Thin-layer chromatography identification method for black prepared aconite in spleen-warming decoction

Abstract

The invention relates to the technical field of traditional Chinese medicine identification, and discloses a thin-layer chromatography identification method for black prepared aconite in spleen-warming decoction. The method comprises the following steps: dispensing a test sample solution and a black prepared tablet reference substance solution on the same silica gel G thin-layer plate for thin-layer chromatography to obtain an identification result; the test solution is from a spleen-warming decoction substance extract solution; a developing solvent in the thin layer chromatography condition is n-hexane-ethyl acetate-methanol. The alkaloid in the spleen-warming decoction can be rapidly separated and purified, the reproducibility is good, and the method is used as the exclusive identification of the black prepared tablet of the variety under the conditions of low temperature and high humidity.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine identification, in particular to a thin layer chromatography identification method of Heishun tablets in Wenpi decoction. Background Art

[0002] The classic prescription Wenpi Decoction is included in the "Catalogue of Ancient Classic Prescriptions (First Batch)" formulated by the State Administration of Traditional Chinese Medicine and the State Food and Drug Administration in 2018. It was first mentioned in the "Prescriptions for Emergencies Worth a Thousand Gold Pieces" written by Sun Simiao in the Tang Dynasty: "4 liang of rhubarb, 2 liang each of ginseng, licorice, and dried ginger, and one large aconite. Grind it, boil it with eight liters of water and take two and a half liters, divide it into three doses. Add rhubarb when it is almost cooked. It has the effect of curing chronic red and white diarrhea, cholera, cold spleen and stomach, and indigestion.

[0003] Aconite is a processed product of the root of Aconitum carmichaelii Debx., a plant of the Ranunculaceae family. The 2020 edition of the Chinese Pharmacopoeia includes three processed products of Aconite, namely Heishunpian, Baifupian and Yanfuzi. Among them, Heishunpian is an irregular longitudinal slice, wide at the top and narrow at the bottom, with slightly raised edges, dark brown skin, dark yellow translucent inside, oily and shiny, odorless, and light taste.

[0004] Studies have shown that the main component of Heishunpian is alkaloids. In the 2020 edition of the "Chinese Pharmacopoeia" and the Heishunpian formula granule standard, benzoylmesaconitine, benzoylmesaconitine and benzoylmesaconitine are used as the determination indicators for thin layer identification of Heishunpian. The extraction method of the test samples is to moisten with ammonia solution, ultrasonically treat with ether, filter, evaporate the filtrate, and add dichloromethane to dissolve the residue.

[0005] The above standards only conducted thin-layer identification research on Heishunpian decoction pieces and Heishunpian formula granules. There is no thin-layer identification method for Heishunpian in Wenpi Decoction. In addition, due to the complex ingredients in the compound, the test sample obtained by extraction with only a single solvent has many impurities other than alkaloids, which greatly interferes with the thin-layer identification of alkaloids. Therefore, in order to ensure the quality control of Wenpi Decoction, it is very important to provide a simple and reliable thin-layer identification method for Heishunpian. Summary of the invention

[0006] In view of the defects in the prior art, a rapid identification method of Heishunpian in Wenpi Decoction based on thin layer chromatography is provided, which can quickly identify Heishunpian in Wenpi Decoction, improve the detection efficiency, and provide a reference for the quality standard of Wenpi Decoction.

[0007] The objective of the present invention is achieved through the following technical solutions:

[0008] A thin layer chromatography identification method for Heishun tablets in Wenpi decoction comprises the following steps:

[0009] The test sample solution and the Heishun Pian reference solution are spotted on the same silica gel G thin layer plate for thin layer chromatography to obtain the identification result. If color is developed, it means that the test sample contains Heishun Pian, and thus it means that Wenpi Decoction contains Heishun Pian;

[0010] The test solution is from the Wenpi Decoction extract solution;

[0011] The developing solvent in the thin layer chromatography conditions is n-hexane-ethyl acetate-methanol.

