Application of amentoflavone in preparing medicine for treating ACLY related diseases or medicine for inhibiting ACLY activity and medicine composition

Abstract

The invention discloses an application of amentoflavone in preparation of an ATP (adenosine triphosphate) citrate lyase (ACLY) inhibitor. In an enzyme activity test, amentoflavone has a very remarkable inhibition effect on ACLY, and the median inhibitory concentration (IC50) is 0.25 + / -0.06 mu M; animal experiments show that amentoflavone can reduce the body weight, blood sugar, liver weight, fat weight, triglyceride and the like of non-alcoholic fatty liver disease mice which are molded by high-fat, high-cholesterol and high-fructose feed diet. Therefore, the amentoflavone has a potential treatment value on ACLY related diseases, such as the non-alcoholic fatty liver disease.

Description

Technical Field

[0001] The invention belongs to the field of drug development, and specifically relates to the use of the natural product aconitum biflavone in the preparation of ATP citrate lyase (ACLY) inhibitors and non-alcoholic fatty liver disease including simple fatty liver, non-alcoholic steatohepatitis and liver fibrosis. Background Art

[0002] ATP citrate lyase (ACLY) catalyzes the cleavage of citrate in the body to produce acetyl-CoA and oxaloacetate. Acetyl-CoA is a precursor molecule for the biosynthesis of fatty acids and cholesterol. Citric acid comes from the tricarboxylic acid cycle. ACLY links carbohydrate metabolism and lipid metabolism, plays a key role in glucose and lipid metabolism, and is an effective target for the treatment of diseases related to lipid metabolism disorders. Currently, ACLY inhibitors have been approved for marketing (Nexletol) for the treatment of hyperlipidemia and related atherosclerotic cardiovascular diseases (Expert Opin Pharmaco, 2019, 20(7):791-803), which has allowed researchers to see the medicinal potential of ACLY inhibitors.

[0003] Currently, there are two main categories of ACLY inhibitors. One category is terminal carboxylic acid inhibitors, represented by hydroxycitric acid (HCA), Medica 16, ETC-1002 and SB-204990 (Eur J Med Chem, 2018, 157: 1276-91). The activity of this type of inhibitor is generally poor, with a half inhibitory concentration (IC 50 ) are all above 1 μM, and the target is not specific, with certain cytotoxicity and poor cell membrane permeability. In in vivo experiments, very high concentrations are required to exert their efficacy; the other type is benzenesulfonamide inhibitors, represented by BMS-303141 and NDI-091143 (Nature, 2019, 568(7753): 566-70). This type of inhibitor has good inhibitory activity against ACLY in non-cellular experiments, but there are few reports on the efficacy of cell experiments and in vivo experiments. In summary, the existing ACLY inhibitors have problems such as poor activity, low safety, few structural types, and limited efficacy. The development of new inhibitors will lay the foundation for drug research targeting ACLY.

[0004] Traditional Chinese medicine is an important source of active compounds. The research team found that aconitum biflavonoids has good ACLY inhibitory activity, IC 50 =0.25±0.06μM. There is no report on the medicinal potential of aconitum biflavonoids in non-alcoholic fatty liver disease including simple fatty liver, non-alcoholic steatohepatitis, and liver fibrosis. Summary of the invention

[0005] One of the purposes of the present invention is to provide azalea biflavone as a highly active natural ACLY inhibitor, and the second purpose of the present invention is to provide a new medical use of azalea biflavone.

[0006]

[0007] Structure of the biflavonoids of Metasequoia gracile.

[0008] Specifically, the new medical use of the present invention is to provide the use of aconitum biflavonoids in the preparation of drugs for treating non-alcoholic fatty liver disease including simple fatty liver, non-alcoholic fatty hepatitis, and liver fibrosis.

[0009] The present invention shows that: sedum biflavone has a very significant inhibitory effect on ACLY and can reduce triglyceride levels. Therefore, sedum biflavone can be used to prepare drugs for non-alcoholic fatty liver disease including simple fatty liver, non-alcoholic fatty hepatitis, and liver fibrosis.

