KR20030043168A - The manufacturing method for ginsenoside compound k with cellulase or lactase composition y-ao - Google Patents

Abstract

PURPOSE: A method of preparing a ginsenoside compound K is provided to make the ginsenoside product at high yield of 80% or more with effects of immuno-stimulation, cancer inhibition, and vascular tumor inhibition. CONSTITUTION: In a method of preparing a ginsenoside compound K, a diol-based saponin extract from ginseng is dissolved in a water solvent or a mixture of a water solvent and an organic solvent, and cellulase isolated from Aspergillus niger or a lactase composition Y-AO where 78-82 wt.% of dextrin powder is mixed with 18-22 wt.% of lactase powder isolated from Aspergillus oryzae is added to the solution. The solution is stirred in a heated water bath during the enzymatic reaction. The reaction solution is mixed with water-saturated butanol to obtain a butanol layer with an eluate of ginsenoside compound K. The resulting product is concentrated, and subjected to column-chromatography to obtain ginsenoside compound K. The ginseng is selected from Panax ginseng, Panax quinquefolium, Panax japonica, or Panax notoginseng.

Description

The manufacturing method of ginsenoside compound k using cellulase or lactase composition Y-AEO {THE MANUFACTURING METHOD FOR GINSENOSIDE COMPOUND K WITH CELLULASE OR LACTASE COMPOSITION Y-AO}

The present invention relates to a process for preparing ginsenoside compound k using cellulase or lactase composition Y-AO.

Ginsenoside compound K is a metabolite of enterobacteriaceae of ginseng saponin.

Ginsenoside Compound K (20 (S) - Prototype wave incident diol -20- 0-beta-D-gluconic nose Llano side) is a compound represented by the formula (1) below, immune enhancing effect, tumor angiogenesis inhibitory action, It is known to have the effect of inhibiting cancer cell invasion, inhibiting cancer cell proliferation.

Cellulase, on the other hand, is an enzyme that hydrolyzes cellulose (EC 3.2.1.4), which can be obtained from shoots, blue mold, yeast mold, and various soil bacteria of higher plants.

In addition, cellulase is contained in the digestive juice of gastrointestinal fluid of the invertebrate snail and digestive vesicle of the caterpillar.

Lactase, also known as beta-galactosidase, is an enzyme (EC 3.2.1.23) that hydrolyzes the glycosidic bonds of β-D-galactosid, including lactose, and E. coli lactase. The agent is a tetramer with a molecular weight of about 540,000.

Conventionally, as a manufacturing method of ginsenoside compound k, 42 mg of ginsenoside compound k was prepared by adding 200 mg of enzyme naringinase to 500 ginsenoside Rb ₂ and reacting (Chem. Pharm. Bull. 30). (7), 2393, Koizumi et al., 1982), oral administration of 300 mg of ginsenoside Rb ₂ to rats followed by separation of 15 mg of ginsenoside compound K produced as a degradation product of Rb _{2 in} the rat colon. Chem. Pharm. Bull. 38,2859, Karikura et al., 1990).

In addition, in Korea, compound k 320 mg was isolated from metabolites after 5 g of a diol-based saponin fraction was reacted with enterobacteriaceae (National Journal of Pharmacognosy 26, 360, Seong Jong-hwan, 1995).

However, these methods are not suitable for the production of ginsenoside compound k efficiently and with high purity because of the extremely low production yield and the generation of various secondary metabolites.

Korean patent application 10-1991-016456 (new material 20 (R) -ginsenoside Rh ₂ ), Korean patent application 10-1996-046168 (20 (S) -ginsenoside R1 and 20 (S) -protopa In the method for preparing Naxatriol, an acid hydrolysis method has been developed as a method for preparing ginsenosides Rh ₁ and Rh ₂ which are trace amounts of saponins contained only in red ginseng. _It is difficult to prepare a ginsenoside compound k in the form of a single molecule of glucose attached to the C ₂₀ position of the protoparanaxadiol because the glycoside bond of the ₂₀ position is decomposed.

