A method for combined extraction of vitexin and aucubin

Abstract

The invention provides a method for jointly extracting vitexin and aucubin, so as to solve the problem that the existing method cannot simultaneously extract vitexin and aucubin. The method comprises the following steps: crushing the dry fruit of vitexin, adding purified water and adjusting the pH, then adding cellulase and pectinase, performing enzyme inactivation after enzymolysis, and obtaining a slurry; adding a first alcohol reagent to the slurry and mixing it to obtain a mixed solution, pumping the mixed solution into a treatment chamber of a high-voltage pulse electric field for extraction, filtering, obtaining an extract and concentrating it to be alcohol-free; preparing a chitosan clarifier; loading a macroporous adsorption resin into a chromatography column; adding a chitosan clarifier to the concentrated extract, heating and stirring, filtering, and after the filtrate is adsorbed by the macroporous adsorption resin, first washing with purified water, then eluting with a first alcohol solution and a second alcohol solution in sequence, collecting the corresponding first eluate and second eluate and extracting; respectively obtaining aucubin and vitexin.

Description

Technical Field

[0001] The invention relates to a method for extracting natural chemical components from plants, and in particular to a method for jointly extracting vitexin and aucubin. Background Art

[0002] Vitex agnus-castus is native to Europe and is mainly distributed in Jiangsu and Shanghai, China. It is also planted in temperate regions of the United States. It is light-loving, relatively cold-resistant, heat-resistant, drought-resistant, and has strong growth potential and resistance, and few diseases and insect pests. Vitex agnus-castus has many pharmacological effects, including antioxidant, anti-cancer, immunomodulatory, antibacterial, anti-inflammatory, insecticidal, liver-protective, bone-protective, kidney-protective, pancreatic-protective, and hypoglycemic. In Europe, Vitex agnus-castus was once used to suppress sexual desire, sober up, and treat symptoms such as bloating, fever, constipation, and uterine cramps. In the 19th century, American herbalists used it as a drug for promoting menstruation and lactation. At present, Vitex agnus-castus is mainly used for female reproductive system diseases caused by hyperprolactinemia or corpus luteum deficiency.

[0003] The fruit of Vitex agnus-castus contains a variety of natural chemical components such as agnus-castus glycosides, vitexin, aucubin, and vitexin. Among them, the molecular formula of agnus-castus glycosides is C 22 H 26 O 11 , the structural formula is as follows:

[0004]

[0005] Aucubin is used to protect the liver and has protective and therapeutic effects on induced liver damage. 15 H 22 O9, the structural formula is as follows:

[0006]

[0007] The chemical name of aucubin is β-D-pyranoglucoside, which is an iridoid ether terpene glycoside compound. It has the activities of clearing dampness and heat, promoting urination, relieving pain, lowering blood pressure, protecting liver and anti-tumor. It can promote stem cell regeneration and significantly inhibit the replication of hepatitis B virus DNA. Its aglycone and effective polymer are antibiotics. Therefore, it is of great significance to extract aucubin and aucubin from the fruit of Vitex agnus-castus.

[0008] A Chinese patent with publication number CN101982471B discloses "a process for extracting aucubin from Eucommia ulmoides fruit". After the Eucommia ulmoides fruit is chopped, it is extracted with water or ethanol solution as the extraction solvent, and the extract is purified by a macroporous resin process to obtain aucubin with a purity greater than 50%.

[0009] The Chinese patent with publication number CN102603828A discloses "a method for extracting aucubin from eucommia leaves", which adopts alcohol extraction, ultrasonic treatment, centrifugation, concentration, extraction, macroporous adsorption resin chromatography, continuous separation, concentration, and crystallization to obtain pure aucubin.

[0010] A Chinese patent with publication number CN102228565B discloses "a preparation method and use of a Vitex agnus-castus extract". The dry fruits of Vitex agnus-castus are extracted by cold soaking, warm soaking, percolation, heating reflux or CO2 supercritical extraction. The Vitex agnus-castus extract is obtained after filtration, concentration, impurity removal and drying. The Vitex agnus-castus extract has a high content of total flavonoids and is detected to contain vitexin.

[0011] However, the common problem of the above prior arts is that it is impossible to extract vitexin and aucubin at the same time. Summary of the invention

[0012] The purpose of the present invention is to solve the technical problem that the existing extraction method cannot simultaneously extract vitexin and aucubin, and to provide a method for jointly extracting vitexin and aucubin.

[0013] In order to achieve the above object, the present invention adopts the following technical solution:

[0014] A method for jointly extracting vitexin and aucubin is special in that it comprises the following steps:

[0015] Step 1, enzymatic hydrolysis and high-voltage pulse electric field extraction;

[0016] Step 1.1, crushing the dry fruits of Vitex agnus-castus, adding purified water in an amount 3-5 times the mass of the dry fruits of Vitex agnus-castus, adjusting the pH to 4.0-5.0, then adding cellulase and pectinase, performing enzymolysis at 45-55° C. for 2-3 hours, and then inactivating the enzymes to obtain a slurry;

[0017] Step 1.2, adding a first alcohol reagent to the slurry until the concentration of the first alcohol reagent is 50-60%, mixing to obtain a mixed solution, and then pumping the mixed solution into a treatment chamber of a high-voltage pulse electric field for extraction, filtering, obtaining an extract and concentrating it to be alcohol-free; the intensity of the high-voltage pulse electric field is 20-30 kV / cm, and the number of pulses is 6-12;

[0018] Step 2, preparing chitosan clarifier;

[0019] Step 3, purification by macroporous adsorption resin;

[0020] Step 3.1, loading the macroporous adsorption resin into the chromatography column;

[0021] Step 3.2, adding the chitosan clarifier prepared in step 2 to the concentrated extract of step 1.2, heating and stirring, filtering, and after the filtrate is adsorbed by the macroporous adsorption resin, first washing with purified water, and then eluting with a first alcohol solution with a concentration of 10%-20% and a second alcohol solution with a concentration of 60%-80% in sequence until the color is light, and collecting the corresponding first eluate and second eluate respectively;

[0022] Step 4, obtaining aucubin;

[0023] Concentrating the first eluate, adding n-butanol to the concentrate for extraction, collecting and concentrating the n-butanol phase, obtaining first crystals after crystallization, then dissolving, decolorizing and crystallizing the first crystals in sequence to obtain second crystals, rinsing the second crystals and drying them to obtain aucubin;

[0024] Step 5, obtaining vitexin;

[0025] The second eluate is concentrated, and ethyl acetate is added to dilute the concentrate for extraction. The ethyl acetate phase is collected and concentrated into a paste. The paste is mixed with silica gel and dried to obtain sample-mixed silica gel. The sample-mixed silica gel is then added to a prepared silica gel chromatography column and eluted with an eluent. The eluate is monitored by thin layer chromatography. When only a single point of vitexin is shown on the silica gel plate, the eluate is collected, and the eluate is successively concentrated, crystallized, rinsed, and dried to obtain vitexin.

[0026] Furthermore, in step 1.1, the dry fruit of Vitex agnus-castus is crushed to 20-40 mesh; the temperature of the enzyme inactivation is 90-95° C., and the time is 15-25 min; the mass of the cellulase is 0.03-0.06% of the mass of the dry fruit of Vitex agnus-castus; the mass of the pectinase is 0.01-0.05% of the mass of the dry fruit of Vitex agnus-castus;

[0027] In step 1.2, the first alcohol reagent is methanol or ethanol; a peristaltic pump is used to pump the mixed solution into a treatment chamber of a high-voltage pulse electric field at a flow rate of 10-15 mL / min for extraction; the extract is concentrated to be alcohol-free at a vacuum degree of -0.08 to -0.06 MPa and a temperature of 60-70°C.

[0028] Further, step 3.1 is specifically, loading a macroporous adsorption resin with a mass of 0.5-1.5 times that of the dry fruit of Vitex agnus-castus into a chromatography column; the macroporous adsorption resin is AB-8 resin, LX-38 resin, LX-18 resin, LX-5 resin or DM130 resin;

[0029] In step 3.2, the volume of the chitosan clarifier is 1%-4% of the volume of the concentrated extract in step 1.2; the heating temperature is 65-75°C; the stirring time is 15-30min; the amount of purified water is 1-2 chromatography column volumes; the first alcohol solution and the second alcohol solution are methanol solution or ethanol solution, the amount of the first alcohol solution is 4-6 chromatography column volumes, and the amount of the second alcohol solution is 5-7 chromatography column volumes.

