Construction method and identification method of characteristic chromatograms of senecio scandens medicinal material as well as decoction pieces, standard decoction and formula granules of senecio scandens medicinal material

Abstract

The invention discloses a construction method and an identification method of characteristic chromatograms of a senecio scandens medicinal material as well as decoction pieces, standard decoction and formula granules thereof, and relates to the technical field of traditional Chinese medicine identification. The construction method of the specific chromatogram comprises the following steps: extracting a to-be-detected sample with methanol to obtain a test solution; the method comprises the following steps: dissolving 5-caffeoylquinic acid, chlorogenic acid, caffeic acid, 4-caffeoylquinic acid, rutin, hyperoside, isoquercitrin and 4, 5-dicaffeoylquinic acid with methanol to obtain a reference solution; and a liquid chromatograph is adopted for determination. A chromatographic column of the liquid chromatograph takes phenyl-methyl polysiloxane as a stationary phase, acetonitrile as a mobile phase A and 0.05 vol%-0.3 vol% formic acid aqueous solution as a mobile phase B for gradient elution. The information of the characteristic chromatogram constructed by the method is rich, and the quality of the senecio scandens medicinal material, decoction pieces, standard decoction and formula granules can be quickly and comprehensively reflected.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine identification, and particularly to a method for constructing characteristic chromatograms and an identification method for Senecio scandens Buch.-Ham. medicinal materials, their cut pieces, standard decoctions, and formula granules. Background Art

[0002] Senecio scandens Buch.-Ham. is included in the Chinese Pharmacopoeia and is the dried aerial part of the plant Senecio scandens Buch.-Ham. of the Compositae family. It is mainly distributed in East China, Central South, and Southwest China, mostly growing in forest edges of hilly mountains, shrubbery grasslands, and roadsides. It is harvested in summer and autumn and is one of the commonly used traditional Chinese medicines with a long application history. The dried whole herb is 60-100 cm long or cut into small sections 2-3 cm long. The stem is cylindrical and its surface is brownish-yellow. The leaves are mostly shrunken and broken, elliptically triangular or ovate-lanceolate in shape, with the base sagittate or truncate, irregular notches on the edge, dark green or grayish-brown in color, and brittle in texture. Sometimes the branch tips carry withered yellow capitula. It can be harvested throughout the year, impurities are removed, and it is dried in the shade. It is cold in nature and bitter in taste, and has the functions of clearing heat and detoxifying, improving eyesight, and promoting diuresis. Clinically, it is used for carbuncles and sores, cold and fever, red and swollen eyes, diarrhea and dysentery, and skin eczema.

[0003] In the prior art, the chemical constituents of Senecio scandens Buch.-Ham. medicinal materials have been studied. For example, in one prior art, acetonitrile - 0.2% phosphoric acid was used as the mobile phase, and the characteristic chromatogram of Senecio scandens Buch.-Ham. formula granules was established by isocratic elution. However, only 6 characteristic peaks were identified, and the chromatographic peak information was not rich enough to effectively achieve comprehensive quality control of Senecio scandens Buch.-Ham. medicinal materials. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for constructing characteristic chromatograms of Senecio scandens Buch.-Ham. medicinal materials, their cut pieces, standard decoctions, and formula granules, with rich characteristic peak information, which can comprehensively reflect the quality characteristics of Senecio scandens Buch.-Ham. medicinal materials, their cut pieces, standard decoctions, and formula granules.

[0005] To solve the above technical problem, the present invention provides a method for constructing characteristic chromatograms of Senecio scandens Buch.-Ham. medicinal materials, their cut pieces, standard decoctions, and formula granules, which includes:

[0006] Extracting Senecio scandens Buch.-Ham. medicinal materials, their cut pieces, standard decoctions, or formula granules with an extraction solvent to obtain a test solution;

[0007] Dissolving 5-caffeoylquinic acid reference substance, chlorogenic acid reference substance, caffeic acid reference substance, 4-caffeoylquinic acid reference substance, rutin reference substance, hyperoside reference substance, isoquercitrin reference substance, and 4,5-dicaffeoylquinic acid reference substance with an extraction solvent to obtain a reference solution;

