A method for preparing isoquercetin by enzymatic hydrolysis of Sophora japonica flowers.

Abstract

The invention discloses a method for preparing isoquercetin by enzymolysis of sophora japonica seed, comprising the following steps: S1, weighing sophora japonica seed, soaking in water, and obtaining soaked sophora japonica seed; S2, pressing the soaked sophora japonica seed to dry, adding water, and sequentially adding a reducing agent, cellulase, pectinase, and rhamnosidase, adjusting the pH to 5.5-7.5, and stirring for enzymolysis; S3, killing the enzyme after enzymolysis, adding activated carbon and borax, adjusting the pH to 10-11, stirring, filtering, collecting the filtrate and adjusting the pH to 4-5, standing, and collecting the filter cake; S4, extracting the filter cake with an alcohol solution, filtering, and collecting the filter cake to obtain isoquercetin. The method uses sophora japonica seed as a raw material, and utilizes cellulase, pectinase, and rhamnosidase to prepare isoquercetin in one step. The yield of the product obtained by the method of the invention is improved, the purity of isoquercetin is high, the environment is friendly, the production process is shortened, and the production cost is reduced.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a method for preparing isoquercetin by enzymatic hydrolysis of sophora japonica seeds. Background Art

[0002] Sophora japonica is the flower bud of the leguminous plant Sophora japonica, which has a high rutin content and is the main raw material for extracting rutin in my country's pharmaceutical industry. Isoquercetin has one less rhamnoside group than rutin and has extremely low solubility in water (95mg / L). The results of relevant pharmacological experiments show that isoquercetin has extremely high medicinal value and its pharmacological activity is much higher than rutin. Therefore, isoquercetin is a hot new drug compound in the pharmaceutical industry at present, and has always been a high-level food additive, auxiliary drug or active ingredient that major international pharmaceutical and food companies are competing to develop. Although isoquercetin is widely distributed in plants in nature, its content is very low. The content of isoquercetin in plants is only a few parts per hundred thousand to a few parts per ten thousand.

[0003] At present, the method for producing isoquercetin is mainly to use rutin enzymatically or hydrolytically desugared, which has the following main shortcomings: 1. In the process of preparing rutin, a large amount of inorganic solution or organic solvent is basically required, and the amount of sewage is very large, causing environmental pollution; 2. Due to the two-step preparation method, rutin is bound to remain in the drug residue easily, and then desugared by enzymatic hydrolysis or acid hydrolysis, resulting in isoquercetin not being 100% extracted, and the longer the process, the lower the loss rate; 3. In the process of using rutin to prepare isoquercetin by traditional methods, only isomers with different rhamnose connection modes are considered to prepare isoquercetin by desugaring, and other flavonoid components with similar structures in Sophora japonica are not considered, resulting in a waste of resources. Summary of the invention

[0004] In order to solve the defects in the prior art, the present invention provides a method for preparing isoquercetin by enzymatic hydrolysis of sophora japonica seeds. The method firstly utilizes cellulase and pectinase to destroy the tissue structure of the sophora japonica seeds so that flavonoids in the sophora japonica seeds are fully exposed, and then utilizes rhamnosidase to directly enzymatically hydrolyze rutin or flavonoid components with a structure similar to rutin (such as quercetin-3-O-glucose-7-O-rhamnoside) to generate isoquercetin. The isoquercetin is prepared from the sophora japonica seeds in one step, the purity and yield of the isoquercetin are improved, and the problem of using a large amount of chemical reagents in the preparation process and generating a large amount of wastewater to pollute the environment can be avoided, the production process is shortened, and the production cost is reduced.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention provides a method for preparing isoquercetin by enzymatic hydrolysis of sophora japonica seeds, comprising the following steps:

[0007] S1, weighing Sophora japonica seeds, adding water to soak, and obtaining the soaked Sophora japonica seeds;

[0008] S2, squeezing the soaked Sophora japonica seeds dry, adding water, and sequentially adding a reducing agent, cellulase, pectinase, and rhamnosidase, adjusting the pH to 5.5-7.5, and stirring for enzymolysis;

[0009] S3, after enzymatic hydrolysis, the enzyme is killed, activated carbon and borax are added, the pH is adjusted to 10-11, stirred, filtered, the filtrate is collected and the pH is adjusted to 4-5, allowed to stand, and the filter cake is collected;

[0010] S4, extracting the filter cake with an alcohol solution, filtering, and collecting the filter cake to obtain isoquercetin.

