Method for screening anti-inflammatory, antioxidant active compounds from natural chinese herbs artemisia based on spectrum-effect relationship

Abstract

The present application relates to a kind of methods for screening anti-inflammatory, antioxidant active compounds from natural Chinese herbal medicine wormwood based on spectrum-effect relationship, the method establishes the method for the common characteristic peak of the fingerprint chromatogram of wormwood extract ultra-high performance liquid chromatography.By grey correlation analysis, Pearson bivariate correlation analysis method and partial least square regression analysis etc. chemometrics method establishes the spectrum-effect relationship of wormwood anti-inflammatory, antioxidant activity, to screen the anti-inflammatory, antioxidant active compounds in wormwood.The analysis result shows that compound chlorogenic acid, isochlorogenic acid A, isochlorogenic acid C is the main anti-inflammatory, antioxidant active compound in wormwood extract.The present application provides scientific and effective method for the research of the medicinal material basis of wormwood and quality control, and provides reference for the development and utilization of wormwood.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for the pharmacodynamic activity of chemical components of traditional Chinese medicine, and relates to a method for screening anti-inflammatory and antioxidant active compounds from natural wormwood based on the spectrum-effect relationship. BACKGROUND

[0002] Wormwood is the dry aboveground part of Artemisia absinthium L. of the family Asteraceae, also known as Central Asian wormwood, European wormwood, wormwood, and beer wormwood. It is native to the Mediterranean region and widely distributed in northwest China, Europe, Siberia and North Africa. Wormwood has a long history of use in traditional medicine systems around the world for the treatment of digestive system diseases, gastrointestinal diseases, nervous system diseases, helminthiasis, bladder diseases, wounds, fever and liver inflammation, etc. Wormwood is listed in the Chinese Pharmacopoeia Uygur Medicine Volume and Chinese Herbal Medicine, and has a long history in the treatment of inflammatory diseases, stomach pain and digestive system diseases. Recent pharmacological studies have found that wormwood has good anticancer, anti-inflammatory, antioxidant, wound healing, cytotoxicity and gastrointestinal protection effects. The main compounds reported in wormwood include volatile oils, sesquiterpene lactones, flavonoids and phenolic acids and their derivatives.

[0003] Spectrum-effect relationship analysis is a method of chemical pattern recognition that combines traditional Chinese medicine fingerprint and pharmacological studies to evaluate the quality of traditional Chinese medicine and screen active compounds in traditional Chinese medicine. It is an exploratory research method that has been recognized by the World Health Organization. Therefore, the present application is the first to use the UPLC fingerprint of wormwood extract and the in vitro anti-inflammatory and antioxidant activity of wormwood extract as the basis for screening anti-inflammatory and antioxidant active compounds in wormwood through grey correlation analysis, bivariate correlation analysis and partial least squares method. The purpose of the present application is to provide a scientific basis for the research of the pharmacodynamic material basis and quality control of wormwood, and to provide a reference for the further development and utilization of wormwood. SUMMARY

[0004] The application aims to provide a method for screening anti-inflammatory and antioxidant active compounds from natural Chinese herbal medicine wormwood based on spectrum-effect relationship, which establishes the ultra performance liquid chromatography (UPLC) fingerprint of wormwood extract and identifies the common characteristic peaks of the wormwood extract. The spectrum-effect relationship of the anti-inflammatory and antioxidant activity of the wormwood is established by grey correlation analysis, Pearson bivariate correlation analysis and partial least squares regression analysis, so as to screen the anti-inflammatory and antioxidant active compounds in the wormwood. The analysis result shows that the compounds chlorogenic acid, isochlorogenic acid A and isochlorogenic acid C are the main anti-inflammatory and antioxidant active compounds in the wormwood extract. The application provides a scientific and effective method for the research on the medicinal material basis and quality control of the wormwood and provides a reference for the development and utilization of the wormwood.

