CN107805245B

CN107805245B - Method for purifying silybin

Info

Publication number: CN107805245B
Application number: CN201711337616.8A
Authority: CN
Inventors: 曹瑞; 任超; 何爽; 谭喜平
Original assignee: Hunan Qianjin Xieli Pharmaceutical Co ltd
Current assignee: Hunan Qianjin Xieli Pharmaceutical Co ltd
Priority date: 2017-12-14
Filing date: 2017-12-14
Publication date: 2020-11-03
Anticipated expiration: 2037-12-14
Also published as: CN107805245A

Abstract

The invention relates to a method for purifying silybin, which comprises the following steps: dissolving the silybin crude product in ethyl acetate to dissolve the silybin crude product; adding silica gel into the solution, and concentrating under reduced pressure to completely volatilize ethyl acetate in the solution; adding the sample into a silica gel column; gradient eluting with chloroform-methanol, and detecting with thin layer chromatography; and concentrating and drying the obtained eluent to obtain the product. The process of the invention has simple and feasible operation, mild conditions, low toxicity of the solvent for purification, and high product purity of over 99.5 percent, and overcomes the problems of low product purity and more impurities of the product obtained by the prior art.

Description

Method for purifying silybin

Technical Field

The invention belongs to the field of traditional Chinese medicine preparation, and particularly relates to a method for purifying silybin.

Background

Silibinin is a flavonoid extracted and separated from fruits of Silybum marianum (L.) Merr. Silibinin can stabilize liver cell membrane, maintain the integrity of liver cell, make toxin unable to penetrate and destroy liver, accelerate the synthesis of deoxyribonucleic acid (DNA) of liver cell, prevent liver cirrhosis, fatty liver, cholangitis, psoriasis and other diseases, and inhibit the growth and differentiation of liver cancer, prostatic cancer, breast cancer and cervical cancer cells, and is the flavonoid found in the world at present with the most curative effect on liver diseases. Silibinin has effects of protecting normal liver cell, and promoting recovery of damaged cell membrane. At present, the refining and purifying processes of silybin reported in the literature mainly comprise ethyl acetate-petroleum ether recrystallization, ethanol recrystallization, acetone-petroleum ether recrystallization and other methods, and the existing literature processes all have the defects of low product quality and many impurities.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the defects and shortcomings mentioned in the background technology and provide a method for purifying silibinin. The method dissolves the crude product of the silybin in the ethyl acetate firstly, and leads the purity of the purified product to reach more than 99.5 percent by a silica gel column method, thereby overcoming the problems of low purity and more impurities of the product obtained by the prior art.

In order to solve the technical problems, the technical scheme provided by the invention is as follows:

a method for purifying silybin comprises the following steps:

(1) dissolving the silybin crude product in ethyl acetate to dissolve the silybin crude product;

(2) adding silica gel into the solution, and concentrating under reduced pressure to completely volatilize ethyl acetate in the solution;

(3) adding the sample into a silica gel column;

(4) gradient eluting with chloroform-methanol, and detecting with thin layer chromatography;

(5) and concentrating and drying the obtained eluent to obtain the product.

In the method, preferably, the adding amount of the ethyl acetate in the step (1) is 5-15 times of the mass of the crude silybin product. The sample can be dissolved with a minimum of solvent.

In the method, preferably, the adding amount of the silica gel in the step (2) is 1-1.5 times of the mass of the crude silybin product. The excessive addition of silica gel can thin the sample; the addition of too little silica gel makes the sample inhomogeneous.

In the method, the mass of the silica gel in the silica gel column in the step (3) is preferably 10 to 15 times of the mass of the silica gel in the sample. The silica gel in the silica gel column is too small, so that the separation is impure; an excessive amount of silica gel may result in a low yield or a slow outflow rate.

In the above method, preferably, the concentration ratio of chloroform-methanol gradient elution in the step (4) is 8-12:1, 4-6:1 and 0.5-1.5: 1. The concentration ratios of 8-12:1, 4-6:1 and 0.5-1.5:1 are selected because the samples which flow out after the first impurity is separated at 10:1 are discovered to be silybin through experiments, the purity is high, but the flowing-out concentration is low, so the polarity is increased, and the samples can flow out rapidly.

In the above method, preferably, in the step (5), the sample concentration temperature is 40-50 ℃ and the drying temperature is 40-50 ℃. The configuration of the silibinin can be changed when the concentration temperature is too high, and the concentration time can be prolonged when the temperature is too low; the drying temperature is selected such that too high a temperature causes a change in the silibinin mechanism and too low a temperature causes a too long drying time.

