A method for constructing an HPLC fingerprint of Mahuang Dingchuan decoction composition

Abstract

The present invention relates to a fingerprint construction method and detection method of an Mahuang Dingchuan Decoction composition. The fingerprint construction method of the present invention comprises the following steps: taking a reference substance and an Mahuang Dingchuan Decoction composition, and preparing a reference substance solution and a test solution respectively; using high performance liquid chromatography to detect the reference substance solution and the test solution, and obtaining corresponding detection patterns and reference patterns; importing the detection patterns into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system for analysis, and obtaining a fingerprint of the Mahuang Dingchuan Decoction composition with identification. The present invention constructs a fingerprint of the Mahuang Dingchuan Decoction composition, which has rich chromatographic peaks and contains effective ingredients belonging to multiple medicinal materials such as ephedra, bitter almond, scutellaria, white mulberry bark, ophiopogon, platycodon, coltsfoot flower, radish seed, ginkgo, etc., which can more comprehensively reflect the overall chemical composition of the Mahuang Dingchuan Decoction composition, provide an effective means for the overall quality control and evaluation of the Mahuang Dingchuan Decoction preparation, can better monitor and evaluate the quality of this product, and has a high application value.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and particularly relates to establishing a high-performance liquid phase fingerprint of an Mahuang Dingchuan decoction composition by using a high-performance liquid chromatography method for controlling the quality of the Mahuang Dingchuan decoction preparation. Background Art

[0002] Bronchial asthma (abbreviated as asthma) is a common chronic respiratory disease worldwide. In recent years, its prevalence has been increasing year by year in countries around the world, making it a serious threat to human health. According to statistics, there are approximately 300 million asthma patients worldwide, with a global prevalence of 1-18%. In my country, there are approximately 10-30 million asthma patients, and the number is expected to continue to increase. Bronchial asthma is an immunological allergic disease characterized by chronic airway inflammation, increased mucus secretion, airway remodeling, and increased airway hyperresponsiveness. Airway inflammation is the primary pathological change and determines the degree of airway obstruction and airway hyperresponsiveness. Currently, Western medicine primarily treats asthma with glucocorticoids. Glucocorticoids are the most effective treatment for both chronic asthma and acute asthma attacks and have become the first-line treatment for bronchial asthma. However, long-term use of glucocorticoids can cause serious side effects in patients with persistent airway hyperresponsiveness or glucocorticoid insensitivity after glucocorticoid treatment. Furthermore, glucocorticoid use in children can have certain effects on their growth and metabolism. In some severe cases, glucocorticoids are inadequately controlled and can develop resistance, leading to the development of a chronic condition. The main treatment options for asthma in Traditional Chinese Medicine are staged treatment, syndrome differentiation and treatment, and specialized prescriptions. While these have achieved certain therapeutic effects in individualized clinical treatment, there has been no breakthrough in the development of drugs suitable for group treatment.

[0003] Mahuang Dingchuan Decoction is composed of ephedra, apricot kernel, scutellaria baicalensis, mulberry bark, ophiopogon japonicus, platycodon grandiflorum, coltsfoot flower, radish seed, and ginkgo.

[0004] Before the completion of the present invention, there has been no report on a method for establishing a fingerprint using the chemical components contained in the Mahuang Dingchuan Decoction composition as indicators. Summary of the Invention

[0005] One of the purposes of the present invention is to provide a fingerprint spectrum detection method and fingerprint spectrum of Mahuang Dingchuan Decoction composition. The fingerprint spectrum constructed by this method can comprehensively reflect the overall chemical composition of Mahuang Dingchuan Decoction composition, and provide an effective means for the overall quality control and evaluation of Mahuang Dingchuan Decoction composition. It aims to solve the defect in the prior art that there is no literature report on the fingerprint spectrum of Mahuang Dingchuan Decoction composition, and effectively control the quality of Mahuang Dingchuan Decoction preparation.

