KR20170025468A - Mehod for producing ginsenoside F1 using bioconversion - Google Patents
Mehod for producing ginsenoside F1 using bioconversion Download PDFInfo
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Abstract
Description
The present invention relates to a method for producing ginsenoside F1 by bioconversion, and more particularly, to a method for producing ginsenoside F1 by biosynthesis, more specifically, a protopanaxatriol-type ginsenoside mixture (hereinafter abbreviated as "PPTGM") derived from ginseng And a step of adding a cellulase to the solution containing the ginsenoside F1 to obtain a bioconversion reaction liquid containing the ginsenoside F1 to produce a ginsenoside F1 using bioconversion, F1.
Ginseng is a well-known medicinal plant and has been used for many thousands of years in traditional medicine in East Asia. In addition, it has been recognized that it can be usefully used in the West for many years (Non-Patent
Many studies have shown that ginseng has a wide range of pharmacological and therapeutic properties. Ginsenoside is a major component of ginseng, and it has a biological and pharmacological function of inhibiting anticancer, antitumor, antiallergic, antiinflammatory, (Non-Patent
Based on the structure of aglycon and dammarane skeleton, ginsenosides can be classified as protopanaxadiol (PPD), protopanaxatriol (PPT), and oleanane ) Saponins (Non-Patent
In addition, glycoside bonds (glycosidic bonds) are made to the prolyl tricholene ginsenoside moiety and aglycone C-6 and C-20. After ingesting ginseng into the mouth, the major ginsenoside is hydrolyzed to become the activated small ginsenoside, which is degraded in the human intestine and absorbed into the blood.
Therefore, the conversion to ginsenosides, in which most of the ginsenosides, which are more than 80% of total ginsenosides, are significantly activated, have a significant impact on the pharmaceutical industry (Non-Patent
In addition, a small amount of ginsenoside F1 exists in Korean ginseng at a low concentration, and ginsenosides Re and Rg1 are desorbed by the intestinal flora and absorbed into blood, which affects the estrogenic action (Non-Patent Document 12) .
The ginsenoside F1 has an anti-cancer effect that strongly inhibits B16 cell proliferation (Non-Patent Document 13). In addition, ginsenoside F1 exhibits anti-aging, antioxidative effects, competitively inhibits CYP3A4 activity, and weakly inhibits the activity of CYP2D6 (Non-Patent Documents 14 and 15).
On the other hand, ginsenoside F1 can be produced by microorganisms and enzymatic methods. As one example, Fusarium memiliforme var. Sublutinas has a beta glucosidase activity, which can convert ginsenoside Rg1 to ginsenoside F1 (Non-Patent Document 16).
As another example, naringinase derived from Penicillium decumbens can be purchased commercially (Non-Patent Document 17), which has the ability to hydrolyze glucose, The rhamnose moiety will be able to produce ginsenoside F1 from ginsenoside Re and ginsenoside Rg1.
However, although only a simple enzyme method has been preceded without technical scale-up or process engineering development, some recombinant enzymes that hydrolyze the protoxyltriol ginsenosides are composed (see Non-patent Document 18 to 22), most of them has a respective ginsenoside Rg2 from the side or Rf1 Re (S) or Rh1 (S) generated valence of glucose residues present in the C20 position of an aglycone in the resolution.
Among these, it is known that two enzymes can produce ginsenoside F1 by hydrolyzing the glucose residue present at the C6 position of the protoporphyrin triol-based aglycon (Non-Patent Documents 19 and 21).
Although ginsenoside F1 has anti-aging, anticancer and antioxidant effects, production of high purity ginsenoside F1 is limited by its economic usefulness. According to the above conventional techniques, There is a problem that the production cost of the side F1 is high and mass production can not be performed.
In order to solve the problems of the prior art as described above, the inventors of the present invention have found that by using a cellulase having high activity so as to transform ginsenoside Re and Rg1 into ginsenoside F1, The present inventors have found that a high purity ginsenoside F1 can be produced in a high yield from a triol based ginsenoside mixture, thereby completing the present invention.
Therefore, an object of the present invention is to provide a production method of ginsenoside F1 capable of producing high purity ginsenoside F1 with high yield by bioconversion.
