A method for constructing a characteristic chromatogram of the fruit of Carpesium abrotanoides L. and its application

Abstract

The present invention provides a method for constructing a characteristic chromatogram of the drug of Carpesium abrotanoides L. and its application. The construction method comprises the following steps: (1) mixing the reference medicinal material of Carpesium abrotanoides L. with an alcohol solution for extraction to obtain a reference solution of the reference medicinal material; (2) mixing a reference substance with an alcohol solution to obtain a reference solution of the reference substance; (3) mixing the drug of Carpesium abrotanoides L. with an alcohol solution and performing ultrasonic treatment to obtain a test solution; (4) respectively performing UPLC detection on the reference solution of the reference medicinal material, the reference solution of the reference substance, and the test solution, screening out characteristic peaks according to the detection results, and selecting, from the characteristic peaks, those with determined components and good peak shapes as the S peak, and calculating the relative retention time of other characteristic peaks relative to the S peak, thereby obtaining the characteristic chromatogram of the drug of Carpesium abrotanoides L. The characteristic chromatogram constructed by the method provided by the present invention has good repeatability, high precision, and good stability, and can effectively identify the quality of the drug of Carpesium abrotanoides L.

Description

Technical Field

[0001] The present invention belongs to the field of identification of traditional Chinese medicines, and particularly relates to a characteristic chromatogram of the drug of Carotae Fructus, a method for constructing the same, and an application thereof, and more particularly to a characteristic chromatogram of the drug of Carotae Fructus with high precision, a method for constructing the same, and an application thereof. Background Art

[0002] Carotae Fructus is the dried ripe fruit of the umbelliferous plant Daucus carota L., which is harvested when the fruit is ripe in autumn. Carotae Fructus is neutral in nature, bitter and pungent in taste, slightly poisonous, and belongs to the spleen and stomach meridians. It has the efficacy of killing parasites and promoting digestion, and is used for the treatment of ascariasis, oxyuriasis, taeniasis, abdominal pain due to parasitic infestation, and infantile malnutrition. It is a commonly used traditional Chinese medicine among the people.

[0003] At present, only the character, microscopic identification, and thin-layer identification items of Carotae Fructus are standardized. There are few studies on Carotae Fructus decoction pieces and Carotae Fructus formula granules in the prior art. Some scholars have used HPLC to study the chemical constituents of Carotae Fructus, and the main material basis is flavonoid components.

[0004] The above individual studies mainly focus on Carotae Fructus medicinal materials. Carotae Fructus formula granules are formula granules obtained by steps such as extraction, concentration, drying, and preparation from Carotae Fructus decoction pieces, losing the original appearance and character features of the medicinal materials. The existing characteristic chromatograms of Carotae Fructus granules are mainly established for flavonoid components, and organic acid components have not been studied, resulting in incomplete quality control. Therefore, how to provide a comprehensive characteristic chromatogram of Carotae Fructus has become an urgent problem to be solved. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a characteristic chromatogram of the drug of Carotae Fructus, a method for constructing the same, and an application thereof, and more particularly to provide a characteristic chromatogram of the drug of Carotae Fructus with high precision, a method for constructing the same, and an application thereof. The characteristic chromatogram constructed by the method provided by the present invention has good repeatability, high precision, and good stability, and can effectively identify the quality of the drug of Carotae Fructus.

[0006] To achieve the purpose of the present invention, the following technical solutions are adopted:

[0007] In the first aspect, the present invention provides a method for constructing a characteristic chromatogram of the drug of Carotae Fructus, and the construction method includes the following steps:

[0008] (1) Mixing the reference medicinal material of Carotae Fructus with an alcohol solution for extraction to obtain a reference solution of the reference medicinal material;

[0009] (2) Mixing the reference substance with an alcohol solution to obtain a reference solution of the reference substance;

[0010] (3) Mixing the drug of Carotae Fructus with an alcohol solution and ultrasonically treating to obtain a test solution;

[0011] (4) Perform UPLC detection on the reference medicinal material solution, reference substance solution, and test solution respectively. Based on the detection results, select the common peaks with consistent retention times, good peak shapes, and high resolution as characteristic peaks, and select the peaks with determined components and good peak shapes among the characteristic peaks as the S peak. Calculate the relative retention times of other characteristic peaks relative to the S peak to obtain the characteristic chromatogram of the said Carpesii Fructus drug.

[0012] Steps (1), (2), and (3) do not distinguish the order.

