Fingerprint spectrum of Huanglong cough-relieving granules and construction method

Abstract

The invention discloses a fingerprint spectrum of Huanglong cough-relieving granules and a construction method, and relates to the technical field of pharmaceutical analysis. The construction method comprises the following steps: (1) preparation of a test solution: taking Huanglong cough-relieving granules, and adding an extraction solvent for extraction; (2) preparing a reference substance solution; (3) preparing a reference medicinal material solution; (4) chromatographic conditions: octadecylsilane chemically bonded silica is used as a filler; the mobile phase A is acetonitrile, the mobile phase B is 0.1-0.2% phosphoric acid, the flow rate is 0.38-0.42 ml / min, the column temperature is 29-31 DEG C, the detection wavelength is 210 nm in 0-17 min, and the detection wavelength is 270 nm in 17-48 min; and (5) absorbing the solution, and adding the solution into a liquid chromatograph for detection. The fingerprint spectrum constructed by the method can systematically reflect the material basis, is relatively good in precision, repeatability and stability, and can be used for quality control of the Huanglong cough-relieving granules.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical analysis, and particularly relates to a fingerprint spectrum of Huanglong Zhike granules and a construction method. BACKGROUND

[0002] As a commonly used traditional Chinese medicine in clinic, Huanglong Zhike granules has the effects of relieving cough and asthma, clearing lung and reducing phlegm, and is widely used for treating respiratory diseases such as common cold, cough, acute bronchitis and acute attack of chronic bronchitis, especially in children, the elderly and patients with weak constitution. The prescription of Huanglong Zhike granules is composed of multiple Chinese medicines such as Astragalus, Earthworm, Herba Epimedii, Platycodon, Belamcanda, Houttuynia cordata, Honey Ephedra, Crataegus and Semen Lepidii, which are complex in composition and rich in active substances. These active ingredients jointly form the basis of the efficacy of the medicine, but also bring challenges to the quality control. At present, the quality control of traditional Chinese medicine mainly relies on the “single component content determination” method specified in the standards such as Chinese Pharmacopoeia. For example, in the current quality standard of Huanglong Zhike granules, the content of baicalin or ephedrine is determined by high performance liquid chromatography (HPLC) to determine whether the product meets the specifications. However, this detection mode based on single or few components has significant limitations: (1) unable to comprehensively reflect the overall composition of the medicine: the efficacy of traditional Chinese medicine is due to the synergistic effect of multiple components, and the single component content cannot represent the overall quality of the medicine, which may result in large differences in efficacy and poor stability of different production batches of medicine; (2) difficult to identify quality problems: some enterprises may add single index components to meet the standard requirements, while ignoring the quality of other medicinal materials or the standardization of production process, which cannot effectively prevent the use of fake and inferior medicinal materials; (3) lack of evaluation basis for batch consistency: with the improvement of drug supervision requirements, the quality consistency between batches of traditional Chinese medicine has become a key to ensure the safety of drug use, but single component detection cannot provide enough characteristic information, and it is difficult to effectively monitor the stability of production process and the difference of medicinal materials.

[0003] Fingerprint spectrum technology, as an analysis method that can comprehensively characterize the chemical composition characteristics of traditional Chinese medicine, has been recognized as an important means of quality control of traditional Chinese medicine. This technology can reflect the existence and proportion of multiple active ingredients in the medicine by collecting the chromatographic or spectral spectrum of all detectable components in the medicine, and directly reflect the overall quality of the medicine; by comparing the similarity of fingerprint spectrum of different batches of medicine, it can be judged whether the production process is stable and the source of medicinal materials is consistent, and the efficacy fluctuation between batches is reduced.

[0004] Currently, fingerprinting technology has been successfully applied to the quality standards of various traditional Chinese medicines. However, there is still a significant gap in the research on the dedicated fingerprint spectrum of Huanglong Zhike Granules. Establishing a set of fingerprint spectra that can comprehensively characterize the component characteristics of Huanglong Zhike Granules, is stable and reliable, and can be widely applied is the key to solving its quality control bottleneck. Summary of the Invention

[0005] The purpose of this invention is to provide a fingerprint spectrum of Huanglong cough granules and its construction method.

[0006] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows: On one hand, the present invention provides a method for constructing the fingerprint spectrum of Huanglong cough granules, comprising the following steps: (1) Preparation of test solution: Take Huanglong cough granules and add extraction solvent for extraction; (2) Preparation of reference solution: Gallic acid, protocatechuic acid, pseudoephedrine hydrochloride, tryptophan, ephedrine hydrochloride, chlorogenic acid, irisin, quercetin, irisin, icariin, hyperoside, guanosine, irisin C, isoquercitrin, cryptochlorogenic acid, neochlorogenic acid, kaempferol-3-O-rutin, stigmoside, irisin B, irisin A, irisin B, irisin A1, and irisin are added to methanol to prepare reference solution; (3) Preparation of medicinal material solution: Take Astragalus membranaceus, earthworm, Epimedium, Platycodon grandiflorus, Belamcanda chinensis, Houttuynia cordata, Ephedra sinica, Crataegus pinnatifida and Lepidium apetalum, add extraction solvent for extraction; (4) Chromatographic conditions: Octadecylsilane bonded silica gel was used as the packing material; mobile phase A was acetonitrile, mobile phase B was 0.1-0.2% phosphoric acid for elution, flow rate was 0.38-0.42 ml / min, column temperature was 29-31℃, and detection wavelength was 210 nm for 0-17 min and 270 nm for 17-48 min. (5) Take the test solution, reference solution and reference medicinal material solution, add them to the liquid chromatograph for detection, and obtain the fingerprint spectrum of Huanglong Zhike Granules.

[0007] Specifically, the extraction solvent in step (1) is methanol or ethanol; Furthermore, the extraction solvent in step (1) is 10%-70% methanol or 10% ethanol; According to some embodiments of the present invention, the extraction solvent in step (1) is 10% methanol, 20% methanol, 30% methanol, 40% methanol, 50% methanol, 60% methanol, or 70% methanol.

[0008] Furthermore, the extraction solvent in step (1) is 10% methanol, 30% methanol, 50% methanol or 70% methanol; Furthermore, the extraction solvent in step (1) is 70% methanol.

[0009] Specifically, in step (1), the mass-volume ratio of Huanglong cough granules to extraction solvent is 2:5-45, with units of g:ml; According to some embodiments of the present invention, the mass-volume ratio of Huanglong cough granules to extraction solvent in step (1) is 2:5, 2:15, 2:25, 2:35, or 2:45; Furthermore, in step (1), the mass-to-volume ratio of Huanglong cough granules to the extraction solvent is 2:15, with units of g:ml.