(A) a solution containing protopanaxyl triol ginsenoside mixture obtained from ginseng has an optimum pH derived from Aspergillus niger of 4.8 to 5.2 and an optimum temperature of 45 to < RTI ID = 0.0 > (B) centrifuging the biotransformation reaction solution; and (c) adding a precipitate formed by the step (b) And separating and purifying the ginsenoside F1 from the supernatant. The present invention also provides a method for producing ginsenoside F1 by bioconversion.
The present invention also provides ginsenoside F1 produced by the above method.
According to the present invention, a ginseng-derived protopanaxadiol-based ginsenoside mixture is bioconverted to ginsenoside F1 by using a cellulase agent, which is advantageous in mass production of ginsenoside F1 at high purity and high yield.
FIG. 1 shows the results of analysis of biosynthesis of ginsenoside by cellulase KN over time by thin layer chromatography.
2 shows the conversion path of ginsenoside Re and Rg1 by the commercial enzyme cellulase KN.
Figure 3 shows the effective concentrations of PPTGM and cellulase KN for the production of ginsenoside F1.
Fig. 4 shows the results of biotransformation high performance liquid chromatography analysis of PPTGM by cellulase KN. Fig. 4A shows the standard ginsenoside, B the substrate (PPTGM) for the production of ginsenoside F1, C the
Figure 5 shows an embodiment of a process for producing ginsenoside F1 by cellulase KN.
The present invention relates to a process for the production of a cellulase comprising the steps of (a) adding a cellulase having an optimum pH derived from Aspergillus niger of 4.8 to 5.2 and an optimum temperature of 45 ° C to 50 ° C to a solution containing a protopanaxyl trihydrate ginsenoside mixture derived from ginseng (B) centrifuging the biotransformation reaction solution; and (c) separating the precipitate and the supernatant from the supernatant by centrifugation to obtain a biosynthesis reaction liquid containing ginsenoside F1, And separating and purifying the F1. The present invention also relates to a method for producing ginsenoside F1 by bioconversion.
In the production method of ginsenoside F1 by the bioconversion of the present invention, the protopanaxyl triol ginsenoside mixture (PPTGM) may contain ginsenoside Re and ginsenoside Rg1.
In the method for producing ginsenoside F1 by the bioconversion of the present invention, the protopanaxyl triol-ginsenoside mixture may be obtained from ginseng roots, but not limited to, ginseng root or ginseng stem have.
In the method for producing ginsenoside F1 by the bioconversion of the present invention, the concentration of the protopanaxadiol-based ginsenoside mixture is 5 mg / mL to 15 mg / mL, preferably 7 mg / mL to 13 mg / mL, more preferably 9 mg / mL to 11 mg / mL, and most preferably 10 mg / mL.
In the method for producing ginsenoside F1 by the bioconversion of the present invention, the cellulase has an optimum pH derived from Aspergillus niger of 4.8 to 5.2, preferably pH 4.9 to 5.1, most preferably pH 5.0, Lt; 0 > C to 50 < 0 > C.
In the method for producing ginsenoside F1 by the bioconversion of the present invention, the concentration of the cellulase in the solution of the protoplanary tridentate ginsenoside mixture is 80 mg / mL to 120 mg / mL, preferably 90 mg / mL to 110 mg / mL, most preferably 110 mg / mL.
In the production method of ginsenoside F1 by the bioconversion of the present invention, the agitation speed of the stirring reaction of step (a) is 100 rpm to 300 rpm, preferably 150 rpm to 250 rpm, most preferably 200 rpm Lt; / RTI >
In the production method of ginsenoside F1 by the bioconversion of the present invention, the reaction pH of the stirring reaction of step (a) may be 4.8 to 5.2, preferably 4.9 to 5.1, and most preferably 5.0.
In the production method of ginsenoside F1 by the bioconversion of the present invention, the reaction temperature of the stirring reaction of step (a) is preferably 48 ° C to 52 ° C, preferably 49 ° C to 51 ° C, most preferably 50 ° C Lt; / RTI >
In the production method of ginsenoside F1 by the bioconversion of the present invention, the reaction time of the stirring reaction of step (a) is 45 to 50 hours, preferably 47 to 49 hours, most preferably 48 hours Lt; / RTI >
In the production method of ginsenoside F1 by the bioconversion of the present invention, the separation of ginsenoside F1 from the precipitate is carried out by dissolving in an organic solvent, preferably an alcohol having 1 to 4 carbon atoms, most preferably ethanol But is not limited thereto.
