Method for preparing ginsenoside Rg3 and Rg5 by converting ginsenoside Rb1
By using a low eutectic solvent-buffered salt system in the conversion reaction of ginsenoside Rb1, ginsenoside Rg3 and Rg5 are efficiently prepared, which solves the problems of low yield of extracting Rg3 and Rg5 in the prior art and low utilization rate of raw medicinal materials. A high purity, high yield and environmentally friendly preparation process is achieved.
Patent Information
- Application Number
- CN202411357934.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-27
- Publication Date
- 2025-01-03
AI Technical Summary
When extracting ginseng saponins Rg3 and Rg5, the existing methods have problems such as low utilization rate of raw medicinal materials, low product yield, and the need for a large number of microbial bacterial species/biological enzymes in large-scale production.
The method of efficiently preparing ginsenoside Rg3 and Rg5 by the conversion reaction of ginsenoside Rb1 includes heating ginsenoside Rb1 in the reaction medium for conversion reaction, followed by cooling and separation. A high purity mixture of Rg3 and Rg5 was obtained and a single component was separated by a column.
It realizes an efficient and simple preparation process, with high yield and high purity, convenient product purification, and low raw material cost, and green and environmentally friendly reaction and purification process.
Smart Images
Figure CN119241627A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biomedicine, and particularly relates to a method for preparing ginsenosides Rg3 and Rg5 by transforming ginsenoside Rb1. Background Art
[0002] The active ingredients of the natural product Panax Ginseng are ginsenosides. The results of modern pharmacological research show that there are as many as 220 kinds of ginsenosides. The constant components of ginsenosides are Rb1, Rb2, Rc, Rd, Rg1, and Re, which account for more than 80-90% of the total saponins, but these constant components often have no or low anti-cancer activity. Rare components of ginsenosides such as ginsenosides Rh1, Rh2, Rg3, Rg5, and Rg6 are increasingly attracting attention due to their significant anti-tumor effects. The extremely rare ginsenoside Rg5 is generally found in red ginseng, which has the pharmacological effects of inhibiting tumor cell growth and anti-liver cancer, gastric cancer, esophageal cancer, and cervical cancer. In addition, ginsenoside Rg5 also has anti-allergic, anti-inflammatory, anti-memory impairment, anti-depression, diabetes prevention, and whitening effects. Conventional methods such as acid hydrolysis, alkaline hydrolysis or direct extraction of Rg3 and Rg5 from red ginseng have problems such as low utilization of raw medicinal materials and low product yield. At present, the more direct method to obtain rare ginsenosides is microbial or enzyme conversion. Compared with chemical conversion, microbial / enzyme conversion conditions are mild, the reaction cycle is short, the specificity is strong, and the reaction does not require a large amount of chemical reagents. However, the preparation of microorganisms / enzymes requires specific reaction hydrolases, and the product of the reaction depends on the activity of the microorganism or enzyme. If large-scale production is required, a large amount of microbial strains / biological enzymes need to be cultivated and consumed. Summary of the invention
[0003] The purpose of the present invention is to solve the problems existing in the existing methods for obtaining and extracting ginsenosides Rg3 and Rg5, and to provide a method for preparing ginsenosides Rg3 and Rg5 by converting ginsenoside Rb1. The method uses a low eutectic solvent-buffered salt as a reaction solvent, has a simple preparation process and downstream processing process, a high product conversion yield, and high purity, and can efficiently prepare a mixture of rare ginsenosides Rg3 and Rg5 and can further separate and obtain single components of ginsenosides Rg3 and Rg5.
[0004] A method for converting ginsenoside Rb1 to prepare ginsenoside Rg3 and Rg5, wherein ginsenoside Rb1 is subjected to a conversion reaction in a reaction solvent to generate ginsenoside Rg3 and Rg5.
[0005] Furthermore, the method for converting the above-mentioned ginsenoside Rb1 into ginsenosides Rg3 and Rg5 comprises the following steps:
[0006] 1) adding the crude extract of ginsenoside Rb1 into a reaction solvent to dissolve it, wherein the mass volume ratio of the crude extract of ginsenoside Rb1 to the reaction solvent is 1-20 mg / ml;
[0007] 2) heating the mixed solution to carry out a conversion reaction;
[0008] 3) After the reaction is completed, the reaction solution is cooled to room temperature and separated to obtain a mixture of ginsenosides Rg3 and Rg5.
