Thin-layer chromatography identification method of Hexishi chrysanthemum

Abstract

The invention provides a thin-layer chromatography identification method of Hexishi chrysanthemum. The identification method comprises the following steps: S1, preparing a test solution: extracting a Hexishi chrysanthemum sample to obtain the Hexishi chrysanthemum test solution; s2, preparing reference substance solutions: respectively preparing the reference substance solutions from aesculin, aesculetin, apigenin and luteolin; s3, detection: carrying out thin-layer chromatography detection on the Hexishi chrysanthemum test solution and the reference substance solution, carrying out sample application on a thin-layer plate, carrying out expansion, and carrying out inspection under ultraviolet light; and S4, result judgment: displaying the same spots at the positions corresponding to the reference substance solution in the chromatogram of the test substance on the thin-layer plate. The method can solve the problem of lack of identification methods for Hexishi chrysanthemum in the prior art, and is suitable for the technical field of traditional Chinese medicinal material identification.

Description

Technical Field

[0001] The invention relates to the technical field of identification of traditional Chinese medicinal materials, and in particular to a thin-layer chromatography identification method of chrysanthemum. Background Art

[0002] Hexinia polydichotoma (Ostenf.) HLYang, a perennial herb belonging to the Asteraceae family, is generally about 15-40 cm tall. It grows in sandy land, sandy land edges, lowlands between dunes, Gobi gullies and sandy field edges at an altitude of -42-1800 meters. Hexinia polydichotoma is a xerophyte with small yellow flowers and degenerate leaves. Because its branches resemble deer antlers, it is also known as deer antler grass. It has extremely high ornamental value and is an excellent ground cover plant and bonsai plant for urban greening in arid areas. Because of its drought and salt-alkali resistance, it is widely distributed in the desert areas of the Tarim Basin in Xinjiang. It is an important windbreak and sand-fixing plant and has important ecological value in the desert edge areas. It is distributed in Gansu, Xinjiang and other places. There are no reports on the chemical composition and medicinal value of this plant. It is used in the folk treatment of skin diseases with special effects, but there is little research on Hexinia polydichotoma at home and abroad. Modern research reports on Hexi chrysanthemum mainly focus on reports on natural plants in the desert, but there are few reports on the identification methods of Hexi chrysanthemum as a medicinal material. Summary of the invention

[0003] The main purpose of the present invention is to provide a thin layer chromatography identification method for Hexi chrysanthemum, so as to solve the problem that there is a lack of identification method for Hexi chrysanthemum in the prior art.

[0004] In order to achieve the above-mentioned purpose, according to the first aspect of the present invention, a thin layer chromatography identification method of Hexi Chrysanthemum is provided, and the above-mentioned identification method comprises: S1, preparing a test solution: extracting a Hexi Chrysanthemum sample to obtain a Hexi Chrysanthemum test solution; S2, preparing a reference solution: using quercetin, quercetin, apigenin and luteolin to prepare quercetin reference solution, quercetin reference solution, apigenin reference solution and luteolin reference solution respectively; S3, detecting: performing thin layer chromatography detection on the Hexi Chrysanthemum test solution, quercetin reference solution and quercetin reference solution, and after spotting on the first thin layer plate After development with the first developing agent, the samples were examined under ultraviolet light; thin layer chromatography was performed on the chrysanthemum test solution, apigenin reference solution and luteolin reference solution, and the samples were spotted on the second thin layer plate and developed with the second developing agent, followed by color development with a color developer and drying, and the samples were examined under ultraviolet light; S4, result determination: in the chromatogram of the test sample on the first thin layer plate, the same spots were displayed at the positions corresponding to the qinpixin reference solution and the qinpixin ethyl reference solution; in the chromatogram of the test sample on the second thin layer plate, the same spots were displayed at the positions corresponding to the apigenin reference solution and the luteolin reference solution.

[0005] Further, S1 includes: mixing the chrysanthemum with methanol, performing heating reflux extraction or ultrasonic extraction, and collecting the liquid to obtain a chrysanthemum test solution; preferably, the chrysanthemum includes chrysanthemum powder; preferably, the ratio of chrysanthemum to methanol is (1-2) g: (5-10) mL; preferably, the heating reflux extraction time is 10-40 min; preferably, the ultrasonic extraction time is 10-40 min.

[0006] Furthermore, the concentration of the aesculin reference solution is 1-10 μg / mL, the concentration of the aesculin reference solution is 1-10 μg / mL, the concentration of the apigenin reference solution is 40-50 μg / mL, and the concentration of the luteolin reference solution is 40-50 μg / mL.

[0007] Further, in S3, the spotting amount on the first thin layer plate is: 1-10 μL of Hexigaceae test solution, 2-20 ng of qinpirin and 10-100 ng of qinpirin; the spotting amount on the second thin layer plate is: 1-10 μL of Hexigaceae test solution, 200-250 ng of apigenin and 120-150 ng of luteolin; preferably, the spotting amount of Hexigaceae test solution on the first thin layer plate and the second thin layer plate are each independently selected from 3-5 μL.

[0008] Furthermore, the first developing agent includes chloroform, methanol and formic acid, wherein the volume ratio of chloroform, methanol and formic acid is (5-10):(1-3):(1-3); preferably 6:1:1.

[0009] Furthermore, the second developing agent includes toluene, ethyl formate and formic acid, wherein the volume ratio of toluene, ethyl formate and formic acid is (1-5):(1-5):(1-3); preferably 3:3:1.

[0010] Furthermore, the wavelength of the ultraviolet light is 320-400 nm, preferably 365 nm.

[0011] Furthermore, the developer comprises an AlCl3-ethanol developer; preferably, the concentration of AlCl3 in the AlCl3-ethanol developer is 1-5wt%; preferably, the drying temperature is 100-120°C.

[0012] Further, the first thin layer plate and / or the second thin layer plate include a silica gel G thin layer plate, a high-efficiency silica gel G thin layer plate, an H thin layer plate or a high-efficiency H thin layer plate; preferably, the first thin layer plate and / or the second thin layer plate are high-efficiency silica gel G thin layer plates.

[0013] Furthermore, when the unfolding is performed in S3, the relative humidity is 15RH%-80RH%; preferably, the relative humidity is 18RH%-42RH%.

[0014] By applying the technical scheme of the present invention, with aesculin, aesculin E, apigenin and luteolin as reference substances, the identification of Hexi chrysanthemum can be achieved through thin layer chromatography, filling the gap in the identification method of Hexi chrysanthemum and providing a basis for the identification of Hexi chrysanthemum. The identification method based on thin layer chromatography has the advantages of easy operation, high development efficiency, good separation effect, clear chromatographic bands, good reproducibility, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0016] Figure 1 The TLC result diagram of 4,5-O-dicaffeoylquinic acid according to Example 2 of the present invention as a reference substance is shown.

[0017] Figure 2 The figure shows the TLC result of Experimental Method 1 using chlorogenic acid according to Example 2 of the present invention as a reference substance.

[0018] Figure 3 The figure shows the TLC result of Experimental Method 2 using chlorogenic acid according to Example 2 of the present invention as a reference substance.

[0019] Figure 4 The TLC result of aesculin according to Example 2 of the present invention as a reference substance is shown.

[0020] Figure 5 The TLC result of aesculetin according to Example 2 of the present invention as a reference substance is shown.

[0021] Figure 6 The TLC result diagram of apigenin-6-O-β-glycoside according to Example 2 of the present invention as a reference substance is shown.

[0022] Figure 7 The TLC result of glycyrrhetinic acid according to Example 2 of the present invention as a reference substance is shown.

[0023] Figure 8 The TLC result of kaempferol according to Example 2 of the present invention as a reference substance is shown.

[0024] Fig. 9 The TLC result diagram of apigenin according to Example 2 of the present invention as a reference substance is shown.

[0025] Fig.10 The TLC result of luteolin according to Example 2 of the present invention as a reference substance is shown.

