A thin layer chromatography identification method for multiple components in traditional Chinese medicine

Abstract

The present invention discloses a thin layer chromatography identification method for multiple components in a Chinese patent medicine, and belongs to the technical field of Chinese medicine detection. The ethyl acetate extract of a Chinese patent medicine containing Cistanche deserticola, hawthorn, loquat leaf, Tibetan calamus, and Forsythia suspensa is used as a test solution; Cistanche deserticola, hawthorn, loquat leaf, Tibetan calamus, and Forsythia suspensa reference medicinal materials are taken separately, slightly boiled with water and decocted, filtered, and the filtrate is concentrated and extracted 2 to 4 times with ethyl acetate shaking. The ethyl acetate solution is combined and concentrated as a reference medicinal material solution, and the test solution and the reference solution are detected by thin layer chromatography to determine the components of Cistanche deserticola, hawthorn, loquat leaf, Tibetan calamus, and Forsythia suspensa. The present invention only needs to prepare a test solution and identify the five medicinal materials at the same time. There is no need for expensive reference substances and cumbersome chromatographic column separation operations, which shortens the identification time, reduces the cost, has strong specificity, and has good reproducibility, providing a new idea for the thin layer chromatography identification of hawthorn inner gold preparations.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a thin-layer chromatography identification method for multiple components in traditional Chinese medicine. Background Art

[0002] Traditional Chinese Medicine (TCM) is a treasure of the Chinese nation. Its safety, effectiveness, and quality control are fundamental requirements. Quality standards, as a voice of authority, are a pioneering and leading factor in the development of TCM. Currently, the traditional quality control model for Chinese patent medicines has certain limitations: different medicinal flavors require different sample preparation methods and thin-layer chromatography conditions. Furthermore, to avoid interference from negative samples, multiple separation and purification methods are often required to eliminate impurities, resulting in complex operations and a heavy workload for inspection and testing.

[0003] Shanzha Nei Jin preparations, including Shanzha Nei Jin oral liquid and Shanzha Nei Jin capsules, are pharmaceutical compositions primarily based on natural plant and animal medicinal ingredients and are exclusively available from Shenwei Pharmaceutical (Kunming) Co., Ltd. Composed of eight herbs: hawthorn, Tibetan calamus, shepherd's purse, scutellaria baicalensis, forsythia suspensa, loquat leaf, cicada slough, and chicken's gizzard lining, these preparations are known to invigorate the spleen and stomach, and eliminate stagnation. They are used to treat pediatric malnutrition, loss of appetite, wrist and abdominal pain, indigestion, and bowel movements caused by food stagnation. The current standard for these preparations is included in the Internal Medicine Spleen and Stomach section of the National Compendium of Traditional Chinese Medicine Standards. The original standard only included TLC identification for scutellaria baicalensis and loquat leaf, but failed to reflect the input of hawthorn, Tibetan calamus, and forsythia suspensa.

[0004] When verifying the TLC identification method of Cistanche deserticola in the original standard, it was found that the color development of the characteristic spots was greatly affected by temperature. After a long period of low-temperature color development, faint characteristic spots appeared at the corresponding positions of the test sample and the control medicinal material, while after heating at 105°C, there were interfering color spots near the characteristic spots, which affected the judgment; in addition, when verifying the identification of loquat leaves in the original standard, the spots at the corresponding positions of the chromatogram of the test sample and the control medicinal material were not obvious, and the background interference was serious. Summary of the Invention

[0005] Aiming at the problems in the prior art that the existing standard TLC detection method for hawthorn inner gold preparations has unclear spots, serious interference, and a single standard detection component, the present invention provides a thin layer chromatography identification method for multiple components in Chinese patent medicines. Only one test solution needs to be prepared, and the five medicinal materials of Cnidium monnieri, hawthorn, loquat leaf, Tibetan calamus, and Forsythia suspensa can be identified simultaneously. The operation is simple, the detection efficiency is improved, and the detection cost is reduced.

