A fingerprint construction method of a traditional Chinese medicine composition containing Atractylodes macrocephala and its application

Abstract

The present invention discloses a method for constructing a fingerprint of a Chinese medicine composition containing Atractylodes macrocephala, 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 Atractylodes macrocephala, 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 containing Atractylodes macrocephala and an application thereof. Background Art

[0002] In-depth research and development of classic Chinese medicine prescriptions is the golden key to tapping 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 containing Atractylodes macrocephala 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 containing Atractylodes macrocephala, and the construction 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 Atractylodes macrocephala, shaking it well, filtering it, and taking a filtrate to obtain a test solution of the Chinese medicine composition; wherein the Chinese medicine composition comprises ginseng, astragalus, Atractylodes macrocephala, licorice, angelica, chuanxiong, white peony root, rehmannia root, chuanxiong, glutinous rice, scutellaria baicalensis, and amomum villosum;

[0008] Preparation of reference solution: weigh appropriate amounts of atractylodes lactone III, paeoniflorin, cryptochlorogenic acid, 4,5-o-dicaffeoylquinic acid, baicalin, octoprolol A, and ammonium glycyrrhizinate reference substances, add methanol to prepare reference solution with concentrations of atractylodes lactone III, paeoniflorin, cryptochlorogenic acid, 4,5-o-dicaffeoylquinic acid, baicalin, octoprolol A, and ammonium glycyrrhizinate of 10 to 100 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: 0-10 min, 97% B→89% B; 10-14 min, 89% B→85% B; 14-18 min, 85% B→80% B; 18-32 min, 80% B→73% B; 32-42 min, 73% B→66% B; 42-44 min, 73% B→66% B; 45-50 min, 80% B→80% B; 47-51 min, 80% B→73% B; 48-64 min, 73% B→66% B; 50-51 min, 80% B→80% B; 51-52 min, 80% B→73% B; 52-53 min, 80% B→80% B; 54-55 min, 80% B→73% B; 56-57 min, 80% B→80% B; 58-59 min, 80% B→73% B; 59-64 min, 80% B→80% B; 60-71 min, 61-64 min, 61-65 min, 66-71 min, 67-71 min, 68-71 min, 69-80 min, 70-80% B; 71-73 min, 71-73 min, 73-73 min, 73-73 min, 73-73 min, 7 in, 66% B→65% B; 44~46min, 65% B→63% B; 46~48min, 63% B→58% B; 48~55min, 58%→50% B; 55~57min, 50%→46% B; 57~62min, 46%→30% B; 62~65min, 30%→97% B; 65~80min, 97% B, the flow rate is 0.5~1.5ml / min, the column temperature is 10~40℃, the detection wavelength is 190~400nm, and the injection volume is 1~20μl.

[0011] Furthermore, the preparation method of the decoction of the Chinese medicine composition containing Atractylodes macrocephala comprises: (a) weighing appropriate amounts of ginseng, astragalus, Atractylodes macrocephala, licorice, angelica, Chuanxiong, white peony root, rehmannia root, chuanxiong, glutinous rice, Scutellaria baicalensis and Amomum villosum into a container, adding a first 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 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, and combining the first filtrate and the second filtrate to obtain the decoction of the Chinese medicine composition containing Atractylodes macrocephala.

[0012] Furthermore, the ratio of the sum of the masses of ginseng, astragalus, atractylodes, liquorice, angelica, chuanxiong, white peony root, rehmannia root, chuanxiong, glutinous rice, scutellaria and amomum to the mass / volume (g / ml) of the first water is 0.05-0.15, for example, about 0.1.

[0013] Furthermore, the soaking time is 20 to 40 minutes, for example, about 30 minutes.

[0014] Furthermore, the first decocting time on low heat is 30 to 90 minutes, for example, about 50 minutes.

[0015] Furthermore, the filter is a 120-mesh screen.

[0016] Furthermore, the ratio of the sum of the masses of ginseng, astragalus, atractylodes, liquorice, angelica, chuanxiong, white peony root, rehmannia root, chuanxiong, glutinous rice, scutellaria and amomum to the mass / volume (g / ml) of the second water is 0.1-0.15, for example, about 0.125.

[0017] Furthermore, the second decocting time on low heat is 30 to 90 minutes, for example, about 45 minutes.

[0018] Furthermore, the volume of the decoction of the traditional Chinese medicine composition containing Atractylodes macrocephala is 300-600 ml, for example, about 450 ml.

[0019] Furthermore, the volume / volume ratio of the first water to the decoction of the traditional Chinese medicine composition containing Atractylodes macrocephala is 0.8-1.0, for example, about 0.837.

[0020] Furthermore, the mass ratios of the ginseng, the astragalus, the atractylodes, the liquorice, the angelica, the ligusticum, the chuanxiong, the white peony root, the rehmannia root, the dipsacus asper, the glutinous rice, the scutellaria and the amomum are (2-4): (2-4): (5-7): (1-3): (2-4): (1.5-3.5): (1.5-3.5): (1.5-3.5): (2-4): (5-7): (2-4): (0.5-2.5).

[0021] Furthermore, the mass ratios of the ginseng, the astragalus, the atractylodes, the liquorice, the angelica, the ligusticum, the chuanxiong, the white peony root, the rehmannia root, the dipsacus asper, the glutinous rice, the scutellaria and the amomum are (2.5-3.5): (2.5-3.5): (5.5-6.5): (1.5-2.5): (2.5-3.5): (2-3): (2-3): (2-3): (2-3): (2.5-3.5): (5.5-6.5): (2.5-3.5): (1-2).

[0022] Furthermore, the mass ratio of the ginseng, the astragalus, the atractylodes, the liquorice, the angelica, the ligusticum, the ligusticum chuanxiong, the white peony root, the rehmannia root, the dipsacus root, the glutinous rice, the scutellaria and the amomum is about 3: about 3: about 6: about 2: about 3: about 2.4: about 2.4: about 2.4: about 3: about 6: about 3: about 1.5.

[0023] Furthermore, the mass of the ginseng ranges from 1 to 10 g, for example, about 3 g.

[0024] Furthermore, the mass range of the Astragalus is 1 to 10 g, for example, about 3 g.

[0025] Furthermore, the mass range of the Atractylodes macrocephala is 1 to 10 g, for example, about 6 g.

[0026] Furthermore, the mass of the liquorice is in the range of 1 to 5 g, for example, about 2 g.

[0027] Furthermore, the weight of the angelica sinensis is in the range of 1 to 10 g, for example, about 3 g.

[0028] Furthermore, the mass range of the Chuanxiong is 1 to 10 g, for example, about 2.4 g.

[0029] Furthermore, the mass range of the white peony root is 1 to 10 g, for example, about 2.4 g.

[0030] Furthermore, the mass of the Rehmannia root is in the range of 1 to 10 g, for example, about 2.4 g.

[0031] Furthermore, the mass range of the Dipsacus asper is 1 to 10 g, for example, about 3 g.

[0032] Furthermore, the mass of the glutinous rice ranges from 1 to 10 g, for example, about 6 g.

[0033] Furthermore, the mass range of the Scutellaria baicalensis is 1 to 10 g, for example, about 3 g.

[0034] Furthermore, the mass range of the Amomum villosum is 0.5 to 5 g, for example, about 1.5 g.

[0035] Furthermore, the licorice is roasted licorice.

[0036] Furthermore, the Rehmannia root is Rehmannia root prepared.

[0037] Furthermore, the Rehmannia root is Rehmannia root prepared by steaming.

[0038] Furthermore, in the preparation of the test solution, the filtration is microporous membrane filtration.

[0039] Furthermore, the pore size of the microporous filter membrane is 0.22 μm.

[0040] Furthermore, the concentration of atractylodes lactone III in the reference solution was about 20.12 μg / ml.

[0041] Furthermore, the concentration of cryptochlorogenic acid in the reference solution is about 20.62 μg / ml.

[0042] Furthermore, the concentration of 4,5-o-dicaffeoylquinic acid in the reference solution is about 22.21 μg / ml.

[0043] Furthermore, the concentration of occidental lactone A in the reference solution is about 10.04 μg / ml.

