Thin-layer identification method for euphorbia kansui formula granules and vinegar euphorbia kansui formula granules

Abstract

The invention provides a thin-layer identification method of euphorbia kansui formula granules and vinegar euphorbia kansui formula granules, which comprises the following steps: preparing a test solution, and the preparation method of the test solution comprises the following steps: extracting an aqueous solution of an extract of a test by using ethyl acetate, recovering an ethyl acetate layer, evaporating to dryness, and adding an organic solvent into residues to dissolve the residues; the thin-layer identification method further comprises the following steps: dripping the test solution on a silica gel G thin-layer plate, and developing by using a developing solvent consisting of methylbenzene, ethyl acetate and formic acid. The method disclosed by the invention can be used for rapidly and accurately identifying the euphorbia kansui formula granules and the vinegar euphorbia kansui formula granules, is simple and convenient to operate and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of detection methods, and particularly relates to a thin-layer identification method of Euphorbia kansui T.N.Liou ex T.P.Wang granules and Euphorbia kansui T.N.Liou ex T.P.Wang vinegar granules. BACKGROUND

[0002] Euphorbia kansui T.N.Liou ex T.P.Wang is first recorded in Shennong's Herbal Classic, and is listed in the Toxic Herbs of the Grass Department in Bencao Gangmu due to its severe medicinal properties and toxicity. Euphorbia kansui T.N.Liou ex T.P.Wang is the dried tuber of Euphorbia kansui T.N.Liou ex T.P.Wang in the Euphorbiaceae family, which is dug up before flowering in spring or after the stems and leaves wither in late autumn, the outer skin is knocked off, and then dried. Euphorbia kansui T.N.Liou ex T.P.Wang has the effects of purging water and reducing edema, resolving swelling and resolving nodules; and can be used for edema, fullness, water retention in the chest and abdomen, accumulation of phlegm and fluid, adverse qi causing cough and asthma, difficulty in urination and defecation, wind-phlegm epilepsy, and abscesses, sores and toxins.

[0003] Euphorbia kansui T.N.Liou ex T.P.Wang vinegar is a processed product of Euphorbia kansui T.N.Liou ex T.P.Wang, and the processing method of Euphorbia kansui T.N.Liou ex T.P.Wang vinegar slices is as follows: clean Euphorbia kansui T.N.Liou ex T.P.Wang, mix with vinegar, soak until soaked, put into a frying container, dry, take out, and cool down. Euphorbia kansui T.N.Liou ex T.P.Wang mainly contains diterpenes and triterpenes, and other components include steroids, phenolic derivatives and flavonoids, as well as fatty acids, sucrose, starch, tannins, palmitic acid and resin, etc. Euphorbia kansui T.N.Liou ex T.P.Wang has strong irritation to the oral cavity, gastrointestinal tract and skin, and has toxic side effects such as inflammation and reproductive cytotoxicity. After being processed with vinegar, the adverse effects of Euphorbia kansui T.N.Liou ex T.P.Wang on the nervous system, cardiovascular system, immune system and energy metabolism of the body are significantly improved. Therefore, Euphorbia kansui T.N.Liou ex T.P.Wang processed products are often used in clinical practice to reduce toxicity and moderate medicinal properties.

[0004] Euphorbia kansui T.N.Liou ex T.P.Wang and Euphorbia kansui T.N.Liou ex T.P.Wang vinegar slices can be distinguished by their properties, but the extracts and granules prepared therefrom have similar characteristics of yellowish white appearance, slight odor and slightly sweet and slightly spicy taste, and are easy to be confused. At present, there are few reports on the identification method of Euphorbia kansui T.N.Liou ex T.P.Wang granules and Euphorbia kansui T.N.Liou ex T.P.Wang vinegar granules, and therefore, there is an urgent need to provide a method for quickly and accurately identifying Euphorbia kansui T.N.Liou ex T.P.Wang granules and Euphorbia kansui T.N.Liou ex T.P.Wang vinegar granules. SUMMARY

[0005] The present application provides a thin-layer identification method of Euphorbia kansui T.N.Liou ex T.P.Wang granules and Euphorbia kansui T.N.Liou ex T.P.Wang vinegar granules, which can quickly and accurately identify Euphorbia kansui T.N.Liou ex T.P.Wang granules and Euphorbia kansui T.N.Liou ex T.P.Wang vinegar granules, and has the advantages of simple operation and good application prospect.

[0006] The present application provides a thin-layer identification method of Euphorbia kansui T.N.Liou ex T.P.Wang granules and Euphorbia kansui T.N.Liou ex T.P.Wang vinegar granules, which can quickly and accurately identify Euphorbia kansui T.N.Liou ex T.P.Wang granules and Euphorbia kansui T.N.Liou ex T.P.Wang vinegar granules, and has the advantages of simple operation and good application prospect. The thin layer identification method further comprises spotting the test sample solution on a silica gel G thin layer plate, and developing using a developing agent consisting of toluene, ethyl acetate, formic acid.

