Quality control method of traditional Chinese medicine composition containing ramulus cinnamomi and application thereof

Abstract

The application discloses a content determination method of a traditional Chinese medicine composition containing Ramulus Cinnamomi, and the method comprises the following steps: detecting test sample solution and control sample solution of the traditional Chinese medicine composition; and obtaining content information of the control sample of the traditional Chinese medicine composition according to the detection result; wherein the traditional Chinese medicine composition comprises Ramulus Cinnamomi, Radix Paeoniae Alba, Radix Glycyrrhizae, Ephedra, Rhizoma Zingiberis Recens, Rhizoma Atractylodis Macrocephalae, Rhizoma Anemarrhenae, Radix Saposhnikoviae and Heishunpian; and the control sample comprises Mangiferin, Paeonoside, Anemonin, 5-O-Methylvisamminol, Cinnamyl Aldehyde, Ammonium Glycyrrhizate and 6-Gingerol. The detection method is simple, accurate and efficient, has good stability, high precision, good reproducibility, is convenient and easy to master, can effectively save energy and reduce analysis cost, and the technical content is not disclosed in the prior art.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to a quality control method of a traditional Chinese medicine composition containing Ramulus Cinnamomi and application thereof. BACKGROUND

[0002] According to the Law of the People's Republic of China on Chinese Medicine, ancient classical famous prescriptions refer to the prescriptions recorded in ancient medical classics that are still widely used, have definite curative effects, and have obvious characteristics and advantages. Ancient classical famous prescriptions have a long history in China, a rich human history, and are still in use today. They are the summary of the clinical practice of generations of physicians and carry the deep accumulation of the brilliant civilization of traditional Chinese medicine over thousands of years. They are the result of thousands of years of refining of traditional Chinese medicine theory, the summary of clinical experience over the ages, and the most precious part of the great treasure trove of traditional Chinese medicine. In-depth research and development of classical famous prescriptions of traditional Chinese medicine is a golden key to tapping the treasure trove of traditional Chinese medicine.

[0003] As the most commonly used dosage form of traditional Chinese medicine clinical drugs, classical famous prescription decoctions have the advantages of reasonable composition, rapid onset, significant efficacy, easy absorption, and are deeply trusted by the majority of patients. However, due to the difficulties in deployment, carrying, temporary decoction, and long-term storage, they are prone to spoilage, bitter taste, and large volume, and the standard is difficult to unify, which seriously affects their clinical efficacy and cannot meet the requirements of modern life. In order to maintain the advantages of decoctions and overcome their shortcomings, the Center for Drug Evaluation of the National Medical Products Administration issued the Technical Guidelines for Pharmaceutical Research of Traditional Chinese Medicine Compound Preparations Managed According to the Directory of Ancient Classical Famous Prescriptions (Trial) on August 31, 2021, which clearly states that the quality of traditional Chinese medicine compound preparations managed according to the directory of ancient classical famous prescriptions should be basically consistent with that of the classical famous prescription reference sample. The reference sample represents the overall internal quality of the preparation, and except for the molding process, the reference sample and the preparation are basically consistent in the rest of the quality control indicators. Therefore, the reference sample is a physical comparison of the internal quality of the preparation and a reference for the optimization of the production process and the development of quality standards. The reference sample is the benchmark for classical famous prescriptions and all traditional Chinese medicine research and development, and is a reference for ensuring the safety and effectiveness of drugs. The production process route, parameter optimization, and quality standard development of classical famous prescription compound preparations should be based on the reference sample. The reference sample is a link between clinical practice, enterprises, and scientific research, and is a benchmark for the inheritance, application, and development of traditional Chinese medicine.

[0004] Fingerprint is based on the understanding of the overall effect of the material group of traditional Chinese medicine, with the aid of spectroscopy and chromatography to obtain the spectrum or chromatogram of the chemical composition of traditional Chinese medicine, which is a feasible mode to realize the identification of the authenticity of traditional Chinese medicine, evaluate the quality consistency and product stability, has the characteristics of large information, strong characteristic, integrity and fuzziness. The fingerprint of traditional Chinese medicine can comprehensively reflect the relative relationship of the chemical components contained in the medicinal materials, embodies the complexity and correlation of the components of traditional Chinese medicine, adapts to the traditional theory of traditional Chinese medicine, can truly effectively characterize, comprehensively evaluate and comprehensively control the internal quality of traditional Chinese medicine, especially suitable for the quality control of traditional Chinese medicinal materials and traditional Chinese medicine products under the condition that the effective components are not completely clear or do not need to be completely clear. The fingerprint of traditional Chinese medicine can be used to investigate the factors such as the origin, harvesting season, harvesting part, processing, storage time of traditional Chinese medicinal materials, so as to provide the basis for identifying the authenticity and quality of raw medicinal materials before production. It can also be used for quality control of the production process of traditional Chinese medicine: tracking the changes of some chemical components in preparations, monitoring the consistency and stability of the quality between raw medicinal materials and finished products, and between batches of finished products. Compared with the quality analysis method of the content of index components, the fingerprint can more comprehensively reflect the types and quantities of chemical components of traditional Chinese medicine. Under the current situation that the effective components of traditional Chinese medicine compound preparations have not been completely elucidated, the comprehensive evaluation of the internal quality of traditional Chinese medicine and the effective control of the overall substance can be realized, which is one of the effective means for the quality control of traditional Chinese medicine and its preparations.

[0005] At present, there is no literature report on the comprehensive analysis, content determination and fingerprint study of the chemical components of the traditional Chinese medicine composition containing cassia twig claimed in the present application. In the prior art, only the quality of a single component in the traditional Chinese medicine compound is analyzed, there is no more comprehensive and systematic quality control method to reflect the quality status of the main reference sample components in the traditional Chinese medicine compound and finished product of the present application, which cannot effectively control the production process and product quality, cannot well guarantee the clinical efficacy, and therefore the fingerprint quality control method which can comprehensively control the overall quality of the traditional Chinese medicine compound of the present application is needed to control the key quality. SUMMARY

[0006] Based on this, the application provides a content determination method of a traditional Chinese medicine composition containing Ramulus Cinnamomi, which comprises the following steps: detecting traditional Chinese medicine composition test sample solution and control sample solution, wherein the chromatographic conditions of the detection are as follows: a chromatographic column with octadecylsilane bonded silica as the filler is used, mobile phase A is selected from one or more of acetonitrile, methanol and tetrahydrofuran, mobile phase B is an acid aqueous solution, an alkali aqueous solution and / or a buffer salt aqueous solution, the gradient elution program is as follows: 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 DEG C, the detection wavelength is 180-360 nm, and the injection amount is 10-30 ul; according to the detection result, the content information of the control sample of the traditional Chinese medicine composition is obtained; wherein the traditional Chinese medicine composition comprises Ramulus Cinnamomi, Radix Paeoniae Alba, Radix Glycyrrhizae, Ephedra sinica, Zingiber officinale, Rhizoma Atractylodis Macrocephalae, Rhizoma Anemarrhenae, Radix Saposhnikoviae and Heishunpian, and the control sample is mangiferin, paeoniflorin, epimedoside A, 5-O-methylvisamminol, cinnamyl aldehyde, ammonium glycyrrhizate and 6-shogaol.

[0007] Further, the information is the content of the control sample in the traditional Chinese medicine composition calculated according to the corresponding peak areas in the chromatogram of the traditional Chinese medicine composition test sample solution and the chromatogram of the control sample solution 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 epimedoside A is y=61489.9671x+28672.7690, R2=0.9999. Further, the linear equation of 5-O-methylvisamminol is y=69455.1451x+80352.1532, R2=0.9999. Further, the linear equation of cinnamyl aldehyde is y=253930.5912x+3832.3004, R2=0.9998. Further, the linear equation of ammonium glycyrrhizate is y=26858.1718x+3266.0328, R2=0.9999. Further, the linear equation of 6-shogaol is y=18804.2424x-2184.2822, R2=0.9999.

[0008] Further, the preparation method of the test solution of the traditional Chinese medicine composition comprises: taking a proper amount of the reference sample of the traditional Chinese medicine composition containing Ramulus Cinnamomi into a container, adding a solvent, shaking, filtering, and obtaining the test solution of the traditional Chinese medicine composition. Further, the container is a volumetric flask. Further, the solvent is methanol. Further, the ratio of the volume of the reference sample of the traditional Chinese medicine composition to the volume of the solvent is 0.1-0.5, for example, about 0.2. Further, the volume of the reference sample of the traditional Chinese medicine composition is 1-5 ml, for example, about 2 ml. Further, the preparation method of the control solution comprises: weighing a proper amount of the controls of mangiferin, paeoniflorin, prim-O-glucosylcimifugin, 5-O-methylvisamminol, cinnamyl aldehyde, ammonium glycyrrhizinate, and 6-shogaol, and adding methanol to prepare the control solution with the concentrations of mangiferin, paeoniflorin, prim-O-glucosylcimifugin, 5-O-methylvisamminol, cinnamyl aldehyde, ammonium glycyrrhizinate, and 6-shogaol being 1-200 μg / ml, respectively. Further, in the preparation method of the control solution, the volume percentage concentration of the methanol is 40%-60%, for example, about 50%. Further, the concentration of mangiferin in the control solution is about 60 μg / ml. Further, the concentration of paeoniflorin in the control solution is about 160 μg / ml. Further, the concentration of prim-O-glucosylcimifugin in the control solution is about 40 μg / ml. Further, the concentration of 5-O-methylvisamminol in the control solution is about 40 μg / ml. Further, the concentration of cinnamyl aldehyde in the control solution is about 10 μg / ml. Further, the concentration of ammonium glycyrrhizinate in the control solution is about 50 μg / ml. Further, the concentration of 6-shogaol in the control solution is about 10 μg / ml.

[0009] Further, the preparation method of the reference sample of the traditional Chinese medicine composition containing Ramulus Cinnamomi comprises: weighing a proper amount of Ramulus Cinnamomi, Radix Paeoniae Alba, Glycyrrhiza uralensis, Ephedra sinica, Zingiber officinale, Atractylodes macrocephala, Anemarrhena asphodeloides, Saposhnikovia divaricata, and Radix Morindae Officinalis, placing them into a container, adding water for soaking, boiling by strong fire, simmering for a period of time, filtering, collecting the filtrate, and obtaining the reference sample of the traditional Chinese medicine composition containing Ramulus Cinnamomi. Further, the volume of the water is 1000-2000 ml, for example, about 1400 ml. Further, the ratio of the sum of the mass of Ramulus Cinnamomi, Radix Paeoniae Alba, Glycyrrhiza uralensis, Ephedra sinica, Zingiber officinale, Atractylodes macrocephala, Anemarrhena asphodeloides, Saposhnikovia divaricata, and Radix Morindae Officinalis to the mass / volume (g / ml) of the water is 0.1-0.5, for example, about 0.31. Further, the soaking time is 30-60 min, for example, about 45 min. Further, the simmering time is 30-90 min, for example, about 60 min. Further, the filtering is 200-mesh nylon cloth filtering.

[0010] Further, the mass ratio between the Ramulus Cinnamomi, the Heishunpian, the Rhizoma Zingiberis Recens, the Saposhnikovia Divaricata, the Ephedra, the Atractylodes Macrocephala, the Radix Paeoniae Alba, the Anemarrhena Asphodeloides, and the Glycyrrhiza Uralensis is (2-6) : (1-3) : (3-7) : (2-6) : (1-3) : (3-7) : (1-5) : (2-6) : (1-3). Further, the mass ratio between the Ramulus Cinnamomi, the Heishunpian, the Rhizoma Zingiberis Recens, the Saposhnikovia Divaricata, the Ephedra, the Atractylodes Macrocephala, the Radix Paeoniae Alba, the Anemarrhena Asphodeloides, and the Glycyrrhiza Uralensis 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 Ramulus Cinnamomi, the Heishunpian, the Rhizoma Zingiberis Recens, the Saposhnikovia Divaricata, the Ephedra, the Atractylodes Macrocephala, the Radix Paeoniae Alba, the Anemarrhena Asphodeloides, and the Glycyrrhiza Uralensis is about 4: about 2: about 5: about 4: about 2: about 5: about 3: about 4: about 2. Further, the mass of the Ramulus Cinnamomi ranges from 40-70g, such as about 55.20g. Further, the mass of the Heishunpian ranges from 20-40g, such as about 27.60g. Further, the mass of the Rhizoma Zingiberis Recens ranges from 50-90g, such as about 69.00g. Further, the mass of the Saposhnikovia Divaricata ranges from 40-70g, such as about 55.20g. Further, the mass of the Ephedra ranges from 20-40g, such as about 27.60g. Further, the mass of the Atractylodes Macrocephala ranges from 50-90g, such as about 69.00g. Further, the mass of the Radix Paeoniae Alba ranges from 30-50g, such as about 41.40g. Further, the mass of the Anemarrhena Asphodeloides ranges from 40-70g, such as about 55.20g. Further, the mass of the Glycyrrhiza Uralensis ranges from 20-40g, such as about 27.60g.

[0011] Further, the detection wavelength of the Mangoiflorin, the Paeoniflorin, the Epimedoside A, the 5-O-Methylvisamminol, and the Glycyrrhizic Acid is 200-300nm, such as about 280nm. Further, the detection wavelength of the Cinnamaldehyde and the 6-Shogaol is 200-300nm, such as about 254nm. Further, the injection volume is 15-25μl, such as about 20μl. Further, the theoretical plate number of the corresponding chromatographic peak of the Paeoniflorin is not less than 2000. Further, the chromatographic column is HALOAQ-C 18 chromatographic column or YMC Meteoric Core C 18Chromatographic column. Further, the specification of the chromatographic column: column length 150 mm, inner diameter 4.6 mm, particle size 2.7 μm. Further, the mobile phase A is acetonitrile. Further, the aqueous acid solution, aqueous alkali solution and / or aqueous buffer salt solution is selected from one or more of weak acids and their salts, weak alkalis and their salts in different concentrations. Further, the aqueous acid solution, aqueous alkali solution and / or aqueous buffer salt 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. Further, the aqueous acid solution is an aqueous acid solution of 0.01% to 1%. Further, the aqueous acid solution is an aqueous phosphoric acid solution of 0.01% to 1%. Further, the aqueous acid solution is an aqueous phosphoric acid solution of 0.05% to 0.15%. Further, the aqueous acid solution is an aqueous phosphoric acid solution of about 0.1%. Further, the aqueous buffer salt solution is an aqueous acetate salt solution and / or an aqueous acetate salt solution. Further, the pH value of the aqueous buffer salt solution is not more than 7.0. Further, the detection limit of the mangiferin is about 13.63 ng / ml. Further, the detection limit of the paeoniflorin is about 0.976 μg / ml. Further, the detection limit of the epimediumin is about 43.55 ng / ml. Further, the detection limit of the 5-O-methylvisamminol is about 46.08 ng / ml. Further, the detection limit of the cinnamaldehyde is about 61.64 ng / ml. Further, the detection limit of the glycyrrhizic acid is about 38.75 ng / ml. Further, the detection limit of the 6-gingerol is about 0.6475 μg / ml. Further, the quantification limit of the mangiferin is about 45.44 ng / ml. Further, the quantification limit of the paeoniflorin is about 4.88 μg / ml. Further, the quantification limit of the epimediumin is about 0.2178 μg / ml. Further, the quantification limit of the 5-O-methylvisamminol is about 92.16 ng / ml. Further, the quantification limit of the cinnamaldehyde is about 0.12328 μg / ml. Further, the quantification limit of the glycyrrhizic acid is about 0.2422 μg / ml. Further, the quantification limit of the 6-gingerol is about 1.295 μg / ml.

