A multi-information thin-layer identification spot fingerprint of Sargentodoxae medicinal material and its method

Abstract

The present invention relates to a multi-information thin-layer identification spot fingerprint of Sargentodoxae medicinal materials and a method thereof. It is characterized in that: a test sample and a control medicinal material solution are obtained by a simple and quick pretreatment method, and more than 30 chemical components of Sargentodoxae medicinal materials from fat-soluble to water-soluble are examined on 5 thin-layer plates under a variety of inspection conditions with gradient developing agents of different polarities, forming a set of thin-layer identification spot fingerprints and a method thereof. The total time is 2 hours, the extraction solvent is 4 ml, and the developing agent is 50 ml. Several thin-layer plates and tens of milliliters of developing agent carry more information than their high-performance liquid fingerprints, but the low cost, ultra-high efficiency, and intuitiveness of spot colors are not available in high-performance liquid fingerprints. It is simple, quick, and efficient, and has the application prospect of greatly improving the identification and subscription rate of Sargentodoxae medicinal materials in compound preparations and supervising their quality through multi-index evaluation.

Description

Technical Field

[0001] The present invention relates to a multi-information thin-layer identification spot fingerprint of Sargentodoxae medicinal materials and a method thereof. That is, a thin-layer identification method is adopted, and a developing agent with gradually increasing polarity is used. In the order of fat-soluble, medium-polar and water-soluble components, dozens of chemical components of Sargentodoxae medicinal materials from fat-soluble to water-soluble are inspected on 5 thin-layer plates under multiple inspection conditions, and a set of thin-layer identification spot fingerprints is formed. It is used to improve the identification and addition rate of Sargentodoxae medicinal materials in compound preparations and to supervise their quality by multi-index evaluation. It belongs to the field of thin-layer identification. Background Art

[0002] The so-called multi-information thin-layer identification spot fingerprint of Chinese medicinal materials and its method refer to the extension of the connotation of high-performance liquid fingerprint spectrum to the field of thin-layer identification of Chinese medicinal materials. The fat-soluble, medium-polar and water-soluble components in Chinese medicinal materials are adsorbed and desorbed repeatedly in the order of non-polar, medium-polar and polar in developing solvents with increasing polarity according to the law of similar dissolution. Most of the effective ingredients with detection information in the medicinal materials are obtained according to the size of polarity on a very small number of thin-layer plates under different inspection conditions. As many spot information spectra as possible are obtained, thus forming a set of multi-information thin-layer identification spot fingerprints from fat-soluble to water-soluble components and their identification methods, providing overall quality supervision of Sargentodoxae medicinal materials from different sources; and compound preparations prepared by various processes as reference spectra and methods for quality evaluation.

[0003] The so-called multi-information means that in the detection part of the fat-soluble components, there is basically no information spot at the forefront of the thin layer plate, and in the detection part of the water-soluble components, there is no obvious spot residue at the baseline of the thin layer plate, indicating that all information spots are already included in the gradient thin layer development range, and then combined with a variety of inspection conditions, the information spots that can be inspected are all presented under different developing agents and inspection conditions, and multi-information from fat-soluble to water-soluble is obtained. The Chinese medicine Sargentodoxae is a variety included in the Chinese Pharmacopoeia, which has the effects of clearing heat and detoxifying, activating blood circulation, and dispelling wind and relieving pain. It is used for abdominal pain caused by intestinal carbuncle, heat-toxic sores, amenorrhea, dysmenorrhea, swelling and pain caused by falls, and rheumatism. It is a commonly used medicinal material for clearing heat and detoxifying, dispelling wind and relieving pain in clinical practice. It contains a variety of chemical components such as tannins, glycosides, cyclic polyphenols, triterpenoid saponins, lignins, flavonoids, glycosides and organic acids. Pharmacological studies have shown that it has multiple pharmacological effects such as anti-radiation, antibacterial, anti-inflammatory, anti-allergic, anti-cancer and cardiovascular system activity. Its numerous chemical components include fat-soluble, medium-polar and water-soluble. There has been a new revision of the thin-layer identification of the Chinese Pharmacopoeia 2020 edition, which has simplified the original thin-layer identification of the test solution processing method, greatly reduced the sampling volume of the test sample, replaced the developing agent system, revised from one identification to two identifications, and revised the fat-soluble components to water-soluble components, that is, the lower layer solution of chloroform-methanol-acetone-water (6:3:1:1) and toluene-ethyl acetate-formic acid-glacial acetic acid-water (0.5:15:1:1:2) as developing agents. After development, the first developing agent is to detect the spots that are colored with iodine vapor; the second developing agent is to detect the fluorescent spots under ultraviolet light at 365nm. From the polarity analysis of the developing agent, all the detected compounds are water-soluble. After consulting the literature, it was found that the method used was the one recorded in the 2015 edition of the Chinese Pharmacopoeia, that is, 5g of crude powder of Sargentodoxae spatholobi was taken, 50ml of methanol was added, ultrasonic treatment was performed for 30 minutes, filtered, the filtrate was evaporated to dryness, 10ml of 2% sodium hydroxide solution was added to the residue to dissolve it, the pH value was adjusted to 2 with hydrochloric acid, and 10ml of ether was shaken and extracted 3 times, each time, the ether solution was combined, evaporated, and 2ml of methanol was added to the residue to dissolve it as the test solution. Another 5g of Sargentodoxae spatholobi was taken, and the control medicinal material solution was prepared in the same way. According to the thin layer chromatography method (Appendix ⅥB), 2μl of each of the above two solutions was taken and spotted on the same silica gel G thin layer plate, and chloroform-acetone-formic acid (8:1:0.8) was used as the developing solvent, developed, taken out, dried, and sprayed with 2% ferric chloride ethanol solution. In the chromatogram of the test sample, spots of the same color appeared at the corresponding position of the chromatogram of the control medicinal material. Not only is the sampling volume large, the preparation of the test sample and control medicinal material solutions is cumbersome and time-consuming, and it pollutes the environment. The detection index is the fat-soluble phenolic components that are colored when exposed to ferric chloride. At present, no other reports on thin-layer identification of Sargassum spatholobi have been found. There is no multi-information thin-layer identification spot fingerprint of Sargassum spatholobi and its method.

