KR100186757B1 - Preparation process of 20(s)-ginsenocide rh1 and 20(s)-protopanaxtrlyol - Google Patents

Abstract

The present invention relates to a process for preparing 20 (S) -ginenoside Rh ₁ of the general formula (I) and 20 (S) -proteinoxatriol of the general formula (II) which is a specific component of red ginseng. . 20 (S) -ginchenoside Rh ₁ and 20 (S) -protopaxnaxetriol are obtained in the form of a mixture when the ginsenoside Re or ginsenoside Rg ₁ is reacted with a lactase, and by silica gel column chromatography Simply separated. According to the present invention, the production method is simple and the yield of 20 (S) -ginsenoside Rh ₁ is high.

Wherein R is Or H, respectively.

Description

Preparation of 20 (S) -ginsenoside Rh1

The present invention relates to the preparation of 20 (S) -proteopaxadcitalol-6-O- beta -D-glucopyranoside [20 (S) -ginchenoside Rh1, represented by the general formula (I) And particularly relates to a method for producing 20 (S) -gincenoside using an enzyme.

Wherein R is .

According to the present invention, 20 (S) -protonaxoxatriol of the above general formula (I) wherein R is H is obtained as a by-product.

The 20 (S) -ginnenoside Rh1 of the above general formula (I) is already known as a component having an experimental hepatic decomposition inhibitory action, a platelet aggregation inhibitory action, a pre-viral activation action and a melanin synthesis promoting action, The paracetamol triol has not been specifically identified for known substances or for its physiological activity.

Ginsenoside derivatives, which are ginsenoside-specific pharmacologically active saponins, have been found to contain saccharides such as glucose, rhamnose, xylulose or arabinose in the alcoholic hydroxyl groups of protanopaxadiol and protopanaxatriol, As a result, about 30 kinds of derivatives have been found in Korean ginseng. In addition, it has already been known that the ginseng saponin has different pharmacological effects depending on the kind of sugar bound to the aglycone, the number of sugar groups bound to it, or the binding position thereof. Especially, the pharmacological effects of major saponins contained in ginseng and white ginseng However, the research on the pharmacological efficacy of trace saponin, which exists only in red ginseng, is relatively small.

Red ginseng is prepared by steam treatment of fresh ginseng. In this process, the glycosidic bond attached to the aglycone C-20 position of the structurally unstable ginsenoside is easily hydrolyzed and the hydroxyl group is inverted and equilibrium is generated to form C-20 (R) An isomer of C-20 (S) is produced. Therefore, red ginseng contains 2- (RS) -ginsenoside Rg2, Rg3, Rh1 and Rh2, which are pro sapogenin, more than ginseng and white ginseng. In particular, ginsenoside Rh1 is a unique component of red ginseng only, but it is difficult to separate only 20 (S) -ginsenoside Rh1 because it exists as a mixture of 20 (RS) isomers. 20 (S) -ginchenoside Rh1 or 20 (S) -protopaxoxathriol can also be prepared from a triol-type saponin which is contained in ginseng by a chemical method. However, (R) and C-20 (S) isomer mixture due to the inverse equilibrium of the C-20 (S) is accompanied by undesirable side reactions such as formation, dehydration, hydroxylation, etc., (Korean Patent No. 4066, No. 8291).

When the ginsenoside Re or Rg1, which is the major triol saponin of ginseng, is reacted with a lactase isolated from a microorganism, the glycoside bond at the C20 position is selectively cleaved within a short period of time to form the native 20 (S) -ginnenoside Rh1 20 (S) -protopyranyl triol in a high yield in a mixture state.

The present invention relates to a process for the preparation of 20 (S) -ginnenoside Rh1 and 20 (S) - (R) -quinoxaline Re or Rg1 by reacting ginsenoside Re or Rg1 with a lactase enzyme in an aqueous solvent such as water or a buffer solution or a mixture of a buffer solution and an organic solvent, To obtain a mixture of protonated triol and isolating 20 (S) -ginnenoside Rh1 from this mixture.

20 (S) -proctopanaxatriol is obtained as a by-product in the process of separating the 20 (S) -ginsenoside Rh1 described above.

