Establishment of HPLC fingerprint of Artemisia indica and analysis of antioxidant activity spectrum

Abstract

This invention discloses a method for establishing HPLC fingerprint chromatograms and analyzing the antioxidant activity of the Tibetan medicine *Artemisia argyi*. The invention establishes an HPLC fingerprint chromatogram for the methanol extract of *Artemisia argyi*, achieving better peak separation, symmetrical peak shapes, and reduced peak tailing. HPLC fingerprint chromatograms were established for 11 batches of *Artemisia argyi* samples from different sources, and 20 common peaks were identified. Cluster analysis and principal component analysis were used to preliminarily evaluate the *Artemisia argyi* samples. Various tests were conducted to assess the antioxidant activity of the samples, and grey relational analysis was used to establish the spectral relationship between the antioxidant activity and the antioxidant activity of *Artemisia argyi*. The results showed that the chromatographic peak P10 had a high correlation with certain antioxidant activity indicators, indicating that it may be a key component for antioxidant activity. This invention provides a scientific basis for distinguishing *Artemisia argyi* from its adulterants and offers a methodological reference for evaluating its antioxidant activity and quality control, which is of great value for advancing research on the quality of this medicinal material.

Description

Technical Field

[0001] This invention belongs to the field of HPLC analysis technology of Tibetan medicine Artemisia argyi, and particularly relates to a method for establishing HPLC fingerprint chromatograms of Tibetan medicine Artemisia argyi and analyzing its antioxidant activity spectrum. Background Technology

[0002] Incarvillea compacta Maxim., a perennial herb belonging to the Bignoniaceae family and the Incarvillea genus, is a Tibetan medicinal plant. Its dried whole herb is known by various transliterations in Tibetan, including "Oche," "Oqu," and "Uqumabo." It grows primarily in damp mountainous areas, mainly distributed in Lhasa, Nagqu, Shigatse, Yadong in Tibet, as well as Qinghai, Gansu, and Yunnan provinces. The *Jingzhu Materia Medica* states that Incarvillea compacta treats ear diseases, relieves bloating, benefits the pulse, and treats ear ailments. Incarvillea compacta is warm in nature and has a bitter and sweet taste. It possesses properties that regulate menstruation and blood circulation, dispel wind and dampness, reduce inflammation, benefit the ears, and improve the pulse. It is commonly used for menstrual disorders, rheumatic pain, otitis media, and hypertension. Incarvillea compacta mainly contains phenylethyl glycosides, flavonoids, terpenes, and alkaloids. Modern pharmacological studies have shown that it has anti-inflammatory, hepatoprotective, anticancer, and antioxidant effects.

[0003] *Houttuynia cordata* is a species listed in the *Drug Standards of the Ministry of Health of the People's Republic of China (Tibetan Medicine)* (1995 edition). The medicinal material is named "Houttuynia cordata," but only a description of its properties and processing methods are provided, which is insufficient for effectively identifying the authenticity of the medicinal material and controlling its quality. Furthermore, due to different regional medicinal practices and the fact that Tibetan medicines often use locally sourced materials or substitutes from plants of the same family and genus, various medicinal materials named *Houttuynia cordata* but with different origins are circulating in the market. For example, the *Standards for Traditional Chinese and Tibetan Medicine in Gansu Province* lists "Houttuynia cordata" as having the dried root of *Houttuynia cordata*, a plant in the Bignoniaceae family. The *Chinese Materia Medica* (Tibetan Medicine Volume) lists "Houttuynia cordata" as having the flowers, seeds, and roots of *Houttuynia cordata*, a plant in the Bignoniaceae family.

[0004] There is currently limited research on Artemisia annua. Existing studies mainly focus on the separation and identification of chemical components and the evaluation of its activity. There is no research on its HPLC fingerprint spectrum, which makes it difficult to clearly distinguish it from other adulterants. The spectrum-effect relationship of its antioxidant activity needs further investigation. Summary of the Invention

[0005] To address the above issues and overcome the shortcomings of existing technologies, this invention establishes an HPLC fingerprint of *Artemisia argyi* and compares it with adulterants on the market. Principal component analysis and cluster analysis are used to study the quality differences of *Artemisia argyi* from different producing areas. The antioxidant capacity of 11 batches of *Artemisia argyi* samples is evaluated, and grey relational analysis is used to correlate them with the fingerprint, aiming to identify characteristic peaks related to the antioxidant properties of *Artemisia argyi*. This provides a reference for enriching the identification and quality evaluation methods of this medicinal material and for the effective development and utilization of its resources.

[0006] To achieve the above objectives, the following technical solution is adopted: This invention provides a method for establishing an HPLC fingerprint of the Tibetan medicine Artemisia argyi and analyzing its antioxidant activity. The establishment of the fingerprint includes the following steps:

[0007] S1. Preparation of the test solution: Take 0.5g of Artemisia annua powder, accurately weigh it, place it in a stoppered conical flask, add 25mL of methanol, weigh it, sonicate it, cool it, make up the weight loss with methanol, filter it through a 0.45μm microporous membrane, and take the filtrate to obtain the test solution.