[0010] Specifically, the extraction method in step (1) is ultrasonic extraction or heating reflux extraction; Furthermore, the conditions for ultrasonic extraction in step (1) are 500-700W and 40-50kHz; Furthermore, the conditions for ultrasonic extraction in step (1) are 600W and 45kHz.

[0011] Furthermore, in step (1), the heating and reflux conditions are 90℃ and extraction time is 50-70 min; Furthermore, in step (1), the heating and reflux conditions are 90°C and extraction time is 60 min.

[0012] Specifically, the extraction time in step (1) is 10-100 min; According to some embodiments of the present invention, the extraction time in step (1) is 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, 65 min, 70 min, 75 min, 80 min, 85 min, 90 min, 95 min, or 100 min.

[0013] Furthermore, the times extracted in step (1) are 10 min, 15 min, 20 min, 25 min, 40 min, 60 min, 80 min, and 100 min.

[0014] Furthermore, the time extracted in step (1) is 10 minutes.

[0015] Specifically, Huanglong cough granules are widely used for respiratory diseases, especially in children, the elderly, and patients with weak constitutions. The prescription consists of Astragalus membranaceus, earthworm, Epimedium, Platycodon grandiflorus, Belamcanda chinensis, Houttuynia cordata, Ephedra sinica, Crataegus pinnatifida, and Lepidium apetalum.

[0016] Specifically, the extraction solvent in step (3) is methanol; Furthermore, the extraction solvent in step (3) is 70% methanol; Specifically, in step (3), the mass-to-volume ratio of Astragalus membranaceus to the extraction solvent is 0.15-0.2:15, in g:ml; Furthermore, in step (3), the mass-to-volume ratio of Astragalus membranaceus to the extraction solvent is 0.18:15, in g:ml; Specifically, in step (3), the mass-to-volume ratio of earthworm to extraction solvent is 0.04-0.08:15, in g:ml; Furthermore, in step (3), the mass-to-volume ratio of earthworm to extraction solvent is 0.06:15, in g:ml; Specifically, in step (3), the mass-to-volume ratio of Epimedium to the extraction solvent is 0.1-0.3:15, in g:ml; Furthermore, in step (3), the mass-to-volume ratio of Epimedium to the extraction solvent is 0.2:15, in g:ml; Specifically, in step (3), the mass-to-volume ratio of Platycodon grandiflorus to the extraction solvent is 0.04-0.08:15, in g:ml; Furthermore, in step (3), the mass-to-volume ratio of Platycodon grandiflorus to the extraction solvent is 0.06:15, in g:ml; Specifically, in step (3), the mass-to-volume ratio of Belamcanda chinensis to the extraction solvent is 0.1-0.14:15, in g:ml; Furthermore, in step (3), the mass-to-volume ratio of Belamcanda chinensis to the extraction solvent is 0.12:15, in g:ml; Specifically, in step (3), the mass-to-volume ratio of Houttuynia cordata to the extraction solvent is 0.3-0.6:15, in g:ml; Furthermore, in step (3), the mass-to-volume ratio of Houttuynia cordata to the extraction solvent is 0.4:15, in g:ml; Specifically, in step (3), the mass-to-volume ratio of ephedra to extraction solvent is 0.04-0.08:15, in g:ml; Furthermore, in step (3), the mass-to-volume ratio of ephedra to extraction solvent is 0.06:15, in g:ml; Specifically, in step (3), the mass-to-volume ratio of hawthorn to extraction solvent is 0.2-0.4:15, in g:ml; Furthermore, in step (3), the mass-to-volume ratio of hawthorn to extraction solvent is 0.3:15, in g:ml; Specifically, in step (3), the mass-to-volume ratio of Lepidium apetalum to the extraction solvent is 0.1-0.14:15, in g:ml; Further, the mass-volume ratio of Platycladi semen to the extraction solvent in step (3) is 0.12:15, unit: g:ml.

[0017] Specifically, the extraction method in step (3) is ultrasonic extraction method. Further, the ultrasonic extraction condition in step (3) is 500-700W, 40-50kHz. Still further, the ultrasonic extraction condition in step (3) is 600W, 45kHz.

[0018] Specifically, the extraction time in step (3) is 10-100min. Still further, the extraction time in step (3) is 10min.

[0019] Specifically, after ultrasonic extraction in step (3), 70% ethanol is added to make up the reduced weight, and the filtrate is taken after filtration.

[0020] Specifically, the column length of the packed column in step (4) is 100mm, the inner diameter is 2.1mm, and the particle size is 1.8μm.

[0021] Preferably, the chromatographic conditions in step (4) are: octadecylsilane bonded silica gel as the filler; the mobile phase A is acetonitrile, the mobile phase B is 0.2% phosphoric acid for elution, the flow rate is 0.40ml / min, the column temperature is 30℃, and the detection wavelength is 210nm for 0-17min and 270nm for 17-48min.

[0022] Specifically, the elution conditions in step (4) are: 0min, mobile phase A: 0%, mobile phase B: 100%; 1min, mobile phase A: 0%, mobile phase B: 100%; 7min, mobile phase A: 7%, mobile phase B: 93%; 14min, mobile phase A: 7%, mobile phase B: 93%; 17min, mobile phase A: 17%, mobile phase B: 83%; 21min, mobile phase A: 17%, mobile phase B: 83%; 28min, mobile phase A: 26%, mobile phase B: 74%; 32min, mobile phase A: 26%, mobile phase B: 74%; 38min, mobile phase A: 39%, mobile phase B: 61%; 43min, mobile phase A: 39%, mobile phase B: 61%; 43.1min, mobile phase A: 0%, mobile phase B: 100%; 48 min, mobile phase A: 0%, mobile phase B: 100%.

[0023] Specifically, in step (4), 1 μl of the control sample solution, the medicinal material reference solution and the sample solution are injected into the liquid chromatograph for determination.

[0024] Specifically, the chromatographic detector in step (4) is a THERMO UHPLC ultra-high performance liquid chromatograph.

[0025] Specifically, the chromatographic column in step (4) is a Waters ACQUITY UPLC ® HSS T3.

