CN110133152B

CN110133152B - Method for screening antioxidant components in honeysuckle

Info

Publication number: CN110133152B
Application number: CN201910507822.1A
Authority: CN
Inventors: 赵恒强; 张敏敏; 刘伟; 崔莉; 王晓; 闫慧娇; 李丽丽
Original assignee: Shandong Analysis and Test Center
Current assignee: BEIJING DAWN AEROSPACE BIO-TECH CO LTD
Priority date: 2019-06-12
Filing date: 2019-06-12
Publication date: 2022-03-18
Anticipated expiration: 2039-06-12
Also published as: CN110133152A

Abstract

The disclosure belongs to the technical field of antioxidant component screening, and particularly relates to a screening method of antioxidant components in honeysuckle. Honeysuckle has various physiological activities of fever reduction, liver protection, virus resistance, oxidation resistance and the like, and related researches in the prior art show that the main oxidation resistant component in the honeysuckle is an organic acid component and is a good source of natural antioxidants. The separation and preparation of the active ingredients with strong antioxidation from the honeysuckle extract, particularly the active ingredients with lower content, has important significance. The invention provides a method for carrying out sectional enrichment analysis on a honeysuckle extract by a two-dimensional chromatographic separation method and combining online antioxidant screening, which is used for separating various trace monomer components with different polarities and strong antioxidant activity from the honeysuckle extract and provides a method and technical support for rapid screening of trace natural antioxidants.

Description

Method for screening antioxidant components in honeysuckle

Technical Field

The invention belongs to the technical field of antioxidant component screening, and particularly relates to a method for rapidly screening trace antioxidant components in honeysuckle by combining two-dimensional high performance liquid chromatography-electrospray flight time mass spectrum with a diphenyltrinitrophenylhydrazine technology (2D-HPLC-ESI-TOF/MS-DPPH).

Background

The information in this background section is only for enhancement of understanding of the general background of the disclosure and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

The radical is an atomic group containing an unpaired electron and is extremely unstable in nature. Excessive free radicals in the body can attack human cells to cause serious injury to the human body, and the maintenance of the balance of the free radicals in the body is an important way for ensuring the health of the human body. Antioxidants are a class of substances that help to trap and neutralize free radicals, thereby removing the damage they do to the body. Research on antioxidants having radical scavenging action has been hot, and natural antioxidants have become important targets in scientific research due to their high safety, strong environmental protection, and the like. The discovery of natural antioxidants is also an important research direction in the field of natural product research.

Honeysuckle is a dry flower bud or a flower with an initial bloom of Lonicera japonica Thunb of Caprifoliaceae [15 edition pharmacopoeia ], has great application ratio in the traditional Chinese medicine prescription in China, is widely used for treating various diseases such as fever, infection, pyocutaneous disease, swelling and the like, is listed in a list of medicines which are not only foods but also medicines published by the original Ministry of health in 2002, and becomes a medicine-food homologous food which is widely applied to the industries of health foods and functional beverages. Modern pharmacological studies show that the honeysuckle extract has wide biological activities of fever reduction, liver protection, antibiosis, anti-inflammation, antioxidation, antivirus and the like. The Heimaxu et al establishes an antioxidant activity fingerprint spectrum, and selects 8 antioxidant components mainly comprising chlorogenic acid and isochlorogenic acid A from honeysuckle, so that the honeysuckle is also a good source of natural antioxidants. Researches find that the honeysuckle contains various chemical components such as phenolic acids, flavonoids, terpenes and the like, the content and polarity of each component are greatly different, the discovery of the antioxidant active component in the target honeysuckle is greatly interfered, and particularly, the activity discovery and the application of trace compounds are greatly difficult.

