A method for identifying and controlling quality of baixin granules based on thin layer chromatography

Abstract

The application discloses a heart-protecting granule identification and quality control method based on a thin layer chromatography, and belongs to the technical field of drug detection. The thin layer chromatography identification method comprises the following steps: taking astragaloside as a control product to identify astragalus membranaceus, taking chrysophanol and emodin as control products to identify largeleaf rhubarb, taking hirudo medicinal materials as a control product to identify hirudo, taking glycyrrhizin and licorice medicinal materials as control products to identify licorice, taking catalpol as a control product to identify radix rehmanniae, taking salvianolic acid B and salvia miltiorrhiza medicinal materials as control products to identify salvia miltiorrhiza, and taking naringin and neohesperidin as control products to identify aurantii fructus. The application establishes the thin layer chromatography identification method of the heart-protecting granule, adopts a special developing agent and a color developing mode, realizes accurate identification according to the spot color, fluorescence characteristics and other characteristics of the corresponding positions of the test product chromatogram, the control product and the control medicinal material chromatogram, the method is simple in operation, low in detection cost, strong in repeatability and specificity, and can effectively control the quality of the heart-protecting granule production process.

Description

Technical Field

[0001] This invention relates to the field of drug detection technology, and in particular to a method for the identification and quality control of Baoxin granules based on thin-layer chromatography. It can be used for the rapid and accurate identification of multiple key medicinal materials in Baoxin granules, providing a reliable means to ensure drug quality and medication safety. Background Technology

[0002] Traditional methods for identifying Chinese medicinal herbs, such as morphological identification and microscopic identification, are greatly affected by subjective factors and have limited accuracy. While modern analytical techniques such as high-performance liquid chromatography (HPLC) and mass spectrometry (MS / MS) offer high accuracy, they suffer from high equipment costs, complex operation, and high testing costs, making them difficult to widely apply in routine quality control. Thin-layer chromatography (TLC) identification technology, with its advantages of simple operation, low cost, and intuitive results, has become one of the commonly used methods for quality control of Chinese medicinal herbs. However, current TLC identification methods for several key medicinal materials in Baoxin granules are not yet perfect, making it impossible to comprehensively and effectively control their quality.

[0003] Therefore, it is urgent to establish a systematic and reliable thin-layer chromatography identification method to achieve accurate identification of key medicinal materials in Baoxin Granules and ensure the stability of drug quality. Summary of the Invention

[0004] The purpose of this invention is to provide a method for the identification and quality control of core-protecting granules based on thin-layer chromatography, so as to solve the problems existing in the prior art.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a thin-layer chromatography (TLC) identification method for Baoxin Granules, wherein the components of Baoxin Granules include Astragalus membranaceus, Salvia miltiorrhiza, Ophiopogon japonicus, Rehmannia glutinosa, Citrus aurantium, Rheum palmatum (processed with wine), Hirudo medicinalis (processed), Angelica sinensis, Poria cocos, Platycodon grandiflorus, Glycyrrhiza uralensis (processed), and Lycopus lucidus; the TLC identification method includes the steps of identifying Astragalus membranaceus using astragaloside A as a reference standard, identifying Rheum palmatum (processed with wine) using rhein and emodin as reference standards, identifying Hirudo medicinalis (processed) using Hirudo medicinalis as a reference standard, identifying Glycyrrhiza uralensis using glycyrrhizin and Glycyrrhiza uralensis as reference standards, identifying Rehmannia glutinosa using catalpol as a reference standard, identifying Salvia miltiorrhiza using salvianolic acid B and Salvia miltiorrhiza as reference standards, and identifying Citrus aurantium using naringin and neohesperidin as reference standards.

[0007] Furthermore, the steps for identifying Astragalus membranaceus are as follows:

[0008] (1) Preparation of test solution: Take 2g of Baoxin granules, add 10mL of ammonia test solution, stir until completely wet, add 30mL of water and mix well, add 50mL of water-saturated n-butanol, heat under reflux for 2 hours, let stand for layering, take 25mL of the upper n-butanol solution, evaporate to dryness in a water bath, dissolve the residue in 5mL of methanol to obtain the test solution.

[0009] (2) Preparation of reference solution: Astragaloside A was dissolved in methanol to obtain a reference solution with a concentration of 1 mg / mL;

[0010] (3) Thin-layer chromatography: Take 5 μL of the test solution and 1 μL of the reference solution and spot them on the same silica gel G thin-layer plate. Use ethyl acetate-acetone-water with a volume ratio of 4:5:1 as the developing solvent. Develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly visible, and examine under ultraviolet light.

[0011] Furthermore, the steps for identifying rhubarb in wine are as follows:

[0012] (1) Preparation of test solution: Take 7g of core-protecting granules, add 50mL of methanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 50mL of 8% hydrochloric acid solution, add 50mL of chloroform, heat under reflux for 2.5 hours, and collect the chloroform layer; wash the aqueous layer twice with chloroform, combine the chloroform washing solution and the chloroform layer, recover the solvent to dryness, dissolve the residue in 3mL of methanol to obtain the test solution;

[0013] (2) Preparation of reference solution: Rhein and emodin were dissolved in methanol to obtain a reference solution containing 0.1 mg / mL rhein and 0.05 mg / mL emodin;

[0014] (3) Thin-layer chromatography: Take 4 μL of the test solution and 2 μL of the reference solution and spot them on the same silica gel G thin-layer plate. Use the upper layer of petroleum ether-ethyl formate-formic acid with a volume ratio of 15:5:1 as the developing solvent. Develop, remove, air dry, and examine under ultraviolet light.

[0015] Furthermore, the steps for identifying scalded leeches are as follows:

[0016] (1) Preparation of test solution: Take 6g of Baoxin granules, add 25mL of ethanol, sonicate for 15 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 0.5mL of ethanol to obtain the test solution;

[0017] (2) Preparation of reference solution: Take 0.25g of leech reference material, add 25mL of ethanol, sonicate for 15 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 0.5mL of ethanol to obtain the reference solution;

[0018] (3) Thin-layer chromatography: Take 10 μL of the test solution and 1 μL of the reference medicinal material solution and spot them on the same silica gel G thin-layer plate. Use cyclohexane-ethyl acetate with a volume ratio of 4:1 as the developing solvent, develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly visible, and examine under sunlight.

[0019] Furthermore, the steps for identifying roasted licorice are as follows:

[0020] (1) Preparation of test solution: Take 2.5g of core-protecting granules, add 10mL of methanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 5mL of methanol to obtain the test solution;

[0021] (2) Preparation of reference herb solution and reference solution: Take 1g of licorice reference herb, add 50mL of water, decoct for 30 minutes, filter, and evaporate the filtrate to dryness; add 10mL of methanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 5mL of methanol to obtain reference herb solution; dissolve glycyrrhizin in methanol to obtain reference solution with a concentration of 0.5mg / mL;

[0022] (3) Thin-layer chromatography: Take 3 μL of the test solution, 1 μL of the reference medicinal material solution, and 1 μL of the reference solution and spot them on the same silica gel G thin-layer plate. Use ethyl acetate-formic acid-glacial acetic acid-water with a volume ratio of 15:1:1:2 as the developing solvent, develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly visible, and examine under ultraviolet light.

[0023] Furthermore, the steps for identifying raw Rehmannia glutinosa are as follows:

[0024] (1) Preparation of test solution: Take 8g of core-protecting granules, add 50mL of methanol, sonicate for 45 minutes, filter, accurately measure 25mL of filtrate, recover the solvent under reduced pressure to dryness, dissolve in water, elute with water and methanol in turn, pass through macroporous resin, collect methanol eluent, concentrate to dryness, dissolve in 3mL of methanol to obtain test solution;

[0025] (2) Preparation of reference solution: Dissolve catalpol in methanol to obtain a reference solution with a concentration of 0.5 mg / mL;

[0026] (3) Thin-layer chromatography: Apply 6 μL of the test solution and 2 μL of the reference solution to the same silica gel GF plate. 254 On a thin-layer plate, a mixture of n-butanol, glacial acetic acid, and water (volume ratio 10:2:1) was used as the developing solvent. The plate was then removed, dried, sprayed with a 10% sulfuric acid ethanol solution, and heated at 105°C until the color development was clear. The plate was then examined under sunlight.

[0027] Furthermore, the steps for identifying Danshen are as follows:

[0028] (1) Preparation of test solution: Take 2g of Baoxin granules, add 5mL of ethanol, sonicate for 15 minutes, filter, and the filtrate is the test solution;

[0029] (2) Preparation of reference herb solution and reference solution: Take 1g of Salvia miltiorrhiza reference herb, add 30mL of water, decoct for 30 minutes, filter, and evaporate the filtrate to dryness; add 5mL of ethanol, sonicate for 15 minutes, filter, and obtain reference herb solution; dissolve saponin B in 60% methanol solution to obtain a reference solution with a concentration of 0.5mg / mL;

[0030] (3) Thin-layer chromatography: Apply 6 μL each of the test solution and the reference medicinal material solution, and 3 μL of the reference solution to the same silica gel GF plate. 254 On a thin-layer plate, a mixture of dichloromethane-toluene-ethyl acetate-methanol-formic acid with a volume ratio of 3:2:7:0.5:2 was used as the developing solvent. The plate was then removed, dried, and examined under ultraviolet light.

[0031] Furthermore, the steps for identifying Citrus aurantium are as follows:

[0032] (1) Preparation of test solution: Take 2.5g of core-protecting granules, add 10mL of methanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 5mL of methanol to obtain the test solution;

[0033] (2) Preparation of reference solutions: Naringin and neohesperidin were dissolved in methanol to prepare reference solutions containing 1 mg / mL naringin and 1.5 mg / mL neohesperidin;

[0034] (3) Thin-layer chromatography: Take 1 μL of the test solution and the reference solution and spot them on the same silica gel G thin-layer plate. Use a chloroform-methanol-water solution with a volume ratio of 13:6:2 as the developing solvent. Develop, remove, air dry, spray with 3% aluminum trichloride ethanol solution, heat at 105℃ for 5 minutes, and examine under ultraviolet light.

[0035] Furthermore, the ultrasonic power is 1100W and the frequency is 40kHz.

[0036] This invention also provides the application of the above-mentioned thin-layer chromatography identification method in the quality control and quality evaluation of core-protecting granules.