[0026] Specifically, the fingerprint spectrum contains 32 common peaks, and the relative retention times of the common peaks 1-15 are as follows: peak 1: 0.246-0.248, peak 2: 0.284-0.286, peak 3: 0.367-0.374, peak 4: 0.477-0.480, peak 5: 0.511-0.514, peak 6: 0.598-0.605, peak 7: 0.799-0.802, peak 8: 0.856-0.858, peak 9: 1.000, peak 10: 1.049-1.051, peak 11: 1.050-1.143, peak 12: 1.252-1.255, peak 13: 1.534-1.537, peak 14: 1.608-1.613, and peak 15: 1.812-1.817, with peak 9 as the reference peak.

[0027] In another aspect, the present application provides a fingerprint spectrum of the Huanglong Cough-Relieving Granules prepared by the above-mentioned construction method.

[0028] Specifically, the fingerprint contains 32 common peaks, with peak No. 9 as the reference peak, and the relative retention times of common peaks No. 1-15 are as follows: peak No. 1: 0.246-0.248, peak No. 2: 0.284-0.286, peak No. 3: 0.367-0.374, peak No. 4: 0.477-0.480, peak No. 5: 0.511-0.514, peak No. 6: 0.598-0.605, peak No. 7: 0.799-0.802, peak No. 8: 0.856-0.858, peak No. 9: 1.000, peak No. 10: 1.049-1.051, peak No. 11: 1.050-1.143, peak No. 12: 1.252-1.255, peak No. 13: 1.534-1.537, peak No. 14: 1.608-1.613, and peak No. 15: 1.812-1.817. With peak No. 27 as the reference peak, the relative retention times of common peaks No. 16-32 are as follows: peak No. 16: 0.653-0.654, peak No. 17: 0.665-0.666, peak No. 18: 0.696-0.697, peak No. 19: 0.719-0.808, peak No. 20: 0.745-0.745, peak No. 21: 0.766-0.767, peak No. 22: 0.772-0.773, peak No. 23: 0.929-0.929, peak No. 24: 0.947-0.947, peak No. 25: 0.963-0.963, peak No. 26: 0.983-0.983, peak No. 27: 1.000, peak No. 28: 1.023-1.023, peak No. 29: 1.084-1.085, peak No. 30: 1.206-1.207, peak No. 31: 1.250-1.251, and peak No. 32: 1.257-1.259.

[0029] The present application has the following advantages: The present application provides a method for constructing a fingerprint of Huanglong Cough Granules, and the constructed fingerprint can systematically reflect the material basis. The similarity of the fingerprints of 15 batches of commercially available Huanglong Cough Granules is greater than the specified lower limit of 0.900 and greater than 0.956, indicating that the fingerprint can be used as a common feature for the quality control of Huanglong Cough Granules, and the established method has good precision, repeatability and stability. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 UPLC chromatograms of Huanglong Cough Granules under different extraction solvents (10% methanol, 30% methanol, 50% methanol, 70% methanol, and methanol).

[0031] Figure 2 UPLC chromatograms of Huanglong Cough Granules under different extraction solvents (10% ethanol, 30% ethanol, 50% ethanol, 70% ethanol, and ethanol).

[0032] Figure 3 UPLC chromatogram of Huanglong Cough-Relieving Granules under ultrasonic extraction with different solvent volumes.

[0033] Figure 4 UPLC chromatogram of Huanglong Cough-Relieving Granules under heating reflux extraction with different solvent volumes.

[0034] Figure 5 UPLC chromatogram of Huanglong Cough-Relieving Granules under different extraction times (20 min, 40 min, 60 min, 80 min, 100 min).

[0035] Figure 6 UPLC chromatogram of Huanglong Cough-Relieving Granules under different extraction times (10 min, 15 min, 20 min, 25 min).

[0036] Figure 7 UPLC chromatogram of Huanglong Cough-Relieving Granules under different mobile phases (methanol-water, ethanol-water).

[0037] Figure 8 UPLC chromatogram of Huanglong Cough-Relieving Granules under different mobile phases (methanol-0.1% phosphoric acid solution, ethanol-0.1% phosphoric acid solution).

[0038] Figure 9 UPLC chromatogram of Huanglong Cough-Relieving Granules under different mobile phases (ethanol-0.1% phosphoric acid solution, 0.1% formic acid solution, 0.1% acetic acid solution).

[0039] Figure 10 UPLC chromatogram of Huanglong Cough-Relieving Granules under different acetonitrile-water conditions with different phosphoric acid concentrations.

[0040] Figure 11 UPLC chromatogram of Huanglong Cough-Relieving Granules under different column temperatures (25℃, 28℃, 30℃, 32℃, 35℃).

[0041] Figure 12 UPLC chromatogram of Huanglong Cough-Relieving Granules under different column temperatures (20℃, 30℃, 31℃).

[0042] Figure 13 UPLC chromatogram of Huanglong Cough-Relieving Granules under different column temperatures (32℃, 33℃, 34℃).

[0043] Figure 14 UPLC chromatogram of Huanglong Cough-Relieving Granules under different flow rates (0.20 ml / min, 0.25 ml / min, 0.30 ml / min).

[0044] Figure 15 UPLC chromatogram of Huanglong Cough-Relieving Granules under different flow rates (0.35 ml / min, 0.40 ml / min, 0.45 ml / min).

[0045] Figure 16 UPLC chromatogram of Huanglong Cough-Relieving Granules under different flow rates (0.36 ml / min, 0.37 ml / min, 0.38 ml / min, 0.39 ml / min, 0.40 ml / min).

[0046] Figure 17 UPLC chromatogram of Huanglong Cough-Relieving Granules under different flow rates (0.41 ml / min, 0.42 ml / min, 0.43 ml / min, 0.44 ml / min).

[0047] Figure 18 UPLC chromatogram of Huanglong Cough-Relieving Granules under different instruments.

[0048] Figure 19 UPLC chromatogram of Huanglong Cough-Relieving Granules under different types of chromatographic columns.

[0049] Figure 20 UPLC chromatogram of Huanglong Cough-Relieving Granules under different types of chromatographic columns.

[0050] Figure 21 UPLC control fingerprint chromatogram of 15 batches of commercially available Huanglong Cough-Relieving Granules.

[0051] Figure 22 UPLC superimposed chromatogram of 15 batches of commercially available Huanglong Cough-Relieving Granules under the preferred chromatographic conditions of the present application. DETAILED DESCRIPTION

[0052] In order to make the technical means, creative features, purposes and effects achieved by the present application easy to understand, the following specific embodiments are further described, but the following embodiments are only preferred embodiments of the present application, not all. Based on the embodiments in the embodiments, other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present application. In the following examples, if not otherwise specified, the operation methods used are conventional operation methods, and the equipment used is conventional equipment, and the equipment materials used in each example are the same.

