Thin-layer chromatography identification method for curcumin in rhizoma coptidis paste

Abstract

The invention relates to the technical field of identification of traditional Chinese medicine preparations, and particularly discloses a thin-layer chromatography identification method for curcumin in rhizoma coptidis paste. The method specifically comprises the following steps: respectively taking a rhizoma coptidis paste test solution, a rhizoma curcumae longae reference medicinal material solution and a curcumin reference substance solution, carrying out strip-shaped sample application on the same silica gel G thin-layer plate, taking ethyl acetate, ethanol and acetic acid in a volume ratio of (7-9): (0.5-1.5): (0.5-1.5) as a developing solvent, developing, taking out, airing and inspecting. By optimizing a pretreatment extraction method and chromatographic conditions, the interference of a complex matrix in the ointment on thin-layer chromatography is effectively reduced, and a thin-layer chromatography analysis method for curcumin in the coptis chinensis ointment is established; the identification method provided by the invention has good specificity, durability and solution stability.

Description

Technical Field

[0001] The present application relates to the technical field of identification of traditional Chinese medicine preparations, and in particular to a thin layer chromatography identification method of curcumin in coptis paste. Background Art

[0002] Curcumin is a phenolic compound found in the rhizome extract of the ginger plant turmeric. It has many pharmacological effects, including anti-inflammatory, anti-platelet aggregation, anti-coagulation, antioxidant, anti-tumor, inhibition of smooth muscle cell proliferation, and lipid-lowering.

[0003] Huanglian ointment is an important external preparation in Chinese medicine hospitals across the country. Its main indications include hairline sores, stye, nasal sores, lip rash, bandages, smelly snails, etc. It has been used for more than 30 years. Its main ingredients include turmeric. At present, the thin layer chromatography identification of turmeric is mainly focused on turmeric medicinal pieces or their water extracts. Due to the interference of the complex matrix in the ointment, there has been no thin layer study on turmeric in the ointment. Summary of the invention

[0004] In order to solve the above technical problems, the present application provides a thin layer chromatography identification method for curcumin in Coptis chinensis paste.

[0005] The present application provides a thin layer chromatography identification method for curcumin in coptis paste, which is characterized by comprising the following steps in sequence: taking coptis paste test solution, turmeric reference medicinal material solution, and curcumin reference solution respectively, spotting them on the same silica gel G thin layer plate in strip shape, using ethyl acetate, ethanol, and acetic acid in a volume ratio of 7-9:0.5-1.5:0.5-1.5 as a developing agent, developing, taking out, drying, and inspecting.

[0006] When performing thin layer chromatography identification, due to the complexity of the components in the Coptis chinensis paste and the different properties of each component, the difficulties that need to be overcome during the identification process include: the reference substance does not show color, the test substance or reference substance solution does not develop completely, the spots are not stretched enough, etc.

[0007] In response to the above problems, this study effectively reduced the interference of complex matrix in the ointment on thin layer chromatography by optimizing the pretreatment extraction method and chromatographic conditions, and established a thin layer chromatography analysis method for the active ingredient curcumin in Coptis chinensis ointment.

[0008] Preferably, the developing agent has a volume ratio of ethyl acetate, ethanol and acetic acid of 7.5-8.5:0.7-1.3:0.7-1.3.

[0009] Preferably, the developing agent volume ratio is 7.8-8.2:0.9-1.1:0.9-1.1 of ethyl acetate, ethanol, acetic acid. In a specific embodiment, the developing agent volume ratio is 8:1:1 of ethyl acetate, ethanol, acetic acid. Preferably, the developing environmental conditions are: temperature of 4-30°C, humidity of 10%-92.5%.

[0010] Preferably, the preparation method of the test solution of Coptis chinensis paste is: take the Coptis chinensis paste test sample, add a methanol aqueous solution with a volume fraction of 40-60% according to the solid-liquid ratio of 1g:4-8mL, heat to melt and stir and reflux for 20-40min, let it reach room temperature, and after solidification, discard the solidified paste, take the supernatant and filter, evaporate the filtrate, and dissolve the residue in methanol until the residue concentration is 3-5mg / mL as the test solution.

