A thin layer chromatography method for identifying Radix Angelicae Dahuricae components in Coptis chinensis paste and its application

Abstract

The present application relates to the field of traditional Chinese medicine detection technology, and specifically discloses a thin layer chromatography method for identifying the Radix Angelicae Pubescentis component in Coptis chinensis paste and its application. The thin layer chromatography method provided by the present application comprises the following steps: preparing a test solution: adding methanol water to the Coptis chinensis paste, heating and melting, and then refluxing with stirring; then storing at 0±5°C; then using dichloromethane-n-butanol with a volume ratio of (2.5-3.5):1 as eluent, filtering through a silica gel column to obtain a filtrate, evaporating and dissolving to obtain a test solution; preparing a reference solution; testing: the above solutions are respectively spotted on the same silica gel G thin layer plate, using ethyl acetate-isopropanol with a volume ratio of (3-5):1 as a developing solvent, placing the silica gel G thin layer plate in a developing cylinder for development, and inspecting under a 360-370nm light source. The thin layer chromatography method provided by the present application can quickly and accurately identify the Radix Angelicae Pubescentis component in the Coptis chinensis paste, providing a theoretical basis for the quality control of the Coptis chinensis 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 Sinensis 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 traditional Chinese medicine preparations are receiving more and more attention. As an important traditional 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. However, the experimental instruments used in these methods are expensive and time-consuming, making them difficult to be widely used in daily quality control work. Thin-layer chromatography research is widely used in the identification of active ingredients of traditional Chinese medicine due to its low cost and fast response time.

[0003] Currently, thin-layer chromatography (TLC) identification methods for Radix Angelicae Sinensis (Radix Guiwei) primarily focus on decoction pieces of Radix Guiwei or its aqueous extracts. Because the complex matrix of ointments can significantly interfere with detection, TLC studies of Radix Guiwei in ointments have yet to be conducted. Furthermore, due to the complexity of the ointment matrix, conventional TLC identification methods for Radix Guiwei in decoction pieces cannot effectively reduce the interference of the matrix on the test results, leading to biased test results.

[0004] Therefore, there is an urgent need 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 work of Coptis chinensis paste. Summary of the Invention

[0005] In order to make up for the defects of the existing technology, a thin layer chromatography method suitable for identifying the Radix Angelicae Dahuricae components in ointments is developed. The present application provides a thin layer chromatography method for identifying the Radix Angelicae Dahuricae 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 Dahuricae component in Coptis chinensis paste, using the following technical solution:

[0007] A thin layer chromatography method for identifying Radix Angelicae Dahuricae in Coptis chinensis paste comprises the following steps:

[0008] (1) Preparation of test solution: methanol water was added to the coptis chinensis paste, heated to melt, then heated under reflux with stirring at 400-450 rpm for 30-40 min, cooled to room temperature; then stored at 0±5°C for 25-35 min; then filtered through a silica gel column using dichloromethane-n-butanol in a volume ratio of (2.5-3.5):1 as eluent to obtain a filtrate, which was evaporated to dryness and dissolved to obtain a test solution;

[0009] (2) preparing a reference solution;

[0010] (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.

[0011] In some embodiments, the volume ratio of dichloromethane to n-butanol may be (2.5-3):1 or (3-3.5):1.

[0012] In a specific embodiment, the volume ratio of dichloromethane to n-butanol can also be 2.5:1, 3:1 or 3.5:1.

[0013] In some embodiments, the volume ratio of ethyl acetate to isopropanol may be (3-4):1 or (4-5):1.

[0014] In a specific embodiment, the volume ratio of ethyl acetate to isopropanol can also be 3:1, 4:1 or 5:1.

[0015] Optionally, the volume ratio of ethyl acetate to isopropanol is 4:1.

[0016] Optionally, the spotting volume of each solution is 3-8 μL.

[0017] Optionally, the eluent is dichloromethane-n-butanol in a volume ratio of 3:1.

[0018] Optionally, the concentration of the methanol water is 75-85%.

[0019] Optionally, the silica gel column filtration adopts normal pressure filtration or suction filtration.

[0020] Optionally, the reference solution is prepared by taking a ligustilide reference substance, adding methanol to dissolve it, and obtaining a reference solution with a concentration of 0.1 mg / mL.

[0021] 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.

