Quality control method and application of traditional Chinese medicine composition containing cassia twig

Abstract

The invention discloses a content determination method of a traditional Chinese medicine composition containing cassia twig. The determination method comprises the following steps: taking a test solution and a reference solution of the traditional Chinese medicine composition for detection; obtaining content information of a reference substance of the traditional Chinese medicine composition according to a detection result; wherein the traditional Chinese medicine composition comprises cassia twig, radix paeoniae alba, liquorice, ephedra, ginger, bighead atractylodes rhizome, rhizoma anemarrhenae, radices sileris and black prepared aconite root, and the reference substances are mangiferin, paeoniflorin, cimicifugin, 5-O-methylvisammioside, cinnamaldehyde, ammonium glycyrrhizinate and 6-gingerol. The detection method is simple, accurate, efficient, good in stability, high in precision, good in reproducibility, convenient and easy to master, energy can be effectively saved, the analysis cost is reduced, and the technical content is not disclosed in the prior art.

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 composition containing cassia twig 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, content determination and fingerprint study of the chemical components of the traditional Chinese medicine composition containing cassia twig claimed by 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 determining the content of a traditional Chinese medicine composition containing cassia twig. The method comprises the following steps: taking a test solution and a reference solution of the traditional Chinese medicine composition for detection, and the chromatographic conditions of the detection are: using a chromatographic column whose filler is octadecylsilane bonded silica gel, a mobile phase A is selected from one or more of acetonitrile, methanol and tetrahydrofuran, a mobile phase B is an acid aqueous solution, an alkaline aqueous solution and / or a buffered saline solution, and the gradient elution program is: 0-2 min, 90% B; 2-8 min, 90% B→87% B; 8-20 min, 87% B→86% B; 20-28 min, 86% B→8 3% B; 28-40min, 83% B→80% B; 40-80min, 80% B→55% B; 80-85min, 55% B; the flow rate is 0.6ml / min, the column temperature is 30°C, the detection wavelength is 180-360nm, and the injection volume is 10-30μl; according to the test results, the content information of the reference substance of the traditional Chinese medicine composition is obtained; wherein the traditional Chinese medicine composition includes cassia twig, white peony root, liquorice, ephedra, ginger, atractylodes macrocephala, rhizoma anemarrhenae, saposhnikovia divaricata and heishun tablets, and the reference substance is mangiferin, paeoniflorin, cimicifuga glycosides, 5-O-methylvisaminol glycosides, cinnamaldehyde, ammonium glycyrrhizinate and 6-gingerol.

[0007] Further, the information is based on the corresponding peak areas in the chromatogram of the test solution of the Chinese medicine composition and the chromatogram of the reference solution, and the content of the reference substance in the Chinese medicine composition is calculated according to the external standard method. Further, the linear equation of mangiferin is y=134118.1290x+61486.8143, R2=0.9999. Further, the linear equation of paeoniflorin is y=3889.5159x+11877.5625, R2=0.9999. Further, the linear equation of cimicifuga glycoside is y=61489.9671x+28672.7690, R2=0.9999. Further, the linear equation of 5-O-methylvisaminol glycoside is y=69455.1451x+80352.1532, R2=0.9999. Further, the linear equation of cinnamaldehyde is y=253930.5912x+3832.3004, R2=0.9998. Further, the linear equation of glycyrrhizic acid is y=26858.1718x+3266.0328, R2=0.9999. Further, the linear equation of 6-gingerol is y=18804.2424x-2184.2822, R2=0.9999.

[0008] Further, the preparation method of the Chinese medicine composition test solution includes: taking an appropriate amount of a Chinese medicine composition reference sample containing cassia twigs and placing it in a container, adding a solvent, shaking it well, filtering it, and obtaining the Chinese medicine composition test solution. Further, the container is a volumetric flask. Further, the solvent is methanol. Further, the volume ratio between the Chinese medicine composition reference sample and the solvent is 0.1 to 0.5, for example, about 0.2. Further, the volume of the Chinese medicine composition reference sample is 1 to 5 ml, for example, about 2 ml. Further, the preparation method of the reference solution includes: weighing an appropriate amount of mangiferin, peony glycoside, cimicifuga glycoside, 5-O-methylvisaminol glycoside, cinnamaldehyde, ammonium glycyrrhizate and 6-gingerol reference substances, adding methanol to prepare the reference solution with a concentration of mangiferin, peony glycoside, cimicifuga glycoside, 5-O-methylvisaminol glycoside, cinnamaldehyde, ammonium glycyrrhizate, and 6-gingerol of 1 to 200 μg / ml, respectively. Further, in the preparation method of the reference solution, the volume percentage concentration of the methanol is 40% to 60%, for example, about 50%. Further, the concentration of mangoside in the reference solution is about 60 μg / ml. Further, the concentration of paeoniflorin in the reference solution is about 160 μg / ml. Further, the concentration of cimicifuga glycoside in the reference solution is about 40 μg / ml. Further, the concentration of 5-O-methylvisaminol glycoside in the reference solution is about 40 μg / ml. Further, the concentration of cinnamaldehyde in the reference solution is about 10 μg / ml. Further, the concentration of ammonium glycyrrhizinate in the reference solution is about 50 μg / ml. Further, the concentration of 6-gingerol in the reference solution is about 10 μg / ml.

[0009] Further, the preparation method of the reference sample of the Chinese medicine composition containing cinnamon twig comprises: weighing an appropriate amount of cinnamon twig, white peony root, licorice, ephedra, ginger, atractylodes, rhizoma anemarrhenae, saposhnikovia divaricata and heishunpian, placing them in a container, adding water for soaking, boiling over high heat, boiling over low heat for a period of time, filtering, collecting the filtrate, and obtaining the reference sample of the Chinese medicine composition containing cinnamon twig. Further, the volume of the water is 1000-2000ml, for example, about 1400ml. Further, the ratio of the sum of the mass of cinnamon twig, white peony root, licorice, ephedra, ginger, atractylodes, rhizoma anemarrhenae, saposhnikovia divaricata and heishunpian to the mass / volume (g / ml) of the water is 0.1-0.5, for example, about 0.31. Further, the soaking time is 30-60min, for example, about 45min. Further, the simmering time is 30-90min, for example, about 60min. Furthermore, the filtration is performed through a 200-mesh nylon filter cloth.

[0010] Further, the mass ratio of the cinnamon twig, the black shunpian, the ginger, the siler, the ephedra, the atractylodes, the white peony root, the rhizoma anemarrhenae and the liquorice is (2-6):(1-3):(3-7):(2-6):(1-3):(3-7):(1-5):(2-6):(1-3). Further, the mass ratio of the cinnamon twig, the black shunpian, the ginger, the siler, the ephedra, the atractylodes, the white peony root, the rhizoma anemarrhenae and the liquorice is (3-5):(1.5-2.5):(4-6):(3-5):(1.5-2.5):(4-6):(2-4):(3-5):(1.5-2.5). Further, the mass ratio between the cinnamon twig, the black shun slice, the ginger, the siler, the ephedra, the atractylodes, the white peony root, the rhizome of anemarrhena and the liquorice is about 4: about 2: about 5: about 4: about 2: about 5: about 3: about 4: about 2. Further, the mass range of the cinnamon twig is 40 to 70 g, for example, about 55.20 g. Further, the mass range of the black shun slice is 20 to 40 g, for example, about 27.60 g. Further, the mass range of the ginger is 50 to 90 g, for example, about 69.00 g. Further, the mass range of the siler is 40 to 70 g, for example, about 55.20 g. Further, the mass range of the ephedra is 20 to 40 g, for example, about 27.60 g. Further, the mass range of the atractylodes is 50 to 90 g, for example, about 69.00 g. Further, the mass range of the white peony root is 30-50g, for example, about 41.40g. Further, the mass range of the anemarrhena root is 40-70g, for example, about 55.20g. Further, the mass range of the liquorice root is 20-40g, for example, about 27.60g.

[0011] Further, the detection wavelength of the mangiferin, the paeoniflorin, the cimicifuga glycoside, the 5-O-methylvisaminol glycoside and the glycyrrhizic acid is 200-300 nm, for example, about 280 nm. Further, the detection wavelength of the cinnamaldehyde and the 6-gingerol is 200-300 nm, for example, about 254 nm. Further, the injection volume is 15-25 μl, for example, about 20 μl. Further, the theoretical plate number of the chromatographic peak corresponding to the paeoniflorin is not less than 2000. Further, the chromatographic column is HALOAQ-C 18 Chromatographic columns or YMC Meteoric Core C 18Chromatographic column. Further, the specifications of the chromatographic column are: column length 150mm, inner diameter 4.6mm, particle size 2.7μm. Further, mobile phase A is acetonitrile. Further, the acid aqueous solution, alkali aqueous solution and / or buffered saline solution are selected from one or more of weak acids and their salts, weak bases and their salts of different concentrations. Further, the acid aqueous solution, alkali aqueous solution and / or buffered saline solution are selected from formic acid, 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 of different concentrations. Further, the acid aqueous solution is 0.01% to 1% acid aqueous solution. Further, the acid aqueous solution is 0.01% to 1% phosphoric acid aqueous solution. Further, the acid aqueous solution is 0.05% to 0.15% phosphoric acid aqueous solution. Further, the acid aqueous solution is an aqueous solution of phosphoric acid of about 0.1%. Further, the buffered saline solution is an aqueous solution of acetate and / or an aqueous solution of acetate. Further, the pH value of the buffered saline solution is not greater than 7.0. Further, the detection limit of the mangoside is about 13.63ng / ml. Further, the detection limit of the paeoniflorin is about 0.976μg / ml. Further, the detection limit of the cimicifuga glycoside is about 43.55ng / ml. Further, the detection limit of the 5-O-methylvisaminol glycoside is about 46.08ng / ml. Further, the detection limit of the cinnamaldehyde is about 61.64ng / ml. Further, the detection limit of the glycyrrhizic acid is about 38.75ng / ml. Further, the detection limit of the 6-gingerol is about 0.6475μg / ml. Further, the limit of quantification of the mangiferin is about 45.44ng / ml. Further, the limit of quantification of the paeoniflorin is about 4.88μg / ml. Further, the limit of quantification of the cimicifuga glycoside is about 0.2178μg / ml. Further, the limit of quantification of the 5-O-methylvisaminol glycoside is about 92.16ng / ml. Further, the limit of quantification of the cinnamaldehyde is about 0.12328μg / ml. Further, the limit of quantification of the glycyrrhizic acid is about 0.2422μg / ml. Further, the limit of quantification of the 6-gingerol is about 1.295μg / ml.

[0012] According to another aspect of the present invention, a method for constructing a fingerprint of a traditional Chinese medicine composition containing cinnamon twig is provided, and the method comprises the following steps: preparing a test solution of the traditional Chinese medicine composition: taking an appropriate amount of a reference sample of the traditional Chinese medicine composition containing cinnamon twig and placing it in a container, adding a solvent, shaking it well, filtering it, and obtaining a test solution of the traditional Chinese medicine composition; wherein the traditional Chinese medicine composition comprises cinnamon twig, white peony root, liquorice, ephedra, ginger, atractylodes macrocephala, rhizoma anemarrhenae, saposhnikovia divaricata and heishun tablets; preparing a reference solution: weighing an appropriate amount of mangiferin, paeoniflorin, cimicifuga glycosides, 5-O-methylvisaminol glycosides, cinnamaldehyde, ammonium glycyrrhizate and 6-gingerol reference substances, and adding methanol to prepare the reference solution with the concentrations of mangiferin, paeoniflorin, cimicifuga glycosides, 5-O-methylvisaminol glycosides, cinnamaldehyde, ammonium glycyrrhizate and 6-gingerol being 1 to 200 μg / ml respectively; and detecting the result of the high performance liquid chromatography detection method. The results of the test solution and the reference solution of the Chinese medicine composition are used to obtain the fingerprint of the Chinese medicine composition; the chromatographic conditions of the high performance liquid phase detection are: a chromatographic column with octadecylsilane bonded silica gel as filler is used, the mobile phase A is selected from one or more of acetonitrile, methanol and tetrahydrofuran, the mobile phase B is an acid aqueous solution, an alkaline aqueous solution and / or a buffered saline solution, and the gradient elution program is: 0-2min, 90% B; 2-8min, 90% B→87% B; 8-20min, 87% B→86% B; 20-28min, 86% B→83% B; 28-40min, 83% B→80% B; 40-80min, 80% B→55% B; 80-85min, 55% B; the flow rate is 0.6ml / min, the column temperature is 27-30℃, the detection wavelength is 180-360nm, and the injection volume is 10-30μl.

[0013] Further, the preparation method of the reference sample of the Chinese medicine composition containing cinnamon twig comprises: weighing an appropriate amount of cinnamon twig, white peony root, licorice, ephedra, ginger, atractylodes, rhizoma anemarrhenae, saposhnikovia divaricata and heishunpian, placing them in a container, adding water for soaking, boiling over high heat, boiling over low heat for a period of time, filtering, collecting the filtrate, and obtaining the reference sample of the Chinese medicine composition containing cinnamon twig. Further, the volume of the water is 1000-2000ml, for example, about 1400ml. Further, the ratio of the sum of the mass of cinnamon twig, white peony root, licorice, ephedra, ginger, atractylodes, rhizoma anemarrhenae, saposhnikovia divaricata and heishunpian to the mass / volume (g / ml) of the water is 0.1-0.5, for example, about 0.31. Further, the soaking time is 30-60min, for example, about 45min. Further, the simmering time is 30-90min, for example, about 60min. Furthermore, the filtration is performed through a 200-mesh nylon filter cloth.

[0014] Further, the mass ratio of the cinnamon twig, the black shunpian, the ginger, the siler, the ephedra, the atractylodes, the white peony root, the rhizoma anemarrhenae and the liquorice is (2-6):(1-3):(3-7):(2-6):(1-3):(3-7):(1-5):(2-6):(1-3). Further, the mass ratio of the cinnamon twig, the black shunpian, the ginger, the siler, the ephedra, the atractylodes, the white peony root, the rhizoma anemarrhenae and the liquorice is (3-5):(1.5-2.5):(4-6):(3-5):(1.5-2.5):(4-6):(2-4):(3-5):(1.5-2.5). Further, the mass ratio between the cinnamon twig, the black shun slice, the ginger, the siler, the ephedra, the atractylodes, the white peony root, the rhizome of anemarrhena and the liquorice is about 4: about 2: about 5: about 4: about 2: about 5: about 3: about 4: about 2. Further, the mass range of the cinnamon twig is 40 to 70 g, for example, about 55.20 g. Further, the mass range of the black shun slice is 20 to 40 g, for example, about 27.60 g. Further, the mass range of the ginger is 50 to 90 g, for example, about 69.00 g. Further, the mass range of the siler is 40 to 70 g, for example, about 55.20 g. Further, the mass range of the ephedra is 20 to 40 g, for example, about 27.60 g. Further, the mass range of the atractylodes is 50 to 90 g, for example, about 69.00 g. Further, the mass range of the white peony root is 30-50g, for example, about 41.40g. Further, the mass range of the anemarrhena root is 40-70g, for example, about 55.20g. Further, the mass range of the liquorice root is 20-40g, for example, about 27.60g.

[0015] Further, in the preparation method of the reference sample of the Chinese medicine composition containing cinnamon twig, the container is a volumetric flask. Further, the solvent is methanol. Further, the volume ratio between the reference sample of the Chinese medicine composition and the solvent is 0.1 to 0.5, for example, about 0.2. Further, the volume of the reference sample of the Chinese medicine composition is 1 to 5 ml, for example, about 2 ml. Further, in the preparation method of the reference solution, the volume percentage concentration of the methanol is 40% to 60%, for example, about 50%. Further, the concentration of mangiferin in the reference solution is about 60 μg / ml. Further, the concentration of paeoniflorin in the reference solution is about 160 μg / ml. Further, the concentration of cimicifuga glycosides in the reference solution is about 40 μg / ml. Further, the concentration of 5-O-methylvisaminol glycosides in the reference solution is about 40 μg / ml. Further, the concentration of cinnamic acid in the reference solution is about 10 μg / ml. Further, the concentration of cinnamaldehyde in the reference solution is about 10 μg / ml. Further, the concentration of ammonium glycyrrhizinate in the reference solution is about 50 μg / ml. Further, the concentration of 6-gingerol in the reference solution is about 10 μg / ml. Further, the detection wavelength is 200-300 nm, for example, about 254 nm.

