A fingerprint construction method of a traditional Chinese medicine composition containing Belamcanda chinensis and Ephedra sinica and its application

Abstract

The present invention discloses a method for constructing a fingerprint of a Chinese medicine composition containing Belamcanda chinensis and Ephedra sinica, and the method comprises the following steps: preparing a test solution of the Chinese medicine composition; preparing a reference solution; and obtaining a fingerprint of the Chinese medicine composition according to the results of high performance liquid phase detection of the test solution of the Chinese medicine composition and the reference solution. The present invention comprehensively and systematically analyzes the chemical components of the Chinese medicine composition containing Belamcanda chinensis and Ephedra sinica, and provides a theoretical basis for in-depth research on quality control and the material basis of drug efficacy.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a fingerprint construction method of a traditional Chinese medicine composition comprising Belamcanda chinensis and Ephedra sinica and an application thereof. Background Art

[0002] According to the Traditional Chinese Medicine Law of the People's Republic of China, ancient classic prescriptions refer to "prescriptions recorded in ancient Chinese medicine classics that are still widely used, have definite efficacy, and have obvious characteristics and advantages". Ancient classic prescriptions have a long and rich history of human use in my country and are still used today. They are the summary of the essence of clinical practice of doctors of all dynasties, carrying the profound accumulation of the splendid civilization of Chinese medicine for thousands of years. They are the result of thousands of years of tempering of Chinese medicine theory, the summary of clinical experience of all dynasties, and the most essential part of the great treasure house of Chinese medicine. In-depth research and development of classic Chinese medicine prescriptions is a golden key to tap into the treasure house of traditional Chinese medicine.

[0003] As the most commonly used dosage form of traditional Chinese medicine clinical medicine, decoctions of classic Chinese medicine prescriptions have the advantages of reasonable formulation, rapid onset, significant efficacy, and easy absorption, and are deeply trusted by the majority of patients. However, due to the shortcomings of easy mold and deterioration during preparation, carrying, temporary decoction, and long-term storage, the bitter taste and large amount of decoction, and the difficulty in standardization, which seriously affects its clinical efficacy, it cannot adapt to the living requirements of modern people. In order to maintain the advantages of decoctions and overcome the various shortcomings of decoctions, the "Technical Guiding Principles for Pharmaceutical Research of Traditional Chinese Medicine Compound Preparations Managed by the Catalog of Ancient Classic Prescriptions (Trial)" issued by the Drug Evaluation Center of the State Food and Drug Administration on August 31, 2021 clearly pointed out that the quality of traditional Chinese medicine compound preparations managed by the catalog of ancient classic prescriptions should be basically consistent with the quality of the classic prescription benchmark samples. The benchmark sample represents the overall intrinsic quality of the preparation. Except for the molding process, the benchmark sample and the other quality control indicators of the preparation are basically consistent. Therefore, the benchmark sample is a physical comparison of the intrinsic quality of the preparation and a reference for the optimization of large-scale production processes and the formulation of its quality standards. Benchmark samples are the benchmark for classic prescriptions and even all Chinese medicine research and development, and are the reference materials to ensure the safety and effectiveness of drugs. The production process route formulation, parameter optimization and quality standard formulation of classic prescription compound preparations should be based on the benchmark samples of classic prescriptions. Benchmark samples are the link between clinic, enterprise and scientific research, and are the benchmark for inheriting, applying and promoting traditional Chinese medicine.

[0004] Fingerprint is based on the understanding of the overall effect of Chinese medicine substance groups. It uses spectral and chromatographic techniques to obtain the spectrum or chromatogram of Chinese medicine chemical components. It is a feasible model for identifying the authenticity of Chinese medicine, evaluating quality consistency and product stability. It has the characteristics of large amount of information, strong characteristic, integrity and fuzziness. Chinese medicine fingerprint can comprehensively reflect the relative relationship of chemical components contained in medicinal materials, embody the complexity and correlation of Chinese medicine components, and adapt to the traditional theory of Chinese medicine. It can truly effectively characterize, comprehensively evaluate and comprehensively control the intrinsic quality of Chinese medicine. It is especially suitable for quality control of Chinese medicine and Chinese medicine products when the effective ingredients are not completely clear or do not need to be completely clear. In addition to being used to investigate the origin, harvesting season, harvesting part, processing, storage time and other factors of Chinese medicine, so as to provide a basis for identifying the authenticity and quality of raw materials before production, Chinese medicine fingerprint can also be used for quality control of Chinese medicine production process: tracking the changes of certain chemical components in preparations, monitoring the consistency and stability of quality between raw materials and finished products, and between batches of finished products. Compared with the quality analysis method of determining the content of index components, fingerprint spectrum can more comprehensively reflect the types and quantities of chemical components of traditional Chinese medicine. At the current situation where the effective ingredients of traditional Chinese medicine compound preparations have not been fully elucidated, it can realize the comprehensive evaluation of the intrinsic quality of traditional Chinese medicine and the effective control of its overall substance. It is one of the effective means of quality control of traditional Chinese medicine and its preparations.

[0005] At present, there is no literature report on a comprehensive analysis of the chemical components and fingerprint study of the Chinese medicine composition comprising Belamcanda chinensis and Ephedra sinica claimed to be protected by the present invention. In the prior art, only a single component in the Chinese medicine compound is analyzed for quality, and there is no comprehensive and systematic quality control method to reflect the quality status of the main reference sample components in the Chinese medicine compound and the finished product of the present invention, and it is impossible to effectively control its production process and product quality, and it is impossible to better guarantee its clinical efficacy. Therefore, it is necessary to adopt a fingerprint quality control method that can comprehensively control the overall quality of the Chinese medicine compound of the present invention to control its key quality. Summary of the invention

[0006] Based on this, the present invention provides a method for constructing a fingerprint of a traditional Chinese medicine composition comprising Belamcanda chinensis and Ephedra sinica, and the method comprises the following steps:

[0007] Preparation of a Chinese medicine composition test solution: taking an appropriate amount of a decoction of a Chinese medicine composition containing Belamcanda chinensis and Ephedra sinica, placing the decoction in a container, adding a first solvent, plugging the container, shaking, filtering, and taking a subsequent filtrate to obtain the Chinese medicine composition test solution; wherein the Chinese medicine composition comprises Belamcanda chinensis, Ephedra sinica, ginger, Asarum, Aster tataricus, Coltsfoot flos, jujube, Pinellia ternata, and Schisandra chinensis;

[0008] Preparation of reference solution: Weigh appropriate amount of neochlorogenic acid and 6-gingerol reference substances, add methanol to prepare reference solutions with concentrations of neochlorogenic acid and 6-gingerol of 5 to 200 μg / ml respectively;

[0009] Obtaining a fingerprint of the Chinese medicine composition according to the results of high performance liquid chromatography detection of the test solution and the reference solution of the Chinese medicine composition;

[0010] The chromatographic conditions of the high performance liquid phase detection are as follows: a chromatographic column with octadecylsilane bonded silica gel as filler is used, mobile phase A is selected from one or more of acetonitrile, methanol and tetrahydrofuran, mobile phase B is an acid aqueous solution, an alkali aqueous solution and / or a buffered saline solution, and the gradient elution program is as follows: 0-8 min, 95% B; 8-13 min, 95% B→90% B; 13-20 min, 90% B→89% B; 20-24 min, 89% B; 24-32 min, 89% B→88% B; 32-34 min, 88% B→78% B; 34-50 min, 78% B; 50-55 min, 78% B→55% B; 55-70 min, 55%→50% B; the flow rate is 0.5-1.5 ml / min, the column temperature is 20-50° C., the detection wavelength is 190-400 nm, and the injection volume is 1-20 μl.

[0011] Furthermore, the preparation method of the decoction of the Chinese medicinal composition containing Belamcanda chinensis and Ephedra sinica comprises: (a) weighing appropriate amounts of Belamcanda chinensis, Ephedra sinica, ginger, Asarum, Aster, Coltsfoot Flos, Jujube, Pinellia ternata and Schisandra chinensis, placing them in a container, adding a first amount of water for soaking, decocting with high heat until boiling, then using a low heat for the first decoction, filtering through a filter to obtain a first filtrate and a filter residue; (b) adding a second amount of water to the filter residue, decocting with high heat until boiling, then using a low heat for the second decoction, filtering through a filter to obtain a second filtrate, combining the first filtrate and the second filtrate to obtain the decoction of the Chinese medicinal composition containing Belamcanda chinensis and Ephedra sinica.

[0012] Further, the ratio of the sum of the masses of the Belamcanda chinensis, Ephedra sinica, Ginger, Asarum, Aster tataricus, Coltsfoot Flos, Jujube, Pinellia ternata and Schisandra chinensis to the mass / volume (g / ml) of the first water is 0.1-0.15, for example, about 0.125.

[0013] Furthermore, the container is a multifunctional medicine decoction pot.

[0014] Furthermore, the soaking time is 20 to 40 minutes, for example, about 30 minutes.

[0015] Furthermore, the first decocting time on low heat is 10 to 30 minutes, for example, about 20 minutes.

[0016] Furthermore, the filter is a 150-mesh screen.

[0017] Further, the ratio of the sum of the masses of the Belamcanda chinensis, Ephedra sinica, Ginger, Asarum, Aster tataricus, Coltsfoot Flos, Jujube, Pinellia ternata and Schisandra chinensis to the mass / volume (g / ml) of the second water is 0.1-0.5, for example, about 0.25.

[0018] Furthermore, the second decocting time on low heat is 5 to 30 minutes, for example, about 15 minutes.

[0019] Furthermore, the volume of the decoction of the Chinese medicinal composition comprising Belamcanda chinensis and Ephedra sinica is 400-700 ml, for example, about 550 ml.

[0020] Furthermore, the volume / volume ratio of the decoction of the traditional Chinese medicine composition comprising Belamcanda chinensis and Ephedra sinica to the first water is 0.5-1.0, for example, about 0.881.

[0021] Furthermore, the mass ratios of the Belamcanda chinensis, the Ephedra sinica, the Ginger, the Asarum, the Aster tataricus, the Coltsfoot Flos, the Ziziphus jujuba, the Pinellia ternata and the Schisandra chinensis are (2-4): (2-4): (1-3): (1-3): (1-3): (1-3): (3-10): (2-4): (0.2-2).

