阅读说明：本技术 金银花提取物在制备atp柠檬酸裂解酶抑制剂中的用途 (Application of honeysuckle extract in preparation of ATP citrate lyase inhibitor ) 是由 胡金锋 万江 熊娟 姜春筱 于 2021-07-30 设计创作，主要内容包括：本申请公开一种金银花提取物在制备ATP柠檬酸裂解酶抑制剂中的用途,本申请经筛选发现,金银花提取物对ACL呈现显著抑制作用,IC-(50)值为2.65±0.83μg/mL,结果显示,该植物提取物可能对高血脂、动脉粥样硬化、脂肪肝、II型糖尿病、癌症及其它由ACL所介导的疾病具有治疗作用,从而在制药领域有巨大的潜在用途。本申请的金银花提取物可经本领域技术人员熟知的方法制备获得。例如,以金银花为原料,可采用溶剂提取法以及层析法制成浓缩的金银花提取物。基于该用途本申请还提供一种ATP柠檬酸裂解酶抑制剂,该抑制剂以金银花提取物为活性成分,金银花提取物独立使用或与药学上可接受的载体或赋形剂制备成药剂。(The application discloses an application of a honeysuckle extract in preparation of an ATP citrate lyase inhibitor 50 The value was 2.65. + -. 0.83. mu.g/mL, and the result was significantThe plant extract has the treatment effect on hyperlipidemia, atherosclerosis, fatty liver, type II diabetes, cancer and other diseases mediated by ACL, so that the plant extract has great potential application in the field of pharmacy. The honeysuckle extract of the present application can be prepared by methods well known to those skilled in the art. For example, honeysuckle flower is used as raw material, and solvent extraction and chromatography can be adopted to prepare concentrated honeysuckle flower extract. Based on the application, the application also provides an ATP citrate lyase inhibitor, which takes honeysuckle extract as an active ingredient, and the honeysuckle extract is independently used or is prepared into a medicament with a pharmaceutically acceptable carrier or excipient.)

1. Use of flos Lonicerae extract in preparing ATP citrate lyase inhibitor is provided.

2. Use of flos Lonicerae extract in preparing medicine for treating diseases mediated by ATP citrate lyase is provided.

3. Use of flos Lonicerae extract in preparing medicine for treating hyperlipidemia, atherosclerosis, non-alcoholic fatty liver, type 2 diabetes, cancer or obesity.

4. The use as claimed in any one of claims 1 to 3, wherein the honeysuckle extract is an ethanol extract of honeysuckle or a fraction obtained by subjecting the ethanol extract to column chromatography.

5. Use according to claim 4, wherein the ethanol extract is a 75% ethanol extract.

6. The use as claimed in claim 4, wherein the ethanol extract is subjected to macroporous resin column chromatography, and the fraction obtained by gradient elution with ethanol-water is the fraction.

7. An ATP citrate lyase inhibitor is characterized in that honeysuckle extract is used as an active ingredient.

8. The ATP citrate lyase inhibitor of claim 7, further comprising a pharmaceutically acceptable carrier and/or excipient.

9. The ATP citrate lyase inhibitor of claim 8, wherein the honeysuckle extract is formulated with excipients as a tablet, pill, capsule, or granule.

10. A medicament for treating hyperlipemia, atherosclerosis, non-alcoholic fatty liver disease, type 2 diabetes, cancer or obesity is characterized in that the medicament takes honeysuckle extract as an effective component.

Technical Field

The application relates to the technical field of medical application, in particular to application of a honeysuckle extract in preparation of an ATP citrate lyase inhibitor.

Background

ATP-citrate lyase (ACL) is a key enzyme in sugar metabolism and fatty acid and cholesterol biosynthesis, and its substrates and products of action are key intermediates in sugar metabolism and can serve as substrates for fatty acid synthesis. Human ACL enzyme proteins are homotetramers of 4 identical 120KDa subunits, each containing 1101 amino residues in the polypeptide chain (Singh et al, j.biol.chem.1976,251, 5254-5250). ACL is a cytosolic enzyme that catalyzes the conversion of citric acid and coa to acetyl-coa and oxaloacetate with concomitant hydrolysis of ATP. ACL is highly expressed in adipogenic cells such as liver, kidney, pancreas, and cholinergic nerve cells (Beigneux et al, j.biol.chem.2004,279,9557-9564), and its catalytically derived acetyl-coa is also used for biosynthesis of cholesterol. ACL, a major source of cytosolic acetyl-coa, is closely related to the synthesis of fatty acids and cholesterol, and changes in its expression are closely related to human cardiovascular diseases, fatty liver, type 2 diabetes, cancer, and the like.