[0011] Preferably, the mass of the water in S1 is 5 times the mass of the Sophora japonica buds.

[0012] Preferably, the soaking time in S1 is 2 hours.

[0013] Preferably, in S2, the mass of the added water is 3 times the mass of the squeezed-dried Sophora japonica seeds.

[0014] Preferably, the mass of the reducing agent is 1 to 1.2% of the mass of the water in S2.

[0015] Preferably, the reducing agent in S2 is sodium pyrosulfite or cysteine.

[0016] Preferably, the mass ratio of cellulase to pectinase in S2 is (2-2.15):1.

[0017] Preferably, the mass ratio of cellulase to rhamnosidase in S2 is (1-3):(2-10).

[0018] Preferably, the pH regulator in S2 is sodium hydroxide solution.

[0019] More preferably, the pH in S2 is adjusted to 6-7.5.

[0020] Preferably, the temperature of the enzymatic hydrolysis in S2 is 40-65°C.

[0021] More preferably, the temperature of enzymatic hydrolysis in S2 is 45-60°C.

[0022] Preferably, the enzymatic hydrolysis time in S2 is 24 to 72 hours.

[0023] More preferably, the enzymatic hydrolysis time in S2 is 48 to 72 hours.

[0024] Preferably, the temperature is raised to 80° C. when inactivating the enzyme in S3.

[0025] Preferably, the mass ratio of activated carbon to borax in S3 is 10:1.

[0026] Preferably, the pH regulator for adjusting the pH to 10-11 in S3 is a sodium hydroxide solution.

[0027] Preferably, the pH regulator for adjusting the pH to 4-5 in S3 is a hydrochloric acid solution.

[0028] Preferably, the standing time in S3 is 12 to 24 hours.

[0029] Preferably, the alcohol solution in S4 is methanol or 90% ethanol.

[0030] More preferably, 90% of the ethanol in S4 is edible alcohol.

[0031] Preferably, the extraction in S4 is reflux extraction.

[0032] Preferably, after filtering in S4, the alcohol solution is recovered.

[0033] Preferably, after collecting the filter cake in S4, the filter cake needs to be washed with water, dried, and crushed.

[0034] The beneficial effects of the present invention are:

[0035] 1. The preparation method of the present invention is to add a reducing agent to the soaked sophora japonica seedlings to protect rutin, inhibit its oxidation, and facilitate the decomposition of subsequent enzymes. In an environment where pH is 5.5 to 7.5, cellulase and pectinase are utilized to destroy the organizational structure of the sophora japonica seedlings, so that flavonoid target components and effective ingredients such as rutin in the sophora japonica seedlings are fully exposed, and insufficient extraction is avoided due to cellulose wrapping, and rhamnosidase is used to directly enzymolyze rutin or a flavonoid component with a similar structure to rutin (such as quercetin-3-O-glucose-7-O-rhamnoside, which is different from the isomers of rhamnose) is generated to generate isoquercetin, then activated carbon and borax are added to decolorize and protect isoquercetin from being oxidized, and pH is adjusted to carry out alkali dissolution and acid precipitation, so as to obtain isoquercetin with a purity of up to 98% and a yield of up to 23.4%.

[0036] 2. The present invention adopts a simple enzyme with rhamnosidic bond, which has strong specificity and will not break the glucosidic bond, thereby ensuring that quercetin will not be produced in the product, improving the purity of isoquercetin, avoiding the problem of reduced yield caused by first preparing rutin and then desugaring to prepare isoquercetin, and also avoiding the problem of environmental pollution caused by a large amount of chemical reagents used in the preparation process and the generation of a large amount of wastewater. The present invention prepares isoquercetin from Sophora japonica in one step, and the obtained product has high yield and high purity of isoquercetin, shortens the production process, and reduces the production cost.

[0037] 3. The present invention has conducted multiple explorations into the enzymatic hydrolysis conditions and determined that in an environment with a pH of 6 to 7.5, the activities of the cellulase and rhamnosidase of the present invention can be maintained at the maximum, and the final product yield and purity can be maintained at a high level.

