Method for preparing high-purity ginsenoside Rh2

Abstract

The invention discloses a method for preparing high-purity ginsenoside Rh2, panoxadiol type total saponins extracted and separated from ginseng roots or ginseng stems and leaves are taken as a substrate, two-step combined enzyme hydrolysis is utilized to convert a diol type ginsenoside mixture into rare ginsenoside Rh2, and the ginsenoside Rh2 with the purity of more than or equal to 98.5% is obtained by recrystallizing the product. The preparation method has industrial prospects, and the product can be used as a raw material of medicines and other products.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a technology for preparing high-purity ginsenoside Rh2 by using combined enzymes to hydrolyze and combining recrystallization with total ginsenoside of the ginsendiol type as a substrate. Background Art

[0002] The rare ginsenoside Rh2 has functions such as antidepressant, anti-aging, anti-allergy, and enhancing body immunity, providing resources for the research, development, and production of candidate drugs by humans. The content of Rh2 in natural plants is very low, and the direct extraction and purification costs are high, which is not conducive to industrial production.

[0003] In the Chinese Patent Application Publication Specification, CN115029279A, Lactiplantibacillus plantarum MB11 was used to convert and prepare rare ginsenosides with relatively high biological activity by using the total saponin extract extracted from ginseng root powder as a raw material, and the converted rare ginsenosides were identified by HPLC. The prepared rare ginsenosides were respectively rare ginsenoside Rg6, rare ginsenoside F4, rare ginsenoside Rk3, rare ginsenoside Rh4, rare ginsenoside 20(R)-Rg3, rare ginsenoside 20(S)-Rg3, rare ginsenoside CK, and rare ginsenoside Rh2. This invention discloses a new optional microorganism, rather than a specific preparation method for Rh2. The products obtained by this method still need to be separated by a silica gel column to obtain different fractions of rare ginsenosides. In the Chinese Patent Application Publication Specification CN102154428A, Lactobacillus delbrueckii subsp. bulgaricus was used to ferment at 37-39 °C for 240-248 h, the fermentation broth was collected and reacted with saponin glycosidase at 88-92 °C for 240-360 h, the reaction solution was collected and filtered, the filtrate was adsorbed by macroporous resin and eluted with 70% ethanol, and then extracted and filtered with 95% ethanol, and the filtrate was dried to obtain ginsenoside Rh2. According to the results of the examples, the highest purity of Rh2 obtained was 97.2%.

[0004] The five enzymes selected in the combined enzyme of the present invention are all reported glycosidases. Those skilled in the art can obtain the preparation methods of glycosidases by referring to the literature. Through the effective combination of glycosidases and combined with the recrystallization method, the present invention can efficiently convert the total ginsenoside of the ginsendiol type into Rh2, and by using the recrystallization purification step of the present invention, Rh2 with a purity ≥ 98.5% can be obtained. The method of the present invention is simple and easy to operate, suitable for industrial production, and the product has a high purity and a wide range of applications. Summary of the Invention

[0005] The technical problem to be solved by the present invention is the preparation of high-purity ginsenoside Rh2, so as to further realize the product application of ginsenoside Rh2.

[0006] To solve the above problems, the present invention provides a method for preparing highly pure ginsenoside Rh2. Using the total ginsenoside of the dammarenediol type extracted and isolated from ginseng roots or ginseng stems and leaves as a substrate, through two-step combined enzyme hydrolysis, the dammarenediol-type ginsenoside mixture is converted into rare ginsenoside Rh2. By recrystallizing the product, ginsenoside Rh2 with a purity ≥ 98.5% is obtained.

[0007] As a preferred embodiment of the method for preparing highly pure ginsenoside Rh2 of the present invention, among which the combined enzyme is added step by step with 5 specific types of glycosidases or their isozymes, that is, in the first step, β-1,6-glucosidase, the β-glucosidase that hydrolyzes the inner glucose of ginsenoside, α-arabinopyranosidase, α-arabinosidase are added. After inactivation, β-1,2-glucosidase is added to complete the reaction.

[0008] As a further preferred embodiment of the method for preparing highly pure ginsenoside Rh2 of the present invention, the mass ratio of the combined enzyme to the substrate is as follows: for β-1,6-glucosidase, 0.1 - 0.5 U / mg; for the β-glucosidase that hydrolyzes the inner glucose of ginsenoside, 0.1 - 0.5 U / mg; for α-arabinopyranosidase, 0.05 - 0.5 U / mg; for α-arabinosidase, 0.05 - 0.5 U / mg; for β-1,2-glucosidase, 0.5 - 2.0 U / mg. The reaction system is a pH 6.0 - 8.0 buffer solution, the reaction temperature is 25 - 37 °C, and the reaction time is 4 - 24 hours.