[0013] The above method can effectively identify the Carpesii Fructus drug by extracting the Carpesii Fructus drug and comparing it with the Carpesii Fructus medicinal material to jointly construct a characteristic chromatogram; and the characteristic chromatogram obtained by performing UPLC detection using a specific method has good repeatability, high precision, and good durability.

[0014] Preferably, the said Carpesii Fructus drug includes any one or a combination of at least two of Carpesii Fructus medicinal material, Carpesii Fructus standard decoction, or Carpesii Fructus formula granules.

[0015] Preferably, the alcohol solution includes methanol aqueous solution, and the volume fraction of the methanol aqueous solution is 60 - 80%.

[0016] Preferably, the material-liquid ratio of the Carpesii Fructus reference medicinal material to the alcohol solution in step (1) is 1:(20 - 30) g / mL.

[0017] Among them, the volume fraction of the methanol aqueous solution can be 60%, 65%, 70%, 75%, or 80%, etc., and the material-liquid ratio of the Carpesii Fructus reference medicinal material to the alcohol solution can be 1:20 g / mL, 1:22 g / mL, 1:24 g / mL, 1:26 g / mL, 1:28 g / mL, or 1:30 g / mL, etc., but is not limited to the above-listed values, and other unlisted values within the above numerical range are equally applicable.

[0018] Preferably, the reference substances in step (2) include any one or a combination of at least two of chlorogenic acid, caffeic acid, ferulic acid, luteolin-7-O-β-D-glucuronide, apigenin-7-O-β-glucoside, or luteolin.

[0019] Preferably, the material-liquid ratio of the Carpesii Fructus drug to the alcohol solution in step (3) is 1:(40 - 50) g / mL, such as 1:40 g / mL, 1:42 g / mL, 1:44 g / mL, 1:46 g / mL, 1:48 g / mL, or 1:50 g / mL, etc., but is not limited to the above-listed values, and other unlisted values within the above numerical range are equally applicable.

[0020] Preferably, the mobile phase for the UPLC detection in step (4) comprises mobile phase A and mobile phase B. Mobile phase A is acetonitrile, and mobile phase B is an aqueous formic acid solution with a volume fraction of 0.08 - 0.12%.

[0021] Preferably, the column temperature for the UPLC detection in step (4) is 33 - 37 °C, and the flow rate is 0.28 - 0.32 mL / min.

[0022] Among them, the volume fraction of the aqueous formic acid solution can be 0.08%, 0.09%, 0.10%, 0.11% or 0.12%, etc.; the column temperature can be 33 °C, 34 °C, 35 °C, 36 °C or 37 °C, etc.; the flow rate can be 0.28 mL / min, 0.29 mL / min, 0.30 mL / min, 0.31 mL / min or 0.32 mL / min, etc. However, it is not limited to the values listed above, and other unlisted values within the above numerical range are equally applicable.

[0023] Preferably, the chromatographic column packing material for the UPLC detection in step (4) is octadecylsilyl silica gel.

[0024] Preferably, gradient elution is used for the UPLC detection, and the specific process of the gradient elution is as follows:

[0025] From 0 - 5 min, the volume fraction of mobile phase A changes uniformly from 5% to 10%, and the volume fraction of mobile phase B changes uniformly from 95% to 90%;

[0026] From 5 - 16 min, the volume fraction of mobile phase A changes uniformly to 12%, and the volume fraction of mobile phase B changes uniformly to 88%;

[0027] From 16 - 20 min, the volume fraction of mobile phase A changes uniformly to 14%, and the volume fraction of mobile phase B changes uniformly to 86%;

[0028] From 20 - 21 min, the volume fraction of mobile phase A changes uniformly to 16%, and the volume fraction of mobile phase B changes uniformly to 84%;

[0029] From 21 - 32 min, the volume fraction of mobile phase A changes uniformly to 23%, and the volume fraction of mobile phase B changes uniformly to 77%;

[0030] From 32 - 42 min, the volume fraction of mobile phase A changes uniformly to 50%, and the volume fraction of mobile phase B changes uniformly to 50%;

[0031] From 42 - 43 min, the volume fraction of mobile phase A changes uniformly to 5%, and the volume fraction of mobile phase B changes uniformly to 95%.

[0032] The above specific elution procedure can effectively distinguish the components in the sample, with high peak separation, obvious peak emergence, and can obtain characteristic peaks with good peak shapes, improving the accuracy of detection and characteristic chromatograms.

