KR20170044828A

KR20170044828A - Preparation method of ginsenoside-Rd using an enzyme

Info

Publication number: KR20170044828A
Application number: KR1020150144410A
Authority: KR
Inventors: 오진환; 허율; 조영래
Original assignee: 주식회사 비티진
Priority date: 2015-10-16
Filing date: 2015-10-16
Publication date: 2017-04-26
Also published as: KR101777673B1

Abstract

Challenge: To provide a safer and more efficient method of producing ginsenoside Rd.
Solution: Reaction of Aspergillus and Penicillium genus-derived alpha-galactosidase with a mixture of ginsenosides Rb1, Rb2, Rc (single or complex ginsenoside) or one or more of them, resulting in ginsenoside Rd (ginsenoside Rd) in high yield. Reaction of Aspergillus and Penicillium genus alpha-galactosidase with ginsenosides Rb1, Rb2, Rc (single or complex) can produce ginsenoside Rd with high conversion.

Description

Preparation method of ginsenoside-Rd using an enzyme "

The present invention relates to a process for preparing ginsenoside Rd from a mixture of one or more of the ginsenosides Rb1, Rb2, Rc or these components with the diol-based saponin in a simple process using enzymes. The process of the present invention is characterized in that the conversion to ginsenoside Rd is high and the process is simple due to the enzymatic reaction and most of the reactions are completed within a short time of 1 to 2 days at room temperature to enable mass production in a short time in a safe and clean condition There are advantages.

The ginsenoside derivatives are a compound in which a protopanaxadiol that has a diterane-based trutherpenoid as a basic structure and a sugar-like substance such as uglycones, rhamnose, arabinose, and xylose of protopanaxatriol, It is known that the pharmacological actions differ depending on the type, number and bonding position of the bound sugar. Among these derivatives, one or more of the ginsenosides Rb1, Rb2, and Rc belonging to the protopanaxadiol is used as a raw material to make ginsenoside Rd. Depending on the respective characteristics, the sugar is degraded by hydrolysis. Ginsenoside Rd is one of the most widely known ginseng saponins together with ginsenosides Rb1, Rb2, Rc, Re and Rg1. However, the content of ginsenoside Rd is very small, about 0.036% based on ginseng (Korean Ginseng and Efficacy 1996, Researchers at the Tobacco Research Institute) Research on pharmacological activities of various angles or commercial applications are not sufficiently performed, and only the structure thereof is revealed.

The pharmacological activity of ginsenoside Rd was compared with that of the inhibitory effect on renal damage (Ren Fail 2000 Mar; 22 (2): 115-27, Nephron 1999 Feb; 81 (2): 200-7) (J Pharm Pharmacol 2004 Jan; 56 (1): 267-74), hepatoprotective effect (anti-liver cancer, patent application 10-2010-0118722) 107-13), neuronal cell protective effects (anti-Alzheimer's, anti-Parkinson's, Int. J. Mol. Sci., 2015, 16, 14395-14408, Alzheimer's Neurochem Res. 2012 Dec; 37 (12): 2738-47, Neural cell protection Neural Regen Res. 2014 Sep 15; 9 (18): 1678-87) have been published and are being shown to have great potential for commercial (functional food, natural medicine, etc.)

Conventionally, the method used for the production of ginsenoside Rd includes thermophilic bacteria ( Thermus Caldophilus method (Biotechnol Lett (2008) using the enzyme β- glucosidase during Days produced by cloning of a DNA) 30: 713 716, the Patent Publication No. 10-2002-0029138). In this method, since the enzyme used in production is an enzyme obtained by gene manipulation, there is a restriction in food processing, a production cost of recombinant enzyme is high, and there is a difficulty in a work process by reacting at high temperature (70-80 ° C).

Another method for producing ginsenoside Rd using enzymes is to use recombinant DNA technology to isolate the soil microorganisms Flavobacterium there is a method using a glucosidase enzyme produced by cloning the DNA during Days of johnsoniae) (Patent No. 1427882 Korea). This method is also disadvantageous in that the enzyme used in production is an enzyme obtained by genetic manipulation, so there is a limitation in food processing and a production cost of recombinant enzyme is high.

It is an object of the present invention to overcome the above problems and provide a safer and more productive process for preparing ginsenoside Rd.