[0009] As an even more preferred embodiment of the method for preparing highly pure ginsenoside Rh2 of the present invention, the mass ratio of the combined enzyme to the substrate is as follows: for β-1,6-glucosidase, 0.1 U / mg; for the β-glucosidase that hydrolyzes the inner glucose of ginsenoside, 0.1 U / mg; for α-arabinopyranosidase, 0.05 U / mg; for α-arabinosidase, 0.05 U / mg; for β-1,2-glucosidase, 0.5 U / mg. The reaction system is a pH 6.0 buffer solution, the reaction temperature is 30 °C, and the reaction time is 4 hours.

[0010] As another preferred embodiment of the method for preparing highly pure ginsenoside Rh2 of the present invention, the product purification includes the following operation steps: Step 1: The product after conversion is dried, dissolved in methanol, and the mass ratio of the product to the volume of methanol is 0.1 g : 10 mL - 0.5 g : 10 mL. Centrifugation is carried out at normal temperature and 4000 rpm, and the supernatant is dried. Step 2: To the sample obtained in Step 1, an equal volume of 50% methanol is added for re-dissolution at 60 °C, and centrifugation is carried out under the same conditions. The precipitate is taken and dried. Step 3: Add an equal volume of 90% methanol to the sample obtained in Step 2 for reconstitution, let it stand at 4°C for 24 h, centrifuge under the same conditions, and dry the precipitate to obtain Rh2 with a purity of ≥98.5%. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The following further describes in detail the specific embodiments of the present invention with reference to the accompanying drawings.

[0012] Figure 1 It is a picture of analyzing the substrate selectivity of glycosidase for ginsenoside by TLC method.

[0013] Figure 2 It is a picture of analyzing ginsenoside products by HPLC method.

[0014] Figure 3 It is the route for preparing Rh2 by converting diol-group ginsenosides.

[0015] Figure 4 It is a picture of determining the optimal conversion conditions of glycosidase by TLC method.

[0016] Figure 5 It is a picture of analyzing the purity of Rh2 by HPLC method. SPECIFIC EMBODIMENTS Example 1 Preparation of Glycosidase

[0017] Inoculate Escherichia coli BL21 carrying the recombinant plasmid into LB liquid medium containing kanamycin, culture overnight at 37°C and 180 rpm to obtain a seed solution. Inoculate the seed solution into a 5 L fermenter containing 3 L of liquid medium and culture at 37°C. When the OD 600nm reaches about 20, cool down to 16°C or 25°C, add 0.5 mM IPTG and induce incubation for 10 h. After the induction ends, centrifuge at 5000 rpm for 15 min to collect the cells, and resuspend them with 20 mM sodium phosphate buffer (pH 6.0 / 7.0). Disrupt the cells with a high-pressure homogenizer, centrifuge at 13000 rpm and 4°C for 30 min to collect the supernatant, and the supernatant is the total protein solution after the recombinant bacteria are induced to express.

[0018] The glycosidases used in the subsequent examples of the present invention are all prepared by this method, including β-1,6-glucosidase (CcGlu3A), β-glucosidase (PsGlu3A) that hydrolyzes the inner glucose of ginsenoside, α-arabinopyranosidase (MeAra2A), α-arabinofuranosidase (BsAra51B), and β-1,2-glucosidase (CcGlu1A). Example 2 Study on the Function of Glycosidase

[0019] The ginsenoside substrates are diol-type ginsenosides Rb1, Rb2, Rb3, Rc, and Rd.

[0020] (1) The reaction system is 1 mL and consists of ginsenoside substrate, 20 mM phosphate buffer (pH 7.0), and purified recombinant glycosidase. The concentration of ginsenoside substrate in the reaction system is 1 mg / mL, and the concentration of purified recombinant glycosidase in the reaction system is 0.1 mg / mL.

[0021] (2) The reaction system prepared in step (1) is placed under the condition of 37 °C and reacted for 2 h.

[0022] (3) After completing step (2), 500 μL of water-saturated n-butanol is added for extraction.

[0023] (4) After completing step (3), 10 - 20 μg of ginsenoside sample, "mixed standard 1 composed of Rh2, Rg3, Rd, Rc, and Rb1 standards", "mixed standard 2 composed of CK, Gyp LXXV, Gyp XVII, and Rb2 standards", and "standard sample 3 composed of Rb3 standard" are spotted on a G60 silica gel plate using a capillary, dried, and then placed in a developing tank containing a developing agent (chloroform: methanol: water = 65:35:10) for development; then the silica gel plate is air-dried, placed in a 10% (v / v) sulfuric acid - ethanol solution for staining; after air-drying again, it is treated at 105 °C for 5 min. According to the migration rate (Rf value) of the standards, the components in the ginsenoside sample are identified.

