Preparation method and application of high-purity rare ginsenoside CK

Abstract

The invention belongs to the technical field of active substance preparation, and particularly relates to a preparation method and application of high-purity rare ginsenoside CK. The high-purity rare ginsenoside CK with the content exceeding 95% is finally produced through the steps of combined enzymolysis, ultrafiltration, nanofiltration, multiple crystallization and the like, the production efficiency and purity of the rare ginsenoside CK are improved through the preparation method, only ethyl alcohol and water are used as solvents in the whole process, it is guaranteed that the technology is green and environmentally friendly, and the method is suitable for industrial production. The method has the advantages of simple operation, no use of harmful chemical solvents, great reduction of environmental pollution, realization of the significant cost advantage in the whole production process due to repeated use of equipment and solvents, suitableness for large-scale industrial application, and wide market prospects and economic benefits.

Description

Technical Field

[0001] The present invention belongs to the technical field of active substance preparation, and particularly relates to a preparation method and application of high-purity rare ginsenoside CK. Background Art

[0002] The enzyme conversion process is currently the main production process for producing rare ginsenoside CK. The crude product of rare ginsenoside CK after enzyme conversion has a complex composition, containing various other saponins and unknown components. Other saponins and CK have similar physical properties, which makes it impossible to purify the CK crude product with a content lower than 70% by relatively simple methods such as extraction, crystallization, ordinary filtration, and centrifugation. It can only be separated and purified by chromatography to obtain CK with a content of more than 80%. Ordinary chromatography such as silica gel column separation requires the use of highly toxic organic solvents such as chloroform and dichloromethane. Moreover, the sample loading volume of silica gel column separation is small, the elution volume is large, and a large amount of organic solvents will be used to obtain a small amount of target product, resulting in high production costs and serious environmental pollution.

[0003] Patent No. (CN 108138212A): 2 kg of ginseng is extracted with 20 L of ethanol, centrifuged, concentrated under reduced pressure, decolorized with 1 L of ether, added with 1 L of butanol, concentrated under reduced pressure, added with a small amount of ethanol, added with a large amount of ethyl acetate, and the precipitate is dried to obtain 40 - 80 g of ginsenoside powder. The ginsenoside powder is enzymatically hydrolyzed with pectinase at 30 °C for 72 h, concentrated with ethyl acetate, and then separated by silica gel column chromatography to obtain CK. Disadvantages: The purification steps of this patent are cumbersome, and a variety of toxic and harmful organic substances are used as extraction media during the preparation process, causing significant damage to the health of production personnel and the environment.

[0004] Patent No. (CN105838770A): First, 200 L of Microbacterium oxydans is liquid-fermented. RB1 with a content of 90% is dissolved in 50% ethanol to prepare 6 L of a 100 g / L saponin solution, added with 10 g of heteropolyacid, enzymatically hydrolyzed at 30 °C for 96 h. The enzymatic hydrolysate is centrifuged to discard the supernatant, the precipitate is washed, extracted with water and ethyl acetate in a ratio of 1:1, concentrated under reduced pressure, and separated and purified by preparative chromatography with a yield of 90% and a purity of 96%. Disadvantages: This patent does not provide a detailed description of the purification steps. Using preparative chromatography to separate and purify CK with a relatively low purity will produce a huge volume of organic solvent eluate, and it is not easy to obtain a large amount of target product. The cost of using preparative chromatography to produce materials is extremely high. Summary of the Invention

[0005] The object of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a preparation method of high-purity rare ginsenoside CK, so as to produce high-purity rare ginsenoside CK with a content of more than 95%, with high production efficiency, less environmental pollution, and the equipment and solvents can be reused, having a huge cost advantage.

[0006] To achieve the above object, the technical solution adopted by the present invention includes: In a first aspect, the present invention provides a method for preparing rare ginsenoside CK, comprising the following steps: S1. Stir and mix ginsenoside Rb1 with a phosphate buffer solution until dissolved to obtain a ginsenoside Rb1 solution. Then, mix a complex enzyme with a phosphate buffer solution to prepare a complex enzyme solution, and add the complex enzyme solution to the ginsenoside Rb1 solution for enzymatic hydrolysis to obtain an enzymatic hydrolysate. S2. Centrifuge the enzymatic hydrolysate, collect the precipitate, stir and mix the obtained precipitate with an ethanol solution to obtain a dissolution solution, and subject the dissolution solution to pressure filtration and microfiltration to obtain filtrate A. S3. Ultrafilter and nanofiltrate the filtrate A obtained in step S2 to obtain a concentrated filtrate B, and then perform a drying treatment on the concentrated filtrate B to obtain a dried product. S4. Mix the dried product with absolute ethanol until dissolved, then add pure water and stir evenly, perform a first recrystallization treatment, filter to obtain crystals, wash and dry the crystals to obtain crystal powder. S5. Repeat the process of step S4 for the crystal powder for a second recrystallization, and after drying, the rare ginsenoside CK is obtained.

[0007] Through a series of process steps, including enzymatic hydrolysis, filtration, redissolution, centrifugation, microfiltration, ultrafiltration, nanofiltration, drying, and recrystallization, etc., the present invention effectively improves the purification efficiency of rare ginsenoside CK. Only ethanol and water are used as solvents throughout the process, which not only ensures the green environmental protection of the process but also avoids the use of harmful chemical solvents. And in the process flow, an efficient membrane filtration technology is applied. Macromolecular and small-molecular impurities in the crude CK product are removed through ultrafiltration and nanofiltration, significantly improving the purity of the product. At the same time, by controlling the recrystallization process parameters, the crude CK product that was originally not suitable for recrystallization is overcome, and its purification is successfully achieved. The present invention finally produces high-purity rare ginsenoside CK with a content exceeding 95% through steps such as combining ultrafiltration, nanofiltration, and multiple recrystallizations. The method not only improves the production efficiency of rare ginsenoside CK but also greatly reduces environmental pollution. The reusable equipment and solvents make the entire production process have significant cost advantages, suitable for large-scale industrial application, and have broad market prospects and economic benefits.

