A method for quality detection of Yinxie No.1 oral granules

Abstract

The application discloses a quality detection method of Yinxie No.1 oral granules, and relates to the field of traditional Chinese medicine analysis. The method adopts a thin layer chromatography method to identify Sophora flavescens, Glycyrrhiza uralensis, Arctium lappa and Saposhnikovia divaricata, and simultaneously adopts a high performance liquid chromatography method to detect the content of paeoniflorin, so that the quality of the Yinxie No.1 oral granules is comprehensively evaluated and controlled, a comprehensive and reliable basis is provided for the authenticity identification and internal quality detection of the granules, and the stability of product quality and the safety and effectiveness of clinical medication are ensured.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine testing, specifically to a method for quality testing of Yin Xie No. 1 oral granules, particularly based on the identification of active ingredients and the determination of the content of indicator components. Background Technology

[0002] Psoriasis No. 1 oral granules are a traditional Chinese medicine preparation derived from a clinical prescription by a medical institution. Composed of multiple herbs including Sophora flavescens, Glycyrrhiza uralensis, Arctium lappa, Saposhnikovia divaricata, and Paeonia lactiflora, it is clinically used to treat psoriasis of the blood-heat type. This preparation has demonstrated definite efficacy through long-term clinical application, and the prescription has become relatively mature. However, as a clinical prescription, it lacks established quality standards and systematic quality control methods. The lack of quality standards for traditional Chinese medicine preparations is a common problem, affecting not only the stability of preparation quality but also posing a threat to clinical medication safety. Establishing scientific and reasonable quality testing methods is of great significance for ensuring the controllable quality and stable efficacy of traditional Chinese medicine preparations.

[0003] Therefore, this paper aims to address the technical problems in existing technologies, such as the lack of a systematic identification scheme for the main medicinal ingredients of psoriasis treatment drugs, including Sophora flavescens, Glycyrrhiza uralensis, Arctium lappa, and Saposhnikovia divaricata, as well as the lack of a specific quantitative determination method for the indicator component paeoniflorin. These problems make it difficult to comprehensively evaluate the quality of compound preparations and provide a comprehensive and reliable basis for the quality control of these preparations. Summary of the Invention

[0004] The purpose of this invention is to provide a method for quality testing of Yin Xie No. 1 oral granules. The method includes thin-layer chromatography to identify Sophora flavescens, Glycyrrhiza uralensis, Arctium lappa, and Saposhnikovia divaricata, while HPLC is used to quantitatively determine the content of paeoniflorin in the granules. The method also examines the extraction method of the test sample solution, the detection wavelength, and the mobile phase conditions, so as to achieve a comprehensive evaluation and control of the quality of Yin Xie No. 1 oral granules. This provides a comprehensive and reliable basis for the identification of the authenticity of the granules and the detection of their intrinsic quality, ensuring the stability of product quality and the safety and effectiveness of clinical use, thereby solving the technical problem of the lack of quality standards for this traditional Chinese medicine granule.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: On one hand, the present invention provides a method for quality testing of Psoriasis No. 1 oral granules, wherein the traditional Chinese medicine granules are made from Rehmannia glutinosa, Lonicera japonica, Scrophularia ningpoensis, Paeonia lactiflora, Gypsum fibrosum, Anemarrhena asphodeloides, Imperata cylindrica, Hedyotis diffusa, Sophora flavescens, Arctium lappa, Schizonepeta tenuifolia, Saposhnikovia divaricata, and Glycyrrhiza uralensis, characterized by comprising the following steps: (1) Thin-layer chromatography was used to identify the following components in the granules: Sophora flavescens, Glycyrrhiza uralensis, Arctium lappa, and Saposhnikovia divaricata. The identification of Sophora flavescens was performed using a toluene-acetone-methanol mixed solvent as the developing solvent, with matrine as a reference standard. The identification of licorice and burdock seed used a chloroform-methanol-water mixture as the developing solvent, and glycyrrhizin and arctiin reference standards were used for comparison. The identification of Saposhnikovia divaricata was performed using a dichloromethane-methanol-water mixed solvent as the developing solvent, with 5-O-methylvisamidol glycoside as a reference standard. (2) The content of paeoniflorin in the particles was determined by high performance liquid chromatography. Acetonitrile-0.1% phosphoric acid mixed solvent was used as the mobile phase and the detection wavelength was 230 nm.

[0006] In some embodiments, the thin-layer chromatographic identification of Sophora flavescens uses a mixed solvent of toluene-acetone-methanol in a volume ratio of 8:3:0.5 as the developing solvent.

[0007] In some embodiments, the thin-layer chromatographic identification of licorice and burdock seeds uses a mixed solvent of chloroform-methanol-water in a volume ratio of 40:9:1 as the developing solvent.

[0008] In some embodiments, the thin-layer chromatographic identification of the wind-proofing uses a mixed solvent of dichloromethane-methanol-water in a volume ratio of 5:1:0.1 as the developing solvent.

[0009] In some embodiments, the determination of paeoniflorin content uses a mixed solvent of acetonitrile and 0.1% phosphate at a volume ratio of 11:89 as the mobile phase.

[0010] In some embodiments, the step of identifying Sophora flavescens by thin-layer chromatography is as follows: 1) Preparation of test sample: Take the Chinese herbal medicine granules, add concentrated ammonia test solution-chloroform mixed solution, sonicate, filter, evaporate the filtrate to dryness, add 1 ml of ethanol to dissolve the residue, and use it as the test sample solution. 2) Preparation of Matrine reference standard: Take matrine reference standard and add ethanol to prepare a solution containing 1 mg per 1 ml, which is used as the reference solution; 3) Identification: Take 10 μL of the test solution and 2 μL of the reference solution and spot them separately on the same silica gel thin-layer plate. Use toluene-acetone-methanol (8:3:0.5) as the developing solvent and develop in a developing tank saturated with ammonia vapor for 20 minutes. Remove the plate, air dry it, and spray it with potassium iodide test solution and sodium nitrite ethanol test solution in sequence.

[0011] Preferably, the weight-to-volume ratio of the particles to ethanol in the preparation of the test sample is 8:0.6-50; Preferably, the ultrasonic treatment lasts for 30 minutes; Preferably, the silicone thin film in the identification process is a silicone G thin film or a silicone HSG thin film; Preferably, the colorimetric solution is potassium iodide test solution and sodium nitrite ethanol test solution; Preferably, the unfolding temperature is 5-35℃, more preferably 10℃~30℃; Preferably, the relative humidity of the unfolded area is 15-30% or 75-90%, more preferably 18%-88%; In some embodiments, the steps for identifying Sophora flavescens using thin-layer chromatography are as follows: 1) Take 8g of Chinese herbal medicine granules, add 0.6ml of concentrated ammonia test solution and 50ml of chloroform, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 1ml of ethanol to dissolve the residue, and use it as the test solution. 2) Take matrine reference standard and add ethanol to prepare a solution containing 1 mg per 1 ml, which is used as the reference solution.

[0012] 3) Take 10 μL of the test solution and 2 μL of the reference solution and spot them separately on the same silica gel G thin-layer plate. Use toluene-acetone-methanol (8:3:0.5) as the developing solvent and develop in a developing tank saturated with ammonia vapor for 20 minutes. Remove the plate, air dry it, and spray it with potassium iodide test solution and sodium nitrite ethanol test solution in sequence.

[0013] In some embodiments, the steps for identifying licorice and burdock seed by thin-layer chromatography are as follows: 1) Preparation of test sample: Take Chinese medicine granules, add ethanol, sonicate, filter, evaporate the filtrate to dryness, dissolve the residue in water, extract with ethyl acetate by shaking, reserve the aqueous solution, combine the ethyl acetate solutions, evaporate to dryness, dissolve the residue in 1 ml of ethanol, and use as the test sample solution. 2) Preparation of glycyrrhizin and arctiin reference standards: Take glycyrrhizin and arctiin reference standards, and add ethanol to prepare solutions containing 0.5 mg per ml and 5 mg per ml, respectively, as reference solutions; 3) Identification: Take 5-10 μL of the test solution, 1 μL of the glycyrrhizin reference solution, and 5 μL of the arctiin reference solution, and spot them separately on the same silica gel G thin layer plate. Use chloroform-methanol-water (40:9:1) as the developing solvent, develop, remove, air dry, and spray with a colorimetric solution for color development. Preferably, the weight-to-volume ratio of particles to ethanol in the preparation of the test sample is 8:50. Preferably, the ultrasonic treatment lasts for 30 minutes; Preferably, the silicone thin-layer plate used in the identification process is a silicone G thin-layer plate; Preferably, the colorimetric solution is 10% sulfuric acid in ethanol; Preferably, the unfolding temperature is 5-35℃, more preferably 10℃~30℃; the unfolding relative humidity is 15-30% or 75-90%, more preferably 18%~88%. More preferably, the steps for identifying licorice and burdock seed by thin-layer chromatography are as follows: 1) Take 8g of Chinese herbal medicine granules, add 50ml of ethanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 20ml of water, extract twice with ethyl acetate, 20ml each time, keep the aqueous solution for later use, combine the ethyl acetate solutions, evaporate to dryness, dissolve the residue in 1ml of ethanol, and use it as the test solution. 2) Take licorice and arctiin reference standards, and add ethanol to prepare solutions containing 0.5 mg per ml and 5 mg per ml, respectively, as reference solutions; 3) Take 5-10 μL of the above test solution, 1 μL of glycyrrhizin reference solution and 5 μL of arctiin reference solution, and spot them separately on the same silica gel G thin layer plate. Use chloroform-methanol-water (40:9:1) as the developing solvent, develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, and heat at 105℃ until the glycyrrhizin spots are clearly visible. After observation, continue heating at 105℃ until the arctiin spots are clearly visible.

