Preparation method of glucosyl puerarin

Abstract

The invention discloses a preparation method of glucosyl puerarin, which belongs to the technical field of pharmaceutical chemicals, and comprises the following steps: S1, adding sugar for reaction, mixing puerarin with pure water, adjusting the pH value to 4.5-5.5, adding beta-cyclodextrin, heating to 45-55 DEG C, adding cyclodextrin glucosyltransferase for reaction for 16-22 hours, then adjusting the pH value to 0.1-0.8, adding activated carbon, filtering filtrate and staying overnight at low temperature; s2, desugaring reaction: adjusting the pH value of the filtrate to 3.8-4.5, heating to 55-65 DEG C, adding saccharifying enzyme and glucose oxidase to react for 25-40 minutes, adjusting the pH value to 0.5-1.2, and filtering; s3, column purification: enabling a reaction solution to pass through a mixed column containing sulfonated modified XAD-16 and amino modified HPD-720 resin, and performing graded elution to collect an ethanol eluent; s4, concentrating and drying to obtain a product. The problems that the water solubility of puerarin is poor, the purity of glucosyl puerarin is low, and impurities are difficult to remove can be solved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine and chemical industry, and particularly relates to a preparation method of glucosyl puerarin. BACKGROUND

[0002] As the core active ingredient of Pueraria lobata, puerarin (chemical name: 4,7-dihydroxy-8-beta-D-glucosyl isoflavone) has the pharmacological effects of dilating blood vessels, improving microcirculation and resisting oxidation, and has broad application prospects in the fields of cardiovascular disease treatment, health product development and cosmetic raw material.

[0003] However, puerarin has a significant physical property defect, that is, it is only soluble in organic solvents such as DMSO and DMF, and has extremely low solubility in water and is slightly soluble in ethanol. This property directly leads to low solubility and absorption efficiency of puerarin in the body, low bioavailability, and greatly limits the application of puerarin in clinical commonly used preparations such as injections and oral solutions.

[0004] In the prior art, in order to improve the water solubility of puerarin, a cosolvent (such as propylene glycol, polysorbate 80, etc.) is usually added, but the introduction of the cosolvent will increase the risk of side effects of the product (such as allergic reactions, liver and kidney function damage, etc.), and may also affect the stability of the pharmacological activity of puerarin.

[0005] Therefore, developing a puerarin derivative with excellent water solubility without relying on a cosolvent and an efficient preparation method thereof has become the key to solving the application bottleneck of puerarin. SUMMARY

[0006] The purpose of the present application is to provide a preparation method of glucosyl puerarin to solve the problem of low purity of glucosyl puerarin derivatives in the prior art.

[0007] The purpose of the present application is achieved by the following technical solutions: A preparation method of glucosyl puerarin, comprising the following steps: S1. Sugar reaction: puerarin and pure water are mixed and uniformly dispersed, then the pH of the system is adjusted to 4.5-5.5, β-cyclodextrin is added and stirred until dissolved, the system is then warmed to 45-55℃, and cyclodextrin glucosyltransferase is added, and the constant temperature reaction is continued for 16-22h

both to ensure the stability of cyclodextrin glucosyltransferase activity (avoiding inactivation at high temperature or low efficiency at low temperature) and to improve the conversion rate of puerarin

[0008] The amino group (-NH2) on the surface of the amino-modified HPD-720 resin is protonated to form -NH3 in an acidic system + (Positive); the glucose puerarin molecule contains multiple hydroxyl groups (-OH), and the oxygen atom of the hydroxyl group has strong electronegativity, making the oxygen atom have weak negative electricity and the hydrogen of the hydroxyl group have weak positive electricity. The overall molecular polarity is much higher than that of isoflavone derivatives. This strong polarity characteristic makes the weak negative electricity of the hydroxyl oxygen atom of glucose puerarin able to form strong electrostatic attraction with the -NH3 + (Positive) of the amino-modified HPD-720 resin, and at the same time, the hydrogen atom in the -NH3 + can also form multiple hydrogen bonds with the hydroxyl oxygen atom, and the double strong forces enable the amino-modified HPD-720 resin to preferentially adsorb the target product glucose puerarin.

