Method for extracting quercetin from sophora flower buds

Abstract

The invention relates to the field of natural product extraction, and particularly discloses a method for extracting quercetin from sophora flower bud, which comprises the following steps: crushing dry sophora flower bud; the method comprises the following steps: by taking an ethanol water solution with the concentration of 70-80% v / v as an extraction solvent, carrying out ultrasonic extraction at 50-60 DEG C, and carrying out solid-liquid separation to obtain an extracting solution (I) containing rutin; adding glycosidase into the obtained extracting solution (I), adjusting the pH value to 3.5-5.5, adjusting the temperature to 35-50 DEG C, carrying out an enzymolysis reaction for 2-4 hours, and then carrying out enzyme deactivation; the liquid after enzyme deactivation is concentrated and filtered while being hot at the temperature of 50-60 DEG C, filter residues are washed with 70-80% hot ethyl alcohol, filtrate is combined to obtain a mixed solution, the mixed solution is loaded to a macroporous adsorption resin column, gradient elution is conducted through an ethyl alcohol-water solution with the concentration increasing progressively, target elution fractions containing quercetin are collected, concentration and freeze drying are conducted, and the quercetin is obtained. The quercetin prepared by the method has the advantages of high purity and good yield.

Description

Technical Field

[0001] The present invention relates to the field of natural product extraction, and more specifically, to a method for extracting quercetin from Sophora japonica seeds. Background Art

[0002] As a traditional medicinal plant resource, Sophora japonica (Sophora japonica) has a long history of application in Traditional Chinese Medicine (TCM). It is rich in a variety of components with important pharmacological activities, with flavonoids being particularly prominent. Among the flavonoids in Sophora japonica, rutin and quercetin are two key active substances. Rutin undergoes metabolic conversion in the body to produce quercetin. Both possess significant pharmacological activities, including antioxidant, anti-inflammatory, and cardiovascular protective properties, and possess extremely high medicinal value. However, the development and utilization of Sophora japonica resources face numerous challenges in existing extraction technologies. Due to the relatively low natural content of quercetin in Sophora japonica, rutin is typically extracted first and then converted to quercetin through a subsequent chemical hydrolysis reaction. Acid / base hydrolysis is currently the most commonly used hydrolysis method, but this method has numerous drawbacks. The acid / base hydrolysis process is poorly controllable, and even slight changes in reaction conditions, such as acid / base concentration, temperature, and time, can lead to over- or under-hydrolysis, resulting in the production of a large number of byproducts. The presence of these byproducts not only reduces the purity of quercetin, but also increases the difficulty of subsequent separation and purification. Existing separation and purification technologies often have difficulty in effectively removing these byproducts, resulting in the final quercetin product having suboptimal purity, which cannot meet the stringent requirements for high-quality raw materials in high-end pharmaceuticals, health products and other fields. At the same time, the cumbersome separation and purification steps further increase production costs and production cycles, limiting the large-scale production and widespread application of quercetin.

[0003] To sum up, the core issue of the development and utilization of Sophora japonica resources lies in how to break through the limitations of existing extraction technology and solve the problem of low quercetin purity. Summary of the Invention

[0004] In order to improve the purity of quercetin extracted from Sophora japonica seeds, the present application provides a method for extracting quercetin from Sophora japonica seeds.

[0005] In the first aspect, the present application provides a method for extracting quercetin from Sophora japonica seeds, which adopts the following technical solution: A method for extracting quercetin from Sophora japonica seeds comprises the following steps: (a) crushing the dried Sophora japonica seeds; (b) using 70-80% v / v ethanol aqueous solution as the extraction solvent, performing extraction at 50-60° C. by ultrasound-assisted extraction, and obtaining an extract (I) containing rutin by solid-liquid separation; (c) adding glycosidase to the extract (I) obtained in step (b), adjusting the pH to 3.5-5.5 and the temperature to 35-50° C., carrying out enzymatic hydrolysis for 2-4 hours, and then inactivating the enzyme; (d) concentrating the liquid after the enzyme inactivation in step (c), filtering while hot at 50-60° C., washing the filter residue with 70-80% hot ethanol, combining the filtrates to obtain a mixed solution, applying the solution to a macroporous adsorption resin column, and performing gradient elution with an ethanol-water solution of increasing concentration to collect the target elution fraction containing quercetin; (e) The target eluted fraction is concentrated and freeze-dried to obtain quercetin.

