KR20030037005A - Manufacturing method of Compound K and Ginsenoside F1 from ginseng ginsenosides - Google Patents

Abstract

PURPOSE: A process for preparing a compound K and ginsenoside F1 from saponin of ginseng is provided, thereby simply mass-producing the compound K and ginsenoside F1 which show main pharmacological activities of ginseng saponin. CONSTITUTION: A process for preparing a compound K of formula(1) and ginsenoside F1 of formula(2) from saponin of ginseng comprises the steps of: dissolving purified saponin of ginseng in an aqueous solvent such as water or a buffer solution or a mixture of an aqueous solvent and an organic solvent; and adding at least one of naringinase and pectinase into the saponin solution, wherein the naringinase is isolated from Penicillium sp. and the pectinase is isolated from Aspergillus; and the organic solvent is lower alcohol such as ethylacetate, toluene, ether, ethanol and methanol and is added in an amount of 60 wt.% or less.

Description

Manufacturing method of Compound K and Ginsenoside F1 from ginseng ginsenosides

The present invention relates to 20-O-β-D-glucopyranosyl-20 (S) -protopanaxadiol (hereinafter referred to as "Compound K") and 20-O-β represented by Formula 2 The present invention relates to a method for preparing -D-glucopyranosyl-20 (S) -protopanaxatriol (hereinafter referred to as "ginsenoside F1"), wherein a purified saponin prepared from ginseng is an aqueous solvent such as water or a buffer solution. Or a high yield of compound K by dissolving in a mixture of an aqueous solvent such as water or a buffer solution and an organic solvent and then reacting with at least one of naringinase isolated from penicillium and pectinase isolated from Aspergillus. The present invention relates to a method for preparing ginsenoside F1.

Compound K represented by Chemical Formula 1 and Ginsenoside F1 represented by Chemical Formula 2 are known to be formed by enterobacteria as a major metabolite of human ginseng saponin (Hasegawa, H., Sung, JH, Matsumiya.S). , Uchiyama. M., (1996) Planta Medica 62,453-457).

Ginsenoside derivatives, the unique pharmacologically active saponins of ginseng, are a compound in which saccharides such as glucose, rhamnose, arabinose or xylose are combined with protoparnaxadiol, a triterpenoid of dammaran, More than 30 derivatives have been found in Korean ginseng. In addition, ginseng saponins have already been shown to have different pharmacological effects depending on the type of sugar bound to aglycone, the number of saccharides bound to each other, or the position of binding.Ginseng saponin has a high content of ginseng and is easy to separate. Although much research has been conducted on the pharmacological efficacy of trace saponins, which are present only in red ginseng, and the metabolite saponins produced by the human body, relatively few studies have been conducted.

Among the saponin components of ginseng, Compound K with one sugar (glucose) attached to ProtoPanaxadiol, Ginsenoside Rh1, Ginsenoside Rh2, Ginsenoside F1, etc. It has been known to have pharmacological effects such as growth inhibition, tumor growth inhibition, and anticancer activity enhancement. Especially, as the recent research on saponin metabolites has been conducted, the effects of ginseng saponin are not enterobacteria but saponin itself. It is known that the metabolite of is an active body.

A useful method of one of the compounds of metabolites K of ginseng, there are soil fungi Aspergillus Nigga (Aspergillus niger), have been reported a method using a jangnaegyun and human feces derived jangnaegyun the rat (Korea Patent No. 178863 No.) A method for obtaining ginsenosides Rh1 and Rh2, which are trace components of red ginseng, has been reported by reacting a saponin mixture with beta galactosidase, naringinase and lactase isolated from microorganisms (Korean Patent No. 186757, Korean Publication). Patent No. 00-45694).

