Coreopsis pinnata extract and spectrum-activity relationship evaluation method

Abstract

The invention discloses a coreopsis tinctoria extract and a spectrum-effect relationship evaluation method, the coreopsis tinctoria extract is obtained by extracting coreopsis tinctoria with water or 10-95% by volume of an ethanol aqueous solution, and the coreopsis tinctoria extract contains 10-35% by weight of total flavonoids, 10-45% by weight of total polysaccharides and 1-30% by weight of total organic acids; according to the method for evaluating the chemical components of the coreopsis tinctoria medicinal material based on the antioxidant spectrum-effect relationship, the chemical components with the antioxidant effect in the coreopsis tinctoria can be rapidly and accurately evaluated, and a scientific and effective method is provided for basic research and quality control of pharmacodynamic substances of the coreopsis tinctoria.

Description

Technical Field

[0001] This application generally belongs to the field of natural medicine analysis, and specifically relates to Acroptilon repens extract and the evaluation method of spectrum-effect relationship. Background Art

[0002] Acroptilon repens (L.) DC., also known as Artemisia bitter, is the aerial part of the plant Acroptilon repens of the Compositae family. It tastes pungent and bitter, and is cold in nature. Its whole herb can be used as medicine, with the effects of clearing heat and detoxifying, promoting blood circulation and reducing swelling. Acroptilon repens mainly contains sesquiterpene lactones, alkaloids, flavonoids, volatile oils and other compounds. At present, there are few research reports on the chemical constituents of Acroptilon repens in China, mainly focusing on flavonoid components. At present, there are few research reports on the pharmacological effects of Acroptilon repens, mainly in the fields of antibacterial, insecticidal, anti-cancer activity, allergy and phytotoxicity. At present, it is urgent to study the effective parts of Acroptilon repens and their spectrum-effect relationship. Summary of the Invention

[0003] One or more embodiments of this application provide an Acroptilon repens extract, which is obtained by extracting Acroptilon repens with water or an ethanol aqueous solution of 10% - 95% (such as 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%) by volume. The total flavonoid content is 10% - 35% (such as 10%, 20%, 30%) by weight, the total polysaccharide content is 10% - 45% (such as 10%, 20%, 30%, 40%) by weight, and the total organic acid content is 1% - 30% (such as 10%, 20%, 30%) by weight.

[0004] In one or more embodiments, the extract is obtained by extracting Acroptilon repens with an ethanol aqueous solution of 30% - 75% by volume, where the total flavonoid content is 10% - 30% by weight, the total polysaccharide content is 10% - 30% by weight, and the total organic acid content is 1% - 20% by weight.

[0005] In one or more embodiments, the extract is obtained by extracting Acroptilon repens with an ethanol aqueous solution of 30% - 60% by volume, where the total flavonoid content is 10% - 30% by weight, the total polysaccharide content is 15% - 30% by weight, and the total organic acid content is 5% - 20% by weight.

[0006] In one or more embodiments, the extract is prepared by the following method: Acroptilon repens is extracted with an aqueous ethanol solution of 45% - 55% by volume, and the extract is obtained by extraction with ethyl acetate or n-butanol, or the ethyl acetate extract and the n-butanol extract are combined to obtain the extract. The total flavonoid content of the extract is 15 - 30% by weight, the total polysaccharide content is 15 - 30% by weight, and the total organic acid content is 10 - 20% by weight.

[0007] In one or more embodiments, in the Acroptilon repens extract:

[0008] The content of neochlorogenic acid is 0.01 - 1% by weight (e.g., 0.5% by weight),

[0009] The content of chlorogenic acid is 0.1 - 3% by weight (e.g., 0.5, 1, 2% by weight),

[0010] The content of cryptochlorogenic acid is 0.1 - 1.5% by weight (e.g., 0.1, 1, 1.5% by weight),

[0011] The content of 1,5-dicaffeoylquinic acid is 0.05 - 1% by weight (e.g., 0.5% by weight),

[0012] The content of isochlorogenic acid C is 0.01 - 0.2% by weight (e.g., 0.1% by weight),

[0013] The content of apigenin is 0.01 - 2% by weight (e.g., 0.1% by weight),

[0014] The content of hispidulin is 0.01 - 2% by weight (e.g., 0.1% by weight).

[0015] In one or more embodiments, the Acroptilon repens extract is prepared by the following method:

[0016] Acroptilon repens is extracted with water or an aqueous ethanol solution of 10% - 95% by volume (e.g., 20% by volume, 30% by volume, 40% by volume, 50% by volume, 60% by volume, 70% by volume, 80% by volume, 90% by volume); for example, it is extracted with an aqueous ethanol solution of 30% - 75% by volume, 30% - 60% by volume, or 45% - 55% by volume.

[0017] In one or more embodiments, the extraction temperature is 50 - 95 °C (e.g., 60, 70, 80 °C), and the extraction is carried out 1, 2, or 3 times, with the time being 1, 2, or 3 hours per time; the Acroptilon repens extract is obtained.

[0018] In one or more embodiments, the filtrate after extraction is concentrated under reduced pressure to a relative density of 1.0 - 1.5 (e.g., 1.2), and the concentrated solution is dried.

[0019] In one or more embodiments, it is dried in a vacuum drying oven with a vacuum degree of -0.08 MPa and a temperature of 50 - 80 °C (such as 60, 70, 80 °C) to obtain a dried extract. The dried extract is suspended in water and extracted with ethyl acetate or n-butanol to obtain an extract, or the ethyl acetate extract and the n-butanol extract are combined to obtain an extract, thereby obtaining the acroptilon repens extract.

[0020] In one or more embodiments, the extract is suspended in water and purified with macroporous resin, polyamide resin or nylon-66 resin to obtain the acroptilon repens extract.

[0021] One or more embodiments of the present application provide a method for preparing the acroptilon repens extract of the present application, which includes:

[0022] In one or more embodiments, acroptilon repens is extracted with water or an ethanol aqueous solution of 10 vol% - 95 vol% (such as 20 vol%, 30 vol%, 40 vol%, 50 vol%, 60 vol%, 70 vol%, 80 vol%, 90 vol%) to obtain the acroptilon repens extract.

[0023] In one or more embodiments, acroptilon repens is extracted with an ethanol aqueous solution of 30 vol% - 75 vol%, 30 vol% - 60 vol%, or 45 vol% - 55 vol%; the extraction temperature is 50 - 95 °C, and the extraction is carried out 1, 2, or 3 times with a time of 1, 2, or 3 hours per time; the acroptilon repens extract is obtained.

[0024] In one or more embodiments, the filtrate after extraction is concentrated under reduced pressure to a relative density of 1.0 - 1.5 (such as 1.2), and the concentrated solution is dried.

