Quality detection method of infantile heat clearing granules and application of quality detection method

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to a quality detection method of children heat clearing granules and application thereof. The quality detection method of the infantile heat-clearing granules provided by the invention comprises the following steps: qualitatively identifying safflower, arbutin, alpine horseradish and liquorice in the infantile heat-clearing granules by adopting a thin layer chromatography, and quantitatively determining total flavonoids and hydroxysafflor yellow A in the infantile heat-clearing granules by adopting an ultraviolet detection method and a high performance liquid chromatography detection method respectively. The quality detection method established by the invention is stable and controllable, and the detection method in the quality standard can exclusively, repeatedly and effectively control the quality of the product, thereby ensuring the safety and effectiveness of clinical medication.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of traditional Chinese medicine, and particularly relates to a quality detection method of pediatric heat-clearing granules and application thereof. BACKGROUND

[0002] The pediatric heat-clearing decoction powder is recorded in the Tibetan Medical Formula Supplement, and the formula includes seven medicinal materials of calcified limestone, safflower, in-vitro cultured ox gall, bonga, ligakadou, glycyrrhiza extract, and high mountain radix euphorbiae, which are prepared into coarse powder and decocted in water, and are used for treating cold shivering, fever, running nose, headache, cough, coughing up phlegm, general body aches, sore throat, chest tightness, and rapid breathing caused by various plagues such as common cold and influenza in children.

[0003] The preparation product of the pediatric heat-clearing decoction powder is only the preparation product of medical institutions in the Tibet Autonomous Region (Tibetan medicine preparation Z20222026000), and the prescription is 250g of calcified limestone, 150g of safflower, 0.1g of in-vitro cultured ox gall, 70g of bonga, 70g of ligakadou, 70g of glycyrrhiza extract, and 100g of high mountain radix euphorbiae.

[0004] The pediatric heat-clearing granule is a modern improved preparation of the pediatric heat-clearing decoction powder, and the preparation process mainly includes taking each medicinal material of the pediatric heat-clearing decoction powder, adding appropriate amount of water for reflux extraction, concentrating the extraction liquid, alcohol precipitation, drying to obtain extraction paste, and then mixing the extraction paste with appropriate amount of medicinal adjuvant, granulating, and drying to obtain the pediatric heat-clearing granule.

[0005] Based on the modern medicine quality control method, the application establishes the quality detection method for the qualitative and quantitative determination of the effective components of the pediatric heat-clearing granule, so as to ensure the stable and controllable quality of the product and provide guarantee for the safety and effectiveness of the product. SUMMARY

[0006] The application aims to provide a quality detection method of pediatric heat-clearing granule, and establish a thin-layer identification method of safflower, arbutin, high mountain radix euphorbiae, and glycyrrhiza in the pediatric heat-clearing granule, an ultraviolet detection method of total flavonoids, and a high performance liquid chromatography detection method of hydroxyl safflower yellow A. The method can comprehensively and accurately analyze the active components of the medicine, is beneficial to the quality control of the effective components of the product, and ensures the clinical curative effect.

[0007] The application is achieved by the following technical scheme. A quality detection method of pediatric heat-clearing granule, the prescription composition of the pediatric heat-clearing granule includes 250 parts of calcified limestone, 150 parts of safflower, 70 parts of ligakadou, 100 parts of high mountain radix euphorbiae, 70 parts of bonga, 70 parts of glycyrrhiza extract, 0.1 part of in-vitro cultured ox gall, and appropriate amount of medicinal adjuvant, and the detection method is to detect the safflower, arbutin, high mountain radix euphorbiae, glycyrrhiza, total flavonoids, and hydroxyl safflower yellow A in the pediatric heat-clearing granule by identification and / or content determination.

[0008] The identification method is thin layer chromatography, and the safflower, arbutin, high mountain radish and licorice in the pediatric heat-clearing granules are identified under 254 nm ultraviolet light or natural light. The content determination method is ultraviolet detection method for determining the total flavonoid content at 510 nm wavelength, and high performance liquid chromatography detection method for determining the content of hydroxysafflor yellow A at 403 nm wavelength.

[0009] The pediatric heat-clearing granules are clinically used for treating various symptoms caused by plague, such as chill, fever, runny nose, headache, cough, sputum, body aches, sore throat, chest tightness and rapid breathing.

[0010] The main medicine safflower contains rich flavonoids, alkaloids, glycosides, sterols, polysaccharides and other components, and has the effects of relieving cough, expectorating sputum, relieving asthma and resisting oxidation. Arbutin is the effective component of rhododendron, which has the effects of anti-inflammatory analgesia, expectorating sputum, relieving asthma, resisting oxidation and resisting tumor. The high mountain radish has the effects of clearing lung heat, relieving cough and expectorating sputum, stopping bleeding and swelling. In addition, the licorice extract, rhododendron and rhododendron all contain various flavonoids, which play the effects of anti-inflammatory, antibacterial and antiviral. Therefore, the safflower, arbutin, high mountain radish, licorice and total flavonoids are selected as the comprehensive quality control indicators of the pediatric heat-clearing granules.

[0011] Preferably, the thin layer chromatography identification method is as follows: (1) The pediatric heat-clearing granules are taken into a conical flask with a plug, acetone solution is added, ultrasonic treatment, centrifugation and concentration are carried out, and the obtained solution is used as a test sample solution; (2) The safflower control medicine is taken, and a control sample solution is prepared according to the preparation method of the test sample solution; (3) The remaining medicines except safflower are weighed according to the prescription, and a negative control sample without safflower is prepared. The negative control sample solution is prepared according to the preparation method of the test sample solution; (4) The above three solutions are respectively spotted on the same silica gel G thin layer plate, n-butanol-methanol-water is used as a developing agent, and the plate is developed, taken out and dried. The plate is observed under ultraviolet light. In the test sample chromatogram, the same color spots appear at the same positions as the control sample chromatogram, and no spots appear at the same positions as the control sample chromatogram in the negative control sample chromatogram.

