Thin-layer chromatography identification method for whole-formula medicines in Houshuning tablet

Abstract

The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a thin-layer chromatography identification method for the flavor of whole medicines in Houshuning tablets. The thin-layer chromatography identification method for the flavor of the whole medicine in the Houshuning tablet, provided by the invention, comprises the following steps: S1, preparing a test solution of the Houshuning tablet; s2, preparing a reference medicinal material solution of spreading hedyotis herb, common andrographis herb and screwtree root; s3, adopting a polyamide thin-layer plate, taking absolute ethyl alcohol-acetic acid-water as a developing solvent, spraying an aluminum trichloride-ethanol solution for inspection, and performing thin-layer chromatography identification. According to the method disclosed by the invention, the method exclusively used for identifying the flavonoid components of the three medicinal materials in the prescription of the Houshuning tablet is established, the improvement of quality control of the Houshuning tablet is promoted, and meanwhile, the method can be used as a reference for researching the quality of the flavonoid components of the single medicinal materials such as oldenlandia diffusa, andrographis paniculata and helicteres angustifolia; and the blank of oldenlandia diffusa, andrographis paniculata and helicteres angustifolia in the aspect of flavone thin-layer chromatography identification is filled.

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 the medicinal flavors of the whole formula of Hou Shuning tablets. Background Art

[0002] Hou Shuning Tablets are a pure Chinese herbal compound preparation, composed of three medicinal materials: Hedyotis diffusa, Andrographis paniculata, and Sesame. They have the functions of clearing away heat and detoxifying, dispersing nodules and relieving pain. They are used for acute and chronic pharyngitis, acute tonsillitis, vocal cord polyps, vocal cord nodules, and can be used as an adjuvant treatment for various inflammations and cancers caused by head and neck cancer during radiotherapy. Hou Shuning preparations approved for marketing in China include Hou Shuning Tablets and Hou Shuning Capsules.

[0003] At present, the identification research on Hedyotis diffusa is mostly focused on terpenoid components (such as oleanolic acid, deacetylated oxalic acid methyl ester, etc.), and Andrographis paniculata is mostly focused on lactone components (such as andrographolide, dehydroandrographolide, etc.), and there are relatively few reports on Sesamum indica, mostly focusing on triterpenoid components (such as sesamic acid methyl ester, etc.). Although there are reports on the research progress of flavonoid components in these three medicinal materials, there are still many gaps in the thin layer chromatography identification research of flavonoid components in these three medicinal materials. Huang Lan et al. mentioned in the journal article "Research Progress on Chemical Composition, Pharmacological Action and Clinical Application of Hedyotis diffusa" ("Chinese Pharmacy" 2023, Vol. 37, No. 12) that Hedyotis diffusa has anti-inflammatory effects in modern pharmacological research. Early studies have shown that Hedyotis diffusa contains a variety of anti-inflammatory active ingredients, among which total flavonoids are the main anti-inflammatory ingredients. Xu Lancheng et al. mentioned in the journal article "Research on the Extraction Process of Active Ingredients from Andrographis Paniculata and Its Application in Animal Husbandry" ("Feed Research" 2024, Vol. 47, No. 5), that andrographolide flavonoids have antibacterial, anti-inflammatory, antioxidant and other pharmacological effects. Zhi Guofan et al. mentioned in the journal article "Progress in Research on the Pharmacological Activity of Sesame" ("Guangdong Chemical Industry" 2023, Vol. 50, No. 20), that the main components of Sesame Paniculata are lignans, flavonoids, saponins, terpenes, alkaloids, etc. These components are the material basis for the efficacy of Sesame Paniculata. Multiple groups of research data have shown that the flavonoid components in Hedyotis diffusa, Andrographis Paniculata and Sesame Paniculata have pharmacological effects such as antibacterial, anti-inflammatory and anti-tumor, and are important effective components for the effectiveness of Hou Shuning Tablets. Effective ingredients are the key to determining the efficacy of traditional Chinese medicine preparations. Therefore, establishing a flavonoid component identification project in these three medicinal materials is of great significance for the overall quality control of Hou Shuning Tablets.

[0004] Hou Shuning preparations have not been included in the Chinese Pharmacopoeia. The current legal standard for Hou Shuning tablets is the 20th volume of the Ministry-issued Standard for Chinese Medicine Formula Preparations, WS3-B-4013-98. The standard includes microscopic identification of Andrographis paniculata and thin-layer chromatography identification of andrographolide, but lacks identification items for Hedyotis diffusa and Sesamum indica, and cannot effectively control product quality. The standards for Hou Shuning capsules are included in the "New Drug Standard 86" (Standard No.: YBZ25372005-2009Z) and "New Drug Standard 87" (Standard No.: YBZ12122005-2009Z). The standards include microscopic identification of Andrographis paniculata, thin-layer chromatography identification of andrographolide and dehydroandrographolide, and thin-layer chromatography identification of oleanolic acid, but lack identification items for Sesamum indica. The standards are still imperfect and need to be improved. Chen Gui et al. established a thin layer chromatography identification project for Hedyotis diffusa, Andrographis paniculata and Sesamum in the journal article "Improvement of Quality Standards of Hou Shuning Tablets" (Journal of Drug Analysis, Vol. 40, No. 7, 2020), but the study required the use of different test sample solutions, thin layer plates, developing agents and colorants to conduct identification tests on individual medicinal materials in the prescription, which was time-consuming and labor-intensive. In addition, the experiment required the use of highly toxic organic solvents such as chloroform, toluene and methanol, which was not conducive to the protection of personnel and the environment.