[0006] By adopting the above technical solution, a 70-80% v / v ethanol-water solution is used as the extraction solvent, which has good solubility for flavonoids such as rutin. Ultrasound-assisted extraction utilizes the cavitation, mechanical, and thermal effects of ultrasound to rupture the cell walls of the Sophora japonica seeds, accelerating the entry of the solvent into the cells and promoting the dissolution of rutin. This significantly shortens the extraction time, thereby obtaining an extract (I) containing rutin in a shorter time. This lays a good foundation for the subsequent preparation of quercetin, improves overall production efficiency, and reduces production costs.

[0007] By adding glycosidase to perform enzymatic hydrolysis, rutin is hydrolyzed into quercetin and rutinose. Subsequent gradient elution through a macroporous adsorption resin column can specifically remove impurities in the extract, such as rutinose, tannins, and polysaccharides. Furthermore, graded elution is used to effectively separate the target product, quercetin, from impurities, ultimately yielding quercetin of higher purity.

[0008] The entire extraction and purification process is carried out under relatively mild conditions. For example, the extraction temperature is controlled at 40-70°C, and the pH and temperature of the enzymatic hydrolysis reaction are also optimized. This avoids the destruction of active ingredients such as rutin and quercetin by extreme conditions such as high temperature, strong acid, and strong alkali, thereby retaining their biological activity and pharmacological effects to the greatest extent, ensuring that the prepared product has good efficacy.

[0009] The present application improves the extraction rate of rutin through low-temperature assisted extraction, further greatly reduces by-products through enzymatic specific conversion, and further greatly improves the recovery rate through resin gradient elution, finally obtaining high-purity quercetin.

[0010] Optionally, the conditions for ultrasound-assisted extraction in step (b) are: frequency 20-60 kHz, power density 200-400 W / L, extraction temperature 50-60° C., and time 20-40 minutes.

[0011] By adopting the above technical solution, 20-60kHz ultrasound produces a cavitation effect, forming tiny bubbles in the liquid and quickly bursting them, generating a strong impact force, destroying the cell walls of the Sophora japonica seeds, and making the rutin in the cells easier to dissolve; 200-400W / L power density ensures sufficient energy input to maintain a stable cavitation effect, while avoiding local overheating caused by excessive power to destroy the active ingredients; 50-60℃ temperature accelerates molecular movement and enhances the solvent's solubility, while preventing high temperature from causing rutin decomposition; 20-40 minutes can ensure the full dissolution of rutin, avoid the introduction of impurities due to excessive extraction time, and achieve efficient and high-quality extraction.

[0012] Optionally, the glycosidase in step (c) is β-glucosidase.

[0013] By adopting the above technical solution, rutin is a glycoside compound formed by the combination of quercetin and rutinose through a β-glycosidic bond. β-glucosidase can specifically recognize and catalyze the hydrolysis of the β-glycosidic bond, accurately cutting the glycosidic bond in the rutin molecule, and decomposing rutin into quercetin and rutinose, thereby increasing the amount of quercetin in the target product and providing a more favorable ingredient basis for the preparation of quercetin.

[0014] Optionally, the amount of enzyme added in step (c) is 0.5-3.0 U / mL.

[0015] By adopting the above technical solution, the enzyme addition amount is controlled at 0.5-3.0U / mL, which can achieve efficient conversion of rutin to quercetin while effectively controlling costs and reducing impurity generation. When the enzyme addition amount is too low, the number of enzyme molecules in the system is insufficient, and the opportunity to contact rutin and catalyze its hydrolysis is limited, resulting in a low rutin conversion rate and an inability to effectively increase the quercetin content; while the enzyme addition amount is too high, not only will it increase production costs, but it may also trigger side reactions due to excessive enzyme concentration and introduce impurities. Within this addition amount range, the number of enzyme molecules is moderate, which can fully contact rutin and efficiently catalyze its hydrolysis, converting rutin to quercetin, while avoiding the above unfavorable conditions and ensuring product quality and economic benefits.

[0016] Optionally, the macroporous adsorption resin in step (d) is HPD-100 or AB-8 type resin.

