KR102056163B1 - Method for producing high amount of ginsenoside Rg1 and compound K - Google Patents

Abstract

The present invention relates to a method for mass production of ginsenoside Rg1 and compound K. Specifically, the present invention relates to a method for mass production of ginsenoside Rg1 and compound K, including a step of inducing a reaction by sequentially adding viscozyme and beta-glucosidase to a ginseng extract.

Description

Method for producing high amount of ginsenoside Rg1 and compound K}

The present invention relates to an optimal production method for obtaining a large amount of ginsenosides Rg1 and compound K from ginseng using a two step enzyme treatment method.

Ginseng saponins have a unique chemical structure that is different from those found in other plants, and because of their unique pharmacological effects, they are also called "ginsenoside", meaning ginseng glycosides. Specific examples of ginseng saponins include ginsenosides Rb1, Rb2, Rc, Rd, compound K, compound Mc, and compound O, which are panaxanadiols, and ginsenosides Re, Rf, Rg1, Rg5, Rh1 and the like, and these ginseng saponins each show different efficacy.

These ginsenosides are metabolized by human intestinal microorganisms after ingestion, and their metabolites are known to have various physiological activities. For example, the protopanaxadiol-based saponins Rb1, Rb2, and Rc are expressed in human intestinal bacteria. Is metabolized to 20-O--D-glucopyranosyl-20 (S) -protopanaxadiol (IH-901, compound K), and Re and Rg1, which are protopanaxatriol-based saponins, are expressed in enterobacteriaceae. It is metabolized by ginsenoside Rh1 or ginsenoside F1, and the converted compounds K, Rh1 and F1 exhibit various physiological activities. Specifically, compound K is known to induce anti-metastatic or anti-cancer effects by preventing tumor invasion or preventing chromosomal alterations and tumorigenesis, and F1 also significantly reduces UV-B-induced cell death, and UV B It is known that human HaCaT keratinocytes can be protected by downregulation of the expression of Bcl-2 and Brn-3a from apoptosis by irradiation. Therefore, in recent years, studies are being actively conducted to produce a large amount of selected ginsenosides for specific pharmacological or foodological purposes.

On the other hand, conventional methods for obtaining ginseng saponins are obtained by extracting various converted ginseng saponins, etc., instead of converting them to specific ginseng saponins as desired, and then purifying them to separate only the desired ginseng saponins as technical solutions. However, since such conventional methods require additional cost and time for purification to obtain pure ginseng saponin, the selling price of ginseng saponin is inevitably increased, and accordingly, it is difficult to apply ginseng saponin in large quantities to related products. There are limitations.

In particular, in the case of compound K, since it corresponds to the last compound produced in the path of the compound converted from ginseng saponin, various enzymes capable of reacting all intermediate metabolites are required, and also in the metabolite produced in the middle of the reaction. This reduces the reactivity between the metabolite and the enzyme. In addition, there is a problem that the yield is lowered due to the aggregation phenomenon in the reaction solution between the intermediate metabolites, there is a limit to the production in large quantities.

In addition, ginsenoside Rg1 enhances immune function, inhibits platelet aggregation, enhances memory and learning function, promotes protein synthesis, anti-fatigue, antistress, central excitability, vasodilation, and promotes neuronal cell survival. It is known to have an inflammatory effect. As such, since ginsenoside Rg1 plays an important role as a functional ingredient, there have been attempts to obtain an extract containing a high content of ginsenoside Rg1. For example, a method of obtaining a ginseng spirit extract by adding spirits to ginseng, adsorbing it to an adsorption resin, and then adding alcohol to the adsorption resin again to obtain an eluate containing Rg1 has been reported. It is necessary to develop a new method that can produce high content of ginsenoside Rg1.