Cardiovascular diseases (CVDs) are the leading cause of death in the world, and are indicated by the World Health Organization (WHO) to account for almost one third of the worldwide deaths (Mendis et al, prog. cardiovascular. dis.2010,53, 10-14). The search for drugs to prevent or treat CDVs has become a global public health priority. ACL is a ubiquitous enzyme linking nutrient metabolism to cholesterol and fatty acid synthesis, and studies have shown that disturbances in cholesterol and triglyceride metabolism contribute to the development of cardiovascular disease in humans. Biosynthetic pathways for nascent cholesterol and fatty acids are dependent on acetyl-CoA provided by catalytic derivation of ACLs, and ACL inhibitors are effective in blocking de novo synthesis of fatty acids and cholesterol and reducing blood lipids (Burke et al, curr. Opin. Lipidol.2017,28, 193-106200; Pinkosky et al, Trends mol. Med.2017,23, 1047-1063). Therefore, finding a potent ACL inhibitor has a very important application prospect for treating or preventing the occurrence of CDVs. In addition, abnormal metabolism of triglycerides also increases risk factors for Nonalcoholic Fatty Liver (NAFLD) and Type II Diabetes Mellitus (T2 DM), and thus ACL can also be a potential target for Nonalcoholic Fatty Liver and Type II Diabetes Mellitus (Cohen et al, science2011,332, 1519-1523; Armstrong et al, Hepatology 2014,59, 1174-1197).

Studies have shown that ACL is also closely related to the development of cancer. The increase in lipid synthesis, which provides the necessary lipids for cell growth and division, is one of the important hallmarks of Cancer, and also an early event in tumorigenesis (Migita et al, Cancer Res.2008,68, 8547-8554). Acetyl coenzyme A is an important component of de novo synthesis of fatty acid, ACL serves as a main source of the acetyl coenzyme A, and inhibition of the expression of genes of the ACL can remarkably inhibit the proliferation of tumor cells and induce the apoptosis of the tumor cells. Therefore, ACL is widely researched as a potential target of anticancer in recent years, and the search for effective inhibitors thereof is expected to become a new anticancer drug (Granchi et al, Eur.J.Med.chem.2018,157, 1276-1291; Zaidi et al, Cancer Res.2012,72: 3709-.

ACL as a new medicinal target has become a hot spot of innovative drug research of glycolipid metabolic disorder diseases in recent years. With the wide application of high-throughput screening technology, a large variety of ACL small-molecule inhibitors have been discovered in succession. However, no ACL inhibitors have yet been successfully marketed, and thus competition in the medical field is very intense. Bempedoic acid (obtained by organic synthesis) is a potent small molecule inhibitor of ACLs and is currently approved by the FDA for the treatment of hypercholesterolemia. In addition to Bempedoic acid, development of other ACL inhibitors has been limited due to their low cell penetration, low affinity for ACL, and poor specificity. Therefore, the search for the small-molecule ACL inhibitor which is efficient and high in selectivity and has good pharmacokinetic properties is of great significance, and the small-molecule ACL inhibitor has a wide application prospect in treatment of cardiovascular diseases, cancers and other diseases.

Natural products have been one of the major sources of drug molecules (Atananov et al, nat. Rev. drug Discov.,2021,20, 200-. Compared with synthetic chemical drugs, the plant extracts and components have the advantages of low toxicity and side effects, no drug resistance, various structures, multiple targets and the like. Therefore, searching for novel ACL small-molecule inhibitors from abundant plant resources (especially traditional medicinal plants such as traditional Chinese medicine) is one of the important directions for the development of modern pharmaceutical industry.

Disclosure of Invention

The application aims to provide medical application of a honeysuckle extract, and multiple pharmacological test researches show that the honeysuckle extract has remarkable ACL (ATP citrate lyase) inhibitory activity.

