CN114209727B - Method for extracting total flavonoids from Hangzhou white chrysanthemum - Google Patents
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Abstract
The invention discloses an extraction method of Hangzhou white chrysanthemum total flavonoids, which comprises the steps of carrying out ultrasonic extraction after enzymolysis of cellulase in Hangzhou white chrysanthemum ethanol aqueous solution, carrying out continuous dynamic countercurrent extraction, microfiltration, ultrafiltration, LS-806B column chromatography and macroporous adsorption resin chromatography to obtain Hangzhou white chrysanthemum total flavonoids. The invention adopts the cellulase pretreatment, can fully hydrolyze the cell wall of the Hangzhou white chrysanthemum, so that the flavonoid substances are fully released; the dynamic continuous countercurrent extraction technology is adopted, the effective components are continuously extracted through the circulated extracting solution, the extraction period is shortened, and the solvent consumption is reduced. And membrane separation is carried out by adopting microfiltration and ultrafiltration technologies, so that the purity of the product is improved. The invention has simple process, simple equipment, easily obtained and safe reagent, low production cost and easy realization of industrialization.
Description
Field of the art
The invention relates to the field of separation and extraction, in particular to a method for extracting total flavonoids of Hangzhou white chrysanthemum.
(II) background art
Flos Chrysanthemi, also known as flos Chrysanthemi, tea flos Chrysanthemi, medicinal flos Chrysanthemi, and small Shang Huang, is the best of flos Chrysanthemi tea, and is the traditional tea flos Chrysanthemi in China. The petals are white like jade, the pistil is bright like gold, the color, the aroma and the taste are elegant, the flavor is sweet and cool, the medicine and the food are homologous, and the flower is a rare product in the chrysanthemum. Hangzhou white chrysanthemum is one of the main economy of Zhejiang province, and although the economic benefit is created, byproducts such as stems and leaves generated in the processes of planting, processing, circulation and consumption cannot be effectively utilized.
For the production of high value-added products from these agricultural wastes, a green and safe extraction method is expected. And (3) carrying out systematic integration on a plurality of separation technologies such as continuous dynamic countercurrent extraction, target column separation, nano-membrane separation and the like, so as to realize high integration, automation and greenization of the production process of the plant active ingredients. Compared with the traditional solvent extraction method, the method can simultaneously recover a plurality of active ingredients by one-step treatment of one raw material, has simple subsequent purification process and high product quality, and simultaneously has the advantages of less use of organic solvents, reduction of emission of organic volatile matters, reduction of energy consumption, reduction of investment of fixed equipment and the like. Integrates multiple technologies and methods into a whole for separation and purification, not only effectively overcomes the pollution of organic solvents to human beings and the environment, but also improves the separation efficiency and the yield of products.
In order to improve the extraction rate of bioactive substances, the invention introduces various separation technologies such as continuous dynamic countercurrent extraction, target column separation, nano-membrane separation and the like into the fields of agricultural byproduct processing and functional natural product extraction, and the implementation of the technology is beneficial to reducing the emission of industrial wastes and has stronger operability.
(III) summary of the invention
The invention aims to provide the method for extracting the total flavonoids of Hangzhou white chrysanthemum, which can not only keep the stability of active substances, but also improve the extraction rate, is beneficial to reducing the emission of industrial wastes and has stronger operability.