[0008] The test solution and the reference solution are determined by a liquid chromatograph to obtain the characteristic chromatograms of the Senecio scandens Buch.-Ham. ex D. Don medicinal material or its cut pieces, the standard decoction, and the formula granules;

[0009] Among them, methanol is selected as the extraction solvent;

[0010] Among them, the chromatographic column of the liquid chromatograph uses phenyl-bonded silica gel as the stationary phase, acetonitrile as mobile phase A, and a 0.05 vol% - 0.3 vol% formic acid aqueous solution as mobile phase B for gradient elution. The gradient elution curve is:

[0011] From 0 min to 15 min, mobile phase A changes from 5% → 8%, and mobile phase B changes from 95% → 92%;

[0012] From 15 min to 22 min, mobile phase A changes from 8% → 12%, and mobile phase B changes from 92% → 88%;

[0013] From 22 min to 38 min, mobile phase A changes from 12% → 16%, and mobile phase B changes from 88% → 84%;

[0014] From 38 min to 50 min, mobile phase A changes from 16% → 25%, and mobile phase B changes from 84% → 75%.

[0015] As an improvement of the above technical solution, the column length of the chromatographic column of the liquid chromatograph is 100 mm - 150 mm, the column diameter is 3 mm - 5 mm, and the particle size of the stationary phase is 2 μm - 3 μm.

[0016] As an improvement of the above technical solution, the chromatographic column is a Waters CORTECS Phenyl chromatographic column, with a column length of 150 mm, a column diameter of 4.6 mm, and a particle size of the stationary phase of 2.7 μm.

[0017] As an improvement of the above technical solution, mobile phase B is a 0.2 vol% formic acid aqueous solution.

[0018] As an improvement of the above technical solution, the column temperature of the chromatographic column of the liquid chromatograph is 28°C - 32°C, the flow rate is 0.7 mL / min - 0.9 mL / min, the injection volume of the test solution is 1 μL - 3 μL, and the injection volume of the reference solution is 1 μL - 3 μL;

[0019] The detection wavelength of the liquid chromatograph is 300 nm - 400 nm.

[0020] As an improvement of the above technical solution, the column temperature of the chromatographic column of the liquid chromatograph is 30°C, the flow rate is 0.8 mL / min, the injection volume of the test solution is 2 μL, and the injection volume of the reference solution is 2 μL;

[0021] The detection wavelength of the liquid chromatograph is 360 nm.

[0022] As an improvement of the above technical solution, in the step of extracting the Senecio scandens Buch.-Ham. herbs or their cut pieces, standard decoctions, and formula granules with an extraction solvent to obtain a test solution, the extraction solvent is 70 vol% - 80 vol% methanol, the extraction method is heating under reflux or ultrasonic extraction, and the extraction time is 20 min - 90 min.

[0023] As an improvement of the above technical solution, when the test sample is Senecio scandens Buch.-Ham. herbs or cut pieces, the ratio of the weight of the test sample to the volume of the extraction solvent is (0.8 - 1.5) g : (20 - 30) mL, the extraction method is heating under reflux, and the extraction time is 40 - 80 min;

[0024] When the test sample is Senecio scandens Buch.-Ham. standard decoction or formula granule, the ratio of the weight of the test sample to the volume of the extraction solvent is (0.8 - 1.5) g : (20 - 30) mL, the extraction method is ultrasonic extraction; the ultrasonic power is 200 - 400 W, the frequency is 20 - 50 kHz, and the extraction time is 20 - 50 min.

[0025] As an improvement of the above technical solution, the characteristic chromatogram includes 10 common peaks. Among them, peak 2 is 5-caffeoylquinic acid, peak 3 is chlorogenic acid, peak 4 is caffeic acid, peak 5 is 4-caffeoylquinic acid, peak 7 is rutin, peak 8 is hyperoside, peak 9 is isoquercitrin, and peak 10 is 4,5-dicaffeoylquinic acid.

