A method for detecting three medicinal materials in Diedazhentonggao by thin layer chromatography

Abstract

The application provides a method for detecting three medicinal materials in Diedazhengtonggao by adopting thin layer chromatography, and belongs to the technical field of effective component detection. Different test sample solutions and control sample solutions are prepared, thin layer chromatography is tested, different developing agents are adopted, rhubarb, huangbai and jiangxiang in Diedazhengtonggao are identified, chemical substances in medicinal material quality control components are used for specific identification, and the method for detecting rhubarb, huangbai and jiangxiang medicinal materials in Diedazhengtonggao by adopting thin layer chromatography is established, and the misjudgment problem caused by similar morphologies of medicinal materials in microscopic identification is solved.

Description

Technical Field

[0001] This invention belongs to the field of active ingredient detection technology, specifically relating to a method for detecting three medicinal materials in a traumatic pain relief plaster using thin-layer chromatography. Background Technology

[0002] The traumatic pain relief plaster is made from finely ground ingredients such as nux vomica, ground beetle, raw aconite, rhubarb, sandalwood, thorny scutellaria, phellodendron bark, and polygonum cuspidatum. It is composed of borneol, menthol oil, camphor, methyl salicylate, menthol, rubber, rosin, and other bases. Approval number: Z44022504. It has the effects of promoting blood circulation and relieving pain, dispersing blood stasis and reducing swelling, and dispelling wind and dampness. Clinically, it is used for acute and chronic sprains and contusions, chronic lumbago and leg pain, and rheumatic joint pain.

[0003] A prior patent, CN105891376A, discloses a quality standard and testing method for a traumatic pain-relieving plaster. This quality standard includes identification items, inspection items, volatile oil items, and content determination items. The identification items include microscopic identification and volatile oil identification. Microscopic identification includes the identification of crystal fibers, bast fibers, calcium oxalate clusters, single cells, and cell walls. Volatile oil identification includes qualitative detection of menthol, borneol, methyl salicylate, and camphor. The inspection items include qualitative testing of strychnine and aconitine, determination of plaster content, and compliance with the relevant provisions for rubber plasters under the section on plasters in the Pharmacopoeia General Rules. The volatile oil item involves the determination of the total volatile oil content. The content determination item involves the quantitative detection of camphor using gas chromatography. The quality standard and testing method established by this invention exhibit strong stability, good linearity, high accuracy, and good repeatability. This method can be used to scientifically evaluate the quality of traumatic pain-relieving plasters and is beneficial for quality control in industrial production.

[0004] Another prior patent, CN118817913A, discloses a method for extracting baicalin from traumatic pain relief plaster, a method for determining its content, and its application. The extraction method of baicalin from traumatic pain relief plaster provided by this invention can more fully extract baicalin from the complex components of traumatic pain relief plaster. The extracted baicalin test solution of traumatic pain relief plaster has good specificity, precision, stability, and repeatability, with a recovery rate of 97.59%-102.80%, and also has good durability.

[0005] However, the bruise and pain relief plaster contains a variety of traditional Chinese medicine ingredients, including nux vomica, ground beetle, raw aconite, rhubarb, sandalwood, thorny scutellaria, scutellaria, phellodendron bark, and polygonum cuspidatum. Among them, rhubarb invigorates blood circulation, removes blood stasis, regulates menstruation, and relieves pain; sandalwood invigorates blood circulation, promotes qi circulation, and relieves pain; and phellodendron bark clears heat, dries dampness, drains fire, detoxifies, reduces swelling, and relieves pain.

[0006] There are various existing technologies for the detection of rhubarb, phellodendron bark, and sandalwood, such as:

[0007] Chinese patent CN118330097A discloses a method for identifying rhubarb in rhubarb laxative tablets. The method includes the following steps: Step (1): preparing a test solution using rhubarb laxative tablets as raw material and a reference solution using rhubarb as raw material; Step (2): determining the test solution and reference solution using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS); wherein the mass spectrometry conditions used in the UHPLC-MS / MS are: mass spectrometry detector, electrospray positive ion mode, and the selected detection ion pairs for mass spectrometry acquisition are m / z 350.7 / 196.7 and m / z 350.7 / 119.7; Step (3): comparing the extracted ion chromatograms of the test solution and the reference solution to identify rhubarb in rhubarb laxative tablets. This method has the advantages of strong detection specificity, stable and reliable detection, and low detection and quantitation limits, and the identification method does not require the use of expensive rhubarb glycosides as a reference solution.

[0008] Chinese patent CN119104670A discloses a thin-layer chromatography method for identifying Dalbergia odorifera and its adulterants. This invention improves the developing system, requiring only one developing solvent to simultaneously identify volatile oils and flavonoids in Dalbergia odorifera, simplifying the operation and shortening the identification time. The invention uses common, non-toxic cyclohexane, ethyl acetate, and acetic acid as developing solvents, avoiding the use of toxic and harmful reagents such as toluene, chloroform, and ether, achieving effective separation of volatile oils and flavonoids while reducing the use of toxic reagents. The invention also provides a new method for detecting volatile oils by spraying a mixed solution of vanillin-sulfuric acid and anhydrous ethanol, heating it, and then placing it under a 365nm ultraviolet lamp for detection, displaying fluorescent spots of different colors, overcoming the problem of weak identification ability caused by relying on color intensity in current methods.

[0009] Chinese patent CN119936291A discloses a thin-layer chromatography method for simultaneously identifying Coptis chinensis and Phellodendron chinense components and its application. The method includes the following steps: (1) preparing a test solution, a Coptis chinensis reference solution, a Phellodendron chinense reference solution, and a reference solution respectively; (2) spotting the above solutions onto the same silica gel G thin-layer plate, using a hexane-butanol-methanol-water mixture with a volume ratio of (2.5-3.5):(2.5-3.5):(1.5-2.5):(0.8-1.2) as the developing solvent, placing the silica gel G thin-layer plate in a developing tank, developing, removing, and drying, and then examining it under a 360-370 nm light source. The thin-layer chromatography method provided in this application can quickly and easily identify Coptis chinensis and Phellodendron chinense components in traditional Chinese medicine external preparations, achieving quality control of traditional Chinese medicine external preparations.

[0010] However, there are few existing technologies for detecting multiple components of traditional Chinese medicine.

[0011] For example, Chinese patent CN119199001A discloses a detection method for Fushenning capsules, which includes microscopic identification of the product, thin-layer chromatography identification of Anemarrhena asphodeloides, thin-layer chromatography identification of geniposide, thin-layer chromatography identification of rhubarb, thin-layer chromatography identification of Phellodendron amurense, and determination of berberine hydrochloride content. Specifically, the thin-layer chromatography identification of Phellodendron amurense is performed as follows: 1-2 μL of each of the above two solutions are applied separately to the same silica gel G thin-layer plate using a 7:2:1 ratio of n-butanol-glacial acetic acid-water as the developing solvent. After development, the plate is removed, dried, and examined under a 365 nm ultraviolet lamp. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions in the chromatogram of the reference medicinal material. The thin-layer chromatography (TLC) identification of rhubarb is as follows: Following the TLC test, apply 2-8 μL of each of the three solutions mentioned above to the same silica gel G TLC plate. Use a 15:5:1 ratio of petroleum ether-ethyl formate-formic acid upper layer solution at 30-60℃ as the developing solvent. Develop, remove, air dry, and examine under ultraviolet light at 365 nm. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatograms of the reference medicinal material and the reference standard. This invention optimizes the TLC identification method for Anemarrhena asphodeloides in Fushenning capsules based on existing quality testing methods. Methodological investigation shows that this detection method is highly specific, robust, accurate, and stable, and is simple and quick to operate, improving the quality testing method for Fushenning capsules and enabling better quality control.

[0012] For example, Chinese patent CN101732463A discloses a quality testing method for Jinhua Xiaozuo Pills. This method improves the quality control standards of Jinhua Xiaozuo Pills, establishes a method for determining the content of the main drug in the preparation, eliminates the identification of poorly specific physicochemical reactions, improves the thin-layer chromatography identification method for geniposide and berberine hydrochloride, making the method simpler and the characteristic spots clearer, and adds thin-layer chromatography identification methods for rhubarb, coptis, phellodendron, and scutellaria, ensuring a high quality standard level for this compound preparation. Specifically, the thin-layer chromatography identification method for rhubarb is as follows: according to thin-layer chromatography (Chinese Pharmacopoeia 2005 Edition, Part I, Appendix VI). B) For the test, take 5-10 μL of each solution and spot them separately on the same silica gel H thin-layer plate with sodium carboxymethyl cellulose as the binder. Use n-hexane-ethyl acetate-formic acid (volume ratio 5-8:1.5-3:0.1-0.2) as the developing solvent. Develop, remove, air dry, and examine under ultraviolet light (365 nm). In the chromatogram of the test sample, five yellow fluorescent spots appear at the same positions as in the chromatogram of the reference medicinal material; in the chromatogram of the reference substance, one yellow spot appears at the same position. After being exposed to ammonia vapor, the spot turns red when examined under sunlight. Thin-layer chromatographic identification method for Coptis chinensis and Phellodendron chinense: Perform the thin-layer chromatography test (Chinese Pharmacopoeia 2005 Edition, Part I, Appendix VIB). Take 5 μL of each solution and spot them separately on the same silica gel G thin-layer plate with sodium carboxymethyl cellulose as binder. Use ethyl acetate-butanone-formic acid-water (volume ratio 8-11:4-7:0.9-1.1:0.9-1.1) as the developing solvent. Develop, remove, air dry, and examine under ultraviolet light (365 nm). In the chromatogram of the test sample, yellow fluorescent spots appear at the same positions as those in the chromatograms of the reference medicinal material and the reference standard.

[0013] However, there are almost no existing technologies that can simultaneously detect multiple samples of rhubarb, phellodendron bark, and sandalwood. The "Traumatic Injury Relief Plaster" section of the Chinese Pharmacopoeia (2020 edition) only identifies the microscopic characteristics of this medicinal material.

[0014] Currently, there is no existing technology for the TLC identification of rhubarb, phellodendron bark, and sandalwood in traumatic pain relief plasters. Therefore, it is necessary to develop an identification and determination method for these three medicinal materials in traumatic pain relief plasters, as this method cannot effectively solve the problem of misidentification caused by the similarity of the medicinal materials. Summary of the Invention

[0015] The prescription for traumatic pain relief plaster contains rhubarb, phellodendron bark, and sandalwood. Current quality standards only identify the microscopic characteristics of these medicinal materials under the microscopic identification section. This method has low precision, cannot utilize the chemical substances in the material control components for specific identification, and suffers from misjudgment due to similar morphological characteristics of the medicinal materials during microscopic identification. Based on the shortcomings of existing technology, this invention aims to utilize the chemical substances in the material control components for specific identification, exploring and establishing a thin-layer chromatography method for detecting rhubarb, phellodendron bark, and sandalwood in traumatic pain relief plaster, thus solving the problem of misjudgment due to similar morphological characteristics of the medicinal materials during microscopic identification.