In the method for producing ginsenoside F1 by the bioconversion of the present invention, the separation of ginsenoside F1 from the supernatant can be carried out using column chromatography, for example, column chromatography packed with octadecylsilane However, the present invention is not limited thereto.
In the method for producing ginsenoside F1 by the bioconversion of the present invention, the purification can be performed using silica-packed column chromatography, but the present invention is not limited thereto.
In the production method of ginsenoside F1 by the bioconversion of the present invention, the production yield of ginsenoside F1 from the protopumpoxyl triol ginsenoside mixture is 25% (w / w) to 30% (w / w) ), Preferably 25% (w / w) to 27% (w / w), more preferably 25.5% (w / w) to 26.5% (w / w).
The present invention also relates to ginsenoside F1 produced by the above method.
The ginsenoside F1 of the present invention may have a high purity, preferably 90% to 95%, more preferably 91% to 93%.
Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the scope of the present invention is not limited to the following examples, but includes modifications of equivalent technical ideas.
<Examples>
1. Materials and Experimental Methods
1-1. Experimental material
Standard grade ginsenosides containing Rg1, Re, Rh1 ( S ), F1 and protopanaxatriol were purchased from Nanjing Zelang Medical Technology Co., Ltd. (Nanjing, China).
The protoporphyrin triol ginsenoside mixture (PPTGM) was extracted from 4kg of Panax ginseng CA Meyer species ginseng and 1kg of Panax quinquefolius species ginseng root.
5 kg of ginseng root powder was extracted twice with 50 L of 70% ethanol. The extract was filtered through a filter paper and then dried with a rotary evaporator.
The dried powder was dissolved in water and placed in a glass column (400 mm L x 100 mm D) filled with a Diaion HP-20 resin (Tokyo, Mitsubishi Chemical).
The free-radical molecules and undesired hydrophilic compounds from the above-mentioned di-ion HP-20 resin were washed with water that was 8 times as high as the column. Finally, the solution was dissolved in ethanol (36%) which was 8 times that of the column.
The ethanol extract was evaporated in vacuo to dryness and the remainder was used as substrate to produce ginsenoside F1.
According to high performance liquid chromatography (HPLC) analysis, the PPTGM was mostly Re (245 mg / g), Rg1 (203 mg / g), Rb1 (32 mg / g), Rb2 53 mg / g), Rd (80 mg / g) and a small amount of other ginsenosides dissolved in 50 mM acetate buffer and PPTGM dissolved up to 100 mg / mL have.
High performance liquid chromatography grades of methanol and acetonitrile are available from SK Chemical Co., Ltd. (Seoul, Korea), and the other chemicals used in this study are of analytical grade or higher.
1-2. Screening of Enzymes Converting Tryptic Ginsenosides
PPTGM can be diluted in 500 μl of acetate buffer (pH 5.0) corresponding to 1% (w / v), and the same volume of enzyme was mixed and then the experiment was continued.
The enzyme was digested with the following enzymes: Novozym 960 (Bagsvaerd, Novozyme), Viscozyme (Novozyme), Pectinex AFPL-4 (Novozyme), Pectinex Ultra SP-L (Novozyme), Fungamyl 800L (Novozyme) and Cellulase KN Chemical).
The mixture of the PPTGM solution and the enzyme was shaken at 150 rpm in an aerobic environment at 50 캜 for 48 hours.
The results were analyzed at regular intervals through thin layer chromatography (TLC) or high performance liquid chromatography (HPLC) after pretreatment for analysis.
1-3. Bioconversion activity of protopanaxyl triol-based ginsenosides using cellulase KN
The activity of the cellulase KN can be measured by determining the specificity and selectivity that can be converted between the ginsenosides Re and Rg1, which means the specificity and selectivity to attach to the C6 position to decompose Rhamnose or glucose residues.