[0009] Furthermore, the method for converting the above-mentioned ginsenoside Rb1 into ginsenosides Rg3 and Rg5 comprises the following steps:
[0010] 1) adding the crude extract of ginsenoside Rb1 into a reaction solvent to dissolve it, wherein the mass volume ratio of the crude extract of ginsenoside Rb1 to the reaction solvent is 1-20 mg / ml;
[0011] 2) heating the mixed solution to carry out a conversion reaction;
[0012] 3) After the reaction is completed, the reaction solution is cooled to room temperature and separated to obtain a mixture of ginsenosides Rg3 and Rg5;
[0013] 4) Separating the mixture of ginsenoside Rg3 and Rg5 to obtain single components of ginsenoside Rg3 and ginsenoside Rg5.
[0014] Furthermore, in the above-mentioned method for converting ginsenoside Rb1 to prepare ginsenoside Rg3 and Rg5, in the step 1), the reaction solvent is a deep eutectic solvent (DES)-acidic buffer solution.
[0015] Furthermore, in the method for converting ginsenoside Rb1 to prepare ginsenoside Rg3 and Rg5, the DES uses choline chloride as a hydrogen bond acceptor and alcohols, urea or a mixture of alcohols and urea as a donor. Preferably, the alcohol is ethylene glycol, propylene glycol or glycerol. The acidic buffer solution is a buffer solution with a pH of 3 to 5.
[0016] Furthermore, in the above-mentioned method for converting ginsenoside Rb1 into ginsenoside Rg3 and Rg5, the mass percentage of DES in the reaction solvent is 1-20wt%.
[0017] Furthermore, in the method for converting ginsenoside Rb1 to prepare ginsenoside Rg3 and Rg5, in step 2), first, the mixed solution is heated to 30-50° C. and reacted for 40-60 min; then, the mixture is heated to 80-85° C. and reacted for 10-30 min;
[0018] Furthermore, in the above method for converting ginsenoside Rb1 into ginsenoside Rg3 and Rg5, in step 3),
[0019] The separation method is:
[0020] Method 1:
[0021] A. Filter the cooled reaction solution with a filter membrane to collect the flocculent precipitate and the supernatant;
[0022] B. Washing the flocculent precipitate with water to remove the salt component in the reaction solution, and then drying the washed precipitate;
[0023] C. dialyzing the supernatant in a dialysis membrane to remove salt components in the supernatant, and then drying the dialyzed supernatant;
[0024] D. Collect the products obtained by drying and drying in steps B and C, which are a mixture of ginsenosides Rg3 and Rg5;
[0025] Or, method 2:
[0026] A. Transferring all the cooled reaction solution to a dialysis membrane for dialysis to remove the salt component in the reaction solution;
[0027] B. The dialyzed reaction solution is filtered to obtain a precipitate and a filtrate, the precipitate and the filtrate are dried separately, and the dried product is collected, which is a mixture of ginsenosides Rg3 and Rg5;
[0028] Or, method three:
[0029] A. Filter the cooled reaction solution with a filter membrane to collect the flocculent precipitate and the supernatant;
[0030] B. Washing the flocculent precipitate with water to remove the salt component in the reaction solution, and then drying the washed precipitate;
[0031] C. The supernatant is adsorbed and desalted by an ion exchange resin column, and then the ginsenosides Rg3 and Rg5 are eluted with an organic solvent, and the eluate is subjected to the removal of the organic solvent and drying; preferably, the supernatant is adsorbed on an ion exchange resin column for a period of time, and then the salt components in the supernatant are removed by deionized water, and then the adsorbed ginsenosides Rg3 and Rg5 are eluted with an organic solvent such as methanol, and the eluate is subjected to the removal of the organic solvent and drying to obtain a mixture of ginsenosides Rg3 and Rg5;
[0032] D. Collect the products obtained by drying and drying in steps B and C, which are a mixture of ginsenosides Rg3 and Rg5.
[0033] Furthermore, in the above method for converting ginsenoside Rb1 to prepare ginsenoside Rg3 and Rg5, step 4) separating the mixture of ginsenoside Rg3 and Rg5 to obtain ginsenoside Rg3 and ginsenoside Rg5 respectively is as follows:
[0034] Method 1: extract the mixture of ginsenosides Rg3 and Rg5 obtained in step 3) with an organic solvent, and pass the extract through a sulfonic acid column or C 18 The chromatographic column was used for gradient elution separation to obtain ginsenoside Rg5 and R-type ginsenoside Rg3 respectively;
[0035] Alternatively, method 2, take the dried precipitate obtained in step B of the separation treatment method 1, 2 or 3, extract it with an organic solvent, and pass the extract through a sulfonic acid chromatography column or C 18 The single component ginsenoside Rg5 and R-type ginsenoside Rg3 were obtained by gradient elution separation of the chromatographic column;
[0036] Alternatively, method 3, taking the dialyzed supernatant of step C in method 1, the filtrate of step B in method 2, or the eluate of step C in method 3, and passing it through a sulfonic acid chromatographic column or C 18 The single components of ginsenoside Rg5 and S-type ginsenoside Rg3 were separated by gradient elution on the chromatographic column.