[0006] The second purpose of the present invention is to comprehensively reflect the current status of each component in the Mahuang Dingchuan Decoction composition through systematic component identification and attribution of single medicinal materials with common peaks in different batches of drugs, so as to provide a reference basis for the quality of Mahuang Dingchuan Decoction preparations.

[0007] The Mahuang Dingchuan Decoction composition of the present invention is prepared from 300g of ephedra, 150g of bitter almond, 100g of scutellaria, 100g of white mulberry bark, 100g of ophiopogon, 100g of platycodon, 100g of coltsfoot flower, 100g of radish seed, and 150g of ginkgo. The specific preparation method is as follows: the above nine ingredients are decocted twice with water, the first time for 2 hours and the second time for 1 hour, the decoctions are combined, allowed to stand, filtered, and the filtrate is concentrated to a thick paste of 1.30-1.35 (60°C), dried, crushed, and sieved to obtain the decoction.

[0008] The present invention adopts the following technical solution: a fingerprint detection method for the Mahuang Dingchuan Decoction composition, which uses high performance liquid chromatography to determine its fingerprint. Specifically, the fingerprint detection method for the Mahuang Dingchuan Decoction composition comprises the following steps:

[0009] a. Preparation of test solution: Grind the Mahuang Dingchuan Decoction composition, take approximately 0.2-2 g, accurately weigh, and place in a stoppered conical flask. Accurately add 20-50 mL of methanol and weigh the solution. Ultrasonicate for 20-40 minutes, cool, and weigh again. Make up the loss with methanol, shake well, filter, and collect the filtrate.

[0010] b. Preparation of reference solution: Accurately weigh appropriate amounts of ephedrine hydrochloride, scutellarin, baicalin, quercetin, baicalein, and wogonin reference substances, and prepare mixed reference solutions with concentrations of 0.1, 0.002, 0.05, 0.002, 0.002, and 0.001 mg / mL, respectively, using methanol as the solvent. These solutions serve as reference solutions.

[0011] c. Determination: Accurately draw 5 to 15 μL of the reference solution and the test solution, inject them into a high performance liquid chromatograph, record the chromatogram within 80 minutes, and process the chromatogram with fingerprint software to obtain the fingerprint of the Mahuang Dingchuan Decoction composition.

[0012] In the above method, the chromatographic conditions for the high performance liquid chromatography determination are: octadecylsilane bonded silica gel as the filler, acetonitrile as the mobile phase A, 0.01-0.2% formic acid solution as the mobile phase B, gradient elution, a flow rate of 0.5-1.5 mL / min, a column temperature of 25-40°C, and a detection wavelength of 210-360 nm; the theoretical plate number calculated based on the baicalin peak should be no less than 4000.

[0013] Preferably, the fingerprint detection method of the Mahuang Dingchuan Decoction composition comprises the following steps:

[0014] a. Preparation of test solution: Grind the Mahuang Dingchuan Decoction composition, take 0.5 g, accurately weigh, place in a stoppered conical flask, accurately add 25 mL of methanol, weigh, sonicate for 30 minutes, let cool, weigh again, make up the loss with methanol, shake well, filter, and take the filtrate to obtain;

[0015] b. Preparation of reference solution: Accurately weigh appropriate amounts of ephedrine hydrochloride, scutellarin, baicalin, quercetin, baicalein, and wogonin reference substances, accurately weigh them, place them in 25 mL volumetric flasks, dissolve them in methanol and dilute them to the mark, shake well, and obtain the reference substance stock solution; then accurately pipette 5, 0.2, 2, 0.2, 0.2, and 0.1 mL of the reference substance stock solution into the same 10 mL volumetric flask, dissolve them in methanol and dilute them to the mark, shake well, and prepare mixed reference substance solutions with mass concentrations of 0.10152, 0.00208, 0.05144, 0.00216, 0.00202, and 0.00108 mg / mL, respectively, as the mixed reference substance solution;

[0016] c. Determination: Accurately draw 10 μl of each reference solution and test solution, inject into high performance liquid chromatograph, record the chromatogram within 80 minutes, and process the map with fingerprint software to obtain the fingerprint of Mahuang Dingchuan Decoction composition.

