A quality detection method for compound isatis root granules

Abstract

The present invention relates to the field of quality control of traditional Chinese medicine preparations, specifically a quality inspection method for compound isatis root granules. The compound isatis root granules are made from isatis root and isatis indigotica. The quality inspection method specifically uses UHPLC-DAD-Q-TOF / MS technology to identify the chemical components of the medicinal material slices, intermediates, and finished granules in the compound isatis root granules to determine the quality control compounds of the compound isatis root granules. The present invention establishes a correlation between the chemical components of the two traditional Chinese medicine slices in the prescription, the water extract paste, the fluid paste, and the granules, providing a basis for the industrial research of compound isatis root granules from the traditional Chinese medicine slices, extract, and granules quality control.

Description

Technical Field

[0001] The present invention relates to the field of quality control of traditional Chinese medicine preparations, in particular to a quality detection method for compound isatis root granules. Background Art

[0002] Compound Isatis Root Granules are a commonly used Chinese patent medicine, made from Isatis Root and Isatis Indigotica (600g: 900g). They have the effects of clearing heat, detoxifying, and cooling blood. They are used for fever caused by febrile diseases, rashes, wind-heat colds, sore throats, epidemic encephalitis B, hepatitis, and mumps. The quality standard for Compound Isatis Root Granules is included in the "Drug Standards of the Ministry of Health of the People's Republic of China" (Volume 12 of Traditional Chinese Medicine Formulas, WS3-B-2377-97). Indigotin and indigo in the preparation are identified by thin-layer chromatography, and a quantitative method for indigotin in Isatis Indigotica has been established using high-performance liquid chromatography (HPLC), with a specified lower limit for its content. In recent years, several research groups have used methods such as high-performance thin-layer chromatography (HPTLC), high-performance liquid chromatography-ultraviolet detection (HPLC-UV), and high-performance liquid chromatography-mass spectrometry (HPLC-MS) to improve the technology of thin-layer identification, content determination, and chemical component identification, aiming to improve its quality standards. Some progress has been made, but reports on the quality control of compound isatis root granules still mainly focus on quantitative analysis of water-soluble components such as (R,S)-galactopyranoside and adenosines, which is insufficient to control the quality of the preparation as a whole. Further improvement is needed in basic research on chemical substances and in mass transfer and traceability.

[0003] Quality evaluation and control of traditional Chinese medicine (TCM) are crucial for ensuring its safety and effectiveness. Current TCM quality control models primarily rely on qualitative and quantitative determination of index components. The TCM production process primarily encompasses two stages, from raw materials to intermediates and finally to finished products, encompassing three product levels. The variation in the quality of each product is integrated one or more times to form the quality variability of the final product. This process involves the transmission or transformation of physical, chemical, and biological information, ultimately impacting the efficacy of the finished preparation. Therefore, quality evaluation and control of TCM preparation production processes are essential. Currently, most studies rely on the qualitative characterization of multiple indicators based on TCM fingerprints. Chemical composition analysis is performed on different research subjects, tailored to the specific research objectives. For example, based on HPLC-UV fingerprints, quantitative transfer studies of prescription medicinal materials, decoction pieces, and benchmark drug efficacy groups were conducted; based on HPLC-UV fingerprints, the main chemical components of decoction pieces, standard decoctions, intermediates, and formula granules were investigated and their quality correlations were evaluated; HPLC-UV fingerprints were used to conduct quality control studies on medicinal materials, intermediates, and finished products, and correlation analysis was performed to ensure the quality and clinical efficacy of the preparations. However, the above studies only calculated common peaks and similarities through HPLC chromatograms, and the attribution of each peak in the chromatogram was not clear. Secondly, Radix Isatidis and Folium Isatidis are the dried roots and leaves of the cruciferous plant Isatis indigotica, and their main chemical components are similar. Since the detection limit of HPLC is lower than that of LC-MS, the chemical component information provided on the chromatogram is limited. Therefore, the fingerprint based on HPLC cannot accurately and comprehensively reflect the specific components of Radix Isatidis and Folium Isatidis in the intermediates and finished products of compound Radix Isatidis granules, and is not suitable for conducting mass transfer and traceability studies of chemical components.

