Quality control method of heart-fire-clearing lotus seed beverage compound preparation

Abstract

The invention relates to the technical field of traditional Chinese medicine detection, in particular to a quality control method of a heart fire clearing lotus seed beverage compound preparation, which comprises the following steps: respectively preparing test solution A-E; based on the test solution A, identifying the scutellaria baicalensis by adopting a thin-layer chromatography; identifying cortex lycii radicis and liquorice based on the test solution B, identifying ginseng based on the test solution C, identifying semen plantaginis based on the test solution D, and identifying lotus seeds based on the test solution E. According to the method, repeated extraction of the reference sample during identification of different medicine flavors can be reduced, and the method has the advantages of clear spots, good separation degree, strong specificity, no background interference and good durability.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine detection, and in particular to a quality control method for a Qingxin Lianziyin compound preparation. Background Art

[0002] Qingxin Lianzi Drink comes from "Taiping Huimin Hejijufang". Its prescription composition is half a liang of Scutellaria baicalensis, Ophiopogon japonicus (without the core), Rehmannia glutinosa, Plantago seed, and licorice (roasted), seven and a half qian of Echeveria seed (without the core), White Poria cocos, Astragalus membranaceus (roasted with honey), and Ginseng. The medicinal property is mild, neither cold nor hot. Regular consumption can clear the heart and nourish the spirit, retain essence and replenish deficiency, nourish the intestines and stomach, and regulate blood and qi. It is an ancient famous prescription for clearing the heart and nourishing yin. It can clear heart fire, connect the heart and kidney, replenish qi and yin, and stop stranguria. It is used to treat white turbid urine, spermatorrhea, bloody stools, five types of stranguria, irritability and thirst.

[0003] In the Qingxin Lianzi Drink recipe, Stone Lotus Seed Meat nourishes the spleen and yin, retains essence and vital energy, calms the mind and nourishes the heart, and is the main medicine; Scutellaria baicalensis clears the heart and moistens the lungs, purges fire and nourishes yin; Rehmannia root bark cools the blood and eliminates steaming, clears the lungs and reduces fire; Plantago seed and White Poria cocos penetrate and eliminate dampness, flow turbid air without stagnation, and relieve heart heat through urination; Ophiopogon japonicus nourishes yin and clears the heart; Ginseng, Astragalus membranaceus, and Roasted Licorice root replenish qi and raise yang, benefit qi and produce body fluid, and restrain floating yang. They are agents for replenishing qi and yin and clearing dampness and heat, and are an effective prescription for the clinical treatment of the three key pathogenesis of "insufficient qi and yin, upward inflammation of heart fire, and downward disturbance of dampness and heat due to kidney deficiency".

[0004] Since agapanthus fragrans is the main ingredient in the Qingxin Lianziyin prescription, and the agapanthus fragrans is used without the core, and the identification of lotus seeds in the 2020 edition of the Chinese Pharmacopoeia uses nelumbosine in the lotus seed core as the identification standard, controlling lotus seeds can more comprehensively evaluate the overall quality of the Qingxin Lianziyin prescription. There are few thin-layer chromatography identification methods related to Qingxin Lianziyin in the existing technology, but their specificity is not strong, and there is a lot of interference in the corresponding positions in actual thin-layer chromatography identification, making it difficult to reproduce. Summary of the Invention

[0005] Therefore, the object of the present invention is to provide a quality control method for a Qingxin Lianziyin compound preparation, comprising preparing a test solution and identifying the corresponding drugs using thin-layer chromatography. This method can reduce the repeated extraction of reference samples when identifying different medicinal flavors, and has clear spots, good separation, strong specificity, no background interference, and good durability.

[0006] To this end, the present invention provides the following technical solutions:

[0007] The present invention provides a quality control method for a Qingxin Lianziyin compound preparation. The Qingxin Lianziyin compound preparation is prepared from raw materials including scutellaria baicalensis, lycium bark, ginseng, licorice, plantain seeds, and lotus seeds. The quality control method comprises the following steps: taking a test sample and preparing a test solution according to at least one of the following methods 1 to 5, and performing a test: method 1: preparing a test solution A, and based on the test solution A, using thin layer chromatography to identify scutellaria baicalensis; method 2: preparing a test solution B, and based on the test solution B, using thin layer chromatography to identify The preparation process of the test solution B includes: taking the test sample of the Qingxin Lianziyin compound preparation, adding an alcohol aqueous solution, ultrasonic extraction, solid-liquid separation, taking the liquid, evaporating to dryness, dissolving the obtained residue in water, extracting with petroleum ether, taking the water layer, extracting with ethyl acetate, taking the ethyl acetate layer, evaporating to dryness, and re-dissolving the obtained residue in alcohol; Method 3: preparing the test solution C, and using thin layer chromatography to identify ginseng based on the test solution C; the preparation process of the test solution C includes: taking the ethyl acetate extracted in the preparation process of the test sample B The aqueous layer was extracted with water-saturated n-butanol, the n-butanol layer extract was washed with ammonia test solution, the n-butanol layer was washed with n-butanol saturated water, the n-butanol layer was evaporated to dryness, and the obtained residue was re-dissolved with alcohol; Method 4: Prepare a test solution D, and identify Plantago seed based on the test solution D by thin layer chromatography; the preparation process of the obtained test solution D includes: taking the n-butanol layer extract obtained in the preparation process of the test sample C, evaporating to dryness, re-dissolving the obtained residue with water, performing column chromatography on a silica gel-based solid phase extraction column, and using a volume fraction of 20% to 40% methanol water The method comprises the following steps: preparing a test solution E, and identifying lotus seeds by thin layer chromatography based on the test solution E; taking a solid phase extraction column eluted with a 20% to 40% methanol aqueous solution in the process of preparing the test solution D, eluting the column with a 40% to 60% methanol aqueous solution by volume as an eluent, evaporating the 40% to 60% methanol aqueous solution layer eluate to dryness, and re-dissolving the residue with alcohol.

[0008] Optionally, the preparation process of the test solution A includes: taking a test sample of the Qingxin Lianzi Drink compound preparation, adding an alcohol aqueous solution, ultrasonic extraction, solid-liquid separation, taking the liquid, evaporating to dryness, and re-dissolving the obtained residue with alcohol; Optionally, the preparation process of the test solution A includes: taking 5-15 g of the Qingxin Lianzi Drink compound preparation test sample, adding 50-150 mL of an alcohol aqueous solution with a volume fraction of 85%-98%, ultrasonic extraction at 35-53 kHz for 20-60 min, solid-liquid separation, taking 5-15 mL of liquid, evaporating to dryness, and re-dissolving the obtained residue with 0.5-1.5 mL of alcohol.

[0009] Optionally, the preparation process of the test solution B includes: taking 5-15 g of the test sample of the Qingxin Lianzi Drink compound preparation, adding 50-150 mL of an aqueous solution of 85% to 98% alcohol by volume, ultrasonically extracting at 35-53 kHz for 20-60 min, separating the solid and liquid, taking 5-100 mL of the liquid, evaporating to dryness, dissolving the resulting residue in 15-45 mL of water, extracting 2-4 times with 15-45 mL of petroleum ether with a boiling range of 60-90° C., taking the aqueous layer, extracting 2-4 times with 15-45 mL of ethyl acetate, taking the ethyl acetate layer, evaporating to dryness, and re-dissolving the resulting residue in 1.5-4.5 mL of alcohol. When the test solution A is prepared, 5-100 mL of the liquid after ultrasonic extraction of the alcohol aqueous solution during the preparation of the test solution A can be directly used for subsequent evaporation to dryness, water dissolution, petroleum ether extraction, etc. At this time, all the remaining liquid can be taken.

[0010] Optionally, the preparation process of the test solution C includes: taking 15-45 mL of the aqueous layer obtained after ethyl acetate extraction in the preparation process of the test sample B, generally taking all the aqueous layer, extracting 2-4 times with 20-40 mL of water-saturated n-butanol, combining the n-butanol layer extracts, taking 10-30 mL of the n-butanol layer extracts, washing 2-4 times with 10-30 mL of ammonia test solution, taking the n-butanol layer and washing it 2-4 times with 20-40 mL of n-butanol-saturated water, taking the n-butanol layer, evaporating it to dryness, and re-dissolving the resulting residue in 0.5-2 mL of alcohol.

[0011] Optionally, the preparation process of the obtained test solution D includes: taking 30-50 mL of the n-butanol layer extract obtained in the preparation process of the test sample C, generally taking all the remaining n-butanol layer extract, evaporating to dryness, adding 1.5-4.5 mL of water to the obtained residue for re-dissolution, and passing through a 500 mg C18 solid phase extraction column for column chromatography, using 1.5-4.5 mL of 20%-40% methanol aqueous solution as an eluent, and evaporating the obtained 20%-40% methanol aqueous solution layer eluate to dryness, and adding 0.5-2 mL of alcohol to the obtained residue for re-dissolution; optionally, using 30% methanol aqueous solution as an eluent.

[0012] Optionally, the preparation process of the obtained test solution E includes: taking the C18 solid phase extraction column eluted with 20% to 40% methanol aqueous solution in the process of preparing the test solution D, continuing to elute with 1.5 to 4.5 mL of 40% to 60% methanol aqueous solution as an eluent, evaporating the obtained 40% to 60% methanol aqueous solution layer eluate to dryness, and re-dissolving the obtained residue with 0.5 to 2 mL of alcohol; optionally, using 50% methanol aqueous solution as an eluent.

[0013] Optionally, based on the test solution A, thin layer chromatography is used to identify Scutellaria baicalensis, comprising the following steps: taking the test solution A, Scutellaria baicalensis control medicinal material solution, and baicalin reference solution, respectively spotting them on the same silica gel thin layer plate, using ethyl acetate-butanone-formic acid-water mixture as the developing agent and ferric chloride ethanol solution as the color developer for inspection.

[0014] In the quality control method of the Qingxin Lianziyin compound preparation provided in the present application, in the step of identifying Scutellaria baicalensis by thin layer chromatography based on the test solution A, it also includes preparing a Scutellaria baicalensis-deficient negative Qingxin Lianziyin compound preparation solution according to the preparation process of the Scutellaria baicalensis control medicinal material solution, and the steps of spotting and inspecting the Scutellaria baicalensis-deficient negative Qingxin Lianziyin compound preparation solution.

[0015] Optionally, based on the test solution B, thin layer chromatography is used to identify the root bark of Chinese wolfberry, comprising the following steps: taking the test solution B and the root bark of Chinese wolfberry control medicinal material solution, respectively spotting them on the same silica gel thin layer plate, using a dichloromethane-methanol mixture as a developing agent, and inspecting under ultraviolet light.

[0016] In the quality control method of the Qingxin Lianzi Drink compound preparation provided in the present application, in the step of identifying the Radix Rehmanniae by thin layer chromatography based on the test solution B, it also includes preparing the Radix Rehmanniae-negative Qingxin Lianzi Drink compound preparation solution according to the preparation process of the test solution B, and the steps of spotting and inspecting the Radix Rehmanniae-negative Qingxin Lianzi Drink compound preparation solution.

[0017] Optionally, based on the test solution B, thin layer chromatography is used to identify licorice, comprising the following steps: taking the test sample B, licorice control medicinal material solution, and liquiritin reference solution, respectively spotting them on the same silica gel thin layer plate, using a dichloromethane-methanol mixture as a developing agent and an ethanol solution of sulfuric acid as a color developer, heating until the spots are clearly colored and then inspecting them under ultraviolet light.

[0018] In the quality control method of the Qingxin Lianzi Drink compound preparation provided in the present application, in the step of identifying licorice by thin layer chromatography based on the test solution B, it also includes the steps of preparing the licorice-deficient negative Qingxin Lianzi Drink compound preparation solution according to the preparation process of the test solution B, and spotting and inspecting the licorice-deficient negative Qingxin Lianzi Drink compound preparation solution.

[0019] Optionally, based on the test solution C, thin layer chromatography is used to identify ginseng, comprising the following steps: taking the test sample C, ginseng control medicinal material solution, and mixed reference solution, respectively spotting them on the same silica gel thin layer plate, using a chloroform-methanol-water mixture as a developing agent and an ethanol solution of sulfuric acid as a color developer, heating until the spots are clearly colored and then inspecting under ultraviolet light; wherein the mixed reference solution contains ginsenoside Re reference, ginsenoside Rg1 reference, and ginsenoside Rb1 reference.

[0020] In the quality control method of the Qingxin Lianzi Drink compound preparation provided in the present application, in the step of identifying ginseng by thin layer chromatography based on the test solution C, it also includes preparing the ginseng-deficient negative Qingxin Lianzi Drink compound preparation solution according to the preparation process of the ginseng control medicinal material solution, and the steps of spotting and inspecting the ginseng-deficient negative Qingxin Lianzi Drink compound preparation solution.

[0021] Optionally, based on the test solution D, thin layer chromatography is used to identify Plantago seed, comprising the following steps: taking the test solution D, Plantago seed control medicinal material solution, and geniposide reference solution, respectively spotting them on the same silica gel thin layer plate, using ethyl acetate-methanol-formic acid-water mixture as the developing agent and vanillin sulfuric acid solution as the color developer, heating until the spots are clearly colored for inspection.