[0011] Preferably, the preparation method of the test solution of Coptis chinensis paste is as follows: take the Coptis chinensis paste test sample, add a methanol aqueous solution with a volume fraction of 40-60% according to a solid-liquid ratio of 1g:5-7mL, heat to melt and stir and reflux for 20-40min, let it reach room temperature, and store it in ice water for 20-40min. After solidification, discard the solidified paste, take the supernatant for filtration, evaporate the filtrate, and dissolve the residue in methanol until the residue concentration is 3-5mg / mL as the test solution.

[0012] Preferably, the preparation method of the Coptidis ointment sample is: mix Coptidis, Phellodendron, Curcuma, Angelica Root, and Rehmannia Glutinosa, add sesame oil, heat to 180-190° C., extract for 10-40 minutes, and filter to obtain oil; weigh beeswax, filter it after melting, mix it with the oil, and cool it to obtain the product.

[0013] Preferably, the preparation method of the turmeric control medicinal material solution is: take turmeric control medicinal material, add a methanol aqueous solution with a volume fraction of 40-60% according to a solid-liquid ratio of 1 mg: 1-2 mL, stir and reflux for 20-40 minutes, let it cool to room temperature, filter, evaporate the filtrate to dryness, add methanol to dissolve the residue to a residue concentration of 4-8 mg / mL as the control medicinal material solution.

[0014] Preferably, the preparation method of the curcumin reference solution is: taking a curcumin reference substance, adding methanol to prepare a solution with a concentration of 0.03-0.08 mg / mL as the reference solution.

[0015] Preferably, the inspection method is: inspecting under ultraviolet light at 365 nm.

[0016] In summary, the technical solution of this application has the following effects:

[0017] The present application effectively reduces the interference of complex matrix in the ointment on thin layer chromatography by optimizing the pretreatment extraction method and chromatographic conditions, and establishes a thin layer chromatography analysis method for the active ingredient curcumin in the coptis paste; and the identification method provided by the present application has good specificity, durability and solution stability.

[0018] The present application improves the sensitivity of the thin layer chromatography identification method of curcumin in coptis paste by specifically selecting a developing agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The figure is the thin layer identification chromatogram of the solution in the specificity investigation result in Example 1.

[0020] Figure 2 This is the thin layer identification chromatogram of the silica gel plate produced by Yantai Huayang New Materials Co., Ltd. in the durability investigation results in Example 1.

[0021] Figure 3 This is the thin layer identification chromatogram of the silica gel plate produced by Qingdao Dingkang Silica Gel Co., Ltd. in the durability investigation results in Example 1.

[0022] Figure 4 This is the thin layer identification chromatogram of the silica gel plate produced by the Qingdao Ocean Chemical Plant Branch in the durability investigation results in Example 1.

[0023] Figure 5 This is the thin layer identification chromatogram under low temperature (4°C) environmental conditions in the durability investigation results in Example 1.

[0024] Figure 6 This is the thin layer identification chromatogram under high humidity (25°C, 90%RH) environmental conditions in the durability investigation results in Example 1.

[0025] Figure 7 This is the thin layer identification chromatogram of the solution in Example 1 when the solution was stored at -20°C for 1 day.

[0026] Figure 8 This is the thin layer identification chromatogram of the solution in Example 1 when the solution was stored at -20°C for 11 days.

[0027] Fig. 9 This is the thin layer identification chromatogram of the solution in Example 1 when the solution was stored at -20°C for 30 days. DETAILED DESCRIPTION

[0028] The present application is further described in detail below in conjunction with examples, comparative examples and performance testing experiments. These examples should not be construed as limiting the scope of protection claimed in the present application.

[0029] Example

[0030] Example 1

[0031] Example 1 provides a thin layer chromatography identification method for curcumin in coptis paste.

