A method for thin-layer identification of Linderae odorata from Zhonghua Dieda Pills

Abstract

The present invention relates to the field of thin layer identification of Chinese medicinal materials in traditional Chinese medicine preparations, and in particular to a method for thin layer identification of Chinese herbal medicines and Chinese herbal medicines. It comprises the following steps: 1) crushing Chinese herbal medicines, soaking them in ethanol, ultrasonic extraction, collecting the filtrate, concentrating it, extracting it with petroleum ether, and evaporating it to obtain a residue; 2) dissolving the residue with petroleum ether and eluting it on a silica gel column three times, eluting it with petroleum ether for the first time, eluting it with petroleum ether-ethyl acetate with a volume ratio of 5 to 15:1 for the second time, and eluting it with petroleum ether-ethyl acetate with a volume ratio of 1 to 5:1 for the third time, collecting the third eluate, evaporating it to obtain a test sample; 3) spotting the test sample and the control medicinal material on a silica gel G thin layer plate, and performing thin layer chromatography identification with n-hexane-ethyl acetate as a developing agent. Chinese herbal medicines are compound preparations, and there is mutual influence between the components. The test solution prepared by the present invention can effectively purify the sample, reduce the interference of other components, and improve the accuracy of detection. At the same time, in the thin layer identification chromatogram, the developing agent is optimized, the color spots are clear, and it has strong specificity. The invention is used for quality control of Zhonghua Dieda Pills, ensuring the quality of the medicine and the safety, effectiveness and reliability of clinical medication.

Description

Technical Field

[0001] The invention relates to the field of thin-layer identification of Chinese medicinal materials in traditional Chinese medicine preparations, and particularly relates to a thin-layer identification method for Chinese medicine Dieda Pills Linderae umbellatae. Background Art

[0002] Zhonghua Dieda Pills are made from 32 traditional Chinese medicines. Due to the large number of ingredients and the complex active ingredients, some ingredients cannot be tested, making quality control of Zhonghua Dieda Pills challenging. Therefore, it is essential to further improve quality standards by controlling the composition of the key ingredients in Zhonghua Dieda Pills. Linderae odorata (Linderae odorata) serves as an adjuvant in Zhonghua Dieda Pills, relieving pain, warming the kidneys and dispelling cold, and guiding the medicine to the chest and abdomen. Its main component is linderae ether lactone.

[0003] The TLC identification method for Linderae odorata is documented in relevant standard research documents, including the "Chinese Pharmacopoeia (2015 Edition)", "Study on the Quality Standards for Fuyanxiao Capsules", "Study on the Quality Standards for Fushu Tablets", "Study on the Quality Standards for Liyou Capsules", "Experimental Study on the Quality Standards for Wenwei Mixture", "Supplementary Study on the Quality Standards for Simotang Oral Liquid", and "Study on the Quality Standards for Fufang Qianliening Capsules". These documents primarily describe the TLC identification method for Linderae odorata as a raw material, but do not mention the identification of Linderae odorata in preparations.

[0004] Chinese patent CN102288722A discloses a fluorescence-enhanced thin-layer chromatography (TLC) identification method for Linderae odorata. The steps are as follows: a. Take 0.3 g of powdered Linderae odorata and a control Linderae odorata, add 4 ml of methanol, and sonicate for 10 minutes. The supernatant is used as the test solution and the reference solution. b. 4 μl of each of the test solution and the reference solution are applied to the same GF254 TLC plate. The plate is developed with cyclohexane-ethyl acetate-formic acid in a volume ratio of 8:2:0.2, removed, and air-dried. c. The plate is sprayed with 10% ethanolic sulfuric acid as a fluorescence enhancer and heated to 105°C until the spots are clearly colored. d. The plate is examined under a 365 nm ultraviolet lamp. The chromatogram of the Linderae odorata test sample will show a fluorescent spot of the same color at the corresponding position in the chromatogram of the Linderae odorata reference sample. However, this method simplifies the preparation of the Linderae odorata test sample. However, Zhonghua Dieda Pills is a traditional Chinese medicine compound preparation with interplay between its components. Simple extraction is prone to interference or incomplete extraction of the active ingredients. Summary of the Invention

[0005] The present invention aims to provide a thin-layer chromatographic identification method for Linderae rutaecarpa of Zhonghua Dieda Pills. By treating the sample using immersion, ultrasonic extraction, extraction, and column chromatography, the interference of other components in Zhonghua Dieda Pills is reduced. Furthermore, the developing agent is adjusted to improve the specificity of the assay.

[0006] The technical solution of the present invention is a method for thin-layer identification of Linderae rutaecarpa of Zhonghua Dieda Pills, characterized by comprising the following steps:

[0007] 1) crushing the Zhonghua Dieda Pills, soaking them in ethanol, and ultrasonically extracting them. The filtrate was collected, concentrated, extracted with petroleum ether, and evaporated to dryness to obtain a residue;

[0008] 2) The residue was dissolved in petroleum ether and eluted on a silica gel column three times, the first elution was with petroleum ether, the second elution was with petroleum ether-ethyl acetate in a volume ratio of 5 to 15:1, and the third elution was with petroleum ether-ethyl acetate in a volume ratio of 1 to 5:1. The third eluate was collected, evaporated to dryness, and the solution was obtained to obtain the test sample;

[0009] 3) Spot the test sample and control medicinal material onto silica gel G thin layer plates respectively and perform thin layer chromatography identification using n-hexane-ethyl acetate as the developing solvent.

