Quality control method for a traditional Chinese medicine compound containing fructus aurantii and mint and application thereof

Abstract

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

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to a quality control method for a traditional Chinese medicine compound containing fructus aurantii and mint and an application thereof. Background Art

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

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

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

[0005] At present, there is no literature report on a comprehensive analysis and fingerprint study of the chemical components of the traditional Chinese medicine compound comprising Citrus aurantium and mint claimed in the present invention. In the prior art, only a single component in the traditional 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 traditional 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 traditional 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 compound containing Fructus Aurantii Immaturus and Mint, and the construction method comprises the following steps:

[0007] Preparation of the test solution: take an appropriate amount of a decoction of a Chinese medicinal compound containing fructus aurantii and mint, shake well, filter, and take the filtrate to obtain the test solution; wherein the Chinese medicinal compound includes ginseng, atractylodes macrocephala, white tuckahoe, angelica sinensis, white peony root, fructus aurantii, amomum villosum, gardenia jasminoides, and mint;

[0008] Preparation of reference solution: Weigh appropriate amounts of paeoniflorin, cryptochlorogenic acid, and hesperidin reference substances, add methanol to prepare reference solutions with concentrations of paeoniflorin, cryptochlorogenic acid, and hesperidin of 10 to 100 μg / ml, respectively;

[0009] According to the results of high performance liquid chromatography detection of the test solution and the reference solution, a fingerprint spectrum of the Chinese medicine compound is obtained;

[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: 0-5 min, 97% B→95% B; 5-13 min, 95% B→94% B; 13-19 min, 94% B→93% B; 19-24 min, 93% B→92% B; 24-33 min, 92% B→89% B; 33-41 min, 89% B→83% B; 41-43 min, 83% B ; 43~49min, 83%B→80%B; 49~55min, 80%B→78%B; 55~65min, 78%→76%B; 65~68min, 76%B; 68~75min, 76%B→71%B; 75~77min, 71%B→56%B; 77~82min, 56%B→32%B; 82~86min, 32%B→97%B; 86~95min, 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 Chinese herbal compound decoction containing fructus aurantii and mint comprises: (a) weighing appropriate amounts of ginseng, atractylodes macrocephala, white poria, angelica sinensis, white peony root, fructus aurantii, amomum villosum, gardenia jasminoides and mint and placing them in 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 (c) combining the first filtrate and the second filtrate to obtain the Chinese herbal compound decoction containing fructus aurantii and mint.

[0012] Furthermore, the container is a multifunctional medicine decoction pot.

[0013] Furthermore, the ratio of the sum of the masses of ginseng, atractylodes macrocephala, white poria, angelica sinensis, white peony root, fructus aurantii, amomum villosum, gardenia jasminoides and mint to the mass / volume (g / ml) of the first water is 0.05-0.2, for example, about 0.125.

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

[0015] Furthermore, the first decocting time on low heat is 20 to 40 minutes, for example, about 30 minutes.

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

[0017] Furthermore, the ratio of the sum of the masses of ginseng, atractylodes macrocephala, white poria, angelica sinensis, white peony root, fructus aurantii, amomum villosum, gardenia jasminoides and mint to the mass / volume (g / ml) of the second water is 0.1-0.3, for example, about 0.167.

[0018] Furthermore, the second decocting time on low heat is 10 to 40 minutes, for example, about 25 minutes.

[0019] Furthermore, the volume of the Chinese herbal compound decoction containing Fructus Aurantii Immaturus and Mint is 400-600 ml, for example, about 500 ml.

[0020] Furthermore, the volume / volume ratio of the Chinese herbal compound decoction comprising Fructus Aurantii Immaturus and Mint to the first water is 0.4-0.8, for example, about 0.587.

[0021] Furthermore, the mass ratios of the ginseng, the atractylodes macrocephala, the white poria, the angelica sinensis, the white peony root, the fructus aurantii, the fructus amomi, the gardenia fruit and the mint are (2-4): (12-18): (8-10): (25-35): (25-35): (0.5-2.5): (2-4): (8-10): (5-7).

[0022] Furthermore, the mass ratios of the ginseng, the atractylodes macrocephala, the white poria, the angelica sinensis, the white peony root, the fructus aurantii, the fructus amomi, the gardenia fruit and the mint are (2.5-3.5): (14-16): (8.5-9.5): (28-32): (28-32): (1-2): (2.5-3.5): (8.5-9.5): (5.5-6.5).

[0023] Furthermore, the mass ratio of the ginseng, the atractylodes macrocephala, the white poria, the angelica sinensis, the white peony root, the fructus aurantii, the fructus amomi, the gardenia fruit and the mint is about 3: about 15: about 9: about 30: about 30: about 1.5: about 3: about 9: about 6.

[0024] Furthermore, the mass of the ginseng ranges from 1 to 10 g, for example, about 3 g.

[0025] Furthermore, the mass range of the Atractylodes macrocephala is 10 to 30 g, for example, about 15 g.

[0026] Furthermore, the mass range of the white Poria cocos is 5 to 15 g, for example, about 9 g.

[0027] Furthermore, the weight of the angelica is in the range of 20 to 40 g, for example, about 30 g.

[0028] Furthermore, the mass range of the white peony root is 20 to 40 g, for example, about 30 g.

[0029] Furthermore, the mass range of the Fructus Aurantii Immaturus is 0.5-5 g, for example, about 1.5 g.

[0030] Furthermore, the mass of the Amomum villosum is in the range of 1 to 10 g, for example, about 3 g.

[0031] Furthermore, the mass of the Gardenia jasminoides is in the range of 5 to 15 g, for example, about 9 g.

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

[0033] Furthermore, the Atractylodes macrocephala is stir-fried Atractylodes macrocephala.

[0034] Furthermore, the angelica is wine-washed angelica.

[0035] Furthermore, the white peony root is white peony root fried with wine.

[0036] Furthermore, the Citrus aurantium is stir-fried Citrus aurantium.

[0037] Furthermore, the Amomum villosum is ground and fried Amomum villosum.

[0038] Furthermore, the gardenia is fried gardenia.

[0039] Furthermore, in the method for preparing the test solution, the filtration is microporous membrane filtration.

[0040] Furthermore, the pore size of the microporous filter membrane is 0.22 μm.

[0041] Furthermore, the concentration of paeoniflorin in the reference solution is about 60.17 μg / ml.

[0042] Furthermore, the concentration of cryptochlorogenic acid in the reference solution is about 20.62 μg / ml.

[0043] Furthermore, the concentration of hesperidin in the reference solution is about 60.02 μg / ml.

[0044] Furthermore, the flow rate of the HPLC detection is 0.8-1.2 ml / min, for example, about 1.0 ml / min.

[0045] Furthermore, the column temperature is 20-30°C, for example, about 25°C.

[0046] Furthermore, the detection wavelength is 260-300 nm, for example, about 280 nm.

[0047] Furthermore, the injection volume is 5-15 μl, for example, about 10 μl.

[0048] Furthermore, the theoretical plate number of the chromatographic peak corresponding to the hesperidin is not less than 2000.

[0049] Furthermore, the chromatographic column is a GL Sciences Inertsustain AQ-C18 chromatographic column.

[0050] Furthermore, the specifications of the chromatographic column are: column length 250 mm, inner diameter 4.6 mm, and particle size 5 μm.

[0051] Furthermore, the mobile phase A is acetonitrile.

[0052] 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.

[0053] 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.

[0054] Furthermore, the acid aqueous solution is a 0.01% to 1% acid aqueous solution.

[0055] Furthermore, the acid aqueous solution is a 0.01% to 1% phosphoric acid aqueous solution.

[0056] Furthermore, the acid aqueous solution is about 0.1% phosphoric acid aqueous solution.