[0025] In one or more embodiments, it is dried in a vacuum drying oven with a vacuum degree of -0.08 MPa and a temperature of 50 - 80 °C (such as 60, 70, 80 °C) to obtain a dried extract. The dried extract is suspended in water and extracted with ethyl acetate or n-butanol to obtain an extract, or the ethyl acetate extract and the n-butanol extract are combined to obtain an extract, thereby obtaining the acroptilon repens extract.

[0026] In one or more embodiments, the extract is suspended in water and purified with macroporous resin, polyamide resin or nylon-66 resin to obtain the acroptilon repens extract.

[0027] One or more embodiments of the present application provide the use of the acroptilon repens extract of the present application in the preparation of a drug for antioxidation.

[0028] One or more embodiments of the present application provide a method for evaluating the extract of Acroptilon repens based on the antioxidant spectrum-activity relationship, which includes the following steps:

[0029] (1) Prepare the extract of Acroptilon repens; for example, extract Acroptilon repens by heating under reflux with water, ethanol at 30% by volume, 50% by volume, 75% by volume, and 95% by volume, respectively, to obtain extracts S1 - S5 in sequence; dissolve extracts S3 and S5 in water respectively, and extract them successively with petroleum ether, ethyl acetate, and n-butanol at 60 - 90 °C, recover the aqueous phase, and obtain extracts S6 - S12; the extracts S1 - S12 are as follows:

[0030]

[0031] (2) Establish the HPLC fingerprint of extracts S1 - S12; for example, the chromatographic column is Agilent Eclipse XDB-C 18 、YMC-Pack ODS-A C 18 or Agilent ZORBAX SB-C 18 , the mobile phase is 0.1% formic acid aqueous solution - acetonitrile, and the detection wavelength is 327 nm; for example, a fingerprint can be established through 22 common peaks in chromatography, and 7 of them can be confirmed.

[0032] (3) Determine the antioxidant effect of extracts S1 - S12; for example, the DPPH radical scavenging rate and total reducing ability of extracts S1 - S12 can be measured.

[0033] (4) Conduct spectrum-activity analysis of extracts S1 - S12 and antioxidant; for example, grey relational analysis and partial least squares analysis can be performed on extracts S1 - S12.

[0034] (5) Based on the analysis results of grey relational analysis and partial least squares analysis, determine and verify the effective parts and effective chemical components of the extract.

[0035] In one or more embodiments, in the above step (2), the chromatographic column is 250 mm × 4.6 mm with a particle size of 5 μm; elution gradient: 0 - 5 min, 5% acetonitrile; 5 - 15 min, 5% - 10% acetonitrile; 15 - 20 min, 10% - 14% acetonitrile; 20 - 80 min, 14% - 26% acetonitrile; 80 - 113 min, 26% - 38% acetonitrile; 113 - 113.01 min, 38% - 5% acetonitrile; 113.01 - 120 min, 5% acetonitrile; column temperature 30 °C, injection volume 10 μL, volume flow rate 1.0 mL / min.

[0036] In one or more embodiments, the chemical components corresponding to the seven peaks are neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, 1,5-dicaffeoylquinic acid, isochlorogenic acid C, apigenin, and homoplantaginin.

[0037] In one or more embodiments, in the above step (3), the method for determining the DPPH free radical scavenging rate includes: preparing a test solution, preparing a vitamin C positive control solution, using 50% methanol plus DPPH solution as blank group A0, different mass concentration sample solutions plus DPPH solution as sample group A1, different mass concentration sample solutions plus methanol as background group A2, reacting in the dark, measuring the absorbance A value at a wavelength of 517 nm using an enzyme-linked immunosorbent assay (ELISA) reader, calculating according to the following formula, DPPH free radical scavenging rate (%) = [1 - (A1 - A2) / A0] × 100%, using concentration as the abscissa and scavenging rate as the ordinate, calculating the linear regression equation, and calculating the half scavenging concentration IC of each sample for DPPH free radicals 50 ;

[0038] The method for determining the total reducing ability includes: preparing a test solution, preparing a vitamin C positive control solution, respectively adding phosphate buffer solution, 1% potassium ferricyanide solution, 10% trichloroacetic acid solution, and 0.1% ferric chloride solution, measuring the absorbance value at 700 nm, using 50% methanol as the blank control, plotting with different mass concentrations of the test sample as the abscissa and absorbance as the ordinate to obtain a linear equation, and calculating the absorbance value at a concentration of 1.0 mg / mL.

[0039] In one or more embodiments, in the above step (4), the calculation formula for the grey relational grade is where r i is the correlation degree between the mother sequence x0 and the child sequence xi, N is the number of data in the child sequence, ξi is the correlation coefficient, k is different extracts, and calculate the correlation degree of the chemical components represented by the common peaks;

[0040] Using the relative peak areas of the common peaks of extracts S1 - S12 as the abscissa, and the DPPH free radical scavenging rate and the absorbance of the total reducing ability as the ordinates respectively, perform partial least squares regression (PLSR) analysis, calculate the standardized regression coefficients and variable projection importance values of the relative peak areas of the common peaks with the DPPH free radical scavenging rate and the absorbance of the total reducing ability, and after standardizing the data, evaluate the prediction and interpretation of the established model.

[0041] In one or more embodiments, in the above step (5), based on the analysis results of grey relational grade analysis and partial least squares analysis, determine the contributions of the extract parts and the chemical components represented by the common peaks to the antioxidant activity of Acroptilon repens.

[0042] One or more embodiments of the present application disclose a method for evaluating the pharmacological activity of the chemical constituents of Acroptilon repens, specifically relating to a method for evaluating the chemical constituents of Acroptilon repens based on the antioxidant spectrum-activity relationship.

[0043] One or more embodiments of the present application disclose a method for evaluating the chemical constituents of Acroptilon repens herbs based on the antioxidant spectrum-activity relationship. The method includes preparing extracts of different polar components of Acroptilon repens by using modern separation techniques; establishing fingerprint chromatograms of each component extract by using high-performance liquid chromatography and calibrating characteristic peaks; evaluating the antioxidant activity of different extracts by using the DPPH free radical scavenging rate and total reducing power as indicators; and bringing the fingerprint chromatogram characteristic peak data and pharmacological activity data into a mathematical model for spectrum-activity correlation analysis to evaluate the pharmacological activity of the characteristic peaks. The method for evaluating the chemical constituents of Acroptilon repens based on the antioxidant spectrum-activity relationship can quickly and accurately evaluate the chemical constituents with antioxidant effects in Acroptilon repens, provide a scientific and effective method for the study of the pharmacological substance basis and quality control of Acroptilon repens, and provide a reference for the further development of drugs or health foods using Acroptilon repens for antioxidant effects.

