KR20110050779A - A method for isolation of antioxidant from glycyrrhiza uralensis - Google Patents

Abstract

PURPOSE: A method for separating antioxidant material from licorice is provided to improve the efficiency of a dehyroglyasperin C, dehyroglyasperin D, and isoangustone A obtaining process from the licorice. CONSTITUTION: A fixed phase solvent is filled in the column of a high speed counter-current chromatography. A mobile phase solvent is injected into the column while the column is being rotated. When the fixed phase solvent is hydromechanically equivalent with the mobile phase solvent, licorice extract is injected through a sample injection hole in order to obtain effluent. Antioxidant materials are separated from the effluent.

Description

A METHOD FOR ISOLATION OF ANTIOXIDANT FROM GLYCYRRHIZA URALENSIS}

The present invention relates to a separation method capable of simultaneously separating three kinds of antioxidants having various functionalities including antioxidant capacity from licorice.

Licorice ( Glycyrrhiza The roots and rhizomes of sp) have long been used as herbs and natural sweeteners worldwide. Licorice root has been used mainly as a traditional medicine to treat peptic ulcers, hepatitis C, pulmonary diseases and skin diseases. Pharmacological studies have reported that they have biological activities such as antioxidants, antiviral agents, anti-inflammatory agents, anticancer agents, immunomodulatory, hepatoprotective and cardioprotective. In addition, many functional substances such as triterpene saponins, flavonoids, isoflavonoids or chalcones have been isolated from licorice. Among these, various flavonoids are known to have major biological activities.

In particular, dehyroglyasperin C, dehyroglyasperin D and isoangustone A have recently been reported toxins such as quinone reductase or glutathione S-transferase. It has been reported to induce detoxifying enzymes, have significant PPAR-gamma ligand-binding activity, and have high anti-inflammatory activity.

Meanwhile, 2,2'-diphenyl-picrylhydrazyl radical (DPPH ·) and 2,2'-axinobis- (3-ethylbenzothiaxoline-6-sulfonate radical cation (ABTS + ·) were used to detect radical scavenging compounds after liquid chromatography separation. Many papers have been published, because they are highly sensitive and easy to handle, and the on-line HPLC bioassay method is an easy way to manipulate individual antioxidants in complex matrices. The advantage of sensitive identification of antioxidants is that the application of ABTS + -HPLC on-line technology enables rapid identification of radical scavenging components in licorice. Or biological activity from natural products using traditional methods such as high-performance liquid chromatography (HPLC). Compared to the method for separating and purifying the components, there is an advantage that it is quick and simple.

High-speed countercurrent chromatography (HSCCC) was first invented by Ito, a type of liquid-liquid partition chromatography without any sorbent, traditional column chromatography. Eliminates the irreversible absorption of the sample on a solid support at. This technique has the best ability to select a wider range of solvent systems compared to HPLC with excellent sample recovery.

In the case of separating the material by using high-speed countercurrent chromatography, the solvent system that will constitute the fixed and mobile phases according to the chemical properties of the sample and the material, ie, the type and composition of the mixed solvent, should be adjusted. In performing high-speed counter chromatography, conditions for improving the purity, separation efficiency and time of the separated material should be established.

In order to solve the problems of the prior art, an object of the present invention is to provide a method for simultaneously separating a number of excellent antioxidant properties from licorice.

More specifically, the present invention is a method for simultaneously separating the dihydrologasperin C, dihydrologasperin D and iso-angostone A having excellent antioxidant capacity from licorice using high-speed countercurrent chromatography to solve the problems of the prior art. The purpose is to provide.

In order to achieve the above object, the present invention provides a method for simultaneously separating dihydrologeriasperin C, dihydrologriasperin D and isoangstone A from the licorice.

Hereinafter, the present invention will be described in more detail.

