CN107098838B

CN107098838B - Preparation method of garlic ajoene reference substance

Info

Publication number: CN107098838B
Application number: CN201710416556.2A
Authority: CN
Inventors: 黄伟昌
Original assignee: Shenzhen Ahuoxi Biotechnology Co ltd
Current assignee: Shenzhen Ahuoxi Biotechnology Co ltd
Priority date: 2017-06-06
Filing date: 2017-06-06
Publication date: 2020-07-10
Anticipated expiration: 2037-06-06
Also published as: CN107098838A

Abstract

The invention relates to a preparation method of a garlic ajoene reference substance, which comprises the following steps: (1) pulverizing Bulbus Allii, adding solvent, homogenizing under high pressure, and filtering to obtain extractive solution; (2) extracting the extract with solvent, standing for layering, and collecting the upper layer to obtain extract; (3) concentrating the extract under reduced pressure, performing silica gel column chromatography, and eluting with solvent to obtain eluate; (4) concentrating the eluate under reduced pressure, and separating and purifying by preparing liquid phase to obtain eluate rich in ajoene; (5) concentrating the eluate under reduced pressure to remove solvent to obtain ajoene reference substance. Finally, all indexes of four spectra and purity of the ajoene reference substance meet the national standard. The process is simple, convenient and stable, can be used for preparing the garlic ajoene reference substance, and provides technical support for the deep research of garlic ajoene.

Description

Preparation method of garlic ajoene reference substance

Technical Field

The invention relates to the technical field of separation and purification, in particular to a preparation method of an ajoene reference substance in garlic.

Background

Garlic generally contains about 1% alliin, and the alliin content in garlic increases during low-temperature storage. Alliinase (Alliinase) rapidly decomposes alliin into thiosulfinate under cutting, crushing, chewing and the like conditions, wherein the main compound is Allicin (Allicin). Allicin and other thiosulfinates are very unstable at room temperature and decompose into the sulfide form, vinyldithiines, ajoenes, for several hours.

Through searching and discovering the prior art documents and published patents, the Chinese patent publication No. CN103525878A, named as a preparation method of a composition rich in ajoene, discloses the following technical scheme: alliin dissolved in buffer solution reacts under the catalysis of alliinase, and natural alliin extracted from garlic is converted into diallyl thiosulfinate to obtain reaction liquid containing the diallyl thiosulfinate; concentrating the reaction solution by a membrane, extracting and enriching diallyl thiosulfinate by using an organic solvent, and then recovering the solvent and extracting for multiple times to obtain the composition with the ajoene content of more than 80%. The technical scheme uses a plurality of organic solvents, and the process is complex, so that the product yield is low. Chinese patent publication No. CN104974068A, entitled "A preparation method of ajoene", discloses the following technical scheme: crushing garlic, adding an extracting solution, mixing to obtain a product A, extracting the product A, then filtering and/or centrifuging, taking the obtained solution as a product B, removing a solvent in the product B, then adding vegetable oil, mixing to obtain a product C, adding the product C into an adsorbent, mixing to obtain a product D, eluting the product D with a solution containing an eluent to obtain a product E, and removing the solvent from the product E to obtain an ajoene product with the concentration of 89.76. However, the method has the problems of large using amount of vegetable oil, complex process and relatively low ajoene purity.

So far, the national food and drug administration and domestic companies have not provided a reference substance of garlic ajoene, and the technology is kept secret at home and abroad. Therefore, the preparation of the ajoene reference substance from the garlic has important significance for the deep development of the garlic.

Disclosure of Invention

The invention aims to provide a preparation method of a garlic ajoene reference substance, which aims to solve the problems of complex process, low ajoene purity and the like caused by excessive organic solvent or vegetable oil used in the process of extracting ajoene from garlic in the prior art.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for preparing a garlic ajoene reference substance, which comprises the following steps:

(1) pulverizing Bulbus Allii, adding first solvent, homogenizing under high pressure, and filtering to obtain extractive solution;

(2) adding a second solvent into the extracting solution for extraction, standing for layering, and taking the upper layer to obtain an extracting solution;

(3) concentrating the extract under reduced pressure, performing silica gel column chromatography, and eluting with a third solvent to obtain a first eluent;

(4) after the first eluent is decompressed and concentrated, the second eluent rich in ajoene is obtained by preparing liquid phase separation and purification;

(5) and (4) concentrating the second eluent rich in ajoene obtained in the step (4) under reduced pressure to remove the solvent, so as to obtain an ajoene reference substance.

Further, in the step (1), the garlic is crushed into garlic granules with the diameter of 50-300 μm. Preferably 100 μm.

Further, in the step (1), the conditions of the high-pressure homogeneous extraction are as follows: the first solvent is purified water, and the material-liquid ratio is 1: 2-1: 10, preferably 1: 5; the pressure is 20-80 MPa, preferably 50 MPa; extracting for 0.5-2 h, preferably 1 h; extracting for 1 time.