[0008] Preferably, in step S3, the cut-off molecular weight during ultrafiltration is 800 - 3000.

[0009] Preferably, in step S3, the cut-off molecular weight during ultrafiltration is 1000.

[0010] Preferably, in step S3, the cut-off molecular weight during nanofiltration is 100 - 300.

[0011] Preferably, in the step S3, the molecular weight cut-off during nanofiltration is 300.

[0012] In the preparation method of the present invention, the precise regulation of the molecular weight cut-off membranes for ultrafiltration and nanofiltration is the key to improving the production efficiency and purity of rare ginsenoside CK. When the molecular weight cut-off during ultrafiltration is too low, it will lead to a high content of CK in the ultrafiltration concentrate, resulting in a low weight of the dried product after the next nanofiltration and ultimately a low overall yield. When ultrafiltration is carried out with too high a molecular weight cut-off and then nanofiltration, it will cause the dried powder to not recrystallize smoothly, and a paste-like substance will form during the recrystallization process, thus unable to effectively improve the product purity. When the molecular weight cut-off during nanofiltration is too low, it will lead to a slow filtration rate, thus significantly affecting the production efficiency of rare ginsenoside CK. If the molecular weight cut-off during nanofiltration is too high, it will cause impurities to not be effectively removed, resulting in poor purification effect. Therefore, by combining the ultrafiltration and nanofiltration processes and using the above-preferred molecular weight cut-off parameter ranges for the purification of rare ginsenoside CK, the present invention significantly improves the production efficiency and purity of the product.

[0013] Preferably, the composite enzyme includes β-glucosidase, β-galactosidase, glucosyltransferase, and xylosidase.

[0014] Preferably, the enzyme activity of β-glucosidase is 11000 - 13000 U / g, the enzyme activity of β-galactosidase is 8000 - 10000 U / g, the enzyme activity unit of glucosyltransferase is 3000 - 5000 U / g, and the enzyme activity of xylosidase is 7000 - 9000 U / g; the mass ratio of β-glucosidase, β-galactosidase, glucosyltransferase, and xylosidase is (0.05 - 0.3):(0.02 - 0.2):(0.01 - 0.1):(0.01 - 0.1).

[0015] During the enzymatic hydrolysis and conversion of ginsenoside Rb1 into rare ginsenoside CK, β-glucosidase and β-galactosidase can effectively remove glycosyl groups, simplifying the original complex sugar chain and generating more easily convertible intermediate products; glucosyltransferase can adjust the affinity and reactivity of the molecule by adding glycosyl groups at appropriate places; xylosidase can help remove impurities and maintain the purity of the reaction system.

[0016] Through experimental exploration, it is found that by using the above four enzymes in a specific quantitative ratio range in combination in the present invention, different enzymes cooperate with each other, and through different hydrolysis and transfer reactions, the glycosyl structure of ginsenoside Rb1 is precisely regulated. Through synergistic effects, unnecessary glycosyl groups can be effectively removed or glycosyl groups can be transferred to specific positions, thereby effectively improving the conversion rate of rare ginsenoside CK.

[0017] Preferably, the mass ratio of the β-glucosidase, β-galactosidase, glucosyltransferase and xylosidase is 0.2:0.1:0.05:0.05.

[0018] Preferably, the mass ratio of the ginsenoside Rb1 to the complex enzyme is 1:(0.09 - 0.7).

[0019] Preferably, the mass ratio of the ginsenoside Rb1 to the complex enzyme is 1:0.4.

[0020] Preferably, in the step S4, the mass ratio of the dry matter to the absolute ethanol is 1:(15 - 25), and the volume ratio of the absolute ethanol to the pure water is 1:(1.5 - 2.5).

[0021] Through experimental exploration, it is found that the addition ratio of the dry matter to the absolute ethanol and water during the recrystallization process has an important influence on the purification effect of the final rare ginsenoside CK. When the above preferred ratio range is adopted for the recrystallization process, it is more helpful to improve the crystallization effect of the rare ginsenoside CK, thereby effectively improving its purity; when the addition amount of water is lower than the above preferred range, it will lead to the inability to effectively form crystals and no crystallization, while when the addition amount of water is higher than the preferred range, it will lead to the formation of a paste-like precipitate, resulting in a significant reduction in the purity of the target product.

[0022] Preferably, in the step S4, the mass ratio of the dry matter to the absolute ethanol is 1:20, and the volume ratio of the absolute ethanol to the water is 1:2.

[0023] Preferably, in the step S4, the temperature for the first recrystallization is -20 - 37°C; in the step S5, the temperature for the second recrystallization is -20 - 25°C.

[0024] Preferably, in the step S4, the temperature for the first recrystallization is 4°C; in the step S5, the temperature for the second recrystallization is 4°C.

