A detection method for traditional Chinese medicine composition

Abstract

The present invention relates to the field of quality testing of traditional Chinese medicine preparations, and specifically provides a method for testing a traditional Chinese medicine composition. The composition of the raw materials of the traditional Chinese medicine composition is described in the instructions. The method prepares a test solution by a specific method and develops it with petroleum ether (60-90°C)-toluene-acetone and cyclohexane-toluene-acetone-formic acid as developing agents; the method achieves identification of Artemisia annua and dried tangerine peel, and furthermore, utilizes HPLC to determine the content of baicalin and chlorogenic acid, the active ingredients in the composition. The method of the present invention has high accuracy and sensitivity, a wide detection range, and achieves effective control of drug quality. The method can be used as a quality monitoring method for the traditional Chinese medicine composition and preparation.

Description

Technical Field

[0001] The present invention relates to the field of quality detection of traditional Chinese medicine preparations, and in particular to a method for detecting a traditional Chinese medicine composition. Background Art

[0002] Colds (also known as acute upper respiratory tract infections) are common childhood exogenous illnesses characterized by fever, chills, headache, nasal congestion, runny nose, cough, and sneezing. Colds, also known as common colds, can be divided into two types: the common cold, caused by wind pathogens, is generally mild, primarily manifests in the lungs, and does not cause epidemics; the epidemic cold, caused by seasonal viruses, is more severe and has epidemic characteristics, known in Western medicine as influenza. The common cold is often accompanied by fever, sore throat, and a mild cough with sputum. A Traditional Chinese Medicine (TCM) symptom analysis of 2,502 patients with upper respiratory tract infection and fever in Guangdong Province found that 45.24% had exogenous wind-heat and dampness syndrome, characterized by fever, chills, headache, poor appetite, cough, heaviness in the head and body, sore throat, body aches, a red or pale red tongue, a white tongue coating, and a floating or rapid pulse (see "Analysis of Traditional Chinese Medicine Symptoms in 2,502 Patients with Upper Respiratory Tract Infection and Fever").

[0003] Over the past thousands of years, traditional Chinese medicine has played an important role in treating diseases. Therefore, giving full play to the advantages of traditional Chinese medicine and developing effective traditional Chinese medicines to treat colds, especially colds caused by exogenous wind-heat and dampness, has become an urgent issue that needs to be addressed.

[0004] In recent years, with the continuous deepening of research into the fundamental theories of Traditional Chinese Medicine (TCM) and the pharmacodynamic studies of various experimental pathological models, combined with years of clinical practice by clinicians, TCM has demonstrated excellent clinical efficacy, multiple therapeutic targets, and a wide range of therapeutic effects. This has offset the shortcomings of chemical drugs, which have limited efficacy, few therapeutic targets, and certain side effects. Given that TCM is a complex system composed of multiple components, and its composition is influenced by multiple factors such as species, origin, harvesting, and processing, the diverse and complex composition of TCM is the material foundation for its excellent efficacy, broad effects, and minimal side effects. However, this material foundation has long been fuzzy and difficult to fully evaluate. Furthermore, after the decoction of multiple medicinal ingredients, the quality of the compound is even more difficult to evaluate using clear, effective, and reasonable indicators. However, the therapeutic effect of TCM compound is directly related to its quality. Poor quality of TCM compound can directly lead to poor therapeutic effects, necessitating quality control of TCM compound to ensure the stability and controllability of its efficacy. Quality control of TCM compound requires monitoring of all active ingredients. Only an established quality control system can ensure the effectiveness, controllability, and safety of TCM compound use. Therefore, establishing a modern quality control system with the characteristics of basic theories of traditional Chinese medicine, solving the difficulties in traditional Chinese medicine analysis, and improving existing quality control methods are hot topics of current research. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to provide a method for detecting a Chinese medicine composition, which overcomes the defect that the quality of the Chinese medicine composition cannot be comprehensively and clearly detected and monitored due to the large number of Chinese medicine flavors, complex ingredients and mutual interference, and improves the stability, consistency and controllability of the quality of the Chinese medicine composition.

[0006] The present invention provides a detection method for a traditional Chinese medicine composition. The traditional Chinese medicine composition comprises the following raw materials, calculated by weight: 100-300 parts of Artemisia annua, 100-300 parts of Scutellaria baicalensis, 100-450 parts of Poria cocos, 100-270 parts of Tangerine peel, 100-270 parts of Rhizoma Anemarrhenae, 580-820 parts of Gypsum, 200-450 parts of Flos Lonicerae, 200-450 parts of Folium Isatidis, 100-300 parts of Rhizoma Pinelliae, 100-270 parts of Periostracum Cicadae, 100-270 parts of Menthol, 100-270 parts of Licorice, and 20-180 parts of Indigo Naturalis.

[0007] The detection method of the Chinese medicine composition includes the steps of identifying Artemisia annua and dried tangerine peel:

[0008] A. Identification of Artemisia annua and Tangerine Peel

[0009] Preparation of the test solution: 1) Weigh the Chinese medicine composition, add the extraction solvent to extract, and separate the solid and liquid to obtain the liquid;

[0010] 2) After drying, dissolving in water, extracting with water-saturated n-butanol, drying the n-butanol solution, and dissolving in an organic solvent;

[0011] 3) The drug-containing solution obtained after dissolution is eluted by column chromatography using a 5-15% ethanol solution as an eluent, the eluate is discarded, and then eluted with a 20-40% ethanol solution as an eluent, the eluate is collected, dried, and then dissolved in an organic solvent to prepare the test solution;

[0012] Preparation of control medicinal material solution: separately extract Artemisia annua and tangerine peel control medicinal material with water, separate the solid and liquid, and obtain the supernatant. The obtained substance after concentration or drying is used instead of the traditional Chinese medicine composition according to the preparation method of the test solution to prepare Artemisia annua control medicinal material solution and tangerine peel control medicinal material solution;

[0013] Identification: According to the thin layer chromatography test, aspirate the test sample solution and the tangerine peel control medicinal material solution, spot them on the same thin layer plate, develop with petroleum ether-toluene-acetone with a boiling range of 60-90℃ as the first developing solvent, spray with a color developer, heat, and inspect under ultraviolet light;

[0014] Pipette the test sample solution and the Artemisia annua control medicinal material solution, spot them on another thin layer plate, develop them with cyclohexane-toluene-acetone-formic acid as the second developing agent, and inspect them under ultraviolet light.

[0015] In certain preferred embodiments, in the identification step, in the first developing solvent, the volume ratio of petroleum ether with a boiling range of 60-90°C, toluene and acetone is 9:1-3:5; and / or, in the second developing solvent, the volume ratio of cyclohexane, toluene, acetone and formic acid is 4-5:3-4:2-3:0.5; and / or, the spotting volume of the test solution, tangerine peel control medicinal material solution or Artemisia annua control medicinal material solution is 1-10 μl; and / or, the color developer is AlCl3 ethanol solution; and / or, the heating temperature is 100-110°C.

[0016] In certain preferred embodiments, during the preparation of the test solution, the extraction solvent in step 1) and the organic solvent in step 2) or step 3) are selected from at least one of methanol, ethanol, and water; and / or the volume ratio of the mass of the traditional Chinese medicine composition to the extraction solvent, water, and the organic solvent in step 2) is 2-6 g: 30-100 ml: 30-50 ml: 3-5 ml; and / or the volume ratio of the drug-containing solution, the eluent, and the organic solvent in step 3) is 1-4: 20-50: 1-3 ml; and / or the solid-liquid separation is centrifugation or filtration; and / or the column chromatography is neutral alumina column chromatography; and / or the number of water-saturated n-butanol extractions is at least 1.

[0017] In certain preferred embodiments, the preparation of the test solution comprises the following steps: 2-6 g of the traditional Chinese medicine composition is added with 30-100 ml of methanol and ultrasonically extracted for 20-60 minutes, filtered, the filtrate is evaporated to dryness, 30-50 ml of water is added to dissolve it, and it is extracted at least once with water-saturated n-butanol, the n-butanol solution is dried and dissolved with 3-5 ml of methanol, 1-4 ml is transferred to a neutral alumina column, eluted with 20-50 ml of 5-15% ethanol solution as an eluent, the eluent is discarded, and eluted with 20-50 ml of 20-40% ethanol solution as an eluent, the eluent is collected, evaporated to dryness, and the residue is dissolved with 1-3 ml of methanol to obtain the product.

[0018] During the preparation of the control medicinal material solution, the extraction is performed by reflux extraction, and the extraction time is 20-60 minutes. The solid-liquid separation is performed by centrifugation or filtration. Specifically, 0.3-1g of the control medicinal material is added with 20-30ml of water, reflux extraction is performed for 30-60 minutes, filtered, and the filtrate is concentrated to 3-5ml. The concentrated solution is used to replace the traditional Chinese medicine composition and prepare the test solution according to the preparation method.

[0019] In certain preferred embodiments, step 2) further comprises extracting with petroleum ether before extracting with water-saturated n-butanol. The petroleum ether extraction is performed at least once, for example, 2 to 3 times, with the mass ratio of the amount of petroleum ether used each time to the amount of the traditional Chinese medicine composition being 30-50 ml: 2-6 g.

[0020] In certain preferred embodiments, the detection method of the traditional Chinese medicine composition further comprises at least one of the following determinations of baicalin content and chlorogenic acid content:

[0021] B. Determination of baicalin content

[0022] Preparation of the test solution: taking the traditional Chinese medicine composition, adding an organic solvent for extraction to obtain a test solution;

[0023] Preparation of reference solution: Take baicalin reference substance, add organic solvent to dissolve it and prepare reference solution;

[0024] Chromatographic conditions and system suitability test: HPLC determination was performed using octadecylsilane bonded silica gel as the filler; acetonitrile as mobile phase A, and 0.1-0.3% phosphoric acid solution as mobile phase B. Gradient elution was performed with the following program: 0-35 min, mobile phase A: 20% by volume, mobile phase B: 80% by volume; 35-40 min, mobile phase A: increasing from 20% to 60% by volume, mobile phase B: decreasing from 80% to 40% by volume; detection wavelength: 260-290 nm.

[0025] Determination method: aspirate the reference solution and the test solution, inject them into the liquid chromatograph, determine and calculate;

[0026] C. Determination of chlorogenic acid content

[0027] Preparation of the test solution: taking the traditional Chinese medicine composition, adding an organic solvent for extraction to obtain a test solution;

[0028] Preparation of reference solution: Take chlorogenic acid reference, add organic solvent to dissolve and prepare reference solution;

[0029] Chromatographic conditions and system suitability test: Determination was performed according to the high performance liquid chromatography method, using octadecylsilane bonded silica gel as the filler; a mixture of tetrahydrofuran, methanol, and acetonitrile as mobile phase A, and 0.1-0.3% phosphoric acid solution as mobile phase B; gradient elution was performed, and the gradient elution program was as follows: 0-60 min, the volume percentage of mobile phase A in the mobile phase increased from 7% to 10%, and the volume percentage of mobile phase B decreased from 93% to 90%; 60-65 min, the volume percentage of mobile phase A in the mobile phase increased from 10% to 60%, and the volume percentage of mobile phase B decreased from 90% to 40%; 65-70 min, the volume percentage of mobile phase A in the mobile phase was 60%, and the volume percentage of mobile phase B was 40%; the detection wavelength was 310-340 nm.

[0030] Determination method: Pipette the reference solution and the test solution, inject them into the liquid chromatograph, measure and calculate.

[0031] In certain preferred embodiments, the detection method further satisfies at least one of the following (1)-(2):

[0032] (1) In the determination of baicalin content, the organic solvent is selected from at least one of methanol, ethanol, and water; and / or the mass ratio of the traditional Chinese medicine composition to the volume of the organic solvent is 0.1-0.6 g: 20-100 ml; and / or the flow rate is 0.8-1.2 ml / min; and / or the column temperature is 25-40°C; and / or the concentration of the reference solution is 10-300 μg / mL; and / or the extraction is ultrasonic extraction, reflux extraction, or shaking extraction; and / or the extraction time is 15-60 min;

[0033] (2) In the determination of the content of chlorogenic acid, the organic solvent is selected from at least one of methanol, ethanol, and water; and / or the volume ratio of the mass of the traditional Chinese medicine composition to the organic solvent is 0.1-0.6 g: 20-100 ml; and / or the flow rate is 0.7-1.2 ml / min; and / or the column temperature is 20-30°C; and / or the volume ratio of tetrahydrofuran, methanol, and acetonitrile in mobile phase A is 1:7-9:15-17; and / or the concentration of the reference solution is 1-400 μg / mL; and / or the extraction is ultrasonic extraction, reflux extraction, or shaking extraction; and / or the extraction time is 15-60 min.

[0034] In the determination of chlorogenic acid content, the extraction solvent of the test solution is preferably 30-75% methanol aqueous solution or 30-75% ethanol aqueous solution.

[0035] In the determination of baicalin content, the extraction solvent of the test solution is preferably 30-70% methanol aqueous solution or 30-70% ethanol aqueous solution.

[0036] Furthermore, the preparation method of the traditional Chinese medicine composition is as follows: after mixing Artemisia annua, Scutellaria baicalensis, Poria cocos, dried tangerine peel, bamboo shavings, gypsum, honeysuckle, isatis indigotica, pinellia ternata, cicada shell, mint and licorice, the mixture is extracted according to a conventional extraction method or after extracting them separately according to a conventional extraction method, the mixture is mixed and then mixed with Indigo naturalis to obtain the composition; or after mixing Artemisia annua, Scutellaria baicalensis, Poria cocos, dried tangerine peel, bamboo shavings, gypsum, honeysuckle, isatis indigotica, pinellia ternata, cicada shell, mint, licorice and Indigo naturalis, the mixture is extracted according to a conventional extraction method or after extracting them separately according to a conventional extraction method, the mixture is mixed to obtain the composition.

[0037] Furthermore, the preparation method of the Chinese medicine composition is: adding excipients to the Chinese medicine composition or not adding excipients and directly or indirectly preparing a clinically acceptable pharmaceutical preparation according to conventional processes:

[0038] Preferably, the pharmaceutical preparation is in the form of micropills, tablets, capsules, powders, mixtures, granules, syrups, gels or decoctions;

[0039] Preferably, the pharmaceutically acceptable excipient is selected from at least one of pharmaceutically acceptable solvents, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, glidants, flavoring agents, preservatives, suspending agents, coating materials, anti-adhesives, penetration enhancers, pH regulators and thickeners.

[0040] Furthermore, the preparation method of the pharmaceutical preparation comprises:

[0041] Decoction step: gypsum is decocted with an extraction solvent, and the decoction liquid and the medicinal residue are collected to obtain A; Artemisia annua, Scutellaria baicalensis, Poria cocos, dried tangerine peel, bamboo shavings, honeysuckle, isatis indigotica, Pinellia ternata, cicada slough, mint, and liquorice are mixed, A is added, and the mixture is soaked with an extraction solvent, decocted and extracted, solid-liquid separation is performed, and the liquid is collected to obtain a medicinal solution;

[0042] The preparation steps of the pharmaceutical preparation include: concentrating and / or drying the medicinal solution, mixing it with Indigo Naturalis, adding or not adding auxiliary materials, and preparing the pharmaceutical preparation according to conventional processes; or including:

[0043] Decoction step: gypsum is decocted with an extraction solvent, and the decoction liquid and the medicinal residue are collected to obtain A; Artemisia annua, Scutellaria baicalensis, Poria cocos, dried tangerine peel, bamboo shavings, honeysuckle, isatis indigotica, Pinellia ternata, cicada slough, mint, liquorice, and indigo naturalis are mixed, A is added, and the mixture is soaked with an extraction solvent, decocted and extracted, solid-liquid separation is performed, and the liquid is collected to obtain a medicinal solution;

[0044] The preparation steps of the pharmaceutical preparation are as follows: the pharmaceutical solution is concentrated and / or dried, excipients are added or not added, and the pharmaceutical preparation is prepared according to conventional processes.

