CN115060813A - Method for screening and purifying antioxidant active ingredients of citrus peel - Google Patents
Method for screening and purifying antioxidant active ingredients of citrus peel Download PDFInfo
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- 241000207199 Citrus Species 0.000 title claims abstract description 26
- 235000020971 citrus fruits Nutrition 0.000 title claims abstract description 26
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- 238000000034 method Methods 0.000 title claims abstract description 17
- 239000003963 antioxidant agent Substances 0.000 title claims abstract description 14
- 238000012216 screening Methods 0.000 title claims abstract description 12
- 239000004480 active ingredient Substances 0.000 title claims abstract description 8
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Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
- C07H17/06—Benzopyran radicals
- C07H17/065—Benzo[b]pyrans
- C07H17/07—Benzo[b]pyran-4-ones
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8624—Detection of slopes or peaks; baseline correction
- G01N30/8631—Peaks
- G01N30/8634—Peak quality criteria
Abstract
The invention discloses a method for screening and purifying an antioxidant active ingredient of citrus peel. The method comprises the steps of performing reflux extraction on dried orange peel by using 70% ethanol, then performing reduced pressure evaporation to remove a solvent to obtain an ethanol crude extract, dispersing a certain amount of the ethanol crude extract by using pure water, and extracting with petroleum ether, ethyl acetate and n-butyl alcohol which have the same volume respectively to obtain an extract of each extraction layer. ABTS free radical scavenging experiments in 96-well plates and IC calculation 50 Value, determination of the best antioxidant ActivityAnd an upper layer (ethyl acetate layer). Determining antioxidant activity peak in ethyl acetate layer by high performance liquid chromatography and micro-fraction activity evaluation method, separating the active substance by screening appropriate two-phase solvent system by liquid-liquid chromatography, and purifying by preparative liquid chromatography to obtain narirutin monomer. The invention utilizes the method of combining high-efficiency liquid-phase micro-fraction active screening with high-speed counter-current chromatography to separate the antioxidant active substances in the orange peel, and has stronger purpose and rapidness and high efficiency.
Description
One, the technical field
The invention relates to a method for screening and purifying antioxidant active ingredients of citrus peel.
Second, background Art
Citrus (Citrus)Particulate Banco), belonging to the family Rutaceae, the subfamily Citrus. The citrus peel has high medicinal value, and contains many bioactive substances such as flavonoids, volatile oil, terpenes, coumarins, carotenoids, phenolic acid, pectin, etc. One of the components with the highest content in the citrus peel is flavonoid, which is mainly divided into two types: one is flavonoid glycoside, such as dihydroflavonoid glycoside like naringin, hesperidin, etc.; the other is polymethoxyflavonoids, such as hesperetin and nobiletin. Citrus flavonoids have effects in reducing blood lipid, enhancing immunity, resisting virus, inflammation and cancer, preventing atherosclerosis and cholesterol increase, resisting oxidation and cancer, preventing cardiovascular diseases, resisting inflammation and allergy, and inhibiting microorganism. Wherein. The antioxidant action is mainly expressed by the ability to scavenge free radicals, using radical cations ABTS + The method of (3) is most commonly used.
China belongs to a large citrus planting country, and annual yield is stable in the third world all the year round. The orange peel is an important byproduct generated in the orange processing and eating process, a small amount of the orange peel is used for producing pectin and orange essential oil, and the rest of the orange peel is directly treated as waste and is buried or thrown into the sea as garbage, so that the environment pollution is caused and the resource waste is caused. Therefore, the research on the antioxidant active substances in the citrus peel is deeply carried out, and the method has important social and economic benefits for environmental protection and comprehensive utilization of natural resources.