[0044] In one or more embodiments, the use of fingerprint chromatograms is to characterize the chemical constituents in traditional Chinese medicine, which can overall control the quality of traditional Chinese medicine. By combining the fingerprint chromatogram and the pharmacological indexes of traditional Chinese medicine, correlation analysis is performed on the fingerprint chromatogram and the pharmacological indexes through some chemometric analysis methods such as partial least squares method, bivariate correlation analysis method, and grey relational analysis method. That is, based on the "spectrum-activity" relationship, the fingerprint chromatogram and the pharmacological activity data are correlated, and the potential pharmacological active components of Acroptilon repens with antioxidant effects are integrally analyzed, realizing the organic combination of the chemical constituents characterized by the traditional Chinese medicine fingerprint chromatogram and the pharmacological research, and enhancing the consistency of the "spectrum" and the "activity".

[0045] In one or more embodiments, the grey relational analysis method and partial least squares method (PLS) are used to evaluate the contribution size and correlation degree of each component of Acroptilon repens to the pharmacological effect, find the pharmacological effect-related components that can reflect its internal quality, determine the effective part of Acroptilon repens by analyzing its spectrum-activity relationship, be used for the study of the material basis and quality evaluation of the effective part of Acroptilon repens, and investigate the spectrum-activity relationship of the antioxidant pharmacological activity of Acroptilon repens.

[0046] In one or more embodiments, the preparation method of the Acroptilon repens extract includes the following steps:

[0047] 1) Take the above-ground part of Acroptilon repens that is dried, crushed, and passed through a 10-mesh sieve, place it in a round-bottom flask, add an ethanol aqueous solution with a volume concentration of 10-95% by weight 15 times the volume for extraction, heat it in a water bath at a temperature of 50-95°C, reflux and extract 1-3 times, with a time of 1-3 hours per extraction, and concentrate the extracted filtrate under reduced pressure to a relative density of 1.0-1.5 for standby;

[0048] 2) drying the extract obtained in step 1) in a vacuum drying oven at a vacuum degree of -0.08 MPa and a temperature of 50 to 80° C. to obtain an extract for later use;

[0049] 3) The extract obtained in step 2) is suspended in purified water, and then extracted with equal volumes of petroleum ether, ethyl acetate and n-butanol respectively to obtain extracts of ethyl acetate and n-butanol, which are concentrated to dryness to obtain extracts of ethyl acetate and n-butanol, and the extracts are suspended in purified water, purified with pretreated macroporous resin, polyamide resin or nylon-66 resin, eluted by adsorption, and the eluted components are collected, concentrated to dryness, and dried in vacuo to obtain the extract of Chrysanthemum vulgare.

[0050] In one or more embodiments, the antioxidant activity experimental results show that the 12 kinds of chrysanthemum extracts of the present application have a scavenging effect on DPPH free radicals, among which the n-butanol part of the 50% ethanol extract of chrysanthemum and the ethyl acetate part of the 50% ethanol extract of chrysanthemum have the strongest activity, and their IC 50 The values ​​were 66.96μg / mL and 67.16μg / mL respectively; the ability of the 12 kinds of chrysanthemum extracts to scavenge free radicals gradually increased with the increase of concentration. All 12 kinds of chrysanthemum extracts had strong reducing ability, among which the ethyl acetate part of the 50% ethanol extract of chrysanthemum had the strongest activity, indicating that the chrysanthemum extract can provide electrons as an antioxidant and has strong reducing properties. The experimental results showed that the ethyl acetate part of the 50% ethanol extract of chrysanthemum had the strongest antioxidant effect.

[0051] In one or more embodiments, the research is conducted by finding the connection between the fingerprint characteristics of the "effective substance group" and the antioxidant effect. Based on the fingerprint spectra and efficacy studies of extracts of different solvents of Chrysanthemum, the peak areas of 22 common peaks in the fingerprint spectra of extracts of Chrysanthemum with different polarities and the quantitative data of in vitro antioxidant efficacy are obtained. Two chemometric methods (GRA and PLSR) are used to associate the "spectrum" and "efficacy" data of extracts of Chrysanthemum with different polarities to model the contribution of the chemical components represented by the common peaks in the fingerprint spectra to the efficacy, and then the antioxidant effect is inferred according to the degree of contribution of each common peak in the fingerprint spectra of extracts of Chrysanthemum with different polarities to the in vitro antioxidant effect, thereby screening out possible antioxidant active ingredient groups.

[0052] In one or more embodiments, the relative retention time method is used to examine the location of the chromatographic peaks. The Agilent Eclipse XDB-C 18 、YMC-Pack ODS-A C 18 、Agilent ZORBAX SB-C 18 、Cosmosil 5C 18-AR-II, Agilent 5TC-C 18 5 columns, discover Cosmosil 5C 18 -AR-II, Agilent5TC-C 18 The chromatographic peaks of neochlorogenic acid, isochlorogenic acid C and apigenin could not be separated well. After repeated experiments, Agilent Eclipse XDB-C 18 、YMC-Pack ODS-A C 18 、Agilent ZORBAX SB-C 18 Three chromatographic columns were used for the test. The relative retention time RSDs of the three chromatographic columns were all less than 5%, and the peak order of the seven components did not change.

[0053] In one or more embodiments, the aqueous phase of the mobile phase of the chromatographic elution method is acid water, and the proportion of the aqueous phase is relatively large. The test results show that the above three chromatographic columns are more suitable for separating the various components in the test solution of the medicinal material of Chrysanthemum indica, and the separation effect is better.

[0054] In one or more embodiments, firstly, HPLC fingerprints of extracts of different polarities of Herba Cynomorii are established, and the chemical components of the Herba Cynomorii extracts are analyzed by UPLC-Q-TOF-MS / MS; secondly, DPPH free radical scavenging rate and total reducing capacity are used as indicators to evaluate the antioxidant activity of different extracts and their antioxidant effects; finally, grey correlation analysis and partial least squares method are used to correlate the common peak data of the fingerprints with in vitro pharmacodynamic tests, and an antioxidant "spectrum-effect" relationship model of Herba Cynomorii is established, and the potential pharmacologically active ingredients of Herba Cynomorii antioxidant are integrated and analyzed, and the contribution of each component of Herba Cynomorii to the pharmacological efficacy and the degree of correlation are evaluated, so as to clarify the active ingredients in Herba Cynomorii that exert antioxidant activity more clearly than in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 This is the superimposed fingerprint of the extract of Chrysanthemum officinale.

[0056] Figure 2 The characteristic peaks of the fingerprint of the extract of Chrysanthemum truncatum are attributed, where A: mixed reference, B: extract S3, C: extract S7; Peak 1 is neochlorogenic acid, Peak 2 is chlorogenic acid (reference peak), Peak 3 is cryptochlorogenic acid, Peak 10 is 1,5-dicaffeoylquinic acid, Peak 15 is isochlorogenic acid C, Peak 20 is apigenin, and Peak 21 is homoplanin.

[0057] Figure 3 The PLSR analysis results of 22 common peaks and antioxidant activity in the extract of Aster strychnifolia, where A1, VIP value of DPPH free radical scavenging rate; B1, R2 Figure; A2, VIP value graph of the absorbance value of the total reducing ability; B2, R of the absorbance value of the total reducing ability 2 Figure.