[0012] According to another aspect of the present application, there is provided a method for constructing a fingerprint of a traditional Chinese medicine composition comprising Ramulus Cinnamomi, the method comprising the following steps: preparing a test solution of the traditional Chinese medicine composition: taking a proper amount of a reference sample of the traditional Chinese medicine composition comprising Ramulus Cinnamomi, placing it in a container, adding a solvent, shaking it well, filtering it, and obtaining the test solution of the traditional Chinese medicine composition; wherein the traditional Chinese medicine composition comprises Ramulus Cinnamomi, Radix Paeoniae Alba, Glycyrrhiza Uralensis, Ephedra Sinica, Zingiber Officinale, Atractylodes Macrocephala, Anemarrhena Asphodeloides, Saposhnikovia Divaricata, and Black Gomphidium; preparing a control solution: taking a proper amount of control samples of mangiferin, paeoniflorin, cimifugin, 5-O-methylvisamminol, cinnamyl aldehyde, ammonium glycyrrhizate, and 6-shogaol, adding methanol to prepare the control solution with the concentrations of mangiferin, paeoniflorin, cimifugin, 5-O-methylvisamminol, cinnamic acid, cinnamyl aldehyde, ammonium glycyrrhizate, and 6-shogaol being 1-200 μg / ml respectively; obtaining the fingerprint of the traditional Chinese medicine composition according to the results of detecting the test solution of the traditional Chinese medicine composition and the control solution by high performance liquid chromatography; and the chromatographic conditions of the high performance liquid chromatography detection are as follows: using a chromatographic column with octadecylsilane bonded silica gel as the filler, mobile phase A being selected from one or more of acetonitrile, methanol, and tetrahydrofuran, mobile phase B being acid aqueous solution, alkali aqueous solution, and / or buffer salt aqueous solution, the gradient elution program being: 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 being 0.6 ml / min, the column temperature being 27-30 °C, the detection wavelength being 180-360 nm, and the injection volume being 10-30 μl.

[0013] Further, the method for preparing the reference sample of the traditional Chinese medicine composition comprising Ramulus Cinnamomi comprises the following steps: taking a proper amount of Ramulus Cinnamomi, Radix Paeoniae Alba, Glycyrrhiza Uralensis, Ephedra Sinica, Zingiber Officinale, Atractylodes Macrocephala, Anemarrhena Asphodeloides, Saposhnikovia Divaricata, and Black Gomphidium, placing them in a container, adding water to soak them, boiling them with strong fire, simmering them for a period of time with weak fire, filtering them, collecting the filtrate, and obtaining the reference sample of the traditional Chinese medicine composition comprising Ramulus Cinnamomi. Further, the volume of the water is 1000-2000 ml, for example, about 1400 ml. Further, the ratio of the sum of the masses of the Ramulus Cinnamomi, Radix Paeoniae Alba, Glycyrrhiza Uralensis, Ephedra Sinica, Zingiber Officinale, Atractylodes Macrocephala, Anemarrhena Asphodeloides, Saposhnikovia Divaricata, and Black Gomphidium to the mass / volume (g / ml) of the water is 0.1-0.5, for example, about 0.31. Further, the soaking time is 30-60 min, for example, about 45 min. Further, the simmering time with weak fire is 30-90 min, for example, about 60 min. Further, the filtering is 200-mesh nylon cloth filtering.

[0014] Further, the mass ratio between the Guizhi, the Heishunpian, the Shengjiang, the Fangfeng, the Mahuang, the Baishao, the Baishao, the Zhimu, and the Gancao is (2-6):(1-3):(3-7):(2-6):(1-3):(3-7):(1-5):(2-6):(1-3). Further, the mass ratio between the Guizhi, the Heishunpian, the Shengjiang, the Fangfeng, the Mahuang, the Baishao, the Baishao, the Zhimu, and the Gancao 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 Guizhi, the Heishunpian, the Shengjiang, the Fangfeng, the Mahuang, the Baishao, the Baishao, the Zhimu, and the Gancao is about 4: about 2: about 5: about 4: about 2: about 5: about 3: about 4: about 2. Further, the mass of the Guizhi ranges from 40-70 g, e.g., about 55.20 g. Further, the mass of the Heishunpian ranges from 20-40 g, e.g., about 27.60 g. Further, the mass of the Shengjiang ranges from 50-90 g, e.g., about 69.00 g. Further, the mass of the Fangfeng ranges from 40-70 g, e.g., about 55.20 g. Further, the mass of the Mahuang ranges from 20-40 g, e.g., about 27.60 g. Further, the mass of the Baishao ranges from 50-90 g, e.g., about 69.00 g. Further, the mass of the Baishao ranges from 30-50 g, e.g., about 41.40 g. Further, the mass of the Zhimu ranges from 40-70 g, e.g., about 55.20 g. Further, the mass of the Gancao ranges from 20-40 g, e.g., about 27.60 g.

[0015] Further, in the preparation method of the reference sample of the traditional Chinese medicine composition containing Ramulus Cinnamomi, the container is a volumetric flask. Further, the solvent is methanol. Further, the ratio of the volume of the reference sample of the traditional Chinese medicine composition to the volume of the solvent is 0.1-0.5, for example, about 0.2. Further, the volume of the reference sample of the traditional Chinese medicine composition is 1-5 ml, for example, about 2 ml. Further, in the preparation method of the control solution, the volume percentage concentration of the methanol is 40%-60%, for example, about 50%. Further, the concentration of mangiferin in the control solution is about 60 μg / ml. Further, the concentration of paeoniflorin in the control solution is about 160 μg / ml. Further, the concentration of epimedoside in the control solution is about 40 μg / ml. Further, the concentration of 5-O-methylvisamminol in the control solution is about 40 μg / ml. Further, the concentration of cinnamic acid in the control solution is about 10 μg / ml. Further, the concentration of cinnamic aldehyde in the control solution is about 10 μg / ml. Further, the concentration of ammonium glycyrrhizate in the control solution is about 50 μg / ml. Further, the concentration of 6-shogaol in the control 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 15-25 μl, for example, about 20 μl. Further, the theoretical plate number of the chromatographic peak corresponding to paeoniflorin is not less than 2000. Further, the chromatographic column is HALO AQ-C 18 chromatographic column or YMC Meteoric Core C 18 chromatographic column. Further, the specification of the chromatographic column is: column length 150 mm, inner diameter 4.6 mm, and particle size 2.7 μm.

[0017] Further, the mobile phase A is acetonitrile. Further, the aqueous acid solution, the aqueous alkali solution, and / or the aqueous buffer salt solution are selected from one or more of weak acids and their salts, weak alkalis and their salts at different concentrations. Further, the aqueous acid solution, the aqueous alkali solution, and / or the aqueous buffer salt 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 at different concentrations. Further, the aqueous acid solution is an aqueous acid solution at a concentration of 0.01%-1%. Further, the aqueous acid solution is an aqueous phosphoric acid solution at a concentration of 0.01%-1%. Further, the aqueous acid solution is an aqueous phosphoric acid solution at a concentration of 0.05%-0.15%. Further, the aqueous acid solution is an aqueous phosphoric acid solution at a concentration of about 0.1%. Further, the aqueous buffer salt solution is an aqueous acetic acid salt solution and / or an aqueous acetic acid salt solution. Further, the pH value of the aqueous buffer salt solution is not more than 7.0.

[0018] Further, the fingerprint spectrum comprises peaks No. 1-13, and the retention time of each of the peaks No. 1-13 corresponds 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, at the detection wavelength of 254 nm. Further, the fingerprint spectrum comprises peaks No. 1-13, wherein the relative retention time average of peak No. 1, peak No. 2, peak No. 3, peak No. 4, peak No. 5, peak No. 6, peak No. 8, peak No. 9, peak No. 10, peak No. 11, peak No. 12, and peak No. 13 corresponds 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, at the detection wavelength of 254 nm, with peak No. 5-O-methyl visamminoside as the reference peak. Further, peak No. 2 is mangiferin, peak No. 4 is paeoniflorin, peak No. 5 is thalidinoside, peak No. 6 is glycyrrhizin, peak No. 7 is 5-O-methyl visamminoside, peak No. 9 is cinnamic acid, peak No. 11 is cinnamic aldehyde, and peak No. 13 is glycyrrhizic acid, at the detection wavelength of 254 nm. Further, peak No. 1 comes from Anemarrhena asphodeloides medicinal material, peak No. 2 comes from Anemarrhena asphodeloides medicinal material, peak No. 3 comes from Anemarrhena asphodeloides medicinal material, peak No. 4 comes from Paeonia lactiflora medicinal material, peak No. 5 comes from Saposhnikovia divaricata medicinal material, peak No. 6 comes from Glycyrrhiza uralensis medicinal material, peak No. 7 comes from Saposhnikovia divaricata medicinal material, peak No. 8 comes from Glycyrrhiza uralensis medicinal material, peak No. 9 comes from Cinnamomum cassia medicinal material, peak No. 10 comes from Saposhnikovia divaricata medicinal material, peak No. 11 comes from Cinnamomum cassia medicinal material, peak No. 12 comes from Glycyrrhiza uralensis medicinal material, and peak No. 13 comes from Glycyrrhiza uralensis medicinal material, at the detection wavelength of 254 nm.

[0019] According to another aspect of the present application, a quality control method of a traditional Chinese medicine composition comprising Cinnamomum cassia is provided, which comprises the following steps: (1) establishing a standard fingerprint spectrum of a reference sample of the traditional Chinese medicine composition according to the above-mentioned fingerprint spectrum construction method; (2) taking a test sample solution of the traditional Chinese medicine composition, and detecting according to the chromatographic conditions in the above-mentioned fingerprint spectrum construction method to obtain a fingerprint spectrum of the test sample of the traditional Chinese medicine composition; and (3) comparing the fingerprint spectrum of the test sample of the traditional Chinese medicine composition obtained in step (2) with the standard fingerprint spectrum of the reference sample of the traditional Chinese medicine composition obtained in step (1), and the product is qualified if it meets the requirements, or unqualified if it does not meet the requirements.

[0020] Further, the requirements include one or more of the following: (1) the test sample of the traditional Chinese medicine composition 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 control chromatographic peak in the standard fingerprint of the traditional Chinese medicine composition reference sample; (2) taking the 5-O-methylvisamminol glycoside peak as the S peak, the relative retention time of each characteristic chromatographic peak in the test sample of the traditional Chinese medicine composition to the S peak is within ±10% of the relative retention time value of each characteristic chromatographic peak in the standard fingerprint of the traditional Chinese medicine composition reference sample; and (3) according to the traditional Chinese medicine chromatographic fingerprint similarity evaluation system, the similarity of the test sample of the traditional Chinese medicine composition to the standard fingerprint of the traditional Chinese medicine composition reference sample is not less than 0.90 after similarity calculation.

[0021] According to another aspect of the present application, there is provided a use of the above-mentioned determination method or the above-mentioned construction method or the above-mentioned quality control method in the quality detection and / or quality evaluation and / or quality control of a traditional Chinese medicine composition containing Ramulus Cinnamomi.

[0022] The present application has the following beneficial effects:

[0023] In short, the present application provides a fingerprint determination and quality control method for a traditional Chinese medicine composition reference sample containing Ramulus Cinnamomi, which identifies 13 common characteristic peaks, has simple conditions and short analysis time, solves the problems of difficulty in separating fingerprint characteristic peaks and interference of impurity peaks, ensures the chemical composition stability and use safety of the reference sample, provides an important reference for subsequent quality control of preparations, ensures the quality stability of products, guarantees the efficacy of traditional Chinese medicine compounds, and makes the traditional Chinese medicine compounds better serve human life and health.

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

[0025] (1) The fingerprint of the traditional Chinese medicine compound reference sample of the present application is established, which overcomes the defect that single component content determination cannot reflect the overall content, can control the internal quality of the traditional Chinese medicine compound reference sample of the present application as a whole and macroscopically, ensures the efficacy of the drug, uses the main means of modern drug research, realizes the inheritance of classics with high quality, and makes the classic famous prescription have more formal quality control;

[0026] (2) The chemical composition of the traditional Chinese medicine compound reference sample of the present application is complex, and it is difficult to separate its characteristic peaks, the present application adopts gradient elution method in the process of establishing the fingerprint, which solves the problems of difficulty in separating fingerprint characteristic peaks and interference of impurity peaks;

[0027] (3) In the process of establishing the fingerprint of the traditional Chinese medicine compound reference sample, 13 common characteristic peaks are confirmed, and the relative retention time, relative peak area and similarity are studied, so as to ensure the stability of the chemical composition of the reference sample and the safety in use, and provide important reference and quality reference for the quality control of the subsequent compound preparation;

[0028] (4) The fingerprint of each effective component in the traditional Chinese medicine compound reference sample is regarded as a whole, and the order and mutual relationship of each characteristic peak are focused on, so as to avoid the one-sidedness of determining the quality of the traditional Chinese medicine compound reference sample as a whole by only determining one or two chemical components, and reduce the possibility of artificial processing for quality standardization, and provide a new method and means for the complete and accurate evaluation of the quality of the traditional Chinese medicine compound reference sample;

[0029] (5) The method has good stability, high precision, good reproducibility, convenience and easy to master. BRIEF DESCRIPTION OF DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without departing from the scope of the present application.

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

[0032] Figure 2 The instrument precision test result schematic diagram 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 technical solutions in the embodiments of the present application will be described clearly and completely in the following with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of the present application.

[0034] Unless otherwise indicated, all technical and scientific terms and abbreviations used herein have the meanings that are commonly understood by one of ordinary skill in the art in the field of the application, or that are given in the specification. Although any methods, conditions, materials, and the like that are similar or equivalent to those described herein can be used in the practice of the present application, the preferred methods, conditions, materials, and the like are described herein.

[0035] The present application is intended to embrace all alternatives, modifications, and equivalents that can fall within the scope of the present application as defined by the claims. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. The application is not intended to be limited to the embodiments 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 this application, the term "comprising" is synonymous with "including," "containing," or "comprising." As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but can include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0038] As described in the background section, there is no literature report on the comprehensive analysis of the chemical components of the traditional Chinese medicine composition containing Ramulus cinnamomi claimed in the present application and the fingerprint research. In order to solve the above problems, the present application provides a content determination method of a traditional Chinese medicine composition containing Ramulus cinnamomi, which comprises the following steps: detecting a test sample solution and a control sample solution of the traditional Chinese medicine composition, wherein the chromatographic conditions of the detection are as follows: a chromatographic column with octadecylsilane bonded silica as the filler, mobile phase A is selected from one or more of acetonitrile, methanol and tetrahydrofuran, mobile phase B is an acid aqueous solution, an alkali aqueous solution and / or a buffer salt aqueous solution, the gradient elution program is as follows: 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 DEG C, the detection wavelength is 180-360 nm, and the injection volume is 10-30 ul; according to the detection result, the content information of the control sample of the traditional Chinese medicine composition is obtained; wherein the traditional Chinese medicine composition comprises Ramulus cinnamomi, Radix paeoniae alba, Radix glycyrrhizae, Ephedra sinica, Zingiber officinale, Rhizoma atractylodis, Anemarrhena asphodeloides, Saposhnikovia divaricata and Radix et Rhizoma strophanthi, and the control sample comprises mangiferin, paeoniflorin, epimedoside A, 5-O-methylvisamminol, cinnamyl aldehyde, ammonium glycyrrhizate and 6-shogaol.

[0039] In the present application, where an item is expressed as a range, a preferred range, or a range of upper and lower preferred values, it is understood that the entire range is specifically disclosed, regardless of whether the range is explicitly disclosed. For example, where a range of "10-30" is disclosed, it is understood that the range "10-30," "10-25," "10-20," "10-15," "15-30," "15-25," "15-20," "20-30," "20-25," "25-30," etc. are specifically disclosed. Where a range of values is recited, unless otherwise stated, the range is intended to include all values between the upper and lower range values, including the upper and lower values, and including integers within the range.

[0040] In a preferred embodiment, the information is the content of the reference substance in the traditional Chinese medicine composition calculated according to the recorded peak area of the chromatogram of the test solution of the traditional Chinese medicine composition and the chromatogram of the reference solution according to the external standard method. In a preferred embodiment, the linear equation of the mangiferin is y = 134118.1290x + 61486.8143, R2= 0.9999. In a preferred embodiment, the linear equation of the paeoniflorin is y = 3889.5159x + 11877.5625, R2= 0.9999. In a preferred embodiment, the linear equation of the epimedoside is y = 61489.9671x + 28672.7690, R2= 0.9999. In a preferred embodiment, the linear equation of the 5-O-methylvisamminol 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 method for preparing the test solution of the traditional Chinese medicine composition comprises: taking an appropriate amount of the reference sample of the traditional Chinese medicine composition containing Ramulus Cinnamomi into a container, adding a solvent, shaking well, and filtering to obtain the test solution of the traditional Chinese medicine composition. In a preferred embodiment, the container is a volumetric flask. In a preferred embodiment, the solvent is methanol.

