Construction method of carpesium abrotanoides fingerprint spectrum

Abstract

The invention discloses a construction method of a carpesium abrotanoides fingerprint spectrum. The construction method comprises the following steps: preparing a carpesium abrotanoides test solution; preparing a mixed reference substance solution; the carpesium abrotanoides fingerprint spectrum is detected by adopting ultra-high performance liquid chromatography, a mobile phase system comprises a mobile phase A and a mobile phase B, the mobile phase A is a phosphoric acid aqueous solution, and the mobile phase B is an acetonitrile solution. The carpesium abrotanoides fingerprint spectrum provided by the invention has 19 common peaks, wherein 10 components are identified. The carpesium abrotanoides UPLC fingerprint spectrum established by the invention is good in repeatability and precision, the method is stable and reliable, the quality of a plurality of components of carpesium abrotanoides can be accurately and quickly monitored at the same time, and a powerful guarantee is provided for controlling the quality and ensuring the clinical curative effect of carpesium abrotanoides.

Description

Technical Field

[0001] The present invention relates to the field of traditional Chinese medicine detection, and specifically relates to a method for constructing a fingerprint of Carpesium abrotanoides L.. Background Art

[0002] Carpesium abrotanoides L. is the dried whole herb of the Compositae plant Carpesium abrotanoides L., and has the effects of clearing heat and detoxifying, removing phlegm and stopping bleeding. Clinically, it is mainly used for treating toothache, oral erosion, herpes zoster, bacterial infection, skin pruritus, folliculitis, hepatitis, epidemic acute viral conjunctivitis and other diseases. Carpesium abrotanoides L. has been included in the provincial standards or processing specifications, indicating the important role it plays in traditional medical practice, such as the "Hunan Province Traditional Chinese Medicine Decoction Pieces Processing Specification" in 2021 edition, the "Jiangsu Province Traditional Chinese Medicine Decoction Pieces Processing Specification" in 2020 edition, the "Hubei Province Chinese Medicinal Materials Quality Standard" in 2018 edition, the "Zhejiang Province Traditional Chinese Medicine Processing Specification" in 2015 edition, the "Hebei Province Traditional Chinese Medicine Decoction Pieces Processing Specification" in 2003 edition and the "Shanghai City Traditional Chinese Medicine Decoction Pieces Processing Specification" in 2018 edition. However, it has not been included in the "Chinese Pharmacopoeia", and there is no established method for determining its index components in the existing standards. At present, the component analysis of Carpesium abrotanoides L. mainly focuses on sesquiterpenes (carabrone, telesterone, etc.).

[0003] At present, there is no research report on the fingerprint of Carpesium abrotanoides L., and there is a lack of relevant research on the quality control of Carpesium abrotanoides L.. Therefore, there is an urgent need to establish a new method for comprehensive quality control of Carpesium abrotanoides L.. Summary of the Invention

[0004] According to the problems raised above, the technical problem to be solved by the present invention is to provide a method for constructing a fingerprint of Carpesium abrotanoides L. to conduct comprehensive quality control on Carpesium abrotanoides L..

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] A method for constructing a fingerprint of Carpesium abrotanoides L. includes the following steps:

[0007] Prepare a test solution of Carpesium abrotanoides L.;

[0008] Prepare a mixed reference substance solution;

[0009] Use ultra-high performance liquid chromatography to detect and obtain the fingerprint of Carpesium abrotanoides L., and the mobile phase system includes mobile phase A and mobile phase B. The mobile phase A is an aqueous phosphoric acid solution, and the mobile phase B is an acetonitrile solution.

[0010] The method of the present invention has good specificity, good linear relationship, good precision, good stability, good repeatability and good accuracy.

[0011] The present invention adopts a phosphoric acid aqueous solution as a mobile phase A, thereby solving the defects of uneven baseline and unsatisfactory peak separation effect that may occur in other mobile phases.

[0012] According to the embodiments of the present invention, the present invention can be further optimized. The following is the technical solution formed after optimization:

[0013] The volume fraction of phosphoric acid in the phosphoric acid aqueous solution is 0.05-0.4%, preferably, the volume fraction of phosphoric acid in the phosphoric acid aqueous solution is 0.05-0.2%, and further preferably, the volume fraction of phosphoric acid in the phosphoric acid aqueous solution is 0.05-0.15%.

[0014] The use of an aqueous phosphoric acid solution with this volume fraction can ensure that more components in Tianmingjing are separated by ultra-high performance liquid chromatography.

