KR0148717B1 - Ginsenoside derivatives and preparation method thereof - Google Patents

Abstract

The present invention relates to a ginsenoside Rg ₁ -alpha-di-glucoside derivative of general formula (I) in which ginsenoside Rg ₁ having excellent anticancer activity is converted into a water-soluble form.

The compound of formula (I) is decomposed into ginsenoside Rg ₁ in the body at the time of ingestion, thus exhibiting the original pharmacological activity of ginsenoside Rg ₁ .

Description

Ginsenoside derivatives and preparation method thereof

The present invention relates to a ginsenoside Rg ₁ -alpha-di-glucoside derivative represented by the following formula (1) and a method for preparing the same, in particular an enzymatic preparation method.

Where R and R 'are respectively (Where n is an integer from 0-6) or H represents one of R and R ' Where R 'and R represent H.

The ginsenoside Rg ₁ -alpha-di-glucoside derivative of Formula 1 is a novel substance that has not been reported in the literature so far, and is a water-soluble substance that is decomposed into ginsenoside Rg ₁ by an alpha-glucosida system.

Ginsenoside derivatives, ginseng's unique pharmacologically active saponins, contain sugars such as glucose, rhamnose, xylose or arabinose in the alcoholic hydroxyl groups of the protoparnasadiol and the protoparnasatriol, which are triterpenoids. As a result of ether-bonded compounds, a total of 29 ginsenoside derivatives have been identified in Korean ginseng. These ginsenosides have already been found to have different pharmacological effects depending on the type of sugars bound to the aglycone, such as ProtoPanaxadiol and ProtoPanaxtriol, and the number or location of the saccharides. Ginsenoside derivatives have been attempted to chemically synthesize some of them. (E.g., Korean Patent 4066; Chen Ingee et al., Japanese Journal of Chemical Pharmacy, Vol. 35, No. 4, pp. 1653-1655, 1987; 6th International Ginseng Symposium, Elia Cove, G, B. et al., 74 -83, 1993).

Ginsenoside compounds are generally soluble in water in the irradiated state obtained by ginseng samples with a solvent such as saturated butanol or methanol, but insoluble in water when separated into a single pure component. There are disadvantages. For example, in the case of ginsenoside Rg ₁ represented by the following Chemical Formula 2, only about 1.8% at 20 ° C. and about 2.0% at 37 ° C. are dissolved, so there is a problem when it is utilized in food, cosmetics or medicine.

In addition, ginsenoside compounds are very unstable under acidic or alkaline conditions, such as desorption, epimation, dehydration, hydroxylation or cyclization of glycosyl groups in the synthesis of ginsenoside derivatives by the above-described method. It is often accompanied by undesirable side reactions, there are disadvantages of having to undergo complex organic reactions of various stages, production yields are extremely low, and in most cases, aglycone sugar is produced in the form of beta.

Therefore, there is a need for converting ginsenoside Rg ₁ to a water-soluble form in which its pharmacological activity is not lowered.

On the other hand, by using the extract of ginseng or ginsenoside to transfer the sugar by enzymatic method to reduce the bitter taste peculiar to saponin, the long-term storage of ginseng extract has been developed to suppress the generation of insoluble matter that is likely to occur in the acidic region (JP-A-5-1 61 481) However, in the above method, no chemical investigation was carried out on exactly which position and form of sugar were bound.

The present inventors have reacted sugars such as starch, cyclodextrin, and ginsenoside Rg1 with cyclomaltodextrin glucanotransferase, which has the activity of transferring sugar in a buffer or a mixture of buffer and organic solvent. at least in combination at least one superior new compound of ginsenoside Rg ₁ out in this more ginsenoside Rg ₁ in water - was the glucoside and accomplished the present invention in view I is produced in a yield of alpha-D. The derivatives developed by the present invention have improved solubility in water compared to ginsenoside Rg ₁ and are degraded by alpha-glucosidase present in the body when using it as a food or medicine or when taking a product containing the same. It is is expressing the original ginsenoside Rg side by being decomposed into _first ginsenoside Rg ₁ is originally having pharmacological activity.