[0008] S2. Precision test: Inject the test solution into the high performance liquid chromatograph, inject 6 times consecutively, record the chromatogram, use the common peak No. 6 as the reference peak, calculate the relative retention time and relative peak area of ​​the common peak, and calculate the RSD value.

[0009] S3. Repeatability test: Take the same batch of Artemisia annua powder and prepare 6 test solutions in parallel according to the method in S1. Inject them into a high performance liquid chromatograph for chromatographic determination, record the chromatogram, take the common peak No. 6 as the reference peak, calculate the relative retention time and relative peak area of ​​the common peak, and calculate the RSD value.

[0010] S4. Stability test: Take Artemisia annua powder and prepare the test solution according to the method in S1. Inject the solution into a high performance liquid chromatograph at 0, 2, 4, 8, 12 and 24 h respectively for chromatographic determination. Record the chromatogram. Take the common peak No. 6 as the reference peak, calculate the relative retention time and relative peak area of ​​the common peak, and calculate the RSD value.

[0011] S5. Establishment of fingerprint chromatograms: The test solutions of 11 batches of Artemisia argyi samples were prepared according to the method in S1. The solutions were injected into a high-performance liquid chromatograph for chromatographic determination. The chromatographic data of each batch of samples were recorded. The data in "cdf" format was imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System". The IC-1 chromatogram was used as a reference chromatogram for matching common peaks. The HPLC fingerprint chromatograms and reference chromatograms of the methanol extracts of 11 batches of Artemisia argyi were generated. The common peaks were identified and similarity analysis was performed.

[0012] S6. Identification of common peaks and comparison of chromatograms of adulterants: Five batches of adulterants were tested using the same method as above, and the chromatographic data of each batch of samples were recorded. The data in "cdf" format was imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System". The common peaks were matched using the JH-1 chromatogram as the reference chromatogram to generate HPLC fingerprint chromatograms and reference chromatograms of methanol extracts of five batches of adulterants, and compared with the reference chromatogram of Artemisia argyi.

[0013] Furthermore, the chromatographic detection conditions are as follows:

[0014] Using Zhongpu Science RD-C 18 The chromatographic column was 250 mm × 4.6 mm, 5 μm; the mobile phase was methanol (A) - 0.1% phosphoric acid aqueous solution (B), the flow rate was 1.0 mL / min; the detection wavelength was 254 nm, the column temperature was 30 ℃, the injection volume was 10 μL, and the gradient elution conditions were as follows:

[0015] 0–5 min, 10–25% A;

[0016] 5–23 min, 25% A;

[0017] 23–25 min, 25–40% A;

[0018] 25–40 min, 40% A;

[0019] 40–50 min, 40–60% A;

[0020] 50–65 min, 60–90% A;

[0021] 65–70 min, 90–10% A;

[0022] 70-75 min, 10% A.

[0023] Furthermore, the ultrasonic treatment conditions in the preparation of the test solution are: power 400W, frequency 40kHz, and time 30min.

[0024] Furthermore, the analysis method includes the following steps:

[0025] S1, Fe 3+ Reducing power determination: Take 1 mL of 10 mg / mL *Artemisia argyi* methanol solution, add 2 mL each of 0.2 mol / L phosphate buffer solution (pH 6.6) and 0.03 mol / L K3Fe(CN)6 solution, mix well, and react at 50℃ for 20 min. Then add 0.2 mL of 0.6 mol / L CCl3COOH solution, mix well, centrifuge at 1000 r / min for 10 min, and take 40 μL of the supernatant, 110 μL of ultrapure water, and 100 μL of 0.006 mol / L FeCl3 solution and place them together in the same well of a 96-well plate, mix well, and use methanol as a blank control. Measure the absorbance at 700 nm. Each sample group is tested three times. 3+ Reducing power (A) = A1 - A0, where A1 is the absorbance of the test sample group and A0 is the absorbance of the blank control group; the larger the A value, the stronger the reducing power of the sample.

[0026] S2, DPPH ·Scavenging capacity determination: Take 100 μL of a 5 mg / mL Artemisia argyi methanol solution and mix it with 0.1 mmol / L DPPH · Mix 3.90 mL of the solution, protect from light, shake at room temperature for 30 min, then take 200 μL of the mixture and place it in a 96-well plate. Measure its absorbance at 517 nm. Use methanol as a blank control. Repeat the test 3 times for each sample. DPPH scavenging ability = (1 - A1 / A0) × 100%, where A1 is the absorbance of the test sample group and A0 is the absorbance of the blank control group.

