Method for identifying cumin and application thereof

Abstract

The application discloses a method for identifying cumin and application thereof, and comprises the following steps: A, adding cumin medicinal material powder or preparation powder into methanol to prepare a test sample solution; B, adding cumin control medicinal material powder into methanol to prepare a control medicinal material solution; C, using cyclohexane-ethyl acetate as a developing agent to develop; D, pattern identification; the method can be applied to identification of cumin medicinal material or cumin-containing medicinal material preparation. Two obvious characteristic spots can be obtained after development, and the separation degree is good; the obtained characteristic spots are stable, the boundary is clear, and can be observed after long-time placement; the treatment process does not need heating, does not need to use flammable, explosive and drug-making ether, and thin layer identification can be directly carried out after ultrasonic treatment and filtration, so that the operation is simple, and the test time is greatly shortened.

Description

TECHNICAL FIELD

[0001] The application relates to a method for identifying Carum carvi L. and application thereof, in particular to a method for identifying Carum carvi L. based on a thin-layer chromatogram, and belongs to the technical field of medicines. BACKGROUND

[0002] Carum carvi L., also known as caraway, is the fruit of Carum carvi L. and is a commonly used medicinal material in Tibetan medicine. The main effective components of Carum carvi L. are volatile oils such as carvone, dihydrocarvone and d-limonene, and the clinical pharmacological effects of Carum carvi L. include antibacterial, antiepileptic, blood lipid regulation, liver protection, cough relieving and asthma relieving, gastrointestinal protection and antioxidant effects.

[0003] At present, the medicinal material of Carum carvi L. is subject to the standard No. WS3-BC-0129-95-2022 (hereinafter referred to as the department standard), and the identification method in the standard is as follows: after the sample and the Carum carvi L. control medicinal material powder are treated with petroleum ether (60-90 DEG C)-diethyl ether (1:1), petroleum ether (60-90 DEG C)-ethyl acetate (17:2.5) is used as the developing agent, and the sample is colored by spraying with dinitrophenylhydrazine, and then observed under daylight. However, it is found through repeated experiments that the pretreatment of the method is slightly cumbersome, there is only one developing spot, the color is relatively light, and the sample needs to be observed immediately after coloring. In addition, the existing technology also provides other thin-layer identification methods for Carum carvi L., such as in the “Research on the Thin-Layer Chromatography Identification Method of Fifteen-Ingredient Luti Mingmu Pill of Tibetan Medicine” (Chinese National and Folk Medicine; No. 15, 2011, Document 1), the sample is extracted and concentrated with diethyl ether, petroleum ether: ethyl acetate (5:2) is used as the developing agent (it is found through repeated experiments that a more expensive and delicate Merck plate is needed), and the sample is observed under a 365 nm lamp. In the “Research on the Quality Standard of Fifteen-Ingredient Luti Mingmu Tablet of Tibetan Medicine” (Qinghai Journal of Medicine, Vol. 40, No. 7, 2010, Document 2) and “Qinghai Province Processing Specification (2010)”, the thin-layer identification of Carum carvi L. is as follows: after the sample and the Carum carvi L. control medicinal material are treated by refluxing with methanol, the developing agent is ethyl acetate-methanol-water-formic acid (15:5:1:1), and the developing spot is observed under ultraviolet light of 365 nm. In the “GC Content Determination and Thin-Layer Identification of Main Components Carvone and Limonene in Spearmint Oil” (Chinese Journal of Chinese Medicine, Vol. 32, No. 3, 2014, Document 3), the sample is treated with n-hexane, n-hexane-ethyl acetate (12:1) is used as the developing agent, the sample is colored by spraying with 5% vanillin-concentrated sulfuric acid and heated. However, no matter the reported literatures or the repeated experiments, the developing spots are not ideal, most of which are in the form of fuzzy long bands, the color is light, the boundary is fuzzy or overlapped, there is one relatively obvious spot, but another spot cannot be effectively separated through careful observation (if a more delicate and expensive M plate is used for development, two spots with different colors can be distinguished which are closely adjacent or overlapped), and the identification chromatogram is not convenient for judgment. SUMMARY

[0004] In order to solve the defects and deficiencies existing in the prior art, the application provides a method for identifying Ferula sinkiangensis K.M.Shen, which is easy to operate, has good separation degree, strong stability, strong specificity, good reproducibility and can effectively identify Ferula sinkiangensis K.M.Shen and its preparations.

[0005] The technical scheme of the application is as follows:

[0006] A method for identifying Ferula sinkiangensis K.M.Shen, comprising the following steps:

[0007] A, preparation of a test sample solution: 1 part by weight of Ferula sinkiangensis K.M.Shen powder or 2 parts by weight of preparation powder of Ferula sinkiangensis K.M.Shen is added to methanol with a volume-weight ratio of 6-18, refluxed for 30 minutes or ultrasonically treated for 10-20 minutes, cooled to room temperature, filtered, and the filtrate of Ferula sinkiangensis K.M.Shen is taken as the test sample solution, or the filtrate of the preparation powder of Ferula sinkiangensis K.M.Shen is further concentrated to a concentrated solution with a volume of half that of the filtrate, and the concentrated solution is taken as the test sample solution;

[0008] B, preparation of a control medicinal material solution: 0.25 parts by weight of Ferula sinkiangensis K.M.Shen control medicinal material powder is added to methanol with a volume-weight ratio of 16-24, ultrasonically treated for 10-20 minutes, cooled to room temperature, filtered, and the filtrate is taken to prepare the control medicinal material solution;

[0009] C, thin layer chromatography development: first, a silica gel plate is dried and activated at 110 DEG C for 30 minutes, then 5 μL or 10 μL of the control medicinal material solution and the test sample solution are taken and spotted on the same silica gel plate, dried by a hair dryer, placed in a development tank for pre-saturation for 15 minutes, and developed with cyclohexane-ethyl acetate with a volume ratio of 8-15:1 as a developing agent, taken out, dried, sprayed with 5% vanillin sulfuric acid solution, and heated at 105 DEG C until the spots are clearly colored;

[0010] D, identification of the atlas: the size, position, color and Rf value of each spot in the identification atlas are observed and compared.

[0011] Preferably, in the preparation process of the test sample solution, methanol with a volume-weight ratio of 10-15 is added.

[0012] Preferably, in the preparation process of the test sample solution, methanol with a volume-weight ratio of 15 is added.

[0013] Preferably, in the preparation process of the control medicinal material solution, methanol with a volume-weight ratio of 20 is added.

[0014] Preferably, in the preparation process of the control medicinal material solution, ultrasonic treatment is adopted, and the time is 10 minutes.

[0015] Preferably, the developing agent is cyclohexane-ethyl acetate with a volume ratio of 10-12:1.

[0016] Further preferably, the developing agent is cyclohexane-ethyl acetate with a volume ratio of 10:1.

[0017] Further preferably, the developing silica gel plate is G plate or M plate.

[0018] The application further provides application of the identification method of the Saussurea lavaulis Maxim in identification of Saussurea lavaulis Maxim or a preparation containing Saussurea lavaulis Maxim.

[0019] Advantages of the application

[0020] 1. Two obvious characteristic spots can be obtained after development, and the resolution is good.

[0021] 2. The characteristic spots obtained after development are stable, and the boundary is clear, which can be observed after long-time placement.