Honeysuckle (Lonicera japonica Flos), also known as honeysuckle stem, honeysuckle flower and the like, is a dry flower bud or an initially-bloomed flower bud of perennial semi-evergreen entwining creeping shrub honeysuckle (Lonicera japonica Thunb.) of Caprifoliaceae. As a traditional Chinese medicinal material commonly used in China, the traditional Chinese medicinal material has a history of thousands of years of use, is listed as a superior product from famous medical records, is recorded in pharmacopoeia of the people's republic of China, and has the effects of clearing heat and removing toxicity, dispelling wind and dissipating heat and the like.

A plurality of pharmacological test researches show that the 75% ethanol extract of the traditional Chinese medicine has obvious ACL inhibitory activity and shows obvious inhibition effect on ACL, IC₅₀The value is 2.65 +/-0.83 mu g/mL, and the result shows that the medicinal material extract has a treatment effect on hyperlipidemia, atherosclerosis, fatty liver, type II diabetes, cancer and other ACL-mediated diseases, so that the medicinal material extract has huge potential application in the field of pharmacy.

The honeysuckle extract of the present application can be prepared by methods well known to those skilled in the art. For example, using honeysuckle as raw material, solvent extraction can be used to make concentrated honeysuckle extract or the honeysuckle extract can be further processed into different fractions by chromatography.

In the solvent extraction method, the organic solvent is selected from water, methanol or ethanol or a mixture of a plurality of solvents, and the honeysuckle extract is obtained by dipping extraction at room temperature or reflux extraction for 5-10 times under heating condition.

Optionally, the solvent is 60-90% ethanol (i.e., a solution with a volume ratio (v/v) of ethanol to water of 60-90/40-10), and the obtained extract is recorded as a 60-90% ethanol extract; preferably, the solvent is 75% ethanol [ i.e., a solution having a volume ratio of ethanol to water (v/v) of 75/25 ]; the obtained extract is 75% ethanol extract.

In the chromatography, the filler is reversed-phase filler, preferably D-101 type macroporous resin, and the elution system is preferably methanol-water or ethanol-water.

Further preferably, ethanol-water is selected as an elution system for gradient elution; ethanol-water (0:100 → 20:80 → 40:60 → 60:40 → 80:20 → 100:0, v/v) gradient elution; the 4 subfractions (JYH-EtOH-F1-JYH-EtOH-F4) were then pooled according to TLC spots.

The obtained honeysuckle extract is subjected to an ATP-citrate lyase (ACL) inhibition activity experiment, and the result shows that the honeysuckle extract has obvious ACL inhibition activity and can be used for preparing medicines for treating ACL mediated diseases

Therefore, the application provides the application of the honeysuckle extract in preparing the ATP citrate lyase inhibitor.

The application also provides application of the honeysuckle extract in preparing a medicine for treating diseases mediated by ATP citrate lyase.

The application also provides application of the honeysuckle extract in preparing a medicament for treating hyperlipidemia, atherosclerosis, non-alcoholic fatty liver disease, type 2 diabetes, cancer or obesity.

The application also provides an ATP citrate lyase inhibitor, which is prepared into oral or non-oral dosage forms by taking the honeysuckle extract as an active ingredient, combining with a pharmaceutically acceptable carrier or excipient and preparing according to a conventional method.

The extract or fraction can be used alone or in combination, or can be combined with pharmaceutically acceptable carriers or excipients to be made into oral or non-oral dosage forms according to conventional method.

Optionally, a pharmaceutically acceptable carrier and/or excipient is also included.

Optionally, the honeysuckle extract and excipient are prepared into tablets, pills, capsules or granules.

The application also provides a medicine for treating hyperlipidemia, atherosclerosis, non-alcoholic fatty liver disease, type 2 diabetes, cancer or obesity, wherein the medicine takes honeysuckle extract as an active ingredient, is combined with a pharmaceutically acceptable carrier or excipient, and is prepared into an oral or non-oral dosage form according to a conventional method.

Optionally, the honeysuckle extract is an ethanol extract of honeysuckle or a fraction of the ethanol extract after column chromatography.

Optionally, the solvent extract is a mixed solvent extract of methanol and/or ethanol and/or water and/or any proportion thereof, and further is an ethanol extract.

Further, the ethanol extract is 60-90% ethanol extract; further, the extract was 75% ethanol extract (JYH-EtOH).

Optionally, subjecting the ethanol extract to macroporous resin column chromatography, and performing gradient elution with ethanol-water to obtain a fraction; ethanol-water (0:100 → 20:80 → 40:60 → 60:40 → 80:20 → 100:0, v/v) gradient elution; the 4 subfractions (JYH-EtOH-F1-JYH-EtOH-F4) were then pooled according to TLC spots.

