Method for preparing D-aronia melanocarpa glycoside from D-amygdalin

Abstract

The invention discloses a method for preparing D-ulcerasin from D-amygdalin. The method comprises the following steps: (1) preparing a D-amygdalin solution and a beta-glucosidase solution; and (2) mixing the D-amygdalin solution and the beta-glucosidase solution, and carrying out an enzymatic reaction under certain conditions to obtain a reaction solution containing D-aronia melanocarpa glycoside. By optimizing the preparation method, the purity of the finally prepared and purified D-ultigenin is larger than or equal to 98%, and isomers are identified through chromatography and nuclear magnetic resonance spectroscopy. The research of the experiment provides a certain possibility for the research on the pharmacological and pharmacodynamic activity, toxicity, pharmacokinetics and the like of the D-aronia melanocarpa, and also provides a certain reference significance for the research on the quality standard of medicinal materials and preparations containing amygdalin.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to a method for preparing D-pyrocalyx glycoside from D-mygdalin. Background Art

[0002] Amygdalin is composed of one molecule of mandelanitrile and two molecules of glucose, and is a cyanogenic glycoside compound. It is widely found in the dried mature seeds of the Rosaceae plants Apricot and Peach. Currently, most of the research and reports on amygdalin are focused on D-amygdalin. The main metabolite of amygdalin in the body is safflower glycoside, which has a chemical composition of one molecule of mandelanitrile and one molecule of glucose, and also has a pair of chiral isomers. The present invention prepares D-safflower glycoside from D-amygdalin through response surface optimization experimental design. The present invention solves the problem that the D-safflower glycoside reference substance is difficult to obtain, provides a certain reference for the optimization of the quality standards of medicinal materials and compound preparations containing amygdalin, and provides a certain basis for the study of the biological activity and pharmacological toxicity of amygdalin isomers. Summary of the invention

[0003] Based on this, the present invention provides a method for preparing D-pyrocalycin from D-mygdalin, the method comprising the following steps:

[0004] (1) preparing a D-amygdalin solution and a β-glucosidase solution; and

[0005] (2) The D-amygdalin solution and the β-glucosidase solution are mixed and subjected to an enzymatic reaction under certain conditions to obtain a reaction solution containing D-pyrocatechol.

[0006] Furthermore, the method further comprises the following steps:

[0007] (3) adding a first solvent to the reaction solution containing D-prunoside to terminate the reaction, and drying to obtain a dried product;

[0008] (4) adding the first solvent to the dried product to redissolve it, adding silica gel powder, and stirring to obtain a product to be eluted;

[0009] (5) gradient eluting the eluted product using a silica gel column, collecting the eluate containing D-prunoside, and drying it to obtain a crude product of D-prunoside;

[0010] (6) re-dissolving the crude D-prunoside product with a first solvent, purifying, detecting and collecting fractions containing D-prunoside, removing the organic solvent, and re-dissolving with an appropriate amount of water to obtain an aqueous solution of D-prunoside; and

[0011] (7) Drying the aqueous solution of D-pranicotin to obtain D-pranicotin.

[0012] Furthermore, the first solvent is alcohol, such as methanol.

[0013] Furthermore, in step (1), the method for preparing the D-amygdalin solution and the β-glucosidase solution comprises weighing appropriate amounts of D-amygdalin powder and β-glucosidase powder, respectively fixing the volumes in containers, to obtain the D-amygdalin solution and the β-glucosidase solution.

[0014] Furthermore, the container is a volumetric flask.

[0015] Furthermore, the concentration of the D-amygdalin solution is 0.2-2.0 mg / ml, such as 0.5-1.5 mg / ml, such as about 1 mg / ml.

[0016] Furthermore, the concentration of the β-glucosidase solution is 0.05 to 2.0 unit / ml, such as 0.1 to 1.0 unit / ml, such as about 0.52 unit / ml or about 0.5 unit / ml.

[0017] Furthermore, in step (2), the enzymatic reaction is carried out in a water bath.

[0018] Furthermore, the temperature of the enzymatic reaction is 30-50°C, such as about 41.2°C or about 40°C.

[0019] Furthermore, the enzymatic reaction time is 5 to 20 min, such as about 7.33 min or about 7 min.

[0020] Furthermore, in step (3), the ratio of the volume (ml / ml) of the first solvent to the volume (ml / ml) of the D-amygdalin solution is 3-5, for example, about 3.63.