[0045] Furthermore, the decoction step includes taking gypsum, adding 3-6 times the weight of gypsum in water or 5-98% alcohol solution and decocting for 30-90 minutes, collecting the decoction and the residue to obtain A; taking Artemisia annua, Scutellaria baicalensis, Poria cocos, Tangerine peel, Rhizoma Anemarrhenae, Honeysuckle, Isatis indigotica, Pinellia ternata, Periostracum Cicadae, Menthol, and Licorice, mixing them, adding A, adding water until the amount of water or 5-98% alcohol solution is 5-12 times the weight of the medicinal materials except Indigo naturalis, soaking for 30-120 minutes, decocting and extracting 1-4 times, the material-liquid mass ratio of each extraction is 1:6-15, the extraction time is 60-120 minutes, solid-liquid separation, collecting the liquid, and obtaining the medicinal solution; or including,

[0046] Take gypsum, add 3-6 times the weight of gypsum water or 5-98% alcohol solution, and boil for 30-90 minutes, collect the decoction and the residue to obtain A; take Artemisia annua, Scutellaria baicalensis, Poria cocos, Tangerine peel, Rhizoma Anemarrhenae, Honeysuckle, Isatis indigotica, Pinellia ternata, Periostracum Cicadae, Menthol, Licorice, and Indigo naturalis, mix, add A, add water to 5-12 times the weight of the medicinal materials in water or 5-98% alcohol solution, soak for 30-120 minutes, boil and extract 1-4 times, the material-liquid mass ratio of each extraction is 1:6-15, the extraction time is 60-120 minutes, separate the solid and liquid, collect the liquid, and obtain a medicinal solution.

[0047] The technical solution of the present invention has the following advantages:

[0048] 1. The detection method of the Chinese medicine composition of the present invention realizes effective monitoring of product quality by identifying the sweet artemisia and dried orange peel of the Chinese medicine combination. In the preparation of the test sample solution of the sweet artemisia and dried orange peel identification method, a suitable extraction method is obtained by multiple screening, and an extraction solvent is added to extract. After solid-liquid separation, the liquid is dissolved in water after drying, extracted with water-saturated n-butanol, dried and dissolved in an organic solvent, the solution obtained after dissolution is eluted with 5-15% ethanol solution as an eluent, the eluent is discarded, and then washed with 20-40% ethanol solution. The method uses an eluent as an eluent, collects the eluate, and then dissolves it in an organic solvent after drying. The method then uses petroleum ether-toluene-acetone and cyclohexane-toluene-acetone-formic acid with a boiling range of 60-90°C as developing agents, so that the effective components in the artemisia annua and dried tangerine peel in the test solution can be separated from the effective components of other medicinal materials, and can be effectively separated from other impurity peaks, which is more conducive to the quality detection of the medicine and makes the chromatogram obtained by detection accurate, stable and reliable. The processing method is convenient and easy to operate, and can not only quickly and easily obtain the quality status of the product, but also effectively monitor the quality performance of the product.

[0049] 2. The detection method of the traditional Chinese medicine composition described in the present invention further improves the comprehensiveness and reliability of the quality monitoring of the traditional Chinese medicine composition by determining the content of baicalin and chlorogenic acid. By extracting the test solution and the reference solution and selecting the appropriate mobile phase and gradient elution program, the detection method is accurate and reliable, with strong specificity, and the linear relationship, precision, and recovery rate meet the requirements, and the operation is simple and convenient. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0051] Figure 1 The figure is the thin layer chromatogram obtained from the control group 1 in Experimental Example 1; numbers 1 and 2 are different batches of test samples; 3 is the control medicinal material of dried tangerine peel; 4 is the control medicinal material of mint; 5 is the control medicinal material of Poria; 6 is the control medicinal material of Scutellaria; 7 is the control medicinal material of Artemisia annua; 8 is the control medicinal material of Pinellia; 9 is the control medicinal material of Licorice;

[0052] Figure 2 This is the thin layer chromatogram obtained from the control group 2 in Experimental Example 1, where number 1 is the test sample; number 2 is the control medicinal material Artemisia annua; number 3 is the control medicinal material Tangerine peel; number 4 is the control medicinal material Licorice; number 5 is the control medicinal material Scutellaria baicalensis;

[0053] Figure 3 This is the thin layer chromatogram obtained from the control group 3 in Experimental Example 1, where number 1 is the test sample; 2 is the control medicinal material Artemisia annua; 3 is the control medicinal material Tangerine peel; 4 is the control medicinal material Licorice; 5 is the control medicinal material Scutellaria baicalensis;

[0054] Figure 4 This is the thin layer chromatogram obtained from the control group 4 in Experimental Example 1, number 1 is the test sample; 2 is the control medicinal material Artemisia annua; 3 is the control medicinal material Tangerine peel; 4 is the control medicinal material Licorice; 5 is Artemisia annua negative; 6 is Tangerine peel negative; 7 is Licorice negative;

[0055] Figure 5 This is the thin layer chromatogram obtained from the control group 5 in Experimental Example 1. Numbers 1, 2, and 3 are three batches of test samples; 4 is the Artemisia annua control medicinal material; 5 is the Artemisia annua negative;

[0056] Figure 6 This is the thin layer chromatogram obtained from the control group 6 in Experimental Example 1, number 1 is the test sample; 2 is the control medicinal material Artemisia annua; 3 is the control medicinal material Tangerine peel; 4 is the control medicinal material Licorice; 5 is Artemisia annua negative; 6 is Tangerine peel negative; 7 is Licorice negative;

[0057] Figure 7 This is the thin layer chromatogram obtained from control group 7 in Experimental Example 1. Numbers 1, 2, and 3 are three batches of test samples; 4 is the Artemisia annua control medicinal material; 5 is the Artemisia annua negative;

[0058] Figure 8 This is the thin layer chromatogram obtained from experimental group 1 in experimental example 1. Numbers 1, 2, and 3 are three batches of test samples; 4 is the control medicinal material of Artemisia annua; 5 is the negative medicinal material of Artemisia annua.

[0059] Figure 9 The thin layer chromatogram obtained from the control group 1 in Experimental Example 2 is shown in Figure 1. Number 1 is the test sample-10% ethanol eluent; 2 is the test sample-30% ethanol eluent; 3 is the test sample-50% ethanol eluent; 4 is the tangerine peel control medicinal material-10% ethanol eluent; 5 is the tangerine peel control medicinal material-30% ethanol eluent; 6 is the tangerine peel control medicinal material-50% ethanol eluent; 7 is the tangerine peel negative sample-10% ethanol eluent;

[0060] Figure 10 The thin layer chromatogram obtained by collecting the 10% ethanol eluate for the control group 2 in Experimental Example 2, numbers 1, 2, and 3 are three batches of test sample-10% ethanol eluate; 4 is the tangerine peel control medicinal material-10% ethanol eluate; 5 is the tangerine peel negative sample-10% ethanol eluate;

[0061] Figure 11 The thin layer chromatogram obtained by collecting the 30% ethanol eluate for the control group 2 in Experimental Example 2, numbers 1, 2, and 3 are three batches of test sample-30% ethanol eluate; 4 is the tangerine peel control medicinal material-30% ethanol eluate; 5 is the tangerine peel negative sample-30% ethanol eluate;

[0062] Figure 12 The thin layer chromatogram obtained by collecting the 10% ethanol eluate for the control group 3 in Experimental Example 2, numbers 1, 2, and 3 are three batches of test sample-10% ethanol eluate; 4 is the tangerine peel control medicinal material-10% ethanol eluate; 5 is the tangerine peel negative sample-10% ethanol eluate;

[0063] Figure 13 The thin layer chromatogram obtained by collecting the 30% ethanol eluate for the control group 3 in Experimental Example 2, numbers 1, 2, and 3 are three batches of test sample-30% ethanol eluate; 4 is the tangerine peel control medicinal material-30% ethanol eluate; 5 is the tangerine peel negative sample-30% ethanol eluate;

[0064] Figure 14 The thin layer chromatogram obtained by collecting the 30% ethanol eluate for the control group 4 in Experimental Example 2, numbers 1 to 5 are the test sample-30% ethanol eluate, and the sample volumes are 2, 4, 6, 8, and 10 μL, respectively; 6 is the tangerine peel control medicinal material-30% ethanol eluate; 7 is the tangerine peel negative sample-30% ethanol eluate;

[0065] Figure 15 This is the thin layer chromatogram obtained by collecting the 30% ethanol eluate from the control group 5 in Experimental Example 2; numbers 1, 2, and 3 are three batches of test sample-30% ethanol eluate; 4 is the tangerine peel control medicinal material-30% ethanol eluate; 5 is the tangerine peel negative sample-30% ethanol eluate;

[0066] Figure 16This is the thin layer chromatogram obtained by collecting the 30% ethanol eluate from test group 1 in Experimental Example 2; numbers 1, 2, and 3 are three batches of test sample-30% ethanol eluate; 4 is the tangerine peel control medicinal material-30% ethanol eluate; 5 is the tangerine peel negative sample-30% ethanol eluate;

[0067] Figure 17 This is the thin layer chromatogram of the sample amount investigation for identification of dried tangerine peel in Experimental Example 5; numbers: 1 to 5 are, respectively, 2 μl, 4 μl, 6 μl, 8 μl, and 10 μl of the test solution (batch number: 2108001); 6. 4 μl of dried tangerine peel control medicinal material solution; 7. 10 μl of the negative sample solution;

[0068] Figure 18-20 This is the thin layer chromatogram obtained from the investigation of developing agents with different ratios for identifying dried tangerine peel in Experimental Example 5; Figure 18 The volume ratio is 1, Figure 19 The volume ratio is 2, Figure 20 The volume ratio is 3, where numbers 1 to 3 are the test solution (batch number: 2108001); 4. Tangerine peel control medicinal material solution; 5. Negative sample solution;

[0069] Figure 21-23 This is the thin layer chromatogram obtained from the temperature and humidity investigation for identification of dried tangerine peel in Experimental Example 5; Figure 21 High temperature and high humidity, Figure 22 For indoor humidity, Figure 23 The temperature is low and the humidity is low. Numbers: 1 to 3 are the test solution (batch number: 2108001); 4. Tangerine peel control medicinal material solution; 5. Negative sample solution;

[0070] Figures 24-28 The thin layer chromatogram obtained from the thin layer plate investigation for identification of dried tangerine peel in Experimental Example 5; Figures 24-28 The plates were silica gel G high-efficiency thin layer plates (Qingdao Ocean Chemical Co., Ltd.), silica gel H thin layer plates (Qingdao Ocean Silica Gel Reagent Factory), Jiyida G thin layer plates (Qingdao Jiyida Chemical Co., Ltd.), silica gel G thin layer plates (made in Germany), and silica gel G thin layer plates (Yantai Jiangyou Silica Gel Development Co., Ltd.); numbers 1 to 3 were test solution (batch number: 2108001); 4. Tangerine peel control medicinal material solution; 5. Negative sample solution;

[0071] Figure 29 This is the thin layer chromatogram of the spotting amount of Artemisia annua in Experimental Example 5; numbers: 1 to 5 are 1, 2, 3, 4, and 5 μl of the test solution (batch number: 2108001); 6. 2 μl of Artemisia annua control medicinal material solution; 7. 5 μl of the negative sample solution;

[0072] Figure 30-32 This is the thin layer chromatogram obtained from the investigation of different ratios of developing agents for identifying Artemisia annua in Experimental Example 5; Figure 30 The volume ratio is 1, Figure 31 The volume ratio is 2, Figure 32 The volume ratio is 3, where numbers 1 to 3 are the test solution (batch number: 2108001); 4. Tangerine peel control medicinal material solution; 5. Negative sample solution;

[0073] Figures 33-35 This is the thin layer chromatogram obtained from the temperature and humidity investigation for identification of Artemisia annua in Experimental Example 5; Figure 33 High temperature and high humidity, Figure 34 For indoor humidity, Figure 35 The temperature is low and the humidity is low. Numbers: 1 to 3 are the test solution (batch number: 2108001); 4. Tangerine peel control medicinal material solution; 5. Negative sample solution;

[0074] Figures 36-38 This is the thin layer chromatogram obtained from the thin layer plate investigation for identification of Artemisia annua in Experimental Example 5; Figures 36-38 The plates were silica gel G thin layer plate (Qingdao Jiyida Silica Gel Reagent Factory), silica gel G thin layer plate (Qingdao Ocean Chemical Co., Ltd.), and high-efficiency silica gel G thin layer plate (Yantai Jiangyou Silica Gel Development Co., Ltd.); numbers 1 to 3 were test solution (batch number: 2108001); 4. Tangerine peel control medicinal material solution; 5. Negative sample solution;

[0075] Figure 39 This is the spectrum of the specificity investigation of baicalin in Experimental Example 6, A is baicalin reference substance; B is test substance; C is scutellaria negative sample; D is blank solvent;

[0076] Figure 40 This is the spectrum of the specificity investigation of chlorogenic acid in Experimental Example 7. A is the chlorogenic acid reference substance; B is the test substance; C is the honeysuckle negative sample; D is the blank solvent. DETAILED DESCRIPTION

[0077] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0078] If the specific experimental steps or conditions are not specified in the examples, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. The reagents or instruments used without indicating the manufacturer are all conventional reagent products that can be obtained commercially. All medicinal materials in this invention have passed the quality inspection and meet the standards of the "Pharmacopoeia of the People's Republic of China". The multiples refer to the mass multiples. The ratios of each reagent in the developing agent are all volume ratios. The reference medicinal material of tangerine peel, batch number: 120969-202011, was provided by the China Food and Drug Inspection Institute.

[0079] Experimental Example 1 Investigation of the Preparation Method of the Test Solution and the Developing Agent in the Identification of Artemisia annua

[0080] Granules prepared according to the composition and preparation method of Example 1 were divided into eight parallel groups, designated experimental and control groups 1-7. The experimental and control groups 1-7 were each subjected to identification of Artemisia annua according to the following method. Furthermore, according to the formulation ratios and preparation method described in Example 1, 1. Artemisia annua negative sample was prepared, differing from Example 1 only in that Artemisia annua was not added during preparation. 2. Tangerine peel negative sample was prepared, differing from Example 1 only in that Tangerine peel was not added during preparation. 3. Licorice negative sample was prepared, differing from Example 1 only in that Licorice was not added during preparation.

[0081] (1) Control group 1: 4 g of the above granules were added to 50 ml of methanol, ultrasonically treated for 30 min, filtered, the filtrate evaporated to dryness, and dissolved in 2 ml of methanol to obtain a test solution; 0.5 g each of Artemisia annua, dried tangerine peel, Poria cocos, Pinellia ternata, Scutellaria baicalensis, Mentha arvense, and Licorice were taken and prepared in the same way as the test solution to obtain a control medicinal solution; 10 μL of the above test solution and control medicinal solution were taken and spotted on the same silica gel G plate, developed with acetone-toluene (1:1), and examined at 365 nm. The results are shown in Table 1. Figure 1 .

[0082] In the chromatogram of the test sample, there are obvious characteristic spots at the corresponding positions in the chromatograms of the control medicinal materials, such as dried tangerine peel, scutellaria baicalensis, artemisia annua, and liquorice, indicating that this method has serious negative interference.