[0010] Preferably, in step 1), the boiling range of petroleum ether is 30-60° C., and the extraction is performed 1-5 times;

[0011] Preferably, in step 2), the silica gel is 60-400 mesh, and the elution volume is 50-200 ml;

[0012] Preferably, in step 2), the silica gel is 100-200 mesh, and the elution volume is 100-150 ml;

[0013] Preferably, in step 2), the inner diameter of the silica gel column is 1.0 to 4.0 cm, the height is 5 to 30 cm, and the silica gel column is dry packed;

[0014] Preferably, in step 2), the boiling range specification of the petroleum ether is 30-60° C.;

[0015] Preferably, in step 2), the volume ratio of petroleum ether to ethyl acetate for the second elution is 8 to 12:1;

[0016] Preferably, in step 2), the volume ratio of petroleum ether to ethyl acetate for the third elution is 1 to 3:1;

[0017] Preferably, in the step 2), the evaporation is followed by dissolution with ethyl acetate;

[0018] Preferably, in step 3), the volume ratio of n-hexane to ethyl acetate is 10-20:4;

[0019] Preferably, in step 3), the volume ratio of n-hexane to ethyl acetate is 15 to 18:4;

[0020] Preferably, in step 3), the detection color developer is a sulfuric acid methanol solution, and the detection conditions are: heating at 100-108° C. until the spots are clearly colored.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. Used for quality control of Zhonghua Dieda Pills to ensure drug quality and the safety, effectiveness and reliability of clinical medication.

[0023] 2. Zhonghua Dieda Pills are a compound preparation, and there are interactions between the components. The test solution prepared by the present invention can effectively purify the sample, reduce interference from other components, and improve the accuracy of detection.

[0024] 3. In the thin layer identification chromatogram, the developing agent is optimized, the color spots are clear, and have strong specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Thin layer chromatograms of each batch of test samples in Examples 1 to 9

[0026] Among them, 1 and 11 are thin layer chromatograms of 10 μL and 15 μL sample loading of Linderae umbellatae reference medicinal material, and 2 to 10 are thin layer chromatograms of 10 μL sample loading of Examples 1 to 9, respectively.

[0027] Figure 2 Chromatograms of different extraction solvents

[0028] Among them, 1 and 4 are control herbs, 2 is the test sample (extracted with petroleum ether at 30-60°C), and 3 is the test sample (extracted with petroleum ether at 60-90°C).

[0029] Figure 3 Extraction and impurity removal chromatogram

[0030] Among them, 1 and 4 are control medicinal materials, 2 is the test solution after extraction, and 3 is the test solution without extraction.

[0031] Figure 4 Chromatogram of elution system investigation

[0032] Among them, 1 and 4 are control medicinal materials, 2 is test solution a, and 3 is test solution b.

[0033] Figure 5 Chromatograms of thin layer plates of different materials

[0034] Among them, the left one is GF 254 Plates, the middle one is G plate, the one on the right is H plate, 1 is the control medicinal material, and 2 is the test solution.

[0035] Figure 6 Chromatograms of thin layer plates of different brands

[0036] Among them, the left picture is a thin layer plate provided by Ocean Chemical, the middle picture is a thin layer plate provided by Merck, and the right picture is a thin layer plate provided by Bangkai. 1 is the control medicinal material, and 2 is the test sample solution.

[0037] Figure 7 Chromatograms of different developing agents

[0038] Among them, a is cyclohexane:ethyl acetate:formic acid (10:1:0.1), b is toluene:ethyl acetate (20:1), c is n-hexane:ethyl acetate (17:4), d is petroleum ether:ethyl acetate (17:4), e is cyclohexane:ethyl acetate:formic acid (5:1:0.1), and f is n-hexane:ethyl acetate (6:1); 1 is the control medicinal material, and 2 is the test solution.

[0039] Figure 8 Chromatographic analysis of colorimetric reagents

[0040] Among them, a is 5% vanillin ethanol solution, b is 5% phosphomolybdic acid solution, c is dinitrophenylhydrazine ethanol solution, d is 3% aluminum chloride ethanol solution, e is 10% sulfuric acid methanol solution, f is 5% vanillin ethanol solution-10% sulfuric acid methanol solution (1:20); 1 is the control medicinal material, and 2 is the test solution.

[0041] Figure 9 Chromatograms at different temperatures

[0042] The left picture is the chromatogram at 8.3℃, the middle picture is the chromatogram at 21.7℃, and the right picture is the chromatogram at 40℃; 1 is the control medicinal material, and 2 is the test solution. DETAILED DESCRIPTION

[0043] The following examples are used to further illustrate the present invention, but the present invention is not limited to these examples.