[0022] In the second aspect, the thin layer chromatography method provided in the present application for identifying the Radix Angelicae Dahuricae component in Coptis chinensis paste is used in identifying the Radix Angelicae Dahuricae component in Coptis chinensis paste.

[0023] In summary, this application has the following beneficial effects:

[0024] 1. In the preparation of the test solution of the present application, the Radix Angelicae Dahuricae component in the Coptis chinensis paste is extracted by using methanol water, and eluted with 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-mentioned 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.

[0025] 2. This application uses ethyl acetate-isopropanol with a volume ratio of (3-5):1 as a developing solvent, and the ratio shift value (RF value) in the chromatogram obtained is in the range of 0.2-0.8, and the characteristic spots are clear, bright, and regular, without impurity spots.

[0026] 3. The thin-layer chromatography method for identifying the Radix Angelicae Dahuricae component in the Coptis chinensis paste provided in this application has strong specificity, good durability, and can be applied to the identification of the Radix Angelicae Dahuricae components in different batches of Coptis chinensis paste, and has a good prospect for promotion and use.

[0027] 4. The thin layer chromatography method for identifying the Radix Angelicae Sinensis components in Coptis chinensis paste provided in this application has the advantages of low cost, simple operation, and short identification time compared with the liquid chromatography and gas chromatography detection methods in related technologies. It can meet the daily work of identifying the Radix Angelicae Sinensis components in Coptis chinensis paste and realize the quality control of Coptis chinensis paste. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a thin-layer chromatogram of the specificity investigation results; from left to right: 1 is the ligustilide reference solution; 2 is the HLG-1002-1073 Coptis chinensis paste test solution (normal pressure filtration); 3 is the HLG-1002-1078 Coptis chinensis paste negative test solution lacking Radix Angelicae Sinensis; 4 is the HLG-1002-1073 Coptis chinensis paste test solution (suction filtration); 5 is the HLG-1002-10788 Coptis chinensis paste negative test solution lacking Radix Angelicae Sinensis;

[0029] 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, 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.

[0030] Figure 3 Thin layer chromatograms obtained by 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; from left to right in each chromatogram are: 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 test solution lacking Radix Angelicae Sinensis; 4 is HLG-1002-1073 coptis paste test solution (suction filtration); 5 is HLG-1002-10788 coptis paste negative test solution lacking Radix Angelicae Sinensis. DETAILED DESCRIPTION

[0031] The present application provides a thin layer chromatography method for identifying the Radix Angelicae Dahuricae component in Coptis chinensis paste, comprising the following steps:

[0032] (1) Preparation of test solution: Add 75-85% methanol water to the coptis chinensis paste, heat to melt, then heat under reflux at 400-450 rpm with stirring 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; the filtration can be atmospheric pressure filtration or suction filtration.

[0033] (2) Prepare a reference solution; take the ligustilide reference solution, add methanol to dissolve it, and obtain a reference solution with a concentration of 0.1 mg / mL.

[0034] (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.

[0035] In this application, the prescription of Coptis chinensis paste is 20g of Coptis chinensis, 20g of Phellodendron chinense, 20g of Turmeric, 33g of Angelica sinensis, and 66g of Rehmannia glutinosa. The preparation method is as follows: mix the above 5 flavors, weigh 792g of sesame oil, heat it to 180-190°C, add the mixed slices and extract for 15 minutes, filter it, weigh 264g of beeswax, filter it after melting, stir it with the oil, let it cool, and make 1000g. The batch number of Coptis chinensis paste used in the examples of this application can be HLG-1002-1073, HLG-100 2-1166, HLG-1002-1167; the Huanglian paste negative sample lacking Radix Angelicae Dahuricae (batch number HLG-1002-1078 or HLG-1002-10788) was prepared by referring to the above-mentioned Huanglian paste prescription and method; Ligustilide was purchased from the China Food and Drug Administration; the 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 KGaA, Germany, and Qingdao Xinchanglai Silica Gel Co., Ltd. The raw materials, reagents, solvents, etc. used in the examples of this application can all be obtained commercially.

[0036] The present application is further described in detail below with reference to the embodiments, test experiments and accompanying drawings.

[0037] Example 1

[0038] Example 1 provides a thin layer chromatography method for identifying Radix Angelicae Dahuricae components in Coptis chinensis paste, comprising the following steps:

[0039] (1) Preparation of test solution: Add 80% methanol water to the coptis chinensis paste, heat to melt, then heat under reflux 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 to obtain a filtrate, evaporate to dryness, and dissolve to obtain the test solution;

[0040] (2) Prepare a reference solution; take the ligustilide reference solution, add methanol to dissolve it, and obtain a reference solution with a concentration of 0.1 mg / mL.

