A multi-region thin-layer chromatography method for identifying Angelica sinensis and Ligusticum chuanxiong

Abstract

This invention provides a multi-region thin-layer chromatography (TLC) identification method for Angelica sinensis and Ligusticum chuanxiong, comprising the following steps: preparation of a test solution; and TLC identification of the test solution. The developing solvent used in the TLC identification is toluene or ethyl acetate; the colorimetric reagent used in the TLC identification is a vanillin-containing phosphate-sulfate ethanol solution. The multi-region TLC identification method for Angelica sinensis and Ligusticum chuanxiong provided by this invention can rapidly and effectively identify Ligusticum chuanxiong and Angelica sinensis, displaying rich bands and exhibiting good separation of the characteristic components of Angelica sinensis and Ligusticum chuanxiong.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine quality control technology, and in particular to a multi-region thin-layer chromatography method for identifying Angelica sinensis and Ligusticum chuanxiong. Background Technology

[0002] Angelica sinensis (Oliv.) Diels, a plant in the Apiaceae family, is the dried root of the herb Angelica sinensis (Oliv.) Diels. It is used to nourish blood, regulate menstruation, and relieve pain. Ligusticum chuanxiong (Ligusticum chuanxiong Hort.), also in the Apiaceae family, is the dried rhizome of the herb Ligusticum chuanxiong Hort., which is used to invigorate blood circulation, promote qi circulation, dispel wind, and relieve pain. Although Angelica sinensis and Ligusticum chuanxiong are from the same family and have very similar chemical compositions, their functions and indications have both similarities and differences. They are often used together in prescriptions for better therapeutic effects. Angelica sinensis is more expensive than Ligusticum chuanxiong in the market. Due to their similar chemical compositions, in the production of prepared Chinese medicines, the proportion of Ligusticum chuanxiong may be increased as a substitute, while the proportion of Angelica sinensis may be reduced. Currently, the same components are often used as the standard for identifying the two herbs; however, there is currently no good thin-layer chromatography method for distinguishing between them. Summary of the Invention

[0003] The technical problem solved by this invention is to provide a multi-region thin-layer chromatography method for identifying Angelica sinensis and Ligusticum chuanxiong. This method can quickly and effectively identify Ligusticum chuanxiong and Angelica sinensis, showing rich bands and good separation of characteristic components of Angelica sinensis and Ligusticum chuanxiong.

[0004] This invention provides a multi-region thin-layer chromatography method for identifying Angelica sinensis and Ligusticum chuanxiong, comprising the following steps:

[0005] (1) Preparation of test solution

[0006] Prepare test solutions from Angelica sinensis and Ligusticum chuanxiong, respectively;

[0007] (2) Thin-layer chromatographic identification of the test solution

[0008] The developing solvent used for the thin-layer chromatography identification is toluene or ethyl acetate.

[0009] The colorimetric reagent used in the thin-layer chromatography identification was an ethanol solution of vanillin containing sulfuric acid and phosphoric acid.

[0010] Preferably, the volume ratio of toluene to ethyl acetate is 33:6.

[0011] Preferably, the colorimetric reagent used in the thin-layer chromatography identification is an ethanol solution containing 1% vanillin, 5% sulfuric acid, and 5% phosphoric acid.

[0012] Preferably, the preparation method of the test solution is as follows: take 1g of Angelica sinensis and Ligusticum chuanxiong respectively, add 5mL of acetone, soak at room temperature for 15min, shake intermittently, centrifuge, take the supernatant, filter through a membrane, and use it as the test solution.

[0013] Preferably, the conditions for the thin-layer chromatography identification are as follows:

[0014] Pre-fabricated silicone thin-layer board: 20cm × 10cm; Merck;

[0015] 4 μL of sample was spotted onto a thin-layer plate in strips, with a strip width of 10 mm. The distance between the origin of the sample and the bottom edge of the thin-layer plate was 10 mm. After spotting, the plate was heated at 50 °C for 15 min and then set aside.

[0016] The solvent system and developing agent are toluene and ethyl acetate, with a volume ratio of 33:6;

[0017] Inspection: Spray with an ethanol solution containing 1% vanillin, 5% sulfuric acid, and 5% phosphoric acid, heat at 105°C for 1 minute, observe and photograph under a UV lamp at a wavelength of 366nm, then heat for another 1.5 minutes and observe and photograph under a fluorescent lamp.

