A rapid thin-layer identification method for multiple information of one plate of Yinao Capsules

Abstract

The present invention relates to a single-plate, multi-information, rapid thin-layer identification method for Yinao Capsules. The method is characterized by using a simple and rapid pretreatment method to obtain test and control medicinal material solutions. Nine medicinal materials were identified on five thin-layer plates using the same test solution. Different medicinal material components were examined under different inspection conditions. Although the components overlapped, they did not interfere with each other under the different inspection conditions, presenting distinct color spots or fluorescent spots on their respective layers. This identification requires only 3g of sample, 1g of Ganoderma lucidum control medicinal material, 0.1-0.3g of other control medicinal materials, 27ml of organic solvent, 60ml of developing agent, and 3 hours. This method can easily, quickly, efficiently, and with low energy consumption identify 90% of the medicinal materials in Yinao Capsules. The fluorescent spots of ginseng, Ganoderma lucidum, and Schisandra chinensis after sulfuric acid color development are new characteristic points. The single-plate identification of ginseng and Poria cocos, Polygala tenuifolia and Ganoderma lucidum, and Schisandra chinensis and Acorus calamus is also reported for the first time.

Description

Technical Field

[0001] The present invention relates to a single-plate, multi-information rapid thin-layer chromatography (TLC) identification method for Yinao Capsules, belonging to the field of TLC testing for traditional Chinese medicines. Multi-information refers to the use of a single TLC plate to detect multiple information spots for one to two medicinal materials or one medicinal material. Rapid refers to the preparation of test or control medicinal material solutions, which bypasses tedious extraction and purification. Only organic solvent ultrasonication or water decoction followed by evaporation is required to dissolve the organic solvent, resulting in the test and control medicinal material solutions. A single test solution can be used for all TLC identification applications. Nine medicinal materials, including ginseng, Poria cocos, tortoise shell glue, Ophiopogon japonicus, Codonopsis pilosula, Polygala tenuifolia, Ganoderma lucidum, Acorus calamus, and Schisandra chinensis, were identified on five TLC plates. Requiring only 3g of sample, 1g of Ganoderma lucidum control medicinal material, 0.1-0.3g of other control medicinal materials, 27ml of organic solvent, 60ml of developing agent, and three hours of processing time, 90% of the medicinal materials in Yinao Capsules can be identified simply, quickly, efficiently, and with low energy consumption, resulting in clear, beautiful spots. Background Art

[0002] In traditional Chinese medicine compound preparations, especially water-extracted compound preparations with more than ten herbs, due to the large number of medicinal flavors, most of which are extracted by water, according to the principle of similar-compatible extraction, fat-soluble components are basically no longer present, and only water-soluble or partially water-soluble components are extracted. For some low-content components, due to the polymerization, decomposition, and destruction of the components during the extraction process, it is basically difficult to detect them. Therefore, the thin layer identification revision rate is low and there are few quantitative determination indicators. After consulting the traditional water-extracted compound preparations included in the 2020 edition of the Chinese Pharmacopoeia, several comparable examples are listed below:

[0003] Example 1: Wushicha Granules are composed of 19 medicinal materials. With hesperidin, forsythin and glycyrrhizic acid as controls, thin layer identification was performed on 4 medicinal materials: dried tangerine peel, immature tangerine peel, forsythia suspensa and licorice. Moreover, hesperidin was used to identify 2 medicinal materials: dried tangerine peel and immature tangerine peel. The identification rate was 21%. The method was cumbersome and time-consuming. The identification of the 4 medicinal materials required 24g of sample, 418ml of organic solvent, 36ml of developing agent and 17.5 hours. Example 2: Yunkang Granules are composed of 23 Chinese medicinal materials. With astragaloside, angelica sinensis, paeoniflorin, baicalin and psoralen as controls, the identification of 5 medicinal materials: astragalus, angelica sinensis, white peony root, scutellaria baicalensis and psoralea corylifolia was performed. The identification rate was 21.7%. The identification required 40g of sample, 382ml of organic solvent, 60ml of developing agent and 14.5 hours. Example 3: Jieyu Anshen Granules are composed of 16 medicinal materials. Example 4 Tianmeng Capsules are composed of 17 medicinal materials. With Polygala tenuifolia, Bupleurum chinense and Licorice as controls, 3 medicinal materials were identified, and the identification and revision rate was 18.8%. The identification required 25g of sample (13g sugar-free), 276ml of organic solvent, 36ml of developing agent, and 11.5 hours. Example 4 Tianmeng Capsules are composed of 17 medicinal materials. With Acanthopanax senticosus, Astragaloside IV, Lycium barbarum, Epimedium and Hesperidin as controls, 5 medicinal materials were identified, and the identification and revision rate was 29.4%. The identification required 12g of sample, 594ml of organic solvent, 60ml of developing agent, and 17 hours.

[0004] The average TLC identification update rate of the above four water-extract preparations is 22.7%. That is, for a preparation composed of 13 medicinal materials, only 3 medicinal materials can be identified as having been added, while the addition of 10 medicinal materials cannot be controlled. The inspection level of quality standards is too low to provide strong support for quality supervision.