[0037] The present invention discloses the following technical effects:

[0038] This invention establishes a thin-layer chromatography identification method for Baoxin Granules. By selecting multiple targeted reference standards such as astragaloside A and rhein, it can systematically and comprehensively identify key medicinal materials such as astragalus and rhubarb in Baoxin Granules. This method is simple to operate, highly specific, and durable. It does not rely on expensive and complex large-scale instruments and equipment, resulting in low detection costs, making it suitable for application in pharmaceutical manufacturing enterprises. Extensive experimental verification has shown that the identification results of this invention are accurate, reliable, and reproducible, effectively controlling the quality of Baoxin Granules during production. In practical applications, it allows for control of medicinal material quality from the source, ensuring consistency between the drug composition and the prescription, providing a solid guarantee for the safety of Baoxin Granules. It also lays an important foundation for improving and enhancing the quality standards of Baoxin Granules, and has positive significance for promoting the standardization and normalization of quality control in traditional Chinese medicine compound preparations. Attached Figure Description

[0039] Figure 1 TLC chromatogram for the identification of Astragalus membranaceus - investigation of the preparation method of the test sample; in which bands 1-4 are astragaloside A, test sample solution 1, test sample solution 2, and test sample solution 3, respectively;

[0040] Figure 2 TLC chromatograms were reproduced for the thin-layer identification of Astragalus membranaceus and preparation of the test sample. Bands 1-4 represent, in order, astragaloside A, Astragalus membranaceus reference material, test sample, and negative sample lacking Astragalus membranaceus.

[0041] Figure 3 TLC chromatograms for the identification of Astragalus membranaceus using a developing solvent system; bands 1-3 represent, in order, astragaloside A, the test sample, and the negative sample lacking Astragalus membranaceus.

[0042] Figure 4 TLC chromatograms for identification and sample quantity determination of Astragalus membranaceus (A) and specificity determination (B) are shown. In A, bands 1-6 represent 1 μL of reference standard, 2 μL of reference standard, 3 μL of reference standard, 3 μL of test sample, 5 μL of test sample, and 6 μL of test sample, respectively. In B, bands 1-6 represent methanol solvent, astragaloside A reference standard, three replicates of the test sample, and a negative sample lacking Astragalus membranaceus.

[0043] Figure 5 Thin-layer chromatography (TLC) for identification of Astragalus membranaceus: TLC images of different manufacturers' plates and TLC images under different temperatures and humidity conditions; among them, bands 1-5 are astragaloside A, 3 replicates of the test sample, and negative sample without Astragalus membranaceus, respectively.

[0044] Figure 6 TLC images of Astragalus membranaceus in six batches of Baoxin Granules are shown. Among them, band 1 is astragaloside A, and bands 2-7 are Baoxin Granules with batch numbers 210601, 221001, 240401, X231102, X231103, and X231104, respectively.

[0045] Figure 7 TLC chromatogram for the identification of wine-processed rhubarb - investigation of the preparation method of the test sample; among them, bands 1-5 are, in order, emodin, chrysophanol, mixed reference standard (upper emodin, lower emodin), test sample, and negative sample of wine-processed rhubarb.

[0046] Figure 8 TLC diagrams (A) and (B) for the identification of rhubarb in wine-based liquor and the determination of sample volume were prepared. In A, bands 1-8 represent, in order, 1 μL of reference standard, 2 μL of reference standard, 3 μL of reference standard, 4 μL of reference standard, 2 μL of test sample, 3 μL of test sample, 4 μL of test sample, and 5 μL of test sample. In B, bands 1-6 represent, in order, blank solvent, mixed reference standard (upper rhein, lower rhein), 3 replicates of test sample, and negative sample of rhubarb in wine-based liquor.

[0047] Figure 9 Thin-layer chromatography (TLC) for identification of rhubarb in wine – TLC images of different manufacturers and TLC images under different temperatures and humidity; among them, bands 1-5 are, in order, a mixed reference standard (upper rhein, lower emodin), three replicates of the test sample, and a negative sample of rhubarb in wine.

[0048] Figure 10 TLC images of rhubarb in six batches of Baoxin Granules are shown. Among them, band 4 is a mixed reference standard (upper rhein and lower emodin), and bands 1-3 and 5-7 are Baoxin Granules with batch numbers 221001, 210601, 240401, X231102, X231103 and X231104, respectively.

[0049] Figure 11 TLC chromatograms for the identification of scalded leeches and investigation of the preparation method of the test sample; among them, bands 1-5 are, in order, leech control material, 3 replicates of the test sample, and negative sample of leech in water deficiency;

[0050] Figure 12 TLC chromatograms for the identification of scalded leeches by heating time; bands 1-5 represent, in order, the leech control material, three replicates of the test sample, and the negative sample of leeches without water;

[0051] Figure 13 TLC diagrams (A) and (B) were prepared for the identification of scalded leeches by sample volume assessment. In A, bands 1-6 represent, in order, 1 μL of control material, 2 μL of control material, 3 μL of control material, 5 μL of test sample, 10 μL of test sample, and 15 μL of test sample. In B, bands 1-6 represent, in order, blank solvent, leech control material, three replicates of test sample, and negative sample of leech in absence of water.

[0052] Figure 14Thin-layer chromatography (TLC) for the identification of scalded leeches - TLC images of different manufacturers' thin-layer plates and TLC images under different temperatures and humidity conditions; among them, bands 1-5 are, in order, leech control material, 3 replicates of the test sample, and leech negative sample in water deficiency;

[0053] Figure 15 TLC images for identifying leeches in six batches of Baoxin Granules; among them, band 4 is the leech control material, and bands 1-3 and 5-7 are Baoxin Granules with batch numbers 221001, 210601, 240401, X231102, X231103, and X231104, respectively.

[0054] Figure 16 TLC images of processed licorice for identification - Method 1 (A) and Method 2 (B); In A, bands 1-5 are, in order, glycyrrhetinic acid, glycyrrhizic acid, glycyrrhizin, test sample, and negative sample lacking processed licorice; In B, bands 1-4 are, in order, glycyrrhizin, licorice reference material, test sample, and negative sample lacking processed licorice.

[0055] Figure 17 TLC chromatograms for identification and systematic investigation of processed licorice root using developing solvent; bands 1-4 represent, in order, glycyrrhizin, licorice reference material, test sample, and negative sample lacking processed licorice root.

[0056] Figure 18 TLC chromatograms for identification of prepared licorice root by sample quantity investigation (A) and specificity investigation (B) are shown. In A, bands 1-12 represent, in order, 1 μL of control material, 2 μL of control material, 3 μL of control material, 4 μL of control material, 1 μL of glycyrrhizin, 2 μL of glycyrrhizin, 3 μL of glycyrrhizin, 4 μL of glycyrrhizin, 1 μL of test sample, 3 μL of test sample, 5 μL of test sample, and 6 μL of test sample. In B, bands 1-7 represent, in order, blank solvent, glycyrrhizin, licorice control material, three replicates of the test sample, and a negative sample lacking prepared licorice root.

[0057] Figure 19 Thin-layer chromatography (TLC) images of different manufacturers and different temperature and humidity conditions were used for the identification of processed licorice. Among them, bands 1-6 represent glycyrrhizin, licorice reference material, three replicates of the test sample, and negative sample without processed licorice, respectively.

[0058] Figure 20 TLC images of processed licorice in six batches of Baoxin Granules are shown. Among them, band 4 is glycyrrhizin, band 5 is licorice reference material, and bands 1-3 and 6-8 are Baoxin Granules with batch numbers 221001, 210601, 240401, X231102, X231103, and X231104, respectively.

[0059] Figure 21TLC images for thin-layer chromatography identification of Rehmannia glutinosa - preparation method of test sample (successful) (A) and preparation method of test sample (failure) (B); In A, bands 1-5 are, in order, catalpol, verbascoside, Rehmannia glutinosa slices, test sample, and negative sample without Rehmannia glutinosa; In B, bands 1-4 are, in order, verbascoside, Rehmannia glutinosa slices, test sample, and negative sample without Rehmannia glutinosa.

[0060] Figure 22 TLC chromatograms for identification of Rehmannia glutinosa using the developing system were examined. In developing system AC, bands 1-5 were, in order, catalpol, the three replicates of the test sample, and the negative sample lacking Rehmannia glutinosa. In developing system DE, bands 1-3 were, in order, catalpol, the test sample, and the negative sample lacking Rehmannia glutinosa.

[0061] Figure 23 The TLC chromatogram for identification of Rehmannia glutinosa by sampling volume determination is shown below; bands 1-8 are respectively 2 μL of catalpol, 3 μL of catalpol, 5 μL of catalpol, 10 μL of catalpol, 3 μL of test sample, 5 μL of test sample, 6 μL of test sample, and 10 μL of test sample.

[0062] Figure 24 TLC chromatograms for the identification of Rehmannia glutinosa by color development and heating time; bands 1-5 are, in order, catalpol, three replicates of the test sample, and negative sample without Rehmannia glutinosa;

[0063] Figure 25 The image shows a specific TLC pattern for the identification of Rehmannia glutinosa; bands 1-6 represent, in order, blank solvent, catalpol, three replicates of the test sample, and a negative sample lacking Rehmannia glutinosa.

[0064] Figure 26 For the identification of Rehmannia glutinosa using thin-layer chromatography (TLC) plates from different manufacturers and under different temperatures and humidity conditions; among them, bands 1-5 are catalpol, three replicates of the test sample, and negative samples without Rehmannia glutinosa, respectively.

[0065] Figure 27 TLC images of Rehmannia glutinosa in six batches of Baoxin Granules are shown. Among them, band 4 is catalpol, and bands 1-3 and 5-7 are Baoxin Granules with batch numbers X231102, X231103, X231104, 221001, 210601 and 240401, respectively.

[0066] Figure 28 TLC chromatogram for the identification of Salvia miltiorrhiza - test sample preparation method investigation; among them, bands 1-4 are, in order, salvianolic acid B, Salvia miltiorrhiza reference material, test sample, and negative sample lacking Salvia miltiorrhiza;

[0067] Figure 29 TLC chromatograms for identification and systematic investigation of Salvia miltiorrhiza; bands 1-4 represent, in order, salvianolic acid B, Salvia miltiorrhiza reference material, test sample, and negative sample lacking Salvia miltiorrhiza.