[0018] The present invention has the following beneficial effects:

[0019] The multi-region thin-layer chromatography method for identifying Angelica sinensis and Ligusticum chuanxiong provided by this invention uses specific colorimetric and developing agents. The combination of the two can quickly and effectively identify Ligusticum chuanxiong and Angelica sinensis, displaying rich bands and good separation of characteristic components of Angelica sinensis and Ligusticum chuanxiong. Attached Figure Description

[0020] The above and other objects, features, and advantages of the present invention will become more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, wherein,

[0021] Figure 1 The TLC spectrum (UV 366nm fluorescence spectrum) for the thin-layer identification of Angelica sinensis and Ligusticum chuanxiong using the multi-region thin-layer identification method of the present invention in Example 1 is shown. In this spectrum, 1 represents Angelica sinensis, 2 represents Angelica sinensis slices, 3 represents Ligusticum chuanxiong slices, and 4 represents Ligusticum chuanxiong.

[0022] Figure 2 The TLC spectrum (UV 366nm fluorescence spectrum reverse phase color) of Angelica sinensis and Ligusticum chuanxiong for thin-layer identification using the multi-region thin-layer identification method of Angelica sinensis and Ligusticum chuanxiong in Example 1 of the present invention is shown. In this spectrum, 1 is Angelica sinensis, 2 is Angelica sinensis slices, 3 is Ligusticum chuanxiong slices, and 4 is Ligusticum chuanxiong.

[0023] Figure 3The TLC (white light spectrum) of Angelica sinensis and Ligusticum chuanxiong for thin-layer identification using the multi-region thin-layer identification method of Angelica sinensis and Ligusticum chuanxiong in Example 1 of the present invention is shown. In the figure, 1 is Angelica sinensis, 2 is Angelica sinensis slices, 3 is Ligusticum chuanxiong slices, and 4 is Ligusticum chuanxiong.

[0024] Figure 4 The TLC scan spectra (UV 366nm fluorescence spectra) of Angelica sinensis and Ligusticum chuanxiong in Example 1 are shown.

[0025] Figure 5 The TLC scan spectrum (UV 366nm fluorescence spectrum inverted color) of Angelica sinensis and Ligusticum chuanxiong in Example 1.

[0026] Figure 6 The TLC scan spectrum (white light spectrum) of Angelica sinensis and Ligusticum chuanxiong in Example 1.

[0027] Figure 7 The TLC spectrum (UV 366nm fluorescence spectrum) of Angelica sinensis and Ligusticum chuanxiong for thin-layer identification using the multi-region thin-layer identification method in Comparative Example 1 is shown. In the TLC spectrum, 1 represents Angelica sinensis, 2 represents Angelica sinensis slices, 3 represents Ligusticum chuanxiong slices, and 4 represents Ligusticum chuanxiong.

[0028] Figure 8 The TLC (white light spectrum) of Angelica sinensis and Ligusticum chuanxiong in Comparative Example 1 is a multi-region thin-layer chromatography identification method for the identification of Angelica sinensis and Ligusticum chuanxiong. In the sample, 1 represents Angelica sinensis, 2 represents Angelica sinensis slices, 3 represents Ligusticum chuanxiong slices, and 4 represents Ligusticum chuanxiong.

[0029] Figure 9 The TLC spectrum (UV 366nm fluorescence spectrum) of Angelica sinensis and Ligusticum chuanxiong for thin-layer identification using the multi-region thin-layer identification method in Comparative Example 2 is shown in Figure 1, where 1 is Angelica sinensis raw material, 2 is Angelica sinensis processed slices, 3 is Ligusticum chuanxiong processed slices, and 4 is Ligusticum chuanxiong raw material.

[0030] Figure 10 The TLC (white light spectrum) of Angelica sinensis and Ligusticum chuanxiong in Comparative Example 2 is a multi-region thin-layer chromatography identification method for the identification of Angelica sinensis and Ligusticum chuanxiong. In the sample, 1 is Angelica sinensis, 2 is Angelica sinensis slices, 3 is Ligusticum chuanxiong slices, and 4 is Ligusticum chuanxiong.

