Method for identifying radix bupleuri in traditional Chinese medicine by thin layer chromatography and application thereof

Abstract

The present application relates to a kind of identifying traditional Chinese medicine in the thin layer chromatography method of Chaihu and its application, belong to traditional Chinese medicine detection technical field, the method includes the following steps: sample preparation: respectively taking sample to be examined, and Chaihu saponin control product and / or Chaihu control medicinal materials, respectively into test solution, and control product solution and / or control medicinal material solution;Thin layer chromatography identification: take above-mentioned sample solution, respectively sample on silica gel thin layer plate, first with volume ratio 4:1 chloroform-methanol as developing agent, development, take out, dry, again with volume ratio 5:65:10:8:8 chloroform-ethyl acetate-acetone-anhydrous formic acid-water as developing agent development, color, namely obtain.The above-mentioned thin layer chromatography method for identifying traditional Chinese medicine in Chaihu, by the way of twice development, four saponin components Chaihu saponin a, d, B1 and B2 in Chaihu can reach the effect of complete separation, can accurately identify the traditional Chinese medicine containing Chaihu.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine detection technology, and in particular to a thin-layer chromatography method and its application for identifying Bupleurum chinense in traditional Chinese medicine preparations. Background Technology

[0002] Bupleurum is a traditional Chinese medicine listed in the Chinese Pharmacopoeia. The medicinal part is the dried root of *Bupleurum chinense* or *Bupleurum angustifolium*, both belonging to the Apiaceae family. It is harvested in spring and autumn, with stems, leaves, and soil removed before drying. Bupleurum is a commonly used diaphoretic, also known as *Di Xun*, *Shan Cai*, *Gu Cao*, and *Chai Cao*. It is bitter and slightly cold in nature, and enters the liver and gallbladder meridians. It has the effects of harmonizing the exterior and interior, soothing the liver, and raising yang. It is used for colds with fever, alternating chills and fever, malaria, liver qi stagnation, chest and rib pain, rectal prolapse, uterine prolapse, and irregular menstruation.

[0003] Bupleurum, a traditional Chinese medicine with a long history of use, is widely used in various prepared Chinese medicines, such as Xiao Chai Hu Granules, Ru He San Jie Tablets, Ganmao Qingre Granules, and Xiao Er Chai Gui Tui Re Granules. Currently, the thin-layer chromatography identification of prepared Chinese medicines containing Bupleurum in the 2020 edition of the Chinese Pharmacopoeia, Part I, uses the following conditions: a silica gel plate is used as the thin-layer plate, ethyl acetate-ethanol-water (12:2:1) is used as the developing solvent, followed by spraying with a 1% solution of p-dimethylaminobenzaldehyde in 10% sulfuric acid ethanol, heating at 105℃ until the spots are clearly visible, and then examining under ultraviolet light (365nm).

[0004] However, the above thin-layer chromatography identification has the problem of being unable to separate the saponin components (saikosaponins a, d, B1 and B2) which are present in high concentrations in Bupleurum chinense. Summary of the Invention

[0005] Therefore, it is necessary to provide a thin-layer chromatography method for identifying Bupleurum in traditional Chinese medicine preparations, addressing the current difficulty in separating saponin components in Bupleurum using thin-layer chromatography. The thin-layer chromatography identification conditions of this method can completely separate the four saponin components in Bupleurum: saikosaponin a, d, B1, and B2.

[0006] A thin-layer chromatography method for identifying Bupleurum chinense in traditional Chinese medicine preparations includes the following steps:

[0007] Sample preparation: Take the sample to be tested, as well as the saikosaponin reference standard and / or the Bupleurum reference material, and prepare the test solution, the reference solution and / or the reference material solution, respectively.

[0008] Thin-layer chromatography identification: Take the above sample solution and spot it onto a silica gel thin-layer plate. First, develop the plate with a 4:1 (v / v) mixture of chloroform and methanol. Remove the plate, air dry it, and then develop it again with a 5:65:10:8:8 (v / v) mixture of chloroform, ethyl acetate, acetone, anhydrous formic acid, and water. The color will then develop.

[0009] The above-mentioned thin-layer chromatography method for identifying Bupleurum in traditional Chinese medicine preparations, through a two-stage development process, enables the complete separation of four saponin components in Bupleurum: saikosaponin a, d, B1, and B2, thus allowing for accurate identification of traditional Chinese medicine preparations containing Bupleurum.

[0010] Understandably, with the above-mentioned two-stage development method, the solvent needs to be completely dried after the first development before the second development can be performed. The second development can be carried out using the conventional thin-layer chromatography development method, with the solvent front developing to near the top of the thin-layer plate.

[0011] In one embodiment, the saikosaponin reference standard is selected from at least one of saikosaponin a, saikosaponin d, saikosaponin B1, and saikosaponin B2.

[0012] Understandably, the above-mentioned saikosaponin reference standard is used in the early stages of method establishment. Once the method is established and the corresponding position of the reference standard on the thin-layer plate is obtained, it is no longer necessary to use the reference standard for positioning in subsequent experiments.

[0013] In one embodiment, the colorimetric conditions in the thin-layer chromatography identification step are as follows: spraying with a 1% p-dimethylaminobenzaldehyde solution in 10% sulfuric acid ethanol, heating at 105°C until the spots are clearly visible, and then examining under a 365nm ultraviolet lamp. It is understood that the aforementioned 1% p-dimethylaminobenzaldehyde solution in 10% sulfuric acid ethanol contains 1g of p-dimethylaminobenzaldehyde in 100ml of 10% sulfuric acid ethanol solution; the concentration unit is mass-volume percentage. The aforementioned 10% sulfuric acid ethanol solution is prepared by adding ethanol to 100ml of 10ml concentrated sulfuric acid solution, and is a commonly used colorimetric reagent in the art.

[0014] In one embodiment, the sample preparation step involves the following method for preparing the test solution:

[0015] Methanol method: Take the test sample, add methanol as solvent and extract by ultrasonication, filter, take the filtrate and evaporate to dryness, dissolve the residue in water-saturated n-butanol, wash with ammonia test solution, take the water-saturated n-butanol solution, evaporate to dryness, add methanol to the residue to obtain the test sample;

[0016] or

[0017] Water decoction method: Take the test sample, add water as a solvent and reflux, filter, take the filtrate, extract with water-saturated n-butanol, take the water-saturated n-butanol layer, wash with ammonia test solution, take the water-saturated n-butanol solution, evaporate to dryness, add methanol to the residue to obtain the test sample solution.