[0016] Further, the injection volume is 15 to 25 μl, for example, about 20 μl. Further, the theoretical plate number of the chromatographic peak corresponding to the paeoniflorin is not less than 2000. Further, the chromatographic column is HALO AQ-C 18 Chromatographic column or YMC Meteoric CoreC 18 Chromatographic column. Further, the specifications of the chromatographic column are: column length 150 mm, inner diameter 4.6 mm, particle size 2.7 μm.

[0017] Further, mobile phase A is acetonitrile. Further, the acid aqueous solution, alkali aqueous solution and / or buffered saline solution are selected from one or more of weak acids and their salts, weak bases and their salts of different concentrations. Further, the acid aqueous solution, alkali aqueous solution and / or buffered saline solution are selected from formic acid, 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 of different concentrations. Further, the acid aqueous solution is 0.01% to 1% acid aqueous solution. Further, the acid aqueous solution is 0.01% to 1% phosphoric acid aqueous solution. Further, the acid aqueous solution is 0.05% to 0.15% phosphoric acid aqueous solution. Further, the acid aqueous solution is about 0.1% phosphoric acid aqueous solution. Further, the buffered saline solution is acetate aqueous solution and / or acetate aqueous solution. Furthermore, the pH value of the buffered saline solution is not greater than 7.0.

[0018] Further, when the detection wavelength is 254 nm, the fingerprint spectrum includes peaks 1-13, and the retention times of peaks 1-13 correspond to about 7.250 min, about 12.917 min, about 14.087 min, about 15.533 min, about 16.590 min, about 26.515 min, about 33.733 min, about 48.726 min, about 50.055 min, about 51.963 min, about 53.086 min, about 63.573 min, and about 70.914 min, respectively. Further, when the detection wavelength is 254 nm, the fingerprint spectrum includes peaks 1-13, wherein peak 7 5-O-methylvisaminose 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 8, peak 9, peak 10, peak 11, peak 12, and peak 13 are approximately 0.21, approximately 0.38, approximately 0.42, approximately 0.46, approximately 0.49, approximately 0.79, approximately 1.44, approximately 1.48, approximately 1.54, approximately 1.57, approximately 1.88, and approximately 2.10, respectively. Further, when the detection wavelength is 254nm, peak No. 2 is mangiferin, peak No. 4 is paeoniflorin, peak No. 5 is cimicifuga glycoside, peak No. 6 is liquiritin, peak No. 7 is 5-O-methylvisaminol glycoside, peak No. 9 is cinnamic acid, peak No. 11 is cinnamaldehyde, and peak No. 13 is glycyrrhizic acid. Further, when the detection wavelength is 254nm, peak No. 1 comes from Rhizoma Anemarrhenae, peak No. 2 comes from Rhizoma Anemarrhenae, peak No. 3 comes from Rhizoma Anemarrhenae, peak No. 4 comes from Paeonia lactiflora, peak No. 5 comes from Radix Saposhnikoviae, peak No. 6 comes from Radix Glycyrrhizae, peak No. 7 comes from Radix Saposhnikoviae, peak No. 8 comes from Radix Glycyrrhizae, peak No. 9 comes from Radix Cinnamomi, peak No. 10 comes from Radix Saposhnikoviae, peak No. 11 comes from Radix Cinnamomi, peak No. 12 comes from Radix Glycyrrhizae, and peak No. 13 comes from Radix Glycyrrhizae.

[0019] According to another aspect of the present invention, a quality control method for a traditional Chinese medicine composition containing cassia twig is provided, the quality control method comprising the following steps: (1) establishing a standard fingerprint of a reference sample of the traditional Chinese medicine composition according to the above-mentioned fingerprint construction method; (2) taking a test solution of the traditional Chinese medicine composition, and testing it according to the chromatographic conditions in the above-mentioned fingerprint construction method to obtain a fingerprint of a sample to be tested of the traditional Chinese medicine composition; and (3) comparing the fingerprint of the sample to be tested of the traditional Chinese medicine composition obtained in step (2) with the standard fingerprint of the reference sample of the traditional Chinese medicine composition obtained in step (1); a product that meets the requirements is a qualified product, and a product that does not meet the requirements is an unqualified product.

[0020] Furthermore, the compliance requirements include one or more of the following: (1) the fingerprint of the Chinese medicine composition sample to be tested presents 13 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 benchmark sample; (2) taking the 5-O-methylvisaminol glycoside 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 benchmark sample; and (3) according to the Chinese medicine chromatographic fingerprint similarity evaluation system, the similarity between the fingerprint of the Chinese medicine composition sample to be tested and the standard fingerprint of the Chinese medicine composition benchmark sample shall not be less than 0.90 after similarity calculation.

[0021] According to another aspect of the present invention, there is provided a use of the above-mentioned determination method, 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 cinnamon twig.

[0022] Beneficial effects of the present invention:

[0023] 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 cassia twig. The method confirms 13 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.

[0024] Specifically, compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) Establishing the fingerprint of the traditional Chinese medicine compound reference sample 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 traditional Chinese medicine compound reference sample of the present invention from a holistic and macroscopic perspective, thereby ensuring the efficacy of the drug, using the main means of modern drug research, realizing the inheritance of classics, and making the classic prescriptions more formally controlled in quality;

[0026] (2) The chemical components in the traditional Chinese medicine compound reference sample 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;

[0027] (3) In the process of establishing the fingerprint spectrum of the traditional Chinese medicine compound reference sample of the present invention, 13 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 the subsequent compound preparation;

[0028] (4) The fingerprints of the effective components in the traditional Chinese medicine compound reference sample of the present invention are viewed as a whole, and the order and relationship of each characteristic peak are emphasized, which not only avoids the one-sidedness of judging the overall quality of the traditional Chinese medicine compound reference sample of the present invention by only measuring one or two chemical components, but also reduces the possibility of artificial processing for the purpose of meeting the quality standard, thus 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;

[0029] (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

[0030] 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.

[0031] Figure 1 The HPLC chromatogram (254 nm) of the optimal chromatographic condition gradient of Example 1. Among them, 1: mangiferin; 2: paeoniflorin; 3: cimicifugo glycoside; 4: 5-O-methylvisaminol glycoside; 5: cinnamaldehyde; 6: glycyrrhizic acid.

[0032] Figure 2 This is a schematic diagram of the instrument precision test results of Example 2. S1: control characteristic spectrum, S2: precision 1, S3: precision 2, S4: precision 3, S5: precision 4, S6: precision 5, S7: precision 6. DETAILED DESCRIPTION

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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 traditional Chinese medicine composition containing cinnamon twigs claimed by the present invention. In order to solve the above problems, the present invention provides a method for determining the content of a traditional Chinese medicine composition containing cinnamon twigs, the determination method comprising the following steps: taking a test solution and a reference solution of the traditional Chinese medicine composition for detection, the chromatographic conditions of the detection are: using a chromatographic column with octadecylsilane bonded silica gel as filler, mobile phase A is selected from one or more of acetonitrile, methanol and tetrahydrofuran, mobile phase B is an acid aqueous solution, an alkaline aqueous solution and / or a buffered saline solution, and the gradient elution program is: 0-2 min, 90% B; 2-8 min, 90% B→87% B; 8-20 min, 87% B→86% B; 20-28 min, 86% B →83%B; 28-40min, 83%B→80%B; 40-80min, 80%B→55%B; 80-85min, 55%B; the flow rate is 0.6ml / min, the column temperature is 30°C, the detection wavelength is 180-360nm, and the injection volume is 10-30μl; according to the test results, the content information of the reference substance of the traditional Chinese medicine composition is obtained; wherein, the traditional Chinese medicine composition includes cassia twig, white peony root, liquorice, ephedra, ginger, atractylodes macrocephala, rhizoma anemarrhenae, saposhnikovia divaricata and heishun tablets, and the reference substance is mangiferin, paeoniflorin, cimicifuga glycosides, 5-O-methylvisaminol glycosides, cinnamaldehyde, ammonium glycyrrhizinate and 6-gingerol.

[0039] In the present invention, when time, concentration, ratio, volume, power, frequency, rotation speed, mass, wavelength, injection amount, temperature, volume percentage concentration, or other values ​​or parameters are expressed as ranges, preferred ranges, or ranges limited 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 range upper limit or preferred value with any range lower limit or preferred value, regardless of whether the range is disclosed separately. For example, when the range "10-30" is disclosed, the described range should be interpreted as including the range "10-30", "10-25", "10-20", "10-15", "15-30", "15-25", "15-20", "20-30", "20-25", "25-30", etc. When a numerical range is described in this article, unless otherwise stated, the range is intended to include its end values ​​and all integers and fractions within the range.

[0040] In a preferred embodiment, the information is calculated according to the corresponding peak areas in the chromatogram of the test solution of the Chinese medicine composition and the chromatogram of the reference solution according to the external standard method. In a preferred embodiment, the linear equation of mangiferin is y=134118.1290x+61486.8143, R2=0.9999. In a preferred embodiment, the linear equation of paeoniflorin is y=3889.5159x+11877.5625, R2=0.9999. In a preferred embodiment, the linear equation of cimicifuga glycoside is y=61489.9671x+28672.7690, R2=0.9999. In a preferred embodiment, the linear equation of the 5-O-methylvisaminol glycoside is y=69455.1451x+80352.1532, R2=0.9999. In a preferred embodiment, the linear equation of the cinnamaldehyde is y=253930.5912x+3832.3004, R2=0.9998. In a preferred embodiment, the linear equation of the glycyrrhizic acid is y=26858.1718x+3266.0328, R2=0.9999. In a preferred embodiment, the linear equation of the 6-gingerol is y=18804.2424x-2184.2822, R2=0.9999.

[0041] In a preferred embodiment, the preparation method of the Chinese medicine composition test solution comprises: taking an appropriate amount of a Chinese medicine composition reference sample containing cinnamon twigs and placing it in a container, adding a solvent, shaking well, filtering, and obtaining the Chinese medicine composition test solution. In a preferred embodiment, the container is a volumetric flask. In a preferred embodiment, the solvent is methanol.

[0042] In a preferred embodiment, the volume ratio between the Chinese medicine composition reference sample and the solvent is 0.1 to 0.5, for example, about 0.2. In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.2" includes ±5% of 0.2, or from 0.19 to 0.21.

[0043] In a preferred embodiment, the volume of the Chinese medicine composition reference sample is 1 to 5 ml, for example, about 2 ml. In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 2" includes ±5% of 2, or from 1.9 to 2.1.

[0044] In a preferred embodiment, the preparation method of the reference solution comprises: weighing appropriate amounts of mangiferin, peony glucoside, cimicifuga glycoside, 5-O-methylvisaminol glycoside, cinnamaldehyde, ammonium glycyrrhizate and 6-gingerol reference substances, and adding methanol to prepare the reference solution having a concentration of 1 to 200 μg / ml for mangiferin, peony glucoside, cimicifuga glycoside, 5-O-methylvisaminol glycoside, cinnamaldehyde, ammonium glycyrrhizate and 6-gingerol, respectively.

[0045] In a preferred embodiment, in the method for preparing the reference solution, the volume percentage concentration of the methanol is 40% to 60%, for example, about 50%. 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%.

[0046] In a preferred embodiment, the concentration of mangiferin in the reference solution is about 60 μg / ml. In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 60" includes ±5% of 60, or from 57 to 63.

[0047] In a preferred embodiment, the concentration of paeoniflorin in the reference solution is about 160 μg / ml. In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 160" includes ±5% of 160, or from 152 to 168.

[0048] In a preferred embodiment, the concentration of cimicifuga glycosides in the reference solution is about 40 μg / ml. In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 40" includes ±5% of 40, or from 38 to 42.

[0049] In a preferred embodiment, the concentration of 5-O-methylvisaminol glycosides in the reference solution is about 40 μg / ml. In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 40" includes ±5% of 40, or from 38 to 42.

[0050] In a preferred embodiment, the concentration of cinnamaldehyde in the reference solution is about 10 μg / ml. In the present invention, "about" refers to a value within a range of ±5% of a particular value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0051] In a preferred embodiment, the concentration of ammonium glycyrrhizinate in the reference solution is about 50 μg / ml. In the present invention, "about" refers to a value within a range of ±5% of a particular value. For example, "about 50" includes ±5% of 50, or from 47.5 to 52.5.

[0052] In a preferred embodiment, the concentration of 6-gingerol in the reference solution is about 10 μg / ml. 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.

[0053] In a preferred embodiment, the preparation method of the reference sample of the traditional Chinese medicine composition containing cinnamon twig comprises: weighing appropriate amounts of cinnamon twig, white peony root, liquorice, ephedra, ginger, atractylodes macrocephala, anemarrhena asphodeloides, saposhnikovia divaricata and heishunpian, placing them in a container, adding water for soaking, decocting over high heat until boiling, decocting over low heat for a period of time, filtering, collecting the filtrate, and obtaining the reference sample of the traditional Chinese medicine composition containing cinnamon twig.

[0054] In a preferred embodiment, the volume of the water is 1000-2000 ml, for example, about 1400 ml. In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1400" includes ±5% of 1400, or from 1330 to 1470.

[0055] In a preferred embodiment, the ratio of the sum of the mass of cinnamon twig, white peony root, liquorice, ephedra, ginger, atractylodes macrocephala, rhizoma anemarrhenae, saposhnikovia divaricata and Heishun Pian to the mass / volume (g / ml) of the water is 0.1 to 0.5, for example, about 0.31. In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 0.31" includes ±5% of 0.31, or from 0.2945 to 0.3255.

[0056] In a preferred embodiment, the soaking time is 30 to 60 minutes, for example, about 45 minutes. 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.

[0057] In a preferred embodiment, the slow-cooking time is 30 to 90 minutes, for example, about 60 minutes. In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 60" includes ±5% of 60, or from 57 to 63.

[0058] In a preferred embodiment, the filtration is 200 mesh nylon filter cloth filtration.

[0059] In a preferred embodiment, the mass ratio of the cinnamon twig, the black shunpian, the ginger, the siler, the ephedra, the atractylodes, the white peony root, the rhizoma anemarrhenae and the liquorice is (2-6):(1-3):(3-7):(2-6):(1-3):(3-7):(1-5):(2-6):(1-3). In a preferred embodiment, the mass ratio of the cinnamon twig, the black shunpian, the ginger, the siler, the ephedra, the atractylodes, the white peony root, the rhizoma anemarrhenae and the liquorice is (3-5):(1.5-2.5):(4-6):(3-5):(1.5-2.5):(4-6):(2-4):(3-5):(1.5-2.5). In a preferred embodiment, the mass ratio between the cinnamon twig, the black shunpian, the ginger, the siler, the ephedra, the atractylodes, the white peony root, the rhizoma anemarrhenae and the liquorice is about 4: about 2: about 5: about 4: about 2: about 5: about 3: about 4: about 2. In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 4" includes ±5% of 4, or from 3.8 to 4.2; "about 2" includes ±5% of 2, or from 1.9 to 2.1; "about 5" includes ±5% of 5, or from 4.75 to 5.25; "about 3" includes ±5% of 3, or from 2.85 to 3.15.

[0060] In a preferred embodiment, the mass range of the cassia twig is 40 to 70 g, for example, about 55.20 g. In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 55.20" includes ±5% of 55.20, or from 52.44 to 57.96.

[0061] In a preferred embodiment, the mass range of the black tablet is 20-40 g, for example, about 27.60 g. In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 27.60" includes ±5% of 27.60, or from 26.22 to 28.98.

[0062] In a preferred embodiment, the weight range of the ginger is 50-90 g, for example, about 69.00 g. In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 69.00" includes ±5% of 69.00, or from 65.55 to 72.45.

[0063] In a preferred embodiment, the mass of the windshield is in the range of 40 to 70 g, for example, about 55.20 g. In the present invention, "about" refers to a value within a range of ±5% of a particular value. For example, "about 55.20" includes ±5% of 55.20, or from 52.44 to 57.96.

[0064] In a preferred embodiment, the mass range of the ephedra is 20 to 40 g, such as about 27.60 g. In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 27.60" includes ±5% of 27.60, or from 26.22 to 28.98.

[0065] In a preferred embodiment, the mass range of the Atractylodes macrocephala is 50 to 90 g, for example, about 69.00 g. In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 69.00" includes ±5% of 69.00, or from 65.55 to 72.45.

[0066] In a preferred embodiment, the mass range of the white peony root is 30 to 50 g, for example, about 41.40 g. In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 41.40" includes ±5% of 41.40, or from 39.33 to 43.47.

