KR20040067022A - A process of extracting glabridine from licorice root - Google Patents

Abstract

PURPOSE: A process of extracting glabridine from Glycyrrhiza uralensis is provided, thereby increasing the content of glabridine in the extract of Glycyrrhiza uralensis, so that it is useful for producing herb medicines, foods, tobaccos or cosmetics. CONSTITUTION: The process of extracting glabridine from Glycyrrhiza uralensis comprises the steps of: mixing pulverized Glycyrrhiza uralensis with solvent selected from ethyl acetate, ethanol, methanol and acetone, and filtering the mixture; concentrating the filtered solution under reduced pressure to obtain an extract of Glycyrrhiza uralensis; and subjecting the extract of Glycyrrhiza uralensis to column chromatography. The process of extracting glabridine from Glycyrrhiza uralensis comprises the steps of: mixing pulverized Glycyrrhiza uralensis with water and heating the mixture at 50 deg.C; adding chloroform into the extract of Glycyrrhiza uralensis and separating a water layer; adding ethyl acetate into the water layer, separating an ethyl acetate layer, and concentrating the ethyl acetate layer under reduced pressure; and subjecting the concentrate to column chromatography.

Description

A PROCESS OF EXTRACTING GLABRIDINE FROM LICORICE ROOT}

The present invention relates to a method for extracting glabridine from licorice.

Licorice (Glycyrrhiza uralensis Fisch, Glycyrrhiza glabra L.) is a perennial herbaceous plant belonging to Leguminosae , whose roots and stems are used for medicinal and edible food. In particular, licorice is a medicinal plant that is used as an antidote, antitussive expectorant, laxative, peptic ulcer, and gastric ulcer in oriental medicine and folk medicine. There is an increase in the effect of diuretic, anti-inflammatory action. Therefore, in order to use the medicinal effect licorice is used as a herbal medicine or licorice extract has been attempted to use in food, tobacco, cosmetics.

Licorice extracts include Glycyrrhizin, Glycyrrhetic acid, Licoricidin, Hispaglabridin A, Hispaglabridin B, and Glabridine (Glycyrrhizin). Glabridin, 4-O-methylglabridin, 4-isoprenylchalcone derivative, Isoliquiritigenin, Formononetin and the like. Glycyrrhizin in licorice extract has anti-inflammatory, detoxification, anti-ulcer, and hydrolyzate hormonal effects, so that conventional methods for extracting and purifying only glycidazine are known. The method of extracting glycyrrhizin from licorice is a method of precipitating impurities by separating soda silicate and aluminum sulfate and then adjusting the pH to 4.5 to 6.5 when licorice root is extracted with water or alkaline aqueous solution. A method of combining or sequentially extracting an methanol extraction process, an anion exchange resin decolorization process, and an ethanol recrystallization process is known. As such, a variety of methods for extracting glycyrrhizin from licorice are known, and the glycyrrhizin extracted therefrom is of high purity.

Glabridine, the main component of the licorice extract, is known to have an inhibitory effect on the oxidation of low density phospholipids (Low Density Lipoprotein) harmful to the human body (Belinky, P., A., Aviram, M., Fuhrman, B., Rosenblat, M., and Vaya, J., "The antioxidative effects of the isoflavan glabridin on endogenous constituents of LDL during its oxidation", Atherosclerosis , Vol. 137, 49, (1998)). Low-density phospholipids are responsible for the delivery of cholesterol throughout the body. Oxidation occurs when excess low-density phospholipids are present in the body, which can cause atherosclerosis, diabetes, and adult disease.

In addition, glabridine is one of the hydrophobic substances having an important effect on the skin, and has an excellent effect on skin whitening, and there are an increasing number of examples of producing functional cosmetics having a whitening effect from the raw material (Korean Patent Publication 1993- 21190).