[0018] or

[0019] Acid decoction method: Take the test sample, add water as a solvent, and add acetic acid at the same time. Reflux, filter, take the filtrate and evaporate to dryness. Dissolve the residue in water-saturated n-butanol, wash with ammonia test solution, take the water-saturated n-butanol solution, evaporate to dryness, and add methanol to the residue to obtain the test sample solution.

[0020] The preparation method of the control herbal solution is as follows:

[0021] Water decoction method: Take Bupleurum reference material, add water as solvent for decoction, filter, take filtrate, extract with water-saturated n-butanol, take water-saturated n-butanol layer, wash with ammonia test solution, take water-saturated n-butanol solution, evaporate to dryness, add methanol to residue to obtain reference material solution.

[0022] or

[0023] Acidic decoction method: Take Bupleurum reference material, add water as solvent, and simultaneously add acetic acid, decoct, filter, take the filtrate and evaporate to dryness, dissolve the residue in water-saturated n-butanol, wash with ammonia test solution, take the water-saturated n-butanol solution, evaporate to dryness, add methanol to the residue to obtain the reference material solution.

[0024] In one embodiment, the sample preparation step involves the following method for preparing the test solution:

[0025] Methanol method: Take a predetermined amount of test sample, add 50±10 ml of methanol as solvent and extract ultrasonically for 30±10 min, filter, evaporate the filtrate to dryness, dissolve the residue in 50±10 ml of water-saturated n-butanol, wash with ammonia test solution 2±1 times, 20±5 ml each time, take the water-saturated n-butanol solution, evaporate to dryness, add 1±0.5 ml of methanol to the residue to obtain the test sample solution;

[0026] or

[0027] Water decoction method: Take a predetermined amount of test sample, add 50±10 ml of water as solvent, reflux for 30±10 min, filter, take the filtrate and extract with water-saturated n-butanol, 30±10 ml each time, combine the water-saturated n-butanol layers, wash with ammonia test solution 2±1 times, 20±5 ml each time, take the water-saturated n-butanol solution, evaporate to dryness, add 1±0.5 ml of methanol to the residue to obtain the test sample solution;

[0028] or

[0029] Acid-water decoction method: Take a predetermined amount of the test sample, add 50±10 ml of water as a solvent, and simultaneously add 0.25-2 ml of acetic acid. Reflux and boil for 30±10 min, filter, take the filtrate and extract with water-saturated n-butanol, 30±10 ml each time, combine the water-saturated n-butanol layers, wash with ammonia test solution 2±1 times, 20±5 ml each time, take the water-saturated n-butanol solution, evaporate to dryness, add 1±0.5 ml of methanol to the residue to obtain the test sample solution;

[0030] Reference herbal solution preparation method:

[0031] Water decoction method: Take 0.5g of Bupleurum reference material, add 50±10ml of water as solvent, decoct for 30±10min, filter, take the filtrate and extract with water-saturated n-butanol, 30±10ml each time, combine the water-saturated n-butanol layers, wash with ammonia test solution 2±1 times, 20±5ml each time, take the water-saturated n-butanol solution, evaporate to dryness, add 1±0.5ml of methanol to the residue to obtain the reference material solution;

[0032] or

[0033] Acidic decoction method: Take 0.5g of Bupleurum reference material, add 50±10ml of water as solvent, and simultaneously add 0.25-2ml of acetic acid. Decoction for 30±10min, filter, take the filtrate and extract with water-saturated n-butanol, 30±10ml each time, combine the water-saturated n-butanol layers, wash with ammonia solution 2±1 times, 20±5ml each time, take the water-saturated n-butanol solution, evaporate to dryness, add 1±0.5ml of methanol to the residue to obtain the reference material solution.

[0034] Understandably, the amount of the test sample used should be determined according to the pharmacopoeia requirements, depending on the specific type of test sample used.

[0035] In one embodiment, a preliminary experimental step is included before the sample preparation step, and the preliminary experimental step is as follows:

[0036] Take the sample to be tested and prepare the pre-test solution of the test sample according to the following method.

[0037] The test sample pre-experimental solution was obtained according to the test sample solution preparation method in the pharmacopoeia;

[0038] Then, perform identification according to the thin-layer chromatography identification steps and observe the spot conditions. If, in the preliminary experimental solutions of each batch of test samples, some batches show fluorescent spots of the same color at the positions of saponins a and B2, and some batches show fluorescent spots of the same color at the positions of saponins B1 and B2, then in the sample preparation steps, both the test sample solution and the reference herbal solution are prepared by acid-water decoction. If, in the preliminary experimental solutions of each batch of test samples, fluorescent spots of the same color are shown at the positions of saponins B1 and B2, then in the sample preparation steps, the test sample solution is prepared by methanol method, and the reference herbal solution is prepared by acid-water decoction method.

[0039] Understandably, the above-mentioned pharmacopoeia method refers to the preparation method of test solution for thin-layer chromatography identification of traditional Chinese medicine containing Bupleurum chinense in the 2020 edition of the Chinese Pharmacopoeia. If the test sample is Xiao Chai Hu granules, the preparation method of the test solution is as follows: Take 5g of Xiao Chai Hu granules sample [specification (1)], 2g [specification (2)] or 1.25g [specification (3)], that is, take half a bag of sample according to different specifications, add 50ml of water, heat under reflux for 30 minutes, cool, filter, extract twice with water-saturated n-butanol, 30ml each time, combine the water-saturated n-butanol solutions, wash twice with ammonia solution, 20ml each time, separate the n-butanol solution, evaporate to dryness, add 1ml of methanol to dissolve the residue, and use it as the test sample pre-experiment solution.

[0040] If the test sample is Ruhusanjie tablets, the test solution is prepared as follows: Take 30 tablets of this product, remove the coating from sugar-coated tablets, grind them into a fine powder, add 50 ml of methanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 50 ml of water-saturated n-butanol, wash twice with 20 ml of ammonia solution each time, evaporate the n-butanol solution to dryness, dissolve the residue in 1 ml of methanol, and use this as the test solution for the preliminary experiment.

