Subject-based quality detection method for semen muscari pills

Abstract

The invention discloses a substance-based quality detection method for a Sui pill, and belongs to the technical field of quality detection of traditional Chinese medicines. According to the method, standardized control of a classic famous prescription is realized by constructing a multi-dimensional quality control system. The method comprises the following steps: firstly, carrying out specific identification on fructus perillae and semen lepidii in the prescription by adopting a thin-layer chromatography; meanwhile, a multi-index component content determination method is established, and the content of the quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside and the content of the rosmarinic acid are synchronously determined by using a high performance liquid chromatography; according to the invention, a systematic and normative substance reference quality standard of the Sui pill is established by constructing a comprehensive quality control system combining qualitative identification and multi-component quantitative analysis, and a reliable basis is provided for quality consistency evaluation and product stability control of a classic famous prescription preparation.

Description

Technical Field

[0001] This invention belongs to the field of quality testing technology for traditional Chinese medicine compound preparations, and specifically relates to a quality testing method for the material reference of Suting Pill. Background Technology

[0002] Su Ting Wan, also known as "Su Ting Ding Chuan Wan," is a prescription derived from Volume 53 of *Yizong Jinjian: Youke Xinfa Yaojue*, compiled by Wu Qian during the Qianlong era of the Qing Dynasty. *Yizong Jinjian* is a comprehensive medical classic from the Qing Dynasty, summarizing medical theories and practical experiences from various dynasties. The main ingredients of Su Ting Wan are perilla seeds and lepidium seeds, with jujube as an excipient. Perilla seeds are pungent and warm, dispersing and entering the lung and large intestine meridians. They have the effects of lowering qi, resolving phlegm, relieving cough and asthma, and are often used for symptoms of phlegm accumulation, shortness of breath, cough, and chest tightness. Their moistening and descending properties also help to clear the intestines and relieve constipation. Lepidium seeds are also widely used in traditional Chinese medicine, having the effects of purging the lungs, relieving asthma, and promoting diuresis and reducing swelling. Combined with perilla seeds, they complement each other, working together to exert a powerful effect of relieving cough and asthma, resolving phlegm, and promoting diuresis. Jujubes play a role in harmonizing the various medicines and moderating their effects, making the entire prescription more mild and reducing its stimulation to the human body.

[0003] Current in-depth research on the overall quality of Suting Pills is still weak. Existing research mostly focuses on the analysis of single medicinal ingredients or the verification of local efficacy, lacking a comprehensive and systematic study of the overall material basis and mechanism of action of Suting Pills. Although the formulation of Suting Pills is simple, the chemical composition of each medicinal ingredient is complex. These components synergistically or antagonistically interact, collectively constituting the unique material basis of Suting Pills' efficacy. At the same time, the quality of Suting Pill preparations is highly susceptible to interference from factors such as planting environment, processing methods, and storage conditions, leading to significant differences in the content of active ingredients and the stability of efficacy between different production batches. Current quality standards mostly remain at the level of physical properties or partial physicochemical indicator testing, lacking a comprehensive understanding of the overall characteristics of complex components, making it difficult to meet the requirements of clinical consistency and modern supervision. Therefore, improving the technical level of Suting Pills' quality control and establishing a comprehensive and systematic quality assessment system are crucial for achieving precise control of drug quality. Constructing a quality evaluation model that can simultaneously determine the content of multiple components and comprehensively assess the efficacy and activity of the drug will not only help to deepen the research on the material basis of Suting Pill, but also strengthen the quality supervision of the entire production chain, establish scientific and standardized quality standards, and provide irreplaceable technical support for the standardized production and clinical application of the preparation. Summary of the Invention

[0004] Given the current weakness in overall quality research on Suting Pills, which focuses primarily on individual medicinal materials and lacks research on the overall material basis and efficacy mechanism, and considering the significant variations in its formulation quality due to multiple factors, the inadequacy of existing standards, and the difficulty in meeting clinical and regulatory requirements, there is an urgent need to improve quality control technology and establish a comprehensive evaluation system. The purpose of this invention is to provide a quality testing method for the material basis of Suting Pills.

