A thin-layer chromatography method for simultaneous identification of patchouli, notopterygium, atractylodes lancea and peppermint in eleven-ingredient Qinggan compound.

Abstract

This invention discloses a thin-layer chromatography method for simultaneously identifying patchouli, notopterygium, atractylodes, and peppermint in the eleven-ingredient Qinggan compound, comprising the following steps: (1) preparation of the test solution; (2) preparation of the reference herb solution; (3) preparation of the negative solution; and (4) thin-layer chromatography analysis. This invention uses only one thin-layer system to simultaneously identify patchouli, notopterygium, atractylodes, and peppermint in the eleven-ingredient Qinggan compound. The operation is simple and quick, greatly improving detection efficiency, reducing the amount of organic solvents and thin-layer plates used, making it environmentally friendly and facilitating efficient control of the product quality of the eleven-ingredient Qinggan compound.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine preparation component detection technology, specifically relating to a thin-layer chromatography method for simultaneously identifying patchouli, notopterygium, atractylodes macrocephala, and peppermint in the eleven-ingredient Qinggan compound. Background Technology

[0002] The Eleven-Ingredient Qinggan Compound prescription was developed by Xiamen Municipal Hospital of Traditional Chinese Medicine based on years of clinical experience. It is derived from four classic prescriptions: Ma Xing Shi Gan Tang (from *Shang Han Lun* by Zhang Zhongjing of the Han Dynasty), Ma Xing Yi Gan Tang (from *Jin Gui Yao Lue* by Zhang Zhongjing of the Han Dynasty), Wei Jing Tang (from *Wai Tai Mi Yao* by Wang Tao of the Tang Dynasty), and Bu Huan Jin Zheng Qi San (from *Taiping Huimin Heji Ju Fang* of the Song Dynasty), with modifications. The prescription consists of: Ephedra, Patchouli, Gypsum, Coix Seed, Reed Rhizome, Fried Atractylodes Rhizome, Notopterygium Root, Artemisia annua, Bitter Almond, Peppermint, and Licorice. Based on the traditional Chinese medicine principle that "the Bladder meridian of Foot Taiyang, the Lung meridian of Hand Taiyin, the Stomach meridian of Foot Yangming, and the Spleen meridian of Foot Taiyin are the body's protective barriers, and all external pathogenic diseases enter from the outside," the Eleven-Ingredient Qinggan Compound prescription uses Ephedra to open the Taiyang and Shaoyin meridians, and Pogostemon cablin to open the Yangming and Taiyin meridians, serving as the principal herbs to expel external pathogens. Atractylodes lancea and Notopterygium incisum disperse dampness from the skin surface, Artemisia annua clears heat, Gypsum clears stagnant heat, Coix lacryma-jobi promotes diuresis and clears heat, and Phragmites communis clears heat and generates fluids, addressing both the exterior and interior, and assisting the principal herbs in expelling pathogens, serving as the assistant herbs. Peppermint, pungent and cool, excels at dispelling wind and clearing the upper and middle jiao, as well as the head, face, and throat; Apricot kernel promotes lung function and stops cough; and Licorice harmonizes the other herbs. In summary, the Eleven-Ingredient Qinggan Compound prescription can relieve exterior symptoms, promote lung function, resolve dampness, and reduce fever. It is suitable for influenza belonging to the damp-heat epidemic type, with symptoms such as fever, chills or shivering, body aches, sore throat, headache, cough, and dry throat.

[0003] The quality of traditional Chinese medicine (TCM) is the fundamental guarantee of its clinical efficacy, and TCM quality standards are an important measure to ensure its quality. Thin-layer chromatography (TLC) identification is a crucial analytical method for controlling TCM quality. The "Technical Guidelines for Research on Quality Standards of New Traditional Chinese Medicines (Trial Implementation)," implemented on a trial basis in May 2020, encourages the establishment of TLC methods that can simultaneously identify multiple medicinal ingredients. A review of the TLC identification methods included in the compound preparations section of the 2020 edition of the Chinese Pharmacopoeia reveals that they are primarily optimized for each medicinal ingredient, with separate optimization of the TLC method, developing solvent, and development conditions. The quality standard for the Shiyiwei Qinggan Heji (Eleven-Ingredient Qinggan Compound) filed with the Fujian Provincial Drug Administration (Quality Standard No.: Min ZYZJBZ2023002) specifies a separate TLC identification method for Notopterygium incisum, but this method only controls the quality of Notopterygium incisum. Therefore, to improve the quality control level of Shiyiwei Qinggan Heji, finding a simple, rapid, and specifically targeted method for detecting multiple components in the aforementioned Shiyiwei Qinggan Compound is a critical challenge that must be overcome in its quality control. Summary of the Invention

[0004] The purpose of this invention is to overcome the defects of the prior art and provide a thin-layer chromatography method for simultaneously identifying patchouli, notopterygium, atractylodes lancea and peppermint in the eleven-ingredient Qinggan compound.

