Chromatographic Identification of Typhae Pollen Charcoal

Abstract

The present invention belongs to the field of traditional Chinese medicine detection, and specifically relates to the chromatographic identification of Typhae Pollen charcoal. Typhae Pollen charcoal in Fuke Duanhongyin capsules is identified using thin-layer chromatography. Identification bands are detected in both the test sample and the reference sample, isorhamnetin-3-O-neohesperidin solution, with good separation between the bands. In the negative control, no characteristic bands are detected in Fuke Duanhongyin capsules lacking Typhae Pollen charcoal, indicating no interference. The chromatographic identification method of Typhae Pollen charcoal has strong specificity and good durability, and is applicable to different thin-layer plates, at different temperatures and humidity levels. It can also be quickly and accurately identified even in high-humidity environments, and can be effectively used to identify Typhae Pollen charcoal in Fuke Duanhongyin capsules.

Description

Technical Field

[0001] The invention belongs to the field of traditional Chinese medicine detection, and particularly relates to chromatographic identification of pollen charcoal. Background Art

[0002] The properties of Chinese herbal medicines often change after charcoal making, and the active ingredients may also change significantly. The composition after charcoal making becomes uncertain. The uncertainty of the material basis of charcoal medicines seriously restricts the formulation of their quality standards. At present, the latest 2020 edition of the Chinese Pharmacopoeia records charcoal medicines with independent identification standards, including Cirsium truncatum charcoal, Sanguisorba officinalis charcoal, Schizonepeta tenuifolia charcoal, Schizonepeta spicate charcoal and Coptis chinensis charcoal. The 2020 edition of the Chinese Pharmacopoeia also discloses the identification methods of Puhuang medicinal materials under Puhuang and Gongning Granules. When identified using the method under Puhuang, although the negative result is without interference, the spots are not clear and there is interference; when identified using the method under Gongning Granules, the negative result is with interference, and neither can be accurately applied to the identification of Puhuang charcoal in Fuke Duanhong Drink. Therefore, it is currently difficult to obtain an identification method for Puhuang charcoal in Fuke Duanhong Drink based on the identification of Puhuang in the pharmacopoeia.

[0003] The prior art discloses a thin layer chromatography identification method for carbonized Chinese medicine formula particles, which uses acid hydrolysis reduction method to 254 Fluorescence quenching thin layer plate is used as a thin layer plate to identify the "non-fluorescent component" part, and then combined with the characteristic component color observation, this method is suitable for identifying most of the charcoal medicine Chinese medicine formula particles containing 254nm fluorescent absorption substances. Although this method shows that it can detect Typha charcoal, it requires a specific silica gel GF 254 Fluorescence quenching thin layer plate can only identify charcoal medicines containing 254nm fluorescent absorption substances. It is not suitable for other different thin layer plates, has limited durability, and is greatly affected by environmental humidity. Therefore, it affects the quality control of Puhuang charcoal formula drugs. It is not suitable for the identification of Puhuang charcoal in Fuke Duanhongyin Capsules and the quality control of Fuke Duanhongyin Capsules. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the shortcomings of the existing identification of Puhuang charcoal in Fuke Duanhongyin capsules, provide a chromatographic identification of Puhuang charcoal, and utilize thin layer chromatography to identify Puhuang charcoal in Fuke Duanhongyin capsules. The thin layer chromatography has strong specificity and good durability for the identification of Puhuang charcoal in Fuke Duanhongyin capsules, and can be used as a quality control standard for Puhuang charcoal in Fuke Duanhongyin capsules.

[0005] The invention aims to provide a developing agent for thin layer chromatography identification and application thereof in detecting Fuke Duanhongyin capsules.

[0006] The present invention also aims to provide a thin layer chromatography identification method for Typhae Pollen Carbon in Fuke Duanhongyin Capsules.

[0007] The above-mentioned purpose of the present invention is achieved by the following technical means:

[0008] The invention provides a developing agent for thin layer chromatography identification. The developing agent is a mixed solution of acetone and water.

[0009] Preferably, the volume ratio of acetone to water is 1-2:1-3.

[0010] Preferably, the volume ratio of acetone to water is 1-2:2-3.

[0011] More preferably, the volume ratio of acetone to water is 2:3.

[0012] The use of the mixed solution of the developing agent acetone and water in the detection of Fuke Duanhongyin Capsules is also within the protection scope of the present invention.

[0013] Preferably, it is used in detecting the charcoal cattail in Fuke Duanhongyin capsules.

[0014] The present invention provides a thin layer chromatography identification method for Typhae Pollen charcoal in Fuke Duanhongyin capsules, comprising the following steps:

[0015] S1. Preparation of test solution

[0016] The contents of Fuke Duanhongyin capsules are mixed with ether, heated under reflux to remove impurities, solid-liquid separation is performed after impurities removal, the liquid is discarded, and the solid is dried. The dried solid is extracted with ethanol, the solid-liquid separation is performed and the solid is discarded, and the liquid is dried to obtain an ethanol extract;

[0017] The aqueous solution of the ethanol extract is then extracted with ethyl acetate, the ethyl acetate layer is discarded, and the aqueous solution layer of the ethanol extract is extracted with water-saturated n-butanol to obtain an n-butanol extract;

[0018] The solvent in the n-butanol extract was removed to obtain a n-butanol extract solid, which was dissolved in methanol and purified by a chromatographic column using methanol as an eluent to obtain a methanol eluent. The methanol eluent was dried and dissolved in methanol again to serve as the test solution.

[0019] S2. Preparation of reference solution

[0020] The methanol solution of isorhamnetin-3-O-neohesperidin was used as the reference solution;

[0021] S3. Thin layer chromatography identification

[0022] The test solution prepared in step S1 and the reference solution prepared in step S2 were spotted on the same polyamide film, respectively. A mixture of acetone and water was used as a developing solvent, and the film was sprayed with an ethanol solution of aluminum chloride. The film was then inspected under 365 nm ultraviolet light for thin layer chromatography identification.