[0058] Figure 4 Results of the DPPH free radical scavenging rate test for vitamin C positive control (A) and monomeric compounds (B - D) in Acroptilon repens; where A is vitamin C; B is chlorogenic acid; C is neochlorogenic acid; D is cryptochlorogenic acid.

[0059] Figure 5 Results of the total reducing ability determination test for vitamin C positive control (A) and monomeric compounds (B - D) in Acroptilon repens; where A is vitamin C; B is chlorogenic acid; C is neochlorogenic acid; D is cryptochlorogenic acid. Detailed implementation manners

[0060] Examples

[0061] The present application will be described below in conjunction with specific examples. The following examples are illustrative descriptions of the present application and do not constitute a limitation on the protection scope of the present application.

[0062] Instruments and materials

[0063] Instruments

[0064] Cytation3 multi-functional microplate reader (Thermo Fisher Scientific, USA); FD-1A 50 freeze dryer (Shanghai Bilang Instrument Manufacturing Co., Ltd.); Agilent-1260 high performance liquid chromatograph (Agilent Technologies, USA); CPA-225D electronic balance (Sartorius, Germany, d = 0.01 mg); JY-5002 electronic balance (Shanghai Tianjing Precision Instruments, d = 0.01 g); AS20500BDT ultrasonic cleaner (frequency 40 kHz, power 500 W, Tianjin Outsen Instruments Co., Ltd.); UPT-10T UltraPure experimental ultrapure water machine (Chengdu UltraPure Technology Co., Ltd.).

[0065] Materials

[0066] The Acroptilon repens medicinal material (batch number: 20210903) was collected from Urumqi, Xinjiang. It was identified by Researcher He Jiang of the Xinjiang Uygur Autonomous Region Institute of Materia Medica as the aerial part of Acroptilon repens (also known as Artemisia santolina L.), a plant of the genus Acroptilon in the Compositae family. The reference standards of neochlorogenic acid (batch number DSTDX001504, mass fraction 98.80%), cryptochlorogenic acid (batch number DST221220-035, mass fraction 99.17%), 1,5-dicaffeoylquinic acid (batch number DSTDE000602, mass fraction 98.42%), and hispidulin (batch number DSTDG002601, mass fraction 99.23%) were all purchased from Chengdu Det Biotechnology Co., Ltd.; chlorogenic acid (batch number 110753-202018, mass fraction 96.1%) and apigenin (batch number 111901-201102, mass fraction 99.6%) were purchased from the National Institutes for Food and Drug Control; isochlorogenic acid C (batch number MUST-21081010, mass fraction 99.77%) was purchased from Chengdu Mansite Biotechnology Co., Ltd.; 1,1-diphenyl-2-picrylhydrazyl (DPPH, batch number 1226J021) was purchased from Solarbio; acetonitrile was of chromatographic grade and was purchased from Fisher Scientific, USA; ultrapure water was pure water from C'estbon, and other chemical reagents were all of analytical grade.

[0067] Example 1 Preparation of Acroptilon repens Extracts with Different Polarities and Determination of Total Content

[0068] Weigh the Acroptilon repens medicinal material. After pulverization, extract it with 6 - 15 times the volume of distilled water, 30% (v / v), 50% (v / v), 75% (v / v), and 95% (v / v) ethanol by heating under reflux for 1 - 4 times, each time for 0.5 - 2.5 h. Filter, combine the extraction solutions respectively, concentrate, and dry to obtain 5 Acroptilon repens extracts, denoted as S1 - S5. Then weigh the Acroptilon repens extracts S3 (50% ethanol extract) and S5 (95% ethanol extract). After dissolving them in water respectively, shake and extract them successively with petroleum ether (60 - 90 °C), ethyl acetate, and n-butanol. Recover the above extraction solutions and the aqueous phase after extraction respectively, and dry to obtain 7 Acroptilon repens extracts, denoted as S6 - S12, as detailed in Table 1-1.

[0069] Table 1-1 Acroptilon repens Extracts with Different Polarities

[0070]

[0071] Determination of Total Flavonoid Content

[0072] Preparation of reference standard solution: Weigh an appropriate amount of rutin reference standard accurately, dissolve it in 50% methanol to make a solution containing 0.2042 mg per 1 mL, and that's it.

[0073] Preparation of test solution: Accurately weigh about 20 mg of each extract, place it in a 25-mL volumetric flask, add an appropriate amount of 50% methanol, ultrasonicate for 30 min to dissolve, cool to room temperature, and dilute to the mark with 50% methanol. Shake well to obtain the solution.

[0074] Determination method: Accurately pipette 6 mL of 50% methanol solution, reference solution, and test solution into a 25-mL volumetric flask respectively. Add 1 mL of 5% sodium nitrite solution, allow to stand for 6 min, add 1 mL of 10% aluminum nitrate solution, allow to stand for 6 min, add 10 mL of sodium hydroxide test solution, dilute to the mark with 50% methanol, allow to stand for 15 min. Using the corresponding solution as the blank, determine the absorbance at the wavelength of 510 nm according to the general rule of ultraviolet-visible spectrophotometry 0401 (Volume IV of Chinese Pharmacopoeia 2020 Edition). Calculate the content of total flavonoids in the test sample.

[0075] Determination of total polysaccharide content

[0076] Preparation of reference solution: Take an appropriate amount of D-anhydroglucose reference substance, accurately weigh it, and dissolve it in 50% methanol to prepare a solution containing 0.08152 mg per 1 mL.

[0077] Preparation of test solution: Accurately weigh about 20 mg of each extract, place it in a 25-mL volumetric flask, add an appropriate amount of 50% methanol, ultrasonicate for 30 min to dissolve, cool to room temperature, and dilute to the mark with 50% methanol. Shake well to obtain the solution.

[0078] Determination method: Accurately pipette 1 mL of 50% methanol solution, reference solution, and test solution into a stoppered test tube respectively. Add 1 mL of water and 6 mL of 0.2% sulfuric acid-anthrone solution, heat in boiling water for 15 min, take out, and place in cold water for 15 min. Using the corresponding solution as the blank, determine the absorbance at the wavelength of 620 nm according to the general rule of ultraviolet-visible spectrophotometry 0401 (Volume IV of Chinese Pharmacopoeia 2020 Edition). Calculate the content of total polysaccharides in the test sample.

[0079] Determination of total organic acid content

[0080] Preparation of reference solution: Take an appropriate amount of chlorogenic acid reference substance, accurately weigh it, and dissolve it in 50% methanol to prepare a solution containing 0.2473 mg per 1 mL.

[0081] Preparation of test solution: Accurately weigh about 20 mg of each extract, place it in a 25-mL volumetric flask, add an appropriate amount of 50% methanol, ultrasonicate for 30 min to dissolve, cool to room temperature, and dilute to the mark with 50% methanol. Shake well to obtain the solution.