However, unlike glycyrrhizin, which has been studied in many extraction methods as described above and used in a high purity form, glabridine has no method of extracting from licorice, and thus has low purity licorice extract in the manufacture of Chinese medicine, food, tobacco or cosmetics. There was a problem that it should be used.

The present invention has been devised to solve the above problems, and an object of the present invention is to provide a method for extracting glabridine from licorice to provide a high purity glabridine extract.

1 shows a chromatogram analyzed by HPLC of an extract according to Example 1 of the present invention,

Figure 2 shows a chromatogram analyzed by HPLC with a fixed injection amount of the extract of the extract according to Example 5 of the present invention,

Figure 3 shows the chromatogram analyzed by HPLC by increasing the injection amount of the sample extract according to Example 5 of the present invention,

Figure 4 shows the chromatogram analyzed by HPLC of the extract according to Example 1 of the present invention,

Figure 5 shows the chromatogram analyzed by HPLC of the standard sample for analysis of glabridine,

FIG. 6 is an HPLC analysis of extracts according to the first aspect of the invention, overlaid on each chromatogram,

Figure 7 shows a chromatogram analyzed by HPLC extract according to Example 5 of the present invention,

8 is a chromatogram analyzed by HPLC of the water layer separated in Example 5 of the present invention,

Figure 9 shows the chromatogram analyzed by HPLC of the standard sample for glabridine analysis.

In order to achieve the above object, the present invention is a method of extracting glabridine by mixing the pulverized licorice with a solvent selected from the group consisting of ethyl acetate, ethanol, methanol and acetone, and then filtering the resulting filtrate under reduced pressure (Hereinafter referred to as "the first sun").

In addition, the present invention is mixed with agitated pulverized licorice and water and extracted with hot water, chloroform is added to the obtained extract, the water layer is separated, ethyl acetate is added to the water layer to separate the ethyl acetate layer and concentrated under reduced pressure It provides a method of extracting a din (hereinafter referred to as a "second sun").

Hereinafter, the present invention will be described in detail.

Specifically, the first aspect of the invention

1) preparing a mixture by mixing pulverized licorice and a solvent selected from the group consisting of ethyl acetate, ethanol, methanol and acetone (step 1);

2) concentrating the obtained filtrate under reduced pressure to obtain an extract (step 2); and

3) Purifying the extract by column chromatography (step 3).

Step 1 is a step of preparing and filtering a mixture by mixing the ground licorice and a solvent selected from the group consisting of ethyl acetate, ethanol, methanol and acetone to elute the glabridine contained in the ground licorice. Preferably ethyl acetate is used as the solvent. In addition, in order to increase the dissolution rate of the glabridine contained in the crushed licorice, the mixture may be sonicated in the process of mixing the crushed licorice and the solvent. The sonication is a process in which a mixture of crushed licorice and water is put into an ultrasonic device and processed.

Step 2 is a step of obtaining a extract by concentrating the obtained filtrate under reduced pressure, concentration under reduced pressure may be carried out by conventional methods known to those skilled in the art.

Step 3 is a step of obtaining the glabridine by purification of the obtained extract by column chromatography. By purifying the extract obtained in step 2 by column chromatography, high content and high purity of the gloridine can be obtained. Column chromatography can be performed by conventional methods.

Also specifically, the second aspect of the present invention

1) mixing and stirring the ground licorice and water to extract hot water (step 1);

2) adding chloroform to the extract and separating the water layer (step 2);

3) adding ethyl acetate to the water layer to separate the ethyl acetate layer and concentrating under reduced pressure to obtain an extract (step 3); and

4) Purifying the extract by column chromatography (step 4).

Step 1 is a step of extracting hot water while mixing and stirring the ground licorice and water. Hot water extraction is to elute the glabridine contained in the crushed licorice by heating the crushed licorice in water, it is possible to increase the dissolution rate of glabridine through this process. Preferably hot water is extracted for 3 hours at a temperature of 50 ℃ while mixing and stirring the ground licorice and water.