[0038] 4. The enzymolysis temperature is a condition for increasing molecular activity and allowing chemical reactions to proceed smoothly. Time is a guarantee for thorough enzymolysis. The inventors have found that it is more appropriate to control the temperature of the present invention to 40-65°C and the time to 24-72h. If the enzymolysis time is too short, the enzymolysis will not be thorough. If the time is too long, the production cycle will be extended and the production cost will be increased. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 is the high performance liquid chromatogram of Example 7;

[0040] Figure 2 is the high performance liquid chromatogram of Example 8;

[0041] Figure 3 is the high performance liquid chromatogram of Example 9;

[0042] Figure 4 It is the high performance liquid chromatogram of Example 10. DETAILED DESCRIPTION

[0043] In order to enable those skilled in the art to better understand the technical solution of the invention, the present invention is further described in detail below in conjunction with specific implementation methods.

[0044] The invention provides a method for preparing isoquercetin by enzymatic hydrolysis of sophora japonica buds. The method uses sophora japonica buds as raw materials and utilizes cellulase, pectinase and rhamnosidase to prepare isoquercetin in one step, thereby avoiding the problems of low yield and purity in the process of first preparing rutin and then desugaring to prepare isoquercetin, and large amounts of organic solvents are used, resulting in large amounts of wastewater polluting the environment. The method of the invention improves the yield of the product obtained, the purity of isoquercetin is high, the production process is shortened, and the production cost is reduced.

[0045] The method comprises the following steps:

[0046] S1, weighing Sophora japonica seeds, adding water to soak, and obtaining the soaked Sophora japonica seeds;

[0047] S2, squeezing the soaked Sophora japonica seeds dry, adding water, and sequentially adding a reducing agent, cellulase, pectinase, and rhamnosidase, adjusting the pH to 5.5-7.5, and stirring for enzymolysis;

[0048] S3, after enzymatic hydrolysis, the enzyme is killed, activated carbon and borax are added, the pH is adjusted to 10-11, stirred, filtered, the filtrate is collected and the pH is adjusted to 4-5, allowed to stand, and the filter cake is collected;

[0049] S4, extracting the filter cake with an alcohol solution, filtering, and collecting the filter cake to obtain isoquercetin.

[0050] Since rutin is easily oxidized, a reducing agent is added to the soaked sophora japonica seedlings to protect rutin, inhibit its oxidation, and facilitate subsequent enzyme decomposition; in an environment with a pH of 5.5 to 7.5, cellulase can destroy the plant fiber of the sophora japonica seedlings, and pectinase can destroy the pectin of the sophora japonica seedlings, so that flavonoid target components and effective components such as rutin in the sophora japonica seedlings are fully exposed, and insufficient extraction due to cellulose wrapping is avoided, thereby improving the yield of the product; rhamnosidase is then used to directly enzymolyze rutin or a flavonoid component with a structure similar to rutin (such as quercetin-3-O-glucose-7-O-rhamnoside, which is different from the isomers of rhamnose) to generate isoquercetin, and then activated carbon and borax are added to decolorize and protect the isoquercetin from being oxidized, and the pH is adjusted again to perform alkali dissolution and acid precipitation, so as to obtain high-purity isoquercetin.

[0051] The structural formula of the quercetin-3-O-glucose-7-O-rhamnoside is:

[0052]

[0053] Among them, the present invention adopts a simple enzyme with rhamnosidic bond, which has strong specificity and will not break the glucoside bond, thereby ensuring that quercetin will not be produced in the product, improving the purity of isoquercetin, avoiding the problem of reduced yield caused by first preparing rutin and then desugaring to prepare isoquercetin, and also avoiding the problem of environmental pollution caused by a large amount of chemical reagents used in the preparation process and the generation of a large amount of wastewater. The present invention prepares isoquercetin from Sophora japonica in one step, and the obtained product has high yield and high isoquercetin content, shortens the production process, and reduces the production cost.

[0054] The mass of water in S1 of the present invention is 5 times the mass of the sophora japonica seeds; and the soaking time in S1 is 2 hours.

[0055] In order to better remove impurities, the soaked Sophora japonica seeds are squeezed dry and then water is added, wherein the mass of the added water is 3 times the mass of the squeezed Sophora japonica seeds.

[0056] The reducing agent in S2 is sodium metabisulfite or cysteine. Sodium metabisulfite has lower cost and is more conducive to industrial production. After many experiments and studies by the inventors, the mass of the reducing agent is most appropriately 1-1.2% of the mass of water in S2. At this time, the solution is light yellow and can have a suitable antioxidant effect on rutin. If it is lower than this level, the color of the sophora japonica enzyme hydrolysis solution will deepen and the protective effect cannot be achieved.

[0057] When the mass ratio of cellulase to pectinase in S2 is (2-2.15):1, and the mass ratio of cellulase to rhamnosidase is (1-3):(2-10), the combined use of cellulase and rhamnosidase can achieve a better synergistic effect.