[0005] To achieve the above objectives, the present invention employs the following technical solution: This invention discloses a method for quality testing of Suting pill material standards, comprising the following steps: 1) Prepare the material standard for Su Ting Wan, the test solution of each medicinal material and the corresponding control medicinal material solution respectively; 2) Qualitative analysis of Lepidium apetalum seeds and Perilla frutescens seeds in the material standard was performed using thin-layer chromatography; 3) Quantitative analysis of quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside and rosmarinic acid in the material standard was performed using high performance liquid chromatography.

[0006] In step 1), the preparation method of the material standard of Su Ting Wan is as follows: weigh perilla seeds and lepidium seeds in sequence, clean them, process them, crush them, sieve them, mix them and then mix them with jujube paste to form a soft material, roll them into strips, cut them into pills, and dry them to obtain the product.

[0007] Preferably, the roasted Lepidium seeds and roasted Perilla seeds are pulverized and passed through a 100-mesh sieve.

[0008] Preferably, the roasted Lepidium seed powder and the roasted Perilla seed powder are mixed in a 1:1 ratio, and the mass ratio of the mixed medicinal materials to the jujube paste is 1:0.5.

[0009] In step 1), the medicinal materials are Lepidium seed and Perilla seed; The preparation method of the Lepidium apetalum test solution is as follows: methanol is added to the Lepidium apetalum standard, ultrasonic treatment is performed, filtration is carried out, the filtrate is evaporated to dryness, and the residue is dissolved in methanol to obtain the Lepidium apetalum test solution; quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside is used as a control, and methanol is added to obtain the control solution. The preparation method of the perilla seed test solution is as follows: methanol is added to the Su Ting Wan material standard, ultrasonic treatment is performed, filtration is carried out, the filtrate is evaporated to dryness, and the residue is dissolved in methanol to obtain the perilla seed test solution; the stir-fried perilla seed reference material is taken and the reference material solution is prepared in the same way.

[0010] Preferably, the ultrasonic treatment time is 30 minutes; In step 2), the specific steps for thin-layer chromatography identification are as follows: take the test solution of Lepidium apetalum and the reference solution of quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside, spot them, use acetone-water as the developing solvent, spray with 2% aluminum trichloride ethanol solution, and examine under a 365 nm ultraviolet lamp. Spot the test solution of perilla seeds and the reference solution of stir-fried perilla seeds, and use hexane-toluene-ethyl acetate-formic acid as the developing solvent. Spray with aluminum trichloride solution and examine under sunlight and 365 nm ultraviolet light, respectively.

[0011] Preferably, during thin-layer chromatography identification, the spotting volume of the Lepidium apetalum test solution and the quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside reference solution is 1.5 µL; The sample volume for the perilla seed test solution and the stir-fried perilla seed reference material solution was 2 µL.

[0012] Preferably, in the acetone-water mixture, the volume ratio of acetone to water is 1:1; in the hexane-toluene-ethyl acetate-formic acid mixture, the volume ratio of hexane, toluene, ethyl acetate, and formic acid is 2:5:2.5:0.5.

[0013] Step 3) In the quantitative analysis, The test solution was prepared by taking Su Ting Wan as a reference, adding methanol, ultrasonically extracting, filtering, and taking the filtrate to obtain the test solution.

[0014] Preferably, the methanol is an 80% aqueous solution.

[0015] Step 3) In the quantitative analysis, The method for preparing the reference solution is as follows: weigh quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside reference standard, add methanol to prepare a single reference solution containing 30 μg of quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside standard per 1 mL; Prepare a single reference solution containing 80 μg of rosmarinic acid per 1 mL by adding methanol to the rosmarinic acid reference standard.

[0016] In step 3), the high-performance liquid chromatography (HPLC) conditions were as follows: Shim-pack VP-ODS column (250 mm × 4.6 mm, 5 μm); mobile phase: acetonitrile-0.1% phosphoric acid aqueous solution; flow rate: 0.8 mL / min; column temperature: 30℃; injection volume: 10 μL; detection wavelength: 254 nm; and the elution gradient is shown in the table below.

