Thin layer chromatography detection method of Chinese medicine preparation for treating mammary gland hyperplasia

Abstract

The application relates to the technical field of traditional Chinese medicine detection, in particular to a thin-layer chromatography detection method of a traditional Chinese medicine preparation for treating mammary gland hyperplasia, which is a rannuclear mass-resolving tablet. The thin-layer chromatography detection method comprises extracting the rannuclear mass-resolving tablet by using dichloromethane to prepare filter residue and a first sample solution, which is used for comparison with angelica sinensis; extracting the filter residue by using n-butanol to prepare a second sample solution, which is used for comparison with epimedium; and extracting the rannuclear mass-resolving tablet by using methanol to prepare a third sample solution, which is used for comparison with astragalus and epimedium. The method simplifies operation steps, makes up for the defect that some medicines cannot be identified, enriches detection information, has strong speciality, is simple in operation, and has wide adaptability.

Description

Technical Field

[0001] This application relates to the field of traditional Chinese medicine detection technology, and in particular to a thin-layer chromatography detection method for a traditional Chinese medicine preparation for treating breast hyperplasia, wherein the traditional Chinese medicine preparation for treating breast hyperplasia is Ruhe Sanjie tablets. Background Technology

[0002] Ruhe Sanjie Tablets are composed of ten herbs: Bupleurum, Angelica sinensis, Astragalus membranaceus, Curcuma longa, Gynostemma pentaphyllum, Echinops latifolius, Laminaria japonica, Sargassum fusiforme, Epimedium brevicornu, and Pyrola rotundifolia. They have the effects of soothing the liver and relieving depression, softening and dispersing nodules, regulating qi and promoting blood circulation. They are used to treat cystic hyperplasia of the breast, mastalgia, fibroadenoma of the breast, and male breast development.

[0003] The current standard for Ruhe Sanjie tablets is included in Part I of the 2020 edition of the Chinese Pharmacopoeia. It specifies the thin-layer chromatographic identification of Angelica sinensis, Epimedium and Astragaloside A (Astragalus membranaceus) and the determination of Epimedoside content. However, it does not limit the detection of other medicinal ingredients. The standard is not perfect and comprehensive enough. In addition, the detection steps are cumbersome and increase the difficulty of detection.

[0004] A method for identifying Bupleurum chinense in traditional Chinese medicine preparations and its application has been described. The traditional Chinese medicine preparations include Xiao Chai Hu granules, Ru He San Jie tablets, Ganmao Qingre granules, and Xiao Er Chai Gui Tui Re granules. The method includes the following steps: Sample preparation: Take the sample to be tested, as well as Bupleurum saponin reference standards and / or Bupleurum reference medicinal materials, and prepare test solution, reference solution, and / or reference medicinal material solution respectively; Thin-layer chromatography identification: Take the above sample solutions and spot them onto a silica gel thin-layer plate. First, develop with a 4:1 (v / v) chloroform-methanol mixture, remove and dry, then develop again with a 5:65:10:8:8 (v / v) chloroform-ethyl acetate-acetone-formic acid-water mixture, and develop the color. The above thin-layer chromatography method for identifying Bupleurum chinense in traditional Chinese medicine preparations, through a two-stage development process, can achieve complete separation of the four saponin components Bupleurum chinense: saponins a, d, B1, and B2, enabling accurate identification of traditional Chinese medicine preparations containing Bupleurum chinense. However, this method can only identify Bupleurum in traditional Chinese medicine, and the detection steps are relatively complicated.

[0005] Therefore, it is necessary to further optimize the detection method of Ruhe Sanjie tablets, simplify the detection steps, and evaluate more medicinal ingredients to make the detection results more comprehensive and objective. Summary of the Invention

[0006] Based on this, the purpose of this application is to provide a thin-layer chromatography detection method for a traditional Chinese medicine preparation for treating breast hyperplasia—Ruhe Sanjie tablets. This method is simple to operate, highly specific, and can accurately identify Angelica sinensis, Astragalus membranaceus, Epimedium brevicornu, and Pyrola rotundifolia, and has wide applicability.

[0007] Specifically, the thin-layer chromatography detection method includes the following steps:

[0008] Dichloromethane was used to extract the agglomerated emulsion cores to prepare filter residue and the first test solution;

[0009] The filter residue was extracted with n-butanol to prepare a second test solution.

[0010] Separately, extract the milk nucleus fragments with methanol to prepare the third test solution;

[0011] Angelica sinensis reference material was extracted with dichloromethane to prepare Angelica sinensis reference solution;

[0012] Pyrola rotundifolia and Epimedium brevicornu reference materials were extracted with water to prepare Pyrola rotundifolia reference solution and Epimedium brevicornu reference solution, respectively.

[0013] Take astragaloside A reference standard, dissolve it in a solvent to prepare astragaloside A reference standard solution;

[0014] The first test solution and the Angelica sinensis reference solution were subjected to thin-layer chromatography on the same silica gel G stationary phase.

[0015] The second test solution and the Pyrola rotundifolia reference solution were subjected to thin-layer chromatography on the same silica gel G stationary phase.

[0016] The third test solution, the epimedium reference solution, and the astragaloside A reference solution were subjected to thin-layer chromatography on the same silica gel G stationary phase.

