KR101276639B1 - A separating method for panaxadiol ginsenosides from panaxatriol ginsenosides - Google Patents

Abstract

The present invention relates to a method for obtaining ginsenoside, which is a ginseng-specific saponin, from ginseng extract or extract, concentrate and concentrate powder, which comprises extracting ginsenosides such as Panaxadiol ginsenoside and Panaxatriol ginsenoside The present invention relates to a method for separating a ginsenoside composite to be separated with a fermentation alcohol (ethanol) at a concentration of 10 to 40% 10 or more basic aqueous solution by stirring to form a precipitate, and separating the solution and the precipitate, thereby separating the panaxadiol-based ginsenoside and the pyrazine triol-based ginsenoside.

Description

[0001] This invention relates to a method for separating diacylglycinoside from panaxadiol ginsenosides from panaxadiol ginsenosides,

The present invention relates to a ginseng containing a panaxadiol system and a panaxatriol ginsenoside (in the present invention, "ginseng" is a comprehensive concept including ginseng and dried ginseng as well as processed ginseng products such as red ginseng and black ginseng) , A ginseng extract or a concentrated solution thereof, and a method for separating prazosinic diol-based ginsenosides and pacificatriol-based ginsenosides by solubility in accordance with pH of two components.

Ginseng has been acknowledged as one of the most important medicines in the world. In particular, research on the pharmacological effect of ginseng has been actively conducted in the 20th century, and the importance of ginseng saponin, ginsenoside, which is the main pharmacologically active substance of ginseng, is highlighted.

Ginsenoside has 37 chemical structures analyzed according to the results of various studies. As a result, basic research has shown that anti-stress, neuronal cell protection, anti-thrombosis, improvement of lipid metabolism, inhibition of cancer cell proliferation, anti- Have been discovered [ Arch . Pharm . Res . 2000, Oct, 23 (5) 518-524, Biochem Pharmacol . Dec 2003, 66 (11): 2213-21. Chin J Physiol . 2003 Mar 31; 46 (1): 1-7., Shibata et al. J. Korean Med Sci . 16 (suppl.) S28-37 (2001)].

Ginsenoside has a bisdesmoside neutral glycoside structure in which sugar molecules such as glucose, arabinose, xylose, and rhamnose are bound to a triterpenoid-based dammarane skeleton, Panaxadiol system, Panaxatrio system, and Oleanane system are classified according to the binding position of sugar molecules and the shape of basic skeleton, and pharmacological actions in each body are distinguished.

Particularly, it has been reported that the pyrazine triazinoside (representative of ginsenosides Rg1 and Re) has a blood pressure rise, a rise in body temperature and a tense up action of the central nervous system, and the pyrazine diol ginsenosides (typically ginsenosides Rb1, Rd) have been reported to have hypotensive effects, body temperature depression, and relaxation of the central nervous system (Saito et al.Jap . Pham . 22: 245-259 (1974), Tagaki K.Proc . One st int'l Ginseng Symp . Korean Office Monopoly; 119-127 (1974)).

Since the action of the pyrazine diol system and the pyrazine triol system ginsenoside are completely different from each other, separation of pyrazine diol system ginsenoside and pyrazine triol system ginsenoside can provide a customized effect according to excellent symptoms, It is possible to see the development possibility of more effective medicines, cosmetics, and health functional foods.

Conventionally, a separation method of two components has not been developed or proposed, but various problems are exposed in terms of efficiency. Examples thereof include a method using a fraction using a solvent such as chloroform, methanol or butanol ( Chem . Pharm . Bull . 53 (2) 177-179 (2005)) or a separation method using chromatography ( J. Chromatography 2009 v31008 no .2 pp. 179-180). However, the former can not be expected to have a high yield, and the latter has a fatal disadvantage that mass production is impossible.

In addition, there is a method of separating through a column packed with a benzene ethylenic synthetic adsorbent resin (Korean Patent No. 315097) and a method of separating through a column packed with a methacrylic synthetic adsorbent resin (Korean Patent No. 10-0678490) Both methods require a long separation time, especially 200 times (v / w) of the amount of crude saponin, which is the sample to be separated, and the excessive consumption of alcohol used for desorption is disadvantageous. The ability is relatively good compared to the former, but it is disadvantageous to use excessive alcohol which is 600 times (v / w) of crude saponin which is the substance to be separated.

Due to the above problems, more efficient and economical separation of pyrazosadiol system and pyrazine triol system ginsenoside is required.

Accordingly, it is an object of the present invention to solve the problems of the conventional separation method as described above, and to provide a method of separating pyrazyltriol system and paraxaxadiol system ginsenoside, which exhibits high separation efficiency without using excessive organic solvent.

Another object of the present invention is to provide a relatively high efficiency and low cost separation method than the conventional separation method of Panaxadiol ginsenoside and Panaxatriol ginsenoside.