[0004] In order to provide a simple, fast, low-cost, and efficient multi-information identification method for Sargentodoxae and its various preparations prepared by different processes, as well as a multi-index quality evaluation system for commercially available medicinal materials, a special thin-layer identification study on Sargentodoxae was conducted. A set of multi-information thin-layer identification spot fingerprints and identification methods from fat-soluble to water-soluble components were obtained. Summary of the invention

[0005] A simple and quick pretreatment method was used to obtain the test sample and control medicinal material solutions. Using gradient developing agents of different polarities, more than 30 chemical components of Sargentodoxae medicinal materials from fat-soluble to water-soluble were examined on 5 thin layer plates under a variety of inspection conditions, forming a set of thin layer identification spot fingerprints and methods. The total time was 2 hours, 4ml of extraction solvent, and 50ml of developing agent. A few thin layer plates and dozens of milliliters of developing agent carry more information than its HPLC fingerprint, but the low cost, ultra-high efficiency, and intuitiveness of spot color are not available in HPLC fingerprints. Simple, fast, and efficient, it has the application prospect of greatly improving the identification and subscription rate of Sargentodoxae medicinal materials in compound preparations and supervising their quality through multi-index evaluation.

[0006] The solution adopted by the present invention to solve the technical problem is:

[0007] (1) Thin layer identification of fat-soluble components of Sargassum ulmoides: 0.2 g of Sargassum ulmoides and Sargassum ulmoides control powder were taken, and 2 ml of methanol was added respectively. The mixture was ultrasonically treated for 10 minutes and centrifuged. The supernatant was used as the test solution and the control solution. 8-10 μl of each of the two solutions was taken and spotted on the same silica gel GF 254 On the thin layer plate, cyclohexane-ethyl acetate-formic acid with a volume ratio of 8:2:0.1 was used as a developing agent, developed, taken out, dried, and examined under an ultraviolet lamp at 365nm. In the chromatogram of the test sample, at the corresponding position of the chromatogram of the control medicinal material, a main fluorescent spot of the same color appeared; sprayed with 10% sulfuric acid ethanol solution, heated at 105℃ until the spots were clearly colored, and examined under an ultraviolet lamp at 365nm. In the chromatogram of the test sample, at the corresponding position of the chromatogram of the control medicinal material, a main fluorescent spot of the same color appeared; in the dark room, through the light, examined, in the chromatogram of the test sample, at the corresponding position of the chromatogram of the control medicinal material, a spot of the same color appeared;

