CN112176008B

CN112176008B - Enzymolysis method for efficiently and quickly preparing icariside II

Info

Publication number: CN112176008B
Application number: CN202011093238.5A
Authority: CN
Inventors: 许卉; 杨昕; 李志勇; 许晓蒙; 孙发鑫; 李艳丽; 孙正
Original assignee: Yantai University
Current assignee: Yantai University
Priority date: 2020-10-13
Filing date: 2020-10-13
Publication date: 2022-12-20
Anticipated expiration: 2040-10-13
Also published as: CN112176008A

Abstract

The invention provides an enzymolysis method for efficiently and quickly preparing icariside II. The method comprises the following steps: using icariin as raw material, adding solvent to dissolve it to obtain high-concentration substrate solution, then adding helicase to make enzymolysis reaction in aqueous buffer solution system to make epimediumGlycoside C ₇ The O-glucoside bond of the position is hydrolyzed quickly and directionally, and the enzymolysis reaction product is purified by conventional post-treatment to obtain the icariside II. The method has the advantages of high conversion rate, simple and convenient operation and mild conditions, and the method has rich and easily obtained material sources, is economic and environment-friendly, is easy for amplified production and overcomes various defects of the prior art.

Description

Enzymolysis method for efficiently and quickly preparing icariside II

Technical Field

The invention relates to an efficient and rapid icariside II enzymolysis preparation method, and belongs to the technical field of foods, health products, cosmetics and medicines.

Background

Herba Epimedii is a traditional Chinese medicine with tonifying effect, and has advantages of pungent and sweet taste, warm nature, kidney yang tonifying, muscle and bone strengthening, and rheumatism eliminating, and can be used for treating kidney yang deficiency, sexual impotence, spermatorrhea, tendons and bones weakness, rheumatalgia, numbness and contracture, etc. The epimedium contains rich flavonoids, comprises various icariin derivatives with flavonol skeletons, has wide pharmacological activities of resisting inflammation, oxidation, tumor, aging and the like, and is an effective component representing the efficacy and the function of the icariin derivatives. The current Chinese pharmacopoeia stipulates that: the total flavone content in herba Epimedii is icariin (C) ₃₃ H ₄₀ O ₁₅ ) Should not be less than 5.0%. Wherein the content of icariin is not less than 0.5%. Icariside II (Icarissidi II, C) ₂₇ H ₃₀ O ₁₀ ) Is a natural mono-desugared metabolite of icariin, also called baohuoside I (Icariside I), and has the following chemical structural formula.

Researches find that the pharmacological activity of icariside II is generally higher than that of other epimedium flavonoids, and the icariside II has good safety and extremely good development and application prospects. However, the natural content of the icariside II is very low, only 0.17mg/g in epimedium, and the requirement is difficult to meet by simple extraction and separation, so the research for preparing the icariside II by taking icariin with rich and easily obtained sources as a raw material is concerned. Due to C in the molecular structure of icariin ₇ -glycosidic linkage atStability in acidic medium lower than C ₃ Glycosidic bonds and sterically hindered enzymatic hydrolysis of C ₃ The activity of the glycosidic bond is extremely low and the biotransformation is undoubtedly a hydrolysis of C of icariin ₇ Best method for obtaining icariside II with free hydroxyl at position 7 by glycosidic bond.

Bioconversion includes microbial fermentation and enzymatic processes. Research reports that icariin II is prepared by incubating icariin and rat intestinal bacteria liquid together, but the problems of complex operation, low transformation capacity and difficult product separation and purification are prominent, and the method is not suitable for industrial preparation. The preparation by the enzyme conversion method is a process for realizing chemical conversion by using enzyme as a catalyst, is a chemical reaction generated by performing structural modification on an exogenous substrate by using an enzyme preparation, has the advantages of mild reaction conditions, strong selectivity, high conversion rate and short flow, is convenient for extraction and separation of a general product, is convenient for process research and control, is environment-friendly and low in cost, and is more beneficial to large-scale preparation.