[0053] Example 1 Huanglong Cough-Relieving Granules has the effects of benefiting qi and tonifying kidney, clearing lung and resolving phlegm, relieving cough and asthma. It is used for cough and asthma due to deficiency of lung and kidney and accumulation of phlegm-heat in lung, as well as acute and chronic bronchitis and bronchial asthma with the above-mentioned symptoms.

[0054] The prescription of Huanglong Cough-Relieving Granules is: Radix Astragali 90 g, Pheretima 30 g, Herba Epimedii 100 g, Platycodonis 30 g, Belamcanda chinensis 60 g, Houttuynia cordata 200 g, Ephedra sinica 30 g, Crataegus pinnatifida 150 g, and Semen Lepidii 60 g.

[0055] The preparation method of Huanglong Cough-Relieving Granules is: the above nine ingredients are added with water to decoct twice, 12 times the amount of water is added in the first time, and 8 times the amount of water is added in the second time, 2 hours each time, the decoction is filtered, the filtrate is concentrated to a clear paste with a relative density of 1.15-1.20 (60℃), the right amount of sucrose: dextrin (2.75:1) is added, granulated, and 1000 g is obtained.

[0056] I. Optimization of fingerprint method 1. Instruments and reagents THERMO UHPLC ultra-high performance liquid chromatograph (Thermo Fisher Scientific (China) Co., Ltd.); THERMO Vanquish ultra-high performance liquid chromatograph (Thermo Fisher Scientific (China) Co., Ltd.); Waters HPLC Hclass ultra-high performance liquid chromatograph (Waters Technology Co., Ltd.); METTLER XSE205DU electronic balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.); MilliQ ultrapure water instrument (Millipore); Constant temperature water bath HH-S6 (Zhengzhou Great Wall Scientific Industrial & Trading Co., Ltd.); SY8200T ultrasonic cleaner (Shanghai Sound Source Ultrasonic Instrument Equipment Co., Ltd.).

[0057] Acetonitrile was chromatographically pure (National Pharmaceutical Group Chemical Reagent Co., Ltd., Dima Technology Co., Ltd.); methanol was chromatographically pure (National Pharmaceutical Group Chemical Reagent Co., Ltd.); phosphoric acid was analytically pure (National Pharmaceutical Group Chemical Reagent Co., Ltd.); acetic acid was analytically pure (National Pharmaceutical Group Chemical Reagent Co., Ltd.); formic acid was analytically pure (National Pharmaceutical Group Chemical Reagent Co., Ltd.); anhydrous ethanol was analytically pure (National Pharmaceutical Group Chemical Reagent Co., Ltd.).

[0058] 2. Source of reference substance and sample The reference substance information is shown in Table 1.

[0059] Table 1 Reference substance information

[0060] The test sample information is Huanglong Cough-Relieving Granules, Shaanxi Dongke Pharmaceutical Co., Ltd., batch number: 20250204.

[0061] 3. Experimental method (initial conditions) 3.1 Chromatographic conditions Octadecylsilane-bonded silica gel as the filler (column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm); mobile phase acetonitrile (A)-0.1% phosphoric acid (B), flow rate 0.4 ml / min, column temperature 30℃, detection wavelength 0-17 min 210 nm, 17-48 min 270 nm, elution according to the following table: Table 2

[0062] 3.2 Preparation of control solution ① Gallic acid control solution: accurately weigh a certain amount of gallic acid control, add methanol to make a solution containing 35 μg per 1 ml, and obtain.

[0063] ② Protocatechuic acid control solution: accurately weigh a certain amount of protocatechuic acid control, add methanol to make a solution containing 40 μg per 1 ml, and obtain.

[0064] ③ Pseudoephedrine hydrochloride control solution: accurately weigh a certain amount of pseudoephedrine hydrochloride control, add methanol to make a solution containing 40 μg per 1 ml, and obtain.

[0065] ④ Tryptophan control solution: accurately weigh a certain amount of tryptophan control, add methanol to make a solution containing 40 μg per 1 ml, and obtain.

[0066] ⑤ Ephedrine hydrochloride control solution: accurately weigh a certain amount of ephedrine hydrochloride control, add methanol to make a solution containing 40 μg per 1 ml, and obtain.

[0067] ⑥ Chlorogenic acid control solution: accurately weigh a certain amount of chlorogenic acid control, add methanol to make a solution containing 40 μg per 1 ml, and obtain.

[0068] ⑦ Cryptochlorogenic acid control solution: accurately weigh a certain amount of cryptochlorogenic acid control, add methanol to make a solution containing 40 μg per 1 ml, and obtain.

[0069] ⑧ Belamcandin control solution: accurately weigh a certain amount of belamcandin control, add methanol to make a solution containing 40 μg per 1 ml, and obtain.

[0070] ⑨ Kaempferol-3-O-rutinoside control solution: accurately weigh a certain amount of kaempferol-3-O-rutinoside control, add methanol to make a solution containing 40 μg per 1 ml, and obtain.

[0071] ⑩ Quercitrin control solution: accurately weigh a certain amount of quercitrin control, add methanol to make a solution containing 40 μg per 1 ml, and obtain.

[0072] Hypogallic acid control solution: take the appropriate amount of hypogallic acid control, accurately weighed, add methanol to make 40 μg per 1 ml of solution, that is.

[0073] Icaritin control solution: take the appropriate amount of icaritin control, accurately weighed, add methanol to make 40 μg per 1 ml of solution, that is.

[0074] Neochlorogenic acid control solution: take the appropriate amount of neochlorogenic acid control, accurately weighed, add methanol to make 45 μg per 1 ml of solution, that is.

[0075] Guanosine control solution: take the appropriate amount of guanosine control, accurately weighed, add methanol to make 45 μg per 1 ml of solution, that is.

[0076] Hyperoside control solution: take the appropriate amount of hyperoside control, accurately weighed, add methanol to make 45 μg per 1 ml of solution, that is.

[0077] Isoquercitrin control solution: take the appropriate amount of isoquercitrin control, accurately weighed, add methanol to make 45 μg per 1 ml of solution, that is.

[0078] Davion C control solution: take the appropriate amount of davion C control, accurately weighed, add methanol to make 45 μg per 1 ml of solution, that is.

[0079] Davion A control solution: take the appropriate amount of davion A control, accurately weighed, add methanol to make 55 μg per 1 ml of solution, that is.

[0080] Davion A1 control solution: take the appropriate amount of davion A1 control, accurately weighed, add methanol to make 60 μg per 1 ml of solution, that is.

