A thin-layer identification chromatographic method for Celosiae Semen and stir-fried Celosiae Semen formula granules

Abstract

The present invention provides a thin-layer identification chromatographic method for Celosiae Semen and stir-fried Celosiae Semen formula granules, including: spotting the test solution on a silica gel G thin-layer plate, developing under thin-layer chromatographic conditions, and inspecting; the thin-layer chromatographic conditions: using toluene-ethyl acetate-methanol as the developing agent. By comparing the thin-layer chromatogram of the stir-fried Celosiae Semen formula granules with that of the Celosiae Semen control medicinal material and the Celosiae Semen granules, the same position with a retention factor value at 0.55-0.67 should not show fluorescent spots, which is used as the identification point 1 of the stir-fried Celosiae Semen granules; by comparing the thin-layer chromatogram of the stir-fried Celosiae Semen formula granules with that of the Celosiae Semen control medicinal material and the Celosiae Semen granules, the same position with a retention factor value at 0.43-0.52 has one more fluorescent spot, which is used as the identification point 2 of the stir-fried Celosiae Semen granules. This method can quickly and effectively identify the stir-fried Celosiae Semen formula granules that have lost the form of the cut crude drug, with simple operation, high precision, high sensitivity, good stability, and has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of detection methods, and particularly relates to a thin-layer identification chromatographic method for Celosiae Semen and fried Celosiae Semen formula granules. Background Art

[0002] Celosiae Semen is the dried and mature seed of the plant Celosia argentea L. of the Amaranthaceae family. It has the effects of clearing the liver and purging fire, and removing nebula and improving eyesight. Clinically, there are usually two types of Celosiae Semen used, namely the raw product and the fried product. The raw product has a stronger effect on calming the liver and is commonly used for redness and swelling of the eyes due to liver heat. After frying, the cold nature is alleviated and it is commonly used for nebula in the eyes, blurred vision, and dizziness due to liver fire. Thus, it can be seen that the chemical components of the two may have changed.

[0003] The Celosiae Semen and fried Celosiae Semen decoction pieces can be distinguished by means of character identification, but there is no reported identification method for their extracts. Therefore, it is necessary to establish a method that can distinguish Celosiae Semen and fried Celosiae Semen formula granules. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a thin-layer identification chromatographic method for Celosiae Semen and fried Celosiae Semen formula granules. This method uses the presence or absence of fluorescent spots within a certain range of the retention factor value in the thin-layer chromatogram as the identification point for quickly and effectively identifying Celosiae Semen and fried Celosiae Semen formula granules.

[0005] The present invention provides a thin-layer identification chromatographic method for Celosiae Semen and fried Celosiae Semen formula granules, including the following steps:

[0006] Spot the test solution on a silica gel G thin-layer plate, develop under thin-layer chromatography conditions, and examine.

[0007] The thin-layer chromatography conditions: using toluene - ethyl acetate - methanol as the developing agent.

[0008] In the present invention, the volume ratio of toluene, ethyl acetate, and methanol in the developing agent is 40:(5.7 - 6.3):(4.8 - 5.2).

[0009] In the present invention, the spotting amount of the test solution is 2 - 7 μL.

[0010] In the present invention, the thin-layer chromatography conditions further include:

[0011] The developing temperature is 4 - 25 °C, and the developing humidity is 32 - 75%.

[0012] In the present invention, compared with the thin-layer chromatogram of Celosiae Semen formula granules, the fried Celosiae Semen formula granules do not show fluorescent spots at the retention factor value of 0.55 - 0.67; there is one more fluorescent spot at the retention factor value of 0.43 - 0.52.

[0013] In the present invention, the test solution is prepared by the following method:

[0014] The test sample is ground, added with n-butanol, refluxed, filtered, the filtrate is evaporated to dryness, and the residue is added with methanol to obtain the test solution;

[0015] The mass of the test sample, the volume of n-butanol, and the volume of methanol are in the ratio of 2 g : (45 - 55) mL : (0.95 - 1.05) mL. The reflux time is 28 - 32 min.

[0016] In the present invention, the inspection is carried out using an ultraviolet lamp, and the ultraviolet wavelength used for inspection is 365 nm.

