CN111116329B

CN111116329B - Preparation method and application of isolariciresinol

Info

Publication number: CN111116329B
Application number: CN201911266989.XA
Authority: CN
Inventors: 陈光英; 娜佳.尼达尔.优素福.马友布; 周学明; 易继凌; 张斌
Original assignee: Hainan Normal University
Current assignee: Hainan Normal University
Priority date: 2019-12-11
Filing date: 2019-12-11
Publication date: 2022-07-19
Anticipated expiration: 2039-12-11
Also published as: CN111116329A

Abstract

The invention discloses a preparation method and application of isolariciresinol, which is extracted from beehive grass, and the beehive grass serving as a raw material has wide sources. The invention discloses a new application of isolariciresinol in the aspects of reducing blood sugar and resisting oxidation, and in-vitro experiments show that the isolariciresinol has better alpha-glycosidase inhibitory activity and anti-free radical activity.

Description

Preparation method and application of isolariciresinol

Technical Field

The invention relates to a preparation method and application of isolariciresinol.

Background

Crepe wheatgrass (academic name:Leucas zeylanica) Is a kind of perennial herb belonging to the genus Saposhnikovia of Labiatae, and is divided into Guangdong, Guangxi, Yunnan, etc.; growing in the sunny open places such as sand and loam coastal areas, fields, roadside areas, gentle slope areas and the like. The herba Melissae axillaris is whole plant of Trifolium pratense, and can be used for treating common cold cough, toothache, gastrointestinal discomfort, pertussis, etc. Through preliminary phytochemical investigation on the beehive grass, the beehive grass contains alkaloid, steroid, tannin, flavonoid, glycoside and other components.

The isolariciresinol is a lignan compound, is used as a standard substance and a reference substance, and has no report about other purposes.

Disclosure of Invention

The invention aims to solve the technical problem of providing a preparation method and application of the isolariciresinol diglucoside, providing a new path for extracting the compound, and the compound has obvious application effects on reducing blood sugar and resisting oxidation.

The first technical scheme for realizing the first purpose of the invention is the application of the isolariciresinol in preparing the hypoglycemic medicament.

The second technical scheme for realizing the first purpose of the invention is the application of the isolariciresinol in preparing the antioxidant drugs.

The technical scheme for realizing the second purpose of the invention is a preparation method of the isolariciresinol, comprising the following steps:

soaking and extracting dry beehive grass with ethanol, combining extracting solutions, and distilling under reduced pressure to obtain an ethanol total extract; and uniformly dispersing the ethanol total extract in distilled water, and sequentially extracting with petroleum ether and ethyl acetate to respectively obtain a petroleum ether part extract and an ethyl acetate part extract.

And (2) subjecting the ethyl acetate part extract obtained in the step (I) to silica gel column chromatography, eluting by adopting a petroleum ether-ethyl acetate solvent system according to a gradient of 100: 1-0: 100 (v/v), collecting fractions per 200 mL, detecting by using TLC (thin layer chromatography), and combining the same fractions to obtain 6 components Fr.1-Fr.6.

And thirdly, carrying out Sephadex LH-20 Sephadex column chromatography (solvent system is chloroform: methanol = 1: 1, V/V) on the fourth large-section component Fr.4 obtained in the step II to separate, and obtaining four sub-components Fr.4-1, Fr.4-2, Fr.4-3 and Fr.4-4.

And fourthly, separating the Fr.4-3 component obtained in the third step by using high performance liquid chromatography and mobile phase acetonitrile-water (35: 75, v/v) to obtain the isolariciresinol diglucoside.

Soaking and extracting with ethanol at room temperature (15-35 ℃), extracting for 2-4 times, and extracting for 2.5-4 days each time.

The invention has the positive effects that:

the invention provides a new source of the isolariciresinol, which can be extracted from beehive grass with wide sources.

The invention discloses a new application of isolariciresinol in the aspects of reducing blood sugar and resisting oxidation, and in-vitro experiments show that the isolariciresinol has better alpha-glycosidase inhibitory activity and anti-free radical activity.

Detailed Description

Example 1 preparation of Isolariciresinol

The preparation method of the isolariciresinol diglucoside of the embodiment comprises the following steps:

soaking and extracting 4kg of dried herb beehive grass with 75% ethanol at room temperature (15-35 ℃) for 2-4 times (3 times in the embodiment), wherein 2.5-4 days (3 days in the embodiment) are extracted each time, and the amount of ethanol used in each extraction is 30L. Filtering after each extraction to obtain extract, mixing the extracts, and distilling under reduced pressure to obtain 351g of ethanol total extract. 351g of ethanol total extract is uniformly dispersed in 1L of distilled water, petroleum ether is firstly used for extraction for 3 times (3 multiplied by 3.5L) to obtain petroleum ether part extract, then ethyl acetate is used for extraction for 3 times (3 multiplied by 4.0L), the color of the upper layer solution is obviously lightened, and ethyl acetate part extract is obtained.

The honeycomb grass used in the embodiment is collected from Haikou city of Hainan province, and the specimens are stored in key laboratories of the chemical education department of tropical medicinal resources of the university of Hainan teachers and universities.

