of ALT III (0-50. mu.g/ml). Cells were collected and assayed for TC, TG, ALT, AST, MDA, GSH-Px and SOD content in the cells according to the instructions.
2.6 determination of oxidative stress
The level of Reactive Oxygen Species (ROS) produced in the cells is determined using a reactive oxygen species assay kit. Fluorescent probe 2', 7' -dichlorodihydrofluorescein diacetate (DCFH-DA) was used to quantify ROS levels in HepG2 cells. DCFH-DA was non-fluorescent and was hydrolyzed to DCFH by esterase after entering the cells. Intracellular ROS react with DCFH to form fluorescent DCF. The DCF fluorescence intensity was determined by a Partec PAS flow cytometer. In addition, oxidative stress can also be detected by measuring Malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px).
2.7Western Blot to detect the expression of AMPK/SIRT1 signal-related protein in HepG2 cells
HepG2 cells were seeded in 6-well plates at a density of 5X 105Individual cells/well. After 12h the medium was discarded and treated with FFA for 24h, and the cells were incubated for 24h with different concentrations of ALT III (0-50. mu.g/ml). The protein is collected by RIPA cell lysis, the protein concentration and sample preparation are carried out, and the expression level of p-LKB1/LKB1, SIRT1, p-AMPK/AMPK, SIRT3, CPT1A, Nrf2 and PGC1 alpha is measured by the Western Blot method.
2.8 inhibitor assay
Compound C is a pyrazolopyrimidine related protein kinase inhibitor and is widely used as a cell-permeable ATP competitive inhibitor of AMPK. EX-527 is a selective SIRT1 inhibitor. HepG2 cells were seeded in 6-well plates (5X 10)5Individual cells/well) for 12 hours, pre-incubated with or without Compound C (2mM) or EX-527 (10. mu.M) for 1h, then treated with FFA for 24h, and incubated with ALT III for 24 h.
2.9 statistical treatment
Experimental data statistical analysis was performed using SPSS 20.0 software. The measured data are expressed by means of the mean +/-standard deviation, the comparison among groups is performed by using a t test, the difference among groups is analyzed by adopting one-factor variance, and the comparison between every two groups is performed by adopting an LSD method. The difference is statistically significant with P < 0.05.
3 results of the experiment
3.1 Effect of FFAs and ALT III on HepG2 cell viability
FFAs and ALT III were applied to HepG2 cells for 24h and 48h at different concentrations, and the results showed that FFAs and ALT III had no significant effect on HepG2 cell viability. According to the experimental results and the reference of the literature, after an experimental scheme is established that 1mM FFA acts for 24 hours, ATL III is added into a drug group to act for 24 hours. The CCK8 experiment result shows that the established experimental scheme has no significant influence on the activity of HepG2 cells, and the figure is shown in figure 5.
3.2HepG2 cell oil Red staining and cell TG, TC
The results of oil red staining indicate that FFA can remarkably increase the number and the volume of lipid deposition lipid drops in cells; ATL iii effect significantly reduced FFA-induced lipid deposition and was dose dependent. The detection result of intracellular TG and TC levels shows that FFA can obviously increase the intracellular TG and TC levels; ATL iii significantly reduced TG, TC levels increased by FFA induction. The result of liver cell function test shows that FFA stimulates HepG2 cells to not produce inflammatory reaction, and neither ALT nor AST is obviously increased, as shown in figure 6.
3.3HepG2 cell oxidative stress level
Flow cytometry is used for measuring intracellular ROS levels of HepG2 cells after FFA stimulation and ATL III action, and results show that FFA can remarkably increase intracellular ROS levels; ATL iii significantly reduces the elevated ROS levels induced by FFA. The biochemical method detects the MDA, SOD and GSH-Px level expressed by the liver cell, and the result shows that the FFA can obviously increase the MDA level in the cell and reduce the SOD and GSH-Px level; ATL iii significantly reduced FFA-induced MDA levels, increased SOD and GSH-Px levels, see figure 7.
3.4 expression of AMPK/SIRT1 Signal related proteins in HepG2 cells
The expression of AMPK/SIRT1 signal related protein in HepG2 cells is detected by a Western Blot method, and the FFA stimulation obviously reduces the expression of P-LKB1, P-AMPK, SIRT1, SIRT3, PGC1 alpha, Nrf2 and CPT1A (P is less than 0.001); ATL III significantly increased the expression levels of these proteins (P < 0.001), as shown in FIG. 8.
3.5 Effect of AMPK and SIRT1 inhibitors on HepG2 cells oil-Red staining
Respectively adding AMPK and SIRT1 inhibitors for pre-incubation for 1h, adding FFA for acting for 24h, adding ATL III for acting for 24h, and performing oil red staining to observe lipid accumulation. The results show that the lipid accumulation of the inhibitor group is more obvious, the lipid drop number is increased, and the volume is larger; lipid accumulation was significantly reduced in group ATL iii, see figure 9.
3.6 intracellular TG, TC levels after AMPK and SIRT1 inhibitors act on HepG2 cells
After the inhibitor acts on HepG2 cells, FFA is added for acting for 24 hours, ATL III is added for acting for 24 hours, and the level of intracellular TG and TC is determined by cracking cells, and the result shows that the level of TG and TC in the inhibitor group is obviously increased; intracellular TG, TC levels were significantly reduced in ATL group iii, see figure 9.
3.7 intracellular oxidative stress levels after AMPK and SIRT1 inhibitors act on HepG2 cells
After two inhibitors are respectively added, the ROS and MDA levels in cells are obviously increased, and the SOD and GSH-Px levels are reduced; ATL III significantly reduced ROS, MDA levels, increased SOD and GSH-Px levels, as shown in FIG. 10.
3.8 intracellular AMPK/SIRT1 Signal-related protein expression following Effect of AMPK and SIRT1 inhibitors on HepG2 cells
After the AMPK inhibitor is dried, the expression levels of p-AMPK, SIRT1, Nrf2, SIRT3, CPT1A and PGC1 alpha are obviously lower than those of the FFA group, and the inhibition effect of the AMPK inhibitor on the signal molecules can be partially recovered by ATL III. p-AMPK expression did not change significantly after the action of the SIRT1 inhibitor, SIRT1, Nrf2, SIRT3, CPT1A and PGC1 α expression was reduced, and the inhibitory action of the SIRT1 inhibitor on the above-mentioned signal molecules was partially restored by ATL iii, see fig. 11, 12.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.