[0012] Preferably, the test solution is prepared by the following steps:

[0013] Take the freeze-dried material of Wenpi Decoction, crush it, add 10.0%-11.2% (wt) ammonia solution to moisten it (the mass volume ratio of the freeze-dried material to the ammonia solution is 3.0-5.0g:7mL), use 40-50mL of ether for ultrasonic extraction for 30 minutes, filter, evaporate to dryness, use 5-10mL of 0.1moL / L hydrochloric acid solution for ultrasonic (300W-400W, 40KHz) for 10min for the residue, centrifuge (5000r) for 5min, transfer the supernatant to a strong cation (MCX) exchange column (pre-activated with methanol and 6mL of water), use water, 1.3%-1.4% (wt) ammonia solution, water, methanol, acetonitrile to elute in sequence, and finally use acetonitrile: 25%-28% (wt) ammonia solution (volume ratio of 9:1) solution to elute and collect the eluate, recover it to dryness under reduced pressure, and finally dissolve it in dichloromethane to obtain the product.

[0014] Preferably, the Wenpi Decoction freeze-dried material is prepared as follows:

[0015] It is made from ginseng, licorice, rhubarb, dried ginger, heishun tablets and water through heating, filtering and freeze-drying processes.

[0016] Preferably, the Heishun tablet reference substance solution is prepared by the following steps:

[0017] Take appropriate amounts of benzoylneaconitine, benzoylaconitine, and benzoylhyaconitine reference substances, and dissolve them in a mixed solution of isopropanol-dichloromethane (volume ratio of 1:1) to prepare a 0.3-0.5 mg / mL mixed reference substance solution.

[0018] Preferably, the method further comprises applying a negative control solution and a positive control solution on a thin layer plate containing silica gel G for thin layer chromatography, wherein the positive control solution shows spots of the same color as the Heishun tablet reference solution at corresponding positions on the thin layer plate, and the negative control solution shows no color on the thin layer plate.

[0019] Preferably, the positive control solution is prepared by the following steps:

[0020] The freeze-dried substance of the water decoction of the single medicinal material of Heishun Tablets is taken, crushed, and moistened with 10.0%-11.2% (wt) ammonia solution (the mass volume ratio of the freeze-dried substance to the ammonia solution is 3.0-5.0 g:7 mL), and ultrasonically extracted with 40-50 mL of ether for 30 minutes, filtered, evaporated, and the residue was ultrasonically treated with 5-10 mL of 0.1 mol / L hydrochloric acid solution (300W-400W, 40KHz) for 10 minutes, centrifuged (5000r) for 5 minutes, and the supernatant was transferred to a strong cation (MCX) exchange column (preliminarily activated with methanol and 6 mL of water), and eluted with 3 mL of water, 1.3%-1.4% (wt) ammonia solution, water, methanol, and 6 mL of acetonitrile in sequence, and finally eluted with acetonitrile: 25%-28% (wt) ammonia solution (volume ratio 9:1) solution and the eluate was collected, recovered under reduced pressure to dryness, and finally dissolved in dichloromethane to obtain the product.

[0021] Preferably, the freeze-dried substance of the single medicinal material decoction of Heishunpian is prepared as follows:

[0022] Heishun tablets and water are made through heating, filtering and freeze-drying processes.

[0023] Preferably, the negative control solution is prepared by the following steps:

[0024] The freeze-dried substance of the decoction of Wenpi Decoction Queheishun Tablets was taken and crushed, and 10.0%-11.2% (wt) ammonia solution was added to moisten it (the mass volume ratio of the freeze-dried substance to the ammonia solution was 3.0-5.0 g:7 mL), and ultrasonic extraction was performed with 40-50 mL of ether for 30 minutes, filtered, evaporated, and the residue was ultrasonically treated with 5-10 mL of 0.1 mol / L hydrochloric acid solution (300W-400W, 40KHz) for 10 min, centrifuged (5000r) for 5 min, and the supernatant was transferred to a strong cation (MCX) exchange column (preliminarily activated with methanol and 6 mL of water), and eluted with 3 mL of water, 1.3%-1.4% (wt) ammonia solution, water, methanol, and 6 mL of acetonitrile in sequence, and finally eluted with acetonitrile: 25%-28% (wt) ammonia solution (volume ratio of 9:1) solution and the eluate was collected, recovered under reduced pressure to dryness, and finally dissolved in dichloromethane to obtain the product.