[0014] In some embodiments, the step of the thin-layer chromatography identification of *Saposhnikovia divaricata* is as follows: 1) Preparation of test sample: Take the aqueous solution prepared under step 1) of claim 4, extract it by shaking with water-saturated n-butanol, combine the n-butanol solutions, evaporate to dryness, dissolve the residue in 1 ml of ethanol, and use it as the test sample solution. 2) Preparation of Saposhnikovia divaricata reference standard: Take 5-O-methylvisamidol glycoside reference standard, add methanol to prepare a solution containing 0.4 mg per 1 ml, and use it as the reference solution; 3) Identification: Take 5 μl of the test solution and 1 μl of the reference solution and spot them separately on the same silica gel G thin layer plate. Use dichloromethane-methanol-water (5:1:0.1) as the developing solvent, develop, remove, air dry, spray with color developing solution, and examine under ultraviolet light. Preferably, the test sample is prepared by shaking with water-saturated n-butanol twice, 20 ml each time; Preferably, the silicone thin film in the identification process is a silicone G thin film or a silicone HSG thin film; Preferably, the colorimetric solution is 10% sulfuric acid ethanol; the development temperature is 10℃~30℃; Preferably, the relative humidity of the unfolded area is 15-30% or 75-90%, more preferably 18%-88%; More preferably, the step of the thin-layer chromatography identification of *Saposhnikovia divaricata* is as follows: 1) Take the prepared aqueous solution from step 1) of claim 4, extract it twice with water-saturated n-butanol, 20 ml each time, combine the n-butanol solutions, evaporate to dryness, dissolve the residue in 1 ml of ethanol, and use it as the test solution. 2) Take 5-O-methylvisamidol glycoside reference standard, add methanol to prepare a solution containing 0.4 mg per 1 ml, and use it as the reference solution; 3) Take 5 μl of the test solution and 2 μl of the reference solution and spot them separately on the same silica gel G thin layer plate. Use dichloromethane-methanol-water (5:1:0.1) as the developing solvent, develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, and examine under a UV lamp at 365 nm.

[0015] In some embodiments, the step of determining the paeoniflorin content in the particles by high performance liquid chromatography is as follows: 1) Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the stationary phase, and acetonitrile-0.1% phosphoric acid solution (11:89) was used as the mobile phase; the detection wavelength was 230 nm; the theoretical plate number calculated based on the paeoniflorin peak should not be less than 2000; 2) Preparation of reference solution: Take an appropriate amount of paeoniflorin reference standard, accurately weigh it, and add 70% methanol to prepare a solution containing 60 μg per ml. 3) Preparation of test solution: Take the granules, grind them finely, weigh them accurately, place them in a stoppered conical flask, add methanol accurately, stopper tightly, weigh them, sonicate them, cool them, weigh them again, make up the weight loss with methanol, shake well, filter them, and take the filtrate to obtain the test solution. 4) Determination method: Accurately pipette 10 μL each of the reference solution and the test solution into the liquid chromatograph and determine the result. Preferably, the flow rate of the mobile phase is 1 ml / min; Preferably, the weight-to-volume ratio of particles to methanol in the preparation of the test solution is 1:25; Preferably, the ultrasonic conditions are 300W-500W power, 45kHz frequency, 20-40min, preferably 300W, 45kHz frequency, 20min; More preferably, the step of determining the paeoniflorin content in the particles by high performance liquid chromatography is as follows: 1) Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel as the stationary phase; mobile phase: acetonitrile-0.1% phosphoric acid solution (11:89); flow rate: 1 ml / min; detection wavelength: 230 nm; theoretical plate number calculated based on paeoniflorin peak should not be less than 2000. 2) Preparation of reference solution: Take an appropriate amount of paeoniflorin reference standard, accurately weigh it, and add 70% methanol to prepare a solution containing 60 μg per ml. 3) Preparation of the test solution: Take Chinese herbal medicine granules, grind them into a fine powder, take about 1g, weigh it accurately, place it in a stoppered conical flask, add 25ml of methanol accurately, stopper tightly, weigh it, sonicate it at 300W power and 45kHz frequency for 20min, cool it, weigh it again, make up the lost weight with methanol, shake it well, filter it, and take the filtrate to obtain the test solution. 4) Determination method: Accurately pipette 10 μL each of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0016] In some embodiments, the linear equation of the method is Y = 1117.3X - 37.856, the correlation coefficient r is 0.9999, the linear range is 0.1821 to 2.2758 µg, the intermediate precision RSD is 2.1%, the sample solution is stable within 20 hours, the repeatability RSD is 1.4%, and the average content is 2.00 mg / g.

[0017] Secondly, the present invention provides a method for detecting the quality of Yin Xie No. 1 oral granules constructed as described above.

[0018] Thirdly, the present invention provides a method for determining the content of indicator components in Yin Xie No. 1 oral granules, wherein the method includes determining the content of paeoniflorin in the granules using the aforementioned high performance liquid chromatography method.

[0019] Preferably, the linear equation of the method is Y=1117.3X-37.856, the correlation coefficient r is 0.9999, the linear range is 0.1821~2.2758µg, the intermediate precision RSD is 2.1%, the sample solution is stable within 20 hours, the repeatability RSD is 1.4%, and the average content is 2.00mg / g.

[0020] Fourthly, the present invention also provides a method for quality testing of the oral granules of Psoriasis No. 1 as described above; Preferably, the quality testing includes the identification of indicator components and the determination of the content of indicator components.

[0021] The beneficial effects of this invention are: 1. This invention establishes a highly specific thin-layer chromatography identification method for Sophora flavescens by using a specific toluene-acetone-methanol volume ratio as the developing solvent system and using matrine as a reference standard. Negative samples lacking Sophora flavescens show no interfering spots at the corresponding positions of the matrine reference standard. Furthermore, this method can be stably reproduced under different humidity levels (18% to 88%), temperatures (10℃ to 30℃), and thin-layer plates from different manufacturers. The test sample spots are clearly separated, and the method has good durability. 2. This invention establishes a specific thin-layer chromatography identification method for licorice and burdock seed by using silica gel G plate as the stationary phase and a ternary developing solvent system with a specific volume ratio of chloroform-methanol-water. The test sample spots are clear and the separation effect is good. There is no interference from negative samples lacking licorice and burdock seed. This method enables the simultaneous systematic qualitative identification of licorice and burdock seed, two main medicinal materials in traditional Chinese medicine granules, and improves the detection efficiency. 3. This invention uses the prepared aqueous solution after identifying licorice and burdock seeds for the identification of Saposhnikovia divaricata. It adopts a ternary developing solvent system with 5-O-methylvisamidol glycoside reference standard and a specific dichloromethane-methanol-water volume ratio, and examines it under ultraviolet light at a wavelength of 365nm. This achieves specific identification of Saposhnikovia divaricata, and there is no interference from negative samples lacking Saposhnikovia divaricata. At the same time, it improves detection efficiency and saves sample volume. 2. This invention also establishes a high-performance liquid chromatography (HPLC) method for the quantitative determination of paeoniflorin as an indicator component by using a mobile phase with an acetonitrile-0.1% phosphoric acid solution volume ratio of 11:89 and a detection wavelength of 230 nm. The method has good linearity, is accurate, reliable and specific, and achieves accurate quantitative determination of paeoniflorin in traditional Chinese medicine granules. 3. The quality testing method established in this invention combines qualitative identification by thin-layer chromatography and quantitative determination by high-performance liquid chromatography, realizing comprehensive evaluation and control of the quality of traditional Chinese medicine granules. It provides a comprehensive and reliable basis for the identification of authenticity and the detection of intrinsic quality of the preparation, ensuring the stability of product quality and the safety and effectiveness of clinical use. Attached Figure Description

[0022] Figure 1 Thin-layer chromatography identification chromatograms of Sophora flavescens; from left to right, the chromatograms are for samples 1-7. Sample 1 - negative sample lacking Sophora flavescens, sample volume 10 μL; Samples 2, 3, 4 - Chinese medicine granules from Preparation Example 1, sample volumes 2, 5, 10 μL; Samples 5, 6 - matrine reference standard, sample volumes 1, 2 μL.