We use Glycyrrhiza While studying how to separate antioxidants from uralensis , the antioxidants dehyroglyasperin C, dehyroglyasperin D and isoangustone A In the case of performing the separation process by high-speed counter current chromatography in order to separate at the same time in a simpler manner, and performing the high-speed countercurrent chromatography under specific conditions using a specific solvent system, It was confirmed that the dihydrologriasperin C, dihydrologriasperin D and isoangustone A of can be effectively separated, to complete the present invention based on this.

The present invention relates to a method for separating an excellent antioxidant activity from licorice.

In the present invention, the licorice is a perennial herb belonging to the Rosaceae legume, and means a representative plant in which different kinds of production are mixed among plants used as natural herbal medicines. The licorice is an example licorice ( Glycyrrhiza uralensis ).

In the present invention, high-speed countercurrent chromatography (HSCCC) is a kind of liquid-liquid partition chromatography without any adsorbent, and is a two-phase solvent system or a two-phase solvent. One of the upper or upper and lower and lower layers is used as the stationary phase, and after filling into the coiled tube column, the other layer is injected and discharged into the mobile phase while the stationary phase is maintained centrifugally in the column. By liquid-liquid fraction chromatography separating the components.

The present invention relates to a method for simultaneously separating dihydrasperin C, dihydrologasperin D and isoangstone A from licorice using high-speed countercurrent chromatography.

Specifically, the present invention comprises the steps of filling a fixed-bed solvent in the column of the high-speed countercurrent chromatography apparatus; Injecting a mobile phase solvent into the column while rotating the column packed with the fixed phase solvent; Dihydrologeriasperin C from licorice using high-speed countercurrent chromatography, comprising injecting a licorice extract through a sample inlet at a time when the stationary phase and the mobile phase are in fluidic equilibrium; The present invention relates to a method for simultaneously separating dihydrologriasperin D and isoanguston A.

The filling of the fixed bed solvent in the column of the high speed counter chromatography device may include filling the fixed bed solvent in a column of the high speed counter chromatography device, for example, a multilayer coil column, and the filling speed of the fixed bed solvent. May be, for example, 10 to 20 ml / min or 10 to 15 ml / min.

Injecting the mobile phase solvent into the column while rotating the column packed with the fixed phase solvent may be performed by a method of injecting the mobile phase solvent while rotating the column filled with the fixed phase solvent at a constant speed. The rotational speed of the column can be determined taking into account the retention of the stationary phase (Sr) related to column efficiency and peak resolution.

The higher the retention of the stationary phase, the better the peak resolution, and if the rotational speed of the column is too low, the amount of stationary phase staying in the column may decrease and the separation efficiency may decrease. Since the violent pulsation occurs in the column due to pressure, and the band of the sample to be separated tends to be too broadening, the rotation speed of the column is preferably 350 rpm, which is excellent in column efficiency and peak resolution. To 550 rpm, preferably 375 rpm to 525 rpm, more preferably 375 rpm to 425 rpm.

If the injection rate of the mobile phase solvent is too low, the elution time is long, there is a problem in the process, if the injection rate is too high, there is a problem in that the retention of the fixed phase is low, column efficiency and peak resolution, The infusion rate may be 6 ml / min to 8 ml / min, more preferably 6.5 ml / min to 7.5 ml / min.

The stationary and mobile phase solvents may be in the upper and lower phases of a two-phase solvent system.

The two-phase solvent system may be a mixed solvent of n-hexane, ethyl acetate, methanol and water. The mixing ratio of the mixed solvent may be determined in consideration of the partition coefficient (K) and the settling time, and satisfactory separation may not be obtained if the first material to be separated is eluted too close to the forward line of the separation peak, If the difference between the values of the distribution coefficients is not equal, the separation of certain substances may not be smooth. In addition, considering the dihydrologasperin C, dihydrologasperin D and isoangustone A, which are substances to be separated, the distribution coefficient is equal to the difference in the value of the distribution coefficient for each substance at a value between 0.3 and 2.5. One is most preferred.