[0025] Preferably, the freeze-dried substance of the decoction of Wenpi Decoction Quehei Shunpian is prepared by the following steps:

[0026] It is made from ginseng, licorice, rhubarb, dried ginger and water through heating, filtering and freeze-drying.

[0027] Preferably, the spot width is 3-5 mm, the spot volume is 10-20 μL; and the volume ratio of n-hexane-ethyl acetate-methanol is 4-8:2-5:1.

[0028] Furthermore, the spot width is 3 mm, the spot volume of the reference solution is 10-15 μL, the spot volume of the positive control solution is 10-20 μL, the spot volume of the test solution is 15-20 μL, and the volume ratio of n-hexane-ethyl acetate-methanol is 6.4:3.6:1.

[0029] Preferably, the thin layer chromatography method is:

[0030] Use an automatic spotter to spot samples. Take 20 μL of the test solution, 15 μL of the positive control solution, 20 μL of the negative control solution and 10 μL of the reference solution and spot them on the same silica gel G thin layer plate (activated at 105°C for 30 to 60 min before use). The spots are strip-shaped and 3 to 5 mm wide. Use n-hexane-ethyl acetate-methanol (volume ratio of 6.4:3.6:1) as the developing agent. Before development, the thin layer plate is pre-saturated with the developing agent in the developing bar for 30 min, developed, taken out, dried, and sprayed with a concentration of 4.0x10 -4 ~5.0x10 - 4 mol / L dilute potassium bismuth iodide solution for color development. Further, the concentration of the dilute potassium bismuth iodide solution is 4.2x10 -4 mol / L.

[0031] Preferably, before step S2, the method further includes the step of thin layer plate activation, wherein the activation temperature is 105° C. and the activation time is 30 to 60 minutes.

[0032] Preferably, the silica gel G thin layer plate is selected from any one of the following: Yinlong high-efficiency silica gel G plate and MERCK high-efficiency silica gel G plate.

[0033] Compared with the prior art, the advantages and positive effects of the present invention are:

[0034] The invention discloses a thin layer identification method for Heishun tablets in Wenpi decoction. The method comprises the following steps: using a solid phase extraction method to separate and purify a solution prepared from a freeze-dried substance of the Wenpi decoction, and then applying a solution prepared from the freeze-dried substance of the Wenpi decoction and a Heishun tablet reference substance solution on the same silica gel G thin layer plate for thin layer chromatography. The method can quickly separate and purify alkaloids in the Wenpi decoction with good reproducibility. Under low temperature (temperature is 3.4 DEG C, humidity is 41%) and high humidity (temperature is 20.2 DEG C, humidity is 76%) conditions, the positive control solution, the test substance and the mixed reference substance solution show spots of the same color at corresponding positions of the thin layer chromatography, and the negative solution has no interference, the separation degree is good, and no tailing occurs. The method can be used as an exclusive identification method for Heishun tablets in the variety. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1The results of the specificity investigation of the Heishun tablet thin layer identification method in Example 1, wherein S is the reference solution, No. 1 is the positive control solution; No. 2 is the test solution; and No. 3 is the negative control solution.

[0036] Figure 2 These are the durability investigation results of different spotting amounts of the Heishun slice thin layer identification method in Example 1, among which, No. 1 to No. 3 are reference solution spotting amounts of 5 μL, 10 μL and 15 μL respectively; No. 4 to No. 6 are positive control solutions, with spotting amounts of 10 μL, 15 μL and 20 μL respectively; No. 7 to No. 9 are test solution, with spotting amounts of 10 μL, 15 μL and 20 μL respectively.

