Method for simultaneously purifying and separating flavonoids of Chinese prickly ash

Abstract

The method comprises the following steps: selecting high-quality zanthoxylum bungeanum raw materials which are zanthoxylum bungeanum fruits and zanthoxylum bungeanum leaves, cleaning the zanthoxylum bungeanum raw materials, removing foreign matters such as impurities, soil and the like, drying the zanthoxylum bungeanum raw materials to constant weight, crushing the zanthoxylum bungeanum raw materials into powder for later use, and performing enzymolysis through a compound enzyme to obtain zanthoxylum bungeanum flavone substances. According to the method, the cell wall structure of the zanthoxylum bungeanum maxim can be destroyed, the flavone substances can be dissolved out more easily, compared with a traditional extraction method, the extraction rate can be increased by 30%-50%, impurities can be effectively removed, the zanthoxylum bungeanum maxim flavone substances with high purity can be obtained by adopting the method of combining primary purification with high-speed counter-current chromatography deep separation, and the method is suitable for industrial production. The purity of the zanthoxylum bungeanum flavone substance can reach 80% or above through detection, the extraction process is mild, damage to the activity of the zanthoxylum bungeanum flavone substance caused by high temperature, strong acid, strong base and the like is avoided, and the obtained zanthoxylum bungeanum flavone substance keeps high biological activity.

Description

Technical Field

[0001] The invention discloses a method for simultaneously purifying and separating zanthoxylum bungeanum flavonoids, and belongs to the technical field of flavonoid extraction. Background Art

[0002] Zanthoxylum flavonoids refer to a class of flavonoid compounds present in Zanthoxylum flavonoids. Flavonoid compounds are a class of natural products with a wide range of biological activities, including antioxidant, anti-inflammatory, antibacterial, antiviral and other biological activities. Flavonoid purification refers to the extraction of flavonoid compounds from plants or other sources, and obtaining high-purity flavonoid compounds through a series of separation and purification steps.

[0003] The existing flavonoid extraction uses alcohol extraction, which is one of the most commonly used flavonoid extraction methods. Commonly used organic solvents include ethanol, ether, methanol and ethyl acetate. However, the simple alcohol extraction method can only extract flavonoid compounds in Zanthoxylum bungeanum. However, Zanthoxylum bungeanum contains many types of flavonoids such as epicatechin, rutin, naringin, etc. The flavonoid extraction method can only extract all flavonoids together, and cannot separate these substances according to their types. Summary of the invention

[0004] The technical problem to be solved by the present invention is that the existing flavonoid extraction method can only extract all flavonoid substances together, but cannot separate these substances according to their types.

[0005] To achieve the above object, the present invention provides the following technical solutions: A method for simultaneously purifying and separating Zanthoxylum bungeanum flavonoids comprises the following steps: S1. Select high-quality Zanthoxylum bungeanum raw materials, which are Zanthoxylum bungeanum fruits and Zanthoxylum bungeanum leaves. Clean the Zanthoxylum bungeanum raw materials, remove impurities, soil and other foreign matter, and then dry the raw materials to constant weight, and then crush them into powder for standby use; S2, adding a buffer solution with a pH value of 5 to the crushed pepper powder at a solid-liquid ratio of 1:20, and then adding a complex enzyme, wherein the amount of the complex enzyme added is 2% of the mass of the pepper powder; S3, enzymatically hydrolyzing the above mixture for 2 hours, and after the enzymatic hydrolysis is completed, inactivating the enzyme at 90-100°C for 15 minutes; S4, select D101 macroporous adsorption resin, soak the resin in ethanol for more than 24 hours, then rinse with deionized water until there is no ethanol smell, load the enzyme-killed hydrolyzate onto a macroporous adsorption resin column for adsorption, then elute with a volume fraction of 60% ethanol solution at a flow rate of 2BV / h, and collect the eluate containing Zanthoxylum bungeanum flavonoids; S5. Mix n-hexane, ethyl acetate, methanol and water in a ratio of 2:3:2:2 to form a two-phase solvent system, and inject the eluate after preliminary purification by macroporous adsorption resin into a high-speed countercurrent chromatograph. The operating parameters of the high-speed countercurrent chromatograph are set to: main engine speed 1000r / min, flow rate 2mL / min, detection wavelength 280nm, and then collect different Zanthoxylum bungeanum flavonoid substance components according to the chromatographic peak value.

