Method for preparing L-amygdalin and L-ulcerasin from D-amygdalin

Abstract

The invention discloses a method for preparing L-amygdalin and L-ulcerasin from D-amygdalin, which comprises the following steps: (1) preparing a D-amygdalin solution, and incubating the D-amygdalin solution under certain conditions to obtain an isomerization solution; and (2) preparing a beta-glucosidase solution, mixing the isomerization solution with the beta-glucosidase solution, and carrying out an enzymatic reaction under certain conditions to obtain a reaction solution containing L-amygdalin and L-ulcerasin. According to the invention, the L-amygdalin is converted from D-amygdalin through SN1 nucleophilic substitution reaction, and it is innovatively found that the L-amygdalin and the L-aronia melanocarpa glycoside are prepared and purified through asymmetric catalytic reaction according to the fact that two pairs of isomers have different selectivity to biological enzyme in space.

Description

Technical Field

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

[0002] Amygdalin is composed of one molecule of mandelanitrile and two molecules of glucose. It is a cyanogenic glycoside compound that 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. In recent years, studies have reported that amygdalin has a pair of chiral isomers. The first carbon atom connected to the benzene ring is a chiral carbon, and the four groups connected to it have different configurations due to different spatial arrangement orders. The main metabolite of amygdalin in the body is sauvignon glycoside, which is chemically composed of one molecule of mandelanitrile and one molecule of glucose, and also has a pair of chiral isomers. When testing the decocted medicinal materials, the coexistence of D / L amygdalin occurs. For medicinal materials with high amygdalin content such as peach kernels and almonds, most studies only focus on the analysis and detection of D-amygdalin and ignore the existence of L-amygdalin. The present invention converts L-amygdalin from D-amygdalin through SN1 nucleophilic substitution reaction, innovatively discovers that the two pairs of isomers have different selectivity for biological enzymes in space, and prepares and purifies L-amygdalin and L-sauropside through asymmetric catalytic reactions. The present invention solves the problem that L-amygdalin and L-sauropside reference substances are 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 L-amygdalin and L-serranoside from D-amygdalin, the method comprising the following steps:

[0004] (1) preparing a D-amygdalin solution, and incubating the D-amygdalin solution under certain conditions to obtain an isomerized solution; and

[0005] (2) preparing a β-glucosidase solution, mixing the isomerization solution and the β-glucosidase solution, and performing an enzymatic reaction under certain conditions to obtain a reaction solution containing L-amygdalin and L-prasinoside.

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

[0007] (3) adding a first solvent to the reaction solution containing L-amygdalin and L-prasinoside to terminate the reaction, and spinning to dry to obtain a dried product;

[0008] (4) adding a first solvent to the dried product for re-dissolution, purifying, collecting a fraction containing L-amygdalin and a fraction containing L-praline glycosides, respectively, to obtain an L-amygdalin collection solution and an L-praline glycoside collection solution, removing the organic solvent, and adding an appropriate amount of water for re-dissolution to obtain an aqueous solution of L-amygdalin and an aqueous solution of L-praline glycosides; and

[0009] (5) The aqueous solution of L-amygdalin and the aqueous solution of L-pranicotin are dried separately to obtain L-amygdalin and L-pranicotin.

[0010] Furthermore, the first solvent is alcohol, such as methanol.

[0011] Furthermore, the method for preparing the D-amygdalin solution comprises weighing an appropriate amount of D-amygdalin powder, placing it in a container, and fixing the volume to obtain the D-amygdalin solution.

[0012] Furthermore, the method for preparing the β-glucosidase solution comprises weighing an appropriate amount of β-glucosidase powder, placing it in a container, and fixing the volume to obtain the β-glucosidase solution.

[0013] Furthermore, the container is a volumetric flask.

[0014] Furthermore, the solvent for fixing the volume is water.

[0015] Furthermore, in step (1), the incubation is carried out in a water bath.

[0016] 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.

