KR20140079892A - A method for simultaneous separation of ginsenoside diols and triols from ginseng extract in one-step by HSCCC isolation - Google Patents

Abstract

The present invention relates to a method for separating ginsenoside from a ginseng extract in one step using a counter-current chromatography. A certain amount and more of high purity of 14 kinds of ginsenoside can be obtained within a short time (100-400 minutes) through one chromatography process without going through a special process. Moreover, an expensive column with a limited reuse is not needed, and the use of a solvent is drastically reduced.

Description

[0001] The present invention relates to a method for separating ginsenoside diol and triol from ginseng extract in one step by means of countercurrent chromatography, and more particularly to a method for simultaneous separation of ginsenoside diol and triol from ginseng extract in one step [

The present invention relates to a method for simultaneous separation of ginsenoside diol and triol saponins from ginseng extract in one step using countercurrent chromatography.

[1] Park, Jong - Dae, A Study on Chemical Components, Latest KOREA Ginseng Research (Korea Ginseng Society) pp.31 ~ 51, 2007

Christensen, L. P, Ginsenosides: Chemistry, Biosynthesis, Analysis, and Potential Health Effects, in Advances in Food Nutritional Reviews, 55, 1-99, 2009

[Patent Document 3] Korean Patent Publication No. 1998-0009275

[Literature 4] Ito Y J. Chromatography, 147 , p. 221, 1978

[Literature 5] Lee Chang-ho, Lee Bu-yong, Korean Food Science Society, 36, 518-521, 2004

[Literature 6] Phytochemical and analytical studies of Panax notoginseng (Burk.) F.H. Chen J. Nat. Med. (2006) 60: 97.106)

[Literature 7] Liquid chromatographic determination of less polar ginsenosides in processed ginseng, Journal of Chromatography A, 921 (2001) 335339

[Literature 8] Developments in the application of counter-current chromatography to plant analysis, Journal of Chromatography A, 1112 (2006) 181194

[Document 9] CCC Review paper, Journal of Chromatography A, 808 (1998) 322

[Literature 10] United States Patent USP patent. 6,444,233

[Document 11] Journal of Liquid Chromatography. & Related Technologies, 18, 1655-1662, 1995

[Document 12] Du et al., Journal of Chromatography A, 1008, 173-200, 2003

[Literature 13] Liu et al., Chromatographia, 71, 267-271, 2010

[14] Ha et al., J. Chromatography A, 1151, 37-44, 2007

[Document 15] Qi et al., J. Chromatography A, 1217, 1955-2001, 2010

[Document 16] Cheng et al., Separation and Purification Technology, 77, 347-354, 2011

[Document 17] Shezad, O et al., J. Separation Science, 34, 1116-1122, 2011

[Literature 18] Journal of Separation Science, 35, 1462-1469, 2012.

The present invention relates to a method for separating ginsenosides from ginseng extracts, and more particularly, to a method for separating ginsenosides from ginseng extracts using counter-current chromatography.

Ginseng ( Panax ginseng CA Meyer) is a perennial herbaceous plant belonging to the genus Ginseng (Araliaceae). It is one of the representative medicinal crops mainly used in oriental medicine. After 4 to 6 years of harvesting according to the post-harvest processing method, the unprocessed raw ginseng is peeled off from fresh ginseng and 4-6 years old ginseng, dried naturally in the sun or dried by hot air at 60 ° C or less, Ginseng, steamed with steam for 4-6 years, without peel, is classified into red ginseng.

     The major physiologically active components of ginseng include saponin, essential oil components, polyacetylene, phenol components, glycosides, and acid peptides [Park Jong Dae, A Study on Chemical Components, Latest KOREA Ginseng Research , 2007]. Among them, saponin, which is the most representative physiologically active ingredient, has been studied from the 1960s to the recent years on the separation of a new saponin component and the physiological activity of a saponin component. Ginseng saponin is a unique model dammarane triterpene glycoside contained only in Panax genus plants. Saponin of ginseng is also called ginsenoside. Until recently, more than 35 kinds of structures have been revealed lost. The ginseng saponin has been studied for its physiological activity in a short period of time. It is believed that the effect on the central nervous system, brain function improving action, anticarcinogenic and anti-cancer action, immune function control action, antidiabetic action, liver function enhancement action, Stress, anti-fatigue, and antioxidant effects have been reported [Christensen, LP, Ginsenosides: Chemistry, Biosynthesis, Analysis, and Potential Health Effects, in Advances in Food Nutritional Reviews, 55, 1-99, 2009].

     Rg1, Re, Rf, Rh1, Rb1, Rb2, Rc, and Rd as major saponins in the ginseng and white ginseng are 80-90% or more of total saponins. The pharmacological activity of ginsenosides As shown in Fig.

     It is known that red ginseng steamed and steamed is superior in its efficacy because it produces a variety of red ginseng-specific components beneficial to the human body during the processing of red ginseng. Rg3, Rg5, Rk1 and F4, which are low polarity ginsenosides, are known to exert their pharmacological effects such as antioxidant activity, vasodilatory action, nerve cell protective action and anti-cancer action. In addition, it contains Rh2, compound K, which is known to have anticancer activity in the diol system, and Rg2 and Rg6, which are triol series, but it is very difficult to separate it very easily.

     Conventionally, the method used to obtain ginsenosides individually in high purity is based on a conventional column chromatography method in which a solid resin is used as a stationary phase and a liquid solvent is used as a mobile phase. For example, in Korean Patent Laid-Open Publication No. 1998-0009275, a silica gel and a C18 resin are fixed on a column of a solid phase, and an appropriate solvent such as methanol and chloroform is selected as a mobile phase solvent, Using conventional chromatography to separate ginsenosides.