[0017] In the present invention, it also includes the preparation of a Celosiae Cristatae control medicinal material solution and a negative solution;

[0018] Using Celosiae Cristatae control medicinal material, adding water, decocting, centrifuging, taking the supernatant and evaporating it to dryness, and adding n-butanol to the residue to obtain the Celosiae Cristatae control medicinal material solution.

[0019] In the present invention, the mass of the Celosiae Cristatae control medicinal material, the volume of water, and the volume of n-butanol are in the ratio of 2 g : 50 ml : 50 ml; the decocting time is 28 - 32 min.

[0020] The present invention provides a thin-layer identification chromatographic method for Celosiae Cristatae and stir-fried Celosiae Cristatae formula granules, including the following steps: spotting the test solution on a silica gel G thin-layer plate, developing under thin-layer chromatographic conditions, and inspecting; the thin-layer chromatographic conditions: using toluene - ethyl acetate - methanol as the developing agent. By comparing the thin-layer chromatogram of the stir-fried Celosiae Cristatae formula granules with the thin-layer chromatograms of the Celosiae Cristatae control medicinal material and Celosiae Cristatae granules, at the same position with a retention factor value of 0.55 - 0.67, no fluorescent spots should appear, which is used as the identification point 1 of the stir-fried Celosiae Cristatae granules; by comparing the thin-layer chromatogram of the stir-fried Celosiae Cristatae formula granules with the thin-layer chromatograms of the Celosiae Cristatae control medicinal material and Celosiae Cristatae granules, at the same position with a retention factor value of 0.43 - 0.52, one more fluorescent spot appears, which is used as the identification point 2 of the stir-fried Celosiae Cristatae granules. This thin-layer method can quickly and effectively identify the stir-fried Celosiae Cristatae formula granules that have lost the form of the cut pieces, with simple operation, high precision, high sensitivity, and good stability, and has good application prospects. Description of the Drawings

[0021] Figure 1 For the chromatogram analysis of different sample application amounts;

[0022] Figure 2 For the specificity investigation;

[0023] Figure 3 For the investigation of different thin-layer plates - Tianjin Silida;

[0024] Figure 4For the investigation of different thin layer plates - Merck;

[0025] Figure 5 For the investigation of different thin layer plates - Qingdao Ocean;

[0026] Figure 6 Spectrum at 4°C;

[0027] Figure 7 Spectrum at 25°C;

[0028] Figure 8 Spectrum at 32% humidity;

[0029] Figure 9 Spectrum at 75% humidity;

[0030] Figure 10 Verification for different batches;

[0031] Figure 11 Test spectrum for the method provided in Comparative Example 1;

[0032] Figure 12 Test spectrum for the method provided in Comparative Example 2. Detailed implementation mode

[0033] In order to further illustrate the present invention, a thin layer identification chromatography method for Celosiae Semen and fried Celosiae Semen formula granules provided by the present invention will be described in detail below in conjunction with examples, but they should not be construed as limiting the protection scope of the present invention.

[0034] Example 1

[0035] 1. Instruments and test drugs

[0036] 1.1 Instruments

[0037] Hot plate, mortar, thin layer imaging system: CAMAG TLC Visualizer, silica gel G thin layer plates (Qingdao Ocean Chemical Factory, batch number: 20180527, Tianjin Sida Technology Co., Ltd., batch number: 191016, Merck, batch number: HX87183353)

[0038] 1.2 Reagents

[0039] Toluene, ethyl acetate, methanol, and n-butanol are all of analytical grade, and water is ultrapure water (self-made in the laboratory).

[0040] 1.3 Test drugs

[0041] Celosiae Semen reference crude drug (National Institutes for Food and Drug Control, batch number: 121765-202001), Celosiae Semen formula granules (Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: 2208001; 2208002; 2208003), stir-fried Celosiae Semen formula granules (Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: 2211001; 2211002; 2211003).

[0042] 2. Establishment of thin-layer method for formula granules

[0043] 2.1 Preparation of test solution

[0044] Take 2 g of this product, grind it finely, add 50 ml of n-butanol, heat under reflux for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of methanol to obtain the test solution. According to the test of thin-layer chromatography (General Principles 0502 of Chinese Pharmacopoeia 2020 Edition), absorb 5 μl of each of the above solutions and spot them on the same silica gel G thin-layer plate respectively. Use toluene-ethyl acetate-methanol (40:6:5) as the developing solvent, develop, take out, dry in air, and examine under ultraviolet light (365 nm). In the chromatogram of the test solution, at the position corresponding to the chromatogram of the reference crude drug, spots of the same color appear.