[0044] Example 1

[0045] 1) Preparation of test sample:

[0046] The Zhonghua Dieda Pills were crushed, 12 g was weighed, 100 ml of 80% by volume ethanol was added, cold soaked for 12 h, ultrasonically treated for 30 min, filtered, evaporated to dryness, dispersed in 50 ml of water, extracted once with petroleum ether with a boiling range of 30-60 ° C, 30 ml each time, the petroleum ether extracts were combined, evaporated to dryness, and a residue was obtained.

[0047] Then, dissolve the residue in 1 ml of petroleum ether with a boiling range of 30-60°C, add 1 g of 60-mesh silica gel, mix well, evaporate the solvent, and place it on a 60-mesh silica gel column with an inner diameter of 1.0 cm and a height of 5 cm. Dry pack the column and elute with 50 ml of petroleum ether, then 50 ml of a 5:1 petroleum ether-ethyl acetate volume ratio, and finally 50 ml of a 1:1 petroleum ether-ethyl acetate volume ratio. Collect the third eluate, evaporate to dryness, and dissolve it in 2 ml of ethyl acetate to prepare the test solution.

[0048] 2) Preparation of Linderae serrata control medicinal material:

[0049] Take 0.5 g of Linderae umbellatae control medicinal material, add 30 ml of 80% by volume ethanol, and prepare the control medicinal material solution in the same way as the test solution.

[0050] 3) Detection:

[0051] Pipette the test solution and the control medicinal material solution onto the silica gel G thin layer plate respectively, use n-hexane-ethyl acetate with a volume ratio of 10:4 as the developing solvent and 10% sulfuric acid methanol solution as the color developer, heat at 100℃ until the spots are clearly colored, and inspect at 365nm or by transmission inspection.

[0052] Example 2

[0053] 1) Preparation of test sample:

[0054] The Zhonghua Dieda Pills were crushed, 12 g was weighed, 100 ml of 80% by volume ethanol was added, cold soaked for 24 h, ultrasonically treated for 30 min, filtered, evaporated to dryness, dispersed in 50 ml of water, extracted five times with petroleum ether with a boiling range of 30-60 ° C, 30 ml each time, the petroleum ether extracts were combined, evaporated to dryness, and a residue was obtained.

[0055] Then, dissolve the residue in 10 ml of petroleum ether with a boiling range of 30-60°C, add 5 g of 400-mesh silica gel, mix well, evaporate the solvent, and place it on a 400-mesh silica gel column with an inner diameter of 4.0 cm and a height of 30 cm. Dry pack the column and elute with 200 ml of petroleum ether, then 200 ml of a 15:1 volume ratio of petroleum ether-ethyl acetate, and finally 200 ml of a 5:1 volume ratio of petroleum ether-ethyl acetate. Collect the third eluate, evaporate to dryness, and dissolve it in 2 ml of ethyl acetate to prepare the test solution.

[0056] 2) Preparation of Linderae serrata control medicinal material:

[0057] Take 0.5 g of Linderae umbellatae control medicinal material, add 30 ml of 80% by volume ethanol, and prepare the control medicinal material solution in the same way as the test solution.

[0058] 3) Detection:

[0059] Pipette the test solution and the control medicinal material solution onto the silica gel G thin layer plate respectively, use n-hexane-ethyl acetate with a volume ratio of 20:4 as the developing solvent and 10% sulfuric acid methanol solution as the color developer, heat at 108℃ until the spots are clearly colored, and inspect at 365nm or transmission light.

[0060] Example 3

[0061] 1) Preparation of test sample:

[0062] The Zhonghua Dieda Pills were crushed, 12 g was weighed, 100 ml of 80% by volume ethanol was added, cold soaked for 18 h, ultrasonically treated for 30 min, filtered, evaporated to dryness, dispersed in 50 ml of water, extracted three times with petroleum ether with a boiling range of 30-60 ° C, 30 ml each time, the petroleum ether extracts were combined, evaporated to dryness, and a residue was obtained.

[0063] Then, dissolve the residue in 5 ml of petroleum ether with a boiling range of 30-60°C, add 2.5 g of 100-mesh silica gel, mix well, evaporate the solvent, and place it on a 100-mesh silica gel column with an inner diameter of 2.0 cm and a height of 15 cm. Dry pack the column and elute it first with 100 ml of petroleum ether, then with 100 ml of a petroleum ether-ethyl acetate ratio of 8:1 by volume, and finally with 100 ml of a petroleum ether-ethyl acetate ratio of 3:1 by volume. Collect the third eluate, evaporate to dryness, and dissolve it in 2 ml of ethyl acetate to prepare the test solution.

[0064] 2) Preparation of Linderae serrata control medicinal material:

[0065] Take 0.5 g of Linderae umbellatae control medicinal material, add 30 ml of 80% by volume ethanol, and prepare the control medicinal material solution in the same way as the test solution.

[0066] 3) Detection:

[0067] Pipette the test solution and the control medicinal material solution onto the silica gel G thin layer plate respectively, use n-hexane-ethyl acetate with a volume ratio of 15:4 as the developing solvent and 10% sulfuric acid methanol solution as the color developer, heat at 105℃ until the spots are clearly colored, and inspect at 365nm or transmission light.