[0058] The pH regulator in S2 of the present invention is sodium hydroxide solution. The inventors have found through multiple experiments that in an environment with a pH of 6 to 7.5, the activities of the two enzymes of the present invention can be maintained at the maximum, and the yield and purity of the final product can be maintained at a high level.

[0059] The temperature of the enzymatic hydrolysis in S2 of the present invention is 40-65°C; the time of the enzymatic hydrolysis is 24-72h. The enzymatic hydrolysis temperature is a condition for increasing the activity of molecules and making the chemical reaction proceed smoothly, and the time is a guarantee for thorough enzymatic hydrolysis. The inventors have found that it is more appropriate to control the temperature and time range of the present invention. If the enzymatic hydrolysis time is too short, the enzymatic hydrolysis is not thorough, and if the time is too long, the production cycle is extended and the production cost is increased.

[0060] The enzyme is inactivated in S3 of the present invention by heating the temperature to 80°C, the purpose of heating is to coagulate the enzyme, so that the enzyme can be separated smoothly and the enzyme is prevented from entering the product; the mass ratio of activated carbon to borax is 10:1, the activated carbon is added for decolorization, and the borax is added to protect the hydroxyl group of isoquercetin from being oxidized, and under this ratio condition, the product can be better protected and the cost can be saved; then, a sodium hydroxide solution is used to adjust the pH to 10-11, after stirring and filtering, a hydrochloric acid solution is used to adjust the pH to 4-5, alkali dissolution and acid precipitation are performed, and high-purity isoquercetin is obtained after standing for 12-24 hours.

[0061] The alcohol solution in S4 of the present invention is methanol or 90% ethanol; further, the 90% ethanol in S4 is edible alcohol. Extraction with edible alcohol further improves the purity of isoquercetin, and edible alcohol has better safety, avoiding the safety hazards brought to the product by the residual toxic and harmful reagents.

[0062] The extraction in S4 of the present invention is reflux extraction twice; after filtration, the alcohol solution can be recovered to save resources; after the filter cake is collected in S4 of the present invention, the filter cake can be washed with water, dried, and crushed to improve the purity of the product.

[0063] The invention uses sophora japonica seeds to directly ferment and prepare isoquercetin, thus shortening the production process, and the solvent alcohol solution can be reused, thereby reducing the production cost.

[0064] The above is a detailed description of the present invention, and the following are embodiments of the present invention.

[0065] Determination conditions of isoquercetin:

[0066] Instruments: High performance liquid chromatograph, electronic analytical balance (not less than 1 / 10,000), ultrasonic cleaner, syringe filter (0.45um for organic system), pipette, volumetric flask, rubber-tipped dropper;

[0067] Reagents and Drugs:

[0068] Tetrahydrofuran----chromatographically pure;

[0069] Sodium dihydrogen phosphate, phosphoric acid--analytical grade;

[0070] Sodium dihydrogen phosphate solution (15.6 g / L): Take 15.6 g of sodium dihydrogen phosphate, dissolve it in water and dilute it to 1000 ml, shake well;

[0071] Isquercetin working reference substance: No treatment is required before use, but its moisture content needs to be determined by the same method. When calculating the content, the moisture content of the reference substance needs to be deducted and the content is calculated based on the dry product.

[0072] Chromatographic conditions:

[0073]

[0074] Use mobile phase A and mobile phase B for gradient elution as shown in the table below;

[0075]

[0076] Preparation of test solution:

[0077] Take 0.05g of the test sample, place it in a 50ml volumetric flask, add 10ml of methanol and sonicate to completely dissolve it, dilute to the scale with mobile phase, and shake well;

[0078] Preparation of reference solution:

[0079] Accurately weigh 0.05 g of isoquercetin working reference substance into a 50 ml volumetric flask, add 10 ml of methanol and sonicate to completely dissolve it, and dilute to the scale with mobile phase B.