[0044] Furthermore, the concentration of paeoniflorin in the reference solution is about 60.17 μg / ml.

[0045] Furthermore, the concentration of baicalin in the reference solution was about 54.92 μg / ml.

[0046] Furthermore, the concentration of ammonium glycyrrhizinate in the reference solution is about 49.94 μg / ml.

[0047] Furthermore, the flow rate of the HPLC detection is 0.8-1.2 ml / min, for example, about 1.0 ml / min.

[0048] Furthermore, the column temperature is 25-35°C, for example, about 30°C.

[0049] Furthermore, the detection wavelength is 200-250 nm, for example, about 230 nm.

[0050] Furthermore, the injection volume is 5-15 μl, for example, about 10 μl.

[0051] Furthermore, the theoretical plate number of the chromatographic peak corresponding to the baicalin is not less than 2500.

[0052] Furthermore, the chromatographic column is a GL Sciences Inertsustain AQ-C18 chromatographic column.

[0053] Furthermore, the specifications of the chromatographic column are: column length 250 mm, inner diameter 4.6 mm, and particle size 5 μm.

[0054] Furthermore, the mobile phase A is acetonitrile.

[0055] 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.

[0056] 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.

[0057] Furthermore, the acid aqueous solution is a 0.01% to 1% acid aqueous solution.

[0058] Furthermore, the acid aqueous solution is a 0.01% to 1% phosphoric acid aqueous solution.

[0059] Furthermore, the acid aqueous solution is about 0.1% phosphoric acid aqueous solution.

[0060] Furthermore, the buffered saline solution is an acetate solution and / or an acetate solution.

[0061] Furthermore, the pH value of the buffered saline solution is not greater than 7.0.

[0062] Further, when the detection wavelength is 230nm, the fingerprint spectrum includes peaks 1-23, and the retention times of peaks 1-23 correspond to about 9.57min, about 14.13min, about 15.338min, about 19.061min, about 19.420min, about 19.781min, about 21.308min, about 21.723min, about 22.760min, about 24.834min, about 25.223min, about 28.220min, about 29.041min, about 33.064min, about 36.546min, about 38.790min, about 40.097min, about 41.687min, about 43.797min, about 51.556min, about 52.919min, about 60.504min and about 61.628min, respectively.

[0063] Furthermore, when the detection wavelength is 230 nm, the fingerprint spectrum includes peaks 1-23, wherein peak 15 is baicalin as a reference peak, peaks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 22 and 23. The relative retention time averages of the peaks correspond to about 0.26, about 0.39, about 0.42, about 0.52, about 0.53, about 0.54, about 0.58, about 0.59, about 0.62, about 0.68, about 0.69, about 0.77, about 0.79, about 0.90, about 1.06, about 1.10, about 1.14, about 1.20, about 1.41, about 1.45, about 1.66 and about 1.69, respectively.

[0064] Furthermore, when the detection wavelength is 230 nm, peak 4 is cryptochlorogenic acid, peak 9 is paeoniflorin, peak 14 is 4,5-o-dicaffeoylquinic acid, peak 15 is baicalin, peak 21 is glycyrrhizic acid, peak 22 is atractylodes lactone III, and peak 23 is ligusticum chuanxiong lactone A.

[0065] Further, when the detection wavelength is 230nm, peak No. 1 comes from white peony root medicinal material, peak No. 2 comes from Rehmannia glutinosa medicinal material, peak No. 3 comes from Dipsacus asper medicinal material, peak No. 4 comes from Ligusticum chuanxiong medicinal material, peak No. 5 comes from Licorice medicinal material, peak No. 6 comes from Dipsacus asper medicinal material, peak No. 7 comes from Amomum villosum medicinal material, peak No. 8 comes from white peony root medicinal material, peak No. 9 comes from white peony root medicinal material, peak No. 10 comes from Scutellaria baicalensis medicinal material, peak No. 11 comes from Astragalus membranaceus medicinal material, peak No. 12 comes from Angelica sinensis medicinal material and Ligusticum chuanxiong medicinal material, peak No. 14 comes from Dipsacus asper medicinal material, peak No. 15 comes from Scutellaria baicalensis medicinal material, peak No. 16 comes from Scutellaria baicalensis medicinal material, peak No. 17 comes from Scutellaria baicalensis medicinal material, peak No. 18 comes from Scutellaria baicalensis medicinal material, peak No. 19 comes from Scutellaria baicalensis medicinal material, peak No. 21 comes from Licorice medicinal material, peak No. 22 comes from Atractylodes macrocephala medicinal material, and peak No. 23 comes from Ligusticum chuanxiong medicinal material.

[0066] According to another aspect of the present invention, a quality control method for a Chinese medicine composition containing Atractylodes macrocephala is provided, the quality control method comprising the following steps:

[0067] (1) establishing a standard fingerprint of a reference sample of a Chinese medicine composition according to the above-mentioned fingerprint construction method;

[0068] (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

[0069] (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.

[0070] Furthermore, the compliance requirement includes one or more of the following:

[0071] (1) The fingerprint of the Chinese medicine composition sample to be tested exhibits 23 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;

[0072] (2) With the baicalin peak 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

[0073] (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.

[0074] 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 containing Atractylodes macrocephala.

[0075] Beneficial effects of the present invention:

[0076] 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 Atractylodes macrocephala. The method confirms 23 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.

[0077] Specifically, compared with the prior art, the present invention has the following beneficial effects:

[0078] (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.

[0079] (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.

[0080] (3) In the process of establishing the fingerprint spectrum of the traditional Chinese medicine compound reference sample of the present invention, 23 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.

[0081] (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.

[0082] (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

[0083] 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.

[0084] Figure 1 This is the fingerprint of the reference sample of the Chinese medicine composition containing Atractylodes macrocephala of the present invention. Among them, peak 4: cryptochlorogenic acid, peak 9: paeoniflorin, peak 14: 4,5-o-dicaffeoylquinic acid, S: baicalin, peak 21: glycyrrhizic acid, peak 22: atractylodes lactone III, peak 23: ligusticum chuanxiong lactone A; GL Sciences InretsustainAQ-C18 (250×4.6mm, 5μm) chromatographic column was used.

[0085] Figure 2 These are multiple batch matching diagrams of 15 batches of the traditional Chinese medicine composition containing Atractylodes macrocephala of the present invention. DETAILED DESCRIPTION

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] As described in the background technology section, there is no literature report on the comprehensive analysis of the chemical components and fingerprint research of the Chinese medicine composition containing Atractylodes macrocephala claimed in the present invention. In order to solve the above problems, the present invention provides a method for constructing a fingerprint of a Chinese medicine composition containing Atractylodes macrocephala, and the construction method comprises the following steps:

[0092] Preparation of a Chinese medicine composition test solution: taking an appropriate amount of a decoction of a Chinese medicine composition containing Atractylodes macrocephala, shaking it well, filtering it, and taking a filtrate to obtain a test solution of the Chinese medicine composition; wherein the Chinese medicine composition comprises ginseng, astragalus, Atractylodes macrocephala, licorice, angelica, chuanxiong, white peony root, rehmannia root, chuanxiong, glutinous rice, scutellaria baicalensis, and amomum villosum;

[0093] Preparation of reference solution: weigh appropriate amounts of atractylodes lactone III, paeoniflorin, cryptochlorogenic acid, 4,5-o-dicaffeoylquinic acid, baicalin, octoprolol A, and ammonium glycyrrhizinate reference substances, add methanol to prepare reference solution with concentrations of atractylodes lactone III, paeoniflorin, cryptochlorogenic acid, 4,5-o-dicaffeoylquinic acid, baicalin, octoprolol A, and ammonium glycyrrhizinate of 10 to 100 μg / ml, respectively;

[0094] 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;

[0095] 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: 0-10 min, 97% B→89% B; 10-14 min, 89% B→85% B; 14-18 min, 85% B→80% B; 18-32 min, 80% B→73% B; 32-42 min, 73% B→66% B; 42-44 min, 73% B→66% B; 45-50 min, 80% B→80% B; 47-51 min, 80% B→73% B; 48-64 min, 73% B→66% B; 50-51 min, 80% B→80% B; 51-52 min, 80% B→73% B; 52-53 min, 80% B→80% B; 54-55 min, 80% B→73% B; 56-57 min, 80% B→80% B; 58-59 min, 80% B→73% B; 59-64 min, 80% B→80% B; 60-71 min, 61-64 min, 61-65 min, 66-71 min, 67-71 min, 68-71 min, 69-80 min, 70-80% B; 71-73 min, 71-73 min, 73-73 min, 73-73 min, 73-73 min, 7 in, 66% B→65% B; 44~46min, 65% B→63% B; 46~48min, 63% B→58% B; 48~55min, 58%→50% B; 55~57min, 50%→46% B; 57~62min, 46%→30% B; 62~65min, 30%→97% B; 65~80min, 97% B, the flow rate is 0.5~1.5ml / min, the column temperature is 10~40℃, the detection wavelength is 190~400nm, and the injection volume is 1~20μl.