[0068] Example 4

[0069] 1) Preparation of test sample:

[0070] The Zhonghua Dieda Pills were crushed, 12 g was weighed, 100 ml of 80% by volume ethanol was added, cold soaked for 20 h, ultrasonically treated for 30 min, filtered, evaporated to dryness, dispersed in 50 ml of water, extracted four times with petroleum ether with a boiling range of 30-60 ° C, 30 ml each time, the petroleum ether extracts were combined, evaporated to dryness, and a residue was obtained.

[0071] Then, dissolve the residue in 8 ml of petroleum ether with a boiling range of 30-60°C, add 1.5 g of 200-mesh silica gel, mix well, evaporate the solvent, and place it on a 200-mesh silica gel column with an inner diameter of 2.0 cm and a height of 20 cm. Dry pack the column and elute with 150 ml of petroleum ether, then 150 ml of a 12:1 petroleum ether-ethyl acetate volume ratio, and finally 100 ml of a 3:1 petroleum ether-ethyl acetate volume ratio. Collect the third eluate, evaporate to dryness, and dissolve it in 2 ml of ethyl acetate to prepare the test solution.

[0072] 2) Preparation of Linderae serrata control medicinal material:

[0073] Take 0.5 g of Linderae umbellatae control medicinal material, add 30 ml of 80% by volume ethanol, and prepare the control medicinal material solution in the same way as the test solution.

[0074] 3) Detection:

[0075] Pipette the test solution and the control medicinal material solution onto the silica gel G thin layer plate respectively, use n-hexane-ethyl acetate with a volume ratio of 18:4 as the developing solvent and 10% sulfuric acid methanol solution as the color developer, heat at 106℃ until the spots are clearly colored, and inspect at 365nm or transmission light.

[0076] Example 5

[0077] 1) Preparation of test sample:

[0078] The Zhonghua Dieda Pills were crushed, 12 g was weighed, 100 ml of 80% by volume ethanol was added, cold soaked for 14 h, ultrasonically treated for 30 min, filtered, evaporated to dryness, dispersed in 50 ml of water, extracted twice with petroleum ether with a boiling range of 30-60 ° C, 30 ml each time, the petroleum ether extracts were combined, evaporated to dryness, and a residue was obtained.

[0079] Then, dissolve the residue in 2 ml of petroleum ether with a boiling range of 30-60°C, add 1.5 g of 150-mesh silica gel, mix well, evaporate the solvent, and place it on a 150-mesh silica gel column with an inner diameter of 2.0 cm and a height of 20 cm. Dry pack the column and elute first with 80 ml of petroleum ether, then with 80 ml of a 10:1 volume ratio of petroleum ether-ethyl acetate, and finally with 80 ml of a 2:1 volume ratio of petroleum ether-ethyl acetate. Collect the third eluate, evaporate to dryness, and dissolve it in 2 ml of ethyl acetate to prepare the test solution.

[0080] 2) Preparation of Linderae serrata control medicinal material:

[0081] Take 0.5 g of Linderae umbellatae control medicinal material, add 30 ml of 80% by volume ethanol, and prepare the control medicinal material solution in the same way as the test solution.

[0082] 3) Detection:

[0083] Pipette the test solution and the control medicinal material solution onto the silica gel G thin layer plate respectively, use n-hexane-ethyl acetate with a volume ratio of 17:4 as the developing solvent and 10% sulfuric acid methanol solution as the color developer, heat at 105℃ until the spots are clearly colored, and inspect at 365nm or transmission light.

[0084] Example 6

[0085] 1) Preparation of test sample:

[0086] The Zhonghua Dieda Pills were crushed, 12 g was weighed, 100 ml of 80% by volume ethanol was added, cold soaked for 20 h, ultrasonically treated for 30 min, filtered, evaporated to dryness, dispersed in 50 ml of water, extracted four times with petroleum ether with a boiling range of 30-60 ° C, 30 ml each time, the petroleum ether extracts were combined, evaporated to dryness, and a residue was obtained.

[0087] Then, dissolve the residue in 8 ml of petroleum ether with a boiling range of 30-60°C, add 4 g of 180-mesh silica gel, mix well, evaporate the solvent, and place it on a 150-mesh silica gel column with an inner diameter of 3.0 cm and a height of 15 cm. Dry pack the column and elute with 150 ml of petroleum ether, then 180 ml of a 10:1 volume ratio of petroleum ether-ethyl acetate, and finally 100 ml of a 2:1 volume ratio of petroleum ether-ethyl acetate. Collect the third eluate, evaporate to dryness, and dissolve it in 2 ml of ethyl acetate to prepare the test solution.

[0088] 2) Preparation of Linderae serrata control medicinal material:

[0089] Take 0.5 g of Linderae umbellatae control medicinal material, add 30 ml of 80% by volume ethanol, and prepare the control medicinal material solution in the same way as the test solution.

[0090] 3) Detection:

[0091] Pipette the test solution and the control medicinal material solution onto the silica gel G thin layer plate respectively, use n-hexane-ethyl acetate with a volume ratio of 16:4 as the developing solvent and 10% sulfuric acid methanol solution as the color developer, heat at 102℃ until the spots are clearly colored, and inspect at 365nm or transmission light.