[0005] The method for screening anti-inflammatory and antioxidant active compounds from natural Chinese herbal medicine wormwood based on spectrum-effect relationship, which establishes the UPLC fingerprint of wormwood extract, identifies the common peaks and uses the chemometrics method to establish the spectrum-effect relationship to screen the compound components in the wormwood, is provided in the application, and the specific operation is performed according to the following steps:

[0006] Preparation of wormwood extract:

[0007] a. 5g of 18 batches of wormwood medicinal material powder is precisely weighed and placed in a round-bottom flask, 100ml of 95% ethanol aqueous solution is added, and the extraction is performed twice by refluxing for 2 hours each time, and then the filtration is performed, the filtrate is concentrated to about 20ml, and then the transfer is performed to an evaporating dish, the water is evaporated by evaporation, and then the vacuum drying box is used to evaporate the water, so as to obtain the wormwood extract;

[0008] Establishment of the ultra performance liquid chromatography (UPLC) fingerprint of wormwood extract:

[0009] b. Ultra-high performance liquid chromatography (UPLC): Chromatographic column: octadecylsilane-bonded silica gel as the filler, particle size 1.7 μm, 2.1 mm x 100 mm reverse-phase chromatographic column, injection volume 2 μL, column temperature 35 °C, flow rate 0.2 ml / min, detection wavelength 330 nm, eluent: formic acid water-acetonitrile or formic acid water-methanol, time 0-95 min, 3-9:97-91 for 0-5 min; time 5-10 min, 9-9.5:91-90.5; time 10-16 min, 9.5-12.5:90.5-87.5; time 16-56 min, 12.5-17.5:87.5-82.5; time 56 min, 17.5-21:82.5-79; time 58 min, 21-30:79-70; time 76 min, 30-38:70-62; time 80 min, 38-52:62-48; time 85 min, 52-90:48-10; time 90-95 min, 90:10, gradient elution;

[0010] c. The 18 batches of wormwood extracts obtained in step a were detected by ultra-high performance liquid chromatography using the chromatographic conditions of step b, the data obtained were analyzed by traditional Chinese medicine chromatographic fingerprint similarity evaluation system, the similarity of the 18 batches of wormwood extracts was calculated, and the common peaks were matched; the similarity of the 18 batches of wormwood extracts was 0.884, 0.959, 0.812, 0.818, 0.959, 0.963, 0.978, 0.936, 0.978, 0.936, 0.983, 0.984, 0.987, 0.948, 0.628, 0.914, 0.721, 0.795, 0.926, 0.954, respectively;

[0011] Identification of common peaks in ultra-high performance liquid chromatography fingerprint:

[0012] d. Qualitative analysis of common characteristic peaks by ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS), 12 common peaks were identified as follows: P1 is hexoside of dihydroxybenzoic acid, P2 is cryptochlorogenic acid, P3 is chlorogenic acid, P4 is apigenin-C-hexoside-C-pentoside, P5 is isochlorogenic acid A, P6 is isochlorogenic acid C, P7 is tri-caffeoyl quinic acid, P8 is oleuropein, P9 is linolenic acid, P10 is 16-hydroxyhexadecanoic acid, P11 is hydroxyoctadecatrienoic acid, and P12 is isomorellic acid;

[0013] Screening of anti-inflammatory and antioxidant active ingredients in wormwood by spectro-effect chemometrics method:

[0014] Anti-inflammatory and antioxidant activity:

[0015] e. The Artemisia vulgaris extract of step a is evaluated by determining its in vitro cyclooxygenase-2 and 15-lipoxygenase enzyme inhibitory activity, and nitric oxide inhibitory activity in lipopolysaccharide-induced mouse microglial cells;

[0016] Antioxidant activity: determined by DPPH 1,1-diphenyl-2-picrylhydrazyl radical scavenging method and ABTS 2,2'-azino-di(3-ethyl-benzothiazoline-6-sulfonic acid) diammonium salt radical scavenging method;