[0022] Furthermore, the mass ratios of the Belamcanda chinensis, the Ephedra sinica, the Ginger, the Asarum, the Aster tataricus, the Coltsfoot Flos, the Ziziphus jujuba, the Pinellia ternata and the Schisandra chinensis are (2.5-3.5): (2.5-3.5): (1.5-2.5): (1.5-2.5): (1.5-2.5): (1.5-2.5): (4-6): (2.5-3.5): (0.5-1.5).

[0023] Further, the mass ratio of the Belamcanda chinensis, the Ephedra sinica, the Ginger, the Asarum, the Aster tataricus, the Coltsfoot Flos, the Ziziphus jujuba, the Pinellia ternata and the Schisandra chinensis is about 3: about 3: about 2: about 2: about 2: about 2: about 5: about 3: about 1.

[0024] Further, the mass range of the Belamcanda is 5 to 15g, for example, about 9g. Further, the mass range of the Ephedra is 5 to 15g, for example, about 9g. Further, the mass range of the ginger is 1 to 10g, for example, about 6g. Further, the mass range of the Asarum is 1 to 10g, for example, about 6g. Further, the mass range of the Aster is 1 to 10g, for example, about 6g. Further, the mass range of the Coltsfoot Flower is 1 to 10g, for example, about 6g. Further, the mass range of the jujube is 10 to 30g, for example, about 15g. Further, the number of the jujube is 2 to 4, for example, 3. Further, the mass range of the Pinellia is 5 to 15g, for example, about 9g. Further, the mass range of the Schisandra is 1 to 10g, for example, about 3g.

[0025] Further, in the preparation of the test solution of the Chinese medicine composition, the container is a conical flask. Further, the conical flask is a stoppered conical flask. Further, the first solvent is an alcohol, such as methanol. Further, the volume percentage concentration of the first solvent is 10% to 90%, such as about 50%. Further, the volume / volume ratio of the first solvent to the decoction of the Chinese medicine composition containing Belamcanda chinensis and Ephedra sinica is 0.8 to 1.2, such as about 1.0. Further, the concentration of neochlorogenic acid in the reference solution is about 96.2 μg / ml. Further, the concentration of 6-gingerol in the reference solution is about 16.9 μg / ml.

[0026] Furthermore, the flow rate of the HPLC detection is 0.8-1.2 ml / min, for example, about 1.0 ml / min.

[0027] Furthermore, the column temperature is 30-40°C, for example, about 35°C.

[0028] Furthermore, the detection wavelength is 250-300 nm, for example, about 280 nm.

[0029] Furthermore, the injection volume is 5-15 μl, for example, about 10 μl.

[0030] Furthermore, the theoretical plate number of the chromatographic peak corresponding to the 6-gingerol is not less than 5000.

[0031] Furthermore, the chromatographic column is an Agilent 5TC-C18 chromatographic column.

[0032] Furthermore, the specifications of the chromatographic column are: column length 250 mm, inner diameter 4.6 mm, and particle size 5 μm.

[0033] Furthermore, the mobile phase A is acetonitrile.

[0034] Furthermore, the acid aqueous solution, alkali aqueous solution and / or buffered saline solution is selected from one or more of weak acids and their salts, weak bases and their salts of different concentrations.

[0035] Further, the acid aqueous solution, alkali aqueous solution and / or buffered saline solution is selected from formic acid, glacial acetic acid, phosphoric acid, trifluoroacetic acid, formic acid and ammonium formate, acetic acid and sodium acetate, acetic acid and ammonium acetate, disodium hydrogen phosphate and sodium dihydrogen phosphate, disodium hydrogen phosphate and potassium dihydrogen phosphate, disodium hydrogen phosphate and citric acid, citric acid and sodium citrate, glycine and hydrochloric acid, or phthalic acid and hydrochloric acid in different concentrations.

[0036] Furthermore, the acid aqueous solution is a 0.01% to 1% acid aqueous solution.

[0037] Furthermore, the acid aqueous solution is a 0.01% to 1% phosphoric acid aqueous solution.

[0038] Furthermore, the acid aqueous solution is about 0.1% phosphoric acid aqueous solution.

[0039] Furthermore, the buffered saline solution is an acetate solution and / or an acetate solution.

[0040] Furthermore, the pH value of the buffered saline solution is not greater than 7.0.

[0041] Further, when the detection wavelength is 280 nm, the fingerprint spectrum includes peaks 1-19, and the retention times of peaks 1-19 correspond to about 7.649 min, about 10.447 min, about 17.212 min, about 18.793 min, about 19.344 min, about 22.597 min, about 23.290 min, about 25.857 min, about 42.484 min, about 43.705 min, about 46.542 min, about 47.170 min, about 57.241 min, about 60.353 min, about 64.758 min, about 65.745 min, about 66.486 min, about 67.620 min and about 68.618 min, respectively.

[0042] Further, when the detection wavelength is 280 nm, the fingerprint spectrum includes peaks 1-19, wherein peak 12 is used as a reference peak, and the relative retention time averages of peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8, peak 9, peak 10, peak 11, peak 13, peak 14, peak 15, peak 16, peak 17, peak 18 and peak 19 are approximately 0.16, approximately 0.22, approximately 0.36, approximately 0.40, approximately 0.41, approximately 0.48, approximately 0.49, approximately 0.55, approximately 0.90, approximately 0.93, approximately 0.99, approximately 1.21, approximately 1.28, approximately 1.37, approximately 1.39, approximately 1.41, approximately 1.43 and approximately 1.45, respectively.

[0043] Furthermore, when the detection wavelength is 280 nm, peak 3 is neochlorogenic acid and peak 17 is 6-gingerol.

[0044] Further, when the detection wavelength is 280nm, peak No. 1 comes from jujube and Belamcanda medicinal materials, peak No. 2 comes from jujube, Pinellia and Schisandra chinensis medicinal materials, peak No. 3 comes from coltsfoot medicinal materials, peak No. 4 comes from Belamcanda medicinal materials, peak No. 5 comes from Belamcanda and Aster medicinal materials, peak No. 6 comes from Ephedra medicinal materials, peak No. 7 comes from Coltsfoot and Aster medicinal materials, peak No. 8 comes from Asarum medicinal materials, peak No. 9 comes from Belamcanda medicinal materials, peak No. 10 comes from Belamcanda medicinal materials, peak No. 11 comes from Belamcanda medicinal materials, peak No. 12 comes from Belamcanda medicinal materials, peak No. 13 comes from Ephedra medicinal materials, peak No. 14 comes from Belamcanda medicinal materials, peak No. 15 comes from Belamcanda medicinal materials, peak No. 16 comes from Aster medicinal materials, peak No. 17 comes from ginger medicinal materials, peak No. 18 comes from Aster medicinal materials, and peak No. 19 comes from Asarum medicinal materials.

[0045] According to another aspect of the present invention, a quality control method for a Chinese medicine composition comprising Belamcanda chinensis and Ephedra sinica is provided, the quality control method comprising the following steps:

[0046] (1) establishing a standard fingerprint of a reference sample of a Chinese medicine composition according to the above-mentioned fingerprint construction method;

[0047] (2) taking a sample solution of the Chinese medicine composition and testing it according to the chromatographic conditions in the fingerprint spectrum construction method to obtain a fingerprint spectrum of the Chinese medicine composition sample to be tested; and

[0048] (3) Compare the fingerprint of the sample of the Chinese medicine composition obtained in step (2) with the standard fingerprint of the reference sample of the Chinese medicine composition obtained in step (1). If the fingerprint meets the requirements, it is a qualified product; if the fingerprint does not meet the requirements, it is an unqualified product.

[0049] Furthermore, the compliance requirement includes one or more of the following:

[0050] (1) The fingerprint of the Chinese medicine composition sample to be tested exhibits 19 characteristic chromatographic peaks, and the retention time of each characteristic chromatographic peak is within ±10% of the retention time value of the corresponding reference chromatographic peak in the standard fingerprint of the Chinese medicine composition reference sample;

[0051] (2) With peak No. 12 as the S peak, the relative retention time of each characteristic chromatographic peak in the fingerprint of the Chinese medicine composition sample to be tested and the S peak is within ±10% of the relative retention time value of each characteristic chromatographic peak in the standard fingerprint of the Chinese medicine composition reference sample; and

[0052] (3) According to the similarity evaluation system of Chinese medicine chromatographic fingerprint, the similarity between the fingerprint of the Chinese medicine composition sample to be tested and the standard fingerprint of the Chinese medicine composition reference sample shall be calculated, and the similarity shall not be less than 0.90.

[0053] According to another aspect of the present invention, there is provided a use of the above-mentioned construction method or the above-mentioned quality control method in quality detection and / or quality evaluation and / or quality control of a traditional Chinese medicine composition comprising Belamcanda chinensis and Ephedra sinica.

[0054] Beneficial effects of the present invention:

[0055] In short, the present invention provides a fingerprint spectrum determination and quality control method for a reference sample of a traditional Chinese medicine composition containing Belamcanda chinensis and Ephedra sinica. The method confirms 19 common characteristic peaks, has simple conditions and short analysis time, solves the problems of difficulty in separating fingerprint characteristic peaks and interference from impurity peaks, ensures the chemical composition stability and safety of use of the reference sample, provides an important reference basis for the quality control of subsequent preparations, ensures the stability of product quality, guarantees the efficacy of the traditional Chinese medicine compound, and allows the traditional Chinese medicine compound to better serve human life and health.

[0056] Specifically, compared with the prior art, the present invention has the following beneficial effects:

[0057] (1) Establishing the fingerprint of the reference sample of the Chinese medicine compound of the present invention overcomes the defect that the determination of the content of a single component is difficult to reflect the overall content, and can control the intrinsic quality of the reference sample of the Chinese medicine compound of the present invention from a holistic and macroscopic perspective, thereby ensuring the efficacy of the drug, utilizing the main means of modern drug research, realizing the inheritance of classics, and enabling classic prescriptions to be subject to more formal quality control.

