Thin-layer chromatography method for identifying radix angelicae lateralis preparata component in rhizoma coptidis paste and application of thin-layer chromatography method

Abstract

The invention relates to the technical field of traditional Chinese medicine detection, and particularly discloses a thin-layer chromatography method for identifying radix angelicae lateralis preparata components in rhizoma coptidis paste and application of the thin-layer chromatography method. The thin-layer chromatography method provided by the invention comprises the following steps: preparing a test solution: adding methanol water into coptis chinensis paste, heating and melting, and then refluxing and stirring; storing at 0 + / -5 DEG C; then, dichloromethane-n-butyl alcohol with the volume ratio of (2.5-3.5): 1 is adopted as an eluent, silica gel column filtration is performed to obtain a filtrate, and the filtrate is evaporated to dryness and dissolved to obtain a test solution; preparing a reference solution; and detection: respectively dripping the solutions on the same silica gel G thin-layer plate, putting the silica gel G thin-layer plate into a developing cylinder to be developed by taking ethyl acetate-isopropanol with the volume ratio of (3-5): 1 as a developing solvent, and inspecting under a light source of 360-370nm. The thin-layer chromatography method provided by the invention can be used for rapidly and accurately identifying the radix angelicae lateralis preparata component in the rhizoma coptidis paste, and a theoretical basis is provided for quality control of the rhizoma coptidis paste.

Description

Technical Field

[0001] The present application relates to the technical field of traditional Chinese medicine detection, and in particular to a thin layer chromatography method for identifying Radix Angelicae Pubescentis components in Coptis chinensis paste and its application. Background Art

[0002] With the acceleration of the modernization of traditional Chinese medicine, the effectiveness and safety of Chinese medicine preparations have received more and more attention. As an important Chinese medicine for external use, Coptis chinensis paste has a long history and extensive clinical application. At present, the identification of the active ingredients of Coptis chinensis paste mainly focuses on liquid chromatography, gas chromatography and other methods, but the experimental instruments used in the above methods are expensive and time-consuming, and it is difficult to be widely used in daily quality control work. Thin layer chromatography research is widely used in the identification of active ingredients of Chinese medicine due to its low cost and fast response time.

[0003] At present, the thin layer chromatography identification method of Radix Angelicae Pubescentis mainly focuses on Radix Angelicae Pubescentis slices or their water extracts. Since the complex matrix of the ointment will have a significant interference effect on the detection, there has been no thin layer study on Radix Angelicae Pubescentis in the ointment. In addition, due to the complexity of the ointment base, the use of the conventional thin layer chromatography identification method of Radix Angelicae Pubescentis in the medicinal slices cannot effectively reduce the interference of the base on the detection results, resulting in deviations in the detection results.

[0004] Therefore, it is urgent to improve the existing pretreatment extraction method and thin layer chromatography method of Coptis chinensis paste, and establish a thin layer chromatography method with high accuracy, good durability and applicable to different batches of Coptis chinensis paste, which is of great significance to the daily quality control of Coptis chinensis paste. Summary of the invention

[0005] In order to make up for the defects of the prior art, a thin layer chromatography method suitable for identifying the Radix Glehniae components in ointments is developed. The present application provides a thin layer chromatography method for identifying the Radix Glehniae components in Coptis chinensis ointment and its application.

[0006] In the first aspect, the present application provides a thin layer chromatography method for identifying the Radix Angelicae Pubescentis component in Coptis chinensis paste, using the following technical scheme: A thin layer chromatography method for identifying the Radix Angelicae Pubescentis component in Coptis chinensis paste comprises the following steps: (1) preparing a test solution: adding methanol water to the coptis root paste, heating to melt, then heating under reflux and stirring at 400-450 rpm for 30-40 min, cooling to room temperature; then storing at 0±5°C for 25-35 min; then using dichloromethane-n-butanol with a volume ratio of (2.5-3.5):1 as an eluent, filtering through a silica gel column to obtain a filtrate, evaporating to dryness, dissolving, and obtaining a test solution; (2) preparing a reference solution; (3) Detection: The above solutions were spotted on the same silica gel G thin layer plate, and ethyl acetate-isopropanol with a volume ratio of (3-5):1 was used as the developing agent. The silica gel G thin layer plate was placed in a developing tank for development and examined under a light source of 360-370 nm.

