KR100947920B1 - Extraction Method for separating active constituent from licorice using reverse phase preparative high performance liquid chromatography - Google Patents

Abstract

The present invention relates to licorice from licorice by adding methanol or aqueous methanol solution to licorice and immersing at room temperature for 20-120 minutes for extraction and by reversed phase liquid chromatography using acetonitrile and 1% (v / v) acetic acid. The present invention relates to an extraction method for separating useful components from licorice, which comprises the steps of separating chitin, glycyrrhizin acid and glabridine.

Licorice, Liquirithin, Glycirizine, Glabridine, Extract

Description

Extraction method for separating active constituent from licorice using reverse phase preparative high performance liquid chromatography

The present invention relates to an extraction method that can simultaneously separate the useful components, such as liqueurin, glycyrrhizin acid and glabridine from licorice.

Licorice is the root of the Glycyrrhiza genus and has been used for over 4000 years. Licorice is a Chinese herb used as an expectorant to stop coughing, lower heat, relax the stomach, relieve urgent situations, and enhance the effectiveness of various other herbs.

Liquiritin (LQ) is one of the most important flavonoids in licorice and has antiviral and antioxidant properties. Glycyrrhizic acid (GA) is the most researched component of licorice, exhibits sweetness and provides 50 times better sweetness than sugar and is widely used as a sweetener additive in the food industry.

GA has anti-inflammatory, anti-ulcer, antihepatic toxicity and antiviral activity, and in many countries GA is used as an important therapeutic agent for treating allergic dermatitis and chronic viral hepatitis.

Glabridin is another active ingredient contained in licorice, antibacterial activity; Cytotoxic activity; Estrogen activity and antiproliferative activity against human breast cancer cells; Effect on low density lipoprotein oxidation, melanocyte production, inflammation; It exhibits various pharmacological activities, including mitochondrial function protection from oxidative stress.

Reversed-phase preparative high-performance liquid chromatography (HPLC) has been applied to purify LQ, GA and glabridine from licorice.

Preparative chromatography is a purification process aimed at identifying pure substances from the mixture.

Several reports have been reported for the isolation of each of LQ, GA and glabridine. However, there are no reports on how to extract and separate these three components simultaneously using a preparative column.

Accordingly, an object of the present invention was to develop a method for simultaneously extracting LQ, GA, and glabridine from licorice using RP-HPLC, and separating three components using a preparative column.

Compared with the prior art, the extraction method according to the present invention is simple, fast and can separate three components from licorice at the same time, which can be used very usefully.

In order to achieve the above object, the present invention comprises the steps of adding methanol or methanol aqueous solution to licorice and immersed for 20-120 minutes at room temperature to extract; And separating liquiritin, glycyrrhizin acid and glabridine from licorice by reverse phase liquid chromatography using a mobile phase composed of acetonitrile and 1% (v / v) acetic acid. To provide.

At this time, the methanol or methanol aqueous solution is preferably added to 15 to 70 parts by weight based on 1 part by weight of licorice. If methanol or an aqueous methanol solution is used in excess of the above range, the extraction solvent is wasted compared to the extraction efficiency, which is uneconomical, and when used in a small amount, the useful components of licorice may not be well extracted.

In addition, the immersion time is higher the extraction efficiency is higher, but the immersion time is more than 120 minutes, since the saturation of the extraction efficiency is generated when the immersion time elapses 120 minutes.

Reverse phase liquid chromatography used in the present invention preferably uses a 12 μm column. In this case, using a 40/63 μm column having a column particle diameter of 12 μm may cause a problem in which three useful components are not clearly separated.

In addition, it is preferable that the maximum injection volume of the sample used for reverse phase liquid chromatography is 50 microliters. Injecting an excess of 50 μL can cause problems that prevent the extraction and separation of LQ.

In addition, the mobile phase preferably uses acetonitrile and 1% (v / v) acetic acid in a volume ratio of 20:80 to 60:40.

According to the extraction method, 0.29 mg / g of liquiritin, 1.43 mg / g of glycyrrhizin acid, and 0.07 mg / g of glabridine were extracted from licorice, and the recovery rates were 80.79%, 89.70%, and 72.51%, respectively.

According to the extraction method of the present invention, it is possible to extract and separate the liquid extract, glycyrrhizin acid and glabridine from licorice at high concentrations, so that useful components can be easily and quickly separated from licorice.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only for the purpose of clarifying the contents of the present invention, and the present invention is not limited by the examples.

Example 1 Preparation

1) Material and Reagent Preparation

Licorice was purchased on the conventional market, liquiditine was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (NICPBP), glycyrrhizinate (the first ammonium salt hydrate) was purchased from Sigma, and glabridine was obtained from Wako Pure. Purchased from Chemical Industries.

Methanol, acetonitrile, chloroform (HPLC grade), acetic acid and n-hexane (Extra pure reagent) were purchased from Duksan Pure Chemical. Water was filtered with secondary distilled water (FH-0.45 μm, Advantec MFS) using a compression pump (Division of Millipore, Waters, USA).