[0067] In a preferred embodiment, the mass range of the Rhizoma Anemarrhenae is 40 to 70 g, for example, about 55.20 g. In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 55.20" includes ±5% of 55.20, or from 52.44 to 57.96.

[0068] In a preferred embodiment, the mass range of the liquorice is 20-40 g, for example, about 27.60 g. In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 27.60" includes ±5% of 27.60, or from 26.22 to 28.98.

[0069] In a preferred embodiment, the detection wavelength of the mangiferin, the paeoniflorin, the cimicifuga glycosides, the 5-O-methylvisaminol glycosides and the glycyrrhizic acid is 200-300 nm, for example, about 280 nm. In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 280" includes ±5% of 280, or from 266 to 294.

[0070] In a preferred embodiment, the detection wavelength of the cinnamaldehyde and the 6-gingerol is 200-300 nm, for example, about 254 nm. In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 254" includes ±5% of 254, or from 241.3 to 266.7.

[0071] In a preferred embodiment, the injection volume is 15 to 25 μl, for example, about 20 μl. In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 20" includes ±5% of 20, or from 19 to 21.

[0072] In a preferred embodiment, the theoretical plate number of the chromatographic peak corresponding to the paeoniflorin is not less than 2000. In a preferred embodiment, the chromatographic column is HALOAQ-C 18 Chromatographic columns or YMC Meteoric Core C 18 Chromatographic column. In a preferred embodiment, the specifications of the chromatographic column are: column length 150 mm, inner diameter 4.6 mm, particle size 2.7 μm.

[0073] In a preferred embodiment, mobile phase A is acetonitrile. In a preferred embodiment, the acid aqueous solution, alkali aqueous solution and / or buffered saline solution are selected from one or more of weak acids and their salts, weak bases and their salts of different concentrations. In a preferred embodiment, the acid aqueous solution, alkali aqueous solution and / or buffered saline solution are selected from formic acid, 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 of different concentrations. In a preferred embodiment, the acid aqueous solution is 0.01% to 1% acid aqueous solution. In a preferred embodiment, the acid aqueous solution is 0.01% to 1% phosphoric acid aqueous solution. In a preferred embodiment, the acid aqueous solution is 0.05% to 0.15% phosphoric acid aqueous solution.

[0074] In a preferred embodiment, the acid aqueous solution is an aqueous solution of about 0.1% phosphoric acid. 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%.

[0075] In a preferred embodiment, the buffered saline solution is an acetate solution and / or an acetate solution. In a preferred embodiment, the pH value of the buffered saline solution is not greater than 7.0.

[0076] In a preferred embodiment, the detection limit of mangiferin is about 13.63 ng / ml. In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 13.63" includes ±5% of 13.63, or from 12.9485 to 14.3115.

[0077] In a preferred embodiment, the detection limit of paeoniflorin is about 0.976 μg / ml. In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.976" includes ±5% of 0.976, or from 0.9272 to 1.0248.

[0078] In a preferred embodiment, the detection limit of the cimicifuga glycoside is about 43.55 ng / ml. In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 43.55" includes ±5% of 43.55, or from 41.3725 to 45.7275.

[0079] In a preferred embodiment, the detection limit of 5-O-methylvisaminol glycoside is about 46.08 ng / ml. In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 46.08" includes ±5% of 46.08, or from 43.776 to 48.384.

[0080] In a preferred embodiment, the detection limit of the cinnamaldehyde is about 61.64 ng / ml. In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 61.64" includes ±5% of 61.64, or from 58.558 to 64.722.

[0081] In a preferred embodiment, the detection limit of the glycyrrhizic acid is about 38.75 ng / ml. In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 38.75" includes ±5% of 38.75, or from 36.8125 to 40.6875.

[0082] In a preferred embodiment, the detection limit of 6-gingerol is about 0.6475 μg / ml. In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.6475" includes ±5% of 0.6475, or from 0.615125 to 0.679875.

[0083] In a preferred embodiment, the limit of quantification of mangiferin is about 45.44 ng / ml. In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 45.44" includes ±5% of 45.44, or from 43.168 to 47.712.

[0084] In a preferred embodiment, the limit of quantification of paeoniflorin is about 4.88 μg / ml. In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 4.88" includes ±5% of 4.88, or from 4.636 to 5.124.

[0085] In a preferred embodiment, the limit of quantification of the cimicifuga glycosides is about 0.2178 μg / ml. In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.2178" includes ±5% of 0.2178, or from 0.20691 to 0.22869.

[0086] In a preferred embodiment, the limit of quantification of the 5-O-methylvisaminol glycoside is about 92.16 ng / ml. In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 92.16" includes ±5% of 92.16, or from 87.552 to 96.768.

[0087] In a preferred embodiment, the limit of quantification of the cinnamaldehyde is about 0.12328 μg / ml. In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.12328" includes ±5% of 0.12328, or from 0.117116 to 0.129444.

[0088] In a preferred embodiment, the limit of quantification of the glycyrrhizic acid is about 0.2422 μg / ml. In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.2422" includes ±5% of 0.2422, or from 0.23009 to 0.25431.

[0089] In a preferred embodiment, the limit of quantification of 6-gingerol is about 1.295 μg / ml. In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1.295" includes ±5% of 1.295, or from 1.23025 to 1.35975.

[0090] According to another aspect of the present invention, a method for constructing a fingerprint of a traditional Chinese medicine composition containing cinnamon twig is provided, and the method comprises the following steps: preparing a test solution of the traditional Chinese medicine composition: taking an appropriate amount of a reference sample of the traditional Chinese medicine composition containing cinnamon twig and placing it in a container, adding a solvent, shaking it well, filtering it, and obtaining a test solution of the traditional Chinese medicine composition; wherein the traditional Chinese medicine composition comprises cinnamon twig, white peony root, liquorice, ephedra, ginger, atractylodes macrocephala, rhizoma anemarrhenae, saposhnikovia divaricata and heishun tablets; preparing a reference solution: weighing an appropriate amount of mangiferin, paeoniflorin, cimicifuga glycosides, 5-O-methylvisaminol glycosides, cinnamaldehyde, ammonium glycyrrhizate and 6-gingerol reference substances, and adding methanol to prepare the reference solution with the concentrations of mangiferin, paeoniflorin, cimicifuga glycosides, 5-O-methylvisaminol glycosides, cinnamaldehyde, ammonium glycyrrhizate and 6-gingerol being 1 to 200 μg / ml respectively; and detecting the result of the high performance liquid chromatography detection method. The results of the test solution and the reference solution of the Chinese medicine composition are used to obtain the fingerprint of the Chinese medicine composition; the chromatographic conditions of the high performance liquid phase detection are: a chromatographic column with octadecylsilane bonded silica gel as filler is used, the mobile phase A is selected from one or more of acetonitrile, methanol and tetrahydrofuran, the mobile phase B is an acid aqueous solution, an alkaline aqueous solution and / or a buffered saline solution, and the gradient elution program is: 0-2min, 90% B; 2-8min, 90% B→87% B; 8-20min, 87% B→86% B; 20-28min, 86% B→83% B; 28-40min, 83% B→80% B; 40-80min, 80% B→55% B; 80-85min, 55% B; the flow rate is 0.6ml / min, the column temperature is 27-30℃, the detection wavelength is 180-360nm, and the injection volume is 10-30μl.

[0091] In a preferred embodiment, the preparation method of the reference sample of the traditional Chinese medicine composition containing cinnamon twig comprises: weighing appropriate amounts of cinnamon twig, white peony root, liquorice, ephedra, ginger, atractylodes macrocephala, anemarrhena asphodeloides, saposhnikovia divaricata and heishunpian, placing them in a container, adding water for soaking, decocting over high heat until boiling, decocting over low heat for a period of time, filtering, collecting the filtrate, and obtaining the reference sample of the traditional Chinese medicine composition containing cinnamon twig.

[0092] In a preferred embodiment, the volume of the water is 1000-2000 ml, for example, about 1400 ml. In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1400" includes ±5% of 1400, or from 1330 to 1470.

[0093] In a preferred embodiment, the ratio of the sum of the mass of cinnamon twig, white peony root, liquorice, ephedra, ginger, atractylodes macrocephala, rhizoma anemarrhenae, saposhnikovia divaricata and Heishun Pian to the mass / volume (g / ml) of the water is 0.1 to 0.5, for example, about 0.31. In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 0.31" includes ±5% of 0.31, or from 0.2945 to 0.3255.

[0094] In a preferred embodiment, the soaking time is 30 to 60 minutes, for example, about 45 minutes. 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.

[0095] In a preferred embodiment, the slow-cooking time is 30 to 90 minutes, for example, about 60 minutes. In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 60" includes ±5% of 60, or from 57 to 63.

[0096] In a preferred embodiment, the filtration is 200 mesh nylon filter cloth filtration.

[0097] In a preferred embodiment, the mass ratio of the cinnamon twig, the black shunpian, the ginger, the siler, the ephedra, the atractylodes, the white peony root, the rhizoma anemarrhenae and the liquorice is (2-6):(1-3):(3-7):(2-6):(1-3):(3-7):(1-5):(2-6):(1-3). In a preferred embodiment, the mass ratio of the cinnamon twig, the black shunpian, the ginger, the siler, the ephedra, the atractylodes, the white peony root, the rhizoma anemarrhenae and the liquorice is (3-5):(1.5-2.5):(4-6):(3-5):(1.5-2.5):(4-6):(2-4):(3-5):(1.5-2.5). In a preferred embodiment, the mass ratio between the cinnamon twig, the black shunpian, the ginger, the siler, the ephedra, the atractylodes, the white peony root, the rhizoma anemarrhenae and the liquorice is about 4: about 2: about 5: about 4: about 2: about 5: about 3: about 4: about 2. In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 4" includes ±5% of 4, or from 3.8 to 4.2; "about 2" includes ±5% of 2, or from 1.9 to 2.1; "about 5" includes ±5% of 5, or from 4.75 to 5.25; "about 3" includes ±5% of 3, or from 2.85 to 3.15.

[0098] In a preferred embodiment, the mass range of the cassia twig is 40 to 70 g, for example, about 55.20 g. In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 55.20" includes ±5% of 55.20, or from 52.44 to 57.96.

[0099] In a preferred embodiment, the mass range of the black tablet is 20-40 g, for example, about 27.60 g. In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 27.60" includes ±5% of 27.60, or from 26.22 to 28.98.

[0100] In a preferred embodiment, the weight range of the ginger is 50-90 g, for example, about 69.00 g. In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 69.00" includes ±5% of 69.00, or from 65.55 to 72.45.

[0101] In a preferred embodiment, the mass of the windshield is in the range of 40 to 70 g, for example, about 55.20 g. In the present invention, "about" refers to a value within a range of ±5% of a particular value. For example, "about 55.20" includes ±5% of 55.20, or from 52.44 to 57.96.

[0102] In a preferred embodiment, the mass range of the ephedra is 20 to 40 g, such as about 27.60 g. In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 27.60" includes ±5% of 27.60, or from 26.22 to 28.98.

[0103] In a preferred embodiment, the mass range of the Atractylodes macrocephala is 50 to 90 g, for example, about 69.00 g. In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 69.00" includes ±5% of 69.00, or from 65.55 to 72.45.

[0104] In a preferred embodiment, the mass range of the white peony root is 30 to 50 g, for example, about 41.40 g. In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 41.40" includes ±5% of 41.40, or from 39.33 to 43.47.

[0105] In a preferred embodiment, the mass range of the Rhizoma Anemarrhenae is 40 to 70 g, for example, about 55.20 g. In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 55.20" includes ±5% of 55.20, or from 52.44 to 57.96.

[0106] In a preferred embodiment, the mass range of the liquorice is 20-40 g, for example, about 27.60 g. In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 27.60" includes ±5% of 27.60, or from 26.22 to 28.98.

[0107] In a preferred embodiment, in the method for preparing the reference sample of the traditional Chinese medicine composition containing cinnamon twig, the container is a volumetric flask. In a preferred embodiment, the solvent is methanol.

[0108] In a preferred embodiment, the volume ratio between the Chinese medicine composition reference sample and the solvent is 0.1 to 0.5, for example, about 0.2. In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.2" includes ±5% of 0.2, or from 0.19 to 0.21.

[0109] In a preferred embodiment, the volume of the Chinese medicine composition reference sample is 1 to 5 ml, for example, about 2 ml. In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 2" includes ±5% of 2, or from 1.9 to 2.1.

[0110] In a preferred embodiment, in the method for preparing the reference solution, the volume percentage concentration of the methanol is 40% to 60%, for example, about 50%. 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%.

[0111] In a preferred embodiment, the concentration of mangiferin in the reference solution is about 60 μg / ml. In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 60" includes ±5% of 60, or from 57 to 63.

[0112] In a preferred embodiment, the concentration of paeoniflorin in the reference solution is about 160 μg / ml. In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 160" includes ±5% of 160, or from 152 to 168.

[0113] In a preferred embodiment, the concentration of cimicifuga glycosides in the reference solution is about 40 μg / ml. In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 40" includes ±5% of 40, or from 38 to 42.

[0114] In a preferred embodiment, the concentration of 5-O-methylvisaminol glycosides in the reference solution is about 40 μg / ml. In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 40" includes ±5% of 40, or from 38 to 42.

[0115] In a preferred embodiment, the concentration of cinnamic acid in the reference solution is about 10 μg / ml. 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.

[0116] In a preferred embodiment, the concentration of cinnamaldehyde in the reference solution is about 10 μg / ml. In the present invention, "about" refers to a value within a range of ±5% of a particular value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0117] In a preferred embodiment, the concentration of ammonium glycyrrhizinate in the reference solution is about 50 μg / ml. In the present invention, "about" refers to a value within a range of ±5% of a particular value. For example, "about 50" includes ±5% of 50, or from 47.5 to 52.5.

[0118] In a preferred embodiment, the concentration of 6-gingerol in the reference solution is about 10 μg / ml. 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.

[0119] In a preferred embodiment, the detection wavelength is 200-300 nm, for example, about 254 nm. In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 254" includes ±5% of 254, or from 241.3 to 266.7.

[0120] In a preferred embodiment, the injection volume is 15 to 25 μl, for example, about 20 μl. In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 20" includes ±5% of 20, or from 19 to 21.

[0121] In a preferred embodiment, the theoretical plate number of the chromatographic peak corresponding to the paeoniflorin is not less than 2000. In a preferred embodiment, the chromatographic column is HALO AQ-C 18 Chromatographic columns or YMC Meteoric Core C 18 Chromatographic column. In a preferred embodiment, the specifications of the chromatographic column are: column length 150 mm, inner diameter 4.6 mm, particle size 2.7 μm.

[0122] In a preferred embodiment, mobile phase A is acetonitrile. In a preferred embodiment, the acid aqueous solution, alkali aqueous solution and / or buffered saline solution are selected from one or more of weak acids and their salts, weak bases and their salts of different concentrations. In a preferred embodiment, the acid aqueous solution, alkali aqueous solution and / or buffered saline solution are selected from formic acid, 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 of different concentrations. In a preferred embodiment, the acid aqueous solution is 0.01% to 1% acid aqueous solution. In a preferred embodiment, the acid aqueous solution is 0.01% to 1% phosphoric acid aqueous solution. In a preferred embodiment, the acid aqueous solution is 0.05% to 0.15% phosphoric acid aqueous solution.

[0123] In a preferred embodiment, the acid aqueous solution is an aqueous solution of about 0.1% phosphoric acid. 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%.

[0124] In a preferred embodiment, the buffered saline solution is an acetate solution and / or an acetate solution. In a preferred embodiment, the pH value of the buffered saline solution is not greater than 7.0.

[0125] In a preferred embodiment, when the detection wavelength is 254 nm, the fingerprint spectrum includes peaks 1-13, and the retention times of the peaks 1-13 correspond to about 7.250 min, about 12.917 min, about 14.087 min, about 15.533 min, about 16.590 min, about 26.515 min, about 33.733 min, about 48.726 min, about 50.055 min, about 51.963 min, about 53.086 min, about 63.573 min, and about 70.914 min, respectively. In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 7.250" includes ±5% of 7.250, or from 6.8875 to 7.6125; "about 12.917" includes ±5% of 12.917, or from 12.27115 to 13.56285; "about 14.087" includes ±5% of 14.087, or from 13.38265 to 14.79135; "about 15.533" includes ±5% of 14.087, or from 13.38265 to 14.79135; "about 16.590" includes ±5% of 16.590, or from 15.7605 to 17.4195; "about 26.515" includes ±5% of 26.515, or from 25.18925 to 27.84075; "about 33.733" includes ±5% of 33.733 %, or from 32.04635 to 35.41965; "about 48.726" includes ±5% of 48.726, or from 46.2897 to 51.1623; "about 50.055" includes ±5% of 50.055, or from 47.55225 to 52.55775; "about 51.963" includes ±5% of 51.963, or from 49.364 85 to 54.56115; "about 53.086" includes ±5% of 53.086, or from 50.4317 to 55.7403; "about 63.573" includes ±5% of 63.573, or from 60.39435 to 66.75165; "about 70.914" includes ±5% of 70.914, or from 67.3683 to 74.4597.