[0022] 3. The method is simple to operate, and the process does not need heating, flammable, explosive and easy-to-poison ether, and thin layer identification can be directly performed after ultrasonic treatment and filtration, which is simple to operate and greatly shortens the test time. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The figure is obtained by repeating the method in literature 1, and the developing agent is petroleum ether: ethyl acetate (5:2), wherein 1, 3 and 5 are samples of Liuwei Mingmu pills, 2 is Saussurea lavaulis Maxim, and 4 is a negative control;

[0024] Figure 2 The figure is obtained by repeating the standard method, and the developing agent is petroleum ether: ethyl acetate (17:2.5), wherein 1-3 are carvone, limonene and anise aldehyde reference substances respectively, 4 and 9 are Saussurea lavaulis Maxim control samples of the same batch, 5-6 are Saussurea lavaulis Maxim samples of different batches, 7 is Liuwei Mingmu pills, and 8 is a negative control;

[0025] Figure 3 The figure is obtained by repeating the method in literature 2, and the developing agent is ethyl acetate-methanol-water-formic acid (15:5:1:1), wherein 1-2 are Saussurea lavaulis Maxim control samples of the same batch, and 3-4 are Saussurea lavaulis Maxim samples of different batches;

[0026] Figure 4 The figure is obtained by repeating the method in literature 3, and the developing agent is n-hexane: ethyl acetate (12:1), G plate, wherein 1-2 are Saussurea lavaulis Maxim control samples of the same batch, and 3-4 are Saussurea lavaulis Maxim samples of different batches;

[0027] Figure 5 The figure is obtained by repeating the method in literature 3, and the developing agent is n-hexane: ethyl acetate (12:1), M plate, wherein 1 is Saussurea lavaulis Maxim control, 2 is carvone control, 3 and 4 are Saussurea lavaulis Maxim samples of different batches treated by centrifugation, and 5 and 6 are Saussurea lavaulis Maxim samples of different batches treated by filtration.

[0028] Figure 6 To improve the method of the literature 3, the developing agent is changed to cyclohexane: ethyl acetate (12:1), in which 1 is the control of Nardostachys chinensis Batal. (treated with ethyl acetate), 2 is the medicinal material of Nardostachys chinensis Batal. (treated with ethyl acetate), 3 and 4 are Liuwei Mingmu pills containing different batches of Nardostachys chinensis Batal. (treated with ethyl acetate), 5 and 6 are Liuwei Mingmu preparations containing different batches of Nardostachys chinensis Batal. (treated with methanol), 7 and 8 are the medicinal material of Nardostachys chinensis Batal. (treated with methanol), and 9 is the control of Nardostachys chinensis Batal. (treated with methanol);

[0029] Figure 7 The spectrum obtained by the identification method of the application is shown in the following table, in which "DZ" at both ends is the control of Nardostachys chinensis Batal. medicinal material, and S1-S9 are Nardostachys chinensis Batal. samples;

[0030] Figure 8 The spectrum obtained by the identification method of the application is shown in the following table, in which 1 is the control of carvone, 2 is the control of Nardostachys chinensis Batal. medicinal material, 3 and 4 are Nardostachys chinensis Batal. medicinal material samples, 5 and 6 are Liuwei Mingmu pills containing Nardostachys chinensis Batal., and 7 is the negative control. DETAILED DESCRIPTION

[0031] The following experimental examples and embodiments are used to further illustrate but not limit the application.

[0032] Embodiment 1, a method for identifying Nardostachys chinensis Batal., the steps are as follows:

[0033] A, preparation of test sample solution: take 1 g of fine powder of Nardostachys chinensis Batal. medicinal material, add 15 mL of methanol, ultrasonic treatment for 10 minutes, cool to room temperature, filter, take the filtrate, as the test sample solution;

[0034] B, preparation of control medicinal material solution: take 0.25 g of fine powder of Nardostachys chinensis Batal. control medicinal material, add 5 mL of methanol, ultrasonic treatment for 10 minutes, cool to room temperature, filter, take the filtrate, as the control medicinal material solution;

[0035] C, thin layer identification: first activate the silica gel plate at 110°C for 30 minutes, then take 5 μL of the control medicinal material solution and the test sample solution respectively, and point them on the same silica gel G plate, blow dry, and then place in the development tank for pre-saturation for 15 minutes, use cyclohexane-ethyl acetate (10:1) as the developing agent, develop, take out, air dry, spray 5% vanillin sulfuric acid solution, and heat at 105°C until the spots are clear;

[0036] D, spectrum identification: observe and compare the size, position, color and Rf value of each spot in the identification spectrum

[0037] Embodiment 2, a method for identifying Nardostachys chinensis Batal., the steps are as follows:

[0038] A. Preparation of test solution: Take 1 g of finely powdered Nardostachys chinensis Batal. as the sample, add 6 mL of methanol, and ultrasonically treat for 10 minutes. Cool to room temperature, filter, and take the filtrate as the test solution.

[0039] B. Preparation of control solution: Take 0.25 g of finely powdered Nardostachys chinensis Batal. as the control sample, add 4 mL of methanol, and ultrasonically treat for 10 minutes. Cool to room temperature, filter, and take the filtrate as the control solution.

[0040] C. Thin-layer identification: Activate the silica gel plate by drying at 110°C for 30 minutes. Take 5 μL of the control solution and the test solution, and spot them on the same silica gel G plate. Dry with a hair dryer, and then place the plate in a developing jar and pre-saturate for 15 minutes. Use a mixture of cyclohexane and ethyl acetate (8:1 by volume) as the developing agent, develop, remove, dry, spray with 5% vanillin sulfuric acid solution, and heat at 105°C until the spots are clearly colored.

[0041] D. Identification by chromatogram: Observe the size, position, color, and Rf value of each spot in the identification chromatogram.

[0042] Example 3: A method for identifying Nardostachys chinensis, comprising the following steps:

[0043] A. Preparation of test solution: Take 1 g of finely powdered Nardostachys chinensis Batal. as the sample, add 18 mL of methanol, and ultrasonically treat for 15 minutes. Cool to room temperature, filter, and take the filtrate as the test solution.

[0044] B. Preparation of control solution: Take 0.25 g of finely powdered Nardostachys chinensis Batal. as the control sample, add 5 mL of methanol, and ultrasonically treat for 15 minutes. Cool to room temperature, filter, and take the filtrate as the control solution.

[0045] C. Thin-layer identification: Activate the silica gel plate by drying at 110°C for 30 minutes. Take 10 μL of the control solution and the test solution, and spot them on the same silica gel plate. Dry with a hair dryer, and then place the plate in a developing jar and pre-saturate for 15 minutes. Use a mixture of cyclohexane and ethyl acetate (15:1 by volume) as the developing agent, develop, remove, dry, spray with 5% vanillin sulfuric acid solution, and heat at 105°C until the spots are clearly colored.

[0046] D. Identification by chromatogram: Observe the size, position, color, and Rf value of each spot in the identification chromatogram.

[0047] Example 4: A method for identifying Nardostachys chinensis, comprising the following steps:

[0048] A. Preparation of test solution: Take 1 g of finely powdered Nardostachys chinensis Batal. as the sample, add 10 mL of methanol, and reflux for 30 minutes. Cool to room temperature, filter, and take the filtrate as the test solution.

