Method for detecting three medicinal materials in traumatic injury analgesic paste by adopting thin-layer chromatography

Abstract

The invention provides a method for detecting three medicinal materials in traumatic injury analgesic paste by adopting thin layer chromatography, and belongs to the technical field of detection of effective components. Different test solution and reference solution are respectively prepared and tested according to thin layer chromatography, different developing solvents are adopted to identify rheum officinale, dalbergia wood and golden cypress in the traumatic injury analgesic ointment, chemical substances in medicine material control components are utilized to perform specific recognition, and the specificity of the traumatic injury analgesic ointment is improved. A thin layer chromatography is explored and established to detect rhubarb, golden cypress and lignum dalbergiae odoriferae medicinal materials in the traumatic injury analgesic paste, and the problem of misjudgment caused by similar forms of the medicinal materials in microscopic identification is solved.

Description

Technical Field

[0001] The invention belongs to the technical field of effective component detection, and particularly relates to a method for detecting three medicinal materials in a tear pain relief plaster by adopting thin layer chromatography. Background Art

[0002] The analgesic plaster for traumatic injuries is made by crushing Strychnos nux vomica, Eupolyphaga sinensis, raw Aconitum kusnezoffii, Rhubarb, Dalbergia odorifera, Zanthoxylum bungeanum, Scutellaria baicalensis, Phellodendron amurense, and Polygonum cuspidatum into fine powder, and combining borneol, peppermint oil, camphor, methyl salicylate, menthol with rubber, rosin and other matrices. The approval number is: Z44022504. It has the effects of promoting blood circulation and relieving pain, dispersing blood stasis and reducing swelling, and dispelling wind and dampness. It is clinically used for acute and chronic sprains and contusions, chronic low back and leg pain, and rheumatic joint pain.

[0003] Prior patent CN105891376A discloses a quality standard for a tui da zhi analgesic plaster and its testing method. This quality standard includes identification items, inspection items, a volatile oil item, and content determination items. Identification items include microscopic identification and volatile oil identification. Microscopic identification includes identification of crystal fibers, bast fibers, calcium oxalate clusters, single cells, and cell walls. Volatile oil identification includes qualitative testing of menthol, borneol, methyl salicylate, and camphor. Inspection items include qualitative testing of strychnine and aconitine, determination of the amount of the plaster, and compliance with various provisions related to rubber plasters under the general provisions of the Pharmacopoeia. The volatile oil item is the determination of the total amount of volatile oil. The content determination item is the quantitative detection of camphor by gas chromatography. The quality standard and its testing method established by this invention are highly stable, have good linearity, high accuracy, and good repeatability. This method can be used to scientifically evaluate the quality of tui da zhi analgesic plasters, facilitating quality control in industrialized production.

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

[0005] However, the pain-relieving ointment contains a variety of Chinese medicinal ingredients, including Strychnos nux vomica, Eupolyphaga sinensis, Radix Aconiti Kusnezoffii, Rhubarb, Dalbergia odorifera, Zanthoxylum bungeanum, Scutellaria baicalensis, Phellodendron chinense, and Polygonum cuspidatum. Among them, Rhubarb promotes blood circulation, removes blood stasis, promotes menstruation and relieves pain; Dalbergia odorifera promotes blood circulation and qi, relieves pain; Phellodendron chinense clears heat and dampness, purges fire and detoxifies, reduces swelling and relieves pain.

[0006] There are many methods for detecting rhubarb, phellodendron chinense and dalbergia odorifera in the prior art, such as:

[0007] Chinese patent CN118330097A discloses a method for identifying Rheum officinale in Dahuang Tongbian Tablets. The method comprises the following steps: step (1): preparing a test solution using Dahuang Tongbian Tablets as raw material, and preparing a reference solution using Rheum officinale as raw material; step (2): determining the test solution and the reference solution by ultra-high performance liquid chromatography-mass spectrometry; wherein the mass spectrometry conditions used in the ultra-high performance liquid chromatography-mass spectrometry are: using a mass spectrometer detector in electrospray positive ion mode, and the detection ion pairs selected 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 Rheum officinale in Dahuang Tongbian Tablets. The method has the advantages of strong detection specificity, stable and reliable detection, low detection limit and quantification limit, and the identification method does not require the use of expensive Rheum officinale glycoside as a reference solution.

[0008] Chinese patent CN119104670A discloses a thin-layer chromatography method for identifying Dalbergia odorifera and its counterfeits. By improving the development system, this invention requires only a single developing agent for simultaneous identification of both volatile oils and flavonoids in Dalbergia odorifera, simplifying the process and shortening identification time. The invention utilizes common, non-toxic cyclohexane, ethyl acetate, and acetic acid as developing agents, avoiding the use of toxic and hazardous reagents such as toluene, chloroform, and ether. This effectively separates volatile oils from flavonoids while reducing the use of toxic reagents. The invention also provides a novel volatile oil detection method. By spraying a mixed solution of vanillin sulfuric acid solution and anhydrous ethanol, heating it, and then examining it under 365nm ultraviolet light, fluorescent spots of varying colors appear, overcoming the current method's reliance on color intensity, which results in weak identification capabilities.

[0009] Chinese patent CN119936291A discloses a thin layer chromatography method for simultaneously identifying the components of Coptis chinensis and Phellodendron chinense and its application. The method comprises 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 on the same silica gel G thin layer plate, using n-hexane-n-butanol-methanol-water with a volume ratio of (2.5-3.5):(2.5-3.5):(1.5-2.5):(0.8-1.2) as a developing solvent, placing the silica gel G thin layer plate in a developing tank, developing, removing, drying, and then inspecting under a 360-370 nm light source. The thin layer chromatography method provided in this application can quickly and easily identify the components of Coptis chinensis and Phellodendron chinense in traditional Chinese medicine external preparations, thereby 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 method for testing Fushenning Capsules, which includes microscopic identification of the product, thin-layer chromatography (TLC) analysis of Anemarrhena Rhizoma, TLC analysis of Gardenia jasminoides, TLC analysis of Rhubarb, TLC analysis of Phellodendron chinense, and determination of berberine hydrochloride. The TLC analysis of Phellodendron chinense is as follows: 1-2 μL of each of the two solutions is applied to the same silica gel G thin-layer plate using a 7:2:1 ratio of n-butanol, glacial acetic acid, and water as the developing solvent. The plate is then developed, removed, air-dried, and examined under ultraviolet light at 365 nm. Fluorescent spots of the same color appear in the chromatogram of the test sample at the corresponding position in the chromatogram of the control medicinal material. The thin layer chromatography identification of rhubarb is as follows: according to the thin layer chromatography test, 2-8 μL of each of the three solutions mentioned above are taken and spotted on the same silica gel G thin layer plate respectively, and the upper layer solution of 30-60°C petroleum ether-ethyl formate-formic acid in a ratio of 15:5:1 is used as the developing solvent, developed, taken out, dried, and inspected under ultraviolet light at 365nm; in the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions of the chromatogram of the reference medicinal material and the chromatogram of the reference sample. This invention optimizes the thin layer chromatography identification method of Anemarrhena asphodeloides in Fushenning Capsules based on the original quality detection method. Through methodological investigation, the detection method has strong specificity, good durability, high accuracy, good stability, simple and quick operation, and has improved the quality detection method of Fushenning Capsules, so that the quality can be better controlled.

[0012] For example, Chinese patent CN101732463A discloses a quality inspection method for Jinhua Xiaozao Pills. This method improves the quality control standard of Jinhua Xiaozao Pills, establishes a method for determining the content of the main drug in the preparation, eliminates the poorly specific physical and chemical reaction identification, improves the thin layer chromatography identification method of gardenia glycoside and berberine hydrochloride, makes the method simpler and the characteristic spots clearer, and adds thin layer chromatography identification methods for rhubarb, coptis chinensis, phellodendron amurense, and scutellaria baicalensis, ensuring the high quality standard level of this compound preparation. Among them, the thin layer chromatography identification method of rhubarb: according to the thin layer chromatography method (Appendix VI of the first volume of the Chinese Pharmacopoeia 2005 edition) B) Test: 5-10 μL of each solution was aspirated and spotted onto the same silica gel H thin layer plate with sodium carboxymethyl cellulose as the adhesive. Developed with n-hexane-ethyl acetate-formic acid (volume ratio 5-8:1.5-3:0.1-0.2). The plate was removed, air-dried, and examined under ultraviolet light (365 nm). In the chromatogram of the test sample, five identical yellow fluorescent spots appeared at the corresponding positions in the chromatogram of the control medicinal material; in the chromatogram of the control, one identical yellow spot appeared at the corresponding position. After fumigation with ammonia vapor and examination under daylight, the spots turned red. Thin layer chromatography identification method of Coptis chinensis and Phellodendron chinense: According to the thin layer chromatography method (Appendix VIB of Part I of the 2005 edition of the Chinese Pharmacopoeia), 5 μL of each solution was aspirated and spotted on the same silica gel G thin layer plate with sodium carboxymethyl cellulose as the adhesive, and ethyl acetate-butanone-formic acid-water (volume ratio 8-11:4-7:0.9-1.1:0.9-1.1) was used as the developing agent. The plates were developed, taken out, dried, and examined under ultraviolet light (365 nm). In the chromatogram of the test sample, the same yellow fluorescent spots appeared at the corresponding positions in the chromatogram of the control medicinal material and the chromatogram of the reference sample.