The traditional natural product active ingredient discovery method generally separates and purifies a large amount of components from an extract, and then performs in vivo or in vitro pharmacological screening, which plays an important role in drug development. However, the method has the defects of time consumption, energy consumption, low efficiency, blindness and the like. In addition, some compounds have low content and unstable properties during separation and purification, so that the antioxidant activity of the compounds is difficult to find and verify. In recent years, an on-line antioxidant component screening technology based on an HPLC-DPPH technology has the advantages of simultaneous performance of chromatographic separation and activity screening, high efficiency, rapidness and the like, and has a good effect when being applied to rapid screening of natural antioxidant active components. The inventor thinks that the existing research shows that the main antioxidant component in honeysuckle is an organic acid component, and the online antioxidant screening can be used for screening the antioxidant component in the medicinal materials widely, but cannot realize the specific screening of certain components or strong antioxidant active components, and has the problems of low sensitivity and the like in the screening of trace components in the medicinal materials.

Disclosure of Invention

In view of the research background, the disclosure provides a method for screening strong antioxidant active ingredients in honeysuckle, and the method is used for carrying out sectional preparation by a two-dimensional chromatographic separation method (2D-HPLC), so as to preliminarily realize the purposes of enrichment and polar section of trace ingredients in honeysuckle. And (3) selecting proper chromatographic separation technology according to the polarity of each section of chemical components to carry out chromatographic separation so as to obtain more comprehensive component information. Meanwhile, the rapid screening of trace antioxidant active ingredients in a honeysuckle complex system is realized by combining an HPLC-DPPH online screening technology. And evaluating the in vitro antioxidant activity of part of the screened monomer compounds. Evaluation results show that the method can screen out various monomer components with different polarities and strong antioxidant activity, and the establishment of the method provides a method and technical support for rapid screening of trace natural antioxidants.

In order to achieve the above technical effects, the present disclosure provides the following technical solutions:

in a first aspect of the present disclosure, a honeysuckle crude extract is provided, and the preparation method of the crude extract is as follows: adding an organic solvent into the honeysuckle for ultrasonic extraction; preferably, the organic solvent is 60-80% of methanol.

In a second aspect of the present disclosure, a method for screening antioxidant components in honeysuckle is provided, the method includes the following steps: performing segmented enrichment and analysis on the honeysuckle extract by adopting a two-dimensional chromatogram, and screening antioxidant components in the honeysuckle on line;

the segmented enrichment is as follows: dividing the honeysuckle extract into a plurality of sections of components by two-dimensional chromatography, drying and redissolving to obtain the concentrated extract.

Preferably, the screening method further comprises analyzing the antioxidant component by mass spectrometry.

Preferably, the honeysuckle extract is the honeysuckle crude extract of the first aspect.

In some embodiments, the honeysuckle flower drug is processed into a powder form, and the powder is mixed with 1 g: adding 80-120 ml of methanol solution into the mixture, and ultrasonically extracting the effective components in the mixture. The extract can be concentrated by drying and redissolving, and then is filtered by a microporous filter membrane for standby.

Preferably, the first dimension of the two-dimensional chromatography is semi-preparative high performance liquid chromatography, which is used for segmenting the honeysuckle extract.

Further preferably, the semi-preparative high performance liquid chromatography is used for segmenting the honeysuckle extract and dividing the honeysuckle extract into three segments by adopting a gradient elution mode: fraction a, fraction B, fraction C.

According to the method, the extract can be conveniently enriched by a way of preparing the extract in sections, so that the concentration of trace components in the extract can reach the sensitivity of a detection device to the greatest extent. On the other hand, the extract is divided into a plurality of fractions according to the polarity, and corresponding mobile phase conditions and elution procedures can be set according to the polarity characteristics of the fractions for separation. Through repeated attempts and continuous researches of the disclosure, an analysis condition capable of screening various strong antioxidant active substances at one time is provided.

In some embodiments, the mobile phase of the gradient elution is: a is water containing 0.2% formic acid and B is acetonitrile.

Preferably, the second dimension of the two-dimensional chromatogram is a reverse phase chromatography column for further analysis of the fragmented components.