In this aspect, the mixing ratio (v / v) of n-hexane, ethylacetic acid, methanol and water is preferably 6 to 6.7: 5 to 5.7: 5.7 to 6.3: 3.7 to 4.3 (n-hexane: ethyl) based on the volume ratio. Acetic acid: methanol: water), more preferably 6.3 to 6.7: 5.3 to 5.5: 5.8 to 6.2: 3.8 to 4.2 (n-hexane: ethyl acetate: methanol: water).

In relation to the stationary phase and the mobile phase, the solvent system, i.e., n-hexane, ethylacetic acid, methanol and water, is mixed in the mixing ratio of the present invention and allowed to stand at room temperature, and then the upper or upper layer is fixed It is a solvent, and lower layer or lower layer is used as a mobile phase solvent.

The injecting the sample may be performed by injecting a sample through a sample inlet at a time when the stationary phase and the mobile phase achieve a fluid equilibrium, and the sample may be a licorice extract.

The licorice extract may be obtained by extracting licorice by a conventional plant extraction method. For example, the licorice, licorice ( Glycyrrhiza uralensis ) or Siberian licorice ( Glycyrrhiza) glabra var . glandifera ), preferably an excellent licorice ( Glycyrrhiza) uralensis ). In addition, the licorice may be any one or more selected from the group consisting of leaves, stems and roots, preferably one or more selected from the group consisting of roots and stems, more preferably may be a root.

Licorice extract of the present invention can be prepared by a conventional plant extraction method using an extraction solvent, for example, solvent extraction or ultrasonic extraction, preferably ultrasonic extraction can be used.

The extractant may be an alcohol having 1 to 5 carbon atoms, hexane, dichloromethane, methylene chloride (methylene chloride), chloroform, ethyl acetate, ethyl ether, or a mixed solvent thereof, and preferably methylene chloride.

The ultrasonic extraction method may be an ultrasonic grinding extraction method, the ultrasonic grinding extraction method is 40 to 60 ℃, preferably 45 to 55 ℃ and 5 to 100 kHZ, preferably 10 to 75 kHZ, more preferably 20 to 40 It can be carried out by the ultrasonic grinding extraction method for 10 to 30 days, preferably 14 to 21 days in the condition of kHZ. The extraction can be extracted by repeating three times, the extract extracted three times it can be concentrated together under reduced pressure conditions to obtain a licorice extract. In addition, the extract prepared by the above method may be concentrated or removed by performing a filtration under reduced pressure, or further concentrated and / or lyophilized.

Specifically, the licorice extract may be prepared by grinding licorice roots washed with running water with a mill, adding methylene chloride, and then performing ultrasonic grinding extraction using an ultrasonic grinding extractor. The extract may be prepared by performing filtration or concentrating and / or lyophilization of the obtained extract. The filtration process may remove residues left in the extract, and may be concentrated and / or lyophilized to remove moisture.

The time point at which the stationary phase and the mobile phase are in fluidic equilibrium may be a time point at which the injected mobile phase solvent is discharged at the outlet of the column. Infusion of the licorice extract, for example, may be performed by the method of injecting the licorice extract into the separation column through the sample inlet.

Separation of the antioxidant from the eluate may be monitored by using a UV detector, an IR detector, a diode array, a fluorescence detector, or by using an HPLC method, and the material corresponding to each peak fraction may be monitored. This can be done by collecting them separately. Specifically, the step of separating the antioxidant may be performed by a method of collecting the eluate discharged through the separation valve at the same speed as the injection speed of the mobile phase.

The antioxidant substance may be dihydrologeriasperin C, dihydrologriasperin D and isoanguston A, and the structural formula of the dihydrologriasperin C, dihydrologriasperin D and isoanguston A is shown in FIG. It is as described in.

Collection of the eluate is, for example, the flow rate of the injection phase of the mobile phase is 7 ml / min, the rotational speed is based on 400 rpm, in the case of the dihydrologeria sprin C 75 minutes to 88 minutes from the eluate is discharged The eluate can be separated by a method of collecting eluate at the point of elapsed time, and the dihydrologriasperin D can be separated by a method of collecting eluate at a point of 189 to 221 minutes after the eluate is discharged. The isoangustone A may be separated by a method of collecting the eluate at the point 284 to 333 minutes have elapsed from the point at which the eluate is discharged. The dihydrologriasperin C, dihydrologriasperin D, and isoangustone A are separated in time intervals from the time when the eluate, which is the separation point, elapsed from the discharge time, can separate each substance with high purity. In addition, the solvent system and the fast reflux chromatography operating conditions of the present invention have an advantageous effect in that three materials can be separated simultaneously by performing one fast reflux chromatography.

