Application of flavonoid compound in preparation of lipid metabolism regulation preparation

Abstract

The invention discloses application of a flavonoid compound in preparation of a lipid metabolism regulation preparation, and belongs to the technical field of biological medicine. The invention discloses an application of a flavonoid compound in preparation of a lipid metabolism regulation preparation. The flavonoid compound is amentoflavone, herba lycopi flavone, isofulvic alcohol, sub-glycoside chalcone, sophora japonica flavanone G or silver forging glycoside. The flavonoid compound disclosed by the invention can improve diseases related to lipid metabolism by inhibiting lipid accumulation. And the compound has the advantages of good safety, simple preparation process, high synthesis yield and the like, so that the compound has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, more particularly to the application of flavonoids in the preparation of lipid metabolism regulator. BACKGROUND

[0002] Carboxylesterase (CES) belongs to the B family of esterases, and the CEs in the human body involved in the metabolism of ester drugs are mainly carboxylesterase 1 and carboxylesterase 2. hCE1 is mainly located in the liver, and the carboxylesterase in the plasma is mainly synthesized in the liver and secreted into the circulatory system, and is highly expressed in the central nervous system, lung, kidney, macrophages and monocytes, testis and placenta, and is rarely expressed in the gastrointestinal tract. hCE2 is mainly distributed in the gastrointestinal tract, especially the small intestine. As an important liver drug metabolizing enzyme, irinotecan can cause severe delayed diarrhea due to excessive secretion of SN-38 in the small intestine, and co-administration with a potent CES2 inhibitor can improve the severe diarrhea caused by CPT-11 in patients, thereby improving the therapeutic effect. Effective hCE2 inhibitors can be used to improve the oral bioavailability and drug half-life. Therefore, hCE2 inhibitors have potential application prospects, which can slow down the catalytic activity of hCE2 in the body, thereby regulating the pharmacokinetic characteristics or reducing the toxicity of hCE2 substrate drugs. Lipids are the energy source for animal storage, and lipid metabolism disorder is related to metabolic disorders, including cardiovascular disease, obesity, non-alcoholic fatty liver and diabetes, and changing the steady state of disordered lipid metabolism can enhance the therapeutic effect. At present, a variety of natural compounds have been found to be able to inhibit fat accumulation in the body, such as anthocyanins and ursolic acid, which can significantly reduce the concentration of triglycerides in the liver and improve insulin levels. Lipolysis is an important lipid metabolism reaction in the body, and studies have shown that the regulation of lipolysis by compounds can improve the related symptoms of type II diabetes patients. In previous studies, many natural compounds have been found to have therapeutic potential for improving lipid metabolism.

[0003] In summary, the development of a safe and potent compound for the regulation of lipid metabolism and as a carboxylesterase 2 inhibitor has potential application prospects. On the one hand, it can slow down the catalytic activity of CES2 in the body, thereby regulating the pharmacokinetic characteristics or reducing the toxicity of CES2 substrate drugs, and on the other hand, it can regulate the metabolism of endogenous compounds in the body (such as triacylglycerol, diacylglycerol, cholesterol ester, etc.) through the main metabolic enzymes in the human body. And this kind of compound has good safety, simple preparation process, high synthesis yield and other advantages, which shows that this kind of compound has good application prospect.

[0004] Therefore, the application of flavonoids in the preparation of lipid metabolism regulator is a problem that those skilled in the art need to solve. SUMMARY

[0005] Therefore, the application provides the application of flavonoids in the preparation of lipid metabolism regulator.

[0006] The compound has the structure of flavonoids, which is coupled by two phenyl rings and a 3-carbon acid heterocyclic ring (C6-C3-C6), and can effectively and selectively inhibit the activity of human carboxylesterase subtype 2, thereby improving the oral bioavailability of carboxylate exogenous prodrugs. Carboxylesterase 2 inhibitors can slow down the catalytic activity of hCE2 in the body, thereby regulating the pharmacokinetic characteristics or reducing the toxicity of carboxylesterase 2 substrate drugs. Human carboxylesterase 2 plays a key role in the metabolic activation of ester drugs (including CPT11 and flutamide). Such inhibitors can also play an important role in the bioavailability of oral ester prodrugs and the efficacy of anticancer drugs such as CPT11 and capecitabine by inhibiting the activity of carboxylesterase 2 in the body. In addition to exogenous metabolism, CES2 enzymes are also involved in endogenous metabolism and lipid production. In vitro activity determination found that the IC50 of the compound (isoflavonol) for inhibiting human carboxylesterase 2 can reach 3.69 micromoles. The IC50 of the compound (amoorastannin) for inhibiting human carboxylesterase 2 can reach 3.54 micromoles.