[0082] Assay: Appropriately pipette the reference solution and the test solution, dilute with 50% methanol to an appropriate concentration, use the corresponding solution as the blank, and determine the absorbance at the wavelength of 327 nm according to the general rules of ultraviolet-visible spectrophotometry 0401 (Volume IV of Chinese Pharmacopoeia 2020 Edition). Calculate the content of total organic acids in the test sample.

[0083] The determination results of the total content of the effective parts and the content of chemical components in the three extracts (determined by HPLC) are shown in the following table:

[0084] Table 1-2 Total content of effective parts and content of chemical components in three extracts

[0085]

[0086] Establishment of fingerprint in Example 2

[0087] Chromatographic conditions: The chromatographic column is Agilent Eclipse XDB-C 18 (250 mm × 4.6 mm, 5 μm particle size); the mobile phase is 0.1% formic acid aqueous solution (A) - acetonitrile (B), elution gradient: 0 - 5 min, 5% B; 5 - 15 min, 5% - 10% B; 15 - 20 min, 10% - 14% B; 20 - 80 min, 14% - 26% B; 80 - 113 min, 26% - 38% B; 113 - 113.01 min, 38% - 5% B; 113.01 - 120 min, 5% B; column temperature 30 °C, injection volume 10 μL, volume flow rate 1.0 mL / min, detection wavelength 327 nm.

[0088] Preparation of test solution: Precisely weigh 100 mg of each of the Acroptilon repens L. extracts S1 - S12, place them in 10 mL volumetric flasks respectively, add an appropriate amount of 50% methanol, ultrasonically treat for 30 minutes, cool to room temperature, dilute to the mark with 50% methanol, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0089] Preparation of mixed reference solution: Precisely weigh appropriate amounts of reference substances of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, 1,5 - dicaffeoylquinic acid, isochlorogenic acid C, apigenin, and homoplantaginin respectively, and prepare a mixed reference solution with methanol at mass concentrations of 4.34 μg / mL, 51.13 μg / mL, 11.04 μg / mL, 51.70 μg / mL, 11.62 μg / mL, 109.35 μg / mL, and 100.23 μg / mL respectively.

[0090] Instrument precision test: The test solution prepared from the 50% ethanol extract of Acroptilon repens (serial number: S3) was continuously injected 6 times under the above chromatographic conditions. Using the 2nd peak as the reference peak, the RSD values of the relative retention time and relative peak area of each common peak were calculated. The results showed that the RSD values of the relative retention time of each common peak were all less than 0.38% (n = 6), and the RSD values of the relative peak area were all less than 2.53% (n = 6), indicating that the precision of this instrument was good.

[0091] Stability test: The test solution prepared from the 50% ethanol extract of Acroptilon repens (serial number: S3) was injected for determination at 0, 2, 4, 6, 8, 10, 12, and 24 hours respectively under the above chromatographic conditions. The RSD values of the relative retention time of each common peak were all less than 0.33% (n = 8), and the RSD values of the relative peak area were all less than 2.90% (n = 8), indicating that the test solution was stable within 24 hours.

[0092] Repeatability test: Six portions of the 50% ethanol extract of Acroptilon repens (serial number: S3) were weighed, and six test solutions were prepared according to the above method. Determination was carried out under the above chromatographic conditions. The RSD values of the relative retention time of each common peak were all less than 0.34%, and the RSD values of the relative peak area were all less than 2.98%, indicating that the method had good repeatability.

[0093] Fingerprint generation and characteristic peak confirmation: The test solutions of 12 Acroptilon repens extracts prepared by the above method were analyzed under the above chromatographic conditions, and the data were imported into the "Similarity Evaluation Software for Traditional Chinese Medicine Chromatographic Fingerprints" (2012 version) in AIA format. The S3 sample was set as the reference chromatogram (median method, time window 0.2). After multi-point calibration and automatic peak matching, a control chromatogram (R) was generated, and a total of 22 common peaks were calibrated, as shown in Figure 1 . By comparing with the reference substances, 7 common peaks were identified, as shown in Figure 2 . Among them, the 1st peak was neochlorogenic acid, the 2nd peak was chlorogenic acid (reference peak), the 3rd peak was cryptochlorogenic acid, the 10th peak was 1,5-dicaffeoylquinic acid, the 15th peak was isochlorogenic acid C, the 20th peak was apigenin, and the 21st peak was homoplantaginin. Similarity analysis was carried out on the control fingerprint and extracts with different polarities. The results are shown in Table 2-1. The similarities of 12 Acroptilon repens extracts with different polarities were 0.202 - 0.981, indicating that there were significant differences in their chemical compositions.

[0094] Table 2 Similarities of Acroptilon repens extracts

[0095]

[0096] Example 3 Antioxidant effect of Acroptilon repens extract

[0097] Determination of DPPH free radical scavenging rate

[0098] Preparation of test solution: Accurately weigh 20 mg each of the acroptilon repens extracts S1 - S12, place them in 10 mL volumetric flasks, add an appropriate amount of 50% methanol, ultrasonically treat for 30 minutes, cool to room temperature, dilute to the mark with 50% methanol, filter, and prepare a test solution stock solution with a concentration of 2 mg / mL. Appropriately pipette the above test solution stock solution into 10 mL volumetric flasks, and dilute with 50% methanol to prepare a series of concentration sample solutions (27.21 μg / mL - 1413.82 μg / mL).

[0099] Preparation of positive control solution: Accurately weigh 8 mg of vitamin C powder into a 100 mL volumetric flask, dissolve with 50% methanol and dilute to the mark to prepare a Vc stock solution with a concentration of 0.08 mg / mL. Appropriately pipette the Vc stock solution into 10 mL volumetric flasks, and dilute with 50% methanol to prepare a series of concentration control solutions (2.69 μg / mL - 13.43 μg / mL).

[0100] Preparation of DPPH solution: Accurately weigh 4 mg of DPPH reference substance, place it in a 100 mL brown volumetric flask, dilute to the mark with methanol to prepare a 0.04 mg / mL solution, and store it as a stock solution in a refrigerator at 4°C for later use.

[0101] Determination: Mix 300 μL of 50% methanol + 750 μL of DPPH solution (denoted as "blank group A0"), 300 μL of sample solutions with different mass concentrations + 750 μL of DPPH solution (denoted as "sample group A1"), and 300 μL of sample solutions with different mass concentrations + 750 μL of methanol (denoted as "background group A2") evenly respectively. Take 200 μL of each mixture and place it in a 96 - well plate, and react at room temperature in the dark for 30 min. Use an enzyme - linked immunosorbent assay (ELISA) reader to measure the absorbance (A) value of each well at a wavelength of 517 nm. Calculate according to the following formula: DPPH free radical scavenging rate (%) = [1 - (A1 - A2) / A0] × 100%. Each sample is determined in parallel 3 times, and the average value is taken. Take the concentration as the abscissa and the scavenging rate as the ordinate for regression. Calculate the half - maximal inhibitory concentration (IC 50 ) of each sample against DPPH free radicals. Use the IC 50 value as an index to evaluate the antioxidant strength of each acroptilon repens extract. The smaller the value, the stronger the in vitro antioxidant activity.

