Method for identifying Chaiqianchang original mixture preparation

Abstract

The invention relates to the field of modernization of traditional Chinese medicines, in particular to a method for identifying a Chaiqianchang original mixture preparation. The invention respectively provides thin-layer chromatography identification methods for glabrous sarcandra herb, radix peucedani and radix scutellariae in the Chaiqiyun original mixture preparation, the thin-layer chromatography identification methods for the three components are good in specificity, repeatability and stability, and effective quality control of the Chaiqiyun original mixture can be realized.

Description

[0001] This application is a divisional application of the Chinese patent application with application date of November 25, 2022 and application number 202211490852.4, entitled "A Chaigechangyuan mixture preparation and its preparation method and detection method". Technical Field

[0002] The invention relates to the field of modernization of traditional Chinese medicine, and in particular to an identification method for a Chaigechangyuan mixture preparation. Background Art

[0003] In recent years, the new coronavirus infection has been rampant around the world. It is an acute respiratory infection infectious disease. The clinical manifestations of the new coronavirus infection are generally fever, dry cough, and fatigue. A small number of patients are accompanied by symptoms such as nasal congestion, runny nose, sore throat, myalgia and diarrhea, which are similar to the symptoms of a cold. Cold is the most common human respiratory infectious disease. It is an exogenous disease caused by wind evil or epidemic viruses, which causes lung dysfunction, nasal congestion, runny nose, sneezing, headache, chills, fever, and general discomfort. Traditional Chinese medicine has a long history and rich successful experience in treating colds and viral influenza diseases. It has the advantages of multi-target and multi-pathway effects that cannot be replaced by Western medicine in preventing and treating viral epidemics, and is particularly prominent in reducing drug side effects and reducing drug resistance.

[0004] The dosage forms of traditional Chinese medicine are mainly decoctions, pills, powders, etc. With the development of science and technology, Chinese medicine preparations such as granules and tablets have emerged. The dosage forms of Chinese medicine preparations are aimed at meeting clinical medical needs, and are selected based on factors such as the composition of the medicinal flavor, clinical needs, the target of the medication, the nature of the medication, the prescription dose, and the safety of the medication. For example, for emergency medication, the drug effect should be fast, so injections, sublingual tablets (pills), aerosols and other dosage forms are used. For chronic medication, the drug effect should be long-lasting, and honey pills, pastes, sustained-release tablets, etc. are often used; drug ingredients that are easily decomposed and ineffective by gastric acid should be made into enteric-coated capsules or enteric-coated tablets: when some drugs are unstable when made into liquid preparations, they can be made into powders, tablets, powder injections or oil solutions, etc.

[0005] Traditional Chinese medicine preparations for preventing and treating colds and novel coronavirus infections are mostly decoctions. Decoctions use water as a solvent, are easy to prepare, and are absorbed and effective quickly, but they are bitter and inconvenient to take, and are not suitable for long-term storage. The extraction rate or retention rate of volatile and insoluble components is low, which affects the efficacy to a certain extent. Summary of the invention

[0006] The technical problem solved by the present invention is that traditional Chinese medicine preparations for treating colds and novel coronavirus infections are mostly decoctions, which are bitter in taste, large in volume, inconvenient to take, difficult to store, and have unstable quality.

[0007] In view of the above problems, the present invention provides a thin layer chromatography identification method for Radix Sarcandrae, Radix Peucedanum and Radix Scutellariae in the preparation of Chaigechangyuan mixture. The Chaigechangyuan mixture prepared by the present invention has good stability and is easy to take, carry and store; the detection method of the content of the index components in the Chaigechangyuan mixture and the thin layer chromatography identification method of some traditional Chinese medicine components provided by the present invention realize the effective quality control of the Chaigechangyuan mixture.

[0008] Specifically, the present invention provides the following technical solutions:

[0009] In the first aspect, the present invention provides a Chaigechangyuan mixture preparation, which contains, by weight, 55-57 parts of Bupleurum chinense, 66-68 parts of Pueraria lobata, 32-34 parts of Andrographis paniculata, 66-68 parts of Saposhnikovia divaricata, 49-51 parts of Peucedanum chinense, 55-57 parts of Perilla frutescens stem, 49-51 parts of Codonopsis pilosula, 32-34 parts of Fried Citrus aurantium wilfordii, 16-18 parts of jujube, 16-18 parts of American ginseng, 55-57 parts of Scutellaria baicalensis, 49-51 parts of Sarcandra chinensis, 16-18 parts of Patchouli and 10-12 parts of ginger.

[0010] In some embodiments, the Chaigechangyuan mixture contains, by weight, 56 parts of Bupleurum chinense, 67 parts of Pueraria lobata, 33 parts of Andrographis paniculata, 67 parts of Saposhnikovia divaricata, 50 parts of Peucedanum chinense, 56 parts of Perilla frutescens stem, 50 parts of Codonopsis pilosula, 33 parts of Fried Citrus aurantium wilfordii, 17 parts of jujube, 17 parts of American ginseng, 56 parts of Scutellaria baicalensis, 50 parts of Sarcandra chinensis, 17 parts of Pogostemon cablin and 11 parts of ginger.

[0011] In some embodiments, the Chaigechangyuan mixture contains, by weight, 10 parts of Bupleurum chinense, 12 parts of Pueraria lobata, 6 parts of Andrographis paniculata, 12 parts of Saposhnikovia divaricata, 9 parts of Peucedanum chinense, 10 parts of Perilla frutescens stem, 9 parts of Codonopsis pilosula, 6 parts of stir-fried Citrus aurantium, 3 parts of jujube, 3 parts of American ginseng, 10 parts of Scutellaria baicalensis, 9 parts of Sarcandra chinensis, 3 parts of Patchouli, and 2 parts of ginger.

[0012] In a second aspect, the present invention provides a method for preparing the Chaigechangyuan mixture preparation, comprising the following steps:

[0013] (1) Soak the medicinal materials of Chaigechangyuan mixture in water, decoct, and filter;

[0014] (2) concentrating the filtrate to obtain a clear paste;

[0015] (3) Add preservatives and flavoring agents to the clear paste, add water, mix well, filter and sterilize to obtain the Chaigechangyuan mixture preparation.

[0016] In some embodiments, in step (1), the soaking time in water is 1 to 3 hours.

[0017] In some embodiments, in step (1), the soaking time in water is 2 hours.

[0018] In some embodiments, in step (1), the decocting is performed 1 to 3 times.

[0019] In some embodiments, in step (1), the decocting step is performed twice.

[0020] In some embodiments, in step (1), the decocting temperature is 95-105°C.

[0021] In some embodiments, in step (1), the amount of water added in the first decoction is 8 to 12 times; preferably, the amount of water added in the first decoction is 12 times; and / or, the amount of water added in the second decoction is 8 to 12 times; preferably, the amount of water added in the second decoction is 10 times; and / or, the amount of water added in the third decoction is 8 to 12 times.

[0022] In some embodiments, in step (1), the first decoction time is 30 to 120 min; preferably, the first decoction time is 30 min; and / or, the second decoction time is 30 to 120 min; preferably, the second decoction time is 30 min; and / or, the third decoction time is 30 to 120 min.

[0023] In some embodiments, in step (1), the mesh size of the sieve used for the filtration is 10 to 300 meshes.

[0024] In some embodiments, in step (2), the concentration temperature is 55 to 75°C;

[0025] In some embodiments, in step (2), the relative density of the concentrated clear paste is 1.0 to 1.2.

[0026] In some embodiments, in step (2), the relative density of the concentrated clear paste is 1.08 to 1.11.

[0027] In some embodiments, in step (3), the amount of flavoring agent added is 2% to 6% of the extract concentrate.

[0028] In some embodiments, in step (3), the flavoring agent includes one or both of xylitol and white sugar.

[0029] In some embodiments, in step (3), the amount of preservative added is 0.04% to 0.1% of the extract concentrate.

[0030] In some embodiments, in step (3), the preservative includes one or both of potassium sorbate and ethylparaben.

[0031] In some embodiments, the decoction obtained in step (1) has a cream yield of 26% to 35%.

[0032] In some embodiments, the cream yield of the clear cream obtained in step (2) is 26% to 35%;

[0033] In some embodiments, the transfer rate of baicalin components in the decoction obtained in step (1) is 33% to 70%.

[0034] In some embodiments, the transfer rate of baicalin components in the decoction obtained in step (1) is 54% to 65%.

[0035] In some embodiments, the transfer rate of baicalin components in the clear paste obtained in step (2) is 30% to 70%.

[0036] In some embodiments, the transfer rate of baicalin components in the clear paste obtained in step (2) is 48% to 65%.

[0037] In some embodiments, the transfer rate of puerarin in the decoction obtained in step (1) is 33% to 80%.

[0038] In some embodiments, the transfer rate of puerarin component in the decoction obtained in step (1) is 56% to 75%.

[0039] In some embodiments, the transfer rate of puerarin component in the clear paste obtained in step (2) is 30% to 80%.

[0040] In some embodiments, the transfer rate of puerarin component in the clear paste obtained in step (2) is 59% to 75%.

[0041] In some embodiments, a Chaigechangyuan mixture preparation is provided, wherein the Chaigechangyuan mixture preparation is prepared by the preparation method of the Chaigechangyuan mixture preparation.

[0042] In a third aspect, the present invention provides a method for detecting the content of puerarin and baicalin in the Chaigechangyuan mixture preparation, comprising the following steps:

[0043] (1) Preparation of reference solution

[0044] Weigh puerarin and add 30% ethanol to prepare a reference solution; weigh baicalin and add methanol to prepare a reference solution;

[0045] (2) Preparation of test solution:

[0046] Take the Chaigechangyuan mixture preparation, add a solvent to extract;

[0047] (3) Ultra-high performance liquid chromatography analysis

[0048] Octadecylsilane bonded silica gel was used as filler, mobile phase A was water, mobile phase B was phosphoric acid aqueous solution, reference solution and test solution were injected into ultra-high performance liquid chromatography, and analyzed using a gradient elution program.

[0049] In some embodiments, the extraction solvent in step (2) is selected from methanol, 80% methanol, 50% methanol, ethanol, 80% ethanol, 50% ethanol or water.

[0050] In some embodiments, the extraction solvent in step (2) is 80% methanol.

[0051] In some embodiments, the extraction method in step (2) is selected from shaking or ultrasound.

[0052] In some embodiments, the extraction time in step (2) is 15 to 45 minutes.

[0053] In some embodiments, the extraction time in step (2) is 30 minutes.

[0054] In some embodiments, in step (3), mobile phase B is 0.1% phosphoric acid aqueous solution.

[0055] In some embodiments, the wavelengths detected are 250 nm and 280 nm.

[0056] In some embodiments, the column temperature is 25-35°C.

[0057] In some embodiments, the column temperature is 25°C;

[0058] In some embodiments, the flow rate is 0.25-0.35 mL / min.

[0059] In some embodiments, the flow rate is 0.3 mL / min.

[0060] In some embodiments, the gradient elution program is as follows:

[0061] 0-25 minutes: 15 vol% mobile phase A increased to 27 vol% mobile phase A, 85 vol% mobile phase B increased to 73 vol% mobile phase B;

[0062] 25-30 minutes: 27 vol% mobile phase A increased to 35 vol% mobile phase A, 73 vol% mobile phase B increased to 65 vol% mobile phase B;

[0063] 30-50 minutes: 35 vol% mobile phase A increased to 48 vol% mobile phase A, 65 vol% mobile phase B increased to 52 vol% mobile phase B.

[0064] In a fourth aspect, the present invention provides a thin layer chromatography identification method of Sarcandra scabra in the Chaigechangyuan mixture preparation, comprising the following steps:

[0065] (1) Preparation of test solution

[0066] Take a sample of Chaigechangyuan mixture, add it to a D101 macroporous resin column (2 g, 1.5 cm*12 cm), and elute it with water, 20% ethanol and 80% ethanol in sequence; collect the 80% ethanol eluate, evaporate it to dryness, add methanol to the residue to dissolve it, filter it, and use it as the test solution;

[0067] (2) Preparation of negative drug solution

[0068] Take a negative sample of Radix Achyranthis Bidentatae, add it to a D101 macroporous resin column (2 g, 1.5 cm*12 cm), and elute it with water, 20% ethanol and 80% ethanol in sequence; collect the 80% ethanol eluate, evaporate it to dryness, add methanol to the residue to dissolve it, filter it, and use it as a negative control solution;

[0069] (3) Preparation of control medicinal material solution

[0070] Take the control medicinal material of Radix Sarcosinae, add water, heat to reflux, filter, evaporate the filtrate to dryness, add water to the residue to dissolve it, add D101 macroporous resin column (2g, 1.5cm*12cm), elute with water, 20% ethanol and 80% ethanol in sequence; collect the 80% ethanol eluate, evaporate to dryness, and dissolve it in methanol as the control medicinal material solution;

[0071] (4) Thin layer chromatography identification method

[0072] The above solutions were absorbed and spotted on the same silica gel G thin layer plate respectively. The developing agent was a mixed solution of toluene, ethyl acetate, methanol and water. Preferably, the volume ratio of toluene, ethyl acetate, alcohol and water was 10:4:2:0.5. The plates were developed, taken out, dried and examined under ultraviolet light.

