Thin-layer identification method for radix aconiti kusnezoffii and / or radix aconiti preparata formula granules

Abstract

The invention relates to a thin-layer identification method and application of kusnezoff monkshood root and / or prepared kusnezoff monkshood root formula granules, and the identification method comprises the following steps: (1) mixing a kusnezoff monkshood root reference medicinal material with water, and carrying out reflux extraction to obtain a reference medicinal material solution; (2) mixing a to-be-detected sample with methanol, and performing ultrasonic treatment to obtain a test solution; and (3) carrying out thin-layer chromatography analysis on the reference medicinal material solution and the test solution. According to the thin-layer identification method provided by the invention, a reference medicinal material identification method is creatively added for identifying the prepared kusnezoff monkshood root formula granules, so that the specificity of the method is greatly improved, the information amount is large, and the kusnezoff monkshood root and the prepared kusnezoff monkshood root formula granules can be identified more accurately; and the used solvent is non-toxic, does not pollute the environment, does not cause harm to the body health of operators, and is easy to popularize and use.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a thin-layer identification method for Kusnezoff Monkshood Root and / or Prepared Kusnezoff Monkshood Root formula granules. Background Art

[0002] Kusnezoff Monkshood Root is the dried tuberous root of Aconitum kusnezoffii Reichb. of the Ranunculaceae family. It is dug in autumn when the stems and leaves wither, the fibrous roots and sediment are removed, and then dried. This product is pungent, bitter, and hot, and highly toxic. It belongs to the heart, liver, kidney, and spleen meridians. It has the effects of dispelling wind and dampness, warming the meridians and relieving pain. It is used for wind-cold-damp arthralgia, joint pain, abdominal cold pain, cold hernia pain, and anesthesia for pain relief. Clinically, it is commonly used for wind-cold-damp arthralgia, joint pain, abdominal cold pain, cold hernia pain, and anesthesia for pain relief. Because of its high toxicity, it must be processed before clinical use, and the dosage must be strictly controlled.

[0003] In the 2020 edition of the Pharmacopoeia of the People's Republic of China (Volume I), in the item of Kusnezoff Monkshood Root, diester-type alkaloids of aconitum are identified, and in the item of Prepared Kusnezoff Monkshood Root, benzoylaconine, benzoylhypaconine, and benzoylmesaconine are identified, all of which are toxic components that need to control the content in Kusnezoff Monkshood Root. The thin-layer development conditions for Kusnezoff Monkshood Root and Prepared Kusnezoff Monkshood Root in the pharmacopoeia method are the same and are applicable to the identification of Prepared Kusnezoff Monkshood Root formula granules. However, the only reference substance identifications in this method are all alkaloids, which are relatively single and lack the specific identification of the medicinal material. At the same time, in the extraction method of the test sample, an isopropanol-chloroform (1:1) mixed solution is used for dissolution, and chloroform is a toxic reagent.

[0004] Currently, there are few relevant research reports on the thin-layer identification of Kusnezoff Monkshood Root medicinal materials. The existing thin-layer identification methods for Prepared Kusnezoff Monkshood Root formula granules only change the proportion of the developing agent on the basis of the pharmacopoeia method. They not only lack the identification method of Kusnezoff Monkshood Root reference medicinal material, but also have the disadvantages of being cumbersome, complex, time-consuming, polluting to the surrounding environment, high cost, and low efficiency.

[0005] Therefore, it has very important practical significance to improve the detection efficiency, reduce the detection cost, reduce environmental pollution, and create a rapid multi-information thin-layer identification method for Kusnezoff Monkshood Root and / or Prepared Kusnezoff Monkshood Root formula granules. Summary of the Invention

[0006] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a thin-layer identification method for Kusnezoff Monkshood Root and / or Prepared Kusnezoff Monkshood Root formula granules.