[0024] (5) After completing step (3), the n-butanol is evaporated to dryness using a metal bath, then 500 μL of methanol is added, and the components of the ginsenoside sample are detected by HPLC-C18 reversed-phase column method. The mobile phase consists of water (A) and acetonitrile (B), the flow rate is 0.4 mL / min, the detection wavelength is 203 nm, the column temperature is 30 °C, and the sample injection volume is 20 μL.

[0025] The detection results are shown in Figure 1 (S1 is mixed standard 1; S2 is mixed standard 2; S3 is standard sample 3) and Figure 2 . The results show that β-glucosidase CcGlu3A can completely convert Rb1 to Rd, only hydrolyzing the β-1,6 glucosidic bond in C20; β-glucosidase CcGlu1A can convert Rb1, Rb2, Rb3, Rc, and Rd to Gyp XVII, CO, CMx1, CMc1, and F2 respectively, only hydrolyzing the β-1,2 glucosidic bond in C3; β-glucosidase PsGlu3A can convert a part of Rb1 and all of Rd to Rg3; α-arabinopyranosidase MeAra2A can only hydrolyze the arabinopyranosidic bond and completely convert Rb2 to Rd; α-arabinofuranosidase BsAra51B can only hydrolyze the arabinofuranosidic bond and completely convert Rc to Rd. Example 3 Optimization of Glycosidase Hydrolysis Conditions

[0026] (1) Determine the conversion pathway. The reaction system is 1 mL, consisting of a diol-type ginsenoside substrate, 20 mM phosphate buffer (pH 7.0), and recombinant glycosidases in the following combination. The concentration of the ginsenoside substrate in the reaction system is 1 mg / mL, and the concentration of the recombinant glycosidase in the reaction system is 0.1 mg / mL.

[0027] Combination 1: Add three enzymes, CcGlu3A, MeAra2A, and BsAra51B, simultaneously.

[0028] Combination 2: Add four enzymes, CcGlu3A, MeAra2A, BsAra51B, and PsGlu3A, simultaneously.

[0029] Combination 3: Add five enzymes, CcGlu3A, MeAra2A, BsAra51B, PsGlu3A, and CcGlu1A, simultaneously.

[0030] Combination 4: Add four enzymes, CcGlu3A, MeAra2A, BsAra51B, and PsGlu3A, simultaneously, and add CcGlu1A after inactivating the reaction at the end.

[0031] (2) Place the reaction system in step (1) under the condition of 37 °C and react overnight.

[0032] (3) After completing step (2), perform thin-layer chromatography according to the method in Example 2.

[0033] The detection results are as Figure 3 , Combinations 1 and 2 can only convert the total saponins into intermediate products, while Combination 3 converts the substrate into ginsenoside PPD. After one-step inactivation, Combination 4 can completely convert the substrate into Rh2 without by-product formation. To adapt to industrial production and reduce experimental steps, Combination 4 is selected for the preparation of Rh2.

[0034] (4) Determine the optimal conditions. The reaction system is 1 mL, consisting of a diol-type ginsenoside substrate, 20 mM phosphate buffer (pH 5.0 - 9.0), and purified recombinant glycosidase. The concentration of the ginsenoside substrate in the reaction system is 1 mg / mL, and the concentration of the purified recombinant glycosidase in the reaction system is 0.1 mg / mL.

[0035] (5) Place the reaction system prepared in step (4) under the condition of 37 °C and detect the conversion by TLC after reacting for 2 h.

[0036] (6) On the basis of the above experiments, fix the buffer solution, which is the same as the above reaction system, set the temperature range to 25 - 45 °C, and detect the conversion by TLC after 2 h; fix the pH and temperature, and add 0.005 U, 0.01 U, 0.05 U, 0.1 U, 0.5 U of the enzyme in the same reaction system as above, and detect the conversion products by TLC.

[0037] The optimal conversion conditions were determined by calculating the gray values of the TLC results at the above optimal pH and temperature using software.

