Method for identifying curcumin in huanglian paste by thin layer chromatography

Abstract

The application relates to the technical field of traditional Chinese medicine preparation identification, and specifically discloses a thin-layer chromatography identification method for curcumin in Huanglian paste. The application specifically comprises the following steps: respectively taking Huanglian paste test sample solution, turmeric control medicine solution and curcumin control sample solution, spotting the solutions on a same silica gel G thin-layer plate in a strip shape, using ethyl acetate, ethanol and acetic acid with a volume ratio of 7-9:0.5-1.5:0.5-1.5 as a developing agent, developing, taking out, air-drying and observing. The application effectively reduces the interference of complex matrix in the paste on the thin-layer chromatography by optimizing the pretreatment extraction method and the chromatography condition, and establishes a thin-layer chromatography analysis method for curcumin in Huanglian paste; and the identification method provided by the application has good specificity, durability and solution stability.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of identification of traditional Chinese medicine preparations, and particularly relates to a thin-layer chromatography identification method for curcumin in Huanglian paste. BACKGROUND

[0002] Curcumin is a phenolic compound in rhizome extracts of Curcuma longa, and has pharmacological effects of anti-inflammatory reaction, anti-platelet aggregation, anticoagulation, antioxidation, antitumor, inhibition of smooth muscle cell proliferation, and blood lipid reduction.

[0003] Huanglian paste is an important external preparation in Chinese medicine hospitals across the country, and is used for treating scalp sores, needle eyes, nasal sores, lip wind, finger, and smelly snails, and has been applied for more than 30 years. The main component of Huanglian paste includes curcumin. At present, the thin-layer chromatography identification of curcumin mainly focuses on curcumin medicinal materials and decoction pieces or water extracts thereof. Due to the interference of complex matrix in the paste, the thin-layer chromatography of curcumin in the paste has not been reported. SUMMARY

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

[0005] The application provides a thin-layer chromatography identification method for curcumin in Huanglian paste, and the method is characterized in that the following steps are sequentially performed: respectively taking a Huanglian paste test sample solution, a curcumin medicinal material solution, and a curcumin reference substance solution, spotting the solutions on a same silica gel G thin-layer plate in a strip shape, using ethyl acetate, ethanol, and acetic acid with a volume ratio of 7-9:0.5-1.5:0.5-1.5 as a developing agent, developing, taking out, air-drying, and observing.

[0006] When the thin-layer chromatography identification is performed, due to the complexity of components in the Huanglian paste, the properties of the components are different, and in the identification process, the difficulties to be overcome include: the reference substance does not develop color, the test sample solution or the reference substance solution is not completely developed, and the spots are not fully expanded, and the like.

[0007] In view of the above problems, the pre-treatment extraction method and the chromatography condition are optimized in the research, the interference of the complex matrix in the paste on the thin-layer chromatography is effectively reduced, and a thin-layer chromatography analysis method for curcumin effective components in Huanglian paste is established.

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

[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 environment conditions are: temperature is 4-30℃, humidity is 10%-92.5%.

[0010] Preferably, the preparation method of the Coptis ointment test sample solution is: taking Coptis ointment test sample, adding 40-60% methanol aqueous solution according to the solid-liquid ratio of 1g:4-8mL, heating and melting, then stirring and refluxing for 20-40min, placing at room temperature, discarding the solidified ointment body after solidification, filtering the supernatant, evaporating the filtrate to dryness, dissolving the residue in methanol to a residue concentration of 3-5mg / mL as the test sample solution.

[0011] Preferably, the preparation method of the Coptis ointment test sample solution is: taking Coptis ointment test sample, adding 40-60% methanol aqueous solution according to the solid-liquid ratio of 1g:5-7mL, heating and melting, then stirring and refluxing for 20-40min, placing at room temperature, storing in ice water for 20-40min, discarding the solidified ointment body after solidification, filtering the supernatant, evaporating the filtrate to dryness, dissolving the residue in methanol to a residue concentration of 3-5mg / mL as the test sample solution.