[0008] (2) Thin layer identification of lipid-soluble components of Sargentodoxae spatholobi: 8-10 μl of the test solution and control solution under (1) were taken and spotted on the same silica gel GF 254On the thin layer plate, cyclohexane-ethyl acetate-formic acid with a volume ratio of 5:6.5:0.15 was used as a developing agent, developed, taken out, dried, and examined under a 365nm ultraviolet lamp. In the chromatogram of the test sample, at the corresponding position of the chromatogram of the control medicinal material, a main fluorescent spot of the same color appeared; sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots were clearly colored, and examined under a 365nm ultraviolet lamp. In the chromatogram of the test sample, at the corresponding position of the chromatogram of the control medicinal material, a main fluorescent spot of the same color appeared; then placed in a dark room and examined through light, the chromatogram of the test sample showed spots of the same color at the corresponding position of the chromatogram of the control medicinal material;

[0009] (3) Thin layer identification of polar components of Sargentodoxae cyrtonema - acidic developing agent: 8-10 μl of the test solution and the control solution under (1) were taken and spotted on the same silica gel GF 254 On the thin layer plate, cyclohexane-ethyl acetate-methanol-formic acid with a volume ratio of 4.2:6.1:2:0.1 was used as a developing solvent, developed, taken out, dried, and examined under an ultraviolet lamp at 365nm. In the chromatogram of the test sample, a main fluorescent spot of the same color appeared at the corresponding position of the chromatogram of the control medicinal material; under an ultraviolet lamp at 254nm, a main fluorescent spot of the same color appeared at the corresponding position of the chromatogram of the test sample and the control medicinal material; sprayed with 10% sulfuric acid ethanol solution, heated at 105℃ until the spots were clearly colored, and examined under an ultraviolet lamp at 365nm. In the chromatogram of the test sample, a main fluorescent spot of the same color appeared at the corresponding position of the chromatogram of the control medicinal material; then sprayed with a mixed solution of 5% vanillin sulfuric acid solution-ethanol with a volume ratio of 1:7, heated at 105℃ until the spots were clearly colored, and examined in a dark room through light. In the chromatogram of the test sample, a spot of the same color appeared at the corresponding position of the chromatogram of the control medicinal material;

[0010] (4) Thin layer identification of polar components of Sargentodoxae cyrtonema - alkaline developing agent: 8-10 μl of the test solution and the control solution under (1) were taken and spotted on the same silica gel GF 254 On the thin layer plate, chloroform-ethyl acetate-methanol-concentrated ammonia with a volume ratio of 4:4:3.8:0.5 was used as a developing solvent, developed, taken out, dried, and examined under a UV lamp at 365nm. In the chromatogram of the test sample, a main fluorescent spot of the same color appeared at the corresponding position of the chromatogram of the reference medicinal material; sprayed with 10% sulfuric acid ethanol solution, heated at 105℃ until the spots were clearly colored, and examined under a UV lamp at 365nm. In the chromatogram of the test sample, a main fluorescent spot of the same color appeared at the corresponding position of the chromatogram of the reference medicinal material;

[0011] (5) Thin layer identification of polar components of Sargentodoxae spatholobi: 5-6 μl of the test solution and the control solution under (1) were taken and spotted on the same silica gel GF 254On the thin layer plate, use butyl acetate-glacial acetic acid-water with a volume ratio of 7.2:3.1:0.2 as the developing agent, develop, take out, dry, and examine under ultraviolet light at 365nm. In the chromatogram of the test sample, a main fluorescent spot of the same color appears at the corresponding position of the chromatogram of the control medicinal material; examine under ultraviolet light at 254nm, and in the chromatogram of the test sample, a main spot of the same color appears at the corresponding position of the chromatogram of the control medicinal material; spray with a mixed solution of 5% vanillin sulfuric acid solution-ethanol in a volume ratio of 1:7, heat at 105°C until the spots are clearly colored, and examine in a dark room through light. In the chromatogram of the test sample, the same red spots appear at the corresponding positions of the chromatogram of the control medicinal material.

[0012] The principle of the present invention is as follows:

[0013] According to the chemical structure and properties of each effective ingredient of traditional Chinese medicine, the principle of similar and compatible extraction is followed, and suitable extraction solvents are used to prepare test and control medicinal material solutions simply and quickly. Then, different developing agents with gradually increasing polarity are used for development. As the polarity of the developing agent increases, the polarity of the developed components will gradually increase. On the same thin layer plate, according to the different abilities of adsorption, desorption, re-adsorption, and re-desorption, the effective ingredients with similar polarity are further separated on their respective thin layer plates. In addition, with the help of various effective ingredients with similar polarity, on the same thin layer plate, under different inspection conditions, although they overlap, they do not interfere with each other at different levels, presenting different spot colors, and obtaining multi-information thin layer chromatograms. Finally, according to the polarity of the developing agent, the well-separated multi-information gradient thin layer chromatograms on each thin layer plate are connected from non-polar to polar, forming a set of multi-information thin layer identification spot fingerprints and corresponding thin layer identification methods.