[0017] Preferably, the content of quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside in the material standard of Suting Pill is ≥0.035%; and the content of rosmarinic acid is ≥0.104%.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The quality detection method for the substance reference of Suting Pills provided by the present invention combines thin-layer chromatography with high-performance liquid chromatography to achieve a comprehensive quality assessment of the substance reference of Suting Pills. Among them, thin-layer chromatography separates the characteristic component spots of Perilla Seed and Lepidium Seed, establishes a specific identification method for medicinal materials, which is simple to operate, has high separation efficiency, can effectively exclude the interference of negative controls, and ensure the authenticity of raw medicinal materials; while high-performance liquid chromatography is used to efficiently and sensitively determine its content. The following optimized liquid chromatography conditions are adopted for this method: the mobile phase is acetonitrile - 0.1% phosphoric acid, the flow rate is 0.8 mL / min, the column temperature is 30 °C, the injection volume is 10 μL, and the detection wavelength is 254 nm, ensuring an ideal chromatographic peak separation effect. This detection method is simple to operate, has high accuracy and good repeatability, provides a convenient and practical way for the quality inspection of the substance reference of Suting Pills, helps to establish its quality standard, and thus guarantees the stability of product quality. Within a specific concentration range, this method can accurately reflect the concentration changes of different components, showing good linear characteristics. The correlation coefficients (R²) of quercetin-3-O-β-D-glucoside-7-O-β-D-gentiobioside and rosmarinic acid are both greater than 0.99; this method evaluates the quality of the substance reference of the classic famous prescription Suting Pills through multiple detection methods, can lay a solid foundation for the quality stability of the product, and achieve effective control of the quality of the substance reference of the classic famous prescription Suting Pills. Description of the Drawings

[0019] Figure 1 It is the HPLC diagram for the particle size investigation of stir-fried Lepidium Seed in the substance reference of Suting Pills of the present invention; Figure 2 It is the HPLC diagram for the particle size investigation of stir-fried Perilla Seed in the substance reference of Suting Pills of the present invention; Figure 3 It is the comparison diagram of the extraction methods for the identification of Lepidium Seed in the thin-layer identification of the substance reference of Suting Pills of the present invention; where 1 - reference substance 3 μL; 2 - test solution 1 3 μL; 3 - test solution 1 6 μL; 4 - test solution 2 3 μL; 5 - test solution 3 3 μL; Figure 4 It is the comparison diagram of different developing agents and sample application amounts for the identification of Lepidium Seed in the thin-layer identification of the substance reference of Suting Pills of the present invention. A: The developing agent is chloroform - methanol (5:1), where 1 - reference solution 1 μL; 2 - test solution 1 μL; 3 - reference solution 3 μL; 4 - test solution 3 μL; B: The developing agent is acetone - water (1:1), where 1 - reference solution 1 μL; 2 - test solution 1 μL; 3 - reference solution 3 μL; 4 - test solution 3 μL; C: The developing agent is acetone - water (1:1), where 1 - reference solution 4.5 μL; 2 - test solution 1.5 μL; Figure 5It is the TLC identification diagram of Lepidium apetalum Willd. in the substance reference of Suting Pills of the present invention; Figure 6 It is the TLC identification diagram of Perilla seed in the substance reference of Suting Pills of the present invention; A is observed under ultraviolet light, and B is observed under daylight; Figure 7 It is the result diagram of the linear relationship investigation of the index components, where A is quercetin-3-O-β-D-glucoside-7-O-β-D-gentiobioside, and B is rosmarinic acid. Detailed implementation manners

[0020] To further understand the present invention, the present invention will be described below in conjunction with embodiments. These descriptions are only to further explain the features and advantages of the present invention and are not used to limit the claims of the present invention.

[0021] It should be noted that the terms "first", "second", etc. in the description and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects and do not necessarily need to describe a specific order or sequence. It should be understood that such data can be interchanged under appropriate circumstances so that the embodiments of the present invention described here can be implemented in an order other than those illustrated or described here. In addition, the terms "comprising" and "having" and any variations thereof are intended to cover non-exclusive inclusion. For example, a process, method, system, product or device comprising a series of steps or units does not necessarily need to be limited to those steps or units clearly listed, but may include other steps or units not clearly listed or inherent to these processes, methods, products or devices.

[0022] To make the purpose, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with specific embodiments and with reference to the drawings. The reagent information used in the following embodiments is shown in Table 1, the reference substances used are shown in Table 2, and the instrument information used is shown in Table 3. For those not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer, and the reagents or instruments used are all conventional products that can be obtained through commercial purchase.