[0004] Compared to HPLC fingerprinting technology, ultra-high performance liquid chromatography coupled with time-of-flight mass spectrometry (UHPLC-Q-TOF / MS) offers advantages such as strong specificity, fast analysis time, high sensitivity, and good reproducibility. It can obtain the precise mass-to-charge ratio of compound ions in a short period of time. By comparing established databases of known chemical components with information such as compound molecular fragments, it can conduct rapid and accurate analysis and identification of components. This technology offers significant advantages in the qualitative analysis of complex multi-component systems in traditional Chinese medicine. Chemical component analysis based on UHPLC-Q-TOF / MS is more suitable for quality control of the entire process from decoction pieces to intermediates to finished products. Furthermore, the use of UHPLC-Q-TOF / MS coupled with UV detection using a diode array detector (DAD) allows for simultaneous relative quantitative analysis of compounds.

[0005] At present, only two articles have studied the chemical components of compound isatis root granules based on liquid chromatography-mass spectrometry (LC-MS), and the number of identified compounds is relatively small [Yan Jun, Liu Shu, Pi Zifeng, Song Fengrui, Liu Zhongying, Liu Zhiqiang. Study on the antiviral components of compound isatis root granules by HPLC-UV fingerprint and LC-ESI-MSn[J]. Acta Chimica Sinica, 2011, 69(2): 204-208. Yan Jun, Liu Shu, Li Boping, Liu Zhiqiang, Guo Dongfa. Study on the APCI-MS fingerprint and LC-APCI-MSn of compound isatis root granules[J]. Journal of Mass Spectrometry, 2011, 69(2): 204-208. , 2017, 38(3): 62-69.], the compounds contained in compound isatis root granules have not been analyzed and identified in detail; on the other hand, the research based on chemical component analysis-biological assay correlation analysis only conducted quality control method research on the production process of isatis root granules [Tan Manrong, Yan Dan, Qiu Lingling, Chen Longhu, Yan Yan, Jin Cheng, Li Hanbing, Xiao Xiaohe. Study on biological evaluation and control methods of quality in the production process of traditional Chinese medicine - taking isatis root granules as an example [J]. China Journal of Traditional Chinese Medicine, 2012, 37(8): 1122-1126.], similar research has not been conducted on the production process of compound isatis root granules. Summary of the Invention

[0006] The present invention believes that the use of UHPLC-DAD-Q-TOF / MS technology can more comprehensively and comprehensively reflect the chemical components contained in medicinal materials, better reflect the complexity of traditional Chinese medicine components, and further carry out research on the analysis and transmission of chemical component information from medicinal pieces to intermediates to finished products based on this technology, improve the quality evaluation system of the preparation, and enhance the controllability and stability of its therapeutic effect.

[0007] The purpose of the present invention is to provide a quality testing method for compound isatis root granules. This invention proposes, for the first time, to establish a model and method for the quality control and evaluation of traditional Chinese medicine (TCM) based on the transfer of chemical components from decoction pieces to intermediates and finished products, drawing on the quality management model for biopharmaceutical production. This method supplements and improves the existing TCM quality control and evaluation system based on TLC identification and HPLC quantitative analysis of multiple chemical components. Based on the research results of this invention, chemical components that can be accurately quantified will be selected as research objects. In combination with technical and economic indicators in the preparation process, the quantitative transfer relationship of the main pharmacological substance groups from decoction pieces to intermediates and finished products will be further studied.

[0008] A first aspect of the present invention provides a quality inspection method for compound isatis root granules. The compound isatis root granules are made of isatis root and isatis indigotica (600g:900g). The quality inspection method specifically uses UHPLC-DAD-Q-TOF / MS technology to identify the chemical components of the medicinal material slices, intermediates, and finished granules in the compound isatis root granules to determine the quality control compounds of the compound isatis root granules.

[0009] The specific steps include:

[0010] Among them, steps A, B, C, and D are the pretreatment methods of different samples (herbal medicines, intermediates, granules, and reference substances), with the purpose of using the best pretreatment method to obtain the best ion response; step E is the processing method of quality control samples (QC), with the purpose of evaluating the quality of data acquisition; step F is the data identification step, which uses the database to identify ions as compounds.

