A processing method of water-soluble components in allium bakeri, a thin-layer identification method and application of developing agent

Abstract

The application provides a thin layer identification method of water-soluble components in Allium bakeri, and provides a train of thought for identifying water-soluble components in traditional Chinese medicine containing Allium bakeri. The water-soluble components in the traditional Chinese medicine containing Allium bakeri, the intermediate and the finished product can be identified by using the processing method provided by the application, and the main feature is the impurity removal process in the sample processing process. The application provides an application of a developing agent in the thin layer identification method of the water-soluble components in the traditional Chinese medicine containing Allium bakeri, the intermediate and the finished product, and the thin layer chromatography spots obtained by the thin layer identification of the water-soluble components in the traditional Chinese medicine containing Allium bakeri, the intermediate and the finished product are clear. In addition, the thin layer chromatography spots obtained by using the specific developing agent, the sample processing method, the silica gel plate and the color developing method are clear, and the detection method has good durability.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pharmaceutical analysis, and particularly relates to a processing method of water-soluble components in Allium bakeri, a thin-layer identification method and application of a developing agent. BACKGROUND

[0002] Allium bakeri is the dried bulb of Allium bakeri or Allium chinense. Allium bakeri has the effects of removing obstruction from meridians and promoting blood circulation, and is used for treating chest pain, abdominal distention, and post-dysentery heaviness.

[0003] In the Chinese Pharmacopoeia, the identification method of Allium bakeri is mainly thin-layer chromatography. The quality standards of Chinese medicine containing Allium bakeri are collected in Table 1.

[0004] Table 1: Sources of thin-layer chromatography methods

[0005]

[0006] In Table 1, the method of the Chinese Pharmacopoeia can identify the fat-soluble components of Allium bakeri medicinal materials and Chinese medicine containing Allium bakeri raw powder, but cannot be used to identify the water decoction of Allium bakeri medicinal materials and Chinese medicine containing Allium bakeri. At present, the most original preparation process of Allium bakeri or Chinese medicine containing Allium bakeri is water decoction process. In order to trace the source and improve the curative effect of Chinese medicine prescriptions, the production process of Chinese medicine (classic prescription) should be as close to the water decoction process as possible. Therefore, it is urgent to provide a thin-layer identification method for the water decoction of Allium bakeri (water-soluble components). SUMMARY

[0007] Therefore, the present application aims to solve the technical problem of providing a processing method of water-soluble components in Allium bakeri, a thin-layer identification method and application of a developing agent.

[0008] The present application provides a processing method of medicinal materials, intermediates and finished products containing water-soluble components in Allium bakeri, comprising the following steps:

[0009] 1) Extracting the water solution of medicinal materials, intermediates and finished products containing water-soluble components in Allium bakeri with n-butanol to obtain a n-butanol extract;

[0010] 2) Dissolving the residue obtained by evaporating the n-butanol extract with methanol to obtain a methanol solution;

[0011] 3) Passing the methanol solution through a neutral alumina column, eluting with methanol, discarding the methanol eluate, and then eluting with water to collect the water eluate;

[0012] 4) Passing the water eluate obtained in step 3) through a macroporous adsorption resin, and then eluting with water, a methanol solution with a volume fraction of 10%, a methanol solution with a volume fraction of 50% and methanol in sequence to collect the methanol eluate;

[0013] 5) The methanol eluent obtained in step 4) is evaporated to dryness to obtain a residue, which is dissolved in methanol to obtain a treated solution for thin layer chromatography identification.

[0014] Preferably, in step 1), the n-butanol extraction is performed 2-4 times.

[0015] The volume ratio of the aqueous solution of the medicinal material, intermediate and finished product containing water-soluble components in Allium bakeri to n-butanol is 100: (30-100).

[0016] Preferably, in step 2), the mass-volume ratio of the residue to methanol is (0.6-1.0) g: (2-10) ml.

[0017] In step 4), the type of macroporous adsorption resin is D101.

[0018] The application also provides an application of a developing agent in a thin layer identification method of a medicinal material, intermediate and finished product containing water-soluble components in Allium bakeri, wherein the developing agent is selected from a mixed solution of ethyl acetate, formic acid, glacial acetic acid and water with a volume ratio of (14-16): (0.8-1.2): (0.8-1.2): (1.5-2.5).

[0019] Preferably, the developing agent is selected from a mixed solution of ethyl acetate, formic acid, glacial acetic acid and water with a volume ratio of 15:1:1:2.

[0020] The application also provides a thin layer identification method of a medicinal material, intermediate and finished product containing water-soluble components in Allium bakeri, comprising the following steps:

[0021] The test sample solution and the Allium bakeri control medicinal material solution are spotted on the same silica gel G thin layer plate, developed with a developing agent, dried and colored, and observed under ultraviolet light. If the same color spots appear in the test sample chromatogram at positions corresponding to the control medicinal material chromatogram, the sample is qualified.

[0022] The test sample solution and the Allium bakeri control medicinal material solution are obtained by the above treatment method.

[0023] The developing agent is selected from the developing agent in the above application.

[0024] Preferably, the spotting amount of the test sample solution and the Allium bakeri control medicinal material solution is 10 μl.

[0025] Preferably, the coloring method is:

[0026] The silica gel G thin layer plate is sprayed with 10% sulfuric acid ethanol solution and then heated.

[0027] Preferably, the heating temperature is 105 DEG C, and the heating time is 4-6 min.

[0028] Preferably, the ultraviolet condition is that the wavelength of the ultraviolet light is 365 nm.

[0029] Compared with the prior art, the present application provides a thin layer identification method of water-soluble components in Baihe, and provides a thought for identifying water-soluble components in Baihe-containing traditional Chinese medicines. The water-soluble components in Baihe-containing medicinal materials, intermediates and finished products can be identified by using the treatment method provided by the present application, and the main feature is the impurity removal process in the sample treatment process. The present application provides an application of a developing agent in the thin layer identification method of Baihe-containing medicinal materials, intermediates and finished products, and the thin layer chromatography spots obtained by the thin layer identification of Baihe-containing medicinal materials, intermediates and finished products are clear. In addition, by using the specific developing agent, sample treatment method, silica gel plate and color development method, the obtained thin layer chromatography spots are clear, and the detection method has good durability. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 The chromatogram obtained by the treatment method provided for Comparative Example 1;

[0031] Figure 2 The chromatogram obtained by the treatment method provided for Comparative Example 1;

[0032] Figure 3 The result chart of five samples under sunlight (left chart) and ultraviolet light (right chart) respectively;

[0033] Figure 4 The result chart of five samples under sunlight (left chart) and ultraviolet light (right chart) respectively;

[0034] Figure 5 The chromatogram obtained by the treatment method provided for Comparative Example 3;

[0035] Figure 6 The chromatogram obtained by the treatment method provided for Comparative Example 4;

[0036] Figure 7 The chromatogram obtained by the treatment method provided for Comparative Example 4;

[0037] Figure 8 The chromatogram obtained by the treatment method provided for Comparative Example 4

[0038] Figure 9 The chromatogram obtained by the treatment method provided for Comparative Example 5;

[0039] Figure 10Chromatogram obtained from the treatment method provided for Comparative Example 6;

[0040] Figure 11 Chromatogram obtained from the treatment method provided for Comparative Example 7;

[0041] Figure 12 Chromatogram obtained from the treatment method provided for Comparative Example 8;

[0042] Figure 13 Chromatogram obtained from the treatment method provided for Comparative Example 9;

[0043] Figure 14 Chromatogram obtained from the treatment method provided for Comparative Example 10;

[0044] Figure 15 Chromatogram obtained from the treatment method provided for Comparative Example 11;

[0045] Figure 16 Chromatogram obtained from the treatment method provided for Example 1;

[0046] Figure 17 Chromatogram obtained from the treatment method provided for Comparative Example 12;

[0047] Figure 18 Chromatogram obtained from the treatment method provided for Comparative Example 13;

[0048] Figure 19 Chromatogram obtained from the treatment method provided for Comparative Example 14 using the developing solvent ethyl acetate-methanol (1 : 1);

[0049] Figure 20 Chromatogram obtained from the treatment method provided for Comparative Example 14 using the developing solvent n-hexane-ethyl acetate (1 : 1);

[0050] Figure 21 Chromatogram obtained from the treatment method provided for Comparative Example 15 using the developing solvent ethyl acetate;

[0051] Figure 22 Chromatogram obtained from the treatment method provided for Comparative Example 16 using the developing solvent methanol;

[0052] Figure 23 Chromatogram obtained from the treatment method provided for Comparative Example 17 using the developing solvent ethyl acetate-glacial acetic acid-formic acid-water (15: 1 : 1 :2);

[0053] Figure 24 Chromatogram obtained from the treatment method provided for Comparative Example 18 using the developing solvent ethyl acetate-methanol-water (15:4:2);

[0054] Figure 25 Chromatogram obtained from the treatment method provided for Comparative Example 17 using the developing solvent ethyl acetate-methanol-0.1% sodium hydroxide (15:4:2);

[0055] Figure 26 Chromatograms obtained with three different eluents provided for Comparative Example 19;

[0056] Figure 27 Chromatograms obtained with two different eluents provided for Comparative Example 20;

[0057] Figure 28 Chromatograms obtained with three different eluents provided for Comparative Example 21;

[0058] Figure 29 Chromatograms obtained with three different eluents provided for Comparative Example 22;

[0059] Figure 30 Chromatogram obtained with the eluent chloroform-ethyl acetate-methanol water (15:40:22:10) provided for Comparative Example 23;

[0060] Figure 31 Chromatograms obtained with three different eluents provided for Comparative Example 24;

[0061] Figure 32 Chromatogram obtained with the eluent ethyl acetate-methanol-water (10:2:1) provided for Comparative Example 25;

[0062] Figure 33 Chromatogram obtained with the eluent ethyl acetate-methanol-ammonia water (10:2:1) provided for Comparative Example 26;

[0063] Figure 34 Chromatograms obtained with three different eluents provided for Comparative Example 27;

[0064] Figure 35 Chromatogram obtained with the eluent ethyl formate-methanol-glacial acetic acid-water (10:2:1:1) provided for Comparative Example 28;

[0065] Figure 36 Chromatograms obtained with four different eluents provided for Comparative Example 29;

[0066] Figure 37 Chromatograms obtained with two different eluents provided for Comparative Example 30;

[0067] Figure 38 Chromatograms obtained with four different eluents provided for Comparative Example 31;

[0068] Figure 39 Chromatogram obtained with the eluent ethyl acetate-methanol-0.1% sodium hydroxide (10:2:1) provided for Comparative Example 32;

[0069] Figure 40 Chromatograms obtained with three different eluents provided for Comparative Example 33;

[0070] Figure 41 The chromatogram obtained using the colorimetric reagent (2% vanillin 10% sulfuric acid ethanol solution) provided in Comparative Example 34;

[0071] Figure 42 The chromatogram obtained using the colorimetric reagent (2% vanillin sulfuric acid solution) provided in Comparative Example 34;

[0072] Figure 43 Chromatogram obtained using the colorimetric reagent (sulfuric acid-acetic anhydride (1:20) solution) provided in Comparative Example 34;

[0073] Figure 44 The chromatogram obtained using the colorimetric reagent (10% sulfuric acid ethanol solution) provided in Comparative Example 34;

[0074] Figure 45 The chromatogram obtained using the colorimetric reagent (2% vanillin sulfuric acid solution) provided in Comparative Example 34;

[0075] Figure 46 The chromatogram obtained using the colorimetric reagent (sulfuric acid-acetic anhydride (1:20) solution) provided in Comparative Example 34;

[0076] Figure 47 The chromatogram obtained using the colorimetric reagent (2% vanillin sulfuric acid solution) provided in Comparative Example 34;

[0077] Figure 48 The chromatogram obtained using the colorimetric reagent (2% vanillin sulfuric acid solution) provided in Comparative Example 34;

[0078] Figure 49 Chromatograms obtained for the two different colorimetric reagents provided in Comparative Example 34;

[0079] Figure 50 The chromatogram obtained from the thin-layer plate (silicone G thin-layer plate) provided for Comparative Example 35;

[0080] Figure 51 The thin-layer plate (GF) provided for Comparative Example 35 254 Chromatograms obtained from thin-layer chromatography (TLC);

[0081] Figure 52 The chromatogram obtained from the thin-layer plate (0.1% sodium hydroxide silica gel G thin-layer plate) provided for Comparative Example 35;

[0082] Figure 53 Chromatogram of the sample amount (10 μl) provided in Example 2;

[0083] Figure 54 Chromatograms of different spotting amounts provided in Example 2;

[0084] Figure 55Specificity results provided for Example 3;

[0085] Figure 56 Results for different manufacturers of thin layer plates;

[0086] Figure 57 Results for thin layer chromatography identification under different relative humidities;

[0087] Figure 58 Results for thin layer chromatography identification under low temperature;

[0088] Figure 59 Specificity results provided for Example 4;

[0089] Figure 60 Results for thin layer chromatography identification under different relative humidities;

[0090] Figure 61 Results for thin layer chromatography identification under different relative humidities;

[0091] Figure 62 Results for thin layer chromatography identification under low temperature;

[0092] Figure 63 Results for thin layer chromatography identification under different sample volumes;

[0093] Figure 64 Chromatogram obtained by the detection method provided for Comparative Example 36. DETAILED DESCRIPTION

[0094] The present application provides an application of a developing agent in a thin layer identification method of a medicinal material, an intermediate and a finished product containing a water-soluble component in Allium bakeri, wherein the developing agent is selected from a mixed solution of ethyl acetate, formic acid, glacial acetic acid and water with a volume ratio of (14-16):(0.8-1.2):(0.8-1.2):(1.5-2.5).

[0095] In some specific embodiments of the present application, the developing agent is selected from a mixed solution of ethyl acetate, formic acid, glacial acetic acid and water with a volume ratio of 15:1:1:2.

[0096] The present application also provides a processing method of a medicinal material, an intermediate and a finished product containing a water-soluble component in Allium bakeri, comprising the following steps:

[0097] 1) extracting a water solution of a medicinal material, an intermediate and a finished product containing a water-soluble component in Allium bakeri with n-butanol to obtain a n-butanol extract;

[0098] 2) dissolving a residue obtained by evaporating the n-butanol extract with methanol to obtain a methanol solution;

[0099] 3) passing the methanol solution through a neutral alumina column and eluting with methanol, discarding the methanol eluate, and then eluting with water, and collecting the water eluate;

[0100] 4) passing the water eluate obtained in step 3) through a macroporous adsorption resin, and then eluting with water, a methanol solution with a volume fraction of 10%, a methanol solution with a volume fraction of 50%, and methanol in sequence, and collecting the methanol eluate;

[0101] 5) evaporating the methanol eluate obtained in step 4) to dryness to obtain a residue, and dissolving the residue with methanol to obtain a processed solution for thin layer chromatography identification.

[0102] The present application first prepares an aqueous solution of a medicinal material, an intermediate, and a finished product containing water-soluble components in Allium bakeri, wherein the present application does not have special restrictions on the preparation method of the aqueous solution of the medicinal material, the intermediate, and the finished product containing water-soluble components in Allium bakeri, and a method known to those skilled in the art can be used. For example, a water extraction method.

[0103] Then, the aqueous solution of the medicinal material, the intermediate, and the finished product containing water-soluble components in Allium bakeri is extracted with n-butanol to obtain an n-butanol extract.

[0104] The volume ratio of the aqueous solution of the medicinal material, the intermediate, and the finished product containing water-soluble components in Allium bakeri to n-butanol is 100: (30-100), preferably 100:30, 100:50, 100:75, 100:100, or any value between 100: (30-100).

[0105] The n-butanol extraction is performed 2-4 times, which can be 2 times, 3 times, or 4 times.

[0106] The n-butanol extracts are combined and evaporated to dryness to obtain a residue.

[0107] The residue is dissolved with methanol, wherein the mass-volume ratio of the residue to methanol is (0.6-1.0) g: (2-10) ml, preferably 0.6 g:10 ml, 1 g:10 ml, 1 g:5 ml, 0.6 g:2 ml.

[0108] Then, the obtained methanol solution is eluted through a neutral alumina column (100-200 mesh, 3-5 g, 1.5 cm in inner diameter), and then eluted with methanol, and the methanol eluate is discarded. The volume ratio of the methanol solution to the methanol used for elution is (5-10):25-50, preferably 5:50, 10:50, 15:50, 20:50, or any value between 5-10:25-50, and preferably 5:50.

[0109] Then, the water eluate obtained in the above step is eluted through a macroporous adsorption resin, and then eluted with water, 10% methanol solution by volume, 50% methanol solution by volume and methanol in sequence. The macroporous adsorption resin is preferably D101 type macroporous adsorption resin, and the volume ratio of the water eluate to the water, 10% methanol solution by volume, 50% methanol solution by volume and methanol used for elution is (25-50):25-50, preferably 25:50, 30:50, 40:50, 50:50, or any value between 25-50:25-50.

[0110] The methanol eluate is evaporated to dryness to obtain a residue. The residue is dissolved in methanol, and the mass-volume ratio of the residue to methanol is (0.1-0.3) g:(1-3) ml, preferably 0.1 g:3 ml, 0.2 g:3 ml, 0.1 g:1 ml, or any value between (0.1-0.3) g:(1-3) ml.

[0111] Finally, a processed solution suitable for thin layer chromatography identification is obtained.

[0112] The above processing method is suitable for test samples of control medicinal materials of Allium bakeri, and medicinal materials, intermediates and finished products containing water-soluble components of Allium bakeri. According to the above processing method, non-Allium bakeri components in Allium bakeri intermediate and finished product samples can be removed better.

[0113] The application also provides a thin layer identification method for medicinal materials, intermediates and finished products containing water-soluble components of Allium bakeri, which comprises the following steps:

[0114] The test sample solution and the control medicinal material solution of Allium bakeri are spotted on the same silica gel G thin layer plate, developed with a developing agent, dried and colored, and then observed under ultraviolet conditions. If the same color spots appear in the test sample chromatogram at positions corresponding to the control medicinal material chromatogram, the sample is qualified.

[0115] The test solution of the water-soluble components in the Allium bakeri and the Allium bakeri control medicinal material solution are obtained according to the above processing method;

[0116] The developing agent is selected from a mixed solution of ethyl acetate, formic acid, glacial acetic acid and water with a volume ratio of (14-16):(0.8-1.2):(0.8-1.2):(1.5-2.5).

[0117] The sample volume of the test solution of the water-soluble components in the Allium bakeri and the Allium bakeri control medicinal material solution is 10 μl.

[0118] The developing method is:

[0119] The silica gel G thin layer plate is sprayed with 10% sulfuric acid ethanol solution and then heated.

[0120] The heating temperature is 105°C, and the heating time is 4-6 min, preferably 4, 5, 6, or any value between 4-6 min.

[0121] The ultraviolet condition is that the ultraviolet wavelength is 365 nm.

[0122] The application provides a thin layer identification method of water-soluble components in Allium bakeri, and provides a thought for identifying water-soluble components in Allium bakeri-containing traditional Chinese medicines. The water-soluble components in the medicinal material, intermediate and finished product containing Allium bakeri and having other interfering components can be identified by using the processing method provided by the application, and the main feature is the impurity removal process in the sample processing process. The thin layer chromatography spot obtained by using the developing agent provided by the application for thin layer identification of the medicinal material, intermediate and finished product containing water-soluble components in Allium bakeri is clear. In addition, by using the specific developing agent, sample processing method, silica gel plate and color developing method, the obtained thin layer chromatography spot is clear, and the detection method has good durability.

[0123] In order to further understand the application, the processing method, developing agent and thin layer identification method of water-soluble components in Allium bakeri provided by the application are described below in combination with examples, and the protection scope of the application is not limited by the following examples.

[0124] Zishixiabai Guizhi Decoction (21030903)

[0125] Zishixiabai Guizhi Decoction (21031601)

[0126] Zishixiabai Guizhi Decoction (21042801)

[0127] Zishixiabai Guizhi Decoction (21051001)

[0128] Zishixiabai Guizhi Decoction (21060201)

[0129] Decoction of Zhishi Xiebai Guizhi Decoction (21060202)

[0130] Decoction of Zhishi Xiebai Guizhi Decoction (21060203)

[0131] Bulbus Allii Macrostemonis powder (YF210101 purchased from Xianyang Jingyang County, Shaanxi Province)

[0132] Zhishi Xiebai Guizhi Decoction without Bulbus Allii Macrostemonis negative sample (21030805)

[0133] Zhishi Xiebai Guizhi Decoction without Bulbus Allii Macrostemonis negative sample (21040604)

[0134] Zhishi Xiebai Guizhi Decoction without Bulbus Allii Macrostemonis negative sample (21042802)

[0135] Zhishi Xiebai Guizhi Decoction without Guizhi negative sample (21042502)

[0136] Zhishi Xiebai Guizhi Decoction without Zhishi negative sample (21042801)

[0137] Zhishi Xiebai Guizhi Decoction without Houpu negative sample (21050702)

[0138] Zhishi Xiebai Guizhi Decoction without Gualou negative sample (21041402)

[0139] Bulbus Allii Macrostemonis decoction sample solution (21040701)

[0140] Bulbus Allii Macrostemonis decoction sample solution (21042803)

[0141] Bulbus Allii Macrostemonis control drug solution (121130-202107)

[0142] Bulbus Allii Macrostemonis decoction (positive sample, batch number: 21042803)

[0143] Bulbus Allii Macrostemonis decoction (positive sample, batch number: 21042803) Bulbus Allii Macrostemonis decoction (positive sample, batch number: 21042803) Preparation method: take 5 kg of Bulbus Allii Macrostemonis medicinal materials (YF210106, Xianyang Jingyang County, Shaanxi Province), manually select impurities, and obtain.

[0144] Bulbus Allii Macrostemonis control drug solution (121130-201906 purchased from China Institute for Drug Control)

[0145] Bulbus Allii Macrostemonis control drug solution (batch number: 121130-202107 purchased from China Institute for Drug Control)

[0146] Among them, (1) Zhishi Xiebai Guizhi Decoction decoction is a Zhishi Xiebai Guizhi Decoction prepared by the laboratory; the specific preparation method is as follows:

[0147] Preparation method: take Fructus Aurantii Immaturus 9g, Magnoliae Officinalis Cortex 12g, soak in 1000ml water for 45min, boil with strong fire, then decoct with gentle fire for 30min, filter, add Fructus Trichosanthis 24g, Bulbus Allii Macrostemonis 25g, Ramulus Cinnamomi 3.1g (soak the three medicinal materials in 1.1 times dose of water for 45min) to the decoction, boil with strong fire, then decoct with gentle fire for 15min, filter to obtain Fructus Aurantii Immaturus, Bulbus Allii Macrostemonis and Ramulus Cinnamomi Decoction.