[0030] Furthermore, step 4 specifically includes the following steps:

[0031] Step 4.1, concentrating the first eluate, adding n-butanol in an amount 4-6 times the mass of the concentrate to the concentrate, extracting 3-5 times, collecting the n-butanol phase and concentrating it to a specific gravity of 1.13-1.18, then placing it for crystallization, and filtering the crystals to obtain first crystals;

[0032] Step 4.2, add a second alcohol reagent in an amount 3-5 times the mass of the first crystal to the first crystal, dissolve at 65-75° C. to obtain a solution, then add activated carbon in an amount of 1%-3% of the mass of the solution to the solution, stir and decolorize at 65-75° C. for 40-60 minutes, then allow the crystal to stand, filter the crystal to obtain a second crystal, rinse the second crystal with a third alcohol reagent, and then dry to obtain aucubin.

[0033] Furthermore, step 5 specifically includes the following steps:

[0034] Step 5.1, concentrating the second eluate until it is free of alcohol, adding purified water in an amount 1-3 times the mass of the concentrate to obtain a diluent, then adding ethyl acetate in an amount 3-5 times the mass of the diluent to the diluent, extracting 3-5 times, each time for 4-6 minutes, collecting the ethyl acetate phase and concentrating it into a paste;

[0035] Step 5.2, mixing the paste with 200-300 mesh silica gel in an amount of 0.5-1.5 times the mass of the paste and drying to obtain the sample mixing silica gel;

[0036] Step 5.3, take 200-300 mesh blank silica gel with a mass of 6 to 10 times the mass of the sample-mixed silica gel for wet column packing to obtain a silica gel chromatography column, and add the sample-mixed silica gel into the silica gel chromatography column, and then elute the silica gel chromatography column with an eluent, and monitor the eluate by thin layer chromatography at the same time, collect the eluate when only a single point of violacein is shown on the silica gel plate, concentrate the eluate in turn, let it stand for crystallization, filter the crystals, rinse, and vacuum dry to obtain violacein.

[0037] Furthermore, step 2 is specifically as follows:

[0038] Add glacial acetic acid in a volume of 3%-5% of the volume of purified water to purified water to obtain an acetic acid aqueous solution, then add chitosan to the acetic acid aqueous solution to prepare a chitosan clarifier; the mass concentration of chitosan in the chitosan clarifier is 1.5%-2.5%.

[0039] Further, step 4.1 is specifically, concentrating the first eluate to a specific gravity of 1.05-1.10 at a vacuum degree of -0.08 to -0.06 MPa and a temperature of 60-70°C, adding n-butanol in an amount 4-6 times the mass of the concentrated solution to the concentrated solution, extracting 3-5 times, collecting the n-butanol phase and concentrating it to a specific gravity of 1.13-1.18, and then placing the crystals at 4-6°C for 18-24 hours, and filtering the crystals to obtain the first crystals;

[0040] In step 4.2, the activated carbon is 320 activated carbon, 767 activated carbon, or KF03 activated carbon; the placing the crystallization specifically refers to placing the crystallization at 4-6°C for 20-26h; the drying specifically refers to drying at a vacuum degree of -0.08 to -0.06MPa and a temperature of 60-70°C; the second alcohol reagent and the third alcohol reagent are methanol or ethanol;

[0041] Step 5.1 is specifically, concentrating the second eluate to be alcohol-free at a vacuum degree of -0.09 to -0.07 MPa and a temperature of 60-70°C, adding purified water in an amount 1-3 times the mass of the concentrated solution to the concentrated solution to obtain a diluent, then adding ethyl acetate in an amount 3-5 times the mass of the diluent to the diluent, extracting 3-5 times, each time for 4-6 minutes, collecting the ethyl acetate phase and concentrating it into a paste at a vacuum degree of -0.09 to -0.07 MPa and a temperature of 60-70°C;

[0042] In step 5.2, the drying specifically refers to drying at a vacuum degree of -0.08 to -0.06 MPa and a temperature of 60-70°C;

[0043] Step 5.3 is as follows: n-hexane is used to wet-pack a 200-300 mesh blank silica gel with a mass of 6 to 10 times the mass of the sample-mixed silica gel to obtain a silica gel chromatography column, and the sample-mixed silica gel is added to the silica gel chromatography column. Then, the silica gel chromatography column is eluted in sequence with a mixed solvent of n-hexane and ethyl acetate in a volume ratio of 10:1, 8:1, 8:2, and 7:3, respectively, and the eluent is monitored by thin layer chromatography. When the silica gel plate shows that only spike flower is present, When vitexin is single point, the eluate is collected, and the eluate is concentrated at a vacuum degree of -0.09 to -0.07 MPa and a temperature of 60-70°C for 30-50 minutes, and then placed at 4-6°C for crystallization for 20-26 hours, and then filtered to obtain wet crystals, and the wet crystals are rinsed with n-hexane in an amount of 0.5 to 1.5 times the mass of the wet crystals, and dried at a vacuum degree of -0.08 to -0.06 MPa and a temperature of 50-60°C to obtain vitexin;

[0044] Alternatively, petroleum ether is used to wet-pack a 200-300 mesh blank silica gel with a mass of 6 to 10 times the mass of the sample-mixed silica gel to obtain a silica gel chromatography column, and the sample-mixed silica gel is added to the silica gel chromatography column, and then the silica gel chromatography column is sequentially eluted with a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 10:1, 8:1, 8:2, and 7:3, respectively, and the eluent is monitored by thin layer chromatography. When the silica gel plate shows that only vitexin is present, The eluate is collected at a single point, concentrated at a vacuum degree of -0.09 to -0.07 MPa and a temperature of 60-70°C for 30-50 min, and then placed at 4-6°C for crystallization for 20-26 h, and then filtered to obtain wet crystals, and the wet crystals are rinsed with petroleum ether in an amount 0.5-1.5 times the mass of the wet crystals, and dried at a vacuum degree of -0.08 to -0.06 MPa and a temperature of 50-60°C to obtain vitexin.

[0045] Further, step 4.1 is specifically, concentrating the first eluate to a specific gravity of 1.08 at a vacuum degree of -0.07 MPa and a temperature of 65°C, adding n-butanol 5 times the mass of the concentrated solution to the concentrated solution, extracting 4 times, collecting the n-butanol phase and concentrating it to a specific gravity of 1.15, then placing it at 5°C for crystallization for 20 hours, and filtering the crystals to obtain the first crystals;

[0046] Step 4.2 is specifically, adding 5 times the mass of the first crystal to the first crystal ethanol, dissolving at 70 ° C to obtain a solution, then adding 767 activated carbon with a mass of 2% of the mass of the solution to the solution, stirring and decolorizing at 70 ° C for 50 minutes, then placing the crystal at 5 ° C for 24 hours, filtering the crystal to obtain a second crystal, rinsing the second crystal with ethanol, and drying at a vacuum degree of -0.07 MPa and a temperature of 65 ° C to obtain aucubin;

[0047] Step 5.1 is specifically, concentrating the second eluate to be free of alcohol at a vacuum degree of -0.07 MPa and a temperature of 65°C, adding purified water twice the mass of the concentrated solution to the concentrated solution to obtain a diluent, then adding ethyl acetate four times the mass of the diluent to the diluent, extracting four times, each time for 5 minutes, collecting the ethyl acetate phase and concentrating it into a paste at a vacuum degree of -0.07 MPa and a temperature of 65°C;

[0048] Step 5.2 is specifically, mixing the paste with 200-300 mesh silica gel in an amount that is 1 times the mass of the paste, and drying the mixture at a vacuum degree of -0.07 MPa and a temperature of 65°C to obtain the sample-mixed silica gel;

[0049] Specifically, step 5.3 is to use n-hexane to wet-pack 200-300 mesh blank silica gel with a mass of 8 times the mass of the sample-mixed silica gel to obtain a silica gel chromatography column, and add the sample-mixed silica gel into the silica gel chromatography column, and then use n-hexane and ethyl acetate mixed solvents in volume ratios of 10:1, 8:1, 8:2, and 7:3 to elute the silica gel chromatography column in sequence, and monitor the eluent by thin layer chromatography. In the eluent section of the mixed solvent with a volume ratio of n-hexane and ethyl acetate of 8:1, the silica gel plate shows that only a single point of violacein is contained. The eluent is collected, and the eluent is concentrated at a vacuum degree of -0.07 MPa and a temperature of 65°C for 40 minutes, and then placed at 5°C for crystallization for 24 hours, and then filtered to obtain wet crystals, and the wet crystals are rinsed with n-hexane with a mass of 1 times the mass of the wet crystals, and dried at a vacuum degree of -0.07 MPa and a temperature of 55°C to obtain violacein.