In order to achieve the above object, the inventors of the present invention have found that by using an alpha-galactosidase derived from Aspergillus spp. And Penicillium sp., An enzyme reaction with ginsenosides Rb ₁ , Rb ₂ , Rc, The present invention has been made.

The present invention relates to a method for producing a ginsenoside Rd by reacting a composition containing at least one of ginsenosides Rb1, Rb2 and Rc or at least one of ginsenosides Rb1, Rb2 and Rc as a substrate with an alpha-galactosidase And a method for manufacturing the same.

In the method of the present invention, the origin of the alpha-galactosidase is not particularly limited. Preferably, the alpha-galactosidase is an enzyme isolated from Aspergillus or Penicillium.

The present invention also relates to a process for preparing the ginsenoside Rd, wherein the reaction is carried out at a temperature of 0 to 60 ° C.

In addition, the composition of the present invention is not particularly limited to a composition containing at least one of ginsenosides Rb1, Rb2 and Rc, but is preferably selected from the group consisting of red ginseng extract, red ginseng concentrate, red ginseng powder, red ginseng concentrated ginseng extract, A concentrated ginseng powder, a ginseng concentrated powder, a Taegeuk gum extract, a Taegeukgang concentrate, a Taegeukgam powder, or a Taegeukgus concentrated powder.

In addition, the ginseng of the present invention is not limited to a particular species of Panax species but is preferably selected from the group consisting of Panaxa ginseng, Panax quinquefolium, Panaxa notoginseng or Panax japonica ). &Lt; / RTI >

Ginsenosides Rd is a widely known component present in unprocessed natural ginseng, such as fresh ginseng and dried ginseng, but its content is only 0.036% (based on dry ginseng), so pharmacological activity studies have not been actively carried out and mass production is difficult Commercialization of large-scale pharmacological studies such as clinical trials or functional food development was not easy.

According to the process of the present invention, mass production of various Rd products from a mixture to a high-purity ginsenoside Rd is possible. Therefore, the identification of pharmacological activities of various angles and the possibility of development of functional foods and new drugs have increased.

According to the method of the present invention, not only the amount of enzyme to be used is small but also the reaction time is short, ginsenoside Rd can be obtained at a very high yield exceeding 90%.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a diagram showing the conversion of ginsenosides Rb1, Rb2, Rc (single or complex) into ginsenoside Rd by the process of the present invention.
Figures 2a and 2b are high pressure liquid chromatography (HPLC) analysis results of the production of Rd converted from Rb1 by alpha-galactosidase. 2a is the result for the pre-reaction composition, and 2b is the result for the post-reaction composition.

Hereinafter, specific methods of the present invention will be described in detail with reference to examples. However, it is apparent to those skilled in the art that the scope of the present invention is not limited to these embodiments. In particular, in Examples 1 to 6 of the present invention, an enzyme reaction was carried out using Aspergillus spp. -Induced alpha-galactosidase. However, the same enzymes were used for the purpose of comparing the yields of each example, Even when the α-galactosidase derived from the genus Silium is used, ginsenoside Rd is obtained from a composition substrate containing at least one of ginsenosides Rb1, Rb2 and Rc or at least one of ginsenosides Rb1, Rb2 and Rc Yields. &Lt; / RTI >

Example One:

50 mg of α-galactosidase derived from Aspergillus in 50 mM citric acid-phosphate buffer (pH 4.5) and 100 mg of complex saponin mixed with ginsenosides Rb1, Rb2 and Rc were mixed with 25 mg The reaction was carried out in a constant temperature water bath for 48 hours. After disappearance of the substrate, the reaction mixture was treated with boiling water for 10 minutes to remove the enzyme activity. The enzyme and the sugar were removed by butanol extraction, and the silica gel column was subjected to silica gel column chromatography (eluent: chloroform: methanol: distilled water = 60:40:10) To obtain 78 mg of ginsenoside Rd.

Example 2:

1 g of ginsenoside Rb1 was reacted with 50 mg of Aspergillus-derived α-galactosidase in 50 mM citric acid-phosphate buffer (pH 4.5) as a substrate for 48 hours in a constant temperature water bath at 25 ° C. After disappearance of the substrate, the reaction mixture was treated with boiling water for 10 minutes to remove the enzyme activity. The enzyme and the sugar were removed by butanol extraction, and the silica gel column was subjected to silica gel column chromatography (eluent: chloroform: methanol: distilled water = 60:40:10) To obtain 81 mg of ginsenoside Rd.

