CN110804079B

CN110804079B - Furocoumarin with DPPIV enzyme inhibitory activity and preparation method thereof

Info

Publication number: CN110804079B
Application number: CN201911072793.7A
Authority: CN
Inventors: 贾栩超; 张名位; 张瑞芬; 刘磊; 池建伟; 黄菲; 董丽红; 马勤
Original assignee: Sericulture and Agri Food Research Institute GAAS
Current assignee: Sericulture and Agri Food Research Institute GAAS
Priority date: 2019-11-05
Filing date: 2019-11-05
Publication date: 2021-02-12
Anticipated expiration: 2039-11-05
Also published as: CN110804079A

Abstract

The invention provides furocoumarin with DPPIV enzyme inhibitory activity and a preparation method thereof. The furocoumarin compound 6-beta-D-glucosyl-6, 7-dihydroxy-5-benzofuran methyl propionate has the structural formula shown in formula (I), and is obtained by separating dried or fresh fruits, roots, stems, branches or leaves of clausena lansium. In vitro pharmacological experiments prove that the compound 6-beta-D-glucosyl-6, 7-dihydroxy-5-benzofuranmethyl propionate has obvious inhibition effect on DPPIV enzyme, and IC of the compound₅₀The value was 117. + -. 8.7. mu.M, indicating good DPPIV enzyme inhibitory activity. The furocoumarin compound 6-beta-D-glucosyl-6, 7-dihydroxy-5-benzofuranmethyl propionate is expected to be used as a lead compound to develop a novel DPPIV enzyme inhibitor drug.

Description

Furocoumarin with DPPIV enzyme inhibitory activity and preparation method thereof

Technical Field

The invention belongs to the technical field of natural medicines, and particularly relates to furocoumarin with DPPIV enzyme inhibitory activity and a preparation method thereof.

Background

Diabetes has developed into the third most serious chronic non-infectious disease after cardiovascular and cerebrovascular diseases and cancer, and is one of three killers harming the health of modern human beings. About 4.25 million people all over the world in 2017 suffer from diabetes, wherein the number of people diagnosed with diabetes in China reaches 1.34 million. The enormous patient population places a heavy economic burden on individuals, families and society, and the prevention and treatment of diabetes has become one of the most serious challenges facing the world. Dipeptidyl peptidase IV (DPPIV), which is a transmembrane serine protease, is distributed in various tissues and organs in the body, and an inhibitor thereof can rapidly activate the biological activity of endogenous substances such as incretins, neuropeptides and cytokines, thereby inhibiting the release of glucagon, reducing fasting and postprandial blood glucose, and possibly improving insulin sensitivity and islet beta cell function, and thus is considered as one of effective targets for treating diabetes, and currently marketed DPPIV enzyme inhibitors are Sitagliptin (Sitagliptin), Saxagliptin (Saxagliptin), Vildagliptin (Vildagliptin), and the like.

Secondary metabolites produced by plants and microorganisms are important sources of drug discovery, such as artemisinin, penicillin, and paclitaxel, among others. Clausena lansium (Lour.) Skeels) is a small arbor of Rutaceae Clausena genus, and has been cultivated in China for 1500 years. The wampee has high medicinal value, and fruits, leaves, roots and seeds of the wampee can be used as medicines. At present, no research report on the coumarin component with DPPIV enzyme inhibition activity in the clausena lansium is found.

Disclosure of Invention

The invention aims to provide furocoumarin with DPPIV enzyme inhibitory activity and a preparation method thereof.

The novel furocoumarin component 6-beta-D-glucosyl-6, 7-dihydroxy-5-benzofuran methyl propionate or its medicinal salt or its esterified derivative has the structure as shown in formula (I):

the novel furocoumarin component 6-beta-D-glucosyl-6, 7-dihydroxy-5-benzofuran methyl propionate is obtained by being separated from a Clausena lansium (Lour.) Skeels plant for the first time by the inventor, and comprises dry products or fresh products of fruits, roots, stems, branches or leaves of the Clausena lansium. Preferably, the extract is obtained by separating dried or fresh product of each tissue part of the wampee fruit, wherein the specific parts can be pericarp and pulp.