[0023] Figure 2 The identification chromatograms were developed to 8 cm using toluene-acetone-methanol (8:3:0.5) as the developing solvent, and then developed in the lower layer solution of toluene-ethyl acetate-methanol-water (2:4:2:1) at below 10 °C. The chromatograms of samples 1-4 are shown from left to right: Sample 1 - negative sample lacking Sophora flavescens, sample volume 10 μl; Sample 2 - Chinese herbal granules from Preparation Example 1, sample volume 10 μl; Sample 3 - matrine reference standard, sample volume 2 μl; Sample 4 - sophoridine reference standard, sample volume 2 μl. Figure 3 Identification chromatograms of the developing solvent cyclohexane-chloroform-methanol (8:4:1); from left to right, the chromatograms are for samples 1-4. Figure 2 ; Figure 4 Silica gel G plate prepared with 2% sodium hydroxide solution; from left to right, the spectra of samples 1-4 are shown. Figure 2 ; Figure 5Silica gel G plate (Qingdao Marine); from left to right, the spectra of samples 1-3 are as follows: Sample 1 - negative sample lacking Sophora flavescens, sample volume 10 μl; Sample 2 - Chinese medicine granules from Preparation Example 1, sample volume 10 μl; Sample 3 - matrine reference standard, sample volume 2 μl. Figure 6 Identification spectra of silica gel HSG plates (Yantai Huanghai brand); from left to right, the spectra are for samples 1-3. Figure 5 ; Figure 7 Identification spectra developed at 18% relative humidity; from left to right, these are spectra for samples 1-3. Figure 5 ; Figure 8 Identification chromatograms developed at 88% relative humidity; from left to right, these are the chromatograms for samples 1-3. Figure 5 ; Figure 9 Identification spectra developed at 10℃; from left to right, these are spectra for samples 1-3. Figure 5 ; Figure 10 Identification spectra developed at 30℃; from left to right, these are spectra for samples 1-3. Figure 5 ; Figure 11 Thin-layer chromatography (TLC) spectra of licorice and burdock seed; from a to c, these are the color spectra of licorice at 365 nm, licorice under sunlight, and burdock seed under sunlight. Each spectrum from a to c represents samples 1-7 from left to right. Sample 1 is a negative sample lacking burdock seed, with a sample volume of 10 μl; Sample 2 is a 5 μl sample of arctiin reference standard; Samples 3, 4, and 5 are the traditional Chinese medicine granules from Preparation Example 1, with sample volumes of 2, 5, and 10 μl respectively; Sample 6 is a negative sample lacking licorice, with a sample volume of 10 μl; and Sample 7 is a 1 μl sample of glycyrrhizin reference standard. Figure 12 Identification spectra of the developing solvent chloroform-methanol-water (12∶4∶1); from a to c, these are the color spectra of licorice at 365 nm, licorice under sunlight, and burdock seed under sunlight. Each spectrum from left to right in a~c represents samples 1-6. Sample 1 - a negative sample lacking burdock seed, sample volume 5 μl; Sample 2 - arctiin reference standard, sample volume 5 μl; Samples 3 and 4 - traditional Chinese medicine granules from Preparation Example 1, sample volumes 2 and 5 μl respectively; Sample 5 - a negative sample lacking licorice, sample volume 5 μl; Sample 6 - glycyrrhizin reference standard, sample volume 1 μl. Figure 13 The identification spectra of the developing solvent dichloromethane-methanol-water (40∶9∶1); from a to c, these are the color spectra of licorice at 365nm, licorice under sunlight, and burdock seed under sunlight, respectively. Each spectrum in a~c, from left to right, represents samples 1-6. Figure 12 ; Figure 14 Identification spectra of silica gel HSG plates (Huanghai brand); from a to c, these are the color spectra of licorice at 365nm, licorice under sunlight, and burdock seed under sunlight. Each spectrum from a to c represents samples 1-7 from left to right. Figure 11 ; Figure 15 Identification spectra of silica gel HSG plates (Yinlong brand); from a to c, these are the color spectra of licorice at 365nm, licorice under sunlight, and burdock seed under sunlight. Spectra a-c, from left to right, represent the spectra of samples 1-7. Figure 11 ; Figure 16 Identification spectra developed at 18% relative humidity; from a to c, these are the color spectra of licorice at 365nm, licorice under sunlight, and burdock seed under sunlight. Spectra a-c, from left to right, represent samples 1-5 respectively. Sample 1 - a negative sample lacking burdock seed, sample volume 10 μl; Sample 2 - arctiin reference standard, sample volume 5 μl; Sample 3 - the traditional Chinese medicine granules from Preparation Example 1, sample volume 10 μl; Sample 4 - a negative sample lacking licorice, sample volume 10 μl; Sample 5 - glycyrrhizin reference standard, sample volume 1 μl. Figure 17 Identification spectra developed at 88% relative humidity; from a to c, these are the color spectra of licorice at 365nm, licorice under sunlight, and burdock seed under sunlight. Each spectrum from a to c represents samples 1-5 from left to right. Figure 16 ; Figure 18 Identification spectra developed at 10℃; from a to c, these are the color spectra of licorice at 365nm, licorice under sunlight, and burdock seed under sunlight. Each spectrum from a to c represents samples 1-5 from left to right. Figure 16 ; Figure 19 Identification spectra developed at 30℃; from a to c, these are the color spectra of licorice at 365nm, licorice under sunlight, and burdock seed under sunlight. Each spectrum from a to c represents samples 1-5 from left to right. Figure 16 ; Figure 20 Thin-layer identification spectrum of Saposhnikovia divaricata; from left to right are the spectra of samples 1-6, sample 1 - negative sample lacking Saposhnikovia divaricata, sample volume 10 μl, samples 2, 3, 4 - Yinxie No. 1 oral granules, sample volumes 2, 5, 10 μl, samples 5, 6 - 5-O-methylvisamidol glycoside reference standard, sample volumes 1, 2 μl, respectively; Figure 21The identification chromatograms of the developing solvent chloroform-methanol (4:1) are, from left to right, the chromatograms of samples 1-3. Sample 1 - negative sample lacking Saposhnikovia divaricata, sample volume 5 μl; Sample 2 - Chinese medicine granules of Preparation Example 1, sample volume 5 μl; Sample 3 - 5-O-methylvisamidol glycoside reference standard, sample volume 2 μl. Figure 22 The identification chromatograms of ethyl acetate-methanol (4:1) as the developing solvent are, from left to right, those of samples 1-3. Figure 21 ; Figure 23 Identification chromatograms of silica gel HSG plates (Yinlong brand); from left to right, the chromatograms are those of samples 1-3. Sample 1 - negative sample lacking Saposhnikovia divaricata, sample volume 5 μl; Sample 2 - Chinese medicine granules from Preparation Example 1, sample volume 5 μl; Sample 3 - 5-O-methylvisamidol glycoside reference standard, sample volume 1 μl. Figure 24 Identification spectra of silica gel HSG plates (Huanghai brand); from left to right, the spectra are for samples 1-3. Figure 23 ; Figure 25 Identification spectra developed at 18% relative humidity; from left to right, these are spectra for samples 1-3. Figure 23 ; Figure 26 Identification chromatograms developed at 88% relative humidity; from left to right, these are the chromatograms for samples 1-3. Figure 23 ; Figure 27 Identification spectra developed at 10℃; from left to right, these are spectra for samples 1-3. Figure 23 ; Figure 28 Identification spectra developed at 30℃; from left to right, these are spectra for samples 1-3. Figure 23 ; Figure 29 High-performance liquid chromatography chromatogram of blank solvent (methanol); Figure 30 High-performance liquid chromatogram of paeoniflorin reference solution; Figure 31 High-performance liquid chromatogram of negative control solution lacking white peony root; Figure 32 High-performance liquid chromatogram of the test solution; Figure 33 Linear relationship graph of paeoniflorin. Detailed Implementation

[0024] The present invention will be further described by way of examples, but the present invention is not limited to the following examples.

[0025] Preparation Example 1: Detection of Psoriasis No. 1 Oral Granules Psoriasis No. 1 Oral Granules are made from 13 kinds of Chinese medicinal herbs, including Rehmannia glutinosa, Lonicera japonica, Scrophularia ningpoensis, Paeonia lactiflora, Gypsum, Anemarrhena asphodeloides, Imperata cylindrica, Hedyotis diffusa, Sophora flavescens, Arctium lappa, Schizonepeta tenuifolia, Saposhnikovia divaricata, and Glycyrrhiza uralensis.

[0026] The analytical methods for the main items in the standard of Psoriasis No. 1 oral granule control agent are as follows: 1. Identification (1) Take 8g of this product, add 0.6ml of concentrated ammonia test solution and 50ml of chloroform, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1ml of ethanol to obtain the test solution. Separately, take matrine reference standard, add ethanol to prepare a solution containing 1mg per ml to obtain the reference solution. Perform thin-layer chromatography (General Chapter 0502 of Chinese Pharmacopoeia 2025) and apply 10μl of the above test solution and 2μl of the reference solution separately to the same silica gel G thin-layer plate. Use toluene-acetone-methanol (8:3:0.5) as the developing solvent, develop in a developing tank saturated with ammonia vapor for 20 minutes, remove, air dry, and spray successively with potassium iodide test solution and sodium nitrite ethanol test solution. In the chromatogram of the test sample, orange spots appear at the corresponding positions as in the chromatogram of the reference standard.

