A method for constructing a fingerprint spectrum of asarum forrestii

Abstract

The application discloses a method for constructing a fingerprint spectrum of radix isatidis, which comprises the following steps: preparing a radix isatidis sample solution; preparing a mixed control sample solution; and detecting the fingerprint spectrum of the radix isatidis by using an ultra performance liquid chromatography (UPLC) method, wherein 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 fingerprint spectrum of the radix isatidis provided by the application has 19 common peaks, and 10 components are identified. The UPLC fingerprint spectrum of the radix isatidis established by the application has good repeatability and precision, and the method is stable and reliable, so that the method can simultaneously and accurately and rapidly monitor the quality of multiple components of the radix isatidis, and provides a strong guarantee for controlling the quality of the radix isatidis and ensuring clinical curative effects.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of traditional Chinese medicine detection, and particularly relates to a method for constructing a fingerprint spectrum of Carpesium abrotanoides. BACKGROUND

[0002] Carpesium abrotanoides L. is the dried whole plant of Carpesium abrotanoides L. in the family Asteraceae, which has the effects of clearing heat and resolving toxins, removing phlegm and stopping bleeding. It is mainly used for treating toothache, oral erosion, herpes zoster, bacterial infection, skin itching, folliculitis, hepatitis, epidemic acute viral conjunctivitis and other diseases. Carpesium abrotanoides L. is included in the provincial standards or processing specifications, which shows that it plays an important role in traditional medical practice, such as the 2021 edition of 'Hunan Province Chinese Medicine Decoction Processing Specification', the 2020 edition of 'Jiangsu Province Chinese Medicine Decoction Processing Specification', the 2018 edition of 'Hubei Province Chinese Medicine Quality Standard', the 2015 edition of 'Zhejiang Province Chinese Medicine Processing Specification', the 2003 edition of 'Hebei Province Chinese Medicine Decoction Processing Specification' and the 2018 edition of 'Shanghai City Chinese Medicine Decoction Processing Specification'. However, it has not been included in 'Chinese Pharmacopoeia', and the determination method of its index components has not been established in the standards that have been included. At present, the component analysis of Carpesium abrotanoides L. mainly focuses on sesquiterpenes (Carpesium abrotanoides ketone, terulactone, etc.).

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

[0004] In view of the above problems, the technical problem to be solved by the present application is to provide a method for constructing a fingerprint spectrum of Carpesium abrotanoides L. for the comprehensive quality control of Carpesium abrotanoides L.

[0005] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

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

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

[0008] Preparing a mixed control solution;

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

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

[0011] The application adopts the mobile phase A of phosphoric acid water solution, solves the defects of baseline unevenness and less ideal peak separation effect that may be caused by other mobile phases.

[0012] According to the embodiments of the application, the application can be further optimized, and the following is the technical scheme formed after optimization:

[0013] The volume fraction of phosphoric acid in the phosphoric acid water solution is 0.05-0.4%; preferably, the volume fraction of phosphoric acid in the phosphoric acid water solution is 0.05-0.2%. Further preferably, the volume fraction of phosphoric acid in the phosphoric acid water solution is 0.05-0.15%.

[0014] The phosphoric acid water solution with the volume fraction can ensure that more components in the asarum sieboldii are separated in the ultra-high performance liquid chromatography.

[0015] In one preferred embodiment, the preparation method of the asarum sieboldii sample solution comprises the following steps: extracting components in the asarum sieboldii by using an extracting agent, the extracting agent comprising 50%-80% methanol or 50%-80% ethanol, preferably, the extracting agent comprising 70%-80% methanol or 70%-80% ethanol.

[0016] The use of the above extracting agent makes the component peak area in the fingerprint spectrum have no large difference.

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

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

[0019] The wavelength range can ensure the separation of 19 characteristic components in the fingerprint spectrum.

[0020] The ultra-high performance liquid chromatogram of the asarum sieboldii sample obtained by the application is more complete and beautiful while meeting the content determination requirements, and the spectrum baseline is stable and each component peak shape is symmetrical.

