KR100877489B1 - Enzymatic Conversion of Ginsenosides - Google Patents

Abstract

The present invention relates to a method for enzymatic conversion of ginsenosides comprising treating ginsenosides with citrozym to obtain compound K and / or ginsenoside F1. According to the method of the present invention, compound K and / or ginsenoside F1 can be obtained more simply with high production rate from ginsenosides of ginseng or red ginseng.

Ginseng, Red ginseng, Conversion, Enzyme, Citrozyme, Compound K, Ginsenoside F1

Description

Enzymatic Conversion of Ginsenosides

1 is a chromatogram showing the results of high performance liquid chromatography (HPLC) analysis of dried ginseng ginsenosides.

Figure 2 is an HPLC chromatogram showing the results of enzyme conversion of ginseng ginsenosides to citrozyme.

Figure 3 is an HPLC chromatogram showing the results of enzymatic conversion of dried ginseng ginsenosides to pectinex.

Figure 4 is an HPLC chromatogram showing the results of enzyme conversion of dried ginseng ginsenosides to cellulose.

5 is an HPLC chromatogram showing the result of enzymatic conversion of dried ginseng ginsenoside to biscozyme.

Figure 6 is an HPLC chromatogram showing the results of enzyme conversion of ginseng ginsenosides to novo perm.

7 is an HPLC chromatogram showing the results of enzymatic conversion of dried ginseng ginsenosides to lactozyme.

The present invention relates to a method for enzymatic conversion of ginseng ginsenosides.

Ginseng is a perennial ripening root belonging to the genus Panax ginseng by plant taxonomy and about 11 species are known on earth. Ginseng has been tested through many pharmacological experiments to reduce cholesterol, inhibit lipid peroxidation, lower blood pressure, increase blood flow, cerebrovascular expansion, increase cardiac function, antiarrhythmia, antithrombotic, platelet aggregation, treatment of chronic renal failure, cytotoxicity and cancer suppression. , Immunomodulatory activity, increased memory, cerebral metabolism, antistress, antioxidant activity, anti-aging, anti-ulcer and gastric secretion, hyperactivity, protection against the gland, antidiabetic, detoxification, hepatocellular enzymes, asthma It is known to have activities such as treatment, anti-inflammatory, analgesic, anemia treatment, enhancement of fertility and sexual performance, decrease in alcohol blood concentration, anti-allergy, anticancer drugs.

화학식 2

Meanwhile, compounds K and ginsenoside F1, known as the major metabolites of human ginsenosides, have been reported to be produced by enteric bacteria (Hasegawa, H. et al., Planta Medica , 62: 453 (1996)). Compound K (Formula 1) and ginsenoside F1 (Formula 2) are known to exhibit cancer cell proliferation inhibition, anti-cancer activity-increasing activity, anti-allergic and skin protective activity of the anticancer agent, many studies have been made to efficiently manufacture them .
Formula 1

Formula 2

There are two broad approaches to the preparation of compound K and ginsenoside F1: (1) bioconversion with lactic acid bacteria or enteric bacteria; And (2) conversion with enzymes.

Korean Patent No. 377546 discloses a method for preparing ginsenoside compound K by enzymatic reaction of compound K by enzymatically reacting diol-based saponins of ginseng in an aqueous solvent. This patent document discloses cellulase and beta-galactosidase as enzymes for preparing compound K.

Korean Patent No. 418604 discloses dissolving ginseng purified saponin 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, and then adding at least one of naringinase and pectinase. A method of producing compound K and ginsenoside F1 by reacting is disclosed.

However, the enzymatic conversion method developed so far to obtain compound K and / or ginsenoside F1 has a low yield (ie, production rate).

Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

The present inventors sought to develop a method for converting saponin, ie, ginsenoside, included in ginseng or red ginseng into a more useful form for the human body, namely, compound K and / or ginsenoside F1. As a result, by treating the ginseng or red ginseng, or extracts obtained from them with citrozyme, the present invention by confirming that ginsenosides unique to ginseng or red ginseng are converted to compound K and / or ginsenoside F1 in a form useful for the human body To complete.

It is therefore an object of the present invention to provide a method for enzymatic conversion of ginsenosides.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to one aspect of the invention, the present invention provides a method for enzymatic conversion of ginsenosides comprising the step of treating the ginsenosides (Citrozym) to obtain a compound K.

According to another aspect of the present invention, the present invention provides a method for enzymatic conversion of ginsenosides comprising treating the ginsenosides with citrozym to obtain compound K and ginsenoside F1. do.

The present inventors sought to develop a method for converting saponin, ie, ginsenoside, included in ginseng or red ginseng into a more useful form for the human body, namely, compound K and / or ginsenoside F1. As a result, it was confirmed that ginseng or red ginseng, or extracts obtained from them, converts ginsenosides unique to ginseng or red ginseng into compound K and / or ginsenoside F1 useful for humans.

The present invention relates to a method for converting ginsenosides (ie, saponins) inherent in ginseng or red ginseng. In particular, the present invention relates to a method for preparing compound K and / or ginsenoside F1, which is a major human metabolite of ginsenosides in ginseng or red ginseng.