      However, such a conventional method has a problem that the solute is irreversibly adsorbed on the stationary phase resin of the column, resulting in the loss of the ginsenoside to be separated, the lifetime of the column is limited, the cost of the column is expensive, It is necessary to repeat the column work at least 2 or 3 times, so that it takes a lot of time and effort, and the solvent must be consumed. That is, in order to obtain a high purity ginsenoside of a certain amount or more by a conventional method, a complicated and cumbersome process must be performed.

     Recently, high-speed liquid chromatography has been widely used for mass separation. Although it has the advantages of high resolution and fast separation time, the amount of sample that can be injected depends on the capacity of the column and is extremely expensive. For example, the amount that can be injected into a 2.5 x 30 cm reversed-phase HPLC is up to 100-200 mg. Due to the price of the equipment and the periodic replacement of the solid stationary phase, it is more expensive than the amount of separation and it is difficult to industrialize the mass separation / production system. Also, when a solid stationary phase is used, loss of the sample occurs due to the problem of adsorption of the sample to the solid stationary phase. Solvents mainly used for the reversed phase column are acetonitrile, methanol and water. Acetonitrile is also an expensive solvent and also limits its separation in large quantities.

In contrast, countercurrent chromatography used in the present invention is a type of liquid-liquid partition chromatography using liquids as both a stationary phase and a mobile phase, and is recently used for separating natural substances. In particular, the high-speed counter-current chromatography (HSCCC) method developed by Ito et al. Uses a solvent system which is separated into two layers after mixing without mixing, Ito Y J. Chromatography, 147 , p. 221, 1978). The distribution process is continuous in a single capillary tube, with one layer as the mobile phase and the other as the stationary phase The column is a system in which three cylindrical holders of polytetrafluoroethylene (PTFE) tubes are wrapped in multiple layers to prevent twisting of the tube through rotation and revolution, and the stationary phase is used as a liquid, It is possible to prevent the loss due to the adsorption to the stationary phase and the deformation of the material due to the reaction with the solid stationary phase, It has many advantages such as low cost and separation of the points you can. In addition, it is not necessary to use expensive columns having limited reuse, and the amount of solvent used is greatly reduced.

Recently, the application of HSCCC has been actively used to mass-purify natural products. Ginsenoside separation of ginseng was also announced.

The prior art related to the present invention is as follows.

Lee et al. Used 5-year-old ginseng to separate and concentrate ginseng saponin using counter-current chromatography. However, Rc, Re, and Rg1 were not separated and were fractionated as a mixture. And that the ginsenosides Rh1, Rb2 and Rc as well as the ginsenosides Rg1, Re, Rf, Rb2 and Rd as well as the eight ginsenosides can be separated at once using countercurrent chromatography There is no. (Lee Chang-ho, Lee Bu-yong, Korean Food Science Journal, 36, 518-521, 2004).

An example of the isolation of various ginsenoside components of Notoginseng is given in Chong-Zhi Wang et al., Phytochemical and Analytical Studies of Panax notoginseng (Burk.) FH Chen J. Nat. Med. (2006) Rb1, Re, Rf, Rb2, Rd as well as ginsenosides Rh1, Rb2, and Rd as well as ginsenosides Rg1, Rb2, Rc There is no description that eight ginsenosides can be separated at once using countercurrent chromatography.

(2001) 335339), a new constituent component of processed ginseng (ginseng) prepared by heat treatment of white ginseng, which is a low-polarity ginseng (20 R ) and (20 S ) stereoisomers using a C 18 -bonded silica column method using the components ginsenoside F, Rg 4, Rg 3, Rg 5, Rk 6, Rk 1, Rs 3, Rs 3, However, the ginsenosides Rh1, Rb2, and Rc as well as the ginsenosides Rg1, Re, Rf, Rb2, and Rd as well as the eight ginsenosides are separated at once using countercurrent chromatography There is no description that it can be.

In Marston, A., et al., A comprehensive description of the separation of natural products using CCC is provided in the Developments in the Application of Counter-Current Chromatography to Plant Analysis, Journal of Chromatography A, 1112 (2006) 181194 (A) CHCl3-MeOH-2-BuOH-H2O; (b) EtOAc-1-BuOH-H2O; (c) n- Hexane-1-BuOH-H2O solvent system to isolate ginsenosides from ginseng (p187, Table 1). However, in this document, ginsenosides Rg1, Re, Rf, Rb2, Rd as well as ginsenosides Rh1, Rb2, and Rc. It is not disclosed that 8 ginsenosides can be separated at once using counter-current chromatography.

AP Foucault et al. (CCC Review paper, Journal of Chromatography A, 808 (1998) 322) disclose that acetogenins, 10-deacetyl-baccatin III, amphotericin amphotericin B and the like is disclosed as a separation method using counter-current chromatography (CCC) and centrifugal partition chromatography (CPC). However, in this document, ginsenosides Rg1, Re, Rf, Rb2 , Rd as well as the ginsenosides Rh1, Rb2, and Rc. It is not disclosed that the eight ginsenosides can be separated at one time by means of countercurrent chromatography.

US Patent of Arntzen (Charles J.) et al. USP patent. 6,444,233 discloses a method of separating new triterpene from Acacia victoriae plants by flash chromatography, DCCC, low-pressure liquid chromatography (LPLC), medium-pressure liquid chromatography (MPLC) The present invention is different from the present invention in terms of the raw material to be extracted and the final separation material, and in this document, ginsenosides Rg1, Re, Rf, Rb2 and Rd as well as ginsenosides Rh1 , Rb2, Rc There is no description that eight ginsenosides can be separated at once using countercurrent chromatography.