[0005] The technical solution of the present invention is as follows:

[0006] A thin-layer chromatography method for simultaneously identifying patchouli, notopterygium, atractylodes lancea, and peppermint in the eleven-ingredient Qinggan compound includes the following steps:

[0007] (1) Preparation of test solution: Take the sample of Eleven Flavors Qinggan Mixture, add petroleum ether with a boiling range of 60-90℃ and shake to extract. After evaporating the petroleum ether extract to dryness, dissolve the residue in ethyl acetate to obtain the test solution.

[0008] (2) Preparation of reference herbal solutions: Take patchouli, notopterygium, atractylodes lancea and peppermint, add petroleum ether with a boiling range of 60-90℃ and sonicate. After evaporating the petroleum ether extracts to dryness, dissolve the residues in ethyl acetate to obtain reference herbal solutions for patchouli, notopterygium, atractylodes lancea and peppermint.

[0009] (3) Preparation of negative solutions: Take negative samples that do not contain patchouli, notopterygium, atractylodes lancea and mint but contain the other ten flavors, add petroleum ether with a boiling range of 60-90℃ and shake to extract. After evaporating the petroleum ether extract to dryness, dissolve the residue in ethyl acetate to obtain negative solutions for patchouli, notopterygium, atractylodes lancea and mint, respectively.

[0010] (4) Thin-layer chromatography analysis: Take the above test solution, reference medicinal material solution, patchouli negative solution, notopterygium negative solution, atractylodes negative solution and peppermint negative solution, spot them on the same thin-layer plate, develop with the developing solvent, take them out, dry them, spray with the color developing agent, heat until the spots are clearly visible, place them under the inspection light source for inspection, and identify patchouli, notopterygium, atractylodes and peppermint;

[0011] The above developing solvent is composed of petroleum ether, ethyl acetate and acetic acid with a boiling range of 60-90℃ in a volume ratio of 4-4.5:1-1.5:0.2-0.5;

[0012] The aforementioned thin-layer plate is a silicone G thin-layer plate;

[0013] The colorimetric reagent mentioned above is a 10% sulfuric acid ethanol solution;

[0014] The light source for identifying Notopterygium incisum and Mentha haplocalyx was a fluorescent lamp, the light source for identifying Pogostemon cablin was a 254nm ultraviolet lamp, and the light source for identifying Atractylodes lancea was a 365nm ultraviolet lamp.

[0015] In a preferred embodiment of the present invention, the preparation method of the eleven-ingredient Qinggan compound sample includes: taking 7-8g of ephedra, 12-13g of patchouli, 20-21g of gypsum, 24-25g of coix seed, 24-25g of reed rhizome, 10-11g of stir-fried Atractylodes lancea, 7-8g of Notopterygium incisum, 8-9g of Artemisia annua, 8-9g of bitter almond, 4-5g of peppermint, and 5-6g of licorice root slices, a total of eleven medicinal slices, adding water to decoct while collecting a certain amount of aromatic water for later use, filtering, combining the filtrates, concentrating to an appropriate amount, adding aromatic water, adding water to 100mL, stirring well, filtering, filling and sealing, sterilizing, and thus obtaining the product.

[0016] More preferably, the preparation of the test solution includes: taking 20 mL of the Eleven Flavors Qinggan Mixture sample, adding 20 mL of petroleum ether with a boiling range of 60-90℃ and shaking to extract twice, each time, combining the obtained petroleum ether extracts and evaporating to dryness, dissolving the residue in 1 mL of ethyl acetate to obtain the test solution.

[0017] More preferably, the preparation of the negative sample includes: taking ten medicinal slices that do not contain patchouli, notopterygium, atractylodes lancea and mint, decocting them with water and collecting a certain amount of aromatic water for later use, filtering, combining the filtrates, concentrating to an appropriate amount, adding aromatic water, adding water to 100mL, stirring well, filtering, filling and sealing, and sterilizing to obtain a negative sample that does not contain patchouli, notopterygium, atractylodes lancea and mint but contains the other ten ingredients.

[0018] More preferably, 20 mL of the negative sample is taken and extracted twice with petroleum ether at a boiling range of 60-90℃, 20 mL each time. The petroleum ether extracts are combined and evaporated to dryness. The residue is dissolved in 1 mL of ethyl acetate to obtain negative solutions for patchouli, notopterygium, atractylodes, and peppermint, respectively.