[0032] The preparation method of the test sample of Coptis chinensis paste is as follows: homemade, weigh 20g of Coptis chinensis, 20g of Phellodendron chinense, 20g of Curcuma longa, 33g of Angelica sinensis root, and 66g of Rehmannia glutinosa, mix them, then add 792g of sesame oil, heat to 180-190℃, extract for 15min, and filter to obtain oil; weigh 264g of beeswax, filter it after melting, stir it with the oil, and cool it to obtain the product.

[0033] The preparation method of the test solution of Coptis chinensis paste is as follows: take 5g of Coptis chinensis paste, add 30ml of 50% methanol aqueous solution, heat to melt, stir (400-450rpm) and reflux for 30min, let it reach room temperature, store it in ice water for 30min, discard the solidified paste, take the supernatant and filter it, evaporate the filtrate, add 2ml of methanol to the residue to dissolve it, and use it as the test solution.

[0034] The preparation method of turmeric control medicinal material solution is as follows: take 20 mg of turmeric control medicinal material, add 30 ml of 50% methanol aqueous solution, stir (400-450 rpm) and reflux for 30 minutes, let it reach room temperature, filter, evaporate the filtrate to dryness, add appropriate amount of methanol to dissolve the residue, and dilute to 5 ml as the control medicinal material solution.

[0035] The preparation method of the curcumin reference solution is as follows: take the curcumin reference substance, add methanol to make a solution containing 0.05 mg per 1 ml, which is used as the reference solution.

[0036] The preparation method of the negative turmeric-deficient test sample is as follows: homemade, weigh 20g of Coptis chinensis, 20g of Phellodendron amurense, 33g of Angelica sinensis root, and 66g of Rehmannia glutinosa, mix them, then add 792g of sesame oil, heat to 180-190℃, extract for 15min, and filter to obtain oil; weigh 264g of beeswax, filter it after melting, mix it with the oil, and cool it to obtain the product.

[0037] The preparation method of the negative turmeric-deficient test solution is as follows: take 5 g of Coptis chinensis paste, add 30 ml of 50% methanol aqueous solution, heat to melt, stir (400-450 rpm) and reflux for 30 minutes, let it cool to room temperature, and store it in ice water for 30 minutes, discard the solidified paste, take the supernatant and filter it, evaporate the filtrate, add 2 ml of methanol to the residue to dissolve it, and use it as the test solution.

[0038] Thin layer chromatography analysis: 5 μl of each of the three solutions mentioned above were taken and spotted in strips on the same silica gel G thin layer plate. Ethyl acetate, ethanol and acetic acid in a volume ratio of 8:1:1 were used as the developing agent. The plate was developed, taken out, dried and examined under ultraviolet light (365 nm).

[0039] (1) Technical effect-specificity investigation

[0040] The silica gel G thin layer plate from Qingdao Ocean Chemical Plant was used for testing at 22-24°C and 65.4% RH. The thin layer identification chromatogram of the solution in the specificity test results is as follows: Figure 1 As shown (1 represents: curcumin reference solution; 2 represents: turmeric reference medicinal material solution; 3 represents: coptis paste test solution; 4 represents: negative turmeric test solution).

[0041] Figure 1 The results showed that the components in the Huanglian paste test solution were well separated, and three fluorescent spots of the same color appeared at the corresponding positions of the chromatogram of the turmeric reference medicinal material; and fluorescent spots of the same color appeared at the corresponding positions of the chromatogram of the curcumin reference substance. There was no interference at the corresponding positions in the chromatogram of the negative test sample lacking turmeric, indicating that the thin layer chromatography identification method provided by the present application has good specificity.