[0081] Davion B control solution: take the appropriate amount of davion B control, accurately weighed, add methanol to make 60 μg per 1 ml of solution, that is.

[0082] Iridin control solution: take the appropriate amount of iridin control, accurately weighed, add methanol to make 60 μg per 1 ml of solution, that is.

[0083] Iridin B control solution: take the appropriate amount of iridin B control, accurately weighed, add methanol to make 75 μg per 1 ml of solution, that is.

[0084] 3.3 Preparation of reference medicinal material and decoction piece solution ①Radix Astragali: Take Radix Astragali decoction piece powder (pass through No. 2 sieve), about 0.18 g, accurately weigh and place in a conical flask with a stopper. Accurately add 70% methanol 15 ml, weigh, ultrasonic treatment (power 600 W, frequency 45 kHz) for 10 minutes, cool, re-weigh, make up the weight loss with 70% methanol, shake well, filter, and take the filtrate, which is obtained.

[0085] ②Earthworm: Take earthworm medicinal material powder (pass through No. 2 sieve), about 0.06 g, accurately weigh and place in a conical flask with a stopper. Accurately add 70% methanol 15 ml, weigh, ultrasonic treatment (power 600 W, frequency 45 kHz) for 10 minutes, cool, re-weigh, make up the weight loss with 70% methanol, shake well, filter, and take the filtrate, which is obtained.

[0086] ③Herba Epimedii: Take Herba Epimedii medicinal material powder (pass through No. 2 sieve), about 0.20 g, accurately weigh and place in a conical flask with a stopper. Accurately add 70% methanol 15 ml, weigh, ultrasonic treatment (power 600 W, frequency 45 kHz) for 10 minutes, cool, re-weigh, make up the weight loss with 70% methanol, shake well, filter, and take the filtrate, which is obtained.

[0087] ④Plumula Nelumbinis: Take Plumula Nelumbinis decoction piece powder (pass through No. 2 sieve), about 0.06 g, accurately weigh and place in a conical flask with a stopper. Accurately add 70% methanol 15 ml, weigh, ultrasonic treatment (power 600 W, frequency 45 kHz) for 10 minutes, cool, re-weigh, make up the weight loss with 70% methanol, shake well, filter, and take the filtrate, which is obtained.

[0088] ⑤Belamcanda Chinensis: Take Belamcanda Chinensis decoction piece powder (pass through No. 2 sieve), about 0.12 g, accurately weigh and place in a conical flask with a stopper. Accurately add 70% methanol 15 ml, weigh, ultrasonic treatment (power 600 W, frequency 45 kHz) for 10 minutes, cool, re-weigh, make up the weight loss with 70% methanol, shake well, filter, and take the filtrate, which is obtained.

[0089] ⑥Houttuynia Cordata: Take Houttuynia Cordata medicinal material powder (pass through No. 2 sieve), about 0.40 g, accurately weigh and place in a conical flask with a stopper. Accurately add 70% methanol 15 ml, weigh, ultrasonic treatment (power 600 W, frequency 45 kHz) for 10 minutes, cool, re-weigh, make up the weight loss with 70% methanol, shake well, filter, and take the filtrate, which is obtained.

[0090]

[0091]

[0092]

[0093] 3.4 Preparation of Test Solution Take Huanglong Zhike Granules, grind finely, take about 2g, accurately weigh, place in a conical flask with a stopper, add 70% methanol 25ml, weigh, ultrasonic treatment (power 600W, frequency 45kHz) for 60 minutes, cool, reweigh, make up the weight loss with 70% methanol, shake well, filter, take the filtrate, and get it.

[0094] 3.5 Determination Method Accurately take 1ul of the control solution, medicinal material, reference solution and test solution respectively, inject into the liquid chromatograph, and determine, and get it.

[0095] 4. Investigation of Extraction Solvent Take Huanglong Zhike Granules, grind finely, take about 2g, accurately weigh, divide into 10 parts, add 10% methanol, 30% methanol, 50% methanol, 70% methanol, methanol, 10% ethanol, 30% ethanol, 50% ethanol, 70% ethanol, and ethanol respectively, and prepare the test solution according to the conditions in item 3.4. Determine according to the chromatographic conditions in item 3.1, and the results are shown in Table 1. Figures 1-2 .

[0096] ​​​The experimental results show that when the extraction solvent is 30% ethanol, 50% ethanol, 70% ethanol and ethanol, the chromatographic peak at 1.5-2.5 min in the chromatogram of the test solution has a poor peak shape; when the extraction solvent is methanol, the chromatographic peak at 26.063 min in the chromatogram of the test solution has a poor peak shape; when the extraction solvent is 10% methanol, 30% methanol, 50% methanol, 70% methanol and 10% ethanol, the chromatographic peaks in the chromatogram of the test solution have no obvious difference; after comprehensive comparison and analysis, the sample extracted by 70% methanol solution has the best chromatographic effect.

[0097] 5. Extraction solvent volume investigation Take an appropriate amount of Huanglong Cough Granules, grind finely, take about 2 g, accurately weigh, 5 parallel, respectively 70% methanol 5 ml, 15 ml, 25 ml, 35 ml, 45 ml, according to the conditions of the test solution under "3.4" preparation. According to the chromatographic conditions under "3.1", the results are shown in Figure 3 .

[0098] The experimental results show that when the extraction solvent volume is 5-45 ml, the number of chromatographic peaks and the peak shape in the chromatogram of the test solution have no obvious difference. Therefore, the extraction solvent volume of 5-45 ml can achieve effective extraction. Considering the operability of the experiment, 15 ml is preferred as the extraction solvent volume of Huanglong Cough Granules.

[0099] 6. Extraction method investigation Take an appropriate amount of Huanglong Cough Granules, grind finely, take about 2 g, accurately weigh, 5 parallel, respectively 70% methanol 5 ml, 15 ml, 25 ml, 35 ml, 45 ml, heated reflux treatment 60 min, preparation of test solution, according to the chromatographic conditions under "3.1", record the chromatogram. The results are shown in Figure 4 .

[0100] The experimental results show that 5 ml, 15 ml, 25 ml, 35 ml, 45 ml chromatography has no obvious abnormality. Combined with the chromatogram of the extraction solvent investigation experiment, the extraction solvent volume is consistent, and the chromatogram of the heating reflux and ultrasonic extraction is basically the same. Considering that the extraction solvent is 5 ml, the experimental process is not easy to filter, 15 ml is preferred as the extraction solvent volume of the test product. When the extraction solvent is 15 ml, the chromatographic peaks corresponding to ultrasonic and heating reflux have little difference, and ultrasonic is preferred as the extraction method of the test product.