Further, in the step (2), the second solvent is one or more of ethyl acetate, n-hexane, isopropanol, n-butanol and chloroform, and preferably ethyl acetate.

Further, in the step (2), the extraction times are 1-3 times, preferably 2 times; the dosage is as follows: the garlic quality is as follows: the volume of the solvent is 1: 0.5-2, preferably 1: 1.

Further, in the step (3), silica gel with 200-300 meshes is selected for silica gel column chromatography.

Further, in the step (3), the third solvent eluted by column chromatography is a mixed solvent of ethyl acetate and petroleum ether.

Further, in the step (3), the chromatography elution flow rate is 1-5 BV/h, preferably 2 BV/h; according to the volume ratio, firstly, mixing ethyl acetate: eluting with a mixed solvent of 20:80 petroleum ether to remove impurities, and then eluting with ethyl acetate: eluting with a mixed solvent of petroleum ether 30: 70-70: 30, preferably ethyl acetate: eluting with 50:50 mixed solvent and collecting the eluent.

Further, in the step (4), the prepared liquid phase separation filler is 10-100 mu m reversed phase C₁₈Preferably 50 μm.

Further, in the step (4), the prepared liquid phase mobile phase is one or more of ethanol, methanol and acetonitrile; the volume fraction is 45-75%, preferably 60%; the flow rate is 5ml/min to 25ml/min, preferably 15 ml/min.

The invention has the advantages that:

1. crushing garlic, adding purified water, homogenizing under high pressure to disperse ajoene in water, and contacting alliin with allinase to convert alliin into ajoene;

2. the ajoene in the garlic water solution is quickly enriched into the extraction solvent through solvent extraction, water-soluble impurities are removed, and the enrichment and purification effects are achieved;

3. the extraction solvent is further purified by silica gel column chromatography after being concentrated, and the silica gel has better separation and purification effects on fat-soluble components. Firstly, removing part of impurities by using a lower proportion of ethyl acetate solvent, and then eluting ajoene by using a high proportion of ethyl acetate solvent;

4. and finally, preparing liquid phase purified ajoene, wherein the prepared liquid phase can be selectively separated and purified.

The ajoene reference substance can be quickly prepared by solvent extraction, silica gel column chromatography and preparation of a liquid phase, the process is simple, the production period is short, and the method can be used for preparing the garlic ajoene reference substance and provides technical support for development of the garlic ajoene.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the principles of the invention:

FIG. 1 is a liquid phase separation purification chromatogram prepared in step (4) of example 1;

FIG. 2 is an Ahodene Infrared Spectroscopy (IR) chart for example 1;

FIG. 3 is a positive ion diagram of the ajoene mass spectrum of example 1;

FIG. 4 is a graph of ajoene in example 1¹H-NMR chart (500 MHz);

FIG. 5 is a graph of ajoene in example 1¹³C-NMR chart (500 MHz);

FIG. 6 is a high performance liquid chromatogram of ajoene in example 1 (HP L C).

Detailed Description

The present invention will be described in detail with reference to the drawings and specific embodiments, which are illustrative of the present invention and are not to be construed as limiting the present invention.

Example 1

1. Peeling 1Kg of Bulbus Allii, pulverizing to 100 μm, adding 5L purified water, homogenizing under 50MPa for 1 hr, extracting for 1 time, and filtering to obtain extractive solution;

2. adding ethyl acetate into the extracting solution, extracting for 2 times, each time 1L, standing for layering, taking an upper layer, combining upper layer liquid to obtain an extracting solution, and concentrating the extracting solution under reduced pressure at 45-65 ℃ to obtain an extract;

3. and (3) taking 100g of 200-300-mesh silica gel, filling the silica gel into a glass column for chromatography, and loading the extract dry powder into a sample. By volume ratio, using 2BV ethyl acetate: eluting with mixed solvent of 20:80 petroleum ether at flow rate of 2BV/h to remove impurities, and eluting with 2BV ethyl acetate: the ajoene is eluted by a mixed solvent of 50:50 petroleum ether at the flow rate of 2 BV/h;

4. concentrating the eluent under reduced pressure at 45-65 ℃, and adding methanol for dissolving. Take 50 μm C₁₈Preparing a column, separating and preparing ajoene by using methanol with the volume fraction of 60% at a speed of 15ml/min, and collecting ajoene chromatographic peak eluent;

5. and (3) concentrating the ajoene chromatographic peak eluent at 55-75 ℃ under reduced pressure to remove the solvent to obtain an ajoene reference substance.

The purity of the ajoene reference substance is detected by four spectra and high performance liquid chromatography, the ajoene purity is 99.5 percent, and the ajoene reference substance meets the national standard.