[0005] At present, there is no report on the simultaneous identification of the medicinal materials of Hou Shuning Tablets using the same test solution on the same thin layer plate. By establishing a simultaneous identification method for the characteristic flavonoid components of each medicinal material in Hou Shuning Tablets, the identification of the medicinal materials of the whole formula can be achieved. It can be used as a fast and simple quality control method. It can also be used as a reference for the quality research of flavonoid components of Hedyotis diffusa, Andrographis paniculata and Sesame, filling the gap in the thin layer chromatography identification of flavonoids of Hedyotis diffusa, Andrographis paniculata and Sesame. Summary of the invention

[0006] In view of the defects of the prior art, the present invention aims to provide a thin layer chromatography identification method for the whole prescription of Hou Shu Ning Tablets. The method takes the flavonoid components in the whole prescription of Hou Shu Ning Tablets as the core entry point, and successfully establishes an identification method for the flavonoid components of the three medicinal flavors (Houttuynia cordata, Andrographis paniculata, Sesame) in the prescription of Hou Shu Ning Tablets. By using the same test solution and conducting a thin layer chromatography test on the same thin layer plate, the effective separation and identification of the three medicinal materials in Hou Shu Ning Tablets can be achieved. The obtained chromatographic spots are clear and easy to identify, with strong specificity and good durability, providing a more scientific and efficient means for the quality control of Hou Shu Ning Tablets.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The first object of the present invention is to provide a thin layer chromatography identification method for the whole prescription of Hou Shu Ning tablets, comprising the following steps:

[0009] S1. Preparation of test solution:

[0010] Take Hou Shu Ning tablets, remove the coating, grind them into powder, add ethanol solution, extract by ultrasonic, filter, evaporate the filtrate to dryness, add ethanol solution to dissolve the residue, and use it as the test solution;

[0011] S2. Preparation of control medicinal material solution:

[0012] Take Hedyotis diffusa, Andrographis paniculata and Sesame as reference medicinal materials respectively, add water to heat and reflux to extract, cool, filter, evaporate the filtrate to dryness, add ethanol solution to dissolve the residues, and use them as Hedyotis diffusa, Andrographis paniculata and Sesame as reference medicinal material solutions;

[0013] S3. Thin layer chromatography identification:

[0014] Pipette the test sample solution and each control medicinal material solution, spot them on the same polyamide film, use anhydrous ethanol-acetic acid-water as the developing agent, develop, take out, dry, spray with aluminum chloride-ethanol solution, heat, take out and inspect;

[0015] Result determination: When examined under ultraviolet light, in the chromatogram of the test sample, the same yellow-green fluorescent spots appeared at the corresponding positions of the chromatograms of the reference medicinal materials Hedyotis diffusa and Andrographis paniculata, indicating that Hedyotis diffusa and Andrographis paniculata can be detected; the same blue fluorescent spots appeared at the corresponding positions of the chromatograms of the reference medicinal materials Sesamum indicum, indicating that Sesamum indicum can be detected.

[0016] Preferably, the concentration of ethanol in step S1 is 60-80%.

[0017] Preferably, the ultrasonic extraction frequency in step S1 is 20-60kHz, the solid-liquid ratio is 1:(40-60), the extraction temperature is room temperature, and the extraction time is 15-25min; the water-adding heating reflux extraction solid-liquid ratio in step S2 is 1:(40-60), the heating temperature is 100-110°C, the reflux temperature is 90-110°C, the extraction time is 50-70min, and the reflux times are 1-2 times; the ethanol concentration is 60-80%.

[0018] Preferably, the specification of the Hou Shu Ning tablets in step S1 is 0.2-0.6 g per tablet; and the residues in step S2 are dissolved in ethanol solution to prepare solutions containing 0.1 g per 1 mL.

[0019] Preferably, the sample spotting volume in the thin layer chromatography identification process in step S3 is 1-2 μl.

[0020] Preferably, in step S3, anhydrous ethanol-acetic acid-water is used as a developing agent, and the volume ratio of anhydrous ethanol, acetic acid and water is (3-6):1:(2-8).

[0021] Preferably, the preparation of the test solution in step S1 can also use methanol as the solvent for ultrasonic extraction and as the final dissolution solvent; the preparation of the control medicinal material solution in S2 can also use methanol as the final dissolution solvent; the developing agent in step S3 also includes methanol-formic acid-water in a volume ratio of (3-6):1:(2-8).

[0022] Preferably, the aluminum chloride-ethanol solution in step S3 is made of aluminum chloride and ethanol with a concentration of 80-95%, and the mass percentage of aluminum chloride in the aluminum chloride ethanol solution is 3-10%.

[0023] Preferably, the heating in step S3 comprises heating at 100-110° C. for 2-5 min.

[0024] Preferably, in step S3, the sample is inspected under a 360-370 nm ultraviolet light.

