A method for separating and purifying ginsenoside F2 from a fermentation broth
The method of gradient elution using macroporous resin YLT810 and ethanol-water solution solves the safety and cost problems of ginsenoside F2 separation and purification in the prior art, achieving separation effect with high purity and high yield, and is suitable for large-scale production of ginsenoside F2.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHENZHEN SIYOMICRO BIO TECH CO LTD
- Filing Date
- 2024-12-30
- Publication Date
- 2026-07-24
AI Technical Summary
Existing technologies for separating and purifying ginsenoside F2 suffer from problems such as the use of flammable, explosive, and toxic organic solvents, high costs, and difficulty in large-scale production. Furthermore, they are not effective in separating ginsenoside F2 from Rb1 and Rd.
The macroporous resin YLT810 was used for loading and elution, and gradient elution with ethanol-water solution was used to reduce the use of organic solvents. Ginsenoside F2 was separated and purified by 10-65% ethanol-water solution. The loading and elution conditions were optimized to improve purity and yield.
It achieves the separation of ginsenoside F2 with high purity (over 85%) and high yield (over 80%), reduces solvent costs, improves safety and environmental friendliness, and is suitable for industrial production.
Smart Images
Figure CN119751536B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a method for separating and purifying ginsenoside F2 from fermentation broth, belonging to the field of separation and purification technology. Background Technology
[0002] Ginsenosides (GS) are the main active components of ginseng. The mechanisms of action and efficacy of individual ginsenoside monomers differ, making extraction necessary to maximize their direct effect. Ginsenoside F2 (GF2) is a ginsenoside diol, relatively rare among natural ginsenosides. GF2 has positive implications for tumor treatment. Its unique chemical structure allows it to replace steroids in liposome synthesis, improving fluidity, while its two exposed glycosidic bonds can bind to GLUT1, directionally inducing glioma cell death. Furthermore, GF2 has a positive effect on lowering blood sugar, improving insulin resistance, and exhibiting some germinal effects.
[0003] Currently, the main methods for separating ginsenoside monomers include single or combined methods such as silica gel column chromatography, preparative chromatography, resin column chromatography, and crystallization, as shown in Chinese patents:
[0004] CN 102718827 A discloses a method for separating and purifying ginsenoside Rb3. This method uses ginseng stem and leaf saponins as raw materials. After dissolving the raw materials in methanol, silica gel column chromatography is performed using a mixed solvent of dichloromethane and methanol as the eluent, with gradient elution to obtain a crude product with the same Rf value as ginsenoside Rb3. This crude product is then separated again using ODS silica gel as the separating gel, with different gradient elutions of methanol and water, ultimately yielding ginsenoside Rb3 with a liquid chromatography purity of 90.17%. This method utilizes a large amount of volatile, flammable, and highly toxic organic solvents for gradient elution, making it highly unsuitable for large-scale production due to safety and environmental concerns.
[0005] CN 106188208 A discloses a method for the industrial preparation of high-purity ginsenoside Re. This method uses ginseng stem and leaf saponins as raw materials, which are dissolved in methanol, subjected to silica gel column chromatography (1% of silica gel), and eluent is a mixture of chloroform and methanol to obtain a high-content crude ginsenoside RE. This crude product is then dissolved in 20% acetonitrile, and further separated by preparative liquid chromatography. After separation, the product is concentrated to dryness and recrystallized from ethanol to obtain pure ginsenoside RE. This method not only introduces a large amount of organic reagents through silica gel column chromatography but also employs preparative liquid chromatography for further purification, significantly increasing costs.
[0006] Extracting ginsenoside F2 monomer from total saponins is quite difficult because ginsenosides have very similar structures, especially ginsenoside Rb1, which differs from F2 only by one glycoside. They are very similar in structure, and it is generally difficult to separate them effectively using resins. Furthermore, there are few reports on the separation of ginsenoside F2 monomer. Summary of the Invention
[0007] In view of the defects and shortcomings of the existing technology, the purpose of this invention is to provide a method for separating and purifying ginsenoside F2 from fermentation broth. This method can effectively separate ginsenoside F2 from ginsenosides Rb1 and Rd; it can also reduce the use of flammable, explosive and toxic organic solvents, and the solvent cost is low, which can be used for the large-scale production of ginsenoside F2.
[0008] To achieve the above objectives, the following technical solution is provided:
[0009] This invention provides a method for separating and purifying ginsenoside F2 from fermentation broth, the method comprising the following steps:
[0010] (1) Take the fermentation liquid from the tank, centrifuge, remove the precipitate, add ethanol and stir to extract, filter after extraction, and evaporate the filtrate to obtain crude product.