[0023] Preferably, the thin layer chromatography identification method of Typhae Pollen Carbon in Fuke Duanhongyin Capsules comprises the following steps:

[0024] S1. Preparation of test solution

[0025] The contents of Fuke Duanhongyin capsules were mixed with ether, heated under reflux for 20 to 30 minutes to remove impurities, and after impurities removal, the solid-liquid separation was performed to discard the liquid, and the solid was dried; the dried solid was extracted by cold soaking with 80% ethanol for 24 to 48 hours, the solid was separated and the solid was discarded, and the liquid was dried to obtain an ethanol extract; then the aqueous solution of the ethanol extract dissolved in water was extracted with ethyl acetate, the ethyl acetate liquid layer was discarded, and the aqueous solution layer of the ethanol extract was extracted with water-saturated n-butanol to obtain an n-butanol extract, and the solvent in the n-butanol extract was removed to obtain an n-butanol extract solid, which was dissolved in methanol and purified by a chromatographic column filled with octadecylsilane bonded silica gel, eluted with methanol as an eluent to obtain a methanol eluent, and the methanol eluent was dried and dissolved in methanol again to serve as a test solution;

[0026] The mass volume ratio of the contents of the Fuke Duanhongyin capsule to ether, ethanol, and ethyl acetate is 2-4 g:15-25 mL:45-55 mL:40-50 mL; the mass volume ratio of the contents of the Fuke Duanhongyin capsule to water-saturated n-butanol is 2-4 g:45-55 mL; the mass volume ratio of the contents of the Fuke Duanhongyin capsule to methanol in the test solution is 2-4 g:1-2.5 mL.

[0027] S2. Preparation of reference solution

[0028] The reference substance isorhamnetin-3-O-neohesperidin was added with methanol to prepare a reference substance solution with a concentration of 0.1-0.5 mg / mL;

[0029] S3. Thin layer chromatography identification

[0030] 1 μL of the test solution prepared in step S1 and 1 μL of the reference solution prepared in step S2 were respectively spotted on the same polyamide film. A mixture of acetone and water was used as the developing solvent in an acetone:water volume ratio of 1-2:1-3. The film was sprayed with 3%-5% aluminum chloride ethanol solution, dried, and examined under ultraviolet light (365 nm) for thin layer chromatography identification.

[0031] Preferably, the mass volume ratio of the contents of the Fuke Duanhongyin capsule to ether, ethanol, and ethyl acetate in step S1 is 2-3 g: 20-25 mL: 50-55 mL: 40-45 mL; the mass volume ratio of the contents of the Fuke Duanhongyin capsule to water-saturated n-butanol is 2-3 g: 50-55 mL; the mass volume ratio of the contents of the Fuke Duanhongyin capsule to methanol in the test solution is 2-3 g: 2-2.5 mL.

[0032] Further preferably, the mass volume ratio of the contents of the Fuke Duanhongyin capsule to ether, ethanol, and ethyl acetate in step S1 is 3g:20mL:50mL:40mL; the mass volume ratio of the contents of the Fuke Duanhongyin capsule to water and water-saturated n-butanol is 3g:50mL; the mass volume ratio of the contents of the Fuke Duanhongyin capsule to methanol in the test solution is 3g:2mL.

[0033] Preferably, the concentration of the reference solution in step S2 is 0.1-0.2 mg / mL.

[0034] Preferably, in step S3, the volume ratio of acetone to water is 1-2:2-3; and the concentration of the ethanol solution of aluminum chloride is 4%-5%.

[0035] Further preferably, the volume ratio of acetone to water in step S3 is 2:3; and the concentration of the ethanol solution of aluminum chloride is 5%.

[0036] Preferably, the developing temperature of the developing agent in step S3 is 4°C to 30°C.

[0037] Preferably, the developing humidity of the developing agent in step S3 is 33% to 70%.

[0038] As a specific embodiment, the thin layer chromatography identification method of Typhae Pollen Carbon in Fuke Duanhongyin Capsule comprises the following steps:

[0039] S1. Preparation of test solution

[0040] Mix 3 g of the contents of Fuke Duanhongyin Capsules with 20 mL of ether, heat and reflux for 20 minutes to remove impurities, separate the solid and liquid after impurities and discard the liquid, and dry the solid; extract the dried solid with 50 mL of 80% ethanol for 24 hours, separate the solid and liquid and discard the solid, and dry the liquid to obtain an ethanol extract; then extract the 15 mL water-soluble ethanol extract aqueous solution with ethyl acetate twice, 20 mL each time, discard the ethyl acetate liquid layer, and shake the remaining ethanol extract aqueous solution with water-saturated n-butanol twice, 25 mL each time. After evaporating the total n-butanol extract, dissolve it in 4 mL of methanol and purify it through a chromatographic column filled with octadecylsilane bonded silica gel, eluting it with 10 mL of methanol as an eluent to obtain a methanol eluent. After drying the methanol eluent, dissolve it in 2 mL of methanol to serve as the test solution.

[0041] S2. Preparation of reference solution

[0042] Take the reference substance isorhamnetin-3-O-neohesperidin and add methanol to make a reference substance solution with a concentration of 0.1 mg / mL;

[0043] S3. Thin layer chromatography identification

[0044] Pipette 1 μL of the test solution and 1 μL of the reference solution, spot them separately on the same polyamide film, use a mixture of acetone and water as the developing agent, and develop with an acetone: water volume ratio of 2:3. Take out, dry, spray with 5% aluminum chloride ethanol solution, dry, and examine under ultraviolet light (365 nm).

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] 1. The thin layer chromatography method of the present invention was used to identify the test sample and the isorhamnetin-3-O-neohesperidin reference solution, and the identification bands were detected. The separation of the bands was good. No characteristic bands were detected in the negative control Puhuangtan Fuke Duanhongyin Capsule, indicating that there was no interference.

[0047] 2. The thin-layer chromatography method of the present invention has strong specificity and good durability for the detection of Puhuang charcoal in Fuke Duanhongyin Capsules, and is suitable for different thin-layer plates, different temperatures and humidity; it can also be quickly and accurately identified in a high-humidity environment, and can be effectively used for the identification of Puhuang charcoal in Fuke Duanhongyin Capsules. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 This is a thin layer chromatography identification chromatogram of Typhae Pollen Carbon in Fuke Duanhongyin Capsule in Example 1 of the present invention; Figure 1 In the middle, 1-blank solvent (methanol), 2-isorhamnetin-3-O-neohesperidin control solution, 3-negative sample solution of Fuke Duanhongyin Capsules lacking Puhuang charcoal, and 4-6 are test solutions of Fuke Duanhongyin Capsules with batch numbers 20200704, 20200906, and 20210102.

[0049] Figure 2 This is a thin layer chromatography identification chromatogram of Typhae Pollen Carbon in Fuke Duanhongyin Capsule in Example 2 of the present invention; Figure 2 1-Isorhamnetin-3-O-neohesperidin control solution, 2-Puhuangtan charcoal-deficient Fuke Duanhongyin Capsule negative sample solution, 3 to 5 are Fuke Duanhongyin Capsule test solutions with batch numbers 20200704, 20200906, and 20210102.