[0017] By adopting the above technical scheme, quercetin can be efficiently separated and purified. HPD-100 is a styrene-type non-polar copolymer with strong adsorption capacity for cyclic aromatic compounds. The adsorption force increases with the increase of the lipophilicity of the adsorbed molecules, and it can effectively adsorb quercetin. AB-8 is a styrene-type weakly polar copolymer with a high specific surface area and suitable pore size. It has good adsorption performance for weakly polar substances and can specifically adsorb quercetin.

[0018] Optionally, the gradient elution step in step (d) includes three elution steps: Primary elution: 10-20% ethanol aqueous solution as mobile phase, elution volume 3-5 BV, flow rate 1.5-3.0 BV / h; Secondary elution: 30-40% ethanol aqueous solution as mobile phase, elution volume 3-5BV, flow rate 2.0-4.0BV / h; Three-stage elution: (i) elution with 60-70% ethanol aqueous solution for 4-6 BV at a flow rate of 2.0-3.0 BV / h, collecting the first target eluate S1; (ii) elution with 80-90% ethanol aqueous solution for 3-4 BV at a flow rate of 1.5-2.5 BV / h, collecting the second target eluate S2; the eluates S1 and S2 were combined to obtain the target fraction containing quercetin.

[0019] By adopting the above technical scheme, the gradient elution step can efficiently separate and purify quercetin, significantly improving the purity and yield of the target product. The first elution uses a 10-20% ethanol aqueous solution to elute polar impurities that are weakly bound to the resin, such as some sugars and acidic impurities; the second elution uses a 30-40% ethanol aqueous solution to further increase the ethanol concentration, which can elute impurities with slightly weaker polarity, such as some proteins and peptides; in the third elution, 60-70% ethanol aqueous solution is first used for elution. At this time, quercetin is eluted due to its relatively weak adsorption effect on the resin and collected as the first target eluent S1. Continuing to elute with 80-90% ethanol aqueous solution further ensures that the residual target product is completely eluted and collected as the second target eluent S2. After S1 and S2 are combined, quercetin and impurities are effectively separated according to the difference in polarity of the substances through graded elution with ethanol aqueous solutions of different concentrations, thereby obtaining a high-purity target fraction containing quercetin.

[0020] Optionally, the gradient elution step is as follows: Primary elution: Use 10-20% ethanol aqueous solution containing 0.03-0.08 mol / L citrate as the mobile phase, adjust the pH to 3.0-4.0, elution volume 3-5 BV, flow rate 1.5-3.0 BV / h; Secondary elution: Use 30-40% ethanol aqueous solution containing 0.05-0.15% (v / v) ammonia as the mobile phase, elution volume 3-5 BV, flow rate 2.0-4.0 BV / h; Three-stage elution: (i) elution with 60-70% ethanol aqueous solution for 4-6 BV at a flow rate of 2.0-3.0 BV / h, collecting the first target eluate S1; (ii) elution with 80-90% ethanol aqueous solution containing 0.1-0.3 mol / L alkali metal salt for 3-4 BV at a flow rate of 1.5-2.5 BV / h, collecting the second target eluate S2; the eluates S1 and S2 were combined to obtain the target fraction containing quercetin.

[0021] By adopting the above technical solution, the first elution uses weakly acidic low-concentration ethanol containing citrate. Under a weakly acidic environment, it can more effectively elute and remove residual water-soluble polysaccharides and possible complex colloidal impurities in the concentrated extract, thereby preventing these substances from clogging the resin pores or interfering with the adsorption of the target product. Compared with low-concentration ethanol elution without the addition of citrate, its ability to remove polysaccharides and colloids is significantly enhanced. The second elution uses weakly alkaline medium-concentration ethanol containing low-concentration ammonia water to create a weak alkaline environment, which can more efficiently remove tannin impurities adsorbed on the resin. The concentration of ammonia is strictly controlled within the above range, which can not only effectively elute tannins, but also ensure the structural stability of important active ingredients such as quercetin.

[0022] The tertiary elution uses a high-concentration ethanol combined with an alkali metal salt. A 60-70% ethanol aqueous solution primarily elutes weakly adsorbed quercetin. The further addition of an alkali metal helps to more fully elute the more strongly adsorbed quercetin, improving its elution efficiency and recovery rate. This graded elution allows for targeted elution and collection of quercetin, effectively reducing the residual amounts of polysaccharides, tannins, and other impurities in the final target fraction and significantly improving the purity of the target product, quercetin.