[0007] To achieve the purpose of this invention, the following technical solutions are adopted:

[0008] In the first aspect, the present invention provides a thin-layer identification method for Kusnezoff Monkshood Root and / or Prepared Kusnezoff Monkshood Root formula granules, and the identification method includes the following steps:

[0009] (1) Mix the radix aconiti kusnezoffii reference medicinal material with water, reflux extract, filter, and after drying the filtrate, mix it with methanol to obtain the reference medicinal material solution;

[0010] (2) Mix the test sample with methanol and perform ultrasonic treatment to obtain the test solution;

[0011] (3) Perform thin-layer chromatography analysis on the reference medicinal material solution and the test solution to identify the radix aconiti kusnezoffii and / or processed radix aconiti kusnezoffii formula granules;

[0012] Steps (1) and (2) do not distinguish the order.

[0013] Based on the chemical structures and properties of the active ingredients in traditional Chinese medicine, following the extraction principle of "like dissolves like", the present invention uses appropriate extraction solvents to simply and quickly prepare the test solution and reference solution. Then, through the movement of the developing agent, due to the different adsorption and desorption abilities of each component on the thin-layer plate, their spots can be separated. With the help of the characteristic functional groups of each component and using the color reaction, the characteristic components in different absorption bands are colored on the same thin-layer plate under the same inspection conditions, obtaining a thin-layer chromatogram with specificity, multiple information, and clear spots.

[0014] The thin-layer identification method of the radix aconiti kusnezoffii and / or processed radix aconiti kusnezoffii formula granules involved in the present invention is studied and improved on the basis of the existing thin-layer chromatography identification method of radix aconiti kusnezoffii in the Chinese Pharmacopoeia according to the physical and chemical properties of radix aconiti kusnezoffii. Creatively, the identification method of the reference medicinal material is added for the identification of processed radix aconiti kusnezoffii granules, greatly increasing the specificity of the method and having a large amount of information. It overcomes the defect that the spot information of the chromatogram obtained by the existing thin-layer identification method is single, and can more accurately identify the radix aconiti kusnezoffii and processed radix aconiti kusnezoffii formula granules. At the same time, the extraction solvent in the identification method involved in the present invention no longer uses the reported trichloromethane with relatively high toxicity, has low toxicity, and can effectively reduce environmental pollution. This method is simple to operate, has strong specificity, high precision and sensitivity, high clarity, good separation effect, obvious spots, and good reproducibility. The solvents used are non-toxic, do not pollute the environment, and will not cause harm to the physical health of operators, and are easy to promote and use.

[0015] Preferably, the material-liquid ratio of the radix aconiti kusnezoffii reference medicinal material to water in step (1) is 1 g:(20 - 30) mL, such as 1 g:20 mL, 1 g:21 mL, 1 g:22 mL, 1 g:23 mL, 1 g:24 mL, 1 g:25 mL, 1 g:26 mL, 1 g:27 mL, 1 g:28 mL, 1 g:29 mL, 1 g:30 mL, etc. Other specific point values within this numerical range can be selected and will not be elaborated one by one here.

[0016] Preferably, the time for reflux extraction is 1 - 3 h (such as 1 h, 1.2 h, 1.4 h, 1.6 h, 1.8 h, 2 h, 2.2 h, 2.4 h, 2.6 h, 2.8 h, 3 h, etc.), and the temperature for reflux extraction is 95 - 100 °C (such as 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 point values within the above numerical ranges can be selected and will not be elaborated here one by one.

[0017] Preferably, after mixing with methanol in step (1), it further includes ultrasonic treatment, filtration, and after drying the filtrate, it is mixed and dissolved with methanol.

[0018] Mix the Aconitum kusnezoffii Reichb. control medicinal material with water, perform reflux extraction, filtration, and after drying the filtrate, mix the residue with methanol, perform ultrasonic treatment, filtration, and after drying the filtrate, mix the residue with methanol for dissolution to obtain the control medicinal material solution.

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

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

[0021] Preferably, after the ultrasonic treatment in step (2), filtration is also included, and the filtrate is dried and then mixed and dissolved with methanol.

[0022] Preferably, in the thin layer chromatography analysis in step (3), the sample application 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 point values within this numerical range can be selected and will not be elaborated one by one here.

[0023] Preferably, in the thin layer chromatography analysis, the sample application volume of the control crude drug 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 point values within this numerical range can be selected and will not be elaborated one by one here.

[0024] Preferably, in the thin layer chromatography analysis in step (3), the developing agent 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 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 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, etc. Other specific point values within the above numerical ranges can be selected and will not be elaborated one by one here.