[0025] The principle of the present invention is as follows:

[0026] The present invention is based on the chemical structure and properties of the whole prescription medicinal flavors of Hedyotis diffusa, Andrographis paniculata and Sesame in Hou Shu Ning Tablets, takes flavonoid components as the starting point, follows the similar and compatible extraction principle, selects a specific extraction solvent to ensure the full extraction of the effective ingredients, and thus simply and quickly prepares a test solution and a control medicinal material solution. Subsequently, using the principle of thin layer chromatography technology, each medicinal material component is effectively separated on a thin layer plate due to the difference in its adsorption and desorption capacity through the movement of a developing agent. In this process, according to the different polarities of each component, an effective component with similar polarity is selected as a basis for identification, and under specific inspection conditions (such as ultraviolet light, visible light or color development with specific chemical reagents, etc.), these components will present their own unique spot colors. Thereby achieving accurate, rapid and intuitive thin layer chromatography identification of the three medicinal materials of Hedyotis diffusa, Andrographis paniculata and Sesame in Hou Shu Ning Tablets, providing a scientific and reliable method for drug quality control.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] Compared with the prior art, the thin layer chromatography identification method of the whole formula of Hou Shu Ning tablets of the present invention adopts thin layer chromatography for identification, and can realize the separation and identification of the three medicinal materials of the whole formula of Hou Shu Ning tablets - Hedyotis diffusa, Andrographis paniculata and Sesamum in only one thin layer chromatography test by using the same test sample solution and the same thin layer plate, the operation is simple and fast, the spots are bright in color and easy to observe, the amount of test samples, control medicinal materials and reagents used is small, the reagents used are safe, non-toxic and environmentally friendly, the number of thin layer chromatography identification tests is reduced, the inspection cost is greatly reduced, the inspection time is saved, the inspection efficiency is improved, and the method is suitable as a quality control method for Hou Shu Ning tablets.

[0029] The present invention takes the flavonoid components of the whole formula of Hou Shuning Tablets as the starting point, establishes an identification method for the flavonoid components of the three medicinal flavors in the prescription of Hou Shuning Tablets, promotes the improvement of the quality control of Hou Shuning Tablets, and can also be used as a reference for the quality research of the flavonoid components of the single medicinal materials of Hedyotis diffusa, Andrographis paniculata and Sesame, filling the gap in the thin layer chromatography identification of flavonoids of Hedyotis diffusa, Andrographis paniculata and Sesame. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The thin layer chromatogram of Example 1; wherein the red frame shows the characteristic spots of Hedyotis diffusa, the blue frame shows the characteristic spots of Andrographis paniculata, and the yellow frame shows the characteristic spots of Sesamum indica;

[0031] Figure 2 The thin layer chromatogram of Example 2; wherein the red frame shows the characteristic spots of Hedyotis diffusa, the blue frame shows the characteristic spots of Andrographis paniculata, and the yellow frame shows the characteristic spots of Sesamum indica;

[0032] Figure 3 This is a thin layer chromatogram of specificity investigation; the red frame shows the characteristic spots of Hedyotis diffusa, the blue frame shows the characteristic spots of Andrographis paniculata, and the yellow frame shows the characteristic spots of Sesamum indica;

[0033] Figure 4 This is a thin layer chromatogram obtained by using tetracarboxylic biochemical polyamide film for thin layer identification in the investigation of thin layer plates from different manufacturers; the red frame shows the characteristic spots of Hedyotis diffusa, the blue frame shows the characteristic spots of Andrographis paniculata, and the yellow frame shows the characteristic spots of Sesamum indica;

[0034] Figure 5 This is a thin layer chromatogram obtained by using Bangkai polyamide film for thin layer identification in the investigation of thin layer plates from different manufacturers; the red frame shows the characteristic spots of Hedyotis diffusa, the blue frame shows the characteristic spots of Andrographis paniculata, and the yellow frame shows the characteristic spots of Sesamum indica;

[0035] Figure 6 This is a thin layer chromatogram obtained by using Xinsrui polyamide film for thin layer identification in the investigation of thin layer plates from different manufacturers; the red frame shows the characteristic spots of Hedyotis diffusa, the blue frame shows the characteristic spots of Andrographis paniculata, and the yellow frame shows the characteristic spots of Sesamum indica;

[0036] Figure 7 The thin layer chromatogram at room temperature (23°C) during the investigation at different temperatures; the red frame shows the characteristic spots of Hedyotis diffusa, the blue frame shows the characteristic spots of Andrographis paniculata, and the yellow frame shows the characteristic spots of Sesamum indica;

[0037] Figure 8 The thin layer chromatogram at low temperature (8℃) in the investigation of different temperatures; the red frame shows the characteristic spots of Hedyotis diffusa, the blue frame shows the characteristic spots of Andrographis paniculata, and the yellow frame shows the characteristic spots of Sesamum indica;

[0038] Fig. 9This is a thin layer chromatogram at low humidity (32%) during the investigation of different humidity levels; the red frame shows the characteristic spots of Hedyotis diffusa, the blue frame shows the characteristic spots of Andrographis paniculata, and the yellow frame shows the characteristic spots of Sesamum indica;

[0039] Fig.10 This is a thin layer chromatogram at high humidity (88%) during the investigation of different humidity levels; the red frame shows the characteristic spots of Hedyotis diffusa, the blue frame shows the characteristic spots of Andrographis paniculata, and the yellow frame shows the characteristic spots of Sesamum indica;

[0040] Fig.11 This is the thin layer chromatogram when anhydrous ethanol-acetic acid-water (5:1:1) was used as the developing solvent in the effect verification of Experiment 3;

[0041] Fig.12 This is the thin layer chromatogram when anhydrous ethanol-acetic acid-water (5:1:10) was used as the developing solvent in the effect verification of Experiment 3;

[0042] Fig.13 This is the thin layer chromatogram when anhydrous ethanol-acetic acid-water (2:1:5) was used as the developing solvent in the effect verification of Experiment 3;

[0043] Fig.14 This is the thin layer chromatogram when anhydrous ethanol-acetic acid-water (8:1:5) was used as the developing solvent in Experiment 3 to verify the effect. DETAILED DESCRIPTION

[0044] The above contents of the present invention are further described in detail below by means of specific implementation methods in the form of examples, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples.