[0148] According to the above method, seven batches of samples were obtained, which were Fructus Aurantii Immaturus, Bulbus Allii Macrostemonis and Ramulus Cinnamomi Decoction decoction (21030903), Fructus Aurantii Immaturus, Bulbus Allii Macrostemonis and Ramulus Cinnamomi Decoction decoction (21031601), Fructus Aurantii Immaturus, Bulbus Allii Macrostemonis and Ramulus Cinnamomi Decoction decoction (21042801), Fructus Aurantii Immaturus, Bulbus Allii Macrostemonis and Ramulus Cinnamomi Decoction decoction (21051001), Fructus Aurantii Immaturus, Bulbus Allii Macrostemonis and Ramulus Cinnamomi Decoction decoction (21060201), Fructus Aurantii Immaturus, Bulbus Allii Macrostemonis and Ramulus Cinnamomi Decoction decoction (21060202), Fructus Aurantii Immaturus, Bulbus Allii Macrostemonis and Ramulus Cinnamomi Decoction decoction (21060203).

[0149] (2) Fructus Aurantii Immaturus, Bulbus Allii Macrostemonis and Ramulus Cinnamomi Decoction without Bulbus Allii Macrostemonis negative sample was prepared according to the preparation method of Fructus Aurantii Immaturus, Bulbus Allii Macrostemonis and Ramulus Cinnamomi Decoction, the only difference was that Bulbus Allii Macrostemonis was not added.

[0150] According to the above method, three batches of samples were obtained, which were Fructus Aurantii Immaturus, Bulbus Allii Macrostemonis and Ramulus Cinnamomi Decoction without Bulbus Allii Macrostemonis negative sample (21030805), Fructus Aurantii Immaturus, Bulbus Allii Macrostemonis and Ramulus Cinnamomi Decoction without Bulbus Allii Macrostemonis negative sample (21040604), Fructus Aurantii Immaturus, Bulbus Allii Macrostemonis and Ramulus Cinnamomi Decoction without Bulbus Allii Macrostemonis negative sample (21042802).

[0151] (3) Fructus Aurantii Immaturus, Bulbus Allii Macrostemonis and Ramulus Cinnamomi Decoction without Ramulus Cinnamomi negative sample was prepared according to the preparation method of Fructus Aurantii Immaturus, Bulbus Allii Macrostemonis and Ramulus Cinnamomi Decoction, the only difference was that Ramulus Cinnamomi was not added.

[0152] According to the above method, Fructus Aurantii Immaturus, Bulbus Allii Macrostemonis and Ramulus Cinnamomi Decoction without Ramulus Cinnamomi negative sample (21042502) was obtained.

[0153] (4) Fructus Aurantii Immaturus, Bulbus Allii Macrostemonis and Ramulus Cinnamomi Decoction without Fructus Aurantii Immaturus negative sample was prepared according to the preparation method of Fructus Aurantii Immaturus, Bulbus Allii Macrostemonis and Ramulus Cinnamomi Decoction, the only difference was that Fructus Aurantii Immaturus was not added.

[0154] According to the above method, Fructus Aurantii Immaturus, Bulbus Allii Macrostemonis and Ramulus Cinnamomi Decoction without Fructus Aurantii Immaturus negative sample (21042801) was obtained.

[0155] (5) Fructus Aurantii Immaturus, Bulbus Allii Macrostemonis and Ramulus Cinnamomi Decoction without Magnoliae Officinalis Cortex negative sample was prepared according to the preparation method of Fructus Aurantii Immaturus, Bulbus Allii Macrostemonis and Ramulus Cinnamomi Decoction, the only difference was that Magnoliae Officinalis Cortex was not added.

[0156] According to the above method, Fructus Aurantii Immaturus, Bulbus Allii Macrostemonis and Ramulus Cinnamomi Decoction without Magnoliae Officinalis Cortex negative sample (21050702) was obtained.

[0157] (6) Fructus Aurantii Immaturus, Bulbus Allii Macrostemonis and Ramulus Cinnamomi Decoction without Fructus Trichosanthis negative sample was prepared according to the preparation method of Fructus Aurantii Immaturus, Bulbus Allii Macrostemonis and Ramulus Cinnamomi Decoction, the only difference was that Fructus Trichosanthis was not added.

[0158] According to the above method, the Zishixiakuibujiazhi Decoction Gualou Yin negative sample (21041402) was obtained.

[0159] (7) The preparation method of the Baiyun Decoction piece single decoction sample solution is as follows: take Baiyun Decoction piece 25 g (soak in water 25 ml for 45 min), add water 800 ml, boil with strong fire, then decoct with gentle fire for 15 min, filter, and the filtrate is obtained.

[0160] According to the above method, two batches of samples were obtained, namely Baiyun Decoction piece single decoction sample solution (21040701) and Baiyun Decoction piece single decoction sample solution (21042803).

[0161] (8) The preparation method of Baiyun control drug material solution (121130-202107) is as follows: take Baiyun control drug material 4.0 g, add water 100 ml, boil for 15 min, cool, filter, add water to 100 ml, extract with n-butanol twice, each time 30 ml, combine the n-butanol liquid, evaporate to dryness, add methanol 5 ml to dissolve, pass through a neutral alumina column (100-200 mesh, 3 g, inner diameter 1.5 cm), elute with methanol 20 ml, discard the eluent, elute with water 25 ml, collect the eluent, pass through a D101 type macroporous adsorption resin column (inner diameter 1.5 cm, column length 10 cm), elute with water 25 ml, 10% methanol 25 ml, 50% methanol 25 ml, methanol 25 ml in turn, collect the methanol eluent, recover the solvent to dryness, add methanol 1 ml to dissolve the residue, and the solution is used as the Baiyun control drug material solution.

[0162] (9) The preparation method of Baiyun decoction liquid (positive sample, batch number: 21042803) is as follows: take Baiyun Decoction piece single decoction liquid 100 ml, extract with n-butanol twice, each time 30 ml, combine the n-butanol liquid, evaporate to dryness, add methanol 5 ml to dissolve the residue, pass through a neutral alumina column (100-200 mesh, 3 g, inner diameter 1.5 cm), elute with methanol 20 ml, discard the eluent, elute with water 25 ml, collect the eluent, pass through a D101 type macroporous adsorption resin column (inner diameter 1.5 cm, column length 10 cm), elute with water 25 ml, 10% methanol 25 ml, 50% methanol 25 ml, methanol 25 ml in turn, collect the methanol eluent, recover the solvent to dryness, add methanol 1 ml to dissolve the residue, and the solution is used as the Baiyun decoction liquid solution.

[0163] (10) The preparation method of Baiyun Decoction piece (YF21012906) is as follows: take Baiyun Decoction piece 25 g (soak in water 25 ml for 45 min), add water 800 ml, boil with strong fire, then decoct with gentle fire for 15 min, filter, and the filtrate is obtained.

[0164] Comparative Example 1

[0165] Test solution ① of Zhishi Xiebai Guizhi Decoction: Take 100ml of Zhishi Xiebai Guizhi Decoction (21030903), extract it twice with n-hexane, 20ml each time, combine the n-hexane extracts, evaporate to dryness, dissolve the residue in 1ml of n-hexane, and use it as test solution ①.

[0166] Allium macrostemon herb solution ①: Take 4g of Allium macrostemon herb powder (YF210101), add 100ml of water, boil for 30 minutes, cool, filter, extract the filtrate twice with n-hexane, 20ml each time, evaporate to dryness, dissolve the residue in 1ml of n-hexane, and use as Allium macrostemon herb solution ①.

[0167] Take two solutions, 5 μl and 10 μl, of each solution and apply them to the same silica gel G thin-layer plate. Use n-hexane-ethyl acetate (10:1) as the developing solvent, develop the plate, remove it, air dry it, spray it with 10% sulfuric acid ethanol solution, heat it at 105℃ until the spots are clearly visible, and examine it under a UV lamp at 365 nm.

[0168] See results Figure 1 , Figure 1 The chromatogram obtained by the processing method provided for Comparative Example 1. Figure 1 In the middle, from left to right, the samples are Allium macrostemon solution ① (5 μl), test sample solution ① (5 μl), Allium macrostemon solution ① (10 μl), and test sample solution ① (10 μl).

[0169] Take 5 μl of each of the two solutions, namely, the sample solution ① and the Allium macrostemon herb solution ①, and spot them separately on the same silica gel G thin-layer plate. Use n-hexane-ethyl acetate (10:1) as the developing solvent, develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly visible, and examine under a UV lamp at 365 nm.

[0170] See results Figure 2 , Figure 2 The chromatogram obtained by the processing method provided for Comparative Example 1. Figure 2 In the middle, from left to right, the samples are Allium macrostemon solution ① (5 μl) and test sample solution ① (5 μl).

[0171] Depend on Figure 1 and Figure 2 It can be seen that the treatment method provided in Comparative Example 1 is to use the identification method of Allium macrostemon in the pharmacopoeia to identify the Allium macrostemon and Allium macrostemon decoction and the Allium macrostemon decoction. No fluorescent spots were found in either case, indicating that the identification method in the pharmacopoeia is not applicable to the Allium macrostemon decoction and the traditional Chinese medicine decoction.

[0172] Comparative Example 2

[0173] Chishishuancizhi Decoction Test Solution 2: 100 ml of Chishishuancizhi Decoction (21031601) was evaporated on a water bath, the residue was dissolved in 20 ml of n-hexane and ultrasonically treated for 20 minutes, the filtrate was evaporated, the residue was dissolved in 1 ml of n-hexane and used as the test solution 2.

[0174] Allium Macrostemonis Solution 2: 4 g of Allium Macrostemonis powder (YF210101) was dissolved in 20 ml of n-hexane and ultrasonically treated for 20 minutes, the filtrate was evaporated, the residue was dissolved in 1 ml of n-hexane and used as the Allium Macrostemonis solution 2.

[0175] Allium Macrostemonis Solution 3: 4 g of Allium Macrostemonis powder (YF210101) was dissolved in 100 ml of water and boiled for 30 minutes, the filtrate was evaporated on a water bath, the residue was dissolved in 20 ml of n-hexane and ultrasonically treated for 20 minutes, the filtrate was evaporated, the residue was dissolved in 1 ml of n-hexane and used as the Allium Macrostemonis solution 3.

[0176] Allium Macrostemonis Solution 4: 4 g of Allium Macrostemonis powder (YF210101) was dissolved in 100 ml of water, 1 ml of n-hexane was added to the volatile oil extractor and heated to reflux for 30 minutes, the n-hexane liquid was collected and used as the Allium Macrostemonis solution 4. The residue of Allium Macrostemonis powder and water in the extractor flask were filtered, the residue of Allium Macrostemonis powder was collected and dried for use.

[0177] Allium Macrostemonis Solution 5: The residue of Allium Macrostemonis powder obtained in the preparation of Allium Macrostemonis Solution 4 was dissolved in 20 ml of n-hexane and ultrasonically treated for 20 minutes, the filtrate was evaporated, the residue was dissolved in 1 ml of n-hexane and used as the Allium Macrostemonis solution 5.

[0178] Ten microliters of Allium Macrostemonis Solution 1, Test Solution 1 and Allium Macrostemonis Solution 2 were spotted on the same silica gel G thin layer plate, developed with n-hexane-ethyl acetate (10:1), taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots were clearly colored, and observed under daylight and ultraviolet light (365 nm).

[0179] The results are shown in Figure 3 , Figure 3 The left and right graphs are the results of observing the three samples under daylight (left graph) and ultraviolet light (right graph). Figure 3 In the left and right graphs, from left to right, the samples are Allium Macrostemonis Solution 1, Test Solution 1 and Allium Macrostemonis Solution 2.

[0180] Take 10 μl of each of the five solutions, Baijiangliangqie medicinal material solution ②, test solution ②, Baijiangliangqie medicinal material solution ③, Baijiangliangqie medicinal material solution ④, Baijiangliangqie medicinal material solution ⑤, and spot them on the same silica gel G thin layer plate. Use n-hexane-ethyl acetate (10:1) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots develop clearly, and observe under daylight and ultraviolet light 365 nm.

[0181] The results are shown in Figure 4 , Figure 4 The results of observing the five samples under daylight (left graph) and ultraviolet light (right graph) are shown in the following figures. Figure 4 In the left and right graphs, the samples from left to right are Baijiangliangqie medicinal material solution ②, test solution ②, Baijiangliangqie medicinal material solution ③, Baijiangliangqie medicinal material solution ④, and Baijiangliangqie medicinal material solution ⑤.

[0182] From Figure 3 and Figure 4 , it can be seen that in the treatment method provided by Comparative Example 2, the treatment process of Baijiangliangqie medicinal material solution ② reproduces the method of the Pharmacopoeia, and using the identification method of the Pharmacopoeia can obtain fluorescent spots, indicating that there is no error in the experiment. The Baijiangliangqie components in test solution ② and Baijiangliangqie medicinal material solution ③ are water decoction components, i.e. water-soluble components, and using the identification method of the Pharmacopoeia does not find the expected fluorescent spots; Baijiangliangqie medicinal material solution ④ is a distillated volatile lipid-soluble component, and using the identification method of the Pharmacopoeia also does not find the expected fluorescent spots; Baijiangliangqie medicinal material solution ⑤ is the residue after water decoction of Baijiangliangqie medicinal material, and using the treatment and identification method of the Pharmacopoeia can obtain the expected fluorescent spots.

[0183] From the experiment of the treatment method provided by Comparative Example 2, it can be seen that the identification method of Baijiangliangqie medicinal material in the Pharmacopoeia identifies lipid-soluble components, while the water decoction process of Chinese patent medicines containing Baijiangliangqie medicinal material and single decoction preparations cannot identify the lipid-soluble components, which remain in the residue of Baijiangliangqie medicinal material and are not extracted by the water decoction process. Therefore, the Pharmacopoeia method cannot identify the water-soluble components in Baijiangliangqie.

[0184] Comparative Example 3

[0185] Baijiangliangqie medicinal material solution: Take Baijiangliangqie medicinal material powder (YF210101) 4.0 g, add water 100 ml, boil for 15 minutes, cool, filter, add water to 100 ml to the filtrate, extract with n-butanol twice, 30 ml each time, combine the n-butanol liquid, evaporate to dryness, add methanol 1 ml to dissolve the residue, and use it as Baijiangliangqie medicinal material solution (referred to as "Baijiangliangqie" solution).

[0186] Zhi Shi Xie Bai Gui Zhi Tang Negative Solution 1 for Lack of Allium macrostemon: Take 100ml of Zhi Shi Xie Bai Gui Zhi Tang negative sample (21030805) for lack of Allium macrostemon, extract twice with ether, 30ml each time, discard the ether solution, extract the aqueous solution twice with n-butanol, 30ml each time, combine the n-butanol solutions, evaporate to dryness, dissolve the residue in 1ml of methanol, and use as Zhi Shi Xie Bai Gui Zhi Tang Negative Solution 1 for lack of Allium macrostemon (hereinafter referred to as "Negative 1").

[0187] Negative Solution 2 for Zhishi Xiebai Guizhi Decoction lacking Xiebai: Take 100ml of negative sample (21030805) of Zhishi Xiebai Guizhi Decoction lacking Xiebai, extract it twice with ethyl acetate, 30ml each time, discard the ethyl acetate solution, extract the aqueous solution twice with n-butanol, 30ml each time, combine the n-butanol solutions, evaporate to dryness, add 1ml of methanol to dissolve the residue, and use it as negative solution 2 for Zhishi Xiebai Guizhi Decoction lacking Xiebai (referred to as "Negative 2").

[0188] Test solution 1 of Zhishi Xiebai Guizhi Decoction: Take 100ml of Zhishi Xiebai Guizhi Decoction (21031601), extract it twice with ether (30ml each time), discard the ether solution, extract the aqueous solution twice with n-butanol (30ml each time), combine the n-butanol solutions, evaporate to dryness, dissolve the residue in 1ml of methanol, and use this as test solution 1 of Zhishi Xiebai Guizhi Decoction (referred to as "Decoction 1").

[0189] Test solution 2 of Zhishi Xiebai Guizhi Decoction: Take 100ml of Zhishi Xiebai Guizhi Decoction (21031601), extract it twice with ethyl acetate, 30ml each time, discard the ethyl acetate solution, extract the aqueous solution twice with n-butanol, 30ml each time, combine the n-butanol solutions, evaporate to dryness, dissolve the residue in 1ml of methanol, and use it as test solution 2 of Zhishi Xiebai Guizhi Decoction (hereinafter referred to as "Decoction 2").

[0190] Test solution 3 of Zhishi Xiebai Guizhi Decoction: Take 100ml of Zhishi Xiebai Guizhi Decoction (21031601), extract it twice with n-butanol, 30ml each time, combine the n-butanol solutions, evaporate to dryness, dissolve the residue in 1ml of methanol, and use it as test solution 3 of Zhishi Xiebai Guizhi Decoction (hereinafter referred to as "Decoction 3").

[0191] Take 10 μl of each of the six solutions, namely "Allium macrostemon", "Anion 1", "Anion 2", "Soup 1", "Soup 2" and "Soup 3", and spot them separately on the same silica gel G thin-layer plate. Use ethyl acetate-glacial acetic acid-formic acid-water (15:1:1:2) as the developing solvent, develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly visible, and examine under sunlight and ultraviolet light at 365 nm.

[0192] See results Figure 5 , Figure 5 The chromatogram obtained by the processing method provided for Comparative Example 3. Figure 5 In the middle, from left to right, the samples are "Allium macrostemon" solution, "Antagon 1" solution, "Antagon 2" solution, "Soup 1" solution, "Soup 2" solution, and "Soup 3" solution.

[0193] Depend on Figure 5 It is known that *Allium macrostemon* mainly contains saponins, and n-butanol is most suitable for extracting saponins from the decoction. Therefore, the preparation method for the test sample was to use n-butanol-water liquid-liquid extraction. For solutions "Soup 1" and "Soup 2," ether and ethyl acetate were used respectively for impurity removal to remove some lipid-soluble impurities from the aqueous solution. Solution "Soup 3" was not pre-treated for impurity removal. Figure 5 It can be seen that in the n-butanol extract of the decoction of Allium macrostemon ("Allium macrostemon" solution), there are several well-separated fluorescent spots, one of which is brighter, indicating that the method (sample processing method, silica gel G thin-layer plate, developing solvent, colorimetric reagent, 365 UV light) can be used to identify the water-soluble components of Allium macrostemon. From the chromatograms of "Antagonist 1", "Antagonist 2", "Decoction 1", "Decoction 2", and "Decoction 3", it can be seen that both the negative solution and the sample solution contain a large number of components that interfere with the fluorescent spots in the "Allium macrostemon" solution.

[0194] Comparative Example 4

[0195] Allium macrostemon decoction solution: Take 100 ml of the single-decoction sample solution of Allium macrostemon (21040701), extract it twice with n-butanol, 30 ml each time, combine the n-butanol solutions, evaporate to dryness, dissolve the residue in a little methanol, add 1 g of silica gel G, mix well, evaporate to dryness, add it to a neutral alumina column (100-200 mesh, 4 g, inner diameter 1.5 cm), elute with 50 ml of n-hexane, collect the n-hexane eluent, evaporate to dryness, dissolve the residue in 1 ml of methanol, and use it as the n-hexane eluent solution of Allium macrostemon (referred to as "Allium macrostemon-n-hexane" solution).

[0196] Take 100 ml of the single-decoction sample solution of Allium macrostemon (21040701), extract it twice with n-butanol (30 ml each time), combine the n-butanol extracts, evaporate to dryness, dissolve the residue in a little methanol, add 1 g of silica gel G, mix well, evaporate to dryness, and add it to a neutral alumina column (100-200 mesh, 4 g, inner diameter 1.5 cm). Elute with 50 ml of ethyl acetate, collect the ethyl acetate eluent, evaporate to dryness, dissolve the residue in 1 ml of methanol, and use it as the ethyl acetate eluent solution of Allium macrostemon (referred to as "Allium macrostemon-ethyl" solution).

[0197] Take 100 ml of the single-decoction sample solution of Allium macrostemon (21040701), extract it twice with n-butanol, 30 ml each time, combine the n-butanol extracts, evaporate to dryness, dissolve the residue in a little methanol, add 1 g of silica gel G, mix well, evaporate to dryness, add it to a neutral alumina column (100-200 mesh, 4 g, inner diameter 1.5 cm), elute with 50 ml of methanol, collect the methanol eluent, recover the solvent to dryness, dissolve the residue in 1 ml of methanol, and use it as the methanol eluent solution of Allium macrostemon (referred to as "Allium macrostemon-methyl" solution).

[0198] Take 100 ml of the single-decoction sample solution of Allium macrostemon (21040701), extract it twice with n-butanol (30 ml each time), combine the n-butanol extracts, evaporate to dryness, dissolve the residue in a little methanol, add 1 g of silica gel G, mix well, evaporate to dryness, and add it to a neutral alumina column (100-200 mesh, 4 g, inner diameter 1.5 cm). Elute with 50 ml of 70% methanol, collect the 70% methanol eluent, recover the solvent to dryness, dissolve the residue in 1 ml of methanol, and use this as the 70% methanol eluent solution of Allium macrostemon (referred to as "Allium-70M" solution).

[0199] Negative solution for Zhishi Xiebai Guizhi Decoction lacking Xiebai: Take 100ml of Zhishi Xiebai Guizhi Decoction negative sample (21040604) and prepare it in the same way as above, starting from "extracted twice with n-butanol". The n-hexane eluent is referred to as "negative-n-", the ethyl acetate eluent is referred to as "negative-ethyl", the methanol eluent is referred to as "negative-methyl", and the 70% methanol eluent is referred to as "negative-70methyl".

[0200] Test solution of Zhishi Xiebai Guizhi Decoction: Take 100ml of Zhishi Xiebai Guizhi Decoction (21031601), and prepare it in the same way as above, starting from "extracted twice with n-butanol". The n-hexane eluent is referred to as "Decoction-n-" solution, the ethyl acetate eluent is referred to as "Decoction-ethyl" solution, the methanol eluent is referred to as "Decoction-A" solution, and the 70% methanol eluent is referred to as "Decoction-70A" solution.