[0026] Correspondingly, the present invention also discloses a method for identifying Senecio scandens Buch.-Ham., which is used to identify Senecio scandens Buch.-Ham. and its counterfeits, and it includes:

[0027] Providing a substance to be identified;

[0028] Constructing the characteristic chromatogram of the substance to be identified by the method for constructing the characteristic chromatogram of the above Senecio scandens Buch.-Ham. herbs, their cut pieces, standard decoctions, and formula granules;

[0029] If the characteristic chromatogram only presents peaks 1 - 10, the substance to be identified includes or is Senecio scandens Buch.-Ham.; otherwise, it is a counterfeit;

[0030] Among them, the counterfeit includes Senecio acuminatus Wall.

[0031] Implementing the present invention has the following beneficial effects:

[0032] The present invention constructs characteristic chromatograms of Senecio scandens Buch.-Ham. ex D. Don herbs, their cut pieces, standard decoctions, and formula granules. The constructed characteristic chromatograms are rich in information and achieve good separation of different chromatographic components. There are 10 common peaks in this characteristic chromatogram, among which 8 chromatographic peaks are identified, namely: peak 2 is 5-caffeoylquinic acid, peak 3 is chlorogenic acid, peak 4 is caffeic acid, peak 5 is 4-caffeoylquinic acid, peak 7 is rutin, peak 8 is hyperoside, peak 9 is isoquercitrin, and peak 10 is 4,5-dicaffeoylquinic acid. Based on the characteristic chromatogram of the present invention, it can provide comprehensive quality control indicators for the quantity and quality transfer of Senecio scandens Buch.-Ham. ex D. Don, and can effectively identify Senecio scandens Buch.-Ham. ex D. Don from common counterfeits. Description of the Drawings

[0033] Figure 1 It is the characteristic chromatogram of Senecio scandens Buch.-Ham. ex D. Don formula granules when different chromatographic columns are used in Example 1;

[0034] Figure 2 It is the characteristic chromatogram of Senecio scandens Buch.-Ham. ex D. Don formula granules when different mobile phase B is used in Example 1;

[0035] Figure 3 It is the characteristic chromatogram of Senecio scandens Buch.-Ham. ex D. Don formula granules when different mobile phase gradients are used in Example 1;

[0036] Figure 4 It is the inspection result diagram of the specificity of the characteristic chromatogram of Senecio scandens Buch.-Ham. ex D. Don formula granules in Example 1;

[0037] Figure 5 It is the inspection result diagram of the durability at different temperatures of the characteristic chromatogram of Senecio scandens Buch.-Ham. ex D. Don formula granules in Example 1;

[0038] Figure 6 It is the inspection result diagram of the durability at different flow rates of the characteristic chromatogram of Senecio scandens Buch.-Ham. ex D. Don formula granules in Example 1;

[0039] Figure 7 It is the superimposed diagram of the characteristic chromatograms of 13 batches of Senecio scandens Buch.-Ham. ex D. Don herbs in Example 2;

[0040] Figure 8 It is the superimposed diagram of the characteristic chromatograms of 13 batches of cut pieces of Senecio scandens Buch.-Ham. ex D. Don in Example 2;

[0041] Figure 9 It is the superimposed diagram of the characteristic chromatograms of 3 batches of Senecio scandens Buch.-Ham. ex D. Don formula granules in Example 3;

[0042] Figure 10 It is the characteristic chromatogram of 2 batches of Senecio scandens Buch.-Ham. ex D. Don var. mollifolius W. W. Smith and Senecio scandens Buch.-Ham. ex D. Don control herbs in Example 4;

[0043] Among them, peak 2 is 5-caffeoylquinic acid, peak 3 is chlorogenic acid, peak 4 is caffeic acid, peak 5 is 4-caffeoylquinic acid, peak 7 is rutin, peak 8 is hyperoside, peak 9 is isoquercitrin, and peak 10 is 4,5-dicaffeoylquinic acid. Detailed implementation manners

[0044] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings and specific implementation manners.