[0007] In some embodiments of the present application, the extract of the test sample is an organic solvent extract of the test sample, preferably an ethanol extract or a methanol extract of the test sample.

[0008] In some embodiments of the present application, the organic solvent used for dissolving the residue is selected from ethanol or methanol.

[0009] In some embodiments of the present application, the number of extractions is selected from 1 to 3 times, for example 1 time, 2 times, or 3 times.

[0010] In some embodiments of the present application, the preparation method of the test sample solution comprises the following steps: finely grinding the test sample, ultrasonically treating with ethanol or methanol for 30 to 60 minutes, filtering, evaporating the filtrate to dryness, adding water to dissolve and then extracting with ethyl acetate for 1 to 3 times, recovering the ethyl acetate layer and evaporating to dryness, and dissolving the residue with ethanol or methanol to obtain the test sample solution.

[0011] In some embodiments of the present application, the ratio of the amount of ethanol or methanol used for ultrasonic treatment to the amount of test sample is (15 to 25) mL:2 g, preferably (18 to 22) mL:2 g, and more preferably 20 mL:2 g.

[0012] In some embodiments of the present application, the ratio of the amount of ethanol or methanol used for ultrasonic treatment to the amount of test sample is (15 to 25) mL:2 g, which means that the ratio of the amount of ethanol or methanol used for ultrasonic treatment to the amount of test sample can be selected from any value or any range of values formed by any two values in the following values: 15 mL:2 g, 16 mL:2 g, 17 mL:2 g, 18 mL:2 g, 19 mL:2 g, 20 mL:2 g, 21 mL:2 g, 22 mL:2 g, 23 mL:2 g, 24 mL:2 g, or 25 mL:2 g.

[0013] In some embodiments of the present application, the ratio of the amount of water used for extraction and the amount of ethyl acetate used for each extraction to the amount of test sample is (15 to 25) mL:(15 to 25) mL:2 g, preferably (18 to 22) mL:(18 to 22) mL:2 g, and more preferably 20 mL:20 mL:2 g.

[0014] In some embodiments of the present application, the ratio of the amount of water used for extraction and the amount of ethyl acetate used for each extraction to the amount of test sample is (15-25) mL:(15-25) mL:2 g, which means that the ratio of the amount of water used for extraction and the amount of ethyl acetate used for each extraction to the amount of test sample can be any value selected from any of the following ratios or any value within the range formed by any two of the following ratios: 15 mL:15 mL:2 g, 16 mL:16 mL:2 g, 17 mL:17 mL:2 g, 18 mL:18 mL:2 g, 19 mL:19 mL:2 g, 20 mL:20 mL:2 g, 21 mL:21 mL:2 g, 22 mL:22 mL:2 g, 23 mL:23 mL:2 g, 24 mL:24 mL:2 g, 25 mL:25 mL:2 g, 15 mL:25 mL:2 g, 16 mL:24 mL:2 g, 17 mL:23 mL:2 g, 18 mL:22 mL:2 g, 19 mL:21 mL:2 g, 25 mL:15 mL:2 g, 24 mL:16 mL:2 g, 23 mL:17 mL:2 g, 22 mL:18 mL:2 g, 21 mL:19 mL:2 g.

[0015] In some embodiments of the present application, the ratio of the amount of ethanol or methanol used for dissolving residue to the amount of test sample is (15-25) mL:2 g, preferably (18-22) mL:2 g, and more preferably 20 mL:2 g.

[0016] In some embodiments of the present application, the ratio of the amount of ethanol or methanol used for dissolving residue to the amount of test sample is (15-25) mL:2 g, which means that the ratio of the amount of ethanol or methanol used for dissolving residue to the amount of test sample can be any value selected from any of the following ratios or any value within the range formed by any two of the following ratios: 15 mL:2 g, 16 mL:2 g, 17 mL:2 g, 18 mL:2 g, 19 mL:2 g, 20 mL:2 g, 21 mL:2 g, 22 mL:2 g, 23 mL:2 g, 24 mL:2 g, 25 mL:2 g.

[0017] In some embodiments of the present application, the organic solvent used for dissolving residue and ultrasonic treatment is the same, i.e., both are methanol or both are ethanol.

[0018] In some embodiments of the present application, the volume of toluene in the developing agent is > the volume of ethyl acetate > the volume of formic acid.

[0019] In some embodiments of the present application, the volume ratio of toluene, ethyl acetate and formic acid in the developing agent is (6-7):(3-4):0.1, preferably 6:4:0.1.