[0201] Take 5 μl of each of the following nine solutions: "Xie-Zheng" solution, "Yin-Zheng" solution, "Tang-Zheng" solution, "Xie-Yi" solution, "Yin-Yi" solution, "Tang-Yi" solution, "Xie-Jia" solution, "Yin-Jia" solution, and "Tang-Jia" solution, and spot them separately on the same silica gel G thin-layer plate. Use ethyl acetate-glacial acetic acid-formic acid-water (15:1:1:2) as the developing solvent, develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly visible, and examine under sunlight and ultraviolet light at 365 nm.

[0202] See results Figure 6 , Figure 6 The chromatogram obtained by the processing method provided in Comparative Example 4. Figure 6In the middle, the left graph is the chromatogram under sunlight, and the right graph is the chromatogram obtained under ultraviolet inspection. Figure 6 In the left and right graphs of Figure 4, from left to right, the samples are "Allium-70A" solution, "Yin-70A" solution, "Tang-70A" solution.

[0203] Take 5 μl of each of "Allium-70A" solution, "Yin-70A" solution, and "Tang-70A" solution, and spot them on the same silica gel G thin layer plate. Use ethyl acetate-glacial acetic acid-formic acid-water (15:1:1:2) as the developing agent, develop, take out, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots develop clearly, and place under sunlight and ultraviolet light 365 nm for inspection.

[0204] The results are shown in Figure 5. Figure 7 , Figure 7 The chromatogram obtained by the treatment method provided for Comparative Example 4. Figure 7 In the middle, the left graph is the chromatogram under sunlight, and the right graph is the chromatogram obtained under ultraviolet inspection. Figure 7 In the left and right graphs of Figure 4, from left to right, the samples are "Allium-70A" solution, "Yin-70A" solution, "Tang-70A" solution.

[0205] Take 5 μl of each of "Allium-70A" solution, "Yin-70A" solution, and "Tang-70A" solution, and spot them on the same silica gel G thin layer plate. Use ethyl acetate-glacial acetic acid-formic acid-water (15:1:1:2) as the developing agent, develop, take out, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots develop clearly, and place under sunlight and ultraviolet light 365 nm for inspection.

[0206] The results are shown in Figure 5. Figure 8 , Figure 8 The chromatogram obtained by the treatment method provided for Comparative Example 4. Figure 8 In the middle, the left graph is the chromatogram under sunlight, and the right graph is the chromatogram obtained under ultraviolet inspection. Figure 8 In the left and right graphs of Figure 4, from left to right, the samples are "Allium-70A" solution, "Yin-70A" solution, "Tang-70A" solution.

[0207] From Figure 6 , Figure 7 and Figure 8It can be seen that the n-butanol extract of the sample aqueous solution is subjected to an alumina chromatographic column, and n-hexane, ethyl acetate, methanol, and 70% methanol are used for elution, respectively. With the increase of the polarity of the eluent, the polarity of the eluted components also increases. By elution with different polar eluents, different eluents are collected to obtain components with different polarities. These components are identified by thin layer chromatography. There is no fluorescent spot in the n-hexane eluent; there is a bright fluorescent spot in the ethyl acetate eluent of the Baiyun decoction and the Baiyun and Citrus fruit Guizhi decoction sample, but the negative solution has interference; there is no fluorescent spot in the methanol eluent of the Baiyun medicinal material solution; and there are two fluorescent spots with smaller brightness in the 70% methanol eluent of the Baiyun medicinal material solution, but it is difficult to exclude interference corresponding to the negative solution.

[0208] Comparative Example 5

[0209] The Baiyun decoction piece solution: 100 ml of the Baiyun decoction piece single decoction sample solution (21040701) was extracted with n-butanol twice, 30 ml each time, the n-butanol liquid was combined and evaporated to dryness, a small amount of methanol was added to the residue to dissolve, 0.5 g of neutral alumina was added and mixed uniformly, evaporated to dryness, and then added to a neutral alumina column (100-200 mesh, 3 g, inner diameter 1.5 cm). The column was eluted with 20 ml of n-hexane-ethyl acetate (1:9) solution, the eluent was collected, evaporated to dryness, and 1 ml of methanol was added to the residue to dissolve, which was used as the Baiyun decoction piece n-hexane-ethyl acetate (1:9) elution solution (referred to as "Baiyun-n-ethyl" solution).

[0210] The Baiyun decoction piece solution: 100 ml of the Baiyun decoction piece single decoction sample solution (21040701) was extracted with n-butanol twice, 30 ml each time, the n-butanol liquid was combined and evaporated to dryness, a small amount of methanol was added to the residue to dissolve, 0.5 g of neutral alumina was added and mixed uniformly, evaporated to dryness, and then added to a neutral alumina column (100-200 mesh, 3 g, inner diameter 1.5 cm). The column was eluted with 20 ml of n-hexane-ethyl acetate (1:9) solution, the eluent was collected, evaporated to dryness, and 1 ml of methanol was added to the residue to dissolve, which was used as the Baiyun decoction piece n-hexane-ethyl acetate (1:9) elution solution (referred to as "Baiyun-n-ethyl" solution).

[0211] The Baiyun decoction piece solution: 100 ml of the Baiyun decoction piece single decoction sample solution (21040701) was extracted with n-butanol twice, 30 ml each time, the n-butanol liquid was combined and evaporated to dryness, a small amount of methanol was added to the residue to dissolve, 0.5 g of neutral alumina was added and mixed uniformly, evaporated to dryness, and then added to a neutral alumina column (100-200 mesh, 3 g, inner diameter 1.5 cm). The column was eluted with 20 ml of n-hexane-ethyl acetate (1:9) solution, the eluent was collected, evaporated to dryness, and 1 ml of methanol was added to the residue to dissolve, which was used as the Baiyun decoction piece n-hexane-ethyl acetate (1:9) elution solution (referred to as "Baiyun-n-ethyl" solution).

[0212] Take the single decoction sample solution of Baiyun decoction pieces (21040701) 100 ml, extract with n-butanol twice, 30 ml each time, combine the n-butanol liquid, evaporate to dryness, add a little methanol to the residue to dissolve, add 0.5 g of neutral alumina, mix evenly, evaporate to dryness, add to the neutral alumina column (100-200 mesh, 3 g, inner diameter 1.5 cm), elute with 70% methanol 20 ml, collect the eluate, recover the solvent to dryness, add 1 ml of methanol to the residue to dissolve, as Baiyun decoction pieces 70% methanol elution solution (referred to as "Xie-70 methanol" solution).

[0213] Take the single decoction sample solution of Baiyun decoction pieces (21040701) 100 ml, extract with n-butanol twice, 30 ml each time, combine the n-butanol liquid, evaporate to dryness, add a little methanol to the residue to dissolve, add 0.5 g of neutral alumina, mix evenly, evaporate to dryness, add to the neutral alumina column (100-200 mesh, 3 g, inner diameter 1.5 cm), elute with 50% methanol 20 ml, collect the eluate, recover the solvent to dryness, add 1 ml of methanol to the residue to dissolve, as Baiyun decoction pieces 50% methanol elution solution (referred to as "Xie-50 methanol" solution).

[0214] Take the single decoction sample solution of Baiyun decoction pieces (21040701) 100 ml, extract with n-butanol twice, 30 ml each time, combine the n-butanol liquid, evaporate to dryness, add a little methanol to the residue to dissolve, add 0.5 g of neutral alumina, mix evenly, evaporate to dryness, add to the neutral alumina column (100-200 mesh, 3 g, inner diameter 1.5 cm), elute with 50% methanol 20 ml, collect the eluate, recover the solvent to dryness, add 1 ml of methanol to the residue to dissolve, as Baiyun decoction pieces 50% methanol elution solution (referred to as "Xie-50 methanol" solution).

[0215] Xishiyin negative solution of Xishiyin decoction: Take Xishiyin decoction without Baiyun (21040604) 100 ml, from "extract with n-butanol twice" as above, prepare by the same method, n-hexane-ethyl acetate (1:9) solution eluate is referred to as "negative-n-ethyl" solution, ethyl acetate-methanol (1:1) solution eluate is referred to as "negative-ethyl-methanol" solution, 90% methanol eluate is referred to as "negative-90 methanol" solution, 70% methanol eluate is referred to as "negative-70 methanol" solution, 50% methanol eluate is referred to as "negative-50 methanol" solution, and water eluate is referred to as "negative-water" solution.

[0216] Jujubae and Radix Bupleuri Cassiae Decoction Test Solution: 100 ml of Jujubae and Radix Bupleuri Cassiae Decoction (21031601) was taken, and prepared in the same manner from "using n-butanol extraction twice" above. The n-hexane-ethyl acetate (1:9) solution eluent was referred to as "decoction-n-ethyl" solution, the ethyl acetate-methanol (1:1) solution eluent was referred to as "decoction-ethyl-methanol" solution, the 90% methanol eluent was referred to as "decoction-90 methanol" solution, the 70% methanol eluent was referred to as "decoction-70 methanol" solution, the 50% methanol eluent was referred to as "decoction-50 methanol" solution, and the water eluent was referred to as "decoction-water" solution.

[0217] Five μl of each of the "Radix Bupleuri-n-ethyl" solution, "negative-n-ethyl" solution, "decoction-n-ethyl" solution, "Radix Bupleuri-ethyl-methanol" solution, "negative-ethyl-methanol" solution, "decoction-ethyl-methanol" solution, "Radix Bupleuri-90 methanol" solution, "negative-90 methanol" solution, "decoction-90 methanol" solution, "Radix Bupleuri-70 methanol" solution, "negative-70 methanol" solution, "decoction-70 methanol" solution, "Radix Bupleuri-50 methanol" solution, "negative-50 methanol" solution, "decoction-50 methanol" solution, and "Radix Bupleuri-water" solution was spotted on the same silica gel G thin layer plate, and developed with ethyl acetate-glacial acetic acid-formic acid-water (15:1:1:2) as the developing agent, taken out, air-dried, sprayed with 10% sulfuric acid ethanol solution, and heated at 105°C until the spots were clearly colored, and observed under daylight and ultraviolet light 365 nm.

[0218] The results are shown in Figure 9 , Figure 9 The chromatogram obtained by the treatment method provided for Comparative Example 5. Figure 9 In the left graph, the chromatogram under daylight, and the right graph, the chromatogram obtained under ultraviolet observation. Figure 9 In the left and right graphs, from left to right, the samples are "Radix Bupleuri-n-ethyl" solution, "negative-n-ethyl" solution, "decoction-n-ethyl" solution, "Radix Bupleuri-ethyl-methanol" solution, "negative-ethyl-methanol" solution, "decoction-ethyl-methanol" solution, "Radix Bupleuri-90 methanol" solution, "negative-90 methanol" solution, "decoction-90 methanol" solution, "Radix Bupleuri-70 methanol" solution, "negative-70 methanol" solution, "decoction-70 methanol" solution, "Radix Bupleuri-50 methanol" solution, "negative-50 methanol" solution, "decoction-50 methanol" solution, and "Radix Bupleuri-water" solution.

[0219] As can be seen from Figure 9 , continuing to use the alumina chromatography column, first using a mixed eluent for elution, and then using different concentrations of methanol for elution, the eluent is also using a solvent with small polarity first and large polarity later, from the chromatogram, it can be seen that the Radix Bupleuri medicinal material, negative solution, and sample solution eluted by 70% methanol have several fluorescent spots, the brightest fluorescent spot of the Radix Bupleuri medicinal material and the sample solution appear at the same position, and have the same color, and the negative solution has no interfering spot at this position, but the background color of each sample chromatogram is obvious.

[0220] The chromatogram obtained in the above TLC identification method was compared under sunlight and UV 365 nm conditions. The spots were clearly distinguishable under UV conditions, and no obvious spots were observed under sunlight.

[0221] Comparative Example 6

[0222] The Baishu decoction piece solution: 100 ml of the Baishu decoction piece single decoction sample solution (21040701) was extracted twice with n-butanol, 30 ml each time, the n-butanol was combined and evaporated to dryness, the residue was dissolved in 10 ml of water, the solution was passed through a polyamide column (60-90 mesh, 2.5 g, inner diameter 1.5 cm), eluted with 50 ml of water, the eluate was collected, evaporated to dryness, the residue was dissolved in 1 ml of methanol, and used as the Baishu decoction piece water elution solution (referred to as "Baishu-water-J" solution).

[0223] The Baishu decoction piece solution: 100 ml of the Baishu decoction piece single decoction sample solution (21040701) was extracted twice with n-butanol, 30 ml each time, the n-butanol was combined and evaporated to dryness, the residue was dissolved in 10 ml of water, the solution was passed through a polyamide column (60-90 mesh, 2.5 g, inner diameter 1.5 cm), eluted with 50 ml of water, the eluate was collected, evaporated to dryness, the residue was dissolved in 1 ml of methanol, and used as the Baishu decoction piece water elution solution (referred to as "Baishu-water-J" solution).

[0224] The Baishu decoction piece solution: 100 ml of the Baishu decoction piece single decoction sample solution (21040701) was extracted twice with n-butanol, 30 ml each time, the n-butanol was combined and evaporated to dryness, the residue was dissolved in 10 ml of water, the solution was passed through a polyamide column (60-90 mesh, 2.5 g, inner diameter 1.5 cm), eluted with 50 ml of water, the eluate was collected, evaporated to dryness, the residue was dissolved in 1 ml of methanol, and used as the Baishu decoction piece water elution solution (referred to as "Baishu-water-J" solution).

[0225] The Baishu decoction piece solution: 100 ml of the Baishu decoction piece single decoction sample solution (21040701) was extracted twice with n-butanol, 30 ml each time, the n-butanol was combined and evaporated to dryness, the residue was dissolved in 10 ml of water, the solution was passed through a polyamide column (60-90 mesh, 2.5 g, inner diameter 1.5 cm), eluted with 50 ml of water, the eluate was collected, evaporated to dryness, the residue was dissolved in 1 ml of methanol, and used as the Baishu decoction piece water elution solution (referred to as "Baishu-water-J" solution).

[0226] Take the sample solution of Bulbus Allii Macrostemonis decoction pieces (21040701) 100 ml, extract with n-butanol for 2 times, 30 ml each time, combine the n-butanol solution, evaporate to dryness, add water 10 ml to dissolve, pass the solution through a polyamide column (60-90 mesh, 2.5 g, inner diameter 1.5 cm), elute with methanol solution 50 ml, collect the eluate, recover the solvent to dryness, add methanol 1 ml to dissolve, as the methanol elution solution of Bulbus Allii Macrostemonis decoction pieces (referred to as "Xie-methanol-J" solution).

[0227] Zishixiebaiguishe Decoction without Bulbus Allii Macrostemonis negative solution: take Zishixiebaiguishe Decoction without Bulbus Allii Macrostemonis negative sample (21040604) 100 ml, from "extract with n-butanol for 2 times" as above, prepare by the same method, the water eluate is referred to as "negative-water-J" solution, the 10% methanol eluate is referred to as "negative-10-methanol-J" solution, the 30% methanol eluate is referred to as "negative-30-methanol-J" solution, the 50% methanol eluate is referred to as "negative-50-methanol-J" solution, and the methanol eluate is referred to as "negative-methanol-J" solution.

[0228] Zishixiebaiguishe Decoction sample solution: take Zishixiebaiguishe Decoction (21031601) 100 ml, from "extract with n-butanol for 2 times" as above, prepare by the same method, the water eluate is referred to as "decoction-water-J" solution, the 10% methanol eluate is referred to as "decoction-10-methanol-J" solution, the 30% methanol eluate is referred to as "decoction-30-methanol-J" solution, the 50% methanol eluate is referred to as "decoction-50-methanol-J" solution, and the methanol eluate is referred to as "decoction-methanol-J" solution.

[0229] Take 5 μl of each of "Xie-water-J" solution, "negative-water-J" solution, "decoction-water-J" solution, "Xie-10-methanol-J" solution, "negative-10-methanol-J" solution, "decoction-10-methanol-J" solution, "Xie-30-methanol-J" solution, "negative-30-methanol-J" solution, "decoction-30-methanol-J" solution, "Xie-50-methanol-J" solution, "negative-50-methanol-J" solution, "decoction-50-methanol-J" solution, "Xie-methanol-J" solution, "negative-methanol-J" solution, and "decoction-methanol-J" solution, and point them on the same silica gel G thin layer plate, use ethyl acetate-glacial acetic acid-formic acid-water (15:1:1:2) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots develop clearly, and observe under daylight and ultraviolet light 365 nm.

[0230] The results are shown in Figure 10 , Figure 10 The chromatogram obtained by the treatment method provided for Comparative Example 6. Figure 10 In the figure, the left graph is the chromatogram under daylight, and the right graph is the chromatogram obtained under ultraviolet observation. Figure 10In the left and right pictures of Figure 1, from left to right, the samples are "Bulbus Allii Macrostemonis-Water-J" solution, "Yin-Water-J" solution, "Tang-Water-J" solution, "Bulbus Allii Macrostemonis-10M-J" solution, "Yin-10M-J" solution, "Tang-10M-J" solution, "Bulbus Allii Macrostemonis-30M-J" solution, "Yin-30M-J" solution, "Tang-30M-J" solution, "Bulbus Allii Macrostemonis-50M-J" solution, "Yin-50M-J" solution, "Tang-50M-J" solution, "Bulbus Allii Macrostemonis-M-J" solution, "Yin-M-J" solution, "Tang-M-J" solution.

[0231] It can be seen that, by using polyamide as the filler of the chromatographic column, using solvents with polarity from large to small for elution, and collecting eluents with different polarities, the components in the eluent with large polarity are relatively large in polarity, and the components in the eluent with small polarity are relatively small in polarity. In the chromatogram, the solution of Bulbus Allii Macrostemonis medicinal material has no fluorescent spots, and the filler is not suitable. Figure 10

[0232] Comparative Example 7

[0233] Bulbus Allii Macrostemonis decoction piece solution: 100 ml of Bulbus Allii Macrostemonis decoction piece single decoction sample solution (21040701) was extracted with n-butanol twice, 30 ml each time, the n-butanol solutions were combined and evaporated to dryness, the residue was dissolved in 30 ml of water, 30 ml of D101 macroporous adsorption resin was added, shaken and left overnight, filtered, the filter residue was washed with 100 ml of water, the washing liquid was discarded, then 100 ml of 10% methanol was used for washing, the washing liquid was discarded, and then the residue was transferred to a stoppered conical flask, 50 ml of methanol was added, ultrasonic treatment was performed for 30 minutes, filtered, and the solvent of the filtrate was recovered to dryness, the residue was dissolved in 1 ml of methanol, and served as the D101 macroporous adsorption resin separation solution of Bulbus Allii Macrostemonis decoction piece (referred to as "Bulbus Allii Macrostemonis-D" solution).

[0234] Yin solution lacking Bulbus Allii Macrostemonis in Cuishei Bulbus Allii Macrostemonis Cassia twig decoction: 100 ml of Yin solution lacking Bulbus Allii Macrostemonis in Cuishei Bulbus Allii Macrostemonis Cassia twig decoction (21040604) was prepared by the same method from "extracted with n-butanol twice" as above, and was referred to as "Yin-D" solution.

[0235] Tang decoction sample solution: 100 ml of Tang decoction sample (21031601) was prepared by the same method from "extracted with n-butanol twice" as above, and the water eluent was referred to as "Tang-D" solution.

[0236] 5 μl of each of "Bulbus Allii Macrostemonis-D" solution, "Yin-D" solution and "Tang-D" solution was taken and spotted on the same silica gel G thin layer plate, ethyl acetate-glacial acetic acid-formic acid-water (15:1:1:2) was used as the developing agent, developed, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots were colored clearly, and observed under daylight and ultraviolet light 365 nm.

[0237] The results are shown in Table 1.​ Figure 11 , Figure 11 The chromatogram obtained by the treatment method provided in Comparative Example 7. Figure 11 In the image, the left image shows the chromatogram under sunlight, and the right image shows the chromatogram obtained under ultraviolet light. Figure 11 In the left and right images, the samples from left to right are "Allium-D" solution, "Anion-D" solution, and "Sodium-D" solution.

[0238] Depend on Figure 11 It can be seen that by using D101 macroporous adsorption resin as the packing material for the chromatography column and eluting with solvents of decreasing polarity, and collecting eluents of different polarities, the components in the highly polar eluent are more polar, while those in the less polar eluent are less polar. In the chromatogram, the Allium macrostemon solution shows a bright fluorescent spot, but the sample solution contains too many interfering components, which interfere with the development of these secondary components.

[0239] Comparative Example 8

[0240] Allium macrostemon slices solution: Take 100 ml of the single-decoction sample solution of Allium macrostemon slices (21040701), extract it twice with n-butanol, 30 ml each time, combine the n-butanol solutions, evaporate to dryness, add 5 ml of methanol to dissolve the residue, pass it through a neutral alumina column (100-200 mesh, 3 g, inner diameter 1.5 cm), elute with 45 ml of methanol, discard the eluent, then elute with 25 ml of 80% methanol, collect the eluent, recover the solvent to dryness, add 1 ml of methanol to dissolve the residue, as Allium macrostemon slices positive solution 1 (abbreviated as "Allium-oxygen-1" solution).

[0241] Take 100 ml of the single-decoction sample solution of Allium macrostemon (21040701), extract it twice with n-butanol, 30 ml each time, combine the n-butanol extracts, evaporate to dryness, dissolve the residue in 5 ml of methanol, pass it through a neutral alumina column (100-200 mesh, 3 g, inner diameter 1.5 cm), elute with 25 ml of 70% methanol, collect the eluent, recover the solvent to dryness, dissolve the residue in 1 ml of methanol, and use it as positive solution 2 of Allium macrostemon (referred to as "Allium-oxygen-2" solution).

[0242] Negative solution for Zhishi Xiebai Guizhi Decoction lacking Xiebai: Take 100ml of Zhishi Xiebai Guizhi Decoction negative sample (21040604) and prepare it in the same way as above, starting from "extracted twice with n-butanol". The 80% methanol solution eluent is referred to as "anion-oxygen-1" solution, and the 70% methanol solution eluent is referred to as "anion-oxygen-2" solution.