[0045] Example 1 Construction of characteristic chromatograms of Senecio scandens Buch.-Ham. ex D. Don herbs, their cut pieces, standard decoctions, and formula granules

[0046] 1. Instruments and reagents

[0047] (1) Instruments: Waters H-Class Plus ultra-high performance liquid chromatograph (Waters); Thermo ultra-high performance liquid chromatograph (Vanquish, Thermo Fisher Scientific); Waters CORTECS Phenyl chromatographic column (4.6 mm × 150 mm, 2.7 μm); ten-thousandth balance (ME204E, Mettler Toledo); hundred-thousandth balance (ABT220-5DM, Guangzhou Dexiang Technology Co., Ltd.); millionth balance (XP26, Mettler Toledo); electronic balance (JJ600, Changshu Shuangjie Testing Instrument Factory); numerical control ultrasonic cleaner (KQ500DE, Kunshan Ultrasonic Instruments Co., Ltd.); constant temperature water bath (model HWS28, Shanghai Yihang Technology Co., Ltd.); ultra-pure water system (Villi-Q Direct, Merck KGaA).

[0048] (2) Reagents: Ethanol (Xilong Scientific Co., Ltd.), methanol (Xilong Scientific Co., Ltd.) are of analytical purity; phosphoric acid (Tianjin Kemiou Chemical Reagent Co., Ltd.), methanol (Merck KGaA), and acetonitrile (Merck KGaA) are of chromatographic grade, and water is self-made ultra-pure water in the laboratory.

[0049] (3) 4-Caffeoylquinic acid (Batch number: wkq18030107, Content: 98.0%, Sichuan Vicibiotech Co., Ltd.); 5-Caffeoylquinic acid (Batch number: DST210427-035, Content: 94.9%, Chengdu Deste Biotechnology Co., Ltd.); Chlorogenic acid (Batch number: 110753-202018, Content: 94.9%, National Institutes for Food and Drug Control); Caffeic acid (Batch number: 110885-201703, Content: 99.7%, National Institutes for Food and Drug Control); Rutin (Batch number: 100080-202012, Content: 91.6%, National Institutes for Food and Drug Control); Hyperoside (Batch number: 111521-201809, Content: 94.9%, National Institutes for Food and Drug Control); Isoquercitrin (Batch number: 111809-201804, Content: 99.4%, National Institutes for Food and Drug Control); 4,5-Dicaffeoylquinic acid (Batch number: 111894-202104, Content: 95.1%, National Institutes for Food and Drug Control); Herba Senecionis Scandentis reference medicinal material (Batch number: 120964-201810, National Institutes for Food and Drug Control); Herba Senecionis Scandentis formula granules (Batch number: KL01, KL02, KL03; Source: Guangdong Yifang Pharmaceutical Co., Ltd.).

[0050] 2. Preparation of test solution

[0051] Medicinal materials and cut pieces: Take the powder of this product (pass through No. 2 sieve), weigh about 1.0 g accurately, place it in a stoppered conical flask, accurately add 25 mL of 75% methanol, weigh, heat under reflux for 1 hour, cool, make up the lost weight with 75% methanol, filter, concentrate the supernatant to an appropriate volume, make up to 5 mL, shake well, filter, and take the subsequent filtrate, that is obtained.

[0052] Standard decoction: Take an appropriate amount of this product, grind it finely, weigh about 1.0 g accurately, place it in a stoppered conical flask, accurately add 25 mL of 75% methanol, weigh, ultrasonically treat (power 300 W, frequency 40 kHz) for 30 minutes, take out, cool, weigh again, make up the lost weight with 75% methanol, shake well, filter, and take the subsequent filtrate, that is obtained.

[0053] Formula granules: Take an appropriate amount of this product, grind it finely, weigh about 1.0 g accurately, place it in a stoppered conical flask, accurately add 25 mL of 75% methanol, weigh, ultrasonically treat (power 300 W, frequency 40 kHz) for 30 minutes, take out, cool, weigh again, make up the lost weight with 75% methanol, shake well, filter, and take the subsequent filtrate, that is obtained.

[0054] 3. Preparation of reference solution

[0055] Take 5-caffeoylquinic acid reference substance, chlorogenic acid reference substance, caffeic acid reference substance, 4-caffeoylquinic acid reference substance, rutin reference substance, hyperoside reference substance, isoquercitrin reference substance, 4,5-dicaffeoylquinic acid reference substance, and add 75% methanol to make a mixed solution containing 25 μg of each per 1 mL as the reference substance solution.

