Method for separating and purifying ginsenoside F2 from fermentation liquor

Abstract

The invention discloses a method for separating and purifying ginsenoside F2 from fermentation liquor, and belongs to the technical field of separation and purification. The method for separating and purifying ginsenoside F2 comprises the following steps: taking fermentation liquor in a tank, centrifuging, taking lower-layer precipitate, adding ethanol, stirring and extracting, filtering after extraction, taking filtrate, and spin-drying to prepare a crude product; dissolving the crude product with an ethanol aqueous solution with the volume fraction of 10-40% to form a sample loading solution; the method comprises the following steps: taking ginsenoside F2 as a raw material, then loading a sample on YLT810 resin, washing impurities by using an ethanol aqueous solution with the volume fraction of 45-50% after the sample loading is finished, eluting by using an ethanol aqueous solution with the volume fraction of 60-65% when the liquid phase purity of a target product reaches 70-75%, collecting an eluent, spin-drying and collecting a solid, thereby obtaining the ginsenoside F2. The purity of ginsenoside F2 obtained through the method reaches 85% or above, the yield can reach 80% or above, and the method is suitable for industrial production.

Description

Technical Field

[0001] The invention relates to a method for separating and purifying ginsenoside F2 from fermentation broth, belonging to the technical field of separation and purification. Background Art

[0002] Ginsenoside (GS) is the main active ingredient of ginseng. The mechanism of action and efficacy of ginsenoside monomers are different. In order to maximize and directly exert their effects, it is necessary to extract the monomers. Ginsenoside F2 (GF2) is a ginsenoside diol. It is present in a relatively low content in natural ginsenosides and is a rare ginsenoside. GF2 has a positive significance in the treatment of tumors. Its unique chemical structure allows it to replace steroids for liposome synthesis to improve its fluidity. At the same time, its two exposed glycosidic bonds can also bind to GLUT1 and induce glioma cell death in a targeted manner. In addition, GF2 also has a positive effect on lowering blood sugar, can improve insulin resistance, and has a certain hair growth effect.

[0003] At present, the main methods for separating ginsenoside monomers are mainly divided into single or combined methods such as silica gel column chromatography, preparative chromatography, resin column chromatography, crystallization, etc., such as the Chinese patent:

[0004] CN 102718827 A discloses a method for separating and purifying ginsenoside Rb3, which uses ginsenosides from ginseng stems and leaves as raw materials, dissolves the raw materials with methanol, and then performs silica gel column chromatography, wherein the eluent is a mixed solvent of dichloromethane and methanol, and gradient elution is performed to obtain a crude product with the same Rf value as ginsenoside Rb3, and then separates again using ODS silica gel as a separation gel, and elutes with methanol and water at different gradients, and finally obtains ginsenoside Rb3 with a liquid phase purity of 90.17%. The method utilizes a large amount of volatile, flammable, and highly toxic organic solvents for gradient elution, and is very unsuitable for large-scale production in terms of safety and environmental protection.

[0005] CN 106188208 A discloses a method for industrial preparation of high-purity ginsenoside RE, which uses ginsenosides from ginseng stems and leaves as raw materials, dissolves them in methanol, and performs silica gel column chromatography, with the sample loading amount being 1% of the silica gel, and the eluent being a mixed solvent of chloroform and methanol, to obtain a high-content crude product of ginsenoside RE, which is dissolved in 20% acetonitrile, and then separated from ginsenoside RE again using a preparation liquid phase, concentrated to dryness after separation, and recrystallized from ethanol to obtain pure ginsenoside RE. This method not only uses silica gel column chromatography to introduce a large amount of organic reagents, but also uses a preparation liquid phase for refining in the further purification process, which greatly increases the cost.

[0006] It is relatively difficult to extract ginsenoside F2 monomer from total saponins because the structures of ginsenosides are very similar, especially for ginsenoside Rb1, which differs from F2 only by a glycoside and is very similar in structure. It is generally difficult to effectively separate them using resins, and 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 prior art, the purpose of the present invention is to provide a method for separating and purifying ginsenoside F2 from fermentation broth, which can effectively separate ginsenoside F2 from ginsenosides Rb1 and Rd; and can also reduce the use of flammable, explosive and toxic organic solvents, and the cost of using solvents is low, so it can be used for large-scale production of ginsenoside F2.