[0068] Figure 30 TLC chromatograms for identification of Salvia miltiorrhiza by sample volume (A) and specificity assessment of Salvia miltiorrhiza (B) are shown. In A, bands 1-9 represent, in order, 1 μL of reference standard, 3 μL of reference standard, 5 μL of reference standard, 5 μL of reference medicinal material, 6 μL of reference medicinal material, 10 μL of reference medicinal material, 5 μL of test sample, 6 μL of test sample, and 10 μL of test sample. In B, bands 1-7 represent, in order, blank solvent, salvianolic acid B, Salvia miltiorrhiza reference medicinal material, three replicates of the test sample, and a negative sample lacking Salvia miltiorrhiza.

[0069] Figure 31 For the identification of Salvia miltiorrhiza by thin-layer chromatography, TLC images of different manufacturers' thin-layer plates and TLC images under different temperatures and humidity were obtained; among them, bands 1-6 are, in order, salvianolic acid B, Salvia miltiorrhiza reference material, 3 replicates of the test sample, and negative sample without Salvia miltiorrhiza.

[0070] Figure 32 TLC images of Danshen in six batches of Baoxin Granules are shown. Among them, band 4 is Danshensu B, band 5 is Danshen reference material, and bands 1-3 and 6-8 are Baoxin Granules with batch numbers X231102, X231103, X231104, 221001, 240401 and 210601 respectively.

[0071] Figure 33 TLC chromatograms for thin-layer identification of Citrus aurantium - investigation of the preparation method of the test sample; among them, bands 1-6 are naringin, neohesperidin, 3 replicates of the test sample, and negative sample without Citrus aurantium, respectively;

[0072] Figure 34 TLC chromatograms for identification and systematic investigation of Citrus aurantium; bands 1-6 are, in order, naringin, neohesperidin, three replicates of the test sample, and negative sample lacking Citrus aurantium;

[0073] Figure 35 TLC chromatograms for identification of Citrus aurantium by sample volume (A) and specificity assessment of Citrus aurantium (B) are shown. In A, bands 1-6 represent, in order, 1 μL of reference standard, 3 μL of reference standard, 5 μL of reference standard, 1 μL of test sample, 3 μL of test sample, and 5 μL of test sample. In B, bands 1-6 represent, in order, blank solvent, mixed reference standard (neopterin and naringin), three replicates of test sample, and negative sample lacking Citrus aurantium.

[0074] Figure 36 Thin-layer chromatography (TLC) was used to identify Citrus aurantium by examining TLC images from different manufacturers and under different temperatures and humidity conditions. Bands 1-5 represent, in order, a mixed control (upper hesperidin and lower naringin), three replicates of the test sample, and a negative sample lacking Citrus aurantium.

[0075] Figure 37TLC images of Citrus aurantium in six batches of Baoxin Granules are shown. Among them, band 4 is a mixed reference standard (upper hesperidin and lower naringin), and bands 1-3 and 5-7 are Baoxin Granules with batch numbers 221001, 210601, 240401, X231102, X231103 and X231104, respectively. Detailed Implementation

[0076] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0077] Example 1: Thin-layer chromatography detection of Astragalus membranaceus

[0078] 1. Investigation of the preparation method of the test sample

[0079] (1) Preparation of test solution:

[0080] Test Solution 1: Take Baoxin Granules (hereinafter referred to as this product, produced by Zhejiang Kang Enbei Pharmaceutical Co., Ltd., according to the following method): Accurately weigh 7g of Astragalus membranaceus, 5g of Salvia miltiorrhiza, 5g of Ophiopogon japonicus, 2g of Poria cocos, 2g of Lycopus lucidus, 1g of Angelica sinensis, 3g of Rehmannia glutinosa, 1g of Citrus aurantium, 1g of Platycodon grandiflorus, 2g of Glycyrrhiza uralensis (processed), 1g of Rheum palmatum (processed with wine), and 1g of Hirudo medicinalis (processed with hot water). Extract the Hirudo medicinalis twice with 70% ethanol, adding 6 times the amount of ethanol each time, and extracting for 1 hour each time. Combine the extracts, recover the ethanol, and concentrate under reduced pressure to a relative density of 1.15-1.25. Extract the remaining 11 herbs, including Astragalus membranaceus, twice with water, adding 10 times the amount of water each time, and extracting for 1 hour each time. Filter the extract, concentrate the filtrate under reduced pressure to a relative density of 1.15-1.25, add 2 times the amount of ethanol to precipitate, let stand overnight, filter, recover the ethanol from the filtrate, and concentrate under reduced pressure to a relative density of 1. 0.15-1.25, combined with leech and other alcohol extracts, dried. The dry extract was mixed with dextrin at a mass ratio of 1:1, granulated, dried, and sized to 1000g, packaged, and then obtained. An appropriate amount was ground finely, 2g was taken, 100mL of methanol was added, and the mixture was ultrasonically treated (power 1100W, frequency 40kHz) for 30 minutes, filtered, the filtrate was evaporated to dryness, the residue was dissolved in 50mL of water, and extracted three times with water-saturated n-butanol, 40, 20, and 20mL respectively. The n-butanol solutions were combined, and the n-butanol solutions were washed three times with 1% sodium hydroxide solution, 25mL each time, and the alkali solution was discarded. The solutions were then washed twice with water-saturated n-butanol, 25mL each time, and the aqueous solution was discarded. The n-butanol solutions were combined, the solvent was recovered to dryness, and the residue was dissolved in 2mL of methanol to obtain the final product.

[0081] Test solution 2: Take an appropriate amount of this product, grind it into a fine powder, take 2g, add 10mL of ammonia test solution, after completely wetting, add 30mL of water, mix well, add 50mL of water-saturated n-butanol, heat under reflux for 2 hours, cool, transfer the extract to a separatory funnel, let stand for 1 hour to allow complete separation, accurately measure 25mL of the upper n-butanol solution, evaporate to dryness in a water bath, dissolve the residue in 5mL of methanol to obtain the final product.

[0082] Test solution 3: Take an appropriate amount of this product, grind it into a fine powder, take 2g, place it in a stoppered conical flask, add 50mL of 80% methanol solution containing 4% concentrated ammonia test solution (take 4mL of concentrated ammonia test solution, add 80% methanol to 100mL, shake well), heat under reflux for 1 hour, cool, filter, accurately measure 25mL of the filtrate, evaporate to dryness, and dissolve the residue in 5mL of 80% methanol to obtain the final product.

[0083] (2) Preparation of reference solution: Take astragaloside A reference standard and add methanol to prepare a 1 mg / mL solution as the reference solution.

[0084] (3) Thin-layer chromatography operation: Take 2 μL of the test solution and the reference solution and spot them on the same high-performance silica gel G thin-layer plate (Yantai Jiangyou Silica Gel Development Co., Ltd.). Perform the test according to the thin-layer chromatography method (General Chapter 0502 of Chinese Pharmacopoeia 2020). Use the lower layer solution of chloroform-methanol-water (13:7:2) as the developing solvent. Develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, and heat at 105℃ until the spots are clearly visible. Examine under sunlight and ultraviolet light (365nm).

[0085] The results are as follows Figure 1 As shown in the figure, the chromatograms of test solutions 1, 2, and 3 all show characteristic spots of the same color at the corresponding positions as the reference chromatogram. Under sunlight, all three show brownish spots, and under ultraviolet light (365 nm), all three show orange-yellow fluorescent spots, indicating that the preparation methods of these three test solutions can preliminarily meet the qualitative requirements for the Astragalus membranaceus flavor in the preparation. However, test solution 3 did not undergo extraction and purification during preparation, resulting in a higher proportion of extracted components and spot tailing, which would affect the accuracy of the results. The preparation process of test solution 1 is cumbersome and prone to emulsification during operation, increasing the complexity of the operation. Therefore, the preparation method of test solution 2 was selected.

[0086] 2. Reproducibility of the test sample preparation method

[0087] Take 1g of Astragalus membranaceus reference material and 1g of Astragalus membranaceus-deficient negative sample. Prepare the reference material and the Astragalus membranaceus-deficient negative solution according to the preparation method of test solution 2 above. Perform thin-layer chromatography (Chinese Pharmacopoeia 2020 Edition, General Chapter 0502) to examine whether the Astragalus membranaceus-deficient negative sample interferes with the test, and simultaneously examine whether the Astragalus membranaceus reference material has other corresponding spots. The results are as follows: Figure 2As shown in the figure, the test sample chromatogram, the reference chromatogram, and the reference medicinal material chromatogram all show the same brownish-brown spots at corresponding positions under sunlight and the same orange-yellow fluorescent spots under ultraviolet light. Negative light causes no interference, and the Rf value of the astragaloside A spots is approximately 0.45 (the regulatory requirement is 0.2~0.8), which is moderate. This indicates that the method has good specificity for identifying Astragalus membranaceus. The reference medicinal material chromatogram shows that, apart from corresponding to the astragaloside A reference standard chromatogram, no other specific spots or fluorescent spots were found. The results observed under sunlight and ultraviolet light show that both viewing conditions can be used for result interpretation, but the results are more obvious under ultraviolet light.

[0088] 3. Systematic investigation of the developing solvent

[0089] Use reference solution, test solution, and negative sample solution to investigate different developing solvent systems. The developing solvents are as follows:

[0090] Developing solvent system A: Considering reagent safety, the initial developing solvent chloroform-methanol-water (13:7:2) lower layer solution was adjusted to dichloroform-methanol-water (13:7:2) lower layer solution; Developing solvent system B: ethyl acetate-acetone-water (5:5:1); Developing solvent system C: ethyl acetate-acetone-water (4:5:1).

[0091] The results are as follows Figure 3 As shown, developing solvent system C has significant advantages. In terms of separation effect, this developing solvent reduces the overlap and tailing of Astragalus membranaceus spots; in terms of safety, it does not contain highly toxic halogenated hydrocarbons. Therefore, ethyl acetate-acetone-water (4:5:1) was chosen as the developing solvent.