The technical scheme adopted by the invention is as follows:
the invention provides a method for extracting total flavonoids from Hangzhou white chrysanthemum, which comprises the following steps:
(1) Pretreatment: drying flos Chrysanthemi, and pulverizing to obtain flos Chrysanthemi powder; adding Hangzhou white inulin powder into ethanol water solution with the volume concentration of 50-70%, stirring at room temperature to fully swell Hangzhou white chrysanthemum cells, adjusting the temperature to 45-65 ℃, adjusting the pH value to 4.0-5.5, adding cellulase, and carrying out enzymolysis for 30-60min at 45-65 ℃ to obtain an enzymolysis material;
(2) Continuous dynamic countercurrent extraction: adopting tank group type dynamic extraction, adding the materials subjected to enzymolysis in the step (1) into each tank on average, extracting 3 times at 40-60 ℃ and 40-100rpm for 30-40min each time by taking ethanol water solution with the volume concentration of 50-70% as a solvent, conveying the extracted solvent along the raw material solute concentration from low to high through each tank for multiple times, and collecting the first extracting solution of each tank to obtain countercurrent extracting solution;
(3) Membrane separation: concentrating the countercurrent extract in the step (2) under reduced pressure to recover ethanol, centrifuging (preferably at 5000rpm for 20 min), collecting supernatant, and sequentially performing microfiltration and ultrafiltration to obtain filtrate;
(4) Removing pesticide residues: performing column chromatography separation by using LS-806B special resin, loading the filtrate obtained in the step (3) at the speed of 2-4BV/h (BV is the bed volume of the resin.BV/h is the volume of the bed flowing out per hour), performing gradient elution by using 30% -70% methanol aqueous solution with the volume concentration of 1-3BV/h and the elution amount of 8-10BV, detecting the content of total flavonoids in the effluent by using a spectrophotometry (rutin is used as a standard substance), collecting the eluent with the mass content of flavonoids of more than 10%, and concentrating under reduced pressure until the volume concentration of methanol is less than 5%, thereby obtaining a concentrated solution for removing pesticide residues;
(5) Column chromatography: loading the concentrated solution prepared in the step (4) into a macroporous adsorption resin chromatographic column at the speed of 2-4BV/h, washing with water until the lower injection is nearly colorless after the adsorption is completed, discarding the water washing liquid, eluting with 60-70% ethanol water solution at the eluting speed of 1-3BV/h and the eluting amount of 8-10BV, collecting ethanol eluent, concentrating under reduced pressure until the ethanol eluent is dry, and obtaining the Hangzhou white chrysanthemum total flavonoids.
Further, the Hangzhou white chrysanthemum in the step (1) comprises low-grade diapause Hangzhou white chrysanthemum, stem and leaf pruning and powder crushing. The Hangzhou white chrysanthemum powder is obtained by baking Hangzhou white chrysanthemum at 105 ℃ for 2-4 hours, crushing (preferably putting the Hangzhou white chrysanthemum powder into a high-speed multifunctional crusher to be ground into powder) and sieving the Hangzhou white chrysanthemum powder with a 80-120-mesh sieve.
Further, the volume consumption of the 50-70% ethanol aqueous solution in the step (1) is 6-12mL/g based on the mass of Hangzhou white inulin powder. The enzyme activity of the cellulase is 10000U/g, and the mass addition amount of the cellulase is 0.1-0.3% of the weight of Hangzhou white inulin powder.
Further, the volume consumption of the ethanol water solution with the volume concentration of 50-70% in the step (2) is 6-12mL/g based on the weight of the Hangzhou white inulin powder in the step (1), and the total volume consumption of the ethanol water solution with the volume concentration of 50-70% in the step (2) and the step (1) is 15-20mL/g based on the weight of the Hangzhou white inulin powder in the step (1).
Further, the tank group type dynamic extraction method in the step (2) comprises the following steps: adding the materials subjected to enzymolysis in the step (1) into each extraction tank on average, extracting three times at 40-60 ℃ for 30-40min each time, stirring at 40-100 r/min, adding 50-70% ethanol water solution by volume concentration into the first extraction tank for the first time, so that the feed-liquid ratio is 1:5-20, and adding the ethanol water solution with the same concentration into the second and third extraction tanks respectively, wherein the total volume is the same as that of the first extraction. Filtering the first extracted extracting solution in the first extracting tank, putting the extracting solution in a reserve tank for standby, taking the extracting solution extracted in the second extracting tank as the first extracted solvent in the second extracting tank, filtering the extracting solution for standby after extraction, taking the extracting solution extracted in the third extracting tank as the second extracted solvent in the second extracting tank, taking the extracting solution obtained in the extraction as the first extracted solvent in the third extracting tank, namely, adding the extracting solution in the N th extracting tank into the extracting solution in the N+1th extracting tank, wherein the ratio of the extracting solution in each extracting tank is 1:5-20, supplementing the extracting solution in the first extracting tank with ethanol water solution with the same concentration, and collecting the extracting solution extracted in the first extracting tank to obtain the countercurrent extracting solution.
Further, 6 tanks are arranged in the tank group type dynamic extraction in the step (2).
And (3) carrying out microfiltration at 40-50 ℃ by adopting a polysulfone membrane or a sulfonated polysulfone membrane with the pore diameter of 0.2-0.45 mu m. The ultrafiltration adopts a polyether sulfone membrane or a polysulfone membrane with the aperture of 5-10nm, and the ultrafiltration is carried out at the temperature of 35-45 ℃ and the pressure of 0.1-0.5Mpa and the membrane surface flow rate of 2-2.5 m/s.