[0042] In a preferred embodiment, the ratio of the volume of the reference sample of the traditional Chinese medicine composition to the volume of the solvent is 0.1-0.5, for example, about 0.2. In the present application, "about" refers to a value within ±5% of a specified 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 reference sample of the traditional Chinese medicine composition is 1-5 ml, for example, about 2 ml. In the present application, "about" refers to a value within ±5% of a specified value. For example, "about 2" includes ±5% of 2, or from 1.9 to 2.1.

[0044] In a preferred embodiment, the method for preparing the reference solution includes: weighing appropriate amounts of mangiferin, paeoniflorin, cimicifugain, 5-O-methylvisamiloside, cinnamaldehyde, ammonium glycyrrhizate, and 6-gingerol reference standards, and adding methanol to prepare a reference solution with concentrations of 1 to 200 μg / ml for mangiferin, paeoniflorin, cimicifugain, 5-O-methylvisamiloside, cinnamaldehyde, ammonium glycyrrhizate, and 6-gingerol.

[0045] In a preferred embodiment, in the preparation method of the reference solution, the volume percentage concentration of methanol is 40% to 60%, for example, about 50%. In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 50%" includes 50% ± 5%, 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 this 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 this 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 cimicifugain glycoside in the reference solution is about 40 μg / ml. In this 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-methylvisamidol in the reference solution is about 40 μg / ml. In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 40" includes 40 ± 5%, or from 38 to 42.

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

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

[0052] In a preferred embodiment, the concentration of 6-shogaol in the control solution is about 10 μg / ml. In the present invention, "about" means a value that is ±5% of a specified value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0053] In a preferred embodiment, the method of preparing the reference sample of the Chinese medicine composition comprising Ramulus Cinnamomi comprises: weighing appropriate amounts of Ramulus Cinnamomi, Radix Paeoniae Alba, Radix Glycyrrhizae, Ephedra, Rhizoma Zingiberis Recens, Radix Poriae, Radix Anemarrhenae, Radix Saposhnikoviae, and Fructus Corni, placing them in a container, adding water to soak, boiling with strong fire, simmering for a period of time with weak fire, filtering, collecting the filtrate, and obtaining the reference sample of the Chinese medicine composition comprising Ramulus Cinnamomi.

[0054] In a preferred embodiment, the volume of water is 1000-2000 ml, for example about 1400 ml. In the present invention, "about" means a value that is ±5% of a specified 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 Ramulus Cinnamomi, Radix Paeoniae Alba, Radix Glycyrrhizae, Ephedra, Rhizoma Zingiberis Recens, Radix Poriae, Radix Anemarrhenae, Radix Saposhnikoviae, and Fructus Corni to the mass / volume (g / ml) of water is 0.1-0.5, for example about 0.31. In the present invention, "about" means a value that is ±5% of a specified value. For example, "about 0.31" includes ±5% of 0.31, or from 0.2945 to 0.3255.

[0056] In a preferred embodiment, the time of soaking is 30-60 min, for example about 45 min. In the present invention, "about" means a value that is ±5% of a specified value. For example, "about 45" includes ±5% of 45, or from 42.75 to 47.25.

[0057] In a preferred embodiment, the time of simmering with weak fire is 30-90 min, for example about 60 min. In the present invention, "about" means a value that is ±5% of a specified value. For example, "about 60" includes ±5% of 60, or from 57 to 63.

[0058] In a preferred embodiment, the filtering is 200-mesh nylon cloth filtering.

[0059] In a preferred embodiment, the mass ratio between the Ramulus Cinnamomi, the Heishunpian, the Rhizoma Zingiberis, the Saposhnikovia Divaricata, the Ephedra, the Atractylodes Macrocephala, the Paeonia Lactiflora, the Anemarrhena Asphodeloides, and the Glycyrrhiza 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 between the Ramulus Cinnamomi, the Heishunpian, the Rhizoma Zingiberis, the Saposhnikovia Divaricata, the Ephedra, the Atractylodes Macrocephala, the Paeonia Lactiflora, the Anemarrhena Asphodeloides, and the Glycyrrhiza 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 Ramulus Cinnamomi, the Heishunpian, the Rhizoma Zingiberis, the Saposhnikovia Divaricata, the Ephedra, the Atractylodes Macrocephala, the Paeonia Lactiflora, the Anemarrhena Asphodeloides, and the Glycyrrhiza is about 4:about 2:about 5:about 4:about 2:about 5:about 3:about 4:about 2. In the present application, "about" refers to a value that is within ±5% of a specified 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 of the Ramulus Cinnamomi is in the range of 40-70 g, for example about 55.20 g. In the present application, "about" refers to a value that is within ±5% of a specified 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 of the Heishunpian is in the range of 20-40 g, for example about 27.60 g. In the present application, "about" refers to a value that is within ±5% of a specified value. For example, "about 27.60" includes ±5% of 27.60, or from 26.22 to 28.98.

[0062] In a preferred embodiment, the mass of the Rhizoma Zingiberis is in the range of 50-90 g, for example about 69.00 g. In the present application, "about" refers to a value that is within ±5% of a specified 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 Saposhnikovia Divaricata is in the range of 40-70 g, for example about 55.20 g. In the present application, "about" refers to a value that is within ±5% of a specified 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 of the Ephedrae is in the range of 20-40 g, for example about 27.60 g. In the present application, "about" means a value in the range of ±5% of a specified 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 of the Atractylodis is in the range of 50-90 g, for example about 69.00 g. In the present application, "about" means a value in the range of ±5% of a specified 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 of the Radix Paeoniae Alba is in the range of 30-50 g, for example about 41.40 g. In the present application, "about" means a value in the range of ±5% of a specified 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 of the Anemarrhena is in the range of 40-70 g, for example about 55.20 g. In the present application, "about" means a value in the range of ±5% of a specified 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 of the Glycyrrhizae is in the range of 20-40 g, for example about 27.60 g. In the present application, "about" means a value in the range of ±5% of a specified 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 Morindin, the Paeonoside, the Cimifugin, the 5-O-Methylvisamminol, and the Glycyrrhizinic Acid is in the range of 200-300 nm, for example about 280 nm. In the present application, "about" means a value in the range of ±5% of a specified 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 in the range of 200-300 nm, for example about 254 nm. In the present application, "about" means a value in the range of ±5% of a specified 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 in the range of 15-25 μl, for example about 20 μl. In the present application, "about" means a value in the range of ±5% of a specified 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 corresponding chromatographic peak of paeoniflorin is not less than 2000. In a preferred embodiment, the chromatographic column is HALOAQ-C 18 chromatographic column or YMC Meteoric Core C 18 chromatographic column. In a preferred embodiment, the specification of the chromatographic column is: column length 150 mm, inner diameter 4.6 mm, particle size 2.7 μm.

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

[0074] In a preferred embodiment, the aqueous acid solution is an aqueous phosphoric acid solution at about 0.1%. In the present application, "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 aqueous buffer salt solution is an aqueous acetic acid salt solution and / or an aqueous acetic acid salt solution. In a preferred embodiment, the pH value of the aqueous buffer salt solution is not more than 7.0.

[0076] In a preferred embodiment, the detection limit of mangiferin is about 13.63 ng / ml. In the present application, "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 application, "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 limit of detection for Cimifugin is about 43.55 ng / ml. In the present invention, "about" means a value that is within ±5% of a specified value. For example, "about 43.55" includes ±5% of 43.55, or from 41.3725 to 45.7275.

[0079] In a preferred embodiment, the limit of detection for 5-O-Methylvisamminol is about 46.08 ng / ml. In the present invention, "about" means a value that is within ±5% of a specified value. For example, "about 46.08" includes ±5% of 46.08, or from 43.776 to 48.384.

[0080] In a preferred embodiment, the limit of detection for Cinnamaldehyde is about 61.64 ng / ml. In the present invention, "about" means a value that is within ±5% of a specified value. For example, "about 61.64" includes ±5% of 61.64, or from 58.558 to 64.722.

[0081] In a preferred embodiment, the limit of detection for Glycyrrhizic Acid is about 38.75 ng / ml. In the present invention, "about" means a value that is within ±5% of a specified value. For example, "about 38.75" includes ±5% of 38.75, or from 36.8125 to 40.6875.

[0082] In a preferred embodiment, the limit of detection for 6-Gingerol is about 0.6475 μg / ml. In the present invention, "about" means a value that is within ±5% of a specified 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 quantitation for Mangiferin is about 45.44 ng / ml. In the present invention, "about" means a value that is within ±5% of a specified 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 quantitation for Paeoniflorin is about 4.88 μg / ml. In the present invention, "about" means a value that is within ±5% of a specified 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 quantitation for Cimifugin is about 0.2178 μg / ml. In the present invention, "about" means a value that is within ±5% of a specified 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 quantitation for the 5-O-methylvisamminol glycoside is about 92.16 ng / ml. In the present application, "about" means a value that is within ±5% of a specified 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 quantitation for the cinnamaldehyde is about 0.12328 μg / ml. In the present application, "about" means a value that is within ±5% of a specified 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 quantitation for the glycyrrhizic acid is about 0.2422 μg / ml. In the present application, "about" means a value that is within ±5% of a specified 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 quantitation for the 6-gingerol is about 1.295 μg / ml. In the present application, "about" means a value that is within ±5% of a specified 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 application, there is provided a method for constructing a fingerprint of a traditional Chinese medicine composition comprising Ramulus Cinnamomi, the method comprising the following steps: preparing a test solution of the traditional Chinese medicine composition: taking a proper amount of a reference sample of the traditional Chinese medicine composition comprising Ramulus Cinnamomi, placing it in a container, adding a solvent, shaking it well, filtering it, and obtaining the test solution of the traditional Chinese medicine composition; wherein the traditional Chinese medicine composition comprises Ramulus Cinnamomi, Radix Paeoniae Alba, Glycyrrhiza Uralensis Fisch, Ephedra Sinica Stapf, Zingiber Officinale Roscoe, Atractylodes Macrocephala Koidz, Anemarrhena Asphodeloides Bunge, Saposhnikovia Divaricata and Black Antlered Deer Antler; preparing a control solution: taking a proper amount of control samples of mangiferin, paeoniflorin, cimifugin, 5-O-methylvisamminol, cinnamyl aldehyde, ammonium glycyrrhizate and 6-shogaol, adding methanol to prepare the control solution with the concentrations of mangiferin, paeoniflorin, cimifugin, 5-O-methylvisamminol, cinnamic acid, cinnamyl aldehyde, ammonium glycyrrhizate and 6-shogaol being 1-200 μg / ml respectively; obtaining the fingerprint of the traditional Chinese medicine composition according to the results of detecting the test solution of the traditional Chinese medicine composition and the control solution by high performance liquid chromatography; the chromatographic conditions of the high performance liquid chromatography detection are as follows: using a chromatographic column with octadecylsilane bonded silica gel as the filler, mobile phase A being selected from one or more of acetonitrile, methanol and tetrahydrofuran, mobile phase B being acid aqueous solution, alkali aqueous solution and / or buffer salt aqueous solution, the gradient elution program being: 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 being 0.6 ml / min, the column temperature being 27-30 °C, the detection wavelength being 180-360 nm, and the injection volume being 10-30 μl.

[0091] In a preferred embodiment, the method for preparing the reference sample of the traditional Chinese medicine composition comprising Ramulus Cinnamomi comprises: taking a proper amount of Ramulus Cinnamomi, Radix Paeoniae Alba, Glycyrrhiza Uralensis Fisch, Ephedra Sinica Stapf, Zingiber Officinale Roscoe, Atractylodes Macrocephala Koidz, Anemarrhena Asphodeloides Bunge, Saposhnikovia Divaricata and Black Antlered Deer Antler, placing them in a container, adding water to soak them, boiling them with strong fire, simmering them for a period of time with weak fire, filtering them, collecting the filtrate, and obtaining the reference sample of the traditional Chinese medicine composition comprising Ramulus Cinnamomi.

[0092] In a preferred embodiment, the volume of water is 1000-2000 ml, for example about 1400 ml. In the present application, "about" means a value within a range of ±5% of a particular 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 the Ramulus Cinnamomi, Radix Paeoniae Alba, Radix Glycyrrhizae, Herba Ephedrae, Rhizoma Zingiberis Recens, Radix Poriae, Radix Anemarrhenae, Radix Saposhnikoviae, and Black Gomishi 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" means a value that is within ±5% of a specified value. For example, "about 0.31" includes ±5% of 0.31, or from 0.2945 to 0.3255.

[0094] In a preferred embodiment, the time of the soaking is 30 to 60 min, for example about 45 min. In the present invention, "about" means a value that is within ±5% of a specified value. For example, "about 45" includes ±5% of 45, or from 42.75 to 47.25.

[0095] In a preferred embodiment, the time of the slow fire decocting is 30 to 90 min, for example about 60 min. In the present invention, "about" means a value that is within ±5% of a specified value. For example, "about 60" includes ±5% of 60, or from 57 to 63.

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

[0097] In a preferred embodiment, the ratio of the mass between the Ramulus Cinnamomi, the Black Gomishi, the Rhizoma Zingiberis Recens, the Radix Saposhnikoviae, the Herba Ephedrae, the Radix Poriae, the Radix Paeoniae Alba, the Radix Anemarrhenae, and the Radix Glycyrrhizae is (2 to 6):(1 to 3):(3 to 7):(2 to 6):(1 to 3):(3 to 7):(1 to 5):(2 to 6):(1 to 3). In a preferred embodiment, the ratio of the mass between the Ramulus Cinnamomi, the Black Gomishi, the Rhizoma Zingiberis Recens, the Radix Saposhnikoviae, the Herba Ephedrae, the Radix Poriae, the Radix Paeoniae Alba, the Radix Anemarrhenae, and the Radix Glycyrrhizae is (3 to 5):(1.5 to 2.5):(4 to 6):(3 to 5):(1.5 to 2.5):(4 to 6):(2 to 4):(3 to 5):(1.5 to 2.5). In a preferred embodiment, the ratio of the mass between the Ramulus Cinnamomi, the Black Gomishi, the Rhizoma Zingiberis Recens, the Radix Saposhnikoviae, the Herba Ephedrae, the Radix Poriae, the Radix Paeoniae Alba, the Radix Anemarrhenae, and the Radix Glycyrrhizae is about 4:about 2:about 5:about 4:about 2:about 5:about 3:about 4:about 2. In the present invention, "about" means a value that is within ±5% of a specified 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 quantity of the Radix Paeoniae Alba is in the range of 30-50 g, for example about 41.40 g. In the present application, "about" means a value in the range of ±5% of a specified value. For example, "about 41.40" includes ±5% of 41.40, or from 39.33 to 43.47.

[0099] In a preferred embodiment, the quantity of the Radix Paeoniae Alba is in the range of 30-50 g, for example about 41.40 g. In the present application, "about" means a value in the range of ±5% of a specified value. For example, "about 41.40" includes ±5% of 41.40, or from 39.33 to 43.47.

[0100] In a preferred embodiment, the quantity of the Radix Paeoniae Alba is in the range of 30-50 g, for example about 41.40 g. In the present application, "about" means a value in the range of ±5% of a specified value. For example, "about 41.40" includes ±5% of 41.40, or from 39.33 to 43.47.

[0101] In a preferred embodiment, the quantity of the Radix Paeoniae Alba is in the range of 30-50 g, for example about 41.40 g. In the present application, "about" means a value in the range of ±5% of a specified value. For example, "about 41.40" includes ±5% of 41.40, or from 39.33 to 43.47.

[0102] In a preferred embodiment, the quantity of the Radix Paeoniae Alba is in the range of 30-50 g, for example about 41.40 g. In the present application, "about" means a value in the range of ±5% of a specified value. For example, "about 41.40" includes ±5% of 41.40, or from 39.33 to 43.47.

[0103] In a preferred embodiment, the quantity of the Radix Paeoniae Alba is in the range of 30-50 g, for example about 41.40 g. In the present application, "about" means a value in the range of ±5% of a specified value. For example, "about 41.40" includes ±5% of 41.40, or from 39.33 to 43.47.

[0104] In a preferred embodiment, the quantity of the Radix Paeoniae Alba is in the range of 30-50 g, for example about 41.40 g. In the present application, "about" means a value in the range of ±5% of a specified value. For example, "about 41.40" includes ±5% of 41.40, or from 39.33 to 43.47.

