A method for extracting sciurocandin A by bioenzymatic pretreatment

Abstract

The invention relates to the technical field of natural plant extraction, and in particular to a method for extracting scutellaria baicalenzyme by pretreatment. The existing scutellaria baicalenzyme extraction technology is improved, and the scutellaria baicalenzyme is pretreated in sequence by amylase, saccharifying enzyme and pectinase, so that the colloid dissolution rate in the extraction process is greatly reduced, thereby increasing the extraction rate of scutellaria baicalenzyme, thereby effectively utilizing wild resources, increasing the utilization rate of raw materials, and improving economic benefits. The pretreated crude product is extracted by a percolation method, which saves the process of separating the extract and the drug residue, reduces energy consumption, greatly reduces the amount of organic reagents, is environmentally friendly, economical, safe, and has a wide market value.

Description

Technical Field

[0001] The invention relates to the technical field of natural plant extraction, and in particular to a method for extracting scutellarin A through biological enzyme pretreatment. Background Art

[0002] The traditional Chinese medicine snow thorn is the dried tuber of the genus Snow thorn, which has medicinal effects such as clearing away heat and detoxifying, anti-inflammatory and bactericidal, stomachic and analgesic. It is often used clinically to treat common infectious diseases such as stomachache, ulcers, bronchitis, pneumonia, enteritis, upper respiratory tract infection, urinary tract infection, etc. It is a type of Chinese herbal medicine commonly used by various ethnic groups in southwest my country.

[0003] Among the active ingredients in snow gall, the content of cucurbitane triterpenes is the highest, including snow gall bladder glycosides A and snow gall bladder glycosides B. This type of ingredient has pharmacological activities such as anti-tumor, antibacterial, anti-inflammatory, and anti-HIV, making snow gall bladder have extremely high medicinal value. At present, the snow gall bladder extract used by pharmaceutical companies to produce snow gall bladder medicines mainly uses snow gall bladder glycosides A as the active ingredient. Since snow gall bladder is an underground tuberous rhizome rich in starch and colloid components, the traditional snow gall bladder glycosides A extraction process is to directly use ethanol reflux extraction after crushing the snow gall bladder, such as the Chinese patent "A method for preparing snow gall bladder glycosides A and snow gall bladder B" with application number 201410279399.1, the method disclosed in the patent is to directly use ethanol to extract the snow gall bladder raw material, and the Chinese patent "An improved method for extracting snow gall" with application number 201010235765.1, the method disclosed in the patent is also to crush the snow gall bladder and extract it with a certain concentration of ethanol. Directly using ethanol extraction will result in a large consumption of organic reagents such as ethanol, ethyl acetate, and acetone during the extraction process, high energy consumption, and no consideration of the comprehensive utilization of starch resources. Furthermore, the crude extract contains a large amount of colloid components, which seriously affects the subsequent purification process. Snow bile is easily wrapped by colloid and difficult to separate, affecting the purity of the final product. Therefore, how to improve the process, reduce the amount of organic solvents, and at the same time improve the utilization rate of resources such as starch, reduce the impact of impurities on product purity, and reduce energy consumption, is an urgent problem to be solved. Summary of the invention

[0004] In order to overcome the shortcomings of the above-mentioned technical defects, the present invention adopts a bio-enzyme method to pretreat the snow gall, and combines it with a room temperature solvent extraction process to solve the problems of high energy consumption, high waste of resources, high glue yield and great difficulty in purification in traditional processes.

[0005] The object of the present invention is to provide a method for extracting sciurocandin A by bioenzymatic pretreatment, which comprises the following steps:

[0006] S1. The mass volume ratio of the snow gallbladder slices and water is 1: (8-15), and heated to gelatinize; in actual operation, the snow gallbladder slices are crushed, soaked in water according to a certain mass volume ratio, and heated to gelatinize. The mass volume ratio of the snow gallbladder slices and water can be 1: 8, 1: 9, 1: 10, 1: 11, 1: 12, 1: 13, 1: 14, 1: 15;

[0007] S2. Under the condition of pH 6-7, α-amylase was added to the gelatinized product of S1 to perform an enzymatic hydrolysis; in actual operation, the iodine solution method was used to determine the reaction end point, and the reaction was stopped until the iodine solution was added dropwise to the reaction solution and the iodine solution did not change to blue;