[0056] 4. Chromatographic conditions

[0057] Chromatographic column: Waters CORTECS Phenyl chromatographic column (column length 150 mm, inner diameter 4.6 mm, particle size 2.7 μm); mobile phase: acetonitrile (A) - 0.2% formic acid aqueous solution (B); elution method: gradient elution; flow rate 0.8 mL / min, column temperature 30 °C, detection wavelength 360 nm. Among them, the mobile phase gradient elution program is shown in Table 1.

[0058] Table 1 Elution program

[0059]

[0060] 5. Determination

[0061] Precisely pipette 2 μL of the reference substance solution and 2 μL of the test solution respectively, inject them into the liquid chromatograph for determination, and that's it.

[0062] 6. Methodology investigation

[0063] (1) Chromatographic column investigation

[0064] This investigation examines the influence of different chromatographic columns on each characteristic peak.

[0065] Chromatographic conditions: Use Waters Cortecs T3 chromatographic column (2.1 mm × 100 mm, 1.6 μm); ThermoAccource C18 chromatographic column (2.1 mm × 100 mm, 1.5 μm); Waters CSH C18 chromatographic column (2.1 mm × 100 mm, 1.7 μm); Waters Cortecs Phenyl chromatographic column (4.6 mm × 150 mm, 2.7 μm); Waters Cortecs C18 chromatographic column (4.6 mm × 150 mm, 2.7 μm) as the chromatographic column; use acetonitrile as mobile phase A and 0.2% formic acid aqueous solution as mobile phase B; perform elution according to the regulations in Table 2, with a flow rate of 0.8 mL per minute; column temperature 30 °C; detection wavelength 360 nm; injection volume 2 μL.

[0066] Table 2 Elution program

[0067]

[0068] The results are shown inFigure 1 As can be seen from the figure, different brands of chromatographic columns have a great influence on the separation of each characteristic peak. When using the Waters CORTECS Phenyl (4.6 mm × 150 mm, 2.7 μm) chromatographic column, the separation effect of each characteristic peak is better. Therefore, the Waters CORTECS Phenyl (4.6 mm × 150 mm, 2.7 μm) chromatographic column was selected.

[0069] (2) Investigation of mobile phase concentration and type

[0070] This investigation examines the influence of different mobile phase B on each characteristic peak in the characteristic chromatogram.

[0071] Chromatographic conditions: Waters Cortecs Phenyl chromatographic column (4.6 mm × 150 mm, 2.7 μm); acetonitrile as mobile phase A, and 0.01% formic acid, 0.05% formic acid, 0.2% formic acid, 0.2% phosphoric acid, 0.2% acetic acid as mobile phase B respectively; elution was carried out according to the provisions of Table 3, the flow rate was 0.8 mL per minute; the column temperature was 30 °C; the detection wavelength was 360 nm; the injection volume was 2 μL.

[0072] Table 3 Elution program

[0073]

[0074]

[0075] The characteristic chromatograms corresponding to different mobile phase B are as Figure 2 shown. As can be seen from the figure, when 0.05% formic acid and 0.2% formic acid are used as mobile phase B, the separation effect of each peak is better. When 0.2% formic acid is used as mobile phase B, the separation effect of each peak is the best.

[0076] (2) Investigation of mobile phase gradient

[0077] This investigation examines the influence of different gradient elution curves on each characteristic peak in the characteristic chromatogram.

[0078] Chromatographic conditions: Waters Cortecs Phenyl chromatographic column (4.6 mm × 150 mm, 2.7 μm); acetonitrile as mobile phase A, and 0.2% formic acid as mobile phase B; elution was carried out according to the provisions of Table 4, the flow rate was 0.8 mL per minute; the column temperature was 30 °C; the detection wavelength was 360 nm; the injection volume was 2 μL.

[0079] Table 4 Gradient elution table

[0080]

[0081] The results are shown in Figure 3, As can be seen from the figure, when gradient 3 is used for programmed elution, the resolution between chromatographic peaks is good.

[0082] 7. Methodological verification

[0083] (1) Specificity investigation

[0084] Take appropriate amounts of the above-mentioned test solution, reference solution, and blank solvent (75% methanol), and inject them for testing according to the chromatographic conditions of the characteristic chromatogram. The results show that the blank solvent has no interference ( Figure 4 ).