Le Men-Olivier et al. Extracted from US ginseng with saturated butanol, dialyzed, and dried residues were dissolved in a solvent system of ethyl acetate: butanol: water to give a gradient of 95: 1: 4 to 40: 46; 1 Re, Rb1 and Gypenoside XVII, F11 were separated in a pure manner over a period of 4 hours while gradually decreasing the concentration of the next butanol and increasing the proportion of water to 90% (Le Men-Olivier et al., Journal of Liquid Chromatography. & Related Technologies, 18, 1655-1662, 1995)

Du et al. Have isolated ginsenoside Rb1, noto ginsenoside R1, ginsenoside Re and ginsenoside Rg1 from Panax notoginseng in China (Du et al., Journal of Chromatography A, 1008, 173- 200, 2003). The solvent system used was a separation of 3: 4: 7 ratio of n-hexane: n-butanol: water to solvent ratio. However, this document also discloses that ginsenosides Rh1, Rb2, and Rc as well as ginsenosides Rg1, Re, Rf, Rb2, and Rd as well as ginsenosides can be separated at one time using countercurrent chromatography There is no content.

Liu et al. Reported that ginsenosides Rc, Rb1, and Re from the American ginseng (Panax quinquefolium) were dissolved in a ratio of solvent ethyl acetate: butanol: water 1: 1: 2 in the solvent using high speed centrifugal partition chromatography similar to countercurrent chromatography (Liu et al., Chromatographia, 71, 267-271, 2010). This document also discloses that ginsenosides Rg1, Re, Rf, Rb2 and Rd as well as ginsenosides Rh1, Rb2 and Rc There is no description that ginsenosides can be separated at once using countercurrent chromatography.

(Ha) et al. Reported that Rg3, Rg5, Rk1, and F4, which are weakly polarized ginsenosides in red ginseng, are converted to methylene chloride-methanol-isopropanol-water 6: 6: 1: 4 Solvent ratio. ≪ / RTI > The characteristic of the solvent system is that it does not use ethyl acetate and butanol, and the evaporative light scattering detector is separated as a detector. However, it is confined to low-polarity saponins. In this document, eight ginsenosides of ginsenosides Rh1, Rb2 and Rc as well as ginsenosides Rg1, Re, Rf, Rb2 and Rd as well as ginsenosides are analyzed by means of countercurrent chromatography There is no indication that it can be removed at once. (Ha et al., J. Chromatography A, 1151, 37-44, 2007)

Rf, Re, Rd and Rb1 were separated by high performance countercurrent chromatography using methylene chloride-methanol-5 mM ammonium acetate-isopropanol 6: 2: 4: 3 solvent system. It was characterized by shortening the separation time using ammonia acetate and keeping the flow rate at 20 ml / min until 67 min and 50 ml / min until 67-120 min. However, it is also limited to two kinds of ginsenoside triols (Rf, Re) and two diols (Rb1, Rd), and this document also discloses that ginsenosides Rg1, Re, Rf, Rb2, There is no description that senosides Rh1, Rb2, and Rc can be separated at one time by using countercurrent chromatography with a total of eight ginsenosides. (Qi et al., J. Chromatography A, 1217, 1955-2001, 2010)

Cheng et al. Reported Re, Rb1, Rb2 and Rc from ginseng in the ratio of 6: 2: 4: 3 methylene chloride-methanol-water-isopropanol as the first stage of high performance countercurrent chromatography, : n-butanol: 0.1% formic acid 6: 2: 4; 3 at a flow rate of 20 mL / min in the first stage and 50 mL / min in the second stage. Finally, the stationary phase was washed with 100 mL / min. The fractions containing senoside Rc were collected and separated by high performance liquid chromatography. Particularly, this method is remarkable in that the separation time is shortened by maintaining the flow velocity of the high-speed countercurrent chromatography at least ten times faster. This is because the equipment of the separation system is different. However, this method is also limited to the above-mentioned ginsenoside triol Re and diols Rb1, Rb2 and Rc. In this document, ginsenosides Rg1, Re, Rf, Rb2 and Rd as well as ginsenosides Rh1, Rb2, Rc There is no description that eight ginsenosides can be separated at once using countercurrent chromatography. (Cheng et al., Separation and Purification Technology, 77, 347-354, 2011).

Thus, the present inventors continued their studies to isolate 8 kinds of ginsenoside diol and triol dinsenoside at a time. First, using a polystyrene column, two of them were divided into chloroform / methanol / water / isopropanol 4: Rz, Rb, Rb, Rb, Rb, Rb and Rd are separated from each other by means of countercurrent chromatography to prepare ginsenosides Rh1, Rb2, Rc (Shezad, O et al., J. Separation Science, 34, 1116-1122, 2011).

However, in order to further improve the step and to separate the ginsenosides of different polarities at once, the separation solvent system is used as a gradient in chloroform / methanol / water / isopropanol, 5: 6; 1: 4; The reaction was carried out in three steps using chloroform / methanol / water / isopropanol 3: 3: 1: 2 in chloroform / methanol / water / isopropanol 4: Rg1, Re, Rf, Rb2, and Rd as well as the ginsenosides Rh1, Rb2, and Rc. Rh1, Rf, Rd and Rg1 were separated in the first stage, Re was in the second stage, Rb2, Rc and Rb1 were separated in the third stage and the flow rate was 4 ml. This method is characterized by a more systematic selection of the solvent system, in particular by separating the ginsenosides of different polarity by the gradient of three different solvents. However, the problem with the above technique is that column chromatography using polystyrene resin is used to remove sugars, and the solvent system contains chloroform known to be toxic. Further, there was a need to separate the ginsenoside triol and the diol separately. (Journal of Separation Science, 35, 1462-1469, 2012).

However, in the above literature, in order to separate ginsenosides regardless of the polarity of ginsenosides from all kinds of ginseng extracts such as white ginseng, processed ginseng, and red ginseng, a specific countercurrent chromatographic separation condition , There is no teaching or explanation about the method of separating ginsenosides by counter-current chromatography.