[0050] Figure 3 The thin layer chromatogram of the identification of Typhae Pollen Carbon in Fuke Duanhongyin Capsules by the method of Comparative Example 1; Figure 3 1-Isorhamnetin, 2-Isorhamnetin-3-O-neohesperidin, 3-Hyperidin, 4-Puhuang charcoal Y108-2003001, 5-Puhuang control medicinal material 121225-201804, 6-Negative sample of Fuke Duanhongyin Capsule lacking Puhuang charcoal, 7-Fuke Duanhongyin Capsule 20200702.

[0051] Figure 4The thin layer chromatogram of the identification of Typhae Pollen Carbon in Fuke Duanhongyin Capsules by the method of Comparative Example 2; Figure 4 1-Isorhamnetin, 2-Isorhamnetin-3-O-neohesperidin, 3-Hyperidin, 4-Puhuang charcoal Y108-2003001, 5-Puhuang control medicinal material 121225-201804, 6-Negative sample of Fuke Duanhongyin Capsule lacking Puhuang charcoal, 7-Fuke Duanhongyin Capsule 20200702.

[0052] Figure 5 This is the thin layer chromatogram of Typha charcoal of Fuke Duanhongyin Capsule identified by the method of Comparative Example 3; Figure 5 1-Isorhamnetin, 2-Isorhamnetin-3-O-neohesperidin, 3-Typhala neoside, 4-Tupiana control medicinal material 121225-201804, 5-Tupiana charcoal Y108-2003001, 6-Tupiana charcoal-deficient Fuke Duanhongyin Capsule negative sample, 7-Fuke Duanhongyin Capsule 20200702.

[0053] Figures 6-8 The thin layer chromatograms of Puhuang charcoal for identifying Fuke Duanhongyin Capsules using the method of Comparative Example 3 under different thin layer chromatography conditions are shown; Figure 6 and Figure 7 1-blank solvent (methanol), 2-xanthopropanoside, 3-isorhamnetin-3-O-neohesperidin, 4-Puhuang control medicinal material, 5-negative sample of Fuke Duanhongyin Capsule lacking Puhuang charcoal, 6-Fuke Duanhongyin Capsule 20200704, 7-Fuke Duanhongyin Capsule 20200906, 8-Fuke Duanhongyin Capsule 20210102; Figure 8 1-blank solvent (methanol), 2-isorhamnetin-3-O-neohesperidin-0.1 mg / mL, 3-isorhamnetin-3-O-neohesperidin-0.2 mg / mL, 4-isorhamnetin-3-O-neohesperidin-0.5 mg / mL, 5-negative sample of Fuke Duanhongyin Capsule lacking Puhuangcharcoal, 6-Fuke Duanhongyin Capsule 20200704, 7-Fuke Duanhongyin Capsule 20200906, 8-Fuke Duanhongyin Capsule 20210102.

[0054] Figure 9 This is the thin layer chromatogram of Typha charcoal of Fuke Duanhongyin Capsule identified by the method of Comparative Example 4; Figure 9 1-negative sample of Puhuang Tan Fuke Duanhongyin Capsule, 2, 6-Puhuang control medicinal material, 3-20200704, 4-20200906, 5-20210102, 7-Isorhamnetin-3-O-neohesperidin.

[0055] Figure 10 Chromatograms of Puhuang charcoal thin layer chromatography identification of Fuke Duanhongyin Capsule under different film plate conditions; Figure 10In the figure, 1-blank solvent (methanol), 2-isorhamnetin-3-O-neohesperidin control solution, 3-negative sample solution of Fuke Duanhongyin Capsules lacking Puhuangcharcoal, 4-6 are test solutions of Fuke Duanhongyin Capsules with batch numbers 20200704, 20200906, and 20210102; A-polyamide film of Zhejiang Taizhou Luqiao Sijia Biochemical Plastic Factory, B-polyamide film of Wuhan Weiqi Boxing Biotechnology Co., Ltd., and C-polyamide film of Shanghai Yiji Industrial Co., Ltd.

[0056] Figure 11 Chromatograms of Puhuang charcoal thin layer chromatography identification of Fuke Duanhongyin Capsule under different temperature conditions; Figure 11 1-blank solvent (methanol), 2-isorhamnetin-3-O-neohesperidin control solution, 3-negative sample solution of Fuke Duanhongyin Capsules lacking Puhuang charcoal, 4-6 are test solutions of Fuke Duanhongyin Capsules with batch numbers 20200704, 20200906, and 20210102; A-20℃, B-4℃, C-30℃.

[0057] Figure 12 This is the chromatogram of the Puhuang charcoal thin layer chromatography identification of Fuke Duanhongyin Capsule under different humidity conditions; Figure 12 1-blank solvent (methanol), 2-isorhamnetin-3-O-neohesperidin control solution, 3-negative sample solution of Fuke Duanhongyin Capsules lacking Puhuang charcoal, 4-6 are test solutions of Fuke Duanhongyin Capsules with batch numbers 20200704, 20200906, and 20210102; A-50%, B-33%, C-70%.

[0058] Figure 13 This is the chromatogram of the 16th batch of Fuke Duanhongyin Capsules with Puhuang charcoal thin layer chromatography identification; Figure 13 1-blank solvent (methanol), 2-isorhamnetin-3-O-neohesperidin, 3-negative sample of Puhuangtan Fuke Duanhongyin Capsule, 4~18-batch numbers are 20200701, 20200702, 20200703, 20200704, 20200705, 20200706, 20200805, 20200806, 20200906, 20201105, 20201202, 20210102, 20210201, 20210301, 20210302 Fuke Duanhongyin Capsule. DETAILED DESCRIPTION

[0059] The present invention is further described below with reference to specific examples, which, however, are not intended to limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the art.

[0060] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0061] Example 1 Thin-layer chromatography identification of Typhae Pollen charcoal in Fuke Duanhongyin capsule

[0062] 1. Experimental Methods

[0063] S1. Preparation of test solution

[0064] Mix 3 g of the contents of Fuke Duanhongyin Capsules with 20 mL of ether, heat and reflux for 20 minutes to remove impurities, separate the solid and liquid after impurities removal and discard the liquid, and dry the solid; then use 50 mL of 80% ethanol to cold soak for 24 hours to extract the dried solid, separate the solid and liquid and discard the solid, and dry the liquid to obtain an ethanol extract; add 15 mL of water to dissolve the ethanol extract, and extract the dissolved ethanol extract aqueous solution with ethyl acetate twice, 20 mL each time, discard the ethyl acetate liquid layer, and shake the remaining ethanol extract aqueous solution with water-saturated n-butanol and extract it twice, 25 mL each time. The total n-butanol extract extracted is evaporated to dryness and dissolved in 4 mL of methanol, and purified by a chromatographic column filled with octadecylsilane bonded silica gel, eluting with 10 mL of methanol as eluent to obtain a methanol eluent. After drying the methanol eluent, add 2 mL of methanol to dissolve it as the test solution.