[0023] Optionally, the particle size of the crushed Sophora japonica seeds is 20-80 mesh, and the mass ratio of the crushed Sophora japonica seeds to the ethanol aqueous solution in step (b) is 1:(20-30).

[0024] In summary, this application has the following beneficial effects: 1. The present application grinds the sophora japonica seeds into a particle size range of 20-80 mesh, which not only ensures that the cell walls of the sophora japonica seeds are sufficiently broken, so that the effective ingredients such as rutin in the cells can be fully exposed, facilitating subsequent solvent extraction; but also avoids the generation of excessive fine powder during the grinding process due to too small a particle size, which increases the difficulty of subsequent solid-liquid separation and the content of impurities. At the same time, the sophora japonica seeds and the ethanol aqueous solution are added at a mass ratio of 1: (20-30). This ratio has been carefully optimized to provide sufficient solvent to dissolve flavonoids such as rutin, ensure that the solvent fully extracts the target ingredients during the extraction process, and improve the extraction rate; and avoid excessive solvent usage causing waste of resources and increased energy consumption in the subsequent concentration process, thereby improving the basic efficiency of the extraction process as a whole and laying a good foundation for the subsequent acquisition of high-purity quercetin.

[0025] 2. This application optimizes the ultrasonic-assisted extraction conditions. The frequency of 20-60kHz produces a cavitation effect to destroy the cell wall. The power density of 200-400W / L ensures a stable cavitation effect and avoids overheating to destroy the active ingredients. The temperature of 50-60°C accelerates molecular movement and avoids rutin decomposition. While ensuring the full dissolution of rutin, it avoids the introduction of impurities due to excessive extraction time, thereby achieving efficient and high-quality extraction.

[0026] 3. In this application, the selection of a suitable extraction solvent lays the foundation, β-glucosidase is used to accurately increase the amount of quercetin, and then gradient elution is performed through a macroporous adsorption resin. By adding substances such as citrate, ammonia water, and alkali metal salts, the residual water-soluble polysaccharides, complex colloidal impurities, tannin impurities, etc. in the concentrated extract are further targeted to be removed, effectively reducing the residual amount of impurities in the final target fraction and significantly improving the purity of the target product quercetin.

[0027] 4. The entire extraction and purification process in this application is carried out under relatively mild conditions, which retains the biological activity and pharmacological effects of active ingredients such as rutin and quercetin to the greatest extent. The extraction temperature is controlled at 40-70°C, the pH of the enzymatic hydrolysis reaction is adjusted to 3.5-5.5, and the temperature is controlled at 35-50°C, avoiding the destruction of active ingredients by extreme conditions such as high temperature, strong acid, and strong alkali. The mild conditions of ultrasound-assisted extraction and the optimized conditions of the enzymatic hydrolysis reaction ensure the stability of rutin during the extraction and conversion process, so that the finally prepared quercetin can maintain good biological activity and pharmacological effects, ensuring that the prepared product has good efficacy, and providing reliable quality assurance for subsequent applications in the fields of medicine, health care, etc. DETAILED DESCRIPTION

[0028] The present application is further described in detail below with reference to the examples. It is particularly noted that if no specific conditions are specified in the following examples, the reactions are carried out according to conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, the raw materials used in the following examples can be obtained from common commercial sources.