[0007] Carboxylesterase is the main metabolic enzyme in the human body, which not only has hydrolytic activity on exogenous drugs, but also plays a key role in the metabolism of endogenous compounds in the body (such as triacylglycerol, diacylglycerol, cholesterol ester, etc.). Lipids are the energy source for animal storage, and lipid metabolism disorder is related to metabolic disorders, including cardiovascular disease, obesity, non-alcoholic fatty liver and diabetes, and changing the steady state of disordered lipid metabolism can enhance the therapeutic effect. At present, it has been found that many natural compounds can inhibit fat accumulation in the body, such as anthocyanins and ursolic acid can significantly reduce the concentration of triglyceride in the liver and improve insulin levels. Basic lipolysis is an important lipid metabolism reaction in the body, and studies have shown that compounds that inhibit lipolysis can improve the related symptoms of type II diabetes patients. In previous studies, many natural compounds have been found to have therapeutic use for improving lipid metabolism. Flavonoids can improve diseases related to lipid metabolism by inhibiting lipid accumulation. And the compound has good safety, simple preparation process, high synthesis yield and other advantages, which shows that the compound has good application prospect.

[0008] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0009] The application provides a flavonoid compound for lipid metabolism regulation and application as a carboxylesterase 2 inhibitor. The compound can effectively and selectively inhibit the activity of human carboxylesterase subtype 2, and the IC50 of the compound for inhibiting human carboxylesterase subtype 2 is 3.69 micromoles. 50Up to 3.69 μM.

[0010] One application of flavonoids for regulating lipid metabolism and as a carboxylesterase 2 inhibitor, which can regulate the metabolism of ester drugs in vivo by inhibiting the activity of carboxylesterase 2, and thus achieve better therapeutic effect on diseases. Carboxylesterase 2 is closely related to metabolic diseases such as obesity, diabetes and non-alcoholic fatty liver disease. Carboxylesterase 2 can also hydrolyze some endogenous substances in the body (such as triacylglycerol and diacylglycerol, etc.). Triglyceride is the main storage form in adipocytes and the core product of lipolysis process, and its generation directly reflects the activity of lipid metabolism. By analyzing the degree of inhibition of compound on the content of triglyceride based on enzymatic reaction, it can be determined whether the compound regulates cell lipid metabolism by blocking triglyceride hydrolysis and reducing glycerol generation

[0011] The application provides one application of flavonoids for regulating lipid metabolism and as a carboxylesterase 2 inhibitor according to claims 1 and 2, and the application is further characterized in that the inhibitor can inhibit the CES2 activity, participate in the metabolic activation process of the anticancer prodrug irinotecan (CPT-11) in vivo, and make the CES2 in the intestinal tract easily convert the CPT-11 into SN-38. CPT-11 is an effective drug for treating colon cancer and has been widely used in the treatment of tumors in the clinic. The flavonoids provided by the application are strong carboxylesterase 2 inhibitors, which can be used in weakening the hydrolysis metabolism of ester drugs and increasing active metabolism. The chemical components of the inhibitor can be used in monomer or compound, mixed with common excipients to form a dosage form, or mixed with esters in different proportions to be used as a drug composition.

[0012] The application of flavonoids in preparing lipid metabolism regulators, wherein the flavonoids are amentoflavone, eupatorium flavone, isoflavucol, isorotundicinnamone, sophoranflavone G or agrimonol.

[0013] Further, the application of flavonoids in preparing CES2 inhibitors, wherein the flavonoids are amentoflavone, eupatorium flavone, isoflavucol, isorotundicinnamone, sophoranflavone G or agrimonol.