Further, the volume concentration of the methanol aqueous solution for gradient elution in the step (4) is 30%, 40%, 50%, 60% and 70%.
Further, the macroporous adsorption resin in the step (4) is ADS-7, AB-8, LK001, polyamide or D-101.
Compared with the prior art, the invention has the beneficial effects that:
(1) The invention adopts cellulase pretreatment, can fully hydrolyze the cell wall of Hangzhou white chrysanthemum, so that flavonoid substances are fully released, and the comparative example 1 is that the extraction rate is reduced from 54.1g to 46.3g on the basis of the example 4 without enzymolysis.
(2) The invention adopts dynamic continuous countercurrent extraction technology, continuously extracts active ingredients through circulating extracting solution, shortens the extraction period, reduces the solvent consumption, and the extraction rate is reduced from 54.1g to 48.7g by changing the dynamic countercurrent extraction into the common tank extraction based on the embodiment 4 in the comparative example 2.
(3) The invention adopts micro-filtration and ultrafiltration technology to carry out membrane separation, thereby improving the purity of the product.
(4) The invention has simple process, simple equipment, easily obtained and safe reagent, low production cost and easy realization of industrialization.
(IV) detailed description of the invention
The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:
unless otherwise indicated, all reagents and starting materials are commercially available or are commonly used in the industry and, unless otherwise indicated, are conventional in the art. The room temperature is 25-30 ℃. The flos Chrysanthemi is selected from Compositae, crowndaisy chrysanthemum, and flos Chrysanthemi, and contains volatile oil 0.13%, chrysin, adenine, choline, stachydrine, trace vitamin A, vitamin B, amino acid, and sophorae radix alkali. The low-grade diapause Hangzhou white chrysanthemum refers to flowers which fall before picking and other products which are smaller and not easy to sell. The chrysanthemum morifolium ramat stem and leaf pruning refers to some fallen leaves and some branches cut off during the period. The flos Chrysanthemi powder refers to some waste petals left after picking and packaging. The enzyme activity of the cellulase is 10000U/g.
The high-speed multifunctional pulverizer is FW10001A, and is used for the national institute of advanced Tauroda in Changzhou city. The tank group type dynamic countercurrent extraction adopts 6 extraction tanks, and the volume of each extraction tank is 20L. LS-806B proprietary resin was purchased from Siemens blue deep specialty resins Co.
In the embodiment of the invention, the spectrophotometry is adopted to detect the light absorption value at 510nm, rutin is used as a standard substance to prepare a standard curve, and the curve equation is y=2.4609x+0.0094 and R 2 =0.9994, total flavone content was obtained according to the curve equation.
Example 1
(1) Pretreatment of raw materials: baking low-grade Hangzhou white chrysanthemum at 105 ℃ for 2 hours, grinding into powder in a high-speed multifunctional grinder, and sieving with a 80-mesh sieve to obtain Hangzhou white chrysanthemum powder; adding 1kg of Hangzhou white inulin powder into 6L of 50% ethanol water solution with volume concentration, wherein the ratio of feed to liquid is 1:6, stirring at room temperature, fully swelling Hangzhou white chrysanthemum cells, heating to 65 ℃, adjusting the pH value to 4.0, adding 1g of cellulase, and carrying out enzymolysis for 40min at 65 ℃ to obtain a material after enzymolysis;
(2) Continuous dynamic countercurrent extraction: dividing the material subjected to enzymolysis in the step (1) into 6 equal parts, respectively adding the equal parts into 6 extraction tanks, extracting each tank for three times at 40 ℃, wherein the extraction time is 40min each time, the stirring frequency is 100 revolutions per minute, adding 1.5L of 50% ethanol aqueous solution by volume concentration into the first extraction tank, and respectively adding the ethanol aqueous solution with the same concentration into the second extraction tank and the third extraction tank, wherein the total volume is the same as that of the first extraction tank. Filtering the first extractive solution of the first extraction tank, placing into a reserve tank for standby, taking the second extractive solution as the first extractive solvent of the second extraction tank, filtering the second extractive solution for standby, taking the third extractive solution as the second extractive solvent of the second extraction tank, taking the obtained extractive solution as the first extractive solvent of the third extraction tank, and the like until the 6 th tank, collecting the first extractive solution of each extraction tank to obtain countercurrent extractive solution.
