KR100671291B1 - A manufacturing method of ginsenoside F1 using beta-galactosidase - Google Patents

Abstract

The present invention relates to a method for producing F ₁ from a triol saponin using beta-galactosidase, which comprises reacting Re or Rg ₁ , which is a triol ginsenoside, with beta-galactosidase, (90% or more) of 20-O-β-D-glucopyranosyl-20 (S) -protopyranosyl triol F ₁ ,

Ginsenoside Re, ginsenoside Rg1, penicillium genus, genus Ubertrich, beta galactosidase, ginsenoside F1

Description

[0001] The present invention relates to a method for producing ginsenoside F1 by using beta-galactosidase (Ginsenoside F1 using beta-galactosidase)             

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a hydrogen nuclear magnetic resonance spectrum of the production of F ₁ converted from Rg ₁ by beta-galactosidase. FIG.

2 is a carbon nuclear magnetic resonance spectrum of the result of the production of F ₁ converted from Rg ₁ by beta-galactosidase.

The present invention relates to a method for producing ginsenoside F ₁ from ginseng extract, ginseng saponin, and more specifically, triol saponin using beta-galactosidase, and more particularly to a method for producing ginsenoside F ₁ from ginsenoside Re, Rg ₁ is selectively hydrolyzed through an enzyme reaction using a β-galactosidase derived from a genus of Penicillium, Aspergillus or Enterobacteriaceae, to produce ginsenoside F ₁ in a high yield ≪ / RTI >

Since ginsenoside F ₁ has only a very small amount of ginseng, studies on the pharmacological activity of various angles are not carried out, and only the structure thereof is known. Recent studies have shown that ginsenoside F ₁ inhibits cell necrosis of epidermal cells induced by ultraviolet light (J. Invest. Dermatology (2003).

Ginsenoside F ₁ is _one of the major metabolites that are degraded by intestinal microorganisms and finally absorbed when the natural triosylated ginsenoside (Re, Rg ₁ ) is absorbed into the body together with PPT (protopanaxatriol) Hasegawa et al., Planta Med (1996), 62, 433), and only trace amounts of ginseng are present in leaves and stems. Accordingly, various methods for producing F ₁ have been studied.

A method using intestinal bacteria and a method using various enzymes. Recently, a method using a double intestinal bacteria has been interested in a method using an enzyme because of a problem that the reaction time is long and the yield of the telephone is low.

Korean Patent Laid-Open Publication No. 2003-94756 discloses a method for producing ginsenoside F ₁ from ginseng saponin using beta-glycosidase.

Korean Patent Laid-Open Publication No. 2003-37005 describes a method for producing compound K and ginsenoside F ₁ by reacting ginseng saponin with at least one of Naringin and pectinase.

Korean Patent Laid-Open Publication No. 2003-43167 discloses a method for producing ginsenoside F ₁ by an enzyme reaction of a triol saponin of ginseng with Aspergillus niger or naringinase from the genus Penicillium, Conversion yields are up to 80%.

Accordingly, an object of the present invention is to provide a method for producing ginsenoside F ₁ at a 90% or higher yield by subjecting ginseng extract or ginseng triol saponin, preferably ginsenoside Re or ginsenoside Rg _1, to an enzyme reaction will be.

In order to achieve the above object, the present inventors have conducted intensive studies on the use of beta-galactosidase (Rg ₎ as a raw material, using Re, which is a trigrosuccinenoside belonging to protopaecoxin triol, and Rg ₁ as a di- And two molecules and one molecule of each molecule were hydrolyzed to produce ginsenoside F ₁ at a conversion yield of 90% or more.

The present invention relates to a method for preparing an enzyme reaction product by dissolving at least one member selected from the group consisting of ginseng extract, ginseng saponin, triol saponin of ginseng and triol saponin in an aqueous solvent or a mixed solution of an aqueous solvent and an organic solvent, And a method for producing ginsenoside F ₁ using beta-galactosidase.

The aqueous solvent is prepared by using water, acetic acid, phosphoric acid, citrate or citrate-phosphate buffer solution and adjusting the pH in the range of 4 to 7. The organic solvent may be a lower alcohol, acetone, acetonitrile, dioxane or dimethyl sulfoxide Use the side.

Further, the present invention is characterized in that the beta-galactosidase is derived from genus Penicillium, genus Aspergillus or genus Eubacterium.

Further, the present invention is characterized in that the triol saponin is ginsenoside Re or ginsenoside Rg ₁ .

In addition, the present invention is characterized in that the enzyme reaction is carried out at 30 to 60 ° C. Magnesium chloride may be added during the enzyme reaction to increase the enzyme activity.

Hereinafter, the present invention will be described in detail.

The following Schemes 1 and 2 illustrate the reaction formula of ginsenoside F ₁ of the present invention, and a method of producing ginsenoside F _{1 according} to the present invention will be described in more detail as follows.

The triol saponin of ginseng, more preferably ginsenoside Re, Rg ₁ or a mixture thereof is dissolved in an aqueous solvent having a pH of 4 to 6 and added to a solution containing a mixture of a pentaerythritol, an Aspergillus or a bovis gallic acid Shidase and magnesium chloride are mixed and reacted at 30 ~ 60 ℃ for 24 ~ 48 hours, treated with boiling water to remove enzyme activity, extracted with saturated butanol and concentrated to obtain ginsenoside mixture. The mixture is separated into a column to obtain ginsenoside F ₁ .