[0038] The detection results are as Figure 4 , and the results show that the optimal conditions for the conversion to prepare Rh2 are pH 6.0 phosphate buffer, 30 °C, and the optimal enzyme addition amounts of the five enzymes are 0.1 U / mg for CcGlu3A, 0.05 U / mg for MeAra2A, 0.05 U / mg for BsAra51B, 0.1 U / mg for PsGlu3A, and 0.5 U / mg for CcGlu1A. Example 4 Preparation of ginsenoside Rh2 from total ginsenosides of the diol type in ginseng leaves and stems

[0039] (1) Dissolve the total ginsenosides of the diol type in ginseng leaves and stems in a buffer solution with pH 6.0, and the final concentration of the substrate is 10 mg / mL. According to the content of each component in the substrate, add 0.1 U / mg of CcGlu3A, 0.05 U / mg of MeAra2A, 0.05 U / mg of BsAra51B, and 0.1 U / mg of PsGlu3A, and react with shaking at 30 °C for 2 h.

[0040] (2) Take 500 μL of the above conversion sample, add 500 μL of n-butanol, shake and extract, and then perform thin-layer chromatography to observe whether the conversion is complete.

[0041] (3) Centrifuge the completely converted ginsenoside sample in step (1), dissolve the precipitate in the buffer solution, and inactivate it at 100 °C for 10 min.

[0042] (4) Wait for the ginsenoside sample in step (3) to cool, add 0.5 U / mg of CcGlu1A to the reaction system, and react for 2 h.

[0043] (5) Take 500 μL of the ginsenoside sample in step (4), add 500 μL of n-butanol, shake and extract, and perform thin-layer chromatography to observe whether the conversion is complete.

[0044] (6) Centrifuge the conversion product in step (5), dissolve the precipitate in 95% ethanol. Let it stand at 70 °C for 2 h, centrifuge and take the supernatant. Repeat the above operation, combine the supernatants twice, and then dry the sample.

[0045] (7) Take the above-mentioned dried sample, dissolve it in methanol according to the ratio of product mass to methanol volume of 1 g: 20 mL, centrifuge to remove insoluble substances, and dry the supernatant.

[0046] (8) Dissolve the sample dried in step (7) in an equal volume of 50% methanol at 60 °C, centrifuge to remove the supernatant, and dry the precipitate.

[0047] (9) Dissolve the sample dried in (8) in an equal volume of 90% methanol, let it stand at 4 °C for 24 h, then centrifuge and dry the precipitate to obtain ginsenoside Rh2 with a purity of 98.5%. Example 5 Preparation of Ginsenoside Rh2 from Total Ginsenosides of the Diol Type in Ginseng Root

[0048] (1) Dissolve the total ginsenosides of the diol type in ginseng stem and root in a buffer solution with a pH of 8.0, and the final concentration of the substrate is 10 mg / mL. According to the content of each component in the substrate, add 0.5 U / mg of CcGlu3A, MeAra2A, BsAra51B, and PsGlu3A, and react with shaking at 30 °C for 12 h.

[0049] (2) Take 500 μL of the above-mentioned transformed sample, add 500 μL of n-butanol, shake and extract, and then perform thin-layer chromatography to observe whether the transformation is complete.

[0050] (3) Centrifuge the completely transformed ginsenoside sample in step (1), redissolve the precipitate in the buffer solution, and inactivate it at 100 °C for 10 min.

[0051] (4) Wait for the ginsenoside sample in step (3) to cool, add 2 U / mg of CcGlu1A to the reaction system, and react for 12 h.

[0052] (5) Take 500 μL of the ginsenoside sample in step (4), add 500 μL of n-butanol, shake and extract, and perform thin-layer chromatography to observe whether the transformation is complete.

[0053] (6) Centrifuge the transformed product in step (5), redissolve the precipitate in 95% ethanol. Let it stand at 70 °C for 2 h, centrifuge and take the supernatant. Repeat the above operation, combine the supernatants twice and dry the sample.

[0054] (7) Take the above-mentioned dried sample, dissolve it in methanol according to the ratio of product mass to methanol volume of 1 g: 20 mL, centrifuge to remove insoluble substances, and dry the supernatant.

[0055] (8) Dissolve the sample dried in step (7) in an equal volume of 50% methanol at 60 °C, centrifuge to remove the supernatant, and dry the precipitate.

[0056] (9) Dissolve the dried sample in (8) an equal volume of 90% methanol, let it stand at 4 °C for 24 h and then centrifuge, and dry the precipitate to obtain ginsenoside Rh2 with a purity of 99.1%. Example 6 Large-scale preparation of ginsenoside Rh2

[0057] (1) Use a 10 L reactor to dissolve 50 g of total ginsenosides from ginseng stems and leaves of the diol type in 5 L of buffer solution with a pH of 6.0, and the final concentration of the substrate is 10 mg / mL. According to the content of each component in the substrate, add 0.1 U / mg of CcGlu3A, 0.05 U / mg of MeAra2A, 0.05 U / mg of BsAra51B, and 0.1 U / mg of PsGlu3A, and react with shaking at 30 °C for 2 h.