[0027] S3, ABTS ·+ Scavenging capacity determination: A suitable amount of 7 mmol / L ABTS solution and 2.45 mmol / L K₂S₂O₈ solution were mixed at a volume ratio of 1:1 and reacted for 14 h in the dark at room temperature to prepare ABTS. ·+ Before use, the stock solution was diluted with 95% ethanol to an absorbance of 0.7 ± 0.02 (at 734 nm). 0.1 mL of a vitamin E (Trolox) solution with a mass concentration of 50–450 μg / mL was mixed with 3.9 mL of the substrate solution, and the reaction was carried out at room temperature for 6 min. 200 μL of the mixture was placed in a 96-well plate, and its absorbance (at 734 nm) was measured. A regression curve was constructed with mass concentration as the x-axis and absorbance as the y-axis to obtain a linear equation. 0.1 mL of a 2 mg / mL Artemisia argyi methanol solution was used instead of the Trolox solution for the reaction, and the results were substituted into the linear equation. ABTS ·+ Scavenging capacity is expressed as Trolox antioxidant capacity equivalent TEAC (mg / g);

[0028] S4. FRAP Determination: Mix appropriate amounts of 0.3 mol / L CH3COONa buffer solution, 10 mmol / L TRTZ solution (solvent: 40 mmol / L HCl solution), and 20 mmol / L FeCl3 at a volume ratio of 10:1:1 to prepare the FRAP working solution. Incubate at 37℃ in a water bath. Prepare and use immediately. Accurately transfer 20 μL of FeSO4·7H2O solution of different concentrations (1–10 mmol / L) to 3 mL of the FRAP working solution and... Mix well and react at 37℃ for 40 min. Take 200 μL of the mixture and place it in a 96-well plate. Measure its absorbance (at 593 nm). Plot a regression curve with concentration as the x-axis and absorbance as the y-axis to obtain a linear equation. Take 20 μL of Artemisia argyi methanol solution with a mass concentration of 10 mg / mL instead of FeSO4·7H2O solution for the reaction and measure the absorbance. Substitute the obtained results into the linear equation and calculate the FRAP value of the sample. The total antioxidant capacity is expressed as FARP (FeSO4 mmol / g).

[0029] Furthermore, using the antioxidant activity levels of different batches of *Artemisia argyi* samples as the parent sequence and the peak areas of the 20 common peaks obtained as the child sequences, grey relational analysis was performed to obtain the Fe... 3+ Reduction ability, DPPH · Clearance ability, ABTS ·+ The scavenging ability and the correlation between FRAP and its common peak were used to preliminarily determine the contribution of the chemical components represented by the common peak to the antioxidant activity by using the correlation level.

[0030] Furthermore, the ABTS ·+ The linear equation obtained from the scavenging capacity determination is Y = 0.2102X - 1.78, r = 0.9982.

[0031] Furthermore, the linear equation obtained by the FRAP determination is Y = 0.0747X + 0.0573, r = 0.9978.

[0032] The beneficial effects of this invention are:

[0033] (1) This invention established an HPLC fingerprint of the methanol extract of Artemisia annua. The wavelength scanning results in the range of 190-400 nm showed that the test solution had the most chromatographic peaks at 254 nm with the highest peak height. Therefore, 254 nm was selected as the detection wavelength. The mobile phase optimization results showed that the gradient elution of methanol-0.1% phosphoric acid aqueous solution could separate most of the chromatographic peaks. Adding 0.1% phosphoric acid to the aqueous phase could effectively improve the peak tailing phenomenon and make the peak shape more symmetrical. Under these conditions, HPLC fingerprints of 11 different batches of Artemisia annua were established and 20 common peaks were matched. However, compared with reference standards such as apigenin and luteolin, no related chromatographic peaks were identified. In the HPLC fingerprint established by this invention, the peak areas of P6, P10 and P11 are large and are the main chemical components contained therein.

[0034] (2) This invention preliminarily classifies and comprehensively evaluates samples of Artemisia argyi through cluster analysis and principal component analysis. The samples used include multiple variables such as different growth stages (flowering and fruiting), different parts (whole herb and above-ground parts), and different sources (self-collected, hospital and market purchases, and different production areas), resulting in sufficient material diversity. The 11 batches of samples were clustered into 3 categories, ranking at the top, middle and bottom in the comprehensive ranking of principal component analysis, respectively. The results of the two analyses were almost consistent. Among them, the samples in category III (IC-10, IC-8) ranked high in the comprehensive ranking, and the peak areas of chromatographic peaks P6, P10 and P11 were large, but whether they are effective components is still unclear. The study of the spectrum-effect relationship of traditional Chinese medicine combines fingerprint spectrum with efficacy research, which can more objectively and comprehensively reflect the intrinsic quality and efficacy material basis of traditional Chinese medicine. Therefore, this invention determined the Fe of the test solution. 3+Reduction ability, DPPH · Clearance ability, ABTS ·+ The antioxidant capacity of the samples was examined by measuring scavenging ability and FRAP. Furthermore, the spectroscopic-efficacy relationship of *Artemisia argyi* was established using grey relational analysis, revealing that the P10 chromatographic peak correlated with ABTS. ·+ The high correlation between scavenging ability and FRAP indicates that the P10 chromatographic peak is a characteristic peak for its antioxidant effect (the two indicators mentioned above);

[0035] (3) This invention establishes an HPLC fingerprint of Artemisia annua, which is significantly different from that of adulterants. The established method can be used to distinguish Artemisia annua from its adulterants. This invention evaluates the antioxidant activity of samples from different sources and analyzes the characteristic peaks that produce antioxidant activity by constructing a spectrophotometric relationship, providing a method reference for the identification and quality evaluation of Artemisia annua. Attached Figure Description

[0036] Figure 1 The HPLC fingerprint chromatograms of 11 batches of Artemisia argyi samples are superimposed.