[0020] In some embodiments of the present invention, the volume ratio of toluene, ethyl acetate and formic acid in the developing solvent is (6-7):(3-4):0.1, which means that the volume ratio of toluene, ethyl acetate and formic acid can be selected from any value of the following volume ratios or any combination thereof: 6:4:0.1, 6.2:3.8:0.1, 6.5:3.5:0.1, 6.8:3.2:0.1, 7:3:0.1.

[0021] In some embodiments of the present invention, the sample volume is 5 to 15 μL, preferably 10 to 15 μL.

[0022] In some embodiments of the present invention, the sample volume of 5 to 15 μL means that the sample volume can be selected from any value of the following volumes or any combination thereof: 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, 10 μL, 11 μL, 12 μL, 13 μL, 14 μL, 15 μL.

[0023] In some embodiments of the present invention, the unfolding temperature is 4–25°C.

[0024] In some embodiments of the present invention, the unfolding temperature of 4 to 25°C means that the unfolding temperature can be selected from any value of the following temperatures or any combination thereof: 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C.

[0025] In some embodiments of the present invention, the relative humidity during the unfolding process is 32% to 63%.

[0026] In some embodiments of the present invention, the unfolded relative humidity of 32% to 63% means that the unfolded relative humidity can be selected from any value of the following relative humidity or any combination thereof: 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%.

[0027] In some embodiments of the present invention, the thin-layer identification method further includes preparing a reference medicinal material solution. The preparation method of the reference medicinal material solution includes water extraction of Euphorbia kansui reference medicinal material and recovery of the water extract by evaporation to dryness. The residue obtained after evaporation is used to prepare the reference medicinal material solution according to the preparation method of the test sample solution.

[0028] In some embodiments of the present invention, the water extraction method is to decoct for 20-40 minutes, preferably 25-35 minutes, and more preferably 30 minutes; preferably, the ratio of water to Euphorbia kansui reference material during the water extraction process is (40-60) mL:2g, preferably (45-55) mL:2g, and more preferably 50 mL:2g.

[0029] In some embodiments of the present invention, the method for preparing the reference medicinal material solution includes water extraction of Euphorbia kansui reference medicinal material and recovery of the water extract by evaporation to dryness, fine grinding of the residue obtained after evaporation, ultrasonic treatment with ethanol or methanol for 30-60 min, filtration, evaporation of the filtrate to dryness, addition of water to dissolve, extraction with ethyl acetate 1-3 times, recovery of the ethyl acetate layer and evaporation to dryness, and dissolution of the residue with ethanol or methanol to obtain the solution.

[0030] In some embodiments of the present invention, the thin-layer identification method further includes spotting the reference herb solution and the test sample solution onto the same silica gel G thin-layer plate.

[0031] In some embodiments of the present invention, the thin-layer identification method further includes drying the reagent on the silica gel G thin-layer plate after it has been developed, then spraying it with a 10% sulfuric acid ethanol solution, heating it at 105°C until the spots are clearly visible, and then examining it under ultraviolet light; preferably, the wavelength of the ultraviolet light used for the examination is 365nm.

[0032] In some embodiments of the present invention, the thin-layer identification method includes the following steps: (1) Take an appropriate amount of the test sample, grind it into a fine powder, add 20 mL of methanol, sonicate for 40 min, filter, evaporate the filtrate to dryness, add 20 mL of water to dissolve, extract twice with ethyl acetate, 20 mL each time, discard the water, evaporate the filtrate to dryness, add 1 mL of methanol to dissolve the residue, and use it as the test sample solution.

[0033] (2) Take 2g of Euphorbia kansui reference material, add 50mL of water, decoct for 30min, centrifuge at 6000rpm for 3min, take the supernatant, place it in a water bath and evaporate to dryness, add 20mL of methanol to the residue, sonicate for 40min, filter, evaporate the filtrate to dryness, add 20mL of water to dissolve, extract twice with ethyl acetate, 20mL each time, discard the water, evaporate the filtrate to dryness, add 1mL of methanol to dissolve the residue, and use it as the reference material solution.

[0034] (3) Perform thin-layer chromatography (Chinese Pharmacopoeia 2020 Edition, Part I, General Chapter 0502). Take 10 μL of the reference medicinal material solution and the test solution and spot them on the same silica gel G thin-layer plate. Use toluene-ethyl acetate-formic acid (volume ratio 6:4:0.1) as the developing solvent. Develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly visible, and examine under ultraviolet light (365nm).

[0035] The steps (1) and (2) above are not in any order; you can perform either step first or both steps simultaneously.

[0036] In some embodiments of the present invention, the criteria for judging the results of the thin-layer identification method include: at a ratio shift value of 0.2 to 0.51, the thin-layer chromatography of Euphorbia kansui formula granules shows significant fluorescent spots, while the thin-layer chromatography of vinegar-treated Euphorbia kansui formula granules shows no significant fluorescent spots.