[0009] S4. Concentration and drying: The collected 75-85% ethanol solution eluate is concentrated under reduced pressure until there is no ethanol smell to obtain a concentrated solution; the concentrated solution is freeze-dried to obtain the glucose puerarin product.

[0010] As some embodiments of the present application, in step S1, the amount ratio of puerarin to pure water is 1g:(35-45)mL; the mass ratio of puerarin to β-cyclodextrin is 1:(3.5-4.5); the enzyme activity of the cyclodextrin glucosyltransferase is 8000-12000U / mL, and the amount used is 0.8-1.2% of the mass of puerarin.

[0011] As some embodiments of the present application, in step S2, the enzyme activity of the saccharifying enzyme is 40000-60000U / g, and the amount used is 0.007-0.01% of the volume of the filtrate; the enzyme activity of the glucose oxidase is 150-250U / mg, and the amount used is 0.008-0.012g / L.

[0012] As some embodiments of the present application, as some embodiments of the present application, the filtration steps in steps S1 and S2 both use 0.2-0.3μm microporous filter membranes.

[0013] As some embodiments of the present application, in the mixed resin, the volume ratio of the sulfonated modified XAD-16 resin to the amino-modified HPD-720 resin is 1:(1.5-2.5).

[0014] As some embodiments of the present application, the preparation method of the sulfonated modified XAD-16 resin is as follows: Take XAD-16 resin, soak in ethanol until completely swollen, after suction filtration, add dichloromethane with a volume of 2-4 times of the resin, under nitrogen protection, at 25-35℃, drop 8-12% of the resin mass of chlorosulfonic acid and stir the reaction; after the reaction is completed, suction filtration, first neutralize with sodium carbonate solution to pH 6.0-7.5, then rinse with pure water until the effluent conductivity is ≤15 μS / cm, finally soak in pH 1.8-2.2 acid water for 10-14 h; after soaking is completed, suction filtration, vacuum drying to obtain the product.

[0015] As some embodiments of the present application, the preparation method of the amino modified HPD-720 resin is as follows: Take HPD-720 resin, soak in KH-550 ethanol solution for 20-28 h, then sequentially rinse with ethanol, hot water, and acid water to remove ungrafted KH-550 and impurities; after rinsing is completed, introduce nitrogen to purge the resin, and finally vacuum dry to obtain the product; wherein the KH-550 ethanol solution contains 0.08-0.12 wt% glucose. The multiple hydroxyl groups in the glucose molecule structure can form hydrogen bonds with the general adsorption sites on the resin surface that are not involved in amino grafting (such as the hydroxyl groups and ether bonds on the resin skeleton), thereby preoccupying these sites; since the size of glucose is smaller than that of glucosyl puerarin, and the adsorption of amino modified HPD-720 resin to glucosyl puerarin is stronger than that to glucose, the glucose occupies the general adsorption sites and does not compete with the target product for other more specific adsorption sites, which can avoid the competition between dehydroxy puerarin and other isoflavone derivatives with the target product through general sites. If glucose is missing, the general adsorption sites are exposed, and impurities can be co-adsorbed with the target product on the amino modified HPD-720 resin through hydrogen bonds or hydrophobic interactions, not only leading to a decrease in the purity of the target product, but also occupying part of the effective adsorption sites, reducing the adsorption capacity of the resin to glucosyl puerarin, and causing the loss of the target product. At the same time, although the sulfonated modified XAD-16 resin can preferentially adsorb most of the isoflavone derivative impurities, a small amount of impurities that are not captured can still compete with the target product, and the pre-occupying effect of glucose can further block this competition path, forming synergy with the impurity capture effect of the sulfonated modified XAD-16 resin, and together ensuring the separation efficiency of the mixed resin column and the purity of the target product.