[0017] In one embodiment, the extraction of the milk nucleus agglomerates using dichloromethane includes: mixing the milk nucleus agglomerates with the dichloromethane, and sonicating; and / or

[0018] Extraction of the filter residue using n-butanol comprises: mixing the filter residue after dichloromethane extraction of the milk core and agglomerated flakes with the n-butanol, followed by ultrasonication; and / or

[0019] Extracting another batch of the breast nucleus agglomerates with methanol involves: mixing the methanol from the separately taken breast nucleus agglomerates with methanol and then sonicating.

[0020] In one embodiment, the frequency of the ultrasound is 40kHz~50kHz and the power is 300W~500W; and / or

[0021] The ultrasound session lasted 20 to 40 minutes.

[0022] In one embodiment, the thin-layer chromatography detection of the first test solution and the Angelica sinensis reference solution on the same silica gel G stationary phase includes:

[0023] Take the first test solution and the Angelica sinensis reference solution, spot them separately onto the same silica gel G stationary phase, develop them in the first developing solvent, remove them, air dry them, and examine them.

[0024] The first developing solvent includes n-hexane and ethyl acetate.

[0025] In one embodiment, the volume ratio of n-hexane to ethyl acetate in the first developing solvent is (8~10):1; and / or

[0026] The inspection includes observing fluorescent spots under a 365nm ultraviolet lamp.

[0027] In one embodiment, the thin-layer chromatography detection of the second test solution and the *Pyrola rotundifolia* reference solution on the same silica gel G stationary phase includes:

[0028] Take the second test solution and the Pyrola rotundi reference solution, spot them separately onto the same silica gel G stationary phase, develop them in the second developing solvent, remove them, air dry them, and examine them.

[0029] The second developing solvent includes chloroform, ethyl acetate, methanol, and formic acid.

[0030] In one embodiment, the volume ratio of the chloroform, ethyl acetate, methanol, and formic acid in the second developing agent is (28~32):10:(14~16):(1~2); and / or

[0031] The inspection includes observing the spots under sunlight.

[0032] In one embodiment, the thin-layer chromatography detection of the third test solution, the epimedium reference solution, and the astragaloside A reference solution on the same silica gel G stationary phase includes:

[0033] Take the third test solution, the epimedium reference solution and the astragaloside A reference solution, spot them on the same silica gel G stationary phase, develop them in the third developing solvent, remove them, air dry them, develop them in the fourth developing solvent, remove them, air dry them, develop the color and examine them.

[0034] The third developing agent includes chloroform, ethyl acetate, methanol, and water;

[0035] The fourth developing agent includes chloroform, methanol, anhydrous formic acid, and water.

[0036] In one embodiment, the volume ratio of the chloroform, ethyl acetate, methanol, and water in the third developing agent is 10:(18~22):(10~12):(4~6); and / or

[0037] In the fourth developing agent, the volume ratio of chloroform, methanol, anhydrous formic acid, and water is (3~5):1:(0.1~0.2):(0.1~0.2); and / or

[0038] The color development includes spraying with an ethanol solution of sulfuric acid at a volume ratio of 8% to 12%, followed by heating at 100°C to 110°C; and / or

[0039] The inspection includes observing fluorescent spots under a 365nm ultraviolet lamp.

[0040] This application also relates to the application of the thin-layer chromatography detection method described in any of the above technical solutions in the qualitative identification of breast nucleus agglomerates.

[0041] The thin-layer chromatography detection method for Ruhe Sanjie tablets, a traditional Chinese medicine preparation for treating breast hyperplasia, proposed in this application simplifies the detection steps, optimizes the detection scheme, makes up for the deficiency that some medicinal materials cannot be identified, enriches the detection information, has strong specificity, is simple to operate, and has wide applicability. Attached Figure Description

[0042] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0043] Figure 1 The thin-layer chromatography results are shown for Angelica sinensis in Ruhe Sanjie tablets according to an embodiment of this application.

[0044] Figure 2 The results of a robustness study of the thin-layer chromatography method for detecting Angelica sinensis in Ruhe Sanjie tablets according to an embodiment of this application;

[0045] Figure 3 The thin-layer chromatography results are shown for the Epimedium and Astragalus medicinal materials in Ruhe Sanjie tablets according to an embodiment of this application.

[0046] Figure 4 The results of a robustness study of the thin-layer chromatography detection method for Epimedium and Astragalus in Ruhe Sanjie tablets according to an embodiment of this application;

[0047] Figure 5 The results of thin-layer chromatography analysis of the herbal medicine *Pyrola rotundifolia* in *Ruhe Sanjie Pian*, an embodiment of this application, are shown below.

[0048] Figure 6 The results of a robustness study of the thin-layer chromatography method for detecting deer antler velvet in Ruhe Sanjie tablets according to an embodiment of this application;

[0049] Figure 7 The results of thin-layer chromatography analysis of Angelica sinensis in the one-to-one ratio of Ruhe Sanjie tablets in this application;

[0050] Figure 8 The results of thin-layer chromatography analysis of a single ratio of Epimedium and Astragalus membranaceus in this application are shown.

[0051] Figure 9 The results of thin-layer chromatography analysis of a proportion of *Pyrola rotundifolia* herb in this application are shown. Detailed Implementation

[0052] The present application is further described below with reference to the embodiments, examples, and accompanying drawings. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the application. Furthermore, it should be understood that after reading the teachings of this application, those skilled in the art can make various alterations or modifications to the application, and these equivalent forms also fall within the protection scope of the appended claims.