Detailed Description

The technical solutions of the present application will be described clearly and completely with reference to the embodiments of the present application, and it should be understood that the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the description of the present application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

Example 1: preparation of honeysuckle extract

Pulverizing dried flos Lonicerae (6.0kg), extracting with 75% ethanol (8L) at room temperature for 5 times, each for 24 hr, mixing extractive solutions, and concentrating under reduced pressure to obtain total extract 738g to obtain 75% ethanol extract (JYH-EtOH) of flos Lonicerae.

The total extract was dispersed in 1.0L water, purified by macroporous resin column chromatography, eluted with a gradient of ethanol-water (0:100 → 20:80 → 40:60 → 60:40 → 80:20 → 100:0, v/v) and combined according to TLC plates to give 4 subfractions (JYH-EtOH-F1-JYH-EtOH-F4).

Example 2: ACL inhibitory Activity assay

The experimental method comprises the following steps: in the experiment, the ATP-dependent citrate lyase ACL can catalyze and convert citric acid into acetyl coenzyme A, and further generate a precursor molecule for synthesizing fatty acid, namely malonyl coenzyme A. This reaction is accompanied by the consumption of ATP, and therefore, the enzyme activity inhibitory effect of the extract on ACL can be indirectly reacted by detecting the change in ATP using an ADP-Glo kinase assay kit.

Specifically, when the 75% ethanol extract (JYH-EtOH) and the 4 fractions (JYH-EtOH-F1-JYH-EtOH-F4) of the honeysuckle flower prepared in example 1 were selected and examined for the percentage inhibition of ACL enzyme activity at a concentration of 20 μ g/mL, the test results showed that the 75% ethanol extract (JYH-EtOH) and the 4 fractions (JYH-EtOH-F1-JYH-EtOH-F4) of the honeysuckle flower had significant inhibition effects on ACL, and the inhibition rates were 52.5%, 90.4%, 98.1%, 91.6% and 93.6%, respectively.

Further determination of IC₅₀The value: samples (75% ethanol extract (JYH-EtOH) prepared in example 1 and 4 fractions (JYH-EtOH-F1-JYH-EtOH-F4)) were dissolved in DMSO to the appropriate concentration just before use, 3-fold diluted, 7 dilutions, triplicate wells, and 2. mu.L of sample solution was added to a standard assay system (40mM Tris, pH 8.0, 10mM MgCl. RTM. MgCl.)₂5mM DTT, ATP, CoA, sodium citrate and ACL), and incubated at 37 ℃ for 30 min. Then, 25. mu.L of ADP-Glo reagent was added to the system, and the reaction was incubated at room temperature for 30min to terminate the reaction,and the remaining ATP is consumed. And adding a kinase detection reagent for incubation for 30min, reading a fluorescence signal by EnVision, and taking the slope of the first-order reaction of a kinetic curve as an activity index of the enzyme. The relative activity was plotted against the concentration of 75% ethanol extract (JYH-EtOH) and 4 fractions (JYH-EtOH-F1-JYH-EtOH-F4) by the formula v/v₀＝100/(1+b*[I]/IC₅₀) Fitting to obtain IC₅₀Value, experiment was repeated three times, results were averaged three times, IC of positive control BMS303141₅₀The value was 0.46. mu.M.

TABLE 1 ACL inhibitory Activity data of 75% ethanol extracts and fractions of Lonicera japonica

ACL inhibiting IC of 75% ethanol extract and fraction of flos Lonicerae₅₀The values are shown in a table 1, and the test results show that the 75% ethanol extract (JYH-EtOH) and 4 fractions (JYH-EtOH-F1-JYH-EtOH-F4) show significant inhibitory activity on ACL, which indicates that the solvent extract can be used for preparing the drugs for treating diseases related to glycolipid metabolic disorders.

In conclusion, in vitro activity experiments show that the 75% ethanol extract of honeysuckle exhibits a remarkable inhibiting effect on ACL, and can be applied to preparation of medicines for preventing, delaying or treating ACL-mediated diseases.

The above-mentioned embodiments only express several embodiments of the present application, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the concept of the present application, which falls within the scope of protection of the present application. Therefore, the protection scope of the present patent shall be subject to the appended claims.