Example 3:

100 mg of ginsenoside Rb2 was reacted with 0.5 g of Aspergillus origin alpha-galactosidase in a 50 mM citric acid-phosphate buffer solution (pH 4.5) for 48 hours in a constant temperature water bath at 25 ° C. After disappearance of the substrate, the reaction mixture was treated with boiling water for 10 minutes to remove the enzyme activity. The enzyme and the sugar were removed by butanol extraction, and the silica gel column was subjected to silica gel column chromatography (eluent: chloroform: methanol: distilled water = 60:40:10) To obtain 0.79 g of ginsenoside Rd.

Example 4:

100 mg of ginsenoside Rc was used as a substrate and reacted with 0.5 g of Aspergillus-derived α-galactosidase in 50 mM citric acid-phosphate buffer (pH 4.5) for 48 hours in a constant temperature water bath at 25 ° C. After disappearance of the substrate, the reaction mixture was treated with boiling water for 10 minutes to remove the enzyme activity. The enzyme and the sugar were removed by butanol extraction, and the silica gel column was subjected to silica gel column chromatography (eluent: chloroform: methanol: distilled water = 60:40:10) To obtain 80 mg of ginsenoside Rd.

Example 5:

1 g of ginseng extract powder (Rb1 = 102 mg, Rb2 = 31 mg, Rc = 52 mg, Rd = 9 mg, etc.) containing ginsenosides Rb1, Rb2 and Rc was dissolved in 50 mM citric acid- (pH 4.5) were reacted with 0.5 g of Aspergillus-derived α-galactosidase in a constant temperature water bath at 25 ° C. for 48 hours. After disappearance of the substrate, the reaction mixture was treated with boiling water for 10 minutes to remove the enzyme activity. The enzyme and the sugar were removed by butanol extraction, and the silica gel column was subjected to silica gel column chromatography (eluent: chloroform: methanol: distilled water = 60:40:10) To obtain 0.5 g of a powder containing a large amount of ginsenoside Rd. The content of ginsenosides Rb1, Rb2, Rc and Rd in the powder was Rb1 = 0 mg, Rb2 = 5 mg, Rc = 9 mg and Rd = 141 mg, respectively.

Example 6:

1 g of red ginseng extract powder (Rb1 = 117 mg, Rb2 = 39 mg, Rc = 50 mg, Rd = 18 mg) containing ginsenosides Rb1, Rb2 and Rc was dissolved in 50 mM citric acid- 4.5) was reacted with 0.5 g of Aspergillus-derived α-galactosidase in a constant temperature water bath at 25 ° C. for 48 hours. After disappearance of the substrate, the reaction mixture was treated with boiling water for 10 minutes to remove the enzyme activity. The enzyme and the sugar were removed by butanol extraction, and the silica gel column was subjected to silica gel column chromatography (eluent: chloroform: methanol: distilled water = 60:40:10) To obtain 0.55 g of powder containing a large amount of ginsenoside Rd. The contents of ginsenosides Rb1, Rb2, Rc and Rd in this powder were Rb1 = 0 mg, Rb2 = 6 mg, Rc = 8 mg and Rd = 172 mg, respectively.

The unit of Table 1 is mg.

Claims

A method of producing ginsenoside Rd by reacting with a composition containing at least one of ginsenosides Rb1, Rb2 and Rc or at least one of ginsenosides Rb1, Rb2 and Rc as a substrate with an alpha-galactosidase .

The method according to claim 1,
Wherein the alpha-galactosidase is an enzyme isolated from Aspergillus or Penicillium genus.

The method according to claim 1,
Wherein the reaction proceeds at a temperature of 0 to 60 < 0 > C.

The method according to claim 1,
The composition containing at least one of ginsenosides Rb1, Rb2 and Rc is selected from the group consisting of red ginseng extract, red ginseng concentrate, red ginseng powder, red ginseng concentrate powder, ginseng extract, ginseng concentrate, ginseng powder, ginseng concentrated powder, Wherein the powder is a Taegeuk gum powder or a Taegeuk gum concentrate powder.

The method of claim 4,
Wherein the ginseng is selected from the group consisting of Panaxa ginseng, Panax quinquefolium, Panaxa notoginseng, and Panax japonica.