A method for identifying traditional Chinese medicinal materials in cross-seating by thin layer chromatography

Abstract

The present invention relates to the field of thin layer identification of Chinese medicinal materials, and specifically to a method for thin layer identification of Hengjingxi. The method comprises the following steps: 1) pre-treating the sample to be identified and the control medicinal material to obtain a test solution and a control medicinal material solution respectively; 2) spotting the test solution and the control medicinal material solution on the same thin layer plate respectively, and performing thin layer chromatography identification using a developing agent. In the past, the properties of Hengjingxi Chinese medicinal materials were identified based solely on experience, and the accuracy and reliability of this method were not high. The present invention, through studying the thin layer spectrum of Hengjingxi, discovered a method for identifying Hengjingxi through thin layer chromatography. By using the developing agent and the color developer of the present invention, the color spots are clear, and the specificity and reproducibility are good. The present invention is used for quality control of Hengjingxi Chinese medicinal materials to ensure the quality of the medicines and the safety of clinical use.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of thin layer identification of traditional Chinese medicinal materials, and particularly relates to a thin layer chromatography identification method for Hengjingchi traditional Chinese medicinal materials. BACKGROUND

[0002] Hengjingchi is the dried whole plant of Pachyrrhizus ziziphioides Hance. The main root is long conical or cylindrical, with uneven thickness, brown to light brownish red surface, and fine longitudinal wrinkles. The bark peeling off part is brownish red. The stem is cylindrical, with grayish green to grayish brown surface. The young branch is quadrangular with wings, yellowish green. The single leaf is opposite, long oval or lanceolate, 6-12 cm long and 1.5-4 cm wide, yellowish green to grayish green, shiny on both sides, glabrous, with acute or cuspidate or caudate acute apex, cuneate base, entire margin, slightly revolute, raised midrib on both sides, and many fine and neat lateral veins nearly vertical to the midrib. The drupe sometimes can be seen in the leaf axil, long oval, about 8 mm in diameter. It has a slight odor and a bitter and astringent taste.

[0003] For a long time, people only identify the characteristics of Hengjingchi by experience, and the related standards of Hengjingchi do not record other identification methods, so there are certain difficulties in the quality control of Hengjingchi traditional Chinese medicinal materials. Therefore, it is of great significance to study an accurate, specific and reproducible detection method for the identification of Hengjingchi traditional Chinese medicinal materials.

[0004] A method for microscopic identification and thin layer chromatography identification of Hengjingchi in medicinal Ai strip is disclosed in "Microscopic Identification and Thin Layer Chromatography Identification of Medicinal Ai Strip", Medicine Frontier, 2017, 7(35): 321-323. The thin layer chromatography identification steps are as follows: taking medicinal Ai strip, tearing off the cotton paper, weighing 10 g of the content, adding 30 ml of methanol, tightly sealing, ultrasonic treating for 30 min, filtering, evaporating the filtrate, adding 1 ml of methanol to the residue to dissolve it to prepare a test sample solution. Preparing medicinal Ai strip without Hengjingchi according to the prescription ratio, taking 10 g, and preparing a negative sample solution lacking Hengjingchi by the same method. Weighing about 0.4 mg of gambogic acid reference substance, adding 2 ml of methanol to prepare a solution containing 0.2 mg per 1 ml as a reference solution. According to the thin layer chromatography method, respectively taking 20 μl of the test sample, 10 μl of the reference solution, and 20 μl of the negative sample solution, and spotting them on the same silica gel G thin layer plate. Using chloroform-methanol-diethylamine (15:1:1) as the developing agent, developing, taking out, drying, placing in an oven at 105℃ for about 20 min, and observing under ultraviolet light (365 nm). But the method has unclear color spots, the developing effect is not good when identifying single Hengjingchi medicinal material, and it cannot distinguish the root, stem, branch and leaf of Hengjingchi.

[0005] The fingerprint of Mallotus philippinensis Muell. Arg. is disclosed in the Journal of Hainan Normal University (Natural Science Edition), 2006(03): 239-242. The steps are as follows: 3 portions of crushed samples of Mallotus philippinensis Muell. Arg. leaves and branches are respectively weighed, each 1.0 g, wrapped with filter paper, and loaded into a Soxhlet extractor, about 200 mL of w=95% ethanol, petroleum ether and ethyl acetate are respectively added, and refluxed until the extract is colorless, and the solvent is recovered under reduced pressure to about 30 mL. The developing agent of the w=95% ethanol extract of Mallotus philippinensis Muell. Arg. is respectively: leaves petroleum ether-ethyl acetate-acetone (7.5:2.5:2.5), branches petroleum ether-acetone (5.0:2.5). The developing agent of the petroleum ether extract is: leaves petroleum ether-ethyl acetate (10:3.5), branches petroleum ether-ethyl acetate (7.0:2.5). The developing agent of the ethyl acetate extract is: leaves petroleum ether-ethyl acetate-acetone (10.5:4.0:1.0), branches petroleum ether-ethyl acetate-acetone (10.5:4.0:1.0). But the author selects the extraction method and developing agent under the premise of not knowing the chemical composition of Mallotus philippinensis Muell. Arg., only analyzes the branches and leaves, and lacks a control sample, and the specificity is poor. SUMMARY

[0006] The purpose of the present application is to provide a method for identifying Mallotus philippinensis Muell. Arg. by TLC, which improves the specificity of detection by adjusting the developing agent and color developing agent through preliminary treatment of the sample by water extraction or alcohol extraction.

[0007] The technical scheme of the present application is a method for identifying Mallotus philippinensis Muell. Arg. by TLC, characterized by comprising the following steps: 1) pre-treating the sample to be identified and the control medicinal material to obtain the test sample solution and the control medicinal material solution; 2) spotting the test sample solution and the control medicinal material solution on the same Merck high-efficiency G plate, performing TLC with a developing agent, spraying with 20% hydrochloric acid ethanol solution after the TLC is completed, heating at 105°C until the spots develop color, and observing under a 365 nm ultraviolet lamp; wherein the developing agent is selected from trichloromethane, ethyl acetate, water, cyclohexane, n-hexane, glacial acetic acid or a mixture thereof.

[0008] Preferably, in step 1), the steps of the pre-treatment method are specifically: taking 1 g of the sample powder to be measured and the control medicinal material powder, respectively, adding 100 ml of purified water, heating and boiling for 30 min, filtering, evaporating the filtrate to dryness, and dissolving with 5 ml of methanol to obtain the test sample solution and the control medicinal material solution.

[0009] Preferably, in step 1), the steps of the pre-treatment method are specifically: taking 1 g of the sample powder to be measured and the control medicinal material powder, respectively, adding 100 ml of purified water, heating and boiling for 30 min, filtering, evaporating the filtrate to dryness, and dissolving with 5 ml of methanol to obtain the test sample solution and the control medicinal material solution.

[0010] Preferably, in the step 1), the pre-treatment method is specifically as follows: 1g of the sample powder and 1g of the control medicinal material powder are respectively added with 30ml of methanol, and then ultrasonic treatment is performed for 30min, filtration is performed, the filtrate is evaporated to dryness, and 5ml of methanol is added for dissolution, so as to obtain the test sample solution and the control medicinal material solution.

[0011] Preferably, in the step 2), the developing agent is a lower solution of chloroform: ethyl acetate: water with a volume ratio of (15-44:22-50:0.5-3), and the sample solution is spotted in an amount of 2ul.

[0012] Preferably, in the step 2), the developing agent is an upper solution of chloroform: ethyl acetate: cyclohexane: water: glacial acetic acid with a volume ratio of (12-30:27-45:30-55:0.1-2:0.1-2), the step 1) is a water extraction process, and the sample solution is spotted in an amount of 3ul; the step 1) is an alcohol extraction process, and the sample solution is spotted in an amount of 2ul.

[0013] Preferably, in the step 2), the developing agent is an upper solution of chloroform: ethyl acetate: cyclohexane: water: glacial acetic acid with a volume ratio of (12-30:27-45:30-55:0.1-2:0.1-2), the step 1) is a water extraction process, and the sample solution is spotted in an amount of 3ul; the step 1) is an alcohol extraction process, and the sample solution is spotted in an amount of 2ul.