[0011] (A) Preparation of test solutions of medicinal materials: Randomly select Radix Isatidis / Leucophyllae from each batch (three samples from each batch) and grind them into powder. 5 g of the powder was accurately weighed and placed in a 50 ml stoppered conical flask. 20 ml of methanol solution (Leucophyllae: 20% methanol, Radix Isatidis: 40% methanol) was accurately added to each flask. Ultrasonic treatment (power 500 W, frequency 40 kHz) was performed for 30 min. The extract was cooled and shaken to mix well. The extract was stirred at 3000 r / min. -1 Centrifuge for 10 min, take the upper layer solution into a 1.5 ml centrifuge tube, and centrifuge at 15000 r min -1 Centrifuge for 10 minutes, let it stand for 10 minutes, and then take the supernatant again at 15000 r / min. -1 Centrifuge for 10 minutes to obtain the product.

[0012] (B) Preparation of test solutions of intermediate water extract paste and fluid paste: Water extract paste and fluid paste (mass calculated based on the transfer of quantity) were randomly taken from each batch (three aliquots from each batch), accurately weighed, and placed in a 50 ml stoppered conical flask. Solvent (water extract paste: 20% methanol, fluid paste: water) was accurately added in a ratio of 1:4 by volume. Ultrasonic treatment (power 500 W, frequency 40 kHz) was performed for 30 min, cooled, and shaken. The extract was stirred at 3000 r / min. -1 Centrifuge for 10 min, take the upper layer solution into a 1.5 ml centrifuge tube, and centrifuge at 15000 r min -1 Centrifuge for 10 minutes, let it stand for 10 minutes, and then take the supernatant again at 15000 r / min. -1 Centrifuge for 10 minutes to obtain the product.

[0013] (C) Preparation of granule test solution: 5 g of each batch of granules (three aliquots from each batch) were accurately weighed and placed in a 50 ml stoppered conical flask. 20 ml of water was accurately added (1:4 extraction ratio). Ultrasonic treatment (power 500 W, frequency 40 kHz) was performed for 30 min, cooled, and shaken. The extract was stirred at 3000 r / min. -1 Centrifuge for 10 min, take the upper layer solution into a 1.5 ml centrifuge tube, and centrifuge at 15000 r min -1 Centrifuge for 10 minutes, let it stand for 10 minutes, and then take the supernatant again at 15000 r / min.-1 Centrifuge for 10 minutes to obtain the product.

[0014] (D) Preparation of reference solutions: Accurately weigh appropriate amounts of indirubin and indigo reference standards, add chloroform to a solution containing 1 mg per 1 mL, sonicate, and vortex to mix. These solutions are stock solutions 1 and 2. Accurately weigh appropriate amounts of leucine, arginine, valine, and isatin reference standards, add 70% ethanol to a solution containing 1 mg per 1 mL, sonicate, and vortex to mix. These solutions are stock solutions 3-6. Accurately weigh (R,S)-epigoitrin and isovitexin, add methanol to a solution containing 1 mg per 1 mL, sonicate, and vortex to mix. These solutions are stock solutions 7-8. Accurately weigh appropriate amounts of L-tryptophan reference standard, add water to a solution containing 1 mg per 1 mL, sonicate, and vortex to mix. These solutions are stock solutions 9. Accurately weigh an appropriate amount of adenosine and uridine reference substances, add water to make a solution containing 1 mg of each reference substance per mL, sonicate to dissolve, and vortex to mix. These solutions are prepared as stock solutions 10 and 11. Accurately weigh an appropriate amount of guanosine reference substance, add water to make a solution containing 100 μg of each reference substance per mL, sonicate to dissolve, and vortex to mix. This is prepared as stock solution 12.

[0015] Accurately pipette 200 μL each of stock solutions 1, 2, 4, 6-9, place in a 2 ml volumetric flask and mix, then add methanol to prepare a mixed standard solution A with a concentration of 100 μg / mL for each standard.

[0016] Accurately pipette 200 μL each of stock solutions 3, 5, 7, and 10-12, and 20 μL of stock solution 4, place them in a 2 ml volumetric flask and mix them. Add methanol to prepare a mixed standard solution B with a standard concentration of 100 μg / mL (arginine and guanosine concentrations of 10 μg / mL).

