KR100403570B1 - Method for the Production of Ginsenoside F2 by Enzymatic Process - Google Patents

Abstract

The present invention relates to a method for preparing ginsenoside F ₂ by an enzymatic method.

Ginsenoside F ₂ is not contained in the root of Korean ginseng, but it contains 0.2% in Korean ginseng leaves, 0.018% in the roots of American ginseng, 0.1% in the flower buds of Champaign, and 0.02% in the brains of Himalayan ginseng. It is known that there is an effect of inhibiting the proliferation of tumor cells, and the effect of reversing the multidrug resistance in tumor cells or bacteria, but it is difficult to obtain a large amount of samples.

Ginsenoside F ₂ of the present invention is a metabolite of enterobacteriaceae of ginseng saponin, and the ginsenoside F ₂ is produced in large quantities by structurally converting diol-based saponins with high content among ginseng components and easily separating. It is about how to. Ginsenoside F ₂ are ginsenoside Rb _1, Rb _2, Rc, Rd, from among the mixture thereof, or buffer solution containing the extract Aspergillus (Aspergillus) enzyme isolated from microorganism of the genus Lactase, cellulase, Reaction with beta-galactosidase or biscozymeel provides simple yields of ginsenoside F ₂ in high yield.

Ginsenoside F ₂ of the present invention can be provided as a therapeutic agent because it has various effective effects such as inhibiting tumor cell proliferation and reversing multidrug resistance in tumor cells or bacteria.

Description

Method for the production of ginsenoside F 2 by enzymatic method {Method for the Production of Ginsenoside F2 by Enzymatic Process}

20 by the present invention, enzymatic methods from ginseng (S) - Prototype wave incident diol-3-0-beta-D-glucoside-pyrazol-shi Figure -20- 0-beta-D-gluconic nose Llano side (20 (S ) -protopanaxadiol-3- O-β- D-glucopyranosido-20- O-β- D-glucopyranoside, hereafter abbreviated as ginsenoside F ₂ ). It contains 0.2% in Korean ginseng leaves, 0.018% in American ginseng roots, 0.1% in the buds of Chinese ginseng, and 0.02% in the brains of Himalayan ginseng, and it is excellent as a metabolite by intestinal bacteria of ginseng saponin. It relates to a new method for preparing ginsenoside F ₂ which has been shown to have efficacy. That is, the present invention relates to a method for producing a large amount of ginsenoside F ₂ , which is an enteric metabolite, by converting a major diol-type saponin contained in ginseng in a large amount and easy to separate by enzymatic method.

The present invention 20 represented by the enzymatic methods from the die olhyeong saponin of ginseng by the following general formula (Ⅰ) (S) - Prototype wave incident diol-3-0-beta-D-glucoside-pyrazol-shi Figure -20- 0 Prepare beta-di-glucopyranoside.

(Ⅰ)

Ginsenoside F ₂ of the general formula (I) is already known as a component having the effect of inhibiting proliferation of tumor cells, and inverting multidrug resistance in tumor cells or bacteria (Korean Journal of Pharmacognosy 28 (1), 35) , Sung Jong-hwan et al., 1997).

Ginsenoside derivatives, which are pharmacologically active saponins unique to ginseng, are compounds in which saccharides such as glucose, rhamnose, arabinose or xylose are bound to the dimaran-based triterpenoids, protopanaxanadiol or protopanaxanatriol. To date, about 34 derivatives of Korean ginseng have been identified. In addition, ginseng saponins have already been shown to have different pharmacological effects depending on the type of sugars bound to aglycone, the number of saccharides bound to each other, or the binding sites.The pharmacological effects of main saponins, which are high in ginseng and easy to separate, are discussed. Although many studies have been conducted, very little research has been conducted on the pharmacological efficacy of trace saponins.

