A method for identifying grass wu and / or grass wu preparation granules by thin layer

Abstract

This invention relates to a thin-layer chromatography (TLC) identification method for Aconitum carmichaelii and / or processed Aconitum carmichaelii granules and its application. The identification method includes the following steps: (1) mixing Aconitum carmichaelii reference material with water and refluxing to extract, obtaining a reference material solution; (2) mixing the test sample with methanol and sonicating to obtain a test solution; (3) performing TLC analysis on the reference material solution and the test solution. The TLC identification method provided by this invention creatively incorporates the identification method of reference material for the identification of processed Aconitum carmichaelii granules, greatly increasing the specificity of the method, providing a large amount of information, and enabling more accurate identification of Aconitum carmichaelii and processed Aconitum carmichaelii granules; the solvent used is non-toxic, does not pollute the environment, and will not harm the health of operators, making it easy to promote and use.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a thin-layer chromatography method for identifying Aconitum carmichaelii and / or processed Aconitum carmichaelii granules. Background Technology

[0002] Aconitum kusnezoffii Reichb., a plant in the Ranunculaceae family, is the dried tuberous root. It is harvested in autumn when the stems and leaves wither, the fibrous roots and soil are removed, and then it is dried. This herb is pungent, bitter, and hot in nature, and highly toxic. It enters the heart, liver, kidney, and spleen meridians. It has the functions of dispelling wind and dampness, warming the meridians, and relieving pain. It is used for wind-cold-dampness arthralgia, joint pain, cold pain in the heart and abdomen, cold hernia pain, and for anesthetic and analgesic effects. Clinically, it is commonly used for wind-cold-dampness arthralgia, joint pain, cold pain in the heart and abdomen, cold hernia pain, and for anesthetic and analgesic effects. Due to its high toxicity, it must be processed before clinical use, and the dosage must be strictly controlled.

[0003] The 2020 edition of the *Pharmacopoeia of the People's Republic of China* (Part I) identifies aconitum diester alkaloids in the section on *Aconitum carmichaelii*, and benzoyl aconitine, benzoyl hypoaconitine, and benzoyl neoaconitine in the section on processed *Aconitum carmichaelii*. These are all toxic components in *Aconitum carmichaelii* whose content needs to be controlled. The thin-layer chromatography (TLC) development conditions for both *Aconitum carmichaelii* and processed *Aconitum carmichaelii* are the same in the pharmacopoeia, making it suitable for the identification of processed *Aconitum carmichaelii* formulation granules. However, this method only provides reference standards for identifying alkaloids, which is relatively limited and lacks specific identification for medicinal materials. Furthermore, the extraction method for the test sample uses a 1:1 mixture of isopropanol and chloroform, which is a toxic reagent.

[0004] There are few research reports on thin-layer chromatography identification of Aconitum carmichaelii. The existing thin-layer chromatography identification methods in processed Aconitum carmichaelii granules are merely changes to the proportion of the developing solvent based on the pharmacopoeia method. They not only lack identification methods for Aconitum carmichaelii reference materials, but also have the disadvantages of being cumbersome, complex, time-consuming, polluting the surrounding environment, and costly and inefficient.

[0005] Therefore, improving detection efficiency, reducing detection costs, and minimizing environmental pollution, as well as creating a rapid, multi-information thin-layer chromatography method for identifying Aconitum carmichaelii and / or processed Aconitum carmichaelii granules, is of great practical significance. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a thin-layer chromatography method for identifying Aconitum carmichaelii and / or processed Aconitum carmichaelii granules.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a thin-layer chromatography method for identifying Aconitum carmichaelii and / or processed Aconitum carmichaelii granules, the method comprising the following steps:

[0009] (1) Mix the aconite reference material with water, reflux to extract, filter, dry the filtrate and mix with methanol to obtain the reference material solution;

[0010] (2) Mix the sample to be tested with methanol and sonicate to obtain the test solution;

[0011] (3) Thin-layer chromatography analysis was performed on the reference herb solution and the test sample solution to identify Aconitum carmichaelii and / or processed Aconitum carmichaelii granules;

[0012] Steps (1) and (2) are not in any particular order.