[0017] The chromatographic conditions for the high performance liquid chromatography determination are preferably: octadecylsilane bonded silica gel as the filler, a Shimadzu C18 250×4.60 mm chromatographic column; acetonitrile as the mobile phase A, 0.1% formic acid solution as the mobile phase B, gradient elution, a flow rate of 1.0 ml / min, a column temperature of 30°C, a detection wavelength of 250 nm, and a theoretical plate number calculated based on the baicalin peak should be no less than 4000.

[0018] In the aforementioned method for constructing the HPLC fingerprint of the Mahuang Dingchuan Decoction composition, during the gradient elution process, the ratio of mobile phases A and B changes as follows: 0-15 min, 5-10% A; 15-20 min, 10-15% A; 20-45 min, 15-25% A; 45-55 min, 25-30% A; 55-70 min, 30-55% A; 70-80 min, 55-65% A.

[0019] The fingerprint has 24 common peaks. The chromatograms of the reference substances are compared with the fingerprints of the samples. The chromatogram peaks at the same retention time as the reference substances are identified. Figure 1 The results showed that among the common peaks, peaks 7, 10, 16, 17, 21, and 22 were identified as ephedrine hydrochloride, scutellarin, baicalin, quercetin, baicalein, and wogonin, respectively.

[0020] The beneficial effects of the present invention are:

[0021] 1. The present invention established HPLC fingerprints of 10 batches of Mahuang Dingchuan Decoction compositions, and the similarities were all greater than 0.90, which can more comprehensively reflect the chemical information contained in the Mahuang Dingchuan Decoction compositions and better characterize their quality.

[0022] 2. 24 common peaks were identified, among which peaks 7, 10, 16, 17, 21, and 22 were identified as ephedrine hydrochloride, scutellarin, baicalin, quercetin, baicalein, and wogonin, respectively;

[0023] 3. The fingerprint of the Mahuang Dingchuan Decoction composition provided by the present invention is systematically optimized through conditions such as the detection wavelength of the fingerprint, the solvent for preparing the test sample, and so on, and a fingerprint method is established. Based on the fingerprint detection results of multiple batches of this traditional Chinese medicine composition, data is gradually accumulated and a control fingerprint is generated as the fingerprint standard of this product, thereby achieving the purpose of more comprehensive and effective control of the preparation quality.

[0024] 3. Since the fingerprint spectrum is not intended to determine the precise content of a certain component, but to fully reflect the information of the chemical composition, the present invention chooses to perform the measurement at a wavelength of 250nm, which has more peaks and reflects more complete information; the absorption value of each peak is good, the baseline is stable, and the large absorption of the near-ultraviolet impurity peak is avoided.

[0025] 4. The high-performance liquid chromatography fingerprint method established in the present invention has realized the quality control of the entire Mahuang Dingchuan Decoction composition for the first time. It does not identify a single compound or medicinal material, but can more effectively guide the feeding of materials, strictly regulate production operations, and ensure the safety and effectiveness of clinical medication.

