CN100415766C - A method for purifying glycyrrhetin - Google Patents

Abstract

The invention discloses a method for separating and preparing high-purity glycyrrhetinic glycoside. The exchange column is filled with domestic nonpolar macroporous adsorption resin. The biological conversion solution of glycyrrhizic acid after centrifugation or filtration is used as crude product solution of the sample, and the crude product solution passes through an exchange column and is dynamically adsorbed to a penetration point. After the acid aqueous solution is washed, the exchange column is dynamically washed by using a low-concentration organic solvent or an alkaline solution with a lower pH value as an eluent, then the exchange column is dynamically eluted by using a high-concentration organic solvent or an alkaline solution with a higher pH value as an eluent, the eluent in the elution process is collected, and the glycyrrhetinic glycoside solid powder with the purity of more than 80 percent is obtained after drying. The separation and purification method disclosed by the invention has the characteristics of simple process operation, good separation effect, low cost consumption and easiness in industrial production.

Description

A method for purifying glycyrrhetin

technical field

The invention relates to a method for separating and purifying glycyrrhetin with a purity greater than 80 percent from a glycyrrhizic acid biotransformation solution by using a nonpolar macroporous adsorption resin.

Background technique

Glycyrrhetin, also known as Glycyrrhetic acid3-O-mono-β-D-glucuronide, is a derivative of glycyrrhizic acid, the main component of licorice. The sweetness of glycyrrhizin is about 5 times that of glycyrrhizic acid and 941 times that of sucrose. It is a natural sweetener, which is better than artificial sweeteners in terms of safety and nutritional functions. Glycyrrhetin and glycyrrhizic acid have similar biological activities, and glycyrrhizin is equal to or surpasses glycyrrhizic acid in anti-inflammatory activity and anti-allergic activity, and has significant anticancer effect. Therefore, Glycyrrhetin has broad application prospects in the fields of food and medicine.

Brieskorn et al. and Mizutani et al first synthesized glycyrrhetinin by chemical method, but this method has a long synthetic route and low yield. Glycyrrhetin can be directly obtained by using hydrolase to selectively hydrolyze glycyrrhizic acid to remove the terminal sugar group. At present, glycyrrhizin is mainly hydrolyzed by various sources of hydrolytic enzymes to obtain a biotransformation liquid containing glycyrrhizin. The biotransformation liquid still contains impurities such as unreacted glycyrrhizic acid, free enzymes, or biological tissue fragments. , using centrifugation or filtration to remove the solid components to obtain crude glycyrrhizin with low purity. Appropriate separation and purification methods must be adopted to obtain glycyrrhetin with high purity. The product on the market is a mixture of glycyrrhizin: glycyrrhizic acid of about 3:7, the purity of glycyrrhizin is about 30%, and it is mainly used as a sweetener in the food field. With the continuous expansion of the application field of glycyrrhetin, it is urgent to solve the problem of preparation of high-purity glycyrrhetin.

The technical problem to be solved in the present invention is to disclose a method for separating and purifying glycyrrhetin with a purity greater than 80%, so as to overcome the above-mentioned defects in the prior art.

Contents of the invention

The inventors found that both glycyrrhizinic acid and glycyrrhetinic acid are composed of non-polar aglycon glycyrrhetinic acid and polar sugar, and the molecular structure differs by one glucuronic acid group, and there is a certain difference in their molecular polarity. Utilizing the difference in molecular polarity, the non-polar macroporous adsorption resin is used to separate and purify the glycyrrhetin, so as to achieve the purpose of preparing the glycyrrhetin with a purity greater than 80%.

The non-polar macroporous adsorption resin used in the present invention has only a porous skeleton because no ion-exchange functional group is introduced, and its properties are similar to those of adsorbents such as activated carbon and silica gel. Compared with classical adsorbents such as activated carbon, nonpolar macroporous resins have the advantages of good selectivity, easy desorption, good mechanical strength, reusability, and low fluid resistance. The preferred non-polar macroporous adsorption resin of the present invention is a large-grid adsorbent polymerized with styrene as a monomer and divinylbenzene as a cross-linking agent. The invention utilizes the non-polar macroporous adsorption resin to separate and purify the glycyrrhetin with a purity greater than 80% from the centrifuged or filtered glycyrrhizic acid biotransformation solution (ie crude glycyrrhetin solution).

The present invention is realized through the following steps:

(1) Filling of the adsorption column:

After the non-polar macroporous adsorption resin is pretreated according to conventional methods, it is loaded into the adsorption column as an exchange column. The adsorption column generally adopts a glass column or a stainless steel column that is resistant to organic solvents, acid and alkali, or ceramic lining;

(2) Adsorption process of resin:

Thick glycyrrhizin solution is carried out dynamic adsorption by the exchange column described in step (1), and the flow rate when the solution is passed through with a peristaltic pump is controlled, until the adsorption of resin in the exchange column reaches the breakthrough point (being 5% of the concentration of the sample solution). -10%).

The conditions to be controlled during operation are:

a. Control the concentration of the solution between 0.001-100g/L;

b. The pH value of the control solution is between 4.5-8, preferably between 5-6, and the optimal pH value is 5.8;

c. Control the flow rate of the solution through the exchange column between 0.5-10BV/h.