[0006] The present invention is achieved through the following technical solutions:

[0007] A thin layer chromatography identification method for multiple components in a Chinese patent medicine comprises the following steps:

[0008] (1) Preparation of test solution: The ethyl acetate extract of Chinese patent medicine containing Cnidium monnieri, Crataegus pinnatifida, Eriobotrya japonica, Acorus calamus, and Forsythia suspensa was used as the test solution;

[0009] (2) Preparation of control medicinal material solution: Take the control medicinal materials of Cistanche deserticola, Crataegus pinnatifida, Loquat leaf, Acorus calamus, and Forsythia suspensa, add water to a slight boil and boil, filter, concentrate the filtrate and shake and extract with ethyl acetate 2 to 4 times, combine the ethyl acetate solution, and concentrate it as the control medicinal material solution;

[0010] (3) Thin layer chromatography detection:

[0011] Detection of Cistanche deserticola: Spot the test sample solution and Cistanche deserticola control medicinal material solution on a thin-layer plate respectively, develop with chloroform-methanol-concentrated ammonia test solution with a volume ratio of 9:2:0.2 as the developing agent, take out the plate after development, dry it, and examine it under a 365nm ultraviolet lamp. Fluorescent spots of the same color should be detected at the corresponding positions of the Cistanche deserticola control medicinal material;

[0012] Detection of hawthorn and loquat leaves: Spot the test sample solution, hawthorn control medicinal material solution, and loquat leaf control medicinal material solution on a thin layer plate respectively, and develop with cyclohexane-ethyl acetate-glacial acetic acid in a volume ratio of 20:6:1. After development, remove the plate, dry it, spray it with 10% sulfuric acid ethanol solution, heat it at 105℃ until the spots are clearly colored, and examine it under sunlight and 365nm ultraviolet light. Spots of the same color and fluorescent spots should be detected at the corresponding positions of the hawthorn control medicinal material and loquat leaf control medicinal material;

[0013] Detection of Tibetan Acorus: Spot the test sample solution and the Tibetan Acorus control medicinal material solution on a thin layer plate, develop with dichloromethane-ethyl acetate in a volume ratio of 9:1. After development, remove the plate, dry it, and examine it under a 365nm ultraviolet lamp. Fluorescent spots of the same color should be detected at the corresponding positions of the Tibetan Acorus control medicinal material.

[0014] Detection of Forsythia suspensa: Spot the test sample solution and Forsythia suspensa control medicinal material solution on a thin layer plate respectively, and develop with chloroform-acetone-methanol-formic acid in a volume ratio of 12:2.5:2:0.2. After development, take out the plate, dry it, spray it with 10% ethanolic sulfuric acid solution, and heat it at 105℃ until the spots are clearly colored. Spots of the same color are detected at the corresponding positions of the Forsythia suspensa control medicinal material.

[0015] Furthermore, the Chinese patent medicine is a hawthorn inner gold preparation, and each milliliter of the test solution corresponds to 3 to 6 grams of the Chinese patent medicine.

[0016] Furthermore, the Hawthorn Gold Preparation is a Hawthorn Gold Capsule or a Hawthorn Gold Oral Liquid.

[0017] Furthermore, the preparation method of the test solution of Hawthorn Nei Jin Capsule is as follows: dissolve the contents of Hawthorn Nei Jin Capsule in methanol, ultrasonically treat for 20 to 50 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in water, extract with ethyl acetate by shaking for 2 to 4 times, combine the ethyl acetate solution, and concentrate it as the test solution of Hawthorn Nei Jin Capsule; the preparation method of the test solution of Hawthorn Nei Jin Oral Liquid is as follows: extract with ethyl acetate by shaking for 2 to 4 times, combine the ethyl acetate solution, and concentrate it as the test solution of Hawthorn Nei Jin Oral Liquid.

[0018] Furthermore, the concentrated ammonia test solution in step (3) is aqueous ammonia with a volume concentration of 25-28%.