[0021] Furthermore, in step (3), the drying is rotary evaporation drying.

[0022] Furthermore, in step (4), the volume of the first solvent is 1-10 ml, for example, about 5 ml.

[0023] Furthermore, in step (4), the silica gel powder is 100-200 mesh silica gel powder.

[0024] Furthermore, in step (4), the mass of the silica gel powder is 5 to 15 g, for example, about 10 g.

[0025] Furthermore, in step (5), the silica gel column is filled with 200-300 mesh silica gel powder.

[0026] Furthermore, in step (5), the mass of the silica gel powder is 40-60 g, for example, about 50 g.

[0027] Furthermore, the mass ratio of the silica gel powder in step (4) to the silica gel powder in step (5) is 0.1 to 0.5, for example, about 0.2.

[0028] Further, the gradient elution was carried out according to the following procedure: (a) ethyl acetate: petroleum ether (1:1), about 3 column volumes; (b) ethyl acetate: petroleum ether (2:5), about 5 column volumes; (c) ethyl acetate, about 10 column volumes; (d) ethyl acetate: methanol (10:1), about 5 column volumes; and (e) ethyl acetate: methanol (10:3), about 5 column volumes.

[0029] Furthermore, the detection method of the eluent is thin layer chromatography.

[0030] Furthermore, the developing solvents of the thin layer chromatography are ethyl acetate and methanol.

[0031] Furthermore, the volume ratio of the ethyl acetate to the methanol is (4-6):(1-3), for example, about 5:about 2.

[0032] Furthermore, the drying is volatile drying or rotary evaporation drying.

[0033] Furthermore, in step (6), the purification is preparative liquid phase purification.

[0034] Furthermore, the chromatographic conditions for the preparative liquid phase purification are: using a CAPCELL PAK C18-AQ chromatographic column, mobile phase A is water, mobile phase B is methanol, and the flow rate is 10 to 20 ml / min.

[0035] Furthermore, the specifications of the chromatographic column are: column length 250 mm, inner diameter 10 mm, and particle size 5 μm.

[0036] Furthermore, the water is pure water.

[0037] Furthermore, the methanol is chromatographic methanol.

[0038] Furthermore, the volume ratio of the pure water to the chromatographic methanol is (10-30):(70-90), for example, about 20:about 80.

[0039] Furthermore, the flow rate is 14-18 ml / min, for example, about 16 ml / min.

[0040] Furthermore, the detection is performed using an ultraviolet diode array detector.

[0041] Furthermore, the drying is rotary evaporation drying.

[0042] Furthermore, in step (7), the drying is freeze-drying.

[0043] Furthermore, the freeze-drying temperature is -20°C.

[0044] According to another aspect of the present invention, a solution containing D-pranicotin prepared by the above method or D-pranicotin is provided.

[0045] Beneficial effects of the present invention:

[0046] The reaction involved in the method for preparing D-pyrocatechol from D-mygdalin of the present invention is a biological enzyme-catalyzed reaction, wherein the enzyme dosage concentration, reaction time, reaction temperature, pH, etc. are important factors affecting the yield of the target compound. After a comprehensive analysis of the reaction, it is found that the Box-Behnken statistical analysis method has certain guiding significance for the analysis of such multivariate functions.

[0047] D-Amygdalin is widely present in nature, and has certain pharmacological activities in antitussive and antiasthmatic, anti-inflammatory and analgesic, liver and kidney fibrosis, etc. Its main active metabolite in the body is D-Prunoside. The present invention finally prepares and purifies D-Prunoside with a purity of ≥98% by optimizing the preparation method, and identifies the isomers by chromatography and nuclear magnetic resonance spectroscopy. The research of this experiment provides certain possibilities for the study of the pharmacological and pharmacodynamic activity, toxicity, and pharmacokinetic of D-Prunoside, and also provides certain reference significance for the study of the quality standards of medicinal materials and preparations containing amygdalin. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without exceeding the scope of protection required by the present invention.

[0049] Figure 1 The figure is a schematic diagram of the single factor investigation results of the preparation of D-Prunoside. Among them, A is the effect of temperature on the yield of D-Prunoside, B is the effect of time on the yield of D-Prunoside, and C is the effect of enzyme concentration on the yield of D-Prunoside.