(3) Membrane separation: recovering ethanol by rotary evaporation of the countercurrent extract in the step (2), centrifuging at 5000rpm for 20min, collecting supernatant, performing microfiltration at 40deg.C by adopting polysulfone membrane with pore diameter of 0.2 μm, and performing ultrafiltration at 40deg.C and pressure of 0.1Mpa by adopting polyether sulfone membrane with pore diameter of 5nm to obtain filtrate;
(4) Removing pesticide residues: performing column chromatography separation (column height is 500mm and diameter is 20 mm) by adopting LS-806B special resin, loading the filtered liquid in the step (3) at the speed of 3BV/h, performing gradient elution by using 30%, 40%, 50%, 60% and 70% methanol water solutions with the volume concentration of 2BV/h, detecting the content of total flavonoids in the effluent liquid by adopting a spectrophotometry method, collecting the eluent with the mass content of flavonoids of more than 10%, concentrating under reduced pressure until the volume content of methanol is 1.1%, and obtaining concentrated liquid for removing pesticide residues, wherein the pesticide residues of the concentrated liquid reach undetected standards (namely <10 ppb).
(5) Column chromatography: and (3) loading the concentrated solution prepared in the step (4) to an ADS-7 macroporous adsorption resin chromatographic column (20 mm multiplied by 500 mm) at the speed of 3BV/h, washing with water until the lower liquid is nearly colorless after the adsorption is finished, discarding the water washing liquid, eluting with 60% ethanol water solution at the volume concentration of 2BV/h, collecting all ethanol eluent at the eluting speed of 9BV, and concentrating under reduced pressure to dryness to obtain 52.7g of Hangzhou white chrysanthemum total flavonoids.
Example 2
(1) Pretreatment of raw materials: taking chrysanthemum morifolium ramat, pruning stems and leaves, baking for 2 hours at 105 ℃, putting into a high-speed multifunctional crusher, grinding into powder, and sieving with a 100-mesh sieve to obtain chrysanthemum morifolium powder; adding 1kg of Hangzhou white inulin powder into 10L of 60% ethanol water solution with volume concentration, wherein the ratio of feed to liquid is 1:10, stirring at room temperature, fully swelling Hangzhou white chrysanthemum cells, adjusting the pH value to 4.5 at 50 ℃, adding 2g of cellulase, and carrying out enzymolysis for 35min at 50 ℃ to obtain an enzymolysis material;
(2) Continuous dynamic countercurrent extraction: dividing the material subjected to enzymolysis in the step (1) into 6 equal parts, respectively adding the equal parts into 6 extraction tanks, extracting each tank for three times at 50 ℃, wherein the extraction time is 40min each time, the stirring frequency is 80 revolutions per minute, adding 1.67L of 60% ethanol aqueous solution by volume concentration into the first extraction tank, and respectively adding the ethanol aqueous solution with the same concentration into the second extraction tank and the third extraction tank, wherein the total volume is the same as that of the first extraction tank. Filtering the first extractive solution of the first extraction tank, placing into a reserve tank for standby, taking the second extractive solution as the first extractive solvent of the second extraction tank, filtering the second extractive solution for standby, taking the third extractive solution as the second extractive solvent of the second extraction tank, taking the obtained extractive solution as the first extractive solvent of the third extraction tank, and the like until the 6 th tank, collecting the first extractive solution of each extraction tank to obtain countercurrent extractive solution.
(3) Membrane separation: recovering ethanol from the countercurrent extraction solution prepared in the step (2) by rotary evaporation, centrifuging at 5000rpm for 20min, collecting supernatant, performing microfiltration at 40deg.C by adopting polysulfone membrane with pore diameter of 0.45 μm, and performing ultrafiltration at 40deg.C and pressure of 0.2Mpa by adopting polysulfone membrane with pore diameter of 10nm to obtain filtrate;
(4) Removing pesticide residues: performing column chromatography separation (column height is 500mm and diameter is 20 mm) by adopting LS-806B special resin, loading the filtered liquid in the step (3) at the speed of 4BV/h, performing gradient elution by using 30%, 40%, 50%, 60% and 70% methanol water solutions with the volume concentration of 3BV/h and the elution amount of 10BV, detecting the content of total flavonoids in the effluent liquid by adopting a spectrophotometry method described in the embodiment 1, collecting the eluent with the flavone mass content of more than 10%, concentrating under reduced pressure until the methanol mass content is 1.4%, and obtaining concentrated liquid for removing pesticide residues, wherein the pesticide residues of the concentrated liquid reach undetected standards (namely <10 ppb).