[0014] According to the technical solution, compared with the prior art, the application provides the application of flavonoids in preparing CES2 inhibitors, which has the following beneficial effects:

[0015] (1) cheap and easy to obtain: the novel carboxylesterase inhibitor provided by the application is obtained by chemical synthesis from cheap flavonoid raw materials, and the synthesis process is simple and easy to operate, and the yield is high.

[0016] (2) High inhibitory activity: the new carboxylesterase inhibitor has a half inhibitory concentration IC50 of 0.25 μM in human tissue microsomes against carboxylesterase 2 50 up to 3.69 μM.

[0017] (3) High lipid-lowering effect: the classic flavone compound amentoflavone has a significant inhibitory effect on lipid accumulation, and its effect at 30 μM in a cell model is very significantly different from that of the control group. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of the provided drawings.

[0019] Figure 1 for amentoflavone inhibition kinetics evaluation;

[0020] Figure 2 for sophoraflavanone G inhibition kinetics evaluation;

[0021] Figure 3 for isohydrocorchole inhibition kinetics evaluation;

[0022] Figure 4 for argyrin inhibition kinetics evaluation;

[0023] Figure 5 for eupafolin inhibition kinetics evaluation;

[0024] Figure 6 for chalcone inhibition kinetics evaluation;

[0025] Figure 7 for amentoflavone inhibition effect on 3T3-L1 lipid droplet accumulation;

[0026] Figure 8 for sophoraflavanone G inhibition effect on 3T3-L1 lipid droplet accumulation;

[0027] Figure 9 for isohydrocorchole inhibition effect on 3T3-L1 lipid droplet accumulation;

[0028] Figure 10 for argyrin inhibition effect on 3T3-L1 lipid droplet accumulation;

[0029] Figure 11 for eupafolin inhibition effect on 3T3-L1 lipid droplet accumulation;

[0030] Figure 12The inhibitory effect of the flavonoid on triglyceride accumulation in 3T3-L1 adipocytes;

[0031] Figures 7-12 In the formula, Con, 10, and 30 correspond to 0, 10, and 30 μM, respectively.

[0032] Figures 13-18 The inhibitory effect of the flavonoid on triglyceride accumulation in 3T3-L1 adipocytes;

[0033] Figure 13 The glycerol content determination trend after the addition of amoorastatin chalcone;

[0034] Figure 14 The glycerol content determination trend after the addition of amoorastatin chalcone;

[0035] Figure 15 The glycerol content determination trend after the addition of amoorastatin chalcone;

[0036] Figure 16 The glycerol content determination trend after the addition of amoorastatin chalcone;

[0037] Figure 17 The glycerol content determination trend after the addition of amoorastatin chalcone;

[0038] Figure 18 The glycerol content determination trend after the addition of amoorastatin chalcone. DETAILED DESCRIPTION

[0039] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of the present application.

[0040] The equipment and its model are as follows: constant temperature mixing instrument (MS-100), fluorescent enzyme label instrument (Synergy), double-person single-face vertical purification workbench (SW-J-2D), and inverted biological microscope (XD-202).

[0041] Example 1: Primary screening of flavonoid inhibitors

[0042] The probe FD (fluorescein diacetate) is used as a substrate, which is added to the reaction system of the test compound (flavonoid) and CES2 (0.1 μM, 1 μM, 10 μM) for hydrolysis. The fluorescence value of the reaction system is detected by using a fluorescent enzyme label instrument, and the residual activity of the enzyme is determined by measuring the concentration of fluorescein in the reaction system. The reaction system is set in a black 96-well plate, and three parallel experiments are set for each group.