[0011] (2) Dissolve the crude product obtained in step (1) in an ethanol aqueous solution with a volume fraction of 10-40% to form a loading solution; then load the sample onto YLT810 resin. After loading, wash the sample with an ethanol aqueous solution with a volume fraction of 45-50% until the liquid phase purity of the target product reaches 70-75%. Then elute the sample with an ethanol aqueous solution with a volume fraction of 60-65%, collect the eluent, and evaporate the solid to obtain ginsenoside F2.
[0012] In one embodiment, the fermentation broth in step (1) is obtained by fermenting crude ginsenoside powder (Rb1 and Rd account for 50% of the total) with Escherichia coli.
[0013] In one embodiment, the centrifugation conditions in step (1) are: 5000-8000 rpm for 5-10 min.
[0014] In one embodiment, the ethanol in step (1) is anhydrous ethanol, and the amount of ethanol added is in a solid-liquid ratio of 1:2 to 5.
[0015] In one embodiment, the stirring extraction parameters in step (1) are: 100-500 rpm, time 20-40 min, and temperature 40-50℃.
[0016] In one embodiment, the solid-liquid ratio of the crude product in step (2) when dissolved in an ethanol aqueous solution with a volume fraction of 10-40% is 1:80-90, g / ml. Ginsenoside F2 is poorly soluble in water, and resin loading requires complete dissolution before loading. The crude ginsenoside F2 is dissolved in a solvent that is miscible with organic solvent and water. However, the solubility of ginsenoside F2 in low-concentration ethanol aqueous solution is low, and the volume of resin loading solution is large. This not only results in a large amount of low-concentration ethanol with high recovery costs, but also leads to excessively long loading time. When loading with high-concentration ethanol aqueous solution, the macroporous resin is in a swollen state in high-concentration alcohol. Although it has a certain adsorption capacity, the adsorption amount will decrease significantly. Therefore, it is further preferred to dissolve the crude product in an ethanol aqueous solution with a volume fraction of 35%.
[0017] In one embodiment, the ratio of YLT810 resin to crude product in step (2) is 0.3-3:15-120; L / g.
[0018] In one embodiment, the ginsenoside F2 in step (2) has a purity of 85-90% and a yield of over 80%.
[0019] In one embodiment, the ethanol aqueous solution used for washing in step (2) has a volume fraction of 50%.
[0020] In one embodiment, the elution in step (2) uses an ethanol aqueous solution with a volume fraction of 60%.
[0021] This invention also provides the application of the above-described method in the monitoring of industrial production of ginsenoside F2.
[0022] Beneficial effects:
[0023] (1) The method of separating and purifying ginsenoside F2 from fermentation broth of the present invention has advantages such as good operability, high safety, low cost, high yield and high purity compared with the existing methods, which will greatly promote the large-scale production of ginsenoside F2.
[0024] (2) The method for separating and purifying ginsenoside F2 in this invention uses a reusable macroporous resin and avoids the use of highly toxic and flammable organic solvents, making the process safer and more environmentally friendly; the operation is simpler, the purity of ginsenoside F2 reaches more than 85%, and the yield can reach more than 80%, which is suitable for industrial production. Attached Figure Description
[0025] Figure 1 Spectrum of ginsenoside F2 standard;
[0026] Figure 2 This is the liquid chromatography spectrum of the crude product before resin treatment in Example 1;
[0027] Figure 3 This is a liquid chromatogram of the pure product obtained after passing through the resin in Example 1. Detailed Implementation
[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. The specific embodiments described below further illustrate the present invention.
[0029] The fermentation broth used in the embodiments and comparative examples of the present invention is a fermentation broth obtained by fermenting crude ginsenoside powder (Rb1 and Rd account for 50% of the total) with Escherichia coli.
[0030] YLT810 resin, a weakly polar resin, was purchased from Purolite (China) Co., Ltd.
[0031] D101 C resin, a non-polar resin, was purchased from Xi'an Lanxiao Technology Materials Co., Ltd.
[0032] AB-8 resin, a weakly polar resin, was purchased from Purolite (China) Co., Ltd.
[0033] Ginsenoside F2, pure product, with a purity of 98%; Aladdin was purchased.