[0006] As a preferred technical solution of the present invention, the drying temperature of the raw material in step S1 needs to be controlled at 40-60° C., and the particle size of the raw material powder needs to be controlled between 40-60 meshes.

[0007] As a preferred technical solution of the present invention, the complex enzyme in step S2 is a mixture of cellulase and pectinase, wherein the mass ratio of cellulase to pectinase is 2:1.

[0008] As a preferred technical solution of the present invention, the enzymatic hydrolysis temperature of the mixture in step S3 needs to be controlled at 50° C., and the mixture is continuously stirred during the enzymatic hydrolysis process at a stirring speed of 200 rpm.

[0009] As a preferred technical solution of the present invention, in step S4, the enzymatic hydrolysate is loaded at a flow rate of 2 BV / h. After loading, the resin column is flushed with deionized water at a flow rate of 3 BV / h to remove impurities.

[0010] The beneficial effects achieved by the present invention are as follows: a method for simultaneously purifying and separating flavonoid substances of Zanthoxylum bungeanum can be provided. The method can destroy the cell wall structure of Zanthoxylum bungeanum through a composite enzymatic hydrolysis step, so that the flavonoid substances can be more easily dissolved. Compared with the traditional extraction method, the extraction rate can be increased by 30%-50%. The method combining the preliminary purification by macroporous adsorption resin and the deep separation by high-speed countercurrent chromatography can effectively remove impurities and obtain high-purity flavonoid substances of Zanthoxylum bungeanum. The purity of the flavonoid substances of Zanthoxylum bungeanum can reach more than 80% after testing. The extraction process is mild, and the destruction of the activity of the flavonoid substances of Zanthoxylum bungeanum by high temperature, strong acid and alkali and the like is avoided. The obtained flavonoid substances of Zanthoxylum bungeanum maintains a relatively high biological activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 It is a schematic diagram of the process of the present invention; Figure 2 It is an implementation flow chart of the present invention. DETAILED DESCRIPTION

[0012] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0013] See also Figure 1-2 The present invention provides a technical solution: a method for simultaneously purifying and separating Zanthoxylum bungeanum flavonoids, comprising the following steps: S1. Select high-quality Zanthoxylum bungeanum raw materials, which are Zanthoxylum bungeanum fruits and Zanthoxylum bungeanum leaves. Clean the Zanthoxylum bungeanum raw materials, remove impurities, soil and other foreign matter, and then dry the raw materials to constant weight, and then crush them into powder for standby use; S2, adding a buffer solution with a pH value of 5 to the crushed pepper powder at a solid-liquid ratio of 1:20, and then adding a complex enzyme, wherein the amount of the complex enzyme added is 2% of the mass of the pepper powder; The complex enzyme can degrade the cell wall of Sichuan pepper, destroy its structure, and promote the dissolution of flavor substances in Sichuan pepper; S3, enzymatically hydrolyzing the above mixture for 2 hours, and after the enzymatic hydrolysis is completed, inactivating the enzyme at 90-100°C for 15 minutes; S4, select D101 macroporous adsorption resin, soak the resin in ethanol for more than 24 hours, then rinse with deionized water until there is no ethanol smell, load the enzyme-killed hydrolyzate onto a macroporous adsorption resin column for adsorption, then elute with a volume fraction of 60% ethanol solution at a flow rate of 2BV / h, and collect the eluate containing Zanthoxylum bungeanum flavonoids; D101 macroporous adsorption resin is a styrene-type non-polar copolymer with a large specific surface area and pore size. It is suitable for adsorbing various traditional Chinese medicine ingredients with a certain hydrophobicity. It has a large adsorption capacity, is easy to elute, and has good adsorption kinetics. It is stable to heat, organic solvents, and acids and alkalis under general use conditions, so it has a long service life; S5. Mix n-hexane, ethyl acetate, methanol and water in a ratio of 2:3:2:2 to form a two-phase solvent system, and inject the eluate after preliminary purification by macroporous adsorption resin into a high-speed countercurrent chromatograph. The operating parameters of the high-speed countercurrent chromatograph are set to: main engine speed 1000r / min, flow rate 2mL / min, detection wavelength 280nm, and then collect different Zanthoxylum bungeanum flavonoid substance components according to the chromatographic peak value.