[0017] Furthermore, the temperature of the water bath is 70-90°C, for example, about 80°C.

[0018] Furthermore, the incubation time is 1 to 3 hours, such as 1.5 to 2 hours.

[0019] Furthermore, in step (2), the concentration of the β-glucosidase solution is 0.2 to 3.0 unit / ml, such as 0.5 to 2.0 unit / ml, such as about 1.06 unit / ml or 1 unit / ml.

[0020] Furthermore, the enzymatic reaction is carried out in a water bath.

[0021] Furthermore, the temperature of the enzymatic reaction is 50-70°C, such as about 58.93°C or 60°C.

[0022] Furthermore, the enzymatic reaction time is 10 to 30 min, such as about 19.67 min or about 20 min.

[0023] Furthermore, in step (3), the ratio of the volume (ml / ml) of the first solvent to the reaction solution is 3 to 5, for example, about 3.8.

[0024] Furthermore, in step (4), the purification is preparative liquid phase purification.

[0025] Furthermore, the mobile phase of the preparative liquid phase is pure water: methanol (75-85): (15-25) isocratic elution.

[0026] Furthermore, the mobile phase of the preparative liquid phase is pure water:methanol at a ratio of about 80:about 20 for isocratic elution.

[0027] Furthermore, the chromatographic column for preparing the liquid phase is a CAPCELLPAK C18-AQ chromatographic column.

[0028] Furthermore, the flow rate of the prepared liquid phase is 10-20 ml / min, for example, about 16 ml / min.

[0029] Furthermore, the detection wavelength of the prepared liquid phase is 200-250 nm, for example, 207 nm.

[0030] Furthermore, the instrument for the preparative liquid phase is an Agilent 1260 preparative liquid chromatograph.

[0031] Furthermore, the method for removing the organic solvent is rotary evaporation.

[0032] Furthermore, in step (5), the drying is freeze-drying.

[0033] According to another aspect of the present invention, there is provided a solution containing L-amygdalin and L-prasinoside prepared by the above method, or L-amygdalin and / or L-prasinoside.

[0034] Beneficial effects of the present invention:

[0035] 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. However, its chiral isomer L-Amygdalin and its metabolite L-Prunoside are relatively rare in nature, difficult to obtain, and there are few reports on biological activity research. The present invention uses biochemical methods such as D-Amygdalin as a raw material, introduces D / L racemate by SN1 nucleophilic substitution reaction, and obtains L-Amygdalin and its metabolite L-Prunoside by chiral asymmetric enzyme catalysis and separation. By optimizing the preparation process, L-Amygdalin and L-Prunoside are finally prepared and purified with a purity of ≥98%, and the two pairs of isomers are identified by chromatography and nuclear magnetic resonance spectroscopy. The research in this experiment provides certain possibilities for studying the pharmacological and pharmacodynamic activity, toxicity, and pharmacokinetic aspects of D / L amygdalin and its metabolite L-pyrocatechol, and also provides certain reference significance for the research on the quality standards of medicinal materials and preparations containing amygdalin. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] 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.

[0037] Figure 1 The figure is a schematic diagram of the results of the single factor investigation on the preparation of L-Prunoside. Among them, A is the effect of enzyme concentration on the yield of L-Prunoside, B is the effect of time on the yield of L-Prunoside, C is the effect of temperature on the yield of L-Prunoside, and D is the effect of pH on the yield of L-Prunoside.

[0038] Figure 2 The figure is a schematic diagram of the effect of interaction factors on the yield of L-sakura glycosides, where A is the interaction factor between temperature and time, B is the interaction factor between enzyme concentration and time, and C is the interaction factor between temperature and enzyme concentration.

[0039] Figure 3 The following is a schematic diagram of the response surface variance analysis results. A is a schematic diagram of prediction vs. actual, B is a normal plot of residuals, and C is a schematic diagram of residuals vs. predictions.