[0013] This invention, based on the chemical structure and properties of the active ingredients of traditional Chinese medicine, follows the extraction principle of "like dissolves like" and uses suitable extraction solvents to prepare test and reference solutions simply and quickly. Then, through the movement of the developing solvent, the different adsorption and desorption capacities of each component on the thin-layer plate allow for spot separation. Utilizing the characteristic functional groups of each component, a colorimetric reaction is employed to develop components with different absorption bands on the same thin-layer plate under the same viewing conditions, obtaining a specific, multi-information, and clearly defined thin-layer chromatogram.

[0014] The thin-layer chromatography (TLC) identification method for Aconitum carmichaelii and / or processed Aconitum carmichaelii granules involved in this invention is based on the physicochemical properties of Aconitum carmichaelii. It is an improvement upon the existing TLC identification method for Aconitum carmichaelii in the Chinese Pharmacopoeia, creatively incorporating a reference herb identification method for processed Aconitum carmichaelii granules. This significantly increases the specificity and information content of the method, overcoming the limitation of existing TLC identification methods that provide only single-spot information in the chromatograms. This allows for more accurate identification of Aconitum carmichaelii and processed Aconitum carmichaelii granules. Furthermore, the extraction solvent used in this method is no longer the highly toxic chloroform previously reported; it is low in toxicity, effectively reducing environmental pollution. This method is simple to operate, highly specific, precise, sensitive, clear, and provides good separation, obvious spots, and good reproducibility. The solvent used is non-toxic, does not pollute the environment, and will not harm the health of operators, making it easy to promote and use.

[0015] Preferably, the ratio of aconite reference material to water in step (1) is 1g:(20-30)mL, for example, 1g:20mL, 1g:21mL, 1g:22mL, 1g:23mL, 1g:24mL, 1g:25mL, 1g:26mL, 1g:27mL, 1g:28mL, 1g:29mL, 1g:30mL, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0016] Preferably, the reflux extraction time is 1-3 hours (e.g., 1 hour, 1.2 hours, 1.4 hours, 1.6 hours, 1.8 hours, 2 hours, 2.2 hours, 2.4 hours, 2.6 hours, 2.8 hours, 3 hours, etc.), and the reflux extraction temperature is 95-100°C (e.g., 95°C, 95.5°C, 96°C, 96.5°C, 97°C, 97.5°C, 98°C, 98.5°C, 99°C, 100°C, etc.). Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0017] Preferably, step (1) further includes ultrasonic treatment, filtration, and drying of the filtrate before mixing and dissolving it with methanol.

[0018] The reference herb, Aconitum carmichaelii, was mixed with water, refluxed for extraction, filtered, and the residue was dried. The residue was then mixed with methanol, sonicated, filtered, and the residue was dried again. The residue was then mixed with methanol to dissolve the residue, thus obtaining the reference herb solution.

[0019] Preferably, the power of the ultrasonic treatment is 200-300W (e.g., 200W, 210W, 220W, 230W, 240W, 250W, 260W, 270W, 280W, 290W, 300W, etc.), the frequency is 30-50kHz (e.g., 30kHz, 32kHz, 34kHz, 36kHz, 38kHz, 40kHz, 42kHz, 44kHz, 46kHz, 48kHz, 50kHz, etc.), and the duration of the ultrasonic treatment is 20-40min (e.g., 20min, 21min, 22min, 23min, 24min, 25min, 26min, 27min, 28min, 29min, 30min, 32min, 35min, 37min, 40min, etc.). Other specific values ​​within the above range can be selected, and will not be elaborated here. Preferably, the ratio of the sample to methanol in step (2) is 1g:(50-70)mL, for example, 1g:50mL, 1g:52mL, 1g:54mL, 1g:56mL, 1g:58mL, 1g:60mL, 1g:62mL, 1g:64mL, 1g:66mL, 1g:70mL, etc. Other specific values ​​within this range can be selected, and will not be described in detail here.

