CN112521434A

CN112521434A - High-efficiency preparation method of stilbene glucoside in polygonum multiflorum

Info

Publication number: CN112521434A
Application number: CN202011418907.1A
Authority: CN
Inventors: 孙蓉; 王岱杰; 刘闰平; 赵恒强; 崔莉; 吴恺怿; 黄娜娜; 邝江莹; 李晓骄阳
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2020-12-07
Filing date: 2020-12-07
Publication date: 2021-03-19

Abstract

The invention discloses a high-efficiency preparation method of stilbene glucoside in polygonum multiflorum, which comprises the following steps: pulverizing Polygoni Multiflori radix, extracting, concentrating to obtain total stilbene glucoside extract; separating and purifying the stilbene glucoside total extract by high-speed counter-current chromatography to obtain cis-stilbene glucoside and trans-stilbene glucoside monomers; the solvent system of the high-speed counter-current chromatography is ethyl acetate-water, 1:1, v/v.

Description

High-efficiency preparation method of stilbene glucoside in polygonum multiflorum

Technical Field

The invention relates to a high-efficiency preparation method of effective components of traditional Chinese medicines, in particular to a high-efficiency preparation method of stilbene glucoside in polygonum multiflorum.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Polygoni Multiflori radix is dried root tuber of Polygonum multiflorum Thunb of Polygonaceae, and has effects of removing toxic substance, resolving carbuncle, preventing malaria, and loosening bowel to relieve constipation, and can be used for treating skin ulcer, carbuncle, lymphoid tuberculosis, rubella pruritus, chronic malaria, asthenia, and constipation due to intestinal dryness. Stilbene glucoside is the main active substance in polygonum multiflorum, which is regulated in Chinese pharmacopoeia to be not less than 1 percent in raw polygonum multiflorum and not less than 0.7 percent in prepared polygonum multiflorum. The stilbene glucoside has cis-trans isomer, and the trans-stilbene glucoside in the polygonum multiflorum can be partially converted into a cis structure under the illumination condition, but the total content is unchanged. Under natural conditions, it exists mainly in the form of trans-stilbene glycoside. Pharmacological research shows that the effect of cis-stilbene glucoside on reducing blood fat of normal mice and acute hyperlipaemia mice is basically the same as that of trans-stilbene glucoside, but the effect of cis-stilbene glucoside on reducing total cholesterol and triglyceride in plasma of chronic hyperlipaemia mice is obviously stronger than that of trans-stilbene glucoside. However, the inventors found that the preparation method in the prior art is difficult to directly separate and prepare cis-stilbene glucoside from polygonum multiflorum due to the low content of cis-stilbene glucoside in polygonum multiflorum, and the yield is low even though some methods can separate and obtain the cis-stilbene glucoside.

High-speed countercurrent chromatography (HSCCC) is a continuous high-efficiency and rapid liquid-liquid distribution chromatography separation technology without any solid support developed in the last 30 years, and avoids various problems of easy dead adsorption, loss, denaturation and the like of a sample caused by a solid support or a carrier. The high-speed counter-current chromatography can directly carry out a large amount of crude extraction samples or synthesis mixtures, the separation result can reach quite high purity, and the method is widely applied to the preparation, separation and purification of chemical substances in the fields of biology, medicine, environmental protection and the like.

The existing solvent systems for preparing stilbene glucoside in polygonum multiflorum through high-speed countercurrent chromatography are ternary or quaternary systems, and the separated waste liquid is difficult to recycle, so that resource waste is easily caused, and the environment is polluted.

Disclosure of Invention

Aiming at the technical problems in the prior art, the invention provides the high-efficiency preparation method of stilbene glucoside in polygonum multiflorum, which can smoothly separate cis-stilbene glucoside and trans-stilbene glucoside and can prepare high-purity cis-stilbene glucoside and trans-stilbene glucoside.

To solve the above technical problem, one or more of the following embodiments of the present invention provide the following technical solutions:

a high-efficiency preparation method of stilbene glucoside in Polygonum multiflorum comprises the following steps:

pulverizing Polygoni Multiflori radix, extracting, concentrating to obtain total stilbene glucoside extract;

separating and purifying the stilbene glucoside total extract by high-speed counter-current chromatography to obtain cis-stilbene glucoside and trans-stilbene glucoside monomers;

the solvent system of the high-speed counter-current chromatography is ethyl acetate-water, 1:1, v/v.

Compared with the prior art, one or more technical schemes of the invention have the following beneficial effects:

the binary system of ethyl acetate/water is adopted for separation, the separation solvent is simple, the ethyl acetate in the separated waste liquid can be recycled, and the method is green, environment-friendly and resource-saving.