(5) Column chromatography: loading the concentrated solution prepared in the step (4) to an AB-8 macroporous adsorption resin chromatographic column (20 mm multiplied by 500 mm) at a speed of 3BV/h, washing with water until the lower liquid is nearly colorless after the adsorption is finished, discarding the water washing liquid, eluting with 60% ethanol water solution with a volume concentration, eluting at a speed of 3BV/h, eluting with an amount of 9BV, collecting ethanol eluent, concentrating under reduced pressure to dryness, and identifying by adopting a method of an example 1 to obtain 27.4g of Hangzhou white chrysanthemum total flavonoids.
Example 3
(1) Pretreatment of raw materials: baking flos Chrysanthemi powder at 105deg.C for 2 hr, grinding into powder in high-speed multifunctional pulverizer, and sieving with 100 mesh sieve to obtain flos Chrysanthemi powder; adding 12L of 70% ethanol water solution with volume concentration of 1:12 into 1kg of Hangzhou white inulin powder, stirring at room temperature, fully swelling Hangzhou white chrysanthemum cells, adjusting the pH value to 4.0 at 45 ℃, adding 3g of cellulase, and carrying out enzymolysis at 45 ℃ for 50min to obtain an enzymolysis material;
(2) Continuous dynamic countercurrent extraction: dividing the material subjected to enzymolysis in the step (1) into 6 equal parts, respectively adding the equal parts into 6 extraction tanks, extracting each tank for three times at 50 ℃, wherein the extraction time is 30min each time, the stirring frequency is 100 revolutions per minute, adding 1L of 70% ethanol aqueous solution by volume concentration into the first extraction tank, and adding the ethanol aqueous solution by the same concentration into the second extraction tank and the third extraction tank respectively, wherein the total volume is the same as that of the first extraction tank. Filtering the first extractive solution of the first extraction tank, placing into a reserve tank for standby, taking the second extractive solution as the first extractive solvent of the second extraction tank, filtering the second extractive solution for standby, taking the third extractive solution as the second extractive solvent of the second extraction tank, taking the obtained extractive solution as the first extractive solvent of the third extraction tank, and the like until the 6 th tank, collecting the first extractive solution of each extraction tank to obtain countercurrent extractive solution.
(3) Membrane separation: recovering ethanol by rotary evaporation of the countercurrent extract in the step (2), centrifuging at 5000rpm for 20min, collecting supernatant, micro-filtering with sulfonated polysulfone membrane with pore diameter of 0.2 μm at 40deg.C, collecting micro-filtrate with polyether sulfone membrane with pore diameter of 10nm, and ultrafiltering at 40deg.C under pressure of 0.5Mpa to obtain filtrate;
(4) Removing pesticide residues: performing column chromatography separation (column height is 500mm and diameter is 20 mm) by adopting LS-806B special resin, loading the filtered liquid in the step (3) at the speed of 4BV/h, performing gradient elution by using 30%, 40%, 50%, 60% and 70% methanol water solutions with the volume concentration of 3BV/h and the elution amount of 10BV, detecting the content of total flavonoids in the effluent liquid by adopting a spectrophotometry method described in the embodiment 1, collecting the eluent with the mass content of flavonoids of more than 10%, concentrating under reduced pressure until the methanol content is 1.4%, and obtaining concentrated liquid for removing pesticide residues, wherein the pesticide residues of the concentrated liquid reach undetected standards (namely <10 ppb).
(5) Column chromatography: loading the concentrated solution prepared in the step (4) to a D-101 macroporous adsorption resin chromatographic column (20 mm multiplied by 500 mm) at the speed of 4BV/h, washing with water until the lower liquid is nearly colorless after the adsorption is finished, discarding the water washing liquid, eluting with 60% ethanol water solution with the volume concentration at the eluting speed of 3BV/h and the eluting amount of 10BV, collecting ethanol eluent, concentrating under reduced pressure until the ethanol eluent is dried, and identifying 48.2g of Hangzhou white chrysanthemum total flavonoids by adopting the method of the embodiment 1.
