KR100249427B1 - Ginsenoside re-beta-d-xylosides and producing method thereof - Google Patents

Abstract

The present invention relates to ginsenosides Re-beta-di-xylosides and methods for their preparation. Ginseng saponins are classified as panaxanadiol (PD) saponins and panaxanatriol (PT) saponins according to the basic structural characteristics of sapongenin. Among the panaxanadiol-based saponins, there is a saponin component in which xylose is bound as a constituent sugar, but a saponin component in which xylose is bound as a constituent sugar has not been found to date.

The present invention relates to the preparation of two new saponin compounds in which xylose is bound to Re by reacting with ginsenoside Re, which is contained in a large amount in the root or ground of ginseng as a substrate, with an enzyme having a sugar transfer activity. In other words, when ginsenoside Re and para-nitrophenyl-beta-xylopyranoside were reacted with the enzyme cellulosease, xylose beta was added to the fourth carbon of glucose bound to the saponenin carbon 6 of Re. Xylose is bound to the sixth carbon of glucose bound to saponenin carbon 6 of Re when reacted with one of the derivatives bound to one form of enzyme, enzyme beta-galactosidase. It relates to the production of one derivative.

Two ginsenosides Re-beta-di-xyloside prepared by the present invention has been found to be a novel saponin, which is simple to react, can be prepared in a short time, and is not known until now.

Description

Ginsenoside R-beta-di-xyloside and its preparation method by enzymatic method

The present invention relates to ginsenoside Re-beta-di-xyloside represented by the following general formula (I) and a preparation method thereof, in particular an enzymatic preparation method.

Where R and R 'are respectively Or one of R and R ' When R 'and R represent H.

Ginsenoside Re-beta-di-xyloxide represented by the above formula (I) is a novel substance that has not been reported in the literature so far.

Ginsenoside derivatives, ginseng's unique pharmacologically active saponins, contain alcohols such as glucose, rhamnose, xylose, or arabinose in the alcoholic hydroxyl groups of the protoparnaxadiol and the protoparnaxatriol. About 30 ginsenoside derivatives from Korean ginseng have been identified as the combined compounds. Ginsenosides have already been found to have different pharmacological effects depending on the types of sugars bound to aglycone, the number of saccharides bound, or the position of the saccharides, and attempts to synthesize these ginsenoside derivatives by chemical methods (E.g., Korean Patent 4066; Chen Ingee et al., Journal of the Japanese Chemical and Pharmaceutical Sciences, Vol. 35, No. 4, pages 1653-1655, 1987; Pp. 74-83, 1993). However, ginsenosides are very unstable under acidic or alkaline conditions, and in the process of synthesizing ginsenoside derivatives by chemical methods, undesirable side reactions such as glycosyl group departure, epimerization, dehydration, hydroxylation or cyclization In many cases, it is necessary to go through complex organic reactions in several stages, and the manufacturing yield is extremely low. In addition, although a saponin component in which xylose is bound as a constituent sugar to protopanaxadiol-based saponins has been found, no saponins in which xylose is bound to protopanasulfa triol-based saponins have been found. No method of binding xylose to triol-based saponins has ever been reported.

The present inventors reacted with a cellulase or beta-galactosidase having a sugar transfer activity in a buffer solution containing a saccharide and ginsenoside Re capable of donating xylose, or a buffer solution and an organic solvent. Ginsenoside Re-beta-di-xyloside, which is a new compound bonded one by one in the form of a beta, was produced in high yield to complete the present invention. Derivatives developed according to the present invention have improved solubility in water compared to ginsenoside Re, and the original ginsenoside Re is produced by an enzyme present in the body when using it as a food or medicine or when taking a product containing the same. Glycenoside Re has the original pharmacological activity.

The present invention is obtained by reacting a saccharide and a ginsenoside Re capable of donating xylose with an enzyme which transfers a sugar to beta in a buffer or a mixture of a buffer and an organic solvent.