[0040] Figure 4 The flowchart of the preparation example of L-amygdalin and L-prunoside is shown in FIG. DETAILED DESCRIPTION

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] As described in the background technology section, when testing the decocted medicinal materials, the coexistence of D / L amygdalin occurs. For medicinal materials with high amygdalin content such as peach kernels and almonds, most studies only focus on the analysis and detection of D-amygdalin and ignore the existence of L-amygdalin and its chiral metabolite L-sakuraside. In order to solve the above problems, the present invention provides a method for preparing L-amygdalin and L-sakuraside from D-amygdalin, the method comprising the following steps:

[0047] (1) preparing a D-amygdalin solution, and incubating the D-amygdalin solution under certain conditions to obtain an isomerized solution; and

[0048] (2) preparing a β-glucosidase solution, mixing the isomerization solution and the β-glucosidase solution, and performing an enzymatic reaction under certain conditions to obtain a reaction solution containing L-amygdalin and L-prasinoside.

[0049] The present invention adopts the SN1 nucleophilic substitution reaction of D-amygdalin, i.e., its configuration is flipped under high temperature conditions to prepare the racemic form of D / L amygdalin, characteristically finds that D / L amygdalin has different selectivity for β-glucosidase, and utilizes the asymmetric catalytic reaction mediated by biological enzymes to prepare L-amygdalin and L-sakuraside.

[0050] Specifically, the present invention has found the following three points:

[0051] ① The conversion of D-amygdalin to L-amygdalin is an SN1 reaction. The isomerization will not proceed 100%. It is found in the experiment that the maximum conversion rate reaches about 50% and the equilibrium is reached. Therefore, the racemic body coexists in the next step of preparation.

[0052] ② In the process of converting amygdalin to cerasin, cerasin is an intermediate product. If the conversion conditions are mild, the yield of the target product is low, the raw material consumption is large, and the purification process is difficult. If the conversion conditions are too drastic, cerasin will be further converted into by-products, and the yield is still not ideal, so it is necessary to explore a more suitable reaction condition.

[0053] ③ There are differences in the spatial configuration of D-amygdalin and L-amygdalin. L-amygdalin is more stable than D-amygdalin under the catalysis of β-glucosidase. L-amygdalin and L-type cerasin can be selectively prepared through the asymmetry of the enzymatic reaction of D-amygdalin and L-amygdalin.

[0054] In a preferred embodiment, the method further comprises the steps of:

[0055] (3) adding a first solvent to the reaction solution containing L-amygdalin and L-prasinoside to terminate the reaction, and spinning to dry to obtain a dried product;

[0056] (4) adding a first solvent to the dried product for re-dissolution, purifying, collecting a fraction containing L-amygdalin and a fraction containing L-praline glycosides, respectively, to obtain an L-amygdalin collection solution and an L-praline glycoside collection solution, removing the organic solvent, and adding an appropriate amount of water for re-dissolution to obtain an aqueous solution of L-amygdalin and an aqueous solution of L-praline glycosides; and

[0057] (5) The aqueous solution of L-amygdalin and the aqueous solution of L-pranicotin are dried separately to obtain L-amygdalin and L-pranicotin.

[0058] In a preferred embodiment, the first solvent is an alcohol, such as methanol.

[0059] In a preferred embodiment, the method for preparing the D-amygdalin solution comprises weighing an appropriate amount of D-amygdalin powder, placing it in a container, and fixing the volume to obtain the D-amygdalin solution.

[0060] In a preferred embodiment, the method for preparing the β-glucosidase solution comprises weighing an appropriate amount of β-glucosidase powder, placing it in a container, and fixing the volume to obtain the β-glucosidase solution.

[0061] In a preferred embodiment, the container is a volumetric flask.

[0062] In a preferred embodiment, the constant volume solvent is water.

[0063] In a preferred embodiment, in step (1), the incubation is carried out in a water bath.

[0064] 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.