[0037] In some embodiments of the present invention, the criteria for judging the results of the thin-layer identification method include: compared with the thin-layer chromatography of Euphorbia kansui reference material, at a ratio shift value of 0.2 to 0.51, the thin-layer chromatography of Euphorbia kansui formula granules has the same fluorescent spots, while the thin-layer chromatography of vinegar-treated Euphorbia kansui formula granules has no significant fluorescent spots.

[0038] In some embodiments of the present invention, when the volume ratio of toluene, ethyl acetate and formic acid in the developing solvent is 6:4:0.1, the result judgment criteria of the thin-layer identification method include: at a ratio shift value of 0.31 to 0.51, preferably at a ratio shift value of 0.36 ± 10%, the thin-layer chromatography of Euphorbia kansui formulation granules has significant fluorescent spots, while the thin-layer chromatography of vinegar Euphorbia kansui formulation granules has no significant fluorescent spots.

[0039] In some embodiments of the present invention, when the volume ratio of toluene, ethyl acetate and formic acid in the developing solvent is 6:4:0.1, the criteria for judging the results of the thin-layer identification method include: compared with the thin-layer chromatography of Euphorbia kansui reference material, at a ratio shift value of 0.31 to 0.51, preferably at a ratio shift value of 0.36 ± 10%, the thin-layer chromatography of Euphorbia kansui formulation granules has the same fluorescent spots, while the thin-layer chromatography of vinegar-processed Euphorbia kansui formulation granules has no significant fluorescent spots.

[0040] The beneficial effects of this invention are: This invention provides a thin-layer chromatography (TLC) method for identifying Euphorbia kansui (Gansui) formula granules and vinegar-processed Euphorbia kansui formula granules. This method effectively reduces water-soluble impurities in the test solution and increases the concentration of the target identification component by extracting the aqueous solution of the test sample with ethyl acetate during sample preparation. Furthermore, this invention proposes using toluene-ethyl acetate-formic acid as the developing solvent. This solvent uses low-polarity toluene as the main developing component, combined with ethyl acetate to increase polarity and improve resolution, and trace amounts of formic acid to reduce tailing. Using this solvent effectively improves the resolution of fluorescent spots in TLC and reduces smearing at the solvent front. Especially when the volume ratio of toluene, ethyl acetate, and formic acid in the developing solvent is 6:4:0.1, the specific gravity is at a more suitable position, resulting in higher resolution of the fluorescent spots. This method can rapidly and accurately identify Euphorbia kansui formula granules and vinegar-processed Euphorbia kansui formula granules. It is simple to operate, highly sensitive, specific, and durable, and has good application prospects. Attached Figure Description

[0041] Figure 1 The images show thin-layer chromatograms for different sample amounts. In the images, 1 to 3 are the reference medicinal materials of Euphorbia kansui with sample amounts of 5 μL, 10 μL, and 15 μL, respectively; 4 to 6 are the Euphorbia kansui formula granules with sample amounts of 5 μL, 10 μL, and 15 μL, respectively; and 7 to 9 are the vinegar-treated Euphorbia kansui formula granules with sample amounts of 5 μL, 10 μL, and 15 μL, respectively. Figure 2 The thin-layer chromatograms are for specificity studies. In the figure, 1 is the negative control formula granules, 2 is the reference medicinal material of Euphorbia kansui, 3 is the formula granules of Euphorbia kansui, and 4 is the vinegar-processed Euphorbia kansui formula granules. Figure 3 The image shows a thin-layer chromatogram developed using a pre-made silica gel G thin-layer plate from Merck, Germany. In the image, 1 represents the reference herb *Euphorbia kansui*, 2 represents the *Euphorbia kansui* formula granules, and 3 represents the vinegar-processed *Euphorbia kansui* formula granules. Figure 4 The image shows a thin-layer chromatogram developed using a pre-made silica gel G thin-layer plate from Tianjin Slida Technology Co., Ltd. In the image, 1 represents the reference herb *Euphorbia kansui*, 2 represents the *Euphorbia kansui* formula granules, and 3 represents the vinegar-processed *Euphorbia kansui* formula granules. Figure 5 The image shows a thin-layer chromatogram developed using silica gel G thin-layer plates prefabricated by Qingdao Marine Chemical Plant. In the image, 1 represents the reference herb *Euphorbia kansui*, 2 represents the *Euphorbia kansui* formula granules, and 3 represents the vinegar-processed *Euphorbia kansui* formula granules. Figure 6 The image shows a thin-layer chromatogram developed at 4℃. In the image, 1 represents the reference herb *Euphorbia kansui*, 2 represents the granulated formula of *Euphorbia kansui*, and 3 represents the granulated formula of *Euphorbia kansui* processed with vinegar. Figure 7 The image shows a thin-layer chromatogram developed at 25℃. In the image, 1 represents the reference herb *Euphorbia kansui*, 2 represents the granulated formula of *Euphorbia kansui*, and 3 represents the granulated formula of *Euphorbia kansui* processed with vinegar. Figure 8 The image shows a thin-layer chromatogram developed at a relative humidity of 32%. In the image, 1 represents the reference herb *Euphorbia kansui*, 2 represents the granulated formula of *Euphorbia kansui*, and 3 represents the granulated formula of *Euphorbia kansui* processed with vinegar. Figure 9 The image shows a thin-layer chromatogram developed at a relative humidity of 75%. In the image, 1 represents the reference herb *Euphorbia kansui*, 2 represents the granulated formula of *Euphorbia kansui*, and 3 represents the granulated formula of *Euphorbia kansui* processed with vinegar. Figure 10 The thin-layer chromatograms obtained by using different batches of test samples to verify the thin-layer identification method are shown in the figure. 1 is the reference herb of Euphorbia kansui, 2 to 4 are Euphorbia kansui formula granules with batch numbers GS2409010, GS2409011, and GS2409012 respectively, and 5 to 7 are vinegar-processed Euphorbia kansui formula granules with batch numbers CGS2412001, CGS2412002, and CGS2412003 respectively. Figure 11 The thin-layer chromatogram obtained by the thin-layer identification method in Example 2 is shown in the figure. In the figure, 1 is the reference medicinal material of Euphorbia kansui, 2 is the granulated formula of Euphorbia kansui, and 3 is the granulated formula of vinegar-treated Euphorbia kansui. Figure 12 The thin-layer chromatograms obtained by the thin-layer identification method in Comparative Example 1 are shown. In the figure, 1 is the reference medicinal material of Euphorbia kansui, 2 is the granulated formula of Euphorbia kansui, and 3 is the granulated formula of vinegar-processed Euphorbia kansui. Detailed Implementation