The core of the micro-fraction activity evaluation method is that after the natural product extract separated by liquid chromatography is shunted, one part of the natural product extract enters a mass spectrum detector to obtain the structural information of a compound, the other part of the natural product extract is collected in a porous plate by a micro-fraction collection system, after a solvent is volatilized, a proper buffer solution is selected to add target protein and corresponding ligand into the porous plate, the signal of biochemical reaction is monitored, and the off-line biological activity detection is completed. Compared with an online bioassay method, the offline activity evaluation method for the micro-fractions has relatively low automation degree, but can relatively improve the stability and accuracy, avoid the influence of the existence of an organic phase on the biological activity of the target protein in an online mode, and can be flexibly combined with more activity detection technologies.
Liquid-liquid chromatography (LLC) is a high-efficiency distribution chromatography technique that has emerged in recent years and is mainly characterized in that both the mobile phase and the stationary phase are liquids, so that the stationary phase does not require a solid carrier, the operating and maintenance costs are relatively low, and preparative separation of samples is easily achieved. No irreversible adsorption, low requirement on sample introduction, and direct sample introduction and separation of the crude product without pretreatment.
Third, the invention
The invention aims to provide a method for separating antioxidant active substances in orange peel by applying high-performance liquid micro-fraction active screening and high-speed counter-current chromatography, which has strong purpose and is rapid and efficient.
The technical scheme of the invention is as follows:
1. extracting dried citrus peel with 70% ethanol solution at a material-to-liquid ratio of 1:10 under reflux for 2 hr for 3 times, mixing extractive solutions, filtering the extractive solution, and vacuum recovering solvent to obtain ethanol crude extract;
2. dispersing the crude ethanol extract with pure water, extracting with petroleum ether, ethyl acetate and n-butanol for three times respectively, mixing the extraction layers, and vacuum recovering solvent to obtain petroleum ether layer, ethyl acetate layer, n-butanol layer and water layer extract;
3. and (3) carrying out ABTS free radical scavenging capacity test on the petroleum ether layer, the ethyl acetate layer, the n-butanol layer and the water layer extract in a 96-well plate, and determining that the ethyl acetate layer is the extraction layer with the strongest antioxidant capacity.
4. And analyzing the active layer by high performance liquid chromatography, and optimizing conditions to obtain a liquid chromatogram with certain repeatability. And determining a target peak with antioxidant activity by micro-fraction activity evaluation.
5. And (3) screening a proper two-phase solvent system by utilizing liquid-liquid extraction and selecting the two-phase solvent system with different solubilities of the target separation substance in two phases, so that the distribution coefficient of the target compound to be separated in the two-phase solvent system is 0.5-2 optimal. A two-phase solvent system consisting of ethyl acetate, n-butanol and water is the optimum separation system.
6. Performing high-speed countercurrent chromatographic separation by using the solvent system screened in the step 5, adding a mixed solvent system consisting of ethyl acetate, n-butyl alcohol and water into a separating funnel, fully shaking, standing for layering, taking an upper phase as a stationary phase of the liquid-liquid chromatography, and taking a lower phase as a mobile phase; the volume parts of ethyl acetate, n-butyl alcohol and water in the mixed solvent system are respectively 3-4 parts, 2-1 parts and 5 parts, preferably 3 parts, 2 parts and 5 parts;
7. dissolving the citrus peel extract sample obtained in the step 3 by using the stationary phase and the mobile phase obtained in the step 6 to obtain a sample solution; the volumes of the stationary phase and the mobile phase are equal, and the dosage is 2mg/ml based on the mass of the orange peel ethyl acetate extraction layer sample;