[0012] Preferably, the preparation method of the Coptis ointment test sample is: mixing Coptis, Cortex Phellodendri, Curcuma longa, Angelica sinensis tail, Radix Rehmanniae, then adding sesame oil, heating and warming to 180-190℃, extracting for 10-40min, filtering to obtain oil; weighing beeswax, filtering after melting, stirring with the oil, and cooling to prepare.

[0013] Preferably, the preparation method of the Curcuma longa reference medicinal material solution is: taking Curcuma longa reference medicinal material, adding 40-60% methanol aqueous solution according to the solid-liquid ratio of 1mg:1-2mL, stirring and refluxing for 20-40min, placing at room temperature, filtering, evaporating the filtrate to dryness, dissolving the residue in methanol to a residue concentration of 4-8mg / mL as the reference medicinal material solution.

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

[0015] Preferably, the method of inspection is: placed under ultraviolet light 365nm for inspection.

[0016] In summary, the technical scheme of the present application has the following effects:

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

[0018] The application improves the sensitivity of the thin layer chromatography identification method for curcumin in Huanglian paste by specific selection of developing agent. BRIEF DESCRIPTION OF DRAWINGS

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

[0020] Figure 2 The thin layer identification chromatogram of the silica gel plate of Yantai Huayang New Material Co., Ltd. in the durability investigation result in Example 1.

[0021] Figure 3 The thin layer identification chromatogram of the silica gel plate of Qingdao Dingkang Silica Gel Co., Ltd. in the durability investigation result in Example 1.

[0022] Figure 4 The thin layer identification chromatogram of the silica gel plate of Qingdao Haizhuan Chemical Factory Branch Factory in the durability investigation result in Example 1.

[0023] Figure 5 The thin layer identification chromatogram under low temperature (4℃) environmental conditions in the durability investigation result in Example 1.

[0024] Figure 6 The thin layer identification chromatogram under high humidity (25℃, 90% RH) environmental conditions in the durability investigation result in Example 1.

[0025] Figure 7 The thin layer identification chromatogram of the solution stored at-20℃ for 1 day in the solution stability investigation result in Example 1.

[0026] Figure 8 The thin layer identification chromatogram of the solution stored at-20℃ for 11 days in the solution stability investigation result in Example 1.

[0027] Figure 9 The thin layer identification chromatogram of the solution stored at-20℃ for 30 days in the solution stability investigation result in Example 1. DETAILED DESCRIPTION

[0028] The application will be further described in detail below in combination with examples, comparative examples and performance test experiments, and these examples cannot be understood as limiting the scope of the application claimed.

[0029] Example

[0030] Example 1

[0031] Example 1 provides a method for identifying curcumin in Huanglian paste by thin layer chromatography.

[0032] The preparation method of Huanglian paste test sample is as follows: Huanglian 20 g, Huangbai 20 g, Jianghuang 20 g, Dangguitai 33 g, Shengdi 66 g are mixed, then sesame oil 792 g is added, heated to 180-190℃, extracted for 15 min, filtered to obtain oil liquid; take beeswax 264 g, melt, filter, mix with oil liquid, cool, and obtain.

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

[0034] The preparation method of Jiaohuang control drug material solution is as follows: take Jiaohuang control drug material 20 mg, add 50% methanol aqueous solution 30 ml, stir (400-450 rpm) for 30 min, cool to room temperature, filter, evaporate the filtrate to dryness, dissolve the residue in appropriate amount of methanol, dilute to 5 ml, and obtain the control drug material solution.

[0035] The preparation method of curcumin control solution is as follows: take curcumin control, add methanol to prepare a solution containing 0.05 mg per 1 ml, and obtain the control solution.

[0036] The preparation method of negative test sample without Jiaohuang is as follows: Huanglian 20 g, Huangbai 20 g, Dangguitai 33 g, Shengdi 66 g are mixed, then sesame oil 792 g is added, heated to 180-190℃, extracted for 15 min, filtered to obtain oil liquid; take beeswax 264 g, melt, filter, mix with oil liquid, cool, and obtain.