[0050] Figure 2 The figure is a schematic diagram of the effect of interaction factors on the yield of D-safflower glycosides, where A is the interaction factor between temperature and time, B is the interaction factor between enzyme concentration and temperature, and C is the interaction factor between enzyme concentration and time.

[0051] Figure 3 A is a schematic diagram of the response surface variance analysis results. A is a schematic diagram of residuals vs. predictions, B is a normal plot of residuals, and C is a schematic diagram of predictions vs. actuals.

[0052] Figure 4 The figure is a flow chart of the preparation example of D-prunin. DETAILED DESCRIPTION

[0053] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0054] Unless otherwise indicated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by those of ordinary skill in the field of the invention or the field in which the terms are used. Although any methods, conditions, substances or materials similar to or equivalent to those disclosed herein may be used in the practice of the present invention, preferred methods, conditions, substances or materials are described herein.

[0055] The present invention is intended to encompass all alternatives, variations and equivalents that may be included in the present invention as defined by the claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

[0056] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0057] In the present invention, the term "comprising" is synonymous with "including". As used herein, the terms "comprising", "including", "having", "containing" or any other variation thereof are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus comprising the listed elements is not necessarily limited to only those elements, but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0058] As described in the background technology section, there is a problem in the prior art that a D-pyrocatechol reference substance is difficult to obtain. In order to solve the above problem, the present invention provides a method for preparing D-pyrocatechol from D-myrtle glycoside, the method comprising the following steps:

[0059] (1) preparing a D-amygdalin solution and a β-glucosidase solution; and

[0060] (2) The D-amygdalin solution and the β-glucosidase solution are mixed and subjected to an enzymatic reaction under certain conditions to obtain a reaction solution containing D-pyrocatechol.

[0061] The present invention uses β-glucosidase to degrade D-amygdalin to prepare D-safflower glycoside. The reaction is an enzymatic reaction, and there are many factors that affect the reaction rate and the yield of the target compound. Therefore, a single factor experiment combined with a response surface optimization experimental design is used to explore the preparation method with the highest yield of D-safflower glycoside. There are three main reactions that affect the yield of D-safflower glycoside in the enzymatic incubation system:

[0062] ① Prunin and monosaccharides generated by enzymatic hydrolysis of amygdalin;

[0063] ② Amygdalin is enzymatically hydrolyzed into benzyl nitrile and disaccharides;

[0064] ③ Enzymatic hydrolysis of rosin produces benzyl alcohol nitrile and monosaccharides;

[0065] If the reaction is not complete, the amount of prussin produced will be small, while if the reaction is too complete, all of it will be converted into by-products. Therefore, there is an optimal reaction condition for the enzymatic hydrolysis kinetics of the intermediate product prussin.

[0066] In a preferred embodiment, the method further comprises the steps of:

[0067] (3) adding a first solvent to the reaction solution containing D-prunoside to terminate the reaction, and drying to obtain a dried product;

[0068] (4) adding the first solvent to the dried product for redissolution, adding silica gel powder, and stirring to obtain a product to be eluted;

[0069] (5) gradient eluting the eluted product using a silica gel column, collecting the eluate containing D-prunoside, and drying it to obtain a crude product of D-prunoside;

[0070] (6) re-dissolving the crude D-prunoside product with a first solvent, purifying, detecting and collecting fractions containing D-prunoside, removing the organic solvent, and re-dissolving with an appropriate amount of water to obtain an aqueous solution of D-prunoside; and

[0071] (7) Drying the aqueous solution of D-pranicotin to obtain D-pranicotin.

[0072] In a preferred embodiment, the first solvent is an alcohol, such as methanol.

[0073] In a preferred embodiment, in step (1), the method for preparing the D-amygdalin solution and the β-glucosidase solution comprises weighing appropriate amounts of D-amygdalin powder and β-glucosidase powder, respectively fixing the volumes in containers to obtain the D-amygdalin solution and the β-glucosidase solution.

[0074] In a preferred embodiment, the container is a volumetric flask.

[0075] In a preferred embodiment, the concentration of the D-amygdalin solution is 0.2-2.0 mg / ml, such as 0.5-1.5 mg / ml, such as about 1 mg / ml.