[0017] (E) Preparation of quality control samples (QC): 10 μL of each test solution was accurately pipetted into a 2 mL centrifuge tube, vortexed and mixed, and centrifuged at 15000 r·min. -1 Centrifuge for 10 minutes, let it stand for 10 minutes, and then take the supernatant.

[0018] (F) Construction of compound analysis library and data analysis: The prescription compound database MS_peaks-clusters_final_processed-doudou0911 was established based on Agilent's own defult file; data analysis was performed using Agilent MassHunter Qualitative Analysis 10.0 software.

[0019] Furthermore, in the quality detection method, the chromatographic conditions are: Agilent Poroshell 120, SB-C18 chromatographic column (4.6 mm×150 mm, 2.7 μm), 0.1% formic acid aqueous solution (A)-methanol (B) as the mobile phase for gradient elution (0-5 min, 2-7% B; 5-10 min, 7%-10% B; 10-20 min, 10-25% B, 20-45 min, 25-50% B, 45-50 min, 50-95% B, 50-55 min, 95% B), column temperature 35°C, flow rate 0.4 mL / min, injection volume 5 μL, and DAD detection wavelengths set to 210, 230, and 260 nm.

[0020] Furthermore, in the mass detection method, the mass spectrometry conditions are as follows: ESI ion source, data acquisition in positive ion mode, data acquisition range m / z 100-1700, ion source temperature 350°C, capillary voltage 3.5 kV (positive ion), 4.0 kV (negative ion), nebulizer gas pressure 45 psi, drying gas flow rate 11 L / min, sheath gas flow rate 11 L / min, sheath gas temperature 350°C, and fragmentor voltage 140 V.

[0021] The second aspect of the present invention provides a quality control compound of compound isatis root granules obtained based on the quality detection method of compound isatis root granules as described above: (R, S)-gaoetin, adenosine, guanosine, uridine, tryptophan, isovitexin, indigo, indigotin and indigo carmine.

[0022] In a third aspect of the present invention, a quality control method for compound isatis root granules is provided, wherein the contents of uridine, adenosine, and guanosine in the preparation are determined by HPLC-UV method, and the contents of indigo, indirubin, isatin, isovitexin, tryptophan, and (R,S)-goitrin in the preparation are determined by TLC method or HPLC-UV method.

[0023] Furthermore, the method for determining the content of indirubin and (R,S)-gaoitone in the preparation by TLC method comprises the following steps:

[0024] (1) Take 45g of this product, add 150mL of water, ultrasonically treat (power 350W, frequency 53kHz) for 15 minutes, add 75mL of chloroform to extract twice, collect and combine the chloroform layers, add 75mL of 0.5% NaOH solution to the chloroform layer and extract twice, combine the two chloroform layers and evaporate to dryness, add 0.5mL of chloroform to dissolve the residue, and use it as the test solution. Take 1g of the control medicinal material of Isatis indigotica, add 10mL of water, ultrasonically treat for 15 minutes to dissolve, add 10mL of chloroform, extract with the test product in the same way, collect and combine the chloroform layers (lower layer), add 10mL of 0.5% NaOH solution to the chloroform collection solution and extract twice (operation as before), combine the two chloroform layer filtrates and evaporate to dryness, add 0.5mL of chloroform to dissolve the residue, and prepare the Isatis indigotica control medicinal material solution. Take 1g of Radix Isatidis reference medicinal material, add 15mL of water, and sonicate for 15 minutes to dissolve it. Add 15mL of chloroform and prepare Radix Isatidis reference medicinal material solution in the same way as the Folium Isatidis reference medicinal material. Take another reference substance of Indirubin and add chloroform to make a solution containing 1mg per 1mL as the reference solution. According to the thin layer chromatography method (General Rule 0502), take 5μL of each of the three solutions and spot them on the same silica gel GF 254 On the thin layer plate, use cyclohexane-chloroform-acetone (5:4:2V / V / V) as the developing solvent, develop, take out, dry, and inspect visually under sunlight. In the chromatogram of the test sample, spots of the same color will appear at the corresponding positions in the chromatogram of the control medicinal material and the chromatogram of the control drug.