Republic of Korea Patent No. 10-0689745

Therefore, the present inventors are expensive to obtain a specific ginseng saponin by the conventional method, in order to improve the problem that it is difficult to obtain a large amount of ginseng saponin, it is possible to mass-produce ginsenoside Rg1 and compound K having excellent physiological activity. As a new manufacturing method that can reduce the cost while completing the present invention by establishing the optimum ginsenoside Rg1 and compound K production conditions using biscozyme and beta-glucosidase.

Therefore, an object of the present invention is the first enzymatic treatment of biscozyme or plantase UF in ginseng extract; And it provides a method for mass production of ginsenoside Rg1 and Compound K comprising the step of sequentially performing the second enzymatic treatment of beta-glucosidase.

In order to achieve the object of the present invention as described above, the present invention comprises the steps of the first enzyme reaction by adding Biscozyme (Picozyme) or plantase UF (Plantase UF) to the ginseng extract; And it provides a method for mass production of ginsenoside Rg1 and Compound K at the same time, comprising the step of adding a beta-glucosidase to the first enzyme reactant and the second enzyme reaction.

In one embodiment of the present invention, the extract may be an extract obtained using water or a lower alcohol having 5 or less carbon atoms.

In one embodiment of the present invention, the Viscozyme or Plantase UF (Plantase UF) is added in an amount of 5-15% (v / v) relative to the total weight of the ginseng extract, pH 4.0 ~ 4.5 , Temperature may be to react for 1 day to 10 days at 40 ~ 45 ℃.

In one embodiment of the present invention, the beta-glucosidase is added in 5 ~ 15% (v / v) relative to the total weight of ginseng extract, pH 6.5 ~ 7.5, temperature 1 ~ 3 days at 40 ~ 50 ℃ May be reacted for a while.

In one embodiment of the present invention, the beta-glucosidase may be TmBG (Thermotoga maritima beta glucosidase).

In one embodiment of the present invention, the compound K may be produced by sequential conversion from ginsenoside Rb1 to Rd, F2 and compound K.

The present invention relates to a method for mass-producing ginsenoside Rg1 and compound K, wherein the ginsenoside Rg1 and compound K comprising a step of sequentially adding biscozyme and beta-glucosidase to the ginseng extract to react It relates to a production method. When applying the method according to the present invention, it is possible to selectively produce a large amount of ginsenoside Rg1 and compound K having important physiological activity, there is an effect that can be utilized in various kinds of functional products containing this useful ingredient.

Figure 1 shows the results of analyzing the type and content of ginsenosides by HPLC chromatography on the ginseng extract prepared in one embodiment of the present invention.
Figure 2 shows the results of measuring the change in the content of ginsenosides contained in the ginseng extract after the first enzyme treatment in one embodiment of the present invention.
Figure 3 shows the results of measuring the change in the content of ginsenosides contained in the ginseng extract after the second enzyme treatment in one embodiment of the present invention.
Figure 4 is a schematic diagram showing the conversion reaction of ginsenosides according to the two-step enzyme treatment for the compound K produced by the method of the present invention.
FIG. 5 shows the results of analyzing the content change of ginsenosides according to reaction time after adding beta-glucosidase (TmBG) and ABG as secondary enzymes to ginseng extracts treated with biscozyme for 10 days. .
Figure 6 shows the results of analyzing the change in the content of ginsenosides by adding biscozyme to the ginseng extract and HPLC chromatography by reaction time (1 day, 5 days and 10 days).
7 is a graph showing the change in content of each ginsenoside according to the reaction time while treating biscozyme for 10 days, and the control shows a control group not treated with enzyme and the enzyme treated with enzyme.

The present invention provides an optimal production method for simultaneously obtaining a large amount of ginsenoside Rg1 and compound K through two steps of enzymatic treatment. Preferably, the method is biscozyme or planta in ginseng extract. Adding a UF (Plantase UF) to the first enzyme reaction; And adding a beta-glucosidase to the first enzyme reactant to perform a second enzyme reaction.