[0005] Even among the varieties with the highest TLC identification update rates, TLC identification is often limited to a single variety. For N identification items, the traditional identification model requires the preparation of N sample solutions, N TLC plates, and N developments to identify N medicinal ingredients. To eliminate interference, sample pretreatment procedures are often complex and tedious, requiring repeated purification with large amounts of organic reagents. This is labor-intensive, time-consuming, and reagent-intensive, polluting the environment, and harmful to health, with a long testing cycle. Consequently, a quality standard test consisting of TLC identification of 7-8 medicinal ingredients and two content determinations typically takes a week to complete. This time can double if repeated testing is required. This testing speed severely constrains the pace of modernized traditional Chinese medicine production. Therefore, finding simple and rapid testing methods, improving testing efficiency, and reducing testing costs have become crucial challenges in the quality testing of traditional Chinese medicine compound preparations. For example, Fangfeng Tongsheng Granules, a preparation with a high TLC identification update rate in the Chinese Pharmacopoeia, contains 17 medicinal ingredients, of which 9 have been identified, resulting in an identification update rate of 52.9%. The specific method is analyzed below:

[0006]

Identification

[0007] (2) Take 6g of this product, grind it into powder, add 30ml of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 10ml of water, adjust the pH value to 10 with 1mol / L sodium hydroxide solution, shake and extract twice with 20ml of chloroform, combine the chloroform extracts, evaporate to dryness at low temperature, dissolve the residue in 2ml of methanol, and use it as the test solution. Separately, take the ephedrine hydrochloride reference substance, add methanol to make a solution containing 1mg per 1ml, and use it as the reference solution. According to the thin layer chromatography method (General Rule 502), take 4-8μl of the test solution and 5μl of the reference solution, and spot them on the same silica gel G thin layer plate, use chloroform-methanol-concentrated ammonia test solution (4:1:0.1) as the developing solvent, develop, remove, dry, spray with ninhydrin test solution, heat at 105℃ until the spots are clearly colored, and inspect under sunlight. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions in the chromatogram of the reference sample.

[0008] (3) Take 6g of this product, grind it into powder, add 10ml of hydrochloric acid solution (3→20), mix well, add 30ml of chloroform, heat and reflux for 1 hour, take the chloroform solution, evaporate to dryness, and dissolve the residue in 2ml of methanol to prepare the test solution. Take 0.5g of rhubarb as a control medicinal material and prepare the control medicinal material solution in the same way. According to the thin layer chromatography method (General Rule 502), take 5μl of each of the above two solutions and spot them on the same silica gel G thin layer plate. Use the upper layer solution of toluene-ethyl formate-methanol-formic acid-water (6:2:0.4:0.1:1) as the developing solvent, develop, remove, dry, and examine under ultraviolet light at 365nm. In the chromatogram of the test product, at the corresponding position of the chromatogram of the control medicinal material, an orange-yellow fluorescent spot of the same color appears; after fumigation with ammonia gas, the spot turns red.

[0009] (4) Take the test solution under [Identification] (2) as the test solution. Separately, take the Gardenia glycoside reference substance and add methanol to make a solution containing 1 mg per 1 ml as the reference solution. According to the thin layer chromatography method (General Rule 502), take 5 μl of the test solution and 2 μl of the reference solution and spot them on the same silica gel G thin layer plate. Use the lower layer solution of chloroform-methanol-water (13:7:2) placed below 10°C as the developing solvent. Develop, remove, dry, spray with 5% vanillin sulfuric acid solution, heat at 105°C until the spots are clearly colored, and inspect under sunlight. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions in the chromatogram of the reference substance.

[0010] (5) Take 15g of this product, grind it into powder, add 40ml of a mixture of 7% ethanolic sulfuric acid solution and water (1:3), heat and reflux for 3 hours, let it cool, and extract it twice with chloroform by shaking, 20ml each time. Combine the chloroform extracts, wash with 30ml of water, discard the water washings, filter the chloroform extracts through a funnel covered with anhydrous sodium sulfate, evaporate the filtrate to dryness, and dissolve the residue in 1ml of methanol to prepare the test solution. Take 1g of Platycodon grandiflorum as a control medicinal material and prepare the control medicinal material solution in the same way. According to the thin layer chromatography method (General Method 502), take 10μl of each of the above two solutions and spot them on the same silica gel G thin layer plate. Use chloroform-ether (1:1) as the developing solvent, develop, remove, dry, spray with 10% ethanolic sulfuric acid solution, heat at 105℃ until the spots are clearly colored, and examine under sunlight. In the chromatogram of the test product, spots of the same color appear at the corresponding positions in the chromatogram of the control medicinal material.

[0011] (6) Take 20g of this product, grind it into powder, add 100ml of ether, heat and reflux for 1 hour, filter, recover the solvent from the filtrate until dry, and dissolve the residue in 1ml of methanol to prepare the test solution. Take 0.5g of Angelica sinensis and Chuanxiong Rhizoma, and prepare the control solution in the same way. According to the thin layer chromatography method (General Rules 502), take 20μl of the test solution and 4μl of the control solution, and spot them on the same silica gel G thin layer plate. Use petroleum ether (30-60℃)-ethyl acetate (9:1) as the developing solvent, develop, remove, dry, and examine under ultraviolet light at 365nm. In the chromatogram of the test sample, at the corresponding position of the chromatogram of the control drug, a fluorescent spot of the same color will appear.

[0012] (7) Take 5 g of this product, grind it into powder, add 30 ml of methanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 15 ml of water, extract twice with 15 ml of ether by shaking, discard the ether solution, adjust the pH of the aqueous solution to 1-2 with dilute hydrochloric acid, extract twice with 10 ml of ethyl acetate by shaking, combine the ethyl acetate extracts, evaporate to dryness, dissolve the residue in 1 ml of methanol, and use it as the test solution. Separately, take the baicalin reference substance and add methanol to make a solution containing 1 mg per 1 ml, which is used as the reference solution. According to the thin layer chromatography method (General Method 502), take 5 μl of each of the above two solutions and spot them on the same silica gel G thin layer plate, use ethyl acetate-butanone-formic acid-water (5:3:1:1) as the developing solvent, develop, remove, dry, spray with 2% ferric chloride ethanol solution, and examine under sunlight. In the chromatogram of the test substance, spots of the same color appear at the corresponding positions in the chromatogram of the reference substance.

[0013] (8) Take 1.5g of this product, grind it into powder, add 5ml of 0.5mol / L hydrochloric acid solution, shake for 10 minutes, centrifuge, add 30ml of 50% ethanol to the residue, adjust the pH value to 7 with sodium carbonate solution, heat and reflux to extract for 1 hour, filter, concentrate the filtrate to dryness, and dissolve the residue in 2ml of 50% ethanol to prepare the test solution. Take another 0.3g of licorice control medicinal material and prepare the control medicinal material solution in the same way. According to the thin layer chromatography method (General Method 502), take 5μl of each of the above two solutions and spot them on the same silica gel G thin layer plate. Use ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) as the developing solvent, develop, remove, dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly colored, and examine under ultraviolet light at 365nm. In the chromatogram of the test product, fluorescent spots of the same color appear at the corresponding positions of the chromatogram of the control medicinal material.