The substance can be obtained by performing the distillation under reduced pressure on the separated eluate to remove the solvent.

Each material obtained from the licorice extract 700 mg by the above method is 72.3 mg for dihydrologersaprine C, purity 95.1%, 73 mg for dihydrologersaprine D, purity 96.2%, Isoanguston A was 47.1 mg with a purity of 99.5%.

According to the separation method of the present invention, since dihydrologriasperin C, dihydrologasperin D, and isoangstone A, which are excellent antioxidant substances, can be simultaneously separated from licorice with high purity and high efficiency, By using domestic licorice which is a cheap and easily available natural product in Korea, it is possible to easily and economically separate high value-added antioxidants such as dihydrasperin C, dihydrologasperin D and isoangstone A. Since the material can be used in a variety of industries associated with the medical industry, food industry or cosmetics industry, etc., the industrial effect will be very large.

Hereinafter, preferred embodiments of the present invention will be described. However, the following examples are only preferred embodiments of the present invention, and the scope of the present invention is not limited to the following examples.

Example 1 Preparation of Sample

Example 1-1. sample

Implementation of the present invention, specifically n-hexane (n-Hexane), ethylacetate, acetonitrile (acetonitrile) used in the preparation of licorice extract and high-speed counter-current chromatography (HSCCC) And methanol were purchased from Fisher Scientific (USA).

Roots of licorice ( G. uralensis ) for the preparation of licorice extract was purchased from the drugstore of Chuncheon, Republic of Korea. The purchased licorice was ground and used before extraction.

For the recording of NMR spectra, tetramethylsilane was used as an internal standard and 600 MHz spectrometer (Bruker) was used.

Example 1-2. Preparation of Licorice Extract

0.5 kg of root of licorice ( G. uralensis ) was extracted with 3 L of methylene chloride (methylene chloride) as a solvent and ultrasonically extracted for 1 hour using Power SONIC 420 ultrasonic cleaner (Hwashin Instrument Co., South Korea). The extract prepared by the above method was concentrated under reduced pressure using an EYELA N-1000 rotary evaporator (Tokyo Rikakikai Co., Japan) to prepare 125 g of crude extract for separation of high-speed countercurrent chromatography (HSCCC). It was.

Example 2 Analysis of Antioxidants in Licorice Extract

In order to analyze the antioxidants contained in licorice, radical scavenging activity was measured using an online HPLC-ABTS + assay.

The on-line HPLC-ABTS + assay stabilized radicals by overnight incubation with 2 mM ABTS + stock solution containing 3.5 mM potassium persulphate at room temperature and dark. The ABTS + reagent was prepared by diluting the stock solution 30 times with water.

Antioxidant isolation was achieved by Agilent Zorbax SB-C18 reversed phase column (150) connected to a 2000 Gradient Pump (Thermo Separation Products, USA), AS3000-021 thermostatted autosampler and Agilent Zorbax Extend C18 guard column (10 mm × 4.6 mm id, 5 μm). mm × 4.6 mm, id, 5 μm) using an SCM 1000-022 degassing device by constant temperature HPLC analysis (analytic Thermo HPLC).