[0073] In some embodiments, in step (1), water, 20% ethanol and 80% ethanol are each eluted at 20 mL.

[0074] In some embodiments, in step (2), water, 20% ethanol and 80% ethanol are eluted at 20 mL each.

[0075] In some embodiments, in step (3), water, 20% ethanol and 80% ethanol are each eluted at 20 mL.

[0076] In a fifth aspect, the present invention provides a thin layer chromatography identification method of Peucedanum peucedanum in the Chaigechangyuan mixture preparation, comprising the following steps:

[0077] (1) Preparation of test solution

[0078] Take a sample of Chaigechangyuan mixture, add chloroform, ultrasonicate, filter, evaporate to dryness, add methanol to the residue to dissolve it, and use it as the test solution;

[0079] (2) Preparation of negative drug solution

[0080] Take a negative sample lacking peucedanum, add chloroform, ultrasonicate, filter, evaporate to dryness, and add methanol to dissolve the residue as a negative control solution;

[0081] (3) Preparation of control medicinal material solution

[0082] Take the control drug of Peucedanum chinense, add water, heat to reflux, filter, evaporate to dryness, add water to dissolve, add chloroform, ultrasonically treat, filter, evaporate to dryness, add methanol to dissolve the residue, and use it as the control drug solution;

[0083] (4) Thin layer chromatography identification method

[0084] The above solutions were absorbed and spotted on the same silica gel G thin layer plate respectively. The developing agent was a mixed solution of petroleum ether and ethyl acetate. Preferably, the volume ratio of petroleum ether to ethyl acetate was 3:1. The plate was developed, taken out, dried, and inspected under ultraviolet light.

[0085] In a sixth aspect, the present invention provides a thin layer chromatography identification method of Scutellaria baicalensis in the Chaigechangyuan mixture preparation, comprising the following steps:

[0086] (1) Preparation of test solution

[0087] Take a sample of Chaigechangyuan mixture, add it to a D101 macroporous resin column (2 g, 1.5 cm*12 cm), and elute it with water, 20% ethanol and 80% ethanol in sequence; collect the 80% ethanol eluate, evaporate it to dryness, add methanol to the residue to dissolve it, filter it, and use it as the test solution;

[0088] (2) Preparation of negative drug solution

[0089] Take a negative sample lacking Scutellaria baicalensis, add it to a D101 macroporous resin column (2 g, 1.5 cm*12 cm), and elute it with water, 20% ethanol and 80% ethanol in sequence; collect the 80% ethanol eluate, evaporate it to dryness, add methanol to the residue to dissolve it, filter it, and use it as a negative control solution;

[0090] (3) Preparation of control medicinal material solution

[0091] Take the reference medicinal material of Scutellaria baicalensis, add methanol, sonicate, filter, evaporate the filtrate to dryness, add water to the residue to dissolve it, add D101 macroporous resin column (2g, 1.5cm*12cm), and elute with water, 20% ethanol and 80% ethanol in sequence; collect the 80% ethanol eluate, evaporate to dryness, and add methanol to the residue to dissolve it as the reference medicinal material solution;

[0092] (4) Thin layer chromatography identification method

[0093] The above solutions were absorbed and spotted on the same silica gel G thin layer plate respectively. The developing agent was a mixed solution of toluene, ethyl acetate and formic acid. Preferably, the volume ratio of toluene, ethyl acetate and formic acid was 10:5:2. The plate was developed, taken out, dried, sprayed with 2% ferric chloride ethanol solution, heated at 105°C until the spots were colored, and inspected under sunlight.

[0094] In some embodiments, in step (1), water, 20% ethanol and 80% ethanol are eluted at 50 mL, 50 mL and 100 mL respectively.

[0095] In some embodiments, in step (2), water, 20% ethanol and 80% ethanol are eluted at 50 mL, 50 mL and 100 mL respectively.

[0096] In some embodiments, in step (3), water, 20% ethanol and 80% ethanol are eluted at 50 mL, 50 mL and 100 mL respectively.

[0097] In some embodiments, the Chaigechangyuan mixture preparation is used in the preparation of medicines for the prevention, treatment and rehabilitation of colds.

[0098] In some embodiments, the Chaigechangyuan mixture preparation is used in the preparation of drugs for preventing new coronavirus infection.

[0099] The present invention provides another set of technical solutions as follows:

[0100] In a first aspect, the present invention provides a thin layer chromatography identification method of Sarcandra scabra in Chaigechangyuan mixture preparation, comprising the following steps:

[0101] (1) Preparation of test solution

[0102] Take a sample of Chaigechangyuan mixture, add it to a D101 macroporous resin column, and elute it with water, 20% ethanol and 80% ethanol in sequence; collect the 80% ethanol eluate, evaporate it to dryness, add methanol to the residue to dissolve it, filter it, and use it as the test solution;

[0103] (2) Preparation of negative drug solution

[0104] Take a negative sample of Radix Achyranthis Bidentatae, add it to a D101 macroporous resin column, and elute it with water, 20% ethanol, and 80% ethanol in sequence; collect the 80% ethanol eluate, evaporate it to dryness, add methanol to the residue to dissolve it, filter it, and use it as a negative control solution;

[0105] (3) Preparation of control medicinal material solution

[0106] Take the control medicinal material of Radix Lycopodiellae, add water, heat to reflux, filter, evaporate the filtrate to dryness, add water to the residue to dissolve it, add it to the D101 macroporous resin column, and elute it with water, 20% ethanol and 80% ethanol in sequence; collect the 80% ethanol eluate, evaporate it to dryness, and add methanol to dissolve it as the control medicinal material solution;

[0107] (4) Thin layer chromatography identification method

[0108] The above solutions were absorbed and spotted on the same silica gel G thin layer plate respectively. The developing agent was a mixed solution of toluene, ethyl acetate, methanol and water. After development, the plate was taken out, dried and examined under ultraviolet light.

[0109] Preferably, in step (1), 20 mL of water, 20% ethanol and 80% ethanol are used for elution.

[0110] Preferably, in step (2), 20 mL of water, 20% ethanol and 80% ethanol are used for elution.

[0111] Preferably, in step (3), 20 mL of water, 20% ethanol and 80% ethanol are used for elution.

[0112] Preferably, in step (4), the volume ratio of toluene, ethyl acetate, alcohol and water is 10:4:2:0.5.

[0113] In a second aspect, the present invention provides a thin layer chromatography identification method of Peucedanum peucedanum in a Chaigechangyuan mixture preparation, comprising the following steps:

[0114] (1) Preparation of test solution

[0115] Take a sample of Chaigechangyuan mixture, add chloroform, ultrasonicate, filter, evaporate to dryness, add methanol to the residue to dissolve it, and use it as the test solution;

[0116] (2) Preparation of negative drug solution

[0117] Take a negative sample lacking peucedanum, add chloroform, ultrasonicate, filter, evaporate to dryness, and add methanol to dissolve the residue as a negative control solution;

[0118] (3) Preparation of control medicinal material solution

[0119] Take the control drug of Peucedanum chinense, add water, heat to reflux, filter, evaporate to dryness, add water to dissolve, add chloroform, ultrasonically treat, filter, evaporate to dryness, add methanol to dissolve the residue, and use it as the control drug solution;

[0120] (4) Thin layer chromatography identification method

[0121] Absorb the above solutions and spot them on the same silica gel G thin layer plate respectively. The developing agent is a mixed solution of petroleum ether and ethyl acetate. Develop, take out, dry and examine under ultraviolet light.

[0122] Preferably, the volume ratio of petroleum ether to ethyl acetate is 3:1.

[0123] In a third aspect, the present invention provides a thin layer chromatography identification method for Scutellaria baicalensis in a Chaigechangyuan mixture preparation, comprising the following steps:

[0124] (1) Preparation of test solution

[0125] Take a sample of Chaigechangyuan mixture, add it to a D101 macroporous resin column, and elute it with water, 20% ethanol and 80% ethanol in sequence; collect the 80% ethanol eluate, evaporate it to dryness, add methanol to the residue to dissolve it, filter it, and use it as the test solution;

[0126] (2) Preparation of negative drug solution

[0127] Take a negative sample lacking Scutellaria baicalensis, add it to a D101 macroporous resin column, and elute it with water, 20% ethanol and 80% ethanol in sequence; collect the 80% ethanol eluate, evaporate it to dryness, add methanol to the residue to dissolve it, filter it, and use it as a negative control solution;

[0128] (3) Preparation of control medicinal material solution

[0129] Take the reference medicinal material of Scutellaria baicalensis, add methanol, sonicate, filter, evaporate the filtrate to dryness, add water to the residue to dissolve it, add D101 macroporous resin column, and elute with water, 20% ethanol and 80% ethanol in sequence; collect the 80% ethanol eluate, evaporate to dryness, and add methanol to the residue to dissolve it as the reference medicinal material solution;

[0130] (4) Thin layer chromatography identification method

[0131] Absorb the above solutions and spot them on the same silica gel G thin layer plate respectively. The developing agent is a mixed solution of toluene, ethyl acetate and formic acid. Develop, take out, dry, spray with 2% ferric chloride ethanol solution, heat at 105℃ until the spots are colored, and examine under sunlight.

[0132] Preferably, in step (1), water, 20% ethanol and 80% ethanol are used for elution at 50 mL, 50 mL and 100 mL respectively.

[0133] Preferably, in step (2), water, 20% ethanol and 80% ethanol are used for elution at 50 mL, 50 mL and 100 mL respectively.

[0134] Preferably, in step (3), water, 20% ethanol and 80% ethanol are preferably used for elution at 50 mL, 50 mL and 100 mL respectively.

[0135] Preferably, in step (4), the volume ratio of toluene, ethyl acetate and formic acid is 10:5:2.

[0136] Preferably, in parts by weight, the Chaigechangyuan mixture contains 55-57 parts of Bupleurum chinense, 66-68 parts of Pueraria lobata, 32-34 parts of Andrographis paniculata, 66-68 parts of Saposhnikovia divaricata, 49-51 parts of Peucedanum chinense, 55-57 parts of Perilla stem, 49-51 parts of Codonopsis pilosula, 32-34 parts of Fried Citrus aurantium wilfordii, 16-18 parts of jujube, 16-18 parts of American ginseng, 55-57 parts of Scutellaria baicalensis, 49-51 parts of Sarcandra sylvestris, 16-18 parts of Patchouli and 10-12 parts of ginger.

[0137] Preferably, in parts by weight, the Chaigechangyuan mixture contains 56 parts of Bupleurum chinense, 67 parts of Pueraria lobata, 33 parts of Andrographis paniculata, 67 parts of Saposhnikovia divaricata, 50 parts of Peucedanum chinense, 56 parts of Perilla frutescens stems, 50 parts of Codonopsis pilosula, 33 parts of stir-fried Qingpi (citrus aurantium), 17 parts of jujube, 17 parts of American ginseng, 56 parts of Scutellaria baicalensis, 50 parts of Sarcandra chinensis, 17 parts of Pogostemon cablin and 11 parts of ginger.

[0138] Beneficial effects of the present invention:

[0139] (1) The Chaigechangyuan mixture of the present invention can be used to prepare medicines for preventing, treating and recovering from colds, giving full play to the role of traditional Chinese medicine in treating colds;

[0140] At the same time, the Chaigechangyuan mixture can also be used to prepare drugs for preventing new coronavirus infection.

[0141] (2) The ultra-high performance liquid chromatography method for detecting puerarin and astragaloside in Chaigechangyuan mixture of the present invention has good specificity, high precision and accuracy, and good stability.

[0142] (3) The thin layer chromatography identification method of Sarcandra scabra, Peucedanum peucedanum and Scutellaria baicalensis in the present invention has good specificity and durability.

[0143] (4) The Chaigechangyuan mixture prepared by the present invention has controllable quality, good stability, is easy to take, carry and store, and is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0144] Figure 1 It is a specificity investigation spectrum in the methodological validation of the content detection method of Chaigechangyuan mixture.

[0145] Figure 2 It is the peak purity inspection spectrum in the methodological validation of the content detection method of Chaigechangyuan mixture.

[0146] Figure 3 This is the standard curve of puerarin in the linear investigation during the methodological validation of the content detection method of Chaigechangyuan mixture.

[0147] Figure 4 This is the standard curve of baicalin in the linear investigation of the methodological validation of the content detection method of Chaigechangyuan mixture.

[0148] Figure 5 These are the chromatograms of different chromatographic columns used in the durability investigation in the methodological validation of the content detection method of Chaigechangyuan mixture.

[0149] Figure 6 It is a chromatogram using different column temperatures in the durability investigation in the methodological validation of the content detection method of Chaigechangyuan mixture.

