Quality detection method of composition for nourishing yin and clearing away lung-heat

Abstract

The invention relates to the technical field of biological medicines, and discloses a quality detection method of a composition for nourishing yin and clearing away lung-heat. The quality detection method comprises the following steps: identifying the radix scrophulariae, the radix paeoniae alba, the bulbus fritillariae cirrhosae, the menthol and the liquorice by adopting a thin layer chromatography; and determining the content of paeoniflorin in the composition by adopting a high performance liquid chromatography. According to the method provided by the invention, a thin-layer chromatography identification method for the five medicinal materials of radix scrophulariae, radix paeoniae alba, bulbus fritillariae cirrhosae, menthol and liquorice is systematically established for the first time, and high performance liquid chromatography content determination of the key active ingredient paeoniflorin is introduced, so that multi-component and multi-dimensional comprehensive evaluation of the product is realized. Each thin-layer identification method realizes clear spots, good separation degree, negative effect and no interference, and has strong specificity and reproducibility, the content determination method has good precision, stability and repeatability, the average adding standard recovery rate reaches 99.87%, the accuracy of a quantitative result is ensured, and the method is suitable for quality control in large-scale production.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a quality detection method of a Yangyin Qingfei composition. BACKGROUND

[0002] The Yangyin Qingfei composition is a traditional Chinese medicine preparation composed of eight Chinese medicinal materials, including Rehmanniae Radix, Scrophulariae Radix, Moutan Cortex, Paeoniae Radix Alba, Ophiopogonis Radix, Fritillariae Cirrhosae Thunb, Menthae Haplocalyx and Glycyrrhizae Radix et Rhizoma. It has the effects of nourishing yin and moistening lung, clearing heat and benefiting throat, and is widely used for treating dry and painful throat, dry cough, little or no sputum, etc. The prepared form of the composition, Yangyin Qingfei Mixture, is subject to the national drug standard with the execution number of WS3-B-3896-98. However, the existing quality standard only includes physicochemical reactions and thin-layer identification of Moutan Cortex, lacks systematic identification of other key medicinal materials (such as Scrophulariae Radix, Paeoniae Radix Alba, Fritillariae Cirrhosae Thunb, Menthae Haplocalyx and Glycyrrhizae Radix et Rhizoma), and content determination of active ingredients, which cannot comprehensively reflect the quality status of the finished product, may lead to batch-to-batch quality fluctuations, and affect the efficacy and safety of the drug.

[0003] In the prior art, Luo et al. published a study on the quality standard of Yangyin Qingfei Syrup, in which they tried to improve the quality control by establishing thin-layer chromatography methods for Scrophulariae Radix, Paeoniae Radix Alba, Moutan Cortex and Glycyrrhizae Radix et Rhizoma, and simultaneously determining the contents of harpagide and paeonol by high performance liquid chromatography (HPLC). Although this study improved the level of quality control, it still did not cover all the key components: the identification methods of Fritillariae Cirrhosae Thunb and Menthae Haplocalyx were missing, and the content determination of paeoniflorin, an important active ingredient in Paeoniae Radix Alba and Moutan Cortex (with effects of anti-inflammatory, immune regulation, etc.), was not included. In addition, the existing method is complicated, consumes a large amount of reagents, and takes a long time to detect, which is not conducive to rapid and environmentally friendly quality control in large-scale production. For example, thin-layer identification requires multiple extractions and elutions, which is time-consuming and labor-intensive, and uses a large amount of organic solvents, which is not in line with the green production concept.

[0004] Therefore, it is urgent to develop a comprehensive, efficient and environmentally friendly quality detection method to adapt to the quality control of Yangyin Qingfei composition in large-scale production, ensure the stability and controllability of product quality, and promote the development of traditional Chinese medicine modernization. SUMMARY

[0005] The present application aims to at least solve one of the above-mentioned technical problems in the prior art. To this end, the purpose of the present application is to provide a quality detection method of a Yangyin Qingfei composition.

[0006] In order to achieve the above-mentioned purpose, the technical solution adopted by the present application is as follows: The application provides a quality detection method of a yin-nourishing and lung-clearing composition, the components of the yin-nourishing and lung-clearing composition include radix rehmanniae, radix scrophulariae, cortex moutan, radix paeoniae alba, radix ophiopogonis, fritillariae cirrhosae, menthol and licorice, and the quality detection method comprises the following steps: adopting thin layer chromatography to identify the radix scrophulariae, radix paeoniae alba, fritillariae cirrhosae, menthol and licorice; adopting high performance liquid chromatography to detect the content of paeoniflorin in the composition.

[0007] In some embodiments of the application, the thin layer chromatography identification of the radix scrophulariae and radix paeoniae alba specifically comprises the following steps: (i) 20-30 mL of the yin-nourishing and lung-clearing composition is concentrated, then 60-90 mL of an alcohol solvent is added for extraction, the extract is collected and concentrated, the obtained residue is dissolved in 20-30 mL of water, then 20-30 mL of a non-alcohol organic solvent is added for extraction to remove impurities, the water phase is collected and purified through a macroporous adsorption resin column, then the eluate is purified through an aluminum oxide column, the purified liquid is concentrated, and then 0.8-1.2 mL of an alcohol solvent is added to dissolve the radix scrophulariae and radix paeoniae alba test sample solution; the harbehobin and paeoniflorin control samples are respectively added into alcohol solvents to prepare control sample solutions with a concentration of 0.8-1.2 mg / mL; (ii) 8-12 µL of the radix scrophulariae and radix paeoniae alba test sample solution, the harbehobin control sample solution and the paeoniflorin control sample solution are spotted on the same silica gel G thin layer plate, a mixed liquid of ethyl acetate, butanone, formic acid and water with a volume ratio of (4-6):(2.4-.6):(0.8-1.2):1 is used as a developing agent for development, the developed thin layer plate is taken out and dried, 8%-12% of sulfuric acid ethanol solution is sprayed, heating is performed until the spots develop clearly, and the thin layer plate is observed under sunlight.