Thus, the present inventors have found that, for the optimal separation in countercurrent chromatography, in order to separate ginsenosides regardless of the polarity of ginsenosides from all kinds of ginseng extracts (white ginseng, processed ginseng, red ginseng) (2) In the case of ginsenosides of white ginseng, the isocratic elution method was used instead of the possible solvent gradient, and in the case of the sample containing low polarity ginsenoside, Since the polarity of the components of ginsenoside is different from that of the straight line, we choose two solvent systems based on the distribution constant and use solvent gradient to separate them. (4) As a result of adjusting the flow rate, the low polarity Jinsetsu Nosai, known as the main pharmacological component of ginseng extract (100-400 minutes) of a high purity 14 kinds of ginsenosides over a certain amount through one chromatography process without going through a special process from a low-purity crude sample (concentration of individual ginsenosides is less than 5%) It is possible to obtain within a few tens to several hundreds of mg units at one time, and to avoid the use of expensive columns having limited reuse, and to greatly reduce the amount of solvent used. An effective separation method has been developed and the present invention has been completed.

(1) extracting ginseng, extracting it with water, alcohol or a mixed solvent thereof, and fractionating it with butanol to obtain a ginseng extract fraction; (2) injecting the mobile phase solvent into the column at a specified moving speed while rotating at a specified centrifugal velocity using a countercurrent chromatography method using a fixed phase solvent and a mobile phase solvent having a specific solvent composition ratio; (3) a step of injecting the ginseng extract of the first stage into the sample inlet at the time when the mobile phase and the stationary phase are in equilibrium, and (4) a step of detecting ginsenoside from the eluent of the column, Thereby effectively separating the side in a short period of time.

In the first step of the separation method,

In order to separate low-polar ginsenosides, the ginseng may be any one containing diol or triol-modified ginsenoside (Rg3, Rg5, R1, F4, etc.) It is preferable to use processed ginseng or red ginseng because the red ginseng contains a low-polar ginsenoside in an abundance. Preferably, white ginseng is used to separate the ginsenoside diol and triol. . More specifically, Korean ginseng ( Panax ginseng , Panax quinquefolia , and Panax notoginseng , Panax vietnamensis , Panax japonicus , Panax elegatior , Panax wangianus , Panax bipinnatifidus , or Panax shudoensis Panax pseudoginseng ) or a processed product thereof.

The ginseng extract fraction may be prepared by extracting a herbal medicine extracting method known to those skilled in the art, for example, extraction with water, alcohol or a mixed solvent thereof, extraction with hot water or room temperature , But is not limited thereto. More specifically, for example, after dried ginseng is pulverized, about 1 to 20 times volume (v / w) of the pulverized product, preferably about 1 to 15 times volume ( preferably in a mixed solvent of water and ethanol or methanol, more preferably 30 to 90% by weight of water, alcohol, lower alcohol of 1 to 4 carbon atoms, v / w of water, alcohol, acetone, chloroform, methylene chloride, Water extraction, ultrasonic extraction, and filtration at 10 ° C to 100 ° C, preferably 30 ° C to 70 ° C, for 1 hour to 1 week, preferably 2 hours to 12 hours, Extraction methods, such as cooling extraction, preferably, a first step of performing ultrasound extraction to give the extract; Filtering the extract obtained above, adding water to the filtered extract and fractionating with butanol to obtain a butanol fraction; It is preferable to use the ginseng extract fraction obtained through the process including the third step of concentrating the butanol fraction under reduced pressure.

The present invention attempts to introduce a systematic method for solvent selection for optimal separation of major ginsenosides of white ginseng in countercurrent chromatography, applying (1) an isocratic elution method instead of the possible solvent gradient, (2) the two solvent systems were selected based on the distribution constant and were separated using a solvent gradient. In addition, since the polarity of the components of the ginsenoside was different, (3) to adjust the flow rate in order to improve the separation efficiency.

In the second step of the separation method,

Specifically, the "fixed-phase solvent" and "mobile phase solvent" specifically include methylene chloride: methanol or ethanol: isopropanol or propanol: water, more specifically methylene chloride: methanol or ethanol: isopropanol or propanol: To 6: 1 to 6: 1 to 3: 2 to 5, and the mixture is allowed to stand at room temperature. The solvent in the lower layer among the two layers produced is used as a mobile phase solvent, and the solvent in the upper phase is used as a fixed phase solvent More specifically, a mixed solvent of methylene chloride: methanol: isopropanol: water is mixed at a volume ratio of 1: 6: 1 to 6: 1 to 3: 2 to 5, The solvent in the lower layer of the layer is used as a mobile phase solvent and the solvent in the upper layer is used as a fixed phase solvent .

Rg2 at Rg6 with low polarity was able to be separated by using a solvent mixture of methylene chloride: methanol: isopropanol: water mixed solvent (5: 4: 1: 3) through the fixed and mobile phase solvent conditions. To S7 using a linear gradient of the solvent.

Specifically, when white gins are separated, the mixing ratio of methylene chloride-methanol-isopropanol-water is 1: 3: 1 to 3: 1 to 2: 2 (v / v), most preferably methylene chloride- A flow rate of 2 to 4 mL / min was passed from 200 minutes to 240 minutes using a solvent such as isopropanol-water in a mixing ratio of 1: 1: 1: 2 (v / v) Rs, Rf, Rd, Rg1 having low Kd values and the highly polar ginsenosides Re, Rc, Rb2, Rb1 are separated by maintaining the flow rate at 3 to 5 ml / min.