[0023] Table 1: Experimental reagents and manufacturers

[0024] Table 2: Reference substances and manufacturers

[0025] Table 3: Model numbers and manufacturers of the instruments used

[0026] Below, taking the substance reference of Suting Pills as an example, the present invention will be described in detail through specific embodiments I. Preparation of Su Ting Wan Material Standard 1. Particle size analysis (1) Investigation on the particle size of stir-fried Lepidium apetalum seeds The same batch of roasted Lepidium seeds with the same dosage were compared at different particle sizes (80 mesh, 100 mesh, and 120 mesh) using quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside as the indicator. The contents of quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside were 0.132%, 0.134%, and 0.130% for particle sizes passing through an 80-mesh sieve, a 100-mesh sieve, and a 120-mesh sieve, respectively. (See Table 4 and...) Figure 1 Data shows that the content of quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside in stir-fried Lepidium apetalum is the highest with a particle size of 100 mesh, and the peak shape is good. Therefore, a particle size of 100 mesh was selected.

[0027] Table 4: Results of Determination of Content in Fried Lepidium Seeds

[0028] (2) Examination of the particle size of perilla seeds Different particle sizes (50 mesh, 60 mesh, 80 mesh, and 100 mesh) were examined using the same batch and dosage of roasted perilla seeds, with the content of quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside as the indicator for comparison. The contents of particles passing through a 50-mesh sieve, 60-mesh sieve, 80-mesh sieve, and 100-mesh sieve were 0.240%, 0.246%, 0.250%, and 0.252%, respectively, as shown in Table 5. Figure 2 It can be seen that the content of quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside is the highest in roasted perilla seeds with a particle size of 100 mesh, and the peak shape is good. Therefore, a particle size of 100 mesh is selected.

[0029] Table 5: Results of Perilla Seed Content Determination

[0030] 2. Optimization of raw material and auxiliary material ratios Processed and sieved powders of Lepidium apetalum and Perilla frutescens were mixed with jujube paste at weight ratios of 1:0.3, 1:0.4, 1:0.5, 1:0.6, and 1:0.7 for 15-20 minutes until a soft material that "can be formed into a clump when grasped but crumbles easily when touched" was formed. Based on the uniformity of mixing, the softness and hardness of the material, and viscosity, Table 6 shows that the optimal mixing ratio of medicinal materials to jujube paste is 1:0.5, resulting in a uniform mixture with moderate softness and viscosity.

[0031] Table 6: Raw and Auxiliary Material Ratios

[0032] 3. Investigation of drying conditions The prepared pills were placed in an electric thermostatic drying oven at 60℃, and different drying times (2h, 3h, 4h, 5h, 6h) were investigated, with moisture content as the evaluation index. The results are shown in Table 7. After drying for 5 hours, the moisture content met the requirement of "moisture content shall not exceed 9%" in the Chinese Pharmacopoeia (2020 edition). Therefore, the drying time was determined to be 5 hours.

[0033] Table 7: Results of Drying Time and Moisture Content Investigation

[0034] II. Qualitative analysis of Lepidium apetalum and Perilla frutescens seeds in a material standard using thin-layer chromatography. 1. The thin-layer chromatography method for identifying Lepidium apetalum seeds in the Su Ting Wan material standard is as follows: (1) Investigation of the extraction method of the test solution Test solution 1: Weigh 2.0 g of Su Ting Wan (a traditional Chinese medicine) as a reference standard, place it in a stoppered conical flask, add 25 mL of methanol, sonicate for 30 min, filter, evaporate the filtrate to dryness, and dissolve the residue in 1 mL of methanol to obtain the test solution.

[0035] Test solution 2: Weigh 2.0 g of Su Ting Wan (a traditional Chinese medicine) as a reference standard, place it in a stoppered conical flask, add 30 mL of 70% methanol, heat under reflux for 1 h, filter, and collect the filtrate.

[0036] Test solution 3: Weigh 2.0 g of Su Ting Wan (a traditional Chinese medicine) as a reference standard, place it in a stoppered conical flask, add 40 mL of 70% methanol, heat under reflux for 1 h, filter, and collect the filtrate.

[0037] Reference solution: Separately, accurately weigh the reference standard quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside, and add 30% methanol to prepare a solution containing 90 μg per 1 mL.