[0025] During the recrystallization process, the control of temperature also has an obvious influence on the purification effect of the final target product. If the temperature is too low, although the crystals form quickly, other impurities in the solution are easily wrapped into the crystals, resulting in low product purity. If the temperature is too high, the crystal formation will be slow and the amount of crystallization will be small, resulting in a low yield of the final target product. Therefore, precise control of the parameter conditions in the recrystallization process can achieve the best purification effect of the rare ginsenoside CK.

[0026] Preferably, in the step S4, stirring treatment is carried out during the first recrystallization, and the stirring rate is 50 - 150 rpm.

[0027] Preferably, in step S4, stirring treatment is performed during the first recrystallization, and the stirring rate is 100 rpm.

[0028] Preferably, in step S1, the specific conditions for enzymatic hydrolysis are: enzymatic hydrolysis at 45 - 55°C for 70 - 72 h, rotation speed of 20 - 800 rpm, and pH of 6.4 - 6.6.

[0029] Preferably, it includes at least one of the following (1) - (5): (1) In step S1, the mass ratio of ginsenoside Rb1 to phosphate buffer solution is 1:(2 - 20), and the concentration of the phosphate buffer solution is 0.5 - 1.5 mM; (2) In step S1, the mass ratio of the complex enzyme preparation to the phosphate buffer solution is 1:(10 - 20), and the concentration of the phosphate buffer solution is 0.5 - 1.5 mM; (3) In step S2, the mass concentration of the ethanol solution is 40 - 95%, and the mass ratio of the precipitate to the ethanol solution is 1:(8 - 12); (4) In step S2, before plate - frame filtration, the dissolved solution is stirred and mixed with diatomaceous earth and then plate - frame filtration is carried out, and the mass ratio of the dissolved solution to diatomaceous earth is 1:(0.01 - 1); (5) In step S2, the filtration pore size of the microfiltration is 0.22 μm.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) In terms of purification effect, traditional purification methods often cannot effectively remove impurities, resulting in low purity. However, the present invention can effectively purify the crude CK product with a content of only 20% - 70% to more than 75% by jointly adopting two - step processes of ultrafiltration and nanofiltration and precisely controlling the parameter conditions in the process. Moreover, the impurities with larger and smaller molecular weights are removed from the crude CK product after ultra - nanofiltration. By carrying out the recrystallization process, the purity can be further improved. The crude CK product without ultra - nanofiltration process cannot smoothly carry out recrystallization and is prone to form paste - like precipitates during recrystallization. Therefore, the process of the present invention can effectively improve the purity and quality of the final product and can achieve kilogram - level mass production.

[0031] (2) In terms of production cost, the price of rare ginsenoside CK with a 90% content on the current market is usually between 130,000 - 400,000 yuan / kg. However, the preparation process of the present invention can increase the content of rare ginsenoside CK to more than 95%, and the production cost per kilogram is controlled within 50,000 yuan. Thus, the production cost of high - purity rare ginsenoside CK can be significantly reduced, and the market competitiveness of the product can be remarkably enhanced.

[0032] (3) In terms of environmental protection, the solvents used in the preparation process of the present invention only include water and ethanol, which can be recycled and reused, greatly reducing environmental pollution during the production process. Compared with traditional purification methods, the present invention greatly reduces the emission of harmful substances during the production process, providing a feasible solution for green production. Description of the Drawings

[0033] Figure 1 It is the HPLC chromatogram of the ginsenoside CK standard product; Figure 2 It is the HPLC chromatogram of the ginsenoside CK sample prepared in Example 1; Figure 3 It is the HPLC chromatogram of the sample before crystallization during the preparation process of Example 1; Figure 4 It is the appearance diagram of the ginsenoside CK prepared in Example 1. Detailed Embodiments

[0034] To better illustrate the purpose, technical solution and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0035] Unless otherwise specified, the raw materials used in the examples and comparative examples are all conventional raw materials in the art and can be obtained through commercial channels. The experimental operations not specifically described in the examples and comparative examples are all conventional operations in the art or operations that can be understood or known by those skilled in the art according to the existing technology or common general knowledge they have mastered.