[0076] In the present invention, when concentration, temperature, time, ratio, particle size, flow rate, mass, or other values ​​or parameters are expressed as a range, a preferred range, or a range defined by a series of upper preferred values ​​and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any upper range limit or preferred value with any lower range limit or preferred value, regardless of whether the range is disclosed separately. For example, when the range "0.5-1.5" is disclosed, the described range should be interpreted as including the range "0.5-1.5", "0.5-1.3", "0.5-1.1", "0.5-0.9", "0.5-0.7", "0.7-1.5", "0.7-1.3", "0.7-1.1", "0.7-0.9", "0.9-1.5", "0.9-1.3", "0.9-1.1", "1.1-1.5", "1.1-1.3", "1.3-1.5", etc. When a numerical range is described herein, unless otherwise indicated, that range is intended to include the endpoints and all integers and fractions within that range.

[0077] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0078] In a preferred embodiment, the concentration of the β-glucosidase solution is 0.05-2.0 unit / ml, such as 0.1-1.0 unit / ml, such as about 0.52 unit / ml or about 0.5 unit / ml.

[0079] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 0.52" includes ±5% of 0.52, or from 0.494 to 0.546; "about 0.5" includes ±5% of 0.5, or from 0.475 to 0.525.

[0080] In a preferred embodiment, in step (2), the enzymatic reaction is carried out in a water bath.

[0081] In a preferred embodiment, the temperature of the enzymatic reaction is 30-50°C, such as about 41.2°C or about 40°C.

[0082] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 41.2" includes ±5% of 41.2, or from 39.14 to 43.26; "about 40" includes ±5% of 40, or from 38 to 42.

[0083] In a preferred embodiment, the enzymatic reaction time is 5 to 20 min, such as about 7.33 min or about 7 min.

[0084] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 7.33" includes ±5% of 7.33, or from 6.9635 to 7.6965; "about 7" includes ±5% of 7, or from 6.65 to 7.35.

[0085] In a preferred embodiment, in step (3), the ratio of the volume (ml / ml) of the first solvent to the volume (ml / ml) of the D-amygdalin solution is 3-5, for example, about 3.63.

[0086] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 3.63" includes ±5% of 3.63, or from 3.4485 to 3.8115.

[0087] In a preferred embodiment, in step (3), the drying is rotary evaporation drying.

[0088] In a preferred embodiment, in step (4), the volume of the first solvent is 1-10 ml, for example, about 5 ml.

[0089] In the present invention, "about" refers to a value within a range of ±5% of a particular value. For example, "about 5" includes ±5% of 5, or from 4.75 to 5.25.

[0090] In a preferred embodiment, in step (4), the silica gel powder is 100-200 mesh silica gel powder.

[0091] In a preferred embodiment, in step (4), the mass of the silica gel powder is 5 to 15 g, for example, about 10 g.

[0092] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0093] In a preferred embodiment, in step (5), the silica gel column is filled with 200-300 mesh silica gel powder.

[0094] In a preferred embodiment, in step (5), the mass of the silica gel powder is 40-60 g, for example, about 50 g.

[0095] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 50" includes ±5% of 50, or from 47.5 to 52.5.

[0096] In a preferred embodiment, the mass ratio of the silica gel powder in step (4) to the silica gel powder in step (5) is 0.1 to 0.5, for example, about 0.2.

[0097] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.2" includes ±5% of 0.2, or from 0.19 to 0.21.

[0098] In a preferred embodiment, the gradient elution is carried out according to the following procedure: (a) ethyl acetate: petroleum ether (1:1), about 3 column volumes; (b) ethyl acetate: petroleum ether (2:5), about 5 column volumes; (c) ethyl acetate, about 10 column volumes; (d) ethyl acetate: methanol (10:1), about 5 column volumes; and (e) ethyl acetate: methanol (10:3), about 5 column volumes.

[0099] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 3" includes ±5% of 3, or from 2.85 to 3.15; "about 5" includes ±5% of 5, or from 4.75 to 5.25; "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0100] In a preferred embodiment, the detection method of the eluent is thin layer chromatography.

[0101] In a preferred embodiment, the developing solvents of the thin layer chromatography are ethyl acetate and methanol.

[0102] In a preferred embodiment, the volume ratio of the ethyl acetate to the methanol is (4-6):(1-3), for example, about 5:about 2.

[0103] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 5" includes ±5% of 5, or from 4.75 to 5.25; "about 2" includes ±5% of 2, or from 1.9 to 2.1.