[0026] Preferably, in the thin-layer chromatography analysis of step (3), the wavelength for inspection under an ultraviolet lamp is 360 - 370 nm, such as 360 nm, 361 nm, 362 nm, 363 nm, 364 nm, 365 nm, 366 nm, 367 nm, 368 nm, 369 nm, 370 nm. Other specific point values within this numerical range can be selected and will not be elaborated one by one 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 Kusnezoff Monkshood and / or processed Kusnezoff Monkshood formula granules.

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

[0030] The thin-layer identification method for Kusnezoff Monkshood and / or processed Kusnezoff Monkshood formula granules involved in the present invention is studied and improved on the basis of the existing thin-layer chromatography identification method of Kusnezoff Monkshood in the Chinese Pharmacopoeia according to the physicochemical properties of Kusnezoff Monkshood. Creatively, a method for identifying reference crude drugs is added to the identification of processed Kusnezoff Monkshood granules, greatly increasing the specificity of the method, with a large amount of information, overcoming the defect of single spot information in the existing thin-layer identification method, and being able to more accurately identify Kusnezoff Monkshood and processed Kusnezoff Monkshood formula granules. At the same time, the extraction solvent in the identification method involved in the present invention no longer uses the reported trichloromethane with relatively high toxicity, has low toxicity, and reduces environmental pollution. This method is simple to operate, highly specific, with high precision, sensitivity, clarity, good separation effect, obvious spots, and good reproducibility. The solvents used are non-toxic, do not pollute the environment, and will not cause harm to the physical health of operators, and are easy to promote and use. Description of the Drawings

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

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

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

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

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

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

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

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

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

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

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

[0042] Figure 12 is a thin-layer chromatogram with the thin-layer plate being Qingdao Dingkang H;

[0043] Figure 13 is a thin-layer chromatogram with the thin-layer plate being Yantai Jiangyou H. Detailed implementation manners

[0044] The technical solutions of the present invention will be further described below through specific implementation manners. Those skilled in the art should understand that the described embodiments are only for helping to understand the present invention and should not be regarded as specific limitations on the present invention.

[0045] In the following embodiments, unless otherwise specified, the reagents and consumables used are purchased from conventional reagent manufacturers in the art; unless otherwise specified, the experimental methods and technical means used are conventional methods and means in the art.

[0046] Instruments: developing 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: processed kusnezoff monkshood formula granules (KL20240711, KL20240712, KL200240713), kusnezoff monkshood reference crude drug (121338 - 201502, National Institutes for Food and Drug Control).

[0048] Reagents: methanol, ethyl acetate, both are of analytical grade (Tianjin Kemiou Chemical Reagent Co., Ltd.), and water is purified water.

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

[0050] Example 1

[0051] This example provides a thin layer identification method for Kusnezoff Monkshood and Prepared Kusnezoff Monkshood formula granules, including the following steps:

[0052] Preparation of test solution: Weigh 0.5 g of the powder of Prepared Kusnezoff Monkshood formula granules KL20240711, KL20240712, and KL200240713 respectively, add 30 mL of methanol, ultrasonically treat (250 W, 40 kHz) for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 5 mL of methanol to obtain the test solution.

[0053] Preparation of control medicinal material solution: Take 5 g of Kusnezoff Monkshood control medicinal material, add 120 mL of water, reflux and extract at 100 °C for 2 hours, filter, spin-dry the filtrate at 65 °C, dissolve the residue in 30 mL of methanol, ultrasonically treat (250 W, 40 kHz) for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 5 mL of methanol to obtain the control medicinal material solution.

[0054] Thin layer chromatography conditions: Pipette 5 μL of the above control medicinal material solution and 10 μL of the test solution respectively, and spot them on the same silica gel H thin layer plate (Yantai Huayang New Material Technology Co., Ltd.). Use ethyl acetate - methanol - water (volume ratio 5:1.5:1) as the developing agent, develop, take out, dry in the air, and examine under 365 nm.