The solvent used in the present invention is not particularly limited as long as it can dissolve the material used in the reaction and does not lower the activity of the enzyme, but a buffer solution in the range of pH 4-8, especially pH 5-7 is preferable. In addition, a mixed solution of a buffer solution and an organic solvent may be used. The organic solvent is not particularly limited as long as it is soluble in water. Among them, methanol, ethanol, propanol, dioxane, acetonitrile, acetone, dimethyl sulfoxide and the like are preferable. The mixing ratio of the buffer solution and the organic solvent is not a big problem as long as it can dissolve the material used in the reaction and does not lower the activity of the enzyme, and the amount of the solvent is particularly limited as long as it can dissolve the material used for the reaction sufficiently. Although it is not received, it is preferable to have a concentration of 0.1-50%, especially 2-10%, based on the ginsenoside Re used.

The saccharide used in the present invention is not particularly limited as long as it is a saccharide capable of donating xylose. Such saccharides include xylan, xyloligosaccharides, para-nitrophenyl-beta-xylose, ortho-nitrophenyl-beta-xylose, beta-phenyl xylose, and the like, and these may be used alone or in combination. The amount of the saccharide used is not particularly limited so long as it is easily dissolved in a solvent, but 0.5-10 times, preferably 1-3 times, is appropriate for the ginsenoside Re used.

The enzyme used in the present invention is not particularly limited as long as the enzyme has beta-xylosidase activity, but especially cellulase, beta-galactosidase, and lactase are useful, and the enzyme addition method does not cause enzyme inactivation. If it does not, it is not particularly limited. For example, a method of adding an enzyme or a solution of an enzyme in an aqueous solution is used.

The reaction temperature should be a temperature condition at which enzyme inactivation does not occur. The temperature is preferably 30-60 ° C. when only a buffer solution is used and 20 ° C. or less when a buffer solution and an organic solvent mixture are used. The reaction time used in the present invention is also not particularly limited as long as the activity of the enzyme is maintained, but 1-72 hours, preferably 24-48 hours is appropriate.

An example of the ginsenoside-Re-beta-di-xyloside preparation method according to the present invention is as follows.

After dissolving saccharides and ginsenosides Re in phosphate buffer solution, cellulose and beta galactosidase were added thereto, reacted at 20-60 ° C. for 1-72 hours, and then heated in a boiling water bath for 5-10 minutes. By inactivating the enzyme, a reaction solution containing 25-45% of ginsenoside Re-beta-di-xyloxide having one xylose bound to ginsenoside-Re in beta form is obtained.

According to the present invention, since the substances used in the reaction are harmless to the human body, the reaction solution containing xyloside may be used as it is for food, cosmetics or medicines, and solvent extraction and silica gel column chromatography may be used if necessary. The reaction products may be separated and used by methods such as graphigraphy, adsorption / desorption using ion exchange resin, or preparative liquid chromatography.

The following examples are provided to illustrate the present invention, and the scope of the present invention is not limited by these examples.

Example 1

1 g of ginsenoside Re and 2 g of para-nitrophenyl-beta-di-xyloxide were dissolved in 20 ml of a mixture of 0.1 M acetic acid buffer solution (pH 5.0) containing 30% acetone, followed by cellulose (Onozuka R- 10) Add 131 U and react for 72 hours with stirring in a 50 degreeC water bath. When the reaction product is no longer generated by thin layer chromatography, the reaction product is no longer produced. The reaction is terminated by heating in hot water for 5-10 minutes, and then the reaction solution is extracted with saturated butanol and concentrated to form xinose. 1.40 g of a powdery reaction product containing 40% or more of Re-beta-di-xyloxide was obtained. The reaction product was separated from Re by silica gel column chromatography (chloroform-methanol-water = 65: 35: 10), followed by licrosorb. NH ₂ Column (eluent: acetonitrile-water-butanol = 8: 2: 1) and Novapack C ₁₈ Separated by column (elution solvent: acetonitrile-water = 12: 88), and added to the formula (I).

West R 20 (S) -protopanaxatriol-6-O-alpha-el-rhamnopyranosyl (1 → 2)-[beta-di-xylpyranosyl (1 → 4)]-wherein R 'is H 226 mg of beta-di-glucopyranosyl-20-O-beta-di-glucopyranoside was obtained.