[0045] 1. Instruments, test drugs, materials and reagents

[0046] 1.1 Instrument:

[0047] Thin layer plate heater (model TCLPLATE HEATERⅢ, Kama Company, Switzerland); thin layer chromatography digital imaging system (model YOKO-ZS, Wuhan Pharmaceutical Technology Development Co., Ltd.); ultrasonic cleaner (model KQ-400DE, Kunshan Ultrasonic Instrument Co., Ltd.); electronic balance (model BS224S, Sartorius Scientific Instrument (Beijing) Co., Ltd.); digital display constant temperature water bath (model HHS-11-4, Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory).

[0048] 1.2 Drug testing

[0049] Test sample: Hou Shuning tablets (produced by Sinopharm Dezhong (Foshan) Pharmaceutical Co., Ltd., batch number: 22001, 23001, 24001);

[0050] Negative control lacking Hedyotis diffusa: Andrographis paniculata and Sesame were prepared according to the prescription and preparation method of Hou Shu Ning Tablets;

[0051] Negative control lacking Andrographis paniculata: Prepare Hou Shu Ning Tablets according to the prescription and preparation method by taking Hedyotis diffusa and Sesame;

[0052] Negative control of Sesame: Prepare Hedyotis diffusa and Andrographis paniculata according to the prescription and preparation method of Hou Shu Ning Tablets;

[0053] The reference medicinal material of Hedyotis diffusa (batch number: 121183-201605); the reference medicinal material of Andrographis paniculata (batch number: 121082-201706); and the reference medicinal material of Sesamum indica (batch number: 121088-202104) were all purchased from China Food and Drug Inspection Institutes.

[0054] 1.3 Materials and reagents

[0055] Polyamide film (produced by Sijia Biochemical Plastic Factory, Luqiao, Taizhou City, Zhejiang Province, hereinafter referred to as Sijia Biochemical); polyamide film (produced by Wuhan Xinsrui Technology Co., Ltd., hereinafter referred to as Xinsrui); polyamide film (produced by Qingdao Bangkai High-tech Materials Co., Ltd., hereinafter referred to as Bangkai); anhydrous ethanol (analytical grade, Guangzhou Chemical Reagent Factory), acetic acid (analytical grade, Guangzhou Chemical Reagent Factory), aluminum chloride (analytical grade, Taishan Yueqiao Reagent Plastic Co., Ltd.), and water are purified water.

[0056] Example 1 Thin layer chromatography identification method of the whole prescription of Hou Shu Ning tablets of the present invention

[0057] The 22001, 23001 and 24001 batches of Hou Shu Ning Tablets (specification: 0.4 g per tablet) of Sinopharm Group Dezhong (Foshan) Pharmaceutical Co., Ltd. were used for thin layer chromatography identification of the medicinal taste of the whole prescription, and negative controls lacking Hedyotis diffusa, lacking Andrographis paniculata and lacking Sesame were used as control groups for comparison, including the following steps:

[0058] S1. Preparation of test solution:

[0059] Take 1 Hou Shu Ning tablet, remove the coating, grind it into powder, add 20 mL of 70% ethanol solution, extract it by ultrasonic for 20 min, filter it, evaporate the filtrate to dryness, add 2 mL of 70% ethanol solution to dissolve the residue, and use it as the test solution;

[0060] S2. Preparation of control medicinal material solution:

[0061] Take 0.2g of Hedyotis diffusa, Andrographis paniculata and Sesamum indica respectively, add 10mL of water respectively, heat and reflux for 1h to extract, cool, filter, evaporate the filtrate to dryness, add 2mL of 70% ethanol solution to dissolve the residue respectively, as the control medicinal material solution;

[0062] S3. Thin layer chromatography identification:

[0063] 1 μl of the test solution and each control drug solution was taken and spotted on the same polyamide film, and developed with anhydrous ethanol-acetic acid-water with a volume ratio of 5:1:5. The film was taken out, dried, sprayed with 5% aluminum chloride-ethanol solution, heated at 105°C for 3 minutes, and taken out for inspection;

[0064] Result determination: When examined under ultraviolet light (365nm), in the chromatogram of the test sample, the same yellow-green fluorescent spots appeared at the corresponding positions of the chromatograms of the reference medicinal materials Hedyotis diffusa and Andrographis paniculata, indicating that Hedyotis diffusa and Andrographis paniculata can be detected; the same blue fluorescent spots appeared at the corresponding positions of the chromatograms of the reference medicinal materials Sesamum indicum, indicating that Sesamum indicum can be detected.

[0065] The experimental results are as follows Figure 1 As shown, in the chromatograms of the three batches of test samples, the same yellow-green fluorescent spots are shown at the positions corresponding to the chromatograms of the control medicinal materials of Hedyotis diffusa and Andrographis paniculata, indicating that Hedyotis diffusa and Andrographis paniculata can be detected; the same blue fluorescent spots are shown at the positions corresponding to the chromatograms of the control medicinal materials of Sesame paniculata, indicating that Sesame paniculata can be detected. The spots are clear and easy to identify, and each negative control has no interference, which can be used as a preferred embodiment of the present invention.