[0036] Example 1 This example provides a method for preparing high-purity rare ginsenoside CK, including the following steps: S1. Weigh 1 kg of ginsenoside Rb1 powder, add 1 mM sodium phosphate (pH 7.0) buffer solution according to a mass ratio of 1:10, start stirring at room temperature until completely dissolved, and the stirring frequency is 200 rpm to obtain a ginsenoside Rb1 solution; S2. Mix β-glucosidase, β-galactosidase, glucosyltransferase and xylosidase according to a mass ratio of 0.2:0.1:0.05:0.05 to obtain a composite enzyme. Mix the composite enzyme with 1 mM sodium phosphate (pH 7.0) buffer at a mass ratio of 1:15 until completely dissolved to obtain a composite enzyme solution. According to the DNS method, the enzyme activity of β-glucosidase is measured to be 12000 U / g, the enzyme activity of β-galactosidase is 9000 U / g, the enzyme activity unit of glucosyltransferase is 4000 U / g, and the enzyme activity of xylosidase is 8000 U / g. The mass ratio of β-glucosidase, β-galactosidase, glucosyltransferase and xylosidase is 0.2:0.1:0.05:0.05; S3. Enzymatic hydrolysis: Add the composite enzyme solution obtained in step S2 to the ginsenoside Rb1 solution obtained in step S1 for enzymatic hydrolysis. After stirring evenly, adjust the pH to 6.5 ± 0.1 with 10% sodium hydroxide solution, carry out enzymatic hydrolysis for 72 h, the enzymatic hydrolysis temperature is 50 °C, and the stirring frequency is 800 rpm. The mass ratio of ginsenoside Rb1 to the composite enzyme is 1:0.4; S4. Centrifuge the enzymatic hydrolysate at a centrifugation speed of 16000 rpm, discard the supernatant, collect the precipitate, wash the precipitate with a small amount of pure water, mix the precipitate and 50% ethanol solution at a mass ratio of 1:10, place them in a reaction kettle, start stirring to 200 rpm, set the temperature to 50 °C, and react until the precipitate is completely dissolved to obtain a dissolved solution; S5. Pressure filtration: After cleaning and installing the pressure filter plate frame, weigh diatomaceous earth into the reaction kettle and mix it evenly with the dissolved solution. The mass ratio of the dissolved solution to diatomaceous earth is 1:0.02. Open the discharge port of the reaction kettle, start the peristaltic pump, and introduce the dissolved solution in the reaction kettle into the plate frame to obtain a clear liquid; S6. Microfiltration: Wash the 0.22 μm PTFE folded filter element with pure water. When the pH of the liquid discharged from the outlet is about 7.0, install a stainless steel filter, start the peristaltic pump, open the exhaust port to relieve pressure, open the outlet of the stainless steel filter to drain the residual liquid inside, introduce the clear liquid obtained in step S5, and carry out self-circulation for 5 min. The filtrate A is obtained by discharging; S7. Ultrafiltration: Wash the ultrafiltration machine with pure water. When the conductivity of the waste liquid discharged from the waste liquid port is lower than 5 us / cm, introduce the filtrate A obtained in step S6 into the ultrafiltration machine for ultrafiltration. The cut-off molecular weight during ultrafiltration is 1000, and the ultrafiltration permeate is collected; S8. Nanofiltration: Wash the nanofiltration machine with pure water. When the conductivity of the waste liquid discharged from the waste liquid port is lower than 5 us / cm, introduce the ultrafiltration permeate into the nanofiltration machine for nanofiltration. The cut-off molecular weight during nanofiltration is 300, and the concentrated filtrate B is obtained; S9, drying: after the concentrated filtrate B is evaporated to a paste by a rotary evaporator, the material is transferred to a vacuum drying oven, the temperature is set to 105°C, the material thickness is less than 1 cm, and the surface of the material is repeatedly flattened with a scraper during the drying process, and finally the paste is dried to a block-like dry material with a moisture content of less than 5%; S10, primary recrystallization: the dried product obtained in step S9 is mixed with anhydrous ethanol in a mass ratio of 1:20, stirred until completely dissolved, and then pure water is added and stirred evenly, wherein the volume ratio of anhydrous ethanol to pure water is 1:2, the solution is placed in a 4°C environment, stirred with a stirrer for 24 hours, and the stirring rate is 100 rpm, and the precipitated crystals are filtered with a Buchner funnel, which needs to be placed in a 4°C refrigerator overnight in advance. After the filtration is completed, the crystals are quickly washed with 1L 35% ice ethanol, and the washing and filtration are repeated twice with 1L 35% ice ethanol, and the crystals are dried at 105°C for 4 hours to obtain crystalline powder; S11. Secondary recrystallization: repeat the above step S10, mix the primary recrystallized crystal powder with anhydrous ethanol in a mass ratio of 1:20, stir until completely dissolved, then add pure water and stir evenly, the volume ratio of anhydrous ethanol to pure water is 1:2, place the solution in a 4°C environment, stir with a stirrer for 24 hours, and the stirring rate is 100 rpm. Filter the precipitated crystals with a Buchner funnel, and quickly wash the crystals with 1L 35% ice ethanol after filtration. Repeat the washing and filtration with 1L 35% ice ethanol twice, and dry the crystals at 105°C for 4 hours to obtain the rare ginsenoside CK.

[0037] Example 2 This example provides a method for preparing high-purity rare ginsenoside CK, which is similar to the method described in Example 1, except that: In step S2, the mass ratio of β-glucosidase, β-galactosidase, glucosyltransferase and xylosidase is 0.05:0.02:0.01:0.01; In step S3, the mass ratio of ginsenoside Rb1 to complex enzyme is 1:0.09.

[0038] Example 3 This example provides a method for preparing high-purity rare ginsenoside CK, which is similar to the method described in Example 1, except that: In step S2, the mass ratio of β-glucosidase, β-galactosidase, glucosyltransferase and xylosidase is 0.3:0.2:0.1:0.1; In step S3, the mass ratio of ginsenoside Rb1 to complex enzyme is 1:0.9.

[0039] Example 4 This embodiment provides a method for preparing highly pure rare ginsenoside CK, which is similar to the method described in Embodiment 1, with the only difference being that: In step S7, the cut-off molecular weight during ultrafiltration is 800; In step S8, the cut-off molecular weight during nanofiltration is 100.

[0040] Embodiment 5 This embodiment provides a method for preparing highly pure rare ginsenoside CK, which is similar to the method described in Embodiment 1, with the only difference being that: In step S7, the cut-off molecular weight during ultrafiltration is 3000.