[0243] Test solution of Zhishi Xiebai Guizhi Decoction: Take 100ml of Zhishi Xiebai Guizhi Decoction (21031601), and prepare it in the same way as above, starting from "extracted twice with n-butanol". The 80% methanol solution eluent is referred to as "Decoction-Oxygen-1" solution, and the 70% methanol solution eluent is referred to as "Decoction-Oxygen-2" solution.

[0244] Bulbus Allii Macrostemi slice solution: take 100 ml of Bulbus Allii Macrostemi slice single decoction sample solution (21040701), extract twice with n-butanol, 30 ml each time, combine the n-butanol solution, evaporate to dryness, add 5 ml of methanol to dissolve the residue, pass the solution through a polyamide column (60-90 mesh, 2.5 g, inner diameter 1.5 cm), elute with 50 ml of water, discard the eluent, then elute with 25 ml of methanol, discard the eluent, evaporate to dryness, elute with 25 ml of ethyl acetate, discard the eluent, evaporate to dryness, add 1 ml of methanol to dissolve the residue, and use the solution as Bulbus Allii Macrostemi slice positive solution 1 (referred to as "Bulbus- poly-1" solution).

[0245] Take 100 ml of Bulbus Allii Macrostemi slice single decoction sample solution (21040701), extract twice with n-butanol, 30 ml each time, combine the n-butanol solution, evaporate to dryness, add 5 ml of methanol to dissolve the residue, pass the solution through a polyamide column (60-90 mesh, 2.5 g, inner diameter 1.5 cm), elute with 25 ml of 10% acetic acid solution, collect the eluent, evaporate to dryness, add 1 ml of methanol to dissolve the residue, and use the solution as Bulbus Allii Macrostemi slice positive solution 2 (referred to as "Bulbus-poly-2" solution).

[0246] Bulbus Allii Macrostemi slice solution: take 100 ml of Bulbus Allii Macrostemi slice single decoction sample solution (21040701), extract twice with n-butanol, 30 ml each time, combine the n-butanol solution, evaporate to dryness, add 5 ml of methanol to dissolve the residue, pass the solution through a polyamide column (60-90 mesh, 2.5 g, inner diameter 1.5 cm), elute with 50 ml of water, discard the eluent, then elute with 25 ml of methanol, discard the eluent, evaporate to dryness, elute with 25 ml of ethyl acetate, discard the eluent, evaporate to dryness, add 1 ml of methanol to dissolve the residue, and use the solution as Bulbus Allii Macrostemi slice positive solution 1 (referred to as "Bulbus- poly-1" solution).

[0247] Bulbus Allii Macrostemi slice solution: take 100 ml of Bulbus Allii Macrostemi slice single decoction sample solution (21040701), extract twice with n-butanol, 30 ml each time, combine the n-butanol solution, evaporate to dryness, add 5 ml of methanol to dissolve the residue, pass the solution through a polyamide column (60-90 mesh, 2.5 g, inner diameter 1.5 cm), elute with 50 ml of water, discard the eluent, then elute with 25 ml of methanol, discard the eluent, evaporate to dryness, elute with 25 ml of ethyl acetate, discard the eluent, evaporate to dryness, add 1 ml of methanol to dissolve the residue, and use the solution as Bulbus Allii Macrostemi slice positive solution 1 (referred to as "Bulbus- poly-1" solution).

[0248] Take 5 μl of each of the "Bulbus-oxygen-1" solution, "negative-oxygen-1" solution, "soup-oxygen-1" solution, "Bulbus-oxygen-2" solution, "negative-oxygen-2" solution, "soup-oxygen-2" solution, "Bulbus-poly-1" solution, "negative-poly-1" solution, "soup-poly-1" solution, "Bulbus-poly-2" solution, "negative-poly-2" solution, and "soup-poly-2" solution, and spot them on the same silica gel G thin layer plate, use ethyl acetate-glacial acetic acid-formic acid-water (15:1:1:2) as the developing agent, develop, take out, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots develop clearly, and observe under daylight and ultraviolet light 365 nm.

[0249] The results are shown in Table 1. Figure 12 , Figure 12 The chromatogram obtained by the treatment method provided for Comparative Example 8. Figure 12 In the image, the left image shows the chromatogram under sunlight, and the right image shows the chromatogram obtained under ultraviolet light. Figure 12 In the left and right images, the samples from left to right are: "Allium-oxygen-1" solution, "Anion-oxygen-1" solution, "Tang-oxygen-1" solution, "Allium-oxygen-2" solution, "Anion-oxygen-2" solution, "Tang-oxygen-2" solution, "Allium-poly-1" solution, "Anion-poly-1" solution, "Tang-poly-1" solution, "Allium-poly-2" solution, "Anion-poly-2" solution, and "Tang-poly-2" solution.

[0250] Depend on Figure 12 It was found that fluorescent spots of Allium macrostemon were present in both the Allium macrostemon and the Citrus aurantium, Allium macrostemon and Cinnamomum cassia decoction solutions eluted by the neutral alumina column, while no fluorescent spots of Allium macrostemon were present in the solutions eluted by the polyamide column. This indicates that the tested polyamide column elution method is not applicable. For the neutral alumina column elution method, 80% methanol is sufficient to elute the Allium macrostemon components from the neutral alumina column.

[0251] Comparative Example 9

[0252] Allium macrostemon decoction solution: Take 100 ml of the single-decoction sample solution of Allium macrostemon (21040701), extract it twice with n-butanol, 30 ml each time, combine the n-butanol solutions, evaporate to dryness, dissolve the residue in 5 ml of methanol, pass the solution through a polyamide column (60-90 mesh, 2.5 g, inner diameter 1.5 cm), elute with 45 ml of methanol, collect the eluent, recover the solvent to dryness, dissolve the residue in 1 ml of methanol, and use this as positive solution 3 of Allium macrostemon decoction (abbreviated as "Allium-poly-3" solution).

[0253] Take 100 ml of the single-decoction sample solution of Allium macrostemon (21040701), extract it twice with n-butanol, 30 ml each time, combine the n-butanol solutions, evaporate to dryness, dissolve the residue in 5 ml of methanol, pass the solution through a polyamide column (60-90 mesh, 2.5 g, inner diameter 1.5 cm), elute with 25 ml of water, collect the eluent, evaporate to dryness, dissolve the residue in 1 ml of methanol, and use this as positive solution 4 of Allium macrostemon (referred to as "Allium-poly-4" solution).

[0254] Take 100 ml of the single-decoction sample solution of Allium macrostemon (21040701), extract it twice with n-butanol (30 ml each time), combine the n-butanol extracts, evaporate to dryness, dissolve the residue in 5 ml of methanol, pass the solution through a polyamide column (60-90 mesh, 2.5 g, inner diameter 1.5 cm), elute with 25 ml of 0.1% sodium hydroxide solution, collect the eluent, evaporate to dryness, dissolve the residue in 1 ml of methanol, and use this as the positive solution of Allium macrostemon (referred to as "Allium-Poly-5" solution).

[0255] Negative solution for Zhishi Xiebai Guizhi Decoction lacking Xiebai: Take 100ml of Zhishi Xiebai Guizhi Decoction negative sample (21040604) and prepare it in the same way as above, starting from "extracted twice with n-butanol". The methanol eluent is referred to as "anion-poly-3" solution, the aqueous solution eluent is referred to as "anion-poly-4" solution, and the 0.1% sodium hydroxide eluent is referred to as "anion-poly-5" solution.

[0256] Test solution of Zhishi Xiebai Guizhi Decoction: Take 100ml of Zhishi Xiebai Guizhi Decoction (21031601), and prepare it in the same way as above, starting from "extracted twice with n-butanol". The methanol eluent is referred to as "Decoction-Poly-3" solution, the aqueous solution eluent is referred to as "Decoction-Poly-4" solution, and the 0.1% sodium hydroxide eluent is referred to as "Decoction-Poly-5" solution.

[0257] Take 5 μl of each of the following twelve solutions: "Allium-Oxygen-1", "Anion-Oxygen-1", "Ton-Oxygen-1", "Allium-Poly-3", "Anion-Poly-3", "Ton-Poly-3", "Allium-Poly-4", "Anion-Poly-4", "Ton-Poly-4", "Allium-Poly-5", "Anion-Poly-5", and "Ton-Poly-5", and spot them separately on the same silica gel G thin-layer plate. Use ethyl acetate-glacial acetic acid-formic acid-water (15:1:1:2) as the developing solvent, develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly visible, and examine under sunlight and ultraviolet light at 365 nm.

[0258] See results Figure 13 , Figure 13 The chromatogram obtained by the processing method provided for Comparative Example 9. Figure 13 In the image, the left image shows the chromatogram under sunlight, and the right image shows the chromatogram obtained under ultraviolet light. Figure 13 In the left and right images, the samples from left to right are: "Allium-Oxygen-1" solution, "Anion-Oxygen-1" solution, "Tang-Oxygen-1" solution, "Allium-Poly-3" solution, "Anion-Poly-3" solution, "Tang-Poly-3" solution, "Allium-Poly-4" solution, "Anion-Poly-4" solution, "Tang-Poly-4", "Allium-Poly-5" solution, "Anion-Poly-5" solution, and "Tang-Poly-5" solution.

[0259] Depend on Figure 13 It can be seen from the fluorescent spots in the "Allium macrostemon-poly-3" solution that methanol can elute the Allium macrostemon component in the polyamide column, but there are too many interfering components in the negative sample and the decoction.

[0260] Comparative Example 10

[0261] BnBuOH extract of B. tubers: 100 ml of the single decoction sample solution of B. tubers (21040701) was extracted with BnBuOH twice, 30 ml each time. The BnBuOH extract was combined and evaporated to dryness. The residue was dissolved in 5 ml of MeOH and passed through a neutral alumina column (100-200 mesh, 3 g, 1.5 cm in diameter). The column was eluted with 20 ml of MeOH, and the eluate was discarded. The column was then eluted with 25 ml of water, and the eluate was collected. The collected eluate was passed through a D101 macroporous adsorption resin column (1.5 cm in diameter, 10 cm in length). The column was eluted with 25 ml of 10% MeOH, and the eluate was collected. The solvent was recovered to dryness. The residue was dissolved in 1 ml of MeOH, and the solution was named "Bt-Big-1" solution.

[0262] BnBuOH extract of B. tubers: 100 ml of the single decoction sample solution of B. tubers (21040701) was extracted with BnBuOH twice, 30 ml each time. The BnBuOH extract was combined and evaporated to dryness. The residue was dissolved in 5 ml of MeOH and passed through a neutral alumina column (100-200 mesh, 3 g, 1.5 cm in diameter). The column was eluted with 20 ml of MeOH, and the eluate was discarded. The column was then eluted with 25 ml of water, and the eluate was collected. The collected eluate was passed through a D101 macroporous adsorption resin column (1.5 cm in diameter, 10 cm in length). The column was eluted with 25 ml of 50% MeOH, and the eluate was collected. The solvent was recovered to dryness. The residue was dissolved in 1 ml of MeOH, and the solution was named "Bt-Big-2" solution.

[0263] BnBuOH extract of B. tubers: 100 ml of the single decoction sample solution of B. tubers (21040701) was extracted with BnBuOH twice, 30 ml each time. The BnBuOH extract was combined and evaporated to dryness. The residue was dissolved in 5 ml of MeOH and passed through a neutral alumina column (100-200 mesh, 3 g, 1.5 cm in diameter). The column was eluted with 20 ml of MeOH, and the eluate was discarded. The column was then eluted with 25 ml of water, and the eluate was collected. The collected eluate was passed through a D101 macroporous adsorption resin column (1.5 cm in diameter, 10 cm in length). The column was eluted with 25 ml of 70% MeOH, and the eluate was collected. The solvent was recovered to dryness. The residue was dissolved in 1 ml of MeOH, and the solution was named "Bt-Big-3" solution.

[0264] BnBuOH extract of B. tubers: 100 ml of the single decoction sample solution of B. tubers (21040701) was extracted with BnBuOH twice, 30 ml each time. The BnBuOH extract was combined and evaporated to dryness. The residue was dissolved in 5 ml of MeOH and passed through a neutral alumina column (100-200 mesh, 3 g, 1.5 cm in diameter). The column was eluted with 20 ml of MeOH, and the eluate was discarded. The column was then eluted with 25 ml of water, and the eluate was collected. The collected eluate was passed through a D101 macroporous adsorption resin column (1.5 cm in diameter, 10 cm in length). The column was eluted with 25 ml of MeOH, and the eluate was collected. The solvent was recovered to dryness. The residue was dissolved in 1 ml of MeOH, and the solution was named "Bt-Big-4" solution.

[0265] Take the single decoction sample solution of Baiyun recipe (21040701) 100 ml, extract with n-butanol for 2 times, 30 ml each time, combine the n-butanol liquid, evaporate to dryness, add methanol 5 ml to dissolve the residue, pass through a neutral alumina column (100-200 mesh, 3 g, inner diameter 1.5 cm), elute with 10% acetic acid solution 25 ml, collect the eluate, evaporate to dryness, add methanol 1 ml to dissolve the residue, and the solution is named "Xie-oxygen-3" solution.

[0266] Take the single decoction sample solution of Baiyun recipe (21040701) 100 ml, extract with n-butanol for 2 times, 30 ml each time, combine the n-butanol liquid, evaporate to dryness, add methanol 5 ml to dissolve the residue, pass through a neutral alumina column (100-200 mesh, 3 g, inner diameter 1.5 cm), elute with 10% acetic acid solution 25 ml, discard the water solution. Continue to elute the neutral alumina column with 0.1% sodium hydroxide solution 25 ml, collect the eluate, evaporate to dryness, add methanol 1 ml to dissolve the residue, and the solution is named "Xie-oxygen-4" solution.

[0267] Baiyun recipe negative solution for lack of Baiyun: Take Baiyun recipe negative sample (21040604) 100 ml, from "extract with n-butanol for 2 times" as above, prepare by the same method, D101 type macroporous adsorption resin column 10% methanol elution solution is named "negative-D-1" solution. D101 type macroporous adsorption resin column 50% methanol elution solution is named "negative-D-2" solution, D101 type macroporous adsorption resin column 70% methanol elution solution is named "negative-D-3" solution, D101 type macroporous adsorption resin column methanol elution solution is named "negative-D-4" solution. Neutral alumina column 10% acetic acid elution solution is named "negative-oxygen-3" solution, neutral alumina column 0.1% sodium hydroxide elution solution is named "negative-oxygen-4" solution.

[0268] Baiyun recipe decoction test solution: Take Baiyun recipe decoction (21031601) 100 ml, from "extract with n-butanol for 2 times" as above, prepare by the same method, D101 type macroporous adsorption resin column 10% methanol elution solution is named "soup-D-1" solution. D101 type macroporous adsorption resin column 50% methanol elution solution is named "soup-D-2" solution, D101 type macroporous adsorption resin column 70% methanol elution solution is named "soup-D-3" solution, D101 type macroporous adsorption resin column methanol elution solution is named "soup-D-4" solution. Neutral alumina column 10% acetic acid elution solution is named "soup-oxygen-3" solution, neutral alumina column 0.1% sodium hydroxide elution solution is named "soup-oxygen-4" solution.

[0269] Absorb 5 μl of each of the eighteen solutions of "Xie - Da - 1" solution, "Yin - Da - 1" solution, "Tang - Da - 1" solution, "Xie - Da - 2" solution, "Yin - Da - 2" solution, "Tang - Da - 2" solution, "Xie - Da - 3" solution, "Yin - Da - 3" solution, "Tang - Da - 3" solution, "Xie - Da - 4" solution, "Yin - Da - 4" solution, "Tang - Da - 4" solution, "Xie - Yang - 3" solution, "Yin - Yang - 3" solution, "Tang - Yang - 3" solution, "Xie - Yang - 4" solution, "Yin - Yang - 4" solution, and "Tang - Yang - 4" solution, and spot them on the same silica gel G thin - layer plate respectively. Use ethyl acetate - glacial acetic acid - formic acid - water (15:1:1:2) as the developing agent, develop, take out, air - dry, spray with 10% sulfuric acid ethanol solution, heat at 105 °C until the spots are clearly colored, and examine under an ultraviolet lamp at 365 nm.

[0270] See Figure 14 , Figure 14 is the chromatogram obtained by the treatment method provided for Comparative Example 10. Figure 14 In

[0271] From Figure 14 it can be seen that the elution effect of a single packing chromatography column is not good. Using a method with two packings, first neutral alumina and then D101 macroporous adsorption resin, in the 70% methanol eluent, there is a spot of the same color at the corresponding position for the sample solution and the Allium macrostemon Bunge medicinal material solution, and the negative solution has no interference at the same position. It shows that using the chromatography column with two packings can obtain a better - separated target fluorescence spot.

[0272] Comparative Example 11

[0273] Bulbus Allii Macrostemi Decoction Solution: 100 ml of the Bulbus Allii Macrostemi Decoction Solution (21042803) was extracted with n-butanol twice, 30 ml each time, and the n-butanol solution was combined and evaporated to dryness. The residue was dissolved in 5 ml of methanol and passed through a neutral alumina column (100-200 mesh, 3 g, 1.5 cm in diameter) eluted with 20 ml of methanol, the eluate was discarded, and then eluted with 25 ml of water, the eluate was collected. The eluate was passed through a D101 macroporous adsorption resin column (1.5 cm in diameter, 10 cm in length) eluted with 25 ml of water, the eluate was discarded, and then eluted with 25 ml of 10% methanol, the eluate was discarded, and then eluted with 25 ml of 50% methanol, the eluate was discarded, and then eluted with 25 ml of methanol, the eluate was collected, the solvent was recovered to dryness, the residue was dissolved in 1 ml of methanol, and the solution was named "Bulbus- D101-5" solution.

[0274] Citrus Aurantium, Bulbus Allii Macrostemi and Ramulus Cinnamomi Decoction Solution: 100 ml of the Citrus Aurantium, Bulbus Allii Macrostemi and Ramulus Cinnamomi Decoction Solution (21042801) was extracted with n-butanol twice, 30 ml each time, and the n-butanol solution was combined and evaporated to dryness. The residue was dissolved in 5 ml of methanol and passed through a neutral alumina column (100-200 mesh, 3 g, 1.5 cm in diameter) eluted with 20 ml of methanol, the eluate was discarded, and then eluted with 25 ml of water, the eluate was collected. The eluate was passed through a D101 macroporous adsorption resin column (1.5 cm in diameter, 10 cm in length) eluted with 25 ml of water, the eluate was discarded, and then eluted with 25 ml of 10% methanol, the eluate was discarded, and then eluted with 25 ml of 50% methanol, the eluate was discarded, and then eluted with 25 ml of methanol, the eluate was collected, the solvent was recovered to dryness, the residue was dissolved in 1 ml of methanol, and the solution was named "Decoction-D101-5" solution.

[0275] Citrus Aurantium, Bulbus Allii Macrostemi and Ramulus Cinnamomi Decoction Solution: 100 ml of the Citrus Aurantium, Bulbus Allii Macrostemi and Ramulus Cinnamomi Decoction Solution (21042801) was extracted with n-butanol twice, 30 ml each time, and the n-butanol solution was combined and evaporated to dryness. The residue was dissolved in 5 ml of methanol and passed through a neutral alumina column (100-200 mesh, 3 g, 1.5 cm in diameter) eluted with 20 ml of methanol, the eluate was discarded, and then eluted with 25 ml of water, the eluate was collected. The eluate was passed through a D101 macroporous adsorption resin column (1.5 cm in diameter, 10 cm in length) eluted with 25 ml of water, the eluate was discarded, and then eluted with 25 ml of 10% methanol, the eluate was discarded, and then eluted with 25 ml of 50% methanol, the eluate was discarded, and then eluted with 25 ml of methanol, the eluate was collected, the solvent was recovered to dryness, the residue was dissolved in 1 ml of methanol, and the solution was named "Decoction-D101-5" solution.

[0276] Bulbus Allii Macrostemi Decoction Solution: 100 ml of the Bulbus Allii Macrostemi Decoction Solution (21042803) was extracted with n-butanol twice, 30 ml each time, and the n-butanol solution was combined and evaporated to dryness. The residue was dissolved in 5 ml of methanol and passed through a neutral alumina column (100-200 mesh, 3 g, 1.5 cm in diameter) eluted with 20 ml of methanol, the eluate was discarded, and then eluted with 25 ml of water, the eluate was collected. The eluate was passed through a D101 macroporous adsorption resin column (1.5 cm in diameter, 10 cm in length) eluted with 25 ml of water, the eluate was discarded, and then eluted with 25 ml of 10% methanol, the eluate was discarded, and then eluted with 25 ml of 50% methanol, the eluate was discarded, and then eluted with 25 ml of methanol, the eluate was collected, the solvent was recovered to dryness, the residue was dissolved in 1 ml of methanol, and the solution was named "Bulbus- D101-5" solution.

[0277] Take the single decoction sample solution of Bai Xie (21042803) 100 ml, extract with n-butanol twice, 30 ml each time, combine the n-butanol liquid, evaporate to dryness, add methanol 5 ml to dissolve the residue, pass the solution through a neutral alumina column (100-200 mesh, 3 g, inner diameter 1.5 cm), elute with methanol 20 ml, discard the eluent, elute with water 25 ml, collect the eluent, pass through a polyamide column (60-90 mesh, 2.5 g, inner diameter 1.5 cm), elute with water 25 ml, discard the eluent, elute with 50% methanol 25 ml, collect the eluent, recover the solvent to dryness, add methanol 1 ml to dissolve the residue, name the solution "Xie-poly-7" solution.

[0278] Take the single decoction sample solution of Bai Xie (21042803) 100 ml, extract with n-butanol twice, 30 ml each time, combine the n-butanol liquid, evaporate to dryness, add methanol 5 ml to dissolve the residue, pass the solution through a neutral alumina column (100-200 mesh, 3 g, inner diameter 1.5 cm), elute with methanol 20 ml, discard the eluent, elute with water 25 ml, collect the eluent, pass through a polyamide column (60-90 mesh, 2.5 g, inner diameter 1.5 cm), elute with water 25 ml, discard the eluent, elute with 70% methanol 25 ml, collect the eluent, recover the solvent to dryness, add methanol 1 ml to dissolve the residue, name the solution "Xie-poly-8" solution.