[0019] Furthermore, the thin layer plate in step (3) is a silica gel G thin layer plate.

[0020] Furthermore, the sample volume in step (3) is 3-10 μL.

[0021] Furthermore, in step (2), 25 ml of water was added to each gram of the control medicinal material, and the decocting time was 20 to 40 minutes.

[0022] The beneficial effects achieved by the present invention are:

[0023] The thin-layer chromatography identification method provided by the present invention only requires the preparation of one test sample solution, and can simultaneously identify five medicinal materials in a traditional Chinese medicine (Hawthorn Gold Preparation) including Cnidium monnieri, Hawthorn, loquat leaf, Acorus calamus, and Forsythia suspensa, without the need for expensive reference substances and cumbersome chromatographic column separation operations, thereby shortening the thin-layer chromatography identification time, reducing the consumption of expensive reference substances and organic reagents, and lowering the detection cost; the operation is relatively simple, the specificity is strong, and the reproducibility is good, providing a new idea for the thin-layer chromatography identification of the Hawthorn Gold Preparation, and can replace or supplement the existing standard content, which is beneficial to the quality control and inspection and detection of the Hawthorn Gold Preparation, and enhances the rationality and reliability. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a TLC analysis of Cistanche deserticola in Shanzha Neijin Capsule in Example 1. 1. Test sample 22031702, 2. Test sample 22031804, 3. Test sample 21122701, 4. Cistanche deserticola control medicinal material, 5. Cistanche deserticola negative control;

[0025] Figure 2 Thin-layer chromatography analysis of hawthorn and loquat leaves in Shanzha Neijin Capsule from Example 1, (a) under sunlight, (b) under 365nm UV light; 1. Test sample 22031702, 2. Test sample 22031804, 3. Test sample 21122701, 4. Loquat leaf control, 5. Hawthorn control, 6. Ursolic acid control, 7. Hawthorn and loquat leaf negative controls;

[0026] Figure 3 This is a thin layer chromatography test chart of Tibetan Acorus calamus in Shanzha Neijin Capsule in Example 1, 1. Test sample 22031702, 2. Test sample 22031804, 3. Test sample 21122701, 4. Tibetan Acorus calamus control medicinal material, 5. Tibetan Acorus calamus negative control;

[0027] Figure 4 This is a thin layer chromatography analysis of Forsythia suspensa in Shanzha Neijin Capsule in Example 1. 1. Test sample 22031702, 2. Test sample 22031804, 3. Test sample 21122701, 4. Forsythia suspensa control medicinal material, 5. Forsythiaside reference substance, 6. Forsythia suspensa negative control medicinal material;

[0028] Figure 5 This is a thin-layer chromatography analysis of Cistanche deserticola in Shanzha Neijin Capsule in Comparative Example 1, (a) at low temperature, (b) after heating at 105°C; 1. Cistanche deserticola control medicinal material, 2. Test sample 22031702, 3. Test sample 22031804, 4. Test sample 21122701;

[0029] Figure 6 This is the TLC test chart of loquat leaves in Shanzha Neijin Capsule in Comparative Example 2, 1. loquat leaves control medicinal material, 2. test sample 22031702, 3. test sample 22031804, 4. test sample 21122701. DETAILED DESCRIPTION

[0030] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is clearly and completely described below. Based on the embodiments in this application, other similar embodiments obtained by ordinary technicians in this field without making any creative work should fall within the scope of protection of this application.

[0031] The Shanzha Neijin Capsules in the following examples were produced by Shenwei Pharmaceutical (Kunming) Co., Ltd., and a total of 10 batches of products were provided, with production batch numbers of 22031701, 22031702, 22031803, 22031804, 21122701, 21122702, 21122803, 21122804, 21122905, and 21122906, respectively.