[0019] In a preferred embodiment of the present invention, the preparation of the reference herb solution of patchouli includes: taking 1g of patchouli reference herb, adding 20mL of petroleum ether with a boiling range of 60-90℃ and ultrasonically treating for 25-35min, evaporating the obtained petroleum ether extract to dryness, and dissolving the residue in 0.5mL of ethyl acetate to obtain the reference herb solution.

[0020] In a preferred embodiment of the present invention, the preparation of the reference herb solution of Notopterygium incisum, Atractylodes lancea and Mentha haplocalyx includes: taking 0.5g of Notopterygium incisum, Atractylodes lancea and Mentha haplocalyx reference herbs respectively, adding 20mL of petroleum ether with a boiling range of 60-90℃ and ultrasonically treating for 25-35min, evaporating the obtained petroleum ether extract to dryness, and dissolving the residue in 0.5mL of ethyl acetate to obtain the reference herb solution.

[0021] In a preferred embodiment of the present invention, the sample volume of the test solution, the control medicinal material solution and the negative solution is 4-8 μL.

[0022] More preferably, the sample volume of the test solution, the control medicinal material solution, and the negative solution is 5 μL.

[0023] In a preferred embodiment of the present invention, the unfolding method is as follows: a double-groove unfolding cylinder is used for unfolding, unfolding agent is slowly added to both sides, filter paper is placed on one side and a dotted thin plate is placed on the other side, and then the cover is put on for unfolding, with an unfolding distance of 7-9cm.

[0024] The beneficial effects of this invention are:

[0025] 1. The thin-layer chromatography method provided by this invention for simultaneously identifying patchouli, notopterygium, atractylodes lancea, and peppermint in the Eleven-Flavor Qinggan Mixture exhibits good sample spot separation and no negative interference. It can simultaneously and exclusively identify four of these herbs. Furthermore, the robustness of the thin-layer chromatography method of this invention was examined through examples, revealing that the detection process of the thin-layer chromatography identification method is not easily affected by changes in external conditions such as the thin-layer plate, temperature, and humidity. This demonstrates that the method is not demanding in terms of external conditions, has good robustness, and is widely applicable.

[0026] 2. This invention uses only a thin-layer system to simultaneously identify patchouli, notopterygium, atractylodes lancea, and peppermint in the Eleven-Flavor Qinggan Mixture. The operation is simple and quick, greatly improving the detection efficiency, reducing the amount of organic solvents and thin-layer plates used, making it green and environmentally friendly, and facilitating efficient control of the quality of the Eleven-Flavor Qinggan Mixture product. Attached Figure Description

[0027] Figures 1 to 16 The top image shows a thin-layer chromatogram under sunlight, the middle image shows a thin-layer chromatogram under 254nm ultraviolet light, and the bottom image shows a thin-layer chromatogram under 365nm ultraviolet light.

[0028] Figure 1 This is a thin-layer chromatogram of three batches of the test solution of the Eleven-Flavor Qinggan Compound prepared in Example 2, as shown in Example 5 of the present invention; wherein, 1: Patchouli reference material, 2: Notopterygium incisum reference material, 3: Atractylodes lancea reference material solution, 4: Mentha haplocalyx reference material, 5: Test solution (QG230601), 6: Test solution (QG230602), 7: Test solution (QG230603).

[0029] Figure 2 This is a thin-layer chromatogram of the test solution preparation method of the Eleven-Flavor Qinggan Compound in Experiment Example 1 of the present invention. In the chromatogram, 1: Patchouli reference material, 2: Patchouli negative solution, 3: Notopterygium reference material, 4: Notopterygium negative solution, 5: Atractylodes lancea reference material solution, 6: Atractylodes lancea negative solution, 7: Peppermint reference material, 8: Peppermint negative solution, 9: Test solution of Example 2 (QG230601), 10: Test solution of Comparative Example 1.

[0030] Figure 3 This is a thin-layer chromatogram for the sample quantity investigation in Experiment Example 2 of the present invention, wherein 1: patchouli reference material, 2: patchouli negative solution, 3: notopterygium reference material, 4: notopterygium negative solution, 5: atractylodes lancea reference material solution, 6: atractylodes lancea negative solution, 7: peppermint reference material, 8: peppermint negative solution, 9-13: test solution (QG230601) 2μL, 4μL, 6μL, 8μL, 10μL.