[0014] The innovative features and beneficial effects of the present invention are as follows:

[0015] 1. After developing the fat-soluble components of Sargentodoxae spatholobi with cyclohexane-ethyl acetate-formic acid in a volume ratio of 8:2:0.1, the thin layer plate was placed under a UV lamp at 365 nm to observe two clear fluorescent spots, one of which was at the front of the developing agent and the other had a moderate Rf value ( Figure 1 ); no spot information under UV light at 254nm; after spraying 10% sulfuric acid for color development, the drug and the control drug both showed 5 fluorescent spots of different colors under UV light at 365nm, and 4 Rf values ​​were moderate ( Figure 2 ); In the dark room, check under the light, in the chromatogram of the test sample, 6 identical brown spots appear at the corresponding positions of the chromatogram of the control medicinal material ( Figure 3 ); Under the three inspection conditions, a total of 13 information spots were detected on the same thin layer plate, belonging to 7 chemical components.

[0016] 2. The lipophilic components of Sargentodoxae were developed with cyclohexane-ethyl acetate-formic acid in a volume ratio of 5:6.5:0.15. Two fluorescent spots were observed under ultraviolet light at 365 nm, one of which had a moderate Rf value ( Figure 4 ), seemingly similar to Figure 1 The same, in fact, is not the same chemical composition, its water solubility is large; under ultraviolet light 254nm, there is no spot information; after spraying 10% sulfuric acid for color development, under ultraviolet light 365nm, the medicinal materials and the control medicinal materials both showed 6 fluorescent spots of different colors, which is consistent with Figure 2 Compared with the original Figure 2 The fluorescent spots in the image all reach the upper part of the thin layer plate ( Figure 5 ); In the dark room, under the light, 7 identical brown spots appeared in the chromatogram of the test sample at the corresponding position of the chromatogram of the control medicinal material ( Figure 6 ); for the same thin layer plate, under three inspection conditions, a total of 15 information spots were detected, excluding Figures 1 to 3 , overlapping spots, belonging to 5 chemical components.

[0017] 3. After the medium polar components were developed by using cyclohexane-ethyl acetate-methanol-formic acid with a volume ratio of 4.2:6.1:2:0.1, two fluorescent spots were observed under ultraviolet light at 365 nm, one of which had a moderate Rf value ( Figure 7 ), seemingly similar to Figure 1 , Figure 4 The spots are the same, but they are not actually of the same chemical composition. Figure 4 than Figure 1 Highly water soluble, Figure 7 The Figure 4 Highly water soluble, Figure 4 There is no spot near the fluorescent spot under the ultraviolet lamp at 254nm; so the thin layer plate is examined under the ultraviolet lamp at 254nm, and a brown spot appears slightly above the fluorescent spot ( Figure 8 ); After spraying 10% sulfuric acid for color development, the samples were examined under a UV lamp at 365 nm. Both the medicinal materials and the control medicinal materials showed 4 fluorescent spots of different colors. Figure 2 , Figure 5 , the colors of these fluorescent spots are different, they are other components ( Fig. 9 ); then spray a 5% vanillin sulfuric acid solution-ethanol mixed solution with a volume ratio of 1:7, heat at 105°C until the spots are clearly colored, place in a dark room and inspect under light. In the chromatogram of the test sample, 8 spots of different colors appear at the corresponding positions of the chromatogram of the control medicinal material, including 3 red, 2 brown-purple, and 3 light brown. The colors are bright and very conducive to quality comparison ( Fig.10); This is the first report. According to the above calculation, under the four inspection conditions, a total of about 15 informative spots were detected on the same thin layer plate, and excluding the spots that overlapped with the previous and next spectra, they were attributed to 7 compounds.

[0018] 4. The medicinal material of Sargentoides cuneata was developed with chloroform-ethyl acetate-methanol-concentrated ammonia in a volume ratio of 4:4:3.8:0.5. After the polar components were developed, three fluorescent spots of different colors were observed under ultraviolet light at 365nm ( Fig.11 ); no clear spots were observed under ultraviolet light at 254 nm; after spraying 10% sulfuric acid for color development, the samples were observed under ultraviolet light at 365 nm. Both the medicinal materials and the control medicinal materials showed 8 fluorescent spots of different colors. Most of the fluorescent spots were on the upper part of the thin layer plate ( Fig.12 ); no clear spots are presented under sunlight and light; in alkaline developing agents, the neutral components are all fluorescent spots, and the spots before color development are at the bottom of the thin layer plate, which are not in the same position as the fluorescent spots after color development. Under the two inspection conditions, 11 fluorescent information spots were detected on the same thin layer plate, belonging to 10 chemical components; the neutral polar component spots in alkaline developing agents are provided.

