Qualitative identification method of water-soluble components of safflower

Abstract

The present application belongs to the technical field of traditional Chinese medicine component detection, and particularly relates to a qualitative identification method of water-soluble components of safflower. The qualitative identification method of water-soluble components of safflower provided by the present application adopts water as an extraction solvent, extracts water-soluble components in safflower, and then adopts a thin layer chromatography method for identification, effectively solves the technical problem that safflower cannot be effectively detected after being boiled in water in the prior art, and is suitable for the inspection of classical famous prescription preparations containing safflower.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine component detection technology, specifically relating to a qualitative identification method for water-soluble components of safflower. Background Technology

[0002] Safflower, also known as safflower or prickly safflower, is a plant belonging to the genus Safflower in the family Asteraceae. It possesses various medicinal properties, including promoting blood circulation, relieving depression, anti-inflammatory and analgesic effects, regulating menstruation, and improving digestion. Safflower contains a large amount of flavonoids, as well as volatile oils, phytosterols, polysaccharides, amino acids, fatty acids, and fatty oils. The volatile oil's main component is a mixture of alkynes, while the safflower polysaccharide is composed of glucose, xylose, arabinose, and galactose linked by β-bonds.

[0003] Current techniques for identifying components in safflower mostly involve extracting the safflower with organic solvents before identification. This method is only suitable for identifying fat-soluble components, while water-soluble components cannot be effectively detected, and the extracted components contain organic solvent residues. For example, the identification method for safflower included in the Chinese Pharmacopoeia is as follows: Take 0.5g of safflower powder, add 5mL of 80% acetone solution, seal tightly, shake for 15min, let stand, and take the supernatant as the test solution. Separately, take 0.5g of safflower reference material and prepare a reference solution using the same method. Perform thin-layer chromatography, applying 50μL of each of the above two solutions to the same silica gel H thin-layer plate, using ethyl acetate-formic acid-water-methanol (7:2:3:0.4) as the developing solvent, develop, remove, and air dry.

[0004] Furthermore, patent publication number CN103940929A discloses a detection method for a traditional Chinese medicine injection for treating cardiovascular and cerebrovascular diseases. This method includes a thin-layer chromatography identification method for Danshen (Salvia miltiorrhiza) and Honghua (Carthamus tinctorius). The identification method for Honghua is as follows: Take 5 ml of the injection, adjust the pH to 2 with dilute hydrochloric acid, extract with 10 ml of ethyl acetate by shaking, separate the ethyl acetate solution, evaporate to dryness, dissolve the residue in 2 ml of anhydrous ethanol, and use this as the test solution. Separately, take 0.5 g of Honghua reference material, add 25 mL of water, reflux for 2 hours, cool, filter, adjust the pH to 2 with dilute hydrochloric acid, extract with 20 mL of ethyl acetate by shaking, separate the ethyl acetate solution, evaporate to dryness, dissolve the residue in 1 mL of anhydrous ethanol, and use this as the reference material solution. According to the thin-layer chromatography test, 10 μL each of the test solution and the reference medicinal material solution from the identification (1) section above were spotted onto the same silica gel GF254 thin-layer plate. A chloroform-acetone-98% formic acid solution (volume ratio 10:0.5:0.5) was used as the developing solvent. After development, the plate was removed, dried, and examined under a UV lamp at 254 nm. In the chromatogram of the test sample, spots of the same color as those in the chromatogram of the reference medicinal material appeared at the corresponding positions. Although this method uses water as the extraction solvent, it is for the identification of injection solutions, not safflower medicinal materials.

[0005] In summary, the existing technology has a technical problem that safflower cannot be effectively detected after boiling in water. Summary of the Invention

[0006] To address the aforementioned technical challenges, this invention provides a qualitative identification method for water-soluble components of safflower. The method uses water as the extraction solvent to extract the water-soluble components from safflower, followed by identification using thin-layer chromatography. This effectively solves the technical problem in existing technologies where safflower cannot be effectively detected after boiling in water, and is suitable for the testing of compound preparations containing safflower.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows:

[0008] A qualitative identification method for water-soluble components of safflower includes the following steps:

[0009] S1. Preparation of test solution: Take safflower, add water, heat and reflux to extract, filter, add organic solvent to the filtrate to extract, combine the extracts and evaporate to dryness to obtain residue, add anhydrous ethanol to the residue to dissolve, and obtain test solution.