[0037] Furthermore, in the above-mentioned method for converting ginsenoside Rb1 to prepare ginsenoside Rg3 and Rg5, the filter membrane specification is 0.45-10 microns.
[0038] Compared with the prior art, the advantages of the present invention are:
[0039] 1. The product obtained by the present invention is single, has high yield and is easy to purify.
[0040] 2. The method of the present invention is simple, the raw material cost is low and the product value is high.
[0041] 3. The reaction and purification process of the product of the present invention is green and environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS
[0042] Figure 1 , schematic diagram of the reaction pathway for the conversion of ginsenoside Rb1 to Rg3 and Rg5;
[0043] Figure 2 , HPLC analysis of the mixture of ginsenosides Rg3 and Rg5 prepared by the method;
[0044] In the figure, (a) is the chromatogram of the reference substance of ginsenoside Rg3; (b) is the chromatogram of the reference substance of ginsenoside Rg5; (c) is the chromatogram of this product;
[0045] Figure 3 , mass spectrometry analysis of the product fractions ginsenosides Rg3 and Rg5 separated by the method;
[0046] In the figure, (a) and (b) are the product and reference quality spectra of ginsenoside Rg3, respectively; (c) and (d) are the product and reference quality spectra of ginsenoside Rg5, respectively. DETAILED DESCRIPTION
[0047] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the protection scope of the present invention.
[0048] In order to further understand the present invention, the present invention is described in detail below in conjunction with the examples. Wherein, unless otherwise specified, the various reaction reagents involved in the examples can be obtained through commercial purchase.
[0049] Example 1
[0050] A method for converting ginsenoside Rb1 to prepare ginsenoside Rg3 and Rg5, the reaction path is shown in Figure 1 :
[0051] In 5 ml of a 10 wt% choline chloride-ethylene glycol-sodium dihydrogen phosphate and citric acid buffer solution reaction medium at pH 3, add 17.5 mg of 90 wt% ginsenoside Rb1 crude extract, ultrasonically vibrate for 5 minutes to dissolve, then place in a constant temperature box at 40 ° C for 1 hour, and then react on a heater at 80 ° C for 20 minutes. After the reaction is completed, cool, filter the precipitate, wash the precipitate with water 3-5 times, and evaporate the water to constant weight in an 80 ° C oven. The filtrate is dialyzed with a dialysis membrane for 2-3 days, and the water is changed every 2-3 hours. Finally, the solution in the dialysis membrane is extracted and placed in a beaker, placed in an oven to evaporate the water to constant weight, and the product is collected. The total yield of the two parts of ginsenoside Rg3 and Rg5 is 80%, and the purity of the two is greater than 95%.
[0052] Example 2
[0053] A method for converting ginsenoside Rb1 into ginsenoside Rg3 and Rg5:
[0054] In 5 ml of a 15 wt% choline chloride-ethylene glycol-sodium dihydrogen phosphate and citric acid buffer solution reaction medium at pH 4, add 25 mg of 80 wt% ginsenoside Rb1 crude extract, ultrasonically vibrate for 5 minutes to dissolve, and then place in a constant temperature box to react for 1 hour at 40 ° C, and then react on a heater at 80 ° C for 20 minutes. After the reaction is completed, cool, filter the precipitate, wash the precipitate with water 3-5 times, and evaporate the water to constant weight in an oven at 80 ° C. The filtrate is dialyzed with a dialysis membrane for 2-3 days, and the water is changed every 2-3 hours. Finally, the solution in the dialysis membrane is extracted and placed in a beaker, placed in an oven to evaporate the water to constant weight, and the product is collected. The total yield of the two parts of ginsenoside Rg3 and Rg5 is 66%, and the purity of the two is greater than 95%.
[0055] Example 3
[0056] A method for converting ginsenoside Rb1 into ginsenoside Rg3 and Rg5:
[0057] In 5 ml of a 15 wt% choline chloride-urea-sodium dihydrogen phosphate and citric acid buffer solution at pH 4, add 17.5 mg of 80 wt% ginsenoside Rb1 crude extract, ultrasonically vibrate for 5 minutes to dissolve, and then place in a constant temperature box to react for 2 hours at 40 ° C, and then react on a heater at 80 ° C for 20 minutes. After the reaction is completed, cool, filter the precipitate, wash the precipitate with water 3-5 times, and evaporate the water to constant weight in an 80 ° C oven. The filtrate is dialyzed with a dialysis membrane for 2-3 days, and the water is changed every 2-3 hours. Finally, the solution in the dialysis membrane is extracted and placed in a beaker, placed in an oven to evaporate the water to constant weight, and the product is collected. The total yield of the two parts of ginsenoside Rg3 and Rg5 is 50%, and the purity of the two is greater than 75%.