[0021] In one preferred embodiment, the ultra-high performance liquid chromatography adopts a gradient elution mode for elution; preferably, the program of the gradient elution comprises:

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

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

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

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

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

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

[0028] In one preferred embodiment, the mixed reference solution comprises chlorogenic acid, caffeic acid, luteolin, 11(13)-dehydroxyevacuolin, isochlorogenic acid A, isochlorogenic acid C, 2,3,4,5-tetra caffeoyl-D-glucaric acid, 2-deoxy-4-epi-euparin, euparin ketone, thumbergide.

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

[0030] Preferably, in the preparation method of the euparin test sample solution, the euparin is obtained from different sources and different batches of the same source; and the ultrasonic extraction is followed by filtration using an organic microporous filter.

[0031] The method further comprises the following steps before detection by ultrahigh performance liquid chromatography:

[0032] The test sample liquid chromatogram is introduced into a traditional Chinese medicine chromatographic characteristic atlas similarity evaluation system to generate a characteristic atlas common mode, and a euparin fingerprint atlas with 19 characteristic peaks is obtained.

[0033] By comparing the retention time of the fingerprint atlas with that of the reference liquid chromatogram, peak 1 is chlorogenic acid, peak 2 is caffeic acid, peak 7 is luteolin, peak 9 is 11(13)-dehydroxyevacuolin, peak 10 is isochlorogenic acid A, peak 12 is isochlorogenic acid C, peak 13 is 2,3,4,5-tetra caffeoyl-D-glucaric acid, peak 15 is 2-deoxy-4-epi-euparin, peak 16 is euparin ketone, and peak 17 is thumbergide.

[0034] After detection by ultra-high performance liquid chromatography, the peak areas of 19 characteristic peaks in the obtained test product chromatogram are introduced into Origin Pro, GraphPad Prism, SIMCA for correlation and chemometrics analysis.

[0035] The application also provides application of the fingerprint of the constructed Teniamina in component detection of Teniamina or identification of Teniamina.

[0036] Traditional Chinese medicine has complex components and various functions, UPLC has shorter analysis time, better separation effect and is more economical and environmentally friendly than HPLC. In addition, traditional Chinese medicine chromatographic fingerprint is a detailed and measurable tool for identifying phytochemicals, which can reflect the characteristics of the 'holism' and 'complexity' of traditional Chinese medicine. Chromatographic fingerprint analysis has become a consensus of the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for evaluating the quality of herbs. The combination of UPLC traditional Chinese medicine fingerprint and multiple chemical components (sesquiterpenes, phenolic acids and flavonoids) in Teniamina can comprehensively reflect the types and quantities of chemical components in the medicinal material, and 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 means of Teniamina cannot well reflect the quality of the medicinal material, therefore, it is of great significance to establish the fingerprint of Teniamina by UPLC for evaluating the quality control of Teniamina.

[0037] The application can promote the perfection of the standard of Teniamina and ensure the efficacy of Teniamina in clinical or preparation. The application finds that Teniamina also contains rich phenolic acids (chlorogenic acid, isochlorogenic acid A, etc.) and flavonoids (acacia glycoside), and the three types of components have pharmacological effects such as anti-inflammatory, antioxidant, antitumor and antibacterial, which can be used as important indicators for measuring the quality of Teniamina.

[0038] The fingerprint of Teniamina provided by the application has 19 characteristic peaks, including the characteristic peaks of chlorogenic acid, caffeic acid, acacia glycoside, 11(13)-dehydroxy-axillary ivacillin, isochlorogenic acid A, isochlorogenic acid C, 2,3,4,5-tetra caffeoyl-D-glucaric acid, 2-deoxy-4-epi-tianrunqulin, teniamin lactone ketone and terenolide. The fingerprint of Teniamina is established for the first time, 10 components are found and identified in Teniamina, and multiple substances are used as quality indicators of Teniamina, which can realize accurate and comprehensive quality control of the quality of Teniamina, and perfect the blank of qualitative identification of index components in the existing quality standard of Teniamina.