[0026] Fig.11 The TLC result diagrams corresponding to different extraction methods according to Example 4 of the present invention are shown.

[0027] Fig.12 The TLC result diagram corresponding to different developing agents according to Example 4 of the present invention is shown, wherein: Fig.12 A in the figure shows the corresponding developing solvent n-butanol: chloroform: formic acid = 8:1:1. Fig.12 In the figure, B corresponds to the developing solvent of toluene:ethyl acetate:formic acid = 3:4:1.

[0028] Fig.13 The TLC result diagram corresponding to different developing agents according to Example 4 of the present invention is shown, wherein: Fig.13 A in the figure corresponds to the developing solvent chloroform: methanol: formic acid = 12:1:1, Fig.13 In the figure, B corresponds to the developing solvent of chloroform: methanol: formic acid = 6:1:1.

[0029] Fig.14 The TLC result diagrams corresponding to different types of thin layer plates according to Example 4 of the present invention are shown, wherein: Fig.14 A in the middle corresponds to silica gel G thin layer plate, Fig.14 B in the middle corresponds to the high-efficiency silica gel G thin layer plate, Fig.14 C in the middle corresponds to H thin layer plate, Fig.14 The D in the middle corresponds to the high-efficiency H thin layer plate.

[0030] Fig.15 The TLC result diagram corresponding to different sample amounts according to Example 4 of the present invention is shown.

[0031] Fig.16 The TLC result diagram corresponding to different development temperatures according to Example 4 of the present invention is shown, wherein: Fig.16 A in the middle corresponds to 4℃, Fig.16 B corresponds to room temperature (28°C). Fig.16 Medium C corresponds to 40℃.

[0032] Fig.17 The TLC result diagram corresponding to different development humidity according to Example 4 of the present invention is shown, wherein: Fig.17 A in the middle corresponds to 18RH%, Fig.17 B corresponds to 42RH%, Fig.17 Medium C corresponds to 72RH%.

[0033] Fig.18 The TLC result graphs corresponding to thin layer plates from different manufacturers according to Example 4 of the present invention are shown, wherein: Fig.18 The A in the middle corresponds to the high-efficiency silica gel G thin layer plate of Anhui Liangchen Silicon Source Material Co., Ltd. Fig.18 The B in the middle corresponds to the high-efficiency silica gel G thin layer plate of Yantai Jiangyou Silica Gel Development Co., Ltd.

[0034] Fig.19 The TLC result graph of 10 different batches of Hexi Chrysanthemum samples according to Example 4 of the present invention is shown.

[0035] Fig. 20 The TLC result graphs corresponding to different extraction methods according to Example 5 of the present invention are shown.

[0036] Fig.21 The TLC result diagram corresponding to different developing agents according to Example 5 of the present invention is shown, wherein: Fig.21 A corresponds to the developing solvent toluene: ethyl acetate: formic acid = 5:3:1, Fig.21 B corresponds to the developing solvent n-hexane: ethyl acetate: acetic acid = 18:4:3, Fig.21 In the middle, C corresponds to the developing agent toluene: ethyl formate: formic acid = 3:3:1.

[0037] Fig. 22 The TLC result diagrams corresponding to different types of thin layer plates according to Example 5 of the present invention are shown, wherein: Fig. 22 A in the middle corresponds to silica gel G thin layer plate, Fig. 22 B in the middle corresponds to the high-efficiency silica gel G thin layer plate, Fig. 22 C in the middle corresponds to H thin layer plate, Fig. 22 The D in the middle corresponds to the high-efficiency H thin layer plate.

[0038] Fig.23 The TLC result graph corresponding to different sample amounts according to Example 5 of the present invention is shown.

[0039] Fig.24 The TLC result diagram corresponding to different development temperatures according to Example 5 of the present invention is shown, wherein: Fig.24 A in the middle corresponds to 4℃, Fig.24 B corresponds to room temperature (28°C). Fig.24 Medium C corresponds to 40℃.

[0040] Fig.25 The TLC result diagram corresponding to different development humidity according to Example 5 of the present invention is shown, wherein: Fig.25 A in the middle corresponds to 18RH%, Fig.25 B corresponds to 42RH%, Fig.25 Medium C corresponds to 72RH%.

[0041] Fig.26 The TLC result graphs corresponding to thin layer plates from different manufacturers according to Example 5 of the present invention are shown, wherein: Fig.26 The A in the middle corresponds to the high-efficiency silica gel G thin layer plate of Yantai Jiangyou Silica Gel Development Co., Ltd. Fig.26 The B in the middle corresponds to the high-efficiency silica gel G thin layer plate of Qingdao Ocean Chemical Co., Ltd.

[0042] Fig. 27 The TLC result graph of 10 different batches of Hexi Chrysanthemum samples according to Example 5 of the present invention is shown.

[0043] Fig.28 The TLC result graph of 10 different batches of Hexi Chrysanthemum samples according to Example 5 of the present invention is shown. DETAILED DESCRIPTION

[0044] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0045] As mentioned in the background technology, there is little research on Hexi chrysanthemum in the prior art, and there is a lack of identification methods for medicinal Hexi chrysanthemum. Therefore, in this application, the inventor attempts to develop a method for identifying Hexi chrysanthemum using thin layer chromatography, and on this basis proposes a series of protection schemes of this application.

[0046] In a first typical embodiment of the present application, a thin layer chromatography identification method for Hexi Chrysanthemum is provided, and the identification method comprises: S1, preparing a test solution: extracting a Hexi Chrysanthemum sample to obtain a Hexi Chrysanthemum test solution; S2, preparing a reference solution: using qinpirin, qinpietrin, apigenin and luteolin to prepare qinpirin reference solution, qinpietrin reference solution, apigenin reference solution and luteolin reference solution respectively; S3, detecting: performing thin layer chromatography detection on the Hexi Chrysanthemum test solution, qinpietrin reference solution and qinpietrin reference solution, and using a first developing agent after spotting on a first thin layer plate After development, inspect under ultraviolet light; perform thin layer chromatography on the chrysanthemum test solution, apigenin reference solution and luteolin reference solution, spot the sample on the second thin layer plate and develop with the second developing agent, develop with AlCl3-ethanol color developer and dry, and inspect under ultraviolet light; S4, result determination: in the test sample chromatogram on the first thin layer plate, the same spots are shown at the positions corresponding to the qinpixin reference solution and the qinpixin ethyl reference solution; in the test sample chromatogram on the second thin layer plate, the same spots are shown at the positions corresponding to the apigenin reference solution and the luteolin reference solution.

[0047] In the above identification method, thin layer chromatography (TLC) is used to identify Hexi chrysanthemum. First, a test solution is prepared by extracting from a Hexi chrysanthemum sample, and the extraction method includes but is not limited to the existing techniques such as immersion, percolation, decoction, reflux extraction, continuous reflux extraction, heating, ultrasound, steam distillation, supercritical fluid extraction, etc.

[0048] Step S2 is performed independently of step S1, and a reference solution is prepared in step S2. In the identification method of the present application, the inventors have selected coumarin compounds quercetin and quercetin, and flavonoid compounds apigenin and luteolin as reference substances through a large number of experimental attempts. The inventors found that Hexi chrysanthemum also contains the above four components, which can form a stable and clear spot pattern in TLC analysis, with a high degree of separation from other components, and there are no other spots nearby to affect the analysis. And the Rf values ​​of the above four components in TLC are not affected by other spots, and the spot positions in the test solution are consistent with the spot positions of the reference substance, which is convenient for subsequent comparison.

[0049] After obtaining the test solution and the reference solution, a thin layer chromatography test is performed. In S3, the coumarin reference substance and the flavonoid reference substance are respectively subjected to thin layer chromatography test. On the first thin layer plate, the Hexi chrysanthemum test solution, the qinpi amine reference solution and the qinpi ethyl reference solution are spotted and developed, and after the development is completed, the corresponding spots can be observed by inspecting under ultraviolet light. On the second thin layer plate, the Hexi chrysanthemum test solution, the apigenin reference solution and the luteolin reference solution are spotted and developed, and after the development is completed, the second thin layer plate is colored with a color developer, and then the second thin layer plate is inspected under ultraviolet light after drying.