The present inventors obtained ginsenosides such as ginseng-specific saponins from ginseng extracts including ginseng or extracts, concentrates, and concentrated liquid powders, and found that ginsenosides such as Panaxadiol ginsenoside and Panaxatriol ginsenoside ) With high solubility by using the difference in solubility of the two components depending on the pH after a long period of research.

The present invention relates to a method for separating prazosinic diol-based ginsenosides and pacificosyl triol-based ginsenosides using the difference in solubility of two components according to pH.

The present invention

a) preparing a ginsenoside solution by dissolving a ginsenoside complex comprising a pyrazine diol ginsenoside and a pyrazine triol ginsenoside in a water-soluble organic solvent, aqueous solution or water; And

b) adding a base solution having a pH of 10 or more to the ginsenoside solution, stirring the mixture, precipitating the mixture, and precipitating and filtering the supernatant, wherein the paraxaxadiol-based ginsenosides and the panaksatriol-based ginsenosides Separation method.

When the pH of the basic solution before mixing with the ginsenoside solution is less than 10, the difference in solubility between the pyrazine triazine ginsenoside and the pyrazine diol ginsenoside is not so large, so that a clear separation is difficult to occur.

The aqueous solution means that the salt is dissolved in purified water, ion-removing water, distilled water, etc. There is no particular limitation on the kind of the aqueous solution, and the aqueous solution does not affect the separation of prazosinic diol ginsenoside and panaxatriol ginsenoside Any solution is acceptable if it is not.

Further, the present invention is characterized in that the base solution of step b) is obtained by dissolving the base in a water-soluble organic solvent, aqueous solution or water. However, in the use of the solvents of a) and b), different solvent polar conditions must be formed by using an organic solvent on one side and a water component (that is, water or an aqueous solution) on the other side.

In addition, the present invention is characterized in that the water-soluble organic solvent in step a) is a fermented juice.

Further, the present invention is characterized in that a step of dissolving the precipitate obtained in the step b) and passing the solution through a synthetic adsorption resin column and purifying the solution is added.

Further, the present invention is characterized in that the step of purifying the supernatant obtained in the step b) by passing through a weakly acidic ion exchange resin column is added.

In the present invention, the solvent used for preparing the ginsenoside solution is a water-soluble organic solvent, an aqueous solution or water. The water-soluble organic solvent refers to an organic solvent that is well miscible with water such as acetonitrile, methanol, ethanol, 2-propanol, propylene glycol and the like. For the preparation of the ginsenoside solution, a water-soluble organic solvent or water may be used as a solvent alone or as a solvent. The solvent for preparing the ginsenoside solution is more preferably fermented ethanol (alcohol alcohol).

The amount of the solvent used for preparing the ginsenoside solution is 12 to 30 times (v / w), preferably 20 times (v / w) of the ginsenoside solution as a separation target material. If the amount of the solvent is too low, it is not easy to separate the pyrazine diol ginsenoside and the pyrazine triazine ginsenoside. When the solvent is too low, the concentration of the ginsenoside solution becomes too low and it is difficult to apply the separation method using the difference in solubility.

The basic substance used for controlling the pH of the ginsenoside solution is not particularly limited as long as it is harmless to the human body. Preferably, sodium diphosphate is used. Sodium phosphate is preferably added to the food additives of the Korean Food and Drug Administration More preferably, sodium phosphate is used as an additive to minimize harm to the human body. The basic substance is used at a ratio of 0.9 to 2 times (w / w) of the ginsenoside solution as the substance to be separated. In this range it is possible to adjust to a pH suitable for separating the two components.

The ion exchange resin and the synthetic adsorbent resin used for the removal of the base remaining in each ginsenoside fraction after the separation of the panaxadiol type ginsenoside and the pyrazine triol ginsenoside are the ginsenoside solution 5 to 20 times (v / w) of the total amount of water. In one embodiment of the present invention, WK-10 resin of Mitsubishi Chemical Co. was used as the ion exchange resin, Amberlite IRC-50 resin of Romh & Haas or TRILITE WK60L of Samyang Co., It is also possible to use a weakly acidic ion exchange resin composed of a group. In addition, as the synthetic adsorbent resin, HP 20 resin which is an aromatic synthetic adsorbent resin of Mitsubishi Chemical Company was used, and HP 10 and HP 21 resin of the same company can be used. Pore volume when measured by nitrogen adsorption method An aromatic synthetic adsorbent resin having a swelling degree of from 0.9 to 1.4 ml / g and a swelling degree of from 3.3 to 3.5 ml / g and having a catalyst swelling ratio of 1.00 in water and 1.2 or more in a water-soluble organic solvent can be used. If the conditions of the resins are satisfied, the kind of the ion-exchange resin and the synthetic adsorption resin is not particularly limited.