[0013] However, there are few existing technologies for simultaneously testing multiple samples of rhubarb, phellodendron, and dalbergia odorifera. The Chinese Pharmacopoeia (2020 edition), under the heading "Dieda Pain Relief Plaster," only identifies the microscopic characteristics of these herbs under the microscopic identification section.

[0014] Currently, there is no existing TLC identification method for the detection of rhubarb, phellodendron, and dalbergia odorifera in the Dieda Pain Relief Plaster. Therefore, it is necessary to develop a method for the identification and determination of rhubarb, phellodendron, and dalbergia odorifera in the Dieda Pain Relief Plaster, but this method cannot effectively solve the problem of misidentification caused by the similar morphology of the herbs. Summary of the Invention

[0015] The prescription of the Dieda Analgesic Plaster contains Rhubarb, Phellodendron and Dalbergia Odorifera. The current quality standard only identifies the microscopic characteristics of these medicinal materials under the microscopic identification item. This method has low accuracy and cannot use the chemical substances in the controlled ingredients of the medicinal materials for specific identification. There is a problem of misjudgment caused by the similarity of the medicinal materials in microscopic identification. Based on the deficiencies in the existing technology, the present invention aims to use the chemical substances in the controlled ingredients of the medicinal materials for specific identification, explore and establish a thin layer chromatography method to detect Rhubarb, Phellodendron and Dalbergia Odorifera in the Dieda Analgesic Plaster, and solve the problem of misjudgment caused by the similarity of the medicinal materials in microscopic identification.

[0016] In order to achieve the above purpose, the present invention uses the following technical solutions:

[0017] A method for detecting three medicinal materials in a dieda analgesic plaster by thin layer chromatography comprises the following steps:

[0018] Thin layer chromatography identification method of rhubarb:

[0019] Preparation of test solution A: Take 1 piece of Dieda Analgesic Plaster, cut it into pieces, remove the cover, add methanol, sonicate, filter, evaporate the filtrate to dryness, dissolve the residue in water, add hydrochloric acid, heat in a water bath, cool immediately, extract by shaking with ether, combine the ether solutions, evaporate to dryness, dissolve the residue in ethyl acetate, and use as the test solution.

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

[0021] More preferably, the amount of methanol 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 3500HZ.

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

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

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

[0029] The ultrasonic time is 15-30 minutes;

[0030] Preferably, the ultrasound duration is 20-25 minutes;

[0031] More preferably, the ultrasonic treatment time is 20 minutes.

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

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

[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 shaking extraction is performed 1-3 times;

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

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

[0041] Preparation of test solution A: Take 1 piece of Dieda Analgesic Plaster, cut it into pieces, remove the cover, add 25 mL of methanol, ultrasonicate 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, and extract with ether by shaking twice, 20 mL each time, combine the ether solutions, evaporate to dryness, and dissolve the residue in 1 mL of ethyl acetate to prepare the test solution.

[0042] Preparation of reference solution: Take rhubarb reference medicinal material and prepare reference solution in the same way.

[0043] Thin layer chromatography identification: According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 edition, Part IV, General Chapter 0502), 5-10 μL of the test solution and 5 μL of the reference solution are respectively spotted on the same silica gel G thin layer plate, developed with a developing agent, removed, dried, and examined under ultraviolet light (254 nm). The chromatogram of the test sample will show a fluorescent spot of the same color at the corresponding position of the chromatogram of the reference sample; place it in ammonia vapor and ammonia fumigation until color develops, and examine it under sunlight, and spots of the same color will appear.

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

[0045] The volume ratio of n-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 n-hexane-ethyl acetate-formic acid is 5:1:0.1.

[0048] As a most preferred embodiment, the thin layer chromatography identification method of rhubarb is:

[0049] Preparation of test solution A: Take 1 piece of Dieda Analgesic Plaster, cut it into pieces, remove the cover, add 25 mL of methanol, ultrasonicate 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, and extract with ether by shaking twice, 20 mL each time, combine the ether solutions, evaporate to dryness, and dissolve the residue in 1 mL of ethyl acetate to prepare the test solution.

[0050] Preparation of reference solution: Take 0.1 g of rhubarb reference medicinal material and prepare reference solution in the same way.

[0051] Identification by thin layer chromatography: According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502), 5-10 μL of the test solution and 5 μL of the reference solution were respectively spotted on the same silica gel G thin layer plate. Using n-hexane-ethyl acetate-formic acid (5:1:0.1) as the developing solvent, the plate was developed, removed, air-dried, and examined under ultraviolet light (254 nm). The test sample chromatogram showed a fluorescent spot of the same color at the corresponding position of the reference chromatogram; the plate was fumigated with ammonia vapor until color developed, and the plate was examined under sunlight to show spots of the same color.

[0052] Thin layer chromatography identification method of Dalbergia odorifera:

[0053] Preparation of test solution B: Take 40cm of Dieda Pain Relief Plaster 2 , cut into pieces, remove the cover lining, add ethanol, ultrasonicate, filter, evaporate the filtrate to dryness, add ethanol to dissolve the residue, and use it as the test solution.

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

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

[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 3500HZ.

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

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

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

[0063] The ultrasonic time is 0.5-2 hours;

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

[0065] More preferably, the ultrasonication time is 1 hour.

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

[0067] Take 40cm of the pain-relieving plaster 2 , cut into pieces, remove the cover lining, add 25 mL of ethanol, ultrasonically treat for 1 hour, filter, evaporate the filtrate to dryness, add 1 mL of ethanol to dissolve the residue, and use it as the test solution.

[0068] Preparation of reference solution: Take Dalbergia odorifera reference medicinal material, add ethanol, reflux extraction, filter, evaporate the filtrate to dryness, add ethanol to dissolve the residue, and use it as the reference solution.

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

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

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

[0072] The temperature of the reflux extraction is 80°C-100°C;

[0073] Preferably, the temperature of the reflux extraction is 90°C-100°C;

[0074] More preferably, the temperature of the reflux extraction 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:

[0079] 0.5 g of Dalbergia odorifera reference medicinal material was added with 40 mL of ethanol, and the mixture was refluxed for 1 h. The mixture was filtered, the filtrate was evaporated to dryness, and 1 mL of ethanol was added to the residue to dissolve it, which was used as the reference solution.

[0080] Identification by thin layer chromatography: According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 edition, Part IV, General Chapter 0502), 2-5 μL of the test solution and 2 μL of the reference solution were applied to the same silica gel G thin layer plate. Develop with a developing agent, remove, dry, and examine under ultraviolet light (254 nm). In the chromatogram of the test sample, a fluorescent spot of the same color should appear at the corresponding position in the chromatogram of the reference sample.

[0081] The developing agent is a mixture solution 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 of Dalbergia woodii is:

[0086] Preparation of test solution B: Take 40cm of Dieda Pain Relief Plaster 2 , cut into pieces, remove the cover lining, add 25 mL of ethanol, ultrasonically treat for 1 hour, filter, evaporate the filtrate to dryness, add 1 mL of ethanol to dissolve the residue, and use it as the test solution.

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

[0088] Identification by thin layer chromatography: According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502), 2-5 μL of the test solution and 2 μL of the reference solution were applied to the same silica gel G thin layer plate. The plate was developed with toluene-ethyl acetate (2:1). The plate was removed, dried, and examined under ultraviolet light (254 nm). In the chromatogram of the test sample, a fluorescent spot of the same color appeared at the corresponding position in the chromatogram of the reference sample.

[0089] Thin layer chromatography identification method of Phellodendron chinense:

[0090] Preparation of test solution C: Take 2 tablets of Dieda Analgesic Plaster, cut them into pieces respectively, remove the cover lining, add methanol solution, ultrasonically treat, filter, evaporate the filtrate to dryness, and dissolve the residue in methanol as the test solution.

[0091] The methanol solution 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] Further 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 3500HZ.

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

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

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

[0102] The ultrasonic time is 10-30 minutes;

[0103] Preferably, the ultrasound duration is 15-20 minutes;

[0104] More preferably, the ultrasonic treatment time is 20 minutes.