[0026] 5. According to the standard fingerprint conditions, the chromatographic peaks were attributed, and the 24 common peaks were attributed to nine medicinal materials, including ephedra, bitter almond, scutellaria, white mulberry bark, ophiopogon, platycodon, coltsfoot flower, radish seed, and ginkgo. Among them, peak 1 was attributed to ginkgo, platycodon, bitter almond, coltsfoot flower, and ephedra; peak 2 was attributed to coltsfoot flower and white mulberry bark; peak 3 was attributed to coltsfoot flower and radish seed; peak 4 was attributed to coltsfoot flower and white mulberry bark; peak 5 was attributed to coltsfoot flower; peak 6 was attributed to ginkgo and coltsfoot flower; peak 7 was attributed to ephedra; peak 8 was attributed to scutellaria and coltsfoot flower. Winter flower, radish seed, peak No. 9 belongs to Scutellaria baicalensis and Coltsfoot Flos, peak No. 10 belongs to Scutellaria baicalensis, peak No. 11 belongs to Coltsfoot Flos, peak No. 12 belongs to Platycodon grandiflorum, peak No. 13 belongs to Coltsfoot Flos, peak No. 14 belongs to Ginkgo biloba and Apricot Kernel, peak No. 15 belongs to Coltsfoot Flos, peak No. 16 belongs to Scutellaria baicalensis, peak No. 17 belongs to Scutellaria baicalensis, peak No. 18 belongs to Scutellaria baicalensis, peak No. 19 belongs to Ephedra, peak No. 20 belongs to Scutellaria baicalensis, peak No. 21 belongs to Scutellaria baicalensis, peak No. 22 belongs to Scutellaria baicalensis, peak No. 23 belongs to Scutellaria baicalensis, and peak No. 24 belongs to Scutellaria baicalensis. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 HPLC chromatogram of mixed reference substance

[0028] Figure 2 The common pattern spectrum of the Mahuang Dingchuan Decoction composition measured by the present invention

[0029] Figure 3 Overlay of fingerprints of 10 batches of Mahuang Dingchuan Decoction compositions

[0030] Figure 4 Comparison of sample fingerprints and chromatograms of various medicinal materials

[0031] Figure 5 Spectrum of Cambogia Bone Strengthening Pills at 360nm wavelength

[0032] Figure 6 Spectrum of Cambogia Bone Strengthening Pills at 320nm wavelength

[0033] Figure 7 Spectrum of Cambogia Bone Strengthening Pills at 280nm wavelength

[0034] Figure 8 Spectrum of Cambogia Bone Strengthening Pills at 250nm wavelength

[0035] Figure 9 Spectrum of Cambogia Bone Strengthening Pills at 230nm wavelength

[0036] Figure 10 Spectrum of Cambogia Bone Strengthening Pills at 210nm wavelength

[0037] Figure 11 Spectrum of Cambogia Bone Strengthening Pills at 203nm wavelength DETAILED DESCRIPTION

[0038] In order to facilitate the understanding of the technical solution of the present invention, the establishment of the HPLC fingerprint of the Mahuang Dingchuan Decoction composition of the present invention and its fingerprint are further explained below in combination with specific test examples.

[0039] Example 1: Detection of fingerprints of different batches of Mahuang Dingchuan Decoction compositions

[0040] 1. Instruments and test drugs

[0041] 1.1 Agilent 1220 high performance liquid chromatograph (Agilent, USA); KQ-250B ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); BSA 124S electronic balance (Sartorius); QUINTIX35-1CN (Sartorius).

[0042] 1.2 Purified water (Wahaha), acetonitrile (chromatographic grade), and all other reagents were of analytical grade. Ephedrine hydrochloride, scutellarin, baicalin, quercetin, baicalein, and wogonin reference substances (batch numbers 171241-201809, 110842-201709, 110715-201821, 100081-201610, 111595-201607, and 111514-201607, with purities of 100.0%, 91.7%, 95.4%, 99.8%, 98.5%, and 95.1%, respectively, were purchased from the China Food and Drug Administration. Mahuang Dingchuan Decoction composition (batch numbers: 20210801, 20210802, 20210803, 20211001, 20211002, 20211003, 20211201, 20211202, 20211203, 20211204).