[0031] Figure 11 The TLC spectrum (UV 366nm fluorescence spectrum) of Angelica sinensis and Ligusticum chuanxiong in Comparative Example 3 is a multi-region thin-layer chromatography identification method for Angelica sinensis and Ligusticum chuanxiong. In the sample, 1 is Angelica sinensis raw material, 2 is Angelica sinensis processed slices, 3 is Ligusticum chuanxiong processed slices, and 4 is Ligusticum chuanxiong raw material.

[0032] Figure 12 The TLC (white light spectrum) of Angelica sinensis and Ligusticum chuanxiong in Comparative Example 3 is a multi-region thin-layer chromatography identification method for the identification of Angelica sinensis and Ligusticum chuanxiong. In the sample, 1 is Angelica sinensis, 2 is Angelica sinensis slices, 3 is Ligusticum chuanxiong slices, and 4 is Ligusticum chuanxiong. Detailed Implementation

[0033] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0034] The sources of the instruments and materials used in the following embodiments are as follows:

[0035] The system includes an ATS 4 fully automated thin-layer chromatography spotter (CAMAG, Switzerland), a dual-chamber thin-layer chromatography developing tank, a TLC visualizer thin-layer chromatography camera (CAMAG, Switzerland), a balance (Sartorius, Germany), and thin-layer silica gel pre-prepared plates (Merck).

[0036] Experimental reagents:

[0037] Acetone, toluene, ethyl acetate, and vanillin were all of analytical grade.

[0038] Preparation method of 1% vanillin ethanol solution containing 5% sulfuric acid and 5% phosphoric acid: Take 5ml sulfuric acid and 5ml phosphoric acid and slowly add them to 90ml ethanol solution, shake well, cool, add 1g vanillin, dissolve, shake well to obtain the solution.

[0039] The proportions of the developing agents involved in this invention are all volume ratios.

[0040] Example 1

[0041] This embodiment provides a multi-region thin-layer chromatography method for identifying Angelica sinensis and Ligusticum chuanxiong.

[0042] (1) Sample preparation:

[0043] Herbal solution: Take 1g each of Angelica sinensis and Ligusticum chuanxiong, add 5mL of acetone to each, soak at room temperature for 15min, shake intermittently, centrifuge for 5min at 4000rpm, take the supernatant, filter through a 0.22μm filter membrane, and use it as the test solution.

[0044] (2) Thin-layer chromatography identification, the conditions are as follows:

[0045] The thin-layer plate uses pre-fabricated silicone ordinary thin-layer plate (20cm×10cm; Merck).

[0046] Spot 4 μL of the sample into thin-layer plates in strips, with a strip width of 10 mm. The distance between the spotting origin and the bottom edge of the thin-layer plate is 10 mm. After spotting, place the plate on a thin-layer heating plate at 50°C for 15 min and set aside.

[0047] Solvent system and developing agent: toluene-ethyl acetate, volume ratio 33:6.

[0048] Inspection: Spray with an ethanol solution containing 1% vanillin, 5% sulfuric acid, and 5% phosphoric acid, heat at 105°C for 1 minute, observe and photograph under a UV lamp (366nm), then heat for another 1.5 minutes and observe and photograph under a fluorescent lamp.

[0049] The results are as follows Figure 1-6 As shown, this is mainly manifested in Figure 1-3 The components within the three dashed boxes are: the components indicated by the three arrows on the right in the spectrum are characteristic components of Ligusticum chuanxiong, and the components indicated by the two arrows on the left are characteristic components of Angelica sinensis. At Rf = 0.27, there is a characteristic component of Angelica sinensis, appearing yellowish-brown in the 366nm fluorescence spectrum, blue in the inverted phase spectrum of the 366nm fluorescence spectrum, and brown in the white light spectrum. At Rf = 0.6, the component indicated by the red arrow, after comparison with the reference standard, is identified as ligustilide A, appearing yellow in the 366nm fluorescence spectrum, dark blue in the inverted phase spectrum of the 366nm fluorescence spectrum, and brown in the white light spectrum. At Rf = 0.68, both Angelica sinensis and Ligusticum chuanxiong have their own characteristic components, among which… In the 366nm fluorescence spectrum, the characteristic component of Angelica sinensis appears light green, while the characteristic component of Ligusticum chuanxiong appears pink. In the inverse color spectrum of the 366nm fluorescence spectrum, the characteristic component of Angelica sinensis appears gray, while the characteristic component of Ligusticum chuanxiong appears green. In the white light spectrum, the characteristic component of Angelica sinensis is colorless, while the characteristic component of Ligusticum chuanxiong appears purple. At Rf = 0.87, the characteristic component of Ligusticum chuanxiong is present, appearing yellowish-brown in the 366nm fluorescence spectrum, blue in the inverse color spectrum of the 366nm fluorescence spectrum, and purple in the white light spectrum.