[0085] (2) Robustness investigation

[0086] (2.1) Investigation of different column temperatures

[0087] Take the sample of Senecio dispensing granules (KL01), prepare the test solution according to the test solution preparation method determined under item "2". Except that the column temperatures are 28°C, 30°C, and 32°C respectively, other chromatographic conditions are the same as those specified under item "4". Inject for analysis. Using peak 8 as the reference peak S, calculate the relative retention time and relative peak area of each characteristic peak with respect to peak S, and calculate the RSD value. The results are as Figure 5 shown. The results show that the RSD values of the relative retention time and relative peak area are both less than 5.0%, indicating that the method has good robustness at different column temperatures.

[0088] (2.2) Investigation of different flow rates

[0089] Take the sample of Senecio dispensing granules (KL01), prepare the test solution according to the test solution preparation method determined under item "2". Except that the flow rates are 0.7 mL / min, 0.8 mL / min, and 0.9 mL / min respectively, other chromatographic conditions are the same as those specified under item "4". Inject for analysis. Using peak 8 as the reference peak S, calculate the relative retention time and relative peak area of each characteristic peak with respect to peak S, and calculate the RSD value. The results are as Figure 6 shown. The RSD values of the relative retention time and relative peak area are both less than 5.0%, indicating that the method has good robustness at different column temperatures.

[0090] (3) Stability investigation

[0091] Take the same batch of Senecio dispensing granules (KL01) sample, prepare the test solution according to the method under item "2", and inject and determine it according to the chromatographic conditions under item "4" at 0 h, 6 h, 8 h, 12 h, 16 h, 20 h, and 24 h respectively. Using peak 8 as the reference peak S, record the relative retention time and relative peak area of each characteristic peak with respect to peak S, and calculate the RSD value. The RSD values of the relative retention time and relative peak area of each characteristic peak are all less than 5%, indicating that the test solution has good stability within 24 hours.

[0092] (4) Precision investigation

[0093] Take the same batch of Senecio scandens formula granules (KL01), prepare the test solution according to the method under item "2" and inject samples continuously for 6 times under the chromatographic conditions under item "4". Take peak 8 as the reference peak S, record the relative retention time and relative peak area of each characteristic peak and peak S, and calculate the RSD value. The results show that the RSD values of the relative retention time and relative peak area of each characteristic peak are all less than 5%, indicating good precision of the instrument.

[0094] (5) Repeatability investigation

[0095] Take 6 portions of the sample powder of the same batch of Senecio scandens formula granules (KL01), prepare 6 portions of test solutions in parallel according to the method under item "2", inject samples for determination under the chromatographic conditions under item "4". Take peak 8 as the reference peak S, record the relative retention time and relative peak area of each characteristic peak and peak S, and calculate the RSD value. The results show that the RSD values of the relative retention time and relative peak area of each characteristic peak are all less than 5%, indicating good repeatability of the method.

[0096] 8. Identification of common peaks

[0097] Take the sample of Senecio scandens formula granules (KL01), prepare the test solution according to the test solution preparation method determined under item "2", inject samples for analysis under the chromatographic conditions specified under item "4", and determine that peak 2 is 5-caffeoylquinic acid, peak 3 is chlorogenic acid, peak 4 is caffeic acid, peak 5 is 4-caffeoylquinic acid, peak 7 is rutin, peak 8 is hyperoside, peak 9 is isoquercitrin, and peak 10 is 4,5-dicaffeoylquinic acid.

[0098] Example 2 Determination of Senecio scandens medicinal materials and cut crude drugs samples

[0099] Take 13 batches of Senecio scandens medicinal materials and cut crude drugs, prepare the test solutions according to the test solution preparation method under item "2" in Example 1; inject samples for determination under the chromatographic conditions under item "4" in Example 1; use the "Similarity Evaluation Software for Traditional Chinese Medicine Chromatographic Fingerprints" to identify the common peaks of the characteristic fingerprints of 13 batches of Senecio scandens medicinal materials and cut crude drugs. The results show that the RSD values of the relative retention time of 13 batches of Senecio scandens medicinal materials are in the range of 0.03% - 0.22%, and the RSD values of the relative peak area are in the range of 40.86% - 95.81%; the RSD values of the relative retention time of 13 batches of Senecio scandens cut crude drugs are in the range of 0.03% - 0.33%, and the RSD values of the relative peak area are in the range of 40.20% - 118.96%; see specifically Figure 7 、 Figure 8 。