[0045] 2.2 Preparation of reference crude drug solution

[0046] Take 2 g of Celosiae Semen reference crude drug, add 50 ml of water, decoct for 30 minutes, centrifuge, take the supernatant and evaporate it to dryness, dissolve the residue in 50 ml of n-butanol, and prepare the reference crude drug solution in the same way.

[0047] 3. Methodology investigation

[0048] 3.1 Investigation of spotting volume

[0049] Under the above-mentioned established experimental conditions, take 2 μl, 5 μl, and 7 μl of Celosiae Semen reference crude drug solution, Celosiae Semen formula granule solution, and stir-fried Celosiae Semen formula granule solution respectively and spot them on the same silica gel G thin-layer plate. Develop according to the established thin-layer chromatography conditions and examine. The results are shown in Figure 1 , among which, 1-3 are the spotting volumes of 2 μl, 5 μl, and 7 μl of Celosiae Semen reference crude drug; 4-6 are the spotting volumes of 2 μl, 5 μl, and 7 μl of Celosiae Semen formula granules; 7-9 are the spotting volumes of 2 μl, 5 μl, and 7 μl of stir-fried Celosiae Semen formula granules; It can be seen from Figure 1 that when the spotting volume of the reference crude drug solution is 2-7 μl and the spotting volume of the test solution is 2-7 μl, the fluorescent spots of the thin-layer chromatography are clearly developed. It is determined that the spotting volume of the reference crude drug solution and the test solution is 2-7 μl. Compared with the reference crude drug chromatogram, there are obvious blue spots at the Rf value of 0.412 for the stir-fried Celosiae Semen formula granules, and there are no spots of the same color at the Rf value of 0.571 for the stir-fried Celosiae Semen formula granules.

[0050] 3.2 Specificity Investigation

[0051] Prepare the Celosiae Semen control medicinal material solution, Celosiae Semen formula granules solution, stir-fried Celosiae Semen formula granules solution, and negative solution according to the above test sample preparation method. Spot them on the same thin-layer plate respectively, develop according to the proposed thin-layer chromatography conditions, and examine. The results are shown in Figure 2 , among which, 1 is the negative solution; 2 is the Celosiae Semen control medicinal material; 3 is the Celosiae Semen formula granules; 4 is the stir-fried Celosiae Semen formula granules; Figure 2 The results show that the negative sample has no interference with the test samples of Celosiae Semen and stir-fried Celosiae Semen formula granules, and the method has good specificity. Compared with the chromatogram of the control medicinal material, there are obvious blue spots at the Rf value of 0.446 for the stir-fried Celosiae Semen formula granules, and there are no spots of the same color at the Rf value of 0.590 for the stir-fried Celosiae Semen formula granules.

[0052] 3.3 Robustness Investigation

[0053] 3.3.1 Comparison of Different Thin-Layer Plates

[0054] Select the Tianjin Sida, Merck, and Qingdao Ocean precast silica gel G plates, and conduct tests according to the proposed test methods respectively. The results are shown in Figures 3 - 5 , among which, 1 is the Celosiae Semen control medicinal material; 2 is the Celosiae Semen formula granules; 3 is the stir-fried Celosiae Semen formula granules; The results show that the thin-layer plates of the 3 brands can all meet the identification requirements, indicating that the method has good robustness. Compared with the chromatogram of the control medicinal material, there are obvious blue spots at the Rf values of 0.480, 0.500, and 0.468 for the stir-fried Celosiae Semen formula granules, and there are no spots of the same color at the Rf values of 0.625, 0.632, and 0.603 for the stir-fried Celosiae Semen formula granules.

[0055] 3.3.2 Comparison of Different Temperatures

[0056] Take the thin-layer plate after spotting, and develop it in the temperature environments of low temperature 4°C and normal temperature 25°C respectively. The results are shown in Figure 6 (4°C) and Figure 7 (25°C). It can be seen from Figures 6 - 7 that the method has good robustness to different temperatures. Compared with the chromatogram of the control medicinal material, there are obvious blue spots at the Rf values of 0.513 and 0.455 for the stir-fried Celosiae Semen formula granules, and there are no spots of the same color at the Rf values of 0.631 and 0.597 for the stir-fried Celosiae Semen formula granules.