Step 2 is adding chloroform to the extract of step 1 and separating the water layer. The extract obtained from step 1 contains not only a lot of hydrophilic and hydrophobic impurities of licorice powder, but also a lot of components other than glabridine. By adding chloroform to the extract and separating the layers and obtaining the water layer, the hydrophobic impurities of the licorice powder can be removed through the chloroform layer.

In step 3, ethyl acetate is added to the separated water layer of step 2, the layers are separated to obtain an ethyl acetate layer, and the obtained ethyl acetate layer is concentrated under reduced pressure to obtain an extract. The extract can be obtained by concentrating the obtained ethyl acetate layer under reduced pressure in a conventional manner.

Step 4 is a step of purifying the extract obtained from step 3 by column chromatography. As shown in Experiment 2, the extract obtained in step 3 contains less impurities than the conventional liquorice extract containing glabridine and has a high content of glabridine. Column chromatography can be performed by conventional methods.

Hereinafter, the present invention will be described in detail by Examples and Experimental Examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.

Example 1 Extract of Glabridine from Licorice According to the First Aspect

20 g of crushed licorice and 100 ml of ethyl acetate were mixed and placed in an ultrasonic device (Bransonic, USA), sonicated for 1 hour, and filtered through a filter paper. The obtained filtrate was concentrated under reduced pressure to separate the extract. The filtrate obtained by filtering the obtained extract with a membrane filter (0.45 μm) was used as a sample for HPLC analysis. 20 μl of the filtrate prepared above was injected into a reverse phase column (15 μm, 250 × 4.6 mm, Licrospher 100RP-18), and a mobile phase solvent (mobile phase solvent was mixed with 50/50 volume / volume of acetonitrile and water). Prepared.). The elution rate of the mobile phase solvent was 1.0 ml / min and the glabridine detection wavelength was maintained at 230 nm. HPLC analysis results are shown in FIG. 1 .

As shown in FIG. 1 , glabridine was detected within 15 minutes of elution time and showed a major peak with a purity of 57.8%.

Example 2 Glabridine Extract from Licorice according to the First Aspect

20 g of crushed licorice and 100 ml of ethanol were mixed and placed in an ultrasonic device (Bransonic, U.S.A), sonicated for 1 hour, and filtered through a filter paper. The obtained filtrate was concentrated under reduced pressure to separate the extract. Column chromatography was performed in the same manner as in Example 1.

Example 3 Extract of Glabridine from Licorice According to the First Aspect

20 g of crushed licorice and 100 ml of methanol were mixed and placed in an ultrasonic device (Bransonic, U.S.A), sonicated for 1 hour, and filtered through a filter paper. The obtained filtrate was concentrated under reduced pressure to obtain an extract. Column chromatography was performed in the same manner as in Example 1.

Example 4 Extract of Glabridine from Licorice According to the First Aspect

20 g of crushed licorice and 100 ml of acetone were mixed and placed in an ultrasonic device (Bransonic, U.S.A), sonicated for 1 hour, and filtered through a filter paper. The obtained filtrate was concentrated under reduced pressure to obtain an extract. Column chromatography was performed in the same manner as in Example 1.

Example 5 Glabridine Extracting from Licorice According to Second Aspect

20 g of the ground licorice and 100 ml of water were mixed and stirred, followed by hot water extraction at 50 ° C. for 3 hours to prepare an extract. 100 ml of chloroform was added to the extract, the layers were separated, and a water layer was obtained. 100 ml of ethyl acetate was added to the water layer and the layers were separated and an ethyl acetate layer was obtained. The obtained ethyl acetate layer was concentrated under reduced pressure to obtain an extract. The obtained extract was column chromatographed in the following manner.