[0041] Embodiment 6 This embodiment provides a method for preparing highly pure rare ginsenoside CK, comprising the following steps: S1. Weigh 1 kg of ginsenoside Rb1 powder, add 1 mM sodium phosphate (pH 7.0) buffer solution in a mass ratio of 1:2, start stirring at room temperature until completely dissolved, and the stirring frequency is 200 rpm to obtain a ginsenoside Rb1 solution; S2. Mix β-glucosidase, β-galactosidase, glucosyltransferase, and xylosidase in a mass ratio of 0.2:0.1:0.05:0.05 to obtain a composite enzyme, and prepare the composite enzyme and 1 mM sodium phosphate (pH 7.0) buffer solution in a mass ratio of 1:20 until completely dissolved to obtain a composite enzyme solution; According to the DNS method, the enzyme activity of β-glucosidase is measured to be 12000 U / g, the enzyme activity of β-galactosidase is 9000 U / g, the enzyme activity unit of glucosyltransferase is 4000 U / g, and the enzyme activity of xylosidase is 8000 U / g. The mass ratio of β-glucosidase, β-galactosidase, glucosyltransferase, and xylosidase is 0.2:0.1:0.05:0.05; S3. Enzymatic hydrolysis: Add the composite enzyme solution obtained in step S2 to the ginsenoside Rb1 solution obtained in step S1 for enzymatic hydrolysis. After stirring evenly, adjust the pH to 6.5 ± 0.1 with 10% sodium hydroxide solution, carry out enzymatic hydrolysis for 74 h, the enzymatic hydrolysis temperature is 45 °C, and the stirring frequency is 20 rpm; The mass ratio of ginsenoside Rb1 to the composite enzyme is 1:0.4; S4. Centrifuge the enzymatic hydrolysate at a centrifugal speed of 16000 rpm, discard the supernatant, collect the precipitate, rinse the precipitate with a small amount of pure water, mix the precipitate and 40% ethanol solution in a mass ratio of 1:8, place them in a reaction kettle, start stirring to 200 rpm, set the temperature to 50 °C, and react until the precipitate is completely dissolved to obtain a dissolved solution; S5, filter press: After cleaning and installing the filter press plate frame, weigh diatomaceous earth into the reactor and mix it evenly with the dissolved liquid, wherein the mass ratio of the dissolved liquid to the diatomaceous earth is 1:0.01, open the feed port of the reactor, start the peristaltic pump, and introduce the dissolved liquid in the reactor into the plate frame to obtain a clear liquid; S6, microfiltration: clean the 0.22 μm PTFE folded filter element with pure water, wait until the pH of the liquid discharged from the outlet is about 7.0, install the stainless steel filter, start the peristaltic pump, open the exhaust port to relieve pressure, open the liquid outlet of the stainless steel filter to drain the internal residual liquid, introduce the clear liquid obtained in step S5, self-circulate for 5 minutes, and discharge the material to obtain filtrate A; S7, ultrafiltration: clean the ultrafiltration machine with pure water, wait until the conductivity of the waste liquid discharged from the waste liquid outlet is lower than 5us / cm, introduce the filtrate A obtained in step S6 into the ultrafiltration machine for ultrafiltration, the molecular weight cutoff during ultrafiltration is 1000, and collect the ultrafiltration permeate; S8, nanofiltration: clean the nanofilter with pure water, wait until the conductivity of the waste liquid discharged from the waste liquid outlet is lower than 5us / cm, introduce the ultrafiltration permeate into the nanofilter for nanofiltration, the molecular weight cutoff during nanofiltration is 300, and obtain concentrated filtrate B; S9, drying: after the concentrated filtrate B is evaporated to a paste by a rotary evaporator, the material is transferred to a vacuum drying oven, the temperature is set to 105°C, the material thickness is less than 1 cm, and the surface of the material is repeatedly flattened with a scraper during the drying process, and finally the paste is dried to a block-like dry material with a moisture content of less than 5%; S10, primary recrystallization: the dried product obtained in step S9 is mixed with anhydrous ethanol in a mass ratio of 1:15, stirred until completely dissolved, and then pure water is added and stirred evenly, wherein the volume ratio of anhydrous ethanol to pure water is 1:1.5, the solution is placed in a -20°C environment, stirred with a stirrer for 24 hours, and the stirring rate is 150rpm, and the precipitated crystals are filtered with a Buchner funnel, which needs to be placed in a 4°C refrigerator overnight in advance. After the filtration is completed, the crystals are quickly washed with 1L 35% ice ethanol, and the washing and filtration are repeated twice with 1L 35% ice ethanol, and the crystals are dried at 105°C for 4 hours to obtain crystalline powder; S11. Secondary recrystallization: repeat the above step S10, mix the primary recrystallized crystal powder with anhydrous ethanol in a mass ratio of 1:15, stir until completely dissolved, then add pure water and stir evenly, the volume ratio of anhydrous ethanol to pure water is 1:1.5, place the solution in a -20°C environment, stir with a stirrer for 24 hours, and the stirring rate is 150rpm. Filter the precipitated crystals with a Buchner funnel, and after filtration, quickly wash the crystals with 1L 35% ice ethanol, repeat the washing and filtration with 1L 35% ice ethanol twice, and dry the crystals at 105°C for 4 hours to obtain the rare ginsenoside CK.