(3) The cleaning process of resin comprises the following continuous steps:

A. in the acidic aqueous solution flushing step (2), the exchange column that reaches the penetration point through dynamic adsorption, controls the pH value of the aqueous solution between 3-5 during operation;

b. Rinse the exchange column washed in the above step a) with deionized water until the effluent is neutral;

c. Low-concentration organic solvents (methanol, ethanol, acetone, etc.) or alkaline solutions (NaOH, KOH, ammonia, etc.) Wash the impurities until there is no glycyrrhizic acid in the effluent. The conditions controlled during operation are:

(a) The concentration of the organic solvent is between 0.001-50%, preferably between 25-50%, and the optimum concentration is 30%;

(b) The pH value of the alkaline solution is between 7.5-9, and the optimum pH is 8.

(4) The elution process of the resin:

High-concentration organic solvents (methanol, ethanol, acetone, etc.) or alkaline solutions (NaOH, KOH, ammonia, etc.) The column is eluted dynamically until there is no glycyrrhetin in the effluent. Collect the eluent in this process, which is a high-purity glycyrrhetin solution, and the conditions to be controlled during operation are:

a. The concentration of the organic solvent is between 50-100%, preferably between 65-100%, and the optimum concentration is 70%;

b. The pH value of the alkaline solution is between 9-14, and the optimum pH value is 12.

(5) Drying of Glycyrrhizin:

The eluent collected in step (4) is dried to remove the solvent therein, and high-purity solid powder of glycyrrhetin is obtained.

The purification process of the glycyrrhizin in the present invention is simple in operation, good in separation effect, low in cost consumption and easy in industrialized production. Moreover, after the resin is processed, it can be used repeatedly without reducing the separation effect, and is suitable for industrial production. Glycyrrhetin with a purity greater than 80% prepared by the method described in the present invention can be widely used in the fields of food, medicine, cosmetics and the like.

Detailed ways

Example 1

The exchange column (Φ2.0×12cm) was filled with non-polar macroporous adsorption resin X-5 (Nankai University Chemical Factory). The 3.6g/L (pH value 5.8) crude glycyrrhetinic solution passed through the exchange column until the adsorption reached the penetration point, and the flow rate of the solution was controlled to be 2BV/h. Wash the exchange column with an acidic aqueous solution with a pH value of 4, and then wash the exchange column with deionized water until the effluent is neutral. Then use 30% ethanol-water solution to dynamically wash the exchange column, until there is no glycyrrhizic acid in the effluent; 70% ethanol-water solution is used for dynamic elution, until there is no glycyrrhizin in the effluent, collect the elution The eluate in the process is dried to obtain about 0.1557 g of high-purity glycyrrhetinin solid powder, with a purity of 80.28% and a yield of 42.36%.

Example 2

Use the non-polar macroporous adsorption resin NKA (Nankai University Chemical Factory) to pack the exchange column (Φ2.0×12cm), and the other steps are the same as in Example 1. The eluate in the elution process was collected and dried to obtain about 0.1891 g of high-purity glycyrrhetin solid powder, with a purity of 80.57% and a yield of 51.45%.

Example 3

Pass through the exchange column packed with NKA resin with 4.8g/L thick glycyrrhizin solution, and other steps are the same as in Example 1. The eluate in the elution process was collected and dried to obtain about 0.2528 g of high-purity glycyrrhetin solid powder, with a purity of about 80.49% and a yield of 51.78%.

Example 4

Pack the exchange column (Φ1.0×20cm) with non-polar macroporous adsorption resin NKA. The 4.8g/L (pH value 5.8) crude glycyrrhetin solution was passed through the exchange column until the adsorption reached the breakthrough point, and the other steps were the same as those described in Example 1. The eluate in the elution process was collected and dried to obtain about 0.9592 g of high-purity glycyrrhetin solid powder, with a purity of 85.58% and a yield of 63.45%.

Example 5

The flow rate of the control solution passing through the exchange column is 3BV/h, and other steps and conditions are as described in Example 4. The eluate in the elution process is collected, and after drying, the high-purity Glycyrrhetin solid powder is about 0.9385 g, the purity is 85.05%, and the yield is about 62.08%.

Example 6

Use 30% methanol-water solution to dynamically wash the exchange column packed with NKA resin until there is no glycyrrhizic acid in the effluent; 70% methanol-water solution is used as the eluent to dynamically elute the exchange column until the effluent There is no glycyrrhetin in it, and other steps and conditions are as described in Example 4. The eluate in the elution process was collected and dried to obtain about 0.9308 g of high-purity glycyrrhetin solid powder with a purity of 86.19% and a yield of about 61.87%.

Example 7

Use 30% acetone-water solution to dynamically wash the exchange column packed with NKA resin until there is no glycyrrhizic acid in the effluent; 70% acetone-water solution is used as eluent to dynamically elute the exchange column until the effluent There is no glycyrrhetin in it, and other steps and conditions are as described in Example 4. The eluate in the elution process was collected and dried to obtain about 0.9416 g of high-purity glycyrrhetin solid powder with a purity of 83.76% and a yield of about 62.45%.