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

[0106] Take 2 pieces of Dieda Analgesic Plaster, cut them into pieces respectively, remove the cover lining, add 40 mL of 1% acetic acid methanol solution, ultrasonically treat at 60℃ for 20 minutes, filter, evaporate the filtrate to dryness, and dissolve the residue in 2 mL of 1% acetic acid methanol solution to prepare the test solution.

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

[0108] The methanol solution is methanol acetate;

[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:

[0114] Take 0.1 g of Phellodendron chinense reference medicinal material, add 20 mL of 1% acetic acid methanol solution, and prepare the reference solution in the same way.

[0115] Identification by thin-layer chromatography: According to the thin-layer chromatography method (Chinese Pharmacopoeia 2020 edition, Part IV, General Chapter 0502), 1-5 μL of the test sample solution and 5 μL of the reference sample solution are applied to the same silica gel G thin-layer plate. Develop with a developing agent, remove, air dry, fumigate with ammonia vapor, and inspect under sunlight. The test sample chromatogram should show spots of the same color at the corresponding positions on the reference sample chromatogram.

[0116] The developing agent 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 of Phellodendron chinense is:

[0121] Preparation of test solution C: Take 2 tablets of Dieda Analgesic Plaster, cut them into pieces respectively, remove the cover lining, add 40 mL of 1% acetic acid methanol solution, ultrasonically treat at 60℃ for 20 minutes, filter, evaporate the filtrate to dryness, and dissolve the residue in 2 mL of 1% acetic acid methanol solution to prepare the test solution.

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

[0123] Identification by thin layer chromatography: According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502), 1-5 μL of the test sample solution and 5 μL of the reference sample solution were applied to the same silica gel G thin layer plate. Develop with ethyl acetate-2-butanone-formic acid-water (10:7:1:1) as the developing solvent. Remove the plate, dry it, fumigate it with ammonia vapor, and inspect it under sunlight. The test sample chromatogram will show spots of the same color at the corresponding positions on the reference sample chromatogram.

[0124] The above method is used to identify the components of rhubarb, dalbergia odorifera and phellodendron in external preparations of traditional Chinese medicine.

[0125] Compared with the prior art, the present invention has the following beneficial effects:

[0126] (1) The specific identification of Radix Rhei, Dalbergia Odorifera, and Phellodendron Bark medicinal materials and decoction pieces all require thin layer identification and microscopic identification. According to the requirements of the "National Drug Standard (China) Research and Preparation Technical Requirements": the identification method of each medicinal flavor in the preparation should, in principle, be consistent with the specific identification method of the medicinal material and decoction piece as much as possible, except for the pre-treatment. Currently, the preparation only has microscopic identification method. The identification method provided by the present invention fills the gap in TLC identification of the preparation.

[0127] (2) The identification method provided by the present invention is not affected by the source of the Chinese medicinal plants in the prescription of the Chinese medicinal preparation. The components in the preparation are separated by differential migration on the thin layer plate based on the different distribution coefficients between the stationary phase and the mobile phase.

[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 the Dida Analgesic Plaster. Not only are the identification results intuitive and easy to identify, but the sensitivity and specificity are higher than those of conventional thin-layer chromatography identification methods. At the same time, this method is not only applicable to the detection of Dida Analgesic Plaster, but also to the detection of other dosage forms, thereby ensuring a more comprehensive detection and providing a convenient and practical way for the qualitative identification of multiple Chinese medicinal materials in the Dida Analgesic Plaster. BRIEF DESCRIPTION OF THE DRAWINGS

[0129] Figure 1 Rhubarb thin layer identification chart;

[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. Test solution 1 5μL;

[0135] 5. Test solution 2 5μL;

[0136] 6. Test solution 3 5μL;

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

[0138] Temperature: 11°C

[0139] Humidity: 72%

[0140] Thin layer plate: silica gel G;

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

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

[0143] Inspection: UV light (254nm), sunlight.

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

[0145] in:

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

[0147] 2. 2 μL of Dalbergia odorifera reference solution;

[0148] 3. Test solution 1 5μL;

[0149] 4. Test solution 2 5μL;

[0150] 5. Test solution 3 5μL;

[0151] Temperature: 28°C

[0152] Humidity: 59%

[0153] Thin layer plate: silica gel G;

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

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

[0156] Figure 3 Phellodendron chinense thin layer identification diagram;

[0157] in:

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

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

[0160] 3. Test solution 1 5μL;

[0161] 4. Test solution 2 5μL;

[0162] 5. Test solution 3 5μL;

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

[0164] Temperature: 27°C

[0165] Humidity: 59%

[0166] Thin layer plate: silica gel G;

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

[0168] Color development: Place ammonia vapor saturated in the expansion cylinder for ammonia fumigation. DETAILED DESCRIPTION

[0169] The present application will be further described below in conjunction with the embodiments, examples and accompanying drawings. It should be understood that these examples are intended only to illustrate the present application and are not intended to limit the scope of the present application. In addition, it should be understood that after reading the content of the present application, those skilled in the art may make various changes or modifications to the present application, and these equivalent forms also fall within the scope of protection of the claims appended hereto.

[0170] Unless defined otherwise, 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 belongs.

[0171] The terms used herein in the description of the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application.

[0172] Terminology: Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0173] The terms "and / or", "or / and", and "and / or" used in this article have a selection scope that includes any one of two or more related listed items, and also includes any and all combinations of the related listed items, and said any and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items.

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

[0175] Herein, "preferred", "better", "better", etc. are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of this application.

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

[0177] In this application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0178] In this application, when a numerical interval (i.e., a numerical range) is referred to, unless otherwise specified, the distribution of the optional values ​​within the numerical interval is considered continuous and includes the two numerical endpoints of the numerical range (i.e., the minimum and maximum values), as well as every value between these two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, it includes the two numerical endpoints of the numerical range, as well as every integer between the two endpoints.

[0179] Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise indicated, the ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein.

[0180] In this application, weight can be mass units known in the chemical industry, such as μg, mg, g, and kg.

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

[0182] The present invention provides a method for detecting three medicinal materials in a didaotongao (analgesic plaster) by thin layer chromatography, comprising the following steps:

[0183] Thin layer chromatography identification method of rhubarb:

[0184] Preparation of test solution: Take 1 piece of Dieda Analgesic Plaster, cut it into pieces, remove the cover, add methanol, sonicate, filter, evaporate the filtrate to dryness, dissolve the residue in water, add hydrochloric acid, heat in a water bath, cool immediately, extract by shaking with ether, combine the ether solution, evaporate to dryness, dissolve the residue in ethyl acetate, and use as the test solution.

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

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

[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 3500HZ.

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

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

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

[0194] The ultrasonic time is 15-30 minutes;

[0195] Preferably, the ultrasound duration is 20-25 minutes;

[0196] More preferably, the ultrasonic treatment time is 20 minutes.

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

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

[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 shaking extraction is performed 1-3 times;

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

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

[0206] Preparation of test solution: Take 1 piece of Dieda Analgesic Plaster, cut it into pieces, remove the cover, add 25 mL of methanol, ultrasonicate 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 with ether by shaking twice, 20 mL each time, combine the ether solutions, evaporate to dryness, and dissolve the residue in 1 mL of ethyl acetate to prepare the test solution.

[0207] Preparation of reference solution: Take rhubarb reference medicinal material and prepare reference solution in the same way.

[0208] Thin layer chromatography identification: According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 edition, Part IV, General Chapter 0502), 5-10 μL of the test solution and 5 μL of the reference solution are respectively spotted on the same silica gel G thin layer plate, developed with a developing agent, removed, dried, and examined under ultraviolet light (254 nm). The chromatogram of the test sample will show a fluorescent spot of the same color at the corresponding position of the chromatogram of the reference sample; place it in ammonia vapor and ammonia fumigation until color develops, and examine it under sunlight, and spots of the same color will appear.

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

[0210] The volume ratio of n-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 n-hexane-ethyl acetate-formic acid is 5:1:0.1.

[0213] As a most preferred embodiment, the thin layer chromatography identification method of rhubarb is:

[0214] Preparation of test solution: Take 1 piece of Dieda Analgesic Plaster, cut it into pieces, remove the cover, add 25 mL of methanol, ultrasonicate 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 with ether by shaking twice, 20 mL each time, combine the ether solutions, evaporate to dryness, and dissolve the residue in 1 mL of ethyl acetate to prepare the test solution.

[0215] Preparation of reference solution: Take 0.1 g of rhubarb reference medicinal material and prepare reference solution in the same way.