[0050] Furthermore, step 2 is specifically as follows:

[0051] Add glacial acetic acid in a volume of 4% of the volume of purified water to purified water to obtain an acetic acid aqueous solution, then add chitosan to the acetic acid aqueous solution to prepare a chitosan clarifier; the mass concentration of chitosan in the chitosan clarifier is 2%.

[0052] Further, step 1.1 is specifically as follows: crushing the dry fruit of Vitex agnus-castus to 40 mesh, adding purified water in an amount of 4 times the mass of the dry fruit of Vitex agnus-castus, and adjusting the pH to 4.5, and then adding 0.05% of the mass of the dry fruit of Vitex agnus-castus and 0.03% of the mass of the dry fruit of Vitex agnus-castus, enzymolysis at 50° C. for 2.5 hours, and then inactivating the enzyme at 95° C. for 20 minutes to obtain a slurry;

[0053] Step 1.2 specifically comprises adding ethanol to the slurry until the concentration of ethanol is 55%, mixing to obtain a mixed solution, and then using a peristaltic pump to pump the mixed solution into a treatment chamber of a high-voltage pulse electric field at a flow rate of 12 mL / min for extraction, filtering, obtaining an extract, and concentrating the extract at a vacuum degree of -0.07 MPa and a temperature of 65°C until there is no alcohol; the intensity of the high-voltage pulse electric field is 25 kV / cm, and the number of pulses is 10;

[0054] Step 3.1 specifically comprises loading LX-38 resin in an amount equal to 1 times the mass of Vitex agnus-castus dry fruit into a chromatography column;

[0055] Specifically, step 3.2 is to add a chitosan clarifier with a volume of 2% of the volume of the concentrated extract in step 1.2 to the concentrated extract in step 1.2, stir at 70°C for 20 minutes, filter, and adsorb the filtrate with LX-38 resin. Then, wash the filtrate with 1 column volume of purified water, and then use 5 column volumes of 15% ethanol solution and 6 column volumes of 75% ethanol solution to elute until the color is light, and collect the corresponding first eluate and second eluate respectively.

[0056] Beneficial effects of the present invention:

[0057] 1. A method for jointly extracting vitexin and aucubin provided by the present invention can simultaneously extract vitexin and aucubin. In the present invention, the dry fruit of Vitex agnuscastus can fully extract aucubin and vitexin in the dry fruit of Vitex agnuscastus through the synergistic effect of enzymolysis and high-voltage pulsed electric field technology. Through enzymolysis and high-voltage pulsed electric field technology, the cell wall and cell membrane of the dry fruit of Vitex agnuscastus are irreversibly damaged. The specificity of the enzyme is utilized to destroy the cell wall of the raw material so that the effective components in the cell wall are extracted. The permeability of the cell membrane can be instantly changed by the high-voltage pulsed electric field technology, so that the treated cells are irreversibly damaged, and the active substances in the cells are rapidly dissolved under the condition of containing the first alcohol reagent, and the dissolution process is completed instantaneously under the flow state.

[0058] 2. The present invention extracts and purifies aucubin and vitexin in the dry fruit of Vitex agnus-castus by a combined extraction and purification method, and finally obtains high-content products. Compared with traditional extraction methods such as water extraction, organic solvent extraction, supercritical CO2 extraction and the like, enzymatic hydrolysis and high-voltage pulse electric field extraction have the advantages of short processing time, extremely low energy consumption, continuous operation, non-thermal processing and high activity of the extracted target product.

[0059] 3. The present invention purifies the clarified extract of the dry fruit of Vitex agnus-castus by a macroporous adsorption resin chromatography column. According to the difference in the properties of aucubin and vitexin, the extract is first eluted with a low-concentration first alcohol solution and then with a high-concentration second alcohol solution, so that aucubin and vitexin in the extract are completely separated, which provides a strong prerequisite for the subsequent extraction of high-content aucubin and vitexin. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 It is a flow chart of Example 1 of a method for jointly extracting vitexin and aucubin of the present invention;

[0061] Figure 2 is the HPLC spectrum of aucubin and vitexin in the first eluate collected in step 3.2 of Example 1 of the present invention;

[0062] Figure 3 is the HPLC spectrum of aucubin and vitexin in the second eluate collected in step 3.2 of Example 1 of the present invention;

[0063] Figure 4 is the HPLC spectrum of aucubin in the second crystal after drying in step 4.2 of Example 1 of the present invention;

[0064] Figure 5 This is the HPLC spectrum of vitexin in the crystals after drying in step 5.3 of Example 1 of the present invention. DETAILED DESCRIPTION

[0065] Embodiment 1

[0066] like Figure 1 As shown, a method for jointly extracting vitexin and aucubin comprises the following steps:

[0067] Step 1, enzymatic hydrolysis and high-voltage pulse electric field extraction;

[0068] Step 1.1, crushing 1 kg of Vitex agnus-castus dried fruit into 40 meshes, adding purified water 4 times the weight of Vitex agnus-castus dried fruit, adjusting the pH to 4.5, then adding 0.05% of cellulase by weight of Vitex agnus-castus dried fruit and 0.03% of pectinase by weight of Vitex agnus-castus dried fruit, performing enzymolysis at 50° C. for 2.5 h, and then inactivating the enzyme at 95° C. for 20 min to obtain a slurry;

[0069] Step 1.2, add ethanol to the slurry until the concentration of ethanol is 55%, mix well to obtain a mixed solution, and then use a peristaltic pump to pump the mixed solution into the treatment chamber of the high-voltage pulse electric field at a flow rate of 12 mL / min for extraction, filter, obtain the extract and concentrate it to be alcohol-free at a vacuum degree of -0.07 MPa and a temperature of 65°C. The intensity of the high-voltage pulse electric field is 25 kV / cm and the number of pulses is 10.

[0070] Step 2, preparing chitosan clarifier;

[0071] Add glacial acetic acid in a volume of 4% of the volume of the purified water to purified water to obtain an acetic acid aqueous solution, then add chitosan to the acetic acid aqueous solution to prepare a chitosan clarifier, wherein the mass concentration of chitosan in the chitosan clarifier is 2%.

[0072] Step 3, purification by macroporous adsorption resin;

[0073] Step 3.1, loading LX-38 resin in an amount equal to 1 times the mass of Vitex agnus-castus dry fruit into a chromatography column;

[0074] Step 3.2, add chitosan clarifier with a volume of 2% of the volume of the concentrated extract in step 1.2 to the concentrated extract in step 1.2, stir at 70°C for 20 minutes, filter, and after the filtrate is adsorbed by LX-38 resin, first wash with 1 column volume of purified water, then use 5 column volumes of 15% ethanol solution and 6 column volumes of 75% ethanol solution to elute in sequence until the color is light, and collect the corresponding first eluate and second eluate respectively. HPLC is used to detect aucubin and vitexin in the first eluate and the second eluate, and the corresponding HPLC spectra are as follows: Figure 2 and Figure 3 The corresponding HPLC test results are shown in Tables 1 and 2.

[0075] Table 1

[0076] Element Retention time / min Peak area / μV*sec Peak height / μV content / % Aucubin 3.760 3253926 327954 33.84 Vitexin 14.975 14212 1520 0.015

[0077] Table 2

[0078] Element Retention time / min Peak area / μV*sec Peak height / μV content / % Aucubin 3.765 52373 5842 0.085 Vitexin 14.962 6681920 921403 21.37

[0079] As shown in Table 1, the content of aucubin in the first eluate was 33.84%, and the content of vitexin was 0.015%; as shown in Table 2, the content of aucubin in the second eluate was 0.085%, and the content of vitexin was 21.37%.