[0012] Preferably, the thin layer chromatography identification method is as follows: (1) The pediatric heat-clearing granules are taken into a conical flask with a plug, acetone solution is added, ultrasonic treatment, centrifugation and concentration are carried out, and the obtained solution is used as a test sample solution; (2) The safflower control medicine is taken, and a control sample solution is prepared according to the preparation method of the test sample solution; (3) Weigh out the remaining medicinal materials except for Ligadu according to the prescription of Xiaor Qingre Granules, prepare a negative control sample without Ligadu, and prepare a negative control sample solution without Ligadu according to the preparation method of the test sample solution; (4) Apply the above three solutions to the same silica gel G thin layer plate, develop with chloroform-methanol as the developing solvent, remove, air dry, spray with phosphomolybdic acid ethanol solution and heat to 105°C until the spots are clear; in the chromatogram of the test sample, spots of the same color appear at the same position as in the chromatogram of the reference sample, and in the chromatogram of the negative control sample, no spots appear at the same position as in the chromatogram of the reference sample.

[0013] Preferably, the thin-layer chromatography identification method is as follows: (1) Take the pediatric heat-clearing granules into an Erlenmeyer flask, add chloroform, sonicate, filter, and concentrate to obtain the test solution; (2) Take the high-altitude horseradish reference material into an Erlenmeyer flask and prepare the reference solution according to the preparation method of the test solution; (3) Weigh out the remaining medicinal materials except for horseradish according to the prescription of Xiaor Qingre Granules, prepare a negative control sample without horseradish, and prepare a negative control sample solution without horseradish according to the preparation method of the test sample solution. (4) Apply the above three solutions separately to the same silica gel G thin layer plate, develop with cyclohexane-ethyl ether-ethyl acetate as the developing solvent, remove, air dry, spray with sulfuric acid ethanol solution, and heat at 105℃ until the spots are clearly visible; in the chromatogram of the test sample, spots of the same color appear at the same position as in the chromatogram of the reference sample, and in the chromatogram of the negative control sample, no spots appear at the same position as in the chromatogram of the reference sample.

[0014] Preferably, the thin-layer chromatography identification method is as follows: (1) Take the children's heat-clearing granules into an Erlenmeyer flask, add water to dissolve, extract with n-butanol, extract with water again, evaporate the n-butanol solution to dryness, add methanol to redissolve, and use as the test solution; (2) Take an appropriate amount of licorice reference material and prepare a reference solution according to the preparation method of the test solution; (3) Weigh out the remaining medicinal materials except for licorice extract according to the prescription of Xiaor Qingre Granules, prepare a negative control sample without licorice extract, and prepare a negative control sample solution without licorice extract according to the preparation method of the test sample solution; (4) Apply the above three solutions separately to the same silica gel G thin layer plate, develop with ethyl acetate-formic acid-glacial acetic acid-water as the developing solvent, remove, air dry, spray with sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly visible, and examine under ultraviolet light; in the chromatogram of the test sample, spots of the same color appear at the same position as in the chromatogram of the reference sample, and in the chromatogram of the negative control sample, no spots appear at the same position as in the chromatogram of the reference sample.

[0015] More preferably, the thin-layer chromatography identification method is as follows: (1) Take 5 g of Xiaor Qingre Granules into a stoppered conical flask, add 20 mL of 80% acetone solution, sonicate (power 250 W, frequency 50 kHz) for 30 min, centrifuge, and evaporate the supernatant to 1 mL as the test solution. (2) Take 0.5 g of safflower reference material, add 20 mL of 80% acetone solution, sonicate (power 250 W, frequency 50 kHz) for 30 min, centrifuge, and evaporate the supernatant to 1 mL as the reference solution; (3) Weigh out the remaining medicinal materials except safflower according to the prescription of Xiaor Qingre Granules, prepare a negative control sample without safflower, and prepare a negative control sample solution without safflower according to the preparation method of the test sample solution; (4) Take 2 μL of each of the above three solutions and spot them separately on the same silica gel G thin-layer plate. Use n-butanol:methanol:water = 6:1:5 as the developing solvent, develop, remove, air dry, and examine under a UV lamp at 365 nm. In the chromatogram of the test sample, spots of the same color appear at the same positions as in the chromatogram of the reference sample. In the chromatogram of the negative control sample, no spots appear at the same positions as in the chromatogram of the reference sample.

[0016] More preferably, the thin-layer chromatography identification method is as follows: (1) Take 5 g of Xiaor Qingre Granules into an Erlenmeyer flask, add 20 mL of methanol, (power 250 W, frequency 50 kHz) for 40 min, centrifuge, and distill the supernatant down to 1 mL to obtain the test solution. (2) Prepare a solution containing 0.2 mg of arbutin reference standard in methanol and use it as the reference solution; (3) Weigh out the remaining medicinal materials except for Ligadu according to the prescription of Xiaor Qingre Granules, prepare a negative control sample without Ligadu, and prepare a negative control sample solution without Ligadu according to the preparation method of the test sample solution; (4) Take 2 μL of each of the above three solutions and spot them separately on the same silica gel G thin-layer plate. Develop the plate using chloroform:methanol = 8:2 as the developing solvent. Remove the plate, air dry it, spray it with 10% phosphomolybdic acid ethanol solution, and heat it to 105℃ until the spots are clear. In the chromatogram of the test sample, spots of the same color appear at the same positions as those in the chromatogram of the reference sample. In the chromatogram of the negative control sample, no spots appear at the same positions as those in the chromatogram of the reference sample.