[0014] The eight TLC tests in the example above identified nine herbs: 5-0-methylvisamidoside as a control for siler, ephedra (Ephedra sinica) as a control for ephedrine hydrochloride, gardenia (Gardenia jasminoides) as a control for jasmine, baicalin as a control for scutellaria (Scutellaria baicalensis), and rhubarb, platycodon, angelica, ligusticum, and liquorice as control herbs. Sample pretreatment alone took 20 hours. With 60g of sample, 755ml of organic extraction solvent, 120ml of developing solvent, and 6 hours of development time, the TLC analysis of one batch of samples took approximately four days and 875ml of organic solvent. This assumes that the identification of each of the four herbs is performed using a control. Using a control herb would increase sample preparation time and solvent consumption. The control provides limited information, hindering quality control.

[0015] If retesting is required, the testing time and organic reagents would need to be doubled, making the testing speed unmatched by mechanized mass production. Quality control methods for traditional water-extracted compound preparations of traditional Chinese medicine, such as those used in the dissection example, require enrichment and purification of the test solution due to the loss of significant amounts of fat-soluble and low-content components. These methods can be even more tedious and complex than those used in the examples, requiring more solvent and time.

[0016] The above summarizes the current status of various TLC identification standards. Specifically, regarding the TLC identification of Yinao Capsules, four TLC reports on the same formula but different dosage forms have been reviewed. Reference 1, the original quality standard for Yinao Capsules, includes TLC identification of two medicinal ingredients, Schisandra chinensis and Ganoderma lucidum. The sample size is 20 capsules (6g), and the reference medicinal ingredients Schisandra chinensis is 1g and Ganoderma lucidum is 8g. The preparation time for the sample and reference medicinal solutions alone is 4–5 hours, and the organic extraction solvent is 93ml. Reference 2, "Study on the Quality Standard of Yinao Capsules," published by Zhao Zhijun et al. in the fifth issue of "Chinese Pharmaceutical Standards" in 2007, identifies Polygala tenuifolia, Panax ginseng, and Radix Ophiopogonis. The TLC identification of these three medicinal ingredients requires 7g of sample, 2g of Polygala tenuifolia reference medicinal ingredient, 1g each of Panax ginseng and Panax ginseng reference medicinal ingredient, 272ml of extraction solvent, 24ml of developing agent, and 5 hours. Document 3: "Study on the Quality Standard of Yinao Tablets" published by Jiang Weike et al. in Volume 30, Issue 11 of Chinese Patent Medicine in 2008, identified four medicinal materials: Schisandra chinensis, Ganoderma lucidum, Ginseng, and Polygala tenuifolia by thin layer chromatography. The identification required 12 g of sample, 1 g each of Schisandra chinensis, Ginseng, and Polygala tenuifolia reference materials, 8 g of Ganoderma lucidum reference material, 450 ml of extraction solvent, 36 ml of developing agent, and 13 hours. Document 4, "Improvement of Quality Standards for Yinao Capsules" published by Jin Yang in the 6th issue of Volume 21 of the West China Journal of Pharmacy in 2006, added thin-layer chromatography identification of Polygala tenuifolia, Codonopsis pilosula, Ophiopogon japonicus, Acorus calamus and Panax ginseng in addition to the thin-layer chromatography identification of Polygala tenuifolia, Codonopsis pilosula, Ophiopogon japonicus, Acorus calamus and Panax ginseng. The thin-layer chromatography of the seven medicinal materials required a total of 18 g of sample, 8 g of Ganoderma lucidum reference medicinal material, 1 g each of Schisandra chinensis, Polygala tenuifolia, Codonopsis pilosula, Ophiopogon japonicus, Acorus calamus and Panax ginseng reference medicinal material, 1065 ml of extraction solvent, 60 ml of developing agent and 28 hours of extraction time.

[0017] In this way, the thin layer situation of Yinao Capsules is very similar to the pharmacopoeia situation described above. Not only is the thin layer identification collection rate too low, and the identification of 2-3 medicinal materials cannot control the feeding situation, but the identification of 7 medicinal materials not only takes a long time, but a large amount of organic reagents evaporate into the atmosphere, causing serious environmental pollution. Moreover, the detection speed seriously restricts the speed of mechanized large-scale production, hinders development, and affects output.

[0018] Therefore, while improving the subscription rate of thin layer identification, we must also take into account improving detection efficiency, reducing detection costs, and reducing environmental pollution. This has become an urgent goal for testers. Under this background, we used traditional water-extracted Yinao Capsules as the research object, and conducted a simple, fast, low-cost, and high-efficiency one-plate multi-information rapid thin layer identification method, and achieved success.

[0019] The prescription composition and preparation process of a Yinao capsule are as follows:

[0020] Prescription: Tortoise Shell Glue 38.6g, Polygala 193.3g, Dragon Bone 387.3g, Ganoderma Lucidum 387.3g

[0021] Schisandra chinensis 49.3g, Ophiopogon japonicus 193.3g, Acorus calamus 193.3g, Codonopsis pilosula 111.0g

[0022] Ginseng 66.6g, Poria 387.3g

[0023] Preparation process: The above ten ingredients, except tortoise shell glue, ginseng and Poria cocos (25%) are crushed into fine powder respectively. The remaining Poria cocos and the other seven ingredients including Polygala tenuifolia are decocted in water twice, the first time for 3 hours and the second time for 2 hours. Filter, combine the filtrate, and concentrate it into a thick paste. Add ginseng, Poria cocos powder and dissolved tortoise shell glue, mix thoroughly, dry, grind into fine powder, sieve, and put into capsules to make 1000 capsules. Summary of the Invention