[0014] As a preferred technical solution of the present invention, the drying temperature of the raw material in step S1 needs to be controlled at 40-60° C., and the particle size of the raw material powder needs to be controlled between 40-60 meshes.

[0015] As a preferred technical solution of the present invention, the complex enzyme in step S2 is a mixture of cellulase and pectinase, wherein the mass ratio of cellulase to pectinase is 2:1.

[0016] As a preferred technical solution of the present invention, the enzymatic hydrolysis temperature of the mixture in step S3 needs to be controlled at 50° C., and the mixture is continuously stirred during the enzymatic hydrolysis process at a stirring speed of 200 rpm.

[0017] As a preferred technical solution of the present invention, in step S4, the enzymatic hydrolysate is loaded at a flow rate of 2 BV / h. After loading, the resin column is flushed with deionized water at a flow rate of 3 BV / h to remove impurities.

[0018] Specifically, the method selects high-quality Zanthoxylum bungeanum raw materials, which are Zanthoxylum bungeanum fruits and Zanthoxylum bungeanum leaves, cleans the Zanthoxylum bungeanum raw materials, removes impurities, soil and other foreign matter, and then dries them to constant weight at a temperature of 40-60°C, and then grinds them into powder with a particle size of 40-60 mesh for standby use, adds a buffer solution with a pH value of 5 to the grinded Zanthoxylum bungeanum powder according to a solid-liquid ratio of 1:20, and then adds a composite enzyme, which is a mixture of cellulase and pectinase, wherein the mass ratio of cellulase to pectinase is 2:1, and the amount of the composite enzyme added is 2% of the mass of the Zanthoxylum bungeanum powder, and enzymolyzes the above mixture at 50°C for 2 hours, continuously stirs during the enzymolysis process, and the stirring speed is 200 rpm. After the enzymolysis is completed, the enzymolysis solution is heated at 90- The enzyme was inactivated at 100°C for 15 minutes, and D101 macroporous adsorption resin was selected. The resin was first soaked in ethanol for more than 24 hours, and then rinsed with deionized water until there was no ethanol smell. The enzymatic hydrolyzate after the enzyme inactivation was loaded onto the macroporous adsorption resin column at a flow rate of 2BV / h for adsorption. After the loading was completed, the resin column was rinsed with deionized water at a flow rate of 3BV / h to remove impurities, and then eluted with a volume fraction of 60% ethanol solution at a flow rate of 2BV / h, and the eluate containing Zanthoxylum bungeanum flavonoids was collected. Hexane, ethyl acetate, methanol and water were mixed in a ratio of 2:3:2:2 to form a two-phase solvent, and the eluate after preliminary purification by the macroporous adsorption resin was injected into a high-speed countercurrent chromatograph. The operating parameters of the high-speed countercurrent chromatograph were set as follows: main engine speed 1000r / min, flow rate 2mL / min, detection wavelength 280nm, and then different Zanthoxylum bungeanum flavonoid material components were collected according to the chromatographic peaks.

[0019] Implementation 1 100 g of Zanthoxylum bungeanum fruit were selected, and the raw material pretreatment steps were followed, such as washing, drying and crushing into 40 mesh powder.