[0092] 4. Sample quantity investigation

[0093] The sample volumes for the test solution were set at 3 μL, 5 μL, and 6 μL, and the sample volumes for the reference solution were set at 1 μL, 2 μL, and 3 μL, respectively. The results are as follows: Figure 4 As shown in Figure A, at different spotting volumes, the test sample chromatogram showed the same orange-yellow fluorescent spots at the corresponding positions as the reference sample chromatogram under ultraviolet light. Considering factors such as fluorescence spot effect, cost, and efficiency, the spotting volume of the test sample solution was determined to be 5 μL, and the spotting volume of the reference sample solution was determined to be 1 μL.

[0094] 5. Specificity assessment

[0095] Take blank methanol solvent, reference solution, test solution, and negative sample solution lacking Astragalus membranaceus, and perform the test according to the thin-layer chromatography method (Chinese Pharmacopoeia 2020 Edition, General Chapter 0502). The results are as follows: Figure 4 As shown in Figure B, the chromatograms of the test sample and the reference sample show the same orange-yellow fluorescent spots at the corresponding positions, while the astragalus-deficient negative sample and the methanol solvent do not show the same colored spots at these positions. This indicates that the method has good specificity.

[0096] 6. Durability test

[0097] 6.1 Thin-layer plate investigation

[0098] Using silica gel G plates already in use, the chromatography performance of thin-layer plates manufactured by Qingdao Ocean and Merck (Germany) on samples was investigated. The results are as follows: Figure 5 As shown, the thin-layer plates from the three manufacturers all met the requirements for sample chromatography, with good separation and no interference from negative samples.

[0099] 6.2 Temperature and Humidity Survey

[0100] The development of samples was investigated under different temperatures (4℃, room temperature) and relative humidity conditions (60%±5%, 90%±5%). The results are as follows: Figure 5 As shown, the chromatographic results of the samples met the requirements under different temperatures and relative humidity conditions, and there was no interference from negative samples.

[0101] 7. Thin-layer chromatography identification of samples

[0102] Following the methods determined in the above experiments, six batches of Baoxin Granules samples (batch numbers 210601, 221001, 240401, X231102, X231103, and X231104, respectively) were prepared and tested. The results are as follows: Figure 6 As shown in the chromatograms, all six batches of Baoxin Granules tested showed the same orange-yellow fluorescent spots at the corresponding positions as the reference standard.

[0103] 8. Conclusion

[0104] The above experimental results demonstrate that the thin-layer chromatography method for Astragalus membranaceus exhibits good specificity and robustness, with a moderate fluorescence main spot shift value, making it effective for the identification of Astragalus membranaceus in Baoxin granules. The final quality standard detection method is as follows:

[0105] Take an appropriate amount of this product, grind it finely, take 2g, add 10mL of ammonia test solution, stir to completely wet, add 30mL of water, mix well, add 50mL of water-saturated n-butanol, heat under reflux for 2 hours, cool, transfer the extract to a separatory funnel, let stand for 1 hour, accurately measure 25mL of the upper n-butanol liquid, evaporate to dryness in a water bath, dissolve the residue in 5mL of methanol, and use this as the test solution. Separately, take astragaloside A reference standard, add methanol to prepare a 1mg / mL solution, and use this as the reference solution. Apply 5μL of the test solution and 1μL of the reference solution separately to the same high-performance silica gel G thin-layer plate, develop with ethyl acetate-acetone-water (4:5:1) as the developing solvent, remove, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly visible, and examine under ultraviolet light (365nm). In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference standard.

[0106] Example 2: Thin-layer chromatographic detection of rhubarb in wine

[0107] 1. Preparation method of test sample

[0108] (1) Preparation of test solution: Take an appropriate amount of this product, grind it into a fine powder, take 7g, add 50mL of methanol, sonicate (power 1100W, frequency 40kHz) for 30 minutes, filter, evaporate the filtrate to dryness, add 50mL of 8% hydrochloric acid solution to the residue and sonicate to dissolve, add 50mL of chloroform and heat under reflux for 2.5 hours, cool, transfer the extract to a separatory funnel, collect the lower chloroform liquid, wash the upper liquid twice with chloroform, 20mL each time, combine the chloroform liquids, recover the solvent to dryness, add 3mL of methanol to dissolve the residue, and use it as the test solution. Take another 7g of rhubarb-deficient negative sample and prepare a rhubarb-deficient negative sample solution in the same way.

[0109] (2) Preparation of reference solutions: Take rhein and emodin reference standards, add methanol to prepare 0.1 mg / mL rhein reference solution, 0.05 mg / mL emodin reference solution and mixed reference solution of the same concentration.

[0110] (3) Thin-layer chromatography operation: Refer to the thin-layer chromatography method (General Chapter 0502 of Chinese Pharmacopoeia 2020 edition) to test. Take 4 μL of the above test solution, 4 μL of the negative sample solution of rhubarb without wine, 1 μL of the reference solution and 1 μL of the mixed reference solution, and spot them on the same high-performance silica gel G thin-layer plate (Yantai Jiangyou Silica Gel Development Co., Ltd.). Use the upper layer of petroleum ether (60~90℃)-ethyl formate-formic acid (15:5:1) as the developing solvent, develop, take out, air dry, and examine under ultraviolet light (365nm).

[0111] The results are as follows Figure 7 As shown in the figure, the chromatogram of the test sample shows the same orange-yellow fluorescent spots at the corresponding positions as the chromatogram of the reference sample, with a spot Rf value of 0.45, which is within the suitable range. Furthermore, there is no interference from the negative control. The chromatogram spots of the mixed reference solution are consistent with those of the separately prepared reference solutions. To improve the efficiency of subsequent experiments, a mixed reference solution will be used.

[0112] 2. Sample quantity investigation

[0113] The sample volumes for the test solution were set at 2 μL, 3 μL, 4 μL, and 5 μL, respectively, and the sample volumes for the mixed reference solution were set at 1 μL, 2 μL, 3 μL, and 4 μL, respectively. The results are as follows: Figure 8 As shown in Figure A, the corresponding positions of the chromatograms of the test sample and the reference sample with different spotting amounts all showed the same orange-yellow fluorescent spots under ultraviolet light. Considering factors such as the effectiveness of the fluorescent spots, cost, and efficiency, the spotting volume of the test sample solution was determined to be 4 μL, and the spotting volume of the reference sample solution was determined to be 2 μL.

[0114] 3. Specificity assessment

[0115] Take blank methanol solvent, mixed reference solution, test solution, and negative sample solution of rhubarb without alcohol, and perform the test according to the thin-layer chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). The results are as follows: Figure 8 As shown in Figure B, the chromatograms of the test sample and the reference sample show the same orange-yellow fluorescent spots at the corresponding positions, while the negative results of rhubarb without wine and the blank solvent show no similar spots at these positions.

[0116] 4. Durability test

[0117] 4.1 Thin-layer plate investigation

[0118] Using silica gel G plates already in use, the chromatography performance of thin-layer plates manufactured by Qingdao Ocean and Merck (Germany) on samples was investigated. The results are as follows: Figure 9 As shown, the thin-layer plate samples from all three manufacturers exhibited good separation, with no interference from negative samples.

[0119] 4.2 Temperature and humidity investigation

[0120] The development of samples was investigated under different temperatures (4℃, room temperature) and relative humidity conditions (60%±5%, 90%±5%). The results are as follows: Figure 9 As shown, the chromatographic results of the samples met the requirements under different temperatures and relative humidity conditions, and there was no interference from negative samples.

[0121] 5. Thin-layer chromatography identification of samples

[0122] Following the methods determined in the above experiments, six batches of Baoxin Granules samples (batch numbers 210601, 221001, 240401, X231102, X231103, and X231104, respectively) were prepared and tested. The results are as follows: Figure 10 As shown in the chromatograms, all six batches of Baoxin Granules tested showed the same orange-yellow fluorescent spots at the corresponding positions as the reference standard.

[0123] 6. Conclusion

[0124] The above experimental results demonstrate that the thin-layer chromatography method for rhubarb in alcohol exhibits good specificity and robustness, with a moderate fluorescence main spot shift value, making it effective for the thin-layer identification of rhubarb in Baoxin granules. The final quality standard detection method is as follows:

[0125] Take an appropriate amount of this product, grind it into a fine powder, take 7g, add 50mL of methanol, sonicate (power 1100W, frequency 40kHz) for 30 minutes, filter, evaporate the filtrate to dryness, add 50mL of 8% hydrochloric acid solution to the residue and sonicate to dissolve, then add 50mL of chloroform and heat under reflux for 2.5 hours, collect the chloroform layer, wash the aqueous layer twice with 20mL of chloroform each time, combine the chloroform solutions, recover the solvent to dryness, add 3mL of methanol to dissolve the residue, and use this as the test solution. Separately, take rhein and emodin reference standards, add methanol to prepare a mixed reference solution containing 0.1mg / mL rhein and 0.05mg / mL emodin. According to the thin-layer chromatography method (General Chapter 0502 of Chinese Pharmacopoeia 2020), 4 μL of the above test solution and 2 μL of the reference solution were spotted separately on the same high-performance silica gel G thin-layer plate. The upper layer of petroleum ether (60~90℃)-ethyl formate-formic acid (15:5:1) was used as the developing solvent. After development, the plate was removed, dried, and examined under ultraviolet light (365nm). In the chromatogram of the test sample, fluorescent spots of the same color appeared at the corresponding positions as in the chromatogram of the reference sample.

[0126] Example 3: Thin-layer chromatographic identification of scalded leeches

[0127] 1. Investigation of the preparation method of the test sample

[0128] (1) Preparation of test solution: Take an appropriate amount of this product, grind it into a fine powder, take 6g, add 25mL of ethanol, sonicate (power 1100W, frequency 40kHz) for 15 minutes, filter, evaporate to dryness, add 0.5mL of ethanol to dissolve the residue, and use it as the test solution.

[0129] Take another 6g of water-deficient leech negative sample and prepare a water-deficient leech negative sample solution using the same method.

[0130] (2) Preparation of reference medicinal material solution: Take 0.25g of leech (leech) reference medicinal material and prepare the reference medicinal material solution in the same way.

[0131] (3) Thin-layer chromatography operation: Refer to the thin-layer chromatography method (General Chapter 0502 of Chinese Pharmacopoeia 2020 edition) to test. Take 5 μL of the test solution and 5 μL of the negative sample solution of dehydrated leech and 1 μL of the control medicinal material solution and spot them on the same high-performance silica gel G thin-layer plate (Yantai Jiangyou Silica Gel Development Co., Ltd.). Use cyclohexane-ethyl acetate (4:1) as the developing solvent, develop, take out, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly displayed, and examine under sunlight.

