Thin-layer chromatography identification method of Xipai gingival fixing liquid and application thereof

Abstract

The invention discloses a thin-layer chromatography identification method of Xipayi gingival fixing liquid and application of the thin-layer chromatography identification method. The thin-layer chromatography identification method comprises the following steps: 1) preparing a reference substance solution; (2) dripping the reference substance solution and the test solution on the same polyamide thin-layer plate, developing by using a developing solvent, and inspecting to obtain a thin-layer chromatogram; (3) in the thin-layer chromatogram, the test solution displays spots with the same color at the spot positions of the reference solution; wherein the test solution is a Xipayi gingival fixing liquid sample; the reference substance solution is selected from any one or more of gallic acid or 1, 3, 6-trigalloylglucose. The method is free of complex sample pretreatment, simple, convenient and rapid to operate, high in specificity and good in reproducibility, and a reliable and efficient identification method is provided for quality control of the Xipayi gingival fixing liquid preparation.

Description

Technical Field

[0001] This invention relates to the field of thin-layer chromatography identification of traditional Chinese medicine preparations, and more specifically, to a thin-layer chromatography identification method for Xipai Guyin Liquid and its application. Background Technology

[0002] Xipay Gums-Strengthening Liquid is a preparation made from gallnut as the main ingredient, with saccharin, peppermint oil, and sodium benzoate as excipients. This preparation has the functions of strengthening teeth and gums, clearing blood stasis, and relieving pain. It is commonly used to treat tooth sensitivity, difficulty chewing, loosening and displacement of teeth, bleeding gums, mouth ulcers, sore throat, bad breath, and tobacco odor caused by periodontal disease.

[0003] Existing thin-layer chromatography (TLC) identification methods are ineffective at separating multiple components in Sipay gingival syrup, exhibiting spot tailing or low resolution, failing to accurately identify multiple components, and involving cumbersome sample pretreatment requiring multiple steps, which is time-consuming and labor-intensive. Therefore, developing a more efficient, accurate, and convenient TLC identification technique has become a common pursuit and an urgent technical challenge in the industry. Summary of the Invention

[0004] The main objective of this invention is to provide a thin-layer chromatography identification method for Sipay gingival solution and its application, so as to solve the problem of poor separation in the existing thin-layer chromatography identification method for Sipay gingival solution.

[0005] To achieve the above objectives, according to a first aspect of the present invention, a thin-layer chromatography (TLC) identification method for Sipay gingival solution is provided. The TLC identification method includes: 1) preparing a reference solution; 2) spotting the reference solution and the test solution onto the same polyamide TLC plate, developing them with a developing solvent, and examining the plate to obtain a TLC chromatogram; 3) in the TLC chromatogram, the test solution shows a spot of the same color at the same location as the spot in the reference solution; wherein the test solution is a Sipay gingival solution sample; and the reference solution is selected from any one or more of the following: gallic acid or 1,3,6-trigalloglucoside.

[0006] Furthermore, the developing solvent is selected from glacial acetic acid, methanol, ethyl acetate, and formic acid.

[0007] Furthermore, the volume ratio of the above-mentioned glacial acetic acid: methanol: ethyl acetate: formic acid is (8~10): (6~10): (2~3): (1~2).

[0008] Furthermore, the sample volume of the above-mentioned test solution is 1 μL to 5 μL.

[0009] Furthermore, the concentration of the above reference solution is 0.5-1.5 mg / mL; the sample volume of the above reference solution is 1 μL-5 μL.

[0010] Furthermore, the temperature range for the above-mentioned expansion is 4℃~40℃;

[0011] Preferably, the humidity during the above-mentioned unfolding is 18% to 88%.

[0012] Furthermore, the above inspection is performed using ultraviolet light or sunlight;

[0013] Preferably, the wavelength of the ultraviolet lamp is 254nm or 366nm.

[0014] Furthermore, the above-mentioned inspection is performed using sunlight, and before the inspection, the polyamide thin film is developed with a color developer.

[0015] Furthermore, the colorimetric reagent mentioned above is selected from ferric chloride ethanol solution;

[0016] Preferably, the mass concentration of the above-mentioned ferric chloride ethanol solution is 1% to 3%.

[0017] To achieve the above objectives, according to a second aspect of the present invention, the application of the above-described thin-layer chromatography identification method in identifying or evaluating the quality of Sipai gingival solution is provided.

[0018] Applying the technical solution of this invention, polyamide thin-layer chromatography (TLC) plates are used to identify Sipay gingival syrup samples. Compared with other types of TLC plates, the amide groups on the surface of the polyamide TLC plate can form hydrogen bonds with Sipay gingival syrup, resulting in better selectivity, clear separation of multiple components, improved resolution, and advantages of economy and reproducibility. This provides a reliable basis for the identification and quality evaluation of Sipay gingival syrup products. Attached Figure Description

[0019] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0020] Figure 1The diagram shows the extraction phases (n-butanol phase) and finished product samples of the silica gel GF254 thin-layer plate using different extractants according to Method 1 of Embodiment 1 of the present invention; wherein, a represents the extraction phase (n-butanol phase) of the finished product of Xipay gingival solution extracted with n-butanol, with a sample volume of 1µL; b represents the extraction phase (n-butanol phase) of the finished product of Xipay gingival solution extracted with n-butanol, with a sample volume of 0.5µL; c represents the extraction phase (ethyl acetate phase) of the finished product of Xipay gingival solution extracted with ethyl acetate, with a sample volume of 1µL; d represents the extraction phase (ethyl acetate phase) of the finished product of Xipay gingival solution extracted with ethyl acetate, with a sample volume of 0.5µL; e represents gallic acid; f represents the raffinate phase (aqueous phase) of the finished product of Xipay gingival solution extracted with n-butanol, with a sample volume of 3µL; g represents the finished product of Xipay gingival solution, with a sample volume of 3µL.