[0053] 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 application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0054] the term:

[0055] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0056] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations encompass any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that, in this application, the technical solution undoubtedly includes solutions connected by "logical AND," and also undoubtedly includes solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0057] In this document, terms such as "preferred," "better," and "more preferred" are merely descriptions of implementation methods or examples that achieve better results, and should be understood as not constituting a limitation on the scope of protection of this application.

[0058] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0059] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0060] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, optional numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0061] In this application, weight can be a well-known unit of mass in the chemical industry, such as μg, mg, g, or kg.

[0062] In this application, "%" represents a percentage, referring to the proportion by weight; however, the percentage of a solution, unless otherwise specified, refers to the amount of solute contained in 100 mL of the solution; the percentage of alcohol refers to the proportion by volume at 20°C. Furthermore, the following symbols may be used as needed: %(g / g) indicates the amount of solute contained in 100 g of solution; %(mL / mL) indicates the amount of solute contained in 100 mL of solution; %(mL / g) indicates the amount of solute contained in 100 g of solution; %(g / mL) indicates the amount of solute contained in 100 mL of solution.

[0063] In one aspect of this application, a thin-layer chromatography method is provided for the detection of Ruhe Sanjie tablets, a traditional Chinese medicine preparation for treating breast hyperplasia. Compared with traditional techniques, this method simplifies the detection steps, optimizes the detection scheme, compensates for the inability to identify some medicinal materials, enriches the detection information, has strong specificity, is simple to operate, and has wide applicability.

[0064] In one embodiment, the thin-layer chromatography detection method includes the following steps:

[0065] S100: Preparation of test solution

[0066] Dichloromethane was used to extract the agglomerated emulsion cores to prepare filter residue and the first test solution;

[0067] The filter residue was extracted with n-butanol to prepare the second test solution;

[0068] Separately, extract the milk nucleus fragments with methanol to prepare the third test solution;

[0069] S200: Preparation of reference solution

[0070] Angelica sinensis reference material was extracted with dichloromethane to prepare Angelica sinensis reference solution;

[0071] Pyrola rotundifolia and Epimedium brevicornu reference materials were extracted with water to prepare Pyrola rotundifolia reference solution and Epimedium brevicornu reference solution, respectively.

[0072] Take astragaloside A reference standard, dissolve it in a solvent to prepare astragaloside A reference standard solution;

[0073] S300: Perform thin-layer chromatography detection

[0074] The first test solution and the Angelica sinensis reference solution were subjected to thin-layer chromatography on the same silica gel G stationary phase.

[0075] The second test solution and the Pyrola rotundifolia reference solution were subjected to thin-layer chromatography on the same silica gel G stationary phase.

[0076] The third test solution, epimedium reference solution, and astragaloside A reference solution were subjected to thin-layer chromatography on the same silica gel G stationary phase.

[0077] S100 Preparation of the test solution:

[0078] This application improves the preparation method of the test sample, and the resulting filtrate and residue can be used to compare different medicinal materials, thus optimizing the detection steps and saving detection costs.

[0079] In one embodiment, dichloromethane was used to extract the agglomerated emulsion core to obtain filter residue and a first test solution;

[0080] The filter residue was extracted with n-butanol to prepare the second test solution.

[0081] Furthermore, the extraction of the milk nucleus agglomerates using dichloromethane includes: mixing the milk nucleus agglomerates with dichloromethane and then sonicating.

[0082] Further, the extraction of the filter residue with n-butanol includes: mixing the filter residue after extracting the milk core and agglomerated flakes with dichloromethane with n-butanol, and then sonicating.

[0083] In one embodiment, a third test solution was prepared by extracting the milk nucleus fragments with methanol.

[0084] Further, extracting the remaining milk nucleus fragments with methanol involves mixing the remaining milk nucleus fragments with methanol and then sonicating.

[0085] In one embodiment, the frequency of the ultrasound is 40KHz~50KHz and the power is 300W~500W.

[0086] Furthermore, the ultrasound duration is 20 to 40 minutes.

[0087] Preparation of reference solution using S200:

[0088] In this application, in step S200, Angelica sinensis reference material is taken, extracted with dichloromethane, and Angelica sinensis reference solution is prepared;

[0089] Pyrola rotundifolia and Epimedium brevicornu reference materials were extracted with water to prepare Pyrola rotundifolia reference solution and Epimedium brevicornu reference solution, respectively.

[0090] Take astragaloside A reference standard, dissolve it in a solvent to prepare astragaloside A reference standard solution.

[0091] S300 for thin-layer chromatography detection:

[0092] In this application, in step S300, the first test solution and the Angelica sinensis reference solution are subjected to thin-layer chromatography detection on the same silica gel G stationary phase;

[0093] The second test solution and the Pyrola rotundifolia reference solution were subjected to thin-layer chromatography on the same silica gel G stationary phase.

[0094] The third test solution, epimedium reference solution, and astragaloside A reference solution were subjected to thin-layer chromatography on the same silica gel G stationary phase.

[0095] In one embodiment, the thin-layer chromatography detection of the first test solution and the Angelica sinensis reference solution on the same silica gel G stationary phase includes: taking the first test solution and the Angelica sinensis reference solution, spotting them separately on the same silica gel G stationary phase, developing them in the first developing solvent, taking them out, air-drying them, and inspecting them;

[0096] Furthermore, the first developing solvent includes n-hexane and ethyl acetate;

[0097] Further examination included observing fluorescent spots under a 365nm ultraviolet lamp.

[0098] Preferably, in the first developing solvent, the volume ratio of n-hexane to ethyl acetate is (8~10):1.