[0025] (2) Take the combined alkali layer under item (1), adjust the pH to 1-2 with hydrochloric acid, add 75 mL of chloroform and extract twice (operation is the same as before), collect the chloroform layers, combine and evaporate to dryness, add 0.5 mL of methanol to dissolve, and use as the test solution. Take the alkali layer under item (1) and prepare the control medicinal material solution in the same way. Take R, S-gaoyichun reference substance and add methanol to make a solution containing 1 mg per 1 mL, which is used as the reference solution. According to the thin layer chromatography method (General Rule 0502), take 5 μL of each of the above three solutions and spot them on the same silica gel GF254 thin layer plate, use n-hexane: ethyl acetate: methanol: ammonia water (6:3.5:0.5:0.1 V / V / V / V) as the developing solvent, develop, remove and dry. Fumigate in iodine cylinder for 2 minutes, and visually inspect under daylight. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions of the chromatogram of the control medicinal material and the chromatogram of the control drug.

[0026] The methods for indigo, isatin, isovitexin and tryptophan refer to the above methods.

[0027] Furthermore, the method for determining the content of uridine, adenosine and guanosine in the preparation by HPLC-UV method comprises the following steps:

[0028] A. Chromatographic Conditions and System Suitability Test: A Diamonsil C18 column (4.6 mm × 250 mm, 5 μm) was used as the chromatographic column; methanol solution was used as mobile phase A, and pure water was used as mobile phase B. Gradient elution was performed (0 min, 3% A; 5 min, 3% A; 15 min, 6% A; 32 min, 7% A; 45 min, 22% A; 50 min, 22% A); column temperature was 25°C; flow rate was 0.8 mL / min; detection wavelength was 254 nm, and acquisition time was 50 min. The number of theoretical plates calculated based on the uridine peak should be no less than 10,000.

[0029] B. Preparation of reference solution: Take appropriate amount of uridine, guanosine and adenosine reference substances respectively, weigh accurately, add pure water to make mixed reference solution containing 0.05, 0.05 and 0.02 mg per 1 mL.

[0030] C. Preparation of test solution: Grind 1 bag of Compound Isatis Root Granules (15 g / bag) and accurately weigh 5.000 g. Pour the mixture into a 10 mL volumetric flask, accurately add 10 mL of pure water, plug the flask, weigh the mass, and ultrasonically treat (power 350 W, frequency 53 kHz) for 30 min. Cool, weigh the mixture, make up the weight loss with water, centrifuge at 14,000 rpm for 10 min, collect the supernatant, filter through a 0.45 μm microporous filter membrane, and obtain the filtrate.

[0031] D. Determination method: Accurately aspirate 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, determine, and record the chromatogram for 50 minutes.

[0032] The advantages of the present invention are:

[0033] 1. The present invention confirms the chemical components contained in the various Chinese herbal medicine slices in the medicinal materials, intermediates, and finished granules of the compound isatis root granules through UHPLC-DAD-Q-TOF / MS, and establishes the correlation of chemical components between the two Chinese herbal medicine slices (isatis root and isatis indigotica) in the prescription, the water extract paste, the flowing paste, and the granules, providing a basis for the quality control of the Chinese herbal medicine slices, the extract, and the granules in the industrial research of the compound isatis root granules.

[0034] 2. The present invention explains from the perspective of mass transfer that the compounds used for quality control of compound isatis root granules are stably present from medicinal materials to intermediates to finished products.

[0035] The first comprehensive quality control of compound isatis granules was carried out using UHPLC-DAD-Q-TOF / MS-based chemical component characterization, TLC-based detection of R,S-gaoichun and indirubin in the preparation, and HPLC-UV-based determination of uridine, adenosine, and guanosine in the preparation.

[0036] 3. This study is the first to perform UHPLC-DAD-Q-TOF / MS analysis on Compound Isatis Root Granules, its raw medicinal materials, and intermediates, and to analyze the transport characteristics of key differential compounds from a mass transfer perspective. This allows for more accurate compound identification and stronger evidence for the selection of quality control compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 TIC plot of the mixed standard based on UHPLC-Q-TOF / MS;

[0038] (A) Mixed standard A solution; (B) Mixed standard B solution; (C) 260 nm chromatogram of mixed standard A solution; (D) 260 nm chromatogram of mixed standard B solution.