Mobile phase was prepared with 0.03% aqueous TFA solution (A) and acetonitrile (B). The gradient program of the present invention is carried out at 0.8 ml / min flow rate using a Thermo 6000 LP detector at 254 nm wavelength and 30% at 5-5 minutes and 30% at 5-13 minutes. 40% at 40% to 65% at 13 to 48 minutes, 65% at 48 to 53 minutes and 65% to 100% at 53 to 58 minutes. The analyte separated by HPLC was reacted with post-column with ABTS +. ABTS reagent was continuously injected at 0.3 ml / min via Pinnacle PCX (Pickering Lab, Germany). The bleaching of the ABTS reagent by the analyte was detected by means of a photometric measurement at 734 nm by a Thermo 2000 LP detector (Thermo-Electron Corp., Waltham, Mass.), Which is indicated by a negative peak in FIG. 1. .

As shown in FIG. 1, when the light peak at 734 nm was measured, the presence of a high radical scavenging compound was confirmed in the licorice extract, and the retention time was 25.47 minutes, 39.48 minutes, and 43.85. Three peaks with min were subsequently specified for HSCCC separation.

Example  3: high-speed countercurrent chromatography high - speed counter current chromatography , HSCCC )

HSCCC for the practice of the present invention used a TBE-1000A HSCCC (Tauto Biotechnique Company, China) having a 50 ml sample loop connected in series with three multi-layered coil columns. The internal diameter of the PTFE (polytetrafluoroethylene) tube column of the instrument is 1.8 mm and the total volume capacity (total capacity) is 1000 ml. The β value of the column varied from 0.42 in the inner layer to 0.63 in the outer layer. The HSCCC instrument of the present invention was equipped with a Merck-Hitachi L-6200 intelligent pump (Hitach, Japan), a TOPAZ dual UV monitor operating at 254 nm and an ECOMAC-ECOM Acquisition and control (Version 0.97). In the HSCCC device of the present invention, the solvent of the present invention is transferred to a column, in particular a PTFE column, via a pump, and the antioxidants separated by the mobile phase in the solvent are continuously monitored.

In addition, HPLC analysis was performed by Agilent technologies 1100 with G1322A gas remover, G1311A quaternary pump, G1313A automatic sample injector, G1315B photodiode array detector, and Chemstation RA 10.02 software.

Example  4: Implementation of Optimal Solvent System

In the practice of the present invention, in order to implement an optimum solvent system for simultaneously separating three target substances having antioxidant activity through the HSCCC method, a two-phase solvent while varying the solvent volume between the mixed solvents. The solvent mixing ratio for the system was obtained. The optimum solvent system is determined by the range of distribution coefficients (K) and the short settling time for the material to be separated.

The upper and lower phases of the two-phase solvent system were measured by HPLC, and the partition coefficient and settling time were determined from the measured values. More specifically, the K value is represented by the ratio of the peak area value of the target compound separated by the lower phase and the peak area value of the target compound separated by the upper phase. In addition, the settling time is a value related to the retention in the column of the fixed phase, and when each layer of the two-phase solvent system is mixed, a distinct layer is formed between the two phases, and the time is divided into two phases. Obtained.

The solvent used in the solvent system was n-hexane, ethyl acetate, methanol and water that can give a variety of hydrophobicity to the solvent system by adjusting the mixing ratio of the solvent was used, the mixing ratio of the solvent is It was determined by the distribution coefficient and the settling time. In order to be able to separate three substances simultaneously, i.e. through one HSCCC, the distribution coefficient was determined to be constant in the distribution coefficient value of each component at a value between 0.3 and 2.5 for each component. The delamination took place quickly within 15 seconds and adjusted to be as short as possible.

Determination of the partition coefficient of the target substance with antioxidant activity from the licorice extract was performed by analyzing the results of analyzing the upper phase and the lower phase by HPLC, and the results are shown in Table 1 below. Shown in The partition coefficients are shown in the following table ratios for the target substances I, II and III having the antioxidant activity described in FIG. 1, respectively.