[0057] Furthermore, the buffered saline solution is an acetate solution and / or an acetate solution.

[0058] Furthermore, the pH value of the buffered saline solution is not greater than 7.0.

[0059] Further, when the detection wavelength is 280 nm, the fingerprint spectrum includes peaks 1-18, and the retention times of peaks 1-18 correspond to about 9.989 min, about 12.167 min, about 14.082 min, about 14.831 min, about 28.774 min, about 32.108 min, about 37.432 min, about 41.433 min, about 48.282 min, about 56.201 min, about 57.78 min, about 61.38 min, about 62.125 min, about 62.924 min, about 64.315 min, about 65.689 min, about 67.886 min and about 71.412 min, respectively.

[0060] Further, when the detection wavelength is 280 nm, the fingerprint spectrum includes peaks 1-18, wherein peak 13 hesperidin is used as a reference peak, and the relative retention time average values ​​of 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 14, peak 15, peak 16, peak 17 and peak 18 are about 0.16, about 0.20, about 0.23, about 0.24, about 0.46, about 0.52, about 0.60, about 0.67, about 0.78, about 0.90, about 0.93, about 0.99, about 1.01, about 1.04, about 1.06, about 1.09 and about 1.15, respectively.

[0061] Furthermore, when the detection wavelength is 280 nm, peak No. 8 is cryptochlorogenic acid, peak No. 9 is paeoniflorin, and peak No. 13 is hesperidin.

[0062] Furthermore, when the detection wavelength is 280nm, peak 1 comes from ginseng, earth-fried atractylodes and wine-washed angelica, peak 2 comes from wine-fried white peony, earth-fried atractylodes and wine-washed angelica, peak 3 comes from wine-washed angelica and wine-fried white peony, peak 4 comes from wine-fried white peony, peak 5 comes from Amomum villosum, peak 6 comes from earth-fried atractylodes, peak 7 comes from mint, peak 8 comes from wine-fried white peony, earth-fried atractylodes and The medicinal material of angelica washed with wine, peak No. 9 is the medicinal material of white peony stir-fried with wine, peak No. 10 comes from the medicinal material of Citrus aurantium, the medicinal material of angelica washed with wine, and the medicinal material of stir-fried Gardenia jasminoides, peak No. 11 comes from the medicinal material of white peony stir-fried with wine, peak No. 12 comes from the medicinal material of angelica washed with wine, peak No. 13 comes from the medicinal material of Citrus aurantium, peak No. 14 comes from the medicinal material of white peony stir-fried with wine and Citrus aurantium, peak No. 15 comes from the medicinal material of angelica washed with wine, peak No. 16 comes from the medicinal material of Citrus aurantium and stir-fried Gardenia jasminoides, peak No. 17 comes from the medicinal material of angelica washed with wine, and peak 18 comes from stir-fried Gardenia jasminoides.

[0063] According to another aspect of the present invention, a quality control method for a traditional Chinese medicine compound containing Fructus Aurantii Immaturus and Mint is provided, the quality control method comprising the following steps:

[0064] (1) Establishing a standard fingerprint of a Chinese medicine compound reference sample according to the above fingerprint construction method;

[0065] (2) taking a test solution and testing it according to the chromatographic conditions in the above fingerprint spectrum construction method to obtain a fingerprint spectrum of the Chinese medicine compound sample to be tested; and

[0066] (3) Compare the fingerprint of the Chinese medicine compound sample obtained in step (2) with the standard fingerprint of the Chinese medicine compound reference sample obtained in step (1). If it meets the requirements, it is a qualified product, and if it does not meet the requirements, it is an unqualified product.

[0067] Furthermore, the compliance requirement includes one or more of the following:

[0068] (1) The fingerprint of the Chinese medicine compound sample to be tested exhibits 18 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 compound reference sample;

[0069] (2) With the hesperidin peak as the S peak, the relative retention time of each characteristic chromatographic peak in the fingerprint of the Chinese medicine compound 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 compound reference sample; and

[0070] (3) According to the similarity evaluation system of Chinese medicine chromatographic fingerprint, the similarity between the fingerprint of the Chinese medicine compound sample to be tested and the standard fingerprint of the Chinese medicine compound reference sample shall be calculated, and the similarity shall not be less than 0.90.

[0071] 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 compound containing Fructus Aurantii Immaturus and Menthus Officinalis.

[0072] Beneficial effects of the present invention:

[0073] In short, the present invention provides a fingerprint spectrum determination and quality control method for a traditional Chinese medicine compound reference sample containing Citrus aurantium and mint. The method confirms 18 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 stable quality of the product, guarantees the efficacy of the traditional Chinese medicine compound, and allows the traditional Chinese medicine compound to better serve human life and health.

[0074] Specifically, compared with the prior art, the present invention has the following beneficial effects:

[0075] (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.

[0076] (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.

[0077] (3) In the process of establishing the fingerprint spectrum of the traditional Chinese medicine compound reference sample of the present invention, 18 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.

[0078] (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.

[0079] (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

[0080] 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.

[0081] Figure 1 This is the reference fingerprint of the test solution of the Chinese medicine compound decoction of the present application at a wavelength of 280nm, wherein peak 8: cryptochlorogenic acid, peak 9: paeoniflorin, peak 13 (S): hesperidin.

[0082] Figure 2 These are the fingerprint spectra of 20 batches of decoction of the Chinese herbal compound decoction of the present application at a wavelength of 280nm. DETAILED DESCRIPTION

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] As described in the background technology section, there is no literature report on a comprehensive analysis of the chemical components and fingerprint study of the Chinese medicine composition comprising Fructus Aurantii Immaturus and Mint 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 compound comprising Fructus Aurantii Immaturus and Mint, and the construction method comprises the following steps:

[0089] Preparation of the test solution: take an appropriate amount of a decoction of a Chinese medicinal compound containing fructus aurantii and mint, shake well, filter, and take the filtrate to obtain the test solution; wherein the Chinese medicinal compound includes ginseng, atractylodes macrocephala, white tuckahoe, angelica sinensis, white peony root, fructus aurantii, amomum villosum, gardenia jasminoides, and mint;

[0090] Preparation of reference solution: Weigh appropriate amounts of paeoniflorin, cryptochlorogenic acid, and hesperidin reference substances, add methanol to prepare reference solutions with concentrations of paeoniflorin, cryptochlorogenic acid, and hesperidin of 10 to 100 μg / ml, respectively;

[0091] According to the results of high performance liquid chromatography detection of the test solution and the reference solution, a fingerprint spectrum of the Chinese medicine compound is obtained;

[0092] 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: 0-5 min, 97% B→95% B; 5-13 min, 95% B→94% B; 13-19 min, 94% B→93% B; 19-24 min, 93% B→92% B; 24-33 min, 92% B→89% B; 33-41 min, 89% B→83% B; 41-43 min, 83% B ; 43~49min, 83%B→80%B; 49~55min, 80%B→78%B; 55~65min, 78%→76%B; 65~68min, 76%B; 68~75min, 76%B→71%B; 75~77min, 71%B→56%B; 77~82min, 56%B→32%B; 82~86min, 32%B→97%B; 86~95min, 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.

[0093] In the present invention, when concentration, time, flow rate, temperature, wavelength, volume, ratio, mass, or other values ​​or parameters are expressed as ranges, preferred ranges, or ranges 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 "10-40" is disclosed, the described range should be interpreted as including the range "10-40", "10-35", "10-30", "10-25", "10-20", "10-15", "15-40", "15-35", "15-30", "15-25", "15-20", "20-40", "20-35", "20-30", "20-25", "25-40", "25-35", "25-30", "30-40", "30-35", "35-40", 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.