[0042] Example 7 This embodiment provides a method for preparing high-purity rare ginsenoside CK, comprising the following steps: S1. Weigh 1 kg of ginsenoside Rb1 powder, add 1 mM sodium phosphate (pH 7.0) buffer solution according to a mass ratio of 1:20, start stirring at room temperature until completely dissolved, and the stirring frequency is 200 rpm to obtain a ginsenoside Rb1 solution; S2. Mix β-glucosidase, β-galactosidase, glucosyltransferase and xylosidase according to a mass ratio of 0.2:0.1:0.05:0.05 to obtain a composite enzyme, and mix the composite enzyme with 1 mM sodium phosphate (pH 7.0) buffer solution according to a mass ratio of 1:20 until completely dissolved to obtain a composite enzyme solution; According to the DNS method, the enzyme activity of β-glucosidase is measured to be 12000 U / g, the enzyme activity of β-galactosidase is 9000 U / g, the enzyme activity unit of glucosyltransferase is 4000 U / g, and the enzyme activity of xylosidase is 8000 U / g. The mass ratio of the β-glucosidase, β-galactosidase, glucosyltransferase and xylosidase is 0.2:0.1:0.05:0.05; S3. Enzymatic hydrolysis: Add the composite enzyme solution obtained in step S2 to the ginsenoside Rb1 solution obtained in step S1 for enzymatic hydrolysis. After stirring evenly, adjust the pH to 6.5±0.1 with 10% sodium hydroxide solution, carry out enzymatic hydrolysis for 70 h, the enzymatic hydrolysis temperature is 55 °C, and the stirring frequency is 800 rpm; The mass ratio of the ginsenoside Rb1 to the composite enzyme is 1:0.4; S4. Centrifuge the enzymatic hydrolysate at a centrifugation speed of 16000 rpm, discard the supernatant, collect the precipitate, rinse the precipitate with a small amount of pure water, mix the precipitate and 95% ethanol solution according to a mass ratio of 1:12, place them in a reaction kettle, start stirring to 200 rpm, set the temperature to 50 °C, and react until the precipitate is completely dissolved to obtain a dissolved solution; S5. Pressure filtration: After cleaning and installing the pressure filter plate frame, weigh diatomaceous earth into the reaction kettle and mix it evenly with the dissolved solution. The mass ratio of the dissolved solution to diatomaceous earth is 1:0.1. Open the discharge port of the reaction kettle, start the peristaltic pump, and introduce the dissolved solution in the reaction kettle into the plate frame to obtain a clear liquid; S6. Microfiltration: Wash the 0.22 μm PTFE folded filter element with pure water. When the pH of the liquid discharged from the liquid outlet is about 7.0, install a stainless steel filter, start the peristaltic pump, open the exhaust port to relieve pressure, open the liquid outlet of the stainless steel filter to drain the residual liquid inside, introduce the clear liquid obtained in step S5, and circulate for 5 min. The filtrate A is obtained by discharging; S7. Ultrafiltration: Wash the ultrafiltration machine with pure water. When the conductivity of the waste liquid discharged from the waste liquid outlet is lower than 5 us / cm, introduce the filtrate A obtained in step S6 into the ultrafiltration machine for ultrafiltration. The cut-off molecular weight during ultrafiltration is 1000, and the ultrafiltration permeate is collected; S8, nanofiltration: clean the nanofilter with pure water, wait until the conductivity of the waste liquid discharged from the waste liquid outlet is lower than 5us / cm, introduce the ultrafiltration permeate into the nanofilter for nanofiltration, the molecular weight cutoff during nanofiltration is 300, and obtain concentrated filtrate B; S9, drying: after the concentrated filtrate B is evaporated to a paste by a rotary evaporator, the material is transferred to a vacuum drying oven, the temperature is set to 105°C, the material thickness is less than 1 cm, and the surface of the material is repeatedly flattened with a scraper during the drying process, and finally the paste is dried to a block-like dry material with a moisture content of less than 5%; S10, primary recrystallization: the dried product obtained in step S9 is mixed with anhydrous ethanol in a mass ratio of 1:25, stirred until completely dissolved, and then pure water is added and stirred evenly, wherein the volume ratio of anhydrous ethanol to pure water is 1:2.5, the solution is placed in a 37°C environment, stirred with a stirrer for 24 hours, and the stirring rate is 20 rpm, and the precipitated crystals are filtered with a Buchner funnel, which needs to be placed in a 4°C refrigerator overnight in advance. After the filtration is completed, the crystals are quickly washed with 1L 35% ice ethanol, and the washing and filtration are repeated twice with 1L 35% ice ethanol, and the crystals are dried at 105°C for 4 hours to obtain crystalline powder; S11. Secondary recrystallization: repeat the above step S10, mix the primary recrystallized crystal powder with anhydrous ethanol in a mass ratio of 1:25, stir until completely dissolved, then add pure water and stir evenly, the volume ratio of anhydrous ethanol to pure water is 1:2.5, place the solution in a 25°C environment, stir with a stirrer for 24 hours, and the stirring rate is 20rpm. Filter the precipitated crystals with a Buchner funnel, and after filtration, quickly wash the crystals with 1L 35% ice ethanol, repeat the washing and filtration with 1L 35% ice ethanol twice, and dry the crystals at 105°C for 4 hours to obtain the rare ginsenoside CK.

[0043] Comparative Example 1 This comparative example provides a method for preparing high-purity rare ginsenoside CK, which is similar to the method described in Example 1, except that: Without adding β-galactosidase, the mass ratio of β-glucosidase, glucosyltransferase and xylosidase is 0.2:0.1:0.1, and the mass ratio of ginsenoside Rb1 to complex enzyme is still 1:0.4.

[0044] Comparative Example 2 This comparative example provides a method for preparing high-purity rare ginsenoside CK, which is similar to the method described in Example 1, except that: Without adding glucosyltransferase, the mass ratio of the β-glucosidase, β-galactosidase and xylosidase is 0.2:0.1:0.1, and the mass ratio of the ginsenoside Rb1 to the complex enzyme is still 1:0.4.

[0045] Comparative Example 3 This comparative example provides a method for preparing high-purity rare ginsenoside CK, which is similar to the method described in Example 1, except that: In step S7, the cut-off molecular weight during ultrafiltration is 500; In step S8, the cut-off molecular weight during nanofiltration is 50.