[0008] In some embodiments of the application, in the thin layer chromatography identification of the radix scrophulariae and radix paeoniae alba, the concentrated mode in step (i) comprises evaporation and concentration to a thick paste.

[0009] In some embodiments of the application, in the thin layer chromatography identification of the radix scrophulariae and radix paeoniae alba, the extraction in step (i) comprises ultrasonic extraction for 24-36 min.

[0010] In some embodiments of the application, in the thin layer chromatography identification of the radix scrophulariae and radix paeoniae alba, the alcohol solvent is at least one selected from methanol and ethanol.

[0011] In some preferred embodiments of the application, in the thin layer chromatography identification of the radix scrophulariae and radix paeoniae alba, the alcohol solvent is methanol.

[0012] In some embodiments of the present application, the thin layer chromatography identification of Scrophularia ningpoensis Hemsley and Radix Paeoniae Alba, in step (i), the inner diameter of the macroporous adsorption resin column is 0.8-1.5 cm, the column height is 8-15 cm, and the interior is filled with AB-8 type resin; the elution process comprises first eluting with 40-60 mL of water, discarding the water eluent, and then eluting with 40-60 mL of methanol, and collecting the methanol eluent.

[0013] In some embodiments of the present application, the thin layer chromatography identification of Scrophularia ningpoensis Hemsley and Radix Paeoniae Alba, in step (i), the inner diameter of the alumina column is 0.8-1.2 cm, the interior is filled with neutral alumina, the particle size is 100-200 mesh, and the amount used is 2-3 g.

[0014] In some embodiments of the present application, the thin layer chromatography identification of Scrophularia ningpoensis Hemsley and Radix Paeoniae Alba, in step (ii), in the developing agent, the volume ratio of ethyl acetate, butanone, formic acid and water is (4.5-5.5):(2.7-3.3):(0.9-1.1):1.

[0015] In some embodiments of the present application, the thin layer chromatography identification of Scrophularia ningpoensis Hemsley and Radix Paeoniae Alba, in step (ii), the heating temperature is 95-115℃.

[0016] In some preferred embodiments of the present application, the thin layer chromatography identification of Scrophularia ningpoensis Hemsley and Radix Paeoniae Alba, in step (ii), the heating temperature is 100-110℃.

[0017] In some embodiments of the present application, the thin layer chromatography identification of Fritillaria cirrhosa Hutsunpe specifically comprises the following steps: (i) 40-60 mL of the Yin-nourishing and Lung-clearing composition is taken, an alkali solution is added to adjust the pH to 10-12, 20-40 mL of an ether solvent is added, and the mixture is shaken to extract 2-3 times, the extract is combined, concentrated, and the residue is collected, 0.4-0.6 mL of an alcohol solvent is added to dissolve the residue, and the Fritillaria cirrhosa test solution is obtained; 1.6-2.4 g of Fritillaria cirrhosa reference substance is taken, moistened with an alkali solution, and ultrasonically extracted with 16-24 mL of an ether solvent, the extract is collected and concentrated, and the residue obtained is dissolved with 0.4-0.6 mL of an alcohol solvent to obtain the Fritillaria cirrhosa reference solution; (ii) 6.4-9.6 μL of the Fritillaria cirrhosa test solution and 16-24 μL of the Fritillaria cirrhosa reference solution are taken and spotted on the same silica gel G thin layer plate, a mixture of n-hexane, ethyl acetate and diethylamine in a volume ratio of (8-12):(8-12):1 is used as the developing agent, the plate is taken out and dried, sprayed with a dilute bismuth potassium iodide solution, and smoked in an iodine jar until the spots are clear, and the same color spots appear in the test chromatogram at the corresponding positions of the reference chromatogram.

[0018] In some embodiments of the present application, the thin layer chromatography identification of the Fritillaria cirrhosa Hsiao et K.C. Hsia, in step (i), the alkaline solution comprises ammonia water.

[0019] In some embodiments of the present application, the thin layer chromatography identification of the Fritillaria cirrhosa Hsiao et K.C. Hsia, in step (i), the ether solvent comprises diethyl ether.

[0020] In some embodiments of the present application, the thin layer chromatography identification of the Fritillaria cirrhosa Hsiao et K.C. Hsia, in step (i), the prepared Fritillaria cirrhosa Hsiao et K.C. Hsia reference solution, the ultrasonic extraction time is 12-18 min; after the ultrasonic extraction is completed, it further comprises standing for 10-14 h.

[0021] In some embodiments of the present application, the thin layer chromatography identification of the Fritillaria cirrhosa Hsiao et K.C. Hsia, in step (ii), in the developing agent, the volume ratio of n-hexane, ethyl acetate and diethylamine is (9-11): (9-11): 1.

[0022] In some embodiments of the present application, the thin layer chromatography identification of menthol specifically comprises the following steps: (i) taking 40-60 mL of the Yin-nourishing and Lung-clearing composition, adding 160-240 mL of water and then performing volatile oil extraction, collecting volatile oil with 1.6-2.4 mL of non-polar organic solvent, and dehydrating the obtained solution to obtain a test solution; taking menthol reference substance, adding alcohol solvent to prepare a reference solution with a concentration of 1.6-2.4 mg / mL; (ii) taking 4.8-7.2 µL of the menthol test solution and 1.6-2.4 µL of the menthol reference solution, spotting them on the same silica gel G thin layer plate, using a mixture of petroleum ether and ethyl acetate with a volume ratio of (4.5-6.8): 1 as the developing agent, taking out and air-drying, spraying with 4%-6% vanillin sulfuric acid solution, heating until the spots develop clearly, and observing under daylight, wherein the test chromatogram shows the same color spots at positions corresponding to the reference chromatogram.