[0010] According to another aspect of the present invention, there is provided a drug combination of aconitum biflavonoids for use in the preparation of a drug for treating or preventing non-alcoholic fatty liver disease including simple fatty liver, non-alcoholic fatty hepatitis, and liver fibrosis.

[0011] The pharmaceutical composition is prepared by using aconitum biflavonoids as an active ingredient and a pharmaceutical carrier.

[0012] The carrier can be any inert material, organic or inorganic, suitable for enteral, percutaneous or parenteral administration, for example: water, gelatin, gum arabic, lactose, microcrystalline cellulose starch, starch, sodium starch glycolate, calcium hydrogen phosphate, magnesium stearate, talc, colloidal silicon dioxide, etc. The composition may also contain other pharmaceutically active agents, and conventional additives such as stabilizers, wetting agents, emulsifiers, flavoring agents, buffers, etc.

[0013] The pharmaceutical composition is in the form of tablets, capsules, granules, suspensions, dripping pills, injections or aerosols.

[0014] The present invention has found a new medical use for the known compound tachyphyllotoxin and has opened up a new application field. Tachyphyllotoxin has a strong pharmacological effect and can be used to prepare drugs for preventing and treating non-alcoholic fatty liver disease including simple fatty liver, non-alcoholic fatty hepatitis, and liver fibrosis.

[0015] Beneficial Effects of the Invention

[0016] The invention discloses a use of aconitum biflavonoids in preparing drugs for ACLY-related diseases. The compound has significant effects of reducing body weight, blood sugar, triglycerides, etc., and has a clear and highly active ACLY inhibitory effect. The compound has good development prospects in the treatment of ACLY-related diseases.

[0017] In the enzyme activity test, the biflavonoids of Achyranthes bidentata had a very significant inhibitory effect on ACLY, with a half inhibitory concentration (IC 50 ) is 0.25±0.06μM; Animal experiments show that tachyphyllotoxin can reduce the body weight, blood sugar, triglyceride levels, etc. of mice with non-alcoholic fatty liver disease modeled by high-fat, high-cholesterol, and high-fructose diets. Therefore, tachyphyllotoxin has potential therapeutic value for ACLY-related diseases such as non-alcoholic fatty liver disease including simple fatty liver, non-alcoholic steatohepatitis, and liver fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The inhibitory activity of aconitum biflavone on ACLY;

[0019] Figure 2 The effect of sedum biflavonoids on body weight and blood sugar;

[0020] Figure 3 The effect of flavonoids of Aconitifolia on liver and adipose tissue;

[0021] Figure 4 This is the effect of aconitum flavonoids on triglycerides. DETAILED DESCRIPTION

[0022] The present invention is further described with reference to examples, which are intended to illustrate the present invention but not to limit the present invention.

[0023] The instruments and reagents used in the experiment mainly include: PE Ensight microplate reader (PerkinElmer, USA), ADP-Glo TMKinase Assay kit (Promega), CulturPlate 384-well plate (PerkinElmer, USA), dimethyl sulfoxide (Shanghai Yuanye Biotechnology Co., Ltd.), sedum biflavonoids (Chengdu Aifa Biotechnology Co., Ltd.), triglyceride (TG) assay kit (Nanjing Jiancheng Bioengineering Institute), blood glucose test strips (Sino), electronic scale (Mettlertoledo), C57BL / 6 mice (Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.), normal feed (Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd., SWS9102), GAN feed (Research Diets, D09100310), castor oil polyoxyethylene ether (MACKLIN) and normal saline (Shandong Qidu Pharmaceutical Co., Ltd.).