[0092] Example 7

[0093] 1) Preparation of test sample:

[0094] The Zhonghua Dieda Pills were crushed, 12 g was weighed, 100 ml of 80% by volume ethanol was added, cold soaked for 22 h, ultrasonically treated for 30 min, filtered, evaporated to dryness, dispersed in 50 ml of water, extracted twice with petroleum ether with a boiling range of 30-60 ° C, 30 ml each time, the petroleum ether extracts were combined, evaporated to dryness, and a residue was obtained.

[0095] Then, dissolve the residue in 8 ml of petroleum ether with a boiling range of 30-60°C, add 4 g of 150-mesh silica gel, mix well, evaporate the solvent, and place it on a 250-mesh silica gel column with an inner diameter of 3.0 cm and a height of 25 cm. Dry pack the column and elute first with 180 ml of petroleum ether, then with 170 ml of a petroleum ether-ethyl acetate ratio of 11:1 by volume, and finally with 170 ml of a petroleum ether-ethyl acetate ratio of 4:1 by volume. Collect the third eluate, evaporate to dryness, and dissolve it in 2 ml of ethyl acetate to prepare the test solution.

[0096] 2) Preparation of Linderae serrata control medicinal material:

[0097] Take 0.5 g of Linderae umbellatae control medicinal material, add 30 ml of 80% by volume ethanol, and prepare the control medicinal material solution in the same way as the test solution.

[0098] 3) Detection:

[0099] Pipette the test solution and the control medicinal material solution onto the silica gel G thin layer plate respectively, use n-hexane-ethyl acetate with a volume ratio of 14:4 as the developing solvent and 10% sulfuric acid methanol solution as the color developer, heat at 106℃ until the spots are clearly colored, and inspect at 365nm or transmission light.

[0100] Example 8

[0101] 1) Preparation of test sample:

[0102] The Zhonghua Dieda Pills were crushed, 12 g was weighed, 100 ml of 80% by volume ethanol was added, cold soaked for 14 h, ultrasonically treated for 30 min, filtered, evaporated to dryness, dispersed in 50 ml of water, extracted twice with petroleum ether with a boiling range of 30-60 ° C, 30 ml each time, the petroleum ether extracts were combined, evaporated to dryness, and a residue was obtained.

[0103] Then, dissolve the residue in 2 ml of petroleum ether with a boiling range of 30-60°C, add 1.5 g of 150-mesh silica gel, mix well, evaporate the solvent, and place it on a 150-mesh silica gel column with an inner diameter of 2.0 cm and a height of 20 cm. Dry pack the column and elute with 80 ml of petroleum ether, then with 80 ml of an 8:1 petroleum ether-ethyl acetate volume ratio, and finally with 80 ml of a 4:1 petroleum ether-ethyl acetate volume ratio. Collect the third eluate, evaporate to dryness, and dissolve it in 2 ml of ethyl acetate to prepare the test solution.

[0104] 2) Preparation of Linderae serrata control medicinal material:

[0105] Take 0.5 g of Linderae umbellatae control medicinal material, add 30 ml of 80% by volume ethanol, and prepare the control medicinal material solution in the same way as the test solution.

[0106] 3) Detection:

[0107] Pipette the test solution and the control medicinal material solution onto the silica gel G thin layer plate respectively, use n-hexane-ethyl acetate with a volume ratio of 12:4 as the developing solvent and 10% sulfuric acid methanol solution as the color developer, heat at 105℃ until the spots are clearly colored, and inspect at 365nm or transmission light.

[0108] Example 9

[0109] 1) Preparation of test sample:

[0110] The Zhonghua Dieda Pills were crushed, 12 g was weighed, 100 ml of 80% by volume ethanol was added, cold soaked for 14 h, ultrasonically treated for 30 min, filtered, evaporated to dryness, dispersed in 50 ml of water, extracted twice with petroleum ether with a boiling range of 30-60 ° C, 30 ml each time, the petroleum ether extracts were combined, evaporated to dryness, and a residue was obtained.

[0111] Then, dissolve the residue in 2 ml of petroleum ether with a boiling range of 30-60°C, add 1.5 g of 150-mesh silica gel, mix well, evaporate the solvent, and place it on a 150-mesh silica gel column with an inner diameter of 2.0 cm and a height of 20 cm. Dry pack the column and elute with 80 ml of petroleum ether, then with 80 ml of a 12:1 petroleum ether-ethyl acetate volume ratio, and finally with 80 ml of a 3:1 petroleum ether-ethyl acetate volume ratio. Collect the third eluate, evaporate to dryness, and dissolve it in 2 ml of ethyl acetate to prepare the test solution.

[0112] 2) Preparation of Linderae serrata control medicinal material:

[0113] Take 0.5 g of Linderae umbellatae control medicinal material, add 30 ml of 80% by volume ethanol, and prepare the control medicinal material solution in the same way as the test solution.

[0114] 3) Detection:

[0115] Pipette the test solution and the control medicinal material solution onto the silica gel G thin layer plate respectively, use n-hexane-ethyl acetate with a volume ratio of 18:4 as the developing solvent and 10% sulfuric acid methanol solution as the color developer, heat at 105℃ until the spots are clearly colored, and inspect at 365nm or transmission light.