The concentration of the enzyme solution in 100 mM acetate buffer is 100 mg / mL, which reacts with a solution of the same volume of Re and Rg1 (
The results were analyzed at regular intervals through thin layer chromatography or high performance liquid chromatography after pretreatment for analysis.
1-4. Optimize bioconversion based on enzyme and substrate concentration
To determine the optimum conditions for bioconversion of PPTGM using cellulase KN, the substrate concentration of PPTGM was adjusted to the best suitability.
The final enzyme concentration was fixed at 50-100 mg / mL, and PPTGM was dissolved in acetate buffer (pH 5.0) to adjust the final substrate concentration to 5, 10, and 15 mg / mL.
These six optimized reactions were carried out in a 2 ml Eppendorf tube for 48 hours at a rate of 200 rpm, a temperature of 50 캜, and a volume of 1 ml.
The results were analyzed at regular intervals through thin layer chromatography or high performance liquid chromatography after pretreatment for analysis.
1-5. Using cellulase KN, PPTGM scale-up bioconversion
The scale-up bioconversion was carried out in a tank type reactor (10 L, Biotron GX, Hanil Science Co., Seoul, Korea) in a volume of 5 L for 48 hours at a speed of 200 rpm. The reaction was carried out at pH 5.0, 50 ° C.
The reaction was initiated when the final concentration of the substrate PPTGM (total 50 g) was to be 10 mg / mL and 500 g of cellulase KN was added.
Samples were periodically collected and analyzed by high performance liquid chromatography for the production of ginsenoside F1 from PPTGM.
1-6. Tablet of Ginsenoside F1
5T of PPTGM and the cellulase KN reaction mixture were centrifuged at 4,000 rpm for 20 minutes at 4 ° C (Component R, Hanil Science Co. Ltd., Korea).
The bioconverted ginsenosides F1 in the supernatant and in the precipitate were subjected to the respective procedures to remove the enzyme, salt and free sugar.
The ginsenoside F1 in the supernatant was separated using a Biotage SNAP flash chromatography cartridge (180 × 70 mm; Biotage, Uppsala, Sweden) filled with 340 g of octadecylsilane (ODS; ZEOprep 60 C 18 , 40-63 μm).
The precipitate was dissolved in 3 L of a 95% ethanol solution, filtered through a filter paper (Advantec, Tokyo, Japan), and evaporated in vacuo.
The ginsenoside powder from the supernatant and the precipitate means raw ginsenoside F1, which was purified with Biotage SNAP flash chromatography cartridge (180 x 70 mm Biotage) filled with 340 g of silica resin (230-400 mesh) .
The cartridge was balanced with chloroform and 13.5 g of the raw ginsenoside F1 powder was filtered through the cartridge twice.
Elution was carried out with 5 fill volumes of chloroform-3 bed volumes (BVs) and chloroform-methanol (85:15, v / v) and the results were analyzed by thin layer chromatography.
The eluent was evaporated in vacuo. The resulting powder was dissolved in 100% methanol and analyzed by high performance liquid chromatography.
1-7. Analysis method
1-7-1. Thin layer chromatography (TLC) analysis
Thin layer chromatography was performed using silica gel plates (60 F 254 silica gel plates, Merck, Darmstadt, Germany) and CHCl 3 -CH 3 OH-H 2 O (65: 35: 10, lower phase) as solvent.
, It can be identified by comparison with a standard ginsenosides was sprayed with a thin layer chromatography plate spot (spot) of 10% sulfuric acid (vol / vol, H 2 SO 4) on, and visualized by heating at 110
1-7-2. High Performance Liquid Chromatography (HPLC) Analysis
High performance liquid chromatographic analysis of ginsenosides (Rb1, Rd, F1, Rh1 ( S ), F2, Rg1, Re, CK, F1 and protopyranosyl triol) was performed using a quaternary pump, Detector and a Younglin ' s AutoChro 3000 software for peak identification and peak integration.
Separation was performed with a guard column (5 μm, 12.5 × 4.6-mm id, Eclipse X DB C 18 ) with Prodigy octadecylsilane (2) C 18 column (5 μm, 150 × 4.6-mm id; Phenomenex, Torrance, . The mobile phase used acetonitrile (A) and water (B).
The gradient elution method was started with 17% solvent A and 83% solvent B, and then proceeded as follows.