[0065] In the present invention, when concentration, temperature, time, ratio, or other value or parameter is 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 range upper limit or preferred value with any range lower 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.

[0066] 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.

[0067] In a preferred embodiment, the temperature of the water bath is 70-90°C, such as about 80°C.

[0068] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 80" includes ±5% of 80, or from 76 to 84.

[0069] In a preferred embodiment, the incubation time is 1 to 3 hours, such as 1.5 to 2 hours.

[0070] In a preferred embodiment, in step (2), the concentration of the β-glucosidase solution is 0.2 to 3.0 unit / ml, such as 0.5 to 2.0 unit / ml, such as about 1.06 unit / ml or 1 unit / ml.

[0071] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1.06" includes ±5% of 1.06, or from 1.007 to 1.113.

[0072] In a preferred embodiment, the enzymatic reaction is carried out in a water bath.

[0073] In a preferred embodiment, the temperature of the enzymatic reaction is 50-70°C, such as about 58.93°C or 60°C.

[0074] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 58.93" includes ±5% of 58.93, or from 55.9835 to 61.8765.

[0075] In a preferred embodiment, the enzymatic reaction time is 10 to 30 min, such as about 19.67 min or about 20 min.

[0076] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 19.67" includes ±5% of 19.67, or from 18.6865 to 20.6535; "about 20" includes ±5% of 20, or from 19 to 21.

[0077] In a preferred embodiment, in step (3), the ratio of the volume (ml / ml) of the first solvent to the reaction solution is 3-5, for example, about 3.8.

[0078] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 3.8" includes ±5% of 3.8, or from 3.61 to 3.99.

[0079] In a preferred embodiment, in step (4), the purification is preparative liquid phase purification.

[0080] In a preferred embodiment, the mobile phase of the preparative liquid phase is pure water:methanol (75-85):(15-25) isocratic elution.

[0081] In a preferred embodiment, the mobile phase of the preparative liquid phase is pure water:methanol at a ratio of about 80:about 20 for isocratic elution.

[0082] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 80" includes ±5% of 80, or from 76 to 84; "about 20" includes ±5% of 20, or from 19 to 21.

[0083] In a preferred embodiment, the chromatographic column for preparing the liquid phase is a CAPCELL PAK C18-AQ chromatographic column.

[0084] In a preferred embodiment, the flow rate of the preparative liquid phase is 10-20 ml / min, such as about 16 ml / min.

[0085] 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 16.2.

[0086] In a preferred embodiment, the detection wavelength of the prepared liquid phase is 200-250 nm, for example, 207 nm.

[0087] In a preferred embodiment, the preparative liquid phase instrument is an Agilent 1260 preparative liquid chromatograph.

[0088] In a preferred embodiment, the method for removing the organic solvent is rotary evaporation.

[0089] In a preferred embodiment, in step (5), the drying is freeze-drying.

[0090] According to another aspect of the present invention, there is provided a solution containing L-amygdalin and L-prasinoside prepared by the above method, or L-amygdalin and / or L-prasinoside.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] Example

[0095] 1. Materials and reagents

[0096] 1.1 Instruments

[0097] 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.

[0098] 1.2 Reagents and test drugs

[0099] 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.).

[0100] 2. Experimental methods

[0101] 2.1. Sample processing method

[0102] Take 200 μL of the incubation sample, add 800 μL of methanol, vortex for 3 min, centrifuge at 13000 r / min for 10 min at 4°C, take the supernatant and filter it through a 0.22 μm microporous membrane, and analyze it according to the chromatographic conditions.

[0103] 2.2. Preparation of solutions and samples

[0104] 2.2.1. Preparation of test solution

[0105] Accurately weigh 1000 mg of D-amygdalin reference substance and dilute it to a 1000 mL volumetric flask with ultrapure water to obtain a D-amygdalin stock solution with a mass concentration of 1 mg / mL.