[0116] The results of Examples 1 to 9 are shown in Figure 1 .

[0117] The results show that each batch of test solution has a corresponding blue fluorescent spot at the corresponding position of the reference solution chromatogram. The homogeneity of each batch of samples is good.

[0118] At the same time, the present invention carries out a series of condition optimization, including: extraction solvent, extraction and impurity removal, elution system, thin layer plates of different materials, developing agent, color developer, temperature, etc.

[0119] 1. Investigation of different extraction solvents

[0120] Preparation of test solution: (1) Take 12g of chopped Zhonghua Dieda Pills, add 100ml of 80% ethanol, soak for 12-24 hours, continue ultrasonic treatment (keep the water temperature below 30℃) for 30 minutes, centrifuge, take the supernatant and evaporate to dryness, add 50ml of water to the residue, extract with petroleum ether (30-60℃) three times, 30ml each time, combine the petroleum ether extracts, and evaporate to dryness. Add 2ml of petroleum ether (30-60℃) to dissolve the residue, add 1.5g of silica gel (100-200 mesh), mix well, evaporate the solvent, and load onto a silica gel column (100-200 mesh, inner diameter 2.0cm, height 20cm, dry packing). Elute with petroleum ether (30-60℃), petroleum ether (30-60℃)-ethyl acetate (20:1), and petroleum ether (30-60℃)-ethyl acetate (10:1) in sequence (discard). Finally, elute with petroleum ether (30-60°C)-ethyl acetate (2:1), collect 100 ml of the eluate, evaporate to dryness, and dissolve the residue in 2 ml of ethyl acetate to serve as the test sample (petroleum ether (30-60°C)).

[0121] (2) Take 12g of chopped Zhonghua Dieda Pills and add 100ml of 80% ethanol to each of the two mixtures. Cold soak for 12-24 hours. Continue ultrasonic treatment (keep the water temperature below 30°C) for 30 minutes. Centrifuge, take the supernatant and evaporate to dryness. Disperse the residue in 50ml of water and extract with petroleum ether (60-90°C) three times, 30ml each time. Combine the petroleum ether (60-90°C) extracts and evaporate to dryness. Dissolve the residue in 2ml of petroleum ether (60-90°C), add 1.5g of silica gel (100-200 mesh), mix well, evaporate the solvent, and load onto a silica gel column (100-200 mesh, inner diameter 2.0cm, height 20cm, dry packing). Elute with 100ml of petroleum ether (60-90°C), petroleum ether (60-90°C)-ethyl acetate (20:1), and petroleum ether (60-90°C)-ethyl acetate (10:1) in sequence (discard). Finally, elute with petroleum ether (60-90°C)-ethyl acetate (2:1), collect 100 ml of the eluate, evaporate to dryness, and dissolve the residue in 2 ml of ethyl acetate to serve as the test sample (petroleum ether (60-90°C)).

[0122] Preparation of control medicinal material solution: Take 0.5 g of Linderae umbellatae control medicinal material, add 30 ml of 80% ethanol, and prepare the control medicinal material solution in the same way.

[0123] Developing solvent: petroleum ether (60-90°C)-ethyl acetate (17:4)

[0124] Color developer: spray 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clear. Figure 2 .

[0125] The results showed that the sample prepared with petroleum ether (60-90°C) had almost no spots, while the sample prepared with petroleum ether (30-60°C) had clear and obvious spots. Therefore, petroleum ether (30-60°C) was selected as the extraction solvent.

[0126] 2. Investigation of extraction and impurity removal

[0127] Preparation of test solution: Take two portions of chopped Zhonghua Dieda Pills, 12g each, add 100ml of 80% ethanol to each portion, soak for 12-24 hours, continue ultrasonic treatment (keep the water temperature below 30°C) for 30 minutes, centrifuge, take the supernatant and evaporate to dryness, disperse the residue in 50ml of water, extract one portion with petroleum ether (30-60°C) three times, 30ml each time, combine the petroleum ether extracts, evaporate to dryness, and do not extract the other portion. Perform the same treatment in the following steps, dissolve the residue in 2ml of petroleum ether (30-60°C), add 1.5g of silica gel (100-200 mesh), mix well, evaporate the solvent, and load onto a silica gel column (100-200 mesh, inner diameter 2.0cm, height 20cm, dry packing). Sequentially elute with petroleum ether (30-60°C), petroleum ether (30-60°C)-ethyl acetate (20:1), and petroleum ether (30-60°C)-ethyl acetate (10:1) (100 ml) (discard). Finally, elute with petroleum ether (30-60°C)-ethyl acetate (2:1). Collect 100 ml of the eluate, evaporate to dryness, and dissolve the residue in 2 ml of ethyl acetate. The extracted sample is used as the test sample (extraction), and the unextracted sample is used as the test sample (no extraction).

[0128] Preparation of control medicinal material solution: Take 0.5 g of Linderae umbellatae control medicinal material, add 30 ml of 80% ethanol, and prepare the control medicinal material solution in the same way.

[0129] Developing solvent: petroleum ether (60-90°C)-ethyl acetate (17:4)

[0130] Color developer: spray 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clear. Figure 3 .