[0046] Comparative Example 4 This comparative example provides a method for preparing high-purity rare ginsenoside CK, which is similar to the method described in Example 1, except that: In step S7, the cut-off molecular weight during ultrafiltration is 5000; In step S8, the cut-off molecular weight during nanofiltration is 400.

[0047] Comparative Example 5 This comparative example provides a method for preparing high-purity rare ginsenoside CK, which is similar to the method described in Example 1, except that: in this comparative example, the ultrafiltration process is not carried out, and the filtrate A obtained in step S6 is directly subjected to subsequent nanofiltration treatment.

[0048] Comparative Example 6 This comparative example provides a method for preparing high-purity rare ginsenoside CK, which is similar to the method described in Example 1, except that: in this comparative example, the nanofiltration process is not carried out, and the ultrafiltration permeate obtained in step S7 is directly subjected to subsequent drying treatment.

[0049] Comparative Example 7 This comparative example provides a method for preparing high-purity rare ginsenoside CK, which is similar to the method described in Example 1, except that: In step S10, the volume ratio of the absolute ethanol to pure water is 1:3.5, and the solution is placed in an environment of -30 °C for recrystallization.

[0050] Comparative Example 8 This comparative example provides a method for preparing high-purity rare ginsenoside CK, which is similar to the method described in Example 1, except that: In step S10, the volume ratio of the absolute ethanol to pure water is 1:0.5, and the solution is placed in an environment of 45 °C for recrystallization.

[0051] Comparative Example 9 This comparative example provides a method for preparing rare ginsenoside CK, which is similar to the method described in Example 1, except that: in this comparative example, the secondary recrystallization process is not carried out.

[0052] Effect Example Taking the rare ginsenoside CK prepared by the preparation method 1 described in Examples 1-7 and Comparative Examples 1-9 as samples, the content and conversion rate of rare ginsenoside CK were detected and calculated by liquid chromatography. The specific operation process of liquid chromatography detection is as follows: Weigh 25 mg of ginsenoside CK reference substance (accurate to 0.0001 g), place it in a 25 mL volumetric flask, add an appropriate amount of methanol to dissolve it, continue to add methanol to the scale line, shake well, and obtain a standard solution (stock solution) with a concentration of 1000 mg / L. It can be stored sealed at 4°C for one month, and the purity of the ginsenoside CK reference substance is ≥98%.

[0053] Respectively take 0.50 mL, 1.0 mL, 2.5 mL, and 5 mL of the stock solution of ginsenoside CK reference substance and place them in 10 mL volumetric flasks, add methanol to the scale, and obtain ginsenoside CK standard series solutions with corresponding concentrations of 50 mg / L, 100 mg / L, 250 mg / L, 500 mg / L, and 1000 mg / L.

[0054] For the solid powder sample of ginsenoside CK with a content ≥10% in the sample to be tested, weigh 0.2 - 0.5 g (accurate to 0.0001 g); for the liquid sample with a content <10%, weigh 2 - 10 g (accurate to 0.0001 g). For samples with a lower content of ginsenoside CK, the sample weighing amount can be appropriately increased. Place the sample in a 25 mL volumetric flask, add methanol to the scale line, ultrasonically oscillate for 30 minutes, shake well, and filter with a 0.22 μm organic filter membrane as the sample to be tested. When the content in the sample exceeds the standard curve range, the sample should be appropriately diluted and a suitable standard curve range should be selected for detection.

[0055] The chromatographic column is a C18 column, the detection wavelength is 311 nm, the flow rate is 1.0 mL / min, the column temperature is 35°C, the injection volume is 10 μL, mobile phase A is primary water meeting the requirements of GB / T 6682, and mobile phase B is chromatographic grade acetonitrile. Gradient elution is carried out, and the elution program is shown in Table 1. Inject the standard series solutions in sequence, record the chromatogram, use the concentration of the standard series solutions as the abscissa and the peak area as the ordinate to draw the standard curve. Inject the samples to be tested in sequence, record the chromatogram, qualitatively analyze by retention time and ultraviolet spectrogram, measure the peak area, obtain the mass concentration of each component in the sample to be tested according to the standard curve, and calculate the content and conversion rate of each component in the sample. The calculation formula is as shown in the following formula: In the formula, ω is the mass fraction of each component in the sample, %; ρ is the mass concentration of each component to be tested obtained from the standard curve, mg / L; V is the sample constant volume, mL; m is the sample sampling amount, g; n is the dilution factor; In Formula 2, n is the amount of ginsenoside CK or ginsenoside Rb1, which is obtained by multiplying the content of ginsenoside by the weight of ginsenoside and then dividing by the relative molecular mass of ginsenoside (1109.29 for ginsenoside Rb1 and 622.87 for ginsenoside CK).

[0056] The liquid chromatogram of ginsenoside CK standard is shown in Figure 1 The liquid chromatogram of ginsenoside CK prepared in Example 1 is shown in Figure 2 The specific results of the content of each component in the sample and the conversion rate are shown in Table 2.