[0279] Take the single decoction sample solution of Bai Xie (21042803) 100 ml, extract with n-butanol twice, 30 ml each time, combine the n-butanol liquid, evaporate to dryness, add methanol 5 ml to dissolve the residue, pass the solution through a neutral alumina column (100-200 mesh, 3 g, inner diameter 1.5 cm), elute with methanol 20 ml, discard the eluent, elute with water 25 ml, collect the eluent, pass through a polyamide column (60-90 mesh, 2.5 g, inner diameter 1.5 cm), elute with water 25 ml, discard the eluent, elute with 70% methanol 25 ml, collect the eluent, recover the solvent to dryness, add methanol 1 ml to dissolve the residue, name the solution "Xie-poly-8" solution.

[0280] Chishixieguizhutang lacks Bai Xie negative solution: take the negative solution (21042802) 100 ml, from "extract with n-butanol twice" as above, prepare by the same method, 10% methanol eluent is named "negative-poly-6" solution, 50% methanol eluent is named "negative-poly-7" solution, 70% methanol eluent is named "negative-poly-8" solution, and methanol eluent is named "negative-poly-9" solution.

[0281] Test solution of Zhishi Xiebai Guizhi Decoction: Take 100ml of Zhishi Xiebai Guizhi Decoction (21042801), and prepare it in the same way as above, starting from "extracted twice with n-butanol". Name the 10% methanol eluent as "Decoction-Poly-6" solution, the 50% methanol eluent as "Decoction-Poly-7" solution, the 70% methanol eluent as "Decoction-Poly-8" solution, and the methanol eluent as "Decoction-Poly-9" solution.

[0282] Take the following solutions: "Xie-Da-5", "Yin-Da-5", "Tang-Da-5", "Xie-Ju-6", "Yin-Ju-6", "Tang-Ju-6", "Xie-Ju-7", "Yin-Ju-7", "Tang-Ju-7", "Xie-Ju-8", "Yin-Ju-8", "Tang-Ju-8", "Xie-Ju-9", "Yin-Ju-9", and "Tang-Ju". Sixteen solutions, including 5 μl each of "-9" solution, "Xie-Da-4" solution and "Tang-Da-4" solution, and 20 μl of "Tang-Da-5" solution, were spotted onto the same silica gel G thin-layer plate. The plate was developed using ethyl acetate-glacial acetic acid-formic acid-water (15:1:1:2) as the developing solvent. The plate was then removed, dried, sprayed with 10% sulfuric acid ethanol solution, and heated at 105°C until the spots were clearly visible. The plates were then examined under a UV lamp at 365 nm.

[0283] See results Figure 15 , Figure 15 The chromatogram obtained by the processing method provided in Comparative Example 11. Figure 15 In the middle, from left to right, the samples are: "Xie-Da-5" solution, "Yin-Da-5" solution, "Tang-Da-5" solution, "Xie-Ju-6" solution, "Yin-Ju-6" solution, "Tang-Ju-6" solution, "Xie-Ju-7" solution, "Yin-Ju-7" solution, "Tang-Ju-7" solution, "Xie-Ju-8" solution, "Yin-Ju-8" solution, "Tang-Ju-8" solution, "Xie-Ju-9" solution, "Yin-Ju-9" solution, "Tang-Ju-9" solution, a mixed solution of "Xie-Da-4" solution and "Tang-Da-4" solution, and 20 μl of "Tang-Da-5" solution.

[0284] Depend on Figure 15 It can be seen that, in the processing method provided in Comparative Example 11, the method of eluting with a mixture of two chromatography columns, first neutral alumina and then D101 macroporous adsorption resin, is significantly better than the method of eluting with a neutral alumina column followed by a polyamide column.

[0285] Example 1

[0286] Bupleurum root decoction solution: 100 ml of Bupleurum root decoction solution (21042803) was taken, extracted with n-butanol twice, 30 ml each time, the n-butanol was combined and evaporated to dryness, the residue was dissolved in 5 ml of methanol, passed through a neutral alumina column (100-200 mesh, 3 g, 1.5 cm in diameter), eluted with 20 ml of methanol, the eluent was discarded, then eluted with 25 ml of water, the eluent was collected, passed through a D101 macroporous adsorption resin column (1.5 cm in diameter, 10 cm in length), eluted with 25 ml of water, 25 ml of 10% methanol, 25 ml of 50% methanol and 25 ml of methanol in sequence, the methanol eluent was collected, the solvent was recovered to dryness, the residue was dissolved in 1 ml of methanol, and the solution was named "Bupleurum-large-6" solution.

[0287] Citrus fruit, Bupleurum root and Cinnamomum twig decoction solution lacking Bupleurum root: 100 ml of Citrus fruit, Bupleurum root and Cinnamomum twig decoction solution lacking Bupleurum root (21042802) was taken, and prepared by the same method from "extracted with n-butanol twice" above, and the methanol eluent of the D101 macroporous adsorption resin column was named "negative-large-6" solution.

[0288] Citrus fruit, Bupleurum root and Cinnamomum twig decoction solution: 100 ml of Citrus fruit, Bupleurum root and Cinnamomum twig decoction (21042801) was taken, and prepared by the same method from "extracted with n-butanol twice" above, and the methanol eluent of the D101 macroporous adsorption resin column was named "decoction-large-6" solution.

[0289] Citrus fruit, Bupleurum root and Cinnamomum twig decoction solution lacking Cinnamomum twig: 100 ml of Citrus fruit, Bupleurum root and Cinnamomum twig decoction solution lacking Cinnamomum twig (21042502) was taken, and prepared by the same method from "extracted with n-butanol twice" above, and the methanol eluent of the D101 macroporous adsorption resin column was named "lack Cinnamomum twig-large-1" solution.

[0290] Citrus fruit, Bupleurum root and Cinnamomum twig decoction solution lacking Citrus fruit: 100 ml of Citrus fruit, Bupleurum root and Cinnamomum twig decoction solution lacking Citrus fruit (21042801) was taken, and prepared by the same method from "extracted with n-butanol twice" above, and the methanol eluent of the D101 macroporous adsorption resin column was named "lack Citrus fruit-large-1" solution.

[0291] Citrus fruit, Bupleurum root and Cinnamomum twig decoction solution lacking Magnolia officinalis: 100 ml of Citrus fruit, Bupleurum root and Cinnamomum twig decoction solution lacking Magnolia officinalis (21050702) was taken, and prepared by the same method from "extracted with n-butanol twice" above, and the methanol eluent of the D101 macroporous adsorption resin column was named "lack Magnolia officinalis-large-1" solution.

[0292] Citrus fruit, Bupleurum root and Cinnamomum twig decoction solution lacking Trichosanthes kirilowii: 100 ml of Citrus fruit, Bupleurum root and Cinnamomum twig decoction solution lacking Trichosanthes kirilowii (21041402) was taken, and prepared by the same method from "extracted with n-butanol twice" above, and the methanol eluent of the D101 macroporous adsorption resin column was named "lack Trichosanthes kirilowii-large-1" solution.

[0293] Neutral alumina column elution solution of Zhishixieguizhitu decoction: 100 ml of Zhishixieguizhitu decoction (21042801) was extracted with n-butanol twice, 30 ml each time, the n-butanol was combined and evaporated to dryness, the residue was dissolved in 5 ml of methanol, and passed through a neutral alumina column (100-200 mesh, 3 g, 1.5 cm in diameter). The eluate was discarded, and then eluted with 25 ml of water. The eluate was collected, evaporated to dryness, the residue was dissolved in 1 ml of methanol, and the solution was named "Xie-oxygen-5" solution.

[0294] Polyamide elution solution of Xiebai decoction pieces: 100 ml of Xiebai decoction piece single decoction sample solution (21042803) was extracted with n-butanol twice, 30 ml each time, the n-butanol was combined and evaporated to dryness, the residue was dissolved in 5 ml of methanol, and passed through a neutral alumina column (100-200 mesh, 3 g, 1.5 cm in diameter). The eluate was discarded, and then eluted with 25 ml of water. The eluate was collected, passed through a polyamide column (60-90 mesh, 2.5 g, 1.5 cm in diameter), eluted with 25 ml of water, and the eluate was collected, evaporated to dryness, the residue was dissolved in 1 ml of methanol, and the solution was named "Xie-poly-10" solution.

[0295] 100 ml of Xiebai decoction piece single decoction sample solution (21042803) was extracted with n-butanol twice, 30 ml each time, the n-butanol was combined and evaporated to dryness, the residue was dissolved in 5 ml of methanol, and passed through a neutral alumina column (100-200 mesh, 3 g, 1.5 cm in diameter). The eluate was discarded, and then eluted with 25 ml of water. The eluate was collected, passed through a polyamide column (60-90 mesh, 2.5 g, 1.5 cm in diameter), eluted with 25 ml of water, and the eluate was collected, evaporated to dryness, the residue was dissolved in 1 ml of methanol, and the solution was named "Xie-poly-10" solution.

[0296] Negative solution of Zhishixieguizhitu decoction without Xiebai: 100 ml of Zhishixieguizhitu decoction without Xiebai (21042802) was prepared by the same method as above from "extracted with n-butanol twice", and the polyamide column water elution solution was named "negative-poly-10" solution, and the polyamide column 50% methanol elution solution was named "negative-poly-11" solution.

[0297] Test solution of Zhishixieguizhitu decoction: 100 ml of Zhishixieguizhitu decoction (21042801) was prepared by the same method as above from "extracted with n-butanol twice", and the polyamide column water elution solution was named "decoction-poly-10" solution, and the polyamide column 50% methanol elution solution was named "decoction-poly-11" solution.

[0298] Take 5 μl of each of the following fifteen solutions: "degreased cotton methanol solution", "Allium macrostemon-oxygen-5" solution, "Allium macrostemon-poly-10" solution, "Anion macrostemon-poly-10" solution, "Tang macrostemon-poly-10" solution, "Allium macrostemon-poly-11" solution, "Anion macrostemon-poly-11" solution, "Tang macrostemon-poly-11" solution, "Allium macrostemon-large-6" solution, "Anion macrostemon-large-6" solution, "Tang macrostemon-large-6" solution, "Immature bitter orange-large-1" solution, "Immature cinnamon twig-large-1" solution, "Immature magnolia bark-large-1" solution, and "Immature trichosanthes fruit-large-1" solution. Spot them separately on the same silica gel G thin-layer plate. Develop the plate using ethyl acetate-glacial acetic acid-formic acid-water (15:1:1:2) as the developing solvent. Remove the plate, air dry it, spray it with 10% sulfuric acid ethanol solution, and heat it at 105℃ until the spots are clearly visible. Examine the plate under a UV lamp at 365 nm.

[0299] See results Figure 16 , Figure 16 The chromatogram obtained by the processing method provided in Example 1. Figure 16 In the middle, from left to right, the samples are: "degreased cotton methanol solution", "Allium macrostemon-oxygen-5" solution, "Allium macrostemon-poly-10" solution, "Anion macrostemon-poly-10" solution, "Tang macrostemon-poly-10" solution, "Allium macrostemon-poly-11" solution, "Anion macrostemon-poly-11" solution, "Tang macrostemon-poly-11" solution, "Allium macrostemon-large-6", "Anion macrostemon-large-6" solution, "Tang macrostemon-large-6" solution, "lacking Citrus aurantium-large-1" solution, "lacking Cinnamomum cassia-large-1" solution, "lacking Magnolia officinalis-large-1" solution, and "lacking Trichosanthes kirilowii-large-1" solution.

[0300] Depend on Figure 16 It can be seen that the separation effect of the solution after treatment with D101 macroporous adsorption resin after passing through the neutral alumina column is better than that of polyamide separation. Only the solution treated with the neutral alumina column is interfered with and cannot identify the Allium macrostemon component. There are no fluorescent spots of Allium macrostemon component in the negative solution lacking Allium macrostemon, while fluorescent spots of Allium macrostemon component are present in all negative solutions that do not lack Allium macrostemon.

[0301] Comparative Example 12

[0302] Test solution 21031601 of Zhishi Xiebai Guizhi Decoction: Take 100ml of Zhishi Xiebai Guizhi Decoction (21031601) and pass it through a D101 macroporous adsorption resin column (inner diameter 1.5cm, column length 10cm). First, elute with 100ml of water and 50ml of 10% methanol, discard the eluent, then elute with 100ml of 50% methanol, collect the last 50ml of eluent, recover the solvent until dry, add 1ml of methanol to dissolve the residue, and name the solution "Decoction 1".

[0303] Take 100 ml of the decoction of Ciliao Xiebai Guizhi Decoction (21031601) and pass it through a D101 macroporous adsorption resin column (1.5 cm in diameter and 10 cm in length). First, elute with 100 ml of water and 50 ml of 10% methanol, discard the eluate, then elute with 50 ml of methanol, collect the eluate, recover the solvent to dryness, add 1 ml of methanol to the residue to dissolve it, and name the solution "Decoction 2" solution.

[0304] For the specificity of the absorption methodology verification, take 10 μl of each of the following four solutions: Baiyunia insignis control drug material solution (121130-202107), Ciliao Xiebai Guizhi Decoction without Baiyunia insignis negative solution (21042802), Ciliao Xiebai Guizhi Decoction (21031601), and "Decoction 2" solution, and spot them on the same silica gel G thin layer plate. Use ethyl acetate-glacial acetic acid-formic acid-water (15:1:1:2) as the developing agent, develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots develop clearly, and observe under a UV lamp at 365 nm.

[0305] See Figure 17 , Figure 17 The chromatogram obtained by the treatment method provided for Comparative Example 12. Figure 17 From left to right, the samples are Baiyunia insignis control drug material solution, Ciliao Xiebai Guizhi Decoction without Baiyunia insignis negative solution, Ciliao Xiebai Guizhi Decoction, and "Decoction 2" solution.

[0306] From Figure 17 It can be seen that Baiyunia insignis components have fluorescent spots in both test solution, and no such fluorescent spots are present in the negative solution. However, the Ciliao Xiebai Guizhi Decoction that has only been treated with a D101 macroporous adsorption resin column has greater interference than the test solution ("Decoction 2" solution) that has been treated with two different chromatography columns.

[0307] Comparative Example 13

[0308] Ciliao Xiebai Guizhi Decoction test solution: Take 100 ml of Ciliao Xiebai Guizhi Decoction (21060201, 21060202, 21060203) and pass it through a D101 macroporous adsorption resin column (1.5 cm in diameter and 10 cm in length). Elute with 100 ml of water, 50 ml of 10% methanol, 100 ml of 50% methanol, and 50 ml of methanol in sequence, collect the methanol eluate, recover the solvent to dryness, add 1 ml of methanol to the residue to dissolve it, and use it as three test solutions (21060201, 21060202, 21060203).

[0309] Ciliao Xiebai Guizhi Decoction without Baiyunia insignis negative solution: Take 100 ml of Ciliao Xiebai Guizhi Decoction without Baiyunia insignis negative sample (21042802) and, from the "passing through a D101 macroporous adsorption resin column" step, prepare the solution in the same manner to obtain Ciliao Xiebai Guizhi Decoction without Baiyunia insignis negative solution.

[0310] Positive solution of Zhi Shi Xie Bai Gui Zhi Tang (Xie Bai decoction alone): Weigh 24g of Xie Bai slices (YF21012906), add 800ml of water, soak for 45 minutes, boil for 15 minutes, filter while hot through a 100-mesh screen, and cool to obtain the Xie Bai decoction alone. Measure 100ml of the Xie Bai decoction alone and pass it through a D101 macroporous adsorption resin column to prepare the positive solution of Zhi Shi Xie Bai Gui Zhi Tang using the same method.

[0311] Allium macrostemon reference material solution: Weigh 4g of Allium macrostemon reference material (batch number: 121130-201906), add 100ml of water, boil for 15 minutes, cool, filter, add water to the filtrate to 100ml, and prepare Allium macrostemon reference material solution in the same way starting from "passing through D101 type macroporous adsorption resin column".

[0312] Blank solution: Take 100 ml of water and prepare a blank solution using the same method, starting from "passing through a D101 macroporous adsorption resin column".

[0313] Take 10 μl of each of the following seven solutions: blank solution, Allium macrostemon control solution, positive solution of Citrus aurantium, Allium macrostemon and Cinnamomum cassia decoction, negative solution of Citrus aurantium, Allium macrostemon and Cinnamomum cassia decoction without Allium macrostemon, and three test sample solutions (21060201, 21060202, 21060203). Spot them separately on the same 0.1% sodium hydroxide silica gel G thin-layer plate. Develop the plate with ethyl acetate-methanol-water (10:2:1). Remove the plate, air dry it, spray it with 10% sulfuric acid ethanol solution, and heat it at 105℃ until the spots are clearly visible. Examine the plate under a UV lamp at 365 nm.

[0314] See results Figure 18 , Figure 18 The chromatogram obtained by the processing method provided in Comparative Example 13. Figure 18 In the middle, from left to right, the samples are blank solution, Allium macrostemon control solution, Zhishi Xiebai Guizhi Decoction positive solution, Zhishi Xiebai Guizhi Decoction negative solution without Allium macrostemon, and test sample solution (21060201, 21060202, 21060203).

[0315] Depend on Figure 18 It can be seen that the processing method provided in Comparative Example 13 failed the methodological verification and contained a lot of impurities, which affected the fluorescent spots of Allium macrostemon.

[0316] Comparative Example 14

[0317] Allium macrostemon ether extract solution: Take 4g of Allium macrostemon powder (YF210101), add 100ml of water, boil for 10 minutes, cool, filter, and extract the filtrate twice with 20ml of ether each time. Combine the ether extracts, evaporate to dryness, and dissolve the residue in 1ml of ethyl acetate to obtain the Allium macrostemon ether extract solution (hereinafter referred to as "ether extract" solution). Collect the aqueous solution for later use.

[0318] Residual solution of the ether extract of Baihe Rhizoma Allii Macrostemonis: The residual water solution of the ether extract was evaporated on a water bath, and the residue was dissolved in 2 ml of methanol to obtain the residual solution of the ether extract of Baihe Rhizoma Allii Macrostemonis (referred to as "ether residual" solution).

[0319] Ethyl acetate extract solution of Baihe Rhizoma Allii Macrostemonis: 4 g of Baihe Rhizoma Allii Macrostemonis powder (YF210101) was added to 100 ml of water, boiled for 10 minutes, cooled, filtered, and the filtrate was extracted with ethyl acetate twice, 20 ml each time. The ethyl acetate extract solution was combined, evaporated, and the residue was dissolved in 1 ml of ethyl acetate to obtain the ethyl acetate extract solution of Baihe Rhizoma Allii Macrostemonis (referred to as "ethyl acetate extract" solution). The water solution was collected for later use.

[0320] Residual solution of the ethyl acetate extract of Baihe Rhizoma Allii Macrostemonis: The residual water solution of the ethyl acetate extract was evaporated on a water bath, and the residue was dissolved in 2 ml of methanol to obtain the residual solution of the ethyl acetate extract of Baihe Rhizoma Allii Macrostemonis (referred to as "ethyl acetate residual" solution).

[0321] n-Butanol extract solution of Baihe Rhizoma Allii Macrostemonis: 4 g of Baihe Rhizoma Allii Macrostemonis powder (YF210101) was added to 100 ml of water, boiled for 10 minutes, cooled, filtered, and the filtrate was extracted with n-butanol twice, 20 ml each time. The n-butanol extract solution was combined, evaporated, and the residue was dissolved in 1 ml of methanol to obtain the n-butanol extract solution of Baihe Rhizoma Allii Macrostemonis (referred to as "n-butanol extract" solution). The water solution was collected for later use.

[0322] Residual solution of the n-butanol extract of Baihe Rhizoma Allii Macrostemonis: The residual water solution of the n-butanol extract was evaporated on a water bath, and the residue was dissolved in 2 ml of methanol to obtain the residual solution of the n-butanol extract of Baihe Rhizoma Allii Macrostemonis (referred to as "n-butanol residual" solution).

[0323] Ten microliters of each of the "ether extract" solution, the "ethyl acetate extract" solution, the "n-butanol extract" solution, the "ether residual" solution, the "ethyl acetate residual" solution, and the "n-butanol residual" solution were taken and spotted on the same silica gel G thin layer plate. Ethyl acetate-methanol (1:1) was used as the developing agent, and the plate was developed, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots were clearly colored, and observed under daylight and ultraviolet light (365 nm).

[0324] The results are shown in Figure 19 , Figure 19 The chromatogram obtained by using the developing agent ethyl acetate-methanol (1:1) provided for Comparative Example 14. Figure 19 In the figure, the left graph is the chromatogram under daylight, and the right graph is the chromatogram under ultraviolet (365 nm) observation. Among them, from left to right, the samples are the "ether extract" solution, the "ethyl acetate extract" solution, the "n-butanol extract" solution, the "ether residual" solution, the "ethyl acetate residual" solution, and the "n-butanol residual" solution.

[0325] Depend on Figure 19 It can be seen that among the six Allium macrostemon solutions, the n-butanol-extracted solution showed more fluorescent components, but the developing solvent ethyl acetate-methanol (1:1) did not separate these components well.

[0326] Take 10 μl of each of the following six solutions: "ether extract", "ethyl acetate extract", "n-butanol extract", "ether residue", "ethyl acetate residue", and "n-butanol residue", and spot them separately on the same silica gel G thin-layer plate. Develop the plate using n-hexane-ethyl acetate (1:1) as the developing solvent. Remove the plate, air dry it, spray it with 10% sulfuric acid ethanol solution, and heat it at 105℃ until the spots are clearly visible. Examine the plate under sunlight and ultraviolet light at 365 nm.

[0327] See results Figure 20 , Figure 20 The chromatogram obtained using the developing solvent n-hexane-ethyl acetate (1:1) provided for Comparative Example 14. Figure 20 The left image shows the chromatogram under sunlight, and the right image shows the chromatogram under ultraviolet (365nm) examination. From left to right, the samples are: ether extract, ethyl acetate extract, n-butanol extract, ether residue, ethyl acetate residue, and n-butanol residue.

[0328] Depend on Figure 20 It can be seen that among the six Allium macrostemon solutions, only the n-butanol-extracted solution had a single fluorescent spot, but this spot was too close to the starting point.

[0329] Comparative Example 15

[0330] The sample was obtained using the same sample processing method as Comparative Example 14.

[0331] Take 10 μl each of the following six solutions: ether extract, ethyl acetate extract, n-butanol extract, residual ether extract, residual ethyl acetate extract, and residual n-butanol extract. Spot each solution onto the same silica gel G thin-layer plate. Develop the plate using ethyl acetate as the developing solvent. Remove the plate, air dry it, spray it with 10% sulfuric acid ethanol solution, and heat it at 105°C until the spots are clearly visible. Examine the plate under sunlight and ultraviolet light at 365 nm.