[0058] Example 4
[0059] A method for converting ginsenoside Rb1 into ginsenoside Rg3 and Rg5:
[0060] Weigh 10 mg of the dried precipitate in Example 1, extract it three times with 0.5 ml of methanol, combine the extracts, and separate it in a sulfonic acid chromatographic column with a particle size of 5 μm, an inner diameter of 4.6 mm, and a length of 250 mm. The mobile phases are acetonitrile-A and water-B, and the gradient elution conditions are: 0-4 min, 93% A, 4-8 min, 87% A; 8-12 min, 70% A; the flow rate is 1.2 ml / min, the detection wavelength is 203 nm, and the injection volume is 20 μl; collect ginsenoside Rg3 and Rg5 fractions multiple times to obtain R-type ginsenosides Rg3 and Rg5 with yields of 35% and 30%, respectively, and purities of 92% and 93%, respectively.
[0061] Example 5
[0062] A method for converting ginsenoside Rb1 into ginsenoside Rg3 and Rg5:
[0063] 2 ml of the filtrate after dialyzed in Example 1 was transferred and separated on a sulfonic acid chromatographic column with a particle size of 5 μm, an inner diameter of 4.6 mm, and a length of 250 mm. The mobile phases were acetonitrile-A and water-B. The gradient elution conditions were: 0-4 min, 93%, 4-8 min, 87%; 8-12 min, 70%; the flow rate was 1.2 ml / min, the detection wavelength was 203 nm, and the injection volume was 20 μl. The fractions of ginsenoside Rg3 and Rg5 were collected separately to obtain S-type ginsenoside Rg3 and Rg5 with yields of 50% and 45%, respectively, and purities of 92% and 90%, respectively.
[0064] Example 6
[0065] A method for converting ginsenoside Rb1 into ginsenoside Rg3 and Rg5:
[0066] Weigh 10 mg of the dried precipitate from Example 1, extract it three times with 0.5 ml of methanol, combine the extracts, and place it on a C 18 Separation was carried out in a chromatographic column with acetonitrile-A and water-B as the mobile phases and gradient elution conditions as follows: 0-13 min, 23% A; 13-32 min, 46% A; 32-45 min, 68% A; 45-55 min; 100% A; the flow rate was 1.0 ml / min, the detection wavelength was 203 nm, and the injection volume was 20 μl; the ginsenoside Rg3 and Rg5 fractions were collected multiple times to obtain R-type ginsenosides Rg3 and Rg5 with yields of 32% and 30% and purities of 91% and 92%, respectively.
[0067] Example 7
[0068] A method for converting ginsenoside Rb1 into ginsenoside Rg3 and Rg5:
[0069] 2 ml of the filtrate after dialysis in Example 1 was taken and placed in a C 18 Separation was carried out in a chromatographic column with acetonitrile-A and water-B as the mobile phases and gradient elution conditions as follows: 0-13 min, 23% A; 13-32 min, 46% A; 32-45 min, 68% A; 45-55 min; 100% A; the flow rate was 1.0 ml / min, the detection wavelength was 203 nm, and the injection volume was 20 μl; the fractions of ginsenoside Rg3 and Rg5 were collected separately to obtain S-type ginsenoside Rg3 and Rg5 with yields of 52% and 47% and purities of 93% and 92%, respectively.
Claims
1. A method for converting ginsenoside Rb1 into ginsenoside Rg3 and Rg5, characterized in that: Ginsenoside Rb1 is converted into ginsenoside Rg3 and Rg5 in a reaction solvent.
2. The method for preparing ginsenoside Rg3 and Rg5 by converting ginsenoside Rb1 according to claim 1, characterized in that: The steps include: 1) adding the crude extract of ginsenoside Rb1 into a reaction solvent to dissolve it, wherein the mass volume ratio of the crude extract of ginsenoside Rb1 to the reaction solvent is 1-20 mg / ml; 2) heating the mixed solution to carry out a conversion reaction; 3) After the reaction is completed, the reaction solution is cooled to room temperature and separated to obtain a mixture of ginsenosides Rg3 and Rg5.