[0032] Example 1

[0033] (1) Preparation of test solution: Take 4 g of the contents of Shanzha Neijin capsule, add 50 ml of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 30 ml of water, and shake and extract with ethyl acetate three times, 20 ml each time, discard the water, combine the ethyl acetate solution, and concentrate to 1 ml as the test solution;

[0034] (2) Preparation of control medicinal material solution: Take 1g of Cistanche deserticola control medicinal material, 2g of Crataegus pinnatifida control medicinal material, 1g of Loquat leaf control medicinal material, 2g of Tibetan Acorus calamus control medicinal material, and 1g of Forsythia suspensa control medicinal material, add 50mL of water and simmer for 30min, filter, concentrate the filtrate to 30mL, shake and extract with ethyl acetate three times, 20ml each time, discard the water, combine the ethyl acetate solution, and concentrate to 0.5mL as the control medicinal material solution;

[0035] (3) Preparation of negative control medicinal material solution: remove the other medicinal ingredients of Cistanche deserticola, hawthorn, loquat leaf, Tibetan calamus, and Forsythia suspensa from the prescription of Shanzha Neijin Capsule, and prepare negative control samples of Cistanche deserticola, hawthorn + loquat leaf, Tibetan calamus, and Forsythia suspensa according to the prescription ratio. Take the negative control samples and prepare Cistanche deserticola negative control solution, hawthorn and loquat leaf negative control solution, Tibetan calamus negative control solution, and Forsythia suspensa negative control solution according to the preparation method of the test solution in step (1);

[0036] (4) Reference solution: Take appropriate amount of ursolic acid and forsythiaside reference substances, add methanol to prepare 0.5 mg ursolic acid reference solution per 1 ml and 1 mg forsythiaside reference solution per 1 ml;

[0037] (5) Thin layer chromatography detection:

[0038] Detection of Cistanche deserticola: 5µl of the test solution (3 batches), 5µl of Cistanche deserticola control medicinal material solution, and 5µl of Cistanche deserticola negative control solution were spotted on a silica gel G thin layer plate, and developed with chloroform-methanol-concentrated ammonia test solution (25%) in a volume ratio of 9:2:0.2. After development, the plate was taken out, dried, and examined under a 365nm ultraviolet lamp. The results were as follows: Figure 1 (1. Test sample 22031702, 2. Test sample 22031804, 3. Test sample 21122701. 4. Cistanche deserticola control medicinal material, 5. Cistanche deserticola negative control) as shown by Figure 1 It can be seen that in the chromatogram of the test sample, at the corresponding position of the control medicinal material of Cnidium monnieri, a fluorescent spot of the same color is shown, and there is no interference from the negative control of Cnidium monnieri.

[0039] Detection of hawthorn and loquat leaves: Spot 10µl of the test solution (3 batches), 3µl of the hawthorn control medicinal material solution, 3µl of the loquat leaf control medicinal material solution, 10µl of the hawthorn and loquat leaf negative control solutions, and 3µl of the ursolic acid reference solution on a silica gel G thin layer plate, develop with cyclohexane-ethyl acetate-glacial acetic acid in a volume ratio of 20:6:1 as the developing solvent, remove from the plate, dry it, spray it with 10% ethanolic sulfuric acid solution, heat it at 105°C until the spots are clearly colored, and examine it under sunlight and 365nm ultraviolet light. The results are as follows: Figure 2((a) Under sunlight, (b) Under 365nm UV light; 1. Test sample 22031702, 2. Test sample 22031804, 3. Test sample 21122701, 4. Loquat leaf control, 5. Hawthorn control, 6. Ursolic acid control, 7. Hawthorn and loquat leaf negative controls) As shown in the chromatogram of the test sample, spots of the same color and fluorescent spots appeared at the corresponding positions of the loquat leaf control, hawthorn control, and ursolic acid control. There was no interference from the loquat leaf and hawthorn negative controls, indicating good separation and clear spots.