[0039] The construction method of the fingerprint of Teniamina provided by the application has high precision, good stability and good reproducibility, and can comprehensively control the quality of Teniamina, so as to better ensure the quality stability, consistency and controllability of Teniamina. The method provided by the application overcomes the problem of no index component detection in the existing quality standard of Teniamina.

[0040] The present application performs chemometrics analysis on different origin of Daphne odora, and screens 12 different quality markers, which provides a theoretical basis for evaluating the quality of Daphne odora. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 The figure is the fingerprint of 17 batches of Daphne odora;

[0042] Figure 2 The figure is the fingerprint of blank (A), control fingerprint (B) and mixed control (C);

[0043] Figure 3 The figure is the correlation coefficient of 17 batches of medicinal materials and control fingerprint;

[0044] Figure 4 The figure is the cluster analysis of 17 batches of Daphne odora samples;

[0045] Figure 5 The figure is the score plot of principal component analysis;

[0046] Figure 6 The figure is the score plot of OPLS-DA;

[0047] Figure 7 The figure is the VIP value;

[0048] Figure 8 The figure is the permutation test plot;

[0049] Figure 9 The figure is the high performance liquid chromatogram of Daphne odora in Comparative Example 1;

[0050] Figure 10 The figure is the ultra performance liquid chromatogram of Daphne odora in Comparative Example 2;

[0051] Figure 11 The figure is the ultra performance liquid chromatogram of Daphne odora in Comparative Example 3;

[0052] Figure 12 The figure is the ultra performance liquid chromatogram of Daphne odora in different extraction solvents in Comparative Example 4;

[0053] Figure 13 The figure is the ultra performance liquid chromatogram of Daphne odora in different wavelengths in Comparative Example 5.

[0054] Notes: Figure 1 , 2 : 1. chlorogenic acid; 2. caffeic acid; 7. lavandulatol; 9. 11 (13) - dehydroxyaxiol; 10. isochlorogenic acid A; 12. isochlorogenic acid C; 13. 2, 3, 4, 5- tetra caffeic acid acyl-D-glucaric acid; 15. 2-deoxy-4-epi-tanacetone; 16. daphne odora lactone ketone; 17. terebinth lactone. DETAILED DESCRIPTION

[0055] The application will be described in detail below with specific examples.

[0056] Example 1

[0057] 1. Sample source

[0058] The specific information of 17 batches of tianmingjing medicinal materials is shown in Table 1, wherein batches S4, 5, 9, 10, 11 and 12 are fresh medicinal materials, which are processed according to the processing method in the 2021 edition of “Hunan Province Chinese Medicine Decoction Processing Standard”, and the obtained products after removing impurities, washing, moisturizing, cutting and drying are identified by Professor Wang Zhi of Hunan University of Chinese Medicine as the dried whole plant of Carpesium abrotanoides Linnaeus of Compositae.

[0059] Table 1 Information table of tianmingjing medicinal materials

[0060]

[0061] 2. Chromatographic conditions

[0062] Waters BEH C 18 Chromatographic column (2.1 x 100 mm, 1.7 μm), mobile phase is 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 is 35℃, flow rate is 0.3 mL·min -1 , detection wavelength is 230 nm, injection amount is 0.5 μL.

[0063] 3. Preparation of solution

[0064] 3.1 Preparation of control solution

[0065] An appropriate amount of chlorogenic acid, caffeic acid, luteolin, 11(13)-dehydroxyaxillaric acid, isochlorogenic acid A, isochlorogenic acid C, 2,3,4,5-tetra caffeoyl-D-glucaric acid, 2-deoxy-4-epi-euparin, euparin ketone, and thumbergide reference substance were precisely weighed, dissolved in 80% ethanol to prepare a single reference substance stock solution with a certain mass concentration. An appropriate amount of the single reference substance stock solution was taken into a same 10 mL volumetric flask, and 80% ethanol was added to constant volume at the calibration mark, and then shaken to obtain a mixed reference substance stock solution with a mass concentration 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] An appropriate amount of the mixed reference substance stock solution was precisely taken into a 10 mL volumetric flask, and 80% ethanol was added to constant volume at the calibration mark, and then shaken to obtain a mixed reference substance solution with a mass concentration 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] An appropriate amount of thumbergide powder (passed through a 60-mesh sieve) was precisely weighed and placed in a conical flask with a stopper. 80% ethanol was precisely added to a total volume of 20 mL, and the weight was determined. After ultrasonic extraction for 60 min, the weight was determined again after cooling, and the weight was made up with 80% ethanol. After shaking, the test solution was obtained by filtering through a 0.22 μm organic microporous filter.