[0065] S2. Preparation of reference solution

[0066] Take the reference substance isorhamnetin-3-O-neohesperidin and add methanol to prepare a reference solution with a concentration of 0.1 mg / mL; the negative control solution is prepared in the same way as the test solution.

[0067] S3. Thin layer chromatography identification

[0068] Refer to the 2020 edition of the Chinese Pharmacopoeia, Part IV, General Chapter 0502. Pipette 1 μL of the test solution and 1 μL of the reference solution onto the same polyamide film. Use a mixture of acetone and water as the developing agent, with an acetone: water volume ratio of 2:3. Remove the film, dry it, spray it with 5% aluminum chloride in ethanol, dry it, and inspect it under ultraviolet light (365 nm).

[0069] 2. Sample and reference material information

[0070]

[0071] 3. Results

[0072] Judgment index: In the chromatogram of the test sample, spots of the same color appear at the corresponding position of the chromatogram of the reference sample. In this example, the above method was used to identify the Fuke Duanhongyin Capsules with production batch numbers of 20200704, 20200906, and 20210102 produced by Zhuzhou Qianjin Pharmaceutical Co., Ltd. The identification results are as follows: Figure 1 shown. Figure 1In the middle, 1-blank solvent (methanol), 2-isorhamnetin-3-O-neohesperidin control solution, 3-negative sample solution of Fuke Duanhongyin Capsules lacking Puhuang charcoal, and 4-6 are test solutions of Fuke Duanhongyin Capsules with batch numbers 20200704, 20200906, and 20210102.

[0073] from Figure 1 It can be seen that the three groups of test samples, Fuke Duanhongyin Capsules and isorhamnetin-3-O-neohesperidin reference substance, can all detect identification bands at Rf=0.30, and the separation of each band is good. In addition, no characteristic bands can be detected in the negative samples of Fuke Duanhongyin Capsules lacking Puhuang charcoal, indicating that this method has strong specificity and can effectively identify Fuke Duanhongyin Capsules and negative samples of Fuke Duanhongyin Capsules lacking Puhuang charcoal, and can be used for thin layer chromatography identification of Puhuang charcoal in Fuke Duanhongyin Capsules.

[0074] Example 2 Thin-layer chromatography identification of Typhae Pollen charcoal in Fuke Duanhongyin capsule

[0075] 1 Experimental methods

[0076] Compared with Example 1, the only difference is that a mixed solution with a volume ratio of acetone to water of 1:2 is used as the developing solvent.

[0077] 2. Sample and reference substance information is the same as in Example 1

[0078] 3 Results

[0079] Judgment index: In the chromatogram of the test solution, a spot of the same color appears at the corresponding position in the chromatogram of the reference solution. Figure 2 shown. Figure 2 1-Isorhamnetin-3-O-neohesperidin control solution, 2-Puhuangtan charcoal-deficient Fuke Duanhongyin Capsule negative sample solution, 3 to 5 are Fuke Duanhongyin Capsule test solutions with batch numbers 20200704, 20200906, and 20210102.

[0080] from Figure 2 It can be seen that the test sample Fuke Duanhongyin Capsule and the isorhamnetin-3-O-neohesperidin reference substance can both detect identification bands at Rf=0.28, and the separation of each band is good. In addition, no characteristic bands can be detected in the negative sample of Fuke Duanhongyin Capsule lacking Puhuangtan, indicating that this method has strong specificity and can effectively identify Fuke Duanhongyin Capsule and the negative sample of Fuke Duanhongyin Capsule lacking Puhuangtan.

[0081] The present invention also performs thin layer chromatography identification of Typhae Pollen Carbon in Fuke Duanhongyin Capsules under different ratios of Fuke Duanhongyin Capsules and solvents, different concentrations of isorhamnetin-3-O-neohesperidin, and different ratios of developing agents. The specific conditions are as follows:

[0082]

[0083] Under conditions No. 1 and 2, identification bands could be detected at Rf=0.26~0.35 for the test sample Fuke Duanhongyin Capsule and isorhamnetin-3-O-neohesperidin reference substance, and the separation degree of each band was good. In addition, no characteristic bands could be detected in the negative sample of Fuke Duanhongyin Capsule lacking Puhuangtan, indicating that this method has strong specificity and can effectively identify Fuke Duanhongyin Capsule and negative samples of Fuke Duanhongyin Capsule lacking Puhuangtan.

[0084] Comparative Example 1 A method for thin layer chromatography of Puhuang charcoal in Fuke Duanhongyin capsule

[0085] 1. Experimental Methods

[0086] Take the contents of Fuke Duanhongyin capsules, add 50 mL of 80% ethanol, soak in cold for 24 hours, filter, evaporate the filtrate to dryness, add 5 mL of water to the residue to dissolve it, filter, add water-saturated n-butanol to the filtrate and shake to extract twice, 5 mL each time, combine the n-butanol liquids, evaporate to dryness, add 2 mL of ethanol to the residue to dissolve it, and use it as the test solution.

[0087] Isorhamnetin, isorhamnetin-3-O-neohesperidin, and cattail neoside were used as reference substances, and Puhuang control medicinal material was used as the control medicinal material; Puhuangtan Fuke Duanhongyin Capsule was used as the negative control;

[0088] Preparation of isorhamnetin solution: Take isorhamnetin reference substance and add methanol to make a solution containing 0.4070 mg per 1 mL, which is used as the reference solution;

[0089] Preparation of isorhamnetin-3-O-neohesperidin solution: Take isorhamnetin-3-O-neohesperidin reference substance and add methanol to make a solution containing 0.9672 mg per 1 mL, which is used as the reference solution;

[0090] Preparation of typhalaenoside solution: Take typhalaenoside reference substance and add methanol to make a solution containing 1.1773 mg per 1 mL, which is used as the reference solution;

[0091] Take 0.5 g of the Puhuang control drug and Puhuang charcoal medicinal materials and prepare them according to the method of the test solution of this comparative example 1; the preparation method of the negative control is the same as that of the test sample of this comparative example 1.