[0094] In a preferred embodiment, the preparation method of the Chinese herbal compound decoction containing Citrus aurantium and mint comprises: (a) weighing appropriate amounts of ginseng, Atractylodes macrocephala, white Poria cocos, Chinese angelica, white peony root, Citrus aurantium, Amomum villosum, Gardenia jasminoides and mint and placing them in 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 (c) combining the first filtrate and the second filtrate to obtain the Chinese herbal compound decoction containing Citrus aurantium and mint.

[0095] In a preferred embodiment, the container is a multifunctional medicine decoction pot.

[0096] In a preferred embodiment, the ratio of the sum of the masses of ginseng, Atractylodes macrocephala, white Poria cocos, Chinese angelica, white peony root, fructus aurantii, fructus amomi, gardenia jasminoides and mint to the mass / volume (g / ml) of the first water is 0.05-0.2, for example, about 0.125.

[0097] 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.

[0098] In a preferred embodiment, the soaking time is 20 to 40 minutes, such as about 30 minutes.

[0099] 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.

[0100] In a preferred embodiment, the first decocting time over low heat is 20 to 40 minutes, for example, 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 filter is a 150 mesh screen.

[0103] In a preferred embodiment, the ratio of the sum of the masses of ginseng, atractylodes, white poria, angelica, white peony root, fructus aurantii, amomum villosum, gardenia jasminoides and mint to the mass / volume (g / ml) of the second water is 0.1-0.3, for example, about 0.167.

[0104] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.167" includes ±5% of 0.167, or from 0.15865 to 0.17535.

[0105] In a preferred embodiment, the second decocting time over low heat is 10 to 40 minutes, for example, about 25 minutes.

[0106] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 25" includes ±5% of 25, or from 23.75 to 26.25.

[0107] In a preferred embodiment, the volume of the Chinese herbal compound decoction containing Fructus Aurantii Immaturus and Mentha is 400-600 ml, for example, about 500 ml.

[0108] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 500" includes ±5% of 500, or from 475 to 525.

[0109] In a preferred embodiment, the volume / volume ratio of the traditional Chinese medicine compound decoction comprising Fructus Aurantii Immaturus and Mint to the first water is 0.4-0.8, for example, about 0.587.

[0110] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.587" includes ±5% of 0.587, or from 0.55765 to 0.61635.

[0111] In a preferred embodiment, the mass ratio of the ginseng, the atractylodes, the white poria, the angelica, the white peony root, the fructus aurantii, the fructus amomi, the gardenia and the mint is (2-4): (12-18): (8-10): (25-35): (25-35): (0.5-2.5): (2-4): (8-10): (5-7).

[0112] In a preferred embodiment, the mass ratio of the ginseng, the atractylodes, the white poria, the angelica, the white peony root, the fructus aurantii, the fructus amomi, the gardenia and the mint is (2.5-3.5): (14-16): (8.5-9.5): (28-32): (28-32): (1-2): (2.5-3.5): (8.5-9.5): (5.5-6.5).

[0113] In a preferred embodiment, the mass ratio of the ginseng, the atractylodes macrocephala, the white poria, the angelica sinensis, the white peony root, the fructus aurantii, the amomum villosum, the gardenia fruit and the mint is about 3: about 15: about 9: about 30: about 30: about 1.5: about 3: about 9: about 6.

[0114] 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 15" includes ±5% of 15, or from 14.25 to 15.75; "about 9" includes ±5% of 9, or from 8.55 to 9.45; "about 30" includes ±5% of 30, or from 28.5 to 31.5; "about 1.5" includes ±5% of 1.5, or from 1.425 to 1.575; "about 6" includes ±5% of 6, or from 5.7 to 6.3.

[0115] In a preferred embodiment, the mass of the ginseng ranges from 1 to 10 g, such as about 3 g.

[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.

[0117] In a preferred embodiment, the mass range of the Atractylodes macrocephala is 10 to 30 g, for example, about 15 g.

[0118] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 15" includes ±5% of 15, or from 14.25 to 15.75.

[0119] In a preferred embodiment, the mass of the white Poria cocos is in the range of 5 to 15 g, for example, about 9 g.

[0120] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 9" includes ±5% of 9, or from 8.55 to 9.45.

[0121] In a preferred embodiment, the weight of the angelica sinensis is in the range of 20 to 40 g, such as about 30 g.

[0122] 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.

[0123] In a preferred embodiment, the mass range of the white peony root is 20-40 g, for example, about 30 g.

[0124] 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.

[0125] In a preferred embodiment, the mass of the Fructus Aurantii Immaturus is in the range of 0.5 to 5 g, for example, about 1.5 g.

[0126] 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.

[0127] In a preferred embodiment, the mass of the Amomum villosum is in the range of 1 to 10 g, such as about 3 g.

[0128] 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.

[0129] In a preferred embodiment, the mass of the Gardenia jasminoides is in the range of 5 to 15 g, for example, about 9 g.

[0130] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 9" includes ±5% of 9, or from 8.55 to 9.45.

[0131] In a preferred embodiment, the mass of the mint ranges from 1 to 10 g, such as about 6 g.

[0132] 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.

[0133] In a preferred embodiment, the Atractylodes macrocephala is stir-fried Atractylodes macrocephala.

[0134] In a preferred embodiment, the angelica is wine-washed angelica.

[0135] In a preferred embodiment, the white peony root is fried with wine.

[0136] In a preferred embodiment, the Fructus Aurantii Immaturus is stir-fried Fructus Aurantii Immaturus.

[0137] In a preferred embodiment, the Amomum villosum is ground and fried Amomum villosum.

[0138] In a preferred embodiment, the gardenia is fried gardenia.

[0139] In a preferred embodiment, in the method for preparing the test solution, the filtration is microporous membrane filtration.

[0140] In a preferred embodiment, the pore size of the microporous filter membrane is 0.22 μm.

[0141] In a preferred embodiment, the concentration of paeoniflorin in the reference solution is about 60.17 μg / ml.

[0142] 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.

[0143] In a preferred embodiment, the concentration of cryptochlorogenic acid in the reference solution is about 20.62 μg / ml.

[0144] 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.

[0145] In a preferred embodiment, the concentration of hesperidin in the reference solution is about 60.02 μg / ml.

[0146] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 60.02" includes ±5% of 60.02, or from 57.019 to 63.021.

[0147] 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.

[0148] 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.

[0149] In a preferred embodiment, the column temperature is 20-30°C, such as about 25°C.

[0150] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 25" includes ±5% of 25, or from 23.75 to 26.25.

[0151] In a preferred embodiment, the detection wavelength is 260-300 nm, such as about 280 nm.

[0152] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 280" includes ±5% of 280, or from 266 to 294.

[0153] In a preferred embodiment, the injection volume is 5-15 μl, such as about 10 μl.

[0154] 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.

[0155] In a preferred embodiment, the theoretical plate number of the chromatographic peak corresponding to the hesperidin is not less than 2000.

[0156] In a preferred embodiment, the chromatographic column is a GL Sciences Inertsustain AQ-C18 chromatographic column.

[0157] 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.

[0158] In a preferred embodiment, mobile phase A is acetonitrile.

[0159] 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.

[0160] 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.

[0161] In a preferred embodiment, the acid aqueous solution is a 0.01% to 1% acid aqueous solution.

[0162] In a preferred embodiment, the acid aqueous solution is a 0.01% to 1% phosphoric acid aqueous solution.

[0163] In a preferred embodiment, the aqueous acid solution is about 0.1% phosphoric acid aqueous solution.

[0164] 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%.

[0165] In a preferred embodiment, the buffered saline solution is an acetate aqueous solution and / or an acetate aqueous solution.

[0166] In a preferred embodiment, the pH value of the buffered saline solution is no greater than 7.0.

