Method for enriching multiple rare ginsenosides by composite enzymolysis combined with composite resin column chromatography and application thereof

Abstract

The present application belongs to the technical field of Chinese medicine active ingredient preparation, and particularly relates to a method for enriching multiple rare ginsenosides by complex enzymolysis combined with composite resin column chromatography and application thereof. Specifically, the present application realizes the continuous completion of ordered glycosidic bond hydrolysis and C-20 dehydration based on time sequence step-by-step enzymolysis combined with synchronous inactivation and dehydration, and simultaneously obtains a mixture of 10 high-purity rare ginsenosides. The total content of the 10 target rare ginsenosides in the rare ginsenoside powder obtained by the present application is greater than or equal to 95%, and the yield is greater than or equal to 2.5%. The process is stable and can be scaled up, and has good industrial application prospect and market economic value.

Description

Technical Field

[0001] This invention belongs to the field of preparation technology of active ingredients of traditional Chinese medicine, specifically involving a method for enriching multiple rare ginsenosides by a combination of enzymatic hydrolysis and composite resin column chromatography, and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Red ginseng is a traditional and precious Chinese medicinal material made from ginseng through steaming and drying. It is rich in various native ginsenosides (such as Rg1, Re, Rd, etc.). Rare ginsenosides (such as Rg3, Rh2, CK, Rk1, Rg5, etc.) have significantly higher biological activity than native ginsenosides, possessing pharmacological activities such as anti-tumor, immune-enhancing, anti-fatigue, and antioxidant effects, but their natural content is extremely low. Literature reports that HPLC was used to determine the content of six rare ginsenosides in 10 batches of commercially available red ginseng, and the content of a single rare ginsenoside did not exceed 0.046%.

[0004] Targeted transformation is an effective means to increase the content of rare ginsenosides. Existing transformation methods mainly include acid-base hydrolysis, single β-glucosidase enzymatic hydrolysis, and one-pot mixed enzymatic hydrolysis. However, acid-base hydrolysis is subject to harsh conditions, easily generating a large number of byproducts and having poor safety; single β-glucosidase cannot cleave rhamnoside bonds, resulting in the ineffective utilization of rhamnosyl native saponins (Re) in the system, causing substrate waste; conventional one-pot mixed enzymatic hydrolysis lacks time control, easily causing excessive degradation of substrates into aglycones, resulting in low product yield. In addition, dehydrated rare saponins (Rk3, Rh4, Rk1, Rg5) require additional high-temperature or acid treatment steps to generate, which is cumbersome and energy-intensive. Although traditional processes can obtain soap powder containing multiple rare ginsenosides, their total content is usually not ideal. For example, the total content of monomers in soap powder containing 15 rare ginsenosides reported to be only 68.5%, which is difficult to meet the requirements of high-purity formulations. Summary of the Invention

[0005] To address the shortcomings of the existing technologies, this invention provides a method for enriching multiple rare ginsenosides using a combination of enzymatic hydrolysis and composite resin column chromatography, and its application. Specifically, this invention is based on sequential stepwise enzymatic hydrolysis combined with simultaneous inactivation and dehydration, thereby achieving the continuous completion of ordered hydrolysis of glycosidic bonds and dehydration at the C-20 position, coupled with gradient elution, to stably obtain a high-purity mixture of 10 rare ginsenosides, which is highly beneficial for industrial production applications. This invention is based on the above research results.

[0006] To achieve the above technical objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for enriching multiple rare ginsenosides by a combination of enzymatic hydrolysis and composite resin column chromatography, the method comprising: S1. The red ginseng is boiled in water to extract and concentrate the solution. S2. Using a time-sequential stepwise enzymatic hydrolysis strategy, α-L-rhamnosidase is added to the concentrate in the first time-sequential stage; β-glucosidase and cellulase are added to it in the second time-sequential stage to obtain the enzymatic hydrolysis reaction solution. S3. The enzymatic hydrolysis reaction solution is heated and filtered to obtain an enzymatic conversion solution; wherein, the heating treatment simultaneously inactivates the enzyme and induces the dehydration of the C-20 hydroxyl group in the enzymatic hydrolysis product; filtration is used to remove insoluble impurities. S4. Various rare ginsenosides are enriched and purified by column chromatography. The column chromatography uses a composite resin column, wherein the composite resin is a mixture of non-polar resin and weakly polar resin.