[0040] Detection of Tibetan Acorus: 10µl of the test solution (3 batches), 5µl of the Tibetan Acorus control medicinal material solution, and 10µl of the Tibetan Acorus negative control medicinal material solution were spotted on a silica gel G thin layer plate, and developed with dichloromethane-ethyl acetate (volume ratio 9:1) as the developing solvent. After development, the plate was taken out, dried, and examined under a 365nm ultraviolet lamp. The results were as follows: Figure 3 As shown in the chromatograms (1. Test sample 22031702, 2. Test sample 22031804, 3. Test sample 21122701, 4. Tibetan calamus control medicinal material, 5. Tibetan calamus negative control), fluorescent spots of the same color were detected at the corresponding positions in the chromatograms of the test samples and the Tibetan calamus control medicinal material, and there was no interference from the Tibetan calamus negative control;

[0041] Forsythia suspensa detection: 5µl of the test solution (3 batches), 5µl of the Forsythia suspensa control medicinal material solution, and 5µl of the Forsythia suspensa negative control medicinal material solution were spotted on a silica gel G thin layer plate, and developed with chloroform-acetone-methanol-formic acid in a volume ratio of 12:2.5:2:0.2. After development, the plate was removed, dried, and sprayed with 10% sulfuric acid ethanol solution. The plate was heated at 105°C until the spots were clearly colored. The results were as follows: Figure 4 As shown in the chromatograms (1. Test sample 22031702, 2. Test sample 22031804, 3. Test sample 21122701. 4. Forsythia suspensa reference material, 5. Forsythiaside reference material, 6. Forsythia suspensa negative control material), spots of the same color were detected in the chromatograms of the test samples at the corresponding positions as those of the Forsythia suspensa reference material and the Forsythia suspensa negative control material, and there was no interference from the Forsythia suspensa negative control material.

[0042] Comparative Example 1

[0043] (1) Preparation of test solution: Take 2 g of the contents of Shanzha Neijin capsule, add 30 ml of water, soak in a 60°C water bath for 30 minutes, centrifuge, take the supernatant, shake and extract 3 times with n-butanol saturated with water, 15 ml each time, combine the n-butanol solution, wash with 30 ml of water saturated with n-butanol, take the n-butanol solution, evaporate to dryness, dissolve the residue in 2 ml of methanol, add it to a neutral alumina column (120 mesh, 3 g, inner diameter 1 cm, wet-packed, pre-washed with methanol), elute with 80 ml of methanol, collect the eluate, evaporate to dryness, dissolve the residue in 1 ml of methanol, and use it as the test solution;

[0044] (2) Preparation of the control medicinal material solution of Cistanche deserticola: 3.5 g of Cistanche deserticola control medicinal material was taken, added with 100 ml of water, and simmered for 30 minutes. The solution was filtered and concentrated to 30 ml. The solution was extracted by shaking with n-butanol saturated with water for 3 times, 15 ml each time. The n-butanol solution was combined and washed with 30 ml of water saturated with n-butanol. The n-butanol solution was taken and evaporated to dryness. The residue was dissolved with 0.5 ml of methanol to prepare the control medicinal material solution of Cistanche deserticola.

[0045] (3) 5µl of the test solution (3 batches) and 5µl of the control medicinal material solution of Cnidium monnieri were spotted on a silica gel G thin layer plate, and developed with chloroform-methanol-concentrated ammonia test solution (25%) in a volume ratio of 9:2:0.2. After development, the plate was taken out, dried, sprayed with 10% sulfuric acid ethanol solution, and heated at 105°C until the spots were clearly colored. The results are as follows: Figure 5 ((a) at low temperature, (b) after heating at 105℃, 1. Ceratonia suffruticosa control, 2. Test sample 22031702, 3. Test sample 22031804, 4. Test sample 21122701) as shown by Figure 5 It can be seen that under sunlight, the color development of characteristic spots is greatly affected by temperature. After a long period of low-temperature color development, faint characteristic spots appear at the corresponding positions of the test sample and the control medicinal material, while after heating at 105°C, interfering color spots appear near the characteristic spots, affecting the judgment.