[0050] In S4, it is determined whether the sample is Hexi Chrysanthemum according to the inspection results in S3. If the same spots as the above four reference substances are shown on the first and second thin layer plates (same Rf value, same color of spots), the sample is determined to be Hexi Chrysanthemum, and the identification is completed. If any one or more spots corresponding to the reference substances are missing on the thin layer plate, it means that the quality of the sample does not meet the regulations, including but not limited to that the sample is not Hexi Chrysanthemum, or is Hexi Chrysanthemum of substandard quality.

[0051] In a preferred embodiment, S1 includes: mixing chrysanthemum with methanol, performing heating reflux extraction or ultrasonic extraction, and collecting the liquid to obtain a chrysanthemum test solution; preferably, the chrysanthemum includes chrysanthemum powder; preferably, the ratio of chrysanthemum to methanol is (1-2) g: (5-10) mL; preferably, the heating reflux extraction time is 10-40 min; preferably, the ultrasonic extraction time is 10-40 min.

[0052] Preferably, in the process of preparing the test solution, methanol is used as the solvent, and coumarins and flavonoids are extracted from the chrysanthemum by heating reflux extraction or ultrasonic extraction, and the liquid is collected by methods including but not limited to centrifugation and filtration. In order to improve the extraction efficiency, chrysanthemum powder is preferably used to increase the contact area between the sample and the solvent and improve the extraction efficiency.

[0053] In a preferred embodiment, the concentration of the aesculin reference solution is 1-10 μg / mL, the concentration of the aesculin reference solution is 1-10 μg / mL, the concentration of the apigenin reference solution is 40-50 μg / mL, and the concentration of the luteolin reference solution is 40-50 μg / mL.

[0054] In a preferred embodiment, in S3, the spotting amount on the first thin layer plate is: 1-10 μL of Hexigaceae test solution, 2-20 ng of qinpirin and 10-100 ng of qinpirin; the spotting amount on the second thin layer plate is: 1-10 μL of Hexigaceae test solution, 200-250 ng of apigenin and 120-150 ng of luteolin; preferably, the spotting amount of Hexigaceae test solution on the first thin layer plate and the second thin layer plate are each independently selected from 3-5 μL.

[0055] By spotting on TLC using the above-mentioned spotting amount, it is possible to obtain clean and identifiable spots during subsequent inspection. If the spotting amount is too small, it is difficult to obtain spots sufficient for identification. If the spotting amount is too large, it will cause TLC overload, inaccurate Rf value of the compound, tailing, etc., resulting in low separation, and spots near the target spot affect the inspection. More preferably, the spotting amount of the Hexi chrysanthemum test solution is 3-5 μL (including but not limited to 3, 3.5, 4, 4.5 or 5 μL), and the spotting amount within this range can ensure that the bands on the TLC are clear and do not tilt.

[0056] In a preferred embodiment, the first developing agent includes chloroform, methanol and formic acid, wherein the volume ratio of chloroform, methanol and formic acid is (5-10):(1-3):(1-3); preferably 6:1:1.

[0057] In the experiment of developing coumarin compounds, the inventors found through a large number of experiments that the above-mentioned first developing agent can make qinpi A and qinpi Et develop well on TLC, with clear and stable bands and suitable Rf values. And the use of the above-mentioned first developing agent to develop the Hexi chrysanthemum test solution can separate the qinpi A and qinpi Et from other chemical components, so that by comparing with the bands of the reference substance, the identification of qinpi A and qinpi Et in the Hexi chrysanthemum test solution can be achieved. In a preferred embodiment, the second developing agent includes toluene, ethyl formate and formic acid, wherein the volume ratio of toluene, ethyl formate and formic acid is (1-5): (1-5): (1-3); preferably 3:3:1.

[0058] Similar to the first developing agent, the inventors have developed the second developing agent, which can develop flavonoids well and identify apigenin and luteolin in the Hexi chrysanthemum test solution. In a preferred embodiment, the wavelength of ultraviolet light is 320-400nm (including but not limited to 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395 or 400nm), preferably 365nm.

[0059] In a preferred embodiment, the concentration of AlCl 3 in the AlCl 3 -ethanol developer is 1-5 wt %.

[0060] In a preferred embodiment, the drying temperature is 100-120°C.

[0061] In a preferred embodiment, the first thin layer plate and / or the second thin layer plate include a silica gel G thin layer plate, a high-efficiency silica gel G thin layer plate, an H thin layer plate or a high-efficiency H thin layer plate; preferably, the first thin layer plate and / or the second thin layer plate are high-efficiency silica gel G thin layer plates.

[0062] In a preferred embodiment, when unfolding is performed in S3, the relative humidity is 15RH%-80RH%, including but not limited to 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or 80RH%; preferably, the relative humidity is 18RH%-42RH%.

[0063] The beneficial effects of the present application will be further explained in detail below in conjunction with specific embodiments.

[0064] Example 1 Sample source

[0065] Sample source: Dried aerial parts of Hexinia polydichotoma (Ostenf.) HLYang.

[0066] 1. Sample collection: A total of 10 batches of Hexi chrysanthemum samples were collected, and detailed information is shown in Table 1.

[0067] Table 1 Hexi Chrysanthemum Sample Information

[0068] serial number name Medicinal Parts Origin Collection time HXJ-1 Hexi Chrysanthemum Dry above ground parts Xinjiang 2019.11 HXJ-2 Hexi Chrysanthemum Dry above ground parts Xinjiang 2020.10 HXJ-3 Hexi Chrysanthemum Dry above ground parts Xinjiang 2022.06 HXJ-4 Hexi Chrysanthemum Dry above ground parts Xinjiang 2022.06 HXJ-5 Hexi Chrysanthemum Dry above ground parts Xinjiang 2023.05 HXJ-6 Hexi Chrysanthemum Dry above ground parts Xinjiang 2023.05 HXJ-7 Hexi Chrysanthemum Dry above ground parts Xinjiang 2023.10 HXJ-8 Hexi Chrysanthemum Dry above ground parts Xinjiang 2023.10 HXJ-9 Hexi Chrysanthemum Dry above ground parts Xinjiang 2024.05 HXJ-10 Hexi Chrysanthemum Dry above ground parts Xinjiang 2024.06

[0069] Among them, the Hexi chrysanthemum sample HXJ-5 was made into a wax leaf specimen, which was identified as the above-ground part of the Hexi chrysanthemum plant Hexinia polydichotoma (Ostenf.) HLYang.

[0070] After the samples were collected, all 10 samples were crushed, sieved and set aside.

[0071] Example 2 Investigation of reference substances

[0072] 1. Phenylpropanoid compounds - 4,5-O--dicaffeoylquinic acid

[0073] 1.1 Experimental methods

[0074] Preparation of reference solution: Weigh 5 mg of 4,5-O-dicaffeoylquinic acid reference solution and place it in a 10 mL volumetric flask. Add appropriate amount of methanol to dissolve it, then add methanol to make up to the mark and shake well.

[0075] Preparation of test solution: Take 0.5 g of the medicinal material and add 10 mL of methanol, heat and reflux for 30 min, filter, evaporate the filtrate, add 3 drops of hydrochloric acid to the residue, then add 8 mL of ethyl acetate, ultrasonically treat for 3 min, filter, evaporate the filtrate, add 1 mL of methanol to dissolve the residue to obtain the test solution.

[0076] Take 5 μL of the reference solution and the test solution respectively, spot them on the same silica gel G thin layer plate, and use toluene-ethyl acetate-formic acid (1:7:1) as the developing solvent. After taking out, let it dry. Spray 1% ferric chloride solution and inspect it under sunlight.

[0077] 1.2 Experimental results: TLC results are as follows Figure 1 As shown, the control solution and the test solution did not have the same spot at the same position.