[0099] In one embodiment, the thin-layer chromatography detection of the second test solution and the Pyrola rotundifolia reference solution on the same silica gel G stationary phase includes: taking the second test solution and the Pyrola rotundifolia reference solution, spotting them separately on the same silica gel G stationary phase, developing them in the second developing solvent, removing them, air-drying them, and inspecting them;

[0100] Furthermore, the second developing solvent includes chloroform, ethyl acetate, methanol, and formic acid;

[0101] Further examination includes observing the spots under sunlight.

[0102] Preferably, in the second developing solvent, the volume ratio of chloroform, ethyl acetate, methanol and formic acid is (28~32):10:(14~16):(1~2).

[0103] In one embodiment, the thin-layer chromatography detection of the third test solution, epimedium reference solution, and astragaloside A reference solution on the same silica gel G stationary phase includes: taking the third test solution, epimedium reference solution, and astragaloside A reference solution, spotting them separately on the same silica gel G stationary phase, developing them in the third developing solvent, removing them, air-drying them, developing them again in the fourth developing solvent, removing them, air-drying them, developing the color, and examining them;

[0104] Furthermore, the third developing solvent includes chloroform, ethyl acetate, methanol, and water;

[0105] Furthermore, the fourth developing agent includes chloroform, methanol, anhydrous formic acid, and water;

[0106] Furthermore, the color development involves spraying an ethanol solution of sulfuric acid at a volume ratio of 8% to 12% and heating it at 100°C to 110°C.

[0107] Further examination included observing fluorescent spots under a 365nm ultraviolet lamp.

[0108] Preferably, in the third developing solvent, the volume ratio of chloroform, ethyl acetate, methanol and water is 10:(18~22):(10~12):(4~6).

[0109] Preferably, in the fourth developing solvent, the volume ratio of chloroform, methanol, anhydrous formic acid and water is (3~5):1:(0.1~0.2):(0.1~0.2).

[0110] This application also relates to the application of the thin-layer chromatography detection method for Ruhu Sanjie tablets described in any of the above technical solutions in the qualitative identification of Ruhu Sanjie tablets.

[0111] The following are some specific examples.

[0112] For experimental parameters not specified in the following specific embodiments, please refer to the guidelines given in this application document first, or refer to experimental manuals or other experimental methods known in the art, or refer to the experimental conditions recommended by the manufacturer.

[0113] The raw materials and reagents involved in the following specific embodiments can be obtained commercially or prepared by those skilled in the art using known methods.

[0114] The samples of Ruhe Sanjie Tablets provided by Guangzhou Baiyunshan Zhongyi Pharmaceutical Co., Ltd. are for the purpose of improving national drug standards. The batch numbers are: A00006, Z00015, Z00029, C00001, B00021, and B00029.

[0115] 1. Identification of Angelica sinensis, Epimedium, Astragalus membranaceus, and Pyrola rotundifolia in Ruhe Sanjie tablets (Example)

[0116] 1.1 Identification of Angelica sinensis

[0117] 1.1.1 Raw materials and supplies

[0118] Samples: Angelica sinensis reference material (120927-202118, National Institutes for Food and Drug Control); Ruhe Sanjie tablets (batch numbers: C00001, B00021, B00029); Angelica sinensis negative control (provided by Guangzhou Baiyunshan Zhongyi Pharmaceutical Co., Ltd.);

[0119] Thin-layer plates: High-efficiency silicone G prefabricated thin-layer plates (Yantai); Silicone prefabricated thin-layer plates (MN).

[0120] 1.1.2 Detection and Identification

[0121] (1) Preparation of test solution: Take 30 tablets of this product, remove the coating of sugar-coated tablets, grind them into a fine powder, add 50 ml of dichloromethane, sonicate for 30 minutes, filter, keep the filter residue for later use, evaporate the filtrate to dryness, add 0.5 ml of methanol to dissolve the residue, and use it as the test solution.

[0122] (2) Preparation of reference medicinal material solution: Take 0.5g of Angelica sinensis reference medicinal material, add 30ml of dichloromethane, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 1ml of methanol to dissolve the residue, and use it as reference medicinal material solution.

[0123] (3) Development system: Take 10 μl of the test solution and 2 μl of the reference drug solution and spot them on the same silica gel G thin layer plate. Use n-hexane-ethyl acetate (9:1) as the developing solvent, develop, remove and air dry.

[0124] (4) Examination: Examine under ultraviolet light (365nm). See the thin-layer chromatogram below. Figure 1 , Figure 1In the table, 1 represents the reference material of Angelica sinensis (120927-202118, National Institutes for Food and Drug Control); 2-7 represent the test samples (batch numbers: A00006, Z00015, Z00029, C00001, B00021, B00029); and 8 represents the negative control of Angelica sinensis (provided by Guangzhou Baiyunshan Zhongyi Pharmaceutical Co., Ltd.). The results showed good correspondence between the main spots of the test samples and the reference material chromatograms, with no interference from the negative control.

[0125] 1.1.3 Durability Assessment

[0126] The results of the durability test can be found in Figure 2 , Figure 2 In the table, 1 is the reference material of Angelica sinensis (120927-202118, National Institutes for Food and Drug Control); 2-7 are the test samples (batch numbers: A00006, Z00015, Z00029, C00001, B00021, B00029); and 8 is the negative control of Angelica sinensis (provided by Guangzhou Baiyunshan Zhongyi Pharmaceutical Co., Ltd.).