Solvent A 17-25%, 12-20 minutes; Solvent A 25-32%, 20-30 minutes; Solvent A 32-55%, 30-35 min; Solvent A 55-60%, 35-40 min; Solvent A 60-80%, 40-45 minutes; Solvent A 80-100%, 45-50 min; Solvent A 100%, 50-54 min; Solvent A 100-17%, 54.0-54.1 min; Solvent A 17%, 54.1-65 min.
The flow rate was 1.0 mL / min, and the injection capacity of 25 ㎕ was measured by monitoring the absorbance at 203 nm.
2. Experimental results
2-1. Selection of the most effective enzyme for production of Ginsenoside F1
Several enzymes were used for 2 days to investigate the production of ginsenoside F1 from PPTGM.
As shown in Table 1 below, the content of ginsenoside F1 produced in the enzymatic treatment reaction was determined by the ratio of ginsenoside F1 in the total ginsenoside peak area measurable using high performance liquid chromatography analysis.
The commercial enzymes Novozym 960, Viscozyme, Pectinex AFPL-4, Pectinex Ultra SP-L and Fungamyl 800 L (Novozyme) failed to convert ginsenosides Re and Rg1 into ginsenoside F1. However, cellulase KN effectively converted ginsenoside Re and Rg1 to ginsenoside F1.
Based on the peak area, PPTGM contained 27.3% of ginsenoside Re and 37.0% of Rg1, and 57.9% of ginsenoside F1 treated with cellulase KN, based on the peak area.
In addition, a small amount of compound K derived from unconverted ginsenoside Re, protopanaxidiol-based ginsenoside F2, ginsenosides Rb1, Rb3 and Rd was detected.
As a result of examining six enzymes, it was found that cellulase KN can produce ginsenoside F1 most effectively.
2-2. Bioconversion of protopanaxyl triol-based ginsenosides using cellulase KN
To determine if bioconversion of the two protopanaxyl triol ginsenosides Re and Rg1 was caused by the cellulase KN, they were analyzed at regular intervals through thin layer chromatography.
When Re (1.0 mg / mL) and Rg1 (1.0 mg / mL) were used as substrates, the bioconversion of ginsenoside F1 to cellulase KN (100 mg / mL) f value (see Fig. 1).
The bioconversion rate of ginsenoside Re by cellulase KN was lower than that of ginsenoside Rg1.
When ginsenoside Rg1 is generated from ginsenoside Re by cellulase KN, it is rapidly converted to ginsenoside F1 so that there is little remaining reaction mixture.
Thus, it can be concluded that the cellulase KN effectively hydrolyzes the Ramanose and glucose residues at the C6 position of ginsenoside Re and bioconverts to ginsenoside F1 (see FIG. 2).
Cellulase KN provides the functionality of cellulase and naringinase. This generally means that there are three cellulolytic enzyme groups in cellulases: cellobiohydrolase, endoglucanase and? -Glucosidase (Trends in Biotechnology 1987; 9: 255261).
Beta glucosidase is considered to be the most suitable enzyme for hydrolyzing beta-glucosidic linkage at the C6 position of the protoporphyrin triol ginsenosides. This suggests that ginsenoside Rg1 can be converted to ginsenoside F1 by beta glucosidase action.
In addition, naringinase attaches to the C 6 hydroxyl group of the aglycon of ginsenoside Re and binds to α-L (2 ← 1) -α-L-rhamnose linkage -rhamnosidase activity, and it is judged that beta glucosidase activity is higher with respect to glucosidic linkage in C6 of ginsenoside Rg1.
The above results indicate that cellulase KN converts ginsenosides Re and Rg1 most effectively to ginsenoside F1.
2-3. Optimization of PPTGM and Enzyme Concentration
(5 mg / mL, 10 mg / mL, and 15 mg / mL) as the final concentration and two coenzyme concentrations (50 mg / mL, 100 mg / mL) was used for the test.
In six test environments, PPTGM and product F1 were analyzed over time using high performance liquid chromatography and the results are shown in FIG.
In the test environment with intermediate substrate concentration (5 mg / mL) and high coenzyme concentration (100 mg / mL), PPTGM was completely converted to ginsenoside F1 within 24 hours. The reaction rate was 1.3 times faster than that of
In the other three reaction conditions, the conversion did not occur completely within 48 hours. Therefore, the following experiment was carried out except for the above three reactions.