Example 8

The NaOH solution with a pH value of 8 is used to dynamically wash the exchange column until there is no glycyrrhizic acid in the effluent, and the NaOH solution with a pH value of 12 is used to dynamically elute the exchange column until there is no glycyrrhetin in the effluent. Other steps and conditions are with embodiment 4. The eluate in the elution process was collected and dried to obtain about 0.9687 g of high-purity glycyrrhizin solid powder, with a purity of about 85.66% and a yield of about 64.08%.

Example 9

Use the KOH solution with a pH value of 8 to dynamically wash the exchange column until there is no glycyrrhizic acid in the effluent, and the KOH solution with a pH value of 12 to dynamically elute the exchange column until there is no glycyrrhizin in the effluent , other steps and conditions are the same as in Example 4. The eluate in the elution process was collected and dried to obtain about 0.9794 g of high-purity glycyrrhetin solid powder, with a purity of about 84.85% and a yield of about 64.79%.

Example 10

Dynamically wash the exchange column with ammonia solution with a pH value of 8, until there is no glycyrrhizic acid in the effluent, and dynamically elute the exchange column with an ammonia solution with a pH value of 12, until there is no glycyrrhizin in the effluent , other steps and conditions are the same as in Example 4. The eluate in the elution process was collected and dried to obtain about 0.9553 g of high-purity glycyrrhetin solid powder, with a purity of about 86.04% and a yield of about 64.96%.

Example 11

Use non-polar macroporous adsorption resin NKA column (Φ5.0×20cm), 4.8g/L (pH value 5.8) crude glycyrrhizin solution is dynamically adsorbed through the exchange column until the adsorption reaches the penetration point, and the solution is controlled. The flow rate is 2BV/h. After washing the exchange column with an acidic aqueous solution with a pH value of 4 and deionized water, use a 30% ethanol-water solution for dynamic washing until there is no glycyrrhizic acid in the effluent, and then 70% ethanol-water solution for dynamic elution. Until there is no glycyrrhetin in the effluent. The eluate in the elution process was collected and dried to obtain about 4.650 g of high-purity glycyrrhetin solid powder with a purity of 86.53% and a yield of about 65.08%.

Claims (9)

1. A method for purifying Glycyrrhetin, characterized in that it comprises the following continuous steps: (1) Packing of the adsorption column: After the non-polar macroporous adsorption resin is pretreated according to the conventional method, it is packed into the adsorption column as an exchange column; (2) The adsorption process of the resin: the thick glycyrrhetin solution is carried out dynamic adsorption by the exchange column described in step (1), until the adsorption of the resin in the exchange column reaches the penetration point, that is, the concentration of the effluent is 1/4 of the concentration of the loading solution. 5%-10%, the conditions to be controlled during operation are: control the concentration of the solution between 0.001-100g/L, the pH value between 4.5-8, and the flow rate of the solution when passing through the exchange column at 0.5-10BV/h between; (3) The cleaning process of resin: including the following continuous steps: A. in the acidic aqueous solution washing step (2), the exchange column that reaches the penetration point by dynamic adsorption, controls the pH value of the aqueous solution between 3-5 during operation; b. Rinse the exchange column washed in the above step a) with deionized water until the effluent is neutral; c. An organic solvent with a concentration between 0.001-50% or an alkaline solution with a pH value between 7.5-9 is used as the eluent used in the washing process of the exchange column, and the exchange column is dynamically washed until it flows out No glycyrrhizic acid in the liquid; (4) The elution process of the resin: the organic solvent between the concentration 50-100% or the alkaline solution between the 9-14 as the eluent used in the exchange column elution process, the exchange column is carried out Dynamic elution until there is no glycyrrhetin in the effluent, and the collected effluent at this time is a high-purity glycyrrhetin solution; (5) Drying of Glycyrrhetin: After drying the eluate collected in step (4), high-purity Glycyrrhetin is obtained, and the purity of the high-purity Glycyrrhetin is greater than 80%. 2. The method according to claim 1, wherein the non-polar macroporous adsorption resin is a large-grid adsorbent formed by polymerization of styrene as a monomer and divinylbenzene as a cross-linking agent. 3. The method according to claim 1, wherein the adsorption column adopts a glass column or an organic solvent-resistant, acid-base resistant or ceramic-lined stainless steel column. 4. method according to claim 1, it is characterized in that the pH value of control solution is between 5-6 in the adsorption process of resin. 5. The method according to claim 1, wherein the organic solvent in the cleaning process of the resin is selected from methanol, ethanol or acetone. 6. The method according to claim 1, characterized in that the concentration of the organic solvent in the cleaning process is between 25-50%. 7. The method according to claim 1, wherein the alkaline solution is selected from NaOH solution, KOH solution or ammoniacal liquor in the washing process. 8. The method according to claim 1, wherein the organic solvent is selected from methanol, ethanol or acetone in the elution process of the resin. 9. The method according to claim 1, wherein the alkaline solution in the elution process of the resin is selected from NaOH solution, KOH solution or ammoniacal liquor.