[0080] Step 4, obtaining aucubin;

[0081] Step 4.1, concentrating the first eluate to a specific gravity of 1.08 at a vacuum degree of -0.07 MPa and a temperature of 65°C, adding n-butanol in an amount 5 times the mass of the concentrate to the concentrate, extracting 4 times, collecting the n-butanol phase and concentrating it to a specific gravity of 1.15, then placing it at 5°C for crystallization for 20 hours, and filtering the crystals to obtain the first crystals;

[0082] Step 4.2, add 5 times the mass of ethanol to the first crystal, dissolve at 70°C to obtain a solution, then add 767 activated carbon with a mass of 2% of the mass of the solution to the solution, stir and decolorize at 70°C for 50 minutes, then place the crystal at 5°C for 24 hours, filter the crystal to obtain a second crystal, rinse the second crystal with ethanol, and dry it at a vacuum degree of -0.07MPa and a temperature of 65°C to obtain aucubin. HPLC is used to detect aucubin in the second crystal after drying, and the obtained HPLC spectrum is as follows: Figure 4 The obtained HPLC test results are shown in Table 3.

[0083] Table 3

[0084] Element Retention time / min Peak area / μV*sec Peak height / μV content / % Aucubin 3.838 4190842 621874 98.27

[0085] As shown in Table 3, the content of aucubin in the second crystal after drying is 98.27%, and the yield is 91.4%.

[0086] Step 5, obtaining vitexin;

[0087] Step 5.1, concentrating the second eluate to be free of alcohol at a vacuum degree of -0.07 MPa and a temperature of 65°C, adding purified water twice the mass of the concentrate to obtain a diluent, then adding ethyl acetate four times the mass of the diluent to the diluent, stirring and extracting four times, each time for 5 minutes, collecting the ethyl acetate phase and concentrating it into a paste at a vacuum degree of -0.07 MPa and a temperature of 65°C;

[0088] Step 5.2, mix the paste with 200-300 mesh silica gel in an amount that is 1 times the mass of the paste, and dry the mixture at a vacuum degree of -0.07 MPa and a temperature of 65°C to obtain the sample-mixed silica gel;

[0089] Step 5.3, using n-hexane to wet-pack 200-300 mesh blank silica gel in an amount 8 times the mass of the sample-mixed silica gel to obtain a silica gel chromatography column, and adding the sample-mixed silica gel into the silica gel chromatography column, and then using n-hexane and ethyl acetate mixed solvents in volume ratios of 10:1, 8:1, 8:2, and 7:3 to elute the silica gel chromatography column in sequence, and simultaneously use thin layer chromatography to monitor the eluent, in the eluent section of the mixed solvent in which the volume ratio of n-hexane and ethyl acetate is 8:1, the silica gel plate shows that only a single point of vitexin is contained, the eluent is collected, and the eluent is concentrated at a vacuum degree of -0.07 MPa and a temperature of 65°C for 40 min, and then placed at 5°C for crystallization for 24 h, and then filtered to obtain wet crystals, and the wet crystals are rinsed with n-hexane in an amount 1 times the mass of the wet crystals, and dried at a vacuum degree of -0.07 MPa and a temperature of 55°C to obtain vitexin. HPLC was used to detect vitexin in the dried crystals, and the obtained HPLC spectrum was as follows: Figure 5 The obtained HPLC test results are shown in Table 4.

[0090] Table 4

[0091] Element Retention time / min Peak area / μV*sec Peak height / μV content / % Vitexin 15.033 4443151 682249 98.41

[0092] As shown in Table 4, the content of vitexin in the dried crystals was 98.41%, and the yield was 86.9%.

[0093] Embodiment 2

[0094] Step 1, enzymatic hydrolysis and high-voltage pulse electric field extraction;

[0095] Step 1.1, crushing 1 kg of Vitex agnus-castus dry fruit into 40 meshes, adding purified water 3 times the weight of Vitex agnus-castus dry fruit, adjusting the pH to 4.5, then adding 0.03% of cellulase by weight of Vitex agnus-castus dry fruit and 0.05% of pectinase by weight of Vitex agnus-castus dry fruit, performing enzymolysis at 50° C. for 3 h, and then inactivating the enzyme at 95° C. for 20 min to obtain a slurry;

[0096] Step 1.2, add ethanol to the slurry until the concentration of ethanol is 55%, mix well to obtain a mixed solution, and then use a peristaltic pump to pump the mixed solution into the treatment chamber of the high-voltage pulse electric field at a flow rate of 12 mL / min for extraction, filter, obtain the extract and concentrate it to be alcohol-free at a vacuum degree of -0.07 MPa and a temperature of 70°C. The intensity of the high-voltage pulse electric field is 30 kV / cm and the number of pulses is 8.

[0097] Step 2, preparing chitosan clarifier;

[0098] Add glacial acetic acid in a volume of 4% of the volume of the purified water to purified water to obtain an acetic acid aqueous solution, then add chitosan to the acetic acid aqueous solution to prepare a chitosan clarifier, wherein the mass concentration of chitosan in the chitosan clarifier is 2%.

[0099] Step 3, purification by macroporous adsorption resin;

[0100] Step 3.1, loading LX-5 resin in an amount 1.5 times the mass of Vitex agnus-castus dry fruit into a chromatography column;

[0101] Step 3.2, add chitosan clarifier with a volume of 2% of the volume of the concentrated extract in step 1.2 to the concentrated extract in step 1.2, stir at 70°C for 20 minutes, filter, and after the filtrate is adsorbed by LX-5 resin, first wash with 1 column volume of purified water, then use 5 column volumes of 10% ethanol solution and 6 column volumes of 75% ethanol solution to elute in sequence until the color is light, and collect the corresponding first eluent and second eluent respectively. HPLC is used to detect aucubin and vitexin in the first eluent and the second eluent respectively, and the corresponding HPLC spectrum obtained is consistent with that in Example 1. Figure 2 and Figure 3 Similarly, the corresponding HPLC test results were as follows: the content of aucubin in the first eluate was 32.49%, and the content of vitexin was 0.027%; the content of aucubin in the second eluate was 0.038%, and the content of vitexin was 21.06%.

[0102] Step 4, obtaining aucubin;

[0103] Step 4.1, concentrating the first eluate to a specific gravity of 1.05 at a vacuum degree of -0.07 MPa and a temperature of 65°C, adding n-butanol in an amount 4 times the mass of the concentrate to the concentrate, extracting 4 times, collecting the n-butanol phase and concentrating it to a specific gravity of 1.15, then placing it at 6°C for crystallization for 20 hours, and filtering the crystals to obtain the first crystals;

[0104] Step 4.2, add ethanol in an amount of 5 times the mass of the first crystal to the first crystal, dissolve at 70°C to obtain a solution, then add 767 activated carbon in an amount of 2% of the mass of the solution to the solution, stir and decolorize at 70°C for 40 minutes, then place the crystal at 6°C for 24 hours, filter the crystal to obtain a second crystal, rinse the second crystal with ethanol, and dry it at a vacuum degree of -0.07MPa and a temperature of 65°C to obtain aucubin. HPLC was used to detect aucubin in the second crystal after drying, and the obtained HPLC spectrum was the same as that in Example 1. Figure 4 Similarly, the HPLC test results obtained were: the content of aucubin in the second crystal after drying was 98.16%, and the yield was 90.8%.

[0105] Step 5, obtaining vitexin;

[0106] Step 5.1, concentrating the second eluate to be free of alcohol at a vacuum degree of -0.07 MPa and a temperature of 65°C, adding purified water twice the mass of the concentrate to obtain a diluent, then adding ethyl acetate five times the mass of the diluent to the diluent, stirring and extracting three times, each time for 5 minutes, collecting the ethyl acetate phase and concentrating it into a paste at a vacuum degree of -0.07 MPa and a temperature of 65°C;

[0107] Step 5.2, mix the paste with 200-300 mesh silica gel in an amount that is 1 times the mass of the paste, and dry the mixture at a vacuum degree of -0.07 MPa and a temperature of 65°C to obtain the sample-mixed silica gel;

[0108] Step 5.3, use petroleum ether to wet-pack 200-300 mesh blank silica gel with a mass of 8 times the mass of the sample-mixed silica gel to obtain a silica gel chromatography column, and add the sample-mixed silica gel into the silica gel chromatography column, and then use petroleum ether and ethyl acetate mixed solvents in volume ratios of 10:1, 8:1, 8:2, and 7:3 to elute the silica gel chromatography column in sequence, and use thin layer chromatography to monitor the eluent. In the eluent section of the mixed solvent with a volume ratio of petroleum ether and ethyl acetate of 8:1, the silica gel plate shows that only a single point of vitexin is contained. The eluent is collected, and the eluent is concentrated at a vacuum degree of -0.07 MPa and a temperature of 65°C for 40 min, and then placed at 6°C for crystallization for 24 h, and then filtered to obtain wet crystals, and the wet crystals are rinsed with petroleum ether in an amount that is 1 times the mass of the wet crystals, and dried at a vacuum degree of -0.07 MPa and a temperature of 55°C to obtain vitexin. HPLC was used to detect vitexin in the dried crystals, and the obtained HPLC spectrum was consistent with that in Example 1. Figure 5 Similarly, the HPLC test results showed that the content of vitexin in the dried crystals was 98.35%, and the yield was 85.4%.

