CN112313342B - Preparation method of rare ginsenoside Rh3 and Rk2 mixture and mixture thereof - Google Patents
Preparation method of rare ginsenoside Rh3 and Rk2 mixture and mixture thereof Download PDFInfo
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Abstract
The method comprises the steps of taking Rg3 as a raw material, carrying out enzymatic preparation by using beta-glucosidase, carrying out acidification reaction after the enzymatic reaction is finished, and then separating by solvent extraction to obtain a crude product of a Rh3 and Rk2 mixture, wherein the proportion of Rk2 in the mixture is higher than that of Rh3.
Description
Technical Field
The invention relates to the technical field of preparation of rare ginsenoside, in particular to a method for preparing Rh3 and Rk2 mixture by a bio-enzyme catalytic technical means and a mixture thereof.
Background
Rare ginsenosides are secondary metabolic derivatives obtained by converting prototype ginsenosides extracted from plants of Araliaceae (e.g., ginseng, notoginseng, american ginseng, korean ginseng, etc.), and are known as rare ginsenosides because they are present only in processed ginseng such as wild ginseng, red ginseng, black ginseng, etc., and their content is very low (in ten thousand parts).
Research shows that compared with prototype ginsenoside, the rare ginsenoside has smaller molecular weight, is easier to be absorbed by human body, has higher bioavailability and thus has stronger anticancer activity, so that the research focus on ginsenoside gradually focuses on rare ginsenoside. After a study for more than half a century, scientists have now found more than 60 rare ginsenosides, including Rg3, rg5, rk1, rk2, rk3, rh1, rh2, rh3, rh4, α PPT, α PPD, etc., which have various anticancer effects. Some rare ginsenosides were tested by Canadian scientists for their anticancer activity and compared as follows: rg3 < Δ (20-22) PPD = Rg5 ≦ α PPT = Rk1 and Rg5 mixture = Rh2 ≦ Δ (20-21) PPD < aPPD = Δ (20-21) PPT = Δ (20-22) PPT = Rh3 and Rk2 mixture ≦ Rk2= Δ (20-21) PPD and Δ (20-22) PPD mixture.
At present, rh2 and Rg3 are the most studied and widely applied to rare ginsenosides in China, but the anticancer activities of Rh2 and Rg3 are not dominant in rare ginsenosides as can be seen from the comparison of the activities, so that the development of other rare ginsenosides with stronger anticancer activity is necessary. However, the stronger the anticancer activity of rare ginsenoside, the more difficult the technology for transforming from prototype ginsenoside is, and it is difficult to realize industrial production, and it is these technological barriers that hinder the development of domestic rare ginsenoside industry, and as a result, no other rare ginsenoside products with higher activity are developed except Rh2 and Rg3.
Summary of The Invention
Technical problem
In view of the above disadvantages of the prior art, the present invention aims to develop a method for preparing a mixture of rare ginsenosides Rh3 and Rk2, which is suitable for industrialization and has low cost, so as to provide a rare ginsenoside product with stronger anticancer activity, thereby solving the technical problem that the rare ginsenoside product in the domestic market is scarce.
Solution to the problem
Technical solution
In order to achieve the aim, the invention provides a preparation method of a rare ginsenoside Rh3 and Rk2 mixture, which comprises the following steps:
(1) Dissolving ginsenoside Rg3 in cosolvent, adding beta-glucosidase, and performing enzyme catalysis reaction at pH of 4-6 and temperature of 20-50 deg.C;
(2) After the enzyme catalysis reaction in the step (1) is finished, adjusting the pH value of the reaction liquid to 2-3, and stirring for 1-2 hours to carry out acidification reaction;
(3) And (3) after the acidification reaction in the step (2) is finished, adding ammonium sulfate into the reaction liquid, fully dissolving, then adding an extracting agent consisting of ethanol and ethyl acetate for shake extraction, standing for layering, collecting an upper layer solution, and recovering the solvent to obtain a crude product of the mixture of Rh3 and Rk2.
In the preparation method provided by the invention, the beta-glucosidase is preferably originated from the coanda bacterium 18JY8-7 (Cohnella sp.18JY8-7). The enzyme can convert Rg3 into Rh3 and Rk2 to the maximum extent under the conditions of pH4-6 and 20-50 ℃.