[0031] Figures 4 to 6 The following are thin-layer chromatograms of developing systems 1, 2, and 3 of Experimental Example 3 of the present invention, wherein 1: patchouli reference material, 2: patchouli negative solution, 3: notopterygium reference material, 4: notopterygium negative solution, 5: atractylodes lancea reference material solution, 6: atractylodes lancea negative solution, 7: peppermint reference material, 8: peppermint negative solution, and 9: test sample (QG230601) solution;

[0032] Figures 7 to 9 The following are thin-layer chromatograms of the Qingdao Ocean G plate, Yantai Yinlong HSG plate, and self-made G plate used in Experimental Example 4 of this invention, respectively. Among them, 1: Patchouli reference material, 2: Patchouli negative solution, 3: Notopterygium reference material, 4: Notopterygium negative solution, 5: Atractylodes lancea reference material solution, 6: Atractylodes lancea negative solution, 7: Peppermint reference material, 8: Peppermint negative solution, 9: Test solution (QG230601).

[0033] Figures 10 to 12 The following are thin-layer chromatograms of Experimental Example 5 of the present invention at low temperature 4℃, normal temperature 24℃, and high temperature 40℃, respectively; wherein, 1: patchouli reference material, 2: patchouli negative solution, 3: notopterygium reference material, 4: notopterygium negative solution, 5: atractylodes lancea reference material solution, 6: atractylodes lancea negative solution, 7: peppermint reference material, 8: peppermint negative solution, 9: test solution (QG230601).

[0034] Figures 13 to 15 The following are thin-layer chromatograms of Experimental Example 6 of the present invention at relative humidity of 29%, 60%, and 75%, respectively, wherein 1: patchouli reference material, 2: patchouli negative solution, 3: notopterygium reference material, 4: notopterygium negative solution, 5: atractylodes lancea reference material solution, 6: atractylodes lancea negative solution, 7: peppermint reference material, 8: peppermint negative solution, and 9: test solution (QG230601).

[0035] Figure 16This is a thin-layer chromatogram for the specificity investigation of Experiment Example 7 of the present invention, wherein 1: patchouli reference material, 2: patchouli negative solution, 3: notopterygium reference material, 4: notopterygium negative solution, 5: atractylodes lancea reference material solution, 6: atractylodes lancea negative solution, 7: peppermint reference material, 8: peppermint negative solution, 9: test solution (QG230601). Detailed Implementation

[0036] The technical solution of the present invention will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0037] Unless otherwise specified, the reagents used in the following examples and comparative examples are conventional reagents, all of which can be purchased from conventional reagent manufacturers and distributors. Information on some material and instrument manufacturers is as follows:

[0038] The instruments and reagents used in this invention are as follows:

[0039] 1. Instruments: CAMAG semi-automatic thin-layer sampling and imaging system; thin-layer heating plate; analytical balance, model XS205D; ultrasonic cleaner, model KQ-500VDB, power 100W, frequency 45KHZ, Kunshan Ultrasonic Instrument Co., Ltd.; low-temperature high-speed centrifuge, model 5810R, Eppendorf; electric heating mantle, model TC-15, Haining Xinhua Medical Instrument Factory; stability test chamber, model Labonce-250SD, Lanbeishi (Beijing) Technology Co., Ltd.

[0040] 2. Thin-layer plates: See the table below for details.

[0041] Silica gel G Qingdao Marine Chemical Factory 20230513 Silica gel HSG Yinlong / Yantai Municipal Chemical Research Institute 20231008 Silica gel G Home-made 20231106

[0042] 3. Reagents: Ethyl acetate, analytical grade, batch number 2306031; petroleum ether (60-90℃), analytical grade, batch number 2305041; n-hexane, analytical grade, batch number 20211028; cyclohexane, analytical grade, batch number 2208222; dichloromethane, analytical grade, batch number 20220511; formic acid, analytical grade, batch number 20201110; acetic acid, analytical grade, batch number 20211130; methanol, analytical grade, batch number 2305261; all purchased from Xiamen Luyin Reagent & Glassware Co., Ltd.

[0043] Reference herbs: Patchouli, batch number 121135-202107, Notopterygium incisum, batch number 120935-201809, Atractylodes lancea, batch number 120983-202106, and Mentha haplocalyx, batch number 120916-202113, purchased from the China National Institutes for Food and Drug Control.