The term "saponin" is used herein in the same sense as ginsenosides, ie ginseng saponins, unless specifically noted otherwise.

For convenience of description, the method of the present invention is described herein as being applied to ginsenosides. Compound K prepared according to the present invention is one glucose bonded to protopanaxadiol. In other words, compound K is a sugar which is separated from the other ginsenosides of the protoparnaxadiol-based ginsenosides having several sugars apart. In addition, ginsenoside F1 prepared by the present invention is one glucose coupled to protopanaxatriol. In other words, the ginsenoside F1 is a sugar from the ginsenoside of the protoparnaxatriol system in which several sugars are combined, except for one glucose.

"Ginsenoside" used as a starting material in the present invention preferably means ginsenosides unique to ginseng or red ginseng. More preferably, ginsenosides used as starting materials in the present invention are (a) ginsenosides of the protopanaxadiol type, that is, ginsenosides Ra1-3, Rb1-3, Rc, Rd, Rg3, Rh2, Ri, Rs1 and Rs2, malonyl-ginsenosides Rb1-2, Rc and Rd; And (b) ginsenosides of the protopananac triol type, ie ginsenosides Re, Rf, Rg1-2 and Rh1.

As ginsenosides used as starting materials in the present invention, ginsenosides in a separated and purified form may be used, but are preferably ginsenosides contained in powder or extract of ginseng or red ginseng. In other words, the powder or extract of ginseng or red ginseng containing ginsenoside is directly used as a starting material. The present invention can be applied to all ginseng processed in various forms, such as ginseng, rice ginseng, white ginseng and red ginseng. As the ginseng used in the present invention, various known ginsengs can be used, and Korean ginseng ( Panax ginseng ), Gyegi ( P. quiquefolius ), Jeonchisam ( P. notoginseng ), Bamboo ginseng ( P. japonicus ), and three leaf ginseng ( P. trifolium ), Himalayan ginseng ( P. pseudoginseng ) and Vietnamese ginseng ( P. vietnamensis ), but are not limited thereto. Red ginseng used in the present invention is prepared by processing the ginseng. Preferably, the ginseng used in the present invention is Panax ginseng or P. notoginseng .

A major feature of the present invention is the use of citrozyme in preparing compounds K and / or ginsenosides F1. Citrozyme used in the present invention shows a very good ginsenoside production rate than other enzymes.

Preferably, the citrozyme used in the present invention is a mixture of pectinase, cellulase and hemicellulase. More preferably, the citrozyme used in the present invention is a mixture of pectinase, cellulase and hemicellulase, which are enzymes that are secreted extracellularly from Aspergillus niger microorganisms. Most preferably, the citrozyme used in the present invention is “Citrozym Cloudy 100L” available from Novozyme Company.

The enzyme conversion reaction in the present invention is preferably carried out in a buffer solution. Buffers that can be used for the enzymatic conversion reaction of the present invention include any buffer used for the enzymatic reaction, preferably acetate buffer, citrate buffer, phosphate buffer or citrate-phosphate buffer.

In the enzymatic conversion reaction of the present invention, the pH is preferably 3.0-7.5, more preferably 4.0-6.0, most preferably about 5.0. The temperature in the enzymatic conversion reaction is preferably 30-65 ° C, more preferably 40-60 ° C, even more preferably 48-52 ° C, most preferably about 50 ° C.

According to the process of the invention, compounds K and / or ginsenosides F1 can be obtained with high production rates. According to a preferred embodiment of the invention, the production rate for compound K of the invention is 20-30%, more preferably 22-30%, most preferably 25-27%. As used herein, the term “production rate” refers to a specific ginsenoside produced from all ginsenosides included in the reaction product when the conversion reaction is performed using a powder solution or extract of ginseng or red ginseng as a starting material. Mean weight percent of compound K).

According to a preferred embodiment of the invention, the production rate for ginsenosides F1 of the invention is 2.5-6%, more preferably 3-5%, most preferably 3.8-4%. The production rate for the ginsenoside F1 of the present invention is lower than that of the compound K, but considering the low content of the ginsenoside of the protoparanaxatriol system in the starting material itself, the production rate is also very excellent.

According to the method of the present invention, compound K and / or ginsenoside F1 can be obtained more simply with high production rate from ginsenosides of ginseng or red ginseng.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example

Example  One: Ginsenoside  Enzyme Conversion 1

5% (w / v) of powdered dried ginseng (Red Ginseng Co., Ltd.) was dissolved in 50 mM Na-acetate buffer (pH 5.0), followed by Citrozym Cloudy 100L (Novozyme; complex of enzyme type, pectinase, cellulase and hemicellulase). Enzyme producing microorganism, Aspergillus niger) 5% (v / v) enzyme was added and reacted at 50 ° C for 72 hours. After the reaction, saturated butanol was added to stop the reaction, and then a supernatant was obtained and dried in vacuo. Methanol was added to the vacuum dry and filtered, followed by HPLC analysis. HPLC analysis was performed using Waters HPLC (600 controller, 717 plus Autosampler, 2487 dual absorbance detector), the column was ZORBAX Edipse XDB-C18 (4.6 x 250 mm, 5 micron), the oven temperature of the column was 30 ℃, The loading amount of the sample was 10 microliters. The mobile phase was obtained by concentration gradient with 100% water and 100% acetonitrile for 75 minutes. The flow rate was 2.5 ml per minute and was detected at 203 nm. The experimental results are shown in FIG.