[0041] In previous experiments, the inventors discovered that the spots on test samples of traditional Chinese medicine containing Bupleurum chinense often showed poor correspondence with the spots on Bupleurum chinense reference materials (extracted using conventional decoction methods). This was particularly evident in the significant differences in characteristic spots among different manufacturers of Xiaochaihu granules. Furthermore, the chromatograms of the reference materials prepared according to the prescribed process (decoction) were consistent with the spots of saikosaponin a and B2, but saikosaponin B1 spots were not observed. These discrepancies easily lead to confusion and doubt among testing personnel regarding the interpretation of results. After repeated experiments and exploration, the inventors discovered that the co-decoction of various herbs and the interactions between components during the preparation of traditional Chinese medicine may lead to the conversion of saikosaponins a and d into saikosaponins B1 and B2.

[0042] Based on this, the inventors attempted to obtain uniform saikosaponin spots with better comparability by adjusting the pretreatment methods of the test sample and the control medicinal material. Through preliminary experiments, the composition of the test sample was predicted, and different pretreatment methods were selected to obtain identification results that could accurately reflect the condition of the test sample and were easy for inspectors to judge.

[0043] In one embodiment, in the acid decoction method, acetic acid is added to make the pH value of the test sample solution and the control herbal solution the same as the lowest pH value of each batch of test sample pre-experiment solutions in the pre-experiment step. For example, when the test sample is Xiao Chai Hu granules, acetic acid is added to make the pH of the test sample solution and the control herbal solution 3-4; when the test sample is Xiao Chai Hu granules or Ru He San Jie tablets, acetic acid is added to make the pH of the test sample solution and the control herbal solution 5.

[0044] The present invention also discloses the application of the above-mentioned thin-layer chromatography method in the identification of traditional Chinese medicines containing Bupleurum chinense.

[0045] In one embodiment, the traditional Chinese medicine includes: Xiao Chai Hu Granules, Ru He San Jie Tablets, Ganmao Qing Re Granules, and Xiao Er Chai Gui Tui Re Granules.

[0046] Through experimental screening and adjustment, this invention has developed a thin-layer chromatography method for identifying Bupleurum in traditional Chinese medicine preparations, which has been successfully applied to Xiao Chai Hu granules, Ru He San Jie tablets, and Xiao Chai Gui antipyretic granules.

[0047] In one embodiment, the traditional Chinese medicine is Xiao Chai Hu granules, and the test solution and reference medicinal material solution are prepared by acid-water decoction; the traditional Chinese medicine is Ru He San Jie tablets, and the test solution is prepared by methanol method, and the reference medicinal material solution is prepared by acid-water decoction method. The inventors found through experiments that the consistency of thin-layer chromatographic spots of Xiao Chai Hu granules from different manufacturers is poor; the consistency of thin-layer chromatographic spots of Ru He San Jie tablets with the chromatogram of Bupleurum reference medicinal material is also poor. The above-mentioned processing method can improve the consistency of the results.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] The present invention provides a thin-layer chromatography method for identifying Bupleurum in traditional Chinese medicine preparations. By using a two-stage thin-layer chromatography development method, the four saponin components, saikosaponins a, d, B1, and B2, in Bupleurum can achieve good separation, enabling accurate identification of traditional Chinese medicine preparations containing Bupleurum.

[0050] Furthermore, experiments revealed that the poor correspondence between the test sample and the control substance in the thin-layer chromatography identification of conventional Bupleurum-containing preparations (using Bupleurum water extraction process) was due to changes in the composition caused by the co-decoction of various herbs and the interaction between components during the preparation of traditional Chinese medicine. Therefore, by adjusting the pretreatment methods of the test sample and the control herbs, Bupleurum saponin spots with good correspondence and comparability were obtained. The results of the preliminary experiments were used to predict the composition of the test sample, thereby selecting different pretreatment methods to obtain identification results that accurately reflect the condition of the test sample and are easy for inspectors to judge.

[0051] At the same time, it solved the problem that the thin-layer chromatography behavior of the same traditional Chinese medicine product, Bupleurum, was not completely consistent between different companies due to differences in the production environment of different companies.

[0052] Furthermore, once the method is established, the identification process can be completed using only Bupleurum chinense reference material, eliminating the need for additional preparation and use of saikosaponin B1 and saikosaponin B2 reference standards. This not only enriches the information available for identification but also reduces the use of reference standards. The reference materials are economical and readily available, and the method is green and environmentally friendly. Attached Figure Description

[0053] Figure 1 The thin-layer identification chromatogram obtained by the 2020 Pharmacopoeia method in Example 1;

[0054] Figure 2 The thin-layer identification spectrum is obtained by the secondary expansion method under condition 1 in Example 2;

[0055] Figure 3 The thin-layer identification spectrum is obtained by the secondary expansion method in condition 2 of Example 2;

[0056] Figure 4 The following are thin-layer chromatograms of Bupleurum reference material obtained under different extraction conditions in Example 3;

[0057] Figure 5 This is a thin-layer chromatogram for identifying Bupleurum in Xiaochaihu granules in Example 5;

[0058] Figure 6 This is a preliminary thin-layer chromatogram for identifying Bupleurum chinense in Ruhe Sanjie tablets in Example 6;

[0059] Figure 7 This is a thin-layer chromatogram for identifying Bupleurum in Xiaochaihu granules in Example 6. Detailed Implementation

[0060] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0061] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0062] Unless otherwise specified, all reagents used in the following examples are commercially available; and all methods used in the following examples are conventional methods unless otherwise specified.

[0063] Saikosaponin d (National Institutes for Food and Drug Control, batch number: 110778-201912), Saikosaponin a (National Institutes for Food and Drug Control, batch number: 110777-201912), Saikosaponin B1 (Shanghai Yuanye Biotechnology Co., Ltd., batch number: A22GB146239), Saikosaponin B2 (Shanghai Yuanye Biotechnology Co., Ltd., batch number: MO5GB140794).