[0044] Chinese medicinal herbs: Ephedra, Patchouli, Gypsum, Coix Seed, Reed Rhizome, Stir-fried Atractylodes Rhizome, Notopterygium Root, Artemisia annua, Stir-fried Bitter Almond, Peppermint, and Licorice Root slices, totaling 11 herbs, all produced in their own production areas or authentic production areas. Upon identification, the following were determined: Ephedra is the dried herbaceous stem of *Ephedra sinica* Stapf. (Ephedrine family); Patchouli is the dried aerial part of *Pogostemon cablin* (Blanco) Benth. (Lamiaceae family); Gypsum is a sulfate mineral, mainly containing hydrated calcium sulfate (CaSO4·2H2O), after which impurities and silt are removed; Coix seed is the dried mature seed kernel of *Coix lacryma-jobi* L. var. *ma-yuen* (Roman.) Stapf. (Poaceae family); Reed rhizome is the dried rhizome of *Phragmites communis* Trin. (Poaceae family); Fried Atractylodes chinensis is a processed product of the dried rhizome of *Atractylodes chinensis* (DC.) Koidz. (Asteraceae family); and Notopterygium incisum is a plant of the Apiaceae family. HTChang's dried rhizome and root; Artemisia annua L.'s dried aerial parts (Asteraceae); Bitter apricot kernel's dried mature seeds (Rosaceae); Mentha haplocalyx Briq.'s dried aerial parts (Lamiaceae); Glycyrrhiza uralensis Fisch.'s dried root and rhizome (Fabaceae). All the above processed medicinal materials comply with the provisions of Part I of the 2020 edition of the Pharmacopoeia of the People's Republic of China.

[0045] Example 1: Preparation of 3 batches of pilot-scale samples of the Eleven-Flavor Qinggan Mixture

[0046] The preparation methods for three batches of pilot-scale samples of the Eleven-Ingredient Qinggan Mixture are as follows (calculated based on 100mL of finished product):

[0047] Take 7.50g of ephedra, 12.50g of patchouli, 20.83g of gypsum, 25.00g of coix seed, 25.00g of reed rhizome, 10.00g of stir-fried Atractylodes lancea, 7.50g of Notopterygium incisum, 8.33g of Artemisia annua, 8.33g of roasted bitter almond, 4.17g of peppermint, and 5.00g of licorice root. Add water and decoct (collect a certain amount of aromatic water at the same time for later use). Filter, combine the filtrates, concentrate to an appropriate amount, add aromatic water, add water to 100mL, stir well, filter, fill and seal, sterilize, and the eleven-ingredient Qinggan compound sample is obtained.

[0048] Three batches of pilot-scale samples of the eleven-ingredient Qinggan compound were prepared: batch numbers QG230601, QG230602, and QG230603, respectively.

[0049] Example 2: Preparation of the test solution

[0050] The preparation method of the test solution is as follows: Take 20 mL of different batches of the Eleven Flavors Qinggan Mixture sample obtained in Example 1, add petroleum ether (60-90℃) and shake to extract twice, 20 mL each time, combine the petroleum ether extracts, evaporate to dryness, dissolve the residue in 1 mL of ethyl acetate to obtain 3 batches of test solution.

[0051] Example 3: Preparation of Control Herbal Solution

[0052] Take 1g of patchouli reference material, add 20mL of petroleum ether (60-90℃), sonicate for 30min, filter, evaporate the filtrate to dryness, and dissolve the residue in 0.5mL of ethyl acetate to prepare the patchouli reference material solution; then take 0.5g each of notopterygium root reference material, atractylodes rhizome reference material, and peppermint reference material, add 20mL of petroleum ether (60-90℃) to each, sonicate for 30min, filter, evaporate the filtrate to dryness, and dissolve the residue in 0.5mL of ethyl acetate to prepare the notopterygium root reference material, atractylodes rhizome reference material, and peppermint reference material solutions respectively.

[0053] Example 4: Preparation of Negative Solution

[0054] Take the medicinal ingredients of the eleven-ingredient Qinggan compound, which do not include patchouli, notopterygium, atractylodes lancea, and peppermint. Prepare negative solutions of patchouli, notopterygium, atractylodes lancea, and peppermint according to the thin-layer chromatography method of Example 1.

[0055] Example 5 Thin-layer chromatography detection

[0056] The thin-layer chromatography method (General Chapter 0502 of the 2020 edition of the Chinese Pharmacopoeia) was used. 5 μL of each of the three batches (QG230601, QG230602, QG230603) of test solution prepared in Example 2 and the reference medicinal material solution prepared in Example 3 were spotted onto the same silica gel G thin-layer plate. Petroleum ether (60-90℃)-ethyl acetate-acetic acid (4∶1∶0.2) was used as the developing solvent. The development method was as follows: a double-groove developing tank was used, with the developing solvent slowly added to both sides. Filter paper was placed on one side and the spotted thin-layer plate was placed on the other side. The plate was covered and developed, with a development distance of 8 cm. Remove the sample, air dry it, spray it with 10% sulfuric acid ethanol solution, and heat it at 105℃ until the spots are clearly visible. When examined under sunlight, the chromatogram of the test sample shows the same brownish-black spots at the corresponding positions as the chromatogram of Notopterygium incisum reference material, and the same purplish-red spots at the corresponding positions as the chromatogram of Mentha haplocalyx reference material. When examined under ultraviolet light (254nm), the chromatogram of the test sample shows the same yellow fluorescent spots at the corresponding positions as the chromatogram of Pogostemon cablin reference material. When examined under ultraviolet light (365nm), the chromatogram of the test sample shows the same blue fluorescent spots at the corresponding positions as the chromatogram of Atractylodes lancea reference material.