[0008] S3. Under the condition of pH 4-5, add saccharifying enzyme to the enzymatic hydrolyzate obtained in S2 to perform second-stage enzymatic hydrolysis; in actual operation, use anhydrous ethanol to test the saccharification degree of the liquid until no white flocculent precipitation appears when the supernatant is dropped into anhydrous ethanol. After the reaction is completed, heat to 80°C to inactivate the enzyme;

[0009] S4. The hydrolyzate obtained in S3 was centrifuged and filtered, the precipitate was taken, mixed with water, the pH was adjusted to 3-4, pectinase was added, and three-stage hydrolysis was performed;

[0010] S5. The enzymatic hydrolyzate obtained in S4 is filtered, the precipitate is taken, ethanol is added for extraction at room temperature, and the extract is concentrated under reduced pressure to obtain a primary extract; in actual operation, the precipitate is dried, placed in a percolation tank, and a certain concentration of ethanol is used for percolation extraction, which reduces energy consumption compared to hot reflux extraction, eliminates the process of separating the extract and the residue, and the amount of anhydrous ethanol and ethyl acetate is relatively reduced, and the colloid dissolution rate during the extraction process is greatly reduced;

[0011] S6. The primary extract obtained in S5 is extracted with ethyl acetate, the extract is collected, and the secondary extract is concentrated under reduced pressure to obtain a secondary extract, and the secondary extract is washed with water to remove impurities to obtain a crude product of snow bile acid; in actual operation, the primary extract is dissolved in ethyl acetate and then filtered to remove fat-soluble insoluble impurities, and the secondary extract is washed with water and then filtered to remove water-soluble impurities;

[0012] S7. Add ethanol to the crude product of scutellariae A for crystallization to obtain a finished product. In actual operation, the solvent used for crystallization can be petroleum ether, ether, n-hexane, dichloromethane, chloroform, acetone, acetonitrile, methanol, or ethanol.

[0013] In some specific embodiments, the finished product obtained in S7 can be further purified by column chromatography to obtain high-purity sciurocandin A.

[0014] Further, in S2, before adding α-amylase, anhydrous calcium chloride is added, and the amount of anhydrous calcium chloride added is 0.1wt%-0.5wt% of the mass of the added water; in actual operation, the amount of anhydrous calcium chloride added can be 0.1wt%, 0.2wt%, 0.3wt%, 0.4wt%, 0.5wt% of the mass of the water;

[0015] Further, in S2, the dosage of α-amylase is 10U / g-20U / g, and the enzymolysis reaction temperature is 80°C-95°C; in actual operation, the dosage of α-amylase can be 10U / g, 12U / g, 14U / g, 16U / g, 18U / g, 20U / g; the enzymolysis reaction temperature can be 80°C, 85°C, 90°C, 95°C;

[0016] Further, in S3, the dosage of saccharifying enzyme is 150U / g-200U / g, and the enzymatic reaction temperature is 48°C-55°C; in actual operation, the dosage of saccharifying enzyme can be 150U / g, 160U / g, 170U / g, 180U / g, 190U / g, 200U / g; the enzymatic reaction temperature can be 48°C, 50°C, 52°C, 55°C;

[0017] Further, in S4, the dosage of pectinase is 0.05%-0.3% of the mass of the precipitate, and the enzymolysis time is 1-2h; in actual operation, the dosage of pectinase can be 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3% of the mass of the precipitate; the enzymolysis reaction temperature can be 48°C, 50°C, 52°C, 55°C;

[0018] Further, in S5, the ethanol concentration is 60%-95%; in actual operation, the ethanol concentration can be 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%;

[0019] Furthermore, in S6, the water washing and impurity removal is specifically as follows: adding an appropriate amount of water to the secondary extract, heating to 50° C. and stirring for 30 minutes, standing at room temperature for 12 hours, removing the supernatant after centrifugation, and drying to obtain the crude product of snow bile acid.

[0020] Furthermore, in S7, the solvent used for crystallization is ethanol, and in a more preferred embodiment, the concentration of ethanol is 50%-70%.