[0020] Preferably, the power of the ultrasonic treatment in step (2) is 200-300W (e.g., 200W, 210W, 220W, 230W, 240W, 250W, 260W, 270W, 280W, 290W, 300W, etc.), the frequency is 30-50kHz (e.g., 30kHz, 32kHz, 34kHz, 36kHz, 38kHz, 40kHz, 42kHz, 44kHz, 46kHz, 48kHz, 50kHz, etc.), and the ultrasonic treatment time is 20-40min (e.g., 20min, 21min, 22min, 23min, 24min, 25min, 26min, 27min, 28min, 29min, 30min, 32min, 35min, 37min, 40min, etc.). Other specific values ​​within the above range can be selected, and will not be described in detail here.

[0021] Preferably, the ultrasonic treatment in step (2) further includes filtration, and the filtrate is dried and then mixed with methanol for dissolution.

[0022] Preferably, in the thin-layer chromatography analysis described in step (3), the sample volume of the test solution is 5-20 μL, such as 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, 10 μL, 11 μL, 12 μL, 13 μL, 14 μL, 15 μL, 16 μL, 17 μL, 18 μL, 19 μL, 20 μL, etc. Other specific spot values ​​within this range can be selected, and will not be described in detail here.

[0023] Preferably, in the thin-layer chromatography analysis, the spotting volume of the reference medicinal material solution is 1-7 μL, such as 1 μL, 1.5 μL, 2 μL, 2.5 μL, 3 μL, 3.5 μL, 4 μL, 4.5 μL, 5 μL, 5.5 μL, 6 μL, 6.5 μL, 7 μL, etc. Other specific spot values ​​within this range can be selected, and will not be elaborated here.

[0024] Preferably, in the thin-layer chromatography analysis described in step (3), the developing solvent includes ethyl acetate, methanol, and water.

[0025] Preferably, the volume ratio of ethyl acetate, methanol, and water is (4-6):(1-2):1, where (4-6) can be selected from 4, 4.2, 4.4, 4.6, 4.8, 5, 5.2, 5.4, 5.6, 5.8, 6, etc., and (1-2) can be selected from 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0026] Preferably, in the thin-layer chromatography analysis described in step (3), the wavelength of the ultraviolet lamp is 360-370nm. For example, 360nm, 361nm, 362nm, 363nm, 364nm, 365nm, 366nm, 367nm, 368nm, 369nm, and 370nm are all possible values ​​within this range, which will not be elaborated here.

[0027] Preferably, in the thin-layer chromatography analysis, the thin-layer plate is a silica gel H thin-layer plate.

[0028] In a second aspect, the present invention provides an application of the thin-layer identification method according to the first aspect in the identification of Aconitum carmichaelii and / or processed Aconitum carmichaelii granules.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] This invention relates to a thin-layer chromatography (TLC) identification method for Aconitum carmichaelii and / or processed Aconitum carmichaelii granules. Based on the physicochemical properties of Aconitum carmichaelii, this method improves upon the existing TLC identification method for Aconitum carmichaelii in the Chinese Pharmacopoeia. It creatively incorporates a reference herb identification method for processed Aconitum carmichaelii granules, significantly increasing the method's specificity and providing a large amount of information. This overcomes the limitation of existing TLC identification methods that produce only single-spot chromatograms, enabling more accurate identification of Aconitum carmichaelii and processed Aconitum carmichaelii granules. Furthermore, the identification method involved in this invention no longer uses chloroform, which has been reported to be highly toxic, as the extraction solvent is low in toxicity, reducing environmental pollution. This method is simple to operate, highly specific, precise, sensitive, clear, and provides good separation, obvious spots, and good reproducibility. The solvent used is non-toxic, does not pollute the environment, and will not harm the health of operators, making it easy to promote and use. Attached Figure Description

[0031] Figure 1 This is the thin-layer chromatogram of Example 1;

[0032] Figure 2 This is the thin-layer chromatogram of Example 2;

[0033] Figure 3 This is the thin-layer chromatogram of Example 3;

[0034] Figure 4 This is the thin-layer chromatogram of Example 4;

[0035] Figure 5 This is the thin-layer chromatogram of Comparative Example 1;

[0036] Figure 6 This is the thin-layer chromatogram of Comparative Example 2;

[0037] Figure 7 This is the thin-layer chromatogram of Comparative Example 3;

[0038] Figure 8 It is a thin-layer chromatogram under normal temperature and humidity conditions;

[0039] Figure 9 It is a thin-layer chromatogram under normal temperature and low humidity conditions;

[0040] Figure 10 It is a thin-layer chromatogram under normal temperature and high humidity conditions;

[0041] Figure 11 It is a thin-layer chromatogram under low temperature and high humidity conditions;

[0042] Figure 12 The image shows the thin-layer chromatogram of Qingdao Dingkang H plate.