[0027] (2) Take 8g of this product, add 50ml of ethanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 20ml of water, extract twice with ethyl acetate, 20ml each time, keep the aqueous solution for later use, combine the ethyl acetate solutions, evaporate to dryness, dissolve the residue in 1ml of ethanol, and use as the test solution. Separately take glycyrrhizin reference standard and arctiin reference standard, and add ethanol to prepare solutions containing 0.5mg per ml and 5mg per ml, respectively, as reference solutions. Perform thin-layer chromatography (General Chapter 0502, Chinese Pharmacopoeia 2025 Edition) using the following method: Apply 5-10 μl of the above-mentioned test solution, 1 μl of glycyrrhizin reference solution, and 5 μl of arctiin reference solution separately to the same silica gel G thin-layer plate. Develop the plate using chloroform-methanol-water (40:9:1) as the developing solvent. Remove the plate, air dry, spray with 10% sulfuric acid ethanol solution, and heat at 105℃ until the glycyrrhizin spots are clearly visible. Examine the plate under sunlight and ultraviolet light (365 nm). In the chromatogram of the test sample, spots of the same color as those in the chromatogram of the glycyrrhizin reference solution appear at the corresponding positions under sunlight and fluorescent spots of the same color under ultraviolet light. Continue heating at 105℃ until the arctiin spots are clearly visible. In the chromatogram of the test sample, spots of the same color as those in the chromatogram of the arctiin reference solution appear at the corresponding positions.

[0028] (3) Take the aqueous solution prepared under [Identification] (2), extract twice with water-saturated n-butanol, 20 ml each time, combine the n-butanol solutions, evaporate to dryness, dissolve the residue in 1 ml of ethanol, and use it as the test solution. Take another 5-O-methylvisamidol glycoside reference standard, add methanol to prepare a solution containing 0.4 mg per ml, and use it as the reference solution. Perform the thin-layer chromatography test (General Chapter 0502 of Chinese Pharmacopoeia 2025 Edition), take 5 μl of the test solution and 1 μl of the reference solution, and spot them separately on the same silica gel G thin-layer plate. Use dichloromethane-methanol-water (5:1:0.1) as the developing solvent, develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, and examine under ultraviolet light (365 nm). In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference standard.

[0029] 2. Content determination The determination was performed according to high performance liquid chromatography (General Chapter 0512 of the Chinese Pharmacopoeia 2025).

[0030] Chromatographic conditions and system suitability tests were conducted using octadecylsilane-bonded silica gel as the stationary phase; acetonitrile-0.1% phosphoric acid solution (11:89) as the mobile phase; and a detection wavelength of 230 nm. The theoretical plate number, calculated based on the paeoniflorin peak, should be no less than 2000.

[0031] Preparation of the reference solution: Take an appropriate amount of paeoniflorin reference standard, accurately weigh it, and add 70% methanol to prepare a solution containing 60 μg per ml.

[0032] Preparation of the test solution: Take the Chinese herbal granules from Preparation Example 1, grind them into a fine powder, take about 1g, weigh it accurately, place it in a stoppered conical flask, accurately add 25ml of methanol, stopper tightly, weigh it, sonicate (power 300W, frequency 45kHz) for 20 minutes, cool it, weigh it again, make up the lost weight with methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0033] The assay involves precisely pipetting 10 μl each of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0034] Each sachet of this product contains paeoniflorin (C) from white peony root. 23 H 28 O 11 The total dose shall not be less than 8.0 mg.

[0035] Validation of analytical methods Example 1: Thin-layer chromatography identification study of Sophora flavescens 1. Thin-layer chromatography identification study of Sophora flavescens 1.1 Method 1.1.1 Preparation of the test solution Take 8g of the Chinese herbal granules from Preparation Example 1, add 0.6ml of concentrated ammonia test solution and 50ml of chloroform, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 1ml of ethanol to dissolve the residue, and use it as the test solution.

[0036] 1.1.2 Preparation of reference solution Take matrine reference standard and sophoridine reference standard (purchased from the National Institutes for Food and Drug Control), and add ethanol to prepare solutions containing 1 mg per ml, as reference solutions.

[0037] 1.1.3 Preparation of negative test solution for Sophora flavescens lacking Sophora flavescens Take 8g of the Chinese herbal granules from Preparation Example 1 (lacking Sophora flavescens) and prepare a negative test solution for Sophora flavescens deficiency according to the method in section "1.1.1".

[0038] 1.1.4 Chromatographic conditions Take 10 μl of the negative test solution for Sophora flavescens (sample 1), 2, 5, and 10 μl of test solutions (samples 2, 3, and 4, respectively), and 2 and 5 μl of reference solutions (samples 5 and 6, respectively), and spot them separately on the same silica gel G thin-layer plate (Qingdao Ocean Chemical Co., Ltd.). Use toluene-acetone-methanol (8:3:0.5) as the developing solvent, develop in a developing tank saturated with ammonia vapor for 20 minutes, remove, air dry, spray with 10% sulfuric acid ethanol solution, and heat at 105℃ until the spots are clearly visible.

[0039] Figure 1 The results show that the test sample chromatogram has the same orange spots at the corresponding positions as the reference sample chromatogram.

[0040] 1.1.5 Specificity Test Take the negative test solution of Sophora flavescens prepared under section "1.1.3". After development, no corresponding spot appears in the chromatogram of the negative test sample at the position corresponding to the reference standard, indicating that the negative sample of Sophora flavescens does not interfere with the experiment (see...). Figure 1 ).

[0041] 1.1.6 Selection of Expanding Systems Three developing solvents were compared: toluene-acetone-methanol (8:3:0.5), first developed to 8 cm in toluene-acetone-methanol (8:3:0.5) and then developed in the lower layer of toluene-ethyl acetate-methanol-water (2:4:2:1) at below 10 °C, and cyclohexane-chloroform-methanol (8:4:1).

[0042] The results showed that the main spot Rf value of the toluene-acetone-methanol (8∶3∶0.5) mixture after development was moderate, the spot was clear, and the separation effect was good (see...). Figure 1 , Figure 2 , Figure 3 Therefore, this developing solvent was ultimately chosen.

[0043] 1.1.7 Selection of Controls Experiments showed that the chromatogram of the test sample showed corresponding spots at the positions corresponding to the chromatograms of matrine and sophoridine reference standards, but the spot corresponding to the sophoridine reference standard was weaker (see...). Figure 2 , Figure 3 , Figure 4 Therefore, sophoridine reference standard was removed, and matrine reference standard was selected as the reference.

[0044] 1.1.8 Investigation of the development effect of thin-layer plates prepared by different manufacturers and different methods According to the chromatographic conditions under section "1.1.4", three different thin-layer plates were used for the experiment: silica gel G plate (Qingdao Ocean Chemical Plant Branch), silica gel G plate (Qingdao Ocean Chemical Co., Ltd.), and silica gel HSG plate (Yantai Chemical Industry Research Institute - Yinlong brand), prepared with 2% sodium hydroxide solution, to investigate the development effect.

[0045] The results showed that the test sample spots achieved good separation when developed using thin-layer plates from three different manufacturers and prepared by different methods. However, the Rf value of the spots on the silica gel G plate prepared with sodium hydroxide solution was larger, and the silica gel G plate is more widely used. Therefore, silica gel G plate or silica gel HSG plate were chosen, as both of these different brands of silica gel showed better development results (see...). Figure 4 , Figure 5 , Figure 6 ).

[0046] 1.1.9 Evaluation of the development effect under different humidity conditions According to the chromatographic conditions under section “1.1.4”, examine one relative humidity at low relative humidity (15-30%) and high relative humidity (75-90%) to assess the development effect.

[0047] The results showed that good separation of the test sample spots was achieved under both 18% and 88% humidity conditions (see [reference needed]). Figure 7 , Figure 8 ).

[0048] 1.1.10 Evaluation of the development effect under different temperature conditions According to the chromatographic conditions under section "1.1.4", the development effect was investigated at two temperatures, 5℃ and 35℃.

[0049] The results showed that good separation of the sample spots was achieved under both 10℃ and 30℃ conditions (see [reference needed]). Figure 9 , Figure 10 ).

[0050] 1.2 Results It has been verified that this thin-layer chromatography identification method can be used for quality control of Yin Xie No. 1 oral granules, and therefore it is included in the main text of the internal control agent standard.

[0051] Example 2: Thin-layer chromatography identification study of licorice and burdock seeds 2. Thin-layer chromatography identification study of licorice and burdock seeds 2.1 Method 2.1.1 Preparation of the test solution Take 8g of the Chinese herbal medicine granules from Preparation Example 1, add 50ml of ethanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 20ml of water, extract twice with ethyl acetate, 20ml each time, keep the aqueous solution for later use, combine the ethyl acetate solutions, evaporate to dryness, dissolve the residue in 1ml of ethanol, and use it as the test solution.