[0132] The results are as follows Figure 11As shown in the chromatogram, the test sample showed a main spot of the same color at the corresponding position as the reference medicinal material, with an Rf value of 0.44. This Rf value is within a suitable range, and there was no negative interference. These results indicate that under the current experimental conditions, there is a good correspondence between the test sample and the reference medicinal material, and the experimental method has a certain degree of specificity, effectively eliminating other interfering factors.

[0133] 2. Examination of color development heating time

[0134] Take the test solution, the reference herb solution, and the negative sample solution of dehydrated leeches, and perform the test according to the thin-layer chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). The heating times were set to 0.5 minutes, 1 minute, 2 minutes, and 3 minutes, respectively. The results are as follows: Figure 12 As shown, color development begins after heating at 105℃ for 0.5 minutes, while the background banding becomes significantly stronger after heating for 3 minutes. Considering both the color development effect and background interference, a heating time of 1 minute was selected.

[0135] 3. Sample quantity investigation

[0136] Take the test sample solution, the reference medicinal material solution, and the negative sample solution of dehydrated leeches, and perform the test according to the thin-layer chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). The sample loading volumes for the test sample solution are set at 5 μL, 10 μL, and 15 μL, respectively, and the sample loading volumes for the reference medicinal material solution are 1 μL, 2 μL, and 3 μL, respectively. The results are as follows: Figure 13 As shown in Figure A, overloading occurred when the sample volume of the test sample reached 15 μL. Considering factors such as spotting effect, cost, and efficiency, the final sample volume for the test sample solution was determined to be 10 μL, and the sample volume for the control herbal material solution was determined to be 1 μL.

[0137] 4. Specificity assessment

[0138] Take blank ethanol solvent, reference medicinal material solution, test solution, and negative sample solution, and perform the test according to the thin-layer chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). The results are as follows: Figure 13 As shown in Figure B, the chromatogram of the test sample shows spots of the same color at the corresponding positions as the chromatogram of the reference medicinal material, while the anhydrous leech negative and blank solvent do not show spots of the same color at these positions. Therefore, this method has good specificity.

[0139] 5. Durability test

[0140] 5.1 Thin-layer plate investigation

[0141] Using silica gel G plates already in use, the chromatography performance of thin-layer plates manufactured by Qingdao Ocean and Merck (Germany) on samples was investigated. The results are as follows: Figure 14 As shown, the thin-layer plate samples from all three manufacturers exhibited good separation, with no interference from negative samples.

[0142] 5.2 Temperature and Humidity Survey

[0143] The development of samples was investigated under different temperatures (4℃, room temperature) and relative humidity conditions (60%±5%, 90%±5%). The results are as follows: Figure 14 As shown, the chromatographic results of the samples met the requirements under different temperatures and relative humidity conditions, and there was no interference from negative samples.

[0144] 6. Thin-layer chromatography identification of samples

[0145] Following the methods determined in the above experiments, six batches of Baoxin Granules samples (batch numbers 210601, 221001, 240401, X231102, X231103, and X231104, respectively) were prepared and tested. The results are as follows: Figure 15 As shown in the chromatograms, all six batches of Baoxin Granules tested showed spots of the same color at the corresponding positions as the reference medicinal material.

[0146] 7. Conclusion

[0147] The above experimental results demonstrate that the thin-layer chromatography method for identifying scalded leeches has good specificity and robustness, and can be effectively applied to the identification of scalded leeches in Baoxin granule formulations. The final quality standard detection method is as follows:

[0148] Take an appropriate amount of this product, grind it into a fine powder, take 6g, add 25mL of ethanol, sonicate (power 1100W, frequency 40kHz) for 15 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 0.5mL of ethanol to prepare the test solution. Separately, take 0.25g of leech reference material and prepare a reference material solution using the same method. Perform the test according to the thin-layer chromatography method (Chinese Pharmacopoeia 2020 Edition, General Chapter 0502), apply 10μL of the above test solution and 1μL of the reference material solution separately to the same high-performance silica gel G thin-layer plate, develop with cyclohexane-ethyl acetate (4:1) as the developing solvent, remove, air dry, spray with 10% sulfuric acid ethanol solution, and heat at 105℃ (about 1 minute) until the spots are clearly visible. Examine under sunlight; the test sample chromatogram should show spots of the same color at the corresponding positions as the reference material chromatogram.

[0149] Example 4 Thin-layer chromatographic detection of roasted licorice

[0150] 1. Investigation of the preparation method of the test solution and the thin-layer chromatography operation method

[0151] Method 1: The test was conducted according to the identification method of roasted licorice in the current quality standard: The test solution under "1. Reproducibility of the preparation method of the test sample" in Example 2 was used as the test solution. 7g of a negative sample lacking roasted licorice was also prepared as a negative sample solution using the same method. Glycyrrhetinic acid reference standard was prepared into a 1mg / mL solution with methanol as the reference solution; glycyrrhizin reference standard and glycyrrhizic acid reference standard were also prepared into a 1mg / mL solution with methanol as the reference solution. According to the thin-layer chromatography method (General Chapter 0502 of the 2020 edition of the Chinese Pharmacopoeia), 5 μL of the test solution and 2 μL of the reference solution were spotted separately on the same high-performance silica gel G thin-layer plate (Yantai Jiangyou Silica Gel Development Co., Ltd.). Petroleum ether (60~90℃)-benzene-ethyl acetate-formic acid (10:20:7:0.5) was used as the developing solvent. After development, the plate was removed and dried, sprayed with phosphomolybdic acid ethanol solution, and heated at 105℃ until the spots were clearly visible. The plates were then examined under sunlight.

[0152] Method 2: Refer to the method under the "Identification" section of the National Drug Standard (First Batch) for Traditional Chinese Medicine Formula Granules: Take an appropriate amount of this product, grind it into a fine powder, take 2.5g, add 10mL of methanol, sonicate (power 1100W, frequency 40kHz) for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 5mL of methanol to prepare the test solution. Take another 2.5g of a negative sample lacking prepared licorice, and prepare a negative sample solution using the same method. Take 1g of licorice reference material, add 50mL of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, and prepare a reference material solution using the same method starting from "add 10mL of methanol". Take glycyrrhizin reference standard, add methanol to prepare a 0.5mg / mL solution as the reference solution. According to the thin-layer chromatography method (General Chapter 0502 of the 2020 edition of the Chinese Pharmacopoeia), 1 μL of each of the above four solutions was applied to the same high-performance silica gel G thin-layer plate (Yantai Jiangyou Silica Gel Development Co., Ltd.). Ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) was used as the developing solvent, and 10% sulfuric acid ethanol solution was sprayed on. The plate was heated at 105°C until the spots were clearly visible and examined under ultraviolet light (365 nm).

[0153] The results of Method 1 and Method 2 are as follows Figure 16 A and Figure 16 As shown in B, in Method 1, spots of the same color appear at the corresponding positions in the chromatograms of the test sample and the reference sample. However, the newly added glycyrrhizin and glycyrrhizic acid do not show spots at the corresponding positions. Method 2 has significant advantages in the thin-layer identification of processed licorice. Compared with the current quality standard method, Method 2, using glycyrrhizin as a reference, is more targeted. Furthermore, adding a reference herbal solution can greatly enhance the specificity of the identification method. Therefore, Method 2 was chosen as the preparation method for the test sample.

[0154] 2. Systematic investigation of the developing solvent

[0155] Take the test solution, reference solution, and negative sample solution from Method 2, and perform thin-layer chromatography (according to General Chapter 0502, Part IV, Chinese Pharmacopoeia 2020 Edition) to investigate different developing solvent systems. The developing solvent systems are as follows:

[0156] Developing solvent system A: ethyl acetate-methanol-formic acid-water (12:1:1:2); Developing solvent system B: lower layer solution of chloroform-methanol-water (13:7:2).

[0157] The results are as follows Figure 17 As shown. Compared with the initial developing solvent, all developing solvent systems were able to identify roasted licorice, and ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) was finally selected as the developing solvent.

[0158] 3. Sample quantity investigation

[0159] Take the test solution, reference herb solution, and reference standard solution from Method 2, and perform the test according to thin-layer chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). The sample loading volumes for the test solution were set at 1 μL, 3 μL, 5 μL, and 6 μL, respectively, and the sample loading volumes for the licorice reference herb solution and glycyrrhizin reference standard solution were 1 μL, 2 μL, 3 μL, and 4 μL, respectively. The results are as follows: Figure 18 As shown in Figure A, the corresponding positions of the chromatograms of the test sample, reference standard, and reference medicinal material with different spotting amounts all showed fluorescent spots of the same color under ultraviolet light (365 nm). Considering factors such as the effect of fluorescent spots, cost, and efficiency, the spotting volume of the test sample solution was determined to be 3 μL, and the spotting volumes of the reference standard solution and the reference medicinal material solution were 1 μL.

[0160] 4. Specificity assessment

[0161] Take the blank methanol solvent, reference solution, test solution, reference medicinal material solution, and negative sample solution of unprocessed licorice root from Method 2, and perform the test according to the thin-layer chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). The results are as follows: Figure 18 As shown in Figure B, in the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatograms of the reference medicinal material and the reference standard, while no fluorescent spots of the same color appear at the same positions in the blank solvent and negative chromatograms of licorice without processing. This indicates that the identification method can specifically identify the target component in licorice without interference from other components in the preparation or the blank solvent, demonstrating good specificity.

[0162] 5. Durability test

[0163] 5.1 Thin-layer plate investigation

[0164] Using the silica gel G plate already used in Method 2, the chromatography performance of thin-layer plates manufactured by Qingdao Ocean and Merck (Germany) on the samples was investigated. The results are as follows: Figure 19 As shown, the thin-layer plate samples from all three manufacturers exhibited good separation, with no interference from negative samples.

[0165] 5.2 Temperature and Humidity Survey

[0166] The development of samples was investigated under different temperatures (4℃, room temperature) and relative humidity conditions (60%±5%, 90%±5%). The results are as follows: Figure 19 As shown, the chromatographic results of the samples met the requirements under different temperatures and relative humidity conditions, and there was no interference from negative samples.