Ginseng is a perennial ripening root belonging to the genus Ginseng of the Aralaiaceae according to the plant taxonomy. About 11 kinds of ginseng are known on the earth, and it has been widely used for health promotion mainly in Korea, China, and Japan for thousands of years. Recently, red ginseng containing a small amount of ginsenoside, which is useful for the human body, is processed by steaming the ginseng several times with steam, and it is known that it is excellent. Saponine, a representative active ingredient of ginseng, is a kind of compound called ginsenoside, and saponin is composed of more than 30 kinds of ginsenosides, and ginsenosides are dammarane-based triterpenoids ( Triterpenoids (Protopanaxadiol) and Protopanaxatriol are compounds that combine sugars such as glucose, rhamnose, arabinose or xylose to date. have. Major protoparanaxadiol derivatives include ginsenosides Rb1, Rb2, Rc, Rd, Rg3, Rh2, compound K and the like. The main protoparanaxatriol derivatives are Re, Rg1, Rh1 and the like.

In addition, ginsenoside derivatives are compounds in which sugars such as glucose, rhamnose, arabinose, and xylose are combined with aglycones of protoparanaxadiol and protoparnaxatriol, which are based on a dimaran-based triterpenoid It is known that pharmacological action is different depending on the type and number of sugars and the position of binding. Of these derivatives, Rd, a trigluco ginsenoside belonging to the protoparnaxadiol, is concentrated to increase the content ratio firstly, and then monoglucose ginseno via F2, a diglucose ginsenoside, through a second enzymatic reaction. The side compound K is made, and in each order, one molecule is dropped by hydrolysis.

However, a small amount of ginsenosides Rg1 and compound K among the ginsenoids could not be produced, and the development of technology for mass production of them was required.

Therefore, in the present invention, a new method for mass production of high content of ginsenoside Rg1 and compound K was established. After the first treatment of biskozyme or plantase UF enzyme in ginseng extract, beta-glucosidase 2 In the case of sequential reaction by treatment with car, it was confirmed that both ginsenoside Rg1 and compound K can be mass produced simultaneously.

Referring to the method established in the present invention in more detail as follows.

First, ginseng extract is prepared using ginseng. At this time, the ginseng extract according to the present invention may be obtained by extraction and separation using extraction and separation methods known in the art, the 'extract' defined in the present invention is extracted from ginseng using a suitable solvent And include, for example, crude extracts, polar solvent soluble extracts or nonpolar solvent soluble extracts.

Suitable solvents for extracting the extract may be water or an organic solvent, preferably water or a lower alcohol having 5 or less carbon atoms, but is not limited thereto. For example, purified water, methanol, ethanol ( alcohols having 1 to 4 carbon atoms, acetone, ether, benzene, chloroform, ethyl acetate, including ethanol, propanol, isopropanol, butanol, etc. Various solvents such as ethyl acetate, methylene chloride, hexane, and cyclohexane may be used alone or in combination.

The ginseng site used to obtain the ginseng extract may use the whole ginseng including root, stem, leaf, or both.

The ginseng extract prepared by the above method is reacted by adding biskozyme or plantase UF as a primary enzyme.

The Vicozyme or Plantase UF is added in an amount of 5 to 15% (v / v) based on the total weight of the ginseng extract, and pH 4.0 to 4.5 and a temperature of 40 to 45 ° C. per day. React for 10 days.

We conducted experiments to establish optimal treatment conditions for biscozyme or plantase UF to produce the largest ginsenosides from ginseng extract, resulting in 10% (v / v) of the ginseng extract's total weight. When added in the amount of), and reacted for 1 day to 10 days at pH 4.0 ~ 4.5, temperature 40 ℃, it was confirmed that Rb1 is quickly converted to F2 via Rd most effectively.

Therefore, it can be seen that the optimum conditions for the first enzyme treatment should be reacted in the above-described amounts of addition, pH, temperature and reaction time.

After the first enzyme treatment is completed, beta-glucosidase is added again to the ginseng extract and a second reaction is performed.