[0017] More preferably, the thin-layer chromatography identification method is as follows: (1) Take 5 g of Xiaor Qingre Granules into an Erlenmeyer flask, add 20 mL of chloroform, sonicate (power 250 W, frequency 50 kHz) for 30 min, filter, and evaporate the filtrate to 1 mL as the test solution. (2) Take 1g of the reference herb horseradish in an Erlenmeyer flask, add 20 mL of chloroform, sonicate (power 250 W, frequency 50 kHz) for 30 min, filter, and evaporate the filtrate to 1 mL as the reference solution. (3) Weigh out the remaining medicinal materials except for horseradish according to the prescription of Xiaor Qingre Granules, prepare a negative control sample without horseradish, and prepare a negative control sample solution without horseradish according to the preparation method of the test sample solution. (4) Apply 2 μL of each of the above three solutions to the same silica gel G thin-layer plate. Develop the plate using cyclohexane:ethyl ether:ethyl acetate = 20:5.5:2.5 as the developing solvent. Remove the plate, air dry, spray with 30% sulfuric acid ethanol solution, and heat at 105℃ until the spots are clearly visible. In the chromatogram of the test sample, spots of the same color appear at the same positions as in the chromatogram of the reference sample. In the chromatogram of the negative control sample, no spots appear at the same positions as in the chromatogram of the reference sample.

[0018] More preferably, the thin-layer chromatography identification method is as follows: (1) Take 5 g of Xiaor Qingre Granules into an Erlenmeyer flask, add 20 mL of water to dissolve, extract with n-butanol 3 times, 20 mL each time (centrifuge if necessary), combine the n-butanol solutions, extract with water 3 times, 30 mL each time, evaporate the n-butanol solution to dryness, add 5 mL of methanol to redissolve, and use as the test solution. (2) Take 1 g of licorice reference material, add 20 mL of water to dissolve it, extract it 3 times with n-butanol, 20 mL each time (centrifuge if necessary), combine the n-butanol solutions, extract it 3 times with water, 30 mL each time, evaporate the n-butanol solution to dryness, add 5 mL of methanol to redissolve it, and use it as the reference solution. (3) Weigh out the remaining medicinal materials except for licorice extract according to the prescription of Xiaor Qingre Granules, prepare a negative control sample without licorice extract, and prepare a negative control sample solution without licorice extract according to the preparation method of the test sample solution; (4) Take 2 μL of each of the above three solutions and spot them separately on the same silica gel G thin-layer plate. Use ethyl acetate:formic acid:glacial acetic acid:water = 15:1:1:2 as the developing solvent. Develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, and heat at 105℃ until the spots are clearly visible. Examine under ultraviolet light (365 nm). In the chromatogram of the test sample, spots of the same color appear at the same positions as in the chromatogram of the reference sample. In the chromatogram of the negative control sample, no spots appear at the same positions as in the chromatogram of the reference sample.

[0019] Preferably, the ultraviolet detection method is as follows: (1) Preparation of reference solution: Accurately weigh 10.43 mg of rutin reference standard into a 50 mL volumetric flask, add an appropriate amount of 30% ethanol, dissolve by sonication, add 30% ethanol to make up to volume, shake well, and a reference stock solution with a concentration of 192.33 μg / mL is obtained. Accurately pipette 4 mL of the reference stock solution into a 25 mL volumetric flask, add 2 mL of 30% ethanol and 1 mL of 5% NaNO2 solution, shake well, let stand for 6 min, add 1 mL of 10% AL(NO3)3 solution, shake well, let stand for 6 min, add 10 mL of 10% NaOH solution, add 30% ethanol to the mark, shake well, and let stand for 15 min to obtain the reference solution. (2) Preparation of test solution: Take 0.6 g of Xiaor Qingre Granules into a 10 mL volumetric flask, add 30% ethanol to make up to volume, accurately pipette 3 mL into a 25 mL volumetric flask, add 3 mL of 30% ethanol and 1 mL of 5% NaNO2 solution, shake well, let stand for 6 min, add 1 mL of 10% AL(NO3)3 solution, shake well, let stand for 6 min, add 10 mL of 10% NaOH solution, add 30% ethanol to the mark, shake well, let stand for 15 min to obtain the test solution; (3) Preparation of negative control solution: Take 6 mL of 30% ethanol into a 25 mL volumetric flask, add 1 mL of 5% NaNO2 solution, shake well, let stand for 6 min, add 1 mL of 10% AL(NO3)3 solution, mix well, let stand for 6 min, add 10 mL of 10% NaOH solution, add 30% ethanol to the mark, shake well, and let stand for 15 min to prepare the negative control solution; (4) The absorbance of the reference solution, test solution and negative control solution was measured at 510 nm using a UV-Vis spectrophotometer and the total flavonoid content was calculated.

[0020] Unless otherwise specified, the component ratios of the developing solvent in this invention are all volume ratios, and NaNO2, Al(NO3)3, and NaOH solutions are all aqueous solutions.

[0021] The detection method of the present invention further includes determining the content of hydroxysaffron yellow A using high performance liquid chromatography (HPLC), wherein the HPLC detection method is as follows: (1) High performance liquid chromatography conditions: A C18 liquid chromatography column with a length of 250 mm, an inner diameter of 4.6 mm, and a packing particle diameter of 5 μm was used; the mobile phase was methanol-0.7% phosphoric acid solution; the volumetric flow rate was 1.0 mL·min-1; the injection volume was 10 μL; the detection wavelength was 403 nm; and the column temperature was 25 ℃.

[0022] (2) Preparation of the reference solution: Accurately weigh 10.03 mg of hydroxysaffron yellow A reference standard into a 50 mL volumetric flask, dilute to volume with 25% methanol, and shake well to obtain a reference standard stock solution with a concentration of 194.18 μg / mL. Accurately pipette 4 mL of the above reference standard stock solution into a 25 mL volumetric flask, dilute to volume with 25% methanol, and shake well to obtain a hydroxysaffron yellow A reference standard solution with a concentration of 31.07 μg / mL.

[0023] (3) Preparation of test solution: Take 1 g of this product into a 10 mL volumetric flask, add 25% methanol to make up to volume, shake well, filter, and take the filtrate to obtain the test solution.

[0024] (4) Preparation of negative control solution: Weigh the other medicinal materials except safflower according to the prescription, prepare negative control sample without safflower, and prepare negative control sample solution without safflower according to the preparation method of test solution.