[0078] 2. Coumarin compounds - Chlorogenic acid

[0079] 2.1 Experimental Methods 1

[0080] Take 0.5g of medicinal powder, add 20mL of 50% methanol, ultrasonically treat for 30 minutes, centrifuge, and take the supernatant as the test solution. Take another chlorogenic acid reference substance, add methanol to make a solution containing 1mg per 1mL and its 10-fold, 20-fold, and 50-fold dilutions as the reference solution. According to the thin layer chromatography method (General Rule 0502), take 2μL of each of the above two solutions and spot them on the same silica gel G thin layer plate, use ethyl acetate-formic acid-water (7:2.5:2.5) as the upper layer solution as the developing agent, develop, take out, dry, and examine under ultraviolet light (365nm). Experimental results: TLC results are as follows Figure 2 As shown, at the position corresponding to the chromatogram of the reference substance, the test substance does not show a fluorescent spot of the same color.

[0081] 2.2 Experimental Method 2

[0082] Take 0.5g of medicinal powder, add 15mL of 50% methanol, ultrasonically treat for 30 minutes, filter, and take the filtrate as the test solution. Prepare the chlorogenic acid reference solution in the same way, add methanol to make it contain 1mg of chlorogenic acid per 1mL, and dilute it with methanol into 10-fold, 20-fold, and 50-fold dilutions as the reference solution. According to the thin layer chromatography method (General Rule 0502), take 2-4μL of each of the above three solutions and spot them on the same polyamide film, use n-butanol-acetic acid-water (5:0.15:4) as the developing agent, develop, take out, dry, spray with 3% aluminum chloride ethanol solution, heat with hot air until the spots are clear, and inspect under ultraviolet light (365nm). Experimental results: TLC results are as follows Figure 3 As shown, in the chromatogram of the test sample, no fluorescent spot of the same color is shown at the corresponding position in the chromatogram of the reference sample.

[0083] 3. Coumarin compounds - aesculin A, aesculin E

[0084] The reference compounds aesculetin and aesculetin selected in the present invention are obtained by re-adjusting the thin layer method after confirming the substance through a single reference test, combining two thin layer conditions, and conducting multiple tests.

[0085] 3.1 Experimental methods

[0086] (1) Take 1g of the powder of this product, add 10ml of methanol, heat and reflux for 10 minutes, cool, filter, and take the filtrate as the test solution. Take another reference substance of esculin, add methanol to make a reference solution containing 2mg per 1ml, as the reference solution. According to the thin layer chromatography method (General Rule 0502), take 10μL of the reference solution and 5, 10, and 15μL of the test solution are respectively spotted on the same silica gel G thin layer plate, and use chloroform-methanol-formic acid (6:1:0.5) as the developing agent, develop, take out, dry, and examine under ultraviolet light (365nm).

[0087] (2) Take 1g of medicinal material powder, add 10mL of methanol, heat and reflux for 10 minutes, cool, filter, and take the filtrate as the test solution. Take the esculetin reference substance, add methanol to make a solution containing 1mg per 1mL and dilute it into 20-fold, 50-fold, and 100-fold dilutions as the reference solution. According to the thin layer chromatography method (General Rule 0502), take 5μL of the reference solution and 5, 10, and 15μL of the test solution on the same silica gel G thin layer plate, respectively, with chloroform-methanol-formic acid (12:2:1) as the developing solvent, develop, take out, dry, and examine under ultraviolet light (365nm).

[0088] 3.2 Experimental results: The TLC results of aesculin are as follows Figure 4As shown in the figure, 0.2 mg / mL, 0.05 mg / mL, and 0.02 mg / mL are the TLC results corresponding to the reference solution, and 10 μL, 15 μL, and 20 μL are the TLC results corresponding to the test solution; the pure number ("0.225") in the figure is the Rf value, the same below. The TLC results of esculetin are as follows Figure 5 As shown. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions of the chromatogram of the reference sample.

[0089] 4. Glycoside compounds - apigenin-6-O-β-glycoside

[0090] 4.1 Experimental methods

[0091] Take 1g of medicinal powder, add 30mL of methanol, ultrasonically treat for 40 minutes, filter, recover the solvent from the filtrate until dry, add 1mL of methanol to the residue to dissolve it, and use it as the test solution. Take apigenin-6-O-β-glycoside reference substance, add methanol to make a solution containing 1mg per 1mL and dilute it into 10 times, 20 times, 50 times, and 100 times dilutions, as reference solution. According to the thin layer chromatography method (General Rule 0502), take 1μL of each of the above four solutions and spot them on the same silica gel G thin layer plate, use toluene-ethyl formate-formic acid (3:3:1) as the developing solvent, develop, take out, dry, spray with 1% aluminum chloride ethanol solution, heat at 105℃ for several minutes, and inspect under ultraviolet light (365nm).

[0092] 4.2 Experimental results: TLC results are as follows Figure 6 As shown, in the chromatogram of the test sample, no fluorescent spot of the same color is shown at the corresponding position in the chromatogram of the reference sample.

[0093] 5. Terpenoids: Glycyrrhetinic Acid

[0094] 5.1 Experimental Methods

[0095] Take 2g of medicinal powder, add 2mL of hydrochloric acid and 20mL of chloroform, heat and reflux for 1 hour, cool, filter, evaporate the filtrate, add 0.5mL of anhydrous ethanol to the residue to dissolve it, as the test solution. Take the glycyrrhetinic acid reference substance, add anhydrous ethanol to make a solution containing 1mg per 1mL, as the reference solution. According to the thin layer chromatography method (General Rule 0502), take 5μL of the test solution and the reference medicinal solution, and 2μL of the reference solution, and spot them on the same silica gel GF254 thin layer plate, use petroleum ether (30-60℃)-ethyl acetate-acetone-formic acid (30:6:5:0.5) as the developing agent, develop, take out, dry, and inspect under ultraviolet light (254nm).

[0096] 5.2 Experimental results: TLC results are as follows Figure 7As shown, in the test sample chromatogram, the fluorescent spot displayed at the position corresponding to the reference sample chromatogram is not clear.

[0097] 6. Flavonoids - Kaempferol

[0098] 6.1 Experimental methods

[0099] Take 1g of medicinal powder, add 25mL of 75% methanol and 1mL of hydrochloric acid, heat and hydrolyze for 1 hour, filter, evaporate the filtrate, dissolve the residue in 10mL of water, extract twice with ethyl acetate, 10mL each time, combine the ethyl acetate solution, evaporate to dryness, and dissolve the residue in 1mL of methanol as the test solution. Take another kaempferol reference substance, add methanol to make a solution containing 1mg per 1mL, as the reference substance solution. According to the thin layer chromatography method (General Rule 0502), take 5μL of the test solution and 2μL of the reference substance solution, respectively, and spot them on the same silica gel G thin layer plate, use cyclohexane-ethyl acetate-formic acid (8:4:0.4) as the developing solvent, develop, take out, dry, spray with 5% aluminum chloride ethanol solution, and immediately place under ultraviolet light (365nm) for inspection. 6.2 Experimental results: TLC results are as follows Figure 8 As shown, in the chromatogram of the test sample, there are spots of the same color at the corresponding positions in the chromatogram of the reference sample, but there is an error in the position of the spots, so it cannot be determined that the HM-001 medicinal material contains kaempferol.

[0100] 7. Flavonoids - Apigenin

[0101] 7.1 Experimental Methods

[0102] Take 1g of the powder of this product, add 30mL of methanol, ultrasonically treat for 40 minutes, filter, recover the solvent from the filtrate until dry, add 1mL of methanol to the residue to dissolve it, and use it as the test solution. Take another 1g of Scutellaria barbata as the reference medicinal material, and prepare the reference medicinal material solution in the same way. Take apigenin reference substance, add methanol to prepare a solution containing 1mg per 1mL, and use it as the reference substance solution. According to the thin layer chromatography method (General Rule 0502), take 1μL of each of the above four solutions, spot them on the same silica gel G thin layer plate, use toluene-ethyl formate-formic acid (3:3:1) as the developing agent, develop, take out, dry, spray with 1% aluminum chloride ethanol solution, heat at 105℃ for several minutes, and inspect under ultraviolet light (365nm). 7.2 Experimental results: The TLC results are as follows Fig. 9 As shown, in the test sample chromatogram, fluorescent spots of the same color appear at the corresponding positions in the reference sample chromatogram.