[0021] Figure 2 The diagram shows the extraction phase (n-butanol phase) and the finished sample based on the polyamide film according to Method 2 of Embodiment 1 of the present invention; Figure 2 In this diagram, A represents the inspection view under 254nm ultraviolet light before color development. Figure 2 In this diagram, B represents the inspection view under 366nm ultraviolet light before color development. Figure 2 In the diagram, C represents the inspection view under sunlight after color development; among A, B, and C, a represents the finished product of Sipaye gingival solution, with a sample volume of 3µL; b represents gallic acid; c represents 1,3,6-trigalloglucoside; d represents the extract phase (n-butanol phase) of the finished product of Sipaye gingival solution extracted with n-butanol, with a sample volume of 3µL; and e represents the raffinate phase (aqueous phase) of the finished product of Sipaye gingival solution extracted with n-butanol, with a sample volume of 3µL.

[0022] Figure 3 The image shows an inspection view of a silica gel GF254 thin-layer plate developed by method three according to embodiment 1 of the present invention, using glacial acetic acid: methanol: ethyl acetate: formic acid = 10:6:2:1 (volume ratio, mL:mL:mL:mL) as the developing solvent. Figure 3 In this diagram, A represents the inspection view under 254nm ultraviolet light before color development. Figure 3 In the diagram, B represents the inspection view under sunlight after color development; among A and B, a represents the finished product of Sipay gingival solution, with a sample volume of 3µL; b represents di-bisgallic acid; c represents gallic acid; d represents 1-galloyl glucose; e represents 1,3,6-trigalloyl glucose; f represents 1,2,3,6-tetragalloyl glucose; g represents 1,2,3,4,6-pentagalloyl glucose; h represents ellagic acid; i represents the extract phase (n-butanol phase) of the finished product of Sipay gingival solution extracted with n-butanol, with a sample volume of 3µL; j represents the raffinate phase (aqueous phase) of the finished product of Sipay gingival solution extracted with n-butanol, with a sample volume of 3µL.

[0023] Figure 4 An inspection view of a polyamide film based on method four of embodiment 1 of the present invention, using glacial acetic acid: methanol: ethyl acetate: formic acid = 10:6:2:1 (volume ratio, mL:mL:mL:mL) as the developing solvent, is shown. Figure 4 In this diagram, A represents the inspection view under 254nm ultraviolet light before color development. Figure 4 In this diagram, B represents the inspection view under 366nm ultraviolet light before color development. Figure 4 In the diagram, C represents the inspection view under sunlight after color development; among A, B, and C, a represents the finished product of Sipaye gingival solution, with a sample volume of 3µL; b represents gallic acid; c represents 1,3,6-trigalloglucoside; d represents the extract phase (n-butanol phase) of the finished product of Sipaye gingival solution extracted with n-butanol, with a sample volume of 3µL; and e represents the raffinate phase (aqueous phase) of the finished product of Sipaye gingival solution extracted with n-butanol, with a sample volume of 3µL.

[0024] Figure 5 The diagram shows a view of a polyamide film using different spreading agents according to method five of embodiment 1 of the present invention; Figure 5 In the diagram, A indicates that the developing solvent is glacial acetic acid:methanol:ethyl acetate:formic acid = 10:6:2:1 (volume ratio, mL:mL:mL:mL). Specifically, a represents the finished product of Sipaye gingival solution, with a sample volume of 3 µL; b represents gallic acid; c represents 1,3,6-trigalloglucoside; d represents the extract phase (n-butanol phase) of the finished product of Sipaye gingival solution extracted with n-butanol, with a sample volume of 3 µL; and e represents the raffinate phase (aqueous phase) of the finished product of Sipaye gingival solution extracted with n-butanol, with a sample volume of 3 µL.

[0025] Figure 5 In the figure, B indicates that the developing solvent is glacial acetic acid: methanol: n-octane: formic acid = 10:6:2:1 (volume ratio, mL:mL:mL:mL:mL); where a represents the extract phase (n-butanol phase) of the finished product of Sipay gingival solution extracted with n-butanol, with a sample volume of 3µL; b represents gallic acid, with a sample volume of 2µL; c represents 1,3,6-trigalloglucoside, with a sample volume of 2µL; and d represents the raffinate phase (aqueous phase) of the finished product of Sipay gingival solution extracted with n-butanol, with a sample volume of 3µL.

[0026] Figure 6 A view showing the effect of methanol dosage on thin-layer chromatography identification in Example 2 of the present invention is shown, wherein A represents the developing solvent being glacial acetic acid:methanol:ethyl acetate:formic acid = 10:6:2:1 (volume ratio, mL:mL:mL:mL); in A, a represents the finished product of Sipay gingival solution; b represents gallic acid; c represents 1,3,6-trigalloglucoside; d represents the extract phase (n-butanol phase) of the finished product of Sipay gingival solution extracted with n-butanol; and e represents the raffinate phase (aqueous phase) of the finished product of Sipay gingival solution extracted with n-butanol.