[0034] Example 1
[0035] A method for separating and purifying ginsenoside F2 from fermentation broth includes the following steps:
[0036] (1) Take 1L of fermentation liquid from the tank. The concentration of ginsenoside F2 in the fermentation liquid is 14g / L. Centrifuge at 7000rpm for 15min, remove the lower layer precipitate, add anhydrous ethanol with a solid-liquid ratio of 1:2 and stir to extract. The stirring speed is 200rpm and the temperature is 50℃. Extract for 30min, then filter, and take the filtrate to dry to obtain crude product. The content of ginsenoside F2 in the crude product is 70%.
[0037] (2) Take 19.14 g of crude product obtained in step (1), add 35% ethanol to dissolve it at a solid-liquid ratio of 1:80 (g / ml), and then load the dissolved solution onto 0.37 L of activated YLT810 resin at a flow rate of 1 column volume per hour. After loading, wash with 50% ethanol until the liquid phase purity of the target product reaches 75% (liquid phase method refers to national standard GB / T41726-2022), elute with 60% ethanol, collect the eluent, and collect the solid after evaporation.
[0038] The final product ginsenoside F2 obtained in this embodiment has a purity of 86.95% and a yield of 82%.
[0039] Example 2
[0040] A method for separating and purifying ginsenoside F2 from fermentation broth includes the following steps:
[0041] (1) Take 6L of fermentation liquid from the tank. The concentration of ginsenoside F2 in the fermentation liquid is 14g / L. Centrifuge at 7000rpm for 15min, remove the lower layer precipitate, add anhydrous ethanol with a solid-liquid ratio of 1:2 and stir to extract. The stirring speed is 200rpm and the temperature is 50℃. Extract for 30min, then filter, and take the filtrate to dry to obtain crude product. The content of ginsenoside F2 in the crude product is 70.2%.
[0042] (2) Take 115.6g of crude product obtained in step (1), add 35% ethanol to dissolve it according to the solid-liquid ratio of 1:80 (g / ml), and then load the solution onto 3L of activated YLT810 resin at a flow rate of 1 column volume per hour. After loading, wash with 50% ethanol until the liquid phase purity of the target product reaches 75%, elute with 60% ethanol, collect the target product, obtain the collected liquid, and collect the solid after evaporation.
[0043] The final product ginsenoside F2 obtained in this embodiment has a purity of 88.34% and a yield of 83%.
[0044] Comparative Example 1
[0045] A method for separating and purifying ginsenoside F2 from fermentation broth includes the following steps:
[0046] (1) Take 3.8L of fermentation liquid from the tank. The concentration of ginsenoside F2 in the fermentation liquid is 14g / L. Centrifuge at 7000rpm for 15min, remove the lower layer precipitate, add anhydrous ethanol with a solid-liquid ratio of 1:2 and stir to extract. The stirring speed is 200rpm and the temperature is 50℃. Extract for 30min, then filter, and take the filtrate to dry to obtain crude product. The content of ginsenoside F2 in the crude product is 72.1%.
[0047] (2) Take 72.81 g of crude product obtained in step (1), add 35% ethanol to dissolve it according to the solid-liquid ratio of 1:80 (g / ml), and then load the solution onto 1 L of activated D101 C resin at a flow rate of 1 column volume per hour. After loading, wash with 40% ethanol and then wash with 50% ethanol until the liquid phase purity of the target product reaches more than 75%. Elute with 60% ethanol, collect the target product, obtain the collected liquid, and collect the solid after evaporation.
[0048] The final product ginsenoside F2 obtained in this comparative example had a purity of 80.34% and a yield of 75.66%.
[0049] Comparative Example 2
[0050] A method for separating and purifying ginsenoside F2 from fermentation broth includes the following steps:
[0051] (1) Take 1L of fermentation liquid from the lower tank. The concentration of ginsenoside F2 in the fermentation liquid is 14g / L. Centrifuge at 7000rpm for 15min, remove the lower layer precipitate, add anhydrous ethanol with a solid-liquid ratio of 1:2 and stir to extract. The stirring speed is 200rpm and the temperature is 50℃. Extract for 30min, then filter, and take the filtrate to dry to obtain crude product. The content of ginsenoside F2 in the crude product is 77.3%.
[0052] (2) Take 17.85g of crude product obtained in step (1), add 35% ethanol to dissolve it at a solid-liquid ratio of 1:80 (g / ml), and load it onto 0.4L of activated AB-8 resin at a flow rate of 1 column volume per hour. After loading, wash with 50% ethanol until the liquid phase purity of the target product reaches more than 75%, elute with 60% ethanol, collect the target product, obtain the collected liquid, and collect the solid after evaporation.
[0053] The final product of this comparative example, ginsenoside F2, had a purity of 81.65% and a yield of 69.6%.