At this time, beta-glucosidase is added in 5 to 15% by weight (v / v) relative to the total weight of ginseng extract, it is preferable to react for 1 to 3 days at pH 6.5 ~ 7.5, temperature 40 ~ 50 ℃, beta -Glucosidase may use TmBG (Thermotoga maritima beta glucosidase).

In one embodiment of the present invention, the changes in the content enhancement of ginsenoside Rg1 and compound K according to the beta-glucosidase treatment conditions were analyzed, the addition amount of 5 ~ 15% by weight (v / v) to the total weight of ginseng extract It was found that the highest content of these ginsenosides can be obtained when used.

When added in less than 5% by weight, the ginsenoside conversion of the compound k to the compound k by beta-glucosidase appeared to have a low problem, when used in excess of 15% by weight of the compound k is further enhanced There was a problem in terms of economics.

In addition, the reaction pH conditions were 6.5 to 7.5 is appropriate, preferably it was confirmed that the pH 7.0, if the pH is outside the 6.5 to 7.5 range is not properly reacted by the enzymes will cause abnormal conversion to the compound k It appeared, there was a problem that the adverse reaction is caused to produce other products, such as precipitate, undesired.

As reaction temperature, it is suitable to react at the temperature of 40-50 degreeC, Preferably it can react at the temperature of 45 degreeC. Beta-glucosidase shows higher activity at higher temperatures, but may react with ginsenoside Rg1 when reacted at higher temperatures. When reacting at a temperature of 60 ° C., the production of Rg1 rapidly decreases. This occurred. On the other hand, when the reaction is less than 40 ℃, the reaction of the enzyme does not occur there was a problem that Rg1 is not produced.

In this regard, when treating beta-glucosidase, it is important to react the reaction temperature at a temperature of 40 to 50 ° C.

In addition, as a result of analyzing the conditions for the reaction time, it can be reacted for 1 day to 7 days, preferably for 1 day to 3 days, more preferably when the reaction for 1 day, the highest yield It can be seen that ginsenoside Rg1 and compound K of can be obtained.

In addition, according to the experimental results of the present invention by treating the ginseng extract-treated ginseng extract beta-glucosidase to produce ginsenoside Rg1 and compound K TmBG (Thermotoga maritima beta glucosidase) as a beta-glucosidase Only when using the compound K can be confirmed that the conversion of ginsenosides.

In one embodiment, Thermotoga as a type of beta-glucosidase maritima beta glucosidase (TmBG) and Agrobacterium sp . Analysis of beta glucosidase (ABG), respectively, showed that ginsenoside Rg1 and compound K (CK) could be produced by saponin conversion in the group treated with TmBG as beta-glucosidase. It was shown that compound K (CK) could not be obtained in the ABG treated group.

From these results, the present inventors found that it is important to use TmBG as a beta-glucosidase for the large amount of compound K (CK) production.

Compound K obtained in the present invention is produced by the sequential conversion from ginsenoside Rb1 to Rd, F2 and compound K. When the method provided by the present invention is applied, the high content of ginsenoside Rg1 and compound K can be obtained.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are intended to illustrate the present invention more specifically, but the scope of the present invention is not limited to these examples.

< Example  1>

Preparation of Ginseng Extract

To prepare the ginseng extract, first, the dried ginseng was ground with a blender (RT-02A; Mill powder tech co., Taiwan), and then 100 ml of 70% MeOH was added to 20 g of the ground ginseng powder, followed by an ultrasonic disperser (Q500). (Culse on time: 20 min) using QSONICA, USA). After removing the solids using a centrifuge (Combi 514R; hanil, Korea) (12,000 rpm, 20 min, 4 ℃), the ginseng extract, that is, ginsenoside extract using a reduced pressure condenser to evaporate methanol at 50 ℃ When about 5 ml remaining, the concentrate was dissolved in distilled water to obtain a concentrated ginseng extract, which was used as a ginseng extract sample for the treatment of biscozyme and beta-glucosidase in the following examples.