[0038] Depend on Figure 3 It is evident that the color development of sample solution one is good and the concentration is high, therefore the extraction method of sample solution one is selected.

[0039] (2) Investigation of developing solvent and spotting amount The composition of the developing solvent and the amount of sample applied were systematically screened and optimized. Based on relevant existing technologies, see Appendix. Figures 4-5, Initially, two developing systems of acetone - water (1:1) and chloroform - methanol (5:1) were preliminarily selected for comparison. First, using chloroform - methanol (5:1) as the developing agent, 1 μL and 3 μL of the test sample and reference solution were spotted respectively. The results showed that the shape of the solvent front was abnormal, indicating that this developing system was not suitable for the identification of this product. Subsequently, the sample application amounts of 1 μL and 3 μL of the test sample and reference were investigated, with acetone - water (1:1) as the developing agent, and it was found that the resolution was good. Using acetone - water (1:1) as the developing agent, the sample application amounts of 4.5 μL of the reference solution and 1.5 μL of the test sample solution were investigated, and it was found that the separation effect did not meet the expectation. It was speculated that the reason might be related to the easy volatilization of acetone, resulting in a change in the actual ratio of the developing agent. Based on this, the operation was improved, and the freshly prepared acetone - water (1:1) developing agent was used for verification under the same sample application amount conditions. The results showed that the chromatographic resolution was significantly improved, and the obtained spots were clear and there was no trailing phenomenon. Finally, it was determined that the freshly prepared acetone - water (1:1) was used as the developing agent, and the sample application amount was 1.5 μL, as the optimized conditions for the thin - layer identification of semen lepidii in the substance reference of Suting Pills.

[0040] 2. In the substance reference of Suting Pills, the thin - layer identification method for perilla seed is as follows: Preparation of test sample solution: Weigh 1 g of the medicinal material powder of the substance reference of Suting Pills, add 25 mL of methanol, ultrasonically treat for 30 min, filter, evaporate the filtrate to dryness, dissolve the residue in 1 mL of methanol to obtain the test sample solution. Take 1 g of the control medicinal material of stir - fried perilla seed, and prepare the control medicinal material solution in the same way for thin - layer identification. See appendix Figure 6 .

[0041] According to the technical specification of 0502 in the Chinese Pharmacopoeia (2020 Edition), accurately pipette 2 μL each of the test sample solution and the control medicinal material solution, and spot them on the same silica gel G thin - layer plate respectively. n - hexane - toluene - ethyl acetate - formic acid (volume ratio 2:5:2.5:0.5) is used as the developing agent. Take it out and let it dry, evenly spray aluminum trichloride test solution, and observe under a 365 nm ultraviolet lamp.

[0042] III. Using high - performance liquid chromatography to determine the contents of the index components (quercetin - 3 - O - β - D - glucopyranoside - 7 - O - β - D - gentiobioside, rosmarinic acid) in the substance reference 1. Preparation of test sample solution Accurately weigh 1.0 g of the substance reference of Suting Pills, place it in a stoppered conical flask, add 25 mL of 80% methanol solution, ultrasonically extract for 30 min (ultrasonic power 250 W, ultrasonic frequency 40 kHz), take the filtrate and pass it through a 0.45 µm microporous filter membrane to obtain the test sample solution.

[0043] 2. Preparation of reference solution Accurately weigh quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside and rosmarinic acid standards into separate stoppered conical flasks, and add methanol to prepare a single reference solution containing 30 μg of quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside standard and 80 μg of rosmarinic acid standard per 1 mL.

[0044] 3. Chromatographic conditions The chromatographic column was a Shim-pack VP-ODS (250 mm × 4.6 mm, 5 μm) column; the mobile phase was acetonitrile-0.1% phosphoric acid aqueous solution; the flow rate was 0.8 mL / min; the column temperature was 30℃; the injection volume was 10 μL; and the detection wavelength was 254 nm. The gradient elution program was as follows:

[0045] The content determination results are shown in the table below. The results show that the content of quercetin disaccharide in the 15 batches of Suting Pill material standard is distributed between 0.045% and 0.055% (mean 0.050%), and the content of rosmarinic acid is between 0.132% and 0.158% (mean 0.148%). The content of each component in the Suting Pill material standard of each batch has small fluctuations and all meet the mean ± 30% tolerance range specified in the "Technical Guidelines for Pharmaceutical Research of Ancient Classic Prescriptions". No discrete values ​​were found, indicating that the quality of each batch of samples is very stable and meets the requirements of the guidelines.