Although there are some differences depending on the parts and types of ginseng, ginseng contains Protoparnaxadiol-based saponins and Protoparnaxatriol-based saponins in a ratio of 1: 1 to 3: 1. These protoparnacindiol-based and proto-parnacetriol-based saponins are known to multiply their effects through complementary actions with different effects. However, the conventional methods are a method of producing compound K by separating most of the protoparanaxadiol-based saponins, and there is a problem in that ginsenoside F1 cannot be prepared from the protoparnaxatriol-based saponins. In addition, when using soil bacteria as a conventional method for preparing compound K requires a culture period of more than two weeks, when using human fecal-derived enterobacterial compounds as well as other intermediates are prepared, beta galacto In the case of simply reacting enzymes such as sidase, naringinase, and lactase, a small amount of compound K and ginsenoside F1 were generated, and ginsenosides Rh1 and Rh2 were mainly produced.

Accordingly, the present invention is to provide a method for producing a high yield of compound K and ginsenoside F1 by a simple process using an enzyme that has a long reaction time and low yield of enterobacteriaceae. More specifically, in the present invention, saponins are dissolved in an aqueous solvent such as water or a buffer solution, or a mixed solution of an aqueous solvent and an organic solvent such as water or a buffer solution, and then separated from the genus Pectina, an enzyme that degrades sugar bonds. It is to provide a method for producing a high yield of compound K and ginsenoside F1 by reacting with at least one of naringinase separated in the agent and penicillium.

1 is a high-performance liquid chromatogram of red ginseng purified saponin

2 is a high-performance liquid chromatogram of reactants after enzyme reaction

The present invention relates to a method for preparing compound K and ginsenoside F1, which are the main metabolites of ginseng, wherein the purified saponin prepared from ginseng is an aqueous solvent such as water or a buffer solution, or an aqueous solvent such as water or a buffer solution, and an organic solvent. The present invention relates to a method for producing a high yield of compound K and ginsenoside F1 by dissolving in a mixture of solvent and reacting with at least one of naringinase isolated from penicillium and pectinase isolated from Aspergillus. .

Hereinafter, the method for preparing Compound K and ginsenoside F1 according to the present invention will be described in detail.

First, 0.1-20% by weight of ginseng purified saponin is dissolved in an aqueous solvent such as water or a buffer solution or a mixture of an aqueous solvent such as water or a buffer solution and an organic solvent.

As a solvent used herein, an aqueous solvent such as water or a buffer solution having a pH of 3 to 8, preferably a pH of 4 to 6, or a mixture of an aqueous solvent such as water or a buffer solution and an organic solvent is used.

In the present invention, a citrate buffer solution is used as an aqueous solvent, and an organic solvent is not particularly limited, and lower alcohols such as methanol, ethanol, propanol, toluene, ethyl acetate, chloroform and the like are used. The organic solvent is preferably mixed in an aqueous solvent of about 0.1 to 50% by weight, but the mixing ratio of the organic solvent is used in the range that can dissolve the high concentration of purified saponin used for the reaction and does not lower the activity of the enzyme. The optimum solvent condition is preferably used by appropriately mixing the organic solvent than using only the aqueous buffer solution. The use of a mixture of organic solvents in this way is believed to improve the yield of compound K and ginsenoside F1 by increasing the solubility of the intermediate reactants produced by the enzymatic reaction. More preferably, an organic solvent containing 5 to 25% by weight of ethanol and ethyl acetate may be used.

Then, 1 to 1000% by weight of naringinase isolated from penicillium and pectinase isolated from Aspergillus was added to the mixed solution relative to the substrate, and then reacted for 1 to 72 hours at 20 to 60 ° C. After heating, the mixture was heated in a boiling water bath for 10 minutes to inactivate the enzyme to obtain a reaction solution containing a large amount of Compound K and ginsenoside F1.

In this case, the enzyme is not particularly limited as long as it does not occur when the enzyme is not inactivated. Narininase, pectinase, or naringinase and pectinase may be simultaneously reacted or one enzyme may be reacted first. In other words, once one enzyme is reacted first and then another enzyme is added one at a time or slowly, the reaction may be performed. Preferably, the pectinase is first reacted and then some time after narininase is reacted. It is good to add slowly.