[0069] 4. Fingerprint study

[0070] 4.1 Precision test

[0071] Thumbergide powder was used to prepare the test solution according to the above method. The sample was continuously injected 6 times according to the above chromatographic conditions, and peak 10 (isochlorogenic acid A) was used as the reference peak. The RSD of the relative retention time of each main common peak was less than 0.15%, and the RSD of the relative peak area was less than 2.65%, indicating that the instrument precision was good. See Table 1.

[0072] Table 1 Precision test results

[0073]

[0074]

[0075] 4.2 Stability test

[0076] The Tianmingjing powder was taken, the test sample solution was prepared according to 1) above, and was injected for detection at 0, 2, 4, 8, 12, 24 h according to the above chromatographic conditions. With peak 10 (isochlorogenic acid A) as the reference peak, the RSD of the relative retention time of each main common peak was less than 0.31%, and the RSD of the relative peak area was less than 2.27%, indicating that the method had good stability, as shown in Table 2.

[0077] Table 2 Stability test results

[0078]

[0079]

[0080] 4.3 Reproducibility test

[0081] Six batches of Tianmingjing powder were taken, the test sample solution was prepared according to 1) above, and was injected for analysis according to the above chromatographic conditions. With peak 10 (isochlorogenic acid A) as the reference peak, the RSD of the relative retention time of each main common peak was less than 0.55%, and the RSD of the relative peak area was less than 2.95%, indicating that the method had good reproducibility, as shown in Table 3.

[0082] Table 3 Reproducibility test results

[0083]

[0084]

[0085] 4.4 Establishment of fingerprint and similarity evaluation

[0086] Seventeen batches of Tianmingjing medicinal materials were taken, the test sample solution was prepared according to "3.2", and was injected for analysis according to the chromatographic conditions in "2". The chromatogram data was imported into the National Pharmacopoeia Committee "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" software, the average number was generated to generate a control chromatogram, the time window width was set to 0.1 min, and multiple point correction Mark peak matching was performed to generate a common mode and a control fingerprint chromatogram, a total of 19 common peaks were identified, as shown in Figure 1 . By comparing with the control, 10 components were identified, as shown in Figure 2 , peak 1 is chlorogenic acid, peak 2 is caffeic acid, peak 7 is osmanthus glycoside, peak 9 is 11 (13) -dehydroxy axillary ivacult, peak 10 is isochlorogenic acid A, peak 12 is isochlorogenic acid C, peak 13 is 2, 3, 4, 5-tetra caffeoyl-D-glucaric acid, peak 15 is 2-deoxy-4-epi-tianranjunling, peak 16 is Tianmingjing lactone ketone, and peak 17 is terenolide.

[0087] The peak areas of 19 common peaks of 17 batches of Tianmingjing medicinal materials were introduced into Origin Pro 2021 software as variables for correlation analysis and correlation heat map was drawn to evaluate the similarity between batches of Tianmingjing medicinal materials from different producing areas, and the results are shown in Figure 3 . In the figure, from red to green, the size of the circle represents the gradual increase of the correlation coefficient, and P value represents the degree of correlation between different producing areas. The results showed that the correlation coefficients of Tianmingjing medicinal materials from different producing areas and the control fingerprint were significantly positively correlated, ranging from 0.64 to 0.95, indicating that the chemical composition of Tianmingjing from different producing areas had high consistency and certain similarity. However, the correlation coefficients between Tianmingjing from the same producing area were generally higher than those between Tianmingjing from different producing areas, indicating that there were differences in the quality of Tianmingjing medicinal materials from different producing areas. Therefore, the establishment of Tianmingjing fingerprint can comprehensively evaluate the quality of Tianmingjing, and the stability and difference of medicinal materials from different producing areas also provide a basis for its research.