[0016] In addition, the amount of glucose added in the KH-550 ethanol solution needs to be strictly controlled at 0.08-0.12wt%, at which the hydrogen bonding between glucose and the amino group of KH-550 is weak, and the grafting reaction between KH-550 and the hydroxyl group on the surface of the HPD-720 resin is not significantly affected; in the subsequent water and ethanol elution process, the glucose can be completely removed with the eluent because the binding force between the glucose and the general adsorption sites of the resin is weaker than that of the target product, and the glucose will not remain in the resin to affect the subsequent adsorption efficiency.

[0017] As some embodiments of the present application, in step S3, the adsorption flow rate is 0.8-1.5BV / h; the pH of the acid water is 1.8-2.5, the temperature is 20-35℃, the flow rate is 1-2BV / h, and 1-2 column volumes are eluted; the flow rate of pure water is 1.5-2.5BV / h, and the elution is performed until the effluent is neutral and the conductivity is reduced to 70-120ppm; the flow rate of the 55-65% ethanol solution is 0.8-1.2BV / h, and 1-2 column volumes are eluted; the flow rate of the 75-85% ethanol solution is 0.6-1.2BV / h, and 1.5-2.5 column volumes are eluted.

[0018] As some embodiments of the present application, in step S4, the temperature for vacuum concentration is 45-60℃, the vacuum degree is -0.07~ -0.1MPa, and the concentration is performed to a density of 1.1-1.4g / cm³. In this scheme, by limiting the parameters of vacuum concentration, the removal of ethanol can be achieved while avoiding the degradation of glucose-based puerarin caused by high temperature; In freeze-drying, the pre-freezing temperature is -55~ -40℃, the pre-freezing time is 1-1.5h, the vacuum degree for sublimation drying is 0.5-8Pa, and the drying time is 10-16h. By limiting the parameters of freeze-drying, the product can be effectively prevented from caking, and the water solubility of the final product is stable.

[0019] Compared with the prior art, the present application has the following advantages: The present application solves the problems of poor water solubility of puerarin, difficult removal of impurities (such as dehydroxyl puerarin and other isoflavone derivatives) and low purity of the product by the whole process of sugar addition reaction, desugar reaction, column purification, concentration and drying. Specifically, in the sugar addition reaction, pure water is used as the solvent, and the donor effect of β-cyclodextrin is combined with the directional catalysis of cyclodextrin glucosyltransferase to lay the foundation for the generation of the target product; in the desugar reaction, the synergistic effect of glucoamylase and glucose oxidase is used to efficiently convert the intermediate product and eliminate feedback inhibition to avoid the generation of impurities; in the column purification stage, sulfonated modified XAD-16 resin and amino modified HPD-720 resin are used to capture isoflavone derivative impurities and target products, and the impurities such as inorganic salts and β-cyclodextrin hydrolysis fragments are effectively removed by fractional elution; finally, low-temperature vacuum concentration and freeze-drying are used to maximize the retention of product properties.

[0020] By using the above preparation process provided by the present application, the solubility of the finally prepared glucosyl puerarin is significantly improved without relying on a cosolvent, compared with the extremely low water solubility of puerarin (only soluble in organic solvents such as DMSO and DMF, with extremely low solubility in water); at the same time, the product purity is stable, effectively solving the problem of impurity residues, and effectively meeting the development needs of injection and oral liquid preparations. DETAILED DESCRIPTION

[0021] Example 1 S1. Sugar addition reaction: 20 g of puerarin was added to 800 mL of pure water at a ratio of 1 g:40 mL, stirred at 300 r / min, and treated with 320 W, 25 kHz ultrasound for 12 min until evenly dispersed; the pH of the system was adjusted to 5.0 with 10 wt% citric acid, 80 g of β-cyclodextrin with a particle size of 90 mesh was added, and stirring and ultrasonic treatment was continued for 6 min until dissolution; the temperature was increased to 50℃ at a rate of 5℃ / min, and after constant temperature for 10 min, 0.2 mL of cyclodextrin glucosyltransferase with an enzyme activity of 10000 U / mL was added, and constant temperature reaction was carried out for 18 h; after the reaction was completed, the pH was adjusted to 0.5 with 1 mol / L hydrochloric acid, 0.5 g of 200 mesh activated carbon was added, stirring was carried out at 32℃ for 2 h, and the filtrate was filtered through a 0.22 μm microporous filter and placed at 4℃ overnight.