[0102] Determination of total reducing power

[0103] Accurately weigh 50 mg of each of the S1 - S12 sample powders, place them in 25 - mL volumetric flasks, add an appropriate amount of 50% methanol, sonicate for 30 minutes, cool to room temperature, dilute to the mark with 50% methanol, filter, and prepare a stock solution of the test substance at a concentration of 2 mg / mL. Respectively pipette 0.5 mL, 2.5 mL, 5 mL, and 7.5 mL of the above - mentioned stock solution of the test substance into 10 - mL volumetric flasks, and dilute with 50% methanol to prepare a series of sample solutions with concentrations (0.1 mg / mL - 2.0 mg / mL). Use vitamin C as a positive control, and prepare the positive control solution in the same way. Respectively take 2.5 mL of each acroptilon repens extract solution with different concentrations (0.1 mg / mL - 2.0 mg / mL) and the positive control solution, add 2.5 mL of phosphate buffer solution with pH = 6.6 and 2.5 mL of 1% potassium ferricyanide solution respectively, mix, incubate in a water bath at 50 °C for 20 min, then cool to room temperature in an ice bath, add 2.5 mL of 10% trichloroacetic acid solution and mix. Take 2.5 mL of the supernatant, successively add 2.5 mL of deionized water and 0.5 mL of 0.1% ferric chloride solution, mix well, let stand for 10 min, take 200 μL of each mixed solution into a 96 - well plate, and measure the absorbance at 700 nm. Use an equal amount of 50% methanol as a blank control, and measure each sample in parallel 3 times and take the average value. Plot a graph with the concentration of each acroptilon repens extract at different mass concentrations as the abscissa and the absorbance as the ordinate to obtain a linear equation, and calculate the absorbance value when the concentration is 1.0 mg / mL. The greater the absorbance, the stronger the reducing ability. The results are shown in Table 3.

[0104] Table 3 Test results of the scavenging of DPPH free radicals and reducing ability of each extract

[0105]

[0106] As shown in the results of Table 3, in the concentration range of 27.21 μg / mL - 1413.82 μg / mL, the different polar extracts of acroptilon repens all have the effect of scavenging DPPH free radicals, among which S8, S7, and S2 have the strongest activities, and their IC 50 values are 66.96 μg / mL, 67.16 μg / mL, and 86.91 μg / mL respectively; the ability of different extracts of acroptilon repens to scavenge free radicals gradually increases with the increase of concentration. Since antioxidants have strong reducing ability, they can reduce ferric iron in the body to ferrous iron, enhance the oxygen - transporting ability of hemoglobin, and can react with free radicals. In the concentration range of 0.1 - 2.0 mg / mL, different extracts of acroptilon repens all have strong reducing ability, among which the S7 and S8 groups have the strongest activities. When the concentration is 1.0 mg / mL, their absorbance values are 0.8615 and 0.6914 respectively; it shows that the acroptilon repens extract can provide electrons as an antioxidant and has strong reducibility.

[0107] Spectrum-Effect Analysis of Different Polar Extracts of Acroptilon repens and Their Antioxidant Activity

[0108] 4.1 Grey Relational Analysis (GRA)

[0109] 4.1.1 Determination of the Analysis Sequence

[0110] First, determine the mother sequence and the son sequence, also known as the reference sequence and the comparison sequence. Among them, the reference sequence is the data sequence reflecting the behavior characteristics of the system, and the comparison sequence is the data sequence composed of the factors affecting the system behavior. In this study, the active ingredients in Acroptilon repens were regarded as a whole, that is, a grey system. The reference sequence was the quantification index of the antioxidant effect of different solvent extracts of Acroptilon repens, and the comparison sequence was the quantification value of the relative peak area of the fingerprint characteristic peaks of different solvent extracts of Acroptilon repens, and the analysis and calculation were carried out.

[0111] First, select the reference sequence, denoted as x0(k)

[0112] x0(k) = [x0(l), x0(2), x0(3),...x0(k)], (k is different solvent extracts of Acroptilon repens, k = l, 2, 3,...n).

[0113] Select the comparison sequence, denoted as x i (k),

[0114] x i (k) = [x i (l), x i (2), x i (3)...x i (k)], (x i is the main characteristic component of different solvent extracts of Acroptilon repens, i = 1, 2,..., m, k is the same as above).

[0115] 4.1.2 Calculation of Grey Relational Coefficient

[0116] Grey relational coefficient:

[0117]

[0118] In the above formula,

[0119] k is different solvent extracts of Acroptilon repens;

[0120] xi is the main characteristic component of different solvent extracts of Acroptilon repens;

[0121] ξi is the relational coefficient between the subsequence xi of the kth Acroptilon repens extract and the mother sequence x0;

[0122] is the minimum difference between the two poles, also denoted as Δmin;

[0123] It is the maximum difference between two poles and is also denoted as Δmax;

[0124] It is the absolute difference between the mother sequence and the child sequence, and is also denoted as Δoi(k);

[0125] ρ is the resolution coefficient. In the calculation, since Δoi(k) is too large, it is likely to cause distortion. To weaken this distortion,

[0126] the resolution coefficient ρ is introduced. Its purpose is to improve the significance of the difference between the correlation coefficients, and its value range is ρ ∈ (0, 1), and generally 0.5 is taken.

[0127] 4.1.3 Method for calculating grey correlation degree

[0128] The arithmetic mean of the correlation coefficients is the correlation degree, and the calculation formula is:

[0129]

[0130] where r i is the correlation degree between the mother sequence x0 and the child sequence x i and N is the number of data in the child sequence.

[0131] 4.1.4 Correlation degree ranking

[0132] The correlation order is to rearrange the correlation degrees of the reference sequence and the comparison sequences according to their magnitudes. It directly reflects the correlation degree between each comparison sequence and the reference sequence, that is, the size of the contribution. The larger the correlation degree, the closer the relationship between the two characteristic components. Based on this, the relationship between the characteristic peaks corresponding to the HPLC fingerprint and the pharmacodynamic effects can be found.

[0133] The peak areas of 22 common peaks in the fingerprint of Acroptilon repens were respectively subjected to initial value processing with the DPPH radical scavenging rate and the absorbance of total reducing ability. Taking the DPPH radical scavenging rate and the absorbance of total reducing ability as the mother sequences and the peak areas as the child sequences, the correlation degrees between each common peak of Acroptilon repens and the DPPH radical scavenging rate and the absorbance of total reducing ability were calculated, and the results were sorted to obtain the correlation order. The results are shown in Table 4.