[0080] Isoquercetin calculation:

[0081]

[0082]

[0083] Example

[0084] The method for preparing isoquercetin from sophora japonica buds by enzymatic hydrolysis comprises the following steps:

[0085] S1, weigh 500g of Sophora japonica seeds, add 2500mL of drinking water and soak for 2h to obtain the soaked Sophora japonica seeds;

[0086] S2, after the soaked Sophora japonica rice is squeezed dry, 1500 mL of purified water is added, 15 g of sodium pyrosulfite, 15 g of cellulase, 7 g of pectinase, and 50 g of rhamnosidase are added in sequence, the pH is adjusted to a certain value, and the enzymolysis is stirred at a set temperature for a certain time;

[0087] S3, after enzymatic hydrolysis, the temperature was raised to 80°C, 10 g of activated carbon and 1 g of borax were added, sodium hydroxide solution was added to adjust the pH to 11, stirred, filtered, the filtrate was collected and hydrochloric acid solution was added to adjust the pH to 4, allowed to stand for 12 h, and the filter cake was collected;

[0088] S4. Reflux extract the filter cake with 300 mL and 200 mL of 90% ethanol twice, filter, recover the ethanol solution, collect the filter cake, wash with 200 mL of water, dry, and crush to obtain isoquercetin.

[0089] Among them, the enzymatic hydrolysis conditions of step 2 were investigated by single factors: Examples 1 to 3 were investigated by single factors on the enzymatic hydrolysis pH value; Examples 4 to 6 were investigated by single factors on the enzymatic hydrolysis temperature; Examples 7 to 9 were investigated by single factors on the enzymatic hydrolysis time, as shown in Tables 1 to 3:

[0090] Table 1 Single factor investigation of enzymatic hydrolysis pH

[0091]

[0092] Table 2 Single factor investigation of enzymatic hydrolysis temperature

[0093]

[0094] Table 3 Single factor investigation of enzymatic hydrolysis time

[0095]

[0096] The HPLC chromatograms of Examples 7, 8 and 9 are shown in the attached Figure 1 , Figure 2 , Figure 3 .

[0097] Example 10

[0098] A method for preparing isoquercetin by enzymatic hydrolysis of Sophora japonica seeds comprises the following steps:

[0099] S1, weigh 500g of Sophora japonica seeds, add 2500mL of drinking water and soak for 2h to obtain the soaked Sophora japonica seeds;

[0100] S2, after squeezing the soaked Sophora japonica rice dry, add 1500 mL of purified water, add 15 g of sodium pyrosulfite, 20 g of cellulase, 10 g of pectinase, and 40 g of rhamnosidase in sequence, adjust the pH to 6, and stir the enzymolysis at 45 ° C for 48 h;

[0101] S3, after enzymatic hydrolysis, the temperature was raised to 80°C, 10 g of activated carbon and 1 g of borax were added, sodium hydroxide solution was added to adjust the pH to 10, the mixture was stirred, filtered, the filtrate was collected, hydrochloric acid solution was added to adjust the pH to 5, the mixture was allowed to stand for 24 h, and the filter cake was collected;

[0102] S4, the filter cake was extracted twice with 300 mL and 200 mL of 90% ethanol respectively, filtered, the ethanol solution was recovered, the filter cake was collected, washed with 300 mL of water, dried, and crushed to obtain 115 g of isoquercetin with a yield of 23% and a purity of 98%. The HPLC chromatogram is shown in FIG. Figure 4 .

[0103] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing isoquercetin by enzymatic hydrolysis of Sophora japonica seeds, characterized in that: The following steps are involved: S1, weighing Sophora japonica seeds, adding water to soak, and obtaining the soaked Sophora japonica seeds; S2, the soaked Sophora japonica seeds are squeezed dry, water is added, and a reducing agent, cellulase, pectinase and rhamnosidase are added in sequence, the pH is adjusted to 6-7.5, and enzymolysis is stirred; the mass ratio of the cellulase to the pectinase is (2-2.15):1, the mass ratio of the cellulase to the rhamnosidase is (1-3):(2-10), the temperature of the enzymolysis is 40-65°C; the time of the enzymolysis is 24-72h; the reducing agent is sodium pyrosulfite or cysteine, and the mass of the reducing agent is 1-1.2% of the mass of the water; S3, after enzymatic hydrolysis, the enzyme is killed, activated carbon and borax are added, the pH is adjusted to 10-11, stirred, filtered, the filtrate is collected and the pH is adjusted to 4-5, allowed to stand, and the filter cake is collected; S4, extracting the filter cake with an alcohol solution, filtering, and collecting the filter cake to obtain isoquercetin.

2. The method for preparing isoquercetin by enzymatic hydrolysis of Sophora japonica seeds according to claim 1, characterized in that: When the enzyme is inactivated in S3, the temperature is raised to 80°C.

3. The method for preparing isoquercetin by enzymatic hydrolysis of Sophora japonica seeds according to claim 1, characterized in that: The mass ratio of activated carbon to borax in S3 is 10:1.

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