KR102591643B1 - Manufacturing method for rare ginsenoside Rh2, Rh3 or Rk2 - Google Patents

Abstract

The present invention relates to a method for producing one or more ginsenosides selected from the group consisting of Rh2, Rh3 and Rk2, and the method of the present invention relates to one or more ginsenosides selected from the group consisting of Rb1, Rb2, Rb3, Rc and Rd. Preparing raw materials containing; acid and heat treatment steps; and an enzyme treatment step derived from the Aspergillus niger C2-2 strain.

Description

{Manufacturing method for rare ginsenoside Rh2, Rh3 or Rk2}

The present invention relates to a method for producing rare ginsenosides Rh2, Rh3 or Rk2. Specifically, the present invention relates to a method for producing rare ginsenosides Rh2, Rh3 or Rk2 from the major ginsenosides of ginseng such as Rb1, Rb2, Rb3, Rc and Rd.

Ginseng is a perennial herb from the Aralia family and has been mainly used as a medicinal herb. Ginseng contains unique saponins that have a different chemical structure from those found in other plants, and are called ginsenosides.

Depending on the structure of the aglycone, ginsenosides are divided into protopanaxadiol-type (PPD type) ginsenoside, protopanaxatriol-type (PPT type) ginsenoside, and oleanolic acid type. (oleanolic acid type) Ginsenosides can be classified into three types.

Currently, more than 40 types of ginsenosides have been isolated, and most of them are PPD type ginsenosides. PPD type ginsenosides include Rb1, Rb2, Rb3, Rc, Rd, gypenoside XVII, compound O, compound Mc1, F2, compound Y, It includes compound Mc, Rg3, Rh2, and compound K (compound K, C-K). PPT type ginsenosides include Re, Rg1, Rf, Rg2, Rh1, etc.

More than 90% of the total ginsenosides contained in ginseng and its processed products, representative red ginseng, are major ginsenosides such as Rg1, Re, Rb1, Rb2, and Rc, and Rd, F2, Rg3, Rh1, Rh2, Rare ginsenosides (also called minor ginsenosides) such as Gyphenoside Major ginsenosides have a very low absorption rate in the body due to their large molecular weight, while rare ginsenosides are known to be relatively easily absorbed and have better medicinal efficacy. Accordingly, various researches are being conducted to produce rare ginsenosides from major ginsenosides.

The present inventor has also continuously conducted research to develop a method for producing rare ginsenosides from major ginsenosides, and in this regard, an enzyme that can efficiently convert major ginsenosides of the ginseng PPD type into Compound K has been developed. A new strain of Aspergillus niger C2-2, which produces Aspergillus niger, has been discovered (Korean Patent No. 10-2258788).

The present inventor did not stop here and studied the possibility of producing other rare ginsenosides using the above strain, and as a result, it was found that rare ginsenosides such as Rh2, Rh3 and Rk2 could be produced using the above strain. After confirming this, the present invention was completed.

Korean Patent No. 10-2258788

Therefore, the main purpose of the present invention is to provide a method for efficiently producing rare ginsenosides Rh2, Rh3 or Rk2 from the major ginsenosides of ginseng.

According to one aspect of the present invention, the present invention includes preparing a raw material containing one or more ginsenosides selected from the group consisting of Rb1, Rb2, Rb3, Rc and Rd; Acid and heat treatment steps of adding acid to the raw material and heat treating it; and an enzyme treatment step of reacting the intermediate product produced in the acid and heat treatment steps with an enzyme derived from Aspergillus niger C2-2 strain. Rh2, Rh3, and Rk2. Provided is a method for producing one or more ginsenosides.

According to another aspect of the present invention, the present invention includes the steps of preparing a raw material containing one or more ginsenosides selected from the group consisting of Rb1, Rb2, Rb3, Rc and Rd; An enzyme treatment step of reacting the raw material with an enzyme derived from Aspergillus niger C2-2 strain; And it provides a method for producing at least one ginsenoside selected from the group consisting of Rh2, Rh3, and Rk2, which includes an acid and heat treatment step of adding an acid to the intermediate product produced in the enzyme treatment step and heat treating it.

According to the present invention, rare ginsenosides Rh2, Rh3 or Rk2 can be efficiently prepared from the major ginsenosides of ginseng such as Rb1, Rb2, Rb3, Rc and Rd.