The novel furocoumarin component 6-beta-D-glucosyl-6, 7-dihydroxy-5-benzofuran methyl propionate provided by the invention has a remarkable inhibitory effect on dipeptidyl peptidase IV (DPPIV) and half Inhibitory Concentration (IC) of the dipeptidyl peptidase IV (DPPIV) proved by in vitro pharmacological experiments₅₀Value) was 117. + -. 8.7. mu.M. Thus indicating that the DPPIV enzyme has good inhibitory activity. The new furocoumarin component 6-beta-D-glucosyl-6, 7-dihydroxy-5-benzofuran methyl propionate, or its medicinal salt, or its esterified derivative can be used for preparing DPPIV enzyme inhibitor medicine.

The new furocoumarin component 6-beta-D-glucosyl-6, 7-dihydroxy-5-benzofuran methyl propionate, or its medicinal salt, or its esterified derivative may be combined with pharmaceutically acceptable excipient or carrier to prepare medicine or medicine composition with DPPIV enzyme inhibiting activity for treating diabetes. The medicine or the pharmaceutical composition can adopt wettable powder, tablets, granules, capsules, oral liquid, dripping pills, injection, aerosol and other formulations; controlled or sustained release formulations or nano-formulations well known in the modern pharmaceutical industry may also be employed.

Therefore, the invention also provides a DPPIV enzyme inhibitor drug which comprises the novel furocoumarin component 6-beta-D-glucosyl-6, 7-dihydroxy-5-benzofuran methyl propionate or medicinal salt thereof or esterified derivative thereof as an active ingredient.

The DPPIV enzyme inhibitor medicine also comprises a preparation or a pharmaceutically allowable excipient or carrier, and can adopt wettable powder, tablets, granules, capsules, oral liquid, dripping pills, injection, aerosol and other dosage forms; controlled or sustained release formulations or nano-formulations well known in the modern pharmaceutical industry may also be employed.

The invention also provides a preparation method of the novel furocoumarin component 6-beta-D-glucosyl-6, 7-dihydroxy-5-benzofuran methyl propionate, which is obtained by separating from Clausena lansium (Lour.) Skeels) plants and comprises dry products or fresh products of fruits, roots, stems, branches or leaves of Clausena lansium. Preferably, the extract is obtained by separating dried or fresh parts of each tissue part of the clausena lansium fruit, wherein the specific parts can be the pericarp and/or the pulp.

The preparation method of the novel furocoumarin component 6-beta-D-glucosyl-6, 7-dihydroxy-5-benzofuran methyl propionate provided by the invention comprises the following specific steps:

homogenizing fresh wampee fruit with 95% ethanol water solution, extracting, concentrating the extractive solution under reduced pressure to remove organic solvent to obtain suspension, sequentially extracting the suspension with ethyl acetate and n-butanol, and concentrating n-butanol extractive solution under reduced pressure to obtain total extract. Subjecting the total extract to normal phase silica gel column chromatography, performing gradient elution with chloroform/methanol from a volume ratio of 100:0 to 60:40, collecting a component F15 eluted with a chloroform/methanol volume ratio of 70:30, subjecting a component F15 to Sephadex LH-20 column chromatography, eluting with methanol, collecting an elution fraction F15-9 in an amount which is 1.2-1.6 times the volume of the column, and subjecting the fraction F15-9 to high performance liquid phase separation and purification to obtain 6-beta-D-glucosyl-6, 7-dihydroxy-5-benzofuranmethyl propionate.

The invention adopts the extraction and separation of the potent DPPIV enzyme inhibitor (drug) from the fruit tissue of wampee, the preparation process conditions are controllable, the extraction is convenient, the plant can be utilized for a long time without being damaged when the fruit is adopted for extraction, the invention is environment-friendly and has potential good economic benefits, and the obtained new furocoumarin compound 6-beta-D-glucosyl-6, 7-dihydroxy-5-benzofuran methyl propionate has stable property and is easy to store. The DPPIV enzyme has good inhibitory activity, and is expected to be used as a lead compound to develop a novel DPPIV enzyme inhibitor medicament for treating diabetes.

Drawings

FIG. 1 is a drawing showing the preparation of methyl 6-beta-D-glucosyl-6, 7-dihydroxy-5-benzofuranpropionate¹H NMR spectrum;

FIG. 2 is a drawing showing the preparation of methyl 6-beta-D-glucosyl-6, 7-dihydroxy-5-benzofuranpropionate¹³A C NMR spectrum;

FIG. 3 is a drawing showing the preparation of methyl 6-beta-D-glucosyl-6, 7-dihydroxy-5-benzofuranpropionate¹H^-1H COSY map;

FIG. 4 is an HSQC spectrum of the compound methyl 6- β -D-glucosyl-6, 7-dihydroxy-5-benzofuranpropionate;

FIG. 5 is an HMBC plot of the compound methyl 6-beta-D-glucosyl-6, 7-dihydroxy-5-benzofuranpropionate;

FIG. 6 is an HR-EI-MS spectrum of compound methyl 6-beta-D-glucosyl-6, 7-dihydroxy-5-benzofuranpropionate.