[0058] (2) Take 500 μL of the above conversion sample, add 500 μL of n-butanol, shake and extract, and then perform thin-layer chromatography to observe whether the conversion is complete.

[0059] (3) Centrifuge the completely converted ginsenoside sample in step (1), dissolve the precipitate in the buffer solution, and inactivate it at 100 °C for 10 min.

[0060] (4) Wait for the ginsenoside sample in step (3) to cool, add 0.5 U / mg of CcGlu1A to the reaction system, and react for 2 h.

[0061] (5) Take 500 μL of the ginsenoside sample in step (4), add 500 μL of n-butanol, shake and extract, and perform thin-layer chromatography to observe whether the conversion is complete.

[0062] (6) Centrifuge the conversion product in step (5), dissolve the precipitate in 95% ethanol. Let it stand at 70 °C for 2 h, centrifuge and take the supernatant. Repeat the above operation, combine the supernatants twice and then dry the sample.

[0063] (7) Take the above dried sample, add 120 mL of methanol to dissolve it, centrifuge to remove the insoluble matter, and dry the supernatant.

[0064] (8) Dissolve the sample dried in step (7) in 120 mL of 50% methanol at 60 °C, centrifuge to remove the supernatant, and dry the precipitate.

[0065] (9) Dissolve the sample dried in (8) in 120 mL of 90% methanol, let it stand at 4 °C for 24 h and then centrifuge, and dry the precipitate to obtain ginsenoside Rh2 with a purity of 98.8%.

Claims

1. A method for preparing high-purity ginsenoside Rh2, the preparation raw material of which is the total ginsenoside of the dammarenediol type obtained by extraction and separation from ginseng roots or ginseng stems and leaves, and the ginsenoside Rh2 is prepared by an enzymatic hydrolysis method combined with a recrystallization method, characterized in that: The combined enzymes used are a specific enzyme combination, and the purity of Rh2 after recrystallization purification is ≥98.5%.

2. The method for preparing high-purity ginsenoside Rh2 according to claim 1, wherein: The combined enzymes are 5 specific types of glycosidases or their isozymes, β-1,6-glucosidase, β-glucosidase that hydrolyzes the inner glucose of ginsenoside, α-arabinopyranosidase, α-arabinofuranosidase, and β-1,2-glucosidase.

3. The method for preparing high-purity ginsenoside Rh2 according to claim 1 or 2, characterized in that: The enzymatic hydrolysis method needs to be completed in 2 steps. In the first step, β-1,6-glucosidase, β-glucosidase that hydrolyzes the inner glucose of ginsenoside, α-arabinopyranosidase, and α-arabinofuranosidase are added. After inactivation, β-1,2-glucosidase is added to complete the reaction.

4. A method for preparing high-purity ginsenoside Rh2 according to claims 1-3, characterized in that: The mass ratio of the combined enzymes to the corresponding components in the substrate is as follows: for β-1,6-glucosidase, 0.1 - 0.5 U / mg; for β-glucosidase that hydrolyzes the inner glucose of ginsenoside, 0.1 - 0.5 U / mg; for α-arabinopyranosidase, 0.05 - 0.5 U / mg; for α-arabinofuranosidase, 0.05 - 0.5 U / mg; for β-1,2-glucosidase, 0.5 - 2.0 U / mg.

5. A method for preparing high-purity ginsenoside Rh2 according to claims 1-4, characterized in that: The reaction system is a buffer solution with pH 6.0 - 8.0, the reaction temperature is 25 - 37°C, and the reaction time is 4 - 24 hours.

6. The method for preparing high-purity ginsenoside Rh2 according to claims 1-5, characterized in that It includes the following operation steps: Step 1: The transformed product is dried, dissolved in methanol, and the mass-to-volume ratio of the product to methanol is 0.1 g:10 mL - 0.5 g:10 mL. Centrifugation is carried out at room temperature and 4000 rpm, and the supernatant is dried. Step 2: The sample obtained in Step 1 is redissolved in an equal volume of 50% methanol at 60°C, centrifuged under the same conditions, and the precipitate is taken and dried. Step 3: The sample obtained in Step 2 is redissolved in an equal volume of 90% methanol, allowed to stand at 4°C for 24 h, centrifuged under the same conditions, and the precipitate is dried to obtain Rh2 with a purity of ≥98.5%.

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