[0109] Embodiment 3

[0110] Step 1, enzymatic hydrolysis and high-voltage pulse electric field extraction;

[0111] Step 1.1, crushing 1 kg of Vitex agnus-castus dried fruit into 40 meshes, adding purified water 5 times the weight of the Vitex agnus-castus dried fruit, adjusting the pH to 4.0, then adding 0.06% of cellulase by weight of the Vitex agnus-castus dried fruit and 0.01% of pectinase by weight of the Vitex agnus-castus dried fruit, performing enzymolysis at 45° C. for 3 h, and then inactivating the enzyme at 90° C. for 20 min to obtain a slurry;

[0112] Step 1.2, add ethanol to the slurry until the concentration of ethanol is 55%, mix well to obtain a mixed solution, and then use a peristaltic pump to pump the mixed solution into the treatment chamber of the high-voltage pulse electric field at a flow rate of 12 mL / min for extraction, filter, obtain the extract and concentrate it to be alcohol-free at a vacuum degree of -0.08 MPa and a temperature of 60°C. The intensity of the high-voltage pulse electric field is 20 kV / cm and the number of pulses is 10.

[0113] Step 2, preparing chitosan clarifier;

[0114] Add glacial acetic acid in a volume of 4% of the volume of the purified water to purified water to obtain an acetic acid aqueous solution, then add chitosan to the acetic acid aqueous solution to prepare a chitosan clarifier, wherein the mass concentration of chitosan in the chitosan clarifier is 2%.

[0115] Step 3, purification by macroporous adsorption resin;

[0116] Step 3.1, loading LX-18 resin in an amount 1.5 times the mass of Vitex agnus-castus dry fruit into a chromatography column;

[0117] Step 3.2, add chitosan clarifier with a volume of 4% of the volume of the concentrated extract in step 1.2 to the concentrated extract in step 1.2, stir at 65°C for 30 minutes, filter, and after the filtrate is adsorbed by LX-18 resin, first wash with 1 column volume of purified water, then use 5 column volumes of ethanol solution with a concentration of 20% and 6 column volumes of ethanol solution with a concentration of 60% to elute in sequence until the color is light, and collect the corresponding first eluent and second eluent respectively. HPLC is used to detect aucubin and vitexin in the first eluent and the second eluent respectively, and the corresponding HPLC spectrum obtained is consistent with that in Example 1. Figure 2 and Figure 3 Similarly, the corresponding HPLC test results were as follows: the content of aucubin in the first eluate was 33.94%, and the content of vitexin was 0.021%; the content of aucubin in the second eluate was 0.026%, and the content of vitexin was 20.83%.

[0118] Step 4, obtaining aucubin;

[0119] Step 4.1, concentrating the first eluate to a specific gravity of 1.05 at a vacuum degree of -0.07 MPa and a temperature of 65°C, adding n-butanol in an amount 4 times the mass of the concentrate to the concentrate, extracting 4 times, collecting the n-butanol phase and concentrating it to a specific gravity of 1.13, then placing it at 4°C for crystallization for 24 hours, filtering the crystals, and obtaining the first crystals;

[0120] Step 4.2, add 5 times the mass of the first crystal to the first crystal in an amount of ethanol, dissolve at 70°C to obtain a solution, then add 320 activated carbon with a mass of 2% of the mass of the solution to the solution, stir and decolorize at 70°C for 50 minutes, then place the crystal at 4°C for 20 hours, filter the crystal to obtain a second crystal, rinse the second crystal with ethanol, and dry it at a vacuum degree of -0.07MPa and a temperature of 60°C to obtain aucubin. HPLC was used to detect aucubin in the second crystal after drying, and the obtained HPLC spectrum was the same as that in Example 1. Figure 4 Similarly, the HPLC test results obtained were: the content of aucubin in the second crystal after drying was 98.07%, and the yield was 89.5%.

[0121] Step 5, obtaining vitexin;

[0122] Step 5.1, concentrating the second eluate to be free of alcohol at a vacuum degree of -0.07 MPa and a temperature of 65°C, adding purified water twice the mass of the concentrate to obtain a diluent, then adding ethyl acetate five times the mass of the diluent to the diluent, stirring and extracting five times, each time for 5 minutes, collecting the ethyl acetate phase and concentrating it into a paste at a vacuum degree of -0.07 MPa and a temperature of 65°C;

[0123] Step 5.2, mix the paste with 200-300 mesh silica gel in an amount that is 1 times the mass of the paste, and dry the mixture at a vacuum degree of -0.07 MPa and a temperature of 65°C to obtain the sample-mixed silica gel;

[0124] Step 5.3, use n-hexane to wet-pack 200-300 mesh blank silica gel with a mass 10 times the mass of the sample-mixed silica gel to obtain a silica gel chromatography column, and add the sample-mixed silica gel into the silica gel chromatography column, and then use n-hexane and ethyl acetate mixed solvents in volume ratios of 10:1, 8:1, 8:2, and 7:3 to elute the silica gel chromatography column in sequence, and use thin layer chromatography to monitor the eluent. In the eluent section of the mixed solvent with a volume ratio of n-hexane and ethyl acetate of 8:1, the silica gel plate shows that only a single point of violacein is contained. The eluent is collected, and the eluent is concentrated at a vacuum degree of -0.07 MPa and a temperature of 70°C for 30 min, and then placed at 4°C for crystallization for 20 h, and then filtered to obtain wet crystals, and the wet crystals are rinsed with n-hexane with a mass 1 times the mass of the wet crystals, and dried at a vacuum degree of -0.07 MPa and a temperature of 60°C to obtain violacein. HPLC was used to detect vitexin in the dried crystals, and the obtained HPLC spectrum was consistent with that in Example 1. Figure 5 Similarly, the HPLC test results showed that the content of vitexin in the dried crystals was 98.25%, and the yield was 85.7%.

[0125] Embodiment 4

[0126] Step 1, enzymatic hydrolysis and high-voltage pulse electric field extraction;

[0127] Step 1.1, crushing 1 kg of Vitex agnus-castus dry fruit into 20 meshes, adding purified water 4 times the weight of Vitex agnus-castus dry fruit, adjusting the pH to 5.0, then adding 0.03% of cellulase by weight of Vitex agnus-castus dry fruit and 0.05% of pectinase by weight of Vitex agnus-castus dry fruit, performing enzymolysis at 55° C. for 3 h, and then inactivating the enzyme at 95° C. for 15 min to obtain a slurry;

[0128] Step 1.2, add ethanol to the slurry until the concentration of ethanol is 60%, mix well to obtain a mixed solution, and then use a peristaltic pump to pump the mixed solution into the treatment chamber of the high-voltage pulse electric field at a flow rate of 10 mL / min for extraction, filter, obtain the extract and concentrate it to be alcohol-free at a vacuum degree of -0.06 MPa and a temperature of 65°C. The intensity of the high-voltage pulse electric field is 30 kV / cm and the number of pulses is 12.

[0129] Step 2, preparing chitosan clarifier;

[0130] Add glacial acetic acid in a volume of 5% of the volume of the purified water to purified water to obtain an acetic acid aqueous solution, then add chitosan to the acetic acid aqueous solution to prepare a chitosan clarifier, wherein the mass concentration of chitosan in the chitosan clarifier is 1.5%.