The key point is to screen cheap, efficient and specific industrial enzyme preparations which are suitable for preparation, generally enzymes with enzymatic glycosidic bonds, such as naringinase, pectinase, cellulase, helicase, lactase and the like. In order to prepare icariside II by taking icariin as a raw material through enzymolysis and conversion, related researches are carried out in recent years. Patent CN105925635A discloses a production process for preparing icariside II by icariin enzymolysis, but the high catalytic activity dextranase in the icariin II needs to be cultured and prepared by specially screening cocci, the cost of the enzyme is high, and the source is limited. Commercial enzymes are more concerned in application, such as beta-glucosaccharase, cellulase and the like (Fitoterapia 81 (2010) 437-442; chinese herbal medicine 41 (2010): 888-892; chinese herbal medicine 35 (2012): 1148-1155; and Chinese patent medicine 42 (2020): 779-782). However, the enzymatic conversion rate in the prior art is low, and in the methods, a solubilizer is generally required to be added into a reaction system in the enzymatic hydrolysis process, and a low-concentration solution is prepared for reaction, so that the enzymatic hydrolysis conversion period is long, the conversion efficiency is low, and the large-scale production is not facilitated.

Disclosure of Invention

In view of the above, the present invention aims to provide a method for rapidly, efficiently and specifically preparing icariside II with stronger biological activity by using icariin as a raw material and performing hydrolysis conversion by using helicase with abundant sources, low price and high enzymolysis efficiency.

An enzymolysis method for efficiently and rapidly preparing icariside II comprises the following steps,

(1) Preparation of a substrate solution: taking a proper amount of icariin, adding a solvent for dissolving to obtain a substrate solution, wherein the solvent is an organic solvent mutually soluble with water or a water-containing solvent system of any proportion, and preferably an aqueous buffer solution containing dimethyl sulfoxide with the volume ratio of not less than 80%;

(2) Preparing an enzyme solution: taking a proper amount of helicase, adding an aqueous buffer solution to dissolve to obtain an enzyme solution, wherein the pH value of the aqueous buffer solution is 4.5-7.0, and preferably the pH value is 5.0-6.0;

(3) And (3) carrying out enzymolysis reaction: adding the enzyme solution obtained in the step (2) into the substrate solution obtained in the step (1), and performing enzymolysis reaction in an aqueous buffer solution medium; the weight ratio of the helicase and the substrate icariin in the enzymolysis reaction is (0.5-2.0): 1, preferably (0.8 to 1.0): 1; the enzymolysis reaction temperature is controlled to be 20-50 ℃, and preferably 30-40 ℃; the enzymolysis reaction time is 0.5 to 12 hours, preferably 2 to 5 hours; in the final enzymolysis reaction system, the substrate concentration is not lower than 2mg/mL, and the substrate contains an organic solvent which is mutually soluble with water and has the volume ratio of not more than 5%, and preferably contains dimethyl sulfoxide with the volume ratio of not more than 1%;

(4) Post-treatment and purification: further processing and purifying the enzymolysis reaction product obtained in the step (3) by adopting a conventional method to obtain icariside II; the conventional treatment and purification method comprises one or more of centrifugation, filtration, column chromatography, recrystallization and solvent extraction.

Compared with the prior art, the invention has the innovation that the icariin with rich natural sources is taken as the raw material, the DSMO which is mutually soluble with water and has high solubility is adopted to dissolve the icariin, firstly, the high-concentration icariin substrate solution with low DSMO amount of an organic solvent is prepared, and then, the enzyme solution is added to carry out enzymolysis conversion reaction, compared with the prior art, the contact chance of the icariin which is a water-insoluble substrate in an aqueous enzymolysis system and the enzyme can be greatly improved under the condition of not influencing the enzyme activity, so that the enzymolysis conversion efficiency is improved, and meanwhile, the use of the organic solvent can be reduced to the maximum extent; the invention adds enzyme solution into high-concentration substrate icariin solution with small volume mutually soluble with water, carries out enzymolysis conversion reaction in a DSMO aqueous buffer medium system under a controlled condition, can ensure that the substrate icariin is fully dispersed and dissolved and fully contacts and reacts with enzyme, and effectively controls the amount of organic solvent DSMO mutually soluble with water in the reaction system at the same time, so that the generated enzyme conversion product has low solubility in the reaction system and is continuously separated out along with the reaction to promote the conversion balance to move towards the product generation direction, thereby further improving the speed and efficiency of the enzymolysis conversion reaction.