[0039] Figure 2 TIC images of Isatis indigotica and Radix Isatidis slices based on UHPLC-Q-TOF / MS;

[0040] Positive ion mode of Radix Isatidis (A), Folium Isatidis (B), water extract paste 260 mm (C), flowing paste (D), and granules (E).

[0041] Figure 3 .PCA diagram of different samples (G-Isatis root, Y-Isatis leaf, S-water extract, L-flowing paste, K-granule, QC-quality control sample).

[0042] Figure 4 .PLSDA diagrams of different samples (G-Isatis root, Y-Isatis leaf, S-water extract, L-flowing paste, K-granules, QC-quality control sample).

[0043] Figure 5 Comparison of the average relative content of compounds in different batches (n=3) (based on peak area quantification by DAD detection); the horizontal axis is the batch, and the vertical axis is the average relative content of each compound (μg / g).

[0044] Figure 6 Comparison of the relative content of compounds in different groups (based on peak area quantification by DAD detection); the horizontal axis represents the different groups, and the vertical axis represents the logarithm of the relative content of each compound in each sample. DETAILED DESCRIPTION

[0045] The specific implementation methods provided by the present invention are described in detail below with reference to the examples.

[0046] Example:

[0047] 1 Experiment and Materials

[0048] 1.1 Preparation of test solution

[0049] 1.1.1 Preparation of test solution of medicinal materials: Randomly take three samples of each batch of Radix Isatidis / Leucophyllae from different batches and grind them into powder. Take 5g of powder, accurately weigh it, and place it in a 50ml conical flask with a stopper. Accurately add 20ml of methanol solution (Leucophyllae: 20% methanol, Radix Isatidis: 40% methanol) to each flask. Ultrasonic treatment (power 500W, frequency 40kHz) for 30min, cool, shake well, and extract at 3000r / min. -1 Centrifuge for 10 min, take the upper layer solution into a 1.5 ml centrifuge tube, and centrifuge at 15000 r min -1 Centrifuge for 10 minutes, let it stand for 10 minutes, and then take the supernatant again at 15000 r·min -1 Centrifuge for 10 minutes to obtain the product.

[0050] 1.1.2 Preparation of test solutions of intermediate water extract paste and flow paste Randomly take water extract paste and flow paste from each batch (three parts from each batch) (the mass is calculated based on the quantity transfer conversion), accurately weigh them, place them in a 50 ml stoppered conical flask, and accurately add 1:4 volume of solvent (water extract paste: 20% methanol, flow paste: water), respectively. Ultrasonic treatment (power 500 W, frequency 40 kHz) for 30 min, cool, shake well, and the extract is stirred at 3000 r·min -1 Centrifuge for 10 min, take the upper layer solution into a 1.5 ml centrifuge tube, and centrifuge at 15000 r min -1 Centrifuge for 10 minutes, let it stand for 10 minutes, and then take the supernatant again at 15000 r / min. -1 Centrifuge for 10 minutes to obtain the product.

[0051] 1.1.3 Preparation of granule test solution 5 g of each batch of granules (three aliquots from each batch) were accurately weighed and placed in a 50 ml stoppered conical flask. 20 ml of water (1:4 extraction) was accurately added to each batch and ultrasonically treated (power 500 W, frequency 40 kHz) for 30 min. The solution was cooled, shaken, and the extract was stirred at 3000 r / min. -1 Centrifuge for 10 min, take the upper layer solution into a 1.5 ml centrifuge tube, and centrifuge at 15000 r min -1 Centrifuge for 10 minutes, let it stand for 10 minutes, and then take the supernatant again at 15000 r / min. -1 Centrifuge for 10 minutes to obtain the product.