[0025] This invention establishes a detection method for pediatric heat-clearing granules. Thin-layer chromatography is used to identify safflower, arbutin, horseradish, and licorice in the granules, offering advantages such as good reproducibility, high specificity, and robustness. Ultraviolet spectrophotometry is used to determine the total flavonoid content in the granules, and high-performance liquid chromatography is used to determine the content of hydroxysafflower yellow A. This method achieves a more comprehensive characterization of the components of pediatric heat-clearing granules, improving drug quality control and making qualitative and quantitative detection more accurate and quality control easier. Attached Figure Description

[0026] Figure 1 Thin-layer chromatography image of safflower in children's heat-clearing granules The labels in the attached figure are as follows: 1, 2, and 3 are children's heat-clearing granules; 4 is safflower control material; and 5 is a negative control material lacking safflower. Figure 2 Thin-layer chromatography image of arbutin in children's heat-clearing granules The labels in the attached figure are described as follows: 1, 2, and 3 are children's heat-clearing granules; 4 is arbutin reference standard; and 5 is a negative control of quercetin. Figure 3 Thin-layer chromatography image of horseradish for children's heat-clearing granules The labels in the attached figure are described as follows: 1, 2, and 3 are children's heat-clearing granules; 4 is high-altitude horseradish control material; 5 is negative control material lacking high-altitude horseradish. Figure 4 Thin-layer chromatography image of licorice for identifying pediatric heat-clearing granules. The labels in the attached figure are described as follows: 1, 2, and 3 are children's heat-clearing granules; 4 is licorice as a reference herb; and 5 is a negative control lacking licorice extract. Figure 5Standard curve of total flavonoid content in children's heat-clearing granules; Figure 6 High performance liquid chromatogram of hydroxysaffron yellow A reference standard; Figure 7 The high-performance liquid chromatogram of the test solution; Figure 8 This is a high-performance liquid chromatogram of a negative sample solution. Figure 9 This is the standard curve diagram for hydroxysaffron yellow pigment A. Detailed Implementation

[0027] The present invention will be further illustrated below with reference to embodiments. These embodiments are for illustrative purposes only and do not limit the scope and nature of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the protection scope of the present invention.

[0028] The pediatric heat-clearing granules of this invention are made from the following components in parts by weight: 250g travertine, 150g safflower, 0.1g cultured bezoar, 70g bangga, 70g ligadu, 70g licorice extract, 100g horseradish, 100g dextrin, 27g povidone K30, 2g sucralose, and 1g sweet orange flavor.

[0029] Example 1: Thin-layer chromatography identification of safflower: Take 5 g of Xiaor Qingre Granules into a stoppered conical flask, add 20 mL of 80% acetone solution, sonicate (power 250 W, frequency 50 kHz) for 30 min, centrifuge, and dilute the supernatant to 1 mL to obtain the test solution. Take 0.5 g of safflower reference material and prepare a reference solution according to the preparation method of the test solution. Weigh the remaining medicinal materials (excluding safflower) according to the prescription of Xiaor Qingre Granules to prepare a negative control sample without safflower, and prepare a negative control sample solution without safflower according to the preparation method of the test solution. Apply 2 μL of each of the above three solutions to the same silica gel G thin-layer plate, develop with n-butanol:methanol:water = 6:1:5 as the developing solvent, remove, air dry, and examine under a UV lamp at 365 nm. The results show that the test sample chromatogram shows spots of the same color at the same positions as the reference chromatogram, while the negative control sample chromatogram shows no spots at the same positions as the reference chromatogram. Figure 1 The established thin-layer chromatography method can be used for the qualitative identification of safflower in pediatric heat-clearing granules.

[0030] Example 2: Thin-layer chromatography identification of arbutin: Take 5 g of Xiaor Qingre Granules into an Erlenmeyer flask, add 20 mL of methanol, centrifuge (power 250 W, frequency 50 kHz) for 40 min, distill the supernatant down to 1 mL, and use this as the test solution. Prepare a solution containing 0.2 mg of arbutin reference standard per 1 mL using methanol, and use this as the reference solution. Weigh the remaining medicinal materials (excluding Ligadu) according to the Xiaor Qingre Granules prescription to prepare a negative control sample without Ligadu, and prepare a negative control sample solution lacking Ligadu according to the preparation method of the test solution. Apply 2 μL of each of the above three solutions to the same silica gel G thin-layer plate, develop using chloroform:methanol = 8:2 as the developing solvent, remove, air dry, spray with 10% phosphomolybdic acid ethanol solution, and heat to 105℃ until the spots are clear. Examine under natural light. The results show that the test sample chromatogram shows spots of the same color at the same positions as the reference chromatogram, while the negative control sample chromatogram shows no spots at the same positions as the reference chromatogram. Figure 2 The established thin-layer chromatography method can be used for the qualitative identification of ligustrazine in children's heat-clearing granules.

[0031] Example 3: Thin-layer chromatography identification of horseradish from high-altitude areas: Take 5 g of Xiaor Qingre Granules into an Erlenmeyer flask, add 20 mL of chloroform, sonicate (power 250 W, frequency 50 kHz) for 30 min, filter, and distill the filtrate down to 1 mL to obtain the test solution. Take 1 g of *Hordeum vulgare* (horseroot) reference material into an Erlenmeyer flask and prepare a reference solution according to the preparation method of the test solution. Weigh the remaining medicinal materials (excluding *Hordeum vulgare*) according to the prescription of Xiaor Qingre Granules to prepare a negative control sample without *Hordeum vulgare*, and prepare a negative control sample solution lacking *Hordeum vulgare* according to the preparation method of the test solution. Take 2 μL of each of the above three solutions and spot them separately on the same silica gel G thin-layer plate. Develop the plate using cyclohexane:ethyl ether:ethyl acetate = 20:5.5:2.5 as the developing solvent. Remove the plate, air dry, spray with 30% sulfuric acid ethanol solution, and heat at 105℃ until the spots are clearly visible. Examine under natural light. The results show that the test sample chromatogram shows spots of the same color at the same positions as the reference sample chromatogram. The negative control sample chromatogram shows no spots at the same positions as the reference sample chromatogram. Figure 3 The established thin-layer chromatography method can be used for the qualitative identification of horseradish in children's heat-clearing granules.

