Targeted purification method of PPD type ginsenoside
The purification of PPD-type ginsenosides using HP-20 macroporous adsorption resin and gradient elution method solves the problem of difficult efficient separation of PPD-type ginsenosides in existing technologies, and achieves efficient and economical purification of PPD-type ginsenosides, meeting the needs of health food and scientific research.
Patent Information
- Application Number
- CN202411489322.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-24
- Publication Date
- 2025-12-02
AI Technical Summary
Existing technologies are insufficient for the efficient separation and extraction of high-value-added PPD-type ginsenosides, which affects their application in health foods and scientific research.
PPD-type ginsenosides were purified stepwise using HP-20 macroporous adsorption resin combined with a specific ethanol gradient elution method. This involved the use of distilled water, aqueous solutions of different concentrations of ethanol, and anhydrous ethanol. The purified PPD-type ginsenoside lyophilized powder was obtained by combining rotary evaporation and freeze-drying techniques.
It significantly improved the purification efficiency and yield of PPD-type ginsenosides, meeting the demand for high value-added products.
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Figure BDA0005099570170000051
Abstract
Description
Technical Field
[0001] This invention relates to the field of health food technology, and in particular to a method for targeted extraction of PPD-type ginsenosides. Background Technology
[0002] Ginseng (Panax ginseng CAMeyer) is an herbaceous plant belonging to the Araliaceae family. The *Shennong Bencao Jing* (Shennong's Classic of Materia Medica) records that ginseng has the effects of "nourishing the five internal organs, calming the spirit, stabilizing the soul, stopping palpitations, dispelling evil influences, improving eyesight, invigorating the mind, enhancing intelligence, and prolonging life with prolonged use." Ginsenosides are the hallmark components of ginseng. Based on the different structures of the sapogenins, they can be divided into oleanolic acid-type pentacyclic triterpenoid saponins and dammarane-type tetracyclic triterpenoid saponins. The latter can be further divided into protopanaxadiol-type ginsenosides (PPD) and protopanatriol-type ginsenosides (PPT). PPD saponins mainly include Rb1, Rb2, Rb3, Rc, and Rd. PPD saponins can be transformed into high-value rare ginsenosides through structural modification. Studies have shown that rare ginsenosides have high anti-tumor, immune-enhancing, cardiovascular-protective, and memory-improving effects. Therefore, it is necessary to separate PPD-type ginsenosides, whether for the preparation of high-value-added rare ginsenosides or for scientific research. Summary of the Invention
[0003] One of the objectives of this invention is to provide a targeted purification method for PPD-type ginsenosides to solve the problems mentioned in the background art.
[0004] To achieve the above objectives, the present invention provides the following technical solution.
[0005] This invention provides a method for targeted purification of PPD-type ginsenosides from ginseng extract, characterized in that...
[0006] S1 Weigh out the total ginsenoside extract and dissolve it in distilled water to prepare a ginsenoside extract sample solution of 0.5-1.5 g / mL;
[0007] S2. Measure the treated HP-20 macroporous adsorption resin and add it to the ginseng total saponin extract sample solution at a ratio of 100mL: 2-6g.
[0008] After the sample is adsorbed on HP-20 macroporous adsorption resin for 12-36 hours, 1-4 times the column volume of distilled water is added to HP-20 macroporous adsorption resin for elution, and the eluent is discarded.
[0009] S4. Add 1-4 column volumes of 15-25% ethanol aqueous solution to HP-20 macroporous adsorption resin for elution, and discard the eluent.
[0010] S5. Add 1-4 times the column volume of 35-45% ethanol aqueous solution to HP-20 macroporous adsorption resin for elution, and discard the eluent.
[0011] S6. Add 1-4 times the column volume of 55-65% ethanol aqueous solution to HP-20 macroporous adsorption resin for elution, and discard the eluent.