[0024] A single-plate, multi-information rapid thin-layer chromatography (TLC) identification method for Yinao Capsules has been established. The method features a simple and rapid pretreatment method to generate test and control herbal solutions. Nine herbs were identified using the same test solution on five TLC plates. Different herbal components were examined under different inspection conditions. Although overlapping, these components did not interfere with each other under the different inspection conditions, appearing as distinct color or fluorescent spots on their respective layers. This identification requires only 3g of sample, 1g of Ganoderma lucidum control herbal solution, 0.1-0.3g of other control herbs, 27ml of organic solvent, 60ml of developing agent, and 3 hours. This method allows for the simple, rapid, efficient, and low-cost identification of 90% of the herbs in Yinao Capsules. The fluorescent spots of ginseng, Ganoderma lucidum, and Schisandra chinensis, which were enhanced by sulfuric acid color development, are novel characteristic points. The single-plate identification of ginseng and Poria cocos, Polygala tenuifolia and Ganoderma lucidum, and Schisandra chinensis and Acorus calamus is also reported for the first time.

[0025] The solution adopted by the present invention to solve the technical problem is:

[0026] (1) Thin layer identification of ginseng and Poria: Take Yinao capsule, remove the capsule shell, weigh 3g of the contents, add 10ml of methanol, ultrasonically treat for 10 minutes, filter, evaporate the filtrate to dryness, add 1ml of methanol to dissolve the residue, and use it as the test solution; take 0.2g of ginseng and Poria powder as control medicinal materials, add 2ml of methanol respectively, and ultrasonically treat for 20 minutes, and the supernatant is used as the control medicinal material solution; take 5μl of the above control medicinal material solution and 5-8μl of the test sample solution, and spot them on the same silica gel GF 254 On a thin layer plate, develop with cyclohexane-ethyl acetate-formic acid in a volume ratio of 10:1:0.1, take out, blow dry with hot air, and examine under a 365nm ultraviolet lamp. In the chromatogram of the test sample, a fluorescent spot of the same color appears at the corresponding position of the chromatogram of the Poria cocos control medicinal material; spray with 10% sulfuric acid ethanol solution, bake at 105℃ until the spots are clearly colored, and examine under a 365nm ultraviolet lamp. 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 ginseng control medicinal material;

[0027] (2) TLC identification of tortoise shell glue: Take 0.1 g of tortoise shell glue control medicinal material, add 2 ml of methanol, and ultrasonically treat for 20 minutes. The supernatant is used as the control medicinal material solution; take 5 μl of the control medicinal material solution and 3 μl of the test sample solution under identification (1), and spot them on the same silica gel G thin layer plate respectively. Use cyclohexane-ethyl acetate-formic acid with a volume ratio of 15:1:0.1 as the developing solvent, develop, take out, blow dry with hot air, spray with 10% sulfuric acid ethanol solution, bake at 105℃ until the spots are clearly colored, and examine under sunlight. In the chromatogram of the test sample, the main spot of the same color appears at the corresponding position of the chromatogram of the control medicinal material;

[0028] (3) Thin layer identification of Radix Ophiopogonis and Radix Codonopsis: Take 0.1 g of Radix Codonopsis and 0.2 g of Radix Ophiopogonis as control medicinal material, add 2 ml of methanol respectively, and ultrasonically treat for 20 minutes, and take the supernatant as the control medicinal material solution; take 3-5 μl of the control medicinal material solution and 2 μl of the test solution under identification (1), and spot them on the same silica gel G thin layer plate, use chloroform-ethyl acetate-methanol-concentrated ammonia with a volume ratio of 1:2:4:0.5 as the developing solvent, develop, take out, blow dry with hot air, spray with 10% sulfuric acid ethanol solution, and heat at 105 ° C until the spots are clearly colored. In the chromatogram of the test sample, the main spot of the same color appears at the corresponding position in the chromatogram of the control medicinal material;

[0029] (4) Thin layer identification of Polygala tenuifolia and Ganoderma lucidum: Take the test solution under identification (1), add 0.3 ml of methanol to dilute, shake well, and use it as the test solution; take 1 g of Ganoderma lucidum control medicinal material, add 50 ml of water, simmer for 30 minutes, filter through cotton, evaporate the filtrate to dryness, add 1 ml of methanol to the residue to dissolve it, and use it as the Ganoderma lucidum control medicinal material solution; take 0.2 g of Polygala tenuifolia control medicinal material, add 2 ml of methanol, ultrasonically treat for 20 minutes, and take the supernatant as the Polygala tenuifolia control medicinal material solution; take 3 μl of Ganoderma lucidum control medicinal material solution, 5 μl of Polygala tenuifolia control medicinal material solution and test solution, and spot them on the same silica gel GF 254 On a thin layer plate, use a lower layer solution of chloroform-methanol-water in a volume ratio of 10:2.5:1 as a developing solvent, develop, remove, dry, and examine under a 254nm ultraviolet lamp. In the chromatogram of the test sample, a main spot of the same color appears at the corresponding position in the chromatogram of the reference medicinal material Polygala tenuifolia. Spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored, and examine under a 365nm ultraviolet lamp. In the chromatogram of the test sample, a main spot of yellow fluorescence appears at the corresponding position in the chromatogram of the reference medicinal material Ganoderma lucidum.