[0008] In order to achieve the above purpose, the technical solutions provided are as follows:

[0009] The present invention provides a method for separating and purifying ginsenoside F2 from a fermentation broth, the method comprising the following steps:

[0010] (1) taking out the fermentation liquid from the tank, centrifuging, taking out the lower layer of precipitate, adding ethanol to stir and extract, filtering after extraction, and taking the filtrate and spinning it to dryness to prepare a crude product;

[0011] (2) dissolving the crude product obtained in step (1) with an ethanol aqueous solution having a volume fraction of 10 to 40% to form a loading solution; then loading the solution onto YLT810 resin; after loading, washing the solution with an ethanol aqueous solution having a volume fraction of 45 to 50% until the target product has a liquid phase purity of 70 to 75%; eluting the solution with an ethanol aqueous solution having a volume fraction of 60 to 65%, collecting the eluate, and collecting the solid by spin drying to obtain ginsenoside F2.

[0012] In one embodiment, the fermentation broth in step (1) is obtained by fermenting ginsenoside crude powder (Rb1 and Rd account for 50% in total) with Escherichia coli.

[0013] In one embodiment, the centrifugation conditions in step (1) are: 5000-8000 rpm, time 5-10 min.

[0014] In one embodiment, the ethanol in step (1) is anhydrous ethanol, and the amount of ethanol added is added in a solid-liquid ratio of 1:2 to 5.

[0015] In one embodiment, the stirring extraction parameters of step (1) are: 100-500 rpm, time is 20-40 min, and temperature is 40-50°C.

[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 to 40% is 1:80 to 90, g / ml; ginsenoside F2 is insoluble in water, and the resin needs to be completely dissolved before loading. The crude ginsenoside F2 is dissolved in an organic solvent and a water-miscible solvent, but the solubility of ginsenoside F2 in a low-concentration ethanol aqueous solution is low, and the volume of the resin loading liquid is large, which will not only cause a large amount of low-concentration ethanol with high recovery cost, but also a large amount of loading liquid volume will cause the loading time to be too long; when loading with a high-concentration ethanol aqueous solution, the macroporous resin is in a swollen state in the high-concentration alcohol, and although it has a certain adsorption capacity, the adsorption amount will be greatly reduced; therefore, it is further preferred that the crude product is dissolved in an ethanol aqueous solution with a volume fraction of 35%.

[0017] In one embodiment, the ratio of the YLT810 resin to the crude product in step (2) is 0.3-3:15-120; L / g.

[0018] In one embodiment, the purity of ginsenoside F2 in step (2) is 85-90%, and the yield is above 80%.

[0019] In one embodiment, the volume fraction of the ethanol aqueous solution used for washing in step (2) is 50%.

[0020] In one embodiment, the volume fraction of the ethanol aqueous solution used for elution in step (2) is 60%.

[0021] The present invention also provides application of the above-mentioned method in industrial production monitoring of ginsenoside F2.

[0022] Beneficial effects:

[0023] (1) Compared with the existing methods, the method of separating and purifying ginsenoside F2 from the fermentation broth of the present invention has the advantages of good operability, high safety, low cost, high yield and high purity, etc., which will greatly promote the large-scale production of ginsenoside F2.

[0024] (2) The method for separating and purifying ginsenoside F2 of the present invention adopts 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the spectrum of the standard product of ginsenoside F2;

[0026] Figure 2 This is the liquid phase spectrum of the crude product before passing through the resin 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 DESCRIPTION

[0028] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The following specific implementation methods further describe the present invention.

[0029] The fermentation liquid used in the examples and comparative examples of the present invention is: a fermentation liquid obtained by fermenting ginsenoside crude powder (Rb1 and Rd account for 50% in total) with Escherichia coli.

[0030] YLT810 resin, a weakly polar resin, was purchased from Purolite (China) Co., Ltd.;

[0031] D101 C resin, non-polar resin, 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] Pure ginsenoside F2, purity 98%; purchased from Aladdin.

[0034] Example 1

[0035] A method for separating and purifying ginsenoside F2 from a fermentation broth comprises the following steps:

[0036] (1) taking 1 L of fermentation liquid from the tank, the concentration of ginsenoside F2 in the fermentation liquid was 14 g / L, centrifuging at 7000 rpm for 15 min, taking the lower precipitate, adding anhydrous ethanol with a solid-liquid ratio of 1:2 for stirring extraction, the stirring speed was 200 rpm, the temperature was 50° C., the extraction was performed for 30 min, and then filtering, taking the filtrate and spin-drying to obtain a crude product, the content of ginsenoside F2 in the crude product was 70%;

[0037] (2) 19.14 g of the crude product obtained in step (1) was dissolved by adding 35% ethanol at a solid-liquid ratio of 1:80 (g / ml), and then the dissolved solution was loaded on 0.37 L of activated YLT810 resin at a flow rate of 1 column volume per hour; after loading, 50% ethanol was used for washing until the liquid phase purity of the target product reached 75% (liquid phase method refers to national standard GB / T41726-2022), 60% ethanol was used for elution, and the eluate was collected to obtain a collected liquid, which was spin-dried and the solid was collected.