[0083] (2) Control group 2: Take 4 g of the above granules, add 50 ml of methanol, ultrasonically treat for 30 min, filter, evaporate the filtrate to dryness, add 40 ml of water, dissolve it with slight heat, 40 ml each time, combine the n-butanol solution, evaporate to dryness in a water bath, and dissolve the residue with 2 ml of methanol to obtain the test solution; take 0.5 g each of Artemisia annua, dried tangerine peel, Scutellaria baicalensis, and Glycyrrhiza uralensis control medicinal materials and prepare the control medicinal material solution in the same way as the above test solution preparation method; take 10 μL of the above test solution and control medicinal material solution, spot them on the same silica gel G plate, develop with acetone-ethyl acetate (1:1), and observe at 365 nm. The results are shown in Table 1. Figure 2 .

[0084] In the chromatogram of the test sample, blue fluorescent spots appeared at positions corresponding to the control herbs Artemisia annua, dried tangerine peel, and licorice, while the spots corresponding to the control herb Scutellaria baicalensis were less obvious. The main spots of the test sample and control herbs were not clearly separated, and each had a significant tail.

[0085] (3) Control group 3: The only difference from control group 2 is that the developing solvent is adjusted to toluene-ethyl acetate-formic acid (3:1:1), and the other conditions remain unchanged. The results are shown in Figure 3 .

[0086] The chromatogram of the test sample showed a blue fluorescent spot at the corresponding position in the Artemisia annua control, while the Artemisia annua control also had a red spot, indicating a discrepancy. To eliminate this discrepancy, the Artemisia annua control was extracted with water and evaporated to dryness, then extracted with methanol and evaporated to dryness. The residue was then dissolved in water and extracted with petroleum ether. The petroleum ether layer was discarded, and the solution was prepared using the same method as the test sample. The results showed that the red spot disappeared, but a fluorescent spot remained at the corresponding position in the tangerine peel or licorice control, indicating that this method could not accurately identify Artemisia annua.

[0087] (4) Control group 4: Take 4 g of the above granules, add 50 ml of methanol, ultrasonically treat for 30 min, filter, evaporate the filtrate to dryness, add 40 ml of water, slightly heat to dissolve, extract with petroleum ether (60-90 ° C) twice, 40 ml each time, discard the petroleum ether layer; then extract with water-saturated n-butanol twice, 40 ml each time, combine the n-butanol solution, evaporate to dryness in a water bath, and dissolve the residue with 4 ml of methanol to obtain the test solution; take 0.5 g each of Artemisia annua, dried tangerine peel, and liquorice as control herbs, add 25 ml of water, reflux extraction for 3 0min, filter, concentrate the filtrate to 3-5ml, and prepare the control medicinal material solution from the "addition of 50ml of methanol" in the same way as the above test solution preparation method; weigh about 3.6g of negative samples of Artemisia annua, dried tangerine peel, and licorice, and prepare negative sample solutions in the same way as the test solution method; aspirate 10μL of the above test solution, control medicinal material solution, and negative sample solution, spot them on the same silica gel G plate, develop with toluene-ethyl acetate-formic acid (3:1:0.5), and inspect under 365nm conditions. The results are shown in Figure 2. Figure 4 .

[0088] In the chromatogram of the test sample, there is a blue fluorescent spot at the position corresponding to the control medicinal material Artemisia annua, and there is slight interference in the negative sample; there is also a light blue fluorescent spot at the position corresponding to the control medicinal material Tangerine peel; there are two fluorescent spots at the position corresponding to the control medicinal material Licorice. This method cannot accurately identify Artemisia annua.

[0089] (5) Control group 5: The only difference from control group 4 is that the developing agent is adjusted to (5:2:0.5). The test solution, Artemisia annua control medicinal material solution and Artemisia annua negative sample solution are prepared according to the method of control group 4; 10 μL of each is aspirated and spotted on the same silica gel G plate, developed with toluene-ethyl acetate-formic acid (5:2:0.5), and inspected at 365 nm. The results are shown in Figure 5 .

[0090] In the chromatogram of the test sample, there is a blue fluorescent spot at the corresponding position of the Artemisia annua control medicinal material, and there is slight interference in the negative sample. This method cannot accurately identify Artemisia annua.

[0091] (6) Control group 6: Take 4 g of the above granules, add 50 ml of methanol, ultrasonically treat for 30 min, filter, evaporate the filtrate to dryness, add 40 ml of water, slightly heat to dissolve, extract twice with petroleum ether (60-90°C), each time 40 ml, discard the petroleum ether layer; then extract twice with water-saturated n-butanol, each time 40 ml, combine the n-butanol solution, evaporate to dryness in a water bath, dissolve the residue with 4 ml of methanol, transfer 2 ml to a neutral alumina column (100-200 mesh, 5 g, inner diameter 1.5 cm), elute with 30 ml of 10% ethanol, collect the eluate, evaporate to dryness, and dissolve the residue with 2 ml of methanol to prepare the test solution. (10% ethanol eluent); take 0.5g each of Artemisia annua, dried tangerine peel and licorice control medicinal materials, add 25ml of water, reflux extraction for 30min, filter, and concentrate the filtrate to 3-5ml. From "add 50ml of methanol", prepare the control medicinal material solution in the same way as the above test solution preparation method; weigh about 3.6g of Artemisia annua, dried tangerine peel and licorice negative samples, and prepare the negative sample solution according to the test solution method; aspirate 10μL of the above test solution, control medicinal material solution and negative sample solution, spot them on the same silica gel G plate, develop with toluene-ethyl acetate-formic acid (3:1:0.5), and inspect under 365nm conditions. The results are shown in FIG. Figure 6 .

[0092] In the chromatogram of the test sample, there is a blue fluorescent spot at the position corresponding to the control medicinal material Artemisia annua, and there is slight interference in the negative sample; there is also a light blue fluorescent spot at the position corresponding to the control medicinal material Tangerine peel; there are two fluorescent spots at the position corresponding to the control medicinal material Licorice. This method cannot accurately identify Artemisia annua.

[0093] (7) Control group 7: The only difference from control group 6 is that the developing agent is adjusted to toluene-ethyl acetate-formic acid (9:2:0.5). The test solution, Artemisia annua control medicinal material solution and Artemisia annua negative sample solution are prepared according to the method of control group 6; 10 μL of each is aspirated and spotted on the same silica gel G plate, developed with toluene-ethyl acetate-formic acid (9:2:0.5), and examined at 365 nm. The other conditions remain unchanged. The results are shown in FIG. Figure 7 .

[0094] In the chromatogram of the test sample, there is a blue fluorescent spot at the corresponding position of the Artemisia annua control medicinal material, and there is slight interference in the negative sample. This method cannot accurately identify Artemisia annua.

[0095] (8) Test Group 1: Take 4 g of the above granules, add 50 ml of methanol, ultrasonically treat for 30 min, filter, evaporate the filtrate to dryness, add 40 ml of water, slightly heat to dissolve, extract twice with petroleum ether (60-90°C), 40 ml each time, discard the petroleum ether layer; then extract twice with water-saturated n-butanol, 40 ml each time, combine the n-butanol solution, evaporate to dryness in a water bath, dissolve the residue with 4 ml of methanol, transfer 2 ml to a neutral alumina column (100-200 mesh, 5 g, inner diameter 1.5 cm), elute with 10% ethanol and 30 ml of 30% ethanol, collect the 30% ethanol eluate, evaporate to dryness, dissolve the residue with 2 ml of methanol, and use as a supply Test solution (30% ethanol eluent); take 0.5g of each Artemisia annua control medicinal material, add 25ml of water, reflux extraction for 30min, filter, concentrate the filtrate to 3-5ml, and prepare the control medicinal material solution in the same way as the preparation method of the test solution starting from "add 50ml of methanol"; weigh about 3.6g of Artemisia annua negative sample, and prepare the negative sample solution according to the test solution method; aspirate 10μL of the above test solution, control medicinal material solution and negative sample solution, spot them on the same silica gel G plate, develop with cyclohexane-toluene-acetone-formic acid (volume ratio of 4:4:2:0.5), and inspect under 365nm conditions. The results are shown in FIG. Figure 8 .

[0096] In the chromatogram of the test sample, there are blue fluorescent spots at the corresponding positions of the control medicinal materials of Artemisia annua, and there is no interference from the negative sample, indicating that this method can accurately identify Artemisia annua.

[0097] Experimental Example 2 Investigation of the Preparation Method of the Sample Solution and the Developing Agent in the Identification of Tangerine Peel

[0098] Granules prepared according to the composition and preparation method of Example 1 were divided into six parallel groups, namely test groups and control groups 1-5, and the test groups and control groups 1-5 were respectively subjected to identification of dried tangerine peel according to the following method. According to the prescription ratio and preparation method recorded in Example 1, the only difference was that dried tangerine peel was not added during preparation, and a dried tangerine peel negative sample was obtained.

[0099] (1) Control group 1: Take 4 g of the above granules, add 50 ml of methanol, ultrasonically treat for 30 min, filter, evaporate the filtrate to dryness, add 40 ml of water, slightly heat to dissolve, extract twice with petroleum ether (60-90 ° C), each time 40 ml, discard the petroleum ether layer; then extract twice with water-saturated n-butanol, each time 40 ml, combine the n-butanol solution, evaporate to dryness in a water bath, dissolve the residue with 4 ml of methanol, transfer 2 ml to a neutral alumina column (100-200 mesh, 5 g, inner diameter 1.5 cm), elute with 30 ml each of 10% ethanol, 30% ethanol, and 5% ethanol, collect the eluents, evaporate to dryness, add 2 ml of methanol to dissolve the residue, and use them as the supply Test solution; take 0.5g of each control medicinal material of tangerine peel, add 25ml of water, reflux extraction for 30min, filter, concentrate the filtrate to 3-5ml, and prepare the control medicinal material solution by the above test solution preparation method starting from "add 50ml of methanol"; weigh about 3.6g of tangerine peel negative sample, and prepare the negative sample solution according to the test solution method; aspirate 10μL of the above test solution, control medicinal material solution and negative sample solution, spot it on the same silica gel G plate, develop it with petroleum ether (60-90℃)-ethyl acetate (2:3), spray it with 3% AlCl3 ethanol solution, heat it at 105℃ for about 2min, and inspect it under 365nm conditions. The results are shown in Figure 2. Figure 9 .

[0100] The experimental results showed that the main spots of the tangerine peel control medicinal material were mainly present in the 10% and 30% ethanol eluents, and there were no obvious characteristic spots in the 50% ethanol eluent. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions of the control medicinal material, the spot separation was poor, and there was no interference in the negative.

[0101] (2) Control group 2: The only difference from control group 1 is that the developing solvent is adjusted to petroleum ether (60-90℃)-acetone (2:1), and the other conditions remain unchanged. The results are shown in Figure 10 and 11 .

[0102] The results showed that under the conditions of this developing agent, the main spots in the test sample and control medicinal material solutions were not effectively separated.

[0103] (3) Control group 3: The only difference from control group 1 is that the developing solvent is adjusted to toluene-acetone (5:3), and the other conditions remain unchanged. The results are shown in Figure 12 and 13 shown.

[0104] The results showed that under the developing solvent conditions, there were more spots in the test sample with 10% ethanol eluent, and there were two obvious main spots in the test sample chromatogram at the corresponding positions with the control medicinal material chromatogram, with no negative interference, but the separation of the control medicinal material spots was poor; there were fewer spots in the test sample with 30% ethanol eluent, and there were main spots of the same color in the test sample chromatogram at the corresponding positions with the control medicinal material, but one spot in the control medicinal material was not effectively separated.

[0105] (4) Control group 4: The only difference from control group 1 is that the developing solvent is adjusted to petroleum ether (60-90℃)-acetone (9:5), and the spotting volume of the test solution is adjusted to 2, 4, 6, 8, and 10 μL respectively. The other conditions remain unchanged. The results are shown in Figure 14 shown.

[0106] The results showed that under the developing solvent conditions, there were two obvious main spots in the chromatogram of the test sample at the corresponding positions in the chromatogram of the control medicinal material, but one spot in the control medicinal material was not effectively separated.

[0107] (5) Control group 5: The only difference from control group 1 is that the developing solvent is adjusted to petroleum ether (60-90℃)-toluene-acetone (9:1:4), the spotting volume of the test solution and the control medicinal material solution is adjusted to 6μL, and the other conditions remain unchanged. The results are shown in Figure 15 shown.

[0108] The results showed that under the developing solvent conditions, there were two obvious main spots in the chromatogram of the test sample at the corresponding positions in the chromatogram of the control medicinal material, but one spot in the control medicinal material was not effectively separated.

[0109] (6) Experimental group 1: The only difference from control group 1 is that the developing solvent was adjusted to petroleum ether (60-90℃)-toluene-acetone (9:2:5), the spotting volume of the test solution and the control medicinal material solution was adjusted to 6μL, and pre-saturation was performed for 15 minutes. The other conditions remained unchanged. The results are shown in Figure 16 shown.

[0110] The results showed that under the conditions of this developing agent, spots of the same color appeared in the chromatogram of the test sample and the corresponding positions in the chromatogram of the control medicinal material. The main spots of the test sample and the control medicinal material could be effectively separated, and there was no negative interference.

[0111] Experimental Example 3 Investigation of mobile phase and gradient elution procedure in the determination of baicalin content

[0112] Since the components of the traditional Chinese medicine composition of the present invention are complex, with flavonoids, saponins and polysaccharides being the majority, the determination of baicalin is somewhat difficult. Therefore, the effects of the mobile phase and gradient elution degree on the chromatographic analysis results were investigated in the preliminary experiments.

[0113] The granules prepared according to the composition and preparation method of Example 1 were divided into three groups in parallel, namely the experimental group and control groups 1-2. The baicalin content of the experimental group and control groups 1-2 was determined according to the following method.

[0114] (1) Test group: The above-mentioned granules were prepared according to the methods of "Preparation of test solution", "Preparation of reference solution" and "Preparation of negative sample solution" in Example 10 to prepare test solution, reference solution and negative sample solution, and then the chromatograms were measured according to the "Chromatographic conditions" and "Assay method" disclosed in Example 10.

[0115] (2) Control group 1: The difference from the experimental group is that the mobile phase and gradient elution are different. This control group uses methanol (A)-0.2% phosphoric acid aqueous solution (volume ratio of 45:55) for isocratic elution.

[0116] (3) Control group 2: The only difference from the experimental group was that acetonitrile was used as mobile phase A, 0.2% phosphoric acid water was used as mobile phase B, and the gradient elution program shown in Table 1 below was used.

[0117] Table 1 Baicalin gradient elution table

[0118] Time / min Mobile phase A (%) Mobile phase B (%) 0 21 79 28 21 79 29 60 40 33 60 40 34 21 79 40 21 79

[0119] (4) Analytical results of the chromatographic results: In the experimental group, the retention time of the chromatographic peak of baicalin in the test solution was consistent with that of the chromatographic peak of baicalin reference substance, the symmetry factor of the chromatographic peak of baicalin was 1.01, the separation degree was 2.27, and the separation degree was greater than 1.5. In addition, there was no interference at the corresponding position of the chromatographic peak of baicalin reference substance in the negative sample solution, and the method had strong specificity. In the control groups 1 and 2, the chromatographic peak of baicalin in the test solution interfered with the peaks of other components, and the peak shape was poor (symmetry factors were 0.87 and 1.09, respectively), and the separation degrees were 1.12 and 1.46, respectively, both of which did not meet the requirements (symmetry factor 0.95-1.05, separation degree R>1.5).

[0120] Experimental Example 4 Investigation of mobile phase and gradient elution procedure in determination of chlorogenic acid content

[0121] Since the components of the traditional Chinese medicine composition of the present invention are complex, with flavonoids, saponins and polysaccharides being the majority, the determination of chlorogenic acid is somewhat difficult. Therefore, the effect of gradient elution degree on chromatographic analysis results was investigated in preliminary experiments.