[0021] Beneficial effects: Compared with the prior art, the method provided by the present invention provides a method for pretreatment of snow bile by bioenzyme to extract snow bile A, in which the snow bile is pretreated in sequence by amylase, saccharifying enzyme and pectinase, thereby destroying the structure of starch and pectin, so that the snow bile encapsulated can be better released, and water-soluble impurities can be removed, the viscosity of the raw materials can be reduced, and the pretreated crude product is extracted by percolation, which eliminates the process of separating the extract and the residue, reduces energy consumption, reduces the amount of organic reagents used, and greatly reduces the colloid dissolution rate in the extraction process. It is environmentally friendly, economical, safe, and has broad market value. DETAILED DESCRIPTION

[0022] In order to make those skilled in the art better understand the technical scheme of the present invention, the present invention is described in detail below in conjunction with specific embodiments. The experimental methods for which specific conditions are not indicated in the following examples are usually carried out under normal conditions or according to the conditions recommended by the manufacturer. The test materials used in the following examples, unless otherwise specified, are purchased from conventional biochemical reagent stores. Unless otherwise specified, percentages and parts are calculated by weight. Unless otherwise defined, all professional and scientific terms used in the text have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar to or equal to the recorded content can all be applied to the present invention. The preferred implementation methods and materials described in the text are only for demonstration purposes.

[0023] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0024] According to the present invention, before extracting sucrose, liquefaction enzyme, saccharification enzyme and pectinase are used to pre-treat the raw material respectively, and the starch and colloid components contained in the raw material are first removed.

[0025] In some specific embodiments, the dried snow gall slices are crushed, soaked in water at a mass volume ratio of 1:8-1:15, and heated for gelatinization; α-amylase is added to the gelatinized product of S1 at a pH of 6-7 to perform a first-stage enzymolysis; saccharifying enzyme is added to the first enzymolysis solution at a pH of 4-5 to perform a second-stage enzymolysis;

[0026] According to the present invention, water is used as the reaction liquid, and liquefying enzyme and saccharifying enzyme are added successively for reaction, and the two enzymes work together to relatively completely hydrolyze the starch substances in the snow gall tuber into water-soluble monosaccharides, and the obtained solid-liquid mixture is separated and the solid and the aqueous phase are retained respectively. The obtained aqueous phase is a high-concentration sugar solution, which can be reused, thereby realizing the full utilization of starch resources. Snow gall has the property of being water-insoluble and will not be removed in the aqueous phase, while most of the water-soluble impurities can be removed during the treatment process;

[0027] In some specific embodiments, the secondary enzymatic hydrolysis solution is centrifuged and filtered, the precipitate is collected, water is added and mixed, the pH is adjusted to 3-4, pectinase is added, and three-stage enzymatic hydrolysis is performed;

[0028] According to the present invention, the solids are then subjected to the action of pectinase to remove part of the colloid, thereby reducing the negative impact of the colloid on the purification of the product, and the solids obtained by finally separating the aqueous phase are used as the raw material for the next step of extraction; and the internal structure of the solids is destroyed to a certain extent by the action of the three enzymes, and most of the components encapsulating the styraclonal glycosides are hydrolyzed, which is more conducive to the dissolution of the styraclonal glycosides during extraction;

[0029] In some specific embodiments, the three-stage enzymatic hydrolyzate is filtered, the precipitate is taken, ethanol is added for extraction at room temperature, and the extract is concentrated under reduced pressure to obtain a primary extract; the primary extract is added with ethyl acetate for extraction, the extract is collected, and concentrated under reduced pressure to obtain a secondary extract, the secondary extract is washed with water to remove impurities, and a crude product of styrene is obtained; the crude product of styrene is added to a solvent for crystallization to obtain refined styrene (the structural formula is shown below).

[0030]

[0031] Based on the present invention, the percolation method is combined to replace the traditional ethanol hot reflux extraction, and the product is extracted at room temperature. On the one hand, the energy consumption during extraction and the amount of organic reagents used are reduced, and on the other hand, the dissolution rate of impurities such as colloids is reduced, the difficulty of purification is reduced, the impurities in the final product are reduced, and the product purity is improved.

[0032] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments and appended tables. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0033] Example 1

[0034] S1. After crushing the dried snow gall slices, soak them in water at a ratio of 1:10 for 24 hours, heat at 85°C for gelatinization, adjust the pH to 6.0, add 0.2% anhydrous calcium chloride powder, and after fully dissolving, add 16U / g α-amylase for enzymolysis, enzymolyze the snow gall at 90°C, and determine the reaction endpoint by iodine solution method, and react until the iodine solution does not turn blue when the reaction solution is added dropwise to the iodine solution;

[0035] S2. The reaction solution obtained in step S1 was adjusted to pH 4.0 to inactivate the enzyme, the pH was adjusted to 4.5, the temperature was 55°C, 180 U / g of saccharifying enzyme was added for enzymatic hydrolysis, and the saccharification degree of the liquid was tested by anhydrous ethanol. The supernatant was dropped into anhydrous ethanol until no white flocculent precipitate appeared. After the reaction was completed, it was heated to 80°C to inactivate the enzyme;

[0036] S3. The saccharification reaction liquid was centrifuged to remove the supernatant, water was added to the precipitate to soak, the pH was adjusted to 3.5, pectinase was added at a dosage of 0.2%, and the mixture was reacted at 50°C for 1.5h.