[0096] 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.

[0097] In a preferred embodiment, the preparation method of the decoction of the traditional Chinese medicine composition containing Atractylodes macrocephala comprises: (a) weighing appropriate amounts of ginseng, astragalus, Atractylodes macrocephala, licorice, angelica, Chuanxiong, white peony root, rehmannia root, chuanxiong, glutinous rice, Scutellaria baicalensis and Amomum villosum into a container, adding a first 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 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, and combining the first filtrate and the second filtrate to obtain the decoction of the traditional Chinese medicine composition containing Atractylodes macrocephala.

[0098] In a preferred embodiment, the ratio of the sum of the masses of ginseng, astragalus, atractylodes, licorice, angelica, chuanxiong, white peony root, rehmannia root, chuanxiong, glutinous rice, scutellaria and amomum to the mass / volume (g / ml) of the first water is 0.05-0.15, for example about 0.1.

[0099] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 0.1" includes ±5% of 0.1, or from 0.095 to 0.105.

[0100] In a preferred embodiment, the soaking time is 20 to 40 minutes, such as about 30 minutes.

[0101] 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.

[0102] In a preferred embodiment, the first decocting time over low heat is 30 to 90 minutes, for example, about 50 minutes.

[0103] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 50" includes ±5% of 50, or from 47.5 to 52.5.

[0104] In a preferred embodiment, the filter is a 120-mesh screen.

[0105] In a preferred embodiment, the ratio of the sum of the masses of ginseng, astragalus, atractylodes, licorice, angelica, chuanxiong, white peony root, rehmannia root, chuanxiong, glutinous rice, scutellaria and amomum to the mass / volume (g / ml) of the second water is 0.1-0.15, for example about 0.125.

[0106] 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.

[0107] In a preferred embodiment, the second decocting time over low heat is 30 to 90 minutes, for example, about 45 minutes.

[0108] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 45" includes ±5% of 45, or from 42.75 to 47.25.

[0109] In a preferred embodiment, the volume of the decoction of the traditional Chinese medicine composition containing Atractylodes macrocephala is 300-600 ml, for example, about 450 ml.

[0110] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 450" includes ±5% of 450, or from 427.5 to 472.5.

[0111] In a preferred embodiment, the volume / volume ratio of the first water to the decoction of the traditional Chinese medicine composition containing Atractylodes macrocephala is 0.8-1.0, for example, about 0.837.

[0112] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.837" includes ±5% of 0.837, or from 0.79515 to 0.87885.

[0113] In a preferred embodiment, the mass ratio of the ginseng, the astragalus, the atractylodes, the liquorice, the angelica, the ligusticum, the chuanxiong, the white peony root, the rehmannia root, the dipsacus asper, the glutinous rice, the scutellaria and the amomum is (2-4): (2-4): (5-7): (1-3): (2-4): (1.5-3.5): (1.5-3.5): (1.5-3.5): (2-4): (5-7): (2-4): (0.5-2.5).

[0114] In a preferred embodiment, the mass ratio between the ginseng, the astragalus, the atractylodes, the liquorice, the angelica, the ligusticum, the chuanxiong, the white peony root, the rehmannia root, the dipsacus asper, the glutinous rice, the scutellaria and the amomum is (2.5-3.5): (2.5-3.5): (5.5-6.5): (1.5-2.5): (2.5-3.5): (2-3): (2-3): (2-3): (2-3): (2.5-3.5): (5.5-6.5): (2.5-3.5): (1-2).

[0115] In a preferred embodiment, the mass ratio of the ginseng, the astragalus, the atractylodes, the liquorice, the angelica, the ligusticum, the chuanxiong, the white peony root, the rehmannia root, the dipsacus asper, the glutinous rice, the scutellaria and the amomum is about 3: about 3: about 6: about 2: about 3: about 2.4: about 2.4: about 2.4: about 3: about 6: about 3: about 1.5.

[0116] 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 6" includes ±5% of 6, or from 5.7 to 6.3; "about 2" includes ±5% of 2, or from 1.9 to 2.1; "about 2.4" includes ±5% of 2.4, or from 2.28 to 2.52; "about 1.5" includes ±5% of 1.5, or from 1.425 to 1.575.

[0117] In a preferred embodiment, the mass of the ginseng ranges from 1 to 10 g, such as about 3 g.

[0118] 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.

[0119] In a preferred embodiment, the mass of the Astragalus is in the range of 1 to 10 g, such as about 3 g.

[0120] 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.

[0121] In a preferred embodiment, the mass range of the Atractylodes macrocephala is 1 to 10 g, for example, about 6 g.

[0122] 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.

[0123] In a preferred embodiment, the weight of the liquorice is in the range of 1 to 5 g, such as about 2 g.

[0124] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 2" includes ±5% of 2, or from 1.9 to 2.1.

[0125] In a preferred embodiment, the mass of the angelica sinensis ranges from 1 to 10 g, such as about 3 g.

[0126] 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.

[0127] In a preferred embodiment, the mass of the Ligusticum chuanxiong ranges from 1 to 10 g, such as about 2.4 g.

[0128] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 2.4" includes ±5% of 2.4, or from 2.28 to 2.52.

[0129] In a preferred embodiment, the mass range of the white peony root is 1 to 10 g, for example, about 2.4 g.

[0130] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 2.4" includes ±5% of 2.4, or from 2.28 to 2.52.

[0131] In a preferred embodiment, the mass of the Rehmannia root is in the range of 1 to 10 g, for example, about 2.4 g.

[0132] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 2.4" includes ±5% of 2.4, or from 2.28 to 2.52.

[0133] In a preferred embodiment, the mass of the Dipsacus asper is in the range of 1 to 10 g, for example, about 3 g.

[0134] 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.

[0135] In a preferred embodiment, the mass of the glutinous rice ranges from 1 to 10 g, such as about 6 g.

[0136] 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.

[0137] In a preferred embodiment, the mass of the Scutellaria baicalensis is in the range of 1 to 10 g, for example, about 3 g.

[0138] 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.

[0139] In a preferred embodiment, the mass of the Amomum villosum is in the range of 0.5 to 5 g, for example, about 1.5 g.

[0140] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1.5" includes ±5% of 1.5, or from 1.425 to 1.575.

[0141] In a preferred embodiment, the licorice is roasted licorice.

[0142] In a preferred embodiment, the Rehmannia root is Rehmannia root prepared.

[0143] In a preferred embodiment, the Rehmannia root is Rehmannia root prepared by steaming.

[0144] In a preferred embodiment, in the preparation of the test solution, the filtration is microporous membrane filtration.

[0145] In a preferred embodiment, the pore size of the microporous filter membrane is 0.22 μm.

[0146] In a preferred embodiment, the concentration of Atractylodes lactone III in the reference solution is about 20.12 μg / ml.

[0147] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 20.12" includes ±5% of 20.12, or from 19.114 to 21.126.

[0148] In a preferred embodiment, the concentration of cryptochlorogenic acid in the reference solution is about 20.62 μg / ml.

[0149] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 20.62" includes ±5% of 20.62, or from 19.589 to 21.651.