3. The method for converting ginsenoside Rb1 into ginsenoside Rg3 and Rg5 according to claim 1, characterized in that: The steps include: 1) adding the crude extract of ginsenoside Rb1 into a reaction solvent to dissolve it, wherein the mass volume ratio of the crude extract of ginsenoside Rb1 to the reaction solvent is 1-20 mg / ml; 2) heating the mixed solution to carry out a conversion reaction; 3) After the reaction is completed, the reaction solution is cooled to room temperature and separated to obtain a mixture of ginsenosides Rg3 and Rg5; 4) separating the mixture of ginsenoside Rg3 and ginsenoside Rg5 to obtain ginsenoside Rg3 and ginsenoside Rg5, respectively.
4. The method for converting ginsenoside Rb1 into ginsenoside Rg3 and Rg5 according to any one of claims 1 to 3, characterized in that: The reaction medium was DES-buffered solution.
5. The method for converting ginsenoside Rb1 into ginsenoside Rg3 and Rg5 according to claim 4, characterized in that: The DES uses choline chloride as a hydrogen bond acceptor and alcohols, urea or a mixture of alcohols and urea as a donor; the buffer solution is a buffer solution with a pH of 3 to 5.
6. The method for converting ginsenoside Rb1 into ginsenoside Rg3 and Rg5 according to claim 4, characterized in that: The mass percentage of DES in the reaction medium is 1-20wt%.
7. The method for converting ginsenoside Rb1 into ginsenoside Rg3 and Rg5 according to claim 2 or 3, characterized in that: In the step 2), first, the mixed solution is heated to 30-50° C. and reacted for 40-60 min; then, the temperature is raised to 80-85° C. and reacted for 10-30 min.
8. The method for converting ginsenoside Rb1 into ginsenoside Rg3 and Rg5 according to claim 2 or 3, characterized in that: The separation method in step 3) is: Method 1: A. Filter the cooled reaction solution with a filter membrane to collect the flocculent precipitate and the supernatant; B. Washing the flocculent precipitate with water to remove the salt component in the reaction solution, and then drying the washed precipitate; C. dialyzing the supernatant in a dialysis membrane to remove salt components in the supernatant, and then drying the dialyzed supernatant; D. Collect the products obtained by drying and drying in steps B and C, which are a mixture of ginsenosides Rg3 and Rg5; Or, method 2: A. Transferring all the cooled reaction solution to a dialysis membrane for dialysis to remove the salt component in the reaction solution; B. The dialyzed reaction solution is filtered to obtain a precipitate and a filtrate, the precipitate and the filtrate are dried separately, and the dried product is collected, which is a mixture of ginsenosides Rg3 and Rg5; Or, method three: A. Filter the cooled reaction solution with a filter membrane to collect the flocculent precipitate and the supernatant; B. Washing the flocculent precipitate with water to remove the salt component in the reaction solution, and then drying the washed precipitate; C. The supernatant is adsorbed and desalted by an ion exchange resin column, and then ginsenosides Rg3 and Rg5 are eluted with an organic solvent, and the eluate is subjected to removal of the organic solvent and drying; D. Collect the products obtained by drying and drying in steps B and C, which are a mixture of ginsenosides Rg3 and Rg5.
9. The method for converting ginsenoside Rb1 into ginsenoside Rg3 and Rg5 according to claim 8, characterized in that: Step 4) Separating the mixture of ginsenoside Rg3 and Rg5 to obtain ginsenoside Rg3 and ginsenoside Rg5 respectively is as follows: Method 1: extract the mixture of ginsenosides Rg3 and Rg5 obtained in step 3) with an organic solvent, and pass the extract through a sulfonic acid column or C 18 The chromatographic column was used for gradient elution separation to obtain ginsenoside Rg5 and R-type ginsenoside Rg3 respectively; Alternatively, method 2, take the dried precipitate obtained in step B of the separation treatment method 1, 2 or 3, extract it with an organic solvent, and pass the extract through a sulfonic acid chromatography column or C 18 The single component ginsenoside Rg5 and R-type ginsenoside Rg3 were obtained by gradient elution separation of the chromatographic column; Alternatively, method 3, taking the dialyzed supernatant of step C in method 1, the filtrate of step B in method 2, or the eluate of step C in method 3, and passing it through a sulfonic acid chromatographic column or C 18 The single component ginsenoside Rg5 and S-type ginsenoside Rg3 were separated by gradient elution on the chromatographic column.
10. The method for converting ginsenoside Rb1 into ginsenoside Rg3 and Rg5 according to claim 8, characterized in that: In the separation treatment method, the filter membrane specification is 0.45-10 microns.