[0134] Table 4 GRA results of 22 common peaks in the extract of Acroptilon repens and antioxidant activity

[0135]

[0136] From the above results, it can be seen that the correlation degrees of the chemical components represented by the 22 common peaks with the DPPH free radical scavenging effect and the total reducing ability are both greater than 0.7, indicating that the antioxidant activity of Acroptilon repens is the result of the combined action of multiple components. Among them, the correlation degrees of peak 2 with both the DPPH free radical scavenging rate and the absorbance of the total reducing ability are greater than 0.9, ranking first, indicating that peak 2 (chlorogenic acid) may play an important role in the antioxidant activity of Acroptilon repens. The correlation degree ranking of the DPPH free radical scavenging rate is 2 > 1 > 5 > 13 > 3 > 7, and the correlation degree ranking of the absorbance of the total reducing ability is 2 > 5 > 13 > 3 > 7 > 1. The peaks that are ranked in the top six in both are 2, 1, 5, 13, 3, and 7.

[0137] 4.2 Partial least squares regression analysis (PLSR)

[0138] Taking the relative peak areas of the 12 common peaks of the Acroptilon repens extracts as the abscissa (X), and the DPPH free radical scavenging rate and the absorbance of the total reducing ability as the ordinates (Y) respectively, import them into the SIMCA 14.0 software for PLSR analysis, and calculate the standardized regression coefficients (R 2 ) and the variable importance in projection (VIP) values of the relative peak areas of the common peaks with the DPPH free radical scavenging rate and the absorbance of the total reducing ability. The results are shown in Figure 3 . After the data is standardized, when the DPPH free radical scavenging rate is the dependent variable, the model fitting parameter R 2 X is 0.946, R 2 Y is 0.754, and Q 2 is 0.66; when the absorbance of the total reducing ability is the dependent variable, the model fitting parameter R 2 X is 0.946, R 2 Y is 0.845, and Q 2 is 0.683. The fitting parameters are all greater than 0.5, indicating that the established model has good prediction and interpretation.

[0139] It is generally considered that when VIP ≥ 1, the independent variable has significant importance in explaining the dependent variable. As can be seen from A1 in Figure 3 , the VIP values of peaks 1, 2, 3, 5, 7, and 13 are all greater than 1, and the error bars do not pass through the origin, with the ranking of peak 1 > 2 > 3 > 13 > 5 > 7. The positive or negative of the regression coefficient represents the positive or negative correlation of each peak with the DPPH free radical scavenging rate. As can be seen from B1 in Figure 3 , peaks 1, 2, 3, 13, 7, and 5 are positively correlated with the DPPH free radical scavenging rate and contribute greatly to the antioxidant activity. Similarly, from A2 in Figure 3 and Figure 3From B2 in Figure 1, we can see that the VIP values ​​of peaks 2, 3, 1, 5, 13, 7, 15, and 10 decrease in sequence, but are all greater than 1, and the error lines do not pass through the origin. The order is peak 2>3>1>5>13>7, and the absolute values ​​of their regression coefficients are also large, indicating that these components have a significant contribution to the total reducing capacity absorbance.

[0140] Based on the regression coefficient of DPPH free radical scavenging rate and total reducing capacity absorbance and VIP value, it is speculated that the chemical components represented by peaks 1, 2, 3, 5, 7, and 13 may contribute significantly to the antioxidant activity of D. truncatum, which is consistent with the GRA results.

[0141] Example 5 In vitro antioxidant validation test of monomeric compounds

[0142] Combined with the results of GRA and PLSR analysis, it was inferred that the chemical components represented by peaks 1, 2, 3, 5, 7, and 13 may contribute significantly to the antioxidant activity of C. In order to verify the reliability of the spectrum-effect analysis results, vitamin C was used as a positive control to determine the in vitro antioxidant activity of the identified neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid.

[0143] 5.1 Determination of DPPH free radical scavenging rate

[0144] Using 50% methanol as solvent, vitamin C and chlorogenic acid were prepared into a series of working solutions with mass concentrations of 3, 6, 11, 14, and 18 μg / mL, and neochlorogenic acid and cryptochlorogenic acid were prepared into a series of working solutions with mass concentrations of 2, 6, 11, 14, and 18 μg / mL, respectively. According to the above method, they were reacted with DPPH solution to determine the free radical scavenging rate. The results were shown in Table 1. Figure 4 Chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid all have strong ability to scavenge DPPH free radicals in a dose-dependent manner, but their scavenging rates for DPPH free radicals are slightly weaker than that of the positive control vitamin C.

[0145] 5.2 Determination of total reducing capacity

[0146] Using 50% methanol as solvent, vitamin C, chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid were prepared into a series of working solutions with mass concentrations of 0.02, 0.04, 0.06, 0.08, and 0.10 mg / mL, respectively. The absorbance values ​​were measured three times in parallel according to the above method. The results are shown in Table 1. Figure 5 Chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid all have strong reducing abilities, and are dose-dependent, but their reducing abilities are slightly weaker than that of the positive control vitamin C.

Claims

1. Acroptilon repens extract, wherein the extract is obtained by extracting Acroptilon repens with water or an ethanol aqueous solution of 10% - 95% by volume, and the total flavonoid content is 10% - 35% by weight, the total polysaccharide content is 10% - 45% by weight, and the total organic acid content is 1% - 30% by weight; Preferably, the extract is obtained by extracting Acroptilon repens with an ethanol aqueous solution of 30% - 75% by volume, and the total flavonoid content is 10% - 30% by weight, the total polysaccharide content is 10% - 30% by weight, and the total organic acid content is 1% - 20% by weight; Preferably, the extract is obtained by extracting Acroptilon repens with an ethanol aqueous solution of 30% - 60% by volume, and the total flavonoid content is 10% - 30% by weight, the total polysaccharide content is 15% - 30% by weight, and the total organic acid content is 5% - 20% by weight; More preferably, it is obtained by the following method: extracting Acroptilon repens with an ethanol aqueous solution of 45% - 55% by volume, and extracting with ethyl acetate or n-butanol to obtain the extract, or combining the ethyl acetate extract and the n-butanol extract to obtain the extract, and the total flavonoid content of the extract is 15% - 30% by weight, the total polysaccharide content is 15% - 30% by weight, and the total organic acid content is 10% - 20% by weight.

2. The Acroptilon repens extract according to claim 1, wherein the content of neochlorogenic acid is 0.01% - 1% by weight, the content of chlorogenic acid is 0.1% - 3% by weight, the content of cryptochlorogenic acid is 0.1% - 1.5% by weight, the content of 1,5-dicaffeoylquinic acid is 0.05% - 1% by weight, the content of isochlorogenic acid C is 0.01% - 0.2% by weight, the content of apigenin is 0.01% - 2% by weight, the content of hispidulin is 0.01% - 2% by weight.