[0066] Example 2 Thin layer chromatography identification method of the whole prescription of Hou Shu Ning tablets of the present invention

[0067] The 22001, 23001 and 24001 batches of Hou Shu Ning Tablets (specification: 0.4 g per tablet) of Sinopharm Group Dezhong (Foshan) Pharmaceutical Co., Ltd. were used for thin layer chromatography identification of the medicinal taste of the whole prescription, and negative controls lacking Hedyotis diffusa, lacking Andrographis paniculata and lacking Sesame were used as control groups for comparison, including the following steps:

[0068] S1. Preparation of test solution:

[0069] Take 1 Hou Shu Ning tablet, remove the coating, grind it into powder, add 20 mL of methanol solution, extract it by ultrasonic for 20 min, filter it, evaporate the filtrate to dryness, add 2 mL of methanol solution to dissolve the residue, and use it as the test solution;

[0070] S2. Preparation of control medicinal material solution:

[0071] Take 0.2g of Hedyotis diffusa, Andrographis paniculata and Sesamum indica respectively, add 10mL of water respectively, heat and reflux for 1h to extract, cool, filter, evaporate the filtrate to dryness, add 2mL of methanol solution to the residue respectively to dissolve, and use as the control medicinal material solution;

[0072] S3. Thin layer chromatography identification:

[0073] 1 μl of the test solution and each control drug solution was taken and spotted on the same polyamide film, and then developed with methanol-formic acid-water with a volume ratio of 5:1:5. The film was taken out, dried, sprayed with 5% aluminum chloride-ethanol solution, heated at 105°C for 3 minutes, and then taken out for inspection.

[0074] Result determination: When examined under ultraviolet light (365nm), in the chromatogram of the test sample, the same yellow-green fluorescent spots appeared at the corresponding positions of the chromatograms of the reference medicinal materials Hedyotis diffusa and Andrographis paniculata, indicating that Hedyotis diffusa and Andrographis paniculata can be detected; the same blue fluorescent spots appeared at the corresponding positions of the chromatograms of the reference medicinal materials Sesamum indicum, indicating that Sesamum indicum can be detected.

[0075] The experimental results are as follows Figure 2 As shown, in the chromatograms of the three batches of test samples, the same yellow-green fluorescent spots appeared at the corresponding positions of the chromatograms of the reference medicinal materials Hedyotis diffusa and Andrographis paniculata, indicating that Hedyotis diffusa and Andrographis paniculata can be detected; the same blue fluorescent spots appeared at the corresponding positions of the chromatograms of the reference medicinal materials Sesame paniculata, indicating that Sesame paniculata can be detected, and there was no interference in the negative controls.

[0076] Example 3 Thin layer chromatography identification method of the medicinal flavors of the whole prescription of Hou Shu Ning tablets of the present invention

[0077] The 22001, 23001 and 24001 batches of Hou Shu Ning Tablets (specification: 0.4 g per tablet) of Sinopharm Group Dezhong (Foshan) Pharmaceutical Co., Ltd. were used for thin layer chromatography identification of the medicinal taste of the whole prescription, and negative controls lacking Hedyotis diffusa, lacking Andrographis paniculata and lacking Sesame were used as control groups for comparison, including the following steps:

[0078] S1. Preparation of test solution:

[0079] Take 1 Hou Shu Ning tablet, remove the coating, grind it into powder, add 20 mL of 70% ethanol solution, extract it by ultrasonic for 20 min, filter it, evaporate the filtrate to dryness, add 2 mL of 70% ethanol solution to dissolve the residue, and use it as the test solution;

[0080] S2. Preparation of control medicinal material solution:

[0081] Take 0.2g of Hedyotis diffusa, Andrographis paniculata and Sesamum indica respectively, add 10mL of water respectively, heat and reflux for 1h to extract, cool, filter, evaporate the filtrate to dryness, add 2mL of 70% ethanol solution to dissolve the residue respectively, as the control medicinal material solution;

[0082] S3. Thin layer chromatography identification:

[0083] 2 μl of the test solution and each control drug solution were taken and spotted on the same polyamide film, respectively, and developed with anhydrous ethanol-acetic acid-water with a volume ratio of 3:1:5, taken out, dried, sprayed with 5% aluminum chloride-ethanol solution, heated at 110℃ for 5 minutes, and taken out for inspection;

[0084] Result determination: When examined under ultraviolet light (365nm), in the chromatogram of the test sample, the same yellow-green fluorescent spots appeared at the corresponding positions of the chromatograms of the reference medicinal materials Hedyotis diffusa and Andrographis paniculata, indicating that Hedyotis diffusa and Andrographis paniculata can be detected; the same blue fluorescent spots appeared at the corresponding positions of the chromatograms of the reference medicinal materials Sesamum indicum, indicating that Sesamum indicum can be detected.