2) HPLC analysis

HPLC systems used in the present invention include M930 solvent delivery pump (Yuong Lin Co.), UV detector (M 720 Absorbance Detector, Young-In Scientific Co.), and integrated data system (Autochrowin. Ver. 1.42, Young Lin Co. ) And two Reodyne injection valves with 25 μL and 200 μL sample loops were used.

The flow rate was 0.5 mL / min and the UV wavelength was fixed at 252 nm. All solvents were filtered and used by a portable syringe filter unit (0.2 μm).

3) Sample Preparation

Licorice roots were dried in an oven, cut, ground and ground to make powders and used for extraction experiments.

Standards of LQ, GA and glabridine were dissolved in methanol to prepare a final concentration of 0.33 mg / mL. All experiments were performed at room temperature.

4) Determination of LQ, GA and Glabridine

Concentrations of LQ, GA and glabridine were analyzed using a column with a mobile phase consisting of acetonitrile / 1% (v: v) acetic acid (C18, 5 μm, 150 × 4.6 mm, RStech). At this time, the gradient composition of the mobile phase of acetonitrile and 1% (v: v) acetic acid was extracted from 20:80 to 60:40 for 0 to 10 minutes.

The peak area values of the analytes were plotted against the relevant concentrations of the analytes and a calibration curve was drawn using the least squares method. Concentrations of 0.2, 0.4, 0.5, 0.8 and 1.0 mg / mL were used for standard solutions of LQ, GA and 0.01, 0.04, 0.08, 0.1 and 0.2 mg / mL for glabridine standard solutions. It was used and injected three times for each concentration.

The calibration curves of the three compounds show good linearity (r ² > 0.998), and each regression equation for LQ, GA and glybridine is Y = 11531x-23.942 (x is 0.01 to 0.1 mg / mL), Y = 9721.7x + 97.429 (x is 0.1-1.0 mg / mL) and Y = 10730x-31.147 (x is 0.01-0.1 mg / mL).

Example 2 Extraction Method

1) Selection of optimal extraction solvent

Various extraction solvents, such as water, methanol, n-hexane and chloroform, were used to extract LQ, GA and glabridine from licorice. 1.0 g of licorice was extracted by immersing for 60 minutes at room temperature using 50 mL of each solvent.

As a result, as shown in Table 1, the extraction efficiency was good in the polar solvent, and in particular, the extraction efficiency increased with the increase of the methanol concentration in the mixed solvent which increased the methanol concentration in water. Since, in the experiment, methanol was used as the extraction solvent.

Extraction solvent LQ (mg / g) GA (mg / g) Glabridine (mg / g) chloroform * * * n-hexane * * 0.006 water 0.15 2.08 0.001 Methanol: Water (25:75) 0.20 1.93 0.008 Methanol: Water (50:50) 0.23 1.76 0.023 Methanol: Water (75:25) 0.25 1.59 0.066 Methanol 0.29 1.43 0.070

*: Not detected

2) Methanol Extraction by Various Extraction Methods

Five samples of 1.0 g licorice powder were mixed with 50 mL methanol and soaked for 20, 30, 60, 120 and 240 minutes at room temperature.

Alternatively, the five samples mixed with methanol were sonicated for 2, 5, 10, 20 and 30 minutes, respectively.

As a result, as shown in FIG. 1, the extraction amount of LQ, GA, and glabridine also increased as the immersion time increased from 20 minutes to 120 minutes, but no apparent increase occurred after 120 minutes. Therefore, the optimum immersion time was confirmed to be 120 minutes.

In addition, the soak-free ultrasonic method also increased the extraction amount of LQ, GA and glabridine as the sonication time increased as shown in FIG.

On the other hand, comparing the two methods, because the ultrasonic method shows a lower extraction amount than the immersion method requires more energy, the immersion method was used in the present invention.

3) Extraction using methanol in various capacities

Five samples of 1.0 g licorice powder were added with 20, 50, 70 and 100 mL of methanol and treated at room temperature without ultrasonic waves.

As a result, as shown in FIG. 3, the extraction amount of LQ, GA, and glabridine also increased as the volume of the extraction solvent increased, but the extraction amount was almost the same in methanol of 70 mL or more. Therefore, the volume of the extraction solvent was used at 70 mL.

4) Feasibility study of extraction method

In order to confirm whether the three components are extracted completely by one extraction, the extraction was carried out in three steps. That is, after mixing 1 g licorice powder with 70 mL methanol for 120 minutes in the first step, the powder was separated from the solvent and dried in an oven.

As a second step and then a third step, the process was repeated once more with 70 mL methanol.

When 1.0 g licorice powder was extracted in three steps using 70 mL of methanol, the extraction amount of LQ and GA was very low in the second step as shown in Table 2, and nothing was extracted from the licorice in the third step. Thus, extraction was performed once with 120 mL of 70 mL methanol.