[0103] 8. Flavonoids - Luteolin

[0104] 8.1 Experimental methods

[0105] Take 1g of the powder of this product, add 30ml of methanol, ultrasonically treat for 40 minutes, filter, recover the solvent from the filtrate until dry, add 1ml of methanol to the residue to dissolve it, and use it as the test solution. Take another luteolin reference substance, add methanol to make a solution containing 1mg per 1ml and dilute it into 10 times, 20 times, 50 times, and 100 times dilutions as the reference solution. According to the thin layer chromatography method (General Rule 0502), take 1μL of the reference solution and 1, 5, and 8μL of the test solution are respectively spotted on the same silica gel G thin layer plate, and toluene-ethyl formate-formic acid (3:3:1) is used as the developing agent. Develop, take out, dry, spray with 1% aluminum chloride ethanol solution, heat at 105℃ for several minutes, and inspect under ultraviolet light (365nm).

[0106] 8.2 Experimental results: The TLC results of luteolin are as follows Fig.10 As shown, the TLC results of aesculetin are as follows Figure 5 As shown. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions of the chromatogram of the reference sample.

[0107] Combined with the investigation results of the above-mentioned reference substances, aesculin, aesculin E, apigenin and luteolin were selected as reference substances.

[0108] Example 3

[0109] A thin layer chromatography identification method for Hexi chrysanthemum, the identification method comprising the following steps:

[0110] Method A: Thin layer chromatography identification method of coumarin substances in Hexi chrysanthemum medicinal materials, the identification method comprises the following steps:

[0111] (1) Preparation of test solution: Take 2 g of Hexi chrysanthemum powder and 10 mL of methanol, heat and reflux for 10 min, cool, filter, and use the filtrate as the test sample.

[0112] (2) Preparation of reference solution: Take esculin and esculin E reference substances, add methanol to prepare solutions containing 1 μg of esculin and 10 μg of esculin per mL, respectively, as reference solutions.

[0113] (3) Identification: According to the thin layer chromatography test, take 2μL of aesculin, 10μL of aesculin, and 3μL of the test solution, and spot them on the high-efficiency G thin layer plate respectively, use chloroform: methanol: formic acid = 6:1:1 as the developing solvent, develop, take out, dry, and place under ultraviolet light (365nm) for inspection. In the chromatogram of the test sample, a spot of the same color should appear at the corresponding position of the chromatogram of the reference sample.

[0114] Method B: Thin layer chromatography identification method of flavonoids in Hexi chrysanthemum medicinal material, the identification method comprises the following steps:

[0115] (1) Preparation of test solution: Take 1 g of Hexi chrysanthemum powder and 5 mL of methanol, sonicate for 10 min, filter, evaporate the filtrate to dryness, and add 2 mL of methanol to dissolve to obtain the test solution.

[0116] (2) Preparation of reference solution: Take apigenin and luteolin reference substances, respectively, and add methanol to prepare reference solutions containing 50 μg of apigenin and luteolin per 1 mL, respectively.

[0117] (3) Identification: According to the thin layer chromatography test, take 5μL of apigenin, 3μL of luteolin, and 3μL of the test solution, and spot them on the G thin layer plate respectively. Use toluene: ethyl formate: formic acid = 3:3:1 as the developing solvent. After developing and drying, spray 1% AlCl3-ethanol color developer in an oven and dry it. Place it under ultraviolet 365nm for inspection. In the chromatogram of the test sample, a spot of the same color should appear at the corresponding position of the chromatogram of the reference sample.

[0118] The above method A was used to perform thin layer chromatography identification on the 10 batches of samples in Table 1. The results showed that the test samples of the 10 batches of samples had good separation effect, clear bands, and good reproducibility, and spots of the same color appeared at the corresponding positions of the chromatograms of the reference samples.

[0119] The above method B was used to perform thin layer chromatography identification on the 10 batches of samples in Table 1. The results showed that the test samples of the 10 batches of samples had good separation effect, clear bands, and good reproducibility, and spots of the same color appeared at the corresponding positions of the chromatograms of the reference samples.

[0120] In the following Examples 4 and 5, the durability experiments were carried out on the thin layer chromatography coumarin and flavonoid identification methods of the Hexi chrysanthemum of the present invention: referring to the fourth volume of the 2020 edition of the "Chinese Pharmacopoeia", different extraction methods, different developer concentrations, different types of thin layer plates, different sample spot amounts, different development temperatures, humidity and thin layer plates from different manufacturers and other factors were investigated.

[0121] The details are as follows:

[0122] Example 4 Investigation of coumarin compounds from Hexi chrysanthemum medicinal material by thin layer chromatography

[0123] 1. Investigation of different extraction methods

[0124] According to method A in Example 3, the experiment was carried out. Under the same other conditions, the samples were heated under reflux for 10 minutes, 20 minutes, 40 minutes and ultrasonic for 10 minutes. Results: The TLC separation effect of the above four extraction methods was good, and the four extraction efficiencies were relatively high. Fig.11 .

[0125] Specific chromatographic conditions: high-efficiency silica gel G thin layer plate, manufacturer: Anhui Liangchen Silicon Source Materials Co., Ltd.; batch number: 20221205; specification: 10cm×10cm; strip spotting, spotting volume: 3μL; temperature: 28℃ (±2℃); relative humidity: 35RH% (±5%); developing agent: chloroform-methanol-formic acid (6:1:1); inspection under ultraviolet light at 365nm.

[0126] 2. Investigation of different developing agents

[0127] According to method A in Example 3, the test was carried out. Under the same other conditions, different developing agents were used, namely, n-butanol: chloroform: formic acid = 8:1:1, toluene: ethyl acetate: formic acid = 3:4:1, chloroform: methanol: formic acid = 12:1:1, and chloroform: methanol: formic acid = 6:1:1. Results: The developing agent was chloroform: methanol: formic acid, and the separation effect was good, among which chloroform: methanol: formic acid = 6:1:1 was the best. Fig.12 and Fig.13 .in, Fig.12 The developing solvent of A is n-butanol: chloroform: formic acid = 8:1:1; Fig.12 The developing solvent of B is toluene:ethyl acetate:formic acid = 3:4:1; Fig.13 The developing solvent of A is chloroform:methanol:formic acid=12:1:1; Fig.13 The developing solvent for B is chloroform: methanol: formic acid = 6:1:1.

[0128] The specific chromatographic conditions are: high-efficiency silica gel G thin layer plate, manufacturer: Anhui Liangchen Silicon Source Materials Co., Ltd.; batch number: 20221205; specification: 10cm×10cm; strip spotting, spotting volume: 3μL; temperature: 28°C (±2°C); relative humidity: 35RH% (±5%); developing solvent: n-butanol: chloroform: formic acid = 8:1:1.

[0129] High-efficiency silica gel G thin layer plate, manufacturer: Anhui Liangchen Silicon Source Materials Co., Ltd.; batch number: 20221205; specification: 10cm×10cm; strip spotting, spotting volume: 3μL; temperature: 28℃ (±2℃); relative humidity: 35RH% (±5%); developing solvent: toluene: ethyl acetate: formic acid = 3:4:1.

[0130] High-efficiency silica gel G thin layer plate, manufacturer: Anhui Liangchen Silicon Source Materials Co., Ltd.; batch number: 20221205; specification: 10cm×10cm; strip spotting, spotting volume: 3μL; temperature: 28℃ (±2℃); relative humidity: 35RH% (±5%); developing solvent: chloroform: methanol: formic acid = 12:1:1.