[0092] Prepare the appropriate solution and perform thin-layer chromatography. Sample pattern: 8 mm strips; Developing solvent: acetone: water (volume ratio 1:2); Spreading distance: 8 cm; Pre-saturate the developing cylinder for 20 minutes; Drying: Air-dry; Color development: Spray with 1% aluminum chloride in ethanol; Observation: UV 365 nm.

[0093] 2. Sample and reference material information

[0094]

[0095] 3 Results

[0096] Fuke Duanhongyin Capsule was identified by the method of this comparative example. The identification results are as follows: Figure 3 . Figure 3 1-Isorhamnetin, 2-Isorhamnetin-3-O-neohesperidin, 3-Hyperidin, 4-Puhuang charcoal Y108-2003001, 5-Puhuang control medicinal material 121225-201804, 6-Negative sample of Fuke Duanhongyin Capsule lacking Puhuang charcoal, 7-Fuke Duanhongyin Capsule 20200702.

[0097] from Figure 3 As can be seen, this method showed no identification spots for isorhamnetin during thin-layer chromatography. Exclusive spots appeared at Rf = 0.36 (isorhamnetin-3-O-neohesperidin) and Rf = 0.58 (typhaneoside), with no interference in the negative test. However, the spots were unclear and showed subtle interference. This method cannot be accurately applied to the identification of Typhae Carbohydrate in Fuke Duanhongyin Capsules. Therefore, the extraction method and reference substance of this method are not suitable for the thin-layer chromatography identification of Typhae Carbohydrate in Fuke Duanhongyin Capsules.

[0098] Comparative Example 2: A method for thin layer chromatography of Typhae Pollen charcoal in Fuke Duanhongyin capsules

[0099] 1. Experimental Methods

[0100] Take the contents of Fuke Duanhongyin Capsules, add 20 mL of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, add 20 mL of water to dissolve the residue, filter, adjust the pH value of the filtrate to 1-2 with dilute hydrochloric acid, shake and extract with ethyl acetate twice, 10 mL each time, combine the extracts, evaporate to dryness, add 2 mL of methanol to dissolve the residue, and use it as the test solution.

[0101] Isorhamnetin, isorhamnetin-3-O-neohesperidin, and cattail neoside were used as control substances, and Puhuang control medicinal material was used as control medicinal material; Puhuang charcoal medicinal material was used as reference medicinal material; Puhuang charcoal Fuke Duanhongyin Capsule was used as negative control;

[0102] Preparation of isorhamnetin solution: Take isorhamnetin reference substance and add methanol to make a solution containing 0.4070 mg per 1 mL, which is used as the reference solution;

[0103] Preparation of isorhamnetin-3-O-neohesperidin solution: Take isorhamnetin-3-O-neohesperidin reference substance and add methanol to make a solution containing 0.9672 mg per 1 mL, which is used as the reference solution;

[0104] Preparation of typhalaenoside solution: Take typhalaenoside reference substance and add methanol to make a solution containing 1.1773 mg per 1 mL, which is used as the reference solution;

[0105] 0.5 g of Puhuang control medicinal material and Puhuang charcoal medicinal material were respectively taken and prepared according to the method of the test solution of this comparative example 2; the preparation method of the negative control was the same as that of the test sample of this comparative example 2.

[0106] After preparing the corresponding solution, perform thin-layer chromatography identification. Sample pattern: 8 mm strips; developer: toluene: ethyl acetate: formic acid (volume ratio: 5:2:0.5); development distance: 8 cm; pre-saturate the developing cylinder for 20 minutes; drying: air-dry; color development: spray with 1% ferric chloride in ethanol; inspection: UV 365 nm.

[0107] 2. Sample and reference material information

[0108]

[0109]

[0110] 3. Results

[0111] Fuke Duanhongyin Capsule was identified by the method of this comparative example 2, and the identification results were as follows: Figure 4 . Figure 4 1-Isorhamnetin, 2-Isorhamnetin-3-O-neohesperidin, 3-Hyperidin, 4-Puhuang charcoal Y108-2003001, 5-Puhuang control medicinal material 121225-201804, 6-Negative sample of Fuke Duanhongyin Capsule lacking Puhuang charcoal, 7-Fuke Duanhongyin Capsule 20200702.

[0112] Figure 4 The results showed that the negative results of this method were interfered by thin layer chromatography, and no traces were found in the reference substance and reference medicinal materials. Therefore, the extraction method and reference substance of this method are not suitable for thin layer chromatography identification of Fuke Duanhongyin Capsules with Puhuang charcoal.

[0113] Comparative Example 3

[0114] 1. A thin layer chromatography method for Puhuang charcoal in Fuke Duanhongyin capsule

[0115] 1.1 Experimental methods

[0116] Mix 3 g of the contents of Fuke Duanhongyin Capsules with 20 mL of ether, heat and reflux for 20 minutes to remove impurities, separate the solid and liquid after impurities removal, and discard the liquid, and dry the solid; then use 50 mL of 80% ethanol to cold soak for 24 hours to extract the dried solid, separate the solid and liquid and discard the solid, and dry the liquid to obtain an ethanol extract; then extract the 15 mL water-soluble ethanol extract aqueous solution with ethyl acetate twice, 20 mL each time, discard the ethyl acetate liquid layer, and shake the remaining ethanol extract aqueous solution with water-saturated n-butanol twice, 25 mL each time, evaporate the total n-butanol extract and dissolve it with 4 mL of methanol, purify it through a chromatographic column filled with octadecylsilane bonded silica gel, and elute it with 10 mL of methanol to obtain a methanol eluent. After the methanol eluent is dried, dissolve it in 2 mL of methanol and use it as the test solution.

[0117] Isorhamnetin-3-O-neohesperidin, typha glycosides, isorhamnetin, and pollen control medicinal materials were used as reference substances, and pollen charcoal medicinal materials were used as reference medicinal materials; pollen charcoal Fuke Duanhongyin Capsules were used as negative controls; isorhamnetin solution was prepared by taking the isorhamnetin reference substance and adding methanol to prepare a solution containing 0.4070 mg per 1 mL as a reference solution; isorhamnetin-3-O-neohesperidin solution was prepared by taking the isorhamnetin-3-O-neohesperidin reference substance and adding methanol to prepare a solution containing 0.9672 mg per 1 mL as a reference solution; typha glycoside solution was prepared by taking the typha glycoside reference substance and adding methanol to prepare a solution containing 1.1773 mg per 1 mL as a reference solution; 0.5 g of pollen control medicinal materials and pollen charcoal medicinal materials were respectively taken and prepared according to the method of the test solution of this comparative example 3; the preparation method of the negative control was the same as that of the test sample of this comparative example 3.