[0017] Screening of anti-inflammatory and antioxidant active ingredients in Artemisia vulgaris by spectrum-effect chemometrics method:

[0018] Grey correlation degree analysis:

[0019] f. The anti-inflammatory activity and antioxidant activity indicators and the common peak area data obtained by the ultra-high performance liquid fingerprint are introduced into the grey system theory and application 7.0.1 software, with the activity data as the mother sequence, the peak area sub-sequence, and the resolution coefficient being 0.5, and Deng's correlation degree analysis is performed on the common peaks and activity data of the sample;

[0020] Pearson bivariate correlation analysis method:

[0021] g. The common peaks obtained by the ultra-high performance liquid fingerprint are used as the independent variable X, and the anti-inflammatory activity and antioxidant activity indicators are used as the dependent variable Y, and Pearson bivariate correlation analysis is performed by using SPSS (25) software;

[0022] Partial least squares regression analysis:

[0023] h. The common peak area obtained by the ultra-high performance liquid fingerprint is used as the independent variable X, and the anti-inflammatory activity and antioxidant activity indicators are used as the dependent variable Y, and partial least squares regression model analysis is performed by using SIMCA (14.1) software;

[0024] i. Combined with the results of the three chemometrics analysis methods, P3 is finally screened out as chlorogenic acid, P5 is isochlorogenic acid A, and P6 is isochlorogenic acid C as the anti-inflammatory and antioxidant active ingredients in Artemisia vulgaris. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The ultra-high performance liquid fingerprint of the Artemisia vulgaris extract of the present application; DETAILED DESCRIPTION

[0026] Preparation of the Artemisia vulgaris extract:

[0027] a, 18 batches of wormwood medicinal material powder was precisely weighed 5 g, placed in a round bottom flask, added 100 ml of 95% ethanol aqueous solution, refluxed for 2 times, 2 hours each time, filtered, the filtrate was concentrated to about 20 ml, transferred to the evaporating dish, evaporated to dryness, then vacuum dried, and the wormwood extract was obtained;

[0028] Establishment of ultra performance liquid chromatography fingerprint of wormwood extract:

[0029] b, ultra performance liquid chromatography fingerprint: chromatographic column: octadecylsilane bonded silica gel as filler, particle size 1.7 μm, 2.1 mm x 100 mm reversed phase chromatographic column, sample volume 2 μL, column temperature: 35℃, flow rate: 0.2 ml / min, detection wavelength: 330 nm, eluent: formic acid water-acetonitrile or formic acid water-methanol see table 1:

[0030] Table 1. Mobile phase ratio of UPLC fingerprint of wormwood

[0031]

[0032] c, using the chromatographic conditions of step b, the 18 batches of wormwood extract obtained in step a were detected by ultra performance liquid chromatography, the data obtained were analyzed by traditional Chinese medicine chromatography fingerprint similarity evaluation system, the similarity of 18 batches of wormwood extract was calculated, and the common peaks were matched; the similarity of 18 batches of wormwood extract is shown in table 2, Figure 1 ;

[0033] Table 2. Similarity of 18 batches of wormwood extract

[0034]

[0035] Identification of common peaks in ultra performance liquid chromatography fingerprint:

[0036] d, using ultra performance liquid chromatography-mass spectrometry technology, 12 common peaks were identified as: the results are shown in table 3:

[0037] Table 3. Identification of common peaks in UPLC fingerprint of wormwood

[0038]

[0039] Screening of anti-inflammatory and antioxidant active ingredients in wormwood by spectro-effect chemometrics method:

[0040] Anti-inflammatory and antioxidant activity:

[0041] e, the wormwood extract described in step a was determined for its cyclooxygenase-2 and 15-lipoxygenase enzyme inhibition activity in vitro, and the nitric oxide inhibition activity in lipopolysaccharide-induced mouse microglial cells was evaluated;