[0015] In one of the preferred embodiments, the method for preparing the Tianmingjing test solution includes the following steps: using an extractant to extract the components in Tianmingjing, the extractant includes 50%-80% methanol or 50%-80% ethanol, preferably, the extractant includes 70%-80% methanol or 70%-80% ethanol.

[0016] The use of the above-mentioned extracting agent results in no significant difference in the peak areas of the components in the fingerprint.

[0017] In one preferred embodiment, the solid-liquid ratio of the Tianmingjing to the extractant is 0.3-0.6 g:15-25 mL; preferably, the solid-liquid ratio of the Tianmingjing to the extractant is 0.45-0.55 g:19.5-20.5 mL.

[0018] In one preferred embodiment, the detection conditions of the ultra-high performance liquid chromatography include: the chromatographic column is an octadecylsilane bonded silica gel chromatographic column, the mobile phase flow rate is 0.2-0.4mL / min, the detection wavelength is 210-230nm, the column temperature is 34-36°C, and the injection volume is 0.5-1μL.

[0019] The separation of 19 characteristic components in the fingerprint spectrum can be guaranteed within this wavelength range.

[0020] The ultra-high performance liquid chromatogram of the Tianmingjing sample obtained by the present invention is more complete and beautiful while meeting the content determination requirements, and the spectrum baseline is stable and the peak shapes of each component are symmetrical.

[0021] In one preferred embodiment, the ultra-high performance liquid chromatography method adopts gradient elution for elution; preferably, the procedure of the gradient elution includes:

[0022] 0-2min, the volume fraction of the mobile phase B is 10%;

[0023] From 2 to 5 min, the volume fraction of mobile phase B is increased from 10% to 14.5%;

[0024] From 5 to 15 min, the volume fraction of mobile phase B is increased from 14.5% to 22%;

[0025] From 15 to 17 min, the volume fraction of mobile phase B is increased from 22% to 26%;

[0026] From 17 to 25 min, the volume fraction of mobile phase B is increased from 26% to 28%;

[0027] From 25 to 30 min, the volume fraction of mobile phase B is increased from 28% to 37%.

[0028] In one preferred embodiment, the mixed reference substance solution includes chlorogenic acid, caffeic acid, luteoloside, 11(13)-dehydroaxillarin, isochlorogenic acid A, isochlorogenic acid C, 2,3,4,5-tetracaffeoyl-D-glucaric acid, 2-deoxy-4-epi-skyrin, carabrone, telekin.

[0029] In one preferred embodiment, the extraction is carried out by ultrasonic extraction; the power of the ultrasonic extraction is 190 - 210 W, the frequency is 35 - 45 kHz, and the time is 50 - 70 min.

[0030] Preferably, in the preparation method of the Carpesium abrotanoides L. test solution, the Carpesium abrotanoides L. includes Carpesium abrotanoides L. from different production areas and different batches from the same production area; after ultrasonic extraction, it is filtered through an organic microporous membrane.

[0031] Before detecting by ultra-high performance liquid chromatography, the following steps are also included:

[0032] Import the test solution liquid chromatogram into the similarity evaluation system of traditional Chinese medicine chromatographic characteristic fingerprints, generate the common mode of the characteristic fingerprints, and obtain the Carpesium abrotanoides L. fingerprint with 19 characteristic peaks;

[0033] By comparing the retention time of the fingerprint with that of the reference substance liquid chromatogram, peak 1 is chlorogenic acid, peak 2 is caffeic acid, peak 7 is luteoloside, peak 9 is 11(13)-dehydroaxillarin, peak 10 is isochlorogenic acid A, peak 12 is isochlorogenic acid C, peak 13 is 2,3,4,5-tetracaffeic acid-D-glucaric acid, peak 15 is 2-deoxy-4-epi-skyrin, peak 16 is carabrone, and peak 17 is telekin.

[0034] After detection by ultra-high performance liquid chromatography, it further includes importing the peak areas of 19 characteristic peaks in the obtained chromatogram of the test sample into Origin Pro, GraphPad Prism, and SIMCA for correlation and chemometric analysis.

[0035] The present invention also provides the application of the Carpesium abrotanoides fingerprint obtained by the construction method in the detection of components in Carpesium abrotanoides or the identification of Carpesium abrotanoides.