[0088] 5. Cluster analysis

[0089] The peak areas of 19 common peaks in 17 batches of Tianmingjing samples were introduced into Origin Pro software, and the system clustering analysis method was used to classify the samples with Euclidean square distance. The results are shown in Figure 3 , and the color of the color block in the figure directly reflects the size of the peak area, which increases from red to blue. The 17 batches of Tianmingjing samples were divided into two categories, S1, S4, S5, S6, S12, S13 and S17 were in one category, and S2, S3, S7, S8, S9, S10, S11, S14, S15 and S16 were in another category, indicating that there were large differences in the quality of Tianmingjing from different producing areas, which may be related to factors such as producing environment and planting technology. It is of great significance to evaluate the quality of Tianmingjing, and can evaluate the quality of Tianmingjing, and provide reliable scientific basis for ensuring the quality of preparations and clinical efficacy.

[0090] 6. Principal component analysis

[0091] With the peak area of 19 common peaks in the fingerprint of 17 batches of Tianmingjing as variables, principal component analysis was performed by using GraphPad Prism 10.1.2 software, and three principal components were obtained with cumulative variance contribution rate > 80% as the extraction standard. The variance contribution rates of the three principal components were 63.22%, 13.66%, and 9.03%, respectively, and the cumulative variance contribution rate was 84.91%, which could represent most of the information of the 19 common components. See Table 4. The principal component 1 with a higher contribution rate included peaks 19, 13, 3, and 14, all of which were loadings. The principal component 2 with a higher contribution rate included peaks 1 and 8, of which peak 8 was a loading. The principal component 3 with a higher contribution rate included peak 17. See Table 5. The scores of the three principal components were denoted as Y1, Y2, and Y3, respectively, and the contribution rates of the respective principal components were used as weight coefficients to establish a principal component comprehensive score model: Y = 0.6222Y1 + 0.1366Y2 + 0.9030Y3

[0092] The score results are shown in Table 6. The score reflects the quality of each batch of Tianmingjing, and 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, respectively. The peak areas of the 19 common peaks in the 17 batches of samples were introduced into SIMCA 14.1 software for PCA processing, and the score plot is shown in Figure 5 which is similar to the clustering result.

[0093] Table 4 Characteristic values and variance contribution rates of principal component analysis of Tianmingjing

[0094]

[0095] Table 5 PCA factor loading matrix of Tianmingjing

[0096]

[0097] Table 6 Principal component factor scores and ranking of 17 batches of Tianmingjing

[0098]

[0099]

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

[0101] In order to further analyze the differences between Tianmingjing from different producing areas, the peak areas of 19 common peaks in the fingerprint of 17 batches of Tianmingjing were introduced into SIMCA 14.1 software for OPLS-DA analysis. The model parameters R 2 X = 0.843, R 2 Y = 0.909, Q 2 = 0.802, which is similar to the results of clustering analysis and principal component analysis. SeeFigure 6 With VIP>1 as the screening standard, the VIP values of peak 10 (isochlorogenic acid A), peak 3, peak 19, peak 13 (2,3,4,5-tetra caffeoyl-D-glucaric acid), peak 1 (chlorogenic acid), peak 12 (isochlorogenic acid C), peak 14, peak 18, peak 15 (2-deoxy-4-epi-tanacetone), peak 6, peak 11, 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, 1.047 4 respectively, all greater than 1, and the 12 components can be used as potential difference quality markers of tanacetum, and the VIP score diagram is shown in Figure 7 The OPLS-DA model is replaced 200 times to obtain a replacement test diagram, and the result R 2 And Q 2 The intercepts on the Y axis are all less than the original values, indicating that the established model is not over-fitted, and the Figure 8 .