[0046] Table 8: Content of components in the traditional Chinese medicine formula Su Ting Wan and its material reference indicators

[0047] IV. Methodological Investigation 1. Examination of linear relationships To verify the linear relationship between quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside and rosmarinic acid, the above-mentioned reference standards were prepared by adding methanol to prepare reference standard solutions. These solutions were then diluted using a two-fold dilution method to obtain reference standard solutions of different mass fractions. Chromatographic analysis was performed under the same conditions as the standard content determination. Linear regression analysis was conducted with the mass concentration of each reference standard solution as the abscissa (X) and the corresponding peak area as the ordinate (Y). A standard curve was plotted (see Appendix). Figure 7 The linear regression equation for quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside is y=243758x+184672 (R0). 2 =0.9994), the linear regression equation for rosmarinic acid is y =139731x+111278 (R = 0.9994). 2=0.9994), and the results all indicate that quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside has a good linear relationship in the range of 15~240 μg / mL and rosmarinic acid in the range of 20~320 μg / mL.

[0048] 2. Precision test Test solutions were prepared using the same batch of Suting Pills as a reference standard. Six consecutive injections were performed under the reference standard chromatographic conditions, and the peak areas of quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside and rosmarinic acid were recorded. The experimental results are shown in Table 9. The RSD values ​​of quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside and rosmarinic acid were 1.137% and 1.423%, respectively, demonstrating good instrument precision.

[0049] Table 9: Precision test results of the two components

[0050] 3. Repeated examination Six test solutions were prepared in parallel using the same batch of Suting Pill as a reference standard. These solutions were injected sequentially under the reference standard chromatographic conditions, and the peak areas of quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside and rosmarinic acid were recorded. The experimental results are shown in Table 10. The RSD values ​​of quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside and rosmarinic acid were 1.253% and 1.041%, respectively, demonstrating the good reproducibility of this method.

[0051] Table 10: Results of repeatability tests for the two components

[0052] 4. Stability assessment The sample solution of Suting Pill (a traditional Chinese medicine) was continuously injected according to the chromatographic conditions for the reference material. Analysis was performed at 0h, 2h, 4h, 8h, 12h, and 24h, and the peak areas of quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside and rosmarinic acid were recorded. The experimental results are shown in Table 11. The RSD values ​​of quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside and rosmarinic acid were 1.006% and 0.976%, respectively, demonstrating the good repeatability of this method.

[0053] Table 11: Results of stability tests on the two components

[0054] 5. Recovery rate analysis Six portions of the same batch of the classic formula Su Ting Wan, with known content, were accurately weighed and divided into two groups based on the index components. Reference standards for each index component were added at a 1:1 ratio. The samples were continuously injected according to the chromatographic conditions of the reference formula, and the peak areas of quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside and rosmarinic acid were recorded. The results are shown in Table 12. The average recoveries of quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside and rosmarinic acid were 99.54% and 99.88%, respectively, with RSD values ​​of 0.304% and 0.175%, respectively. The good recovery range indicates that the methods for Su Ting Wan have good accuracy.

[0055] Table 12: Results of the Recovery Test

[0056] 6. Measurement transfer examination The results of the value transfer are shown in Table 13. The average transfer rates of quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside and rosmarinic acid from raw materials to the reference material were 32.137% and 31.132%, respectively. The experimental results were stable, with no discrete data, and the experimental data were good.

[0057] Table 13: Results of the Quantitative Transfer Experiment of the Classic Formula Su Ting Wan

[0058] The above experimental results demonstrate that the characteristic map construction method for Suting Pill provided by this invention has the characteristics of good stability, high precision, and good repeatability. It can comprehensively and objectively evaluate the quality of Suting Pill, providing quality assurance for clinical efficacy. The above embodiments are merely exemplary embodiments of this invention and are not intended to limit the invention. The scope of protection of this invention is defined by the claims.

[0059] The above description is merely an example and illustration of the concept of the present invention. Any modifications or additions made by those skilled in the art to the specific embodiments described, or any substitutions made in a similar manner, as long as they do not deviate from the inventive concept or exceed the scope defined by the claims, shall fall within the protection scope of the present invention.

