A thin layer chromatography method for identifying the medicinal properties of Hou Shuning tablets

Abstract

The present invention belongs to the technical field of traditional Chinese medicine detection, and in particular to a thin layer chromatography identification method for the whole prescription of Hou Shuning tablets. The thin layer chromatography identification method for the whole prescription of Hou Shuning tablets provided by the present invention comprises the following steps: S1, preparation of Hou Shuning tablet test solution; S2, preparation of control medicinal material solutions of Hedyotis diffusa, Andrographis paniculata and Sesame; S3, using a polyamide thin layer plate, with anhydrous ethanol-acetic acid-water as a developing agent, spraying with aluminum chloride-ethanol solution for inspection, and performing thin layer chromatography identification. The present invention establishes an identification method for the three medicinal flavonoid components of Hou Shuning tablets, promotes the improvement of Hou Shuning tablets quality control, and can be used as a reference for the quality study of flavonoid components of 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.

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 entire Hou Shuning tablet. Background Art

[0002] Hou Shuning Tablets are a pure traditional Chinese medicine compound preparation, made from Hedyotis diffusa, Andrographis paniculata, and Sesame. They clear heat and detoxify, dissipate nodules, and relieve pain. They are indicated for acute and chronic pharyngitis, acute tonsillitis, vocal cord polyps, and vocal cord nodules. They can also be used as an adjunct treatment for various inflammations and cancers caused by radiotherapy for head and neck cancer. Approved Hou Shuning preparations 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, deacetylcarbamate methyl ester, etc.), and Andrographis paniculata is mostly focused on lactone components (such as andrographolide, dehydroandrographolide, etc.). There are relatively few reports on Sesamum indicum, and most of them are focused on triterpenoid components (such as sesamonic acid methyl ester, etc.). Although there are reports on the progress of research on 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 Pharmaceutical Affairs" 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 of Andrographis Paniculata and Its Application in Animal Husbandry" ("Feed Research" 2024, Vol. 47, No. 5) that andrographis paniculata flavonoids have antibacterial, anti-inflammatory, antioxidant and other pharmacological effects. Zhi Guofan et al. mentioned in the journal article "Progress in Research on Pharmacological Activity of Sesamum Indicum" ("Guangdong Chemical Industry" 2023, Vol. 50, No. 20) that the main components of Sesamum Indicum are lignans, flavonoids, saponins, terpenes, alkaloids, etc. These components are the material basis for the medicinal effects of Sesamum Indicum. Multiple groups of research data have shown that the flavonoids in Houttuynia cordata, Andrographis Paniculata and Sesamum Indicum have pharmacological effects such as antibacterial, anti-inflammatory and anti-tumor, and are important effective ingredients 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 identification project in these three medicinal materials is of great significance to the overall quality control of Hou Shuning Tablets.

[0004] Hou Shuning preparations are not included in the Chinese Pharmacopoeia. The current legal standard for Hou Shuning tablets is WS3-B-4013-98, Volume 20 of the Ministry-issued Standard for Traditional Chinese Medicine Formula Preparations. This standard includes microscopic identification of Andrographis paniculata and thin-layer chromatography identification of andrographolide, but lacks identification of Hedyotis diffusa and Sesamum indicum, thus failing to effectively control product quality. The standards for Hou Shuning capsules are included in "New Drug Conversion Standard 86" (Standard Number: YBZ25372005-2009Z) and "New Drug Conversion Standard 87" (Standard Number: YBZ12122005-2009Z). These 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 of Sesamum indicum. The standards remain incomplete and need improvement. Chen Gui et al. established a thin layer chromatography identification project for Houttuynia cordata, Andrographis paniculata and Sesamum in the journal article "Improvement of Quality Standards of Hou Shuning Tablets" (Journal of Pharmaceutical Analysis, Vol. 40, No. 7, 2020). However, 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] Currently, there are no reports on the simultaneous identification of all the medicinal ingredients in Hou Shuning Tablets using the same test solution on the same thin-layer plate. By establishing a method for the simultaneous identification of the characteristic flavonoid components of each medicinal ingredient in Hou Shuning Tablets, the identification of all the medicinal ingredients in the whole formula can be achieved. This can be used as a rapid and simple quality control method. It can also serve as a reference for the quality research of flavonoid components of Hedyotis diffusa, Andrographis paniculata, and Sesamum indica, filling the gap in the thin-layer chromatography identification of flavonoids from Hedyotis diffusa, Andrographis paniculata, and Sesamum indica. Summary of the Invention