Figure 1 shows a process for producing rare ginsenosides according to an embodiment of the present invention.
Figure 2 shows the results of TLC analysis of the raw material (1) and the acid and heat treatment intermediate product (2) of the raw material according to an embodiment of the present invention. S: Mixture of standard ginsenosides Rb1, Rb2, Rb3, Rc, Rd, Rg3, F2, Rk1 and Rg5 (control).
Figure 3 shows the results of TLC analysis of the final product (Rh2-mix) obtained through acid and heat treatment followed by enzyme treatment according to an embodiment of the present invention. S: Mixture of standard ginsenosides Rb1, Rb2, Rb3, Rc, Rd, Rg3, F2, Rk1 and Rg5 (control).
Figure 4 shows the results of HPLC analysis of the raw material (A), the intermediate product (B) and the final product (C) of the acid and heat treatment of this raw material according to an embodiment of the present invention, which is subjected to enzyme treatment after acid and heat treatment.
Figure 5 shows the results of HPLC analysis of the raw material (A), the intermediate product (B) of enzyme treatment of this raw material, and the final product (C) according to an embodiment of the present invention subjected to acid and heat treatment after enzyme treatment.

The method of the present invention is a method for producing rare ginsenosides such as Rh2 (S type or R type), Rh3 and Rk2 from major ginsenosides such as Rb1, Rb2, Rb3, Rc and Rd, using acid and It is characterized by comprising a heat treatment step and an enzyme treatment step derived from the Aspergillus niger C2-2 strain.

In one aspect, the method of the present invention includes preparing a raw material containing one or more ginsenosides selected from the group consisting of Rb1, Rb2, Rb3, Rc, and Rd; Acid and heat treatment steps of adding acid to the raw material and heat treating it; And an enzyme treatment step of reacting the intermediate product produced in the acid and heat treatment steps with an enzyme derived from the Aspergillus niger C2-2 strain.

In another aspect, the method of the present invention includes preparing a raw material containing one or more ginsenosides selected from the group consisting of Rb1, Rb2, Rb3, Rc and Rd; An enzyme treatment step of reacting the raw material with an enzyme derived from Aspergillus niger C2-2 strain; and an acid and heat treatment step of adding acid and heat treatment to the intermediate product produced in the enzyme treatment step.

The raw material, that is, the raw material containing one or more ginsenosides selected from the group consisting of Rb1, Rb2, Rb3, Rc, and Rd, may be ginseng, an extract thereof, or a purified product thereof. In one embodiment, the raw material is obtained through a process of purifying PPD type ginsenosides from an extract of ginseng. Ginseng may be, for example, Chinese ginseng, Panax ginseng, or Korean ginseng. Ginseng may be undried (fresh ginseng), dried (dried ginseng), or steamed and dried (e.g., red ginseng). Methods for obtaining extracts containing the above PPD-type ginsenosides from ginseng and processes for purifying PPD-type ginsenosides from such extracts are well known to those skilled in the art. For example, it can be extracted using a 40 to 60% (v/v) aqueous ethanol solution as a solvent, and purified through adsorption using a porous hydrophobic resin. In a preferred embodiment, the raw material contains all of Rb1, Rb2, Rb3, Rc and Rd.

The acid and heat treatment step is performed by adding acid to raw materials (raw materials containing one or more ginsenosides selected from the group consisting of Rb1, Rb2, Rb3, Rc, and Rd) or intermediate products (intermediate products produced in the enzyme treatment step). This can be done by heating. In one embodiment, the acid and heat treatment step is a step of adding acid to the raw material or intermediate product to adjust the pH to 1.9 to 2.5 and heat treating the raw material or intermediate product at 70 to 90° C. for 10 hours or more. According to this, the desired rare ginsenoside, that is, one or more ginsenosides selected from the group consisting of Rh2, Rh3, and Rk2, can be produced more efficiently. For this purpose, the pH is more preferably adjusted to 2.0 to 2.3 and heat treated at 75 to 85°C for more than 12 hours. The heat treatment time is preferably 24 hours or less, more preferably 20 hours or less, and even more preferably 15 hours or less. If the heat treatment time exceeds the above time, the amount of the desired intermediate product (an intermediate product that can be converted to the desired final rare ginsenoside by the enzyme treatment of the present invention) or the desired final rare ginsenoside increases compared to the elapsed time. The degree may decrease, resulting in lower efficiency.

In one embodiment, the acid is an organic acid, preferably citric acid, propionic acid, lactic acid, tartaric acid, acetic acid and mixtures thereof. According to this, the desired rare ginsenoside can be manufactured more efficiently. For this purpose, citric acid is more preferably used.