[0131] Step 3, purification by macroporous adsorption resin;

[0132] Step 3.1, loading AB-8 resin in an amount equal to 1 times the mass of Vitex agnus-castus dry fruit into a chromatography column;

[0133] Step 3.2, add chitosan clarifier with a volume of 3% of the volume of the concentrated extract in step 1.2 to the concentrated extract in step 1.2, stir at 70°C for 20 minutes, filter, and after the filtrate is adsorbed by AB-8 resin, first wash with 2 column volumes of purified water, then use 4 column volumes of ethanol solution with a concentration of 20%, and 7 column volumes of ethanol solution with a concentration of 80% to elute in sequence until the color is light, and collect the corresponding first eluent and second eluent respectively. HPLC is used to detect aucubin and vitexin in the first eluent and the second eluent respectively, and the corresponding HPLC spectrum obtained is consistent with that in Example 1. Figure 2 and Figure 3 Similarly, the corresponding HPLC test results were as follows: the content of aucubin in the first eluate was 31.79%, and the content of vitexin was 0.027%; the content of aucubin in the second eluate was 0.094%, and the content of vitexin was 20.85%.

[0134] Step 4, obtaining aucubin;

[0135] Step 4.1, concentrating the first eluate to a specific gravity of 1.10 at a vacuum degree of -0.06 MPa and a temperature of 70°C, adding n-butanol in an amount 4 times the mass of the concentrate to the concentrate, extracting three times, collecting the n-butanol phase and concentrating it to a specific gravity of 1.13, then placing it at 5°C for crystallization for 24 hours, filtering the crystals, and obtaining the first crystals;

[0136] Step 4.2, add ethanol in an amount 3 times the mass of the first crystal to the first crystal, dissolve at 65°C to obtain a solution, then add 767 activated carbon in an amount of 1% of the mass of the solution to the solution, stir and decolorize at 65°C for 60 minutes, then place the crystal at 5°C for 24 hours, filter the crystal to obtain a second crystal, rinse the second crystal with ethanol, and dry it at a vacuum degree of -0.06MPa and a temperature of 70°C to obtain aucubin. HPLC was used to detect aucubin in the second crystal after drying, and the obtained HPLC spectrum was the same as that in Example 1. Figure 4 Similarly, the HPLC test results obtained were: the content of aucubin in the second crystal after drying was 98.15%, and the yield was 90.7%.

[0137] Step 5, obtaining vitexin;

[0138] Step 5.1, concentrating the second eluate to be free of alcohol at a vacuum degree of -0.08 MPa and a temperature of 70°C, adding purified water in an amount of 1 times the mass of the concentrate to obtain a dilution, then adding ethyl acetate in an amount of 3 times the mass of the dilution to the dilution, stirring and extracting 5 times, each time for 6 minutes, collecting the ethyl acetate phase and concentrating it into a paste at a vacuum degree of -0.08 MPa and a temperature of 70°C;

[0139] Step 5.2, mix the paste with 200-300 mesh silica gel in an amount 0.5 times the mass of the paste, and dry the mixture at a vacuum degree of -0.06 MPa and a temperature of 70°C to obtain the mixed silica gel;

[0140] Step 5.3, use n-hexane to wet-pack 200-300 mesh blank silica gel with a mass 10 times that of the sample-mixed silica gel to obtain a silica gel chromatography column, and add the sample-mixed silica gel into the silica gel chromatography column, and then use n-hexane and ethyl acetate mixed solvents in volume ratios of 10:1, 8:1, 8:2, and 7:3 to elute the silica gel chromatography column in sequence, and use thin layer chromatography to monitor the eluent. In the eluent section of the mixed solvent with a volume ratio of n-hexane and ethyl acetate of 8:1, the silica gel plate shows that only a single point of vitexin is contained. The eluent is collected, and the eluent is concentrated at a vacuum degree of -0.08 MPa and a temperature of 65°C for 40 min, and then placed at 5°C for crystallization for 24 h, and then filtered to obtain wet crystals, and the wet crystals are rinsed with n-hexane in an amount 0.5 times the mass of the wet crystals, and dried at a vacuum degree of -0.08 MPa and a temperature of 55°C to obtain vitexin. HPLC was used to detect vitexin in the dried crystals, and the obtained HPLC spectrum was consistent with that in Example 1. Figure 5 Similarly, the HPLC test results showed that the content of vitexin in the dried crystals was 98.36%, and the yield was 85.4%.

[0141] Embodiment 5

[0142] Step 1, enzymatic hydrolysis and high-voltage pulse electric field extraction;

[0143] Step 1.1, crushing 1 kg of Vitex agnus-castus dried fruit into 30 meshes, adding purified water 3 times the weight of Vitex agnus-castus dried fruit, adjusting the pH to 5.0, then adding 0.05% of cellulase by weight of Vitex agnus-castus dried fruit and 0.03% of pectinase by weight of Vitex agnus-castus dried fruit, performing enzymolysis at 55° C. for 2 h, and then inactivating the enzyme at 93° C. for 25 min to obtain a slurry;

[0144] Step 1.2, add methanol to the slurry until the concentration of methanol is 50%, mix well to obtain a mixed solution, and then use a peristaltic pump to pump the mixed solution into the treatment chamber of the high-voltage pulse electric field at a flow rate of 15 mL / min for extraction, filter, obtain the extract and concentrate it to be alcohol-free at a vacuum degree of -0.07 MPa and a temperature of 70°C. The intensity of the high-voltage pulse electric field is 25 kV / cm and the number of pulses is 6.

[0145] Step 2, preparing chitosan clarifier;

[0146] Add glacial acetic acid in a volume of 3% of the volume of the purified water to purified water to obtain an acetic acid aqueous solution, and then add chitosan to the acetic acid aqueous solution to prepare a chitosan clarifier, wherein the mass concentration of chitosan in the chitosan clarifier is 2.5%.

[0147] Step 3, purification by macroporous adsorption resin;

[0148] Step 3.1, loading DM130 resin in an amount 0.5 times the mass of Vitex agnus-castus dry fruit into a chromatography column;

[0149] Step 3.2, add chitosan clarifier with a volume of 1% of the volume of the concentrated extract in step 1.2 to the concentrated extract in step 1.2, stir at 75°C for 15 minutes, filter, and after the filtrate is adsorbed by DM130 resin, wash with 2 column volumes of purified water, and then use 6 column volumes of methanol solution with a concentration of 20% and 5 column volumes of methanol solution with a concentration of 70% to elute in sequence until the color is light, and collect the corresponding first eluent and second eluent respectively. HPLC is used to detect aucubin and vitexin in the first eluent and the second eluent respectively, and the corresponding HPLC spectrum obtained is consistent with that in Example 1. Figure 2 and Figure 3 Similarly, the corresponding HPLC test results were as follows: the content of aucubin in the first eluate was 32.92%, and the content of vitexin was 0.048%; the content of aucubin in the second eluate was 0.097%, and the content of vitexin was 21.41%.

[0150] Step 4, obtaining aucubin;

[0151] Step 4.1, concentrating the first eluate to a specific gravity of 1.10 at a vacuum degree of -0.08 MPa and a temperature of 60°C, adding n-butanol 6 times the mass of the concentrate to the concentrate, extracting 5 times, collecting the n-butanol phase and concentrating it to a specific gravity of 1.18, then placing it at 5°C for crystallization for 18 hours, and filtering the crystals to obtain the first crystals;

[0152] Step 4.2, add methanol 4 times the mass of the first crystal to the first crystal, dissolve at 75°C to obtain a solution, then add KF03 activated carbon with a mass of 3% of the mass of the solution to the solution, stir and decolorize at 75°C for 60 minutes, then place the crystal at 5°C for 26 hours, filter the crystal to obtain a second crystal, rinse the second crystal with methanol, and dry it at a vacuum degree of -0.08MPa and a temperature of 60°C to obtain aucubin. HPLC was used to detect aucubin in the second crystal after drying, and the obtained HPLC spectrum was the same as that in Example 1. Figure 4 Similarly, the HPLC test results obtained were: the content of aucubin in the second crystal after drying was 98.27%, and the yield was 89.3%.