[0332] See results Figure 21 , Figure 21 The chromatogram obtained using ethyl acetate as the developing solvent for Comparative Example 15. Figure 21 The left image shows the chromatogram under sunlight, and the right image shows the chromatogram under ultraviolet (365nm) examination. From left to right, the samples are: ether extract, ethyl acetate extract, n-butanol extract, ether residue, ethyl acetate residue, and n-butanol residue.

[0333] It can be seen that the first three solutions have fluorescent spots, which are in a suitable position, but the spots are relatively blurred. Figure 21

[0334] Comparative Example 16

[0335] The sample was obtained according to the sample processing method of Comparative Example 14.

[0336] Ten μl of each of the "ethyl ether extract" solution, the "ethyl acetate extract" solution, the "n-butanol extract" solution, the "ethyl ether residue" solution, the "ethyl acetate residue" solution and the "n-butanol residue" solution was spotted on the same silica gel G thin layer plate, and developed with methanol as the developing agent. After taking out, air-drying, spraying with 10% sulfuric acid ethanol solution and heating at 105°C until the spots were clearly colored, the plate was observed under daylight and ultraviolet light 365 nm.

[0337] The results are shown in Figure 22 , Figure 22 The chromatogram obtained by using methanol as the developing agent for Comparative Example 16. Figure 22 The left graph is the chromatogram under daylight, and the right graph is the chromatogram under ultraviolet (365 nm) observation. Among them, from left to right, the samples are the "ethyl ether extract" solution, the "ethyl acetate extract" solution, the "n-butanol extract" solution, the "ethyl ether residue" solution, the "ethyl acetate residue" solution and the "n-butanol residue" solution.

[0338] It can be seen that the last four solutions all have fluorescent spots, but the spots are too close to the front, and the developing agent needs to be further adjusted. Figure 22

[0339] Comparative Example 17

[0340] The sample was obtained according to the sample processing method of Comparative Example 14.

[0341] Ten μl of each of the "ethyl ether extract" solution, the "ethyl acetate extract" solution, the "n-butanol extract" solution, the "ethyl ether residue" solution, the "ethyl acetate residue" solution and the "n-butanol residue" solution was spotted on the same silica gel G thin layer plate, and developed with ethyl acetate-glacial acetic acid-formic acid-water (15:1:1:2) as the developing agent. After taking out, air-drying, spraying with 10% sulfuric acid ethanol solution and heating at 105°C until the spots were clearly colored, the plate was observed under daylight and ultraviolet light 365 nm.

[0342] The results are shown in Figure 23 , Figure 23 The chromatogram obtained by using ethyl acetate-glacial acetic acid-formic acid-water (15:1:1:2) as the developing agent for Comparative Example 17. Figure 23 ​​The left image shows the chromatogram under sunlight, and the right image shows the chromatogram under ultraviolet (365nm) examination. From left to right, the samples are: ether extract, ethyl acetate extract, n-butanol extract, ether residue, ethyl acetate residue, and n-butanol residue.

[0343] Depend on Figure 23 It can be seen that there are many fluorescent spots in the "ethyl acetate extract" solution and the separation is good, indicating that the developing solvent ethyl acetate-glacial acetic acid-formic acid-water (15:1:1:2) is good for separating the Allium macrostemon herb solution.

[0344] Comparative Example 18

[0345] The sample was obtained using the same sample processing method as in Comparative Example 8.

[0346] Take 5 μl of each of the three solutions, namely "Xie-Yang-1", "An-Yang-1" and "Tang-Yang-1", and spot them separately on the same silica gel G thin-layer plate. Use ethyl acetate-methanol-water (15:4:2) as the developing solvent, develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, and heat at 105℃ until the spots are clearly visible. Examine under sunlight and ultraviolet light at 365 nm.

[0347] See Figure 24 , Figure 24 The chromatogram obtained using the developing solvent ethyl acetate-methanol-water (15:4:2) provided for Comparative Example 18. Figure 24 In the image, the left image shows the chromatogram under sunlight, and the right image shows the chromatogram under ultraviolet (365nm) examination. From left to right, the samples are "Allium-Oxygen-1" solution, "Anion-Oxygen-1" solution, and "Ton-Oxygen-1" solution.

[0348] Depend on Figure 24 It can be seen that the developing solvent ethyl acetate-methanol-water (15:4:2) is not good at separating the Allium macrostemon component in the decoction of Citrus aurantium, Allium macrostemon and Cinnamomum cassia.

[0349] Take 5 μl of each of the three solutions, namely "Xan-O-1", "An-O-1" and "Tang-O-1", and spot them separately on the same silica gel G thin-layer plate. Use ethyl acetate-methanol-0.1% sodium hydroxide (15:4:2) as the developing solvent, develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, and heat at 105℃ until the spots are clearly visible. Examine under sunlight and ultraviolet light at 365 nm.

[0350] See Figure 25 , Figure 25 The chromatogram obtained using the developing solvent ethyl acetate-methanol-0.1% sodium hydroxide (15:4:2) provided for Comparative Example 17. Figure 25In the image, the left image shows the chromatogram under sunlight, and the right image shows the chromatogram under ultraviolet (365nm) examination. From left to right, the samples are "Allium-Oxygen-1" solution, "Anion-Oxygen-1" solution, and "Ton-Oxygen-1" solution.

[0351] Depend on Figure 25 It can be seen that the developing solvent ethyl acetate-methanol-0.1% sodium hydroxide (15:4:2) is not good at separating the Allium macrostemon component in the Zhishi Xiebai Guizhi Decoction.

[0352] Comparative Example 19

[0353] Samples were obtained according to the sample preparation methods of Example 1 and Comparative Example 12.

[0354] Four solutions (10 μl each) were taken from the method validation specificity test: Allium macrostemon control herb solution (121130-202107, batch number of Allium macrostemon control herb: 121130-202107), negative solution for Allium macrostemon lacking in Zhishi Allium macrostemon and Cinnamon Twig Decoction (21042802), test solution 21031601 (i.e. decoction of Zhishi Allium macrostemon and Cinnamon Twig Decoction (21031601)), and "Decoction 2" solution. They were spotted onto the same silica gel G thin-layer plate. Three plates were spotted using the same method. The developing solvents were ethyl acetate-methanol-water (15:4:2), ethyl acetate-methanol-10% acetic acid (15:4:2), and ethyl acetate-methanol-0.1% sodium hydroxide (15:4:2), respectively. After development, the plates were removed, dried, sprayed with 10% sulfuric acid ethanol solution, and heated at 105℃ until the spots were clearly visible. The spots were then examined under a UV lamp at 365 nm.

[0355] See Figure 26 , Figure 26 Chromatograms obtained using three different developing solvents provided for Comparative Example 19. Figure 26 In the figures, the developing solvent in the left figure is ethyl acetate-methanol-water, the developing solvent in the middle figure is ethyl acetate-methanol-10% acetic acid, and the developing solvent in the right figure is ethyl acetate-methanol-0.1% sodium hydroxide. From left to right, the samples in the figures are: Allium macrostemon control solution, Allium macrostemon negative solution, test solution 21031601, and "Tang 2" solution.

[0356] Depend on Figure 26 It can be seen that the developing solvent ethyl acetate-methanol-water (15:4:2) did not develop the target spot. The developing solvent ethyl acetate-methanol-10% acetic acid (15:4:2) separated the Allium macrostemon component in the Zhi Shi Xie Bai Gui Zhi Tang decoction in the sample relatively well, but it was too close to the impurity spots, indicating room for improvement. The two test solutions of Zhi Shi Xie Bai Gui Zhi Tang decoction with the developing solvent ethyl acetate-methanol-0.1% sodium hydroxide (15:4:2) did not show the target fluorescent spot due to uneven spraying of the developing solvent.

[0357] Comparative Example 20

[0358] The sample was prepared according to the sample preparation method of Example 1 and Comparative Example 12.

[0359] Ten μl of the Allium tuberosum control drug material solution (121130-202107), the negative solution without Allium tuberosum in the Allium tuberosum, Fructus Aurantii and Ramulus Cinnamomi Decoction, the test sample solution 21031601, the "Decoction 1" solution and the "Decoction 2" solution were taken and spotted on the same silica gel G thin layer plate. Two plates were spotted in the same way and developed with ethyl acetate-methanol-0.1% sodium hydroxide (15:4:2) and ethyl acetate-methanol-water (15:4:2) respectively. The developed plates were taken out, dried, sprayed with 10% sulfuric acid ethanol solution and heated at 105°C until the spots were clearly colored. The plates were observed under ultraviolet light at 365 nm.

[0360] Reference is made to Figure 27 , Figure 27 The chromatograms obtained using two different developing agents provided for Comparative Example 20. Figure 27 In the figure, the developing agent for the left plate is ethyl acetate-methanol-0.1% sodium hydroxide and the developing agent for the right plate is ethyl acetate-methanol-water. From left to right in the figure, the samples are the Allium tuberosum control drug material solution, the negative solution without Allium tuberosum, the test sample solution 21031601 and the "Decoction 2" solution.

[0361] As can be seen from Figure 27 , both developing agents are suitable. There is no fluorescent spot of the Allium tuberosum component in the "Decoction 1" solution. The test sample solution (21031601) and the "Decoction 2" solution both have fluorescent spots of the Allium tuberosum component. However, the separation of the test sample solution (21031601) is obviously better and the spots are brighter, while the spots in the "Decoction 2" solution are dimmer, possibly interfered by impurity components.

[0362] Comparative Example 21

[0363] The sample was prepared according to the sample preparation method of Comparative Example 12.

[0364] Ten μl of the "Decoction 2" solution was taken and spotted on a silica gel G thin layer plate. Three plates were spotted in the same way and developed with ethyl acetate-methanol-formic acid-water (8:1:1:1), toluene-glacial acetic acid-water (7.5:10:0.3) and n-butanol-ethanol-glacial acetic acid-water (4:1:2:1) respectively. The developed plates were taken out, dried, sprayed with 10% sulfuric acid ethanol solution and heated at 105°C until the spots were clearly colored. The plates were observed under ultraviolet light at 365 nm.

[0365] Reference is made to Figure 28 , Figure 28 The chromatograms obtained using three different developing agents provided for Comparative Example 21. Figure 28The developing agent of the left picture is ethyl acetate-methanol-formic acid-water, the developing agent of the middle picture is toluene-glacial acetic acid-water, and the developing agent of the right picture is n-butanol-ethanol-glacial acetic acid-water.

[0366] It can be seen that the three developing agents above cannot separate the components of the "Tang 2" solution. Figure 28

[0367] Comparative Example 22

[0368] The sample was prepared according to the sample preparation method of Comparative Example 12.

[0369] 10 μl of the "Tang 2" solution was taken and spotted on a silica gel G thin layer plate, and three plates were spotted in the same way. The lower layer solution was developed with chloroform-ethyl acetate-methanol-water (15:40:22:10) at 10°C below, the upper layer solution was developed with n-butanol-ethyl acetate-water (4:1:5), and the upper layer solution was developed with n-butanol-acetic acid-water (3:2:1). After development, the plate was taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots were clearly colored, and observed under a UV light lamp at 365 nm.

[0370] Reference is made to Figure 29 , Figure 29 The chromatograms obtained by the three different developing agents provided for Comparative Example 22. Figure 29 The developing agent of the left picture is chloroform-ethyl acetate-methanol-water, the developing agent of the middle picture is n-butanol-ethyl acetate-water, and the developing agent of the right picture is n-butanol-acetic acid-water.

[0371] It can be seen that the three developing agents above cannot separate the components of the "Tang 2" solution. Figure 29

[0372] Comparative Example 23

[0373] The sample was prepared according to the sample preparation method of Example 1 and Comparative Example 12.

[0374] 10 μl of each of the Baijiangqian Guizhi Tang solution without Baijiangqian (21042802), the test sample solution (21031601), the "Tang 2" solution, and the Baijiangqian control drug material solution (121130-202107) in the method validation specificity were taken and spotted on the same silica gel G thin layer plate. The developing agent was chloroform-ethyl acetate-methanol-water (15:40:22:10). After development, the plate was taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots were clearly colored, and observed under a UV light lamp at 365 nm.

[0375] Reference is made to Figure 30 , Figure 30 ​​The chromatogram obtained using the developing solvent chloroform-ethyl acetate-methanol-water (15:40:22:10) provided for Comparative Example 23. Figure 30 In the middle, from left to right, the samples are: Allium macrostemon control solution, Allium macrostemon negative solution, test sample solution (21031601), and "Tang 2" solution.

[0376] Depend on Figure 30 It can be seen that the developing solvent chloroform-ethyl acetate-methanol-water (15:40:22:10) is not good at separating the Allium macrostemon components in Zhishi Xiebai Guizhi Decoction.

[0377] Comparative Example 24

[0378] Samples were obtained according to the sample preparation methods of Example 1 and Comparative Example 12.

[0379] Take 10 μl of each of the following four solutions from the method validation specificity test: Allium macrostemon control solution (121130-202107), negative solution for Allium macrostemon lacking in Zhishi Xiebai Guizhi Decoction (21042802), test solution (21031601), and "Tang 2" solution. Spot them on the same silica gel G thin-layer plate. Spot three plates in the same way. Use ethyl acetate-methanol-water (10:2:1), ethyl acetate-methanol-0.1% sodium hydroxide (10:2:1), and ethyl acetate-methanol-10% acetic acid (10:2:1) as developing solvents, respectively. Remove the plates, air dry them, spray them with 10% sulfuric acid ethanol solution, heat them at 105℃ until the spots are clearly visible, and examine them under a UV lamp at 365 nm.

[0380] See Figure 31 , Figure 31 Chromatograms obtained using the three developing solvents provided in Comparative Example 24. Figure 31 In the diagram, the left image shows ethyl acetate-methanol-water, the middle image shows ethyl acetate-methanol-0.1% sodium hydroxide, and the right image shows ethyl acetate-methanol-10% acetic acid. Figure 31 In the middle, from left to right, the samples are: Allium macrostemon control solution, Allium macrostemon negative solution, Zhishi Allium macrostemon and Cinnamon Twig Decoction (21031601), and "Decoction 2" solution.

[0381] Depend on Figure 31 It can be seen that the developing solvents ethyl acetate-methanol-water (10:2:1) and ethyl acetate-methanol-0.1% sodium hydroxide (10:2:1) are good at separating the Allium macrostemon components in Zhishi Xiebai Guizhi Decoction, while the developing solvent ethyl acetate-methanol-10% acetic acid (10:2:1) is poor at separation.

[0382] Comparative Example 25

[0383] The sample was obtained according to the sample preparation method of Comparative Example 13.

[0384] Take 10 μl of each of the following seven solutions: blank solution, Allium macrostemon control solution (121130-202107), Allium macrostemon positive solution, Zhi Shi Xie Bai Gui Zhi Tang (Zhi Shi Xie Bai Gui Zhi Tang) negative solution (21042802), and Zhi Shi Xie Bai Gui Zhi Tang decoction (21060201, 21060202, 21060203). Spot them separately on the same 0.1% sodium hydroxide silica gel G thin-layer plate. Develop the plate with ethyl acetate-methanol-water (10:2:1). Remove the plate, air dry it, spray it with 10% sulfuric acid ethanol solution, and heat it at 105℃ until the spots are clearly visible. Examine the plate under a UV lamp at 365 nm.

[0385] See Figure 32 , Figure 32 The chromatogram obtained using the developing solvent ethyl acetate-methanol-water (10:2:1) provided for Comparative Example 25. Figure 32 In the middle, from left to right, the samples are blank solution, Allium macrostemon control solution, Allium macrostemon positive solution, Zhishi Allium macrostemon and Cinnamon Twig Decoction without Allium macrostemon negative solution, and Zhishi Allium macrostemon and Cinnamon Twig Decoction decoction (21060201, 21060202, 21060203).

[0386] Depend on Figure 32 It can be seen that the developing solvent ethyl acetate-methanol-water (10:2:1) is not good at separating the Allium macrostemon components in Zhishi Xiebai Guizhi Decoction.

[0387] Comparative Example 26

[0388] The sample was obtained according to the sample preparation method of Comparative Example 13.

[0389] Take 10 μl of each of the following seven solutions: blank solution, Allium macrostemon control solution (121130-202107), Allium macrostemon positive solution, Zhishi Xiebai Guizhi Tang (Zhishi Xiebai Guizhi Tang) negative solution (21042802), and Zhishi Xiebai Guizhi Tang decoction (21060201, 21060202, 21060203), and spot them on the same silica gel G thin-layer plate. Develop the plate using ethyl acetate-methanol-ammonia water (10:2:1) as the developing solvent. Remove the plate, air dry it, spray it with 10% sulfuric acid ethanol solution, and heat it at 105℃ until the spots are clearly visible. Examine the plate under a UV lamp at 365 nm.

[0390] See Figure 33 , Figure 33 The chromatogram obtained using the developing solvent ethyl acetate-methanol-ammonia (10:2:1) provided for Comparative Example 26. Figure 33 In the middle, from left to right, the samples are blank solution, Allium macrostemon control solution, Allium macrostemon positive solution, Zhishi Allium macrostemon and Cinnamon Twig Decoction without Allium macrostemon negative solution, and Zhishi Allium macrostemon and Cinnamon Twig Decoction decoction (21060201, 21060202, 21060203).

[0391] Depend on Figure 33It can be seen that the developing solvent ethyl acetate-methanol-ammonia (10:2:1) is not good at separating the Allium macrostemon components in Zhishi Xiebai Guizhi Decoction.

[0392] Comparative Example 27

[0393] The sample was obtained according to the sample preparation method of Comparative Example 13.

[0394] Take 10 μl of each of the following seven solutions: blank solution, Allium macrostemon control solution (121130-202107), Allium macrostemon positive solution, Zhi Shi Xie Bai Gui Zhi Tang (Zhi Shi Xie Bai Gui Zhi Tang) negative solution (21042802), and Zhi Shi Xie Bai Gui Zhi Tang decoction (21060201, 21060202, 21060203). Spot each solution onto the same silica gel G thin-layer plate. Spot three plates using the same method. Develop the plates using ethyl acetate-methanol-water (10:2:1), ethyl acetate-methanol-0.1% sodium hydroxide (10:2:1), and ethyl formate-methanol-water-ammonia (10:2:1:0.1), respectively. Remove the plates, air dry them, spray them with 10% sulfuric acid ethanol solution, and heat them at 105℃ until the spots are clearly visible. Examine them under a UV lamp at 365 nm.

[0395] See Figure 34 , Figure 34 Chromatograms obtained using the three developing solvents provided in Comparative Example 27. Figure 34 In the middle figure, the developing solvent in the left figure is ethyl acetate-methanol-water, the developing solvent in the middle figure is ethyl acetate-methanol-0.1% sodium hydroxide, and the developing solvent in the right figure is ethyl formate-methanol-water-ammonia. Figure 34 In the middle, from left to right, the samples are blank solution, Allium macrostemon control solution, Allium macrostemon positive solution, Zhishi Allium macrostemon and Cinnamon Twig Decoction without Allium macrostemon negative solution, and Zhishi Allium macrostemon and Cinnamon Twig Decoction decoction (21060201, 21060202, 21060203).

[0396] Depend on Figure 34 It can be seen that the developing solvent ethyl acetate-methanol-water (10:2:1) separates the components of Allium macrostemon well, but the negative solution has fluorescent spots of the same color at the target spot position. The other two developing solvents have poor development effects.

[0397] Comparative Example 28

[0398] The sample was obtained according to the sample preparation method of Comparative Example 13.

[0399] Take 10 μl of each of the following seven solutions: blank solution, Baijiu control drug material solution (121130-202107), Baijiu positive solution, Zhishu Baijiu Guizhi Decoction without Baijiu negative solution (21042802), Zhishu Baijiu Guizhi Decoction extract (21060201, 21060202, 21060203), and spot them on the same silica gel G thin layer plate. Use formic acid ethyl ester-methanol-glacial acetic acid-water (10:2:1:1) as the developing agent, develop, take out, air dry, spray with 10% sulfuric acid ethanol solution, heat to 105°C until the spots develop clearly, and observe under ultraviolet light at 365 nm.

[0400] See Figure 35 , Figure 35 The chromatogram obtained by using the developing agent formic acid ethyl ester-methanol-glacial acetic acid-water (10:2:1:1) provided for Comparative Example 28. Figure 35 From left to right, the samples are blank solution, Baijiu control drug material solution, Baijiu positive solution, Zhishu Baijiu Guizhi Decoction without Baijiu negative solution, and Zhishu Baijiu Guizhi Decoction extract (21060201, 21060202, 21060203).

[0401] As can be seen from Figure 35 , each sample solution does not have a target spot.

[0402] Comparative Example 29

[0403] According to the sample preparation method of Comparative Example 13, a sample is obtained.

[0404] Take 10 μl of each of the following seven solutions: blank solution, Baijiu control drug material solution (121130-202107), Baijiu positive solution, Zhishu Baijiu Guizhi Decoction without Baijiu negative solution (21042802), Zhishu Baijiu Guizhi Decoction extract (21060201, 21060202, 21060203), and spot them on the same silica gel G thin layer plate. Spot 4 pieces in the same way, and use formic acid ethyl ester-methanol-water (10:2:1), formic acid ethyl ester-methanol-water-ammonia (10:2:1:0.02), formic acid ethyl ester-methanol-water-ammonia (15:2:1:0.05), and formic acid ethyl ester-methanol-water-ammonia (15:2:1:0.02) as the developing agents, develop, take out, air dry, spray with 10% sulfuric acid ethanol solution, heat to 105°C until the spots develop clearly, and observe under ultraviolet light at 365 nm.

[0405] See Figure 36 , Figure 36 The chromatogram obtained by using the four developing agents provided for Comparative Example 29. Figure 36In the diagram, the developing solvent in the upper left image is ethyl formate-methanol-water (10:2:1), the developing solvent in the upper right image is ethyl formate-methanol-water-ammonia (10:2:1:0.02), the developing solvent in the lower left image is ethyl formate-methanol-water-ammonia (15:2:1:0.05), and the developing solvent in the lower right image is ethyl formate-methanol-water-ammonia (15:2:1:0.02). Figure 36 In the middle, from left to right, the samples are blank solution, Allium macrostemon control solution, Allium macrostemon positive solution, Zhishi Allium macrostemon and Cinnamon Twig Decoction without Allium macrostemon negative solution, and Zhishi Allium macrostemon and Cinnamon Twig Decoction decoction (21060201, 21060202, 21060203).