[0022] In the quality control method of the Qingxin Lianzi Drink compound preparation provided in the present application, in the step of identifying Plantago seed by thin layer chromatography based on the test solution D, it also includes preparing the Plantago seed-deficient negative Qingxin Lianzi Drink compound preparation solution according to the preparation process of the test solution D, and the steps of spotting and inspecting the Plantago seed-deficient negative Qingxin Lianzi Drink compound preparation solution.

[0023] Optionally, thin layer chromatography is used to identify lotus seeds based on the test solution E, comprising the following steps: taking the test solution E and the lotus seed control medicinal material solution, respectively spotting them on the same silica gel thin layer plate, using a dichloromethane-methanol mixture as a developing agent, and inspecting under ultraviolet light.

[0024] In the quality control method of the Qingxin Lianzi Drink compound preparation provided in the present application, in the step of identifying lotus seeds by thin layer chromatography based on the test solution E, it also includes preparing the lotus seed-deficient negative Qingxin Lianzi Drink compound preparation solution according to the preparation process of the lotus seed control medicinal material solution, and the steps of spotting and inspecting the lotus seed-deficient negative Qingxin Lianzi Drink compound preparation solution.

[0025] Optionally, the preparation process of the Scutellaria baicalensis reference medicinal material solution includes: taking 0.5-1 g of Scutellaria baicalensis reference medicinal material, adding 10-40 mL of an aqueous solution of alcohol with a volume fraction of 85%-98%, ultrasonic extraction at 35-53 kHz for 10-30 minutes, solid-liquid separation, taking the liquid, evaporating it to dryness, and re-dissolving the obtained residue in 0.5-1.5 mL of alcohol.

[0026] Optionally, the preparation process of the baicalin reference solution includes: taking a baicalin reference, adding an organic solvent to prepare a baicalin reference solution; optionally, each 1 mL of the baicalin reference solution contains 0.5 to 1.5 mg of the baicalin reference; optionally, the organic solvent includes methanol.

[0027] Optionally, the spotting volume of the test solution A, the scutellaria baicalensis control medicinal material solution, and the baicalin reference solution is 2 to 10 μL; optionally, the spotting volume is 5 μL.

[0028] Optionally, in the step of identifying Scutellaria baicalensis by thin layer chromatography based on the test solution A, the volume ratio of ethyl acetate, butanone, formic acid and water in the developing solvent is 6 to 4:2 to 4:1:1; optionally, the volume ratio is 5:3:1:1.

[0029] Optionally, in the step of identifying Scutellaria baicalensis by thin layer chromatography based on the test solution A, the mass fraction of the ferric chloride ethanol solution is 1% to 3%.

[0030] Optionally, the preparation process of the Radix Rehmanniae control medicinal material solution includes: taking 0.5-1.5 g of the Radix Rehmanniae control medicinal material, adding 10-30 mL of ethyl acetate, ultrasonic extraction at 35-53 kHz for 15-60 min, solid-liquid separation, taking the liquid, evaporating it to dryness, and adding 0.5-1.5 mL of alcohol to dissolve the obtained residue.

[0031] Optionally, the spotting volume of the test solution B and the Cortex Lycii control medicinal material solution is 2 to 10 μL; optionally, the spotting volume is 5 to 10 μL.

[0032] Optionally, in the step of identifying the root bark of Chinese wolfberry by thin layer chromatography based on the test solution B, the volume ratio of dichloromethane and methanol in the developing agent is 20:0.5~1.5; optionally, the volume ratio is 20:1.

[0033] Optionally, in the step of identifying the cortex rehmanniae by thin layer chromatography based on the test solution B, the wavelength of the ultraviolet light is 365 nm.

[0034] Optionally, the preparation process of the licorice control medicinal material solution includes: taking 0.5-1.5g of licorice control medicinal material, adding 10-30mL of ethyl acetate, ultrasonic extraction at 35-53kHz for 15-60min, solid-liquid separation, taking the liquid, evaporating it to dryness, and re-dissolving the obtained residue in 0.5-1.5mL of alcohol.

[0035] Optionally, the preparation process of the liquiritin reference solution includes: taking a liquiritin reference, adding an organic solvent to prepare a liquiritin reference solution; optionally, each 1 mL of the liquiritin reference solution contains 0.5 to 1.5 mg of the liquiritin reference; optionally, the organic solvent includes methanol.

[0036] Optionally, the spotting volume of the test solution B is 2 to 10 μL.

[0037] Optionally, the spotting volume of the licorice control medicinal material solution and the liquiritin reference solution is 1 to 3 μL.

[0038] Optionally, in the step of identifying licorice by thin layer chromatography based on the test solution B, the volume ratio of dichloromethane and methanol in the developing solvent is 10:3 to 5:1; optionally, the volume ratio is 4:1.

[0039] Optionally, in the step of identifying licorice by thin layer chromatography based on the test solution B, the volume fraction of the ethanol solution of sulfuric acid is 5% to 15%.

[0040] Optionally, in the step of identifying licorice by thin layer chromatography based on the test solution B, the wavelength of the ultraviolet light is 365 nm.

[0041] Optionally, the preparation process of the ginseng control medicinal material solution includes: taking 0.5-1.5 g of ginseng control medicinal material, adding 10-30 mL of an aqueous solution with a volume fraction of 85%-98% alcohol, ultrasonic extraction at 35-53 kHz for 15-45 min, solid-liquid separation, taking the liquid, evaporating to dryness, adding 20-30 mL of water to the resulting residue, extracting 2-4 times with 20-30 mL of petroleum ether with a boiling range of 60-90°C, taking the water layer, extracting 2-4 times with 20-30 mL of water-saturated n-butanol, combining the n-butanol layer extracts, washing 2-4 times with 10-30 mL of ammonia test solution, taking the n-butanol layer and washing 2-4 times with 20-30 mL of n-butanol-saturated water, taking the upper layer liquid, evaporating to dryness, and adding 0.5-2 mL of alcohol to the resulting residue.

[0042] Optionally, the preparation process of the mixed reference solution includes: taking ginsenoside Re reference, ginsenoside Rg1 reference, and ginsenoside Rb1 reference, and adding an organic solvent to prepare a mixed reference solution; optionally, the content of ginsenoside Re reference, ginsenoside Rg1 reference, and ginsenoside Rb1 reference in each 1 mL of the mixed reference solution is 0.5 to 1.5 mg, respectively; optionally, the organic solvent includes methanol.

[0043] Optionally, the spotting volume of the test sample C is 2 to 10 μL; optionally, the spotting volume is 5 to 10 μL.

[0044] Optionally, the spotting volume of the ginseng control medicinal material solution and the mixed reference substance solution is 1 to 3 μL.

[0045] Optionally, in the step of identifying ginseng by thin layer chromatography based on the test solution C, the volume ratio of chloroform, methanol and water in the developing agent is 13:6 to 8:2; optionally, the volume ratio is 13:7:2; optionally, the lower layer solution refrigerated overnight using the developing agent has a refrigeration temperature of ≤10°C.

[0046] Optionally, in the step of identifying ginseng by thin layer chromatography based on the test solution C, the volume fraction of the ethanol solution of sulfuric acid is 5% to 15%.

[0047] Optionally, in the step of identifying ginseng by thin layer chromatography based on the test solution C, the wavelength of the ultraviolet light is 365 nm.

[0048] Optionally, the preparation process of the Plantago seed control medicinal material solution includes: taking 0.5-1.5 g of Plantago seed control medicinal material, adding 10-30 mL of alcohol, ultrasonic extraction at 35-53 kHz for 15-45 min, solid-liquid separation, taking the liquid, evaporating it to dryness, adding 2-4 mL of water to the obtained residue to dissolve it, and performing column chromatography through a C18 solid phase extraction column with a specification of 500 mg, using 3-5 mL of 20%-40% methanol aqueous solution as an eluent, evaporating the obtained 20%-40% methanol aqueous solution layer eluate to dryness, and adding 0.5-2 mL of alcohol to dissolve the obtained residue.

[0049] Optionally, the preparation process of the geniposide reference solution includes: taking a geniposide reference, adding an organic solvent to prepare a geniposide reference solution; optionally, each 1 mL of the geniposide reference solution contains 0.5 to 1 mg of the geniposide reference; optionally, the organic solvent includes methanol.

[0050] Optionally, the spotting volume of the test solution D, the plantago seed reference medicinal material solution, and the geniposide reference solution is 5 to 15 μL.

[0051] Optionally, in the step of identifying Plantago seed by thin layer chromatography based on the test solution D, the volume ratio of ethyl acetate, methanol, formic acid and water in the developing solvent is 18:1 to 3:1.5:1; optionally, the volume ratio is 18:2:1.5:1.

[0052] Optionally, in the step of identifying Plantago seed by thin layer chromatography based on the test solution D, the mass fraction of the vanillin sulfuric acid solution is 1% to 3%.

[0053] Optionally, the preparation process of the lotus seed control medicinal material solution includes: taking 3-7 g of lotus seed control medicinal material, adding 40-60 mL of an aqueous solution with a volume fraction of 85%-98% alcohol, ultrasonic extraction at 35-53 kHz for 15-45 min, solid-liquid separation, taking the liquid, evaporating to dryness, adding 20-40 mL of water to the resulting residue to dissolve it, extracting it 2-4 times with 20-40 mL of petroleum ether with a boiling range of 60-90° C., taking the water layer, extracting it 2-4 times with 20-40 mL of ethyl acetate, taking the water layer, extracting it 2-4 times with 20-40 mL of water-saturated n-butanol, taking the n-butanol layer, evaporating to dryness, and adding 0.5-2 mL of alcohol to dissolve the resulting residue.

[0054] Optionally, the spotting volume of the test solution B is 2 to 10 μL; optionally, the spotting volume is 5 to 10 μL.

[0055] Optionally, the spotting volume of the lotus seed control medicinal material solution is 7 to 13 μL.

[0056] Optionally, in the step of identifying lotus seeds by thin layer chromatography based on the test solution E, the volume ratio of dichloromethane to methanol in the developing solvent is 100:23-27; optionally, the volume ratio is 4:1.

[0057] Optionally, in the step of identifying lotus seeds using thin layer chromatography based on the test solution E, the wavelength of the ultraviolet light is 365 nm.

[0058] Optionally, the Qingxin Lianzi Drink compound preparation includes a solid preparation, semi-solid preparation or liquid preparation of Qingxin Lianzi Drink; optionally, the Qingxin Lianzi Drink compound preparation includes a Qingxin Lianzi Drink benchmark sample decoction, a Qingxin Lianzi Drink benchmark sample concentrated extract, a Qingxin Lianzi Drink benchmark sample freeze-dried powder, a Qingxin Lianzi Drink preparation, a Qingxin Lianzi Drink intermediate extract or a Qingxin Lianzi Drink intermediate inclusion compound.

[0059] Optionally, the alcohol aqueous solution includes one or more of a methanol aqueous solution and an ethanol aqueous solution; optionally, the alcohol aqueous solution is an ethanol aqueous solution.

[0060] Optionally, the alcohol includes one or more of methanol and ethanol; optionally, the alcohol is methanol.

[0061] The technical solution of the present invention has the following advantages:

[0062] 1. The present invention provides a quality control method for a Qingxin Lianziyin compound preparation, comprising preparing test solutions A to E, and using thin-layer chromatography to identify Scutellaria baicalensis based on test solution A, identifying Rehmannia glutinosa and Licorice root based on test solution B, identifying Panax ginseng based on test solution C, identifying Plantago seed based on test solution D, and identifying Lotus seed based on test solution E. Furthermore, in the preparation method of the test samples, based on the polarity differences of the characteristic chemical components of the medicinal flavors in the prescription, the Qingxin Lianziyin compound preparation test sample can be extracted only once for identification of the six medicinal flavors, thereby reducing the need for repeated extraction of reference samples when identifying different medicinal flavors, reducing the amount of reagents used, making the operation more convenient, and improving the efficiency of thin-layer chromatography identification, making it more systematic. Furthermore, test solution B can also be used to identify Rehmannia glutinosa and Licorice root, which significantly reduces the sample preparation process and costs.

[0063] 2. The quality control method of the Qingxin Lianziyin compound preparation provided by the present invention has the following characteristics: the obtained thin layer test results of Scutellaria baicalensis, Rehmannia root bark, Licorice root, Ginseng, Plantago seed and Lotus seed have clear spots, good separation, strong specificity, no background interference and good durability.

[0064] 3. The quality control method of the Qingxin Lianziyin compound preparation provided by the present invention mostly uses alcohol aqueous solution, ethyl acetate, n-butanol, etc. as extraction solvents, uses a small amount of toxic reagents, and is environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0066] Figures 1 to 7 (b) is a diagram showing the methodological validation and multi-batch testing results in Example 1 of the present invention;

[0067] Figures 8 to 14 (b) is a diagram showing the methodological validation and multi-batch testing results in Example 2 of the present invention;

[0068] Figures 15-21 (b) is a diagram showing the methodological validation and multi-batch testing results in Example 3 of the present invention;

[0069] Figures 22-28 (b) is a diagram showing the methodological validation and multi-batch testing results in Example 4 of the present invention;

[0070] Figures 29-35(b) is a diagram showing the methodological validation and multi-batch testing results in Example 5 of the present invention;

[0071] Figures 36-42 (b) is a diagram showing the methodological validation and multi-batch testing results in Example 6 of the present invention;

[0072] Figure 43 (a) to (d) are the results obtained in Comparative Examples 1 to 4 of the present invention;

[0073] Figure 44 This is a flow chart of the preparation of test solutions A to E in the present invention. DETAILED DESCRIPTION

[0074] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0075] In the following examples and comparative examples, reagents not otherwise specified are conventional reagents and can be purchased from conventional reagent production and sales companies.