[0085] Example 4 Thin layer chromatography identification method of the whole prescription of Hou Shu Ning tablets of the present invention

[0086] The 22001, 23001 and 24001 batches of Hou Shu Ning Tablets (specification: 0.4 g per tablet) of Sinopharm Group Dezhong (Foshan) Pharmaceutical Co., Ltd. were used for thin layer chromatography identification of the medicinal taste of the whole prescription, and negative controls lacking Hedyotis diffusa, lacking Andrographis paniculata and lacking Sesame were used as control groups for comparison, including the following steps:

[0087] S1. Preparation of test solution:

[0088] Take 1 Hou Shu Ning tablet, remove the coating, grind it into powder, add 20 mL of 70% ethanol solution, extract it by ultrasonic for 25 min, filter it, evaporate the filtrate to dryness, add 2 mL of 70% ethanol solution to dissolve the residue, and use it as the test solution;

[0089] S2. Preparation of control medicinal material solution:

[0090] Take 0.2g of Hedyotis diffusa, Andrographis paniculata and Sesamum indica respectively, add 10mL of water respectively, heat and reflux for 70min to extract, cool, filter, evaporate the filtrate to dryness, add 2mL of 70% ethanol solution to dissolve the residue respectively, as the control medicinal material solution;

[0091] S3. Thin layer chromatography identification:

[0092] 2 μl of the test solution and each control drug solution were taken and spotted on the same polyamide film, respectively, and developed with anhydrous ethanol-acetic acid-water with a volume ratio of 6:1:5, taken out, dried, sprayed with 5% aluminum chloride-ethanol solution, heated at 100°C for 5 minutes, and taken out for inspection;

[0093] Result determination: When examined under ultraviolet light (365nm), in the chromatogram of the test sample, the same yellow-green fluorescent spots appeared at the corresponding positions of the chromatograms of the reference medicinal materials Hedyotis diffusa and Andrographis paniculata, indicating that Hedyotis diffusa and Andrographis paniculata can be detected; the same blue fluorescent spots appeared at the corresponding positions of the chromatograms of the reference medicinal materials Sesamum indicum, indicating that Sesamum indicum can be detected.

[0094] Example 5 Thin layer chromatography identification method of the medicinal flavors of the whole prescription of Hou Shu Ning tablets of the present invention

[0095] The 22001, 23001 and 24001 batches of Hou Shu Ning Tablets (specification: 0.4 g per tablet) of Sinopharm Group Dezhong (Foshan) Pharmaceutical Co., Ltd. were used for thin layer chromatography identification of the medicinal taste of the whole prescription, and negative controls lacking Hedyotis diffusa, lacking Andrographis paniculata and lacking Sesame were used as control groups for comparison, including the following steps:

[0096] S1. Preparation of test solution:

[0097] Take 1 Hou Shu Ning tablet, remove the coating, grind it into powder, add 20 mL of 70% ethanol solution, extract it by ultrasonic for 25 min, filter it, evaporate the filtrate to dryness, add 2 mL of 70% ethanol solution to dissolve the residue, and use it as the test solution;

[0098] S2. Preparation of control medicinal material solution:

[0099] Take 0.2g of Hedyotis diffusa, Andrographis paniculata and Sesamum indica respectively, add 10mL of water respectively, heat and reflux for 70min to extract, cool, filter, evaporate the filtrate to dryness, add 2mL of 70% ethanol solution to dissolve the residue respectively, as the control medicinal material solution;

[0100] S3. Thin layer chromatography identification:

[0101] 2 μl of the test solution and each control drug solution were taken and spotted on the same polyamide film, respectively, and developed with anhydrous ethanol-acetic acid-water with a volume ratio of 5:1:3, taken out, dried, sprayed with 5% aluminum chloride-ethanol solution, heated at 100°C for 5 minutes, and taken out for inspection;

[0102] Result determination: When examined under ultraviolet light (365nm), in the chromatogram of the test sample, the same yellow-green fluorescent spots appeared at the corresponding positions of the chromatograms of the reference medicinal materials Hedyotis diffusa and Andrographis paniculata, indicating that Hedyotis diffusa and Andrographis paniculata can be detected; the same blue fluorescent spots appeared at the corresponding positions of the chromatograms of the reference medicinal materials Sesamum indicum, indicating that Sesamum indicum can be detected.

[0103] Example 6 Thin layer chromatography identification method of the medicinal flavors of the whole prescription of Hou Shu Ning tablets of the present invention

[0104] The 22001, 23001 and 24001 batches of Hou Shu Ning Tablets (specification: 0.4 g per tablet) of Sinopharm Group Dezhong (Foshan) Pharmaceutical Co., Ltd. were used for thin layer chromatography identification of the medicinal taste of the whole prescription, and negative controls lacking Hedyotis diffusa, lacking Andrographis paniculata and lacking Sesame were used as control groups for comparison, including the following steps:

[0105] S1. Preparation of test solution:

[0106] Take 1 Hou Shu Ning tablet, remove the coating, grind it into powder, add 20 mL of 70% ethanol solution, extract it by ultrasonic for 25 min, filter it, evaporate the filtrate to dryness, add 2 mL of 70% ethanol solution to dissolve the residue, and use it as the test solution;

[0107] S2. Preparation of control medicinal material solution:

[0108] Take 0.2g of Hedyotis diffusa, Andrographis paniculata and Sesamum indica respectively, add 10mL of water respectively, heat and reflux for 70min to extract, cool, filter, evaporate the filtrate to dryness, add 2mL of 70% ethanol solution to dissolve the residue respectively, as the control medicinal material solution;

[0109] S3. Thin layer chromatography identification:

[0110] 2 μl of the test solution and each control drug solution were taken and spotted on the same polyamide film, respectively, and developed with anhydrous ethanol-acetic acid-water with a volume ratio of 5:1:7, taken out, dried, sprayed with 5% aluminum chloride-ethanol solution, heated at 100°C for 5 minutes, and taken out for inspection;

[0111] Result determination: When examined under ultraviolet light (365nm), in the chromatogram of the test sample, the same yellow-green fluorescent spots appeared at the corresponding positions of the chromatograms of the reference medicinal materials Hedyotis diffusa and Andrographis paniculata, indicating that Hedyotis diffusa and Andrographis paniculata can be detected; the same blue fluorescent spots appeared at the corresponding positions of the chromatograms of the reference medicinal materials Sesamum indicum, indicating that Sesamum indicum can be detected.