[0150] In a preferred embodiment, the concentration of 4,5-o-dicaffeoylquinic acid in the reference solution is about 22.21 μg / ml.

[0151] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 22.21" includes ±5% of 22.21, or from 21.0995 to 23.3205.

[0152] In a preferred embodiment, the concentration of cypermethrin A in the reference solution is about 10.04 μg / ml.

[0153] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10.04" includes ±5% of 10.04, or from 9.538 to 10.542.

[0154] In a preferred embodiment, the concentration of paeoniflorin in the reference solution is about 60.17 μg / ml.

[0155] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 60.17" includes ±5% of 60.17, or from 57.1615 to 63.1785.

[0156] In a preferred embodiment, the concentration of baicalin in the reference solution is about 54.92 μg / ml.

[0157] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 54.92" includes ±5% of 54.92, or from 52.174 to 57.666.

[0158] In a preferred embodiment, the concentration of ammonium glycyrrhizinate in the reference solution is about 49.94 μg / ml.

[0159] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 49.94" includes ±5% of 49.94, or from 47.443 to 52.437.

[0160] 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.

[0161] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1.0" includes ±5% of 1.0, or from 0.95 to 1.05.

[0162] In a preferred embodiment, the column temperature is 25-35°C, such as about 30°C.

[0163] 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.

[0164] In a preferred embodiment, the detection wavelength is 200-250 nm, such as about 230 nm.

[0165] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 230" includes ±5% of 230, or from 218.5 to 241.5.

[0166] In a preferred embodiment, the injection volume is 5-15 μl, such as about 10 μl.

[0167] 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.

[0168] In a preferred embodiment, the theoretical plate number of the chromatographic peak corresponding to baicalin is not less than 2500.

[0169] In a preferred embodiment, the chromatographic column is a GL Sciences Inertsustain AQ-C18 chromatographic column.

[0170] 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.

[0171] In a preferred embodiment, mobile phase A is acetonitrile.

[0172] 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.

[0173] 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.

[0174] In a preferred embodiment, the acid aqueous solution is a 0.01% to 1% acid aqueous solution.

[0175] In a preferred embodiment, the acid aqueous solution is a 0.01% to 1% phosphoric acid aqueous solution.

[0176] In a preferred embodiment, the aqueous acid solution is about 0.1% phosphoric acid aqueous solution.

[0177] 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%.

[0178] In a preferred embodiment, the buffered saline solution is an acetate aqueous solution and / or an acetate aqueous solution.

[0179] In a preferred embodiment, the pH value of the buffered saline solution is no greater than 7.0.

[0180] In a preferred embodiment, when the detection wavelength is 230 nm, the fingerprint spectrum includes peaks 1-23, and the retention times of peaks 1-23 correspond to about 9.57 min, about 14.13 min, about 15.338 min, about 19.061 min, about 19.420 min, about 19.781 min, about 21.308 min, about 21.723 min, about 22.760 min, about 24.834 min, about 25.223 min, about 28.220 min, about 29.041 min, about 33.064 min, about 36.546 min, about 38.790 min, about 40.097 min, about 41.687 min, about 43.797 min, about 51.556 min, about 52.919 min, about 60.504 min and about 61.628 min, respectively.

[0181] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 9.57" includes ±5% of 9.57, or from 9.0915 to 10.0485; "about 14.13" includes ±5% of 14.13, or from 13.4235 to 14.8365; "about 15.338" includes ±5% of 15.338, or from 14.5711 to 16.1049; "about 19.061" includes ±5% of 19.061, or from 18.10795 to 20.01405; "about 19.420" includes ±5% of 19.420, or from 18.449 to 20.391; "about 19.781" includes ±5% of 19.781, or from 18.79195 to 20. 77005; "about 21.308" includes ±5% of 21.308, or from 20.2426 to 22.3734; "about 21.723" includes ±5% of 21.723, or from 20.63685 to 22.80915; "about 22.760" includes ±5% of 22.760, or from 21.622 to 23.898; "about 24.834" includes ±5% of 24.834, or from 23.5923 to 26.0757; "about 25.223" includes ±5% of 25.223, or from 23.96185 to 26.48415; "about 28.220" includes ±5% of 28.220, or from 26.809 to 29.631; "about 29.041" includes ±5% of 29.041, or from 27.58895 to 30.49305; "about 33.064" includes ±5% of 33.064, or from 31.4108 to 34.7172; "about 36.546" includes ±5% of 36.546, or from 34.7187 to 38.3733; "about 38.790" includes ±5% of 38.790, or from 36.8505 to 40.7295; "about 40.097" includes ±5% of 40.097, or from 38.09215 to 42.10185; "about 41.687" includes 41. "about 43.797" includes ±5% of 43.797, or from 41.60715 to 45.98685; "about 51.556" includes ±5% of 51.556, or from 48.9782 to 54.1338; "about 52.919" includes ±5% of 52.919, or from 50.27305 to 55.56495; "about 60.504" includes ±5% of 60.504, or from 57.4788 to 63.5292; "about 61.628" includes ±5% of 61.628, or from 58.5466 to 64.7094.

[0182] In a preferred embodiment, when the detection wavelength is 230nm, the fingerprint spectrum includes peaks 1-23, wherein peak 15 is baicalin as a reference peak, peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8, peak 9, peak 10, peak 11, peak 12, peak 13, peak 14, peak 16, peak 17, peak 18, peak 19, peak 20, peak 21, peak 22 and peak 23. The relative retention time averages of Peak 23 correspond to about 0.26, about 0.39, about 0.42, about 0.52, about 0.53, about 0.54, about 0.58, about 0.59, about 0.62, about 0.68, about 0.69, about 0.77, about 0.79, about 0.90, about 1.06, about 1.10, about 1.14, about 1.20, about 1.41, about 1.45, about 1.66 and about 1.69, respectively.

[0183] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.26" includes ±5% of 0.26, or from 0.247 to 0.273; "about 0.39" includes ±5% of 0.39, or from 0.3705 to 0.4095; "about 0.42" includes ±5% of 0.42, or from 0.399 to 0.441; "about 0.52" includes ±5% of 0.52, or from 0.494 to 0.546; "about 0.53" includes ±5% of 0.53, or from 0.5035 to 0.5565; "about 0.54" includes ±5% of 0.54 "about 0.58" includes ±5% of 0.58, or from 0.551 to 0.609; "about 0.59" includes ±5% of 0.59, or from 0.5605 to 0.6195; "about 0.62" includes ±5% of 0.62, or from 0.589 to 0.651; "about 0.68" includes ±5% of 0.68, or from 0.646 to 0.714; "about 0.69" includes ±5% of 0.69, or from 0.6555 to 0.724 5; "about 0.77" includes ±5% of 0.77, or from 0.7315 to 0.8085; "about 0.79" includes ±5% of 0.79, or from 0.7505 to 0.8295; "about 0.90" includes ±5% of 0.90, or from 0.855 to 0.945; "about 1.06" includes ±5% of 1.06, or from 1.007 to 1.113; "about 1.10" includes ±5% of 1.10, or from 1.045 to 1.155; "about 1.14" includes ±5% of 1.14 5%, or from 1.083 to 1.197; "about 1.20" includes ±5% of 1.20, or from 1.14 to 1.26; "about 1.41" includes ±5% of 1.41, or from 1.3395 to 1.4805; "about 1.45" includes ±5% of 1.45, or from 1.3775 to 1.5225; "about 1.66" includes ±5% of 1.66, or from 1.577 to 1.743; "about 1.69" includes ±5% of 1.69, or from 1.6055 to 1.7745.

[0184] In a preferred embodiment, when the detection wavelength is 230 nm, peak No. 4 is cryptochlorogenic acid, peak No. 9 is paeoniflorin, peak No. 14 is 4,5-o-dicaffeoylquinic acid, peak No. 15 is baicalin, peak No. 21 is glycyrrhizic acid, peak No. 22 is atractylodes lactone III, and peak No. 23 is ligusticum chuanxiong lactone A.