[0167] In a preferred embodiment, when the detection wavelength is 280 nm, the fingerprint spectrum includes peaks 1-18, and the retention times of peaks 1-18 correspond to about 9.989 min, about 12.167 min, about 14.082 min, about 14.831 min, about 28.774 min, about 32.108 min, about 37.432 min, about 41.433 min, about 48.282 min, about 56.201 min, about 57.78 min, about 61.38 min, about 62.125 min, about 62.924 min, about 64.315 min, about 65.689 min, about 67.886 min and about 71.412 min, respectively.

[0168] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 9.989" includes ±5% of 9.989, or from 9.48955 to 10.48845; "about 12.167" includes ±5% of 12.167, or from 11.55865 to 12.77535; "about 14.082" includes ±5% of 14.082, or from 13.3779 to 14.7861; "about 14.831" includes ±5% of 14.831, or from 14.08945 to 15.57255; "about 28.774" includes 28.77 4 ± 5%, or from 27.3353 to 30.2127; "about 32.108" includes ± 5% of 32.108, or from 30.5026 to 33.7134; "about 37.432" includes ± 5% of 37.432, or from 35.5604 to 39.3036; "about 41.433" includes ± 5% of 41.433, or from 39.36135 to 43.50465; "about 48.282" includes ± 5% of 48.282, or from 45.8679 to 50.6 961; "about 56.201" includes ±5% of 56.201, or from 53.39095 to 59.01105; "about 57.78" includes ±5% of 57.78, or from 54.891 to 60.669; "about 61.38" includes ±5% of 61.38, or from 58.311 to 64.449; "about 62.125" includes ±5% of 62.125, or from 59.01875 to 65.23125; "about 62.924" includes ±5% of 62.924, Or from 59.7778 to 66.0702; "about 64.315" includes ±5% of 64.315, or from 61.09925 to 67.53075; "about 65.689" includes ±5% of 65.689, or from 62.40455 to 68.97345; "about 67.886" includes ±5% of 67.886, or from 64.4917 to 71.2803; "about 71.412" includes ±5% of 71.412, or from 67.8414 to 74.9826.

[0169] In a preferred embodiment, when the detection wavelength is 280 nm, the fingerprint spectrum includes peaks 1-18, wherein peak 13 hesperidin is used as a reference peak, and the relative retention time averages of peaks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17 and 18 are about 0.16, about 0.20, about 0.23, about 0.24, about 0.46, about 0.52, about 0.60, about 0.67, about 0.78, about 0.90, about 0.93, about 0.99, about 1.01, about 1.04, about 1.06, about 1.09 and about 1.15, respectively.

[0170] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.16" includes ±5% of 0.16, or from 0.152 to 0.168; "about 0.20" includes ±5% of 0.20, or from 0.19 to 0.21; "about 0.23" includes ±5% of 0.23, or from 0.2185 to 0.2415; "about 0.24" includes ±5% of 0.24, or from 0.228 to 0.252; "about 0.46 ... "about 0.52" includes ±5% of 0.52, or from 0.494 to 0.546; "about 0.60" includes ±5% of 0.60, or from 0.57 to 0.63; "about 0.67" includes ±5% of 0.67, or from 0.6365 to 0.7035; "about 0.78" includes ±5% of 0.78, or From 0.741 to 0.819; "about 0.90" includes ±5% of 0.90, or from 0.855 to 0.945; "about 0.93" includes ±5% of 0.93, or from 0.8835 to 0.9765; "about 0.99" includes ±5% of 0.99, or from 0.9405 to 1.0395; "about 1.01" includes ±5% of 1.01, or from 0.9595 to 1.0605; “about 1.04” includes ±5% of 1.04, or from 0.988 to 1.092; “about 1.06” includes ±5% of 1.06, or from 1.007 to 1.113; “about 1.09” includes ±5% of 1.09, or from 1.0355 to 1.1445; “about 1.15” includes ±5% of 1.15, or from 1.0925 to 1.2075.

[0171] In a preferred embodiment, when the detection wavelength is 280 nm, peak No. 8 is cryptochlorogenic acid, peak No. 9 is paeoniflorin, and peak No. 13 is hesperidin.

[0172] In a preferred embodiment, when the detection wavelength is 280nm, peak 1 comes from ginseng medicinal materials, earth-fried atractylodes medicinal materials and wine-washed angelica medicinal materials, peak 2 comes from wine-fried white peony medicinal materials, earth-fried atractylodes medicinal materials and wine-washed angelica medicinal materials, peak 3 comes from wine-washed angelica medicinal materials and wine-fried white peony medicinal materials, peak 4 comes from wine-fried white peony medicinal materials, peak 5 comes from Amomum villosum medicinal materials, peak 6 comes from earth-fried atractylodes medicinal materials, peak 7 comes from mint medicinal materials, peak 8 comes from wine-fried white peony medicinal materials, earth-fried atractylodes medicinal materials Medicinal materials and wine-washed angelica medicinal materials, Peak No. 9 comes from wine-fried white peony medicinal materials, Peak No. 10 comes from Citrus aurantium medicinal materials, wine-washed angelica medicinal materials, and fried Gardenia jasminoides medicinal materials, Peak No. 11 comes from wine-fried white peony medicinal materials, Peak No. 12 comes from wine-washed angelica medicinal materials, Peak No. 13 comes from Citrus aurantium medicinal materials, Peak No. 14 comes from wine-fried white peony medicinal materials and Citrus aurantium medicinal materials, Peak No. 15 comes from wine-washed angelica medicinal materials, Peak No. 16 comes from Citrus aurantium medicinal materials and fried Gardenia jasminoides medicinal materials, Peak No. 17 comes from wine-washed angelica medicinal materials, and Peak 18 comes from fried Gardenia jasminoides medicinal materials.

[0173] According to another aspect of the present invention, a quality control method for a traditional Chinese medicine compound containing Fructus Aurantii Immaturus and Mint is provided, the quality control method comprising the following steps:

[0174] (1) Establishing a standard fingerprint of a Chinese medicine compound reference sample according to the above fingerprint construction method;

[0175] (2) taking a test solution and testing it according to the chromatographic conditions in the above fingerprint spectrum construction method to obtain a fingerprint spectrum of the Chinese medicine compound sample to be tested; and

[0176] (3) Compare the fingerprint of the Chinese medicine compound sample obtained in step (2) with the standard fingerprint of the Chinese medicine compound reference sample obtained in step (1). If it meets the requirements, it is a qualified product, and if it does not meet the requirements, it is an unqualified product.

[0177] In a preferred embodiment, the compliance requirements include one or more of the following:

[0178] (1) The fingerprint of the Chinese medicine compound sample to be tested exhibits 18 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 compound reference sample;

[0179] (2) With the hesperidin peak as the S peak, the relative retention time of each characteristic chromatographic peak in the fingerprint of the Chinese medicine compound 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 compound reference sample; and

[0180] (3) According to the similarity evaluation system of Chinese medicine chromatographic fingerprint, the similarity between the fingerprint of the Chinese medicine compound sample to be tested and the standard fingerprint of the Chinese medicine compound reference sample shall be calculated, and the similarity shall not be less than 0.90.

[0181] 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 compound containing Fructus Aurantii Immaturus and Menthus Officinalis.

[0182] 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.

[0183] 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.

[0184] 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.

[0185] Example

[0186] 1. Instruments and test drugs

[0187] Instruments: High performance liquid chromatograph Waters 2695 chromatography system, including quaternary gradient infusion pump (Alliance2695), 120-position high performance autosampler, imported column oven, Waters 2996 DAD diode array detector, Empower 3 chromatography management system; 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; Chromatographic columns: Chromatographic column 1: GL Sciences InretSustain AQ-C18 5μm4.6x250mm Serial No.23J0443221; Chromatographic column 2: GL Sciences InretSustain AQ-C18 5.0μm4.6x250mm Serial No. 23J0443146; Chromatographic column 3: GL Sciences InretSustain AQ-C185μm4.6x250mm 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.