[0036] Traditional Chinese medicines have complex components and diverse effects. UPLC has a shorter analysis time, better separation effect, and is more economical and environmentally friendly than HPLC. At the same time, the chromatographic fingerprint of traditional Chinese medicine is a detailed and measurable tool for identifying phytochemicals, which can reflect the characteristics of "holism" and "complexity" of traditional Chinese medicine. Chromatographic fingerprint analysis has become the consensus of the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for the quality assessment of herbal medicines. The combination of multiple types of chemical components (sesquiterpenes, phenolic acids, and flavonoids) in Carpesium abrotanoides and the UPLC fingerprint of traditional Chinese medicine can comprehensively reflect the types and quantities of chemical components in the medicinal materials. At the same time, it can also be used for comparative analysis of different batches, which is of great significance for describing and evaluating the quality of medicinal materials. The existing quality control methods of Carpesium abrotanoides cannot well reflect the quality of the medicinal materials. Therefore, establishing the fingerprint of Carpesium abrotanoides by UPLC method is of great significance for evaluating the quality control of Carpesium abrotanoides.

[0037] The present invention can promote the improvement of the standards of Carpesium abrotanoides and ensure the efficacy of Carpesium abrotanoides in clinical or preparation. The present invention discovers that Carpesium abrotanoides also contains rich phenolic acid components (such as chlorogenic acid, isochlorogenic acid A, etc.) and flavonoid components (such as luteoloside). These three types of components all have pharmacological effects such as anti-inflammatory, antioxidant, anti-tumor, and antibacterial, which can be used as important indicators for measuring the quality of Carpesium abrotanoides.

[0038] The Carpesium abrotanoides fingerprint provided by the present invention has 19 characteristic peaks, including the characteristic peaks of chlorogenic acid, caffeic acid, luteoloside, 11(13)-dehydroabrotanin, isochlorogenic acid A, isochlorogenic acid C, 2,3,4,5-tetracaffeoyl-D-glucaric acid, 2-deoxy-4-epi-helianthenin, carabrone, and telesterone. The present invention establishes the Carpesium abrotanoides fingerprint for the first time, discovers and identifies 10 components in Carpesium abrotanoides, and uses multiple substances as quality indicators for Carpesium abrotanoides, which can achieve accurate and comprehensive quality control of the quality of Carpesium abrotanoides and perfects the blank of qualitative identification of index components in the currently implemented quality standard of Carpesium abrotanoides.

[0039] The construction method of the Carpesium abrotanoides fingerprint provided by the present invention has high precision, good stability, and good reproducibility, can comprehensively control the quality of Carpesium abrotanoides, and thus better ensure the quality stability, consistency, and controllability of Carpesium abrotanoides. The method provided by the present invention overcomes the problem of no detection of index components in the existing quality standard of Carpesium abrotanoides.

[0040] The present invention conducts chemometric analysis on Carpesium abrotanoides from different origins, screens out 12 differential quality markers, and provides a theoretical basis for evaluating the quality of Carpesium abrotanoides. Description of the Drawings

[0041] Figure 1 is the fingerprint of 17 batches of Carpesium abrotanoides;

[0042] Figure 2 are the maps of blank (A), reference fingerprint (B) and mixed reference substances (C);

[0043] Figure 3 is the correlation coefficient map of 17 batches of medicinal materials and reference maps;

[0044] Figure 4 is the cluster analysis map of 17 batches of Carpesium abrotanoides samples;

[0045] Figure 5 is the score map of principal component analysis;

[0046] Figure 6 is the score map of OPLS-DA;

[0047] Figure 7 is the VIP value;

[0048] Figure 8 is the permutation test map;

[0049] Figure 9 is the high performance liquid chromatography map of Carpesium abrotanoides in Comparative Example 1;

[0050] Figure 10 is the ultra high performance liquid chromatography map of Carpesium abrotanoides in Comparative Example 2;

[0051] Figure 11 is the ultra high performance liquid chromatography map of Carpesium abrotanoides in Comparative Example 3;

[0052] Figure 12 is the ultra high performance liquid chromatography map of Carpesium abrotanoides with different extraction solvents in Comparative Example 4;

[0053] Figure 13 is the ultra high performance liquid chromatography map of Carpesium abrotanoides with different wavelengths in Comparative Example 5.

[0054] Note: Figure 1 、 2 : 1. Chlorogenic acid; 2. Caffeic acid; 7. Luteoloside; 9. 11(13)-Dehydroaxillarin; 10. Isochlorogenic acid A; 12. Isochlorogenic acid C; 13. 2,3,4,5-Tetracaffeoyl-D-glucaric acid; 15. 2-Deoxy-4-epi-heliangolide; 16. Carabrone; 17. Telekin. Detailed implementation manners

[0055] The present invention will be described in detail below in conjunction with specific embodiments.