The solvent system of the high-speed counter-current chromatography is adopted to separate and purify the stilbene glucoside total extract, so that cis-stilbene glucoside and trans-stilbene glucoside can be successfully separated, high-purity cis-stilbene glucoside and trans-stilbene glucoside can be prepared, and the yield of the cis-stilbene glucoside can be effectively improved.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a process flow diagram of an embodiment of the invention;

FIG. 2 is a schematic diagram of high-speed countercurrent chromatographic separation of crude stilbene glucoside extract in the embodiment of the present invention;

FIG. 3 is a high performance liquid chromatogram of a crude stilbene glycoside extract and cis-stilbene glycoside and trans-stilbene glycoside monomers in an embodiment of the invention.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. 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 invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

In some embodiments, the total extract is obtained by reflux extraction of pulverized polygonum multiflorum with 65% to 75% ethanol (% by volume).

Further, the concentration of ethanol was 70%.

Further, ultrasonic extraction is carried out by adopting ethanol solution.

Further, ultrasonic extracting with ethanol solution for 2-3 times, each for 20-40 min.

In some embodiments, the method of extraction is: firstly, petroleum ether is adopted for extraction, the pH value of the extracted water phase is adjusted to 9-11, and then ethyl acetate is used for extraction.

Further, the pH value of the extracted water phase is adjusted to 10, and then the water phase is extracted by ethyl acetate.

Further, the method also comprises the steps of combining, concentrating and drying the total extract.

In some embodiments, the method further comprises the step of performing purity detection on the prepared cis-stilbene glucoside and trans-stilbene glucoside monomers, and the high performance liquid chromatography adopts Waters C₁₈Chromatography column (250 mm. times.4.6 mm, 5 μm) at a flow rate of 1.0 mL/min; the column temperature is 25 ℃; detection wavelength: 280nm, and the sample injection amount is 10 mu L. Mobile phase: acetonitrile-B water (25:75, v/v), isocratic elution.

Examples

1. Sample extraction

500g of polygonum multiflorum medicinal material is crushed, ultrasonically extracted for 2 times by 70 percent ethanol, each time lasts for 30 minutes, filtered, combined filtrate and concentrated under reduced pressure until no alcohol smell exists. Extracting the concentrated solution with isovolumetric petroleum ether for 3 times, adjusting pH of the aqueous phase to 10 with ammonia water, extracting with isovolumetric ethyl acetate for 3 times, concentrating the ethyl acetate phase under reduced pressure to obtain 5.2g of total crude extract of stilbene glucoside, and refrigerating in refrigerator for storage.

2. Solvent system screening

This example provides K at different solvent compositions_DThe value is obtained.

TABLE 1K of the target Compounds of different solvent systems_DValue of

The selection of a suitable two-phase solvent system for countercurrent chromatographic separation is a very critical step, and a suitable two-phase solvent system can successfully complete the separation experiment. In this study, three biphasic mixed solvent systems comprising two, or three solvent mixtures were tested in different ratios. A good solvent system can provide a desired partition coefficient (K) for the target compound in the mixture_D) The value is obtained. K_DThe values describe the ratio of solute distribution between two-phase solvent systems in equilibrium with each other. As shown in Table 1, the K of adenosine was determined in a biphasic ethyl acetate/n-butanol/water (2:2:3, v/v), n-butanol/water (1:1, v/v) solvent system_DAll values are greater than 5.0 and are therefore unsuitable for use in countercurrent chromatographic separations. Finally, ethyl acetate/water (1:1, v/v) is most suitable.

3. High-speed countercurrent chromatography separation and purification

In the high-speed countercurrent chromatography, the solvent system is ethyl acetate/water (1:1, v/v), the upper phase solution is selected as the stationary phase, the lower phase solution is selected as the mobile phase, and ultrasonic degassing is carried out. The stationary phase was pumped into a separation column of countercurrent chromatography at a flow rate of 20 mL/min. The rotation speed is adjusted to 800rpm clockwise, the temperature is controlled to 25 ℃, and the mobile phase starts to balance at the flow rate of 2.0mL/min in a head-to-tail mode. After the fluid dynamics balance is achieved, sample solution (500mg of crude stilbene glucoside extract is dissolved in 5mL of upper phase and 5mL of lower phase) is injected into a sample loop manually, the absorbance of an ultraviolet detector is set to be 280nm, a recorder is started, separated components are collected after sample injection, a spectrogram is collected, and elution fractions are collected according to a tube. And after the separation process is finished, closing the instrument, ejecting the components remained in the countercurrent chromatographic separation column by using a vacuum pump, and obtaining the retention rate of the stationary phase according to the proportion of the volume of the stationary phase in the tail blowing to the total volume. And carrying out subsequent liquid phase detection by reduced pressure concentration.

High performance liquid chromatography using Waters C₁₈Chromatography column (250 mm. times.4.6 mm, 5 μm) at a flow rate of 1.0 mL/min; the column temperature is 25 ℃; detection wavelength: 280nm, and the sample injection amount is 10 mu L. Mobile phase: acetonitrile-B water (25:75, v/v), isocratic elution.

Finally, cis-stilbene glycoside (50mg, yield 92%, purity 98.5%) and trans-stilbene glycoside (328mg, yield 94%, purity 99%) were isolated.