[0105] In a preferred embodiment, the quantity of the Radix Paeoniae Alba is in the range of 30-50 g, for example about 41.40 g. In the present application, "about" means a value in the range of ±5% of a specified value. For example, "about 41.40" includes ±5% of 41.40, or from 39.33 to 43.47.

[0106] In a preferred embodiment, the mass of the liquorice is in the range of 20-40 g, for example about 27.60 g. In the present application, "about" means a value in the range of ±5% of a specified 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 of preparing the reference sample of the Chinese medicine composition comprising cassia twig, the container is a volumetric flask. In a preferred embodiment, the solvent is methanol.

[0108] In a preferred embodiment, the ratio of the volume of the reference sample of the Chinese medicine composition to the volume of the solvent is in the range of 0.1-0.5, for example about 0.2. In the present application, "about" means a value in the range of ±5% of a specified 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 reference sample of the Chinese medicine composition is in the range of 1-5 ml, for example about 2 ml. In the present application, "about" means a value in the range of ±5% of a specified value. For example, "about 2" includes ±5% of 2, or from 1.9 to 2.1.

[0110] In a preferred embodiment, in the method of preparing the reference sample of the Chinese medicine composition comprising cassia twig, the volume percentage concentration of the methanol is in the range of 40%-60%, for example about 50%. In the present application, "about" means a value in the range of ±5% of a specified 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 application, "about" means a value in the range of ±5% of a specified 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 application, "about" means a value in the range of ±5% of a specified value. For example, "about 160" includes ±5% of 160, or from 152 to 168.

[0113] In a preferred embodiment, the concentration of epimedusin in the reference solution is about 40 μg / ml. In the present application, "about" means a value in the range of ±5% of a specified value. For example, "about 40" includes ±5% of 40, or from 38 to 42.

[0114] In a preferred embodiment, the concentration of 5-O-methylvisamminol in the control solution is about 40 μg / ml. In the present invention, "about" means a value within ±5% of a specified 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 control solution is about 10 μg / ml. In the present invention, "about" means a value within ±5% of a specified value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0116] In a preferred embodiment, the concentration of cinnamic acid in the control solution is about 10 μg / ml. In the present invention, "about" means a value within ±5% of a specified 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 glycyrrhizate in the control solution is about 50 μg / ml. In the present invention, "about" means a value within ±5% of a specified 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 control solution is about 10 μg / ml. In the present invention, "about" means a value within ±5% of a specified 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" means a value within ±5% of a specified 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 15-25 μl, for example about 20 μl. In the present invention, "about" means a value within ±5% of a specified 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 paeoniflorin is not less than 2000. In a preferred embodiment, the chromatographic column is HALO AQ-C 18 chromatographic column 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, and particle size 2.7 μm.

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

[0123] In a preferred embodiment, the aqueous acid solution is an aqueous phosphoric acid solution at about 0.1%. In the present application, "about" means a value within a range of ±5% of a particular value. For example, "about 0.1%" includes ±5% of 0.1%, or from 0.095% to 0.105%.

[0124] In a preferred embodiment, the aqueous buffer salt solution is an aqueous acetate salt solution and / or an aqueous acetate salt solution. In a preferred embodiment, the aqueous buffer salt solution has a pH value of no more than 7.0.

[0125] In a preferred embodiment, the fingerprint spectrum comprises peaks No. 1-13 at the detection wavelength of 254 nm, the retention time of the peaks No. 1-13 are 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, about 70.914 min, respectively. In the present application, "about" means a value within ±5% of a specified 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 15.533, or from 14.75635 to 16.30965; "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 49.36485 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, the fingerprint spectrum comprises peaks No. 1-13 at the detection wavelength of 254 nm, wherein the peak No. 7, 5-O-methylvisamminol glycoside, is taken as a reference peak, and the relative retention time average values of the peak No. 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 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, about 2.10, respectively. In the present application, "about" refers to a value within a range of ±5% of a particular 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 0.8295; "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, the peak No. 2 is mangiferin, the peak No. 4 is paeoniflorin, the peak No. 5 is epimedium glycoside, the peak No. 6 is glycyrrhizin, the peak No. 7 is 5-O-methylvisamminol glycoside, the peak No. 9 is cinnamic acid, the peak No. 11 is cinnamic aldehyde, and the peak No. 13 is glycyrrhizic acid at the detection wavelength of 254 nm. In a preferred embodiment, the peak No. 1 comes from Anemarrhena asphodeloides medicinal material, the peak No. 2 comes from Anemarrhena asphodeloides medicinal material, the peak No. 3 comes from Anemarrhena asphodeloides medicinal material, the peak No. 4 comes from Paeonia lactiflora medicinal material, the peak No. 5 comes from Saposhnikovia divaricata medicinal material, the peak No. 6 comes from Glycyrrhiza uralensis medicinal material, the peak No. 7 comes from Saposhnikovia divaricata medicinal material, the peak No. 8 comes from Glycyrrhiza uralensis medicinal material, the peak No. 9 comes from Cinnamomum cassia medicinal material, the peak No. 10 comes from Saposhnikovia divaricata medicinal material, the peak No. 11 comes from Cinnamomum cassia medicinal material, the peak No. 12 comes from Glycyrrhiza uralensis medicinal material, and the peak No. 13 comes from Glycyrrhiza uralensis medicinal material.

[0128] According to another aspect of the present application, there is provided a quality control method of a traditional Chinese medicine composition comprising Ramulus Cinnamomi, which comprises 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) detecting a test sample solution of the traditional Chinese medicine composition according to the chromatographic conditions in the above-mentioned fingerprint construction method to obtain a fingerprint of the traditional Chinese medicine composition to be tested; and (3) comparing the fingerprint of the traditional Chinese medicine composition to be tested obtained in step (2) with the standard fingerprint of the reference sample of the traditional Chinese medicine composition obtained in step (1), and if the comparison is satisfactory, the product is qualified, otherwise, the product is unqualified.

[0129] In a preferred embodiment, the satisfactory comparison comprises one or more of the following: (1) the fingerprint of the traditional Chinese medicine composition 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 control chromatographic peak in the standard fingerprint of the reference sample of the traditional Chinese medicine composition; (2) taking the peak of 5-O-methylvisamminol as the S peak, the relative retention time of each characteristic chromatographic peak in the fingerprint of the traditional Chinese medicine composition to be tested is within ±10% of the relative retention time value of each characteristic chromatographic peak in the standard fingerprint of the reference sample of the traditional Chinese medicine composition; and (3) according to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint, the similarity of the fingerprint of the traditional Chinese medicine composition to be tested with the standard fingerprint of the reference sample of the traditional Chinese medicine composition is not less than 0.90.

[0130] According to another aspect of the present application, there is provided a use of the above-mentioned determination method or the above-mentioned construction method or the above-mentioned quality control method in the quality detection and / or quality evaluation and / or quality control of a traditional Chinese medicine composition comprising Ramulus Cinnamomi.

[0131] The present application is further described in conjunction with the following specific examples. It should be understood that these examples are only used to illustrate the present application and not intended to limit the scope of the present application. The experimental methods in the following examples, if not otherwise specified, are generally carried out according to the conventional conditions or the conditions recommended by the manufacturers.

[0132] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. In addition, any method and material similar or equivalent to those described herein can be used in the practice of the present application. The preferred methods and materials described herein are illustrative only and not intended to be limiting.

[0133] The features mentioned above in this invention, or the features mentioned in the embodiments, can be combined arbitrarily. All features disclosed in this patent specification can be used in any compositional form, and each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0134] Instruments and reagents

[0135] 1. Instruments

[0136] Decoction pot (model: 2.5L, manufacturer: Kangya Shun Electric Co., Ltd.); Liquid heater (model: 30B2, manufacturer: Kangya Shun Electric Co., Ltd.); Electronic balance (specification: 0.0001%, model: MS105DU, manufacturer: Mettler); Electronic balance (specification: 0.0001%, model: BCE124I-1CCN, manufacturer: Sartorius Scientific Instruments Co., Ltd.); Electronic balance (specification: 0.0001%, model: BCE224-1CCN, manufacturer: Sartorius Scientific Instruments Co., Ltd.); High-performance liquid chromatograph (model: Waters e2695-2998, manufacturer: Waters Technologies Inc.); High-performance liquid chromatograph (model: Agilent 1100-DAD, manufacturer: Agilent Technologies Inc.); High-performance liquid chromatography column (model: HLAO AQ-C). 18 4.6mm × 150mm, 2.7μm, serial number: USAQT001492, manufacturer: Spectrum Technology Co., Ltd.; High-performance liquid chromatography column (model: YMCMeteoric Core C 18 4.6mm×150mm, 2.7μm, serial number: MC004488, manufacturer: YMC / Weimei Company); High performance liquid chromatography column (model: HLAO AQ-C) 18 4.6mm×150mm, 2.7μm, serial number: USAQT001472, manufacturer: Spectrum 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 Science and Technology Co., Ltd.); acetonitrile (grade: HPLC, batch number: WXBF1104V, manufacturer: Sigma-Aldrich); phosphoric acid (grade: HPLC, batch number: 20230110, manufacturer: Komiyue); methanol (grade: HPLC, batch number: WXBF1223V / WXBF1598V, manufacturer: Sigma-Aldrich); anhydrous ethanol (grade: analytical pure, batch number: 20230719, manufacturer: National Pharmaceutical Group Chemical Reagent Co., Ltd.).

[0140] 2.2, Reference substance

[0141] Paeoniflorin reference substance (batch number: 110736-202246, China National Institute for Food and Drug Control, content 96.7%); Mangiferin reference substance (batch number: 111607-202305, China National Institute for Food and Drug Control, content 98.3%); Cimifugin reference substance (batch number: 111522-202214, China National Institute for Food and Drug Control, content 95.7%); Glycyrrhizin reference substance (batch number: 111610-202209, China National Institute for Food and Drug Control, content 95.2%); 5-O-methylvisamminol reference substance (batch number: 111523-202212, China National Institute for Food and Drug Control, content 97.8%); Cinnamic acid reference substance (batch number: 110786-202305, China National Institute for Food and Drug Control, content 99.8%); Cinnamaldehyde reference substance (batch number: 110710-202223, China National Institute for Food and Drug Control, content 98.8%); Ammonium glycyrrhizinate reference substance (batch number: 110731-202122, China National Institute for Food and Drug Control, content 94.4%); 6-Gingerol reference substance (batch number: 111833-202007, China National Institute for Food and Drug Control, content 99.3%).

[0142] 2.3, Information of decoction pieces

[0143] Ramulus cinnamomi (batch number: ZA00922082505, origin: Deqing County, Zhaoqing City, Guangdong Province, source: self-made by Ynneng); Ramulus cinnamomi (batch number: ZA00923070704, origin: Rong County, Yulin City, Guangxi Zhuang Autonomous Region, source: self-made by Ynneng); Radix paeoniae alba (batch number: ZA00121031813, origin: Lin'an County, Hangzhou City, Zhejiang Province, source: self-made by Ynneng); Radix paeoniae alba (batch number: ZA00123070503, origin: Qiaocheng District, Bozhou City, Anhui Province, source: self-made by Ynneng); Rhizoma atractylodis (batch number: ZA04921031805, origin: Xianju County, Taizhou City, Zhejiang Province, source: self-made by Ynneng); Rhizoma atractylodis (batch number: ZA04923070707, origin: Shexian County, Huangshan City, Anhui Province, source: self-made by Ynneng); Rhizoma anemarrhenae (batch number: ZA08021122901, origin: Qiaocheng District, Bozhou City, Anhui Province, source: self-made by Ynneng); Rhizoma anemarrhenae (batch number: ZA08023070704, origin: Anguo City, Baoding City, Hebei Province, source: self-made by Ynneng); Colla corii asini (batch number: ZA17322082301, origin: Jiangyou City, Mianyang City, Sichuan Province, source: self-made by Ynneng); Colla corii asini (batch number: ZA17323071001, origin: Jiangyou City, Mianyang City, Sichuan Province, source: self-made by Ynneng); Radix glycyrrhizae (batch number: ZA00421031608, origin: Yumen County, Jiuquan City, Gansu Province, source: self-made by Ynneng); Radix glycyrrhizae (batch number: ZA00423071001, origin: Guazhou County, Jiuquan City, Gansu Province, source: self-made by Ynneng); Radix saposhnikoviae (batch number: ZA03921090101, origin: Zhalute Banner, Tongliao City, Inner Mongolia Autonomous Region, source: self-made by Ynneng); Radix saposhnikoviae (batch number: ZA03923070703, origin: Tailai County, Qiqihar City, Heilongjiang Province, source: self-made by Ynneng); Ephedra sinica (batch number: Z1110101, origin: Gansu Province, source: Hunan Zhensheng Chinese Medicine Co., Ltd.); Ephedra sinica (batch number: 01-21030201, origin: Ar Horqin Banner, Chifeng City, Inner Mongolia Autonomous Region, source: Anhui Runfuryang Pharmaceutical Co., Ltd.); Zingiber officinale (batch number: ZA00523071007, origin: Qianwei County, Leshan City, Sichuan Province, source: self-made by Ynneng); Zingiber officinale (batch number: ZA00523080901, origin: Qianwei County, Leshan City, Sichuan Province, source: self-made by Ynneng); Zingiber officinale (batch number: ZA00523080905, origin: Qianwei County, Leshan City, Sichuan Province, source: self-made by Ynneng); Zingiber officinale (batch number: ZA00523091101, origin: Qianwei County, Leshan City, Sichuan Province, source: self-made by Ynneng).

[0144] 2.4, Preparation of reference samples

[0145] Reference sample: Take cassia 55.20 g, white peony root 41.40 g, licorice 27.60 g, Ephedra 27.60 g, ginger 69.00 g, white atractylodes 69.00 g, Anemarrhena 55.20 g, silvertree 55.20 g and black smooth tablet 27.60 g, put them in 3.0L automatic ceramic medicine pot, add water 1400ml, soak for 45 minutes, heat to boiling with strong fire, keep boiling with weak fire for 60 minutes, filter through 200 mesh nylon filter cloth while hot, collect the filtrate, and get it. Lack of black smooth tablet negative control decoction: lack of black smooth tablet in the prescription, prepare according to the preparation method of reference sample, and get it. Black smooth tablet single decoction: take black smooth tablet 27.60 g, and prepare according to the preparation method of reference sample, and get it.

[0146] 2.5, decoction information

[0147] Lack of cassia twig negative control decoction (batch number: GZSYZMKLST-2023112001-L); Lack of white peony root negative control decoction (batch number: GZSYZMKLST-2023112002-L); Lack of licorice negative control decoction (batch number: GZSYZMKLST-2023112003-L); Lack of raw ginger negative control decoction (batch number: GZSYZMKLST-2023112004-L); Lack of atractylodes negative control decoction (batch number: GZSYZMKLST-2023112005-L); Lack of know mother negative control decoction (batch number: GZSYZMKLST-2023112006-L); Lack of windproof negative control decoction (batch number: GZSYZMKLST-2023112007-L); Lack of know mother negative control decoction (batch number: GZSYZMKLST-2023112801-L); Cassia twig single decoction (batch number: GZSYZMKLST-2023112008-L); White peony root single decoction (batch number: GZSYZMKLST-2023112009-L); Licorice single decoction (batch number: GZSYZMKLST-2023112010-L); Raw ginger single decoction (batch number: GZSYZMKLST-2023112101-L); Atractylodes single decoction (batch number: GZSYZMKLST-2023112102-L); Know mother single decoction (batch number: GZSYZMKLST-2023112103-L); Windproof single decoction (batch number: GZSYZMKLST-2023112104-L); Reference sample test solution (batch number: GZSYZMKLST-2023112105-L); Reference sample test solution (batch number: GZSYZMKLST-2024010701-L); Reference sample test solution (batch number: GZSYZMKLST-20230912-L); Reference sample test solution (batch number: GZSYZMKLST-20231101-L); Reference sample test solution (batch number: GZSYZMKLST-2023111001-L).

[0148] Example one content determination

[0149] 1. Determination of chromatographic conditions

[0150] 1.1. Investigation of elution gradient

[0151] The following method was used for preliminary trials:

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

[0153] Table 1 Chromatographic condition optimization gradient table

[0154] Time (min) 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] From the analysis of each identified peak under this chromatographic condition, the overall chromatographic behavior was acceptable, but the separation effect of some index components such as paeoniflorin and 5-O-methylvisamminol glycoside around 15 min still had certain optimization space, and the 6-gingerol chromatographic peak response was not good, so the chromatographic condition needed to be further optimized.