[0033] Preferably, in the characteristic chromatogram, the number of characteristic peaks is 13, and the number of S peaks is 2, namely S1 peak and S2 peak. Sorted in ascending order of retention time, taking peak 6 as S1 peak, the relative retention times of peak 1, peak 2, peak 3, peak 4, peak 5, and peak 7 relative to S1 peak are 0.50±10%, 0.54±10%, 0.62±10%, 0.75±10%, 0.90±10%, 1.29±10%; taking peak 8 as S2 peak, calculate the relative retention times of peak 9, peak 10, peak 11, peak 12, and peak 13 relative to S2 peak are 1.10±10%, 1.11±10%, 1.12±10%, 1.13±10%, 1.46±10%.

[0034] The component of peak 1 is chlorogenic acid, the component of peak 2 is caffeic acid, the component of peak 6 is ferulic acid, the component of peak 8 is luteolin-7-O-β-D-glucuronide, the component of peak 10 is apigenin-7-O-β-glucoside, and peak 13 is luteolin.

[0035] In the second aspect, the present invention provides the application of the method for constructing the characteristic chromatogram of the above-mentioned Daucus carota L. fruit drug in the quality control of Daucus carota L. fruit drugs.

[0036] In the third aspect, the present invention also provides a method for quality control of Daucus carota L. fruit drugs. Different batches of Daucus carota L. fruit drugs are detected by the above-mentioned UPLC detection method, the detection results are processed, and a standard characteristic chromatogram is obtained in combination with the characteristic chromatogram; the sample to be tested is detected by the above-mentioned UPLC detection method, the characteristic peaks are judged according to the characteristic chromatogram, and then the similarity is judged to determine whether the sample to be tested is a qualified product or an unqualified product.

[0037] In the similarity judgment, calculate the similarity between the characteristic peaks of the sample to be tested and the characteristic peaks in the standard characteristic chromatogram; when the similarity is not less than 0.85, the sample to be tested is a qualified product, and when the similarity is less than 0.85, the sample to be tested is an unqualified product.

[0038] The Daucus carota L. fruit drugs include any one or at least two combinations of Daucus carota L. fruit herbs, Daucus carota L. fruit standard decoctions, or Daucus carota L. fruit formula granules.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] The present invention provides a method for constructing a characteristic chromatogram of the drug of Carpesium abrotanoides L., which extracts the drug of Carpesium abrotanoides L. and compares it with the medicinal material of Carpesium abrotanoides L. to jointly construct a characteristic chromatogram. The obtained characteristic chromatogram can effectively identify the drug of Carpesium abrotanoides L., which is beneficial to realizing the overall quality control of Carpesium abrotanoides L. from the raw medicinal material to its related preparations; and the characteristic chromatogram obtained by UPLC detection using a specific method has good repeatability, high precision and good durability; in addition, by adopting a specific elution program, the components in the sample can be effectively separated, the peak shape resolution is high, the peaks appear obviously, and characteristic peaks with good peak shapes can be obtained, thereby improving the accuracy of detection and the characteristic chromatogram. Description of the Drawings

[0041] Figure 1 is the superimposed chromatogram of the test solution in Example 1;

[0042] Figure 2 is the characteristic chromatogram in Example 1;

[0043] Figure 3 is the liquid phase detection result diagram in Comparative Example 1;

[0044] Figure 4 is the liquid phase detection result diagram in Comparative Example 2;

[0045] Figure 5 is the specificity chromatogram;

[0046] Figure 6 is the sample investigation chromatogram. Detailed Embodiments

[0047] The technical solutions of the present invention will be further described below through specific embodiments. Those skilled in the art should understand that the described embodiments are only for helping to understand the present invention and should not be regarded as specific limitations on the present invention.

[0048] Example 1

[0049] This example provides a method for constructing a characteristic chromatogram of the drug of Carpesium abrotanoides L. and its application, and the specific steps are as follows:

[0050] Preparation of the reference substance solution: Take 1.0398 g of the reference medicinal material of Carpesium abrotanoides L., accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of 70% methanol, tightly stopper it, heat under reflux for 30 minutes, take it out, let it cool, shake well, filter, and take the continuous filtrate as the reference medicinal material solution.