TABLE 1

No Solvent system Mixing ratio
(v / v) Distribution coefficient (K) Settlement
Time (s) I II III One n-Heptane: ethylacetate: methanol: water 6: 4: 6: 4 0.18 0.64 0.84 11 2 n-Heptane: ethylacetate: methanol: water 6.5: 3.5: 6: 4 0.18 0.71 0.92 15 3 n-Hexane: ethylacetate: methanol: water 8: 3: 6: 4 0.14 0.78 1.42 15 4 n-Hexane: ethylacetate: methanol: water 7: 4: 6: 4 0.24 1.04 1.89 17 5 n-Hexane: ethylacetate: methanol: water 7: 3: 6: 4 0.12 0.74 1.35 17 6 n-Hexane: ethylacetate: methanol: water 7: 3: 5: 4 0.28 1.51 3.28 16 7 n-Hexane: ethylacetate: methanol: water 6.5: 5.5: 6: 4 0.42 1.19 2.00 13 8 n-Hexane: ethylacetate: methanol: water 6: 5.5: 6: 4 0.37 1.25 2.13 13 9 n-Hexane: ethylacetate: methanol: water: isopropanol 6: 5: 4: 4: 2 0.62 1.62 2.89 15 10 n-Hexane: ethylacetate: methanol: water: isopropanol 6: 5: 5: 4: 1 0.47 1.36 2.36 15

As shown in Table 1, when the mixing ratio of the n-heptane ethylacetate methanolwater mixed solvent is 6: 4: 6: 4 (v / v), the mobile phase has a strong polarity, As the first active substance I eluted near the forward line, satisfactory separation could not be obtained, and the distribution coefficients of the second active substance Ⅱ and the third active substance III were close to each other, resulting in overlapping peaks, resulting in decreased separation efficiency. It was confirmed that it is not preferable. In addition, in the case of a mixed solvent of n-heptaneethylacetatemethanolwater-isopropanol (HEMWiP), the partition coefficient of I, the first active substance, was improved, but the partition coefficient of III, the third active substance, was improved. The value was high and found to be unfavorable.

Based on the results, the mixing ratio of n-heptane-ethyl acetate-methanol-water (HEMW) mixed solvent was adjusted by increasing the volume ratio of ethyl acetate and decreasing the volume ratio of n-hexane. In the 6.5: 5.5: 6: 4 volume ratio, the partition coefficient of each active substance had a value between 0.3 and 2.5, and the difference in partition coefficient value of each component was not only constant, but also the settling time was short and the retention rate in the column was It was confirmed that it was high and preferable.

Example  5: optimal HSCCC  Confirmation of operating conditions

Stationary phase retention (Sr) in HSCCC is an important parameter related to column efficiency, peak resolution and derivation of solute retention. More specifically, the successful separation of the material of interest in the HSCCC is determined by the retention of the stationary phase, ie the stationary phase, which is retained in the column, and in general, the higher the stationary phase, the better the peak resolution. .

Therefore, in order to confirm the optimum HSCCC operating conditions, Sr was measured while varying the flow rates and rotational speeds of the stationary phase, and the optimum operating conditions were determined in consideration of the dissolution time according to the measured flow rates of Sr and the stationary phase. In the experiments on the operating conditions of the HSCCC, the solvent system is a mixture ratio of n-hexane-ethylacetic acid-methanol-water (HEMW) which is confirmed as the optimum solvent system in Example 4 is 6.5: 5.5: 6: 4 based on the volume ratio The experiment was carried out by dissolving 700 mg of the licorice extract prepared in Example 1 in 50 ml of the solvent system with respect to the sample. More specifically, first, the upper layer (fixed phase) is filled in a multilayer coil column, and then the lower layer (mobile phase) is injected into the column using a pump while rotating at a constant rotational speed. It was determined that the hydrodynamic equilibrium was established when the mobile phase solvent was discharged from the outlet of the column, and the sample solution was injected into the separation column using the sample inlet. Emissions from the separation column were continuously monitored using a UV detector at 254 nm. Each of the peak fractions were collected separately to identify the separated compound through HPLC, and the retention of the stationary phase (Sr) was measured. The results are shown in FIG. 2.