[0150] Figure 7 It is a chromatogram using different flow rates in the durability investigation in the methodological validation of the content detection method of Chaigechangyuan mixture.

[0151] Figure 8 This is the thin layer chromatogram of different sample amounts of Sarcandra suspensa in Chaigechangyuan mixture.

[0152] Fig. 9 It is the thin layer chromatography specific for Radix Aconiti Lateralis Preparata in Chaigechangyuan mixture.

[0153] Fig.10 This is an investigation on different thin layer chromatography plates of Sarcandra glabra in the identification of Sarcandra glabra in Chaigechangyuan mixture.

[0154] Fig.11 This is an investigation on different development temperatures of thin layer chromatography for the identification of Radix Sarcandrae in Chaigechangyuan mixture.

[0155] Fig.12 The humidity of the Radix Adenophorae in Chaigechangyuan mixture was investigated by thin layer chromatography.

[0156] Fig.13 This is a thin layer chromatogram of different sample amounts of Peucedanum chinense in Chaigechangyuan mixture.

[0157] Fig.14 It is the thin layer chromatography specific for Peucedanum chinense in Chaigechangyuan mixture.

[0158] Fig.15 This is an investigation on different thin layer chromatography plates of Peucedanum chinense in the identification of Peucedanum chinense in Chaigechangyuan mixture.

[0159] Fig.16 This is an investigation on different development temperatures of thin layer chromatography of Peucedanum praeparata in the identification of Peucedanum praeparata in Chaigechangyuan mixture.

[0160] Fig.17 The humidity of the peucedanum chinense in Chaigechangyuan mixture was investigated by thin layer chromatography.

[0161] Fig.18 This is a thin layer chromatogram of different amounts of Scutellaria baicalensis in Chaigechangyuan mixture.

[0162] Fig.19 It is the thin layer chromatography specific for Scutellaria baicalensis in Chaige Changyuan mixture.

[0163] Fig. 20 This is an investigation on different thin layer chromatography plates of Scutellaria baicalensis in the identification of Scutellaria baicalensis in Chaigechangyuan mixture.

[0164] Fig.21 This is an investigation on different development temperatures of Scutellaria baicalensis by thin layer chromatography in the identification of Scutellaria baicalensis in Chaigechangyuan mixture.

[0165] Fig. 22 The humidity of Scutellaria baicalensis was investigated by thin layer chromatography at different levels in the identification of Scutellaria baicalensis in Chaigechangyuan mixture. DETAILED DESCRIPTION

[0166] As described above, the object of the present invention is to provide a Chaigechangyuan mixture preparation, which contains, by weight, 55-57 parts of Bupleurum chinense, 66-68 parts of Pueraria lobata, 32-34 parts of Andrographis paniculata, 66-68 parts of Saposhnikovia divaricata, 49-51 parts of Peucedanum chinense, 55-57 parts of Perilla frutescens stem, 49-51 parts of Codonopsis pilosula, 32-34 parts of Fried Citrus aurantium wilfordii, 16-18 parts of jujube, 16-18 parts of American ginseng, 55-57 parts of Scutellaria baicalensis, 49-51 parts of Sarcandra chinensis, 16-18 parts of Pogostemon cablin and 10-12 parts of ginger.

[0167] Bupleurum chinense: Functions and indications: dispersing fever, relieving liver depression, and raising Yang Qi. Pharmacological effects: anti-inflammatory, antipyretic, sedative, analgesic, antitussive, hypolipidemic, antibacterial, antiviral, anti-tumor, etc.

[0168] Pueraria root: Functions and indications: relieves muscle and fever, promotes the production of body fluid and quenches thirst, promotes the exfoliation of rashes, promotes the yang and stops diarrhea, promotes the circulation of blood and activates collaterals, and relieves alcohol poisoning. Pharmacological effects: improves myocardial ischemia, cerebral ischemia, diabetes and complications, has estrogen-like effects, resists osteoporosis, improves intelligence, resists tumors, lowers blood lipids, resists oxidation, relieves alcohol poisoning, etc.

[0169] Andrographis paniculata: Functions and indications: clearing away heat and detoxifying, cooling blood, and reducing swelling. Pharmacological effects: antipyretic, anti-inflammatory, anti-cancer, improving the body's immune system, anti-platelet activation and anti-thrombotic, liver protection and gallbladder promotion, anti-oxidation and protection of vascular endothelial cells, lipid regulation and reduction of atherosclerosis, blood pressure reduction, etc.

[0170] Saposhnikovia divaricata: Functions and indications: dispelling wind and relieving exterior symptoms, eliminating dampness and relieving pain, and stopping spasms. Pharmacological effects: antipyretic and cooling, sedative, analgesic, anticonvulsant, anti-inflammatory, antibacterial, anti-tumor, etc.

[0171] Radix Peucedanum: Functions and indications: descending qi and resolving phlegm, dispersing wind and clearing away heat. Pharmacological effects: cardiovascular system (anti-arrhythmia), heart, vasodilation, effect on platelet aggregation, expectorant, etc.

[0172] Perilla stem: Functions and indications: regulating qi and relieving fullness, relieving pain, and stabilizing pregnancy. Pharmacological action: progesterone-like effect, components in wild perilla stem inhibit type I cyclooxygenase activity in vitro.

[0173] Codonopsis: Functions and indications: Strengthens the spleen and lungs, nourishes blood and produces body fluids. Pharmacological effects: enhances the body's stress resistance, enhances the body's immune function, delays aging, resists ulcers, affects the central nervous system (improves intelligence), affects blood and hematopoietic function (inhibits in vitro thrombosis), improves cardiovascular function, anti-tumor auxiliary effect, and restores blood sugar, etc.

[0174] Fried green peel with bran: Function: soothes the liver and relieves qi, eliminates stagnation. Pharmacological effects: regulates gastrointestinal function, promotes bile secretion, relieves asthma, protects cardiovascular and cerebrovascular vessels, etc.

[0175] Jujube: Functions and indications: tonifying the middle and replenishing qi, nourishing blood and calming the mind. Pharmacological effects: central nervous system inhibition, liver protection, muscle strength enhancement, immune system (anti-allergic reaction, immune excitement), anti-oxidation and anti-aging, anti-tumor.

[0176] American ginseng: Functions and indications: replenishing qi and yin, clearing away heat and promoting the production of body fluids. Pharmacological effects: central nervous system (central inhibition, improving learning and memory), improving immune function, cardiovascular system, anti-fatigue, anti-tumor, reproductive function, etc.

[0177] Scutellaria baicalensis: Functions and indications: clearing away heat and dampness, purging fire and detoxifying, stopping bleeding, and stabilizing pregnancy. Pharmacological effects: antimicrobial, effects on immune function, lowering blood pressure and diuresis, lowering blood lipids, protective effect against ischemia-reperfusion injury, antiplatelet aggregation and anticoagulation, antioxidant, and anticancer.

[0178] Herba Lycopodii: Functions and indications: clearing away heat and cooling blood, promoting blood circulation and removing spots, dispelling wind and unblocking meridians. Pharmacological effects: anti-tumor, anti-bacterial, anti-viral, promoting fracture healing, affecting platelets, etc.

[0179] Patchouli: Functions and indications: aromatic to clear turbidity, soothe the stomach and stop vomiting, relieve heatstroke. Pharmacological effects: antibacterial, calcium antagonist.

[0180] Ginger: Functions and indications: dispelling cold, warming the middle and stopping vomiting, resolving phlegm and relieving cough, and detoxifying fish and crab. Pharmacological effects: effects on the digestive system (affecting gastrointestinal digestive function, protecting gastric mucosa, stopping vomiting and preventing motion sickness, protecting the liver and promoting bile secretion), cardiovascular effects, sedation and anticonvulsant, antioxidant, antimicrobial, antiplatelet aggregation, anti-5-hydroxytryptamine, antipyretic, analgesic and anti-inflammatory, etc.

[0181] Table 1 Raw material sources and harvesting seasons

[0182]

[0183]

[0184] The bupleurum root, kudzu root, andrographis paniculata, siler, peucedanum chinense, perilla stem, codonopsis pilosula, stir-fried green peel with bran, jujube, American ginseng, scutellaria baicalensis, scutellaria scabra, patchouli and ginger used in the present invention can be selected from conventional Chinese medicinal materials sold on the market.

[0185] In a second aspect, the present invention provides a method for preparing a Chaigechangyuan mixture preparation, comprising the following steps:

[0186] (1) Soak the medicinal materials of Chaigechangyuan mixture in water for 1 to 3 hours, decoct for 1 to 3 times at a temperature of 95 to 105° C., combine the decoctions, and filter;

[0187] (2) concentrating the filtrate at a temperature of 55 to 75° C. to obtain a clear paste having a relative density of 1.08 to 1.11;

[0188] (3) adding preservatives and flavoring agents to the clear paste, adding water, mixing, filtering, filling, and sterilizing to obtain the Chaigechangyuan mixture preparation.

[0189] In a third aspect, the present invention provides a method for detecting the content of puerarin and baicalin in a Chaigechangyuan mixture preparation, comprising the following steps:

[0190] (1) Preparation of reference solution

[0191] Weigh puerarin and add 30% ethanol to prepare a reference solution; weigh baicalin and add methanol to prepare a reference solution;

[0192] (2) Preparation of test solution:

[0193] Take the Chaigechangyuan mixture preparation, add a solvent to extract;

[0194] (3) Ultra-high performance liquid chromatography analysis

[0195] Octadecylsilane bonded silica gel was used as filler, mobile phase A was water, mobile phase B was phosphoric acid aqueous solution, reference solution and test solution were injected into ultra-high performance liquid chromatography, and analyzed using a gradient elution program.

[0196] Baicalin comes from the scutellaria in the prescription, which is the dried root of Scutellaria baicalensis Georgi, a plant of the Labiatae family. So far, more than 40 chemical components of scutellaria have been discovered, mainly including flavonoids (baicalin, wogonin, baicalein, wogonin), volatile oils (acetophenone, palmitic acid and oleic acid), terpenoids and other components (benzoic acid, benzyl alcohol, β-sitosterol). Among them, flavonoids are the main active substances of scutellaria. Baicalin has pharmacological effects such as protecting the nervous system, anti-inflammatory, antioxidant and protecting the liver. Therefore, baicalin in scutellaria was selected as one of the indicator components.

[0197] Puerarin comes from the pueraria root in the prescription, which is the dried root of Pueraria lobata (Willd.) Ohwi, a leguminous plant. It is mainly composed of isoflavones (puerarin, daidzein, daidzein), among which puerarin is the main active ingredient of pueraria root. Pueraria root has a good therapeutic effect on sudden deafness and cardiovascular and cerebrovascular diseases. It can be used to dilate blood vessels, improve blood circulation; reduce myocardial oxygen consumption, inhibit cancer cells; increase coronary flow, adjust blood microcirculation; treat sudden deafness at all ages; and reduce the risk of cardiovascular and cerebrovascular diseases. Therefore, puerarin in pueraria root was selected as one of the indicator components.

[0198] In a fourth aspect, the present invention provides a thin layer chromatography identification method of Radix Sarcandrae in a Chaigechangyuan mixture preparation, comprising the following steps:

[0199] (1) Preparation of test solution

[0200] Take 5 mL of Chaigechangyuan mixture sample, add it to the treated D101 macroporous resin column (2 g, 1.5 cm*12 cm), and elute it with 20 mL each of water, 20% ethanol, and 80% ethanol in sequence, collect the 80% ethanol eluate, evaporate it on a water bath, add 2 mL of methanol to the residue to dissolve it, filter it, and use it as the test solution.

[0201] (2) Preparation of negative drug solution

[0202] Take 5 mL of the negative sample of Scutellaria baicalensis, add it into the treated D101 macroporous resin column (2 g, 1.5 cm*12 cm), and elute it with 20 mL each of water, 20% ethanol and 80% ethanol in sequence, collect the 80% ethanol eluate, evaporate it on a water bath, add 2 mL of methanol to the residue to dissolve it, filter it, and use it as the negative control solution.

[0203] (3) Preparation of control medicinal material solution

[0204] Take 2g of Radix Sarcosioides control medicinal material, add 20mL of water, heat and reflux for 30min, filter, evaporate the filtrate on a water bath, add 5mL of water to the residue to dissolve it, add it to a treated D101 macroporous resin column (2g, 1.5cm*12cm), elute with 20mL of water, 20% ethanol, and 80% ethanol in turn, collect the 80% ethanol eluate, evaporate to dryness, and add 3mL of methanol to dissolve it as the control medicinal material solution.

[0205] (4) Thin layer chromatography identification method

[0206] According to the thin layer chromatography method (General Rule 0502), the above solutions were taken and spotted on the same silica gel G thin layer plate, and toluene: ethyl acetate: methanol: water (10:4:2:0.5) was used as the developing solvent. The plate was developed, taken out, dried, and examined under ultraviolet light (365nm).