[0185] In a preferred embodiment, when the detection wavelength is 230nm, peak No. 1 comes from white peony root medicinal material, peak No. 2 comes from Rehmannia root medicinal material, peak No. 3 comes from Dipsacus asper medicinal material, peak No. 4 comes from Ligusticum chuanxiong medicinal material, peak No. 5 comes from Licorice root medicinal material, peak No. 6 comes from Dipsacus asper medicinal material, peak No. 7 comes from Amomum villosum medicinal material, peak No. 8 comes from white peony root medicinal material, peak No. 9 comes from white peony root medicinal material, peak No. 10 comes from Scutellaria baicalensis medicinal material, peak No. 11 comes from Astragalus membranaceus medicinal material, peak No. 12 comes from Angelica sinensis medicinal material and Ligusticum chuanxiong medicinal material, peak No. 14 comes from Dipsacus asper medicinal material, peak No. 15 comes from Scutellaria baicalensis medicinal material, peak No. 16 comes from Scutellaria baicalensis medicinal material, peak No. 17 comes from Scutellaria baicalensis medicinal material, peak No. 18 comes from Scutellaria baicalensis medicinal material, peak No. 19 comes from Scutellaria baicalensis medicinal material, peak No. 21 comes from Licorice root medicinal material, peak No. 22 comes from Atractylodes macrocephala medicinal material, and peak No. 23 comes from Ligusticum chuanxiong medicinal material.

[0186] According to another aspect of the present invention, a quality control method for a Chinese medicine composition containing Atractylodes macrocephala is provided, the quality control method comprising the following steps:

[0187] (1) establishing a standard fingerprint of a reference sample of a Chinese medicine composition according to the above-mentioned fingerprint construction method;

[0188] (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

[0189] (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.

[0190] In a preferred embodiment, the compliance requirements include one or more of the following:

[0191] (1) The fingerprint of the Chinese medicine composition sample to be tested exhibits 23 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;

[0192] (2) With the baicalin peak 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

[0193] (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.

[0194] 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 containing Atractylodes macrocephala.

[0195] 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.

[0196] 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.

[0197] 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.

[0198] Example

[0199] The prescription of the Chinese medicine composition containing Atractylodes macrocephala of the present invention is: 3g of ginseng; 3g of astragalus; 6g of Atractylodes macrocephala; 2g of roasted liquorice root; 3g of angelica; 2.4g of chuanxiong; 2.4g of white peony root; 2.4g of prepared rehmannia root (steamed); 3g of chuanxiong; 6g of glutinous rice; 3g of scutellaria; 1.5g of amomum; a total of 37.7g.

[0200] The preparation method of the Chinese medicine composition containing Atractylodes macrocephala of the present invention is as follows: take the above twelve herbs, put them in a multifunctional decoction pot, add 10 times the amount of water to cover the herbs by 2-5 cm, soak for 30 minutes, decoct once, boil over high heat, then simmer over low heat for about 50 minutes, filter with a 120-mesh sieve, add 8 times the amount of water to the residue, boil over high heat, then simmer again over low heat for 45 minutes, combine the decoctions, and the final decoction volume is about 450 ml.

[0201] 1. Instruments and test drugs

[0202] 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;

[0203] 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;

[0204] 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: GL Sciences InretSustain AQ-C18 5μm4.6x250mm Serial No.23J0443221; Column 4: GLSciences InretSustain AQ-C18 5μm 4.6x250mm Serial No.23J0443226; Methanol, acetonitrile, and phosphoric acid were of chromatographic grade; glacial acetic acid and formic acid were of analytical grade; and water was purified water.

[0205] Reference Material Information:

[0206] Atractylodes macrocephala L. III (batch number 111978-202302, content 100%, China Food and Drug Inspection Institute); paeoniflorin (batch number 110736-202246, content 96.7%, China Food and Drug Inspection Institute); cryptochlorogenic acid (batch number AYA030012-202207, content 98.77%, China Food and Drug Inspection Institute); 4,5-o-dicaffeoylquinic acid (batch number 111894-202 205, content 94.9%, China Food and Drug Inspection Institutes); baicalin (batch number 110715-202122, content 94.2%, China Food and Drug Inspection Institutes); yangchuanxiong lactone A (batch number AY190013-202208, content 99.65%, China Food and Drug Inspection Institutes); ammonium glycyrrhizinate (batch number 110731-202122, content 94.4%, China Food and Drug Inspection Institutes).

[0207] 2. Solution preparation

[0208] 2.1. Preparation of reference solution

[0209] Accurately weigh appropriate amounts of Atractylodes macrolide III, paeoniflorin, cryptochlorogenic acid, 4,5-o-dicaffeoylquinic acid, baicalin, octopyrin A, and ammonium glycyrrhizic acid reference substances, and add methanol to prepare solutions of Atractylodes macrolide III 20.12 μg / ml, paeoniflorin 60.17 μg / ml, cryptochlorogenic acid 20.62 μg / ml, 4,5-o-dicaffeoylquinic acid 22.21 μg / ml, baicalin 54.92 μg / ml, octopyrin A 10.04 μg / ml, and ammonium glycyrrhizic acid 49.94 μg / ml; prepare the reference substance solution.

[0210] 2.2 Preparation of test solution

[0211] Take an appropriate amount of a sample (decoction) of the traditional Chinese medicine composition containing Atractylodes macrocephala, shake well, filter, and take the filtrate to obtain the product.

[0212] 2.3 Preparation of negative solution

[0213] 2.3.1. Preparation of negative sample solution lacking Astragalus: Weigh the other medicinal ingredients except Astragalus according to the prescription, and prepare the negative sample solution lacking Astragalus according to the “Preparation of test solution”.

[0214] 2.3.2. Preparation of negative sample solution lacking ginseng: Weigh the other medicinal ingredients except ginseng according to the prescription, and prepare the negative sample solution lacking ginseng according to the “Preparation of test solution”.

[0215] 2.3.3. Preparation of negative sample solution lacking Atractylodes macrocephala: Weigh other medicinal ingredients except Atractylodes macrocephala according to the prescription, and prepare negative sample solution lacking Atractylodes macrocephala according to the “Preparation of test solution”.

[0216] 2.3.4. Preparation of negative sample solution lacking roasted licorice: Weigh other medicinal ingredients except roasted licorice according to the prescription, and prepare the negative sample solution lacking roasted licorice according to the “Preparation of test solution”.

[0217] 2.3.5. Preparation of negative sample solution lacking Angelica sinensis: Weigh other medicinal ingredients except Angelica sinensis according to the prescription, and prepare negative sample solution lacking Angelica sinensis according to the “Preparation of test solution”.

[0218] 2.3.6. Preparation of negative sample solution lacking Chuanxiong: Weigh other medicinal ingredients except Chuanxiong according to the prescription, and prepare negative sample solution lacking Chuanxiong according to the “Preparation of test solution”.

[0219] 2.3.7. Preparation of negative sample solution lacking white peony root: Weigh other medicinal ingredients except white peony root according to the prescription, and prepare negative sample solution lacking white peony root according to the “Preparation of test solution”.

[0220] 2.3.8. Preparation of negative sample solution lacking Rehmannia glutinosa: Weigh other medicinal ingredients except Rehmannia glutinosa according to the prescription, and prepare the negative sample solution lacking Rehmannia glutinosa according to the “Preparation of test solution”.

[0221] 2.3.9. Preparation of negative sample solution lacking Dipsacus asper: weigh other medicinal ingredients except Dipsacus as per the prescription, and prepare negative sample solution lacking Dipsacus as per the “Preparation of test solution”.

[0222] 2.3.10. Preparation of negative sample solution lacking glutinous rice: Weigh other medicinal ingredients except glutinous rice according to the prescription, and prepare negative sample solution lacking glutinous rice according to the “Preparation of test solution”.

[0223] 2.3.11. Preparation of negative sample solution lacking Scutellaria baicalensis: Weigh the other medicinal ingredients except Scutellaria baicalensis according to the prescription, and prepare the negative sample solution lacking Scutellaria baicalensis according to the “Preparation of test solution”.

[0224] 2.3.12. Preparation of negative sample solution lacking Amomum villosum: Weigh other medicinal ingredients except Amomum villosum according to the prescription, and prepare negative sample solution lacking Amomum villosum according to the “Preparation of test solution”.