[0102] The method of tanacetum established in the application is different from related technologies, including the chromatographic instrument, the chromatographic column, the mobile phase, the gradient elution program, the variable wavelength detection program, the flow rate, the column temperature, and the injection amount, and the component types of tanacetum established by using the chromatographic conditions are also different from related technologies, the identified components include 5 phenolic acids (chlorogenic acid, caffeic acid, isochlorogenic acid A, isochlorogenic acid C, 2,3,4,5-tetra caffeoyl-D-glucaric acid), 4 sesquiterpenes (11 (13)-dehydro-axillary ivacic acid, 2-deoxy-4-epi-tanacetone, tanacetum lactone ketone, terenolide) and 1 flavonoid (luteoloside), and the obtained tanacetum sample ultra-high performance liquid chromatogram is more complete and beautiful while meeting the requirements of the fingerprint, and the baseline of the spectrum is stable and the shape of each component peak is symmetrical.

[0103] Comparative Example 1

[0104] The present comparative example is a kind of tanacetum HPLC separation method, and the specific liquid phase condition is as follows:

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

[0106] The high performance liquid chromatogram of the tanacetum test solution in the present comparative example is shown in Figure 9As shown in the figure, HPLC is time-consuming and requires a large amount of mobile phase and sample. Therefore, UPLC was subsequently used for separation.

[0107] Comparative Example 2

[0108] This comparative example demonstrates a Tianmingjing UPLC separation method, with the specific liquid phase conditions as follows:

[0109] The instrument used was a Waters H-Class ultra-high performance liquid chromatograph with a PDA detector; the chromatographic column was a Waters ACQUITY UPLC. BEH C 18 (2.1×100mm, 1.7μm); mobile phase: 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); column temperature: 30℃; wavelength: 230nm; injection volume: 1μL; flow rate: 0.3mL / min.

[0110] The UPLC chromatogram of the Tianmingjing test solution in this comparative example is shown below. Figure 10 ,Depend on Figure 10 It was found that this method, compared to Comparative Example 1, has a shorter separation time and is faster, achieving near-complete peak elution within 20 minutes with a greater number of peaks. However, the use of formic acid in this method leads to an uneven baseline, and the peak separation effect is not yet ideal; the liquid chromatography conditions need further optimization. Comparative Example 3

[0111] This comparative example demonstrates a Tianming-based UPLC separation method, with the following specific gradient:

[0112] The instrument used was a Waters H-Class ultra-high performance liquid chromatograph with a PDA detector; the chromatographic column was a Waters ACQUITY UPLC. BEH C 18 (2.1×100mm, 1.7μm); mobile phase: 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); column temperature: 30℃; wavelength: 230nm; injection volume: 0.5μL; flow rate: 0.3mL / min.

[0113] The UPLC chromatogram of the Tianmingjing test solution in this comparative example is shown below. Figure 11 ,Depend on Figure 11 It was learned that when the method was changed to phosphoric acid water, the baseline was more stable than that of formic acid water, and most components had been separated, but some components still needed to be separated further.

[0114] Comparative Example 4

[0115] This comparative example, which investigates the extraction solvent of the Tianmingjing test sample, consists of the following steps:

[0116] 1) Preparation of the test solution

[0117] Accurately weigh 0.5 g of Tianmingjing powder (passed through a 60-mesh sieve) and 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 the solution, and extract with ultrasound for 60 min. After cooling, weigh the solution again and make up the weight with the corresponding extraction solvent. Shake well and filter through a 0.22 μm microporous membrane to obtain the test solution.

[0118] 2) Liquid phase conditions

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

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

[0121] The ultra-high performance liquid chromatography (UHPLC) chromatogram of the Tianmingjing test solution in this comparative example is shown below. Figure 12 ,Depend on Figure 12 It was found that when the extraction solvent was water, 30% methanol / ethanol, or methanol / ethanol, the number of components was small or the peak area was small. When the extraction solvent was 50-80% methanol / ethanol, there was no significant difference in the number of components and the peak area.