[0207] In a fifth aspect, the present invention provides a thin layer chromatography identification method of Peucedanum peucedanum in a Chaigechangyuan mixture preparation, comprising the following steps:

[0208] (1) Preparation of test solution

[0209] Take 5 mL of Chaigechangyuan mixture sample, add 10 mL of chloroform, ultrasonically treat for 10 minutes, filter, evaporate to dryness on a water bath, add 5 mL of methanol to the residue to dissolve it, and use it as the test solution.

[0210] (2) Preparation of negative drug solution

[0211] Take 5 mL of the negative sample lacking peucedanum, add 10 mL of chloroform, ultrasonically treat for 10 minutes, filter, evaporate to dryness on a water bath, add 5 mL of methanol to the residue to dissolve it, and use it as the negative control solution.

[0212] (3) Preparation of control medicinal material solution

[0213] Take 2 g of Peucedanum chinense reference medicinal material, add 20 mL of water, heat and reflux for 30 min, filter, evaporate to dryness on a water bath, add 5 mL of water to dissolve, add 10 mL of chloroform, ultrasonically treat for 10 minutes, filter, evaporate to dryness, add 6 mL of methanol to the residue to dissolve, and use it as the reference medicinal material solution.

[0214] (4) Thin layer chromatography method

[0215] According to the thin layer chromatography method (General Rule 0502), the above solutions were taken and spotted on the same silica gel G thin layer plate, and petroleum ether (60-90°C)-ethyl acetate (3:1) was used as the developing solvent. The plate was developed, taken out, dried, and examined under ultraviolet light (365nm).

[0216] In a sixth aspect, the present invention provides a thin layer chromatography identification method for Scutellaria baicalensis in a Chaigechangyuan mixture preparation, comprising the following steps:

[0217] (1) Preparation of test solution

[0218] Take 5 mL of Chaigechangyuan mixture sample, add it to the treated D101 macroporous resin column (2 g, 1.5 cm*12 cm), and elute with water, 20% ethanol, 80% ethanol at 50 mL, 50 mL, and 100 mL respectively. Collect the 80% ethanol eluate, evaporate it on a water bath, add 1 mL of methanol to the residue to dissolve it, filter it, and use it as the test solution.

[0219] (2) Preparation of negative sample solution

[0220] Take 5 mL of the negative sample lacking Scutellaria baicalensis, add it to the treated D101 macroporous resin column (2 g, 1.5 cm*12 cm), and elute it with water, 20% ethanol, 80% ethanol at 50 mL, 50 mL, and 100 mL respectively. Collect the 80% ethanol eluate, evaporate it on a water bath, add 1 mL of methanol to the residue to dissolve it, and filter it as the negative control solution.

[0221] (3) Preparation of control medicinal material solution

[0222] Take 1 g of Scutellaria baicalensis reference medicinal material, add 30 mL of methanol, ultrasonicate for 30 min, filter, evaporate the filtrate on a water bath, add 5 mL of water to the residue to dissolve it, add it to a treated D101 macroporous resin column (2 g, 1.5 cm*12 cm), and elute with water, 20% ethanol, 80% ethanol at 50 mL, 50 mL, and 100 mL in sequence, collect the 80% ethanol eluate, evaporate to dryness, and add 2 mL of methanol to the residue to dissolve it as the reference medicinal material solution.

[0223] (4) Thin layer chromatography method

[0224] According to the thin layer chromatography method (General Rule 0502), the above solutions were taken and spotted on the same silica gel G thin layer plate, and toluene-ethyl acetate-formic acid (10:5:2) was used as the developing solvent. The plate was taken out, dried, sprayed with 2% ferric chloride ethanol solution, heated at 105°C until the spots were colored, and examined under sunlight.

[0225] Unless otherwise specified, the various reagents / instruments used in the examples of the present invention are conventional commercially available products. The experimental materials and instrument information used in the present invention are shown in the following table:

[0226] Table 1 Experimental materials / instruments and manufacturers

[0227]

[0228]

[0229] 1. Determination of the content of decoction pieces

[0230] (1) Determination of the content of Pueraria root slices

[0231] Pueraria root was determined according to the method of high performance liquid chromatography (General Chapter 0512 of Part IV of the 2020 edition of the Chinese Pharmacopoeia), and the results are shown in the table below.

[0232] Table 2 Determination of the content of Pueraria root medicinal materials

[0233]

[0234] From the above results, it can be seen that the content determination result of Pueraria lobata slices is 2.6%, which is in line with the requirements of the pharmacopoeia.

[0235] (2) Determination of content of Scutellaria baicalensis slices

[0236] Scutellaria baicalensis was determined according to the method of high performance liquid chromatography (General Chapter 0512 of Part IV of the 2020 edition of the Chinese Pharmacopoeia), and the results are shown in the table below.

[0237] Table 3 Results of content determination of Scutellaria baicalensis slices

[0238]

[0239]

[0240] From the above results, it can be seen that the content determination result of Scutellaria baicalensis slices is 12.8%, which is in line with the requirements of the pharmacopoeia.

[0241] 2. Determination method

[0242] (1) Determination of solid content of extract

[0243] Referring to the "hot immersion method" in the general rules of Part 4 (2201) of the 2020 edition of the "Chinese Pharmacopoeia", 25 g of the extract was accurately aspirated and placed in an evaporating dish with constant weight. After evaporation on a water bath, it was dried at 105°C for 3 hours and cooled in a desiccator for 30 minutes. The weight was quickly and accurately weighed to calculate the solid content of the extract.

[0244] The calculation formula of the extract yield is:

[0245]

[0246] (2) Determination method of solid content of concentrated liquid

[0247] Refer to the "hot leaching method" in the general rules of Part 4 (2201) of the 2020 edition of the "Chinese Pharmacopoeia" for the determination of extracts: accurately weigh 10 g of the concentrate, place it in an evaporating dish with constant weight, evaporate it to dryness on a water bath, dry it at 105°C for 3 hours, cool it in a desiccator for 30 minutes, quickly and accurately weigh the weight, and calculate the solid content of the concentrate.

[0248] The calculation formula of concentrate cream yield is:

[0249]

[0250] (3) Transfer rate calculation method

[0251]

[0252] In order to better understand the technical solution of the present invention, the technical solution of the present invention is described in detail below in conjunction with specific embodiments.

[0253] Example 1: Preparation of Chaigechangyuan mixture preparation

[0254] Example 1-1

[0255] (1) Take 56g of Bupleurum chinense, 67g of Pueraria root, 33g of Andrographis paniculata, 67g of Saposhnikovia divaricata, 50g of Peucedanum chinense, 56g of Perilla stem, 50g of Codonopsis pilosula, 33g of Rhizoma Cibotii Root, 17g of Jujube, 17g of American ginseng, 56g of Scutellaria baicalensis, 50g of Radix Salviae Miltiorrhizae, 17g of Patchouli and 11g of ginger, and decoct them twice. The decoction temperature is 95-105°C. The amount of water added for the first time is 12 times the amount of the decoction pieces, the soaking time is 2.0 hours, and the extraction time after boiling is 0.5 hours. The amount of water added for the second time is 10 times the amount of the decoction pieces, and the extraction time after boiling is 0.5 hours. Use a filter screen with a mesh number of 9 (200 mesh) to filter.

[0256] (2) The obtained filtrate is concentrated under reduced pressure at a temperature of 60 to 70°C and a vacuum degree of -0.04 to -0.08 MPa to obtain a clear paste having a relative density of 1.08 to 1.11 (20±5°C).

[0257] (3) Add 2% xylitol and 0.05% potassium sorbate to the clear paste, add water to 1000 mL, mix well, refrigerate and let stand for 12 hours, filter, fill and sterilize to obtain Chaigechangyuan mixture.

[0258] After testing, the cream yield of decoction was 29.51%, and the cream yield of clear cream was 30.08%;

[0259] The transfer rates of astragaloside in the decoction were 59.78%, and puerarin were 59.68%; the transfer rates of astragaloside in the paste were 61.01%, and puerarin were 59.57%;

[0260] The content of astragaloside in Chaigechangyuan mixture is 2.68 mg / mL, and the content of puerarin is 0.66 mg / mL.

[0261] Among them, the method for determining the content of astragaloside and puerarin in the intermediate (decoction or clear paste) is as follows:

[0262] (1) Preparation of test solution

[0263] Weigh appropriate amounts of sample solutions respectively, evaporate to dryness in a water bath (90°C), add 25 mL of 50% methanol to dissolve, sonicate for 30 min, make up with 50% methanol, filter, and filter through a 0.22 μm microporous filter membrane to obtain the product.

[0264] (2) Preparation of reference solution

[0265] Take an appropriate amount of puerarin reference substance, accurately weigh it, and add 30% ethanol to make a solution containing 75 μg per 1 mL; take an appropriate amount of baicalin reference substance, accurately weigh it, and add methanol to make a solution containing 60 μg per 1 mL.

[0266] (3) Measurement method

[0267] Accurately pipette 4 μL of reference solution and test solution respectively, inject into liquid chromatograph, and measure to obtain the result.

[0268] (4) Chromatographic conditions

[0269] According to the HPLC method (General Rule 0512) of Part IV of the 2020 edition of the Chinese Pharmacopoeia, acetonitrile was used as mobile phase A, 0.2% phosphoric acid water was used as mobile phase B, the detection wavelength was 250nm for puerarin and 280nm for baicalin, and the elution gradient was as shown in Table 4:

[0270] Table 4 Gradient elution program

[0271]

[0272]

[0273] Example 1-2

[0274] (1) Take 56g of Bupleurum chinense, 67g of Pueraria root, 33g of Andrographis paniculata, 67g of Saposhnikovia divaricata, 50g of Peucedanum chinense, 56g of Perilla stem, 50g of Codonopsis pilosula, 33g of Rhizoma Cibotii Root, 17g of Jujube, 17g of American ginseng, 56g of Scutellaria baicalensis, 50g of Radix Salviae Miltiorrhizae, 17g of Patchouli and 11g of ginger, and decoct them twice at a temperature of 95-105°C. The amount of water added for the first time is 8 times the amount of the decoction pieces, the soaking time is 0.5 hour, and the extraction time after boiling is 0.5 hour. The amount of water added for the second time is 8 times the amount of the decoction pieces, the extraction time after boiling is 0.5 hour, and the mixture is filtered through a No. 9 sieve (200 mesh).

[0275] (2) The obtained filtrate is concentrated under reduced pressure at a temperature of 60 to 70°C and a vacuum degree of -0.04 to -0.08 MPa to obtain a clear paste with a relative density of 1.07 (25°C).

[0276] (3) Add 2% xylitol, 0.5% white sugar and 0.10% potassium sorbate to the clear paste, add water to 1000 mL, mix well, refrigerate and let stand for 12 hours, filter, fill and sterilize to obtain Chaigechangyuan mixture.

[0277] After testing, the cream yield of decoction was 26.89%, and the cream yield of clear cream was 26.38%;

[0278] The transfer rates of astragaloside in the decoction were 54.59%, and puerarin were 56.43%; the transfer rates of astragaloside in the clear paste were 48.91%, and puerarin were 63.96%;

[0279] The content of astragaloside in Chaigechangyuan mixture is 1.10 mg / mL, and the content of puerarin is 0.47 mg / mL.

[0280] Examples 1-3

[0281] (1) Take 56g of Bupleurum chinense, 67g of Pueraria root, 33g of Andrographis paniculata, 67g of Saposhnikovia divaricata, 50g of Peucedanum chinense, 56g of Perilla stem, 50g of Codonopsis pilosula, 33g of Rhizoma Cibotii Root, 17g of Jujube, 17g of American ginseng, 56g of Scutellaria baicalensis, 50g of Radix Salviae Miltiorrhizae, 17g of Patchouli and 11g of ginger, and decoct three times at a decoction temperature of 95-105°C. The first water addition amount is 10 times the amount of the decoction piece, the soaking time is 0.5 hour, and the extraction time after boiling is 0.5 hour. The second water addition amount is 8 times the amount of the decoction piece, the extraction time after boiling is 0.5 hour, and the third water addition amount is 8 times the amount of the decoction piece, the extraction time after boiling is 0.5 hour, and the filter is filtered through a filter screen with a mesh number of 9 (200 mesh).

[0282] (2) The obtained filtrate is concentrated under reduced pressure at a temperature of 60 to 70°C and a vacuum degree of -0.04 to -0.08 MPa to obtain a clear paste having a relative density of 1.11 (21°C).

[0283] (3) Add 2% xylitol and 0.05% ethylparaben to the clear paste, add water to 1000 mL, mix well, refrigerate and let stand for 12 hours, filter, fill and sterilize to obtain Chaigechangyuan mixture.

[0284] After testing, the cream yield of decoction was 27.37%, and the cream yield of clear cream was 27.09%;

[0285] The transfer rates of astragaloside in the decoction were 58.39%, and puerarin were 68.58%; the transfer rates of astragaloside in the paste were 55.31%, and puerarin were 66.12%;

[0286] The content of astragaloside in Chaigechangyuan mixture is 2.48 mg / mL, and the content of puerarin is 0.68 mg / mL.