[0106] 2.2.2. Isomerization conditions of D-amygdalin

[0107] Take D-amygdalin with a mass concentration of 1 mg / mL to investigate

[0108] Temperature: The degree of isomerization was investigated at 50℃, 60℃, 70℃, 80℃ and 90℃ for 60 min respectively.

[0109] Treatment time: The isomerization degree was investigated at 80°C for 30 min, 40 min, 50 min, 60 min, 70 min, 80 min and 90 min respectively.

[0110] PH: Isomerization degree at PH 3, PH 5, PH 7, PH 8, and PH 9 for 60 min.

[0111] 2.3 Factors affecting the preparation of L-Prunin

[0112] 2.3.1. Study on the factors affecting the yield of L-prunin at different times

[0113] The production rate of L-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.

[0114] 2.3.2. Study on the factors affecting the yield of L-prunin at different temperatures

[0115] The production rate of L-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℃.

[0116] 2.3.3. Study on the factors affecting the yield of L-prunin at different enzyme concentrations

[0117] The production rates of L-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.

[0118] 2.3.4 Response surface optimization experimental design

[0119] The Box-Behnken response surface method was used to optimize the experimental conditions by screening out the appropriate interval through the results of single-factor investigation. The temperature range for L-prunoside preparation was 30-80°C, the reaction time was 10-30min, and the enzyme concentration was 0.5-2unit / ml.

[0120] 3. Experimental results

[0121] 3.1. Isomerization of D-amygdalin

[0122] The isomerization experiment of D-amygdalin investigated the isomerization degree of amygdalin when the temperature was 30-80°C and the heat treatment time was 60-120min. The results showed that isomerization occurred after the temperature exceeded 50°C, and this isomerization intensified with the increase of temperature and heat treatment time, and finally stabilized at an isomerization rate of about 50%. The present invention investigated that when the temperature was 80°C and the heat treatment time was 60min, 90min, and 120min, the isomerization ratios were 43.35%, 48.26%, and 48.33%, respectively.

[0123] The experimental results show that when the temperature exceeds 50°C, D-type amygdalin will be converted into L-type amygdalin, and acidic conditions can inhibit its conversion. As the treatment time increases, the reaction will reach equilibrium, and the ratio of D-type and L-type amygdalin will no longer change. However, under alkaline conditions (such as pH 11), the structure of D-type amygdalin will be destroyed. It was finally determined that the isomerization conditions were 80°C, the heat treatment time was 90 to 120 minutes, and the conversion rate of L-amygdalin was about 48%.

[0124] 3.2. Single factor investigation results of L-Prunoside preparation

[0125] 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 L-prunin will increase with the increase of incubation temperature. When a certain temperature is reached, 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 10-30min, the yield R will increase with the increase of incubation time. When a certain time is reached, 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 0.5-2Unit / m, the yield R of L-prunin will increase with the increase of enzyme concentration. When a certain time is reached, the yield R will decrease with the increase of enzyme concentration. Since the single-factor pH experiment found that the fluctuation of yield R caused by pH changes was only within 1%, the four-factor response surface process optimization investigation was more difficult. Therefore, the subsequent investigation of the preparation process selected the three more critical factors of temperature, reaction time, and enzyme concentration as variables, and then the optimal reaction conditions were screened through response surface optimization.

[0126] 3.3 Response surface design of L-prunin

[0127] 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.

[0128] Table 1 Experimental design of independent variable response surface

[0129]

[0130]

[0131] Table 2 Experimental statistical analysis of independent variable responses