And (2) performing silica gel column chromatography on 90g of ethyl acetate part extract obtained in the step (I), eluting by adopting a petroleum ether-ethyl acetate solvent system according to the gradient of 100: 1-0: 100 (v/v), collecting fractions per 200 mL, detecting by TLC (thin layer chromatography), and combining the same fractions to obtain 6 components (Fr.1-Fr.6).

And fourthly, separating the Fr.4-3 component in the third step by using high performance liquid chromatography and mobile phase acetonitrile-water (35: 75, v/v) to obtain 5.8mg of target extract.

Analysis of target extract product:

process for preparing extract¹H and¹³the C-NMR data are as follows (Brooks (Beijing) science and technology Ltd, model AV-III 400 MHz nuclear magnetic resonance apparatus, TMS internal standard,¹H 400 Hz ，¹³C 100 Hz）：

and (4) comparing and confirming that the extract obtained in the step (iv) is the isolariciresinol diglucoside.

Test example 1 evaluation of alpha-glucosidase inhibitory Activity of Compound

The α -glucosidase inhibitory activity was performed in 96-well plates using the PNPG method.

1.1 preparation of reaction solution.

Preparing an enzyme solution of alpha-glucosidase: dissolving the freeze-dried enzyme powder with 0.01M PBS buffer solution, wherein the PBS buffer solution contains 0.2 percent of bovine serum albumin, and obtaining 0.2U/mL enzyme solution of alpha-glucosidase for later use.

Test sample solution: preparing the isolarch resin solution with the concentration range of 10 mu M to 1000 mu M for standby, wherein the solvent is 50 percent methanol water solution.

1.2 measurement of alpha-glucosidase inhibitory activity.

Accurately transferring 20 mu L of 0.1 mol.L^-1Adding 20 mu L of test sample into a 96-well plate, respectively, uniformly mixing, reacting at 37 ℃ for 5min, adding 20 mu L of 2.5mmol/L PNPG solution into each well of the 96-well plate, reacting at 37 ℃ for 15 min, and adding 0.2 mol/L Na₂CO₃The reaction was stopped with 80. mu.L of the solution, and the absorbance was measured at a wavelength of 405 nm.

Acarbose was dissolved in double distilled water, filtered through filter paper, and diluted with methanol to various concentrations as a positive control.

Calculating the inhibition rate and IC of alpha-glucosidase according to the formula₅₀The value is obtained.

Inhibition (%) = [ (AS-ASB)/(AC-ACB) ] × 100%.

Alpha-glucosidase inhibition IC of the isolariciresinol is measured₅₀The value was 2.76 mM.

Alpha-glucosidase inhibition IC of acarbose as control₅₀The value was 5.17 mM.

(test example 2, detection of ABTS radical scavenging Activity)

A stock solution of ABTS was prepared by adding 1.7mL of 140mmol/L potassium persulfate solution to 100mL of 7mmol/L ABTS solution and reacting at 25 ℃ in the dark for 12 hours. The ABTS stock solution was diluted with phosphate buffer (0.05 mol/L, pH 7.4) just before use, and the absorbance at 734 nm was adjusted to 0.70. + -. 0.02 to prepare an ABTS working solution.

Preparing 5-60 μ g/mL solution of iso-lariciresinol with methanol, adding 150 μ L iso-lariciresinol solution into 2.85mL ABTS working solution, mixing, reacting at 25 deg.C in dark environment for 10min, and detecting absorbance A with ethanol at 734 nm as blank_sample. The absorbance A of a negative control was determined_controlThe clearance is then calculated according to the formula.

ABTS free radical clearance = [ (A)_control-A _sample）/A_control]× 100%。

Determination of ABTS free radical scavenging IC of isolariciresinol₅₀The value is 0.1712.

ABTS free radical scavenging IC of Trolox₅₀The value is 0.4712.

Claims

1. A preparation method of isolariciresinol is characterized by comprising the following steps:

soaking and extracting dry beehive grass with ethanol, combining extracting solutions, and distilling under reduced pressure to obtain an ethanol total extract; uniformly dispersing the ethanol total extract in distilled water, and sequentially extracting with petroleum ether and ethyl acetate to obtain a petroleum ether part extract and an ethyl acetate part extract respectively;

secondly, subjecting the ethyl acetate part extract obtained in the step I to silica gel column chromatography, adopting a petroleum ether-ethyl acetate solvent system, eluting according to the gradient of 100: 1-0: 100 (v/v), collecting fractions per 200 mL, detecting by TLC, and combining the same fractions to obtain 6 components Fr.1-Fr.6;

thirdly, carrying out Sephadex LH-20 Sephadex column chromatography separation on the fourth large-section component Fr.4 obtained in the second step to obtain four sub-components Fr.4-1, Fr.4-2, Fr.4-3 and Fr.4-4; performing Sephadex LH-20 Sephadex column chromatography with chloroform and methanol at volume ratio of 1: 1;

fourthly, the Fr.4-3 component in the third step is separated by high performance liquid chromatography and mobile phase acetonitrile-water to obtain the isolariciresinol diglucoside; the volume ratio of acetonitrile to water in the mobile phase was 35: 75.

2. The method for producing isolariciresinol according to claim 1, characterized in that: soaking and extracting with ethanol at room temperature for 2-4 times, wherein each extraction lasts for 2.5-4 days.