[0037] S4. Centrifuge the reaction solution to obtain the precipitate, and dry it.

[0038] S5. Add the dried snow gall residue into a percolation tank and use 90% ethanol for percolation extraction. After the extraction is completed, the percolation liquid is reduced in pressure and concentrated to an extract.

[0039] S6. Add ethyl acetate to the extract to dissolve, filter to remove insoluble impurities, concentrate under reduced pressure and evaporate to dryness, add appropriate amount of water, heat to 50°C and stir for 30 minutes, and stand at room temperature for 12 hours to precipitate. After centrifugation of the precipitate, remove the supernatant and dry to obtain the crude product of sciurosin A.

[0040] S7. Crystallize the sclerostin A with 60% ethanol to obtain a refined sclerostin A product.

[0041] Example 2

[0042] S1. After crushing the dried snow gall slices, soak them in water at a ratio of 1:9 for 36 hours, heat at 85°C for gelatinization, adjust the pH to 6.2, add 0.2% anhydrous calcium chloride powder, and after fully dissolving, add 14U / g α-amylase for enzymolysis, enzymolyze the snow gall at 85°C, and determine the reaction endpoint by iodine solution method, and react until the iodine solution does not turn blue when the reaction solution is added dropwise to the iodine solution;

[0043] S2. The reaction solution obtained in step S1 was adjusted to pH 4.0 to inactivate the enzyme, the pH was adjusted to 4.2, the temperature was 60°C, 200 U / g of saccharifying enzyme was added for enzymatic hydrolysis, and the saccharification degree of the liquid was tested by anhydrous ethanol. The supernatant was dropped into anhydrous ethanol until no white flocculent precipitate appeared. After the reaction was completed, it was heated to 80°C to inactivate the enzyme;

[0044] S3. The saccharification reaction liquid was centrifuged to remove the supernatant, water was added to the precipitate to soak, the pH was adjusted to 3.5, pectinase was added at a dosage of 0.25%, and the mixture was reacted at 50°C for 2h.

[0045] S4. Centrifuge the reaction solution to obtain the precipitate, and dry it.

[0046] S5. Add the dried snow gall residue into a percolation tank and use 90% ethanol for percolation extraction. After the extraction is completed, the percolation liquid is reduced in pressure and concentrated to an extract.

[0047] S6. Add ethyl acetate to the extract to dissolve, filter to remove insoluble impurities, concentrate under reduced pressure and evaporate to dryness, add appropriate amount of water, heat to 50°C and stir for 30 minutes, and stand at room temperature for 12 hours to precipitate. After centrifugation of the precipitate, remove the supernatant and dry to obtain the crude product of sciurosin A.

[0048] S7. Crystallize the sclerostin A with 60% ethanol to obtain a refined sclerostin A product.

[0049] Example 3

[0050] S1. After crushing the dried snow gall slices, soak them in water at a ratio of 1:12 for 48 hours, heat at 85°C for gelatinization, adjust the pH to 6.8, add 0.2% anhydrous calcium chloride powder, and after fully dissolving, add 20U / g α-amylase for enzymolysis, enzymolyze the snow gall at 85°C, and determine the reaction endpoint by iodine solution method, and react until the iodine solution does not turn blue when the reaction solution is added dropwise to the iodine solution;

[0051] S2. The reaction solution obtained in step S1 was adjusted to pH 4.0 to inactivate the enzyme, the pH was adjusted to 4.8, the temperature was 50°C, 150 U / g of saccharifying enzyme was added for enzymatic hydrolysis, and the saccharification degree of the liquid was tested with anhydrous ethanol. The supernatant was dropped into anhydrous ethanol until no white flocculent precipitate appeared. After the reaction was completed, it was heated to 80°C to inactivate the enzyme;

[0052] S3. The saccharification reaction liquid was centrifuged to remove the supernatant, water was added to the precipitate to soak, the pH was adjusted to 3.5, pectinase was added at a dosage of 0.15%, and the mixture was reacted at 50°C for 1 hour.