[0188] Reference substance information: 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); hesperidin (batch number 110721-202220, content 97.2%, China Food and Drug Inspection Institute).

[0189] 2. Solution preparation

[0190] 2.1. Preparation of reference solution

[0191] Accurately weigh appropriate amounts of paeoniflorin, cryptochlorogenic acid, and hesperidin reference substances, and add methanol to prepare solutions of 60.17 μg / ml paeoniflorin, 20.62 μg / ml cryptochlorogenic acid, and 60.02 μg / ml hesperidin; prepare reference solutions.

[0192] 2.2 Preparation of test solution

[0193] 3g ginseng; 15g atractylodes (fried in soil); 9g white poria; 30g angelica (washed with wine); 30g white peony root (fried with wine); 1.5g fructus aurantii (fried); 3 grains of amomum villosum (fried and ground); 9g gardenia (fried); 6g mint, a total of 106.5g. Take the above nine flavors, put them in a multi-functional decoction pot, add 8 times the amount of water to cover the medicinal materials by 2-5cm, soak for 30 minutes, boil over high heat, then simmer for about 30 minutes with slow heat, filter with a 150-mesh sieve, add 6 times the amount of water to the residue, boil over high heat, simmer again for 25 minutes with slow heat, combine the decoction, and the final decoction volume is about 500ml. Take an appropriate amount of the above decoction, shake well, filter, and take the filtrate.

[0194] 2.3 Preparation of negative solution

[0195] 2.3.1. Preparation of negative sample solution lacking ginseng

[0196] 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".

[0197] 2.3.2 Preparation of negative sample solution of Atractylodes macrocephala with lack of soil

[0198] Weigh the other medicinal ingredients except the stir-fried Atractylodes macrocephala according to the prescription, and prepare the negative sample solution lacking the stir-fried Atractylodes macrocephala according to the "Preparation of test solution".

[0199] 2.3.3 Preparation of negative sample solution lacking white Poria cocos

[0200] Weigh the other medicinal ingredients except white Poria cocos according to the prescription, and prepare the negative sample solution lacking white Poria cocos according to the "Preparation of test solution".

[0201] 2.3.4. Preparation of negative sample solution of Angelica sinensis without alcohol

[0202] Weigh the other medicinal ingredients except Angelica sinensis wine according to the prescription, and prepare the negative sample solution lacking Angelica sinensis wine according to the "Preparation of test solution".

[0203] 2.3.5. Preparation of negative sample solution of Paeonia lactiflora with lack of alcohol

[0204] Weigh the other medicinal ingredients except white peony root with wine according to the prescription, and prepare the negative sample solution lacking white peony root with wine according to the "Preparation of test solution".

[0205] 2.3.6 Preparation of negative sample solution without stir-fried Fructus Aurantii Immaturus

[0206] Weigh the other medicinal ingredients except stir-fried Fructus Aurantii Immaturus according to the prescription, and prepare the negative sample solution without stir-fried Fructus Aurantii Immaturus according to the "Preparation of test solution".

[0207] 2.3.7 Preparation of negative sample solution lacking stir-fried Amomum villosum

[0208] Weigh the other medicinal ingredients except stir-fried Amomum villosum according to the prescription, and prepare the negative sample solution without stir-fried Amomum villosum according to the "Preparation of test solution".

[0209] 2.3.8 Preparation of negative sample solution lacking fried Gardenia jasminoides

[0210] Weigh the other medicinal ingredients except stir-fried gardenia according to the prescription, and prepare the negative sample solution without stir-fried gardenia according to the "Preparation of test solution".

[0211] 2.4 Preparation of single-flavor solutions

[0212] Take appropriate amounts of the nine single-ingredient pieces respectively, put them into a stoppered conical flask, add 50 ml of water, heat and reflux for 45 minutes, cool, filter, and obtain the filtrate.

[0213] 3. Screening of chromatographic conditions

[0214] 3.1. Initial chromatographic conditions

[0215] Determined by high performance liquid chromatography (General Rules 0512 of Part IV of the 2020 edition of the Chinese Pharmacopoeia).

[0216] Chromatographic conditions and system suitability test: Octadecyl bonded silica gel was used as filler (GL Sciences Inertsustain AQ-C18 4.6x250 mm); acetonitrile was used as mobile phase A, and 0.1% phosphoric acid was used as mobile phase B; initial gradient elution was performed according to the provisions in Table 1; and the detection wavelength was 190-400 nm.

[0217] Table 1 Initial gradient

[0218] Time (min) Acetonitrile (%) 0.1% Phosphoric acid (%) 0～120 5→95 95→5

[0219] According to the separation of components in the compound, the best absorption wavelength is selected and the gradient conditions are optimized, and finally a better detection method is obtained.

[0220] The results show that the isobsorptivity diagram of GL Sciences Inertsustain AQ-C18 at 190-400 nm shows that there is obvious absorption fluorescence at 200-300 nm, and in the liquid chromatogram at 210 nm, there is a characteristic peak within the first 75 minutes. Therefore, considering that the acetonitrile ratio is within 60%, gradient 1 in Table 2 is designed.

[0221] 3.2. Mobile phase gradient selection

[0222] In order to make each main chromatographic peak in the chromatogram have better separation parameters and present better chromatographic behavior, the test solution in "2.2, Preparation of test solution" was taken to optimize the elution gradient. The optimized gradient program is as follows.

[0223] Table 2 Gradient 1

[0224] Time (min) Acetonitrile (%) 0.1% Phosphoric acid (%) 0→10 5→11 95→89 10→20 11→20 89→80 20→25 20→23 80→77 25→30 23→28 77→72 30→35 28→30 72→70 35→40 30→38 70→62 40→50 38→60 62→40

[0225] The results show that from the isobsorption line diagram of gradient 1, it can be seen that the Chinese herbal compound decoction containing Citrus aurantium and mint has a chromatographic peak absorption fluorescence point in the range of 200-280nm. At a wavelength of 250nm, there is a large accumulation of characteristic peaks in the first 25 minutes and no characteristic peaks in 37-42 minutes. Therefore, optimization was carried out to design gradient 2 in Table 3.

[0226] Table 3 Gradient 2

[0227] Time (min) Acetonitrile (%) 0.1% Phosphoric acid (%) 0→6 3→7 97→93 6→10 7→8 93→92 10→16 8→11 92→89 16→26 11→20 89→80 26→31 20→23 80→77 31→36 23→28 77→72 36→41 28→30 72→70 41→43 30→38 70→62 43→45 38→44 62→56 45→50 44→68 56→32 50→55 68→3 32→97 55→70 3 97

[0228] Under the liquid phase spectrum at wavelengths of 250nm and 280nm, the peak response values ​​at 250nm are higher in the first 35 minutes (acetonitrile ratio is about 28%), and the peak response values ​​at 280nm are higher after 35 minutes. In view of the concentration of characteristic peaks in the entire spectrum, the comparison is carried out by extending the time, so the gradient 3 of Table 4 is designed.

[0229] Table 4 Gradient 3

[0230]

[0231]

[0232] Under the liquid phase spectrum with a wavelength of 250nm, the characteristic peaks are more accumulated in the first 14 minutes, and the time is extended and the acetonitrile gradient is refined to separate them. The chromatographic peaks are well separated from 14 to 35 minutes, and the acetonitrile ratio remains unchanged. Under the liquid phase spectrum with a wavelength of 280nm, the characteristic peaks are accumulated after 45 to 55 minutes, and the time is extended and the acetonitrile gradient is refined to separate them, so the gradient 4 in Table 5 is designed.