In addition, the reaction temperature is a temperature at which enzyme inactivation does not occur, that is, 20 to 80 ° C for naringinase, 20 to 60 ° C for pectinase, preferably 30 to 50 ° C, pectina The agent is reacted with stirring at 30 to 40 ° C for 1 to 120 hours, preferably 36 to 72 hours.

Finally, ethyl acetate was added 1: 1 in the reaction solution, extracted three times, and concentrated. Then, compound K and ginsenoside F1 were obtained by silica gel column chromatography (chloroform: methanol = 9: 1).

The products prepared by the present invention exhibited the following characteristics and were identified as compound K and ginsenoside F1.

Physical and chemical properties of Compound K

Appearance: White fine crystal

Positive FAB-MS: 623 [M + H] +

<Physicochemical Properties of Ginsenoside F1>

Appearance: White fine crystal

Positive FAB-MS: 639 [M + H] +

1 H, 13 C-NMR Data of Compound K with Ginsenoside F1 Compound K Ginsenoside F1 13C 1H 13C 1H C-1 40.030 0.774 (3H, S, Me-19) C-1 40.121 0.955 (6H, S, Me-18, 19) C-2 27.999 0.913 (3H, S, Me-18) C-2 27.748 1.080 (3H, S, Me-29) C-3 78.212 0.923 (3H, S, Me-30) C-3 78.22 1.283 (3H, S, Me-28) C-4 40.030 0.959 (3H, S, Me-28) C-4 40.493 1.393 (3H, S, Me-21) C-5 57.272 1.016 (3H, S, Me-29) C-5 62.515 1.616 (6H, S, Me-27, 30) C-6 19.416 1.280 (3H, S, Me-21) C-6 68.871 1.673 (3H, S, Me-26) C-7 35.853 1.616 (3H, S, Me-27) C-7 36.596 4.020 (1H, ddd-like, H-6) C-8 40.968 1.676 (3H, S, Me-26) C-8 41.996 4.585 (1H, d, 8.1 Hz, H-1´-20-Glu) C-9 49.77 4.612 (1H, doublet, 7.8 Hz, H-1´-20-Glu) C-9 49.383 5.077 (1H, t, 6.9 Hz, H-24) C-10 38.171 5.102 (1H, t, 6.9 Hz, H-24) C-10 40.121 - C-11 31.641 - C-11 30.894 - C-12 71.185 - C-12 71.174 - C-13 51.064 - C-13 50.442 - C-14 52.491 - C-14 52.347 - C-15 30.765 - C-15 30.894 - C-16 27.194 - C-16 27.183 - C-17 53.147 - C-17 53.09 - C-18 16.244 - C-18 16.134 - C-19 16.126 - C-19 16.134 - C-20 84.936 - C-20 84.871 - C-21 22.854 - C-21 22.808 - C-22 36.653 - C-22 31.611 - C-23 24.254 - C-23 24.212 - C-24 125.853 - C-24 125.842 - C-25 132.288 - C-25 132.288 - C-26 25.878 - C-26 25.885 - C-27 17.948 - C-27 17.951 - C-28 28.625 - C-28 31.448 - C-29 16.718 - C-29 17.223 - C-30 17.189 - C-30 17.674 - C-1´ 98.303 - C-1´ 98.28 - C-2´ 75.397 - C-2´ 75.37 - C-3´ 79.529 - C-3´ 79.506 - C-4´ 71.925 - C-4´ 71.784 - C-5´ 77.947 - C-5´ 77.92 - C-6´ 62.519 - C-6´ 62.109 -

Hereinafter, the configuration and effects of the present invention will be described in more detail with reference to Examples.