[0127] Under the same temperature and humidity conditions (20℃~25℃, relative humidity 45%~55%), tests were conducted using different types of silicone rubber sheets. Figure 2 Image 2A shows the test results for the Yantai high-efficiency silicone G precast panel. Figure 2 The test results for MN silicone prefabricated plate in Figure 2B show that both types of thin-layer plates meet the requirements for identification.

[0128] Tests were conducted under the same thin-layer plate and temperature conditions (Yantai high-efficiency silica gel G prefabricated thin-layer plate, 20℃~25℃) but different relative humidity conditions. Figure 2 The result for 2C is based on a relative humidity of 32%. Figure 2 The results in 2D are from a relative humidity of 72%. The comparison shows that different relative humidity conditions have little impact on the thin-layer chromatography effect.

[0129] Tests were conducted under the same thin-layer plate and relative humidity conditions (Yantai high-efficiency silica gel G prefabricated thin-layer plate, 65%~75%), but under different relative temperature conditions. Figure 2 The results for 2D are from a test conducted at 24℃. Figure 2 The results in 2E are from the detection at 7℃. The comparison shows that the development under different relative temperatures has little effect on the thin-layer chromatography effect.

[0130] 1.2 Identification of Epimedium and Astragalus

[0131] 1.2.1 Raw materials and materials

[0132] Samples: Astragaloside A reference standard (110781-202118, National Institutes for Food and Drug Control); Astragalus membranaceus (Mongolian Astragalus) reference medicinal material (120974-201813, National Institutes for Food and Drug Control); Epimedium reference medicinal material (121632-201502, National Institutes for Food and Drug Control); Ruhe Sanjie tablets (batch numbers: C00001, B00021, B00029); Astragalus membranaceus negative control (provided by Guangzhou Baiyunshan Zhongyi Pharmaceutical Co., Ltd.); Epimedium negative control (provided by Guangzhou Baiyunshan Zhongyi Pharmaceutical Co., Ltd.).

[0133] Thin-layer board: High-efficiency silicone G prefabricated thin-layer board (Yantai).

[0134] 1.2.2 Detection and Identification

[0135] (1) Preparation of test solution: Take 30 tablets of this product, remove the coating of 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 test solution each time, evaporate the n-butanol solution to dryness, add 1 ml of methanol to dissolve the residue, and use it as the test solution.

[0136] (2) Preparation of the reference herb solution: Take 1g of Epimedium reference herb, add 50ml of water, decoct for 30 minutes, filter, extract the filtrate twice with water-saturated n-butanol, 30ml each time, combine the n-butanol extracts, wash with 20ml of ammonia solution, evaporate the n-butanol extract to dryness, dissolve the residue in 1ml of methanol, and use as the reference herb solution. Separately, take astragaloside A reference standard, add methanol to prepare a solution containing 1mg per 1ml, and use as the reference standard solution.

[0137] (3) Development system: Take 2 μl each of the test solution and the above-mentioned reference medicinal material solution and reference solution, and spot them on the same silica gel G thin layer plate. Use the lower layer solution of chloroform-ethyl acetate-methanol-water (10:20:11:5) that has been left at below 10°C overnight as the developing solvent, develop, remove, and air dry. Then use chloroform-methanol-anhydrous formic acid-water (8:2:0.3:0.3) as the developing solvent, develop, remove, and air dry.

[0138] (4) Inspection: Spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly visible, and examine under ultraviolet light (365nm). The thin-layer chromatogram is shown below. Figure 3 Medium 3A, visual inspection under daylight, thin-layer chromatogram (see [reference image]). Figure 3 3B. Figure 3In the sample, 1 is astragaloside A reference standard (110781-202118, National Institutes for Food and Drug Control); 2 is Epimedium reference material (121632-201502, National Institutes for Food and Drug Control); 3-7 are test samples (batch numbers: A00006, Z00015, C00001, B00021, B00029); 8 is Astragalus negative control (provided by Guangzhou Baiyunshan Zhongyi Pharmaceutical Co., Ltd.); 9 is Epimedium negative control (provided by Guangzhou Baiyunshan Zhongyi Pharmaceutical Co., Ltd.). The results showed good correspondence between the chromatograms of the test samples and the chromatograms of Epimedium and astragaloside A reference standards, with no interference from either the Epimedium negative control or the Astragalus negative control.

[0139] 1.2.3 Durability Assessment

[0140] The results of the durability test are as follows Figure 4 , Figure 4 Among them, 1 is astragaloside A reference standard (110781-202118, National Institutes for Food and Drug Control); 2 is epimedium reference medicinal material (121632-201502, National Institutes for Food and Drug Control); 3-7 are test samples (batch numbers: A00006, Z00015, C00001, B00021, B00029); 8 is astragalus negative control (provided by Guangzhou Baiyunshan Zhongyi Pharmaceutical Co., Ltd.); 9 is epimedium negative control (provided by Guangzhou Baiyunshan Zhongyi Pharmaceutical Co., Ltd.).

[0141] Under the same temperature and humidity conditions (20℃~25℃, relative humidity 45%~55%), tests were conducted using different types of silicone rubber sheets. Figure 4 4A-1 and 4A-2 are the test results for Merck silicone 60 precast panels. Figure 4 The results of tests on Yantai high-efficiency silicone G precast panels are shown in 4B-1 and 4B-2. The comparison shows that both types of thin-layer panels can meet the identification requirements.