[0043] 2. Fingerprint determination

[0044] 2.1 Chromatographic conditions: Octadecylsilane bonded silica gel was used as the filler, acetonitrile was used as the mobile phase A, 0.1% formic acid solution was used as the mobile phase B, gradient elution was performed, the flow rate was 1.0 ml / min, the column temperature was 30°C, the detection wavelength was 250 nm, and the mobile phase gradient elution program was as follows: 0-15 min, 5-10% A; 15-20 min, 10-15% A; 20-45 min, 15-25% A; 45-55 min, 25-30% A; 55-70 min, 30-55% A; 70-80 min, 55-65% A.

[0045] 2.2 Preparation of test solution: Take the composition of Mahuang Dingchuan Decoction, grind it into powder, take 0.5 g, weigh it accurately, put it into a stoppered conical flask, add 25 mL of methanol accurately, weigh it, and treat it ultrasonically (power 80 W, frequency 250 kHz) for 30 minutes. Let it cool, weigh it again, make up the lost weight with methanol, shake it well, filter it, and take the filtrate to obtain the product.

[0046] 2.3 Preparation of control medicinal materials and control extracts: According to the prescription ratio and preparation method, weigh the control extract or control medicinal material and prepare it according to the preparation method of the test sample.

[0047] 2.4 Preparation of reference solution: accurately weigh appropriate amount of ephedrine hydrochloride, scutellarin, baicalin, quercetin, baicalein, and wogonin reference substances, accurately weigh them, place them in a 25 mL volumetric flask, and use methanol as solvent to prepare reference substance stock solutions with mass concentrations of 0.30304, 0.1040, 0.2572, 0.1080, 0.1010, and 0.1080 mg / mL respectively; then accurately pipette the reference solution of Place 5, 0.2, 2, 0.2, 0.2, and 0.1 mL of the reference stock solution in the same 10 mL volumetric flask, dissolve with methanol and dilute to the mark, shake well to prepare mixed reference solution with mass concentrations of 0.10152, 0.00208, 0.05144, 0.00216, 0.00202, and 0.00108 mg / mL, respectively. Pass through a 0.45 μm microporous filter membrane, and take the filtrate as the mixed reference solution;

[0048] 2.4 Determination: Accurately pipette 10 μL of reference solution and test solution respectively, inject into high performance liquid chromatograph, record the chromatogram within 80 minutes, see Figure 1 、 Figure 2 .

[0049] Example 2: Fingerprint analysis of 10 batches of Mahuang Dingchuan decoction compositions

[0050] 1. Fingerprint similarity analysis

[0051] Ten batches of Mahuang Dingchuan Decoction compositions were prepared according to the test sample preparation method, sampled and analyzed under chromatographic conditions, and the fingerprints of the Mahuang Dingchuan Decoction compositions were recorded. The "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine 2012A Edition" issued by the State Pharmacopoeia Committee was used to perform similarity analysis on the fingerprints of the 10 batches of Mahuang Dingchuan Decoction compositions. S1 was set as the reference spectrum, the median method was used, the time window width was set to 0.1 min, and a total of 24 common peaks were calibrated. The similarities of the 10 batches of Mahuang Dingchuan Decoction compositions were all greater than 0.90. The similarity evaluation results are shown in Table 1. The relative retention times of the 24 common peaks were basically consistent, while the relative peak areas were quite different, as shown in Table 2. The fingerprints of the 10 batches of Mahuang Dingchuan Decoction compositions are shown in Table 2. Figure 3 ( Figure 3S1-S10 correspond to batch numbers 20210801, 20210802, 20210803, 20211001, 20211002, 20211003, 20211201, 20211202, 20211203, and 20211204, respectively).

[0052] Table 1 Similarity evaluation results of 10 batches of Mahuang Dingchuan Decoction composition samples

[0053]

[0054]

[0055] Table 2 Standard fingerprint data

[0056]

[0057]

[0058] 2. Common peak identification: Compare the chromatogram of the reference substance with the fingerprint of the sample. The chromatographic peak at the same retention time as the reference substance is identified. Figure 1 The results showed that among the common peaks, peaks 7, 10, 16, 17, 21, and 22 were identified as ephedrine hydrochloride, scutellarin, baicalin, quercetin, baicalein, and wogonin, respectively.