[0049] B. Preparation of the reference medicinal material solution: 0.25 g of finely powdered Nardostachys chinensis reference medicinal material was added to 6 mL of methanol, and ultrasonic treatment was performed for 10 minutes. After cooling to room temperature, filtration was performed, and the filtrate was collected to prepare the reference medicinal material solution;

[0050] C. Thin layer identification: the silica gel plate was activated by drying at 110°C for 30 minutes. Then, 10 μL of the reference medicinal material solution and the test sample solution were spotted on the same silica gel M plate, which was dried by a hair dryer and then placed in a developing jar for 15 minutes of pre-saturation. The developing agent was a mixture of cyclohexane and ethyl acetate (12:1 by volume), and the plate was developed, removed, dried, and sprayed with 5% vanillin sulfuric acid solution, and heated at 105°C until the spots were clearly colored.

[0051] D. Identification by chromatogram: the size, position, color, and Rf value of each spot in the identification chromatogram were observed.

[0052] Example 5: A method for identifying Nardostachys chinensis, the steps of which are as follows:

[0053] A. Preparation of the test sample solution: 1 g of finely powdered Nardostachys chinensis medicinal material was added to 15 mL of methanol, and reflux was performed for 30 minutes. After cooling to room temperature, filtration was performed, and the filtrate was collected as the test sample solution.

[0054] B. Preparation of the reference medicinal material solution: 0.25 g of finely powdered Nardostachys chinensis reference medicinal material was added to 5 mL of methanol, and ultrasonic treatment was performed for 15 minutes. After cooling to room temperature, filtration was performed, and the filtrate was collected to prepare the reference medicinal material solution.

[0055] C. Thin layer identification: the silica gel plate was activated by drying at 110°C for 30 minutes. Then, 5 μL of the reference medicinal material solution and the test sample solution were spotted on the same silica gel G plate, which was dried by a hair dryer and then placed in a developing jar for 15 minutes of pre-saturation. The developing agent was a mixture of cyclohexane and ethyl acetate (14:1 by volume), and the plate was developed, removed, dried, and sprayed with 5% vanillin sulfuric acid solution, and heated at 105°C until the spots were clearly colored.

[0056] D. Identification by chromatogram: the size, position, color, and Rf value of each spot in the identification chromatogram were observed.

[0057] Example 6: A method for identifying Nardostachys chinensis, the steps of which are as follows:

[0058] A. Preparation of the test sample solution: 1 g of finely powdered Nardostachys chinensis medicinal material was added to 15 mL of methanol, and ultrasonic treatment was performed for 10 minutes. After cooling to room temperature, filtration was performed, and the filtrate was collected as the test sample solution.

[0059] B. Preparation of the reference medicinal material solution: 0.25 g of finely powdered Nardostachys chinensis reference medicinal material was added to 5 mL of methanol, and ultrasonic treatment was performed for 10 minutes. After cooling to room temperature, filtration was performed, and the filtrate was collected to prepare the reference medicinal material solution.

[0060] C. TLC identification: First, activate the silica gel plate at 110°C for 30 minutes, then take 10 μL of the control medicinal material solution and the test solution and point them on the same silica gel M plate, dry them with a hair dryer, and then place them in the developing tank for 15 minutes of pre-saturation. Use cyclohexane-ethyl acetate (12:1 by volume) as the developing agent, develop, take out, dry, spray 5% vanillin sulfuric acid solution, and heat at 105°C until the spots are clearly colored;

[0061] D. Spectrum identification: Observe the size, position, color, and Rf value of each spot in the comparative identification spectrum.

[0062] Example 7. A method for identifying Cnidium monnieri in Liuwei Mingmu Pills, the steps of which are as follows:

[0063] A. Preparation of the test solution: Take 2 g of Liuwei Mingmu Pills containing Cnidium monnieri medicinal material, add 10 mL of methanol, and ultrasonically treat for 10 minutes. Cool to room temperature, filter, and take the filtrate, which is concentrated to half the volume to serve as the test solution.

[0064] B. Preparation of the control medicinal material solution: Take 0.25 g of Cnidium monnieri control medicinal material powder, add 4 mL of methanol, and ultrasonically treat for 15 minutes. Cool to room temperature, filter, and take the filtrate to prepare the control medicinal material solution.

[0065] C. TLC identification: First, activate the silica gel plate at 110°C for 30 minutes, then take 10 μL of the control medicinal material solution and the test solution and point them on the same silica gel G plate, dry them with a hair dryer, and then place them in the developing tank for 15 minutes of pre-saturation. Use cyclohexane-ethyl acetate (10:1 by volume) as the developing agent, develop, take out, dry, spray 5% vanillin sulfuric acid solution, and heat at 105°C until the spots are clearly colored.

[0066] D. Spectrum identification: Observe the size, position, color, and Rf value of each spot in the comparative identification spectrum.

[0067] Example 8. A method for identifying Cnidium monnieri in Liuwei Mingmu Pills, the steps of which are as follows:

[0068] A. Preparation of the test solution: Take 1 g of Liuwei Mingmu Pills containing Cnidium monnieri medicinal material, add 15 mL of methanol, and ultrasonically treat for 15 minutes. Cool to room temperature, filter, and take the filtrate to serve as the test solution.

[0069] B. Preparation of the control medicinal material solution: Take 0.25 g of Cnidium monnieri control medicinal material powder, add 5 mL of methanol, and ultrasonically treat for 10 minutes. Cool to room temperature, filter, and take the filtrate to prepare the control medicinal material solution.

[0070] C. TLC identification: First, activate the silica gel plate at 110°C for 30 minutes, then take 5 μL of the control medicinal material solution and the test solution and point them on the same silica gel M plate, dry them with a hair dryer, and then place them in the developing tank for 15 minutes of pre-saturation. Use cyclohexane-ethyl acetate (12:1 by volume) as the developing agent, develop, take out, dry, spray 5% vanillin sulfuric acid solution, and heat at 105°C until the spots are clearly colored;

[0071] D. Spectrum identification: Observe the size, position, color, and Rf value of each spot in the comparative identification spectrum.

[0072] Example 9, a method for identifying Cnidium monnieri in Liuwei Mingmu Pills, the steps are as follows:

[0073] A. Preparation of test solution: Take 2 g of Liuwei Mingmu Pills containing Cnidium monnieri medicinal material, add 18 mL of methanol, and reflux for 30 minutes. Cool to room temperature, filter, take the filtrate, and concentrate to half the volume as the test solution;

[0074] B. Preparation of control medicinal material solution: Take 0.25 g of Cnidium monnieri control medicinal material powder, add 5 mL of methanol, and ultrasonically treat for 10 minutes. Cool to room temperature, filter, take the filtrate, and prepare the control medicinal material solution;

[0075] C. TLC identification: First, activate the silica gel plate at 110°C for 30 minutes, then take 5 μL of the control medicinal material solution and the test solution and point them on the same silica gel G plate, dry them with a hair dryer, and then place them in the developing tank for 15 minutes of pre-saturation. Use cyclohexane-ethyl acetate (8:1 by volume) as the developing agent, develop, take out, dry, spray 5% vanillin sulfuric acid solution, and heat at 105°C until the spots are clearly colored;

[0076] D. Spectrum identification: Observe the size, position, color, and Rf value of each spot in the comparative identification spectrum.