The enzyme treatment step is performed on raw materials (raw materials containing one or more ginsenosides selected from the group consisting of Rb1, Rb2, Rb3, Rc, and Rd) or intermediate products (intermediate products produced in the acid and heat treatment steps). This can be done by adding an enzyme derived from the Niger C2-2 strain and incubating it. In one embodiment in which the enzyme treatment step is performed after the acid and heat treatment steps, the enzyme treatment step involves adding an enzyme derived from the Aspergillus niger C2-2 strain to the raw material or intermediate product and reacting at 40 to 60 ° C. for more than 1 day. It's a step. According to this, the desired rare ginsenoside can be manufactured more efficiently. For this purpose, more preferably, an enzyme derived from the Aspergillus niger C2-2 strain is added and reacted at 45 to 55° C. for more than 2 days. The incubation period is preferably 5 days or less, more preferably 4 days or less, and even more preferably 3 days or less. If the incubation period exceeds the above period, the degree of increase in the amount of the desired rare ginsenoside finally produced may decrease compared to the elapsed period, and as a result, the efficiency may decrease. In one embodiment in which the acid and heat treatment steps are performed after the enzyme treatment step, the enzyme treatment step is performed by adding an enzyme derived from the Aspergillus niger C2-2 strain to the raw material or intermediate product and heating at 40 to 60° C. for 10 to 20 hours. This is the reaction step. According to this, the desired rare ginsenoside can be manufactured more efficiently. On the other hand, if the above temperature conditions and incubation time are exceeded, the production amount of the desired intermediate product (an intermediate product that can be converted to the desired final rare ginsenoside by the acid and heat treatment of the present invention) is reduced, or even the desired intermediate product is reduced. If this is not produced or the amount of unwanted intermediate products produced may increase, the production efficiency of the desired rare ginsenoside may decrease. For this purpose, more preferably, an enzyme derived from the Aspergillus niger C2-2 strain is added and reacted at 45 to 55° C. for 12 to 15 hours.

Aspergillus niger C2-2 strain has been deposited at the Agricultural Genetic Resources Center (KACC) of the National Academy of Agricultural Sciences under the accession number KACC 93333P, and for specific details, Korean Patent No. 10-2258788 (refer to this specification in its entirety) It is disclosed in (referenced by). This strain can be cultured at a temperature of 25 to 33°C using, for example, a medium containing malt extract or wheat bran, and can be cultured in liquid and solid phases.

The enzyme derived from the Aspergillus niger C2-2 strain can be obtained from the Aspergillus niger C2-2 strain by a conventional enzyme purification method. In one embodiment, the enzyme from the Aspergillus niger C2-2 strain is a crude enzyme solution obtained from a culture of the Aspergillus niger C2-2 strain. The crude enzyme solution can be obtained, for example, from a culture of Aspergillus niger C2-2 strain by a conventional enzyme purification method, preferably a method of purifying extracellular enzymes. For example, it can be obtained by adding a buffer to the Aspergillus niger C2-2 strain culture, removing the cells and solids, and then adding alcohol to precipitate the enzyme. Preferably, the Aspergillus niger C2-2 strain is cultured on a solid medium containing wheat bran to obtain a culture, acetic acid buffer is added to the culture, mixed, and then centrifuged or membrane separated to separate the bacterial cells and solids. It is obtained by removing and adding alcohol to precipitate the enzyme. Preferably, 10 to 100mM acetic acid buffer with a pH of 4.0 to 6.0 is used. Add the acetic acid buffer and mix at 5 to 15°C for 1 to 10 hours to ensure that the enzyme is well mixed in the buffer, and then remove the bacteria and solids. Carry out the process. Also, it is preferable to use alcohol with an alcohol content of 80 to 100% (v/v), and to precipitate it by adding alcohol and leaving it to stand at 1 to 10°C for 10 to 20 hours. Using the crude enzyme solution obtained in this way, the desired rare ginsenoside can be produced more efficiently.

The step of purifying the intermediate or final product may be further included after the acid and heat treatment step and/or enzyme treatment step of the present invention. For example, purification may be accomplished through a conventional PPD type ginsenoside purification process, for example, a purification process using a porous hydrophobic resin.

Hereinafter, the present invention will be described in more detail through examples. The examples below are merely for illustrating the present invention and should not be construed as limiting the scope of the present invention.