[0057] Test results are shown Figure 1 The results showed that the test sample chromatogram showed spots of the same color at the corresponding positions as the reference herb chromatogram. This indicates that the method can specifically identify patchouli, notopterygium, atractylodes, and peppermint in the Eleven-Ingredient Qinggan Compound sample.

[0058] Preparation of test solution in Comparative Example 1

[0059] Take 20 mL of the eleven-ingredient Qinggan compound sample (QG220601) prepared in Example 1, add 80 mL of methanol, sonicate for 30 min, filter, evaporate the filtrate to dryness, dissolve the residue in 20 mL of water, extract twice with 20 mL of petroleum ether (60-90℃), combine the petroleum ether extracts, evaporate to dryness, dissolve the residue in 1 mL of ethyl acetate, and use as the test solution.

[0060] Experimental Example 1: Investigation of Thin-Layer Chromatography of Test Sample Solution

[0061] Take 5 μL each of the control herbal solution from Example 3, the negative solution from Example 4, the test solution from Example 2 (QG220601), and the test solution from Comparative Example 1, and spot them separately on the same silica gel G thin-layer plate for thin-layer chromatography detection. Use petroleum ether (60-90℃)-ethyl acetate-acetic acid (4∶1∶0.2) 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 a fluorescent lamp to identify Notopterygium incisum and Mentha haplocalyx; examine under an ultraviolet lamp (254nm) to identify Pogostemon cablin; examine under an ultraviolet lamp (365nm) to identify Atractylodes lancea.

[0062] Test results are shown Figure 2 The results showed that although spots of the same color appeared at the corresponding positions in the chromatograms of Example 2 and Comparative Example 1, the preparation of the test solution obtained in Comparative Example 1 involved more steps and took longer.

[0063] Experimental Example 2: Investigation of Sample Size

[0064] Take 2 μL, 4 μL, 6 μL, 8 μL, and 10 μL of the control herbal solution from Example 3, the negative solution from Example 4, and the test solution (QG220601) from Example 2, respectively, and spot them onto the same silica gel G thin-layer plate. Develop the plate using petroleum ether (60-90℃)-ethyl acetate-acetic acid (4:1:0.2) as the developing solvent. Remove the plate, air dry, spray with 10% sulfuric acid ethanol solution, and heat at 105℃ until the spots are clearly visible. Examine under a fluorescent lamp to identify Notopterygium incisum and Mentha haplocalyx; examine under an ultraviolet lamp (254 nm) to identify Pogostemon cablin; and examine under an ultraviolet lamp (365 nm) to identify Atractylodes lancea. Results are shown below. Figure 3 .

[0065] The results showed that when the sample volume of the test solution was 4-8 μL, the spots in the chromatogram of the test sample at the corresponding positions as those in the chromatogram of the reference medicinal material were clearer and more appropriate in color. Therefore, the sample volume of the test solution was selected as 4-8 μL.

[0066] Experimental Example 3: Systematic Investigation

[0067] The effects of three different developing systems on the thin-layer chromatography identification of patchouli, notopterygium root, atractylodes rhizome, and peppermint were investigated. 5 μL each of the control solution from Example 3, the negative solution from Example 4, and the test solution (QG220601) from Example 2 were spotted onto the same silica gel G thin-layer plate. The plates were developed using the three different developing systems, removed, dried, sprayed with 10% sulfuric acid ethanol solution, and heated at 105°C until the spots were clearly visible. The plates were then examined under daylight to identify notopterygium root and peppermint, under ultraviolet light (254 nm) to identify patchouli, and under ultraviolet light (365 nm) to identify atractylodes rhizome.

[0068] The three deployment systems are as follows:

[0069] Developing solvent 1: Petroleum ether (60-90℃) - ethyl acetate - acetic acid (4:1:0.2)

[0070] Developing solvent 2: n-hexane-ethyl acetate-acetic acid (4:1:0.2)

[0071] Developing solvent 3: Cyclohexane-dichloromethane-ethyl acetate-formic acid (5:1:2:0.2)

[0072] See resultsFigures 4 to 6 The results showed that when the developing system was petroleum ether (60-90℃)-ethyl acetate-acetic acid (4∶1∶0.2), the spots of the test sample were clearer, the specific shift value was moderate, and there was no interference from negative samples. This indicates that the developing system used in this invention can better identify the specificity of patchouli, notopterygium, atractylodes, and peppermint in this product.