[0055] The results are as Figure 1 shown, where 1 is the control medicinal material, and 2 - 4 are the Prepared Kusnezoff Monkshood formula granules KL20240711, KL20240712, and KL200240713 respectively. In the chromatogram of the test solution, at the positions corresponding to the chromatogram of the control medicinal material, fluorescent spots of the same color appear.

[0056] Example 2

[0057] This example provides a thin layer identification method for Kusnezoff Monkshood and Prepared Kusnezoff Monkshood formula granules. The difference from Example 1 is only the sample application amount in the thin layer chromatography conditions. The sample application amounts of the control medicinal material solution are 1 μL, 3 μL, 5 μL, and 7 μL respectively, and the sample application amounts of the test solution (Prepared Kusnezoff Monkshood formula granules KL20240711) are 5 μL, 10 μL, 15 μL, and 20 μL respectively, and other conditions remain unchanged.

[0058] The results are as follows Figure 2 shown in which 1 - 4 are 1 μL, 3 μL, 5 μL, and 7 μL of the control crude Kusnezoff Monkshood root respectively, and 5 - 8 are 5 μL, 10 μL, 15 μL, and 20 μL of the processed Kusnezoff Monkshood root formula granules respectively. When the sample application volume is 5 μL for the control crude drug solution and 10 μL for the test sample solution, the sample application volume can clearly and accurately identify the processed Kusnezoff Monkshood root formula granules.

[0059] Example 3

[0060] This example provides a thin - layer chromatography identification method for crude Kusnezoff Monkshood root and processed Kusnezoff Monkshood root formula granules:

[0061] Preparation of the test sample solution: The same as in Example 1.

[0062] Preparation of the control crude drug solution: The same as in Example 1.

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

[0064] Thin - layer chromatography conditions: Pipette 5 μL of the above - mentioned control crude drug solution and 10 μL of the test sample solution, and spot them on the same silica gel H thin - layer plate (Yantai Huayang New Material Technology Co., Ltd.) respectively. Use ethyl acetate - methanol - water (volume ratio 10:1.5:1) as the developing agent, develop, take out, dry in the air, and examine under 365 nm.

[0065] The results are as follows Figure 3 shown in which 1 is the excipient dextrin solution, 2 - 3 are the control crude drug solutions, and 4 - 6 are the processed Kusnezoff Monkshood root formula granules KL20240711.

[0066] Example 4

[0067] This example provides a thin - layer chromatography identification method for crude Kusnezoff Monkshood root and processed Kusnezoff Monkshood root formula granules, and the difference from Example 3 is only that the silica gel H thin - layer plate is replaced with a silica gel G thin - layer plate (Merck, product number: 1.05553.0001) in the thin - layer chromatography conditions, and other conditions remain unchanged.

[0068] The results are as follows Figure 4 shown in which 1 is the excipient dextrin solution, 2 - 3 are the control crude drug solutions, and 4 - 5 are the processed Kusnezoff Monkshood root formula granules KL20240711.

[0069] Comparative Example 1

[0070] This comparative example provides a thin - layer chromatography identification method for crude Kusnezoff Monkshood root and processed Kusnezoff Monkshood root formula granules, including the following steps:

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

[0072] Preparation of the control crude drug solution 1: Weigh 5 g of the powder of Aconitum kusnezoffii Reichb. as the control crude drug, add 30 mL of methanol, and ultrasonically treat (250 W, 40 kHz) for 30 minutes. Filter, evaporate the filtrate to dryness, dissolve the residue in 1 mL of methanol to obtain the control crude drug solution 1.

[0073] Preparation of the control crude drug solution 2: Weigh 5 g of the powder of Aconitum kusnezoffii Reichb. as the control crude drug, add 30 mL of methanol, reflux in a water bath at 80 °C for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 mL of methanol to obtain the control crude drug solution 2.

[0074] Thin-layer chromatography conditions: Pipette 5 μL of the above-mentioned control crude drug solution and 15 μL of the test solution respectively, and spot them on the same silica gel H thin-layer plate (Yantai Huayang New Materials Technology Co., Ltd.). Use ethyl acetate - methanol - water (volume ratio 10:1.5:1) as the developing agent, develop, take out, air dry, and examine under 365 nm.