[0216] Identification by thin layer chromatography: According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502), 5-10 μL of the test solution and 5 μL of the reference solution were respectively spotted on the same silica gel G thin layer plate. Using n-hexane-ethyl acetate-formic acid (5:1:0.1) as the developing solvent, the plate was developed, removed, air-dried, and examined under ultraviolet light (254 nm). The chromatogram of the test sample showed a fluorescent spot of the same color at the corresponding position of the chromatogram of the reference sample; after fumigation with ammonia vapor until color developed, the plate was examined under sunlight and spots of the same color were observed.

[0217] Thin layer chromatography identification method of Dalbergia odorifera:

[0218] Preparation of test solution: Take 40cm of Dieda Pain Relief Plaster 2 , cut into pieces, remove the cover lining, add ethanol, ultrasonicate, filter, evaporate the filtrate to dryness, add ethanol to dissolve the residue, and use it as the test solution.

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

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

[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 3500HZ.

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

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

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

[0228] The ultrasonic time is 0.5-2 hours;

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

[0230] More preferably, the ultrasonication time is 1 hour.

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

[0232] Take 40cm of the pain-relieving plaster 2 , cut into pieces, remove the cover lining, add 25 mL of ethanol, ultrasonically treat for 1 hour, filter, evaporate the filtrate to dryness, add 1 mL of ethanol to dissolve the residue, and use it as the test solution.

[0233] Preparation of reference solution: Take Dalbergia odorifera reference medicinal material, add ethanol, reflux extraction, filter, evaporate the filtrate to dryness, add ethanol to dissolve the residue, and use it as the reference solution.

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

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

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

[0237] The temperature of the reflux extraction is 80°C-100°C;

[0238] Preferably, the temperature of the reflux extraction is 90°C-100°C;

[0239] More preferably, the temperature of the reflux extraction 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:

[0244] 0.5 g of Dalbergia odorifera reference medicinal material was added with 40 mL of ethanol, and the mixture was refluxed for 1 h. The mixture was filtered, the filtrate was evaporated to dryness, and 1 mL of ethanol was added to the residue to dissolve it, which was used as the reference solution.

[0245] Identification by thin layer chromatography: According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 edition, Part IV, General Chapter 0502), 2-5 μL of the test solution and 2 μL of the reference solution were applied to the same silica gel G thin layer plate. Develop with a developing agent, remove, dry, and examine under ultraviolet light (254 nm). In the chromatogram of the test sample, a fluorescent spot of the same color should appear at the corresponding position in the chromatogram of the reference sample.

[0246] The developing agent is a mixture solution 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 of Dalbergia woodii is as follows:

[0251] Preparation of test solution: Take 40cm of Dieda Pain Relief Plaster 2 , cut into pieces, remove the cover lining, add 25 mL of ethanol, ultrasonically treat for 1 hour, filter, evaporate the filtrate to dryness, add 1 mL of ethanol to dissolve the residue, and use it as the test solution.

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

[0253] Identification by thin layer chromatography: According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502), 2-5 μL of the test solution and 2 μL of the reference solution were applied to the same silica gel G thin layer plate. The plate was developed with toluene-ethyl acetate (2:1). The plate was removed, dried, and examined under ultraviolet light (254 nm). In the chromatogram of the test sample, a fluorescent spot of the same color appeared at the corresponding position in the chromatogram of the reference sample.

[0254] Thin layer chromatography identification method of Phellodendron chinense:

[0255] Preparation of the test solution: Take 2 tablets of Dieda Analgesic Plaster, cut them into pieces respectively, remove the cover lining, add methanol solution, ultrasonically treat, filter, evaporate the filtrate to dryness, and dissolve the residue in methanol as the test solution.

[0256] The methanol solution is methanol acetate;

[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 3500HZ.

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

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

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

[0267] The ultrasonic time is 10-30 minutes;

[0268] Preferably, the ultrasound duration is 15-20 minutes;

[0269] More preferably, the ultrasonic treatment time is 20 minutes.

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

[0271] Take 2 pieces of Dieda Analgesic Plaster, cut them into pieces respectively, remove the cover lining, add 40 mL of 1% acetic acid methanol solution, ultrasonically treat at 60℃ for 20 minutes, filter, evaporate the filtrate to dryness, and dissolve the residue in 2 mL of 1% acetic acid methanol solution to prepare the test solution.

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

[0273] The methanol solution 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] Further 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:

[0279] Take 0.1 g of Phellodendron chinense reference medicinal material, add 20 mL of 1% acetic acid methanol solution, and prepare the reference solution in the same way.

[0280] Identification by thin-layer chromatography: According to the thin-layer chromatography method (Chinese Pharmacopoeia 2020 edition, Part IV, General Chapter 0502), 5 μL of the test sample solution and 5 μL of the reference sample solution were spotted separately on the same silica gel G thin-layer plate. Developed with a developing agent, the plate was removed, air-dried, and fumigated with ammonia vapor before inspection under sunlight. The test sample chromatogram should show spots of the same color at the corresponding positions on the reference sample chromatogram.

[0281] The developing agent 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 of Phellodendron chinense is:

[0286] Preparation of the test solution: Take 2 tablets of Dieda Analgesic Plaster, cut them into pieces respectively, remove the cover lining, add 40 mL of 1% acetic acid methanol solution, ultrasonically treat at 60℃ for 20 minutes, filter, evaporate the filtrate to dryness, and dissolve the residue in 2 mL of 1% acetic acid methanol solution to prepare the test solution.

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

[0288] Identification by thin-layer chromatography: According to the thin-layer chromatography method (Chinese Pharmacopoeia 2020 edition, Part IV, General Chapter 0502), 5 μL of the test sample solution and 5 μL of the reference sample solution were spotted separately on the same silica gel G thin-layer plate. Develop with ethyl acetate-2-butanone-formic acid-water (10:7:1:1) as the developing solvent. Remove the plate, air-dry, fumigate with ammonia vapor, and inspect under sunlight. The test sample chromatogram will show spots of the same color at the corresponding positions on the reference chromatogram.

[0289] Example 1-1 Thin layer chromatography identification method of rhubarb:

[0290] Preparation of reference solution:

[0291] Take 2.0 g of the negative paste without rhubarb and prepare the rhubarb negative sample solution in the same way.

[0292] Take 2.0 g of the negative paste lacking Polygonum cuspidatum and Rhubarb, and prepare a double-negative sample solution lacking Rhubarb and Polygonum cuspidatum by the same method.

[0293] Take 0.1 g of rhubarb reference medicinal material and prepare the reference solution in the same way.

[0294] Take the rhein reference substance and add methanol to make a solution containing 1 mg per 1 mL.

[0295] Preparation of test solution:

[0296] Take 1 piece of Dieda Analgesic Plaster, cut it into pieces, remove the cover, add 25 mL of methanol, ultrasonicate 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 minutes, cool immediately, shake and extract with ether twice, 20 mL each time, combine the ether solution, evaporate to dryness, add 1 mL of ethyl acetate to dissolve the residue, and use it as the test solution.

[0297] 5 μL of each of the above solutions was taken and spotted on the same silica gel G thin layer plate, and developed with n-hexane-ethyl acetate-formic acid (5:1:0.1) as the developing agent. The plate was taken out, dried, and examined under ultraviolet light (365 nm, 254 nm).

[0298] In the chromatogram of the test sample, at the corresponding position with the chromatogram of the control medicinal material, the same two orange-yellow fluorescent spots appeared, with no interference under negative conditions, and the characteristic spots were more obvious when inspected at 254nm than at 365nm; after being fumigated with ammonia vapor and inspected under sunlight, the same red spots appeared, with no interference under negative conditions, and good specificity.

[0299] Comparative Example 1-1:

[0300] Preparation of reference solution:

[0301] Take 2.0 g of the negative paste lacking Polygonum cuspidatum and Rhubarb, and prepare a double-negative sample solution lacking Rhubarb and Polygonum cuspidatum by the same method.

[0302] Take 0.1 g of rhubarb reference medicinal material and prepare the reference solution in the same way.

[0303] Take 2.0 g of the negative paste without rhubarb and prepare the rhubarb negative sample solution in the same way.

[0304] Preparation of test solution:

[0305] Take 1 piece of Dieda Analgesic Plaster, cut it into pieces, remove the cover, add 25 mL of methanol, ultrasonicate 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 minutes, cool immediately, shake and extract with ether twice, 20 mL each time, combine the ether solution, evaporate to dryness, add 1 mL of ethyl acetate to dissolve the residue, and use it as the test solution.

[0306] 2 μL of each of the above four solutions were taken and spotted on the same silica gel G thin layer plate. The upper layer solution of petroleum ether (30-60℃)-ethyl formate-formic acid (15:5:1) was used as the developing agent. The plate was developed, taken out, dried, and examined under ultraviolet light (365nm).

[0307] In the chromatogram of the test sample, the same two orange-yellow fluorescent spots appeared at the corresponding positions of the chromatogram of the control medicinal material; after being fumigated with ammonia vapor and examined under sunlight, the same orange-red spots appeared, but the negative result had interference.