Reference Example 1 Preparation of Ginseng Purified Saponin

Red ginseng, white ginseng, fresh ginseng, rice ginseng or 4 g of these ginseng leaves were added with 4 liters of methanol or methanol containing water and water, extracted three times under reflux, and then deposited at 15 ° C. for 6 days. Thereafter, the residue and the filtrate were separated through filter cloth filtration and centrifugation. The extract obtained by concentrating the separated filtrate under reduced pressure was suspended in water, and then extracted five times with 1 L of ether to remove the pigment, and the aqueous layer was 1-butanol. Extracted three times with 500 ml. The total 1-butanol layer obtained therefrom was treated with 5% KOH, washed with distilled water, and then concentrated under reduced pressure to obtain 1-butanol extract, which was dissolved in a small amount of methanol and added to a large amount of ethyl acetate. By drying the precipitate, 40 to 80 g of ginseng purified saponin extract was obtained.

Example 1

10 g of ginseng purified saponin was dissolved in 1000 ml of citrate buffer (pH 4.0), and 15 g of pectinase enzyme was added thereto and reacted with stirring in a 30 ° C water bath. The reaction was allowed to proceed for 48 hours while stirring 100 ml of ethanol and 2.5 g of naringinase every 12 hours from 12 hours to 36 hours. After the reaction was completed, the mixture was heated for 10 minutes, and the reaction solution was extracted three times with the same amount of ethyl acetate and concentrated. The obtained product was separated by silica gel column chromatography (chromoform: methanol = 9: 1) to obtain 3.2 g of compound K and 1.5 g of ginsenoside F1. The yield of the resulting compound K and ginsenoside F1 was 87.8%. It was.

Example 2

5 g of ginseng purified saponin was dissolved in 500 ml of citrate buffer (pH 5.5), and 10 g of pectinase was added thereto to react with stirring in a 30 ° C. water bath for 24 hours, followed by heating in a boiling water bath to inactivate the enzyme. . 85 ml of ethanol and 5 g of naringinase were added thereto, and the reaction proceeded while stirring for 24 hours in a 40 DEG C water bath. After the reaction was completed, the mixture was heated for 10 minutes, and the reaction solution was extracted and concentrated three times with the same amount of ethyl acetate. The obtained product was separated by silica gel column chromatography (chromoform: methanol = 9: 1) to obtain 1.25 g of compound K and 0.68 g of ginsenoside F1. The yield of compound K and ginsenoside F1 was 72%. It was.

Example 3

2 g of ginseng purified saponin was dissolved in 200 ml of citrate buffer solution (pH 4.0) containing 20 ml of ethyl acetate, and 4 g of naringinase was added thereto and reacted with stirring for 24 hours in a 40 DEG C water bath. Thereafter, 4 g of pectinase was added thereto, and the reaction was performed while stirring for 24 hours in a 30 ° C water bath. After the reaction was completed, the mixture was heated for 10 minutes, and the reaction solution was extracted and concentrated three times with the same amount of ethyl acetate. The obtained product was separated by silica gel column chromatography (chromoform: methanol = 9: 1) to obtain 360 mg of compound K and 240 mg of ginsenoside F1, wherein the yield of compound K and ginsenoside F1 was 56%.

Example 4

2 g of ginseng purified saponin was dissolved in 200 ml of citrate buffer (pH 4.0), and 2 g of naringinase and 4 g of pectinase were added thereto and reacted with stirring in a 37 ° C. water bath. The reaction was allowed to proceed for 56 hours while adding 15 ml of ethyl acetate to the reaction solution at 8 hour intervals, and the reaction solution was extracted and concentrated three times with the same amount of ethyl acetate. The obtained product was separated by silica gel column chromatography (chromoform: methanol = 9: 1) to obtain 400 mg of Compound K and 200 mg of ginsenoside F1, wherein the yield of Compound K and ginsenoside F1 was 56%.

Example 5

1 g of ginseng purified saponin was dissolved in 100 ml of citrate buffer solution (pH 4.0) containing 15 ml of ethanol, and 0.2 g of naringinase and 0.4 g of pectinase were added thereto. g and 0.4 g each were added and reacted with stirring for 48 hours on a 37 DEG C water bath. After the reaction was completed, the reaction was terminated by heating for 10 minutes, and then the reaction solution was extracted and concentrated three times with the same amount of ethyl acetate. The obtained product was separated by silica gel column chromatography (chromoform: methanol = 9: 1) to obtain 120 mg of Compound K and 80 mg of ginsenoside F1, wherein the yield of Compound K and ginsenoside F1 was 37.4%.