[0057] 3.3.3 Comparison of Different Humidities

[0058] Take the thin-layer plate after spotting, and develop it in the humidity environments of 32% and 75% respectively. The results are shown in Figure 8 and Figure 9, where 1 is the reference medicinal material of Celosiae Cristatae Semen; 2 is the formula granule of Celosiae Cristatae Semen, and 3 is the stir-fried formula granule of Celosiae Cristatae Semen; from Figures 8 - 9 It can be seen that this method has good durability for different humidities. Compared with the chromatogram of the reference medicinal material, there are obvious blue spots at the Rf values of 0.481 and 0.507 for the stir-fried formula granule of Celosiae Cristatae Semen, and there are no spots of the same color at the Rf values of 0.623 and 0.627 for the stir-fried formula granule of Celosiae Cristatae Semen.

[0059] 3.3.4 Verification

[0060] Thin-layer identification verification was carried out on the spots of 3 batches of formula granules of Celosiae Cristatae Semen and 3 batches of stir-fried formula granules of Celosiae Cristatae Semen. The experimental results are shown in Figure 10 , where 1 is the reference medicinal material of Celosiae Cristatae Semen; 2 - 4 are the 2208001 batch, 2208002 batch, and 2208003 batch of formula granules of Celosiae Cristatae Semen; 5 - 7 are the 2211001 batch, 2211002 batch, and 2211003 batch of stir-fried formula granules of Celosiae Cristatae Semen; the results show that compared with the chromatogram of the reference medicinal material, there is an obvious blue spot at the Rf value of 0.505 for the stir-fried formula granule of Celosiae Cristatae Semen, and there is no spot of the same color at the Rf value of 0.616 for the stir-fried formula granule of Celosiae Cristatae Semen.

[0061] 2.4 Determination of the Rf value of the identification point of the stir-fried formula granule of Celosiae Cristatae Semen

[0062] The Rf values of each item in the thin-layer chromatography methodology investigation were summarized, and the results are shown in Table 1. According to the summary results, it was determined that the Rf value of the identification spot 1 of the stir-fried formula granule of Celosiae Cristatae Semen should be within the range of 10%, and the specified value is 0.612; the Rf value of the identification point 2 of the stir-fried formula granule of Celosiae Cristatae Semen should be within the range of 10%, and the specified value is 0.477.

[0063] Table 1 Summary of Rf values in the methodology investigation

[0064]

[0065] Comparative Example 1

[0066] Take 2 g of Celosiae Semen formula granules and stir-fried Celosiae Semen formula granules, grind them finely, add 40 ml of 50% ethanol, ultrasonically treat for 30 minutes, centrifuge for 10 minutes, take the supernatant, evaporate to dryness, dissolve the residue in 10 ml of water, pass through a D101 macroporous adsorption resin column (inner diameter 1 cm, column height 10 cm), elute with 50 ml of water, discard the aqueous solution; then elute with 50 ml of 60% ethanol solution, collect the eluate, evaporate to dryness, dissolve the residue in 5 ml of 50% ethanol solution to prepare the test solution. Additionally, take 1 g of Celosiae Semen reference medicinal material and prepare the reference medicinal material solution in the same manner. According to the thin-layer chromatography method (General Principles 0502), absorb 2 - 5 μl of each of the above three solutions and spot them on the same silica gel GF254 thin-layer plate respectively. Use ethyl acetate - methanol - water - formic acid (volume ratio 13:7:2:0.1) as the developing solvent, develop, take out, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly visible, and examine under daylight. The experimental results are shown in Figure 11 , where 1 - 3 are the spotted amounts of 2 μl, 5 μl, and 7 μl of the Celosiae Semen reference medicinal material; 4 - 6 are the spotted amounts of 2 μl, 5 μl, and 7 μl of the Celosiae Semen formula granules; 7 - 9 are the spotted amounts of 2 μl, 5 μl, and 7 μl of the stir-fried Celosiae Semen formula granules.