[0022] S2. Desugar reaction: 1 mol / L sodium hydroxide was added to the overnight filtrate to adjust the pH to 4.2, and the temperature was increased to 60℃; 80 μL of glucoamylase with an enzyme activity of 50000 U / g was added, and 0.01 g / L of glucose oxidase with an enzyme activity of 200 U / mg was added at the same time (the amount added was calculated based on the filtrate), and constant temperature reaction was carried out for 30 min; the pH was adjusted to 1.0 with 1 mol / L hydrochloric acid, cooled to room temperature in an ice bath for 12 min, and filtered through a 0.22 μm microporous filter.

[0023] S3. Column purification: S31. Mixed resin preparation: Take sulfonated modified XAD-16 resin (40 mL) and amino modified HPD-720 resin (80 mL), mix thoroughly, and then pack into a glass chromatographic column with an inner diameter of 2.5 cm and a column height of 30 cm; when packing, use wet packing (with pure water as a suspending agent), slowly pour the mixed resin into the column while stirring, and avoid generating air bubbles; after packing is completed, rinse with pure water at a flow rate of 1.5 BV / h for 1 column volume to make the resin bed compact and flat.

[0024] The preparation method of the sulfonated modified XAD-16 resin is as follows: soak the XAD-16 resin in 95% ethanol (v / v) for 24 h to swell the resin, then filter and add 3 volumes of anhydrous dichloromethane (previously dehydrated), drop 10% chlorosulfonic acid by mass of the resin at a rate of 1 mL / min under ice water bath temperature control (0-5°C) and nitrogen protection, after the drop is completed, warm to 30°C and stir for 4 h, then adjust the pH of the system to 7.0 with 5wt% sodium carbonate solution, rinse with pure water until the conductivity is ≤15 μS / cm, and finally soak in acid water with a pH of 2.0 for 12 h. Finally, vacuum dry (−0.07 MPa) at 42°C for 5 h to obtain the sulfonated modified XAD-16 resin.

[0025] The preparation method of the amino modified HPD-720 resin is as follows: soak the HPD-720 resin in 3wt% KH-550 ethanol solution (containing 0.1wt% glucose) for 24 h, then rinse with 95% ethanol (v / v) at a speed of 1.5 BV / h for 4 column volumes, rinse with 50°C pure water at a speed of 2 BV / h for 2 column volumes, and finally rinse with acid water with a pH of 2.0 at a speed of 1.5 BV / h for 1 column volume. After rinsing is completed, blow with nitrogen gas with a purity of ≥99.9% at a flow rate of 1 BV / h for 1.5 h, and vacuum dry (−0.06 MPa) at 40°C for 4 h to obtain the amino modified HPD-720 resin, which has no KH-550 residue detected by GC.

[0026] S32. Adsorption and elution: pass the filtered reaction liquid of step S2 through the column at a flow rate of 1.0 BV / h, then sequentially elute with acid water with a pH of 2.0 at 28°C at a flow rate of 1.5 BV / h for 1.5 column volumes, pure water at a flow rate of 2 BV / h until the conductivity is ≤100 ppm, 60% ethanol at a flow rate of 1 BV / h for 1.5 column volumes, 80% ethanol at a flow rate of 0.8 BV / h for 2 column volumes, and collect the eluent of 80% ethanol.

[0027] S4. Concentration and drying: The eluent of 80% ethanol by volume was concentrated to a density of 1.2 g / cm3 at 48℃ and -0.08 MPa reduced pressure, and the concentrated solution was divided into trays (1 cm in thickness) and cooled to -50℃ at a rate of 5℃ / min for pre-freezing for 1.5 h, and then sublimation dried at 1 Pa for 13 h to obtain the gluco-panoxin product.