[0142] Tests were conducted under the same thin-layer plate and temperature conditions (Yantai high-efficiency silica gel G prefabricated thin-layer plate, 20℃~25℃) but different relative humidity conditions. Figure 4 The results for 4C-1 and 4C-2 are based on a relative humidity of 32%. Figure 4 The results for 4D-1 and 4D-2 are from a relative humidity of 72%. The comparison shows that different relative humidity conditions have little impact on the thin-layer chromatography effect.

[0143] Tests were conducted under the same thin-layer plate and relative humidity conditions (Yantai high-efficiency silica gel G prefabricated thin-layer plate, 65%~75%), but under different relative temperature conditions. Figure 4 The results for 4D-1 and 4D-2 are from tests conducted at 24℃. Figure 4 The results for 4E-1 and 4E-2 are from the detection at 6℃. The comparison shows that the development under different relative temperatures has little effect on the thin-layer chromatography effect.

[0144] 1.3 Identification of Pyrola rotundifolia

[0145] 1.3.1 Raw materials and materials

[0146] Samples: Pyrola rotundifolia negative control (provided by Guangzhou Baiyunshan Zhongyi Pharmaceutical Co., Ltd.); test samples (batch numbers: C00001, B00021, B00029); Pyrola rotundifolia reference material (121211-201202, National Institutes for Food and Drug Control).

[0147] Thin-layer plates: Silicone 60 prefabricated thin-layer plates (Merck); High-efficiency silicone G prefabricated thin-layer plates (Yantai).

[0148] 1.3.2 Detection and Identification

[0149] (1) Preparation of test solution: Take 30 tablets of this product, remove the coating of sugar-coated tablets, grind them into a fine powder, add 50 ml of dichloromethane, sonicate for 30 minutes, filter, add 50 ml of n-butanol to the filter residue, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 1 ml of methanol to dissolve the residue, and use it as the test solution.

[0150] (2) Preparation of reference herb solution: Take 1g of Pyrola rotundifolia reference herb, add 50ml of water, decoct for 30 minutes, filter, extract the filtrate twice with water-saturated 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 reference herb solution.

[0151] (3) Development system: Take 15 μl of each of the above two solutions and spot them on the same silica gel G thin layer plate. Use chloroform-ethyl acetate-methanol-formic acid (30:10:15:1.5) as the developing solvent, develop, remove and air dry.

[0152] (4) Inspection: Spray with 5% vanillin-sulfuric acid solution and heat at 105℃ until the spots are clearly visible. See the thin-layer chromatogram. Figure 5 . Figure 5 In the chromatograms, 1 is the negative control of *Pyrola rotundifolia* (provided by Guangzhou Baiyunshan Zhongyi Pharmaceutical Co., Ltd.); 2-4 are the test samples (batch numbers: C00001, B00021, B00029); and 5 is the reference material of *Pyrola rotundifolia* (121211-201202, National Institutes for Food and Drug Control). The results showed that the chromatograms of the test samples showed a single purplish-red main spot at the same position as the chromatograms of the reference material of *Pyrola rotundifolia*, while the negative control of *Pyrola rotundifolia* showed no interference.

[0153] 1.3.3 Durability Assessment

[0154] The results of the durability test are as follows Figure 6 , Figure 6In the table, 1 is a negative control of *Pyrola rotundifolia* (provided by Guangzhou Baiyunshan Zhongyi Pharmaceutical Co., Ltd.); 2-4 are test samples (batch numbers: C00001, B00021, B00029); and 5 is the reference material of *Pyrola rotundifolia* (121211-201202, National Institutes for Food and Drug Control). Detection was performed using different types of silica gel plates under the same temperature and humidity conditions (20℃~25℃, relative humidity 65%~75%). Figure 4 The result for Merck silicone 60 prefabricated board is shown in section 6A. Figure 6 The result of the test for Yantai high-efficiency silicone G precast board is shown in Figure 6B. The comparison shows that both types of thin-layer boards can meet the requirements for identification.

[0155] Tests were conducted under the same thin-layer plate and temperature conditions (Yantai high-efficiency silica gel G prefabricated thin-layer plate, 20℃~25℃) but different relative humidity conditions. Figure 6 The result for 6B is based on a relative humidity of 70%. Figure 6 The result for 6C is based on a relative humidity of 32%. The comparison shows that different relative humidity conditions have little impact on the thin-layer chromatography effect.

[0156] Tests were conducted under the same thin-layer plate and relative humidity conditions (Yantai high-efficiency silica gel G prefabricated thin-layer plate, 65%~75%), but under different relative temperature conditions. Figure 6 The result in 6B is the test result at 24℃. Figure 6 The results for 6D are from the test at 6℃. The comparison shows that different relative temperatures have little effect on the thin-layer chromatography effect.

[0157] 2. Identification of Angelica sinensis, Epimedium, and Astragalus membranaceus in Ruhe Sanjie tablets using other methods (comparative example)

[0158] 2.1 Identification of Angelica sinensis

[0159] 2.1.1 Raw materials and supplies

[0160] Samples: Angelica sinensis reference material (120927-202118, National Institutes for Food and Drug Control); Ruhe Sanjie tablets (batch numbers: C00001, B00021, B00029); Angelica sinensis negative control (provided by Guangzhou Baiyunshan Zhongyi Pharmaceutical Co., Ltd.);

[0161] Thin-layer plates: High-efficiency silicone G prefabricated thin-layer plates (Yantai); Silicone prefabricated thin-layer plates (MN).