[0058] (2) The chemical components in the reference sample of the traditional Chinese medicine compound of the present invention are complex, and it is difficult to separate its characteristic peaks. In the process of establishing the fingerprint spectrum, the present invention adopts a gradient elution method to solve the problem of difficulty in separating the fingerprint characteristic peaks and interference of impurity peaks.

[0059] (3) In the process of establishing the fingerprint spectrum of the traditional Chinese medicine compound reference sample of the present invention, 19 common characteristic peaks were confirmed, and their relative retention time, relative peak area and similarity were studied, thereby ensuring the chemical composition stability and safety of the reference sample, and providing an important reference basis and quality reference for the quality control of subsequent compound preparations.

[0060] (4) The fingerprint spectra of each effective ingredient in the traditional Chinese medicine compound reference sample of the present invention are regarded as a whole, and the order and relationship of each characteristic peak are emphasized. This not only avoids the one-sidedness of judging the overall quality of the traditional Chinese medicine compound reference sample of the present invention due to only measuring one or two chemical components, but also reduces the possibility of artificial processing to meet the quality standards, thereby providing a new method and means for completely and accurately evaluating the quality of the traditional Chinese medicine compound reference sample of the present invention.

[0061] (5) The method of the present invention has good stability, high precision, good reproducibility, is convenient and easy to master. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without exceeding the scope of protection required by the present invention.

[0063] Figure 1 The fingerprint spectrum of the reference sample of the Chinese medicine composition containing Belamcanda chinensis and Ephedra sinica of the present invention is shown in FIG. Among them, peak 3: neochlorogenic acid; peak 17: 6-gingerol; Aglient 5TC C18 (250×4.6mm, 5μm) chromatographic column is used.

[0064] Figure 2 These are multiple batch matching diagrams of 20 batches of the Chinese medicinal composition comprising Belamcanda chinensis and Ephedra sinica of the present invention. DETAILED DESCRIPTION

[0065] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0066] Unless otherwise indicated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by those of ordinary skill in the field of the invention or the field in which the terms are used. Although any methods, conditions, substances or materials similar to or equivalent to those disclosed herein may be used in the practice of the present invention, preferred methods, conditions, substances or materials are described herein.

[0067] The present invention is intended to encompass all alternatives, variations and equivalents that may be included in the present invention as defined by the claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

[0068] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0069] In the present invention, the term "comprising" is synonymous with "including". As used herein, the terms "comprising", "including", "having", "containing" or any other variation thereof are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus comprising the listed elements is not necessarily limited to only those elements, but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0070] As described in the background technology section, there is no literature report on the comprehensive analysis of the chemical components and fingerprint study of the Chinese medicine composition comprising Belamcanda chinensis and Ephedrae rhizome claimed in the present invention. In order to solve the above problems, the present invention provides a method for constructing a fingerprint of the Chinese medicine composition comprising Belamcanda chinensis and Ephedrae rhizome, and the construction method comprises the following steps:

[0071] Preparation of a Chinese medicine composition test solution: taking an appropriate amount of a decoction of a Chinese medicine composition containing Belamcanda chinensis and Ephedra sinica, placing the decoction in a container, adding a first solvent, plugging the container, shaking, filtering, and taking a subsequent filtrate to obtain the Chinese medicine composition test solution; wherein the Chinese medicine composition comprises Belamcanda chinensis, Ephedra sinica, ginger, Asarum, Aster tataricus, Coltsfoot flos, jujube, Pinellia ternata, and Schisandra chinensis;

[0072] Preparation of reference solution: Weigh appropriate amount of neochlorogenic acid and 6-gingerol reference substances, add methanol to prepare reference solutions with concentrations of neochlorogenic acid and 6-gingerol of 5 to 200 μg / ml respectively;

[0073] Obtaining a fingerprint of the Chinese medicine composition according to the results of high performance liquid chromatography detection of the test solution and the reference solution of the Chinese medicine composition;

[0074] The chromatographic conditions of the high performance liquid phase detection are as follows: a chromatographic column with octadecylsilane bonded silica gel as filler is used, mobile phase A is selected from one or more of acetonitrile, methanol and tetrahydrofuran, mobile phase B is an acid aqueous solution, an alkali aqueous solution and / or a buffered saline solution, and the gradient elution program is as follows: 0-8 min, 95% B; 8-13 min, 95% B→90% B; 13-20 min, 90% B→89% B; 20-24 min, 89% B; 24-32 min, 89% B→88% B; 32-34 min, 88% B→78% B; 34-50 min, 78% B; 50-55 min, 78% B→55% B; 55-70 min, 55%→50% B; the flow rate is 0.5-1.5 ml / min, the column temperature is 20-50° C., the detection wavelength is 190-400 nm, and the injection volume is 1-20 μl.

[0075] In the present invention, when concentration, time, flow rate, temperature, wavelength, injection volume, ratio, volume, mass, or other values ​​or parameters are expressed as a range, a preferred range, or a range defined by a series of upper preferred values ​​and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether the range is disclosed separately. For example, when the range "0.5-1.5" is disclosed, the described range should be interpreted as including the range "0.5-1.5", "0.5-1.3", "0.5-1.1", "0.5-0.9", "0.5-0.7", "0.7-1.5", "0.7-1.3", "0.7-1.1", "0.7-0.9", "0.9-1.5", "0.9-1.3", "0.9-1.1", "1.1-1.5", "1.1-1.3", "1.3-1.5", etc. When a numerical range is described herein, unless otherwise indicated, that range is intended to include the endpoints and all integers and fractions within that range.

[0076] In a preferred embodiment, the preparation method of the decoction of the traditional Chinese medicine composition containing Belamcanda chinensis and Ephedra sinica comprises: (a) weighing appropriate amounts of Belamcanda chinensis, Ephedra sinica, ginger, Asarum, Aster, Coltsfoot Flos, Jujube, Pinellia ternata and Schisandra chinensis and placing them in a container, adding a first amount of water for soaking, decocting with high heat until boiling, then using a low heat for the first decoction, filtering through a filter to obtain a first filtrate and a filter residue; (b) adding a second amount of water to the filter residue, decocting with high heat until boiling, then using a low heat for the second decoction, filtering through a filter to obtain a second filtrate, combining the first filtrate and the second filtrate to obtain the decoction of the traditional Chinese medicine composition containing Belamcanda chinensis and Ephedra sinica.

[0077] In a preferred embodiment, the ratio of the sum of the masses of the Belamcanda chinensis, Ephedra sinica, Ginger, Asarum, Aster tataricus, Coltsfoot Flos, Jujube, Pinellia ternata and Schisandra chinensis to the mass / volume (g / ml) of the first water is 0.1-0.15, for example about 0.125.

[0078] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.125" includes ±5% of 0.125, or from 0.11875 to 0.13125.

[0079] In a preferred embodiment, the container is a multifunctional medicine decoction pot.

[0080] In a preferred embodiment, the soaking time is 20 to 40 minutes, such as about 30 minutes.

[0081] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 30" includes ±5% of 30, or from 28.5 to 31.5.

[0082] In a preferred embodiment, the first decocting time over low heat is 10 to 30 minutes, for example, about 20 minutes.

[0083] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 20" includes ±5% of 20, or from 19 to 21.

[0084] In a preferred embodiment, the filter is a 150 mesh screen.

[0085] In a preferred embodiment, the ratio of the sum of the masses of the Belamcanda chinensis, Ephedra sinica, Ginger, Asarum, Aster tataricus, Coltsfoot Flos, Jujube, Pinellia ternata and Schisandra chinensis to the mass / volume (g / ml) of the second water is 0.1-0.5, for example about 0.25.

[0086] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.25" includes ±5% of 0.25, or from 0.2375 to 0.2625.

[0087] In a preferred embodiment, the second decocting time over low heat is 5 to 30 minutes, for example, about 15 minutes.

[0088] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 15" includes ±5% of 15, or from 14.25 to 15.75.

[0089] In a preferred embodiment, the volume of the decoction of the traditional Chinese medicine composition comprising Belamcanda chinensis and Ephedra sinica is 400-700 ml, for example, about 550 ml.

[0090] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 550" includes ±5% of 550, or from 522.5 to 577.5.

[0091] In a preferred embodiment, the volume / volume ratio of the decoction of the traditional Chinese medicine composition comprising Belamcanda chinensis and Ephedra sinica to the first water is 0.5-1.0, for example, about 0.881.

[0092] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.881" includes ±5% of 0.881, or from 0.83695 to 0.92505.

[0093] In a preferred embodiment, the mass ratio between the Belamcanda chinensis, the Ephedra sinica, the Ginger, the Asarum, the Aster tataricus, the Coltsfoot Flos, the Ziziphus jujuba, the Pinellia ternata and the Schisandra chinensis is (2-4): (2-4): (1-3): (1-3): (1-3): (1-3): (3-10): (2-4): (0.2-2).

[0094] In a preferred embodiment, the mass ratio between the Belamcanda chinensis, the Ephedra sinica, the Ginger, the Asarum, the Aster tataricus, the Coltsfoot Flos, the Ziziphus jujuba, the Pinellia ternata and the Schisandra chinensis is (2.5-3.5): (2.5-3.5): (1.5-2.5): (1.5-2.5): (1.5-2.5): (1.5-2.5): (4-6): (2.5-3.5): (0.5-1.5).

[0095] In a preferred embodiment, the mass ratio of the Belamcanda chinensis, the Ephedra sinica, the Zingiber officinale, the Asarum sieboldii, the Aster tataricus, the Coltsfoot flos, the Ziziphus jujuba, the Pinellia ternata and the Schisandra chinensis is about 3: about 3: about 2: about 2: about 2: about 2: about 5: about 3: about 1.

[0096] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 3" includes ±5% of 3, or from 2.85 to 3.15; "about 2" includes ±5% of 2, or from 1.9 to 2.1; "about 5" includes ±5% of 5, or from 4.75 to 5.25; "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0097] In a preferred embodiment, the mass of the Belamcanda chinensis is in the range of 5 to 15 g, for example, about 9 g.

[0098] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 9" includes ±5% of 9, or from 8.55 to 9.45.

[0099] In a preferred embodiment, the mass of the ephedra is in the range of 5 to 15 g, such as about 9 g.