[0308] Comparative Example 1-2:

[0309] Preparation of reference solution:

[0310] Take 2.0 g of the negative paste without rhubarb and prepare the rhubarb negative sample solution in the same way.

[0311] Take 2.0 g of the negative paste lacking Polygonum cuspidatum and Rhubarb, and prepare a double-negative sample solution lacking Rhubarb and Polygonum cuspidatum by the same method.

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

[0313] Take the rhein reference substance and add methanol to make a solution containing 1 mg per 1 mL.

[0314] Preparation of test solution:

[0315] Take 1 piece of Dieda Analgesic Plaster, cut it into pieces, remove the cover, add 20 mL of a mixed solution of 30% ethanol-hydrochloric acid (10:1), heat under reflux in a water bath for 1 hour, cool immediately, and extract with chloroform by shaking twice, 20 mL each time. Combine the chloroform liquid, evaporate to dryness, and dissolve the residue in 5 mL of a mixed solution of anhydrous ethanol-ethyl acetate (2:1) to prepare the test solution.

[0316] 5 μL of each of the above solutions was taken and spotted on the same silica gel G thin layer plate. The plate was developed with the upper layer solution of petroleum ether (30-60°C)-ethyl formate-formic acid (15:5:1). The plate was taken out, dried, and examined under ultraviolet light (365 nm).

[0317] In the chromatogram of the test sample, the same orange-yellow fluorescent spots appeared at the corresponding positions in the chromatogram of the control medicinal material and the chromatogram of the reference sample, and the negative sample had interference; after being fumigated with ammonia vapor and inspected under sunlight, the spots were not obvious and there were no corresponding identical spots.

[0318] Comparative Examples 1-3:

[0319] Preparation of reference solution:

[0320] Take 2.0 g of the negative paste without rhubarb and prepare the rhubarb negative sample solution in the same way.

[0321] Take 2.0 g of the negative paste lacking Polygonum cuspidatum and Rhubarb, and prepare a double-negative sample solution lacking Rhubarb and Polygonum cuspidatum by the same method.

[0322] Take 0.5 g of rhubarb reference medicinal material and prepare reference solution in the same way.

[0323] Take the rhein reference substance and add methanol to make a solution containing 1 mg per 1 mL.

[0324] Preparation of test solution:

[0325] Take 1 piece of Dieda Analgesic Plaster, cut it into pieces, remove the cover, add 20 mL of methanol, soak it in cold for 1 hour, filter, evaporate the filtrate to dryness, add 10 mL of water to the residue to dissolve it, add 1 mL of hydrochloric acid dropwise, heat it in a water bath for 30 minutes, cool it immediately, and extract it twice with ether, 10 mL each time, combine the ether solutions, evaporate them to dryness, and add 1 mL of chloroform to dissolve the residue to use as the test solution.

[0326] 5 μL of each of the above solutions was taken and spotted on the same silica gel G thin layer plate, and developed with n-hexane-ethyl acetate-formic acid (6:2:0.1) as the developing agent. The plate was taken out, dried, and examined under ultraviolet light (365 nm).

[0327] The test sample chromatogram showed the same orange-yellow fluorescent spot at the corresponding position on the control herb chromatogram. After exposure to ammonia vapor, the spot turned red. However, the Rf value of the target spot was too high and required further adjustment.

[0328] Example 1-2 Thin layer chromatography identification method of rhubarb:

[0329] Preparation of reference solution:

[0330] Take 2.0 g of the negative paste without rhubarb and prepare the rhubarb negative sample solution in the same way.

[0331] Take 2.0 g of the negative paste lacking Polygonum cuspidatum and Rhubarb, and prepare a double-negative sample solution lacking Rhubarb and Polygonum cuspidatum by the same method.

[0332] Take 0.1 g of rhubarb reference medicinal material and prepare the reference solution in the same way.

[0333] Take the rhein reference substance and add methanol to make a solution containing 1 mg per 1 mL.

[0334] Preparation of test solution:

[0335] Take 1 piece of Dieda Analgesic Plaster, cut it into pieces, remove the cover, add 20 mL of methanol, ultrasonicate 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 minutes, cool immediately, shake and extract with ether 3 times, 20 mL each time, combine the ether solution, evaporate to dryness, add 1 mL of ethyl acetate to dissolve the residue, and use it as the test solution.

[0336] 5 μL of each of the above solutions was taken and spotted on the same silica gel G thin layer plate, and developed with n-hexane-ethyl acetate-formic acid (5:1:0.1) as the developing agent. The plate was taken out, dried, and examined under ultraviolet light (365 nm, 254 nm).

[0337] The test sample chromatogram showed two identical orange-yellow fluorescent spots at corresponding positions in the control herb chromatogram, with no interference from negative analysis. The characteristic spots were more pronounced at 254 nm than at 365 nm. After exposure to ammonia vapor and subsequent observation under sunlight, the same red spots were observed, with no interference from negative analysis, indicating good specificity. Compared to Example 1-1, the spots were lighter, possibly due to incomplete extraction.

[0338] Example 1-3 Thin layer chromatography identification method of rhubarb:

[0339] Preparation of reference solution:

[0340] Take 2.0 g of the negative paste without rhubarb and prepare the rhubarb negative sample solution in the same way.

[0341] Take 2.0 g of the negative paste lacking Polygonum cuspidatum and Rhubarb, and prepare a double-negative sample solution lacking Rhubarb and Polygonum cuspidatum by the same method.

[0342] Take 0.1 g of rhubarb reference medicinal material and prepare the reference solution in the same way.

[0343] Take the rhein reference substance and add methanol to make a solution containing 1 mg per 1 mL.

[0344] Preparation of test solution:

[0345] Take 1 piece of Dieda Analgesic Plaster, cut it into pieces, remove the cover, add 30 mL of methanol, ultrasonicate 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, shake and extract with ether twice, 20 mL each time, combine the ether solution, evaporate to dryness, add 1 mL of ethyl acetate to dissolve the residue, and use it as the test solution.

[0346] 5 μL of each of the above solutions was taken and spotted on the same silica gel G thin layer plate, and developed with n-hexane-ethyl acetate-formic acid (5:1:0.1) as the developing agent. The plate was taken out, dried, and examined under ultraviolet light (365 nm, 254 nm).

[0347] In the chromatogram of the test sample, at the corresponding position with the chromatogram of the control medicinal material, the same two orange-yellow fluorescent spots appeared, with no interference under negative conditions, and the characteristic spots were more obvious when inspected at 254nm than at 365nm; after being fumigated with ammonia vapor and inspected under sunlight, the same red spots appeared, with no interference under negative conditions, and good specificity.

[0348] Example 1-4 Thin layer chromatography identification method of rhubarb:

[0349] Preparation of reference solution:

[0350] Take 2.0 g of the negative paste without rhubarb and prepare the rhubarb negative sample solution in the same way.

[0351] Take 2.0 g of the negative paste lacking Polygonum cuspidatum and Rhubarb, and prepare a double-negative sample solution lacking Rhubarb and Polygonum cuspidatum by the same method.

[0352] Take 0.1 g of rhubarb reference medicinal material and prepare the reference solution in the same way.

[0353] Take the rhein reference substance and add methanol to make a solution containing 1 mg per 1 mL.

[0354] Preparation of test solution:

[0355] Take 1 piece of Dieda Analgesic Plaster, cut it into pieces, remove the cover, add 20 mL of methanol, ultrasonicate 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 minutes, cool immediately, shake and extract with ether twice, 20 mL each time, combine the ether solution, evaporate to dryness, add 1 mL of ethyl acetate to dissolve the residue, and use it as the test solution.

[0356] 5 μL of each of the above solutions was taken and spotted on the same silica gel G thin layer plate, and developed with n-hexane-ethyl acetate-formic acid (5:1:0.1) as the developing agent. The plate was taken out, dried, and examined under ultraviolet light (365 nm, 254 nm).

[0357] The test sample chromatogram showed two identical orange-yellow fluorescent spots at corresponding positions in the control herb chromatogram, with no interference from negative analysis. The characteristic spots were more pronounced at 254 nm than at 365 nm. After exposure to ammonia vapor and subsequent observation under sunlight, the same red spots were observed, with no interference from negative analysis, indicating good specificity. Compared to Example 1-1, the spots were lighter, possibly due to incomplete extraction.

[0358] Example 1-5 Thin layer chromatography identification method of rhubarb:

[0359] Preparation of reference solution:

[0360] Take 2.0 g of the negative paste without rhubarb and prepare the rhubarb negative sample solution in the same way.

[0361] Take 2.0 g of the negative paste lacking Polygonum cuspidatum and Rhubarb, and prepare a double-negative sample solution lacking Rhubarb and Polygonum cuspidatum by the same method.