[0037] Figure 3 The results of the thin-layer identification method of Heishun tablets under low temperature conditions in Example 1 are shown in Figure 1, wherein No. S is a mixed reference solution, No. 1 is a positive control solution, No. 2 is a test solution, and No. 3 is a negative control solution.

[0038] Figure 4 The results of the thin layer identification method of Heishun tablets under high humidity conditions in Example 1 are shown in Figure 1, wherein No. S is a mixed reference solution, No. 1 is a positive control solution, No. 2 is a test solution, and No. 3 is a negative control solution.

[0039] Figure 5 The results of the thin layer identification method of Heishun tablets using the MERCK thin layer plate in Example 1 are shown in Figure 1, wherein No. S is a mixed reference solution, No. 1 is a positive control solution, No. 2 is a test solution, and No. 3 is a negative control solution. DETAILED DESCRIPTION

[0040] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several changes and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0041] The instruments and materials used in the following examples are as follows. Other materials are commercially available unless otherwise specified.

[0042] 1. Instruments and test drugs

[0043] Table 1 is the information table of medicinal materials.

[0044] Table 1.

[0045] name Decoction piece batch number Origin Ginseng RS307Y1 Qingyuan County, Fushun City, Liaoning Province Black Straight Film 220201 Yunnan Lijiang, Dali, Kunming Dried ginger J20221001 Yuanmou County, Chuxiong City, Yunnan Province Fried Licorice C-GC228Y1 Guazhou County, Jiuquan City, Gansu Province rhubarb J20221001 Lichuan, Enshi City, Hubei Province

[0046] Table 2 is the reagent information table.

[0047] Table 2.

[0048] Reagent name Specification factory Ammonia solution 25%~28%(wt) GENERAL-REAGENT Ether AR (99.5%) GENERAL-REAGENT Isopropyl alcohol AR (99.7%) HoneyweLL Dichloromethane AR (99.5%) GENERAL-REAGENT n-Hexane AR (97.0%) Damas-beta Ethyl acetate AR (99.5%) GENERAL-REAGENT Methanol AR (99.5%) Damas-beta Bismuth Subnitrate 71.5%-74.5% (wt) Damas-beta Acetic acid AR (99.5%) GENERAL-REAGENT Potassium iodide AR (99.0%) GENERAL-REAGENT Acetonitrile AR (99.9%) Damas-beta hydrochloric acid 36%-38% (wt) GENERAL-REAGENT

[0049] Table 3 is the reference material information table.

[0050] Table 3.

[0051] Reference substance name batch number source Benzoyl aconitine 111795-202106 China Food and Drug Inspection Institute Benzoylhypaconitine 111796-201906 China Food and Drug Inspection Institute Benzoylaconitine 111794-202006 China Food and Drug Inspection Institute

[0052] Table 4 is the instrument information table.

[0053] Table 4.

[0054]

[0055]

[0056] Example 1

[0057] The thin layer chromatography identification method of Heishun tablets in Wenpi decoction comprises the following steps:

[0058] 1. Preparation of freeze-dried material of Wenpi Decoction:

[0059] Take 27.6g of ginseng, 27.6g of licorice, 27.6g of dried ginger, and 27.6g of Heishun tablets, add 1300mL of water, and soak in a decoction pot for 60min. Bring to a boil over high heat, open the lid and keep it at a slight boil for 90min. In the last 20min, add rhubarb (soaked in 300mL of water for 60min), control the liquid volume to 500mL by weighing, filter through a 400-mesh filter cloth while hot, concentrate under reduced pressure at 60℃ to 200mL, pre-freeze at -40℃ for 24h, and freeze-dry in a freeze dryer for 48h (cold trap temperature -55℃, vacuum degree 0.4mbar).