3. The Acroptilon repens extract according to claim 1, which is obtained by the following method: extracting Acroptilon repens with water or an ethanol aqueous solution of 10% - 95% by volume; preferably, extracting with an ethanol aqueous solution of 30% - 75%, 30% - 60%, or 45% - 55% by volume; more preferably, the extraction temperature is 50 - 95 °C, extracting 1 - 3 times, and the time is 1 - 3 hours per time; obtaining the Acroptilon repens extract; Optionally, the extracted filtrate is concentrated under reduced pressure to a relative density of 1.0 - 1.5, and the concentrated solution is dried; preferably, it is dried in a vacuum drying oven with a vacuum degree of -0.08 MPa and a temperature of 50 - 80 °C to obtain a dry extract. The dry extract is suspended in water and extracted with ethyl acetate or n-butanol to obtain an extract, or the ethyl acetate extract and the n-butanol extract are combined to obtain an extract, thereby obtaining the acroptilon repens extract. Optionally, the extract is suspended in water and purified with macroporous resin, polyamide resin or nylon-66 resin to obtain the acroptilon repens extract.

4. The preparation method of the acroptilon repens extract according to any one of claims 1 - 3, which comprises: Acroptilon repens is extracted with water or an ethanol aqueous solution of 10% - 95% by volume to obtain the Acroptilon repens extract; or Acroptilon repens is extracted with an ethanol aqueous solution of 30% - 75% by volume, 30% - 60% by volume, or 45% - 55% by volume; more preferably, the extraction temperature is 50 - 95 °C, and the extraction is carried out 1 - 3 times for 1 - 3 hours each time; the Acroptilon repens extract is obtained; Optionally, the filtrate after extraction is concentrated under reduced pressure to a relative density of 1.0 - 1.5, and the concentrated solution is dried; preferably, it is dried in a vacuum drying oven with a vacuum degree of -0.08 MPa and a temperature of 50 - 80 °C to obtain a dried extract. The dried extract is suspended in water and extracted with ethyl acetate or n-butanol to obtain an extract, or the ethyl acetate extract and the n-butanol extract are combined to obtain an extract, and the Acroptilon repens extract is obtained; Optionally, the extract is suspended in water and purified with macroporous resin, polyamide resin, or nylon-66 resin to obtain the Acroptilon repens extract.

5. The use of the acroptilon repens extract according to any one of claims 1 - 3 in the preparation of a drug for antioxidation.

6. The evaluation method of the acroptilon repens extract based on the spectrum-effect relationship of antioxidation, which comprises the following steps: (1) Prepare the extract of acroptilon repens; preferably, heat and reflux extract acroptilon repens with water, ethanol at 30% (v / v), 50% (v / v), 75% (v / v), 95% (v / v) respectively to obtain extracts S1 - S5 in sequence; dissolve extracts S3 and S5 in water respectively, and extract them with petroleum ether, ethyl acetate, n-butanol at 60 - 90 °C in sequence, and recover the aqueous phase to obtain extracts S6 - S12; extracts S1 - S12 are as follows: (2) Establish the HPLC fingerprint of extracts S1 - S12; preferably, the chromatographic column is Agilent Eclipse XDB-C 18 、YMC-Pack ODS-A C 18 or Agilent ZORBAX SB-C 18 , the mobile phase is 0.1% formic acid aqueous solution - acetonitrile, and the detection wavelength is 327 nm; more preferably, establish the fingerprint through 22 common peaks in chromatography and confirm 7 of them; (3) Determine the antioxidation of extracts S1 - S12; preferably, measure the DPPH free radical scavenging rate and total reducing ability of extracts S1 - S12; (4) Spectrum-effect analysis of extracts S1 - S12 and antioxidation; preferably, conduct grey relational analysis and partial least squares analysis on extracts S1 - S12; (5) Determine and verify the effective parts and effective chemical components of the extract based on the analysis results of grey relational analysis and partial least squares analysis.

7. The evaluation method according to claim 6, wherein in step (2), the chromatographic column is 250 mm × 4.6 mm with a particle size of 5 μm; the elution gradient is as follows: 0 - 5 min, 5% acetonitrile; 5 - 15 min, 5% - 10% acetonitrile; 15 - 20 min, 10% - 14% acetonitrile; 20 - 80 min, 14% - 26% acetonitrile; 80 - 113 min, 26% - 38% acetonitrile; 113 - 113.01 min, 38% - 5% acetonitrile; 113.01 - 120 min, 5% acetonitrile; the column temperature is 30°C, the injection volume is 10 μL, and the volume flow rate is 1.0 mL / min; preferably, the chemical components corresponding to the 7 peaks are neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, 1,5-dicaffeoylquinic acid, isochlorogenic acid C, apigenin, and homoplantaginin.

8. The evaluation method according to claim 6, wherein in step (3), the method for determining the DPPH free radical scavenging rate includes: Prepare the test solution, prepare the vitamin C positive control solution, use 50% methanol plus DPPH solution as the blank group A0, different mass concentration sample solutions plus DPPH solution as the sample group A1, different mass concentration sample solutions plus methanol as the background group A2, react in the dark, use a microplate reader to measure the absorbance A value at a wavelength of 517 nm, calculate according to the following formula, DPPH free radical scavenging rate (%) = [1 - (A1 - A2) / A0] × 100%, use the concentration as the abscissa and the scavenging rate as the ordinate, calculate the linear regression equation, and calculate the half scavenging concentration IC of each sample for DPPH free radicals 50 ; The method for determining the total reducing ability includes: preparing a test solution and a vitamin C positive control solution, adding phosphate buffer solution, 1% potassium ferricyanide solution, 10% trichloroacetic acid solution, and 0.1% ferric chloride solution respectively, measuring the absorbance at 700 nm, using 50% methanol as a blank control, plotting the absorbance against the test samples with different mass concentrations as the abscissa to obtain a linear equation, and calculating the absorbance value at a concentration of 1.0 mg / mL.

9. The evaluation method according to claim 6, wherein in step (4), the calculation formula for grey relational degree is where r i is the correlation degree between the mother sequence x0 and the son sequence x i ; N is the number of data of the son sequence, ξi is the correlation coefficient, k is different extracts, and calculate the correlation degree of the chemical components represented by the common peaks; Taking the relative peak areas of the common peaks of extracts S1 - S12 as the abscissa, and the DPPH free radical scavenging rate and the absorbance of total reducing power as the ordinates respectively, perform PLSR analysis, calculate the standardized regression coefficients and variable projection importance values of the relative peak areas of the common peaks with the DPPH free radical scavenging rate and the absorbance of total reducing power. After standardizing the data, evaluate the prediction and interpretation of the established model.

10. The evaluation method according to claim 6, wherein in step (5), based on the analysis results of grey relational analysis and partial least squares analysis, determine the contribution of the extract part and the chemical components represented by the common peaks to the antioxidant activity of Acroptilon repens.

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