[0051] Take 2.991 mg of chlorogenic acid reference substance, 1.616 mg of caffeic acid reference substance, and 1.578 mg of ferulic acid reference substance, and add a methanol aqueous solution with a volume fraction of 70% to obtain a mixed reference substance solution containing 29.91 μg / mL of chlorogenic acid, 16.11 μg / mL of caffeic acid, and 15.69 μg / mL;

[0052] Take 2.675 mg of luteolin-7-O-β-D-glucuronide reference substance, add methanol aqueous solution with a volume fraction of 70%, and obtain a luteolin-7-O-β-D-glucuronide reference substance solution with a concentration of 52.27 μg / mL;

[0053] Take 2.032 mg of apigenin-7-O-β-glucoside reference substance, add methanol solution, and obtain an apigenin-7-O-β-glucoside reference substance solution with a concentration of 19.91 μg / mL.

[0054] Take 2.330 mg of luteolin reference substance, add methanol solution, and obtain a luteolin reference substance solution with a concentration of 23.30 μg / mL.

[0055] Preparation of test solution: Take 0.5 g of the sample of Carpesium abrotanoides L., accurately weigh it, place it in a stoppered conical flask, accurately add 25 mL of methanol aqueous solution with a volume fraction of 70%, stopper it, treat it by ultrasonic wave (power 500 W, frequency 40 kHz) for 30 min, take it out, let it cool, shake well, filter, and take the subsequent filtrate to obtain a test solution with a concentration of 20 mg / ml.

[0056] Chromatographic conditions and system suitability test: Determine according to the high performance liquid chromatography method. Use a Waters CORTECS UPLC Shield RP18 (2.1×150 mm, 1.6 μm) chromatographic column. Use acetonitrile as mobile phase A and formic acid solution with a volume fraction of 0.1% as mobile phase B. Perform chromatographic analysis according to the gradient elution program. The gradient elution program is as follows:

[0057] From 0 to 5 min, the volume fraction of mobile phase A changes uniformly from 5% to 10%, and the volume fraction of mobile phase B changes uniformly from 95% to 90%;

[0058] From 5 to 16 min, the volume fraction of mobile phase A changes uniformly to 12%, and the volume fraction of mobile phase B changes uniformly to 88%;

[0059] From 16 to 20 min, the volume fraction of mobile phase A changes uniformly to 14%, and the volume fraction of mobile phase B changes uniformly to 86%;

[0060] From 20 to 21 min, the volume fraction of mobile phase A changes uniformly to 16%, and the volume fraction of mobile phase B changes uniformly to 84%;

[0061] From 21 to 32 min, the volume fraction of mobile phase A changes uniformly to 23%, and the volume fraction of mobile phase B changes uniformly to 77%;

[0062] From 32 to 42 min, the volume fraction of mobile phase A changes uniformly to 50%, and the volume fraction of mobile phase B changes uniformly to 50%;

[0063] At 42 - 43 min, the volume fraction of mobile phase A uniformly changes to 5%, and the volume fraction of mobile phase B uniformly changes to 95%.

[0064] At 0 - 21 min, the detection wavelength is 305 nm; at 21 - 43 min, the detection wavelength is 347 nm, the column temperature is 35 °C, the flow rate is 0.3 mL / min, and the injection volume is 2 μL.

[0065] Establishment of characteristic chromatogram:

[0066] Take 15 batches of freeze - dried powder of standard decoction of Carpesium abrotanoides L. as test samples, prepare the test solution according to the above method, absorb 2 μL and inject it into an ultra - high performance liquid chromatograph, and determine according to the above chromatographic conditions to obtain the characteristic chromatograms of 15 batches of freeze - dried powder of standard decoction of Carpesium abrotanoides L. Analyze using the software "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines (2012 Edition)", set the time window to 0.1 and use the median method. Take the 4th peak as the reference peak, and after multi - point correction, automatically match to obtain the superimposed chromatogram of 15 batches of test samples and generate the reference characteristic chromatogram. The superimposed chromatograms of the test solutions of 15 batches are shown in Figure 1 (S1(13) - S15(13) refer to 15 batches of samples respectively), and the generated characteristic chromatogram is shown in Figure 2 (R(13) refers to the characteristic chromatogram, and the numbers 1 - 13 in the chromatogram represent characteristic peaks 1 - 13), and the similarity evaluation results are shown in Table 1.