[0023] In some embodiments of the present application, the thin layer chromatography identification of menthol, in step (i), the non-polar organic solvent is selected from at least one of cyclohexane, n-hexane and petroleum ether.

[0024] In some preferred embodiments of the present application, the thin layer chromatography identification of menthol, in step (i), the non-polar organic solvent is cyclohexane.

[0025] In some embodiments of the present application, the thin layer chromatography identification of menthol, in step (i), the collection of volatile oil with non-polar organic solvent comprises heating and refluxing for 1.6-2.4 h.

[0026] In some embodiments of the present application, the menthol is identified by thin layer chromatography, and in step (i), the dehydration comprises using anhydrous sodium sulfate.

[0027] In some embodiments of the present application, the menthol is identified by thin layer chromatography, and in step (i), the alcohol solvent is ethanol.

[0028] In some embodiments of the present application, the menthol is identified by thin layer chromatography, and in step (ii), the temperature of petroleum ether in the developing agent is 60-90℃.

[0029] In some embodiments of the present application, the menthol is identified by thin layer chromatography, and in step (ii), the volume ratio of petroleum ether to ethyl acetate in the developing agent is (5.1-6.3): 1.

[0030] In some embodiments of the present application, the menthol is identified by thin layer chromatography, and in step (ii), the temperature of heating is 95-115℃.

[0031] In some preferred embodiments of the present application, the menthol is identified by thin layer chromatography, and in step (ii), the temperature of heating is 100-110℃.

[0032] In some embodiments of the present application, the glycyrrhiza is identified by thin layer chromatography, which specifically comprises the following steps: (i) 24-36 mL of the Yin-nourishing and Lung-clearing composition is taken and extracted with an alcohol solvent saturated with an equal volume of water for 2-4 times, the extract is combined and washed with 16-24 mL of water for 2-4 times, the water is discarded, the alcohol solvent layer is concentrated to dryness, the residue is collected and dissolved with 1.6-2.4 mL of alcohol solvent to obtain a glycyrrhiza test solution; 0.8-1.2 g of glycyrrhiza reference substance is taken and extracted with 32-28 mL of non-alcohol organic solvent by reflux, the residue is collected and extracted with 24-36 mL of alcohol solvent by reflux, the obtained extract is concentrated, dissolved with 32-48 mL of water, and then extracted with 24-36 mL of alcohol solvent saturated with water for 2-4 times, the extract is washed with water, concentrated, and the residue is collected and dissolved with 1.6-2.4 mL of alcohol solvent to obtain a glycyrrhiza reference solution; (ii) 1.6-2.4 μL of the glycyrrhiza test solution and 0.8-1.2 μL of the glycyrrhiza reference solution are spotted on the same silica gel G thin layer plate prepared with a basic solution with a concentration of 0.8 wt%-1.2 wt%, a mixture of n-ethyl acetate, formic acid, glacial acetic acid, and water with a volume ratio of (12-18):(0.8-1.2):1:(1.6-2.4) is used as the developing agent, the plate is taken out and dried, sprayed with 8%-12% sulfuric acid ethanol solution, heated until the spots develop clearly, and observed under 365 nm ultraviolet light, the same color spots appear in the test chromatogram at the corresponding positions of the reference chromatogram.

[0033] In some embodiments of the present application, the thin layer chromatography identification of liquorice, in step (i), the alcohol solvent is selected from at least one of methanol, n-butanol, isoamyl alcohol.

[0034] In some preferred embodiments of the present application, the thin layer chromatography identification of liquorice, in step (i), the liquorice test solution is prepared, the alcohol solvent for extraction is n-butanol; the alcohol solvent for dissolution is methanol.

[0035] In some preferred embodiments of the present application, the thin layer chromatography identification of liquorice, in step (i), the liquorice reference solution is prepared, the alcohol solvent for reflux extraction of the residue is methanol; the alcohol solvent for extraction after concentration and dissolution of the extract is n-butanol; the alcohol solvent for dissolution of the residue is methanol.

[0036] In some embodiments of the present application, the thin layer chromatography identification of liquorice, in step (i), the non-alcohol organic solvent is selected from at least one of petroleum ether, diethyl ether.

[0037] In some embodiments of the present application, the thin layer chromatography identification of liquorice, in step (i), the non-alcohol organic solvent is diethyl ether.

[0038] In some embodiments of the present application, the thin layer chromatography identification of liquorice, in step (i), the liquorice reference solution is prepared, the non-alcohol organic solvent is reflux extracted for 0.8-1.2 h; the alcohol solvent is reflux extracted for 0.8-1.2 h.

[0039] In some embodiments of the present application, the thin layer chromatography identification of liquorice, in step (ii), in the developing agent, the volume ratio of ethyl acetate, formic acid, glacial acetic acid and water is (14-16):(0.9-1.1):1:(1.8-2.2).

[0040] In some embodiments of the present application, the thin layer chromatography identification of liquorice, in step (ii), the heating temperature is 95-115℃.

[0041] In some preferred embodiments of the present application, the thin layer chromatography identification of liquorice, in step (ii), the heating temperature is 100-110℃.

[0042] In some embodiments of the present application, the determination of the content of paeoniflorin by high performance liquid chromatography specifically comprises the following steps: (i) Take 1.6-2.4 mL of the Yang-yin-Qing-fei composition, dilute 4-6 times with 40%-60% alcohol solvent, filter, and collect the filtrate as the test sample solution; take the paeoniflorin control sample, and prepare a control sample solution with a concentration of 48-72 µg / mL by adding 40%-60% alcohol solvent; (ii) Take 8-12 mL of the test sample solution and the control sample solution, inject into a liquid chromatograph, use octadecylsilane-bonded silica gel as the filler, use acetonitrile-phosphoric acid solution as the mobile phase, and detect at a wavelength of 220-240 nm.