[0104] In a preferred embodiment, the drying is volatile drying or rotary evaporation drying.

[0105] In a preferred embodiment, in step (6), the purification is preparative liquid phase purification.

[0106] In a preferred embodiment, the chromatographic conditions for the preparative liquid phase purification are: using a CAPCELL PAKC18-AQ chromatographic column, mobile phase A is water, mobile phase B is methanol, and the flow rate is 10-20 ml / min.

[0107] In a preferred embodiment, the specifications of the chromatographic column are: column length 250 mm, inner diameter 10 mm, and particle size 5 μm.

[0108] In a preferred embodiment, the water is pure water.

[0109] In a preferred embodiment, the methanol is chromatographic methanol.

[0110] In a preferred embodiment, the volume ratio of the pure water to the chromatographic methanol is (10-30):(70-90), such as about 20:about 80.

[0111] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 20" includes ±5% of 20, or from 19 to 21; "about 80" includes ±5% of 80, or from 76 to 84.

[0112] In a preferred embodiment, the flow rate is 14-18 ml / min, such as about 16 ml / min.

[0113] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 16" includes ±5% of 16, or from 15.2 to 16.8.

[0114] In a preferred embodiment, the detection is performed using an ultraviolet diode array detector.

[0115] In a preferred embodiment, the drying is rotary evaporation drying.

[0116] In a preferred embodiment, in step (7), the drying is freeze-drying.

[0117] In a preferred embodiment, the freeze-drying temperature is -20°C.

[0118] According to another aspect of the present invention, a solution containing D-pranicotin or D-pranicotin prepared by the above method is provided.

[0119] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions or conditions recommended by the manufacturer.

[0120] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0121] The above features mentioned in the present invention or the features mentioned in the embodiments can be combined in any way. All the features disclosed in this patent specification can be used in combination with any combination form, and each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equal or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equal or similar features.

[0122] Example

[0123] 1. Materials and reagents

[0124] 1.1 Instruments

[0125] Agilent 1260 Infinity II high performance liquid chromatograph (Agilent, USA, equipped with quaternary pump, autosampler, DAD detector, Agilent ChemStation workstation); Eyela bath SB-9 constant temperature incubator (Tokyo Rikkigi Co., Ltd., Japan); VX-200 vortex mixer (Labnet, USA); BT-25S electronic analytical balance (Beijing Sartorius Scientific Instrument Co., Ltd.); CT15RE desktop refrigerated centrifuge (Hitachi KOKi Co., Japan); FE28 pH meter (Mettler-Toledo Instrument Shanghai Co., Ltd.), Milli-Q water purifier (Millipore, USA) volumetric flasks (several specifications), conical flasks (several specifications), test tubes (several), glass rods, silica gel columns, double balls.

[0126] 1.2 Reagents and test drugs

[0127] D-Amygdalin (Chengdu Aifa Biotechnology Co., Ltd., batch number: AFBG1435), β-D-glucosidase (Shanghai Yuanye Biotechnology Co., Ltd., batch number: Y30A10H85903), acetonitrile (Fisher-HPLC grade), methanol (Wokai-HPLC grade), analytical grade methanol (Sinopharm Chemical Reagent Co., Ltd.), water (Wahaha Pure Water), petroleum ether (Sinopharm Chemical Reagent Co., Ltd.), ethyl acetate (Sinopharm Chemical Reagent Co., Ltd.), acetonitrile (chromatographic grade, Fisher Co., USA, batch number F23NCE202); formic acid (chromatographic grade, CNW Technologies GmbH, Germany, batch number Q1510120), deuterated DMSO (CIL Reagent Co., USA); ultrapure water was purified by Milli-Q Academic System was prepared; hydrochloric acid (analytical grade, Sinopharm Chemical Reagent Co., Ltd., batch number 20200731); sodium hydroxide (analytical grade, Sinopharm Chemical Reagent Co., Ltd., batch number 20070307); silica gel 200-300 mesh (Qingdao Ocean Chemical Co., Ltd.), silica gel 100-200 mesh (Qingdao Ocean Chemical Co., Ltd.), and thin layer plate (Yantai Yinlong Silica Gel Co., Ltd.).