[0153] Step 5, obtaining vitexin;

[0154] Step 5.1, concentrating the second eluate to be free of alcohol at a vacuum degree of -0.09 MPa and a temperature of 60°C, adding purified water 3 times the mass of the concentrate to obtain a dilution, then adding ethyl acetate 5 times the mass of the dilution to the dilution, stirring and extracting 3 times, each time for 4 minutes, collecting the ethyl acetate phase and concentrating it into a paste at a vacuum degree of -0.09 MPa and a temperature of 60°C;

[0155] Step 5.2, mix the paste with 200-300 mesh silica gel in an amount 1.5 times the mass of the paste, and dry the mixture at a vacuum degree of -0.08 MPa and a temperature of 60°C to obtain the mixed silica gel;

[0156] Step 5.3, use petroleum ether to wet-pack 200-300 mesh blank silica gel with a mass of 6 times the mass of the sample-mixed silica gel to obtain a silica gel chromatography column, and add the sample-mixed silica gel into the silica gel chromatography column, and then use petroleum ether and ethyl acetate mixed solvents in volume ratios of 10:1, 8:1, 8:2, and 7:3 to elute the silica gel chromatography column in sequence, and use thin layer chromatography to monitor the eluent. In the eluent section of the mixed solvent with a volume ratio of petroleum ether and ethyl acetate of 8:1, the silica gel plate shows that only a single point of vitexin is contained. The eluent is collected, and the eluent is concentrated at a vacuum degree of -0.09 MPa and a temperature of 60°C for 50 min, and then placed at 5°C for crystallization for 26 h, and then filtered to obtain wet crystals, and the wet crystals are rinsed with petroleum ether in an amount of 1.5 times the mass of the wet crystals, and dried at a vacuum degree of -0.06 MPa and a temperature of 50°C to obtain vitexin. HPLC was used to detect vitexin in the dried crystals, and the obtained HPLC spectrum was consistent with that in Example 1. Figure 5 Similarly, the HPLC test results showed that the content of vitexin in the dried crystals was 98.27% and the yield was 85.1%.

[0157] In Examples 1 to 5, cellulase and pectinase were provided by Angel Enzyme Co., Ltd., where the cellulase model was CE-10 and the pectinase model was DM. The high-voltage pulse electric field device was provided by Changchun Huadi Biotechnology Development Co., Ltd., and its model was LDT-100 / 4-25 Chinese medicine high-voltage pulse electric field extraction device.

[0158] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A method for jointly extracting vitexin and aucubin, characterized in that: The following steps are involved: Step 1, enzymatic hydrolysis and high-voltage pulse electric field extraction; Step 1.1, crushing the dry fruits of Vitex agnus-castus, adding purified water in an amount 3-5 times the mass of the dry fruits of Vitex agnus-castus, adjusting the pH to 4.0-5.0, then adding cellulase and pectinase, performing enzymolysis at 45-55° C. for 2-3 hours, and then inactivating the enzymes to obtain a slurry; Step 1.2, adding a first alcohol reagent to the slurry until the concentration of the first alcohol reagent is 50-60%, mixing to obtain a mixed solution, and then pumping the mixed solution into a treatment chamber of a high-voltage pulse electric field for extraction, filtering, obtaining an extract and concentrating it to be alcohol-free; the intensity of the high-voltage pulse electric field is 20-30 kV / cm, and the number of pulses is 6-12; Step 2, preparing chitosan clarifier; Step 3, purification by macroporous adsorption resin; Step 3.1, loading the macroporous adsorption resin into the chromatography column; Step 3.2, adding the chitosan clarifier prepared in step 2 to the concentrated extract in step 1.2, heating and stirring, filtering, and after the filtrate is adsorbed by the macroporous adsorption resin, first washing with purified water, and then eluting with a first alcohol solution with a concentration of 10%-20% and a second alcohol solution with a concentration of 60%-80% in sequence until the color is light, and collecting the corresponding first eluate and second eluate respectively; Step 4, obtaining aucubin; Concentrating the first eluate, adding n-butanol to the concentrate for extraction, collecting and concentrating the n-butanol phase, obtaining first crystals after crystallization, then dissolving, decolorizing and crystallizing the first crystals in sequence to obtain second crystals, rinsing the second crystals and drying them to obtain aucubin; Step 5, obtaining vitexin; The second eluate is concentrated, and ethyl acetate is added to dilute the concentrate for extraction. The ethyl acetate phase is collected and concentrated into a paste. The paste is mixed with silica gel and dried to obtain sample-mixed silica gel. The sample-mixed silica gel is then added to a prepared silica gel chromatography column and eluted with an eluent. The eluate is monitored by thin layer chromatography. When only a single point of vitexin is shown on the silica gel plate, the eluate is collected, and the eluate is successively concentrated, crystallized, rinsed, and dried to obtain vitexin.

2. The method for jointly extracting vitexin and aucubin according to claim 1, characterized in that: In step 1.1, the dry fruit of Vitex agnus-castus is crushed to 20-40 mesh; the temperature of the enzyme inactivation is 90-95° C., and the time is 15-25 min; the mass of the cellulase is 0.03-0.06% of the mass of the dry fruit of Vitex agnus-castus; the mass of the pectinase is 0.01-0.05% of the mass of the dry fruit of Vitex agnus-castus; In step 1.2, the first alcohol reagent is methanol or ethanol; a peristaltic pump is used to pump the mixed solution into a treatment chamber of a high-voltage pulse electric field at a flow rate of 10-15 mL / min for extraction; the extract is concentrated to be alcohol-free at a vacuum degree of -0.08 to -0.06 MPa and a temperature of 60-70°C.

3. The method for jointly extracting vitexin and aucubin according to claim 2, characterized in that: Step 3.1 specifically comprises loading a macroporous adsorption resin in an amount 0.5-1.5 times the mass of the dry fruit of Vitex agnus-castus into a chromatography column; the macroporous adsorption resin is AB-8 resin, LX-38 resin, LX-18 resin, LX-5 resin or DM130 resin; In step 3.2, the volume of the chitosan clarifier is 1%-4% of the volume of the concentrated extract in step 1.2; the heating temperature is 65-75°C; the stirring time is 15-30min; the amount of purified water is 1-2 chromatography column volumes; the first alcohol solution and the second alcohol solution are methanol solution or ethanol solution, the amount of the first alcohol solution is 4-6 chromatography column volumes, and the amount of the second alcohol solution is 5-7 chromatography column volumes.

4. The method for jointly extracting vitexin and aucubin according to any one of claims 1 to 3, characterized in that: Step 4 specifically includes the following steps: Step 4.1, concentrating the first eluate, adding n-butanol in an amount 4-6 times the mass of the concentrate to the concentrate, extracting 3-5 times, collecting the n-butanol phase and concentrating it to a specific gravity of 1.13-1.18, then placing it for crystallization, and filtering the crystals to obtain first crystals; Step 4.2, add a second alcohol reagent in an amount 3-5 times the mass of the first crystal to the first crystal, dissolve at 65-75° C. to obtain a solution, then add activated carbon in an amount of 1%-3% of the mass of the solution to the solution, stir and decolorize at 65-75° C. for 40-60 minutes, then allow the crystal to stand, filter the crystal to obtain a second crystal, rinse the second crystal with a third alcohol reagent, and then dry to obtain aucubin.

5. The method for jointly extracting vitexin and aucubin according to claim 4, characterized in that: Step 5 specifically includes the following steps: Step 5.1, concentrating the second eluate until it is free of alcohol, adding purified water in an amount 1-3 times the mass of the concentrate to obtain a diluent, then adding ethyl acetate in an amount 3-5 times the mass of the diluent to the diluent, extracting 3-5 times, each time for 4-6 minutes, collecting the ethyl acetate phase and concentrating it into a paste; Step 5.2, mixing the paste with 200-300 mesh silica gel in an amount of 0.5-1.5 times the mass of the paste and drying to obtain the sample mixing silica gel; Step 5.3, take 200-300 mesh blank silica gel with a mass of 6 to 10 times the mass of the sample-mixed silica gel for wet column packing to obtain a silica gel chromatography column, and add the sample-mixed silica gel into the silica gel chromatography column, and then elute the silica gel chromatography column with an eluent, and monitor the eluate by thin layer chromatography at the same time, collect the eluate when only a single point of violacein is shown on the silica gel plate, concentrate the eluate in turn, let it stand for crystallization, filter the crystals, rinse, and vacuum dry to obtain violacein.

6. The method for jointly extracting vitexin and aucubin according to claim 5, characterized in that: Step 2 is as follows: Add glacial acetic acid in a volume of 3%-5% of the volume of purified water to purified water to obtain an acetic acid aqueous solution, then add chitosan to the acetic acid aqueous solution to prepare a chitosan clarifier; the mass concentration of chitosan in the chitosan clarifier is 1.5%-2.5%.