[0010] S2. Preparation of medicinal material reference solution: Take safflower reference medicinal material, add water, heat and reflux to extract, filter, add organic solvent to the filtrate for extraction, combine the extracts and evaporate to dryness to obtain residue, add anhydrous ethanol to the residue to dissolve, and obtain medicinal material reference solution;

[0011] S3. Identification by thin-layer chromatography: Take the test solution prepared in step S1 and the reference solution prepared in step S2, spot them on the same silica gel thin-layer plate, develop them in the developing solvent, remove them, air dry them, and examine them under ultraviolet light.

[0012] Furthermore, in steps S1 and S2, the mass ratio of safflower to water is 1:20-80.

[0013] Furthermore, in steps S1 and S2, the reflux extraction temperature is 80-100℃, and the reflux extraction time is 1-3h.

[0014] Furthermore, after filtration in steps S1 and S2, the filtrate is cooled to room temperature.

[0015] Furthermore, in steps S1 and S2, the organic solvent is diethyl ether, the amount of organic solvent used is 0.2-5 times the volume of the filtrate, the extraction method is shaking extraction, and the number of extractions is 1-5 times.

[0016] Furthermore, in steps S1 and S2, the amount of anhydrous ethanol used is 1-6 times the mass of safflower.

[0017] Furthermore, the evaporation in steps S1 and S2 can be replaced by steaming.

[0018] Furthermore, in step S3, the amount of the test solution and the medicinal material control solution taken is 10-30 μL.

[0019] Furthermore, the silicone sheet in step S3 is a high-efficiency silicone G sheet or a high-efficiency silicone GF254 sheet.

[0020] Furthermore, in step S3, the developing agent is a mixture of cyclohexane, dichloromethane, and ethyl acetate.

[0021] Furthermore, the mass ratio of cyclohexane, dichloromethane, and ethyl acetate in the developing solvent is 2-6:1:1.

[0022] Furthermore, in step S3, the wavelength of the ultraviolet lamp is 245nm or 365nm.

[0023] Compared with existing technologies, the qualitative identification method for water-soluble components of safflower provided by this invention has the following technical advantages:

[0024] (1) The qualitative identification method for water-soluble components of safflower provided by the present invention can effectively detect safflower decoction and is applicable to compound preparations containing safflower.

[0025] (2) The qualitative identification method for water-soluble components of safflower provided by the present invention avoids the occurrence of multiple solvent fronts during the development process, ensures that the spots are round during the development process, and improves the accuracy of identification.

[0026] (3) The qualitative identification method for water-soluble components of safflower provided by the present invention does not require the addition of a colorimetric reagent for colorimetric reaction during thin-layer chromatography;

[0027] (4) The qualitative identification method for water-soluble components of safflower provided by the present invention is simple to operate, has a fast and accurate separation effect, and good reproducibility, providing a basis for the quality control of compound preparations containing safflower components. Attached Figure Description

[0028] Figure 1 This is an inspection view of Example 1;

[0029] Figure 2 This is an inspection view of Example 2;

[0030] Figure 3 This is an inspection view of Example 3;

[0031] Figure 4 This is the inspection view for Comparative Example 1;

[0032] Figure 5 This is the inspection view for Comparative Example 2;

[0033] Figure 6 This is the inspection view for Comparative Example 3;

[0034] Figure 7 This is the inspection view for Comparative Example 4;

[0035] In the figure, 1 is the chromatographic band of the medicinal material reference solution, and 2 is the chromatographic band of the test sample solution. Detailed Implementation

[0036] The present invention is further described below through specific embodiments, but the present invention is not limited to the following embodiments. Those skilled in the art can make various modifications based on the basic idea of ​​the present invention, but all modifications are within the scope of the present invention as long as they do not depart from the basic idea of ​​the present invention.

[0037] Example 1: A qualitative identification method for water-soluble components of safflower.