[0287] Examples 1-4

[0288] (1) Take 56g of Bupleurum chinense, 67g of Pueraria root, 33g of Andrographis paniculata, 67g of Saposhnikovia divaricata, 50g of Peucedanum chinense, 56g of Perilla stem, 50g of Codonopsis pilosula, 33g of Rhizoma Cibotii radix with bran stir-fry, 17g of Jujube, 17g of American ginseng, 56g of Scutellaria baicalensis, 50g of Radix Achyranthis Bidentatae, 17g of Pogostemon cablin and 11g of ginger, decoct three times, the decoction temperature is 95-105°C, the first water addition amount is 8 times the amount of the decoction piece, the soaking time is 2.0 hours, and the extraction time after boiling is 1.0 hour. The second water addition amount is 6 times the amount of the decoction piece, the extraction time after boiling is 1.0 hour, the third water addition amount is 6 times the amount of the decoction piece, the extraction time after boiling is 1.0 hour, and the filter is filtered through a No. 9 sieve (200 mesh).

[0289] (2) The obtained filtrate is concentrated under reduced pressure at a temperature of 60 to 70°C and a vacuum degree of -0.04 to -0.08 MPa to obtain a clear paste having a relative density of 1.08 (24°C).

[0290] (3) Add 6% xylitol and 0.05% potassium sorbate to the clear paste, add water to 1000 mL, mix well, refrigerate and let stand for 12 hours, filter, fill and sterilize to obtain Chaigechangyuan mixture.

[0291] After testing, the cream yield of decoction was 30.44%, and the cream yield of clear paste was 31.60%;

[0292] The transfer rates of astragaloside in the decoction were 59.41%, and puerarin were 74.07%; the transfer rates of astragaloside in the clear paste were 59.31%, and puerarin were 73.21%;

[0293] The content of astragaloside in Chaigechangyuan mixture is 2.60 mg / mL, and the content of puerarin is 0.70 mg / mL.

[0294] The data obtained from Examples 1-1 to 1-4 are summarized in the following table:

[0295] Table 5 Summary of data obtained in Example 1

[0296]

[0297]

[0298] Study on the stability of Chaigechangyuan mixture

[0299] The preparation method of Example 1-1 was scaled up to 90 doses to prepare three batches of pilot samples of Chaigechangyuan mixture, which were named CG211213, CG211220 and CG211227, respectively. The pilot samples of Chaigechangyuan mixture were used as the detection objects in the stability study experiment of Chaigechangyuan mixture.

[0300] (1) High temperature test

[0301] Take the pilot sample of Chaigechangyuan mixture (CG211227), open it and place it in a constant temperature device at 60℃±2℃, take samples on the 0th day, 5th day, 10th day and 30th day respectively, and test according to the key items of stability inspection. And compare the test results with the established quality standards to determine whether they meet the established quality standards.

[0302] Table 6 High temperature test results

[0303]

[0304] (2) Strong light irradiation test

[0305] Take the pilot sample of Chaigechangyuan mixture (CG211227), place the opening in a container with an illumination of 4500Lx±500Lx, and the total illumination of the light source should not be less than 1.2×106Lux·hr, and the energy of the near-ultraviolet lamp should not be less than 200W·hr / m 2 , samples are taken on the 0th, 5th, 10th and 30th days respectively, and tested according to the key stability inspection items. The test results are compared with the established quality standards to determine whether they meet the established quality standards.

[0306] Table 7 Strong light irradiation test results

[0307]

[0308] (3) Accelerated stability test results

[0309] Take 3 batches of pilot production samples and place them at a temperature of 40℃±2℃ and a relative humidity of 75%±5% for 6 months. At the end of the 1st month, 2nd month, 3rd month and 6th month respectively during the test period, and test them according to the specified stability inspection items. Compare the test results with the established quality standards to determine whether they meet the established quality standards.

[0310] Table 8 Results of accelerated stability test of Chaigechangyuan mixture Sample packaging conditions: soda-lime glass tube oral liquid bottle, storage conditions: 40℃±2℃, humidity 75%±5%

[0311]

[0312]

[0313] (4) Long-term stability test

[0314] Take 3 batches of pilot production samples and place them at a temperature of 30℃±2℃ and a relative humidity of 65%±5% for 12 months. Take samples every 3 months, and take samples at 0 months, 3 months, 6 months, 9 months, and 12 months respectively, and test them according to the key stability inspection items. After 12 months, it is still necessary to continue the inspection, and take samples for inspection at 18 months, 24 months, and 36 months respectively, and compare the inspection results with 0 months to determine the shelf life of the drug.

[0315] Table 9 Results of long-term stability test of Chaigechangyuan mixture

[0316] Sample packaging conditions: Soda-lime glass controlled oral liquid bottle, storage conditions: temperature 30℃±2℃, relative humidity 65%±5%

[0317]

[0318]

[0319] Example 2: Study on the content determination of Chaigechangyuan mixture

[0320] 2.1 Detection Method

[0321] (1) Preparation of reference solution

[0322] Take an appropriate amount of puerarin reference substance, accurately weigh it, and add 30% ethanol to make a solution containing 75 μg per 1 mL; take an appropriate amount of baicalin reference substance, accurately weigh it, and add methanol to make a reference substance solution containing 100 μg per 1 mL.

[0323] (2) Preparation of test solution

[0324] Accurately measure 3 mL of the pilot product of Chaigechangyuan mixture, put it in a 25 mL volumetric flask, add an appropriate amount of 80% methanol, shake well, add 80% methanol to make up to 25 mL, shake well, filter, and take the filtrate to obtain.

[0325] (3) Ultra-high performance liquid chromatography analysis

[0326] Octadecylsilane bonded silica gel is used as a filler (column length is 150 mm, inner diameter is 2.1 mm, particle size is 1.8 μm); methanol is used as mobile phase A, 0.1% phosphoric acid water is used as mobile phase B, and elution is performed according to the following table; the column temperature is 25° C., the flow rate is 0.3 mL / min; the detection wavelengths are 250 nm and 280 nm (when calculating the content and transfer rate, puerarin is calculated at a detection wavelength of 250 nm, and baicalin is calculated at a peak area at a wavelength of 280 nm. At wavelengths of 250 and 280 nm, there is no obvious difference in the peak shape of the chromatogram, so the present invention only attaches the chromatogram at a wavelength of 250 nm), the injection volume is 1 μL; the number of theoretical plates calculated based on puerarin should not be less than 25,000.

[0327] Table 10 Mobile phase elution gradient

[0328]

[0329] 2.2 Investigation of experimental conditions in the detection method

[0330] A. Determination of detection wavelength

[0331] The ultraviolet absorption spectra of puerarin, baicalin reference solution and sample solution at 190nm-400nm were recorded respectively. The results showed that puerarin had maximum ultraviolet absorption at around 250nm and baicalin had maximum ultraviolet absorption at around 280nm, so 250nm and 280nm were selected as the maximum absorption wavelengths for the determination of puerarin and baicalin content, respectively.

[0332] B. Preparation of Test Solution

[0333] Ⅰ. Investigation of different extraction solvents

[0334] Accurately measure 3mL of Chaigechangyuan mixture (CG211227) and place it in a 25mL volumetric flask. Add appropriate amounts of extraction solvents such as methanol, 80% methanol, 50% methanol, ethanol, 80% ethanol, 50% ethanol, and water in turn, shake well, add appropriate amounts of solvents to 25mL in turn, shake well, and filter. Accurately draw 1μL of each sample solution and inject it into the liquid chromatograph to calculate the content of puerarin and baicalin. The experimental results are shown in the table below.

[0335] Table 11 Extraction solvent investigation results

[0336]

[0337] In the experiment, the test sample solution extracted with water as solvent was difficult to filter and the test sample would produce precipitation after filtration. Considering the impact on the chromatographic column, water was not considered in the comparison range. When 80% methanol solvent was used for extraction, the content of puerarin and baicalin was relatively high, so 80% methanol was selected as the best extraction solvent.

[0338] II. Investigation of extraction methods

[0339] Accurately measure 3mL of Chaigechangyuan mixture (CG211227), divide into 2 groups, 2 portions in each group, place in a 25mL volumetric flask, add appropriate amount of 80% methanol to 1 group, shake well, ultrasonicate for 30min, add 80% methanol to 25mL, shake well; add appropriate amount of 80% methanol to the other group, shake well, make up to 25mL, shake well, filter, and obtain. Accurately draw 1μL, inject into liquid chromatograph, determine according to the chromatographic conditions under 2.1, calculate the content of puerarin and baicalin, and the experimental results are shown in the table below.

[0340] Table 12 Results of the extraction method investigation

[0341]

[0342] There was no significant effect of ultrasonic extraction content on shaking. Considering that the direct volume extraction method by shaking was simpler, the direct volume extraction by shaking was chosen.

[0343] 2.3 Methodological validation of the detection method

[0344] A. Exclusiveness Investigation

[0345] Take Chaigechangyuan mixture, kudzu root negative sample, and scutellaria negative sample, and prepare the test solution according to the above method. Take puerarin and baicalin reference solution, 80% methanol blank solvent and 1 μL of each test solution, inject into the liquid chromatograph, and determine according to the chromatographic conditions under 2.1. The results are shown in Figure 1 .

[0346] The experimental results showed that there were no chromatographic peaks at the retention times corresponding to puerarin and baicalin in the blank solvent chromatogram, indicating that the solvent had no interference with the determination of puerarin and baicalin; there were no other medicinal materials in the sample that interfered with baicalin and puerarin, and puerarin and baicalin in the sample came from Pueraria lobata and Scutellaria baicalensis slices, respectively. Therefore, the determination of puerarin and baicalin in Chaigechangyuan mixture by this method has specificity.

[0347] B. Peak Purity Investigation

[0348] Accurately pipette 1 μL of the Chaigechangyuan mixture sample solution, 1 μL of puerarin and baicalin reference solution, inject into liquid chromatograph, and measure according to the chromatographic conditions under 2.1 to investigate the purity of the chromatographic peak.

[0349] Depend on Figure 2 The experimental results show that the purity of the chromatographic peak of each index component is greater than 990, indicating that the chromatographic peak of each measured component is relatively pure.

[0350] C. Linear Investigation

[0351] Accurately weigh appropriate amounts of puerarin and baicalin reference substances, place them in 50mL volumetric flasks, add 30% ethanol and methanol respectively to dissolve and dilute to the scale to prepare puerarin and baicalin reference substance solutions containing 0.15751mg and 0.49819mg per 1mL as mother solutions.

[0352] Use the same pipette to accurately pipette 8mL, 7mL, 6mL, 5mL, 4mL, and 2mL of the puerarin reference substance mother solution into a 10mL volumetric flask, add 30% ethanol to dilute to the scale, and obtain puerarin reference substance solutions with a concentration of 0.12601mg, 0.11026mg, 0.09451mg, 0.07876mg, 0.06300mg, and 0.03150mg per 1mL; take 4mL, 3.5mL, 3mL, 2.5mL, 2mL, 1.5mL, and 1mL of the baicalin reference substance mother solution, respectively, and dilute to a 5mL volumetric flask with methanol to obtain baicalin reference substance solutions with a concentration of 0.39855mg, 0.34873mg, 0.29891mg, 0.24909mg, 0.19927mg, and 0.09964mg per 1mL. Accurately pipette 1 μL of each into the liquid chromatograph and determine according to the chromatographic conditions in 2.1. Draw a standard curve with the peak area as the ordinate and the concentration of puerarin and baicalin (mg / mL) as the abscissa. The results are shown in the table below. Figure 3 , Figure 4 .

[0353] Table 13 Puerarin linearity test results

[0354]

[0355] Table 14 Results of linear investigation of baicalin

[0356]

[0357]

[0358] The results in the table are plotted and the linear regression equation of puerarin is y=14146x-28.238, with a correlation coefficient of R 2 =0.9997. The experimental results showed that the concentration of puerarin had a good linear relationship with the peak area in the concentration range of 0.03150-0.15751 mg / mL. The linear regression equation of baicalin was y=4156.1x+1.5867, and the correlation coefficient R 2 =0.9991. The experimental results showed that the concentration of baicalin had a good linear relationship with the peak area in the concentration range of 0.09964-0.49819 mg / mL.

[0359] D. Precision inspection

[0360] Take Chaigechangyuan mixture (CG211227), prepare the test solution according to the method under 2.1, repeat the injection 6 times according to the chromatographic conditions under 2.1, with an injection volume of 1 μL, determine the contents of puerarin and baicalin in the test solution, and calculate the RSD% value. The determination results are shown in the table below.

[0361] Table 15 Precision investigation results

[0362]

[0363] The experimental results showed that the RSD% of puerarin was less than 3%, and the RSD% of baicalin was less than 2%. According to the "Guidelines for Validation of Analytical Methods" of the 2020 edition of the "Chinese Pharmacopoeia", when the content of the tested component in the sample is greater than 0.1 mg / g, the precision RSD% limit is less than 3%; when the content is greater than 1 mg / g, the precision RSD% limit is less than 2.0%; the results show that the instrument has good precision.