[0112] Experiment 1: Specificity Investigation

[0113] Take 1 tablet of each of three batches of Hou Shuning Tablets (batch numbers 22001, 23001, and 24001) of Sinopharm Dezhong (Foshan) Pharmaceutical Co., Ltd., and take 1 tablet of each of the negative controls lacking Hedyotis diffusa, lacking Andrographis paniculata, and lacking Sesame, to prepare the test solution and the corresponding negative control solution. Take 1 μl of each of the test solution, negative control solution, and each control medicinal material solution, and spot them on the same polyamide film, use anhydrous ethanol-acetic acid-water (5:1:5, v / v / v) as the developing agent, develop, take out, dry, spray with 5% aluminum chloride ethanol solution, heat at 105°C for 3 minutes, take out, and inspect under ultraviolet light (365nm).

[0114] The experimental results are as follows Figure 3 As shown, in the chromatogram of the test sample, the same yellow-green fluorescent spots are shown at the corresponding positions of the chromatograms of the reference medicinal materials of Hedyotis diffusa and Andrographis paniculata, indicating that Hedyotis diffusa and Andrographis paniculata can be detected; the same blue fluorescent spots are shown at the corresponding positions of the chromatograms of the reference medicinal materials of Sesamum indicum, indicating that Sesamum indicum can be detected. The spots are clear and easy to observe, and there is no interference in each negative control solution, indicating that the thin layer chromatography method of the present invention has good specificity and can be used to simultaneously identify all the medicinal flavors in Hou Shu Ning Tablets.

[0115] Experiment 2: Durability test

[0116] (1) Inspection of thin-layer boards from different manufacturers

[0117] Polyamide films produced by different manufacturers (Luqiao Sijia Biochemical Plastic Factory, Taizhou City, Zhejiang Province, Qingdao Bangkai High-tech Materials Co., Ltd. and Wuhan Xinsrui Technology Co., Ltd.) were used to perform thin layer chromatography on the three batches of Hou Shuning Tablets test solutions obtained in Example 1. The experimental results are as follows: Figures 4 to 6 shown. Figure 4 In order to use tetramethyl biochemical polyamide film as thin layer plate, Figure 5 In order to use Bangkai polyamide film as thin layer board, Figure 6 Xinsrui polyamide film was used as the thin layer plate. The results showed that the test solution could obtain ideal thin layer chromatography effect when developed with polyamide films from three manufacturers. In the test sample chromatogram, the same yellow-green fluorescent spots appeared at the corresponding positions of the chromatograms of the control medicinal materials Hedyotis diffusa and Andrographis paniculata; the same blue fluorescent spots appeared at the corresponding positions of the chromatograms of the control medicinal materials Sesame. The spots were clear and easy to observe, and there was no interference with the negative control solutions, indicating that the thin layer identification method described in the present invention is suitable for polyamide films from different manufacturers and has good durability.

[0118] (2) Investigation at different temperatures

[0119] According to the method described in Example 1, the expansion cylinder was placed in a low temperature (8°C) and room temperature (23°C) environment for expansion. The experimental results are as follows: Figures 7-8 shown. Figure 7 This is the thin layer chromatogram at room temperature (23°C). Figure 8 The figure is a thin layer chromatogram at low temperature (8°C). The results show that ideal thin layer chromatography effects can be obtained at both low temperature and room temperature. In the chromatogram of the test sample, the same yellow-green fluorescent spots are shown at the corresponding positions of the chromatograms of Hedyotis diffusa and Andrographis paniculata control medicinal materials; the same blue fluorescent spots are shown at the corresponding positions of the chromatograms of Sesame control medicinal materials. This shows that the thin layer identification method described in the present invention has good durability at different temperatures.

[0120] (3) Investigation at different humidity levels

[0121] According to the method described in Example 1, the expansion cylinder was placed in a low humidity (relative humidity 32%) and high humidity (relative humidity 88%) environment for expansion. The experimental results are as follows: Figures 9-10 shown. Fig. 9 This is the thin layer chromatogram at low humidity (relative humidity 32%). Fig.10 The results show that ideal thin layer chromatography can be obtained under low humidity and high humidity conditions. In the chromatogram of the test sample, the same yellow-green fluorescent spots are shown at the corresponding positions of the chromatograms of Hedyotis diffusa and Andrographis paniculata, and the same blue fluorescent spots are shown at the corresponding positions of the chromatograms of Sesame paniculata. This shows that the thin layer identification method described in the present invention has good durability under different humidity conditions.

[0122] It can be seen that the thin layer chromatography identification method for the whole medicinal flavor of Hou Shu Ning Tablets established by the present invention has good durability and strong applicability to the above-mentioned thin layer plates of different brands, development temperature and relative humidity.

[0123] Experiment 3 Effect Verification

[0124] According to the thin layer chromatography identification method of Example 1, under the same other conditions, chromatographic investigations were carried out on anhydrous ethanol-acetic acid-water (5:1:1), anhydrous ethanol-acetic acid-water (5:1:10), anhydrous ethanol-acetic acid-water (2:1:5), and anhydrous ethanol-acetic acid-water (8:1:5) with different volume ratios of developing agents.