[0016] To achieve the above objectives, the present invention further employs the following technical solution:

[0017] A method for detecting three medicinal materials in a traumatic pain relief plaster using thin-layer chromatography includes the following steps:

[0018] Thin-layer chromatography identification method for rhubarb:

[0019] Preparation of test solution A: Take one piece of liniment for bruises and pain relief, cut it into small pieces, remove the cap, add methanol, sonicate, filter, evaporate the filtrate to dryness, add water to dissolve the residue, add hydrochloric acid, heat in a water bath, cool immediately, extract with ether by shaking, combine the ether extracts, evaporate to dryness, add ethyl acetate to dissolve the residue, and use as the test solution.

[0020] The amount of methanol used is 20-30 mL;

[0021] More preferably, the amount of methanol used is 20mL-25mL;

[0022] More preferably, the amount of methanol used is 25 mL.

[0023] The power of the ultrasound is 3000Hz-4000Hz;

[0024] Preferably, the power of the ultrasound is 3000Hz-3500Hz;

[0025] More preferably, the power of the ultrasound is 3500 Hz.

[0026] The temperature of the ultrasound is 20℃-30℃;

[0027] Preferably, the temperature of the ultrasound is 25℃-30℃;

[0028] More preferably, the temperature of the ultrasound is 25°C.

[0029] The ultrasound session lasted 15-30 minutes.

[0030] Preferably, the ultrasound session lasts for 20-25 minutes;

[0031] More preferably, the ultrasound duration is 20 minutes.

[0032] The amount of hydrochloric acid used is 1 mL to 5 mL;

[0033] More preferably, the amount of hydrochloric acid used is 2 mL to 3 mL;

[0034] More preferably, the amount of hydrochloric acid used is 2 mL.

[0035] The water bath heating time is 20-40 minutes;

[0036] Preferably, the water bath heating time is 25-35 minutes;

[0037] More preferably, the water bath heating time is 30 minutes.

[0038] The ether extraction is performed 1-3 times by shaking.

[0039] Preferably, the ether shaking extraction is performed twice.

[0040] As a preferred embodiment, the preparation of the test solution is as follows:

[0041] Preparation of test solution A: Take one piece of liniment for bruises and pain relief, cut it into small pieces, remove the cap, add 25 mL of methanol, sonicate for 20 minutes, filter, evaporate the filtrate to dryness, add 20 mL of water to dissolve the residue, add 2 mL of hydrochloric acid, heat in a water bath for 30 minutes, cool immediately, extract twice with 20 mL of ether each time, combine the ether extracts, evaporate to dryness, add 1 mL of ethyl acetate to dissolve the residue, and use as the test solution.

[0042] Preparation of reference solution: Take rhubarb reference material and prepare reference solution using the same method.

[0043] Thin-layer chromatography identification: Perform the thin-layer chromatography test (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). Apply 5-10 μL of the above test solution and 5 μL of the reference solution separately to the same silica gel G thin-layer plate. Develop with the developing solvent, remove, air dry, and examine under ultraviolet light (254 nm). The chromatogram of the test sample shows fluorescent spots of the same color at the corresponding positions as the chromatogram of the reference sample. After fuming with ammonia vapor until color development is achieved, examine under sunlight; spots of the same color will appear.

[0044] The developing solvent is a mixed solution of n-hexane, ethyl acetate, and formic acid.

[0045] The volume ratio of hexane-ethyl acetate-formic acid is (4-6):(0.5-1.5):(0.05-0.15);

[0046] Preferably, the volume ratio of n-hexane-ethyl acetate-formic acid is (4.5-5.5):(0.8-1.2):(0.08-0.1);

[0047] More preferably, the volume ratio of hexane-ethyl acetate-formic acid is 5:1:0.1.

[0048] As a preferred embodiment, the thin-layer chromatography identification method for rhubarb is as follows:

[0049] Preparation of test solution A: Take one piece of liniment for bruises and pain relief, cut it into small pieces, remove the cap, add 25 mL of methanol, sonicate for 20 minutes, filter, evaporate the filtrate to dryness, add 20 mL of water to dissolve the residue, add 2 mL of hydrochloric acid, heat in a water bath for 30 minutes, cool immediately, extract twice with 20 mL of ether each time, combine the ether extracts, evaporate to dryness, add 1 mL of ethyl acetate to dissolve the residue, and use as the test solution.

[0050] Preparation of reference solution: Take 0.1g of rhubarb reference material and prepare a reference solution using the same method.

[0051] Thin-layer chromatography identification: Perform the thin-layer chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). Apply 5-10 μL of the above test solution and 5 μL of the reference solution separately to the same silica gel G thin-layer plate. Use n-hexane-ethyl acetate-formic acid (5:1:0.1) as the developing solvent. Develop, remove, air dry, and examine under ultraviolet light (254 nm). The chromatogram of the test sample shows fluorescent spots of the same color at the corresponding positions as the chromatogram of the reference sample. After fuming with ammonia vapor until color development is achieved, examine under sunlight; spots of the same color will appear.

[0052] Thin-layer chromatography identification method for Dalbergia odorifera:

[0053] Preparation of test solution B: Take 40cm of liniment for bruises and sprains. 2 Cut into small pieces, remove the liner, add ethanol, sonicate, filter, evaporate the filtrate to dryness, dissolve the residue in ethanol to obtain the test solution.

[0054] The amount of ethanol used is 20mL-30mL;

[0055] More preferably, the amount of ethanol used is 25 mL to 30 mL;

[0056] More preferably, the amount of ethanol used is 25 mL.

[0057] The power of the ultrasound is 3000Hz-4000Hz;

[0058] Preferably, the power of the ultrasound is 3500Hz-4000Hz;

[0059] More preferably, the power of the ultrasound is 3500 Hz.

[0060] The temperature of the ultrasound is 20℃-30℃;

[0061] Preferably, the temperature of the ultrasound is 25℃-30℃;

[0062] More preferably, the temperature of the ultrasound is 25°C.

[0063] The duration of the ultrasound is 0.5-2 hours;

[0064] Preferably, the ultrasound duration is 1-1.5 hours;

[0065] More preferably, the ultrasound duration is 1 hour.

[0066] As a preferred embodiment, the preparation of test solution B is as follows:

[0067] Take 40cm of liniment for bruises and sprains. 2 Cut the contents into small pieces, remove the liner, add 25 mL of ethanol, sonicate for 1 hour, filter, evaporate the filtrate to dryness, dissolve the residue in 1 mL of ethanol, and use it as the test solution.

[0068] Preparation of reference solution: Take the reference herb *Dalbergia odorifera*, add ethanol, reflux to extract, filter, evaporate the filtrate to dryness, dissolve the residue in ethanol to obtain the reference solution.

[0069] The amount of ethanol used is 35mL-50mL;

[0070] More preferably, the amount of ethanol used is 40mL-45mL;

[0071] More preferably, the amount of ethanol is 40 mL.

[0072] The reflux extraction temperature is 80℃-100℃;

[0073] Preferably, the reflux extraction temperature is 90℃-100℃;

[0074] More preferably, the reflux extraction temperature is 100°C.

[0075] The reflux extraction time is 40-90 minutes;

[0076] Preferably, the reflux extraction time is 60-90 minutes;

[0077] More preferably, the reflux extraction time is 60 minutes.

[0078] Preferably, the preparation of the reference solution is as follows:

[0079] 0.5g of Dalbergia odorifera reference material was extracted by reflux for 1 hour with 40mL of ethanol, filtered, the filtrate was evaporated to dryness, and the residue was dissolved in 1mL of ethanol to obtain the reference solution.

[0080] Thin-layer chromatography identification: Perform the thin-layer chromatography test (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). Apply 2-5 μL of the test solution and 2 μL of the reference solution separately to the same silica gel G thin-layer plate. Develop with the developing solvent, remove, air dry, and examine under ultraviolet light (254 nm). In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample.

[0081] The developing solvent is a mixture of toluene and ethyl acetate.

[0082] The volume ratio of toluene to ethyl acetate is (1-3):1;

[0083] Preferably, the volume ratio of toluene to ethyl acetate is (1.5-2.5):1;

[0084] More preferably, the volume ratio of toluene to ethyl acetate is 2:1.

[0085] As a preferred embodiment, the thin-layer chromatography identification method for Dalbergia odorifera is as follows:

[0086] Preparation of test solution B: Take 40cm of liniment for bruises and sprains. 2 Cut the contents into small pieces, remove the liner, add 25 mL of ethanol, sonicate for 1 hour, filter, evaporate the filtrate to dryness, dissolve the residue in 1 mL of ethanol, and use it as the test solution.

[0087] Preparation of reference solution: 0.5g of Dalbergia odorifera reference material was added to 25mL of ethanol and refluxed for 30min. The mixture was filtered, the filtrate was evaporated to dryness, and the residue was dissolved in 2mL of ethanol to obtain the reference solution.

[0088] Thin-layer chromatography identification: Perform the thin-layer chromatography test (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). Apply 2-5 μL of the above test solution and 2 μL of the reference solution separately to the same silica gel G thin-layer plate. Develop the plate using toluene-ethyl acetate (2:1) as the developing solvent. Remove the plate, air-dry it, and examine it under ultraviolet light (254 nm). In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample.

[0089] Thin-layer chromatography identification method for Phellodendron bark:

[0090] Preparation of test solution C: Take 2 pieces of bruise and pain relief plaster, cut them into small pieces, remove the caps, add methanol solution, sonicate, filter, evaporate the filtrate to dryness, dissolve the residue in methanol, and use it as the test solution.

[0091] The methanol solution mentioned is a methanol solution of acetic acid;

[0092] The concentration of acetic acid in the acetic acid-methanol solution is 0.8-2%;

[0093] Preferably, the concentration of acetic acid in the acetic acid-methanol solution is 1-1.5%;

[0094] More preferably, the concentration of acetic acid in the acetic acid-methanol solution is 1-1.2%;

[0095] More preferably, the concentration of acetic acid in the acetic acid-methanol solution is 1%.

[0096] The power of the ultrasound is 3000Hz-4000Hz.

[0097] Preferably, the power of the ultrasound is 3500Hz-4000Hz.

[0098] More preferably, the power of the ultrasound is 3500 Hz.

[0099] The temperature of the ultrasound is 50-70℃;

[0100] Preferably, the temperature of the ultrasound is 55-65°C;

[0101] More preferably, the temperature of the ultrasound is 60°C.