[0007] In some embodiments, the volume ratio of dichloromethane to n-butanol may be (2.5-3):1 or (3-3.5):1.

[0008] In a specific embodiment, the volume ratio of dichloromethane to n-butanol can also be 2.5:1, 3:1 or 3.5:1.

[0009] In some embodiments, the volume ratio of ethyl acetate to isopropanol may be (3-4):1 or (4-5):1.

[0010] In a specific embodiment, the volume ratio of ethyl acetate to isopropanol can also be 3:1, 4:1 or 5:1.

[0011] Optionally, the volume ratio of ethyl acetate to isopropanol is 4:1.

[0012] Optionally, the spotting volume of each solution is 3-8 μL.

[0013] Optionally, the eluent is dichloromethane-n-butanol in a volume ratio of 3:1.

[0014] Optionally, the concentration of the methanol water is 75-85%.

[0015] Optionally, the silica gel column filtration adopts normal pressure filtration or suction filtration.

[0016] Optionally, the preparation method of the reference solution is: take a ligustilide reference substance, add methanol to dissolve it, and obtain a reference solution with a concentration of 0.1 mg / mL.

[0017] Optionally, the inspection standard is: check whether a characteristic fluorescent spot of the same color appears at a position corresponding to the chromatogram of the ligustilide reference substance in the chromatogram of the test solution, and whether there is interference from the negative sample; if the chromatogram of the test solution has the characteristic fluorescent spot and the negative sample has no interference, then the detected Coptis chinensis paste contains Radix Angelicae Pubescentis components. In the second aspect, the thin layer chromatography method provided in the present application for identifying the Guiwei component in the Coptis chinensis paste is used in identifying the Guiwei component in the Coptis chinensis paste.

[0018] In summary, this application has the following beneficial effects: 1. In the preparation of the test solution of the present application, the Radix Angelicae Pubescentis component in the Coptis chinensis paste is extracted by using methanol water, and eluted by using dichloromethane-n-butanol with a volume ratio of (2.5-3.5):1. The obtained test solution has high purity and is free of other impurities. Using the above test solution for detection can avoid the interference of other complex matrices in the Coptis chinensis paste on the test results, and the obtained test results are highly accurate.

[0019] 2. The present application adopts ethyl acetate-isopropanol with a volume ratio of (3-5):1 as a developing solvent, and the ratio shift value (RF value) in the obtained chromatogram is in the range of 0.2-0.8, and the characteristic spots are clear, bright and regular, without impurity spots.

[0020] 3. The thin layer chromatography method for identifying the Radix Angelicae Pubescentis component in the Coptis chinensis paste provided in the present application has strong specificity, good durability, and can be applied to the identification of the Radix Angelicae Pubescentis components in different batches, and has a good prospect for promotion and use.

[0021] 4. Compared with the liquid chromatography and gas chromatography detection methods in the related art, the thin layer chromatography method for identifying the Radix Angelicae Pubescentis component in the Coptis chinensis paste provided in the present application has the advantages of low cost, simple operation, and short identification time. It can meet the daily work of identifying the Radix Angelicae Pubescentis component in the Coptis chinensis paste and realize the quality control of the Coptis chinensis paste. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a thin layer chromatogram of the specificity investigation result; from left to right: 1 is the ligustilide reference solution; 2 is the HLG-1002-1073 coptis paste test solution (normal pressure filtration); 3 is the HLG-1002-1078 coptis paste negative lack of Radix Angelicae Sinensis test solution; 4 is the HLG-1002-1073 coptis paste test solution (suction filtration); 5 is the HLG-1002-10788 coptis paste negative lack of Radix Angelicae Sinensis test solution; Figure 2 It is a thin layer chromatogram obtained by using silica gel G thin layer plates produced by different manufacturers; Figure 2 In the figure, (a) is Merck of Germany, (b) is Yantai Huayang, (c) is Yantai Jiangyou, and (d) is Qingdao Xinchanglai; from left to right in each chromatogram are: 1 is ligustilide reference solution; 2 is HLG-1002-1166 coptis paste test solution 1; 3 is HLG-1002-1167 coptis paste test solution 2; Figure 3 Thin layer chromatograms obtained for silica gel G thin layer plate detection under different environmental conditions; Figure 3In the figure, (a) is Yantai Jiangyou, 4℃ low temperature and 67% humidity; (b) is Yantai Jiangyou, 25℃ and 92% humidity; in each chromatogram, from left to right: 1 is ligustilide reference solution; 2 is HLG-1002-1073 coptis paste test solution (normal pressure filtration); 3 is HLG-1002-1078 coptis paste negative lack of Radix Angelicae Pubescentis test solution; 4 is HLG-1002-1073 coptis paste test solution (suction filtration); 5 is HLG-1002-10788 coptis paste negative lack of Radix Angelicae Pubescentis test solution. DETAILED DESCRIPTION