※ 20 (S) -protopa-anaxtriol-6-O-alpha-el-ramnopyranosyl (1 → 2)-[beta-di-xylopyranosyl (1 → 4)]-beta-di-glucose Pyranosyl-20-O-beta-di-glucopyranoside

Rf: 0.21 (silica gel, chloroform-methanol-water = 65:35:10, lower layer)

mp: 185-187 ° C (decomposition)

[α] D ²² : -7.5 (C = 0.40, MeOH)

IR ( cm ^-1 ): 3420 (OH), 1636 (olefinic)

Positive electrospray MS: m / z 1079.5 (M + 1) ⁺ , m / z 1102.1 (M + Na) ^{+ 1} H-NMR (δ): 5.02 (lH, d, J = 7.60 Hz, β-anomeric, 6-glucose), 5.18 (1H, d, J = 7.70 Hz, β-anomeric, 20-glucose), 4.79 (lH, br s, α-anomeric, rhamnose), 5.16 (lH, d, J = 6.75 Hz, β-anomeric, β-gyros).

¹³ C-NMR (ppm): 17.13 17.39, 17.51, 17.67, 17.67, 18.69, 22.22, 23.14, 25.69, 26,52, 27.65, 30.59, 30.82, 32.07, 35.93, 39.30, 39.54, 39.87, 41.07, 45.73, 48 , 96, 49.41, 51.27, 51.55, 60.68, 62.20, 62.77, 67.26, 69.47, 70.06, 70.64, 71.52, 72.15, 72.24, 74.02, 74.82, 74.97, 75.06, 76.19, 78.23, 78.23, 77.05, 78.16, 78.16, 79.14 , 82.28, 83.15, 98.18, 101.49, 101.78, 105.61, 125.90, 130.82

Example 2

1 g of ginsenoside Re and 2 g of para-nitrphenyl-beta-di-xyloxide were dissolved in 20 ml of a 0.1 M mechirubine buffer solution (pH 5.0) containing 40% methanol, followed by cellulase (ono). Zhuka R-10) 131 U was added and reacted for 48 hours with stirring in a 40 ° C water bath. When the reaction product is no longer generated by thin layer chromatography, the reaction product is no longer produced. The reaction is terminated by heating for 5-10 minutes in hot water, and then concentrated under reduced pressure to remove methanol. The concentrate was extracted with saturated butanol and concentrated to obtain 1.35 g of a powdery reaction product containing 45% of ginsenoside Re-beta-di-xyloxide having one xylose bound. The reaction product was separated from Re by silica gel column chromatography (chloroform-methanol-water = 65: 35: 10). C ₁₈ 207 mg of new saponin as in Example 1 was obtained by column chromatography (elution solvent: acetonitrile-water = 15: 85).

Example 3

1 g of ginsenoside Re and 2 g of phenyl-beta-di-xyloside were dissolved in 20 ml of a 0.1 M mechirubine buffer solution (pH 5.0) mixture containing 30% acetonitrile and then beta-galactosidase 148 U The reaction was added for 48 hours with stirring in a 50 ° C. water bath and then heated for 5-10 minutes in a boiling water bath to inactivate the enzyme. The reaction solution was passed through a column filled with diion ACP-20 resin preactivated with methanol to remove unreacted sugars by distilled water, and the ginsenoside Re and the reaction product were eluted with methanol and concentrated. The concentrate was separated by silica gel column chromatography (chloroform-methanol-water = 65: 35: 10, lower layer), and the ginsenoside-Re beta xyloside product was purified using a lysosorb NH ₂ column (eluent solvent: acetonitrile-). Separated by water-butanol = 8: 2: 1), where R is H and R 'is Phosphorus 20 (S) -protopanaxatriol-6-О-beta-di-ramnopyranosyl I (1 → 2)-[beta-di-xylopyranosyl (1 → 6)]-beta-di- 158 mg of glucopyranosyl-20-О-beta-di-glucopyranoside was obtained.