[0102] The ultrasound session lasted 10-30 minutes.

[0103] Preferably, the ultrasound session lasts for 15-20 minutes;

[0104] More preferably, the ultrasound duration is 20 minutes.

[0105] As a preferred embodiment, the preparation of the test solution C is as follows:

[0106] Take two pieces of bruise and pain relief plaster, cut them into small pieces, remove the caps, add 40 mL of 1% acetic acid methanol solution, sonicate at 60℃ for 20 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 2 mL of 1% acetic acid methanol solution, and use it as the test solution.

[0107] Preparation of reference solution: Take Phellodendron bark reference material, add methanol solution, and prepare reference solution in the same way.

[0108] The methanol solution mentioned is methanol acetic acid;

[0109] The concentration of the acetic acid-methanol solution is 0.8-2%;

[0110] Preferably, the concentration of the acetic acid-methanol solution is 1-1.5%;

[0111] More preferably, the concentration of the acetic acid-methanol solution is 1-1.2%;

[0112] More preferably, the concentration of the acetic acid-methanol solution is 1%.

[0113] As a preferred embodiment, the preparation of the reference solution is as follows:

[0114] Take 0.1g of Phellodendron bark reference material and add 20mL of 1% acetic acid methanol solution to prepare a reference solution using the same method.

[0115] Thin-layer chromatography identification: Perform the thin-layer chromatography test (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). Apply 1-5 μL of the test solution and 5 μL of the reference solution separately to the same silica gel G thin-layer plate. Develop with the developing solvent, remove, air dry, fumigate with ammonia vapor, and then examine under sunlight. The chromatogram of the test solution should show spots of the same color at the corresponding positions as the chromatogram of the reference solution.

[0116] The developing solvent is a mixed solution of ethyl acetate, 2-butanone, formic acid and water.

[0117] The volume ratio of ethyl acetate, 2-butanone, formic acid and water is (8-12):(6-8):(1-2):(1-2);

[0118] Preferably, the volume ratio of ethyl acetate, 2-butanone, formic acid and water is (9-11):(6-7):(1-2):(1-2);

[0119] More preferably, the volume ratio of ethyl acetate, 2-butanone, formic acid, and water is 10:7:1:1.

[0120] As a preferred embodiment, the thin-layer chromatography identification method for Phellodendron amurense is as follows:

[0121] Preparation of test solution C: Take 2 pieces of bruise and pain relief plaster, cut them into small pieces, remove the caps, add 40 mL of 1% acetic acid methanol solution, sonicate at 60℃ for 20 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 2 mL of 1% acetic acid methanol solution, and use it as the test solution.

[0122] Preparation of reference solution: Take 0.1g of Phellodendron bark reference material, add 20mL of 1% acetic acid methanol solution, and prepare the reference solution in the same way.

[0123] Thin-layer chromatography identification: Perform the thin-layer chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). Apply 1-5 μL of the test solution and 5 μL of the reference solution separately to the same silica gel G thin-layer plate. Use ethyl acetate-2-butanone-formic acid-water (10:7:1:1) as the developing solvent. Develop, remove, air-dry, fumigate with ammonia vapor, and then examine under sunlight. The chromatogram of the test sample should show spots of the same color at the corresponding positions as the chromatogram of the reference sample.

[0124] The above method is used to identify rhubarb, sandalwood, and phellodendron components in topical Chinese medicine preparations.

[0125] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0126] (1) Thin-layer chromatography (TLC) and microscopic identification are used for the specific identification of rhubarb, sandalwood, and phellodendron bark medicinal materials and processed slices. According to the requirements of the "National Drug Standard (China) Research and Manufacturing Technical Requirements", in addition to the pretreatment of each medicinal ingredient in the preparation, the identification method should, in principle, be as consistent as possible with the specific identification method of the medicinal material and processed slice. Currently, only microscopic identification method is available for this preparation. The identification method provided by this invention fills the gap in TLC identification of this preparation.

[0127] (2) The identification method provided by the present invention is not affected by the source of Chinese medicinal plants in the prescription of Chinese medicine preparations. Based on the different distribution coefficients of each component in the preparation between the stationary phase and the mobile phase, differential migration occurs on the thin-layer plate to achieve separation.

[0128] (3) The identification method provided by the present invention does not require complicated reagents, instruments or equipment, and can quickly identify multiple Chinese medicinal materials in traumatic pain relief plaster. Not only are the identification results vivid and easy to identify, but the sensitivity and specificity are also higher than conventional thin-layer chromatography identification methods. In addition, this method is not only applicable to the detection of traumatic pain relief plaster, but also applicable to the detection of other dosage forms, thus ensuring more comprehensive detection and providing a convenient and practical way for qualitative identification of multiple Chinese medicinal materials in traumatic pain relief plaster. Attached Figure Description

[0129] Figure 1 Thin-layer chromatography identification diagram of rhubarb;

[0130] in:

[0131] 1. 5 μL of negative sample solution lacking rhubarb;

[0132] 2. 5 μL of rhubarb reference solution;

[0133] 3. 5 μL of rhein reference solution;

[0134] 4. 1.5 μL of test solution;

[0135] 5. 25 μL of test solution;

[0136] 6. 35 μL of test solution;

[0137] 7. 5 μL of double-negative sample solution lacking rhubarb and Polygonum cuspidatum;

[0138] Temperature: 11℃;

[0139] Humidity: 72%

[0140] Thin-layer plate: Silicone G;

[0141] Developing solvent: n-hexane-ethyl acetate-formic acid (5:1:0.1); Development time: 16:58-17:50;

[0142] Color development: Place in ammonia vapor until color develops;

[0143] Inspection: Under ultraviolet light (254nm) and sunlight.

[0144] Figure 2 Thin-layer chromatography identification diagram of Dalbergia odorifera;

[0145] in:

[0146] 1. 5 μL of negative sample solution lacking Dalbergia odorifera;

[0147] 2. 2 μL of the frankincense reference solution;

[0148] 3. 1.5 μL of test solution;

[0149] 4. 25 μL of test solution;

[0150] 5. 35 μL of test solution;

[0151] Temperature: 28℃;

[0152] Humidity: 59%;

[0153] Thin-layer plate: Silicone G;

[0154] Developing solvent: Toluene-ethyl acetate (2:1);

[0155] Inspection: Under ultraviolet light (254nm).

[0156] Figure 3 Thin-layer chromatography identification diagram of Phellodendron bark;

[0157] in:

[0158] 1. 5 μL of negative sample solution lacking Phellodendron bark;

[0159] 2. 5 μL of Phellodendron bark reference solution;

[0160] 3. 1.5 μL of test solution;

[0161] 4. 25 μL of test solution;

[0162] 5. 35 μL of test solution;

[0163] 6. 10 μL of berberine hydrochloride reference solution;

[0164] Temperature: 27℃;

[0165] Humidity: 59%;

[0166] Thin-layer plate: Silicone G;

[0167] Developing solvent: ethyl acetate-2-butanone-formic acid-water (10:7:1:1);

[0168] Color development: Ammonia fumigate in a developing tank saturated with ammonia vapor. Detailed Implementation

[0169] The present application is further described below with reference to the embodiments, examples, and accompanying drawings. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the application. Furthermore, it should be understood that after reading the contents of this application, those skilled in the art can make various alterations or modifications to this application, and these equivalent forms also fall within the protection scope of the appended claims.

[0170] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0171] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of this application.

[0172] Terminology: Unless otherwise stated or in case of conflict, the terms or phrases used herein have the following meanings:

[0173] As used herein, the terms “and / or,” “or / and,” and “and / or” encompass any one of two or more of the related listed items, as well as any and all combinations of the related listed items, including any two related listed items, any more related listed items, or a combination of all the related listed items.

[0174] It should be understood that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", the technical solution in this application undoubtedly includes technical solutions connected by "logical AND" and also undoubtedly includes technical solutions connected by "logical OR". For example, "A and / or B" includes three parallel solutions: A, B, and A+B. As another example, the technical solution "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (i.e., technical solutions connected by "logical OR"), any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (i.e., technical solutions connected by "logical AND").

[0175] In this document, terms such as "preferred," "better," and "more preferred" are merely descriptions of implementation methods or examples that achieve better results, and should be understood as not constituting a limitation on the scope of protection of this application.

[0176] In this application, terms such as "further," "even further," and "particularly" are used for descriptive purposes and to indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0177] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0178] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints of the numerical range (i.e., the minimum and maximum values), as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints.

[0179] Furthermore, when multiple scopes are provided to describe a feature or characteristic, these scopes may be merged. In other words, unless otherwise specified, the scopes disclosed herein should be understood to include any and all subscopes to which they are included.

[0180] In this application, weight can be a well-known unit of mass in the chemical industry, such as μg, mg, g, or kg.

[0181] In this application, "%" represents a percentage, referring to the proportion by weight; however, the percentage of a solution, unless otherwise specified, refers to the number of grams of solute contained in 100 mL of the solution; the percentage of alcohol refers to the proportion by volume at 20°C. Furthermore, the following symbols may be used as needed: % (g / g) indicates the number of grams of solute contained in 100 g of solution; % (mL / mL) indicates the number of milliliters of solute contained in 100 mL of solution; % (mL / g) indicates the number of milliliters of solute contained in 100 g of solution; % (g / mL) indicates the number of grams of solute contained in 100 mL of solution.

[0182] This invention provides a method for detecting three medicinal materials in a traumatic pain relief plaster using thin-layer chromatography, comprising the following steps:

[0183] Thin-layer chromatography identification method for rhubarb:

[0184] Preparation of test solution: Take one piece of liniment for bruises and pain relief, cut it into small pieces, remove the cap, add methanol, sonicate, filter, evaporate the filtrate to dryness, add water to dissolve the residue, add hydrochloric acid, heat in a water bath, cool immediately, extract with ether by shaking, combine the ether extracts, evaporate to dryness, add ethyl acetate to dissolve the residue, and use as the test solution.

[0185] The amount of methanol used is 20-30 mL;

[0186] More preferably, the amount of methanol used is 20mL-25mL;

[0187] More preferably, the amount of methanol used is 25 mL.

[0188] The power of the ultrasound is 3000Hz-4000Hz;

[0189] Preferably, the power of the ultrasound is 3000Hz-3500Hz;

[0190] More preferably, the power of the ultrasound is 3500 Hz.

[0191] The temperature of the ultrasound is 20℃-30℃;

[0192] Preferably, the temperature of the ultrasound is 25℃-30℃;

[0193] More preferably, the temperature of the ultrasound is 25°C.

[0194] The ultrasound session lasted 15-30 minutes.