[0100] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 9" includes ±5% of 9, or from 8.55 to 9.45.

[0101] In a preferred embodiment, the weight of the ginger is in the range of 1 to 10 g, for example, about 6 g.

[0102] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 6" includes ±5% of 6, or from 5.7 to 6.3.

[0103] In a preferred embodiment, the mass of the asarum is in the range of 1 to 10 g, for example, about 6 g.

[0104] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 6" includes ±5% of 6, or from 5.7 to 6.3.

[0105] In a preferred embodiment, the mass of the Aster is in the range of 1 to 10 g, for example, about 6 g.

[0106] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 6" includes ±5% of 6, or from 5.7 to 6.3.

[0107] In a preferred embodiment, the mass of the coltsfoot flower ranges from 1 to 10 g, such as about 6 g.

[0108] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 6" includes ±5% of 6, or from 5.7 to 6.3.

[0109] In a preferred embodiment, the mass of the jujube is in the range of 10 to 30 g, such as about 15 g.

[0110] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 15" includes ±5% of 15, or from 14.25 to 15.75.

[0111] In a preferred embodiment, the number of the jujubes is 2 to 4, for example, 3.

[0112] In a preferred embodiment, the mass of the Pinellia ternata ranges from 5 to 15 g, such as about 9 g.

[0113] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 9" includes ±5% of 9, or from 8.55 to 9.45.

[0114] In a preferred embodiment, the mass of Schisandra chinensis is in the range of 1 to 10 g, such as about 3 g.

[0115] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 3" includes ±5% of 3, or from 2.85 to 3.15.

[0116] In a preferred embodiment, in the preparation of the test solution of the traditional Chinese medicine composition, the container is a conical flask.

[0117] In a preferred embodiment, the conical flask is a stoppered conical flask.

[0118] In a preferred embodiment, the first solvent is an alcohol, such as methanol.

[0119] In a preferred embodiment, the volume percentage concentration of the first solvent is 10% to 90%, for example, about 50%.

[0120] In the present invention, "about" refers to a value within a range of ±5% of a particular value. For example, "about 50%" includes ±5% of 50%, or from 47.5% to 52.5%.

[0121] In a preferred embodiment, the volume / volume ratio of the first solvent to the decoction of the traditional Chinese medicine composition comprising Belamcanda chinensis and Ephedra sinica is 0.8-1.2, such as about 1.0.

[0122] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0123] In a preferred embodiment, the concentration of neochlorogenic acid in the reference solution is about 96.2 μg / ml.

[0124] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 96.2" includes ±5% of 96.2, or from 91.39 to 101.01.

[0125] In a preferred embodiment, the concentration of 6-gingerol in the reference solution is about 16.9 μg / ml.

[0126] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 16.9" includes ±5% of 16.9, or from 16.055 to 17.745.

[0127] In a preferred embodiment, the flow rate of the HPLC detection is 0.8-1.2 ml / min, for example, about 1.0 ml / min.

[0128] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0129] In a preferred embodiment, the column temperature is 30-40°C, such as about 35°C.

[0130] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 35" includes ±5% of 35, or from 33.25 to 36.75.

[0131] In a preferred embodiment, the detection wavelength is 250-300 nm, such as about 280 nm.

[0132] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 280" includes ±5% of 280, or from 266 to 294.

[0133] In a preferred embodiment, the injection volume is 5-15 μl, such as about 10 μl.

[0134] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0135] In a preferred embodiment, the theoretical plate number of the chromatographic peak corresponding to the 6-gingerol is not less than 5,000.

[0136] In a preferred embodiment, the chromatographic column is an Agilent 5TC-C18 chromatographic column.

[0137] In a preferred embodiment, the specifications of the chromatographic column are: column length 250 mm, inner diameter 4.6 mm, and particle size 5 μm.

[0138] In a preferred embodiment, mobile phase A is acetonitrile.

[0139] In a preferred embodiment, the acid aqueous solution, base aqueous solution and / or buffered saline solution is selected from one or more of weak acids and their salts, weak bases and their salts of different concentrations.

[0140] In a preferred embodiment, the acid aqueous solution, alkali aqueous solution and / or buffered saline solution is selected from formic acid, glacial acetic acid, phosphoric acid, trifluoroacetic acid, formic acid and ammonium formate, acetic acid and sodium acetate, acetic acid and ammonium acetate, disodium hydrogen phosphate and sodium dihydrogen phosphate, disodium hydrogen phosphate and potassium dihydrogen phosphate, disodium hydrogen phosphate and citric acid, citric acid and sodium citrate, glycine and hydrochloric acid, or phthalic acid and hydrochloric acid in different concentrations.

[0141] In a preferred embodiment, the acid aqueous solution is a 0.01% to 1% acid aqueous solution.

[0142] In a preferred embodiment, the acid aqueous solution is a 0.01% to 1% phosphoric acid aqueous solution.

[0143] In a preferred embodiment, the aqueous acid solution is about 0.1% phosphoric acid aqueous solution.

[0144] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.1%" includes ±5% of 0.1%, or from 0.095% to 0.105%.

[0145] In a preferred embodiment, the buffered saline solution is an acetate aqueous solution and / or an acetate aqueous solution.

[0146] In a preferred embodiment, the pH value of the buffered saline solution is no greater than 7.0.

[0147] In a preferred embodiment, when the detection wavelength is 280 nm, the fingerprint spectrum includes peaks 1-19, and the retention times of peaks 1-19 correspond to about 7.649 min, about 10.447 min, about 17.212 min, about 18.793 min, about 19.344 min, about 22.597 min, about 23.290 min, about 25.857 min, about 42.484 min, about 43.705 min, about 46.542 min, about 47.170 min, about 57.241 min, about 60.353 min, about 64.758 min, about 65.745 min, about 66.486 min, about 67.620 min and about 68.618 min, respectively.

[0148] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 7.649" includes ±5% of 7.649, or from 7.26655 to 8.03145; "about 10.447" includes ±5% of 10.447, or from 9.92465 to 10.96935; "about 17.212" includes ±5% of 17.212, or from 16.3514 to 18.0726; "about 18.793" includes ±5% of 18.793, or from 17.85335 to 19.73265; "about 19.344" includes ±5% of 19.344, or from 18.37 68 to 20.3112; "about 22.597" includes ±5% of 22.597, or from 21.46715 to 23.72685; "about 23.290" includes ±5% of 23.290, or from 22.1255 to 24.4545; "about 25.857" includes ±5% of 25.857, or from 24.56415 to 27.14985; "about 42.484" includes ±5% of 42.484, or from 40.3598 to 44.6082; "about 43.705" includes ±5% of 43.705 5%, or from 41.51975 to 45.89025; "about 46.542" includes ±5% of 46.542, or from 44.2149 to 48.8691; "about 47.170" includes ±5% of 47.170, or from 44.8115 to 49.5285; "about 57.241" includes ±5% of 57.241, or from 54.37895 to 60.10305; "about 60.353" includes ±5% of 60.353, or from 57.33535 to 63.37065; "about 64.7 “58” includes ±5% of 64.758, or from 61.5201 to 67.9959; “about 65.745” includes ±5% of 65.745, or from 62.45775 to 69.03225; “about 66.486” includes ±5% of 66.486, or from 63.1617 to 69.8103; “about 67.620” includes ±5% of 67.620, or from 64.239 to 71.001; “about 68.618” includes ±5% of 68.618, or from 65.1871 to 72.0489.

[0149] In a preferred embodiment, when the detection wavelength is 280 nm, the fingerprint spectrum includes peaks 1-19, wherein peak 12 is used as a reference peak, and the relative retention time averages of peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8, peak 9, peak 10, peak 11, peak 13, peak 14, peak 15, peak 16, peak 17, peak 18 and peak 19 are about 0.16, about 0.22, about 0.36, about 0.40, about 0.41, about 0.48, about 0.49, about 0.55, about 0.90, about 0.93, about 0.99, about 1.21, about 1.28, about 1.37, about 1.39, about 1.41, about 1.43 and about 1.45, respectively.

[0150] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.16" includes ±5% of 0.16, or from 0.152 to 0.168; "about 0.22" includes ±5% of 0.22, or from 0.209 to 0.231; "about 0.36" includes ±5% of 0.36, or from 0.342 to 0.378; "about 0.40" includes ±5% of 0.40, or from 0.38 to 0.42; "about 0.41" includes ±5% of 0.41, or from 0.3895 to 0.4305; "about 0.48" includes ±5% of 0.48, or from 0.456 to 0.504; "about 0.49" includes ±5% of 0.49, or from 0.4655 to 0.5145; "about 0.55" includes ±5% of 0.55, or from 0.5225 to 0.5775; "about 0.90" includes ±5% of 0.90, or from 0.855 to 0.945; "about 0. "93" includes ±5% of 0.93, or from 0.8835 to 0.9765; "about 0.99" includes ±5% of 0.99, or from 0.9405 to 1.0395; "about 1.21" includes ±5% of 1.21, or from 1.1495 to 1.2705; "about 1.28" includes ±5% of 1.28, or from 1.216 to 1.344; "about 1.37" includes ±5% of 1.37, or from 1.3015 to 1.4385; “about 1.39” includes ±5% of 1.39, or from 1.3205 to 1.4595; “about 1.41” includes ±5% of 1.41, or from 1.3395 to 1.4805; “about 1.43” includes ±5% of 1.43, or from 1.3585 to 1.5015; “about 1.45” includes ±5% of 1.45, or from 1.3775 to 1.5225.

[0151] In a preferred embodiment, when the detection wavelength is 280 nm, peak No. 3 is neochlorogenic acid, and peak No. 17 is 6-gingerol.