[0012] S7. Add 2-4 column volumes of anhydrous ethanol-water solution to HP-20 macroporous adsorption resin for elution and collect the eluent.
[0013] In one specific embodiment, in S1, a ginseng total saponin extract sample solution of 1.0 g / mL is prepared by dissolving in distilled water.
[0014] In one specific embodiment, ginseng total saponin extract sample solution is added to S2 at a ratio of 100mL:4g.
[0015] In one specific embodiment, in step S3, distilled water with a volume twice the column volume is added to the HP-20 macroporous adsorption resin for elution; in step S4, 20% ethanol aqueous solution with a volume twice the column volume is added to the HP-20 macroporous adsorption resin for elution.
[0016] In one specific embodiment, in step S5, two column volumes of 40% ethanol aqueous solution are added to the HP-20 macroporous adsorption resin for elution.
[0017] In one specific embodiment, in step S6, two times the column volume of 60% ethanol aqueous solution is added to the HP-20 macroporous adsorption resin for elution.
[0018] In one specific embodiment, in step S7, an anhydrous ethanol aqueous solution with a volume twice the column volume is added to the HP-20 macroporous adsorption resin for elution.
[0019] In one specific embodiment, the HP-20 macroporous adsorption resin treated in step S2 is subjected to the following treatment:
[0020] A1. After soaking HP-20 macroporous adsorption resin in anhydrous ethanol solution for 12-36 hours, wash with anhydrous ethanol until the effluent is not turbid when mixed with water at a 1:2 ratio.
[0021] A2 After washing the HP-20 macroporous adsorption resin with distilled water until there is no alcohol odor, it is soaked in 2% hydrochloric acid for 0.5-1.5 hours, washed with distilled water until neutral, and then soaked in 5% sodium hydroxide solution for 0.5-1.5 hours. It is then washed with distilled water until neutral and can be used directly.
[0022] Optionally, the following steps may also be included:
[0023] After filtering, the A3 resin is soaked in anhydrous ethanol. Before use, the resin is washed until there is no alcohol odor.
[0024] The purification method further includes the following steps:
[0025] S8 concentrated the collected eluent using a rotary evaporator at 45-55℃ until no alcohol odor remained. The concentrated solution was then transferred to a freeze dryer for freeze drying to finally obtain PPD type ginsenoside freeze-dried powder.
[0026] The targeted purification method for PPD-type ginsenosides provided by this invention utilizes HP-20 macroporous adsorption resin and adopts specific elution solvents and elution conditions to effectively increase the content of PPD-type ginsenosides in ginseng extracts, and has application value. Detailed Implementation
[0027] The present invention will be described below through specific embodiments in order to better understand the present invention, but this does not constitute a limitation on the present invention.
[0028] Treatment with HP-20 macroporous adsorption resin:
[0029] After soaking the HP-20 macroporous adsorption resin in anhydrous ethanol solution for 12-36 hours, wash it with anhydrous ethanol until the effluent mixed with water at a 1:2 ratio is clear. Next, wash the HP-20 macroporous adsorption resin with distilled water until there is no alcohol odor, then soak it in 2% hydrochloric acid for 0.5-1.5 hours, wash it with distilled water until neutral, and then soak it in 5% sodium hydroxide solution for 0.5-1.5 hours, washing it with distilled water until neutral. After acid and alkali treatment, filter the resin, soak it in anhydrous ethanol, and wash it until there is no alcohol odor before use.