[0122] Comparative Example 5

[0123] This comparative example, which investigates the wavelength of the UPLC chromatographic method of Tianming Precision, consists of the following steps:

[0124] 1) Preparation of the test solution

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

[0126] 2) Liquid phase conditions

[0127] The chromatographic column was Waters ACQUITY UPLC. BEH C 18 (2.1 x 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 amount was 0.5 μL; and the flow rate was 0.3 mL / min.

[0128] The ultrahigh performance liquid chromatography detection diagram of the test sample solution of the thymol in the present comparative example at different wavelengths is shown in Figure 2. Figure 13 It can be known from Figure 2 that the peaks 9, 15, 16 and 17 among the 19 characteristic peaks are at 210-230 nm, and the rest of the components are at 201-380 nm. Figure 13

[0129] In summary, the construction method of the thymol fingerprint provided by the present application has high precision, good stability and good reproducibility, can comprehensively control the quality of the thymol, and thus better ensures the quality stability, consistency and controllability of the thymol.

[0130] The above only describes the preferred embodiments of the present application, and it should be noted that, for those skilled in the art, some improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.​

Claims

1. A method for constructing a fingerprint spectrum of a specific type of fingerprint, characterized in that, Includes the following steps: Prepare the Tianmingjing test solution; Prepare a mixed reference solution; The fingerprint spectrum of Tianming was obtained by ultra-high performance liquid chromatography. The mobile phase system included mobile phase A and mobile phase B, wherein mobile phase A was an aqueous solution of phosphoric acid and mobile phase B was an acetonitrile solution. The volume fraction of phosphoric acid in the phosphoric acid aqueous solution is 0.05–0.4%. The preparation method of the Tianmingjing test solution includes the following steps: extracting the components in Tianmingjing using an extractant, wherein the extractant includes 50%-80% methanol or 50%-80% ethanol; The detection conditions for the ultra-high performance liquid chromatography method include: an octadecylsilane-bonded silica column, a mobile phase flow rate of 0.2-0.4 mL / min, a detection wavelength of 210-230 nm, a column temperature of 33-37 °C, and an injection volume of 0.5-1 μL. The ultra-high performance liquid chromatography method employs gradient elution, and the gradient elution procedure includes: From 0 to 2 minutes, the volume fraction of the mobile phase B is 10%. Over 2–5 minutes, the volume fraction of the mobile phase B increased from 10% to 14.5%. Over 5–15 minutes, the volume fraction of the mobile phase B increased from 14.5% to 22%. Within 15–17 minutes, the volume fraction of the mobile phase B increased from 22% to 26%. Within 17–25 minutes, the volume fraction of the mobile phase B increased from 26% to 28%. Over 25–30 minutes, the volume fraction of the mobile phase B increased from 28% to 37%. The mixed reference solution includes chlorogenic acid and caffeic acid and luteolin and 11(13)-dehydroaxillary isochlorogenic acid A and isochlorogenic acid C and 2,3,4,5-tetracaffeoyl-D-gluconic acid and 2-deoxy-4-epiota-glucan and terbinafine lactone and terbinafine lactone.

2. The method for constructing the Tianming fingerprint spectrum according to claim 1, characterized in that, The volume fraction of phosphoric acid in the phosphoric acid aqueous solution is 0.05–0.2%.

3. The method for constructing the Tianming fingerprint spectrum according to claim 1, characterized in that, The extractant comprises 70%-80% methanol or 70%-80% ethanol.

4. The method for constructing the Tianming fingerprint spectrum according to claim 3, characterized in that, The solid-liquid ratio of the tianmingjing to the extractant is 0.3-0.6g:15-25mL.

5. The method for constructing the Tianming fingerprint spectrum according to claim 4, characterized in that, The solid-liquid ratio of the extract to the extractant is 0.45-0.55g: 19.5-20.5mL.

6. The method for constructing a Tianming fingerprint spectrum according to any one of claims 1-5, characterized in that, The extraction was performed using ultrasound; the power of the ultrasound extraction was 190–210 W, the frequency was 35–45 kHz, and the time was 50–70 min.

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