[0029] Preparation examples of raw materials and / or intermediates Example

[0030] Example 1 A method for extracting quercetin from Sophora japonica seeds comprises the following steps: (a) crushing the dried Sophora japonica seeds to 40-60 mesh and sieving for later use; (b) taking 1 kg of the sieved sophora japonica rice flour, adding 25 kg of 75% v / v ethanol aqueous solution as an extraction solvent, ultrasonically extracting at a frequency of 40 kHz, a power density of 300 W / L, and a temperature of 55° C. for 25 min, centrifuging at 4000 rpm for 10 min, and separating the solid and liquid to obtain an extract (I) containing rutin; (c) adding β-glucosidase to the extract (I) obtained in step (b), adding citric acid buffer to adjust the pH to 4.5 and the temperature to 42°C, and carrying out enzymatic hydrolysis for 3 hours, followed by heating to 60°C and keeping warm for 15 minutes to inactivate the enzyme; the amount of the enzyme added is 1.8 U / mL; (d) concentrating the liquid after the enzyme inactivation in step (c) by rotary evaporation at 60°C to 1 / 5 of the original volume, filtering with a Buchner funnel preheated to 60°C and equipped with a 0.45 μm polyethersulfone filter membrane, washing the filter residue twice with 10 mL of 75% v / v hot ethanol (60°C), combining the filtrates to obtain a mixed solution, applying the sample to a pre-activated HPD-100 macroporous adsorption resin column, and performing gradient elution with an ethanol-water solution of increasing concentration: Primary elution: 15% ethanol in water as mobile phase, elution volume 4 BV, flow rate 2.2 BV / h; Secondary elution: 35% ethanol in water as mobile phase, elution volume 4 BV, flow rate 3.0 BV / h; Three-stage elution: (i) elution with 65% ethanol in water for 5 BV at a flow rate of 2.5 BV / h, collecting the first target eluate S1; (ii) eluting with 85% ethanol in water for 3.5 BV at a flow rate of 2 BV / h to collect the second target eluate S2; combining the eluates S1 and S2 to obtain the target fraction containing quercetin; (e) The target eluted fraction was concentrated by rotary evaporation at -0.09 MPa to remove ethanol, and then freeze-dried to obtain quercetin.

[0031] Example 2 A method for extracting quercetin from Sophora japonica seeds comprises the following steps: (a) crushing the dried Sophora japonica seeds to 20-40 mesh and sieving for later use; (b) taking 1 kg of the sieved sophora japonica rice flour, adding 20 kg of 70% v / v ethanol aqueous solution as an extraction solvent, ultrasonically extracting at a frequency of 20 kHz, a power density of 400 W / L, and a temperature of 50° C. for 40 min, centrifuging at 4000 rpm for 10 min, and separating the solid and liquid to obtain an extract (I) containing rutin; (c) adding β-glucosidase to the extract (I) obtained in step (b), adjusting the pH to 3.5 and the temperature to 50° C., carrying out enzymatic hydrolysis for 2 hours, and then heating to 60° C. and keeping the temperature for 15 minutes to inactivate the enzyme; the enzyme addition amount is 3 U / mL; (d) The liquid after the enzyme inactivation in step (c) was concentrated to 1 / 5 of its original volume by rotary evaporation at 50°C, and filtered through a Buchner funnel preheated to 50°C with a 0.45 μm polyethersulfone filter membrane. The filter residue was washed twice with 10 mL of 70% v / v hot ethanol (60°C). The filtrates were combined to obtain a mixed solution, which was loaded onto an HPD-100 macroporous adsorption resin column and gradient eluted with an ethanol-water solution of increasing concentrations: Primary elution: 10% ethanol in water as mobile phase, elution volume 5 BV, flow rate 3.0 BV / h; Secondary elution: 30% ethanol aqueous solution as mobile phase, elution volume 5 BV, flow rate 4.0 BV / h; Three-stage elution: (i) elution with 60% ethanol in water for 6 BV at a flow rate of 3.0 BV / h, collecting the first target eluate S1; (ii) eluting with 80% ethanol in water for 4 BV at a flow rate of 2.5 BV / h, collecting the second target eluate S2; combining the eluates S1 and S2 to obtain the target fraction containing quercetin; (e) The target eluted fraction was concentrated by rotary evaporation at -0.09 MPa to remove ethanol, and then freeze-dried to obtain quercetin.