[0030] Example 1
[0031] Dissolve the ginseng total saponin extract sample solution in distilled water to prepare a 0.5 g / mL solution. Then, accurately measure 0.5 L of the treated HP-20 macroporous adsorption resin and add a certain amount of the ginseng total saponin extract sample solution at a ratio of 100 mL: 4 g. After adsorbing the sample onto the HP-20 macroporous adsorption resin for 12 hours, first add 2 column volumes of distilled water to the HP-20 macroporous adsorption resin for elution, and discard the eluent. Next, add 2 column volumes of 20% ethanol aqueous solution to the HP-20 macroporous adsorption resin for elution, and discard the eluent. Then, add 2 column volumes of 40% ethanol aqueous solution to the HP-20 macroporous adsorption resin for elution, and discard the eluent. Finally, add 2 column volumes of 60% ethanol aqueous solution to the HP-20 macroporous adsorption resin for elution, and discard the eluent. Two column volumes of anhydrous ethanol-water solution were then added to the HP-20 macroporous adsorption resin for elution, and the eluent was collected. The anhydrous ethanol eluent was concentrated using a rotary evaporator at 50 degrees Celsius until no alcohol odor remained. The concentrate was then transferred to a freeze dryer and freeze-dried for 24 hours to finally obtain PPD-type ginsenoside freeze-dried powder.
[0032] Example 2
[0033] Dissolve the ginseng total saponin extract in distilled water to prepare a 1 g / mL sample solution. Then, accurately measure 1 L of the treated HP-20 macroporous adsorption resin and add a certain amount of the ginseng total saponin extract sample solution at a ratio of 100 mL: 4 g. After adsorbing the sample onto the HP-20 macroporous adsorption resin for 12 hours, first add 3 column volumes of distilled water to the HP-20 macroporous adsorption resin for elution, and discard the eluent. Next, add 3 column volumes of 20% ethanol aqueous solution to the HP-20 macroporous adsorption resin for elution, and discard the eluent. Then, add 3 column volumes of 40% ethanol aqueous solution to the HP-20 macroporous adsorption resin for elution, and discard the eluent. Next, add 3 column volumes of 60% ethanol aqueous solution to the HP-20 macroporous adsorption resin for elution, and discard the eluent. Finally, add 3 column volumes of anhydrous ethanol aqueous solution to the HP-20 macroporous adsorption resin for elution, and collect the eluent. The anhydrous ethanol eluent was concentrated using a rotary evaporator at 50 degrees Celsius until no alcohol odor remained. The concentrate was then transferred to a freeze dryer and freeze-dried for 24 hours to obtain PPD-type ginsenoside freeze-dried powder.
[0034] Example 3
[0035] Dissolve the ginseng total saponin extract sample in distilled water to prepare a 1.5 g / mL solution. Then, accurately measure 1.5 L of the treated HP-20 macroporous adsorption resin and add a certain amount of the ginseng total saponin extract sample solution at a ratio of 100 mL: 4 g. After adsorbing the sample onto the HP-20 macroporous adsorption resin for 12 hours, first add 4 column volumes of distilled water to the HP-20 macroporous adsorption resin for elution, and discard the eluent. Next, add 4 column volumes of 20% ethanol aqueous solution to the HP-20 macroporous adsorption resin for elution, and discard the eluent. Then, add 4 column volumes of 40% ethanol aqueous solution to the HP-20 macroporous adsorption resin for elution, and discard the eluent. Finally, add 4 column volumes of 60% ethanol aqueous solution to the HP-20 macroporous adsorption resin for elution, and discard the eluent. Four times the column volume of anhydrous ethanol aqueous solution was added to the HP-20 macroporous adsorption resin for elution, and the eluent was collected. The anhydrous ethanol eluent was concentrated using a rotary evaporator at 50 degrees Celsius until no alcohol odor remained. The concentrate was then transferred to a freeze dryer and freeze-dried for 24 hours to finally obtain PPD type ginsenoside freeze-dried powder.
[0036] The content of the lyophilized powders obtained by the purification methods in Examples 1-3 was determined.
[0037] Table 1. High Performance Liquid Chromatography (HPLC) Analysis Conditions
[0038] Time (min) 0.1% Formic Acid-Water (%) Acetonitrile (%) 0 80 20 10 50 50 30 10 90 35 80 20 45 80 20
[0039] The chromatographic column model was Xbridge BEH C18 (3.0×150mm, 2.5μm), the column temperature was 30℃, the flow rate was 0.5mL / min, and the injection volume was 5μL.