[0067] The characteristic chromatogram of Carpesium abrotanoides L. samples includes 13 common characteristic peaks; through the chromatogram of the reference substance solution for identification, chlorogenic acid is peak 1, caffeic acid is peak 2, ferulic acid is peak 6, luteolin - 7 - O - β - D - glucuronide is peak 8, apigenin - 7 - O - β - glucoside is peak 10, and luteolin is peak 13; take ferulic acid at peak 6 as reference peak 1 (S1), calculate the relative retention times of characteristic peaks 1, 2, 3, 4, 5, 7 and S1, take luteolin - 7 - O - β - D - glucuronide at peak 8 as reference peak 2 (S2), calculate the relative retention times of characteristic peaks 9, 10, 11, 12, 13 and S2, and take the average value of the relative retention times of each characteristic peak in 15 batches of samples as the specified value, with the allowable error within ±10% of the specified value. The specified values of relative retention times are:

[0068] The relative retention time of peak 1 is 0.50; the relative retention time of peak 2 is 0.54; the relative retention time of peak 3 is 0.62; the relative retention time of peak 4 is 0.75; the relative retention time of peak 5 is 0.90; the relative retention time of peak 7 is 1.29; the relative retention time of peak 9 is 1.10; the relative retention time of peak 10 is 1.11; the relative retention time of peak 11 is 1.12; the relative retention time of peak 12 is 1.13; the relative retention time of peak 13 is 1.46.

[0069] Table 1 Evaluation results of similarity of characteristic chromatograms of 15 batches of standard decoction freeze-dried powder of Carpesium abrotanoides L.

[0070]

[0071]

[0072] In Table 1, the similarity values between the characteristic chromatograms of the test solutions of 15 batches of Carpesium abrotanoides L. and the reference characteristic chromatogram are in the range of 0.988 - 0.999, indicating that the differences between the characteristic chromatograms of these 15 batches are small, and the generated reference characteristic chromatogram is representative, and all are qualified products.

[0073] Comparative Example 1

[0074] This comparative example provides a method for constructing a characteristic chromatogram of Carpesium abrotanoides L. drug and its application. In the specific steps, except that the gradient elution process is as follows and the detection wavelength is 347 nm, the rest are the same as in Example 1.

[0075] From 0 to 6 min, the volume fraction of mobile phase A changes uniformly from 5% to 12%, and the volume fraction of mobile phase B changes uniformly from 95% to 88%;

[0076] From 6 to 18 min, the volume fraction of mobile phase A changes uniformly to 16%, and the volume fraction of mobile phase B changes uniformly to 84%;

[0077] From 18 to 19 min, the volume fraction of mobile phase A changes uniformly to 23%, and the volume fraction of mobile phase B changes uniformly to 77%;

[0078] From 19 to 32 min, the volume fraction of mobile phase A changes uniformly to 26%, and the volume fraction of mobile phase B changes uniformly to 74%;

[0079] From 32 to 34 min, the volume fraction of mobile phase A changes uniformly to 33%, and the volume fraction of mobile phase B changes uniformly to 67%;

[0080] From 34 to 40 min, the volume fraction of mobile phase A changes uniformly to 65%, and the volume fraction of mobile phase B changes uniformly to 35%.

[0081] The results are asFigure 3 As shown, it can be found that in Comparative Example 1, there is an obvious peak at 15 min, and the exact component represented by this peak could not be identified, making other chromatographic peaks appear smaller, affecting the calculation accuracy, and the resolution of the chromatographic peaks at 18 - 26 min is poor.

[0082] Comparative Example 2

[0083] This comparative example provides a method for constructing the characteristic chromatogram of Carpesium abrotanoides L. drugs and its application. In the specific steps, except that the gradient elution process is as follows and from 0 - 28 min, the detection wavelength is 305 nm, and from 40 - 55 min, the detection wavelength is 347 nm, the rest are the same as in Example 1.

[0084] From 0 - 2 min, the volume fraction of mobile phase A changes uniformly from 5% to 8%, and the volume fraction of mobile phase B changes uniformly from 95% to 92%;

[0085] From 2 - 25 min, the volume fraction of mobile phase A changes uniformly to 12%, and the volume fraction of mobile phase B changes uniformly to 88%;

[0086] From 25 - 28 min, the volume fraction of mobile phase A changes uniformly to 15%, and the volume fraction of mobile phase B changes uniformly to 85%;

[0087] From 28 - 40 min, the volume fraction of mobile phase A changes uniformly to 22%, and the volume fraction of mobile phase B changes uniformly to 78%;

[0088] From 40 - 44 min, the volume fraction of mobile phase A changes uniformly to 30%, and the volume fraction of mobile phase B changes uniformly to 70%;

[0089] From 44 - 54 min, the volume fraction of mobile phase A changes uniformly to 50%, and the volume fraction of mobile phase B changes uniformly to 50%.

[0090] From 54 - 55 min, the volume fraction of mobile phase A changes uniformly to 5%, and the volume fraction of mobile phase B changes uniformly to 95%.