Example 4
(1) Pretreatment of raw materials: baking low-grade Hangzhou white chrysanthemum at 105 ℃ for 2 hours, grinding into powder in a high-speed multifunctional grinder, and sieving with a 100-mesh sieve to obtain Hangzhou white chrysanthemum byproduct powder; adding 1kg of Hangzhou white inulin powder into 10L of 70% ethanol water solution with volume concentration, wherein the ratio of feed to liquid is 1:20, stirring at room temperature, fully swelling Hangzhou white chrysanthemum cells, adjusting the pH value to 4.5 at 50 ℃, adding 3g of cellulase, and carrying out enzymolysis for 40min at 50 ℃ to obtain an enzymolysis material;
(2) Continuous dynamic countercurrent extraction: dividing the material obtained after ultrasonic treatment in the step (1) into 6 equal parts, respectively adding the equal parts into 6 extraction tanks, extracting each tank for three times at 60 ℃, wherein the time of each extraction is 40min, the stirring frequency is 80 r/min, the first extraction tank is used for adding 1.67L of ethanol aqueous solution with the volume concentration of 50-70%, and the second and third extraction are respectively used for adding ethanol aqueous solution with the same concentration, and the total volume is the same as that of the first extraction. Filtering the first extractive solution of the first extraction tank, placing into a reserve tank for standby, taking the second extractive solution as the first extractive solvent of the second extraction tank, filtering the second extractive solution for standby, taking the third extractive solution as the second extractive solvent of the second extraction tank, taking the obtained extractive solution as the first extractive solvent of the third extraction tank, and the like until the 6 th tank, collecting the first extractive solution of each extraction tank to obtain countercurrent extractive solution.
(3) Membrane separation: recovering ethanol from the countercurrent extract prepared in the step (2) by rotary evaporation, centrifuging at 5000rpm for 20min, collecting supernatant, performing microfiltration at 40deg.C by adopting polysulfone membrane with pore diameter of 0.45 μm, collecting micro-filtrate, and performing ultrafiltration at 40deg.C and pressure of 0.5Mpa by adopting polysulfone membrane with pore diameter of 10nm to obtain filtrate;
(4) Removing pesticide residues: performing column chromatography separation (column height is 500mm and diameter is 20 mm) by adopting LS-806B special resin, loading the filtered liquid in the step (3) at the speed of 3BV/h, performing gradient elution by using 30%, 40%, 50%, 60% and 70% methanol water solutions with the volume concentration of 2BV/h and the elution amount of 9BV, detecting the content of total flavonoids in the effluent liquid by adopting a spectrophotometry method described in the embodiment 1, collecting the eluent with the mass content of flavonoids of more than 10%, concentrating under reduced pressure until the volume content of methanol is 1.6%, and obtaining concentrated liquid for removing pesticide residues, wherein the pesticide residues of the concentrated liquid reach undetected standards (namely <10 ppb).
(5) Column chromatography: loading the concentrated solution prepared in the step (4) to an AB-8 macroporous adsorption resin chromatographic column (20 mm multiplied by 500 mm) at the speed of 4BV/h, washing with water until the lower liquid is nearly colorless after the adsorption is finished, discarding the water washing liquid, eluting with 70% ethanol water solution at the volume concentration of 3BV/h, eluting with the amount of 9BV, collecting ethanol eluent, concentrating under reduced pressure until the ethanol eluent is dry, and identifying 54.1g of Hangzhou white chrysanthemum total flavonoids by adopting the method of the embodiment 1.
Comparative example 1
(1) Pretreatment of raw materials: baking low-grade Hangzhou white chrysanthemum at 105 ℃ for 2 hours, grinding into powder in a high-speed multifunctional grinder, and sieving with a 100-mesh sieve to obtain Hangzhou white chrysanthemum powder; adding 1kg of Hangzhou white inulin powder into 10L of 70% ethanol water solution with volume concentration, wherein the ratio of feed to liquid is 1:20, stirring at room temperature, and fully swelling Hangzhou white chrysanthemum cells;
(2) Continuous dynamic countercurrent extraction: dividing the swelled material in the step (1) into 6 equal parts, respectively adding the equal parts into 6 extraction tanks, extracting three times at 60 ℃ in each tank, wherein the extraction time is 40min each time, the stirring frequency is 80 revolutions per minute, the first extraction tank is used for adding 1.67L of ethanol aqueous solution with the volume concentration of 50-70%, the second extraction tank and the third extraction tank are used for adding ethanol aqueous solution with the same concentration respectively, and the total volume is the same as that of the first extraction tank. Filtering the first extractive solution of the first extraction tank, placing into a reserve tank for standby, taking the second extractive solution as the first extractive solvent of the second extraction tank, filtering the second extractive solution for standby, taking the third extractive solution as the second extractive solvent of the second extraction tank, taking the obtained extractive solution as the first extractive solvent of the third extraction tank, and the like until the 6 th tank, collecting the first extractive solution of each extraction tank to obtain countercurrent extractive solution.