[0122] The granules prepared according to the composition and preparation method of Example 1 were divided into three groups in parallel, namely, a test group and a control group. The chlorogenic acid content of the test groups and control groups 1-3 was determined according to the following method.

[0123] (1) Test group: The above-mentioned granules were prepared according to the methods of "Preparation of test solution", "Preparation of reference solution" and "Preparation of negative sample solution" in Example 11 to prepare test solution, reference solution and negative sample solution, and then the chromatogram was determined according to the "Chromatographic conditions" and "Assay method" disclosed in Example 11.

[0124] (2) Control group 1: The difference from the experimental group is that acetonitrile was used as mobile phase A, 0.2% phosphoric acid water was used as mobile phase B, the flow rate was 0.7 ml / min, and the gradient elution program shown in Table 2 below was used.

[0125] Table 2 Chlorogenic acid gradient elution table

[0126] Time / min Mobile phase A (%) Mobile phase B (%) 0 14 86 8 19 81 14 19 81 34 31 69 35 90 10 40 90 10 41 14 86 55 14 86

[0127] (3) Control group 2: The difference from the experimental group is that acetonitrile was used as mobile phase A, 0.2% phosphoric acid water was used as mobile phase B, the flow rate was 0.7 ml / min, the column temperature was 20°C, and the gradient elution program shown in Table 3 below was used.

[0128] Table 3 Chlorogenic acid gradient elution table

[0129] Time / min Mobile phase A (%) Mobile phase B (%) 0 5 95 50 10 90 60 20 80 61 60 40 66 60 40 67 5 95 80 5 95

[0130] (4) Control group 3: The difference from the experimental group is that tetrahydrofuran:methanol:acetonitrile (1:8:16) was used as mobile phase A, 0.2% phosphoric acid water was used as mobile phase B, and the gradient elution program shown in Table 4 below was used.

[0131] Table 4 Chlorogenic acid gradient elution table

[0132]

[0133]

[0134] (5) Chromatographic analysis results: In the experimental group, the retention time of the chlorogenic acid chromatographic peak in the test solution was consistent with the retention time of the chlorogenic acid reference chromatographic peak, the symmetry factor of the chlorogenic acid chromatographic peak was 1.03, and the separation was 21.05, which was much greater than 1.5. There was no interference at the corresponding position of the chlorogenic acid reference chromatographic peak in the negative sample solution, and the method had strong specificity. In the control groups 1, 2, and 3, the chlorogenic acid chromatographic peak in the test solution interfered with the peaks of other components, and the peak shape was poor (symmetry factors were 1.04, 0.94, and 0.92, respectively), and the separation (0.8, 1.23, and 3.89, respectively) did not meet the relevant requirements (symmetry factor 0.95-1.05, separation R>1.5).

[0135] Experimental Example 5 Methodological Investigation of the Identification Method of Tangerine Peel Thin Layer

[0136] 1. Methodological Investigation of the Identification Method of Tangerine Peel in Example 9

[0137] 1. Investigation of sample quantity

[0138] 2, 4, 6, 8, 10 μl of the test solution obtained in Example 9 were drawn, and 4 μl of tangerine peel control medicinal material solution and 10 μl of tangerine peel negative sample solution were drawn and respectively spotted on the same silica gel G high-efficiency thin layer plate soaked with 0.5% sodium hydroxide, with petroleum ether (60-90°C)-toluene-acetone (9:2:5) as a developing agent, placed in a developing cylinder for pre-saturation for 15 minutes, developed, taken out, dried, sprayed with 3% AlCl3 ethanol solution, heated at 105°C, and inspected under an ultraviolet lamp (365nm). When the test sample spotting amount was 6 μl and the control medicinal material 4 μl, in the test sample chromatogram, at the position corresponding to the control medicinal material chromatogram, fluorescent spots of the same color were observed, the spots were well separated, and the negative was not interfered with, and the Rf value was moderate, so the test sample spotting amount was selected to be 6 μl and the control medicinal material 4 μl. The results are shown in the attached Figure 17 shown.

[0139] 2. Investigation of developing agent

[0140] 6 μl, 4 μl and 6 μl of the test solution, tangerine peel control medicinal material solution and tangerine peel negative sample solution prepared in Example 9 were respectively spotted on the same silica gel G high efficiency thin layer plate soaked with 0.5% sodium hydroxide, and petroleum ether (60-90 ℃)-toluene-acetone (volume ratio of 1:9:1:5), petroleum ether (60-90 ℃)-toluene-acetone (volume ratio of 2:9:2:5) and petroleum ether (60-90 ℃)-toluene-acetone (volume ratio of 3:9:3:5) were used as developing agents. The plates were pre-saturated in the developing cylinder for 15 minutes, developed, taken out, dried, sprayed with 3% AlCl3 ethanol solution, heated at 105 ℃, and inspected under ultraviolet light (365 nm). Under the conditions of three different developing solvent polarities, in the chromatogram of the test sample, fluorescent spots of the same color appeared at the corresponding positions in the chromatogram of the control medicinal material, and the spots were effectively separated. However, petroleum ether (60-90℃)-toluene-acetone (9:2:5) was the most ideal, with good spot separation, no negative interference, and moderate Rf value. Therefore, petroleum ether (60-90℃)-toluene-acetone (9:2:5) was determined to be the best developing solvent. Figure 18-20 shown.

[0141] 3. Investigation of temperature and humidity

[0142] 6 μl, 4 μl and 6 μl of the test solution, tangerine peel control medicinal material solution and tangerine peel negative sample solution prepared in Example 9 were taken, respectively, and spotted on the same silica gel G high-efficiency thin-layer plate soaked with 0.5% sodium hydroxide. Petroleum ether (60-90 ℃)-toluene-acetone (9:2:5) was used as a developing agent. The plate was pre-saturated in the developing cylinder for 15 minutes and developed under the environmental conditions of high temperature and high humidity (35 ℃, RH: 75%), room temperature and humidity (16 ℃, RH: 10%) and low temperature and low humidity (4 ℃, 10%). The plate was taken out, dried, sprayed with 3% AlCl3 ethanol solution, heated at 105 ℃, and inspected under ultraviolet light (365 nm). Under each environmental condition, the test sample chromatogram showed fluorescent spots of the same color at the corresponding position of the control medicinal material chromatogram. The spots were well separated, and the negative was not interfered with. The Rf value was moderate, indicating that when the development ambient temperature changed slightly, the development results had no significant effect. The established method had good durability. Figure 21-23 shown.

[0143] 4. Inspection of thin layer boards from different manufacturers

[0144] 6 μl, 4 μl and 6 μl of the test solution, tangerine peel control medicinal material solution and tangerine peel negative sample solution prepared in Example 9 were taken and applied to silica gel G high-efficiency plates, silica gel G plates, H plates and Yantai G plates produced by different manufacturers, respectively. Petroleum ether (60-90 ℃)-toluene-acetone (9:2:5) was used as a developing agent. The plates were pre-saturated in the developing cylinder for 15 minutes, developed, sprayed with 3% AlCl3 ethanol solution, heated at 105 ℃, and inspected under ultraviolet light (365nm). The results showed that the silica gel G plates and H plates of different manufacturers had slightly different development results. Among them, Qingdao Ocean G high-efficiency thin-layer plate had a slightly better development effect. Fluorescent spots of the same color were observed at the corresponding positions of the control medicinal material chromatograms. The spots were well separated, and the negative group had no interference. The Rf value was moderate. Figures 24-28 shown.

[0145] 2. Methodological Investigation of the Identification Method of Artemisia annua in Example 9

[0146] 1. Investigation of sample quantity

[0147] 1, 2, 3, 4, 5 μl of the test solution prepared in Example 9 was taken, and 2 μl of the Artemisia annua control medicinal material solution and 5 μl of the Artemisia annua negative sample solution were taken respectively on the same silica gel G high-efficiency thin layer plate, and cyclohexane-toluene-acetone-formic acid (4:4:2:0.5) was used as the developing agent. The plate was developed, taken out, dried, and inspected under an ultraviolet lamp (365 nm). When the amount of the test sample spotted was 2 μl and the control medicinal material was 2 μl, fluorescent spots of the same color appeared in the test sample chromatogram at the corresponding position of the control medicinal material chromatogram, and there was no interference from the negative solution. The spots were well separated and the Rf value was moderate. Therefore, the amount of the test sample spotted was 2 μl and the control medicinal material was 2 μl. The results are shown in the attached figure. Figure 29 shown.

[0148] 2. Investigation of developing agent

[0149] 2 μl of the test solution, Artemisia annua control medicinal material solution, and Artemisia annua negative sample solution prepared in Example 9 were respectively spotted on the same silica gel G high-performance thin layer plate, and cyclohexane-toluene-acetone-formic acid (volume ratio 1:5:3:2:0.5), cyclohexane-toluene-acetone-formic acid (volume ratio 2:4:4:3:0.5), and cyclohexane-toluene-acetone-formic acid (volume ratio 3:4:4:2:0.5) were used as developing agents, respectively. The plates were developed, taken out, dried, and examined under ultraviolet light (365 nm). Under the conditions of three different developing solvent polarities, in the chromatogram of the test sample, fluorescent spots of the same color appeared at the corresponding positions in the chromatogram of the control medicinal material. The spots were effectively separated, but cyclohexane-toluene-acetone-formic acid (4:4:2:0.5) was the most ideal, with good spot separation, no negative interference, and moderate Rf value. Therefore, cyclohexane-toluene-acetone-formic acid (4:4:2:0.5) was determined to be the best developing solvent. The results are shown in the attached Figure 30-32 shown.

[0150] 3. Investigation of temperature and humidity

[0151] 2 μl, 2 μl, and 2 μl of the test solution, Artemisia annua control medicinal material solution, and Artemisia annua negative sample solution prepared in Example 9 were taken and spotted on the same silica gel G high-efficiency thin layer plate, respectively, with cyclohexane-toluene-acetone-formic acid (4:4:2:0.5) as the developing solvent, and developed under high temperature and high humidity (35°C, 75%), room temperature and humidity (16°C, 10%), and low temperature and low humidity (4°C, 10%) environmental conditions, respectively. The plate was taken out, dried, and examined under ultraviolet light (365 nm). Under each environmental condition, fluorescent spots of the same color appeared in the chromatogram of the test sample at the corresponding position of the chromatogram of the control medicinal material. The spots were well separated, and there was no interference from the negative sample. The Rf value was moderate, indicating that when the development environment temperature changed slightly, there was no significant effect on the development results. The established method had good durability. The results are shown in the attached Figures 33-35 shown.

[0152] 4. Inspection of thin layer boards from different manufacturers

[0153] 2 μL of each of the test solution, Artemisia annua control medicinal material solution, and Artemisia annua negative sample solution prepared in Example 9 were taken and spotted on silica gel G high-efficiency plates, silica gel G plates, and Yantai G plates produced by different manufacturers, respectively. Cyclohexane-toluene-acetone-formic acid (4:4:2:0.5) was used as the developing agent for development. The plates were placed under ultraviolet light (365 nm) for inspection. The results showed that there was no significant difference in the development results of silica gel G plates from different manufacturers. Fluorescent spots of the same color appeared at the corresponding positions of the control medicinal material chromatograms. The spots were well separated, and there was no negative interference. The Rf value was moderate. The results are shown in the attached Figures 36-38 shown.

[0154] Experimental Example 6 Methodological Study on Quantitative Determination of Baicalin

[0155] 1. Determination of detection wavelength

[0156] The 3D spectral absorption spectrum of the baicalin reference solution at wavelengths of 190-400 nm shows that baicalin has a maximum absorption peak at 278 nm. Since the maximum absorption values ​​are basically consistent within the range of 270-275 nm, 278 nm of baicalin in the granules is preferably used as the detection wavelength.

[0157] 2. Preparation of test solution

[0158] (1) Investigation of different extraction solvents

[0159] Approximately 0.5 g of the granules prepared in Example 1 (Batch No. 2108001) was accurately weighed and placed in a stoppered conical flask. 50 ml of 30% methanol, 50% methanol, 70% methanol, methanol, 30% ethanol, 50% ethanol, 70% ethanol, and ethanol were accurately added in duplicate. The mixture was ultrasonically treated for 30 minutes. The mixture was cooled, weighed again, and the weight loss was made up with the corresponding solvent. The mixture was shaken and filtered. 10 μl of the filtrate was accurately measured and injected into a liquid chromatograph. The peak area was measured under the chromatographic conditions of Example 10. The results are shown in Table 5.

[0160] Table 5 Extraction solvent investigation

[0161]

[0162] The above experimental results show that the content of the extract obtained by 70% ethanol is the highest, and 70% ethanol is preferably used as the extraction solvent.

[0163] (2) Investigation of different extraction methods

[0164] Approximately 0.5 g of the granules prepared in Example 1 (Batch No. 2108001) were accurately weighed and placed in a stoppered conical flask. 50 ml of 70% ethanol was accurately added and weighed. The mixture was then ultrasonically extracted for 30 minutes, refluxed for 30 minutes, and shaken for 30 minutes in duplicate. The mixture was cooled and weighed. The remaining weight was then supplemented with 70% ethanol. The mixture was shaken and filtered. Accurately measure 10 μl of the filtrate and inject it into a liquid chromatograph. The peak area was measured under the chromatographic conditions of Example 10. The results are shown in Table 6.

[0165] Table 6 Comparison of different extraction methods (n=2)

[0166]

[0167]

[0168] The above experimental results show that there is no difference in the content between ultrasonic extraction and reflux extraction, so the ultrasonic extraction method was finally selected.

[0169] (3) Investigation of different extraction times

[0170] About 0.5 g of the granules prepared in Example 1 (batch number: 2108001) was accurately weighed and placed in a stoppered conical flask. 50 ml of 70% ethanol was accurately added and weighed. The extracts were ultrasonically extracted for 15 min, 30 min, 45 min, and 60 min in duplicate. The extracts were cooled and weighed. The lost weight was supplemented with 70% ethanol, shaken, filtered, and the filtrate was accurately measured. 10 μl was injected into a liquid chromatograph and the peak area was measured under the chromatographic conditions of Example 10. The results are shown in Table 7.

[0171] Table 7 Comparison of different extraction times (n=2)

[0172]

[0173] The above experimental results show that there is no significant difference in the content of ultrasonic extraction for 30min, 45min and 60min. In accordance with the principles of simplicity, sensitivity and rapidity of pharmaceutical quality standards, the ultrasonic extraction method for 30min was adopted.

[0174] (4) Investigation of different solvent amounts

[0175] About 0.5 g of the granules prepared in Example 1 (batch number: 2108001) was accurately weighed and placed in a stoppered conical flask. 25 ml, 50 ml, and 100 ml of 70% ethanol were accurately added in duplicate, and the weight was weighed. Ultrasonic extraction was performed for 30 min, and the mixture was cooled and weighed. The lost weight was supplemented with 70% ethanol, shaken, filtered, and the filtrate was accurately measured. 10 μl was injected into a liquid chromatograph, and the peak area was measured under the chromatographic conditions of Example 10. The results are shown in Table 8.

[0176] Table 8 Comparison of different solvent amounts (n=3)

[0177]

[0178]

[0179] The above results show that the baicalin content is the highest after weighing 0.5g of sample and adding 50ml of 70% ethanol for extraction. Considering resource and economic issues, ultrasonic extraction is finally carried out by weighing 0.5g of sample and adding 50ml of 70% ethanol.

[0180] 3. Specificity inspection

[0181] ① Granules were prepared according to the prescription and preparation method described in Example 1, and then the test solution and the reference solution were prepared according to the preparation method of the test solution and the preparation method of the reference solution in Example 10, respectively; the concentration of baicalin in the reference solution was 250 μg / ml; ② Granules not containing Scutellaria baicalensis were prepared according to the prescription ratio and preparation method described in Example 1, i.e., Scutellaria baicalensis negative samples, and then Scutellaria baicalensis negative sample solutions were prepared according to the test solution preparation method described in Example 10.