[0132] Sources of variance sum of squares Degrees of Freedom Mean square sum F-number P-value Significance Model 140.27 9 15.59 8.40 0.0052 Significant A-Reaction time 0.94 1 0.94 0.51 0.4989 B-Reaction temperature 4.79 1 4.79 2.58 0.1522 C-enzyme concentration 22.55 1 22.55 12.16 0.0102 AB 0.014 1 0.014 <![CDATA[7.36×10 -3 ]]> 0.9341 AC 14.36 1 14.36 7.74 0.0272 BC 3.97 1 3.97 2.14 0.1868 <![CDATA[A 2 ]]> 9.52 1 9.52 5.13 0.0579 <![CDATA[B 2 ]]> 0.056 1 0.056 0.03 0.8670 <![CDATA[C 2 ]]> 55.88 1 55.88 30.13 0.0009 Residual 12.98 7 1.85 Lack of Fit 3.65 3 1.22 0.52 0.6897 Not significant Pure Error 9.33 4 2.33 sum 153.25 16

[0133] 3.4. Influence of interaction factors on the yield of L-prunasin

[0134] To study the effects of reaction time, reaction temperature, and enzyme concentration on the yield of L-prunasin, the designexpert 10.0 software was used to draw 3D response surface contour plots to show the relationship between the yield of L-prunasin and any two independent variables, and to more intuitively observe the interaction between variables from Figure 2 them. Three-dimensional response surface plots and two-dimensional contour plots were drawn while controlling the third factor at 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 pairwise interaction influence 3D diagram, it can be seen that the steepness between AC is higher than that between AB and BC. This also indicates that the two factors of enzyme concentration and reaction time have a greater impact on the production of L-prunasin.

[0135] Enzymatic hydrolysis kinetic equation: Yield of L-prunasin = -1.90 + 0.89A - 0.01B + 18.58C - 0.25AC + 0.05BC - 0.02A 2 -7.504C 2

[0136] 3.5. Response surface variance analysis

[0137] The designexpert 10.0 software was used to perform statistical and variance analysis on the experimental data. 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 the model of this experiment, P < 0.05, and the lack-of-fit term P = 0.0052 < 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 = 0.9153, R 2 adj = 0.8064, indicating that the model can explain 80.64% 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 L-prunasin.

[0138] 3.6. Verification of the response surface experimental model

[0139] Results: According to the conditions recommended by the response surface optimization equation, the yield was the highest at 27% when the reaction temperature was 58.93℃, the enzyme concentration was 1.06unit / ml, and the reaction time was 19.67min with L-amygdalin as the substrate. The verification results of this condition are as follows: Due to the high cost of preparation using L-amygdalin as the substrate, we used 2g of D-amygdalin as the raw material, first subjected it to racemization treatment to obtain a racemization system in which D / L-amygdalin coexisted, and then rounded off the above conditions to integer values ​​to obtain 106mg of L-sucrose glycoside under the conditions of 60℃, 1unit / ml, and 20min. The yield of L-sucrose glycoside calculated using D-amygdalin as the raw material was 5.3%, and the yield of L-sucrose glycoside 230mg was 11.5%.

[0140] 4. Examples of the preparation of L-amygdalin and L-chernoside

[0141] Preparation Example Flow Chart Figure 4 shown.

[0142] Accurately weigh 2000 mg of D-amygdalin powder and dilute it into a 2000 ml volumetric flask to obtain a solution with a concentration of 1 mg / ml. Incubate it in an 80°C water bath for 90 min to isomerize it. Take another β-glucosidase powder and dilute it into a 100 ml volumetric flask to obtain a solution with a concentration of 1 unit / ml. Mix the two and incubate them in a 60°C water bath for 20 min. Then use 8000 ml of methanol to stop the reaction, spin dry, and obtain a dry product. Dissolve the dry product in methanol and then perform preparative liquid chromatography (the preparative liquid phase conditions are pure water: methanol 80:20 isocratic elution, CAPCELL PAK C18-AQ chromatographic column, flow rate 16 ml / min, detection wavelength 207 nm, instrument Agilent 1260 preparative liquid chromatography) purification, collect the fractions containing L-amygdalin and L-prasinoside, and spin-dry the organic solvent, add an appropriate amount of water to re-dissolve and then dry in a freeze dryer to obtain pure L-amygdalin and L-prasinoside powder.