[0195] Preferably, the ultrasound session lasts for 20-25 minutes;

[0196] More preferably, the ultrasound duration is 20 minutes.

[0197] The amount of hydrochloric acid used is 1 mL to 5 mL;

[0198] More preferably, the amount of hydrochloric acid used is 2 mL to 3 mL;

[0199] More preferably, the amount of hydrochloric acid used is 2 mL.

[0200] The water bath heating time is 20-40 minutes;

[0201] Preferably, the water bath heating time is 25-35 minutes;

[0202] More preferably, the water bath heating time is 30 minutes.

[0203] The ether extraction is performed 1-3 times by shaking.

[0204] Preferably, the ether shaking extraction is performed twice.

[0205] As a preferred embodiment, the preparation of the test solution is as follows:

[0206] Preparation of test solution: Take one piece of liniment for bruises and pain relief, cut it into small pieces, remove the cap, add 25 mL of methanol, sonicate for 20 minutes, filter, evaporate the filtrate to dryness, add 20 mL of water to dissolve the residue, add 2 mL of hydrochloric acid, heat in a water bath for 30 minutes, cool immediately, extract twice with 20 mL of ether each time, combine the ether extracts, evaporate to dryness, add 1 mL of ethyl acetate to dissolve the residue, and use as the test solution.

[0207] Preparation of reference solution: Take rhubarb reference material and prepare reference solution using the same method.

[0208] Thin-layer chromatography identification: Perform the thin-layer chromatography test (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). Apply 5-10 μL of the above test solution and 5 μL of the reference solution separately to the same silica gel G thin-layer plate. Develop with the developing solvent, remove, air dry, and examine under ultraviolet light (254 nm). The chromatogram of the test sample shows fluorescent spots of the same color at the corresponding positions as the chromatogram of the reference sample. After fuming with ammonia vapor until color development is achieved, examine under sunlight; spots of the same color will appear.

[0209] The developing solvent is a mixed solution of n-hexane, ethyl acetate, and formic acid.

[0210] The volume ratio of hexane-ethyl acetate-formic acid is (4-6):(0.5-1.5):(0.05-0.15);

[0211] Preferably, the volume ratio of n-hexane-ethyl acetate-formic acid is (4.5-5.5):(0.8-1.2):(0.08-0.1);

[0212] More preferably, the volume ratio of hexane-ethyl acetate-formic acid is 5:1:0.1.

[0213] As a preferred embodiment, the thin-layer chromatography identification method for rhubarb is as follows:

[0214] Preparation of test solution: Take one piece of liniment for bruises and pain relief, cut it into small pieces, remove the cap, add 25 mL of methanol, sonicate for 20 minutes, filter, evaporate the filtrate to dryness, add 20 mL of water to dissolve the residue, add 2 mL of hydrochloric acid, heat in a water bath for 30 minutes, cool immediately, extract twice with 20 mL of ether each time, combine the ether extracts, evaporate to dryness, add 1 mL of ethyl acetate to dissolve the residue, and use as the test solution.

[0215] Preparation of reference solution: Take 0.1g of rhubarb reference material and prepare a reference solution using the same method.

[0216] Thin-layer chromatography identification: Perform the thin-layer chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). Apply 5-10 μL of the above test solution and 5 μL of the reference solution separately to the same silica gel G thin-layer plate. Use n-hexane-ethyl acetate-formic acid (5:1:0.1) as the developing solvent. Develop, remove, air dry, and examine under ultraviolet light (254 nm). The chromatogram of the test sample shows fluorescent spots of the same color at the corresponding positions as the chromatogram of the reference sample. After fuming with ammonia vapor until color development is achieved, and examining under sunlight, spots of the same color should appear.

[0217] Thin-layer chromatography identification method for Dalbergia odorifera:

[0218] Preparation of the test solution: Take 40cm of liniment for bruises and sprains. 2 Cut into small pieces, remove the liner, add ethanol, sonicate, filter, evaporate the filtrate to dryness, dissolve the residue in ethanol to obtain the test solution.

[0219] The amount of ethanol used is 20mL-30mL;

[0220] More preferably, the amount of ethanol used is 25 mL to 30 mL;

[0221] More preferably, the amount of ethanol used is 25 mL.

[0222] The power of the ultrasound is 3000Hz-4000Hz;

[0223] Preferably, the power of the ultrasound is 3500Hz-4000Hz;

[0224] More preferably, the power of the ultrasound is 3500 Hz.

[0225] The temperature of the ultrasound is 20℃-30℃;

[0226] Preferably, the temperature of the ultrasound is 25℃-30℃;

[0227] More preferably, the temperature of the ultrasound is 25°C.

[0228] The duration of the ultrasound is 0.5-2 hours;

[0229] Preferably, the ultrasound duration is 1-1.5 hours;

[0230] More preferably, the ultrasound duration is 1 hour.

[0231] As a preferred embodiment, the preparation of the test solution is as follows:

[0232] Take 40cm of liniment for bruises and sprains. 2 Cut the contents into small pieces, remove the liner, add 25 mL of ethanol, sonicate for 1 hour, filter, evaporate the filtrate to dryness, dissolve the residue in 1 mL of ethanol, and use it as the test solution.

[0233] Preparation of reference solution: Take the reference herb *Dalbergia odorifera*, add ethanol, reflux to extract, filter, evaporate the filtrate to dryness, dissolve the residue in ethanol to obtain the reference solution.

[0234] The amount of ethanol used is 35mL-50mL;

[0235] More preferably, the amount of ethanol used is 40mL-45mL;

[0236] More preferably, the amount of ethanol is 40 mL.

[0237] The reflux extraction temperature is 80℃-100℃;

[0238] Preferably, the reflux extraction temperature is 90℃-100℃;

[0239] More preferably, the reflux extraction temperature is 100°C.

[0240] The reflux extraction time is 40-90 minutes;

[0241] Preferably, the reflux extraction time is 60-90 minutes;

[0242] More preferably, the reflux extraction time is 60 minutes.

[0243] Preferably, the preparation of the reference solution is as follows:

[0244] 0.5g of Dalbergia odorifera reference material was extracted by reflux for 1 hour with 40mL of ethanol, filtered, the filtrate was evaporated to dryness, and the residue was dissolved in 1mL of ethanol to obtain the reference solution.

[0245] Thin-layer chromatography identification: Perform the thin-layer chromatography test (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). Apply 2-5 μL of the test solution and 2 μL of the reference solution separately to the same silica gel G thin-layer plate. Develop with the developing solvent, remove, air dry, and examine under ultraviolet light (254 nm). In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample.

[0246] The developing solvent is a mixture of toluene and ethyl acetate.

[0247] The volume ratio of toluene to ethyl acetate is 1-(3:1);

[0248] Preferably, the volume ratio of toluene to ethyl acetate is (1.5-2.5):1;

[0249] More preferably, the volume ratio of toluene to ethyl acetate is 2:1.

[0250] As a preferred embodiment, the thin-layer chromatography identification method for Dalbergia odorifera is as follows:

[0251] Preparation of the test solution: Take 40cm of liniment for bruises and sprains. 2 Cut the contents into small pieces, remove the liner, add 25 mL of ethanol, sonicate for 1 hour, filter, evaporate the filtrate to dryness, dissolve the residue in 1 mL of ethanol, and use it as the test solution.

[0252] Preparation of reference solution: 0.5g of Dalbergia odorifera reference material was added to 25mL of ethanol and refluxed for 30min. The mixture was filtered, the filtrate was evaporated to dryness, and the residue was dissolved in 2mL of ethanol to obtain the reference solution.

[0253] Thin-layer chromatography identification: Perform the thin-layer chromatography test (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). Apply 2-5 μL of the above test solution and 2 μL of the reference solution separately to the same silica gel G thin-layer plate. Develop the plate using toluene-ethyl acetate (2:1) as the developing solvent. Remove the plate, air-dry it, and examine it under ultraviolet light (254 nm). In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample.

[0254] Thin-layer chromatography identification method for Phellodendron bark:

[0255] Preparation of the test solution: Take two pieces of bruise and pain relief plaster, cut them into small pieces, remove the caps, add methanol solution, sonicate, filter, evaporate the filtrate to dryness, dissolve the residue in methanol to obtain the test solution.

[0256] The methanol solution mentioned is methanol acetic acid;

[0257] The concentration of the acetic acid-methanol solution is 0.8-2%;

[0258] Preferably, the concentration of the acetic acid-methanol solution is 1-1.5%;

[0259] More preferably, the concentration of the acetic acid-methanol solution is 1-1.2%;

[0260] More preferably, the concentration of the acetic acid-methanol solution is 1%.

[0261] The power of the ultrasound is 3000Hz-4000Hz.

[0262] Preferably, the power of the ultrasound is 3500Hz-4000Hz.

[0263] More preferably, the power of the ultrasound is 3500 Hz.

[0264] The temperature of the ultrasound is 50-70℃;

[0265] Preferably, the temperature of the ultrasound is 55-65°C;

[0266] More preferably, the temperature of the ultrasound is 60°C.

[0267] The ultrasound session lasted 10-30 minutes.

[0268] Preferably, the ultrasound session lasts for 15-20 minutes;

[0269] More preferably, the ultrasound duration is 20 minutes.

[0270] As a preferred embodiment, the preparation of the test solution is as follows:

[0271] Take two pieces of bruise and pain relief plaster, cut them into small pieces, remove the caps, add 40 mL of 1% acetic acid methanol solution, sonicate at 60℃ for 20 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 2 mL of 1% acetic acid methanol solution, and use it as the test solution.

[0272] Preparation of reference solution: Take Phellodendron bark reference material, add methanol solution, and prepare reference solution in the same way.

[0273] The methanol solution mentioned is a methanol solution of acetic acid;

[0274] The concentration of acetic acid in the acetic acid-methanol solution is 0.8-2%;

[0275] Preferably, the concentration of acetic acid in the acetic acid-methanol solution is 1-1.5%;

[0276] More preferably, the concentration of acetic acid in the acetic acid-methanol solution is 1-1.2%;

[0277] More preferably, the concentration of acetic acid in the acetic acid-methanol solution is 1%.

[0278] As a preferred embodiment, the preparation of the reference solution is as follows:

[0279] Take 0.1g of Phellodendron bark reference material and add 20mL of 1% acetic acid methanol solution to prepare a reference solution using the same method.

[0280] Thin-layer chromatography identification: Perform the thin-layer chromatography test (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). Apply 5 μL of the test solution and 5 μL of the reference solution separately to the same silica gel G thin-layer plate. Develop with the developing solvent, remove, air dry, fumigate with ammonia vapor, and then examine under sunlight. The chromatogram of the test solution should show spots of the same color at the corresponding positions as the chromatogram of the reference solution.