Further preferably, the mobile phase of fraction A is A (0.8% formic acid, 10mM ammonium formate), B: and (3) acetonitrile.

In some embodiments, fraction a is separated using a gradient elution.

Further preferably, the mobile phase of fraction B is a: water (0.6% formic acid), B: acetonitrile (10% water).

In some embodiments, fraction B is separated by gradient elution.

Further preferably, the mobile phase of fraction C is a: water (0.8% formic acid, 10mM ammonium formate), B: and (3) acetonitrile.

In some embodiments, fraction C is separated by gradient elution.

In some embodiments, fractions A, B, and C are collected, dried, and redissolved with an organic solvent.

Preferably, the on-line screening of the antioxidant component comprises the following steps: mixing the separated components with DPPH, and detecting with DAD detector to obtain peak-inversion component as antioxidant component.

Compared with the prior art, the beneficial effect of this disclosure is:

36 components with antioxidant activity are screened from the honeysuckle extract by the method, 12 components are identified by high-resolution mass spectrometry, and the antioxidant activity of 7 components is superior to that of a positive control (vitamin C) as shown by an in-vitro DPPH free radical scavenging activity verification experiment. The method overcomes the difficulty that trace active compounds in the honeysuckle are difficult to find due to complex components and large difference of content and polarity of each component in the honeysuckle. Provides a new research idea and technical support for screening antioxidant active ingredients from a complex system.

Drawings

The accompanying drawings, which are included to provide a further understanding of the disclosure, illustrate embodiments of the disclosure and together with the description serve to explain the disclosure and are not to limit the disclosure.

FIG. 1 is a HPLC chromatogram (280nm) of a staged preparation of honeysuckle flower in example 1;

FIG. 2 is a HPLC chromatogram of the step-screening of the honeysuckle flower in example 1.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present disclosure. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

As introduced in the background art, honeysuckle contains a plurality of components with strong antioxidant activity, and is a good natural antioxidant source. However, the honeysuckle contains more components and has larger content and polarity difference, which brings great difficulty to the activity discovery and extraction of trace compounds. In order to solve the technical problems, the method for rapidly finding trace antioxidant active ingredients in honeysuckle by combining a two-dimensional liquid phase with an online antioxidant technology is established in the disclosure.

In order to make the technical solutions of the present disclosure more clearly understood by those skilled in the art, the technical solutions of the present disclosure will be described in detail below with reference to specific examples and comparative examples.

Example 1

1 materials and methods

1.1 instruments and materials

A Saimei fly Unimats 3000 series high performance liquid chromatography system (American Saimei fly science and technology); high speed centrifuges (Shanghai Anting). Tecan M20 series multifunctional microplate reader (Switzerland Ken group Co., Ltd.); brooks IMPACT II ESI-Q-TOF/MS mass spectrometer (Broks, USA); MICROTEST^TMA 96-well microplate (boston diekinson); SB-5200D series high power ultrasonic cleaning apparatus (Ningbo New Ganoderma Co.).

DPPH (2, 2-Diphenyl-1-piperidinylhydrazy) is purchased from Sigma, and acetonitrile (chromatographically pure) is purchased from Merch; the honeysuckle sample is purchased from the big drugstore of Jade Cisco, and identified as the dry bud of honeysuckle of Caprifoliaceae by the university of Qilu Industrial university (Shandong province analysis and test center of Shandong academy of sciences). The methanol is chromatographically pure, and other reagents are analytically pure. The experimental water was ultrapure water (Millipore Q-Plus system, USA).

1.2 methods

1.2.1 preparation of test solutions

Weighing flos Lonicerae powder 10g precisely, adding 70% methanol 1000mL, ultrasonic extracting for 30min, evaporating to dryness under reduced pressure, redissolving with 70% methanol, and filtering with 0.45 microporous membrane for use.

Accurately weighing DPPH, and preparing with 80% acetonitrile to obtain a solution with a concentration of 6 × 10^-5M in DPPH free radical, and stored at 4 ℃.