The distribution of ginsenoside F ₂ of the present invention is known to contain 0.2% in Korean ginseng leaves, 0.018% in the roots of American ginseng, 0.1% in the flower buds of Champaign ginseng, and 0.02% in the brains of Himalayan ginseng (Asia Chemical Studies of the Plants of the Genus Panax, 森 田俊信, 1986). Oral administration of ginseng saponins is metabolized and absorbed by intestinal bacteria, especially Prevotellaris , and these metabolites have been shown to be mediators of the body that indicate the effects of ginseng saponins (The Ginseng Review 10, Kobayashi et al., 1994). ). Ginsenoside F ₂ has been shown to have various effective effects such as suppressing tumor cell proliferation and reversing multidrug resistance in tumor cells and bacteria through efficacy experiments, but it is not contained in the root of Korean ginseng species and ingested ginseng saponin. If so, it is difficult to obtain a large amount of samples at a time as the yield is low as an intestinal metabolite.

Diol-based saponins in ginseng saponins are high in content and have a structure that can be structurally converted to ginsenoside Rd, thereby revealing a method of enzymatically converting diol-based saponins to obtain ginsenoside Rd in high yield. (Korean Patent Application No. 16212, 1998). The ginsenoside F ₂ is a protocol wave incident die with C ₃ and C ₂₀ positions glucose one minutes characters each coupled each component in the all-sapogenin binary glucose molecules bonded to the C ₂₀ position of ginsenoside Rd components, leaving C ₃ This study was carried out with the idea that selective cleavage of one terminal glucose of two molecules of glucose bound to position converts to ginsenoside F ₂ .

Is a method for producing a small amount of saponins is ginsenoside Rh ₁ and Rh ₂ are contained only in red ginseng acid hydrolysis method has been developed, but (Republic of Korea Patent No. 82 730 No.: Republic of Korea Patent No. 186 757 No.) Gene agglomerans of ginsenoside silicone C ₂₀ position It is difficult to prepare ginsenoside F ₂ in which glucose is bonded to each molecule of C ₃ and C ₂₀ of the protoparanaxadiol by acid hydrolysis.

Conventionally, a method for preparing ginsenoside F ₂ is hydrolyzed 500 mg of ginsenoside Rb ₂ standard with enzyme naringinase, and compound ginseng, Y, K (compound O, Y, K) together with ginsenoside F ₂ 115 mg. Ginsenoside Ra ₁ 200 mg was reacted with enzyme naringinase to prepare 2.6 mg of F ₂ with compound K and Mc (Chem. Pharm. Bull. 30 (7), 2393, Koizumi). Et al., 1982). In addition, 5 mg of ginsenoside F ₂ produced as a degradation product in the large intestine after oral administration of 300 mg of Rb ₂ (Chem. Pharm. Bull. 38 (10). 2859, Karikura et al., 1990) . As described above, ginsenoside F ₂ has a very low production yield and is difficult to purify ginsenoside F ₂ with high purity because various secondary metabolites are produced.

The present invention is a ginsenoside by ginsenoside Rb ₁ , Rb ₂ , Rc, Rd, a major diol-based saponin contained in ginseng, ginsenoside Rb ₁ , Rb ₂ , Rc, Rd and mixtures thereof or various ginseng extracts with high diol saponin content It is an object to provide a method for producing a large amount of side F ₂ simply.

1 is a structural formula of ginsenoside F ₂ of the present invention.

Lactase isolated from ginsenosides Rb ₁ , Rb ₂ , Rc, Rd or a mixture thereof, which is a major diol-based saponin of ginseng, in an aqueous solvent or a mixture of an aqueous solvent and an organic solvent in the genus Aspergillus (Lactase), Aspergillus (Aspergillus) a cell separation in the (Cellulase), Aspergillus (Aspergillus) separating the beta in-galactosidase (β-galactosidase) or Aspergillus (Aspergillus) in Reaction with the isolated Viscozyme L readily cleaves the glycoside bonds except for one molecule of glucose at the aglycone C ₃ position and one molecule of glucose at the C ₂₀ position in a short time, and is represented by the following general formula (Ⅰ). Ginsenoside F ₂ to be produced in high yield.