[0006] In response to the shortcomings of the prior art, the present invention aims to provide a thin-layer chromatography (TLC) identification method for the full range of medicinal ingredients in Hou Shuning Tablets. This method, focusing on the flavonoids present in the full range of medicinal ingredients in Hou Shuning Tablets, successfully establishes a method for identifying the flavonoids specific to the three medicinal ingredients in the Hou Shuning Tablets formula (Hedychium diffusum, Andrographis paniculata, and Sesamum indicum). By using the same sample solution and performing a single TLC experiment on the same TLC plate, effective separation and identification of these three medicinal ingredients in Hou Shuning Tablets can be achieved. The resulting chromatographic spots are clear, easily identifiable, highly specific, and durable, providing a more scientific and efficient means for quality control of Hou Shuning 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 Shuning tablets, remove the coating, grind them into powder, add ethanol solution, extract by ultrasonic, filter, evaporate the filtrate to dryness, and dissolve the residue in ethanol solution to prepare the test solution;

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

[0012] Taking Hedyotis diffusa, Andrographis paniculata and Sesamum indicum reference medicinal materials respectively, adding water, heating under reflux extraction, cooling, filtering, evaporating the filtrate to dryness, and adding ethanol solution to dissolve the residues respectively as Hedyotis diffusa, Andrographis paniculata and Sesamum indicum reference medicinal material solutions;

[0013] S3. Thin layer chromatography identification:

[0014] Pipette the test sample solution and each control medicinal material solution onto the same polyamide film, respectively, and use anhydrous ethanol-acetic acid-water as the developing agent. Develop, remove, dry, spray with aluminum chloride-ethanol solution, heat, and remove for inspection.

[0015] Result determination: When inspected 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 Houttuynia cordata and Andrographis paniculata, indicating that Houttuynia cordata 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 20-60 kHz, the solid-liquid ratio is 1:(40-60), the extraction temperature is room temperature, and the extraction time is 15-25 min; the water-adding, heating, and reflux extraction 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 50-70 min, and the reflux times are 1-2 times; and the ethanol concentration is 60-80%.

[0018] Preferably, the specification of the Hou Shu Ning tablets in step S1 is 0.2 to 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 volume during the thin layer chromatography identification process in step S3 is 1 to 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 with a volume ratio of (3-6):1:(2-8).

[0022] Preferably, 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%.

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

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

[0025] The principles of the present invention are as follows:

[0026] The present invention is based on the chemical structure and properties of the full-form medicinal ingredients of Hedyotis diffusa, Andrographis paniculata and Sesamum in Houshuning Tablets, takes flavonoid components as the starting point, follows the principle of similar and compatible extraction, selects a specific extraction solvent to ensure the full extraction of the effective ingredients, thereby simply and quickly preparing a test solution and a control medicinal material solution. Subsequently, utilizing the principle of thin-layer chromatography technology, the movement of the developing agent allows each medicinal material component to be effectively separated on the thin-layer plate due to the difference in its adsorption and desorption capacity. In this process, according to the difference in polarity of each component, active ingredients with similar polarity are selected as the basis for identification. Under specific inspection conditions (such as ultraviolet light, visible light or specific chemical reagent color development, etc.), these components will show 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 Sesamum in Houshuning Tablets, a scientific and reliable method is provided 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 for the whole formula of the Hou Shuning 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 the Hou Shuning Tablets - Hedyotis diffusa, Andrographis paniculata and Sesamum indicum medicinal materials - through only one thin-layer chromatography test using the same test sample solution and the same thin-layer plate. The operation is simple and rapid, and the spots are bright in color and easy to observe. The amount of test sample, control medicinal materials and reagents used is small, and the reagents used are safe, non-toxic and environmentally friendly, thereby reducing the number of thin-layer chromatography identification tests, greatly reducing the inspection cost, saving the inspection time, and improving the inspection efficiency. The method is suitable as a quality control method for the Hou Shuning 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 ingredients in the prescription of Hou Shuning Tablets, promotes the improvement of the quality control of Hou Shuning Tablets, and can also serve as a reference for the quality research of the flavonoid components of the single medicinal ingredients of Hedyotis diffusa, Andrographis paniculata and Sesamum indica, filling the gap in the thin-layer chromatography identification of flavonoids of Hedyotis diffusa, Andrographis paniculata and Sesamum indica. 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 indicum;