[0007] The specific steps of the extraction by boiling water in step S1 include: boiling red ginseng in water, cooling, and filtering to obtain the extract; preferably, a two-stage extraction method is used, in which 8-12 times the mass of red ginseng is added to the extract, and the extract is boiled for 1.5-2.5 hours to obtain the filtrate and the residue; in the second stage, 6-10 times the mass of the residue is added to the extract and the extract is boiled for 1-2 hours; after cooling, the extract is filtered through a 100-300 mesh sieve and the filtrates are combined.

[0008] In step S2, α-L-rhamnosidase is added in the first time-series stage to remove the C-6 terminal rhamnose from Re, converting Re to Rg1. β-glucosidase is not added in this stage to avoid premature hydrolysis of the glucosinolate bond by β-glucosidase, which would generate intermediates and competitively inhibit subsequent rhamnosidase activity.

[0009] While red ginseng concentrate naturally contains Rg1 (which can be directly hydrolyzed by β-glucosidase), it also contains a large amount of Re. The C-6 position of Re is linked to a rhamnose-glucose disaccharide, and the terminal rhamnose hinders the effective action of β-glucosidase, causing Re to be ineffectively utilized in a system containing only β-glucosidase, resulting in substrate waste. Therefore, this invention introduces α-L-rhamnosidase in the first time-series step to hydrolyze Re into Rg1, allowing this previously wasted substrate to also serve as a reaction substrate for β-glucosidase, additionally contributing to the yield of Rh1, F1, Rk3, and Rh4.

[0010] In the second time step, this invention does not require the separation of α-L-rhamnosidase, but instead directly adds β-glucosidase and cellulase, and continues the reaction for 3 hours to directionally hydrolyze the glucosinolate bond.

[0011] This invention, monitored in real-time by HPLC, shows that under conditions of pH 5.0 and 55℃, α-L-rhamnosidase can remove more than 95% of the terminal rhamnose sugar within 2 hours, while the β-glucosidic bond retention rate is >95%. If β-glucosidase is added prematurely, it will competitively hydrolyze unprotected glucosidic bonds, generating non-natural intermediates and inhibiting subsequent rhamnosidase activity. Therefore, the first stage is set to 1.5~3 h (preferably 2 h), and the second stage to 2~5 h (preferably 3 h), thereby achieving a temporal decoupling effect between barrier removal and main-chain hydrolysis.

[0012] Meanwhile, to maintain optimal enzyme activity, the pH is preferably maintained at 4.9–5.1 during enzymatic hydrolysis. Specifically, since the hydrolysis of glycosidic bonds releases free sugars and organic acids, the pH of the reaction solution may decrease (experimental monitoring shows that the pH can decrease by 0.3–0.5 within 5 hours). To maintain optimal enzyme activity, the pH is measured every hour after the start of enzymatic hydrolysis. If the pH is lower than 4.8, it is adjusted back to 5.0 ± 0.1 using 0.1 M trisodium citrate solution.

[0013] In this invention, the composition of the complex enzyme system (based on per kilogram of red ginseng): β-glucosidase (100 U / g, derived from Aspergillus niger): 20~30g; α-L-rhamnosidase (40,000 U / g, derived from Aspergillus niger): 0.07~0.20g; Cellulase (400 U / mg, derived from Trichoderma): 2.5~5.0g.

[0014] In step S3, the specific conditions for heat treatment are: heat treatment at 75~95℃ (preferably 80℃) for 20~60min, thereby completely inactivating the enzyme and terminating the enzymatic hydrolysis reaction; at the same time, it induces the dehydration of the C-20 hydroxyl group in the enzymatic hydrolysis product to generate Rk3, Rh4, Rk1, and Rg5.

[0015] Chemical mechanism of dehydrated saponin formation: Under weakly acidic heating conditions at pH 5.0 and 80℃, the exposed tertiary hydroxyl group at the C-20 position undergoes protonation dehydration (following the E1 elimination mechanism), generating a stable carbocation intermediate. This intermediate then further eliminates the proton to form a Δ20(21) or Δ20(22) double bond, specifically generating Rk3 / Rh4 (from Rh1) and Rk1 / Rg5 (from Rg3). Dehydration is extremely difficult when the C-20 position of native saponins is protected by a glycosyl group. This invention, through sequential enzymatic hydrolysis, fully exposes the C-20 hydroxyl group of Rh1 and Rg3, providing a specific substrate for dehydration. Therefore, this dehydration step is substrate-dependent, does not trigger random dehydration at other sites, and the product profile is controllable.