[0057] Table 1 Elution procedure for ginsenoside CK by liquid chromatography Time / min Volume (mobile phase A) / % Volume (mobile phase B) / % 0 80 20 4 50 50 12 20 80 16 0 100 16.5 60 40 17 80 20 20 80 20 Table 2 From Table 2 and Figure 1 - 3 The results show that the preparation methods described in Examples 1-7 of the present invention can effectively improve the production efficiency and purity of rare ginsenoside CK, especially the method described in Example 1 can increase the content of ginsenoside CK in the product to 97.16%, and the purification effect of ginsenoside CK is the best. When the β-galactosidase and glucosyltransferase in the specific composite enzymes described in Examples 1-2 are missing, the conversion rate of ginsenoside CK is significantly reduced, indicating that the four enzymes selected in the present invention cooperate with each other to better accurately regulate the glycosyl structure of ginsenoside Rb1 and thus synergistically improve the conversion rate of ginsenoside CK; when the molecular weight cutoff parameter conditions in the ultrafiltration and nanofiltration processes described in Examples 3-4 exceed the preferred range of the present invention, the purity of ginsenoside CK is also significantly reduced, and when the ultrafiltration and nanofiltration processes are missing in Examples 5-6, the purity of the obtained ginsenoside CK decreases more significantly. The above results show that the present invention combines ultrafiltration and nanofiltration to obtain ginsenoside CK. The nanofiltration process is used to purify ginsenosides, and the key parameter of the molecular weight cutoff in the process is precisely controlled to achieve better production efficiency and purity of ginsenoside CK. Once the molecular weight cutoff deviates slightly from the specified range of the present invention, the purification effect of ginsenoside CK will be significantly deteriorated. In comparative examples 7-9, the important parameters involved in the recrystallization process in the preparation method described in the present invention are explored, and it is found that the recrystallization temperature and the amount of water added during crystallization have an impact on the purification effect of the final ginsenoside CK, and a single recrystallization process is not sufficient to fully improve the purity. Therefore, the present invention proposes a strategy of combining multiple processes to improve the purity and stability of the final product.

[0058] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing rare ginsenoside CK, characterized in that: The following steps are involved: S1, stirring and mixing ginsenoside Rb1 and phosphate buffer until dissolved to obtain ginsenoside Rb1 solution, then mixing composite enzyme with phosphate buffer to obtain composite enzyme solution, adding composite enzyme solution to ginsenoside Rb1 solution for enzymolysis to obtain enzymolysis solution; S2, centrifuging the enzymatic hydrolyzate, collecting the precipitate, stirring and mixing the precipitate with the ethanol solution to obtain a dissolved solution, and filtering the dissolved solution by pressure and microfiltration to obtain a filtrate A; S3, ultrafiltration and nanofiltration are performed on the filtrate A in step S2 to obtain a concentrated filtrate B, and then the concentrated filtrate B is dried to obtain a dried product; S4, mixing the dried product with anhydrous ethanol until dissolved, adding pure water and stirring evenly, performing a recrystallization treatment, filtering to obtain crystals, washing the crystals, and drying to obtain crystalline powder; S5, repeating step S4 to perform secondary recrystallization on the crystal powder, and obtaining the rare ginsenoside CK after drying; The complex enzyme comprises beta-glucosidase, beta-galactosidase, glucosyltransferase and xylosidase.

2. The preparation method according to claim 1, characterized in that The enzyme activity of the β-glucosidase is 11000-13000U / g, the enzyme activity of the β-galactosidase is 8000-10000U / g, the enzyme activity unit of the glucosyltransferase is 3000-5000U / g, and the enzyme activity of the xylosidase is 7000-9000U / g; The mass ratio of the β-glucosidase, β-galactosidase, glucosyltransferase and xylosidase is (0.05-0.3):(0.02-0.2):(0.01-0.1):(0.01-0.1).

3. The preparation method according to claim 1, characterized in that: The mass ratio of the ginsenoside Rb1 to the complex enzyme is 1:(0.09-0.7).

4. The preparation method according to claim 1, characterized in that: In the step S3, the molecular weight cut-off during ultrafiltration is 800-3000.

5. The preparation method according to claim 1, characterized in that: In the step S3, the molecular weight cut-off during nanofiltration is 100-300.

6. The preparation method according to claim 1, characterized in that: In the step S4, the mass ratio of the dried product to anhydrous ethanol is 1:(15-25), and the volume ratio of anhydrous ethanol to pure water is 1:(1.5-2.5).

7. The preparation method according to claim 1, characterized in that: In step S1, the specific conditions of the enzymatic hydrolysis are: enzymatic hydrolysis at 45-55°C for 70-74h, a rotation speed of 20-800rpm, and a pH of 6.4-6.

6.

8. The preparation method according to claim 1, characterized in that: In the step S4, the temperature for the first recrystallization is -20-37°C; in the step S5, the temperature for the second recrystallization is -20-25°C.

9. The preparation method according to claim 1, characterized in that: Include at least one of the following (1)-(5): (1) In step S1, the mass ratio of ginsenoside Rb1 to phosphate buffer is 1:(2-20), and the concentration of the phosphate buffer is 0.5-1.5 mM; (2) In step S1, the mass ratio of the complex enzyme to the phosphate buffer is 1:(10-20), and the concentration of the phosphate buffer is 0.5-1.5 mM; (3) In step S2, the mass concentration of the ethanol solution is 40-95%, and the mass ratio of the precipitate to the ethanol solution is 1:(8-12); (4) In the step S2, the dissolved liquid and diatomaceous earth are stirred and mixed before the plate and frame filter pressing, and the mass ratio of the dissolved liquid to the diatomaceous earth is 1: (0.01-0.1); (5) In step S2, the filtration pore size of the microfiltration is 0.22 μm.

10. Use of the preparation method according to any one of claims 1 to 9 in the preparation of rare ginsenoside CK.

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