[0143] 5. Identification of L-amygdalin and L-pyrocatechol

[0144] HPLC area normalization showed that the yields of L-amygdalin and L-prunin were both ≥98%. Mass spectrometry showed that the fragment ions of L-amygdalin and L-prunin were consistent with L-amygdalin and L-prunin, respectively. Nuclear magnetic resonance spectra showed that the chemical structures of L-amygdalin and L-prunin were consistent and there were no obvious impurities.

[0145] 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 L-amygdalin and L-rutin from D-amygdalin, characterized in that: The method comprises the following steps: (1) preparing a D-amygdalin solution, and incubating the D-amygdalin solution under certain conditions to obtain an isomerized solution; and (2) preparing a β-glucosidase solution, mixing the isomerization solution and the β-glucosidase solution, and performing an enzymatic reaction under certain conditions to obtain a reaction solution containing L-amygdalin and L-prasinoside.

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 L-amygdalin and L-prasinoside to terminate the reaction, and spinning to dry to obtain a dried product; (4) adding a first solvent to the dried product for re-dissolution, purifying, collecting a fraction containing L-amygdalin and a fraction containing L-praline glycosides, respectively, to obtain an L-amygdalin collection solution and an L-praline glycoside collection solution, removing the organic solvent, and adding an appropriate amount of water for re-dissolution to obtain an aqueous solution of L-amygdalin and an aqueous solution of L-praline glycosides; and (5) Drying the aqueous solution of L-amygdalin and the aqueous solution of L-pranicotin, respectively, to obtain L-amygdalin and L-pranicotin.

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 The method for preparing the D-amygdalin solution comprises weighing an appropriate amount of D-amygdalin powder and placing it in a container, and fixing the volume to obtain the D-amygdalin solution; Preferably, the method for preparing the β-glucosidase solution comprises weighing an appropriate amount of β-glucosidase powder and placing it in a container, and fixing the volume to obtain the β-glucosidase solution; More preferably, the container is a volumetric flask; Also preferably, the constant volume solvent is water.

5. The method according to claim 1, characterized in that In step (1), the incubation is carried out in a water bath; 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; Preferably, the temperature of the water bath is 70-90°C, for example about 80°C; More preferably, the incubation time is 1 to 3 hours, such as 1.5 to 2 hours.

6. The method according to claim 1, characterized in that In step (2), the concentration of the β-glucosidase solution is 0.2 to 3.0 unit / ml, such as 0.5 to 2.0 unit / ml, such as about 1.06 unit / ml or 1 unit / ml; More preferably, the enzymatic reaction is carried out in a water bath; Also preferably, the temperature of the enzymatic reaction is 50-70°C, such as about 58.93°C or 60°C; Preferably, the enzymatic reaction time is 10 to 30 min, such as about 19.67 min or about 20 min.

7. 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 reaction solution is 3 to 5, for example, about 3.

8.

8. The method according to claim 2, characterized in that: In step (4), the purification is preparative liquid phase purification; Preferably, the mobile phase of the preparative liquid phase is pure water:methanol (75-85):(15-25) isocratic elution; Preferably, the mobile phase of the preparative liquid phase is pure water:methanol at a ratio of about 80:about 20 for isocratic elution; Preferably, the chromatographic column for preparing the liquid phase is a CAPCELL PAKC18-AQ chromatographic column; Preferably, the flow rate of the preparation liquid phase is 10 to 20 ml / min, for example about 16 ml / min; Preferably, the detection wavelength of the prepared liquid phase is 200-250 nm, for example 207 nm; Preferably, the instrument for preparing the liquid phase is an Agilent 1260 preparative liquid chromatograph; Preferably, the method of removing the organic solvent is rotary evaporation.

9. The method according to claim 2, characterized in that: In step (5), the drying is freeze-drying.

10. A solution containing L-amygdalin and L-prasinoside prepared by the method according to any one of claims 1 to 9, or L-amygdalin and / or L-prasinoside.

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