As shown in FIG. 2, as a result of measuring at the same flow rate of 7 ml / min, it was confirmed that Sr is about 4% higher than 71% when the rotational speed is 400 rpm compared to the rotational speed of 500 rpm. , It was found to be more preferred. In addition, when the flow rate of the stationary phase was changed from 5 ml / min to 9 ml / min during the same rotational speed of 400 rpm, Sr decreased from 75% to 65% as the flow rate increased. In particular, when the flow rate is increased from 5 ml / min to 7 ml / min, Sr decreases by 4%, while when the flow rate is increased from 7 ml / min to 9 ml / min, Sr decreases by 6% to increase the same flow rate. In comparison, it was found that the degree of reduction of Sr was remarkable.

Considering the above results and the problem that the dissolution time is long when the flow rate is low, the conditions of rotation speed 400 rpm and flow rate 7 ml / min were confirmed as the most suitable conditions in Sr, dissolution time and peak resolution.

Example  6: identification of active substances

The fraction of each peak separated in Example 5 was analyzed via HPLC. More specifically, the HPLC was carried out as shown in Example 2, Agilent Zorbax SB-C18 reversed phase column (150 mm × 4.6 mm, id, 5 μm) connected to the Agilent Zorbax Extend C18 guard column (10 mm × 4.6 mm id, 5 μm) ) Was performed. The mobile phase consisted of trifluoroacetic acid (TFA, A) and acetonitrile (acetonitrile, B) in 0.1% water, and the gradient program of HPLC was 0.8 ml / using a Thermo 6000 LP detector at 254 nm wavelength. min flow rate at 42% at 0-10 minutes, 42% at 55% at 10-12 minutes, 55% at 60% at 12-17 minutes, 60, 25 at 17-25 minutes 60 to 100% in minutes to 28 minutes. The injection amount was 10 mL, analysis was performed at room temperature, and the identification of the peak fraction was performed by ESI-Mass, ¹ H-NMR and ¹³ C-NMR, and the results are shown in FIGS. 3 to 5.

As shown in FIG. 3, 72.3 mg of the antioxidant active substance I obtained through simple purification from 700 mg of the licorice extract as a sample was obtained for 75 to 88 minutes, and 73 mg of the active substance II was obtained for 189 to 221 minutes. The active substance III obtained 47.1 mg in 284-333 minutes. Purity of the material was 95.1%, Activator II was 96.2%, and Activator III was 99.5%, respectively, as determined by HPLC.

The peak fractions analyzed by HPLC were identified by performing NMR using ESI-Mass and ¹ H-NMR and ¹³ C-NMR. 4, the active substance I corresponding to the peak I is dehydroglyasperin C, and the active substance II corresponding to the peak II is Dehydroglyasperin D (dehydroglyasperin D), the active material III corresponding to the peak III was confirmed to be isoangustone A (isoangustone A).

Peak I: IR (KBr) 3351, 2967, 1699, 1616, 1514, 1167, 839 cm ¹ ; UV max 289, 329 nm; ESI-MS, C ₂₁ H ₂₂ O ₅ , 355 [M] ⁺ , 354 [M ^○ + ^○ H] ⁺ .

Peak II: IR (KBr) 3375, 2929, 1613, 1516, 1458, 1308, 1198, 1164, 978, 837 cm ¹ ; UV max 243, 330 nm; ESI-MS, C ₂₂ H ₂₄ O ₅ , 368 [M] ⁺ , 369 [M ^○ + ^○ H] ⁺ .

Peak III: IR (KBr) 3315, 2918, 1687, 1515, 1169, 831 cm ¹ ; UV max 225, 264, 306 nm; ESI-MS, C ₂₅ H ₂₆ O ₆ , 422 [M] ⁺ , 423 [M ^○ + ^○ H] ⁺ .

Dihydrogliasperin C, dihydrogliasperin D, and isoangostone A identified as the active substances I to III are shown in FIG. 5.