[0225] 2.4. Preparation of single-flavor solution

[0226] Take appropriate amounts of the twelve single-ingredient pieces of medicine respectively, put them into a stoppered conical flask, add 50 ml of water, heat and reflux for 50 minutes, cool, filter, and obtain the filtrate.

[0227] 3. Investigation of chromatographic conditions

[0228] 3.1. Initial chromatographic conditions

[0229] Determined by high performance liquid chromatography (General Rules 0512 of Part IV of the 2020 edition of the Chinese Pharmacopoeia).

[0230] The chromatographic conditions and system suitability test used octadecyl bonded silica gel as the filler (Agilent 5TC-C18 5.0μm 4.6x250mm, GL Sciences Inertsustain AQ-C184.6x250mm); acetonitrile as mobile phase A, 0.1% phosphoric acid as mobile phase B; gradient elution was performed according to the provisions in Table 1; the detection wavelength was 190-400nm.

[0231] Table 1 Initial gradient

[0232]

[0233] 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.

[0234] The results show that, by comparing the isosbesty graphs of Agilent 5TC-C18 and GL Sciences Inertsustain AQ-C18, it can be seen that there is basically no chromatographic peak absorption fluorescence point under the Agilent 5TC-C18 column, so GL Sciences Inertsustain AQ-C18 was selected for this project research. From the isosbesty graph of GL Sciences Inertsustain AQ-C18 at 190-400nm, it can be seen that there are basically absorption fluorescence points at the low-end wavelength, so 230nm is temporarily set for detection and analysis; in the 230nm liquid chromatogram, there are chromatographic peaks in the first 60 minutes, there are characteristic peaks around 90 minutes, and there are basically no characteristic peaks within 60-90 minutes and after 90 minutes, so considering that the acetonitrile ratio is within 70%, gradient 1 is designed.

[0235] 3.2. Mobile phase gradient selection

[0236] 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.

[0237] Table 2 Gradient 1

[0238] Time (min) Acetonitrile (%) 0.1 Phosphoric acid (%) 0→12 3→11 97→89 12→26 11→20 89→80 26→40 20→27 80→73 40→50 27→35 73→65 50→65 35→50 65→50 65→70 50→70 50→30 62→65 70→0 30→100 65→80 0 100

[0239] The results show that the isobsorptiometry of gradient 1 shows that the decoction of the Chinese medicine composition containing Atractylodes macrocephala has a chromatographic peak absorption fluorescence point in the range of 190-260nm, so the liquid chromatograms of 198nm, 230nm and 254nm are selected for comparison. At 190nm and 230nm, the information of the chromatographic peak is more comprehensive; considering the response value of each characteristic peak, it is better at 230nm and 198nm is the terminal wavelength. There is contingency and instrument compatibility, so the detection wavelength of this project is 230nm. In the 230nm liquid chromatogram, the peaks are well separated, the running time is 90 minutes, and the chromatographic peaks are not separated between 30 and 35 minutes, so considering optimization, gradients 2, 3 and 4 are designed.

[0240] Table 3 Gradient 2

[0241] Time (min) Acetonitrile (%) 0.1 Phosphoric acid (%) 0→12 3→11 97→89 12→26 11→20 89→80 26→30 20→22 80→78 30→40 22→27 78→73 40→50 27→35 73→65 50→65 35→50 65→50 65→70 50→70 50→30 62→65 70→0 30→100 65→80 0 100

[0242] Table 4 Gradient 3

[0243] Time (min) Acetonitrile (%) 0.1 Phosphoric acid (%) 0→10 3→11 97→89 10→14 11→15 89→85 14→26 15→20 85→80 26→30 20→22 80→78 30→40 22→27 78→73 40→50 27→35 73→65 50→65 35→50 65→50 65→70 50→70 50→30 62→65 70→0 30→100 65→80 0 100

[0244] Table 5 Gradient 4

[0245]

[0246]

[0247] The results show that under gradient 2, the characteristic peaks did not meet the separation requirements at around 15 minutes; under gradient 3, the characteristic peaks did not meet the separation requirements at around 30 minutes; under the elution program of gradient 4 in Table 5, the baseline was stable, the main chromatographic peaks were well separated, and the analysis time was within 80 minutes, so the gradient 4 chromatographic conditions were temporarily selected for fingerprint determination.

[0248] Determined by high performance liquid chromatography (General Rules 0512 of Part IV of the 2020 edition of the Chinese Pharmacopoeia).

[0249] The chromatographic conditions and system suitability test used octadecylsilane bonded silica gel as the filler; acetonitrile as mobile phase A, 0.1% phosphoric acid as mobile phase B, and gradient elution as specified in Table 5; the flow rate was 1.0 ml per minute, the detection wavelength was 230 nm; the column temperature was 30°C; and the theoretical plate number calculated based on the baicalin peak should be no less than 2500.

[0250] Preparation of test solution: Take an appropriate amount of decoction and filter it through a 0.22μm microporous membrane.

[0251] Determination method: Accurately pipette 10μl of the test solution, inject it into the liquid chromatograph, and determine the result.

[0252] 4. Validation of analytical methods

[0253] 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).

[0254] 4.1. Exclusivity

[0255] Prepare negative blank sample solution according to the test solution preparation method, test it according to the fingerprint method, and record the chromatogram.

[0256] The system parameters of the traditional Chinese medicine composition containing Atractylodes macrocephala are shown in Table 6.

[0257] Table 6 System parameters of the Chinese medicine composition containing Atractylodes macrocephala

[0258]

[0259] The results show that the chromatogram of the reference sample of the Chinese medicine composition containing Atractylodes macrocephala (eg Figure 1) mainly presents 23 peaks, peak 1, peak 8, peak 9 belong to white peony flavor, peak 9 is paeoniflorin; peak 2 belongs to Rehmannia glutinosa; peaks 3, 6, 14 belong to Dipsacus asper, among which peak 14 is 4,5-o-dicaffeoylquinic acid; peaks 4 and 23 belong to Ligusticum chuanxiong, among which peak 4 is cryptochlorogenic acid, and peak 23 is yangchuanqiong lactone A; peaks 5 and 21 belong to Licorice flavor, among which peak 21 is glycyrrhizic acid; peak 7 belongs to Amomum villosum; peaks 10 and peaks 15-19 belong to Scutellaria baicalensis, among which peak 15 is baicalin; peak 11 belongs to Astragalus membranaceus; peak 12 belongs to Angelica sinensis and Ligusticum chuanxiong; peak 22 belongs to Atractylodes macrocephala, among which peak 22 is Atractylodes lactone III; peaks 12 and 22 are generated by the reaction during the decoction process, negative without interference, and the method has good specificity. The specific summary is shown in Table 7.

[0260] Table 7 Chromatographic peak summary

[0261]

[0262] 4.2 Precision

[0263] 4.2.1. Instrument precision test

[0264] 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.

[0265] 4.2.2 Intermediate precision

[0266] 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.

[0267] 4.2.3 Repeatability test

[0268] Take 6 samples from the same batch, prepare the test sample according to the method of "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.

[0269] 4.3 Durability

[0270] 4.3.1 Stability Study

[0271] Take the same test solution and inject it at 0, 2h, 4h, 6h, 9h, 12h, 18h and 26h. Record the chromatogram, calculate the similarity of each characteristic peak, and examine the stability of the test solution. The results show that within 46h of stability, the similarity of each characteristic peak is greater than 0.90, which meets the requirements, indicating that it is stable within 26h.

[0272] 4.3.2 Investigation of different flow rates

[0273] 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.

[0274] 4.3.3 Investigation of different column temperatures

[0275] Take the same test solution, test at 30±5℃, 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 regulations.

[0276] 4.3.4 Different chromatographic columns

[0277] The same test solution was tested under different chromatographic columns (chromatographic column 1: GL Sciences InretSustainAQ-C185μm 4.6x250mm Serial No.23J0443146), (chromatographic column 2: GL Sciences InretSustain AQ-C18 5μm 4.6x250mm Serial No.23J0443221), (chromatographic column 3: GL Sciences InretSustain AQ-C185μm4.6x250mm Serial No.23J0443226), and the chromatograms were recorded and the similarity matching calculation was performed with the standard reference spectrum of the traditional Chinese medicine composition containing Atractylodes macrocephala. The results showed that the similarity results of different chromatographic columns were all greater than 0.90, which met the requirements.