[0014] The application also provides a thin layer identification method for different parts of the roots, stems, branches and leaves of the Heteropanax fragilis. The Heteropanax fragilis is taken as the test medicinal material, and a lower solution of chloroform: ethyl acetate: water with a volume ratio of (15-44:22-50:0.5-3) is used as the developing agent for thin layer chromatography. If there are yellow, sky blue and blue spots at RF values of 0.80±0.04, 0.71±0.07 and 0.45±0.05 respectively, the sample is the root of the Heteropanax fragilis; if there is a blue spot at an RF value of 0.26±0.04, the sample is the stem of the Heteropanax fragilis; if there is no brown spot in the range of RF values of 0.8-0.9 in the observation result, the sample is the branch of the Heteropanax fragilis; and if there is a tailing spot at an RF value of 0.2±0.05 in the observation result, the sample is the leaf of the Heteropanax fragilis.

[0015] Preferably, take the medicine to be tested, using the developing agent is the volume ratio (12-30:27-45:30-55:0.1-2:0.1-2) chloroform: ethyl acetate: cyclohexane: water: glacial acetic acid of the upper solution carries on thin layer chromatography; RF value is at 0.65±0.03, 0.54±0.04, 0.27±0.04 respectively there are yellow, sky blue, blue spots, then it is the root of Hengjingxi; RF value is at 0.25±0.05 there is a blue spot, then it is the stem of Hengjingxi; RF value is in the range of 0.7-0.8 without brown spot, then it is the branch of Hengjingxi; RF value is at 0.18±0.04 there is a tailing spot, no corresponding blue spot is the leaf of Hengjingxi.

[0016] Preferably, take the medicine to be tested, using the developing agent is the volume ratio (12-30:27-45:30-50:0.1-2:0.1-2) chloroform: ethyl acetate: n-hexane: water: glacial acetic acid of the upper solution carries on thin layer chromatography; RF value is at 0.65±0.03, 0.54±0.04, 0.27±0.04 respectively there are yellow, sky blue, blue spots, then it is the root of Hengjingxi; RF value is at 0.24±0.04 there is a blue spot, then it is the stem of Hengjingxi; RF value is in the range of 0.7-0.8 without brown spot, then it is the branch of Hengjingxi; carries on thin layer chromatography; RF value is at 0.18±0.04 there is a tailing spot, no corresponding blue spot is the leaf of Hengjingxi.

[0017] The present application has the following advantages: 1. for the quality control of Hengjingxi traditional Chinese medicinal materials, ensure the safety of clinical medication; 2. solve the problem of big error in traditional method identification, improve the accuracy of detection; 3. in the thin layer identification chromatogram, the developing agent and the color developing agent are optimized, the color developing spot is clear, and it has strong specificity. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The thin layer chromatogram of the pre-treatment (1) result of example 2, wherein 1-5 are respectively the root, stem, branch, leaf and whole plant solution of the test sample, and 6 is the control medicinal material solution.

[0019] Figure 2 The thin layer chromatogram of the pre-treatment (2) result of example 2, wherein 1-5 are respectively the root, stem, branch, leaf and whole plant solution of the test sample, and 6 is the control medicinal material solution.

[0020] Figure 3 The thin layer chromatogram of the pre-treatment (1) result of example 3, wherein 1-5 are respectively the root, stem, branch, leaf and whole plant solution of the test sample, and 6 is the control medicinal material solution.

[0021] Figure 4The result of TLC of pre-treatment (1) of Example 3, wherein 1-5 are root, stem, branch, leaf and whole plant solution of the test sample respectively, and 6 is the solution of the control medicinal material.

[0022] Figure 5 The result of TLC of pre-treatment (1) of Example 4, wherein 1-5 are root, stem, branch, leaf and whole plant solution of the test sample respectively, and 6 is the solution of the control medicinal material.

[0023] Figure 6 The result of TLC of pre-treatment (2) of Example 4, wherein 1-5 are root, stem, branch, leaf and whole plant solution of the test sample respectively, and 6 is the solution of the control medicinal material.

[0024] Figure 7 The result of TLC of pre-treatment (1) of Example 5, wherein 1-5 are root, stem, branch, leaf and whole plant solution of the test sample respectively, and 6 is the solution of the control medicinal material.

[0025] Figure 8 The result of TLC of pre-treatment (2) of Example 5, wherein 1-5 are root, stem, branch, leaf and whole plant solution of the test sample respectively, and 6 is the solution of the control medicinal material.

[0026] Figure 9 The result of TLC of pre-treatment (1) of Example 6, wherein 1-5 are root, stem, branch, leaf and whole plant solution of the test sample respectively, and 6 is the solution of the control medicinal material.

[0027] Figure 10 The result of TLC of pre-treatment (2) of Example 6, wherein 1-5 are root, stem, branch, leaf and whole plant solution of the test sample respectively, and 6 is the solution of the control medicinal material.

[0028] Figure 11 The result of TLC of pre-treatment (1) of Example 7, wherein 1-5 are root, stem, branch, leaf and whole plant solution of the test sample respectively, and 6 is the solution of the control medicinal material.

[0029] Figure 12 The result of TLC of pre-treatment (2) of Example 7, wherein 1-5 are root, stem, branch, leaf and whole plant solution of the test sample respectively, and 6 is the solution of the control medicinal material.

[0030] Figure 13 The result of TLC of pre-treatment (1) of Example 8, wherein 1-5 are root, stem, branch, leaf and whole plant solution of the test sample respectively, and 6 is the solution of the control medicinal material.

[0031] Figure 14 The result of TLC of pre-treatment (2) of Example 8, wherein 1-5 are root, stem, branch, leaf and whole plant solution of the test sample respectively, and 6 is the solution of the control medicinal material.

[0032] Figure 15 Pre-treatment (1), thin layer chromatogram with 10% sulfuric acid ethanol as color developing agent, wherein 1 is the test sample whole plant solution, and 2 is the control medicinal material solution.

[0033] Figure 16 Pre-treatment (1), thin layer chromatogram with 20% perchloric acid ethanol as color developing agent, wherein 1 is the test sample whole plant solution, and 2 is the control medicinal material solution.

[0034] Figure 17 Pre-treatment (2), thin layer chromatogram with 10% sulfuric acid ethanol as color developing agent, wherein 1 is the test sample whole plant solution, and 2 is the control medicinal material solution.

[0035] Figure 18 Pre-treatment (2), thin layer chromatogram with 20% perchloric acid ethanol as color developing agent, wherein 1 is the test sample whole plant solution, and 2 is the control medicinal material solution.

[0036] Figure 19 Pre-treatment (1), thin layer chromatogram with G plate as thin layer plate, wherein 1 is the test sample whole plant solution, and 2 is the control medicinal material solution.

[0037] Figure 20 Pre-treatment (1), thin layer chromatogram with H plate as thin layer plate, wherein 1 is the test sample whole plant solution, and 2 is the control medicinal material solution.

[0038] Figure 21 Pre-treatment (1), thin layer chromatogram with Merck high efficiency G plate as thin layer plate, wherein 1 is the test sample whole plant solution, and 2 is the control medicinal material solution.

[0039] Figure 22 Pre-treatment (2), thin layer chromatogram with G plate as thin layer plate, wherein 1 is the test sample whole plant solution, and 2 is the control medicinal material solution.

[0040] Figure 23 Pre-treatment (2), thin layer chromatogram with H plate as thin layer plate, wherein 1 is the test sample whole plant solution, and 2 is the control medicinal material solution.

[0041] Figure 24 Pre-treatment (2), thin layer chromatogram with Merck high efficiency G plate as thin layer plate, wherein 1 is the test sample whole plant solution, and 2 is the control medicinal material solution.

[0042] Figure 25 Pre-treatment (1), thin layer chromatogram at a temperature of 22.8 ℃, wherein 1 is the test sample whole plant solution, and 2 is the control medicinal material solution.

[0043] Figure 26 Pre-treatment (1), thin layer chromatogram at a temperature of 8.1 ℃, wherein 1 is the test sample whole plant solution, and 2 is the control medicinal material solution.

[0044] Figure 27 Pre-treatment (2), thin layer chromatogram at temperature 8.1℃, wherein 1 is the test solution of whole plant, 2 is the control solution of medicinal material.

[0045] Figure 28 Pre-treatment (2), thin layer chromatogram at temperature 8.1℃, wherein 1 is the test solution of whole plant, 2 is the control solution of medicinal material.

[0046] Figure 29 Pre-treatment (1), thin layer chromatogram of different sample application amount optimization, wherein 1-4 are the test solution of whole plant with sample application amount of 2ul, 4ul, 6ul, 8ul respectively, and 5 is the control solution of medicinal material.