[0042] (2) Technical effect-durability inspection

[0043] (2.1) Durability study of silicone sheets from different manufacturers

[0044] The silica gel G thin layer plates produced by Yantai Huayang New Materials Co., Ltd., Qingdao Dingkang Silica Gel Co., Ltd., and Qingdao Ocean Chemical Plant Branch were inspected under the environmental conditions of 22-24°C and 28-34% RH. The thin layer identification chromatogram of the silica gel plate produced by Yantai Huayang New Materials Co., Ltd. is shown in the following figure: Figure 2 As shown in the durability test results, the thin layer identification chromatogram of the silica gel plate produced by Qingdao Dingkang Silica Gel Co., Ltd. is as follows Figure 3 As shown in the durability test results, the thin layer identification chromatogram of the silica gel plate produced by Qingdao Ocean Chemical Plant Branch is as follows: Figure 4 As shown, (1 represents: curcumin reference solution; 2 represents: turmeric reference medicinal material solution; 3 represents: coptis paste test solution No. 1; 4 represents: coptis paste test solution No. 2).

[0045] Figure 2-4 The results showed that the components in the test solution of Coptis chinensis paste were well separated on silica gel G thin layer plates from different manufacturers.

[0046] (2.2) Environmental durability study

[0047] We selected the silica gel G thin layer plate from Qingdao Ocean Chemical Plant Branch and inspected it under low temperature (4°C) and high humidity (25°C, 90% RH) environmental conditions to investigate its environmental durability. The results of the durability inspection showed that the thin layer identification chromatogram under low temperature (4°C) environmental conditions was as follows: Figure 5As shown in the durability test results, the thin layer identification chromatogram under high humidity (25°C, 90%RH) environment conditions is as follows Figure 6 As shown (1 represents: curcumin reference solution; 2 represents: turmeric reference medicinal material solution; 3 represents: coptis paste test solution No. 1; 4 represents: coptis paste test solution No. 2).

[0048] Figure 5 The results showed that the spots under low temperature (4°C) were slightly smaller, and the Rf value of the spots was reduced, and the components in the Huanglian paste test solution were well separated; the results under high humidity (25°C, 90% RH) showed that the components in the Huanglian paste test solution were well separated. The results showed that the three components related to turmeric could be effectively separated under two extreme conditions.

[0049] The durability tests on silica gel plates from different manufacturers and the environment showed that the thin layer chromatography identification method provided in the present application has good durability.

[0050] (3) Technical effect - solution stability study

[0051] The Huanglian paste test solution, turmeric reference medicinal material solution, and curcumin reference solution were stored at -20°C, and development experiments were carried out at 1 day, 11 days, and 30 days (development and inspection were carried out at 22-24°C, 40-50% RH) to observe the stability of each solution. The thin layer identification chromatogram of the solution stored at -20°C for 1 day is as follows: Figure 7 As shown in the results of the solution stability test, the thin layer identification chromatogram of the solution stored at -20℃ for 11 days is as follows Figure 8 As shown in the results of the solution stability test, the thin layer identification chromatogram of the solution stored at -20°C for 30 days is as follows Fig. 9 As shown (1 represents: curcumin reference solution; 2 represents: turmeric reference medicinal material solution; 3 represents: coptis paste test solution).

[0052] Figure 7 , Figure 8 , Fig. 9 The results showed that the test solution of Coptis chinensis paste, the reference medicinal material solution of turmeric, and the reference solution of curcumin had good stability when stored at -20℃ for 30 days.

[0053] Embodiment 2-5

[0054] Examples 2-5 respectively provide a thin layer chromatography identification method for curcumin in Coptis chinensis paste.

[0055] The difference between the above embodiment and embodiment 1 is that the composition of the developing agent is different, as shown below.

[0056] In Example 2, ethyl acetate, ethanol and acetic acid in a volume ratio of 7:1.5:0.5 were used as developing agents.

[0057] In Example 3, ethyl acetate, ethanol and acetic acid in a volume ratio of 9:0.5:1.5 were used as developing solvents.

[0058] In Example 4, ethyl acetate, ethanol and acetic acid in a volume ratio of 7.5:0.7:1.3 were used as developing solvents.

[0059] In Example 5, ethyl acetate, ethanol and acetic acid in a volume ratio of 8.5:1.3:0.7 were used as developing solvents.

[0060] The remaining process parameters in the above embodiment are the same as those in embodiment 1.