[0037] Figure 2 The superimposed HPLC fingerprint chromatograms of five batches of Artemisia argyi adulterants;

[0038] Figure 3 The reference fingerprint chromatograms of Artemisia argyi (A) and its adulterant (B);

[0039] Figure 4 Cluster heatmap (A) and principal component analysis scatter plot (B) of 11 batches of Artemisia densiflora.

[0040] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof. Detailed Implementation

[0041] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.

[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the experimental materials used in the following examples are all purchased from commercial channels.

[0044] Instruments used in this invention:

[0045] BS224S electronic balance (10,000 ppm, Sartorius, Germany); LPCD-E3000 forced-air drying oven (Shanghai Longyue Instrument Equipment Co., Ltd.); e2695-2998 high-performance liquid chromatograph (Waters Technologies, USA, PAD detector); UPR-II-20L pure water system (Sichuan Youpu Ultrapure Technology Co., Ltd.); VGT-2013QT ultrasonic cleaner (250W power, 40kHz frequency, Guangdong Gote Ultrasonic Co., Ltd.).

[0046] Reagents used in this invention:

[0047] Methanol (chromatographic grade, TEDIA, USA); phosphoric acid (chromatographic grade, TEDIA, USA); all other reagents were of analytical grade, and water was ultrapure water.

[0048] The medicinal materials used in this invention are:

[0049] Eleven batches of Artemisia argyi samples were collected. All samples were identified by researcher Dawa Zhuoma of the Tibet Autonomous Region Food and Drug Inspection Institute as dried whole herb or aerial parts of Artemisia argyi, a plant of the Bignoniaceae family. The other five batches of Artemisia argyi samples were adulterants. Specific information is shown in Table 1.

[0050] Table 111 Sample Information

[0051]

[0052] Example 1

[0053] Establishment of HPLC fingerprint

[0054] Chromatographic conditions: Zhongpu Science RD-C 18 The chromatographic column was 250 mm × 4.6 mm, 5 μm; the mobile phase was methanol (A) - 0.1% phosphoric acid aqueous solution (B), with gradient elution: 0–5 min, 10–25% A; 5–23 min, 25% A; 23–25 min, 25–40% A; 25–40 min, 40% A; 40–50 min, 40–60% A; 50–65 min, 60–90% A; 65–70 min, 90–10% A; 70–75 min, 10% A; the flow rate was 1.0 mL / min; the detection wavelength was 254 nm; the column temperature was 30 °C; and the injection volume was 10 μL.

[0055] Preparation of the test solution: Take 0.5 g of Artemisia annua powder, accurately weigh it, place it in a stoppered conical flask, add 25 mL of methanol, weigh it, sonicate it, cool it, make up the weight loss with methanol, filter it through a 0.45 μm microporous membrane, and take the filtrate to obtain the test solution.

[0056] Precision test: The test solution was injected into the high performance liquid chromatograph and injected 6 times consecutively. The chromatogram was recorded. The relative retention time and relative peak area of ​​the common peak were calculated using the 6th common peak as the reference peak, and the RSD value was calculated.

[0057] Repeatability test: Take the same batch of Artemisia annua powder and prepare 6 test solutions in parallel according to the method in S1. Inject them into a high performance liquid chromatograph for chromatographic determination, record the chromatogram, take the common peak No. 6 as the reference peak, calculate the relative retention time and relative peak area of ​​the common peak, and calculate the RSD value.

[0058] Stability study: Take Artemisia annua powder and prepare the test solution according to the method in S1. Inject the solution into a high performance liquid chromatograph at 0, 2, 4, 8, 12 and 24 h for chromatographic determination. Record the chromatogram. Using the common peak No. 6 as the reference peak, calculate the relative retention time and relative peak area of ​​the common peak and calculate the RSD value.

[0059] The results of precision, repeatability, and stability studies showed that the RSD of retention time and peak area for each peak was less than 2%, which met the analytical requirements.

[0060] Fingerprint chromatogram establishment: Eleven batches of Artemisia argyi samples were prepared into test solutions according to the method in S1. The solutions were injected into a high-performance liquid chromatograph for chromatographic determination. The chromatographic data of each batch of samples were recorded. The data in "cdf" format was imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System". The IC-1 chromatogram was used as a reference chromatogram for common peak matching. The HPLC fingerprint chromatograms and reference chromatograms of the methanol extracts of eleven batches of Artemisia argyi were generated. The common peaks were identified and similarity analysis was performed.