[0362] Take 0.1 g of rhubarb reference medicinal material and prepare the reference solution in the same way.

[0363] Take the rhein reference substance and add methanol to make a solution containing 1 mg per 1 mL.

[0364] Preparation of test solution:

[0365] Take 1 piece of Dieda Analgesic Plaster, cut it into pieces, remove the cover, add 25 mL of methanol, ultrasonicate 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 minutes, cool immediately, shake and extract with ether twice, 20 mL each time, combine the ether solution, evaporate to dryness, add 1 mL of ethyl acetate to dissolve the residue, and use it as the test solution.

[0366] 5 μL of each of the above solutions was taken and spotted on the same silica gel G thin layer plate, and developed with n-hexane-ethyl acetate-formic acid (5:1:0.1) as the developing agent. The plate was taken out, dried, and examined under ultraviolet light (365 nm, 254 nm).

[0367] In the chromatogram of the test sample, at the corresponding position with the chromatogram of the control medicinal material, the same two orange-yellow fluorescent spots appeared, with no interference under negative conditions, and the characteristic spots were more obvious when inspected at 254nm than at 365nm; after being fumigated with ammonia vapor and inspected under sunlight, the same red spots appeared, with no interference under negative conditions, and good specificity.

[0368] Example 2-1 Thin layer chromatography identification method of Dalbergia odorifera:

[0369] Preparation of reference solution:

[0370] Take 1 g of the Dalbergia odorifera negative paste, add 25 mL of ethanol, and prepare the Dalbergia odorifera negative sample solution in the same way.

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

[0372] Preparation of test solution:

[0373] Take 40cm of the pain-relieving plaster 2, cut into pieces, remove the cover lining, add 25ml of ethanol, ultrasonically treat at 25℃ for 1 hour, filter, evaporate the filtrate to dryness, and dissolve the residue in 1mL of ethanol as the test solution.

[0374] According to the thin layer chromatography method (General Method 0502), 5 μL of the test solution and 2 μL of the reference solution were respectively spotted on the same silica gel G thin layer plate and the homemade plate, and developed with toluene-ethyl acetate (2:1) as the developing solvent. The plates were taken out, dried, and examined under ultraviolet light.

[0375] The test sample chromatogram showed a fluorescent spot of the same color at the corresponding position in the reference sample chromatogram, and there was no negative interference, indicating that the method has good specificity. In addition, the chromatographic information under 254nm UV light is richer than that under 365nm.

[0376] Comparative Example 2-1:

[0377] Preparation of reference solution:

[0378] Take 1 g of the Dalbergia odorifera negative paste, add 25 mL of ethanol, and prepare the Dalbergia odorifera negative sample solution in the same way.

[0379] Take 1 g of Dalbergia odorifera reference medicinal material, add 25 mL of ethanol, ultrasonically treat for 1 hour, filter, evaporate the filtrate to dryness, and dissolve the residue in 1 mL of ethanol to serve as the reference solution.

[0380] Preparation of test solution:

[0381] Take 40cm of the pain-relieving plaster 2 , cut into pieces, remove the cover lining, add 25ml of ethanol, ultrasonically treat for 1 hour, filter, evaporate the filtrate to dryness, add 1mL of ethanol to dissolve the residue, and use it as the test solution.

[0382] 10 μL of the test solution, negative sample solution and 2 μL of the reference solution were taken and spotted on the same silica gel G thin layer plate respectively. The plate was developed with toluene-ether-chloroform (7:2:1) as the developing agent. The plate was taken out and dried in air. The plate was sprayed with a mixed solution of 1% vanillin sulfuric acid solution and anhydrous ethanol (1:9), and heated at 105°C until the spots were clearly colored.

[0383] Example 2-2 Thin layer chromatography identification method of Dalbergia odorifera:

[0384] Preparation of reference solution:

[0385] Take 1 g of the Dalbergia odorifera negative paste, add 25 mL of ethanol, and prepare the Dalbergia odorifera negative sample solution in the same way.

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

[0387] Preparation of test solution:

[0388] Take 40cm of the pain-relieving plaster 2 , cut into pieces, remove the cover lining, add 20ml of ethanol, ultrasonically treat at 30℃ for 2 hours, filter, evaporate the filtrate to dryness, and dissolve the residue in 1mL of ethanol as the test solution.

[0389] According to the thin layer chromatography method (General Method 0502), 5 μL of the test solution and 2 μL of the reference solution were respectively spotted on the same silica gel G thin layer plate and the homemade plate, and developed with toluene-ethyl acetate (2:1) as the developing solvent. The plates were taken out, dried, and examined under ultraviolet light.

[0390] The test sample chromatogram showed a fluorescent spot of the same color at the corresponding position in the control chromatogram, with no negative interference, demonstrating the good specificity of this method. Furthermore, the chromatographic information was richer when examined under 254nm UV light than at 365nm. Compared to Example 2-1, the spot was lighter, possibly due to incomplete extraction.

[0391] Example 2-3 Thin layer chromatography identification method of dalbergia woodii:

[0392] Preparation of reference solution:

[0393] Take 1 g of the Dalbergia odorifera negative paste, add 25 mL of ethanol, and prepare the Dalbergia odorifera negative sample solution in the same way.

[0394] Take 1 g of Dalbergia odorifera reference medicinal material, add 50 mL of ethanol, ultrasonically treat for 40 minutes, filter, evaporate the filtrate to dryness, and dissolve the residue in 1 mL of ethanol to serve as the reference solution.

[0395] Preparation of test solution:

[0396] Take 40cm of the pain-relieving plaster 2 , cut into pieces, remove the cover lining, add 30ml of ethanol, ultrasonically treat at 20℃ for 1.5 hours, filter, evaporate the filtrate to dryness, and dissolve the residue in 1mL of ethanol as the test solution.

[0397] According to the thin layer chromatography method (General Method 0502), 5 μL of the test solution and 2 μL of the reference solution were respectively spotted on the same silica gel G thin layer plate and the homemade plate, and developed with toluene-ethyl acetate (2:1) as the developing solvent. The plates were taken out, dried, and examined under ultraviolet light.

[0398] The test sample chromatogram showed a fluorescent spot of the same color at the corresponding position in the reference sample chromatogram, and there was no negative interference, indicating that the method has good specificity. In addition, the chromatographic information under 254nm UV light is richer than that under 365nm.

[0399] Example 2-4 Thin layer chromatography identification method of dalbergia woodii:

[0400] Preparation of reference solution:

[0401] Take 1 g of the Dalbergia odorifera negative paste, add 25 mL of ethanol, and prepare the Dalbergia odorifera negative sample solution in the same way.

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

[0403] Preparation of test solution:

[0404] Take 40cm of the pain-relieving plaster 2 , cut into pieces, remove the cover lining, add 25ml of ethanol, ultrasonically treat at 25℃ for 1.5 hours, filter, evaporate the filtrate to dryness, and dissolve the residue in 1mL of ethanol as the test solution.

[0405] According to the thin layer chromatography method (General Method 0502), 5 μL of the test solution and 2 μL of the reference solution were respectively spotted on the same silica gel G thin layer plate and the homemade plate, and developed with toluene-ethyl acetate (2:1) as the developing solvent. The plates were taken out, dried, and examined under ultraviolet light.

[0406] 5 μL of the test solution and 2 μL of the reference solution were respectively spotted on the same silica gel G thin layer plate and the homemade plate, and developed with toluene-ethyl acetate (2:1). The plates were taken out, dried, and inspected under UV light.

[0407] The test sample chromatogram showed a fluorescent spot of the same color at the corresponding position in the reference sample chromatogram, and there was no negative interference, indicating that the method has good specificity. In addition, the chromatographic information under 254nm UV light is richer than that under 365nm.

[0408] Example 2-5 Thin layer chromatography identification method of dalbergia woodii:

[0409] Preparation of reference solution:

[0410] Take 1 g of the Dalbergia odorifera negative paste, add 25 mL of ethanol, and prepare the Dalbergia odorifera negative sample solution in the same way.

[0411] Take 1 g of Dalbergia odorifera reference medicinal material, add 40 mL of ethanol, ultrasonically treat for 80 minutes, filter, evaporate the filtrate to dryness, and dissolve the residue in 1 mL of ethanol to serve as the reference solution.

[0412] Preparation of test solution:

[0413] Take 40cm of the pain-relieving plaster 2 , cut into pieces, remove the cover lining, add 30ml of ethanol, ultrasonically treat at 25℃ for 1 hour, filter, evaporate the filtrate to dryness, and dissolve the residue in 1mL of ethanol as the test solution.