[0156] Further optimization was initially tried by using the following method:

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

[0158] Table 2 Chromatographic condition optimization gradient table

[0159] Time (min) 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 this chromatographic condition, the overall chromatographic behavior was acceptable, but the separation effect of some index components such as glycyrrhizin around 26 min was not obviously improved, and the separation effect of some index components such as cimifugin and cinnamic acid around 17 min and 45 min was not obviously improved, and tailing phenomenon was found, so the chromatographic condition was re-optimized.

[0161] Further optimization was initially tried by using the following method:

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

[0163] Table 3 Chromatographic condition optimization gradient table

[0164] Time (min) 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 was well separated, but the response of some marker components such as 6-gingerol was weak near 70 min. The overall response of the 6-gingerol chromatographic peak at 280 nm was improved, so the wavelength was switched at 280 nm to improve the response of 6-gingerol. In order to ensure the stability of the chromatographic system, the elution time was extended to 85 min from 80 min, and the chromatographic conditions were further optimized.

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

[0167] Table 4 Final gradient table of chromatographic conditions

[0168] Time (min) 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 each marker component in the reference sample of the traditional Chinese medicine composition containing Ramulus Cinnamomi according to the present application (254 nm)

[0170] Name Peak area Resolution Symmetry factor Theoretical plates Mangiferin 30781329 - 1.92 40356 Paeoniflorin 3014205 6.74 1.59 45591 Epimedoside 8726497 3.83 1.15 82138 5-O-methylvisamminol glycoside 12271936 18.69 1.15 261390 Cinnamaldehyde 2220704 5.47 0.98 108013 Glycyrrhizic acid 5410450 38.61 1.04 2529293

[0171] The results show that under this elution gradient condition, the chromatographic behavior of each chromatographic peak is good, and the separation degree is better, so that subsequent research can 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 determine the ultraviolet absorption curve of the reference solution of mangiferin, paeoniflorin, cimifugin, 5-O-methylvisamminol, cinnamaldehyde, glycyrrhizic acid, and 6-gingerol.

[0174] It was found that the response of mangiferin, paeoniflorin, cimifugin, 5-O-methylvisamminol, and glycyrrhizic acid chromatographic peaks was better at 254 nm wavelength, which was suitable for quantitative analysis; while the response of cinnamaldehyde chromatographic peak was better at 280 nm wavelength, and 6-gingerol could be monitored at 280 nm. Therefore, the detection wavelength for the determination of mangiferin, paeoniflorin, cimifugin, glycyrrhizin, 5-O-methylvisamminol, and glycyrrhizic acid was determined to be 254 nm, and the detection wavelength for the determination of cinnamaldehyde and 6-gingerol was determined to be 280 nm.

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

[0176] 2.1. Selection of different solvents

[0177] Because the compound decoction sample containing cinnamon twig of this invention is relatively concentrated and the content of each component in the compound is high, direct injection may result in a large sample loading on the chromatographic column, potentially leading to the loss of some component information or unstable chromatographic conditions. To ensure quantitative analysis without losing the decoction information, this study selected solvents such as pure water, methanol, ethanol, and acetonitrile for quantitative dilution of the compound decoction for comparison to determine the appropriate solvent for the test sample.

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

[0179] The results show that ethanol and acetonitrile have strong elution capabilities, resulting in significant solvent effects on the chromatographic peaks of the test solution within 30 minutes. This leads to poor peak shapes for mangiferin, paeoniflorin, and other chromatographic peaks, making them unsuitable for quantitative analysis. Methanol and pure water dilution resulted in better chromatographic behavior for each indicator component, with smaller differences in peak area. However, methanol has a certain purification effect on macromolecular components, which can effectively reduce the sample loading of the chromatographic column, improve the analytical efficiency of the chromatographic column, and extend the 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 factors

[0181] To further optimize the preparation method of the test solution, the methanol dilution ratio was also investigated. 5 ml, 2 ml and 1 ml of the reference sample were placed in 10 ml volumetric flasks, methanol was added to the mark, and the solution was filtered to obtain the final product.

[0182] The results show that the peak shapes of each chromatographic peak were poor before 30 minutes after methanol was diluted 10 times, indicating a certain solvent effect. The chromatographic behavior of each peak was similar after methanol was diluted 2 times and 5 times, but the peak shapes of components such as mangiferin and paeoniflorin were slightly better at 5 times dilution than at 2 times dilution. Considering factors such as experimental operability, methanol was chosen as the preparation method for subsequent experimental samples.

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

[0184] Determined by high performance liquid chromatography (General Chapter 0512, Chinese Pharmacopoeia 2020 Edition).

[0185] Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the stationary phase; acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B, with gradient elution performed according to the specifications in Table 4; the flow rate was 0.6 ml per minute; the column temperature was 30℃; the detection wavelengths for mangiferin, paeoniflorin, cimicifugin, 5-O-methylvisamilol, and glycyrrhizic acid were 254 nm, and the detection wavelengths for cinnamaldehyde and 6-gingerol were 280 nm.

[0186] Preparation of the control solution: Take the reference substance of morindin, the reference substance of paeoniflorin, the reference substance of prim-O-glucosylcimifugin, the reference substance of 5-O-methylvisamminol, the reference substance of cinnamaldehyde, the reference substance of ammonium glycyrrhizinate, and the reference substance of 6-shogaol, accurately weigh and determine the amount, and add 50% methanol to prepare a mixed solution containing 60 μg of morindin, 0.16 mg of paeoniflorin, 40 μg of prim-O-glucosylcimifugin, 40 μg of 5-O-methylvisamminol, 10 μg of cinnamaldehyde, 50 μg of ammonium glycyrrhizinate, and 10 μg of 6-shogaol per 1 ml, and the control solution is obtained.

[0187] Preparation of the test solution: accurately take 2 ml of the product, place it in a 10 ml volumetric flask, add methanol to the mark, shake and filter, and the test solution is obtained.

[0188] Determination method: accurately take 20 μl of the control solution and the test solution respectively, inject into the liquid chromatograph, and determine, and the test solution is obtained.

[0189] 4. Methodology investigation

[0190] 4.1. Specificity test

[0191] Take the reference sample test solution of the traditional Chinese medicine composition containing Ramulus Cinnamomi of the present application, the negative control solutions prepared in the same way without Ramulus Cinnamomi, without Radix Paeoniae Alba, without Radix Glycyrrhizae, without Radix Anemarrhenae, without Radix Saposhnikoviae, and without Rhizoma Zingiberis Recens, and single decoction and blank solvent (50% methanol), investigate whether the negative control solution will cause interference, perform HPLC analysis according to the proposed chromatographic conditions, and record the chromatogram.

[0192] The results show that the UV absorption wavelength of each identified component peak is consistent, the negative control and the blank solution have no interference, and the specificity is good; the separation degrees of morindin, paeoniflorin, prim-O-glucosylcimifugin, 5-O-methylvisamminol, cinnamaldehyde, glycyrrhizic acid, and 6-shogaol in the test solution are 12.31, 3.72, 1.70, 17.94, 43.92, 5.29, and 17.24 respectively, which meets the requirements of high performance liquid chromatography content determination; and it is shown that morindin in the test solution is derived from the Radix Anemarrhenae medicinal flavor in the reference sample of the traditional Chinese medicine composition containing Ramulus Cinnamomi of the present application, paeoniflorin is derived from the Radix Paeoniae Alba medicinal flavor, prim-O-glucosylcimifugin and 5-O-methylvisamminol are derived from the Radix Saposhnikoviae medicinal flavor, glycyrrhizic acid is derived from the Radix Glycyrrhizae medicinal flavor, cinnamaldehyde is derived from the Ramulus Cinnamomi medicinal flavor, and 6-shogaol is derived from the Rhizoma Zingiberis Recens medicinal flavor.

[0193] 4.2. Precision test

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

[0195] Take three different concentrations of control solution, according to the above determination of chromatographic conditions, continuous injection 6 times, determination of mango glycoside, paeonol glycoside, cimicifugoside, 5-O-methyl visamminol glycoside, cinnamyl aldehyde, glycyrrhizic acid, 6-gingerol chromatographic peak area and calculate RSD. The first needle of three different concentrations of control solution continuous injection 6 needles as the first day of determination value, according to the chromatographic conditions, in the second day, the third day, each injection 1 times, determination of mango glycoside, paeonol glycoside, cimicifugoside, 5-O-methyl visamminol glycoside, cinnamyl aldehyde, glycyrrhizic acid, 6-gingerol chromatographic peak peak area and calculate RSD.

[0196] The results showed that the intra-day precision and inter-day precision of mango glycoside, paeonol glycoside, cimicifugoside, 5-O-methyl visamminol glycoside, cinnamyl aldehyde, glycyrrhizic acid, 6-gingerol were less than 2.0%, indicating that the intra-day precision and inter-day precision were good.

[0197] 4.2.2, repeatability test

[0198] Take the same batch of the invention of traditional Chinese medicine composition containing cassia reference sample (batch number: GZSYZMKLST-2024010701-L), according to the proposed test solution preparation method parallel preparation of 6, according to the proposed chromatographic conditions determination of mango glycoside, paeonol glycoside, cimicifugoside, 5-O-methyl visamminol glycoside, cinnamyl aldehyde, glycyrrhizic acid and 6-gingerol content and calculate SD and RSD.

[0199] The results showed that the SD of the content of mango glycoside, paeonol glycoside, cimicifugoside, 5-O-methyl visamminol glycoside, cinnamyl aldehyde, glycyrrhizic acid and 6-gingerol in the 6 repeated test solutions were 0.2, 0.1, 0.1, 0.2, 0.1, 0.4 and 0.1 respectively, and the RSD of the content of mango glycoside, paeonol glycoside, cimicifugoside, 5-O-methyl visamminol glycoside, cinnamyl aldehyde, glycyrrhizic acid and 6-gingerol was not more than 4.0%, indicating that the repeatability of the method was good.

[0200] 4.2.3, intermediate precision test

[0201] Take the same batch of the invention of traditional Chinese medicine composition containing cassia reference sample (batch number: GZSYZMKLST-2024010701-L), according to the proposed test solution preparation method preparation sample, respectively by A, B two experimenters in different instruments independent operation, A experimenters use Waters e2695-2998 high performance liquid chromatograph, B experimenters use Agilent 1100-DAD high performance liquid chromatograph, in different time, different laboratory according to the proposed chromatographic conditions determination of mango glycoside, paeonol glycoside, cimicifugoside, 5-O-methyl visamminol glycoside, cinnamyl aldehyde, glycyrrhizic acid, 6-gingerol content and calculate SD and RSD.

[0202] The results showed that the SD of the contents of mangiferin, paeoniflorin, epimedoside, 5-O-methylvisamminol, 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 RSD of the contents of mangiferin, paeoniflorin, epimedoside, 5-O-methylvisamminol, cinnamaldehyde, glycyrrhizic acid and 6-gingerol were not more than 4.0%, indicating that the intermediate precision of the method was good.

[0203] 4.3, Detection limit test

[0204] According to the detection limit in the General Principles of Analysis Method Validation of Chinese Pharmacopoeia 2020 Edition, the signal of the known concentration sample was compared with the signal of the blank sample, and the signal-to-noise ratio was 3:1.

[0205] The results showed that the minimum concentrations of mangiferin, paeoniflorin, epimedoside, 5-O-methylvisamminol, 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, Quantitative limit test

[0207] According to the quantitative limit in the General Principles of Analysis Method Validation of Chinese Pharmacopoeia 2020 Edition, the signal of the known concentration sample was compared with the signal of the blank sample, and the signal-to-noise ratio was 10:1.

[0208] The results showed that the minimum concentrations of mangiferin, paeoniflorin, epimedoside, 5-O-methylvisamminol, 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, Linear test

[0210] Accurately weigh a certain amount of mangiferin reference substance, paeonol reference substance, episesamin reference substance, 5-O-methylvisamminol reference substance, cinnamaldehyde reference substance, ammonium glycyrrhizinate reference substance and 6-gingerol reference substance, and add 50% methanol to prepare seven different concentration reference substance mixed solutions containing mangiferin, paeonol, episesamin, 5-O-methylvisamminol, cinnamaldehyde, ammonium glycyrrhizinate and 6-gingerol, respectively. Accurately pipette 20 μl of the reference substance mixed solutions with different concentrations, inject into a liquid chromatograph, measure the peak area, and draw a standard curve with the concentration of mangiferin, paeonol, episesamin, 5-O-methylvisamminol, cinnamaldehyde, ammonium glycyrrhizinate and 6-gingerol as the horizontal coordinate and the peak area as the vertical coordinate, respectively.

[0211] The results show that the linear regression equation of mangiferin is Y = 134,118.1290X + 61,486.8143, the correlation coefficient r is 0.9999, and the residual sum of squares RSS is 36333756483.60, indicating that mangiferin has a good linear relationship in the range of 0.04544 μg / ml to 142 μg / ml; the linear regression equation of paeoniflorin is Y = 3,889.5159X + 11,877.5625, the correlation coefficient r is 0.9999, and the residual sum of squares RSS is 335053024.13, indicating that paeoniflorin has a good linear relationship in the range of 4.88 μg / ml to 488 μg / ml; the linear regression equation of epimedoside 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 epimedoside has a good linear relationship in the range of 0.2178 μg / ml to 272.2 μg / ml; the linear regression equation of 5-O-methylvisamminol is Y = 69,455.1451X + 80,352.1532, the correlation coefficient r is 0.9999, and the residual sum of squares RSS is 40201771117.42, indicating that 5-O-methylvisamminol has a good linear relationship in the range of 0.09216 μg / ml to 288 μg / 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 0.12328 μg / ml to 30.82 μg / ml; the linear regression equation of glycyrrhizic acid is Y = 26,858.1718X + 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 0.2422 μg / ml to 121.0934 μg / 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 1.295 μg / ml to 12.95 μg / ml.

[0212] 4.6. Accuracy test

[0213] That is, the sample recovery test, take the same batch of the invention containing Ramulus cinnamomi traditional Chinese medicine composition reference sample (batch number: GZSYZMKLST-2023112105-L) 9 parts, respectively, 1ml of supernatant is accurately measured in 10ml volumetric flask, high, medium and low three different concentrations are respectively added to the mixed control solution according to the proportion of 1:1.5, 1:1, 1:0.5, add methanol, dilute to the mark, shake well, get, each concentration is prepared 3 test solution for determination. At the same time, 2 test solution is prepared for determination. The content of the test solution is calculated, mangiferin, paeonol, cimifugin, 5-O-methylvisamminol, cinnamaldehyde, glycyrrhizic acid and 6-gingerol.

[0214] The results show that the sample recovery rate of mangiferin in the test solution is between 98.7% and 104.3%, the RSD is 1.9%; the sample recovery rate of paeonol is between 99.7% and 103.4%, the RSD of the recovery rate is 1.1%; the sample recovery rate of cimifugin is between 101.7% and 104.6%, the RSD is 0.9%; the sample recovery rate of 5-O-methylvisamminol is between 100.1% and 105.4%, the RSD is 1.7%; the sample recovery rate of cinnamaldehyde is between 100.3% and 106.4%, the RSD is 1.8%; the sample recovery rate of glycyrrhizic acid is between 98.3% and 104.0%, the RSD is 2.3%; the sample recovery rate of 6-gingerol is between 100.2% and 107.0%, the RSD is 2.4%; according to the requirements of the recovery rate limit in the 2020 edition of Chinese Pharmacopoeia four parts 9101 drug quality standard analysis method verification guide principle, it can be judged that the method has good accuracy.

[0215] 4.7, durability test

[0216] 4.7.1, stability test

[0217] Take the same batch of test solution, according to the proposed chromatographic conditions, respectively, at 0h, 3h, 5h, 10h, 20h, 30h, 48h, 62h, 72h, the peak area of mangiferin, paeonol, cimifugin, 5-O-methylvisamminol, cinnamaldehyde, glycyrrhizic acid and 6-gingerol chromatographic peak is determined to investigate the stability of the solution.