More specifically, the method for preparing ginsenoside F ₂ by enzymatic method is to dissolve the diol-based saponin of ginseng in an aqueous solvent or a mixture of an aqueous solvent and an organic solvent and add an enzyme isolated from Aspergillus . Reacting with stirring in a water bath of a heated state,

Periodically checking the reaction solution by thin layer chromatography to terminate the reaction by heating in hot water when the substrate is completely lost;

Adsorbing the reaction solution to a column packed with an ion exchange resin, and then removing saccharides and enzymes by distilled water;

The reaction product is characterized by obtaining a compound represented by the following general formula (I), including eluting with an organic solvent and concentrating to obtain a powdery reaction product containing high concentration of ginsenoside F ₂ .

As the diol-based saponin, any one selected from ginsenosides Rb ₁ , Rb ₂ , Rc, Rd or a diol-based saponin mixture may be used.

(Ⅰ)

As the aqueous solvent used in the present invention, it is preferable to use one that does not lower the activity of the enzyme, such as water, acetic acid, citrate, phosphoric acid or citrate-phosphate buffer solution. As the most preferred aqueous solvent, a buffer solution having a pH of 4 to 8, particularly a pH of 4 to 6 is preferable. The above-mentioned aqueous solvent may be used alone, or may be used in combination with other aqueous solvents and organic solvents. The organic solvents that may be used in admixture should not be degrading the activity of the enzyme while being mixed with water. Preferred organic solvents include acetonitrile, dioxane, dimethyl sulfoxide, methanol, ethanol, 1-propanol, 2-propanol, and the like. The amount of the organic solvent used is 1-50% based on the substrate used, particularly 2-30. When used in a concentration of% or when used with an aqueous solvent, it is preferable to use 40% or less based on the weight of the aqueous solvent in view of increasing the yield of the reaction product.

The reaction temperature should be a temperature condition at which enzyme inactivation does not occur. When only an aqueous solvent is used, 60 ° C. or lower is preferable, and when a mixed solution of an aqueous solvent and an organic solvent is used, 50 ° C. or lower is preferable. The overall temperature is preferably maintained at 10 to 50 ℃.

The reaction time used in the present invention is not particularly limited as long as the activity of the enzyme is maintained, but is preferably 1 to 72 hours, preferably 24 to 48 hours, in which the activity of the enzyme is actively maintained.

Referring to the ginsenoside F ₂ production method according to the present invention in detail. Ginsenosides Rb ₁ , Rb ₂ , Rc, Rd or mixtures thereof and ginseng extracts having a high content ratio thereof are dissolved in an aqueous solvent or a mixed solution of an aqueous solvent and an organic solvent in a ratio of 19: 1 to 5: 1. Add lactase, cellulase, beta-galactosidase or biscozyme derived from the genus Aspergillus , react for 1 to 72 hours at 20 to 60 ° C, and heat in a boiling water bath for 2 to 15 minutes. The enzyme is inactivated to obtain a reaction solution containing a large amount of ginsenoside F ₂ . According to the enzymatic method, conventionally, a high-purity ginsenoside standard was prepared by reacting with enzyme naringinase or obtained separately from intestinal metabolite of saponin, but in this method, ginsenoside Rb ₁ is a substrate of reaction. , Lb ₂ , Rc, Rd, etc., without the difficult process of separating high purity products, saponin mixture from the genus Lactase isolated from Aspergillus, enzyme cellulase from the genus Aspergillus , Aspergillus When reacted with enzyme beta-galactosidase isolated from the genus or enzyme biscozymeel isolated from the genus Aspergillus , only ginsenoside F ₂ is produced within a short period of time, so it is easy to separate. The product prepared by the present invention exhibited the following physical and chemical properties and was identified as ginsenoside F ₂ .