[0037] The preparation method of negative test sample solution without Jiaohuang is as follows: take Huanglian paste 5 g, add 50% methanol aqueous solution 30 ml, heat and melt, stir (400-450 rpm) for 30 min, cool to room temperature, store in ice water for 30 min, discard the solidified paste, filter the supernatant, evaporate the filtrate to dryness, add methanol 2 ml to dissolve, and obtain the test sample solution.

[0038] Thin layer chromatography analysis: take 5 μl of each of the above three solutions, spot on the same silica gel G thin layer plate in strip shape, use ethyl acetate, ethanol, and acetic acid in a volume ratio of 8:1:1 as developing agent, develop, take out, dry, and observe under ultraviolet light (365 nm).

[0039] (1) Technical effect - specificity investigation

[0040] The thin layer chromatography was performed on the silica gel G thin layer plate of Qingdao Marine Chemical Plant Branch at 22-24℃ and 65.4% RH. The thin layer identification chromatogram of the solution in the specificity investigation results is shown in Figure 1 (1 represents curcumin reference solution; 2 represents turmeric reference medicinal material solution; 3 represents Huanglian paste test solution; 4 represents negative test solution without turmeric).

[0041] Figure 1 The results show that the components in the Huanglian paste test solution are well separated, and three fluorescent spots of the same color appear at the positions corresponding to the turmeric reference medicinal material chromatogram; the same color fluorescent spot appears at the position corresponding to the curcumin reference chromatogram. There is no interference at the corresponding position in the negative test chromatogram without turmeric, indicating that the thin layer chromatography identification method provided in the present application has good specificity.

[0042] (2) Technical effect - durability investigation

[0043] (2.1) Durability investigation of silica gel plates from different manufacturers

[0044] The thin layer chromatography was performed on the silica gel G thin layer plate of Qingdao Marine Chemical Plant Branch at 22-24℃ and 65.4% RH. The thin layer identification chromatogram of the solution in the specificity investigation results is shown in Figure 2 , the thin layer identification chromatogram of the solution in the specificity investigation results is shown in Figure 3 , and the thin layer identification chromatogram of the solution in the specificity investigation results is shown in Figure 4 (1 represents curcumin reference solution; 2 represents turmeric reference medicinal material solution; 3 represents Huanglian paste test solution No. 1; 4 represents Huanglian paste test solution No. 2).

[0045] Figures 2-4 The results show that the components in the Huanglian paste test solution are well separated on the silica gel G thin layer plate from different manufacturers.

[0046] (2.2) Environmental durability investigation

[0047] The silica gel G thin layer plate of Qingdao Marine Chemical Plant Branch was selected and investigated under low temperature (4℃) and high humidity (25℃, 90% RH) environmental conditions to investigate the environmental durability. The thin layer identification chromatogram under low temperature (4℃) environmental conditions is shown in Figure 5The thin layer chromatogram of the result in the high humidity (25℃, 90% RH) environment condition in the durability investigation is shown in Figure 2. Figure 6 The thin layer chromatogram of the result in the high humidity (25℃, 90% RH) environment condition in the durability investigation is shown in Figure 2.

[0048] Figure 5 The result shows that the spots are slightly smaller and the Rf values of the spots are reduced under the low temperature (4℃) environment condition, and the components in the Huanglian Gao test solution are well separated; the result under the high humidity (25℃, 90% RH) environment condition shows that the components in the Huanglian Gao test solution are well separated. The results show that the three components related to turmeric can be effectively separated under the two extreme conditions.

[0049] Through the silica gel plates from different manufacturers and the environment durability investigation, it is shown that the thin layer chromatography identification method provided in the application has good durability.

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

[0051] The Huanglian Gao test solution, the turmeric reference drug solution and the curcumin reference solution were stored at -20℃, and the development experiments were carried out at 1 day, 11 days and 30 days respectively (the development and observation were carried out under the environment condition of 22-24℃, 40-50% RH), and the stability of each solution was observed. The thin layer chromatogram of the solution stability investigation result when the solution was stored at -20℃ for 1 day is shown in Figure 3. Figure 7 The thin layer chromatogram of the result in the high humidity (25℃, 90% RH) environment condition in the durability investigation is shown in Figure 2. Figure 8 The thin layer chromatogram of the result in the high humidity (25℃, 90% RH) environment condition in the durability investigation is shown in Figure 2. Figure 9 The thin layer chromatogram of the result in the high humidity (25℃, 90% RH) environment condition in the durability investigation is shown in Figure 2.