1 is HPLC analysis data for dry ginseng (control) without enzyme treatment. The retention time, 17 minutes, 24.5 minutes, 26.5 minutes, 28.3 minutes and 31 minutes correspond to ginsenosides Rg1 (Re), Rb1, Rc, Rb2 and Rd, respectively.

In the HPLC chromatogram of FIG. 2, the retention times 29 minutes, 55.8 minutes and 68.7 minutes correspond to ginsenoside F1, compound K and protopanaxadiol (PPD), respectively.

As can be seen in Figure 2, it can be seen that the peak of 55.8 minutes of the retention time corresponding to Compound K was greatly increased by the treatment with Citrozym Cloudy 100L (the ratio of Compound K in the detected ginsenosides was 26.3%). As a result, it can be seen that the enzymatic conversion of ginsenosides to ginseng in ginseng is very excellent by Citrozym. In addition, it can be seen that the peak of the retention time 29 minutes corresponding to ginsenoside F1 was greatly increased by the treatment with Citrozym Cloudy 100L (the ratio of ginsenoside F1 among the detected ginsenosides was 3.88%). Thus, it can be seen that ginsenosides in ginseng are efficiently converted to ginsenoside F1 by Citrozym.

Example  2: Ginsenoside  Enzyme Conversion 2

Enzymatic conversion was performed in the same manner as in Example 1, but Pectinex 5XL (Novozyme; enzyme type, polygalacturonase; enzyme-producing microorganism, Aspergillus aculatatus, Aspergillus niger) was used.

As can be seen in Figure 3, by the Pectinex enzyme it was possible to obtain a compound K of 0.8% (the ratio of the compound K in the ginsenosides detected). On the other hand, almost no peak corresponding to ginsenoside F1 was formed.

Example  3: Ginsenoside  Enzyme Conversion 3

Enzyme conversion was carried out in the same manner as in Example 1, but Celluclast 1.5L (Novozyme; enzyme type, cellulase; enzyme-producing microorganism, tricoderma essays) was used.

As can be seen in Figure 4, by the Celluclast 1.5L enzyme it was possible to obtain a compound K of 2.6% (the ratio of the compound K in the ginsenosides detected). On the other hand, almost no peak corresponding to ginsenoside F1 was formed.

Example  4: Ginsenoside  Enzyme Conversion 4

Enzyme conversion was carried out in the same manner as in Example 1, but Viscozyme L (Novozyme; enzyme type, mixture of carbohydratase; enzyme producing microorganism, Aspergillus) was used.

As can be seen in Figure 5, it was confirmed that the compound K can not be obtained by the Viscozyme L enzyme. On the other hand, the peak corresponding to ginsenoside F1 was hardly formed.

Example  5: Ginsenoside  Enzyme Conversion 5

Enzyme conversion was carried out in the same manner as in Example 1, but Novoferm 61 (Novozyme; enzyme type, polygalacturonase; enzyme-producing microorganism, Aspergillus aculeatus, Aspergillus niger) was used.

As can be seen in Figure 6, it was confirmed that the compound K can not be obtained by the Novoferm 61 enzyme. On the other hand, almost no peak corresponding to ginsenoside F1 was formed.

Example  6: Ginsenoside  Enzyme Conversion 6

Enzyme conversion was performed in the same manner as in Example 1, but Lactozym 3000L HP-G (Novozyme; enzyme type, beta-galactosidase; enzyme producing microorganism, Kluyveromyces lactis) was used.

As can be seen in Figure 7, it was confirmed that the compound K can not be obtained by the Lactozym 3000L HP-G enzyme. On the other hand, almost no peak corresponding to ginsenoside F1 was formed.

As described in detail above, the present invention provides a method for enzymatic conversion of ginsenosides which exhibits an improved production rate. According to the method of the present invention, compound K and / or ginsenoside F1 can be obtained more simply with high production rate from ginsenosides of ginseng or red ginseng.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (7)

A method for enzymatic conversion of ginsenosides comprising treating ginsenosides with pectinase, cellulase and hemicellulase to obtain compound K. A method of enzymatic conversion of ginsenosides comprising treating ginsenosides with pectinase, cellulase and hemicellulase to obtain compound K and ginsenoside F1. The method of claim 1 or 2, wherein the ginsenoside is contained in a powder or extract of ginseng. The method according to claim 1 or 2, wherein the method is carried out at pH 4.0-6.0. The method of claim 1 or 2, wherein the method is carried out at 48-52 ° C. A process according to claim 1 or 2, characterized in that the production rate for compound K of the process is 20-30% by weight. The method of claim 2, wherein the production rate for ginsenosides F1 of the method is 3-5% by weight.

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