[0061] Identification of common peaks and comparison of chromatograms of adulterants: Five batches of adulterants were tested using the same method, and the chromatographic data of each batch of samples were recorded. The data in "cdf" format was imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System". The common peaks were matched using the JH-1 chromatogram as the reference chromatogram to generate HPLC fingerprint chromatograms and reference chromatograms of methanol extracts of five batches of adulterants, and compared with the reference chromatogram of Artemisia argyi.

[0062] HPLC fingerprints of Artemisia annua: Overlayed fingerprint chromatograms and control chromatograms of 11 batches of Artemisia annua methanol extracts are shown below. Figure 1 and Figure 3A. Twenty common peaks were matched. The similarity of the 11 batches of samples with the control spectrum was 0.978, 0.982, 0.939, 0.663, 0.733, 0.620, 0.810, 0.962, 0.833, 0.921, and 0.881, respectively. Among them, IC-4, IC-5, and IC-6 had lower similarity, indicating that these three batches differed significantly from the other batches.

[0063] The fingerprint overlay and reference fingerprints of methanol extracts from five batches of Artemisia argyi adulterants are shown below. Figure 2 and Figure 3 B, matching 22 common peaks. Figure 3 It can be seen that there are significant differences between the control chromatogram of the adulterant and that of *Artemisia argyi*, including differences in peak shape, number of peaks, and retention time. This indicates that this fingerprinting method can distinguish *Artemisia argyi* from its adulterants.

[0064] Example 2

[0065] Cluster analysis

[0066] Using the area of ​​20 common peaks from 11 batches of Artemisia argyi as the raw data, the data was imported into the scientific mapping cloud platform (https: / / cloud.metware.cn) for Z-score standardization and clustering heatmaps were generated.

[0067] Cluster analysis results can reflect the similarity between different batches of samples. See the cluster analysis heatmap results below. Figure 4 (A). As shown in the figure, the 11 batches of *Artemisia argyi* can be roughly divided into three categories. Samples from the Tibetan Hospital of Linzhi, Tibet (IC-5, IC-7, IC-11) were clustered into Category I, with all three batches being whole plants (fruiting stage). Samples from Jiali County, Tibet (IC-1, IC-2, IC-3), Yala Xiangbu Mountain in Nedong County, Tibet (IC-4), and Qinghai Province (IC-9) were clustered into Category II, with all five batches being whole plants, including two at the flowering stage and three at the fruiting stage. Samples from Nagqu City, Tibet (IC-6), Changdu City, Tibet (IC-8), and Shannan City, Tibet (IC-10) were clustered into Category III, all being fruiting samples, including one batch of aerial parts and two batches of whole plants. It is evident that the characteristic chromatographic peak areas in *Artemisia argyi* are influenced by multiple factors, including the place of origin, growth stage, and harvested part.

[0068] Example 3

[0069] Principal component analysis

[0070] Using the peak areas of 20 common peaks in the fingerprint spectra of 11 batches of *Artemisia argyi* as variables, principal component analysis was performed in SPSS 26.0 software. Eigenvalues ​​and cumulative contribution rates were used as the criteria for extraction, resulting in the identification of three principal components. The scores PCA1, PCA2, and PCA3 of principal components 1, 2, and 3 were calculated respectively, where α is the factor score, λ is the eigenvalue, and PCA is the principal component score. Then, the comprehensive score F of the 11 batches of samples was calculated according to F = 0.501PCA1 + 0.279PCA2 + 0.220PCA3. The larger the F value, the higher the ranking, indicating that the content of common components in the sample is higher.

[0071] Based on the dimensionality reduction results, three principal components, PC1, PC2, and PC3, were extracted, with a cumulative variance contribution of 74.015%, indicating strong representativeness and representing the common peak information of *Artemisia argyi*. The overall scores of different samples were ranked as follows: IC-10 > IC-8 > IC-9 > IC-4 > IC-1 = IC-3 > IC-2 > ​​IC-6 > IC-11 > IC-7 > IC-5. Samples from Shannan City and Changdu City in Tibet had higher overall scores. Combined with the cluster analysis heatmap, it was found that the common peaks P10, P15, P3, P6, P8, and P11 of these two batches of samples had high peak area thermal response values, indicating that these common peaks were the main factors contributing to their higher overall scores. The overall scores of IC-9, IC-4, IC-1, IC-3, and IC-2 were in the middle position, consistent with the clustering results. IC-5, IC-7, and IC-11 had low overall scores and were clustered into one class in cluster analysis. Since the samples came from the same source, the heatmap from the cluster analysis revealed that the common peaks P1, P10, P13, P15, P3, P6, P12, P7, P8, and P5 of these three batches of samples all had low peak area thermal response values. A scatter plot using PC1, PC2, and PC3 as coordinates is shown below. Figure 4 (B) As shown in the figure, the distributions of IC-5, IC-7, IC-11 and IC-6 are also located at different extreme edges.