Detailed Description

The following examples are further illustrative of the present invention and are not intended to be limiting thereof, and all simple modifications of the invention which are within the spirit of the invention are intended to be within the scope of the invention as claimed.

Example 1: preparation of furocoumarin compound 6-beta-D-glucosyl-6, 7-dihydroxy-5-benzofuranmethyl propionate

1.1 plant origin and identification

Fruit samples of plant material wampee (claur.) Skeels for extraction were collected in southwestern county of Guangdong province 8 months in 2018 and identified by Chenhongfeng researchers in the south China plant Garden of China academy of sciences.

1.2 extraction and separation

Homogenizing fresh seedless wampee fruit with 95% ethanol water solution, extracting, concentrating the extract under reduced pressure to remove organic solvent to obtain suspension, sequentially extracting the suspension with ethyl acetate and n-butanol, and concentrating n-butanol extract under reduced pressure to obtain total extract. Subjecting the total extract to normal phase silica gel column chromatography, performing gradient elution with chloroform/methanol from a volume ratio of 100:0 to 60:40, collecting chloroform/methanol 70:30, performing v/v elution on a component F15, subjecting a component F15 to Sephadex LH-20 column chromatography, performing elution with methanol, and collecting an elution fraction F15-9 in a volume 1.2-1.6 times that of the column. Subfraction F15-9 was further chromatographically isolated and purified by preparative HPLC using 50% aqueous methanol (v/v) as the mobile phase at a flow rate of 7mL/min and using a Shim-pack PRC-ODS C-18 column (5 μm, 20X 250mm) to give Compound 1 (Compound 6-. beta. -D-glucosyl-6, 7-dihydroxy-5-benzofuranpropanoic acid methyl ester, 10mg, tR 50 min).

1.3 structural identification of novel furocoumarins

FIG. 1 is a drawing showing the preparation of methyl 6-beta-D-glucosyl-6, 7-dihydroxy-5-benzofuranpropionate¹H NMR spectrum; FIG. 2 is a drawing showing the preparation of methyl 6-beta-D-glucosyl-6, 7-dihydroxy-5-benzofuranpropionate¹³A C NMR spectrum; FIG. 3 is a drawing showing the preparation of methyl 6-beta-D-glucosyl-6, 7-dihydroxy-5-benzofuranpropionate¹H-¹H COSY map; FIG. 4 is an HSQC spectrum of the compound methyl 6- β -D-glucosyl-6, 7-dihydroxy-5-benzofuranpropionate; FIG. 5 is an HMBC plot of the compound methyl 6-beta-D-glucosyl-6, 7-dihydroxy-5-benzofuranpropionate; FIG. 6 is an HR-EI-MS spectrum of the compound methyl 6- β -D-glucosyl-6, 7-dihydroxy-5-benzofuranpropionate; 6-beta-D-glucosyl-6, 7-dihydroxy-5-benzofuranpropionic acid methyl ester, colorless oily substance;

HR-ESI-MS:m/z 397.0811[M-H]^-(calcd 397.1248,C₁₈H₂₀O₁₀)；¹H NMR(CDCl₃600MHz) and¹³C NMR(CDCl₃150MHz) data array is shown in table 1 below.

TABLE 1 NMR data (in CD) for the compound methyl 6-beta-D-glucosyl-6, 7-dihydroxy-5-benzofuranpropionate₃OD)

According to the comprehensive analysis of the related data of the spectra such as ultraviolet spectrum, mass spectrum, one-dimensional nuclear magnetism and two-dimensional nuclear magnetism, the structural formula of the compound 1 is analytically deduced to be shown as the formula (I), and the compound is named as 6-beta-D-glucosyl-6, 7-dihydroxy-5-benzofuranmethyl propionate:

example 2: DPPIV enzyme inhibition activity detection of furocoumarin compound 6-beta-D-glucosyl-6, 7-dihydroxy-5-benzofuranmethyl propionate

1. Apparatus and materials

Dipeptide ground-state enzyme-4 (DPPIV), sitagliptin phosphate, glycine-proline-p-nitroanilide, dimethyl sulfoxide (DMSO) were all purchased from sigma, USA. The microplate reader is a Genois microplate reader (Tecan GENios, Swizerland).