[0052] Figure 7 、 Figure 8 、 Figure 9 The results show that the Huanglian Gao test solution, the turmeric reference drug solution and the curcumin reference solution are stable when stored at -20℃ for 30 days.

[0053] Example 2-5

[0054] Example 2-5 respectively provides a thin layer chromatography identification method of curcumin in Huanglian Gao.

[0055] The difference between the above examples and Example 1 is that the composition of the developing agent is different, which is shown as follows.

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

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

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

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

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

[0061] It can be known from the thin layer chromatogram results that the components in the Huanglian Gao test sample solution are well separated, and there is no interference in the corresponding position in the negative test sample chromatogram of turmeric, indicating that the thin layer chromatography identification method provided by the application has good specificity.

[0062] Example 6

[0063] Example 6 investigates the sensitivity of the thin layer chromatography identification method of curcumin in Huanglian Gao.

[0064] The Huanglian Gao test sample solution prepared in Example 1 was used as the sample to be tested, which was diluted by 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, and 1:256, respectively, and then the thin layer chromatography identification method provided in Examples 1-5 was used to observe the spots of the Huanglian Gao test sample solution (the scoring details refer to the following points), and the sensitivity of the thin layer chromatography identification method was investigated.

[0065] (1) Spot clarity:

[0066] The sharpness of the edge of the spot, whether there are phenomena such as blur and tailing.

[0067] The uniformity of the spot, whether there are ring-shaped objects, spot splitting or multiple spots overlapping.

[0068] (2) Spot brightness:

[0069] The color depth of the spot, whether it is bright and the contrast with the background color.

[0070] Under ultraviolet light, whether the spot emits fluorescence and its fluorescence intensity.

[0071] (3) Spot shape and size:

[0072] Whether the spot presents a regular circle or an approximate circle.

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

[0074] (4) Background interference:

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

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

[0077] (5) Scoring details:

[0078] 5 points: The spot is clear, the edge is sharp, the color is bright and the contrast is high, there is no background interference, the shape is regular and the size is moderate.

[0079] 4 points: The spot is relatively clear, the color is relatively bright, there is slight background interference or deviation in shape and size.

[0080] 3 points: The spot is generally clear, the color is slightly pale, there is a certain degree of background interference or shape and size inconsistency.

[0081] 2 points: The spot is blurred, the color is dark, the background interference is serious, or the shape and size are obviously inconsistent with the standard.

[0082] 1 point: The spot is extremely unclear and almost impossible to identify, or it is completely interfered by other factors.

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

[0084] Table 1 Sensitivity of the thin layer chromatography identification method of curcumin in Huanglian paste in Examples 1-5

[0085]

[0086]

[0087] Comparative Examples

[0088] Comparative Examples 1-4

[0089] Comparative Examples 1-4 each provide a thin layer chromatography identification method of curcumin in Huanglian paste.

[0090] The difference between the above comparative examples 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 the developing agent. From the thin layer chromatography identification results, it can be seen that the test sample solution of Huanglian paste and the curcumin control drug material solution in this comparative example were not fully developed, and the spots were not fully stretched; the spots of other components in the test sample solution of Huanglian paste were seriously interfered.

[0092] In the comparative example 2: chloroform, ethyl acetate, formic acid, water were used as the developing agent with the volume ratio of 12:24:7:4. The results of the thin-layer chromatography showed that the test solution of Huanglian Gao and the control drug solution of Curcuma longa L. were not completely developed and the spots were not fully stretched. The spots of other components in the test solution of Huanglian Gao were interfered.

[0093] In the comparative example 3: ethyl acetate, methanol, formic acid were used as the developing agent with the volume ratio of 7:1.5:0.5. The results of the thin-layer chromatography showed that the test solution of Huanglian Gao and the control drug solution of Curcuma longa L. were not completely developed and the spots were not fully stretched.