[0041] (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; then ethyl acetate-isopropanol with a volume ratio of 4:1 was used as the developing agent, and the silica gel G thin layer plate was placed in a developing tank for development and examined under a 365 nm light source.

[0042] Exclusive inspection

[0043] The samples of Coptis chinensis paste and Coptis chinensis paste negative and lacking Radix Angelicae Dahuricae were used as test samples and tested according to the method of Example 1. The results are as follows: Figure 1 shown.

[0044] Figure 1 This is the thin layer chromatogram of the specificity investigation results. ( Figure 1 In the table, 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 test solution lacking Radix Angelicae Sinensis; 4 is the HLG-1002-1073 coptis paste test solution (filtered by suction); 5 is the HLG-1002-10788 coptis paste negative test solution lacking Radix Angelicae Sinensis).

[0045] 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 negative sample solution of the Huanglian paste lacking Radix Angelicae Dahuricae, which indicates that the thin layer chromatography method provided in this application for identifying the Radix Angelicae Dahuricae component in the Huanglian paste has good specificity.

[0046] Durability inspection

[0047] (1) Durability inspection of thin layer board manufacturers

[0048] 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 chinensis paste was investigated. According to the method in Example 1, ligustilide reference solution, coptis chinensis paste test solution 1 (batch number HLG-1002-1166) and coptis chinensis paste test solution 2 (batch number HLG-1002-1167) were prepared respectively. 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.

[0049] 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.

[0050] Figure 2 The results show that the silica gel G thin layer plates produced by four manufacturers, namely Merck, Yantai Huayang, Yantai Jiangyou, and Qingdao Xinchanglai, can effectively separate Radix Angelicae Dahuricae from Coptis chinensis paste with good resolution and no impurity spots. Therefore, the thin layer chromatography method provided in this application for identifying Radix Angelicae Dahuricae in Coptis chinensis paste has good durability.

[0051] (2) Environmental durability assessment

[0052] The separation effect of the silica gel G thin layer plate produced by Yantai Jiangyou on Radix Angelicae Dahuricae in the coptis paste was investigated under the conditions of low temperature of 4°C and humidity of 92%. According to the method in Example 1, the ligustilide reference solution, HLG-1002-1073 coptis paste test solution (normal pressure filtration), HLG-1002-1078 coptis paste negative Radix Angelicae Dahuricae lacking Radix Angelicae Dahuricae test solution, HLG-1002-1073 coptis paste test solution (suction filtration) and HLG-1002-10788 coptis paste negative Radix Angelicae Dahuricae lacking Radix Angelicae Dahuricae test solution were prepared respectively. The above solutions were spotted on the silica gel G thin layer plate produced by Yantai Jiangyou, and separation and detection were carried out according to the method in Example 1. The results are as follows. Figure 3 shown.

[0053] Figure 3 Thin layer chromatograms obtained by 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; from left to right in each chromatogram are: 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 test solution lacking Radix Angelicae Sinensis; 4 is HLG-1002-1073 coptis paste test solution (suction filtration); 5 is HLG-1002-10788 coptis paste negative test solution lacking Radix Angelicae Sinensis.

[0054] 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 were slightly smaller, while the characteristic spots were normal in a 92% humidity environment, and the separation effect between adjacent components was good. Therefore, it shows that the thin layer chromatography method provided by this application for identifying the Radix Angelicae Dahuricae component in Huanglian Paste has good environmental durability.

[0055] Example 2-3

[0056] Examples 2-3 respectively provide a thin layer chromatography method for identifying Radix Angelicae Dahuricae components in Coptis chinensis paste.

[0057] The difference between the above embodiment and embodiment 1 lies in the type and ratio of the eluent, as shown in Table 1 below.

[0058] Comparative Example 1-2

[0059] Comparative Examples 1-2 respectively provide a thin layer chromatography method for identifying the Radix Angelicae Dahuricae component in Coptis chinensis paste.

[0060] The difference between the comparative example and Example 1 is: the type and ratio of the eluent, as shown in Table 1 below.