[0152] In a preferred embodiment, when the detection wavelength is 280nm, peak No. 1 comes from jujube and Belamcanda medicinal materials, peak No. 2 comes from jujube, Pinellia and Schisandra chinensis medicinal materials, peak No. 3 comes from coltsfoot medicinal materials, peak No. 4 comes from Belamcanda medicinal materials, peak No. 5 comes from Belamcanda and Aster medicinal materials, peak No. 6 comes from Ephedra medicinal materials, peak No. 7 comes from Coltsfoot and Aster medicinal materials, peak No. 8 comes from Asarum medicinal materials, peak No. 9 comes from Belamcanda medicinal materials, peak No. 10 comes from Belamcanda medicinal materials, peak No. 11 comes from Belamcanda medicinal materials, peak No. 12 comes from Belamcanda medicinal materials, peak No. 13 comes from Ephedra medicinal materials, peak No. 14 comes from Belamcanda medicinal materials, peak No. 15 comes from Belamcanda medicinal materials, peak No. 16 comes from Aster medicinal materials, peak No. 17 comes from ginger medicinal materials, peak No. 18 comes from Aster medicinal materials, and peak No. 19 comes from Asarum medicinal materials.

[0153] According to another aspect of the present invention, a quality control method for a Chinese medicine composition comprising Belamcanda chinensis and Ephedra sinica is provided, the quality control method comprising the following steps:

[0154] (1) establishing a standard fingerprint of a reference sample of a Chinese medicine composition according to the above-mentioned fingerprint construction method;

[0155] (2) taking a sample solution of the Chinese medicine composition and testing it according to the chromatographic conditions in the fingerprint spectrum construction method to obtain a fingerprint spectrum of the Chinese medicine composition sample to be tested; and

[0156] (3) Compare the fingerprint of the sample of the Chinese medicine composition obtained in step (2) with the standard fingerprint of the reference sample of the Chinese medicine composition obtained in step (1). If the fingerprint meets the requirements, it is a qualified product; if the fingerprint does not meet the requirements, it is an unqualified product.

[0157] In a preferred embodiment, the compliance requirements include one or more of the following:

[0158] (1) The fingerprint of the Chinese medicine composition sample to be tested exhibits 19 characteristic chromatographic peaks, and the retention time of each characteristic chromatographic peak is within ±10% of the retention time value of the corresponding reference chromatographic peak in the standard fingerprint of the Chinese medicine composition reference sample;

[0159] (2) With peak No. 12 as the S peak, the relative retention time of each characteristic chromatographic peak in the fingerprint of the Chinese medicine composition sample to be tested and the S peak is within ±10% of the relative retention time value of each characteristic chromatographic peak in the standard fingerprint of the Chinese medicine composition reference sample; and

[0160] (3) According to the similarity evaluation system of Chinese medicine chromatographic fingerprint, the similarity between the fingerprint of the Chinese medicine composition sample to be tested and the standard fingerprint of the Chinese medicine composition reference sample shall be calculated, and the similarity shall not be less than 0.90.

[0161] According to another aspect of the present invention, there is provided a use of the above-mentioned construction method or the above-mentioned quality control method in quality detection and / or quality evaluation and / or quality control of a traditional Chinese medicine composition comprising Belamcanda chinensis and Ephedra sinica.

[0162] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions or conditions recommended by the manufacturer.

[0163] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0164] The above features mentioned in the present invention or the features mentioned in the embodiments can be combined in any way. All the features disclosed in this patent specification can be used in combination with any combination form, and each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equal or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equal or similar features.

[0165] Example

[0166] The prescription of the Chinese medicinal composition containing Belamcanda chinensis and Ephedra sinica of the present invention is as follows: 9g Belamcanda chinensis; 9g Ephedra sinica; 6g ginger; 6g Asarum; 6g Aster; 6g Coltsfoot Flos; 15g Jujube; 9g Pinellia tuber; 3g Schisandra chinensis.

[0167] The preparation method of the Chinese medicine composition containing Belamcanda chinensis and Ephedra sinica is as follows: the above nine herbs are taken, placed in a multifunctional decoction pot, 8 times the amount of water is added to cover the herbs by 2-5 cm, soaked for 30 minutes, decocted once, boiled over high heat, then slowly decocted over low heat for 20 minutes, filtered through a 150-mesh sieve, 4 times the amount of water is added to the residues again, boiled over high heat, then slowly decocted over low heat for 15 minutes, the decoctions are combined, and the final decoction volume needs to be about 550 ml.

[0168] 1. Instruments and test drugs

[0169] Instruments: High performance liquid chromatograph Agilent 1290 chromatography system, including G1322A degasser, G1311A quaternary pump, G1313A autosampler, G1315B DAD diode array detector, G1316A column oven, ChemStation chromatography workstation;

[0170] High performance liquid chromatograph Waters 2695 chromatography system, including quaternary gradient infusion pump (Alliance 2695 model), 120-position high performance autosampler, imported column oven, Waters 2996 DAD diode array detector, Empower 3 chromatography management system;

[0171] Chromatographic columns: Column 1: Agilent 5TC-C18 5.0μm 4.6x250mm Serial No.731424; Column 2: GL Sciences InretSustain AQ-C18 5μm 4.6x250mm Serial No.23J0443146; Column 3: Agilent 5TC-C18 5.0μm 4.6x250mm Serial No.735516; Column 4: Agilent 5TC-C18 5.0μm4.6x250mm Serial No.735519; Methanol, acetonitrile, and phosphoric acid were of chromatographic grade, glacial acetic acid and formic acid were of analytical grade, and water was purified water.

[0172] Reference Material Information:

[0173] Neochlorogenic acid (batch number AY140002-202209, content 99.39%, China Food and Drug Inspection Institute); 6-gingerol (batch number 111833-202007, content 99.3%, China Food and Drug Inspection Institute).

[0174] 2. Solution preparation

[0175] 2.1. Preparation of reference solution

[0176] Accurately weigh appropriate amounts of neochlorogenic acid and 6-gingerol reference substances, respectively, and add methanol to prepare solutions of 96.2 μg / ml neochlorogenic acid and 16.9 μg / ml 6-gingerol; prepare reference substance solutions.

[0177] 2.2 Preparation of test solution

[0178] Take 10 ml of the sample (decoction) of the traditional Chinese medicine composition containing Belamcanda chinensis and Ephedra sinica, accurately measure it, put it in a stoppered conical flask, accurately add 10 ml of 50% methanol, seal it, shake it well, filter it, and take the filtrate to obtain the product.

[0179] 2.3 Preparation of negative solution

[0180] 2.3.1. Preparation of negative sample solution lacking Belamcanda chinensis: Weigh other medicinal ingredients according to the prescription, prepare negative sample lacking Belamcanda chinensis according to the test sample preparation method, and prepare negative sample solution according to "2.2. Preparation of test sample solution".

[0181] 2.3.2. Preparation of negative sample solution lacking ephedra: Weigh other medicinal ingredients according to the prescription, prepare a negative sample lacking ephedra according to the test sample preparation method, and prepare the negative sample solution according to "2.2. Preparation of test sample solution".

[0182] 2.3.3. Preparation of negative sample solution lacking ginger: Weigh other medicinal ingredients according to the prescription, prepare a negative sample lacking ginger according to the test sample preparation method, and prepare the negative sample solution according to "2.2. Preparation of test sample solution".

[0183] 2.3.4. Preparation of negative sample solution lacking Asarum: Weigh other medicinal ingredients according to the prescription, prepare a negative sample lacking Asarum according to the test sample preparation method, and prepare the negative sample solution according to "2.2. Preparation of test sample solution".

[0184] 2.3.5. Preparation of negative sample solution lacking Aster tataricus: Weigh other medicinal ingredients according to the prescription, prepare a negative sample lacking Aster tataricus according to the test sample preparation method, and prepare the negative sample solution according to "2.2. Preparation of test sample solution".

[0185] 2.3.6. Preparation of negative sample solution lacking coltsfoot: Weigh other medicinal ingredients according to the prescription, prepare a negative sample lacking coltsfoot according to the test sample preparation method, and prepare the negative sample solution according to "2.2. Preparation of test sample solution".

[0186] 2.3.7. Preparation of negative sample solution lacking Schisandra chinensis: Weigh other medicinal ingredients according to the prescription, prepare a negative sample lacking Schisandra chinensis according to the test sample preparation method, and prepare the negative sample solution according to "2.2. Preparation of test sample solution".

[0187] 2.3.8. Preparation of negative sample solution lacking jujube: Weigh other medicinal ingredients according to the prescription, prepare a negative sample lacking jujube according to the test sample preparation method, and prepare the negative sample solution according to "2.2. Preparation of test sample solution".

[0188] 2.3.9. Preparation of negative sample solution lacking Pinellia: Weigh other medicinal ingredients according to the prescription, prepare a negative sample lacking Pinellia according to the test sample preparation method, and prepare the negative sample solution according to "2.2. Preparation of test sample solution".

[0189] 2.4. Preparation of single-flavor solution

[0190] Take appropriate amounts of the nine single-ingredient slices respectively, put them into a stoppered conical flask, add 50 ml of water, heat and reflux for 30 minutes, cool, filter, and obtain the filtrate.

[0191] 3. Investigation of chromatographic conditions

[0192] 3.1. Initial chromatographic conditions

[0193] Determined by high performance liquid chromatography (General Rules 0512 of Part IV of the 2020 edition of the Chinese Pharmacopoeia).

[0194] The chromatographic conditions and system suitability test used octadecyl bonded silica gel as the filler (Agilent 5TC-C18 5.0 μm 4.6x250 mm); acetonitrile as mobile phase A, 0.1% phosphoric acid as mobile phase B; the flow rate was 1.0 mi / min, the column temperature was 25°C, and gradient elution was performed as specified in Table 1; the detection wavelength was 190-400 nm.

[0195] Table 1 Initial gradient

[0196] Time (min) Acetonitrile (%) 0.1% Phosphoric acid (%) 0～120 5→95 95→5

[0197] According to the separation of components in the compound, the chromatographic column, the best absorption wavelength and the optimized gradient conditions are selected, and finally a better detection method is obtained.

[0198] The results show that from the isobsorption line diagram of Agilent 5TC-C18 at 190-400nm, there are basically absorption fluorescence points at the middle and low end wavelengths. 254nm was selected for detection and analysis. In the liquid chromatogram at 254nm, there are chromatographic peaks in the first 90 minutes, and there are basically no characteristic peaks after 90 minutes. Therefore, considering that the acetonitrile ratio is within 68%, gradient 1 is designed.