[0060] 2. Preparation of freeze-dried samples of Heishunpian single-ingredient decoction solution:

[0061] Add 1300 mL of water to 27.6 g of Heishun tablets and soak in a decoction pot for 60 min. Bring to a boil over high heat and keep the lid open to keep it at a slight boil for 90 min. Control the liquid volume to 500 mL by weighing, filter through a 400-mesh filter cloth while hot, concentrate to 200 mL at 60°C under reduced pressure, pre-freeze at -40°C for 24 h, and freeze-dry in a freeze dryer for 48 h (cold trap temperature -55°C, vacuum degree 0.4 mbar).

[0062] 3. Preparation of freeze-dried sample of Wenpi Decoction Quehei Shun Tablets decoction:

[0063] Take 27.6g of ginseng, 27.6g of licorice, 27.6g of dried ginger, add 1300mL of water in a decoction pot and soak for 60min. Bring to a boil over high heat, open the lid and keep it at a slight boil for 90min. In the last 20min, add rhubarb (soaked in 300mL of water for 60min), control the liquid volume to 500mL by weighing, filter through a 400-mesh filter cloth while hot, concentrate under reduced pressure at 60℃ to 200mL, pre-freeze at -40℃ for 24h, and freeze-dry in a freeze dryer for 48h (cold trap temperature -55℃, vacuum degree 0.4mbar).

[0064] 4. Preparation of test solution:

[0065] Take 5.0g of the freeze-dried material of Wenpi Decoction, crush it, add 7mL of 10.0%~11.2% (wt) ammonia solution to moisten it, use 50mL of ether for ultrasonic extraction for 30 minutes, filter, evaporate to dryness, use 10mL of 0.1moL / L hydrochloric acid solution for ultrasonic (400W, 40KHz) for 10min for the residue, centrifuge (5000r) for 5min, transfer the supernatant to a strong cation (MCX) exchange column (pre-activated with methanol and 6mL of water), and use water, 1.3%~1.4% (wt) ammonia solution, water, methanol, and acetonitrile to elute in sequence; finally, use acetonitrile: 25%~28% (wt) ammonia solution (volume ratio of 9:1) to elute and collect the eluate, recover it to dryness under reduced pressure, and dissolve it in dichloromethane to obtain the product.

[0066] 5. Preparation of positive control solution:

[0067] Take 5.0g of the freeze-dried substance of the water decoction of Heishunpian single medicinal material, crush it, add 7mL of 10.0%~11.2% (wt) ammonia solution to moisten it, use 50mL of ether for ultrasonic extraction for 30 minutes, filter, evaporate to dryness, use 10mL of 0.1moL / L hydrochloric acid solution for ultrasonic (400W, 40KHz) for 10min for the residue, centrifuge (5000r) for 5min, transfer the supernatant to a strong cation (MCX) exchange column (pre-activated with methanol and water), use water, 1.3%~1.4% (wt) ammonia solution, water, methanol, acetonitrile for elution in sequence, and finally use acetonitrile: 25%~28% (wt) ammonia solution (volume ratio of 9:1) for elution and collect the eluate, recover it to dryness under reduced pressure, and dissolve it in dichloromethane to obtain.

[0068] 6. Preparation of negative control solution:

[0069] Take 5g of the lyophilized material of the decoction of Wenpi Decoction Queheishun Tablets, crush it, add 7mL of 10.0%-11.2% (wt) ammonia solution to moisten it, use 50mL of ether for ultrasonic extraction for 30 minutes, filter, evaporate to dryness, use 10mL of 0.1 mol / L hydrochloric acid solution for ultrasonic (400W, 40KHz) for 10min for the residue, centrifuge (5000r) for 5min, transfer the supernatant to a strong cation (MCX) exchange column (pre-activated with methanol and water), and elute with water, 1.3%-1.4% (wt) ammonia solution, water, methanol, and acetonitrile in sequence, and finally elute with acetonitrile: 25%-28% (wt) ammonia solution (volume ratio of 9:1) and collect the eluate, recover it to dryness under reduced pressure, and dissolve it in dichloromethane to obtain the product.