[0128] 2. Experimental methods

[0129] 2.1. Preparation of solutions and samples

[0130] Preparation of the test solution: Accurately weigh 1000 mg of D-amygdalin reference substance and dilute to 1000 mL volumetric flask with ultrapure water to obtain a D-amygdalin stock solution with a mass concentration of 1 mg / mL.

[0131] 2.2 Factors affecting the preparation of D-prunin

[0132] 2.2.1. Study on the factors affecting the yield of D-prunin at different times

[0133] The production rate of D-mygdalin was investigated at a temperature of 40°C, an enzyme concentration of 0.5 unit / ml, and reaction times of 4 min, 5 min, 6 min, 7 min, 8 min, and 10 min.

[0134] 2.2.2. Study on the factors affecting the yield of D-prunin at different temperatures

[0135] The production rate of D-mygdalin was investigated when the enzyme concentration was 0.5unit / ml, the reaction time was 5min and the temperatures were 30℃, 40℃, 50℃, 60℃, 70℃ and 80℃.

[0136] 2.2.3. Study on the factors affecting the yield of D-pyrocatechol at different enzyme concentrations

[0137] The production rates of D-mygdalin were investigated at reaction times of 5 min, temperatures of 40°C, and enzyme concentrations of 0.05unit / ml, 0.1unit / ml, 0.5unit / ml, 1unit / ml, and 2unit / ml.

[0138] 2.2.4 Response surface optimization experimental design

[0139] The Box-Behnken response surface method was used to optimize the experimental conditions through screening out the appropriate intervals through the results of single factor investigation. Finally, the temperature range for D-prunoside preparation was selected as 30-80°C, the reaction time was selected as 4-10min, and the enzyme concentration was selected as 0.1-1unit / ml.

[0140] 3. Experimental results

[0141] 3.1. Results of single factor investigation on the preparation of D-prunin

[0142] The experimental results are as follows Figure 1 As shown, through the temperature single factor experiment, it was found that when the temperature range is 30-80℃, the yield R of D-wild black cherry glycoside will increase with the increase of incubation temperature, and when it reaches a certain temperature, the yield R will decrease with the increase of temperature. Through the incubation time single factor experiment, it was found that when the incubation time is 3-20min, the yield R will increase with the increase of incubation time, and when it reaches a certain time, the yield R will decrease with the increase of temperature. Through the catalytic enzyme concentration single factor experiment, it was found that when the enzyme concentration is 0.1-1Unit / m, the yield R of D-wild black cherry glycoside will increase with the increase of enzyme concentration, and when it reaches a certain time, the yield R will decrease with the increase of enzyme concentration.

[0143] 3.2 Response surface design of D-prunin

[0144] The experimental design of the independent variable response surface is shown in Table 1. The statistical analysis of the model is shown in Table 2.

[0145] Table 1 Experimental design of independent variable response

[0146]

[0147]

[0148] Table 2 Experimental statistical analysis of independent variable responses

[0149]

[0150]

[0151] 3.3 Influence of Interaction Factors on the Yield of D-Prunasin

[0152] To study the influence of reaction time, reaction temperature, and enzyme concentration on the yield of D-prunasin, the 3D response surface contour map was drawn using Design-Expert 10.0 software to show the relationship between the yield of D-prunasin and any two independent variables, and the interaction between variables was observed more intuitively from Figure 2 it. The three-dimensional response surface map and two-dimensional contour map were drawn under the condition of controlling the third factor to be 0. Combining with the table, it can be seen that PB < PA < PC. Therefore, the influence degree of the three factors on the production rate of prunasin is C (enzyme concentration) > A (time) > B (temperature). From the 3D diagrams of the pairwise interactions, it can be seen that the steepness between AC is higher than that between AB and BC, indicating that the two factors of enzyme concentration and reaction time have a greater impact on the production of D-prunasin.

[0153] 3.4 Response Surface Variance Analysis

[0154] The experimental data were statistically analyzed and variance analyzed using Design-Expert 10.0 software. From Figure 3 it can be seen that the residual normal distribution is close to a straight line and its fitting effect is relatively good. For this experimental model, P < 0.05, and the lack-of-fit term P = 0.0006 < 0.05, indicating that the selected model in this study is true and reliable. Since PB < PA < PC, the influence degree of the three factors on the production rate of prunasin is C (enzyme concentration) > A (time) > B (temperature). R 2 = 0.9549, R 2 adj = 0.8969, indicating that the model can explain 89.69% of the variation in the response value, proving that the model has a good fitting degree and can be used for predicting and analyzing the production rate of D-prunasin.