[0131] High-efficiency silica gel G thin layer plate, manufacturer: Anhui Liangchen Silicon Source Materials Co., Ltd.; batch number: 20221205; specification: 10cm×10cm; strip spotting, spotting volume: 3μL; temperature: 28℃; relative humidity: 35RH% (±5%); developing solvent: chloroform: methanol: formic acid = 6:1:1.

[0132] 3. Investigation of different types of thin layer plates

[0133] According to method A in Example 3, the experiment was conducted using silica gel G thin layer plate, high-efficiency silica gel G thin layer plate, H thin layer plate, and high-efficiency H thin layer plate respectively under the same other conditions. Results: All four thin layer plates can have certain spots, and the best separation effect is the high-efficiency silica gel G thin layer plate, as shown in Figure 2. Fig.14 . Fig.14 A in the middle is a silica gel G thin layer plate; Fig.14 Middle B is on the high-efficiency silica gel G thin layer plate; Fig.14 C in the middle is H thin layer plate; Fig.14 D in the middle is a high-efficiency H thin layer plate.

[0134] Specific chromatographic conditions:

[0135] Silica gel G thin layer plate, manufacturer: Qingdao Ocean Chemical Co., Ltd.; batch number: 20240102; specification: 10cm×10cm; strip spotting, spotting volume: 3μL; temperature: 28℃(±2℃); relative humidity: 35RH%(±5%); developing solvent: chloroform:methanol:formic acid=6:1:1.

[0136] High-efficiency silica gel G thin layer plate, manufacturer: Anhui Liangchen Silicon Source Materials Co., Ltd.; batch number: 20221205; specification: 10cm×10cm; strip spotting, spotting volume: 3μL; temperature: 28℃; relative humidity: 35RH% (±5%); developing solvent: chloroform: methanol: formic acid = 6:1:1.

[0137] Silica gel H thin layer plate, manufacturer: Qingdao Ocean Chemical Co., Ltd.; batch number: 20220308; specification: 10cm×10cm; strip spotting, spotting volume: 3μL; temperature: 28℃; relative humidity: 35RH% (±5%); developing solvent: chloroform: methanol: formic acid = 6:1:1.

[0138] High-efficiency silica gel H thin layer plate, manufacturer: Anhui Liangchen Silicon Source Material Co., Ltd.; batch number: 20230215; specification: 10cm×10cm; strip spotting, spotting volume: 3μL; temperature: 28℃; relative humidity: 35RH% (±5%); developing agent:

[0139] Chloroform:methanol:formic acid=6:1:1.

[0140] 4. Investigation of different sampling quantities

[0141] According to method A in Example 3, the test was conducted with the sample solution of 1 μL, 3 μL, 5 μL, 8 μL, and 10 μL under the same other conditions. Results: When the sample volume was greater than 5 μL, the band on the thin layer was tilted; when the sample volume was 1 μL, the sample band was not clear; when the sample volume was 3 μL, the band was clear and the separation effect was good. Fig.15 .

[0142] Specific chromatographic conditions: high-efficiency silica gel G thin layer plate, manufacturer: Anhui Liangchen Silicon Source Materials Co., Ltd.; batch number: 20221205; specification: 10cm×10cm; temperature 28°C (±2°C); relative humidity: 35RH% (±5%); developing solvent: chloroform: methanol: formic acid = 6:1:1.

[0143] 5. Investigation of different development temperatures

[0144] The experiment was carried out according to method A in Example 3. Under the same other conditions, the development was investigated at different temperatures: 4°C, room temperature (28°C), and 40°C. Results: There was no significant difference in the sample thin layer at different temperatures. The sample TLC diagram is shown in Fig.16 ,in, Fig.16 A in the middle corresponds to 4℃, Fig.16 B corresponds to room temperature (28°C). Fig.16 Medium C corresponds to 40℃.

[0145] Specific chromatographic conditions: high-efficiency silica gel G thin layer plate, manufacturer: Anhui Liangchen Silicon Source Materials Co., Ltd.; batch number: 20221205; specification: 10cm×10cm; relative humidity: 35RH% (±5%); developing solvent: chloroform: methanol: formic acid = 6:1:1.

[0146] 6. Investigation of different development humidity

[0147] The test was carried out according to method A in Example 3, and carried out at relative humidity of 18RH%, 42RH% and 72RH% under the same other conditions. Results: Under the above humidity conditions of 18RH%, 42RH%, the TLC separation effect was good, and the separation effect was poor at 72RH%. The results are shown in Table 1. Fig.17 , Fig.17 A in the middle corresponds to 18RH%, Fig.17 B corresponds to 42RH%, Fig.17 Medium C corresponds to 72RH%.

[0148] Specific chromatographic conditions: high-efficiency silica gel G thin layer plate, manufacturer: Anhui Liangchen Silicon Source Materials Co., Ltd.; batch number: 20221205; specification: 10cm×10cm; temperature 28°C (±2°C); developing solvent: chloroform: methanol: formic acid = 6:1:1.

[0149] 7. Inspection of thin-layer boards from different manufacturers

[0150] According to the method A in Example 3, the experiment was carried out, and the high-efficiency silica gel G thin layer plates of two brands, Anhui Liangchen Silicon Source Material Co., Ltd. and Yantai Jiangyou Silica Gel Development Co., Ltd., were respectively used for chromatography inspection under the same other conditions. Results:

[0151] Both brands of silicone G plates can achieve the separation effect.

[0152] The TLC chart of Hexi chrysanthemum sample obtained by testing with high-efficiency silica gel G thin layer plate of Anhui Liangchen Silicon Source Material Co., Ltd. is shown in Fig.18 Middle A.

[0153] Specific chromatographic conditions: high-efficiency silica gel G thin layer plate, manufacturer: Anhui Liangchen Silicon Source Materials Co., Ltd.; batch number: 20221205; specification: 10cm×10cm; temperature 28°C (±2°C); developing solvent: chloroform: methanol: formic acid = 6:1:1.

[0154] The TLC chart of Hexi chrysanthemum sample obtained by testing with high-efficiency silica gel G thin layer plate produced by Yantai Jiangyou Silica Gel Development Co., Ltd. is shown in Fig.18 Middle B.

[0155] Specific chromatographic conditions: high-efficiency silica gel G thin layer plate, manufacturer: Yantai Jiangyou Silica Gel Development Co., Ltd.; batch number: 20230510; specification: 10cm×10cm; strip spotting, spotting volume: 3μL; temperature: 28℃ (±2℃); relative humidity: 35RH% (±5%) developing solvent: chloroform: methanol: formic acid = 6:1:1.

[0156] From the durability test results, it can be seen that the thin layer chromatography identification method for coumarin compounds of Hexi Chrysanthemum medicinal materials established in the present invention has good durability, strong applicability and good separation effect.

[0157] TLC images of 10 different batches of Hexi Chrysanthemum samples are shown in Fig.19 .

[0158] Specific chromatographic conditions: high-efficiency silica gel G thin layer plate, manufacturer: Anhui Liangchen Silicon Source Material Co., Ltd.; batch number: 20221205; specification: 10cm×10cm; strip spotting, spotting volume: 3μL; temperature: 28℃ (±2℃); relative humidity: 35RH% (±5%); developing agent: chloroform-methanol-formic acid (6:1:1); inspection under ultraviolet light at 365nm. From the thin layer identification spectra of 10 batches of Hexi chrysanthemum samples, it can be seen that each batch has the same color spots at the same position as the reference.

[0159] Example 5 Investigation of the Thin Layer Chromatography Method for Flavonoid Compounds in Chrysanthemum officinale

[0160] 1. Investigation of different extraction methods

[0161] According to method B in Example 3, the experiment was conducted. Under the same other conditions, the samples were heated under reflux for 10min, 20min, 40min and ultrasonic for 10min, 20min. The samples were sprayed with 1% AlCl3-ethanol developer and dried in an oven. The samples were then placed under ultraviolet light at 365nm. The results showed that the TLC separation effect was good under the above different extraction methods. Fig. 20 .