[0281] The developing solvent is a mixed solution of ethyl acetate, 2-butanone, formic acid and water.

[0282] The volume ratio of ethyl acetate, 2-butanone, formic acid and water is (8-12):(6-8):(1-2):(1-2);

[0283] Preferably, the volume ratio of ethyl acetate, 2-butanone, formic acid and water is (9-11):(6-7):(1-2):(1-2);

[0284] More preferably, the volume ratio of ethyl acetate, 2-butanone, formic acid, and water is 10:7:1:1.

[0285] As a preferred embodiment, the thin-layer chromatography identification method for Phellodendron amurense is as follows:

[0286] Preparation of the test solution: Take 2 pieces of bruise and pain relief plaster, cut them into small pieces, remove the cap, add 40 mL of 1% acetic acid methanol solution, sonicate at 60℃ for 20 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 2 mL of 1% acetic acid methanol solution, and use it as the test solution.

[0287] Preparation of reference solution: Take 0.1g of Phellodendron bark reference material, add 20mL of 1% acetic acid methanol solution, and prepare the reference solution in the same way.

[0288] Thin-layer chromatography identification: Perform the thin-layer chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). Apply 5 μL of the test solution and 5 μL of the reference solution separately to the same silica gel G thin-layer plate. Use ethyl acetate-2-butanone-formic acid-water (10:7:1:1) as the developing solvent. Develop, remove, air-dry, fumigate with ammonia vapor, and then examine under sunlight. The chromatogram of the test sample should show spots of the same color at the corresponding positions as the chromatogram of the reference medicinal material.

[0289] Example 1-1 Thin-layer chromatographic identification method for rhubarb:

[0290] Preparation of reference solution:

[0291] Take 2.0g of the negative ointment lacking rhubarb and prepare a rhubarb negative sample solution using the same method.

[0292] Take 2.0g of negative ointment lacking both Polygonum cuspidatum and Rheum palmatum, and prepare a double negative sample solution lacking both Rheum palmatum and Polygonum cuspidatum using the same method.

[0293] Take 0.1g of rhubarb reference material and prepare a reference solution using the same method.

[0294] Prepare a solution containing 1 mg of rhein reference standard in 1 mL of methanol.

[0295] Preparation of test solution:

[0296] Take one piece of liniment for bruises and sprains, cut it into small pieces, remove the cap, add 25 mL of methanol, sonicate at 25 °C for 20 min, filter, evaporate the filtrate to dryness, add 20 mL of water to dissolve the residue, add 2 mL of hydrochloric acid, heat in a water bath for 30 min, cool immediately, extract twice with 20 mL of ether each time, combine the ether extracts, evaporate to dryness, add 1 mL of ethyl acetate to dissolve the residue, and use this as the test solution.

[0297] Take 5 μL of each of the above solutions and spot them separately on the same silica gel G thin-layer plate. Develop the plate using n-hexane-ethyl acetate-formic acid (5:1:0.1) as the developing solvent. Remove the plate, air dry it, and examine it under ultraviolet light (365 nm, 254 nm).

[0298] In the chromatogram of the test sample, two identical orange-yellow fluorescent spots appear at the corresponding positions as in the chromatogram of the reference medicinal material. There is no interference from negative samples, and the characteristic spots are more obvious when viewed at 254 nm than at 365 nm. After being fumigated in ammonia vapor, the same red spots appear when viewed under sunlight. There is no interference from negative samples, indicating good specificity.

[0299] Comparative Example 1-1:

[0300] Preparation of reference solution:

[0301] Take 2.0g of negative ointment lacking both Polygonum cuspidatum and Rheum palmatum, and prepare a double negative sample solution lacking both Rheum palmatum and Polygonum cuspidatum using the same method.

[0302] Take 0.1g of rhubarb reference material and prepare a reference solution using the same method.

[0303] Take 2.0g of the negative ointment lacking rhubarb and prepare a rhubarb negative sample solution using the same method.

[0304] Preparation of test solution:

[0305] Take one piece of liniment for bruises and sprains, cut it into small pieces, remove the cap, add 25 mL of methanol, sonicate at 25 °C for 20 min, filter, evaporate the filtrate to dryness, add 20 mL of water to dissolve the residue, add 2 mL of hydrochloric acid, heat in a water bath for 30 min, cool immediately, extract twice with 20 mL of ether each time, combine the ether extracts, evaporate to dryness, add 1 mL of ethyl acetate to dissolve the residue, and use this as the test solution.

[0306] Take 2 μL of each of the above four solutions and spot them separately on the same silica gel G thin-layer plate. Use the upper layer of petroleum ether (30-60℃)-ethyl formate-formic acid (15:5:1) as the developing solvent, develop, remove, air dry, and examine under ultraviolet light (365nm).

[0307] In the chromatogram of the test sample, two identical orange-yellow fluorescent spots appear at the corresponding positions in the chromatogram of the reference medicinal material; after being fumigated in ammonia vapor and examined under sunlight, identical orange-red spots appear, but there is interference from the negative test.

[0308] Comparative Examples 1-2:

[0309] Preparation of reference solution:

[0310] Take 2.0g of the negative ointment lacking rhubarb and prepare a rhubarb negative sample solution using the same method.

[0311] Take 2.0g of negative ointment lacking both Polygonum cuspidatum and Rheum palmatum, and prepare a double negative sample solution lacking both Rheum palmatum and Polygonum cuspidatum using the same method.

[0312] Take 1g of rhubarb reference material, add 30mL of methanol, heat under reflux in a water bath for 30 minutes, filter, evaporate the filtrate to dryness, and prepare the reference solution in the same way.

[0313] Prepare a solution containing 1 mg of rhein reference standard in 1 mL of methanol.

[0314] Preparation of test solution:

[0315] Take one piece of liniment for bruises and pain relief, cut it into small pieces, remove the cap, add 20 mL of a 30% ethanol-hydrochloric acid (10:1) mixture, heat in a water bath under reflux for 1 hour, cool immediately, and extract twice with 20 mL of chloroform each time. Combine the chloroform extracts, evaporate to dryness, and dissolve the residue in 5 mL of anhydrous ethanol-ethyl acetate (2:1) mixture to obtain the test solution.

[0316] Take 5 μL of each of the above solutions and spot them separately on the same silica gel G thin-layer plate. Use the upper layer of petroleum ether (30-60℃)-ethyl formate-formic acid (15:5:1) as the developing solvent, develop, remove, air dry, and examine under ultraviolet light (365nm).

[0317] In the chromatogram of the test sample, orange-yellow fluorescent spots appear at the corresponding positions as in the chromatograms of the reference medicinal material and the reference standard, indicating interference from negative samples; after being fumigated in ammonia vapor and examined under sunlight, the spots are not obvious and there are no corresponding identical spots.

[0318] Comparative Examples 1-3:

[0319] Preparation of reference solution:

[0320] Take 2.0g of the negative ointment lacking rhubarb and prepare a rhubarb negative sample solution using the same method.

[0321] Take 2.0g of negative ointment lacking both Polygonum cuspidatum and Rheum palmatum, and prepare a double negative sample solution lacking both Rheum palmatum and Polygonum cuspidatum using the same method.

[0322] Take 0.5g of rhubarb reference material and prepare a reference solution using the same method.

[0323] Prepare a solution containing 1 mg of rhein reference standard in 1 mL of methanol.

[0324] Preparation of test solution:

[0325] Take one piece of liniment for bruises and sprains, cut it into small pieces, remove the cap, add 20 mL of methanol, soak in cold water for 1 hour, filter, evaporate the filtrate to dryness, add 10 mL of water to dissolve the residue, add 1 mL of hydrochloric acid, heat in a water bath for 30 minutes, cool immediately, extract twice with 10 mL of ether each time, combine the ether extracts, evaporate to dryness, add 1 mL of chloroform to dissolve the residue, and use this as the test solution.

[0326] Take 5 μL of each of the above solutions and spot them separately on the same silica gel G thin-layer plate. Develop the plate using n-hexane-ethyl acetate-formic acid (6:2:0.1) as the developing solvent. Remove the plate, air dry it, and examine it under a UV lamp (365 nm).

[0327] In the chromatogram of the test sample, orange-yellow fluorescent spots appeared at the same positions as those in the chromatogram of the reference medicinal material; after being exposed to ammonia vapor, the spots turned red. However, the Rf value of the target spot was too high and further adjustment is needed.

[0328] Example 1-2: Thin-layer chromatographic identification method for rhubarb:

[0329] Preparation of reference solution:

[0330] Take 2.0g of the negative ointment lacking rhubarb and prepare a rhubarb negative sample solution using the same method.

[0331] Take 2.0g of negative ointment lacking both Polygonum cuspidatum and Rheum palmatum, and prepare a double negative sample solution lacking both Rheum palmatum and Polygonum cuspidatum using the same method.

[0332] Take 0.1g of rhubarb reference material and prepare a reference solution using the same method.

[0333] Prepare a solution containing 1 mg of rhein reference standard in 1 mL of methanol.

[0334] Preparation of test solution:

[0335] Take one piece of liniment for bruises and sprains, cut it into small pieces, remove the cap, add 20 mL of methanol, sonicate at 30 °C for 30 min, filter, evaporate the filtrate to dryness, add 20 mL of water to dissolve the residue, add 1 mL of hydrochloric acid, heat in a water bath for 40 min, cool immediately, extract with 20 mL of ether three times, combine the ether extracts, evaporate to dryness, add 1 mL of ethyl acetate to dissolve the residue, and use this as the test solution.

[0336] Take 5 μL of each of the above solutions and spot them separately on the same silica gel G thin-layer plate. Develop the plate using n-hexane-ethyl acetate-formic acid (5:1:0.1) as the developing solvent. Remove the plate, air dry it, and examine it under ultraviolet light (365 nm, 254 nm).

[0337] In the chromatogram of the test sample, two identical orange-yellow fluorescent spots appeared at the corresponding positions as in the chromatogram of the reference medicinal material. These spots showed no interference from negative samples, and were more clearly visible at 254 nm than at 365 nm. After fumigation in ammonia vapor, identical red spots appeared under sunlight, again showing no interference from negative samples, indicating good specificity. Compared to Examples 1-1, the spots were paler, possibly due to incomplete extraction.

[0338] Examples 1-3: Thin-layer chromatographic identification method for rhubarb:

[0339] Preparation of reference solution:

[0340] Take 2.0g of the negative ointment lacking rhubarb and prepare a rhubarb negative sample solution using the same method.

[0341] Take 2.0g of negative ointment lacking both Polygonum cuspidatum and Rheum palmatum, and prepare a double negative sample solution lacking both Rheum palmatum and Polygonum cuspidatum using the same method.