[0162] 2.1.2 Detection and Identification

[0163] (1) Preparation of test solution: Take 20 tablets of this product, remove the coating, grind finely, add 30ml of petroleum ether (60-90℃), sonicate for 20 minutes, filter, keep the filter residue for later use, concentrate the filtrate to about 0.5ml, and use it as the test solution.

[0164] (2) Preparation of reference medicinal material solution: Take 0.5g of Angelica sinensis reference medicinal material, add 15ml of petroleum ether (60-90℃), sonicate for 20 minutes, filter, concentrate the filtrate to about 1ml, and use it as reference medicinal material solution.

[0165] (3) Development system: Take 10 μl of the test solution and 1 μl of the reference drug solution and spot them on the same silica gel G thin layer plate. Use n-hexane-ethyl acetate (9:1) as the developing solvent, develop, remove and air dry.

[0166] (4) Examination: Examine under ultraviolet light (365nm). See the thin-layer chromatogram. Figure 7 . Figure 7 In the table, 1 represents the reference material of Angelica sinensis (120927-202118, National Institutes for Food and Drug Control); 2-4 represent the test samples (batch numbers: C00001, B00021, B00029); and 5 represents the negative control of Angelica sinensis (provided by Guangzhou Baiyunshan Zhongyi Pharmaceutical Co., Ltd.). The results showed that the main spots of the test samples and the reference material chromatograms corresponded well, and there was no interference from the negative control.

[0167] 2.2 Identification of Epimedium and Astragalus membranaceus medicinal materials

[0168] 2.2.1 Raw materials and supplies

[0169] Samples: Astragaloside A reference standard (110781-202118, National Institutes for Food and Drug Control); Astragalus membranaceus (Mongolian Astragalus) reference medicinal material (120974-201813, National Institutes for Food and Drug Control); Epimedium reference medicinal material (121632-201502, National Institutes for Food and Drug Control); Ruhe Sanjie tablets (batch numbers: C00001, B00021, B00029); Astragalus membranaceus negative control (provided by Guangzhou Baiyunshan Zhongyi Pharmaceutical Co., Ltd.); Epimedium negative control (provided by Guangzhou Baiyunshan Zhongyi Pharmaceutical Co., Ltd.).

[0170] Thin-layer board: High-efficiency silicone G prefabricated thin-layer board (Yantai).

[0171] 2.2.2 Detection and Identification

[0172] (1) Preparation of test solution: Take 30 tablets of this product, remove the coating, grind finely, add 50 ml of methanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 30 ml of water to dissolve the residue, take the supernatant and place it in a separatory funnel, add water-saturated n-butanol and shake to extract twice, 40 ml each time, combine the n-butanol liquids, evaporate to dryness, add 2 ml of methanol to dissolve the residue, add 1 g of polyamide, mix well, place on a water bath to evaporate the methanol, add to a polyamide column (100-200 mesh, 5 g, inner diameter 2 cm), elute with 150 ml of water, discard the aqueous liquid, elute again with 150 ml of 30% ethanol, collect the eluent, evaporate to dryness, dissolve in 20 ml of water-saturated n-butanol, wash twice with 1% potassium hydroxide solution, 10 ml each time, discard the alkali solution, evaporate the n-butanol liquid to dryness, add 1 ml of methanol to dissolve the residue, and use as the test solution.

[0173] (2) Preparation of the reference herb solution: Take 2g of Epimedium reference herb, add 100ml of water, decoct for 0.5 hours, filter, concentrate the filtrate to about 20ml, extract twice with water-saturated n-butanol, 20ml each time, combine the n-butanol extracts, wash twice with 1% potassium hydroxide solution, 10ml each time, discard the alkali, evaporate the n-butanol extract to dryness, dissolve the residue in 1ml of methanol, and use as the reference herb solution. Take astragaloside A reference standard, add methanol to prepare a solution containing 1mg per 1ml, and use as the reference standard solution.

[0174] (3) Development system: chloroform-ethyl acetate-methanol-water (10:20:11:5) The lower layer solution placed below 10℃ is used as the developing solvent. Develop to 12cm, remove and air dry.

[0175] (4) Inspection: Spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly visible, and examine under ultraviolet light (365nm). The thin-layer chromatogram is shown below. Figure 8 8A, visual inspection under sunlight, thin-layer chromatogram (see [image]). Figure 8 8B. Although this image can achieve good separation results, the preprocessing is too complex, which greatly affects the detection efficiency. Figure 8 Among them, 1 is astragaloside A reference standard (110781-202118, National Institutes for Food and Drug Control); 2 is Astragalus membranaceus (Mongolian Astragalus) reference medicinal material (120974-201813, National Institutes for Food and Drug Control); 3 is Epimedium reference medicinal material (121632-201502, National Institutes for Food and Drug Control); 4-6 are test samples (batch numbers: C00001, B00021, B00029); 7 is Astragalus membranaceus negative control (provided by Guangzhou Baiyunshan Zhongyi Pharmaceutical Co., Ltd.); 8 is Epimedium negative control (provided by Guangzhou Baiyunshan Zhongyi Pharmaceutical Co., Ltd.).

[0176] 2.3 Raw materials and supplies

[0177] Samples: Pyrola rotundifolia negative control (provided by Guangzhou Baiyunshan Zhongyi Pharmaceutical Co., Ltd.); test sample (batch number: C00001); Pyrola rotundifolia reference material (121211-201202, National Institutes for Food and Drug Control).