[0032] Example 3 A method for extracting quercetin from Sophora japonica seeds comprises the following steps: (a) crushing the dried sophora japonica seeds to 60-80 mesh and sieving for later use; (b) taking 1 kg of the sieved sophora japonica rice flour, adding 30 kg of 80% v / v ethanol aqueous solution as an extraction solvent, ultrasonically extracting at a frequency of 60 kHz, a power density of 200 W / L, and a temperature of 60° C. for 20 min, centrifuging at 4000 rpm for 10 min, and separating the solid and liquid to obtain an extract (I) containing rutin; (c) adding β-glucosidase to the extract (I) obtained in step (b), adjusting the pH to 5.5 and the temperature to 35° C., and performing enzymatic hydrolysis for 4 hours, followed by heating to 60° C. and keeping the temperature for 15 minutes to inactivate the enzyme; the enzyme addition amount was 0.5 U / mL; (d) The liquid after the enzyme inactivation in step (c) was concentrated to 1 / 5 of its original volume by rotary evaporation at 60°C, and filtered through a Buchner funnel preheated to 60°C with a 0.45 μm polyethersulfone filter membrane. The filter residue was washed twice with 10 mL of 80% v / v hot ethanol (60°C). The filtrates were combined to obtain a mixed solution, which was loaded onto an HPD-100 macroporous adsorption resin column and gradient eluted with an ethanol-water solution of increasing concentrations. Primary elution: 20% ethanol aqueous solution as mobile phase, elution volume 3BV, flow rate 1.5BV / h; Secondary elution: 40% ethanol aqueous solution as mobile phase, elution volume 3BV, flow rate 2.0BV / h; Three-stage elution: (i) elution with 70% ethanol in water (4 BV) at a flow rate of 2.0 BV / h, collecting the first target eluate S1; (ii) eluting with 90% ethanol in water for 3 BV at a flow rate of 1.5 BV / h, collecting the second target eluate S2; combining the eluates S1 and S2 to obtain the target fraction containing quercetin; (e) The target eluted fraction was concentrated by rotary evaporation at -0.09 MPa to remove ethanol, and freeze-dried to obtain quercetin.

[0033] Example 4 A method for extracting quercetin from Sophora japonica seeds, which differs from Example 1 in that the gradient elution in this embodiment comprises the following steps: Primary elution: Use 15% ethanol aqueous solution containing 0.05 mol / L trisodium citrate as the mobile phase, adjust the pH to 3.5, elution volume 4 BV, flow rate 2.2 BV / h; Secondary elution: 35% ethanol aqueous solution containing 0.1% (v / v) ammonia as the mobile phase, elution volume 4 BV, flow rate 3.0 BV / h; Tertiary elution: (i) elution with 65% ethanol aqueous solution 5 BV, flow rate 2.5 BV / h, collecting the first target eluate S1; (ii) eluting with 85% ethanol aqueous solution containing 0.2 mol / L sodium acetate for 3.5 BV at a flow rate of 2 BV / h to collect the second target eluate S2; combining the eluates S1 and S2 to obtain the target fraction containing quercetin; The remaining steps are the same as in Example 1.

[0034] Example 5 A method for extracting quercetin from Sophora japonica seeds, which differs from Example 1 in that the gradient elution in this embodiment comprises the following steps: Primary elution: Use 15% ethanol aqueous solution containing 0.03 mol / L trisodium citrate as the mobile phase, adjust the pH to 3.5, elution volume 4 BV, flow rate 2.2 BV / h; Secondary elution: using 35% ethanol aqueous solution containing 0.05% (v / v) ammonia as the mobile phase, elution volume 4 BV, flow rate 3.0 BV / h; Tertiary elution: (i) elution with 65% ethanol aqueous solution 5 BV, flow rate 2.5 BV / h, collecting the first target eluate S1; (ii) eluting with 85% ethanol aqueous solution containing 0.3 mol / L sodium acetate for 3.5 BV at a flow rate of 2 BV / h to collect the second target eluate S2; combining the eluates S1 and S2 to obtain the target fraction containing quercetin; The remaining steps are the same as in Example 1.

[0035] Example 6 A method for extracting quercetin from Sophora japonica seeds, which differs from Example 1 in that the gradient elution in this embodiment comprises the following steps: Primary elution: Use 15% ethanol aqueous solution containing 0.08 mol / L trisodium citrate as the mobile phase, adjust the pH to 3.5, elution volume 4 BV, flow rate 2.2 BV / h; Secondary elution: using 35% ethanol aqueous solution containing 0.15% (v / v) ammonia as the mobile phase, elution volume 4 BV, flow rate 3.0 BV / h; Tertiary elution: (i) elution with 65% ethanol aqueous solution 5 BV, flow rate 2.5 BV / h, collecting the first target eluate S1; (ii) eluting with 85% ethanol aqueous solution containing 0.1 mol / L sodium acetate for 3.5 BV at a flow rate of 2 BV / h to collect the second target eluate S2; combining the eluates S1 and S2 to obtain the target fraction containing quercetin; The remaining steps are the same as in Example 1.