4. Structural identification

And performing structural characterization by methods such as ultraviolet spectroscopy, infrared spectroscopy, mass spectrometry, nuclear magnetic resonance technology and the like. The testing solvent of the UV method is methanol, and the scanning range is 200-400 nm; the MS analysis adopts an electrospray ion source, the temperature of carrier gas is 300 ℃, the flow rate of the carrier gas is 10L/min, the carrier gas is common nitrogen, the voltage of a capillary tube is 4.0kV, the scanning range is m/z 100-1000, and the deuterated reagent is DMSO.

Ultraviolet spectrum analysis is carried out on the prepared cis-stilbene glucoside sample to obtain

I.e. the maximum absorption wavelength is 284 nm. Mass spectrometric analysis was performed, M/z 405.11827[ M-H]^-I.e., a molecular weight of 406.11827. Nuclear magnetic resonance analysis¹In the H-NMR spectrum, the chemical shifts delta 8.35(1H, s) and 8.14(1H, s) show proton signals at the 2-position and 8-position, and the chemical shift delta 7.35(2H, s) shows an amino proton signal.¹³In the C-NMR spectrum, the chemical shifts delta: 156.6 show a 6-carbon signal, delta: 152.9 shows a 2-carbon signal, and the chemical shifts delta: 88.4, 86.4, 73.9, 71.1 and 62.2 indicate that the compound contains a five-carbon sugar. The data of the compound are consistent with the comparison result of the literature, and the compound is identified as cis-stilbene glucoside.

Ultraviolet spectrum analysis is carried out on the prepared trans-stilbene glucoside sample to obtain

I.e. a maximum absorption wavelength of 320 nm. Mass spectrometric analysis was performed, M/z 405.11848[ M-H]^-I.e., a molecular weight of 406.11848. Nuclear magnetic resonance analysis¹Chemical shift δ in H-NMR spectrum8.35(1H, s) and 8.14(1H, s) show proton signals at the 2-position and 8-position, and chemical shifts delta: 7.35(2H, s) show amino proton signals.¹³In the C-NMR spectrum, the chemical shifts delta: 156.6 show a 6-carbon signal, delta: 152.9 shows a 2-carbon signal, and the chemical shifts delta: 88.4, 86.4, 73.9, 71.1 and 62.2 indicate that the compound contains a five-carbon sugar. The data of the compound are consistent with the comparison result of the literature, and the compound is identified as trans-stilbene glucoside.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. an efficient preparation method of stilbene glycosides in Polygonum multiflorum, is characterized in that: comprise the steps:

After crushing, extracting, extracting and concentrating Polygonum multiflorum, the total extract of stilbene glycosides is obtained;

The total extract of stilbene glycosides is separated and purified by high-speed countercurrent chromatography to obtain cis-stilbene glycosides and trans-stilbene glycosides monomers;

The solvent system for high-speed countercurrent chromatography was ethyl acetate-water, 1:1, v/v.

2. the efficient preparation method of stilbene glycosides in Polygonum multiflorum according to claim 1, is characterized in that: adopt the ethanol of 65%-75%, % is volume fraction, carry out reflux extraction to the Polygonum multiflorum after pulverization, obtain total extraction liquid.

3. the efficient preparation method of stilbene glycosides in Polygonum multiflorum according to claim 2, is characterized in that: the concentration of ethanol is 70%.

4. the efficient preparation method of stilbene glycosides in Polygonum multiflorum according to claim 3, is characterized in that: adopts ethanol solution ultrasonic extraction.

5. the efficient preparation method of stilbene glycosides in Polygonum multiflorum according to claim 4, is characterized in that: adopt ethanol solution ultrasonic extraction 2-3 times, extract 20-40min each time.

6. the efficient preparation method of stilbene glycosides in Polygonum multiflorum according to claim 5, is characterized in that: the method for described extraction is: at first adopt petroleum ether extraction, the water phase after extraction adjusts pH value to 9-11, It was then extracted with ethyl acetate.

7. the efficient preparation method of stilbene glycosides in Polygonum multiflorum according to claim 6, is characterized in that: the number of times that adopts equal volume ethyl acetate extraction is 2-4 times.

8. the efficient preparation method of stilbene glycosides in Polygonum multiflorum according to claim 6, is characterized in that: the aqueous phase after extraction is adjusted to pH value to 10, and then extracted with ethyl acetate.

9 . The efficient preparation method of stilbene glycosides in Polygonum multiflorum according to claim 8 , wherein the method further comprises the steps of combining, concentrating and drying the total extracts. 10 .

10. the efficient preparation method of stilbene glycosides in Polygonum multiflorum according to claim 1, is characterized in that: also comprises the step of carrying out purity detection to the prepared cis-stilbene glycosides and trans-stilbene glycoside monomers, High performance liquid chromatography adopts Waters C ₁₈ column, 250mm×4.6mm, 5μm, flow rate 1.0mL/min; column temperature 25℃; detection wavelength: 280nm, injection volume 10μL, mobile phase: A acetonitrile-B water, 25: 75, v/v, isocratic elution.