[0077] Example 10, a method for identifying Cnidium monnieri preparations, the steps are as follows:

[0078] A. Preparation of test solution: Take 2 g of Liuwei Mingmu Pills containing Cnidium monnieri medicinal material, add 18 mL of methanol, and reflux for 30 minutes. Cool to room temperature, filter, take the filtrate, and concentrate to half the volume as the test solution;

[0079] B. Preparation of control medicinal material solution: Take 0.25 g of Cnidium monnieri control medicinal material powder, add 5 mL of methanol, and ultrasonically treat for 10 minutes. Cool to room temperature, filter, take the filtrate, and prepare the control medicinal material solution;

[0080] C. Thin-layer identification: First, activate the silica gel plate at 110°C for 30 minutes, then take 10 μL of the control medicinal material solution and the test sample solution and apply them to the same silica gel G plate, dry them with a hair dryer, and then place them in the development tank for 15 minutes of pre-saturation. Use cyclohexane-ethyl acetate (10:1 by volume) as the developing agent, develop, remove, dry, and spray with 5% vanillin sulfuric acid solution, and heat at 105°C until the spots are clearly colored;

[0081] D. Spectrum identification: Observe the size, position, color, and Rf value of each spot in the identification spectrum.

[0082] The following experimental examples are used to further illustrate the present application.

[0083] Experimental Example 1: Exploration of the developing agent for Cuminum cyminum thin-layer identification

[0084] 1. Experimental instruments, reagents, and control samples

[0085] 1.1 Instruments: Electronic balance: YP20002 (2 kg, 0.01 g); double-column four-hole water bath, HH-ZK4.

[0086] 1.2 Reagents (all chromatographic grade):

[0087] Methanol (Yu test reagent), formic acid (National Pharmaceutical Group), ethyl acetate (Tianjin Fuyu), petroleum ether (60-90°C, National Pharmaceutical Group), n-hexane (National Pharmaceutical Group), cyclohexane (National Pharmaceutical Group), anhydrous ethanol (Yu test reagent)

[0088] 1.3 Control medicinal material: Cuminum cyminum control medicinal material (Beijing Puxi), batch number: CH03099; 1 g / bottle.

[0089] 1.4 Control samples: Carvone (L16J12Z138140, 98.0%) from Beijing Puxi Standard Technology Co., Ltd., limonene control sample (100470-201503, 96.0%), and anisaldehyde control sample (110838-202108 0.2 mL / branch) from the China Institute for Control.

[0090] 1.5 Samples: Nine kinds of Cuminum cyminum medicinal materials from Bozhou, Guangxi, and Qinghai, respectively; Six-Mingmu pills were provided by the Jinhu Tibetan Medicine Stock Company, and the negative control was self-made by the laboratory according to the Six-Mingmu pill formula by excluding Cuminum cyminum medicinal materials.

[0091] 2. Preparation of control sample solvent

[0092] Take 0.25 g of Cuminum cyminum control medicinal material powder, add 5 mL of methanol, heat and reflux for 30 minutes, cool, and filter to prepare the control medicinal material solution;

[0093] Take 66.34 mg of carvone, dissolve in 50 mL of methanol to obtain a carvone control solution of 1.30264 mg / mL;

[0094] Take 117.4 mg of limonene, dissolve in 100 mL of methanol to obtain a limonene control solution of 1.703 mg / mL;

[0095] Take 0.2 mL of anisaldehyde, dilute to 10 mL with 95% ethanol. Take 0.5 mL, continue to dilute to 5 mL with 95% ethanol to obtain an anisaldehyde control solution of 2 μg / mL.

[0096] 3 Identification method and results

[0097] 3.1 Method 1: Repeat Literature 1, developing agent is petroleum ether: ethyl acetate (5:2), developing distance 8 cm

[0098] 3.1.1 Sample treatment: Take 10 g of Nardostachys chinensis Batal. medicinal material sample (Qinghai 202210) powder, cold soak in 25 mL of diethyl ether for 4 h, filter, and concentrate the filtrate to 1 mL to obtain a test solution; take 20 g of Liuwei Mingmu pill sample powder and 20 g of negative control powder, and prepare Liuwei Mingmu sample solution and negative control solution by the same method.

[0099] 3.1.2 Development: Petroleum ether: ethyl acetate (5:2) is the developing agent, the temperature is 23.1 ℃ and the humidity is 67% when the plate is developed, and the sample volume is 5 uL.

[0100] The thin layer plate is a silica gel G plate, which is activated by drying at 110 ℃ for 30 min according to the method of the Pharmacopoeia before use. Before development, it is first pre-saturated in the development tank for 15 min, and then dried by a hair dryer after spotting. After development, it is dried and observed under a UV light (365 nm).

[0101] 3.1.3 Results: See Figure 1 The spectrum has two developed spots of light blue and orange red, but the two spots are very close in distance, and are very close to the nearby developed band, with Rf of 0.62 and 0.65 respectively, which is not ideal in separation degree, and requires higher experimental operation.

[0102] 3.2 Method 2: Repeat the published standard, developing agent is petroleum ether: ethyl acetate (17:2.5), developing distance 8 cm

[0103] 3.2.1 Sample treatment: Take 2 g of Nardostachys chinensis Batal. medicinal material sample (Qinghai 202210) powder, seal and shake in 25 mL of petroleum ether-diethyl ether (1:1) mixed solution for 15 min, filter, and evaporate the filtrate at low temperature to dryness. Immediately add 2 mL of anhydrous ethanol to dissolve to obtain a test solution. Take 20 g of Liuwei Mingmu pill sample powder and 20 g of negative control powder, and prepare Liuwei Mingmu sample solution and negative control solution by the same method.

[0104] 3.2.2 Development: Petroleum ether: ethyl acetate (17:2.5) as developing solvent, temperature of developing chamber 23.6°C, humidity 64%, sample size 5 uL, TLC plate silica gel G, activated at 110°C for 30 min before use, pre-saturated in the developing chamber for 15 min before development, and dried by a hair dryer after spotting. After development, the plate was dried and sprayed with the dinitrosophenylhydrazine reagent, and examined under daylight.

[0105] 3.2.3 Results: See Figure 1. Figure 2 Only one orange-yellow spot was observed, which was very faint in color. This spot was not carvone, limonene or anisaldehyde. Moreover, the spot was almost invisible after about half an hour, and disappeared completely after 50 min. Upon closer inspection, the spot was actually two spots overlapped but not separated.

[0106] 3.3 Method 3: Replicate of Literature 2, developing solvent ethyl acetate-methanol-water-formic acid (15:5:1:1), developing distance 8 cm

[0107] 3.3.1 Sample preparation: 1 g of the powder of the sample of Cuminum cyminum (Qinghai 202210) was finely ground, and 15 mL of methanol was added. The mixture was heated to reflux for 30 min, and then cooled. The mixture was filtered to obtain the test solution.

[0108] 3.3.2 Development: ethyl acetate-methanol-water-formic acid (15:5:1:1) as developing solvent, temperature of developing chamber 24.1°C, humidity 69%, sample size 5 uL (1, 3) and 10 uL (2, 4), TLC plate silica gel G, activated at 110°C for 30 min before use, pre-saturated in the developing chamber for 15 min before development, and dried by a hair dryer after spotting. After development, the plate was dried and examined under UV light (365 nm).