Precisely weighing 1.0mg of each standard reference substance in a 1mL volumetric flask respectively, dissolving, and diluting to constant volume with methanol to prepare a 1.0mg/mL standard substance test solution.

1.2.2 one-dimensional chromatographic conditions

And (3) adopting a RIGOL-3245 semi-preparative liquid chromatography system and combining a reverse-phase preparative chromatographic column to carry out preliminary sectional preparation on the honeysuckle crude extract. The gradient elution procedure was: 0min, 10% of B, 0-20min and 10% -40% of B; 20-30min, 40% -50% B; 30-31 min, 50% -100% B. The mobile phase is as follows: a (water with 0.2% formic acid) and B (acetonitrile). The flow rate was 3.0mL/min and the detection wavelength was 280 nm. The sample size was 100. mu.L. The three fractions (fraction A: fraction 1, fraction B: fraction 2, fraction C: fraction 3) were collected, freeze-dried, reconstituted with 70% methanol, filtered through a 0.22 μm microfiltration membrane and used for subsequent analysis, identification and screening.

1.2.3 two-dimensional chromatographic conditions

And the step of performing segmented analysis on the sample by adopting a Saimeifeng U3000 ultra-performance liquid chromatography system. In view of the difference of the polarity and the content of the components, different liquid phase separation conditions and mass spectrum identification conditions need to be optimized for the three fractions respectively so as to obtain more chromatographic separation peaks and better separation effect.

1.2.3.1 chromatographic analysis of fraction A

Chromatographic conditions are as follows: watt Xbridge Amide column (4.6X 250mm, 3.5 μm); mobile phase A: water (0.8% formic acid, 10mM ammonium formate), B: acetonitrile; elution gradient: 96% -93% B in 0-5min, 93% -93% B in 5-20min, 93% -85% B in 20-30min, 85% -82% B in 30-40min, 82% -78% B in 40-50min, and 50% B in 51 min; flow rate: 1.0 mL/min; detection wavelength: 280 nm; column temperature: 35 ℃ is carried out.

1.2.3.2 chromatographic analysis of fraction B

Chromatographic conditions are as follows: agilent Eclipse Plus C18 column (3.0X 100mm, 1.8 μm); mobile phase A: water (0.6% formic acid), B: acetonitrile (10% water); elution gradient: 0-5min 5% -6% B,5-15min 6% -8% B,15-25min 8% -10% B,25-40min 10% -13% B,40-45min 13% -15% B,45-55min 15% -17% B,55-65min 17% -30% B,65-70min 30% -45% B; flow rate: 0.5 mL/min; detection wavelength: 254 nm; column temperature: 35 ℃ is carried out.

1.2.3.3 fraction C chromatography

Chromatographic conditions are as follows: watcht XSELET HST 3 chromatography column (3.0X 150mm, 3.5 μm); mobile phase A: water (0.8% formic acid, 10mM ammonium formate), B: acetonitrile; elution gradient: 14% -17% of B in 0-10min, 17% -18% of B in 10-20min, 18% -19% of B in 20-30min, 19% -23% of B in 30-50min, 23% -28% of B in 50-65min, 28% -35% of B in 65-75 min; flow rate: 0.5 mL/min; detection wavelength: 345 nm; column temperature: at 40 ℃.

1.2.4 Online antioxidant screening

And (3) finishing the on-line antioxidant screening of each component by using a Saimeishafio U3000 dual-ternary liquid chromatography system. And (3) carrying out sample injection analysis on each fraction by using a right pump under an optimized chromatographic condition, separating a sample by using a chromatographic column, mixing the sample with a DPPH solution pumped by a left pump, reacting the mixed solution in a reaction tank, and then flowing into a DAD detector of an HPLC system for detection. Whereas the characteristic peak of DPPH at a wavelength of 517nm forms a peak inversion due to scavenging of DPPH radicals by the antioxidant active ingredient. And comparing the HPLC spectrums of the sample under the optimal absorption wavelength and the characteristic wavelength of the DPPH free radical to determine and screen out the antioxidant active ingredients.