[0233] Table 5 Gradient 4

[0234] Time (min) Acetonitrile (%) 0.1% Phosphoric acid (%) 0→5 3→5 97→95 5→9 5→6 95→94 9→13 6→7 94→93 13→18 7→8 93→92 18→27 8→11 92→89 27→35 11→17 89→83 35→46 17→22 83→78 46→54 22→24 78→76 54→65 24→29 76→71 65→67 29→41 71→59 67→69 41→44 59→56 69→71 44→68 56→32 71→75 68→3 32→97 75→90 3 97

[0235] In the liquid phase spectrum at a wavelength of 250 nm, the characteristic peaks accumulated more in the first 14 minutes, and the optimization was carried out by extending the time. In the liquid phase spectrum at a wavelength of 280 nm, a split peak appeared at the peak time of 55.997, and isocratic optimization was adopted, so the gradient 5 in Table 6 was designed.

[0236] Table 6 Gradient 5

[0237]

[0238]

[0239] Through the liquid phase spectrum at wavelengths of 250nm and 280nm, in the first 51 minutes, the response values ​​of each peak at 250nm were high and the separation effect of each peak was good; after 51 minutes, the response values ​​of each peak at 280nm were high and met the separation requirements of the fingerprint spectrum; considering the differences in instruments, Waters 2695_2996 high performance liquid chromatograph was used to further verify gradient 5 and determine its detection wavelength.

[0240] The results showed that under the gradient 5 elution procedure, the 280nm baseline was relatively smoother, the separation was relatively better, and the separation degree was greater than 1.5, which met the fingerprint requirements. Therefore, the gradient 5 chromatographic conditions and the detection wavelength of 280nm were selected for the fingerprint determination of the traditional Chinese medicine compound containing Citrus aurantium and mint.

[0241] 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 gradient 5 specified in Table 6; the flow rate is 1.0 ml per minute, and the detection wavelength is 280 nm; the column temperature is 25°C; the number of theoretical plates calculated based on the hesperidin peak should be not less than 2000.

[0242] Preparation of test solution: Take an appropriate amount of decoction and filter it through a 0.22μm microporous membrane.

[0243] Determination method: Accurately pipette 10μl of the test solution, inject it into the liquid chromatograph, and determine it.

[0244] 4. Validation of analytical methods

[0245] 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).

[0246] 4.1. Exclusivity

[0247] Prepare negative blank sample solution according to the test solution preparation method, test according to the fingerprint method, and record the chromatograms of the reference solution and negative blank sample solution.

[0248] By comparing the absorption spectra with those of the reference solution, it was determined that peaks 8, 9 and 13 were chlorogenic acid, paeoniflorin and hesperidin, respectively, as shown in Table 7.

[0249] Table 7 System parameters of the fingerprint of the traditional Chinese medicine compound containing Fructus Aurantii Immaturus and Mentha spicata of the present invention

[0250] Characteristic peak Retention time area high width Separation Symmetry Factor USP Theoretical Plate Number Remark Peak 1 9.989 1252394 120115 43 / 1.54 23906 / Peak 2 12.167 4767563 401588 43 7.65 1.08 23822 / Peak 3 14.082 261144 23597 24 6.14 1.23 34278 / Peak 4 14.831 420295 27332 49 2.09 1.05 21159 / Peak 5 28.774 57195 2912 46 29.81 1.00 47918 / Peak 6 32.108 225806 10217 65 6.00 1.01 48835 / Peak 7 37.432 35064 1835 43 9.75 0.84 88103 / Peak 8 41.433 186438 16823 25 9.82 0.88 300167 Cryptochlorogenic acid Peak 9 48.282 1090902 64759 62 19.04 1.12 210570 Paeoniflorin Peak 10 56.201 677351 40347 52 18.35 1.15 250412 / Peak 11 57.78 483037 26382 49 3.36 0.93 211491 / Peak 12 61.38 2142377 134729 49 7.88 1.02 334573 / Peak 13 62.125 1695153 100177 50 1.71 1.07 306313 Hesperidin Peak 14 62.924 214268 8128 55 1.35 1.33 144460 / Peak 15 64.315 1403957 67863 77 2.17 1.01 221071 / Peak 16 65.689 1442668 77026 59 2.65 0.97 279220 / Peak 17 67.886 293188 13442 54 4.06 1.02 211101 / Peak 18 71.412 39917 1994 42 6.15 1.13 260163 /

[0251] The results show that the chromatogram of the traditional Chinese medicine compound benchmark sample containing Citrus aurantium and mint of the present invention mainly presents 18 peaks, peak 1 belongs to ginseng, earth-fried Atractylodes macrocephala and wine Angelica sinensis; peaks 2 and 8 belong to wine white peony, earth-fried Atractylodes macrocephala and wine Angelica sinensis, where peak 8 is cryptochlorogenic acid; peak 3 belongs to wine Angelica sinensis and wine white peony; peaks 4, 9, and 11 belong to wine white peony, where peak 9 is paeoniflorin; peak 5 belongs to Amomum villosum; peak 6 belongs to earth-fried Atractylodes macrocephala; peak 7 belongs to mint; peaks 12, 15, and 17 belong to wine Angelica sinensis; peak 13 belongs to Citrus aurantium, which is hesperidin; peak 18 belongs to Gardenia jasminoides; negative without interference, the method has good specificity. The specific summary is shown in Table 8.

[0252] Table 8 Summary of specific chromatographic peaks of the Chinese medicinal compound containing Fructus Aurantii Immaturus and Peppermint of the present invention

[0253]

[0254]

[0255] 4.2 Precision

[0256] 4.2.1. Instrument precision test

[0257] The same test solution was sampled 6 times continuously, the chromatogram was recorded, and the similarity of each characteristic peak was calculated.

[0258] The results show that the similarities of the instrument precision of this method are all greater than 0.90, indicating that the instrument precision of this method is good.

[0259] 4.2.2 Intermediate precision

[0260] Two personnel prepared 6 test sample solutions according to the above conditions, respectively, injected and tested them on Agilent HPLC and Waters HPLC, recorded the chromatograms, and calculated the similarity of each characteristic peak.

[0261] The results showed that the similarity of the intermediate precision of each characteristic peak was greater than 0.90, which was in line with the regulations.

[0262] 4.2.3 Repeatability test

[0263] Take 6 samples from the same batch and prepare the test samples according to the method of “2.2, Preparation of test solution”, record the chromatograms, and calculate the similarity of each characteristic peak.

[0264] The results show that the similarity results of the repeatability of each characteristic peak are all greater than 0.90, which meets the requirements.

[0265] 4.3 Durability

[0266] 4.3.1 Stability Study

[0267] Take the same test solution and inject it at 0, 3h, 6h, 9h, 12h, 18h, 24h and 48h. Record the chromatogram, calculate the similarity of each characteristic peak, and examine the stability of the test solution.

[0268] The results showed that its stability was within 48 hours, and the similarity results of each characteristic peak were greater than 0.90, which met the requirements, indicating that it was stable within 48 hours.

[0269] 4.3.2 Investigation of different flow rates

[0270] Take the same test solution and conduct tests at different flow rates of 1.0±0.1ml / min, record the chromatograms, and calculate the similarity of each characteristic peak.

[0271] 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.

[0272] 4.3.3 Investigation of different column temperatures

[0273] Take the same test solution, test it at 25±3℃ respectively, and record the chromatogram.

[0274] The results showed that under the column temperature of 22°C, the characteristic peaks were not well separated when the retention time was around 62-63 minutes, and under the column temperature of 28°C, the characteristic peaks were not well separated when the retention time was around 11-12 minutes, so it is recommended to determine the column temperature to be 25°C.