[0076] 1. Experimental Materials

[0077] (1) Baicalin (110715-202223), ginsenoside Re (110754-202330), ginsenoside Rg1 (110703-202436), ginsenoside Rb1 (110704-202331), glycyrrhizin (111610-202209), geniposide (111828-201805), Scutellaria baicalensis reference material (12 0955-201810), Radix Rehmanniae (121087-201707), Ginseng (120927-201712), Licorice (120904-202021), Plantago (121628-201702), and Lotus (121121-201205) were provided by the China Food and Drug Inspection Institutes.

[0078] (2) Qingxin Lianzi Drink granules (Z20241001, Z20241002, Z20241003) and Qingxin Lianzi Drink reference samples (lyophilized powder) (JZ-QXLZY240701~JZ-QXLZY240715) were all produced by China Resources Sanjiu.

[0079] The Qingxin Lianzi Drink sample lacking Scutellaria baicalensis, the Qingxin Lianzi Drink sample lacking Rehmannia glutinosa, the Qingxin Lianzi Drink sample lacking Licorice root, the Qingxin Lianzi Drink sample lacking Ginseng, the Qingxin Lianzi Drink sample lacking Plantago seed, and the Qingxin Lianzi Drink sample lacking Lotus seed were prepared using the same method as the Qingxin Lianzi Drink benchmark sample (lyophilized powder). The only difference in the preparation method is the lack of corresponding medicinal materials.

[0080] 2. Thin layer board

[0081] The manufacturers and batch numbers of the thin layer plates used are shown in the following table.

[0082] name batch number Specification factory <![CDATA[TLC Silica gel 60F 254 ]]> HX30745729 20cm×10cm Merck TLC Silica gel 60 HX02721141 20cm×10cm Merck Thin layer chromatography silica gel precast plate (GF254) 2024-05-09 10cm×10cm Yantai Huayang New Material Technology Co., Ltd. Thin layer chromatography silica gel precast plate (G) 2024-02-27 10cm×10cm Yantai Huayang New Material Technology Co., Ltd. Thin layer chromatography silica gel precast plate (GF254) 2024-07-23 20cm×10cm Yantai Huayang New Material Technology Co., Ltd. Thin layer chromatography silica gel precast plate (G) 2024-02-21 20cm×10cm Yantai Huayang New Material Technology Co., Ltd. Silicone plate (GF254) 2024-04-02 10cm×10cm Qingdao Ocean Chemical Co., Ltd. Silicone plate (G) 2024-04-05 10cm×10cm Qingdao Ocean Chemical Co., Ltd.

[0083] Example 1 Thin layer chromatography identification of Scutellaria baicalensis in Qingxin Lianziyin granules

[0084] 1. Identification method

[0085] (1) Preparation of test solution A: 10 g of Qingxin Lianziyin granules (Z20241001, Z20241002, Z20241003) was added to 100 mL of 95% ethanol aqueous solution, and ultrasonically extracted at 35 kHz for 30 min. The mixture was filtered through filter paper, and 10 mL of the filtrate was taken and evaporated to dryness. The residue was re-dissolved in 1 mL of methanol to prepare the test solution A.

[0086] (2) Preparation of Scutellaria baicalensis reference medicinal material solution: 0.5 g of Scutellaria baicalensis reference medicinal material was added to 20 mL of 95% ethanol aqueous solution, and ultrasonically extracted at 35 kHz for 15 min. The mixture was filtered, the filtrate was collected, and evaporated to dryness. The resulting residue was re-dissolved in 1 mL of methanol to prepare the Scutellaria baicalensis reference medicinal material solution.

[0087] (3) Preparation of baicalin reference solution: Take baicalin reference solution and add methanol to prepare a reference solution containing 1 mg of baicalin per 1 ml, which is used as the baicalin reference solution.

[0088] (4) Chromatographic conditions: According to the thin layer chromatography method (General Method 0502), 5 μl of each of the test solution A, the scutellaria baicalensis reference medicinal material solution, and the baicalin reference solution were spotted on the same silica gel G thin layer plate (Yantai 10×10) and developed with ethyl acetate-butanone-formic acid-water (5:3:1:1). The plate was removed, dried, sprayed with 2% ferric chloride ethanol solution, and examined. It can be seen that in the chromatogram of the test sample, spots of the same color appeared at the corresponding positions in the chromatogram of the reference medicinal material and the chromatogram of the reference substance.

[0089] 2. Methodological Validation

[0090] 1. Specificity test

[0091] (1) Preparation of the compound preparation solution of Qingxin Lianzi Drink for the deficiency of scutellaria baicalensis negative: Take 1 g of Qingxin Lianzi Drink for the deficiency of scutellaria baicalensis negative and prepare the compound preparation solution of Qingxin Lianzi Drink for the deficiency of scutellaria baicalensis negative by the same method as that of preparing the control medicinal material solution of scutellaria baicalensis.

[0092] (2) Chromatographic conditions: According to the thin layer chromatography method (General Rule 0502), 5 μl of the test solution A, the reference medicinal material solution of Scutellaria baicalensis, the reference solution of baicalin, and the compound preparation solution of Qingxin Lianziyin lacking Scutellaria baicalensis were respectively spotted on the same silica gel G thin layer plate (Yantai 10×10), and ethyl acetate-butanone-formic acid-water (5:3:1:1) was used as the developing solvent. The plate was taken out, dried, sprayed with 2% ferric chloride ethanol solution, and inspected. Figure 1 In the figure, 1 is the solution of Qingxin Lianziyin compound preparation lacking Scutellaria baicalensis, 2 to 4 are the test solution A of Z20241001, Z20241002 and Z20241003 granules respectively, 5 is the reference medicinal material solution of Scutellaria baicalensis, and 6 is the reference substance solution of baicalin ( Figure 2 , Figures 4-6 It can be seen that in the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions in the chromatograms of the control medicinal material and the reference sample, and there is no interference from the negative test solution, indicating that this method has good specificity.

[0093] 2. Durability test

[0094] (1) Investigation of different developing agent ratios

[0095] 5 μl of each of the test solution A, scutellaria baicalensis control medicinal material solution, baicalin reference solution, and scutellaria baicalensis negative Qingxin Lianziyin compound preparation solution were taken and spotted on the same silica gel G thin layer plate (Yantai 10×10). The same method was used to spot two thin layer plates. The two plates were respectively coated with ethyl acetate-butanone-formic acid-water (5:2:1:1) ( Figure 2 (a)), ethyl acetate-butanone-formic acid-water (5:4:1:1) Figure 2 (b)) was used as the developing solvent for development, and the other chromatographic conditions were the same as those above. Figure 1 It can be seen that different developing agent ratios have a certain influence on the Rf value of each spot, but there is no obvious difference in the separation effect, which shows that a slight fluctuation in the developing agent ratio of this method has no obvious effect on its development and separation.

[0096] (2) Investigation of different sample quantities

[0097] 5 μl of each of the above-mentioned Scutellaria baicalensis control medicinal material solution, baicalin reference solution, and Scutellaria baicalensis negative Qingxin Lianziyin compound preparation solution were taken, and 2 μl, 5 μl, and 10 μl of three test solution A (Z20241001, Z20241002, and Z20241003) were taken, respectively, and spotted on the same silica gel G thin layer plate (Yantai 20×10). The other chromatographic conditions were the same as those mentioned above. The results are shown in the figure. Figure 3 , where 1 is the solution of the Qingxin Lianziyin compound preparation lacking Scutellaria baicalensis, 2-4 are the sample volumes of Z20241001 test solution A (2μl, 5μl, and 10μl), 5-7 are the sample volumes of Z20241002 test solution A (2μl, 5μl, and 10μl), 8-10 are the sample volumes of Z20241003 test solution A (2μl, 5μl, and 10μl), 11 is the Scutellaria baicalensis reference solution, and 12 is the baicalin reference solution. It can be seen that the 2μl sample volume produces unclear spots, the 10μl sample volume has a higher concentration and a tailing effect, and the 5μl sample volume produces clear spots and good chromatographic results, making 5μl the preferred sample volume.

[0098] (3) Investigation at different temperatures

[0099] 5 μl of each of the test solution A, scutellaria baicalensis control medicinal material solution, baicalin reference solution, and scutellaria baicalensis negative Qingxin Lianziyin compound preparation solution were taken and spotted on the same silica gel G thin layer plate (Yantai 10×10). The same method was used to spot two thin layer plates, and the plates were incubated at 4°C ( Figure 4 (a)) and 30℃( Figure 4 (b)) and the rest of the chromatographic conditions were the same as above. Figure 1 It can be seen that different temperatures have a certain influence on the Rf value of each spot, but there is no obvious difference in the separation effect and spot display, which shows that this method has good durability at an ambient temperature of 4 to 30°C.

[0100] (4) Investigation at different humidity levels

[0101] 5 μl of each of the test solution A, scutellaria baicalensis control medicinal material solution, baicalin reference solution, and scutellaria baicalensis negative Qingxin Lianziyin compound preparation solution were taken and spotted on the same silica gel G thin layer plate (Yantai 10×10). The same method was used to spot two thin layer plates, and the plates were heated at RH 32% ( Figure 5 (a)) and RH 88% ( Figure 5 (b)) and the rest of the chromatographic conditions were the same as above. Figure 1 It can be seen that different humidity has a certain effect on the Rf value of each spot, but there is no obvious difference in the separation effect and spot display, which shows that this method has good durability under an ambient humidity of 32% to 88%.

[0102] (5) Inspection of different brands of thin layer boards

[0103] 5 μl of each of the test solution A, scutellaria baicalensis control medicinal material solution, baicalin reference solution, and scutellaria baicalensis negative Qingxin Lianziyin compound preparation solution were taken, and the above operation was repeated twice. The samples were spotted on Merck plates (G plate 10×10) ( Figure 6 (a)) and Qingdao board (G board 10×10) ( Figure 6 (a)) and the rest of the chromatographic conditions were the same as those above. Figure 1 It can be seen that the Rf values of each spot are different after development of different thin layer plates, but there is no obvious difference in the separation effect and spot display.

[0104] 3. Multi-batch inspection of benchmark samples

[0105] (1) Preparation of test solution A: Take 10 g of Qingxin Lianzi Drink reference sample (JZ-QXLZY240701~15), add 100 mL of 95% ethanol aqueous solution, ultrasonically extract at 35 kHz for 30 min, filter through filter paper, take 10 mL of filtrate, evaporate to dryness, and re-dissolve the obtained residue in 1 mL of methanol to prepare test solution A.

[0106] (2) Chromatographic conditions: According to the thin layer chromatography method (General Rule 0502), 5 μl of each of the test solution A, the above-mentioned Scutellaria baicalensis reference medicinal material solution, and the above-mentioned baicalin reference solution were taken and spotted in batches on two silica gel G thin layer plates (Yantai 20×10), and ethyl acetate-butanone-formic acid-water (5:3:1:1) was used as the developing solvent. The plates were taken out, dried, sprayed with 2% ferric chloride ethanol solution, and inspected. Figure 7 (a), (b), Figure 7 In (a), 1 is the reference medicinal material solution of Scutellaria baicalensis, 2 is the reference solution of baicalin, and 3 to 10 are the test solution A of the JZ-QXLZY240701 to 08 reference samples; Figure 7 In (b), 1 is the Scutellaria baicalensis reference solution, 2 is the baicalin reference solution, and 3-9 are the test solution A of the JZ-QXLZY240709-15 benchmark samples. It can be seen that in the test solution chromatogram, spots of the same color appear at the corresponding positions in the control solution chromatogram and the reference solution chromatogram.

[0107] Example 2 Thin layer identification of the bark of Chinese wolfberry in Qingxin Lianziyin granules

[0108] 1. Identification method

[0109] (1) Preparation of test solution B: 80 mL of the filtrate after ultrasonic extraction of ethanol-water solution during the preparation of test solution A was taken and evaporated to dryness. The resulting residue was dissolved in 30 mL of water and extracted twice with 30 mL of petroleum ether with a boiling range of 60-90°C. The aqueous layer was extracted twice with 30 mL of ethyl acetate. The ethyl acetate layer was taken and evaporated to dryness. The resulting residue was redissolved in 3 mL of methanol to prepare test solution B.

[0110] (2) Preparation of Radix Rehmanniae control medicinal material solution: 1 g of Radix Rehmanniae control medicinal material was taken, 20 mL of ethyl acetate was added, and ultrasonic extraction was performed at 35 kHz for 30 min. The solid-liquid separation was performed, and the filtrate was taken and evaporated to dryness. The obtained residue was re-dissolved in 1 mL of methanol to prepare the Radix Rehmanniae control medicinal material solution.

[0111] (3) Chromatographic conditions: According to the thin layer chromatography method (General Principle 0502), 5 μl of the test solution B and the Radix Lycii control medicinal material solution were taken and spotted on the same silica gel GF 254 A thin layer plate (Yantai 10×10) was developed with dichloromethane-methanol (20:1), removed, air-dried, and examined under a UV lamp (365 nm). It can be seen that the chromatogram of the test sample has a fluorescent spot of the same color at the corresponding position in the chromatogram of the control medicinal material.