1.2.5ESI-Q-TOF/MS analysis

Carrying out electrospray positive and negative ion full-scanning detection; the full scanning range m/z is 50-1500; taper hole voltage: 60V; capillary voltage: 5.0 kV; cleavage voltage: 120V; spraying air pressure: 45 psi; flow rate of drying gas: 8.0L/min; temperature of the drying gas: the three fractions were each subjected to mass spectrometric identification analysis at 200 ℃ under the above mass spectrometric conditions.

1.2.6 Activity validation

Standard stocks of compound were diluted to 7 different concentrations (0.50mg/mL, 0.25mg/mL, 0.10mg/mL, 0.05mg/mL, 0.02mg/mL, 0.01mg/mL, and 0.005mg/mL) as test samples for use.

And adding 50 mu L of solution to be detected and 100 mu L of 0.04mg/mL DPPH solution into a 96-well plate, uniformly mixing, incubating at room temperature in a dark place for 30min, and then determining under the condition of 517nm of a microplate reader. The control group contained 50. mu.L of methanol and 100. mu.L of 0.04mg/mL DPPH solution, and the blank group contained 150. mu.L of methanol. The DPPH free radical clearance rate calculation formula is as follows: (A)_Control-A_Sample(s))/(A_Control-A_{Blank space})×100％，A_Control、A_Sample(s)、A_{Blank space}The absorbance of the control group, the experimental group and the blank group are respectively. The concentration of the sample half cleared of DPPH free radicals was taken as IC₅₀. Calculating IC of each standard substance by drawing standard curve with clearance rate as ordinate and concentration as abscissa₅₀The value is obtained.

2 results and analysis

2.1 two-dimensional chromatographic enrichment and analysis of Trace active ingredients

In the subject group, a method for analyzing the active ingredients of honeysuckle by an RPLC-DAD method is established in earlier researches, and the content difference of the active ingredients in the honeysuckle extract is found to be large, and the contents of most other ingredients are low except chlorogenic acid and isochlorogenic acid. In addition, some chemical components have high polarity, and are difficult to separate by using a reverse phase chromatographic column. Significant differences in content and polarity place higher demands on the resolution and sensitivity of online screening. In order to solve the problems, the research adopts a semi-preparative liquid chromatography technology to carry out primary separation on the honeysuckle extract according to the appearance sequence and the content characteristics of each chromatographic peak in a chromatogram, thereby realizing the enrichment of low-content components and the primary classification of components with different polarities. Different types of chromatographic columns are further adopted for analysis so as to improve the resolution and sensitivity of online screening.

2.1.1 one-dimensional chromatographic Condition optimization

The reversed-phase preparative high performance liquid chromatography system is widely applied to separation and purification of natural products, and has the advantages of high efficiency, high separation degree and high recovery rate. The honeysuckle extract is subjected to sectional analysis by using a reversed-phase preparative liquid chromatography technology. In the present study, a gradient elution method was used for the initial separation, taking into account the complexity of the honeysuckle extract. The sample eluted by the semi-preparative liquid chromatography column is collected into three components A, B and C according to comprehensive evaluation of polarity, content and the like (the preparation and separation results are shown in figure 1). Wherein, the component A has stronger polarity and lower content, and the component C has lower polarity and content, and needs to be enriched for many times.

In order to find more trace antioxidant ingredients from honeysuckle, the research focuses on the preparation and concentration of the component A and the component C for multiple times and the preparation and concentration of the component B for proper times.