More preferably, the temperature of the enzyme-catalyzed reaction is 30 ℃.
In the preparation method provided by the invention, the addition amount of the beta-glucosidase is preferably 0.5-3 times of the weight of Rg3.
More preferably, the amount of the beta-glucosidase derived from the above is the same as the weight of Rg3.
In the above preparation method provided by the present invention, DMSO is preferably used as the cosolvent.
Preferably, DMSO is added in an amount of 0.5-3ml/g Rg3.
More preferably, DMSO is added in an amount of 1ml/g Rg3.
Preferably, the present invention provides the above preparation method, wherein the enzyme-catalyzed reaction of step (1) is performed in the presence of a phosphate buffer. More preferably, the phosphate buffer is a sodium phosphate buffer.
In the preparation method provided by the invention, the acidification reaction in the step (2) aims to promote the reaction to be more favorable for generating Rk2, so that a mixture of Rh3 and Rk2 with more Rk2 is obtained.
In the preparation method provided by the invention, hydrochloric acid is preferably added to adjust the pH value in the step (2), and the preparation method has the advantages of low cost of hydrochloric acid, mild reaction process, more stable Rk2 generation and minimum impurity generation amount.
In the step (3) of the preparation method provided by the invention, the beta-glucosidase in the reaction solution is settled and separated by adding ammonium sulfate, wherein the adding amount of the ammonium sulfate is preferably 0.01-0.03 time of the weight of the beta-glucosidase; more preferably, the amount of ammonium sulfate added is 0.02 times the weight of the β -glucosidase.
In the preparation method provided by the invention, the adding amount of the extracting agent is preferably 2 times of the weight of Rg3.
In the preparation method provided by the invention, the volume ratio of the ethanol to the ethyl acetate is preferably 1: 0.5-5. More preferably, the volume ratio of ethanol to ethyl acetate is preferably 1: 3.
The crude Rh3 and Rk2 mixture prepared by the method of the present invention may be further purified to obtain a higher purity Rh3 and Rk2 mixture, and the purification method may be any known and applicable separation and purification method.
Preferably, the preparation method provided by the invention further comprises the following refining steps:
(4) And (3) performing gradient elution on the crude product of the mixture of Rh3 and Rk2 by preparative liquid chromatography, wherein the mobile phase is acetonitrile and water, the chromatographic column is a polystyrene column, collecting a target peak, and drying to obtain a pure product of the mixture of Rh3 and Rk2.
In the refining step of the preparation method provided by the invention, the elution gradient is preferably 10min for 30-50% acetonitrile aqueous solution and 20min for 75-85% acetonitrile aqueous solution.
In the refining step of the above-mentioned production method of the present invention, the elution gradient is more preferably 10min for 40% acetonitrile aqueous solution and 20min for 80% acetonitrile aqueous solution. Under the elution condition, the separation degree of the target substance from other impurities is higher, and the peak shape of the target peak is better.
In addition, the invention also provides a rare ginsenoside Rh3 and Rk2 mixture, and the mixture is prepared by the preparation method provided by the invention.
Advantageous effects of the invention
Advantageous effects
Compared with the prior art, the invention has the following advantages:
1. the preparation method of the rare ginsenoside Rh3 and Rk2 mixture provided by the invention has the advantages of low cost and simple operation, and is suitable for industrial production, and the Rh3 and Rk2 mixture with higher Rk2 ratio can be prepared by the method, so that the obtained mixture product has stronger anticancer activity;
2. the rare ginsenoside Rh3 and Rk2 mixture provided by the invention has stronger anticancer activity.
Examples of the invention
Modes for carrying out the invention
The present invention will be described in further detail with reference to specific examples, which are illustrative of the present invention and are not to be construed as being limited thereto.
The beta-glucosidase in the following examples is obtained by self-preparation by a biological fermentation method, and after biological sequencing, the self-prepared enzyme of the inventor is the same as the amino acid sequence (Genbank accession number: CP 033433.1) of the beta-glucosidase from the coanda bacterium 18JY8-7 (Cohnella sp.18JY8-7). The other reagents and raw materials are all commercial products.