[0278] 4.3.5. Investigation of mobile phases with different acid types

[0279] The results showed that the baseline of the spectrum was uneven and the separation was poor under the conditions of 0.1% formic acid and glacial acetic acid, so phosphoric acid was used as the acid type of the mobile phase.

[0280] 4.3.6. Investigation of mobile phases with different acid concentrations

[0281] The same test solution was tested at 0.09%, 0.10% and 0.11% phosphoric acid solution concentration in the mobile phase, and the chromatogram was recorded and the similarity matching calculation was performed with the standard reference spectrum of the traditional Chinese medicine composition containing Atractylodes macrocephala. The results showed that under different acid concentration conditions, the similarity results of each common peak were greater than 0.90, which met the requirements.

[0282] 5. Fingerprint method confirmation

[0283] Based on the above methodological investigation results, the fingerprint spectrum (such as Figure 1 Among the 23 common peaks (as shown in the figure), 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 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.

[0284] Based on the above results, the fingerprint method is as follows:

[0285] Determined by high performance liquid chromatography (General Rules 0512 of Part IV of the 2020 edition of the Chinese Pharmacopoeia).

[0286] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel is used as filler; acetonitrile is used as mobile phase A, 0.1% phosphoric acid is used as mobile phase B, and gradient elution is performed according to the provisions in Table 5; the flow rate is 1.0 ml per minute, and the detection wavelength is 230 nm; the column temperature is 30°C; the theoretical plate number calculated based on the baicalin peak should be not less than 2500.

[0287] Preparation of test solution: Take an appropriate amount of decoction and filter it through a 0.22μm microporous membrane.

[0288] Determination method: Accurately pipette 10μl of the test solution, inject it into the liquid chromatograph, and determine it.

[0289] 6. Determination of fingerprint standard regulations

[0290] 15 batches of the Chinese medicine composition containing Atractylodes macrocephala of the present invention are matched as shown in the figure Figure 2 shown.

[0291] It was determined that 23 characteristic peaks were present in the chromatogram of the test sample, among which the peak corresponding to the baicalin reference peak was the S peak. According to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprints, the similarity between the test sample fingerprint and the reference fingerprint should not be less than 0.90.

[0292] 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 containing Atractylodes macrocephala, 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 Atractylodes macrocephala, shaking it well, filtering it, and taking a filtrate to obtain the Chinese medicine composition test solution; wherein the Chinese medicine composition comprises ginseng, astragalus, Atractylodes macrocephala, licorice, angelica, Chuanxiong, white peony root, rehmannia root, Sichuan teasel, glutinous rice, Scutellaria baicalensis and Amomum villosum; The preparation method of the decoction of the Chinese medicine composition containing Atractylodes macrocephala comprises: (a) weighing appropriate amounts of ginseng, astragalus, Atractylodes macrocephala, liquorice, angelica, Chuanxiong, white peony root, rehmannia root, chuanxiong, glutinous rice, scutellaria baicalensis and amomum villosum into a container, adding a first water for soaking, boiling over high heat, then boiling over low heat for the first time, filtering through a filter to obtain a first filtrate and a filter residue; (b) adding a second water to the filter residue, boiling over high heat, then boiling over low heat for the second time, filtering through a filter to obtain a second filtrate, combining the first filtrate and the second filtrate to obtain the decoction of the Chinese medicine composition containing Atractylodes macrocephala; Preparation of reference solution: weigh appropriate amounts of atractylodes lactone III, paeoniflorin, cryptochlorogenic acid, 4,5-o-dicaffeoylquinic acid, baicalin, octopyrin A, and ammonium glycyrrhizic acid reference substances, and add methanol to prepare reference solutions with concentrations of atractylodes lactone III, paeoniflorin, cryptochlorogenic acid, 4,5-o-dicaffeoylquinic acid, baicalin, octopyrin A, and ammonium glycyrrhizic acid being 10 to 100 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 HPLC detection are as follows: the chromatographic column is GL Sciences Inertsustain AQ-C18, acetonitrile is used as mobile phase A, 0.09% to 0.11% phosphoric acid solution is used as mobile phase B, and the gradient elution program is as follows: 0 to 10 min, 97% B→89% B; 10 to 14 min, 89% B→85% B; 14 to 18 min, 85% B→80% B; 18 to 32 min, 80% B→73% B; 32 to 42 min, 73% B→66% B; 42 to 44 min, 66% B→65% B; 44-46 min, 65% B→63% B; 46-48 min, 63% B→58% B; 48-55 min, 58%→50% B; 55-57 min, 50%→46% B; 57-62 min, 46%→30% B; 62-65 min, 30%→97% B; 65-80 min, 97% B, the flow rate is 0.9-1.1 ml / min, the column temperature is 25-35°C, the detection wavelength is 230 nm, and the injection volume is 1-20 μl.

2. The construction method according to claim 1, characterized in that: The ratio of the sum of the mass of the ginseng, astragalus, atractylodes, liquorice, angelica, chuanxiong, white peony root, rehmannia root, chuanxiong, glutinous rice, scutellaria and amomum to the mass / volume (g / ml) of the first water is 0.05-0.

15.

3. The construction method according to claim 2, characterized in that: The ratio of the sum of the masses of the ginseng, astragalus, atractylodes, liquorice, angelica, chuanxiong, white peony root, rehmannia root, chuanxiong, glutinous rice, scutellaria and amomum to the mass / volume (g / ml) of the first water is 0.095-0.

105.

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

5. The construction method according to claim 4, characterized in that: The soaking time is 28.5 to 31.5 minutes.

6. The construction method according to claim 1, characterized in that: The time for the first decocting over low heat is 30 to 90 minutes.

7. The construction method according to claim 6, characterized in that: The time for the first decocting over low heat is 47.5 to 52.5 minutes.

8. The construction method according to claim 1, characterized in that: The filter is a 120-mesh screen.

9. The construction method according to claim 1, characterized in that: The ratio of the sum of the mass of the ginseng, astragalus, atractylodes, liquorice, angelica, chuanxiong, white peony root, rehmannia root, chuanxiong, glutinous rice, scutellaria and amomum to the mass / volume (g / ml) of the second water is 0.1-0.

15.

10. The construction method according to claim 9, characterized in that: The ratio of the sum of the masses of the ginseng, astragalus, atractylodes, liquorice, angelica, chuanxiong, white peony root, rehmannia root, chuanxiong, glutinous rice, scutellaria and amomum to the mass / volume (g / ml) of the second water is 0.11875-0.13125.

11. The construction method according to claim 1, characterized in that: The second decocting time over low heat is 30 to 90 minutes.

12. The construction method according to claim 11, characterized in that: The second decocting time over low heat is 42.75 to 47.25 minutes.

13. The construction method according to claim 1, characterized in that: The volume of the decoction of the traditional Chinese medicine composition containing Atractylodes macrocephala is 300-600 ml.

14. The construction method according to claim 13, characterized in that: The volume of the decoction of the traditional Chinese medicine composition containing Atractylodes macrocephala is 427.5-472.5 ml.

15. The construction method according to claim 1, characterized in that: The volume / volume ratio of the first water to the decoction of the traditional Chinese medicine composition containing Atractylodes macrocephala is 0.8 to 1.

0.

16. The construction method according to claim 15, characterized in that: The volume / volume ratio of the first water to the decoction of the traditional Chinese medicine composition containing Atractylodes macrocephala is 0.79515 to 0.87885.

17. The construction method according to claim 1, characterized in that: The mass ratios of the ginseng, the astragalus, the atractylodes, the liquorice, the angelica, the ligusticum, the white peony root, the rehmannia root, the dipsacus asper, the glutinous rice, the scutellaria and the amomum are (2-4): (2-4): (5-7): (1-3): (2-4): (1.5-3.5): (1.5-3.5): (1.5-3.5): (2-4): (5-7): (2-4): (0.5-2.5).