[0126] In a preferred embodiment, when the detection wavelength is 254 nm, the fingerprint spectrum includes peaks 1-13, wherein peak 7 5-O-methylvisaminol glycoside is used as a reference peak, and the relative retention time averages of peaks 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, and 13 correspond to about 0.21, about 0.38, about 0.42, about 0.46, about 0.49, about 0.79, about 1.44, about 1.48, about 1.54, about 1.57, about 1.88, and about 2.10, respectively. In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 0.21" includes ±5% of 0.21, or from 0.1995 to 0.2205; "about 0.38" includes ±5% of 0.38, or from 0.361 to 0.399; "about 0.42" includes ±5% of 0.42, or from 0.399 to 0.441; "about 0.46" includes ±5% of 0.46, or from 0.437 to 0.483; "about 0.49" includes ±5% of 0.49, or from 0.4655 to 0.5145; "about 0.79" includes ±5% of 0.79, or from 0.7505 to 0.8 295; “about 1.44” includes ±5% of 1.44, or from 1.368 to 1.512; “about 1.48” includes ±5% of 1.48, or from 1.406 to 1.554; “about 1.54” includes ±5% of 1.54, or from 1.463 to 1.617; “about 1.57” includes ±5% of 1.57, or from 1.4915 to 1.6485; “about 1.88” includes ±5% of 1.88, or from 1.786 to 1.974; “about 2.10” includes ±5% of 2.10, or from 1.995 to 2.205.

[0127] In a preferred embodiment, when the detection wavelength is 254nm, peak No. 2 is mangiferin, peak No. 4 is paeoniflorin, peak No. 5 is cimicifuga glycoside, peak No. 6 is liquiritin, peak No. 7 is 5-O-methylvisaminol glycoside, peak No. 9 is cinnamic acid, peak No. 11 is cinnamaldehyde, and peak No. 13 is glycyrrhizic acid. In a preferred embodiment, when the detection wavelength is 254nm, peak No. 1 comes from the medicinal material of Anemarrhena asphodeloides, peak No. 2 comes from the medicinal material of Anemarrhena asphodeloides, peak No. 3 comes from the medicinal material of Anemarrhena asphodeloides, peak No. 4 comes from the medicinal material of Paeonia lactiflora, peak No. 5 comes from the medicinal material of Saposhnikovia divaricata, peak No. 6 comes from the medicinal material of Licorice, peak No. 7 comes from the medicinal material of Saposhnikovia divaricata, peak No. 8 comes from the medicinal material of Licorice, peak No. 9 comes from the medicinal material of Cinnamomum cassiae, peak No. 10 comes from the medicinal material of Saposhnikovia divaricata, peak No. 11 comes from the medicinal material of Cinnamomum cassiae, peak No. 12 comes from the medicinal material of Licorice, and peak No. 13 comes from the medicinal material of Licorice.

[0128] According to another aspect of the present invention, a quality control method for a traditional Chinese medicine composition containing cassia twig is provided, the quality control method comprising the following steps: (1) establishing a standard fingerprint of a reference sample of the traditional Chinese medicine composition according to the above-mentioned fingerprint construction method; (2) taking a test solution of the traditional Chinese medicine composition, and testing it according to the chromatographic conditions in the above-mentioned fingerprint construction method to obtain a fingerprint of a sample to be tested of the traditional Chinese medicine composition; and (3) comparing the fingerprint of the sample to be tested of the traditional Chinese medicine composition obtained in step (2) with the standard fingerprint of the reference sample of the traditional Chinese medicine composition obtained in step (1); a product that meets the requirements is a qualified product, and a product that does not meet the requirements is an unqualified product.

[0129] In a preferred embodiment, the compliance requirements include one or more of the following: (1) the fingerprint of the Chinese medicine composition sample to be tested presents 13 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 benchmark sample; (2) taking the 5-O-methylvisaminol glycoside 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 benchmark sample; and (3) according to the Chinese medicine chromatographic fingerprint similarity evaluation system, the similarity between the fingerprint of the Chinese medicine composition sample to be tested and the standard fingerprint of the Chinese medicine composition benchmark sample shall not be less than 0.90 after similarity calculation.

[0130] According to another aspect of the present invention, there is provided a use of the above-mentioned determination method, 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 cinnamon twig.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] Instruments and reagents

[0135] 1. Instrument

[0136] Decoction pot (model: 2.5L, manufacturer: Kangyashun Electric Co., Ltd.); liquid heater (model: 30B2, manufacturer: Kangyashun Electric Co., Ltd.); electronic balance (specification: one hundred thousandth, model: MS105DU, manufacturer: Mettler); electronic balance (specification: one ten thousandth, model: BCE124I-1CCN, manufacturer: Sartorius Scientific Instrument Co., Ltd.); electronic balance (specification: one ten thousandth, model: BCE224-1CCN, manufacturer: Sartorius Scientific Instrument Co., Ltd.); high performance liquid chromatograph (model: Waters e2695-2998, manufacturer: Waters Technology Co., Ltd.); high performance liquid chromatograph (model: Agilent1100-DAD, manufacturer: Agilent Technology Co., Ltd.); high performance liquid chromatography column (model: HLAO AQ-C 18 4.6mm×150mm, 2.7μm, serial number: USAQT001492, manufacturer: Spectrum Technology Co., Ltd.); HPLC column (model: YMCMeteoric Core C 18 4.6mm×150mm, 2.7μm, serial number: MC004488, manufacturer: YMC / Vimex); HPLC column (model: HLAO AQ-C 18 4.6mm×150mm, 2.7μm, serial number: USAQT001472, manufacturer: Puzhidao Technology Co., Ltd.); centrifuge (model: Yingtai-TD4N, manufacturer: Changsha Yingtai Instrument Co., Ltd.).

[0137] 2. Drug testing

[0138] 2.1 Reagents

[0139] Water (grade: purified water, batch number: 20231027 / 20231205, manufacturer: Hunan Xiangwa Beverage Technology Co., Ltd.); acetonitrile (grade: HPLC, batch number: WXBF1104V, manufacturer: Sigma-Aldrich); phosphoric acid (grade: HPLC, batch number: 20230110, manufacturer: Komiou); methanol (grade: HPLC, batch number: WXBF1223V / WXBF1598V, manufacturer: Sigma-Aldrich); anhydrous ethanol (grade: analytical grade, batch number: 20230719, manufacturer: Sinopharm Chemical Reagent Co., Ltd.).

[0140] 2.2. Reference substances

[0141] Paeoniflorin reference substance (batch number: 110736-202246, China Food and Drug Inspection Institute, content is 96.7%); mangiferin reference substance (batch number: 111607-202305, China Food and Drug Inspection Institute, content is 98.3%); cimicifugin glycoside reference substance (batch number: 111522-202214, China Food and Drug Inspection Institute, content is 95.7%); liquiritin reference substance (batch number: 111610-202209, China Food and Drug Inspection Institute, content is 95.2%); 5-O-methylvisaminol glycoside reference substance (batch number: 111523-202 212, China Food and Drug Inspection Institutes, content is 97.8%); cinnamic acid reference substance (batch number: 110786-202305, China Food and Drug Inspection Institutes, content is 99.8%); cinnamaldehyde reference substance (batch number: 110710-202223, China Food and Drug Inspection Institutes, content is 98.8%); ammonium glycyrrhizinate reference substance (batch number: 110731-202122, China Food and Drug Inspection Institutes, content is 94.4%); 6-gingerol reference substance (batch number: 111833-202007, China Food and Drug Inspection Institutes, content is 99.3%).

[0142] 2.3. Medicinal pieces information

[0143] Cassia twig (batch number: ZA00922082505, origin: Deqing County, Zhaoqing City, Guangzhou Province, source: Yineng Homemade); Cassia twig (batch number: ZA00923070704, origin: Rong County, Yulin City, Guangxi Zhuang Autonomous Region, source: Yineng Homemade); White peony root (batch number: ZA00121031813, origin: Lin'an County, Hangzhou City, Zhejiang Province, source: Yineng Homemade); White peony root (batch number: ZA00123070503, origin: Qiaocheng District, Bozhou City, Anhui Province, source: Yineng Homemade); Atractylodes macrocephala (batch number: ZA04921031805, origin: Xianju County, Taizhou City, Zhejiang Province, source: Yineng Homemade); Atractylodes macrocephala (batch number: ZA04923070707, origin: She County, Huangshan City, Anhui Province, source: Yineng Homemade); Anemarrhena asphodeloides (batch number: ZA08021122901, origin: Qiaocheng District, Bozhou City, Anhui Province, source: Yineng Homemade); Anemarrhena asphodeloides (ZA08023070704, origin: Anguo City, Baoding City, Hebei Province, source: Yineng Homemade); Heishun Tablets (batch number: ZA17322082301, origin: Jiangyou City, Mianyang City, Sichuan Province, source: Yineng Homemade); Heishun Tablets (batch number: ZA17323071001, origin: Jiangyou City, Mianyang City, Sichuan Province, source: Yineng Homemade); Licorice (batch number: ZA00421031608, origin: Yumen County, Jiuquan City, Gansu Province, source: Yineng Homemade); Licorice (Batch No.: ZA00423071001, origin: Guazhou County, Jiuquan City, Gansu Province, source: Yineng Homemade); Saposhnikovia divaricata (Batch No.: ZA03921090101, origin: Zhalute Banner, Tongliao City, Inner Mongolia Autonomous Region, source: Yineng Homemade); Saposhnikovia divaricata (Batch No.: ZA03923070703, origin: Tailai County, Qiqihar City, Heilongjiang Province, source: Yineng Homemade); Ephedra (Batch No.: Z1110101, origin: Gansu Province, source: Hunan Zhenxing Chinese Medicine Co., Ltd.); Ephedra (Batch No.: 0 1-21030201, origin: Alukeerqin Banner, Chifeng City, Inner Mongolia Autonomous Region, source: Anhui Runfurong Pharmaceutical Co., Ltd.); ginger (batch number: ZA00523071007, origin: Qianwei County, Leshan City, Sichuan Province, source: Yineng Homemade); ginger (batch number: ZA00523080901, origin: Qianwei County, Leshan City, Sichuan Province, source: Yineng Homemade); ginger (batch number: ZA00523080905, origin: Qianwei County, Leshan City, Sichuan Province, source: Yineng Homemade); ginger (batch number: ZA00523091101, origin: Qianwei County, Leshan City, Sichuan Province, source: Yineng Homemade).

[0144] 2.4 Preparation of reference samples

[0145] Benchmark sample: weigh 55.20g of cinnamon twig, 41.40g of white peony root, 27.60g of liquorice, 27.60g of ephedra, 69.00g of ginger, 69.00g of atractylodes, 55.20g of rhizoma anemarrhenae, 55.20g of saposhnikovia divaricata and 27.60g of Heishunpian, put them in a 3.0L fully automatic ceramic decoction pot, add 1400ml of water, soak for 45 minutes, heat to boiling over high heat, keep boiling for 60 minutes over low heat, filter with 200 mesh nylon filter cloth while hot, collect the filtrate, and obtain. Negative control decoction lacking Heishunpian: The prescription lacks the taste of Heishunpian, and is prepared in the same way as the preparation method of the benchmark sample. Heishunpian single decoction: weigh 27.60g of Heishunpian, and prepare it in the same way as the preparation method of the benchmark sample.

[0146] 2.5. Decoction Information

[0147] The negative control decoction of cinnamon twig (batch number: GZSYZMKLST-2023112001-L) was missing; the negative control decoction of white peony root (batch number: GZSYZMKLST-2023112002-L) was missing; the negative control decoction of licorice root (batch number: GZSYZMKLST-2023112003-L) was missing; the negative control decoction of ginger (batch number: GZSYZMKLST-2023112004-L) was missing; the negative control decoction of atractylodes macrocephala (batch number: GZSYZMKLST-2023112005-L) was missing. 005-L); negative control decoction lacking Anemarrhena asphodeloides (batch number: GZSYZMKLST-2023112006-L); negative control decoction lacking Saposhnikovia divaricata (batch number: GZSYZMKLST-2023112007-L); negative control decoction lacking Anemarrhena asphodeloides (batch number: GZSYZMKLST-2023112801-L); single decoction of Cinnamomum cassia twig (batch number: GZSYZMKLST-2023112008-L); single decoction of Paeonia lactiflora (batch number: GZSYZMKLST-2023112 009-L); Licorice decoction (batch number: GZSYZMKLST-2023112010-L); Ginger decoction (batch number: GZSYZMKLST-2023112101-L); Atractylodes macrocephala decoction (batch number: GZSYZMKLST-2023112102-L); Anemarrhena asphodeloides decoction (batch number: GZSYZMKLST-2023112103-L); Saposhnikovia divaricata decoction (batch number: GZSYZMKLST-2023112104-L); Benchmark Sample test solution (batch number: GZSYZMKLST-2023112105-L); benchmark sample test solution (batch number: GZSYZMKLST-2024010701-L); benchmark sample test solution (batch number: GZSYZMKLST-20230912-L); benchmark sample test solution (batch number: GZSYZMKLST-20231101-L); benchmark sample test solution (batch number: GZSYZMKLST-2023111001-L).

[0148] Example 1 Content Determination

[0149] 1. Determination of chromatographic conditions

[0150] 1.1. Investigation of elution gradient

[0151] The following methods were used for preliminary attempts:

[0152] Octadecylsilane bonded silica as filler (HALO AQ-C 18, 4.6mm×150mm, 2.7μm); acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in Table 1; the flow rate was 0.6ml per minute; the column temperature was 30°C; and the detection wavelength was 254nm.

[0153] Table 1 Chromatographic conditions optimization gradient table

[0154] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～2 10 90 2～8 10→12 90→88 8～30 12→22 88→78 30～60 22→45 78→55 60～70 45→70 55→30

[0155] Analyzing the identified peaks under the chromatographic conditions, the overall chromatographic behavior is acceptable, but the separation effect of some index components near 15 min, such as paeoniflorin and 5-O-methylvisaminol, still has room for improvement, and the chromatographic peak response of 6-gingerol is not good, so the chromatographic conditions need to be further optimized.

[0156] Further optimization was attempted using the following methods:

[0157] Octadecylsilane bonded silica as filler (HALO AQ-C 18 , 4.6mm×150mm, 2.7μm); acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in Table 2; the flow rate was 0.6ml per minute; the column temperature was 30°C; and the detection wavelength was 254nm.

[0158] Table 2 Chromatographic conditions optimization gradient table

[0159] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～2 10 90 2～8 10→13 90→87 8～22 13→15 87→85 22～30 15→17 85→83 30～40 17→20 83→80 40～60 20→45 80→55 60～70 45→70 55→30

[0160] From the analysis of each identified peak under the chromatographic conditions, the overall chromatographic behavior is acceptable, but the separation effect of some indicator components near 26 minutes, such as liquiritin, has not been significantly improved. At the same time, the separation effect of some indicator components near 17 minutes and 45 minutes, such as cimicifuga glycosides and cinnamic acid, has not been significantly improved. At the same time, tailing phenomenon was found, so the chromatographic conditions were re-optimized.

[0161] Further optimization was attempted using the following methods:

[0162] Octadecylsilane bonded silica as filler (HALO AQ-C 18 , 4.6mm×150mm, 2.7μm); acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in Table 3; the flow rate was 0.6ml per minute; the column temperature was 30°C; and the detection wavelength was 254nm.

[0163] Table 3 Chromatographic conditions optimization gradient table

[0164] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～2 10 90 2～8 10→13 90→87 8～20 13→14 87→86 20～28 14→17 86→83 28～40 17→20 83→80 40～80 20→45 80→55

[0165] From the analysis of each identified peak under this chromatographic condition, the overall chromatographic behavior is well separated, but the response of some index components such as 6-gingerol near 70min is weak. The overall response of the chromatographic peak of 6-gingerol at 280nm is improved, so the wavelength is switched at 280nm to improve the response of 6-gingerol. In order to ensure the stability of the chromatographic system, the elution time of 80min is extended to 85min to further optimize the chromatographic conditions.