Example 6

3 g of ginseng purified saponin was dissolved in 100 ml of citrate buffer solution (pH 4.0) containing 20 ml of ethyl acetate, and 3 g of naringinase and 3 g of pectinase were added thereto, followed by reaction for 64 hours in a 37 ° C water bath. I was. After the reaction was completed, the mixture was heated in hot water for 10 minutes, and the reaction solution was extracted and concentrated three times with the same amount of ethyl acetate. The obtained product was separated by silica gel column chromatography (chromoform-methanol = 9: 1) to obtain 300 mg of Compound K and 190 mg of ginsenoside F1, wherein the yield of Compound K and ginsenoside F1 was 30%.

Example 7

2 g of ginseng purified saponin was dissolved in 100 ml of citrate buffer (pH 5.5), and 6 g of pectinase was added thereto and reacted with stirring for 30 hours in a 30 ° C water bath. After periodic confirmation by thin layer chromatography, when the substrate was completely lost, the reaction was terminated by heating in hot water for 10 minutes, and then the reaction solution was extracted and concentrated three times with the same amount of ether. The obtained product was separated by silica gel column chromatography (chromoform: methanol = 9: 1) to obtain 50 mg of Compound K and 20 mg of ginsenoside F1, wherein the yield of Compound K and ginsenoside F1 was 6.5%.

Example 8

2 g of ginseng purified saponin was dissolved in 100 ml of citrate buffer solution (pH 4.0) containing 10 ml toluene, and 2 g of pectinase was added thereto and reacted with stirring for 40 hours in a 40 DEG C water bath. After periodic confirmation by thin layer chromatography, when the substrate was completely lost, the reaction was terminated by heating in hot water for 10 minutes, and then the reaction solution was extracted and concentrated three times with the same amount of ethyl acetate. The obtained product was separated by silica gel column chromatography (chromoform: methanol = 9: 1) to obtain 50 mg of compound K, wherein the yield of the resulting compound K and ginsenoside F1 was 4.7%.

Example 9

1 g of ginseng purified saponin was dissolved in 100 ml of citrate buffer solution (pH 4.0) containing 10 ml ethanol, and 1 g of naringinase was added thereto and reacted with stirring for 40 hours in a 40 ° C water bath. After periodic confirmation by thin layer chromatography, when the substrate was completely lost, the reaction was terminated by heating in hot water for 10 minutes, and then the reaction solution was extracted and concentrated three times with the same amount of ethyl acetate. The obtained product was separated by silica gel column chromatography (chromoform: methanol = 9: 1) to obtain 60 mg of Compound K and 20 mg of ginsenoside F1, wherein the yield of Compound K and ginsenoside F1 was 14.9%.

Example 10

1 g of ginseng purified saponin was dissolved in 100 ml of citrate buffer (pH 5.5), and 2 g of naringinase was added thereto and reacted with stirring for 50 hours on a 50 ° C water bath. After periodic confirmation by thin layer chromatography, when the substrate was completely lost, the reaction was terminated by heating in hot water for 10 minutes, and then the reaction solution was extracted and concentrated three times with the same amount of ethyl acetate. The obtained product was separated by silica gel column chromatography (chloroform: methanol = 9: 1) to obtain 40 mg of compound K and 15 mg of ginsenoside F1, wherein the yield of compound K and ginsenoside F1 was 10.3%.