[0112] 2. Methodological Validation

[0113] 1. Specificity test

[0114] (1) Preparation of the compound preparation solution of the negative Qingxin Lianzi Drink with the missing Rehmannia root bark: Take the negative Qingxin Lianzi Drink granules with the missing Rehmannia root bark and prepare the compound preparation solution of the negative Qingxin Lianzi Drink with the missing Rehmannia root bark according to the same preparation method as the test solution B.

[0115] (2) Chromatographic conditions: According to the thin layer chromatography method (General Principle 0502), 5 μl of the test solution B, the Radix Rehmanniae control medicinal material solution, and the Radix Rehmanniae negative Qingxin Lianziyin compound preparation solution were taken and spotted on the same silica gel GF 254 On a thin layer plate (Yantai 10×10), dichloromethane-methanol (20:1) was used as the developing agent, the plate was developed, taken out, dried, and examined under an ultraviolet lamp (365 nm). Figure 8 In the figure, 1 is the negative Qingxin Lianziyin compound preparation solution lacking the root bark, 2 to 4 are the test solution B of Z20241001, Z20241002, and Z20241003 granules respectively, and 5 is the control medicinal material solution of the root bark. ( Figure 9 , Figures 11-13 It can be seen that in the chromatogram of the test sample, there are fluorescent spots of the same color at the corresponding positions of the control medicinal material, and there is no interference from the negative test solution, indicating that this method has good specificity.

[0116] 2. Durability test

[0117] (1) Investigation of different developing agent ratios

[0118] 5 μl of each of the test solution B, the Radix Rehmanniae control medicinal material solution, and the Radix Rehmanniae negative Qingxin Lianziyin compound preparation solution were taken and spotted on the same silica gel GF. 254 On a thin layer plate (Yantai 10×10), two thin layer plates were spotted in the same way, and the two plates were respectively saturated with dichloromethane-methanol (20:0.5) ( Figure 9 (a)) and dichloromethane-methanol (20:1.5) ( Figure 9 (b)) was used as the developing solvent for development, and the other chromatographic conditions were the same as those above. Figure 7 It can be seen that different developing agent ratios have a certain influence on the Rf value of each spot, but there is no obvious difference in the separation effect, which shows that a slight fluctuation in the developing agent ratio of this method has no obvious effect on its development and separation.

[0119] (2) Investigation of different sample quantities

[0120] 5 μl of the above-mentioned Radix Rehmanniae control medicinal material solution and Radix Rehmanniae negative Qingxin Lianziyin compound preparation solution were respectively aspirated, and 2 μl, 5 μl and 10 μl of three test solution B (Z20241001, Z20241002 and Z20241003) were aspirated respectively, and spotted on the same silica gel GF 254 The other chromatographic conditions were the same as those above. Figure 10 , where 1 is the Qingxin Lianziyin compound preparation solution lacking the root bark of Radix Rehmanniae, 2-4 are the Z20241001 test solution B with a spotting volume of 2μl, 5μl, and 10μl, 5-7 are the Z20241002 test solution B with a spotting volume of 2μl, 5μl, and 10μl, 8-10 are the Z20241003 test solution B with a spotting volume of 2μl, 5μl, and 10μl, and 11 is the root bark of Radix Rehmanniae control medicinal material solution. It can be seen that the 2μl spot is not obvious, while the 5μl and 10μl spotting volumes have clear spots and good chromatographic effects. The 5-10μl spotting volume can be used for the thin layer identification method study of the root bark of Radix Rehmanniae.

[0121] (3) Investigation at different temperatures

[0122] 5 μl of each of the test solution B, the Radix Rehmanniae control medicinal material solution, and the Radix Rehmanniae negative Qingxin Lianziyin compound preparation solution were taken and spotted on the same silica gel GF. 254 On a thin layer plate (Yantai 10×10), spot two thin layer plates in the same way, and place the two plates at 4℃ ( Figure 11 (a)) and 30℃( Figure 11(b)) and the rest of the chromatographic conditions were the same as above. Figure 8 It can be seen that different temperatures have a certain influence on the Rf value of each spot, but there is no obvious difference in the separation effect and spot display, which shows that this method has good durability at an ambient temperature of 4 to 30°C.

[0123] (4) Investigation at different humidity levels

[0124] 5 μl of each of the test solution B, the Radix Rehmanniae control medicinal material solution, and the Radix Rehmanniae negative Qingxin Lianziyin compound preparation solution were taken and spotted on the same silica gel GF. 254 On a thin layer plate (Yantai 10×10), two thin layer plates were spotted in the same way and respectively at RH 32% ( Figure 12 (a)) and RH 88% ( Figure 12 (b)) and the rest of the chromatographic conditions were the same as above. Figure 8 It can be seen that different humidity has a certain effect on the Rf value of each spot, but there is no obvious difference in the separation effect and spot display, which shows that this method has good durability under an ambient humidity of 32% to 88%.

[0125] (5) Inspection of different brands of thin layer boards

[0126] 5 μl of each of the test solution B, the Radix Lycii control medicinal material solution, and the Radix Lycii negative Qingxin Lianziyin compound preparation solution were taken, and the above operation was repeated twice. The samples were spotted on Merck plates (GF 254 Plate 10×10)( Figure 13 (a)) and Qingdao board (GF 254 Plate 10×10)( Figure 13 (a)) and the rest of the chromatographic conditions were the same as those above. Figure 8 It can be seen that the Rf values of each spot are different after development of different thin layer plates, but there is no obvious difference in the separation effect and spot display.

[0127] 3. Multi-batch inspection of benchmark samples

[0128] (1) Preparation of test solution B: Take 80 mL of the filtrate after ultrasonic extraction of ethanol-water solution during the preparation of the corresponding test solution A of the Qingxin Lianzi Drink reference sample (JZ-QXLZY240701-15), evaporate to dryness, dissolve the obtained residue in 30 mL of water, extract twice with petroleum ether with a boiling range of 60-90 ° C, 30 mL each time, take the water layer, extract twice with 30 mL of ethyl acetate, take the ethyl acetate layer, evaporate to dryness, and re-dissolve the obtained residue in 3 mL of methanol to prepare the test solution B.

[0129] (2) Chromatographic conditions: According to the thin layer chromatography method (General Principle 0502), 5 μl of the test solution B and the above-mentioned Radix Rehmanniae control medicinal material solution were taken and spotted on the same silica gel GF 254 On a thin layer plate (Yantai 20×10), dichloromethane-methanol (20:1) was used as the developing agent, the plate was developed, taken out, dried, and examined under an ultraviolet lamp (365nm). Figure 14 (a), (b), Figure 14 In (a), 1 is the control medicinal material solution of Radix Lycii, and 2 to 9 are the test solution B of the JZ-QXLZY240701 to 08 benchmark samples; Figure 14 In (b), 1 is the Radix Lycii control medicinal material solution, and 2 to 8 are the test solution B of the JZ-QXLZY240709-15 benchmark sample. It can be seen that in the chromatogram of the test sample, there are fluorescent spots of the same color at the corresponding positions of the control medicinal material.

[0130] Example 3 Thin layer chromatography identification of licorice in Qingxin Lianziyin granules

[0131] 1. Identification method

[0132] (1) The preparation of test solution B is the same as in Example 2.

[0133] (2) Preparation of licorice control medicinal material solution: 1 g of licorice control medicinal material was added with 20 mL of ethyl acetate, and ultrasonically extracted at 35 kHz for 30 min. The solid and liquid were separated, and the filtrate was taken and evaporated to dryness. The obtained residue was re-dissolved in 1 mL of methanol to prepare the licorice control medicinal material solution.

[0134] (3) Preparation of liquiritin reference solution: Take liquiritin reference solution and add methanol to prepare a reference solution containing 0.5 mg of liquiritin per 1 ml, which is used as the liquiritin reference solution.

[0135] (4) Chromatographic conditions: According to the thin layer chromatography method (General Method 0502), 5 μl of test sample B, 2 μl of licorice control medicinal material solution, and 2 μl of liquiritin reference solution were respectively spotted on the same silica gel GF 254 A thin layer plate (Yantai 10×10) was developed with dichloromethane-methanol (4:1) as the developing solvent. The plate was removed, air-dried, sprayed with 10% sulfuric acid-ethanol test solution, heated until the spots were clear, and examined under a UV lamp (365 nm). Fluorescent spots of the same color were observed in the chromatogram of the test sample, the control herb, and the reference substance at the corresponding positions.

[0136] 2. Methodological Validation

[0137] 1. Specificity test

[0138] (1) Preparation of the solution of the compound preparation of the Qingxin Lianzi Drink with the deficiency of licorice and negative effect: Take the Qingxin Lianzi Drink granules with the deficiency of licorice and negative effect, and prepare the solution of the compound preparation of the Qingxin Lianzi Drink with the deficiency of licorice and negative effect according to the same preparation method as the test solution B.

[0139] (2) Chromatographic conditions: According to the thin layer chromatography method (general rule 0502), 5 μl of the test solution B, 5 μl of the Qingxin Lianziyin compound preparation solution lacking licorice, 2 μl of the licorice control medicinal material solution, and 2 μl of the liquiritin reference solution were taken and spotted on the same silica gel GF. 254 On a thin layer plate (Yantai 10×10), dichloromethane-methanol (4:1) was used as the developing agent, the plate was developed, taken out, dried, sprayed with 10% sulfuric acid ethanol test solution, heated until the spots were clear, and examined under ultraviolet light (365nm). Figure 15 In the figure, 1 is the solution of Qingxin Lianziyin compound preparation lacking licorice, 2 to 4 are the test solution B of Z20241001, Z20241002 and Z20241003 granules respectively, 5 is the licorice control medicinal material solution, 6 is the liquiritin reference solution ( Figure 16 , Figures 18-20 It can be seen that in the chromatogram of the test sample, there are fluorescent spots of the same color at the corresponding positions of the control medicinal material, and there is no interference from the negative test solution, indicating that this method has good specificity.

[0140] 2. Durability test

[0141] (1) Investigation of different developing agent ratios

[0142] 5 μl of the test solution B, 5 μl of the Qingxin Lianziyin compound preparation solution lacking licorice, 2 μl of the licorice control medicinal material solution, and 2 μl of the liquiritin reference solution were respectively taken and spotted on the same silica gel GF. 254 On a thin layer plate (Yantai 10×10), two thin layer plates were spotted in the same way, and the two plates were respectively stained with dichloromethane-methanol (10:3) ( Figure 16 (a)) and dichloromethane-methanol (5:1) ( Figure 16 (b)) was used as the developing solvent for development, and the other chromatographic conditions were the same as those above. Figure 15 It can be seen that different developing agent ratios have a certain influence on the Rf value of each spot, but there is no obvious difference in the separation effect, which shows that a slight fluctuation in the developing agent ratio of this method has no obvious effect on its development and separation.

[0143] (2) Investigation of different sample quantities

[0144] 5 μl of the above-mentioned Qingxin Lianziyin compound preparation solution lacking licorice, 2 μl of licorice control medicinal material solution and liquiritin reference solution were respectively taken, and 2 μl, 5 μl and 10 μl of three test solution B (Z20241001, Z20241002 and Z20241003) were taken respectively, and spotted on the same silica gel GF 254 The other chromatographic conditions were the same as those above. Figure 17 , where 1 is the Qingxin Lianziyin compound preparation solution lacking licorice, 2-4 are the Z20241001 test solution B with a spot size of 2μl, 5μl, and 10μl, 5-7 are the Z20241002 test solution B with a spot size of 2μl, 5μl, and 10μl, 8-10 are the Z20241003 test solution B with a spot size of 2μl, 5μl, and 10μl, 11 is the licorice control solution, and 12 is the liquiritin reference solution. It can be seen that the spots of 2μl, 5μl, and 10μl spot sizes are all clear and have good chromatographic effects. Therefore, 2-10μl spot sizes can be used for TLC identification of licorice.

[0145] (3) Investigation at different temperatures

[0146] 5 μl of the test solution B, 5 μl of the Qingxin Lianziyin compound preparation solution lacking licorice, 2 μl of the licorice control medicinal material solution, and 2 μl of the liquiritin reference solution were respectively taken and spotted on the same silica gel GF. 254 On a thin layer plate (Yantai 10×10), spot two thin layer plates in the same way, and place the two plates at 4℃ ( Figure 18 (a)) and 30℃( Figure 18 (b)) and the rest of the chromatographic conditions were the same as above. Figure 15 It can be seen that different temperatures have a certain influence on the Rf value of each spot, but there is no obvious difference in the separation effect and spot display, which shows that this method has good durability at an ambient temperature of 4 to 30°C.

[0147] (4) Investigation at different humidity levels

[0148] 5 μl of the test solution B, 5 μl of the Qingxin Lianziyin compound preparation solution lacking licorice, 2 μl of the licorice control medicinal material solution, and 2 μl of the liquiritin reference solution were respectively taken and spotted on the same silica gel GF. 254 On a thin layer plate (Yantai 10×10), two thin layer plates were spotted in the same way and respectively at RH 32% ( Figure 19 (a)) and RH 88% ( Figure 19 (b)) and the rest of the chromatographic conditions were the same as above. Figure 15It can be seen that different humidity has a certain effect on the Rf value of each spot, but there is no obvious difference in the separation effect and spot display, which shows that this method has good durability under an ambient humidity of 32% to 88%.