[0155] Enzymatic Hydrolysis Kinetics Equation:

[0156] Yield of D-Prunasin = 0.42 + 1.48A + 0.03B + 55.14C + 0.01AB - 0.99AC - 0.06BC - 0.07A 2 - 42.94C 2

[0157] 3.5 Verification of the Response Surface Experimental Model

[0158] Results: Under the conditions recommended by the response surface optimization equation, when the reaction temperature is 41.2 °C, the enzyme concentration is 0.52 unit / ml, and the reaction time is 7.33 min, the highest yield is 25%. The verification results under this condition are as follows: Using 1 g of D-amygdalin raw material, at a temperature of 40 °C, an enzyme concentration of 0.5 unit / ml, and a reaction time of 7 min, 220 mg of D-prunasin is obtained, and the yield is 22.00%.

[0159] 4. Example of Preparation of D-Prunoside

[0160] Preparation Example Flow Chart Figure 4 shown.

[0161] Accurately weigh 1000 mg of D-amygdalin powder and dilute it into a 1000 ml volumetric flask to obtain a solution with a concentration of 1 mg / ml. Separately weigh β-glucosidase powder and dilute it into a 100 ml volumetric flask to obtain a solution with a concentration of 0.5 unit / ml. Mix the two and incubate them in a 40°C water bath for 7 minutes. Then, use 4000 ml of methanol to terminate the reaction. Then, use a rotary evaporator to dry the solvent. Take 5 ml of methanol to dissolve it evenly in 10 g of 100-200 mesh silica gel powder. Take another 50 g of 200-300 mesh silica gel was loaded into a chromatographic column, and gradient elution was performed at the eluent ratio in Table 3 below. The eluent was collected and spotted on a silica gel thin layer plate, and developed with a developing solvent of ethyl acetate: methanol in a ratio of 5:2. The eluent containing prunin was combined and dried, and after being re-dissolved in water, it was frozen into a solid at -20°C, and dried in a freeze dryer to finally obtain 280 mg of powder. The purity of D-prunin was determined to be about 90%. The dried powder was re-dissolved in pure water, and after re-dissolution, a CAPCELL PAK C18-AQ (250×10mm, 5μm) preparative column was used, and ultrapure water: chromatographic methanol was 20:80 as the mobile phase, the flow rate was 16ml / min, and ultraviolet DAD was used as the detector for online detection. The preparative liquid was used for further purification, the collected liquid was re-dissolved in pure water after being dried, and dried in a freeze dryer to finally obtain 220 mg of powder, that is, pure D-prunin powder (purity ≥98%) was obtained.

[0162] Table 3 Silica gel column chromatography separation conditions

[0163] Eluent ratio Column volume Ethyl acetate: petroleum ether (1:1) 3 Ethyl acetate: petroleum ether (2:5) 5 Ethyl acetate 10 Ethyl acetate: methanol (10:1) 5 Ethyl acetate: methanol (10:3) 5

[0164] 5. Identification of D-Prunin

[0165] HPLC area normalization showed that the yield of D-prunin was ≥98%, the mass spectrometry showed that the fragment ions of D-prunin were consistent with D-prunin, and the nuclear magnetic resonance spectrum showed that the chemical structure of D-prunin was consistent with that of D-prunin and there were no obvious impurities.

[0166] The embodiments of the present invention are described in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, changes or deformations made by those skilled in the art based on the ideas of the present invention, the specific implementation methods and application scope of the present invention, all belong to the scope of protection of the present invention. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A method for preparing D-pyrocatechol from D-myrtle glycoside, characterized in that: The method comprises the following steps: (1) preparing a D-amygdalin solution and a β-glucosidase solution; and (2) The D-amygdalin solution and the β-glucosidase solution are mixed, and an enzymatic reaction is performed under certain conditions to obtain a reaction solution containing D-amygdalin.