[0118] Prepare the appropriate solution and perform thin-layer chromatography. Sample pattern: 8 mm strips; developer: acetone: water (volume ratio 1:2); development distance: 8 cm; pre-saturate the developing cylinder for 20 minutes; air-dry; color development: spray with 5% aluminum chloride in ethanol; inspection: UV 365 nm.

[0119] 1.2 Sample and reference substance information

[0120]

[0121] 1.3 Results

[0122] Fuke Duanhongyin Capsule was identified by the method of this comparative example 3, and the identification results are as follows: Figure 5 . Figure 51-Isorhamnetin, 2-Isorhamnetin-3-O-neohesperidin, 3-Typhala neoside, 4-Tupiana control medicinal material 121225-201804, 5-Tupiana charcoal Y108-2003001, 6-Tupiana charcoal-deficient Fuke Duanhongyin Capsule negative sample, 7-Fuke Duanhongyin Capsule 20200702.

[0123] Figure 5 The results show that the method is subjected to thin-layer chromatography, and the negative result is free of interference. The interference point near the reference substance (isorhamnetin-3-O-neohesperidin) disappears, and the impurity removal effect is good. The four main spots of the Puhuang reference medicinal material and the Puhuang charcoal medicinal material are vaguely aligned. It can be seen from the chromatogram that the selected exclusive point is exclusive to the Puhuang medicinal material. After processing, it is transferred to the Puhuang charcoal medicinal material, so it can be used as a qualitative identification exclusive point. Because the Puhuang charcoal control medicinal material is not collected by the China National Institute for Inspection and Quarantine, this method uses the Puhuang control medicinal material as a reference. Therefore, the extraction method of this comparative example 3, with isorhamnetin-3-O-neohesperidin, champroglucosidin and Puhuang control medicinal material as reference substances, can be used to perform thin-layer chromatography identification of Puhuang charcoal in Fuke Duanhongyin Capsules according to the chromatographic conditions of this comparative example 3.

[0124] 2. Effects of different TLC conditions on the TLC identification of Puhuang charcoal in Fuke Duanhongyin Capsules

[0125] 2.1 Experimental methods

[0126] Isorhamnetin-3-O-neohesperidin, champroglucosidin, and pollen control medicinal materials were used as reference substances; pollen charcoal fukeduanhongyin capsules were used as negative control. Each solution was prepared according to the method of comparative example 3, and thin layer chromatography identification was performed according to the chromatographic conditions in Table 1.

[0127] Table 1

[0128]

[0129] Note: Chromatographic condition 2 is to replace the thin layer plate with a polyamide film of a different batch number from the same manufacturer as that of chromatographic condition 1 (the batch number of the polyamide film in condition 1 is 20181106, and the batch number of the polyamide film in condition 2 is 20210312).

[0130] 2.2 Sample, reference substance and reference medicinal material information

[0131]

[0132] 2.3 Results

[0133] The results of thin layer chromatography identification using chromatographic condition 1 are as follows Figure 6 As shown, Figure 61-blank solvent (methanol), 2-xanthopropanoside, 3-isorhamnetin-3-O-neohesperidin, 4-Puhuang control medicinal material, 5-negative sample of Fuke Duanhongyin Capsule lacking Puhuang charcoal, 6-Fuke Duanhongyin Capsule 20200704, 7-Fuke Duanhongyin Capsule 20200906, 8-Fuke Duanhongyin Capsule 20210102.

[0134] from Figure 6 It can be seen that when thin-layer chromatography was performed under chromatographic condition 1, the result was negative and without interference, one main spot of the Puhuang reference medicinal material was covered, and the low-humidity test failed, indicating that isorhamnetin-3-O-neohesperidin and champroglucosidin can be used as reference substances to perform thin-layer chromatography identification of Puhuang charcoal in Fuke Duanhongyin Capsules.

[0135] The results of thin layer chromatography identification using chromatographic condition 2 are as follows Figure 7 As shown, Figure 7 1-blank solvent (methanol), 2-xanthopropanoside, 3-isorhamnetin-3-O-neohesperidin, 4-Puhuang control medicinal material, 5-negative sample of Fuke Duanhongyin Capsule lacking Puhuang charcoal, 6-Fuke Duanhongyin Capsule 20200704, 7-Fuke Duanhongyin Capsule 20200906, 8-Fuke Duanhongyin Capsule 20210102.

[0136] from Figure 7 It can be seen that when thin layer chromatography was performed on the thin layer plate under chromatographic condition 2, the negative result was without interference, one main spot of the Puhuang reference medicinal material was covered, and the spot of the Typha neohesperidin reference substance was obscured, indicating that isorhamnetin-3-O-neohesperidin can be used as the reference substance to perform thin layer chromatography identification of Puhuang charcoal in Fuke Duanhongyin Capsules.

[0137] The results of thin layer chromatography identification using chromatographic condition 3 are as follows Figure 8 As shown, Figure 8 1-blank solvent (methanol), 2-isorhamnetin-3-O-neohesperidin-0.1 mg / mL, 3-isorhamnetin-3-O-neohesperidin-0.2 mg / mL, 4-isorhamnetin-3-O-neohesperidin-0.5 mg / mL, 5-negative sample of Fuke Duanhongyin Capsule lacking Puhuangcharcoal, 6-Fuke Duanhongyin Capsule 20200704, 7-Fuke Duanhongyin Capsule 20200906, 8-Fuke Duanhongyin Capsule 20210102.

[0138] from Figure 8 As can be seen from the figure, the thin layer chromatography was negative without interference when the chromatographic condition 3 was used, and the identification of the reference substance isorhamnetin-3-O-neohesperidin by thin layer chromatography was performed at a spot concentration of 0.1 mg / mL. That is, the method of Example 1 can accurately perform thin layer chromatography identification on the Typhae Pollen Carbon in Fuke Duanhongyin Capsules.

[0139] Comparative Example 4

[0140] Thin layer chromatography (TLC) was performed on the charcoal in Fuke Duanhongyin capsules using the method disclosed in patent CN105241997A. 1.0 g of the contents of Fuke Duanhongyin capsules were placed in a conical flask, dissolved by ultrasonication with 25 mL of water, and then 5 mL of 15% hydrochloric acid was added. The mixture was heated in a 98°C water bath for 1 hour. The mixture was cooled, filtered, and the filtrate was extracted twice with 20 mL of ethyl acetate each time. The ethyl acetate solutions were combined and evaporated to dryness. The residue was dissolved in 1 mL of methanol to prepare the test solution. Separately, 2.0 g of the control herb Typhae was added to 80 mL of water, boiled for 30 minutes, filtered, and the filtrate was concentrated to 25 mL. 5 mL of 15% hydrochloric acid was added, and the remainder was prepared using the same method as the test sample to prepare the control herb solution.