[0167] 6. Thin-layer chromatography identification of samples

[0168] Following the methods determined in the above experiments, six batches of Baoxin Granules samples (batch numbers 210601, 221001, 240401, X231102, X231103, and X231104, respectively) were prepared and tested. The results are as follows: Figure 20 As shown in the chromatograms, all six batches of Baoxin Granules tested showed spots of the same color at the corresponding positions as the reference medicinal material.

[0169] 7. Conclusion

[0170] The above experimental results indicate that the thin-layer chromatography method for processing licorice root exhibits good specificity and robustness, with a moderate fluorescence main spot shift value, making it effective for the identification of processed licorice root in Baoxin granules. The final quality standard detection method is as follows:

[0171] Take an appropriate amount of this product, grind it into a fine powder, take 2.5g, add 10mL of methanol, sonicate (power 1100W, frequency 40kHz) for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 5mL of methanol to prepare the test solution. Separately, take 1g of licorice (licorice) reference material, add 50mL of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, and prepare the reference material solution using the same method starting from "add 10mL of methanol". Also, take glycyrrhizin reference standard, add methanol to prepare a 0.5mg / mL solution to prepare the reference solution. According to the thin-layer chromatography method (General Chapter 0502, Chinese Pharmacopoeia 2020 Edition), 3 μL of the above-mentioned test solution, 1 μL each of the reference medicinal material solution and the reference standard solution were spotted separately onto the same high-performance silica gel G thin-layer plate. The plate was developed using ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) as the developing solvent. After development, the plate was removed, dried, sprayed with 10% sulfuric acid ethanol solution, and heated at 105℃ until the spots were clearly visible. The plate was then examined under ultraviolet light (365 nm). In the chromatogram of the test sample, fluorescent spots of the same color appeared at the corresponding positions as in the chromatograms of the reference medicinal material and the reference standard.

[0172] Example 5: Thin-layer chromatographic identification of Rehmannia glutinosa

[0173] 1. Investigation of the preparation method of the test sample

[0174] (1) Preparation of test solution: Refer to the thin-layer chromatography identification item of Rehmannia glutinosa in the reference "Research on the Improvement of Quality Standards of Rehmannia glutinosa and Liuwei Dihuang Capsules": Take an appropriate amount of this product, grind it into a fine powder, take 8g, add 50mL of methanol, sonicate (power 1100W, frequency 40kHz) for 45 minutes, let it stand at room temperature, filter, accurately measure 25mL of the filtrate, recover the solvent under reduced pressure to dryness, dissolve it in water, pass the solution through a macroporous resin column (10g, inner diameter 15~20mm), first elute with 100mL of water, then elute with 100mL of methanol, recover the methanol eluent to dryness, dissolve it with 2mL of methanol, and use it as the test solution. Take another 2g of Rehmannia glutinosa slices and prepare a slice solution in the same way.

[0175] (2) Preparation of reference solutions: Take verbascoside and catalpol reference standards, add methanol to prepare 0.5 mg / mL solutions respectively, and use them as reference solutions.

[0176] (3) Thin-layer chromatography procedure: Perform the thin-layer chromatography test (General Chapter 0502 of the 2020 edition of the Chinese Pharmacopoeia). Take 10 μL of the test solution, 15 μL of the decoction piece solution, and 5 μL of the reference solution, and spot them separately on the same high-performance silica gel GF. 254 On a thin-layer plate (Yantai Jiangyou Silica Gel Development Co., Ltd.), ethyl acetate-methanol-formic acid-water (14:6:1) was used as the developing solvent. The plate was then removed, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105℃, and examined under sunlight.

[0177] The results are as follows Figure 21 As shown in Figure A, the spots in the test sample at the corresponding positions in the chromatograms of the prepared slices and the reference standard are the same color, indicating that this method can detect the relevant components in Rehmannia glutinosa. However, it was also found that the content of verbascoside in the test sample may be low, with the corresponding spots being dull and poorly separated. To perform thin-layer chromatography identification of Rehmannia glutinosa more accurately and stably, catalpol reference standard is tentatively used for subsequent studies.

[0178] This embodiment also verified the method under the "Identification" section of the National Drug Standard (First Batch) for "Rehmannia glutinosa Formula Granules," and the results are as follows. Figure 21 As shown in B, the test sample did not exhibit fluorescent spots of the same color at the corresponding positions as the Rehmannia glutinosa slices and the reference sample, and its chromatogram was essentially indistinguishable from that of the negative sample lacking Rehmannia glutinosa. Therefore, this method is not suitable for the detection of Rehmannia glutinosa in this product.

[0179] 2. Systematic investigation of the developing solvent

[0180] Prepare the catalpol reference solution, test solution, and negative sample solution, and simultaneously investigate different developing solvent systems. The developing solvent systems are as follows:

[0181] Developing solvent system A: dichloromethane-methanol-glacial acetic acid (20:10:3); Developing solvent system B: ethyl acetate-methanol-formic acid-water (14:6:1); Developing solvent system C: ethyl acetate-methanol-formic acid-water (17:5:1:2); Developing solvent system D: n-butanol-glacial acetic acid-water (10:2:1); Developing solvent system E: n-butanol-glacial acetic acid-water (10:1:1).

[0182] The results are as follows Figure 22 As shown, developing solvent system D has significant advantages. Regarding spot location, its Rf value is moderate, indicating that the target component migrates at an appropriate distance on the thin-layer plate. In terms of resolution, this system effectively separates the target component from impurities in the sample, with few adjacent impurities for the target component spot, which helps improve the accuracy and reliability of qualitative analysis. In summary, n-butanol-glacial acetic acid-water (10:2:1) was selected as the developing solvent.

[0183] 3. Sample quantity investigation

[0184] Take the catalpol reference solution, test solution, and negative sample solution, and perform the test according to the thin-layer chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). The sample loading volumes for the test solution were set at 3 μL, 5 μL, 6 μL, and 10 μL, respectively, and the sample loading volumes for the reference solution were set at 2 μL, 3 μL, 5 μL, and 10 μL, respectively. The results are as follows: Figure 23 As shown, at different spotting volumes, the corresponding positions of the test sample and the reference sample chromatograms all showed the same color spots under sunlight. Considering factors such as cost and efficiency, the final spotting volume for the test sample solution was determined to be 6 μL, and the spotting volume for the reference sample was 2 μL.

[0185] 4. Effect of heating time on color development

[0186] Take the catalpol reference solution, test solution, and negative sample solution, and perform thin-layer chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). The heating times were set to 1 minute, 4 minutes, 7 minutes, and 10 minutes, respectively. The results are as follows: Figure 24 As shown, no color development was observed in either the reference standard or the test sample chromatogram at a heating time of 1 minute. Color development began in both after 4 minutes of heating. With further extension of heating time to 7 and 10 minutes, although the spot color deepened, the background bands also became more prominent. Considering factors such as color development and background interference, a heating time of 4 minutes is preferred.

[0187] 5. Specificity assessment

[0188] Take the reference solution, test solution, negative sample solution, and blank methanol solvent, and perform the test according to the thin-layer chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). The specificity of this method for identifying Rehmannia glutinosa is evaluated by examining the chromatograms of the methanol solvent, the negative sample lacking Rehmannia glutinosa, the reference solution, and the test sample. The results are as follows: Figure 25 As shown in the chromatogram, the test sample shows a spot of the same color at the corresponding position as the reference sample. However, in the negative chromatogram lacking Rehmannia glutinosa, no spot of the same color appears at that position, and the blank solvent also shows no interfering spots. This indicates that the identification method can specifically identify the target component in Rehmannia glutinosa, unaffected by other components in the preparation or by the blank solvent; therefore, this method has good specificity.

[0189] 6. Durability test

[0190] 6.1 Thin-layer plate investigation

[0191] Combined with the silicone GF already in use 254 Thin-layer chromatography (TLC) was used to investigate the chromatographic performance of samples on TLC plates produced by Qingdao Marine Products Co., Ltd. The results are as follows: Figure 26 As shown, the thin-layer plate samples from both manufacturers exhibited good separation, with moderate spot Rf values ​​and no interference from negative samples.

[0192] 6.2 Temperature and Humidity Survey

[0193] The development of samples was investigated under different temperature (4℃, room temperature) and humidity (60%±5%, 90%±5%) conditions. The results are as follows: Figure 26 As shown, the chromatographic results of the samples met the requirements under different temperatures and relative humidity conditions, and there was no interference from negative samples.

[0194] 7. Thin-layer chromatography identification of samples

[0195] Following the methods determined in the above experiments, six batches of Baoxin Granules samples (batch numbers 210601, 221001, 240401, X231102, X231103, and X231104, respectively) were prepared and tested. The results are as follows: Figure 27 As shown in the chromatograms, the six batches of Baoxin Granules test samples showed spots of the same color at the corresponding positions as the reference standard chromatograms.

[0196] 8. Conclusion

[0197] The above experimental results show that the thin-layer chromatography method for Rehmannia glutinosa has good specificity and robustness, and can be effectively applied to the thin-layer identification of Rehmannia glutinosa in Baoxin granules. The final quality standard detection method is as follows:

[0198] Take an appropriate amount of this product, grind it into a fine powder, take 8g, add 50mL of methanol, sonicate (power 1100W, frequency 40kHz) for 45 minutes, let it stand at room temperature, filter, accurately measure 25mL of the filtrate, recover the solvent under reduced pressure to dryness, dissolve in water, pass the solution through a macroporous resin column (10g, inner diameter 15~20mm), first elute with 100mL of water, then elute with 100mL of methanol, recover the methanol eluent to dryness, dissolve in 3mL of methanol, and use as the test solution. Separately take catalpol reference standard, add methanol to prepare a 0.5mg / mL solution, as the reference solution. Perform the test according to the thin-layer chromatography method (Chinese Pharmacopoeia 2020 General Chapter 0502), take 6μL of the test solution and 2μL of the reference solution, and spot them separately on the same high-performance silica gel GF. 254 On a thin-layer plate, n-butanol-glacial acetic acid-water (10:2:1) was used as the developing solvent. The developing tank was pre-equilibrated for 15 minutes before development, followed by upward development at a distance of 8 cm. After development, the plate was removed, air-dried, sprayed with 10% sulfuric acid ethanol solution, and heated at 105℃ for 4 minutes until the spots were clearly visible. The plate was then examined under sunlight. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions as in the chromatogram of the reference sample.