[0030] (5) Thin layer identification of Acorus tatarinowii and Schisandra chinensis: Take 0.3 g of Schisandra chinensis, add 2 ml of methanol, and ultrasonically treat for 20 minutes. Take the supernatant as the Schisandra chinensis control medicinal material solution; take another 0.3 g of Acorus tatarinowii, add 50 ml of water, and simmer for 30 minutes. Filter through cotton, evaporate the filtrate to dryness, and dissolve the residue in 1 ml of methanol to make it a control medicinal material solution of Acorus tatarinowii; take 5 μl of the control medicinal material solution and the test sample under identification (4) and apply them to the same silica gel GF gel. 254 The thin layer plate was developed with cyclohexane-ethyl acetate-formic acid in a volume ratio of 8:3:0.1, and the plate was taken out, dried with hot air, and examined under a 365 nm ultraviolet lamp. 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 Acorus tatarinowii control medicinal material; the plate was sprayed with a 10% sulfuric acid ethanol solution, heated at 105°C until the spots were clearly colored, and examined under a 365 nm ultraviolet lamp. In the chromatogram of the test sample, at least three identical bright blue fluorescent spots appeared at the corresponding position of the chromatogram of the chromatogram of the schizandra chinensis control medicinal material; and when examined under daylight, at least three identical spots of the same color appeared at the corresponding position of the chromatogram of the test sample as those of the chromatogram of the schizandra chinensis control medicinal material.

[0031] The principles of the present invention are as follows:

[0032] Based on the chemical structure and properties of each active ingredient of traditional Chinese medicine, following the extraction principle of like dissolves like, and using appropriate extraction solvents, we can easily and quickly obtain test sample and control herbal solutions with complete ingredients. Then, based on the different chemical structures and polarities of each active ingredient, as the developing agent moves, the adsorption and desorption abilities on the thin layer plate vary, so that the spots of each active ingredient can be separated. At the same time, after development, the active ingredients of each herbal medicine with similar polarity will show different color spots under different inspection conditions. Although they overlap, they do not interfere with each other, realizing the rapid multi-information thin layer identification of multiple herbs on the same thin layer plate.

[0033] The innovative features and beneficial effects of the present invention are as follows:

[0034] (1) It breaks through the traditional identification method of Chinese herbal compound preparations. There are N items to be identified, and N test sample solutions, N thin-layer plates, are developed N times to identify N medicinal materials. A method was established using the same test sample solution or its dilution, methanol ultrasonic or water decoction, and methanol to make up the volume of each control medicinal material solution. Nine medicinal materials were identified on five thin-layer plates. Different Chinese medicinal components were inspected under different inspection conditions. Each spot was clear and intertwined, but the components at different levels did not interfere with each other. Not only did the identification rate of Yinao Capsules increase from the original standard of 2 medicinal materials to 9 medicinal materials, reaching 90% of the input medicinal materials, but it also saved 2 hours, 66 ml of organic reagent, 6.5 g of control medicinal materials, and 3 g of sample compared to the thin-layer identification of 2 medicinal materials. This demonstrates the novelty, creativity, high-quality control, low cost, high efficiency, and practicality of energy conservation and emission reduction of this application.

[0035] (2) On the same thin layer plate, after development, both Poria cocos and the sample showed bright blue fluorescent spots when directly detected under a 365nm UV lamp, while ginseng showed no spots under this inspection condition (see Figure 1 After spraying 10% sulfuric acid ethanol solution for color development, not only did the bright blue fluorescence of Poria disappear, but the original fluorescent spots directly observed under 365nm ultraviolet light disappeared, and a group of new fluorescent spots of ginseng control herbs and samples appeared (see Figure 2 Ginseng and Poria cocos showed two distinct groups of fluorescent spots under different inspection conditions. These spots did not overlap, were clear, sensitive, and well-separated, which is the first discovery reported.

[0036] (3) There is no thin layer identification item under the tortoise shell glue in the 2020 edition of the Chinese Pharmacopoeia. There is only one qualitative item of ion pair MRM chromatographic peak detection by high performance liquid chromatography-mass spectrometry with gradient elution. Not only is the instrument expensive, at least more than 1 million, but the method is cumbersome and time-consuming. It takes at least 2 days to complete the test. Just imagine how many production units have such testing conditions? How can its simplicity and speed be reflected in its value of implementation and promotion? No reports of sensitive and high-content thin layer identification of exclusive ingredients have been found in the literature. The content of tortoise shell glue medicinal material in the Yinao Capsule formula is low, only 38.6g, accounting for 1.9%. Finding its thin layer identification method is not an easy task. Therefore, the qualitative item of ion pair MRM chromatographic peak detection by high performance liquid chromatography-mass spectrometry in the pharmacopoeia appeared. The thin layer identification of this application has high sensitivity. Only 0.1g of the control medicinal material is needed. It is ultrasonically dissolved in 2ml of methanol, the supernatant is spotted, developed, and colored with sulfuric acid, which will present a clear high-content exclusive spot ( Figure 3 ), which is simple, fast, and spot-specific, is reported for the first time.

[0037] (4) Codonopsis pilosula and Ophiopogon japonicus were developed with chloroform-ethyl acetate-methanol-concentrated ammonia in a volume ratio of 1:2:4:0.5. The brown spots detected on the same plate were water-soluble components with high content. Only 0.1g of Codonopsis pilosula and 0.2g of Ophiopogon japonicus were needed. After ultrasonic extraction with 2ml of methanol, the supernatant was spotted and developed to obtain the identification effect of high-concentration spots. Compared with the sampling amount of 2g of Ophiopogon japonicus reference medicinal materials included in the current pharmacopoeia and its fixed volume of 0.5ml, the difference is 40 times. It can also be said that the content of the detection index is 40 times higher. Compared with the detection index of Codonopsis pilosula and the reference substance index of codonopsis glycoside, the content of the detection index in the sample is also 20 times higher, and the sample preparation time is saved by 92%. It must be said that the detection index of Codonopsis pilosula and Ophiopogon japonicus is also innovative, with high content and small sampling amount. Only 0.1g to 0.2g of reference medicinal materials, methanol ultrasonication, and supernatant spotting are needed to control the quality of Yinao Capsules simply, quickly and with high content. Figure 4 This identification is similar to the aforementioned identification of Tangerine Peel and Citrus aurantium using hesperidin, and is a double-negative identification.