[0024] Example 1: Determination of ACLY Inhibitory Activity of Azadirachta indica

[0025] The stock solution concentration of the sedum biflavonoid compound is 50mM, and the solvent is dimethyl sulfoxide. Take 1μL of the stock solution and add it to 61.5μL of aqueous buffer to obtain a 0.8mM compound sedum biflavonoid solution; the composition of the aqueous buffer is: in water, a final concentration of 40mM tris (tris), a final concentration of 4mM dithiothreitol, a final mass concentration of 0.01% lauryl alcohol polyoxyethylene ether, 10mM MgCl2, a final volume fraction of 1% dimethyl sulfoxide, pH = 8.0.

[0026] The 0.8 mM compound sedum biflavonoids solution was gradiently diluted with the aqueous buffer to prepare 14 sedum biflavonoids working solutions with concentration gradients, and the concentrations were 0.8 mM, 0.4 mM, 0.2 mM, 0.1 mM, 0.05 mM, 0.025 mM, 0.0125 mM, 0.00625 mM, 0.003125 mM, 0.0015625 mM, 0.00078125 mM, 0.000390625 mM, 0.0001953125 mM and 0.00009765625 mM respectively;

[0027] The ACLY stock solution (1.5 mg / mL) was diluted to 30 nM with aqueous buffer to obtain a diluted ACLY solution, and 2.5 μL of the diluted ACLY solution was placed in the wells of a 384-well plate, and then 2.5 μL of the above-mentioned aconitum biflavonoids working solution of different concentrations were placed in the wells of the 384-well plate containing the diluted ACLY solution; the aqueous stock solution of ATP citrate lyase (ACLY) was composed of: in water, a final concentration of 50 mM tris, a final concentration of 400 mM NaCl, and a final volume fraction of 10% glycerol.

[0028] The well plate was flicked to mix the liquid in the 384-well plate system evenly, and incubated at room temperature for 15 minutes. Then 5 μL of substrate mixture solution was added to the wells; the substrate mixture solution (final concentration) was: 600 μM citric acid, 150 μM adenosine triphosphate (Promega), 150 μM coenzyme A, and the solvent was water; low-speed centrifugation (1200 rpm, 1 min, 20°C) was performed to mix evenly.

[0029] Then, the 384-well plate was placed in a 37°C incubator for 55 minutes, taken out, and allowed to stand at room temperature for 5 minutes. After returning to room temperature, 10 μL of ADP-Glo ​​reagent in the ADP-GloTmKinase Assay kit was added to each well, and the reaction was carried out at room temperature for 40 minutes. Subsequently, 20 μL of kinase detection reagent in the ADP-GloTmKinase Assay kit was added to each well, incubated at room temperature for 40 minutes, and then the fluorescence intensity was detected on a PE Ensight microplate reader. GraphPad Prism 6.0 was used to fit the relationship between the concentration and fluorescence intensity of tassel-flowered flavonoids, and the dose-effect curve of the compound tassel-flowered flavonoids was obtained ( Figure 1 ), the horizontal axis is the logarithmic value of the molar concentration of the compound sedum biflavonoids, and the vertical axis is the fluorescence intensity value measured by the instrument. The inhibitory activity of the compound sedum biflavonoids targeting ACLY is IC 50 =0.25±0.06 μM, indicating that the compound aconitum biflavone is a highly active inhibitor of ACLY.

[0030] Example 2: Aconitum biflavonoids reduce body weight and blood sugar in mice induced by GAN diet

[0031] The dosage of sedum biflavonoids for mice is 20 mg (mass of sedum biflavonoids) / kg (mouse weight), and the volume ratio of the solvent is dimethyl sulfoxide: anhydrous ethanol: a mixture of castor oil polyoxyethylene ether and normal saline = 1:2:97, wherein the volume ratio of castor oil polyoxyethylene ether and normal saline in the mixture is 1:9. The concentration of sedum biflavonoids in the solvent is 2 mg / mL.