[0053] S4. Centrifuge the reaction solution to obtain the precipitate, and dry it.

[0054] S5. Add the dried snow gall residue into a percolation tank and use 90% ethanol for percolation extraction. After the extraction is completed, the percolation liquid is reduced in pressure and concentrated to an extract.

[0055] S6. Add ethyl acetate to the extract to dissolve, filter to remove insoluble impurities, concentrate under reduced pressure and evaporate to dryness, add appropriate amount of water, heat to 50°C and stir for 30 minutes, and stand at room temperature for 12 hours to precipitate. After centrifugation of the precipitate, remove the supernatant and dry to obtain the crude product of sciurosin A.

[0056] S7. Crystallize the sclerostin A with 60% ethanol to obtain a refined sclerostin A product.

[0057] Example 4

[0058] S1. After crushing the dried snow gall slices, soak them in water at a ratio of 1:11 for 20 hours, heat at 85°C for gelatinization, adjust the pH to 6.0, add 0.2% anhydrous calcium chloride powder, and after fully dissolving, add 10U / g α-amylase for enzymolysis, enzymolyze the snow gall at 85°C, and determine the reaction endpoint by iodine solution method, and react until the iodine solution does not turn blue when the reaction solution is added dropwise to the iodine solution;

[0059] S2. The reaction solution obtained in step S1 was adjusted to pH 4.0 to inactivate the enzyme, the pH was adjusted to 5.0, the temperature was 45°C, 180 U / g of saccharifying enzyme was added for enzymatic hydrolysis, and the saccharification degree of the liquid was tested by anhydrous ethanol. The supernatant was dropped into anhydrous ethanol until no white flocculent precipitate appeared. After the reaction was completed, it was heated to 80°C to inactivate the enzyme;

[0060] S3. The saccharification reaction liquid was centrifuged to remove the supernatant, water was added to the precipitate to soak, the pH was adjusted to 3.5, pectinase was added at a dosage of 0.2%, and the mixture was reacted at 50°C for 1.5h.

[0061] S4. Centrifuge the reaction solution to obtain the precipitate, and dry it.

[0062] S5. Add the dried snow gall residue into a percolation tank and use 90% ethanol for percolation extraction. After the extraction is completed, the percolation liquid is reduced in pressure and concentrated to an extract.

[0063] S6. Add ethyl acetate to the extract to dissolve, filter to remove insoluble impurities, concentrate under reduced pressure and evaporate to dryness, add appropriate amount of water, heat to 50°C and stir for 30 minutes, and stand at room temperature for 12 hours to precipitate. After centrifugation of the precipitate, remove the supernatant and dry to obtain the crude product of sciurosin A.

[0064] S7. Crystallize the sclerostin A with 60% ethanol to obtain a refined sclerostin A product.

[0065] Example 5

[0066] S1. After crushing the dried snow gall slices, soak them in water at a ratio of 1:10 for 36 hours, heat at 85°C for gelatinization, adjust the pH to 6.5, add 0.2% anhydrous calcium chloride powder, and after fully dissolving, add 15U / g α-amylase for enzymolysis, enzymolyze the snow gall at 85°C, and determine the reaction endpoint by iodine solution method, and react until the iodine solution does not turn blue when the reaction solution is added dropwise to the iodine solution;

[0067] S2. The reaction solution obtained in step S1 was adjusted to pH 4.0 to inactivate the enzyme, the pH was adjusted to 4.5, the temperature was 55°C, 160 U / g of saccharifying enzyme was added for enzymatic hydrolysis, and the saccharification degree of the liquid was tested with anhydrous ethanol. The supernatant was dropped into anhydrous ethanol until no white flocculent precipitate appeared. After the reaction was completed, it was heated to 80°C to inactivate the enzyme;

[0068] S3. The saccharification reaction liquid was centrifuged to remove the supernatant, water was added to the precipitate to soak, the pH was adjusted to 3.5, pectinase was added at a dosage of 0.1%, and the mixture was reacted at 50°C for 2h.

[0069] S4. Centrifuge the reaction solution to obtain the precipitate, and dry it.

[0070] S5. Add the dried snow gall residue into a percolation tank and use 90% ethanol for percolation extraction. After the extraction is completed, the percolation liquid is reduced in pressure and concentrated to an extract.