[0100] Example 3 Determination of Senecio scandens formula granules samples

[0101] Take 3 batches of Senecio scandens formula granules (batch numbers: KL01, KL02, KL03), and prepare the test solution according to the preparation method of the test solution under item "2" in Example 1; inject and determine according to the chromatographic conditions under item "4" in Example 1; use the "Similarity Evaluation Software for Traditional Chinese Medicine Chromatographic Fingerprints" to identify the common peaks of the characteristic fingerprints of 3 batches of Senecio scandens formula granules. The results are shown in Tables 5 to 6. Figure 9 。

[0102] Table 5 Determination of the characteristic fingerprints of Senecio scandens formula granules (relative retention time)

[0103]

[0104] Table 6 Determination of the characteristic fingerprints of Senecio scandens formula granules (relative peak area)

[0105]

[0106] The above are the preferred embodiments of the invention. It should be noted 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 are also regarded as the protection scope of the present invention.

[0107] Example 4 Application of distinguishing Senecio scandens and its adulterants

[0108] Respectively take the reference medicinal material of Senecio scandens and the medicinal material of Senecio scandens var. subdentatus, and prepare the test solution according to the preparation method of the test solution under item "2" in Example 1; inject and determine according to the chromatographic conditions under item "4" in Example 1; use the "Similarity Evaluation Software for Traditional Chinese Medicine Chromatographic Fingerprints" to conduct a specificity study on the characteristic fingerprints of Senecio scandens and its adulterants. The results show that Senecio scandens var. subdentatus lacks peak 1 and peak 6, and presents its characteristic peaks at the retention time of 38 min to 45 min, indicating that this characteristic fingerprint method can effectively distinguish Senecio scandens and its adulterants. The specific results are shown in Figure 10 。

Claims

1. A method for constructing characteristic fingerprints of Senecio scandens Buch.-Ham. herbs, their cut pieces, standard decoctions, and formula granules, characterized in that, Comprising: Extracting the Senecio scandens Buch.-Ham. ex D. Don medicinal material or its cut crude drug, standard decoction, and formula granule with an extraction solvent to obtain a test solution; Dissolving reference substances of 5-caffeoylquinic acid, chlorogenic acid, caffeic acid, 4-caffeoylquinic acid, rutin, hyperoside, isoquercitrin, and 4,5-dicaffeoylquinic acid with an extraction solvent to obtain a reference substance solution; Determining the test solution and the reference substance solution using a liquid chromatograph to obtain the characteristic chromatogram of the Senecio scandens Buch.-Ham. ex D. Don medicinal material or its cut crude drug, standard decoction, and formula granule; Wherein, the extraction solvent is methanol; Wherein, the chromatographic column of the liquid chromatograph uses phenyl-bonded silica gel as the stationary phase, acetonitrile as mobile phase A, and a 0.05 vol% - 0.3 vol% formic acid aqueous solution as mobile phase B for gradient elution, and the gradient elution curve is: 0 min - 15 min, mobile phase A from 5% → 8%, mobile phase B from 95% → 92%; 15 min - 22 min, mobile phase A from 8% → 12%, mobile phase B from 92% → 88%; 22 min - 38 min, mobile phase A from 12% → 16%, mobile phase B from 88% → 84%; 38 min - 50 min, mobile phase A from 16% → 25%, mobile phase B from 84% → 75%.

2. The method for constructing the characteristic fingerprint of the Senecio scandens Buch.-Ham. ex D. Don medicinal material, its cut pieces, standard decoction, and formula granules as claimed in claim 1, wherein, The column length of the chromatographic column of the liquid chromatograph is 100 mm - 150 mm, the column diameter is 3 mm - 5 mm, and the particle size of the stationary phase is 2 μm - 3 μm.