(3) Membrane separation: recovering ethanol from the countercurrent extract prepared in the step (2) by rotary evaporation, centrifuging at 5000rpm for 20min, collecting supernatant, performing microfiltration at 40deg.C by adopting polysulfone membrane with pore diameter of 0.45 μm, collecting micro-filtrate, and performing ultrafiltration at 40deg.C and pressure of 0.5Mpa by adopting polysulfone membrane with pore diameter of 10nm to obtain filtrate;
(4) Removing pesticide residues: performing column chromatography separation (column height is 500mm and diameter is 20 mm) by adopting LS-806B special resin, loading the filtered liquid in the step (3) at the speed of 3BV/h, performing gradient elution by using 30%, 40%, 50%, 60% and 70% methanol water solutions with the volume concentration of 2BV/h and the elution amount of 9BV, detecting the content of total flavonoids in the effluent liquid by adopting a spectrophotometry method described in the embodiment 1, collecting the eluent with the mass content of flavonoids of more than 10%, concentrating under reduced pressure until the volume content of methanol is 1.6%, and obtaining concentrated liquid for removing pesticide residues, wherein the pesticide residues of the concentrated liquid reach undetected standards (namely <10 ppb).
(5) Column chromatography: loading the concentrated solution prepared in the step (4) to an AB-8 macroporous adsorption resin chromatographic column (20 mm multiplied by 500 mm) at the speed of 4BV/h, washing with water until the lower liquid is nearly colorless after the adsorption is finished, discarding the water washing liquid, eluting with 70% ethanol water solution at the volume concentration of 3BV/h, eluting with the amount of 9BV, collecting ethanol eluent, concentrating under reduced pressure until the ethanol eluent is dry, and identifying 46.3g of Hangzhou white chrysanthemum total flavonoids by adopting the method of the embodiment 1.
Comparative example 2
(1) Pretreatment of raw materials: baking low-grade Hangzhou white chrysanthemum at 105 ℃ for 2 hours, grinding into powder in a high-speed multifunctional grinder, and sieving with a 100-mesh sieve to obtain Hangzhou white chrysanthemum byproduct powder; adding 1kg of Hangzhou white inulin powder into 10L of 70% ethanol water solution with volume concentration, wherein the ratio of feed to liquid is 1:20, stirring at room temperature, fully swelling Hangzhou white chrysanthemum cells, adjusting the pH value to 4.5 at 50 ℃, adding 3g of cellulase, and carrying out enzymolysis for 40min at 50 ℃ to obtain an enzymolysis material;
(2) Extracting: and (3) adding all the materials subjected to enzymolysis in the step (1) into a multifunctional extraction tank (YC-020, shanghai elegance equipment Co., ltd.), adding 10L of 70% ethanol water solution with volume concentration to enable the feed-liquid ratio to be 1:20, and extracting at 60 ℃ for 2 hours to obtain an extracting solution.
(3) Membrane separation: recovering ethanol from the extract prepared in the step (2) by rotary evaporation, centrifuging at 5000rpm for 20min, collecting supernatant, performing microfiltration at 40deg.C by adopting polysulfone membrane with pore diameter of 0.45 μm, collecting micro-filtrate, and performing ultrafiltration at 40deg.C and pressure of 0.5Mpa by adopting polysulfone membrane with pore diameter of 10nm to obtain filtrate;
(4) Removing pesticide residues: performing column chromatography separation (column height is 500mm and diameter is 20 mm) by adopting LS-806B special resin, loading the filtered liquid in the step (3) at the speed of 3BV/h, performing gradient elution by using 30%, 40%, 50%, 60% and 70% methanol water solutions with the volume concentration of 2BV/h and the elution amount of 9BV, detecting the content of total flavonoids in the effluent liquid by adopting a spectrophotometry method described in the embodiment 1, collecting the eluent with the mass content of flavonoids of more than 10%, concentrating under reduced pressure until the volume content of methanol is 1.6%, and obtaining concentrated liquid for removing pesticide residues, wherein the pesticide residues of the concentrated liquid reach undetected standards (namely <10 ppb).