[0071] S6. Add ethyl acetate to the extract to dissolve, filter to remove insoluble impurities, concentrate under reduced pressure and evaporate to dryness, add appropriate amount of water, heat to 50°C and stir for 30 minutes, and stand at room temperature for 12 hours to precipitate. After centrifugation of the precipitate, remove the supernatant and dry to obtain the crude product of sciurosin A.

[0072] S7. Crystallize the sclerostin A with 60% ethanol to obtain a refined sclerostin A product.

[0073] Comparative Example 1 Traditional Process for Extracting Snow Gallbladder A

[0074] S1. The powder of snow gall is extracted with 95% ethanol in a constant temperature water bath at 80℃ for 1h, and the ethanol extract is retained by vacuum filtration. The residue is further added with 95% ethanol and refluxed for 1h. The reflux is repeated 3 times, and the ethanol extract is combined. The extract is allowed to stand at room temperature for 12h to precipitate impurities, and then vacuum filtered again. The filtrate is subjected to vacuum rotary evaporation at 50℃ to obtain a syrupy ethanol concentrate.

[0075] S2. Add appropriate amount of hot water to the ethanol concentrate to dilute it, and add 1:1 volume of petroleum ether to degrease it three times, add appropriate amount of anhydrous sodium sulfate to break the emulsion, and discard the petroleum ether part.

[0076] S3. Add 250 mL of ethyl acetate to the suspension, condense and reflux at 78°C for 1 h, repeat the reflux for 3 times, combine the ethyl acetate extracts, and concentrate under reduced pressure at 45°C to obtain a concentrate. Add a certain proportion of distilled water to the concentrate, heat to 50°C and stir for 30 min, let stand at room temperature for 12 h to precipitate, and filter to obtain the crude product of snow bile.

[0077] S4. Add appropriate amount of acetone to the precipitate, heat under reflux to fully dissolve it, filter the solution and retain the filtrate, concentrate under reduced pressure and add distilled water until turbidity appears, let it stand at room temperature for crystallization, filter out the crystals and dry them to obtain the final product.

[0078] Comparative Example 2

[0079] The same as Example 1, except that only α-amylase and saccharifying enzyme are used to treat the raw materials.

[0080] Comparative Example 3

[0081] The same as Example 2, except that only α-amylase and saccharifying enzyme are used to treat the raw materials.

[0082] The content of sutfollicularisin A in the refined sutfollicularisin A prepared in each embodiment was tested:

[0083] 1.1 Chromatographic conditions: Use C18 chromatographic column, acetonitrile: water as mobile phase for gradient elution, detection wavelength of 202nm, flow rate of 1mL / min, column temperature of 30℃, injection volume of 20μL. The specific elution method is shown in Table 1:

[0084] Table 1 HPLC elution method

[0085] Time (min) Flow rate (mL / min) Acetonitrile water 0 1.0 25％ 75％ 20 1.0 50％ 50％ 30 1.0 80％ 20％ 35 1.0 25％ 75％ 40 1.0 25％ 75％

[0086] 1.2 Draw the standard curve of sedative choline A: The peak time of sedative choline A is about 18 minutes, and the peak areas corresponding to the standard solutions of different concentrations are shown in Table 2:

[0087] Table 2 Peak areas corresponding to standard solutions of various concentrations

[0088] Injection concentration (mg / mL) Peak area 0.10 1593.2331 0.20 3051.7875 0.30 4552.4156 0.40 6202.6875 0.50 7729.8597 0.60 8979.7835

[0089] With the injection concentration of the standard sample of sedative as the abscissa (x) and the peak area as the ordinate (y), the regression equation was obtained: y=15033x+89.904, and a sedative standard curve was drawn, with a good linear relationship.

[0090] 1.3 Precision experiment: Accurately measure the standard solution of serotonin, measure it three times continuously, and calculate the precision. RSD = 1.2%, indicating good precision.

[0091] 1.4 Stability experiment: The same standard solution was measured three times at intervals of 4h, 8h and 12h, and the RSD was calculated to be 1.0%, indicating that the sample was stable at room temperature.

[0092] 1.5 Weigh 0.2 g of snow gall sample and place it in a 25 mL volumetric flask. After making up to volume with chromatographic methanol, ultrasonicate for 1 h, replenish with chromatographic methanol, and perform liquid phase detection after passing through a microporous filter membrane. The test results are shown in Table 3.