Claims

1. A method for quality testing of Su Ting Wan material reference, characterized in that, Includes the following steps: 1) Prepare the material standard for Su Ting Wan, the test solution of each medicinal material and the corresponding control medicinal material solution respectively; 2) Qualitative analysis of perilla seeds and lepidium seeds in the material standard was performed using thin-layer chromatography; 3) Quantitative analysis of quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside and rosmarinic acid in the material standard was performed using high performance liquid chromatography.

2. The method for quality testing of the material standard of Suting Pill according to claim 1, characterized in that, In step 1), the preparation method of the material basis of Su Ting Wan is as follows: weigh perilla seeds and lepidium seeds in sequence, clean, process, crush, sieve, mix and then mix with jujube paste to form a soft material, roll into strips, cut into pills, and dry to obtain the product.

3. The method for quality testing of the material standard of Suting Pill according to claim 1, characterized in that, In step 1), the medicinal materials are Lepidium seed and Perilla seed; The preparation method of the Lepidium apetalum test solution is as follows: methanol is added to the Lepidium apetalum standard, ultrasonicated, filtered, the filtrate is evaporated to dryness, and the residue is dissolved in methanol to obtain the Lepidium apetalum test solution; quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside is used as a control, and methanol is added to obtain the control solution; The preparation method of the perilla seed test solution is as follows: methanol is added to the Su Ting Wan material standard, ultrasonic treatment is performed, the filtrate is evaporated to dryness after filtration, and the residue is dissolved in methanol to obtain the perilla seed test solution; the stir-fried perilla seed reference material is taken and the reference material solution is prepared in the same way.

4. The method for quality testing of the material standard of Suting Pill according to claim 1, characterized in that, In step 2), the specific steps for thin-layer chromatography identification are as follows: Spot the test solution of Lepidium apetalum and the reference solution of quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside, and examine them under a 365 nm UV lamp using acetone-water as the developing solvent. Spot the test solution of perilla seeds and the reference solution of stir-fried perilla seeds, and examine them under sunlight and 365 nm ultraviolet light using hexane-toluene-ethyl acetate-formic acid as the developing solvent.

5. The method for quality testing of the material standard of Suting Pill according to claim 4, characterized in that, When using thin-layer chromatography for identification, The sample volume for the Lepidium apetalum test solution and the quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside reference solution was 1.5 µL. The sample volume for both the perilla seed test solution and the stir-fried perilla seed reference solution was 2 µL.

6. The method for quality testing of the material standard of Suting Pill according to claim 4, characterized in that, In the acetone-water mixture, the volume ratio of acetone to water is 1:1; in the hexane-toluene-ethyl acetate-formic acid mixture, the volume ratio of hexane, toluene, ethyl acetate, and formic acid is 2:5:2.5:0.

5.

7. The method for quality testing of the material standard of Suting Pill according to claim 1, characterized in that, Step 3) In the quantitative analysis, The test solution was prepared by taking Su Ting Wan as a reference, adding methanol, ultrasonically extracting, filtering, and taking the filtrate to obtain the test solution.

8. The method for quality testing of the material standard of Suting Pill according to claim 1, characterized in that, In step 3) of the quantitative analysis, the preparation method of the reference solution is as follows: Take quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside reference standard and add methanol to prepare a single reference solution containing 30 μg of quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside standard per 1 mL. Prepare a single reference solution containing 80 μg of rosmarinic acid per 1 mL by adding methanol to the rosmarinic acid reference standard.

9. The method for quality testing of the material standard of Suting Pill according to claim 1, characterized in that, In step 3), the high-performance liquid chromatography (HPLC) conditions were as follows: Column type: Shim-pack VP-ODS, 250 mm × 4.6 mm, 5 μm; Mobile phase: acetonitrile-0.1% phosphoric acid aqueous solution; Flow rate: 0.8 mL / min; Column temperature: 30℃; Injection volume: 10 μL; Detection wavelength: 254 nm; Elution gradient is shown in the table below.

10. The method for quality testing of the material standard of Suting Pill according to claim 1, characterized in that, In step 3), the content of quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside in the material standard of Suting Pill is ≥0.035%; the content of rosmarinic acid is ≥0.104%.

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