[0042] To further illustrate the present invention, the thin-layer chromatography identification method for Euphorbia kansui formula granules and vinegar-based Euphorbia kansui formula granules provided by the present invention will be described in detail below with reference to the embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0043] Instruments and reagents 1. Instruments Heating plate; mortar and pestle; thin-layer imaging system: CAMAG TLC Visualizer; silicone G thin-layer plates (Merck, Germany, batch number: HX46121653; Tianjin Slida Technology Co., Ltd., batch number: 20250529; Qingdao Ocean Chemical Plant, batch number: 20240504).

[0044] 2. Reagents Toluene, ethyl acetate, methanol, ethanol, petroleum ether, acetone, and formic acid were all of analytical grade, and water was ultrapure water (prepared in the laboratory).

[0045] 3. Drug testing Euphorbia kansui reference material (China National Institutes for Food and Drug Control, batch number: 121042-202006), Euphorbia kansui formula granules (Sichuan Xinlvse Pharmaceutical Technology Development Co., Ltd., batch numbers: GS2409010; GS2409011; GS2409012), and Euphorbia kansui vinegar formula granules (Sichuan Xinlvse Pharmaceutical Technology Development Co., Ltd., batch numbers: CGS2412001; CGS2412002; CGS2412003).

[0046] Example 1 Thin-layer chromatography identification methods include the following steps: (1) Preparation of test solution: Take an appropriate amount of test sample, grind it into a fine powder, about 2g, add 20mL of methanol, sonicate for 40min, filter, evaporate the filtrate to dryness, add 20mL of water to dissolve, extract twice with ethyl acetate, 20mL each time, discard the water, evaporate the filtrate to dryness, add 1mL of methanol to dissolve the residue, and use it as the test solution.

[0047] (2) Preparation of reference medicinal material solution: Take 2g of Euphorbia kansui reference medicinal material, add 50mL of water, decoct for 30min, centrifuge at 6000rpm for 3min, take the supernatant, place it in a water bath and evaporate to dryness, add 20mL of methanol to the residue, sonicate for 40min, filter, evaporate the filtrate to dryness, add 20mL of water to dissolve, extract twice with ethyl acetate, 20mL each time, discard the water, evaporate the filtrate to dryness, add 1mL of methanol to dissolve the residue, and use it as the reference medicinal material solution.

[0048] (3) Identification: Perform thin-layer chromatography (Chinese Pharmacopoeia 2020 Edition, Part I, General Chapter 0502). Take 10 μL of the reference medicinal material solution and the test solution and spot them separately on the same silica gel G thin-layer plate. Use toluene-ethyl acetate-formic acid (volume ratio 6:4:0.1) as the developing solvent. Develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly visible, and examine under ultraviolet light (365nm).