[0056] Example 1

[0057] 1. Source of samples

[0058] Specific information of 17 batches of Carpesium abrotanoides L. herbs is shown in Table 1. Among them, batches S4, 5, 9, 10, 11, and 12 are fresh herbs, which are obtained by removing impurities, washing, moistening thoroughly, cutting, and air-drying according to the processing method of "Hunan Province Traditional Chinese Medicine Decoction Pieces Processing Specification" (2021 edition), and are identified as the dried whole herb of the Compositae plant Carpesium abrotanoides Linnaeus by Associate Professor Wang Zhi of Hunan University of Chinese Medicine.

[0059] Table 1 Information table of Carpesium abrotanoides L. herbs

[0060]

[0061] 2. Chromatographic conditions

[0062] Waters BEH C 18 Chromatographic column (2.1×100 mm, 1.7 μm), mobile phase: 0.1% phosphoric acid water (A) - acetonitrile (B), gradient elution (0 - 2 min, 10% B; 2 - 5 min, 10% - 14.5% B; 5 - 15 min, 14.5% - 22% B; 15 - 17 min, 22% - 26% B; 17 - 25 min, 26% - 28% B; 25 - 30 min, 28% - 37% B), column temperature: 35°C, flow rate: 0.3 mL·min -1 , detection wavelength: 230 nm, injection volume: 0.5 μL.

[0063] 3. Preparation of solutions

[0064] 3.1 Preparation of reference substance solution

[0065] Weigh appropriate amounts of chlorogenic acid, caffeic acid, luteoloside, 11(13)-dehydroaxillarin, isochlorogenic acid A, isochlorogenic acid C, 2,3,4,5-tetracaffeoyl-D-glucaric acid, 2-deoxy-4-epihelenalin, carabrone, and telupetalin reference substances accurately, dissolve them in 80% ethanol to prepare single reference substance stock solutions with certain mass concentrations. Take appropriate amounts of the above single reference substance stock solutions, place them in the same 10 mL volumetric flask, dilute to the mark with 80% ethanol, and shake well to obtain mixed reference substance stock solutions with mass concentrations of 158.88, 19.86, 149.76, 95.70, 445.20, 19.96, 72.94, 173.50, 63.84, and 67.50 μg·mL -1 respectively.

[0066] Precisely pipette 2 mL of the above mixed reference substance stock solution into a 10 mL volumetric flask, dilute to the mark with 80% ethanol, and shake well to obtain a mixed reference substance solution with mass concentrations of 31.78, 3.97, 29.95, 19.14, 89.04, 3.99, 14.59, 34.70, 12.77, and 13.50 μg·mL -1 respectively.

[0067] 3.2 Preparation of the test solution

[0068] Take 0.5 g of Carpesium abrotanoides L. powder (passed through a 60-mesh sieve), weigh it accurately, place it in a stoppered conical flask, accurately add 20 mL of 80% ethanol, weigh it, ultrasonically extract for 60 min, let it cool, weigh it again, make up the weight with 80% ethanol, shake well, and filter through a 0.22 μm organic microporous filter membrane to obtain the test solution.

[0069] 4. Fingerprint study

[0070] 4.1 Precision test

[0071] Take Carpesium abrotanoides L. powder, prepare the test solution according to the above 1), inject samples continuously for 6 times under the above chromatographic conditions, use peak 10 (isochlorogenic acid A) as the reference peak, calculate that the RSDs of the relative retention times of each main common peak are all < 0.15%, and the RSDs of the relative peak areas are all < 2.65%, indicating that the precision of the instrument is good, as shown in Table 1.

[0072] Table 1 Results of precision investigation

[0073]

[0074]

[0075] 4.2 Stability test

[0076] Take the powder of Carpesium abrotanoides L., prepare the test solution according to 1) above, inject samples for detection at 0, 2, 4, 8, 12, and 24 h respectively under the above chromatographic conditions. Using peak 10 (isochlorogenic acid A) as the reference peak, the RSDs of the relative retention times of each main common peak are all <0.31%, and the RSDs of the relative peak areas are all <2.27%, indicating that this method has good stability, as shown in Table 2.