[0109] 3.3.3 Results: See Figure 2. Figure 3 Two spots were observed, one of which was faint in color and close to another spot with Rf of about 0.75. Moreover, the overall Rf of the method was too large, and one spot had Rf close to or exceeding 0.9. Even if the ratio of the developing solvent was adjusted, or Merck plates were used, the results were not satisfactory.

[0110] 3.4 Method 4: Replicate of Literature 3, developing solvent n-hexane-ethyl acetate (12:1), developing distance 8 cm

[0111] 3.4.1 Sample preparation: 1 g of the powder of the sample of Cuminum cyminum (Qinghai 202210) was finely ground, and 15 mL of n-hexane was added. The mixture was ultrasonicated for 15 min, and then filtered to obtain the test solution.

[0112] 3.4.2 Development: n-hexane-ethyl acetate (12:1) as developing solvent, temperature 23.5°C, humidity 67%, sample size 5uL (1, 3) and 10uL (2, 4), TLC plate: silica gel G plate, activated at 110°C for 30min before use, pre-saturated in the developing jar for 15min before development, dried by air blower after spotting. After development, air dry, spray 5% vanillin-sulfuric acid solution, heat at 105°C until spots are clear, observe under daylight.

[0113] 3.4.3 Results: see Figure 4 . One light brownish red spot was shown, and the spot was more obvious with 10uL sample size. But careful observation, the spot was actually two spots overlapped, but not separated.

[0114] 3.5 Method 5: change to M plate, developing solvent: n-hexane-ethyl acetate (12:1)

[0115] 3.5.1 Sample treatment: take 2g powder of Qinghai and Bozhou samples (Qinghai 202210, Bozhou 20210824) respectively, add 30mL n-hexane, ultrasonic treatment for 10min, centrifuge at 5000rpm for 20min, take the supernatant as sample solution 3 and 4 respectively; take one of the two supernatants to continue normal filtration, and the other to filter through a microporous membrane, take the filtrate as sample solution 5 and 6 respectively; sample solution 1 is the control of Cuminum cyminum, and sample solution 2 is the control of carvone.

[0116] 3.5.2 Development: n-hexane-ethyl acetate (12:1) as developing solvent, temperature 24.2°C, humidity 75%, sample size 10uL, TLC plate: silica gel M (Merck) plate, activated at 110°C for 30min before use, pre-saturated in the developing jar for 15min before development, dried by air blower after spotting. After development, air dry, spray 5% vanillin-sulfuric acid solution, heat at 105°C until spots are clear, observe under daylight.

[0117] 3.5.3 Results: see Figure 5 . The spots on M plate are more detailed, two overlapped brown spots with Rf around 0.4 were shown, and two overlapped spots can be distinguished by UV or color observation (different colors), which needs to be further improved. In addition, there is almost no difference between the spots obtained by filtration and centrifugation.

[0118] 3.6 Method 6: improvement, developing solvent changed to cyclohexane-ethyl acetate (12:1), samples treated with ethyl acetate and methanol respectively for comparison

[0119] 3.6.1 Sample processing: 1 g of each of the control material of Nardostachys chinensis, Nardostachys chinensis medicinal material sample (Qinghai 202210), and two kinds of Nardostachys chinensis Six-Ingredient Eye-Sight Decoction Powder containing different batches (Qinghai, Bozhou) were finely ground, 15 mL of ethyl acetate was added respectively, and ultrasonic treatment was performed for 20 min, and the filtrate was taken as sample solutions 1-4; 1 g of each of the two kinds of Nardostachys chinensis Six-Ingredient Eye-Sight Decoction Powder containing different batches (Qinghai, Bozhou), Nardostachys chinensis medicinal material (Qinghai 202210), Nardostachys chinensis medicinal material (Bozhou 20210824), and the control material of Nardostachys chinensis was finely ground, 15 mL of methanol was added respectively, and ultrasonic treatment was performed for 20 min, and the filtrate was taken as sample solutions 5-9.

[0120] 3.6.2 Development: n-hexane-ethyl acetate (12:1) was used as the developing agent, the temperature of the developing plate was 24.7℃, the humidity was 68%, the sample amount was 10uL, the thin layer plate was a silica gel G plate, which was activated by drying at 110℃ for 30 min according to the method of the Pharmacopoeia before use, and was pre-saturated in the developing cylinder for 15 min before development, and was dried by a hair dryer after sample spotting. After development, it was dried, 5% vanillin sulfuric acid solution was sprayed, heated to 105℃ until the spots were clearly colored, and observed under daylight.

[0121] 3.6.3 Results: see Figure 6 . The two spots had Rf values of 0.62 and 0.78 respectively, and the separation degree was good. In this experiment, it was found that the spot with Rf value of about 0.75 (suspected to be limonene) was relatively dispersed (such as 1-4 in Figure 6 , while the two spots treated with methanol were relatively focused and not dispersed (such as 5-9 in Figure 6 . Therefore, the sample treated with methanol had a more excellent chromatogram.

[0122] 3.7 Method 7: n-hexane-ethyl acetate (10:1) was used as the developing agent, and nine Nardostachys chinensis samples from different origins were tested

[0123] 3.7.1 Sample processing: 1 g of each of the Nardostachys chinensis medicinal material samples from different batches (Qinghai, Guangxi, Bozhou) was finely ground and added with 15 mL of methanol, and ultrasonic treatment was performed for 10 min, and the filtrate was taken as sample solutions S1-S9; 1 g of the control material of Nardostachys chinensis was finely ground, 15 mL of methanol was added, ultrasonic treatment was performed for 10 min, and the filtrate was taken as the control material solution DZ.

[0124] 3.7.2 Development: n-hexane-ethyl acetate (10:1) was used as the developing agent, the temperature of the developing plate was 23.2℃, the humidity was 57%, the sample amount was 10uL, the thin layer plate was a silica gel G plate, which was activated by drying at 110℃ for 30 min according to the method of the Pharmacopoeia before use, and was pre-saturated in the developing cylinder for 15 min before development, and was dried by a hair dryer after sample spotting. After development, it was dried, 5% vanillin sulfuric acid solution was sprayed, heated to 105℃ until the spots were clearly colored, and observed under daylight.

[0125] 3.7.3 Results: see Table 3.7.3 Figure 7 The sample and the control drug material both showed two obvious brown spots with good separation degree. The method was stable and simple to operate.

[0126] 3.8 Method 8: The developing solvent was n-hexane-ethyl acetate (10:1), and the test was performed on the Liuwei Mingmu pills containing Cuminum cyminum

[0127] 3.8.1 Sample processing: 1 g of Cuminum cyminum samples from Qinghai and Bozhou and Liuwei Mingmu pills containing Cuminum cyminum from different producing areas were ground, 15 mL of methanol was added, and the mixture was ultrasonically treated for 10 min. The filtrate was collected and used as sample solutions 3-6. The negative control solution was prepared by the same method. 1 was the control solution of carvone, 2 was the Cuminum cyminum control drug solution prepared in 3.7, and 7 was the Liuwei Mingmu pill solution without Cuminum cyminum.