[0027] Wherein, B indicates that the developing solvent is glacial acetic acid:methanol:ethyl acetate:formic acid = 10:8:2:1; D indicates that the developing solvent is glacial acetic acid:methanol:ethyl acetate:formic acid = 10:10:2:1 (volume ratio, mL:mL:mL:mL); in B and D, a represents the extract phase (n-butanol phase) of the finished Sipay gingival solution extracted with n-butanol; b represents gallic acid; c represents 1,3,6-trigalloglucoside; d represents the raffinate phase (aqueous phase) of the finished Sipay gingival solution extracted with n-butanol.

[0028] Wherein, C indicates that the developing solvent is glacial acetic acid: methanol: ethyl acetate: formic acid = 10:9:2:1 (volume ratio, mL:mL:mL:mL); in C, a represents the extract phase (n-butanol phase) of the finished Sipay gingival solution extracted with n-butanol; b represents 1-galloyl glucose; c represents 1,2,3,6-tetragalloyl glucose; d represents corilagin; and e represents the raffinate phase (aqueous phase) of the finished Sipay gingival solution extracted with n-butanol.

[0029] Figure 7 A view showing the effect of ethyl acetate dosage on thin-layer chromatography identification in Example 2 of the present invention is shown; wherein, A indicates that the developing solvent is glacial acetic acid:methanol:ethyl acetate:formic acid = 10:8:2:1 (volume ratio, mL:mL:mL:mL); in A, a represents the extract phase (n-butanol phase) of the Sipaye gingival solution product extracted with n-butanol; b represents gallic acid; c represents 1,3,6-trigalloglucoside; d represents the raffinate phase (aqueous phase) of the Sipaye gingival solution product extracted with n-butanol.

[0030] Wherein, B indicates that the developing solvent is glacial acetic acid: methanol: ethyl acetate: formic acid = 10:8:3:1 (volume ratio, mL:mL:mL:mL); in B, a represents the extract phase (n-butanol phase) of the finished Sipay gingival solution extracted with n-butanol; b represents 1-galloyl glucose; c represents 1,2,3,6-tetragalloyl glucose; d represents corilagin; and e represents the raffinate phase (aqueous phase) of the finished Sipay gingival solution extracted with n-butanol.

[0031] Figure 8The following is a view showing the effect of different amounts of glacial acetic acid on thin-layer chromatography identification in Example 2 of the present invention; wherein, A represents the developing solvent being glacial acetic acid:methanol:ethyl acetate:formic acid = 10:8:2:1 (volume ratio, mL:mL:mL:mL); B represents the developing solvent being glacial acetic acid:methanol:ethyl acetate:formic acid = 8:8:2:1 (volume ratio, mL:mL:mL:mL); wherein, in A and B, a represents the extract phase (n-butanol phase) of the Sipaye gingival solution product extracted with n-butanol; b represents gallic acid; c represents 1,3,6-trigalloglucoside; d represents the raffinate phase (aqueous phase) of the Sipaye gingival solution product extracted with n-butanol.

[0032] Figure 9 A view showing the effect of different amounts of formic acid on thin-layer chromatography identification in Example 2 of the present invention is shown.

[0033] Where A represents the developing solvent as glacial acetic acid:methanol:ethyl acetate:formic acid = 10:8:2:1 (volume ratio, mL:mL:mL:mL); 'a' in A represents the extract phase (n-butanol phase) of the Sipaye gingival solution product extracted with n-butanol; 'b' represents gallic acid; 'c' represents 1,3,6-trigalloglucoside; and 'd' represents the raffinate phase (aqueous phase) of the Sipaye gingival solution product extracted with n-butanol.

[0034] B indicates that the developing solvent is glacial acetic acid:methanol:ethyl acetate:formic acid = 10:8:2:2 (volume ratio, mL:mL:mL:mL); in B, a represents the extract phase (n-butanol phase) of the finished Sipay gingival solution extracted with n-butanol; b represents 1-galloyl glucose; c represents 1,2,3,6-tetragalloyl glucose; d represents corilagin; e represents the raffinate phase (aqueous phase) of the finished Sipay gingival solution extracted with n-butanol.

[0035] Figure 10 The following is a view showing the effect of different spotting amounts on thin-layer chromatography identification according to Example 3.1 of the present invention; wherein, A represents the finished product of Sipay gingival solution with different spotting amounts (batch number 250405); B represents the finished product of Sipay gingival solution with different spotting amounts (batch number 220408); and C represents the view of the finished product of Sipay gingival solution with different spotting amounts (batch number 210608).

[0036] Figure 11The following is a view showing the effect of polyamide films from different manufacturers on thin-layer chromatography identification in Example 3.2 of the present invention, where A represents manufacturer Taizhou, Zhejiang; B represents manufacturer Xinsirui, Wuhan; and C represents manufacturer Sinopharm Group. In A, B, and C, a represents the finished product of Xipayi Gingival Solution (batch number 250412); b represents gallic acid; c represents 1,3,6-trigalloglucoside; and d represents the finished product of Xipayi Gingival Solution (batch number 250412).