[0093] Table 3 Snow bile acid A content determination results

[0094] category Content of refined product sucrose acetylcholine A (%) Content of crude bile bile A (%) Example 1 84.53 64.52 Example 2 82.20 62.19 Example 3 80.50 63.24 Example 4 83.65 60.59 Example 5 82.30 64.11 Comparative Example 1 70.20 50.21 Comparative Example 2 73.52 54.12 Comparative Example 3 74.49 53.25

[0095] As can be seen from the above table, the crude product of the present invention 1-5 has a content of 60-65% of stygocandin A, and the content of the refined product of stygocandin A obtained in Examples 1-5 of the present invention is 80-85%, wherein the content of the refined product of stygocandin A prepared in Example 1 reaches 84.53%, while the content of stygocandin A in the refined product of stygocandin A prepared in Comparative Examples 1-3 is 70-75%, which shows that the method of the present invention increases the content of stygocandin A by 15-20%. In addition, compared with Comparative Example 1, the present invention reduces the amount of ethanol by an average of 21.44%, and the amount of ethyl acetate by an average of 80.52%, and compared with Comparative Example 2, the amount of ethanol by an average of 17.82%, and the amount of ethyl acetate by an average of 76.45%.

[0096] Finally, it should be noted that the above description is only a preferred embodiment of the present invention. Under the guidance of the present invention, those skilled in the art can make a variety of similar expressions without violating the purpose and claims of the present invention, and such changes all fall within the protection scope of the present invention.

Claims

1. A method for extracting sciurocandin by bioenzymatic pretreatment, characterized in that The following steps are involved: S1. Heat and gelatinize the snow gallbladder tablets and water in a volume ratio of 1: (8-15); S2. Add α-amylase to the gelatinized material of S1 at a pH value of 6-7 for enzymatic hydrolysis; S3. Under the condition of pH 4-5, saccharifying enzyme is added to the enzymatic hydrolyzate obtained in S2 to perform a second enzymatic hydrolysis; S4. The hydrolyzate obtained in S3 was centrifuged and filtered, the precipitate was taken, mixed with water, the pH was adjusted to 3-4, pectinase was added, and three-stage hydrolysis was performed; S5. The enzymatic hydrolyzate obtained in S4 is filtered, the precipitate is taken, ethanol is added for extraction at room temperature, and the extract is concentrated under reduced pressure to obtain a primary extract; S6. The primary extract obtained in S5 was extracted with ethyl acetate, the extract was collected, and concentrated under reduced pressure to obtain a secondary extract, and the secondary extract was washed with water to remove impurities to obtain a crude product of snow bile acid; S7. Add the crude product of sciurocandin A into a solvent for crystallization to obtain a finished product.

2. The method for extracting sciurocandin A by bioenzymatic pretreatment according to claim 1, characterized in that In S2, before adding α-amylase, anhydrous calcium chloride is added first, and the addition amount of the anhydrous calcium chloride is 0.1wt% to 0.5wt% based on the added mass of water.

3. The method for extracting sciurocandin A by bioenzymatic pretreatment according to claim 2, characterized in that In the S2, the dosage of α-amylase is 10U / g-20U / g, and the enzymatic reaction temperature is 80°C-95°C.

4. The method for extracting sciurocandin A by bioenzymatic pretreatment according to claim 2, characterized in that In the S3, the dosage of saccharifying enzyme is 150U / g-200U / g, and the enzymatic reaction temperature is 48°C-55°C.

5. The method for extracting sciurocandin A by bioenzymatic pretreatment according to claim 2, characterized in that In the S4, the dosage of pectinase is 0.05%-0.3% of the precipitate mass, and the enzymolysis time is 1-2h.

6. The method for extracting sciurocandin A by bioenzymatic pretreatment according to claim 2, characterized in that In the S5, the ethanol concentration is 60%-95%.

7. The method for extracting sciurocandin A by bioenzymatic pretreatment according to claim 2, characterized in that In S6, the water washing and impurity removal is specifically as follows: adding an appropriate amount of water to the secondary extract, heating to 50° C. and stirring for 30 minutes, standing at room temperature for 12 hours, removing the supernatant after centrifugation, and drying to obtain the crude product of scutellariae flavus.

8. The method for extracting sciurocandin A by bioenzymatic pretreatment according to claim 2, characterized in that In the step S7, the solvent used for crystallization is ethanol.

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