[0052] 2.1.2 Preparation of reference solution Glycyrrhizin and arctiin reference standards (purchased from the National Institutes for Food and Drug Control) were prepared into solutions containing 0.5 mg / ml and 5 mg / ml, respectively, by adding ethanol. These solutions were used as reference solutions.

[0053] 2.1.3 Preparation of negative test solutions for licorice and burdock seed deficiencies Take 8g of Yin Xie No. 1 oral granules powder lacking licorice and burdock seeds, and prepare licorice-deficient negative test solution and burdock seed-deficient negative test solution respectively according to the method in section "2.1.1".

[0054] 2.1.4 Chromatographic conditions Take 5-10 μl of the test solution, 1 μl of glycyrrhizin reference solution and 5 μl of arctiin reference solution, and spot them separately on the same silica gel G thin-layer plate. Develop the plate using chloroform-methanol-water (40:9:1) as the developing solvent. Remove the plate, air dry it, spray it with 10% sulfuric acid ethanol solution, and heat it at 105℃ until the glycyrrhizin spots are clearly visible. Examine the plate under sunlight and ultraviolet light (365nm).

[0055] In the chromatogram of the test sample, spots of the same color as those in the chromatogram of glycyrrhizin reference standard appear under sunlight and under ultraviolet light. Continue heating at 105℃ until the arctiin spots are clearly visible, and spots of the same color appear in the chromatogram of the test sample at the same positions as those in the chromatogram of arctiin reference standard.

[0056] 2.1.5 Specificity Test The licorice-deficient negative test solution and the burdock-deficient negative test solution prepared under section "2.1.3" were analyzed. After development, no corresponding spots were found in the negative test solutions at the positions corresponding to the glycyrrhizin and arctiin reference standards, indicating that the licorice-deficient negative test solution and the burdock-deficient negative sample did not interfere with the experiment (see...). Figure 11 ).

[0057] 2.1.6 Selection of sample size Experiments showed that when the sample volume of the test solution was 5-10 μl, the sample volume of the glycyrrhizin reference solution was 1 μl, and the sample volume of the arctiin reference solution was 5 μl, the spots were clearer (see...). Figure 11 Therefore, this sampling amount was ultimately selected.

[0058] 2.1.7 Selection of Expanding System In the early stages of exploration, it was found that the separation of licorice was better developed in chloroform-methanol-water (40:10:1), and the separation of burdock seed was better developed in chloroform-methanol-water (40:8:1). Since both used the same test solution and the same colorimetric method, the two identification methods were combined for investigation, which saved time and cost.

[0059] The lower layer solutions of chloroform-methanol-water (40:9:1), chloroform-methanol-water (12:4:1), and dichloroform-methanol-water (40:9:1) were compared.

[0060] The results showed that, compared with the corresponding spots of glycyrrhizin and arctiin reference standards, the main spot after development with chloroform-methanol-water (40:9:1) was clearer and the separation effect was better (see...). Figure 11 , Figure 12 , Figure 13 Therefore, this developing solvent was ultimately chosen.

[0061] 2.1.8 Selection of color development conditions Experiments showed that after the sample was developed, dried, and sprayed with 10% sulfuric acid ethanol solution, and heated at 105°C, the glycyrrhizin reference standard and its corresponding spots showed clear color development in a short time. When examined under sunlight and ultraviolet light (365nm), the test sample chromatogram showed clear spots and fluorescent spots at the corresponding positions as the glycyrrhizin reference standard chromatogram. However, the arctiin reference standard and its corresponding spots only showed clear color development after being heated at 105°C for a slightly longer time. At this time, the glycyrrhizin and its corresponding spots were no longer obvious. Therefore, it was chosen to spray with 10% sulfuric acid ethanol solution and examine the samples after heating at 105°C for different times.

[0062] 2.1.9 Examination of the unfolding effect of thin-layer plates from different manufacturers According to the chromatographic conditions under section “2.1.4”, different thin-layer plates, such as silica gel G plate (Qingdao Ocean Chemical Co., Ltd.), silica gel HSG plate (Yantai Chemical Industry Research Institute - Yinlong brand), and silica gel HSG plate (Yantai Jiangyou Silica Gel Development Co., Ltd. - Huanghai brand), were used to conduct experiments to examine the development effect.

[0063] The results showed that using thin-layer plates from three different manufacturers, the test sample spots could all achieve good separation (see...). Figure 11 , Figure 14 , Figure 15 ).

[0064] 2.1.10 Evaluation of the development effect under different humidity conditions According to the chromatographic conditions under section “2.1.4”, one relative humidity was examined at both low relative humidity (15-30%) and high relative humidity (75-90%) to assess the development effect.

[0065] The results showed that good separation of the test sample spots was achieved under both 18% and 88% humidity conditions (see [reference needed]). Figure 16 , Figure 17 ).

[0066] 2.1.11 Evaluation of the development effect under different temperature conditions According to the chromatographic conditions under section “2.1.4”, the development effect was investigated at two temperatures, 5℃ and 35℃ respectively.

[0067] The results showed that good separation of the sample spots was achieved under both 10℃ and 30℃ conditions (see [reference needed]). Figure 18 , Figure 19 ).

[0068] 2.2 Results It has been verified that this thin-layer chromatography identification method can be used for quality control of Yin Xie No. 1 oral granules, and therefore it is included in the main text of the internal control agent standard.

[0069] Example 3: Thin-layer identification study of windproofing 3.1 Method 3.1.1 Preparation of the test solution Take the aqueous solution prepared in Example 2, extract it twice with water-saturated n-butanol, 20 ml each time, combine the n-butanol solutions, evaporate to dryness, dissolve the residue in 1 ml of ethanol, and use it as the test solution.

[0070] 3.1.2 Preparation of reference standards and reference herbal solutions Take 5-O-methylvisamidol glycoside reference standard (purchased from the National Institutes for Food and Drug Control), add methanol to prepare a solution containing 0.4 mg per 1 ml, and use this solution as the reference standard.

[0071] 3.1.3 Preparation of negative test solution for Saposhnikovia divaricata Take 8g of the Chinese herbal granules from Example 1 (for the preparation of Saposhnikovia divaricata), and prepare a negative test solution for Saposhnikovia divaricata according to the method in section "3.1.1".

[0072] 3.1.4 Chromatographic conditions Take 5 μl of the test solution and 1 μl of the reference solution and spot them separately on the same silica gel G thin-layer plate. Develop the plate using dichloromethane-methanol-water (5:1:0.1) as the developing solvent. Remove the plate, air dry it, spray it with 10% sulfuric acid ethanol solution, and examine it under ultraviolet light (365 nm).

[0073] In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample.

[0074] 3.1.5 Selection of Extraction Method for Test Sample Solution Experiments showed that the test solution prepared according to the method in section "3.1.1" produced clear spots corresponding to the reference standard, without interference from impurities, and saved on sample and solvent. Therefore, this method 1 was selected for the preparation of the test solution.

[0075] 3.1.6 Specificity Test Take the negative test solution of Saposhnikovia divaricata (prepared under section 3.1.3). After development, no corresponding spots appear at the positions on the chromatogram corresponding to the reference standard, indicating that the negative sample lacking Saposhnikovia divaricata does not interfere with the experiment (see...). Figure 20 ).

[0076] 3.1.7 Selection of sample amount Experiments showed that when the sample volume of the test solution was 5 μl and the sample volume of the reference solution was 1 μl, the spots were clearer and the development effect was better (see...). Figure 20 Therefore, this sampling amount was ultimately selected.

[0077] 3.1.8 Selection of System Three developing solvents were compared: dichloromethane-methanol-water (5:1:0.1), chloroform-methanol (4:1), and ethyl acetate-methanol (4:1).

[0078] The results showed that the separation of chloroform-methanol (4:1) was poor (see...). Figure 21 ), Ethyl acetate-methanol (4:1) spots are more scattered (see Figure 22 The main spot after development of dichloromethane-methanol-water (5∶1∶0.1) was clear, indicating good separation (see...). Figure 20 Therefore, this developing solvent was ultimately chosen.

[0079] 3.1.9 Selection of color development conditions The experiment showed that after the sample was developed, dried, and sprayed with 10% sulfuric acid ethanol solution, it was examined under a UV lamp (365nm). The test sample chromatogram showed clear spots at the corresponding positions as the reference chromatogram. If the sample was heated to 105℃ and then examined under a UV lamp (365nm), the spots would be interfered with by impurities. Therefore, it was chosen to spray with 10% sulfuric acid ethanol solution before examination.

[0080] 3.1.10 Examination of the unfolding effect of thin-layer plates from different manufacturers According to the chromatographic conditions under section "3.1.4", different thin-layer plates, such as silica gel G plate (Qingdao Ocean Chemical Co., Ltd.), silica gel HSG plate (Yantai Chemical Industry Research Institute - Yinlong brand), and silica gel HSG plate (Yantai Jiangyou Silica Gel Development Co., Ltd. - Huanghai brand), were used to conduct experiments to examine the development effect.