18. The construction method according to claim 17, characterized in that: The mass ratios of the ginseng, the astragalus, the atractylodes, the liquorice, the angelica, the ligusticum, the white peony root, the rehmannia root, the dipsacus asper, the glutinous rice, the scutellaria and the amomum are (2.5-3.5): (2.5-3.5): (5.5-6.5): (1.5-2.5): (2.5-3.5): (2-3): (2-3): (2-3): (2-3): (2.5-3.5): (5.5-6.5): (2.5-3.5): (1-2).

19. The construction method according to claim 18, characterized in that: The mass ratios of the ginseng, the astragalus, the atractylodes, the liquorice, the angelica, the ligusticum, the white peony root, the rehmannia root, the dipsacus asper, the glutinous rice, the scutellaria and the amomum are (2.85-3.15): (2.85-3.15): (5.7-6.3): (1.9-2.1): (2.85-3.15): (2.28-2.52): (2.28-2.52): (2.28-2.52): (2.85-3.15): (5.7-6.3): (2.85-3.15): (1.425-1.575).

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

21. The construction method according to claim 20, characterized in that: The mass range of the ginseng is 2.85 to 3.15 g.

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

23. The construction method according to claim 22, characterized in that: The mass range of the astragalus is 2.85 to 3.15 g.

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

25. The construction method according to claim 24, characterized in that: The mass range of the Atractylodes macrocephala is 5.7 to 6.3 g.

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

27. The construction method according to claim 26, characterized in that: The mass range of the licorice is 1.9 to 2.1 g.

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

29. The construction method according to claim 28, characterized in that: The mass range of the angelica is 2.85 to 3.15 g.

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

31. The construction method according to claim 30, characterized in that: The mass range of the Chuanxiong is 2.28-2.52g.

32. The construction method according to claim 1, characterized in that: The mass range of the white peony root is 1 to 10 g.

33. The construction method according to claim 32, characterized in that: The mass range of the white peony root is 2.28-2.52g.

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

35. The construction method according to claim 34, characterized in that: The mass range of the Rehmannia root is 2.28-2.52g.

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

37. The construction method according to claim 36, characterized in that: The mass range of the Dipsacus asper is 2.85 to 3.15 g.

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

39. The construction method according to claim 38, characterized in that: The mass range of the glutinous rice is 5.7-6.3g.

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

41. The construction method according to claim 40, characterized in that: The mass range of the Scutellaria baicalensis is 2.85 to 3.15 g.

42. The construction method according to claim 1, characterized in that: The mass range of the Amomum villosum is 0.5 to 5 g.

43. The construction method according to claim 42, characterized in that: The mass range of the Amomum villosum is 1.425-1.575 g.

44. The construction method according to claim 1, characterized in that: The licorice is roasted licorice.

45. The construction method according to claim 1, characterized in that: The Rehmannia root is Rehmannia root prepared.

46. ​​The construction method according to claim 1, characterized in that The Rehmannia root is Rehmannia root prepared by steaming.

47. The construction method according to claim 1, characterized in that In the preparation of the test solution, the filtration is microporous membrane filtration.

48. The construction method according to claim 47, characterized in that: The pore size of the microporous filter membrane is 0.22 μm.

49. The construction method according to claim 1, characterized in that: The concentration of Atractylodes lactone III in the reference solution is 19.114-21.126 μg / ml.

50. The construction method according to claim 1, characterized in that: The concentration of cryptochlorogenic acid in the reference solution is 19.589-21.651 μg / ml.

51. The construction method according to claim 1, characterized in that: The concentration of 4,5-o-dicaffeoylquinic acid in the reference solution is 21.0995-23.3205 μg / ml.

52. The construction method according to claim 1, characterized in that: The concentration of occidental lactone A in the reference solution is 9.538-10.542 μg / ml.

53. The construction method according to claim 1, characterized in that: The concentration of paeoniflorin in the reference solution is 57.1615-63.1785 μg / ml.

54. The construction method according to claim 1, characterized in that: The concentration of baicalin in the reference solution is 52.174-57.666 μg / ml.

55. The construction method according to claim 1, characterized in that: The concentration of ammonium glycyrrhizinate in the reference solution is 47.443-52.437 μg / ml.

56. The construction method according to claim 1, characterized in that: The flow rate of the high performance liquid phase detection is 0.95-1.05 ml / min.

57. The construction method according to claim 1, characterized in that: The column temperature is 28.5-31.5°C.

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

59. The construction method according to claim 58, characterized in that: The injection volume is 9.5-10.5 μl.

60. The construction method according to claim 1, characterized in that: The theoretical plate number of the chromatographic peak corresponding to the baicalin is not less than 2500.

61. 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.

62. The construction method according to claim 1, characterized in that: The mobile phase B is a 0.095% to 0.105% phosphoric acid aqueous solution.

63. The construction method according to claim 1, characterized in that: When the detection wavelength is 230nm, the fingerprint spectrum includes peaks 1-23, and the retention times of peaks 1-23 correspond to 9.0915-10.0485min, 13.4235-14.8365min, 14.5711-16.1049min, 18.10795-20.01405min, 18.449-20.391min, 18.79195-20.77005min, 20.2426-22.3734min, 20.63685-22.80915min, 21.622-23.898min, 23.5923-26.0757min, 23.96185-24. 26.48415min, 26.809~29.631min, 27.58895~30.49305min, 31.4108~34.7172min, 34.7187~38.3733min, 36.8505~40.7295min, 38.09215~42.10185min, 39.60265~43.77135min, 41.60715~45.98685min, 48.9782~54.1338min, 50.27305~55.56495min, 57.4788~63.5292min and 58.5466~64.7094min.

64. The construction method according to claim 1, characterized in that When the detection wavelength is 230 nm, the fingerprint spectrum includes peaks 1-23, wherein peak 15 is baicalin as a reference peak, and the average relative retention times of peaks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 22 and 23 are 0.247-0.273, 0.3705-0.4095, 0.399-0.441, 0.494-0.546, 0.5035-0.5 565, 0.513~0.567, 0.551~0.609, 0.5605~0.6195, 0.589~0.651, 0.646~0.714, 0.6555~0.7245, 0.7315~0.8085, 0.7505~0.8295, 0.855~0.945, 1.007~1.113, 1.045~1.155, 1.083~1.197, 1.14~1.26, 1.3395~1.4805, 1.3775~1.5225, 1.577~1.743 and 1.6055~1.7745.

65. The construction method according to claim 1, characterized in that When the detection wavelength is 230nm, peak 4 is cryptochlorogenic acid, peak 9 is paeoniflorin, peak 14 is 4,5-o-dicaffeoylquinic acid, peak 15 is baicalin, peak 21 is glycyrrhizic acid, peak 22 is atractylodes lactone III, and peak 23 is ligusticum chuanxiong lactone A.

66. The construction method according to claim 1, characterized in that When the detection wavelength is 230nm, peak 1 comes from white peony root, peak 2 comes from Rehmannia root, peak 3 comes from Dipsacus asper, peak 4 comes from Ligusticum chuanxiong, peak 5 comes from Licorice root, peak 6 comes from Dipsacus asper, peak 7 comes from Amomum villosum, peak 8 comes from white peony root, peak 9 comes from white peony root, peak 10 comes from Scutellaria baicalensis, peak 11 comes from Astragalus membranaceus, peak 12 comes from Angelica sinensis and Ligusticum chuanxiong, peak 14 comes from Dipsacus asper, peak 15 comes from Scutellaria baicalensis, peak 16 comes from Scutellaria baicalensis, peak 17 comes from Scutellaria baicalensis, peak 18 comes from Scutellaria baicalensis, peak 19 comes from Scutellaria baicalensis, peak 21 comes from Licorice root, peak 22 comes from Atractylodes macrocephala, and peak 23 comes from Ligusticum chuanxiong.

67. Use of the construction method according to any one of claims 1 to 66 in quality detection and / or quality evaluation and / or quality control of a Chinese medicine composition comprising Atractylodes macrocephala.

Images