Compared with the prior art, the invention has the advantages that:

1. the method has simple and feasible process operation and mild conditions, and the solvent for purification can be recycled;

2. the solvent ethyl acetate for purification has low toxicity and high solubility to samples;

3. the separation method of the silica gel column has the advantages of high sample separation speed and high sample purity;

4. the method selects chloroform-methanol for elution, and has the advantages of low toxicity, high sample separation speed and high purity;

5. the invention does not need decoloring and recrystallization and has high yield. When in decolorization, not only the pigment is adsorbed on the active carbon, but also the sample is adsorbed; the loss of the sample by recrystallization is larger;

6. the purity of the product reaches more than 99.5 percent, the product is white-like, and the problems of low purity and more impurities of the product obtained by the prior art are solved.

Detailed Description

In order to facilitate an understanding of the present invention, the present invention will be described more fully and in detail with reference to the preferred embodiments, but the scope of the present invention is not limited to the specific embodiments below.

Unless otherwise defined, all terms of art used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.

Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.

Example 1:

adding 0.5Kg of crude silybin into a reaction kettle, adding 3L of ethyl acetate to dissolve, and adding 0.5Kg of silica gel. Then, the mixture was concentrated under reduced pressure to evaporate ethyl acetate as completely as possible. Adding 5Kg of silica gel into a silica gel column, adding a sample into the silica gel column, eluting with chloroform-methanol at a ratio of 10:1, adjusting to 5:1 until the 1:1 is completely eluted, monitoring by TLC, concentrating the eluent under reduced pressure in a water bath at 45 ℃, and drying in a vacuum drying oven at 50 ℃ to obtain 0.42Kg of a product, wherein the yield is 84%, and the color of the product is off-white. The content was 99.9% by HPLC.

Example 2:

adding 0.5Kg of crude silybin into a reaction kettle, adding 3L of ethyl acetate to dissolve, and adding 0.5Kg of silica gel. Then, the mixture was concentrated under reduced pressure to evaporate ethyl acetate as completely as possible. Adding 5Kg of silica gel into a silica gel column, adding a sample into the silica gel column, eluting with chloroform-methanol at a ratio of 10:1, adjusting to 5:1 until the 1:1 is completely eluted, monitoring by TLC, concentrating the eluent under reduced pressure in a water bath at 45 ℃, and drying in a vacuum drying oven at 50 ℃ to obtain 0.41Kg of a product, wherein the yield is 82%, and the color of the product is off-white. The content was 99.8% by HPLC.

Example 3:

adding 0.5Kg of crude silybin into a reaction kettle, adding 3L of ethyl acetate to dissolve, and adding 0.5Kg of silica gel. Then, the mixture was concentrated under reduced pressure to evaporate ethyl acetate as completely as possible. Adding 5Kg of silica gel into a silica gel column, adding a sample into the silica gel column, eluting with chloroform-methanol at a ratio of 10:1, adjusting to 5:1 until the 1:1 is completely eluted, monitoring by TLC, concentrating the eluent under reduced pressure in a water bath at 45 ℃, and drying in a vacuum drying oven at 50 ℃ to obtain 0.41Kg of a product, wherein the yield is 82%, and the color of the product is off-white. The content was 99.9% by HPLC.

Comparative example 1:

adding 0.5Kg of crude silybin into a reaction kettle, adding 2L of 95% ethanol, heating to completely dissolve, cooling, stirring, crystallizing, cooling to room temperature, filtering, purifying and washing a small amount of 95% to obtain a white solid, drying in a vacuum drying oven at 50 ℃ to obtain 0.2Kg of product with the yield of 40%, and detecting by HPLC to obtain the product with the content of 98.7%.

The product yields and purity ratios for examples 1-3 and comparative examples are shown in table 1 below:

TABLE 1 comparison of product yield and purity of examples 1-3 with comparative examples

Group of	Yield of	HPLC purity
			Example 1	84％	99.9％
Example 2	82％	99.8％
			Example 3	82％	99.9％
Comparative example 1	40％	98.7％

As can be seen from Table 1, the purity of the product obtained by the purification method of the invention reaches more than 99.5%, and the product yield is also very high.

Claims

1. A method for purifying silybin is characterized by comprising the following steps:

(2) adding silica gel into the solution, and concentrating under reduced pressure to completely volatilize ethyl acetate in the solution; the adding mass of the silica gel is 1-1.5 times of the mass of the crude product of the silibinin;

(3) adding the sample into a silica gel column; the mass of the silica gel in the silica gel column is 10-15 times of that of the silica gel in the sample;

(4) gradient eluting with chloroform-methanol, and detecting with thin layer chromatography; the concentration ratio of chloroform-methanol gradient elution is 8-12:1, 4-6:1 and 0.5-1.5:1 respectively;

(5) and concentrating and drying the obtained eluent to obtain the product.

2. The purification method according to claim 1, wherein the amount of ethyl acetate added in step (1) is 5-15 times the mass of the crude silybin.

3. The purification method according to claim 1, wherein the sample concentration temperature in the step (5) is 40 to 50 ℃ and the drying temperature is 40 to 50 ℃.