[0091] The results are as Figure 4 shown. It can be found that in Comparative Example 2, the resolution of the chromatographic peaks at 7 - 9 min is relatively low, and there are no obvious chromatographic peaks for up to 9 min at 23 - 31 min.

[0092] Thus, it can be seen that the characteristic chromatogram obtained by the gradient elution program adopted in the present invention is more beautiful, has a larger amount of information, better resolution of each chromatographic peak, and better peak shape, showing significant advantages compared with other gradient elution programs.

[0093] Methodological investigation:

[0094] Specificity:

[0095] Inject 2 μL of blank solvent, an aqueous solution of methanol with a volume fraction of 70%, into the ultra-high performance liquid chromatograph. According to the chromatographic conditions in Example 1, measure. There are no chromatographic peaks in the obtained chromatogram, indicating that the solvent has no effect on the chromatographic peaks of the characteristic chromatogram of Carpesium abrotanoides L. The chromatogram is shown in Figure 5 .

[0096] Stability:

[0097] Take the Carpesium abrotanoides L. sample to be tested of the same batch (batch number: KL202105-24-13), prepare the test solution according to the method in Example 1, and place it at 20 °C for 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 18 h, and 24 h respectively. Then, according to the chromatographic conditions in Example 1, measure to obtain the chromatogram. Take the 6th peak as reference peak 1 and calculate the relative retention times of peaks 1-5, 7 and reference peak 1; take the 8th peak as reference peak 2 and calculate the relative retention times of peaks 9-13 and reference peak 2, as shown in Table 2. The results show that the relative standard deviation (RSD) of the relative retention times of the common peaks is less than 0.09%, indicating that the sample is relatively stable within 24 h.

[0098] Table 2 Relative retention times of characteristic peaks

[0099]

[0100]

[0101]

[0102] Repeatability:

[0103] Take the Carpesium abrotanoides L. sample to be tested of the same batch (batch number: KL202105-24-13), prepare 6 test solutions according to the method in Example 1, numbered 1-6 respectively, and measure according to the chromatographic conditions in Example 1. Take the 6th peak as reference peak 1 and calculate the relative retention times of peaks 1-5, 7 and reference peak 1; take the 8th peak as reference peak 2 and calculate the relative retention times of peaks 9-13 and reference peak 2, as shown in Table 3. The calculation results show that the relative standard deviation (RSD) of the relative retention times of the common peaks is less than 0.07%, indicating that the repeatability of this method is good.

[0104] Table 3 Relative retention times of characteristic peaks

[0105]

[0106]

[0107]

[0108] Intermediate precision:

[0109] The same batch of samples of southern crane lice to be tested (batch number: KL202105-24-13) were taken, and different analysts conducted intermediate precision tests on different instruments (Agilent 1290InfinityⅡ ultra-high performance liquid chromatograph, DAD detector, Empower 3 chromatography workstation). Six test solutions were prepared according to the method in Example 1, numbered 1-6 respectively, and measured according to the chromatographic conditions in Example 1. Taking peak 6 as reference peak 1, the relative retention time of peaks 1-5 and 7 with reference peak 1 was calculated; taking peak 8 as reference peak 2, the relative retention time of peaks 9-13 with reference peak 2 was calculated, as shown in Table 4. The calculation results show that the relative retention time RSD of the common peaks in the intermediate precision is less than 0.06%, and the relative retention time RSD of the common peaks compared with the repeatability study is less than 0.06%. 12 All of them were less than 1.41%, indicating that the intermediate precision of this method was good.

[0110] Table 4 Relative retention time of characteristic peaks

[0111]

[0112]

[0113]

[0114] Column temperature investigation:

[0115] Take the same batch of samples of southern crane lice to be tested (batch number: KL202105-24-13), prepare the test solution according to the method in Example 1, and measure it under different column temperatures (33°C, 35°C and 37°C) according to the chromatographic conditions in Example 1. Take peak 6 as reference peak 1, calculate the relative retention time of peaks 1-5 and 7 with reference peak 1; take peak 8 as reference peak 2, calculate the relative retention time of peaks 9-13 with reference peak 2, see Table 5. The calculation results show that the relative retention time RSD of the common peaks is less than 0.99%, indicating that the method has good durability for different column temperatures. The peak area is more stable when the column temperature is 33-35°C.