[0038] The qualitative identification method for the water-soluble components of safflower includes the following steps:

[0039] S1. Preparation of test solution: Take 0.5g of safflower and add 10g of water, heat and reflux at 80℃ for 3h, filter while hot, cool the filtrate, and extract once with ether by shaking. The amount of ether used is 0.2 times the volume of the filtrate. Combine the extracts and evaporate to dryness to obtain the residue. Add 0.5g of anhydrous ethanol to the residue to dissolve it to obtain the test solution.

[0040] S2. Preparation of medicinal material reference solution: Take 0.5g of safflower reference material and add 10g of water. Heat and reflux at 80℃ for 3h. Filter while hot. After the filtrate cools, extract once with ether by shaking. The amount of ether used is 0.2 times the volume of the filtrate. Combine the extracts and evaporate to dryness to obtain the residue. Add 0.5g of anhydrous ethanol to the residue to dissolve it to obtain the medicinal material reference solution.

[0041] S3. Thin-layer chromatography test (General rule 0502): Take 10 μL of the test solution prepared in step S1 and the medicinal material reference solution prepared in step S2, respectively, and spot them on the same high-performance silica gel G thin-layer plate. Develop the plate in a developing solvent composed of cyclohexane, dichloromethane, and ethyl acetate in a mass ratio of 2:1:1. Remove the plate, air dry it, and examine it under a UV lamp with a wavelength of 365 nm.

[0042] Example 2: A qualitative identification method for water-soluble components of safflower.

[0043] The qualitative identification method for the water-soluble components of safflower includes the following steps:

[0044] S1. Preparation of test solution: Take 0.5g of safflower and add 37.5g of water, heat and reflux at 100℃ for 1h, filter while hot, cool the filtrate, and extract 5 times with ether by shaking. The amount of ether used is 5 times the volume of the filtrate. Combine the extracts and evaporate to dryness to obtain the residue. Add 3g of anhydrous ethanol to the residue to dissolve it and obtain the test solution.

[0045] S2. Preparation of medicinal material reference solution: Take 0.5g of safflower reference material and add 37.5g of water. Heat and reflux at 100℃ for 1h. Filter while hot. After the filtrate cools, extract with ether by shaking 5 times. The amount of ether used is 5 times the volume of the filtrate. Combine the extracts and evaporate to dryness to obtain the residue. Add 3g of anhydrous ethanol to the residue to dissolve it and obtain the medicinal material reference solution.

[0046] S3. Thin-layer chromatography test (General rule 0502): Take 30 μL of the test solution prepared in step S1 and the medicinal material reference solution prepared in step S2, respectively, and spot them on the same high-performance silica gel G thin-layer plate. Develop the plate in a developing solvent composed of cyclohexane, dichloromethane, and ethyl acetate in a mass ratio of 6:1:1. Remove the plate, air dry it, and examine it under a UV lamp with a wavelength of 365 nm.

[0047] Example 3: A qualitative identification method for water-soluble components of safflower.

[0048] The qualitative identification method for the water-soluble components of safflower includes the following steps:

[0049] S1. Preparation of test solution: Take 0.5g of safflower and add 40g of water, heat and reflux at 90℃ for 2h, filter while hot, cool the filtrate, and extract 4 times with ether by shaking. The amount of ether used is 3 times the volume of the filtrate. Combine the extracts and evaporate to dryness to obtain the residue. Add 2.5g of anhydrous ethanol to the residue to dissolve it to obtain the test solution.

[0050] S2. Preparation of medicinal material reference solution: Take 0.5g of safflower reference material and add 40g of water. Heat and reflux at 90℃ for 2h. Filter while hot. After the filtrate cools, extract with ether by shaking 4 times. The amount of ether used is 3 times the volume of the filtrate. Combine the extracts and evaporate to dryness to obtain the residue. Add 2.5g of anhydrous ethanol to the residue to dissolve it to obtain the medicinal material reference solution.

[0051] S3. Thin-layer chromatography test (General rule 0502): Take 20 μL of the test solution prepared in step S1 and the medicinal material reference solution prepared in step S2, respectively, and spot them on the same high-performance silica gel G thin-layer plate. Develop the plate in a developing solvent composed of cyclohexane, dichloromethane and ethyl acetate in a mass ratio of 4:1:1. Remove the plate, air dry it, and examine it under a UV lamp with a wavelength of 365 nm.