[0342] Take 0.1g of rhubarb reference material and prepare a reference solution using the same method.

[0343] Prepare a solution containing 1 mg of rhein reference standard in 1 mL of methanol.

[0344] Preparation of test solution:

[0345] Take one piece of liniment for bruises and sprains, cut it into small pieces, remove the cap, add 30 mL of methanol, sonicate at 20 °C for 15 min, filter, evaporate the filtrate to dryness, add 20 mL of water to dissolve the residue, add 5 mL of hydrochloric acid, heat in a water bath for 20 minutes, cool immediately, extract twice with 20 mL of ether each time, combine the ether extracts, evaporate to dryness, add 1 mL of ethyl acetate to dissolve the residue, and use this as the test solution.

[0346] Take 5 μL of each of the above solutions and spot them separately on the same silica gel G thin-layer plate. Develop the plate using n-hexane-ethyl acetate-formic acid (5:1:0.1) as the developing solvent. Remove the plate, air dry it, and examine it under ultraviolet light (365 nm, 254 nm).

[0347] In the chromatogram of the test sample, two identical orange-yellow fluorescent spots appear at the corresponding positions as in the chromatogram of the reference medicinal material. There is no interference from negative samples, and the characteristic spots are more obvious when viewed at 254 nm than at 365 nm. After being fumigated in ammonia vapor, the same red spots appear when viewed under sunlight. There is no interference from negative samples, indicating good specificity.

[0348] Examples 1-4: Thin-layer chromatographic identification method for rhubarb:

[0349] Preparation of reference solution:

[0350] Take 2.0g of the negative ointment lacking rhubarb and prepare a rhubarb negative sample solution using the same method.

[0351] Take 2.0g of negative ointment lacking both Polygonum cuspidatum and Rheum palmatum, and prepare a double negative sample solution lacking both Rheum palmatum and Polygonum cuspidatum using the same method.

[0352] Take 0.1g of rhubarb reference material and prepare a reference solution using the same method.

[0353] Prepare a solution containing 1 mg of rhein reference standard in 1 mL of methanol.

[0354] Preparation of test solution:

[0355] Take one piece of liniment for bruises and sprains, cut it into small pieces, remove the cap, add 20 mL of methanol, sonicate at 25 °C for 20 min, filter, evaporate the filtrate to dryness, add 20 mL of water to dissolve the residue, add 3 mL of hydrochloric acid, heat in a water bath for 25 min, cool immediately, extract twice with 20 mL of ether each time, combine the ether extracts, evaporate to dryness, add 1 mL of ethyl acetate to dissolve the residue, and use this as the test solution.

[0356] Take 5 μL of each of the above solutions and spot them separately on the same silica gel G thin-layer plate. Develop the plate using n-hexane-ethyl acetate-formic acid (5:1:0.1) as the developing solvent. Remove the plate, air dry it, and examine it under ultraviolet light (365 nm, 254 nm).

[0357] In the chromatogram of the test sample, two identical orange-yellow fluorescent spots appeared at the corresponding positions as in the chromatogram of the reference medicinal material. These spots showed no interference from negative samples, and were more clearly visible at 254 nm than at 365 nm. After fumigation in ammonia vapor, identical red spots appeared under sunlight, again showing no interference from negative samples, indicating good specificity. Compared to Examples 1-1, the spots were paler, possibly due to incomplete extraction.

[0358] Examples 1-5: Thin-layer chromatographic identification method for rhubarb:

[0359] Preparation of reference solution:

[0360] Take 2.0g of the negative ointment lacking rhubarb and prepare a rhubarb negative sample solution using the same method.

[0361] Take 2.0g of negative ointment lacking both Polygonum cuspidatum and Rheum palmatum, and prepare a double negative sample solution lacking both Rheum palmatum and Polygonum cuspidatum using the same method.

[0362] Take 0.1g of rhubarb reference material and prepare a reference solution using the same method.

[0363] Prepare a solution containing 1 mg of rhein reference standard in 1 mL of methanol.

[0364] Preparation of test solution:

[0365] Take one piece of liniment for bruises and sprains, cut it into small pieces, remove the cap, add 25 mL of methanol, sonicate at 25 °C for 30 min, filter, evaporate the filtrate to dryness, add 20 mL of water to dissolve the residue, add 3 mL of hydrochloric acid, heat in a water bath for 25 min, cool immediately, extract twice with 20 mL of ether each time, combine the ether extracts, evaporate to dryness, add 1 mL of ethyl acetate to dissolve the residue, and use this as the test solution.

[0366] Take 5 μL of each of the above solutions and spot them separately on the same silica gel G thin-layer plate. Develop the plate using n-hexane-ethyl acetate-formic acid (5:1:0.1) as the developing solvent. Remove the plate, air dry it, and examine it under ultraviolet light (365 nm, 254 nm).

[0367] In the chromatogram of the test sample, two identical orange-yellow fluorescent spots appear at the corresponding positions as in the chromatogram of the reference medicinal material. There is no interference from negative samples, and the characteristic spots are more obvious when viewed at 254 nm than at 365 nm. After being fumigated in ammonia vapor, the same red spots appear when viewed under sunlight. There is no interference from negative samples, indicating good specificity.

[0368] Example 2-1 Thin-layer chromatographic identification method for Dalbergia odorifera:

[0369] Preparation of reference solution:

[0370] Take 1g of the anti-Dalbergia odorifera paste, add 25mL of ethanol, and prepare an anti-Dalbergia odorifera sample solution using the same method.

[0371] Take 1g of Dalbergia odorifera reference material, add 40mL of ethanol, sonicate for 1 hour, filter, evaporate the filtrate to dryness, add 1mL of ethanol to dissolve the residue, and use it as the reference solution.

[0372] Preparation of test solution:

[0373] Take 40cm of liniment for bruises and sprains. 2Cut the contents into small pieces, remove the liner, add 25 mL of ethanol, sonicate at 25 °C for 1 hour, filter, evaporate the filtrate to dryness, dissolve the residue in 1 mL of ethanol, and use it as the test solution.

[0374] Perform the thin-layer chromatography test (General Rule 0502). Take 5 μL of each of the above test solutions and 2 μL of the reference solution and spot them separately on the same silica gel G thin-layer plate and the self-made plate. Use toluene-ethyl acetate (2:1) as the developing solvent, develop, remove, air dry, and examine under ultraviolet light.

[0375] In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample, and there is no interference from negative samples, indicating that this method has good specificity. Furthermore, the chromatographic information is richer when viewed under 254 nm ultraviolet light than under 365 nm.

[0376] Comparative Example 2-1:

[0377] Preparation of reference solution:

[0378] Take 1g of the anti-Dalbergia odorifera paste, add 25mL of ethanol, and prepare an anti-Dalbergia odorifera sample solution using the same method.

[0379] Take 1g of Dalbergia odorifera reference material, add 25mL of ethanol, sonicate for 1 hour, filter, evaporate the filtrate to dryness, add 1mL of ethanol to dissolve the residue, and use it as the reference solution.

[0380] Preparation of test solution:

[0381] Take 40cm of liniment for bruises and sprains. 2 Cut into small pieces, remove the liner, add 25 ml of ethanol, sonicate for 1 hour, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of ethanol, and use as the test solution.

[0382] Take 10 μL each of the above test solution and negative sample solution, and 2 μL each of the reference solution, and spot them separately on the same silica gel G thin layer plate. Develop the plate using toluene-diethyl ether-chloroform (7:2:1) as the developing solvent. Remove the plate, air dry it, spray it with a mixed solution of 1% vanillin sulfuric acid solution and anhydrous ethanol (1:9), and heat it at 105°C until the spots are clearly visible.

[0383] Example 2-2 Thin-layer chromatographic identification method for Dalbergia odorifera:

[0384] Preparation of reference solution:

[0385] Take 1g of the anti-Dalbergia odorifera paste, add 25mL of ethanol, and prepare an anti-Dalbergia odorifera sample solution using the same method.

[0386] Take 1g of Dalbergia odorifera reference material, add 35mL of ethanol, sonicate for 1.5h, filter, evaporate the filtrate to dryness, add 1mL of ethanol to dissolve the residue, and use it as the reference solution.

[0387] Preparation of test solution:

[0388] Take 40cm of liniment for bruises and sprains. 2 Cut the contents into small pieces, remove the liner, add 20 mL of ethanol, sonicate at 30 °C for 2 hours, filter, evaporate the filtrate to dryness, dissolve the residue in 1 mL of ethanol, and use it as the test solution.

[0389] Perform the thin-layer chromatography test (General Rule 0502). Take 5 μL of each of the above test solutions and 2 μL of the reference solution and spot them separately on the same silica gel G thin-layer plate and the self-made plate. Use toluene-ethyl acetate (2:1) as the developing solvent, develop, remove, air dry, and examine under ultraviolet light.

[0390] In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample, and there is no negative interference, indicating that the method has good specificity. Furthermore, the chromatographic information is richer under 254 nm UV light than under 365 nm. Compared to Example 2-1, the spots are fainter, possibly due to incomplete extraction.

[0391] Example 2-3 Thin-layer chromatographic identification method for Dalbergia odorifera:

[0392] Preparation of reference solution:

[0393] Take 1g of the anti-Dalbergia odorifera paste, add 25mL of ethanol, and prepare an anti-Dalbergia odorifera sample solution using the same method.

[0394] Take 1g of Dalbergia odorifera reference material, add 50mL of ethanol, sonicate for 40 minutes, filter, evaporate the filtrate to dryness, add 1mL of ethanol to dissolve the residue, and use it as the reference solution.

[0395] Preparation of test solution:

[0396] Take 40cm of liniment for bruises and sprains. 2 Cut the contents into small pieces, remove the liner, add 30 mL of ethanol, sonicate at 20 °C for 1.5 hours, filter, evaporate the filtrate to dryness, dissolve the residue in 1 mL of ethanol, and use it as the test solution.

[0397] Perform the thin-layer chromatography test (General Rule 0502). Take 5 μL of each of the above test solutions and 2 μL of the reference solution and spot them separately on the same silica gel G thin-layer plate and the self-made plate. Use toluene-ethyl acetate (2:1) as the developing solvent, develop, remove, air dry, and examine under ultraviolet light.

[0398] In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample, and there is no interference from negative samples, indicating that this method has good specificity. Furthermore, the chromatographic information is richer when viewed under 254 nm ultraviolet light than under 365 nm.

[0399] Example 2-4 Thin-layer chromatographic identification method for Dalbergia odorifera:

[0400] Preparation of reference solution:

[0401] Take 1g of the anti-Dalbergia odorifera paste, add 25mL of ethanol, and prepare an anti-Dalbergia odorifera sample solution using the same method.