[0364] E. Stability test

[0365] Take Chaigechangyuan mixture (CG211227), prepare the test solution according to the method under 2.1, and inject samples at 0, 2, 4, 6, 8, 16, and 24 hours according to the chromatographic conditions under 2.1, with an injection volume of 1 μL. Determine the contents of puerarin and baicalin in the test solution, and calculate the RSD% values. The determination results are shown in the table below.

[0366] Table 16 Stability test results

[0367]

[0368] The test results showed that the RSD% of puerarin content was less than 3%, and the RSD% of baicalin content was less than 2%. According to the "Guidelines for Validation of Analytical Methods" of the 2020 edition of the "Chinese Pharmacopoeia", when the content of the tested component in the sample is ≥ 0.1 mg / g, the RSD% limit is ≤ 3%; when the content is ≥ 1 mg / g, the RSD% limit is ≤ 2.0%; the results showed that the test solution of Chaigechangyuan mixture was relatively stable within 24 hours.

[0369] F. Repeatability test

[0370] Take Chaigechangyuan mixture (CG211227), prepare 6 test solutions according to the method in 2.1, and use the chromatographic conditions in 2.1 with an injection volume of 1 μL. Determine the contents of puerarin and baicalin in the test solution, and calculate the RSD% value. The test results are shown in the table below.

[0371] Table 17 Repeatability test results

[0372]

[0373]

[0374] The test results showed that the RSD% of puerarin content was less than 3%, and the RSD% of baicalin content was less than 2%. According to the "Guidelines for Validation of Analytical Methods" of the 2020 edition of the "Chinese Pharmacopoeia", when the content of the tested component in the sample is ≥ 0.1 mg / g, the RSD% limit is ≤ 3%; when the content is ≥ 1 mg / g, the RSD% limit is ≤ 2.0%; the results show that the sample preparation method has good repeatability.

[0375] G. Intermediate precision study

[0376] Other analysts of the project team operated on different dates and under different chromatographs, accurately measured 3 mL of Chaigechangyuan mixture (CG211227), prepared the test solution according to the method in 2.1, and measured 6 replicates according to the chromatographic conditions in 2.1. The contents of puerarin and baicalin in the test solution were determined, and the RSD% values ​​were calculated. The results are shown in the table below.

[0377] Table 18 Intermediate precision inspection results

[0378]

[0379]

[0380] The experimental results showed that the RSD% of puerarin content was less than 3%, and the RSD% of baicalin content was less than 2%. According to the "Guidelines for Validation of Analytical Methods" of the 2020 edition of the "Chinese Pharmacopoeia", when the content of the tested component in the sample is ﹥1mg / g, the RSD% limit is ﹤2.0%; when the content is ﹥0.1mg / g, the RSD% limit is ﹤3%. The results show that the intermediate precision of this analytical method is good.

[0381] H. Accuracy Test

[0382] Take 1.5mL of the sample of Chaigechangyuan mixture with known content (CG211227, puerarin 0.800mg / g, baicalin 3.37mg / g), accurately measure, and accurately add 5mL and 10mL of puerarin and baicalin reference solutions (concentrations are 0.24373mg / mL and 0.50722mg / mL, respectively). At the same time, take 6 portions and make test solutions according to the test solution preparation method. Inject 1μL of each sample, determine the peak area, calculate the content, and calculate the recovery rate and RSD (%) according to the following formula. The results are shown in the table below.

[0383]

[0384] Table 19 Accuracy test results

[0385]

[0386]

[0387] According to the "Guidelines for Validation of Analytical Methods" in Part IV of the 2020 edition of the Chinese Pharmacopoeia, when the content of the tested component in the sample is >1 mg / g, the recovery rate limit is 92% to 105%; when the content of the tested component is >0.1 mg / g, the recovery rate limit is 90% to 108%. The experimental results showed that the recovery rates of puerarin and baicalin were 96% and 101%, respectively, which were within the specified range, indicating that the accuracy of the determination method was good.

[0388] I. Durability test

[0389] (1) Investigation of different chromatographic columns

[0390] The effects of Agilent, Shimadzu and Waters columns on the determination of puerarin and baicalin in Chaigechangyuan mixture were compared. Column 1-Agilent ZORBAX SB C18 (2.1×150mm, 1.8μm); Column 2-GL Sciences Inertsil ODS-3 (2.1×150mm, 2μm); Column 3-ACQUITY BEH C18 (2.1×150mm, 1.7μm).

[0391] Take 3mL of Chaigechangyuan mixture (DY20211227), prepare the test solution according to the method in 2.1, accurately take 1μL and put it into the high performance liquid chromatograph, according to the chromatographic conditions in 2.1, the experimental results are shown in the following table and Figure 5 .

[0392] Table 20 Results of investigation on different chromatographic columns

[0393]

[0394]

[0395] Depend on Figure 5 The data in the table show that different chromatographic columns have different effects on the content of puerarin and baicalin. Columns 2 and 3 have poor symmetry, while column 1 has good separation and symmetry for each component, and the content of the index component is high and the baseline is relatively stable. Therefore, it is recommended to use column 1-AgiLentZORBAX SB-C18.

[0396] (2) Investigation of different column temperatures

[0397] The effects of different column temperatures (25℃, 30℃, and 35℃) on the contents of puerarin and baicalin in Chaigechangyuan mixture were compared.

[0398] Accurately measure 3 mL of Chaigechangyuan mixture (GC211227), prepare the test solution according to the method in 2.1, accurately measure 1 μL and inject it into the high performance liquid chromatograph, change the column temperature to 25℃, 30℃, and 35℃ respectively according to the chromatographic conditions in 2.1. The experimental results are shown in the following table and Figure 6 .

[0399] Table 21 Results of investigation at different column temperatures

[0400]

[0401] The experimental results show that different column temperatures have certain differences in the content of puerarin and baicalin. When the column temperature is 25℃, the content of the index components is higher, and the chromatographic peak separation and symmetry of each component are better, so it is recommended to choose the best column temperature of 25℃.

[0402] (3) Investigation of different flow rates

[0403] Compare the effects of different flow rates of 0.25mL / min, 0.30mL / min, and 0.35mL / min on the contents of puerarin and baicalin in Chaigechangyuan mixture.

[0404] Take 3mL of Chaigechangyuan mixture (GC211227), prepare the test solution according to the method in 2.1, accurately take 1μL and put it into the high performance liquid chromatograph, change the flow rate to 0.25mL / min, 0.30mL / min, and 0.35mL / min according to the chromatographic conditions in 2.1, respectively. The experimental results are shown in the following table and Figure 7 .

[0405] Table 22 Results of investigation at different flow rates

[0406]

[0407] The experimental results show that different flow rates have a certain effect on paeoniflorin and baicalin. When the flow rate is 0.30 mL / min, the separation and symmetry of the index components are better. Therefore, it is recommended to select the optimal flow rate of 0.30 mL / min.

[0408] (4) Content determination of three batches of Chaigechangyuan mixture

[0409] Weigh 3 mL of the pilot product of Chaigechangyuan mixture, accurately measure, prepare the test solution according to the method under 2.1, inject 1 μL according to the chromatographic conditions under 2.1, record the peak area, calculate the content of puerarin and baicalin, and the results are shown in the table below.

[0410] Table 23 Results of determination of puerarin and baicalin in three batches of Chaigechangyuan mixture

[0411]

[0412] Analysis results: According to the content determination results of three batches of Chaigechangyuan mixture, it can be seen that there is no significant difference in the content of puerarin and baicalin in Chaigechangyuan mixture. The mean value of puerarin is 0.73 mg / L, the range of mean ±30% is 0.51-0.95 mg / L, and the range of mean ±3SD is 0.60-0.87 mg / mL; the mean value of baicalin is 3.0 mg / mL, the range of mean ±30% is 2.1-3.9 mg / mL, and the range of mean ±3SD is 2.3-3.7 mg / mL.

[0413] Conclusion: Due to the small number of sample batches in this study, the standard was set based on the minimum limit, that is, 1 mL of pueraria root contains puerarin (C 21 H 20 O9) should not be less than 0.51 mg, and each 1 mL contains baicalin (C 21 H 18 O 11 ) should not be less than 2.1 mg.

[0414] Example 3: TLC Identification Study of Radix Sarcandrae in Chaigechangyuan Mixture

[0415] 3.1 Preparation of test solution

[0416] Take 5 mL of Chaigechangyuan mixture sample and add it to a treated D101 macroporous resin column (2 g, 1.5 cm*12 cm) (plug the column with cotton and take 2 g of macroporous resin and mix it with water and load it onto the column. After the gas is exhausted, plug it with cotton and glass beads again), and elute it with 20 mL each of water, 20% ethanol and 80% ethanol in sequence. Collect the 80% ethanol eluate, evaporate it on a water bath, add 2 mL of methanol to the residue to dissolve it, filter it, and use it as the test solution.

[0417] 3.2 Preparation of negative sample solution

[0418] Take 5 mL of the negative sample of Scutellaria baicalensis, add it into the treated D101 macroporous resin column (2 g, 1.5 cm*12 cm), and elute it with 20 mL each of water, 20% ethanol and 80% ethanol in sequence, collect the 80% ethanol eluate, evaporate it on a water bath, add 2 mL of methanol to the residue to dissolve it, filter it, and use it as the negative control solution.

[0419] 3.3 Preparation of control medicinal material solution

[0420] Take 2g of Radix Sarcosioides control medicinal material, add 20mL of water, heat and reflux for 30min, filter, evaporate the filtrate on a water bath, add 5mL of water to the residue to dissolve it, add it to a treated D101 macroporous resin column (2g, 1.5cm*12cm), elute with 20mL of water, 20% ethanol, and 80% ethanol in turn, collect the 80% ethanol eluate, evaporate to dryness, and add 3mL of methanol to dissolve it as the control medicinal material solution.

[0421] 3.4 Thin layer chromatography method

[0422] According to the thin layer chromatography method (General Rule 0502), the above solutions were taken and spotted on the same silica gel G thin layer plate, and toluene: ethyl acetate: methanol: water (10:4:2:0.5) was used as the developing solvent. The plate was developed, taken out, dried, and examined under ultraviolet light (365nm).

[0423] 3.5 Investigation of different spotting amounts

[0424] Take Chaigechangyuan mixture with batch number CG211227, prepare the corresponding test sample according to the method of "3.1", examine the sample spot amount (1, 2, 4, 6 μL) respectively, develop it according to the thin layer development system of "3.4", and check the results. Figure 8 As shown. Figure 8 The horizontal axis 1 represents 1 μL of the test sample; 2 represents 2 μL of the test sample; 3 represents 4 μL of the test sample; 4 represents 6 μL of the test sample; and 5 represents 2 μL of the control drug. Figure 8 It can be seen that there is no significant difference in the results of the thin layer identification test with different spotting amounts. The spots in the range of 1-6μL are clearly visible, and the larger the spotting amount, the more obvious the spots. Taking all factors into consideration, 2μL is taken as the spotting amount in thin layer identification.

[0425] 3.6 Exclusivity Investigation

[0426] Take Chaigechangyuan mixture with batch number CG211227, prepare the corresponding test sample, negative sample and control medicinal material according to the method of "3.1", take 2μL of each of the above solutions, spot them on the same silica gel G thin layer plate, develop them according to the thin layer chromatography conditions of "3.4", and check. The results are as follows Fig. 9 As shown in the figure, the horizontal axis 1 represents the negative sample; 2 represents the test sample; and 3 represents the control medicinal material. Fig. 9 It can be seen that the same color fluorescent spots appear at the same position of the test sample chromatogram and the control medicinal material chromatogram, and there is no negative interference, which shows that the thin layer method has good specificity.

[0427] 3.7 Durability test

[0428] 3.7.1 Investigation of different thin layer plates

[0429] Take Chaigechangyuan mixture with batch number CG211227, prepare the corresponding test sample, negative sample and control medicinal materials according to the "3.1" method, take 2μL of each of the above solutions, and conduct durability study on thin layer plates of different manufacturers (Sinopharm Group silica gel G plate, Qingdao Ocean silica gel G plate, Qingdao Ocean high-efficiency silica gel G plate, German Merck G plate), develop and inspect according to the thin layer chromatography conditions of "3.4", and the results are as follows Fig.10 As shown. Among them, Fig.10a is the Sinopharm silica gel G plate, 1-2 on the horizontal axis represent the test products; 3 represents the control medicinal materials; Fig.10 b is Qingdao Ocean Silica G Plate, 1-2 on the horizontal axis represent the test products; 3 represents the control medicinal materials; Fig.10 c is Qingdao Ocean High-efficiency Silica G Plate, 1-2 on the horizontal axis represent the test products; 3 represents the control medicinal materials; Fig.10 d is a Merck G plate, with 1-2 on the horizontal axis representing the test sample and 3 representing the control drug. Fig.10 It can be seen that there is no obvious difference in the thin layer identification of the four thin layer plates. In the test sample chromatogram, spots of the same color appear at the corresponding position in the control medicinal material chromatogram, and the spots of the test sample and the control medicinal material are very clear. The separation degree of the test samples meets the requirements, and the Rf value is moderate, indicating that this identification method has good durability when carried out on different thin layer plates.