8. injecting a stationary phase into a separation column of a liquid full-liquid chromatograph, starting a speed controller to enable the separation column to rotate forwards, injecting a mobile phase at the flow rate of 2mL/min at the rotation speed of 500-1000 r/min, and when the mobile phase flows out from the tail end of the column, indicating that the two phases are balanced, injecting a sample solution into the separation column through a sample injection valve, detecting by using an ultraviolet detector with the wavelength of 254-280 nm, collecting eluent by using an automatic part collector, detecting the collected eluent by using a high-efficiency liquid phase, merging the eluent containing the highest compound, evaporating the solvent under reduced pressure, and purifying and separating by using a semi-preparative chromatograph to obtain a naringin monomer;
the sample injection volume of the sample solution is generally less than 30% of the volume of the separation column, and preferably the sample injection volume is 5% -10% of the volume of the separation column;
description of the drawings
Table 1: IC for eliminating ABTS free radical in different extraction layers of citrus peel 50 Value of
FIG. 1 is a High Performance Liquid Chromatography (HPLC) spectrum of ethyl acetate layer of citrus peel
FIG. 2 is an activity spectrum of 5mg ABTS free radical scavenging micro-fraction
FIG. 3 is the activity spectrum of 10mg ABTS free radical scavenging micro-fraction
FIG. 4 shows a high-speed countercurrent chromatography (HSCCC) spectrum of ethyl acetate, n-butanol, water (4: 1: 5) (v/v), a sample size of 20mg
FIG. 5 shows HSCCC spectrum of 20mg of ethyl acetate, n-butanol, water, 3:2:5 (v/v)
FIG. 6 shows HSCCC spectrum of 35mg of ethyl acetate, n-butanol, water, 3:2:5 (v/v)
FIG. 7 is an HPLC chart of the main peak in FIGS. 3 and 4
Fifth, detailed description of the invention
The technical solution of the present invention is further illustrated by the following specific examples, but the scope of the present invention is not limited thereto.
In the embodiment of the invention, an analytical liquid chromatograph (the volume of a separation column is 120mL) is adopted, and a separation system of the liquid chromatograph comprises a constant flow pump, an ultraviolet detector, a recorder and the like.
Example 1:
1. taking 100g of dried citrus peel, crushing, extracting for 3 times by refluxing with 1000mL of 70% ethanol solution, performing ultrasonic extraction for 2h, mixing the extracting solutions, filtering the extracting solution while the extracting solution is hot, and recovering the solvent in vacuum to obtain 45.8g of ethanol crude extract; 10g of crude ethanol extract is dispersed by 500ml of pure water, extracted by petroleum ether, ethyl acetate and n-butanol with equal volumes for three times respectively, extraction layers are combined, and the solvent is recovered in vacuum to obtain 70.0mg of petroleum ether extract, 316mg of ethyl acetate extract, 1.267g of n-butanol extract and 8.135g of water layer respectively.
ABTS free radical scavenging experiments. Solution preparation: ABTS source solution: 28mg of ABTS powder was weighed and dissolved in 16mL of purified water. ② K 2 S 2 O 8 Solution: weighing K 2 S 2 O 8 Powder 9.6mg, dissolved in 4mL of purified water. Take 0.5mL K 2 S 2 O 8 Dissolving the solution in ABTS solution, adding 16.9mL of pure water to prepare ABTS source solution, and placing in a dark refrigerator for 12-16h for later use. ③ ABTS working solution: 5mL of the ABTS source solution was dissolved in 20mL of PBS (pH 7.4) buffer. Of sample solution: precisely weighing 1.00mg of the total extract, petroleum ether extract, ethyl acetate extract, n-butanol extract and water layer sample of Mandarin orange peel, respectively, and dissolving in 1mL of methanolIn the method, 1mg/mL solution is obtained, and 100ug/mL, 50ug/mL, 25ug/mL, 12.5ug/mL and 0.125ug/mL sample source solution are diluted. Preparing a positive reference substance (VC) solution: weighing 1mg of VC solution, dissolving the VC solution in 1mL of methanol solution to obtain 1mg/mL of VC solution, and diluting the VC solution into 0.5mg/mL, 0.25mg/mL, 0.125mg/mL, 0.0625mg/mL, 0.0375mg/mL, 0.01875mg/mL and 0.009375mg/mL of VC solution.