[0019] 5. The medicinal material of Sargentophyllum sphaerocephalum was developed with butyl acetate-glacial acetic acid-water in a volume ratio of 7.2:3.1:0.2. After the water-soluble components were developed, a bright blue fluorescent spot was observed under ultraviolet light at 365 nm ( Fig.13 ); A brown spot was observed under UV light at 254 nm. Comparing the spot shapes under the two inspection conditions, it is not the same compound ( Fig.14 ); spray with a 5% vanillin sulfuric acid solution-ethanol mixed solution with a volume ratio of 1:7, heat at 105°C until the spots are clearly colored, place in a dark room and inspect under light. In the chromatogram of the test sample, 4 clear blood-red spots appear at the corresponding positions of the chromatogram of the control medicinal material ( Fig.15 ). Based on the mechanism of the reaction with vanillin sulfuric acid, the blood-red spots should belong to saponins, and the content is relatively high. From the analysis of spot shape and Rf value, it is consistent with Fig.13 The fluorescent spots are not the same component. Under three inspection conditions, a total of 6 information spots were detected on the same thin layer plate, belonging to 5 chemical components. And these blood-red spots just reflect the blood in the name of the big blood vine, with a unique color, which is reported for the first time.

[0020] 6. In summary, using the same test sample and control medicinal material solution, with 5 different polarity developing agents, on 5 thin layer plates, under various inspection conditions, about 60 information spots of Sargentodoxae medicinal materials were inspected, and some connecting spots were removed, which were attributed to about 34 chemical components. A set of thin layer identification spot fingerprints was formed. The total time taken was 2 hours, 4 ml of extraction solvent, and 50 ml of developing agent. A few thin layer plates, tens of milliliters of developing agent, carry more information than its high performance liquid fingerprint spectrum. 【 1 】 More Fig.16 ), but the low cost, ultra-high efficiency and intuitiveness of spot color are not possessed by HPLC fingerprint.

[0021] 7. To evaluate and compare the quality of Sargentoides chinensis from different sources, different origins, and different harvesting seasons, as well as compound preparations with different processes and formulations, it provides optional multi-information chromatograms and methods for thin-layer identification of Sargentoides chinensis. The method is simple, fast, and efficient, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the thin layer TLC image of the fat-soluble components of Sargentodoxae chinensis under ultraviolet light at 365nm.

[0023] Figure 2 This is a thin layer TLC chart of the fat-soluble components of Sargentodoxae cyrtonema after being developed with 10% sulfuric acid ethanol solution and examined under ultraviolet light at 365nm.

[0024] Figure 3 This is a thin layer TLC chart of the fat-soluble components of Sargentodoxae cyrtonema developed with 10% sulfuric acid ethanol solution and then observed under light in a dark room.

[0025] Figure 4 This is the thin layer TLC image of the fat-soluble components of Sargentodoxae chinensis under ultraviolet light at 365nm.

[0026] Figure 5 This is the thin layer TLC chart of the fat-soluble components of Sargentodoxae cyrtonema under ultraviolet light at 365nm after being developed with 10% sulfuric acid ethanol solution.

[0027] Figure 6 This is a thin layer TLC chart of the partially fat-soluble components of Sargentodoxae cyrtonema developed with 10% sulfuric acid ethanol solution and then observed under light in a dark room.

[0028] Figure 7 This is the thin layer TLC chart of the polar components of the Sargentophyllum grandiflorum developed with an acidic developer under ultraviolet light at 365nm.

[0029] Figure 8This is the thin layer TLC chart of the polar components of the Sargentophyllum grandiflorum developed with an acidic developer under ultraviolet light at 254nm.

[0030] Fig. 9 The mid-polar components of Sargentophyllum cuneata developed with an acidic developer were visualized with 10% sulfuric acid ethanol solution and then analyzed by TLC under ultraviolet light at 365 nm.

[0031] Fig.10 The polar components of Sargentodon chinensis developed with an acidic developer were then developed with ethanolic sulfate and then with vanillin ethanolic sulfate solution. This is a thin layer TLC image observed through light.