The ginseng used as a raw material for obtaining ginsenoside used in the present invention is ginseng, which contains Korean ginseng, Panax notoginseng, Panax quinquefolius, and all ginseng containing pyaxanthiol and pyrazine triol ginsenosides , A ginseng extract and / or a concentrate thereof may be used. In the present specification, the term "ginseng" is used to mean a variety of processed ginseng such as red ginseng, dried ginseng, white ginseng, red ginseng, black ginseng, and oriental ginseng obtained by processing ginseng including dried ginseng, dried ginseng and white ginseng.

The raw material ginseng can be used as a sample of the separation method of the present invention by preparing a ginsenoside solution in which paxxadiol and panaxatriol are mixed in the form of ginseng powder, ginseng extract and / or concentrate as described above, The basic solution can be directly added to the ginsenoside solution in which the diol and pyrazole triol are mixed, but it is also possible to add a purification step for removing substances other than ginsenoside as a preliminary step. Various purification methods known to those skilled in the art can be applied. For example, as a process for removing a substance other than ginsenoside from a ginsenoside solution, an extract obtained by extracting ginseng, The concentrated solution is passed through a synthetic adsorption resin, washed with 10 times as much water as the resin, passed through 5 times as much of the resin as the fermentation alcohol, and the like. The obtained ginsenoside solution can be used as a sample of the present invention.

As a first step of the method of the present invention, a ginsenoside complex containing a pyrazine diol system and a pyrazine triol system ginsenoside is dissolved in a concentration of 10 to 40% (v / w), more preferably 30% Dissolved in water, an aqueous solution or a water-soluble organic solvent to prepare a ginsenoside solution.

A basic solution in which a basic substance such as sodium phosphate is dissolved is prepared. The solvent is water, an aqueous solution or a water-soluble organic solvent.

As a cautionary note in the preparation of the solution, solvent polar conditions must be established in the use of solvents, one of which is an organic solvent and the other is water or an aqueous solution. That is, when the solvent used in the preparation of the ginsenoside solution is an organic solvent, the solvent of the basic solution is water or an aqueous solution, and vice versa.

The prepared basic solution is added to the pre-prepared ginsenoside solution at a speed of 500 to 1,000 RPM with stirring. At this time, when precipitation occurs, stirring is stopped at the time of precipitation, and the mixture is allowed to stand for about 2 to 10 minutes to maximize the precipitation. At this time, if the settling time exceeds 10 minutes, the settling dissolves again, so be careful not to exceed 10 minutes, preferably not more than 5 minutes. At this time, there is a paraxadiol-based ginsenoside in the precipitation, and the supernatant triazine ginsenoside exists in the supernatant.

After separating the precipitate and the solution by using a filter, water was added to the precipitate to dissolve it. Then, the precipitate was passed through a column filled with a synthetic adsorbent resin to adsorb the ginsenoside to the resin, do. The point at which the pH of the washing liquid passing through the column becomes 6 to 7 is determined as the end point of washing, and a sufficient amount of a water-soluble organic solvent, preferably alcohol, is passed through the column to desorb the adsorbed ginsenoside. If necessary, the solution in which ginsenosides are desorbed and dissolved is analyzed to confirm the presence of the remaining basic substance. The ginsenoside obtained from the precipitation which is insoluble in the basic solution is paraxaxadiol ginsenoside.

After the basic solution filtration, the obtained solution (supernatant) is passed through a weakly acidic ion exchange resin column to desorb and remove a basic substance from the resin, and obtain a solution through the column. The ginsenoside obtained from the supernatant dissolved in the basic solution is prazosin triazinecinoside.

The present invention relates to a method for separating saponin in ginseng extract, that is, ginsenosides, from the paraxaxadiol system and the pyrazoxyrid system ginsenoside, using the difference in solubility according to pH, In other words, the use of fermented ethanol and sodium phosphate to minimize the harmful effects on the human body, and it is meaningful that the work progress time is greatly reduced.

In particular, the present invention is more economical as compared with Korean Patent No. 315097 and Korean Patent No. 10-0678490, because the amount of resin consumed in the column process and the amount of organic solvent used throughout the entire separation process are small, The process was presented to expand the application range of ginsenoside for edible and medicinal use.

1 shows the results of HPLC analysis of the ginsenoside standard product (Rg1, Rb1, Rb2, Rc: 99.2% Wako Pure Chemical Industries, Ltd. Rd, Re: 95.6% Herb Pioneer Co., Ltd).
FIG. 2 shows the results of HPLC analysis of prazosinic diol ginsenoside contained in the precipitate.
Fig. 3 shows the result of HPLC analysis of the pyrazolopyrimidine ginsenoside contained in the supernatant.
The HPLC system conditions in FIGS. 1, 2 and 3 are as follows: pump - waters 600, detector - waters 486 (UV), column - 4.6 x 250 mm agilent TC-C18.
FIG. 4 is a flowchart showing a method of separating a paraxaxidol ginsenoside and a pyrazine triol ginsenoside according to an embodiment of the present invention.
Fig. 5 is a structural formula showing the basic skeleton of paraxaxyl diol ginsenoside and pyrazine triol ginsenoside.