[0199] Example 6: Thin-layer chromatography identification of Salvia miltiorrhiza

[0200] 1. Investigation of the preparation method of the test sample

[0201] (1) Preparation of test solution:

[0202] According to the identification method (2) of "Danshen" in the 2020 edition of the Chinese Pharmacopoeia, Part I, medicinal materials and decoction pieces, take an appropriate amount of this product, grind it into a fine powder, take 2g, add 5mL of ethanol, sonicate (power 1100W, frequency 40kHz) for 15 minutes, filter, and use the filtrate as the test solution. Take another 2g of Danshen-deficient negative sample and prepare a negative sample solution in the same way.

[0203] (2) Preparation of reference solution: Take 1g of Salvia miltiorrhiza reference material, add 30mL of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, and prepare the reference material solution from the residue starting from "add 5mL of ethanol". Take salvianolic acid B reference standard, add 60% methanol to prepare a 0.5mg / mL solution, as the reference solution.

[0204] (3) Thin-layer chromatography operation

[0205] Following the thin-layer chromatography method (General Chapter 0502, Chinese Pharmacopoeia 2020 Edition), 5 μL of each of the above four solutions were spotted onto the same silica gel GF plate. 254 On a thin-layer plate (Yantai Jiangyou Silica Gel Development Co., Ltd.), a mixture of chloroform-toluene-ethyl acetate-methanol-formic acid (3:2:4:0.5:2) was developed, removed, dried, and examined under ultraviolet light (254nm).

[0206] The results are as follows Figure 28 As shown in the chromatogram, the test sample chromatogram shows spots of the same color at the corresponding positions as the chromatograms of the reference medicinal material and the reference standard, with no negative interference. This indicates that the test solution prepared according to this method can effectively detect the target components in Danshen.

[0207] 2. Systematic investigation of the developing solvent

[0208] Take the reference medicinal material solution, reference standard solution, test solution, and negative sample solution, and perform thin-layer chromatography (according to Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). Investigate different developing solvent systems. The developing solvent systems are as follows:

[0209] Developing solvent system A: chloroform-toluene-ethyl acetate-methanol-formic acid (3:2:4:0.5:2). Considering reagent safety and availability, it was adjusted to dichloroform-toluene-ethyl acetate-methanol-formic acid (3:2:4:0.5:0.2); Developing solvent system B: dichloroform-toluene-ethyl acetate-methanol-formic acid (3:2:4:1:2); Developing solvent system C: dichloroform-toluene-ethyl acetate-methanol-formic acid (3:2:6:0.5:2); Developing solvent system D: dichloroform-toluene-ethyl acetate-methanol-formic acid (3:2:7:0.5:2).

[0210] The results are as follows Figure 29 As shown. Replacing chloroform with dichloromethane did not result in a significant difference compared to the original developing solvent system, although the Rf value was lower. Next, the proportions of system A were adjusted. Considering various factors, system D (dichloromethane-toluene-ethyl acetate-methanol-formic acid, 3:2:7:0.5:2) was selected as the developing solvent, as this solvent allows for better separation and development of the target components on the thin-layer plate.

[0211] 3. Sample quantity investigation

[0212] Take the reference medicinal material solution, reference standard solution, test solution, and negative sample solution, and perform the test according to the thin-layer chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). The sample loading volumes for the test solution are 3 μL, 5 μL, 6 μL, and 10 μL, respectively, and the sample loading volumes for the reference standard solution are 3 μL, 5 μL, 6 μL, and 10 μL, respectively. The results are as follows: Figure 30 As shown in Figure A. Under different spotting volumes, the test sample chromatogram showed spots of the same color at the corresponding positions as the reference medicinal material chromatogram and the reference standard chromatogram. Considering factors such as cost and efficiency, the final determination was 6 μL each for the test sample solution and the reference medicinal material solution, and 3 μL for the reference standard solution.

[0213] 4. Specificity assessment

[0214] Take blank ethanol solvent, reference medicinal material solution, reference solution, test solution, and negative sample solution for *Salvia miltiorrhiza* deficiency, and perform the test according to thin-layer chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). The results are as follows: Figure 30 As shown in B. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatograms of the reference medicinal material and the reference standard, while no spots of the same color appear at the same positions in the ethanol solvent and tanshinone-deficient chromatograms.

[0215] 5. Durability test

[0216] 5.1 Thin-layer plate investigation

[0217] Combined with the silicone GF already in use 254 Thin-layer chromatography (TLC) was used to investigate the chromatographic performance of samples on TLC plates produced by Qingdao Marine Products Co., Ltd. The results are as follows: Figure 31 As shown, the thin-layer plate samples from both manufacturers exhibited good separation, with moderate spot Rf values ​​and no interference from negative samples.

[0218] 5.2 Temperature and Humidity Survey

[0219] The development of samples was investigated under different temperatures (4℃, room temperature) and relative humidity conditions (60%±5%, 90%±5%). The results are as follows: Figure 31 As shown, the chromatographic results of the samples met the requirements under different temperatures and relative humidity conditions, and there was no interference from negative samples.

[0220] 6. Thin-layer chromatography identification of samples

[0221] Following the methods determined in the above experiments, six batches of Baoxin Granules samples (batch numbers 210601, 221001, 240401, X231102, X231103, and X231104, respectively) were tested. The results are as follows: Figure 32 As shown in the chromatograms, in the six batches of Baoxin Granules test samples, spots of the same color appeared at the corresponding positions as in the chromatograms of the reference medicinal material and the reference standard.

[0222] 7. Conclusion

[0223] The above experimental results show that the thin-layer chromatography method for Danshen (Salvia miltiorrhiza) has good specificity and robustness, and can be effectively applied to the identification of Danshen in Baoxin granules. The final quality standard detection method is as follows:

[0224] Take an appropriate amount of this product, grind it finely, take 2 g, add 5 mL of ethanol, and ultrasonically treat (power 1100 W, frequency 40 kHz) for 15 minutes. Filter, and use the filtrate as the test solution. Separately take 1 g of the control crude drug of Salvia miltiorrhiza, add 30 mL of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, and prepare the control crude drug solution in the same method starting from "add 5 mL of ethanol" for the residue. Then take the reference substance of salvianolic acid B, dissolve it in 60% methanol to prepare a solution containing 0.5 mg per 1 mL as the reference substance solution. According to the thin-layer chromatography method (General Principles 0502 of the Chinese Pharmacopoeia 2020 Edition), take 6 μL of each of the above-mentioned test solution and control crude drug solution and 3 μL of the reference substance solution, and spot them on the same high-efficiency silica gel GF 254 thin-layer plate. Use dichloromethane-toluene-ethyl acetate-methanol-formic acid (3:2:7:0.5:2) as the developing solvent, develop, take out, dry in air, and examine under ultraviolet light (254 nm). In the chromatogram of the test solution, spots of the same color should appear at the corresponding positions as in the chromatograms of the control crude drug and the reference substance.

[0225] Example 7 Identification of Fructus Aurantii by Thin-Layer Chromatography

[0226] 1. Investigation on the Preparation Method of the Test Solution

[0227] (1) Preparation of the test solution

[0228] According to the method of "Identification of Fructus Aurantii" (2) in Part I of the Chinese Pharmacopoeia 2020 Edition: Take an appropriate amount of this product, grind it finely, take 2.5 g, add 10 mL of methanol, ultrasonically treat (power 1100 W, frequency 40 kHz) for 30 minutes, filter, evaporate the filtrate to dryness, and dissolve the residue in 5 mL of methanol to obtain the test solution.

[0229] (2) Preparation of the reference substance solution

[0230] Take the reference substances of naringin and neohesperidin, and dissolve them in methanol respectively to prepare solutions containing 1 mg of naringin and 1.5 mg of neohesperidin per 1 mL as the reference substance solution.

[0231] (3) Operation of the thin-layer chromatography method

[0232] Refer to the thin-layer chromatography method (General Principles 0502 of the Chinese Pharmacopoeia 2020 Edition) for the test. Take 3 μL of the test solution and 2 μL of the reference substance solution, and spot them on the same high-efficiency silica gel G thin-layer plate (Yantai Jiangyou Silica Gel Development Co., Ltd.) respectively. Use the lower layer solution of chloroform-methanol-water (13:6:2) as the developing solvent, develop, take out, dry in air, spray with 3% aluminum trichloride ethanol solution, heat at 105 °C for about 5 minutes, and examine under ultraviolet light (365 nm).

[0233] The results are as Figure 33As shown in the figure, the chromatogram of the test sample shows fluorescent spots of the same color at the corresponding positions as the chromatogram of the reference sample, and there is no interference from negative samples. This indicates that the preparation method of the test sample can effectively detect the target components in Citrus aurantium, and preliminarily proves the feasibility of the method.

[0234] 2. Conduct a systematic investigation

[0235] Take the reference medicinal material solution, reference standard solution, test solution, and negative sample solution, and perform thin-layer chromatography (according to Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). Investigate different developing solvent systems. The developing solvent systems are as follows:

[0236] Initial developing solvent system: chloroform-methanol-water (13:6:2) lower layer solution; developing solvent system A: chloroform-methanol-ammonia solution (3:3:1); developing solvent system B: ethyl acetate-formic acid-water (10:2:3).

[0237] The results are as follows Figure 34 As shown. The developing solvent systems A and B were not as effective as the initial developing solvent. Therefore, a chloroform-methanol-water (13:6:2) lower layer solution was chosen as the developing solvent. To reduce workload and improve the aesthetics of the thin-layer chromatograms, mixed standard solutions containing 1 mg / mL naringin and 1.5 mg / mL neohesperidin were prepared for subsequent experiments.

[0238] 3. Sample quantity investigation

[0239] The sample volumes for the test solution and the mixed reference solution were set to 1 μL, 3 μL, and 5 μL, respectively. The results are as follows: Figure 35 As shown in Figure A. Considering factors such as color development, separation effect, and sample volume, the spotting volume for both the test solution and the mixed reference solution was selected to be 1 μL.