[0037] Figure 12 The following is a view showing the effect of different humidity levels on thin-layer chromatography identification according to Example 3.3 of the present invention; wherein, A represents a humidity of 18%, B represents a humidity of 32%, C represents a humidity of 58%, and D represents a humidity of 88%; wherein, in A, B, C, and D, a represents the finished product of Sipay gingival solution (batch number 250412); b represents gallic acid; c represents 1,3,6-trigalloglucoside; and d represents the finished product of Sipay gingival solution (batch number 250412).

[0038] Figure 13 The following is a view showing the effect of different temperatures on thin-layer chromatography identification in Example 3.4 of the present invention; wherein, A represents a temperature of 40°C, B represents a temperature of 4°C, and C represents a temperature of 27°C; wherein, in A, B, and C, a represents the finished product of Sipay gingival solution (batch number 250412); b represents gallic acid; c represents 1,3,6-trigalloglucoside; and d represents the finished product of Sipay gingival solution (batch number 250412).

[0039] Figure 14 The images show inspection views of different batches of the finished Sipay gingival solution according to Embodiment 3 of the present invention; wherein, a represents the finished Sipay gingival solution (batch number 220403); b represents gallic acid; c represents 1,3,6-trigalloglucoside; d represents the finished Sipay gingival solution (batch number 230301); and e represents the finished Sipay gingival solution (batch number 230507).

[0040] Figure 15 The images show inspection views of other batches of Sipay gingival solution products according to Embodiment 3 of the present invention; wherein a to e represent Sipay gingival solution products with batch numbers 210407, 220408, 230507, 240402 and 250408 respectively. Detailed Implementation

[0041] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0042] Terminology Explanation:

[0043] Thin-layer chromatography identification refers to developing and examining the sample to be tested and the corresponding reference standard or reference medicinal material on the same thin-layer plate under the same conditions, and comparing the color and position (R0) of the chromatographic spots obtained by the two methods. f An analytical method for determining the authenticity of a sample by comparing its properties (such as the value of the sample with the actual value of the medicinal material) to determine the authenticity of a specific component or medicinal material.

[0044] Thin-layer chromatography (TLC) is an analytical technique that separates components in a sample based on differences in interactions such as adsorption, desorption, or partition between a stationary phase (a thin layer made of adsorbents such as silica gel, alumina, or polyamide) and a mobile phase (developing solvent). The separated components are then located and identified by irradiation with visible light or ultraviolet light, or by a chromogenic reaction.

[0045] As mentioned in the background section, existing thin-layer chromatography (TLC) methods for identifying multiple components in Sipaye gingival syrup often result in unclear separation, streaking, or low resolution, failing to accurately identify multiple components. Furthermore, sample pretreatment is cumbersome, requiring multiple steps (e.g., extraction, evaporation, and dissolution), which is time-consuming and labor-intensive. In this invention, the inventors discovered that the material of the TLC plate is a crucial factor affecting separation efficiency and selected a polyamide TLC plate suitable for separating Sipaye gingival syrup. This achieves effective, economical, and reproducible separation of multiple components, and requires no special sample pretreatment, making the operation convenient. Therefore, the protection scheme of this invention is proposed.

[0046] In a first typical embodiment of the present invention, a thin-layer chromatography (TLC) identification method for Sipay gingival solution is provided. The TLC identification method includes: 1) preparing a reference solution; 2) spotting the reference solution and the test solution on the same polyamide TLC plate, developing them with a developing solvent, and examining them to obtain a TLC chromatogram; 3) in the TLC chromatogram, the test solution shows a spot of the same color at the spot position of the reference solution; wherein the test solution is a Sipay gingival solution sample; and the reference solution is selected from any one or more of the following: gallic acid or 1,3,6-trigalloglucoside.

[0047] Xipayi Gingival Solution is a traditional Chinese medicine for treating oral diseases. Its main components include gallic acid and 1,3,6-trigalloglucoside. These components form specific spots on a thin-layer chromatography plate. By observing the location and color of these spots, it is possible to quickly and accurately determine whether the test solution contains these key components. The aforementioned reference solution contains gallic acid or 1,3,6-trigalloglucoside as a reference standard for the active ingredients in Xipayi Gingival Solution, while the test solution is the finished Xipayi Gingival Solution to be tested.

[0048] The inventors discovered that the silica gel thin-layer chromatography plates used in the existing technology for Sipaye gingival syrup have a highly polar surface, resulting in excessive adsorption of polar components in the syrup, leading to spot tailing or diffusion, poor separation, and low resolution. In contrast, the polyamide thin-layer plate of this application has amide groups on its surface, which form hydrogen bonds with the syrup, resulting in better selectivity and clear separation of multiple components (especially 1,3,6-trigalloglucoside), improving resolution and offering better reproducibility.

[0049] In a preferred embodiment of the present invention, the above-mentioned polyamide thin film was purchased from Luqiao Sijia Biochemical Plastics Factory in Taizhou City, Zhejiang Province, Wuhan Xinsirui Technology Co., Ltd., or Sinopharm Chemical Reagent Co., Ltd.

[0050] This invention investigated the composition of the developing solvent in detail and ultimately determined that a mixed system comprising glacial acetic acid, methanol, ethyl acetate, and formic acid should be used. In a preferred embodiment of this invention, the developing solvent is selected from glacial acetic acid, methanol, ethyl acetate, and formic acid.