[0182] According to the chromatographic conditions in Example 10, blank solvent (70% ethanol), test solution, reference solution, and Scutellaria baicalensis negative sample solution were injected into the chromatographic column. The results are shown in Table 1. Figure 39 As shown in the figure, the separation degrees of baicalin and other impurity peaks in the test solution are all greater than 1.5, indicating that the separation is good. The scutellaria negative sample solution does not show a chromatographic peak at the same retention time as baicalin, so it is considered to have no interference. At the same time, the blank solvent has no interference and has strong specificity.

[0183] 4. Linear relationship investigation

[0184] A 530.91 μg / ml baicalin reference solution was used as stock solution No. 1. 10 ml of the solution was taken from No. 2 and diluted to 25 ml to form No. 2. 10 ml of the solution was taken from No. 2 and diluted to 25 ml to form No. 3. A series of concentrations of 5.44, 13.59, 33.98, 84.95, 212.36, and 530.91 μg / ml were prepared in sequence. 10 μl was accurately measured and injected into a liquid chromatograph, and the peak area was measured. The results are shown in Table 9. The peak area of ​​baicalin in the range of 5.44 to 530.91 μg / ml showed a good linear relationship with the concentration of the reference substance, and the regression equation was Y = 36,240,998.05X + 17,149.43, R 2 =1.00. The results showed that the concentration of baicalin was linear in the range of 5.44-530.91 μg / ml.

[0185] Table 9 Baicalin reference substance concentration and peak area results

[0186]

[0187] 5. Precision test

[0188] The reference solution was accurately measured and injected six times using the chromatographic conditions described in Example 10. The baicalin peak area was measured and the relative standard deviation (RSD) of the peak area was calculated. The RSD value was 0.1%. This indicates that the instrument has good precision under these chromatographic conditions.

[0189] Table 10 Precision test results

[0190]

[0191] 6. Stability test

[0192] The above granules were prepared according to the method described in Example 10 to obtain a test solution. 10 μl of the same test solution was accurately aspirated and the chromatographic conditions were the same as in Example 10. Samples were injected at 0, 2, 4, 8, 12, and 24 hours after preparation. The peak areas were measured and the relative standard deviation (RSD) of the peak areas was calculated. The RSD value was 0.8%. The results demonstrated good stability under these chromatographic conditions.

[0193] Table 11 Stability test results

[0194]

[0195] 7. Repeatability test

[0196] Six test solutions were prepared using the granules described above in parallel with the method described in Example 10. The peak area of ​​baicalin was measured and the content was calculated. The average peak area of ​​baicalin in the six samples was 8656138.2, with an RSD of 0.8%. The results demonstrated good reproducibility of this method.

[0197] Table 12 Repeatability test results

[0198]

[0199] 8. Intermediate precision

[0200] Six sample solutions were prepared under the same repeatability test. These solutions were then measured in the same laboratory by different researchers at different times using different instruments. Accurately measure 10 μl of each solution and inject it into a liquid chromatograph using the same chromatographic conditions as in Example 10. Peak areas were measured and the RSD of baicalin in the sample was calculated. The RSD% was 0.6%. The results demonstrate that this method has good intermediate precision.

[0201] Table 13 Intermediate precision test results

[0202]

[0203]

[0204] 9. Recovery rate test

[0205] Approximately 0.25 g of the above granules were accurately weighed, totaling nine portions. A baicalin reference substance was added at a mass ratio of 150%, 100%, and 50% for the high, medium, and low concentration reference substances to the test substance to be measured, respectively. Six test solutions were prepared according to the method for preparing the test solution in Example 10. The solution was assayed under the chromatographic conditions described in Example 10, and the recovery was calculated as follows. The average recovery for the nine samples was 101%, with an RSD of 0.9%. The results demonstrated that the recovery rate of this method met the requirements.

[0206] Table 14 Recovery test

[0207]

[0208] 10. Durability test

[0209] (1) Column temperature investigation

[0210] Test and reference solutions were prepared according to Example 10, and the chromatography was performed at column temperatures of 28°C, 30°C, and 32°C, respectively. The remaining chromatographic conditions were the same as those in Example 10. The test results showed that when the column temperature varied within the range of 28°C to 32°C, the retention time varied slightly, but there was no significant difference in the baicalin content, with an RSD% of 0.4%. The resolution (greater than or equal to 2.34), tailing factor (all 1.11), and number of theoretical plates (all greater than or equal to 11872) all met the requirements, indicating that the method was robust even with slight changes in column temperature.

[0211] Table 15 Investigation results of different column temperatures

[0212]

[0213]

[0214] (2) Flow rate investigation

[0215] A test solution and a reference solution were prepared according to Example 10, and the chromatography was performed at flow rates of 0.9 ml / min, 1.0 ml / min, and 1.1 ml / min, respectively. The remaining chromatographic conditions were the same as those in Example 10. The test results showed that when the flow rate varied within the range of 0.9 to 1.1 ml / min, the retention time and peak area of ​​baicalin changed, and the column pressure also changed, but there was no significant difference in the sample content, with an RSD% of 0.3%. The resolution (greater than or equal to 2.19), tailing factor (all 1.11), and number of theoretical plates (all greater than or equal to 10716) all met the requirements. Therefore, when the instrument flow rate varied slightly, the method had good durability and had little effect on the test results.

[0216] Table 16 Investigation results of different flow rates of baicalin

[0217]

[0218] (3) Investigation of phosphoric acid aqueous solution concentration

[0219] According to Example 10, test sample solutions and reference sample solutions were prepared, and the mobile phase B was 0.18%, 0.2%, and 0.22% phosphoric acid aqueous solution, respectively. The other chromatographic conditions were the same as in Example 10. The test results showed that when the concentration of the phosphoric acid solution in the mobile phase of the finished product of Xiaoer Reliqing changed slightly, the retention time changed, while there was no significant difference in the sample content, and the RSD% was 0.1%. The resolution (greater than or equal to 2.36), tailing factor (all 1.10), and number of theoretical plates (all greater than or equal to 11665) all met the requirements, indicating that the method has good durability when the mobile phase ratio changes slightly, and has little effect on the test results.

[0220] Table 17 Investigation results of phosphoric acid aqueous solution concentration

[0221]

[0222] (4) Chromatographic column inspection

[0223] A test solution and a reference solution were prepared according to Example 10 and assayed using Agilent Eclipse Plus C18, SVEA, and Dima chromatographic columns, respectively. The remaining chromatographic conditions were the same as in Example 10. The test results showed that when the baicalin content was assayed using different chromatographic columns, the retention time and peak area changed, but there was no significant difference in the sample content. The RSD% was 0.2%, and the resolution (greater than or equal to 2.40), tailing factor (between 1.02 and 1.10), and number of theoretical plates (all greater than or equal to 12075) showed good method robustness, indicating that the established method for assaying the content of the finished product of Xiaoer Reliqing was robust on different chromatographic columns.

[0224] Table 18 Investigation results of different chromatographic columns

[0225]

[0226] 11. Multi-batch content determination

[0227] Four batches of pilot samples of granules were prepared according to Example 10 to obtain test solutions and the results showed that the content of baicalin was 23.47-24.94 mg / g, with an average of 24.12 mg / g. Combined with the baicalin transfer rate during the batch production process and taking into account various influencing factors in the actual production process, it is tentatively determined that each 1g of this product contains baicalin (C 21 H 18 O 11 ) shall not be less than 9.0 mg.

[0228] Experimental Example 7 Methodological Investigation of Quantitative Determination of Chlorogenic Acid

[0229] 1. Determination of detection wavelength

[0230] Weigh an appropriate amount of chlorogenic acid reference substance accurately, then add 75% methanol to prepare a 1ml reference solution containing 60 μg of chlorogenic acid. A 3D spectral absorption spectrum of the chlorogenic acid glycoside reference solution at wavelengths between 190 and 400 nm reveals a maximum absorption peak for chlorogenic acid at 326 nm. Since the maximum absorption values ​​are generally consistent within the 325-330 nm range, and considering the detection wavelength specified for honeysuckle in the 2020 edition of the Chinese Pharmacopoeia, 327 nm was selected as the detection wavelength for chlorogenic acid in the granules.

[0231] 2. Preparation of test solution

[0232] (1) Investigation of different extraction solvents

[0233] About 0.5 g of the granules (batch number: 2108001) were accurately weighed and placed in a stoppered conical flask. 30% methanol, 50% methanol, 75% methanol, methanol, 30% ethanol, 50% ethanol, 75% ethanol, and 50 ml of ethanol were accurately added respectively in parallel, and the weight was weighed. Ultrasonic treatment was performed for 30 minutes. The mixture was cooled and weighed again. The lost weight was supplemented with the corresponding solvent, shaken, filtered, and the filtrate was taken. 10 μl of the filtrate was accurately measured and injected into a liquid chromatograph respectively. The peak area was measured under the chromatographic conditions of Example 11. The results are shown in Table 19.

[0234] Table 19 Extraction solvent investigation (n=2)

[0235]

[0236]

[0237] The above experimental results show that the content of 75% methanol extraction is the highest, and 75% methanol is finally selected as the extraction solvent.

[0238] (2) Investigation of different extraction methods

[0239] Take about 0.5 g of the granules (batch number: 2108001), accurately weigh it, place it in a stoppered conical flask, accurately add 50 ml of 75% methanol, weigh the weight, and extract it by ultrasonication for 30 minutes, reflux extraction for 30 minutes, and shaking extraction for 45 minutes, respectively, in parallel. Cool it, weigh it, and make up the lost weight with 75% methanol. Shake it well, filter it, take the filtrate, accurately measure 10 μl, and inject it into the liquid chromatograph respectively. Measure the peak area under the chromatographic conditions of Example 11. The results are shown in Table 20.

[0240] Table 20 Comparison of different extraction methods (n=2)

[0241]

[0242] The above experimental results show that there is no difference in the content between ultrasonic extraction and reflux extraction, so the ultrasonic extraction method was finally selected.

[0243] (3) Investigation of different extraction times

[0244] Take about 0.5 g of granules (batch number: 2108001), accurately weigh, place in a stoppered conical flask, accurately add 50 ml of 75% methanol, weigh the weight, and ultrasonically extract for 15 minutes, 30 minutes, 45 minutes, and 60 minutes, in parallel. Cool, weigh, and make up the lost weight with 75% methanol. Shake well, filter, take the filtrate, accurately measure 10 μl, and inject it into the liquid chromatograph respectively. Measure the peak area under the chromatographic conditions of Example 11. The results are shown in Table 21.

[0245] Table 21 Results of the investigation on extraction time (n=2)

[0246]

[0247] The results showed that the contents of reflux extraction for 45 minutes and 60 minutes were consistent and the highest. Considering the resource saving and cost issues, the ultrasonic extraction method for 45 minutes was adopted.

[0248] (4) Investigation of different solvent amounts

[0249] Take about 0.5 g of granules (batch number: 2108001), accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml, 50 ml, and 100 ml of 75% methanol, each in parallel, weigh the weight, and ultrasonically extract for 30 minutes. Let cool, weigh the weight, make up the lost weight with 75% methanol, shake well, filter, take the filtrate, accurately measure 10 μl, and inject it into the liquid chromatograph respectively. Measure the peak area under the chromatographic conditions of Example 11. The results are shown in Table 22.

[0250] Table 22 Results of investigation on the amount of extraction solvent (n=2)

[0251]

[0252] The above results show that the chlorogenic acid content is the highest after weighing 0.5g of sample and adding 50ml and 100ml of 75% methanol for extraction. Considering resource and economic issues, ultrasonic extraction is finally carried out by weighing 0.5g of sample and adding 50ml of 75% methanol.

[0253] 3. Specificity inspection

[0254] ① Granules were prepared according to the prescription and preparation method described in Example 1, and then the test solution and the reference solution were respectively prepared according to the preparation method of the test solution and the preparation method of the reference solution in Example 11; the concentration of chlorogenic acid in the reference solution was 60 μg / ml; ② Granules not containing honeysuckle, i.e., honeysuckle negative samples, were prepared according to the prescription ratio and preparation method described in Example 11, and honeysuckle negative sample solutions were respectively prepared according to the test solution preparation method described in Example 11.

[0255] According to the chromatographic conditions in Example 11, blank solvent (75% methanol), test solution, reference solution, and honeysuckle negative sample solution were injected into the chromatographic column respectively. The results are shown in Table 1. Figure 40 As shown in the figure, the separation degrees of chlorogenic acid and other impurity peaks in the test solution are all greater than 1.5, indicating that the separation is good. The negative sample solution of honeysuckle has no chromatographic peak at the same retention time as chlorogenic acid, so it is considered to have no interference. At the same time, the blank solvent has no interference and has strong specificity.

[0256] 4. Linear relationship investigation

[0257] A 396.70 μg / ml chlorogenic acid reference solution was used as the stock solution, with the mother solution designated as No. 1. 10 ml of No. 1 was diluted to 25 ml for No. 2, and 10 ml of No. 2 was diluted to 25 ml for No. 3. A series of concentrations of 1.62, 4.06, 10.16, 25.39, 63.47, 158.68, and 396.70 μg / ml was prepared sequentially. Accurately measure 10 μl and inject each into a liquid chromatograph for peak area measurement. The results are shown in Table 23. The peak area of ​​chlorogenic acid within the range of 1.62 to 396.70 μg / ml showed a good linear relationship with the concentration of the reference solution, with the regression equation being Y = 42,344.75X + 4,149.04R. 2 =1.00. The results showed that chlorogenic acid had a linear relationship in the range of 1.62-396.70 μg / ml.

[0258] Table 23 Chlorogenic acid reference substance concentration and peak area results

[0259]

[0260] 5. Precision test

[0261] Accurately measure the reference solution and repeat the injection six times using the chromatographic conditions described in Example 11. The chlorogenic acid peak area was measured and the relative standard deviation (RSD) of the peak area was calculated. The RSD value was 0.2%. This result demonstrates good instrument precision under these chromatographic conditions.

[0262] Table 24 Precision test results

[0263]

[0264] 6. Stability test

[0265] The above granules were prepared according to the method described in Example 11 to obtain a test solution. 10 μl of the same test solution was accurately aspirated and the chromatographic conditions were the same as in Example 11. Samples were injected 0, 2, 4, 8, 12, and 24 hours after preparation. The peak areas were measured and the relative standard deviation (RSD) of the peak areas was calculated. The RSD value was 0.3%. The results demonstrated good stability under these chromatographic conditions.

[0266] Table 25 Stability test results

[0267]

[0268] 7. Repeatability test

[0269] Six test solutions were prepared using the granules described above in parallel with the method described in Example 11. The peak area of ​​chlorogenic acid was measured and the content was calculated. The average chlorogenic acid content in the six samples was 5.6 mg / g, with an RSD of 0.1%. These results demonstrate good reproducibility of this method.

[0270] Table 26 Repeatability test results

[0271]

[0272] 8. Intermediate precision

[0273] Six sample solutions were prepared under the same repeatability test. These solutions were measured in the same laboratory by different researchers at different times using different instruments. Accurately measure 10 μl of each solution and inject it into a liquid chromatograph using the same chromatographic conditions as in Example 11. Peak areas were measured and the RSD of chlorogenic acid in the sample was calculated. The RSD% was 0.8%. The results demonstrate good intermediate precision.