[0149] (5) Inspection of different brands of thin layer boards

[0150] 5 μl of the test solution B, 5 μl of the Qingxin Lianziyin compound preparation solution lacking licorice, 2 μl of the licorice control medicinal material solution, and 2 μl of the liquiritin reference solution were taken respectively, and the above operation was repeated twice. The samples were spotted on Merck plates (GF 254 Plate 10×10)( Figure 20 (a)) and Qingdao board (GF 254 Plate 10×10)( Figure 20 (a)) and the rest of the chromatographic conditions were the same as those above. Figure 15 It can be seen that the Rf values of each spot are different after development of different thin layer plates. The separation effect and spot display of Qingdao plate are different from those of Merck plate and Yantai plate, but it can also be used for identification.

[0151] 3. Multi-batch inspection of benchmark samples

[0152] (1) The preparation of test solution B is the same as in Example 2.

[0153] (2) Chromatographic conditions: According to the thin layer chromatography method (General Principle 0502), 5 μl of the test solution B, 2 μl of the licorice control medicinal material solution, and 2 μl of the liquiritin reference solution were respectively spotted on the same silica gel GF 254 On a thin layer plate (Yantai 20×10), dichloromethane-methanol (4:1) was used as the developing agent, the plate was developed, taken out, dried, sprayed with 10% sulfuric acid ethanol test solution, heated until the spots were clear, and examined under ultraviolet light (365nm). Figure 21 (a), (b), Figure 21 In (a), 1 is the reference liquorice medicinal material solution, 2 is the reference liquiritin solution, and 3 to 10 are the test solution B of the JZ-QXLZY240701 to 08 reference samples; Figure 21 In (b), 1 is the licorice control medicinal material solution, 2 is the liquiritin reference solution, and 3 to 9 are the test solution B of the JZ-QXLZY240709 to 15 benchmark samples. It can be seen that in the chromatogram of the test sample, there are fluorescent spots of the same color at the corresponding positions of the control medicinal material.

[0154] Example 4 Thin-layer chromatography identification of ginseng in Qingxin Lianziyin granules

[0155] 1. Identification method

[0156] (1) Preparation of test solution C: Take 30 mL of the aqueous layer obtained after ethyl acetate extraction during the preparation of test sample B, extract it twice with saturated n-butanol with water, each time 30 mL, combine the n-butanol layer extracts, take 20 mL of the n-butanol layer extracts, wash it twice with ammonia test solution, each time 20 mL, take the n-butanol layer and wash it twice with saturated n-butanol water, each time 30 mL, take the n-butanol layer, evaporate it to dryness, and re-dissolve the resulting residue in 1 mL of methanol to prepare test solution C.

[0157] (2) Preparation of ginseng control medicinal material solution: Take 1 g of ginseng control medicinal material, add 20 mL of 95% ethanol aqueous solution, ultrasonically extract at 35 kHz for 30 min, filter, take the filtrate, evaporate to dryness, re-dissolve the obtained residue in 30 mL of water, extract twice with petroleum ether with a boiling range of 60-90 ° C, each time 30 mL, take the water layer, extract twice with water-saturated n-butanol, each time 30 mL, combine the n-butanol layer extracts, wash twice with 20 mL of ammonia test solution, take the n-butanol layer and wash twice with n-butanol saturated water, each time 30 mL, take the upper layer liquid, evaporate to dryness, and re-dissolve the obtained residue in 1 mL of methanol as ginseng control medicinal material solution.

[0158] (3) Preparation of mixed reference solution: Take ginsenoside Re reference, ginsenoside Rg1 reference, and ginsenoside Rb1 reference, and add methanol to prepare a mixed reference solution containing 0.5 mg of each reference substance per 1 ml.

[0159] (4) Chromatographic conditions: According to the thin layer chromatography method (General Principle 0502), 5 μl of the test solution C, 2 μl of the ginseng control medicinal material solution, and 2 μl of the mixed reference solution were respectively spotted on the same silica gel G thin layer plate (Yantai 10×10). The lower layer solution of chloroform-methanol-water (13:7:2) refrigerated overnight was used as the developing solvent. The plate was developed, taken out, dried, sprayed with 10% sulfuric acid ethanol test solution, heated until the spots were clear, and examined under ultraviolet light (365 nm). It can be seen that in the chromatogram of the test sample, there are fluorescent spots of the same color at the corresponding positions of the control medicinal material and the reference substance.

[0160] 2. Methodological Validation

[0161] 1. Specificity test

[0162] (1) Preparation of the compound preparation solution of the Qingxin Lianzi Drink for Lack of Ginseng Yin: Take the Qingxin Lianzi Drink granules for Lack of Ginseng Yin and prepare the compound preparation solution of the Qingxin Lianzi Drink for Lack of Ginseng Yin by the same preparation method as the ginseng control medicinal material solution.

[0163] (2) Chromatographic conditions: According to the thin layer chromatography method (General Rule 0502), 5 μl of the test solution C, 5 μl of the ginseng-deficient negative Qingxin Lianziyin compound preparation solution, 2 μl of the ginseng control medicinal material solution, and 2 μl of the mixed reference solution were respectively spotted on the same silica gel G thin layer plate (Yantai 10×10), and the lower layer solution refrigerated overnight in chloroform-methanol-water (13:7:2) was used as the developing agent. The plate was developed, taken out, dried, sprayed with 10% sulfuric acid ethanol test solution, heated until the spots were clear, and examined under ultraviolet light (365 nm). Figure 22 In the figure, 1 is the solution of the Qingxin Lianziyin compound preparation lacking ginseng, 2 to 4 are the test solution C of Z20241001, Z20241002, and Z20241003 granules respectively, 5 is the ginseng control medicinal material solution, and 6 is the mixed reference solution ( Figure 23 , Figures 25-27 It can be seen that in the chromatogram of the test sample, there are fluorescent spots of the same color at the corresponding positions of the control medicinal material, and there is no interference from the negative test solution, indicating that this method has good specificity.

[0164] 2. Durability test

[0165] (1) Investigation of different developing agent ratios

[0166] 5 μl of the test solution C, ginseng-deficient negative Qingxin Lianziyin compound preparation solution, 2 μl of ginseng control medicinal material solution, and mixed reference solution were respectively taken and spotted on the same silica gel G thin layer plate (Yantai 10×10). The same method was used to spot two thin layer plates. The two plates were respectively stained with chloroform-methanol-water (13:6:2) ( Figure 23 (a)) and chloroform-methanol-water (13:8:2) ( Figure 23 (b) The lower layer solution refrigerated overnight was used as the developing solvent for development, and the other chromatographic conditions were the same as those above. Figure 22 It can be seen that different developing agent ratios have a certain influence on the Rf value of each spot, but there is no obvious difference in the separation effect, which shows that a small fluctuation in the developing agent ratio of this method has no obvious effect on its development and separation.

[0167] (2) Investigation of different sample quantities

[0168] 2 μl of the above-mentioned ginseng control medicinal material solution and mixed reference solution, 5 μl of the ginseng-deficient negative Qingxin Lianziyin compound preparation solution, and 2 μl, 5 μl, and 10 μl of three test solution C (Z20241001, Z20241002, and Z20241003) were respectively taken and spotted on the same silica gel G thin layer plate (Yantai 20×10). The other chromatographic conditions were the same as those mentioned above. The results are shown in the figure. Figure 24, where 1 is the ginseng-deficient negative Qingxin Lianziyin compound preparation solution, 2-4 are the Z20241001 test solution C with a spotting volume of 2μl, 5μl, and 10μl, 5-7 are the Z20241002 test solution C with a spotting volume of 2μl, 5μl, and 10μl, 8-10 are the Z20241003 test solution C with a spotting volume of 2μl, 5μl, and 10μl, 11 is the ginseng control medicinal material solution, and 12 is the mixed reference solution. It can be seen that the 2μl spotting volume has unclear spots, while the 5μl and 10μl spotting volumes have clear spots and good chromatographic effects. Therefore, the 5-10μl spotting volume can be used for thin-layer chromatography identification of ginseng.

[0169] (3) Investigation at different temperatures

[0170] 5 μl of the test solution C, 5 μl of the ginseng-negative Qingxin Lianziyin compound preparation solution, 2 μl of the ginseng control medicinal material solution, and 2 μl of the mixed reference solution were respectively taken and spotted on the same silica gel G thin layer plate (Yantai 10×10). The same method was used to spot two thin layer plates. The two plates were placed at 4°C ( Figure 25 (a)) and 30℃( Figure 25 (b)) and the rest of the chromatographic conditions were the same as above. Figure 22 It can be seen that different temperatures have a certain influence on the Rf value of each spot, but there is no obvious difference in the separation effect and spot display, which shows that this method has good durability at an ambient temperature of 4 to 30°C.

[0171] (4) Investigation at different humidity levels

[0172] 5 μl of the test solution C, 5 μl of the ginseng-negative Qingxin Lianziyin compound preparation solution, 2 μl of the ginseng control medicinal material solution, and 2 μl of the mixed reference solution were respectively taken and spotted on the same silica gel G thin layer plate (Yantai 10×10). The same method was used to spot two thin layer plates, and the plates were respectively heated at RH 32% ( Figure 26 (a)) and RH 88% ( Figure 26 (b)) and the rest of the chromatographic conditions were the same as above. Figure 22 It can be seen that different humidity has a certain effect on the Rf value of each spot, but there is no obvious difference in the separation effect and spot display, which shows that this method has good durability under an ambient humidity of 32% to 88%.

[0173] (5) Inspection of different brands of thin layer boards

[0174] 5 μl of the test solution C, 5 μl of the ginseng-deficient negative Qingxin Lianziyin compound preparation solution, 2 μl of the ginseng control medicinal material solution, and 2 μl of the mixed reference solution were taken respectively, and the above operation was repeated twice, and the samples were spotted on Merck plates (G plate 10×10) ( Figure 27 (a)) and Qingdao board (G board 10×10) ( Figure 27 (a)) and the rest of the chromatographic conditions were the same as those above. Figure 22 It can be seen that the Rf values of each spot are different after development of different thin layer plates. The spots on Qingdao plate appear more blurred, but can also be used for identification.

[0175] 3. Multi-batch inspection of benchmark samples

[0176] (1) Preparation of test solution C: Take 30 mL of the aqueous layer obtained after ethyl acetate extraction during the preparation of the corresponding test solution B of the Qingxin Lianzi Drink reference sample (JZ-QXLZY240701~15), extract it twice with 30 mL of saturated n-butanol with water, combine the n-butanol layer extracts, take 20 mL of the n-butanol layer extracts, wash it twice with 20 mL of ammonia test solution, take the n-butanol layer and wash it twice with 30 mL of saturated n-butanol water, take the n-butanol layer, evaporate it to dryness, and re-dissolve the resulting residue in 1 mL of methanol to prepare the test solution C.

[0177] (2) Chromatographic conditions: According to the thin layer chromatography method (General Principle 0502), 5 μl of the test solution C, 2 μl of the ginseng control medicinal material solution, and 2 μl of the mixed reference solution were respectively spotted on the same silica gel G thin layer plate (Yantai 20×10), and the lower layer solution refrigerated overnight in chloroform-methanol-water (13:7:2) was used as the developing agent. The plate was developed, taken out, dried, sprayed with 10% sulfuric acid ethanol test solution, heated until the spots were clear, and examined under ultraviolet light (365 nm). Figure 28 (a), (b), Figure 28 In (a), 1 is the ginseng control medicinal material solution, 2 is the mixed reference solution, and 3 to 10 are the test solution C of the JZ-QXLZY240701 to 08 benchmark samples; Figure 28 In (b), 1 is the ginseng control medicinal material solution, 2 is the mixed reference solution, and 3 to 9 are the test solution C of the JZ-QXLZY240709-15 benchmark sample. It can be seen that in the chromatogram of the test sample, there are fluorescent spots of the same color at the corresponding positions of the control medicinal material.

[0178] Example 5 Thin-layer chromatography identification of Plantago seeds in Qingxin Lianziyin granules

[0179] 1. Identification method

[0180] (1) Preparation of test solution D: 40 mL of the n-butanol extract from the preparation of test sample C was evaporated to dryness. The resulting residue was reconstituted with 3 mL of water and passed through activated Wates Sep-Pak Vac 6 cc (500 mg) C18 Cartridges for column chromatography using 3 mL of 30% aqueous methanol as the eluent. The resulting 30% aqueous methanol layer was evaporated to dryness, and the resulting residue was reconstituted with 1 mL of methanol to prepare test solution D.

[0181] (2) Preparation of Plantago seed control medicinal material solution: 1 g of Plantago seed control medicinal material was added with 20 mL of methanol, and ultrasonically extracted at 35 kHz for 30 min. The solution was filtered through qualitative filter paper, and the filtrate was taken and evaporated to dryness. The resulting residue was reconstituted with 3 mL of water and subjected to column chromatography using activated Wates Sep-Pak Vac 6 cc (500 mg) C18 Cartridges. 4 mL of 30% aqueous methanol solution was used as the eluent. The resulting 30% aqueous methanol layer was evaporated to dryness, and the resulting residue was reconstituted with 1 mL of methanol to prepare the Plantago seed control medicinal material solution.