Example 1. Preparation of rare ginsenosides using acid and heat treatment followed by enzyme treatment.

1-1. Raw material preparation

PPD-type ginsenosides were purified and powdered from American ginseng to obtain PPD-type crude ginsenosides, which were used as raw materials for manufacturing rare ginsenosides.

PPD type roughasponin powder was obtained by the following method.

First, add about 1 kg of powdered American ginseng to about 10 liters of 50% ethanol and extract it three times for about 24 hours at about 50°C (approximately 10 liters of 50% ethanol is added to the extraction residue to proceed with the next extraction) method was used). Afterwards, the three extracts were collected and concentrated to about half the volume using a vacuum concentrator to have an ethanol content of 5% or less. Pass the extracted concentrate through an HP20 porous resin column, remove impurities by flowing about 40 liters or more of distilled water through the column, and flow about 32 liters of 29% ethanol to first elute and remove PPT-type ginsenosides, and then remove about 32 liters of 29% ethanol. PPD type ginsenoside was obtained by elution by flowing 20 liters of 80% ethanol. The obtained eluate containing PPD type ginsenoside was powdered.

1-2. Preparation of crude enzyme solution

First, Aspergillus niger C2-2 strain (KACC 93333P) was inoculated into malt extract broth (BD Difco, cat. 218630) and cultured at about 30°C for about 3 days to obtain a culture medium.

Next, about 250 g of wheat bran was placed in a tray (80x100x320 mm), the water content was adjusted to about 20%, sterilized under high pressure, and about 75 ml of the culture medium was added to achieve a final water content of about 50%, followed by solid-phase culture. At this time, solid-state culture was performed in a constant temperature and humidity chamber at approximately 30°C and humidity of approximately 90% for approximately 8 days.

Approximately 10 times the volume of acetic acid buffer solution (pH 5.0, 50mM) was added to the solid-phase culture obtained through the solid-state culture, mixed by shaking in a shaking incubator at approximately 10°C for approximately 3 hours, and then centrifuged or membrane separated. An extract from which bacteria were removed was obtained. About three times the volume of alcohol (alcohol content about 95% (v/v)) was mixed with this extract and left to stand overnight at about 4°C to perform enzyme precipitation. After precipitation, the supernatant was removed by centrifugation, and acetic acid buffer solution was added to the enzyme precipitate to prepare a crude enzyme solution concentrated about 10 times.

1-3. Acid and heat treatment

Mix 20 g of the PPD type josaponin powder of 1-1 with 20 times (v/w) water, add citric acid at a rate of about 2% (w/v) to adjust the pH to about 2.0, and then cool to about 80°C. It was heat treated for about 12 hours.

1-4. Enzyme treatment

Add 1,400 ml of acetic acid buffer solution (pH 5.0, 50mM) and 200 ml of the crude enzyme solution prepared in 1-2 to 400 ml of the reaction solution for which acid and heat treatment was completed in 1-3, and adjust the pH to 5.0 with NaOH. The enzyme reaction was performed by shaking at about 50°C for 48 to 72 hours.

After the enzyme reaction was completed, it was powdered through a purification process using an HP20 porous resin column and used in the experiment. Specifically, the reaction solution in which the enzyme reaction was completed was passed through an HP20 porous resin column, distilled water in a volume of about 10 times the column volume or more was flowed through the column to remove impurities, and about 100% ethanol in a volume of about 5 times the column volume was added. The eluate was obtained by flowing, and it was powdered.

Example 2. Preparation of rare ginsenosides using enzyme treatment followed by acid and heat treatment.

Rare ginsenosides were prepared using the same method as in Example 1, except that enzyme treatment was followed by acid and heat treatment.

2-1. Enzyme treatment

The PPD type crude enzyme solution prepared in 1-1 of Example 1 and the crude enzyme solution prepared in 1-2 of Example 1 were added to an acetic acid buffer solution (pH 5.0, 50mM), and incubated at about 50°C for 12 to 12 minutes in a shaking incubator. The enzyme reaction was performed by shaking for 15 hours.

At this time, the PPD type crude enzyme solution was adjusted to a concentration of approximately 10,000 ppm (10 mg/ml) based on the total volume of the reaction solution, and the crude enzyme solution was adjusted to a volume of approximately 1/10 based on the total volume of the reaction solution. did.