Compared with the prior art, the method provided by the invention has the advantages that,

(1) The icariin has good solubility in an aqueous buffer solution containing dimethyl sulfoxide with the volume ratio not less than 80%, the prepared solution has the advantages of high substrate concentration and low organic solvent amount, an enzyme solution is added into the solution for enzymolysis conversion reaction, the contact chance of a water-insoluble substrate in an aqueous enzymolysis system and enzyme can be greatly improved under the condition of not influencing the enzyme activity, and the enzymolysis conversion efficiency is improved;

(2) Adding an enzyme-containing aqueous buffer solution into a small-volume high-concentration substrate solution which is mutually soluble with water, and carrying out enzymolysis conversion reaction in an aqueous buffer medium system under a control condition, so that the quantity of an organic solvent which is mutually soluble with water in the reaction system can be effectively controlled while the substrate is fully dispersed and dissolved and fully contacted with the enzyme for reaction, and the solubility of an enzyme conversion product in the reaction system is greatly reduced, so that the product is continuously separated out along with the reaction, thereby promoting the conversion balance to move towards the product generation direction, and further ensuring the improvement of the speed and the efficiency of the enzymolysis conversion reaction;

(3) The helicase is a mixed enzyme prepared from the snail craw and the digestive tract, contains more than 20 enzymes such as cellulase, pectinase, amylase, protease and the like, has strong biological transformation capability, is economical and easily obtained, and is widely applied in the related fields of biological engineering. Compared with most pure enzymes used in the prior art, the helicase has lower cost, higher reaction specificity and high product purity;

(4) The enzymolysis conversion process provided by the invention does not need to add any surfactant, can reduce the use of organic solvent to the maximum extent, realizes environmental friendliness, greatly facilitates post-treatment purification, is easy to amplify and industrialize, and integrally realizes resource saving and green and high-efficiency production.

Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Detailed Description

The method is characterized in that a small-volume high-concentration substrate solution is prepared by utilizing the DSMO with strong dissolving property to the icariin for enzymolysis conversion, and the reaction is carried out in an aqueous buffer system for controlling the content of the DSMO, so that the enzyme activity and the contact reaction efficiency of the substrate and the enzyme are fully ensured, and meanwhile, the converted product icariside II is continuously separated out, thereby promoting the enzymolysis conversion efficiency and the reaction speed under the condition of reducing the use of an organic solvent to the maximum extent.

In the interest of clarity, not all features of an actual implementation are described. In the following description, well-known functions or constructions are not described in detail since they would obscure the invention in unnecessary detail. It will of course be appreciated that in the development of any such actual embodiment, numerous implementation-specific details must be set forth in order to achieve the developer's specific goals. The present invention will be described in further detail with reference to examples below for the purpose of facilitating understanding of the present invention, but the scope of the present invention is not limited to these examples at all.

The experimental operation methods used in the examples of the present invention are all conventional methods unless otherwise specified. Except for specific description, various materials, reagents and the like used in the embodiments of the present invention are commercially known products, wherein the selected icariin and icariside II reference substances (purity > 98.0%) are provided by shanghai pluronic ltd, and the snailase (> 300U/mg) is provided by shanghai gao biotech ltd.

Example 1:

this example provides a preferred mode for preparing icariside II by enzymolysis of icariin using the method of the present invention, which is further described in detail.

(1) And (3) carrying out enzymolysis conversion reaction: placing 500mg of icariin in a triangular flask, adding 1mL of DSMO to dissolve to obtain a substrate solution, adding 100mL of aqueous buffer solution containing 500mg of helicase, namely acetic acid-sodium acetate buffer solution (pH 5.0), mixing well, placing in a constant temperature gas bath oscillator, and reacting at 37 +/-1 ℃ for 2 hours. HPLC analysis monitoring showed 98.5% conversion of the substrate icariin.