[0182] (3) Preparation of Geniposide Reference Solution: Take Geniposide Reference Solution and add methanol to prepare Geniposide Reference Solution containing 0.5 mg of reference solution per 1 ml.

[0183] (4) Chromatographic conditions: According to the thin layer chromatography method (General Principle 0502), 10 μl of each of the test solution D, the plantago seed control medicinal material solution, and the geniposide 9 reference solution were taken and spotted on the same silica gel GF 254 A thin layer plate (Yantai 10×10) was developed with ethyl acetate-methanol-formic acid-water (18:2:1.5:1). The plate was removed, air-dried, and sprayed with a 1% vanillin-sulfuric acid solution. The plate was heated until the spots developed clearly and then examined. The test sample chromatogram showed spots of the same color at the corresponding positions in the control herb and reference substance.

[0184] 2. Methodological Validation

[0185] 1. Specificity test

[0186] (1) Preparation of the Plantago Seed-Deficient Negative Qingxin Lianzi Drink Compound Preparation Solution: Take the Plantago Seed-Deficient Negative Qingxin Lianzi Drink granules and prepare the Plantago Seed-Deficient Negative Qingxin Lianzi Drink Compound Preparation Solution according to the same preparation method as the test solution D.

[0187] (2) Chromatographic conditions: According to the thin layer chromatography method (General Rule 0502), 10 μl of each of the test solution D, the Qingxin Lianzi Decoction compound preparation solution lacking Plantago seed, the Plantago seed control medicinal material solution, and the geniposide reference solution were taken and spotted on the same silica gel GF 254 On a thin layer plate (Yantai 10×10), ethyl acetate-methanol-formic acid-water (18:2:1.5:1) was used as the developing agent, the plate was taken out, dried, sprayed with 1% vanillin sulfuric acid solution, heated until the spots were clearly colored, and examined. Figure 29 In the figure, 1 is the negative Qingxin Lianziyin compound preparation solution lacking Plantago seed, 2 to 4 are the test solution D of Z20241001, Z20241002, and Z20241003 granules respectively, 5 is the Plantago seed control medicinal material solution, and 6 is the geniposide acid reference substance solution ( Figure 30, Figures 32-34 It can be seen that in the chromatogram of the test sample, there are spots of the same color at the corresponding positions of the control medicinal material, and there is no interference from the negative test solution, indicating that this method has good specificity.

[0188] 2. Durability test

[0189] (1) Investigation of different developing agent ratios

[0190] 10 μl of each of the test solution D, the Qingxin Lianziyin compound preparation solution lacking Plantago seed negative, the Plantago seed control medicinal material solution, and the geniposide reference solution were taken and spotted on the same silica gel GF. 254 On a thin layer plate (Yantai 10×10), two thin layer plates were spotted in the same way, and the two plates were respectively stained with ethyl acetate-methanol-formic acid-water (18:1:1.5:1) ( Figure 30 (a)) and ethyl acetate-methanol-formic acid-water (18:3:1.5:1) ( Figure 30 (b)) was used as the developing solvent for development, and the other chromatographic conditions were the same as those above. Figure 29 It can be seen that different developing agent ratios have a certain influence on the Rf value of each spot, but there is no obvious difference in the separation effect, which shows that a slight fluctuation in the developing agent ratio of this method has no obvious effect on its development and separation.

[0191] (2) Investigation of different sample quantities

[0192] 10 μl of each of the above-mentioned Qingxin Lianziyin compound preparation solution lacking Plantago seed negative, Plantago seed control medicinal material solution, and Geniposide acid reference solution were taken, and 5 μl, 10 μl, and 15 μl of three test solution D (Z20241001, Z20241002, and Z20241003) were taken, respectively, and spotted on the same silica gel GF. 254 The other chromatographic conditions were the same as those above. Figure 31 1 represents the negative Qingxin Lianziyin compound preparation solution lacking Plantago seed; 2-4 represent the Z20241001 test solution D with spot sizes of 5μl, 10μl, and 15μl, respectively; 5-7 represent the Z20241002 test solution D with spot sizes of 5μl, 10μl, and 15μl, respectively; 8-10 represent the Z20241003 test solution D with spot sizes of 5μl, 10μl, and 15μl, respectively; 11 represents the Plantago seed control solution; and 12 represents the geniposide reference solution. As can be seen, the 5μl, 10μl, and 15μl spot sizes all exhibit clear spots and excellent chromatographic results. Therefore, the 5-15μl spot size was selected for the TLC identification method study of Plantago seed.

[0193] (3) Investigation at different temperatures

[0194] 10 μl of each of the test solution D, the Qingxin Lianziyin compound preparation solution lacking Plantago seed negative, the Plantago seed control medicinal material solution, and the geniposide reference solution were taken and spotted on the same silica gel GF. 254 On a thin layer plate (Yantai 10×10), spot two thin layer plates in the same way, and place the two plates at 4℃ ( Figure 32 (a)) and 30℃( Figure 33 (b)) and the rest of the chromatographic conditions were the same as above. Figure 29 It can be seen that different temperatures have a certain influence on the Rf value of each spot, but there is no obvious difference in the separation effect and spot display, which shows that this method has good durability at an ambient temperature of 4 to 30°C.

[0195] (4) Investigation at different humidity levels

[0196] 10 μl of each of the test solution D, the Qingxin Lianziyin compound preparation solution lacking Plantago seed negative, the Plantago seed control medicinal material solution, and the geniposide reference solution were taken and spotted on the same silica gel GF. 254 On a thin layer plate (Yantai 10×10), two thin layer plates were spotted in the same way and respectively at RH 32% ( Figure 33 (a)) and RH 88% ( Figure 33 (b)) and the rest of the chromatographic conditions were the same as above. Figure 29 It can be seen that different humidity has a certain effect on the Rf value of each spot, but there is no obvious difference in the separation effect and spot display, which shows that this method has good durability under an ambient humidity of 32% to 88%.

[0197] (5) Inspection of different brands of thin layer boards

[0198] 10 μl of each of the test solution D, the Qingxin Lianziyin compound preparation solution lacking Plantago seed negative, the Plantago seed control medicinal material solution, and the geniposide acid reference solution were taken, and the above operation was repeated twice. The samples were spotted on Merck plates (GF 254 Plate 10×10)( Figure 34 (a)) and Qingdao board (GF 254 Plate 10×10)( Figure 34 (a)) and the rest of the chromatographic conditions were the same as those above. Figure 29 It can be seen that the Rf values of each spot are different after development of different thin layer plates, but there is no obvious difference in the separation effect, and there are some differences in the spot colors.

[0199] 3. Multi-batch inspection of benchmark samples

[0200] (1) Preparation of test solution D: Take 40 mL of the n-butanol layer extract from the preparation process of the corresponding test solution C of the Qingxin Lianzi Drink reference sample (JZ-QXLZY240701~15), evaporate it to dryness, reconstitute the obtained residue with 3 mL of water, and perform column chromatography on activated Wates Sep-Pak Vac 6cc (500 mg) C18 Cartridges using 3 mL of 30% methanol aqueous solution as eluent. Evaporate the obtained 30% methanol aqueous solution layer eluate to dryness, and reconstitute the obtained residue with 1 mL of methanol to prepare the test solution D.

[0201] (2) Chromatographic conditions: According to the thin layer chromatography method (General Principle 0502), 10 μl of the test solution D, the plantago seed control medicinal material solution, and the geniposide acid reference solution were taken and spotted on the same silica gel GF 254 On a thin layer plate (Yantai 20×10), use ethyl acetate-methanol-formic acid-water (18:2:1.5:1) as the developing agent, develop, take out, dry, spray with 1% vanillin sulfuric acid solution, heat until the spots are clearly colored, and inspect. Figure 35 (a), (b), Figure 35 In (a), 1 is the ginseng reference medicinal material solution, 2 is the geniposide reference solution, and 3-10 are the test solution D of the JZ-QXLZY240701-08 reference sample; Figure 35 In (b), 1 is the ginseng reference medicinal material solution, 2 is the geniposide acid reference solution, and 3 to 9 are the test solution D of the JZ-QXLZY240709-15 benchmark sample. It can be seen that in the chromatogram of the test sample, there are spots of the same color at the corresponding positions of the reference medicinal material.

[0202] Example 6 Thin-layer chromatography identification of lotus seeds in Qingxin Lianziyin granules

[0203] 1. Identification method

[0204] (1) Preparation of test solution E: Take 6cc (500mg) of Wates Sep-Pak Vac C18 Cartridges after elution with 30% methanol in water during the preparation of test solution D, and continue eluting with 3mL of 50% methanol in water as the eluent. The resulting 50% methanol in water layer is evaporated to dryness, and the resulting residue is re-dissolved in 1mL of methanol to prepare test solution E.

[0205] (2) Preparation of lotus seed control medicinal material solution: 5 g of lotus seed control medicinal material was added to 50 mL of 95% ethanol aqueous solution, and ultrasonically extracted at 35 kHz for 30 min. The solution was filtered through qualitative filter paper, and the filtrate was evaporated to dryness. The residue was redissolved in 30 mL of water, and extracted twice with petroleum ether at a boiling range of 60-90 ° C, 30 mL each time. The aqueous layer was extracted twice with ethyl acetate, 30 mL each time. The aqueous layer was extracted twice with water-saturated n-butanol, 30 mL each time. The n-butanol layer was evaporated to dryness, and the residue was redissolved in 1 mL of methanol.

[0206] (3) Chromatographic conditions: According to the thin layer chromatography method (General Method 0502), 5 μl of the test solution E and 10 μl of the lotus seed control medicinal material solution were taken and spotted on the same silica gel GF 254 On a thin layer plate (Yantai 10×10), dichloromethane-methanol (4:1) was used as the developing solvent, the plate was taken out, dried, and examined under a UV lamp (365 nm). It can be seen that in the chromatogram of the test sample, there are fluorescent spots of the same color at the corresponding positions of the control medicinal material.

[0207] 2. Methodological Validation

[0208] 1. Specificity test

[0209] (1) Preparation of the compound preparation solution of the lotus seed deficiency negative Qingxin Lianziyin: Take the lotus seed deficiency negative Qingxin Lianziyin granules and prepare the compound preparation solution of the lotus seed deficiency negative Qingxin Lianziyin according to the same preparation method as the lotus seed control medicinal material solution.

[0210] (2) Chromatographic conditions: According to the thin layer chromatography method (General Rule 0502), 5 μl of the test solution E, 5 μl of the lotus seed-deficient negative Qingxin Lianziyin compound preparation solution, and 10 μl of the lotus seed control medicinal material solution were taken and spotted on the same silica gel GF 254 On a thin layer plate (Yantai 10×10), dichloromethane-methanol (4:1) was used as the developing agent, the plate was developed, taken out, dried, and examined under an ultraviolet lamp (365nm). Figure 36 In the figure, 1 is the solution of the Qingxin Lianziyin compound preparation lacking lotus seeds, 2 to 4 are the test solution E of Z20241001, Z20241002, and Z20241003 granules respectively, and 5 is the lotus seed control medicinal material solution ( Figure 37 , Figures 39-41 It can be seen that in the chromatogram of the test sample, there are fluorescent spots of the same color at the corresponding positions of the control medicinal material, and there is no interference from the negative test solution, indicating that this method has good specificity.

[0211] 2. Durability test

[0212] (1) Investigation of different developing agent ratios

[0213] 5 μl of the test solution E, 5 μl of the lotus seed negative Qingxin Lianziyin compound preparation solution, and 10 μl of the lotus seed control medicinal material solution were respectively taken and spotted on the same silica gel GF 254 On a thin layer plate (Yantai 10×10), two thin layer plates were spotted in the same way, and the two plates were respectively saturated with dichloromethane-methanol (100:23) ( Figure 37 (a)) and dichloromethane-methanol (100:27) ( Figure 37 (b)) was used as the developing solvent for development, and the other chromatographic conditions were the same as those above. Figure 36 It can be seen that different developing agent ratios have a certain influence on the Rf value of each spot, but there is no obvious difference in the separation effect, which shows that a slight fluctuation in the developing agent ratio of this method has no obvious effect on its development and separation.

[0214] (2) Investigation of different sample quantities

[0215] 5 μl of the above-mentioned lotus seed-deficient negative Qingxin Lianziyin compound preparation solution and 10 μl of lotus seed control medicinal material solution were respectively taken, and 2 μl, 5 μl and 10 μl of three test sample solutions E (Z20241001, Z20241002 and Z20241003) were taken respectively and spotted on the same silica gel GF 254 The other chromatographic conditions were the same as those above. Figure 38 1 represents the lotus seed-negative Qingxin Lianziyin compound preparation solution; 2-4 represent the Z20241001 test solution D with spot sizes of 2μl, 5μl, and 10μl, respectively; 5-7 represent the Z20241002 test solution D with spot sizes of 2μl, 5μl, and 10μl, respectively; 8-10 represent the Z20241003 test solution D with spot sizes of 2μl, 5μl, and 10μl, respectively; and 11 represents the lotus seed control medicinal material solution. As can be seen, the 2μl spot is unclear, while the 5μl and 10μl spot sizes provide clear spots and excellent chromatographic results. Therefore, 5-10μl spot sizes are suitable for studying the thin-layer chromatography identification method for lotus seeds.

