CN109358153B - Thin-layer identification method for fat-soluble components in radix pseudostellariae medicinal material - Google Patents
Thin-layer identification method for fat-soluble components in radix pseudostellariae medicinal material Download PDFInfo
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- CN109358153B CN109358153B CN201811609989.0A CN201811609989A CN109358153B CN 109358153 B CN109358153 B CN 109358153B CN 201811609989 A CN201811609989 A CN 201811609989A CN 109358153 B CN109358153 B CN 109358153B
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention provides a thin-layer identification method of fat-soluble components in a radix pseudostellariae medicinal material, which takes petroleum ether-chloroform-ethyl acetate solution with the volume ratio of 10:2:1 as a developing agent, under the optimized condition of thin-layer chromatography analysis, various fat-soluble components in the radix pseudostellariae medicinal material are effectively separated, and the thin-layer chromatography identification method has good distinguishing capability on pseudo-mixed medicinal materials such as radix ophiopogonis, radix stemonae, lophatherum gracile and the like. The method expands the quality evaluation indexes of the radix pseudostellariae and enriches the quality evaluation content.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine material identification, and particularly relates to a thin-layer identification method for fat-soluble components in a radix pseudostellariae medicinal material.
Background
Radix Pseudostellariae is Caryophyllaceae plant radix PseudostellariaePseudostellaria heterophylla(Miq.) Pax ex Paxet Hoffm. Is a Chinese traditional medicine (national pharmacopoeia committee, pharmacopoeia of people's republic of China, 2015 edition, one part, Beijing: chinese medical science and technology press, 2015: 68.]. Sweet, slightly bitter and neutral in taste. It enters spleen and lung meridians. Has the effects of replenishing qi to invigorate the spleen, promoting the production of body fluid and moistening the lung. Can be used for treating spleen deficiency, asthenia, deficiency of both qi and yin, spontaneous perspiration, thirst, and dry cough due to lung dryness.
The existing radix pseudostellariae thin-layer chromatography identification method in pharmacopoeia of the people's republic of China (2015 edition one part) is mainly based on amino acid identification. However, the radix pseudostellariae contains abundant chemical components, and contains polysaccharide, cyclic peptide and fatty acid chemical substances besides amino acid. At present, no content determination index is formulated in the quality standard of the radix pseudostellariae medicinal material in pharmacopoeia of 2015 edition.
Therefore, the method has certain limitation on evaluating the quality of the radix pseudostellariae medicinal material only by the amino acid thin-layer chromatography characteristic. Common false mixing products of the radix pseudostellariae comprise radix ophiopogonis, radix stemonae, lophatherum gracile root tuber and the like. And the quality evaluation index is expanded, and the content of quality evaluation is enriched.
Disclosure of Invention
The invention aims to provide a thin-layer identification method for fat-soluble components in a radix pseudostellariae medicinal material, which expands the quality evaluation indexes of the radix pseudostellariae and enriches the quality evaluation content.
A thin-layer identification method for fat-soluble components in a radix pseudostellariae medicinal material comprises the following steps:
(1) preparation of test and reference substances
Taking 5g of test sample powder, adding 150ml of 60-90 ℃ petroleum ether, carrying out ultrasonic treatment for 30min, filtering, concentrating the filtrate to dryness, adding 2ml of 60-90 ℃ petroleum ether, transferring to a 10 ml round-bottom flask, concentrating to dryness, adding 2ml of 0.5 mol/L potassium hydroxide-methanol solution, carrying out water bath at 60 ℃ for 30min, cooling, adding 2ml of 14wt% boron trifluoride-methanol solution, carrying out water bath at 60 ℃ for 30min, cooling, precisely adding 2ml of n-hexane, adding 2ml of saturated sodium chloride solution, shaking uniformly, and taking the upper layer as a test sample solution;
preparing 5g of radix pseudostellariae reference medicinal material powder into a radix pseudostellariae reference medicinal material solution according to the same method;
respectively taking 5g of pseudo-mixed medicinal materials of radix ophiopogonis, radix stemonae and lophatherum gracile powder, and preparing an radix ophiopogonis solution, a radix stemonae solution and a lophatherum gracile solution according to the same method;
(2) thin layer authentication
Respectively dropping 10 μ l of test solution, radix Pseudostellariae reference medicinal material solution, radix Ophiopogonis solution, radix Stemonae solution and folium Bambusae solution on the same silica gel G thin layer plate, pre-saturating with petroleum ether-chloroform-ethyl acetate solution at volume ratio of 10:2:1 as developing agent in a double-tank developing cylinder for 15min, developing, taking out, air drying, soaking the plate in 10wt% phosphomolybdic acid ethanol solution, heating at 105 deg.C for 3min, and inspecting.