[0072] Example 4

[0073] In vitro antioxidant activity assessment

[0074] Fe 3+ Reducing power determination: Take 1 mL of 10 mg / mL *Artemisia argyi* methanol solution, add 2 mL each of 0.2 mol / L phosphate buffer solution (pH 6.6) and 0.03 mol / L K3Fe(CN)6 solution, mix well, and react at 50℃ for 20 min. Then add 0.2 mL of 0.6 mol / L CCl3COOH solution, mix well, centrifuge at 1000 r / min for 10 min, and take 40 μL of the supernatant, 110 μL of ultrapure water, and 100 μL of 0.006 mol / L FeCl3 solution and place them together in the same well of a 96-well plate, mix well, and use methanol as a blank control. Measure the absorbance at 700 nm. Each sample group is tested three times. 3+Reducing power (A) = A1 - A0, where A1 is the absorbance of the test sample group and A0 is the absorbance of the blank control group; the larger the A value, the stronger the reducing power of the sample.

[0075] DPPH · Scavenging capacity determination: Take 100 μL of a 5 mg / mL Artemisia argyi methanol solution and mix it with 0.1 mmol / L DPPH · Mix 3.90 mL of the solution, protect from light, shake at room temperature for 30 min, then take 200 μL of the mixture and place it in a 96-well plate. Measure its absorbance at 517 nm. Use methanol as a blank control. Repeat the test 3 times for each sample. DPPH scavenging ability = (1 - A1 / A0) × 100%, where A1 is the absorbance of the test sample group and A0 is the absorbance of the blank control group.

[0076] ABTS ·+ Scavenging capacity determination: A suitable amount of 7 mmol / L ABTS solution and 2.45 mmol / L K₂S₂O₈ solution were mixed at a volume ratio of 1:1 and reacted for 14 h in the dark at room temperature to prepare ABTS. ·+ Before use, the stock solution was diluted with 95% ethanol to an absorbance of 0.7 ± 0.02 (at 734 nm). 0.1 mL of a vitamin E (Trolox) solution with a mass concentration of 50–450 μg / mL was mixed with 3.9 mL of the substrate solution, and the reaction was carried out at room temperature for 6 min. 200 μL of the mixture was placed in a 96-well plate, and its absorbance (at 734 nm) was measured. A regression curve was plotted with mass concentration on the x-axis and absorbance on the y-axis, yielding the linear equation Y = 0.2102X - 1.78, r = 0.9982. Alternatively, 0.1 mL of a 2 mg / mL Artemisia argyi methanol solution was used instead of the Trolox solution for the reaction, and the results were substituted into the above linear equation. ·+ Scavenging capacity is expressed as Trolox antioxidant capacity equivalent TEAC (mg / g);

[0077] FRAP determination: Take an appropriate amount of 0.3 mol / L CH3COONa buffer solution, 10 mmol / L TRTZ solution (solvent is 40 mmol / L HCl solution) and 20 mmol / L FeCl3 in a volume ratio of 10:1:1 to generate FRAP working solution, keep it in a 37℃ water bath, and use it immediately after preparation. Accurately transfer 20 μL of FeSO4·7H2O solutions of different concentrations (1–10 mmol / L), add 3 mL of FRAP working solution and mix well. React at 37 °C for 40 min. Take 200 μL of the mixture and place it in a 96-well plate, and measure its absorbance (at 593 nm). Plot a regression curve with concentration as the abscissa and absorbance as the ordinate. The linear equation obtained is Y = 0.0747X + 0.0573, r = 0.9978. Take 20 μL of Artemisia argyi methanol solution with a mass concentration of 10 mg / mL to replace the FeSO4·7H2O solution for the reaction and measure the absorbance. Substitute the obtained results into the linear equation and calculate the FRAP value of the sample. The total antioxidant capacity is expressed as FARP (FeSO4 mmol / g).

[0078] Using the antioxidant activity levels of different batches of *Artemisia argyi* samples as the parent sequence, and the peak areas of the 20 common peaks obtained as the child sequences, grey relational analysis was performed to obtain the Fe... 3+ Reduction ability, DPPH · Clearance ability, ABTS ·+ The scavenging ability and the correlation between FRAP and its common peak were used to preliminarily determine the contribution of the chemical components represented by the common peak to the antioxidant activity by using the correlation level.