[0430] 3.7.2 Different development temperatures

[0431] Take Chaigechangyuan mixture with batch number CG211227, prepare the corresponding test sample, negative sample and control medicinal material according to the method of "3.1", take 2μL of each of the above solutions, and conduct durability inspection at different development temperatures (5℃, 25℃, 35℃), develop according to the thin layer chromatography conditions of "3.4", and check. The results are as follows Fig.11 As shown. Among them, Fig.11 a Development temperature: 5°C, 1-2 on the horizontal axis represent the test products; 3 represents the control herbal medicine; Fig.11 b Development temperature: 25°C, 1-2 on the horizontal axis represent the test products; 3 represents the control herbal medicine; Fig.11 c development temperature: 35°C, 1-2 on the horizontal axis represent the test sample; 3 represents the control herb. Fig.11 It can be seen that there is no significant difference in the results of the thin layer identification test at the above three development temperatures. In the chromatogram of the test sample, spots of the same color appear at the corresponding position of the chromatogram of the control medicinal material, and the spots of the test sample and the control medicinal material are very clear. The separation degree of the test sample meets the requirements and the Rf value is moderate. This shows that the identification method has good durability when developed at different temperatures.

[0432] 3.7.3 Different development humidity

[0433] Take Chaigechangyuan mixture with batch number CG211227, prepare the corresponding test sample, negative sample and control medicinal material according to the method of "3.1", take 2μL of each of the above solutions, and conduct durability inspection at different development humidity (33%, 66%, 88%). Different relative humidity is controlled by configuring different concentrations of sulfuric acid. Develop according to the thin layer chromatography conditions of "3.4" and check. The results are as follows Fig.12 As shown. Among them, Fig.12a. Development humidity (relative humidity): 33%, 1-2 on the horizontal axis represent the test sample; 3 represents the control medicinal material; Fig.12 b) Development humidity (relative humidity): 66%, 1-2 on the horizontal axis represent the test sample; 3 represents the control medicinal material; Fig.12 c development humidity (relative humidity): 88%, 1-2 on the horizontal axis represent the test sample; 3 represents the control medicinal material. Fig.12 It can be seen that there is no significant difference in the results of the above three TLC identification tests at different humidity levels. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions of the chromatogram of the control medicinal material, and the spots of the test sample and the control medicinal material are very clear. The separation of the test samples meets the requirements, and the Rf value is moderate. This shows that the identification method has good durability under different humidity levels.

[0434] Example 4: TLC Identification Study of Peucedanum in Chaigechangyuan Mixture

[0435] 4.1 Preparation of test solution

[0436] Take 5 mL of Chaigechangyuan mixture sample, add 10 mL of chloroform, ultrasonically treat for 10 minutes, filter, evaporate to dryness on a water bath, add 5 mL of methanol to the residue to dissolve it, and use it as the test solution.

[0437] 4.2 Preparation of negative sample solution

[0438] Take 5 mL of the negative sample lacking peucedanum, add 10 mL of chloroform, ultrasonically treat for 10 minutes, filter, evaporate to dryness on a water bath, add 5 mL of methanol to the residue to dissolve it, and use it as the negative control solution.

[0439] 4.3 Preparation of control medicinal material solution

[0440] Take 2 g of Peucedanum chinense reference medicinal material, add 20 mL of water, heat and reflux for 30 min, filter, evaporate to dryness on a water bath, add 5 mL of water to dissolve, add 10 mL of chloroform, ultrasonically treat for 10 minutes, filter, evaporate to dryness, add 6 mL of methanol to the residue to dissolve, and use it as the reference medicinal material solution.

[0441] 4.4 Thin layer chromatography method

[0442] According to the thin layer chromatography method (General Rule 0502), the above solutions were taken and spotted on the same silica gel G thin layer plate, and petroleum ether (60-90°C)-ethyl acetate (3:1) was used as the developing solvent. The plate was developed, taken out, dried, and examined under ultraviolet light (365nm).

[0443] 4.5 Investigation of different spotting amounts

[0444] Take Chaigechangyuan mixture with batch number CG211227, prepare the corresponding test sample by "4.2" method, examine the sample spot amount (1, 3, 6, 8 μL) respectively, and develop it according to "4.4" thin layer development system. The results are as follows Fig.13 As shown. Fig.13 The horizontal axis 1 represents 1 μL of the test sample; 2 represents 3 μL of the test sample; 3 represents 6 μL of the test sample; 4 represents 8 μL of the test sample; and 5 represents 1 μL of the control drug. Fig.13 It can be seen that there is no obvious difference in the results of the thin layer identification test with different spotting amounts. The spots in the range of 1-8μL are clearly visible. The larger the spotting amount, the more deformed the spots. Taking all factors into consideration, 3μL is taken as the spotting amount in thin layer identification.

[0445] 4.6 Specificity Investigation

[0446] Take Chaigechangyuan mixture with batch number CG211227, prepare the corresponding test sample, negative sample and control medicinal material according to the method "4.1", take 3μL of the test sample and negative sample solution, and 1μL of the control medicinal material, and spot them on the same silica gel G thin layer plate, and develop them according to the thin layer chromatography conditions of "4.4". The results are as follows Fig.14 As shown in the figure, the horizontal axis 1 represents the negative sample; 2 represents the test sample; and 3 represents the control medicinal material. Fig.14 It can be seen that the same color fluorescent spots appear at the same position of the test sample chromatogram and the control medicinal material chromatogram, and there is no negative interference. This shows that the thin layer method has good specificity.

[0447] 4.7 Durability test

[0448] 4.7.1 Investigation of different thin layer plates

[0449] Take Chaigechangyuan mixture with batch number CG211227, prepare the corresponding test sample, negative sample and control medicinal material according to the "4.1" method, take 3μL of test sample and negative sample solution, and 1μL of control medicinal material, and conduct durability study on thin layer plates of different manufacturers (Sinopharm Group silica gel G plate, Qingdao Ocean silica gel G plate, Qingdao Ocean high-efficiency silica gel G plate, German Merck G plate), and develop them according to the "4.4" thin layer development system. The results are as follows Fig.15 As shown. Among them, Fig.15 a is the Sinopharm silica gel G plate, 1-2 on the horizontal axis represent the test products; 3 represents the control medicinal materials; Fig.15 b is Qingdao Ocean Silica G Plate, 1-2 on the horizontal axis represent the test products; 3 represents the control medicinal materials; Fig.15 c is Qingdao Ocean High-efficiency Silica G Plate, 1-2 on the horizontal axis represent the test products; 3 represents the control medicinal materials; Fig.15d is Merck G plate, 1-2 on the horizontal axis represent the test sample; 3 represents the control medicinal material. There is no obvious difference in the thin layer identification of the above four thin layer plates. In the chromatogram of the test sample, spots of the same color appear at the corresponding position of the chromatogram of the control medicinal material, and the spots of the test sample and the control medicinal material are very clear. The separation degree of the test sample meets the requirements, and the Rf value is moderate, indicating that the durability of this identification method is good when carried out on different thin layer plates.

[0450] 4.7.2 Different development temperatures

[0451] Take Chaigechangyuan mixture with batch number CG211227, prepare the corresponding test sample, negative sample and control medicinal material according to the "4.1" method, take 3μL of test sample and negative sample solution, and 1μL of control medicinal material, and conduct durability test at different development temperatures (5℃, 25℃, 35℃), and develop according to the "4.4" thin layer development system. The results are as follows Fig.16 As shown. Among them, Fig.16 a Development temperature: 5°C, 1-2 on the horizontal axis represent the test products; 3 represents the control herbal medicine; Fig.16 b Development temperature: 25°C, 1-2 on the horizontal axis represent the test products; 3 represents the control herbal medicine; Fig.16 c development temperature: 35℃, 1-2 on the horizontal axis represent the test sample; 3 represents the control medicinal material. There is no significant difference in the results of the thin layer identification test at the above three development temperatures. In the chromatogram of the test sample, spots of the same color appear at the corresponding position of the chromatogram of the control medicinal material, and the spots of the test sample and the control medicinal material are very clear. The separation degree of the test sample meets the requirements, and the Rf value is moderate. This shows that this identification method has good durability when developed at different temperatures.

[0452] 4.7.3 Different development humidity

[0453] Take Chaigechangyuan mixture with batch number CG211227, prepare the corresponding test sample, negative sample and control medicinal material according to the "4.1" method, take 3μL of the test sample and negative sample solution, and 1μL of the control medicinal material, and conduct durability inspection at different development humidity (33%, 66%, 88%). Different relative humidity is controlled by configuring different concentrations of sulfuric acid, and the development is carried out according to the "4.4" thin layer development system. The results are as follows Fig.17 As shown. Among them, Fig.17 a. Development humidity (relative humidity): 33%, 1-2 on the horizontal axis represent the test sample; 3 represents the control medicinal material; Fig.17 b) Development humidity (relative humidity): 66%, 1-2 on the horizontal axis represent the test sample; 3 represents the control medicinal material; Fig.17c development humidity (relative humidity): 88%, 1-2 on the horizontal axis represent the test sample; 3 represents the control medicinal material. There is no significant difference in the results of the thin layer identification test at 33% and 66% humidity. In the chromatogram of the test sample, spots of the same color appear at the corresponding position of the chromatogram of the control medicinal material, and the spots of the test sample and the control medicinal material are very clear. The separation degree of the test sample meets the requirements, and the Rf value is moderate. The results of the thin layer identification test at 88% humidity are not clear. In the chromatogram of the test sample, spots of the same color appear at the corresponding position of the chromatogram of the control medicinal material, and the spots of the test sample and the control medicinal material are not clear. The separation degree of the test sample does not meet the requirements, indicating that this identification method has good development durability at medium and low humidity, and poor development at high humidity. The humidity should be strictly controlled at 33% to 66% when examining the chromatogram.

[0454] Example 5: Study on TLC Identification of Scutellaria Baicalensis in Chaigechangyuan Mixture

[0455] 5.1 Preparation of test solution

[0456] Take 5 mL of Chaigechangyuan mixture sample, add it to the treated D101 macroporous resin column (2 g, 1.5 cm*12 cm), and elute with water, 20% ethanol, 80% ethanol at 50 mL, 50 mL, and 100 mL respectively. Collect the 80% ethanol eluate, evaporate it on a water bath, add 1 mL of methanol to the residue to dissolve it, filter it, and use it as the test solution.

[0457] 5.2 Preparation of negative sample solution

[0458] Take 5 mL of the negative sample lacking Scutellaria baicalensis, add it to the treated D101 macroporous resin column (2 g, 1.5 cm*12 cm), and elute it with water, 20% ethanol, 80% ethanol at 50 mL, 50 mL, and 100 mL respectively. Collect the 80% ethanol eluate, evaporate it on a water bath, add 1 mL of methanol to the residue to dissolve it, and filter it as the negative control solution.

[0459] 5.3 Preparation of control medicinal material solution

[0460] Take 1 g of Scutellaria baicalensis reference medicinal material, add 30 mL of methanol, ultrasonicate for 30 min, filter, evaporate the filtrate on a water bath, add 5 mL of water to the residue to dissolve it, add it to a treated D101 macroporous resin column (2 g, 1.5 cm*12 cm), and elute with water, 20% ethanol, 80% ethanol at 50 mL, 50 mL, and 100 mL in sequence, collect the 80% ethanol eluate, evaporate to dryness, and add 2 mL of methanol to the residue to dissolve it as the reference medicinal material solution.

[0461] 5.4 Thin layer chromatography method

[0462] According to the thin layer chromatography method (General Rule 0502), the above solutions were taken and spotted on the same silica gel G thin layer plate, and toluene-ethyl acetate-formic acid (10:5:2) was used as the developing solvent. The plate was taken out, dried, sprayed with 2% ferric chloride ethanol solution, heated at 105°C until the spots were colored, and examined under sunlight.

[0463] 5.5 Investigation of different spotting amounts

[0464] Take Chaigechangyuan mixture with batch number CG211227, prepare the corresponding test sample according to the "5.1" method, examine the sample spot volume (1, 3, 6, 8 μL) respectively, and develop it according to the "5.4" thin layer development system. The results are as follows Fig.18 As shown. Fig.18 The horizontal axis 1 represents 1μL of test sample; 2 represents 3μL of test sample; 3 represents 6μL of test sample; 4 represents 8μL of test sample; 5 represents 1μL of control medicinal material. There is no significant difference in the results of the above thin layer identification test with different sample amounts. The spots are clearly visible in the range of 2-6μL. The larger the sample amount, the more obvious the spots. Considering comprehensively, 3μL is taken as the sample amount in thin layer identification.