[0043] In some embodiments of the present application, the paeoniflorin content is determined by high performance liquid chromatography, and the alcohol solvent is at least one selected from methanol and ethanol.

[0044] In some embodiments of the present application, the paeoniflorin content is determined by high performance liquid chromatography, and the alcohol solvent is methanol.

[0045] In some embodiments of the present application, the paeoniflorin content is determined by high performance liquid chromatography, and in the mobile phase, the volume concentration of the phosphoric acid solution is 0.08%-0.12%; and the volume ratio of acetonitrile to the phosphoric acid solution is (8-12):(88-92).

[0046] In some preferred embodiments of the present application, the paeoniflorin content is determined by high performance liquid chromatography, and in the mobile phase, the volume concentration of the phosphoric acid solution is 0.09%-0.11%; and the volume ratio of acetonitrile to the phosphoric acid solution is (9-11):(89-91).

[0047] In some embodiments of the present application, the paeoniflorin content is determined by high performance liquid chromatography, and the detection wavelength is 230 nm.

[0048] In some embodiments of the present application, the paeoniflorin content is determined by high performance liquid chromatography, and the theoretical plate number of the chromatographic system calculated based on the paeoniflorin peak is not less than 5000.

[0049] In some embodiments of the present application, the Yang-yin-Qing-fei composition is a Yang-yin-Qing-fei mixture.

[0050] Compared with the prior art, the present application has the following beneficial effects: 1) The quality detection method of the Yangyin Qingfei composition provided by the application firstly systematically establishes the thin layer chromatography identification method of five kinds of medicinal materials of Scrophularia ningpoensis, Radix Paeoniae Alba, Fritillaria cirrhosa, menthol and Glycyrrhiza uralensis, and introduces the high performance liquid chromatography content determination of the key active ingredient paeoniflorin, so that the comprehensive evaluation of multiple components and multiple dimensions of the product is realized. The thin layer identification method realizes clear spots, good separation degree and no interference of negative, has strong specificity and reproducibility, the content determination method has good precision, stability and repeatability, the average standard addition recovery rate reaches 99.87%, and the accuracy of the quantitative result is ensured, so that the quality control in large-scale production is suitable; 2) The identification of Scrophularia ningpoensis and Radix Paeoniae Alba is integrated in the same thin layer plate operation, and the purification step is optimized, so that the detection time and the amount of organic reagent are greatly reduced, and the advantages of green environmental protection and cost saving are obtained. BRIEF DESCRIPTION OF DRAWINGS

[0051] Figure 1 It is a thin layer identification spectrum of Scrophularia ningpoensis and Radix Paeoniae Alba in the example; Figure 2 It is a thin layer identification spectrum of Fritillaria cirrhosa in the example; Figure 3 It is a thin layer identification spectrum of menthol in the example; Figure 4 It is a thin layer identification spectrum of Glycyrrhiza uralensis in the example; Figure 5 It is a spectrum of paeoniflorin reference solution in the example; Figure 6 It is a chromatogram of paeoniflorin reference solution in the example; Figure 7 It is a double negative control solution of Radix Paeoniae Alba and Cortex Moutan; Figure 8 It is a chromatogram of paeoniflorin negative control solution. DETAILED DESCRIPTION

[0052] The content of the application will be further described in detail through specific examples. Unless otherwise specified, the raw materials, reagents or devices used in the examples can be obtained from conventional commercial channels, or can be obtained by existing technical methods. Unless otherwise specified, the test or test method is a conventional method in the art.

[0053] EXAMPLE In this embodiment, the quality detection of the Yangyin Qingfei mixture (batch number: S05009C) of Guangzhou Baiyunshan Pang Gaoshou Pharmaceutical Co., Ltd. is carried out: 1. Refer to the Chinese Pharmacopoeia 2020 edition four general rules 0502, the main components of the mixture of Yin and lung, radix scrophulariae, radix paeoniae alba, fritillariae cirrhosae, menthol and licorice are identified by thin layer chromatography, table 1 is the information table of instrument and equipment used in thin layer chromatography identification, table 2 is the information table of reagent and thin layer plate used in thin layer chromatography identification, table 3 is the information of control drug / control sample used in thin layer chromatography identification.

[0054] Table 1 information table of instrument and equipment used in thin layer chromatography identification

[0055] Table 2 information table of reagent and thin layer plate used in thin layer chromatography identification

[0056] Table 3 information of control drug / control sample used in thin layer chromatography identification

[0057] The thin layer chromatography identification of radix scrophulariae and radix paeoniae alba is as follows: (i) take 25 mL of the mixture of Yin and lung, evaporate to thick paste, put it in a conical flask with plug, add 75 mL of methanol, ultrasonic treatment for 30 min, filter, recover and concentrate the filtrate to thick paste at low temperature, collect the residue, add 30 mL of water to dissolve it, extract with diethyl ether for 2 times, the single use amount of diethyl ether is 30 mL, discard the diethyl ether liquid, combine the water solution, purify by AB-8 type macroporous adsorption resin column (inner diameter 1 cm, column height 10 cm), first elute with 50 mL of water, discard the eluent, then elute with 50 mL of methanol, collect the eluent, add neutral alumina column (100-200 mesh, 2 g, inner diameter 1 cm), collect the effluent, concentrate to dryness, add 1 mL of methanol to dissolve it as radix scrophulariae and radix paeoniae alba test solution; the negative sample solution is prepared by the same method; take haibao glycoside and paeoniflorin control solution, add methanol to prepare 1 mg / mL solution as control solution; (ii) take 10 μL of radix scrophulariae and radix paeoniae alba test solution, radix paeoniae alba negative sample solution, radix scrophulariae negative sample solution, radix paeoniae alba and cortex moutan double negative sample solution, haibao glycoside control solution and paeoniflorin control solution, and point them on the same silica gel G thin layer plate, develop with ethyl acetate-butyron-methanoic acid-water (5:3:1:1, v / v / v / v) as developing agent, take out and dry, spray with 10% sulfuric acid ethanol solution, heat at 105 ℃ until the spots develop clearly, observe under daylight, in the test sample chromatogram, the same colored spots appear at the corresponding position of the control sample chromatogram, and the negative sample has no interference.