In the case of processed ginseng, red ginseng, ginsenosides with low Kd values, Rg ₆ , Rg ₅ , Rk _1, Rg3, low flow rates the Rd (1 ml / min) at 130 to 160 minutes, the eight kinds of ginsenoside polarity such as Rg1, Re, Rf, Rg2, Rb1, Rb2, Rc, Rd high after separation in a The mixing ratio of methylene chloride-methanol-isopropanol-water is 4: 6 to 3: 5: 1 to 2: 3 (v / v), most preferably methylene chloride-methanol-isopropanol- Methanol / isopropanol-water in a low polar solvent system of 1: 3 (v / v) is 1: 3: 1 to 3: 1 to 2: 2 (v / v) (200-700 min) at a flow rate of 1.0 to 1.5 mL / min by a solvent gradient which converts to a solvent system of methylene chloride-methanol-isopropanol-water mixing ratio of 1: 1: 1: 2 (v / v) Lt; / RTI >

The preferred centrifugal velocity is preferably 300 to 1,500 rpm, more preferably 500 to 1,000 rpm, the preferred moving velocity of the mobile phase solvent is 0.5 to 10 mL / min, more preferably 1 to 5 mL / Min. ≪ / RTI >

In the third step, the ginseng extract is injected into the sample inlet at a time when the mobile phase and the stationary phase are in equilibrium, and the time when the mobile phase solvent exits from the outlet of the column is determined as a time when the mobile phase and the stationary phase are in equilibrium.

In the fourth step, as time elapses, the eluent is continuously discharged through the separation valve to the outlet of the column at the same rate as the injection rate of the mobile phase. The ginsenosides are detected from the separated eluent. In the present invention, it is particularly preferable to use an evaporative light scattering detector (ELSD) for ginsenoside detection.

     According to the present invention, the low-polarity ginsenoside, which is known as the main pharmacological component of ginseng extract, is separated from a low-purity crude sample (concentration of individual ginsenosides of less than 5%) through a single chromatography process Rg1, Re, Rf, Rg2, Rb1, Rb2, Rc, Rd, Rg3, Rk1, Rg5, Rg6 and F4 of the high purity 14 kinds of ginsenosides can be separated.

In the case of white ginseng, the solvent was distilled in TBE-1000A using a solvent system No. 7 (methylene chloride-methanol-isopropanol-water 1: 1: 1: 2) The flow rate is 3 mL / min, and then the flow rate is maintained at 4 ml / min. As a result, ginsenosides Rh1, Rf, Rd, and Rg1 having low Kd values are separated at a flow rate of 3 ml / min, and highly polar ginsenosides Re, Rc, Rb2 and Rb1 are separated at a flow rate of 4 ml / (Fig. 3A).

In the case of red ginseng, TBE-300A was used to measure ginsenosides having low Kd values, Rg ₆ , Rg ₅ , Rk _1, Rg3, low flow rates the Rd (1 ml / min) can be isolated within 150 minutes after a high eight kinds of ginsenoside (Rg1, Re, Rf, Rg2 , Rb1, Rb2, Rc, Rd,) polarity was from Was dissolved in a solvent system No. 7 (methylene chloride-methanol-isopropanol-water 1: 1: 1) at a low polarity of the solvent system No. 3 (methylene chloride-methanol-isopropanol-water 5: 4: (200-700 min) at a flow rate of 1.2 mL / min by a solvent gradient which converts to a polar solvent system of 1: 2 (1: 2) (Figure 3B).

The present invention provides a method for separating ginsenoside from white ginseng or red ginseng using HSCCC and a method for separating 13 kinds of ginsenosides by one step derived from the method.

Hereinafter, the specific configuration of the present invention will be described in detail with reference to examples. However, the scope of the present invention is not limited to the description of these embodiments.

The separation process for separating ginsenosides from ginseng using HCCC of the present invention is characterized in that low polarity ginsenosides, which are known as the main pharmacological components of ginseng extract, are separated from low-purity crude samples (concentration of individual ginsenosides is less than 5%) Through a single chromatography process without any process, it is possible to obtain 14 kinds of high purity ginsenosides over a certain amount in a short time (100-400 minutes). In addition, there is no need to use an expensive column having limited reuse, and an advantageous effect such as a drastically reduced amount of solvent is exhibited. Thus, an industrial separation method in which an effective ingredient is efficiently extracted from a ginseng extract and a ginsenoside ingredient is mass- .

1 is a diagram showing an experimental procedure for selecting a solvent system system;
2 is an HPLC-ELSD chromatogram of butanol fractions of white ginseng (A) and red ginseng (B);
3 is a chart showing HSCCC chromatograms of the butanol fraction of white ginseng (A) and red ginseng (B);
Figure 4 shows HPLC-ELSD chromatograms of HSCCC chromatograms of ginsenosides isolated from white and red ginseng samples.

Hereinafter, the present invention will be described in detail with reference to the following examples and experimental examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the contents of the present invention are not limited by the following Examples, Reference Examples and Experimental Examples.

Reference Example  1. Experimental preparation

 1-1. Experimental equipment

Two different sizes of HSCCC instruments were used and were used in the preparative TBE-1000A (1-1.5 g sample injection, Tauto Biotechinique Co, China) and semi-preparative TBE-300A (200-300 mg injection, Tauto Biotechinique, Co China) were used.

The preparative TBE-1000A was a pump (TBP-50A constant-flow pump, Tauto Biotechnique Co., Ltd.) using a coil of 1000 ml and 3.0 mm polytetrafluoroethylene tube.

The speed of the machine was 0-600 rpm. The semi-preparative TBE-300A was prepared by using a coil of 260 ml and 1.8 mm polytetrafluoroethylene tube and a pump (Hitachi L-6200A Intelligent pump).

The speed of the device was 0-1000 rpm. The eluent flowing through the valve was continuously retrieved with the coiled column connected to a detector (Evaporative Light Scattering Detector, SEDERE, Sedex 5, France).

