Phenolic components with lipase inhibitory activity, methods for their preparation, salts, lipidated derivatives, medicaments and uses

Abstract

The application belongs to the technical field of natural medicine, and particularly relates to a phenolic component with lipase inhibitory activity and a preparation method, salt, lipidated derivative, medicine and application thereof. The phenolic component with lipase inhibitory activity is separated from leaves of Castanopsis chinensis plants. In vitro pharmacological experiments prove that the phenolic component has good lipase inhibitory activity, wherein an IC 50 value is 13.26+ / -1.1 uM. The new phenolic component Chinensin A, or a medicinal salt thereof, or an ester derivative thereof can be used for preparing a lipase inhibitor medicine.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of natural medicine, and particularly relates to a phenolic component with lipase inhibitory activity, a preparation method, a salt, a lipidated derivative, a medicine and application thereof. BACKGROUND

[0002] Overweight and obesity can cause hyperlipidemia, diabetes, hypertension, cardiovascular and cerebrovascular diseases, bone and joint abnormalities and other diseases, which seriously threaten the physical and mental health of patients. The causes of obesity are complex, mainly including genetic, lifestyle, disease and other factors. According to statistics, the main factor affecting obesity is the influence of lifestyle, such as increase in total food intake and preference for high-sugar and high-fat food, which can easily lead to excessive calories and thus obesity. In addition, some people lack exercise and energy consumption is reduced, which can also cause excess energy in the body every day and thus obesity. At present, orlistat, as an obesity treatment drug, is most widely used in clinical practice at home and abroad. Orlistat acts on the central nervous system, inhibits gastric and intestinal lipase, prevents the hydrolysis of triacylglycerol into free fatty acids and monoacylglycerol, reduces the absorption of intestinal mucosa to dietary fat (triacylglycerol), and promotes the excretion of fat from the body. Although orlistat has been approved by the US FDA, the European Union EMA and the China Drug Administration, it still has adverse reactions such as gastrointestinal reactions, respiratory tract infections and urinary tract infections. Therefore, it is urgent to find safe and efficient lipase inhibitor drugs from natural products.

[0003] Castanopsis chinensis, also known as Guilin chestnut, chestnut, small plate chestnut, etc., is a tree of Fagaceae Castanopsis, with a height of 20 meters and a diameter at breast height of 0.6 centimeters. The bark is longitudinally cracked and flaky, and the branches and leaves are not hairy. It is distributed in Guangdong, Guangxi, southwest of Guizhou and southeast of Yunnan, China. The wood is light brown or light gray yellow, with coarse grain and solid texture, and is a good material for making furniture, buildings and farm tools. In folk, it is mainly used for the treatment of diarrhea, hemostasis and gastrointestinal inflammation. At present, there is no report on the chemical composition of Castanopsis chinensis, and there is no report on the Chinensin A component with lipase inhibitory activity in Castanopsis chinensis.

[0004] Therefore, it is necessary to provide a preparation method and application of a lipase inhibitory active component prepared from the plant Castanopsis chinensis, so as to provide a new choice for obesity treatment drugs and make up for the deficiencies of the prior art. SUMMARY

[0005] In order to solve the deficiencies of the prior art, the present application provides a phenolic component with lipase inhibitory activity, a preparation method, a salt, a lipidated derivative, a medicine and application thereof.

[0006] The technical scheme provided by the present application is as follows:

[0007] A phenolic component with lipase inhibitory activity, which structure is shown as formula (I):

[0008]

[0009] In the above technical solution, the phenolic component with lipase inhibitory activity provided by the application is first isolated from the leaves of Castanopsis chinensis by the inventors of the application. The new phenolic component can be named as Chinensin A.

[0010] The new phenolic component Chinensin A provided by the application has good lipase inhibitory activity, which is verified by in-vitro pharmacological experiments, and the IC 50 value is 13.26±1.1 μM. The new phenolic component Chinensin A, or a pharmaceutically acceptable salt thereof, or an ester derivative thereof can be used for preparing a lipase inhibitor drug.