[0216] (3) Investigation at different temperatures

[0217] 5 μl of the test solution E, 5 μl of the lotus seed negative Qingxin Lianziyin compound preparation solution, and 10 μl of the lotus seed control medicinal material solution were respectively taken and spotted on the same silica gel GF 254 On a thin layer plate (Yantai 10×10), spot two thin layer plates in the same way, and place the two plates at 4℃ ( Figure 39 (a)) and 30℃( Figure 39 (b)) and the rest of the chromatographic conditions were the same as above. Figure 36It can be seen that different temperatures have a certain influence on the Rf value of each spot, but there is no obvious difference in the separation effect and spot display, which shows that this method has good durability at an ambient temperature of 4 to 30°C.

[0218] (4) Investigation at different humidity levels

[0219] 5 μl of the test solution E, 5 μl of the lotus seed negative Qingxin Lianziyin compound preparation solution, and 10 μl of the lotus seed control medicinal material solution were respectively taken and spotted on the same silica gel GF 254 On a thin layer plate (Yantai 10×10), two thin layer plates were spotted in the same way and respectively at RH 32% ( Figure 40 (a)) and RH 88% ( Figure 40 (b)) and the rest of the chromatographic conditions were the same as above. Figure 36 It can be seen that different humidity has a certain effect on the Rf value of each spot, but there is no obvious difference in the separation effect and spot display, which shows that this method has good durability under an ambient humidity of 32% to 88%.

[0220] (5) Inspection of different brands of thin layer boards

[0221] 5 μl of the test solution E, 5 μl of the lotus seed negative Qingxin Lianziyin compound preparation solution, and 10 μl of the lotus seed control medicinal material solution were taken respectively, and the above operation was repeated twice, and the samples were spotted on Merck plates (GF 254 Plate 10×10)( Figure 41 (a)) and Qingdao board (GF 254 Plate 10×10)( Figure 41 (a)) and the rest of the chromatographic conditions were the same as those above. Figure 36 It can be seen that the Rf values of the spots on different thin layer plates are different after development. The separation effect and spot display show that the characteristic points of Merck plate and Qingdao plate are difficult to identify, while those of Yantai plate are clearer.

[0222] 3. Multi-batch inspection of benchmark samples

[0223] (1) Preparation of test solution E: Take the Wates Sep-Pak Vac 6cc (500mg) C18 Cartridges of the Qingxin Lianzi Drink reference sample (JZ-QXLZY240701~15) after elution with 30% methanol aqueous solution during the preparation of test solution D, and continue to elute with 3mL of 50% methanol aqueous solution as the eluent. The resulting 50% methanol aqueous solution layer eluate is evaporated to dryness, and the resulting residue is re-dissolved in 1mL of methanol to prepare the test solution E.

[0224] (2) Chromatographic conditions: According to the thin layer chromatography method (General Method 0502), 5 μl of the test solution E and 10 μl of the lotus seed control medicinal material solution were taken and spotted on the same silica gel GF254 On a thin layer plate (Yantai 10×10), use dichloromethane-methanol (4:1) as the developing solvent, develop, take out, dry, and examine under ultraviolet light (365nm). Figure 42 (a), (b), (c), Figure 42 In (a), 1 is the lotus seed control medicinal material solution, and 2 to 6 are the test solution E of the JZ-QXLZY240701 to 05 benchmark samples; Figure 42 In (b), 1 is the lotus seed control medicinal material solution, and 2 to 6 are the test solution E of samples JZ-QXLZY240706 to 10; Figure 42 In (c), 1 is the lotus seed control medicinal material solution, and 2 to 6 are the test solution E of samples JZ-QXLZY240711 to 15. It can be seen that in the chromatogram of the test sample, there are fluorescent spots of the same color at the corresponding positions of the control medicinal material.

[0225] Comparative Example 1

[0226] This comparative example is different from Example 2 only in that, when preparing the test solution B, there is no petroleum ether extraction step. The obtained test solution is recorded as test solution 1. The other preparation steps and chromatographic conditions are the same. The results are shown in FIG. Figure 43 (a) In the figure, 1 is test solution 1 (JZ-QXLZY240701), 2 is test solution B (JZ-QXLZY240701) prepared in Example 2, and 3 is the Cortex Lycii reference solution. It can be seen that other spots appear in test solution 1, which may affect the characteristic main spots.

[0227] Comparative Example 2

[0228] Compared with Example 4, this comparative example differs only in that the preparation method of the test solution is as follows: 1 g of the Qingxin Lianzi Drink reference sample (JZ-QXLZY240701) is taken, 25 ml of water is added, and heated to dissolve, and extracted twice by shaking with water-saturated n-butanol, 25 ml each time, the n-butanol solution is combined, and extracted twice by shaking with a mass fraction of 0.5% sodium hydroxide solution, 25 ml each time, and the obtained n-butanol solution is washed twice with n-butanol saturated water having a pH value of 3 to 4, 25 ml each time, and the water washing liquid is discarded, and then washed 3 times with n-butanol saturated water, 25 ml each time, and the water washing liquid is discarded. The n-butanol solution is evaporated to dryness, and the residue is dissolved in 1 ml of methanol, which is recorded as the test solution 2. The preparation method and chromatographic conditions of the remaining mixed reference solutions are the same, and the results are shown in FIG. Figure 43 (b) In the figure, 1 is test solution 2, 2 is test solution C (JZ-QXLZY240701) prepared in Example 4, 3 is ginseng control herbal solution, and 4 is the mixed reference solution. It can be seen that the first characteristic point in test solution 2 has significant interference, and the presence of components such as liquiritin and astragaloside IV can affect the main spot.

[0229] Comparative Example 3

[0230] In this comparative example, three different methods were used to prepare the test solution. The test sample used was the Qingxin Lianzi Drink reference sample (JZ-QXLZY240701). (1) The Qingxin Lianzi Drink reference sample (JZ-QXLZY240701) was directly extracted twice with n-butanol, 30 ml each time. The obtained test solution was recorded as test solution 3a. (2) Compared with the steps of preparing test solution D in Example 5, the only difference was that 10% methanol aqueous solution was used as the eluent. The obtained test solution was recorded as test solution 3b. (3) Compared with the steps of preparing test solution D in Example 5, the only difference was that 50% methanol aqueous solution was used as the eluent. The obtained test solution was recorded as test solution 3c. The preparation method and chromatographic conditions of Plantago seed control medicinal material solution and geniposide reference solution were the same as those in Example 5. The results are shown in Table. Figure 43 (c). In the figure, 1 is test solution 3a, 2 is test solution 3b, 3 is test solution 3c, 4 is test solution D prepared in Example 5, 5 is the Plantago seed control medicinal material solution, and 6 is the geniposide acid reference solution. It can be seen that in test solutions 3a-c, the yellow spots interfere severely, and the purple characteristic spots of geniposide acid cannot be identified.

[0231] Comparative Example 4

[0232] In this comparative example, three different methods were used to prepare the test solution. The test sample used was the Qingxin Lianzi Drink reference sample (JZ-QXLZY240701). (1) The Qingxin Lianzi Drink reference sample (JZ-QXLZY240701) was directly extracted twice with n-butanol, 30 ml each time. The obtained test solution was recorded as test solution 4a. (2) Compared with the steps of preparing test solution E in Example 6, the only difference was that 30% methanol aqueous solution was used as eluent. The obtained test solution was recorded as test solution 4b. (3) Compared with the steps of preparing test solution E in Example 6, the only difference was that 70% methanol aqueous solution was used as eluent. The obtained test solution was recorded as test solution 4c. The preparation method and chromatographic conditions of the lotus seed control medicinal material solution were the same as those in Example 6. The results are shown in FIG. Figure 43 (d) In the figure, 1 is test solution 4a, 2 is test solution 4b, 3 is test solution 4c, 4 is test solution E prepared in Example 6, and 5 is a lotus seed control medicinal material solution. It can be seen that in test solutions 4a and b, there are no characteristic points corresponding to lotus seeds, while in test solution 4c, the characteristic points are blurred and have a lot of interference.

[0233] Figure 44This is a flow chart of the preparation of test solutions A to E in Examples 1 to 6 of the present invention. It can be intuitively seen from the flow chart that the quality control method provided by the present invention can achieve the extraction of the Qingxin Lianziyin compound preparation test sample only once for the identification of 6 medicinal flavors, reducing the repeated extraction of reference samples when identifying different medicinal flavors, reducing the amount of reagents used, making the operation more convenient, and being able to improve the efficiency of thin layer identification, making it more systematic.

[0234] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A quality control method for a Qingxin Lianziyin compound preparation, wherein the Qingxin Lianziyin compound preparation is prepared from raw materials including scutellaria baicalensis, lycium bark, ginseng, liquorice, plantain seeds, and lotus seeds, characterized in that: The quality control method comprises the following steps, Take the test sample and prepare a test solution according to at least one of the following methods 1 to 5 and perform the test: Method 1: Prepare a test solution A, and identify Scutellaria baicalensis by thin layer chromatography based on the test solution A; Method 2: Prepare a test solution B, and use thin layer chromatography to identify the root bark of Lycium bark and Licorice root based on the test solution B; The preparation process of the test solution B comprises: taking a test sample of the Qingxin Lianziyin compound preparation, adding an alcohol aqueous solution, ultrasonically extracting, solid-liquid separation, taking the liquid, evaporating to dryness, dissolving the resulting residue in water, extracting with petroleum ether, taking the water layer, extracting with ethyl acetate, taking the ethyl acetate layer, evaporating to dryness, and re-dissolving the resulting residue in alcohol; Method 3: Prepare a test solution C, and identify ginseng by thin layer chromatography based on the test solution C; the preparation process of the test solution C comprises: extracting the aqueous layer obtained after ethyl acetate extraction in the preparation process of the test sample B with water-saturated n-butanol, taking the n-butanol layer extract, washing it with ammonia test solution, washing the n-butanol layer with n-butanol-saturated water, taking the n-butanol layer, evaporating it to dryness, and re-dissolving the resulting residue in alcohol; Method 4: Prepare a test solution D, and identify Plantago seed based on the test solution D by thin layer chromatography; the preparation process of the obtained test solution D comprises: taking the n-butanol layer extract obtained in the preparation process of the test sample C, evaporating it to dryness, re-dissolving the obtained residue in water, performing column chromatography on a silica gel-based solid phase extraction column, using a 20% to 40% methanol aqueous solution as an eluent, evaporating the obtained 20% to 40% methanol aqueous solution layer eluate to dryness, and re-dissolving the obtained residue in alcohol; Method 5: Prepare a test solution E, and identify lotus seeds based on the test solution E using thin layer chromatography; the preparation process of the obtained test solution E includes: taking a solid phase extraction column eluted with a 20% to 40% methanol aqueous solution in the process of preparing the test solution D, eluting with a 40% to 60% methanol aqueous solution by volume as an eluent, evaporating the obtained 40% to 60% methanol aqueous solution layer eluate to dryness, and re-dissolving the obtained residue in alcohol.

2. The quality control method of the Qingxin Lianziyin compound preparation according to claim 1, characterized in that: The preparation process of the test solution A comprises: taking a test sample of the Qingxin Lianzi Drink compound preparation, adding an alcohol aqueous solution, ultrasonic extraction, solid-liquid separation, taking the liquid, evaporating to dryness, and re-dissolving the obtained residue with alcohol; optionally, the preparation process of the test solution A comprises: taking 5-15 g of the Qingxin Lianzi Drink compound preparation test sample, adding 50-150 mL of an 85%-98% alcohol aqueous solution, ultrasonic extraction at 35-53 kHz for 20-60 min, solid-liquid separation, taking 5-15 mL of the liquid, evaporating to dryness, and re-dissolving the obtained residue with 0.5-1.5 mL of alcohol; And / or, the preparation process of the test solution B comprises: taking 5-15 g of the test sample of the Qingxin Lianziyin compound preparation, adding 50-150 mL of an aqueous solution with a volume fraction of 85%-98% alcohol, ultrasonically extracting at 35-53 kHz for 20-60 min, separating the solid and liquid, taking 5-100 mL of the liquid, evaporating to dryness, dissolving the resulting residue in 15-45 mL of water, extracting 2-4 times with 15-45 mL of petroleum ether with a boiling range of 60-90° C., taking the aqueous layer, extracting 2-4 times with 15-45 mL of ethyl acetate, taking the ethyl acetate layer, evaporating to dryness, and re-dissolving the resulting residue in 1.5-4.5 mL of alcohol; And / or, the preparation process of the test solution C includes: taking 15-45 mL of the aqueous layer obtained after ethyl acetate extraction in the preparation process of the test sample B, extracting 2-4 times with 20-40 mL of water-saturated n-butanol, combining the n-butanol layer extracts, taking 10-30 mL of the n-butanol layer extracts, washing 2-4 times with 10-30 mL of ammonia test solution, taking the n-butanol layer and washing it 2-4 times with 20-40 mL of n-butanol-saturated water, taking the n-butanol layer, evaporating it to dryness, and re-dissolving the resulting residue in 0.5-2 mL of alcohol; And / or, the preparation process of the obtained test solution D includes: taking 30-50 mL of the n-butanol layer extract obtained in the preparation process of the test sample C, evaporating it to dryness, re-dissolving the obtained residue with 1.5-4.5 mL of water, and performing column chromatography on a 500 mg C18 solid phase extraction column, using 1.5-4.5 mL of 20%-40% methanol aqueous solution as an eluent, evaporating the obtained 20%-40% methanol aqueous solution layer eluent to dryness, and re-dissolving the obtained residue with 0.5-2 mL of alcohol; optionally, using 30% methanol aqueous solution as an eluent; And / or, the preparation process of the obtained test solution E includes: taking the C18 solid phase extraction column after elution with 20% to 40% methanol aqueous solution in the process of preparing the test solution D, continuing to elute with 1.5 to 4.5 mL of 40% to 60% methanol aqueous solution as an eluent, evaporating the obtained 40% to 60% methanol aqueous solution layer eluate to dryness, and re-dissolving the obtained residue with 0.5 to 2 mL of alcohol; optionally, using 50% methanol aqueous solution as an eluent.