2. The method according to claim 1, characterized in that The method further comprises the steps of: (3) adding a first solvent to the reaction solution containing D-prunoside to terminate the reaction, and drying to obtain a dried product; (4) adding the first solvent to the dried product to re-dissolve it, adding silica gel powder, stirring, and obtaining a product to be eluted; (5) gradient eluting the eluted product using a silica gel column, collecting the eluate containing D-prunoside, and drying to obtain a crude product of D-prunoside; (6) re-dissolving the crude D-pranicotin product with a first solvent, purifying, detecting and collecting fractions containing D-pranicotin, removing the organic solvent, and re-dissolving with an appropriate amount of water to obtain an aqueous solution of D-pranicotin; and (7) Drying the aqueous solution of D-prunoside to obtain D-prunoside.

3. The method according to claim 2, characterized in that The first solvent is an alcohol, such as methanol.

4. The method according to claim 1, characterized in that: In step (1), the method for preparing the D-amygdalin solution and the β-glucosidase solution comprises weighing appropriate amounts of D-amygdalin powder and β-glucosidase powder, respectively fixing the volumes in containers to obtain the D-amygdalin solution and the β-glucosidase solution; Preferably, the container is a volumetric flask; More preferably, the concentration of the D-amygdalin solution is 0.2 to 2.0 mg / ml, such as 0.5 to 1.5 mg / ml, such as about 1 mg / ml; More preferably, the concentration of the β-glucosidase solution is 0.05 to 2.0 unit / ml, such as 0.1 to 1.0 unit / ml, such as about 0.52 unit / ml or about 0.5 unit / ml.

5. The method according to claim 1, characterized in that In step (2), the enzymatic reaction is carried out in a water bath; Preferably, the temperature of the enzymatic reaction is 30-50°C, such as about 41.2°C or about 40°C; More preferably, the enzymatic reaction time is 5 to 20 min, such as about 7.33 min or about 7 min.

6. The method according to claim 2, characterized in that In step (3), the ratio of the volume (ml / ml) of the first solvent to the D-amygdalin solution is 3 to 5, for example, about 3.63; Preferably, in step (3), the drying is rotary evaporation drying.

7. The method according to claim 2, characterized in that: In step (4), the volume of the first solvent is 1 to 10 ml, for example, about 5 ml; Preferably, in step (4), the silica gel powder is 100-200 mesh silica gel powder; Preferably, in step (4), the mass of the silica gel powder is 5 to 15 g, for example, about 10 g; Preferably, in step (5), the silica gel column is filled with 200-300 mesh silica gel powder; Preferably, in step (5), the mass of the silica gel powder is 40 to 60 g, for example, about 50 g; More preferably, the mass ratio of the silica gel powder of step (4) to the silica gel powder of step (5) is 0.1 to 0.5, for example, about 0.2; More preferably, the gradient elution is performed according to the following procedure: (a) ethyl acetate: petroleum ether (1:1), about 3 column volumes; (b) ethyl acetate: petroleum ether (2:5), about 5 column volumes; (c) ethyl acetate, about 10 column volumes; (d) ethyl acetate: methanol (10:1), about 5 column volumes; and (e) ethyl acetate: methanol (10:3), about 5 column volumes; More preferably, the detection method of the eluent is thin layer chromatography; More preferably, the developing solvent of the thin layer chromatography is ethyl acetate and methanol; More preferably, the volume ratio of the ethyl acetate to the methanol is (4-6):(1-3), such as about 5:about 2; Also preferably, the drying is volatile drying or rotary evaporation drying.

8. The method according to claim 1, characterized in that In step (6), the purification is preparative liquid phase purification; Preferably, the chromatographic conditions for the preparative liquid phase purification are: using a CAPCELL PAK C18-AQ chromatographic column, mobile phase A is water, mobile phase B is methanol, and the flow rate is 10 to 20 ml / min; Preferably, the specifications of the chromatographic column are: column length 250 mm, inner diameter 10 mm, particle size 5 μm; More preferably, the water is pure water; More preferably, the methanol is chromatographic methanol; Also preferably, the volume ratio of the pure water to the chromatographic methanol is (10-30):(70-90), for example, about 20:about 80; Also preferably, the flow rate is 14-18 ml / min, for example about 16 ml / min; Still more preferably, the detection is performed using an ultraviolet diode array detector; Particularly preferably, the drying is rotary evaporation drying.

9. The method according to claim 2, characterized in that: In step (7), the drying is freeze-drying; Particularly preferably, the freeze-drying temperature is -20°C.

10. A solution containing D-pranin prepared by the method according to any one of claims 1 to 9, or D-pranin.

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