[0101] 7. Extraction time investigation Take Huanglong Zhike Granules appropriate amount, grind, take about 2g, accurately weigh, add 70% methanol 15ml, ultrasonic treatment (power 600W, frequency 45kHz) 10min, cool, reweigh, with 70% methanol to make up the loss, shake up, filter, take the filter liquor, namely. According to the chromatographic conditions under "3.1", the influence of different types of elution system on the chromatogram of Huanglong Zhike Granules was investigated. The results are as follows Figures 5-6 .

[0102] The experimental results show that when the extraction time is 10min-100min, the number of chromatographic peaks, peak type and peak area in the chromatogram of the test solution have no obvious difference. Therefore, the extraction time of 10min-100min can realize effective extraction, and 10min is preferred as the extraction time of Huanglong Zhike Granules.

[0103] 8. Mobile phase investigation 8.1 Different types of elution system Take Huanglong Zhike Granules appropriate amount, grind, take about 2g, accurately weigh, add 70% methanol 15ml, ultrasonic treatment (power 600W, frequency 45kHz) 10min, cool, reweigh, with 70% methanol to make up the loss, shake up, filter, take the filter liquor, namely. According to the chromatographic conditions under "3.1", the influence of different types of elution system on the chromatogram of Huanglong Zhike Granules was investigated. The results are as follows Figures 7-8 .

[0104] Table 3 Different types of elution system

[0105] The experimental results show that when the extraction time is 10min-100min, the number of chromatographic peaks, peak type and peak area in the chromatogram of the test solution have no obvious difference. Therefore, the extraction time of 10min-100min can realize effective extraction, and 10min is preferred as the extraction time of Huanglong Zhike Granules.

[0106] 8.2 Acid type investigation Take Huanglong Zhike Granules, grind finely, take about 2g, accurately weigh, add 70% methanol 15ml, ultrasonic treatment (power 600W, frequency 45kHz) 10min, cool, re-weigh, make up the lost amount with 70% methanol, shake well, filter, take the filtrate, and get it. According to the chromatographic conditions under item "3.1" except the mobile phase, the effects of different proportions of phosphoric acid concentration on the chromatogram of Huanglong Zhike Granules in Table 5 were investigated, and the results were as follows Figure 9 .

[0107] Table 4 Different types of acids

[0108] Result analysis: When the mobile phase is acetonitrile-0.1% acetic acid, acetonitrile-0.1% formic acid, the baseline fluctuation of the test solution chromatogram is more obvious within 0-18min. Therefore, acetonitrile-phosphoric acid aqueous solution is selected as the mobile phase of Huanglong Zhike Granules.

[0109] 8.3 Investigation of phosphoric acid concentration Take Huanglong Zhike Granules, grind finely, take about 2g, accurately weigh, add 70% methanol 15ml, ultrasonic treatment (power 600W, frequency 45kHz) 10min, cool, re-weigh, make up the lost amount with 70% methanol, shake well, filter, take the filtrate, and get it. According to the chromatographic conditions under item "3.1" except the mobile phase, the effects of different proportions of phosphoric acid concentration on the chromatogram of Huanglong Zhike Granules in Table 5 were investigated, and the results were as follows Figure 10 .

[0110] Table 5 Different proportions of phosphoric acid concentration

[0111] Result analysis: Compared with the mobile phase of acetonitrile-0.1% phosphoric acid solution, when the mobile phase is acetonitrile-0.05% phosphoric acid solution, acetonitrile-0.3% phosphoric acid solution, the number of chromatographic peaks in the test solution chromatogram is relatively small, and the peak type at 10.9min and 15.4min is relatively poor; when the mobile phase is acetonitrile-0.2% phosphoric acid solution, the number of chromatographic peaks in the test solution chromatogram is basically the same, and the peak type is better than that of acetonitrile-0.1% phosphoric acid solution. Therefore, 0.2% phosphoric acid solution is selected as the water phase of the mobile phase of Huanglong Zhike Granules.

[0112] In summary, the method of Huanglong Zhike Granules is as follows: Chromatographic conditions: Octadecylsilane bonded silica gel was used as the packing material (column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm); the mobile phase was acetonitrile (A)-0.2% phosphoric acid (B), the flow rate was 0.4 ml / min, the column temperature was 30℃, the detection wavelength was 210 nm at 0-17 min and 270 nm at 17-48 min, and the elution was performed according to the following table: Table 6

[0113] Preparation of test solution: a certain amount of Huanglong Zhike granules was finely ground, about 2 g was accurately weighed into a conical flask with a plug, 70% methanol 15 ml was added, the weight was determined, ultrasonic treatment (power 600 W, frequency 45 kHz) was performed for 10 minutes, the flask was cooled, the weight was determined again, 70% methanol was added to make up for the weight loss, shaken well, filtered, and the filtrate was taken, which was obtained.

[0114] Determination method: 1 μl of the test solution was accurately taken and injected into the liquid chromatograph for determination.

[0115] 9. Column temperature investigation After the elution system, the type of acid, and the concentration of phosphoric acid were determined, the influence of different column temperatures (29℃, 30℃, 31℃, 32℃, 33℃, 34℃) on the UPLC chromatogram of Huanglong Zhike granules was investigated, and the results are shown in Figures 11-13 .

[0116] Result analysis: in the chromatograms obtained at different column temperatures, when the column temperature was 32℃, the separation effect of the chromatographic peaks at 34.2 min and 34.5 min was not good; when the column temperature was 33℃ and 34℃, the chromatographic peak at 34.9 min was less than that corresponding to the chromatogram at 30℃; when the column temperature was 29℃, 30℃, and 31℃, the chromatographic peaks were evenly distributed and the separation effect was good. When the column temperature was 30℃, the separation effect of the chromatographic peaks was better.

[0117] 10. Flow rate investigation After the elution system, the type of acid, the concentration of phosphoric acid, and the column temperature were determined, the influence of different flow rates (0.36 ml / min, 0.37 ml / min, 0.38 ml / min, 0.39 ml / min, 0.40 ml / min, 0.41 ml / min, 0.42 ml / min, 0.43 ml / min, 0.44 ml / min) on the UPLC chromatogram of Huanglong Zhike granules was investigated, and the results are shown in Figures 14-17 .