[0051] The above-mentioned developing agent combination has been optimized in proportion to ensure the best separation and band clarity of gallic acid and 1,3,6-trigalloglucopyranoside on polyamide plates, effectively avoiding problems such as spot tailing or insufficient resolution.

[0052] In a preferred embodiment of the present invention, the volume ratio (mL / mL / mL / mL) of the above-mentioned glacial acetic acid:methanol:ethyl acetate:formic acid is (8~10):(6~10):(2~3):(1~2), for example, 8:6:2:1, 9:6:2:1, 10:6:2:1, 8:7:2:1, 8:8:2:1, 8:9:2:1, 8:10:2:1, 8:6:3:2, 9:6:3:2, 10:6:3:2, 8:7:3:2, 8:8:3:2, 8:9:3:2, 10:8:2:2, or 8:10:3:2, etc. The development operation is performed using a specific ratio of developing solvent, and this combination of developing solvents ensures sufficient resolution and band clarity.

[0053] In a more preferred embodiment of the present invention, the volume ratio of glacial acetic acid:methanol:ethyl acetate:formic acid is 10:8:2:2. Using this ratio of developing solvent helps to resolve spot tailing and improve chromatographic resolution.

[0054] To obtain the best chromatographic results, the method of this invention explored the amount of sample applied, which ensured the clarity of the spots and avoided inaccurate component detection due to insufficient sample amount or spot diffusion and tailing caused by excessive sample amount.

[0055] In a preferred embodiment of the present invention, the sample volume of the test solution is 1 μL to 5 μL. For example, the sample volume is 1 μL, 1.5 μL, 2 μL, 2.5 μL, 3 μL, 3.5 μL, 4 μL, 4.5 μL, or 5 μL. In a more preferred embodiment of the present invention, the sample volume of the test solution is 3 μL to 4 μL.

[0056] In a preferred embodiment of the present invention, the concentration of the reference solution is 0.5-1.5 mg / mL; the sample volume of the reference solution is 1 μL to 5 μL. For example, the sample volume is 1 μL, 1.5 μL, 2 μL, 2.5 μL, 3 μL, 3.5 μL, 4 μL, 4.5 μL, or 5 μL.

[0057] The control of temperature and humidity during the development process ensures the stability and separation efficiency of the components on the thin-layer plate, and consistent analytical results can be obtained even under different laboratory conditions, thus improving the versatility and practicality of the method.

[0058] In a preferred embodiment of the present invention, the temperature for the above-described unfolding is 4°C to 40°C. For example, the temperature is 4°C, 10°C, 20°C, 27°C, 30°C, 36°C, or 40°C.

[0059] In a preferred embodiment of the present invention, the humidity range described above is 18% to 88%. For example, the humidity is 18%, 28%, 32%, 38%, 48%, 58%, 68%, 78%, or 88%. The humidity described above is relative humidity, expressed as a percentage (%). Relative humidity is the percentage obtained by multiplying the actual amount of water vapor in the air to the maximum amount of water vapor that the air can reach at the same temperature by 100.

[0060] Since some components may have spots that only appear under ultraviolet light or only under sunlight, this application used both ultraviolet light and sunlight for inspection during the investigation. The study found that the spots in this application can appear under both sunlight and ultraviolet light. Therefore, in a preferred embodiment of the invention, ultraviolet light or sunlight is used for the above inspection; in a more preferred embodiment of the invention, the wavelength of the ultraviolet light is 254 nm or 366 nm.

[0061] In a preferred embodiment of the present invention, the above-mentioned inspection is performed using sunlight. Before the inspection, a colorimetric agent is used to develop the polyamide thin-layer plate. In a more preferred embodiment of the present invention, the colorimetric agent is selected from ferric chloride ethanol solution. This colorimetric agent can produce a specific reaction with phenolic compounds, generating a color change, which facilitates the observation and comparison of spot positions and colors under sunlight, thereby achieving accurate identification of the active ingredients in the test sample solution.

[0062] The amount of color developer used is selected according to actual needs, ensuring that the color developer is evenly sprayed onto the thin-layer plate until the spots are completely colored. In a further preferred embodiment of the present invention, the mass concentration of the above-mentioned ferric chloride ethanol solution is 1% to 3%, for example, 1%, 1.5%, 2%, 2.5% or 3%.

[0063] In a second typical embodiment of the present invention, the application of the above-described thin-layer chromatography identification method in identifying or evaluating the quality of Sipai gingival solution is provided.

[0064] The thin-layer chromatography identification method of the present invention is not limited to the identification of components, but can also be applied to the quality control of Sipai gingival solution, including the qualitative analysis of components and the consistency evaluation between production batches, providing reliable technical support for the standardized production and market access of this drug.

[0065] The present invention will be further described in detail below with reference to specific embodiments, which should not be construed as limiting the scope of protection claimed by the present invention.

[0066] Unless otherwise specified, in the embodiments of this application, the proportions of each component in the developing solvent are volume ratios, all in mL; the developing temperature is 22℃~30℃, and the developing humidity is 25%~40%; the finished Xipay gingival solution was purchased from Xinqikang Pharmaceutical Co., Ltd.; the batch number of the finished Xipay gingival solution is 250412. GF254 silica gel thin-layer plates were purchased from Qingdao Ocean Chemical Plant Branch, and polyamide film was purchased from Luqiao Sijia Biochemical Plastics Factory, Taizhou City, Zhejiang Province.