[0011] The new phenolic component Chinensin A provided by the application, or a pharmaceutically acceptable salt thereof, or an ester derivative thereof can be combined with a formulation or a pharmaceutically acceptable excipient or carrier to prepare a drug or a pharmaceutical composition with lipase inhibitory activity for controlling body weight. The drug or the pharmaceutical composition can have a dosage form of tablets, granules, capsules, etc.; and can also adopt a formulation known in the modern pharmaceutical industry such as nano, or controlled release, or sustained release formulation, etc.

[0012] The application further provides a preparation method of the phenolic component with lipase inhibitory activity, comprising the following steps: isolating the phenolic component with lipase inhibitory activity from the plant Castanopsis chinensis.

[0013] Specifically, the Castanopsis chinensis is selected from the group consisting of dry or fresh leaves, branches, stems or barks of Castanopsis chinensis.

[0014] Preferably, the Castanopsis chinensis is selected from the group consisting of leaves of Castanopsis chinensis.

[0015] In the above technical solution, the leaves of Castanopsis chinensis are used as the object for extraction, which has the advantages of rich resources and little impact on the environment, etc.

[0016] The preparation method of the phenolic component with lipase inhibitory activity comprises the following steps:

[0017] a. preparing an extraction solution: crushing the leaves of Castanopsis chinensis, adding a 75-85% ethanol aqueous solution by volume fraction for room temperature extraction, filtering the extraction solution after removing the organic solvent by reduced pressure concentration to obtain the extraction solution;

[0018] b. Gel column chromatography: the extract was purified by dextran gel column chromatography, and eluted with 0%, 20%, 40%, 60%, 80%, and 100% methanol aqueous solution in sequence, and the fractions containing phenolic components with lipase inhibitory activity were collected and concentrated to obtain a crude extract;

[0019] c. Isolation and purification: the crude extract was prepared and purified by semi-preparative high performance liquid chromatography, and eluted with 25-35% methanol aqueous solution in sequence, and the fractions containing phenolic components with lipase inhibitory activity were collected and concentrated to obtain phenolic components with lipase inhibitory activity.

[0020] The above extraction method has the advantages of simple process, easy operation, and environmental friendliness.

[0021] The application further provides a pharmaceutical salt of the phenolic component with lipase inhibitory activity.

[0022] The application further provides a pharmaceutical ester derivative of the phenolic component with lipase inhibitory activity.

[0023] The application further provides an application of the phenolic component with lipase inhibitory activity in the preparation of a lipase inhibitor.

[0024] Specifically, the application is used for the preparation of a medicine for treating obesity.

[0025] The application further provides a lipase inhibitor medicine, comprising an effective dose of an active ingredient, characterized in that the active ingredient is selected from any one or more of the phenolic component with lipase inhibitory activity, the pharmaceutical salt, or the pharmaceutical ester derivative.

[0026] The above technical solution uses the new phenolic component Chinensin A, or a pharmaceutical salt thereof, or an ester derivative thereof as an effective ingredient.

[0027] Further, the application further includes a pharmaceutical composition of a pharmaceutically acceptable pharmaceutical excipient or carrier.

[0028] The lipase inhibitor medicine further includes a pharmaceutically acceptable excipient or carrier.

[0029] The preparation can be in the form of tablets, granules, capsules, etc. Modern pharmaceutical industry known preparations such as nano, or controlled release, or sustained release preparations, etc. can also be used.

[0030] The application uses cone leaf as raw material to extract and separate potent lipase inhibiting components, and has the advantages of simple preparation method, easy operation, rich resources of leaf as raw material for extraction, good potential economic benefits, and stable and easy-to-store new phenolic component Chinensin A. The lipase inhibiting activity is good, and it is expected to be used as a lead compound to develop new obesity treatment drugs. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 is the H-NMR (500 MHz, CD3OD) spectrum of compound Chinensin A. 1 H-NMR (500 MHz, CD3OD) spectrum;

[0032] Figure 2 is the C-NMR (125 MHz, CD3OD) spectrum of compound Chinensin A. 13 C-NMR (125 MHz, CD3OD) spectrum;

[0033] Figure 3 is the HSQC spectrum of compound Chinensin A.