[0166] The final elution gradient is shown in Table 4, and the result spectrum is shown in Figure 1 And as shown in Table 5.

[0167] Table 4 Final gradient table of chromatographic conditions

[0168] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～2 10 90 2～8 10→13 90→87 8～20 13→14 87→86 20～28 14→17 86→83 28～40 17→20 83→80 40～80 20→45 80→55 80～85 45 55

[0169] Table 5 Parameter values ​​of various index components in the reference sample of the Chinese medicine composition containing cinnamon twig of the present invention (254nm)

[0170] name Peak area Separation Symmetry Factor Theoretical plates Mangiferin 30781329 - 1.92 40356 Paeoniflorin 3014205 6.74 1.59 45591 Cimicifuga glycosides 8726497 3.83 1.15 82138 5-O-methylvisaminol 12271936 18.69 1.15 261390 Cinnamaldehyde 2220704 5.47 0.98 108013 Glycyrrhizic acid 5410450 38.61 1.04 2529293

[0171] The results showed that under the elution gradient conditions, the chromatographic behavior of each chromatographic peak was good and the separation was good, and subsequent investigation and research could be carried out based on this elution gradient.

[0172] 1.2. Investigation of detection wavelength

[0173] In this study, the DAD detector was used to measure the UV absorption curves of reference solutions of ingredients such as mangiferin, paeoniflorin, cimicifugo glycosides, 5-O-methylvisaminol glycosides, cinnamaldehyde, glycyrrhizic acid, and 6-gingerol.

[0174] The results showed that the chromatographic peak responses of mangiferin, paeoniflorin, cimicifuga glycosides, 5-O-methylvisaminol glycosides, and glycyrrhizic acid at 254nm were good, which was suitable for quantitative analysis; while the chromatographic peak response of cinnamaldehyde was good at 280nm, and 6-gingerol could be monitored at 280nm. Therefore, the detection wavelength for the determination of mangiferin, paeoniflorin, cimicifuga glycosides, glycyrrhizic acid was determined to be 254nm, and the detection wavelength for the determination of cinnamaldehyde and 6-gingerol was determined to be 280nm.

[0175] 2. Determination of the preparation method of the test solution

[0176] 2.1. Selection of different solvents

[0177] Because the sample of the compound decoction of the Chinese medicine composition containing cinnamon twig of the present invention is relatively concentrated, and the content of each component of the compound is relatively high, direct injection is likely to cause a large sample load on the chromatographic column, and there may be some component information loss or unstable chromatographic conditions. In order to perform quantitative analysis in the early stage without losing the decoction information, this study selected pure water, methanol, ethanol, acetonitrile and other solvents to perform quantitative dilution treatment on the compound decoction for comparison, and determine the type of test sample solvent.

[0178] Take 2 ml of the reference sample, place it in a 10 ml volumetric flask, add pure water, methanol, ethanol and acetonitrile to the scale respectively, filter and obtain.

[0179] The results show that ethanol and acetonitrile have strong elution ability, and the chromatographic peaks of the test solution before 30 minutes have a significant solvent effect, resulting in poor peak shapes of chromatographic peaks such as mangiferin and paeoniflorin, which are not suitable for quantitative analysis; the chromatographic peaks of each index component after dilution with methanol and pure water have good chromatographic behavior, and the peak area difference is small, but methanol has a certain purification effect on macromolecular components, which can effectively reduce the sample loading of the chromatographic column, improve the analysis efficiency of the chromatographic column, and extend the service life of the chromatographic column. Therefore, after comprehensive comparison, methanol was selected as the solvent for subsequent experiments.

[0180] 2.2. Selection of different dilution multiples

[0181] In order to further optimize the preparation method of the test solution, the methanol dilution ratio was also investigated accordingly. 5 ml, 2 ml, and 1 ml of the reference sample were taken and placed in 10 ml volumetric flasks respectively. Methanol was added to the scale line and filtered to obtain the solution.

[0182] The results show that the peak shapes of the chromatographic peaks are poor before 30 minutes after 10-fold dilution with methanol, and there is a certain solvent effect; the chromatographic behaviors of the peaks after 2-fold and 5-fold dilution with methanol are slightly different, but the peak shapes of components such as mangiferin and paeoniflorin when diluted 5-fold are slightly better than those when diluted 2-fold. Considering the experimental operability and other factors, 5-fold dilution with methanol was selected as the preparation method for the test samples in subsequent experiments.

[0183] 3. Preparation method of test solution and chromatographic conditions

[0184] Determined by high performance liquid chromatography (General Rules 0512 of the Chinese Pharmacopoeia 2020 Edition).

[0185] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel was used as filler; acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in Table 4; the flow rate was 0.6 ml per minute; the column temperature was 30°C; the detection wavelength for mangiferin, paeoniflorin, cimicifugo glycosides, 5-O-methylvisaminol glycosides, and glycyrrhizic acid was 254 nm, and the detection wavelength for cinnamaldehyde and 6-gingerol was 280 nm.

[0186] Preparation of reference solution: Take appropriate amount of mangiferin reference substance, paeoniflorin reference substance, cimicifuga glycoside reference substance, 5-O-methylvisaminol glycoside reference substance, cinnamaldehyde reference substance, ammonium glycyrrhizate reference substance, and 6-gingerol reference substance, accurately weigh, and add 50% methanol to make a mixed solution containing 60 μg of mangiferin, 0.16 mg of paeoniflorin, 40 μg of cimicifuga glycoside, 40 μg of 5-O-methylvisaminol glycoside, 10 μg of cinnamaldehyde, 50 μg of ammonium glycyrrhizate, and 10 μg of 6-gingerol per 1 ml.

[0187] Preparation of test solution: Accurately measure 2 ml of the product, place it in a 10 ml volumetric flask, add methanol to the scale, shake well, filter, and obtain.

[0188] Determination method: Accurately pipette 20μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0189] 4. Methodological investigation

[0190] 4.1. Specificity test

[0191] Take the reference sample test solution of the traditional Chinese medicine composition containing cinnamon twig of the present invention, the negative control solution lacking cinnamon twig, lacking white peony root, lacking liquorice, lacking rhizoma anemarrhenae, lacking saposhnikovia divaricata and lacking ginger prepared in the same way, and the single-flavor decoction as well as the blank solvent (50% methanol), and examine whether the negative control solution will cause interference, perform HPLC analysis according to the proposed chromatographic conditions, and record the chromatogram.

[0192] The results showed that the ultraviolet absorption wavelengths of the peaks of the identified components were relatively consistent, there was no interference between the negative control and the blank solution, and the specificity was good; the separation degrees of mangiferin, paeoniflorin, cimicifuga glycosides, 5-O-methylvisaminol glycosides, cinnamaldehyde, glycyrrhizic acid and 6-gingerol in the test solution were 12.31, 3.72, 1.70, 17.94, 43.92, 5.29 and 17.24, respectively, which met the requirements of high performance liquid chromatography content determination; and it was shown that the mangiferin in the test solution was derived from the medicinal taste of Anemarrhena asphodeloides in the benchmark sample of the traditional Chinese medicine composition containing cassia twig of the present invention, paeoniflorin was derived from the medicinal taste of white peony, cimicifuga glycosides and 5-O-methylvisaminol glycosides were derived from the medicinal taste of Saposhnikovia divaricata, glycyrrhizic acid was derived from the medicinal taste of liquorice, cinnamaldehyde was derived from the medicinal taste of cassia twig, and 6-gingerol was derived from the medicinal taste of ginger.

[0193] 4.2 Precision test

[0194] 4.2.1 Intra-day and inter-day precision

[0195] Take three different concentrations of reference solution, according to the above determined chromatographic conditions, inject 6 times continuously, determine the peak area of ​​mangiferin, peony glycoside, cimicifuga glycoside, 5-O-methylvisaminol glycoside, cinnamaldehyde, glycyrrhizic acid, 6-gingerol chromatographic peaks and calculate RSD. Take the first injection of 6 injections of three different concentrations of reference solution as the first day's measurement value, according to the chromatographic conditions, inject once on the second day and the third day respectively, determine the peak area of ​​mangiferin, peony glycoside, cimicifuga glycoside, 5-O-methylvisaminol glycoside, cinnamaldehyde, glycyrrhizic acid, 6-gingerol chromatographic peaks and calculate RSD.

[0196] The results showed that the peak area RSDs of the intra-day precision and inter-day precision of mangiferin, paeoniflorin, cimicifugo glycosides, 5-O-methylvisaminol glycosides, cinnamaldehyde, glycyrrhizic acid, and 6-gingerol were all less than 2.0%, indicating that their intra-day precision and inter-day precision within three consecutive days were good.

[0197] 4.2.2 Repeatability test

[0198] The same batch of the Chinese medicine composition benchmark sample containing cassia twig of the present invention (batch number: GZSYZMKLST-2024010701-L) was taken, and 6 portions were prepared in parallel according to the proposed test solution preparation method, and the contents of mangiferin, paeoniflorin, cimicifugo glycosides, 5-O-methylvisamin glucoside, cinnamaldehyde, glycyrrhizic acid and 6-gingerol were determined according to the proposed chromatographic conditions, and the SD and RSD were calculated.

[0199] The results showed that the SDs of mangiferin, paeoniflorin, cimicifuga glycosides, 5-O-methylvisaminol glycosides, cinnamaldehyde, glycyrrhizic acid and 6-gingerol in 6 repeated test solutions were 0.2, 0.1, 0.1, 0.2, 0.1, 0.4 and 0.1, respectively, and the RSDs of mangiferin, paeoniflorin, cimicifuga glycosides, 5-O-methylvisaminol glycosides, cinnamaldehyde, glycyrrhizic acid and 6-gingerol were no more than 4.0%, indicating that the method had good repeatability.

[0200] 4.2.3 Intermediate precision test

[0201] The same batch of Chinese medicine composition reference samples containing cassia twig of the present invention (batch number: GZSYZMKLST-2024010701-L) were taken, and samples were prepared according to the proposed test solution preparation method. The samples were independently operated by two experimenters, A and B, on different instruments. Experimenter A used Waters e2695-2998 high performance liquid chromatograph and experimenter B used Agilent 1100-DAD high performance liquid chromatograph. The contents of mangiferin, paeoniflorin, cimicifugo glycosides, 5-O-methylvisaminol glycosides, cinnamaldehyde, glycyrrhizic acid, and 6-gingerol were determined at different times and in different laboratories according to the proposed chromatographic conditions, and the SD and RSD were calculated.

[0202] The results showed that the SDs of mangiferin, paeoniflorin, cimicifugo glycosides, 5-O-methylvisaminol glycosides, cinnamaldehyde, glycyrrhizic acid and 6-gingerol were 1.2, 0.1, 0.1, 0.4, 0.1, 1.8 and 0.2, respectively, and the RSDs of mangiferin, paeoniflorin, cimicifugo glycosides, 5-O-methylvisaminol glycosides, cinnamaldehyde, glycyrrhizic acid and 6-gingerol were no more than 4.0%, indicating that the intermediate precision of the method was good.

[0203] 4.3 Detection limit test

[0204] The determination was carried out in accordance with the detection limit in the guidelines for validation of analytical methods for general pharmaceutical quality standards in the 2020 edition of the "Chinese Pharmacopoeia". The signal measured from the sample with known concentration was compared with the signal measured from the blank sample, and the signal-to-noise ratio was 3:1.

[0205] The results showed that the minimum concentrations of mangiferin, paeoniflorin, cimicifugo glycoside, 5-O-methylvisaminol glycoside, cinnamaldehyde, glycyrrhizic acid and 6-gingerol that could be reliably detected were 13.63 ng / ml, 0.976 μg / ml, 43.55 ng / ml, 46.08 ng / ml, 61.64 ng / ml, 38.75 ng / ml and 0.6475 μg / ml, respectively.

[0206] 4.4. Limit of Quantitation Test

[0207] The determination was carried out in accordance with the quantitative limit in the guidelines for validation of analytical methods for general pharmaceutical quality standards in the 2020 edition of the "Chinese Pharmacopoeia". The signal measured from the sample of known concentration was compared with the signal measured from the blank sample, and the signal-to-noise ratio was 10:1.

[0208] The results showed that the minimum concentrations of mangiferin, paeoniflorin, cimicifugo glycoside, 5-O-methylvisaminol glycoside, cinnamaldehyde, glycyrrhizic acid and 6-gingerol that could be reliably quantified were 45.44 ng / ml, 4.88 μg / ml, 0.2178 μg / ml, 92.16 ng / ml, 0.12328 μg / ml, 0.2422 μg / ml and 1.295 μg / ml, respectively.

[0209] 4.5 Linearity test

[0210] Take appropriate amount of mangiferin reference substance, peonyrin reference substance, cimicifuga glycoside reference substance, 5-O-methylvisaminol glycoside reference substance, cinnamaldehyde reference substance, ammonium glycyrrhizinate reference substance and 6-gingerol reference substance, accurately weigh, add 50% methanol to prepare 7 reference substance mixed solutions of different concentrations containing mangiferin, peonyrin, cimicifuga glycoside, 5-O-methylvisaminol glycoside, cinnamaldehyde, glycyrrhizic acid and 6-gingerol, accurately draw 20 μl of the reference substance mixed solution of different concentrations, inject into liquid chromatograph, determine the peak area, and draw standard curve with the concentration of mangiferin, peonyrin, cimicifuga glycoside, 5-O-methylvisaminol glycoside, cinnamaldehyde, glycyrrhizic acid and 6-gingerol as the horizontal coordinate and the peak area as the vertical coordinate.

[0211] The results showed that the linear regression equation of mangiferin was Y=134,118.1290X+61,486.8143, the correlation coefficient r was 0.9999, and the residual sum of squares RSS was 36333756483.60, indicating that mangiferin had a good linear relationship in the range of 0.04544μg / ml to 142μg / ml; the linear regression equation of peony root glycoside was Y=3,889.5159X+11,877.5625, the correlation coefficient r was 0.9999, and the residual sum of squares RSS was 335053024.13, indicating that peony root glycoside had a good linear relationship in the range of 4.88μg / ml. / ml~488μg / ml has a good linear relationship; the linear regression equation of cimicifuga glycoside is Y=61,489.9671X+28,672.7690, the correlation coefficient r is 0.9999, and the residual sum of squares RSS is 21849531305.22, indicating that the linear relationship of cimicifuga glycoside is good in the range of 0.2178μg / ml~272.2μg / ml; the linear regression equation of 5-O-methylvisaminol glycoside is Y=69,455.1451X+80,352.1532, the correlation coefficient r is 0.9999, and the residual sum of squares RSS is 21849531305.22. The RSS is 40201771117.42, indicating that 5-O-methylvisaminol glycoside has a good linear relationship in the range of 0.09216μg / ml to 288μg / ml; the linear regression equation of cinnamaldehyde is Y=253,930.5912X+3832.3004, the correlation coefficient r is 0.9998, and the residual sum of squares RSS is 15195561421.52, indicating that cinnamaldehyde has a good linear relationship in the range of 0.12328μg / ml to 30.82μg / ml; the linear regression equation of glycyrrhizic acid is Y=26,858.1718 X+3,266.0328, the correlation coefficient r is 0.9999, and the residual sum of squares RSS is 1959644085.14, indicating that glycyrrhizic acid has a good linear relationship in the range of 0.2422μg / ml to 121.0934μg / ml; the linear regression equation of 6-gingerol is Y=18,804.2424X-2,184.2822, the correlation coefficient r is 0.9999, and the residual sum of squares RSS is 2213726.25, indicating that 6-gingerol has a good linear relationship in the range of 1.295μg / ml to 12.95μg / ml.

[0212] 4.6 Accuracy test

[0213] That is, the sample recovery test, take 9 parts of the same batch of the Chinese medicine composition benchmark sample containing cinnamon twigs of the present invention (batch number: GZSYZMKLST-2023112105-L), accurately measure 1 ml of the supernatant and put it in a 10 ml volumetric bottle, accurately add the mixed reference solution at the ratio of 1:1.5, 1:1, and 1:0.5 for high, medium, and low concentrations, add methanol, dilute to the scale, shake well, and prepare 3 test solutions for each concentration for determination. At the same time, prepare 2 accompanying test solutions for content determination. Use the content of the accompanying test solution to calculate the sample recovery of mangiferin, paeoniflorin, cimicifuga glycosides, 5-O-methylvisaminol glycosides, cinnamaldehyde, glycyrrhizic acid and 6-gingerol.