3.96-well plate free radical scavenging assay, set up sample (sample), sample blank (blank sample), blank (blank), control (control), positive control (positive control), respectively. For the sample group, 180. mu.l of ABTS working solution and 20. mu.l of sample solution were added to a 96-well plate, 180. mu.l of methanol solution and 20. mu.l of sample solution were added to a sample blank group, 200. mu.l of methanol solution was added to a blank group, 180. mu.l of ABTS and 20. mu.l of sample solution were added to a control group, and 180. mu.l of ABTS working solution and 20. mu.l of positive control solution were added to a positive control group. Incubating in dark for 6min, measuring ultraviolet absorbance at 734nm for 20min, and measuring once in 1 min. Each sample group horizontal test was repeated three times. ABTS free radical clearance was calculated as equation 1, as follows: experimental creating capacity [ [1- (Asample-Ablanksample)/(Acontrol-Ablank) × 100% (equation 1)
4. Optimizing the liquid phase condition of the ethyl acetate active layer of the citrus peel, and determining an active peak by a micro-fraction activity evaluation method. The sampling amount of the liquid phase is 5mg, 20 mu l, the sampling frequency is 10 s/hole, and fractions of 2-85min in the liquid phase diagram are collected. Each plate was set up with 84 sample sets, 12 control sets. After the solvent of the sample solution in the sample group is removed in a solvent volatilizer, 100. mu.l of methanol solution and 100. mu.l of ABTS working solution are added, wherein the dilution factor of the ABTS working solution is 1/2 in step 3, and the absorbance is measured and the radical clearance is calculated according to the method of step 3. Obtaining an activity spectrogram and comparing the activity spectrogram with a liquid phase spectrogram.
The optimized high performance liquid chromatography conditions are as follows: ufavor ODS-C 18 column (250X 4.6mm,5 μm); the column temperature is 25 ℃; the mobile phase is acetonitrile (A) -water (B) solution, the gradient elution mode (0-15 min, 10-16% A, 15-55 min, 16-30% A, 55-65 min, 30-60% A, 65-80 min, 60-85% A) is that the flow rate is as follows: 0.8 mL/min; detection wavelength: 280nm and 254 nm; intoSample size: 20 mu L of the solution;
example 2
1. Micro-fraction activity experiment, wherein the liquid phase sample amount is 10mg, 20 mu l, the sampling frequency is 10 s/hole, and fractions of 2-85min in a liquid phase diagram are collected. Each plate was set up with 84 sample sets, 12 control sets. After the solvent was removed from the sample solution in the sample group in the solvent volatilizer, 100. mu.l of methanol solution and 100. mu.l of ABTS working solution, wherein the dilution ratio of ABTS working solution was 1/2 in step 3 of example 1, and the absorbance was measured and the radical clearance was calculated according to the method of step 3 of example 1. Obtaining an active spectrogram and comparing the active spectrogram with a liquid phase spectrogram;
example 3
1. Liquid-liquid chromatography, namely preparing ethyl acetate, n-butyl alcohol and water in a separating funnel according to the volume ratio of 4:1:5, fully shaking, standing and layering, wherein an upper phase is used as a stationary phase, and a lower phase is used as a mobile phase; weighing 20mg of ethyl acetate extract, and dissolving with 10mL of fixed phase to obtain a sample solution;
2. and separating the orange peel ethyl acetate layer sample by adopting a preparative liquid chromatograph, wherein the column volume of a separation column is 125 mL. Filling the stationary phase into the separation column at a flow rate of 10mL/min, starting a speed controller to enable the separation column to rotate forwards, setting the flow rate of the mobile phase to be 2mL/min, starting pumping the mobile phase, and when the mobile phase flows out from the tail end of the column, indicating that the two phases are balanced, and then injecting the sample into the separation column through a six-way valve; receiving the eluate at the speed of 3 min/tube with an automatic fraction collector, detecting by liquid chromatography (mobile phase is acetonitrile and water) until no component with highest compound content is present in the eluate, and stopping collection. Detecting the collected eluate with high performance liquid chromatography, mixing eluates containing only narirutin (retention time is 39.3min, and evaporating the solvent under reduced pressure to obtain narirutin;