[0058] Figure 1For the Scrophularia ningpoensis, Paeonia lactiflora thin-layer identification atlas in the examples, 1 is the negative sample without Paeonia lactiflora, 2 is the double negative sample without Paeonia lactiflora and Paeonia suffruticosa, 3 is the negative sample without Scrophularia ningpoensis, 4, 5 and 6 are different batches of test samples, 7 is the harbaroside control, 8 is the paeoniflorin control, and the chromatogram is shown in Figure 1. Figure 1 It can be seen that the determined sample amount is 10 μL for the control solution, the test sample solution and the negative sample solution, the paeoniflorin spot in channel 1 disappears, and the harbaroside spot in channel 3 disappears, indicating that the spots in the test sample are indeed derived from the target medicinal material. This method can effectively distinguish Scrophularia ningpoensis and Paeonia lactiflora / Paeonia suffruticosa, and other medicinal materials in the prescription have no interference in identification. The method has specificity and feasibility. In addition, according to this method, a durability test was conducted to investigate the influence factors such as different silica gel G thin-layer plates (self-made, Merck and Qingdao Haoyang Chemical Factory), different development temperatures (30°C and 4.0°C) and different relative humidities (35.0% and 75.0%). The results show that the development condition has good durability, and the spots are clear.

[0059] The thin-layer chromatography identification of Fritillaria cirrhosa Hsiao et K.C.Hsia is as follows: (i) Take 50 mL of Yin-Ying-Qingfei Mixture, add appropriate amount of concentrated ammonia test solution to adjust the pH to 12, and extract with ether for 2 times, with a single use amount of 25 mL. Combine the ether extract, evaporate to dryness, add 0.5 mL of anhydrous ethanol to the residue to dissolve it, and use it as the test sample solution. The negative sample solution is prepared in the same way. Take 2 g of Fritillaria cirrhosa Hsiao et K.C.Hsia control medicinal material, add appropriate amount of concentrated ammonia test solution to make it evenly wet, add 20 mL of ether, ultrasonic treatment for 15 min, stand for 12 h, filter, evaporate the filtrate to dryness, collect the residue and add 0.5 mL of anhydrous ethanol to dissolve it, and use it as the control medicinal material solution; (ii) Take 8 μL of sample test solution, 8 μL of negative sample solution and 20 μL of control medicinal material solution, and point them on the same silica gel G thin-layer plate. Use n-hexane-ethyl acetate-diethylamine (10:10:1, v / v / v) as the developing agent, take it out, dry, spray with dilute bismuth potassium iodide solution, and then place it in an iodine cylinder to fumigate until the spots develop clearly. In the test sample chromatogram, the same color main spot appears at the position corresponding to the control medicinal material chromatogram, and the negative sample has no interference.

[0060] Figure 2 For the Fritillaria cirrhosa Hsiao et K.C.Hsia thin-layer identification atlas in the examples, 1 is the negative sample without Fritillaria cirrhosa Hsiao et K.C.Hsia, 2, 3 and 4 are different batches of test samples, and 5 is the Fritillaria cirrhosa Hsiao et K.C.Hsia control medicinal material, as shown in Figure 2. Figure 2It can be seen that the determined sample size is 8 μL for sample test solution and negative sample solution, and 20 μL for control medicinal material solution. In channels 2, 3 and 4, the same color main spot appears at the corresponding position of the control medicinal material, proving the presence of Fritillaria cirrhosa Hutsun, and the corresponding main spot in channel 1 disappears, proving that the spot in the test sample is indeed from Fritillaria cirrhosa Hutsun without interference from other components, so the method has specificity. In addition, according to this method, a durability test was conducted to investigate the influence factors such as different manufacturers of silica gel G thin layer plates (self-made, Merck and Qingdao Haoyang Chemical Factory), different development temperatures (30°C and 4.0°C) and different relative humidities (35.0% and 75.0%), and the results showed that this development condition has good durability and clear spots.

[0061] The thin layer chromatography identification of menthol is as follows: (i) Take 50 mL of Yin-Qing Fei mixture and place it in a 500 mL round-bottom flask. Add 200 mL of water and a few glass beads, connect the volatile oil tester, add water to the top of the tester to fill the scale part, and overflow into the flask. Then add 2 mL of cyclohexane, connect the reflux condenser, heat and reflux for 2 h, cool, take the cyclohexane liquid, add an appropriate amount of anhydrous sodium sulfate, shake, and take the supernatant as the test sample solution. The negative sample is prepared in the same way to obtain the negative sample solution. Take an appropriate amount of menthol reference substance, add anhydrous ethanol to prepare a solution with a concentration of 2 mg / mL as the reference solution. (ii) Take 6 μL of sample test solution, 6 μL of negative sample solution and 2 μL of reference solution and spot them on the same silica gel G thin layer plate. Use petroleum ether (60-90°C)-ethyl acetate (17:3, v / v) as the developing agent, take it out and dry, spray with 5% vanillin sulfuric acid solution, heat at 105°C until the spots develop clearly, and observe under daylight. The same color main spot appears at the corresponding position of the reference chromatogram, and the negative sample has no interference.