[0199] 3.2. Mobile phase gradient selection

[0200] In order to make each main chromatographic peak in the chromatogram have better separation parameters and present better chromatographic behavior, the test sample solution was taken and the elution gradient was optimized. The optimized gradient program is shown in Table 2.

[0201] Table 2 Gradient 1

[0202] Time (min) Acetonitrile (%) 0.1 Phosphoric acid (%) 0→5 5 95 5→15 5→10 95→90 15→20 10→12 90→88 20→40 12→20 88→80 40→50 20→30 80→70 50→60 30→40 70→60 60→70 40→45 60→55 70→80 45→50 55→50 80→85 50→55 50→45 85→100 55→68 45→32 100→102 68→5 32→95 102→120 5 95

[0203] The results show that the isobsorptiometry of gradient 1 shows that the decoction of the Chinese medicine composition containing Belamcanda chinensis and Ephedra sinica has a chromatographic peak absorption fluorescence point in the range of 190-300nm, so the liquid chromatograms of 210nm, 230nm, 254nm, 280nm and 300nm are selected for comparison. At 280nm, the information of the chromatographic peak is more comprehensive, so the detection wavelength of this project is 280nm. In the 280nm liquid chromatogram, the separation of each peak is poor, and there is basically no characteristic peak after 80min. Considering that the proportion of acetonitrile is within 50%, optimization is considered and gradient 2 is designed.

[0204] Table 3 Gradient 2

[0205]

[0206]

[0207] The results showed that the separation of the characteristic peaks in the gradient 2 chromatogram was poor, so we continued to optimize the gradient and designed gradient 3.

[0208] Table 4 Gradient 3

[0209] Time (min) Acetonitrile (%) 0.1 Phosphoric acid (%) 0→5 3 97 5→15 3→5 97→95 15→20 5→10 95→90 20→40 10→15 90→85 40→45 15→22 85→78 45→60 22→27 78→73 60→70 27→40 73→60 70→95 40→50 60→50

[0210] The results showed that the characteristic peaks in the 50-60 minute range of the gradient 3 chromatogram were not separated, so we continued to optimize the gradient and designed gradient 4.

[0211] Table 5 Gradient 4

[0212]

[0213] The results showed that the characteristic peaks in the gradient 4 chromatogram from 53 to 55 minutes were not separated, and there were basically no characteristic peaks from 55 to 65 minutes, so the gradient was further optimized and gradient 5 was designed.

[0214] Table 6 Gradient 5

[0215] Time (min) Acetonitrile (%) 0.1 Phosphoric acid (%) 0→10 5 95 10→14 5→10 95→90 14→34 10→15 90→85 34→39 15→22 85→78 39→47 22→23 78→77 47→55 23 77 55→60 23→35 77→65 60→65 35→43 65→57 65→75 43→50 57→50

[0216] The results showed that the characteristic peaks in the gradient 5 chromatogram from 50 to 52 minutes were not separated, and there were basically no characteristic peaks from 52 to 62 minutes. Therefore, the gradient was further optimized, and the column temperature was appropriately increased to 30°C to design gradient 6.

[0217] Table 7 Gradient 6 (30°C)

[0218] Time (min) Acetonitrile (%) 0.1 Phosphoric acid (%) 0→10 5 95 10→14 5→10 95→90 14→34 10→15 90→85 34→39 15→22 85→78 39→47 22→23 78→77 47→53 23 77 53→60 23→43 77→57 60→70 43→50 57→50

[0219] The results showed that the separation of characteristic peaks at 48-50 minutes in the chromatogram of gradient 6 was better than that of gradient 5, but its separation was still poor, and there were basically no characteristic peaks at 34-40 minutes and 52-60 minutes. Therefore, the gradient was further optimized, and the column temperature was appropriately increased to 35°C to design gradient 7.

[0220] Table 8 Gradient 7 (35°C)

[0221] Time (min) Acetonitrile (%) 0.1 Phosphoric acid (%) 0→8 5 95 8→13 5→10 95→90 13→32 10→15 90→85 32→34 15→22 85→78 34→50 22 78 50→55 22→45 78→55 55→70 45→50 55→50

[0222] The results showed that the characteristic peaks in the gradient 7 chromatogram had good separation at 44-48 minutes, but poor separation at 18-30 minutes, and basically no characteristic peaks at 48-54 minutes. Therefore, we continued to optimize the gradient, considered lowering the column temperature to 30°C, and designed gradient 8.

[0223] Table 9 Gradient 8 (30°C)

[0224] Time (min) Acetonitrile (%) 0.1 Phosphoric acid (%) 0→8 5 95 8→13 5→10 95→90 13→19 10→11 90→89 19→22 11 89 22→30 11→12 89→88 30→33 12→15 88→85 33→35 15→22 85→78 35→45 22→23 78→77 45→53 23 77 53→57 23→43 77→57 57→67 43→50 57→50 67→70 50→54 50→46

[0225] The results showed that the separation of characteristic peaks at 46-48 minutes and 66-70 minutes in the chromatogram of gradient 8 was worse than that of gradient 7, but the separation of characteristic peaks at 18-30 minutes was better than that of gradient 7. Therefore, the gradient was further optimized and gradient 9 was designed.

[0226] Table 10 Gradient 9 (30°C)

[0227] Time (min) Acetonitrile (%) 0.1 Phosphoric acid (%) 0→8 5 95 8→13 5→10 95→90 13→19 10→11 90→89 19→22 11 89 22→30 11→12 89→88 30→32 12→22 88→78 32→42 22 78 42→44 22→23 78→77 44→49 23 77 49→51 23→43 77→57 51→61 43→50 57→50 61→64 50→53 50→47 64→67 53→55 47→45 67→70 55→5 45→95

[0228] The results showed that the separation of characteristic peaks at 44-48 minutes and 62-66 minutes in the chromatogram of gradient 9 was poor. After comprehensive comparison of the chromatograms of gradients 7, 8, and 9, the column temperature was 35°C, and the gradient was further optimized to design gradient 10.

[0229] Table 11 Gradient 10 (35°C)

[0230]

[0231]

[0232] The results show that under the gradient 10 elution program in Table 11, the baseline is stable, the main chromatographic peaks are well separated, which has met the requirements of the fingerprint spectrum, and the analysis time is within 70 minutes. Therefore, the gradient 10 chromatographic conditions are temporarily selected for fingerprint spectrum determination.

[0233] Determined by high performance liquid chromatography (General Rules 0512 of Part IV of the 2020 edition of the Chinese Pharmacopoeia).

[0234] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler; acetonitrile was used as mobile phase A, 0.1% phosphoric acid was used as mobile phase B, and gradient elution was performed according to the provisions in Table 11; the flow rate was 1.0 ml / min, the column temperature was 35°C, and the detection wavelength was 280 nm. The theoretical plate number calculated based on the 6-gingerol peak should not be less than 5000.

[0235] Preparation of test solution: Take 10 ml of decoction, accurately measure, place in a stoppered conical flask, accurately add 10 ml of 50% methanol, stopper, shake well, filter, and take the filtrate to obtain.

[0236] Determination method: Accurately pipette 10μl of the test solution, inject it into the liquid chromatograph, and determine the result.

[0237] 4. Validation of analytical methods

[0238] The fingerprint spectrum established above was systematically methodologically validated in accordance with the 9101 Analytical Method Validation Guidelines of Part IV of the 2020 Edition of the Chinese Pharmacopoeia and the National Drug Standard Work Manual (Fourth Edition).

[0239] 4.1. Exclusivity

[0240] Prepare negative blank sample solution according to the test solution preparation method, test it according to the fingerprint method, and record the chromatogram.

[0241] The system parameters of the fingerprint spectrum of the whole Chinese medicine composition containing Belamcanda chinensis and Ephedra sinica are shown in Table 13.

[0242] Table 13 System parameters of the fingerprint of the whole prescription of the Chinese medicine composition containing Belamcanda chinensis and Ephedra sinica

[0243]

[0244]

[0245] The results show that the chromatogram of the Chinese medicine composition benchmark sample containing Belamcanda and Ephedra mainly presents 19 peaks, peak 1 belongs to jujube and Belamcanda; peak 2 belongs to jujube, Pinellia and Schisandra; peak 3 belongs to coltsfoot, peak 3 is new chlorogenic acid; peak 4, peak 9-12, peak 14-15 belong to Belamcanda, peak 6 and peak 13 belong to Ephedra; peak 5 belongs to Belamcanda and Aster; peak 7 belongs to coltsfoot and Aster; peak 8 and peak 19 belong to Asarum; peak 16 and peak 18 belong to Aster; peak 17 belongs to ginger; peak 17 is 6-gingerol, negative without interference, and the method has good specificity. The specific summary is shown in Table 14.

[0246] Table 14 Chromatographic peak summary

[0247]

[0248]

[0249] 4.2 Precision

[0250] 4.2.1. Instrument precision test

[0251] The same test solution was sampled 6 times continuously, the chromatogram was recorded, and the similarity of each characteristic peak was calculated. The results showed that the similarity of the instrument precision of this method was greater than 0.90, indicating that the instrument precision of this method was good.

[0252] 4.2.2 Intermediate precision

[0253] Two personnel prepared 6 test solutions according to the above conditions, respectively, injected and tested on Agilent HPLC and Waters HPLC, recorded chromatograms, and calculated the similarity of each characteristic peak. The results showed that the similarity of the intermediate precision of each characteristic peak was greater than 0.90, which met the requirements.

[0254] 4.2.3 Repeatability test

[0255] Take 6 samples from the same batch, prepare the test solution according to the method of "2.2, Preparation of Test Solution", record the chromatogram, and calculate the similarity of each characteristic peak. The results show that the similarity of the repeatability of each characteristic peak is greater than 0.90, which meets the requirements.

[0256] 4.3 Durability

[0257] 4.3.1 Stability Study

[0258] Take the same test solution and inject it at 0, 3h, 6h, 9h, 12h, 24h, 36h, 48h and 54h. Record the chromatogram, calculate the similarity of each characteristic peak, and examine the stability of the test solution. The results show that within 54h of stability, the similarity of each characteristic peak is greater than 0.90, which meets the requirements, indicating that it is stable within 54h.