[0032] Fig.11 This is the thin layer TLC chart of the polar components of the Sargentophyllum grandiflorum developed with an alkaline developer under ultraviolet light at 365nm.

[0033] Fig.12 The mid-polar components of Sargentophyllum cuneata developed with an alkaline developer were visualized with 10% ethanolic sulfuric acid solution and then analyzed by TLC under an ultraviolet lamp at 365 nm.

[0034] Fig.13 This is the thin layer TLC diagram of the water-soluble components of Sargentodoxae medicinal materials under ultraviolet light at 365nm.

[0035] Fig.14 This is the thin layer TLC diagram of the water-soluble components of Sargentodoxae medicinal materials under ultraviolet light at 254nm.

[0036] Fig.15 This is a thin layer TLC image observed through light after the water-soluble component of Sargentodoxae cyrtonema was developed with the ethanolic solution of vanillin sulfate.

[0037] Fig.16 This is the HPLC fingerprint of Sargentodoxae chinensis reported in the literature.

[0038] Figure 1 , Figure 2 , Figure 3 These are TLC images of the same TLC plate under different inspection conditions. In the figure, 1.2.3.4.5 are Sargassum spatholobi medicinal materials; 6. Sargassum spatholobi control medicinal material.

[0039] Figure 4 , 5 , 6 are TLC pictures under different inspection conditions for the same TLC plate, in which 1.2.3.4.5 are Sargentodoxae medicinal materials; 6. Sargentodoxae control medicinal materials.

[0040] Figure 7 , 8 , 9, and 10 are TLC images of the same TLC plate under different inspection conditions. In the figure, 1. 2. 3. 4. 5. Sargassum spatholobi medicinal material; 6. Sargassum spatholobi control medicinal material.

[0041] Fig.11 , 12 These are TLC images of the same TLC plate under different inspection conditions. In the figure, 1.2.3.4.5 are Sargassum spatholobi medicinal materials; 6. Sargassum spatholobi control medicinal material.

[0042] Fig.13 , 14 , 15 are TLC images of the same TLC plate under different inspection conditions, in which 1.2.3.4 are Sargentodoxa chinensis medicinal materials; 5. Sargentodoxa chinensis control medicinal material.

[0043] Fig.16 In the figure, A. is Sargentodoxae spatholobi; B. is a mixed reference substance, 1 to 22 are peak numbers, including 8. protocatechuic aldehyde, 9. salidroside, 22. rhein, and the others are unknown component peaks; the ordinate is the peak area; the abscissa is the retention time (minutes).

[0044] The specific implementation methods of the present invention are as follows:

[0045] (1) Thin layer identification of fat-soluble components of Sargassum ulmoides: 0.2 g of Sargassum ulmoides and Sargassum ulmoides control powder were taken, and 2 ml of methanol was added respectively. The mixture was ultrasonically treated for 10 minutes and centrifuged. The supernatant was used as the test solution and the control solution. 8-10 μl of each of the two solutions was taken and spotted on the same silica gel GF 254 On the thin layer plate, cyclohexane-ethyl acetate-formic acid with a volume ratio of 8:2:0.1 was used as a developing agent, developed, taken out, dried, and examined under an ultraviolet lamp at 365nm. In the chromatogram of the test sample, at the corresponding position of the chromatogram of the control medicinal material, a main fluorescent spot of the same color appeared; sprayed with 10% sulfuric acid ethanol solution, heated at 105℃ until the spots were clearly colored, and examined under an ultraviolet lamp at 365nm. In the chromatogram of the test sample, at the corresponding position of the chromatogram of the control medicinal material, a main fluorescent spot of the same color appeared; in the dark room, through the light, examined, in the chromatogram of the test sample, at the corresponding position of the chromatogram of the control medicinal material, a spot of the same color appeared;

[0046] (2) Thin layer identification of lipid-soluble components of Sargentodoxae spatholobi: 8-10 μl of the test solution and control solution under (1) were taken and spotted on the same silica gel GF 254On the thin layer plate, cyclohexane-ethyl acetate-formic acid with a volume ratio of 5:6.5:0.15 was used as a developing agent, developed, taken out, dried, and examined under a 365nm ultraviolet lamp. In the chromatogram of the test sample, at the corresponding position of the chromatogram of the control medicinal material, a main fluorescent spot of the same color appeared; sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots were clearly colored, and examined under a 365nm ultraviolet lamp. In the chromatogram of the test sample, at the corresponding position of the chromatogram of the control medicinal material, a main fluorescent spot of the same color appeared; then placed in a dark room and examined through light, the chromatogram of the test sample showed spots of the same color at the corresponding position of the chromatogram of the control medicinal material;