[0023] The present application provides a thin layer chromatography method for identifying the Radix Angelicae Pubescentis component in Coptis chinensis paste, comprising the following steps: (1) Preparation of a test solution: Add 75-85% methanol water to the coptis root paste, heat to melt, then heat under reflux and stir at 400-450 rpm for 30-40 min, cool to room temperature; then store at 0±5°C for 25-35 min; then use dichloromethane-n-butanol with a volume ratio of (2.5-3.5):1 as an eluent, filter through a silica gel column to obtain a filtrate, evaporate to dryness, dissolve, and obtain a test solution; the filtration can be atmospheric pressure filtration or suction filtration.

[0024] (2) Prepare a reference solution; take the ligustilide reference substance, add methanol to dissolve it, and obtain a reference solution with a concentration of 0.1 mg / mL.

[0025] (3) Detection: Spot the above solutions on the same silica gel G thin layer plate, with a spot volume of 3-8 μL; then use ethyl acetate-isopropanol with a volume ratio of (3-5:1) as the developing agent, place the silica gel G thin layer plate in a developing tank for development, and inspect it under a light source of 360-370 nm.

[0026] In the present application, the prescription of the coptis paste is 20g of coptis root, 20g of phellodendron, 20g of turmeric, 33g of angelica root, and 66g of raw rehmannia. The preparation method is: mix the above 5 flavors, weigh 792g of sesame oil, heat to 180-190°C, add the mixed pieces to extract for 15 minutes, filter, weigh 264g of beeswax, filter after melting, stir with the oil, cool, and make 1000g, that is; the batch number of the coptis paste used in the embodiment of the present application can be HLG-1002-1073, HLG-100 2-1166, HLG-1002-1167; refer to the above-mentioned prescription and method of Huanglian paste to prepare the Huanglian paste negative sample lacking Radix Angelicae Pubescentis (batch number HLG-1002-1078 or HLG-1002-10788); Ligustilide was purchased from China Food and Drug Inspection Institute; Silica gel G thin layer plates used in this application were purchased from Qingdao Ocean Chemical Plant Branch, Yantai Huayang New Materials Technology Co., Ltd., Yantai Jiangyou Silica Gel Development Co., Ltd., Merck, Germany, and Qingdao Xinchanglai Silica Gel Co., Ltd. The raw materials, reagents, solvents, etc. used in the examples of this application can be obtained commercially.

[0027] The present application is further described in detail below in conjunction with embodiments, test experiments and accompanying drawings.

[0028] Example 1 Embodiment 1 provides a thin layer chromatography method for identifying the Radix Angelicae Pubescentis component in Coptis chinensis paste, comprising the following steps: (1) Preparation of test solution: Add 80% methanol water to the coptis root paste, heat to melt, then heat under reflux and stir at 420 rpm for 30 min, cool to room temperature; then store at 0±5°C for 30 min; then use dichloromethane-n-butanol with a volume ratio of 3:1 as eluent, filter through a silica gel column, obtain a filtrate, evaporate to dryness, and dissolve to obtain a test solution; (2) Prepare a reference solution; take the ligustilide reference substance, add methanol to dissolve it, and obtain a reference solution with a concentration of 0.1 mg / mL.