[0218] The results show that under room temperature conditions, within 72 hours, the RSDs of the peak areas of the chromatographic peaks of mangiferin, paeoniflorin, thalidomide glycoside, 5-O-methyl visamminol glycoside, glycyrrhizic acid and 6-gingerol are all less than 2.0%, indicating that the mangiferin, paeoniflorin, thalidomide glycoside, 5-O-methyl visamminol glycoside, glycyrrhizic acid and 6-gingerol in the test solution are basically stable under room temperature conditions within 72 hours, meeting the requirements for content determination. Within 72 hours, the RSD of the peak area of cinnamaldehyde is 5.5%. Because cinnamaldehyde is a volatile component, it is unstable and changes with environmental factors. Within 30 hours, the peak area of cinnamaldehyde is relatively stable, and the RSD of the peak area of cinnamaldehyde is 2.9%. However, after 30 hours, the content of cinnamaldehyde shows a certain downward trend. Therefore, in this method, after the preparation of the test solution is completed, the detection should be carried out as soon as possible within 30 hours to obtain more accurate cinnamaldehyde content.

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

[0220] The same batch of test solution was taken and injected under the proposed chromatographic conditions with the concentration of phosphoric acid solution in the mobile phase being 0.05%, 0.10% and 0.15% respectively, the influence of different acid concentrations on the index components was investigated, the contents of mangiferin, paeoniflorin, thalidomide glycoside, 5-O-methyl visamminol glycoside, cinnamaldehyde, glycyrrhizic acid and 6-gingerol were determined and the SD and RSD were calculated.

[0221] The results show that under the conditions of the concentration of phosphoric acid solution in the mobile phase being 0.05%-0.15%, the SDs of the contents of mangiferin, paeoniflorin, thalidomide glycoside, 5-O-methyl visamminol glycoside, cinnamaldehyde, glycyrrhizic acid and 6-gingerol in the test solution are 0.4, 0.1, 0.3, 0.6, 0.2, 1.0 and 0.1 respectively, the RSDs of the contents of mangiferin, paeoniflorin, thalidomide glycoside, 5-O-methyl visamminol glycoside, cinnamaldehyde, glycyrrhizic acid and 6-gingerol are not more than 4.0%, meeting the requirements of General Test 9101 in Chinese Pharmacopoeia 2020 Edition; it is indicated that the method has good durability (different acid concentrations of mobile phase).

[0222] 4.7.3. Investigation of different flow rates

[0223] The same batch of test solution was taken and injected under the proposed chromatographic conditions with different flow rates of 0.5 ml / min, 0.6 ml / min and 0.7 ml / min respectively, the contents of mangiferin, paeoniflorin, thalidomide glycoside, 5-O-methyl visamminol glycoside, cinnamaldehyde, glycyrrhizic acid and 6-gingerol were determined and the SD and RSD were calculated.

[0224] The results show that under the condition of flow rate 0.5ml / min-0.7ml / min, the SD of the contents of mangiferin, paeoniflorin, thujin, 5-O-methylvisamminol, cinnamaldehyde, glycyrrhizic acid and 6-shogaol in the test solution of the sample is 1.0, 0.1, 0.6, 0.7, 0.4, 0.7 and 0.1 respectively, the RSD of the contents of mangiferin, paeoniflorin, thujin, 5-O-methylvisamminol, cinnamaldehyde, glycyrrhizic acid and 6-shogaol is not more than 4.0%, which meets the requirements of General Test 9101 in Chinese Pharmacopoeia 2020 edition; under the condition of flow rate 0.5ml / min, the content of paeoniflorin in the test solution of the reference sample of the traditional Chinese medicine composition containing Ramulus Cinnamomi of the application is greatly affected by the flow rate, so it is suggested to fix the flow rate at 0.6ml / min.

[0225] 4.7.4, Investigation of different column temperatures

[0226] The same batch of test solution is taken, and the contents of mangiferin, paeoniflorin, thujin, 5-O-methylvisamminol, cinnamaldehyde, glycyrrhizic acid and 6-shogaol are determined and the SD and RSD are calculated under the condition of different column temperatures 27℃, 30℃ and 33℃.

[0227] The results show that under the condition of column temperature 27℃-33℃, the SD of the contents of mangiferin, paeoniflorin, thujin, 5-O-methylvisamminol, cinnamaldehyde, glycyrrhizic acid and 6-shogaol in the test solution is 0.7, 0.1, 0.6, 1.1, 0.4, 0.5 and 0.2 respectively, the RSD of the contents of mangiferin, paeoniflorin, thujin, 5-O-methylvisamminol, cinnamaldehyde and glycyrrhizic acid is not more than 4.0%, which meets the requirements of General Test 9101 in Chinese Pharmacopoeia 2020 edition; under the condition of column temperature 27℃, the content of 6-shogaol in the test solution of the reference sample of the traditional Chinese medicine composition containing Ramulus Cinnamomi of the application is greatly affected by the temperature, so it is suggested to fix the temperature at 30℃.

[0228] 4.7.5, Investigation of different chromatographic columns

[0229] The same batch of test solution is taken, and the different brand and batch of chromatographic columns are investigated under the chromatographic conditions: chromatographic column 1 (HALO AQ-C 18 (4.6mmx150mm, 2.7um, S.N. USA QT001492), chromatographic column 2 (YMC Meteoric Core C 18 (4.6mmx150mm, 2.7um, S.N. MC004488), chromatographic column 3 (HALO AQ-C 18(4.6mm×150mm, 2.7um, S.N. USAQT001472) to determine the contents of mangiferin, paeonol, epimedoside, 5-O-methylvisamminol, cinnamyl aldehyde, glycyrrhizic acid and 6-gingerol and calculate SD and RSD.

[0230] The results show that under the conditions of different brands and different batches of chromatographic columns, the SDs of the contents of mangiferin, paeonol, epimedoside, 5-O-methylvisamminol, cinnamyl aldehyde, glycyrrhizic acid and 6-gingerol in the test solution are 0.2, 0.1, 0.3, 0.3, 0.2, 1.5 and 0.1 respectively, and the RSDs of the contents of mangiferin, paeonol, epimedoside, 5-O-methylvisamminol, cinnamyl aldehyde, glycyrrhizic acid and 6-gingerol are not more than 4.0%, which meets the requirements of General Test 9101 in Chinese Pharmacopoeia 2020 Edition; it is indicated that the method has good durability (different brands and different batches of chromatographic columns).

[0231] 4.8, Verification summary

[0232] Through the above methodological verification tests, it is indicated that the method can accurately, reliably, stably and effectively detect the contents of mangiferin, paeonol, epimedoside, 5-O-methylvisamminol, cinnamyl aldehyde, glycyrrhizic acid and 6-gingerol in the reference sample of the traditional Chinese medicine composition containing Ramulus Cinnamomi of the application.

[0233] 5, Summary

[0234] The multi-index content determination and analysis method of the reference sample of the traditional Chinese medicine composition containing Ramulus Cinnamomi of the application is determined as follows:

[0235] Determination by high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition General Test 0512).

[0236] Chromatographic conditions and system suitability test: octadecylsilane-bonded silica gel is used as the filler; acetonitrile is used as the mobile phase A, and 0.1% phosphoric acid solution is used as the mobile phase B, which is gradient eluted 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 of mangiferin, paeonol, epimedoside, 5-O-methylvisamminol and glycyrrhizic acid is 254 nm, and the detection wavelength of cinnamyl aldehyde and 6-gingerol is 280 nm. The theoretical plate number calculated according to the paeonol peak should not be less than 2000

[0237] Preparation of the control solution: Take the control sample of morindin, the control sample of paeonol, the control sample of thalidomide, the control sample of 5-O-methyl visamminol, the control sample of cinnamyl aldehyde, the control sample of ammonium glycyrrhizinate, and 6- gingerol, accurately weigh and determine, add 50% methanol to prepare a mixed solution containing 60 μg of morindin, 0.16 mg of paeonol, 40 μg of thalidomide, 40 μg of 5-O-methyl visamminol, 10 μg of cinnamyl aldehyde, 50 μg of ammonium glycyrrhizinate, and 10 μg of 6-gingerol per 1 ml.

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

[0239] Determination method: accurately take 20 μl of the control solution and the test solution respectively, inject into the liquid chromatograph, and determine, to obtain the result.

[0240] Example two fingerprint

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

[0242] The same as the determination method of the multi-index content of the traditional Chinese medicine composition containing cassia twig in the present application: take 2 ml of the product, place it in a 10 ml volumetric flask, add methanol to the mark, shake well, and filter, to obtain the test solution.

[0243] 2. Investigation of the elution gradient

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

[0245] 3. Investigation of the detection wavelength

[0246] Referring to the detection wavelength of each medicinal ingredient in the determination method of the content in the Chinese Pharmacopoeia 2020 edition and the standard formula granules, and combining the development results of the determination method, it is found that there is more peak information at the wavelength of 254 nm, and the information of the reference sample components is rich, and in order to ensure convenient operation and economic savings, after comprehensive evaluation, the detection wavelength of the reference sample characteristic spectrum of the traditional Chinese medicine composition containing cassia twig in the present application is determined to be 254 nm.

[0247] 4. Determination of the characteristic peak of the characteristic spectrum

[0248] From the chemical composition analysis of the above-mentioned medicinal ingredients, it can be known that the main indicator components in Guizhi are cinnamic acid and cinnamyl aldehyde, the main indicator component in Baishao is paeoniflorin, the main indicator components in Gancao are glycyrrhizin and glycyrrhizic acid, the main indicator component in Zhimu is mangiferin, and the main indicator components in Fangfeng are episesamin and 5-O-methylvisamminol. In the Traditional Chinese Medicine Dispensing Granules Drug Standard (Guangxi Zhuang Autonomous Region), cinnamyl aldehyde and cinnamic acid in Guizhi are characterized in the characteristic spectrum, and other medicinal ingredients are characterized in the characteristic spectrum by the above-mentioned corresponding indicator components in the national drug standard of Traditional Chinese Medicine Dispensing Granules. Combined with the previous experimental results, it is found that the separation degree of the corresponding indicator components of each medicinal ingredient is good, so the known components mangiferin, paeoniflorin, episesamin, glycyrrhizin, 5-O-methylvisamminol, cinnamic acid, cinnamyl aldehyde and glycyrrhizic acid are used as characteristic peaks in the characteristic spectrum of the reference sample.

[0249] 5. Two times of time investigation

[0250] The research results show that under the chromatographic conditions, the separation of each indicator component chromatographic peak is good, and the proportion of delayed mobile phase is 45:55 for 180 min, and no other significant component information is found for 85 min running time, indicating that each monitoring indicator component of the reference sample is reflected in the effective time spectrum, and the maximum amount of effective information is satisfied within the monitoring time, and the information has been characterized comprehensively.

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

[0252] Determined by high performance liquid chromatography (Chinese Pharmacopoeia 2020 edition general 0512).

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

[0254] Preparation of reference solution: take appropriate amount of mangiferin reference substance, paeoniflorin reference substance, episesamin reference substance, 5-O-methylvisamminol reference substance, cinnamyl aldehyde reference substance, ammonium glycyrrhizinate reference substance, 6- gingerol reference substance, cinnamic acid reference substance, accurately weigh, add 50% methanol to prepare a mixed solution containing 60μg of mangiferin, 0.16mg of paeoniflorin, 40μg of episesamin, 40μg of 5-O-methylvisamminol, 10μg of cinnamyl aldehyde, 50μg of ammonium glycyrrhizinate, 10μg of 6-gingerol and 10μg of cinnamic acid per 1ml, and then obtain.

[0255] Preparation of test solution: accurately take 2ml of the product, put it in a 10ml volumetric flask, add methanol to the mark, shake well, filter, and then obtain.

[0256] Determination: 20 μl of the reference solution and the test solution, respectively, was injected into the liquid chromatograph, and determination was made, and the result was obtained.

[0257] 7. Methodological investigation

[0258] 7.1. Specificity test

[0259] The reference sample test solution of the traditional Chinese medicine composition containing Ramulus Cinnamomi of the present application, the negative control decoction prepared in the same manner without Ramulus Cinnamomi, without Radix Paeoniae Alba, without Radix Glycyrrhizae, without Radix Anemarrhenae and without Radix Saposhnikoviae, and the single-ingredient decoction prepared in the same manner were taken, and it was investigated whether the negative control solution would cause interference. HPLC analysis was performed according to the proposed chromatographic conditions, and the chromatogram was recorded.

[0260] The results showed that the blank solution had no interference. HPLC-DAD was used to scan the chromatographic peaks of the reference sample and the control samples, and the ultraviolet absorption spectra were compared to determine some characteristic peaks, which were peak 2 for mangiferin, peak 4 for paeoniflorin, peak 5 for thalidomide glycoside, peak 6 for glycyrrhizin, peak 7 for 5-O-methyl visamminol glycoside, peak 9 for cinnamic acid, peak 11 for cinnamyl aldehyde, and peak 13 for glycyrrhizic acid. By comparing the test solution of the single-ingredient decoction with the negative control decoction, it was determined that peak 9 (cinnamic acid) and peak 11 (cinnamyl aldehyde) were derived from Ramulus Cinnamomi, peak 4 (paeoniflorin) was derived from Radix Paeoniae Alba, peak 6 (glycyrrhizin), peak 8, peak 12 and peak 13 (glycyrrhizic acid) were derived from Radix Glycyrrhizae, peak 1, peak 2 (mangiferin) and peak 3 were derived from Radix Anemarrhenae, and peak 5 (thalidomide glycoside), peak 7 (5-O-methyl visamminol glycoside) and peak 10 were derived from Radix Saposhnikoviae. This showed that the method had good specificity.

[0261] 7.2. Precision test

[0262] 7.2.1. Instrument precision test

[0263] The test solution of the reference sample (batch number: GZSYZMKLST-2023112105-L) of the traditional Chinese medicine composition containing Ramulus Cinnamomi of the present application was taken, and 6 injections were repeated according to the proposed chromatographic conditions. The relative retention time, relative peak area and RSD of each characteristic peak were determined and calculated, and the similarity of the test characteristic spectrum and the control characteristic spectrum was calculated according to the traditional Chinese medicine chromatographic fingerprint similarity evaluation system. The experimental results are shown in Table 1. Figure 2The results show that the test sample characteristic spectrum presents 13 characteristic peaks corresponding to the control characteristic spectrum, the relative retention time RSD of each characteristic peak to the reference S peak (peak 7) is less than 2.0%; the relative peak area RSD of each characteristic peak to the reference S peak (peak 7) is less than 3.0%; according to the Chinese medicine chromatographic fingerprint similarity evaluation system, the similarity of the test sample characteristic spectrum and the control characteristic spectrum is calculated, and the similarity is above 0.95. It shows that the instrument precision of the method is good, which meets the requirements of the characteristic spectrum.

[0264] 7.2.2, repeatability test

[0265] The same batch of the Chinese medicine composition containing Ramulus cinnamomi reference sample of the application (batch number: GZSYZMKLST-2024010701-L) was taken, 6 test sample solutions were prepared in parallel according to the proposed test sample solution preparation method, the relative retention time, relative peak area and RSD of each characteristic peak were determined and calculated, and the similarity of the test sample characteristic spectrum and the control characteristic spectrum was calculated according to the Chinese medicine chromatographic fingerprint similarity evaluation system.

[0266] The results show that the test sample characteristic spectrum presents 13 characteristic peaks corresponding to the control characteristic spectrum, the relative retention time RSD of each characteristic peak to the reference S peak (peak 7) is less than 2.0%; the relative peak area RSD of each characteristic peak to the reference S peak (peak 7) is less than 2.0%; according to the Chinese medicine chromatographic fingerprint similarity evaluation system, the similarity of the test sample characteristic spectrum and the control characteristic spectrum is calculated, and the similarity is above 0.95. It shows that the repeatability of the method is good, which meets the requirements of the characteristic spectrum.

[0267] 7.2.3, intermediate precision

[0268] The same batch of the Chinese medicine composition containing Ramulus cinnamomi reference sample of the application (batch number: GZSYZMKLST-2024010701-L) was taken, the sample was prepared according to the proposed test sample solution preparation method, and was 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 at different times and in different laboratories according to the proposed chromatographic conditions, and the similarity of the test sample characteristic spectrum and the control characteristic spectrum was calculated according to the Chinese medicine chromatographic fingerprint similarity evaluation system.