[0034] Figure 4 is the HMBC spectrum of compound Chinensin A.

[0035] Figure 5 is the H- 1 H- 1 H COSY spectrum.

[0036] Figure 6 is the HREIMS spectrum of compound Chinensin A. DETAILED DESCRIPTION

[0037] The principles and characteristics of the application are described below, and the examples are only used to explain the application and not to limit the scope of the application.

[0038] Example 1:

[0039] Preparation of phenolic compound Chinensin A

[0040] 1.1 Plant source

[0041] The leaf sample of the plant cone (Castanopsis chinensis) used for extraction was collected from Guilin City, Guangxi Zhuang Autonomous Region.

[0042] 1.2 Extraction and separation

[0043] Fresh cone leaves were pulverized and extracted with 80% ethanol aqueous solution at room temperature. The extract was concentrated under reduced pressure to remove organic solvents and then filtered to obtain the extract. The extract was purified by Sephadex LH-20 dextran gel chromatography, eluting with 0%, 20%, 40%, 60%, 80%, and 100% methanol aqueous solutions to remove impurities and purify. The component containing Chinensin A was collected and concentrated to obtain the crude extract. The crude extract was further purified by semi-preparative high-performance liquid chromatography (HPLC) using 30% methanol aqueous solution as the mobile phase at a flow rate of 2 mL / min with an SB-C18 Agilent ZORBAX RRHD column (9.4 × 250 mm, 5 μm) to obtain the compound Chinensin A(t R (12.5 min).

[0044] 1.3 Identification of new phenolic compounds

[0045] Figure 1 It is the compound Chinensin A 1 H-NMR spectrum; Figure 2 It is the compound Chinensin A 13 C-NMR spectrum; Figure 3 This is the HSQC spectrum of compound Chinensin A; Figure 4 This is the HMBC spectrum of the compound Chinensin A; Figure 5 It is the compound Chinensin A 1 H- 1 H COSY spectrum; Figure 6 This is the HREIMS spectrum of compound Chinensin A. Chinensin A is a white amorphous powder with a bright spot under 254 nm UV light. It reacts with 1% FeCl3-EtOH colorimetric reagent to form brown spots. The HREIMS spectrum shows its quasi-molecular ion peak at m / z: 479.1189 [MH]. - (Calculated value 479.1195, C) 22 H 24 O 12 The molecular formula is C. 22 H 24 O 12 The degree of unsaturation is 11. 1 H-NMR (500MHz, CD3OD), 13 C-NMR (125MHz, CD3OD) data are shown in Table 1 below.

[0046] Table 1. NMR data of compound Chinensin A (in CD3OD)

[0047]

[0048]

[0049] According to the comprehensive analysis of the above mass spectrum, one-dimensional and two-dimensional nuclear magnetic resonance spectrum data, the structure of the compound is deduced as shown in formula (I), and named as Chinensin A:

[0050]

[0051] Example 2:

[0052] Detection of lipase inhibitory activity of phenolic compound Chinensin A

[0053] 1. Instruments and materials

[0054] The substrate 4-methlumbelliferyl oleate (4-MUO 75164) and porcine pancreatic amylase (EC 3.1.1.3, L3126) were purchased from Sigma Company, and phosphate buffer PBS was purchased from Shanghai Yuanye Biological Technology Co., Ltd. DMSO, MeOH were analytical pure, and citric acid and sodium citrate were purchased from Xilong Chemical Co., Ltd.; enzyme reader SpectraMax iD5 (Sunnyvale, USA); 96-well plate (Thermo Fisher Scientific China Co., Ltd.).

[0055] 2. Evaluation of lipase inhibitory activity

[0056] The colorimetric method (Ercan et al, 2016) was used to determine the inhibition rate of the test compound on pancreatic lipase by 96-well cell culture plate. The substrate 4-MUO generates 4-methyl umbelliferone with fluorescence under the action of pancreatic lipase, and the fluorescence intensity of each well after reaction is determined to judge the inhibitory activity of the compound on lipase.