[0128] 3.8.2 Development: The developing solvent was cyclohexane-ethyl acetate (10:1), the temperature was 23.2℃, the humidity was 57%, the sample volume was 10 uL, the thin layer plate was silica gel G plate, which was activated at 110℃ for 30 min before use. The developing tank was pre-saturated for 15 min before development. The sample was dried by a hair dryer after spotting. After development, the plate was dried, sprayed with 5% vanillin sulfuric acid solution, heated at 105℃ until the spots were clear, and observed under daylight.

[0129] 3.8.3 Results: see Table 3.8.3 Figure 8 The Cuminum cyminum control drug (2), the drug material samples (3 and 4), and the Liuwei Mingmu pills (5 and 7) all showed two obvious brown spots with good separation degree. The method was stable and simple to operate. The negative control (7) did not have the two spots.

[0130] 4. Experimental summary

[0131] 4.1 The patterns obtained by repeating the standard or literature method were all one spot, which was overlapped and had a fuzzy boundary, or the spot was unstable, or the separation degree Rf was not appropriate, which was not convenient for identification.

[0132] 4.2 Experimental research found that for Cuminum cyminum drug material powder, there was no difference in HPLC pattern or thin layer identification pattern between reflux and ordinary ultrasonic treatment of the sample with methanol or n-hexane or ethyl acetate. However, ultrasonic treatment was faster and simpler, and ordinary ultrasonic treatment could be completed in 10 min. In addition, centrifugation or filtration of the sample solution had little effect on the results.

[0133] 4.3 The development agent is improved to a mixed solution of cyclohexane and ethyl acetate, which can obtain two development spots, and the identification effect is better; but when the sample is treated with ethyl acetate, one of the spots is more dispersed; when the sample is treated with methanol, the two spots are similar in size, not dispersed, and have good separation degree.

[0134] 4.4 The experiment also found that the ratio of cyclohexane and ethyl acetate can be 8-15:1 to obtain two spots with good separation degree, the larger the proportion of cyclohexane, the closer the distance between the two spots, but 10-12:1 is the best.

[0135] 4.5 The sample solution is better concentrated, or the amount of sample can be increased.

[0136] Experimental Example 2, Identification Methodology Verification of Cuminum cyminum L. Thin Layer Chromatography

[0137] Reference "Quality Standard Research of Tibetan Medicine Fifteen Leodi Name Piece (Huang Zhongquan)"; "GC Content Determination and Identification of Main Components of Spearmint Oil, Carvone and Limonene (Kang Yanlei)"; The identification method of Cuminum cyminum L. Thin layer chromatography was studied, including the study of the preparation method of the test solution, the study of the developing agent, etc. The results showed that the method was simple and feasible, had good durability, and could effectively control the quality.

[0138] I. Methodology verification summary

[0139] Methodology verification summary table

[0140]

[0141] II. Experimental instruments, reagents, reference materials and reference substances

[0142] 2.1 Instruments: electronic balance: YP20002 (2 kg, 0.01 g); double column four hole water bath, HH-ZK4; ultrasonic cleaner, SK5200HP; electric heating air drying oven: 101-2AB type.

[0143] 2.2 Reagents:

[0144] The reagents used in this experiment are chromatographically pure methanol, other reagents are analytically pure, and the water used in the experiment is purified water.

[0145] 2.2.1: Methanol (CH3OH): 20210805316, Yuzhi Reagent

[0146] 2.2.2: Sulfuric acid (H2SO4): 20200902, Tianjin Fuyu

[0147] 2.2.3: Ethyl acetate (C4H8O2): 20191125, Tianjin Fuyu

[0148] 2.2.4: Cyclohexane (C6H 12 ) : 20210211, Tianjin Fuyu

[0149] 2.2.5: Vanillin (C8H8O3): 20190819, Tianjin Guangfu

[0150] 2.2.6: 5% Vanillin Sulfuric Acid Solution: Take vanillin 0.5 g, add sulfuric acid 10 mL to dissolve.

[0151] Controlled Drug Material: Ferula Sinkiangensis Controlled Drug Material (Beijing Pu Fen), batch number: CH03099; 1 g / bottle.

[0152] III. Preparation of Test Product Solution, Controlled Drug Material Solution, and Controlled Product Solution

[0153] 3.1 Preparation of Test Product Solution: Take 1 g of the product (Ferula Sinkiangensis controlled drug material, same below), finely grind (pass through a No. 5 sieve), add 15 mL of methanol, ultrasonic for 10 min, cool, filter through a filter head, take the subsequent filtrate as the test product solution.

[0154] 3.2 Preparation of Controlled Drug Material Solution: Take another 0.25 g of Ferula Sinkiangensis controlled drug material powder, add 5 mL of methanol, and prepare the controlled drug material solution in the same way.

[0155] According to the thin layer chromatography test, take the above three solutions, and control the drug material and test product solution sample size to 5 μL, and point them on the same silica gel G plate. Before use, activate the plate at 110°C for 30 min according to the pharmacopoeia method, and pre-saturate in the development cylinder for 15 min before development. After spotting, dry with a hair dryer, develop with cyclohexane-ethyl acetate (10:1) as the developing agent, take it out and dry, spray 5% vanillin sulfuric acid solution, heat to 105°C until the spots develop clearly, and observe under daylight. The test product chromatogram shows the same color spots at the corresponding positions of the controlled drug material.

[0156] IV. Test Research

[0157] (I) Sample size selection

[0158] 1. Sample processing

[0159] Test product solution preparation: Take 0.5 g, 1 g, and 1.5 g of the product, respectively, finely grind (pass through a No. 5 sieve), add 15 mL of methanol, heat and reflux for 30 min, cool, filter through a filter head, and take the subsequent filtrate as the test product solution.

[0160] Controlled drug material solution preparation: Take another 0.25 g of Ferula Sinkiangensis controlled drug material powder, add 5 mL of methanol, and prepare the controlled drug material solution in the same way.

[0161] Take 5 μL of each of the above-mentioned solutions and apply them on the same silica gel G plate. Dry the plate at 110 ℃ for 30 min before use. Saturate the developing tank with the developing solvent for 15 min before use. Dry the sample spots with a hair dryer. Use cyclohexane-ethyl acetate (12:1) as the developing solvent. Dry the plate after development. Spray 5% vanillin-sulfuric acid solution on the plate and heat it at 105 ℃ until the spots are clear. Observe the plate under daylight.

[0162] 2. Test results

[0163] In the test sample chromatogram, two spots of the same color appear at the positions corresponding to the control drug chromatogram.

[0164] In addition, the sample weight screening summary is as follows:

[0165] In the thin layer chromatogram, through observation and analysis, when the sample weight is 1 g, the test sample spot is basically the same size and color as the control drug spot. When the sample weight is 0.5 g, the spot is larger. When the sample weight is 1.5 g, the spot is larger. When the sample weight is 1 g, the identification requirement can be met. Therefore, the sample weight for the thin layer chromatographic identification of Canghuixiang medicinal materials is determined to be 1 g.

[0166] (II) Sample processing method screening

[0167] 1. Sample processing

[0168] Test sample solution preparation: Take 1 g of the product, grind it finely (pass through a No. 5 sieve), add 15 mL of methanol, and ultrasonicate for 30 min. Cool, filter through a filter head, and take the filtrate as test sample solution 1.