The conditions that provide the advantage of the conversion of small amounts of enzyme and complete Re are at 10 mg / mL substrate concentration and 100 mg / mL crude enzyme concentration, and the above conditions were also used in the next scale up biotransformation experiments.
2-4. Gin Senocide F1 scale-up production and refining
The enzyme reaction was generated by cellulase KN for the PPTGM substrate at a concentration of 10 mg / mL, and the cellulase KN was adjusted to a final concentration of 100 mg / mL of 5 L to produce ginsenoside F1.
The ginsenosides Re and Rg1 of PPTGM gradually changed in proportion to time, and within 48 hours, 98% of ginsenoside Rg1 and 92% of ginsenoside Re were converted to ginsenoside F1.
Bioconversion of ginsenosides was well illustrated by high performance liquid chromatography analysis, as shown in Figures 4A, 4B, 4C, 4D and 4E. This theoretically means that 11.3 g of 12.3 g of ginsenoside Re of 50 g of PPTGM and 9.9 g of 10.2 g of ginsenoside Rg1 can be converted to produce 17.1 g of 17.9 g of ginsenoside F1.
In order to obtain ginsenoside F1 with high purity, enzymes, salts and free sugars were removed from the reaction product of 5 L reaction of PPTGM containing cellulase KN.
About half of the ginsenoside F1 precipitated as a solid while the other half was dissolved in the supernatant. Using 3 L of a 95% ethanol solution twice, the precipitated ginsenoside F1 was completely dissolved twice.
The ginsenoside F1 in the supernatant was purified using column chromatography packed with octadecylsilane (about 4 L of a 95% methanol solvent).
The eluant of the precipitate and supernatant fraction was evaporated in vacuo to yield 26.9 g of the crude ginenoside F1. At this time, the chromatographic purity was 60.8 ± 1.1%, which was measured by high performance liquid chromatography.
Silica column, and finally 13.0 g of high-purity ginsenoside F1 (91.5 ± 1.1%) was finally obtained from 50 g of PPTGM (see FIG. 4F).
PPTGM was mainly composed of ginsenosides Re and Rg1 and contained ginsenosides Rb1, Rb2, Rb3, Rd and Rg3 ( S ).
In these ginsenosides, the total molar amount of ginsenosides Re and Rg1 can be bioconverted to ginsenoside F1 using cellulase KN, which was 24.3 mmol corresponding to 22.5 g of 50 g.
The residue (27.5 g) consisted of other series of ginsenosides and unknown impurities, and the molar amount of the prepared high purity ginsenoside F1 (13.0 g) was 18.6 mmol.
The recovery rate of the PPTGM ginsenoside Re and Rg1 during the whole bioconversion to ginsenoside F1 was 72.7%.
It will be understood by those skilled in the art that various modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims. And changes may be made without departing from the spirit and scope of the invention.
According to the present invention, because the ginsenoside mixture derived from ginseng is bioconverted to ginsenoside F1 by using a cellulase, ginsenoside F1 can be mass-produced at a high purity and a high yield, The present invention can be applied to a technical field to which the present invention belongs.
Claims (11)
(b). Centrifuging the biotransformation reaction solution; And
(c). And separating and purifying the ginsenoside F1 from the precipitate and the supernatant formed by the above step.
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| KR20200018047A (en) * | 2018-08-10 | 2020-02-19 | 한국생명공학연구원 | Aspergillus oryzae GBE174 and method for preparing fermentated extract of ginseng berry using the same |
| KR102590463B1 (en) * | 2023-04-07 | 2023-10-18 | 아미코젠주식회사 | Novel Aspergillus niger strain and method of preparing ginsenoside F1 using the same |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20200018047A (en) * | 2018-08-10 | 2020-02-19 | 한국생명공학연구원 | Aspergillus oryzae GBE174 and method for preparing fermentated extract of ginseng berry using the same |
| KR102590463B1 (en) * | 2023-04-07 | 2023-10-18 | 아미코젠주식회사 | Novel Aspergillus niger strain and method of preparing ginsenoside F1 using the same |
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