[0029] (3) Detection: The above solutions were spotted on the same silica gel G thin layer plate (Qingdao Ocean, 25°C, 67% humidity) with a spot volume of 5 μL. Ethyl acetate-isopropanol with a volume ratio of 4:1 was then used as a developing agent. The silica gel G thin layer plate was placed in a developing tank for development and examined under a 365 nm light source.

[0030] Exclusive inspection The samples of Coptis chinensis paste and Coptis chinensis paste negative and lacking Radix Angelicae Pubescentis were used as test samples, and the test was carried out according to the method of Example 1. The results are as follows: Figure 1 shown.

[0031] Figure 1 This is a thin layer chromatogram showing the specificity investigation results. Figure 1Among them, 1 is the ligustilide reference solution; 2 is the HLG-1002-1073 coptis paste test solution (filtered at normal pressure); 3 is the HLG-1002-1078 coptis paste negative lacking Radix Angelicae Pubescentis test solution; 4 is the HLG-1002-1073 coptis paste test solution (filtered by suction); 5 is the HLG-1002-10788 coptis paste negative lacking Radix Angelicae Pubescentis test solution).

[0032] Depend on Figure 1 It can be seen that the separation between the components in the test sample is good, and there is no interference from other impurities in the chromatogram of the test sample solution of the negative lacking Radix Angelicae Sinensis of Coptis chinensis paste, which indicates that the thin layer chromatography method for identifying the Radix Angelicae Sinensis components in the Coptis chinensis paste provided in the present application has good specificity.

[0033] Durability test (1) Durability inspection of thin layer board manufacturers The separation effect of silica gel G thin layer plates produced by four manufacturers, namely Merck, Yantai Huayang, Yantai Jiangyou and Qingdao Xinchanglai, on coptis paste was investigated. According to the method in Example 1, ligustilide reference solution, coptis paste test solution 1 (batch number HLG-1002-1166) and coptis paste test solution 2 (batch number HLG-1002-1167) were prepared respectively, and the above solutions were spotted on the above three silica gel G thin layer plates respectively, and separation and detection were carried out according to the method in Example 1. The results are as follows: Figure 2 shown.

[0034] Figure 2 It is a thin layer chromatogram obtained by using silica gel G thin layer plates produced by different manufacturers; Figure 2 In the figure, (a) is Merck of Germany, (b) is Yantai Huayang, (c) is Yantai Jiangyou, and (d) is Qingdao Xinchanglai; from left to right in each chromatogram: 1 is ligustilide reference solution; 2 is HLG-1002-1166 coptis paste test solution 1; 3 is HLG-1002-1167 coptis paste test solution 2.

[0035] Figure 2 The results show that the silica gel G thin layer plates produced by the four manufacturers, Merck, Yantai Huayang, Yantai Jiangyou and Qingdao Xinchanglai, can effectively separate Radix Angelicae Sinensis from Coptis chinensis paste, with good separation and no impurity spots. Therefore, it is shown that the thin layer chromatography method for identifying Radix Angelicae Sinensis in Coptis chinensis paste provided by the present application has good durability.

[0036] (2) Environmental durability assessment The separation effect of the silica gel G thin layer plate produced by Yantai Jiangyou on Radix Angelicae Pubescentis in the environment of low temperature of 4°C and humidity of 92% was investigated. According to the method in Example 1, the ligustilide reference solution, HLG-1002-1073 Rhizoma Coptidis paste test solution (normal pressure filtration), HLG-1002-1078 Rhizoma Coptidis paste negative Radix Angelicae Pubescentis test solution, HLG-1002-1073 Rhizoma Coptidis paste test solution (suction filtration) and HLG-1002-10788 Rhizoma Coptidis paste negative Radix Angelicae Pubescentis test solution were prepared respectively. The above solutions were spotted on the silica gel G thin layer plate produced by Yantai Jiangyou, and the separation and detection were carried out according to the method in Example 1. The results are shown in the figure. Figure 3 shown.