The invention has the obvious advantages that
The method determines the type of the developing solvent and gropes out the optimal proportion. The application of petroleum ether-chloroform-ethyl acetate, wherein the petroleum ether is a nonpolar solvent, the chloroform is a medium polar solvent, and the polarity of the ethyl acetate is similar to that of the chloroform, and the multi-component developing agents with different types and different polarities are adopted, so that different types of fat-soluble components in the radix pseudostellariae are separated according to the similarity and intermiscibility principle; the polarity of fat-soluble substances in the radix pseudostellariae is low, and the dosage of non-polar substances petroleum ether in the developing agent system is relatively large. Ethyl acetate served to adjust the component ratio shift values and reduce the speckle tailing in the present system. Under the optimized thin-layer chromatography analysis condition, various fat-soluble components in the radix pseudostellariae medicinal material are more effectively separated, and the pseudostellariae medicinal material has good distinguishing capability on pseudo-mixed medicinal materials such as radix ophiopogonis, radix stemonae, lophatherum gracile and the like.
Selection of the developer system:
firstly, an ethyl acetate-acetone-formic acid-water developing agent system is used for determining the polarity range of fatty acid in the radix pseudostellariae, and experimental results show that thin-layer spots are gathered at the front edge of a solvent, which indicates that the polarity of liposoluble substances of the radix pseudostellariae is low and the polarity of the developing agent system is relatively large; further removing water, formic acid and acetone components in the developing agent system, adding a weak polarity developing agent such as petroleum ether or chloroform and the like, taking petroleum ether-chloroform-ethyl acetate and chloroform-methanol-water as the developing agent system, and still not realizing the complete separation of each spot through a plurality of tests, wherein the Rf value of each thin spot in the chloroform-methanol-water developing agent system is larger; cyclohexane-ethyl acetate is used as a developing agent, and thin layer spots are few; the phenomenon of trailing of thin-layer spots is serious when petroleum ether-chloroform is used as a developing agent; but separation of spots was achieved with the petroleum ether-chloroform-ethyl acetate developer system. When petroleum ether-chloroform-ethyl acetate =5-1-1, the components can not develop color, and still two spots have larger Rf value, on the basis, the use amount of petroleum ether is increased, and the polarity of the developing agent system is further reduced. Repeated tests and verifications show that petroleum ether-chloroform-ethyl acetate =10-2-1 is finally selected as a developing agent for thin-layer identification of fatty acid components in radix pseudostellariae, thin-layer spots in a thin-layer chromatogram obtained by the developing agent system are abundant, the separation degree of each spot is good, and the Rf value of most spots is 0.2-0.8. The identification effect of the type of developing solvent and the ratio optimization process is shown in fig. 1.
Drawings
FIG. 1 is a thin layer chromatogram of radix Pseudostellariae based on the type of developing solvent and its different ratios;
FIG. 2 is a thin-layer chromatogram of radix Pseudostellariae medicinal material and pseudolites mixture, wherein 1-6 are radix Pseudostellariae samples produced by Anhui, Fujian (Cudrania tricuspidata No. 2), Guizhou, Jiangsu, Shandong, Fujian (Cudrania tricuspidata No. 3), respectively; 7. radix Pseudostellariae as reference material; 8. radix Stemonae; 9. lophatherum gracile root tuber; 10. radix Ophiopogonis.
Detailed Description
The invention will now be further described by way of examples, which are not intended to limit the scope of the invention. It will be understood by those skilled in the art that the equivalent substitutions and modifications made in the present disclosure are within the scope of the present invention.
Example 1
A thin-layer identification method for fat-soluble components in a radix pseudostellariae medicinal material comprises the following steps:
preparing a test solution: respectively taking 5g of radix pseudostellariae medicinal material powder produced by Anhui, Fujian (Cudrania tricuspidata No. 2), Guizhou, Jiangsu, Shandong and Fujian (Cudrania tricuspidata No. 3), adding 150ml of petroleum ether (60-90 ℃), carrying out ultrasonic treatment for 30min, filtering, concentrating filtrate until 2ml of petroleum ether (60-90 ℃) is added, transferring the filtrate to a 10 ml round-bottom flask, concentrating until the filtrate is dried, adding 2ml of 0.5 mol/L potassium hydroxide-methanol solution, carrying out water bath at 60 ℃ for 30min, cooling, adding 2ml of 14wt% boron trifluoride-methanol solution, carrying out water bath at 60 ℃ for 30min, cooling, precisely adding 2ml of n-hexane, adding 2ml of saturated sodium chloride solution, shaking uniformly, and taking the upper layer to obtain radix pseudostellariae sample solutions of different production places.