[0059] 3. Medicinal material identification: According to the standard fingerprint conditions, the chromatographic peaks are identified. Figure 4 (S1 is the sample, S2-S10 are nine medicinal materials, namely, scutellaria, ginkgo, platycodon, bitter almond, coltsfoot flower, radish seed, ephedra, ophiopogon, and white mulberry bark). The 24 common peaks are respectively attributed to the nine medicinal materials, namely, ephedra, bitter almond, scutellaria, white mulberry bark, ophiopogon, platycodon, coltsfoot flower, radish seed, and ginkgo; among them, peak 1 is attributed to ginkgo, platycodon, bitter almond, coltsfoot flower, and ephedra; peak 2 is attributed to coltsfoot flower and white mulberry bark; peak 3 is attributed to coltsfoot flower and radish seed; peak 4 is attributed to coltsfoot flower and white mulberry bark; peak 5 is attributed to coltsfoot flower; peak 6 is attributed to ginkgo and coltsfoot flower; peak 7 is attributed to ephedra; peak 8 is attributed to scutellaria, coltsfoot flower, and radish seed; peak 9 is attributed to Belongs to Scutellaria baicalensis and Coltsfoot Flos. Peak 10 belongs to Scutellaria baicalensis, peak 11 belongs to Coltsfoot Flos, peak 12 belongs to Platycodon grandiflorum, peak 13 belongs to Coltsfoot Flos, peak 14 belongs to Ginkgo biloba and Apricot kernel, peak 15 belongs to Coltsfoot Flos, peak 16 belongs to Scutellaria baicalensis, peak 17 belongs to Scutellaria baicalensis, peak 18 belongs to Scutellaria baicalensis, peak 19 belongs to Ephedra sinica, peak 20 belongs to Scutellaria baicalensis, peak 21 belongs to Scutellaria baicalensis, peak 22 belongs to Scutellaria baicalensis, peak 23 belongs to Scutellaria baicalensis, and peak 24 belongs to Scutellaria baicalensis.

[0060] Example 3: Wavelength selection

[0061] The 20210801 batch of samples were tested, and the overall effect of the chromatograms at different wavelengths such as 360, 320, 280, 250, 230, 210, and 203 were investigated. The other chromatographic conditions were the same as those in Example 1. Figure 5 、 6 , 7, 8, 9, 10, 11. From the measured chromatogram, we know that when the wavelength is 360nm, all the peaks cannot fully reflect the characteristics of each medicinal material ( Figure 5 ); When the wavelength is 320nm, all peaks cannot fully reflect the characteristics of each medicinal material ( Figure 6 ); When the wavelength is 280nm, all peaks cannot fully reflect the characteristics of each medicinal material ( Figure 7 ); When the wavelength is 250nm, there are more chromatographic peaks, each chromatographic peak can fully reflect the characteristics of each medicinal material, the chromatogram baseline is flat, and the peak shape is better, so this is the best condition ( Figure 8 When the wavelength is 230nm, the chromatogram baseline is not used and the chromatographic peak fluctuates greatly ( Figure 9 When the wavelength is 210, the chromatogram baseline is not flat and the chromatographic peak fluctuates greatly ( Figure 10 ); When the wavelength is 203nm, the chromatogram baseline is not in the standard position ( Figure 11 ). Comprehensively selected, the fingerprint spectrum with 250nm as the detection wavelength can most comprehensively reflect the composition of the preparation, and the peak shape is the best, so 230nm is preferred as the optimal detection wavelength.

[0062] Example 4: Methodological Investigation

[0063] The 20210801 batch of samples was tested, and the chromatographic conditions and the preparation method of the test sample were the same as those in Example 1.