[0020] A buffer solution with a pH value of 5.5 was added to the powder at a solid-liquid ratio of 1:20, and then a complex enzyme of 0.6 g of cellulase and 0.3 g of pectinase was added. The mixture was enzymolyzed at 50°C for 2 hours with a stirring speed of 200 rpm. After the enzymolysis was completed, the enzyme was inactivated at 95°C for 15 minutes.

[0021] Select D101 macroporous adsorption resin, follow the above-mentioned macroporous adsorption resin preliminary purification steps, elute with 60% ethanol solution, and collect the eluate.

[0022] Hexane, ethyl acetate, methanol and water were mixed in a ratio of 2:3:2:2 to form a two-phase solvent system, and the eluent was injected into a high-speed countercurrent chromatograph, and deep separation was performed according to the set operating parameters to collect the Zanthoxylum bungeanum flavonoids components. After testing, the extraction rate of Zanthoxylum bungeanum flavonoids was 23%, the purity was 80%, and it had good antioxidant activity.

[0023] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for simultaneously purifying and separating Zanthoxylum bungeanum flavonoids, characterized in that: The following steps are involved: S1. Select high-quality Zanthoxylum bungeanum raw materials, which are Zanthoxylum bungeanum fruits and Zanthoxylum bungeanum leaves. Clean the Zanthoxylum bungeanum raw materials, remove impurities, soil and other foreign matter, and then dry the raw materials to constant weight, and then crush them into powder for standby use; S2, adding a buffer solution with a pH value of 5 to the crushed pepper powder at a solid-liquid ratio of 1:20, and then adding a complex enzyme, wherein the amount of the complex enzyme added is 2% of the mass of the pepper powder; S3, enzymatically hydrolyzing the above mixture for 2 hours, and after the enzymatic hydrolysis is completed, inactivating the enzyme at 90-100°C for 15 minutes; S4, select D101 macroporous adsorption resin, soak the resin in ethanol for more than 24 hours, then rinse with deionized water until there is no ethanol smell, load the enzyme-killed hydrolyzate onto a macroporous adsorption resin column for adsorption, then elute with a volume fraction of 60% ethanol solution at a flow rate of 2BV / h, and collect the eluate containing Zanthoxylum bungeanum flavonoids; S5. Mix n-hexane, ethyl acetate, methanol and water in a ratio of 2:3:2:2 to form a two-phase solvent system, and inject the eluate after preliminary purification by macroporous adsorption resin into a high-speed countercurrent chromatograph. The operating parameters of the high-speed countercurrent chromatograph are set to: main engine speed 1000r / min, flow rate 2mL / min, detection wavelength 280nm, and then collect different Zanthoxylum bungeanum flavonoid substance components according to the chromatographic peak value.

2. A method for simultaneously purifying and separating Zanthoxylum bungeanum flavonoids according to claim 1, characterized in that: In step S1, the drying temperature of the raw material needs to be controlled at 40-60° C., and the particle size of the raw material powder needs to be controlled between 40-60 meshes.

3. A method for simultaneously purifying and separating Zanthoxylum bungeanum flavonoids according to claim 1, characterized in that: The complex enzyme in step S2 is a mixture of cellulase and pectinase, wherein the mass ratio of cellulase to pectinase is 2:

1.

4. The method for simultaneously purifying and separating Zanthoxylum bungeanum flavonoids according to claim 1, characterized in that: The enzymatic hydrolysis temperature of the mixture in step S3 needs to be controlled at 50° C. and the mixture is stirred continuously during the enzymatic hydrolysis process at a stirring speed of 200 rpm.

5. The method for simultaneously purifying and separating Zanthoxylum bungeanum flavonoids according to claim 1, characterized in that: In the step S4, the enzymatic hydrolysate is loaded at a flow rate of 2 BV / h. After loading, the resin column is washed with deionized water at a flow rate of 3 BV / h to remove impurities.

Images