On the other hand, Korean Patent Application No. 10-2000-0043170 (method of preparing ginsenoside compound K by enzymatic method), which the applicant of the present invention is filed on July 26, 2000, and is not disclosed, the diol-based saponin fraction of ginseng A method for preparing ginsenoside compound K by reacting with enzyme cellulase isolated from silium microorganism or enzyme beta-galactosidase isolated from genus Aspergillus, but has a low yield of 75%.

An object of the present invention is a ginsenoside Rb ₁ , Rb ₂ , Rc, Rd and mixtures thereof, or ginseng extracts having a high diol saponin content, ginsenosides which are diol saponins contained in ginseng in a large amount. It is to provide a method for producing a compound k in high yield in a short time.

1 is a manufacturing process of the ginsenoside compound k of the present invention.

The present invention relates to a process for preparing ginsenoside compound k using cellulase derived from Aspergillus neiser, or lactase composition Y-AO.

The inventors of the present application, the ginsenoside compound K is a component in which a molecule of glucose is bonded to the C ₂₀ position of the protopanaxadiol sapogenin as shown by the formula (1) is bound to the C ₂₀ position of the ginsenoside Rd component The experiment was repeated with the idea that cleavage of two glucose molecules bound to the C ₃ position, leaving one molecule intact, converts it to ginsenoside compound k.

In order to develop a method for increasing the production yield of ginsenoside compound K using diol-based saponin, which is high in ginseng, various tests were carried out using enzymes isolated from various microorganisms. Or when the lactase composition Y-AO is reacted with ginsenosides Rb _1, Rb ₂ , Rc, Rd or mixtures thereof, the production yield of ginsenoside compound k is 80% or more, compared to the conventional production method. It was found to produce in high yield, and the experiment was completed through several experiments.

The lactase composition Y-AO used in the present invention is prepared by mixing 18 to 22 wt% of powdered lactase separated from Aspergillus duckase and 78 to 82 wt% of dextrin powder.

The present invention employs any one of three methods of producing a compound k using cellulase derived from Aspergillus niger, or lactase composition Y-AO, and then separating the product.

The structure of the method 1 of this invention dissolves the diol-type saponin or the mixture of diol-type saponins extracted from ginseng in the aqueous solvent or the mixed solution of the aqueous solvent and the organic solvent, The cellulase derived from Aspergillus niger, or lactase Adding the composition Y-AO, reacting with stirring on a warm water 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, and reacting The product consists of mixing with the saturated butanol to obtain the upper butanol layer from which the ginsenoside compound k is eluted, and then concentrating the separated one by column chromatography to obtain the ginsenoside compound k.

The composition of Method 2 of the present invention is to dissolve a diol-based saponin or a mixture of diol-based saponins extracted from ginseng in an aqueous solvent or a mixture of an aqueous solvent and an organic solvent, and the cellulase or lacta derived from Aspergillus niger. Adding the composition Y-AO and reacting with stirring in a warm water bath; periodically checking the reaction solution by thin layer chromatography; and heating the substrate in hot water to terminate the reaction when the substrate is completely lost; The reaction solution was adsorbed on a column filled with an ion exchange resin, and then the sugars and enzymes were washed off with distilled water. The reaction product was eluted with ethanol and concentrated, followed by column chromatography to obtain a ginsenoside compound k. It consists of steps.

The structure of the method 3 of this invention is the cellulase or lactase composition Y which originates in Aspergillus niger, dissolving the diol-type saponin or the mixture of diol-type saponins extracted from ginseng in the aqueous solvent or the mixed solution of the aqueous solvent and the organic solvent. Adding -AO and reacting with stirring in a warm water bath; periodically checking the reaction solution by thin layer chromatography; terminating the reaction by heating in hot water when the substrate is completely lost; Mixing with saturated butanol to obtain the upper butanol layer from which the ginsenoside compound k eluted was concentrated and then concentrated again to obtain a ginsenoside compound k high content until powdery.