2.1.2 two-dimensional chromatographic analysis Condition optimization

The A component peaks faster on the semi-preparative liquid phase. The contained chemical components have stronger polarity, the effective separation is difficult to realize by adopting a common reversed phase chromatographic column,therefore, a hydrophilic chromatographic column is selected for analysis. Examine Xbridge^TM HILIC column(3.5μm,2.1×150mm；Waters Technologies,USA)，SeQuant^TMZIC-HILIC column (3.5 μm, 2.1X 150 mm; Merck KGaA, DE) and Xbridge^TMAmide column (3.5 μm, 4.6X 250 mm; Waters Technologies, USA) three different hydrophilic columns have separation effects on this component. The results show that the utilization of Xbridge^TMWhen Amide column (3.5 μm,4.6 × 250 mm; Waters Technologies, USA) is used for separation, the chromatographic column is selected for chromatographic separation of component a, because the number of peaks produced is large, the degree of separation is good, and the peak pattern is sharp. The mobile phase is yet another important factor affecting chromatographic separations. According to the investigation, the acetonitrile-water system and the gradient elution are adopted to realize the better separation of the chemical components in the component A. In order to improve the phenomenon of tailing of chromatographic peaks, the addition of 0.8% formic acid and 10mM ammonium formate in the water phase is optimized to obtain better chromatographic peak patterns. The separation effect of the HILIC chromatographic column is greatly influenced by the column temperature, so the research investigates the influence of four different column temperatures (25 ℃,30 ℃,35 ℃ and 40 ℃) on the chromatographic separation. The results show that: when the column temperature is 35 ℃, the peak shape is sharp, the separation degree is high, and the symmetry is good. The optimized chromatogram is shown in FIG. 2A.

And (4) selecting a reverse phase chromatographic column for analysis according to the peak-out time of the component B on the semi-preparative liquid chromatography. In addition, because the component B contains more chemical components and has small polarity difference among the components, effective separation is difficult to realize by only adopting an acetonitrile-water system and gradient elution. Further investigating the separation effect of adding water (5%, 10%, 15%) in different proportions into acetonitrile, and the result shows that better separation of the component B can be realized by gradient elution by adopting an acetonitrile (10% water) -water system. Meanwhile, considering that the main chemical components contained in the honeysuckle are phenolic acid and flavonoid compounds, which easily cause the phenomenon of tailing of a C18 column, through optimization and investigation, a certain proportion of acid (0.6% formic acid) is added into a water phase to achieve the effect of improving the chromatographic peak type. Under the optimized chromatographic conditions, the chromatographic separation pattern of the B component is shown in FIG. 2B.

The peak time of component C on the semi-preparative chromatographic column was later, and the reverse phase chromatographic column was used for its analytical study. The chromatographic conditions of the component C are systematically optimized by adopting the same research method and thought as the component B, and the finally obtained chromatogram is shown in figure 2C.

2.2 Online screening Condition optimization

The online screening conditions used in this study were slightly modified based on the online antioxidant screening model established in the early stage of the subject group. The optimization investigation is mainly carried out aiming at DPPH solvent, DPPH concentration, DPPH flow rate, reaction tank specification (inner diameter and length) and the like.

Since the chromatographic analysis of A, B, C three components all uses acetonitrile-water as mobile phase, in order to ensure the baseline stability, three different concentrations (50%, 80% and 100%) of acetonitrile were selected as DPPH dissolution solvent for comparative optimization. The test result shows that the baseline is relatively stable when 80% acetonitrile water is used as a solvent in the experimental system. By comparing three DPPH solutions of different concentrations (6X 10)^-4M，6×10^-5M and 6X 10^-6M), it can be seen that the screening effect increases with increasing concentration in a certain range until it is stable. But too large a concentration affects the stability of the baseline. Thus, 6 × 10 is selected^-5M was used as the optimum concentration for screening. In addition, the pumping flow rate of the DPPH solution needs to be adjusted optimally according to different chromatographic separation conditions, the research is used for analyzing the pumping flow rate of five DPPH solutions of 0.2mL/min, 0.4mL/min, 0.6mL/min, 0, 8mL/min and 1.0mL/min by combining the chromatographic conditions of all components, and the result shows that the peak inversion height is continuously increased and gradually becomes stable along with the increase of the pumping flow rate in a proper range. It is correspondingly known that excessive flow rates can cause strong fluctuations in baseline that affect screening sensitivity. Thus, the component A screening flow rate was determined to be 0.8mL/min, and the component B and component C screening flow rates were determined to be 0.4 mL/min.