[0064] Example 1

[0065] Currently, the identification method for traditional Chinese medicine preparations containing Bupleurum chinense under the thin-layer chromatography identification section of the 2020 edition of the Chinese Pharmacopoeia, Part I, is as follows:

[0066] I. Solution Preparation Method

[0067] 1. Preparation of mixed reference solution: Take appropriate amounts of saikosaponins a, d, B1 and B2 reference standards, dissolve them in methanol, and prepare a mixed reference solution containing 0.5 mg of each per 1 ml;

[0068] 2. Preparation of test solution ①: Take one bag of Xiaochaihu granules (one bag each according to different specifications: 6g, 2.5g or 1.5g), add 50ml of water, heat under reflux for 30 minutes, cool, filter, and extract twice with water-saturated n-butanol, 30ml each time. Combine the water-saturated n-butanol solutions and wash twice with ammonia test solution (take 400ml of concentrated ammonia water, dilute with water to 1000ml to obtain the solution), 20ml each time. Separate the water-saturated n-butanol layer, evaporate to dryness, and dissolve the residue in 1ml of methanol to obtain the test solution ①.

[0069] 3. Preparation of Bupleurum reference herb solution ②: Take 0.5g of Bupleurum reference herb, add 50ml of water, decoct for 30 minutes, cool, filter, add water-saturated n-butanol to the filtrate and shake to extract twice, 30ml each time, combine the water-saturated n-butanol solutions, wash twice with ammonia solution, 20ml each time, take the water-saturated n-butanol solution and evaporate to dryness, add 1ml of methanol to dissolve the residue, as the reference herb solution ②.

[0070] II. Thin-layer chromatography identification

[0071] Take the above sample solution and spot it onto a high-efficiency silica gel pre-prepared plate (Yantai). Use a mixed solvent of ethyl acetate-ethanol-water (12:2:1, volume ratio) as the developing solvent. After development, spray with a 1% p-dimethylaminobenzaldehyde solution in 10% sulfuric acid ethanol, heat at 105℃ until the spots are clearly visible, and examine under ultraviolet light (365nm).

[0072] The results are as follows Figure 1 As shown, Figure 1 The samples for orbitals 1-9 are as follows:

[0073] 1. Mixed control (from top to bottom: saikosaponin d, saikosaponin a, saikosaponin B1 and saikosaponin B2);

[0074] 2. Bupleurum reference herb solution ② (decocted in water; National Institutes for Food and Drug Control (120992-201509));

[0075] 3. Xiaochaihu granules test solution ① (Company A);

[0076] 4. Xiaochaihu granules test solution ① (Company B);

[0077] 5. Xiaochaihu granules test solution ① (Company C);

[0078] 6. Xiaochaihu granules test solution ① (Company D);

[0079] 7. Xiaochaihu granules test solution ① (Company D);

[0080] 8. Test solution of Xiaochai Gui antipyretic granules for children ① (Company 1);

[0081] 9. Test solution of Xiaochai Gui antipyretic granules for children ① (Company 2).

[0082] The results above show that the four different saponins in the mixed control sample of track 1 could not be separated, and the developing system had difficulty separating the saponin components (saikosaponins a, d, B1 and B2) that were present in high concentrations in Bupleurum chinense.

[0083] Example 2

[0084] To address the problem in Example 1 above that conventional developing systems struggle to separate the high-content saponin components in Bupleurum chinense, we explored and optimized developing solvents, examining the development performance of various solvents. Some of the screening conditions are as follows:

[0085] Condition 1: Two-stage development method. First, develop with chloroform-methanol (4:1) as the developing solvent, remove and air dry, then develop again with chloroform-ethyl acetate-acetone-anhydrous formic acid-water (5:65:10:8:8) as the developing solvent. The chromatogram is shown below. Figure 2 As shown.

[0086] Condition 2: The chromatogram was developed using ethyl acetate-acetone-anhydrous formic acid-water (7:1:1:1) as the developing solvent. Figure 3 As shown.

[0087] Figure 3 The samples for orbitals 1-5 are as follows:

[0088] 1. Bupleurum negative control;

[0089] 2. Bupleurum reference herb solution ② (decocted in water; National Institutes for Food and Drug Control, batch number: 120992-201509);

[0090] 3-5. Test solution of Ruhusanjuan tablets ③ (Company I)

[0091] The chromatogram developed under the above conditions 1 is as follows: Figure 2 As shown, Figure 2The sample and orbit numbering in this example are the same as in Example 1. Figure 1 Same, from Figure 2 As can be seen, the four different saponins in the mixed reference sample of track 1 can be separated, showing good separation performance; while other conditions, such as condition 2, show poor separation performance. Figure 3 It can be seen that under these development conditions, only one spot of saikosaponin was visible in the Bupleurum reference material, indicating that the separation effect was still poor.

[0092] However, after optimizing the development system, it was found that the spots of the test samples containing Bupleurum chinense were poorly correlated with the spots of the Bupleurum chinense reference material (decocted in water), especially for Xiao Chai Hu granules. The characteristic spots of products from different companies varied greatly. Among them, the test sample from company A was consistent with the spots of saikosaponins B1 and B2. Figure 2 In the middle track 3), the three spots in the chromatograms of the test samples from companies B and D corresponded to saikosaponins a, B1, and B2. Figure 2 The chromatograms of the test sample from Company C (tracks 4, 6, and 7) were consistent with those of the reference standards for saikosaponin a and B2. Figure 2 The chromatogram of the reference material prepared according to the prescription process (decoction) was consistent with that of the reference materials for saikosaponin a and B2, but the spot of saikosaponin B1 was not observed. Figure 2 In the case of the middle track 2), the above situation can easily cause confusion and doubt among the inspectors regarding the judgment of the results.

[0093] Example 3

[0094] After repeated testing and comparison of various possible influencing factors and situations, the inventors have preliminarily discovered that the problem may be caused by the interaction between the ingredients during the decoction of traditional Chinese medicine.

[0095] Further research revealed that the acid-base conditions during the extraction of traditional Chinese medicine may have affected the saikosaponin components. Based on this, the inventors designed the following experiment to investigate the influence of extraction acid-base conditions on subsequent thin-layer chromatography.

[0096] 1. Establish thin-layer chromatograms of Bupleurum reference material obtained under different extraction conditions.

[0097] Preparation of Bupleurum reference herb solution ① (methanol method): Take 0.5g of Bupleurum reference herb, add 10ml of methanol, extract by ultrasonication for 30 minutes, cool, filter, evaporate the filtrate to dryness, dissolve the residue in 50ml of water-saturated n-butanol, wash twice with 20ml of ammonia test solution each time, evaporate the water-saturated n-butanol solution to dryness, dissolve the residue in 1ml of methanol, and use as reference herb solution ①.