[0116] Table 5 Retention time and relative retention time of characteristic peaks

[0117]

[0118]

[0119] Flow rate investigation

[0120] The same batch of samples of southern crane lice to be tested (batch number: KL202105-24-13) were taken, and the test solution was prepared according to the method in Example 1, and the test solution was determined at different flow rates (0.28ml / min, 0.30ml / min and 0.32ml / min) according to the chromatographic conditions in Example 1. Taking peak 6 as reference peak 1, the relative retention time of peaks 1-5 and 7 with reference peak 1 was calculated; taking peak 8 as reference peak 2, the relative retention time of peaks 9-13 with reference peak 2 was calculated, as shown in Table 6. The calculation results show that the relative retention time RSD of the common peaks is less than 1.10%, indicating that the method has good durability for different flow rates.

[0121] Table 6 Retention time and relative retention time of characteristic peaks

[0122]

[0123]

[0124]

[0125] Sample inspection:

[0126] Experimental group 1: Take 1.0 g of crude powder of the medicinal material of Southern Heshi (batch number: YC202105-24-12), accurately weigh it, put it in a stoppered conical flask, accurately add 25 mL of 70% methanol by volume, stopper it, weigh it, heat it under reflux for 30 minutes, cool it to room temperature, make up for the lost weight, shake it well, filter it, and take the filtrate to obtain the test solution of the medicinal material of Southern Heshi.

[0127] Experimental group 2: Take 0.5 g of freeze-dried powder of standard decoction of Southern Hechi (batch number: YP202105-24-12), grind it into powder, weigh it accurately, put it in a stoppered conical flask, accurately add 25 mL of 70% methanol by volume, stopper it, treat it with ultrasound (power 500 W, frequency 40 kHz) for 30 min, cool it to room temperature, shake it well, filter it, and take the filtrate to obtain the test solution of standard decoction of Southern Hechi.

[0128] Experimental group 3: Take 0.5 g of the formula granules of Nanheshi (batch number: KL202105-24-12), grind it into powder, weigh it accurately, put it in a stoppered conical flask, accurately add 25 mL of 70% methanol by volume, stopper it, treat it with ultrasound (power 500 W, frequency 40 kHz) for 30 min, cool it to room temperature, shake it well, filter it, and take the filtrate to obtain the test solution of the standard decoction of Nanheshi.

[0129] The test solution prepared in the experimental groups 1-3 was injected according to the chromatographic conditions of Example 1, with an injection volume of 2 μL, and the characteristic spectrum was obtained. Figure 6(S1 - S3 represent the fruit of Carpesium abrotanoides L., freeze - dried powder of the standard decoction of the fruit of Carpesium abrotanoides L., and formula granules of the fruit of Carpesium abrotanoides L., respectively), and the relative retention times are shown in Table 7.

[0130] Table 7 Relative retention times of characteristic peaks of the fruit of Carpesium abrotanoides L., standard decoction, and granules

[0131]

[0132]

[0133] As can be seen from the above table and the figure, the construction method provided by the present invention is applicable to the quality control of the fruit of Carpesium abrotanoides L., freeze - dried powder of the standard decoction of the fruit of Carpesium abrotanoides L., and formula granules of the fruit of Carpesium abrotanoides L.

[0134] The applicant declares that the present invention uses the above - mentioned embodiments to illustrate the construction method and its application of the characteristic chromatogram of the Carpesium abrotanoides L. drug of the present invention. However, the present invention is not limited to the above - mentioned embodiments, that is, it does not mean that the present invention must rely on the above - mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent substitution of each raw material of the product of the present invention, addition of auxiliary components, selection of specific methods, etc. all fall within the protection scope and the disclosure scope of the present invention.

[0135] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above - mentioned embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0136] In addition, it should be noted that, in the case of no contradiction, the various specific technical features described in the above - mentioned specific embodiments can be combined in any appropriate way. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