Table 1 shows the results of the infrared spectroscopy (IR) analysis of ajoene in example 1;

TABLE 1 Infrared Spectroscopy analysis

Example 2

1. Peeling 1Kg of single garlic, pulverizing to 50 μm, adding 2L purified water, homogenizing under 80MPa for 0.5 hr, extracting for 1 time, and filtering to obtain extractive solution;

2. extracting the extract with 2L n-butanol for 1 time, standing for layering, collecting the upper layer to obtain extract, and concentrating the extract at 45-65 deg.C under reduced pressure to obtain extract;

3. and (3) taking 100g of 200-300-mesh silica gel, filling the silica gel into a glass column for chromatography, and loading the extract dry powder into a sample. By volume ratio, using 2BV ethyl acetate: eluting with mixed solvent of 20:80 petroleum ether at flow rate of 2BV/h to remove impurities, and eluting with 2BV ethyl acetate: the ajoene is eluted by a mixed solvent of petroleum ether 30:70 at the flow rate of 2 BV/h;

4. eluentConcentrating under reduced pressure at 45-65 ℃, and adding methanol for dissolving. Take 100 mu m C₁₈Preparing a column, separating and preparing ajoene by using methanol with the volume fraction of 45% at the flow rate of 25ml/min, and collecting ajoene chromatographic peak eluent;

The ajoene reference substance is detected by high performance liquid chromatography, and the ajoene purity is 99.1%.

Example 3

1. Peeling 1Kg of Bulbus Allii, pulverizing to 300 μm, adding 2L purified water, homogenizing under 20MPa for 2 hr, extracting for 1 time, and filtering to obtain extractive solution;

2. extracting the extractive solution with chloroform for 3 times (500m L each time), standing for layering, collecting the upper layer, mixing the upper layer solutions to obtain extractive solution, and concentrating under reduced pressure at 45-65 deg.C to obtain extract;

3. and (3) taking 100g of 200-300-mesh silica gel, filling the silica gel into a glass column for chromatography, and loading the extract dry powder into a sample. By volume ratio, using 2BV ethyl acetate: eluting with mixed solvent of 20:80 petroleum ether at flow rate of 2BV/h to remove impurities, and eluting with 2BV ethyl acetate: the ajoene is eluted by a mixed solvent of 70:30 petroleum ether at the flow rate of 2 BV/h;

4. concentrating the eluent under reduced pressure at 45-65 ℃, and adding methanol for dissolving. Take 10. mu. m C₁₈Preparing a column, separating and preparing ajoene by using 75% methanol by volume fraction at 5ml/min, and collecting ajoene chromatographic peak eluent;

The ajoene reference substance is detected by high performance liquid chromatography, and the ajoene purity is 99.3%.

The technical solutions provided by the embodiments of the present invention are described in detail above, and the principles and embodiments of the present invention are explained herein by using specific examples, and the descriptions of the embodiments are only used to help understanding the principles of the embodiments of the present invention; meanwhile, for a person skilled in the art, according to the embodiments of the present invention, there may be variations in the specific implementation manners and application ranges, and in summary, the content of the present description should not be construed as a limitation to the present invention.

Claims

1. A preparation method of a garlic ajoene reference substance is characterized by comprising the following steps: the method comprises the following steps:

(1) crushing garlic into garlic granules with the particle size of 50-300 mu m, adding a first solvent, carrying out high-pressure homogeneous extraction, and filtering to obtain an extracting solution;

the conditions of high-pressure homogeneous extraction are as follows: the first solvent is purified water, the material-liquid ratio is 1: 2-1: 10, the pressure is 20-80 MPa, the extraction is carried out for 0.5-2 h, and the extraction is carried out for 1 time;

(3) concentrating the extract under reduced pressure, performing silica gel column chromatography, selecting 200-300 mesh silica gel for the silica gel column chromatography, eluting with a third solvent to obtain a first eluent, wherein the third solvent for the column chromatography elution is a mixed solvent of ethyl acetate and petroleum ether, and the flow rate of the chromatography elution is 1-5 BV/h; according to the volume ratio, firstly, mixing ethyl acetate: eluting with a mixed solvent of 20:80 petroleum ether to remove impurities, and then eluting with ethyl acetate: eluting with a mixed solvent of petroleum ether 30: 70-70: 30, and collecting the eluate;

the prepared liquid phase separation filler is 10-100 mu m reversed phase C₁₈；

The prepared liquid phase mobile phase is one or more of ethanol, methanol and acetonitrile, and the volume fraction is 45-75%; the flow rate is 5ml/min to 25 ml/min;

2. The method for preparing a garlic ajoene control substance according to claim 1, wherein the method comprises the following steps: in the step (2), the second solvent is one or more of ethyl acetate, n-hexane, isopropanol, n-butanol and chloroform.

3. The method for preparing a garlic ajoene control substance according to claim 1, wherein the method comprises the following steps: in the step (2), the extraction times are 1-3; the dosage is as follows: the garlic quality is as follows: the volume of the solvent is 1: 0.5-2.