Preparation of a reference solution: 5g of radix pseudostellariae reference medicinal material powder, and preparing a radix pseudostellariae reference medicinal material solution according to the same method for preparing the test solution; taking 5g of each of the pseudo-mixed medicinal materials of radix ophiopogonis, radix stemonae and lophatherum gracile powder, and preparing an radix ophiopogonis solution, a radix stemonae solution and a lophatherum gracile solution according to the same method for preparing the test solution;
thin-layer plate: silica gel thin layer plates (TLC Silica gel 60, Merck) were preformed.
Sample application: 10 mul, strip sample application, strip width 10mm, strip interval 8-10mm, and origin 10mm from bottom edge.
Unfolding the cylinder: the double tank deployment cylinder.
Developing agent: petroleum ether-chloroform-ethyl acetate (10: 2:1, volume ratio), 13 ml.
Unfolding: the thin layer plate is saturated with the spreading agent for 15min, and then spread upwards for 7 cm.
Color development: the plate was immersed in 10wt% ethanol phosphomolybdate solution and heated at 105 ℃ for 3 min.
And (6) inspection: the chromatographic image was observed under visible light. As a result, as shown in FIG. 2, the pseudostellaria root medicinal material sample chromatogram in different producing areas shows the spot with the same pigment color at the position corresponding to the control medicinal material chromatogram. The thin-layer chromatography characteristics of pseudo-mixture such as radix ophiopogonis, radix stemonae, lophatherum gracile and the like are obviously different from those of radix pseudostellariae.
The thin-layer identification method can identify fat-soluble components in the radix pseudostellariae medicinal material, and can also distinguish mixed and counterfeit medicinal materials of radix ophiopogonis, radix stemonae and lophatherum gracile. The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Claims (1)
1. A thin-layer identification method for distinguishing radix pseudostellariae medicinal materials and false-mixing products thereof based on fat-soluble components is characterized by comprising the following steps:
(1) preparation of test and reference substances
Taking 5g of test sample powder, adding 150ml of 60-90 ℃ petroleum ether, carrying out ultrasonic treatment for 30min, filtering, concentrating the filtrate to dryness, adding 2ml of 60-90 ℃ petroleum ether, transferring to a 10 ml round-bottom flask, concentrating to dryness, adding 2ml of 0.5 mol/L potassium hydroxide-methanol solution, carrying out water bath at 60 ℃ for 30min, cooling, adding 2ml of 14wt% boron trifluoride-methanol solution, carrying out water bath at 60 ℃ for 30min, cooling, precisely adding 2ml of n-hexane, adding 2ml of saturated sodium chloride solution, shaking uniformly, and taking the upper layer as a test sample solution;
preparing 5g of radix pseudostellariae reference medicinal material powder into a radix pseudostellariae reference medicinal material solution according to the same method;
respectively taking 5g of pseudo-mixed medicinal materials of radix ophiopogonis, radix stemonae and lophatherum gracile powder, and preparing an radix ophiopogonis solution, a radix stemonae solution and a lophatherum gracile solution according to the same method;
(2) thin layer authentication
Respectively dropping 10 μ l of test solution, radix Pseudostellariae reference medicinal material solution, radix Ophiopogonis solution, radix Stemonae solution and folium Bambusae solution on the same silica gel G thin layer plate, placing into a double-tank developing cylinder pre-saturated with developing agent for 15min with petroleum ether-chloroform-ethyl acetate solution at volume ratio of 10:2:1, developing, taking out, air drying, soaking the plate with 10wt% phosphomolybdic acid ethanol solution, heating at 105 deg.C for 3min, and inspecting;
the result shows that spots with the same pigment color appear in the chromatogram of the radix pseudostellariae test sample at the positions corresponding to the chromatogram of the reference drug; the thin-layer chromatography characteristics of the pseudo-mixed products of radix ophiopogonis, radix stemonae, lophatherum gracile and radix pseudostellariae are obviously different.
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