[0067] The manufacturers and batch numbers of the reference standards are as follows: gallic acid (China National Institutes for Food and Drug Control, 110831-202408), 1-galloyl glucose (Chengdu Desite Biotechnology Co., Ltd., DST241010-291), meta-digallic acid (Shanghai Yuanye Biotechnology Co., Ltd., KS371277), 1,3,6-trigalloyl glucose (Shanghai Hongyong Biotechnology Co., Ltd., 230031-202308), 1,2,3,6-tetragalloyl glucose (Shanghai Hongyong Biotechnology Co., Ltd., 190213-202109), 1,2,3,4,6-pentagalloyl glucose (Shanghai Hongyong Biotechnology Co., Ltd., 130118-202208), ellagic acid (China National Institutes for Food and Drug Control, 111959-201903), and corilagin (China National Institutes for Food and Drug Control, 1623-200302).

[0068] Preparation method of reference standards: Various reference standards were dissolved in ethanol, and the concentration of each reference standard was 1 mg / ml.

[0069] Example 1: Experiments using different thin-layer methods

[0070] Method 1 conditions: Apply 1-3 μL of the test solution and 2 μL of gallic acid reference solution to the same GF254 silica gel thin-layer plate. Use toluene:ethyl acetate:formic acid = 6:4.5:2 as the developing solvent. Develop, remove, and air-dry to allow the organic phase to evaporate. Examine under ultraviolet light (254 nm). Experimental results are shown in [Figure number missing]. Figure 1 .

[0071] Preparation of the test sample: Sipay's gingival sclerosing solution was extracted with ethyl acetate and n-butanol. The organic phase after extraction was used as the extract phase. Water was added.

[0072] The phase is the remaining phase.

[0073] It should be noted that concentration is required before spotting the sample using the above-mentioned extractive phase. The specific steps are as follows: evaporate the extractive phase to dryness.

[0074] The extract phase was then dissolved in 1 mL of ethanol.

[0075] Conclusion: Using silica gel GF254 plates and developing under conditions of toluene: ethyl acetate: formic acid = 6:4.5:2, the separation of each component was not good.

[0076] Method 2 conditions: Apply 1-3 μL of the test solution and 2 μL of gallic acid and 1,3,6-trigalloglucopyranoside reference solution to the same polyamide film. Develop using toluene:ethyl acetate:formic acid = 6:4.5:2 as the developing solvent. Remove the film, air dry, and examine under ultraviolet light (254 nm and 366 nm). Then spray with a 2% ferric chloride ethanol solution (2 g of ferric chloride dissolved in ethanol and diluted to 100 mL) and examine under sunlight. Experimental results are shown below. Figure 2 . Figure 2 In this diagram, A represents the inspection view at 254 nm before color development. Figure 2 In this diagram, B represents the inspection view at 366nm before color development. Figure 2 In the diagram, C represents the inspection view under daylight conditions after color development. Conclusion: This method produces poor unfolding results.

[0077] Method 3 conditions: Apply 1-3 μL of the test solution and 2 μL of the reference solution to the same GF254 silica gel thin-layer plate. Develop using glacial acetic acid:methanol:ethyl acetate:formic acid = 10:6:2:1 as the developing solvent. Remove the plate, air dry, and examine under UV light at 254 nm. Then spray with 2% ferric chloride ethanol solution and examine under sunlight. Experimental results are shown below. Figure 3 . Figure 3 In this diagram, A represents the inspection view at 254 nm before color development. Figure 3 In this diagram, B represents the inspection view under daylight conditions after color development.

[0078] Conclusion: This method is not very effective.

[0079] Method 4 conditions: Apply 1-3 μL of the test solution and 2 μL of the reference solution to the same polyamide film. Develop using glacial acetic acid:methanol:ethyl acetate:formic acid = 10:6:2:1 as the developing solvent. Remove the film, air dry, and examine under ultraviolet light (254 nm and 366 nm). Then spray with 2% ferric chloride ethanol solution and examine under sunlight. Experimental results are shown below. Figure 4 . Figure 4 In this diagram, A represents the inspection view at 254 nm before color development. Figure 4 In this diagram, B represents the inspection view at 366nm before color development. Figure 4 In this diagram, C represents the inspection view under daylight conditions after color development.

[0080] Conclusion: After color development, there are many spots and the separation effect is good. Each test sample and the reference sample show fluorescent spots. Based on this method, the experimental conditions should be optimized.

[0081] Method 5 conditions: Apply 1-3 μL of the test solution and 2 μL of the reference solution to the same polyamide film. Develop using glacial acetic acid:methanol:n-octane:formic acid = 10:6:2:1 as the developing solvent. Remove the film, air dry, spray with 2% ferric chloride ethanol solution, and examine under sunlight. Experimental results are shown below. Figure 5 . Figure 5 In this context, A represents a ratio of glacial acetic acid:methanol:ethyl acetate:formic acid = 10:6:2:1. Figure 5 In this context, B represents a ratio of glacial acetic acid: methanol: n-octane: formic acid = 10:6:2:1.