(2) And (3) post-treatment and purification: extracting the reaction liquid with ethyl acetate for three times, combining organic phases, washing with water and saturated saline solution in sequence, drying with anhydrous sodium sulfate, decompressing, removing the solvent until the solution is dry to obtain a yellow-green powdery solid, performing HPLC determination on the yellow-green powdery solid and an icariside II reference substance under the same condition, and keeping the chromatographic retention time consistent.

Wherein, the HPLC determination conditions comprise: a Waters e2695 type high performance liquid chromatograph, agilent TC-C18 column (4.6X 250mm,5 μm), water (containing 0.1% formic acid) -acetonitrile (35, 65, v/v) mixed solvent system as a mobile phase for isocratic elution, the flow rate of 0.8mL/min, the detection wavelength of 270nm, the column temperature of 35 ℃, and the sample injection amount of 10 μ L. The conversion was determined as follows: and (3) after the enzymolysis reaction reaches a set time, taking out the reaction vessel, extracting with ethyl acetate, measuring the generation amount of a conversion product icariside II in the reaction system by an HPLC (high performance liquid chromatography) external standard method, and calculating the substrate conversion rate (%, n = 3).

The normalized purity of example 1 was calculated to be 99.3%.

Example 2:

(1) And (3) carrying out enzymolysis conversion reaction: putting 1000mg of icariin into a triangular flask, adding 2mL of DSMO to dissolve to obtain a substrate solution, then adding 400mL of aqueous buffer solution containing 1000mg of helicase, namely acetic acid-sodium acetate buffer solution (pH 5.0), fully mixing uniformly, putting into a constant temperature gas bath oscillator, and reacting for 2 hours at the temperature of (35 +/-2) ° C. HPLC analysis monitored the conversion of the substrate icariin to 97.6%.

(2) And (3) post-treatment and purification: centrifuging the reaction solution, collecting precipitate, recrystallizing twice with ethanol to obtain yellow-green needle crystal, performing HPLC determination with icariside II control under the same conditions and with the same chromatographic retention time and normalized purity of 99.8% in the same operation as example 1.

Example 3:

this example provides a preferred mode of using the method of the present invention for enzymatic hydrolysis of icariin, and further describes the enzymatic hydrolysis method of the present invention in detail.

(1) And (3) carrying out enzymolysis conversion reaction: placing 100mg of icariin in a triangular flask, adding 0.2mL of DSMO to dissolve to obtain substrate solution, adding 20mL of acetic acid-sodium acetate buffer solution (pH 5.0) containing 100mg of helicase, mixing well, placing in a constant temperature gas bath oscillator, and performing enzymolysis reaction at 37 deg.C for 2 hr. HPLC analysis monitored the conversion of the substrate icariin to 99.2%.

(2) And (3) post-treatment and purification: centrifuging the reaction solution, collecting precipitate, recrystallizing twice with ethanol to obtain yellow-green needle crystal, performing HPLC determination with icariside II reference substance under the same conditions and the same operation as example 1, with chromatographic retention time and normalized purity of 99.5%.

The product of examples 1-3 was identified by comprehensive spectroscopic analysis showing: HRESIMS: m/z 515.1911 ([ M + H)] ⁺ ,100％)，C ₂₇ H ₃₀ O ₁₀ ；UVλmax(in MeOH-H ₂ O)：269.7nm； ¹ H NMR(600MHz)、 ¹³ The results of C NMR (150 MHz) measurement are shown in Table 1.

Table 1 NMR measurement results of the product (delta, in DMSO-d) ₆ )

The results of the comprehensive spectrum analysis are consistent with those of the literature, and show that the product obtained in examples 1-3 is icariside II.

The enzymolysis effect of the existing method is different by taking the example 3 as a reference, as shown in comparative examples 1-4.