< Example  2>

Component Analysis of Ginsenosides in Ginseng Extract

First, the inventors performed HLPC analysis to investigate the components and contents of ginsenosides contained in the ginseng extract obtained in Example 1 in the step before the enzyme treatment. HLPC analysis was performed by loading a sample filtered ginseng extract on a column using a 0.45 um PVDF filter, column: YMC-Triart C18 (5 um, 250 X 4.6 mm), mobile phase A: DW, B: Acetonitrile, flow rate : 1 ml / min, detector: DAD detector (203 nm), injection volume: 20 ul, the concentration gradient of the solvent was performed as shown in Table 1.

As a result, as a result of analyzing the ginseng extract obtained by adding methanol and sonication without any enzyme treatment by HPLC, ginsenosides Rg1 and Rb1 were detected as shown in Table 2 above, and the ginsenoside content contained in the ginseng extract. Silver Rg1 was 0.48 mg / ml and Rb1 was 0.72 mg / ml. In addition, the detection graph of ginsenosides analyzed through HPLC is shown in FIG. 1. In particular, ginsenoside CK was not detected in the ginseng extract obtained without enzyme treatment.

< Example  3>

By primary enzyme treatment Ginsenoside t analysis

<3-1> Viscozyme  or Plantase UF  Ginsenoside content analysis by single treatment

The ginseng extract obtained in Example 1 was treated with biscozyme or Plantase UF enzyme alone, and analyzed for the change of ginsenoside content according to the enzyme treatment through HLPC analysis. To this end, the treatment conditions of biscozyme were added for 24 hours by adding biscozyme in an amount of 10% (v / v) based on the total volume of ginseng extract at a temperature of pH 4.0 and 40 ° C, and the treatment condition of Plantase was pH 4.5. And at a temperature of 40 ° C. for 24 hours in an amount of 10% (v / v) based on the total volume of the ginseng extract. After completion of the reaction, the reaction was terminated by adding methanol, and then diluted with 200 times the volume of distilled water of the reaction solution, and then analyzed the components and contents of ginsenoside by HPLC. HPLC analysis conditions were performed under the same conditions as in Example 2.

* ND; Not detected

As a result of the analysis, as shown in Table 3 and FIG. 2, the first ginseng extract was treated with Viscozyme and Plantase UF for 24 hours. Ginsenoside Rb1 was disappeared and F2 was generated. The amount was 1913.68 ug when treated with Viscozyme. / ml, 4012.08 ug / ml when treated with Plantase UF. These results suggest that Viscozyme and Plantase UF hydrolyzed glucose at the C3 and C20 positions of ginsenoside Rb1 and converted to F2.

On the other hand, during the primary enzyme reaction, no compound K was produced, and it was found that the conversion of ginsenoside F2 to K was limited only by these primary enzyme treatments.

<3-2> Viscozyme  Ginsenoside content analysis according to treatment time

In the biscozyme treatment method of <3-1>, the reaction time was reacted to 1 day, 3 days, 5 days, 7 days, 9 days, and 10 days, respectively, and the content of ginsenoside was analyzed through HPLC analysis.

As a result of the analysis, as shown in Table 4 and FIG. 6 and FIG. 7, it was found that after the biscozyme treatment, all ginsenosides Rb1 disappeared from day 1 and quickly converted to F2 via Rd. However, no compound K was produced, and the conversion of ginsenoside F2 to K was found to be limited only by biscozyme alone.