[0043] Figure 13 The image shows the thin-layer chromatogram of Yantai Jiangyou H. Detailed Implementation

[0044] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0045] Unless otherwise specified, the reagents and consumables used in the following embodiments were purchased from conventional reagent manufacturers in the art; unless otherwise specified, the experimental methods and techniques used were conventional methods and techniques in the art.

[0046] Instruments: Development tank, GOODLOOK-1000 thin-layer imaging system (Shanghai Kezhe Biochemical Technology Co., Ltd.), JY20002 electronic balance (Shanghai Shunyu Hengping Scientific Instrument Co., Ltd.), KQ-5200V ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.), ME155DU electronic balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.).

[0047] Test drugs: Prepared Aconitum carmichaelii granules (KL20240711, KL20240712, KL200240713) and Aconitum carmichaelii reference material (121338-201502, China National Institutes for Food and Drug Control).

[0048] Reagents: Methanol and ethyl acetate, both analytical grade (Tianjin Kemeio Chemical Reagent Co., Ltd.), and purified water.

[0049] Thin-layer chromatography plates: Thin-layer chromatography H silica gel plate (manufacturer: Yantai Jiangyou Silica Gel Development Co., Ltd., size 10×20cm, batch number: 20150506); Thin-layer chromatography H silica gel plate (manufacturer: Yantai Huayang New Material Technology Co., Ltd., size 100×200mm, batch number: 2024-04-08); Thin-layer chromatography H silica gel plate (manufacturer: Qingdao Dingkang Silica Gel Co., Ltd., size 100×200mm, batch number: 20221021).

[0050] Example 1

[0051] This embodiment provides a thin-layer chromatography method for identifying Aconitum carmichaelii and processed Aconitum carmichaelii granules, including the following steps:

[0052] Preparation of test solution: Weigh 0.5g of powder from each of the following formulations of Aconitum carmichaelii: KL20240711, KL20240712, and KL200240713. Add 30mL of methanol and sonicate (250W, 40kHz) for 30 minutes. Filter the solution, evaporate the filtrate to dryness, and dissolve the residue in 5mL of methanol to obtain the test solution.

[0053] Preparation of the reference herb solution: Take 5g of Aconitum carmichaelii reference herb, add 120mL of water, reflux at 100℃ for 2 hours, filter, evaporate the filtrate to dryness at 65℃, add 30mL of methanol to the residue, sonicate (250W, 40kHz) for 30 minutes, filter, evaporate the filtrate to dryness, add 5mL of methanol to dissolve the residue, and use as the reference herb solution.

[0054] Thin-layer chromatography conditions: Take 5 μL of the above reference herb solution and 10 μL of the test solution and spot them separately on the same silica gel H thin-layer plate (Yantai Huayang New Material Technology Co., Ltd.). Use ethyl acetate-methanol-water (volume ratio of 5:1.5:1) as the developing solvent, develop, remove, air dry, and examine at 365 nm.

[0055] The results are as follows Figure 1 As shown, 1 is the reference medicinal material, and 2-4 are the prepared Aconitum carmichaelii granules KL20240711, KL20240712, and KL200240713, respectively. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as those in the chromatogram of the reference medicinal material.

[0056] Example 2

[0057] This embodiment provides a thin-layer chromatography method for identifying Aconitum carmichaelii and processed Aconitum carmichaelii granules. The only difference between this method and Example 1 is the amount of sample applied in the thin-layer chromatography conditions. The amount of sample applied to the reference herbal solution is 1 μL, 3 μL, 5 μL, and 7 μL, respectively, and the amount of sample applied to the test solution (processed Aconitum carmichaelii granules KL20240711) is 5 μL, 10 μL, 15 μL, and 20 μL, respectively. All other conditions remain unchanged.