[0047] Figure 30 Pre-treatment (2), thin layer chromatogram of different sample application amount optimization, wherein 1-4 are the test solution of whole plant with sample application amount of 1ul, 2ul, 3ul, 4ul respectively, and 5 is the control solution of medicinal material.

[0048] Figure 31 Pre-treatment (3) of Example 9, thin layer chromatogram of the results, wherein 1-5 are the solution of root, stem, branch, leaf and whole plant of the test product respectively, and 6 is the control solution of medicinal material.

[0049] Figure 32 Pre-treatment (2) of Example 9, thin layer chromatogram of the results, wherein 1-5 are the solution of root, stem, branch, leaf and whole plant of the test product respectively, and 6 is the control solution of medicinal material.

[0050] Figure 33 Pre-treatment (3) of Example 10, thin layer chromatogram of the results, wherein 1-5 are the solution of root, stem, branch, leaf and whole plant of the test product respectively, and 6 is the control solution of medicinal material.

[0051] Figure 34 Pre-treatment (2) of Example 10, thin layer chromatogram of the results, wherein 1-5 are the solution of root, stem, branch, leaf and whole plant of the test product respectively, and 6 is the control solution of medicinal material.

[0052] Figure 35 Pre-treatment (3) of Example 11, thin layer chromatogram of the results, wherein 1-5 are the solution of root, stem, branch, leaf and whole plant of the test product respectively, and 6 is the control solution of medicinal material.

[0053] Figure 36 Pre-treatment (2) of Example 11, thin layer chromatogram of the results, wherein 1-5 are the solution of root, stem, branch, leaf and whole plant of the test product respectively, and 6 is the control solution of medicinal material.

[0054] Figure 37Thin layer chromatogram of the result of pre-treatment (3) of Example 12, wherein 1-5 are root, stem, branch, leaf, and whole plant solution of the test sample, respectively, and 6 is the solution of the control medicinal material.

[0055] Figure 38 Thin layer chromatogram of the result of pre-treatment (2) of Example 12, wherein 1-5 are root, stem, branch, leaf, and whole plant solution of the test sample, respectively, and 6 is the solution of the control medicinal material.

[0056] Figure 39 Thin layer chromatogram of the result of pre-treatment (3) of Example 13, wherein 1-5 are root, stem, branch, leaf, and whole plant solution of the test sample, respectively, and 6 is the solution of the control medicinal material.

[0057] Figure 40 Thin layer chromatogram of the result of pre-treatment (2) of Example 13, wherein 1-5 are root, stem, branch, leaf, and whole plant solution of the test sample, respectively, and 6 is the solution of the control medicinal material.

[0058] Figure 41 Thin layer chromatogram of pre-treatment (3) with 10% sulfuric acid ethanol as the color developing agent, wherein 1 is the whole plant solution of the test sample, and 2 is the solution of the control medicinal material.

[0059] Figure 42 Thin layer chromatogram of pre-treatment (3) with 20% perchloric acid ethanol as the color developing agent, wherein 1 is the whole plant solution of the test sample, and 2 is the solution of the control medicinal material.

[0060] Figure 43 Thin layer chromatogram of pre-treatment (2) with 10% sulfuric acid ethanol as the color developing agent, wherein 1 is the whole plant solution of the test sample, and 2 is the solution of the control medicinal material.

[0061] Figure 44 Thin layer chromatogram of pre-treatment (2) with 20% perchloric acid ethanol as the color developing agent, wherein 1 is the whole plant solution of the test sample, and 2 is the solution of the control medicinal material.

[0062] Figure 45 Thin layer chromatogram of pre-treatment (3) with G plate as the thin layer plate, wherein 1 is the whole plant solution of the test sample, and 2 is the solution of the control medicinal material.

[0063] Figure 46 Thin layer chromatogram of pre-treatment (3) with H plate as the thin layer plate, wherein 1 is the whole plant solution of the test sample, and 2 is the solution of the control medicinal material.

[0064] Figure 47 Thin layer chromatogram of pre-treatment (3) with Merck high efficiency G plate as the thin layer plate, wherein 1 is the whole plant solution of the test sample, and 2 is the solution of the control medicinal material.

[0065] Figure 48Pre-treatment (2), thin layer chromatogram of G plate, wherein 1 is the test solution of whole plant, and 2 is the control solution of medicinal material.

[0066] Figure 49 Pre-treatment (2), thin layer chromatogram of H plate, wherein 1 is the test solution of whole plant, and 2 is the control solution of medicinal material.

[0067] Figure 50 Pre-treatment (2), thin layer chromatogram of Merck high-performance G plate, wherein 1 is the test solution of whole plant, and 2 is the control solution of medicinal material.

[0068] Figure 51 Pre-treatment (3), thin layer chromatogram at a temperature of 22.4℃, wherein 1 is the test solution of whole plant, and 2 is the control solution of medicinal material.

[0069] Figure 52 Pre-treatment (3), thin layer chromatogram at a temperature of 8.3℃, wherein 1 is the test solution of whole plant, and 2 is the control solution of medicinal material.

[0070] Figure 53 Pre-treatment (2), thin layer chromatogram at a temperature of 22.4℃, wherein 1 is the test solution of whole plant, and 2 is the control solution of medicinal material.

[0071] Figure 54 Pre-treatment (2), thin layer chromatogram at a temperature of 8.3℃, wherein 1 is the test solution of whole plant, and 2 is the control solution of medicinal material.

[0072] Figure 55 Pre-treatment (3), thin layer chromatogram of different sample amounts, wherein 1-4 are the test solutions of whole plant with sample amounts of 1ul, 3ul, 5ul and 7ul respectively, and 5 is the control solution of medicinal material.

[0073] Figure 56 Pre-treatment (2), thin layer chromatogram of different sample amounts, wherein 1-4 are the test solutions of whole plant with sample amounts of 1ul, 2ul, 3ul and 4ul respectively, and 5 is the control solution of medicinal material.

[0074] Figure 57 Thin layer chromatogram of the results of pre-treatment (3) in Example 14, wherein 1-5 are the solutions of roots, stems, branches, leaves and whole plant of the test product respectively, and 6 is the control solution of medicinal material.

[0075] Figure 58 Thin layer chromatogram of the results of pre-treatment (2) in Example 14, wherein 1-5 are the solutions of roots, stems, branches, leaves and whole plant of the test product respectively, and 6 is the control solution of medicinal material.

[0076] Figure 59Thin layer chromatogram of the result of pre-treatment (3) of Example 15, wherein 1-5 are root, stem, branch, leaf, and whole plant solution of the test sample, respectively, and 6 is the solution of the control medicinal material.

[0077] Figure 60 Thin layer chromatogram of the result of pre-treatment (2) of Example 15, wherein 1-5 are root, stem, branch, leaf, and whole plant solution of the test sample, respectively, and 6 is the solution of the control medicinal material.

[0078] Figure 61 Thin layer chromatogram of the result of pre-treatment (3) of Example 16, wherein 1-5 are root, stem, branch, leaf, and whole plant solution of the test sample, respectively, and 6 is the solution of the control medicinal material.

[0079] Figure 62 Thin layer chromatogram of the result of pre-treatment (2) of Example 16, wherein 1-5 are root, stem, branch, leaf, and whole plant solution of the test sample, respectively, and 6 is the solution of the control medicinal material.

[0080] Figure 63 Thin layer chromatogram of the result of pre-treatment (3) of Example 17, wherein 1-5 are root, stem, branch, leaf, and whole plant solution of the test sample, respectively, and 6 is the solution of the control medicinal material.

[0081] Figure 64 Thin layer chromatogram of the result of pre-treatment (2) of Example 17, wherein 1-5 are root, stem, branch, leaf, and whole plant solution of the test sample, respectively, and 6 is the solution of the control medicinal material.

[0082] Figure 65 Thin layer chromatogram of the result of pre-treatment (3) of Example 18, wherein 1-5 are root, stem, branch, leaf, and whole plant solution of the test sample, respectively, and 6 is the solution of the control medicinal material.

[0083] Figure 66 Thin layer chromatogram of the result of pre-treatment (2) of Example 18, wherein 1-5 are root, stem, branch, leaf, and whole plant solution of the test sample, respectively, and 6 is the solution of the control medicinal material.

[0084] Figure 67 Pre-treatment (3), thin layer chromatogram with 10% sulfuric acid ethanol as the color developing agent, wherein 1 is the whole plant solution of the test sample, and 2 is the solution of the control medicinal material.