The present invention includes a method of reacting sugars such as starch, cyclodextrin, and ginsenoside Rg ₁ with an enzyme which transfers sugars in alpha form in a buffer or a mixture of buffer and 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 particularly preferred solvent is a buffer solution in the range of pH 4-8, especially pH 5-7. . 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, 1-propanol, 2-propanol, dioxane, acetonitrile, dimethyl sulfoxide and the like are preferable. Do. The mixing ratio of the buffer solution and the organic solvent is not a 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 used can be sufficient to dissolve the material used for the reaction. Although not particularly limited, it is preferable to have a concentration of 0.1-50%, especially 2-10%, based on the weight of ginsenoside Rg ₁ used.

The sugars used in the present invention are not particularly limited as long as the sugars are oligosaccharides or polysaccharides in which two or more sugars are bound in the form of alpha. Such saccharides include starch and starch hydrolysates, malto oligosaccharides, dextrins and cyclodextrins, and these may be used alone or in combination. The amount of the saccharide is not particularly limited as long as it is easily dissolved in a solvent, but 0.5-10 times, preferably 1-3 times, is appropriate for the weight of ginsenoside Rg1 used.

According to the present invention, as the enzyme, cyclomaltodextrin glucanotransferase (CGTase) derived from various fines is used. The method of adding the enzyme is not particularly limited as long as it is a method in which the enzyme inactivation does not occur. For example, a method in which an enzyme is added to a solution in which sugars and ginsenosides Rg1 are dissolved or an enzyme is added in an aqueous solution is used. The enzyme used in the present invention may be immobilized using an ion exchange resin or a polymer gel in some cases.

The reaction temperature should be a temperature condition in which enzyme inactivation does not occur. It 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 a method for preparing a ginsenoside Rg ₁ -alpha-di-glucoside derivative according to the present invention is as follows.

To the acetate buffer solution, an aqueous solution of calcium chloride, sugars and ginsenosides Rg ₁ were added and dissolved, followed by addition of cyclomaltodextrin glucanotransferase (EC 2. 4. 1. 19) and 1-72 at 20-60 ° C. After the reaction was conducted for 5-10 minutes in a boiling water bath to inactivate the enzyme, the di-glucoside product of Formula 1, in which at least one sugar in the alpha form is bound to ginsenoside Rg ₁ of Formula 2, is 75-80. A reaction solution containing% is obtained.

The one-bonded ginsenoside -Rg ₁ of the formula 3, 4 to each Rg ₁ - - alpha, if necessary according to the invention ginsenosides represented by the formula (2) by processing the article Lokomotiv amylase to the above reaction mixture It is also possible to obtain di-monoglucoside.

According to the present invention, since the substances used in the reaction are harmless to the human body, the reaction solution containing glucoside may be used as it is for food, cosmetics or medicine. However, if desired, the reaction product may be separated and used by methods such as solvent extraction, silica gel column chromatography, adsorption and desorption using ion exchange resin, or preparative liquid chromatography.

The embodiment is described below.

Example 1

1.25 g of dextrin, 1.0 g of ginsenoside Rg ₁ , and 0.20 ml of calcium chloride (0.1 mole) aqueous solution were dissolved in acetic acid buffer solution (pH 6.0), and then the whole was made up to 20 ml, followed by cyclo maltodextrin glucanotransferase (Japan 500 U of the executive biochemistry laboratory materials are added and reacted for 48 hours while stirring in a 50 degreeC water bath. After periodic confirmation by thin layer chromatography, when 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. Glucoside was the powdery reaction product containing 2.3g 76.5% is obtained in the form of a mixture in the proportion of Table 1 - ginsenoside Rg ₁ in the ginsenoside Rg binding at least one sugar, at least _one-alpha-D.

The composition of the reaction product is shown in Table 1.