[0067] Comparative Example 2

[0068] Take 1 g of Celosiae Semen formula granules and stir-fried Celosiae Semen formula granules, grind them finely, add 25 ml of 50% methanol, macerate for 30 minutes, ultrasonically treat for 25 minutes, let it cool, shake well, transfer to a 50 ml centrifuge tube, centrifuge (rotation speed 3000 revolutions per minute) for 10 minutes, and take the supernatant as the test solution. According to the thin-layer chromatography method (General Principles 0502), absorb 5 μl of the test solution and 15 μl of the reference solution and spot them on the same thin-layer silica gel plate respectively. Use n-butanol - acetic acid - water (4:1:1) as the developing solvent, develop, take out, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly visible, air dry, and examine under an ultraviolet lamp (365 nm). The experimental results are shown in Figure 12 , where 1 - 3 are the spotted amounts of 2 μl, 5 μl, and 7 μl of the Celosiae Semen formula granules; 4 - 6 are the spotted amounts of 2 μl, 5 μl, and 7 μl of the stir-fried Celosiae Semen formula granules.

[0069] It can be seen from Figure 11 and Figure 12 that by using the above thin-layer identification method, there are no obvious differences in the spots of Celosiae Semen and stir-fried Celosiae Semen formula granules, and the two cannot be distinguished.

[0070] As can be seen from the above embodiments, in the thin-layer chromatogram of the stir-fried Celosiae Semen formula granules of the present invention, compared with the thin-layer chromatogram of the Celosiae Semen control medicinal material and the Celosiae Semen granules, the same position is at the retention factor value of 0.55 - 0.67, and no fluorescent spots shall appear, which is used as the identification point 1 of the stir-fried Celosiae Semen granules; in the thin-layer chromatogram of the stir-fried Celosiae Semen formula granules, compared with the thin-layer chromatogram of the Celosiae Semen control medicinal material and the Celosiae Semen granules, the same position is at the retention factor value of 0.43 - 0.52, and one more fluorescent spot appears, which is used as the identification point 2 of the stir-fried Celosiae Semen granules. This thin-layer method can quickly and effectively identify the stir-fried Celosiae Semen formula granules that have lost the form of the cut pieces, with simple operation, high precision, high sensitivity and good stability, and has good application prospects.

[0071] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A thin-layer chromatography identification method for Celosiae Semen and stir-fried Celosiae Semen formula granules, comprising the following steps: Spot the test solution on a silica gel G thin-layer plate, develop under thin-layer chromatography conditions, and examine; The thin-layer chromatography conditions: using toluene-ethyl acetate-methanol as the developing agent; The volume ratio of toluene, ethyl acetate and methanol in the developing agent is 40:(5.7 - 6.3):(4.8 - 5.2); Compared with the thin-layer chromatogram of Celosiae Semen formula granules, the stir-fried Celosiae Semen formula granules do not show fluorescent spots at the retention factor value of 0.55 - 0.67; there is one more fluorescent spot at the retention factor value of 0.43 - 0.52; The test solution is prepared according to the following method: Grind the test sample, add n-butanol, reflux, filter, evaporate the filtrate to dryness, and dissolve the residue in methanol to obtain the test solution.

2. The thin-layer chromatography identification method for Celosiae Semen and stir-fried Celosiae Semen formula granules according to claim 1, characterized in that the spotting amount of the test solution is 2 - 7 μL.

3. The thin-layer chromatography identification method for Celosiae Semen and stir-fried Celosiae Semen formula granules according to claim 1, characterized in that the thin-layer chromatography conditions further include: the developing temperature is 4 - 25 °C, and the developing humidity is 32 - 75%.

4. The thin-layer chromatography identification method for Celosiae Semen and stir-fried Celosiae Semen formula granules according to claim 1, characterized in that the mass ratio of the test sample, the volume of n-butanol and the volume of methanol is 2 g:(45 - 55) mL:(0.95 - 1.05) mL.

5. The thin-layer chromatography identification method for Celosiae Semen and stir-fried Celosiae Semen formula granules according to claim 1, characterized in that the examination is carried out using an ultraviolet lamp, and the ultraviolet wavelength used for examination is 365 nm.

6. The thin-layer chromatography identification method for Celosiae Semen and stir-fried Celosiae Semen formula granules according to claim 1, characterized in that it further includes preparing a control medicinal material solution and a negative solution of Celosiae Semen; Using the control medicinal material of Celosiae Semen, add water, decoct, centrifuge, take the supernatant and evaporate it to dryness, and dissolve the residue in n-butanol to obtain the control medicinal material solution of Celosiae Semen.

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