According to the present invention, the antioxidants dihydrogliasperin C, dihydrogliasperin D and isoangstone A contained in licorice extract extracted from G. uralensis can be separated in one separation process using HSCCC. In particular, using specific solvent systems and specific HSCCC operating conditions established in the present invention, 72.3 mg (about 10.3%) of dihydrogliasperin C with a purity of 95.1% (about 10.3%) and dihydrogliasperin with a purity of 96.2% from 700 mg of crude extract. It was found that 73.0 mg (about 10.4%) of D and 47.1 mg (about 6.8%) of isoangustone A with a purity of 99.5% were obtained, which separated antioxidants from certain active substances, especially licorice, from natural materials. It has been found to be an effective method, and by the above method, the separation of antioxidants from natural products is facilitated, and thus the medical industry, food industry and cosmetics where antioxidants are used. It is expected to be applied to various industries, including industry.

1 is a graph showing the results obtained by performing On-line HPLC-ABTS ⁺ radical scavenging chromatography from licorice extract according to an embodiment of the present invention, the upper trace (positive trace) at 254 nm HPLC (gradient reverse Licorice extract was analyzed by phase HPLC), and the lower trace (negative trace) was analyzed by antioxidant activity using ABTS ⁺ radical at 734 nm.

FIG. 2 is a graph measuring separation results of licorice extract according to flow rate and revolution speeds of a mobile phase according to one embodiment of the present invention at 254 nm, where Fr represents a flow rate and Rs Denotes the rotational speed and retention of the stationary phase retention in the coil (Sr).

3 is a graph (chromatograms) analyzed by HPLC for HSCCC separation results according to an embodiment of the present invention, FIG. 3a is a result of HPLC analysis of licorice extract, and FIG. 3b is left in the coil after HSCCC separation. HPLC analysis of the residual sample, FIG. 3C is the HPLC result of the first peak, FIG. 3D is the HPLC result of the second peak, and FIG. 3E is the HPLC result of the third peak.

FIG. 4 is a diagram showing an NMR analysis result (NMR chmical shlft) of a compound included in an eluate corresponding to a first peak, a second peak, and a third peak according to an embodiment of the present invention.

Figure 5 shows the structural formula of the compound contained in the eluate corresponding to each peak, according to an embodiment of the present invention, Figure 5a is a chemical structural formula of the dihydrologasperin C corresponding to the first peak, 5b is the chemical structural formula of dihydrologasperine D corresponding to the second peak, and FIG. 5C is a chemical structural formula of isoanguston A corresponding to the third peak.

Claims (4)

Filling a fixed bed solvent in a column of a high speed counter chromatography device; Injecting a mobile phase solvent into the column while rotating the column packed with the fixed phase solvent; Injecting licorice extract through a sample inlet at a time when the stationary phase and the mobile phase are in fluidic equilibrium; and Separating the antioxidant from the eluate, The fixed-phase solvent and the mobile phase solvent is n-hexane, ethyl acetate, methanol and water is 6 to 6.5: 5 to 5.5: 5.7 to 6.3: 3.7 to 4.3 based on the volume ratio (v / v) (n-hexane: ethyl acetate: After the mixed solvent mixed with methanol: water) was allowed to stand at room temperature, the upper layer was a fixed phase solvent and the lower layer was a mobile phase solvent. A method for simultaneously separating dihydrologriasperin C, dihydrologriasperin D, and isoangostone A from licorice using high-speed countercurrent chromatography. The method of claim 1, The licorice extract is obtained by using any one selected from the group consisting of alcohols having 1 to 5 carbon atoms, hexane, dichloromethane, methylene chloride, chloroform, ethyl acetate, ethyl ether, and a mixed solvent thereof as a solvent. A method of simultaneously separating hydroliasperin C, dihydrologriasperin D and isoanguston A. The method of claim 1, The step of injecting the mobile phase solvent is dihydrologeriasperin C, dihydrologeriasperin D and isoanguston A from licorice, characterized in that the mobile phase solvent is injected at a flow rate of 6 ml / min to 8 ml / min How to separate at the same time. The method of claim 1, Rotation of the column in the step of injecting the mobile phase solvent is a method for simultaneously separating the dihydrologasperin C, dihydrologasperin D and isoangstone A from licorice, characterized in that the rotation of the rotation at 350 rpm to 550 rpm.

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