[0141] The chromatographic conditions of Example 1 were the same, except that a mixture of toluene: ethyl formate: formic acid in a volume ratio of 5:5:0.5 was used as the developing solvent. 254 On thin layer plate.

[0142] The results of thin layer chromatography identification of Typhae Pollen Carbon in Fuke Duanhongyin Capsule are as follows: Figure 9 shown. Figure 9 1-negative sample of Puhuang Tan Fuke Duanhongyin Capsule, 2, 6-Puhuang control medicinal material, 3-20200704, 4-20200906, 5-20210102, 7-Isorhamnetin-3-O-neohesperidin.

[0143] The identification results showed that the method failed to pass the specificity test and could not effectively distinguish between the control medicinal material Puhuang, isorhamnetin-3-O-neohesperidin, the negative sample of Fuke Duanhongyin Capsule lacking Puhuang charcoal, and the Fuke Duanhongyin Capsule sample.

[0144] Experimental Example 1 Effects of different film plates on the identification of Puhuang charcoal in Fuke Duanhongyin capsules by thin-layer chromatography

[0145] 1. Experimental Methods

[0146] Polyamide film plates from different manufacturers were used to perform thin layer chromatography identification of Puhuang charcoal in Fuke Duanhongyin Capsules according to the method of Example 1.

[0147] 2. Sample and control information is the same as in Example 1. Thin layer plate information is as follows:

[0148] Serial number factory model batch number Specification 1 Zhejiang Taizhou Luqiao Sijia Biochemical Plastic Factory Polyamide film 20210312 100*200mm 2 Wuhan Weiqi Boxing Biotechnology Co., Ltd. Polyamide film 20201120-WQ 100*200mm 3 Shanghai Yiji Industrial Co., Ltd. Polyamide film 20201025 100*200mm

[0149] 3. Results

[0150] In the chromatogram of the test sample, spots of the same color appear at the corresponding positions of the chromatogram of the reference sample. The results of thin layer chromatography identification of Fuke Duanhongyin Capsule Puhuang Charcoal using different thin layer plates are as follows: Figure 10 shown. Figure 10In the figure, 1-blank solvent (methanol), 2-isorhamnetin-3-O-neohesperidin control solution, 3-negative sample solution of Fuke Duanhongyin Capsules lacking Puhuangcharcoal, 4-6 are test solutions of Fuke Duanhongyin Capsules with batch numbers 20200704, 20200906, and 20210102; A-polyamide film of Zhejiang Taizhou Luqiao Sijia Biochemical Plastic Factory, B-polyamide film of Wuhan Weiqi Boxing Biotechnology Co., Ltd., and C-polyamide film of Shanghai Yiji Industrial Co., Ltd.

[0151] from Figure 10 It can be seen that the Rf of the identification bands under the thin layer plates of different manufacturers A to C are 0.30, 0.31, and 0.41, respectively. Although the band identified by the polyamide film of C-Shanghai Yiji Industrial Co., Ltd. has moved upward as a whole, the thin layer chromatography behavior of Fuke Duanhongyin Capsules is basically the same. This shows that the thin layer chromatography method of Example 1 of this application can be applied to different polyamide films and has good durability; the durability and feasibility of using the thin layer chromatography method of Example 1 to identify Fuke Duanhongyin Capsules and Fuke Duanhongyin Capsules negative samples are good.

[0152] Experimental Example 2 Effect of different temperatures on the identification of Puhuang charcoal in Fuke Duanhongyin capsules by thin-layer chromatography

[0153] 1. Experimental Methods

[0154] TLC identification of Puhuang charcoal in Fuke Duanhongyin Capsule was performed according to the method of Example 1 at 4°C, 20°C and 30°C respectively.

[0155] 2. Sample and control information are the same as in Example 1.

[0156] 3. Results

[0157] In the chromatogram of the test sample, spots of the same color appear at the corresponding positions in the chromatogram of the control. The results of thin layer chromatography of Fuke Duanhongyin Capsule Puhuang Charcoal at different temperatures are as follows Figure 11 shown. Figure 11 1-blank solvent (methanol), 2-isorhamnetin-3-O-neohesperidin control solution, 3-negative sample solution of Fuke Duanhongyin Capsules lacking Puhuang charcoal, 4-6 are test solutions of Fuke Duanhongyin Capsules with batch numbers 20200704, 20200906, and 20210102; A-20℃, B-4℃, C-30℃.

[0158] from Figure 11It can be seen that the Rf of the identification bands at different temperatures A to C are 0.30, 0.45, and 0.42, respectively. In the temperature range of 4°C to 30°C, the thin layer chromatography behavior of Fuke Duanhongyin Capsules is basically the same, indicating that the thin layer chromatography method of Example 1 of the present application has good durability in the temperature range of 4°C to 30°C; the durability and feasibility of identifying the negative samples of Fuke Duanhongyin Capsules and Fuke Duanhongyin Capsules lacking Puhuangtan using the thin layer chromatography method of Example 1 are good.

[0159] Experimental Example 3 Effect of Different Humidities on TLC Identification of Puhuang Charcoal in Fuke Duanhongyin Capsules

[0160] 1. Experimental Methods

[0161] Thin layer chromatography identification of the Puhuang charcoal in Fuke Duanhongyin Capsules was performed according to the method of Example 1 at relative humidity of 33%, 50% and 70%.

[0162] 2. Sample and control information are the same as in Example 1.

[0163] 3. Results

[0164] In the chromatogram of the test sample, spots of the same color appear at the corresponding positions of the chromatogram of the reference sample. Figure 12 shown. Figure 12 1-blank solvent (methanol), 2-isorhamnetin-3-O-neohesperidin control solution, 3-negative sample solution of Fuke Duanhongyin Capsules lacking Puhuang charcoal, 4-6 are test solutions of Fuke Duanhongyin Capsules with batch numbers 20200704, 20200906, and 20210102; A-50%, B-33%, C-70%.

[0165] from Figure 12 It can be seen that the Rf of the identification bands at different humidity levels A to C are 0.30, 0.37, and 0.36, respectively. Within the relative humidity range of 33% to 70%, the thin-layer chromatography behavior of Fuke Duanhongyin Capsules is essentially the same. This indicates that the thin-layer chromatography method of Example 1 of the present application has good durability within the humidity range of 33% to 70%, and that the thin-layer chromatography method of Example 1 has good durability and feasibility in distinguishing negative samples of Fuke Duanhongyin Capsules from those of Fuke Duanhongyin Capsules lacking Puhuangtan.