[0085] Figure 68 Pre-treatment (3), thin layer chromatogram with 20% perchloric acid ethanol as the color developing agent, wherein 1 is the whole plant solution of the test sample, and 2 is the solution of the control medicinal material.

[0086] Figure 69 Pre-treatment (2), thin layer chromatogram with 10% sulfuric acid ethanol as the color developing agent, wherein 1 is the whole plant solution of the test sample, and 2 is the solution of the control medicinal material.

[0087] Figure 70 Pre-treatment (2), TLC with color reagent of 20% perchloric acid ethanol, 1 is the test sample of whole plant solution, 2 is the control medicinal material solution.

[0088] Figure 71 Pre-treatment (3), TLC with G plate, 1 is the test sample of whole plant solution, 2 is the control medicinal material solution.

[0089] Figure 72 Pre-treatment (3), TLC with H plate, 1 is the test sample of whole plant solution, 2 is the control medicinal material solution.

[0090] Figure 73 Pre-treatment (3), TLC with Merck high performance G plate, 1 is the test sample of whole plant solution, 2 is the control medicinal material solution.

[0091] Figure 74 Pre-treatment (2), TLC with G plate, 1 is the test sample of whole plant solution, 2 is the control medicinal material solution.

[0092] Figure 75 Pre-treatment (2), TLC with H plate, 1 is the test sample of whole plant solution, 2 is the control medicinal material solution.

[0093] Figure 76 Pre-treatment (2), TLC with Merck high performance G plate, 1 is the test sample of whole plant solution, 2 is the control medicinal material solution.

[0094] Figure 77 Pre-treatment (3), TLC at temperature of 22.4℃, 1 is the test sample of whole plant solution, 2 is the control medicinal material solution.

[0095] Figure 78 Pre-treatment (3), TLC at temperature of 8.3℃, 1 is the test sample of whole plant solution, 2 is the control medicinal material solution.

[0096] Figure 79 Pre-treatment (2), TLC at temperature of 22.4℃, 1 is the test sample of whole plant solution, 2 is the control medicinal material solution.

[0097] Figure 80 Pre-treatment (2), TLC at temperature of 8.3℃, 1 is the test sample of whole plant solution, 2 is the control medicinal material solution.

[0098] Figure 81 Pre-treatment (3), TLC with different sample application amount, 1-4 are the test sample of whole plant solution with sample application amount of 1ul, 3ul, 5ul and 7ul respectively, 5 is the control medicinal material solution.

[0099] Figure 82 Pre-treatment (2), TLC of different sample application volume optimization, wherein 1-4 are test sample whole plant solution with sample application volume of 1 ul, 2 ul, 3 ul, 4 ul respectively, and 5 is the control medicinal material solution. DETAILED DESCRIPTION

[0100] Example 1

[0101] Pre-treatment (1): 1 g of test sample whole plant / root / stem / branch / leaf and 1 g of control medicinal material whole plant powder were weighed respectively, 100 ml of purified water was added, heated and boiled for 30 min, filtered, the filtrate was evaporated to dryness, 5 ml of methanol was added for dissolution, to obtain test sample whole plant / root / stem / branch / leaf solution and control medicinal material solution.

[0102] Pre-treatment (2): 1 g of test sample whole plant / root / stem / branch / leaf and 1 g of control medicinal material whole plant powder were weighed respectively, 30 ml of methanol was added, ultrasonic treatment was performed for 30 min, filtered, the filtrate was evaporated to dryness, 5 ml of methanol was added for dissolution, to obtain test sample whole plant / root / stem / branch / leaf solution and control medicinal material solution.

[0103] Pre-treatment (3): 1 g of test sample whole plant / root / stem / branch / leaf and 1 g of control medicinal material whole plant powder were weighed respectively, 100 ml of purified water was added, heated and boiled for 30 min, filtered, the filtrate was evaporated to dryness, 0.5 ml of methanol was added for dissolution, to obtain test sample whole plant / root / stem / branch / leaf solution and control medicinal material solution.

[0104] Example 2

[0105] 2 ul of test sample whole plant / root / stem / branch / leaf solution and control medicinal material solution of pre-treatment (1) and pre-treatment (2) were respectively taken and spotted on the same Merck high-performance silica gel G plate, chloroform-ethyl acetate-water (15:22:0.5) lower solution was used as developing agent for development, taken out, air-dried, sprayed with 20% hydrochloric acid ethanol solution, heated at 105°C until the spots developed color, and observed under 365 nm ultraviolet light.

[0106] The results of pre-treatment (1) are shown in Table 1. Figure 1 The root had yellow, sky blue and blue spots at Rf values of 0.79, 0.66 and 0.43 respectively, the stem had a blue spot at Rf value of 0.25, the branch had no brown spot in the range of Rf value of 0.8-0.9, and the leaf had a tailing spot at Rf value of 0.2.

[0107] The results of pre-treatment (2) are shown in Table 2. Figure 2, roots have yellow, sky blue, blue spots at Rf values of 0.81, 0.7, 0.49 respectively, stems have blue spots at Rf value of 0.3, branches have no brown spots at Rf values of 0.8-0.9, leaves have a trailing spot at Rf value of 0.25.

[0108] Example 3

[0109] Take 2ul of the whole plant / root / stem / branch / leaf solution of the test sample of pretreatment (1) and pretreatment (2) and the control medicinal material solution respectively, and point them on the same Merck high-performance silica gel G plate. Use the lower solution of chloroform-ethyl acetate-water (20:25:0.5) as the developing agent to develop, take out, air dry, spray with 20% perchloric acid ethanol solution, heat to spot coloration at 105°C, and observe under 365nm ultraviolet light.

[0110] The results of pretreatment (1) are shown in Figure 3 , roots have yellow, sky blue, blue spots at Rf values of 0.78, 0.73, 0.45 respectively, stems have blue spots at Rf value of 0.25, branches have no brown spots at Rf values of 0.8-0.9, leaves have a trailing spot at Rf value of 0.24.

[0111] The results of pretreatment (2) are shown in Figure 4 , roots have yellow, sky blue, blue spots at Rf values of 0.81, 0.66, 0.42 respectively, stems have blue spots at Rf value of 0.26, branches have no brown spots at Rf values of 0.8-0.9, leaves have a trailing spot at Rf value of 0.2.

[0112] Example 4

[0113] Take 2ul of the whole plant / root / stem / branch / leaf solution of the test sample of pretreatment (1) and pretreatment (2) and the control medicinal material solution respectively, and point them on the same Merck high-performance silica gel G plate. Use the lower solution of chloroform-ethyl acetate-water (20:25:0.5) as the developing agent to develop, take out, air dry, spray with 20% perchloric acid ethanol solution, heat to spot coloration at 105°C, and observe under 365nm ultraviolet light.

[0114] The results of pretreatment (1) are shown in Figure 5 , roots have yellow, sky blue, blue spots at Rf values of 0.78, 0.73, 0.45 respectively, stems have blue spots at Rf value of 0.25, branches have no brown spots at Rf values of 0.8-0.9, leaves have a trailing spot at Rf value of 0.24.

[0115] The results of pretreatment (2) are shown in Figure 6, roots have yellow, sky blue, blue spots at Rf values of 0.8, 0.66, 0.4 respectively, stems have blue spots at Rf value of 0.25, branches have no brown spots at Rf values of 0.8-0.9, leaves have a trailing spot at Rf value of 0.15.

[0116] Example 5

[0117] Take 2ul of the test sample whole plant / root / stem / branch / leaf solution of pretreatment (1) and pretreatment (2) and the control medicinal material solution respectively, and point them on the same Merck high-performance silica gel G plate respectively. Develop with the lower solution of chloroform-ethyl acetate-water (38:42:3) as the developing agent, take out, air dry, spray with 20% perchloric acid ethanol solution, heat to spot coloration at 105°C, and observe under 365nm ultraviolet light.

[0118] The results of pretreatment (1) are shown in Figure 7 , roots have yellow, sky blue, blue spots at Rf values of 0.8, 0.66, 0.4 respectively, stems have blue spots at Rf value of 0.25, branches have no brown spots at Rf values of 0.8-0.9, leaves have a trailing spot at Rf value of 0.15.

[0119] The results of pretreatment (2) are shown in Figure 8 , roots have yellow, sky blue, blue spots at Rf values of 0.8, 0.66, 0.4 respectively, stems have blue spots at Rf value of 0.25, branches have no brown spots at Rf values of 0.8-0.9, leaves have a trailing spot at Rf value of 0.15.