[0070] 7. Preparation of reference solution:

[0071] Take 5.205 mg of benzoylmeaconitine and dilute it to 5 mL with a mixed solution of isopropanol-dichloromethane (volume ratio of 1:1); take 10.62 mg of benzoylmeaconitine and dilute it to 10 mL with a mixed solution of isopropanol-dichloromethane (volume ratio of 1:1); take 10.10 mg of benzoylhyaconitine and dilute it to 10 mL with a mixed solution of isopropanol-dichloromethane (volume ratio of 1:1), take 1 mL of the above reference solution respectively, combine and mix well to obtain.

[0072] 8. Thin layer chromatography:

[0073] Before use, the thin layer plate was activated in an electric hot air drying oven at 105°C for 30 min. The sample was spotted using an automatic spotter. 20 μL of the test solution, 15 μL of the positive control solution, 20 μL of the negative control solution, and 10 μL of the reference solution were respectively spotted on the same silica gel G thin layer plate with a spot width of 3 mm. Hexane-ethyl acetate-methanol (volume ratio of 6.4:3.6:1) was used as the developing agent. The plate was developed, taken out, dried, and sprayed with a concentration of 4.2×10 -4 mol / L dilute potassium bismuth iodide solution for color development.

[0074] 9. Determination results:

[0075] The positive control solution, test sample and mixed reference solution show spots of the same color at the corresponding positions on the thin layer chromatography, while the negative control solution does not show any color.

[0076] Example 2

[0077] Methodological validation:

[0078] (I) Specificity inspection

[0079] 1. Prepare the test solution, positive control solution, negative control solution and reference solution according to Example 1.

[0080] 2. Before using the thin layer plate, activate it in an electric hot air drying oven at 105°C for 30 minutes. Use an automatic spotter to spot the sample. Take 20 μL of the test solution, 15 μL of the positive control solution, 20 μL of the negative control solution and 10 μL of the reference solution and spot them on the same Yinlong high-efficiency silica gel G thin layer plate. The spot width is 3 mm. Use n-hexane-ethyl acetate-methanol (volume ratio of 6.4:3.6:1) as the developing agent. Add the developing agent to the developing bar to pre-saturate the thin layer plate for 30 minutes, develop it, take it out, dry it for 30 minutes, and spray it with a concentration of 4.2x10 -4 mol / L dilute potassium bismuth iodide solution for color development.

[0081] 3. The results are as follows Figure 1 As shown, the color spots of the reference substance spots from bottom to top are benzoylmeaconitine, benzoylaconitine and benzoylhyaconitine, the positive control solution, the test sample and the reference substance solution show spots of the same color at the corresponding positions of the thin layer chromatography, and there is no interference with the negative solution, indicating that the method has good specificity.

[0082] (II) Durability inspection

[0083] 1. Investigation of different sample quantities

[0084] 5 μL, 10 μL and 15 μL of the mixed reference solution; 10 μL, 15 μL and 20 μL of the positive control solution; 10 μL, 15 μL and 20 μL of the test solution were respectively taken, and the test was carried out at room temperature (temperature of 19.2°C, humidity of 47%) according to the thin layer chromatography conditions of Example 1 to compare the color development effects of different sample amounts.

[0085] The results are as follows Figure 2 As shown, the color development effect is good when the sample volume of the control solution is 10-15 μL, the sample volume of the positive control solution is 10-20 μL, and the sample volume of the test solution is 15-20 μL.

[0086] 2. Temperature durability test

[0087] 10 μL of the mixed reference solution, 15 μL of the positive control solution, 20 μL of the test solution and 20 μL of the negative control solution were respectively taken and tested under low temperature conditions (temperature of 3.4°C and humidity of 41%) according to the thin layer chromatography conditions of Example 1 to observe the separation and color development effects.

[0088] The results are as follows Figure 3 As shown, the positive control solution, the test sample and the mixed reference solution show spots of the same color at the corresponding positions of the thin layer chromatogram, and the negative sample has no interference, the separation is good, and there is no tailing.