[0169] Take 1 g of the product, grind it finely, add 15 mL of methanol, heat and reflux for 30 min, cool, filter through a filter head, and take the filtrate as test sample solution 2.

[0170] Control drug solution preparation: Take another 0.25 g of Canghuixiang control drug powder, add 5 mL of methanol, and prepare the control drug solution in the same way.

[0171] Take 5 μL of each of the above-mentioned solutions and apply them on the same silica gel G plate. Dry the plate at 110 ℃ for 30 min before use. Saturate the developing tank with the developing solvent for 15 min before use. Dry the sample spots with a hair dryer. Use cyclohexane-ethyl acetate (12:1) as the developing solvent. Dry the plate after development. Spray 5% vanillin-sulfuric acid solution on the plate and heat it at 105 ℃ until the spots are clear. Observe the plate under daylight.

[0172] 2. Test results

[0173] In the test sample chromatogram, two spots of the same color appear at the positions corresponding to the control drug chromatogram.

[0174] 3. Processing method screening summary

[0175] In thin layer chromatography, through observation and analysis, the sample treated by ultrasonic method and the sample treated by reflux method have the same color chromatographic spots in the corresponding position with the control medicinal material. The ultrasonic treatment is more convenient than the reflux treatment method, so the sample processing method is determined as ultrasonic.

[0176] (Three) sample processing ultrasonic time screening

[0177] 1. Sample preparation

[0178] Preparation of test solution: take 1 g of the product, finely grind (pass through a No. 5 sieve), add 15 mL of methanol, ultrasonic for 10 min, 20 min, and 30 min, cool, filter through a filter head, take the subsequent filtrate as the test solution.

[0179] Preparation of control medicinal material solution: take another 0.25 g of finely powdered Nardostachys chinensis control medicinal material, add 5 mL of methanol, and prepare the control medicinal material solution in the same way.

[0180] Take the above four solutions, and control medicinal material and test solution sample 5 μL, respectively, and point them on the same silica gel G plate. Before use, activate at 110°C for 30 min according to the pharmacopoeia method, and pre-saturate in the development cylinder for 15 min before development. After spotting, dry with a hair dryer, use cyclohexane-ethyl acetate (12:1) as the developing agent, develop, take out and air dry, spray 5% vanillin sulfuric acid solution, heat to 105°C until the spots develop clearly, and observe under daylight.

[0181] 2. Test results

[0182] In the test solution chromatogram, two spots of the same color are shown in the corresponding position with the control medicinal material chromatogram.

[0183] 3. Ultrasonic time screening summary

[0184] In thin layer chromatography, through observation and analysis, the samples treated by ultrasonic for 10, 20, and 30 minutes have the same color spots with the control medicinal material. Ultrasonic treatment for 10 minutes makes the test more convenient. Therefore, the ultrasonic time is determined as 10 minutes.

[0185] (Four) sample amount screening

[0186] Preparation of test solution: take 1 g of the product, finely grind (pass through a No. 5 sieve), add 15 mL of methanol, ultrasonic for 10 min, cool, filter through a filter head, take the subsequent filtrate as the test solution.

[0187] Preparation of control medicinal material solution: take another 0.25 g of finely powdered Nardostachys chinensis control medicinal material, add 5 mL of methanol, and prepare the control medicinal material solution in the same way.

[0188] Take the above two solutions, 5 μL of the sample solution, 2 μL, 5 μL, 10 μL respectively on the same silica gel G plate, before use according to the method of drying 30 min at 110 ℃ activation, before development first in the development cylinder pre-saturation 15 min, after spotting the hair dryer dry, with cyclohexane-ethyl acetate (12:1) as developing agent, development, take out and dry, spray 5% vanillin sulfuric acid solution, heated to 105 ℃ until the spots color clear, in the sunlight view.

[0189] 2. Test results

[0190] The test sample chromatogram shows two spots of the same color at the corresponding position of the control drug chromatogram.

[0191] 3. Spotting amount screening summary

[0192] In thin layer chromatography, by observation and analysis, the spot color is light when the spotting amount is 2 μL, and the spot color is deep when the spotting amount is 10 μL, so the spotting amount is determined to be 5 μL or 10 μL.

[0193] (Five) Thin layer plate screening

[0194] 1. Sample preparation

[0195] Test sample solution preparation: take 1 g of the product, grind finely (pass through a No. 5 sieve), add 15 mL of methanol, ultrasonic for 10 min, cool, filter through a filter head, take the filtrate, as the test sample solution.

[0196] Control drug solution preparation: take another 0.25 g of the fine powder of the control drug, add 5 mL of methanol, and prepare the control drug solution in the same way.

[0197] Take the above four solutions, 5 μL of the control drug and the test sample solution respectively on the silica gel G plate and M plate, before use according to the method of drying 30 min at 110 ℃ activation, before development first in the development cylinder pre-saturation 15 min, after spotting the hair dryer dry, with cyclohexane-ethyl acetate (12:1) as developing agent, development, take out and dry, spray 5% vanillin sulfuric acid solution, heated to 105 ℃ until the spots color clear, in the sunlight view.

[0198] 2. Test results

[0199] The test sample chromatogram shows two spots of the same color at the corresponding position of the control drug chromatogram.

[0200] 3. Thin layer plate screening summary

[0201] Through observation and analysis, compared with ordinary G plate, the spot showed more clearly using M plate, but considering the high cost of M plate and ordinary plate can meet the identification of Ferula sinkiangensis, so the thin layer plate is determined as ordinary silica gel G plate.

[0202] (VI) Developing agent screening

[0203] 1. Sample preparation

[0204] Preparation of test solution: Take 1g of the product, grind it finely (pass through a No. 5 sieve), add 15 mL of methanol, ultrasonic for 10 min, cool, filter through a filter head, take the filtrate, and use it as the test solution.

[0205] Preparation of control drug solution: Take another 0.25g of Ferula sinkiangensis control drug powder, add 5 mL of methanol, and prepare the control drug solution in the same way.

[0206] Take the above four solutions, control drug and test solution, and spot 5 μL of each on a silica gel G plate. Before use, activate the plate at 110°C for 30 min according to the pharmacopoeia method, and pre-saturate in the developing tank for 15 min before developing. After spotting, dry with a hair dryer, use cyclohexane-ethyl acetate (8:1), cyclohexane-ethyl acetate (10:1), cyclohexane-ethyl acetate (12:1), cyclohexane-ethyl acetate (14:1), and cyclohexane-ethyl acetate (15:1) as developing agents, develop, take out and air dry, spray 5% vanillin sulfuric acid solution, heat at 105°C until the spots develop clearly, and observe under daylight.

[0207] 2. Test results

[0208] In the test solution chromatogram, two spots of the same color were shown at the positions corresponding to the control drug chromatogram.

[0209] 3. Thin layer plate screening summary

[0210] In thin layer chromatography, through observation and analysis, when the developing agent is cyclohexane-ethyl acetate (10:1), the spot shows most clearly, and the shape is regular. When the ratio of cyclohexane-ethyl acetate is lower than 8:1 or higher than 15:1, the upper spot is not good, is relatively dispersed, and the greater the proportion of cyclohexane, the smaller the distance between the two spots. Therefore, the developing agent for the identification of Ferula sinkiangensis is determined to be cyclohexane-ethyl acetate with a ratio of 8-15:1, and the resulting chromatogram has two separate spots. The separation degree of the two spots is better when the ratio is 10-12:1, and the separation degree is best when the ratio is 10:1.