[0062] Figure 3 In the thin layer chromatography identification spectrum of menthol in the examples, 1 is a negative sample without menthol, 2, 3 and 4 are different batches of test samples, and 5 is a menthol reference substance. Figure 3 It can be seen that the determined sample size is 6 μL for sample test solution and negative sample solution, and 2 μL for reference solution. Channel 5 is consistent with channels 2, 3 and 4 at the main spot position, and channel 1 has no such spot, so the method has specificity. In addition, according to this method, a durability test was conducted to investigate the influence factors such as different manufacturers of silica gel G thin layer plates (self-made, Merck and Qingdao Haoyang Chemical Factory), different development temperatures (30°C and 4.0°C) and different relative humidities (35.0% and 75.0%), and the results showed that this development condition has good durability and clear spots.

[0063] The thin layer chromatography identification of licorice is as follows: (i) Take 30 mL of Yangyin Qingfei Mixture, extract with water-saturated n-butanol for 3 times, with a single dosage of 30 mL, combine the n-butanol extract, wash with 20 mL of water for 3 times, discard the water wash, evaporate the n-butanol liquid, add 2 mL of methanol to the residue to dissolve it as the test sample solution; prepare the negative sample solution in the same way; take another 1 g of licorice control medicinal material, add 40 mL of ether, heat and reflux for 1 h, filter, add 30 mL of methanol to the residue, heat and reflux for 1 h, filter, evaporate the filtrate, add 40 mL of water to the residue to dissolve it, extract with water-saturated n-butanol for 3 times, with a single dosage of 30 mL, combine the n-butanol extract, wash with 20 mL of water for 3 times, discard the water wash, evaporate the n-butanol liquid, add 2 mL of methanol to the residue to dissolve it as the control medicinal material solution; (ii) Take 2 ul of the test sample solution, 2 µL of the negative sample solution, and 1 µL of the control medicinal material solution, and spot them on the same silica gel G thin layer plate prepared with 1 wt% sodium hydroxide solution, develop them with n-ethyl acetate-formic acid-glacial acetic acid-water (15: 1: 1: 2, v / v / v / v) as the developing agent, take them out and air dry, spray them with 10% sulfuric acid ethanol solution, heat them at 105°C until the spots develop clear color, and place them under the ultraviolet lamp (365 nm) for inspection. In the test sample chromatogram, at the positions corresponding to the control medicinal material chromatogram, the same color fluorescent spots appear, and the negative sample does not interfere.

[0064] Figure 4 In the licorice thin layer identification spectrum in the examples, 1 is the negative sample without licorice, 2, 3, and 4 are different batches of test samples, and 5 is the licorice control. As shown in the figure, Figure 4 It can be seen that the determined sample size is 2 µL for the test sample solution and the negative sample solution, and 1 µL for the control medicinal material solution. Channel 5 shows the same color fluorescent spots as channels 2, 3, and 4 under the ultraviolet lamp, and channel 1 has no such fluorescent spots. The method has specificity. In addition, according to this method, a durability test was conducted to investigate the influence factors such as different silica gel G thin layer plates (self-made, Merck, and Qingdao Haoyang Chemical Factory), different developing temperatures (30°C and 4.0°C), and different relative humidities (35.0% and 75.0%). The results show that the developing conditions are good in durability, and the spots are clear.

[0065] 2. The content of paeoniflorin in the Yangyin Qingfei Mixture was determined by high performance liquid chromatography: (1) Instruments and reagents: Agilent 1260 liquid chromatograph (workstation software version B.03.02), VWD detector; Acetonitrile (American Tianqi Company, chromatographic pure); Phosphoric acid (Guangzhou Chemical Reagent Factory, analytical pure); Methanol (Guangzhou Chemical Reagent Factory, chromatographic pure); The paeoniflorin control sample was provided by the China Institute for Drug and Biological Products (batch number: 110736-201741).

[0066] (2) Chromatographic conditions and system suitability test: Chromatographic column: Agilent Hypersil ODS column (150 mm x 4.6 mm, 5 μm); Mobile phase: 0.1% phosphoric acid solution-acetonitrile (90:10, v / v); flow rate: 1 mL / min; Detection wavelength: 230 nm; Column temperature: 25°C; Injection volume: 10 μL.

[0067] (3) The detection steps are as follows: (i) Take 2 mL of Yangyin Qingfei Mixture and place it in a 10 mL volumetric flask. Dilute to the mark with 50% methanol, shake well, filter, filter through a 0.45 μL filter membrane, and take the filtrate as the test sample solution. Take the bai-sha and mudanpi double-negative samples and prepare double-negative control solutions in the same way. Take 1.954 mg of paeoniflorin control sample and place it in a 10 mL volumetric flask. Dissolve it with 50% methanol, then dilute to the mark, shake well, and obtain a stock solution. Precisely take 3 mL of the stock solution and place it in a 10 mL volumetric flask. Dilute to the mark with 50% methanol, shake well, filter through a 0.45 μL filter membrane, and obtain a control sample solution with a concentration of 60 μg / mL; (ii) Take 10 mL of the test sample solution, double-negative control solution, paeoniflorin negative control solution, and control sample solution, and inject them into the liquid chromatograph for detection.

[0068] Figure 5 For the paeoniflorin control sample solution spectrum in the example, Figure 6 For the paeoniflorin control sample chromatogram in the example, Figure 7 For the bai-sha and mudanpi double-negative control solution, Figure 8 For the paeoniflorin negative control solution chromatogram, from Figures 5-8 It can be seen that paeoniflorin has a large absorption near 230 nm, so 230 nm is used as the detection wavelength. Under this test condition, there is a corresponding peak in the paeoniflorin peak position in the test sample chromatogram, and the paeoniflorin peak can also be seen in the bai-sha negative control sample chromatogram. The bai-sha and mudanpi double-negative samples do not have a paeoniflorin peak, indicating that both bai-sha and mudanpi in the product formula contain paeoniflorin. This shows that the method is specific for determining the paeoniflorin content in bai-sha and mudanpi in Yangyin Qingfei Mixture, and there is no interference from the negative. The bai-sha and mudanpi contained in this product can be counted as paeoniflorin.