[0081] The results showed that using thin-layer plates from three different manufacturers, the test sample spots could all achieve good separation (see...). Figure 20 , Figure 23 , Figure 24 ).

[0082] 3.1.11 Evaluation of the development effect under different humidity conditions According to the chromatographic conditions under section “3.1.4”, examine one relative humidity at low relative humidity (15-30%) and high relative humidity (75-90%) to assess the development effect.

[0083] The results showed that good separation of the test sample spots was achieved under both 18% and 88% humidity conditions (see [reference needed]). Figure 25 , Figure 26 ).

[0084] 3.1.12 Evaluation of the development effect under different temperature conditions According to the chromatographic conditions under section “3.1.4”, the development effect was investigated at two temperatures, 5℃ and 35℃.

[0085] The results showed that good separation of the sample spots was achieved under both 10℃ and 30℃ conditions (see [reference needed]). Figure 27 , Figure 28 ).

[0086] 3.2 Results It has been verified that this thin-layer chromatography identification method can be used for quality control of Yin Xie No. 1 oral granules, and therefore it is included in the main text of the internal control agent standard.

[0087] Example 4: Determination of paeoniflorin content 4.1 Selection of chromatographic conditions 1) Medicines and reagents: Preparation of the traditional Chinese medicine granules in Example 1 2) Experimental instruments: Shimadzu 2030C high performance liquid chromatograph, PDA detector.

[0088] 4.1.1 Selection of detection wavelength Take an appropriate amount of paeoniflorin reference standard, accurately weigh it, add methanol to prepare a solution containing 60 μg per ml, scan the full wavelength, and find that the maximum absorption is at 230 nm. Finally, select 230 nm as the detection wavelength.

[0089] 4.1.2 Selection of mobile phase Acetonitrile-water (14:86) showed negative interference; ratios of (13:87), (12:88), and (11:89) improved negative interference, but the sample baseline fluctuated significantly, affecting the calculation of the paeoniflorin peak area.

[0090] Acetonitrile-0.1% phosphoric acid solution [ratio: (15:85), (16:84)], the paeoniflorin peak was not separated from the previous small peak; [ratio: (14:86), (13:87), (12:88)], there was interference in the negative test; (11:89), the paeoniflorin peak in the sample was well separated, and there was no interference in the negative test.

[0091] 4.1.3 Chromatographic conditions Octadecylsilane-bonded silica gel was used as the packing material; the mobile phase was acetonitrile-0.1% phosphoric acid solution (11:89); the flow rate was 1 ml / min; and the detection wavelength was 230 nm. The theoretical plate number, calculated based on the paeoniflorin peak, should be no less than 2000.

[0092] 4.2 Research on Sample Processing Methods 4.2.1 Comparison of extraction solvents Take the Chinese herbal granules from Preparation Example 1, grind them finely, take about 1g, accurately weigh it, place it in a stoppered conical flask, and accurately add 25ml each of methanol, 70% methanol, ethanol, and water, respectively. Seal the flask tightly, weigh it, and sonicate it (500W power, 45kHz frequency) for 30 minutes. After cooling, weigh it again, make up the weight loss with the appropriate solvent, shake well, filter, and collect the filtrate. According to the chromatographic conditions under section "4.1.3", accurately pipette 10 μL of each of the above test solutions, inject them into the liquid chromatograph, record the peak area values, and calculate the paeoniflorin content.

[0093] The results showed that the paeoniflorin content was significantly lower when ethanol was used as the extraction solvent; when methanol or 70% methanol was used as the extraction solvent, there was no significant difference in the paeoniflorin content. The theoretical plate number of paeoniflorin extraction with methanol was higher, so methanol was finally chosen as the extraction solvent.

[0094] 4.2.2 Comparison of Extraction Methods Take the herbal granules from Preparation Example 1, grind them finely, take about 1g, accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of methanol, seal tightly, weigh, heat under reflux and sonicate (power 500W, frequency 45kHz) for 30 minutes respectively, cool, weigh again, make up the lost weight with methanol, shake well, filter, and collect the filtrate. According to the chromatographic conditions under section "4.1.3", accurately pipette 10 μL of each of the above test solutions, inject into the liquid chromatograph, record the peak area values, and calculate the paeoniflorin content.

[0095] The results showed that there was no significant difference in paeoniflorin content between the ultrasonically treated and the heated refluxed samples, so the simpler ultrasonic treatment was ultimately chosen.

[0096] 4.2.3 Comparison of extraction times Take approximately 1g of the oral granules of Silver Psoriasis No. 1, grind them into a fine powder, and accurately weigh it. Place the 1g powder in a stoppered conical flask, accurately add 25ml of methanol, seal tightly, and weigh. Sonicate the flask (500W power, 45kHz frequency) for 20 minutes, 30 minutes, and 40 minutes respectively. After cooling, weigh the flask again, replenish the lost weight with methanol, shake well, filter, and collect the filtrate. According to the chromatographic conditions in section "4.1.3", accurately inject 10µL of each of the above test solutions into the liquid chromatograph, record the peak area values, and calculate the paeoniflorin content.

[0097] The results showed that there was no significant difference in the paeoniflorin content measured at different extraction times. After 20 minutes of ultrasonic treatment, the sample was basically completely extracted, so 20 minutes was finally selected as the extraction time.

[0098] 4.2.4 Comparison of Ultrasonic Power Take approximately 1g of the oral granules of Silver Powder No. 1, grind them into a fine powder, and accurately weigh it. Place the granules in a stoppered conical flask, accurately add 25ml of methanol, seal tightly, and weigh. Sonicate the flask (powers of 300W, 400W, and 500W, frequency 45kHz) for 20 minutes, cool, and weigh again. Make up the lost weight with methanol, shake well, filter, and collect the filtrate. According to the chromatographic conditions in section "4.1.3", accurately inject 10µL of each of the above test solutions into the liquid chromatograph, record the peak area values, and calculate the paeoniflorin content.

[0099] The results showed that there was no significant difference in the paeoniflorin content of samples treated with different ultrasonic powers, so the final ultrasonic power was 300W.

[0100] 4.2.5 Preparation of the test solution Take the Chinese herbal granules from Preparation Example 1, grind them into a fine powder, take about 1g, weigh it accurately, place it in a stoppered conical flask, add 25ml of methanol accurately, seal tightly, weigh it, sonicate (power 300W, frequency 45kHz) for 20 minutes, cool it, weigh it again, make up the lost weight with methanol, shake well, filter it, and take the filtrate to obtain the final product.

[0101] 4.2.6 Preparation of reference solution Take an appropriate amount of paeoniflorin reference standard (purchased from the National Institutes for Food and Drug Control), weigh it accurately, and add 70% methanol to prepare a solution containing 60 μg per ml.

[0102] 4.2.7 Preparation of negative control solution Take about 0.5g of the dried herbal extract powder from Example 1 (which lacks white peony root) and follow the procedure in section “4.2.5” to prepare a negative control solution lacking white peony root.

[0103] 4.3 Methodological Examination 4.3.1 Specificity According to the chromatographic conditions under section “4.1.3”, accurately pipette 10 μL each of the above-mentioned reference solution, test solution, negative control solution and blank solvent, and inject them into the liquid chromatograph to obtain the corresponding chromatograms.

[0104] In the blank solvent and negative control chromatograms, no chromatographic peaks appeared at the retention times corresponding to the reference and test sample chromatograms, proving that other components in the traditional Chinese medicine granule sample of Preparation Example 1 did not interfere with the determination of paeoniflorin. This indicates that the method has good specificity and the system suitability meets the requirements. Results are shown below. Figures 29-32 .

[0105] 4.3.2 Linear Accurately measure 0.5 ml, 0.9 ml, and 1.6 ml of paeoniflorin stock solution (concentration 0.9103 mg / ml), place them in 25 ml volumetric flasks, dilute to the mark with 70% methanol, and shake well to obtain L1 to L3 linear solutions with concentrations of 0.01821 mg / ml, 0.03277 mg / ml, and 0.05826 mg / ml.

[0106] Accurately measure 0.9 ml, 2.0 ml, and 2.5 ml of the above stock solution and place them in 10 ml volumetric flasks respectively. Dilute to the mark with 70% methanol and shake well to obtain L4 to L6 linear solutions with concentrations of 0.08193 mg / ml, 0.18206 mg / ml, and 0.22758 mg / ml.

[0107] Following the chromatographic conditions described in section "4.1.3", precisely pipette 10 μL of each of the L1–L6 linear solutions of different concentrations and inject them into the liquid chromatograph for analysis. Record the peak area of ​​paeoniflorin at each injection volume. Plot a standard curve with injection volume (X) on the x-axis and peak area (Y) on the y-axis, and calculate the linear correlation coefficient. The results are shown in Table 1. Figure 33 .