[0043] The reaction was carried out in a 50 μL PBS (pH 7.4) reaction system. A background group, a blank group, a positive inhibitor group, and an experimental group were set up, each with PBS (pH 7.4) added. The following concentration gradients were set up according to the experiment: flavonoid compound concentration gradient (0.5, 50, 500 μM, initial concentration), BNPP (10 μL, 100 μM, initial concentration), and HLM human liver microsomes (10 μL, 10 μg / mL, initial concentration); background group (PBS 40 μL); blank group (PBS 30 μL, HLM 10 μL). Simultaneously, a positive inhibitor group (PBS 20 μL, HLM 10 μL, BNPP 10 μL) was set up using BNPP (bis-p-nitrophenyl phosphate) as a positive inhibitor, and an experimental group (PBS 20 μL, HLM 10 μL, test compound 10 μL) was set up. The reaction system was then incubated at 37°C for 5 minutes. The reaction was then initiated by adding probe FD (10 μL, 75 μM, initial concentration). After shaking the 96-well plate at 37°C for 30 minutes in a constant temperature incubator, 50 μL of ice-cold acetonitrile was added to the reaction solution (50 mL) to terminate the experiment. Finally, fluorescence was detected using a Synergy HTXISILFTA fluorescent microplate reader with an excitation wavelength of 480 nm, an emission wavelength of 525 nm, and a gain of 50. All flavonoid inhibitors used in the inhibition assay were prepared in chromatographic grade DMSO, with a final DMSO volume ratio <1% (v / v). Preliminary screening revealed several CES2 inhibitors with good inhibitory effects, such as cephalotaxine biflavonoids, eupatorium flavonoids, isoflavone, chalcone hypoglycinate, cypermethrin, and sophoranone G.

[0044] Example 2: Inhibition of FD hydrolysis mediated by flavonoids (IC50) 50 Measurement

[0045] Based on the initial screening results, the half-maximal inhibitory concentration (IC50) of the flavonoid compounds with inhibitory effects on CES2 was determined. 50 The range was determined, and a series of concentrations of the test compound were set to plot its dose-inhibition curve, which was then evaluated using Gaphpadprism 10.0 software for nonlinear regression.

[0046] The reaction was carried out in a 50 mL PBS reaction system. The background group, blank group and experimental group were set up, and PBS (pH 7.4) was added. According to the experimental setting, the concentration gradient of flavonoid compounds and HLM human liver microsomes (10 μL, 10 μg / mL, initial concentration) were set; the background group (PBS 40 μL); the blank group (PBS 30 μL, HLM 10 μL). The experimental group (PBS 20 μL, HLM 10 μL, test compound 10 μL) was set, and the compound concentration was set to 40, 20, 10, 5, 2, 1, 0.2 μM. Next, the reaction system was incubated at 37°C for 5 minutes. Then, the reaction was started by adding probe FD (10 μL, 75 μM, initial concentration). After shaking the 96-well plate in a constant temperature incubator at 37°C for 30 minutes, 50 μL of ice acetonitrile was added to the reaction solution (50 mL) to terminate the experiment. Finally, the fluorescence was detected in the fluorescence enzyme marker (SynergyHTXISILFTA), the excitation wavelength was 480 nm, the emission wavelength was 525 nm, and the gain was 50. The experimental results were measured and evaluated by non-linear regression in gaphpadprism 10.0 software.

[0047] Table 1 Inhibition IC of flavonoid compounds on CES2 50 Value

[0048]

[0049] Example 3 Kinetic analysis of flavonoid compounds on carboxylesterase 2 inhibition

[0050] After determining the inhibition type of the compound with inhibitory effect on CES2 and the half-inhibitory concentration (IC 50 ) of the compound on CES2, the compound concentration value range was determined according to (IC 50 ), a variety of reaction rates were obtained by adding different concentrations of inhibitors, and a variety of reaction rates were used to determine the inhibition type and inhibition constant of the inhibitor on CES2. Using Lineweaver-Burk plot, the inhibition type includes: competitive inhibition, non-competitive inhibition, and mixed inhibition.

[0051] The reaction was carried out in 50 μL of PBS (pH 7.4) reaction system. The background group, blank group and experimental group were set up, and PBS (pH 7.4) was added respectively. The concentration gradient of flavonoid compounds was set according to the IC50 value experiment, HLM human liver microsomes (10 μL, 10 μg / mL, initial concentration); the background group (PBS 40 μL); the control group (PBS 30 μL, HLM 10 μL). The experimental group (PBS 20 μL, HLM 10 μL, test compound 10 μL) was set up, and then the reaction was started by adding the probe FD (different concentrations). After shaking the 96-well plate in the constant temperature incubator at 37°C for 30 minutes, 50 μL of ice acetonitrile was added to the reaction solution (50 mL) to terminate the experiment. Finally, the fluorescence was detected in the fluorescence enzyme label instrument (SynergyHTXISILFTA), the excitation wavelength was 480 nm, the emission wavelength was 525 nm, and the gain was 50. The experimental results were measured and evaluated by non-linear regression in gaphpadprism 10.0 software. Amentoflavone, sophoranflavanone G, and naringenin chalcone were non-competitive inhibitors, and isoflavucrol, geniflavin, and zerlinflavone were non-competitive inhibitors. The results are shown in Figures 1-6 .