[0079] Fe in 11 batches of Artemisia argyi samples 3+ Reduction ability, DPPH · Clearance ability, ABTS ·+ Scavenging capacity and FARP are shown in Table 2. The results show that IC-1, IC-2, and IC-3 have Fe... 3+ Reduction ability, DPPH · Clearance capabilities and ABTS ·+ The clearance ability was the strongest in all groups; IC-10 showed significantly better FARP than other groups. * (P < 0.05). The four antioxidant activity indices, IC-5, IC-6, IC-7, and IC-11, were all the weakest, consistent with the ranking results of the principal component analysis (PCA) comprehensive score, indicating a certain correlation between the 20 common peaks (PCA variables) and their antioxidant capacity. Notably, IC-10 showed the strongest FARP, but DPPH... · However, its scavenging ability is relatively weak; IC-1 has extremely strong Fe... 3+ Restoration ability and ABTS ·+The scavenging ability was good, but FARP was slightly insufficient, and the two batches of samples were clustered into different categories, indicating that different common peak components contributed differently to the four antioxidant activity indicators.

[0080] Table 2 Antioxidant Results of Each Efficacy Index

[0081]

[0082] Example 5

[0083] Grey relational analysis of fingerprint spectroscopy and antioxidant activity

[0084] With Fe 3+ Reduction ability, DPPH · Clearance ability, ABTS ·+ Using scavenging ability and FRAP as reference sequences, the peak areas of 20 common peaks were used as comparison sequences for grey relational analysis. The correlation between the 20 common HPLC peaks of *Artemisia argyi* and four antioxidant activity indicators was calculated, and the results are shown in Table 3. The magnitude of the correlation reflects the degree of correlation between the chemical components represented by each common peak and antioxidant activity. The closer the correlation is to 1, the greater the influence of the component represented by the peak on antioxidant activity, indicating a stronger connection between chemical components and antioxidant capacity.

[0085] Table 3. Correlation between common peaks and antioxidant activity

[0086] P1 0.6708 0.5988 0.6766 0.7243 P2 0.6730 0.5999 0.6805 0.6861 P3 0.5681 0.6471 0.6085 0.6878 P4 0.6225 0.6291 0.6267 0.6359 P5 0.6590 0.6113 0.6728 0.6859 P6 0.5786 0.6169 0.6457 0.7385 P7 0.6658 0.6514 0.6386 0.6349 P8 0.6232 0.6759 0.6046 0.6955 P9 0.6480 0.7021 0.4923 0.5326 P10 0.6212 0.5277 0.8490 0.8512 P11 0.6250 0.5854 0.6070 0.6091 P12 0.6832 0.6375 0.7183 0.7474 P13 0.6136 0.5814 0.7775 0.7393 P14 0.5480 0.7027 0.5307 0.5563 P15 0.5576 0.6964 0.6820 0.7280 P16 0.6678 0.6518 0.5179 0.5921 P17 0.6028 0.6843 0.5003 0.5522 P18 0.6197 0.6312 0.5961 0.6045 P19 0.7029 0.5738 0.5613 0.5647 P20 0.6984 0.5569 0.6024 0.5970

[0087] As can be seen from the table, Fe 3+ The correlation between reducing power and DPPH ranged from 0.5480 to 0.7029. · The correlation coefficients for scavenging ability ranged from 0.5277 to 0.7027, and the correlation coefficients with all common peaks were <0.8, indicating that the material basis for the two antioxidant capabilities of *Artemisia argyi* is not the prominent contribution of any single common peak, but is likely the result of the combined effects of multiple components. ABTS ·+ The correlation between scavenging ability and FRAP ranged from 0.4923 to 0.8490, while the correlation between FRAP and FRAP ranged from 0.5522 to 0.8512. Peak P10 showed a correlation greater than 0.8 with both peaks, indicating that peak P10 correlates with ABTS. ·+ Scavenging ability and FRAP contributed the most; peak P9 had the least correlation with both.

[0088] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0089] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention. The actual application is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar methods and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A method for establishing an HPLC fingerprint of the Tibetan medicine Artemisia argyi, characterized in that, The establishment of the fingerprint spectrum includes the following steps: S1. Preparation of the test solution: Take 0.5g of Artemisia annua powder, accurately weigh it, place it in a stoppered conical flask, add 25mL of methanol, weigh it, sonicate it, cool it, make up the weight loss with methanol, filter it through a 0.45µm microporous membrane, and take the filtrate to obtain the test solution. S2. Establishment of fingerprint spectrum: The test solution of 11 batches of Artemisia argyi samples was prepared according to the method in S1, and injected into the high performance liquid chromatograph for chromatographic determination. The chromatographic detection conditions were as follows: a Zhongpu Science RD C-18 column (250 mm × 4.6 mm, 5 µm) was used; methanol was used as mobile phase A, and 0.1% phosphoric acid aqueous solution was used as mobile phase B; the flow rate was 1.0 mL / min; the detection wavelength was 254 nm; the column temperature was 30 °C; the injection volume was 10 µL; and gradient elution conditions were used. From 0 to 5 minutes, the volume percentage of mobile phase A increased from 10% to 25%. The volume percentage of mobile phase A remains at 25% for 5–23 min. Over 23–25 minutes, the volume percentage of mobile phase A increased from 25% to 40%. For 25–40 minutes, the volume percentage of mobile phase A is maintained at 40%. Over 40–50 minutes, the volume percentage of mobile phase A increased from 40% to 60%. Within 50–65 minutes, the volume percentage of mobile phase A increased from 60% to 90%. Over 65–70 minutes, the volume percentage of mobile phase A decreased from 90% to 10%. For 70–75 minutes, the volume percentage of mobile phase A is maintained at 10%.