[0275] 4.3.4 Different chromatographic columns

[0276] The same test solution was tested on different chromatographic columns (chromatographic column 1: GL Sciences InretSustainAQ-C18 5μm 4.6x250mm Serial No.23J0443146), (chromatographic column 2: GL Sciences InretSustain AQ-C18 5μm 4.6x250mm SerialNo.23J0443221), and (chromatographic column 3: GL Sciences InretSustain AQ-C18 5μm4.6x250mm SerialNo.23J0443226). The chromatograms were recorded and the similarity matching was calculated with the standard reference spectrum of the traditional Chinese medicine compound containing Fructus Aurantii Immaturus and Mentha.

[0277] The results showed that the similarity results of different chromatographic columns were all greater than 0.90, which was in line with the regulations.

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

[0279] The results show that the similarity results of different acid types under this method are all greater than 0.9, indicating that this method has good durability in 0.1% formic acid, phosphoric acid, and acetic acid.

[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 concentrations in the mobile phase, and the chromatograms were recorded and the similarity matching calculation was performed with the standard reference spectrum of the traditional Chinese medicine compound containing Citrus aurantium and mint.

[0282] The results show that under different acid concentrations, the similarity of each common peak is greater than 0.90, which meets the requirements.

[0283] 4.4. Fingerprint method confirmation

[0284] According to the results of the above methodological investigation, among the 18 common peaks of the established fingerprint, each chromatographic peak is less affected by factors such as different brands of HPLC, different chromatographic columns, different acid types and flow rates, and the similarities of each characteristic peak of the instrument precision, intermediate precision, repeatability test and stability test are all greater than 0.9, indicating that the method has good durability, is stable and reliable, and can be used for fingerprint determination.

[0285] Based on the above results, the fingerprint method is as follows:

[0286] Determined by high performance liquid chromatography (General Rules 0512 of Part IV of the 2020 edition of the Chinese Pharmacopoeia).

[0287] 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 gradient 5 specified in Table 6; the flow rate is 1.0 ml per minute, and the detection wavelength is 280 nm; the column temperature is 25°C; the number of theoretical plates calculated based on the hesperidin peak should be not less than 2000.

[0288] Preparation of reference solution: Take appropriate amount of paeoniflorin, cryptochlorogenic acid, and hesperidin reference substances, accurately weigh them, add methanol to make 1 ml of a mixed solution containing 60.17 μg of paeoniflorin, 20.62 μg of cryptochlorogenic acid, and 60.02 μg of hesperidin, respectively.

[0289] Preparation of test solution: Take an appropriate amount of decoction and filter it through a 0.22μm microporous membrane.

[0290] Determination method: Accurately pipette 10μl of the test solution, inject it into the liquid chromatograph, and determine it.

[0291] The reference fingerprint of the test solution of the Chinese medicine compound decoction of the present application at a wavelength of 280nm is as follows Figure 1 shown.

[0292] 5. Determination of fingerprint standard

[0293] 20 batches of multi-batch matching pictures Figure 2 It was determined that there were 18 characteristic peaks in the chromatogram of the test sample, among which the peak corresponding to the hesperidin reference peak was the S peak. According to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint, the similarity between the fingerprint of the test sample and the reference fingerprint should not be less than 0.90.

[0294] 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 traditional Chinese medicine compound containing Fructus Aurantii Immaturus and Mentha Piperita, characterized in that: The construction method comprises the following steps: Preparation of the test solution: take an appropriate amount of a decoction of a Chinese medicinal compound containing fructus aurantii and mint, shake well, filter, and take a subsequent filtrate to obtain the test solution; wherein the Chinese medicinal compound is composed of ginseng, atractylodes macrocephala, white tuckahoe, angelica sinensis, white peony root, fructus aurantii, amomum villosum, gardenia jasminoides, and mint; Preparation of a Chinese herbal compound decoction containing Citrus aurantium and mint: (a) weighing appropriate amounts of ginseng, Atractylodes macrocephala, white Poria, Chinese angelica, white peony root, Citrus aurantium, Amomum villosum, Gardenia jasminoides and mint into a container, adding a first water for soaking, decocting with high heat until boiling, then decocting with 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, decocting with high heat until boiling, then decocting with low heat for the second time, filtering through a filter to obtain a second filtrate; and (c) combining the first filtrate and the second filtrate to obtain the Chinese herbal compound decoction containing Citrus aurantium and mint; Preparation of reference solution: Weigh appropriate amounts of paeoniflorin, cryptochlorogenic acid, and hesperidin reference substances, add methanol to prepare reference solutions with concentrations of paeoniflorin, cryptochlorogenic acid, and hesperidin of 10 to 100 μg / ml, respectively; Obtaining a fingerprint of the Chinese medicine compound according to the results of high performance liquid chromatography detection of the test solution and the reference solution; The chromatographic conditions of the high performance liquid phase detection are as follows: a chromatographic column with octadecylsilane bonded silica as filler is used, mobile phase A is acetonitrile, mobile phase B is 0.1% phosphoric acid aqueous solution, and the gradient elution program is as follows: 0-5 min, 97% B→95% B; 5-13 min, 95% B→94% B; 13-19 min, 94% B→93% B; 19-24 min, 93% B→92% B; 24-33 min, 92% B→89% B; 33-41 min, 89% B→83% B; 41-43 min, 83% B; 43-49 min, 83% B→80% B; 49~55min, 80% B→78% B; 55~65min, 78%→76% B; 65~68min, 76% B; 68~75min, 76% B→71% B; 75~77min, 71% B→56% B; 77~82min, 56% B→32% B; 82~86min, 32% B→97% B; 86~95min, 97% B, the flow rate is 0.5~1.5ml / min, the column temperature is 10~40℃, the detection wavelength is 280nm, and the injection volume is 1~20μl.

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

3. The construction method according to claim 1, characterized in that: The ratio of the sum of the masses of the ginseng, atractylodes macrocephala, white poria, angelica sinensis, white peony root, fructus aurantii, fructus amomi, gardenia jasminoides and mint to the mass / volume (g / ml) of the first water is 0.05-0.

2.

4. The construction method according to claim 3, characterized in that: The ratio of the sum of the masses of the ginseng, atractylodes macrocephala, white tuckahoe, angelica sinensis, white peony root, fructus aurantii, fructus amomi, gardenia jasminoides and mint to the mass / volume (g / ml) of the first water is 0.

125.

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

6. The construction method according to claim 5, characterized in that: The soaking time is 30 minutes.

7. The construction method according to claim 1, characterized in that: The first decocting time over low heat is 20 to 40 minutes.

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

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

10. The construction method according to claim 1, characterized in that: The ratio of the sum of the masses of the ginseng, atractylodes macrocephala, white poria, angelica sinensis, white peony root, fructus aurantii, amomum villosum, gardenia jasminoides and mint to the mass / volume (g / ml) of the second water is 0.1-0.

3.

11. The construction method according to claim 10, characterized in that: The ratio of the sum of the masses of the ginseng, atractylodes macrocephala, white tuckahoe, angelica sinensis, white peony root, fructus aurantii, fructus amomi, gardenia jasminoides and mint to the mass / volume (g / ml) of the second water is 0.

167.

12. The construction method according to claim 1, characterized in that: The second decocting time over low heat is 10 to 40 minutes.

13. The construction method according to claim 12, characterized in that: The second decocting time over low heat is 25 min.

14. The construction method according to claim 1, characterized in that: The volume of the traditional Chinese medicine compound decoction containing fructus aurantii and mint is 400-600 ml.