[0120] Example 6

[0121] Take 2ul of the test sample whole plant / root / stem / branch / leaf solution of pretreatment (1) and pretreatment (2) and the control medicinal material solution respectively, and point them on the same Merck high-performance silica gel G plate respectively. Develop with the lower solution of chloroform-ethyl acetate-water (40:44:3) as the developing agent, take out, air dry, spray with 20% perchloric acid ethanol solution, heat to spot coloration at 105°C, and observe under 365nm ultraviolet light.

[0122] The results of pretreatment (1) are shown in Figure 9 , roots have yellow, sky blue, blue spots at Rf values of 0.8, 0.66, 0.4 respectively, stems have blue spots at Rf value of 0.25, branches have no brown spots at Rf values of 0.8-0.9, leaves have a trailing spot at Rf value of 0.15.

[0123] The results of pretreatment (2) are shown in Figure 10, roots have yellow, sky blue, blue spots at Rf values of 0.8, 0.75, 0.45 respectively, stems have blue spots at Rf value of 0.3, branches have no brown spots at Rf values of 0.8-0.9, leaves have a trailing spot at Rf value of 0.19.

[0124] Example 7

[0125] Take 2ul of the test sample whole plant / root / stem / branch / leaf solution of pretreatment (1) and pretreatment (2) and the control medicinal material solution respectively, and spot them on the same Merck high-performance silica gel G plate. Develop with the lower solution of chloroform-ethyl acetate-water (42:47:3) as the developing agent, take out, air dry, spray with 20% perchloric acid ethanol solution, heat to spot coloration at 105°C, and observe under 365nm ultraviolet light.

[0126] The results of pretreatment (1) are shown in Figure 11 , roots have yellow, sky blue, blue spots at Rf values of 0.8, 0.75, 0.45 respectively, stems have blue spots at Rf value of 0.3, branches have no brown spots at Rf values of 0.8-0.9, leaves have a trailing spot at Rf value of 0.19.

[0127] The results of pretreatment (2) are shown in Figure 12 , roots have yellow, sky blue, blue spots at Rf values of 0.8, 0.75, 0.45 respectively, stems have blue spots at Rf value of 0.3, branches have no brown spots at Rf values of 0.8-0.9, leaves have a trailing spot at Rf value of 0.19.

[0128] Example 8

[0129] Take 2ul of the test sample whole plant / root / stem / branch / leaf solution of pretreatment (1) and pretreatment (2) and the control medicinal material solution respectively, and spot them on the same Merck high-performance silica gel G plate. Develop with the lower solution of chloroform-ethyl acetate-water (42:47:3) as the developing agent, take out, air dry, spray with 20% perchloric acid ethanol solution, heat to spot coloration at 105°C, and observe under 365nm ultraviolet light.

[0130] The results of pretreatment (1) are shown in Figure 13 , roots have yellow, sky blue, blue spots at Rf values of 0.8, 0.75, 0.45 respectively, stems have blue spots at Rf value of 0.3, branches have no brown spots at Rf values of 0.8-0.9, leaves have a trailing spot at Rf value of 0.19.

[0131] The results of pretreatment (2) are shown in Figure 14, the roots have yellow, sky blue and blue spots at Rf values of 0.77, 0.7 and 0.43 respectively, the stems have blue spots at Rf value of 0.24, the branches have no brown spots at Rf values of 0.8-0.9, and the leaves have a tailing spot at Rf value of 0.16.

[0132] Meanwhile, the present application carries out a series of condition optimization, including: color developing agent, different material thin layer plate, temperature, sample quantity, and the used developing agent is the lower solution of chloroform-ethyl acetate-water.

[0133] 1. Color developing agent optimization

[0134] 2ul of the sample solutions and the control medicinal material solutions of the pretreatment (1) and the pretreatment (2) are respectively taken and spotted on the same Merck high-efficiency silica gel G plate, developed, taken out, dried, sprayed with 20% hydrochloric acid ethanol solution and 10% sulfuric acid ethanol, heated to spot coloration at 105℃, and observed under 365nm ultraviolet light to investigate the influence of different color developing agents on the thin layer identification effect.

[0135] The results are shown in Figures 15-18 It can be seen from the chromatogram that the color developing effect of 20% hydrochloric acid ethanol solution is better, so 20% hydrochloric acid ethanol solution is used for color development.

[0136] 2. Different material thin layer plate optimization

[0137] 2ul of the sample solutions and the control medicinal material solutions of the pretreatment (1) and the pretreatment (2) are respectively developed by using silica gel G plate, H plate and high-efficiency G plate, taken out, dried, sprayed with 20% hydrochloric acid ethanol solution, heated to spot coloration at 105℃, and observed under 365nm ultraviolet light to investigate the influence of different material thin layer plates on the thin layer separation effect.

[0138] The results are shown in Figures 19-24 It can be seen from the chromatogram that the spots of the Merck high-efficiency G plate are the clearest and most obvious, and the separation degree is the best, so the Merck high-efficiency G plate is selected.

[0139] 3. Temperature optimization

[0140] 2ul of the sample solutions and the control medicinal material solutions of the pretreatment (1) and the pretreatment (2) are respectively spotted on the same Merck high-efficiency silica gel G plate, developed under temperature conditions of 8.1℃ and 22.8℃ respectively, taken out, dried, sprayed with 20% hydrochloric acid ethanol solution, heated to spot coloration at 105℃, and observed under 365nm ultraviolet light to investigate the influence of different laboratory temperature conditions on the thin layer separation effect.

[0141] The results are shown in Figures 25-28From the atlas, it can be seen that the control medicinal material and the test solution are developed under different laboratory temperature conditions, and there is no obvious difference in the profile of the thin layer chromatogram.

[0142] 4. Optimization of sample application amount

[0143] 2ul, 4ul, 6ul, 8ul of the test solution and the control medicinal material solution of the pretreatment (1) were respectively applied to the same Merck high-performance silica gel G plate; 1ul, 2ul, 3ul, 4ul of the test solution and the control medicinal material solution of the pretreatment (2) were respectively applied to the same Merck high-performance silica gel G plate, developed, taken out, dried, sprayed with 20% perchloric acid ethanol solution, heated to color development at 105°C, and observed under 365nm ultraviolet light to investigate the sample application amount.

[0144] The results are shown in Table 1 and Table 2. Figures 29-30 From the atlas, it can be seen that the test solution has the clearest spots when the sample application amount is 2ul, and therefore the sample application amount of the test solution is selected as 2ul.

[0145] Example 9

[0146] 3ul of the test solution and the control medicinal material solution of the pretreatment (3) and 2ul of the test solution and the control medicinal material solution of the pretreatment (2) were respectively applied to the same Merck high-performance silica gel G plate, developed with the upper solution of chloroform: ethyl acetate: cyclohexane: water: glacial acetic acid (12:27:30:0.1:0.1) as the developing agent, taken out, dried, sprayed with 20% perchloric acid ethanol solution, heated to color development at 105°C, and observed under 365nm ultraviolet light.

[0147] The results of the pretreatment (3) are shown in Table 3 and Table 4. Figure 31 The root has yellow, sky blue and blue spots at Rf values of 0.68, 0.56 and 0.24 respectively, the stem has a blue spot at Rf value of 0.22, the branch has no brown spot in the range of Rf values of 0.7-0.8, and the leaf has a trailing spot at Rf value of 0.15 without the corresponding blue spot.

[0148] The results of the pretreatment (2) are shown in Table 5 and Table 6. Figure 32 The root has yellow, sky blue and blue spots at Rf values of 0.68, 0.56 and 0.24 respectively, the stem has a blue spot at Rf value of 0.22, the branch has no brown spot in the range of Rf values of 0.7-0.8, and the leaf has a trailing spot at Rf value of 0.15 without the corresponding blue spot.

[0149] Example 10

[0150] Take 3 μl of the test sample whole plant / root / stem / branch / leaf solution and the control medicinal material solution of pre-treatment (3) and 2 μl of the test sample whole plant / root / stem / branch / leaf solution and the control medicinal material solution of pre-treatment (2) respectively, and spot them on the same Merck high-performance silica gel G plate. Use the upper solution of chloroform: ethyl acetate: cyclohexane: water: glacial acetic acid (16:33:40:0.3:0.3) as the developing agent to develop, take out, dry, spray with 20% perchloric acid ethanol solution, heat to color the spots at 105°C, and observe under 365 nm ultraviolet light.