Hereinafter, a method for preparing ginsenoside compound k according to the present invention will be described in detail with reference to FIG. 1.

<Process of Method 1>

First step diol-based saponin dissolution

Ginsenosides Rb ₁ , Rb ₂ , Rc, Rd and mixtures thereof, which are diol-based saponins of ginseng, or a mixture thereof, are dissolved in an aqueous solvent or a mixture of an aqueous solvent and an organic solvent.

The diol-based saponins of ginseng used herein are those obtained by extracting and separating from Korean ginseng, Samchil ginseng, American ginseng, bamboo shoot ginseng, Himalayan ginseng or Vietnamese ginseng.

The aqueous solvent is selected from buffers of water, acetic acid, phosphoric acid, citrate, citrate-phosphoric acid, and a buffer solution having a pH in the range of 3 to 7, especially pH 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 solvent used for mixing should be one that does not lower the activity of the enzyme while being mixed with water.

Preferred organic solvents include acetone, acetonitrile, dioxane, dimethyl sulfoxide, methanol, ethanol, 1-propanol, 2-propanol, etc., and the amount of the organic solvent is 2 to 30% based on the substrate used. .

2nd process enzyme reaction

Cellulase or lactase composition Y-AO derived from a microorganism Aspergillus niger is added to a diol-type saponin dissolution solution, and it stirs for 1 to 72 hours in 20-50 degreeC water bath, and performs an enzyme reaction.

The reaction temperature should be a temperature condition at which enzyme inactivation does not occur, preferably 50 ° C. or less when only an aqueous solvent is used, and 40 ° C. or less when a mixed solution of an aqueous solvent and an organic solvent is used.

The reaction time is not particularly limited as long as the activity of the enzyme is maintained, but 1 to 72 hours, preferably 24 to 48 hours is appropriate.

Termination of the third process enzyme reaction

After periodic confirmation by thin layer chromatography, the substrate is completely lost, and heated in a boiling water bath for 10 minutes to inactivate the enzyme to obtain a reaction solution containing mainly ginsenoside compound k.

4th process mixed with saturated butanol

The reaction solution obtained in the third step is mixed with saturated butanol to elute the ginsenoside compound k.

Obtained the fifth process butanol layer

In the solution divided into the upper butanol layer and the lower water layer, the water layer containing sugars and enzymes is discarded to obtain a butanol layer in which the ginsenoside compound k is eluted.

6th process concentration

The butanol layer is concentrated to give a concentrate.

Seventh Process Column Chromatography

The concentrate is separated by column chromatography to give the ginsenoside compound k.

<Process of Method 2>

The first to third processes of method 1 are performed in the same way.

4th process sugar and enzyme removal

After the reaction solution is adsorbed on a column packed with ion exchange resin, sugars and enzymes are removed by washing with distilled water.

Elution in Ethanol 5th Process

The reaction solution from which sugars and enzymes are removed is eluted in ethanol.

The sixth process concentration of the method 1 and the seventh process column chromatography were performed to obtain the ginsenoside compound k.

<Step 3 of the process>

The first to sixth steps of method 1 are performed in the same way.

7th process reconcentration

The concentrate obtained in the sixth step is reconcentrated until it is powdered to obtain a ginsenoside compound k high content.

Hereinafter, the present invention will be described in more detail with reference to Examples, but these are not intended to limit the scope of the present invention.

Example 1 Preparation of Ginsenoside Compound K of the Present Invention Using Ginsenoside Rc (Method 1)

1 g of ginsenoside Rc was dissolved in a phosphate buffer solution (pH 5.0), and the whole amount was adjusted to 50 ml, and then, to 18 wt% of powdered lactase separated from Aspergillus duck, dextrin powder 82 3 g of lactase composition Y-AO mixed with the weight% was added and reacted for 72 hours with stirring on a 37 ° C water bath.