[0118] Result analysis: in the chromatogram obtained by using different flow rates, when the flow rate is 0.36 ml / min, 0.37 ml / min, 0.43 ml / min, 0.44 ml / min, the separation effect of part of the chromatographic peaks is poor. When the flow rate is 0.38 ml / min-0.42 ml / min, the chromatographic peaks are uniformly distributed and well separated. And when the flow rate is 0.40 ml / min, the separation effect of the chromatographic peaks is better.

[0119] 10. Instrument investigation After determining the elution system, the type of acid, the concentration of phosphoric acid, the column temperature and the flow rate, the influence of different instruments on the UPLC chromatogram of Huanglong Cough-Relieving Granules was investigated, and the results were as follows Figure 18 .

[0120] Result analysis: in the chromatogram obtained by using different instruments, when the instrument is THERMO Vanquish, the separation effect of the chromatographic peak at 3.9 min is poor. When the instrument is Waters HPLC Hclass, the baseline of the chromatographic peaks at 15.8 min and 16.7 min is uneven. When the instrument is THERMO UHPLC, the chromatographic peaks are uniformly distributed and well separated. Therefore, THERMO UHPLC ultra-high performance liquid chromatograph is preferred for the determination of Huanglong Cough-Relieving Granules.

[0121] 10. Column investigation After determining the elution system, the type of acid, the concentration of phosphoric acid, the column temperature and the instrument, the influence of different chromatographic columns on the UPLC chromatogram of Huanglong Cough-Relieving Granules was investigated, and the results were as follows Figures 19-20 .

[0122] Table 7

[0123] Result analysis: in the chromatogram obtained by using different types of chromatographic columns, compared with Waters ACQUITY UPLC HSS T3 chromatographic column, the separation effect of the chromatographic peak at 1.87 min in the chromatogram of the test sample was poor when using Waters UPLC CSH C18 type chromatographic column; the separation effect of the chromatographic peak at 3.21 min in the chromatogram of the test sample was poor when using Agilent ZORBAX RRHD SB-C18 type chromatographic column; the separation effect of the chromatographic peaks at 1.56 min, 1.69 min, 8.18 min and 8.30 min in the chromatogram of the test sample was poor when using Waters ACQOITY UPLC BEH C18 type chromatographic column; the separation effect of the chromatographic peaks at 6.94 min, 10.81 min and 11.42 min in the chromatogram of the test sample was poor when using Waters CORTECS UPLC T3 type chromatographic column.

[0124] The chromatographic peaks of the four same model chromatographic columns were evenly distributed and well separated in the chromatogram obtained using the Waters ACQUITY UPLC® HSS T3 same model chromatographic column. Therefore, the Waters ACQUITY UPLC® HSS T3 chromatographic column is preferred for the determination of Huanglong Zhike Granules.

[0125] 11. Chromatographic peak positioning Take Huanglong Zhike Granules, grind finely, take about 2 g, accurately weigh, add 70% methanol 15 ml, ultrasonic treat (power 600 W, frequency 45 kHz) for 10 min, cool, re-weigh, make up the lost amount with 70% methanol, shake well, filter, take the filtrate, and obtain. According to the chromatographic conditions under item "3.1" except the mobile phase, the mobile phase is acetonitrile (A)-0.2% phosphoric acid (B), accurately pipette 1 μl of each of the control solution, each of the reference medicinal material solution and the test sample solution, inject into the liquid chromatograph, and determine. The results are shown in Tables 8-9.

[0126] Table 8 Chromatographic peak positioning attribution table of Huanglong Zhike Granules

[0127] Table 9 Chromatographic peak positioning attribution table of Huanglong Zhike Granules

[0128] II. Methodology verification 1. Precision investigation Accurately pipette 1 μl of the same test sample solution, inject into the liquid chromatograph, and determine continuously for 6 times. Take peak No. 9 (ephedrine hydrochloride) as S1 peak and peak No. 27 (icariin) as S2 peak, and calculate the relative retention time and relative peak area of other peaks. The relative retention time and relative peak area of other peaks are all <2.0%, indicating that the instrument precision is good, as shown in Tables 10-13: Table 10 Relative retention time of Huanglong Zhike Granules for precision

[0129] Table 11 Relative retention time of Huanglong Zhike Granules for precision

[0130] Table 12 Relative peak area of Huanglong Zhike Granules for precision

[0131] Table 13 Relative peak area of Huanglong Zhike Granules for precision

[0132] 2. Reproducibility investigation Take the same test sample, parallel weighing 6, precision weighing, respectively, add 70% methanol 15 ml, ultrasonic treatment (power 600 W, frequency 45 kHz) 10 min, cool, reweigh, with 70% methanol to make up for the loss, shake, filter, take the filtrate, that is. Under the chromatographic conditions of "3.1", except for the mobile phase, the mobile phase is acetonitrile (A)-0.2% phosphoric acid (B). Peak 9 (ephedrine hydrochloride) is S1 peak, peak 27 (icariin) is S2 peak, and the relative retention time and relative retention peak area of other peaks are all less than 3.0%, indicating that the repeatability of the method is good, see Tables 14-17: Table 14 Relative retention time of Huanglong Cough Granules

[0133] Table 15 Relative retention time of Huanglong Cough Granules

[0134] Table 16 Relative peak area of Huanglong Cough Granules

[0135] Table 17 Relative peak area of Huanglong Cough Granules

[0136] 3. Stability test Precisely take the same test sample solution, respectively, at 0h, 2h, 4h, 6h, 8h, 12h, 18h, 24h, 36h, 48h, take peak 9 (ephedrine hydrochloride) as S1 peak, peak 27 (icariin) as S2 peak, calculate the relative retention time and relative retention peak area of other peaks, all less than 5.0%, indicating that the solution is stable within 48h, see Tables 18-21: Table 18 Relative retention time of Huanglong Cough Granules

[0137] Table 19 Relative retention time of Huanglong Cough Granules

[0138] Table 20 Relative peak area of Huanglong Cough Granules

[0139] Table 21 Relative peak area of Huanglong Cough Granules

[0140] The UPLC chromatograms of the test solution prepared from 15 batches of commercially available Huanglong Zhike Granules (purchased from Shaanxi Dongke Pharmaceutical Co., Ltd.) were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" for processing, multi-point correction and peak matching were performed Figure 21 . The UPLC superimposed chromatograms of the 15 batches of commercially available Huanglong Zhike Granules are shown in Figure 22 . The similarity of the chromatograms of each batch of sample to the control fingerprint was calculated, and the similarity evaluation results are shown in Table 22. The relative peak areas are shown in Tables 23-26.