The yields of Examples 1 to 10 above were when the final product (Compound K and ginsenoside F1) was 5.35 g (yield 100%) when 100 g of ginseng purified saponin was converted to 100%. The final product of 10 was calculated by converting 10 g of ginseng purified saponin into the reaction. For example, in Example 1, the final product produced by the reaction of 10 g of ginseng purified saponin was 4.7 g, so the yield was 4.7 / 5.35 X 100 = 87.8%. In Example 2, 5 g of ginseng purified saponin reacted to give a final product. It was 1.93g, so the yield was 3.86g in terms of 10g of ginseng purified saponin, so the yield was 3.86 / 5.35 X 100 = 72%.

Test Example 1 Change of Red Ginseng Saponin Content after Enzyme Reaction

Red ginseng saponin was prepared from red ginseng by the method of Reference Example 1, and the enzyme reaction was carried out by the method of Example 1, and the change before and after the enzymatic reaction was measured by high performance liquid chromatography.

Standard: Purified saponin 5mg / 1ml (5000ppm) (HPLC) Holding time (min) Content (ppm) Ginsenoside Rg1 + Re 12.302 914 (18.28%) Ginsenoside Rb1 17.708 1306 (26.12%) Ginsenoside Rc 19.550 388 (7.76%) Ginsenoside Rb2 18.580 810 (16.2%) Ginsenoside Rd 21.933 180 (3.6%) Total (including other saponins) 4200 (84%)

The results of measuring the red ginseng tablet saponin content before the enzyme reaction are shown in Table 2 and FIG. 1, and the results of measuring the red ginseng tablet saponin content after the enzyme reaction are shown in FIG. 2.

As shown in FIGS. 1 and 2, ginsenosides Rb1, Rb2, Rc, Rd, Rg1, and Re, which constitute most of red ginseng saponin, were converted to compound K and ginsenoside F1 after the enzymatic reaction. Able to know.

As described above, the present invention was able to produce a large amount of compound K and ginsenoside F1, which are metabolites of ginseng saponin, exhibiting major pharmacological effects from ginseng purified saponins, through a simple process using enzymes.

Claims (7)

Ginseng Purified Saponin is dissolved in an aqueous solvent such as water or a buffer solution, or an aqueous solvent such as water or a buffer solution and an organic solvent, and then reacted by addition of at least one of naringinase and pectinase. To prepare a compound K and ginsenoside F1 represented by the formula (2). [Formula 1] [Formula 2] The method for producing compound K and ginsenoside F1 according to claim 1, wherein naringinase is an enzyme isolated from penicillium and pectinase is an enzyme isolated from Aspergillus. A compound K according to claim 1, wherein lower organic alcohols such as ethyl acetate, toluene, ether, ethanol and methanol are used as organic solvents, and the content thereof is 60% by weight or less based on the weight of the buffer solution or the aqueous solvent. Process for preparing ginsenoside F1. The method for preparing compound K and ginsenoside F1 according to claim 3, wherein the organic solvent is an organic solvent containing 5 to 25% by weight of ethanol and ethyl acetate. The method according to claim 1, wherein 1 to 1000% by weight of pectinase is added to the aqueous buffer solution at pH 3.0 to 8.0 and reacted at 20 to 60 ° C for 1 to 48 hours, and then 1 to 60% by weight of the reaction solution. To prepare compound K and ginsenoside F1, which is produced by adding 1 to 500 wt% of naringinase to the organic solvent and the substrate 1 to 10 times at intervals of 1 to 24 hours to react for 1 to 48 hours. Way. The method according to claim 1, wherein 1 to 500% by weight of naringinase is added to the aqueous buffer solution having a pH of 3.0 to 8.0 and reacted at 30 to 50 ° C. for 1 to 48 hours. To prepare compound K and ginsenoside F1, which is produced by adding 1 to 1000 wt% of pectinase 1 to 10 times at an interval of 1 to 24 hours and reacting for 1 to 48 hours. Way. The reaction solution according to claim 1, wherein 1 to 500% by weight of naringinase and 1 to 1000% by weight of pectinase are simultaneously added to the aqueous buffer solution having a pH of 3.0 to 8.0 for 1 to 48 hours. To produce Compound K and ginsenoside F1.