[0274] Table 27 Intermediate precision test results

[0275]

[0276] 9. Recovery rate test

[0277] Approximately 0.25 g of the above-mentioned granules were accurately weighed, totaling nine portions. A chlorogenic acid reference substance was added at a mass ratio of 150%, 100%, and 50% for the high, medium, and low concentration reference substances to the test substance component, respectively. Six test solutions were prepared according to the preparation method of the test solution in Example 11. The chromatographic conditions described in Example 11 were used for analysis, and the recovery was calculated as follows. The average recovery for the nine samples was 99%, with an RSD of 1.6%. These results demonstrate that the recovery rate of this method meets the required standards.

[0278] Table 28 Chlorogenic acid recovery test

[0279]

[0280]

[0281] 10. Durability test

[0282] (1) Column temperature investigation

[0283] The test solution and the reference solution were prepared according to Example 11, and the column temperatures were 23°C, 25°C, and 27°C, respectively. The other chromatographic conditions were the same as in Example 11. The test results showed that when the column temperature changed within the range of 23-27°C, the retention time was slightly different, but there was no significant difference in the chlorogenic acid content, the RSD% was 0.2%, and the resolution (greater than or equal to 8.72), tailing factor (all between 1.11-114), and number of theoretical plates (all greater than or equal to 21661) all met the requirements, indicating that the chlorogenic acid content determination method has good durability when the column temperature changes slightly.

[0284] Table 29 Investigation results of different column temperatures

[0285]

[0286] (2) Flow rate investigation

[0287] The test solution and the reference solution were prepared according to Example 11, and the analysis was performed at flow rates of 0.7 ml / min, 0.8 ml / min, and 0.9 ml / min, respectively. The other chromatographic conditions were the same as those in Example 11. The test results showed that when the flow rate changed within the range of 0.7 to 0.9 ml / min, the retention time and peak area of ​​chlorogenic acid changed, and the column pressure also changed, but there was no significant difference in the sample content, the RSD% was 0.3%, and the resolution (greater than or equal to 20.92), tailing factor (all between 1.12 and 1.14), and number of theoretical plates (all greater than or equal to 19483) all met the requirements. Therefore, when the instrument flow rate changed slightly, the method was robust and had little effect on the test results.

[0288] Table 30 Investigation results of chlorogenic acid at different flow rates

[0289]

[0290] (3) Investigation of phosphoric acid aqueous solution concentration

[0291] According to Example 11, test solutions and reference solutions were prepared, and the assay was performed using 0.18%, 0.2%, and 0.22% aqueous phosphoric acid as mobile phase B, respectively. The remaining chromatographic conditions were the same as in Example 11. The test results showed that when the concentration of aqueous phosphoric acid in the mobile phase changed slightly, the retention time of the finished product of Xiaoer Reliqing changed, while there was no significant difference in the sample content, and the RSD% was 0.2%. The resolution (greater than or equal to 18.85), tailing factor (all between 1.02-1.06), and number of theoretical plates (all greater than or equal to 18143) all met the requirements, indicating that the method has good durability and has little effect on the test results when the mobile phase ratio changes slightly.

[0292] Table 31 Investigation results of phosphoric acid aqueous concentration

[0293]

[0294] (4) Chromatographic column inspection

[0295] The test solution and the reference solution were prepared according to Example 11 and determined using Agilent Eclipse Plus C18, SVEA, and Dima chromatographic columns, respectively. The remaining chromatographic conditions were the same as in Example 11. The test results showed that when different chromatographic columns were used to determine the chlorogenic acid content, the retention time and peak area changed, but there was no significant difference in the sample content, and the RSD% was 3.0%. The resolution (greater than or equal to 19.15), tailing factor (all between 0.96-1.07), and number of theoretical plates (all greater than or equal to 18676) all met the requirements. The method was robust, indicating that the established method for determining the content of the finished product of Xiaoer Reliqing was robust for different chromatographic columns.

[0296] Table 32 Investigation results of different chromatographic columns

[0297]

[0298] 11. Multi-batch content determination

[0299] Four batches of pilot samples of granules were taken to prepare test solutions according to Example 11 and measured. The results showed that the content of chlorogenic acid was 5.06-5.63 mg / g, with an average of 5.30 mg / g. Combined with the chlorogenic acid transfer rate during batch production and taking into account various influencing factors in the actual production process, it is tentatively estimated that each 1g of this product contains chlorogenic acid (C 16 H 18 O9), not less than 1.0 mg.

[0300] Example 1 Particles

[0301] This embodiment provides a traditional Chinese medicine composition, comprising the following raw materials: 250g of Artemisia annua, 250g of Scutellaria baicalensis, 375g of Poria cocos, 150g of dried tangerine peel, 150g of Rhizoma Anemarrhenae, 750g of Gypsum, 375g of Honeysuckle, 375g of Isatis indigotica, 250g of Pinellia ternata, 150g of Periostracum cicadae, 150g of Menthol, 150g of Licorice and 50g of Indigo Naturalis.

[0302] This embodiment also provides granules of the above-mentioned Chinese medicine composition, and the preparation method thereof is as follows:

[0303] Add 6 times the amount of water to gypsum and boil for 30 minutes. Collect the decoction and residue to obtain A; mix Artemisia annua, Scutellaria baicalensis, Poria cocos, dried tangerine peel, Rhizoma Anemarrhenae, Flos Lonicerae, Folium Isatidis, Rhizoma Pinelliae, Periostracum Cicadae, Mentha, and Glycyrrhizae, add A, add water to 11.5 times the weight of the prescribed medicinal materials (except Indigo), soak for 2 hours, boil for 1.5 hours, filter the decoction, and collect the filtrate; add 10 times the amount of water to the residue, boil for 1.5 hours, filter, combine the filtrates, and concentrate under reduced pressure to an extract with a relative density of 1.05-1.12 (60°C), spray-dry into a fine powder, add the prescribed amount of Indigo, 5g of stevioside, 40g of citric acid, and an appropriate amount of dextrin, mix evenly, and dry granulate to make 1000g of granules.

[0304] Example 2 Syrup

[0305] This embodiment provides a traditional Chinese medicine composition, comprising the following raw materials: 100g of Artemisia annua, 100g of Scutellaria baicalensis, 150g of Poria cocos, 100g of dried tangerine peel, 100g of Rhizoma Anemarrhenae, 580g of Gypsum, 200g of Honeysuckle, 200g of Isatis indigotica, 150g of Pinellia ternata, 100g of Periostracum cicadae, 100g of Menthol, 100g of Licorice and 20g of Indigo Naturalis.

[0306] This embodiment also provides a syrup of the above-mentioned Chinese medicine composition, and its preparation method is as follows:

[0307] Add 6 times the weight of water to gypsum and boil for 30 minutes. Collect the decoction and residue to obtain A. Mix Artemisia annua, Scutellaria baicalensis, Poria cocos, Tangerine peel, Rhizoma Anemarrhenae, Flos Lonicerae, Folium Isatidis, Rhizoma Pinelliae, Periostracum Cicadae, Menthol, Glycyrrhizae uralensis, and Indigo Naturalis, add A, add water to 11.5 times the weight of the prescribed medicinal materials (excluding Indigo Naturalis), soak for 2 hours, boil for 1.5 hours, filter the decoction, and collect the filtrate. Add 10 times the weight of water to the residue, boil for 1.5 hours, filter, combine the filtrates, and concentrate under reduced pressure to an extract with a relative density of 1.05-1.12 (60°C). Separately, take 2000 g of sucrose, add water, boil, dissolve, filter, and concentrate to obtain a syrup. Mix the mixture with the above concentrate, boil, cool, add a preservative and flavoring, and dilute with an appropriate amount of cold water to obtain a syrup.

[0308] Example 3 Tablets

[0309] This embodiment provides a traditional Chinese medicine composition, comprising the following raw materials: 300g of Artemisia annua, 300g of Scutellaria baicalensis, 450g of Poria cocos, 270g of dried tangerine peel, 270g of Rhizoma Anemarrhenae, 820g of Gypsum, 450g of Honeysuckle, 450g of Isatis indigotica, 300g of Pinellia ternata, 270g of Periostracum cicadae, 270g of Menthol, 270g of Licorice and 60g of Indigo Naturalis.

[0310] This embodiment also provides tablets of the above-mentioned Chinese medicine composition, and the preparation method thereof is as follows:

[0311] Add 6 times the amount of water to gypsum, decoct for 30 minutes, collect the decoction and residue to obtain A; mix Artemisia annua, Scutellaria baicalensis, Poria cocos, dried tangerine peel, Rhizoma Anemarrhenae, Flos Lonicerae, Folium Isatidis, Rhizoma Pinelliae, Periostracum Cicadae, Mentha, and Glycyrrhizae, add A, add water to 11.5 times the weight of the prescribed medicinal materials (except Indigo Naturalis), soak for 2 hours, decoct for 1.5 hours, filter the decoction, and collect the filtrate; add 10 times the amount of water to the residue, decoct for 1.5 hours, filter, combine the filtrates, and concentrate under reduced pressure to an extract with a relative density of 1.05-1.12 (60° C.), spray-dry into a fine powder, add the prescribed amount of Indigo Naturalis and an appropriate amount of dextrin, mix evenly, dry granulate, and then add 0.5% magnesium stearate and 5% talc based on the mass of the granules, mix evenly, compress into tablets, and coat to obtain the product.

[0312] Example 4 Micropellets

[0313] This embodiment provides a traditional Chinese medicine composition, comprising the following raw materials: 200g of Artemisia annua, 200g of Scutellaria baicalensis, 250g of Poria cocos, 170g of dried tangerine peel, 170g of Rhizoma Anemarrhenae, 650g of Gypsum, 350g of Honeysuckle, 250g of Folium Isatidis, 200g of Pinellia ternata, 170g of Periostracum Cicadae, 170g of Menthol, 170g of Licorice and 40g of Indigo Naturalis.

[0314] This embodiment also provides pellets of the above-mentioned Chinese medicine composition, which are prepared as follows:

[0315] Add 6 times the amount of water to gypsum, decoct for 30 minutes, collect the decoction and residue to obtain A; mix Artemisia annua, Scutellaria baicalensis, Poria cocos, dried tangerine peel, Rhizoma Anemarrhenae, Honeysuckle, Folium Isatidis, Pinellia ternata, Periostracum Cicadae, Menthol, and Licorice, add A, add water to 11.5 times the weight of the prescribed medicinal materials (except Indigo), soak for 2 hours, decoct for 1.5 hours, filter the decoction, and collect the filtrate; add 10 times the amount of water to the residue, decoct for 1.5 hours, filter, combine the filtrate, and concentrate under reduced pressure to an extract with a relative density of 1.05-1.12 (60°C), spray-dry into a fine powder, add the prescribed amount of Indigo and an appropriate amount of starch, mix evenly, use ethanol as a wetting agent, make micropills, and sieve; dry below 60°C to obtain micropills.

[0316] Example 5 Powder

[0317] This embodiment provides a traditional Chinese medicine composition, comprising the following raw materials: 12 kg of Artemisia annua, 12 kg of Scutellaria baicalensis, 18 kg of Poria cocos, 7.2 kg of dried tangerine peel, 7.2 kg of Rhizoma Anemarrhenae, 36 kg of Gypsum, 18 kg of Honeysuckle, 18 kg of Isatis indigotica, 12 kg of Pinellia ternata, 7.2 kg of Cicada Periostracum, 7.2 kg of Menthol, 7.2 kg of Licorice, and 2.4 kg of Indigo Naturalis.

[0318] This embodiment also provides a powder of the above-mentioned Chinese medicine composition, and its preparation method is as follows:

[0319] Add 6 times the amount of water to gypsum and boil for 30 minutes. Collect the decoction and residue to obtain A. Mix Artemisia annua, Scutellaria baicalensis, Poria cocos, Tangerine peel, Rhizoma Anemarrhenae, Flos Lonicerae, Folium Isatidis, Pinellia ternata, Periostracum Cicadae, Mentha, and Licorice, add A, add water to 11.5 times the weight of the prescribed medicinal materials (except Indigo), soak for 2 hours, boil for 1.5 hours, filter the decoction, and collect the filtrate; add 10 times the amount of water to the residue, boil for 1.5 hours, filter, combine the filtrates, and concentrate under reduced pressure to an extract with a relative density of 1.05-1.12 (60°C), spray-dry into a fine powder, and mix evenly with Indigo to obtain a powder.

[0320] Example 6 Powder

[0321] This embodiment provides a traditional Chinese medicine composition, comprising the following raw materials: 250g of Artemisia annua, 250g of Scutellaria baicalensis, 375g of Poria cocos, 150g of dried tangerine peel, 150g of Rhizoma Anemarrhenae, 750g of Gypsum, 375g of Honeysuckle, 375g of Isatis indigotica, 250g of Pinellia ternata, 150g of Periostracum cicadae, 150g of Menthol, 150g of Licorice and 50g of Indigo Naturalis.

[0322] This embodiment also provides a powder of the above-mentioned Chinese medicine composition, and its preparation method is as follows:

[0323] Add 6 times the amount of 30% ethanol to gypsum, decoct for 30 minutes, collect the decoction and residue to obtain A; mix Artemisia annua, Scutellaria baicalensis, Poria cocos, dried tangerine peel, Rhizoma Anemarrhenae, Flos Lonicerae, Folium Isatidis, Rhizoma Pinelliae, Periostracum Cicadae, Menthol, and Glycyrrhizae, add A, add 30% ethanol to 11.5 times the weight of the prescribed medicinal materials (except Indigo Naturalis), soak for 2 hours, decoct for 1.5 hours, filter the decoction, and collect the filtrate; add 10 times the amount of 30% ethanol to the residue, decoct for 1.5 hours, filter, combine the filtrate, and concentrate under reduced pressure to an extract with a relative density of 1.05-1.12 (60°C), spray-dry into a fine powder, and mix evenly with Indigo Naturalis to obtain a powder.

[0324] Example 7 Powder

[0325] This embodiment provides a traditional Chinese medicine composition, comprising the following raw materials: 250g of Artemisia annua, 250g of Scutellaria baicalensis, 375g of Poria cocos, 150g of dried tangerine peel, 150g of Rhizoma Anemarrhenae, 750g of Gypsum, 375g of Honeysuckle, 375g of Isatis indigotica, 250g of Pinellia ternata, 150g of Periostracum cicadae, 150g of Menthol, 150g of Licorice and 50g of Indigo Naturalis.

[0326] This embodiment also provides a powder of the above-mentioned Chinese medicine composition, and its preparation method is as follows:

[0327] Add 6 times the amount of 50% ethanol to gypsum, decoct for 30 minutes, collect the decoction and residue to obtain A; mix Artemisia annua, Scutellaria baicalensis, Poria cocos, dried tangerine peel, Rhizoma Anemarrhenae, Flos Lonicerae, Folium Isatidis, Rhizoma Pinelliae, Periostracum Cicadae, Menthol, and Glycyrrhizae, add A, add 50% ethanol to 11.5 times the weight of the prescribed medicinal materials (except Indigo Naturalis), soak for 2 hours, decoct for 1.5 hours, filter the decoction, and collect the filtrate; add 10 times the amount of 50% ethanol to the residue, decoct for 1.5 hours, filter, combine the filtrate, and concentrate under reduced pressure to an extract with a relative density of 1.05-1.12 (60°C), spray-dry into a fine powder, and mix evenly with Indigo Naturalis to obtain a powder.

[0328] Example 8 Powder

[0329] This embodiment provides a traditional Chinese medicine composition, comprising the following raw materials: 250g of Artemisia annua, 250g of Scutellaria baicalensis, 375g of Poria cocos, 150g of dried tangerine peel, 150g of Rhizoma Anemarrhenae, 750g of Gypsum, 375g of Honeysuckle, 375g of Isatis indigotica, 250g of Pinellia ternata, 150g of Periostracum cicadae, 150g of Menthol, 150g of Licorice and 50g of Indigo Naturalis.