[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 indicum;

[0032] Figure 3 This is a thin-layer chromatogram for 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 indicum.

[0033] Figure 4 This is a thin layer chromatogram obtained by thin layer identification using tetramethyl biochemical polyamide film during 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 indicum.

[0034] Figure 5 This is a thin layer chromatogram obtained by thin layer identification using Bangkai polyamide film during 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 indicum.

[0035] Figure 6 This is a thin layer chromatogram obtained by using Xinsrui polyamide film for thin layer identification during 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 indicum.

[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 indicum.

[0037] Figure 8 The following is a thin layer chromatogram at low temperature (8°C) during 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 indicum.

[0038] Figure 9The following 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 indicum.

[0039] Figure 10 The figure 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 indicum.

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

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

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

[0043] Figure 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. DETAILED DESCRIPTION

[0044] The following is a further detailed description of the present invention through specific embodiments in the form of examples, but it should not be understood that the scope of the present invention is limited to the following examples.

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

[0046] 1.1 Instruments:

[0047] Thin layer plate heater (model TCLPLATE HEATER III, Kama, 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 numbers: 22001, 23001, 24001);

[0050] Negative control without Hedyotis diffusa: Andrographis paniculata and Sesamum indicum were prepared according to the prescription and preparation method of Hou Shuning Tablets;

[0051] Negative control lacking Andrographis paniculata: Prepare Hou Shuning Tablets according to the prescription and preparation method by taking Hedyotis diffusa and Sesamum indicum.

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

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

[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 purified water.

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

[0057] Thin layer chromatography identification of the medicinal properties of the whole prescription was performed using batches 22001, 23001, and 24001 of Sinopharm Group Dezhong (Foshan) Pharmaceutical Co., Ltd. Houshuning tablets (specification: 0.4 g per tablet), and negative controls lacking Hedyotis diffusa, lacking Andrographis paniculata, and lacking Sesamum indicum were used as control groups for comparison, including the following steps:

[0058] S1. Preparation of test solution:

[0059] Take one Hou Shuning 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, and dissolve the residue in 2 mL of 70% ethanol solution to prepare the test solution;

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

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

[0062] S3. Thin layer chromatography identification:

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

[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 Houttuynia cordata and Andrographis paniculata, indicating that Houttuynia cordata 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, the chromatograms of the three test sample batches all showed the same yellow-green fluorescent spot at the corresponding position in the chromatograms of the control medicinal materials Hedyotis diffusa and Andrographis paniculata, indicating that Hedyotis diffusa and Andrographis paniculata were detectable. The same blue fluorescent spot appeared at the corresponding position in the chromatogram of the control medicinal material Sesamum indicum, indicating that Sesamum indicum was detectable. The spots were clear and easy to identify, and each negative control showed no interference, making it a preferred embodiment of the present invention.

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

[0067] Thin layer chromatography identification of the medicinal properties of the whole prescription was performed using batches 22001, 23001, and 24001 of Sinopharm Group Dezhong (Foshan) Pharmaceutical Co., Ltd. Houshuning tablets (specification: 0.4 g per tablet), and negative controls lacking Hedyotis diffusa, lacking Andrographis paniculata, and lacking Sesamum indicum were used as control groups for comparison, including the following steps:

[0068] S1. Preparation of test solution:

[0069] Take one Hou Shuning 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, and dissolve the residue in 2 mL of methanol solution to prepare the test solution.