Ginsenoside F ₂ Physical and chemical properties of

Appearance: White amorphous powder (butanol)

Melting Point: 186-188 ℃

[alpha]_D ²⁰: + 29.6 ° (c = 1.01, MeOH)

Rf value: 0.49 when developed with chloroform-methanol-water (= 65: 35: 10, lower layer) using Kieselgel 60 F ₂₅₄ thin layer chromatography

High speed atom impact mass spectrum (positive): m / z 807.4 [M ⁺ + Na] ⁺

Hydrogen nuclear magnetic resonance spectrum (δ): 4.92 (1H, d, J = 7.02 Hz, beta-anomeric), 5.17 (1H, d, J = 7.27 Hz, beta-anomeric)

Carbon nuclear magnetic resonance spectra (δ): 15.8, 16.1, 16.6, 17.1, 17.6, 18.2, 22.3, 23.1, 25.6, 26.4, 26.5, 27.9, 29.8, 30.6, 34.9, 35.8, 36.7, 38.9, 39.5, 39.8, 49.5 , 49.9, 51.2, 51.5, 56.1, 62.5, 62.8, 70.1, 71.3, 71.7, 75.0, 75.6, 78.1, 78.5, 79.0, 79.6, 83.1, 88.6, 98.1, 106.7, 125.7, 130.8

Hereinafter, the present invention will be described in detail with reference to Examples. However, these examples do not limit the technical scope of the present invention.

<Example 1>

₁ g of ginsenoside Rb ₁ was dissolved in 0.1 M acetic acid buffer solution (pH 4.5), and the whole was basified to 50 ml. Then, 3 g of lactase isolated from Aspergillus was added thereto, followed by stirring in a 37 ° C. water bath. React for 48 hours. After periodic confirmation by thin layer chromatography, when the substrate is completely lost, the reaction is terminated by heating in hot water for 10 minutes, and then adsorbed on the column filled with DIION HP-20 resin, and then the saccharides and enzymes are removed by washing with distilled water. The product was eluted with ethanol and concentrated to give a powdered reaction product containing 90% or more of ginsenoside F ₂ .

<Example 2>

1 g of ginsenoside Rb ₂ was dissolved in phosphate buffer (pH 4.8), and the whole was made up to 50 ml. Then, 3 g of beta-galactosidase isolated in Aspergillus was added and stirred in a 45 ° C. water bath. The reaction is carried out for 72 hours. The reaction solution is heated in hot water for 10 minutes to complete the reaction, followed by extraction with saturated butanol and concentrated under reduced pressure. The concentrate was purified by silica gel column chromatography (chloroform-methanol = 9: 1) to obtain 554 mg of ginsenoside F ₂ .

<Example 3>

1 g of ginsenoside Rc was dissolved in 0.1 M acetic acid buffer solution (pH 4.5), and the whole solution was made up to 50 ml. Then, 3 g of cellulase isolated from Aspergillus was added thereto, and stirred in a 37 ° C. water bath. React for 48 hours. After periodic confirmation by thin layer chromatography, when the substrate is completely lost, the reaction is terminated by heating in hot water for 10 minutes, and then adsorbed on a column filled with DIION HP-20 resin. The product was eluted with ethanol and concentrated to give a powdered reaction product containing 90% or more of ginsenoside F ₂ .

<Example 4>

1 g of ginsenoside Rd was dissolved in phosphate-citrate buffer solution (pH 5.0), and the whole was basified to 50 ml. Then, 4 g of biscozymeel isolated from Aspergillus was added thereto, and the mixture was heated in a 40 ° C water bath. The reaction is carried out for 72 hours while stirring. The reaction solution was heated in hot water for 10 minutes to terminate the reaction, followed by extraction with saturated butanol to give 760 mg of powdered reaction product. The concentrate was separated by silica gel column chromatography (chloroform-methanol-water = 70: 30: 5) and reverse phase RP-8 column chromatography (85% methanol) to obtain 525 mg of ginsenoside F ₂ .