Example 1 to example 3
From the perspective of industrial production, for the purpose of reducing production time and production cost, the following examples were used to prepare rare ginsenoside Rh3 and Rk2 mixtures using the following preparation methods of rare ginsenoside Rh3 and Rk2 mixtures provided by the present invention:
(1) Weighing ginsenoside Rg3, adding into DMSO and sodium phosphate buffer solution with pH of 4-6, stirring thoroughly until Rg3 is completely dissolved, wherein the addition amount of DMSO is 1ml/g ginsenoside Rg3, and the addition amount of sodium phosphate buffer solution is 5 times volume of DMSO; then adding beta-glucosidase with the same weight as Rg3, and carrying out enzyme catalytic reaction for 10 hours at the temperature of 30 ℃;
(2) After the enzyme catalysis reaction in the step (1) is finished, adding dilute hydrochloric acid to adjust the pH value of the reaction solution to 2-3, stirring at room temperature for 1-2 hours to carry out acidification reaction, measuring the contents of Rh3 and Rk2 in the acidification reaction solution through HPLC after the acidification reaction is finished, and calculating the proportion of Rk2 in the mixture of Rh3 and Rk2 and the substrate conversion rate calculated by the mixture of Rh3 and Rk2, wherein the results are shown in Table 2;
(3) Adding ammonium sulfate into the acidified reaction liquid in the step (2), and stirring to dissolve the ammonium sulfate, wherein the adding amount of the ammonium sulfate is 0.02 time of the weight of the beta-glucosidase; after the ammonium sulfate is fully dissolved, adding an extracting agent with the weight of 2 times of that of the Rg3, wherein the extracting agent consists of V Ethanol ∶V Ethyl acetate Sufficiently shaking up, standing for layering, distilling the upper solution to 1/3 of the original volume at 70 ℃, recovering the solvent, filtering while hot, washing the filter cake with 1% volume of pure water, and drying to obtain a crude product of the mixture of Rh3 and Rk 2;
(4) Dissolving the crude product of the mixture of Rh3 and Rk2 in acetonitrile, filtering with a 0.45-micron membrane, performing gradient elution on the filtrate by preparative liquid chromatography according to the conditions in the table 1, collecting a target peak, and drying to obtain a pure product of the mixture of Rh3 and Rk2, wherein the total yield of the product is shown in the table 2.
TABLE 1
[ Table 1]
Examples 4 to 5
Examples 4-5 differ from examples 1-3 in that: and (3) after the enzyme catalysis reaction in the step (1) is finished, the acidification reaction in the step (2) is not carried out, the content of each substance in the reaction liquid of the enzyme catalysis reaction is directly measured through HPLC, and then the operation of the step (3) and the operation of the step (4) are sequentially carried out. The data of examples 4 to 5 are shown in Table 2.
TABLE 2
[ Table 2]
Structure confirmation of Rh3 and Rk2
1. Dissolving the pure Rh3 and Rk2 mixture in methanol, and detecting by high-resolution liquid mass spectrometryWhen the peak is measured, all the peaks in the liquid phase mass spectrogram have no difference, and the peak with M/z of 663.4411 is [ M + CH3COO ]] - Peak, M is exactly Rh 3 Or Rk 2 The molecular weight of (2).
2. The pure Rh3 and Rk2 mixture was dissolved in deuterated pyridine and measured on a 400MHz Bruker NMR spectrometer with the results shown in Table 3.
TABLE 3
Claims (9)
1. A method for preparing a mixture of rare ginsenosides Rh3 and Rk2, characterized in that the method comprises the following steps:
(1) Dissolving ginsenoside Rg3 in a cosolvent, adding beta-glucosidase, and performing enzyme catalytic reaction at pH of 4-6 and temperature of 20-50 deg.C, wherein the beta-glucosidase is obtained by fermenting Endothia chrysolepis 18JY 8-7;
(2) After the enzyme catalysis reaction in the step (1) is finished, adjusting the pH value of the reaction liquid to 2-3, and stirring for 1-2 hours to carry out an acidification reaction;
(3) And (3) after the acidification reaction in the step (2) is finished, adding ammonium sulfate into the reaction liquid, fully dissolving, then adding an extracting agent consisting of ethanol and ethyl acetate, performing oscillation extraction, standing for layering, collecting an upper-layer solution, and recovering the solvent to obtain a crude product of the mixture of Rh3 and Rk2.