[0259] 4.3.2 Investigation of different flow rates

[0260] Take the same test solution and test it at different flow rates of 1.0±0.1ml / min, record the chromatogram, and calculate the similarity of each characteristic peak. The results show that the similarity results of different flow rates are all greater than 0.90, and the flow rate durability is good and meets the regulations.

[0261] 4.3.3 Investigation of different column temperatures

[0262] Take the same test solution, test at 35±3℃, record the chromatogram, calculate the similarity of each characteristic peak, and perform similarity matching calculation with the standard reference spectrum. The results show that the similarity of each characteristic peak is greater than 0.90, and the column temperature durability is good and meets the requirements.

[0263] 4.3.4 Different chromatographic columns

[0264] The same test solution was taken and tested under different chromatographic columns (chromatographic column 1: Agilent 5TC-C18 5.0μm 4.6x250mm No.731424), (chromatographic column 2: Agilent 5TC-C18 5.0μm 4.6x250mm No.735516), (chromatographic column 3: Agilent 5TC-C18 5.0μm 4.6x250mm No.735519), and (chromatographic column 4: GL Sciences InretSustain AQ-C18 5μm 4.6x250mm SerialNo.23J0443146). The chromatograms were recorded and the similarity matching calculation was performed with the standard reference spectrum of the traditional Chinese medicine composition containing Belamcanda chinensis and Ephedra sinica. The results showed that the AQ-C18 chromatogram had poor separation of chromatographic peaks at 42-50 minutes and 65-70 minutes, so the Aglient 5TC C18 column was still used for verification. The similarity results of different chromatographic columns were all greater than 0.90, which met the requirements.

[0265] 4.3.5. Investigation of mobile phases with different acid types

[0266] The results show that under the conditions of 0.1% formic acid and 0.1% acetic acid, the spectrum t R =23.290 was lost, so phosphoric acid was used as the acid type of the mobile phase.

[0267] 4.3.6. Investigation of mobile phases with different acid concentrations

[0268] Take the same test solution, test it at the concentration of 0.09%, 0.10% and 0.11% of phosphoric acid solution in the mobile phase, record the chromatogram, and perform similarity matching calculation with the standard reference spectrum of the Chinese medicine composition containing Belamcanda and Ephedra. The results show that under different acid concentration conditions, the similarity results of each common peak are greater than 0.90, which meets the requirements.

[0269] 5. Fingerprint method confirmation

[0270] According to the results of the above methodological investigation, among the 19 common peaks of the established fingerprint, each chromatographic peak is less affected by factors such as different brands of HPLC, different chromatographic columns, different column temperatures and flow rates, and the similarities of each characteristic peak of the instrument precision, intermediate precision, repeatability test and stability test are all greater than 0.9, indicating that the method has good durability, is stable and reliable, and can be used for fingerprint determination.

[0271] Based on the above results, the fingerprint method is as follows:

[0272] Determined by high performance liquid chromatography (General Rules 0512 of Part IV of the 2020 edition of the Chinese Pharmacopoeia).

[0273] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel was used as filler; acetonitrile was used as mobile phase A, and 0.1% phosphoric acid was used as mobile phase B; gradient elution was performed according to the provisions in Table 11; the flow rate was 1.0 ml / min, the column temperature was 35°C, and the detection wavelength was 280 nm.

[0274] Preparation of reference solution: Take appropriate amount of neochlorogenic acid and 6-gingerol reference substances, weigh them accurately, add methanol to make 1 ml of reference solution containing 50 μg of neochlorogenic acid and 50 μg of 6-gingerol respectively.

[0275] Preparation of test solution: Accurately measure 10 ml of decoction, place in a stoppered conical flask, accurately add 10 ml of 50% methanol, stopper tightly, shake well, filter, and obtain the filtrate.

[0276] Determination method: Accurately pipette 10μl of the test solution, inject it into the liquid chromatograph, and determine it.

[0277] 6. Fingerprint standard regulations are determined

[0278] 20 batches of the Chinese medicine composition containing Belamcanda and Ephedra of the present invention are matched in batches as shown in FIG. Figure 2 It was determined that there were 19 characteristic peaks in the chromatogram of the test sample, of which peak 12 was the S peak. According to the similarity evaluation system of the chromatographic fingerprint of traditional Chinese medicine, the similarity between the fingerprint of the test sample and the fingerprint of the control should not be less than 0.90.

[0279] The embodiments of the present invention are described in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, changes or deformations made by those skilled in the art based on the ideas of the present invention, the specific implementation methods and application scope of the present invention, all belong to the scope of protection of the present invention. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A method for constructing a fingerprint of a Chinese medicine composition comprising Belamcanda chinensis and Ephedra sinica, characterized in that: The construction method comprises the following steps: Preparation of a Chinese medicine composition test solution: taking an appropriate amount of a decoction of a Chinese medicine composition containing Belamcanda chinensis and Ephedra sinica, placing the decoction in a container, adding an alcohol solution, plugging the container, shaking, filtering, and taking a subsequent filtrate to obtain the Chinese medicine composition test solution; wherein the Chinese medicine composition comprises Belamcanda chinensis, Ephedra sinica, ginger, Asarum, Aster tataricus, Coltsfoot flos, jujube, Pinellia ternata, and Schisandra chinensis; Preparation of reference solution: weigh appropriate amounts of neochlorogenic acid and 6-gingerol reference substances, add methanol to prepare reference solutions with neochlorogenic acid and 6-gingerol concentrations of 5 to 200 μg / ml, respectively; Obtaining a fingerprint of the Chinese medicine composition according to the results of high performance liquid chromatography detection of the Chinese medicine composition test solution and the reference solution; The chromatographic conditions of the high performance liquid phase detection are as follows: using an Agilent 5TC-C18 chromatographic column, the mobile phase A is acetonitrile, the mobile phase B is a 0.095% to 0.105% phosphoric acid aqueous solution, the gradient elution program is: 0 to 8 min, 95% B; 8 to 13 min, 95% B→90% B; 13 to 20 min, 90% B→89% B; 20 to 24 min, 89% B; 24 to 32 min, 89% B→88% B; 32 to 34 min, 88% B→78% B; 34 to 50 min, 78% B; 50 to 55 min, 78% B→55% B; 55 to 70 min, 55%→50% B; the flow rate is 0.5 to 1.5 ml / min, the column temperature is 20 to 50° C., the detection wavelength is 280 nm, and the injection volume is 1 to 20 μl; Wherein, when the detection wavelength is 280nm, the fingerprint spectrum includes peaks 1-19, wherein peak 12 is used as a reference peak, and the average relative retention times of peaks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18 and 19 are 0.152-0.168, 0.209-0.231, 0.342-0.378, 0.38-0.42, 0. 3895-0.4305, 0.456-0.504, 0.4655-0.5145, 0.5225-0.5775, 0.855-0.945, 0.8835-0.9765, 0.9405-1.0395, 1.1495-1.2705, 1.216-1.344, 1.3015-1.4385, 1.3205-1.4595, 1.3395-1.4805, 1.3585-1.5015 and 1.3775-1.5225; Among them, when the detection wavelength is 280nm, peak 3 is neochlorogenic acid and peak 17 is 6-gingerol; The preparation method of the decoction of the Chinese medicinal composition containing Belamcanda chinensis and Ephedra sinica comprises: (a) weighing appropriate amounts of Belamcanda chinensis, Ephedra sinica, ginger, Asarum, Aster, Coltsfoot Flos, Jujube, Pinellia ternata and Schisandra chinensis and placing them in a container, adding a first amount of water for soaking, decocting with high heat until boiling, then using a low heat for the first decoction, filtering through a filter to obtain a first filtrate and a filter residue; (b) adding a second amount of water to the filter residue, decocting with high heat until boiling, then using a low heat for the second decoction, filtering through a filter to obtain a second filtrate, combining the first filtrate and the second filtrate to obtain the decoction of the Chinese medicinal composition containing Belamcanda chinensis and Ephedra sinica.

2. The construction method according to claim 1, characterized in that: The ratio of the sum of the masses of the Belamcanda chinensis, Ephedra sinica, ginger, Asarum, Aster tataricus, Coltsfoot flos, Jujube, Pinellia ternata and Schisandra chinensis to the mass / volume of the first water is 0.1-0.15, in g / ml.

3. The construction method according to claim 2, characterized in that: The ratio of the sum of the masses of the Belamcanda chinensis, Ephedra sinica, Ginger, Asarum, Aster tataricus, Coltsfoot flos, Jujube, Pinellia ternata and Schisandra chinensis to the mass / volume of the first water is 0.11875-0.13125, in g / ml.

4. The construction method according to claim 1, characterized in that: The container is a multifunctional medicine decocting pot.

5. The construction method according to claim 1, characterized in that: The soaking time is 20 to 40 minutes.

6. The construction method according to claim 5, characterized in that: The soaking time is 28.5 to 31.5 minutes.

7. The construction method according to claim 1, characterized in that: The first decocting time over low heat is 10 to 30 minutes.

8. The construction method according to claim 7, characterized in that: The time for the first decocting over low heat is 19 to 21 minutes.

9. The construction method according to claim 1, characterized in that: The filter is a 150-mesh screen.

10. The construction method according to claim 1, characterized in that: The ratio of the sum of the masses of the Belamcanda chinensis, Ephedra sinica, Ginger, Asarum, Aster tataricus, Coltsfoot Flos, Jujube, Pinellia ternata and Schisandra chinensis to the mass / volume of the second water is 0.1-0.5, in g / ml.

11. The construction method according to claim 10, characterized in that: The ratio of the sum of the masses of the Belamcanda chinensis, Ephedra sinica, ginger, Asarum, Aster tataricus, Coltsfoot flos, Jujube, Pinellia ternata and Schisandra chinensis to the mass / volume of the second water is 0.2375-0.2625, in g / ml.

12. The construction method according to claim 1, characterized in that: The second decocting time over low heat is 5 to 30 minutes.

13. The construction method according to claim 12, characterized in that: The second decocting time over low heat is 14.25 to 15.75 minutes.

14. The construction method according to claim 1, characterized in that: The volume of the decoction of the Chinese medicinal composition containing Belamcanda chinensis and Ephedra sinica is 400-700 ml.