[0240] 4. Specificity assessment

[0241] Take the test solution, negative sample solution, blank methanol solvent, and mixed reference solution, and perform the test according to the thin-layer chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). The results are as follows: Figure 35 As shown in Figure B, the chromatogram of the test sample shows a fluorescent spot of the same color at the corresponding position as the chromatogram of the reference sample, while the chromatograms of the methanol solvent and the negative chromatogram lacking Citrus aurantium do not show a fluorescent spot of the same color at that position. This indicates that the identification method can specifically identify the target component in Citrus aurantium, unaffected by other components in the preparation or by the methanol solvent, demonstrating good specificity.

[0242] 5. Durability test

[0243] 5.1 Thin-layer plate investigation

[0244] Using silica gel G plates already in use, the chromatography performance of thin-layer plates manufactured by Qingdao Ocean and Merck (Germany) on samples was investigated. The results are as follows: Figure 36 As shown, the thin-layer plate samples from all three manufacturers exhibited good separation, with no interference from negative samples.

[0245] 5.2 Temperature and Humidity Survey

[0246] The development of samples was investigated under different temperatures (4℃, room temperature) and relative humidity conditions (50%±5%, 90%±5%). The results are as follows: Figure 36 As shown, the chromatographic results of the samples met the requirements under different temperatures and relative humidity conditions, and there was no interference from negative samples.

[0247] 6. Thin-layer chromatography identification of samples

[0248] Following the methods determined in the above experiments, six batches of Baoxin Granules samples (batch numbers 210601, 221001, 240401, X231102, X231103, and X231104, respectively) were tested. The results are as follows: Figure 37 As shown in the figure, the chromatograms of the six batches of Baoxin Granules test samples all showed fluorescent spots of the same color at the corresponding positions as the reference chromatograms.

[0249] 7. Conclusion

[0250] The above experimental results show that the thin-layer chromatography method for detecting Citrus aurantium has good specificity and robustness, and can be effectively applied to the identification of Citrus aurantium in Baoxin granule preparations. The final quality standard detection method is as follows:

[0251] Take an appropriate amount of this product, grind it into a fine powder, take 2.5g, add 10mL of methanol, sonicate (power 1100W, frequency 40kHz) for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 5mL of methanol to obtain the test solution. Separately, take naringin and neohesperidin reference standards, add methanol to prepare a mixed solution containing 1mg of naringin and 1.5mg of neohesperidin per 1mL, as the reference solution. Perform thin-layer chromatography (Chinese Pharmacopoeia 2020 Edition, General Chapter 0502), apply 1μL of each of the above two solutions to the same high-performance silica gel G thin-layer plate, use the lower layer of chloroform-methanol-water (13:6:2) as the developing solvent, develop, remove, air dry, spray with 3% aluminum trichloride ethanol solution, heat at 105℃ (about 5 minutes), and examine under ultraviolet light (365nm). In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference standards.

[0252] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A thin-layer chromatographic identification method for core-protecting granules, characterized in that, The ingredients of the Baoxin Granules include Astragalus membranaceus, Salvia miltiorrhiza, Ophiopogon japonicus, Rehmannia glutinosa, Citrus aurantium, Rheum palmatum (processed with wine), Hirudo medicinalis (processed), Angelica sinensis, Poria cocos, Platycodon grandiflorus, Glycyrrhiza uralensis (processed), and Lycopus lucidus; the thin-layer chromatography identification method includes the steps of identifying Astragalus membranaceus using astragaloside A as a reference standard, identifying Rheum palmatum (processed with wine) using rhein and emodin as reference standards, identifying Hirudo medicinalis (processed) using Hirudo medicinalis as a reference standard, identifying Glycyrrhizin and Glycyrrhiza uralensis medicinalis as reference standards, identifying Rehmannia glutinosa using catalpol as a reference standard, identifying Salvia miltiorrhiza using salvianolic acid B and Salvia miltiorrhiza medicinalis as reference standards, and identifying Citrus aurantium using naringin and neohesperidin as reference standards; The steps for identifying rhubarb in wine are as follows: (1) Preparation of test solution: Take 7g of core-protecting granules, add 50mL of methanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 50mL of 8% hydrochloric acid solution, add 50mL of chloroform, heat under reflux for 2.5 hours, and collect the chloroform layer; wash the aqueous layer twice with chloroform, combine the chloroform washing solution and the chloroform layer, recover the solvent to dryness, dissolve the residue in 3mL of methanol to obtain the test solution; (2) Preparation of reference solution: Rhein and emodin were dissolved in methanol to obtain a reference solution containing 0.1 mg / mL rhein and 0.05 mg / mL emodin; (3) Thin-layer chromatography: Take 4 μL of the test solution and 2 μL of the reference solution and spot them on the same silica gel G thin-layer plate. Use the upper layer solution of petroleum ether-ethyl formate-formic acid with a volume ratio of 15:5:1 as the developing solvent. Develop, remove, air dry, and examine under ultraviolet light. The steps for identifying scalded leeches are as follows: (1) Preparation of test solution: Take 6g of Baoxin granules, add 25mL of ethanol, sonicate for 15 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 0.5mL of ethanol to obtain the test solution; (2) Preparation of reference solution: Take 0.25g of leech reference material, add 25mL of ethanol, sonicate for 15 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 0.5mL of ethanol to obtain the reference solution; (3) Thin-layer chromatography: Take 10 μL of the test solution and 1 μL of the reference medicinal material solution and spot them on the same silica gel G thin-layer plate. Use cyclohexane-ethyl acetate with a volume ratio of 4:1 as the developing solvent, develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly visible, and examine under sunlight. The steps for identifying processed licorice root are as follows: (1) Preparation of test solution: Take 2.5g of core-protecting granules, add 10mL of methanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 5mL of methanol to obtain the test solution; (2) Preparation of reference herb solution and reference solution: Take 1g of licorice reference herb, add 50mL of water, decoct for 30 minutes, filter, and evaporate the filtrate to dryness; add 10mL of methanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 5mL of methanol to obtain reference herb solution; dissolve glycyrrhizin in methanol to obtain reference solution with a concentration of 0.5mg / mL; (3) Thin-layer chromatography: Take 3 μL of the test solution, 1 μL of the reference medicinal material solution, and 1 μL of the reference solution and spot them on the same silica gel G thin-layer plate. Use ethyl acetate-formic acid-glacial acetic acid-water with a volume ratio of 15:1:1:2 as the developing solvent, develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly visible, and examine under ultraviolet light.

2. The thin-layer chromatography identification method according to claim 1, characterized in that, The steps for identifying Astragalus membranaceus are as follows: (1) Preparation of test solution: Take 2g of Baoxin granules, add 10mL of ammonia test solution, stir until completely wet, add 30mL of water and mix well, add 50mL of water-saturated n-butanol, heat under reflux for 2 hours, let stand for layering, take 25mL of the upper n-butanol solution, evaporate to dryness in a water bath, dissolve the residue in 5mL of methanol to obtain the test solution. (2) Preparation of reference solution: Astragaloside A was dissolved in methanol to obtain a reference solution with a concentration of 1 mg / mL; (3) Thin-layer chromatography: Take 5 μL of the test solution and 1 μL of the reference solution and spot them on the same silica gel G thin-layer plate. Use ethyl acetate-acetone-water with a volume ratio of 4:5:1 as the developing solvent. Develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly visible, and examine under ultraviolet light.

3. The thin-layer chromatography identification method according to claim 1, characterized in that, The steps for identifying raw Rehmannia glutinosa are as follows: (1) Preparation of test solution: Take 8g of core-protecting granules, add 50mL of methanol, sonicate for 45 minutes, filter, accurately measure 25mL of filtrate, recover the solvent under reduced pressure to dryness, dissolve in water, elute with water and methanol in turn, pass through macroporous resin, collect methanol eluent, concentrate to dryness, dissolve in 3mL of methanol to obtain test solution; (2) Preparation of reference solution: Dissolve catalpol in methanol to obtain a reference solution with a concentration of 0.5 mg / mL; (3) Thin-layer chromatography: Take 6 μL of the test solution and 2 μL of the reference solution and spot them on the same silica gel GF254 thin-layer plate. Use n-butanol-glacial acetic acid-water with a volume ratio of 10:2:1 as the developing solvent. Develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the color is clear, and examine under sunlight.

4. The thin-layer chromatography identification method according to claim 1, characterized in that, The steps for identifying Salvia miltiorrhiza are as follows: (1) Preparation of test solution: Take 2g of Baoxin granules, add 5mL of ethanol, sonicate for 15 minutes, filter, and the filtrate is the test solution; (2) Preparation of reference herb solution and reference solution: Take 1g of Salvia miltiorrhiza reference herb, add 30mL of water, decoct for 30 minutes, filter, and evaporate the filtrate to dryness; add 5mL of ethanol, sonicate for 15 minutes, filter, and obtain reference herb solution; dissolve saponin B in 60% methanol solution to obtain a reference solution with a concentration of 0.5mg / mL; (3) Thin-layer chromatography: Take 6 μL of the test solution, 6 μL of the reference medicinal material solution and 3 μL of the reference solution and spot them on the same silica gel GF254 thin-layer plate. Use dichloromethane-toluene-ethyl acetate-methanol-formic acid in a volume ratio of 3:2:7:0.5:2 as the developing solvent. Develop, remove, air dry and examine under ultraviolet light.

5. The thin-layer chromatography identification method according to claim 1, characterized in that, The steps for identifying Citrus aurantium are as follows: (1) Preparation of test solution: Take 2.5g of core-protecting granules, add 10mL of methanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 5mL of methanol to obtain the test solution; (2) Preparation of reference solution: Naringin and neohesperidin were dissolved in methanol to obtain reference solutions containing 1 mg / mL naringin and 1.5 mg / mL neohesperidin; (3) Thin-layer chromatography: Take 1 μL of the test solution and the reference solution and spot them on the same silica gel G thin-layer plate. Use a chloroform-methanol-water solution with a volume ratio of 13:6:2 as the developing solvent. Develop, remove, air dry, spray with 3% aluminum trichloride ethanol solution, heat at 105℃ for 5 minutes, and examine under ultraviolet light.

6. The thin-layer chromatography identification method according to claim 1 or any one of claims 3-5, characterized in that, The ultrasound has a power of 1100W and a frequency of 40kHz.

7. The application of the thin-layer chromatography identification method according to any one of claims 1-6 in the quality control and quality evaluation of core-protecting granules.

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