[0406] Depend on Figure 36 It can be seen that none of the four developing solvents can develop the target spot.

[0407] Comparative Example 30

[0408] The sample was obtained according to the sample preparation method of Comparative Example 13.

[0409] Take 10 μl of each of the following seven solutions: blank solution, Allium macrostemon control solution (121130-202107), Allium macrostemon positive solution, Zhi Shi Xie Bai Gui Zhi Tang (Zhi Shi Xie Bai Gui Zhi Tang) negative solution (21042802), and Zhi Shi Xie Bai Gui Zhi Tang decoction (21060201, 21060202, 21060203). Spot them separately on the same silica gel G thin-layer plate. Spot two plates in the same way. Use ethyl formate-methanol-water-ammonia (12:2:1:0.02) and ethyl formate-methanol-water-ammonia (10:1.5:1:0.02) as developing solvents, respectively. Remove the plates, air dry them, spray them with 10% sulfuric acid ethanol solution, heat them at 105℃ until the spots are clearly visible, and examine them under a UV lamp at 365 nm.

[0410] See Figure 37 , Figure 37 Chromatograms obtained using the two developing solvents provided in Comparative Example 30. Figure 37 In the middle, the developing solvent of the left figure is ethyl formate-methanol-water-ammonia (12:2:1:0.02), and the developing solvent of the right figure is ethyl formate-methanol-water-ammonia (10:1.5:1:0.02). Figure 37 In the middle, from left to right, the samples are blank solution, Allium macrostemon control solution, Allium macrostemon positive solution, Zhishi Allium macrostemon and Cinnamon Twig Decoction without Allium macrostemon negative solution, and Zhishi Allium macrostemon and Cinnamon Twig Decoction decoction (21060201, 21060202, 21060203).

[0411] Depend on Figure 37 It can be seen that neither of the two developing solvents can develop the target spot.

[0412] Comparative Example 31

[0413] The sample was obtained according to the sample preparation method of Comparative Example 13.

[0414] Ten μl of each of the three solutions of Baihe reference material solution (121130-202107), Jishi Baihe Guizhi Decoction minus Baihe negative solution (21042802), and Jishi Baihe Guizhi Decoction (21060201) were spotted on the same silica gel G thin layer plate, and four plates were prepared in the same manner. Ethyl acetate-methanol-water-ammonia (10:2:1:0.02), ethyl acetate-methanol-water-ammonia (10:3:1:0.02), ethyl acetate-methanol-water-ammonia (10:2:1.5:0.02), and ethyl acetate-methanol-0.1% sodium hydroxide (10:2:1) were used as the developing agents, and the plate was developed, removed, air-dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots developed clearly, and observed under ultraviolet light at 365 nm.

[0415] See Figure 38 , Figure 38 The chromatogram obtained using the four developing agents provided for Comparative Example 31. Figure 38 In the figure, the developing agent of the upper left graph is ethyl acetate-methanol-water-ammonia (10:2:1:0.02), the developing agent of the upper right graph is ethyl acetate-methanol-water-ammonia (10:3:1:0.02), the developing agent of the lower left graph is ethyl acetate-methanol-water-ammonia (10:2:1.5:0.02), and the developing agent of the lower right graph is ethyl acetate-methanol-0.1% sodium hydroxide (10:2:1). Figure 38 In each of the figures, the samples from left to right are Baihe reference material solution, Jishi Baihe Guizhi Decoction minus Baihe negative solution, and Jishi Baihe Guizhi Decoction (21060201).

[0416] As can be seen from Figure 38 , none of the four developing agents can develop the target spots.

[0417] Comparative Example 32

[0418] The sample was obtained according to the sample preparation method of Comparative Example 13.

[0419] Ten μl of each of the four solutions of Baihe reference material solution (121130-202107), Baihe positive solution, Jishi Baihe Guizhi Decoction minus Baihe negative solution (21042802), and test sample solution (21060201) were spotted on the same silica gel G thin layer plate, and ethyl acetate-methanol-0.1% sodium hydroxide (10:2:1) was used as the developing agent, and the plate was developed, removed, air-dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots developed clearly, and observed under ultraviolet light at 365 nm.

[0420] See Figure 39 , Figure 39The chromatogram obtained using the developing solvent ethyl acetate-methanol-0.1% sodium hydroxide (10:2:1) provided for Comparative Example 32. Figure 39 In the middle, from left to right, the samples are: Allium macrostemon control solution, Allium macrostemon positive solution, Zhi Shi Xie Bai Gui Zhi Tang (without Allium macrostemon negative solution), and test sample solution (21060201).

[0421] Depend on Figure 39 It can be seen that the developing solvent ethyl acetate-methanol-0.1% sodium hydroxide (10:2:1) provides good separation for the sample.

[0422] Comparative Example 33

[0423] The sample was obtained according to the sample preparation method of Comparative Example 13.

[0424] Take 10 μl of each of the following three solutions: Allium macrostemon control solution (121130-202107), negative solution for Allium macrostemon deficiency in Citrus aurantium and Allium macrostemon decoction (21042802), and test solution (21060201), and spot them separately on the same silica gel G thin-layer plate. Spot three plates in the same way. Use ethyl acetate-methanol-water-ammonia (10:2:1:0.02), ethyl acetate-methanol-water-ammonia (10:3:1:0.02), and ethyl acetate-methanol-water-ammonia (10:2:1.5:0.02) as developing solvents, respectively. After development, remove the plates, air dry them, spray them with 10% sulfuric acid ethanol solution, heat them at 105℃ until the spots are clearly visible, and examine them under a UV lamp at 365 nm.

[0425] See Figure 40 , Figure 40 Chromatograms obtained using the three developing solvents provided in Comparative Example 33. Figure 40 In the middle, the developing solvent of the left figure is ethyl acetate-methanol-water-ammonia (10:2:1:0.02), the developing solvent of the middle figure is ethyl acetate-methanol-water-ammonia (10:3:1:0.02), and the developing solvent of the bottom figure is ethyl acetate-methanol-water-ammonia (10:2:1.5:0.02). Figure 40 In the middle, from left to right, the samples are: Allium macrostemon control solution, Zhishi Allium macrostemon and Cinnamon Twig Decoction without Allium macrostemon negative solution, and test sample solution (21060201).

[0426] Depend on Figure 40 It can be seen that the developing solvents ethyl acetate-methanol-water-ammonia (10:2:1:0.02) and ethyl acetate-methanol-water-ammonia (10:3:1:0.02) are better for separating the samples, while ethyl acetate-methanol-water-ammonia (10:2:1.5:0.02) are worse.

[0427] From the above, it can be seen from the figure that the developing agent ethyl acetate-methanol-10% acetic acid (15:4:2), ethyl acetate-methanol-0.1% sodium hydroxide (15:4:2), ethyl acetate-methanol-water (15:4:2), ethyl acetate-methanol-water (10:2:1), ethyl acetate-methanol-0.1% sodium hydroxide (10:2:1), ethyl acetate-methanol-water-ammonia water (10:2:1:0.02), ethyl acetate-methanol-water-ammonia water (10:3:1:0.02), ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) are all good for separating the allium tuberosum components in the allium tuberosum Guizhifuling decoction, and the Rf value of the target spot on the thin layer plate, the separation degree from the adjacent spot, and the brightness of the fluorescent spot of several developing agents are compared, and it is found that the developing agent ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) is the best, so the developing agent is finally selected as the developing agent of the final method.

[0428] Comparative Example 34

[0429] 1. The sample was prepared according to the sample preparation method of Comparative Example 3.

[0430] Take 5 μl of each of the "allium tuberosum" solution, the "Yin 2" solution, and the "soup 3" solution, and point them on the same silica gel G thin layer plate, use ethyl acetate-glacial acetic acid-formic acid-water (15:1:1:2) as the developing agent, develop, take out, dry, spray with 2% vanillin 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored, and observe under daylight and ultraviolet light 365 nm.

[0431] See Figure 41 , Figure 41 The chromatogram obtained by the color developing agent (2% vanillin 10% sulfuric acid ethanol solution) provided for Comparative Example 34. Figure 41 In the figure, the left graph is the chromatogram under daylight, and the right graph is the chromatogram under ultraviolet (365 nm) observation. Figure 41 In each graph of the figure, from left to right, the samples are "allium tuberosum" solution, "Yin 2" solution, and "soup 3" solution.

[0432] Take 5 μl of each of the "allium tuberosum" solution, the "Yin 2" solution, and the "soup 3" solution, and point them on the same silica gel G thin layer plate, use ethyl acetate-glacial acetic acid-formic acid-water (15:1:1:2) as the developing agent, develop, take out, dry, spray with 2% vanillin 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored, and observe under daylight and ultraviolet light 365 nm.

[0433] See Figure 42 , Figure 42 The chromatogram obtained by the color developing agent (2% vanillin 10% sulfuric acid ethanol solution) provided for Comparative Example 34. Figure 42In the figure, the left is the chromatogram under sunlight, and the right is the chromatogram under ultraviolet (365nm) inspection. Figure 42 In each figure of the present application, the samples from left to right are "Bulbus Allii Macrostemonis" solution, "Yin 2" solution, and "Tang 3" solution.

[0434] Take 5 μl of each of "Bulbus Allii Macrostemonis" solution, "Yin 2" solution, and "Tang 3" solution, and spot them on the same silica gel G thin layer plate. Use ethyl acetate-glacial acetic acid-formic acid-water (15:1:1:2) as the developing agent, develop, take out, dry, spray with sulfuric acid-acetic anhydride (1:20) solution, heat at 105°C until the spots are clearly colored, and inspect under sunlight and ultraviolet light 365nm.

[0435] See Figure 43 , Figure 43 The chromatogram obtained by using the color developing agent (sulfuric acid-acetic anhydride (1:20) solution) provided for Comparative Example 34. Figure 43 In the figure, the left is the chromatogram under sunlight, and the right is the chromatogram under ultraviolet (365nm) inspection. Figure 43 In each figure of the present application, the samples from left to right are "Bulbus Allii Macrostemonis" solution, "Yin 2" solution, and "Tang 3" solution.

[0436] 2. Obtain the sample according to the sample preparation method of Comparative Example 7.

[0437] Take 5 μl of each of "Bulbus Allii Macrostemonis" solution, "Yin 2" solution, and "Tang 3" solution, and spot them on the same silica gel G thin layer plate. Use ethyl acetate-glacial acetic acid-formic acid-water (15:1:1:2) as the developing agent, develop, take out, dry, spray with sulfuric acid-acetic anhydride (1:20) solution, heat at 105°C until the spots are clearly colored, and inspect under sunlight and ultraviolet light 365nm.

[0438] See Figure 44 , Figure 44 The chromatogram obtained by using the color developing agent (sulfuric acid-acetic anhydride (1:20) solution) provided for Comparative Example 34. Figure 44 In the figure, the left is the chromatogram under sunlight, and the right is the chromatogram under ultraviolet (365nm) inspection. Figure 44 In each figure of the present application, the samples from left to right are "Bulbus Allii Macrostemonis" solution, "Yin 2" solution, and "Tang 3" solution.

[0439] Take 5 μl of each of "Bulbus Allii Macrostemonis" solution, "Yin 2" solution, and "Tang 3" solution, and spot them on the same silica gel G thin layer plate. Use ethyl acetate-glacial acetic acid-formic acid-water (15:1:1:2) as the developing agent, develop, take out, dry, spray with sulfuric acid-acetic anhydride (1:20) solution, heat at 105°C until the spots are clearly colored, and inspect under sunlight and ultraviolet light 365nm.

[0440] See Figure 45 , Figure 45 The chromatogram obtained using the colorimetric reagent (2% vanillin sulfuric acid solution) provided for Comparative Example 34. Figure 45 In the image, the left image shows the chromatogram under sunlight, and the right image shows the chromatogram under ultraviolet (365nm) examination. Figure 45 In the figures, the samples from left to right are "Allium-D" solution, "Anion-D" solution, and "Sodium-D" solution.

[0441] Take 5 μl of each of the three solutions, namely "Xie-D", "Yin-D", and "Tang-D", and spot them separately on the same silica gel G thin-layer plate. Use ethyl acetate-glacial acetic acid-formic acid-water (15:1:1:2) as the developing solvent, develop, remove, air dry, spray with sulfuric acid-acetic anhydride (1:20) solution, heat at 105℃ until the spots are clearly visible, and examine under sunlight and ultraviolet light at 365 nm.

[0442] See Figure 46 , Figure 46 The chromatogram obtained for the colorimetric reagent (sulfuric acid-acetic anhydride (1:20) solution) provided in Comparative Example 34. Figure 46 In the image, the left image shows the chromatogram under sunlight, and the right image shows the chromatogram under ultraviolet (365nm) examination. Figure 46 In the figures, the samples from left to right are "Allium-D" solution, "Anion-D" solution, and "Sodium-D" solution.

[0443] 3. The sample was obtained according to the sample preparation method of Comparative Example 5.

[0444] Take aspirate the following solutions: "Allium macrostemon-N-B" solution, "Anion macrostemon-N-B" solution, "Sodium-N-B" solution, "Allium macrostemon-B-A" solution, "Anion macrostemon-B-A" solution, "Sodium-B-A" solution, "Allium macrostemon-90A" solution, "Anion macrostemon-90A" solution, "Sodium-90A" solution, "Allium macrostemon-70A" solution, "Anion macrostemon-70A" solution, "Sodium-70A" solution, "Allium macrostemon-50A" solution, and "Anion macrostemon-50A". Eighteen solutions, including "Tang-50A", "Xie-Shui", "Xieyin-Shui", and "Tang-Shui", were spotted separately onto the same silica gel G thin-layer plate. The plate was developed using ethyl acetate-glacial acetic acid-formic acid-water (15:1:1:2) as the developing solvent. The plate was then removed, dried, sprayed with 2% vanillin-sulfuric acid solution, and heated at 105°C until the spots were clearly visible. The plates were then examined under sunlight and ultraviolet light at 365 nm.

[0445] See Figure 47 , Figure 47 The chromatogram obtained using the colorimetric reagent (2% vanillin sulfuric acid solution) provided for Comparative Example 34. Figure 47 In the image, the left image shows the chromatogram under sunlight, and the right image shows the chromatogram under ultraviolet (365nm) examination. Figure 47In the figures, from left to right, the samples are: "Allium macrostemon-positive-B" solution, "Anion macrostemon-positive-B" solution, "Sodium macrostemon-positive-B" solution, "Allium macrostemon-B-A" solution, "Anion macrostemon-B-A" solution, "Sodium macrostemon-B-A" solution, "Allium macrostemon-90A" solution, "Anion macrostemon-90A" solution, "Sodium macrostemon-90A" solution, "Allium macrostemon-70A" solution, "Anion macrostemon-70A" solution, "Sodium macrostemon-70A" solution, "Allium macrostemon-50A" solution, "Anion macrostemon-50A" solution, "Sodium macrostemon-50A" solution, "Allium macrostemon-water" solution, "Anion macrostemon-water" solution, and "Sodium macrostemon-water" solution.

[0446] 4. The sample was obtained according to the sample preparation method of Comparative Example 6.

[0447] Take samples of the following solutions: "Allium macrostemon-water-J", "Anion-water-J", "Decoction-water-J", "Allium macrostemon-10A-J", "Anion-10A-J", "Decoction-10A-J", "Allium macrostemon-30A-J", "Anion-30A-J", "Decoction-30A-J", "Allium macrostemon-50A-J", "Anion-50A-J", and "Decoction-50A-J". Fifteen different solutions, including the "Allium macrostemon-methyl-J" solution, the "Anion-methyl-J" solution, and the "Sodium-methyl-J" solution, were each 5 μl of and spotted onto the same silica gel G thin-layer plate. The plate was developed using ethyl acetate-glacial acetic acid-formic acid-water (15:1:1:2) as the developing solvent. The plate was then removed, dried, sprayed with 2% vanillin-sulfuric acid solution, and heated at 105°C until the spots were clearly visible. The plates were then examined under sunlight and ultraviolet light at 365 nm.

[0448] See Figure 48 , Figure 48 The chromatogram obtained using the colorimetric reagent (2% vanillin sulfuric acid solution) provided for Comparative Example 34. Figure 48 In the image, the left image shows the chromatogram under sunlight, and the right image shows the chromatogram under ultraviolet (365nm) examination. Figure 48 In the figures, from left to right, the samples are: "Allium macrostemon-water-J" solution, "Anion-water-J" solution, "Soup-water-J" solution, "Allium macrostemon-10methyl-J" solution, "Anion-10methyl-J" solution, "Soup-10methyl-J" solution, "Allium macrostemon-30methyl-J" solution, "Anion-30methyl-J" solution, "Soup-30methyl-J" solution, "Allium macrostemon-50methyl-J" solution, "Anion-50methyl-J" solution, "Soup-50methyl-J" solution, "Allium macrostemon-methyl-J" solution, "Anion-methyl-J" solution, and "Soup-methyl-J" solution.

[0449] 5. The sample was obtained according to the sample preparation method of Comparative Example 12.

[0450] Take 10 μl of each of the four solutions, i.e. the Baiyunshu reference material solution, the Baiyunshu-free negative solution of the Zhishibaiyunshu Guizheng Decoction, the test sample solution 21031601 and the "Decoction 2" solution, and spot them on the same silica gel G thin layer plate. Spot three plates in the same way, and use ethyl acetate-glacial acetic acid-formic acid-water (15:1:1:2) as the developing agent to develop, remove and dry. Then, spray with 10% antimony trichloride chloroform solution and 10% sulfuric acid ethanol solution, and heat at 105°C until the spots develop clear color. Then, observe under ultraviolet light at 365 nm.

[0451] See Figure 49 , Figure 49 The chromatograms obtained by using the two different color developing agents provided for Comparative Example 34. Figure 49 In the figure, the color developing agent for the left graph is 10% antimony trichloride chloroform solution, and the color developing agent for the right graph is 10% sulfuric acid ethanol solution. Figure 49 In each graph, the samples from left to right are the Baiyunshu reference material solution, the Baiyunshu-free negative solution, the test sample solution 21031601 and the "Decoction 2" solution.

[0452] As can be seen from Figures 41-49 , the fluorescent spots are most obvious and easy to distinguish when using 10% sulfuric acid ethanol as the color developing agent.

[0453] Comparative Example 35

[0454] 1. The sample was obtained according to the sample preparation method of Comparative Example 5.

[0455] Take 5 μl of each of the three solutions, i.e. the "Baiyun-70A" solution, the "Negative-70A" solution and the "Decoction-70A" solution, and spot them on the same silica gel G thin layer plate. Use ethyl acetate-glacial acetic acid-formic acid-water (15:1:1:2) as the developing agent to develop, remove and dry. Then, spray with 10% sulfuric acid ethanol solution, and heat at 105°C until the spots develop clear color. Then, observe under sunlight and ultraviolet light at 365 nm.

[0456] See Figure 50 , Figure 50 The chromatogram obtained by using the thin layer plate (silica gel G thin layer plate) provided for Comparative Example 35. Figure 50 In the figure, the left graph is the chromatogram under sunlight, and the right graph is the chromatogram observed under ultraviolet light at 365 nm. Figure 50 In each graph of the figure, the samples from left to right are the "Baiyun-70A" solution, the "Negative-70A" solution and the "Decoction-70A" solution.

[0457] 2. The sample was obtained according to the sample preparation method of Comparative Example 3.

[0458] Take 5 μl of each of "Baiyunao" solution, "Yin-2" solution, "Tang-2" solution, "Baiyunao-70A" solution, "Yin-70A" solution and "Tang-70A" solution, and spot them on the same silica gel GF254 thin layer plate. Take ethyl acetate-glacial acetic acid-formic acid-water (15:1:1:2) as developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored, and observe under daylight and ultraviolet light lamp 254 nm.

[0459] See Figure 51 , Figure 51 The chromatogram obtained from the thin layer plate (0.1% sodium hydroxide silica gel G thin layer plate) provided for Comparative Example 35. 254 Thin layer plate) provided for Comparative Example 35. Figure 51 In FIG. 6, the left graph is the chromatogram under daylight, and the right graph is the chromatogram observed under ultraviolet light lamp 365 nm. Figure 51 In FIG. 6, the samples from left to right are "Baiyunao" solution, "Yin-2" solution, "Tang-2" solution, "Baiyunao-70A" solution, "Yin-70A" solution and "Tang-70A" solution.

[0460] 3. The sample was obtained according to the sample preparation method of Comparative Example 13.

[0461] Take 10 μl of each of blank solution, Baiyunao reference drug material solution, Baiyunao positive solution, Baiyunao-free negative solution of Jishibaiyunao Guizhiting Decoction, test sample solutions 21060201, 21060202 and 21060203, and spot them on the same 0.1% sodium hydroxide silica gel G thin layer plate. Take ethyl acetate-methanol-water (10:2:1) as developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored, and observe under ultraviolet light lamp 365 nm.

[0462] See Figure 52 , Figure 52 The chromatogram obtained from the thin layer plate (0.1% sodium hydroxide silica gel G thin layer plate) provided for Comparative Example 35. Figure 52 In FIG. 6, the samples from left to right are blank solution, Baiyunao reference drug material solution, Baiyunao positive solution, Baiyunao-free negative solution of Jishibaiyunao Guizhiting Decoction, test sample solutions 21060201, 21060202 and 21060203.

[0463] It can be known from Figures 50-52 that by comparing the chromatograms of silica gel G, GF 254 , G (1% sodium hydroxide) thin layer plates, it is found that the spots on the silica gel G thin layer plate are clearly colored and well separated, so the silica gel G thin layer plate is selected.

[0464] Example 2

[0465] Thin layer identification method:

[0466] Extract 100 ml of sample twice with 30 ml of n-butanol each time. Combine the n-butanol extracts, evaporate to dryness, dissolve the residue in 5 ml of methanol, and pass the solution through a neutral alumina column (100-200 mesh, 3 g, 1.5 cm inner diameter). Elute with 20 ml of methanol, discard the eluent, and then elute with 25 ml of water. Collect the eluent and pass it through a D101 macroporous adsorption resin column (1.5 cm inner diameter, 10 cm length). Elute sequentially with 25 ml of water, 25 ml of 10% methanol, 25 ml of 50% methanol, and 25 ml of methanol. Collect the methanol eluent, recover the solvent to dryness, and dissolve the residue in 1 ml of methanol to obtain the sample solution.