Claims (8)

1. Application of atractylenolide III in preparing medicine for treating non-alcoholic fatty liver disease is provided.
2. The use of claim 1, wherein the non-alcoholic fatty liver disease is Simple Fatty Liver (SFL), non-alcoholic steatohepatitis (NASH), or cirrhosis associated therewith.
3. Application of atractylenolide III in preparation of drugs for inhibiting and inducing non-alcoholic fatty liver disease cells.
4. The use of claim 3, wherein the non-alcoholic fatty liver disease-inducing cell is a HepG2 cell.
5.A pharmaceutical composition for treating non-alcoholic fatty liver disease comprises atractylenolide III as an active ingredient, and further comprises a pharmaceutically acceptable carrier.
6. The pharmaceutical composition of claim 5, wherein the pharmaceutical composition is in the form of an external preparation or an internal preparation.
7. The pharmaceutical composition of claim 6, wherein the pharmaceutical composition is in the form of a patch, a paste, an ointment, a gel, a film coating agent, a cataplasm, a tablet, an oral liquid, an injection, a capsule or a granule.
8. Application of atractylenolide III as a target spot in screening and preparing medicines for treating non-alcoholic fatty liver disease.
CN202111088737.XA 2021-09-16 2021-09-16 Application of atractylenolide III in preparation of drug for treating non-alcoholic fatty liver disease Pending CN113577057A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111088737.XA CN113577057A (en) 2021-09-16 2021-09-16 Application of atractylenolide III in preparation of drug for treating non-alcoholic fatty liver disease

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111088737.XA CN113577057A (en) 2021-09-16 2021-09-16 Application of atractylenolide III in preparation of drug for treating non-alcoholic fatty liver disease

Publications (1)

Publication Number Publication Date
CN113577057A true CN113577057A (en) 2021-11-02

Family

ID=78242044

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111088737.XA Pending CN113577057A (en) 2021-09-16 2021-09-16 Application of atractylenolide III in preparation of drug for treating non-alcoholic fatty liver disease

Country Status (1)

Country Link
CN (1) CN113577057A (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102218056A (en) * 2011-04-19 2011-10-19 陕西省食品药品检验所 Use of butenolide I in preparing drugs for controlling immunological liver injury
WO2016167444A1 (en) * 2015-04-14 2016-10-20 동국대학교 경주캠퍼스 산학협력단 Pharmaceutical composition for preventing or treating obesity or lipid-related metabolic diseases, containing atractylodis macrocephalae rhizoma extract
CN109276566A (en) * 2018-11-21 2019-01-29 浙江中医药大学 Application of the atractylenolide Ⅰ in preparation prevention or treatment liver injury medicament