[0046] Comparative Example 2

[0047] (1) Preparation of test solution: Take 2 g of the contents of Shanzha Neijin capsule, add 30 ml of water, soak in a 60°C water bath for 30 minutes, centrifuge, take the supernatant, shake and extract with water-saturated n-butanol 3 times, 15 ml each time, combine the n-butanol solution, wash with 30 ml of water saturated with n-butanol, take the n-butanol solution, evaporate to dryness, add 1 ml of methanol to dissolve the residue, and use it as the test solution;

[0048] (2) Preparation of loquat leaf control medicinal material solution: 3 g of loquat leaf control medicinal material was added to 100 ml of water, and the mixture was simmered for 30 minutes. The mixture was filtered and concentrated to 30 ml. The mixture was extracted by shaking with n-butanol saturated with water for 3 times, 15 ml each time. The n-butanol solution was combined and washed with 30 ml of water saturated with n-butanol. The n-butanol solution was evaporated to dryness, and 1 ml of methanol was added to the residue to dissolve it. This was used as the loquat leaf control medicinal material solution.

[0049] (3) 5µl of the test solution (3 batches) and 5µl of the loquat leaf control medicinal material solution were spotted on a silica gel G thin layer plate, and developed with cyclohexane-ethyl acetate-glacial acetic acid in a volume ratio of 16:8:0.1. After development, the plate was taken out, dried, sprayed with 10% sulfuric acid ethanol solution, and heated at 105°C until the spots were clearly colored. The results are as follows: Figure 6 (1. loquat leaf control medicinal material, 2. test sample 22031702, 3. test sample 22031804, 4. test sample 21122701) As shown, under sunlight, the spots of the test sample chromatogram at the corresponding positions of the loquat leaf control medicinal material are not obvious, and the background interference is serious.

[0050] Comparative Example 3

[0051] The preparation method of the test solution, loquat leaf control medicinal material solution, ursolic acid reference solution, and loquat leaf negative control solution was the same as in Example 1; the developing solvent was dichloromethane-ethyl acetate in a volume ratio of 8:2:0.2:0.2. After the spot plate was developed, it was taken out, dried, sprayed with 10% ethanolic sulfuric acid solution, and heated at 105°C until the spots were clearly colored; the results showed that under sunlight, in the chromatogram of the test sample, spots of the same color appeared at the corresponding positions of the loquat leaf control medicinal material and the ursolic acid reference, but the spots were faint, and the loquat leaf negative control had interference.

[0052] Comparative Example 4

[0053] The preparation method of the test solution, loquat leaf control medicinal material solution, ursolic acid reference solution, and loquat leaf negative control solution was the same as in Example 1; the developing solvent was chloroform-methanol-water-formic acid with a volume ratio of 8:2:0.2:0.2. After the plate was developed, it was taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots were clearly colored, and inspected under a 365nm ultraviolet lamp; the results showed that in the chromatogram of the test sample, no spots appeared at the positions corresponding to the loquat leaf control medicinal material and the ursolic acid reference, and the reference spot R f The value is too high.

[0054] Comparative Example 5

[0055] (1) Preparation of test solution: Take 4 g of the contents of Shanzha Neijin capsule, add 50 ml of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 30 ml of water, shake and extract with ether three times, 20 ml each time, discard the water, combine the ether solution, evaporate to dryness, and dissolve the residue in 1 ml of methanol to prepare the test solution;

[0056] (2) Preparation of the reference medicinal material solution of Tibetan Acorus calamus: 2 g of the reference medicinal material of Tibetan Acorus calamus was added to 50 ml of water and simmered for 30 minutes. The solution was filtered and concentrated to 30 ml. The solution was extracted with ether by shaking for 3 times, 20 ml each time. The aqueous solution was discarded, the ether solution was combined, evaporated to dryness, and the residue was dissolved with 1 ml of methanol to prepare the reference medicinal material solution of Tibetan Acorus calamus.