[0040] The specific data on the content determination of PPD-type ginsenoside freeze-dried powder are as follows:
[0041] Table 2. Determination of PPD-type ginsenoside content in freeze-dried powder
[0042]
[0043] It is evident that the method of this invention can effectively separate PPD-type ginsenosides, and the yield is relatively high.
Claims
1. A method for targeted purification of PPD-type ginsenosides from ginseng extract, characterized in that, S1 Weigh out the total ginsenoside extract and dissolve it in distilled water to prepare a ginsenoside extract sample solution of 0.5-1.5 g / mL; S2. Measure the treated HP-20 macroporous adsorption resin and add it to the ginseng total saponin extract sample solution at a ratio of 100mL: 2-6g. After the sample is adsorbed on HP-20 macroporous adsorption resin for 12-36 hours, 1-4 times the column volume of distilled water is added to HP-20 macroporous adsorption resin for elution, and the eluent is discarded. S4. Add 1-4 column volumes of 15-25% ethanol aqueous solution to HP-20 macroporous adsorption resin for elution, and discard the eluent. S5. Add 1-4 times the column volume of 35-45% ethanol aqueous solution to HP-20 macroporous adsorption resin for elution, and discard the eluent. S6. Add 1-4 column volumes of 55-65% ethanol aqueous solution to HP-20 macroporous adsorption resin for elution, and discard the eluent. S7. Add 2-4 column volumes of anhydrous ethanol-water solution to HP-20 macroporous adsorption resin for elution and collect the eluent.
2. The method as described in claim 1, characterized in that, In S1, the total ginsenoside extract sample solution was prepared by dissolving it in distilled water to a concentration of 1.0 g / mL.
3. The method as described in claim 1, characterized in that, Add the ginseng total saponin extract sample solution to S2 at a ratio of 100mL:4g.
4. The method as described in claim 1, characterized in that, In S3, two column volumes of distilled water are added to the HP-20 macroporous adsorption resin for elution; in S4, two column volumes of 20% ethanol aqueous solution are added to the HP-20 macroporous adsorption resin for elution.
5. The method as described in claim 1, characterized in that, In S5, two column volumes of 40% ethanol aqueous solution are added to HP-20 macroporous adsorption resin for elution.
6. The method as described in claim 1, characterized in that, In S6, two column volumes of 60% ethanol aqueous solution are added to HP-20 macroporous adsorption resin for elution.
7. The method as described in claim 1, characterized in that, In S7, anhydrous ethanol aqueous solution with a volume twice the column volume is added to HP-20 macroporous adsorption resin for elution.
8. The method as described in claim 1, characterized in that, The HP-20 macroporous adsorption resin treated in step S2 is then subjected to the following treatment: A1. After soaking HP-20 macroporous adsorption resin in anhydrous ethanol solution for 12-36 hours, wash with anhydrous ethanol until the effluent is not turbid when mixed with water at a ratio of 1:
2. A2 After washing the HP-20 macroporous adsorption resin with distilled water until there is no alcohol odor, it is soaked in 2% hydrochloric acid for 0.5-1.5 hours, washed with distilled water until neutral, and then soaked in 5% sodium hydroxide solution for 0.5-1.5 hours. It is then washed with distilled water until neutral and can be used directly.
9. The method as described in claim 8, characterized in that, Optionally, the following steps may also be included: After filtering, the A3 resin is soaked in anhydrous ethanol. Before use, the resin is washed until there is no alcohol odor.
10. The method as described in claim 1, characterized in that, It also includes the following steps: S8 concentrated the collected eluent using a rotary evaporator at 45-55℃ until no alcohol odor remained. The concentrated solution was then transferred to a freeze dryer for freeze drying to finally obtain PPD type ginsenoside freeze-dried powder.