The reaction cell specification affects the length of the reaction time, thereby affecting the screening effect. The specifications of the reaction tank include both the inner diameter of the reaction tube and the length of the reaction tube. The experiment was optimized for two inner diameters (0.18mm and 0.25mm) and three lengths (5m, 10m and 15 m). The research result shows that: the shorter the reaction tube is, the narrower the peak width is, and the higher the negative peak separation degree is; the longer the reaction tube, the higher the peak height and the poorer the degree of negative peak separation. The smaller the inner diameter of the reaction tube, the narrower the peak width and the higher the negative peak separation degree; the larger the inner diameter of the reaction tube, the higher the peak height and the poorer the resolution. By comprehensively considering the factors, the PEEK tube with the inner diameter of 0.25nm and the length of 10m is determined as the optimal reaction tank for the component A, and the PEEK tube with the inner diameter of 0.25nm and the length of 10m is determined as the optimal reaction tank for the component B and the component C. And (3) carrying out online antioxidant screening on the three components of the honeysuckle extract according to the optimized screening conditions, wherein the screening results are shown in figure 2.

As can be seen from fig. 2, in the component a, 7 compounds with antioxidant activity were screened out, including 6 compounds with weak spectral absorption intensity or low content and difficult to screen; in the component B, 12 compounds with antioxidant activity are screened out, wherein 9 compounds which are not easy to screen due to weak spectral absorption intensity, low content or large-content compound masking influence are screened out; as can be seen by combining the preparation chromatogram of the honeysuckle total sample in FIG. 1, 17 antioxidant active ingredients screened from the component C are not easy to observe and screen in the non-grouped enrichment chromatogram. The screening results are combined to discover that 36 chemical components with antioxidant activity are screened out from different polarity components of honeysuckle after enrichment, and the research result is obviously superior to the quantity of the antioxidant components in honeysuckle in literature reports.

2.3 ESI-TOF/MS identification of antioxidant ingredients

The three components are respectively subjected to liquid mass analysis according to the optimized HPLC condition and TOF-MS condition, and the screened antioxidant active ingredients are identified according to reference documents, and the results are shown in Table 1. Among them, 12 active ingredients were preliminarily identified. In addition, as shown in table 1 by combining the spectral characteristics of various compounds, the antioxidant component selected from honeysuckle flower is mainly organic acids and contains partial flavonoids, terpenes and the like.

TABLE 1 identification of antioxidant component in honeysuckle

Table 1 Identification of Antioxidant components in honeysuckle

2.4 Activity validation

Evaluation of the activity of individual compounds was achieved by compound in vitro DPPH free radical scavenging assay. And verifying the established screening method of the trace antioxidant active ingredients in the honeysuckle by utilizing the evaluation result. Combining screening results and economic applicability, this study chose to determine antioxidant activity and IC of 9 of these compounds₅₀Values, see table 2. The measurement result shows that the 9 compounds have antioxidant activity and show good concentration dependence in a certain range. Wherein IC of isochlorogenic acid B₅₀The value was the smallest (19.0. mu.M), indicating the best antioxidant activity. Comparison of IC of the Compounds₅₀The antioxidant activity sequence obtained by the values is as follows: isochlorogenic acid B > isochlorogenic acid A > 1, 3-dicaffeoylquinic acid > isochlorogenic acid C > rutin > cryptochlorogenic acid > chlorogenic acid > vitamin C > neochlorogenic acid > luteolin. From the evaluation result of the antioxidant activity, the honeysuckle contains a plurality of antioxidant active ingredients with better activity, and in addition, the antioxidant activity of the honeysuckle is the result of the combined action of the plurality of antioxidant ingredients contained in the honeysuckle.