[0077] Table 2 Results of stability investigation

[0078]

[0079]

[0080] 4.3 Repeatability test

[0081] Take 6 batches of Carpesium abrotanoides L. powder, prepare the test solution according to 1) above, inject samples for analysis under the above chromatographic conditions. Using peak 10 (isochlorogenic acid A) as the reference peak, the RSDs of the relative retention times of each main common peak are all <0.55%, and the RSDs of the relative peak areas are all <2.95%, indicating that this method has good repeatability, as shown in Table 3.

[0082] Table 3 Results of repeatability investigation

[0083]

[0084]

[0085] 4.4 Establishment of fingerprint and similarity evaluation

[0086] Take 17 batches of Carpesium abrotanoides L. medicinal materials, prepare the test solution according to the method under "3.2", inject samples under the chromatographic conditions under "2", and record the chromatograms. Import the chromatogram data into the software "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (2012 Edition)" of the National Pharmacopoeia Commission, generate a reference chromatogram using the average value, set the time window width to 0.1 min, perform multi-point calibration and Mark peak matching to generate the common pattern of the fingerprint and the reference fingerprint, and a total of 19 common peaks are confirmed, as shown in Figure 1 . By comparing with the reference substances, 10 components are identified, as shown in Figure 2 , peak 1 is chlorogenic acid, peak 2 is caffeic acid, peak 7 is luteoloside, peak 9 is 11(13)-dehydroaxillarin, peak 10 is isochlorogenic acid A, peak 12 is isochlorogenic acid C, peak 13 is 2,3,4,5-tetracaffeoyl-D-glucaric acid, peak 15 is 2-deoxy-4-epi-heliangolide, peak 16 is carabrone, peak 17 is telekinolide.

[0087] The peak areas of 19 common peaks in 17 batches of Carpesium abrotanoides L. herbs were used as variables and imported into Origin Pro 2021 software for correlation analysis, and a correlation heat map was drawn to evaluate the similarity among Carpesium abrotanoides L. herbs from different origins. The results are shown in Figure 3 . In the figure, from red to green, and the size of the circles, all indicate that the correlation coefficient gradually increases, and the P value represents the significance degree of the correlation between different origins. The results show that the correlation coefficients between Carpesium abrotanoides L. herbs from different origins and the reference fingerprint are all significantly positively correlated, between 0.64 and 0.95, indicating that the chemical compositions of Carpesium abrotanoides L. from different origins are highly consistent and have certain similarities. However, by comparing different origins, it is found that the correlation coefficients among Carpesium abrotanoides L. from the same origin are generally higher than those between different origins, indicating that there are differences in the quality of Carpesium abrotanoides L. herbs from different origins. Therefore, establishing the fingerprint of Carpesium abrotanoides L. can comprehensively evaluate the quality of Carpesium abrotanoides L. as a whole. At the same time, the stability and differences of its herbs from different origins also provide a basis for its research.

[0088] 5. Cluster analysis

[0089] The peak areas of 19 common peaks in 17 batches of Carpesium abrotanoides L. samples were imported into Origin Pro software, and the systematic cluster analysis method was used for classification with Euclidean squared distance. The results are shown in Figure 3 . In the figure, the color of the color blocks intuitively reflects the size of the peak area, increasing from red to blue. The 17 batches of Carpesium abrotanoides L. samples are divided into 2 categories. S1, S4, S5, S6, S12, S13, and S17 are one category, and S2, S3, S7, S8, S9, S10, S11, S14, S15, and S16 are another category, showing that there are significant differences in the quality of Carpesium abrotanoides L. among different origin batches, which may be related to factors such as the origin environment and planting technology. It is of great significance to evaluate its quality, which can evaluate the quality of Carpesium abrotanoides L. and provide a reliable scientific basis for ensuring the preparation and clinical efficacy.

[0090] 6. Principal component analysis

[0091] Taking the peak areas of 19 common peaks in the fingerprint of 17 batches of Carpesium abrotanoides L. as variables, principal component analysis was carried out using GraphPad Prism 10.1.2 software. Three principal components were obtained with the cumulative variance contribution rate > 80% as the extraction standard. Their variance contribution rates were 63.22%, 13.66%, and 9.03% respectively, and the cumulative variance contribution rate was 84.91%, which can represent most of the information of 19 common components, as shown in Table 4. The peaks with higher contribution rates in principal component 1 are peak 19, peak 13, peak 3, and peak 14, all of which are negative loadings. The peaks with higher contribution rates in principal component 2 are peak 1 and peak 8, among which peak 8 is a negative loading. The peak with higher contribution rate in principal component 3 is peak 17, as shown in Table 5. The scores of the three principal components are recorded as Y 1 、Y 2 、Y 3, taking the contribution rate corresponding to each main component as the weight coefficient, a comprehensive score model of the main components was established: Y = 0.6222Y 1 + 0.1366Y 2 + 0.9030Y 3