[0038] The final product ginsenoside F2 obtained in this example has a purity of 86.95% and a yield of 82%.

[0039] Example 2

[0040] A method for separating and purifying ginsenoside F2 from a fermentation broth comprises the following steps:

[0041] (1) 6 L of fermentation liquid from the tank was taken out, the concentration of ginsenoside F2 in the fermentation liquid was 14 g / L, and the mixture was centrifuged at 7000 rpm for 15 min. The lower precipitate was taken out, and anhydrous ethanol with a solid-liquid ratio of 1:2 was added for stirring extraction. The stirring speed was 200 rpm, the temperature was 50° C., and the extraction was performed for 30 min. The mixture was then filtered, and the filtrate was spin-dried to obtain a crude product. The content of ginsenoside F2 in the crude product was 70.2%;

[0042] (2) 115.6 g of the crude product obtained in step (1) was dissolved by adding 35% ethanol at a solid-liquid ratio of 1:80 (g / ml), and then the solution was loaded on 3 L of activated YLT810 resin at a flow rate of 1 column volume per hour. After loading, the mixture was washed with 50% ethanol until the liquid phase purity of the target product reached 75%, and eluted with 60% ethanol to collect the target product to obtain a collected liquid, which was then spin-dried and the solid was collected.

[0043] The purity of the final product ginsenoside F2 obtained in this example is 88.34%, and the yield is 83%.

[0044] Comparative Example 1

[0045] A method for separating and purifying ginsenoside F2 from a fermentation broth comprises the following steps:

[0046] (1) 3.8 L of fermentation liquid from the tank was taken out, the concentration of ginsenoside F2 in the fermentation liquid was 14 g / L, and the mixture was centrifuged at 7000 rpm for 15 min. The lower precipitate was taken out, and anhydrous ethanol with a solid-liquid ratio of 1:2 was added for stirring extraction. The stirring speed was 200 rpm, the temperature was 50° C., and the extraction was performed for 30 min. The mixture was then filtered, and the filtrate was spin-dried to obtain a crude product. The content of ginsenoside F2 in the crude product was 72.1%;

[0047] (2) Take 72.81 g of the crude product obtained in step (1), add 35% ethanol at a solid-liquid ratio of 1:80 (g / ml) to dissolve it, and then load the solution on 1 L of activated D101 C resin at a flow rate of 1 column volume per hour. After the loading is completed, wash with 40% ethanol, then wash with 50% 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, spin dry and collect the solid.

[0048] The purity of the final product ginsenoside F2 obtained in this comparative example is 80.34%, and the yield is 75.66%.

[0049] Comparative Example 2

[0050] A method for separating and purifying ginsenoside F2 from a fermentation broth comprises the following steps:

[0051] (1) taking 1 L of fermentation liquid from the tank, the concentration of ginsenoside F2 in the fermentation liquid was 14 g / L, centrifuging at 7000 rpm for 15 min, taking the lower precipitate, adding anhydrous ethanol with a solid-liquid ratio of 1:2 for stirring extraction, the stirring speed was 200 rpm, the temperature was 50° C., the extraction was performed for 30 min, and then filtering, taking the filtrate and spin-drying to obtain a crude product, the content of ginsenoside F2 in the crude product was 77.3%;

[0052] (2) 17.85 g of the crude product obtained in step (1) was dissolved by adding 35% ethanol at a solid-liquid ratio of 1:80 (g / ml), and loaded onto 0.4 L of activated AB-8 resin at a flow rate of 1 column volume per hour; after loading, the mixture was washed with 50% ethanol until the liquid phase purity of the target product reached more than 75%, and eluted with 60% ethanol to collect the target product to obtain a collected liquid, which was then spin-dried to collect the solid.

[0053] The purity of the final product ginsenoside F2 of this comparative example is 81.65%, and the yield is 69.6%.