[0131] As a result, the sample without extraction treatment had almost no spots, while the sample with extraction treatment had clear and obvious spots, so extraction treatment was chosen.

[0132] 3. Elution system investigation

[0133] Preparation of test solution: Take two portions of chopped Zhonghua Dieda Pills, 12 g each, and add 100 ml of 80% ethanol to each portion. Cold soak for 12 to 24 hours. Continue ultrasonic treatment (maintain the water temperature below 30°C) for 30 minutes. Centrifuge, remove the supernatant, evaporate to dryness, disperse the residue in 50 ml of water, and extract with petroleum ether (30 to 60°C) three times, 30 ml each time. Combine the petroleum ether extracts and evaporate to dryness. Dissolve the residue in 2 ml of petroleum ether (30 to 60°C), add 1.5 g of silica gel (100 to 200 mesh), mix well, evaporate the solvent, and load onto a silica gel column (100 to 200 mesh, 2.0 cm inner diameter, 20 cm height, dry packing). One portion was eluted sequentially with 100 ml of petroleum ether (30-60°C), 100 ml of petroleum ether (30-60°C)-ethyl acetate (20:1), and 100 ml of petroleum ether (30-60°C)-ethyl acetate (10:1). The solution was discarded and then eluted again with 100 ml of petroleum ether (30-60°C)-ethyl acetate (2:1). The eluate was collected and evaporated to dryness. The residue was dissolved in 2 ml of ethyl acetate and used as test solution a. The other portion was eluted sequentially with 100 ml of petroleum ether (30-60°C), 100 ml of petroleum ether (30-60°C)-ethyl acetate (10:1), discarded and then eluted again with 100 ml of petroleum ether (30-60°C)-ethyl acetate (2:1). The eluate was collected and evaporated to dryness. The residue was dissolved in 2 ml of ethyl acetate and used as test solution b.

[0134] Preparation of control medicinal material solution: Take 0.5 g of Linderae umbellatae control medicinal material, add 30 ml of 80% ethanol, and prepare the control medicinal material solution in the same way.

[0135] Developing solvent: petroleum ether (60-90°C)-ethyl acetate (17:4)

[0136] Color developer: spray 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clear. Figure 4 .

[0137] As a result, the spots of sample a and sample b were clearer and more obvious, indicating that reducing the eluent of petroleum ether (30-60℃)-ethyl acetate (20:1) did not affect the thin layer identification effect. Therefore, the elution systems were selected as petroleum ether (30-60℃) 100ml, petroleum ether (30-60℃)-ethyl acetate (10:1) 100ml, and petroleum ether (30-60℃)-ethyl acetate (2:1) elution, and the petroleum ether (30-60℃)-ethyl acetate (2:1) eluent was collected.

[0138] A comprehensive analysis of the results of the investigation of the preparation methods of all test samples was conducted, and the preparation method of the thin-layer identification test sample of Zhonghua Dieda Pills was determined: 12 g of chopped Zhonghua Dieda Pills were added with 100 ml of 80% ethanol respectively, cold soaked for 12 to 24 hours, and continued ultrasonic treatment (keeping the water temperature below 30°C) for 30 minutes, centrifuged, the supernatant was evaporated to dryness, the residue was dispersed in 50 ml of water, extracted with petroleum ether (30 to 60°C) for 3 times, 30 ml each time, the petroleum ether extracts were combined, and evaporated to dryness. Dissolve the residue in 2 ml of petroleum ether (30-60°C), add 1.5 g of silica gel (100-200 mesh), mix well, evaporate the solvent, and load onto a silica gel column (100-200 mesh, 2.0 cm inner diameter, 20 cm height, dry packing). Elute with 100 ml of petroleum ether (30-60°C), then 100 ml of petroleum ether (30-60°C)-ethyl acetate (10:1), discard the solution, and then elute with 100 ml of petroleum ether (30-60°C)-ethyl acetate (2:1). Collect the eluate and evaporate to dryness. Dissolve the residue in 2 ml of ethyl acetate to prepare the test solution.

[0139] 4. Thin layer board inspection

[0140] Investigation of thin layer plates of different materials

[0141] Take the reference medicinal material solution of Linderae atractylodes and the test solution and use silica gel G plate, H plate and GF plate respectively. 254 Three types of thin layer plates were developed. The effects of different brands of thin layer plates on thin layer separation were investigated. The results are shown in Figure 5 .

[0142] As a result, the spots on the three thin layer plates of different materials were clear and obvious, and the separation of spots on the G plate was slightly better than that of the other two thin layer plates, so the silica gel G plate was selected.

[0143] Inspection of different brands of thin layer boards

[0144] The control medicinal material solution of Linderae rutinae and the test sample solution were developed using three brands of thin layer plates: Silica G plate from Qingdao Ocean Chemical Co., Ltd., Silica G plate from Merck, and Silica G plate from Qingdao Bangkai Separation Materials Co., Ltd. The effects of different brands of thin layer plates on thin layer separation were investigated. The results are shown in Figure 6 .

[0145] The results showed that the spots on the three different brands of thin layer plates were clear and obvious, and the identification pattern outlines were basically the same, indicating that different brands of thin layer plates did not affect the identification effect of Linderae odorifera thin layer.