[0166] Experimental Example 4: Detection of Puhuang Charcoal in Different Batches of Fuke Duanhongyin Capsules

[0167] 1. Experimental Methods

[0168] Sixteen batches of Fuke Duanhongyin Capsules produced by Zhuzhou Qianjin Pharmaceutical Co., Ltd. (including one batch of negative samples lacking Typhae Pollen Carbon) were taken and thin layer chromatography identification was performed according to the method of Example 1.

[0169] 2. Sample and reference material information

[0170]

[0171]

[0172] 3. Results

[0173] The results of thin layer chromatography of Puhuang charcoal of different batches of Fuke Duanhongyin capsules are as follows Figure 13 . Figure 13 1-blank solvent (methanol), 2-isorhamnetin-3-O-neohesperidin, 3-negative sample of Puhuangtan Fuke Duanhongyin Capsule, 4~18-batch numbers are 20200701, 20200702, 20200703, 20200704, 20200705, 20200706, 20200805, 20200806, 20200906, 20201105, 20201202, 20210102, 20210201, 20210301, 20210302 Fuke Duanhongyin Capsule.

[0174] Figure 13 The results showed that when examined at 365 nm, among the chromatograms of the 16 batches of Fuke Duanhongyin Capsules test samples, except for one batch of Fuke Duanhongyin Capsules negative samples lacking Puhuang charcoal, the other 15 batches of samples all showed spots of the same color at the corresponding positions with the control chromatograms, indicating that the thin layer chromatography method of Example 1 can accurately and quickly identify Puhuang charcoal in Fuke Duanhongyin Capsules.

[0175] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A thin layer chromatography identification method for Typhae Pollen Carbon in Fuke Duanhongyin Capsules, characterized in that: The method comprises the following steps: S1. preparing a test solution: mixing the contents of Fuke Duanhongyin capsules with ether, heating under reflux to remove impurities, separating the solid and liquid after the impurities removal and discarding the liquid, drying the solid, extracting the dried solid with ethanol, separating the solid and liquid and discarding the solid, and drying the liquid to obtain an ethanol extract; then extracting the aqueous solution of the ethanol extract with ethyl acetate, discarding the ethyl acetate liquid layer, and extracting the aqueous solution layer of the ethanol extract with water-saturated n-butanol to obtain an n-butanol extract; removing the solvent from the n-butanol extract to obtain an n-butanol extract solid, dissolving the n-butanol extract solid with methanol, and purifying the solution by passing it through a chromatographic column filled with octadecylsilane bonded silica gel, eluting with methanol as an eluent to obtain a methanol eluent, and drying the methanol eluent and dissolving it in methanol again to serve as the test solution; S2. Preparation of reference solution: A methanol solution of the reference substance isorhamnetin-3-O-neohesperidin is used as the reference solution; S3 thin layer chromatography identification: the test solution prepared in step S1 and the reference solution prepared in step S2 were spotted on the same polyamide film, a mixture of acetone and water as a developing agent, sprayed with an ethanol solution of aluminum chloride, and placed under 365nm ultraviolet light for thin layer chromatography identification; The volume ratio of acetone to water in the developing agent in step S3 is 1-2:1-3.

2. The method according to claim 1, characterized in that The volume ratio of acetone to water in the developing agent in step S3 is 1-2:2-3.

3. The method according to claim 2, characterized in that The volume ratio of acetone to water in the developing solvent in step S3 is 2:

3.

4. The method according to claim 1, characterized in that The method comprises the following steps: S1. preparing a test solution: mixing the contents of the Fuke Duanhongyin capsule with ether, heating and refluxing for 20 to 30 minutes to remove impurities, separating the solid and liquid after removing impurities and discarding the liquid, and drying the solid; extracting the dried solid by cold soaking in 80% ethanol for 24 to 48 hours, separating the solid and liquid and discarding the solid, and drying the liquid to obtain an ethanol extract; then extracting the aqueous solution of the ethanol extract dissolved in water with ethyl acetate, discarding the ethyl acetate liquid layer, extracting the aqueous solution layer of the ethanol extract with water-saturated n-butanol to obtain an n-butanol extract, removing the solvent in the n-butanol extract to obtain an n-butanol extract solid, and dissolving the n-butanol extract with methanol. The solid is purified by a chromatographic column using octadecylsilane bonded silica gel as a filler, and eluted with methanol to obtain a methanol eluate. The methanol eluate is dried and then dissolved in methanol to serve as a test solution; the mass volume ratio of the contents of the Fuke Duanhongyin capsule to ether, ethanol, and ethyl acetate is 2-4 g: 15-25 mL: 45-55 mL: 40-50 mL; the mass volume ratio of the contents of the Fuke Duanhongyin capsule to water-saturated n-butanol is 2-4 g: 45-55 mL; the mass volume ratio of the contents of the Fuke Duanhongyin capsule to methanol in the test solution is 2-4 g: 1-2.5 mL; S2. Preparation of reference solution: Add methanol to the reference substance isorhamnetin-3-O-neohesperidin to prepare a reference solution with a concentration of 0.1-0.5 mg / mL; S3. Identification by thin layer chromatography: Pipette 1 μL of the test solution prepared in step S1 and 1 μL of the reference solution prepared in step S2, spot them separately on the same polyamide film, use a mixture of acetone and water as the developing solvent, and use the acetone:water volume ratio of 1-2:1-3. Spray with 3%-5% aluminum chloride ethanol solution, dry it, and inspect it under ultraviolet light at 365 nm for thin layer chromatography identification.

5. The method according to claim 4, characterized in that: The mass volume ratio of the contents of the Fuke Duanhongyin capsule to ether, ethanol, and ethyl acetate in step S1 is 2-3 g: 20-25 mL: 50-55 mL: 40-45 mL; the mass volume ratio of the contents of the Fuke Duanhongyin capsule to water-saturated n-butanol is 2-3 g: 50-55 mL; the mass volume ratio of the contents of the Fuke Duanhongyin capsule to methanol in the test solution is 2-3 g: 2-2.5 mL.

6. The method according to claim 4, characterized in that: The concentration of the reference solution in step S2 is 0.1-0.2 mg / mL.

7. The method according to claim 4, characterized in that: The concentration of the aluminum chloride ethanol solution in step S3 is 4% to 5%.

8. The method according to claim 4, characterized in that: The developing temperature of the developing agent in step S3 is 4°C to 30°C.

9. The method according to claim 4, characterized in that: The developing humidity of the developing agent in step S3 is 33% to 70%.

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