[0082] Conclusion: Replacing ethyl acetate with n-octane resulted in blurred spots during development. By comparing the above experimental conditions, method four was selected for subsequent optimization experiments.

[0083] Example 2: Optimization of Thin-Layer Test Method

[0084] 2.1 Methanol dosage

[0085] Conditions: Apply 1 μL of the test solution and 2 μL of the reference solution to the same polyamide film. Develop the film using glacial acetic acid:methanol:ethyl acetate:formic acid at ratios of 10:6:2:1, 10:8:2:1, 10:9:2:1, and 10:10:2:1, respectively. Remove the film, air dry, spray with 2% ferric chloride ethanol solution, and examine under sunlight. Experimental results are shown below. Figure 6 Conclusion: Increasing the amount of methanol and using a developing solvent ratio of glacial acetic acid:methanol:ethyl acetate:formic acid = 10:8:2:1 resulted in clear bands, distinct spots, and good separation.

[0086] 2.2 Ethyl acetate dosage

[0087] Conditions: Apply 1 μL of the test solution and 2 μL of the reference solution to the same polyamide film. Develop the film using glacial acetic acid:methanol:ethyl acetate:formic acid at ratios of 10:8:2:1 and 10:8:3:1, respectively. Remove the film, air dry, spray with 2% ferric chloride ethanol solution, and examine under sunlight. Experimental results are shown below. Figure 7 Conclusion: Increasing the amount of ethyl acetate resulted in longer separation time and poor development performance.

[0088] It should be noted that the raw materials used to prepare the Sipay gingival solution may contain corilagin. Therefore, this study compared the samples with corilagin, but the spots did not appear in the samples, thus confirming that the samples did not contain corilagin under the experimental conditions.

[0089] 2.3 Different Doses of Glacial Acetic Acid

[0090] Conditions: Apply 1 μL of the test solution and 2 μL of the reference solution to the same polyamide film. Develop the film using glacial acetic acid:methanol:ethyl acetate:formic acid at ratios of 10:8:2:1 and 8:8:2:1, respectively. Remove the film, air dry, spray with 2% ferric chloride ethanol solution, and examine under sunlight. Experimental results are shown below. Figure 8 Conclusion: Reducing the amount of glacial acetic acid did not improve the separation effect, and the spots were not clear.

[0091] 2.4 Different dosages of formic acid

[0092] Conditions: Apply 1 μL of the test solution and 2 μL of the reference solution to the same polyamide film. Develop the film using glacial acetic acid:methanol:ethyl acetate:formic acid at ratios of 10:8:2:1 and 10:8:2:2, respectively. Remove the film, air dry, spray with 2% ferric chloride ethanol solution, and examine under sunlight. Experimental results are shown below. Figure 9 Conclusion: Increasing the amount of formic acid improved the separation effect.

[0093] Example 3: Thin-layer methodology

[0094] Based on the above optimization test results, a methodological investigation test was conducted according to the following thin-layer chromatography method.

[0095] Apply the finished Sipay gingival solution to the same polyamide film, develop it using glacial acetic acid: methanol: ethyl acetate: formic acid = 10:8:2:2 as the developing solvent, air dry, spray with 2% ferric chloride ethanol solution for color development, air dry, and examine under fluorescent light.

[0096] 3.1 Investigation of different sample sizes

[0097] Methods: 1 μL, 2 μL, 3 μL, 4 μL, and 5 μL of the Sipaye gingival fixation solution (250405, 220408, and 210608) were spotted onto the same polyamide film. Developing solvent was glacial acetic acid:methanol:ethyl acetate:formic acid = 10:8:2:2. After development, the film was dried, sprayed with 2% ferric chloride ethanol solution for color development, dried again, and examined under fluorescent light. Experimental results are shown below. Figure 10 . Figure 10 In the diagram, AC represents the experimental results of the finished products with batch numbers 250405, 220408, and 210608, respectively.

[0098] Conclusion: All three batches of samples showed the same spots at different spotting volumes. The spots were less clear at spotting volumes of 1 μL and 2 μL, and tailing occurred at a spotting volume of 5 μL. A spotting volume of 3-4 μL was better.

[0099] 3.2 Investigation of Polyamide Films from Different Manufacturers

[0100] Methods: 3 μL of the finished Sipay gingival solution and 2 μL each of gallic acid and 1,3,6-trigalloglucopyranoside were spotted onto the same polyamide film from different manufacturers. Developing solvent was glacial acetic acid:methanol:ethyl acetate:formic acid = 10:8:2:2. The films were dried, sprayed with 2% ferric chloride ethanol solution for color development, dried again, and examined under fluorescent light. Experimental results are shown below. Figure 11 . Figure 11 In the diagram, AC represents the experimental results from Taizhou, Zhejiang; Xinsirui, Wuhan; and Sinopharm Group, respectively.

[0101] Conclusion: The polyamide film was produced by Luqiao Sijia Biochemical Plastics Factory in Taizhou City, Zhejiang Province. Figure 11 A) Wuhan Xinsirui Technology Co., Ltd. Figure 11 B in the text), Sinopharm Chemical Reagent Co., Ltd. Figure 11 The C in the sample can be developed well, and spots of the same color appear at the corresponding positions as the reference standards (gallic acid, 1,3,6-trigalloglucoside).