[0162] Specific chromatographic conditions: high-efficiency silica gel G thin layer plate, manufacturer: Anhui Liangchen Silicon Source Materials Co., Ltd.; batch number: 20221205; specification: 10cm×10cm; strip spotting, spotting volume: 3μL; temperature: 26℃(±2℃); relative humidity: 38RH%(±5%); the developing agent ratio is toluene: ethyl formate: formic acid = 3:3:1. After development and drying, spray 1% AlCl3-ethanol color developer and bake in a 105℃ oven for 5 minutes, and inspect at UV 365nm.

[0163] 2. Investigation of different developing agents

[0164] According to method B in Example 3, the experiment was carried out. Under the same other conditions, different developing agents were used, namely, toluene: ethyl acetate: formic acid = 5:3:1; n-hexane: ethyl acetate: acetic acid = 18:4:3; toluene: ethyl formate: formic acid = 3:3:1. Results: The developing agent was toluene: ethyl formate: formic acid = 3:3:1, and the separation effect was good. Fig.21 .in, Fig.21 A corresponds to the developing solvent toluene: ethyl acetate: formic acid = 5:3:1, Fig.21 B corresponds to the developing solvent n-hexane: ethyl acetate: acetic acid = 18:4:3, Fig.21 In the middle, C corresponds to the developing agent toluene: ethyl formate: formic acid = 3:3:1.

[0165] The specific chromatographic conditions are: high-efficiency silica gel G thin layer plate, manufacturer: Anhui Liangchen Silicon Source Materials Co., Ltd.; batch number: 20230215; specification: 10cm×10cm; strip spotting, spotting volume: 3μL; temperature: 28°C (±2°C); relative humidity: 35RH% (±5%); developing solvent: toluene: ethyl acetate: formic acid = 5:3:1.

[0166] High-efficiency silica gel G thin layer plate, manufacturer: Anhui Liangchen Silicon Source Materials Co., Ltd.; batch number: 20230215; specification: 10cm×10cm; strip spotting, spotting volume: 3μL; temperature: 28℃ (±2℃); relative humidity: 35RH% (±5%); developing solvent: n-hexane: ethyl acetate: acetic acid = 18:4:3.

[0167] High-efficiency silica gel G thin layer plate, manufacturer: Anhui Liangchen Silicon Source Materials Co., Ltd.; batch number: 20230215; specification: 10cm×10cm; strip spotting, spotting volume: 3μL; temperature: 28℃(±2℃); relative humidity: 35RH%(±5%); developing agent: toluene: ethyl formate: formic acid=3:3:1.

[0168] 3. Investigation of different types of thin layer plates

[0169] According to method B in Example 3, the experiment was conducted. Under the same other conditions, silica gel G thin layer plate, high-efficiency silica gel G thin layer plate, H thin layer plate, and high-efficiency H thin layer plate were used for investigation. Results: Among the above four thin layer plates, certain spots can appear. The best separation effect is the high-efficiency silica gel G thin layer plate. Fig. 22 .in, Fig. 22 A in the middle corresponds to silica gel G thin layer plate, Fig. 22 B in the middle corresponds to the high-efficiency silica gel G thin layer plate, Fig. 22 C in the middle corresponds to H thin layer plate, Fig. 22 The D in the middle corresponds to the high-efficiency H thin layer plate.

[0170] Specific chromatographic conditions:

[0171] Silica gel G thin layer plate, manufacturer: Qingdao Ocean Chemical Co., Ltd.; batch number: 20240102; specification: 10cm×10cm; strip spotting, spotting volume: 3μL; temperature: 30℃(±2℃); relative humidity: 38RH%(±5%); developing agent: toluene: ethyl formate: formic acid = 3:3:1. After developing and drying, spray 1% AlCl3-ethanol color developer and bake in a 105℃ oven for 5min, and inspect at UV 365nm.

[0172] High-efficiency silica gel G thin layer plate, manufacturer: Anhui Liangchen Silicon Source Materials Co., Ltd.; batch number: 20221205; specification: 10cm×10cm; strip spotting, spotting volume: 3μL; temperature: 30℃(±2℃); relative humidity: 38RH%(±5%); developing agent: toluene: ethyl formate: formic acid = 3:3:1. After developing and drying, spray 1% AlCl3-ethanol color developer and bake in a 105℃ oven for 5min, and inspect at UV 365nm.

[0173] Silica gel H thin layer plate, manufacturer: Qingdao Ocean Chemical Plant; batch number: 20210308; specification: 10cm×10cm; strip spotting, spotting volume: 3μL; temperature: 30℃ (±2℃); relative humidity: 38RH% (±5%); developing agent: toluene: ethyl formate: formic acid = 3:3:1. After developing and drying, spray 1% AlCl3-ethanol color developer and bake in a 105℃ oven for 5min, and inspect at UV 365nm.

[0174] High-efficiency silica gel H thin layer plate, manufacturer: Anhui Liangchen Silicon Source Materials Co., Ltd.; batch number: 20230215; specification: 10cm×10cm; strip spotting, spotting volume: 3μL; temperature: 30℃(±2℃); relative humidity: 38RH%(±5%); toluene: ethyl formate: formic acid = 3:3:1, after unfolding and drying, spray 1% AlCl3-ethanol color developer and bake in a 105℃ oven for 5min, and inspect at UV 365nm.

[0175] 4. Investigation of different sampling quantities

[0176] According to method B in Example 3, the test was conducted with 1 μL, 3 μL, 5 μL, 7 μL, and 10 μL sample volumes of the test solution under the same other conditions. Results: As the sample volume increased, the upper part of the thin layer showed uneven separation, so 3 μL sample volume was selected as the thin layer sample volume. Fig.23 .

[0177] Specific chromatographic conditions: high-efficiency silica gel G thin layer plate, manufacturer: Anhui Liangchen Silicon Source Materials Co., Ltd.; batch number: 20221205; specification: 10cm×10cm; temperature 30℃ (±2℃); relative humidity: 38RH% (±5%); developing agent: toluene: ethyl formate: formic acid = 3:3:1. After development and drying, spray with 1% AlCl3-ethanol color developer and bake in a 105℃ oven for 5 minutes, and inspect at UV 365nm.

[0178] 5. Investigation of different development temperatures

[0179] The experiment was carried out according to method B in Example 3. Under the same other conditions, the development was investigated at different temperatures: 4°C, room temperature (28°C), and 40°C. Results: There was no significant difference in the sample thin layer at different temperatures. The sample TLC diagram is shown in Fig.24 .in, Fig.24 A in the middle corresponds to 4℃, Fig.24 B corresponds to room temperature (28°C). Fig.24 Medium C corresponds to 40℃.

[0180] Specific chromatographic conditions: high-efficiency silica gel G thin layer plate, manufacturer: Anhui Liangchen Silicon Source Materials Co., Ltd.; batch number: 20221205; specification: 10cm×10cm; relative humidity: 38RH% (±5%); developing agent: toluene: ethyl formate: formic acid = 3:3:1. After development and drying, spray with 1% AlCl3-ethanol color developer and bake in a 105℃ oven for 5min, and inspect at UV 365nm.

[0181] 6. Investigation of different development humidity

[0182] The test was carried out according to method B in Example 3, and the thin layer was developed at relative humidity of 18RH%, 42RH% and 72RH% under the same other conditions. Results: When the humidity was 72%, the thin layer developed slowly and the separation effect was slightly poor; when the humidity was 18% and 42%, the thin layer separation effect was better, as shown in FIG. Fig.25 .in, Fig.25 A in the middle corresponds to 18RH%, Fig.25 B corresponds to 42RH%, Fig.25 Medium C corresponds to 72RH%.

[0183] Specific chromatographic conditions: high-efficiency silica gel G thin layer plate, manufacturer: Anhui Liangchen Silicon Source Materials Co., Ltd.; batch number: 20221205; specification: 10cm×10cm; temperature 30℃ (±2℃); developing agent: toluene: ethyl formate: formic acid = 3:3:1. After development and drying, spray with 1% AlCl3-ethanol color developer and bake in a 105℃ oven for 5 minutes, and inspect at UV 365nm.