[0042] Eighteen batches of wormwood extract were prepared into solutions with final concentrations of 50 μg / mL and 100 μg / mL, and their in vitro COX-2 enzyme and 15-LOX enzyme inhibitory activities were determined. The eighteen batches of wormwood extract had significant (P<0.01) COX-2 enzyme inhibitory activity, with inhibition rates ranging from 85.77±1.34% to 97.97±0.44%, higher than the positive control group at a concentration of 100 nM. The 15-LOX enzyme inhibition rates of the eighteen batches of wormwood extract were 7.97±0.83%-25.25±2.24%, significantly lower than the COX-2 enzyme inhibitory activity, indicating that wormwood extract participates in the anti-inflammatory process by inhibiting COX-2 enzyme rather than 15-LOX enzyme. The results are shown in Table 4.

[0043] Table 4. COX-2 and 15-LOX enzyme inhibitory activities of eighteen batches of wormwood extract

[0044]

[0045] LPS-induced BV-2 cell NO content determination:

[0046] 1. After confirming that the cell viability was not significantly affected by the drug concentration by MTT method, different concentrations of wormwood extract 6.25-100 μg / mL were added, incubated for 2 h, and then 1 μg / mL of lipopolysaccharide (LPS) was added for a total of 22 h of incubation. After incubation, the cell supernatant was collected, and the Griess method was used to determine the content of nitric oxide in the cell supernatant. Before measurement, Griess Reagent I and II were taken out and allowed to recover to room temperature;

[0047] 2. The standard (1-100 μM) was diluted with complete medium, and the concentration of the standard was taken as 0, 1, 2, 5, 10, 20, 40, 60, 100 μM;

[0048] 3. The standard and collected culture supernatant were added to the 96-well plate at 50 μL / well, shaken and mixed for 5 min, and then the absorbance was measured at 540 nm to draw a standard curve. The NO content in the culture supernatant was calculated according to the standard curve, and the inhibition rate was calculated according to the formula: inhibition rate (%) = [(A LPS -A 样品 ) / A LPS] x 100%; the cytotoxicity results showed that wormwood extract 100 μg / mL had no toxicity to BV-2 cells, therefore, further detection of 18 batches of wormwood extract on LPS-induced BV-2 cell NO production under different concentration gradient. The results showed that, in addition to S9, S10 and S11, other batches of wormwood extract at a concentration of 100 μg / mL can inhibit the production of NO, the inhibition rate is more than 50%, indicating that the wormwood extract has strong anti-inflammatory activity. Therefore, further determination of the NO inhibitory activity of different concentrations of 6.25 μg / mL-100 μg / mL wormwood extract; the results showed that the wormwood extract inhibited the production of LPS-induced BV-2 cell NO in a concentration-dependent manner, showing good inhibitory activity, IC 50 The value is 14.21±1.27 μg / mL-34.74±2.84 μg / mL, the results are shown in Table 5:

[0049] Table 5 LSP-induced BV-2 cell NO inhibitory activity of 18 batches of wormwood extract

[0050]

[0051] Detection of antioxidant activity of 18 batches of wormwood extract:

[0052] Determination of DPPH free radical scavenging ability DPPH free radical (1,1-diphenyl-2-trinitrobenzene hydrazine) is a stable free radical centered on nitrogen. The test was carried out by gradient dilution method, first 5 different concentrations of wormwood extract solution was prepared with anhydrous ethanol, the initial concentration of the sample was 400 ug / mL, 100 μL sample solution and 100 μL DPPH free radical solution 2 mM were added in 96 well plate, then gradient dilution, the concentration range was 400-12.5 μg / mL, mixed well and placed in the dark at room temperature for 30 min, the absorbance of the solution at wavelength 517 nm was measured, and the inhibition rate was calculated; the positive control group (vitamin C) was tested by the same method; the calculation formula of inhibition rate (I%) is: I (%) = [A0-(AI-As)] / A0 x 100, wherein A0 is the absorbance value of the blank solution, AI is the absorbance of the sample and free radical coexistence, and As is the absorbance of the sample without free radical; the half inhibition rate IC 50All tests and analysis were performed in triplicate and the mean values were taken. The ABTS radical scavenging assay was performed according to the method of V Hui' with slight modifications; ABTS radical solution was prepared by mixing 7 mM ABTS with 2.45 mM potassium persulfate and allowing the mixture to stand in the dark at room temperature for 12-16 h. The ABTS+solution was then diluted with absolute ethanol to an absorbance of 0.70 ± 0.2 at 734 nm. In a 96-well plate, 100 μL of sample solution was added to 100 μL of ABTS+solution, and the concentration range was 200-3.125 μg / mL. After mixing, the solution was allowed to stand in the dark at room temperature for 10 min. The absorbance of the solution at 734 nm was measured using a SpectraMax-MD5, and the inhibition rate was calculated. The positive control group (vitamin C) was tested in the same way. Eighteen batches of wormwood extract had good antioxidant activity, with DPPH radical scavenging IC 50 values ranging from 14.19 ± 3.2 to 284.1 ± 7.31 μg / mL, and DPPH radical scavenging IC 50 values ranging from 25.66 ± 4.12 to 105.2 ± 7.32 μg / mL. The results are shown in Table 6.

[0053] Table 6. Antioxidant activity of 18 batches of wormwood extract

[0054]

[0055] Screening of anti-inflammatory and antioxidant active ingredients in wormwood using spectrum-effect chemometrics:

[0056] Grey correlation analysis:

[0057] f、The anti-inflammatory activity and antioxidant activity indicators and the 12 common peak peak area data obtained by ultra-high performance liquid chromatography fingerprint were introduced into the gray system theory and application 7.0.1 software, and the activity data was used as the mother sequence, the peak area subsequence, and the resolution coefficient was 0.5. The common peaks and activity data of the samples were analyzed by Deng's correlation degree analysis. Table 7 lists the results of gray correlation degree correlation analysis between common peaks and five pharmacodynamic indicators. The gray correlation coefficients between 12 common peaks and pharmacodynamic indicators are between 0.8161-0.9903, indicating that all common peaks have an impact on the anti-inflammatory and antioxidant activity of wormwood extract; The correlation coefficient order of NO inhibition activity is as follows: P1>P10>P8>P11>P9>P12>P4>P2>P3>P5>P6>P7; The correlation coefficient order of COX-2 enzyme inhibition activity is: P10>P1>P11>P8>P9>P12>P4>P2>P3>P5>P6>P7; The correlation coefficient order of 15-LOX enzyme inhibition activity is: P1>P8>P10>P12>P4>P2>P3>P5>P6>P7; The correlation coefficient order of DPPH free radical scavenging is: P10>P1>P11>P9>P12>P4>P2>P3>P5>P6>P7; The correlation coefficient order of ABTS free radical scavenging is the same as that of DPPH;

[0058] Table 7. Gray correlation degree correlation analysis results

[0059]

[0060]

[0061] Pearson bivariate correlation analysis method:

[0062] g、The 12 common peaks obtained by ultra-high performance liquid chromatography fingerprint were used as independent variables X, and the anti-inflammatory activity and antioxidant activity indicators were used as dependent variables Y. Pearson bivariate correlation analysis was performed using SPSS (25) software. The Pearson correlation analysis results are shown in Table 8. The larger the Pearson correlation coefficient, the stronger the correlation between the chemical composition and the activity. As shown in Table 8, P5, P1, P6, P3 and P4 have a strong correlation with NO inhibition activity. Among them, P5, P1 and P6 have significant correlation (P<0.05); P8, P6, P5 and P7 have significant correlation with COX-2 inhibition activity (P<0.05). Similarly, P3, P6, P5, P2 and P7 have significant correlation with DPPH free radical scavenging activity and ABTS free radical scavenging activity (P<0.05);