3. The method for constructing the characteristic fingerprint of the Senecio scandens Buch.-Ham. ex D. Don medicinal material, its cut crude drug, standard decoction and formula granules as claimed in claim 1 or 2, characterized in that, The chromatographic column is a Waters CORTECS Phenyl chromatographic column with a column length of 150 mm, a column diameter of 4.6 mm, and a particle size of the stationary phase of 2.7 μm.

4. The method for constructing the characteristic fingerprint of the Senecio scandens Buch.-Ham. ex D. Don medicinal material, its cut crude drug, standard decoction and formula granules as claimed in claim 1, characterized in that, The mobile phase B is a 0.2 vol% formic acid aqueous solution.

5. The method for constructing the characteristic chromatogram of the Senecio scandens Buch.-Ham. ex D. Don medicinal material, its cut crude drug, standard decoction, and formula granules as claimed in claim 1, wherein, The column temperature of the chromatographic column of the liquid chromatograph is 28°C - 32°C, the flow rate is 0.7 mL / min - 0.9 mL / min, the injection volume of the test solution is 1 μL - 3 μL, and the injection volume of the reference substance solution is 1 μL - 3 μL; The detection wavelength of the liquid chromatograph is 300 nm - 400 nm.

6. The method for constructing the characteristic fingerprint of the Senecio scandens Buch.-Ham. ex D. Don medicinal material, its cut pieces, standard decoction, and formula granules as claimed in claim 1 or 5, characterized in that, The column temperature of the chromatographic column of the liquid chromatograph is 30°C, the flow rate is 0.8 mL / min, the injection volume of the test solution is 2 μL, and the injection volume of the reference substance solution is 2 μL; The detection wavelength of the liquid chromatograph is 360 nm.

7. The method for constructing the characteristic fingerprint of the Senecio scandens Buch.-Ham. ex D. Don medicinal material, its cut crude drug, standard decoction, and formula granules as claimed in claim 1, wherein In the step of extracting the Senecio scandens Buch.-Ham. ex D. Don medicinal material or its cut crude drug, standard decoction, and formula granule with an extraction solvent to obtain a test solution, the extraction solvent is 70 vol% - 80 vol% methanol, the extraction method is heating under reflux extraction or ultrasonic extraction, and the extraction time is 20 min - 90 min.

8. The method for constructing the characteristic fingerprint of the Senecio scandens Buch.-Ham. ex D. Don medicinal material, its cut pieces, standard decoction, and formula granules as claimed in claim 1 or 7, characterized in that, When the test sample is the Senecio scandens Buch.-Ham. ex D. Don medicinal material or cut crude drug, the ratio of the weight of the test sample to the volume of the extraction solvent is (0.8 - 1.5) g : (20 - 30) mL, the extraction method is heating under reflux extraction, and the extraction time is 40 - 80 min; When the test sample is the standard decoction of Senecio scandens or the formula granules of Senecio scandens, the ratio of the weight of the test sample to the volume of the extraction solvent is (0.8 - 1.5) g : (20 - 30) mL, and the extraction method is ultrasonic extraction; the ultrasonic power is 200 - 400 W, the frequency is 20 - 50 kHz, and the extraction time is 20 - 50 min.

9. The method for constructing the characteristic chromatogram of the Senecio scandens Buch.-Ham. ex D. Don medicinal material, its cut pieces, standard decoction and formula granules as claimed in claim 1, wherein, The characteristic chromatogram includes 10 common peaks. Among them, peak 2 is 5-caffeoylquinic acid, peak 3 is chlorogenic acid, peak 4 is caffeic acid, peak 5 is 4-caffeoylquinic acid, peak 7 is rutin, peak 8 is hyperoside, peak 9 is isoquercitrin, and peak 10 is 4,5-dicaffeoylquinic acid.

10. A method for identifying Senecio scandens, used to distinguish Senecio scandens from its counterfeits, characterized in that, including: providing the substance to be identified; constructing the characteristic chromatogram of the substance to be identified by the method for constructing the characteristic chromatogram of the medicinal material of Senecio scandens and its cut pieces, standard decoction, and formula granules according to any one of claims 1 - 9; if only peaks 1 - 10 are presented in the characteristic chromatogram, the substance to be identified includes or is Senecio scandens; otherwise, it is a counterfeit; wherein, the counterfeit includes Senecio scandens Buch.-Ham.

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