[0402] Take 1g of Dalbergia odorifera reference material, add 40mL of ethanol, sonicate for 50 minutes, filter, evaporate the filtrate to dryness, add 1mL of ethanol to dissolve the residue, and use it as the reference solution.

[0403] Preparation of test solution:

[0404] Take 40cm of liniment for bruises and sprains. 2 Cut the contents into small pieces, remove the liner, add 25 mL of ethanol, sonicate at 25 °C for 1.5 hours, filter, evaporate the filtrate to dryness, dissolve the residue in 1 mL of ethanol, and use it as the test solution.

[0405] Perform the thin-layer chromatography test (General Rule 0502). Take 5 μL of each of the above test solutions and 2 μL of the reference solution and spot them separately on the same silica gel G thin-layer plate and the self-made plate. Use toluene-ethyl acetate (2:1) as the developing solvent, develop, remove, air dry, and examine under ultraviolet light.

[0406] Take 5 μL of each of the above test solutions and 2 μL of the reference solution, and spot them on the same silica gel G thin-layer plate and the self-made plate, respectively. Use toluene-ethyl acetate (2:1) as the developing solvent, develop, remove, air dry, and examine under ultraviolet light.

[0407] In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample, and there is no interference from negative samples, indicating that this method has good specificity. Furthermore, the chromatographic information is richer when viewed under 254 nm ultraviolet light than under 365 nm.

[0408] Example 2-5 Thin-layer chromatographic identification method for Dalbergia odorifera:

[0409] Preparation of reference solution:

[0410] Take 1g of the anti-Dalbergia odorifera paste, add 25mL of ethanol, and prepare an anti-Dalbergia odorifera sample solution using the same method.

[0411] Take 1g of Dalbergia odorifera reference material, add 40mL of ethanol, sonicate for 80 minutes, filter, evaporate the filtrate to dryness, add 1mL of ethanol to dissolve the residue, and use it as the reference solution.

[0412] Preparation of test solution:

[0413] Take 40cm of liniment for bruises and sprains. 2 Cut the contents into small pieces, remove the liner, add 30 mL of ethanol, sonicate at 25 °C for 1 hour, filter, evaporate the filtrate to dryness, dissolve the residue in 1 mL of ethanol, and use it as the test solution.

[0414] Perform the thin-layer chromatography test (General Rule 0502). Take 5 μL of each of the above test solutions and 2 μL of the reference solution and spot them separately on the same silica gel G thin-layer plate and the self-made plate. Use toluene-ethyl acetate (2:1) as the developing solvent, develop, remove, air dry, and examine under ultraviolet light.

[0415] In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample, and there is no interference from negative samples, indicating that this method has good specificity. Furthermore, the chromatographic information is richer when viewed under 254 nm ultraviolet light than under 365 nm.

[0416] Example 3-1 Thin-layer chromatographic identification method for Phellodendron bark:

[0417] Preparation of reference solution:

[0418] Take 3.14g of Phellodendron bark negative control paste and 1.08g of rubber, and prepare a negative control solution lacking Phellodendron bark according to the preparation method of the test solution.

[0419] Take 0.1g of Phellodendron bark reference material, add 20mL of 1% acetic acid methanol, and prepare a reference solution using the same method.

[0420] Take berberine hydrochloride reference standard, add methyl methacrylate to prepare a solution containing 0.5 mg per 1 mL, and use it as the reference solution.

[0421] Preparation of test solution:

[0422] Take two pieces of traumatic pain relief plaster, cut them into small pieces, remove the caps and liner, add 40 mL of 1% acetic acid methanol solution, sonicate at 60℃ for 20 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 2 mL of methanol, and use it as the test solution.

[0423] Take 5 μL of each of the above solutions and spot them separately on the same silica gel G thin-layer plate. Use ethyl acetate-2-butanone-formic acid-water (10:7:1:1) as the developing solvent, develop, remove, air dry, fumigate in ammonia vapor, and then examine under sunlight.

[0424] The chromatogram of the test sample shows spots of the same color at the corresponding positions as the chromatograms of the reference medicinal material and the reference standard, while the negative sample shows no spots of the same color at the same positions.

[0425] Comparative Example 3-1:

[0426] Preparation of reference solution:

[0427] Take 3.14g of Phellodendron bark negative control paste and 1.08g of rubber, and prepare a negative control solution lacking Phellodendron bark according to the preparation method of the test solution.

[0428] Take another 0.1g of Phellodendron bark reference material, add 20mL of 1% acetic acid methanol, and prepare a reference solution using the same method.

[0429] Preparation of test solution:

[0430] Take two pieces of traumatic pain relief plaster, cut them into small pieces, remove the caps and liner, add 40 mL of 1% acetic acid methanol solution, sonicate at 60℃ for 20 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 2 mL of methanol, and use it as the test solution.

[0431] Apply 5 μL and 10 μL of the above four solutions to the same silica gel G thin-layer plate, using the lower layer of chloroform-methanol-water (30:15:4) as the developing solvent; place the plate in a developing tank saturated with ammonia vapor, develop, remove, air dry, and spray with dilute potassium bismuth iodide solution.

[0432] During ammonia fumigation, the test sample chromatogram shows spots of the same color at the corresponding positions as the reference medicinal material chromatogram and the reference standard chromatogram. After spraying the colorimetric reagent, the color of the reference medicinal material chromatogram is inconsistent with that of the test sample, and therefore it is not included in the standard for the time being.

[0433] Comparative Example 3-2:

[0434] Preparation of reference solution:

[0435] Take 3.14g of Phellodendron bark negative control paste and 1.08g of rubber, and prepare a negative control solution lacking Phellodendron bark according to the preparation method of the test solution.

[0436] Take another 0.1g of Phellodendron bark reference material, add 20mL of 1% acetic acid methanol, and prepare a reference solution using the same method.

[0437] Take berberine hydrochloride reference standard, add methyl methacrylate to prepare a solution containing 0.5 mg per 1 mL, and use it as the reference solution.

[0438] Preparation of test sample:

[0439] Take two pieces of traumatic pain relief plaster, cut them into small pieces, remove the caps and liner, add 40 mL of 1% acetic acid methanol solution, sonicate at 60℃ for 20 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 2 mL of methanol, and use it as the test solution.

[0440] Apply 5 μL and 10 μL of the above four solutions to the same silica gel G thin-layer plate, using the lower layer of dichloromethane-methanol-water (30:15:4) as the developing solvent; place the plate in a developing tank saturated with ammonia vapor, develop, remove, air dry, and spray with dilute potassium bismuth iodide reagent into the chromatogram of the test sample.

[0441] Spots of the same color appear at the corresponding positions as in the chromatograms of the reference medicinal material and the reference standard. However, the Rf value is relatively high, and inclusion in the standard is not recommended at this time.

[0442] Comparative Example 3-3:

[0443] Preparation of reference solution:

[0444] Take 3.14g of Phellodendron bark negative control paste and 1.08g of rubber, and prepare a negative control solution lacking Phellodendron bark according to the preparation method of the test solution.

[0445] Take 0.1g of Phellodendron bark reference material, add 20mL of 1% acetic acid methanol, and prepare a reference solution using the same method.

[0446] Preparation of test solution:

[0447] Take two pieces of traumatic pain relief plaster, cut them into small pieces, remove the caps and liner, add 40 mL of 1% acetic acid methanol solution, sonicate at 60℃ for 20 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 2 mL of methanol, and use it as the test solution.

[0448] Take 5 μL of each of the above three solutions and spot them separately on the same silica gel G thin-layer plate. Use toluene-ethyl acetate-methanol-isopropanol-water (6:3:2:1.5:0.3) as the developing solvent. Place the plate in a developing tank saturated with ammonia vapor, develop, remove and air dry.

[0449] The test sample chromatogram did not show any spots at the corresponding positions as the reference medicinal material chromatogram, therefore it was not included in the standard.

[0450] Example 3-2 Thin-layer chromatographic identification method for Phellodendron bark:

[0451] Preparation of reference solution:

[0452] Take 3.14g of Phellodendron bark negative control paste and 1.08g of rubber, and prepare a negative control solution lacking Phellodendron bark according to the preparation method of the test solution.

[0453] Take 0.1g of Phellodendron bark reference material and add 20mL of 0.8% acetic acid methanol to prepare a reference solution using the same method.

[0454] Take berberine hydrochloride reference standard, add methyl methacrylate to prepare a solution containing 0.5 mg per 1 mL, and use it as the reference solution.

[0455] Preparation of test solution:

[0456] Take two pieces of liniment for bruises and sprains, cut them into small pieces, remove the caps and liner, add 40 mL of 0.8% acetic acid methanol solution, sonicate at 70℃ for 10 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 2 mL of methanol, and use it as the test solution.

[0457] Take 5 μL of each of the above solutions and spot them separately on the same silica gel G thin-layer plate. Use ethyl acetate-2-butanone-formic acid-water (10:7:1:1) as the developing solvent, develop, remove, air dry, fumigate in ammonia vapor, and then examine under sunlight.

[0458] The chromatogram of the test sample shows spots of the same color at the corresponding positions as the chromatograms of the reference medicinal material and the reference standard, while the negative sample shows no spots of the same color at the same positions.

[0459] Example 3-3 Thin-layer chromatographic identification method for Phellodendron bark:

[0460] Preparation of reference solution:

[0461] Take 3.14g of Phellodendron bark negative control paste and 1.08g of rubber, and prepare a negative control solution lacking Phellodendron bark according to the preparation method of the test solution.

[0462] Take 0.1g of Phellodendron bark reference material, add 20mL of 2% acetic acid methanol, and prepare a reference solution using the same method.

[0463] Take berberine hydrochloride reference standard, add methyl methacrylate to prepare a solution containing 0.5 mg per 1 mL, and use it as the reference solution.

[0464] Preparation of test solution:

[0465] Take two pieces of bruise and pain relief plaster, cut them into small pieces, remove the caps and liner, add 40 mL of 2% acetic acid methanol solution, sonicate at 70℃ for 10 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 2 mL of methanol, and use it as the test solution.

[0466] Take 5 μL of each of the above solutions and spot them separately on the same silica gel G thin-layer plate. Use ethyl acetate-2-butanone-formic acid-water (10:7:1:1) as the developing solvent, develop, remove, air dry, fumigate in ammonia vapor, and then examine under sunlight.

[0467] The chromatogram of the test sample shows spots of the same color at the corresponding positions as the chromatograms of the reference medicinal material and the reference standard, while the negative sample shows no spots of the same color at the same positions.

[0468] Example 3-4 Thin-layer chromatographic identification method for Phellodendron bark:

[0469] Preparation of reference solution:

[0470] Take 3.14g of Phellodendron bark negative control paste and 1.08g of rubber, and prepare a negative control solution lacking Phellodendron bark according to the preparation method of the test solution.