2. The method of preparing a mixture of rare ginsenosides Rh3 and Rk2 according to claim 1, characterized in that: the addition amount of the beta-glucosidase is 0.5-3 times of the weight of Rg3.
3. The method of preparing a mixture of rare ginsenosides Rh3 and Rk2 according to claim 1, characterized in that: the cosolvent is DMSO.
4. The method of preparing a mixture of rare ginsenosides Rh3 and Rk2 according to claim 1, characterized in that: and (3) adjusting the pH value by adding hydrochloric acid in the step (2).
5. The method of preparing a mixture of rare ginsenosides Rh3 and Rk2 according to claim 1, characterized in that: the addition amount of the ammonium sulfate is 0.01-0.03 time of the weight of the beta-glucosidase.
6. The method of preparing a mixture of rare ginsenosides Rh3 and Rk2 according to claim 1, characterized in that: the addition amount of the extracting agent is 2 times of the weight of Rg3.
7. The method of preparing a mixture of rare ginsenosides Rh3 and Rk2 according to claim 1, characterized in that: the volume ratio of the ethanol to the ethyl acetate is 1: 0.5-5.
8. The method of preparing a mixture of rare ginsenosides Rh3 and Rk2 according to claim 1, characterized in that the method further comprises the following refining steps: (4) And (3) performing gradient elution on the crude product of the mixture of Rh3 and Rk2 by preparative liquid chromatography, wherein the mobile phase is acetonitrile and water, the chromatographic column is a polystyrene column, collecting a target peak, and drying to obtain a pure product of the mixture of Rh3 and Rk2.
9. The method of preparing a mixture of rare ginsenosides Rh3 and Rk2 according to claim 8, characterized in that: the elution gradient in the step (4) is that 30-50% acetonitrile aqueous solution is eluted for 10min, and 75-85% acetonitrile aqueous solution is eluted for 20min.
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| PCT/CN2019/084173 WO2020215265A1 (en) | 2019-04-25 | 2019-04-25 | Method for preparing mixture of rare ginsenosides rh3 and rk2 and mixture thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20170027367A (en) * | 2015-09-01 | 2017-03-10 | 셀루스원 주식회사 | Production of ginsenoside Rh2, Rk2 and Rh3 through combinative usage of enzymes and organic acids |
| CN107338280A (en) * | 2017-06-30 | 2017-11-10 | 肖永坤 | A kind of high activity low sugar base ginseng saponin group and its preparation method of aglycon |
| CN107337704A (en) * | 2017-06-15 | 2017-11-10 | 吉林大学 | A kind of rare ginsenoside Rk2 and Rh3 separation method |
| CN108430596A (en) * | 2016-12-14 | 2018-08-21 | 邦泰生物工程(深圳)有限公司 | A kind of purification process of ginseng saponin Rh 2 |
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| CN1269835C (en) * | 2002-12-13 | 2006-08-16 | 中国科学院大连化学物理研究所 | Method for preparing low-polarity ginseng saponin and its aglycone by catalytic pyrolysis |
| KR101968004B1 (en) * | 2017-10-13 | 2019-04-10 | 재단법인 지능형 바이오 시스템 설계 및 합성 연구단 | Mass producing method of ginsenoside Rh2 mix |
| WO2019104608A1 (en) * | 2017-11-30 | 2019-06-06 | 邦泰生物工程(深圳)有限公司 | Method for enzyme catalysis synthesis of ginsenoside rh2 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20170027367A (en) * | 2015-09-01 | 2017-03-10 | 셀루스원 주식회사 | Production of ginsenoside Rh2, Rk2 and Rh3 through combinative usage of enzymes and organic acids |
| CN108430596A (en) * | 2016-12-14 | 2018-08-21 | 邦泰生物工程(深圳)有限公司 | A kind of purification process of ginseng saponin Rh 2 |
| CN107337704A (en) * | 2017-06-15 | 2017-11-10 | 吉林大学 | A kind of rare ginsenoside Rk2 and Rh3 separation method |
| CN107338280A (en) * | 2017-06-30 | 2017-11-10 | 肖永坤 | A kind of high activity low sugar base ginseng saponin group and its preparation method of aglycon |
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