3. the detection conditions of the high performance liquid chromatography are as follows: ufavor ODS-C 18 column (250X 4.6mm,5 μm); the column temperature is 25 ℃; the mobile phase is acetonitrile (A) -water (B) solution, the gradient elution mode (0-15 min, 10-16% A, 15-55 min, 16-30% A, 55-65 min, 30-40% A, 65-80 min, 40-85% A) is that the flow rate is as follows: 0.8 mL/min; detection wavelength: 280nm of,254 nm; sample introduction amount: 20 mu L of the solution;
example 4
1. Liquid-liquid chromatography, namely preparing ethyl acetate, n-butanol and water in a separating funnel according to the volume ratio of 3:2:5, fully shaking, standing and layering, wherein the upper phase is used as a stationary phase, and the lower phase is used as a mobile phase; weighing 20mg of ethyl acetate extract, and dissolving with 10mL of fixed phase to obtain a sample solution;
2. separating the sample of the ethyl caproate layer of the citrus peel by adopting a preparative liquid chromatograph, wherein the column volume of a separation column is 125 mL. Filling the stationary phase into the separation column at a flow rate of 10mL/min, starting a speed controller to enable the separation column to rotate forwards, setting the flow rate of the mobile phase to be 2mL/min, starting pumping the mobile phase, and when the mobile phase flows out from the tail end of the column, indicating that the two phases are balanced, and then injecting the sample into the separation column through a six-way valve; receiving the eluate at the speed of 3 min/tube with an automatic fraction collector, detecting by liquid chromatography (mobile phase is acetonitrile and water) until no component with highest compound content is present in the eluate, and stopping collection. Detecting the collected eluate with high performance liquid chromatography, mixing eluates containing only narirutin (retention time is 39.3min, and evaporating the solvent under reduced pressure to obtain narirutin;
the detection conditions of the high performance liquid chromatography are as follows: ufavor ODS-C 18 column (250X 4.6mm,5 μm); the column temperature is 25 ℃; the mobile phase is acetonitrile (A) -water (B) solution, the gradient elution mode (0-15 min, 10-16% A, 15-55 min, 16-30% A, 55-65 min, 30-40% A, 65-80 min, 40-85% A) is that the flow rate is as follows: 0.8 mL/min; detection wavelength: 280nm and 254 nm; sample introduction amount: 20 mu L of the solution;
example 5
1. Liquid-liquid chromatography, namely preparing ethyl acetate, n-butanol and water in a separating funnel according to the volume ratio of 3:2:5, fully shaking, standing and layering, wherein the upper phase is used as a stationary phase, and the lower phase is used as a mobile phase; weighing 35mg of ethyl acetate extract, and dissolving with 10mL of fixed phase to obtain a sample solution;
2. separating the sample of the ethyl caproate layer of the citrus peel by adopting a preparative liquid chromatograph, wherein the column volume of a separation column is 125 mL. Filling the stationary phase into the separation column at a flow rate of 10mL/min, starting a speed controller to enable the separation column to rotate forwards, setting the flow rate of the mobile phase to be 2mL/min, starting pumping the mobile phase, and when the mobile phase flows out from the tail end of the column, indicating that the two phases are balanced, and then injecting the sample into the separation column through a six-way valve; receiving the eluate at the speed of 3 min/tube with an automatic fraction collector, detecting by liquid chromatography (mobile phase is acetonitrile and water) until no component with highest compound content is present in the eluate, and stopping collection. Detecting the collected eluate with high performance liquid chromatography, mixing eluates containing only narirutin with retention time of 39.3min, and evaporating solvent from the eluates under reduced pressure to obtain narirutin;
the detection conditions of the high performance liquid chromatography are as follows: ufavor ODS-C 18 column (250X 4.6mm,5 μm); the column temperature is 25 ℃; the mobile phase is acetonitrile (A) -water (B) solution, the gradient elution mode (0-15 min, 10-16% A, 15-55 min, 16-30% A, 55-65 min, 30-40% A, 65-80 min, 40-85% A) is that the flow rate is as follows: 0.8 mL/min; detection wavelength: 280nm and 254 nm; sample introduction amount: 20 μ L.