[0214] The results showed that the recovery rate of mangiferin in the test solution was between 98.7% and 104.3%, with an RSD of 1.9%; the recovery rate of paeoniflorin was between 99.7% and 103.4%, with an RSD of 1.1%; the recovery rate of cimicifuga glycoside was between 101.7% and 104.6%, with an RSD of 0.9%; the recovery rate of 5-O-methylvisaminol glycoside was between 100.1% and 105.4%, with an RSD of 1.7% ; The recovery rate of cinnamaldehyde was between 100.3% and 106.4%, with an RSD of 1.8%; the recovery rate of glycyrrhizic acid was between 98.3% and 104.0%, with an RSD of 2.3%; the recovery rate of 6-gingerol was between 100.2% and 107.0%, with an RSD of 2.4%; referring to the requirements for recovery limit in the Guidelines for Validation of Analytical Methods for Pharmaceutical Quality Standards in Part IV 9101 of the 2020 Edition of the "Chinese Pharmacopoeia", it can be judged that this method has good accuracy.

[0215] 4.7 Durability test

[0216] 4.7.1 Stability test

[0217] Take the same batch of test solution and inject it at 0h, 3h, 5h, 10h, 20h, 30h, 48h, 62h and 72h according to the proposed chromatographic conditions, and determine the peak areas of mangiferin, paeoniflorin, cimicifugo glycoside, 5-O-methylvisaminol glycoside, cinnamaldehyde, glycyrrhizic acid and 6-gingerol chromatographic peaks to investigate the stability of the solution.

[0218] The results showed that under room temperature, within 72 hours, the RSD of the peak areas of mangiferin, peony glycoside, cimicifuga glycoside, 5-O-methylvisaminol glycoside, glycyrrhizic acid and 6-gingerol chromatographic peaks were all less than 2.0%, indicating that mangiferin, peony glycoside, cimicifuga glycoside, 5-O-methylvisaminol glycoside, glycyrrhizic acid and 6-gingerol in the test solution were basically stable within 72 hours under room temperature, meeting the requirements of content determination. The RSD of the peak area of ​​cinnamaldehyde within 72 hours was 5.5%, because cinnamaldehyde is a volatile component and is easily unstable due to changes in environmental factors. The peak area of ​​cinnamaldehyde was relatively stable within 30 hours, and the RSD of the peak area of ​​cinnamaldehyde was 2.9%. However, after 30 hours, the content of cinnamaldehyde showed a certain downward trend. Therefore, in this method, after the preparation of the test sample, in order to obtain a more accurate content of cinnamaldehyde, it is necessary to detect it as soon as possible within 30 hours.

[0219] 4.7.2. Investigation of different mobile phase acid concentrations

[0220] The same batch of test solutions were taken and injected according to the proposed chromatographic conditions when the concentration of mobile phase phosphoric acid solution was 0.05%, 0.10% and 0.15%, respectively. The effects of different acid concentrations on the index components were investigated, and the contents of mangiferin, paeoniflorin, cimicifugo glycosides, 5-O-methylvisaminol glycosides, cinnamaldehyde, glycyrrhizic acid and 6-gingerol were determined, and the SD and RSD were calculated.

[0221] The results showed that under the condition of 0.05%-0.15% concentration of mobile phase phosphoric acid solution, the SD of the contents of mangiferin, peony root glycoside, cimicifuga glycoside, 5-O-methylvisaminol glycoside, cinnamaldehyde, glycyrrhizic acid and 6-gingerol in the test solution were 0.4, 0.1, 0.3, 0.6, 0.2, 1.0 and 0.1, respectively, and the RSD of the contents of mangiferin, peony root glycoside, cimicifuga glycoside, 5-O-methylvisaminol glycoside, cinnamaldehyde, glycyrrhizic acid and 6-gingerol were not more than 4.0%, which met the requirements of 9101 of the general rules of Part IV of the 2020 edition of the Chinese Pharmacopoeia; it showed that the method had good durability (different mobile phase acid concentrations).

[0222] 4.7.3. Investigation of different flow rates

[0223] The same batch of test solution was taken and injected at different flow rates of 0.5 ml / min, 0.6 ml / min and 0.7 ml / min according to the proposed chromatographic conditions to determine the contents of mangiferin, paeoniflorin, cimicifugo glycoside, 5-O-methylvisaminol glycoside, cinnamaldehyde, glycyrrhizic acid and 6-gingerol, and the SD and RSD were calculated.

[0224] The results showed that under the flow rate of 0.5ml / min~0.7ml / min, the SD of the contents of mangoside, paeoniflorin, cimicifuga glycoside, 5-O-methylvisaminol glycoside, cinnamaldehyde, glycyrrhizic acid and 6-gingerol in the test solution were 1.0, 0.1, 0.6, 0.7, 0.4, 0.7 and 0.1 respectively, and the RSD of the contents of mangoside, cimicifuga glycoside, 5-O-methylvisaminol glycoside, cinnamaldehyde, glycyrrhizic acid and 6-gingerol were not more than 4.0%, which met the requirements of 9101 of the general rules of Part IV of the 2020 edition of the "Chinese Pharmacopoeia"; under the flow rate of 0.5ml / min, the content of paeoniflorin in the test solution of the benchmark sample of the traditional Chinese medicine composition containing cassia twig of the present invention was greatly affected by the flow rate, so it is comprehensively recommended that the fixed flow rate be 0.6ml / min.

[0225] 4.7.4. Investigation of different column temperatures

[0226] The same batch of test solution was taken and injected at different column temperatures of 27℃, 30℃ and 33℃ according to the proposed chromatographic conditions to determine the contents of mangiferin, paeoniflorin, cimicifugo glycoside, 5-O-methylvisaminol glycoside, cinnamaldehyde, glycyrrhizic acid and 6-gingerol, and the SD and RSD were calculated.

[0227] The results showed that under the condition of column temperature of 27℃~33℃, the SD of the contents of mangiferin, peony glucoside, cimicifuga glycoside, 5-O-methylvisaminol glycoside, cinnamaldehyde, glycyrrhizic acid and 6-gingerol in the test solution were 0.7, 0.1, 0.6, 1.1, 0.4, 0.5 and 0.2 respectively, and the RSD of the contents of mangiferin, peony glucoside, cimicifuga glycoside, 5-O-methylvisaminol glycoside, cinnamaldehyde and glycyrrhizic acid was not more than 4.0%, which met the requirements of 9101 of the general rules of Part IV of the 2020 edition of the "Chinese Pharmacopoeia"; under the condition of column temperature of 27℃, the content of 6-gingerol in the test solution of the benchmark sample of the traditional Chinese medicine composition containing cassia twig of the present invention was greatly affected by temperature, so it is comprehensively recommended to fix the temperature at 30℃.

[0228] 4.7.5. Investigation of different chromatographic columns

[0229] Take the same batch of test solution and examine different brands and batches of chromatographic columns according to the proposed chromatographic conditions: Column 1 (HALO AQ-C 18 (4.6 mm × 150 mm, 2.7 μm, SNUSAQT001492), chromatographic column 2 (YMC Meteoric CoreC 18 (4.6 mm × 150 mm, 2.7 μm, SNMC004488), chromatographic column 3 (HALO AQ-C 18(4.6 mm × 150 mm, 2.7 um, SNUSAQT001472) on the index components, the contents of mangiferin, paeoniflorin, cimicifugo glycoside, 5-O-methylvisaminol glycoside, cinnamaldehyde, glycyrrhizic acid and 6-gingerol were determined and the SD and RSD were calculated.

[0230] The results showed that under the conditions of chromatographic columns of different brands and batches, the SD of the contents of mangiferin, peonyrin, cimicifuga glycosides, 5-O-methylvisaminol glycosides, cinnamaldehyde, glycyrrhizic acid and 6-gingerol in the test solution were 0.2, 0.1, 0.3, 0.3, 0.2, 1.5 and 0.1, respectively, and the RSD of the contents of mangiferin, peonyrin, cimicifuga glycosides, 5-O-methylvisaminol glycosides, cinnamaldehyde, glycyrrhizic acid and 6-gingerol were not more than 4.0%, which met the requirements of 9101 of the general rules of Part IV of the 2020 edition of the Chinese Pharmacopoeia; it showed that this method has good durability (chromatographic columns of different brands and batches).

[0231] 4.8 Verification Summary

[0232] The above-mentioned methodological verification related experiments show that the method is accurate, reliable, stable and effective in detecting the contents of mangiferin, paeoniflorin, cimicifugo glycosides, 5-O-methylvisaminol glycosides, cinnamaldehyde, glycyrrhizic acid and 6-gingerol in the benchmark sample of the traditional Chinese medicine composition containing cassia twig of the present invention.

[0233] 5. Summary

[0234] The method for determining the multi-index content of the benchmark sample of the Chinese medicine composition containing cinnamon twig of the present invention is as follows:

[0235] Determined by high performance liquid chromatography (General Rules 0512 of the Chinese Pharmacopoeia 2020 Edition).

[0236] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel as filler; acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution according to the provisions in Table 4; flow rate of 0.6 ml per minute; column temperature of 30°C; detection wavelength of mangiferin, paeoniflorin, cimicifuga glycoside, 5-O-methylvisaminol glycoside, glycyrrhizic acid at 254 nm, cinnamaldehyde, 6-gingerol at 280 nm. The theoretical plate number calculated based on the paeoniflorin peak should be no less than 2000

[0237] Preparation of reference solution: Take appropriate amount of mangiferin reference substance, paeoniflorin reference substance, cimicifuga glycoside reference substance, 5-O-methylvisaminol glycoside reference substance, cinnamaldehyde reference substance, ammonium glycyrrhizate reference substance, and 6-gingerol reference substance, accurately weigh, and add 50% methanol to make a mixed solution containing 60 μg of mangiferin, 0.16 mg of paeoniflorin, 40 μg of cimicifuga glycoside, 40 μg of 5-O-methylvisaminol glycoside, 10 μg of cinnamaldehyde, 50 μg of ammonium glycyrrhizate, and 10 μg of 6-gingerol per 1 ml.

[0238] Preparation of test solution: Accurately measure 2 ml of the product, place it in a 10 ml volumetric flask, add methanol to the scale, shake well, filter, and obtain.

[0239] Determination method: Accurately pipette 20μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0240] Example 2 Fingerprint

[0241] 1. Determination of the preparation method of the test solution

[0242] Same as the method for determining the multi-index content of the traditional Chinese medicine composition containing cinnamon twig of the present invention: take 2 ml of the product, put it in a 10 ml volumetric bottle, add methanol to the scale, shake well, filter, and obtain.

[0243] 2. Investigation of elution gradient

[0244] This study intends to develop chromatographic conditions in combination with the content determination chromatographic conditions. The chromatographic conditions are as follows: octadecylsilane bonded silica gel is used as the filler (HALO AQ-C 18 , 4.6mm×150mm, 2.7μm); acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution according to the provisions in Table 4; flow rate of 0.6ml per minute; column temperature of 30℃; detection wavelength of mangiferin, paeoniflorin, cimicifuga glycoside, 5-O-methylvisaminol glycoside, glycyrrhizic acid is 254nm, cinnamaldehyde, 6-gingerol detection wavelength is 280nm. The theoretical plate number calculated based on the paeoniflorin peak should not be less than 2000.

[0245] 3. Investigation of detection wavelength

[0246] With reference to the 2020 edition of the "Chinese Pharmacopoeia" and the detection wavelengths of each medicinal flavor in the content determination methods under the standard items of each medicinal formula granule, and combined with the analysis of the content determination method development results, it was found that there was more peak information at a wavelength of 254nm, and the benchmark sample component information was richer. In order to ensure convenient operation and economic savings, after comprehensive evaluation, the characteristic spectrum detection wavelength of the benchmark sample of the traditional Chinese medicine composition containing cinnamon twig of the present invention was proposed to be 254nm.

[0247] 4. Determination of characteristic peaks of characteristic spectrum

[0248] From the analysis of the chemical components of the above-mentioned medicinal flavors, it can be seen that the main index components in cinnamon twig are cinnamic acid and cinnamaldehyde, the main index component in white peony root is paeoniflorin, the main index components in liquorice are liquiritin and glycyrrhizic acid, the main index component in anemarrhena asphodeloides is mangiferin, and the main index components in siler are cimicifuga glycoside and 5-O-methylvisaminol glycoside. In the Chinese medicine formula granule drug standard (Guangxi Zhuang Autonomous Region), cinnamaldehyde and cinnamic acid in cinnamon twig medicinal flavor are characterized in the characteristic spectrum, and other medicinal flavors are characterized in the characteristic spectrum with the above corresponding index components in the national drug standard of Chinese medicine formula granules. Combined with the previous experimental results, it is found that the corresponding index components of each medicinal flavor have good separation, so it is proposed that the known components mangiferin, paeoniflorin, cimicifuga glycoside, liquiritin, 5-O-methylvisaminol glycoside, cinnamic acid, cinnamaldehyde and glycyrrhizic acid are characterized as characteristic peaks in the characteristic spectrum of the benchmark sample.

[0249] 5. Double the time for inspection

[0250] The research results show that under this chromatographic condition, the chromatographic peaks of each index component are well separated, and the mobile phase ratio is delayed to 45:55 for 180 minutes. No significant other component information is found after 85 minutes of running time, indicating that each monitoring index component of the benchmark sample is reflected in the effective time spectrum, and the maximum amount of effective information is met within the monitoring time, and the information has been characterized more comprehensively.

[0251] 6. Preparation method of test solution and chromatographic conditions

[0252] Determined by high performance liquid chromatography (General Rules 0512 of the Chinese Pharmacopoeia 2020 Edition).

[0253] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel was used as filler; acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in Table 4; the flow rate was 0.6 ml per minute; the column temperature was 30°C; and the detection wavelength was 254 nm.

[0254] Preparation of reference solution: Take appropriate amount of mangiferin reference substance, paeoniflorin reference substance, cimicifuga glycoside reference substance, 5-O-methylvisaminol glycoside reference substance, cinnamic aldehyde reference substance, ammonium glycyrrhizate reference substance, 6-gingerol reference substance, and cinnamic acid reference substance, accurately weigh, and add 50% methanol to make a mixed solution containing 60 μg of mangiferin, 0.16 mg of paeoniflorin, 40 μg of cimicifuga glycoside, 40 μg of 5-O-methylvisaminol glycoside, 10 μg of cinnamic aldehyde, 50 μg of ammonium glycyrrhizate, 10 μg of 6-gingerol, and 10 μg of cinnamic acid per 1 ml.

[0255] Preparation of test solution: Accurately measure 2 ml of the product, place it in a 10 ml volumetric flask, add methanol to the scale, shake well, filter, and obtain.

[0256] Determination method: Accurately pipette 20μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0257] 7. Methodological investigation

[0258] 7.1. Specificity test

[0259] Take the benchmark sample test solution of the traditional Chinese medicine composition containing cinnamon twig of the present invention, the negative control decoction lacking cinnamon twig, lacking white peony root, lacking liquorice, lacking rhizoma anemarrhenae and lacking saposhnikovia divaricata and the single decoction as well as the blank solution prepared in the same way, examine whether the negative control solution will cause interference, perform HPLC analysis according to the proposed chromatographic conditions, and record the chromatogram.

[0260] The results showed that the blank solution had no interference. The chromatographic peaks of the reference sample and the control variety were scanned by HPLC-DAD, and their UV absorption spectra were compared to determine some characteristic peaks, namely, peak 2 was mangiferin, peak 4 was paeoniflorin, peak 5 was cimicifuga glycoside, peak 6 was licorice glycoside, peak 7 was 5-O-methylvisaminol glycoside, peak 9 was cinnamic acid, peak 11 was cinnamaldehyde, and peak 13 was glycyrrhizic acid. By comparing the test solution of the single decoction with the negative control decoction, it was clear that peak 9 (cinnamic acid) and peak 11 (cinnamaldehyde) came from the medicinal taste of cassia twig, peak 4 (peoniflorin) came from the medicinal taste of white peony, peak 6 (licorice glycoside), peak 8, peak 12, peak 13 (glycyrrhizic acid) came from the medicinal taste of licorice, peak 1, peak 2 (mangiferin), peak 3 came from the medicinal taste of Anemarrhena asphodeloides, peak 5 (cimicifuga glycoside), peak 7 (5-O-methylvisaminol glycoside), peak 10 came from the medicinal taste of Saposhnikovia divaricata. This showed that the method had good specificity.