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

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

[0072] S3. Thin layer chromatography identification:

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

[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 Houttuynia cordata and Andrographis paniculata, indicating that Houttuynia cordata 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 Houttuynia cordata and Andrographis paniculata, indicating that Houttuynia cordata 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, and there was no interference in the negative controls.

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

[0077] Thin layer chromatography identification of the medicinal properties of the whole prescription was performed using batches 22001, 23001, and 24001 of Sinopharm Group Dezhong (Foshan) Pharmaceutical Co., Ltd. Houshuning tablets (specification: 0.4 g per tablet), and negative controls lacking Hedyotis diffusa, lacking Andrographis paniculata, and lacking Sesamum indicum were used as control groups for comparison, including the following steps:

[0078] S1. Preparation of test solution:

[0079] Take one Hou Shuning 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, and dissolve the residue in 2 mL of 70% ethanol solution to prepare the test solution;

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

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

[0082] S3. Thin layer chromatography identification:

[0083] 2 μl of the test solution and each control herbal solution were taken and spotted on the same polyamide film. The film was developed with anhydrous ethanol-acetic acid-water in a volume ratio of 3:1:5. The film was taken out and dried. The film was sprayed with 5% aluminum chloride-ethanol solution and heated at 110°C for 5 minutes. The film was taken out and examined.

[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 Houttuynia cordata and Andrographis paniculata, indicating that Houttuynia cordata 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 for the medicinal flavors of the whole prescription of Hou Shu Ning tablets of the present invention

[0086] Thin layer chromatography identification of the medicinal properties of the whole prescription was performed using batches 22001, 23001, and 24001 of Sinopharm Group Dezhong (Foshan) Pharmaceutical Co., Ltd. Houshuning tablets (specification: 0.4 g per tablet), and negative controls lacking Hedyotis diffusa, lacking Andrographis paniculata, and lacking Sesamum indicum were used as control groups for comparison, including the following steps:

[0087] S1. Preparation of test solution:

[0088] Take one Hou Shuning tablet, remove the coating, grind it into powder, add 20 mL of 70% ethanol solution, extract it by ultrasonic for 25 minutes, filter it, evaporate the filtrate to dryness, and dissolve the residue in 2 mL of 70% ethanol solution to prepare the test solution.

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

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

[0091] S3. Thin layer chromatography identification:

[0092] 2 μl of the test solution and each control herbal solution were respectively spotted on the same polyamide film. Developed with anhydrous ethanol-acetic acid-water in a volume ratio of 6:1:5. The film was removed and air-dried. Sprayed with 5% aluminum chloride-ethanol solution, heated at 100°C for 5 minutes, and removed 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 Houttuynia cordata and Andrographis paniculata, indicating that Houttuynia cordata 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 for the medicinal flavors of the whole prescription of Hou Shu Ning tablets of the present invention

[0095] Thin layer chromatography identification of the medicinal properties of the whole prescription was performed using batches 22001, 23001, and 24001 of Sinopharm Group Dezhong (Foshan) Pharmaceutical Co., Ltd. Houshuning tablets (specification: 0.4 g per tablet), and negative controls lacking Hedyotis diffusa, lacking Andrographis paniculata, and lacking Sesamum indicum were used as control groups for comparison, including the following steps:

[0096] S1. Preparation of test solution:

[0097] Take one Hou Shuning tablet, remove the coating, grind it into powder, add 20 mL of 70% ethanol solution, extract it by ultrasonic for 25 minutes, filter it, evaporate the filtrate to dryness, and dissolve the residue in 2 mL of 70% ethanol solution to prepare the test solution.