[0069] (4) Linear range investigation: Take paeoniflorin control stock solution, precision take 6mL to 10mL amount bottle, add 50% methanol to the scale, shake up, make paeoniflorin concentration is 117.24 μg / mL control solution, shake up, through 0.45 μm filter membrane filtration, take continue filter 1 μL, 2 μL, 4 μL, 8 μL, 16 μL and 20 μL, according to the preceding chromatographic conditions analysis, with control solution injection amount as the abscissa X, corresponding peak area as ordinate Y, linear regression, get regression equation y=1426.7565x-40.6421, R 2 =0.9998, control solution injection amount in 0.11724-2.34480 μg range and peak area response value show good linear relationship.

[0070] Table 4 paeoniflorin injection amount and paeoniflorin peak area

[0071] (5) injection precision test: Take the same batch of sample test solution, according to the preceding chromatographic conditions, continuous injection 6 times for determination, the results show that the average peak area is 751.446, RSD=1.12% (n=6), indicating that the injection precision is good.

[0072] Table 5 precision test results

[0073] (6) stability test: Take sample (batch number: S05009C), according to the preparation method of test solution, take test solution respectively at 0, 1, 2, 4, 8, 12, 18 and 24h, 10 μL injection liquid chromatograph for determination, the results show that RSD=0.42% (n=8), indicating good stability.

[0074] Table 6 stability test results

[0075] (7) repeatability test: Take 6 batches of sample S05009C, according to the preparation method of test solution, according to the law, the results show that the average content of paeoniflorin is 0.3034 mg / mL, RSD=0.85% (n=6), indicating good repeatability.

[0076] Table 7 repeatability test results

[0077] (8) accuracy test: Precisely pipette 1 mL of sample (batch number S05009C, paeoniflorin content 0.3034 mg / mL) into a 10 mL volumetric flask, precisely add 1 mL of paeoniflorin control solution (paeoniflorin concentration 0.3036 mg / mL), dissolve with 50% methanol, filter, and precisely pipette 10 μL into the liquid chromatograph for determination according to the aforementioned chromatographic conditions. The average recovery rate was 99.87% and RSD = 1.48 (n = 6), indicating that the method has high precision.

[0078] Table 8 Precision test results

[0079] (9) Chromatographic column suitability test: Take the sample with batch number S05009C, prepare the test solution according to the preparation method of the test solution, and use different chromatographic columns for determination, and calculate the sample content. The results show that the average value of the paeoniflorin content determination results using different chromatographic columns is 0.2764 mg / mL, and RSD is 1.02%, indicating that the established method has good suitability for chromatographic columns.

[0080] Table 9 Chromatographic column suitability test results

Claims

1. A quality detection method of a Yangyin Qingfei composition, the components of the Yangyin Qingfei composition including Rehmannia glutinosa, Scrophularia ningpoensis, Cortex Moutan, Radix Paeoniae Alba, Ophiopogon japonicus, Fritillaria walujin, menthol and licorice, characterized in that, The quality detection method comprises the following steps: adopting thin layer chromatography to identify Scrophulariae Radix, Radix Paeoniae Alba, Fritillariae Cirrhosae Bulbus, menthol and Glycyrrhiza; adopting high performance liquid chromatography to detect the content of paeoniflorin in the composition.

2. The quality detection method according to claim 1, characterized in that, The thin layer chromatography identification of Scrophulariae Radix and Radix Paeoniae Alba specifically comprises the following steps: (i) 20-30 mL of the Yin-nouring and Lung-clearing composition is concentrated, and then added with 60-90 mL of an alcohol solvent for extraction; the extraction liquid is collected and concentrated; the obtained residue is dissolved with 20-30 mL of water, and then added with 20-30 mL of a non-alcohol organic solvent for extraction to remove impurities; the water phase is collected and purified through a macroporous adsorption resin column; the eluate is collected and purified through an aluminum oxide column; the purified liquid is concentrated, and then dissolved with 0.8-1.2 mL of an alcohol solvent to obtain a Scrophulariae Radix and Radix Paeoniae Alba test sample solution; Scrophularin and paeoniflorin reference substances are taken, and an alcohol solvent is added to prepare reference substance solutions with a concentration of 0.8-1.2 mg / mL; (ii) 8-12 μL of the Scrophulariae Radix and Radix Paeoniae Alba test sample solution, the Scrophularin reference substance solution and the paeoniflorin reference substance solution are spotted on the same silica gel G thin layer plate; a mixture of ethyl acetate, butanone, formic acid and water with a volume ratio of (4-6):(2.4-3.6):(0.8-1.2):1 is used as a developing agent for development; the developed thin layer plate is taken out and dried; 8%-12% sulfuric acid ethanol solution is sprayed; heating is performed until the spots develop clearly; the thin layer plate is observed under sunlight; the same color spots appear in the test sample chromatogram at positions corresponding to the reference substance chromatogram.

3. The quality detection method according to claim 2, characterized in that, In the thin layer chromatography identification of Scrophulariae Radix and Radix Paeoniae Alba, the alcohol solvent is at least one selected from methanol and ethanol; And / or, the non-alcohol organic solvent is at least one selected from diethyl ether and ethyl acetate; And / or, the aluminum oxide column is filled with neutral aluminum oxide with a particle size of 100-200 meshes and a dosage of 2-3 g; And / or, the heating temperature is 95-115 ℃.