[0073] Experimental Example 4: Investigation of Thin-Layer Plates

[0074] The Qingdao Ocean G thin-layer plate, the Yantai Yinlong HSG thin-layer plate, and the laboratory-made G thin-layer plate were investigated respectively.

[0075] Take 5 μL each of the control herbal solution from Example 3, the negative solution from Example 4, and the test solution (QG220601) from Example 2, and spot them on Qingdao Marine G thin-layer plates, Yantai Yinlong HSG thin-layer plates, and laboratory-made G thin-layer plates, respectively. Use petroleum ether (60-90℃)-ethyl acetate-acetic acid (4∶1∶0.2) as the developing solvent, develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly visible, examine under a fluorescent lamp to identify Notopterygium incisum and Mentha haplocalyx; examine under an ultraviolet lamp (254nm) to identify Pogostemon cablin; examine under an ultraviolet lamp (365nm) to identify Atractylodes lancea.

[0076] See results Figures 7 to 9 All three thin-layer plates were able to specifically identify patchouli, notopterygium root, atractylodes rhizome, and peppermint in this product, indicating that the invention has good durability and wide applicability. Subsequent research of this invention will use the Qingdao Marine G thin-layer plate.

[0077] Investigation of temperature in Experiment Example 5

[0078] Take 5 μL each of the control herbal solution from Example 3, the negative solution from Example 4, and the test solution (QG220601) from Example 2, and spot them separately on the same silica gel G thin-layer plate. Use petroleum ether (60-90℃)-ethyl acetate-acetic acid (4∶1∶0.2) as the developing solvent, and develop them at different temperature environments (low temperature 4℃, room temperature 24℃, high temperature 40℃). After development, remove the plate, air dry it, spray it with 10% sulfuric acid ethanol solution, and heat it at 105℃ until the spots are clearly visible. Examine it under a fluorescent lamp to identify Notopterygium incisum and Mentha haplocalyx; examine it under an ultraviolet lamp (254nm) to identify Pogostemon cablin; examine it under an ultraviolet lamp (365nm) to identify Atractylodes lancea.

[0079] See results Figures 10 to 12 The spot visualization results of the chromatogram of the test sample showed little difference at different temperatures. This indicates that the thin-layer chromatography identification method provided by this invention is less affected by temperature changes and has good robustness.

[0080] Experiment 6: Investigation of Relative Humidity

[0081] Take 5 μL each of the control herbal solution from Example 3, the negative solution from Example 4, and the test solution (QG220601) from Example 2, and spot them separately on the same silica gel G thin-layer plate. Use petroleum ether (60-90℃)-ethyl acetate-acetic acid (4∶1∶0.2) as the developing solvent, and develop them under different relative humidity environments (29%, 60%, and 75%). Remove the plates, air dry them, spray them with 10% sulfuric acid ethanol solution, and heat them at 105℃ until the spots are clearly visible. Examine them under a fluorescent lamp to identify Notopterygium incisum and Mentha haplocalyx; examine them under an ultraviolet lamp (254nm) to identify Pogostemon cablin; and examine them under an ultraviolet lamp (365nm) to identify Atractylodes lancea.

[0082] See results Figures 13 to 15 The spot visualization results of the test sample chromatograms showed little difference under different relative humidity levels. This indicates that the thin-layer chromatography identification method provided by this invention is less affected by humidity changes and has good durability.

[0083] Specificity investigation in Experiment Example 7

[0084] Take 5 μL each of the control herbal solution from Example 3, the negative solution from Example 4, and the test solution (QG220601) from Example 2, and spot them separately on the same silica gel G thin-layer plate. Develop the plate using petroleum ether (60-90℃)-ethyl acetate-acetic acid (4∶1∶0.2) as the developing solvent. Remove the plate, air dry it, spray it with 10% sulfuric acid ethanol solution, and heat it at 105℃ until the spots are clearly visible. Examine the plate under a fluorescent lamp to identify Notopterygium incisum and Mentha haplocalyx; examine the plate under an ultraviolet lamp (254nm) to identify Pogostemon cablin; and examine the plate under an ultraviolet lamp (365nm) to identify Atractylodes lancea.

[0085] See results Figure 16 In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference medicinal material. This indicates that the thin-layer chromatography detection method provided by this invention has good robustness and can specifically identify patchouli, notopterygium, atractylodes, and peppermint in the Eleven-Ingredient Qinggan Compound sample.