[0038] (5) Using the lower layer solution of chloroform-methanol-water with a volume ratio of 10:2.5:1 as the developing agent, thin layer identification of two medicinal materials, Polygala tenuifolia and Ganoderma lucidum, was performed on the same plate. First, the brown spots of Polygala tenuifolia were examined under a UV lamp at 254 nm. There was no interference in the blank, indicating exclusive identification (5); then, after spraying with 10% sulfuric acid ethanol solution for color development and fluorescence enhancement, the yellow fluorescent main spot of Ganoderma lucidum was examined under a UV lamp at 365 nm. This spot was exclusive to Ganoderma lucidum and was not found in other medicinal materials. It was exclusive to identification ( Figure 6); Furthermore, only 1g of Ganoderma lucidum is required as a control herb, only 1 / 8 the amount reported in the literature. This demonstrates that this application detects a higher concentration of a water-soluble component of Ganoderma lucidum, making it valuable for monitoring the quality of traditional water-extracted preparations. Furthermore, the identification of Polygala tenuifolia and Ganoderma lucidum on the same plate is reported for the first time, demonstrating novelty, creativity, and practicality in achieving twice the result with half the effort.

[0039] (6) The thin layer chromatography identification of Acorus tatarinowii and Schisandra chinensis currently included in the Chinese Pharmacopoeia are all fat-soluble components extracted with chloroform or petroleum ether. After decoction, the amount of fat-soluble components extracted is very low. If the identification is to be carried out, the sampling volume must be quite large and enrichment treatment must be carried out. Even if the identification is revised with great effort, it is of no significance to control the decoction process. After reviewing the literature, there are no reports on the thin layer chromatography identification of the decoction components of Acorus tatarinowii and Schisandra chinensis. Unfortunately, after a lot of discussion on the ratio of developing agent components, thin layer plates and color development conditions, cyclohexane-ethyl acetate-formic acid with a volume ratio of 8:3:0.1 was finally used as the developing agent. After development, the sample was placed under ultraviolet light at 365nm and examined. The sample showed an identical blue-purple fluorescent main spot at the corresponding position of the chromatogram of the Acorus tatarinowii control medicinal material. The blank sample had no interference, and the identification was exclusive ( Figure 7 ); After spraying with 10% sulfuric acid ethanol solution for color development and fluorescence enhancement, the sample was examined under a 365nm ultraviolet lamp. The sample showed several fluorescent spots of the same color at the corresponding position on the chromatogram of the Schisandra chinensis control medicinal material. Among them, there was a large pink fat-soluble spot. Because the sample was a water extract and its content was low, the fluorescence color was inconsistent. Therefore, the requirement of "at least 3 identical bright blue fluorescent spots" was set to eliminate the interference of the pink fat-soluble spot ( Figure 8 ); then the thin layer plate was placed under sunlight for inspection. The three spots of the same color of the sample and the control medicinal material were clearer. Similarly, the limitation of "at least three spots of the same color" eliminated the interference of high-content fat-soluble spots and enhanced the accuracy of the result judgment ( Figure 9 The use of highly water-soluble components to identify Acorus calamus and Schisandra chinensis on the same plate is also reported for the first time, demonstrating novelty, creativity, and practicality in monitoring water extraction processes. This method also provides a reference for identifying water extract preparations containing these two medicinal herbs, and holds broad application prospects.

[0040] (7) Compared with the current thin-layer identification method for Chinese herbal medicine decoction preparations with similar formula and preparation process, this application not only detects the most medicinal flavors, but also takes the shortest time, uses the least amount of organic reagents, and has no environmental pollution. It has formed a set of simple, fast, efficient, low-consumption, multi-drug flavor, multi-information rapid thin-layer identification methods, which plays a demonstration and leading role. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 After sample development, the fluorescent spot TLC pattern of Poria cocos was observed under UV light at 365 nm.

[0042] Figure 2 After the sample was developed, it was sprayed with 10% sulfuric acid ethanol solution for color development, and the TLC pattern of ginseng fluorescence spots was observed under a UV lamp at 365 nm.

[0043] Figure 3 After the sample was developed, it was sprayed with 10% sulfuric acid ethanol solution for color development, and the TLC pattern of the tortoise shell glue color spots was observed under sunlight.

[0044] Figure 4 After the samples were developed, they were sprayed with 10% sulfuric acid ethanol solution for color development. The TLC images of the color spots of Ophiopogon japonicus and Codonopsis pilosula were observed under sunlight.

[0045] Figure 5 After sample development, the TLC chart of brown spots on Polygala tenuifolia was observed under UV light at 254 nm.

[0046] Figure 6 After the sample was developed, it was sprayed with 10% sulfuric acid ethanol solution for color development, and the TLC pattern of the yellow fluorescent spots of Ganoderma lucidum was observed under a UV lamp at 365 nm.

[0047] Figure 7 After sample development, the TLC chart of blue fluorescent spots of Acorus tatarinowii was observed under UV light at 365 nm.

[0048] Figure 8 After the sample was developed, it was sprayed with 10% sulfuric acid ethanol solution for color development, and the TLC pattern of Schisandra chinensis fluorescence spots was observed under a UV lamp at 365 nm.

[0049] Figure 9 After the sample was developed, it was sprayed with 10% sulfuric acid ethanol solution for color development, and the TLC pattern of Schisandra chinensis color spots was observed under sunlight.

[0050] Figure 1 、 2 These are chromatograms of the same thin layer plate under different inspection conditions. In the figure, 1. Blank sample without ginseng; 2. Ginseng control medicinal material; 3. 4.5 samples; 6. Poria control medicinal material; 7. Blank sample without Poria.

[0051] Figure 3 Among them, 1.6. Tortoise shell glue control medicinal material; 2.3.4. Sample; 5. Blank sample without tortoise shell glue.

[0052] Figure 4 In the table, 1. Radix Ophiopogonis as control material; 2. 3. 4. Samples; 5. Codonopsis pilosula as control material; 6. Blank sample without Radix Ophiopogonis and Codonopsis pilosula.

[0053] Figure 5 、 6These are chromatograms of the same thin layer plate under different inspection conditions. In the figure, 1. Blank without Polygala tenuifolia; 2. Polygala tenuifolia reference medicinal material; 3. 4. 5 samples; 6. Ganoderma lucidum reference medicinal material; 7. Blank sample without Ganoderma lucidum.