[0261] 7.2 Precision test

[0262] 7.2.1. Instrument precision test

[0263] Take the same batch of the traditional Chinese medicine composition benchmark sample containing cinnamon twig of the present invention (batch number: GZSYZMKLST-2023112105-L) test solution, repeat the injection 6 times according to the proposed chromatographic conditions, measure and calculate the relative retention time, relative peak area and RSD of each characteristic peak, and calculate the similarity between the test sample characteristic spectrum and the control characteristic spectrum according to the traditional Chinese medicine chromatographic fingerprint similarity evaluation system. The experimental results are as follows Figure 2As shown. The results show that the characteristic spectrum of the test sample presents 13 characteristic peaks corresponding to the characteristic spectrum of the control, and the relative retention time RSD of each characteristic peak and the reference S peak (peak 7) is less than 2.0%; the relative peak area RSD of each characteristic peak and the reference S peak (peak 7) is less than 3.0%; according to the similarity evaluation system of Chinese medicine chromatographic fingerprint, the characteristic spectrum of the test sample and the characteristic spectrum of the control are calculated by similarity, and the similarity is above 0.95. This shows that the instrument precision of this method is good and meets the requirements of the characteristic spectrum.

[0264] 7.2.2 Repeatability test

[0265] Take the same batch of Chinese medicine composition reference samples containing cinnamon twigs of the present invention (batch number: GZSYZMKLST-2024010701-L), prepare 6 test solutions in parallel according to the proposed test solution preparation method, determine and calculate the relative retention time, relative peak area and RSD of each characteristic peak, and calculate the similarity between the test sample characteristic spectrum and the control characteristic spectrum according to the Chinese medicine chromatographic fingerprint similarity evaluation system.

[0266] The results showed that there were 13 characteristic peaks in the characteristic spectrum of the test sample corresponding to the characteristic spectrum of the control, and the relative retention time RSD of each characteristic peak and the reference S peak (peak 7) was less than 2.0%; the relative peak area RSD of each characteristic peak and the reference S peak (peak 7) was less than 2.0%; according to the similarity evaluation system of Chinese medicine chromatographic fingerprint, the characteristic spectrum of the test sample and the characteristic spectrum of the control were calculated to have a similarity of more than 0.95. This shows that the method has good repeatability and meets the requirements of the characteristic spectrum.

[0267] 7.2.3 Intermediate precision

[0268] The same batch of Chinese medicine composition reference samples containing cassia twig of the present invention (batch number: GZSYZMKLST-2024010701-L) were taken, and samples were prepared according to the proposed test solution preparation method. The samples were independently operated by two experimenters, A and B, on different instruments. Experimenter A used Waters e2695-2998 high performance liquid chromatograph and experimenter B used Agilent 1100-DAD high performance liquid chromatograph. The relative retention time and RSD of each characteristic peak were determined and calculated according to the proposed chromatographic conditions at different times and in different laboratories, and the similarity between the test sample characteristic spectrum and the control characteristic spectrum was calculated according to the traditional Chinese medicine chromatographic fingerprint similarity evaluation system.

[0269] The results showed that the characteristic spectrum of the test sample presented 13 characteristic peaks corresponding to the characteristic spectrum of the control, and the relative retention time RSD of each characteristic peak and the reference S peak (peak 7) was less than 2.0%; according to the similarity evaluation system of Chinese medicine chromatographic fingerprint, the characteristic spectrum of the test sample and the characteristic spectrum of the control were calculated to have a similarity of more than 0.95, indicating that the intermediate precision of this method is good and meets the requirements of the characteristic spectrum.

[0270] 7.3 Durability

[0271] 7.3.1 Stability test

[0272] Take the same batch of test solution and inject it at 0h, 3h, 5h, 10h, 20h, 30h, 48h, 62h and 72h according to the proposed chromatographic conditions. Determine and calculate the relative retention time, relative peak area and RSD of each characteristic peak, and calculate the similarity between the characteristic spectrum of the test sample and the characteristic spectrum of the control according to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint.

[0273] The results showed that the characteristic spectrum of the test sample presented 13 characteristic peaks corresponding to the characteristic spectrum of the control, and the relative retention time RSD of each characteristic peak and the reference S peak (peak 7) was less than 2.0%; the relative peak area RSD of each characteristic peak and the reference S peak (peak 7) was less than 6.0%. According to the similarity evaluation system of Chinese medicine chromatographic fingerprint, the characteristic spectrum of the test sample and the characteristic spectrum of the control were calculated to have a similarity of more than 0.95, indicating that the method has good stability and meets the requirements of the characteristic spectrum.

[0274] 7.3.2. Investigation of different mobile phase acid concentrations

[0275] The same batch of test sample solutions were taken and injected according to the proposed chromatographic conditions when the concentration of mobile phase phosphoric acid solution was 0.05%, 0.10% and 0.15%, respectively. The influence of different acid concentrations of the mobile phase on the characteristic peaks was investigated, and the relative retention time and RSD of each characteristic peak were determined and calculated. The similarity between the characteristic spectrum of the test sample and the characteristic spectrum of the control was calculated according to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint.

[0276] The results showed that the characteristic spectrum of the test sample presented 13 characteristic peaks corresponding to the characteristic spectrum of the control, and the relative retention time RSD of each characteristic peak and the reference S peak (peak 7) was less than 2.0%; according to the similarity evaluation system of Chinese medicine chromatographic fingerprint, the characteristic spectrum of the test sample and the characteristic spectrum of the control were calculated to have a similarity of more than 0.95, indicating that the method has good durability (different mobile phase acid concentrations) and meets the requirements of the characteristic spectrum.

[0277] 7.3.3. Investigation of different flow rates

[0278] Take the same batch of test solution and inject it at different flow rates of 0.5ml / min, 0.6ml / min and 0.7ml / min according to the proposed chromatographic conditions. Determine and calculate the relative retention time and RSD of each characteristic peak, and calculate the similarity between the characteristic spectrum of the test sample and the characteristic spectrum of the control according to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint.

[0279] The results showed that when the flow rate was 0.5ml / min, the separation degree of peak 9 (cinnamic acid) and its adjacent unknown chromatographic peak (peak 10) was 0.85; when the flow rate was 0.7ml / min, the separation degree of peak 11 (cinnamaldehyde) and its adjacent unknown chromatographic peak (peak 10) was 1.19; it did not meet the requirements of the chromatographic peak separation of the characteristic spectrum, so the relative retention time and characteristic spectrum similarity of each characteristic peak under the conditions of 0.5ml / min and 0.7ml / min were not further analyzed; when the flow rate was 0.6ml / min, the separation of each characteristic peak was better, so it was comprehensively recommended to fix the flow rate to 0.6ml / min.

[0280] 7.3.4. Investigation of different column temperatures

[0281] Take the same batch of test sample solutions and inject them at different column temperatures of 27℃, 30℃ and 33℃ according to the proposed chromatographic conditions. Determine and calculate the relative retention time and RSD of each characteristic peak, and calculate the similarity between the characteristic spectrum of the test sample and the characteristic spectrum of the control according to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint.

[0282] The results show that the separation of peaks 10 and 11 (cinnamaldehyde) is greatly affected by column temperature. When the column temperature is 33℃, the separation degree of peaks 10 and 11 (cinnamaldehyde) is 0.89, which does not meet the requirements of the separation degree of the characteristic spectrum chromatographic peaks. Therefore, the relative retention time and characteristic spectrum similarity of the characteristic peaks under this condition are not further analyzed; when the column temperature is 27℃ and 30℃, the relative retention time RSD of each characteristic peak and the reference S peak (peak 7) are less than 2.0%; according to the similarity evaluation system of Chinese medicine chromatographic fingerprint, the characteristic spectrum of the test sample and the control characteristic spectrum are calculated by similarity, and the similarity is above 0.95. This shows that the method has good durability under the column temperature of 27℃~30℃ and meets the requirements of the characteristic spectrum.

[0283] 7.3.5. Investigation of different chromatographic columns

[0284] Take the same batch of test solution and examine different brands and batches of chromatographic columns according to the proposed chromatographic conditions: Column 1 (HALO AQ-C 18 (4.6 mm × 150 mm, 2.7 μm, SNUSAQT001492), chromatographic column 2 (YMC Meteoric CoreC 18(4.6 mm × 150 mm, 2.7 μm, SNMC004488), chromatographic column 3 (HALO AQ-C 18 The influence of (4.6mm×150mm, 2.7um, SNUSAQT001472) on each characteristic peak was determined and calculated. The relative retention time and RSD of each characteristic peak were determined and calculated. According to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint, the similarity between the characteristic spectrum of the test sample and the characteristic spectrum of the control was calculated.

[0285] The results showed that the characteristic spectrum of the test sample presented 13 characteristic peaks corresponding to the characteristic spectrum of the control, and the relative retention time RSD of each characteristic peak and the reference S peak (peak 7) was less than 2.0%; according to the similarity evaluation system of Chinese medicine chromatographic fingerprint, the characteristic spectrum of the test sample and the characteristic spectrum of the control were calculated to have a similarity of more than 0.95, indicating that the method has good durability (chromatographic columns of different brands and batches) and meets the requirements of the characteristic spectrum.

[0286] 7.4 Verification Summary

[0287] The above-mentioned methodological verification related experiments show that the method for the characteristic spectrum of the benchmark sample of the traditional Chinese medicine composition containing cinnamon twig established in this experiment is simple, feasible, and durable, and meets the requirements of 9101 of the General Rules of Part Four of the 2020 Edition of the "Chinese Pharmacopoeia".

[0288] 8. Summary

[0289] Through the above-mentioned methodology to verify the relevant test results, it was found that the retention time of the chromatographic peak of 5-O-methylvisaminol glycoside was relatively central, the peak area accounted for a large proportion, about 15%, and 5-O-methylvisaminol glycoside was an indicative component of the medicinal taste of Fangfeng, and the peak was relatively stable, so it was determined that the chromatographic peak of 5-O-methylvisaminol glycoside was the S peak. The characteristic spectrum analysis method of the benchmark sample of the Chinese medicine composition containing cinnamon twigs of the present invention is determined as follows: according to the high performance liquid chromatography (General Rules 0512 of the 2020 Edition of the Chinese Pharmacopoeia). Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel is used as a filler; acetonitrile is used as mobile phase A, and 0.1% phosphoric acid solution is used as mobile phase B, and gradient elution is performed according to the provisions in Table 4; the flow rate is 0.6 ml per minute; the column temperature is 30°C; the detection wavelength is 254nm. The number of theoretical plates calculated according to the paeoniflorin peak should not be less than 2000.

[0290] Preparation of reference solution: Take appropriate amount of mangiferin reference substance, paeoniflorin reference substance, cimicifuga glycoside reference substance, 5-O-methylvisaminol glycoside reference substance, cinnamic aldehyde reference substance, ammonium glycyrrhizate reference substance, 6-gingerol reference substance, and cinnamic acid reference substance, accurately weigh, and add 50% methanol to make a mixed solution containing 60 μg of mangiferin, 0.16 mg of paeoniflorin, 40 μg of cimicifuga glycoside, 40 μg of 5-O-methylvisaminol glycoside, 10 μg of cinnamic aldehyde, 50 μg of ammonium glycyrrhizate, 10 μg of 6-gingerol, and 10 μg of cinnamic acid per 1 ml.

[0291] Preparation of test solution: Accurately measure 2 ml of the product, place it in a 10 ml volumetric flask, add methanol to the scale, shake well, filter, and obtain.

[0292] Determination method: Accurately pipette 20μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0293] 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 determining the content of a Chinese medicine composition containing cinnamon twig, characterized in that: The determination method comprises the following steps: Take the test solution and the reference solution of the Chinese medicine composition for testing. The chromatographic conditions of the detection are: 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 acid aqueous solution, alkali aqueous solution and / or buffered saline solution, and the gradient elution program is: 0-2 min, 90% B; 2-8 min, 90% B→87% B; 8-20 min, 87% B→86% B; 20-28 min, 86% B→83% B; 28-40 min, 83% B→80% B; 40-80 min, 80% B→55% B; 80-85 min, 55% B; the flow rate is 0.6 ml / min, the column temperature is 30°C, the detection wavelength is 180-360 nm, and the injection volume is 10-30 μl; According to the test results, obtaining the content information of the reference substance of the Chinese medicine composition; The Chinese medicine composition includes cinnamon twig, white peony root, liquorice, ephedra, ginger, atractylodes macrocephala, rhizoma anemarrhenae, saposhnikovia divaricata and heishunpian, and the reference substances include mangoside, paeoniflorin, cimicifuga glycoside, 5-O-methylvisaminol glycoside, cinnamaldehyde, ammonium glycyrrhizinate and 6-gingerol.

2. The measuring method according to claim 1, characterized in that The information is the content of the reference substance in the Chinese medicine composition calculated according to the external standard method based on the corresponding peak areas in the recorded chromatogram of the test solution of the Chinese medicine composition and the chromatogram of the reference substance solution; Preferably, the linear equation of mangiferin is y=134118.1290x+61486.8143, R2=0.9999; Preferably, the linear equation of the paeoniflorin is y=3889.5159x+11877.5625, R2=0.9999; Preferably, the linear equation of the cimicifuga glycoside is y=61489.9671x+28672.7690, R2=0.9999; Preferably, the linear equation of the 5-O-methylvisaminol glycoside is y=69455.1451x+80352.1532, R2=0.9999; Preferably, the linear equation of cinnamaldehyde is y=253930.5912x+3832.3004, R2=0.9998; Preferably, the linear equation of glycyrrhizic acid is y=26858.1718x+3266.0328, R2=0.9999; Preferably, the linear equation of 6-gingerol is y=18804.2424x-2184.2822, R2=0.9999.

3. The measuring method according to claim 1, characterized in that The preparation method of the Chinese medicine composition test solution comprises: taking an appropriate amount of a Chinese medicine composition reference sample containing cassia twig and placing it in a container, adding a solvent, shaking well, filtering, and obtaining the Chinese medicine composition test solution; Preferably, the container is a volumetric flask; Preferably, the solvent is methanol; Preferably, the volume ratio between the Chinese medicine composition reference sample and the solvent is 0.1 to 0.5, for example, about 0.2; Preferably, the volume of the Chinese medicine composition reference sample is 1 to 5 ml, for example, about 2 ml; More preferably, the preparation method of the reference solution comprises: weighing appropriate amounts of mangiferin, paeoniflorin, cimicifuga glycosides, 5-O-methylvisaminol glycosides, cinnamaldehyde, ammonium glycyrrhizate and 6-gingerol reference substances, and adding methanol to prepare the reference solution having a concentration of mangiferin, paeoniflorin, cimicifuga glycosides, 5-O-methylvisaminol glycosides, cinnamaldehyde, ammonium glycyrrhizate and 6-gingerol of 1 to 200 μg / ml, respectively; Also preferably, in the method for preparing the reference solution, the volume percentage concentration of the methanol is 40% to 60%, for example, about 50%; Also preferably, the concentration of mangiferin in the reference solution is about 60 μg / ml; Also preferably, the concentration of paeoniflorin in the reference solution is about 160 μg / ml; Also preferably, the concentration of cimicifuga glycoside in the reference solution is about 40 μg / ml; Also preferably, the concentration of 5-O-methylvisaminol glycoside in the reference solution is about 40 μg / ml; Also preferably, the concentration of cinnamaldehyde in the reference solution is about 10 μg / ml; Also preferably, the concentration of ammonium glycyrrhizinate in the reference solution is about 50 μg / ml; Also preferably, the concentration of 6-gingerol in the reference solution is about 10 μg / ml; Still more preferably, the preparation method of the reference sample of the traditional Chinese medicine composition containing cinnamon twig comprises: weighing appropriate amounts of cinnamon twig, white peony root, liquorice, ephedra, ginger, atractylodes macrocephala, rhizoma anemarrhenae, saposhnikovia divaricata and aconite root, placing them in a container, adding water for soaking, decocting with high heat until boiling, decocting with low heat for a period of time, filtering, collecting the filtrate, and obtaining the reference sample of the traditional Chinese medicine composition containing cinnamon twig; Particularly preferably, the volume of the water is 1000-2000 ml, for example about 1400 ml; Particularly preferably, the ratio of the sum of the mass of the cinnamon twig, white peony root, liquorice, ephedra, ginger, atractylodes macrocephala, rhizoma anemarrhenae, saposhnikovia divaricata and Heishun tablets to the mass / volume (g / ml) of the water is 0.1 to 0.5, for example, about 0.31; Particularly preferably, the soaking time is 30 to 60 minutes, for example, about 45 minutes; Particularly preferably, the slow-cooking time is 30 to 90 minutes, for example, about 60 minutes; Particularly preferably, the filtration is 200 mesh nylon filter cloth filtration; Particularly preferably, the mass ratio of the cinnamon twig, the black shunpian, the ginger, the siler, the ephedra, the atractylodes, the white peony root, the rhizome of anemarrhena and the liquorice is (2-6):(1-3):(3-7):(2-6):(1-3):(3-7):(1-5):(2-6):(1-3); Particularly preferably, the mass ratio of the cinnamon twig, the black shunpian, the ginger, the siler, the ephedra, the atractylodes, the white peony root, the rhizome of anemarrhena and the liquorice is (3-5):(1.5-2.5):(4-6):(3-5):(1.5-2.5):(4-6):(2-4):(3-5):(1.5-2.5); Most preferably, the mass ratio of the cinnamon twig, the black shunpian, the ginger, the siler, the ephedra, the atractylodes, the white peony root, the anemarrhena and the liquorice is about 4: about 2: about 5: about 4: about 2: about 5: about 3: about 4: about 2; Most preferably, the mass of the cassia twig ranges from 40 to 70 g, for example, about 55.20 g; Most preferably, the mass of the black shun tablets ranges from 20 to 40 g, for example, about 27.60 g; Most preferably, the mass of the ginger ranges from 50 to 90 g, for example, about 69.00 g; Most preferably, the mass of the windshield is in the range of 40 to 70 g, for example about 55.20 g; Most preferably, the mass of the ephedra is in the range of 20 to 40 g, for example, about 27.60 g; Most preferably, the mass range of the Atractylodes macrocephala is 50 to 90 g, for example, about 69.00 g; Most preferably, the mass range of the white peony root is 30 to 50 g, for example, about 41.40 g; Most preferably, the mass range of the Anemarrhena Rhizoma is 40 to 70 g, for example, about 55.20 g; Most preferably, the mass of the liquorice is in the range of 20 to 40 g, such as about 27.60 g.