[0414] According to the thin layer chromatography method (General Method 0502), 5 μL of the test solution and 2 μL of the reference solution were respectively spotted on the same silica gel G thin layer plate and the homemade plate, and developed with toluene-ethyl acetate (2:1) as the developing solvent. The plates were taken out, dried, and examined under ultraviolet light.

[0415] The test sample chromatogram showed a fluorescent spot of the same color at the corresponding position in the reference sample chromatogram, and there was no negative interference, indicating that the method has good specificity. In addition, the chromatographic information under 254nm UV light is richer than that under 365nm.

[0416] Example 3-1 Thin layer chromatography identification method of Phellodendron chinense:

[0417] Preparation of reference solution:

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

[0419] Take 0.1 g of Phellodendron chinense reference medicinal material, add 20 mL of 1% acetic acid methanol, and prepare the reference solution in the same way.

[0420] Then take the phellodendrine hydrochloride reference substance and add A to make a solution containing 0.5 mg per 1 mL as the reference substance solution.

[0421] Preparation of test solution:

[0422] Take 2 pieces of Dieda Analgesic Plaster, cut them into pieces respectively, remove the cover and lining, add 40 mL of 1% acetic acid methanol solution, ultrasonically treat at 60 ° C for 20 minutes, filter, evaporate the filtrate to dryness, and dissolve the residue in 2 mL of methanol as the test solution.

[0423] 5 μL of each of the above solutions was spotted on the same silica gel G thin layer plate, and developed with ethyl acetate-2-butanone-formic acid-water (10:7:1:1). The plate was taken out, dried, fumigated with ammonia vapor, and examined under sunlight.

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

[0425] Comparative Example 3-1:

[0426] Preparation of reference solution:

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

[0428] Take another 0.1 g of Phellodendron chinense reference medicinal material, add 20 mL of 1% acetic acid methanol, and prepare the reference solution in the same way.

[0429] Preparation of test solution:

[0430] Take 2 pieces of Dieda Analgesic Plaster, cut them into pieces respectively, remove the cover and lining, add 40 mL of 1% acetic acid methanol solution, ultrasonically treat at 60 ° C for 20 minutes, filter, evaporate the filtrate to dryness, and dissolve the residue in 2 mL of methanol as the test solution.

[0431] 5 μL and 10 μL of the above four solutions were respectively spotted on the same silica gel G thin layer plate, and the lower layer solution of chloroform-methanol-water (30:15:4) was used as the developing agent; the plate was placed in a developing cylinder saturated with ammonia vapor, developed, taken out, dried, and sprayed with dilute potassium bismuth iodide test solution.

[0432] During ammonia fumigation, spots of the same color appear in the chromatogram of the test solution at the corresponding positions in the chromatogram of the control medicinal material and the chromatogram of the reference substance. After spraying the color developer, if the chromatogram of the control medicinal material is inconsistent with the test sample, it will not be included in the standard for the time being.

[0433] Comparative Example 3-2:

[0434] Preparation of reference solution:

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

[0436] Take another 0.1 g of Phellodendron chinense reference medicinal material, add 20 mL of 1% acetic acid methanol, and prepare the reference solution in the same way.

[0437] Then take the phellodendrine hydrochloride reference substance and add A to make a solution containing 0.5 mg per 1 mL as the reference substance solution.

[0438] Preparation of test sample:

[0439] Take 2 pieces of Dieda Analgesic Plaster, cut them into pieces respectively, remove the cover and lining, add 40 mL of 1% acetic acid methanol solution, ultrasonically treat at 60 ° C for 20 minutes, filter, evaporate the filtrate to dryness, and dissolve the residue in 2 mL of methanol as the test solution.

[0440] 5 μL and 10 μL of the above four solutions were respectively spotted on the same silica gel G thin layer plate, and the lower layer solution of dichloromethane-methanol-water (30:15:4) was used as the developing solvent; the plate was placed in a developing cylinder saturated with ammonia vapor, developed, taken out, dried, and sprayed with dilute potassium bismuth iodide test solution for the test sample chromatograph.

[0441] The same color spots are shown at the corresponding positions of the chromatograms of the reference medicinal materials and the reference substance. However, the Rf value is too large and it is not recommended to be included in the standard.

[0442] Comparative Example 3-3:

[0443] Preparation of reference solution:

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

[0445] Take 0.1 g of Phellodendron chinense reference medicinal material, add 20 mL of 1% acetic acid methanol, and prepare the reference solution in the same way.

[0446] Preparation of test solution:

[0447] Take 2 pieces of Dieda Analgesic Plaster, cut them into pieces respectively, remove the cover and lining, add 40 mL of 1% acetic acid methanol solution, ultrasonically treat at 60 ° C for 20 minutes, filter, evaporate the filtrate to dryness, and dissolve the residue in 2 mL of methanol as the test solution.

[0448] 5 μL of each of the three solutions was taken and spotted on the same silica gel G thin layer plate, and toluene-ethyl acetate-methanol-isopropanol-water (6:3:2:1.5:0.3) was used as the developing solvent; the plate was placed in a developing cylinder saturated with ammonia vapor, developed, taken out, and dried.

[0449] In the chromatogram of the test sample, there is no spot corresponding to the position in the chromatogram of the control medicinal material, so it is not included in the standard.

[0450] Example 3-2 Thin layer chromatography identification method of Phellodendron chinense:

[0451] Preparation of reference solution:

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

[0453] Take 0.1 g of Phellodendron chinense reference medicinal material, add 20 mL of 0.8% acetic acid methanol, and prepare the reference solution in the same way.

[0454] Then take the phellodendrine hydrochloride reference substance and add A to make a solution containing 0.5 mg per 1 mL as the reference substance solution.

[0455] Preparation of test solution:

[0456] Take 2 pieces of Dieda Analgesic Plaster, cut them into pieces respectively, remove the cover and lining, add 40 mL of 0.8% acetic acid methanol solution, ultrasonically treat at 70 ° C for 10 minutes, filter, evaporate the filtrate to dryness, and dissolve the residue in 2 mL of methanol as the test solution.

[0457] 5 μL of each of the above solutions was spotted on the same silica gel G thin layer plate, and developed with ethyl acetate-2-butanone-formic acid-water (10:7:1:1). The plate was taken out, dried, fumigated with ammonia vapor, and examined under sunlight.

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

[0459] Example 3-3 Thin layer chromatography identification method of Phellodendron chinense:

[0460] Preparation of reference solution:

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

[0462] Take 0.1 g of Phellodendron chinense reference medicinal material, add 20 mL of 2% acetic acid methanol, and prepare the reference solution in the same way.

[0463] Then take the phellodendrine hydrochloride reference substance and add A to make a solution containing 0.5 mg per 1 mL as the reference substance solution.

[0464] Preparation of test solution:

[0465] Take 2 pieces of Dieda Analgesic Plaster, cut them into pieces respectively, remove the cover and lining, add 40 mL of 2% acetic acid methanol solution, ultrasonically treat at 70 ° C for 10 minutes, filter, evaporate the filtrate to dryness, and dissolve the residue in 2 mL of methanol to prepare the test solution.

[0466] 5 μL of each of the above solutions was spotted on the same silica gel G thin layer plate, and developed with ethyl acetate-2-butanone-formic acid-water (10:7:1:1). The plate was taken out, dried, fumigated with ammonia vapor, and examined under sunlight.

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

[0468] Example 3-4 Thin layer chromatography identification method of Phellodendron chinense:

[0469] Preparation of reference solution:

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

[0471] Take 0.1 g of Phellodendron chinense reference medicinal material, add 20 mL of 1.5% acetic acid methanol, and prepare the reference solution in the same way.

[0472] Then take the phellodendrine hydrochloride reference substance and add A to make a solution containing 0.5 mg per 1 mL as the reference substance solution.

[0473] Preparation of test solution:

[0474] Take 2 tablets of Dieda Analgesic Plaster, cut them into pieces respectively, remove the cover and lining, add 40 mL of 1.5% acetic acid methanol solution, ultrasonically treat at 65 ° C for 15 minutes, filter, evaporate the filtrate to dryness, and dissolve the residue in 2 mL of methanol as the test solution.

[0475] 5 μL of each of the above solutions was spotted on the same silica gel G thin layer plate, and developed with ethyl acetate-2-butanone-formic acid-water (10:7:1:1). The plate was taken out, dried, fumigated with ammonia vapor, and examined under sunlight.

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

[0477] Example 3-5 Thin layer chromatography identification method of Phellodendron chinense:

[0478] Preparation of reference solution:

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

[0480] Take 0.1 g of Phellodendron chinense reference medicinal material, add 20 mL of 1.8% acetic acid methanol, and prepare the reference solution in the same way.

[0481] Then take the phellodendrine hydrochloride reference substance and add A to make a solution containing 0.5 mg per 1 mL as the reference substance solution.