[0032] Example 4: Thin-layer chromatography identification of licorice extract: Take 5 g of Xiaor Qingre Granules into an Erlenmeyer flask, add 20 mL of water to dissolve, extract three times with n-butanol, 20 mL each time (centrifuge if necessary), combine the n-butanol solutions, and extract three more times with water, 30 mL each time. Evaporate the n-butanol solution to dryness, add 5 mL of methanol to redissolve, and use as the test solution. Take 1 g of licorice reference material and prepare a reference solution according to the preparation method of the test solution. Weigh the remaining medicinal materials except for licorice extract according to the prescription of Xiaor Qingre Granules to prepare a negative control sample without licorice extract, and prepare a negative control sample solution without licorice extract according to the preparation method of the test solution. Take 2 μL of each of the above three solutions and spot them separately on the same silica gel G thin-layer plate. Use ethyl acetate:formic acid:glacial acetic acid:water = 15:1:1:2 as the developing solvent, develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly visible, and examine under ultraviolet light (365 nm). The results showed that the test sample chromatogram showed spots of the same color at the same positions as the reference sample chromatogram, while the negative control sample chromatogram showed no spots at the same positions as the reference sample chromatogram. Figure 4 The established thin-layer chromatography method can be used for the qualitative identification of licorice extract in children's heat-clearing granules.

[0033] Example 5: Determination of total flavonoid content 1. Preparation of sample solution 1.1 Preparation of reference stock solution Accurately weigh 10.43 mg of rutin reference standard into a 50 mL volumetric flask, add an appropriate amount of 30% ethanol, dissolve by sonication, then add 30% ethanol to make up to volume, shake well, and the reference standard stock solution with a concentration of 192.33 μg / mL is obtained.

[0034] 1.2 Preparation of the test solution Take 0.6 g of Xiaor Qingre Granules into a 10 mL volumetric flask, add 30% ethanol to make up to volume, accurately pipette 3 mL into a 25 mL volumetric flask, add 3 mL of 30% ethanol and 1 mL of 5% NaNO2 solution, shake well, let stand for 6 min, add 1 mL of 10% Al(NO3)3 solution, shake well, let stand for 6 min, add 10 mL of 10% NaOH solution, add 30% ethanol to the mark, shake well, let stand for 15 min to obtain the test solution; 1.3 Preparation of negative control sample solution Take 6 mL of 30% ethanol into a 25 mL volumetric flask, add 1 mL of 5% NaNO2 solution, shake well, let stand for 6 min, add 1 mL of 10% AL(NO3)3 solution, mix well, let stand for 6 min, add 10 mL of 10% NaOH solution, add 30% ethanol to the mark, shake well, and let stand for 15 min as a negative control solution. 2. Examination of linear relationships Accurately measure 0, 1, 2, 3, 4, 5, and 6 mL of the reference stock solution under section "1.1" into 25 mL volumetric flasks, add 30% ethanol to 6 mL, add 1 mL of 5% NaNO2 solution, shake well, let stand for 6 min, add 1 mL of 10% Al(NO3)3 solution, mix well, let stand for 6 min, add 10 mL of 10% NaOH solution, add 30% ethanol to the mark, shake well, let stand for 15 min, using the negative control solution under section "1.3" as a blank, and measure the absorbance at a wavelength of 510 nm using ultraviolet-visible spectrophotometry (General Rule 0401). Plot a standard curve with concentration and absorbance as the x and y axes. The results are shown below. Figure 5 The regression equation was Y=0.0125X-0.0028 (r=0.9998, n=6), indicating that rutin has a good linear relationship in the range of 0~46.16 μg / mL.

[0035] 3. Precision test Accurately transfer 4 mL of the reference stock solution from section 1.1 into 25 mL volumetric flasks, for a total of 6 portions. Following the method in section “2”, starting from “add 30% ethanol to 6 mL”, measure the absorbance sequentially. The RSD of the result is 0.14%, indicating that the instrument has good precision.

[0036] 4. Stability Test The same reference solution and test solution were taken, and the absorbance was measured at 0, 10, 20, 30, 40 and 60 min, respectively. The RSDs were 0.14% and 0.58%, respectively, indicating that the content of the reference solution and the test solution was stable within 60 min.

[0037] 5 Repeatability Test Six test solutions were prepared in parallel according to the method described in section 1.2, and the absorbance was determined according to the method. The results showed that the RSD was 0.64% and RSD < 2%, indicating that the method has good repeatability.

[0038] 6. Spiking recovery test Take 0.6 g of Xiaor Qingre Granules and put them into a 10 mL volumetric flask. Add 30% ethanol to make up to volume. Prepare 6 parallel aliquots. Take 1.5 mL of the sample solution into a 25 mL volumetric flask, add 2 mL of the reference stock solution under section "1.1", and determine the absorbance according to the method under section "2", starting from "add 30% ethanol to 6 mL". The results are shown in Table 1. The average recovery rate of total flavonoids was calculated to be 96.61%, and the RSD was 1.50%. n =6). A recovery rate of 92% to 105% and an RSD of <2% indicate good recovery.

[0039] Table 1 Results of the recovery test ( n =6) Tab. 1 Results of sampling recovery test ( n =6)

[0040] 7. Content determination of samples Three batches of self-made pediatric heat-clearing granules were taken, two samples from each batch. Test solutions were prepared according to the method described in section 1.2. The absorbance was measured sequentially, and the concentration of total flavonoids in the samples was calculated using the external standard method. The concentrations were 4222.0462, 4351.6214, and 4389.4006 μg·g⁻¹, respectively. -1 .

[0041] Example 6: Determination of the content of hydroxysaffron yellow pigment A 1. Chromatographic conditions Chromatographic column: Ultimate XB-C18 HPLC column (250 mm × 4.6 mm, 5 μm); mobile phase: methanol-0.7% phosphoric acid solution (27:73, V / V) (wherein the 0.7% phosphoric acid solution was adjusted to pH 6.0 with triethylamine); flow rate: 1.0 mL / min; column temperature: 25 °C; detection wavelength: 403 nm; injection volume: 10 μL. The theoretical plate number, calculated based on the peak of hydroxysaffron yellow pigment A, should be no less than 2000.