[0211] (VII) Specificity

[0212] Preparation of test solution: 1 g of each of the samples of Nardostachys chinensis DC, 2 batches of samples of Nardostachys chinensis DC, 2 g of each of 2 batches of Liuwei Mingmu pills containing Nardostachys chinensis DC and Liuwei Mingmu negative samples not containing Nardostachys chinensis DC were taken, the above samples were finely ground (through a No. 5 sieve), 15 mL of methanol was added, ultrasonic treatment was performed for 10 min, cooling was performed, filtration was performed on a filter head, and the filtrate was taken, and the filtrate of Liuwei Mingmu pills and the negative control sample was further concentrated to half the volume as the test solution.

[0213] Preparation of control drug solution: 0.25 g of finely powdered Nardostachys chinensis DC control drug was taken, 5 mL of methanol was added, ultrasonic treatment was performed for 10 min, cooling was performed, filtration was performed on a filter head, and the filtrate was taken to prepare the control drug solution.

[0214] According to the thin layer chromatography test, 5 μL of each of the above six solutions was taken and spotted on the same silica gel G plate, which was activated by drying at 110 ℃ for 30 min according to the method in the Pharmacopoeia before use, and was pre-saturated in the developing tank for 15 min before development, and was dried by a hair dryer after spotting, and was developed with cyclohexane-ethyl acetate (10:1) as the developing agent, and was taken out and dried, and was sprayed with 5% vanillin sulfuric acid solution, and was heated at 105 ℃ until the spots developed color clearly, and was observed under daylight.

[0215] 2. Test results

[0216] In the test sample chromatogram, the samples of Nardostachys chinensis DC and Liuwei Mingmu pills showed two spots of the same color at the positions corresponding to the control drug chromatogram, and the negative control showed no spot at the same position.

[0217] 3. Summary of specificity: according to the observation and analysis in the thin layer chromatography, the negative blank showed no interference, and the method had strong specificity.

[0218] (Eight) Repetition (repeatability) of the identification method of Nardostachys chinensis

[0219] Preparation of test solution: 1 g of each of the samples of Nardostachys chinensis DC, 2 batches of samples of Nardostachys chinensis DC, 2 g of each of 2 batches of Liuwei Mingmu pills containing Nardostachys chinensis DC and Liuwei Mingmu negative samples not containing Nardostachys chinensis DC were taken, the above samples were finely ground (through a No. 5 sieve), 15 mL of methanol was added, ultrasonic treatment was performed for 10 min, cooling was performed, filtration was performed on a filter head, and the filtrate was taken, and the filtrate of Liuwei Mingmu pills and the negative control sample was further concentrated to half the volume as the test solution.

[0220] Preparation of control drug solution: 0.25 g of finely powdered Nardostachys chinensis DC control drug was taken, 5 mL of methanol was added, ultrasonic treatment was performed for 10 min, cooling was performed, filtration was performed on a filter head, and the filtrate was taken to prepare the control drug solution.

[0221] According to the thin layer chromatography test, 5 μL of each of the above six solutions was taken and spotted on the same silica gel G plate, which was activated by drying at 110 ℃ for 30 min according to the method in the Pharmacopoeia before use, and was pre-saturated in the developing tank for 15 min before development, and was dried by a hair dryer after spotting, and was developed with cyclohexane-ethyl acetate (10:1) as the developing agent, and was taken out and dried, and was sprayed with 5% vanillin sulfuric acid solution, and was heated at 105 ℃ until the spots developed color clearly, and was observed under daylight.

[0222] 2. Test results

[0223] The test sample chromatography shows two same color spots at the positions corresponding to the chromatography of the control medicinal material.

[0224] 3. Reproducibility summary: In the thin layer chromatography, the method is well reproducible by observation and analysis by different people or different samples operated at different times.

[0225] In summary, the identification method of the Cuminum cyminum of the application can obtain two obvious characteristic spots after development, and the separation degree is good; the obtained characteristic spots are stable, the boundary is clear, and can be observed after long time placement; the treatment method is simple to operate, and the treatment process does not need heating, does not need to use flammable, explosive and easy-to-poison ether, and can be directly filtered after ultrasonic treatment to carry out thin layer identification, which is simple to operate and greatly shortens the test time.

[0226] The above-mentioned embodiments of the application are only illustrative, not the only ones, and all changes within the scope of the application or equivalent to the scope of the application are covered by the application.

Claims

1. A method for identifying Tibetan fennel, characterized in that, Includes the following steps: A. Preparation of test solution: Take 1 part by weight of fine powder of Tibetan fennel or 2 parts by weight of fine powder of its preparation, add methanol with a volume-to-weight ratio of 6-18, reflux for 30 minutes or sonicate for 10-20 minutes, cool to room temperature, filter, and take the filtrate of Tibetan fennel as the test solution, or further concentrate the filtrate of fine powder of Tibetan fennel preparation to half the volume of concentrate as the test solution. B. Preparation of reference herb solution: Take 0.25 parts by weight of fine powder of Tibetan fennel reference herb, add methanol with a volume-to-weight ratio of 16-24, sonicate for 10-20 minutes, cool to room temperature, filter, and take the filtrate to prepare the reference herb solution. C. Thin-layer chromatography development: First, dry the silica gel plate at 110℃ for 30 minutes to activate it. Then, take 5 μL or 10 μL of the reference herb solution and the test solution and spot them on the same silica gel plate. After drying with a hair dryer, place it in the developing tank for pre-saturation for 15 minutes. Use cyclohexane-ethyl acetate with a volume ratio of 8-15:1 as the developing solvent. Develop, remove, air dry, spray with 5% vanillin-sulfuric acid solution, and heat at 105℃ until the spots are clearly visible. D. Spectrum identification: Observe and compare the size, position, color and Rf value of each spot in the spectrum.

2. The method for identifying Tibetan anise according to claim 1, characterized in that, In the preparation of the test solution in step A, the volume-to-weight ratio is 10-15.

3. The method for identifying Tibetan anise according to claim 1, characterized in that, In the preparation of the test solution in step A, the volume-to-weight ratio is 15.

4. The method for identifying Tibetan anise according to claim 1, characterized in that, In the preparation of the control herbal solution in step B, methanol with a volume-to-weight ratio of 20 is added.

5. The method for identifying Tibetan anise according to claim 1, characterized in that, In the preparation of the control herbal solution in step B, the ultrasonic treatment time is 10 minutes.

6. The method for identifying Tibetan anise according to claim 1, characterized in that, In the thin-layer chromatography development in step C, the developing solvent is cyclohexane-ethyl acetate with a volume ratio of 10-12:

1.

7. The method for identifying Tibetan anise according to claim 1, characterized in that, In the thin-layer chromatography development in step C, the developing solvent is cyclohexane-ethyl acetate in a volume ratio of 10:

1.

8. The method for identifying Tibetan anise according to any one of claims 1-7, characterized in that, In the thin-layer chromatography development in step C, the silica gel plate is either a G plate or an M plate.

9. The application of the method for identifying Tibetan fennel as described in any one of claims 1-8 in the identification of Tibetan fennel medicinal materials or preparations containing Tibetan fennel medicinal materials.

Images