[0151] The results of pre-treatment (3) are shown in Table 3. Figure 33 The root has yellow, sky blue and blue spots at Rf values of 0.68, 0.58 and 0.28 respectively, the stem has a blue spot at an Rf value of 0.27, the branch has no brown spot in the range of Rf values of 0.7-0.8, and the leaf has a trailing spot at an Rf value of 0.18 and no corresponding blue spot.

[0152] The results of pre-treatment (2) are shown in Table 2. Figure 34 The root has yellow, sky blue and blue spots at Rf values of 0.68, 0.56 and 0.28 respectively, the stem has a blue spot at an Rf value of 0.26, the branch has no brown spot in the range of Rf values of 0.7-0.8, and the leaf has a trailing spot at an Rf value of 0.16 and no corresponding blue spot.

[0153] Example 11

[0154] Take 3 μl of the test sample whole plant / root / stem / branch / leaf solution and the control medicinal material solution of pre-treatment (3) and 2 μl of the test sample whole plant / root / stem / branch / leaf solution and the control medicinal material solution of pre-treatment (2) respectively, and spot them on the same Merck high-performance silica gel G plate. Use the upper solution of chloroform: ethyl acetate: cyclohexane: water: glacial acetic acid (18:40:50:0.5:0.5) as the developing agent to develop, take out, dry, spray with 20% perchloric acid ethanol solution, heat to color the spots at 105°C, and observe under 365 nm ultraviolet light.

[0155] The results of pre-treatment (3) are shown in Table 3. Figure 35 The root has yellow, sky blue and blue spots at Rf values of 0.64, 0.51 and 0.23 respectively, the stem has a blue spot at an Rf value of 0.2, the branch has no brown spot in the range of Rf values of 0.7-0.8, and the leaf has a trailing spot at an Rf value of 0.19 and no corresponding blue spot.

[0156] The results of pre-treatment (2) are shown in Table 2. Figure 36, root at Rf value of 0.65, 0.57, 0.31 respectively have yellow, sky blue, blue spots, stem at Rf value of 0.3 have blue spots, branch at Rf value of 0.7-0.8 range have no brown spots, leaves at Rf value of 0.22 have trailing spots, no corresponding blue spots.

[0157] Example 12

[0158] Take the test sample whole plant / root / stem / branch / leaf solution of pre-treatment (3) and control medicinal material solution 3ul, test sample whole plant / root / stem / branch / leaf solution of pre-treatment (2) and control medicinal material solution 2ul, respectively, on the same Merck high-performance silica gel G plate, with chloroform: ethyl acetate: cyclohexane: water: glacial acetic acid (22:37:45:1.5:2) upper solution as developing agent, take out, dry, spray with 20% perchloric acid ethanol solution, heat to spot color at 105°C, and observe under 365nm ultraviolet light.

[0159] The results of pre-treatment (3) are shown in Figure 37 , root at Rf value of 0.65, 0.57, 0.31 respectively have yellow, sky blue, blue spots, stem at Rf value of 0.3 have blue spots, branch at Rf value of 0.7-0.8 range have no brown spots, leaves at Rf value of 0.22 have trailing spots, no corresponding blue spots.

[0160] The results of pre-treatment (2) are shown in Figure 38 , root at Rf value of 0.63, 0.52, 0.29 respectively have yellow, sky blue, blue spots, stem at Rf value of 0.27 have blue spots, branch at Rf value of 0.7-0.8 range have no brown spots, leaves at Rf value of 0.2 have trailing spots, no corresponding blue spots.

[0161] Example 13

[0162] Take the test sample whole plant / root / stem / branch / leaf solution of pre-treatment (3) and control medicinal material solution 3ul, test sample whole plant / root / stem / branch / leaf solution of pre-treatment (2) and control medicinal material solution 2ul, respectively, on the same Merck high-performance silica gel G plate, with chloroform: ethyl acetate: cyclohexane: water: glacial acetic acid (30:45:55:2:2) upper solution as developing agent, take out, dry, spray with 20% perchloric acid ethanol solution, heat to spot color at 105°C, and observe under 365nm ultraviolet light.

[0163] The results of pre-treatment (3) are shown in Figure 39, the root has yellow, sky blue and blue spots at Rf values of 0.68, 0.58 and 0.3 respectively, the stem has a blue spot at Rf value of 0.28, the branch has no brown spot at Rf value of 0.7-0.8, and the leaf has a tailing spot at Rf value of 0.2 without a corresponding blue spot.

[0164] The results of the pre-treatment (2) are shown in Figure 40 , the root has yellow, sky blue and blue spots at Rf values of 0.68, 0.58 and 0.3 respectively, the stem has a blue spot at Rf value of 0.28, the branch has no brown spot at Rf value of 0.7-0.8, and the leaf has a tailing spot at Rf value of 0.2 without a corresponding blue spot.

[0165] Meanwhile, the present application carries out a series of condition optimization, including: color developing agent, different material thin layer plate, temperature, sample amount, and the used developing agent is the upper solution of chloroform: ethyl acetate: cyclohexane: water: glacial acetic acid.

[0166] 1. Color developing agent optimization

[0167] 3ul of the sample solution and the control medicinal material solution of the pre-treatment (3) and 2ul of the sample solution and the control medicinal material solution of the pre-treatment (2) are respectively spotted on the same Merck high-efficiency silica gel G plate, developed, taken out, dried, respectively sprayed with 20% perchloric acid ethanol solution and 10% sulfuric acid ethanol, heated to spot coloration at 105 DEG C, and observed under 365nm ultraviolet light, so as to investigate the influence of different color developing agents on the thin layer identification effect.

[0168] The results are shown in Figures 41-44 It can be known from the chromatogram that the color developing effect of 20% perchloric acid ethanol solution is better, so the color developing is carried out by using 20% perchloric acid ethanol solution.

[0169] 2. Different material thin layer plate optimization

[0170] 3ul of the sample solution and the control medicinal material solution of the pre-treatment (3) and 2ul of the sample solution and the control medicinal material solution of the pre-treatment (2) are respectively developed by using silica gel G plate, H plate and high-efficiency G plate, taken out, dried, sprayed with 20% perchloric acid ethanol solution, heated to spot coloration at 105 DEG C, and observed under 365nm ultraviolet light, so as to investigate the influence of different material thin layer plates on the thin layer separation effect.

[0171] The results are shown in Figures 45-50 It can be known from the chromatogram that the spots of the Merck high-efficiency G plate are the clearest and most obvious, and the separation degree is the best, so the Merck high-efficiency G plate is selected.

[0172] 3. Temperature optimization

[0173] Take 3ul of the sample solution and the control drug solution of pre-treatment (3), 2ul of the sample solution and the control drug solution of pre-treatment (2) and spot them on the same Merck high-performance silica gel G plate, respectively, and develop them at 8.1℃ and 22.8℃, respectively, take them out, dry them, spray them with 20% perchloric acid ethanol solution, heat them at 105℃ until the spots develop color, and observe them under 365nm ultraviolet light to investigate the influence of different laboratory temperature conditions on the thin-layer separation effect.

[0174] The results are shown in Figures 51-54 As can be seen from the atlas, there is no obvious difference in the profile of the thin-layer chromatogram between the control drug and the sample solution developed at different laboratory temperature conditions.

[0175] 4. Spotting volume optimization

[0176] Take 1ul, 3ul, 5ul, 7ul of the sample solution and the control drug solution of pre-treatment (3) and spot them on the same Merck high-performance silica gel G plate, take 1ul, 2ul, 3ul, 4ul of the sample solution and the control drug solution of pre-treatment (2) and spot them on the same Merck high-performance silica gel G plate, develop them, take them out, dry them, spray them with 20% perchloric acid ethanol solution, heat them at 105℃ until the spots develop color, and observe them under 365nm ultraviolet light to investigate the spotting volume.

[0177] The results are shown in Figures 55-56 As can be seen from the atlas, the spots are clearest when the spotting volume of the sample solution of pre-treatment (3) is 3ul and the spotting volume of the sample solution of pre-treatment (2) is 2ul, so the spotting volume of the sample solution of pre-treatment (3) is selected to be 3ul and the spotting volume of the sample solution of pre-treatment (2) is selected to be 2ul.