[0467] Perform the thin-layer chromatography test (General Chapter 0502 of the Chinese Pharmacopoeia). Take 10 μl of the sample solution and spot it separately on the same silica gel G thin-layer plate. Use ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) as the developing solvent. Develop the plate, remove it, air dry it, spray it with 10% sulfuric acid ethanol solution, and heat it at 105℃ until the spots are clearly visible. Examine it under ultraviolet light (365nm).

[0468] Specifically, take 10 μl of each of the following seven solutions: blank (pure water), Allium macrostemon control herb solution (121130-202107), negative sample solution of Zhishi Xiebai Guizhi Decoction without Allium macrostemon (21042802), positive sample solution (21042803), and decoction of Zhishi Xiebai Guizhi Decoction (21031601, 21042801, 21051001), and conduct identification experiments according to the proposed thin-layer chromatography identification method.

[0469] See results Figure 53 , Figure 53 The chromatogram of the sample amount (10 μl) provided in Example 2. Figure 53 In the middle, from left to right, are blank, Allium macrostemon control herb solution (121130-202107), negative sample solution of Zhishi Xiebai Guizhi Decoction without Allium macrostemon (21042802), positive sample solution (21042803), and decoction of Zhishi Xiebai Guizhi Decoction (21031601, 21042801, 21051001).

[0470] The following samples were tested using the proposed thin-layer chromatography method under different spotting conditions: Allium macrostemon reference material solution (121130-202107), negative sample solution of Zhishi Allium macrostemon and Cinnamon Twig Decoction (21042802), and decoction of Zhishi Allium macrostemon and Cinnamon Twig Decoction (21031601, 21042801, 21051001). The results for 5 μl and 15 μl samples are as follows.

[0471] See results Figure 54 , Figure 54 Chromatograms of different spotting amounts provided in Example 2. Figure 54 In the middle, from left to right, are the Allium macrostemon control solution, negative solution, and decoction of Citrus aurantium, Allium macrostemon and Cinnamomum cassia (21031601, 21042801, 21051001) (all 5 μl); Allium macrostemon control solution, negative solution, and decoction of Citrus aurantium, Allium macrostemon and Cinnamomum cassia (21031601, 21042801, 21051001) (all 15 μl).

[0472] The results showed that when the sample volume was 10 μl, the fluorescent spots of the reference solution and the test solution on the silica gel G plate were clearly visible, while when the sample volume was 5 μl, the fluorescent spots of the test solution on the silica gel G plate were dimmer and difficult to observe.

[0473] Example 3 - Validation of Thin-Layer Chromatography Identification Method (Method 1)

[0474] Method 1: The thin-layer chromatographic identification method for the decoction of Zhi Shi Xie Bai Gui Zhi Tang is as follows:

[0475] Take 100 ml of sample and extract twice with n-butanol, 30 ml each time. Combine the n-butanol extracts, evaporate to dryness, dissolve the residue in 5 ml of methanol, and pass the solution through a neutral alumina column (100-200 mesh, 3 g, inner diameter 1.5 cm). Elute with 20 ml of methanol, discard the eluent, and then elute with 25 ml of water. Collect the eluent and pass it through a D101 macroporous adsorption resin column (inner diameter 1.5 cm, column length 10 cm). Elute sequentially with 25 ml of water, 25 ml of 10% methanol, 25 ml of 50% methanol, and 25 ml of methanol. Collect the methanol eluent, recover the solvent to dryness, and dissolve the residue in 1 ml of methanol to obtain the sample solution.

[0476] According to the above thin-layer chromatography method (General Chapter 0502 of the Chinese Pharmacopoeia), 10 μl of the above solution was applied separately to the same silica gel G thin-layer plate. Ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) was used as the developing solvent. The plate was developed, removed, and dried. It was then sprayed with 10% sulfuric acid ethanol solution and heated at 105°C until the spots were clearly visible. The plates were then examined under ultraviolet light (365 nm).

[0477] In accordance with the relevant requirements of the "Guiding Principles for Validation of Analytical Methods for Drug Quality Standards" in General Chapter 9101 of the Chinese Pharmacopoeia, the thin-layer chromatography identification method 1 for Allium macrostemon in the reference sample of Zhishi Xiebai Guizhi Decoction was validated. Specificity and robustness tests showed that this method is robust to thin-layer identification using Allium macrostemon as a reference standard.

[0478] 1. Exclusivity

[0479] Allium macrostemon decoction (positive sample, batch number: 21042803), Allium macrostemon and Cinnamomum cassia decoction (negative sample lacking Allium macrostemon, batch number: 21042802), and Allium macrostemon and Cinnamomum cassia decoction (reference sample, batch numbers: 21031601, 21042801, 21051001) were prepared according to the standard decoction process.

[0480] Take appropriate amounts of pure water (blank sample), reference sample, negative sample, positive sample, and Allium macrostemon (batch number: 121130-202107) as reference materials, and prepare the test solution and control solution according to the thin-layer chromatography method described in Method 1 above. Take the above solutions and perform identification experiments according to the proposed thin-layer identification method. The results are shown in [Figure 1]. Figure 55 .

[0481] Figure 55 This is a specificity result diagram provided for Example 3. Figure 55 In the middle, from left to right, are: blank, Allium macrostemon control material (batch number: 121130-202107), negative sample of Zhi Shi Xie Bai Gui Zhi Tang without Allium macrostemon (batch number: 21042802), Allium macrostemon decoction (positive sample, batch number: 21042803), and Zhi Shi Xie Bai Gui Zhi Tang decoction (reference samples: 21031601, 21042801, 21051001).

[0482] The results showed that the control sample, the positive sample, and the three batches of reference samples all had a fluorescent spot of the same color at the same location. The blank solution did not have a fluorescent spot of the same color at this location, and the negative solution did not have a significant fluorescent spot of the same color at this location. This indicates that the method has good specificity for identifying reference samples.

[0483] 2. Durability

[0484] (1) Thin-layer boards from different manufacturers

[0485] Thin-layer chromatography plates produced by Qingdao Hailang Silica Gel Desiccant Co., Ltd. and Qingdao Ocean Chemical Plant Branch (see specificity results figure) were used. After activation treatment, the following samples were taken: Allium macrostemon reference solution (121130-202107), negative sample of Zhishi Allium macrostemon and Cinnamon Twig Decoction lacking Allium macrostemon (batch number: 21042802), and decoction of Zhishi Allium macrostemon and Cinnamon Twig Decoction (reference samples, batch numbers: 21031601, 21042801, 21051001). The results of Qingdao Hailang plates are shown in the figure. Figure 56 .

[0486] Figure 56 Images showing the results of thin-layer laminates from different manufacturers. Figure 56 In the middle, from left to right, are the control material of Allium macrostemon, the negative solution, and the decoction of Citrus aurantium, Allium macrostemon and Cinnamomum cassia (reference samples: 21031601, 21042801, 21051001).

[0487] The results showed that different brands of silica gel G plate did not show significant difference in the identification of the method, indicating that the method was suitable for different brands of silica gel plate.

[0488] (2) Different humidity

[0489] Considering that the results of thin-layer identification experiments are often affected by temperature and humidity, the humidity conditions that may occur in the laboratory in different seasons were investigated. Relative humidity of 32%, 65%, and 88% were selected for research. The Baijiang decoction of Fructus Aurantii, Bulbus Allii Macrostemonis, and Ramulus Cinnamomi (reference samples: 21031601, 21042801, 21051001) were investigated under the proposed thin-layer chromatography at three different humidity conditions, and the results are shown in Figure 57 .

[0490] Figure 57 The thin-layer chromatography identification results under different relative humidity. Figure 57 In the above figures, from left to right are Baijiang decoction of Fructus Aurantii, Bulbus Allii Macrostemonis, and Ramulus Cinnamomi (reference samples: 21031601, 21042801, 21051001). Figure 57

[0491] The results showed that the thin-layer identification results under different humidity conditions could be reproduced, indicating that the method was not sensitive to humidity and had good environmental humidity resistance.

[0492] (3) Temperature

[0493] The study continued to investigate the temperature that may occur in the laboratory in different seasons, and refrigerators (about 5°C) and room temperature (about 25°C, see specificity chromatogram) were selected for research. Baijiang decoction of Fructus Aurantii, Bulbus Allii Macrostemonis, and Ramulus Cinnamomi (reference samples: 21031601, 21042801, 21051001) were investigated under the proposed thin-layer chromatography at different temperature conditions, and the low-temperature development results are shown in Figure 58 .

[0494] Figure 58 The thin-layer chromatography identification results under low-temperature temperature. Figure 58 In the above figures, from left to right are Baijiang decoction of Fructus Aurantii, Bulbus Allii Macrostemonis, and Ramulus Cinnamomi (reference samples: 21031601, 21042801, 21051001).

[0495] ​The results showed that the thin layer identification results at different temperatures had no difference, and the reproducibility was good, indicating that the method had good temperature tolerance.

[0496] (4) Spotting volume

[0497] The specific method can be seen in Example 2. The results showed that a spotting volume of 10 μl could ensure that the fluorescence spots in the chromatogram were clear and distinguishable, so the spotting volume of the Allium bakeri component thin layer chromatography identification was selected to be 10 μl.

[0498] In the robustness experiment, the effects of different brands of silica gel G plates, different temperatures, humidities and different spotting volumes on the identification results were investigated. The comparison test of different spotting volumes determined that the optimal spotting volume was 10 μl. It was found that other factors had good reproducibility, and the reference sample test solution showed the same colored spots at the corresponding positions of the Allium bakeri control drug material chromatogram, indicating that the method had good robustness.

[0499] Example 4 - Methodology verification (Method 2)

[0500] According to the relevant requirements of the "Guiding Principles for the Verification of Analysis Methods for Drug Quality Standards" of the Chinese Pharmacopoeia 9101, the Allium bakeri component thin layer chromatography identification method 2 of the reference sample of Zhishixi Allium bakeri Guizhi Decoction was verified. The specificity and robustness test showed that the method had poor specificity and robustness for thin layer identification using Allium bakeri control drug material as the control drug material.

[0501] The specific verification process is as follows:

[0502] Method 2 is as follows:

[0503] Take 100 ml of sample solution and pass it through a D101 macroporous adsorption resin column (inner diameter 1.5 cm, column length 10 cm). Elute with water 100 ml, 10% methanol 50 ml, 50% methanol 100 ml and methanol 50 ml, respectively. Collect the methanol eluate, recover the solvent to dryness, add 1 ml of methanol to dissolve the residue, and use it as the sample solution.

[0504] According to the thin layer chromatography method (Chinese Pharmacopoeia General 0502), take 10 μl of the above solution and spot it on the same silica gel G thin layer plate. Use ethyl acetate-methanol-ammonia water-water (10:2:0.02:1) as the developing agent, develop, take out, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored, and observe under ultraviolet light (365 nm).

[0505] 1 Specificity

[0506] The Baijiu decoction (positive sample, batch number: 21060301), negative decoction of Ciguizhi decoction without Baijiu (negative sample, batch number: 21042802), and Ciguizhi decoction (reference sample, batch numbers: 21060201, 21060202, and 21060203) were prepared according to the standard decoction process. Appropriate amounts of water, reference sample, negative sample, positive sample, and Baijiu control medicinal materials (batch number: 121130-202107) were taken to prepare the test sample solution and control solution according to the proposed thin layer chromatography method. The above solutions were taken and subjected to identification experiments according to the proposed thin layer identification method. The results are shown in Figure 59 .

[0507] Figure 59 The specificity results of Example 4 are shown in the figure. Figure 59 From left to right in the figure are blank, Baijiu control medicinal materials, positive solution, negative solution, and Ciguizhi decoction (reference samples 21060201, 21060202, and 21060203).

[0508] The results show that the Baijiu control medicinal materials, Baijiu positive sample, and 3 batches of reference samples have a fluorescent spot of the same color at the same position. The blank solution has no fluorescent spot of the same color at this position, and the negative solution has no obvious fluorescent spot of the same color at this position. However, the negative sample has a relatively obvious fluorescent band with background color from the starting point to the solvent front. This indicates that the method has poor specificity for identifying reference samples.

[0509] 2. Durability

[0510] (1) Thin layer plates from different manufacturers

[0511] Thin layer chromatography plates produced by Qingdao Hailang Silica Gel Drier Co., Ltd. and Qingdao Marine Chemical Factory Branch (see the specificity results figure) were selected and activated. Baijiu control medicinal materials, negative solution, and Ciguizhi decoction (reference samples: 21060201, 21060202, and 21060203) were determined according to the proposed thin layer chromatography method. The results of Qingdao Hailang plates are shown in Figure 59 . Figure 59 The chromatogram of the thin layer plate is a Qingdao Hailang plate. Figure 59 From left to right in the figure are Baijiu control medicinal materials, negative solution, and Ciguizhi decoction (reference samples: 21060201, 21060202, and 21060203).

[0512] (2) Different humidity

[0513] Considering that the results of thin-layer identification experiments are often affected by temperature and humidity, the humidity conditions that may occur in the laboratory in different seasons were investigated, and relative humidity of 32%, 65%, and 88% were selected for research. The Baihe reference materials, negative solution, and Zhishixie Baihe Guizhi Decoction (benchmark samples: 21060201, 21060202, and 21060203) were investigated under the proposed thin-layer chromatography at three different humidity conditions, and the results are shown in Figure 60 and Figure 61 , Figure 60 and Figure 61 are the thin-layer chromatography identification results under different relative humidity. Among them, Figure 60 and Figure 61 are two repeated tests, Figure 61 is the retest result. Figure 60 and Figure 61 , the left graph is the thin-layer chromatography identification result under relative humidity of 88%, the middle graph is the thin-layer chromatography identification result under relative humidity of 65%, and the right graph is the thin-layer chromatography identification result under relative humidity of 32%. Figure 60 and Figure 61 In each graph, from left to right are Baihe reference materials, negative solution, and Zhishixie Baihe Guizhi Decoction (benchmark samples: 21031601, 21042801, and 21051001).

[0514] The results show that the thin-layer identification results under different humidity environments cannot be reproduced, indicating that this method is not suitable for humidity investigation.

[0515] (3) Temperature

[0516] The study continued to investigate the temperature that may occur in the laboratory in different seasons, and the refrigerator (about 5°C) and room temperature (about 25°C, see specificity chromatogram) were selected for research. The Baihe reference materials, negative solution, and Zhishixie Baihe Guizhi Decoction (benchmark samples: 21060201, 21060202, and 21060203) were investigated under the proposed thin-layer chromatography at different temperature conditions, and the low-temperature development results are shown in Figure 62 . Figure 62 is the thin-layer chromatography identification result under low temperature. Figure 62 In the graph, from left to right are Baihe reference materials, negative solution, and Zhishixie Baihe Guizhi Decoction (benchmark samples: 21060201, 21060202, and 21060203).

[0517] The results show that under low temperature, the test solution does not show the same color fluorescent spots at the same position as the Baihe reference material solution, indicating that the method is not suitable for low temperature conditions.

[0518] (4) Sample size

[0519] Comparing different sample application amounts, 5 μl, 10 μl and 15 μl were selected for comparative study (10 μl see the specificity chromatogram). The Baijianggui Decoction (reference samples: 21060201, 21060202 and 21060203) was taken as the sample, and the negative solution and the control Allium bakeri were taken as the samples. The Baijianggui Decoction (reference samples: 21060201, 21060202 and 21060203) was taken as the sample, and the negative solution and the control Allium bakeri were taken as the samples. The results of 5 μl and 15 μl are shown in Figure 63 . Figure 63 The identification results of different sample application amounts of thin layer chromatography. Figure 63 In the above figures, the left graph is the identification result of 5 μl sample application amount of thin layer chromatography, and the right graph is the identification result of 15 μl sample application amount of thin layer chromatography. Figure 63 In the above figures, the left graph is the identification result of 5 μl sample application amount of thin layer chromatography, and the right graph is the identification result of 15 μl sample application amount of thin layer chromatography.

[0520] In the durability experiment, the effects of different brands of silica gel G plates, different temperatures, humidities and different sample application amounts on the identification results were investigated. The comparative test of different sample application amounts determined that the optimal sample application amount was 10 μl. It was found that other factor tests could not reproduce the method, indicating that the method had poor durability.

[0521] Comparative Example 36

[0522] 1. Preparation of sample

[0523] 1.1 Preparation of the Baijianggui Decoction test solution: 50 ml of the Baijianggui Decoction extract (21060203) (equivalent to 1.7 g of Allium bakeri) was taken, 3 ml of hydrochloric acid was added, heated for reflux for 1 hour, cooled, and extracted with ethyl acetate twice, 30 ml each time. The ethyl acetate liquid was combined and concentrated to 5 ml to serve as the Baijianggui Decoction test solution.

[0524] 1.2 Preparation of the Allium bakeri test solution: 1.5 g of Allium bakeri (YF210101) was taken, finely ground, 50 ml of water and 3 ml of hydrochloric acid were added, heated for reflux for 1 hour, cooled, and extracted with ethyl acetate twice, 30 ml each time. The ethyl acetate liquid was combined and concentrated to 5 ml to serve as the Allium bakeri test solution.

[0525] 1.3 Preparation of the Allium bakeri control solution: 1.5 g of the Allium bakeri control (121130-202107) was taken, finely ground, 50 ml of water and 3 ml of hydrochloric acid were added, heated for reflux for 1 hour, cooled, and extracted with ethyl acetate twice, 30 ml each time. The ethyl acetate liquid was combined and concentrated to 5 ml to serve as the Allium bakeri control solution.

[0526] 2. Process of thin layer identification:

[0527] 2 μl of the test sample solution and 1 μl of the control medicinal material were spotted on the same silica gel G thin layer plate, developed with petroleum ether (60-90 °C)-ethyl acetate-dichloromethane-formic acid (5:3.5:1:0.8) as the developing agent, taken out, dried, and observed under a 365 nm ultraviolet light.

[0528] 3 Results and analysis

[0529] See Figure 64 , Figure 64 The chromatogram obtained by the detection method provided for Comparative Example 36. It can be seen from Figure 64 that the Baishu medicinal material and the Baishu control medicinal material have a same color fluorescent spot at the same position, indicating that the patent is suitable for identifying the Baishu medicinal material. The Zhishibaibaiguizhitou decoction and the Baishu control medicinal material have a spot at the same position of the main fluorescent spot, but the color of the spot is lighter, and the brightness of the main fluorescent spot of the Baishu control medicinal material is too different, and it is not easy to distinguish whether the spot is the same yellow-green color as the main fluorescent spot. Therefore, the thin layer chromatography identification of the Zhishibaibaiguizhitou decoction Baishu medicinal flavor in Comparative Example 36 is not practical.

[0530] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered within the protection scope of the present application.

Claims

1. A processing method of a medicinal material, an intermediate and a finished product containing a water-soluble component of Typhonium giganteum Engl., the processing method being used for thin-layer identification of the medicinal material, the intermediate and the finished product containing the water-soluble component of Typhonium giganteum Engl., characterized in that, The method comprises the following steps: 1) extracting the water-soluble component-containing medicinal material, intermediate and finished product with n-butanol to obtain a n-butanol extract; 2) dissolving the residue obtained by evaporating the n-butanol extract with methanol to obtain a methanol solution; 3) passing the methanol solution through a neutral alumina column, eluting with methanol, discarding the methanol eluate, and then eluting with water to collect the water eluate; 4) passing the water eluate obtained in step 3) through a macroporous adsorption resin, and then eluting with water, a 10% (by volume) methanol solution, a 50% (by volume) methanol solution and methanol in sequence to collect the methanol eluate; 5) dissolving the residue obtained by evaporating the methanol eluate obtained in step 4) with methanol to obtain a processed solution which can be used for thin layer chromatography identification, wherein the water-soluble component-containing medicinal material, intermediate and finished product is a decoction of Fructus Aurantii and Bulbus Allii Macrostemonis, a solution of Bulbus Allii Macrostemonis medicinal material or a single decoction sample solution of Bulbus Allii Macrostemonis decoction pieces; The developing agent for the thin layer chromatography identification is selected from a mixture of ethyl acetate, formic acid, glacial acetic acid and water with a volume ratio of (14-16):(0.8-1.2):(0.8-1.2):(1.5-2.5).

2. The treatment method according to claim 1, characterized in that, In step 1), the n-butanol extraction is performed for 2-4 times. The volume ratio of the water-soluble component-containing medicinal material, intermediate and finished product to n-butanol is 100:(30-100).

3. The treatment method of claim 1, wherein In step 2), the mass-volume ratio of the residue to methanol is (0.6-1.0) g:(2-10) ml. In step 4), the macroporous adsorption resin is D101.

4. Use of the developing agent in a thin layer identification method of medicinal materials, intermediates and finished products containing water-soluble components of Typhonium giganteum Engl., which are treated according to the treatment method of any one of claims 1 to 3, characterized in that, The developing agent is selected from a mixture of ethyl acetate, formic acid, glacial acetic acid and water with a volume ratio of (14-16):(0.8-1.2):(0.8-1.2):(1.5-2.5).

5. Use according to claim 4, characterized in that, The developing agent is selected from a mixture of ethyl acetate, formic acid, glacial acetic acid and water with a volume ratio of 15:1:1:

2.

6. A method for identifying the thin layer of a medicinal material containing the water-soluble components of Typhonium giganteum Engl, intermediate and finished product, characterized in that, The method comprises the following steps: The test sample solution and the control medicinal material solution are spotted on the same silica gel G thin layer plate, developed with a developing agent, dried and colored, and then observed under ultraviolet conditions. If the same color spots appear in the test sample chromatogram at positions corresponding to the control medicinal material chromatogram, the sample is qualified. The test sample solution and the control medicinal material solution are obtained by the processing method according to any one of claims 1-3. The developing agent is selected from the developing agent used in the application of claims 4 or 5.

7. The thin layer discrimination method according to claim 6, wherein The spotting amount of the test sample solution and the control medicinal material solution is 10 μl.

8. The method of claim 6, wherein, The coloring method is: The silica gel G thin layer plate is sprayed with a 10% sulfuric acid ethanol solution and then heated.

9. The thin layer discrimination method of claim 8, wherein, The heating temperature is 105°C, and the heating time is 4-6 min.

10. The thin layer discrimination method of claim 6, wherein, The ultraviolet condition is that the ultraviolet wavelength is 365 nm.

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