[0075] The results are as Figure 5 shown, where 1 - 2 are the test solutions, 3 is the control crude drug solution 1, and 4 is the control crude drug solution 2.

[0076] Comparative Example 2

[0077] This comparative example provides a method for identifying Aconitum kusnezoffii Reichb. (a method disclosed in the first volume of the Chinese Pharmacopoeia 2020 edition), including the following steps:

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

[0079] Preparation of the reference substance solution: Take the reference extract of aconitine diester alkaloids, add the mixed solution of isopropanol - chloroform (1:1) to make a mixed solution containing 3 mg per 1 mL as the reference substance solution.

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

[0081] The results are as Figure 6 shown, where 1 is the reference extract of aconitine diester alkaloids, and 2 - 3 are the Aconitum kusnezoffii Reichb.

[0082] Comparative Example 3

[0083] This comparative example provides a method for identifying processed Kusnezoff Monkshood Formula Granules (a method disclosed in Part I of the Chinese Pharmacopoeia 2020 Edition), including the following steps:

[0084] Preparation of the test solution: Take 2 g of the powder of processed Kusnezoff Monkshood Formula Granules, moisten it with 2 mL of ammonia test solution, add 20 mL of ether, ultrasonically treat (250 W, 40 kHz) for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 mL of dichloromethane to obtain the test solution.

[0085] Preparation of the reference solution: Take reference standards of benzoylaconine, benzoylhypaconine, and benzoylmesaconine, and prepare a mixed solution containing 20 μg of benzoylaconine reference standard, 0.1 mg of benzoylhypaconine reference standard, and 80 μg of benzoylmesaconine reference standard per 1 mL with an isopropanol-chloroform (1:1) mixed solution as the reference solution.

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

[0087] Thin-layer chromatography conditions: 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 on the same silica gel G thin-layer plate respectively. Use hexane-ethyl acetate-methanol (6.4:3.6:1) as the developing solvent, place it in a developing tank pre-saturated with ammonia vapor for 20 minutes, develop, take out, dry, spray with dilute bismuth potassium iodide test solution, and examine under daylight.

[0088] The results are as Figure 7 shown, where 1 is the excipient dextrin solution, 2 is the mixed reference standard of aconitum monoester alkaloids, and 3 is the processed Kusnezoff Monkshood Formula Granules KL20240711.

[0089] Comparing Example 1 and Example 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, in the chromatogram of the test solution, there is a missing bright spot at the corresponding position of the chromatogram of the reference medicinal material.

[0090] Comparing Example 3 and Example 4, it can be seen that the silica gel H thin-layer plate has a better effect than the silica gel G thin-layer plate.

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

[0092] Comparing Example 1 with Comparative Examples 2-3, it can be seen that the thin-layer identification method provided by the present invention has clearer spots, more information, better resolution, a moderate Rf value of the main spot on the thin-layer chromatogram compared with the method disclosed in the pharmacopoeia, and can show the main spot of the same color as the control medicinal material, and can more accurately identify Radix Aconiti Kusnezoffii Preparata.

[0093] Test Example 1

[0094] Durability investigation:

[0095] (1) Investigation under different temperature and humidity conditions

[0096] The method in Example 1 was repeated under different temperature and humidity conditions, and the results are shown in Figures 8-11, where Figure 8 is the case of normal temperature and normal humidity, with the conditions of 21.7 °C and a relative humidity of 30%; Figure 9 is the case of normal temperature and low humidity, with the conditions of 21.7 °C and a relative humidity of 15%; Figure 10 is the case of normal temperature and high humidity, with the conditions of 21.7 °C and a relative humidity of 80%; Figure 11 is the case of low temperature and high humidity, with the conditions of 6 °C and a relative humidity of 70%. In the figure, 1 is the control medicinal material, and 2-4 are the Radix Aconiti Kusnezoffii Preparata formula granules KL20240711, KL20240712, and KL200240713 respectively.