[0042] 2. Solution preparation 2.1 Preparation of the reference solution Accurately weigh 10.03 mg of hydroxysaffron yellow A reference standard into a 50 mL volumetric flask, dilute to volume with 25% methanol, and shake well to obtain a reference standard stock solution with a concentration of 194.18 μg / mL. Accurately pipette 4 mL of the above reference standard stock solution into a 25 mL volumetric flask, dilute to volume with 25% methanol, and shake well to obtain a hydroxysaffron yellow A reference standard solution with a concentration of 31.07 μg / mL.

[0043] 2.2 Preparation of the test solution Take 1 g of Xiaor Qingre Granules into a 10 mL volumetric flask, add 25% methanol to dilute to volume, shake well, filter, and collect the filtrate to obtain the product.

[0044] 2.3 Preparation of negative control solution Prepare a negative sample lacking safflower according to the prescription ratio. It is prepared using the same method as the test solution preparation method described in section 2.2.

[0045] 3. Exclusivity 3.1 Blank Interference Test Accurately measure 10 μL each of the negative control solution, reference solution, and test solution, and inject them according to the chromatographic conditions described in section "1". Record the chromatograms. The results show that absorption peaks appear at the same retention time position for hydroxysaffron yellow A in the chromatograms of the test solution and reference solution, while no absorption peak appears at the same position in the chromatogram of the negative control solution. This indicates that the method has strong specificity and the negative control does not interfere. (See section "1"). Figures 5-7 .

[0046] 4. Examination of linear relationships Accurately transfer 1, 2, 4, 6, and 8 mL of the reference standard stock solution from section 2.1 into 25 mL volumetric flasks, dilute to volume with 25% methanol, and mix well to obtain hydroxysaffron yellow A series reference standard solutions with mass concentrations of 7.77, 15.53, 31.07, 46.60, and 62.14 μg / mL. Inject the above series of reference standard solutions and the reference standard stock solution according to the chromatographic conditions in section "1". Plot a standard curve with the injection concentration (X, μg / mL) and peak area (Y) as the x and y axes, as shown in the figure. Figure 8 The regression equation obtained is Y=32220.3045X-1766.2839 (r=1.0000, n=6), indicating that hydroxysaffron yellow pigment A has a good linear relationship in the range of 7.77~194.18 μg / mL.

[0047] 5. Precision test Accurately pipette 10 μL of the reference solution under section 2.1, and inject it 6 times consecutively under the chromatographic conditions under section “1”. Record the peak area and calculate the RSD value. The result shows that the RSD is 0.05%, indicating that the instrument has good precision.

[0048] 6. Stability Test The same reference solution and test solution were injected at 0, 2, 4, 6, 8, 10, 12, 14, 24, 30, 42, 54, 70, and 72 h, respectively, and the peak areas were measured. The RSDs were 0.38% and 1.67%, respectively, indicating that the contents of the reference solution and test solution were stable within 72 h.

[0049] 7 Repeatability Test Six test solutions were prepared in parallel according to the method in section 2.2, and the peak area was determined according to the chromatographic conditions in section "1". The RSD of the content of hydroxysaffron yellow pigment A was 1.63%, indicating that the method has good repeatability.

[0050] 8. Spiking recovery test Take 1 g of granules into a 20 mL volumetric flask, add 1.6 mL of the reference stock solution from section 2.1, and dilute to volume with 25% methanol. Perform six parallel operations, and determine the peak area under the chromatographic conditions in section "1". The average recovery rate of hydroxysaffron yellow A was calculated to be 99.99%, with an RSD of 2.12%. n =6). RSD < 3%, indicating good recovery rate of this method. Results are shown in Table 2.

[0051] Table 2 Results of the recovery test of hydroxysaffron yellow pigment A ( n =6) Table 2 Results of recovery of hydroxysafflor yellow A ( n =6)

[0052] 9. Durability Under the chromatographic conditions specified in section "1", with single-factor variation detection wavelength ±2 nm, flow rate ±0.2 ml·min⁻¹, column temperature ±2℃, organic phase ratio ±2%, and pH ±0.2 for 0.7% phosphoric acid, and by replacing the column with a ShimNex CS C18 (5 μm, 4.6×250 mm) column manufactured by Shimadzu (Shanghai) Experimental Equipment Co., Ltd., the RSD of hydroxysaffron yellow A content was not greater than 2.0%, indicating that the method has good robustness under single-factor variation conditions.

[0053] Content determination of 10 samples Three batches of self-made pediatric heat-clearing granules were taken, two samples from each batch. Test solutions were prepared according to the method described in section 2.2. Peak areas were determined under the chromatographic conditions described in section "1". The content of hydroxysaffron yellow A in the three batches was calculated using the external standard method, and the results were 314.548, 310.232, and 300.133 μg·g⁻¹, respectively. -1 .

[0054] This invention is stable and accurate, and can be used for the quality evaluation of children's heat-clearing granules, providing a reference for improving the quality standards of children's heat-clearing granules.

[0055] Finally, it should be noted that the above embodiments are merely preferred embodiments of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be pointed out that those skilled in the art can make various modifications and improvements without departing from the principles and concepts of the present invention, and these modifications and improvements all fall within the protection scope of the present invention.

Claims

1. A method for quality testing of pediatric heat-clearing granules, characterized in that, The prescription composition of the pediatric heat-clearing granules is as follows: 250 parts travertine, 150 parts safflower, 70 parts ligadu, 100 parts alpine horseradish, 70 parts bangga, 70 parts licorice extract, 0.1 parts in vitro cultured bezoar, and appropriate amount of pharmaceutical excipients. The detection method is to use identification and / or content determination methods to detect safflower, arbutin, alpine horseradish, licorice, total flavonoids, and hydroxysafflower yellow A in the pediatric heat-clearing granules. The identification method is to use thin-layer chromatography to examine under 254 nm ultraviolet light or natural light to identify safflower, arbutin, alpine horseradish, and licorice in the pediatric heat-clearing granules. The content determination method described above is to use rutin standard as a reference and determine the total flavonoid content at a wavelength of 510 nm using ultraviolet detection method; The content determination method is to use hydroxysaffron yellow A as a control and determine the content of hydroxysaffron yellow A by high performance liquid chromatography at a wavelength of 403 nm.