(5) Column chromatography: loading the concentrated solution prepared in the step (4) into an AB-8 macroporous adsorption resin chromatographic column (20 mm multiplied by 500 mm) at the speed of 4BV/h, washing with water until the lower liquid is nearly colorless after the adsorption is finished, discarding the water washing liquid, eluting with 70% ethanol water solution at the volume concentration of 3BV/h, eluting with the amount of 9BV, collecting ethanol eluent, concentrating under reduced pressure until the ethanol eluent is dry, and identifying 48.7g of Hangzhou white chrysanthemum total flavonoids by adopting the method of the embodiment 1.
Claims (5)
1. The method for extracting the total flavonoids of Hangzhou white chrysanthemum is characterized by comprising the following steps of:
(1) Pretreatment: drying flos Chrysanthemi, and pulverizing to obtain flos Chrysanthemi powder; adding Hangzhou white inulin powder into ethanol water solution with the volume concentration of 50-70%, stirring at room temperature to fully swell, adjusting the temperature to 45-65 ℃, adjusting the pH value to 4.0-5.5, adding cellulase, and carrying out enzymolysis for 30-60min to obtain a material after enzymolysis;
(2) Continuous dynamic countercurrent extraction: adopting tank group type dynamic extraction, adding the materials subjected to enzymolysis in the step (1) into each tank on average, extracting 3 times at 40-60 ℃ and 40-100rpm for each tank by taking 50-70% ethanol water solution as a solvent, and collecting the first extracting solution of each tank to obtain countercurrent extracting solution;
(3) Membrane separation: concentrating the countercurrent extract in the step (2) under reduced pressure to recover ethanol, centrifuging, collecting supernatant, and sequentially performing microfiltration and ultrafiltration to obtain filtrate; the microfiltration is carried out at the temperature of 40-50 ℃ by adopting a polysulfone membrane or a sulfonated polysulfone membrane with the pore diameter of 0.2-0.45 mu m; the ultrafiltration adopts a polyether sulfone membrane or a polysulfone membrane with the aperture of 5-10nm, and the ultrafiltration is carried out under the temperature of 35-45 ℃ and the pressure of 0.1-0.5 Mpa;
(4) Removing pesticide residues: performing column chromatography separation by using LS-806B resin, loading the filtered liquid in the step (3) at the speed of 2-4BV/h, performing gradient elution by using 30% -70% methanol aqueous solution with the volume concentration of 1-3BV/h, eluting the filtered liquid with the elution amount of 8-10BV, collecting eluent with the flavone mass content of more than 10%, and concentrating the eluent under reduced pressure until the methanol volume concentration is less than 5%, thereby obtaining concentrated liquid for removing pesticide residues;
(5) Column chromatography: loading the concentrated solution prepared in the step (4) into a macroporous adsorption resin chromatographic column at the speed of 2-4BV/h, washing with water until the lower injection is nearly colorless after the adsorption is completed, discarding the water washing liquid, eluting with 60-70% ethanol water solution at the eluting speed of 1-3BV/h and the eluting amount of 8-10BV, collecting ethanol eluent, concentrating under reduced pressure until the ethanol eluent is dry to obtain Hangzhou white chrysanthemum total flavonoids; the macroporous adsorption resin is ADS-7, AB-8, LK001, polyamide or D-101.
2. The method of claim 1, wherein the chrysanthemum morifolium powder of step (1) is obtained by baking chrysanthemum morifolium at 105 ℃ for 2-4 hours, crushing, and sieving with a 80-120 mesh sieve.
3. The method of claim 1, wherein the volume of the 50-70% aqueous ethanol solution in step (1) is 6-12mL/g based on the mass of Hangzhou white inulin powder; the enzyme activity of the cellulase is 10000U/g, and the mass addition amount of the cellulase is 0.1-0.3% of the weight of Hangzhou white inulin powder.
4. The method according to claim 1, wherein the volume amount of the ethanol aqueous solution with the volume concentration of 50-70% in the step (2) is 6-12mL/g based on the weight of the Hangzhou white inulin powder in the step (1).
5. The method of claim 1, wherein the aqueous methanol solution for gradient elution in step (4) has a volume concentration of 30%, 40%, 50%, 60% and 70%.
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