The reaction solution was heated in hot water for 10 minutes to terminate the reaction.

50 ml of saturated butanol was mixed with the reaction solution, and the mixture was extracted.

An upper butanol layer was obtained and concentrated to give 710 mg of concentrate.

The concentrate was separated and purified by silica gel column chromatography (chloroform: methanol: water = 7: 3: 1) and reverse phase C ₁₈ column chromatography (85% methanol) to obtain 440 mg of ginsenoside compound k.

Example 2 Preparation of Ginsenoside Compound K of the Invention Using Mixture of Ginsenosides Rb ₁ , Rb ₂ , Rc, and Rd (Method 1)

1 g of a mixture of the same amount of ginsenosides Rb ₁ , Rb ₂ , Rc, and Rd was dissolved in citrate-phosphate buffer solution (pH 5.0), the whole was basified to 50 ml, and then powdered lacta separated from Aspergillus duckase. To the 18th weight%, 4 g of lactase composition Y-AO which mixed 82 weight% of dextrin powders was added, and it was made to react for 72 hours, stirring in a 50 degreeC water bath.

The reaction solution was heated in hot water for 10 minutes to terminate the reaction.

50 ml of saturated butanol was mixed with the reaction solution, and the mixture was extracted.

An upper butanol layer was obtained and then concentrated.

The concentrate was separated by silica gel column chromatography (chloroform: methanol = 10: 1), and then purified by reverse phase C ₁₈ column chromatography (85% methanol) to obtain 463 mg of ginsenoside compound k.

<Example 3> Preparation of ginsenoside compound k of the present invention using ginseng root mainly containing diol-based saponin (method 1)

Dissolve 1 g of the irradiated saponin fraction in ginseng root containing mainly diol-based saponin in acetic acid buffer solution (pH 4.8) to make 50 ml of the whole, and then add 4 g of enzyme cellulase isolated from Aspergillus niger. It was added and reacted for 48 hours, stirring on a 37 degreeC water bath.

The reaction solution was heated in hot water to terminate the reaction.

50 ml of saturated butanol was mixed with the reaction solution, and the mixture was extracted.

An upper butanol layer was obtained and then concentrated.

The concentrate was purified by silica gel column chromatography (chloroform: methanol = 9: 1) to obtain 335 mg of ginsenoside compound k.

Example 4 Preparation of Ginsenoside Compound K of the Present Invention Using Ginsenoside Rb ₁ (Method 2)

1 g of ginsenoside Rb ₁ was dissolved in 0.1 M acetic acid buffer solution (pH 4.5) to make the whole 50 ml.

3 g of enzyme cellulase separated from Aspergillus niger was added thereto and reacted for 48 hours with stirring on a 37 ° C water bath.

After periodic confirmation by thin layer chromatography, when the substrate disappeared completely, the reaction was terminated by heating in hot water for 10 minutes.

After adsorbing to the column packed with DIION HP-20 resin, sugars and enzymes were removed by washing with distilled water, and the reaction product was eluted with ethanol and concentrated.

The concentrate was separated by silica gel column chromatography (chloroform: methanol = 9: 1) to give 428 mg of ginsenoside compound k.

Example 5 Preparation of Ginsenoside Compound K of the Present Invention Using Ginsenoside Rd (Method 2)

1 g of ginsenoside Rd was dissolved in citrate buffer (pH 4.5), and the whole was basified to 50 ml, followed by addition of 3 g of enzyme cellulase isolated from Aspergillus niger, and stirred in a 45 ° C. water bath for 72 hours. Reacted.

The reaction solution was heated in hot water for 10 minutes to terminate the reaction, and then adsorbed onto a column filled with DIION HP-20 resin, washed with distilled water, eluted with ethanol and concentrated.

The concentrate was purified by silica gel column chromatography (chloroform: methanol = 85: 15) to obtain 485 mg of ginsenoside compound k.