[0057] a. Prepare 0.1M citric acid-sodium citrate buffer (pH=4.2) with ultrapure water; prepare 1mg / mL pancreatic enzyme solution, 0.1mM 4-MUO solution, and 0.2M Na2CO3 solution with PBS (pH=7.4).

[0058] b. Take part of the prepared lipase solution (1mg / mL) and heat it in a 90℃ water bath for 20 minutes to inactivate the enzyme.

[0059] c. The positive control of the compound is diluted with DMSO to an initial concentration of 10, 5, 2.5, 1.25, 0.625, and 0.3125mM, respectively.

[0060] d. Sample (DMSO concentration is not more than 5% of the whole system) as follows:

[0061] Sample group: 1 μL sample + 24 μL PBS + 25 μL enzyme

[0062] Sample blank group: 1 μL sample + 24 μL PBS + 25 μL inactivated enzyme

[0063] Negative group: 1 μL DMSO + 24 μL PBS + 25 μL enzyme

[0064] Negative blank group: 1 μL DMSO + 24 μL PBS + 25 μL inactivated enzyme

[0065] After mixing, 50 μL of reaction substrate 4-MUO (0.1 mM) was added to each well, and the reaction was incubated in a 25°C constant temperature incubator for 20 minutes. Then, 100 μL of citric acid-sodium citrate solution (0.1 M, pH = 4.2) was added to each sample well to terminate the reaction. The fluorescence absorption value of each well was measured by an enzyme marker, and the excitation wavelength was 320 nm. Each sample was repeated at least 3 times, and the inhibition rate was calculated according to the following formula:

[0066] Inhibition rate (%) = [1 - (A sample - A sample blank) / (A negative - A negative blank)] x 100

[0067] The SPSS 19.0 statistical analysis software was used to calculate the half inhibitory concentration IC 50 value.

[0068] 3. Experimental data are shown in Table 2:

[0069] Table 2. Lipase inhibitory activity of phenolic compound Chinensin A

[0070]

[0071] 4. Experimental conclusion:

[0072] The experiment shows that the phenolic compound Chinensin A has good lipase inhibitory activity, and is expected to be used as a lead compound to develop a new obesity treatment drug.

[0073] The above only describes the preferred embodiments of the present application, and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A phenolic compound with lipase inhibitory activity, characterized in that, Its structure is shown in equation (Ⅰ): Equation (Ⅰ).

2. A method for preparing a phenolic component with lipase inhibitory activity according to claim 1, characterized in that, Includes the following steps: a. Preparation of extract: Take cone leaves, crush them, add 75-85% ethanol aqueous solution (volume fraction) for extraction at room temperature, concentrate the extract under reduced pressure to remove organic solvent, and then filter to obtain the extract. b. Gel column chromatography for impurity removal: The extract was purified by dextran gel column chromatography, and eluted successively with methanol aqueous solution of 0%, 20%, 40%, 60%, 80%, and 100% (v / v). The phenolic component containing lipase inhibitory activity was collected and concentrated to obtain its crude extract. c. Separation and purification: The crude extract was prepared and purified by semi-preparative high performance liquid chromatography. It was eluted with a gradient of 25-35% methanol aqueous solution. The phenolic components containing lipase inhibitory activity were collected, concentrated, and the phenolic components with lipase inhibitory activity were obtained.

3. A medicinal salt, characterized in that: It is a pharmaceutical salt of the phenolic component with lipase inhibitory activity as described in claim 1.

4. The application of a phenolic component with lipase inhibitory activity according to claim 1, characterized in that: Used to prepare lipase inhibitors.

5. The application of the phenolic component with lipase inhibitory activity according to claim 4, characterized in that: Used to prepare drugs for treating obesity.

6. A drug having lipase inhibitory activity, comprising an effective dose of the active ingredient, characterized in that: The active ingredient is selected from any one or a mixture of the phenolic components with lipase inhibitory activity described in claim 1 or the pharmaceutical salts described in claim 3.

7. The drug with lipase inhibitory activity according to claim 6, characterized in that: It also includes pharmaceutically acceptable drug excipients or carriers.

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