The physical and chemical properties of the resultant product obtained by the present invention are shown below.

mp: 172-174 [deg.] C

_{^{1 H-NMR (d 5 -Py}} ) δ: 5.21 (. 1H, d, J = 7.67Hz, 20-glu), 5.26 (1H, t, J = 6.34Hz, H-24)

_{^{13 C-NMR (d 5 -Py}} ) δ: 16.9, 17.8, 17.9, 18.0, 18.1, 22.7, 23.6, 26.1, 27.0, 28.5, 31.2, 31.4, 32.4, 36.6, 39.8, 39.8, 40.7, 41.6, 47.9, 49.6, 50.3, 51.8, 52.0, 62.2, 63.3, 68.1, 70.5, 72.1, 75.5, 78.7, 78.9, 79.7, 83.7, 98.6, 126.4, 131.3

Hereinafter, the specific configuration of the present invention will be described in detail with reference to examples. However, the scope of the present invention is not limited to the description of these embodiments.

Example 1: Preparation of Ginsenoside F by Penicillium betagalactosidase _One Gt;

₁ g of ginsenoside Re and ₁ g of Rg were used as a substrate in 200 ml of 50 mM sodium acetate buffer solution (pH 4.5) in the presence of 1 g of beta-galactosidase derived from Penicillium spp. And a trace amount of magnesium chloride (0.1% React for 24 hours in a constant-temperature water bath. After the disappearance of the substrate, the reaction mixture was treated with boiling water for 10 minutes to remove the enzyme activity. The reaction solution was then diluted with an equal volume of water-saturated butanol to give 3 (3-chloro-2-methoxyphenyl) The extract was concentrated and concentrated to obtain a ginsenoside mixture. The obtained product was separated by silica gel column chromatography (chloroform: methanol: water = 15: 3: 0.3) to obtain 0.61 g of ginsenoside F ₁ (at Re reaction) and 0.72 g (at Rg ₁ reaction) : 90%, 90.2%, respectively).

Example 2 Preparation of Ginsenoside F by Aspartyl Beta-Galactosidase _One Gt;

₁ g of beta-galactosidase derived from Aspergillus sp. And 200 g of magnesium chloride (0.1%) were dissolved in 200 ml of 50 mM sodium acetate buffer (pH 4.5) using ₁ g of ginsenoside Re and ₁ g of Rg as substrates, For 24 hours each. After the disappearance of the substrate, the reaction mixture was treated with boiling water for 10 minutes to remove the enzyme activity. The reaction solution was then diluted with an equal volume of water-saturated butanol to give 3 (3-chloro-2-methoxyphenyl) The extract was concentrated and concentrated to obtain a ginsenoside mixture. The obtained product was separated by silica gel column chromatography (chloroform: methanol: water = 15: 3: 0.3) to obtain 0.62 g of ginsenoside F ₁ (at Re reaction) and 0.73 g (at Rg ₁ reaction) : 90% and 92%, respectively).

≪ Example 3: Synthesis of Ginsenoside F < RTI ID = 0.0 > _One Gt;

Using ₁ g of ginsenoside Re and ₁ g of Rg as substrates, ₁ g of beta-galactosidase derived from the genus Ustwoeterium and the presence of a small amount of magnesium chloride (0.1%) were mixed in 200 ml of 50 mM phosphate buffer solution (pH 6.5) For 24 hours each. After the disappearance of the substrate, the reaction mixture was treated with boiling water for 10 minutes to remove the enzyme activity. The reaction solution was then diluted with an equal volume of water-saturated butanol to give 3 (3-chloro-2-methoxyphenyl) The extract was concentrated and concentrated to obtain a ginsenoside mixture. The obtained product was separated by silica gel column chromatography (chloroform: methanol: water = 15: 3: 0.3) to obtain 0.61 g of Ginsenoside F1 (at Re reaction) and 0.72 g (at Rg ₁ reaction) 90% and 90%, respectively).

The present invention can produce ginsenoside F ₁ with high efficiency because it is not present in ginseng and its pharmacological activity studies can not be carried out and its structure has been revealed. Therefore, it is possible to increase the possibility of mass production and development of new drugs according to various pharmacological activities have. In addition, since ginsenosides Re and Rg ₁ , which are used as raw materials, can be obtained from ginseng in a large amount, the supply and demand of raw materials is smooth, so the possibility of development of new drugs is high.

In addition, the present invention can obtain a ginsenoside F ₁ having a very high conversion yield in spite of a small amount of added enzyme as well as a short reaction time.

Claims (4)

At least one member selected from the group consisting of ginseng extract, ginseng saponin, ginseng triol saponin and triol saponin mixture containing at least one member selected from the group consisting of ginsenoside Re and ginsenoside Rg ₁ in an aqueous solvent or an aqueous solvent Wherein the ginsenoside F ₁ is dissolved in a mixed solution of an organic solvent and an enzyme reaction is carried out by mixing with a beta-galactosidase. 2. The method according to claim 1, wherein the beta galactosidase is derived from a selected one of the group consisting of genus Penicillium, genus Aspergillus and genus Eubacterium, using beta galactosidase A process for producing ginsenoside F ₁ . delete The method of claim 1, wherein the enzymatic reaction process for preparing ginsenoside F ₁ using, beta-galactosidase, comprising a step of reacting at 30 ~ 60 ℃.

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