(1) When the injection amount of the sample is fixed

The extract obtained from step 3 of Example 5 was filtered through a membrane filter (0.45 μm) and used as a sample for HPLC analysis. 20 μl of the prepared analytical sample was injected into a semipreparative column (15 μm, 250 × 4.6 mm, Lichrospher 100RP-18) and a mobile phase solvent (mobile phase solvent was acetonitrile and water of 60/40 (volume / volume). Prepared by mixing in proportion). HPLC analysis results are shown in FIG. 2 .

As shown in FIG. 2 , glabridine was detected within 15 minutes of elution time and showed a major peak with a purity of 95.35%.

(2) When the injection amount of the sample is increased

The extract obtained from step 3 of Example 5 was filtered through a membrane filter (0.45 μm) and used as a sample for HPLC analysis. 50, 100, 150 μl of the prepared analytical sample was injected into a semipreparative column (15 μm, 250 × 4.6 mm, Lichrospher 100RP-18) and linearly increased the amount of acetonitrile by about 5% by volume (mobile phase solvent). Was prepared using acetonitrile and water prepared from 0/100 (volume / volume) to 5/95 (volume / volume). In the same manner, analytical samples were prepared from the water layer of Example 5 and analyzed by HPLC. HPLC analysis results are shown in FIG. 3 .

As shown in FIG. 3 , it can be seen that the elution amount of glabridine increases relatively as the injection amount increases. However, when the injection amount is 100 μl or more, the effect of glabridine according to the injection amount is similar.

Experimental Example 1 Analysis of Glabridine Extracted According to the First Sun

(1) In the first aspect, the extract contained glybridine

The filtrate obtained by filtering the extract of Example 1 with a membrane filter (0.45 μm) was used as a sample for HPLC analysis. 20 μl of the filtrate prepared above was introduced into an analytical column, a reversed phase column (5 μm, 250 × 4.6 mm, Chrompack Omnispher C ₁₈ , Varian, USA) and a mobile phase solvent (acetonitrile and water in a volume ratio of 50:50). Prepared by mixing). The elution rate of the mobile phase solvent was 1.0 ml / min and the glabridine detection wavelength was maintained at 230 nm. HPLC analysis results are shown in FIG. 4 .

In addition, in order to confirm that the extract of Example 1 is glabridine, an analytical standard sample was prepared by glabridine (purchased from Wako Pure Chemical Industries, LTd) by the same method as described above, and the same method as described above. HPLC analysis. The result is shown in Fig.

As shown in FIG. 4 , the material extracted with the first aspect of the present invention showed a major peak with a purity of 68.48% at 20 minutes of retention time.

As shown in Figure 5 , the standard sample for glabridine analysis also showed a peak at the same position. Therefore, it was confirmed that the glabridine is contained in the extract according to the first aspect of the present invention.

(2) Comparison of extraction efficiency according to extraction solvent in the first embodiment

When extracting glabridine from licorice according to the first aspect of the present invention, to compare the extraction efficiency of glabridine according to the extraction solvent to prepare an analysis sample of the same concentration with the extract extracted in Examples 1 to 4 After analyzing the prepared samples by HPLC, the chromatograms obtained therefrom were compared.

Specifically, the filtrate obtained by filtering the whole extract obtained in Example 1 with a membrane filter (0.45 μm) was used as a sample for HPLC analysis. 20 μl of the prepared analytical sample was injected into an analytical column, a reversed phase column (5 μm, 250 × 4.6 mm, Chrompack Omnispher C ₁₈ , Varian, USA) and a mobile phase solvent (mobile phase solvent was acetonitrile and water 50 / Eluted at 50 (volume / volume). The elution rate of the mobile phase solvent was 1.0 ml / min and the glabridine detection wavelength was maintained at 230 nm. The sample for analysis was prepared in the same manner for the extract obtained in Examples 2 to 4, and HPLC analysis was performed using the same HPLC analysis method. The content of glabridine obtained therefrom is shown in Table 1 below.