[0037] Figure 3 Thin layer chromatograms obtained for silica gel G thin layer plate detection under different environmental conditions; Figure 3 In the figure, (a) is Yantai Jiangyou, 4℃ low temperature and 67% humidity; (b) is Yantai Jiangyou, 25℃ and 92% humidity; in each chromatogram, from left to right: 1 is ligustilide reference solution; 2 is HLG-1002-1073 coptis paste test solution (normal pressure filtration); 3 is HLG-1002-1078 coptis paste negative lack of Radix Angelicae Pubescentis test solution; 4 is HLG-1002-1073 coptis paste test solution (suction filtration); 5 is HLG-1002-10788 coptis paste negative lack of Radix Angelicae Pubescentis test solution.

[0038] Figure 3 The results show that the characteristic spots detected by the silica gel G thin layer plate produced by Yantai Jiangyou at a low temperature of 4°C are slightly smaller, and the characteristic spots are normal under a humidity environment of 92%, and the separation effect between adjacent components is good. Therefore, it is shown that the thin layer chromatography method for identifying the Radix Angelicae Pubescentis component in the Coptis chinensis paste provided by the present application has good environmental durability.

[0039] Example 2-3 Examples 2-3 respectively provide a thin layer chromatography method for identifying the Radix Angelicae Pubescentis component in Coptis chinensis paste.

[0040] The difference between the above embodiment and embodiment 1 is: the type and ratio of the eluent, as shown in Table 1 below.

[0041] Comparative Example 1-2 Comparative Examples 1-2 respectively provide a thin layer chromatography method for identifying the Radix Angelicae Pubescentis component in Coptis chinensis paste.

[0042] The difference between the comparative example and Example 1 is that the type and ratio of the eluent are shown in Table 1 below.

[0043] Table 1 Types and ratios of eluents used in Examples 2-3 and Comparative Examples 1-3 / Type and ratio of eluent Example 1 Dichloromethane-n-butanol in a volume ratio of 3:1 Example 2 Dichloromethane-n-butanol with a volume ratio of 2.5:1 Example 3 Dichloromethane-n-butanol with a volume ratio of 3.5:1 Comparative Example 1 Dichloromethane-n-butanol in a volume ratio of 2:1 Comparative Example 2 Dichloromethane-n-butanol in a volume ratio of 4:1 Embodiment 4-5 Examples 4-5 respectively provide a thin layer chromatography method for identifying the Radix Angelicae Pubescentis component in Coptis chinensis paste.

[0044] The difference between the above embodiment and embodiment 1 is: the type and ratio of the developing agent, as shown in Table 2 below.

[0045] Comparative Examples 3-5 Comparative Examples 3-5 respectively provide a thin layer chromatography method for identifying the Radix Angelicae Pubescentis component in Coptis chinensis paste.

[0046] The difference between the comparative example and Example 1 is: the type and ratio of the developing agent, as shown in Table 2 below.

[0047] Table 2 Types and ratios of developing agents used in Examples 4-5 and Comparative Examples 3-5 / Type and ratio of developing agent Example 1 Ethyl acetate-isopropanol in a volume ratio of 4:1 Example 4 Ethyl acetate-isopropanol in a volume ratio of 3:1 Example 5 Ethyl acetate-isopropanol in a volume ratio of 5:1 Comparative Example 3 Ethyl acetate-isopropanol in a volume ratio of 2:1 Comparative Example 4 Ethyl acetate-isopropanol in a volume ratio of 6:1 Comparative Example 5 Toluene-ethyl acetate-formic acid in a volume ratio of 10:4:0.1 Test results The HLG-1002-1073 Coptis chinensis paste test solution was tested according to the thin layer chromatography method provided in Examples 1-5 and Comparative Examples 1-5, and the separation effect, the number of characteristic spots and the ratio shift value RF value in the chromatogram were obtained. The results are shown in Table 3 below.

[0048] (1) Separation effect: Observe whether the characteristic spots of the test solution of Coptis chinensis paste are clear and bright, and whether the shapes are regular. Count the number of characteristic spots and check whether there are impurity spots.

[0049] (2) Ratio shift value (RF value): the ratio of the distance from the origin to the center of the main spot to the distance from the origin to the solvent front.