[0178] Thin-layer plates: Silicone 60 prefabricated thin-layer plates (Merck); High-efficiency silicone G prefabricated thin-layer plates (Yantai).

[0179] 2.3.2 Detection and Identification

[0180] (1) Preparation of test solution: Take 20 tablets of this product, remove the coating, grind finely, add 30 ml of petroleum ether (60-90℃), sonicate for 20 minutes, filter, add 50 ml of n-butanol to the filter residue, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 1 ml of methanol to dissolve the residue, and use it as the test solution.

[0181] (2) Preparation of reference herb solution: Take 1g of Pyrola rotundifolia reference herb, add 50ml of water, decoct for 30 minutes, filter, extract the filtrate twice with water-saturated 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 reference herb solution.

[0182] (3) Development system: Take 15 μl of each of the above two solutions and spot them on the same silica gel G thin layer plate. Use chloroform-ethyl acetate-methanol-formic acid (30:10:15:1.5) as the developing solvent, develop, remove and air dry.

[0183] (4) Inspection: Spray with 5% vanillin-sulfuric acid solution and heat at 105℃ until the spots are clearly visible. See the thin-layer chromatogram. Figure 9 . Figure 9 In the sample, 1 is the reference material of *Pyrola rotundifolia* (121211-201202, National Institutes for Food and Drug Control), 2 is the reference standard of crystalline glycoside, 3 is the negative control of *Pyrola rotundifolia* (provided by Guangzhou Baiyunshan Zhongyi Pharmaceutical Co., Ltd.), and 4 is the test sample (batch number: C00001). The results showed that the chromatogram of the test sample did not show the corresponding purplish-red main spot as the reference material of *Pyrola rotundifolia*, nor did it contain the marker component crystalline glycoside found in *Pyrola rotundifolia*, thus making identification impossible.

[0184] All references to this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the purpose and / or technical solution of this application, all cited references are incorporated herein by reference in their entirety and for all purposes. When references are cited in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0185] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples 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.

[0186] The embodiments described above merely illustrate several implementation methods of this application and should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Furthermore, it should be understood that after reading the above teachings, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the protection scope of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the protection scope of the appended claims. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A thin-layer chromatography detection method for a traditional Chinese medicine preparation for treating breast hyperplasia, wherein the traditional Chinese medicine preparation for treating breast hyperplasia is Ruhe Sanjie tablets, characterized in that, Includes the following steps: Mix the milk nucleus dispersing tablets with dichloromethane and sonicate to obtain filter residue and the first test solution; The filter residue was mixed with n-butanol and sonicated to prepare a second test solution. Separately, mix the milk nucleus dispersing tablets with methanol and sonicate to prepare the third test solution; Angelica sinensis reference material was extracted with dichloromethane to prepare Angelica sinensis reference solution; Pyrola rotundifolia and Epimedium brevicornu reference materials were extracted with water to prepare Pyrola rotundifolia reference solution and Epimedium brevicornu reference solution, respectively. Take astragaloside A reference standard, dissolve it in a solvent to prepare astragaloside A reference standard solution; The first test solution and the Angelica sinensis reference solution were subjected to thin-layer chromatography detection on the same silica gel G stationary phase, including: taking the first test solution and the Angelica sinensis reference solution, spotting them separately on the same silica gel G stationary phase, developing them in the first developing solvent, removing them, air-drying them, and inspecting them; the first developing solvent was hexane and ethyl acetate in a volume ratio of (8~10):1, and the inspection was performed by observing the fluorescent spots under a UV lamp at 365nm; The second test solution and the *Pyrola rotundifolia* reference solution were subjected to thin-layer chromatography on the same silica gel G stationary phase, including: taking the second test solution and the *Pyrola rotundifolia* reference solution, spotting them separately on the same silica gel G stationary phase, developing them in the second developing solvent, removing them, air-drying them, developing the color, and examining them; the second developing solvent was a mixture of chloroform, ethyl acetate, methanol, and formic acid in a volume ratio of (28~32):10:(14~16):(1~2); the color development was performed by spraying with 5% vanillin-sulfuric acid solution and heating at 105℃; the examination was performed by observing the spots under sunlight; The third test solution, the epimedium reference solution, and the astragaloside A reference solution were subjected to thin-layer chromatography detection on the same silica gel G stationary phase, including: taking the third test solution, the epimedium reference solution, and the astragaloside A reference solution, spotting them separately on the same silica gel G stationary phase, developing them in the third developing solvent, removing them, air-drying them, developing them again in the fourth developing solvent, removing them, air-drying them, developing the color, and examining them; the third developing solvent was a mixture of chloroform, ethyl acetate, methanol, and water in a volume ratio of 10:(18~22):(10~12):(4~6); the fourth developing solvent was a mixture of chloroform, methanol, anhydrous formic acid, and water in a volume ratio of (3~5):1:(0.1~0.2):(0.1~0.2); the color development was performed by spraying with an ethanol solution of sulfuric acid in a volume ratio of 8%~12% and heating at 100℃~110℃; the examination was performed by observing the fluorescent spots under a UV lamp at 365nm.

2. The thin-layer chromatography detection method as described in claim 1, characterized in that, The frequency of the ultrasound is 40KHz~50KHz and the power is 300W~500W.

3. The thin-layer chromatography detection method according to any one of claims 1-2, characterized in that, The ultrasound session lasted 20 to 40 minutes.

4. The application of the thin-layer chromatography detection method according to any one of claims 1-3 in the qualitative identification of milk nucleus agglomerates.

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