Claims (4)
1. A method for screening and purifying antioxidant active ingredients of orange peels is characterized by comprising the following steps: s1 extracting dried citrus peel with 70% ethanol solution at a ratio of 1:10 under reflux for 2 hr for 3 times, mixing extractive solutions, filtering, and vacuum recovering solvent to obtain crude ethanol extract; s2 dispersing the crude ethanol extract with pure water, extracting with petroleum ether, ethyl acetate and n-butanol for three times respectively, mixing the extraction layers, and vacuum recovering solvent to obtain petroleum ether layer, ethyl acetate layer, n-butanol layer and water layer extract; s3, carrying out ABTS free radical scavenging capacity test on the petroleum ether layer, the ethyl acetate layer, the n-butanol layer and the water layer extract in a 96-well plate, and determining that the ethyl acetate layer is the extraction layer with the strongest antioxidant capacity; s4, analyzing the active layer by high performance liquid chromatography, and optimizing conditions to obtain a liquid chromatogram with certain repeatability. Determining a target peak with antioxidant activity by micro-fraction activity evaluation; s5 liquid-liquid extraction is utilized, the solubility of a target separation substance in two phases is different, and a proper two-phase solvent system is screened, so that the distribution coefficient of the target compound to be separated in the two-phase solvent system is 0.5-2 optimal. S6, performing high-speed counter-current chromatography separation by using the solvent system screened in the step S5, adding the mixed solvent system into a separating funnel, fully shaking, standing for layering, taking the upper phase as the stationary phase of liquid-liquid chromatography, and taking the lower phase as the mobile phase; s7, dissolving the citrus peel extract sample obtained in the step S3 by using the stationary phase and the mobile phase obtained in the step S6 to obtain a sample solution; the volumes of the stationary phase and the mobile phase are equal, and the dosage is 2mg/ml based on the mass of the orange peel ethyl acetate extraction layer sample; s8, taking a stationary phase, filling a separation column of a liquid full-liquid chromatograph, starting a speed controller to enable the separation column to rotate forwards, filling a mobile phase at a flow rate of 2mL/min at a rotation speed of 500-1000 r/min, when the mobile phase flows out from the tail end of the column, indicating that the two phases are balanced, filling a sample solution into the separation column through a sample injection valve, detecting by an ultraviolet detector with a wavelength of 280nm, collecting eluent by an automatic part collector, detecting the collected eluent by a high-efficiency liquid phase, merging the eluent with the highest compound content, evaporating the solvent under reduced pressure, and purifying and separating by semi-preparative chromatography to obtain the naringin monomer.
2. The method for screening and purifying antioxidant active ingredients in citrus peel as claimed in claim 1, wherein the liquid phase micro-fraction experiment of step S4 is performed with a sample amount of 5 mg/ml.
3. The method for screening and purifying antioxidant active ingredients in citrus peel as claimed in claim 1, wherein the liquid chromatography analysis mobile phase of the ethyl acetate extraction layer of citrus peel in step S3 is acetonitrile (a) -water (B) solution, and the gradient elution mode (0-15 min, 10% -16% a, 15-55 min, 16% -30% a, 55-65 min, 30% -60% a, 65-80 min, 60% -85% a) is as follows, the flow rate: 0.8 mL/min; detection wavelength: 280nm and 254 nm; sample introduction amount: 20 μ L.
4. The method for screening and purifying antioxidant active ingredients in citrus peel as claimed in claim 1, wherein the two-phase solvent system of high speed counter current chromatography of step S5 is ethyl acetate, n-butanol, water in a volume ratio of 3:2: 5.
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