[0047] (3) Thin layer identification of polar components of Sargentodoxae cyrtonema - acidic developing agent: 8-10 μl of the test solution and the control solution under (1) were taken and spotted on the same silica gel GF 254 On the thin layer plate, cyclohexane-ethyl acetate-methanol-formic acid with a volume ratio of 4.2:6.1:2:0.1 was used as a developing solvent, developed, taken out, dried, and examined under an ultraviolet lamp at 365nm. In the chromatogram of the test sample, a main fluorescent spot of the same color appeared at the corresponding position of the chromatogram of the control medicinal material; under an ultraviolet lamp at 254nm, a main fluorescent spot of the same color appeared at the corresponding position of the chromatogram of the test sample and the control medicinal material; sprayed with 10% sulfuric acid ethanol solution, heated at 105℃ until the spots were clearly colored, and examined under an ultraviolet lamp at 365nm. In the chromatogram of the test sample, a main fluorescent spot of the same color appeared at the corresponding position of the chromatogram of the control medicinal material; then sprayed with a mixed solution of 5% vanillin sulfuric acid solution-ethanol with a volume ratio of 1:7, heated at 105℃ until the spots were clearly colored, and examined in a dark room through light. In the chromatogram of the test sample, a spot of the same color appeared at the corresponding position of the chromatogram of the control medicinal material;

[0048] (4) Thin layer identification of polar components of Sargentodoxae cyrtonema - alkaline developing agent: 8-10 μl of the test solution and the control solution under (1) were taken and spotted on the same silica gel GF 254 On the thin layer plate, chloroform-ethyl acetate-methanol-concentrated ammonia with a volume ratio of 4:4:3.8:0.5 was used as a developing solvent, developed, taken out, dried, and examined under a UV lamp at 365nm. In the chromatogram of the test sample, a main fluorescent spot of the same color appeared at the corresponding position of the chromatogram of the reference medicinal material; sprayed with 10% sulfuric acid ethanol solution, heated at 105℃ until the spots were clearly colored, and examined under a UV lamp at 365nm. In the chromatogram of the test sample, a main fluorescent spot of the same color appeared at the corresponding position of the chromatogram of the reference medicinal material;

[0049] (5) Thin layer identification of polar components of Sargentodoxae spatholobi: 5-6 μl of the test solution and the control solution under (1) were taken and spotted on the same silica gel GF 254On the thin layer plate, use butyl acetate-glacial acetic acid-water with a volume ratio of 7.2:3.1:0.2 as the developing agent, develop, take out, dry, and examine under ultraviolet light at 365nm. In the chromatogram of the test sample, a main fluorescent spot of the same color appears at the corresponding position of the chromatogram of the control medicinal material; examine under ultraviolet light at 254nm, and in the chromatogram of the test sample, a main spot of the same color appears at the corresponding position of the chromatogram of the control medicinal material; spray with a mixed solution of 5% vanillin sulfuric acid solution-ethanol in a volume ratio of 1:7, heat at 105°C until the spots are clearly colored, and examine in a dark room through light. In the chromatogram of the test sample, the same red spots appear at the corresponding positions of the chromatogram of the control medicinal material.

[0050] References

[0051] 【1】Xu Feng, Yang Ye, Shu Qiu'e, et al. HPLC fingerprint of Sargentodoxae cyrtonema[J]. Chinese Journal of Experimental Traditional Chinese Medicine, 2013, 19(13): 145-148

Claims

1. A method for constructing a multi-information thin-layer identification spot fingerprint of Sargentodoxae medicinal materials, characterized by: (1) Thin layer identification of fat-soluble components of Sargassum ulmoides: Take 0.2 g of Sargassum ulmoides and Sargassum ulmoides control medicinal material powder, add 2 ml of methanol respectively, treat with ultrasound for 10 minutes, centrifuge, and use the supernatant as the test solution and control medicinal material solution; take 8-10 μl of each of the above two solutions, spot them on the same silica gel GF254 thin layer plate, use cyclohexane-ethyl acetate-formic acid with a volume ratio of 8:2:0.1 as the developing agent, develop, take out, dry, and examine under ultraviolet light at 365 nm. In the chromatogram of the test sample, at the corresponding position of the chromatogram of the control medicinal material, a main fluorescent spot of the same color appears; spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored, and examine under ultraviolet light at 365 nm. In the chromatogram of the test sample, at the corresponding position of the chromatogram of the control medicinal material, a main fluorescent spot of the same color appears; examine under light in a dark room, and in the chromatogram of the test sample, at the corresponding position of the chromatogram of the control medicinal material, a spot of the same color appears; (2) Thin layer identification of lipid-soluble components of Sargentodoxae sphaerocarpa: 8-10 μl of the test solution and control solution under (1) are taken and spotted on the same silica gel GF254 thin layer plate respectively. The plate is developed with cyclohexane-ethyl acetate-formic acid in a volume ratio of 5:6.5:0.