[0108] Table 1 Linear relationship of paeoniflorin The results showed that paeoniflorin exhibited a good linear relationship between 0.1821 and 2.2758 µg.

[0109] 4.3.3 Repeatability Following the method described in section "4.2.5", prepare six parallel test solutions using Yin Xie No. 1 oral granules. Under the chromatographic conditions described in section "4.1.3", accurately pipette 10 μL each of the reference solution described in section "4.2.6" and the above-mentioned test solution, and inject them into the liquid chromatograph for analysis. Record the peak areas of paeoniflorin in the test solutions and reference solutions. Calculate the paeoniflorin content in the six test solutions using the external standard method, based on the peak areas, and report the average value and RSD. The results are shown in Table 2.

[0110] Table 2 Repeatability Results The results showed that the RSD value of the six parallel test solutions was 1.4%, which met the requirement of RSD≤3.0%.

[0111] 4.3.4 Intermediate Precision Different personnel used different instruments on different days to prepare test solutions in parallel, following the method described in section "4.2.5", by taking the Chinese herbal granules from Preparation Example 1. Following the method described in section "4.2.7", a negative test solution for peony-deficient herbs was prepared from the dried herbal extract powder of Preparation Example 1, which lacked peony root.

[0112] Following the chromatographic conditions described in section "4.1.3", precisely pipette 10 μL each of the reference solution described in section "4.2.6" and the above-mentioned test solution, and inject them into the liquid chromatograph for analysis. Record the chromatographic peak areas of paeoniflorin in the test solution and the reference solution. Calculate the paeoniflorin content in the six test solutions using the external standard method, based on the peak areas.

[0113] The results of repeatability and intermediate precision determinations were compared, and the average value and RSD of the content determination results of 12 test samples were reported. The results showed that, when the experimental date, instrument, and operator conditions were changed, no chromatographic peaks appeared at the retention times corresponding to the chromatograms of the reference standard and the test samples in the negative solution lacking white peony. When comparing the results of the intermediate precision and repeatability determinations, the RSD value was 2.1%, which meets the requirement of RSD≤3.0%, indicating that the intermediate precision of the method is good.

[0114] 4.3.5 Accuracy Take the same known content of the Chinese herbal granules from Preparation Example 1, and according to the method under "4.2.5", take 1 / 2 of the sample amount of the test sample (about 0.5 g), and accurately add 1.1 ml of paeoniflorin reference solution with a concentration of 0.9103 mg / ml to prepare a 100% concentration level spiking recovery test sample solution. Prepare 6 parallel samples.

[0115] Following the chromatographic conditions described in section "4.1.3", precisely pipette 10 μL each of the reference solution described in section "4.2.6" and the above six recovered test solutions, and inject them into the liquid chromatograph for analysis. Record the peak areas of paeoniflorin in the test solutions and reference solutions. Calculate the recoveries of the six test solutions using the external standard method, based on the peak areas, and report the average value and RSD. The results are shown in Table 3.

[0116] Table 3 Accuracy Results The results showed that the recovery rate of the test solution was 98.3%, with an RSD of 1.9%, which meets the requirement that the recovery rate should be between 90% and 108% and the RSD should be ≤3.0%.

[0117] 4.3.6 Solution stability Prepare one aliquot of the reference solution and one aliquot of the test solution according to the methods in sections “4.2.5” and “4.2.6”. Incubate at room temperature and perform at least five injections within 0–42 hours to assess their stability. Under the chromatographic conditions in section “4.1.3”, accurately pipette 10 μL of each of the above-mentioned reference solution and test solution and inject them into the liquid chromatograph for analysis. Record the peak area of ​​paeoniflorin in the reference solution and test solution, and calculate the RAD value of the peak area at 0°C.

[0118] The results showed that the longest continuous injection time with the peak area at each time point and the peak area at 0 time point both ≤2.0% and the RSD value of all peak areas ≤4.0% was taken as the longest stable time of the solution. Therefore, the shelf life of the reference solution was 42 hours and the shelf life of the test solution was 20 hours.

[0119] 4.3.7 Durability Following the method described in section "4.2.5", the Chinese herbal granules from Preparation Example 1 were used to prepare the test solution in parallel, and the reference solution described in section "4.2.6" was used. By changing the flow rate, column temperature, chromatographic column, and liquid chromatograph, the system suitability and content determination results after changing the conditions were examined.

[0120] Under the modified conditions, precisely pipette 10 μL each of the reference solution from section "4.2.6" and the test solution described above, and inject them into the liquid chromatograph for analysis. The reference solution was injected five times consecutively. The peak areas of paeoniflorin in the test and reference solutions under different conditions were recorded. The paeoniflorin content of the test sample was calculated using the external standard method, based on the peak area. The average value and RSD were reported, along with the RAD value compared to the result determined using the standard method in section "4.3.3". The results are shown in Table 4.

[0121] Table 4 Durability Results The results showed that at 35℃, the resolution between the paeoniflorin peak and the subsequent smaller peak in the test sample was <1.5. The paeoniflorin content measured under other conditions and the RAD value of the results measured under the selected conditions all met the requirement of ≤3.0%. This method requires controlling the temperature below 35℃ or adjusting the mobile phase ratio appropriately as specified.

[0122] 4.3.8 Scope Based on the results of the above examinations on linearity, accuracy, and precision, the concentration range (calculated as paeoniflorin) used in this method is limited to 18.21 µg / ml to 227.58 µg / ml.

[0123] 4.3.9 Validation results of the method for determining paeoniflorin content To effectively control the quality of the herbal granules prepared in Example 1, a study was conducted on the content determination of white peony root, one of the main ingredients in the formula. A high-performance liquid chromatography (HPLC) method was established to determine the paeoniflorin content. Verification showed that this content determination method can be used for quality control of Yin Xie No. 1 oral granules, and therefore it was included in the internal control standard.

[0124] 4.4 Sample testing and establishment of limit standards The smallest packaging specification for the Chinese herbal granules in Preparation Example 1 is 14g per bag (equivalent to 32.62g of processed medicinal slices), meaning that each bag (14g granules) contains paeoniflorin (C) as a component of white peony root. 23 H 28 O 11 The content limit and usual limit control value per bag of granules shall not be less than 20.16 mg (based on a 100% transfer rate). Based on the average transfer rate (55%) of existing prepared samples, the content limit per bag of granules shall be calculated as 70%. Each bag (14g granules) of this product contains paeoniflorin (C... 23 H 28 O 11 The total dose shall not be less than 8.0 mg.

[0125] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations included in the claims. Various modifications and changes can be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments can be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only illustrate several implementations of the present invention and do not limit the scope of protection of this patent.

Claims

1. A method for quality testing of Psoriasis No. 1 oral granules, wherein the traditional Chinese medicine granules are made from Rehmannia glutinosa, Lonicera japonica, Scrophularia ningpoensis, Paeonia lactiflora, Gypsum fibrosum, Anemarrhena asphodeloides, Imperata cylindrica, Hedyotis diffusa, Sophora flavescens, Arctium lappa, Schizonepeta tenuifolia, Saposhnikovia divaricata, and Glycyrrhiza uralensis, characterized in that, Includes the following steps: (1) Thin-layer chromatography was used to identify the following components in the granules: Sophora flavescens, Glycyrrhiza uralensis, Arctium lappa, and Saposhnikovia divaricata. The identification of Sophora flavescens was performed using a toluene-acetone-methanol mixed solvent as the developing solvent, with matrine as a reference standard. The identification of licorice and burdock seed used a chloroform-methanol-water mixed solvent as the developing solvent, and glycyrrhizin and arctiin reference standards were used for comparison. The identification of Saposhnikovia divaricata was performed using a dichloromethane-methanol-water mixed solvent as the developing solvent, with 5-O-methylvisamidol glycoside as a reference standard. (2) The content of paeoniflorin in the particles was determined by high performance liquid chromatography. Acetonitrile-0.1% phosphoric acid mixed solvent was used as the mobile phase and the detection wavelength was 230 nm.

2. The method according to claim 1, characterized in that, The toluene-acetone-methanol volume ratio is 8:3:0.5; the chloroform-methanol-water volume ratio is 40:9:1; the dichloroform-methanol-water volume ratio is 5:1:0.1; and the paeoniflorin content is determined using an acetonitrile-0.1% phosphoric acid solution with a volume ratio of 11:

89.