[0052] 1.2 Preparation of Reference Solution: Accurately weigh appropriate amounts of indirubin and indigo reference standards, add chloroform to make a solution containing 1 mg per 1 mL, sonicate, and vortex to mix thoroughly. These solutions are stock solutions 1 and 2. Accurately weigh appropriate amounts of leucine, arginine, valine, and isatin reference standards, add 70% ethanol to make a solution containing 1 mg per 1 mL, sonicate, and vortex to mix thoroughly. These solutions are stock solutions 3-6. Accurately weigh (R,S)-epigoitrin and isovitexin, add methanol to make a solution containing 1 mg per 1 mL, sonicate, and vortex to mix thoroughly. These solutions are stock solutions 7-8. Accurately weigh appropriate amounts of L-tryptophan reference standard, add water to make a solution containing 1 mg per 1 mL, sonicate, and vortex to mix thoroughly. This solution is stock solution 9. Accurately weigh an appropriate amount of adenosine and uridine reference substances, add water to make a solution containing 1 mg of each reference substance per mL, sonicate to dissolve, and vortex to mix. These solutions are prepared as stock solutions 10 and 11. Accurately weigh an appropriate amount of guanosine reference substance, add water to make a solution containing 100 μg of each reference substance per mL, sonicate to dissolve, and vortex to mix. This is prepared as stock solution 12.

[0053] Accurately pipette 200 μL each of stock solutions 1, 2, 4, 6-9, place in a 2 ml volumetric flask and mix, then add methanol to prepare a mixed standard solution A with a concentration of 100 μg / mL for each standard.

[0054] Accurately pipette 200 μL each of stock solutions 3, 5, 7, and 10-12, and 20 μL of stock solution 4, place them in a 2 ml volumetric flask and mix them. Add methanol to prepare a mixed standard solution B with a standard concentration of 100 μg / mL (arginine and guanosine concentrations of 10 μg / mL).

[0055] 1.3 Preparation of QC: Accurately pipette 10 μL of each test solution into a 2 mL centrifuge tube, vortex mix, and centrifuge at 15000 r·min. -1 Centrifuge for 10 minutes, let it stand for 10 minutes, and then take the supernatant.

[0056] 1.4 Compound Analysis Library Construction and Data Analysis: The compositional information for each herb in this prescription was obtained from existing literature on the compositional analysis of medicinal materials. This ensured that the compounds included in the database were searchable in the published articles. Thus, the prescription compound database MS_peaks-clusters_final_processed-doudou0911 (Huang Doudou. Chemical Characterization of Isatis Plants and Chemical Evolution of Isatis Indigotica [D], Naval Medical University 2020 PhD Dissertation) was established based on the Agilent defult file. Data analysis was performed using Agilent MassHunter Qualitative Analysis 10.0 software.

[0057] 1.4 Chromatographic conditions: An Agilent Poroshell 120, SB-C18 column (4.6 mm × 150 mm, 2.7 μm) was used with a 0.1% formic acid aqueous solution (A)-methanol (B) as the mobile phase for gradient elution (0-5 min, 2-7% B; 5-10 min, 7%-10% B; 10-20 min, 10-25% B, 20-45 min, 25-50% B, 45-50 min, 50-95% B, 50-55 min, 95% B). The column temperature was 35°C, the flow rate was 0.4 mL / min, the injection volume was 5 μL, and the DAD detection wavelengths were set at 210, 230, and 260 nm.

[0058] 1.5 Mass spectrometry conditions: ESI ion source, data acquisition in positive ion mode. Data acquisition range: m / z 100–1700, ion source temperature: 350°C, capillary voltage: 3.5 kV (positive ion), 4.0 kV (negative ion), nebulizer gas pressure: 45 psi, drying gas flow rate: 11 L / min, sheath gas flow rate: 11 L / min, sheath gas temperature: 350°C, and fragmentor voltage: 140 V.

[0059] 2 Results

[0060] 2.1 Identification of chemical composition

[0061] First, the MS DIAL software was used to analyze the components of each group. In the positive ion mode, the format-converted data of each group and the blank group data were imported into the software, and the adduct ion selection [M+H] was used. + 、[M+NH4] + 、[M+Na] + 、[M+K] +The minimum peak height was set to 5000, the data acquisition time range was set to 0-55 min, the m / z scanning range was set to 100-1500, the QC sample was calibrated, the RT error range was set to 0.15 min, and normalized analysis was performed using the warfarin internal standard (positive ion: m / z 309.1121, RT50.011 min). After deducting the blank, a total of 165,207 ions were screened as candidate ions for further identification.