[0052] Example 4 Inhibition of Lipid Droplet Accumulation in 3T3-L1 Cells by Flavonoid Compounds

[0053] 3T3-L1 cells were seeded in 6-well plates (2 x 10 5 cells) and cultured in DMEM / F12 medium. When the cells grew to 100% confluence, they were cultured for another 2 days. Flavonoid compounds at concentrations (0-30 μM) were prepared with induction medium I and cultured for 2 days. Flavonoid compounds at concentrations (0-30 μM) were prepared with induction medium II and cultured for 2 days. Flavonoid compounds at concentrations (0-30 μM) were prepared with DMEM / F12 medium and cultured for 4 days, and the medium was changed every 2 days. After staining with oil red O dye, the lipid droplets were observed under a biological inverted microscope. The results are shown in Figures 7-12 . It was shown that amentoflavone, sophoranflavanone G and other series of compounds had different degrees of inhibition ability on the induced differentiation of 3T3-L1 cells at 10 μM, and the inhibition ability on differentiation was particularly obvious at 30 μM compared with the control group at 0 concentration.

[0054] Reagent preparation:

[0055] (1) Insulin stock solution (Ins): 2.5 mg of insulin was dissolved in 2.5 ml of dilute hydrochloric acid (pH = 2) to prepare a stock solution with a final concentration of 1 mg / ml, which was stored at -20°C.

[0056] (2) 3-isobutyl-1-methylxanthine stock solution (IBMX, 1000x): 100 mg of IBMX was dissolved in 900 μl of DMSO to prepare a stock solution with a final concentration of 0.5 M, which was stored at -20°C.

[0057] (3) Dexamethasone stock solution (DEX, 1000x): 3047 μl DMSO was added to 11.96 mg DEM to give a final concentration of 10 mM.

[0058] (4) Induction medium I: 50 ml DMEM / F12 medium + 500 μl insulin stock solution (1 mg / ml) + 50 μl 3-isobutyl-l-methylxanthine stock solution (0.5 M) + 5 μl dexamethasone stock solution (10 mM).

[0059] (5) Induction medium II: 50 ml DMEM / F12 + 500 μl insulin stock solution (1 mg / ml).

[0060] Example 5 Determination of the effect of flavonoids on glycerol content after inhibition of differentiation of 3T3-L1 cells

[0061] 3T3-L1 cells were seeded in 6-well plates (2 x 10 5 cells) and cultured in DMEM / F12 medium. When the cells reached 100% confluency, they were cultured for a further 2 days in induction medium I with flavonoids at concentrations ranging from 0 to 30 μM. The cells were then cultured for a further 2 days in induction medium II with flavonoids at concentrations ranging from 0 to 30 μM. The cells were then cultured for a further 2 days in DMEM / F12 medium without any additives. Finally, the cells were cultured for a further 2 days in DMEM / F12 medium with flavonoids at concentrations ranging from 0 to 30 μM. The glycerol content was determined using a glycerol kit. The glycerol content decreased with increasing concentrations of flavonoids. The results are shown in Figure 2. Figures 13-18 .

[0062] The foregoing description of the disclosed embodiments enables a person skilled in the art to make or use the application. Numerous modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein can be applied to other embodiments without departing from the spirit or scope of the application. Therefore, the present application is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Use of flavonoids for the preparation of a regulator of lipid metabolism, characterized in that, The flavonoids are amentoflavone, eupatorin, isofukinol, merensin chalcone, sophoranflavone G or thujopsin.

2. Use of flavonoids for the preparation of a formulation for inhibiting CES2, characterized in that, The flavonoids are amentoflavone, eupatorin, isofukinol, merensin chalcone, sophoranflavone G or thujopsin.

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