[0049] The steps (1) and (2) above are not in any order; you can perform either step first or both steps simultaneously.

[0050] The methodological examination of the thin-layer identification method in Example 1 above is as follows: 1. Sample quantity investigation Prepare the test solution and the reference herb solution according to steps (1) and (2) above. Take 5 μL, 10 μL, and 15 μL of each solution and spot them onto the same silica gel G thin-layer plate (Merck, Germany, batch number: HX46121653). Identify the sample according to the development and inspection conditions in step (3) above. The results are shown in […]. Figure 1The results showed that when the sample volume of the test solution was 5–15 μL, the thin-layer chromatographic spots were clearly visible, and the optimal sample volume for both the reference herb solution and the test solution was determined to be 10 μL. Compared with the thin-layer chromatography of the Euphorbia kansui reference herb, at a ratio shift (Rf) of 0.31, the thin-layer chromatography of the Euphorbia kansui formula granules showed the same fluorescent spots, while the thin-layer chromatography of the vinegar-processed Euphorbia kansui formula granules showed no significant fluorescent spots.

[0051] 2. Specificity Examination Prepare the following solutions according to step (1): Euphorbia kansui formula granule solution, vinegar-prepared Euphorbia kansui formula granule solution, and negative control formula granule solution (the negative control formula granule is a formula granule that does not contain Euphorbia kansui and vinegar-prepared Euphorbia kansui, but whose other components and production methods are the same as those of Euphorbia kansui formula granule and vinegar-prepared Euphorbia kansui formula granule). At the same time, prepare the control medicinal material solution according to step (2). Then, take 10 μL each of the Euphorbia kansui formula granule solution, vinegar-prepared Euphorbia kansui formula granule solution, negative control formula granule solution, and control medicinal material solution and spot them on the same silica gel G thin-layer plate (Merck, Germany, batch number: HX46121653). Identify the results according to the development and inspection conditions in step (3). See below for the results. Figure 2 The results showed that the negative control formulation granule solution did not interfere with either the Euphorbia kansui formulation granules or the vinegar-processed Euphorbia kansui formulation granules, indicating that the method of this invention has good specificity. Compared with the thin-layer chromatography of Euphorbia kansui reference material, at a ratio shift (Rf) of 0.35, the thin-layer chromatography of the Euphorbia kansui formulation granules showed the same fluorescent spots, while the thin-layer chromatography of the vinegar-processed Euphorbia kansui formulation granules showed no significant fluorescent spots.

[0052] 3. Durability assessment 3.1 Investigation of different silicone G thin-layer plates Pre-fabricated silica gel G thin-layer plates from three companies—Merck (Germany), Tianjin Silida Technology Co., Ltd., and Qingdao Ocean Chemical Plant (batch number: 20240504)—were selected. Tests were conducted using the thin-layer identification method described above. The results are shown below. Figures 3-5The results showed that all three brands of silica gel G thin-layer plates met the expected identification requirements, indicating that the method of the present invention has good durability. Compared with the thin-layer chromatography of Euphorbia kansui reference material, on the silica gel G thin-layer plate of Merck GmbH (Germany), the thin-layer chromatography of Euphorbia kansui formulation granules at a ratio shift (Rf) of 0.32 showed obvious fluorescent spots, while the thin-layer chromatography of vinegar-processed Euphorbia kansui formulation granules did not show fluorescent spots of the same color; on the silica gel G thin-layer plate of Tianjin Slida Technology Co., Ltd., the thin-layer chromatography of Euphorbia kansui formulation granules at a ratio shift (Rf) of 0.51 showed obvious fluorescent spots, while the thin-layer chromatography of vinegar-processed Euphorbia kansui formulation granules did not show fluorescent spots of the same color; on the silica gel G thin-layer plate of Qingdao Ocean Chemical Plant, the thin-layer chromatography of Euphorbia kansui formulation granules at a ratio shift (Rf) of 0.49 showed obvious fluorescent spots, while the thin-layer chromatography of vinegar-processed Euphorbia kansui formulation granules did not show significant fluorescent spots.

[0053] 3.2 Investigation at different temperatures Prepare the test solution and the reference herb solution according to steps (1) and (2) above. Take 10 μL of each of the test solution and the reference herb solution and spot them on the same silica gel G thin-layer plate (Merck, Germany, batch number: HX46121653). Take the spotted silica gel G thin-layer plate and place it at a low temperature of 4℃ and a normal temperature of 25℃ respectively for development and examination according to the conditions of step (3) above. The results are shown in the figure below. Figure 6 (4℃) and Figure 7 (25℃). By Figure 6 and Figure 7 It can be seen that the method of the present invention has good durability at different temperatures. Compared with the thin-layer chromatography of Euphorbia kansui reference material, at a temperature of 4°C, the thin-layer chromatography of Euphorbia kansui formulation granules showed the same fluorescent spots at a ratio shift (Rf) of 0.33, while the thin-layer chromatography of vinegar-processed Euphorbia kansui formulation granules showed no fluorescent spots; at a temperature of 25°C, at a ratio shift (Rf) of 0.32, the thin-layer chromatography of Euphorbia kansui formulation granules showed the same fluorescent spots, while the thin-layer chromatography of vinegar-processed Euphorbia kansui formulation granules showed no significant fluorescent spots.