[0184] 7. Inspection of thin-layer boards from different manufacturers

[0185] According to method B in Example 3, the experiment was conducted. Under the same other conditions, in addition to the high-efficiency silica gel G thin layer plate produced by Anhui Liangchen Silicon Source Material Co., Ltd. used in the above examples, the high-efficiency silica gel G thin layer plates of two brands, Yantai Jiangyou Silica Gel Development Co., Ltd. and Qingdao Ocean Chemical Co., Ltd., were also used for chromatography. The results showed that silica gel G plates of different brands can achieve effective separation effects.

[0186] The TLC chart of Hexi chrysanthemum sample obtained by testing with high-efficiency silica gel G thin layer plate produced by Yantai Jiangyou Silica Gel Development Co., Ltd. is shown in Fig.26 Middle A.

[0187] Specific chromatographic conditions: high-efficiency silica gel G thin layer plate, manufacturer: Yantai Jiangyou Silica Gel Development Co., Ltd.; batch number: 20230510; specification: 10cm×10cm; strip spotting, spotting volume: 3μL; temperature: 30℃(±2℃); relative humidity: 38RH%(±5%); developing agent: toluene: ethyl formate: formic acid = 3:3:1. After developing and drying, spray 1% AlCl3-ethanol color developer and bake in a 105℃ oven for 5min, and inspect at UV 365nm.

[0188] The TLC chart of Hexi Chrysanthemum sample obtained by using high-efficiency silica gel G thin layer plate of Qingdao Ocean Chemical Co., Ltd. is shown in Fig.26 Middle B.

[0189] Specific chromatographic conditions: high-efficiency silica gel G thin layer plate, manufacturer: Qingdao Ocean Chemical Co., Ltd.; batch number: 20200407; specification: 10cm×10cm; temperature 30℃ (±2℃); 38RH% (±5%); developing agent: toluene: ethyl formate: formic acid = 3:3:1. After development and drying, spray with 1% AlCl3-ethanol color developer and bake in a 105℃ oven for 5 minutes, and inspect at UV 365nm.

[0190] From the durability test results, it can be seen that the thin layer chromatography identification method of flavonoid compounds in Chrysanthemum officinale medicinal material established in the present invention has good durability, strong applicability and good separation effect.

[0191] TLC images of 10 different batches of Hexi chrysanthemum samples are shown in Fig. 27 and Fig.28 .

[0192] Specific chromatographic conditions: Samples 1-6 use high-efficiency silica gel G thin layer plates, manufacturer: Anhui Liangchen Silicon Source Material Co., Ltd.; batch number: 20221205; specification: 10cm×10cm; samples 7-10 use Yantai Jiangyou Silica Gel Development Co., Ltd.; batch number: 20230510; specification: 10cm×10cm; strip spotting, spotting volume: 3μL; temperature: 30℃ (±2℃); relative humidity: 38RH% (±5%); developing agent: toluene: ethyl formate: formic acid = 3:3:1; after developing and drying, spray 1% AlCl3-ethanol color developer and bake in a 105℃ oven for 5min, and examine at ultraviolet 365nm. From the thin layer identification spectra of 10 batches of Hexi chrysanthemum samples, it can be seen that each batch has the same color spots at the same position as the reference.

[0193] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: in the above identification method, quercetin, quercetin, apigenin and luteolin are used as reference substances, and thin layer chromatography is used to identify Hexi chrysanthemum, which specifically includes the preparation of test solution, the preparation of reference solution, thin layer chromatography detection test and result determination. The above identification method is used to detect and analyze Hexi chrysanthemum samples, which has the advantages of convenient operation, high development efficiency, good separation effect, clear chromatographic bands, good reproducibility, etc. It can quickly and accurately identify Hexi chrysanthemum medicinal materials, fill the gap in Hexi chrysanthemum thin layer chromatography identification method, and provide a basis for the identification of Hexi chrysanthemum.

[0194] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A thin layer chromatography identification method of Hexi chrysanthemum, characterized in that: The identification method comprises: S1, preparing a test solution: extracting a Hexi chrysanthemum sample to obtain a Hexi chrysanthemum test solution; S2, preparing reference solutions: using aesculin, aesculetin, apigenin and luteolin to prepare aesculin reference solution, aesculetin reference solution, apigenin reference solution and luteolin reference solution respectively; S3, detection: performing thin layer chromatography detection on the chrysanthemum test solution, the aesculin A reference solution and the aesculin B reference solution, after spotting on a first thin layer plate, developing with a first developing agent, and inspecting under ultraviolet light; performing thin layer chromatography detection on the chrysanthemum test solution, the apigenin reference solution and the luteolin reference solution, after spotting on a second thin layer plate, developing with a second developing agent, developing with a color developer and drying, and inspecting under ultraviolet light; S4, result determination: in the chromatogram of the test sample on the first thin layer plate, the same spots are shown at the positions corresponding to the quercetin reference solution and the quercetin reference solution; in the chromatogram of the test sample on the second thin layer plate, the same spots are shown at the positions corresponding to the apigenin reference solution and the luteolin reference solution.

2. The identification method according to claim 1, characterized in that: The S1 comprises: mixing the chrysanthemum with methanol, performing heating reflux extraction or ultrasonic extraction, and collecting the liquid to obtain the chrysanthemum test solution; Preferably, the Chrysanthemum indicum comprises Chrysanthemum indicum powder; Preferably, the ratio of Hexi chrysanthemum to methanol is (1-2) g: (5-10) mL; Preferably, the heating reflux extraction time is 10-40min; Preferably, the ultrasonic extraction time is 10-40 min.

3. The identification method according to claim 1, characterized in that: The concentration of the aesculin reference solution is 1 to 10 μg / mL. The concentration of the aesculetin reference solution is 1 to 10 μg / mL. The concentration of the apigenin reference solution is 40-50 μg / mL. The concentration of the luteolin reference solution is 40-50 μg / mL.

4. The identification method according to any one of claims 1 to 3, characterized in that: In S3, the amount of samples spotted on the first thin layer plate is: 1-10 μL of the Chrysanthemum morifolium test solution, 2-20 ng of the aesculetin, and 10-100 ng of the aesculetin; The amount of samples spotted on the second thin layer plate is: 1-10 μL of the Hexi Chrysanthemum test solution, 200-250 ng of the apigenin, and 120-150 ng of the luteolin; Preferably, the spotting volume of the Chrysanthemum morifolium test solution on the first thin layer plate and the second thin layer plate is independently selected from 3-5 μL.

5. The identification method according to claim 1, characterized in that: The first developing agent includes chloroform, methanol and formic acid, wherein the volume ratio of chloroform, methanol and formic acid is (5-10):(1-3):(1-3); preferably 6:1:

1.

6. The identification method according to claim 1, characterized in that: The second developing agent includes toluene, ethyl formate and formic acid, wherein the volume ratio of toluene, ethyl formate and formic acid is (1-5):(1-5):(1-3); preferably 3:3:

1.

7. The identification method according to claim 1, characterized in that: The wavelength of the ultraviolet light is 320-400 nm, preferably 365 nm.

8. The identification method according to claim 1, characterized in that: The developer includes AlCl3-ethanol developer; Preferably, the concentration of AlCl3 in the AlCl3-ethanol developer is 1 to 5 wt%; Preferably, the drying temperature is 100-120°C.

9. The identification method according to claim 1, characterized in that: The first thin layer plate and / or the second thin layer plate include a silica gel G thin layer plate, a high-efficiency silica gel G thin layer plate, an H thin layer plate or a high-efficiency H thin layer plate; Preferably, the first thin layer plate and / or the second thin layer plate is a high-efficiency silica gel G thin layer plate.

10. The identification method according to claim 1, characterized in that: When the unfolding is performed in S3, the relative humidity is 15RH%-80RH%; Preferably, the relative humidity is 18RH%-42RH%.

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