Comparative example 1:

direct mixing and dispersing reaction method of substrate and enzyme

Putting 100mg of icariin and 100mg of helicase into a triangular flask, adding 20mL of acetic acid-sodium acetate buffer solution with the pH value of 5.0, putting the triangular flask into a constant-temperature air bath oscillator, performing enzymolysis reaction at 37 ℃ for 2 hours, sampling and performing HPLC (high performance liquid chromatography) determination, and obtaining the substrate icariin, wherein the enzymolysis conversion rate is only 9.87%.

Comparative example 2:

enzymolysis reaction method of cyclodextrin inclusion substrate

Substrate inclusion was carried out according to the literature method (Chinese medicinal materials, 2012, 35 vol. 7, pages 1148-1153), and the other conditions were the same as in example 3.

Dissolving 100mg of icariin in 50% ethanol with a certain volume, dripping a proper amount of hydroxypropyl-beta-cyclodextrin aqueous solution with equal molar quantity, fixing the volume, adding 100mg of helicase, adjusting the pH value to 5.0, uniformly mixing, placing in a constant-temperature air bath oscillator, carrying out enzymolysis reaction for 2 hours at 37 ℃, sampling, and carrying out HPLC (high performance liquid chromatography) determination to obtain a substrate icariin with the conversion rate of 16.3%.

Comparative example 3:

enzymolysis reaction method for solubilizing surfactant

The solubilization of the substrate was carried out by adding a surfactant according to the literature method (Chinese medicinal material, 2012, vol. 35, no. 7, page 1148-1153), and the other conditions were the same as in example 3.

100mg of icariin, 100mg of helicase and a proper amount of surfactant (poloxamer 188 or tween-80) are taken, placed in a triangular flask, added with acetic acid-sodium acetate buffer solution with pH of 5.0, the volume fraction of the surfactant is adjusted to be 0.20%, evenly mixed, placed in a constant-temperature air bath oscillator, subjected to enzymolysis reaction for 2 hours at 37 ℃, sampled and subjected to HPLC determination, and the substrate icariin is obtained, wherein the enzymolysis conversion rates of the icariin in a system containing 0.2% of poloxamer 188 and 0.2% of tween-80 are respectively 24.1% and 27.0%.

Comparative example 4:

enzymolysis reaction method for solubilizing organic solvent

According to the method disclosed in CN201610289040.1, an organic solvent is added to the enzymatic conversion system for solubilization, and other conditions are the same as those in example 3.

Taking 100mg of icariin, adding a proper amount of acetic acid-sodium acetate buffer solution (pH 5.0) for suspension dispersion, adding 5% of glycerol or dimethyl sulfoxide by volume of a reaction system and 100mg of helicase into the reaction system, uniformly mixing, placing the mixture into a constant-temperature air bath oscillator, performing enzymolysis reaction for 2 hours at 37 ℃, sampling and performing HPLC (high performance liquid chromatography) determination to obtain a substrate icariin, wherein the enzymolysis conversion rates of the icariin in a system containing 5% of glycerol and 5% of dimethyl sulfoxide are 23.5% and 26.2% respectively.