[0054] Comparative Example 3
[0055] A method for separating and purifying ginsenoside F2 from fermentation broth includes the following steps:
[0056] (1) Take 6L of fermentation liquid from the tank. The concentration of ginsenoside F2 in the fermentation liquid is 14g / L. Centrifuge at 7000rpm for 15min, remove the precipitate, add anhydrous ethanol with a solid-liquid ratio of 1:2 and stir to extract. The stirring speed is 200rpm and the temperature is 50℃. Extract for 30min, then filter to obtain the filtrate.
[0057] (2) Add twice the mass of 300-mesh silica gel to the filtrate obtained in step (2), evaporate to a state with good fluidity, and then dry-load the sample onto the pre-packed 300-mesh silica gel column. Wash the sample with dichloromethane containing 5% methanol by volume, and then wash it with dichloromethane containing 10% methanol by volume. At the same time, monitor the sample by TLC spotting (the ratio of developing solvent refers to the national standard GB / T 41726-2022) until the target product appears. Finally, collect and elute the sample with dichloromethane containing 15% methanol by volume to obtain the collected liquid. After evaporation, collect the solid.
[0058] The final product ginsenoside F2 obtained in this comparative example had a purity of 83.88% and a yield of 40%.
[0059] Comparative Example 4
[0060] A method for separating and purifying ginsenoside F2 from fermentation broth includes the following steps:
[0061] (1) Take 1.2L of fermentation liquid from the tank. The concentration of ginsenoside F2 in the fermentation liquid is 14g / L. Centrifuge at 7000rpm for 15min, remove the lower layer precipitate, add anhydrous ethanol with a solid-liquid ratio of 1:2 and stir to extract. The stirring speed is 200rpm and the temperature is 50℃. Extract for 30min, filter, and take the filtrate to dry it to obtain crude product. The content of ginsenoside F2 in the crude product is 72.2%.
[0062] (2) Take 24.93g of crude product obtained in step (1), add 35% ethanol to dissolve it at a solid-liquid ratio of 1:80 (g / ml), and then load the solution onto 0.5L of activated YLT810 resin at a flow rate of 1 column volume per hour. After loading, wash with 40% ethanol until the liquid phase purity of the target product reaches 75%, then elute with 70% ethanol, collect the target product, obtain the collected liquid, and collect the solid after evaporation.
[0063] The final product of this comparative example, ginsenoside F2, had a purity of 78.5% and a yield of 76.5%.
[0064] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.
Claims
1. A method for separating and purifying ginsenoside F2 from fermentation broth, characterized in that, The method includes the following steps: (1) Take the fermentation liquid from the tank, centrifuge, remove the precipitate, add ethanol and stir to extract, filter after extraction, and evaporate the filtrate to obtain crude product. The fermentation broth is obtained by fermenting crude ginsenoside powder with Escherichia coli; wherein, the total proportion of Rb1 and Rd in the crude ginsenoside powder is 50%. (2) Dissolve the crude product obtained in step (1) in an ethanol aqueous solution with a volume fraction of 10-40% to form a loading solution; then load the sample onto YLT810 resin. After loading, wash the sample with an ethanol aqueous solution with a volume fraction of 45-50% until the liquid phase purity of the target product reaches 70-75%. Then elute the sample with an ethanol aqueous solution with a volume fraction of 60-65%, collect the eluent, and evaporate the solid to obtain ginsenoside F2.
2. The method according to claim 1, characterized in that, The centrifugation conditions described in step (1) are: 5000~8000 rpm, time 5~10 min.
3. The method according to claim 1, characterized in that, The stirring extraction parameters in step (1) are: 100~500 rpm, time is 20~40 min, and temperature is 40~50℃.
4. The method according to claim 1, characterized in that, In step (2), the solid-liquid ratio of the crude product dissolved in an ethanol aqueous solution with a volume fraction of 10-40% is 1:80-90, g / ml.
5. The method according to claim 1, characterized in that, In step (2), the ratio of YLT810 resin to crude product is 0.3~3:15~120; L / g.
6. The method according to claim 1, characterized in that, The purity of ginsenoside F2 in step (2) is 85-90%, and the yield is over 80%.
7. The method according to claim 1, characterized in that, The ethanol aqueous solution used for washing impurities in step (2) has a volume fraction of 50%.
8. The method according to claim 1, characterized in that, The elution in step (2) uses an ethanol aqueous solution with a volume fraction of 60%.
9. The application of the method according to any one of claims 1 to 8 in the monitoring of industrial production of ginsenoside F2.