15. The construction method according to claim 14, characterized in that: The volume of the decoction of the traditional Chinese medicine composition containing Belamcanda chinensis and Ephedra sinica is 522.5-577.5 ml.

16. The construction method according to claim 1, characterized in that: The volume / volume ratio of the decoction of the traditional Chinese medicine composition containing Belamcanda chinensis and Ephedra sinica to the first water is 0.5 to 1.

0.

17. The construction method according to claim 16, characterized in that: The volume / volume ratio of the decoction of the traditional Chinese medicine composition containing Belamcanda chinensis and Ephedra sinica to the first water is 0.83695-0.92505.

18. The construction method according to claim 1, characterized in that: The mass ratios of the Belamcanda chinensis, the Ephedra sinica, the Ginger, the Asarum, the Aster tataricus, the Coltsfoot Flos, the Jujube, the Pinellia ternata and the Schisandra chinensis are (2-4): (2-4): (1-3): (1-3): (1-3): (1-3): (3-10): (2-4): (0.2-2).

19. The construction method according to claim 18, characterized in that: The mass ratios of the Belamcanda chinensis, the Ephedra sinica, the Ginger, the Asarum, the Aster tataricus, the Coltsfoot Flos, the Jujube, the Pinellia ternata and the Schisandra chinensis are (2.5-3.5): (2.5-3.5): (1.5-2.5): (1.5-2.5): (1.5-2.5): (1.5-2.5): (4-6): (2.5-3.5): (0.5-1.5).

20. The construction method according to claim 19, characterized in that: The mass ratios of the Belamcanda chinensis, the Ephedra sinica, the Ginger, the Asarum, the Aster tataricus, the Coltsfoot Flos, the Jujube, the Pinellia ternata and the Schisandra chinensis are (2.85-3.15): (2.85-3.15): (1.9-2.1): (1.9-2.1): (1.9-2.1): (1.9-2.1): (4.75-5.25): (2.85-3.15): (0.95-1.05).

21. The construction method according to claim 20, characterized in that: The mass range of the Belamcanda chinensis is 5 to 15 g.

22. The construction method according to claim 21, characterized in that: The mass range of the Belamcanda chinensis is 8.55-9.45 g.

23. The construction method according to claim 1, characterized in that: The mass range of the ephedra is 5 to 15 g.

24. The construction method according to claim 23, characterized in that: The mass range of the ephedra is 8.55 to 9.45 g.

25. The construction method according to claim 1, characterized in that: The weight range of the ginger is 1 to 10 g.

26. The construction method according to claim 25, characterized in that: The weight range of the ginger is 5.7-6.3 g.

27. The construction method according to claim 1, characterized in that: The mass range of the asarum is 1 to 10 g.

28. The construction method according to claim 27, characterized in that: The mass range of the asarum is 5.7 to 6.3 g.

29. The construction method according to claim 1, characterized in that: The mass range of the Aster is 1 to 10 g.

30. The construction method according to claim 29, characterized in that: The mass range of the Aster is 5.7-6.3 g.

31. The construction method according to claim 1, characterized in that: The mass range of the coltsfoot flower is 1 to 10 g.

32. The construction method according to claim 31, characterized in that: The mass range of the coltsfoot flower is 5.7 to 6.3 g.

33. The construction method according to claim 1, characterized in that: The mass range of the jujube is 10 to 30 g.

34. The construction method according to claim 33, characterized in that: The mass range of the jujube is 14.25-15.75g.

35. The construction method according to claim 1, characterized in that: The number of the jujubes is 2 to 4.

36. The construction method according to claim 35, characterized in that: The number of the jujubes is 3.

37. The construction method according to claim 1, characterized in that: The mass range of the pinellia is 5 to 15 g.

38. The construction method according to claim 37, characterized in that: The mass range of the pinellia is 8.55 to 9.45 g.

39. The construction method according to claim 1, characterized in that: The mass range of the Schisandra chinensis chinensis is 1 to 10 g.

40. The construction method according to claim 39, characterized in that: The mass range of the Schisandra chinensis chinensis is 2.85-3.15 g.

41. The construction method according to claim 1, characterized in that: In the preparation of the test solution of the traditional Chinese medicine composition, the container is a conical flask.

42. The construction method according to claim 41, characterized in that: The conical flask is a conical flask with a stopper.

43. The construction method according to claim 1, characterized in that: The alcohol solution is a methanol solution.

44. The construction method according to claim 1, characterized in that: The volume percentage concentration of the alcohol solution is 10% to 90%.

45. The construction method according to claim 44, characterized in that: The volume percentage concentration of the alcohol solution is 47.5% to 52.5%.

46. ​​The construction method according to claim 1, characterized in that: The volume / volume ratio of the alcohol solution to the decoction of the traditional Chinese medicine composition containing Belamcanda chinensis and Ephedra sinica is 0.8-1.

2.

47. The construction method according to claim 46, characterized in that: The volume / volume ratio of the alcohol solution to the decoction of the traditional Chinese medicine composition containing Belamcanda chinensis and Ephedra sinica is 0.95-1.

05.

48. The construction method according to claim 1, characterized in that: The concentration of neochlorogenic acid in the reference solution is 91.39-101.01 μg / ml.

49. The construction method according to claim 1, characterized in that: The concentration of 6-gingerol in the reference solution is 16.055-17.745 μg / ml.

50. The construction method according to claim 1, characterized in that: The flow rate of the high performance liquid phase detection is 0.8-1.2 ml / min.

51. The construction method according to claim 50, characterized in that: The flow rate of the high performance liquid phase detection is 0.95-1.05 ml / min.

52. The construction method according to claim 1, characterized in that: The column temperature is 30-40°C.

53. The construction method according to claim 52, characterized in that: The column temperature is 33.25-36.75°C.

54. The construction method according to claim 1, characterized in that: The injection volume is 5 to 15 μl.

55. The construction method according to claim 54, characterized in that: The injection volume is 9.5-10.5 μl.

56. The construction method according to claim 1, characterized in that: The theoretical plate number of the chromatographic peak corresponding to the 6-gingerol is not less than 5000.

57. The construction method according to claim 1, characterized in that: The specifications of the chromatographic column are: column length 250 mm, inner diameter 4.6 mm, and particle size 5 μm.

58. The construction method according to claim 1, characterized in that When the detection wavelength is 280 nm, the fingerprint spectrum includes peaks 1 to 19, and the retention times of peaks 1 to 19 correspond to 7.26655 to 8.03145 min, 9.92465 to 10.96935 min, 16.3514 to 18.0726 min, 17.85335 to 19.73265 min, 18.3768 to 20.3112 min, 21.46715 to 23.72685 min, 22.1255 to 24.4545 min, 24.56415 to 27.14985 min, 40.3 598~44.6082min, 41.51975~45.89025min, 44.2149~48.8691min, 44.8115~49.5285min, 54.37895~60.10305min, 57.33535~63.37065min, 61.5201~67.9959min, 62.45775~69.03225min, 63.1617~69.8103min, 64.239~71.001min and 65.1871~72.0489min.

59. The construction method according to claim 1, characterized in that: When the detection wavelength is 280nm, peak No. 1 comes from jujube and Belamcanda medicinal materials, peak No. 2 comes from jujube, Pinellia and Schisandra chinensis medicinal materials, peak No. 3 comes from coltsfoot medicinal materials, peak No. 4 comes from Belamcanda medicinal materials, peak No. 5 comes from Belamcanda and Aster medicinal materials, peak No. 6 comes from Ephedra medicinal materials, peak No. 7 comes from Coltsfoot and Aster medicinal materials, peak No. 8 comes from Asarum medicinal materials, peak No. 9 comes from Belamcanda medicinal materials, peak No. 10 comes from Belamcanda medicinal materials, peak No. 11 comes from Belamcanda medicinal materials, peak No. 12 comes from Belamcanda medicinal materials, peak No. 13 comes from Ephedra medicinal materials, peak No. 14 comes from Belamcanda medicinal materials, peak No. 15 comes from Belamcanda medicinal materials, peak No. 16 comes from Aster medicinal materials, peak No. 17 comes from ginger medicinal materials, peak No. 18 comes from Aster medicinal materials, and peak No. 19 comes from Asarum medicinal materials.

60. A quality control method for a Chinese medicine composition comprising Belamcanda chinensis and Ephedra sinica, characterized in that: The quality control method comprises the following steps: (1) Establishing a standard fingerprint of a reference sample of a Chinese medicine composition according to the fingerprint construction method described in any one of claims 1 to 59; (2) taking a sample solution of the Chinese medicine composition, and testing it according to the chromatographic conditions in the fingerprint construction method according to any one of claims 1 to 59 to obtain a fingerprint of the Chinese medicine composition sample to be tested; as well as (3) Compare the fingerprint of the Chinese medicine composition sample obtained in step (2) with the standard fingerprint of the Chinese medicine composition reference sample obtained in step (1). If it meets the requirements, it is a qualified product, and if it does not meet the requirements, it is an unqualified product.

61. The quality control method according to claim 60, characterized in that: The compliance requirements include one or more of the following: (1) The fingerprint of the Chinese medicine composition sample to be tested presents 19 characteristic chromatographic peaks, and the retention time of each characteristic chromatographic peak is within ±10% of the retention time value of the corresponding reference chromatographic peak in the standard fingerprint of the Chinese medicine composition reference sample; (2) With peak No. 12 as the S peak, the relative retention time of each characteristic chromatographic peak in the fingerprint of the Chinese medicine composition sample to be tested and the S peak is within ±10% of the relative retention time value of each characteristic chromatographic peak in the standard fingerprint of the Chinese medicine composition reference sample; as well as (3) According to the similarity evaluation system of Chinese medicine chromatographic fingerprint, the similarity between the fingerprint of the Chinese medicine composition sample to be tested and the standard fingerprint of the Chinese medicine composition reference sample is calculated, and the similarity shall not be less than 0.

90.

62. Use of the construction method according to any one of claims 1 to 59 or the quality control method according to claim 60 or 61 in quality detection and / or quality evaluation and / or quality control of a Chinese medicine composition comprising Belamcanda chinensis and Ephedra sinica.

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