[0125] The experimental results are as follows Figures 11 to 14 As shown, among which, Fig.11 As shown, the overall Rf value of the characteristic spots is too large, the characteristic spots of Hedyotis diffusa are not well separated, the characteristic spots of Sesamum indica are not obvious, and the development effect is worse than that of Example 1; Fig.12 The characteristic spots corresponding to Andrographis paniculata and Sesamum indica are not clear and neat, and the unfolding effect is worse than that of Example 1; Fig.13 The characteristic spots of Sesame in Zhongshan overlap with those of Andrographis paniculata, which affects the observation. The characteristic spots of Hedyotis diffusa and Andrographis paniculata partially overlap, which interferes with the negative. Fig.14 The spots of Hedyotis diffusa, Andrographis paniculata and Sesamum in the middle overlapped and were difficult to distinguish, and the development effect was poor; the separation degree of Example 1 of the present invention (i.e., the technical solution of the present invention) was the best, and the chromatogram was the clearest. It can be seen that the developing agent of the thin layer chromatography identification method of the whole prescription of Hou Shu Ning Tablets provided by the present invention is more suitable under the condition of the limited volume ratio of the present invention.

[0126] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A thin layer chromatography identification method for the whole prescription of Hou Shu Ning tablets, characterized in that: The steps include: S1. Preparation of test solution: Take Hou Shu Ning tablets, remove the coating, grind them into powder, add ethanol solution, extract by ultrasonic, filter, evaporate the filtrate to dryness, add ethanol solution to dissolve the residue, and use it as the test solution; S2. Preparation of control medicinal material solution: Take Hedyotis diffusa, Andrographis paniculata and Sesame as reference medicinal materials respectively, add water to heat and reflux to extract, cool, filter, evaporate the filtrate to dryness, add ethanol solution to dissolve the residues, and use them as Hedyotis diffusa, Andrographis paniculata and Sesame as reference medicinal material solutions; S3. Thin layer chromatography identification: Pipette the test sample solution and each control medicinal material solution, spot them on the same polyamide film, use anhydrous ethanol-acetic acid-water as the developing agent, develop, take out, dry, spray with aluminum chloride-ethanol solution, heat, take out and inspect; Result determination: When examined under ultraviolet light, in the chromatogram of the test sample, the same yellow-green fluorescent spots appeared at the corresponding positions of the chromatograms of the reference medicinal materials Hedyotis diffusa and Andrographis paniculata, indicating that Hedyotis diffusa and Andrographis paniculata can be detected; the same blue fluorescent spots appeared at the corresponding positions of the chromatograms of the reference medicinal materials Sesamum indicum, indicating that Sesamum indicum can be detected.

2. The thin layer chromatography identification method of the whole formula of Hou Shu Ning Tablets according to claim 1 is characterized in that: The concentration of ethanol in step S1 is 60-80%.

3. The thin layer chromatography identification method of the whole formula of Hou Shu Ning Tablets according to claim 1 is characterized in that: The ultrasonic extraction frequency in step S1 is 20-60kHz, the solid-liquid ratio is 1:(40-60), the extraction temperature is room temperature, and the extraction time is 15-25min; the water-adding heating reflux extraction in step S2 has a solid-liquid ratio of 1:(40-60), a heating temperature of 100-110°C, a reflux temperature of 90-110°C, an extraction time of 50-70min, and a reflux number of 1-2 times; and the ethanol concentration is 60-80%.

4. The thin layer chromatography identification method of the whole formula of Hou Shu Ning Tablets according to claim 1 is characterized in that: The specification of the Hou Shu Ning tablets in step S1 is 0.2-0.6 g per tablet; the residues in step S2 are dissolved in ethanol solution to prepare solutions containing 0.1 g per 1 mL.

5. The thin layer chromatography identification method of the whole formula of Hou Shu Ning Tablets according to claim 1 is characterized in that: The sample spotting volume in the thin layer chromatography identification process in step S3 is 1-2 μl.

6. The thin layer chromatography identification method of the whole formula of Hou Shu Ning Tablets according to claim 1 is characterized in that: In step S3, anhydrous ethanol-acetic acid-water is used as a developing agent, and the volume ratio of anhydrous ethanol, acetic acid and water is (3-6):1:(2-8).

7. The thin layer chromatography identification method of the whole formula of Hou Shu Ning Tablets according to claim 1 is characterized in that: The preparation of the test solution in step S1 can also use methanol as the solvent for ultrasonic extraction and as the final dissolving solvent; the preparation of the control medicinal material solution in S2 can also use methanol as the final dissolving solvent; the developing agent in step S3 also includes methanol-formic acid-water in a volume ratio of (3-6):1:(2-8).

8. The thin layer chromatography identification method of the whole formula of Hou Shu Ning Tablets according to claim 1 is characterized in that: The aluminum chloride-ethanol solution in step S3 is prepared from aluminum chloride and ethanol with a concentration of 80-95%, and the mass percentage of aluminum chloride in the aluminum chloride ethanol solution is 3-10%.

9. The thin layer chromatography identification method of the whole formula of Hou Shu Ning Tablets according to claim 1, characterized in that: The heating in step S3 includes heating at 100-110° C. for 2-5 min.

10. The thin layer chromatography identification method of the whole formula of Hou Shu Ning Tablets according to claim 1, characterized in that: In step S3, the sample is inspected under a 360-370 nm ultraviolet light.

Images