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102218056A (en) * 2011-04-19 2011-10-19 陕西省食品药品检验所 Use of butenolide I in preparing drugs for controlling immunological liver injury
WO2016167444A1 (en) * 2015-04-14 2016-10-20 동국대학교 경주캠퍼스 산학협력단 Pharmaceutical composition for preventing or treating obesity or lipid-related metabolic diseases, containing atractylodis macrocephalae rhizoma extract
CN109276566A (en) * 2018-11-21 2019-01-29 浙江中医药大学 Application of the atractylenolide Ⅰ in preparation prevention or treatment liver injury medicament

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JIASHUO WU等: "Integrating Network Pharmacology and RT-qPCR Analysis to Investigate the Mechanisms Underlying ZeXie Decoction-Mediated Treatment of Non-alcoholic Fatty Liver Disease", 《FRONTIERS IN PHARMACOLOGY》 *
鲁晓岚等: "《简明实用肝脏病学》", 31 May 2014, 世界图书西安出版公司 *

Similar Documents

Publication Publication Date Title
Huang et al. Ethnopharmacology, phytochemistry, and pharmacology of Cornus officinalis Sieb. et Zucc
Dashora et al. Antitumor activity of Dendrophthoe falcata against ehrlich ascites carcinoma in swiss albino mice
Yu et al. In vitro and in vivo anti-inflammatory effects of ethanol extract from Acer tegmentosum
Tao et al. Regulation effects on abnormal glucose and lipid metabolism of TZQ-F, a new kind of Traditional Chinese Medicine
Cho et al. An herbal formula consisting of Schisandra chinensis (Turcz.) Baill, Lycium chinense Mill and Eucommia ulmoides Oliv alleviates disuse muscle atrophy in rats
Muthusamy et al. Inhibition of protein tyrosine phosphatase 1B and regulation of insulin signalling markers by caffeoyl derivatives of chicory (Cichorium intybus) salad leaves
Long et al. Mulberry anthocyanins improves thyroid cancer progression mainly by inducing apoptosis and autophagy cell death
US20080233220A1 (en) Further Medical Use Of A Botanical Drug Or Dietary Supplement
Nakayama et al. Effects of three Chinese herbal medicines on plasma and liver lipids in mice fed a high-fat diet
KR100912243B1 (en) Compositions for Relieving Hangover
CN116234561B (en) Pharmaceutical composition for preventing and treating respiratory diseases in winter
CN102091083B (en) Petroleum ether extract of traditional Chinese medicine for preventing and treating glucose and lipid metabolic disturbance and preparation method thereof
Tseng et al. Protective effects of Liuwei dihuang water extracts on diabetic muscle atrophy
CN110833595A (en) Traditional Chinese medicine composition for treating gout and preparation method thereof
Liu et al. Atractylenolide III from Atractylodes macrocephala Koidz promotes the activation of brown and white adipose tissue through SIRT1/PGC-1α signaling pathway
KR20180107439A (en) Compositions for anti-obesity comprising extract of Cyperus microiria Steud.
Malematja et al. Potential hypoglycaemic and antiobesity effects of Senna italica leaf acetone extract
TWI733458B (en) Composition for preventing, improving or treating metabolic syndrome accompanying obesity and/or diabetes comprising a complex of emblica officinalis extract and hordeum vulgare extract(ib complex) as an active ingredient
CN103860706B (en) A kind of pure Chinese medicine health preparation with protection alcoholic liver injury function
KR20000007345A (en) Medicine of extract from long aged platycodon grandiflorum(korean name &#34;doraji&#34;)
US20180193401A1 (en) Anti-obesity composition comprising natural complex
CN102526227A (en) Use of Rheum emodi extract for preparing drugs for preventing and treating fatty liver diseases
CN110656083A (en) Pre-adipocyte brown induction kit
CN113577057A (en) Application of atractylenolide III in preparation of drug for treating non-alcoholic fatty liver disease
Venkateshwarlu et al. Antitumor activity of Alangium salvifolium against Dalton’s ascitic Lymphoma

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
WD01 Invention patent application deemed withdrawn after publication

Application publication date: 20211102

WD01 Invention patent application deemed withdrawn after publication