[0054] Figure 7 、 8 9 are chromatograms of the same thin layer plate under different inspection conditions. In the figure, 1. blank sample without Schisandra chinensis; 2. Schisandra chinensis reference medicinal material; 3. 4. 5. sample; 6. Acorus tatarinowii reference medicinal material; 7. blank sample without Acorus tatarinowii.

[0055] Specific embodiments of the present invention

[0056] (1) Thin layer identification of ginseng and Poria: Take Yinao capsule, remove the capsule shell, weigh 3g of the contents, add 10ml of methanol, ultrasonically treat for 10 minutes, filter, evaporate the filtrate to dryness, add 1ml of methanol to dissolve the residue, and use it as the test solution; take 0.2g of ginseng and Poria powder as control medicinal materials, add 2ml of methanol respectively, and ultrasonically treat for 20 minutes, and the supernatant is used as the control medicinal material solution; take 5μl of the above control medicinal material solution and 5-8μl of the test sample solution, and spot them on the same silica gel GF 254 On a thin layer plate, develop with cyclohexane-ethyl acetate-formic acid in a volume ratio of 10:1:0.1, take out, blow dry with hot air, and examine under a 365nm ultraviolet lamp. In the chromatogram of the test sample, a fluorescent spot of the same color appears at the corresponding position of the chromatogram of the Poria cocos control medicinal material; spray with 10% sulfuric acid ethanol solution, bake at 105℃ until the spot is clearly colored, and examine under a 365nm ultraviolet lamp. 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 ginseng control medicinal material;

[0057] (2) TLC identification of tortoise shell glue: Take 0.1 g of tortoise shell glue control medicinal material, add 2 ml of methanol, and ultrasonically treat for 20 minutes. The supernatant is used as the control medicinal material solution; take 5 μl of the control medicinal material solution and 3 μl of the test sample solution under identification (1), and spot them on the same silica gel G thin layer plate respectively. Use cyclohexane-ethyl acetate-formic acid with a volume ratio of 15:1:0.1 as the developing solvent, develop, take out, blow dry with hot air, spray with 10% sulfuric acid ethanol solution, bake at 105℃ until the spots are clearly colored, and examine under sunlight. In the chromatogram of the test sample, the same color main spot appears at the corresponding position of the chromatogram of the control medicinal material;

[0058] (3) Thin layer identification of Radix Ophiopogonis and Radix Codonopsis: Take 0.1g of Radix Codonopsis and 0.2g of Radix Ophiopogonis as control medicinal material, add 2ml of methanol respectively, and ultrasonicate for 20 minutes, and take the supernatant as the control medicinal material solution; take 3-5μl of the control medicinal material solution and 2μl of the test solution under identification (1), and spot them on the same silica gel G thin layer plate, use chloroform-ethyl acetate-methanol-concentrated ammonia with a volume ratio of 1:2:4:0.5 as the developing solvent, develop, take out, blow dry with hot air, spray with 10% sulfuric acid ethanol solution, and heat at 105℃ until the spots are clearly colored. In the chromatogram of the test sample, spots of the same color appear at the corresponding position of the chromatogram of the control medicinal material;

[0059] (4) Thin layer identification of Polygala tenuifolia and Ganoderma lucidum: Take the test solution under identification (1), add 0.3 ml of methanol to dilute, shake well, and use it as the test solution; take 1 g of Ganoderma lucidum control medicinal material, add 50 ml of water, simmer for 30 minutes, filter through cotton, evaporate the filtrate to dryness, add 1 ml of methanol to the residue to dissolve it, and use it as the Ganoderma lucidum control medicinal material solution; take 0.2 g of Polygala tenuifolia control medicinal material, add 2 ml of methanol, ultrasonically treat for 20 minutes, and take the supernatant as the Polygala tenuifolia control medicinal material solution; take 3 μl of Ganoderma lucidum control medicinal material solution, 5 μl of Polygala tenuifolia control medicinal material solution and test solution, and spot them on the same silica gel GF 254 On a thin layer plate, use a lower layer solution of chloroform-methanol-water in a volume ratio of 10:2.5:1 as a developing solvent, develop, remove, dry, and examine under a 254nm ultraviolet lamp. In the chromatogram of the test sample, a main spot of the same color appears at the corresponding position in the chromatogram of the reference medicinal material Polygala tenuifolia. Spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored, and examine under a 365nm ultraviolet lamp. In the chromatogram of the test sample, a main spot of yellow fluorescence appears at the corresponding position in the chromatogram of the reference medicinal material Ganoderma lucidum.

[0060] (5) Thin layer identification of Acorus tatarinowii and Schisandra chinensis: Take 0.3 g of Schisandra chinensis, add 2 ml of methanol, and ultrasonically treat for 20 minutes. Take the supernatant as the Schisandra chinensis control medicinal material solution; take another 0.3 g of Acorus tatarinowii, add 50 ml of water, and simmer for 30 minutes. Filter through cotton, evaporate the filtrate to dryness, and dissolve the residue in 1 ml of methanol to make it a control medicinal material solution of Acorus tatarinowii; take 5 μl of the control medicinal material solution and the test sample under identification (4) and apply them to the same silica gel GF gel. 254The thin layer plate was developed with cyclohexane-ethyl acetate-formic acid in a volume ratio of 8:3:0.1, and the plate was taken out, dried with hot air, and examined under a 365 nm ultraviolet lamp. 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 Acorus tatarinowii control medicinal material; the plate was sprayed with a 10% sulfuric acid ethanol solution, heated at 105°C until the spots were clearly colored, and examined under a 365 nm ultraviolet lamp. In the chromatogram of the test sample, at least three identical bright blue fluorescent spots appeared at the corresponding position of the chromatogram of the chromatogram of the schizandra chinensis control medicinal material; and when examined under daylight, at least three identical spots of the same color appeared at the corresponding position of the chromatogram of the test sample as those of the chromatogram of the schizandra chinensis control medicinal material.