Samples were analyzed by HPLC using a Hitachi L-6200 instrument and equipped with an automatic injector (Sil-9A, Shimadzu, Japan) and a column (Zorbax SB-Aq C18 column, Agilent Technologies, USA).

GC was analyzed with a YL 6100 GC equipped with a flame ionization detector, a 6000 series auto sampler (YL Instrument LTD, Anyang, Korea) and a 10 μL syringe (SGE Analytical Science, Australia). The device was also connected to a cylinder containing hydrogen, oil-free air and carrier gas.

 1-2. Reagents and samples

(JT Baker, Phillipsburg, NJ, USA) methylene chloride, methanol, ethanol, and isopropanol (for HPLC) were purchased from commercial sources and red ginseng (Korean ginseng corporation, Seoul, Korea) and white gins (Analytical) was purchased from Fisher Scientific (Pittsburgh, Pa., USA) and distilled water (NANO pure diamond, Barnstead, USA) was used for all solution preparation and dilution.

Example  1. Preparation of Ginseng Extract Samples

 1-1. White ginseng Butanol  extraction Fraction  pharmacy

The total content of saponins in white ginseng is about 4-5% of the dry weight. For extracting ginsenosides, 50 g of white ginseng powder was extracted with 150 ml of 70% ethanol and extracted with ultrasonic extraction method (Branson 5510, Danbury, CT, USA) for 3 hours. The supernatant was filtered through a filter paper (HYUNDAI Micro Co.). The mixture was filtered through a rotary evaporator (EYELA, N-1000S-W Rotary Evaporator) and concentrated under reduced pressure to remove the solvent. The extract was dissolved in water and extracted three times with butanol to obtain 70% ethanol- The polar substances were removed by centrifugation using a centrifugal separator and lyophilized with a freeze dryer (FD5505, Ilshin, Korea) to obtain 5 g of a white ginseng butanol extract fraction (hereinafter referred to as WGE) And stored in a freezer for isolation.

 1-1. Red ginseng Butanol  extraction Fraction  pharmacy

The supernatant was filtered with a filter paper (HYUNDAI Micro Co., Ltd.), and the filtrate was concentrated under reduced pressure (50 mL) The concentrate was concentrated under reduced pressure using a concentrator (EYELA, N-1000S-W Rotary Evaporator) to remove the solvent. After dissolving in water, the extract was extracted three times with butanol to obtain 70% ethanol soluble extract of red ginseng. Using a centrifugal separator The polar material was removed and lyophilized with a freeze dryer (FD5505, Ilshin, Korea) to obtain 3 g (hereinafter referred to as "RGE") of a dried fraction of red ginseng butanol. The dried material was stored in a freezer for subsequent HSCCC separation.

Example  2. HSCCC On by Gin Senocide  detach

 2-1. HPLC

The fraction of ginsenoside was confirmed by high performance liquid chromatography (HPLC). High-performance liquid chromatography (HPLC) was performed using a HITACHI L-6200 pump, a Shimadzu SIL-9A autoinjector, and an ELS detector (Sedex 75 ELS detector). Solvent A was water and solvent B was acetonitrile min at 18-23% B, 6-48.5 min at 23-40% B, 48.5-55 min at 40-100% B and 18% B over the last 5 min at 1 mL / min. Separate ginsenosides were separated using a reverse phase Zorbax SB-Aq C ₁₈ column (150 mm x 4.6 mm, 5 μm particle size, Agilent Technologies, Palo Alto, Calif., USA) and analyzed using an ELS detector (SEDERE, Sedex 55, France) The probe temperature was fixed at 70 ° C, the gain was 7.0, and the nitrogen gas pressure of the nebulizer was fixed at 2.5 bar.

2-2. Solvent system  selection

The solvent system for the target material has a distribution constant ( K _D ) And this value is measured by HPLC as follows.

Add 3 mg of ginsenoside butanol extract to a test tube and add 2 ml of each layer of the equilibrated two-phase system. After thoroughly stirring in a test tube and dividing into two layers, separate each layer and dry with nitrogen gas. The residue of each layer is dissolved in methanol and the HPLC process is carried out. The distribution constant is calculated by dividing the peak area of the target material contained in the supernatant by the peak area of the supernatant (see FIG. 1).

As shown in Table 1, a mixed solvent system (methylene chloride / methanol / isopropanol / water, v / v) having different mixing ratios as shown in Table 1 is suitable for separating CCC of ginsenoside. And the solvent system was selected. Solvent systems with various solvent gradients (S1 - S7) were selected based on their polarity and measured for target separation, and the distribution constant ( K _D ) The values were reduced by increasing the volume ratio of isopropanol in the solvent system. The solvent gradients S1 to S4 exhibited K _D values suitable for the ginsenoside compounds (Rg ₆ to Rg ₂ ) having a high polarity. On the other hand, solvent gradients S5 through S7 showed suitable K _D values for the more polar ginsenoside compounds (Rf through Rb ₁ ). In conclusion, the solvent gradient method was chosen as the solvent system composition for the separation of the 14 ginsenosides of the sample.