TABLE 2 verification of the Activity of antioxidant ingredients in Lonicera japonica

Table 2 Verification the Antioxidant activity of component in honeysuckle

a represents the clearance rate of DPPH when the sample concentration is 20mg/L

The above description is only a preferred embodiment of the present disclosure and is not intended to limit the present disclosure, and various modifications and changes may be made to the present disclosure by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present disclosure should be included in the protection scope of the present disclosure.

Claims

1. A method for screening antioxidant components in honeysuckle is characterized by comprising the following steps: performing segmented enrichment and analysis on the honeysuckle extract by adopting a two-dimensional chromatogram, and screening antioxidant components in the honeysuckle through online antioxidant;

the segmented enrichment is as follows: dividing the honeysuckle extract into a plurality of sections of components by two-dimensional chromatography, drying and redissolving to obtain a concentrated extract; the crude extract is in powder form and is prepared by the following steps: mixing the raw materials in a ratio of 1 g: adding 80-120 ml of methanol solution, ultrasonically extracting active ingredients in the methanol solution, and concentrating the extract in a redissolution mode after drying;

the first dimension of the two-dimensional chromatogram is a semi-preparative high performance liquid chromatogram which is used for segmenting the honeysuckle extract; the semi-preparative high performance liquid chromatography is used for segmenting the honeysuckle extract and dividing the honeysuckle extract into three segments by adopting a gradient elution mode: fraction a, fraction B, fraction C; the mobile phase of the gradient elution is: a-water containing 0.2% formic acid and B-acetonitrile; the gradient elution degree is as follows: 0min, 10% B₁，0~20 min，10%~40%B₁；20-30 min，40%~50%B₁；30~31 min，50%~100%B₁；

The second dimension of the two-dimensional chromatogram is a reverse phase chromatographic column used for analyzing fraction A, fraction B and fraction C; the mobile phase of the fraction A is A: water-0.8% formic acid, 10mM ammonium formate, B: acetonitrile; separating the fraction A by adopting a gradient elution mode; the column was XBridge Amide, elution gradient: 96% -93% B in 0-5min, 93% -93% B in 5-20min, 93% -85% B in 20-30min, 85% -82% B in 30-40min, 82% -78% B in 40-50min, and 50% B in 51 min;

the mobile phase of the fraction B is A: water-0.6% formic acid, B: acetonitrile-10% water; separating the fraction B by adopting a gradient elution mode; the column was an Eclipse Plus C18 column, elution gradient: 0-5min 5% -6% B,5-15min 6% -8% B,15-25min 8% -10% B,25-40min 10% -13% B,40-45min 13% -15% B,45-55min 15% -17% B,55-65min 17% -30% B,65-70min 30% -45% B;

the mobile phase of the fraction C is A: water-0.8% formic acid, 10mM ammonium formate, B: acetonitrile; separating the fraction C by adopting a gradient elution mode; the column was an XSELECT HST 3 column, elution gradient: 14% -17% of B in 0-10min, 17% -18% of B in 10-20min, 18% -19% of B in 20-30min, 19% -23% of B in 30-50min, 23% -28% of B in 50-65min, and 28% -35% of B in 65-75 min.

2. The method for screening the anti-oxidation ingredients in the honeysuckle according to claim 1, wherein the screening method further comprises analyzing the fraction A, the fraction B and the fraction C respectively by mass spectrometry; the mass spectrum adopts electrospray positive and negative ion full-scanning detection; the full scanning range m/z is 50-1500; taper hole voltage: 60V; capillary voltage: 5.0 kV; cleavage voltage: 120V; spraying air pressure: 45 psi; flow rate of drying gas: 8.0L/min; temperature of the drying gas: at 200 ℃.