[0028] Example 2 S1. Sugar-added reaction: 20 g of panoxin was added to 760 mL of pure water at a ratio of 1 g:38 mL, and stirred at 220 r / min and treated with ultrasonic waves at 350 W and 30 kHz for 10 min to disperse uniformly; 75 g of β-cyclodextrin with a particle size of 80 mesh was added after adjusting the pH to 4.8 with 10 wt% citric acid, and the stirring and ultrasonic treatment was continued for 5 min; the temperature was increased to 48℃ at a rate of 5℃ / min, and 0.18 mL of cyclodextrin glucosyltransferase with an enzyme activity of 9000 U / mL was added, and the reaction was carried out at constant temperature for 20 h; after the reaction, the pH was adjusted to 0.4, 0.5 g of activated carbon with a mesh size of 180 was added, and stirred at 35℃ for 2.2 h, and then filtered through a 0.25 μm filter membrane, and the filtrate was placed at 2℃ overnight.

[0029] S2. Deglucosylation reaction: 1 mol / L sodium hydroxide was added to the overnight filtrate to adjust the pH to 4.0, and the temperature was increased to 58℃; 78 μL of saccharifying enzyme with an enzyme activity of 45000 U / g was added, and 0.009 g / L of glucose oxidase with an enzyme activity of 200 U / mg was added at the same time, and the reaction was carried out at constant temperature for 35 min; the pH was adjusted to 1.0 with 1 mol / L hydrochloric acid, and cooled to room temperature in an ice bath for 10 min, and then filtered through a 0.25 μm microfiltration membrane.

[0030] S3. Column purification: S31. Preparation of mixed resins: sulfonated modified XAD-16 resin (40 mL) and amino modified HPD-720 resin (88 mL) were mixed and then packed into a glass chromatographic column with an inner diameter of 2.5 cm and a column height of 30 cm; wet packing was used during packing (pure water was used as a suspending agent), and the mixed resins were slowly poured into the column while stirring to avoid the generation of air bubbles; after packing, the resins were rinsed with pure water at a flow rate of 1.5 BV / h for 1 column volume to make the resin bed compact and flat.

[0031] The preparation methods of the two resins were the same as in Example 1.

[0032] S32. Adsorption and elution: the same as in Example 1.

[0033] S4. Concentration and drying: the same as in Example 1.

[0034] It is worth mentioning that the acid water mentioned in the above embodiments 1-2 is a dilute hydrochloric acid solution.

[0035] Comparative Example 1 Compared with Example 1, no glucose oxidase was added in the S2 desugar reaction, and the remaining steps and parameters were the same as those in Example 1.

[0036] Comparative Example 2 Compared with Example 1, neither XAD-16 resin nor HPD-720 resin was modified in S3, and the remaining steps and parameters were the same as those in Example 1.

[0037] Comparative Example 3 Compared with Example 1, the amino-modified HPD-720 resin was replaced by unmodified AB-8 resin, and the remaining steps and parameters were the same as those in Example 1.

[0038] Comparative Example 4 Compared with Example 1, 60% ethanol was changed to 70% ethanol and 80% ethanol was changed to 65% ethanol during elution in S3, and the remaining steps and parameters were the same as those in Example 1.

[0039] Comparative Example 5 Compared with Example 1, the KH-550 ethanol solution did not contain glucose during the preparation of the amino-modified HPD-720 resin, and the remaining steps and parameters were the same as those in Example 1.

[0040] Yield (%) = [ (actual product mass x purity of glucose-based puerarin in product) ÷ (initial mass of puerarin x mass conversion coefficient) ] x 100%. The mass conversion coefficient is 1.39.

[0041] Experimental Example The products prepared in Examples 1-2 and Comparative Examples 1-5 were tested for relevant indicators, and the test data are shown in Table 1.

[0042] Table 1: Note: “—” in Table 1 means that the relevant experiment was not performed.