[0178] Example 14

[0179] Take 3ul of the sample whole plant / root / stem / branch / leaf solution and the control drug solution of pre-treatment (3), 2ul of the sample whole plant / root / stem / branch / leaf solution and the control drug solution of pre-treatment (2) and spot them on the same Merck high-performance silica gel G plate, develop them with trichloromethane: ethyl acetate: n-hexane: water: glacial acetic acid (12:27:30:0.1:0.1) upper solution as the developing agent, take them out, dry them, spray them with 20% perchloric acid ethanol solution, heat them at 105℃ until the spots develop color, and observe them under 365nm ultraviolet light.

[0180] The results of pre-treatment (3) are shown in Figure 57 , the root has yellow, sky blue and blue spots at Rf values of 0.68, 0.56 and 0.24, respectively, the stem has a blue spot at Rf value of 0.22, the branch has no brown spot in the range of Rf value of 0.7-0.8, and the leaf has a trailing spot at Rf value of 0.15 without the corresponding blue spot.

[0181] The results of the pre-treatment (2) are shown in Table 2. Figure 58 The root had yellow, sky blue and blue spots at Rf values of 0.68, 0.55 and 0.25, respectively, the stem had a blue spot at an Rf value of 0.23, the branch had no brown spot in the range of Rf values of 0.7-0.8, and the leaf had a trailing spot at an Rf value of 0.14 without a corresponding blue spot.

[0182] Example 15

[0183] The test sample whole plant / root / stem / branch / leaf solution and the control medicinal material solution of 3 μΐ of the pre-treatment (3) and the test sample whole plant / root / stem / branch / leaf solution and the control medicinal material solution of 2 μΐ of the pre-treatment (2) were spotted on the same Merck high-performance silica gel G plate, respectively, and developed with the upper solution of chloroform: ethyl acetate: n-hexane: water: glacial acetic acid (16:33:40:0.3:0.3) as the developing agent, taken out, air-dried, sprayed with 20% perchloric acid ethanol solution, heated to spot coloration at 105°C, and observed under a 365 nm ultraviolet light.

[0184] The results of the pre-treatment (3) are shown in Table 3. Figure 59 The root had yellow, sky blue and blue spots at Rf values of 0.68, 0.58 and 0.28, respectively, the stem had a blue spot at an Rf value of 0.27, the branch had no brown spot in the range of Rf values of 0.7-0.8, and the leaf had a trailing spot at an Rf value of 0.18 without a corresponding blue spot.

[0185] The results of the pre-treatment (2) are shown in Table 2. Figure 60 The root had yellow, sky blue and blue spots at Rf values of 0.68, 0.56 and 0.28, respectively, the stem had a blue spot at an Rf value of 0.26, the branch had no brown spot in the range of Rf values of 0.7-0.8, and the leaf had a trailing spot at an Rf value of 0.16 without a corresponding blue spot.

[0186] Example 16

[0187] The test sample whole plant / root / stem / branch / leaf solution and the control medicinal material solution of 3 μΐ of the pre-treatment (3) and the test sample whole plant / root / stem / branch / leaf solution and the control medicinal material solution of 2 μΐ of the pre-treatment (2) were spotted on the same Merck high-performance silica gel G plate, respectively, and developed with the upper solution of chloroform: ethyl acetate: n-hexane: water: glacial acetic acid (18:40:50:0.5:0.5) as the developing agent, taken out, air-dried, sprayed with 20% perchloric acid ethanol solution, heated to spot coloration at 105°C, and observed under a 365 nm ultraviolet light.

[0188] The results of the pre-treatment (3) are shown in Table 3. Figure 61, root at Rf value of 0.64, 0.51, 0.23, respectively, yellow, sky blue, blue spots, stem at Rf value of 0.2, blue spot, branches at Rf value of 0.7-0.8, no brown spot, leaves at Rf value of 0.19, tailing spot, no corresponding blue spot.

[0189] The results of pre-treatment (2) are shown in Figure 62 , root at Rf value of 0.66, 0.52, 0.23, respectively, yellow, sky blue, blue spots, stem at Rf value of 0.2, blue spot, branches at Rf value of 0.7-0.8, no brown spot, leaves at Rf value of 0.16, tailing spot, no corresponding blue spot.

[0190] Example 17

[0191] 3ul of the test sample whole plant / root / stem / branch / leaf solution of pre-treatment (3) and the control medicinal material solution and 2ul of the test sample whole plant / root / stem / branch / leaf solution of pre-treatment (2) and the control medicinal material solution are spotted on the same Merck high-performance silica gel G plate, developed with chloroform: ethyl acetate: n-hexane: water: glacial acetic acid (22:37:45:1.5:2) upper solution as developing agent, taken out, air-dried, sprayed with 20% perchloric acid ethanol solution, heated at 105°C until the spots develop color, and observed under 365nm ultraviolet light.

[0192] The results of pre-treatment (3) are shown in Figure 63 , root at Rf value of 0.65, 0.57, 0.31, respectively, yellow, sky blue, blue spots, stem at Rf value of 0.3, blue spot, branches at Rf value of 0.7-0.8, no brown spot, leaves at Rf value of 0.22, tailing spot, no corresponding blue spot.

[0193] The results of pre-treatment (2) are shown in Figure 64 , root at Rf value of 0.63, 0.52, 0.29, respectively, yellow, sky blue, blue spots, stem at Rf value of 0.27, blue spot, branches at Rf value of 0.7-0.8, no brown spot, leaves at Rf value of 0.2, tailing spot, no corresponding blue spot.

[0194] Example 18

[0195] Take 3ul of the test sample whole plant / root / stem / branch / leaf solution and the control medicinal material solution of the pretreatment (3) and 2ul of the test sample whole plant / root / stem / branch / leaf solution and the control medicinal material solution of the pretreatment (2) respectively, and point them on the same Merck high-performance silica gel G plate, and develop them with the upper solution of chloroform: ethyl acetate: n-hexane: water: glacial acetic acid (30:45:55:2:2) as the developing agent, take them out, dry them, spray them with 20% perchloric acid ethanol solution, heat them to color the spots at 105 DEG C, and observe them under the light of 365nm ultraviolet lamp.

[0196] The results of the pretreatment (3) are shown in Figure 65 The root has yellow, sky blue and blue spots at Rf values of 0.68, 0.58 and 0.3 respectively, the stem has a blue spot at Rf value of 0.28, the branch has no brown spot in the range of Rf values of 0.7-0.8, and the leaf has a trailing spot at Rf value of 0.2 and no corresponding blue spot.

[0197] The results of the pretreatment (2) are shown in Figure 66 The root has yellow, sky blue and blue spots at Rf values of 0.65, 0.55 and 0.28 respectively, the stem has a blue spot at Rf value of 0.26, the branch has no brown spot in the range of Rf values of 0.7-0.8, and the leaf has a trailing spot at Rf value of 0.18 and no corresponding blue spot.

[0198] Meanwhile, the present application carries out a series of condition optimization, including: color developing agent, different material thin layer plate, temperature, sample amount, and the used developing agent is the upper solution of chloroform: ethyl acetate: n-hexane: water: glacial acetic acid.

[0199] 1. Color developing agent optimization

[0200] Take 3ul of the test sample whole plant / root / stem / branch / leaf solution and the control medicinal material solution of the pretreatment (3) and 2ul of the test sample whole plant / root / stem / branch / leaf solution and the control medicinal material solution of the pretreatment (2) respectively, and point them on the same Merck high-performance silica gel G plate, develop them, take them out, dry them, spray them with 20% perchloric acid ethanol solution and 10% sulfuric acid ethanol respectively, heat them to color the spots at 105 DEG C, and observe them under the light of 365nm ultraviolet lamp, so as to investigate the influence of different color developing agents on the thin layer identification effect.

[0201] The results are shown in Figures 11-14 It can be known from the atlas that the color developing effect of 20% perchloric acid ethanol solution is better, so the color developing is carried out with 20% perchloric acid ethanol solution.

[0202] 2. Different material thin layer plate optimization

[0203] Take 3ul of the test sample whole plant / root / stem / branch / leaf solution and the control medicinal material solution of pretreatment (3) and 2ul of the test sample whole plant / root / stem / branch / leaf solution and the control medicinal material solution of pretreatment (2), respectively, and develop them on silica gel G plate, H plate and high-performance G plate, take them out, dry, spray with 20% perchloric acid ethanol solution, heat to color the spots at 105°C, and observe under 365nm ultraviolet light to investigate the influence of different materials on thin layer separation effect.

[0204] The results are shown in Figures 15-20 As shown in the atlas, the spots on the Merck high-performance G plate are the clearest and most obvious, and the separation degree is the best, so the Merck high-performance G plate is selected.