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

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

[0100] S3. Thin layer chromatography identification:

[0101] 2 μl of the test solution and each control herbal solution were respectively spotted on the same polyamide film. Developed with anhydrous ethanol-acetic acid-water in a volume ratio of 5:1:3. The film was removed and air-dried. Sprayed with 5% aluminum chloride-ethanol solution, heated at 100°C for 5 minutes, and removed 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 Houttuynia cordata and Andrographis paniculata, indicating that Houttuynia cordata 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 for the medicinal flavors of the whole prescription of Hou Shu Ning tablets of the present invention

[0104] Thin layer chromatography identification of the medicinal properties of the whole prescription was performed using batches 22001, 23001, and 24001 of Sinopharm Group Dezhong (Foshan) Pharmaceutical Co., Ltd. Houshuning tablets (specification: 0.4 g per tablet), and negative controls lacking Hedyotis diffusa, lacking Andrographis paniculata, and lacking Sesamum indicum were used as control groups for comparison, including the following steps:

[0105] S1. Preparation of test solution:

[0106] Take one Hou Shuning tablet, remove the coating, grind it into powder, add 20 mL of 70% ethanol solution, extract it by ultrasonic for 25 minutes, filter it, evaporate the filtrate to dryness, and dissolve the residue in 2 mL of 70% ethanol solution to prepare the test solution.

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

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

[0109] S3. Thin layer chromatography identification:

[0110] 2 μl of the test solution and each control herbal solution were respectively spotted on the same polyamide film. Developed with anhydrous ethanol-acetic acid-water in a volume ratio of 5:1:7. The film was removed and air-dried. Sprayed with 5% aluminum chloride-ethanol solution, heated at 100°C for 5 minutes, and removed 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 Houttuynia cordata and Andrographis paniculata, indicating that Houttuynia cordata 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] Prepare test solution and corresponding negative control solutions using one tablet each of three batches of Hou Shuning Tablets (lot numbers 22001, 23001, and 24001) from Sinopharm Dezhong (Foshan) Pharmaceutical Co., Ltd., and one tablet each of the negative controls lacking Hedyotis diffusa, Andrographis paniculata, and Sesamum indicum. Pipette 1 μl of each of the test solution, negative control solution, and each control herb solution onto the same polyamide film. Develop with anhydrous ethanol-acetic acid-water (5:1:5, v / v / v), remove, air dry, spray with 5% aluminum chloride ethanol solution, heat at 105°C for 3 minutes, remove, and examine under ultraviolet light (365 nm).

[0114] The experimental results are as follows Figure 3 As shown, the chromatogram of the test sample shows the same yellow-green fluorescent spot at the corresponding position in the chromatogram of the control medicinal materials Hedyotis diffusa and Andrographis paniculata, indicating that Hedyotis diffusa and Andrographis paniculata can be detected. The same blue fluorescent spot appears at the corresponding position in the chromatogram of the control medicinal material Sesamum indicum, indicating that Sesamum indicum can be detected. The spots are clear and easy to observe, and there is no interference from the negative control solutions, 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 Shuning Tablets.

[0115] Experiment 2: Durability test

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

[0117] Three batches of Hou Shuning tablets test solution obtained in Example 1 were subjected to thin layer chromatography using 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.). The experimental results are as follows: Figures 4-6 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 chromatography plate. Results showed that ideal thin layer chromatography (TLC) results were achieved when the test sample solution was developed using polyamide films from all three manufacturers. The test sample chromatogram showed the same yellow-green fluorescent spots at positions corresponding to those in the chromatograms of the control medicinal materials Hedyotis diffusa and Andrographis paniculata; and the same blue fluorescent spots at positions corresponding to those in the chromatogram of the control medicinal material Sesamum indicum. The spots were clear and easy to observe, and none of the negative control solutions showed any interference, demonstrating that the TLC identification method described herein is applicable to polyamide films from different manufacturers and exhibits 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 is the thin layer chromatogram at room temperature (23°C), Figure 8 The following is a TLC image at low temperature (8°C). The results show that ideal TLC results can be achieved at both low and room temperature. In the chromatogram of the test sample, the same yellow-green fluorescent spot appears at the corresponding position in the chromatogram of the control medicinal materials Hedyotis diffusa and Andrographis paniculata; the same blue fluorescent spot appears at the corresponding position in the chromatogram of the control medicinal material Sesamum indicum. This demonstrates that the TLC identification method described in the present invention is highly durable 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. Figure 9 This is the thin layer chromatogram at low humidity (relative humidity 32%). Figure 10 The following is a TLC chromatogram obtained under high humidity (88% relative humidity). The results show that ideal TLC results can be achieved under both low and high humidity conditions. In the chromatogram of the test sample, the same yellow-green fluorescent spots appear at positions corresponding to those in the chromatograms of the control medicinal materials Hedyotis diffusa and Andrographis paniculata; the same blue fluorescent spots appear at positions corresponding to those in the chromatogram of the control medicinal material Sesamum indicum. This demonstrates the robustness of the TLC identification method described herein under varying humidity conditions.