[0269] The results show that the test sample characteristic spectrum presents 13 characteristic peaks corresponding to the control characteristic spectrum, the relative retention time RSD of each characteristic peak to the reference S peak (peak 7) is less than 2.0%; according to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint, the similarity of the test sample characteristic spectrum to the control characteristic spectrum is calculated, and the similarity is more than 0.95. It shows that the intermediate precision of the method is good, which meets the requirements of the characteristic spectrum.

[0270] 7.3, Durability

[0271] 7.3.1, Stability test

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

[0273] The results show that the test sample characteristic spectrum presents 13 characteristic peaks corresponding to the control characteristic spectrum, the relative retention time RSD of each characteristic peak to the reference S peak (peak 7) is less than 2.0%; the relative peak area RSD of each characteristic peak to the reference S peak (peak 7) is less than 6.0%, according to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint, the similarity of the test sample characteristic spectrum to the control characteristic spectrum is calculated, and the similarity is more than 0.95. It shows that the stability of the method is good, which meets the requirements of the characteristic spectrum.

[0274] 7.3.2, Investigation of different mobile phase acid concentration

[0275] Take the same batch of test sample solution, and sample at 0h, 3h, 5h, 10h, 20h, 30h, 48h, 62h, 72h respectively under the proposed chromatographic conditions, determine and calculate the relative retention time, relative peak area and RSD of each characteristic peak, and calculate the similarity of the test sample characteristic spectrum to the control characteristic spectrum according to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint.

[0276] The results show that the test sample characteristic spectrum presents 13 characteristic peaks corresponding to the control characteristic spectrum, the relative retention time RSD of each characteristic peak to the reference S peak (peak 7) is less than 2.0%; according to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint, the similarity of the test sample characteristic spectrum to the control characteristic spectrum is calculated, and the similarity is more than 0.95. It shows that the intermediate precision of the method is good, which meets the requirements of the characteristic spectrum.

[0277] 7.3.3, Different flow rate investigation

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

[0279] The results show that, at a flow rate of 0.5ml / min, the separation degree of peak 9 (cinnamic acid) and its adjacent unknown chromatographic peak (peak 10) is 0.85; at a flow rate of 0.7ml / min, the separation degree of peak 11 (cinnamaldehyde) and its adjacent unknown chromatographic peak (peak 10) is 1.19; which does not meet the separation degree requirement of characteristic peaks, so further analysis of the relative retention time and similarity of characteristic peaks under the conditions of 0.5ml / min and 0.7ml / min is not carried out; at a flow rate of 0.6ml / min, the separation of each characteristic peak is better, so the fixed flow rate is recommended to be 0.6ml / min.

[0280] 7.3.4, different column temperature investigation

[0281] Take the same batch of test solution, according to the proposed chromatographic conditions, respectively, with different column temperature 27℃, 30℃ and 33℃ sample, determine and calculate the relative retention time and RSD of each characteristic peak, and according to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint, calculate the similarity of test characteristic spectrum and control characteristic spectrum.

[0282] The results show that the separation of peaks 10 and 11 (cinnamaldehyde) is greatly affected by column temperature, at a column temperature of 33℃, the separation degree of peaks 10 and 11 (cinnamaldehyde) is 0.89, which does not meet the separation degree requirement of characteristic peaks, so further analysis of the relative retention time and similarity of characteristic peaks under this condition is not carried out; at a column temperature of 27℃ and 30℃, the relative retention time RSD of each characteristic peak and reference substance S peak (peak 7) is less than 2.0%; according to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint, the similarity of test characteristic spectrum and control characteristic spectrum is calculated, and the similarity is more than 0.95. It shows that the method has good durability under the column temperature condition of 27℃-30℃, which meets the requirement of characteristic spectrum.

[0283] 7.3.5, different chromatographic column investigation

[0284] Take the same batch of test solution, according to the proposed chromatographic conditions, investigate different brands and different batches of chromatographic columns: chromatographic column 1 (HALO AQ-C 18 (4.6mm×150mm, 2.7μm, S.N. USA QT001492), chromatographic column 2 (YMC Meteoric Core C 18(4.6 mm x 150 mm, 2.7 μm, S.N. MC004488), Column 3 (HALO AQ-C 18 (4.6 mm x 150 mm, 2.7 μm, S.N. MC004488), Column 3 (HALO AQ-C

[0285] The results show that the test sample characteristic spectrum presents 13 characteristic peaks corresponding to the reference characteristic spectrum, and the relative retention time RSD of each characteristic peak to the reference peak (peak 7) is less than 2.0%; according to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint, the similarity of the test sample characteristic spectrum and the reference characteristic spectrum is calculated, and the similarity is above 0.95. It is shown that the method has good durability (different brands and different batches of chromatographic columns), and meets the requirements of the characteristic spectrum.

[0286] 7.4, Verification summary

[0287] The above methodological validation tests show that the method for establishing the characteristic spectrum of the reference sample of the traditional Chinese medicine composition containing Ramulus Cinnamomi of the present application is simple, feasible, and has good durability, and meets the requirements of General Test 9101 of Chinese Pharmacopoeia 2020 Edition.

[0288] 8, Summary

[0289] According to the above methodological validation test results, it is found that the retention time of 5-O-methylvisamminol glycoside chromatographic peak is relatively in the middle, the peak area is relatively large, about 15%, and 5-O-methylvisamminol glycoside is an index component of the flavor of Saposhnikovia divaricata, and the peak is relatively stable, so it is determined that the 5-O-methylvisamminol glycoside chromatographic peak is the S peak. The analysis method of the reference sample characteristic spectrum of the traditional Chinese medicine composition containing Ramulus Cinnamomi of the present application is as follows: determined by high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition General Test 0512). Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel as the filler; acetonitrile as the mobile phase A, 0.1% phosphoric acid solution as the mobile phase B, gradient elution 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 254 nm. The theoretical plate number should not be less than 2000 calculated by the peak of paeoniflorin.

[0290] Preparation of reference solution: mango glycoside control, paeonol control, thalamus glycoside control, 5-O-methyl visamminol glycoside control, cinnamic aldehyde control, ammonium glycyrrhizinate control, 6-gingerol control, cinnamic acid control, appropriate amount, accurately weighed, 50% methanol was prepared into a mixture solution containing 60 μg of mango glycoside, 0.16 mg of paeonol, 40 μg of thalamus glycoside, 40 μg of 5-O-methyl visamminol glycoside, 10 μg of cinnamic aldehyde, 50 μg of ammonium glycyrrhizinate, 10 μg of 6-gingerol, and 10 μg of cinnamic acid per 1 ml, namely.

[0291] Preparation of test solution: 2 ml of the product was accurately measured and placed in a 10 ml volumetric flask, and methanol was added to the mark, shaken and filtered, and the test solution was obtained.

[0292] Determination method: 20 μl of the reference solution and the test solution were accurately taken respectively, injected into the liquid chromatograph, and determined, and the test solution was obtained.

[0293] The above describes the embodiments of the present application in detail, and the principles and implementation modes of the present application are described by applying specific examples. The above description of the embodiments is only used to help understand the method of the present application and its core idea. Meanwhile, the changes or deformations made by the skilled in the art according to the principles of the present application, based on the specific implementation modes and application scope of the present application, all belong to the scope of protection of the present application. In summary, the content of the present description should not be understood as a limitation of the present application.

Claims

1. A method for determining the content of a traditional Chinese medicine composition containing cinnamon twig, characterized in that, The determination method includes the following steps: Take the test solution and reference solution of the traditional Chinese medicine composition for testing. The chromatographic conditions for the detection were as follows: a column packed with octadecylsilane-bonded silica gel was used; mobile phase A was acetonitrile; mobile phase B was approximately 0.1% aqueous phosphoric acid solution; and the gradient elution program was as follows: 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 was 0.6 ml / min; the column temperature was 30 °C; the detection wavelength was 180–360 nm; and the injection volume was 10–30 μl. Based on the test results, the content information of the reference standard of the traditional Chinese medicine composition is obtained; The traditional Chinese medicine composition includes cinnamon twig, white peony root, licorice root, ephedra, ginger, atractylodes macrocephala, anemarrhena rhizome, saposhnikovia root, and aconite root slices; the reference standards are mangiferin, paeoniflorin, cimicifugain, 5-O-methylvisammiol, cinnamaldehyde, ammonium glycyrrhizate, and 6-gingerol. The preparation method of the test solution of the traditional Chinese medicine composition includes: taking an appropriate amount of the reference sample of the traditional Chinese medicine composition containing cinnamon twig and placing it in a container, adding methanol, shaking well, filtering, and obtaining the test solution of the traditional Chinese medicine composition.

2. The determination method according to claim 1, characterized in that, The information is obtained by calculating the content of the reference standard in the traditional Chinese medicine composition according to the external standard method based on the corresponding peak areas in the chromatograms of the test solution and the reference solution of the recorded traditional Chinese medicine composition.

3. The determination method according to claim 1, characterized in that, The linear equation for mangiferin is y = 134118.1290x + 61486.8143, R² = 0.9999.

4. The determination method according to claim 1, characterized in that, The linear equation for paeoniflorin is y = 3889.5159x + 11877.5625, R² = 0.9999.

5. The determination method according to claim 1, characterized in that, The linear equation for the cimicifugain glycoside is y = 61489.9671x + 28672.7690, R² = 0.9999.

6. The determination method according to claim 1, characterized in that, The linear equation for the 5-O-methylvisamidol glycoside is y = 69455.1451x + 80352.1532, R² = 0.9999.

7. The determination method according to claim 1, characterized in that, The linear equation for cinnamaldehyde is y = 253930.5912x + 3832.3004, R² = 0.9998.

8. The determination method according to claim 1, characterized in that, The linear equation for the glycyrrhizic acid is y = 26858.1718x + 3266.0328, with R² = 0.9999.

9. The determination method according to claim 1, characterized in that, The linear equation for 6-gingerol is y = 18804.2424x - 2184.2822, R² = 0.9999.

10. The determination method according to claim 1, characterized in that, The container is a volumetric flask.

11. The determination method according to claim 1, characterized in that, The volume ratio between the reference sample of the traditional Chinese medicine composition and the methanol is 0.1 to 0.

5.

12. The determination method according to claim 11, characterized in that, The volume ratio between the reference sample of the traditional Chinese medicine composition and the methanol is approximately 0.

2.

13. The determination method according to claim 1, characterized in that, The volume of the reference sample of the traditional Chinese medicine composition is 1-5 ml.

14. The determination method according to claim 13, characterized in that, The volume of the reference sample of the traditional Chinese medicine composition is approximately 2 ml.

15. The determination method according to claim 1, characterized in that, The method for preparing the reference solution includes: weighing appropriate amounts of mangiferin, paeoniflorin, cimicifugain, 5-O-methylvisamiloside, cinnamaldehyde, ammonium glycyrrhizate, and 6-gingerol reference standards, and adding methanol to prepare the reference solution with concentrations of 1-200 μg / ml for mangiferin, paeoniflorin, cimicifugain, 5-O-methylvisamiloside, cinnamaldehyde, ammonium glycyrrhizate, and 6-gingerol.

16. The determination method according to claim 15, characterized in that, In the method for preparing the reference solution, the volume percentage concentration of methanol is 40% to 60%.

17. The determination method according to claim 16, characterized in that, The volume percentage concentration of methanol is approximately 50%.

18. The determination method according to claim 15, characterized in that, The concentration of mangiferin in the reference solution was approximately 60 μg / ml.

19. The determination method according to claim 15, characterized in that, The concentration of paeoniflorin in the reference solution was approximately 160 μg / ml.

20. The determination method according to claim 15, characterized in that, The concentration of cimicifugoside in the reference solution was approximately 40 μg / ml.

21. The determination method according to claim 15, characterized in that, The concentration of 5-O-methylvisamidol in the reference solution was approximately 40 μg / ml.

22. The determination method according to claim 15, characterized in that, The concentration of cinnamaldehyde in the reference solution was approximately 10 μg / ml.

23. The determination method according to claim 15, characterized in that, The concentration of ammonium glycyrrhizate in the reference solution was approximately 50 μg / ml.

24. The determination method according to claim 15, characterized in that, The concentration of 6-gingerol in the reference solution was approximately 10 μg / ml.

25. The determination method according to claim 1, characterized in that, The preparation method of the reference sample of the traditional Chinese medicine composition containing cinnamon twig includes: weighing appropriate amounts of cinnamon twig, white peony root, licorice root, ephedra, ginger, atractylodes macrocephala, anemarrhena rhizome, saposhnikovia root, and aconite root, placing them in a container, adding water to soak them, boiling them over high heat, simmering them over low heat for a period of time, filtering them, collecting the filtrate, and obtaining the reference sample of the traditional Chinese medicine composition containing cinnamon twig.

26. The determination method according to claim 25, characterized in that, The volume of the water is 1000-2000 ml.

27. The determination method according to claim 26, characterized in that, The volume of the water is approximately 1400 ml.

28. The determination method according to claim 25, characterized in that, The ratio of the sum of the masses of cinnamon twig, white peony root, licorice root, ephedra, ginger, atractylodes macrocephala, anemarrhena asphodeloides, saposhnikovia divaricata, and aconite root slices to the mass / volume of water is 0.1 to 0.5, in g / ml.

29. The determination method according to claim 28, characterized in that, The ratio of the sum of the masses of cinnamon twig, white peony root, licorice root, ephedra, ginger, atractylodes macrocephala, anemarrhena asphodeloides, saposhnikovia divaricata, and aconite root slices to the mass / volume of the water is approximately 0.31, in g / ml.

30. The determination method according to claim 25, characterized in that, The soaking time is 30 to 60 minutes.

31. The determination method according to claim 30, characterized in that, The soaking time is approximately 45 minutes.

32. The determination method according to claim 25, characterized in that, The simmering time is 30 to 90 minutes.

33. The determination method according to claim 32, characterized in that, The simmering time is approximately 60 minutes.

34. The determination method according to claim 25, characterized in that, The filtration is performed using 200-mesh nylon filter cloth.

35. The determination method according to claim 25, characterized in that, The mass ratio of the cinnamon twig, the black aconite slices, the ginger, the saposhnikovia root, the ephedra, the atractylodes rhizome, the white peony root, the anemarrhena rhizome, and the licorice root is (2-6):(1-3):(3-7):(2-6):(1-3):(3-7):(1-5):(2-6):(1-3).

36. The determination method according to claim 35, characterized in that, The mass ratio of the cinnamon twig, the black aconite slices, the ginger, the saposhnikovia root, the ephedra, the atractylodes rhizome, the white peony root, the anemarrhena rhizome, and the licorice root 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).

37. The determination method according to claim 36, characterized in that, The mass ratio of the cinnamon twig, the black aconite root slices, the ginger, the saposhnikovia root, the ephedra, the atractylodes rhizome, the white peony root, the anemarrhena rhizome, and the licorice root is approximately 4:2:5:4:2:5:3:4:

2.

38. The determination method according to claim 37, characterized in that, The weight range of the cinnamon twigs is 40–70g.

39. The determination method according to claim 38, characterized in that, The weight of the cinnamon twig is approximately 55.20g.

40. The determination method according to claim 37, characterized in that, The weight range of the black shiitake slices is 20-40g.

41. The determination method according to claim 40, characterized in that, The weight of the black shiitake slices is approximately 27.60g.

42. The determination method according to claim 37, characterized in that, The weight range of the ginger is 50-90g.

43. The determination method according to claim 42, characterized in that, The ginger weighs approximately 69.00g.

44. The determination method according to claim 37, characterized in that, The weight range of the windproof material is 40–70g.

45. The determination method according to claim 44, characterized in that, The windproof material weighs approximately 55.20g.

46. ​​The determination method according to claim 37, characterized in that, The mass range of the ephedra is 20–40 g.

47. The determination method according to claim 46, characterized in that, The mass of the ephedra is approximately 27.60g.

48. The determination method according to claim 37, characterized in that, The weight range of Atractylodes macrocephala is 50-90g.

49. The determination method according to claim 48, characterized in that, The mass of the Atractylodes macrocephala is approximately 69.00g.

50. The determination method according to claim 37, characterized in that, The weight range of the white peony root is 30-50g.

51. The determination method according to claim 50, characterized in that, The weight of the white peony root is approximately 41.40g.