G: ginsenoside Rg ₁ ; GG ₁ : ginsenoside Rg ₁ -alpha-di-monoglucoside; GG ₂ : ginsenoside Rg ₁ -alpha-di-diglucoside; G-oligo: Ginsenoside Rg ₁ -alpha-di-oligoglucoside

Example 2

2.5 g of Finedex (DE 8), 2.0 g of ginsenoside Rg ₁ , 0.20 ml of calcium chloride (0.1 mol) solution was dissolved in 20 ml of acetic acid buffer solution (pH 6.0) containing 50% methanol, followed by cyclomaltodextrin glucano. 500 U of transferase is added and reacted for 72 hours with stirring at 20 ° C. The reaction solution is heated in hot water for 5-10 minutes to complete the reaction, and then concentrated under reduced pressure to remove methanol. The glucoside is the reaction product of a powder containing 64% 2.8g - alpha-D concentrate is at least one or more combined ginsenoside Rg ₁ in alpha form per the ginsenoside Rg ₁ binary extracts with saturated butanol and concentrated Got it.

The composition of the reaction product is shown in Table 1.

Example 3

Dextrin 1.25g, binary cycles the entire 20㎖ dissolved in ginsenoside Rg ₁ 1.0g, of calcium chloride (0.1 mol) of acetic acid buffer solution containing an aqueous solution 0.20㎖ (pH 6.0) and then in jeongyonghan here maltodextrin glue Kano trans flops cyclase After 500 U was added and reacted for 48 hours while stirring in a 50 ° C. water bath, the enzyme was inactivated by heating for 5-10 minutes in a boiling water bath. The reaction solution was adjusted to pH 4.5 with 4N acetic acid solution, and 36U of glucoamylase (manufactured by Nippon Biochemistry Co., Ltd.) was added thereto, followed by reacting with stirring at 50 ° C for 24 hours. The reaction solution was heated in a boiling water bath for 10 minutes to inactivate the enzyme, and then passed through a column filled with diion Api-20 resin, which had been previously activated with methanol, to remove unreacted sugars by distilled water, and to remove ginsenosides. Rg ₁ and the reaction product are concentrated by elution with methanol.

The concentrate was separated by silica gel column chromatography (chloroform-methanol-water = 65: 35: 10, lower layer) to separate ginsenoside Rg ₁ -alpha-di-monoglucoside 620 mg and unreacted ginsenoside Rg ₁ 480. Mg mg of ginsenoside Rg ₁ -alpha-di-monoglucoside product is isolated by means of Lycroprep C ₁₈ column chromatography (eluent: 65% methanol solution), where R is represented by 20 (S) -protopanaxatriol-6-0-alpha-di-glucopyranosyl (1 → 4) -beta-di-glucopyranosyl-20-beta-di-glocopy with R 'being H 310 mg of ranoside and in Formula 1, R is H and R 'is Phosphorus 20 (S) -protopanaxatriol-6-0-beta-di-glocopyranosyl-20-0-alpha-di-glocopinanosyl (1 → 3) -beta-di-glocopyrano 280 mg of side were obtained.

20 (S) -Plutopanaxatriol-6-0-alpha-di-glocopyranosyl (1 → 4) -beta-di-glucopyranosyl-20-beta-di-glucopyranoside

Rf: 0.29 (silica gel, chloroform-methanol-water = 65:35:10, lower layer)

0.56 (RP C ₁₈ , 65% Methanol)

mp: 210-212 ° C (decomposition)

IR cm ^-1 ): 3400 (OH), 1630 (olepic)

FAB-MS: Positive: mz 985.6 (M + Na) ⁺

Negative: m / z 961.8 (MH) ^-

¹ H-NMR (δ): 4.91 (1H, d, J + 7.76 μs, β-anomeric), 5.18 (1H, d, J = 7.81 μs, β-anomeric), 5.91 (1H, d, J = 3.77 ms, α-anomeric).