[0330] This embodiment also provides a powder of the above-mentioned Chinese medicine composition, and its preparation method is as follows:

[0331] Add 6 times the amount of 75% ethanol to gypsum, decoct for 30 minutes, collect the decoction and residue to obtain A; mix Artemisia annua, Scutellaria baicalensis, Poria cocos, dried tangerine peel, Rhizoma Anemarrhenae, Flos Lonicerae, Folium Isatidis, Pinellia ternata, Periostracum Cicadae, Menthol, and Licorice, add A, add 75% ethanol to 11.5 times the weight of the prescribed medicinal materials (except Indigo Naturalis), soak for 2 hours, decoct for 1.5 hours, filter the decoction, and collect the filtrate; add 10 times the amount of 75% ethanol to the residue, decoct for 1.5 hours, filter, combine the filtrate, and concentrate under reduced pressure to an extract with a relative density of 1.05-1.12 (60°C), spray-dry into a fine powder, and mix evenly with Indigo Naturalis to obtain a powder.

[0332] Example 9 Quality Inspection

[0333] The granules prepared in Example 1 were subjected to quality testing in the following steps:

[0334] A. Identification of Artemisia annua and Tangerine Peel

[0335] Preparation of the test solution: Take 4g of the granules, add 50ml of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate in a water bath to dryness, add 40ml of water to the residue, gently heat to dissolve, shake and extract twice with 40ml of water-saturated n-butanol, combine the n-butanol solutions, evaporate to dryness in a water bath, and dissolve the residue in 4ml of methanol. Pipette 2ml onto a neutral alumina column (100-200 mesh, 5g, inner diameter 1.5cm), elute with 30ml of 10% ethanol, discard the eluate, and elute with 30ml of 30% ethanol again. Collect the eluate, evaporate to dryness, and dissolve the residue in 2ml of methanol to prepare the test solution.

[0336] Preparation of negative sample solution: Take tangerine peel negative sample and Artemisia annua negative sample and prepare tangerine peel negative sample solution and Artemisia annua negative sample solution in the same way as the preparation of the test solution;

[0337] Preparation of a control medicinal solution: 0.25g of Artemisia annua and 0.5g of Tangerine Peel were added to 50ml of water, heated under reflux for 30 minutes, and centrifuged. The supernatant was evaporated to 3ml in a water bath, 50ml of methanol was added, and ultrasonic treatment was performed for 30 minutes. The filtrate was evaporated to dryness in a water bath, and the residue was dissolved by adding 40ml of water and slightly warmed. The mixture was extracted twice with 40ml of water-saturated n-butanol by shaking. The n-butanol solutions were combined and evaporated to dryness in a water bath. The residue was dissolved in 2ml of methanol. The mixture was added to a neutral alumina column (100-200 mesh, 5g, inner diameter 1.5cm), and eluted with 30ml of 10% ethanol. The eluate was discarded and then eluted with 30ml of 30% ethanol. The eluate was collected and evaporated to dryness. The residue was dissolved in 2ml of methanol to serve as the control medicinal solution.

[0338] Identification: According to the thin-layer chromatography method (General Method 0502), 6 μl of the test sample solution and 4 μl of the tangerine peel control medicinal material solution are spotted separately onto the same silica gel G high-performance thin-layer plate soaked in 0.5% sodium hydroxide. Using petroleum ether (60-90°C)-toluene-acetone (9:2:5) as the developing solvent, presaturate in a developing cylinder for 15 minutes. Develop, remove, air-dry, spray with 3% AlCl₃ ethanol solution, heat at 105°C, and examine under ultraviolet light (365nm). In the chromatogram of the test sample, a fluorescent spot of the same color will appear at the corresponding position in the chromatogram of the control medicinal material.

[0339] According to the thin-layer chromatography method (General Method 0502), 2 μl of the test sample solution and 2 μl of the Artemisia annua solution were spotted separately on silica gel G plates. Developed with cyclohexane-toluene-acetone-formic acid (4:4:2:0.5), the plates were removed, air-dried, and examined under ultraviolet light (365 nm). Fluorescent spots of the same color should appear in the chromatogram of the test sample at the corresponding positions in the chromatogram of the control.

[0340] Example 10 Quality Inspection

[0341] The granules prepared in Example 1 were subjected to quality testing in the following steps:

[0342] C. Determination of baicalin content

[0343] Preparation of test solution: Take 0.5 g of the granules, accurately weigh, place in a stoppered conical flask, accurately add 50 ml of 70% ethanol, weigh, treat with ultrasound (power 250 W, frequency 40 kHz) for 30 minutes, let cool, weigh again, make up the loss with 70% ethanol, shake well, filter, and take the filtrate;

[0344] Preparation of reference solution: Weigh an appropriate amount of baicalin reference solution accurately, add 70% ethanol to prepare a reference solution containing 250 μg of baicalin per ml;

[0345] Preparation method of negative sample solution: weigh the medicinal pieces according to the prescription ratio and prepare the negative sample of Scutellaria baicalensis using the same method, and prepare the negative sample solution according to the treatment method of the test sample.

[0346] Chromatographic conditions: Determine by HPLC using octadecyl silica gel as the filler; acetonitrile as mobile phase A, 0.2% phosphoric acid as mobile phase B, gradient elution as specified in the table below, flow rate 1.0 ml / min; column temperature 30°C; detection wavelength 278 nm; theoretical plate number calculated based on the baicalin peak should be no less than 5000.

[0347]

[0348] Determination method: Accurately aspirate 10 μl of reference solution and test solution respectively, inject into liquid chromatograph and determine.

[0349] Example 11 Quality Inspection

[0350] The granules prepared in Example 1 were subjected to quality testing in the following steps:

[0351] D. Determination of chlorogenic acid content

[0352] Preparation of test solution: Take 0.5 g of the granules, accurately weigh, place in a stoppered conical flask, accurately add 50 ml of 75% methanol, weigh the weight, ultrasonicate (250 W, frequency 40 kHz) for 45 minutes, let cool, weigh again, make up the loss with 75% methanol, shake well, filter, and collect the filtrate;

[0353] Preparation of reference solution: Weigh an appropriate amount of chlorogenic acid reference solution accurately, add 75% methanol to prepare a reference solution containing 60 μg of chlorogenic acid in 1 ml;

[0354] Preparation method of negative sample solution: weigh the medicinal pieces according to the prescription ratio and prepare the honeysuckle negative sample in the same way, and prepare the negative sample solution according to the treatment method of the test sample.

[0355] Chromatographic conditions: Determine by high performance liquid chromatography using octadecyl silica bonded phase as the filler; tetrahydrofuran-methanol-acetonitrile (1:8:16) as mobile phase A, 0.2% phosphoric acid as mobile phase B, gradient elution as specified in the table below, flow rate 0.8 ml / min; column temperature 25°C; detection wavelength 327 nm; theoretical plate number calculated based on the chlorogenic acid peak should be no less than 8000.

[0356]

[0357] Determination method: Accurately aspirate 10 μl of reference solution and test solution respectively, inject into liquid chromatograph and determine.

[0358] Example 12 Capsules

[0359] This embodiment provides a traditional Chinese medicine composition, comprising the following raw materials: 100g of Artemisia annua, 120g of Scutellaria baicalensis, 125g of Poria cocos, 100g of dried tangerine peel, 110g of Rhizoma Anemarrhenae, 650g of Gypsum, 275g of Honeysuckle, 225g of Folium Isatidis, 280g of Pinellia ternata, 250g of Periostracum Cicadae, 110g of Menthol, 120g of Licorice and 30g of Indigo Naturalis.

[0360] This embodiment also provides a powder of the above-mentioned Chinese medicine composition, and its preparation method is as follows:

[0361] Add 3 times the amount of 50% ethanol to gypsum, decoct for 30 minutes, collect the decoction and residue to obtain A; mix Artemisia annua, Scutellaria baicalensis, Poria cocos, dried tangerine peel, Rhizoma Anemarrhenae, Flos Lonicerae, Folium Isatidis, Rhizoma Pinelliae, Periostracum Cicadae, Mentha, and Glycyrrhizae, add A, add 50% ethanol to 10 times the weight of the prescribed medicinal materials (except Indigo Naturalis), soak for 1.5 hours, decoct for 1 hour, filter the decoction, and collect the filtrate; add 10 times the amount of 50% ethanol to the residue, decoct for 1 hour, filter, combine the filtrates, and concentrate under reduced pressure to an extract with a relative density of 1.05-1.12 (60°C), spray-dry into a fine powder, mix evenly with Indigo Naturalis, granulate, and make into capsules.

[0362] Example 13 Composition

[0363] This embodiment provides a traditional Chinese medicine composition, comprising the following raw materials: 280g of Artemisia annua, 300g of Scutellaria baicalensis, 425g of Poria cocos, 260g of dried tangerine peel, 250g of Rhizoma Anemarrhenae, 800g of Gypsum, 425g of Honeysuckle, 425g of Isatis indigotica, 120g of Pinellia ternata, 110g of Periostracum cicadae, 250g of Menthol, 270g of Licorice and 170g of Indigo Naturalis.

[0364] This embodiment also provides a powder of the above-mentioned Chinese medicine composition, and its preparation method is as follows:

[0365] Add 3 times the amount of water to gypsum, decoct for 30 minutes, collect the decoction and residue to obtain A; mix Artemisia annua, Scutellaria baicalensis, Poria cocos, dried tangerine peel, Rhizoma Anemarrhenae, Flos Lonicerae, Folium Isatidis, Rhizoma Pinelliae, Periostracum Cicadae, Mentha, Glycyrrhizae uralensis, and Indigo Naturalis, add A, add water to 10 times the weight of the prescribed medicinal materials, soak for 2 hours, decoct for 1.5 hours, filter the decoction, and collect the filtrate; add 10 times the amount of water to the residue, decoct for 1 hour, filter, combine the filtrates, concentrate under reduced pressure to an appropriate amount, add an appropriate amount of ethanol for precipitation, remove the supernatant, recover the ethanol under reduced pressure, add water, stir, and let stand; remove the supernatant, filter, concentrate the filtrate to 4000 ml, add an appropriate amount of preservative, and stir well to obtain the product.

[0366] Pharmacodynamic experiments

[0367] 1. Drug testing and animals

[0368] The powders prepared in Example 5, Example 6, Example 7, and Example 8 were used as test drugs.

[0369] An appropriate amount of the powder prepared in Example 5 was weighed, ground uniformly, and then a small amount of ultrapure water was added to continue grinding. Finally, the volume was adjusted to the desired concentration with ultrapure water.

[0370] Weigh appropriate amounts of the powders prepared in Example 6, Example 7, and Example 8, respectively, grind them uniformly, then add a small amount of ultrapure water and continue grinding until the desired concentration is achieved with ultrapure water.

[0371] Ibuprofen suspension is available in 2g per 100mL, or 20mg / mL. Add an appropriate volume of ibuprofen suspension to the desired concentration in ultrapure water. Prepare under normal conditions and prepare immediately before use.

[0372] Weigh an appropriate amount of Xiaoer Chiqiao Qingre Granule powder, grind it evenly, then add a small amount of ultrapure water and continue grinding until the desired concentration is achieved with ultrapure water.

[0373] Angel highly active dry yeast, properties: light yellow thin strips, storage conditions: cool and dry, specifications: 15g / bag, batch number: 20230103, shelf life: 12 months, manufacturer: Angel Yeast Co., Ltd., provider: Tianjin Tiancheng New Drug Evaluation Co., Ltd.

[0374] SD rats, grade: SPF grade, weight / age: 140-160 g / 4-5 weeks old, sex: half male and half female, quantity: 200, quality certificate number 110011231105520161 (male), 110011231105520075 (female), supplier: Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., license number: SCXK (Beijing) 2021-0006.

[0375] 2. Experimental methods

[0376] (1) Construction of animal model

[0377] Two hundred healthy Sprague-Dawley rats, 4-5 weeks old, weighing 140-160 g (half male and half female), were selected and acclimated for four days. After the acclimation period, rectal temperature was measured twice daily to acclimate the rats to the environment and the temperature stimulus. Rats with rectal temperatures between 36.6 and 37.9°C (with a rectal temperature difference of no more than 0.5°C) for two consecutive days were selected and retained.

[0378] The retained rats were fasted overnight before the experiment. Rectal temperatures were measured twice on the day of the experiment, and the average was used as the baseline rectal temperature (before modeling). After the experiment, 10 rats were randomly selected as a normal control group based on the baseline rectal temperature. The remaining animals were subcutaneously injected with 10 mL / kg of a 20% yeast suspension for modeling. The injection site was massaged to ensure uniform subcutaneous diffusion of the suspension. Six hours after modeling, rectal temperatures were measured, and rats with a rectal temperature increase of 1.2°C or more were selected as qualified rats for modeling.

[0379] (2) Experimental groups

[0380] Qualified rats were selected and randomly divided into groups according to the rectal temperature difference. See Table 33 and Table 34 for details.

[0381] Table 33 Effect of the powder of Example 5 on the fever model of rats induced by dry yeast solution Experimental groups and administration methods

[0382]

[0383] Note: n: number of animals

[0384] Table 34 Effects of the powders of Examples 5, 6, 7, and 8 on the fever model of rats induced by dry yeast solution Experimental groups and administration methods

[0385]

[0386] Note: n: number of animals

[0387] (3) Dosage setting and administration

[0388] Example 5: Example 5 is clinically applicable to children aged 3 to 12 years, with a daily dose of 137 g of crude drug / kg. Based on the average weight of 26.4 kg for eight-year-old children, the clinical equivalent dose in rats, calculated using the Meeh-Rubner formula based on body surface area, is 23.6 g of crude drug / kg. Based on the results of previous experiments, the doses of Example 5 in this experiment were set at 2, 1, and 0.5 times the clinical equivalent dose, to 47.2, 23.6, and 11.8 g of crude drug / kg, respectively.

[0389] Example 6: Referring to the experimental results of Example 5, the dosage setting was consistent with the high dose of 47.2 crude drug / kg in Example 5.

[0390] Example 7: Referring to the experimental results of Example 5, the dosage setting is consistent with the high dose of 47.2 crude drug / kg in Example 5.

[0391] Example 8: Referring to the experimental results of Example 5, the dosage setting was consistent with the high dose of 47.2 crude drug / kg in Example 5.

[0392] Ibuprofen suspension: The clinical daily dose of ibuprofen suspension for children aged 7 to 9 years is 0.64g. Based on the average weight of 26.4kg for eight-year-old children, the clinical equivalent dose for rats is 0.11g / kg, estimated by the Meeh-Rubner formula based on body surface area.

[0393] Pediatric Chiqiao Qingre Granules: The clinical daily dosage of Pediatric Chiqiao Qingre Granules for children aged 7 to 9 years is 15g. Based on the average weight of 26.4kg for eight-year-old children, the clinical equivalent dose for rats is 2.60g / kg estimated by the Meeh-Rubner formula based on body surface area.

[0394] The drug was administered orally in a manner similar to that used in clinical practice. Each dose group was given the drug by gavage, with a volume of 10 mL / kg per gavage. The normal control group and the model control group were given an equal volume of ultrapure water by gavage, once after the experimental grouping and 5 hours after the first dose.

[0395] (4) Detection indicators

[0396] Research indicators: Measure the basal rectal temperature (before modeling), before administration, and 1 to 11 hours after the first administration of rats. Measure once every hour and calculate the rectal temperature difference at each time point (rectal temperature difference = rectal temperature at each time point after administration - basal rectal temperature).

[0397] (5) Data processing

[0398] The measurement data were expressed as mean ± standard error (SEM) and statistically analyzed using SPSS 25.0 software. One-way ANOVA was performed when the variances were equal. When there were significant differences in the one-way ANOVA, pairwise comparisons were performed using the LSD test. When there were unequal variances, the Kruskal-Wallis nonparametric test was performed, and pairwise comparisons were performed when there were significant differences.