[0063] Table 8. Pearson bivariate correlation analysis results

[0064]

[0065] *P<0.05,**P<0.01

[0066] Partial least squares regression analysis:

[0067] h, the common peak area obtained by ultra-high performance liquid chromatography fingerprint was set as the independent variable X, and the anti-inflammatory activity and antioxidant activity indicators were set as the dependent variable Y. Partial least squares regression model analysis was performed using SIMCA (14.1) software;

[0068] Partial least squares regression analysis can intuitively analyze the comprehensive contribution of each common peak in the spectrum to the efficacy. The greater the absolute value of the regression coefficient, the greater the correlation with the efficacy. A positive correlation coefficient indicates a positive correlation with the efficacy, and a negative correlation coefficient indicates the opposite. In addition, the analysis results give the importance index of the variable, VIP value. When VIP is greater than 1, it indicates that the independent variable X is important to the model. Generally, common peaks with a VIP value greater than 1 are considered effective active substances, and the greater the correlation coefficient, the greater the contribution rate to the activity. In this study, the common peak area was set as the independent variable (X), and the anti-inflammatory activity and antioxidant activity indicators were set as the dependent variable (Y). Partial least squares regression analysis was performed using SIMCA (14.1) software, and the results are shown in Table 9. In all five partial least squares regression models, the VIP values of the same three common peaks were greater than 1, namely P3, P5, and P6. These three common peaks were positively correlated with the five activity indicators;

[0069] Table 9. Partial least squares regression analysis results

[0070]

[0071] i. Based on the results of the three metrology analysis methods, P3 was finally selected as the anti-inflammatory and antioxidant active ingredient in Artemisia vulgaris, P5 was selected as iso-chlorogenic acid A, and P6 was selected as iso-chlorogenic acid C. Based on the results of the three correlation analyses, the common peaks P3, P5, and P6 were finally selected as the anti-inflammatory and antioxidant active compounds in Artemisia vulgaris extract;

[0072] Experimental verification: Finally, the anti-inflammatory activity of the three active compounds was verified, and the results are shown in Table 10:

[0073] Table 10. In vitro COX-2 and 15-LOX enzyme inhibition activity of three compounds

[0074] As can be seen from Table 10, iso-chlorogenic acid A (P5) and iso-chlorogenic acid C (P6) have good in vitro COX-enzyme inhibition activity, with IC 50 values of 682.93±18.33 and 489.5±27.58 nM, respectively. Among the three compounds, iso-chlorogenic acid C (P6) has good inhibition activity on 15-LOX enzyme, with an IC50 The value is 111.3 ± 11.88 μM; the enzyme inhibition activity of chlorogenic acid (P3) at a concentration of 50 μM is less than 50%, and the activity is less than the other two compounds; the results of the verification experiment are consistent with the results of the spectrum-effect relationship, and two compounds, isochlorogenic acid A (P5) and isochlorogenic acid C (P6), can be used as quality control substances of Artemisia vulgaris. This study provides a scientific basis for the establishment of quality standards and the safe and effective use of Artemisia vulgaris.