[0102] 3.3 Investigation of different humidity levels

[0103] Methods: 3 μL of the prepared Sipay gingival solution and 2 μL each of gallic acid and 1,3,6-trigalloglucopyranoside were spotted onto the same polyamide film. The developing solvent was glacial acetic acid:methanol:ethyl acetate:formic acid = 10:8:2:2. The humidity levels were 18%, 32%, 58%, and 88%, respectively. After development, the films were air-dried, sprayed with 2% ferric chloride ethanol solution for color development, air-dried again, and examined under fluorescent light. Experimental results are shown below. Figure 12 . Figure 12 In the diagram, A, B, C, and D represent the experimental results with humidity levels of 18%, 32%, 58%, and 88%, respectively.

[0104] Conclusion: The samples were developed well at all four humidity levels, and spots of the same color appeared at the corresponding positions on the chromatograms of gallic acid and 1,3,6-trigalloglucopyranoside reference standards.

[0105] 3.4 Investigation at different temperatures

[0106] Methods: 3 μL of the prepared Sipay gingival solution and 2 μL each of gallic acid and 1,3,6-trigalloglucopyranoside were spotted onto the same polyamide film. The developing solvent was glacial acetic acid:methanol:ethyl acetate:formic acid = 10:8:2:2. The films were developed at 4℃, room temperature (27℃), and 40℃, respectively. After drying, the films were sprayed with 2% ferric chloride ethanol solution for color development, dried, and examined under fluorescent light. Experimental results are shown below. Figure 13 . Figure 13 In the figure, A, B and C represent the experimental results at temperatures of 40℃, 4℃ and room temperature (27℃), respectively.

[0107] Conclusion: The samples were developed well at all three temperatures, and spots of the same color appeared at the corresponding positions on the chromatograms of gallic acid and 1,3,6-trigalloglucopyranoside reference standards.

[0108] Example 4 Thin-layer chromatography of samples

[0109] Methods: 3 μL of the prepared Sipay gingival solution and 2 μL each of gallic acid and 1,3,6-trigalloglucopyranoside were spotted onto the same polyamide film. The film was developed using glacial acetic acid:methanol:ethyl acetate:formic acid = 10:8:2:2 as the developing solvent. After drying, the film was sprayed with 2% ferric chloride ethanol solution for color development, dried, and examined under fluorescent light. Experimental results are shown below. Figure 14 and Figure 15 .

[0110] Conclusion: All 7 batches of samples showed the same spots, which were the same color at the corresponding positions as the reference standard chromatograms.

[0111] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects:

[0112] This application provides a highly efficient and accurate thin-layer chromatography (TLC) identification method for Sipay gingival fixation solution. This method not only effectively identifies key active ingredients in Sipay gingival fixation solution but also significantly overcomes the limitations of existing identification techniques, such as poor separation and complex sample pretreatment, thereby significantly improving the accuracy and consistency of component identification. It also simplifies the sample pretreatment process, reduces analytical costs, and shows significant application prospects in identifying or evaluating the quality of Sipay gingival fixation solution. This application is applicable to pharmaceutical quality control and helps improve the overall quality and market competitiveness of pharmaceuticals.

[0113] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A thin-layer chromatographic identification method for Sipay gingival solution, characterized in that, The thin-layer chromatography identification method includes: 1) Prepare the reference solution; 2) Spot the reference solution and the test solution onto the same polyamide thin-layer plate, develop with the developing solvent, examine, and obtain a thin-layer chromatogram; 3) In the thin-layer chromatogram, the test solution shows spots of the same color at the same locations as the spots in the reference solution; The test solution is a sample of Sipay gingival fixation solution; The reference solution is selected from any one or more of the following: gallic acid or 1,3,6-trigalloglucoside.

2. The thin-layer chromatography identification method according to claim 1, characterized in that, The developing solvent is selected from glacial acetic acid, methanol, ethyl acetate and formic acid.

3. The thin-layer chromatography identification method according to claim 2, characterized in that, The volume ratio of glacial acetic acid: methanol: ethyl acetate: formic acid is (8~10): (6~10): (2~3): (1~2).

4. The thin-layer chromatography identification method according to claim 1, characterized in that, The sample volume of the test solution is 1 μL to 5 μL.

5. The thin-layer chromatography identification method according to claim 1, characterized in that, The concentration of the reference solution is 0.5-1.5 mg / mL; the sample volume of the reference solution is 1 μL-5 μL.

6. The thin-layer chromatography identification method according to claim 1, characterized in that, The temperature for the unfolding process is 4℃~40℃; Preferably, the humidity of the unfolded part is 18% to 88%.

7. The thin-layer chromatography identification method according to claim 1, characterized in that, The inspection was performed using ultraviolet light or sunlight. Preferably, the wavelength of the ultraviolet lamp is 254nm or 366nm.

8. The thin-layer chromatography identification method according to claim 7, characterized in that, The inspection is performed using sunlight, and prior to the inspection, the polyamide thin film is developed with a color developer.

9. The thin-layer chromatography identification method according to claim 8, characterized in that, The colorimetric reagent is selected from ferric chloride ethanol solution; Preferably, the mass concentration of the ferric chloride ethanol solution is 1% to 3%.

10. The application of the thin-layer chromatography identification method according to any one of claims 1-9 in identifying or evaluating the quality of Sipay gingival solution.

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