Claims

1. A one-plate multi-information rapid thin-layer identification method for Yinao capsules, characterized by: (1) Thin layer identification of ginseng and Poria: Take Yinao capsule, remove the capsule shell, weigh 3g of the contents, add 10ml of methanol, ultrasonically treat for 10 minutes, filter, evaporate the filtrate to dryness, add 1ml of methanol to dissolve the residue, and use it as the test solution; take 0.2g of ginseng and Poria powder as control medicinal materials, add 2ml of methanol respectively, and ultrasonically treat for 20 minutes, and the supernatant is used as the control medicinal material solution; take 5μl of the above control medicinal material solution and 5-8μl of the test solution, respectively, and spot them on the same silica gel GF 254 On a thin layer plate, develop with cyclohexane-ethyl acetate-formic acid in a volume ratio of 10:1:0.1, take out, blow dry with hot air, and examine under a 365nm ultraviolet lamp. In the chromatogram of the test sample, a fluorescent spot of the same color appears at the corresponding position of the chromatogram of the Poria cocos control medicinal material; spray with 10% sulfuric acid ethanol solution, bake at 105℃ until the spots are clearly colored, and examine under a 365nm ultraviolet lamp. 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 ginseng control medicinal material; (2) TLC identification of tortoise shell glue: Take 0.1 g of tortoise shell glue control medicinal material, add 2 ml of methanol, and ultrasonically treat for 20 minutes. Take the supernatant as the control medicinal material solution; take 5 μl of the control medicinal material solution and 3 μl of the test sample solution under identification (1), and spot them on the same silica gel G thin layer plate respectively. Use cyclohexane-ethyl acetate-formic acid with a volume ratio of 15:1:0.1 as the developing solvent, develop, take out, blow dry with hot air, spray with 10% sulfuric acid ethanol solution, bake at 105℃ until the spots are clearly colored, and examine under sunlight. In the chromatogram of the test sample, the main spot of the same color appears at the corresponding position of the chromatogram of the control medicinal material; (3) Thin layer identification of Radix Ophiopogonis and Radix Codonopsis: Take 0.1 g of Radix Codonopsis and 0.2 g of Radix Ophiopogonis as control medicinal material, add 2 ml of methanol respectively, and ultrasonically treat for 20 minutes, and take the supernatant as the control medicinal material solution; take 3-5 μl of the control medicinal material solution and 2 μl of the test solution under identification (1), and spot them on the same silica gel G thin layer plate respectively, and use chloroform-ethyl acetate-methanol-concentrated ammonia with a volume ratio of 1:2:4:0.5 as the developing agent, develop, take out, blow it with hot air, spray it with 10% sulfuric acid ethanol solution, and heat it at 105°C until the spots are clearly colored. In the chromatogram of the test sample, the main spot of the same color appears at the corresponding position of the chromatogram of the control medicinal material; (4) Thin layer identification of Polygala tenuifolia and Ganoderma lucidum: Take the test solution under identification (1), add 0.3 ml of methanol to dilute, shake well, and use it as the test solution; take 1 g of Ganoderma lucidum reference medicinal material, add 50 ml of water, simmer for 30 minutes, filter through cotton, evaporate the filtrate to dryness, add 1 ml of methanol to dissolve the residue, and use it as the Ganoderma lucidum reference medicinal material solution; take 0.3 ml of Polygala tenuifolia reference medicinal material .2g, add 2ml of methanol, and ultrasonically treat for 20 minutes, and take the supernatant as the Polygala control medicinal material solution; take 3μl of the Ganoderma lucidum control medicinal material solution, 5μl of the Polygala control medicinal material solution and the test sample solution, respectively, and spot them on the same silica gel GF254 thin layer plate, use the lower layer solution of chloroform-methanol-water with a volume ratio of 10:2.5:1 as the developing solvent, develop, take out, dry, and examine under a UV lamp at 254nm. In the chromatogram of the test sample, at the corresponding position of the chromatogram of the Polygala control medicinal material, a main spot of the same color appears; spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spot is clearly colored, and examine under a UV lamp at 365nm. In the chromatogram of the test sample, at the corresponding position of the chromatogram of the Ganoderma lucidum control medicinal material, a main spot of the same yellow fluorescence appears; (5) Thin layer chromatography identification of Acorus tatarinowii and Schisandra chinensis: Take 0.3 g of Schisandra chinensis as the control medicinal material, add 2 ml of methanol, and ultrasonicate for 20 minutes. The supernatant is used as the Schisandra chinensis control medicinal material solution; take another 0.3 g of Acorus tatarinowii, add 50 ml of water, and simmer for 30 minutes. Filter through cotton, evaporate the filtrate to dryness, and dissolve the residue in 1 ml of methanol to use as the Acorus tatarinowii control medicinal material solution; Take 5 μl of the control medicinal material solution and the test solution under identification (4) and apply them to the same silica gel GF 254 On a thin layer plate, develop with cyclohexane-ethyl acetate-formic acid in a volume ratio of 8:3:0.1, remove, blow dry with hot air, and examine under a 365nm ultraviolet lamp. 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 Acorus tatarinowii control medicinal material; spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored, and examine under a 365nm ultraviolet lamp. In the chromatogram of the test sample, at least three identical bright blue fluorescent spots appear at the corresponding position of the chromatogram of the chromatogram of the chromatogram of the chinensis control medicinal material; when examined under sunlight, at least three identical spots of the same color appear at the corresponding position of the chromatogram of the test sample and the chromatogram of the chinensis control medicinal material; Yinao Capsules are made from 10 medicinal herbs including tortoise shell glue, Polygala tenuifolia, Dragon Bone, Ganoderma lucidum, Schisandra chinensis, Ophiopogon japonicus, Acorus calamus, Codonopsis pilosula, Ginseng, and Poria cocos through extraction and processing. Each capsule is equivalent to 2g of raw herbs.

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