3. The method according to claim 1, characterized in that, The steps for identifying Sophora flavescens using thin-layer chromatography are as follows: 1) Preparation of test sample: Take Chinese medicine granules, add concentrated ammonia test solution-chloroform mixed solution, sonicate, filter, evaporate the filtrate to dryness, add 1 ml of ethanol to dissolve the residue, and use it as the test sample solution; 2) Preparation of matrine reference standard: Take matrine reference standard and add ethanol to prepare a solution containing 1 mg per 1 ml, which is used as the reference solution; 3) Identification: Take 10 μL of the test solution and 2 μL of the reference solution and spot them separately on the same silica gel thin-layer plate. Use toluene-acetone-methanol (8:3:0.5) as the developing solvent, develop, remove, air dry, and spray with colorimetric solution. Preferably, the weight-to-volume ratio of the particles to ethanol in the preparation of the test sample is 8:0.6-50; the ultrasonication is preferably performed for 30 minutes. Preferably, the silica gel thin-layer plate used in the identification process is a silica gel G thin-layer plate or a silica gel HSG thin-layer plate; the colorimetric solution is potassium iodide test solution and sodium nitrite ethanol test solution; the developing temperature is 5-35℃, preferably 10℃~30℃; the relative humidity during the developing process is 15-30% or 75-90%, preferably 18%~88%. More preferably, the steps for identifying Sophora flavescens using thin-layer chromatography are as follows: 1) Take 8g of Chinese herbal medicine granules, 0.6ml of concentrated ammonia test solution, and 50ml of chloroform, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 1ml of ethanol to dissolve the residue, and use it as the test solution. 2) Take matrine reference standard, add ethanol to prepare a solution containing 1 mg per 1 ml, and use it as the reference solution; 3) Take 10 μL of the test solution and 2 μL of the reference solution and spot them separately on the same silica gel G thin-layer plate. Use toluene-acetone-methanol (8:3:0.5) as the developing solvent and develop in a developing tank saturated with ammonia vapor for 20 minutes. Remove the plate, air dry it, and spray it with potassium iodide test solution and sodium nitrite ethanol test solution in sequence.

4. The method according to claim 1, characterized in that, The steps for identifying licorice and burdock seed using thin-layer chromatography are as follows: 1) Preparation of test sample: Take Chinese medicine granules, add ethanol, sonicate, filter, evaporate the filtrate to dryness, dissolve the residue in water, extract with ethyl acetate by shaking, reserve the aqueous solution, combine the ethyl acetate solutions, evaporate to dryness, dissolve the residue in 1 ml of ethanol, and use as the test sample solution. 2) Preparation of glycyrrhizin and arctiin reference standards: Take glycyrrhizin and arctiin reference standards, and add ethanol to prepare solutions containing 0.5 mg per ml and 5 mg per ml, respectively, as reference solutions; 3) Identification: Take 5-10 μL of the test solution, 1 μL of the glycyrrhizin reference solution, and 5 μL of the arctiin reference solution, and spot them separately on the same silica gel G thin layer plate. Use chloroform-methanol-water (40:9:1) as the developing solvent, develop, remove, air dry, and spray with a colorimetric solution for color development. Preferably, the weight-to-volume ratio of particles to ethanol in the preparation of the test sample is 8:50; the ultrasonication is preferably performed for 30 minutes. Preferably, the silica gel thin-layer plate used in the identification process is a silica gel G thin-layer plate; the colorimetric solution is 10% sulfuric acid ethanol; the developing temperature is 5-35℃, preferably 10℃~30℃; and the relative humidity during developing is 15-30% or 75-90%, preferably 18%~88%. More preferably, the steps for identifying licorice and burdock seed by thin-layer chromatography are as follows: 1) Take 8g of Chinese herbal medicine granules, add 50ml of ethanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 20ml of water, extract twice with ethyl acetate, 20ml each time, keep the aqueous solution for later use, combine the ethyl acetate solutions, evaporate to dryness, dissolve the residue in 1ml of ethanol, and use it as the test solution. 2) Take glycyrrhizin reference standard and arctiin reference standard, and add ethanol to prepare solutions containing 0.5 mg per 1 ml and 5 mg per 1 ml, respectively, as reference solutions; 3) Take 5-10 μL of the test solution, 1 μL of glycyrrhizin reference solution, and 5 μL of arctiin reference solution, and spot them separately on the same silica gel G thin-layer plate. Use chloroform-methanol-water (40:9:1) as the developing solvent, develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, and heat at 105℃ until the glycyrrhizin spots are clearly visible. Examine under sunlight and ultraviolet light (365nm) respectively.

5. The method according to claim 1, characterized in that, The steps for identifying the windproof thin-layer chromatography method are as follows: 1) Preparation of the test sample: Take the prepared aqueous solution as described in step 1) of claim 4, extract it by shaking with water-saturated n-butanol, combine the n-butanol solutions, evaporate to dryness, dissolve the residue in 1 ml of ethanol, and use it as the test sample solution. 2) Preparation of Saposhnikovia divaricata reference standard: Take 5-O-methylvisamidol glycoside reference standard, add methanol to prepare a solution containing 0.4 mg per 1 ml, as the reference solution; 3) Identification: Take 5 μl of the test solution and 1 μl of the reference solution and spot them separately on the same silica gel G thin layer plate. Use dichloromethane-methanol-water (5:1:0.1) as the developing solvent, develop, remove, air dry, spray with color developing solution, and examine under ultraviolet light. Preferably, the test sample is prepared by shaking with water-saturated n-butanol twice, 20 ml each time; Preferably, the silica gel thin-layer plate used in the identification process is a silica gel G thin-layer plate or a silica gel HSG thin-layer plate; the colorimetric solution is 10% sulfuric acid ethanol; the developing temperature is 5~35℃, preferably 10℃~30℃; and the relative humidity during developing is 15~30% or 75~90%, preferably 18%~88%. More preferably, the step of the thin-layer chromatography identification of *Saposhnikovia divaricata* is as follows: 1) Take the prepared aqueous solution from step 1) of claim 4, extract it twice with water-saturated n-butanol, 20 ml each time, combine the n-butanol solutions, evaporate to dryness, dissolve the residue in 1 ml of ethanol, and use it as the test solution. 2) Take 5-O-methylvisamidol glycoside reference standard, add methanol to prepare a solution containing 0.4 mg per 1 ml, and use it as the reference solution; 3) Take 5 μl of the test solution and 1 μl of the reference solution and spot them separately on the same silica gel G thin layer plate. Use dichloromethane-methanol-water (5:1:0.1) as the developing solvent, develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, and examine under a UV lamp at 365 nm.

6. The method according to claim 1, characterized in that, The steps for determining the paeoniflorin content in granules using high performance liquid chromatography are as follows: 1) Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the stationary phase, and acetonitrile-0.1% phosphoric acid solution (11:89) was used as the mobile phase; the detection wavelength was 230 nm; the theoretical plate number calculated based on the paeoniflorin peak should not be less than 2000; 2) Preparation of reference solution: Take an appropriate amount of paeoniflorin reference standard, accurately weigh it, and add 70% methanol to prepare a solution containing 60 μg per ml. 3) Preparation of test solution: Take the granules, grind them finely, weigh them accurately, place them in a stoppered conical flask, add methanol accurately, stopper tightly, weigh them, sonicate them, cool them, weigh them again, make up the weight loss with methanol, shake well, filter them, and take the filtrate to obtain the test solution. 4) Determination method: Accurately pipette 10 μL each of the reference solution and the test solution into the liquid chromatograph and determine the result. Preferably, the flow rate of the mobile phase is 1 ml / min; Preferably, the weight-to-volume ratio of particles to methanol in the preparation of the test solution is 1:25; the ultrasonic conditions are 300W-500W power, 45kHz frequency, 20-40min, preferably 300W, 45kHz frequency, 20min; More preferably, the step of determining the paeoniflorin content in the particles by high performance liquid chromatography is as follows: 1) Chromatographic conditions and system suitability test: C18 column, 250mm×4.6mm, 5μm; mobile phase: acetonitrile-0.1% phosphoric acid solution (11∶89); flow rate: 1ml / min; detection wavelength: 230nm; theoretical plate number calculated based on paeoniflorin peak should not be less than 2000; 2) Preparation of reference solution: Take an appropriate amount of paeoniflorin reference standard, accurately weigh it, and add 70% methanol to prepare a solution containing 60 μg per ml. 3) Preparation of the test solution: Take Chinese herbal medicine granules, grind them into a fine powder, take about 1g, weigh it accurately, place it in a stoppered conical flask, add 25ml of methanol accurately, stopper tightly, weigh it, sonicate it at 300W power and 45kHz frequency for 20min, cool it, weigh it again, make up the lost weight with methanol, shake it well, filter it, and take the filtrate to obtain the test solution. 4) Determination method: Accurately pipette 10 μL each of the reference solution and the test solution into the liquid chromatograph and determine the result.

7. A method for quality testing of the oral granules of Psoriasis No. 1 obtained by the method described in any one of claims 1 to 6.

8. A method for determining the content of indicator components in Psoriasis No. 1 oral granules, characterized in that, The determination method includes determining the paeoniflorin content in the particles using the high performance liquid chromatography method as described in claim 6, and the concentration range of paeoniflorin used in the method is limited to 18.21 µg / ml to 227.58 µg / ml.

9. The method according to any one of claims 1 to 6, the quality detection of Yin Xie No. 1 oral granules according to claim 7, or the determination method according to claim 8 in the quality detection of Yin Xie No. 1 oral granules; Preferably, the quality testing includes one or more of the following: identification of indicator components and determination of the content of indicator components.

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