2. The method for quality testing of pediatric heat-clearing granules according to claim 1, characterized in that, The thin-layer chromatography identification method is as follows: (1) Take the pediatric heat-clearing granules into a stoppered conical flask, add acetone solution, sonicate, centrifuge, and concentrate to obtain the test solution; (2) Take safflower reference material and prepare a reference solution according to the preparation method of the test solution; (3) Weigh out the remaining medicinal materials except safflower according to the prescription of Xiaor Qingre Granules, prepare a negative control sample without safflower, and prepare a negative control sample solution without safflower according to the preparation method of the test sample solution; (4) Apply the above three solutions to the same silica gel G thin layer plate, develop with n-butanol-methanol-water as the developing solvent, remove, air dry, and examine under ultraviolet light; in the chromatogram of the test sample, spots of the same color appear at the same position as in the chromatogram of the reference medicinal material, and in the chromatogram of the negative control sample, no spots appear at the same position as in the chromatogram of the reference substance.

3. The method for quality testing of pediatric heat-clearing granules according to claim 1, characterized in that, The thin-layer chromatography identification method is as follows: (1) Take the pediatric heat-clearing granules into an Erlenmeyer flask, add methanol, sonicate, centrifuge, and concentrate to obtain the test solution; (2) Take arbutin reference standard, add methanol to prepare the reference solution; (3) Weigh out the remaining medicinal materials except for Ligadu according to the prescription of Xiaor Qingre Granules, prepare a negative control sample without Ligadu, and prepare a negative control sample solution without Ligadu according to the preparation method of the test sample solution; (4) Apply the above three solutions to the same silica gel G thin layer plate, develop with chloroform-methanol as the developing solvent, remove, air dry, spray with phosphomolybdic acid ethanol solution and heat to 105°C until the spots are clear; in the chromatogram of the test sample, spots of the same color appear at the same position as in the chromatogram of the reference sample, and in the chromatogram of the negative control sample, no spots appear at the same position as in the chromatogram of the reference sample.

4. The method for quality testing of pediatric heat-clearing granules according to claim 1, characterized in that, The thin-layer chromatography identification method is as follows: (1) Take the pediatric heat-clearing granules into an Erlenmeyer flask, add chloroform, sonicate, filter, and concentrate to obtain the test solution; (2) Take the high-altitude horseradish reference material into an Erlenmeyer flask and prepare the reference solution according to the preparation method of the test solution; (3) Weigh out the remaining medicinal materials except for horseradish according to the prescription of Xiaor Qingre Granules, prepare a negative control sample without horseradish, and prepare a negative control sample solution without horseradish according to the preparation method of the test sample solution. (4) Apply the above three solutions separately to the same silica gel G thin layer plate, develop with cyclohexane-ethyl ether-ethyl acetate as the developing solvent, remove, air dry, spray with sulfuric acid ethanol solution, and heat at 105℃ until the spots are clearly visible; in the chromatogram of the test sample, spots of the same color appear at the same position as in the chromatogram of the reference sample, and in the chromatogram of the negative control sample, no spots appear at the same position as in the chromatogram of the reference sample.

5. The method for quality testing of pediatric heat-clearing granules according to claim 1, characterized in that, The thin-layer chromatography identification method is as follows: (1) Take the children's heat-clearing granules into an Erlenmeyer flask, add water to dissolve, extract with n-butanol, extract with water again, evaporate the n-butanol solution to dryness, add methanol to redissolve, and use as the test solution; (2) Take an appropriate amount of licorice reference material and prepare a reference solution according to the preparation method of the test solution; (3) Weigh out the remaining medicinal materials except for licorice extract according to the prescription of Xiaor Qingre Granules, prepare a negative control sample without licorice extract, and prepare a negative control sample solution without licorice extract according to the preparation method of the test sample solution; (4) Apply the above three solutions separately to the same silica gel G thin layer plate, develop with ethyl acetate-formic acid-glacial acetic acid-water as the developing solvent, remove, air dry, spray with sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly visible, and examine under ultraviolet light; in the chromatogram of the test sample, spots of the same color appear at the same position as in the chromatogram of the reference sample, and in the chromatogram of the negative control sample, no spots appear at the same position as in the chromatogram of the reference sample.

6. The method for quality testing of pediatric heat-clearing granules according to claim 1, characterized in that, The thin-layer chromatography identification method is as follows: (1) Take 5 g of Xiaor Qingre Granules into a stoppered conical flask, add 20 mL of 80% acetone solution, sonicate (power 250 W, frequency 50 kHz) for 30 min, centrifuge, and dilute the supernatant to 1 mL; (2) Take 0.5 g of safflower reference material, add 20 mL of 80% acetone solution, sonicate (power 250 W, frequency 50 kHz) for 30 min, centrifuge, and evaporate the supernatant to 1 mL as the reference solution; (3) Take the remaining medicinal materials except safflower according to the prescription of Xiaor Qingre Granules, and prepare negative control samples without safflower. Prepare negative control sample solutions without safflower according to the preparation method of test solution. (4) Take 2 μL of each of the above three solutions and spot them separately on the same silica gel G thin-layer plate. Use n-butanol:methanol:water = 6:1:5 as the developing solvent, develop, remove, air dry, and examine under a UV lamp at 365 nm. In the chromatogram of the test sample, spots of the same color appear at the same positions as in the chromatogram of the reference sample. In the chromatogram of the negative control sample, no spots appear at the same positions as in the chromatogram of the reference sample.