15. The construction method according to claim 14, characterized in that: The volume of the traditional Chinese medicine compound decoction containing fructus aurantii and mint is 500 ml.

16. The construction method according to claim 1, characterized in that: The volume / volume ratio of the traditional Chinese medicine compound decoction containing fructus aurantii and mint to the first water is 0.4-0.

8.

17. The construction method according to claim 16, characterized in that: The volume / volume ratio of the Chinese medicinal compound decoction containing Fructus Aurantii Immaturus and Mint to the first water is 0.

587.

18. The construction method according to claim 1, characterized in that: The mass ratios of the ginseng, the atractylodes, the white poria, the angelica, the white peony root, the fructus aurantii, the fructus amomi, the gardenia and the mint are (2-4): (12-18): (8-10): (25-35): (25-35): (0.5-2.5): (2-4): (8-10): (5-7).

19. The construction method according to claim 18, characterized in that: The mass ratios of the ginseng, the atractylodes, the white poria, the angelica, the white peony root, the fructus aurantii, the amomum villosum, the gardenia and the mint are (2.5-3.5): (14-16): (8.5-9.5): (28-32): (28-32): (1-2): (2.5-3.5): (8.5-9.5): (5.5-6.5).

20. The construction method according to claim 19, characterized in that: The mass ratio of the ginseng, the atractylodes, the white poria, the angelica, the white peony root, the fructus aurantii, the fructus amomi, the gardenia and the mint is 3:15:9:30:30:1.5:3:9:

6.

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

22. The construction method according to claim 21, characterized in that: The mass of the ginseng is 3g.

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

24. The construction method according to claim 23, characterized in that: The mass of the Atractylodes macrocephala is 15g.

25. The construction method according to claim 1, characterized in that: The mass range of the white Poria cocos is 5 to 15 g.

26. The construction method according to claim 25, characterized in that: The mass of the white Poria cocos is 9g.

27. The construction method according to claim 1, characterized in that: The mass range of the angelica is 20 to 40 g.

28. The construction method according to claim 27, characterized in that: The mass of the angelica sinensis is 30g.

29. The construction method according to claim 1, characterized in that: The mass range of the white peony root is 20 to 40 g.

30. The construction method according to claim 29, characterized in that: The mass of the white peony root is 30g.

31. The construction method according to claim 1, characterized in that: The mass range of the Fructus Aurantii Immaturus is 0.5 to 5 g.

32. The construction method according to claim 31, characterized in that: The mass of the Fructus Aurantii Immaturus is 1.5 g.

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

34. The construction method according to claim 33, characterized in that: The mass of the Amomum villosum is 3g.

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

36. The construction method according to claim 35, characterized in that: The mass of the Gardenia jasminoides is 9 g.

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

38. The construction method according to claim 37, characterized in that: The mass of the mint is 6g.

39. The construction method according to claim 1, characterized in that: The Atractylodes macrocephala is stir-fried Atractylodes macrocephala.

40. The construction method according to claim 1, characterized in that: The angelica sinensis described is wine-washed angelica sinensis.

41. The construction method according to claim 1, characterized in that: The white peony root is fried with wine.

42. The construction method according to claim 1, characterized in that: The Citrus aurantium is stir-fried Citrus aurantium.

43. The construction method according to claim 1, characterized in that: The Amomum villosum is ground and fried Amomum villosum.

44. The construction method according to claim 1, characterized in that: The gardenia is fried gardenia.

45. The construction method according to claim 1, characterized in that: In the method for preparing the test solution, the filtration is microporous membrane filtration.

46. ​​The construction method according to claim 45, characterized in that The pore size of the microporous filter membrane is 0.22 μm.

47. 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.

48. 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.

49. The construction method according to claim 1, characterized in that: The concentration of hesperidin in the reference solution is 57.019-63.021 μg / ml.

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

51. The construction method according to claim 50, characterized in that: The flow rate of the HPLC detection is 1.0 ml / min.

52. The construction method according to claim 1, characterized in that: The column temperature is 20-30°C.

53. The construction method according to claim 52, characterized in that: The column temperature was 25°C.

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

55. The construction method according to claim 54, characterized in that: The injection volume was 10 μl.

56. The construction method according to claim 1, characterized in that: The theoretical plate number of the chromatographic peak corresponding to the hesperidin is not less than 2000.

57. The construction method according to claim 1, characterized in that: The chromatographic column is a GL Sciences Inertsustain AQ-C18 chromatographic column.

58. 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.

59. The construction method according to claim 1, characterized in that: When the detection wavelength is 280 nm, the fingerprint spectrum includes peaks 1 to 18, and the retention times of peaks 1 to 18 correspond to 9.48955 to 10.48845 min, 11.55865 to 12.77535 min, 13.3779 to 14.7861 min, 14.08945 to 15.57255 min, 27.3353 to 30.2127 min, 30.5026 to 33.7134 min, 35.5604 to 39.3036 min, 39.36135 to 43.5036 min, respectively. 465min, 45.8679~50.6961min, 53.39095~59.01105min, 54.891~60.669min, 58.311~64.449min, 59.01875~65.23125min, 59.7778~66.0702min, 61.09925~67.53075min, 62.40455~68.97345min, 64.4917~71.2803min and 67.8414~74.9826min.

60. The construction method according to claim 1, characterized in that: When the detection wavelength is 280 nm, the fingerprint spectrum includes peaks 1-18, wherein peak 13 hesperidin is used as a reference peak, and the relative retention time average values ​​of peaks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17 and 18 are 0.152-0.168, 0.19-0.21, 0.2185-0.2415, 0.2 28~0.252, 0.437~0.483, 0.494~0.546, 0.57~0.63, 0.6365~0.7035, 0.741~0.819, 0.855~0.945, 0.8835~0.9765, 0.9405~1.0395, 0.9595~1.0605, 0.988~1.092, 1.007~1.113, 1.0355~1.1445 and 1.0925~1.2075.

61. The construction method according to claim 1, characterized in that: When the detection wavelength is 280 nm, peak No. 8 is cryptochlorogenic acid, peak No. 9 is paeoniflorin, and peak No. 13 is hesperidin.

62. The construction method according to claim 1, characterized in that: When the detection wavelength is 280nm, peak 1 comes from ginseng, earth-fried atractylodes and wine-washed angelica, peak 2 comes from wine-fried white peony, earth-fried atractylodes and wine-washed angelica, peak 3 comes from wine-washed angelica and wine-fried white peony, peak 4 comes from wine-fried white peony, peak 5 comes from Amomum villosum, peak 6 comes from earth-fried atractylodes, peak 7 comes from mint, peak 8 comes from wine-fried white peony, earth-fried atractylodes and wine-washed Angelica medicinal materials, Peak No. 9 comes from white peony medicinal materials stir-fried with wine, Peak No. 10 comes from Citrus aurantium medicinal materials, wine-washed Angelica medicinal materials, and stir-fried Gardenia jasminoides medicinal materials, Peak No. 11 comes from white peony medicinal materials stir-fried with wine, Peak No. 12 comes from Angelica medicinal materials washed with wine, Peak No. 13 comes from Citrus aurantium medicinal materials, Peak No. 14 comes from white peony medicinal materials stir-fried with wine and Citrus aurantium medicinal materials, Peak No. 15 comes from Angelica medicinal materials washed with wine, Peak No. 16 comes from Citrus aurantium medicinal materials and stir-fried Gardenia jasminoides medicinal materials, Peak No. 17 comes from Angelica medicinal materials washed with wine, and Peak 18 comes from stir-fried Gardenia jasminoides medicinal materials.

63. Use of the construction method according to any one of claims 1 to 62 in quality detection, quality evaluation or quality control of a traditional Chinese medicine compound comprising Fructus Aurantii Immaturus and Mentha Piperita.

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