< Example  4>

Analysis of Ginsenoside Components and Contents according to Second Stage Enzyme Treatment

<4-1> Ginsenosides by Secondary Enzyme Treatment Rg1  And Compound  K mass production analysis

As confirmed in Example 3, in the mass production of ginsenoside Rg1 and Compound K alone, only treatment with Viscozyme or Plantase UF enzyme showed a limitation in yield and yield. After treatment with the enzyme, beta-glucosidase was further treated. Beta-glucosidase is a Thermotoga from Megazyme maritima beta glucosidase (TmBG) and Agrobacterium sp . Beta glucosidase (ABG) was used and experiments were performed to determine the optimum pH and temperature conditions for these enzymes. Specifically, after adding beta-glucosidase to the ginseng extract treated with the primary enzyme in Example 3, the type and content of ginsenosides were analyzed through HLPC at a temperature of pH 7.0 and 45 ° C. for 24 hours. It was. In addition, the amount of beta-glucosidase was added to be 10% (v / v) based on the total volume of the ginseng extract.

As a result, as shown in Table 5 and FIG. 3, the F2 generated by Viscozyme and Plantase UF disappeared and CK was generated as the secondary enzyme was treated, and the content of Vicozyme → TmBG was treated. 1369.88 ug / ml, and 1426.03 ug / ml when treated with Plantase UF → TmBG. This production of CK is expected to be hydrolyzed and converted to CK glucose at the C3 position of ginsenoside F2 when treated with TmBG. The bioconversion pathways between these ginsenosides are shown in FIG. 4.

On the other hand, the present inventors analyzed whether compound K (CK) can be obtained when treating ABG other than TmBG as a secondary enzyme. As a result, as shown in FIG. 5, compound K was obtained in the group treated with ABG. I could not see.

<4-2> Analysis of Ginsenoside Components and Contents According to the Treatment Time of Secondary Enzymes

The present inventors add 10 ml corresponding to 10% of the volume of the extract of TmBG and ABG as secondary enzymes to 100 ml of ginseng extract treated with Biscozyme for 10 days in Example 3, and the temperature of pH 7 and 45 ° C. The reaction was carried out under the conditions, and the change in the content of ginsenoside with the reaction time was measured.

As a result, as shown in Table 6, 5 and 7, ginsenoside Rg1 and compound K (CK) can be produced through the saponin conversion reaction in the group treated with TmBG as beta-glucosidase On the other hand, it was found that compound K (CK) could not be obtained in the ABG treatment group, and the treatment of beta-glucosidase was not time dependent, but treatment for one day was the best.

Based on the above results, the present inventors found that the optimum conditions for mass production of ginsenoside Rg1 and compound K (CK) at the same time require enzymatic treatment in two stages. Specifically, the present inventors treated biscozyme or Plantase UF. In addition to the addition of beta-glucosidase TmBG to one reaction, the second reaction at a temperature of pH 7 and 45 ℃ for one day, the highest amount of ginsenoside Rg1 and compound K (CK) at the same time It was found that it can be obtained.

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

Claims (6)

Adding a plantase UF, which is a pectinase, to the ginseng extract for primary enzyme reaction; And
To include the second enzyme reaction by adding a TmBG (Thermotoga maritima beta glucosidase) as a beta-glucosidase to the primary enzyme reactant;
Method for mass production of ginsenosides Rg1 and compound K simultaneously. The method of claim 1,
The extract is a method of mass production of ginsenoside Rg1 and compound K at the same time, characterized in that the extract obtained by using water or lower alcohol having 5 or less carbon atoms. The method of claim 1,
The plantase UF, which is the pectinase, is added in an amount of 5-15% (v / v) based on the total weight of the ginseng extract, at pH 4.0-4.5, at a temperature of 40-45 ° C. Method for mass production of ginsenoside Rg1 and compound K simultaneously, characterized in that the reaction for 1 to 10 days. The method of claim 1,
As the beta-glucosidase, TmBG (Thermotoga maritima beta glucosidase) is added at 5-15% (v / v) based on the ginseng extract total weight, and at pH 6.5-7.5, temperature 40-50 ° C. for 1 to 3 days. Reacting, mass production of ginsenoside Rg1 and compound K simultaneously. delete The method of claim 1,
Compound K is produced by sequential conversion from ginsenoside Rb1 to Rd, F2 and compound K, mass production of ginsenoside Rg1 and compound K simultaneously.

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