[0058] The results are as follows Figure 2 As shown, 1-4 represent 1 μL, 3 μL, 5 μL, and 7 μL of aconite reference material, respectively, and 5-8 represent 5 μL, 10 μL, 15 μL, and 20 μL of processed aconite granules, respectively. When the sample volume is 5 μL of reference material solution and 10 μL of test sample solution, the sample volume can clearly and accurately identify the processed aconite granules.

[0059] Example 3

[0060] This embodiment provides a thin-layer chromatography method for identifying aconite and processed aconite granules:

[0061] Preparation of the test solution: Same as in Example 1.

[0062] Preparation of the control herbal solution: Same as in Example 1.

[0063] Preparation of excipient dextrin solution: Weigh 0.5g of dextrin powder, add 30mL of methanol, sonicate (250W, 40kHz) for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 5mL of methanol to obtain the test solution.

[0064] Thin-layer chromatography conditions: Take 5 μL of the above reference herb solution and 10 μL of the test solution and spot them separately on the same silica gel H thin-layer plate (Yantai Huayang New Material Technology Co., Ltd.). Use ethyl acetate-methanol-water (volume ratio of 10:1.5:1) as the developing solvent, develop, remove, air dry, and examine at 365 nm.

[0065] The results are as follows Figure 3 As shown, 1 is the excipient dextrin solution, 2-3 are the control herbal solutions, and 4-6 are the processed Aconitum carmichaelii granules KL20240711.

[0066] Example 4

[0067] This embodiment provides a thin-layer chromatography method for identifying Aconitum carmichaelii and processed Aconitum carmichaelii granules. The only difference between this method and Example 3 is that the silica gel H thin-layer plate is replaced with a silica gel G thin-layer plate (Merck, catalog number: 1.05553.0001) in the thin-layer chromatography conditions, while all other conditions remain unchanged.

[0068] The results are as follows Figure 4 As shown, 1 is the excipient dextrin solution, 2-3 are the control herbal solution, and 4-5 are the processed Aconitum carmichaelii granules KL20240711.

[0069] Comparative Example 1

[0070] This comparative example provides a thin-layer chromatography method for identifying Aconitum carmichaelii and processed Aconitum carmichaelii granules, including the following steps:

[0071] Preparation of the test solution: Same as in Example 1.

[0072] Preparation of reference herb solution 1: Weigh 5g of Aconitum carmichaelii reference herb powder, add 30mL of methanol and sonicate (250W, 40kHz) for 30 minutes, filter, evaporate the filtrate to dryness, add 1mL of methanol to dissolve the residue, and use it as reference herb solution 1.

[0073] Preparation of reference herb solution 2: Weigh 5g of Aconitum carmichaelii reference herb powder, add 30mL of methanol, reflux in a water bath at 80℃ for 30 minutes, filter, evaporate the filtrate to dryness, add 1mL of methanol to dissolve the residue, and use it as reference herb solution 2.

[0074] Thin-layer chromatography conditions: Take 5 μL of the above reference herb solution and 15 μL of the test solution and spot them separately on the same silica gel H thin-layer plate (Yantai Huayang New Material Technology Co., Ltd.). Use ethyl acetate-methanol-water (volume ratio of 10:1.5:1) as the developing solvent, develop, remove, air dry, and examine at 365 nm.

[0075] The results are as follows Figure 5 As shown, 1-2 are the test solution, 3 is the reference herb solution 1, and 4 is the reference herb solution 2.

[0076] Comparative Example 2

[0077] This comparative example provides a method for identifying Aconitum carmichaelii (a method disclosed in Part I of the 2020 edition of the Chinese Pharmacopoeia), including the following steps:

[0078] Preparation of the test solution: Take 1g of Aconitum carmichaelii powder, moisten with 2mL of ammonia test solution, add 20mL of ether, sonicate (250W, 40kHz) for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1mL of isopropanol-chloroform (1:1) mixed solution to obtain the test solution.