Claims

1. A method for screening anti-inflammatory, antioxidant active compounds from natural Chinese herb, Artemisia absinthium based on the spectrum-effect relationship, characterized by The method establishes the UPLC fingerprint of the extract of Artemisia vulgaris, identifies the common peaks, and uses chemometrics to establish the spectrum-effect relationship to screen the active ingredients in Artemisia vulgaris. The specific operation is carried out according to the following steps: Preparation of Artemisia vulgaris extract: a. Select 18 batches of Artemisia vulgaris powder, accurately weigh 5 g, and place it in a round-bottom flask. Add 100 ml of 95% ethanol aqueous solution, reflux extract for 2 times, each for 2 hours, filter, concentrate the filtrate to 20 ml, transfer to an evaporating dish, evaporate to dryness, and then evaporate the water in a vacuum drying box to obtain the extract of Artemisia vulgaris; Establishment of UPLC fingerprint of Artemisia vulgaris extract: b. UPLC fingerprint: Chromatographic column: octadecylsilane bonded silica gel as filler, particle size 1.7 μm, 2.1 mm x 100 mm reverse phase chromatographic column, injection volume 2 μL, column temperature 35 ℃, flow rate 0.2 ml / min, detection wavelength 330 nm, eluent formic acid water-acetonitrile, gradient elution according to Table 1; Table 1. Mobile phase ratio column for UPLC fingerprint analysis of Artemisia vulgaris c. The chromatographic conditions of step b are used to detect the 18 batches of Artemisia vulgaris extract obtained in step a by UPLC, the data obtained are analyzed by traditional Chinese medicine chromatographic fingerprint similarity evaluation system, the similarity of the 18 batches of Artemisia vulgaris extract is calculated, and the common peaks are matched; the similarity of the 18 batches of Artemisia vulgaris extract is 0.884, 0.959, 0.812, 0.818, 0.959, 0.963, 0.978, 0.936, 0.978, 0.936, 0.983, 0.984, 0.987, 0.948, 0.628, 0.914, 0.721, 0.795, 0.926, 0.954, respectively; Identification of common peaks in UPLC fingerprint: d. Qualitative analysis of common characteristic peaks by UPLC-MS, 12 common peaks are identified as: P1 is hexose glycoside of dihydroxybenzoic acid, P2 is cryptomycin, P3 is chlorogenic acid, P4 is apigenin-C-hexose glycoside-C-pentose glycoside, P5 is isochlorogenic acid A, P6 is isochlorogenic acid C, P7 is tri-caffeoyl quinine, P8 is vitexin, P9 is linolenic acid, P10 is 16-hydroxyhexadecanoic acid, P11 is hydroxyoctadecatrienoic acid, and P12 is isomolate; Spectrum-effect chemometrics method for screening anti-inflammatory and antioxidant active ingredients in Artemisia vulgaris: Anti-inflammatory and antioxidant activity: e. The extract of Artemisia vulgaris in step a is used to determine the in vitro cyclooxygenase-2 and 15-lipoxygenase enzyme inhibition activity, and the nitric oxide inhibition activity in lipopolysaccharide-induced mouse microglial cells to evaluate; Antioxidant activity: determined by DPPH 1,1-diphenyl-2-trinitrobenzene hydrazine radical scavenging method and ABTS 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt radical scavenging method; Spectrum-effect chemometrics method for screening anti-inflammatory and antioxidant active ingredients in Artemisia vulgaris: Grey correlation degree analysis: f. Import the common peak area data obtained from the anti-inflammatory and antioxidant activity indicators and the ultra-high performance liquid chromatography fingerprint into the Grey System Theory and Application 7.0.1 software. Use the activity data as the parent sequence and the peak area subsequence as the resolution coefficient. Perform Dunk correlation analysis on the common peaks and activity data of the sample. Pearson bivariate correlation analysis: g. Using the common peaks obtained from the ultra-high performance liquid chromatography fingerprint as independent variable X and the anti-inflammatory and antioxidant activity indices as dependent variable Y, Pearson bivariate correlation analysis was performed using SPSS25 software. Partial least squares regression analysis: h. The common peak area obtained from the ultra-high performance liquid chromatography fingerprint spectrum was set as the independent variable X, and the anti-inflammatory and antioxidant activity indicators were set as the dependent variable Y. Partial least squares regression model analysis was performed using SIMCA 14.1 software. i. Combining the results of three quantitative analysis methods, P3 was finally screened as chlorogenic acid, P5 as isochlorogenic acid A and P6 as isochlorogenic acid C, which are the anti-inflammatory and antioxidant active ingredients in Artemisia argyi.

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