Claims

1. A method for constructing a characteristic chromatogram of the drug of Carpesium abrotanoides L., characterized in that, the construction method comprises the following steps: (1) Mix and extract the reference medicinal material of Carpesium abrotanoides L. with an alcohol solution to obtain a reference solution of the reference medicinal material; (2) Mix the reference substance with an alcohol solution to obtain a reference solution of the reference substance; (3) Mix the drug of Carpesium abrotanoides L. with an alcohol solution and ultrasonicate to obtain a test solution; (4) Perform UPLC detection on the reference solution of the reference medicinal material, the reference solution of the reference substance and the test solution respectively. According to the detection results, select the common peaks with consistent retention time, good peak shape and high resolution as characteristic peaks, and select the one with determined components and better peak shape among the characteristic peaks as the S peak, and calculate the relative retention time of other characteristic peaks relative to the S peak, thus obtaining the characteristic chromatogram of the drug of Carpesium abrotanoides L.; Steps (1), (2) and (3) do not distinguish the order; The reference substances in step (2) include a combination of chlorogenic acid, caffeic acid, ferulic acid, luteolin-7-O-β-D-glucuronide, apigenin-7-O-β-glucoside and luteolin; The UPLC detection in step (4) adopts gradient elution, and the specific process of the gradient elution is as follows: From 0 to 5 min, the volume fraction of mobile phase A changes uniformly from 5% to 10%, and the volume fraction of mobile phase B changes uniformly from 95% to 90%; From 5 to 16 min, the volume fraction of mobile phase A changes uniformly to 12%, and the volume fraction of mobile phase B changes uniformly to 88%; From 16 to 20 min, the volume fraction of mobile phase A changes uniformly to 14%, and the volume fraction of mobile phase B changes uniformly to 86%; From 20 to 21 min, the volume fraction of mobile phase A changes uniformly to 16%, and the volume fraction of mobile phase B changes uniformly to 84%; From 21 to 32 min, the volume fraction of mobile phase A changes uniformly to 23%, and the volume fraction of mobile phase B changes uniformly to 77%; From 32 to 42 min, the volume fraction of mobile phase A changes uniformly to 50%, and the volume fraction of mobile phase B changes uniformly to 50%; From 42 to 43 min, the volume fraction of mobile phase A changes uniformly to 5%, and the volume fraction of mobile phase B changes uniformly to 95%; The mobile phase for the UPLC detection in step (4) includes mobile phase A and mobile phase B. Mobile phase A is acetonitrile, and mobile phase B is an aqueous formic acid solution, and the volume fraction of the aqueous formic acid solution is 0.08 - 0.12%; The chromatographic column for the UPLC detection in step (4) is Waters CORTECS UPLC Shield RP18, with a specification of 2.1×150mm, 1.6μm. The detection wavelength from 0 to 21 min is 305nm, and the detection wavelength from 21 to 43 min is 347nm; The drug of Carpesium abrotanoides L. is the medicinal material of Carpesium abrotanoides L., the standard decoction of Carpesium abrotanoides L. and the formula granules of Carpesium abrotanoides L.; The alcohol solution includes an aqueous methanol solution, and the volume fraction of the aqueous methanol solution is 60 - 80%.

2. According to the method for constructing a characteristic chromatogram of the drug of Carpesium abrotanoides L. as claimed in claim 1, characterized in that, The solid-liquid ratio of the southern helictid control medicinal material to the alcohol solution in step (1) is 1:(20-30) g / mL.

3. The method for constructing the characteristic spectrum of the drug of southern crane louse according to claim 1, It is characterized in that In step (3), the solid-liquid ratio of the southern crane lice drug to the alcohol solution is 1:(40-50) g / mL.

4. The method for constructing the characteristic spectrum of the drug of southern crane louse according to claim 1, It is characterized in that The column temperature of the UPLC detection in step (4) is 33-37° C., and the flow rate is 0.28-0.32 mL / min.

5. The method for constructing the characteristic spectrum of the drug of southern crane louse according to claim 1, It is characterized in that In the characteristic spectrum, the number of characteristic peaks is 13, the number of S peaks is 2, which are S1 peak and S2 peak respectively, and are sorted from small to large by retention time. Peak 6 is the S1 peak, and the relative retention times of peak 1, peak 2, peak 3, peak 4, peak 5, and peak 7 relative to the S1 peak are 0.50±10%, 0.54±10%, 0.62±10%, 0.75±10%, 0.90±10%, and 1.29±10%; Peak 8 is the S2 peak, and the relative retention times of peak 9, peak 10, peak 11, peak 12, and peak 13 relative to the S2 peak are calculated to be 1.10±10%, 1.11±10%, 1.12±10%, 1.13±10%, and 1.46±10%; The peak 1 component is chlorogenic acid, the peak 2 component is caffeic acid, the peak 6 component is ferulic acid, the peak 8 component is luteolin-7-O-β-D-glucuronide, the peak 10 component is apigenin-7-O-β-glucoside, and the peak 13 is luteolin.

6. Application of a method for constructing a characteristic map of a drug for the southern crane louse according to any one of claims 1 to 5 in the quality control of the drug for the southern crane louse.

Images