※ 20 (S) -protopanaxatriol-6-О-beta-di-ramnopyranosyl [(1 → 2) -beta-di-xylpyranosyl (1 → 6)]-beta-di-glucose Pyranosyl-20-О-beta-di-glucopyranoside

Rf: 0.15 (silica gel, chloroform-methanol-water = 65: 35: 10, lower layer)

mp: 189-192 ° C

[α] D ²² : -12 (C = 0.40, MeOH)

IR (cm ^-1 ): 3419 (OH), 1636 (olefinic)

Positive e1ectrospray MS: m / z 1079.5 (M + 1) ⁺ m / z 1102.1 (M + Na) ⁺

¹ H-NMR (δ): 4.87 (1H, d, J = 7.40 Hz, β-anomeric, 6-glucose), 5.17 (1H, d, J = 7.71 Hz, β-anomeric, 20-g1ucose), 4.75 ( 1H, br s, α-anomeric, rhamnose), 5.24 (1H, d, J = 6.61 Hz, β-anomeric, xylose).

¹³ C-NMR (ppm): 17.32, 17.32, 17.32, 17.63, 17.82, 18.68, 22.32, 23.25, 25.75, 26.66, 27.71, 30.86, 30.86, 32.14, 35.91, 39.44, 39.66, 39.90, 41.22, 46.40, 49.03, 49.59, 51.47, 51.81, 60.83, 62.79, 67.04, 69.27, 70.29, 71.01, 71.37, 71.52, 72.24, 72.36, 72.82, 73.54, 74.13, 74.81, 75.17, 76.56, 78.13, 78.22, 78.38, 78.43, 79.13, 79.13, 79.13 , 83.28, 98.26, 101.52, 101.94, 106.47, 125.95, 130.92

Example 4

1 g of ginsenoside Re and 2 g of phenyl-beta-di-xyloxide were dissolved in 20 ml of a mixture of 0.1 M acetic acid buffer solution (pH 5.0) containing 20% dioxane, followed by addition of lactase Y-AO 164 U. After reacting for 48 hours while stirring in a 40 ° C. water bath, the enzyme is inactivated by heating for 5-10 minutes in a boiling water bath. The reaction solution was passed through a column packed with DIION AHPI-20 resin previously activated with methanol to remove unreacted sugars by distilled water, and the ginsenoside Re and the reaction product were eluted with methanol and concentrated. Concentrate was purified by silica gel column chromatography (chloroform-methanol-water = 65: 35: 10, lower layer) to separate ginsenoside -Re beta xylene as a side product in the Nova Pack C ₁₈ column (eluent: acetonitrile-water = 12 : 88) to give 145 mg of new saponin as in Example 3.

Ginsenoside Re-beta-di-xylosides developed by the present invention are novel saffins that can be easily reacted and prepared in a short period of time. When using or taking a product containing the same, it is degraded to the original ginsenoside Re by an enzyme present in the body, thereby expressing the original pharmacological activity of the ginsenoside Re.

Claims (6)

Ginsenoside Re-beta-di-xyloside represented by formula (1) Where R and R 'are respectively Or at least one of R and R ' When R 'or R represents H. Method for preparing ginsenoside Re-beta-di-xyloxide by enzymatic reaction of ginsenoside Re and saccharides in a solvent to form a sugar represented by the general formula (I) in the form of beta. Where R and R 'are respectively Or at least one of R and R ' When R 'or R represents H. The method of claim 2, A process for preparing ginsenoside Re-beta-di-xyloxide by enzymatic method characterized in that the enzyme is cellulase, beta-glucosidase or lactase. The method of claim 2, Characterized in that the saccharide is xylan, xyloligosaccharide, para-nitrophenyl-beta-di-xyloside, ortho-nitrophenyl-beta-di-xyloside, phenyl-beta-di-xyloside or mixtures thereof The manufacturing method of ginsenoside Re-beta-di-xyloside by the enzymatic method. The method of claim 2, Ginsenoside Re-beta- by enzymatic method characterized in that the solvent is selected from acetate buffer, methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, dioxane, dimethylsulfoxide or a mixture thereof. Method for preparing di-xyloxide. The method of claim 2, A process for preparing ginsenosides Re-beta-di-xyloxide by an enzymatic method, characterized in that the reaction proceeds at 10-60 ℃.