[0089] 3. Humidity durability test

[0090] 10 μL of the mixed reference solution, 15 μL of the positive control solution, 20 μL of the test solution and 20 μL of the negative control solution were respectively taken and tested under high humidity conditions (temperature of 20.2°C and humidity of 76%) according to the thin layer chromatography conditions of Example 1 to observe the separation and color development effects.

[0091] The results are as follows Figure 4 As shown, the positive control solution, the test sample and the mixed reference solution show spots of the same color at the corresponding positions of the thin layer chromatogram, and the negative sample has no interference, the separation is good, and there is no tailing.

[0092] 3. Inspection of different thin layer boards

[0093] 10 μL of the mixed reference solution, 15 μL of the positive control solution, 20 μL of the test solution and 20 μL of the negative control solution were respectively taken and tested at room temperature (temperature of 19.2°C and humidity of 47%) according to the thin layer chromatography conditions of Example 1 to observe the separation and color development effects.

[0094] The results are as follows Figure 5 As shown, the positive control solution, the test sample and the mixed reference solution show spots of the same color at the corresponding positions of the thin layer chromatogram, and the negative sample has no interference. Figure 1 The Yinlong high-efficiency silica gel G plate and MERCK high-efficiency silica gel G plate used have better separation and no tailing occurs.

[0095] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various changes or modifications within the scope of the claims, which does not affect the essence of the present invention. In the absence of conflict, the embodiments of the present application and the features in the embodiments can be combined with each other arbitrarily.

Claims

1. A thin layer chromatography identification method for Heishun tablets in Wenpi Decoction, characterized in that: The steps include: The test solution and the Heishun tablet reference solution were spotted on the same silica gel G thin layer plate and developed with a developing agent for thin layer chromatography to obtain the identification results; The test solution is from the Wenpi Decoction extract solution; The developing solvent is n-hexane-ethyl acetate-methanol.

2. The thin layer chromatography identification method according to claim 1, characterized in that: The steps also include applying the negative control solution and the positive control solution on a thin layer plate containing silica gel G for thin layer chromatography, wherein the positive control solution shows spots of the same color as the black Shun tablet reference solution at corresponding positions on the thin layer plate, and the negative control solution does not show color on the thin layer plate.

3. The thin layer chromatography identification method according to claim 1 or 2, characterized in that: The spot width is 3-5 mm, and the spot volume is 10-20 μL; the volume ratio of n-hexane-ethyl acetate-methanol is 4-8:2-5:

1.

4. The thin layer chromatography identification method according to claim 1, characterized in that: The test solution is prepared by the following steps: Ammonia solution was added to the freeze-dried material of Wenpi Decoction, extracted with ether, filtered, evaporated, the residue was ultrasonically centrifuged with hydrochloric acid solution, the supernatant was passed through a column, eluted, recovered to dryness under reduced pressure, and finally dissolved in dichloromethane.

5. The thin layer chromatography identification method according to claim 4, characterized in that: The mass volume ratio of the freeze-dried substance of Wenpi Decoction to the ammonia solution is 3-5g:7mL.

6. The thin layer chromatography identification method according to claim 1, characterized in that: The Heishun Tablets reference substance solution is prepared by the following steps: Take benzoylmeaconitine, benzoylaconitine and benzoylhyaconitine reference substances respectively, and dissolve them in a mixed solution of isopropanol and dichloromethane with a volume ratio of 1:1; the content of each substance is 0.3-0.5 mg / mL.

7. The thin layer chromatography identification method according to any one of claims 1 to 6, characterized in that: The steps S2 and S4 include the following operations: After developing with the developing agent, use dilute potassium bismuth iodide test solution to develop color.

8. The thin layer chromatography identification method according to claim 1, characterized in that: The thin layer plate is activated before spotting, wherein the activation temperature is 100-105°C and the activation time is 30-60 minutes.

9. The thin layer chromatography identification method according to claim 1, characterized in that: The silica gel G thin layer plate is selected from any one of the following: Yinlong high-efficiency silica gel G plate and MERCK high-efficiency silica gel G plate.

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