[0057] (3) Preparation of negative control solution of Tibetan calamus: Take the other medicinal ingredients in the prescription except for Tibetan calamus, and prepare a negative control sample without Tibetan calamus according to the prescription ratio. Add 50 ml of water, simmer for 30 minutes, filter, concentrate the filtrate to about 30 ml, and extract with ether by shaking for 3 times, 20 ml each time. Discard the water solution, combine the ether solution, evaporate to dryness, and dissolve the residue with 1 ml of methanol to prepare the negative control solution of Tibetan calamus.

[0058] (4) 5µl of the test solution, 5µl of the Tibetan Acorus calamus control solution, and 10µl of the Tibetan Acorus calamus negative control solution were spotted on a silica gel G thin layer plate, and developed with petroleum ether (60-90°C)-ethyl acetate in a volume ratio of 4:1. After development, the plate was taken out, dried, and examined under a 254nm ultraviolet lamp. In the chromatogram of the test sample, no spots of the same color were found at the corresponding positions of the Tibetan Acorus calamus control.

[0059] Comparative Example 6

[0060] (1) Preparation of test solution: Take 4 g of the contents of Shanzha Neijin capsule, add 50 ml of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 30 ml of water, shake and extract with ether three times, 20 ml each time, discard the water, combine the ether solution, evaporate to dryness, and dissolve the residue in 1 ml of methanol to prepare the test solution;

[0061] (2) Preparation of Forsythia suspensa control medicinal material solution: Take 1 g of Forsythia suspensa control medicinal material, add 50 ml of water, simmer for 30 minutes, filter, concentrate the filtrate to 30 ml, shake and extract with ether three times, 20 ml each time, discard the water, combine the ether solution, evaporate to dryness, and dissolve the residue with 1 ml of methanol to prepare the Forsythia suspensa control medicinal material solution;

[0062] (3) Preparation of forsythiaside reference solution: Take forsythiaside reference solution and add methanol to make a solution containing 1 mg per 1 ml, which is used as forsythiaside reference solution;

[0063] (4) Preparation of the negative control solution of Forsythia suspensa: Remove the other medicinal ingredients of Forsythia suspensa from the prescription and prepare a negative control sample without Forsythia suspensa according to the prescription ratio. Add 50 ml of water and simmer for 30 minutes. Filter and concentrate the filtrate to about 30 ml. Shake and extract with ether three times, 20 ml each time. Discard the water solution, combine the ether solution, evaporate to dryness, and dissolve the residue with 1 ml of methanol to prepare the negative control solution of Forsythia suspensa.

[0064] (5) 5µl of the test sample solution, 5µl of the Forsythia suspensa control medicinal material solution, 5µl of the Forsythia suspensa negative control solution were spotted on a silica gel G thin layer plate, and developed with chloroform-acetone-methanol-98% formic acid in a volume ratio of 12:2.5:2:0.2. After development, the plate was taken out, dried, sprayed with 10% ethanolic sulfuric acid solution, and heated at 105°C until the spots were clearly colored. In the chromatogram of the test sample, no spots of the same color were seen at the corresponding positions of the Forsythia suspensa control medicinal material and the Forsythia suspensa negative control.

[0065] Comparative Example 7

[0066] Preparation of test solution, preparation of Forsythia control medicinal material solution, preparation of forsythiaside reference solution, preparation of Forsythia negative control solution and the same as comparative example 6, 5µl of test solution, 5µl of Forsythia control medicinal material solution, 5µl of forsythiaside reference solution, and 5µl of Forsythia negative control solution were spotted on silica gel G thin layer plate, and cyclohexane-ethyl formate-formic acid with a volume ratio of 15:10:0.25 was used as the developing solvent. After development, the plate was taken out, dried, sprayed with 10% ethanolic sulfuric acid solution, and heated at 105°C until the spots were clearly colored. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions of the Forsythia control medicinal material, but the Forsythia negative control had interference.