[0092] The scoring results are shown in Table 6. The scores reflect the quality of Carpesium abrotanoides in each batch. The higher the score, the better the quality. The results show that the top five batches in terms of quality are S8, S17, S6, S11, and S10. The peak areas of 19 common peaks in the 17 batches of samples were imported into SIMCA 14.1 software for PCA processing. The score plot is shown in Figure 5 , which is similar to the clustering result.

[0093] Table 4 Eigenvalues and variance contribution rates of the main components analysis of Carpesium abrotanoides

[0094]

[0095] Table 5 PCA factor loading matrix of Carpesium abrotanoides

[0096]

[0097] Table 6 Main component factor scores and rankings of 17 batches of Carpesium abrotanoides

[0098]

[0099]

[0100] 7. Orthogonal partial least squares-discriminant analysis (OPLS-DA)

[0101] In order to further analyze the differences between Carpesium abrotanoides from different origins, the peak areas of 19 common peaks in the fingerprint spectra of 17 batches of Carpesium abrotanoides were imported into SIMCA 14.1 software for OPLS-DA analysis. The model parameters are R 2 X = 0.843, R 2 Y = 0.909, Q 2 = 0.802. The results of its score plot are similar to those of the cluster analysis and the main component analysis, as shown in Figure 6Using VIP > 1 as the screening criterion, the VIP values of peak 10 (isochlorogenic acid A), peak 3, peak 19, peak 13 (2,3,4,5-tetracaffeoyl-D-glucaric acid), peak 1 (chlorogenic acid), peak 12 (isochlorogenic acid C), peak 14, peak 18, peak 15 (2-deoxy-4-epi-helianthenin), peak 6, peak 11, and peak 7 are 1.233 4, 1.164 9, 1.117 4, 1.098 3, 1.079 0, 1.073 5, 1.076 7, 1.073 5, 1.063 3, 1.063 1, 1.054 7, and 1.047 4 respectively, all of which are greater than 1. These 12 components can be used as potential differential quality markers of Carpesium abrotanoides L. The VIP score chart is shown in Figure 7 The OPLS-DA model was permuted 200 times to obtain the permutation test chart, and the results of R 2 and Q 2 The intercepts on the Y-axis are both smaller than the original values, indicating that the established model is not overfitted. See Figure 8 .

[0102] The method for Carpesium abrotanoides L. established in the present invention is different from the related technologies, including the chromatographic instrument, chromatographic column, mobile phase, gradient elution program, variable wavelength detection program, flow rate, column temperature, and injection volume used. The types of components of Carpesium abrotanoides L. established under these chromatographic conditions are also different from the related technologies. The identified components include 5 phenolic acids (chlorogenic acid, caffeic acid, isochlorogenic acid A, isochlorogenic acid C, 2,3,4,5-tetracaffeoyl-D-glucaric acid), 4 sesquiterpenoids (11(13)-dehydroaxillarin, 2-deoxy-4-epi-helianthenin, carabrone, telesterone), and 1 flavonoid (luteoloside) component. The ultra-high performance liquid chromatography map of the Carpesium abrotanoides L. sample obtained is more complete and beautiful while meeting the requirements of the fingerprint. The baseline of the map is stable and the peak shapes of each component are symmetrical.

[0103] Comparative Example 1

[0104] This comparative example is a HPLC separation method for Carpesium abrotanoides L., and the specific liquid phase conditions are as follows:

[0105] The instrument is an Agilent high performance liquid chromatograph and a DAD detector; the chromatographic column is SunFire TM C 18 (250 mm × 4.6 mm, 5 μm); the mobile phase is water (A) - acetonitrile (B), with gradient elution (0 - 30 min, 5% - 95% B); the column temperature is 35°C; the wavelength is 230 nm; the injection volume is 10 μL; the flow rate is 1 mL / min.

[0106] The high performance liquid chromatography detection map of the test solution of Carpesium abrotanoides L. in this comparative example is shown in Figure 9, As can be seen from the figure, HPLC takes a long time and requires a large amount of mobile phase and sample. Subsequently, UPLC was used for separation.