[0016] In step S4, the non-polar resin can be D101 resin, and the weakly polar resin can be AB-8 resin; therefore, the composite resin is a mixture of D101 resin and AB-8 resin, with a mass ratio of D101 resin to AB-8 resin of (1~5):1, more preferably 3:1. The adsorption capacity of the mixed resin for Rh1, F1, Rk3, and Rh4 is increased by more than 20% compared to that of D101 resin alone.

[0017] The sample loading volume is 0.1~0.3 g of the corresponding enzymatic hydrolysis solution of crude drug per milliliter of resin; the elution flow rate is 0.5~2 BV / h; the specific gradient elution procedure includes: eluting sequentially with 0.5~2 BV of pure water, 3~5 BV of 20% ethanol, 3~5 BV of 60% ethanol, 3~5 BV of 70% ethanol, and 2~4 BV of 95% ethanol, and combining the 60%~95% ethanol eluent.

[0018] Furthermore, the method also includes the steps of concentrating, drying and pulverizing the product of step S4 to obtain rare saponin powder of red ginseng.

[0019] A second aspect of the present invention provides the application of the above-described method in the preparation of products rich in various rare red ginseng saponins. The rare red ginseng saponins include ginsenoside Rh1, ginsenoside F1, ginsenoside F2, 20(S)-ginsenoside Rg3, 20(S)-ginsenoside Rh2, ginsenoside CK, ginsenoside Rk3, ginsenoside Rh4, ginsenoside Rk1, and ginsenoside Rg5.

[0020] The product may be food or medicine, and the food may be health food, without specific limitations.

[0021] Compared with existing technical solutions, one or more of the above technical solutions have the following beneficial effects: The above-mentioned technical solution hydrolyzes Re to Rg1 using α-L-rhamnosidase in the first stage, making Re, which was previously unavailable in a single β-glucosidase system, an effective substrate for subsequent enzymatic hydrolysis reactions. This increases the total yield of 10 target rare saponins from approximately 1.89% in a single enzyme system to 2.92%, significantly improving raw material utilization. Simultaneously, the above-mentioned technical solution, through a coupled design of sequential stepwise enzymatic hydrolysis and simultaneous inactivation and dehydration by heating, achieves continuous completion of ordered hydrolysis of glycosidic bonds and dehydration at the C-20 position, enabling the stable and simultaneous acquisition of 10 target rare saponins in a single process. The saponins were obtained with a complete product spectrum. Based on this, gradient elution was performed using a composite resin of D101 and AB-8 (3:1) to further leverage the synergistic adsorption effect of the composite resin, effectively removing interfering impurities. This resulted in a total content of over 95% of the 10 rare ginsenosides in the final product, while the yield of rare saponin powder remained stable at over 2.5% (based on red ginseng), achieving a synergistic balance between high purity and high yield. Furthermore, the results of three batch scale-up verification showed that the yield RSD was 2.3% and the total content RSD was 0.5%, indicating that the process is stable and scalable, with promising prospects for industrial application. Attached Figure Description

[0022] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0023] Figure 1 The figure shows the HPLC chromatograms of the 13 ginsenoside reference standards of the present invention; in the figure, 1. Ginsenoside Re; 2. Ginsenoside Rg1; 3. Ginsenoside Rh1; 4. Ginsenoside Rd; 5. Ginsenoside F1; 6. Ginsenoside F2; 7. Ginsenoside Rk3; 8. Ginsenoside Rh4; 9. Ginsenoside 20(S)-Rg3; 10. Ginsenoside Rk1; 11. Ginsenoside Rg5; 12. Ginsenoside CK; 13. Ginsenoside 20(S)-Rh2.

[0024] Figure 2 The figure shows the HPLC chromatogram of the rare saponin powder obtained in Example 1 of the present invention; in the figure, 1. Ginsenoside Rh1; 2. Ginsenoside F1; 3. Ginsenoside F2; 4. Ginsenoside Rk3; 5. Ginsenoside Rh4; 6. Ginsenoside 20(S)-Rg3; 7. Ginsenoside Rk1; 8. Ginsenoside Rg5; 9. Ginsenoside CK; 10. Ginsenoside 20(S)-Rh2.

[0025] Figure 3 This is a schematic diagram of the reaction in this invention. Detailed Implementation

[0026] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0027] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0028] The present invention will be further illustrated below with specific examples. These examples are for illustrative purposes only and do not limit the scope of the invention. Unless otherwise specified in the examples, experimental conditions are generally performed under conventional conditions or as recommended by the sales company. Components not specifically limited in this invention can be commercially available. The methods for calculating the yield of rare saponin powder and the total content of the 10 target rare saponins in the various examples and comparative examples are as follows: (1) Yield (%) = (mass of rare saponin powder obtained / mass of red ginseng) × 100%; (2) Total content of 10 target rare saponins (%) = (total mass of 10 target saponins) / (total mass of rare saponin powder) × 100%.