[0094] In the comparative example 4: ethyl acetate, ethanol, acetic acid were used as the developing agent with the volume ratio of 6:3:3. The results of the thin-layer chromatography showed that the test solution of Huanglian Gao and the control drug solution of Curcuma longa L. were not completely developed and the spots were not fully stretched.

[0095] In the above comparative examples, other process parameters were the same as those in the example 1.

[0096] Although the present application has been described in detail with general description and specific embodiments above, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, are within the scope of the present application.

Claims

1. A method for identifying curcumin in Huanglian paste by thin layer chromatography, characterized in that, Specifically comprising the following steps in sequence: taking the Coptis chinensis ointment test sample solution, turmeric control medicinal material solution and curcumin control sample solution respectively, spotting in strip shape on the same silica gel G thin layer plate, using ethyl acetate, ethanol and acetic acid in a volume ratio of 7-9: 0.5-1.5:0.5-1.5 as the developing agent, developing, taking out, airing and drying, and observing; The preparation method of the Coptis chinensis ointment test sample solution is as follows: taking the Coptis chinensis ointment test sample, adding methanol aqueous solution with a volume fraction of 40-60% according to a solid-liquid ratio of 1g:4-8mL, heating and melting, stirring and refluxing for 20-40min, and then placing at room temperature; after solidification, the solidified ointment is discarded, the supernatant is filtered, the filtrate is evaporated to dryness, the residue is dissolved in methanol to a residue concentration of 3-5mg / mL, and the solution is used as the test sample solution; The method of observation is as follows: placing under an ultraviolet lamp with a wavelength of 365nm for observation.

2. The method as claimed in claim 1, wherein the said method is characterized by, The developing agent is ethyl acetate, ethanol and acetic acid in a volume ratio of 7.5-8.5:0.7-1.3:0.7-1.

3.

3. The method as claimed in claim 2, wherein the said method is characterized by, The developing agent is ethyl acetate, ethanol and acetic acid in a volume ratio of 7.8-8.2:0.9-1.1:0.9-1.

1.

4. The method as claimed in claim 1, wherein the said method is characterized by, The environmental conditions for development are as follows: a temperature of 4-30℃ and a humidity of 10%-92.5%.

5. The method as claimed in claim 1, wherein the said method is characterized by, The preparation method of the Coptis chinensis ointment test sample solution is as follows: taking the Coptis chinensis ointment test sample, adding methanol aqueous solution with a volume fraction of 40-60% according to a solid-liquid ratio of 1g:5-7mL, heating and melting, stirring and refluxing for 20-40min, and then placing at room temperature; after solidification, the solidified ointment is discarded, the supernatant is filtered, the filtrate is evaporated to dryness, the residue is dissolved in methanol to a residue concentration of 3-5mg / mL, and the solution is used as the test sample solution.

6. The method as claimed in claim 5, wherein the said method is characterized by, The preparation method of the Coptis chinensis ointment test sample is as follows: mixing Coptis chinensis, Cortex Phellodendri, turmeric, angelica tail and raw Rehmannia, adding sesame oil, heating and warming to 180-190℃, extracting for 10-40min, and filtering to obtain oil; weighing beeswax, filtering after melting, stirring with the oil, and cooling to obtain the product.

7. The method as claimed in claim 1, wherein the said method is characterized by, The preparation method of the turmeric control medicinal material solution is as follows: taking the turmeric control medicinal material, adding methanol aqueous solution with a volume fraction of 40-60% according to a solid-liquid ratio of 1mg:1-2mL, stirring and refluxing for 20-40min, and then placing at room temperature; filtering, evaporating the filtrate to dryness, dissolving the residue in methanol to a residue concentration of 4-8mg / mL, and using the solution as the control medicinal material solution.

8. The method as claimed in claim 1, wherein the said method is characterized by, The preparation method of the curcumin control sample solution is as follows: taking curcumin control sample, adding methanol to prepare a solution with a concentration of 0.03-0.08mg / mL, and using the solution as the control sample solution.

Images