Hereinafter, the configuration of the present invention will be described in more detail with reference to specific embodiments. However, it is to be understood that the scope of the present invention is not limited to the scope of the embodiments, and that the present invention can be variously modified and changed without departing from the scope of the present invention.

Example 1: Preparation of ginsenoside solution containing panaxadiol and panaxatriol

The ginseng extract containing dianthraquinone and triacylglycinoside is adsorbed through a synthetic adsorbent resin, washed with water 10 times as much as the resin to remove foreign matter, and subjected to 5 times of fermentation After passing through alcohol, fermentation alcohol was added or concentrated as needed to prepare a senenoside solution dissolved in fermentation alcohol so as to have a concentration of 30%.

Example 2: Separation of paraxanthiol and pyruvate triol components in a base solution

1.1 times (w / v) sodium phosphate of water was added to 1-fold (v / w) water of the ginsenoside solution to prepare a saturated aqueous solution. A saturated aqueous solution of sodium phosphate was added to the previously prepared ginsenoside solution at a speed of 800 RPM with stirring. At this time, when precipitation occurred, stirring was stopped at the point of precipitation, and the precipitation was maximized by allowing to stand for 5 minutes. The precipitate and the supernatant were separated using a filter.

Example 3: Ginsenoside purification included in the precipitate

The precipitate obtained in Example 2 was dissolved in water (v / w) twice as much as the precipitate. The synthetic adsorbent resin was passed through a column packed with an amount of ginsenoside solution 15 times to adsorb the ginsenoside to the resin, Was passed through water to wash out substances other than ginsenosides. The point of time when the pH of the washing liquid passing through the column became 7 was determined as the end point of washing, and 10 times as much ethanol as the resin was passed through the column to desorb the adsorbed ginsenoside.

Example 4: Ginsenoside purification in supernatant

The solution (the supernatant) obtained after the basic solution filtration in Example 2 was passed through a column packed with the weakly acidic ion exchange resin WK-10 15 times the ginsenoside solution to attach the basic substance used in the basic solution preparation to the resin And the solution passed through the column is obtained.

Experimental Example 1: Analysis of each ginsenoside

The components of the ginsenosides obtained in Examples 3 and 4 were analyzed by HPLC column chromatography. The results are shown in Table 1, FIG. 2, and FIG.

Gin Senocide Purity Rg1 78% Re 12% Total pike triol 91.4% Rb1 74% Rc 13% Rb2 2% Rd 2.3% Total Panax Dior 92.1%

As shown in Table 1, after pouring and column purification of the supernatant, purity of 90% or more of the prazosinic diol-based ginsenoside and the prazosin triol-based ginsenoside is contained.

Claims (7)

a) preparing a ginsenoside solution by dissolving a ginsenoside complex comprising a pyrazine diol ginsenoside and a pyrazine triol ginsenoside in at least one solvent selected from the group consisting of methanol and ethanol;
b) adding to the ginsenoside solution a base aqueous solution having a pH of not less than 10 obtained by dissolving a base in water, stirring the mixture, precipitating, precipitating and filtering the supernatant, and parasadzidiol ginsenoside Method for separating diazotized ginsenosides.
a) preparing a ginsenoside solution by dissolving a ginsenoside complex, comprising a pyrazine diol ginsenoside and a pyrazine triol ginsenoside, in water;
b) adding to the ginsenoside solution a base solution having a pH of at least 10 in which a base is dissolved in at least one solvent selected from the group consisting of methanol and ethanol, stirring the mixture, precipitating, and filtering the precipitate and supernatant A method of separating dianionic ginsenoside and pyrazine triazine ginsenosides from pyrazine.
delete 3. The method according to claim 1 or 2,
and dissolving the precipitate obtained in step b) and passing it through a column of a synthetic adsorbent resin to thereby purify the resulting mixture. The method of separating pyrazine diol ginsenoside from pyrazine triol ginsenoside.
3. The method according to claim 1 or 2,
a step of purifying the supernatant obtained in step b) through a weakly acidic ion exchange resin column; and a step of adding the paraxanthiol-based ginsenoside and the pyrazine triol-based ginsenoside.
3. The method according to claim 1 or 2,
A process for separating pyrazine diol ginsenoside and pyrazine triol ginsenoside, characterized by adding a step of removing substances other than ginsenoside prior to step a).
The method according to claim 6,
Wherein the step of removing the substance other than the ginsenoside is a step of separating the pyrazyldiol ginsenoside and the pyrazine triol ginsenoside using an adsorption resin.

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