[0205] 3. Temperature optimization

[0206] Take 3ul of the test sample whole plant / root / stem / branch / leaf solution and the control medicinal material solution of pretreatment (3) and 2ul of the test sample whole plant / root / stem / branch / leaf solution and the control medicinal material solution of pretreatment (2), respectively, and develop them on the same Merck high-performance silica gel G plate under temperature conditions of 8.1°C and 22.8°C, take them out, dry, spray with 20% perchloric acid ethanol solution, heat to color the spots at 105°C, and observe under 365nm ultraviolet light to investigate the influence of different laboratory temperature conditions on thin layer separation effect.

[0207] The results are shown in Figures 21-24 As shown in the atlas, there is no obvious difference in the profile of the thin layer chromatogram between the control medicinal material and the test sample solution developed under different laboratory temperature conditions.

[0208] 4. Sample volume optimization

[0209] Take 1ul, 3ul, 5ul and 7ul of the test sample solution and the control medicinal material solution of pretreatment (3) and 1ul, 2ul, 3ul and 4ul of the test sample solution and the control medicinal material solution of pretreatment (2), respectively, and develop them on the same Merck high-performance silica gel G plate, take them out, dry, spray with 20% perchloric acid ethanol solution, heat to color the spots at 105°C, and observe under 365nm ultraviolet light to investigate the sample volume.

[0210] The results are shown in Figures 25-26 As shown in the atlas, the spots are the clearest when the sample volume of the test sample solution of pretreatment (3) is 3ul and the sample volume of the test sample solution of pretreatment (2) is 2ul, so the sample volume of the test sample solution of pretreatment (3) is selected to be 3ul and the sample volume of the test sample solution of pretreatment (2) is selected to be 2ul.

Claims

1. A thin layer chromatography identification method for Hengjingxi Chinese medicinal materials, characterized in that: The steps include: 1) Take 1 g of the sample powder and the control medicinal material powder respectively, add 100 ml of purified water, heat and boil for 30 minutes, filter, evaporate the filtrate to dryness, and dissolve it in 5 ml of methanol to obtain the test solution and the control medicinal material solution; 2) The test sample solution and the control medicinal material solution were spotted onto the same Merck High-Performance G plate and subjected to thin-layer chromatography using a developing solvent. After chromatography, the plate was sprayed with 20% perchloric acid-ethanol solution and heated at 105°C until the spots developed. The plate was then inspected under 365nm ultraviolet light; the developing solvent was a lower layer solution of chloroform:ethyl acetate:water in a volume ratio of (15-44:22-50:0.5-3). The sample volume of the test solution was 2 μl.

2. A method for identifying thin layers at different locations on a horizontal warp mat, characterized in that: Take the medicinal material to be tested and perform thin layer chromatography using the method according to claim 1. If there are yellow, sky blue and blue spots at RF values ​​of 0.80±0.04, 0.71±0.07 and 0.45±0.05, respectively, it is the root of the horizontal meridian mat; if there are blue spots at the RF value of 0.26±0.04, it is the stem of the horizontal meridian mat; if there are no brown spots within the RF value range of 0.8-0.9, it is the branch of the horizontal meridian mat; if there are trailing spots at the RF value of 0.2±0.05, it is the leaf of the horizontal meridian mat.

3. A thin layer chromatography identification method for Hengjingxi Chinese medicinal materials, characterized in that: The steps include: 1) Take 1 g of the sample powder and the control medicinal material powder respectively, add 100 ml of purified water, heat and boil for 30 minutes, filter, evaporate the filtrate to dryness, and add 0.5 ml of methanol to dissolve to obtain the test solution and the control medicinal material solution; 2) The test sample solution and the control medicinal material solution were spotted on the same Merck high-performance G plate and subjected to thin-layer chromatography using a developing solvent. After chromatography, the plate was sprayed with 20% perchloric acid-ethanol solution, heated at 105°C until the spots were colored, and the plate was inspected under 365nm ultraviolet light; wherein the developing solvent is an upper layer solution with a volume ratio of (12-30:27-45:30-55:0.1-2:0.1-2) chloroform:ethyl acetate:cyclohexane:water:glacial acetic acid, and the sample volume of the test sample solution was 3 μl; or an upper layer solution with a volume ratio of (12-30:27-45:30-50:0.1-2:0.1-2) chloroform:ethyl acetate:n-hexane:water:glacial acetic acid, and the sample volume of the test sample solution was 3 μl.

4. A method for identifying thin layers at different locations on a transverse warp mat, characterized in that: Take the medicinal material to be tested and perform thin layer chromatography using the method according to claim 3; if there are yellow, sky blue and blue spots at the RF values ​​of 0.65±0.03, 0.54±0.04 and 0.27±0.04 respectively, it is the root of the horizontal mat; if there are blue spots at the RF value of 0.25±0.05, it is the stem of the horizontal mat; if there are no brown spots in the RF value within the range of 0.7-0.8, it is the branch of the horizontal mat; if there are trailing spots at the RF value of 0.18±0.04 and no corresponding blue spots If there are yellow spots at RF values ​​of 0.65±0.03, 0.54±0.04 and 0.27±0.04 respectively, it is the root of the horizontal warp mat; if there are blue spots at RF value of 0.24±0.04, it is the stem of the horizontal warp mat; if there are no brown spots within the RF value range of 0.7-0.8, it is the branch of the horizontal warp mat; thin layer chromatography was performed; if there are trailing spots at RF value of 0.18±0.04 and no corresponding blue spots, it is the leaf of the horizontal warp mat.

5. A thin layer chromatography identification method for Hengjingxi Chinese medicinal materials, characterized in that: The steps include: 1) Take 1 g of the sample powder and the control medicinal material powder respectively, add 30 ml of methanol, ultrasonically treat for 30 min, filter, evaporate the filtrate to dryness, and dissolve it in 5 ml of methanol to obtain the test solution and the control medicinal material solution; 2) The test solution and the control medicinal material solution were spotted on the same Merck high-performance G plate, and thin-layer chromatography was performed with a developing agent. After the chromatography was completed, 20% perchloric acid ethanol solution was sprayed and heated at 105°C until the spots were colored. The thin-layer plate was inspected under 365nm ultraviolet light; wherein the developing agent was a lower layer solution of chloroform: ethyl acetate: water in a volume ratio of (15-44: 22-50: 0.5-3), and the amount of the test solution spotted was The sample volume of the test solution is 2 μl; or the upper layer solution of (12-30:27-45:30-55:0.1-2:0.1-2) chloroform:ethyl acetate:cyclohexane:water:glacial acetic acid, the sample volume of the test solution is 2 μl; or the upper layer solution of (12-30:27-45:30-50:0.1-2:0.1-2) chloroform:ethyl acetate:n-hexane:water:glacial acetic acid, the sample volume of the test solution is 2 μl.

6. A method for identifying thin layers at different locations on a transverse warp mat, characterized in that: Take the medicinal material to be tested, and perform thin layer chromatography using the method according to claim 5. If the RF values ​​are 0.80±0.04, 0.71±0.07, and 0.45±0.05 with yellow, sky blue, and blue spots, respectively, it is the root of the horizontal mat; if the RF value is 0.26±0.04 with blue spots, it is the stem of the horizontal mat; if the RF value is within the range of 0.8-0.9 and there are no brown spots, it is the branch of the horizontal mat; if the RF value is 0.2±0.05 with trailing spots, it is the leaf of the horizontal mat; or if the RF values ​​are 0.65±0.03, 0.54±0.04, and 0.27±0.04 with yellow, sky blue, and blue spots, respectively, it is the root of the horizontal mat; if the RF value is 0.25±0.0 If there are blue spots at 5 places, it is the stem of the horizontal warp mat; if there are no brown spots within the RF value range of 0.7-0.8, it is the branch of the horizontal warp mat; if there are trailing spots at the RF value of 0.18±0.04 and no corresponding blue spots, it is the leaf of the horizontal warp mat; or if there are yellow, sky blue and blue spots at the RF values ​​of 0.65±0.03, 0.54±0.04 and 0.27±0.04 respectively, it is the root of the horizontal warp mat; if there are blue spots at the RF value of 0.24±0.04, it is the stem of the horizontal warp mat; if there are no brown spots within the RF value range of 0.7-0.8, it is the branch of the horizontal warp mat; perform thin layer chromatography; if there are trailing spots at the RF value of 0.18±0.04 and no corresponding blue spots, it is the leaf of the horizontal warp mat.

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