¹³ C-NMR (ppm): 39.4, 27.9, 78.4, 40.3, 61.2, 76.3, 44.8, 41.0, 49.9, 39.6, 30.9, 70.1, 49.1, 51.3, 30.6, 26.6, 51.4, 17.6, 17.6, 83.1, 22.3, 36.1, 23.2, 125.9, 130.8, 25.7, 17.8, 31.7, 16.4, 17.2, 105.5, 75.4, 80.2, 81.2, 78.7, 62.8, 102.8, 74.3, 75.2, 71.8, 74.7, 62.1, 98.0, 75.0, 79.2, 71.5, 78.1, 62.7

20 (S) -protopanaxatriol-6-0-beta-di-glucopyranosyl-20-0-alpha-di-glucopyranosyl (1 → 3) -beta-di-glucopyranoside

Rf: 0.29 (silica gel, chloroform-methanol-water = 65:35:10, lower layer)

0.61 (RP C ₁₈ , 65% Methanol)

mp: 208-210 ° C (decomposition)

IR cm ^-1 ): 3300 (OH), 1620 (Olefinic)

FAB-MS: Positive: m.z 985.6 (M + Na) +

Negative: m / z 961.9 (MH) ^-

¹ H-NMR (δ): 4.94 (1H, d, J + 7.65 μs, β-anomeric), 5.18 (1H, d, J = 7.76 μs, β-anomeric), 5.90 (1H, d, J = 3.90 ms, α-anomeric).

¹³ C-NMR (ppm): 39.4, 27.9, 78.4, 40.3, 61.3, 78.1, 45.0, 41.1, 49.9, 39.6, 30.9, 70.0, 49.1, 51.3, 30.6, 26.6, 51.4, 17.5, 17.5, 83.1, 22.3, 36.1, 23.2, 125.9, 130.8, 25.7, 17.7, 31.7, 16.3, 17.1, 105.5, 75.4, 80.2, 71.7, 79.2, 62.7, 98.0, 75.0, 88.9, 71.5, 77.4, 62.4, 102.0, 73.6, 74.6, 70.8, 74.1, 62.2

Claims (11)

Ginsenoside Rg1-alpha-di-glucoside derivative represented by general formula (I) Where R and R 'are respectively (Where n is an integer from 0-6) or H, where at least one of R and R ' When R 'or R represents H. Producing a glucoside derivative-alpha-D Gene The ginsenoside Rg ₁ represented by the formula (I) bonded at least two in the form of alpha per the ginsenoside Rg ₁ Gene by enzyme reaction in ginsenosides Rg solvent ₁ and sugars How to. Where R and R 'are respectively (Where n is an integer from 1-5) or H, where at least one of R and R ' When R 'or R represents H. The method of claim 2 wherein the enzyme is cyclomaltodextrin glucanotransferase (CGTase). The method of claim 2, wherein the saccharide is selected from starch hydrolyzate, dextrin, cyclodextrin, and mixtures thereof. The method according to claim 2, wherein the solvent is selected from acetate buffer, methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, dioxane, dimethylsulfoxide or a mixture thereof. The method of claim 2, wherein the reaction proceeds at 10-60 ° C. 4. Ginsenoside Rg to the party of the alpha form and the _first saccharide in the ginsenoside Rg ₁ Gene by reacting a bicyclo maltodextrin with glucoamylase in a solvent of one binary combination represented by the formula 3, 4, ginsenoside -Rg ₁ - alpha A method of making a mono-glucoside derivative. 8. The method of claim 7, wherein the enzyme is cyclomaltodextrin glucanotransferase (CGTase). 8. The method of claim 7, wherein the saccharide is selected from starch hydrolyzate, dextrin, cyclodextrin, or mixtures thereof. 8. The method of claim 7, wherein the solvent is selected from acetate buffer, methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, dioxane, dimethylsulfoxide or mixtures thereof. 8. The method according to claim 7, wherein the glucoamylase is subjected to the enzymatic reaction of ginsenoside Rg ₁ with saccharides.