[0054] 3.3 Investigation of different humidity levels Prepare the test solution and the control herbal solution according to steps (1) and (2) above. Take 10 μL of each of the test solution and the control herbal solution and spot them on the same silica gel G thin-layer plate (Merck, Germany, batch number: HX46121653). Take the spotted silica gel G thin-layer plate and place it in an environment with relative humidity of 32% and 75% respectively. Develop it according to the conditions of step (3) above and then examine it. The results are shown in the figures below. Figure 8 (32%) and Figure 9 (75%). By Figure 8 and Figure 9It can be seen that the method of the present invention has good durability under different humidity levels. Compared with the thin-layer chromatography of Euphorbia kansui reference material, when the relative humidity is 63%, the thin-layer chromatography of Euphorbia kansui formula granules has the same fluorescent spot at a ratio shift (Rf) of 0.32, while the thin-layer chromatography of vinegar-processed Euphorbia kansui formula granules has no fluorescent spot; when the relative humidity is 32%, the thin-layer chromatography of Euphorbia kansui formula granules has the same fluorescent spot at a ratio shift (Rf) of 0.31, while the thin-layer chromatography of vinegar-processed Euphorbia kansui formula granules has no significant fluorescent spot.

[0055] 4. Validation of different batches of test samples Thin-layer chromatography (TLC) was used to validate the identification method using three batches of Euphorbia kansui formula granules (Sichuan Xinlvse Pharmaceutical Technology Development Co., Ltd., batch numbers: GS2409010; GS2409011; GS2409012) and three batches of vinegar-processed Euphorbia kansui formula granules (Sichuan Xinlvse Pharmaceutical Technology Development Co., Ltd., batch numbers: CGS2412001; CGS2412002; CGS2412003). The experimental results are shown in the table below. Figure 10 The results showed that, compared with the thin-layer chromatography of Euphorbia kansui reference material, the thin-layer chromatography of Euphorbia kansui formula granules had the same fluorescent spots at a ratio shift (Rf) of 0.33, while the thin-layer chromatography of vinegar-processed Euphorbia kansui formula granules had no significant fluorescent spots.

[0056] Based on the identification method of Example 1, the ratio shift (Rf) of the identification spots of Euphorbia kansui formula granules and vinegar-processed Euphorbia kansui formula granules was determined. The Rf values ​​of various parameters in the methodological investigation of the thin-layer identification method in Example 1 are summarized, and the results are shown in Table 1. Based on the summarized results, it is determined that the Rf value (Rf) of the identification spots of Euphorbia kansui formula granules and vinegar-processed Euphorbia kansui formula granules should be within 10%, with a specified value of 0.36.

[0057] Table 1 Summary of Methodological Examinations and Shift Values

[0058] Example 2 The thin-layer chromatography identification method is basically the same as in Example 1, except that toluene-ethyl acetate-formic acid (volume ratio 7:3:0.1) is used as the developing solvent. The obtained thin-layer chromatogram is shown below. Figure 11 The results showed that, compared with the thin-layer chromatography of Euphorbia kansui reference material, the thin-layer chromatography of Euphorbia kansui formula granules had the same fluorescent spots at a ratio shift (Rf) of 0.21, while the thin-layer chromatography of vinegar-processed Euphorbia kansui formula granules had no significant fluorescent spots.

[0059] Comparative Example 1 The thin-layer chromatography identification method is basically the same as in Example 2, except that petroleum ether is used instead of ethyl acetate for extraction in steps (1) and (2). The obtained thin-layer chromatogram is shown in [Figure 2]. Figure 12The results showed that the thin-layer chromatography of the reference herb *Euphorbia kansui* and the formulation granules showed the same fluorescent spot at a ratio shift of 0.23. However, the fluorescent spot at this point was very faint and difficult to distinguish from the thin-layer chromatography of the vinegar-processed *Euphorbia kansui* granules. Therefore, this thin-layer identification method cannot accurately distinguish between the formulation granules and the vinegar-processed *Euphorbia kansui* granules.

[0060] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A thin-layer chromatography method for identifying Euphorbia kansui formula granules and vinegar-processed Euphorbia kansui formula granules, comprising preparing a test sample solution, characterized in that, The method for preparing the test solution includes extracting an aqueous solution of the extract of the test sample with ethyl acetate, recovering the ethyl acetate layer and evaporating it to dryness, and dissolving the residue with an organic solvent. The thin-layer identification method further includes spotting the test solution onto a silica gel G thin-layer plate and developing it using a developing solvent composed of toluene, ethyl acetate, and formic acid.