[0061] The results of the thin layer identification chromatogram show that the components in the Coptis chinensis paste test solution are well separated, and there is no interference at the corresponding positions in the chromatogram of the negative test sample lacking turmeric, indicating that the thin layer chromatography identification method provided in this application has good specificity.

[0062] Example 6

[0063] Example 6 investigates the sensitivity of the thin layer chromatography identification method for curcumin in Coptis chinensis paste.

[0064] The test solution of the coptis chinensis paste prepared in Example 1 was used as the sample to be tested, and gradient dilution was performed according to 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, and 1:256 times, respectively. Then, the spots of the coptis chinensis paste test solution were inspected using the thin layer chromatography identification method provided in Examples 1-5 (the scoring criteria were formulated with reference to the following points) to examine the sensitivity of the thin layer chromatography identification method.

[0065] (1) Spot clarity:

[0066] The sharpness of the spot edge, whether blurring, tailing, etc. occur.

[0067] The uniformity of the spots within, whether there are rings, spot splitting, or multiple spots overlapping.

[0068] (2) Brightness of spots:

[0069] The depth of the spots' color, whether they are vivid, and the contrast with the background color.

[0070] Under UV light, whether the spot fluoresces and its fluorescence intensity.

[0071] (3) Spot shape and size:

[0072] Whether the spots are regular circles or nearly circular.

[0073] Whether the spot size is moderate, too large or too small may affect the accuracy of the score.

[0074] (4) Background interference:

[0075] Check whether the color is uniform and whether there are impurities or stains that interfere with the observation of the spots.

[0076] The degree of interference of other surrounding spots on the observation of the target spot.

[0077] (5) Scoring details:

[0078] 5 points: The spots are clear, with sharp edges, bright colors and high contrast, without background interference, regular shapes and moderate sizes.

[0079] 4 points: The spots are relatively clear and bright in color, with slight background interference or deviation in shape and size.

[0080] 3 points: The spot clarity is average, the color is slightly light, there is a certain degree of background interference or the shape and size are inconsistent.

[0081] 2 points: The spots are blurred, the color is dull, the background interference is serious, or the shape and size obviously do not meet the standards.

[0082] 1 point: The spots are extremely unclear, almost unrecognizable, or completely obscured by other factors.

[0083] The test results are shown in Table 1.

[0084] Table 1 Sensitivity of TLC identification method of curcumin in Huanglian paste in Examples 1-5

[0085]

[0086]

[0087] Comparative Example

[0088] Comparative Examples 1-4

[0089] Comparative Examples 1-4 respectively provide a thin layer chromatography identification method for curcumin in Coptis chinensis paste.

[0090] The difference between the comparative example and Example 1 is that the composition of the developing agent is different, as shown below.

[0091] In comparative example 1, chloroform, methanol and formic acid in a volume ratio of 6:0.8:0.1 were used as developing solvents. The results of the thin layer identification chromatogram showed that the Rhizoma Coptidis Ointment test solution and the Rhizoma Curcumae control medicinal material solution in this comparative example were not fully developed and the spots were not stretched enough; the spots of other components in the Rhizoma Coptidis Ointment test solution had serious interference.

[0092] In comparative example 2, chloroform, ethyl acetate, formic acid and water in a volume ratio of 12:24:7:4 were used as developing solvents. The results of the thin layer identification chromatogram showed that the Rhizoma Coptidis Ointment test solution and the Rhizoma Curcumae control medicinal material solution in this comparative example were not fully developed and the spots were not stretched enough; the spots of other components in the Rhizoma Coptidis Ointment test solution were interfering.

[0093] In comparative example 3, ethyl acetate, methanol and formic acid were used as developing solvents in a volume ratio of 7:1.5:0.5. The results of the thin layer identification chromatogram showed that the Huanglian paste test solution and the turmeric control medicinal material solution in this comparative example were not fully developed and the spots were not stretched enough.

[0094] In comparative example 4, ethyl acetate, ethanol and acetic acid in a volume ratio of 6:3:3 were used as developing solvents. The results of the thin layer identification chromatogram showed that the Huanglian paste test solution and the turmeric control medicinal material solution in this comparative example were not fully developed and the spots were not stretched enough.