3. The quality control method of the Qingxin Lianziyin compound preparation according to claim 1 or 2, characterized in that: Based on the test solution A, thin layer chromatography is used to identify Scutellaria baicalensis, comprising the following steps: taking the test solution A, Scutellaria baicalensis reference medicinal material solution, and baicalin reference solution, respectively spotting them on the same silica gel thin layer plate, using ethyl acetate-butanone-formic acid-water mixture as a developing solvent and ferric chloride ethanol solution as a color developer, and inspecting them; And / or, based on the test solution B, using thin layer chromatography to identify the root bark of Chinese wolfberry, comprising the following steps: taking the test solution B and the root bark of Chinese wolfberry control medicinal material solution, respectively spotting them on the same silica gel thin layer plate, using a dichloromethane-methanol mixture as a developing solvent, and inspecting under ultraviolet light; And / or, based on the test solution B, using thin layer chromatography to identify licorice, comprising the following steps: taking the test sample B, the licorice control medicinal material solution, and the liquiritin reference solution, respectively spotting them on the same silica gel thin layer plate, using a dichloromethane-methanol mixture as a developing solvent and an ethanol solution of sulfuric acid as a color developer, heating until the spots are clearly colored and then inspecting them under ultraviolet light; And / or, based on the test solution C, thin layer chromatography is used to identify ginseng, comprising the following steps: taking the test sample C, the ginseng control medicinal material solution, and the mixed reference solution, spotting them respectively on the same silica gel thin layer plate, using a chloroform-methanol-water mixture as a developing solvent and an ethanolic solution of sulfuric acid as a color developer, heating until the spots are clearly colored, and then inspecting under ultraviolet light; wherein the mixed reference solution contains a ginsenoside Re reference substance, a ginsenoside Rg1 reference substance, and a ginsenoside Rb1 reference substance; And / or, based on the test solution D, identifying Plantago seed by thin layer chromatography, comprising the following steps: taking the test solution D, the Plantago seed control medicinal material solution, and the geniposide reference solution, spotting them respectively on the same silica gel thin layer plate, using an ethyl acetate-methanol-formic acid-water mixture as a developing solvent and a vanillin sulfuric acid solution as a color developer, heating until the spots are clearly colored for inspection; And / or, based on the test solution E, thin layer chromatography is used to identify lotus seeds, comprising the following steps: taking the test solution E and the lotus seed control medicinal material solution, respectively spotting them on the same silica gel thin layer plate, using a dichloromethane-methanol mixture as a developing solvent, and inspecting under ultraviolet light.

4. The quality control method of the Qingxin Lianziyin compound preparation according to claim 3, characterized in that: The preparation process of the scutellaria baicalensis reference medicinal material solution comprises: taking 0.5-1 g of the scutellaria baicalensis reference medicinal material, adding 10-40 mL of an aqueous solution with a volume fraction of 85%-98% alcohol, ultrasonically extracting at 35-53 kHz for 10-30 minutes, separating the solid and the liquid, taking the liquid, evaporating it to dryness, and re-dissolving the resulting residue in 0.5-1.5 mL of alcohol; And / or, the preparation process of the baicalin reference solution comprises: taking a baicalin reference, adding an organic solvent to prepare a baicalin reference solution; optionally, each 1 mL of the baicalin reference solution contains 0.5 to 1.5 mg of the baicalin reference; optionally, the organic solvent comprises methanol; And / or, the sample volume of the test solution A, the scutellaria baicalensis reference medicinal material solution, and the baicalin reference solution is 2 to 10 μL; optionally, the sample volume is 5 μL; And / or, in the step of identifying Scutellaria baicalensis by thin layer chromatography based on the test solution A, the volume ratio of ethyl acetate, butanone, formic acid, and water in the developing solvent is 6 to 4:2 to 4:1:1; optionally, the volume ratio is 5:3:1:1; And / or, in the step of identifying Scutellaria baicalensis by thin layer chromatography based on the test solution A, the mass fraction of the ferric chloride ethanol solution is 1% to 3%.

5. The quality control method of the Qingxin Lianziyin compound preparation according to claim 3, characterized in that: The preparation process of the Chinese wolfberry bark control medicinal material solution comprises: taking 0.5-1.5g of Chinese wolfberry bark control medicinal material, adding 10-30mL of ethyl acetate, ultrasonically extracting at 35-53kHz for 15-60min, separating the solid and the liquid, taking the liquid, evaporating it to dryness, and re-dissolving the obtained residue in 0.5-1.5mL of alcohol; And / or, the sample volume of the test solution B and the Radix Lycii control medicinal material solution is 2 to 10 μL; optionally, the sample volume is 5 to 10 μL; And / or, in the step of identifying the root bark of Lycium bark by thin layer chromatography based on the test solution B, the volume ratio of dichloromethane to methanol in the developing solvent is 20:0.5-1.5; optionally, the volume ratio is 20:1; And / or, in the step of identifying the cortex rehmanniae by thin layer chromatography based on the test solution B, the wavelength of the ultraviolet light is 365nm.

6. The quality control method of the Qingxin Lianziyin compound preparation according to claim 3, characterized in that: The preparation process of the licorice control medicinal material solution includes: taking 0.5-1.5g of the licorice control medicinal material, adding 10-30mL of ethyl acetate, ultrasonically extracting at 35-53kHz for 15-60min, separating the solid and liquid, taking the liquid, evaporating it to dryness, and re-dissolving the obtained residue in 0.5-1.5mL of alcohol; And / or, the preparation process of the liquiritin reference solution comprises: taking a liquiritin reference substance and adding an organic solvent to prepare a liquiritin reference solution; optionally, each 1 mL of the liquiritin reference solution contains 0.5 to 1.5 mg of the liquiritin reference substance; optionally, the organic solvent comprises methanol; And / or, the sample volume of the test solution B is 2 to 10 μL; And / or, the spotting volume of the licorice control medicinal material solution and the liquiritin reference solution is 1 to 3 μL; And / or, in the step of identifying licorice by thin layer chromatography based on the test solution B, the volume ratio of dichloromethane to methanol in the developing solvent is 10:3 to 5:1; optionally, the volume ratio is 4:1; And / or, in the step of identifying licorice by thin layer chromatography based on the test solution B, the volume fraction of the ethanol solution of sulfuric acid is 5% to 15%; And / or, in the step of identifying licorice by thin layer chromatography based on the test solution B, the wavelength of the ultraviolet light is 365nm.

7. The quality control method of the Qingxin Lianziyin compound preparation according to claim 3, characterized in that: The preparation process of the ginseng control medicinal material solution includes: taking 0.5-1.5 g of ginseng control medicinal material, adding 10-30 mL of an aqueous solution with a volume fraction of 85%-98% alcohol, ultrasonic extraction at 35-53 kHz for 15-45 minutes, solid-liquid separation, taking the liquid, evaporating to dryness, adding 20-30 mL of water to the obtained residue, extracting 2-4 times with 20-30 mL of petroleum ether with a boiling range of 60-90° C., taking the water layer, extracting 2-4 times with 20-30 mL of water-saturated n-butanol, combining the n-butanol layer extracts, washing 2-4 times with 10-30 mL of ammonia test solution, taking the n-butanol layer, washing 2-4 times with 20-30 mL of n-butanol-saturated water, taking the upper layer liquid, evaporating to dryness, and adding 0.5-2 mL of alcohol to the obtained residue; And / or, the preparation process of the mixed reference solution comprises: taking ginsenoside Re reference substance, ginsenoside Rg1 reference substance, and ginsenoside Rb1 reference substance, and adding an organic solvent to prepare a mixed reference solution; optionally, the content of ginsenoside Re reference substance, ginsenoside Rg1 reference substance, and ginsenoside Rb1 reference substance in each 1 mL of the mixed reference solution is 0.5-1.5 mg, respectively; optionally, the organic solvent comprises methanol; And / or, the sample C is spotted in an amount of 2 to 10 μL; optionally, the sample is spotted in an amount of 5 to 10 μL; And / or, the spotting volume of the ginseng control medicinal material solution and the mixed reference substance solution is 1 to 3 μL; And / or, in the step of identifying ginseng by thin layer chromatography based on the test solution C, the volume ratio of chloroform, methanol, and water in the developing solvent is 13:6 to 8:2; optionally, the volume ratio is 13:7:2; optionally, the lower layer solution refrigerated overnight using the developing solvent is refrigerated at a temperature of ≤10°C; And / or, in the step of identifying ginseng by thin layer chromatography based on the test solution C, the volume fraction of the ethanol solution of sulfuric acid is 5% to 15%; And / or, in the step of identifying ginseng by thin layer chromatography based on the test solution C, the wavelength of the ultraviolet light is 365 nm.

8. The quality control method of the Qingxin Lianziyin compound preparation according to claim 3, characterized in that: The preparation process of the plantago seed control medicinal material solution includes: taking 0.5-1.5 g of the plantago seed control medicinal material, adding 10-30 mL of alcohol, ultrasonically extracting at 35-53 kHz for 15-45 minutes, separating the solid and liquid, taking the liquid, evaporating it to dryness, re-dissolving the obtained residue in 2-4 mL of water, performing column chromatography on a 500 mg C18 solid phase extraction column, using 3-5 mL of a 20%-40% methanol aqueous solution as an eluent, evaporating the obtained 20%-40% methanol aqueous solution layer to dryness, and re-dissolving the obtained residue in 0.5-2 mL of alcohol; And / or, the preparation process of the geniposide acid reference solution comprises: taking a geniposide acid reference substance and adding an organic solvent to prepare a geniposide acid reference solution; optionally, each 1 mL of the geniposide acid reference solution contains 0.5 to 1 mg of the geniposide acid reference substance; optionally, the organic solvent comprises methanol; And / or, the spotting volume of the test solution D, the plantago seed reference medicinal material solution, and the geniposide reference solution is 5 to 15 μL; And / or, in the step of identifying Plantago seed by thin layer chromatography based on the test solution D, the volume ratio of ethyl acetate, methanol, formic acid, and water in the developing solvent is 18:1 to 3:1.5:1; optionally, the volume ratio is 18:2:1.5:1; And / or, in the step of identifying Plantago seed by thin layer chromatography based on the test solution D, the mass fraction of the vanillin sulfuric acid solution is 1% to 3%.

9. The quality control method of the Qingxin Lianziyin compound preparation according to claim 3, characterized in that: The preparation process of the lotus seed control medicinal material solution comprises: taking 3-7 g of lotus seed control medicinal material, adding 40-60 mL of an aqueous solution with a volume fraction of 85%-98% alcohol, performing ultrasonic extraction at 35-53 kHz for 15-45 minutes, separating the solid and liquid, taking the liquid, evaporating to dryness, adding 20-40 mL of water to the obtained residue, extracting 2-4 times with 20-40 mL of petroleum ether with a boiling range of 60-90° C., taking the water layer, extracting 2-4 times with 20-40 mL of ethyl acetate, taking the water layer, extracting 2-4 times with 20-40 mL of water-saturated n-butanol, taking the n-butanol layer, evaporating to dryness, and adding 0.5-2 mL of alcohol to the obtained residue; And / or, the sample volume of the test solution B is 2 to 10 μL; optionally, the sample volume is 5 to 10 μL; And / or, the spotting volume of the lotus seed control medicinal material solution is 7 to 13 μL; And / or, in the step of identifying lotus seeds by thin layer chromatography based on the test solution E, the volume ratio of dichloromethane to methanol in the developing solvent is 100:23-27; optionally, the volume ratio is 4:1; And / or, in the step of identifying lotus seeds by thin layer chromatography based on the test solution E, the wavelength of the ultraviolet light is 365 nm.

10. The quality control method for the Qingxin Lianziyin compound preparation according to any one of claims 1 to 9, characterized in that: The Qingxin Lianzi Drink compound preparation includes a solid preparation, a semisolid preparation or a liquid preparation of Qingxin Lianzi Drink; optionally, the Qingxin Lianzi Drink compound preparation includes a Qingxin Lianzi Drink reference sample decoction, a Qingxin Lianzi Drink reference sample concentrated extract, a Qingxin Lianzi Drink reference sample freeze-dried powder, a Qingxin Lianzi Drink preparation, a Qingxin Lianzi Drink intermediate extract or a Qingxin Lianzi Drink intermediate inclusion compound; And / or, the alcohol aqueous solution includes one or more of a methanol aqueous solution and an ethanol aqueous solution; optionally, the alcohol aqueous solution is an ethanol aqueous solution; And / or, the alcohol includes one or more of methanol and ethanol; optionally, the alcohol is methanol.

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