[0399] The test level was α=0.05, and P<0.05 indicated that the difference was statistically significant.

[0400] (6) Handling unexpected situations

[0401] During the experiment, the rectal temperature of the rats in the model control group showed a downward trend over time. To prevent the observation period from being shortened after the second drug administration, the second drug administration time was adjusted to 5 hours after the first administration. The original plan was to have a 6-hour interval between the two administrations.

[0402] 3. Test results

[0403] (1) Experimental results of the powder of Example 5 on the fever model of rats induced by dry yeast solution

[0404] The test results showed that 6 hours after the rats were subcutaneously injected with 20% yeast suspension, the rectal temperature of the rats in each dosage group was significantly increased compared with the normal group (P < 0.01), indicating that the model was successfully established. Compared with the model control group, Example 5 (23.6g, 47.2g crude drug / kg) can significantly reduce the rectal temperature of rats 3h after the first administration (P < 0.05, P < 0.01); Example 5 (11.8g crude drug / kg) can significantly reduce the rectal temperature of rats 7h to 9h after the first administration (P < 0.05); Ibuprofen suspension 0.11g / kg has a very significant effect of reducing the rectal temperature of rats 1h to 11h after the first administration (P < 0.01); Children's Chiqiao Qingre Granules 2.6g / kg can significantly reduce the rectal temperature of rats 5h after the first administration (P < 0.05, P < 0.01); Compared with Children's Chiqiao Qingre Granules, the rectal temperature of rats in Example 5 was significantly reduced at 6h to 10h (P < 0.05, P < 0.01). The test results are shown in Tables 35 and 36 for details.

[0405] Table 35 Effect of the powder of Example 5 on yeast-induced fever in young rats (Mean ± SD, n)

[0406]

[0407] Note: Compared with the normal control group, ##P<0.01; compared with the model control group, *P<0.05; **P<0.01; compared with the Xiaoer Chiqiao Qingre Granule group, △P<0.05; △△P<0.05; n: number of animals

[0408] Table 36 Effect of the powder of Example 5 on yeast-induced fever in young rats (Mean±SD, n)

[0409]

[0410] Note: Compared with the normal control group, ##P<0.01; compared with the model control group, *P<0.05; **P<0.01; compared with the Xiaoer Chiqiao Qingre Granule group, △P<0.05; △△P<0.05; n: number of animals

[0411] (2) Experimental results of the powders of Examples 5, 6, 7, and 8 on fever in rats induced by dry yeast solution

[0412] The test results showed that 6 hours after subcutaneous injection of 20% yeast suspension in rats, the rectal temperature of rats in each dose group was significantly increased compared with the normal group (P < 0.01), indicating that the model was successfully established. Compared with the model control group, the powders of Example 5, Example 6, Example 7, and Example 8 were able to significantly reduce the rectal temperature of rats 4 hours to 11 hours after the first administration (P < 0.05, P < 0.01); Ibuprofen suspension 0.11g / kg had a very significant effect of reducing the rectal temperature of rats 1 hour to 11 hours after the first administration (P < 0.01); There was no significant difference in the antipyretic effect between the powders of Example 5, Example 6, Example 7, and Example 8 (P > 0.05). The test results are shown in Tables 37 and 38.

[0413] Table 37 Effects of the powders of Examples 5, 6, 7, and 8 on yeast-induced fever in young rats (Mean ± SD, n)

[0414]

[0415] Note: Compared with the normal control group, ##P<0.01; compared with the model control group, *P<0.05; **P<0.01;

[0416] Table 38 Effects of the powders of Examples 5, 6, 7, and 8 on yeast-induced fever in young rats (Mean ± SD, n)

[0417]

[0418] Note: Compared with the normal control group, ##P<0.01; compared with the model control group, *P<0.05; **P<0.01;

[0419] The study showed that the powder of Example 5 (11.8, 23.6, 47.2 g crude drug / kg) had a significant antipyretic effect on yeast-induced fever in young rats. The powder of Example 5 had a faster onset of antipyretic effect and a better antipyretic effect than the Children's Chiqiao Qingre Granules. The powders of Example 5, Example 6, Example 7, and Example 8 all had a significant cooling effect on yeast-induced fever in young rats, and there was no significant difference between the different powders.

[0420] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for detecting a Chinese medicine composition, characterized in that: The Chinese medicine composition comprises the following raw materials in parts by weight: 100-300 parts of Artemisia annua, 100-300 parts of Scutellaria baicalensis, 100-450 parts of Poria cocos, 100-270 parts of dried tangerine peel, 100-270 parts of bamboo shavings, 580-820 parts of gypsum, 200-450 parts of honeysuckle, 200-450 parts of isatis indigotica, 100-300 parts of pinellia ternata, 100-270 parts of cicada slough, 100-270 parts of mint, 100-270 parts of liquorice and 20-180 parts of indigo naturalis; The detection method of the Chinese medicine composition includes the following steps of identifying Artemisia annua and dried tangerine peel: A. Identification of Artemisia annua and Tangerine Peel Preparation of test solution: 2-6 g of the traditional Chinese medicine composition is added with 30-100 ml of methanol and ultrasonically extracted for 20-60 minutes, filtered, the filtrate is evaporated to dryness, 30-50 ml of water is added to dissolve, and the solution is extracted at least once with water-saturated n-butanol. The n-butanol solution is dried and dissolved with 3-5 ml of methanol. 1-4 ml is transferred to a neutral alumina column and eluted with 20-50 ml of 5-15% ethanol solution as eluent. The eluate is discarded and eluted with 20-50 ml of 20-40% ethanol solution as eluent. The eluate is collected and evaporated to dryness. The residue is dissolved with 1-3 ml of methanol to obtain the product. Preparation of control medicinal material solution: separately extract Artemisia annua and tangerine peel control medicinal material with water, separate the solid and liquid, and obtain the supernatant. The obtained substance after concentration or drying is used instead of the traditional Chinese medicine composition according to the preparation method of the test solution to prepare Artemisia annua control medicinal material solution and tangerine peel control medicinal material solution; Identification: According to the thin layer chromatography test, aspirate the test sample solution and the tangerine peel control medicinal material solution, spot them on the same thin layer plate, develop with petroleum ether-toluene-acetone with a boiling range of 60-90℃ as the first developing solvent, spray with a color developer, heat, and inspect under ultraviolet light; Pipette the test sample solution and the Artemisia annua control medicinal material solution, spot them on another thin layer plate, develop them with cyclohexane-toluene-acetone-formic acid as the second developing solvent, and inspect them under ultraviolet light; In the identification step, in the first developing solvent, the volume ratio of petroleum ether with a boiling range of 60-90° C., toluene and acetone is 9:1-3:5; and / or, in the second developing solvent, the volume ratio of cyclohexane, toluene, acetone and formic acid is 4-5:3-4:2-3:0.

5.

2. The method for detecting the Chinese medicine composition according to claim 1, wherein The spotting volume of the test solution, tangerine peel control medicinal material solution or artemisia annua control medicinal material solution is 1-10 μl; and / or the color developer is AlCl3 ethanol solution; and / or the heating temperature is 100-110°C.

3. The method for detecting the Chinese medicine composition according to claim 1, wherein The detection method of the traditional Chinese medicine composition further includes at least one of the following determinations of baicalin content and chlorogenic acid content: B. Determination of baicalin content Preparation of the test solution: taking the traditional Chinese medicine composition, adding an organic solvent for extraction to obtain a test solution; Preparation of reference solution: Take baicalin reference substance, add organic solvent to dissolve it and prepare reference solution; Chromatographic conditions and system suitability test: HPLC determination was performed using octadecylsilane bonded silica gel as the filler; acetonitrile as mobile phase A, and 0.1-0.3% phosphoric acid solution as mobile phase B. Gradient elution was performed with the following program: 0-35 min, mobile phase A: 20% by volume, mobile phase B: 80% by volume; 35-40 min, mobile phase A: increasing from 20% to 60% by volume, mobile phase B: decreasing from 80% to 40% by volume; detection wavelength: 260-290 nm. Determination method: aspirate the reference solution and the test solution, inject them into the liquid chromatograph, determine and calculate; C. Determination of chlorogenic acid content Preparation of the test solution: taking the traditional Chinese medicine composition, adding an organic solvent for extraction to obtain a test solution; Preparation of reference solution: Take chlorogenic acid reference, add organic solvent to dissolve and prepare reference solution; Chromatographic conditions and system suitability test: HPLC determination was performed using octadecylsilane bonded silica gel as the filler; a mixture of tetrahydrofuran, methanol, and acetonitrile was used as mobile phase A, and 0.1-0.3% phosphoric acid solution was used as mobile phase B. Gradient elution was performed with the following program: 0-60 min, the volume percentage of mobile phase A in the mobile phase increased from 7% to 10%, and the volume percentage of mobile phase B decreased from 93% to 90%; 60-65 min, the volume percentage of mobile phase A in the mobile phase increased from 10% to 60%, and the volume percentage of mobile phase B decreased from 90% to 40%; 65-70 min, the volume percentage of mobile phase A in the mobile phase was 60%, and the volume percentage of mobile phase B was 40%; the detection wavelength was 310-340 nm. Determination method: Pipette the reference solution and the test solution, inject them into the liquid chromatograph, measure and calculate.

4. The method for detecting the Chinese medicine composition according to claim 3, wherein The detection method also satisfies at least one of the following (1)-(2): (1) In the determination of baicalin content, the organic solvent is selected from at least one of methanol, ethanol, and water; and / or the mass ratio of the traditional Chinese medicine composition to the volume of the organic solvent is 0.1-0.6 g: 20-100 ml; and / or the flow rate is 0.8-1.2 ml / min; and / or the column temperature is 25-40°C; and / or the concentration of the reference solution is 10-300 μg / mL; and / or the extraction is ultrasonic extraction, reflux extraction, or shaking extraction; and / or the extraction time is 15-60 min; (2) In the determination of the content of chlorogenic acid, the organic solvent is selected from at least one of methanol, ethanol, and water; and / or the volume ratio of the mass of the traditional Chinese medicine composition to the organic solvent is 0.1-0.6 g: 20-100 ml; and / or the flow rate is 0.7-1.2 ml / min; and / or the column temperature is 20-30°C; and / or the volume ratio of tetrahydrofuran, methanol, and acetonitrile in mobile phase A is 1:7-9:15-17; and / or the concentration of the reference solution is 1-400 μg / mL; and / or the extraction is ultrasonic extraction, reflux extraction, or shaking extraction; and / or the extraction time is 15-60 min.

5. The method for detecting the Chinese medicine composition according to any one of claims 1 to 4, characterized in that: The preparation method of the traditional Chinese medicine composition comprises the following steps: mixing Artemisia annua, Scutellaria baicalensis, Poria cocos, dried tangerine peel, Rhizoma Anemarrhenae, Gypsum, Flos Lonicerae, Folium Isatidis, Rhizoma Pinelliae, Periostracum Cicadae, Mentha and licorice, extracting the mixture according to a conventional extraction method or extracting the mixture separately according to a conventional extraction method, and then mixing the mixture with Indigo Naturalis to obtain the traditional Chinese medicine composition; or mixing Artemisia annua, Scutellaria baicalensis, Poria cocos, dried tangerine peel, Rhizoma Anemarrhenae, Gypsum, Flos Lonicerae, Folium Isatidis, Rhizoma Pinelliae, Periostracum Cicadae, Mentha, Glycyrrhizae and Indigo Naturalis, extracting the mixture according to a conventional extraction method or extracting the mixture separately according to a conventional extraction method, and then mixing the mixture to obtain the traditional Chinese medicine composition.

6. The method for detecting the Chinese medicine composition according to any one of claims 1 to 4, characterized in that: The preparation method of the traditional Chinese medicine composition comprises: adding excipients to the traditional Chinese medicine composition or not adding excipients to directly or indirectly prepare a clinically acceptable pharmaceutical preparation according to a conventional process.

7. The method for detecting the Chinese medicine composition according to claim 6, wherein: The pharmaceutical preparation is in the form of micropills, tablets, capsules, powders, mixtures, granules, syrups, gels or decoctions.

8. The method for detecting the Chinese medicine composition according to claim 6, wherein: The pharmaceutically acceptable excipient is selected from at least one of pharmaceutically acceptable solvents, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, glidants, flavoring agents, preservatives, suspending agents, coating materials, anti-adhesives, penetration enhancers, pH regulators and thickeners.

9. The method for detecting the Chinese medicine composition according to claim 6, wherein: The preparation method of the pharmaceutical preparation comprises: Decoction step: gypsum is decocted with an extraction solvent, and the decoction liquid and the medicinal residue are collected to obtain A; Artemisia annua, Scutellaria baicalensis, Poria cocos, dried tangerine peel, bamboo shavings, honeysuckle, isatis indigotica, Pinellia ternata, cicada slough, mint, and liquorice are mixed, A is added, and the mixture is soaked with an extraction solvent, decocted and extracted, solid-liquid separation is performed, and the liquid is collected to obtain a medicinal solution; The preparation steps of the pharmaceutical preparation include: concentrating and / or drying the medicinal solution, mixing it with Indigo Naturalis, adding or not adding auxiliary materials, and preparing the pharmaceutical preparation according to conventional processes; or including: Decoction step: gypsum is decocted with an extraction solvent, and the decoction liquid and the medicinal residue are collected to obtain A; Artemisia annua, Scutellaria baicalensis, Poria cocos, dried tangerine peel, bamboo shavings, honeysuckle, isatis indigotica, Pinellia ternata, cicada slough, mint, liquorice, and indigo naturalis are mixed, A is added, and the mixture is soaked with an extraction solvent, decocted and extracted, solid-liquid separation is performed, and the liquid is collected to obtain a medicinal solution; The preparation steps of the pharmaceutical preparation are as follows: the pharmaceutical solution is concentrated and / or dried, excipients are added or not added, and the pharmaceutical preparation is prepared according to conventional processes.

10. The method for detecting the Chinese medicine composition according to claim 9, characterized in that: The decoction step comprises: taking gypsum, adding 3-6 times the weight of gypsum in water or 5-98% alcohol solution, decoct for 30-90 minutes, collecting the decoction and the residue to obtain A; taking Artemisia annua, Scutellaria baicalensis, Poria cocos, Tangerine peel, Rhizoma Anemarrhenae, Honeysuckle, Folium Isatidis, Pinellia ternata, Periostracum Cicadae, Mentha, and Licorice, mixing, adding A, adding water until the weight of the medicinal materials except Indigo naturalis is 5-12 times the weight of the water or 5-98% alcohol solution, soaking for 30-120 minutes, decoct and extract 1-4 times, the material-liquid mass ratio of each extraction being 1:6-15, the extraction time being 60-120 minutes, solid-liquid separation, collecting the liquid, and obtaining the medicinal solution; or comprising: Take gypsum, add 3-6 times the weight of gypsum water or 5-98% alcohol solution, and boil for 30-90 minutes, collect the decoction and the residue to obtain A; take Artemisia annua, Scutellaria baicalensis, Poria cocos, Tangerine peel, Rhizoma Anemarrhenae, Honeysuckle, Isatis indigotica, Pinellia ternata, Cicadae Periostracum, Menthol, Licorice, and Indigo naturalis, mix, add A, add water to 5-12 times the weight of the medicinal materials in water or 5-98% alcohol solution, soak for 30-120 minutes, boil and extract 1-4 times, the material-liquid mass ratio of each extraction is 1:6-15, the extraction time is 60-120 minutes, separate the solid and liquid, collect the liquid, and obtain a medicinal solution.

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