[0095] The other process parameters in the above comparative example are the same as those in Example 1.

[0096] 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 identification method for curcumin in coptis paste, characterized in that: Specifically, the following steps are performed in sequence: respectively taking the Huanglian paste test solution, the turmeric reference medicinal material solution, and the curcumin reference solution, and spotting them in strips on the same silica gel G thin layer plate, using a volume ratio of 7-9: 0.5-1.5: Use 0.5-1.5 ethyl acetate, ethanol, or acetic acid as the developing agent, develop, take out, dry, and inspect.

2. The thin layer chromatography identification method of curcumin in the coptis chinensis paste according to claim 1, characterized in that: The developing agent has a volume ratio of ethyl acetate, ethanol and acetic acid of 7.5-8.5:0.7-1.3:0.7-1.

3.

3. The thin layer chromatography identification method of curcumin in the coptis chinensis paste according to claim 2, characterized in that: The developing agent has a volume ratio of ethyl acetate, ethanol and acetic acid of 7.8-8.2:0.9-1.1:0.9-1.

1.

4. The thin layer chromatography identification method of curcumin in the coptis chinensis paste according to claim 1, characterized in that: The environmental conditions for the development are: temperature of 4-30° C. and humidity of 10%-92.5%.

5. The thin layer chromatography identification method of curcumin in the coptis chinensis paste according to claim 1, characterized in that: The preparation method of the test solution of Coptis chinensis paste is as follows: take the test sample of Coptis chinensis paste, add a methanol aqueous solution with a volume fraction of 40-60% according to a solid-liquid ratio of 1g:4-8mL, heat to melt, stir and reflux for 20-40min, let it cool to room temperature, and after solidification, discard the solidified paste, take the supernatant for filtration, evaporate the filtrate, and dissolve the residue in methanol until the residue concentration is 3-5mg / mL as the test solution.

6. The thin layer chromatography identification method of curcumin in Huanglian paste according to claim 1, characterized in that: The preparation method of the test solution of Coptis chinensis paste is as follows: take the test sample of Coptis chinensis paste, add a methanol aqueous solution with a volume fraction of 40-60% according to a solid-liquid ratio of 1g:5-7mL, heat to melt and stir and reflux for 20-40min, let it reach room temperature, and store it in ice water for 20-40min. After solidification, discard the solidified paste, take the supernatant for filtration, evaporate the filtrate, and dissolve the residue in methanol until the residue concentration is 3-5mg / mL, which is used as the test solution.

7. The thin layer chromatography identification method of curcumin in Huanglian paste according to any one of claims 5-6, characterized in that: The preparation method of the test sample of the coptis paste is as follows: mix coptis root, phellodendron, turmeric, angelica root, and raw rehmannia, add sesame oil, heat to 180-190° C., extract for 10-40 minutes, and filter to obtain oil; weigh beeswax, filter it after it is melted, mix it with the oil, and cool it to obtain the product.

8. The thin layer chromatography identification method of curcumin in Huanglian paste according to claim 1, characterized in that: The preparation method of the turmeric control medicinal material solution is as follows: taking turmeric control medicinal material, adding a methanol aqueous solution with a volume fraction of 40-60% according to a solid-liquid ratio of 1 mg: 1-2 mL, stirring and refluxing for 20-40 minutes, cooling to room temperature, filtering, evaporating the filtrate, and dissolving the residue with methanol to a residue concentration of 4-8 mg / mL as the control medicinal material solution.

9. The thin layer chromatography identification method of curcumin in Huanglian paste according to claim 1, characterized in that: The preparation method of the curcumin reference solution is as follows: taking a curcumin reference, adding methanol to prepare a solution with a concentration of 0.03-0.08 mg / mL as the reference solution.

10. The thin layer chromatography identification method of curcumin in Huanglian paste according to claim 1, characterized in that: The inspection method is: inspect under ultraviolet light at 365 nm.

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