[0036] Example 7 A method for extracting quercetin from Sophora japonica seeds is different from Example 1 in that the macroporous adsorption resin used in this example is AB-8 type resin.

[0037] Comparative Example Comparative Example 1 A method for extracting quercetin from Sophora japonica seeds, which differs from Example 1 in that gradient elution is not used in this comparative example. Instead, 85% ethanol aqueous solution is used for elution at a flow rate of 2 BV / h for 3.5 BV, and the target eluate is collected to obtain a target fraction containing quercetin; the remaining steps are the same as in Example 1.

[0038] Comparative Example 2 A method for extracting quercetin from Sophora japonica seeds, which differs from Example 1 in that gradient elution is not used in this comparative example. Instead, 5 BV of 65% ethanol aqueous solution is used for elution at a flow rate of 2.5 BV / h, and the target eluate is collected to obtain a target fraction containing quercetin; the remaining steps are the same as in Example 1.

[0039] Comparative Example 3 A method for extracting quercetin from Sophora japonica seeds is provided, which differs from Example 4 in that sodium citrate is not added in the gradient elution (d) in this comparative example, and the remaining steps are the same as Example 4.

[0040] Comparative Example 4 A method for extracting quercetin from Sophora japonica seeds is different from Example 4 in that no ammonia water is added in the gradient elution (d) in this comparative example, and the remaining steps are the same as Example 4.

[0041] Comparative Example 5 A method for extracting quercetin from Sophora japonica seeds, which is different from Example 1 in that the method comprises the following preparation steps: (a) grinding the dried Sophora japonica seeds into 40-60 mesh and sieving for later use; (b) using 70% v / v ethanol as the solvent, extracting under reflux at 70°C for 2 hours (without ultrasound), and performing solid-liquid separation to obtain an extract (I); The remaining steps are the same as in Example 1.

[0042] Performance testing Weigh 10 mg of freeze-dried quercetin sample, dissolve it in methanol and dilute to 10 ml. HPLC detection was performed using a C18 column, a column temperature of 30°C, a mobile phase of methanol-0.1% phosphoric acid aqueous solution, a flow rate of 1.0 mL / min, a detection wavelength of 370 nm, and an injection volume of 10 μL. Calculate the quercetin peak area in the sample to obtain the purity (%). The yield is the mass conversion efficiency of the raw material Sophora japonica to the final quercetin product, which is calculated using the following formula: The theoretical conversion coefficient is 2.02.

[0043] Table 1 Test data Combining Examples 1-3 and Comparative Examples 1-2 and the data in Table 1, it can be seen that the experimental data of Examples 1-3 are better than those of Comparative Examples 1-2, indicating that the gradient elution of the present application can significantly improve the purity of quercetin while ensuring a high yield, while eluting with only a single concentration of ethanol cannot effectively separate quercetin from impurities, and collecting a single target eluate is not conducive to obtaining high yield and high purity quercetin. The method of using different concentrations of ethanol for elution in the three-stage elution of the present application and collecting the target eluates separately is more effective.

[0044] Combining Example 4 with Comparative Examples 3-4 and the data in Table 1, it can be seen that the experimental data of Example 4 are better than those of Comparative Examples 3-4, indicating that the addition of citrate and ammonia water helps to improve the purity of quercetin. They effectively remove impurities during the elution process and improve the purity of the final product.

[0045] Combining Examples 1-3 and Comparative Example 5 and the data in Table 1, it can be seen that the experimental data of Examples 1-3 are all better than those of Comparative Example 5, indicating that ultrasonic extraction has obvious advantages in terms of quercetin yield and purity compared with traditional hot reflux extraction, and ultrasonic extraction is more conducive to the dissolution of the active ingredient without damaging the target component.

[0046] Combining Example 1 with Examples 4-6 and the data in Table 1, it can be seen that the experimental data of Examples 4-6 are all better than those of Example 1, indicating that the addition of trisodium citrate in the primary elution, the addition of ammonia water in the secondary elution, and the addition of sodium acetate in the tertiary elution, and adjusting their concentrations within a certain range, enhances the adsorption-desorption selectivity of quercetin and the resin, and can further improve the yield and purity of quercetin.