[0064] (1) Precision test

[0065] A test solution was prepared using the Mahuang Dingchuan Decoction composition. Six consecutive injections were made, and the retention times and areas of the 24 major chromatographic peaks were recorded. Using baicalin as the reference peak, the relative peak area RSD was <3.0% (n=6) and the relative retention time RSD was <1.0% (n=6), demonstrating good instrument precision. The experimental results are shown in Table 3.

[0066] (2) Stability test

[0067] A test solution was prepared from the Mahuang Dingchuan Decoction composition. Tests were performed at 0, 2, 4, 8, 12, and 24 hours, recording the retention times and areas of the 24 main chromatographic peaks. Using baicalin as the reference peak, the relative peak area RSD was <3.0% (n=6) and the relative retention time RSD was <1.0% (n=6), demonstrating good stability within 24 hours. The experimental results are shown in Table 3.

[0068] (3) Repeatability test

[0069] Six test solutions of the Mahuang Dingchuan Decoction composition were prepared and tested, recording the retention times and areas of the 24 main chromatographic peaks. Using baicalin as the reference peak, the relative peak area RSD was <3.0% (n=6) and the relative retention time RSD was <1.0% (n=6), demonstrating good reproducibility. The experimental results are shown in Table 3.

[0070] Table 3 Methodological investigation experimental results

[0071]

[0072]

Claims

1. A method for constructing an HPLC fingerprint of a Mahuang Dingchuan Decoction composition, characterized in that: The following steps are involved: a. Preparation of test solution: Grind the Mahuang Dingchuan Decoction composition, take 0.5 g, accurately weigh, and place in a stoppered conical flask. Accurately add 25 mL of methanol and weigh the solution. Ultrasonicate for 20–40 minutes, cool, and weigh again. Make up the loss with methanol, shake well, filter, and collect the filtrate. b. Preparation of reference solution: Accurately weigh appropriate amounts of ephedrine hydrochloride, scutellarin, baicalin, quercetin, baicalein, and wogonin reference substances, and prepare mixed reference solutions with concentrations of 0.1, 0.002, 0.05, 0.002, 0.002, and 0.001 mg / mL, respectively, using methanol as the solvent. These solutions serve as reference solutions. c. Determination: Precision draw reference solution and test solution 5 ~ 15μL each, injected into the high performance liquid chromatograph, record the chromatogram within 80 minutes, use fingerprint software to process the map, that is, the fingerprint of the Mahuang Dingchuan Decoction composition; The chromatographic conditions of the high performance liquid chromatography determination are as follows: octadecylsilane bonded silica gel as the filler, acetonitrile as the mobile phase A, 0.1% formic acid solution as the mobile phase B, gradient elution, flow rate 1 mL / min, column temperature 25-40°C, detection wavelength 250 nm; the theoretical plate number calculated based on the baicalin peak should be no less than 4000; During the gradient elution process, the ratio of mobile phase A and B changed as follows: 0-15 min, 5-10% A; 15-20 min, 10-15% A; 20-45 min, 15-25% A; 45-55 min, 25-30% A; 55-70 min, 30-55% A; 70-80 min, 55-65% A; The Mahuang Dingchuan Decoction composition is composed of 300g of ephedra, 150g of bitter almond, 100g of scutellaria, 100g of mulberry bark, The herb is prepared from 100g of Rhizoma Coptidis, 100g of Radishes, 100g of Radishes and 150g of Ginkgo Biloba.