4. The quality detection method of claim 1, wherein, The thin layer chromatography identification of Fritillariae Cirrhosae Bulbus specifically comprises the following steps: (i) 40-60 mL of the Yin-nouring and Lung-clearing composition is added with an alkali solution to adjust the pH to 10-12; 20-40 mL of an ether solvent is added for shaking extraction for 2-3 times; the extraction liquid is combined and concentrated; the obtained residue is dissolved with 0.4-0.6 mL of an alcohol solvent to obtain a Fritillariae Cirrhosae Bulbus test sample solution; 1.6-2.4 g of Fritillariae Cirrhosae Bulbus reference substance is added with an alkali solution for wetting; 16-24 mL of an ether solvent is used for ultrasonic extraction; the extraction liquid is collected and concentrated; the obtained residue is dissolved with 0.4-0.6 mL of an alcohol solvent to obtain a Fritillariae Cirrhosae Bulbus reference substance solution; (ii) 6.4-9.6 μL of the Fritillariae Cirrhosae Bulbus test sample solution and 16-24 μL of the Fritillariae Cirrhosae Bulbus reference substance solution are spotted on the same silica gel G thin layer plate; a mixture of n-hexane, ethyl acetate and diethylamine with a volume ratio of (8-12):(8-12):1 is used as a developing agent for development; the developed thin layer plate is taken out and dried; dilute bismuth potassium iodide solution is sprayed; the thin layer plate is smoked in an iodine jar until the spots develop clearly; the same color spots appear in the test sample chromatogram at positions corresponding to the reference substance chromatogram.

5. The quality detection method of claim 1, wherein The thin layer chromatography identification of menthol specifically comprises the following steps: (i) Take 40-60 mL of the Yin-nourishing and Lung-clarifying Composition, add 160-240 mL of water, and then perform volatile oil extraction, collect the volatile oil with 1.6-2.4 mL of a non-polar organic solvent, and dehydrate the obtained solution to obtain a test solution; take the menthol reference substance, add an alcohol solvent to prepare a reference solution with a concentration of 1.6-2.4 mg / mL; (ii) Take 4.8-7.2 µL of the menthol test solution and 1.6-2.4 µL of the menthol reference solution, and spot them on the same silica gel G thin layer plate, use a mixture of petroleum ether and ethyl acetate with a volume ratio of (4.5-6.8): 1 as the developing agent, take out and air dry, spray with 4%-6% vanillin sulfuric acid solution, heat until the spots develop clearly, and then observe under daylight, the test chromatogram shows the same color spots at the corresponding positions of the reference chromatogram.

6. The quality detection method according to claim 5, characterized in that, In the thin layer chromatography identification of the menthol, the non-polar organic solvent is at least one selected from cyclohexane, n-hexane, and petroleum ether; And / or, in the developing agent, the temperature of petroleum ether is 60-90 ℃; And / or, the heating temperature is 95-115 ℃.

7. The quality detection method of claim 1, wherein, The thin layer chromatography identification of the liquorice specifically includes the following steps: (i) Take 24-36 mL of the Yin-nourishing and Lung-clarifying Composition, extract it with an alcohol solvent saturated with an equal volume of water for 2-4 times, combine the extract and wash it with 16-24 mL of water for 2-4 times, discard the water, concentrate the alcohol solvent layer to dryness, collect the residue, and dissolve it with 1.6-2.4 mL of an alcohol solvent to obtain a liquorice test solution; take 0.8-1.2 g of liquorice reference substance, extract it with a non-alcohol organic solvent by reflux, collect the residue, extract it with an alcohol solvent saturated with 24-36 mL of water by reflux, concentrate the obtained extract, add 32-48 mL of water to dissolve it, extract it with an alcohol solvent saturated with 24-36 mL of water for 2-4 times, wash the extract with water, concentrate it, collect the residue, and dissolve it with 1.6-2.4 mL of an alcohol solvent to obtain a liquorice reference solution; (ii) Take 1.6-2.4 µL of the liquorice test solution and 0.8-1.2 µL of the liquorice reference solution, and spot them on the same silica gel G thin layer plate prepared with a basic solution with a concentration of 0.8wt%-1.2wt%, use a mixture of n-ethyl acetate, formic acid, glacial acetic acid, and water with a volume ratio of (12-18): (0.8-1.2): 1: (1.6-2.4) as the developing agent, take out and air dry, spray with 8%-12% sulfuric acid ethanol solution, heat until the spots develop clearly, and then observe under 365 nm ultraviolet light, the test chromatogram shows the same color spots at the corresponding positions of the reference chromatogram.

8. The quality detection method according to claim 7, characterized in that, The alcohol solvent is at least one selected from methanol, n-butanol, and isoamyl alcohol; And / or, the non-alcohol organic solvent is at least one selected from petroleum ether and diethyl ether; And / or, the heating temperature is 95-115 ℃.

9. The quality detection method of claim 1, wherein, The determination of the content of paeoniflorin by high performance liquid chromatography specifically includes the following steps: (i) Take 1.6-2.4 mL of the Yangyin Qingfei composition, dilute 4-6 times with 40%-60% alcohol solvent, filter, and collect the filtrate as the test sample solution; take the paeonol reference substance, and prepare a reference substance solution with a concentration of 48-72 µg / mL by adding 40%-60% alcohol solvent; (ii) Take 8-12 mL of the test sample solution and the reference substance solution, inject them into a liquid chromatograph, use octadecylsilane-bonded silica gel as the filler, use acetonitrile-phosphoric acid solution as the mobile phase, and detect at a wavelength of 220-240 nm.

10. The quality detection method of claim 9, wherein, The alcohol solvent is at least one selected from methanol and ethanol; And / or, in the mobile phase, the volume concentration of the phosphoric acid solution is 0.08%-0.12%; the volume ratio of acetonitrile to the phosphoric acid solution is (8-12):(88-92).

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