[0141] Table 22 Similarity evaluation results of commercially available Huanglong Zhike Granules (15 batches)

[0142] Table 23 Relative peak areas of commercially available Huanglong Zhike Granules (15 batches)

[0143] Table 24 Relative peak areas of commercially available Huanglong Zhike Granules (15 batches)

[0144] Table 25 Relative peak areas of commercially available Huanglong Zhike Granules (15 batches)

[0145] Table 26 Relative peak areas of commercially available Huanglong Zhike Granules (15 batches)

[0146] Results analysis: The similarity of the fingerprints of the 15 batches of commercially available Huanglong Zhike Granules was all greater than the specified lower limit of 0.900, and was all greater than 0.956, indicating that the fingerprint can be used as a common feature for the quality control of Huanglong Zhike Granules.

[0147] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for constructing a fingerprint spectrum of Huanglong Cough-Relieving Granules, characterized in that, Includes the following steps: (1) Preparation of test solution: Take Huanglong cough granules and add extraction solvent for extraction; (2) Preparation of reference solutions: Gallic acid, protocatechuic acid, pseudoephedrine hydrochloride, tryptophan, ephedrine hydrochloride, chlorogenic acid, irisin, quercetin, irisin, icariin, hyperoside, guanosine, irisin C, isoquercetin, cryptochlorogenic acid, neochlorogenic acid, kaempferol-3-O-rutin, stigmoside, irisin B, irisin A, irisin B, irisin A1, and irisin were respectively added to methanol to prepare reference solutions. (3) Preparation of medicinal material solution: Take Astragalus membranaceus, earthworm, Epimedium, Platycodon grandiflorus, Belamcanda chinensis, Houttuynia cordata, Ephedra sinica, Crataegus pinnatifida and Lepidium apetalum, and add extraction solvent to each for extraction; (4) Chromatographic conditions: Octadecylsilane bonded silica gel was used as the packing material; mobile phase A was acetonitrile, mobile phase B was 0.1-0.2% phosphoric acid for elution, flow rate was 0.38-0.42 ml / min, column temperature was 29-31℃, and detection wavelength was 210 nm for 0-17 min and 270 nm for 17-48 min. (5) Take the test solution, reference solution, and reference medicinal material solution, add them to the liquid chromatograph for detection, and obtain the fingerprint spectrum of Huanglong Zhike Granules; The extraction solvent in step (1) is 10%-70% methanol or 10% ethanol; the extraction solvent in step (3) is methanol.

2. The construction method of claim 1, wherein, The extraction solvent in step (1) is 70% methanol.

3. The construction method of claim 1, wherein, In step (1), the mass-to-volume ratio of Huanglong cough granules to the extraction solvent is 2:5-45, with units of g:ml.

4. The construction method according to claim 3, characterized in that, In step (1), the mass-volume ratio of Huanglong cough granules to the extraction solvent is 2:15, with units of g:ml.

5. The construction method of claim 1, wherein, The extraction method in step (1) is ultrasonic extraction or heating reflux extraction.

6. The construction method of claim 1, wherein, The extraction time in step (1) is 10-100 min.

7. The construction method of claim 1, wherein, The chromatographic conditions in step (4) are as follows: octadecylsilane-bonded silica gel is used as the packing material; mobile phase A is acetonitrile, mobile phase B is 0.2% phosphoric acid for elution, flow rate is 0.40 ml / min, column temperature is 30℃, and detection wavelength is 210 nm for 0-17 min and 270 nm for 17-48 min.

8. The construction method of claim 1, wherein, The elution conditions in step (4) are: 0 min, mobile phase A: 0%, mobile phase B: 100%; 1 min, mobile phase A: 0%, mobile phase B: 100%; 7 min, mobile phase A: 7%, mobile phase B: 93%; 14 min, mobile phase A: 7%, mobile phase B: 93%; 17 min, mobile phase A: 17%, mobile phase B: 83%; 21 min, mobile phase A: 17%, mobile phase B: 83%; 28 min, mobile phase A: 26%, mobile phase B: 74%; 32 min, mobile phase A: 26%, mobile phase B: 74%; 38 min, mobile phase A: 39%, mobile phase B: 61%; 43 min, mobile phase A: 39%, mobile phase B: 61%; 43.1 min, mobile phase A: 0%, mobile phase B: 100%; 48 min, mobile phase A: 0%, mobile phase B: 100%.

9. The construction method of claim 1, wherein, The chromatographic detector instrument in step (4) is a THERMO UHPLC ultra-high performance liquid chromatograph, and the chromatographic column model in step (4) is Waters ACQUITY UPLC HSS T3.

10. The construction method according to any one of claims 1 to 9, characterized in that, The Huanglong cough-relieving granules are composed of Astragalus, Earthworm, Herba Epimedii, Platycodon, Belamcanda, Houttuynia cordata, honey Ephedra, Crataegus and Eutrema.

11. The construction method according to any one of claims 1 to 9, characterized in that, The fingerprint spectrum contains 32 common peaks, with peak No. 9 as a reference peak, and the relative retention times of common peaks No. 1-15 are as follows: peak No. 1: 0.246-0.248, peak No. 2: 0.284-0.286, peak No. 3: 0.367-0.374, peak No. 4: 0.477-0.480, peak No. 5: 0.511-0.514, peak No. 6: 0.598-0.605, peak No. 7: 0.799-0.802, peak No. 8: 0.856-0.858, peak No. 9: 1.000, peak No. 10: 1.049-1.051, peak No. 11: 1.050-1.143, peak No. 12: 1.252-1.255, peak No. 13: 1.534-1.537, peak No. 14: 1.608-1.613, and peak No. 15: 1.812-1.817; with peak No. 27 as a reference peak, the relative retention times of common peaks No. 16-32 are as follows: peak No. 16: 0.653-0.654, peak No. 17: 0.665-0.666, peak No. 18: 0.696-0.697, peak No. 19: 0.719-0.808, peak No. 20: 0.745-0.745, peak No. 21: 0.766-0.767, peak No. 22: 0.772-0.773, peak No. 23: 0.929-0.929, peak No. 24: 0.947-0.947, peak No. 25: 0.963-0.963, peak No. 26: 0.983-0.983, peak No. 27: 1.000, peak No. 28: 1.023-1.023, peak No. 29: 1.084-1.085, peak No. 30: 1.206-1.207, peak No. 31: 1.250-1.251, and peak No. 32: 1.257-1.

259.

12. The Huanglong cough-relieving granules obtained by the construction method of any one of claims 1-11.

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