2. The method for constructing an HPLC fingerprint of the Mahuang Dingchuan Decoction composition according to claim 1, characterized in that: The following steps are involved: a. Preparation of test solution: Grind the Mahuang Dingchuan Decoction composition, take 0.5 g, accurately weigh, and place in a stoppered conical flask. Accurately add 25 mL of methanol and weigh the solution. Ultrasonicate for 30 minutes, cool, and weigh again. Make up the loss with methanol, shake well, filter, and collect the filtrate. b. Preparation of reference solution: Accurately weigh appropriate amounts of ephedrine hydrochloride, scutellarin, baicalin, quercetin, baicalein, and wogonin reference substances, accurately weigh them, and place them in 25 mL volumetric flasks. Dissolve them in methanol and dilute to the mark. Shake well to obtain reference substance stock solutions. Accurately pipette 5, 0.2, 2, 0.2, 0.2, and 0.1 mL of the reference substance stock solutions into the same 10 mL volumetric flask, dissolve them in methanol and dilute to the mark. Shake well to prepare mixed reference substance solutions with mass concentrations of 0.10152, 0.00208, 0.05144, 0.00216, 0.00202, and 0.00108 mg / mL, respectively, as mixed reference substance solutions. c. Determination: Accurately pipette 10 μL of the reference solution and the test solution respectively, inject them into the high performance liquid chromatograph, record the chromatogram within 80 minutes, and process the chromatogram with fingerprint software to obtain the fingerprint of the Mahuang Dingchuan Decoction composition.

3. The method for constructing an HPLC fingerprint of the Mahuang Dingchuan Decoction composition according to claim 2, characterized in that: The chromatographic conditions for the high performance liquid chromatography determination are as follows: octadecylsilane bonded silica gel as the filler, the chromatographic column is: Shimadzu C18 250×4.60 mm; acetonitrile is used as the mobile phase A, 0.1% formic acid solution is used as the mobile phase B, gradient elution, flow rate 1.0 mL / min, column temperature 30°C, detection wavelength 250 nm, and the theoretical plate number calculated based on the baicalin peak should be no less than 4000.

4. The method for constructing an HPLC fingerprint of the Mahuang Dingchuan Decoction composition according to any one of claims 1 to 3, characterized in that: The fingerprint has a total of 24 common peaks, among which chromatographic peaks 7, 10, 16, 17, 21 and 22 are respectively identified as ephedrine hydrochloride, scutellarin, baicalin, quercetin, baicalein and wogonin; according to the standard fingerprint conditions, the chromatographic peaks are attributed to nine medicinal materials, namely, ephedra, bitter almond, scutellaria, mulberry bark, ophiopogon, platycodon, coltsfoot flower, radish seed and ginkgo; Peak 1 belongs to Ginkgo biloba, Platycodon grandiflorum, Bitter almond, Coltsfoot flower, and Ephedra sinica, Peak 2 belongs to Coltsfoot flower and Morus alba bark, Peak 3 belongs to Coltsfoot flower and Radish seed, Peak 4 belongs to Coltsfoot flower and Morus alba bark, Peak 5 belongs to Coltsfoot flower, Peak 6 belongs to Ginkgo biloba and Coltsfoot flower, Peak 7 belongs to Ephedra sinica, Peak 8 belongs to Scutellaria baicalensis, Coltsfoot flower, and Radish seed, Peak 9 belongs to Scutellaria baicalensis and Coltsfoot flower, Peak 10 belongs to Scutellaria baicalensis, Peak 11 Peak No. 11 belongs to Coltsfoot, Peak No. 12 belongs to Platycodon grandiflorum, Peak No. 13 belongs to Coltsfoot, Peak No. 14 belongs to Ginkgo biloba and Apricot kernel, Peak No. 15 belongs to Coltsfoot, Peak No. 16 belongs to Scutellaria baicalensis, Peak No. 17 belongs to Scutellaria baicalensis, Peak No. 18 belongs to Scutellaria baicalensis, Peak No. 19 belongs to Ephedra, Peak No. 20 belongs to Scutellaria baicalensis, Peak No. 21 belongs to Scutellaria baicalensis, Peak No. 22 belongs to Scutellaria baicalensis, Peak No. 23 belongs to Scutellaria baicalensis, and Peak No. 24 belongs to Scutellaria baicalensis.

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