[0029] The individual ginsenosides in the rare saponin powder were analyzed by HPLC. Specific detection conditions included: The chromatographic column was a ZORBAX SB-C18 (4.6 mm × 250 mm, 5 μm); the column temperature was 30℃, the detection wavelength was 203 nm, the sample volume was 10 μL, and the flow rate was 1.0 mL·min. 1 The mobile phase A is acetonitrile, and the mobile phase B is water. The gradient elution program is shown in Table 1 below.

[0030] Table 1 Gradient elution procedure for HPLC detection of rare ginsenosides

[0031] Standard curve establishment method: Each target saponin was quantified using its corresponding external standard method with its corresponding reference standard to ensure the accuracy of the quantitative results. Accurately weigh an appropriate amount of each reference standard, dissolve it in methanol, and dilute it serially to prepare a series of mixed reference standard solutions at five concentration levels. Inject and determine the solution according to the above HPLC conditions. The HPLC chromatogram is shown below. Figure 1As shown, a standard curve was plotted by performing linear regression on the peak area (y) of each reference standard against its mass concentration (x, μg / mL). The linear range, linear equation, and correlation coefficient of rare ginsenosides used for quantification are shown in Table 2 below.

[0032] Table 2. Linear range, linear equation, and correlation coefficient of 10 rare ginsenoside reference standards.

[0033] The reference standards ginsenoside Rh1 (purity > 98.0%), ginsenoside F1 (purity > 98.0%), ginsenoside F2 (purity > 98.0%), ginsenoside CK (purity > 98.0%), ginsenoside Rh4 (purity > 99.0%), ginsenoside Rk3 (purity > 98.0%), and 20(S)-ginsenoside Rg3 (purity ≥ 98.0%) were all purchased from Chengdu Pusi Biotechnology Co., Ltd. Limited Company; 20(S)-Ginsenoside Rh2 (purity 95.8%), ginsenoside Rd (purity 96.9%), ginsenoside Re (purity 95.5%), and ginsenoside Rg1 (purity 97.0%) were purchased from the China National Institutes for Food and Drug Control; ginsenoside Rk1 (purity > 98.0%) and ginsenoside Rg5 (purity > 99.0%) were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0034] Example 1 Boil 1 kg of red ginseng in 10 L of water for 2 hours. Add 8 L of water to the dregs and boil for 1.5 hours. Cool the decoction to 25°C, filter through a 200-mesh sieve, and combine the filtrates. Concentrate the filtrate under reduced pressure (50°C) to 2.9 L, and cool to 25°C. Adjust the pH to 5.0 with citric acid.

[0035] First stage of enzymatic hydrolysis: Add 0.10 g (40000 U / g) of α-L-rhamnosidase and hydrolyze at 55℃ for 2 hours.

[0036] The second stage of enzymatic hydrolysis involved directly adding 24 g (100 U / g) of β-glucosidase and 4.0 g (400 U / mg) of cellulase, followed by 3 hours of hydrolysis. The total hydrolysis time was 5 hours, with a stirring speed of 45 rpm. The pH was measured every hour during the hydrolysis process, and adjusted to pH 5.0 at the end of the 2nd and 4th hours using 0.1 M trisodium citrate.

[0037] The temperature was raised to 80℃ and held for 30 min to simultaneously achieve enzyme inactivation and dehydration at the C-20 position. The enzyme hydrolysis solution was obtained by filtering through a Buchner funnel filter paper.

[0038] Composite resin was packed into a column (column volume 1 L, column diameter-to-height ratio 1:6). Resin pretreatment: soaking in 95% ethanol for 24 h, then washing with pure water until no alcohol odor remained. Sample loading: the enzymatic conversion buffer was loaded at a flow rate of 1 BV / h (the loading amount was equivalent to 0.2 g crude drug / mL resin). Gradient elution (flow rate 1 BV / h): 1 L pure water → 4 L 20% ethanol → 4 L 60% ethanol → 4 L 70% ethanol → 3 L 95% ethanol. The 60%~95% eluents were combined, concentrated, and dried to obtain 29.2 g of product, yield 2.92%. 0.1 g of the product was dissolved in methanol and diluted to 100 mL. HPLC analysis was performed, and the HPLC chromatogram is shown below. Figure 2 As shown in Table 3 below, the data indicates that the above method achieves a synergistic balance between high purity and high yield of rare ginsenosides.