2. The thin-layer identification method according to claim 1, characterized in that, The extract of the test sample is an organic solvent extract of the test sample, preferably an ethanol extract or a methanol extract of the test sample. And / or, the organic solvent used to dissolve the residue is selected from ethanol or methanol; And / or, the number of extractions is selected from 1 to 3 times.

3. The thin-layer identification method according to claim 1 or 2, characterized in that, The preparation method of the test sample solution includes the following steps: take the test sample, grind it into a fine powder, sonicate it with ethanol or methanol for 30-60 minutes, filter it, evaporate the filtrate to dryness, add water to dissolve it, add ethyl acetate to extract it 1-3 times, recover the ethyl acetate layer and evaporate it to dryness, add ethanol or methanol to dissolve the residue to obtain the test sample solution.

4. The thin-layer identification method according to claim 3, characterized in that, The ratio of ethanol or methanol used in ultrasonic treatment to the test sample is (15-25) mL:2g, preferably (18-22) mL:2g, and more preferably 20 mL:2g. And / or, the ratio of water used for extraction and ethyl acetate used in each extraction to the amount of test sample is (15-25) mL:(15-25) mL:2 g, preferably (18-22) mL:(18-22) mL:2 g, more preferably 20 mL:20 mL:2 g; And / or, the ratio of ethanol or methanol used to dissolve the residue to the test sample is (15-25) mL: 2 g, preferably (18-22) mL: 2 g, more preferably 20 mL: 2 g; And / or, the solvent used to dissolve the residue is the same as the organic solvent used in the ultrasonic treatment.

5. The thin-layer identification method according to any one of claims 1 to 4, characterized in that, The volume ratio of toluene, ethyl acetate and formic acid in the developing solvent is (6-7): (3-4):0.1, preferably 6:4:0.

1.

6. The thin-layer chromatography identification method according to any one of claims 1 to 5, characterized in that, The sample loading volume is 5–15 μL, preferably 10–15 μL; And / or, the unfolding temperature is 4–25°C; And / or, the relative humidity during the unfolding process is 32% to 63%.

7. The thin-layer chromatography identification method according to any one of claims 1 to 6, characterized in that, The thin-layer identification method further includes preparing a reference medicinal material solution. The preparation method of the reference medicinal material solution includes water extraction of Euphorbia kansui reference medicinal material and evaporation of the water extract to dryness. The residue obtained after evaporation is used to prepare the reference medicinal material solution according to the preparation method of the test sample solution. Preferably, the water extraction method is decoction for 20-40 minutes. More preferably, the ratio of water to Euphorbia kansui reference medicinal material used in the water extraction process is (40-60) mL:2g.

8. The thin-layer identification method according to claim 7, characterized in that, The preparation method of the reference medicinal material solution includes water extraction of Euphorbia kansui reference medicinal material and recovery of the water extract by evaporation to dryness. The residue obtained after evaporation is finely ground, ultrasonically treated with ethanol or methanol for 30-60 min, filtered, the filtrate is evaporated to dryness, water is added to dissolve it, ethyl acetate is added to extract 1-3 times, the ethyl acetate layer is recovered and evaporated to dryness, and the residue is dissolved in ethanol or methanol to obtain the solution. And / or, the thin-layer identification method further includes spotting the reference herb solution and the test sample solution onto the same silica gel G thin-layer plate.

9. The thin-layer chromatography identification method according to any one of claims 1 to 8, characterized in that, The thin-layer identification method further includes drying the reagent on the silica gel G thin-layer plate after it has been developed, then spraying it with 10% sulfuric acid ethanol solution, heating it at 105°C until the spots are clearly visible, and then examining it under ultraviolet light; preferably, the wavelength of the ultraviolet light used for examination is 365nm.

10. The thin-layer identification method according to any one of claims 1 to 9, characterized in that, The results of the thin-layer identification method include the following criteria: at a ratio shift value of 0.2 to 0.51, the thin-layer chromatography of Euphorbia kansui formula granules shows significant fluorescent spots, while the thin-layer chromatography of vinegar-treated Euphorbia kansui formula granules does not show significant fluorescent spots. Alternatively, the criteria for judging the results of the thin-layer identification method include: compared with the thin-layer chromatography of Euphorbia kansui reference material, at a ratio shift value of 0.2 to 0.51, the thin-layer chromatography of Euphorbia kansui formula granules has the same fluorescent spots, while the thin-layer chromatography of vinegar-treated Euphorbia kansui formula granules has no significant fluorescent spots.

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