[0052] Comparative Example 1

[0053] This comparative example is similar to Example 3, except that methanol is used instead of cyclohexane in the developing solvent in this comparative example.

[0054] Comparative Example 2

[0055] This comparative example is similar to Example 3, except that toluene is used instead of dichloromethane in the developing solvent in this comparative example.

[0056] Comparative Example 3

[0057] This comparative example is similar to Example 3, except that acetone is used instead of ethyl acetate as the developing solvent in this comparative example.

[0058] Comparative Example 4

[0059] This comparative example is similar to Example 3, except that the developing agent in this comparative example is composed of cyclohexane, dichloromethane and ethyl acetate in a mass ratio of 1:3:4.

[0060] Experimental results: Figure 1 , Figure 2 and Figure 3 It can be seen that in the chromatogram of the test sample solution, spots and fluorescent main spots of the same color appear at the corresponding positions as in the chromatogram of the medicinal material reference solution. The spots are round and uniform, which improves the accuracy of the identification process. At the same time, in the above embodiment, a clean chromatogram can be obtained by only one spotting, one development and one inspection, which significantly improves the success rate of identifying water-soluble components of safflower in one attempt.

[0061] Depend on Figure 4 , Figure 5 , Figure 6 and Figure 7 It can be seen that, compared with the chromatogram of the reference solution, the spots in the chromatogram of the test solution are blurry, incomplete, and misaligned. This indicates that the changes in the formulation and dosage of the developing solvent in Comparative Examples 1-4 will affect the identification results of the water-soluble components of safflower.

[0062] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A method for the qualitative identification of water-soluble components of safflower, characterized in that, The method comprises the following steps: S1, preparation of the test sample solution: take safflower and add water, heat and reflux to extract, filter, add organic solvent to the filtrate to extract, combine the extract and evaporate to dryness, obtain residue, add anhydrous ethanol to the residue to dissolve, and obtain the test sample solution; S2, preparation of the medicinal material control solution: take safflower control medicinal material and add water, heat and reflux to extract, filter, add organic solvent to the filtrate to extract, combine the extract and evaporate to dryness, obtain residue, add anhydrous ethanol to the residue to dissolve, and obtain the medicinal material control solution; S3, identification by thin layer chromatography: take the test sample solution prepared in step S1 and the medicinal material control solution prepared in step S2, spot on the same silica gel thin layer plate, place in the developing agent to develop, take out, air dry, and place under ultraviolet light to observe; The organic solvent in steps S1 and S2 is diethyl ether; The wavelength of the ultraviolet light in step S3 is 365 nm, and the silica gel thin layer plate is high-efficiency silica gel G thin layer plate; The developing agent in step S3 is a mixture of cyclohexane, dichloromethane and ethyl acetate, and the mass ratio of cyclohexane, dichloromethane and ethyl acetate is 2-6:1:

1.

2. The method for the qualitative identification of water-soluble components of Safflower according to claim 1, characterized in that, The mass ratio of safflower to water in steps S1 and S2 is 1:20-80.

3. The method for the qualitative identification of water-soluble components of Safflower according to claim 1, characterized in that, The reflux extraction temperature in steps S1 and S2 is 80-100°C, and the reflux extraction time is 1-3 h.

4. The method for the qualitative identification of water-soluble components of Safflower according to claim 1, characterized in that, After filtration in steps S1 and S2, the filtrate is cooled to room temperature.

5. The method for qualitative identification of water-soluble components of safflower according to claim 1, characterized in that, The amount of organic solvent used in steps S1 and S2 is 0.2-5 times the volume of the filtrate, the extraction method is shaking extraction, and the extraction times is 1-5 times.

6. The method for qualitative identification of water-soluble components of safflower according to claim 1, characterized in that, The amount of anhydrous ethanol used in steps S1 and S2 is 1-6 times the mass of safflower.

7. The method for qualitative identification of water-soluble components of safflower according to claim 1, characterized in that, The amount of test sample solution and medicinal material control solution taken in step S3 is 10-30 μL.

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