[0098] Preparation of Bupleurum reference herb solution ② (water decoction method): Take 0.5g of Bupleurum reference herb, add 50ml of water, decoct for 30 minutes, cool, filter, add water-saturated n-butanol to the filtrate and shake to extract twice, 30ml each time, combine the water-saturated n-butanol solutions, wash twice with ammonia test solution, 20ml each time, take the water-saturated n-butanol solution and evaporate to dryness, add 1ml of methanol to dissolve the residue, as the reference herb solution ②.

[0099] Preparation of Bupleurum reference herb solution ③ (acid-water decoction method): Take three portions of Bupleurum reference herb, 0.5g each, add 50ml of water and 0.25ml, 0.75ml and 2ml of acetic acid respectively, the pH values ​​of the solutions are 3.7, 3.3 and 3.0 respectively, decoct for 30 minutes each, cool, filter, add water-saturated n-butanol to the filtrate and shake to extract twice, 30ml each time, combine the water-saturated n-butanol solutions, wash twice with ammonia test solution, 20ml each time, take the n-butanol solution and evaporate to dryness, add 1ml of methanol to dissolve the residue, as the reference herb solution ③.

[0100] The control herbal solutions prepared by the different methods described above were subjected to secondary development using the optimized developing solvent system of Example 2. The results are as follows: Figure 3 As shown.

[0101] Figure 4 The samples for orbitals 1-6 are as follows:

[0102] 1. Mixed control (from top to bottom: saikosaponin d, saikosaponin a, saikosaponin B1 and saikosaponin B2);

[0103] 2. Bupleurum reference herb solution ① (methanol extraction; National Institutes for Food and Drug Control, batch number: 120992-201509);

[0104] 3. Bupleurum reference herb solution ② (decocted in water; National Institutes for Food and Drug Control, batch number: 120992-201509);

[0105] 4. Bupleurum reference herb solution ③ (decocted with 0.25ml of acid and water; National Institutes for Food and Drug Control, batch number: 120992-201509);

[0106] 5. Bupleurum reference herb solution ③ (decocted with 0.75ml of acid and water; National Institutes for Food and Drug Control, batch number: 120992-201509);

[0107] 6. Bupleurum reference herb solution ③ (decocted with 2.0 ml of acid and water; National Institutes for Food and Drug Control, batch number: 120992-201509);

[0108] The results above show that the methanol extraction of Bupleurum reference material mainly contains saponins a and d (track 2) in the chromatogram. However, the water decoction extraction of Bupleurum reference material yields saponins a and B2 as the main spot components (track 3), indicating that saponin d can be converted to saponin B2 during the water decoction process. If a small amount of acetic acid is added to the Bupleurum reference material before water decoction extraction, the main spot components in the chromatogram are saponins B1 and B2 (tracks 4-6). This suggests that adding a small amount of acid can partially convert saponin a into saponin B1 (track 4, which shows a faint spot of saponin a above the B1 spot). When sufficient acid is added, all saponin a can be converted to saponin B1 (tracks 5-6).

[0109] Example 4

[0110] Based on the experimental results of Example 3, the strategy for identifying Bupleurum in traditional Chinese medicine is as follows: First, through preliminary experiments, the composition of the test sample to be identified is predicted, considering the form of saikosaponins present therein. Then, different and matching pretreatment methods are selected to obtain identification results that can accurately reflect the condition of the test sample and are easy for testing personnel to judge.

[0111] Specifically, the following methods can be used:

[0112] 1. Preliminary Experiment

[0113] Take an appropriate amount of the sample to be identified (sampled according to the pharmacopoeia requirements), add 50 ml of water, heat under reflux for 30 minutes, cool, filter, and extract twice with water-saturated n-butanol, 30 ml each time. Combine the water-saturated n-butanol solutions, wash twice with ammonia solution, 20 ml each time, separate the n-butanol solution, evaporate to dryness, and dissolve the residue in 1 ml of methanol to obtain the pre-test solution for the sample.

[0114] Perform the thin-layer chromatography test (General Rule 0502). Take an appropriate amount of the test solution and spot it on a silica gel thin-layer plate. Develop the plate using chloroform-methanol (4:1) as the developing solvent. Remove the plate and air dry it. Then develop the plate again using chloroform-ethyl acetate-acetone-anhydrous formic acid-water (5:65:10:8:8) as the developing solvent. Remove the plate and air dry it. Spray the plate with a 1% solution of 1% p-dimethylaminobenzaldehyde in 10% sulfuric acid ethanol. Heat the plate at 105°C until the spots are clearly visible. Examine the plate under ultraviolet light (365nm).

[0115] 2. Sample preparation

[0116] Based on the preliminary experimental results, if some batches of the test sample solutions show fluorescent spots of the same color at the positions of saponins a and B2, and some batches show fluorescent spots of the same color at the positions of saponins B1 and B2, then in the sample preparation step, both the test sample solution and the control herbal solution shall be prepared by acid-water decoction. If all batches of the test sample solutions show fluorescent spots of the same color at the positions of saponins B1 and B2, then in the sample preparation step, the test sample solution shall be prepared by methanol method, and the control herbal solution shall be prepared by acid-water decoction.

[0117] Preparation of test solution:

[0118] A. Methanol Method: Take an appropriate amount of the test sample, add 50 ml of methanol as a solvent, extract ultrasonically for 30 min, filter, take the filtrate and evaporate to dryness, dissolve the residue in 50 ml of water-saturated n-butanol, wash twice with 20 ml of ammonia test solution each time, take the water-saturated n-butanol solution, evaporate to dryness, add 1 ml of methanol to the residue to prepare the test sample solution.

[0119] B. Water decoction method: Take an appropriate amount of sample, add 50 ml of water, heat under reflux for 30 minutes, cool, filter, and extract twice with water-saturated n-butanol, 30 ml each time. Combine the n-butanol solutions, wash twice with ammonia solution, 20 ml each time, separate the n-butanol solution, evaporate to dryness, dissolve the residue in 1 ml of methanol, and use it as the test solution.