[0047] Combining Example 1 with Example 7 and the data in Table 1, it can be seen that the experimental data are similar, so AB-8 type resin can be used to replace HPD-100 macroporous adsorption resin.

[0048] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A method for extracting quercetin from Sophora japonica seeds, characterized in that: The following steps are involved: (a) crushing the dried Sophora japonica seeds; (b) using 70-80% v / v ethanol aqueous solution as the extraction solvent, performing extraction by ultrasound-assisted extraction at 50-60° C., and performing solid-liquid separation to obtain an extract (I) containing rutin; (c) adding glycosidase to the extract (I) obtained in step (b), adjusting the pH to 3.5-5.5 and the temperature to 35-50° C., carrying out enzymatic hydrolysis for 2-4 hours, and then inactivating the enzyme; (d) concentrating the liquid after the enzyme inactivation in step (c), filtering while hot at 50-60° C., washing the filter residue with 70-80% hot ethanol, combining the filtrates to obtain a mixed solution, applying the solution to a macroporous adsorption resin column, and performing gradient elution with an ethanol-water solution of increasing concentration to collect the target elution fraction containing quercetin; (e) The target eluted fraction is concentrated and freeze-dried to obtain quercetin.

2. A method for extracting quercetin from Sophora japonica seeds according to claim 1, characterized in that, The conditions for ultrasonic-assisted extraction in step (b) are: frequency 20-60 kHz, power density 200-400 W / L, extraction temperature 50-60° C., and time 20-40 minutes.

3. A method for extracting quercetin from Sophora japonica seeds according to claim 1, characterized in that: The glycosidase in step (c) is β-glucosidase.

4. A method for extracting quercetin from Sophora japonica seeds according to claim 1, characterized in that: The amount of enzyme added in step (c) is 0.5-3.0 U / mL.

5. A method for extracting quercetin from Sophora japonica seeds according to claim 1, characterized in that: The macroporous adsorption resin in step (d) is HPD-100 or AB-8 type resin.

6. A method for extracting quercetin from Sophora japonica seeds according to claim 1, characterized in that: The gradient elution step in step (d) includes three elution steps: Primary elution: 10-20% ethanol aqueous solution as mobile phase, elution volume 3-5BV, flow rate 1.5-3.0BV / h; Secondary elution: 30-40% ethanol aqueous solution as mobile phase, elution volume 3-5BV, flow rate 2.0-4.0BV / h; Three-stage elution: (i) elution with 60-70% ethanol in water for 4-6 BV at a flow rate of 2.0-3.0 BV / h, collecting the first target eluate S1; (ii) eluting with 80-90% ethanol aqueous solution for 3-4 BV at a flow rate of 1.5-2.5 BV / h, collecting the second target eluate S2; combining the eluates S1 and S2 to obtain the target fraction containing quercetin.

7. A method for extracting quercetin from Sophora japonica seeds according to claim 6, characterized in that: The gradient elution steps are as follows: Primary elution: Use 10-20% ethanol aqueous solution containing 0.03-0.08 mol / L citrate as the mobile phase, adjust the pH to 3.0-4.0, elution volume 3-5 BV, flow rate 1.5-3.0 BV / h; Secondary elution: Use 30-40% ethanol aqueous solution containing 0.05-0.15% (v / v) ammonia as the mobile phase, elution volume 3-5 BV, flow rate 2.0-4.0 BV / h; Three-stage elution: (i) elution with 60-70% ethanol in water for 4-6 BV at a flow rate of 2.0-3.0 BV / h, collecting the first target eluate S1; (ii) eluting with 80-90% ethanol aqueous solution containing 0.1-0.3 mol / L alkali metal salt for 3-4 BV at a flow rate of 1.5-2.5 BV / h, collecting the second target eluate S2; combining the eluates S1 and S2 to obtain the target fraction containing quercetin.

8. A method for extracting quercetin from Sophora japonica seeds according to claim 1, characterized in that: The particle size of the crushed Sophora japonica seeds is 20-80 meshes, and the mass ratio of the crushed Sophora japonica seeds to the ethanol aqueous solution in step (b) is 1:(20-30).

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