2. The method for establishing an HPLC fingerprint of the Tibetan medicine Artemisia annua according to claim 1, characterized in that: The ultrasonic treatment conditions for preparing the test solution were: power 400W, frequency 40kHz, and time 30min.

3. A method for analyzing the antioxidant activity of the Tibetan medicine Artemisia argyi using an HPLC fingerprint established based on the method described in any one of claims 1 or 2, characterized in that, The analytical method includes the following steps: S1、 Reducing power determination: Take 1 mL of a 10 mg / mL *Artemisia argyi* methanol solution, and add 0.2 mol / L phosphate buffer solution (pH=6.6) and 0.03 mol / L... Add 2 mL of each solution, mix well, and react at 50 °C for 20 min. Then add 0.6 mol / L of [a specific solution / concentrate]. Mix 0.2 mL of solution, centrifuge at 1000 rpm for 10 min, and take 40 μL of the supernatant, 110 μL of ultrapure water, and 0.006 mol / L of [a specific solution / concentrate]. 100 μL of the solution was placed in the same well of a 96-well plate, mixed well, and methanol was used as a blank control. The absorbance at 700 nm was measured. Each group of samples was tested three times. The reducing power is expressed as the A value. ,in The absorbance of the test sample group. The absorbance is for the blank control group; the larger the A value, the stronger the reducing power of the sample. S2, DPPH · Scavenging capacity determination: Take 100 μL of a 5 mg / mL Artemisia argyi methanol solution and mix it with 0.1 mmol / L DPPH · Mix 3.90 mL of the solution, protect from light, shake at room temperature for 30 min, then transfer 200 μL of the mixture to a 96-well plate and measure its absorbance at 517 nm. Use methanol as a blank control. Each sample group was measured three times. DPPH · Clearance ability = ,in The absorbance of the test sample group. The absorbance of the blank control group; S3 Scavenging capacity assay: Take an appropriate amount of 7 mmol / L ABTS solution and mix it with 2.45 mmol / L... The solutions were mixed at a volume ratio of 1:1 and reacted for 14 hours in the dark at room temperature to prepare... Before use, the stock solution was diluted with 95% ethanol to an absorbance of 0.7 ± 0.02 at 734 nm. 0.1 mL of Trolox solution (50–450 μg / mL) was mixed with 3.9 mL of substrate solution, and the mixture was reacted at room temperature for 6 min. 200 μL of the mixture was placed in a 96-well plate, and the absorbance at 734 nm was measured. A regression curve was constructed with Trolox concentration on the x-axis and absorbance on the y-axis to obtain a linear equation. 0.1 mL of 2 mg / mL Artemisia argyi methanol solution was used instead of the Trolox solution for the reaction, and the results were substituted into the linear equation. Scavenging capacity is expressed as Trolox antioxidant capacity equivalent (TEAC), in mg / g. S4, FRAP determination: Take an appropriate amount of 0.3 mol / L Buffer solution, 10 mmol / L TPTZ solution and 20 mmol / L The solutions were mixed at a volume ratio of 10:1:1 to prepare the FRAP working solution, which was then incubated in a 37°C water bath and prepared immediately before use. 1–10 mmol / L of the solution was accurately transferred. Add 20 μL of solution to 3 mL of FRAP working solution and mix well. Incubate at 37 °C for 40 min. Transfer 200 μL of the mixture to a 96-well plate and measure the absorbance at 593 nm. A regression curve was constructed with concentration on the x-axis and absorbance on the y-axis, yielding a linear equation. 20 μL of a 10 mg / mL *Artemisia argyi* methanol solution was used instead. The solution was reacted and the absorbance was measured. The results were substituted into a linear equation to calculate the FRAP value of the sample. The total antioxidant capacity was expressed as FRAP, with units of [missing information]. mmol / g; Using the antioxidant activity levels of different batches of *Artemisia argyi* samples as the parent sequence, and the peak areas of 20 common peaks obtained based on the method of claim 1 or 2 as the child sequences, grey relational analysis was performed to obtain the *Artemisia argyi*... Reduction ability, DPPH · Clearance ability The scavenging ability and the correlation between FRAP and its common peak were used to preliminarily determine the contribution of the chemical components represented by the common peak to the antioxidant activity by using the correlation level.

4. The analytical method according to claim 3, characterized in that: The The linear equation obtained from the scavenging capacity determination is Y = 0.2102X - 1.78, r = 0.9982.

5. The analytical method according to claim 3, characterized in that: The linear equation obtained by the FRAP determination is Y=0.0747X+0.0573, r=0.9978.

Images