52. The determination method according to claim 37, characterized in that, The weight range of Anemarrhena asphodeloides is 40-70g.

53. The determination method according to claim 52, characterized in that, The mass of the Anemarrhena asphodeloides is approximately 55.20g.

54. The determination method according to claim 37, characterized in that, The weight range of the licorice is 20-40g.

55. The determination method according to claim 54, characterized in that, The weight of the licorice is approximately 27.60g.

56. The determination method according to claim 1, characterized in that, The detection wavelengths for mangiferin, paeoniflorin, cimicifugoside, 5-O-methylvisamidolol and glycyrrhizic acid are 200–300 nm.

57. The determination method according to claim 56, characterized in that, The detection wavelength for mangiferin, paeoniflorin, cimicifugoside, 5-O-methylvisamidolol and glycyrrhizic acid is approximately 280 nm.

58. The determination method according to claim 1, characterized in that, The detection wavelengths for cinnamaldehyde and 6-gingerol are 200–300 nm.

59. The determination method according to claim 58, characterized in that, The detection wavelength for cinnamaldehyde and 6-gingerol is approximately 254 nm.

60. The determination method according to claim 1, characterized in that, The injection volume is 15–25 μl.

61. The determination method according to claim 60, characterized in that, The injection volume is approximately 20 μl.

62. The determination method according to claim 1, characterized in that, The theoretical plate number of the chromatographic peak corresponding to the paeoniflorin is not less than 2000.

63. The determination method according to claim 1, characterized in that, The chromatographic column was HALOAQ-C. 18 Column or YMC Meteoric Core C 18 Chromatographic column.

64. The determination method according to claim 1, characterized in that, The specifications of the chromatographic column are as follows: column length 150 mm, inner diameter 4.6 mm, particle size 2.7 μm.

65. The determination method according to claim 1, characterized in that, The detection limit for mangiferin was approximately 13.63 ng / ml.

66. The determination method according to claim 1, characterized in that, The detection limit for paeoniflorin was approximately 0.976 μg / ml.

67. The determination method according to claim 1, characterized in that, The detection limit for the cimicifugoside is approximately 43.55 ng / ml.

68. The determination method according to claim 1, characterized in that, The detection limit for the 5-O-methylvisamidol glycoside is approximately 46.08 ng / ml.

69. The determination method according to claim 1, characterized in that, The detection limit for cinnamaldehyde is approximately 61.64 ng / ml.

70. The determination method according to claim 1, characterized in that, The detection limit for glycyrrhizic acid is approximately 38.75 ng / ml.

71. The determination method according to claim 1, characterized in that, The detection limit for 6-gingerol was approximately 0.6475 μg / ml.

72. The determination method according to claim 1, characterized in that, The limit of quantification for mangiferin is approximately 45.44 ng / ml.

73. The determination method according to claim 1, characterized in that, The limit of quantification for paeoniflorin is approximately 4.88 μg / ml.

74. The determination method according to claim 1, characterized in that, The limit of quantification for the cimicifugoside is approximately 0.2178 μg / ml.

75. The determination method according to claim 1, characterized in that, The limit of quantification for the 5-O-methylvisamidol glycoside is approximately 92.16 ng / ml.

76. The determination method according to claim 1, characterized in that, The limit of quantification for cinnamaldehyde is approximately 0.12328 μg / ml.

77. The determination method according to claim 1, characterized in that, The limit of quantification for the glycyrrhizic acid is approximately 0.2422 μg / ml.

78. The determination method according to claim 1, characterized in that, The limit of quantification for 6-gingerol is approximately 1.295 μg / ml.

79. A method for constructing a fingerprint spectrum of a traditional Chinese medicine composition containing cinnamon twig, characterized in that, The construction method includes the following steps: Preparation of the test solution of the traditional Chinese medicine composition: Take an appropriate amount of the reference sample of the traditional Chinese medicine composition containing cinnamon twig and place it in a container, add methanol, shake well, filter, and obtain the test solution of the traditional Chinese medicine composition; wherein, the traditional Chinese medicine composition includes cinnamon twig, white peony root, licorice root, ephedra, ginger, atractylodes macrocephala, anemarrhena rhizome, saposhnikovia root, and black aconite root slices; Preparation of reference solutions: Weigh appropriate amounts of mangiferin, paeoniflorin, cimicifugain, 5-O-methylvisamiloside, cinnamaldehyde, ammonium glycyrrhizate, and 6-gingerol reference standards, and add methanol to prepare reference solutions with concentrations of 1–200 μg / ml for mangiferin, paeoniflorin, cimicifugain, 5-O-methylvisamiloside, cinnamic acid, cinnamaldehyde, ammonium glycyrrhizate, and 6-gingerol. Based on the results of high-performance liquid chromatography (HPLC) detection of the test solution and the reference solution of the traditional Chinese medicine composition, a fingerprint spectrum of the traditional Chinese medicine composition was obtained. The chromatographic conditions for high-performance liquid chromatography (HPLC) detection were as follows: a column packed with octadecylsilane-bonded silica gel was used; mobile phase A was acetonitrile; mobile phase B was approximately 0.1% aqueous phosphoric acid solution; and the gradient elution program was as follows: 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 was 0.6 ml / min; the column temperature was 27–30 °C; the detection wavelength was 180–360 nm; and the injection volume was 10–30 μl.

80. The construction method according to claim 79, characterized in that, The preparation method of the reference sample of the traditional Chinese medicine composition containing cinnamon twig includes: weighing appropriate amounts of cinnamon twig, white peony root, licorice root, ephedra, ginger, atractylodes macrocephala, anemarrhena rhizome, saposhnikovia root, and black aconite root slices, placing them in a container, adding water to soak them, boiling them over high heat, simmering them over low heat for a period of time, filtering them, collecting the filtrate, and obtaining the reference sample of the traditional Chinese medicine composition containing cinnamon twig.

81. The construction method according to claim 80, characterized in that, The volume of the water is 1000-2000 ml.

82. The construction method according to claim 81, characterized in that, The volume of the water is approximately 1400 ml.

83. The construction method according to claim 80, characterized in that, The ratio of the sum of the masses of cinnamon twig, white peony root, licorice root, ephedra, ginger, atractylodes macrocephala, anemarrhena asphodeloides, saposhnikovia divaricata, and aconite root slices to the mass / volume of water is 0.1 to 0.5, in g / ml.

84. The construction method according to claim 83, characterized in that, The ratio of the sum of the masses of cinnamon twig, white peony root, licorice root, ephedra, ginger, atractylodes macrocephala, anemarrhena asphodeloides, saposhnikovia divaricata, and aconite root slices to the mass / volume of the water is approximately 0.31, in g / ml.

85. The construction method according to claim 80, characterized in that, The soaking time is 30 to 60 minutes.

86. The construction method according to claim 85, characterized in that, The soaking time is approximately 45 minutes.

87. The construction method according to claim 80, characterized in that, The simmering time is 30 to 90 minutes.

88. The construction method according to claim 87, characterized in that, The simmering time is approximately 60 minutes.

89. The construction method according to claim 80, characterized in that, The filtration is performed using 200-mesh nylon filter cloth.

90. The construction method according to claim 80, characterized in that, The mass ratio of the cinnamon twig, the black aconite slices, the ginger, the saposhnikovia root, the ephedra, the atractylodes rhizome, the white peony root, the anemarrhena rhizome, and the licorice root is (2-6):(1-3):(3-7):(2-6):(1-3):(3-7):(1-5):(2-6):(1-3).

91. The construction method according to claim 90, characterized in that, The mass ratio of the cinnamon twig, the black aconite slices, the ginger, the saposhnikovia root, the ephedra, the atractylodes rhizome, the white peony root, the anemarrhena rhizome, and the licorice root 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).

92. The construction method according to claim 91, characterized in that, The mass ratio of the cinnamon twig, the black aconite root slices, the ginger, the saposhnikovia root, the ephedra, the atractylodes rhizome, the white peony root, the anemarrhena rhizome, and the licorice root is approximately 4:2:5:4:2:5:3:4:

2.

93. The construction method according to claim 92, characterized in that, The weight range of the cinnamon twigs is 40–70g.

94. The construction method according to claim 93, characterized in that, The weight of the cinnamon twig is approximately 55.20g.

95. The construction method according to claim 92, characterized in that, The weight range of the black shiitake slices is 20-40g.

96. The construction method according to claim 95, characterized in that, The weight of the black shiitake slices is approximately 27.60g.

97. The construction method according to claim 92, characterized in that, The weight range of the ginger is 50-90g.

98. The construction method according to claim 97, characterized in that, The ginger weighs approximately 69.00g.

99. The construction method according to claim 92, characterized in that, The weight range of the windproof material is 40–70g.

100. The construction method according to claim 99, characterized in that, The windproof material weighs approximately 55.20g.

101. The construction method according to claim 92, characterized in that, The mass range of the ephedra is 20–40 g.

102. The construction method according to claim 101, characterized in that, The mass of the ephedra is approximately 27.60g.

103. The construction method according to claim 92, characterized in that, The weight range of Atractylodes macrocephala is 50-90g.

104. The construction method according to claim 103, characterized in that, The mass of the Atractylodes macrocephala is approximately 69.00g.

105. The construction method according to claim 92, characterized in that, The weight range of the white peony root is 30-50g.

106. The construction method according to claim 105, characterized in that, The weight of the white peony root is approximately 41.40g.

107. The construction method according to claim 92, characterized in that, The weight range of Anemarrhena asphodeloides is 40-70g.

108. The construction method according to claim 107, characterized in that, The mass of the Anemarrhena asphodeloides is approximately 55.20g.

109. The construction method according to claim 92, characterized in that, The weight range of the licorice is 20-40g.

110. The construction method according to claim 109, characterized in that, The weight of the licorice is approximately 27.60g.

111. The construction method according to claim 80, characterized in that, In the method for preparing the reference sample of the traditional Chinese medicine composition containing cinnamon twig, the container is a volumetric flask.

112. The construction method according to claim 79, characterized in that, The volume ratio between the reference sample of the traditional Chinese medicine composition and the methanol is 0.1 to 0.

5.

113. The construction method according to claim 112, characterized in that, The volume ratio between the reference sample of the traditional Chinese medicine composition and the methanol is approximately 0.

2.

114. The construction method according to claim 79, characterized in that, The volume of the reference sample of the traditional Chinese medicine composition is 1-5 ml.

115. The construction method according to claim 114, characterized in that, The volume of the reference sample of the traditional Chinese medicine composition is approximately 2 ml.

116. The construction method according to claim 79, characterized in that, In the method for preparing the reference solution, the volume percentage concentration of methanol is 40% to 60%.

117. The construction method according to claim 116, characterized in that, The volume percentage concentration of methanol is approximately 50%.

118. The construction method according to claim 79, characterized in that, The concentration of mangiferin in the reference solution was approximately 60 μg / ml.

119. The construction method according to claim 79, characterized in that, The concentration of paeoniflorin in the reference solution was approximately 160 μg / ml.

120. The construction method according to claim 79, characterized in that, The concentration of cimicifugoside in the reference solution was approximately 40 μg / ml.

121. The construction method according to claim 79, characterized in that, The concentration of 5-O-methylvisamidol in the reference solution was approximately 40 μg / ml.

122. The construction method according to claim 79, characterized in that, The concentration of cinnamic acid in the reference solution was approximately 10 μg / ml.

123. The construction method according to claim 79, characterized in that, The concentration of cinnamaldehyde in the reference solution was approximately 10 μg / ml.

124. The construction method according to claim 79, characterized in that, The concentration of ammonium glycyrrhizate in the reference solution was approximately 50 μg / ml.

125. The construction method according to claim 79, characterized in that, The concentration of 6-gingerol in the reference solution was approximately 10 μg / ml.

126. The construction method according to claim 79, characterized in that, The detection wavelength is 200–300 nm.

127. The construction method according to claim 126, characterized in that, The detection wavelength is approximately 254 nm.

128. The construction method according to claim 79, characterized in that, The injection volume is 15–25 μl.

129. The construction method according to claim 128, characterized in that, The injection volume is approximately 20 μl.

130. The construction method according to claim 79, characterized in that, The theoretical plate number of the chromatographic peak corresponding to the paeoniflorin is not less than 2000.

131. The construction method according to claim 79, characterized in that, The chromatographic column was HALOAQ-C. 18 Column or YMC Meteoric Core C 18 Chromatographic column.

132. The construction method according to claim 79, characterized in that, The specifications of the chromatographic column are as follows: column length 150 mm, inner diameter 4.6 mm, particle size 2.7 μm.

133. The construction method according to claim 79, characterized in that, When the detection wavelength is 254 nm, the fingerprint spectrum includes peaks 1-13, and the retention times of peaks 1-13 are approximately 7.250 min, approximately 12.917 min, approximately 14.087 min, approximately 15.533 min, approximately 16.590 min, approximately 26.515 min, approximately 33.733 min, approximately 48.726 min, approximately 50.055 min, approximately 51.963 min, approximately 53.086 min, approximately 63.573 min, and approximately 70.914 min, respectively.

134. The construction method according to claim 79, characterized in that, When the detection wavelength is 254 nm, the fingerprint spectrum includes peaks 1-13, with peak 7 (5-O-methylvisamidol glycoside) serving as a reference peak. The average relative retention times of peaks 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, and 13 are approximately 0.21, 0.38, 0.42, 0.46, 0.49, 0.79, 1.44, 1.48, 1.54, 1.57, 1.88, and 2.10, respectively.

135. The construction method according to claim 79, characterized in that, At the detection wavelength of 254 nm, peak 2 is mangiferin, peak 4 is paeoniflorin, peak 5 is cimicifugoside, peak 6 is glycyrrhizin, peak 7 is 5-O-methylvisamidol, peak 9 is cinnamic acid, peak 11 is cinnamaldehyde, and peak 13 is glycyrrhizic acid.

136. The construction method according to claim 79, characterized in that, When the detection wavelength is 254nm, peak 1 comes from Anemarrhena asphodeloides, peak 2 comes from Anemarrhena asphodeloides, peak 3 comes from Anemarrhena asphodeloides, peak 4 comes from Paeonia lactiflora, peak 5 comes from Saposhnikovia divaricata, peak 6 comes from Glycyrrhiza uralensis, peak 7 comes from Saposhnikovia divaricata, peak 8 comes from Glycyrrhiza uralensis, peak 9 comes from Cinnamomum cassia, peak 10 comes from Saposhnikovia divaricata, peak 11 comes from Cinnamomum cassia, peak 12 comes from Glycyrrhiza uralensis, and peak 13 comes from Glycyrrhiza uralensis.

137. A method for quality control of a traditional Chinese medicine composition containing cinnamon twig, characterized in that, The quality control method includes the following steps: (1) Establish a standard fingerprint spectrum of a traditional Chinese medicine composition reference sample using the fingerprint spectrum construction method according to any one of claims 79 to 136; (2) Take the test solution of the traditional Chinese medicine composition and detect it under the chromatographic conditions in the fingerprint spectrum construction method according to any one of claims 79 to 136 to obtain the fingerprint spectrum of the traditional Chinese medicine composition to be tested; as well as (3) Compare the fingerprint spectrum of the Chinese medicine composition to be tested obtained in step (2) with the standard fingerprint spectrum of the Chinese medicine composition reference sample obtained in step (1). If the fingerprint spectrum meets the requirements, the product is qualified; if it does not meet the requirements, the product is unqualified.

138. The quality control method according to claim 137, characterized in that, The compliance requirements include one or more of the following: (1) The fingerprint spectrum of the Chinese herbal composition sample to be tested shows 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 spectrum of the Chinese herbal composition reference sample. (2) Taking the 5-O-methylvisamidol glycoside peak as the S peak, the relative retention time of each characteristic chromatographic peak in the fingerprint spectrum 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 spectrum of the Chinese medicine composition reference sample. as well as (3) According to the similarity evaluation system of chromatographic fingerprint of traditional Chinese medicine, the similarity between the fingerprint of the sample to be tested of the traditional Chinese medicine composition and the standard fingerprint of the reference sample of the traditional Chinese medicine composition shall not be less than 0.

90.

139. The use of the determination method according to any one of claims 1 to 78, the construction method according to any one of claims 79 to 136, or the quality control method according to claims 137 or 138 in the quality detection and / or quality evaluation and / or quality control of a traditional Chinese medicine composition containing cinnamon twig.

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