7. The method for jointly extracting vitexin and aucubin according to claim 6, characterized in that: Step 4.1 is specifically, concentrating the first eluate to a specific gravity of 1.05-1.10 at a vacuum degree of -0.08 to -0.06 MPa and a temperature of 60-70°C, adding n-butanol in an amount 4-6 times the mass of the concentrated solution to the concentrated solution, extracting 3-5 times, collecting the n-butanol phase and concentrating it to a specific gravity of 1.13-1.18, then placing the crystals at 4-6°C for 18-24 hours, and filtering the crystals to obtain the first crystals; In step 4.2, the activated carbon is 320 activated carbon, 767 activated carbon, or KF03 activated carbon; the placing the crystallization specifically refers to placing the crystallization at 4-6°C for 20-26h; the drying specifically refers to drying at a vacuum degree of -0.08 to -0.06MPa and a temperature of 60-70°C; the second alcohol reagent and the third alcohol reagent are methanol or ethanol; Step 5.1 is specifically, concentrating the second eluate to be alcohol-free at a vacuum degree of -0.09 to -0.07 MPa and a temperature of 60-70°C, adding purified water in an amount 1-3 times the mass of the concentrated solution to the concentrated solution to obtain a diluent, then adding ethyl acetate in an amount 3-5 times the mass of the diluent to the diluent, extracting 3-5 times, each time for 4-6 minutes, collecting the ethyl acetate phase and concentrating it into a paste at a vacuum degree of -0.09 to -0.07 MPa and a temperature of 60-70°C; In step 5.2, the drying specifically refers to drying at a vacuum degree of -0.08 to -0.06 MPa and a temperature of 60-70°C; Step 5.3 is as follows: n-hexane is used to wet-pack a 200-300 mesh blank silica gel with a mass of 6 to 10 times the mass of the sample-mixed silica gel to obtain a silica gel chromatography column, and the sample-mixed silica gel is added to the silica gel chromatography column. Then, the silica gel chromatography column is eluted in sequence with a mixed solvent of n-hexane and ethyl acetate in a volume ratio of 10:1, 8:1, 8:2, and 7:3, respectively, and the eluent is monitored by thin layer chromatography. When the silica gel plate shows that only spike flower is present, When vitexin is single point, the eluate is collected, and the eluate is concentrated at a vacuum degree of -0.09 to -0.07 MPa and a temperature of 60-70°C for 30-50 minutes, and then placed at 4-6°C for crystallization for 20-26 hours, and then filtered to obtain wet crystals, and the wet crystals are rinsed with n-hexane in an amount of 0.5 to 1.5 times the mass of the wet crystals, and dried at a vacuum degree of -0.08 to -0.06 MPa and a temperature of 50-60°C to obtain vitexin; Alternatively, petroleum ether is used to wet-pack a 200-300 mesh blank silica gel with a mass of 6 to 10 times the mass of the sample-mixed silica gel to obtain a silica gel chromatography column, and the sample-mixed silica gel is added to the silica gel chromatography column, and then the silica gel chromatography column is sequentially eluted with a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 10:1, 8:1, 8:2, and 7:3, respectively, and the eluent is monitored by thin layer chromatography. When the silica gel plate shows that only vitexin is present, The eluate is collected at a single point, concentrated at a vacuum degree of -0.09 to -0.07 MPa and a temperature of 60-70°C for 30-50 min, and then placed at 4-6°C for crystallization for 20-26 h, and then filtered to obtain wet crystals, and the wet crystals are rinsed with petroleum ether in an amount 0.5-1.5 times the mass of the wet crystals, and dried at a vacuum degree of -0.08 to -0.06 MPa and a temperature of 50-60°C to obtain vitexin.

8. The method for jointly extracting vitexin and aucubin according to claim 7, characterized in that: Step 4.1 is specifically, concentrating the first eluate to a specific gravity of 1.08 at a vacuum degree of -0.07 MPa and a temperature of 65°C, adding n-butanol in an amount 5 times the mass of the concentrated solution to the concentrated solution, extracting 4 times, collecting the n-butanol phase and concentrating it to a specific gravity of 1.15, then placing it at 5°C for crystallization for 20 hours, and filtering the crystals to obtain the first crystals; Step 4.2 is specifically, adding 5 times the mass of the first crystal to the first crystal ethanol, dissolving at 70 ° C to obtain a solution, then adding 767 activated carbon with a mass of 2% of the mass of the solution to the solution, stirring and decolorizing at 70 ° C for 50 minutes, then placing the crystal at 5 ° C for 24 hours, filtering the crystal to obtain a second crystal, rinsing the second crystal with ethanol, and drying at a vacuum degree of -0.07 MPa and a temperature of 65 ° C to obtain aucubin; Step 5.1 is specifically, concentrating the second eluate to be free of alcohol at a vacuum degree of -0.07 MPa and a temperature of 65°C, adding purified water twice the mass of the concentrated solution to the concentrated solution to obtain a diluent, then adding ethyl acetate four times the mass of the diluent to the diluent, extracting four times, each time for 5 minutes, collecting the ethyl acetate phase and concentrating it into a paste at a vacuum degree of -0.07 MPa and a temperature of 65°C; Step 5.2 is specifically, mixing the paste with 200-300 mesh silica gel in an amount that is 1 times the mass of the paste, and drying the mixture at a vacuum degree of -0.07 MPa and a temperature of 65°C to obtain the sample-mixed silica gel; Specifically, step 5.3 is to use n-hexane to wet-pack 200-300 mesh blank silica gel with a mass of 8 times the mass of the sample-mixed silica gel to obtain a silica gel chromatography column, and add the sample-mixed silica gel into the silica gel chromatography column, and then use n-hexane and ethyl acetate mixed solvents in volume ratios of 10:1, 8:1, 8:2, and 7:3 to elute the silica gel chromatography column in sequence, and monitor the eluent by thin layer chromatography. In the eluent section of the mixed solvent with a volume ratio of n-hexane and ethyl acetate of 8:1, the silica gel plate shows that only a single point of violacein is contained. The eluent is collected, and the eluent is concentrated at a vacuum degree of -0.07 MPa and a temperature of 65°C for 40 minutes, and then placed at 5°C for crystallization for 24 hours, and then filtered to obtain wet crystals, and the wet crystals are rinsed with n-hexane with a mass of 1 times the mass of the wet crystals, and dried at a vacuum degree of -0.07 MPa and a temperature of 55°C to obtain violacein.

9. The method for jointly extracting vitexin and aucubin according to claim 8, characterized in that: Step 2 is as follows: Add glacial acetic acid in a volume of 4% of the volume of purified water to purified water to obtain an acetic acid aqueous solution, then add chitosan to the acetic acid aqueous solution to prepare a chitosan clarifier; the mass concentration of chitosan in the chitosan clarifier is 2%.

10. The method for combined extraction of vitexin and aucubin according to claim 3, characterized in that: Step 1.1 is specifically as follows: crushing the dry fruit of Vitex agnus-castus into 40 meshes, adding purified water in an amount of 4 times the mass of the dry fruit of Vitex agnus-castus, adjusting the pH to 4.5, then adding 0.05% of cellulase by mass of the dry fruit of Vitex agnus-castus and 0.03% of pectinase by mass of the dry fruit of Vitex agnus-castus, performing enzymolysis at 50° C. for 2.5 hours, and then inactivating the enzyme at 95° C. for 20 minutes to obtain a slurry; Step 1.2 specifically comprises adding ethanol to the slurry until the concentration of ethanol is 55%, mixing to obtain a mixed solution, and then using a peristaltic pump to pump the mixed solution into a treatment chamber of a high-voltage pulse electric field at a flow rate of 12 mL / min for extraction, filtering, obtaining an extract, and concentrating the extract at a vacuum degree of -0.07 MPa and a temperature of 65°C until there is no alcohol; the intensity of the high-voltage pulse electric field is 25 kV / cm, and the number of pulses is 10; Step 3.1 specifically comprises loading LX-38 resin in an amount equal to 1 times the mass of Vitex agnus-castus dry fruit into a chromatography column; Specifically, step 3.2 is to add a chitosan clarifier with a volume of 2% of the volume of the concentrated extract in step 1.2 to the concentrated extract in step 1.2, stir at 70°C for 20 minutes, filter, and adsorb the filtrate with LX-38 resin. Then, wash the filtrate with 1 column volume of purified water, and then use 5 column volumes of 15% ethanol solution and 6 column volumes of 75% ethanol solution to elute until the color is light, and collect the corresponding first eluate and second eluate respectively.

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