[0122] It can be seen that the thin layer chromatography identification method for the whole medicinal flavor of Hou Shuning Tablets established in the present invention has good durability and strong applicability for the above-mentioned different brands of thin layer plates, development temperatures 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 11-14 As shown, among them, Figure 11 As shown, the overall Rf value of the characteristic spots is too large, the characteristic spots of Hedyotis diffusa are poorly separated, the characteristic spots of Sesamum indicum are not obvious, and the expansion effect is worse than that of Example 1; Figure 12 The characteristic spots corresponding to Andrographis paniculata and Sesamum indicum were not clear and neat, and the unfolding effect was worse than that of Example 1; Figure 13 The characteristic spots of Sesame in Zhongshan Mountain overlap with those of Andrographis paniculata, which affects the observation. The characteristic spots of Hedyotis diffusa in Baihua and Andrographis paniculata partially overlap, which interferes with the negative results. Figure 14 The spots of Hedyotis diffusa, Andrographis paniculata, and Sesamum indicum overlapped, making them difficult to distinguish, and the development effect was poor; Example 1 of the present invention had the best separation (i.e., the technical solution of the present invention) and the clearest chromatogram. Thus, the thin-layer chromatography method for identifying the medicinal flavors of the whole formula in Hou Shuning Tablets provided by the present invention is more suitable for the developing agent under the conditions 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 skilled in 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 one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be covered by the claims of the present invention.

Claims

1. A thin layer chromatography identification method for the whole medicinal flavor of Hou Shuning Tablets, characterized in that: The steps include: S1. Preparation of test solution: Take Hou Shuning tablets, remove the coating, grind them into powder, add ethanol solution, extract by ultrasonic, filter, evaporate the filtrate to dryness, and dissolve the residue in ethanol solution to prepare the test solution; S2. Preparation of control medicinal material solution: Taking Hedyotis diffusa, Andrographis paniculata and Sesamum indicum reference medicinal materials respectively, adding water, heating under reflux extraction, cooling, filtering, evaporating the filtrate to dryness, and adding ethanol solution to dissolve the residues respectively as Hedyotis diffusa, Andrographis paniculata and Sesamum indicum reference medicinal material solutions; S3. Thin layer chromatography identification: Pipette the test sample solution and each control medicinal material solution onto the same polyamide film, respectively, and use anhydrous ethanol-acetic acid-water as the developing agent. Develop, remove, dry, spray with aluminum chloride-ethanol solution, heat, and remove for inspection. Result determination: Check under ultraviolet light. In the chromatogram of the test sample, the same yellow-green fluorescent spots appear at the corresponding positions of the chromatograms of Hedyotis diffusa and Andrographis paniculata, indicating that Hedyotis diffusa and Andrographis paniculata can be detected; the same blue fluorescent spots appear at the corresponding positions of the chromatograms of the control herbal medicine Sesamum indicum, indicating that Sesamum indicum can be detected. 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).

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

4. The thin layer chromatography identification method for the whole prescription 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 prescription of Hou Shu Ning tablets according to claim 1 is characterized in that: The sample volume during the thin layer chromatography identification process in step S3 is 1-2 μl.

6. The thin layer chromatography identification method for the whole formula of Hou Shuning 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 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 with a volume ratio of (3~6):1:(2~8).

7. The thin layer chromatography identification method for the whole formula of Hou Shuning 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%.

8. The thin layer chromatography identification method for the whole formula of Hou Shuning Tablets according to claim 1 is characterized in that: The heating in step S3 includes heating at 100-110° C. for 2-5 minutes.

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

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