Content of extracted glabridine Extraction solvent Glabridine (mg / kg) Example 1 Ethyl acetate 1260.0 Example 2 ethanol 1213.4 Example 3 Methanol 990.2 Example 4 Acetone 886.8

As shown in Table 1 , when ethyl acetate was used as the extraction solvent in the first aspect according to the present invention, it can be seen that the content of the extracted glabridine is high.

In addition, it is shown in Figure 6 superimposed on each chromatogram obtained therefrom.

As shown in Figure 6 , when extracting glabridine from licorice according to the first aspect of the present invention, it can be seen that the extraction efficiency of glabridine is high when using ethyl acetate as the extraction solvent, the purity is also methanol It was found that the purity of ethyl acetate was high as 73.80%, acetone 69.65% and ethyl acetate 80.06%.

Experimental Example 2 Analysis of Glabridine Extracted According to Second Aspect

Confirmed that the extract according to the second aspect contains glabridine

The extract obtained in Example 5 was filtered through a membrane filter (0.45 μm) and used as a sample for HPLC analysis. 20 μl of the analytical sample prepared above was injected into a reversed phase column (5 μm, 250 × 4.6 mm, Chrompack Omnispher C ₁₈ , Varian, USA) and the mobile phase solvent (mobile phase solvent was acetonitrile and water 60/40 (volume / Volume) to elute. The mobile phase solvent was kept at 1.0 mL / min and the detection wavelength was kept at 230 nm. The results are shown in Table 2 and FIG. 7 .

In the same way, an analytical sample was prepared from the water layer separated with the ethyl acetate layer in Example 5 and analyzed by HPLC. The results are shown in Table 2 and FIG. 8 .

In addition, standard samples were prepared and analyzed by HPLC using glabridine (purchased from Wako Pure Chemical Industries, LTd) in the same manner. The results are shown in Table 2 and FIG. 9 .

Time (min) Area (uVｘsec) Height (uV) Butterfly (min) Example 5 2.1333 97011168 1463684 1.1046 Water layer 2.2833 5224168 1429694 0.6090 Standard sample 2.5533 29920114 1471680 0.3388

As shown in Table 2 and FIGS. 7 to 9 , samples of the material and the water layer obtained in Example 5 exhibited material peaks at 2 minutes of retention time in the chromatogram. In addition, existing glabridine standard samples also showed peaks at the same elution time. As a result, it was confirmed that the glabridine separated by the extraction method of the present invention is the same substance as the existing glabridine. And, in the case of the sample of the water layer and the ethyl acetate layer, glabridine was eluted at a similar time in time, but in the case of the ethyl acetate layer, the amount of elution increased almost twice.

As described above, the present invention relates to a method for extracting glabridine from licorice, which contains less impurities than other licorice extracts containing conventional glabridine, and has a high content of glabridine. And the extract was purified by column chromatography to extract a high content of high gloridine and high degree of bribridine, so that it can be efficiently used in the manufacture of herbal, food, tobacco or cosmetics.

Claims (5)

1) mixing and filtering the ground licorice with a solvent selected from the group consisting of ethyl acetate, ethanol, methanol and acetone (step 1); 2) concentrating the obtained filtrate under reduced pressure to obtain an extract (step 2); and 3) A method for extracting glabridine from licorice consisting of purifying the extract by column chromatography (step 3). The extraction method according to claim 1, wherein the solvent is ethyl acetate. 2. Extraction method according to claim 1, wherein the mixture is further sonicated. 1) mixing and stirring the ground licorice and water to extract hot water (step 1); 2) adding chloroform to the extract and separating the water layer (step 2); 3) adding ethyl acetate to the water layer to separate the ethyl acetate layer and concentrating under reduced pressure to obtain an extract (step 3); and 4) A method for extracting glabridine from licorice, comprising the step of purifying the extract by column chromatography (step 4). 4. The extraction method according to claim 3, wherein the hot water extraction is performed at 50 ° C.