[0120] C. Acid-water decoction method: Take an appropriate amount of sample, add 50 ml of water and 0.25-2 ml of acetic acid, heat under reflux for 30 minutes, cool, filter, and extract twice with water-saturated n-butanol, 30 ml each time. Combine the n-butanol solutions, wash twice with ammonia solution, 20 ml each time, separate the n-butanol solution, evaporate to dryness, and dissolve the residue in 1 ml of methanol to obtain the test solution.

[0121] Preparation of Bupleurum reference herb solution:

[0122] A. Water decoction method: Take 0.5g of Bupleurum reference material, add 50ml of water, decoct for 30 minutes, cool, filter, add water-saturated n-butanol to the filtrate and shake to extract twice, 30ml each time, combine the n-butanol solutions, wash twice with ammonia solution, 20ml each time, take the n-butanol solution and evaporate to dryness, add 1ml of methanol to dissolve the residue, and use it as the reference material solution.

[0123] B. Acid-water decoction method: Take 0.5g of Bupleurum reference material, add 50ml of water and 0.25-2ml of acetic acid, decoct for 30 minutes, cool, filter, add water-saturated n-butanol to the filtrate and shake to extract twice, 30ml each time, combine the n-butanol solutions, wash twice with ammonia solution, 20ml each time, take the n-butanol solution and evaporate to dryness, add 1ml of methanol to dissolve the residue, and use it as the reference material solution.

[0124] 3. Thin-layer chromatography identification

[0125] Perform the thin-layer chromatography test (General Rule 0502). Take appropriate amounts of the test solution and the reference medicinal material solution and spot them on the same silica gel thin-layer plate. Develop the plate using chloroform-methanol (4:1) as the developing solvent. Remove the plate and air dry it. Then develop the plate again using chloroform-ethyl acetate-acetone-anhydrous formic acid-water (5:65:10:8:8) as the developing solvent. Remove the plate and air dry it. Spray the plate with a 1% solution of 1% p-dimethylaminobenzaldehyde in 10% sulfuric acid ethanol. Heat the plate at 105°C until the spots are clearly visible. Examine the plate under ultraviolet light (365nm).

[0126] Example 5

[0127] A method for identifying Bupleurum in Xiao Chai Hu granules includes the following steps:

[0128] 1. Preliminary Experiment

[0129] A preliminary experiment was conducted according to the method in Example 4 to obtain a sample solution of Xiaochaihu granules ①. It was found that the differences in extraction process conditions among different companies led to different degrees of conversion of saikosaponins.

[0130] 2. Sample preparation

[0131] Therefore, in this embodiment, acetic acid was added uniformly during sample extraction to maintain the pH of the sample solution at 3-4, so that samples from different companies could be extracted under the same conditions, and Bupleurum reference material was treated with the same acidic decoction conditions.

[0132] Preparation of test solution:

[0133] Take 5g of Xiaochaihu granules (specification (1)), 2g (specification (2)) or 1.25g (specification (3)), i.e., take half a bag of each according to different specifications, add 50ml of water and 1ml of acetic acid, the pH of the solution is 3.2, heat under reflux for 30 minutes, cool, filter, extract the filtrate with water-saturated n-butanol by shaking twice, 30ml each time, combine the water-saturated n-butanol solutions, continue to wash with ammonia test solution twice, 30ml each time, separate the water-saturated n-butanol solution, evaporate to dryness, add 1ml of methanol to dissolve the residue, and use it as the test solution ②.

[0134] Preparation of Bupleurum reference herb solution:

[0135] Take another 0.5g of Bupleurum reference material, add 50ml of water and 0.75ml of acetic acid, the pH of the solution is 3.3, decoct for 30 minutes, filter, and prepare the reference material solution from the filtrate by the same method as the test solution preparation method, starting from "extracted twice with water-saturated n-butanol".

[0136] Simultaneously, a control herbal solution without acetic acid was prepared in parallel for comparison.

[0137] 3. Thin-layer chromatography identification

[0138] Perform the thin-layer chromatography test (General Rule 0502). Take 2 μl of each of the two solutions mentioned above and spot them separately on the same silica gel G thin-layer plate (high-efficiency silica gel pre-made plate, Yantai). Use chloroform-methanol (4:1) as the developing solvent, develop, remove, dry with hot air and let cool. Then use chloroform-ethyl acetate-acetone-anhydrous formic acid-water (5:65:10:8:8) as the developing solvent, develop, remove and air dry.

[0139] Spray with a 1% solution of p-dimethylaminobenzaldehyde in 10% sulfuric acid ethanol, heat at 105℃ until the spots are clearly visible, and examine under ultraviolet light (365nm). In the chromatogram of the test sample, fluorescent spots of the same color appear at the positions corresponding to those in the chromatogram of the reference medicinal material.

[0140] The results are as follows Figure 5 As shown, Figure 5 The samples for orbitals 1-11 are as follows:

[0141] 1. Mixed control (from top to bottom: saikosaponin d, saikosaponin a, saikosaponin B1 and saikosaponin B2);

[0142] 2. Bupleurum reference herb solution ② (decocted in water; National Institutes for Food and Drug Control, batch number: 120992-201509);

[0143] 3. Xiaochaihu granules test solution ① (Company A); pre-experimental preparation without acid addition

[0144] 4. Xiaochaihu granules test solution ① (Company B); pre-experimental preparation without acid addition

[0145] 5. Xiaochaihu granules test solution ① (Company C); Preliminary experimental preparation without acid addition

[0146] 6. Xiaochaihu granules test solution ① (Company D); Preliminary experimental preparation without acid addition

[0147] 7. Bupleurum reference herb solution ③ (with 0.75 ml of acid added, National Institutes for Food and Drug Control (120992-201509));

[0148] 8. Xiaochaihu granules test solution ② (Company A); prepared by acid decoction.

[0149] 9. Xiaochaihu granules test solution ② (Company B); prepared by acid decoction.

[0150] 10. Xiaochaihu granules test solution ② (Company C); prepared by acid decoction.

[0151] 11. Xiaochaihu granules test solution ② (Company D); prepared by acid decoction.

[0152] The above results indicate that by conducting preliminary experiments to predict the composition of the test sample, and then treating the Bupleurum reference material and the test sample under the same acidic decoction conditions, the chromatogram of the test sample was found to be completely consistent with the spots of the Bupleurum reference material obtained under the same extraction conditions. This results accurately reflect the condition of the test sample and are easy for testing personnel to judge.