4. The measuring method according to claim 1, characterized in that The detection wavelength of the mangiferin, the paeoniflorin, the cimicifuga glycoside, the 5-O-methylvisaminol glycoside and the glycyrrhizic acid is 200 to 300 nm, for example, about 280 nm; Preferably, the detection wavelength of the cinnamaldehyde and the 6-gingerol is 200 to 300 nm, for example, about 254 nm; Preferably, the injection volume is 15 to 25 μl, for example, about 20 μl; More preferably, the theoretical plate number of the chromatographic peak corresponding to the paeoniflorin is not less than 2000; More preferably, the chromatographic column is HALOAQ-C 18 Chromatographic columns or YMC Meteoric Core C 18 Chromatographic columns; Preferably, the specifications of the chromatographic column are: column length 150 mm, inner diameter 4.6 mm, particle size 2.7 μm; Still more preferably, mobile phase A is acetonitrile; Still more preferably, 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; Still more preferably, the acid aqueous solution, alkali aqueous solution and / or buffered saline solution is selected from formic acid, 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; More preferably, the acid aqueous solution is a 0.01% to 1% acid aqueous solution; More preferably, the acid aqueous solution is a 0.01% to 1% phosphoric acid aqueous solution; More preferably, the acid aqueous solution is a 0.05% to 0.15% phosphoric acid aqueous solution; Still more preferably, the acid aqueous solution is about 0.1% phosphoric acid aqueous solution; Still more preferably, the buffered saline solution is an acetate aqueous solution and / or an acetate aqueous solution; Still more preferably, the pH value of the buffered saline solution is no greater than 7.0; Particularly preferably, the detection limit of mangiferin is about 13.63 ng / ml; Particularly preferably, the detection limit of the paeoniflorin is about 0.976 μg / ml; Particularly preferably, the detection limit of the cimicifuga glycoside is about 43.55 ng / ml; Particularly preferably, the detection limit of the 5-O-methylvisaminol glycoside is about 46.08 ng / ml; Particularly preferably, the detection limit of cinnamaldehyde is about 61.64 ng / ml; Particularly preferably, the detection limit of glycyrrhizic acid is about 38.75 ng / ml; Particularly preferably, the detection limit of 6-gingerol is about 0.6475 μg / ml; Particularly preferably, the limit of quantification of mangiferin is about 45.44 ng / ml; Particularly preferably, the limit of quantification of paeoniflorin is about 4.88 μg / ml; Particularly preferably, the limit of quantification of the cimicifuga glycoside is about 0.2178 μg / ml; Particularly preferably, the limit of quantification of the 5-O-methylvisaminol glycoside is about 92.16 ng / ml; Particularly preferably, the limit of quantification of cinnamaldehyde is about 0.12328 μg / ml; Particularly preferably, the limit of quantification of the glycyrrhizic acid is about 0.2422 μg / ml; Particularly preferably, the quantification limit of 6-gingerol is about 1.295 μg / ml.

5. A method for constructing a fingerprint of a traditional Chinese medicine composition containing cinnamon twig, characterized in that: The construction method comprises the following steps: Preparation of a Chinese medicine composition test solution: taking an appropriate amount of a Chinese medicine composition reference sample containing cinnamon twigs and placing it in a container, adding a solvent, shaking it well, filtering it, and obtaining the Chinese medicine composition test solution; wherein the Chinese medicine composition comprises cinnamon twigs, white peony root, liquorice, ephedra, ginger, atractylodes macrocephala, rhizoma anemarrhenae, saposhnikovia divaricata and heishun tablets; Preparation of reference solution: weigh appropriate amounts of mangiferin, paeoniflorin, cimicifuga glycosides, 5-O-methylvisaminol glycosides, cinnamaldehyde, ammonium glycyrrhizate and 6-gingerol reference substances, add methanol to prepare reference solutions with concentrations of mangiferin, paeoniflorin, cimicifuga glycosides, 5-O-methylvisaminol glycosides, cinnamic acid, cinnamaldehyde, ammonium glycyrrhizate and 6-gingerol of 1 to 200 μg / ml, respectively; Obtaining a fingerprint of the Chinese medicine composition according to the results of high performance liquid chromatography detection of the Chinese medicine composition test solution and the reference solution; The chromatographic conditions of the high performance liquid phase detection are as follows: 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 alkaline aqueous solution and / or a buffered saline solution, and the gradient elution program is: 0-2 min, 90% B; 2-8 min, 90% B→87% B; 8-20 min, 87% B→86% B; 20-28 min, 86% B→83% B; 28-40 min, 83% B→80% B; 40-80 min, 80% B→55% B; 80-85 min, 55% B; the flow rate is 0.6 ml / min, the column temperature is 27-30° C., the detection wavelength is 180-360 nm, and the injection volume is 10-30 μl.

6. The construction method according to claim 5, characterized in that: The preparation method of the reference sample of the traditional Chinese medicine composition containing cinnamon twig comprises: weighing appropriate amounts of cinnamon twig, white peony root, liquorice, ephedra, ginger, atractylodes macrocephala, rhizoma anemarrhenae, saposhnikovia divaricata and heishunpian, placing the mixture in a container, adding water for soaking, boiling over high heat, boiling over low heat for a period of time, filtering, collecting the filtrate, and obtaining the reference sample of the traditional Chinese medicine composition containing cinnamon twig; Preferably, the volume of the water is 1000-2000 ml, for example about 1400 ml; Preferably, the ratio of the sum of the mass of the cinnamon twig, white peony root, liquorice, ephedra, ginger, atractylodes macrocephala, rhizoma anemarrhenae, saposhnikovia divaricata and Heishun tablets to the mass / volume (g / ml) of the water is 0.1 to 0.5, for example, about 0.31; Preferably, the soaking time is 30 to 60 minutes, for example, about 45 minutes; Preferably, the slow-cooking time is 30 to 90 minutes, for example, about 60 minutes; Preferably, the filtration is 200 mesh nylon filter cloth filtration; Preferably, the mass ratio of the cinnamon twig, the heishunpian, the ginger, the siler, the ephedra, the atractylodes, the white peony root, the rhizome of anemarrhena and the liquorice is (2-6):(1-3):(3-7):(2-6):(1-3):(3-7):(1-5):(2-6):(1-3); More preferably, the mass ratio of the cinnamon twig, the heishunpian, the ginger, the siler, the ephedra, the atractylodes, the white peony root, the rhizome of anemarrhena and the liquorice is (3-5):(1.5-2.5):(4-6):(3-5):(1.5-2.5):(4-6):(2-4):(3-5):(1.5-2.5); Preferably, the mass ratio of the cinnamon twig, the black shunpian, the ginger, the siler, the ephedra, the atractylodes, the white peony root, the rhizome of anemarrhena and the liquorice is about 4: about 2: about 5: about 4: about 2: about 5: about 3: about 4: about 2; Preferably, the mass of the cassia twig is in the range of 40 to 70 g, for example, about 55.20 g; Preferably, the mass of the black slices is in the range of 20 to 40 g, for example, about 27.60 g; Preferably, the weight of the ginger is in the range of 50 to 90 g, for example, about 69.00 g; Also preferably, the mass of the windproof is in the range of 40 to 70 g, for example, about 55.20 g; Preferably, the mass of the ephedra is in the range of 20 to 40 g, for example, about 27.60 g; Preferably, the mass of the Atractylodes macrocephala is in the range of 50 to 90 g, for example, about 69.00 g; Preferably, the mass of the white peony root is in the range of 30 to 50 g, for example, about 41.40 g; Preferably, the mass range of the Rhizoma Anemarrhenae is 40 to 70 g, for example, about 55.20 g; Preferably, the mass of the licorice is in the range of 20 to 40 g, for example, about 27.60 g; Still more preferably, in the method for preparing a reference sample of a traditional Chinese medicine composition comprising cinnamon twig, the container is a volumetric flask; Still more preferably, the solvent is methanol; More preferably, the volume ratio between the Chinese medicine composition reference sample and the solvent is 0.1 to 0.5, such as about 0.2; Still more preferably, the volume of the Chinese medicine composition reference sample is 1 to 5 ml, for example, about 2 ml; Particularly preferably, in the method for preparing the reference solution, the volume percentage concentration of the methanol is 40% to 60%, for example, about 50%; Particularly preferably, the concentration of mangiferin in the reference solution is about 60 μg / ml; Particularly preferably, the concentration of paeoniflorin in the reference solution is about 160 μg / ml; Particularly preferably, the concentration of cimicifuga glycoside in the reference solution is about 40 μg / ml; Particularly preferably, the concentration of 5-O-methylvisaminol glycoside in the reference solution is about 40 μg / ml; Particularly preferably, the concentration of cinnamic acid in the reference solution is about 10 μg / ml; Particularly preferably, the concentration of cinnamaldehyde in the reference solution is about 10 μg / ml; Particularly preferably, the concentration of ammonium glycyrrhizinate in the reference solution is about 50 μg / ml; Particularly preferably, the concentration of 6-gingerol in the reference solution is about 10 μg / ml; Particularly preferably, the detection wavelength is 200 to 300 nm, for example, about 254 nm; Particularly preferably, the injection volume is 15 to 25 μl, for example, about 20 μl; Particularly preferably, the theoretical plate number of the chromatographic peak corresponding to the paeoniflorin is not less than 2000; Particularly preferably, the chromatographic column is HALO AQ-C 18 Chromatographic columns or YMC Meteoric Core C 18 Chromatographic columns; Particularly preferably, the specifications of the chromatographic column are: column length 150 mm, inner diameter 4.6 mm, and particle size 2.7 μm.

7. The construction method according to claim 5, characterized in that: Mobile phase A was acetonitrile; Preferably, 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; Preferably, the acid aqueous solution, alkali aqueous solution and / or buffered saline solution is selected from formic acid, 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; Preferably, the acid aqueous solution is a 0.01% to 1% acid aqueous solution; More preferably, the acid aqueous solution is a 0.01% to 1% phosphoric acid aqueous solution; More preferably, the acid aqueous solution is a 0.05% to 0.15% phosphoric acid aqueous solution; Also preferably, the acid aqueous solution is about 0.1% phosphoric acid aqueous solution; Also preferably, the buffered saline solution is an acetate solution and / or an acetate solution; Also preferably, the pH value of the buffered saline solution is not greater than 7.0; Still more preferably, when the detection wavelength is 254 nm, the fingerprint spectrum includes peaks 1-13, and the retention times of the peaks 1-13 correspond to about 7.250 min, about 12.917 min, about 14.087 min, about 15.533 min, about 16.590 min, about 26.515 min, about 33.733 min, about 48.726 min, about 50.055 min, about 51.963 min, about 53.086 min, about 63.573 min, and about 70.914 min, respectively; Particularly preferably, when the detection wavelength is 254 nm, the fingerprint spectrum includes peaks 1-13, wherein peak 7 5-O-methylvisaminol glycoside is used as a reference peak, and the relative retention time averages of peaks 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, and 13 are about 0.21, about 0.38, about 0.42, about 0.46, about 0.49, about 0.79, about 1.44, about 1.48, about 1.54, about 1.57, about 1.88, and about 2.10, respectively; Particularly preferably, when the detection wavelength is 254 nm, peak 2 is mangiferin, peak 4 is paeoniflorin, peak 5 is cimidocosapentaenoic acid glycoside, peak 6 is liquiritigenin, peak 7 is 5-O-methylvisaminol glycoside, peak 9 is cinnamic acid, peak 11 is cinnamaldehyde, and peak 13 is glycyrrhizic acid; Particularly preferably, when the detection wavelength is 254 nm, peak No. 1 comes from the medicinal material Anemarrhena asphodeloides, peak No. 2 comes from the medicinal material Anemarrhena asphodeloides, peak No. 3 comes from the medicinal material Anemarrhena asphodeloides, peak No. 4 comes from the medicinal material Paeonia lactiflora, peak No. 5 comes from the medicinal material Saposhnikovia divaricata, peak No. 6 comes from the medicinal material Glycyrrhiza uralensis, peak No. 7 comes from the medicinal material Saposhnikovia divaricata, peak No. 8 comes from the medicinal material Glycyrrhiza uralensis, peak No. 9 comes from the medicinal material Cinnamomum cassiae, peak No. 10 comes from the medicinal material Saposhnikovia divaricata, peak No. 11 comes from the medicinal material Cinnamomum cassiae, peak No. 12 comes from the medicinal material Glycyrrhiza uralensis, and peak No. 13 comes from the medicinal material Glycyrrhiza uralensis.

8. A quality control method for a Chinese medicine composition containing cinnamon twig, characterized in that: The quality control method comprises the following steps: (1) Establishing a standard fingerprint of a reference sample of a Chinese medicine composition according to the fingerprint construction method described in any one of claims 5 to 7; (2) taking a sample solution of the Chinese medicine composition, and testing it according to the chromatographic conditions in the fingerprint construction method according to any one of claims 5 to 7 to obtain a fingerprint of the Chinese medicine composition sample to be tested; as well as (3) Compare the fingerprint of the Chinese medicine composition sample obtained in step (2) with the standard fingerprint of the Chinese medicine composition reference sample obtained in step (1). If it meets the requirements, it is a qualified product, and if it does not meet the requirements, it is an unqualified product.

9. The quality control method according to claim 8, characterized in that: The compliance requirements include one or more of the following: (1) The fingerprint of the Chinese medicine composition sample to be tested exhibits 13 characteristic chromatographic peaks, and the retention time of each characteristic chromatographic peak is within ±10% of the retention time value of the corresponding reference chromatographic peak in the standard fingerprint of the Chinese medicine composition reference sample; (2) With the 5-O-methylvisaminol glycoside 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; as well as (3) According to the similarity evaluation system of Chinese medicine chromatographic fingerprint, the similarity between the fingerprint of the Chinese medicine composition sample to be tested and the standard fingerprint of the Chinese medicine composition reference sample is calculated, and the similarity shall not be less than 0.

90.

10. Use of the assay method according to any one of claims 1 to 4, the construction method according to any one of claims 5 to 7, or the quality control method according to claim 8 or 9 in quality detection and / or quality evaluation and / or quality control of a Chinese medicine composition comprising Cassia twig.

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