[0107] Comparative Example 2

[0108] This comparative example is a method for separating Carpesium abrotanoides by UPLC. The specific liquid phase conditions are as follows:

[0109] The instrument is a Waters H-Class ultra-high performance liquid chromatograph and a PDA detector; the chromatographic column is a Waters ACQUITY UPLC BEH C 18 (2.1×100 mm, 1.7 μm); the mobile phase is 0.1% formic acid water (A) - acetonitrile (B), gradient elution (0 - 2 min, 5% - 22% B; 2 - 25 min, 22% - 65% B; 25 - 30 min, 65% - 5% B); the column temperature is 30 °C; the wavelength is 230 nm; the injection volume is 1 μL; the flow rate is 0.3 mL / min.

[0110] The UPLC chromatogram of the Carpesium abrotanoides test solution in this comparative example is shown in Figure 10 , from Figure 10 it can be seen that compared with Comparative Example 1, this method has a shorter separation time and faster speed. The peaks basically come out completely within 20 minutes, and the number of peaks is large. However, this method uses formic acid water, resulting in an uneven baseline and less than ideal peak separation effect. The liquid phase conditions still need to be optimized. Comparative Example 3

[0111] This comparative example is a method for separating Carpesium abrotanoides by UPLC. The specific gradient is as follows:

[0112] The instrument is a Waters H-Class ultra-high performance liquid chromatograph and a PDA detector; the chromatographic column is a Waters ACQUITY UPLC BEH C 18 (2.1×100 mm, 1.7 μm); the mobile phase is 0.1% phosphoric acid (A) - acetonitrile (B), gradient elution (0 - 2 min, 10% - 10% B; 2 - 5 min, 10% - 14.5% B; 5 - 10 min, 14.5% - 18% B; 10 - 15 min, 18% - 22% B; 15 - 30 min, 22% - 50% B); the column temperature is 30 °C; the wavelength is 230 nm; the injection volume is 0.5 μL; the flow rate is 0.3 mL / min.

[0113] The UPLC diagram of the Carpesium abrotanoides test solution in this comparative example is shown in Figure 11 , from Figure 11 it can be seen that when this method is changed to phosphoric acid water, the baseline is more stable than that of formic acid water, and most components have been separated, but some components still need to be further explored for separation.

[0114] Comparative Example 4

[0115] This comparative example is for the investigation of the extraction solvent of the Carpesium abrotanoides L. test sample, and consists of the following steps:

[0116] 1) Preparation of the test sample solution

[0117] Take 0.5 g of Carpesium abrotanoides L. powder (passed through a 60-mesh sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 20 mL of the corresponding extraction solvent (water, 30% methanol / ethanol, 50% methanol / ethanol, 70% methanol / ethanol, 80% methanol / ethanol, methanol / ethanol), weigh it, ultrasonically extract for 60 min, let it cool and then weigh it again, make up the weight with the corresponding extraction solvent, shake well, filter through a 0.22-μm microporous membrane to obtain the test sample solution.

[0118] 2) Liquid phase conditions

[0119] The chromatographic column is Waters ACQUITY UPLC BEH C 18 (2.1×100 mm,

[0120] 1.7 μm); the mobile phase is 0.1% phosphoric acid water (A) - acetonitrile (B), gradient elution (0 - 30 min, 10% - 65% B); the column temperature is 30 °C; the wavelength is 230 nm; the injection volume is 1 μL; the flow rate is 0.3 mL / min.

[0121] The ultra-high performance liquid chromatography detection chart of the Carpesium abrotanoides L. test sample solution in this comparative example is shown in Figure 12 , and it can be known from Figure 12 that when the extraction solvent is water, 30% methanol / ethanol, and methanol / ethanol, the components are few or the peak areas are small, and there is no significant difference in the number and peak areas of the components of 50 - 80% methanol / ethanol.

[0122] Comparative Example 5

[0123] This comparative example is for the investigation of the wavelength of the Carpesium abrotanoides L. UPLC chromatographic method, and consists of the following steps:

[0124] 1) Preparation of the test sample solution

[0125] Take 0.5 g of Carpesium abrotanoides L. powder (passed through a 60-mesh sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 20 mL of 80% ethanol, weigh it, ultrasonically extract for 60 min, let it cool and then weigh it again, make up the weight with 80% ethanol, shake well, filter through a 0.22-μm microporous membrane to obtain the test sample solution.