The solvent used in the present invention is preferably a buffer solution having a pH in the range of 4 to 8, particularly 4 to 6, but other acceptable solvents that do not deteriorate the activity of the enzyme may also be used. In addition, a mixed solvent of an aqueous solvent and an organic solvent may be used as a reaction solvent. The organic solvent is not particularly limited as long as it is miscible with water. Among them, a lower alcohol such as methanol, ethanol, propanol, acetonitrile, dioxane, Side, and acetone. It is preferable to mix the organic solvent in an amount not exceeding 30% in the aqueous solvent. However, if the mixing ratio of the organic solvent is within a range that dissolves the ginsenoside used in the reaction and does not lower the activity of the enzyme, Or more. The amount of the solvent to be used is preferably 1 to 50%, particularly 2 to 30%, based on the substrate used.

According to the present invention, as the enzyme, a lactase isolated from the genus Penicillium is used, and the method of adding the enzyme is not particularly limited as long as it does not cause inactivation of the enzyme. For example, a method in which an enzyme is added to a solution in which ginsenoside Re or Rg1 is dissolved, or an enzyme is mixed with ginsenoside Re or Rg1 in an aqueous solution may be used.

The reaction temperature should be a temperature condition that does not cause inactivation of the enzyme, preferably 60 ° C or less when using only an aqueous solvent or 40 ° C or less when using a mixture of a water-soluble solvent and an organic solvent. The reaction time used in the present invention is not particularly limited as long as the activity of the enzyme is maintained, but it is suitably 1 to 72 hours, preferably 24 to 48 hours.

An example of the method for producing 20 (S) -ginnenoside-Rh1 and 20 (S) -proteopaxatriol according to the present invention is as follows.

Ginsenoside Re or Rg1 was dissolved in a phosphate buffer solution or acetic acid buffer solution. Then, the lactase separated from the genus Penicillium was added thereto, reacted at 20 to 60 ° C for 1 to 72 hours, and then incubated in a boiling water bath for 10 minutes The reaction is heated to inactivate the enzyme to obtain a mixed reaction mixture of 20 (S) -ginchenoides Rh1 and 20 (S) -protopaxnoxetriol. According to such an enzymatic method, the yield is higher than that of the conventional chemical method, the reaction takes place in a short period of time, and only the 20 (S) type ginsenoside derivative is produced. Thus, silica gel column chromatography (chloroform- (S) -ginchenoside Rh1 from the 20 (S) -protonacoxytriol can be easily isolated by the method described in J. Org. The ginsenoside derivatives prepared according to the present invention were identified as 20 (S) -ginenoside Rh1 and 20 (S) -protonacoxat triol, respectively, after the following physicochemical properties.

Physicochemical Properties of 20 (S) -ginsenoside Rh1

mp: 210-212 占 폚 (decomposition)

IR (cm -1): 3420 (hydroxyl), 1636 (olephenic)

Positive FAB-MS: m / z 639.5 (M + 1) < + &

1 H-NMR (?): 5.04 (1H, d, J = 7.78 Hz,? - anomeric)

39.1 > C NMR (ppm): 16.4, 16.7, 17.3, 17.6, 23.0, 25.8, 26.8, 27.0, 27.8, 31.2, 31.7, 32.0, 35.8, 39.4, 39.6, 40.3, 41.1, 45.2, 48.2, , 61.4, 63.1, 71.0, 71.8, 73.0, 75.4, 78.1, 78.6, 79.5, 80.0, 105.9, 126.3, 130.8

20 (S) - Physicochemical Properties of Protopanaxatriol

mp: 219-222 [deg.] C

IR (cm -1): 3420 (hydroxyl), 1636 (olephenic)

Positive FAB-MS: m / z 477.4 (M + 1) < + >

39.1 > C NMR (ppm): 16.4, 16.7, 17.3, 17.6, 23.0, 25.8, 26.8, 27.0, 27.8, 31.2, 31.7, 32.0, 35.8, 39.4, 39.6, 40.3, 41.1, 45.2, 48.2, , 61.4, 71.0, 73.0, 78.1, 78.6, 126.3, 130.8

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

[Example 1]

1 g of ginsenoside Re was dissolved in 0.1 M acetic acid buffer solution (pH 5.0), and the whole was adjusted to 50 ml. Then, 5 g of the lactase separated from the penicillium solution was added and reacted for 48 hours while stirring in a water bath at 50 캜. The reaction was terminated by heating for 10 minutes in hot water when the substrate completely disappeared by thin layer chromatography. The reaction solution was extracted with water saturated butanol and concentrated to give 20 (S) -ginnenoside Rh1 and 20 Lt; / RTI > -protopyranyl triol in an amount of at least 90%. 432 mg of 20 (S) -ginnenoside Rh1 and 59 mg of 20 (S) -protonacoxatriol were obtained from the reaction product by silica gel column chromatography (chloroform-methanol-water = 90: 10: 5).