[0079] Preparation of reference solution: Take aconite diester type alkaloid reference extract and add isopropanol-chloroform (1:1) mixed solution to prepare a mixed solution containing 3mg of each per 1mL, as the reference solution.

[0080] Thin-layer chromatography conditions: Perform the test according to the thin-layer chromatography method (General Rule 0502). Take 5 μL of the test solution and 10 μL of the reference extract solution and spot them separately on the same silica gel G thin-layer plate. Use n-hexane-ethyl acetate-methanol (6.4:3.6:1) as the developing solvent. Place the plate in a developing tank pre-saturated with ammonia vapor for 20 minutes. Develop, remove, air dry, spray with dilute potassium bismuth iodide test solution, and examine under sunlight.

[0081] The results are as follows Figure 6 As shown, 1 is a control extract of aconite diester alkaloids, and 2-3 are aconite medicinal materials.

[0082] Comparative Example 3

[0083] This comparative example provides a method for identifying processed Aconitum carmichaelii granules (a method disclosed in Part I of the 2020 edition of the Chinese Pharmacopoeia), including the following steps:

[0084] Preparation of the test solution: Take 2g of prepared Aconitum carmichaelii granules powder, moisten with 2mL of ammonia test solution, add 20mL of diethyl ether, sonicate (250W, 40kHz) for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1mL of dichloromethane to obtain the test solution.

[0085] Preparation of reference solution: Take benzoyl aconitine reference standard, benzoyl hypoaconitine reference standard, and benzoyl neoaconitine reference standard, add isopropanol-chloroform (1:1) mixed solution to prepare a mixed solution containing 20 μg benzoyl aconitine reference standard, 0.1 mg benzoyl hypoaconitine reference standard, and 80 μg benzoyl neoaconitine reference standard per 1 mL, as the reference solution.

[0086] Preparation of dextrin solution: Take 2g of dextrin powder, moisten with 2mL of ammonia test solution, add 20mL of diethyl ether, sonicate (250W, 40kHz) for 30 minutes, filter, evaporate the filtrate to dryness, and dissolve the residue in 1mL of dichloromethane.

[0087] Thin-layer chromatography conditions: Perform the test according to the thin-layer chromatography method (General Rule 0502). Take 5 μL of the test solution and 10 μL of the reference extract solution and spot them separately on the same silica gel G thin-layer plate. Use n-hexane-ethyl acetate-methanol (6.4:3.6:1) as the developing solvent. Place the plate in a developing tank pre-saturated with ammonia vapor for 20 minutes. Develop, remove, air dry, spray with dilute potassium bismuth iodide test solution, and examine under sunlight.

[0088] The results are as follows Figure 7 As shown, 1 is the excipient dextrin solution, 2 is the aconitine monoester type alkaloid mixed reference standard, and 3 is the prepared aconitine formula granules KL20240711.

[0089] Comparing Examples 1 and 3, it can be seen that the volume ratio of ethyl acetate, methanol and water in the developing solvent will affect the thin-layer identification method. When the volume ratio of ethyl acetate, methanol and water in Example 3 is 10:1.5:1, the chromatogram of the test sample solution lacks a bright spot at the corresponding position as the chromatogram of the reference medicinal material.

[0090] Comparing Examples 3 and 4, it can be seen that the silicone H thin film is superior to the silicone G thin film.

[0091] Comparing Example 1 and Comparative Example 1, it can be seen that after changing the sample preparation method and developing system, simply using methanol to extract the control medicinal material does not result in each spot corresponding to the test sample.

[0092] Comparing Example 1 and Comparative Examples 2-3, it can be seen that the thin-layer identification method provided by the present invention, compared with the method disclosed in the pharmacopoeia, produces thin-layer chromatograms with clear spots, more information, better separation, and moderate Rf values ​​of the main spots. It can display the same color main spots as the reference medicinal materials and can more accurately identify processed Aconitum carmichaelii.