[0061] Table 1 Types and ratios of eluents used in Examples 2-3 and Comparative Examples 1-3

[0062] / Type and ratio of eluent Example 1 Dichloromethane-n-butanol with 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 with a volume ratio of 2:1 Comparative Example 2 Dichloromethane-n-butanol with a volume ratio of 4:1

[0063] Examples 4-5

[0064] Examples 4-5 respectively provide a thin layer chromatography method for identifying the Radix Angelicae Dahuricae component in Coptis chinensis paste.

[0065] The difference between the above embodiment and embodiment 1 lies in the type and ratio of the developing agent, as shown in Table 2 below.

[0066] Comparative Examples 3-5

[0067] Comparative Examples 3-5 respectively provide a thin layer chromatography method for identifying the Radix Angelicae Dahuricae component in Coptis chinensis paste.

[0068] The difference between the comparative example and Example 1 lies in the type and ratio of the developing agent, as shown in Table 2 below.

[0069] Table 2 Types and ratios of developing agents used in Examples 4-5 and Comparative Examples 3-5

[0070] / Type and ratio of developing agent Example 1 Ethyl acetate-isopropanol with a volume ratio of 4:1 Example 4 Ethyl acetate-isopropanol with a volume ratio of 3:1 Example 5 Ethyl acetate-isopropanol with a volume ratio of 5:1 Comparative Example 3 Ethyl acetate-isopropanol with a volume ratio of 2:1 Comparative Example 4 Ethyl acetate-isopropanol with a volume ratio of 6:1 Comparative Example 5 Toluene-ethyl acetate-formic acid with a volume ratio of 10:4:0.1

[0071] Test results

[0072] 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, number of characteristic spots and ratio shift value RF value in the chromatogram were obtained. The results are shown in Table 3 below.

[0073] (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.

[0074] (2) 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.

[0075] Table 2 Detection results of Huanglian paste obtained by thin layer chromatography method of Examples 1-7 and Comparative Examples 1-5

[0076]

[0077]

[0078] 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 was used as the eluent for preparing the test solution, and ethyl acetate-isopropanol with a volume ratio of (3-5): 1 was used as the developing solvent. The obtained chromatogram had a clear characteristic spot, and the characteristic spot was clear, bright, and regular, without impurity spots. Therefore, it is shown that the thin layer chromatography method provided in the present application can effectively identify the Radix Angelicae Pubescentis components in Coptis chinensis paste, and the separation and identification effect is good.

[0079] 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 two impurity spots in the obtained chromatogram, indicating that the test solution prepared with the above eluent contained an impurity matrix and its purity was slightly poor, which affected the identification effect of the Radix Angelicae Pubescentis component in the Coptis chinensis paste.

[0080] In the thin-layer chromatography methods provided in Comparative Examples 3-5, using ethyl acetate-isopropanol (volume ratio: 2:1), ethyl acetate-isopropanol (volume ratio: 6:1), or toluene-ethyl acetate-formic acid (volume ratio: 10:4:0.1) as the developing solvent, the resulting chromatograms exhibited unclear spot boundaries and tailing, and in addition to the characteristic spots, there was also one impurity spot. Therefore, none of the three developing solvents described above were effective in identifying the Radix Glycyrrhizae Radix in Coptis chinensis paste.

[0081] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A thin layer chromatography method for identifying Radix Angelicae Pubescentis components in Coptis chinensis paste, characterized in that: The following steps are involved: (1) Prepare the test solution: add 75-85% methanol water to the coptis chinensis paste, heat to melt, then heat under reflux at 400-450 rpm with stirring 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, obtain the filtrate, evaporate to dryness, and dissolve to obtain the 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 inspect 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 silica gel column filtration adopts normal pressure filtration or suction filtration.

6. The thin layer chromatography method according to claim 1, characterized in that The preparation method of the reference solution is as follows: taking a ligustilide reference substance, adding methanol to dissolve it, and obtaining a reference solution with a concentration of 0.1 mg / mL.

7. The thin layer chromatography method according to any one of claims 1 to 6, characterized in that The inspection criteria are: checking whether the characteristic fluorescent spot of the same color appears in the chromatogram of the test solution at the corresponding position of the chromatogram of the ligustilide reference 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, then the tested Coptis chinensis paste contains Radix Angelicae Pubescentis components.

8. Use of the thin layer chromatography method for identifying the Radix Angelicae Dahuricae component in Coptis chinensis paste according to any one of claims 1 to 7 in identifying the Radix Angelicae Dahuricae component in Coptis chinensis paste.

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