15. The plate is taken out and dried. The plate is examined under a UV lamp at 365 nm. In the chromatogram of the test sample, a main fluorescent spot of the same color is shown at the corresponding position of the chromatogram of the control drug. The plate is sprayed with 10% sulfuric acid ethanol solution and heated at 105°C until the spots are clearly colored. The plate is examined under a UV lamp at 365 nm. In the chromatogram of the test sample, a main fluorescent spot of the same color is shown at the corresponding position of the chromatogram of the control drug. The plate is then placed in a dark room and examined under light. In the chromatogram of the test sample, a spot of the same color is shown at the corresponding position of the chromatogram of the control drug. (3) Thin-layer identification of polar components in Sargentopsis ulmoides - acidic developing agent: 8-10 μl of the test solution and control solution under (1) were taken and spotted on the same silica gel GF254 thin-layer plate, respectively. Cyclohexane-ethyl acetate-methanol-formic acid with a volume ratio of 4.2:6.1:2:0.1 was used as the developing agent. The plate was developed, taken out, dried, and examined under ultraviolet light at 365 nm. In the chromatogram of the test sample, a main fluorescent spot of the same color appeared at the corresponding position in the chromatogram of the control drug. In the chromatogram of the test sample, a main fluorescent spot of the same color appeared. In the chromatogram of the control drug, a main fluorescent spot of the same color appeared. In the chromatogram of the test sample, a main fluorescent spot of the same color appeared at the corresponding position in the chromatogram of the control drug ... In the spectrum, the main spot of the same color appears at the position corresponding to the chromatogram of the control medicinal material; spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spot is clearly colored, and examine under ultraviolet light at 365nm, and in the chromatogram of the test sample, the main fluorescent spot of the same color appears at the position corresponding to the chromatogram of the control medicinal material; then spray with a mixed solution of 5% vanillin sulfuric acid solution-ethanol at a volume ratio of 1:7, heat at 105℃ until the spot is clearly colored, and examine under light in a dark room, and in the chromatogram of the test sample, the spot of the same color appears at the position corresponding to the chromatogram of the control medicinal material; (4) Thin-layer identification of polar components of Sargentodoxae spatholobi - alkaline developing agent: 8-10 μl of the test solution and control solution under (1) were taken and spotted on the same silica gel GF254 thin-layer plate respectively. The plate was developed with chloroform-ethyl acetate-methanol-concentrated ammonia in a volume ratio of 4:4:3.8:0.5 as the developing agent. The plate was taken out and dried. The plate was examined under a UV lamp at 365 nm. In the chromatogram of the test sample, a main fluorescent spot of the same color appeared at the corresponding position of the chromatogram of the control drug. The plate was sprayed with 10% sulfuric acid ethanol solution and heated at 105°C until the spots were clearly colored. The plate was examined under a UV lamp at 365 nm. In the chromatogram of the test sample, a main fluorescent spot of the same color appeared at the corresponding position of the chromatogram of the control drug. (5) Thin layer identification of polar components of Sargentodoxae sphaerocarpa: 5 μl of the test sample solution and the control drug solution under (1) were taken and spotted on the same silica gel GF254 thin layer plate respectively. The plate was developed with butyl acetate-glacial acetic acid-water in a volume ratio of 7.2:3.1:0.

2. The plate was taken out and dried in the air. The plate was examined under a UV lamp at 365 nm. The test sample chromatogram showed a main fluorescent spot of the same color at the corresponding position of the control drug chromatogram. The plate was examined under a UV lamp at 254 nm. The test sample chromatogram showed a main fluorescent spot of the same color at the corresponding position of the control drug chromatogram. The plate was sprayed with a mixed solution of 5% vanillin sulfuric acid solution-ethanol in a volume ratio of 1:

7. The plate was heated at 105°C until the spots were clearly colored. The plate was examined in a dark room under light. The test sample chromatogram showed a main red spot at the corresponding position of the control drug chromatogram.

Images