[0097] From Figures 8 - 11 it can be seen that under different temperature and humidity conditions, the chromatographic spots of the test solution and the control medicinal material solution are clear, and the corresponding property and separation effect are both good, indicating that the thin-layer identification method has good durability to 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 are as shown in Figure 12 , 13 where Figure 12 , 13 were developed using silica gel H plates from two manufacturers, Qingdao Dingkang H and Yantai Jiangyou H respectively. In the figure, 1 is the control medicinal material, and 2-4 are the Radix Aconiti Kusnezoffii Preparata formula granules KL20240711, KL20240712, and KL200240713 respectively.

[0100] From Figure 12 , 13 it can be seen that when developing using different silica gel H thin-layer plates, the chromatographic spots of the test solution and the control medicinal material solution are clear, and the corresponding property and separation effect are both good, indicating 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 present invention illustrates the technical solution of the present invention through the above embodiments, but the present invention is not limited to the above embodiments, that is, it does not mean that the present invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvement to the present invention, the equivalent replacement of each raw material of the present invention's products, the addition of auxiliary components, the selection of specific methods, etc., all fall within the protection scope and the disclosure scope of the present 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 belong to the protection scope of the present invention.

[0103] In addition, it should be noted that, in the case of no contradiction, the various specific technical features described in the above specific embodiments can be combined in any appropriate manner. In order to avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

Claims

1. A thin-layer identification method for Aconitum kusnezoffii Reichb. and / or processed Aconitum kusnezoffii Reichb. formula granules, characterized in that, The identification method includes the following steps: (1) Mix the radix aconiti kusnezoffii preparata reference medicinal material with water, carry out reflux extraction, filter, and after drying the filtrate, mix it with methanol to obtain a reference medicinal material solution; (2) Mix the sample to be tested with methanol and perform ultrasonic treatment to obtain a test solution; (3) Perform thin-layer chromatography analysis on the reference medicinal material solution and the test solution to identify the radix aconiti kusnezoffii and / or processed radix aconiti kusnezoffii formula granules; Steps (1) and (2) do not distinguish the order.

2. The thin layer identification method according to claim 1, wherein In step (1), the material-liquid ratio of the radix aconiti kusnezoffii preparata reference medicinal material to water is 1 g:(20 - 30) mL; Preferably, the time for the reflux extraction is 1 - 3 h, and the temperature for the reflux extraction is 95 - 100 °C.

3. The thin layer identification method according to claim 1 or 2, characterized in that After mixing with methanol in step (1), it further includes ultrasonic treatment, filtration, and after drying the filtrate, mixing and dissolving it with methanol; Preferably, the power of the ultrasonic treatment is 200 - 300 W, the frequency is 30 - 50 kHz, and the time for the ultrasonic treatment is 20 - 40 min.

4. The thin layer identification method according to any one of claims 1-3, characterized in that, In step (2), the material-liquid ratio of the sample to be tested to methanol is 1 g:(50 - 70) mL.

5. The thin layer identification method according to any one of claims 1-4, characterized in that, In step (2), the power of the ultrasonic treatment is 200 - 300 W, the frequency is 30 - 50 kHz, and the time for the ultrasonic treatment is 20 - 40 min.

6. The thin-layer identification method according to any one of claims 1-4, characterized in that, After the ultrasonic treatment in step (2), it further includes filtration, and after drying the filtrate, mixing and dissolving it with methanol.

7. The thin layer identification method according to any one of claims 1-5, characterized in that In the thin-layer chromatography analysis in step (3), the sample application amount of the test solution is 5 - 20 μL; Preferably, in the thin-layer chromatography analysis, the sample application amount of the reference medicinal material solution is 1 - 7 μL.

8. The thin layer identification method according to any one of claims 1-7, characterized in that In the thin-layer chromatography analysis in step (3), the developing agent includes ethyl acetate, methanol, and water; Preferably, the volume ratio of ethyl acetate, methanol, and water is (4 - 6):(1 - 2):

1.

9. The thin layer identification method according to any one of claims 1-7, characterized in that, In the thin-layer chromatography analysis in step (3), the wavelength for ultraviolet lamp inspection is 360 - 370 nm; Preferably, in the thin-layer chromatography analysis, the thin-layer plate is a silica gel H thin-layer plate.

10. Use of the thin-layer identification method according to any one of claims 1 - 9 in the identification of radix aconiti kusnezoffii and / or processed radix aconiti kusnezoffii formula granules.

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