[0039] Table 3. HPLC detection results of rare ginsenoside powder obtained in Example 1

[0040] Example 2 Add 5 kg of red ginseng to 45 L of water and decoct for 2 hours. Add 35 L of water to the dregs and decoct for 1.5 hours. Cool the decoction to 25°C, filter through a 200-mesh sieve, and combine the filtrates. Concentrate the filtrate to 14 L (2.8 times the weight of the ginseng), cool to 25°C, and adjust the pH to 5.0.

[0041] Phase 1: α-L-rhamnosidase 0.45 g (40000 U / g), enzymatic hydrolysis at 55℃ for 2 h.

[0042] Second stage: β-glucosidase 115 g (100 U / g) and cellulase 19 g (400 U / mg) were hydrolyzed at 55℃ for 3 h.

[0043] Keep warm at 80℃ for 30 minutes to simultaneously inactivate and dehydrate.

[0044] A composite resin (5 L column volume, column diameter to height ratio 1:6) was used. The loading flow rate was 1 BV / h, and the elution gradient was: 5 L pure water → 20 L 20% ethanol → 20 L 60% ethanol → 20 L 70% ethanol → 15 L 95% ethanol. The 60%–95% eluents were combined, concentrated, and dried to obtain 136 g of product, with a yield of 2.72%. 0.1 g of the product was dissolved in methanol and diluted to 100 mL. HPLC analysis data are shown in Table 4 below.

[0045] Table 4. HPLC detection results of rare ginsenoside powder obtained in Example 2

[0046] Two batch scale-up experiments were conducted using the same method to verify the stability of the method, ultimately yielding 138 g and 132 g of product, respectively. 0.1 g of each product was dissolved in methanol and diluted to 100 mL. HPLC analysis data are shown in Table 5 below.

[0047] Table 5. HPLC detection results of rare ginsenoside powder obtained from batches 1 and 2 for scale-up verification.

[0048] Analysis showed that the yields of the three batches verified by scale-up were 2.72%, 2.76%, and 2.64%, with an RSD of 2.3%. The mass fractions of the 10 rare ginsenosides in the product were 95.6%, 96.5%, and 96.2%, with an RSD of 0.5%. These data indicate that the process has good stability.

[0049] Comparative Example 1: No α-L-rhamnosidase was used; all other steps were the same as in Example 1. Results: 18.9 g of product was obtained, with a yield of 1.89%, a significant decrease compared to Example 1. Because Re was not hydrolyzed by rhamnosidase, it could not be effectively hydrolyzed by β-glucosidase, resulting in complete waste of this substrate. The sharp decrease in yield directly reflects the product loss caused by the unutilized Re.

[0050] Comparative Example 2: The gradient was changed to pure water → 30% ethanol → 50% ethanol → 80% ethanol → 95% ethanol. Each step of the gradient used 4 BV of eluent, and the rest was the same as in Example 1. Results: 24.6 g of product was obtained, with a yield of 2.46%. However, there were many impurities, making accurate quantification impossible. This demonstrates that the gradient elution procedure of this invention has higher selectivity for the target saponins.

[0051] Comparative Example 3: No enzymatic hydrolysis or incubation was performed; the concentrate was directly loaded onto the column. Results: Only 2.4 g of product was obtained, with a yield of only 0.24%, and the total content of the 10 target rare ginsenosides was 0%. These results indicate that the natural content of the 10 target rare ginsenosides in the unenzymatically converted red ginseng extract is extremely low (below the HPLC detection limit), and cannot be effectively enriched by simple resin column chromatography, further highlighting the necessity of the enzymatic conversion step in this invention.

[0052] Comparative Example 4: All enzymes were added at once, and enzymatic hydrolysis was performed for 5 hours, with the rest of the process the same as in Example 1. Results: 11.6 g of product was obtained, with a yield of 1.16%, significantly lower than in Example 1. This is because the temporal disruption led to the excessive degradation of some substrates into aglycones. The aglycones, due to changes in polarity, altered their adsorption capacity on the resin, resulting in some loss. The decreased yield reflects the direct impact of temporal disruption on the total product volume.

[0053] Comparative Example 5: After enzymatic hydrolysis, the temperature was rapidly raised to 80℃ and immediately cooled (within 2 min), without holding for 30 min. Results: 30.1 g of product was obtained, with a yield of 3.01%, but only 6 target rare ginsenosides were obtained.