[0465] 5.6 Exclusivity Examination

[0466] Take Chaigechangyuan mixture with batch number CG211227, prepare the corresponding test sample, negative sample and control medicinal material according to the method of "5.1", take 3μL of test sample and negative sample solution, 1μL of control medicinal material, spot them on the same silica gel G thin layer plate, and develop them according to the thin layer chromatography conditions of "5.4". The results are as follows Fig.19 As shown in the figure, the horizontal axis 1 represents the negative sample; 2 represents the test sample; and 3 represents the control medicinal material. The same color fluorescent spots appear at the same position of the test sample chromatogram and the control medicinal material chromatogram, and there is no negative interference. This shows that the thin layer method has good specificity.

[0467] 5.7 Durability test

[0468] 5.7.1 Investigation of different thin layer plates

[0469] Take Chaigechangyuan mixture with batch number CG211227, prepare the corresponding test sample, negative sample and control medicinal material according to the "5.1" method, take 3μL of test sample and negative sample solution, and 1μL of control medicinal material, and conduct durability study on thin layer plates of different manufacturers (Sinopharm Group silica gel G plate, Qingdao Ocean silica gel G plate, Qingdao Ocean high-efficiency silica gel G plate, German Merck G plate), and develop them according to the "5.4" thin layer development system. The results are as follows Fig. 20 As shown. Among them, Fig. 20 a is the Sinopharm silica gel G plate, 1-2 on the horizontal axis represent the test products; 3 represents the control medicinal materials; Fig. 20b is Qingdao Ocean Silica G Plate, 1-2 on the horizontal axis represent the test products; 3 represents the control medicinal materials; Fig. 20 c is Qingdao Ocean High-efficiency Silica G Plate, 1-2 on the horizontal axis represent the test products; 3 represents the control medicinal materials; Fig. 20 d is Merck G plate, 1-2 on the horizontal axis represent the test sample; 3 represents the control medicinal material. Except for Merck G plate, there is no obvious difference in the thin layer identification of the above three thin layer plates. In the chromatogram of the test sample, spots of the same color appear at the corresponding position of the chromatogram of the control medicinal material. Among them, the spots of Qingdao Ocean High-efficiency Silica G Plate and Qingdao Ocean Silica G Plate are clearer, and the separation of Qingdao Ocean Silica G Plate is better. The separation of the test samples meets the requirements, and the Rf value is moderate, indicating that the durability of this identification method under Qingdao Ocean Silica G Plate is good, and Merck G plate is not considered when investigating this chromatogram.

[0470] 5.7.2 Different development temperatures

[0471] Take Chaigechangyuan mixture with batch number CG211227, prepare the corresponding test sample, negative sample and control medicinal material according to the "5.1" method, take 3μL of the test sample and negative sample solution, and 1μL of the control medicinal material, and conduct durability inspection at different development temperatures (5℃, 25℃, 35℃), and develop it according to the "5.4" thin layer development system. The results are as follows Fig.21 As shown. Among them, Fig.21 a Development temperature: 5°C, 1-2 on the horizontal axis represent the test products; 3 represents the control herbal medicine; Fig.21 b Development temperature: 25°C, 1-2 on the horizontal axis represent the test products; 3 represents the control herbal medicine; Fig.21 c development temperature: 35℃, 1-2 on the horizontal axis represent the test sample; 3 represents the control medicinal material. In the chromatograms of the test sample at the above three temperatures, spots of the same color appear at the corresponding positions of the chromatograms of the control medicinal material, and the separation of the test samples meets the requirements. There is no significant difference in the results of the thin layer identification test at 25℃ and 35℃. The spots of the test sample and the control medicinal material are relatively blurred, and the spots of the test sample and the control medicinal material are clearer at 5℃, and the Rf value is better. This shows that the durability of this identification method is better when developed at low temperatures, and the temperature should be strictly controlled at 5℃~25℃ when examining the chromatogram.

[0472] 5.7.3 Different development humidity

[0473] Take Chaigechangyuan mixture with batch number CG211227, prepare the corresponding test sample, negative sample and control medicinal material according to the "5.1" method, take 3μL of the test sample and negative sample solution, and 1μL of the control medicinal material, and conduct durability inspection at different development humidity (33%, 66%, 88%). Different relative humidity is controlled by configuring different concentrations of sulfuric acid, and the development is carried out according to the "5.4" thin layer development system. The results are as follows Fig. 22As shown. Among them, Fig. 22 a. Development humidity (relative humidity): 33%, 1-2 on the horizontal axis represent the test sample; 3 represents the control medicinal material; Fig. 22 b) Development humidity (relative humidity): 66%, 1-2 on the horizontal axis represent the test sample; 3 represents the control medicinal material; Fig. 22 c development humidity (relative humidity): 88%, 1-2 on the horizontal axis represent the test sample; 3 represents the control medicinal material. There is no significant difference in the TLC identification test results of the above three development humidity levels. In the chromatogram of the test sample, spots of the same color appear at the corresponding position of the chromatogram of the control medicinal material, and the spots of the test sample and the control medicinal material are very clear. The separation degree of the test sample meets the requirements, and the Rf value is moderate. This shows that the identification method has good durability under different humidity conditions.

[0474] Application Examples

[0475] The effect of the Chaigechangyuan mixture prepared in Example 1-1 was verified. As of May 2022, there were 37 cold patients who used "Chaigechangyuan mixture" in the outpatient clinic of the Second Affiliated Hospital of Guizhou University of Traditional Chinese Medicine. Most of the patients had coughs and sputum symptoms. Some patients had physical fatigue, dizziness, discomfort, low fever, nasal congestion, runny nose or stomach distension, nausea, loss of appetite and other symptoms. After taking Chaigechangyuan mixture, after follow-up and return visits, it was found that 35 patients were effective after taking the medicine, accounting for about 94.59% of the total observed medical records. Among them, 33 patients had symptoms disappearing after taking the medicine, tending to clinical cure, accounting for about 89.18% of the total observed medical records; 2 patients had no symptoms after taking the medicine, no obvious relief, but no aggravation, accounting for about 5.40% of the total observed medical records; 2 patients felt that the medicine was ineffective, and the symptoms were progressive, accounting for about 5.40% of the total observed medical records. The above data show that the Chaigechangyuan mixture of the present invention has a significant therapeutic effect on patients with colds and can improve symptoms such as cough and sputum, loss of appetite and mental fatigue.

[0476] Although the above describes the specific implementation methods of the present invention, it does not limit the scope of protection of the present invention. Based on the technical solution of the present invention, various modifications or variations that can be made by those skilled in the art without creative work are still within the scope of protection of the present invention.

Claims

1. A thin layer chromatography identification method for Radix Sarcandrae in Chaigechangyuan mixture preparation, characterized in that: The steps include: (1) Preparation of test solution Take a sample of Chaigechangyuan mixture, add it to a D101 macroporous resin column, and elute it with water, 20% ethanol and 80% ethanol in sequence; collect the 80% ethanol eluate, evaporate it to dryness, add methanol to the residue to dissolve it, filter it, and use it as the test solution; (2) Preparation of negative drug solution Take a negative sample of Radix Achyranthis Bidentatae, add it to a D101 macroporous resin column, and elute it with water, 20% ethanol, and 80% ethanol in sequence; collect the 80% ethanol eluate, evaporate it to dryness, add methanol to the residue to dissolve it, filter it, and use it as a negative control solution; (3) Preparation of control medicinal material solution Take the control medicinal material of Radix Lycopodiellae, add water, heat to reflux, filter, evaporate the filtrate to dryness, add water to the residue to dissolve it, add it to the D101 macroporous resin column, and elute it with water, 20% ethanol and 80% ethanol in sequence; collect the 80% ethanol eluate, evaporate it to dryness, and add methanol to dissolve it as the control medicinal material solution; (4) Thin layer chromatography identification method The above solutions were absorbed and spotted on the same silica gel G thin layer plate respectively. The developing agent was a mixed solution of toluene, ethyl acetate, methanol and water. After development, the plate was taken out, dried and examined under ultraviolet light.

2. The thin layer chromatography identification method according to claim 1, wherein: In step (1), 20 mL of water, 20% ethanol and 80% ethanol were used for elution; and / or, in step (2), eluting with 20 mL each of water, 20% ethanol and 80% ethanol; And / or, in step (3), 20 mL of water, 20% ethanol and 80% ethanol are used for elution.

3. The thin layer chromatography identification method according to claim 1 or 2, wherein: In step (4), the volume ratio of toluene, ethyl acetate, alcohol and water is 10:4:2:0.

5.

4. A thin layer chromatography identification method for Peucedanum peucedanum in Chaigechangyuan mixture preparation, characterized in that: The steps include: (1) Preparation of test solution Take a sample of Chaigechangyuan mixture, add chloroform, ultrasonicate, filter, evaporate to dryness, add methanol to the residue to dissolve it, and use it as the test solution; (2) Preparation of negative drug solution Take a negative sample lacking peucedanum, add chloroform, ultrasonicate, filter, evaporate to dryness, and add methanol to dissolve the residue as a negative control solution; (3) Preparation of control medicinal material solution Take the control drug of Peucedanum chinense, add water, heat to reflux, filter, evaporate to dryness, add water to dissolve, add chloroform, ultrasonically treat, filter, evaporate to dryness, add methanol to dissolve the residue, and use it as the control drug solution; (4) Thin layer chromatography identification method Absorb the above solutions and spot them on the same silica gel G thin layer plate respectively. The developing agent is a mixed solution of petroleum ether and ethyl acetate. Develop, take out, dry and examine under ultraviolet light.

5. The thin layer chromatography identification method according to claim 4, wherein: The volume ratio of petroleum ether to ethyl acetate is 3:

1.

6. A thin layer chromatography identification method for Scutellaria baicalensis in Chaigechangyuan mixture preparation, characterized in that: The steps include: (1) Preparation of test solution Take a sample of Chaigechangyuan mixture, add it to a D101 macroporous resin column, and elute it with water, 20% ethanol and 80% ethanol in sequence; collect the 80% ethanol eluate, evaporate it to dryness, add methanol to the residue to dissolve it, filter it, and use it as the test solution; (2) Preparation of negative drug solution Take a negative sample lacking Scutellaria baicalensis, add it to a D101 macroporous resin column, and elute it with water, 20% ethanol and 80% ethanol in sequence; collect the 80% ethanol eluate, evaporate it to dryness, add methanol to the residue to dissolve it, filter it, and use it as a negative control solution; (3) Preparation of control medicinal material solution Take the reference medicinal material of Scutellaria baicalensis, add methanol, sonicate, filter, evaporate the filtrate to dryness, add water to the residue to dissolve it, add D101 macroporous resin column, and elute with water, 20% ethanol and 80% ethanol in sequence; collect the 80% ethanol eluate, evaporate to dryness, and add methanol to the residue to dissolve it as the reference medicinal material solution; (4) Thin layer chromatography identification method Absorb the above solutions and spot them on the same silica gel G thin layer plate respectively. The developing agent is a mixed solution of toluene, ethyl acetate and formic acid. Develop, take out, dry, spray with 2% ferric chloride ethanol solution, heat at 105℃ until the spots are colored, and examine under sunlight.

7. The thin layer chromatography identification method according to claim 6, wherein: In step (1), water, 20% ethanol and 80% ethanol were used for elution at 50 mL, 50 mL and 100 mL respectively; and / or, in step (2), eluting with water, 20% ethanol and 80% ethanol at 50 mL, 50 mL and 100 mL respectively; And / or, in step (3), preferably, water, 20% ethanol and 80% ethanol are used for elution at 50 mL, 50 mL and 100 mL respectively.

8. The thin layer chromatography identification method according to claim 6 or 7, wherein: In step (4), the volume ratio of toluene, ethyl acetate and formic acid is 10:5:

2.

9. The thin layer chromatography identification method according to any one of claims 1 to 8, wherein: In parts by weight, the Chaigechangyuan mixture contains 55-57 parts of Bupleurum chinense, 66-68 parts of Pueraria lobata, 32-34 parts of Andrographis paniculata, 66-68 parts of Saposhnikovia divaricata, 49-51 parts of Peucedanum chinense, 55-57 parts of Perilla stem, 49-51 parts of Codonopsis pilosula, 32-34 parts of fried green peel of bran, 16-18 parts of jujube, 16-18 parts of American ginseng, 55-57 parts of Scutellaria baicalensis, 49-51 parts of Sarcandra chinensis, 16-18 parts of Patchouli and 10-12 parts of ginger.

10. The thin layer chromatography identification method according to claim 9, wherein: In parts by weight, the Chaigechangyuan mixture contains 56 parts of northern bupleurum, 67 parts of kudzu root, 33 parts of andrographis paniculata, 67 parts of siler, 50 parts of peucedanum, 56 parts of perilla stem, 50 parts of codonopsis pilosula, 33 parts of fried green peel with bran, 17 parts of jujube, 17 parts of American ginseng, 56 parts of scutellaria baicalensis, 50 parts of swollen knotweed, 17 parts of patchouli and 11 parts of ginger.

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