[0153] Example 6

[0154] A method for identifying Bupleurum chinense in breast nodule powder includes the following steps:

[0155] 1. Preliminary Experiment

[0156] A preliminary experiment was conducted following the method described in Example 4:

[0157] Preparation of mixed reference solution: Take appropriate amounts of saikosaponin B1 and B2 reference standards, dissolve them in methanol, and prepare a reference solution containing 0.5 mg per 1 ml.

[0158] Preparation of Bupleurum reference herb solution: Take 0.5g of Bupleurum reference herb, add 50ml of water, decoct for 30 minutes, cool, filter, add water-saturated n-butanol to the filtrate and shake to extract twice, 30ml each time, combine the n-butanol solutions, wash twice with ammonia solution, 20ml each time, take the n-butanol solution and evaporate to dryness, add 1ml of methanol to dissolve the residue, and use as the reference herb solution.

[0159] Preparation of test solution: Take 30 tablets of this product, remove the coating from sugar-coated tablets, grind them into a fine powder, add 50 ml of methanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 50 ml of water-saturated n-butanol to dissolve the residue, wash twice with 20 ml of ammonia solution each time, evaporate the n-butanol solution to dryness, add 1 ml of methanol to dissolve the residue, and use this as the test solution for the preliminary experiment ③.

[0160] The results are as follows Figure 6 As shown, Figure 6 The samples for orbitals 1-7 are as follows:

[0161] 1. Bupleurum reference herb solution ② (decocted in water; National Institutes for Food and Drug Control, batch number: 120992-201509);

[0162] 2. Saikosaponin B1 (Shanghai Yuanye Biotechnology Co., Ltd., batch number: A22GB146239);

[0163] 3. Saikosaponin B2 (Shanghai Yuanye Biotechnology Co., Ltd., batch number: MO5GB140794);

[0164] 4-7. Test solution ③ (Company I);

[0165] A discrepancy was found between the chromatogram of the test sample and the chromatogram of the Bupleurum reference herb (decocted in water). The main saponin components in the chromatogram of the Bupleurum reference herb were saikosaponin a and B2 (track 1), while the main saikosaponin components in the chromatogram of the test sample were saikosaponin B1 and B2 (tracks 4-7).

[0166] 2. Sample preparation

[0167] In the experiment, the pH of the aqueous solution obtained after the concentration of the compound extract of Ruhe Sanjie Pian was determined to be approximately 5.0, which is acidic. Therefore, when preparing the Bupleurum reference material, the thin-layer chromatography identification method of Bupleurum in Example 5 was also referred to. When preparing the Bupleurum reference material, an appropriate amount of acetic acid was added to make its pH 5.0, and the extraction was carried out by acid decoction.

[0168] Preparation of Bupleurum reference herb solution: Take 0.5g of Bupleurum reference herb, add 50ml of water and 0.75ml of acetic acid to make the pH 5.0, decoct for 30 minutes, cool, filter, add water-saturated n-butanol to the filtrate and shake to extract twice, 30ml each time, combine the n-butanol extracts, wash twice with ammonia solution, 20ml each time, take the n-butanol extract and evaporate to dryness, add 1ml of methanol to dissolve the residue, and use as the reference herb solution.

[0169] Preparation of test solution: Take 30 tablets of this product, remove the coating from sugar-coated tablets, grind them into a fine powder, add 50 ml of methanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 50 ml of water-saturated n-butanol to dissolve the residue, wash twice with 20 ml of ammonia solution each time, evaporate the n-butanol solution to dryness, add 1 ml of methanol to dissolve the residue, and use this as test solution ③ (i.e., prepared by methanol method).

[0170] 3. Thin-layer chromatography identification

[0171] Perform the thin-layer chromatography test (General Rule 0502). Take 2 μl of each of the two solutions mentioned above and spot them separately on the same silica gel G thin-layer plate (high-efficiency silica gel pre-made plate, Yantai). Use chloroform-methanol (4:1) as the developing solvent, develop, remove, dry with hot air and let cool. Then use chloroform-ethyl acetate-acetone-anhydrous formic acid-water (5:65:10:8:8) as the developing solvent, develop, remove and air dry.

[0172] Spray with a 1% solution of p-dimethylaminobenzaldehyde in 10% sulfuric acid ethanol, heat at 105℃ until the spots are clearly visible, and examine under ultraviolet light (365nm). In the chromatogram of the test sample, fluorescent spots of the same color appear at the positions corresponding to those in the chromatogram of the reference medicinal material.

[0173] The results are as follows Figure 7 As shown, Figure 7 The samples for orbitals 1-7 are as follows:

[0174] 1. Bupleurum reference herb solution ③ (decocted with acid water; National Institutes for Food and Drug Control, batch number: 120992-201509);

[0175] 2-6. Test solution ③ (Company I);

[0176] 7. Bupleurum negative control (provided by Company I).

[0177] The above results show that by conducting preliminary experiments to predict the composition of the test sample, and then treating the Bupleurum reference material and the test sample under the same acidic decoction conditions, the chromatogram of the test sample was found to be completely consistent with the spots of the Bupleurum reference material obtained under the same extraction conditions. This results accurately reflect the condition of the test sample and are easy for testing personnel to judge.

[0178] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0179] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A thin layer chromatography method for identifying Bupleurum in Chinese patent medicines, characterized in that, Comprising the following steps: Sample preparation: taking the sample to be tested, and the saikosaponin control and the bupleurum control drug material respectively, and preparing the test solution, and the control solution and the control drug material solution, wherein the saikosaponin control is selected from the group consisting of saikosaponin a, saikosaponin d, saikosaponin B1 and saikosaponin B2; Thin layer chromatography identification: taking the test solution, the control solution and the control drug material solution, and spotting them on silica gel thin layer plates, first using chloroform-methanol (4:1 by volume) as the developing agent, developing, taking out, and air-drying, and then using chloroform-ethyl acetate-acetone-anhydrous formic acid-water (5:65:10:8:8 by volume) as the developing agent, developing, and coloring, and then obtaining the result; the coloring condition is: spraying with 1% p-dimethylaminobenzaldehyde in 10% sulfuric acid ethanol solution, heating at 105°C until the spots are clearly colored, and observing under ultraviolet light at 365nm.