The results of comparative experiments between example 3 and comparative examples 1-4 show that, under the same condition of icariin of 100mg, HPLC analysis and monitoring show that the conversion rate of icariin as a substrate in the enzymolysis method of the invention reaches more than 97%, and the enzymolysis effect is far superior to the conversion rate of 20% in the prior art. It is known that icariin is difficult to dissolve in water, and the contact chance of icariin and enzyme in an aqueous enzymolysis system is very limited, so that the method generally needs to add a solubilizer into a reaction system to prepare a low-concentration solution for reaction in the enzymolysis process, but the enzymolysis conversion efficiency is still low, and the method is not beneficial to large-scale production. Compared with the closest prior art comparative example 4, the key point of the enzymolysis of the icariin is that the DSMO which is mutually soluble with water and has high solubility to the icariin firstly dissolves the icariin, and then the enzymolysis reaction is carried out in a water-based buffer system containing the DSMO so as to separate out icariside II which is not dissolved in water/DSMO, so that the irreversible icariin enzymolysis reaction is carried out in the water-based buffer system containing the DSMO until the enzymolysis is complete. Correspondingly, in comparative example 4, due to the difference of the adding time of the DSMO, the extremely low solubility of the icariin in the acetic acid-sodium acetate buffer solution can only form a suspension solution, and the low-concentration DSMO added at this time cannot enable the icariin to be dissolved in the DSMO and to be fully contacted with the helicase for enzymolysis as in the enzymolysis method of the present invention, and the enzymolysis reaction at this time does not enable the icariin to be subjected to enzymolysis in the DSMO as in the icariin suspension of the present invention, but the icariin suspension is subjected to enzymolysis reaction with the helicase in a mixed system, so that the contact probability of the icariin and the helicase is influenced, and finally, the conversion rate is still low. Compared with the prior various substrate solubilization technologies, the method provided by the invention fundamentally improves the contact probability of icariin and enzyme in an aqueous enzymolysis system, thereby greatly improving the enzymolysis conversion efficiency.

Meanwhile, in order to verify the analysis, the method for inspecting the DMSO and icariin powder solvent is as follows:

taking a proper amount of icariin, respectively dissolving or dispersing the icariin in aqueous buffer solutions (pH 5.0) containing DMSO with different concentrations, then adding enzyme solutions, respectively carrying out enzymolysis reaction for 2h at 30 ℃ in buffer solutions (pH 5.0) with the weight ratio of enzyme to substrate being 1.0, and measuring the conversion rate, wherein the results are shown in Table 2.

TABLE 2 influence of DMSO concentration in substrate Dispersion solvent on enzymatic conversion

The results confirm the above analysis: namely, when the volume fraction of DMSO in the dispersion solvent reaches more than 80%, the enzymolysis conversion rate is high and stable (> 90%). Considering the tolerance of the helicase to organic solvents, reducing the solubility of the product in the reaction system and facilitating the post-treatment and purification of the reaction system, the invention preferably adopts an aqueous buffer solution containing not less than 80 percent by volume of dimethyl sulfoxide as a dispersing solvent to fully dissolve the icariin, controls the volume fraction of the dimethyl sulfoxide not to exceed 1 percent in the final enzymolysis reaction system, and simultaneously controls the concentration of the substrate not to be less than 2mg/mL.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An enzymolysis method for efficiently and quickly preparing icariside II is characterized by comprising the following steps:

(1) Preparation of a substrate solution: dissolving a proper amount of icariin in a solvent to obtain a substrate solution, wherein the solvent is an aqueous buffer solution containing dimethyl sulfoxide with volume ratio not less than 80%;

(2) Preparation of enzyme solution: taking a proper amount of helicase, adding an aqueous buffer solution into the helicase to dissolve the helicase to obtain an enzyme solution, wherein the aqueous buffer solution is an acetic acid-sodium acetate buffer solution, and the pH value of the aqueous buffer solution is 5.0-6.0;

(3) And (3) carrying out enzymolysis reaction: adding the enzyme solution obtained in the step (2) into the substrate solution obtained in the step (1) to perform an enzymolysis reaction in an aqueous buffer solution medium, wherein the volume ratio of dimethyl sulfoxide contained in an enzymolysis reaction system of the aqueous buffer solution medium is not more than 1%, and the concentration of substrate icariin in the enzymolysis reaction system of the aqueous buffer solution medium is not less than 2mg/mL; the weight ratio of the helicase to the substrate icariin in the enzymolysis reaction is 0.8-1.0: 1; controlling the temperature of the enzymolysis reaction to be 30-40 ℃; the enzymolysis reaction time is 2 hours;

(4) Post-treatment and purification: and (4) purifying the enzymolysis reaction product obtained in the step (3) to obtain icariside II.

2. The enzymolysis method according to claim 1, wherein the purification method in step (4) is one or more of centrifugation, filtration, column chromatography, recrystallization and solvent extraction.