[0043] From Table 1, it can be seen that: Examples 1-2 strictly followed the full-process operation of the present application, i.e., sugar addition reaction → desugar reaction → column purification → concentration and drying, and the final product had excellent performance, with a product purity ≥82.8%, a dehydroxy puerarin residue ≤0.4%, and 1g of the product being soluble in no more than 9.2mL of water under the test conditions of 25℃ and 300r / min stirring for 30min, without the need for a cosolvent to meet the water solubility requirements of preparations such as injections and oral solutions.

[0044] In the comparative example 1, the accumulation of glucose in the system due to the absence of glucose oxidase reduces the relative concentration of glucosan-based puerarin, indirectly slows down the catalytic efficiency of saccharifying enzyme, reduces the efficiency of the conversion of glucosan-based puerarin into glucosan-based puerarin, and further leads to the decrease in product purity and yield; at the same time, the decrease in reaction efficiency may prolong the reaction time and increase the risk of degradation of the target product, further affecting the product quality.

[0045] In the comparative example 2, the unmodified XAD-16 resin lacks sulfonic acid groups and cannot effectively adsorb isoflavone derivative impurities such as dehydroxy puerarin; the unmodified HPD-720 resin has no amino modification and has poor adsorption selectivity for glucosan-based puerarin, and is easy to co-adsorb impurities. Finally, the impurities in the obtained product cannot be effectively removed, and the product purity is significantly reduced.

[0046] In the comparative example 3, the unmodified AB-8 resin is a general adsorption resin, which has no adsorption sites (such as -NH3 + ) for glucosan-based puerarin, and is easy to co-adsorb impurities such as β-cyclodextrin hydrolysis fragments and residual glucose during adsorption, leading to the decrease in purity of the target product; at the same time, the adsorption capacity and selectivity of AB-8 resin are lower than those of amino-modified HPD-720 resin, which may cause the loss of the target product and reduce the yield.

[0047] In the comparative example 4, the 70% ethanol concentration is too high, which can elute part of the target product together with β-cyclodextrin hydrolysis fragments, leading to the mixing of impurities in the target product; the 65% ethanol concentration is too low, which can only elute part of the target product, and the uneluted target product remains in the resin together with the impurities, which leads to the decrease in product purity and yield under the double effects.

[0048] In the comparative example 5, the absence of glucose exposes the general adsorption sites, which are easy to co-adsorb impurities such as dehydroxy puerarin, leading to the decrease in product purity; at the same time, co-adsorption may affect the adsorption capacity of the resin for the target product, causing the loss of the target product and reducing the yield.

Claims

1. A process for the preparation of glucosylpuerarin, characterized by, Comprising the following steps: S1. Sugar-added reaction: puerarin and pure water are mixed and uniformly dispersed, after which the system pH is adjusted to 4.5-5.5 and β-cyclodextrin is added and stirred until dissolved, then the system is warmed to 45-55℃, and cyclodextrin glucosyltransferase is added, and the constant temperature reaction is continued for 16-22h; after the reaction is completed, the system pH is adjusted to 0.1-0.8, and activated carbon is added, and stirred at 30-40℃ for 1.5-2.5h, and the activated carbon is removed by filtration, and the filtrate is placed at 2-6℃ overnight; S2. Sugar-removing reaction: the pH of the overnight filtrate is adjusted to 3.8-4.5, after which it is warmed to 55-65℃, and saccharifying enzyme and glucose oxidase are added, and the constant temperature reaction is continued for 25-40min; after the reaction is completed, the system pH is adjusted to 0.5-1.2, and cooled to room temperature, and the precipitate is removed by filtration; S3. Column purification: the reaction liquid after removing the precipitate is adsorbed through a mixed resin column, after adsorption is complete, first eluted with acid water, then eluted with pure water until the effluent is neutral and the conductivity is reduced to 70-120ppm, then eluted with a volume fraction of 55-65% ethanol solution, and finally eluted with a volume fraction of 75-85% ethanol solution, and the eluate of the volume fraction of 75-85% ethanol solution is collected; the mixed resin column is packed with mixed resin, and the mixed resin is composed of sulfonated modified XAD-16 resin and amino modified HPD-720 resin; S4. Concentration and drying: the collected eluate of the volume fraction of 75-85% ethanol solution is concentrated under reduced pressure until there is no ethanol smell, to obtain a concentrated liquid; the concentrated liquid is freeze-dried to produce a glucosyl puerarin product.