  Methanol extraction LQ (mg / g) GA (mg / g) Glabridine (mg / g) Step 1 (70 mL) Step 2 (70 mL) Step 3 (70 mL) 0.29 0.001 0.0 1.43 0.03 0.0 0.07 0.0 0.0

The RSD of the accuracy test, limit of detection (LOD) and recovery for the standard solution are shown in Table 3. These values represent acceptable accuracy for actual sample analysis.

Reproducibility analysis, measured as a relative standard deviation (RSD), was performed by injecting LQ, GA, and glabridine standards five times a day. This experiment was repeated for 5 days. The concentration of the standard solution was 0.33 mg / mL, and the injection volume was fixed at 10 μL.

Each of the three standard concentrations, LQ (0.15, 0.20, 0.30 mg / mL), GA (0.5, 0.6, 0.8 mg / mL), and Glybridine (0.05, 0.06, 0.07 mg / mL), was used for 3 mL of actual sample. Was added to and filled with 6 mL.

The measured concentrations were compared with theoretical concentrations for recovery recovery. LQ, GA, and globridine standard solutions were diluted and injected with a detection limit (LOD) of which signal / noise ratio was obtained at 3.

ingredient RSD (%) Recovery LOD (ng / mL) Intra-day Inter-day Added (mg / mL) Recovery (%) RSD (%) LQ 0.34 0.37 0.15 0.20 0.30 81.33 79.99 81.04 0.38 365 GA 0.54 0.59 0.5 0.6 0.8 88.73 90.05 90.32 0.66 464 Glabridine 0.83 0.90 0.05 0.06 0.07 74.45 69.89 73.17 0.77 229

By the extraction method of the present invention, 0.29 mg / g of liqueurine, 1.43 mg / g of glycyrrhizin acid and 0.07 mg / g of glycridine are extracted from licorice, and the recovery rates are 80.79%, 89.70% and 72.51%, respectively. The useful components of licorice can be extracted and separated, respectively. The method according to the invention is simple, fast and can separate three components simultaneously from licorice, which can be very useful.

Example 3 Extraction Separation by Different Grain Size Columns

In order to determine the maximum injection dose for the assay column, 10, 25, 50, 75 and 100 μL injection doses were used, respectively.

As shown in FIG. 4, when the injection dose was injected in excess of 50 μL, GA and glybridine separated well, but LQ could not. Therefore, the maximum injection volume of the analytical column was 50 μL.

Particle diameters of ODS (12 μm, 40/63 μm, YMC Co.) were used in a preparative column (250 × 4.6 mm). 10, 20, 50, 100 and 200 μL of licorice extract were injected into each column.

5 and 6 show chromatograph analysis at particle diameters of 40/63 and 12 μm, respectively. GA is well separated in both preparative columns when the results of the 10 μL injection volume are compared. Peaks were disturbed on 40/63 μm columns.

As the injection volume is increased to 100 μL, as shown in Table 4, GA is not purely separated and the other two components are hardly detected in 40/63 μm columns.

Larger particle sizes reduce column efficiency and resolution due to the smaller contact area, wider thermal diffusivity, and longer outflow path of the sample with a solid packing surface. From these results, a preparative column with a particle diameter of 12 μm should be used.

Injection volume (μL) Peak width 40/63 μm 12 μm LQ GA Glabridine LQ GA Glabridine 10 1.38 1.54 1.02 0.87 0.78 0.50 30 1.78 1.61 1.12 1.23 0.88 0.63 50 1.84 1.66 1.52 1.38 1.14 0.77 100 * 1.68 * 1.78 1.61 * 150 * 1.70 * 1.96 1.79 * 200 * 1.82 * * 1.84 *

*: Not detected

Figure 1 shows the extraction concentration of LQ, GA and glabridine with immersion time,

Figure 2 shows the extraction concentration of LQ, GA and glabridine with sonication time,

Figure 3 shows the extraction concentrations of LQ, GA and glabridine according to the methanol capacity,

Figure 4 shows the chromatograph according to the injection capacity of the licorice extract using a column of 5μm (red line: 10μL, black line: 75μL),

Figure 5 shows the chromatograph according to the injection capacity of the licorice extract using a column of 40 / 63μm (black line: 10μL, red line: 100μL, green line: 200μL),

Figure 6 shows the chromatograph according to the injection capacity of the licorice extract using a column of 12μm (black line: 10μL, red line: 100μL, green line: 200μL).

Claims (3)

Adding methanol or aqueous methanol solution to licorice and immersing at room temperature for 20-120 minutes to extract; And Separation of Liquiritin, Glycirizinic Acid, and Glybridine from Licorice by Reversed-Phase Liquid Chromatography Using Mobile Phase Composed of Acetonitrile and 1% (v / v) Acetic Acid Extraction method comprising a. The method according to claim 1, wherein the methanol or aqueous methanol solution is added in an amount of 15 to 70 parts by weight based on 1 part by weight of licorice. The method of claim 1, wherein the reversed phase liquid chromatography uses a 12 μm column.

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