[0093] Test Example 1

[0094] Durability assessment:

[0095] (1) Investigation of different temperatures and humidity

[0096] The method in Example 1 was repeated under different temperature and humidity conditions, and the results are shown in Figures 8-11. Figure 8 Under normal temperature and humidity conditions, the conditions are 21.7℃ and 30% relative humidity; Figure 9 The conditions are: ambient temperature and low temperature, 21.7℃, and relative humidity of 15%. Figure 10 The conditions were normal temperature and high humidity, specifically 21.7℃ and 80% relative humidity. Figure 11 The conditions were low temperature and high humidity, specifically 6℃ and 70% relative humidity. In the figure, 1 is the control herb, and 2-4 are the processed Aconitum carmichaelii granules KL20240711, KL20240712, and KL200240713, respectively.

[0097] Depend on Figure 8-11 It can be seen that under different temperature and humidity conditions, the chromatographic spots of the test sample solution and the reference medicinal material solution are clear, and the correspondence and separation effects are good, indicating that the thin-layer identification method has good durability under temperature and humidity.

[0098] (2) Investigation of different thin-layer plates

[0099] The method in Example 1 was repeated using different thin-layer plates, and the results were as follows: Figure 12 , 13 As shown, where Figure 12 , 13 Developing was performed using silica gel plates from two manufacturers: Qingdao Dingkang (H) and Yantai Jiangyou (H). In the figure, 1 represents the control herb, and 2-4 represent the processed Aconitum carmichaelii granules KL20240711, KL20240712, and KL200240713, respectively.

[0100] Depend on Figure 12 , 13 It can be seen that when different silica gel H thin-layer plates are used for development, the chromatographic spots of the test solution and the reference drug solution are clear, and the correspondence and separation effects are good. This indicates that the thin-layer identification method has good compatibility with silica gel H thin-layer plates from different manufacturers and the method is reliable.

[0101] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0102] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0103] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A thin-layer chromatography method for identifying aconite-based formula granules, characterized in that, The identification method includes the following steps: (1) Mix the aconite reference material with water, reflux to extract, filter, dry the filtrate and mix with methanol to obtain the reference material solution; (2) Mix the sample to be tested with methanol and sonicate to obtain the test solution; (3) Thin-layer chromatography analysis was performed on the reference herb solution and the test sample solution to identify the processed Aconitum carmichaelii formula granules; in the thin-layer chromatography analysis, the developing solvent was ethyl acetate, methanol and water in a volume ratio of (4-6):(1-2):1, the wavelength of the ultraviolet lamp was 360-370 nm, and the thin-layer plate was a silica gel H thin-layer plate. Steps (1) and (2) are not in any particular order.

2. The thin-layer identification method according to claim 1, characterized in that, In step (1), the ratio of the aconite reference material to water is 1 g : (20-30) mL.

3. The thin-layer identification method according to claim 1, characterized in that, The reflux extraction time is 1-3 h, and the reflux extraction temperature is 95-100℃.

4. The thin-layer identification method according to claim 1, characterized in that, Step (1) further includes ultrasonic treatment, filtration, drying of the filtrate, and mixing and dissolving it with methanol.

5. The thin-layer identification method according to claim 4, characterized in that, The ultrasonic treatment has a power of 200-300W, a frequency of 30-50 kHz, and a duration of 20-40 min.

6. The thin-layer identification method according to claim 1, characterized in that, In step (2), the ratio of the sample to methanol is 1 g : (50-70) mL.

7. The thin-layer identification method according to claim 1, characterized in that, The ultrasonic treatment in step (2) has a power of 200-300 W, a frequency of 30-50 kHz, and a duration of 20-40 min.

8. The thin-layer identification method according to claim 1, characterized in that, Step (2) includes filtration after ultrasonic treatment, and the filtrate is dried and then mixed with methanol to dissolve it.

9. The thin-layer identification method according to claim 1, characterized in that, In the thin-layer chromatography analysis described in step (3), the sample volume of the test solution is 5-20 μL.

10. The thin-layer identification method according to claim 1, characterized in that, In the thin-layer chromatography analysis described in step (3), the amount of the reference medicinal material solution spotted is 1-7 μL.

11. The application of the thin-layer identification method according to any one of claims 1-10 in the identification of processed Aconitum carmichaelii granules.

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