[0054] Comparative Example 3

[0055] A method for separating and purifying ginsenoside F2 from a fermentation broth comprises the following steps:

[0056] (1) 6 L of fermentation liquid from the tank was taken out, the concentration of ginsenoside F2 in the fermentation liquid was 14 g / L, and the mixture was centrifuged at 7000 rpm for 15 min. The precipitate was taken out and anhydrous ethanol with a solid-liquid ratio of 1:2 was added for stirring extraction. The stirring speed was 200 rpm and the temperature was 50° C. The extraction was performed for 30 min, and then filtered to obtain a filtrate;

[0057] (2) The filtrate obtained in step (2) is added with twice the mass of 300 mesh silica gel, and the mixture is spin-dried to a state with good fluidity, and then dry-loaded on a loaded 300 mesh silica gel column, washed with dichloromethane containing 5% methanol by volume, and then washed with dichloromethane containing 10% methanol by volume, and at the same time, a TLC spot plate (the proportion of the developing solvent refers to the national standard GB / T 41726-2022) is used for monitoring until the target product appears, and finally, dichloromethane containing 15% methanol by volume is used for collecting and eluting to obtain a collected liquid, and the solid is collected after spin-drying.

[0058] The purity of the final product ginsenoside F2 obtained in this comparative example is 83.88%, and the yield is 40%.

[0059] Comparative Example 4

[0060] A method for separating and purifying ginsenoside F2 from a fermentation broth comprises the following steps:

[0061] (1) 1.2 L of fermentation liquid from the tank was taken out, the concentration of ginsenoside F2 in the fermentation liquid was 14 g / L, and the mixture was centrifuged at 7000 rpm for 15 min. The lower precipitate was taken out, and anhydrous ethanol with a solid-liquid ratio of 1:2 was added for stirring extraction. The stirring speed was 200 rpm, the temperature was 50° C., and the extraction was performed for 30 min. The mixture was filtered, and the filtrate was spin-dried to obtain a crude product. The content of ginsenoside F2 in the crude product was 72.2%;

[0062] (2) Take 24.93 g of the crude product obtained in step (1), add 35% ethanol at a solid-liquid ratio of 1:80 (g / ml) to dissolve it, and then load the solution on 0.5 L of activated YLT810 resin at a flow rate of 1 column volume per hour. After loading, wash with 40% by volume ethanol until the liquid phase purity of the target product reaches 75%, and then elute with 70% by volume ethanol to collect the target product to obtain a collected liquid, which is then spin-dried to collect the solid.

[0063] The purity of the final product ginsenoside F2 of this comparative example is 78.5%, and the yield is 76.5%.

[0064] The embodiments provided above are not intended to limit the scope of the present invention, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present invention in combination with existing common knowledge, which also fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for separating and purifying ginsenoside F2 from a fermentation broth, characterized in that: The method comprises the following steps: (1) taking out the fermentation liquid from the tank, centrifuging, taking out the lower layer of precipitate, adding ethanol to stir and extract, filtering after extraction, and taking the filtrate and spinning it to dryness to prepare a crude product; (2) dissolving the crude product obtained in step (1) in an ethanol aqueous solution with a volume fraction of 10 to 40% to form a loading solution; then loading the solution on YLT810 resin, and after loading, washing the solution with an ethanol aqueous solution with a volume fraction of 45 to 50% until the target product has a liquid phase purity of 70 to 75%, and then eluting the solution with an ethanol aqueous solution with a volume fraction of 60 to 65%, collecting the eluate, and collecting the solid by spin drying to obtain ginsenoside F2.

2. The method according to claim 1, characterized in that The fermentation liquid in step (1) is obtained by fermenting ginsenoside crude powder (Rb1 and Rd account for 50% in total) with Escherichia coli.

3. The method according to claim 1, characterized in that The centrifugal conditions in step (1) are: 5000-8000 rpm, and the time is 5-10 min.

4. The method according to claim 1, characterized in that: The stirring extraction parameters of step (1) are: 100-500 rpm, time is 20-40 min, and temperature is 40-50° C.

5. The method according to claim 1, characterized in that The solid-liquid ratio of the crude product in step (2) when dissolved in an ethanol aqueous solution with a volume fraction of 10 to 40% is 1:80 to 90, g / ml.

6. The method according to claim 1, characterized in that The ratio of the YLT810 resin to the crude product in step (2) is 0.3-3:15-120; L / g.

7. The method according to claim 1, characterized in that The purity of ginsenoside F2 in step (2) is 85-90%, and the yield is more than 80%.

8. The method according to claim 1, characterized in that The volume fraction of the ethanol aqueous solution used for washing in step (2) is 50%.

9. The method according to claim 1, characterized in that: The volume fraction of the ethanol aqueous solution used for elution in step (2) is 60%.

10. Use of the method according to any one of claims 1 to 9 in industrial production monitoring of ginsenoside F2.

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