[0126] 2) Liquid phase conditions

[0127] The chromatographic column is Waters ACQUITY UPLC BEH C 18 (2.1×100 mm, 1.7 μm); The mobile phase was 0.1% phosphoric acid (A) - acetonitrile (B), gradient elution (0 - 2 min, 10% B; 2 - 5 min, 10% - 14.5% B; 5 - 15 min, 14.5% - 22% B; 15 - 17 min, 22% - 26% B; 17 - 25 min, 26% - 28% B; 25 - 30 min, 28% - 37% B); The column temperature was 30 °C; The wavelength was 230 nm; The injection volume was 0.5 μL; The flow rate was 0.3 mL / min.

[0128] The ultra - high performance liquid chromatography detection graphs of the Carpesium abrotanoides L. test solution at different wavelengths in this comparative example are shown in Figure 13 , and it can be known from Figure 13 that among the 19 characteristic peaks, peaks 9, 15, 16, and 17 eluted at 210 - 230 nm, and the remaining components eluted at 201 - 380 nm.

[0129] In summary, the method for constructing the fingerprint of Carpesium abrotanoides L. provided by the present invention has high precision, good stability and good reproducibility, can comprehensively control the quality of Carpesium abrotanoides L., and thus better ensure the quality stability, consistency and controllability of Carpesium abrotanoides L.

[0130] The above - mentioned are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A method for constructing a Tianmingjing fingerprint spectrum, characterized in that: The following steps are involved: Prepare the Tianmingjing test solution; Prepare mixed reference solution; The fingerprint of Tianmingjing was obtained by ultra-high performance liquid chromatography, and the mobile phase system included mobile phase A and mobile phase B, wherein the mobile phase A was a phosphoric acid aqueous solution, and the mobile phase B was an acetonitrile solution.

2. The method for constructing the Tianmingjing fingerprint spectrum according to claim 1, characterized in that: The volume fraction of phosphoric acid in the phosphoric acid aqueous solution is 0.05-0.4%; preferably, the volume fraction of phosphoric acid in the phosphoric acid aqueous solution is 0.05-0.2%.

3. The method for constructing the Tianmingjing fingerprint spectrum according to claim 1, characterized in that: The method for preparing the Tianmingjing test solution comprises the following steps: extracting the components in Tianmingjing with an extractant, wherein the extractant comprises 50%-80% methanol or 50%-80% ethanol, and preferably, the extractant comprises 70%-80% methanol or 70%-80% ethanol.

4. The method for constructing the Tianmingjing fingerprint spectrum according to claim 3, characterized in that: The solid-liquid ratio of the Tianmingjing to the extractant is 0.3-0.6 g:15-25 mL; preferably, the solid-liquid ratio of the Tianmingjing to the extractant is 0.45-0.55 g:19.5-20.5 mL.

5. The method for constructing the Tianmingjing fingerprint spectrum according to claim 1, characterized in that: The detection conditions of the ultra-high performance liquid chromatography method include: the chromatographic column is an octadecylsilane bonded silica gel chromatographic column, the mobile phase flow rate is 0.2-0.4mL / min, the detection wavelength is 210-230nm, the column temperature is 33-37°C, and the injection volume is 0.5-1μL.

6. The method for constructing the Tianmingjing fingerprint spectrum according to claim 1, characterized in that: The ultra-high performance liquid chromatography method adopts gradient elution for elution; preferably, The procedure of the gradient elution includes: 0-2min, the volume fraction of the mobile phase B is 10%; 2-5 min, the volume fraction of the mobile phase B increases from 10% to 14.5%; 5-15 min, the volume fraction of the mobile phase B increases from 14.5% to 22%; 15-17 min, the volume fraction of the mobile phase B increased from 22% to 26%; 17-25 min, the volume fraction of the mobile phase B increased from 26% to 28%; From 25 to 30 minutes, the volume fraction of the mobile phase B increases from 28% to 37%.

7. The method for constructing the Tianmingjing fingerprint spectrum according to claim 1, characterized in that: The mixed reference substance solution includes chlorogenic acid, caffeic acid, luteolin, 11(13)-dehydro-axillin, isochlorogenic acid A, isochlorogenic acid C, 2,3,4,5-tetra-caffeoyl-D-glucaric acid, 2-deoxy-4-epimeryl, tianaquinone, and telolactone.

8. The method for constructing the Tianmingjing fingerprint spectrum according to any one of claims 1 to 7, characterized in that: The extraction adopts ultrasonic extraction; the power of the ultrasonic extraction is 190-210W, the frequency is 35-45kHz, and the time is 50-70min.

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