[0482] Preparation of test solution:

[0483] Take 2 pieces of Dieda Analgesic Plaster, cut them into pieces respectively, remove the cover and lining, add 40 mL of 1.8% acetic acid methanol solution, ultrasonically treat at 55 ° C for 15 minutes, filter, evaporate the filtrate to dryness, and dissolve the residue in 2 mL of methanol as the test solution.

[0484] 5 μL of each of the above solutions was spotted on the same silica gel G thin layer plate, and developed with ethyl acetate-2-butanone-formic acid-water (10:7:1:1). The plate was taken out, dried, fumigated with ammonia vapor, and examined under sunlight.

[0485] The test sample chromatogram shows spots of the same color at the corresponding positions of the control medicinal material chromatogram and the control sample chromatogram, while the negative sample has 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 quantities

[0490] Take 5-10μL of the test sample solution and 5μL of the control sample solution, and perform thin layer chromatography identification according to the above method to investigate the effect of different sample size on thin layer chromatography identification. According to the test results, 5-10μL sample size can achieve rhubarb identification, that is, the sample size of the test solution for rhubarb identification in Dieda Analgesic Plaster is 5-10μL.

[0491] 1.2 Comparison of different thin layer plates

[0492] The reference solution and the test solution (prepared according to the method given in the main text) were spotted on a homemade silica gel G thin layer plate and an ordinary prefabricated silica gel G thin layer plate (Qingdao Ocean Chemical Co., Ltd.), respectively. The results showed that the test sample could achieve good separation effect on both different thin layer plates, and the results were basically consistent.

[0493] 1.3 Comparison of different temperatures and humidity

[0494] Take the reference solution and the test solution (prepared according to the method in the text) and investigate the separation under two conditions: normal temperature and humidity (recording the temperature and humidity of the air-conditioned room) and low temperature and humidity (recording the temperature and humidity of the refrigerator). The results show that the test sample can achieve good separation effect under the above conditions, and temperature has little effect on the separation.

[0495] 1.4. Measurement results of different samples

[0496] Four batches of samples were selected and the test solution was prepared according to the method proposed in the main text. The reference solution and the test solution were spotted on the same silica gel G thin layer plate, and developed according to the conditions proposed in the main text. As a result, the chromatograms of the four batches of test samples showed the same yellow fluorescent spots at the corresponding positions of the chromatograms of the reference medicinal materials, and the development effect was good, which can be used as a control indicator 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 on a homemade silica gel G thin layer plate and an ordinary prefabricated silica gel G thin layer plate (Qingdao Ocean Chemical Co., Ltd.), respectively. The results showed that the test sample could achieve good separation effect on both different thin layer plates, and the results were basically consistent.

[0500] 2.2 Comparison of different temperatures

[0501] The reference sample solution and the test sample solution were taken to investigate the separation under two conditions: normal temperature and normal humidity and low temperature and normal humidity. 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 amurense

[0503] 3.1 Comparison of different thin layer plates

[0504] The reference solution and the test solution were spotted on a homemade silica gel G thin layer plate and an ordinary prefabricated silica gel G thin layer plate (Qingdao Ocean Chemical Co., Ltd.), respectively. The results showed that the test sample could achieve good separation effect on both different thin layer plates, and the results were basically consistent.

[0505] 3.2 Comparison of different temperatures and humidity

[0506] The reference solution and the test solution were taken and the separation conditions under three conditions: normal temperature and humidity (A: T = 25 ° C, RH: = 57%), low temperature and humidity (T = 3 ° C, RH = 65%), normal temperature and high humidity (T = 17 ° C, RH = 81%) and normal temperature and humidity (C: T = 25 ° C, RH = 39%) were investigated. The results showed that the test sample was prone to produce edge effect under the above low temperature conditions, but had no effect on identification; humidity had little effect on separation, and the test sample could achieve good separation effect under the above humidity conditions.

[0507] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A method for detecting three medicinal materials in a dihetong ointment by thin layer chromatography, characterized in that: The steps include: Thin layer chromatography identification method of rhubarb: Thin layer chromatography identification: Pipette the test solution A and the rhubarb reference solution onto the same silica gel G thin layer plate, develop with a developing agent, remove, dry, and examine under a 254nm ultraviolet lamp; the test sample chromatogram will show fluorescent spots of the same color at the corresponding positions of the reference chromatogram; place in ammonia vapor and fumigate until color develops, and examine under sunlight to show spots of the same color; the developing agent is a mixed solution of n-hexane-ethyl acetate-formic acid; Thin layer chromatography identification method of Dalbergia odorifera: Thin layer chromatography identification: aspirate the test solution B and the dalbergia odorifera reference solution onto the same silica gel G thin layer plate, develop with a developing agent, remove, dry, and examine under a UV lamp at 254nm; in the chromatogram of the test sample, a fluorescent spot of the same color appears at the corresponding position in the chromatogram of the reference; the developing agent is a mixture of toluene and ethyl acetate; Thin layer chromatography identification method of Phellodendron chinense: Identification by thin layer chromatography: aspirate the test solution C and the Phellodendron chinense reference solution and spot them separately on the same silica gel G thin layer plate, develop with a developing agent, take out, dry, fumigate in ammonia vapor, and inspect under sunlight; the test sample chromatogram will show spots of the same color at the corresponding positions of the reference chromatogram; the developing agent is a mixed solution of ethyl acetate, 2-butanone, formic acid and water.

2. The method according to claim 1, wherein: Preparation of the test solution A: Take 1 piece of Dieda Analgesic Plaster, cut it into pieces, remove the cover liner, 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 by shaking with ether, combine the ether solutions, evaporate to dryness, and dissolve the residue in ethyl acetate to prepare the test solution.

3. The method according to claim 2, wherein: The water bath heating time is 20-40 minutes.

4. The method according to claim 3, wherein: The water bath heating time is 30 minutes.

5. The method according to claim 1, wherein: Preparation of the test solution A: Take 1 piece of Dieda Analgesic Plaster, cut it into pieces, remove the cover liner, add 25 mL of methanol, ultrasonicate 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, and extract twice with ether by shaking, each time with 20 mL, combine the ether solutions, evaporate to dryness, and add 1 mL of ethyl acetate to dissolve the residue to serve as the test solution.

6. The method according to claim 1, wherein: Preparation of the test solution B: Take 40cm of the analgesic plaster 2 , cut into pieces, remove the cover lining, add ethanol, ultrasonicate, filter, evaporate the filtrate to dryness, add ethanol to dissolve the residue, and use it as the test solution.

7. The method according to claim 1, wherein: Preparation of the test solution B: Take 40cm of the pain-relieving plaster 2 , cut into pieces, remove the cover lining, add 25 mL of ethanol, ultrasonically treat for 1 hour, filter, evaporate the filtrate to dryness, add 1 mL of ethanol to dissolve the residue, and use it as the test solution.

8. The method according to claim 1, wherein: Preparation of the test solution C: Take 2 tablets of Dieda Analgesic Plaster, cut them into pieces respectively, remove the cover lining, add methanol solution, ultrasonically treat, filter, evaporate the filtrate to dryness, dissolve the residue in methanol, and use it as the test solution.

9. The method according to claim 8, characterized in that: The methanol solution is a methanol solution of acetic acid; the concentration of acetic acid in the acetic acid methanol solution is 0.8-2%.

10. The method according to claim 9, characterized in that: The concentration of acetic acid in the acetic acid methanol solution is 1%.

11. The method according to claim 1, wherein: Preparation of the test solution C: Take 2 pieces of Dieda Analgesic Plaster, cut them into pieces respectively, remove the cover lining, add 40 mL of 1% acetic acid methanol solution, ultrasonically treat at 60℃ for 20 minutes, filter, evaporate the filtrate to dryness, and dissolve the residue in 2 mL of 1% acetic acid methanol solution to prepare the test solution.

12. The method according to claim 1, wherein: The volume ratio of n-hexane-ethyl acetate-formic acid is 4-6:0.5-1.5:0.05-0.

15.

13. The method according to claim 12, wherein: The volume ratio of n-hexane-ethyl acetate-formic acid is 5:1:0.

1.

14. The method according to claim 1, wherein: The volume ratio of toluene to ethyl acetate is 1-3:

1.

15. The method according to claim 14, characterized in that: The volume ratio of toluene to ethyl acetate is 2:

1.

16. The method according to claim 1, wherein: The volume ratio of the ethyl acetate, 2-butanone, formic acid and water is 8-12:6-8:1-2:1-2.

17. The method according to claim 16, wherein: The volume ratio of ethyl acetate, 2-butanone, formic acid and water is 10:7:1:

1.

18. Use of the method according to any one of claims 1 to 17 in identifying the components of rhubarb, dalbergia odorifera and phellodendron amurense in external Chinese medicine preparations.

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