2-2. Acid and heat treatment

Citric acid was added at a ratio of about 2% (w/v) to the reaction solution in which the enzyme treatment was completed in 2-1 to adjust the pH to about 2.0, and then heat-treated at about 80°C for about 12 hours.

After the acid and heat treatment reactions were completed, it was powdered through a purification process using an HP20 porous resin column and used in the experiment. Specifically, the reaction solution in which the enzyme reaction was completed was passed through an HP20 porous resin column, distilled water in a volume of about 10 times the column volume or more was flowed through the column to remove impurities, and about 100% ethanol in a volume of about 5 times the column volume was added. The eluate was obtained by flowing, and it was powdered.

Experimental Example 1. TLC analysis

Using thin layer chromatography (TLC), the raw material (PPD type josaponin powder of 1-1 of Example 1 above), the intermediate product (the reaction solution in which acid and heat treatment were completed in 1-3 of Example 1 above) ) and the final product (powder purified after completion of enzyme treatment in 1-4 of Example 1 above) were analyzed for ginsenosides.

The raw materials and final product were dissolved in methanol and used in the experiment, and the intermediate product was extracted with butanol and used in the experiment.

For TLC, a silica gel 60F254 plate was used, and the lower layer of a mixture of chloroform, methanol, and water (65:35:10, v/v/v) was used as a developing solvent. After development, 0.2% p-anisaldehyde was sprayed and heated to develop color to confirm the developed spot. As a control, a mixture of standard ginsenosides Rb1, Rb2, Rb3, Rc, Rd, Rg3, F2, Rk1, and Rg5 was used.

As a result, the raw material of Example 1 contained major ginsenosides such as Rb1, Rb2, Rb3, Rc and Rd (Figure 2), and through acid and heat treatment, ginsenosides such as Rg3, Rg5 and Rk1 were produced. It was shown that Rh2, Rh3, and Rk2 were produced through enzyme treatment after acid and heat treatment (Figure 2) (Figure 3).

Experimental Example 2. HPLC analysis

Using high performance liquid chromatography (HPLC), raw materials (PPD type josaponin powder of Example 1 1-1), intermediate products (acid and heat treatment completed in Example 1 1-3) were obtained using high performance liquid chromatography (HPLC). Reaction solution; and reaction solution after completion of enzyme treatment in 2-1 of Example 2) and final product (powder purified after completion of enzyme treatment in 1-4 of Example 1; and 2-2 of Example 2) Ginsenosides (powder purified after completion of acid and heat treatment) were analyzed.

Raw materials and final products were dissolved in methanol and used in the experiment, and intermediate products were diluted with methanol and used in the experiment. Before the experiment, each sample was filtered using a membrane filter (0.45㎛).

The column used was a Prodigy ODS(2) C18 column (5㎛, 150x4.6㎜ i.d.; Phenomenex), the mobile phase used acetonitrile and distilled water, the sample injection volume was 10㎕, and the flow rate was 1㎖/ minutes, the temperature was 30°C, and the absorbance was measured at 203nm with a UV detector. More specific HPLC analysis conditions are shown in Table 1 below.

As a result, in Example 1, the raw material contained major ginsenosides such as Rb1, Rb2, Rb3, Rc and Rd (A in Figure 4), and through acid and heat treatment, ginsenosides such as Rg3, Rg5 and Rk1 were produced. Ginsenosides were produced (B in Figure 4), and Rh2, Rh3, and Rk2 were found to be produced through enzyme treatment after acid and heat treatment (C in Figure 4). In Example 2, the raw materials included Rb1, It contains major ginsenosides such as Rb2, Rb3, Rc, and Rd (A in Figure 5), and ginsenoside F2 is produced through enzyme treatment (B in Figure 5), and acid and heat treatment after enzyme treatment It was shown that Rh2, Rh3, and Rk2 were produced through (C in Figure 5).

Claims (4)

Preparing a raw material containing one or more ginsenosides selected from the group consisting of Rb1, Rb2, Rb3, Rc, and Rd;
An acid and heat treatment step of adding acid to the raw materials to adjust pH to 2.0 to 2.3 and heat treating at 75 to 85°C for 12 to 24 hours; and
A crude enzyme solution obtained from a culture of Aspergillus niger C2-2 strain is added to the intermediate product produced in the acid and heat treatment steps and reacted at 45 to 55° C. for 2 to 5 days. Enzyme treatment step
A method for producing one or more ginsenosides selected from the group consisting of Rh2, Rh3 and Rk2. delete delete delete