[0050] Comparative Example 1

[0051] Everything else was the same as in Example 1, except for the colorimetric reagent, which was a 10% sulfuric acid ethanol solution. The results were as follows: Figure 7-8 The chromatographic bands are monochromatic, lacking color richness, and the clarity of the chromatographic bands is inferior to the chromatographic effect of the colorimetric reagent used in Example 1 of this invention.

[0052] Comparative Example 2

[0053] Everything else was the same as in Example 1, except for the colorimetric reagent, which was 1% vanillin and 10% ethanol phosphate. The results were as follows... Figure 9-10 The chromatographic bands are monochromatic, lacking color richness, and the clarity of the chromatographic bands is inferior to the chromatographic effect of the colorimetric reagent used in Example 1 of this invention.

[0054] Comparative Example 3

[0055] Everything else was the same as in Example 1, except for the colorimetric reagent, which was 1% vanillin and 10% sulfuric acid in ethanol. The results were as follows: Figure 11-12 The chromatographic bands are monochromatic, lacking color richness, and the clarity of the chromatographic bands is inferior to the chromatographic effect of the colorimetric reagent used in Example 1 of this invention.

[0056] Comparative Example 4

[0057] Everything else was the same as in Example 1, except that the developing solvent was different, which was hexane-ethyl acetate-formic acid (5:5:0.4). The chromatographic results could not clearly separate the characteristic components of Angelica sinensis and Ligusticum chuanxiong, and could not achieve the effect of effectively identifying Ligusticum chuanxiong and Angelica sinensis separately.

[0058] Comparative Example 5

[0059] The process was the same as in Example 1, except for the developing solvent, which was chloroform-methanol (8:1). The chromatographic results could not clearly separate the characteristic components of Angelica sinensis and Ligusticum chuanxiong, and could not effectively distinguish between Ligusticum chuanxiong and Angelica sinensis.

[0060] Therefore, it can be seen that the present invention, in Example 1, uses a specific combination of colorimetric agent and developing agent to quickly and effectively identify Ligusticum chuanxiong and Angelica sinensis, displaying rich bands, and the characteristic components of Ligusticum chuanxiong and Angelica sinensis are well separated.

[0061] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A multi-region thin-layer chromatography method for identifying Angelica sinensis and Ligusticum chuanxiong, characterized in that, The steps include: (1) Preparation of test solution Prepare test solutions from Angelica sinensis and Ligusticum chuanxiong, respectively; (2) Thin-layer chromatographic identification of the test solution The developing solvent used for the thin-layer chromatography identification was toluene or ethyl acetate. The colorimetric reagent used in the thin-layer chromatography identification was an ethanol solution containing vanillin-phosphate sulfate. The preparation method of the test solution is as follows: take 1g of Angelica sinensis and Ligusticum chuanxiong respectively, add 5mL of acetone, soak at room temperature for 15min, shake intermittently, centrifuge, take the supernatant, filter membrane, and use as the test solution; The conditions for the thin-layer chromatography identification are as follows: Pre-fabricated silicone thin-layer board: 20cm × 10cm; Merck; 4 μL of sample was spotted onto a thin-layer plate in strips, with a strip width of 10 mm. The distance between the origin of the sample and the bottom edge of the thin-layer plate was 10 mm. After spotting, the plate was heated at 50 °C for 15 min and then set aside. The developing solvent is toluene and ethyl acetate, with a volume ratio of 33:6; Inspection: Spray with an ethanol solution containing 1% vanillin, 5% sulfuric acid, and 5% phosphoric acid, heat at 105°C for 1 minute, observe and photograph under a UV lamp at a wavelength of 366nm, then heat for another 1.5 minutes and observe and photograph under a fluorescent lamp.

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