<Example 5>

₁ g of a mixture of ginsenosides Rb ₁ , Rb ₂ , Rc, and Rd was dissolved in citrate buffer (pH 5.0), the whole was basified to 50 ml, and then 4 g of lactase isolated from Aspergillus was added. It is made to react for 72 hours, stirring in a 50 degreeC water bath. The reaction solution is heated in hot water for 10 minutes to complete the reaction, followed by extraction with saturated butanol and concentration. The concentrate was subjected to silica gel column chromatography (chloroform-methanol = 9: 1) to obtain 545 mg of ginsenoside F ₂ .

<Example 6>

1 g of the irradiated saponin fraction from ginseng root containing predominantly diol-based saponin was dissolved in phosphate buffer (pH 4.5), and the whole was basified to 50 ml, and then beta-galacto was isolated from Aspergillus . 3 g of sidase is added and reacted for 48 hours with stirring in a 42 degreeC water bath. The reaction solution is heated in hot water to terminate the reaction, extracted with saturated butanol and concentrated under reduced pressure. The concentrate was purified by silica gel column chromatography (chloroform-methanol = 9: 1) to obtain 436 mg of ginsenoside F ₂ .

<Example 7>

1 g of ginseng extract extract was dissolved in water, and the whole was applied to 50 ml, and then, 4 g of cellulase separated in Aspergillus was added thereto, and reacted at 37 ° C. for 48 hours. The reaction solution was heated in hot water to inactivate the enzyme, adsorbed onto a column filled with DIION HP-20 resin, washed with distilled water to remove sugars and enzymes, and the reaction product was eluted with ethanol and concentrated. A powdered reaction product containing 70% or more of F ₂ was obtained.

As described above, the present inventors conducted a multi-faceted test using enzymes isolated from various microorganisms for the purpose of developing a method for preparing ginsenoside F ₂ from diol-based saponins having high content in ginseng by enzymatic methods. (Aspergillus) a lactase separated in the first, Aspergillus (Aspergillus) a cell separation in the second, Aspergillus (Aspergillus), remove the beta from the in-galactosidase and Asda separation in Aspergillus Scotland (Aspergillus) Ginsenosides F ₂ can be produced in a high yield of 85% or more when the biscozymeel is reacted with ginsenosides Rb _1, Rb ₂ , Rc, Rd or a mixture thereof.

Claims (8)

Ginseng diol-based saponins are dissolved in an aqueous solvent or a mixture of an aqueous solvent and an organic solvent, and warmed in water by adding cellulase or biskozyme isolated from Aspergillus as an enzyme. Reacting with stirring in a bath, Periodically checking the reaction solution by thin layer chromatography to terminate the reaction by heating in hot water when the substrate is completely lost; Adsorbing the reaction solution to a column packed with an ion exchange resin, and then removing saccharides and enzymes by distilled water; The reaction product was eluted with an organic solvent and concentrated to obtain a powdery reaction product containing ginsenoside F ₂ , thereby obtaining a compound represented by the following general formula (I). Method for preparing ginsenoside F ₂ . (Ⅰ) The method according to claim 1, wherein the diol-based saponin is any one selected from ginsenoside Rb ₁ , ginsenoside Rb ₂ , ginsenoside Rc, ginsenoside Rd or a mixture thereof. Process for the preparation of cenoside F ₂ . delete delete The method of claim 1, wherein the enzymatic reaction process for producing a ginsenoside F ₂ by the enzymatic method, comprising a step of reacting at 10~50 ℃ range. The method according to claim 1, wherein the organic solvent is any one selected from acetonitrile, dioxane, dimethyl sulfoxide, methanol, ethanol, 1-propanol, 2-propanol of the ginsenoside F ₂ by the enzymatic method Manufacturing method. The method of claim 1, wherein the aqueous solvent is any one of water, acetic acid, citrate, phosphoric acid or citrate-phosphate buffer solution of any one selected from mixed with an organic solvent to be used in the range of pH 4-8 Method for producing ginsenoside F _{2 by} the method. delete