[0146] 5. Investigation of different developing agents

[0147] Take the control medicinal material solution of Linderae rutinae and the test solution, and use different developing agents to develop them respectively to investigate the effect of different developing agents on the thin layer separation effect. Figure 7 .

[0148] The results showed that the developing agent ethyl acetate-acetone-formic acid-water (16:2:2:1) was too polar. The developing agents cyclohexane:ethyl acetate:formic acid (5:1:0.1), cyclohexane:ethyl acetate:formic acid (10:1:0.1), toluene:ethyl acetate (20:1), and n-hexane:ethyl acetate (6:1) could not completely separate the characteristic spots from other impurity spots. Only the developing agent n-hexane:ethyl acetate (17:4) had the best separation effect, so n-hexane:ethyl acetate (17:4) was selected as the developing agent.

[0149] 6. Color development agent inspection

[0150] The control medicinal material solution of Linderae rutaecarpa and the test sample solution were developed with different color developers. The effects of different color developers on the thin layer separation effect were investigated. Figure 8 .

[0151] The results showed that the color development effects of 5% vanillin ethanol solution, 5% phosphomolybdic acid solution, dinitrophenylhydrazine ethanol solution, and 3% aluminum chloride ethanol were extremely poor, while the color development effects of 10% sulfuric acid methanol solution and 5% vanillin ethanol solution-10% sulfuric acid methanol solution (1:20) were almost the same. However, the preparation method of the color developer 10% sulfuric acid methanol solution is relatively simple, so the color developer 10% sulfuric acid methanol solution was selected.

[0152] 7. Effect of different temperatures

[0153] The control medicinal material solution of Linderae rutinae and the test solution were developed under different laboratory temperature conditions. The effect of different laboratory temperature conditions on the thin layer separation effect was investigated. Figure 9 .

[0154] The results showed that there was no significant difference in the TLC profiles between the control herbal medicine and the test sample solutions developed under different laboratory temperature conditions.

Claims

1. A method for thin-layer identification of Linderae rutaecarpa of Zhonghua Dieda Pills, characterized in that: The steps include: 1) crushing the Zhonghua Dieda Pills, soaking them in ethanol, and ultrasonically extracting them. The filtrate was collected, concentrated, extracted with petroleum ether, and evaporated to dryness to obtain a residue; 2) The residue was dissolved in petroleum ether and eluted on a silica gel column three times, the first elution was with petroleum ether, the second elution was with petroleum ether-ethyl acetate in a volume ratio of 5 to 15:1, and the third elution was with petroleum ether-ethyl acetate in a volume ratio of 1 to 5:

1. The third eluate was collected, evaporated to dryness, and the solution was obtained to obtain the test sample; 3) Spot the test sample and control medicinal material onto a silica gel G thin layer plate and perform thin layer chromatography using n-hexane-ethyl acetate as the developing solvent; In the step 3), the volume ratio of n-hexane to ethyl acetate is 10 to 20:4; In the step 3), the detection color developing agent is a sulfuric acid methanol solution, and the detection conditions are: heating at 100-108° C. until the spots are clearly colored.

2. The method for thin-layer identification of Linderae rutaecarpa of Zhonghua Dieda Pills according to claim 1, characterized in that: In the step 1), the boiling range of the petroleum ether is 30-60° C., and the extraction is performed 1-5 times.

3. The method for thin-layer identification of Linderae rutaecarpa of Zhonghua Dieda Pills according to claim 1, characterized in that: In the step 2), the silica gel has a mesh size of 60 to 400, and the elution volume is 50 to 200 ml.

4. The method for thin-layer identification of Linderae rutaecarpa of Zhonghua Dieda Pills according to claim 1, characterized in that: In the step 2), the silica gel has a mesh size of 100 to 200, and the elution volume is 100 to 150 ml.

5. The method for thin-layer identification of Linderae rutaecarpa of Zhonghua Dieda Pills according to claim 1, characterized in that: In the step 2), the inner diameter of the silica gel column is 1.0 to 4.0 cm, and the height is 5 to 30 cm. The silica gel column is dry packed.

6. The method for thin-layer identification of Linderae rutaecarpa of Zhonghua Dieda Pills according to claim 1, characterized in that: In the step 2), the boiling range of the petroleum ether is 30-60°C.

7. The method for thin-layer identification of Linderae rutaecarpa of Zhonghua Dieda Pills according to claim 1, characterized in that: In the step 2), the volume ratio of petroleum ether to ethyl acetate for the second elution is 8 to 12:

1.

8. The method for thin-layer identification of Linderae rutaecarpa of Zhonghua Dieda Pills according to claim 1, characterized in that: In the step 2), the volume ratio of petroleum ether to ethyl acetate for the third elution is 1 to 3:

1.

9. The method for thin-layer identification of Linderae rutaecarpa of Zhonghua Dieda Pills according to claim 1, characterized in that: In the step 2), the evaporation is followed by dissolution with ethyl acetate.

10. The method for thin-layer identification of Linderae rutaecarpa of Zhonghua Dieda Pills according to claim 1, characterized in that: In the step 3), the volume ratio of n-hexane to ethyl acetate is 15 to 18:4.

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