Example 6 Preparation of Ginsenoside Compound K of the Present Invention Using Ginsenoside Rb ₂ (Method 3)

1 g of ginsenoside Rb ₂ was dissolved in acetic acid buffer solution (pH 5.0), and then the whole amount was adjusted to 50 ml, followed by 18 weight% of powdered lactase separated from Aspergillus duck, and 82 weight of dextrin powder. 3 g of lactase composition Y-AO mixed with% was added and reacted for 72 hours with stirring on a 45 ° C. water bath.

The reaction solution was heated in hot water for 10 minutes to complete the reaction, followed by extraction with saturated butanol, and concentrated to give 650 mg of a reaction product containing 90% or more of ginsenoside compound k.

According to the present invention there is provided a process for producing ginsenoside compound k in high yield of at least 80%.

Ginsenoside compound k prepared according to the present invention is used for the purpose of immunopotentiation, cancer cell invasion and proliferation inhibition, tumor angiogenesis inhibitory action and the like.

Claims (8)

Diol-based saponin or a mixture of diol-based saponins extracted from ginseng, It is dissolved in an aqueous solvent or a mixture of an aqueous solvent and an organic solvent, and it is 18% to 22% by weight of cellulase isolated from Aspergillus niger, or powdered lactase separated from Aspergillus duck, dextrin. Adding one selected from the lactase composition Y-AO mixed with 78 to 82% by weight of the powder, followed by enzymatic reaction with stirring on a heated water 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; Mixing the reaction solution with saturated butanol and obtaining a butanol layer in the upper layer in which the ginsenoside compound k is eluted, Concentrating the obtained product, The method of producing a ginsenoside compound k, comprising the step of obtaining a ginsenoside compound k by column chromatography. The method according to claim 1, wherein the ginseng is selected from Korean ginseng, Samchil ginseng, American ginseng, bamboo shoot ginseng, Himalayan ginseng, and Vietnamese ginseng. The method according to claim 1, wherein the diol-based saponin is any one selected from ginsenosides Rb ₁ , Rb ₂ , Rc, Rd or a diol-based saponin mixture. The method for preparing ginsenoside compound k according to claim 1, wherein the temperature during the enzymatic reaction is 20 to 50 ° C. The method according to claim 1, wherein the aqueous solvent is one selected from buffers of water, acetic acid, phosphoric acid, citrate, and citrate-phosphate. The method according to claim 1, wherein the organic solvent is one selected from acetone, acetonitrile, dioxane, dimethyl sulfoxide, and lower alcohols. Diol-based saponin or a mixture of diol-based saponins extracted from ginseng, Dextrin powder is dissolved in an aqueous solvent or a mixture of an aqueous solvent and an organic solvent and powdered lactase 18 to 22% by weight separated from cellulase or Aspergillus duckase separated from Aspergillus niger. Adding one selected from the lactase composition Y-AO mixed with 78 to 82% by weight, and performing an enzymatic reaction while stirring in a warm water 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 on a column packed with an ion exchange resin, saccharides and enzymes are washed off with distilled water, and the reaction product is eluted with ethanol, Concentrating the eluate, The method of producing a ginsenoside compound k, comprising the step of obtaining a ginsenoside compound k by column chromatography. Diol-based saponin or a mixture of diol-based saponins extracted from ginseng, Dextrin powder is dissolved in an aqueous solvent or a mixture of an aqueous solvent and an organic solvent and powdered lactase 18 to 22% by weight separated from cellulase or Aspergillus duckase separated from Aspergillus niger. Adding one selected from the lactase composition Y-AO mixed with 78 to 82% by weight, and performing an enzymatic reaction while stirring in a warm water 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; Mixing the reaction solution with saturated butanol and obtaining a butanol layer in the upper layer in which the ginsenoside compound k is eluted, Concentrating the obtained product, Reconcentrating the concentrate to obtain a powdery ginsenoside compound kae content.