[0054] Comparative Example 6: Cellulase was not used, and all other conditions remained unchanged. Results: 10.2 g of product was obtained, with a yield of 1.02%, which was much lower than that in Example 1, and the total content of the 10 target substances was 88.9%. This indicates that the lack of cellulase caused the plant fiber residue to encapsulate the substrate, reducing substrate accessibility and resulting in incomplete enzymatic hydrolysis.

[0055] Comparative Example 7 (single D101 resin, without AB-8 mixing): Only D101 resin was used for column packing, and the rest was the same as in Example 1. Results: 25.4 g of product was obtained, with a yield of 2.54%, but there were many impurities, making accurate quantification impossible. The specific effect data of each example and comparative example are summarized in Table 6 below.

[0056] Table 6 Summary of Effect Data for Each Embodiment and Comparative Example

[0057] In summary, the method for enriching rare ginsenosides using a combination of enzymatic hydrolysis and composite resin column chromatography provided by this invention features clear process steps, simple operation, mild conditions, and is environmentally friendly. The enzymes and resins used are commercially available, making it suitable for large-scale industrial production. The rare ginsenoside powder obtained by this invention has a total content of 10 target rare ginsenosides ≥95%, a yield ≥2.5%, and the process is stable and scalable, demonstrating good prospects for industrial application and market economic value.

[0058] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for enriching multiple rare ginsenosides using a combination of enzymatic hydrolysis and composite resin column chromatography, characterized in that, The method includes: S1. The red ginseng is boiled in water to extract and concentrate the solution. S2. Using a time-sequential stepwise enzymatic hydrolysis strategy, α-L-rhamnosidase is added to the concentrate in the first time-sequential stage; β-glucosidase and cellulase are added to it in the second time-sequential stage to obtain the enzymatic hydrolysis reaction solution. S3. The enzymatic hydrolysis reaction solution is heated and filtered to obtain an enzymatic conversion solution; S4. Enrichment and purification of various rare ginsenosides by column chromatography. The column chromatography uses a composite resin column, which is a mixture of D101 resin and AB-8 resin, with a mass ratio of D101 resin to AB-8 resin of (1~5):

1. The sample loading volume is 0.1~0.3 g of the enzymatic conversion solution corresponding to the crude drug per milliliter of resin. The elution flow rate is 0.5~2 BV / h. The gradient elution procedure includes: sequential elution with 0.5~2 BV of pure water, 3~5 BV of 20% ethanol, 3~5 BV of 60% ethanol, 3~5 BV of 70% ethanol, and 2~4 BV of 95% ethanol, and combining the 60%~95% ethanol eluent.

2. The method as described in claim 1, characterized in that, In step S1, the specific steps of decoction extraction include: adding water to the red ginseng medicinal material and decocting to extract, cooling, and filtering to obtain the extract; preferably, a two-stage extraction method is used, in which 8-12 times the mass of the red ginseng medicinal material is added for the first time, and the decoction is carried out for 1.5-2.5 hours and filtered to obtain the filtrate and the residue; in the second time, 6-10 times the mass of the residue is added for the second time, and the decoction is carried out for 1-2 hours; after the decoction is cooled, it is filtered through a 100-300 mesh sieve, and the filtrates are combined.

3. The method as described in claim 1, characterized in that, In step S2, the first time stage is 1.5~3 h, and the second time stage is 2~5 h; the pH is maintained at 4.9~5.1 during the enzymatic hydrolysis process.

4. The method as described in claim 1, characterized in that, In step S2, the amount of α-L-rhamnosidase added is 0.07~0.20g per kilogram of red ginseng, with an enzyme activity of 40000U / g; the amount of β-glucosidase added is 20~30g per kilogram of red ginseng, with an enzyme activity of 100U / g; and the amount of cellulase added is 2.5~5.0g per kilogram of red ginseng, with an enzyme activity of 400U / mg.

5. The method as described in claim 1, characterized in that, In step S3, the specific conditions for heat treatment are: heat treatment at 75~95℃ for 20-60 min.

6. The application of the method according to any one of claims 1-5 in the preparation of a product rich in various rare red ginseng saponins; wherein the rare red ginseng saponins include ginsenoside Rh1, ginsenoside F1, ginsenoside F2, 20(S)-ginsenoside Rg3, 20(S)-ginsenoside Rh2, ginsenoside CK, ginsenoside Rk3, ginsenoside Rh4, ginsenoside Rk1 and ginsenoside Rg5.

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