[Example 2]

1 g of ginsenoside Re was dissolved in an acetic acid buffer solution (pH 5.0) containing 20% acetonitrile, and the whole was adjusted to 50 ml. Then, 5 g of the lactase isolated from the genus Penicillium was added, and 72 Time reaction. The reaction solution was heated in hot water for 10 minutes to terminate the reaction, followed by extraction with water-saturated butanol and concentration to obtain 0.87 g of a powdery reaction product. 420 mg of 20 (S) -ginenoside Rh1 and 56 mg of 20 (S) -protonaconsatriol were obtained from the reaction product by silica gel column chromatography (chloroform-methanol-water = 90: 10: 5)

[Example 3]

1 g of ginsenoside Re was dissolved in 0.1 M phosphate buffer solution (pH 4.8), and the whole was adjusted to 50 ml. Then, 5 g of the lactase isolated in the penicillium solution was added and reacted for 72 hours while stirring in a 37 ° C water bath. 20 (S) -ginchenoside Rh1 and 20 (S) were obtained by extraction with water-saturated butanol and concentrated to obtain 20 (S) -ginenoside Rh1 and 20 - The powdery reaction product containing the protopanaxyl triol was obtained. 406 mg of 20 (S) -ginenoside Rh1 and 51 mg of 20 (S) -protonaconsaltriol were obtained from the reaction product by silica gel column chromatography (chloroform-methanol-water = 90: 10: 5).

[Example 4]

1 g of ginsenoside Rg1 was dissolved in citrate-phosphate buffer solution (pH 5.0) containing 30% ethanol, and the whole was adjusted to 50 ml. Then, 3 g of lactase isolated from the genus Penicillium was added, While reacting for 72 hours. The reaction mixture is heated in hot water to terminate the reaction. The reaction mixture is adsorbed on a column packed with Diaion HP-20 resin. The saccharides and enzymes are washed with distilled water to remove them, and the reaction product is eluted with methanol and concentrated. 409 mg of 20 (S) -ginnenoside Rh1 and 53 mg of 20 (S) -protonaconsaltriol were obtained from the concentrate by silica gel column chromatography (chloroform-methanol-water = 90: 10: 5).

[Example 5]

1 g of ginsenoside Rg1 was dissolved in a citrate buffer solution (pH 5.0), and the whole was adjusted to 50 ml. Then, 5 g of the lactase separated in the penicillium solution was added and reacted for 72 hours at 20 ° C with stirring. The reaction solution is heated in hot water to terminate the reaction and adsorbed on a column packed with Diaion HP-20 resin. The saccharides and enzyme are washed off with distilled water and the reaction product is eluted with methanol and concentrated. 395 mg of 20 (S) -ginenoside Rh1 and 48 mg of 20 (S) -protonaconsatriol were obtained from the concentrate by silica gel column chromatography (chloroform-methanol-water = 90: 10: 5).

Claims (6)

(S) -ginnenoside Rh1 represented by the general formula (I) by reacting a compound selected from the group consisting of glycosides, Reenosines, Reenosines, Re, Rg1 and mixtures thereof with a lactase which is an enzyme in a buffer solution having a pH of 4 to 7 . Wherein R is . The process according to claim 1, wherein 20 (S) -ginnenoside Rh1 is obtained in the form of a mixture of 20 (S) -protonacoxatriol and 20 (S) -ginnenoside Rh1 is separated from the mixture by column chromatography ≪ / RTI > The method according to claim 1, wherein the lactase is an enzyme isolated from the genus Penicillium. The process of claim 1, wherein the buffer solution is used in combination with an organic solvent selected from the group consisting of lower alcohols, acetone dioxane, dimethyl sulfone, acetonitrile. The method of claim 4, wherein the organic solvent is used at 30% or less based on the weight of the buffer solution. The process of claim 1, wherein the reaction proceeds in the range of 10-50 < 0 > C.