Solvent (S) ratio K _D value Rg ₆ Rg ₅ Rk ₁ F ₄ Rg ₃ Rg ₂ / Rh ₁ Rf Rd Rg ₁ Re Rc Rb ₂ Rb ₁ S1 6: 6: 1: 4 0.35 0.49 0.78 2.12 2.65 3.5 4.6 S2 5: 6: 1: 4 0.28 0.60 0.69 1.44 2 3.1 4.2 S3 5: 4: 1: 3 0.18 0.35 0.52 0.78 1.56 2.1 3.5 S4 6: 6: 2: 5 0.18 0.29 0.4 0.61 0.96 1.67 3.3 4.45 13.8 19 18.6 25 NA S5 6: 6: 3: 5 0.06 0.1 0.1 0.14 0.21 0.61 1.2 2.5 2.6 3.9 4 5.7 8 S6 2: 2: 1: 2 0.1 0.16 0.22 0.31 0.54-0.71 1.2 2.1 2.9 3.1 4.6 4.9 6.5 7 S7 1: 1: 1: 2 0.15 0.29 0.6 0.9 1.5 1.9 2.6 3.2

(K _D of the target substance in a mixture of various non- ) shame

Table  One. K _D values of the target compounds in various ratios of CH ₂ Cl ₂ : MeOH: PrOH : DW

2-3. For analysis of the constituents of solvents GC

A two-phase solvent system was prepared as needed without pre-saturating the solvents. GC-FID was used to analyze the constituents of the selected solvent system. The analysis used tetrahydrofuran (THF) as the internal standard (IS). The experimental conditions are as follows: column temperature 50 캜; Injector temperature, 190 캜; Detector temperature, 250 ° C and carrier gas (helium) are used. The column was HP-5 (30 m × 0.32 mm × 0.25 μm) and the injection volume was 1 μL. Initially, calibration curves were plotted against the individual solvents at different concentrations of IS and phase composition analysis was performed with concentrations of the individual solvents previously used. Finally, the volume concentrations of methylene chloride, methanol and isopropanol were measured as V ₁ , V ₂ and V _3, respectively. Since FID does not react to DW, the volume concentration (V ₄ ) in all of the above phases was calculated by the following equation (1).

2-4. Two phase solvent system and preparation of sample solution

Mixed solvents of different mixing ratios (methylene chloride / methanol / isopropanol / water, v / v) were used as a two phase solvent system for HSCCC separation. The solvent mixture was equilibrated in a fraction flask at room temperature and the two phases were separated before use. As a sample solution, 1000 mg of a separation sample WGE was dissolved in 60 mL of a separating solvent system S6 and used in TBE-1000A. 300 mg of RGE was dissolved in 20 mL of a separating solvent system S3 and used in TBE-300A. However, the HSCCC system (TBE-1000A, TBE-300A) is not limited to samples.

2-5. Isolation using HSCCC

Even if the solvent system selected in the previous step has a proper distribution constant, if the stationary phase has a low rate of retention in the HSCCC separation process, there is a high probability of failure. Therefore, it is necessary to confirm the retention rate of stationary phase.

It is reported that the flow rate of the mobile phase and the centrifugal velocity of the coil wound in the coil shape influence the resolution during the HSCCC separation process. Generally, the lower the flow rate of the mobile phase, the better the resolution, but the longer the total separation time. Also, as the centrifugal velocity of the column increases, the resolution increases. Therefore, by adjusting these factors, we are trying to find the HSCCC-optimized resolution with the previously selected solvent system.

The separation conditions using HSCCC are as follows.

In this example, a large capacity model TBE-1000A (Tauto Biotech, Shanghai, China) having a polytetrafluoroethylene (PTFE) tube column having an inner diameter of 3.0 mm and a total capacity of 1000 ml was used, , And TBA-300A having a total capacity of 260 ml were used. In the individual separation process, a multi-layered coiled column was filled with the upper layer forming the stationary phase and the device was rotated (TBE-1000A at 500 rpm and TBE-300A at 850 rpm) to form the lower organic layer mobile phase was pumped toward the column end (flow rate: 3 mL min ^-1 - TBE-1000 A and flow rate 1 mL min ^-1 - TBE-300 A).

    The solvent of methylene chloride: methanol or ethanol: isopropanol or propanol: water is preliminarily prepared so that the lower layer is divided into a fixed phase and the upper layer is divided into a mobile phase to remove gas dissolved immediately before use. First, the column is wound in the form of a coil, and the column is rotated at a speed of 500 rpm (TBE-1000A) or 850 rpm (TBE-300A). When the rotation of the column is stabilized, the mobile phase is flowed at a flow rate of 3 ml / min (TBE-1000A) or 1.0 ml / min. When equilibrium is established between the stationary phase and the mobile phase in the column, the sample is dissolved in 60 ml (TBE-1000A) at a volume ratio of 1000 mg of each solvent in a two-phase system, or dissolved in 20 ml of the same solvent (TBE-300A) It works. The eluent of HSCCC was detected using a separation valve and an ELS detector and fractionated using a fraction collector. As a condition, the temperature is adjusted to 70 ° C, the gain is 6, and the nitrogen is used as the nebulizer gas to 2.2 bar.

When isolating white ginseng, use TBE-1000A with solvent system No. 7 (methylene chloride-methanol-isopropanol-water 1: 1: 1: 2) And the flow rate was maintained at 4 ml / min thereafter.

As a result, the ginsenosides Rh1, Rf, Rd, and Rg1 having low Kd values were separated at a flow rate of 3 ml / min and the highly polar ginsenosides Re, Rc, Rb2 and Rb1 were separated at a flow rate of 4 ml / Could. (Figure 3A)

In the case of red ginseng containing a large amount of modified ginsenosides, 400 mg was injected into the system of TBE-300A to obtain ginsenosides Rg ₆ , Rg _{5 ,} Rk _{1 and} F ₄ having a low Kd value with a low polarity _, Rg ₃ could be separated in 150 minutes at low flow rate (1 ml / min) using solvent system 3 (methylene chloride-methanol-isopropanol-water 5: 4: 1: 3) (Rg1, Re, Rf, Rg2, Rb1, Rb2, Rc, Rd) of the 8 kinds of ginsenosides were dissolved in solvent system No. 3 of solvent system No. 7 (methylene chloride-methanol-isopropanol- : 1: 2) in a short time (200-700 min) at a flow rate of 1.2 mL / min by solvent gradient (Fig. 3B).