[0086] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A thin-layer chromatography method for simultaneously identifying patchouli, notopterygium, atractylodes lancea, and peppermint in an eleven-ingredient Qinggan compound, characterized in that: The preparation method of the eleven-ingredient Qinggan compound sample includes: taking 7-8 g of ephedra, 12-13 g of patchouli, 20-21 g of gypsum, 24-25 g of coix seed, 24-25 g of reed rhizome, 10-11 g of stir-fried Atractylodes lancea, 7-8 g of Notopterygium incisum, 8-9 g of Artemisia annua, 8-9 g of bitter almond, 4-5 g of peppermint, and 5-6 g of licorice root slices, a total of eleven medicinal slices, adding water to decoct while collecting a certain amount of aromatic water for later use, filtering, combining the filtrates, concentrating to an appropriate amount, adding aromatic water, adding water to 100 mL, stirring well, filtering, filling and sealing, sterilizing, and the product is obtained; This thin-layer chromatography method includes the following steps: (1) Preparation of test solution: Take 20 mL of the eleven-flavor Qinggan compound sample, add petroleum ether with a boiling range of 60-90 ℃ and shake to extract twice, 20 mL each time. Combine the petroleum ether extracts and evaporate to dryness. Dissolve the residue in 1 mL of ethyl acetate to obtain the test solution. (2) Preparation of reference medicinal material solutions: Take patchouli, notopterygium, atractylodes lancea and peppermint, respectively add petroleum ether with a boiling range of 60-90 ℃ and sonicate. After evaporating the petroleum ether extracts to dryness, the residues are dissolved in ethyl acetate and used as reference medicinal material solutions for patchouli, notopterygium, atractylodes lancea and peppermint respectively. (3) Preparation of negative solutions: Take the ten medicinal slices that do not contain patchouli, notopterygium, atractylodes lancea and mint respectively, decoct them with water and collect a certain amount of aromatic water for later use, filter, combine the filtrates, concentrate to an appropriate amount, add aromatic water, add water to 100 mL, stir well, filter, fill and sterilize to obtain negative samples that do not contain patchouli, notopterygium, atractylodes lancea and mint but contain the other ten ingredients; take 20 mL of the negative sample, add petroleum ether with a boiling range of 60-90 ℃ and shake to extract twice, 20 mL each time, combine the petroleum ether extracts and evaporate to dryness, dissolve the residue with 1 mL of ethyl acetate, and use them as negative solutions for patchouli, notopterygium, atractylodes lancea and mint respectively. (4) Thin-layer chromatography analysis: Take the above test solution, reference medicinal material solution, patchouli negative solution, notopterygium negative solution, atractylodes negative solution and peppermint negative solution, spot them on the same thin-layer plate, develop with the developing solvent, take them out, dry them, spray with the color reagent, heat until the spots are clearly visible, place them under the inspection light source for inspection, and identify patchouli, notopterygium, atractylodes and peppermint; the spotting volume of the test solution, reference medicinal material solution and negative solution is 5 μL; the development method is: use a double-groove developing tank, slowly add the developing solvent on both sides, put filter paper on one side and the spotted thin-layer plate on the other side, cover and develop, the development distance is 7-9 cm; The preparation of the reference herb solution for patchouli includes: taking 1 g of patchouli reference herb, adding 20 mL of petroleum ether with a boiling range of 60-90 ℃ and ultrasonically treating for 25-35 min, evaporating the obtained petroleum ether extract to dryness, and dissolving the residue in 0.5 mL of ethyl acetate to obtain the reference herb solution; the preparation of the reference herb solutions for notopterygium root, atractylodes rhizome, and peppermint includes: taking 0.5 g of notopterygium root, atractylodes rhizome, and peppermint reference herb respectively, adding 20 mL of petroleum ether with a boiling range of 60-90 ℃ and ultrasonically treating for 25-35 min, evaporating the obtained petroleum ether extract to dryness, and dissolving the residue in 0.5 mL of ethyl acetate to obtain the reference herb solution; The above developing solvent is composed of petroleum ether, ethyl acetate and acetic acid with a boiling range of 60-90 °C in a volume ratio of 4-4.5: 1-1.5: 0.2-0.5; The aforementioned thin-layer plate is a silicone G thin-layer plate; The colorimetric reagent mentioned above is a 10% sulfuric acid ethanol solution; The light source for identifying Notopterygium incisum and Mentha haplocalyx was a fluorescent lamp, the light source for identifying Pogostemon cablin was a 254 nm ultraviolet lamp, and the light source for identifying Atractylodes lancea was a 365 nm ultraviolet lamp.

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