[0050] Table 2 Detection results of Huanglian ointment obtained by thin layer chromatography method of Examples 1-7 and Comparative Examples 1-5 According to the test results in Table 2, in the thin layer chromatography method provided in Examples 1-5 of the present application, dichloromethane-n-butanol with a volume ratio of (2.5-3.5): 1 is used as the eluent for preparing the test solution, and ethyl acetate-isopropanol with a volume ratio of (3-5): 1 is used as the developing solvent. There is an obvious characteristic spot in the obtained chromatogram, and the characteristic spot is clear, bright, and regular, without impurity spots. Therefore, it is explained that the thin layer chromatography method provided in the present application can realize the effective identification of the Radix Glehniae components in the Coptis chinensis paste, and the separation and identification effect is good.

[0051] In the thin layer chromatography method provided in Comparative Example 1-2, dichloromethane-n-butanol with a volume ratio of 2:1 or 4:1 was used as the eluent for preparing the test solution. There were 2 impurity spots in the obtained chromatogram, indicating that the test solution prepared by the above eluent contained an impurity matrix, and its purity was slightly poor, which affected the identification effect of the Radix Glycyrrhizae Radix in Coptis chinensis paste.

[0052] In the thin layer chromatography method provided in Comparative Examples 3-5, ethyl acetate-isopropanol with a volume ratio of 2:1 or ethyl acetate-isopropanol with a volume ratio of 6:1 or toluene-ethyl acetate-formic acid with a volume ratio of 10:4:0.1 was used as the developing agent, and the obtained chromatogram had unclear spot boundaries and tailing phenomenon, and in addition to the characteristic spots, there was also an impurity spot. Therefore, it is shown that the above three developing agents are unable to efficiently identify the Guiwei component in the Huanglian paste.

[0053] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A thin layer chromatography method for identifying the Radix Angelicae Pubescentis component in Coptis chinensis paste, characterized in that: The following steps are involved: (1) Preparation of test solution: Add methanol water to the coptis root paste, heat to melt, then heat under reflux and stir at 400-450 rpm for 30-40 min, cool to room temperature; then store at 0±5°C for 25-35 min; then use dichloromethane-n-butanol with a volume ratio of (2.5-3.5):1 as eluent, filter through a silica gel column to obtain a filtrate, evaporate to dryness, and dissolve to obtain a test solution; (2) Prepare reference solution; (3) Detection: Spot the above solutions on the same silica gel G thin layer plate, use ethyl acetate-isopropanol with a volume ratio of (3-5):1 as the developing agent, place the silica gel G thin layer plate in a developing tank for development, and examine it under a light source of 360-370nm.

2. The thin layer chromatography method according to claim 1, characterized in that The volume ratio of ethyl acetate to isopropanol is 4:

1.

3. The thin layer chromatography method according to claim 1, characterized in that: The spotting volume of each solution was 3-8 μL.

4. The thin layer chromatography method according to claim 1, characterized in that The eluent is dichloromethane-n-butanol in a volume ratio of 3:

1.

5. The thin layer chromatography method according to claim 1, characterized in that: The concentration of the methanol water is 75-85%.

6. The thin layer chromatography method according to claim 1, characterized in that: The silica gel column filtration adopts normal pressure filtration or suction filtration.

7. The thin layer chromatography method according to claim 1, characterized in that: The preparation method of the reference solution is: take a ligustilide reference substance, add methanol to dissolve it, and obtain a reference solution with a concentration of 0.1 mg / mL.

8. The thin layer chromatography method according to any one of claims 1 to 7, characterized in that The inspection criteria are: checking whether the characteristic fluorescent spots of the same color appear at the corresponding positions of the chromatogram of the ligustilide reference substance in the chromatogram of the test solution, and whether there is interference with the negative sample; If the chromatogram of the test solution has the characteristic fluorescent spot and the negative sample has no interference, the detected Coptis chinensis paste contains Radix Angelicae Pubescentis components.

9. Use of the thin layer chromatography method for identifying the Radix Angelicae Pubescentis component in Rhizoma Coptidis according to any one of claims 1 to 8 in identifying the Radix Angelicae Pubescentis component in Rhizoma Coptidis.

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