Evaluation of inhibitory Activity of DPPIV enzyme

DPPIV was dissolved in 1mL of 100mM Tris buffer solution (pH 0.8), and diluted 50-fold to prepare a DPPIV working solution. Both the sample and sitagliptin phosphate (positive control) were dissolved in DMSO. Samples of different concentrations and sitagliptin phosphate were added to a 96-well plate, 50 μ L of DPPIV working solution was added, the 96-well plate was placed in an microplate reader and incubated at 37 ℃ for 10min, then 50 μ L of 1mM glycine-proline-p-nitroaniline and 50 μ L of 100mM Tris buffer solution (pH 0.8) were added to each well, respectively, and the 96-well plate was placed in an microplate reader and incubated at 37 ℃ for 60 min. The reaction was stopped by adding 50. mu.L of 3% acetic acid solution to each well. DMSO was used as a blank instead of the sample, and the sample was mixed with Tris buffer only (pH 0.8) as a control to eliminate interference with the background color of the sample. After the reaction was terminated, the absorbance at 405nm was measured. DPPIV inhibition (%) [ a405 blank- (a405 sample-a 405 background) ]/a405 blank × 100%

3. See table 2 for experimental data:

TABLE 2 DPPIV enzyme IC for the compound methyl 6-beta-D-glucosyl-6, 7-dihydroxy-5-benzofuranpropionate₅₀，μmol/L

4. And (4) experimental conclusion:

the experiment shows that the furocoumarin compound 6-beta-D-glucosyl-6, 7-dihydroxy-5-benzofuranmethyl propionate has good inhibition effect on dipeptidyl peptidase IV (DPPIV), represents a new type of DPPIV enzyme inhibition active compound, is expected to be developed and used for preparing novel and effective DPPIV enzyme inhibitor drugs, and has wide application potential and good prospect.

The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.

Claims

1. The furocoumarin compound 6-beta-D-glucosyl-6, 7-dihydroxy-5-benzofuran methyl propionate or medicinal salt thereof is characterized in that the structure of the 6-beta-D-glucosyl-6, 7-dihydroxy-5-benzofuran methyl propionate is shown as a formula (I):

2. a method for preparing 6-beta-D-glucosyl-6, 7-dihydroxy-5-benzofuranpropionic acid methyl ester of furocoumarin compound as claimed in claim 1, wherein said 6-beta-D-glucosyl-6, 7-dihydroxy-5-benzofuranpropionic acid methyl ester is separated from Clausena lansium.

3. The preparation method according to claim 2, comprising the following steps:

a. preparing a total extract: homogenizing wampee with 95% ethanol water solution by volume, extracting, concentrating the extract under reduced pressure to remove organic solvent to obtain suspension, sequentially extracting the suspension with ethyl acetate and n-butanol, and concentrating n-butanol extract under reduced pressure to obtain total extract;

b. separation and purification: subjecting the total extract to normal phase silica gel column chromatography, performing gradient elution with chloroform/methanol from a volume ratio of 100:0 to 60:40, collecting a component F15 eluted with a chloroform/methanol volume ratio of 70:30, subjecting a component F15 to Sephadex LH-20 column chromatography, eluting with methanol, collecting an elution fraction F15-9 in an amount which is 1.2-1.6 times the volume of the column, and subjecting the fraction F15-9 to high performance liquid phase separation and purification to obtain 6-beta-D-glucosyl-6, 7-dihydroxy-5-benzofuranmethyl propionate.

4. The method according to claim 2 or 3, wherein the wampee comprises dried or fresh fruit, root, stem, branch or leaf of wampee.

5. The method of claim 4, wherein the wampee is wampee fruit, including dried or fresh products of the flesh and/or peel of the fruit.

6. The use of the furocoumarin compound methyl 6- β -D-glucosyl-6, 7-dihydroxy-5-benzofuranpropionate or its pharmaceutically acceptable salt as claimed in claim 1 in the preparation of DPPIV enzyme inhibitor medicament.

7. A DPPIV enzyme inhibitor drug comprising an effective amount of methyl 6- β -D-glucosyl-6, 7-dihydroxy-5-benzofuranpropionate according to claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

8. The DPPIV enzyme inhibitor medicament as claimed in claim 7, further comprising formulation or pharmaceutically acceptable excipients or carriers.