2. The process for the preparation of glucosyl puerarin as claimed in claim 1 wherein, In step S1, the amount ratio of puerarin to pure water is 1g:(35-45)mL; the mass ratio of puerarin to β-cyclodextrin is 1:(3.5-4.5); and the enzyme activity of cyclodextrin glucosyltransferase is 8000-12000U / mL, and the amount is 0.8-1.2% of the mass of puerarin.

3. The process for the preparation of glucosyl puerarin as claimed in claim 1 wherein, In step S2, the enzyme activity of saccharifying enzyme is 40000-60000U / g, and the amount is 0.007-0.01% of the volume of the filtrate; and the enzyme activity of glucose oxidase is 150-250U / mg, and the amount is 0.008-0.012g / L.

4. The process for the preparation of glucosyl puerarin as claimed in claim 1 wherein, The filtration steps in steps S1 and S2 both use 0.2-0.3μm microporous filter membranes.

5. The process for the preparation of glucosyl puerarin as claimed in claim 1, wherein, In the mixed resin, the volume ratio of sulfonated modified XAD-16 resin to amino modified HPD-720 resin is 1:(1.5-2.5).

6. The process for the preparation of glucosyl puerarin as claimed in claim 5 wherein, The preparation method of the sulfonated modified XAD-16 resin is as follows: Take XAD-16 resin, soak in ethanol until completely swollen, after filtration, add dichloromethane with 2-4 times the volume of the resin, under nitrogen protection, drop 8-12% of the mass of the resin of chlorosulfonic acid and stir the reaction; after the reaction is completed, filter, first neutralize to pH 6.0-7.5 with sodium carbonate solution, then rinse with pure water until the effluent conductivity is ≤15 μS / cm, finally soak in pH 1.8-2.2 acid water for 10-14 h; after soaking is complete, filter, and vacuum dry to obtain the product.

7. The process for the preparation of glucosyl puerarin as claimed in claim 6, wherein, The preparation method of the amino-modified HPD-720 resin is as follows: Take HPD-720 resin, soak in KH-550 ethanol solution for 20-28 h, then rinse with ethanol, hot water, and acid water in sequence; after rinsing is complete, introduce nitrogen to purge the resin, and finally vacuum dry to obtain the product; wherein the KH-550 ethanol solution contains 0.08-0.12 wt% glucose.

8. The process for the preparation of glucosyl puerarin as claimed in claim 1, wherein, In step S3, the adsorption flow rate is 0.8-1.5 BV / h; the acid water pH is 1.8-2.5, the temperature is 20-35℃, the flow rate is 1-2 BV / h, and 1-2 column volumes are eluted; the pure water flow rate is 1.5-2.5 BV / h, and elution is performed until the effluent is neutral and the conductivity is reduced to 70-120 ppm; the flow rate of the 55-65% ethanol solution is 0.8-1.2 BV / h, and 1-2 column volumes are eluted; the flow rate of the 75-85% ethanol solution is 0.6-1.2 BV / h, and 1.5-2.5 column volumes are eluted.

9. The process for the preparation of glucosyl puerarin as claimed in claim 1, wherein, In step S4, the temperature for vacuum concentration is 45-60℃, the vacuum degree is -0.07 ~ -0.1 MPa, and the concentration is performed until the density is 1.1-1.4 g / cm³; In freeze-drying, the pre-freezing temperature is -55~ -40℃, the pre-freezing time is 1-1.5 h, the sublimation drying vacuum degree is 0.5-8 Pa, and the drying time is 10-16 h.

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