2-6. Determination of purity and structure of isolated ginsenosides

The isolated single ginsenosides of high purity were identified by using spectroscopic methods such as ESI-MS, ESI-MS / MS, ¹ H NMR, ¹³ C-NMR and MS, and fractions of each peak collected from HSCCC The purity is confirmed using the above HPLC method. First, the molecular weight was determined by performing a negative and positive mode of ESI-MS, and structural determination was performed by ESI-MS / MS.

    When the solvent remaining on the stationary phase after the separation was analyzed, it was found that the ginsenoside was efficiently separated from the red ginseng because no sugar component was detected. In Fig. 4, Rg2 and Rg3 are separated from the chemical structure isomers (S) and (R). In this case, a small amount of water was added to purify the main component (S), and the water-insoluble (R) form was removed by centrifugation. In Fig. 4, the ginsenosides Rg5 and F4 were 91%, while the other 12 ginsenosides were 97% or more. The recovery rate was 70% for red ginseng and 80% for white ginseng. The structure was determined by ESI-MS / MS (Finnigan LCQ ion trap mass spectrometer from Thermo Finnigan, San Jose, Calif.) Equipped with an electrospray (ESI) probe, H-NMR (Nuclear Magnetic Resonance, Bruker AVANCE 500 NMR spectrometer) - NMR and consistent with the published results.

According to the present invention, low-polarity ginsenoside, which is known as the main pharmacological component of ginseng extract, can be separated from a low-purity crude sample (concentration of individual ginsenosides of less than 5%) through a single chromatography process 14 high-purity ginsenosides can be obtained in a short time (100-400 minutes). In addition, it is not necessary to use expensive columns having limited reuse, and the amount of solvent used is greatly reduced.

Claims (8)

(1) extracting ginseng, extracting it with water, alcohol or a mixed solvent thereof, and fractionating it with butanol to obtain a ginseng extract fraction; (2) The stationary phase solvent and the mobile phase solvent are prepared by mixing methylene chloride: methanol or ethanol: isopropanol or propanol: water at room temperature, and then the solvent in the lower layer among the two layers produced is used as a mobile phase solvent. A second step of injecting the mobile phase solvent into the column at a moving speed ranging from 0.5 to 10 ml / min while rotating at a centrifugal speed ranging from 300 to 1,500 rpm using a countercurrent chromatography method using a solvent as a fixed phase solvent; (3) a step of injecting the ginseng extract of the first stage into the sample inlet at the time when the mobile phase and the stationary phase are in equilibrium, and (4) a step of detecting ginsenoside from the eluent of the column, A method of separating a side effectively within a short period of time. The method according to claim 1,
Wherein the ginseng is ginseng, white ginseng, red ginseng, or processed ginseng. The method according to claim 1,
The ginseng was ginseng ( Panax ginseng , Panax quinquefolia), jeonchilsam (thirty-seven, Panax notoginseng , Panax vietnamensis), jukjeol three (Panax japonicus), Panax elegans tee climb (Panax elegatior), Panax Wan Jia Augustine (Panax wangianus), Panax non-typhoid pinna (Panax bipinnatifidus ), or Panax pseudoginseng ) or a processed product thereof. The method according to claim 1,
The ginseng extract fraction may be prepared by pulverizing dried ginseng and pulverizing the dried ginseng with 1 to 20 times volume (v / w) water, alcohol, lower alcohol having 1 to 4 carbon atoms, acetone, chloroform, methylene chloride, A first step of adding the mixed solvent thereof to the extract to obtain an extract by performing cold extraction, hot water extraction, ultrasonic extraction, or reflux cooling extraction at 10 to 100 ° C for 1 hour to 1 week; Filtering the extract obtained above, adding water to the filtered extract and fractionating with butanol to obtain a butanol fraction; And a third step of concentrating the butanol fraction under reduced pressure to obtain a ginseng extract fraction. The method according to claim 1,
In the second step, when white ginseng is separated, a mixture of methylene chloride-methanol-isopropanol-water in a solvent having a mixing ratio of 1: 3: 1 to 3: 1 to 2: 2 (v / v) The ginsenosides Rh1, Rf, Rd, and Rg1 having a low Kd value by flowing the flow rate at 2 to 4 mL / min from 200 to 240 minutes by the method, and then maintaining the flow rate at 3 to 5 ml / And isolating the highly polar ginsenosides Re, Rc, Rb2, Rb1. The method according to claim 1,
In the second step, ginsenosides having a low Kd value in the case of red ginseng, Rg ₆ , Rg ₅ , Rk _1, Rg3, low flow rates the Rd (1 ml / min) at 130 to 160 minutes, the eight kinds of ginsenoside polarity such as Rg1, Re, Rf, Rg2, Rb1, Rb2, Rc, Rd high after separation in a The mixing ratio of methylene chloride-methanol-isopropanol-water in the low polar solvent system of methylene chloride-methanol-isopropanol-water mixture of 4: 6: 3 to 5: 1 to 2: 3 (v / Characterized in that the solvent is obtained at a flow rate of from 1.0 to 1.5 mL / min in a period of from 200 minutes to 700 minutes by means of a solvent gradient which converts to a polar solvent system of from 3: 1 to 3: 1 to 2: 2 (v / v). The method according to claim 1, wherein, in the third step, the ginseng extract is injected into the sample inlet at a time when the mobile phase and the stationary phase are in equilibrium, and a time point at which the mobile phase solvent exits from the outlet of the column is a time when the mobile phase and the stationary phase are in equilibrium ≪ / RTI > The method according to claim 1,
Wherein in the fourth step, the ginsenoside detection uses an evaporative light scattering detector (ELSD).

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