[0471] Take 0.1g of Phellodendron bark reference material and add 20mL of 1.5% acetic acid methanol to prepare a reference solution using the same method.

[0472] Take berberine hydrochloride reference standard, add methyl methacrylate to prepare a solution containing 0.5 mg per 1 mL, and use it as the reference solution.

[0473] Preparation of test solution:

[0474] Take two pieces of bruise and pain relief plaster, cut them into small pieces, remove the caps and liner, add 40 mL of 1.5% acetic acid methanol solution, sonicate at 65℃ for 15 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 2 mL of methanol, and use it as the test solution.

[0475] Take 5 μL of each of the above solutions and spot them separately on the same silica gel G thin-layer plate. Use ethyl acetate-2-butanone-formic acid-water (10:7:1:1) as the developing solvent, develop, remove, air dry, fumigate in ammonia vapor, and then examine under sunlight.

[0476] The chromatogram of the test sample shows spots of the same color at the corresponding positions as the chromatograms of the reference medicinal material and the reference standard, while the negative sample shows no spots of the same color at the same positions.

[0477] Examples 3-5: Thin-layer chromatographic identification method for Phellodendron bark:

[0478] Preparation of reference solution:

[0479] Take 3.14g of Phellodendron bark negative control paste and 1.08g of rubber, and prepare a negative control solution lacking Phellodendron bark according to the preparation method of the test solution.

[0480] Take 0.1g of Phellodendron bark reference material and add 20mL of 1.8% acetic acid methanol to prepare a reference solution using the same method.

[0481] Take berberine hydrochloride reference standard, add methyl methacrylate to prepare a solution containing 0.5 mg per 1 mL, and use it as the reference solution.

[0482] Preparation of test solution:

[0483] Take two pieces of bruise and pain relief plaster, cut them into small pieces, remove the caps and liner, add 40 mL of 1.8% acetic acid methanol solution, sonicate at 55℃ for 15 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 2 mL of methanol, and use it as the test solution.

[0484] Take 5 μL of each of the above solutions and spot them separately on the same silica gel G thin-layer plate. Use ethyl acetate-2-butanone-formic acid-water (10:7:1:1) as the developing solvent, develop, remove, air dry, fumigate in ammonia vapor, and then examine under sunlight.

[0485] The chromatogram of the test sample shows spots of the same color at the corresponding positions as the chromatograms of the reference medicinal material and the reference standard, while the negative sample shows no spots of the same color at the same positions.

[0486] Effect detection:

[0487] Durability monitoring

[0488] 1. Identification of rhubarb

[0489] 1.1 Investigation of different sample sizes

[0490] Take 5-10 μL of the test sample solution and spot it onto the control herb solution, then spot 5 μL of the control herb solution. Perform thin-layer chromatography (TLC) identification as described above to investigate the effect of different spotting amounts of the test sample on TLC identification. The test results indicate that spotting amounts of 5-10 μL are sufficient for rhubarb identification; therefore, the optimal spotting amount for rhubarb identification in liniment is 5-10 μL.

[0491] 1.2 Comparison of different thin-layer plates

[0492] The reference solution and the test solution (prepared according to the method outlined in the text) were spotted onto a self-made silica gel G thin-layer plate and a common pre-made silica gel G thin-layer plate (Qingdao Haiyang Chemical Co., Ltd.), respectively. The results showed that the test sample could achieve good separation on both types of thin-layer plates, and the results were basically consistent.

[0493] 1.3 Comparison of different temperatures and humidity levels

[0494] The reference solution and the test solution (prepared according to the method outlined in the text) were tested for separation under two conditions: normal temperature and humidity (recording the temperature and humidity of an air-conditioned room) and low temperature and humidity (recording the temperature and humidity of a refrigerator). The results showed that the test solution achieved good separation under both conditions, and temperature had little effect on the separation.

[0495] 1.4 Measurement results of different samples

[0496] Four batches of samples were selected, and test solutions were prepared according to the method described in the main text. The reference solution and test solutions were spotted separately onto the same silica gel G thin-layer plate, and developed under the conditions described in the main text. The results showed that the chromatograms of the four batches of test samples exhibited the same yellow fluorescent spots at the corresponding positions as the reference medicinal material chromatograms, and the development effect was good. These can be used as control indicators to control the quality of this product.

[0497] 2. Identification of Dalbergia odorifera

[0498] 2.1 Comparison of different thin-layer plates

[0499] The reference solution and the test solution were spotted onto a self-made silica gel G thin-layer plate and a common pre-made silica gel G thin-layer plate (Qingdao Ocean Chemical Co., Ltd.), respectively. The results showed that the test sample could achieve good separation on both types of thin-layer plates, and the results were basically consistent.

[0500] 2.2 Comparison of different temperatures

[0501] The separation of the reference solution and the test solution under normal temperature and humidity and low temperature and humidity conditions was investigated. The results showed that the test sample could achieve the identification purpose under the above conditions. Temperature had little effect on the separation, and the separation effect was better at room temperature than at low temperature.

[0502] 3. Identification of Phellodendron bark

[0503] 3.1 Comparison of different thin-layer plates

[0504] The reference solution and the test solution were spotted onto a self-made silica gel G thin-layer plate and a common pre-made silica gel G thin-layer plate (Qingdao Ocean Chemical Co., Ltd.), respectively. The results showed that the test sample could achieve good separation on both types of thin-layer plates, and the results were basically consistent.

[0505] 3.2 Comparison of different temperatures and humidity levels

[0506] The separation of the reference solution and the test solution was investigated under three conditions: normal temperature and humidity (A: T = 25℃, RH = 57%), low temperature and normal humidity (T = 3℃, RH = 65%), normal temperature and high humidity (T = 17℃, RH = 81%), and normal temperature and humidity (C: T = 25℃, RH = 39%). The results showed that the test solution was prone to edge effects under the above low temperature conditions, but this had no effect on identification. Humidity had little effect on separation, and the test solution achieved good separation under all the above humidity conditions.

[0507] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for detecting three medicinal materials in Diedazhengtong paste by thin layer chromatography, characterized in that: It comprises the following steps: The thin layer chromatography identification method of Rhubarb: Thin layer chromatography identification: take the test solution A and the Rhubarb control solution respectively, and point them on the same silica gel G thin layer plate. Develop, take out, dry, and observe under UV light 254 nm. The test chromatogram shows the same color fluorescent spots as the control chromatogram. Fumigate in ammonia vapor, and observe under sunlight. The developing agent is a mixture of n-hexane, ethyl acetate, and formic acid. The volume ratio of n-hexane, ethyl acetate, and formic acid is 4-6:0.5-1.5:0.05-0.

15. The preparation of the test solution A: take one piece of the Dian Dui Zhen Tong ointment, cut it into pieces, remove the cover, add methanol, ultrasonic, filter, evaporate the filtrate, add water to dissolve the residue, add hydrochloric acid, heat in water bath, cool immediately, extract with ether, combine the ether extracts, evaporate, add ethyl acetate to dissolve the residue, and use it as the test solution. The thin layer chromatography identification method of Jdown Xiang: Thin layer chromatography identification: take the test solution B and the Jdown Xiang control solution respectively, and point them on the same silica gel G thin layer plate. Develop, take out, dry, and observe under UV light 254 nm. The test chromatogram shows the same color fluorescent spots as the control chromatogram. The developing agent is a mixture of toluene and ethyl acetate. The volume ratio of toluene and ethyl acetate is 1-3:

1. Preparation of Test Solution B: Take 40 cm of Didi Zhen Tonggao, cut into pieces, remove the cover liner, add ethanol, ultrasonic, filter, evaporate the filtrate to dryness, add ethanol to dissolve the residue as the test solution; 2 ​ The thin layer chromatography identification method of Huang Bai: Thin layer chromatography identification: take the test solution C and the Huang Bai control solution respectively, and point them on the same silica gel G thin layer plate. Develop with the developing agent, take out, dry, fumigate in ammonia vapor, and observe under sunlight. The test chromatogram shows the same color spots as the control chromatogram. The developing agent is a mixture of ethyl acetate, 2-butanone, formic acid, and water. The volume ratio of ethyl acetate, 2-butanone, formic acid, and water is 8-12:6-8:1-2:1-2. The preparation of the test solution C: Take two pieces of the Dian Dui Zhen Tong ointment, cut them into pieces, remove the cover, add methanol solution, ultrasonic, filter, evaporate the filtrate, add methanol to dissolve the residue, and use it as the test solution. The methanol solution is acetic acid methanol solution. The concentration of acetic acid in the acetic acid methanol solution is 0.8-2%.

2. The method of claim 1, wherein: The water bath heating time is 20-40 minutes.

3. The method of claim 2, wherein: The water bath heating time is 30 minutes.

4. The method of claim 1, wherein: The preparation of the test solution A: take one piece of the Dian Dui Zhen Tong ointment, cut it into pieces, remove the cover, add 25 mL of methanol, ultrasonic for 20 minutes, filter, evaporate the filtrate, add 20 mL of water to dissolve the residue, add 2 mL of hydrochloric acid, heat in water bath for 30 minutes, cool immediately, extract with ether twice, each time 20 mL, combine the ether extracts, evaporate, add 1 mL of ethyl acetate to dissolve the residue, and use it as the test solution.

5. The method of claim 1, wherein: The preparation of the test solution B: Take 40 cm of Didingzhen Tonggao 2 , cut into pieces, remove the cover, add 25 mL of ethanol, and ultrasonically treat for 1 hour. Filter, evaporate the filtrate, add 1 mL of ethanol to the residue to dissolve, and use as the test solution.

6. The method of claim 1, wherein: The concentration of acetic acid in the acetic acid methanol solution is 1%.

7. The method of claim 1, wherein: The preparation of the test solution C: Take 2 tablets of Dieda Zhen Tong Gao, respectively cut into pieces, remove the cover, add 40 mL of 1% acetic acid methanol solution, ultrasonic treat at 60 ℃ for 20 minutes, filter, evaporate the filtrate, dissolve the residue with 2 mL of 1% acetic acid methanol solution as the test solution.

8. The method of claim 1, wherein: The volume ratio of the n-hexane-ethyl acetate-formic acid is 5:1:0.

1.

9. The method of claim 1, wherein: The volume ratio of the toluene-ethyl acetate is 2:

1.

10. The method of claim 1, wherein: The volume ratio of the ethyl acetate, 2-butanone, formic acid and water is 10:7:1:

1.

11. The use of the method according to any one of claims 1-10 in identifying the components of Rhubarb, Dangxiang and Huangbai in Chinese medicine external preparation.

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