[0032] In this experiment, 8-week-old healthy male C57BL / 6 mice were selected and purchased from Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd. The experiment was divided into 3 groups, namely the control group (given normal feed), the model group (GAN feed), and the drug-treated group (GAN feed + sedum flavonoids group). During the experiment, the mice were induced by 14 weeks of GAN feed feeding (free feeding) + 8 weeks of sedum flavonoids (20 mg / kg). The drug-treated group was given 8 weeks of sedum flavonoids, and the control group and the model group were given 8 weeks of solvent induction (intraperitoneal injection) with the same volume ratio, once a day. During the modeling process, the mice were weighed and their blood sugar was tested every week. Figure 2 At the 22nd week, the body weight of mice in the model group was significantly increased compared with that in the control group, indicating that the high-fat model induced by GAN feed was successful; the body weight and blood sugar of the drug-treated group were significantly decreased compared with those in the model group, indicating that sedum biflavonoids can reduce the body weight and blood sugar of mice after GAN feed induction.

[0033] Example 3: Aquifolium biflavonoids reduces the weight of liver, mesenteric fat, gonadal fat, and perirenal fat in mice induced by GAN diet.

[0034] At the end of 22 weeks of mouse breeding in Example 2, the experimental mice in Example 2 were killed, and the liver, mesenteric fat, gonadal fat, and perirenal fat were weighed. Figure 3 Compared with the control group mice, the weights of the liver, mesenteric fat, gonadal fat, and perirenal fat of the model group mice were significantly increased, indicating that the high-fat model induced by GAN feed was successful and caused liver hypertrophy in mice; compared with the model group mice, the weights of the liver, mesenteric fat, gonadal fat, and perirenal fat of the group treated with tasselflower biflavonoids were significantly decreased, indicating that tasselflower biflavonoids can reduce lipid accumulation and liver hypertrophy induced by GAN feed.

[0035] Example 4: Azadirachta biflavonoids reduce triglyceride levels in mice induced by GAN diet

[0036] Accurately weigh the liver tissue of the experimental mice in Example 2 (at the end of the 22-week breeding of the mice in Example 2, the experimental mice in Example 2 were killed), add 9 volumes of homogenization medium (anhydrous ethanol) at a weight (g): volume (ml) ratio of 1:9, mechanically homogenize under ice-water bath conditions, centrifuge at 2500 rpm for 10 minutes, and take the supernatant to measure the triglyceride according to the kit instructions. Figure 4 Compared with the control group, the triglyceride level in the model group was significantly increased, indicating that the blood lipid level in mice was significantly increased and metabolic disorders occurred after GAN feed feeding; compared with the model group, the triglyceride level in the flavonoids group was significantly decreased, indicating that flavonoids has a potential therapeutic effect on non-alcoholic fatty liver disease including simple fatty liver, non-alcoholic fatty hepatitis, and liver fibrosis.

Claims

1. Use of aconitum biflavonoids in the preparation of drugs for ACLY-related diseases or drugs for inhibiting ACLY activity.

2. The use according to claim 1, It is characterized in that The structure of the flavonoids of Achyranthes bidentata is as follows: it has significant inhibitory activity on ATP citrate lyase, Structure of the biflavonoids from Metasequoia gracile.

3. The use according to claim 1, It is characterized in that The ATP citrate lyase-related disease is non-alcoholic fatty liver disease, including one or more diseases or conditions of simple fatty liver, non-alcoholic fatty liver hepatitis, and liver fibrosis, and the drug is a drug for treating and / or preventing related diseases.

4. The use according to claim 3, It is characterized in that The drug for inhibiting the activity of ATP citrate lyase can reduce the synthesis of triglycerides by inhibiting the activity of ATP citrate lyase.

5. A pharmaceutical composition, It is characterized in that The invention discloses a pharmaceutical composition which is prepared by using aconitum biflavonoids as an active ingredient and a pharmaceutical carrier or a pharmaceutically acceptable carrier or auxiliary material.

6. The pharmaceutical composition according to claim 5, Features: The pharmaceutical composition is in the form of tablets, capsules, granules, suspensions, dropping pills, injections or aerosols.

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