7. The method for quality testing of pediatric heat-clearing granules according to claim 3, characterized in that, The thin-layer chromatography identification method is as follows: (1) Take 5 g of Xiaor Qingre Granules into an Erlenmeyer flask, add 20 mL of methanol, (power 250 W, frequency 50 kHz) for 40 min, centrifuge, and distill the supernatant down to 1 mL to obtain the test solution. (2) Prepare a solution containing 0.2 mg of arbutin per 1 mL by adding methanol to the arbutin reference standard; (3) Weigh out the remaining medicinal materials except for Ligadu according to the prescription of Xiaor Qingre Granules, prepare a negative control sample without Ligadu, and prepare a negative control sample solution without Ligadu according to the preparation method of the test solution; (4) Take 2 μL of each of the above three solutions and spot them separately on the same silica gel G thin-layer plate. Develop the plate using chloroform:methanol = 8:2 as the developing solvent. Remove the plate, air dry it, spray it with 10% phosphomolybdic acid ethanol solution, and heat it to 105℃ until the spots are clear. In the chromatogram of the test sample, spots of the same color appear at the same positions as those in the chromatogram of the reference sample. In the chromatogram of the negative control sample, no spots appear at the same positions as those in the chromatogram of the reference sample.

8. The method for quality testing of pediatric heat-clearing granules according to claim 4, characterized in that, The thin-layer chromatography identification method is as follows: (1) Take 5 g of Xiaor Qingre Granules into an Erlenmeyer flask, add 20 mL of chloroform, sonicate (power 250 W, frequency 50 kHz) for 30 min, filter, and evaporate the filtrate to 1 mL as the test solution. (2) Take 1g of highland horseradish reference material into an Erlenmeyer flask, add 20 mL of chloroform, sonicate (power 250 W, frequency 50 kHz) for 30 min, filter, and evaporate the filtrate to 1 mL as the reference solution. (3) Weigh out the remaining medicinal materials except for horseradish according to the prescription of Xiaor Qingre Granules, prepare a negative control sample without horseradish, and prepare a negative control sample solution without horseradish according to the preparation method of the test sample solution. (4) Apply 2 μL of each of the above three solutions to the same silica gel G thin-layer plate. Develop the plate using cyclohexane:ethyl ether:ethyl acetate = 20:5.5:2.5 as the developing solvent. Remove the plate, air dry, spray with 30% sulfuric acid ethanol solution, and heat at 105℃ until the spots are clearly visible. In the chromatogram of the test sample, spots of the same color appear at the same positions as in the chromatogram of the reference sample. In the chromatogram of the negative control sample, no spots appear at the same positions as in the chromatogram of the reference sample.

9. The method for quality testing of pediatric heat-clearing granules according to claim 5, characterized in that, The thin-layer chromatography identification method is as follows: (1) Take 5 g of Xiaor Qingre Granules into an Erlenmeyer flask, add 20 mL of water to dissolve, extract with n-butanol 3 times, 20 mL each time, combine the n-butanol solutions, extract with water 3 times, 30 mL each time, evaporate the n-butanol solution to dryness, add 5 mL of methanol to redissolve, and use as the test solution. (2) Take 1 g of licorice reference material, add 20 mL of water to dissolve it, extract it 3 times with n-butanol, 20 mL each time, combine the n-butanol solutions, extract it 3 times with water, 30 mL each time, evaporate the n-butanol solution to dryness, add 5 mL of methanol to redissolve it, and use it as the reference solution. (3) Weigh out the remaining medicinal materials except for licorice extract according to the prescription of Xiaor Qingre Granules, prepare a negative control sample without licorice extract, and prepare a negative control sample solution without licorice extract according to the preparation method of the test sample solution; (4) Take 2 μL of each of the above three solutions and spot them separately on the same silica gel G thin-layer plate. Use ethyl acetate:formic acid:glacial acetic acid:water = 15:1:1:2 as the developing solvent. Develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, and heat at 105℃ until the spots are clearly visible. Examine under ultraviolet light (365 nm). In the chromatogram of the test sample, spots of the same color appear at the same positions as in the chromatogram of the reference sample. In the chromatogram of the negative control sample, no spots appear at the same positions as in the chromatogram of the reference sample.

10. The method for quality testing of pediatric heat-clearing granules according to claim 1, characterized in that, The ultraviolet detection method is as follows: (1) Preparation of reference solution: Accurately weigh 10.43 mg of rutin reference standard into a 50 mL volumetric flask, add an appropriate amount of 30% ethanol, dissolve by sonication, add 30% ethanol to make up to volume, shake well, and a reference stock solution with a concentration of 192.33 μg / mL is obtained. Accurately pipette 4 mL of the reference stock solution into a 25 mL volumetric flask, add 2 mL of 30% ethanol and 1 mL of 5% NaNO2 solution, shake well, let stand for 6 min, add 1 mL of 10% AL(NO3)3 solution, shake well, let stand for 6 min, add 10 mL of 10% NaOH solution, add 30% ethanol to the mark, shake well, and let stand for 15 min to obtain the reference solution. (2) Preparation of test solution: Take 0.6 g of Xiaor Qingre Granules into a 10 mL volumetric flask, add 30% ethanol to make up to volume, accurately pipette 3 mL into a 25 mL volumetric flask, add 3 mL of 30% ethanol and 1 mL of 5% NaNO2 solution, shake well, let stand for 6 min, add 1 mL of 10% AL(NO3)3 solution, shake well, let stand for 6 min, add 10 mL of 10% NaOH solution, add 30% ethanol to the mark, shake well, let stand for 15 min to obtain the test solution; (3) Preparation of negative control solution: Take 6 mL of 30% ethanol into a 25 mL volumetric flask, add 1 mL of 5% NaNO2 solution, shake well, let stand for 6 min, add 1 mL of 10% AL(NO3)3 solution, mix well, let stand for 6 min, add 10 mL of 10% NaOH solution, add 30% ethanol to the mark, shake well, and let stand for 15 min to prepare the negative control solution; (4) The absorbance of the reference solution, test solution and negative control solution was measured at 510 nm using a UV-Vis spectrophotometer and the total flavonoid content was calculated.

11. The application of a quality testing method as described in any one of claims 1 to 10 in testing the quality of pediatric heat-clearing granules.

Images