Claims

1. A thin layer chromatography identification method for multiple components in Chinese patent medicine, characterized in that: The Chinese patent medicine is a Shanzha Neijin capsule or a Shanzha Neijin oral liquid, and the thin layer chromatography identification method comprises the following steps: (1) Preparation of test solution: When the Chinese patent medicine is Hawthorn Gold Capsule, the preparation of Hawthorn Gold Capsule test solution includes: adding methanol to dissolve the contents of Hawthorn Gold Capsule, ultrasonic treatment for 20 to 50 minutes, filtering, evaporating the filtrate to dryness, dissolving the residue in water, extracting with ethyl acetate 2 to 4 times, combining the ethyl acetate solution, and concentrating as the Hawthorn Gold Capsule test solution; when the Chinese patent medicine is Hawthorn Gold Oral Liquid, the preparation method of Hawthorn Gold Oral Liquid test solution includes: extracting with ethyl acetate 2 to 4 times, combining the ethyl acetate solution, and concentrating as the Hawthorn Gold Oral Liquid test solution; each milliliter of test solution corresponds to 3 to 6 g of Chinese patent medicine; (2) Preparation of control medicinal material solution: Take the control medicinal materials of Cistanche deserticola, Crataegus pinnatifida, Loquat leaf, Acorus calamus, and Forsythia suspensa, add water to a slight boil and boil, filter, concentrate the filtrate and shake and extract with ethyl acetate 2 to 4 times, combine the ethyl acetate solution, and concentrate it as the control medicinal material solution; (3) Thin layer chromatography detection: Detection of Cistanche deserticola: Spot the test sample solution and Cistanche deserticola control medicinal material solution on a thin-layer plate respectively, develop with chloroform-methanol-concentrated ammonia test solution with a volume ratio of 9:2:0.2 as the developing agent, take out the plate after development, dry it, and examine it under a 365nm ultraviolet lamp. Fluorescent spots of the same color should be detected at the corresponding positions of the Cistanche deserticola control medicinal material; Detection of hawthorn and loquat leaves: Spot the test sample solution, hawthorn control medicinal material solution, and loquat leaf control medicinal material solution on a thin layer plate respectively, and develop with cyclohexane-ethyl acetate-glacial acetic acid in a volume ratio of 20:6:

1. After development, remove the plate, dry it, spray it with 10% sulfuric acid ethanol solution, heat it at 105℃ until the spots are clearly colored, and examine it under sunlight and 365nm ultraviolet light. Spots of the same color and fluorescent spots should be detected at the corresponding positions of the hawthorn control medicinal material and loquat leaf control medicinal material; Detection of Tibetan Acorus: Spot the test sample solution and the Tibetan Acorus control medicinal material solution on a thin layer plate, develop with dichloromethane-ethyl acetate in a volume ratio of 9:

1. After development, remove the plate, dry it, and examine it under a 365nm ultraviolet lamp. Fluorescent spots of the same color should be detected at the corresponding positions of the Tibetan Acorus control medicinal material. Detection of Forsythia suspensa: Spot the test sample solution and Forsythia suspensa control medicinal material solution on a thin layer plate respectively, develop with chloroform-acetone-methanol-formic acid in a volume ratio of 12:2.5:2:0.2, remove from the plate after development, dry it, spray it with 10% sulfuric acid ethanol solution, heat it at 105℃ until the spots are clearly colored, and detect spots of the same color at the corresponding positions of the Forsythia suspensa control medicinal material; The thin layer plate is a silica gel G thin layer plate.

2. The thin layer chromatography identification method for multiple components in Chinese patent medicine according to claim 1, characterized in that: The concentrated ammonia test solution described in step (3) is aqueous ammonia with a volume concentration of 25-28%.

3. The thin layer chromatography identification method for multiple components in Chinese patent medicine according to claim 1, characterized in that: The sample volume in step (3) is 3~10μL.

4. The thin layer chromatography identification method for multiple components in Chinese patent medicine according to claim 1, characterized in that: In step (2), 25 ml of water was added to each gram of the control medicinal material, and the decocting time was slightly boiled for 20 to 40 minutes.

Images