2. The method according to claim 1, wherein the method is characterized in that, In the sample preparation step, the test solution preparation method is as follows: Methanol method: taking the test sample, adding methanol as the solvent for ultrasonic extraction, filtering, taking the filtrate, evaporating to dryness, dissolving the residue in water-saturated n-butanol, washing with ammonia solution, taking the water-saturated n-butanol solution, evaporating to dryness, and adding methanol to the residue to obtain the test solution; or Water decoction method: taking the test sample, adding water as the solvent for refluxing, filtering, taking the filtrate, extracting with water-saturated n-butanol, washing with ammonia solution, taking the water-saturated n-butanol solution, evaporating to dryness, and adding methanol to the residue to obtain the test solution; or Acid water decoction method: taking the test sample, adding water as the solvent and simultaneously adding acetic acid for refluxing, filtering, taking the filtrate, evaporating to dryness, dissolving the residue in water-saturated n-butanol, washing with ammonia solution, taking the water-saturated n-butanol solution, evaporating to dryness, and adding methanol to the residue to obtain the test solution; The control drug material solution preparation method is as follows: Water decoction method: taking the bupleurum control drug material, adding water as the solvent for decoction, filtering, taking the filtrate, extracting with water-saturated n-butanol, washing with ammonia solution, taking the water-saturated n-butanol solution, evaporating to dryness, and adding methanol to the residue to obtain the control drug material solution; or Acid water decoction method: taking the bupleurum control drug material, adding water as the solvent and simultaneously adding acetic acid for decoction, filtering, taking the filtrate, evaporating to dryness, dissolving the residue in water-saturated n-butanol, washing with ammonia solution, taking the water-saturated n-butanol solution, evaporating to dryness, and adding methanol to the residue to obtain the control drug material solution.

3. The method according to claim 2, wherein the method is characterized in that, In the sample preparation step, the test solution preparation method is as follows: Methanol method: taking a predetermined amount of test sample, adding 50±10ml of methanol as the solvent for ultrasonic extraction for 30±10min, filtering, taking the filtrate, evaporating to dryness, dissolving the residue in 50±10ml of water-saturated n-butanol, washing with ammonia solution 2±1 times, each time 20±5ml, taking the water-saturated n-butanol solution, evaporating to dryness, and adding 1±0.5ml of methanol to the residue to obtain the test solution; or Water decoction method: take the predetermined amount of test product, add water 50±10ml as solvent, reflux 30±10min, filter, take the filtrate add water saturated n-butanol extraction, each 30±10ml, combined water saturated n-butanol layer, with ammonia test solution washing, 2±1 times, each 20±5ml, take water saturated n-butanol solution, dry, residue add methanol 1±0.5ml, get test product solution; Or Acid water decoction method: take the predetermined amount of test product, add water 50±10ml as solvent, and at the same time add acetic acid 0.25-2ml, reflux 30±10min, filter, take the filtrate add water saturated n-butanol extraction, each 30±10ml, combined water saturated n-butanol layer, with ammonia test solution washing, 2±1 times, each 20±5ml, take water saturated n-butanol solution, dry, residue add methanol 1±0.5ml, get test product solution; The preparation method of the control medicinal material solution is as follows: Water decoction method: take 0.5g of radix bupleuri control medicinal material, add water 50±10ml as solvent, decoct 30±10min, filter, take the filtrate add water saturated n-butanol extraction, each 30±10ml, combined water saturated n-butanol layer, with ammonia test solution washing, 2±1 times, each 20±5ml, take water saturated n-butanol solution, dry, residue add methanol 1±0.5ml, get control medicinal material solution; or Acid water decoction method: take 0.5g of radix bupleuri control medicinal material, add water 50±10ml as solvent, and at the same time add acetic acid 0.25-2ml, decoct 30±10min, filter, take the filtrate add water saturated n-butanol extraction, each 30±10ml, combined water saturated n-butanol layer, with ammonia test solution washing, 2±1 times, each 20±5ml, take water saturated n-butanol solution, dry, residue add methanol 1±0.5ml, get control medicinal material solution.

4. The method according to claim 2 or 3, wherein the method is characterized in that, Before the sample preparation step, a pre-experiment step is further included, and the pre-experiment step is as follows: Take the sample to be tested, and prepare the test product pre-experiment solution according to the following method: take the test product to be identified, add water 50ml, heat and reflux for 30 minutes, cool, filter, shake with water saturated n-butanol for extraction 2 times, each 30ml, wash with ammonia test solution 2 times, each 20ml, separate the n-butanol liquid, dry, add methanol 1ml to dissolve, as the test product pre-experiment solution; Then, identify according to the thin layer chromatography identification step, and observe the spot conditions. If some batches of test product pre-experiment solution present the same color fluorescent spots at the positions of saponin a and B2, and some batches present the same color fluorescent spots at the positions of saponin B1 and B2, then in the sample preparation step, the test product solution and the control medicinal material solution are both prepared by the acid water decoction method. If all batches of test product pre-experiment solution present the same color fluorescent spots at the positions of saponin B1 and B2, then in the sample preparation step, the test product solution is prepared by the methanol method, and the control medicinal material solution is prepared by the acid water decoction method.

5. The method according to claim 4, wherein the method is characterized in that, In the acid decoction method, acetic acid is added to make the pH value of the test sample solution and the control medicinal material solution the same as the lowest pH value of the pre-experiment solution of each batch of test sample in the pre-experiment step.

6. The thin layer chromatography method of any one of claims 1-4 for identifying Chinese patent medicines containing Bupleurum.

7. Use according to claim 6, characterized in that: The Chinese patent medicines include Xiaochaihu Granules, Ruhn Scattering Knot Tablets, Cold Clearing Heat Granules, and Infantile Chai Gui Fever-Relieving Granules.

8. Use according to claim 7, characterized in that: The Chinese patent medicine is Xiaochaihu Granules, and the test sample solution and the control medicinal material solution are prepared by the acid decoction method. The Chinese patent medicine is Ruhn Scattering Knot Tablets, the test sample solution is prepared by the methanol method, and the control medicinal material solution is prepared by the acid decoction method.

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