[0062] The collected UHPLC-Q-TOF / MS data were imported into Agilent MassHunter QualitativeAnalysis 10.0 software. The "Find by Formula" function under the "Target / Suspect Screening" option was selected. The collected data were preliminarily analyzed using the previously obtained formula compound database MS_peaks-clusters_final_processed-doudou0911 (Huang Doudou. Chemical Characterization of Isatis Plants and Chemical Evolution of Isatis [D]. Naval Medical University 2020 Doctoral Dissertation). Based on the baseline intensity, the absolute peak height was set to ≥1500, the mass number matching tolerance was <±5ppm, and the positive ion mode ion species were selected as [M+H]+, [M+NH4]+, [M+Na]+, and [M+K]+ to preliminarily identify the chemical components contained in the various components of Isatis Radix. The preliminarily identified chemical components were then further confirmed using the method. The corresponding compounds can be identified by comparing the retention time and fragment information with those of the reference substances; the compounds without reference substances can be attributed by importing the secondary fragment ion information into the MS FINDER software and using the literature and online databases such as Massbank and Mzcloud.

[0063] 2.2 Multivariate statistical analysis and variance analysis

[0064] The 165,207 ions exported by MSDial were further screened, with the number of data values ​​in the 32 QC samples not equal to 0 set as 100% as the screening criterion. 97,780 ions were eliminated, and the remaining 67,427 ions were imported into the metaboAnalyst website in .csv format. After data normalization, 34,815 ions were screened out using the RSD < 0.5 standard in the QC samples, and then multivariate statistical analysis was performed.

[0065] The corresponding ions of the identified compounds were found in the MSdial data and imported into the metaboAnalyst website in .csv format for differential analysis. t-tests were performed on the SL and LK groups, respectively.

[0066] Table 1 List of differential compounds

[0067]

[0068]

[0069]

[0070]

[0071]

[0072] “-” represents P>0.05; SL refers to the transfer from water extract paste to fluid paste, and LK refers to the transfer from fluid paste to granules.

[0073] The main components of Radix Isatidis include alkaloids, organic acids, lignans, amino acids, nucleosides, and others. Finding exclusive and specific compounds as quality evaluation criteria and mass transfer analysis of these compounds are crucial. The present invention sampled 10 batches of medicinal materials, intermediates, and granules with transfer characteristics according to the actual production transfer coefficients. Based on UHPLC-Q-TOF / MS analysis of the chemical composition differences of the samples, 133 compounds (Table 1) were obtained that showed statistically significant differences in content during the transfer process from the intermediates to the granules. These included alkaloids represented by (R,S)-gaoitin and nucleosides represented by adenosine and uridine, which have antiviral activity; amino acids represented by arginine, leucine, valine, and tryptophan, which have immunomodulatory effects; and flavonoids represented by isovitexin and indole components represented by indigo, indirubin, and isatin, which have antioxidant activity.

[0074] Since arginine, leucine, and valine have high polarity and do not have good separation under the present chromatographic analysis conditions, the relative content changes of the above nine typical compounds ((R,S)-gaoitin, adenosine, guanosine, uridine, tryptophan, isovitexin, indigo, indirubin, and indigo carmine) from medicinal materials to water extract paste to liquid paste to granules were investigated based on the peak areas obtained by DAD.

[0075] from Figure 6It can be seen that indirubin only exists in the leaves of Isatis indigotica, and after being transferred, it is basically undetectable in the intermediates and granules. The contents of the remaining compounds in Radix Isatidis are as follows: (R,S)-gaoipin>indigo>adenosine>uridine>guanosine>L-tryptophan>indigotin>isovitexin; the contents in the leaves of Isatis indigotica are as follows: vitexin>uridine>guanosine>indigo>L-tryptophan>indigotin>(R,S)-gaoipin>adenosine; the contents in the water extract paste are as follows:indigo>uridine>isovitexin>indigotin>(R,S)-gaoipin>guanosine>L-tryptophan>adenosine; the contents in the fluid paste and granules are as follows:uridine>isovitexin>adenosine>guanosine>(R,S)-gaoipin>indigotin>L-tryptophan, and indigo is below the detection limit. In addition, Figure 5 It can be seen from the content values ​​(μg / g) of the compounds that in the process from medicinal materials to water-extracted paste, flowing paste and granules, the content differences of various compounds between batches gradually become smaller, and the content proportions in the flowing paste and granules are highly consistent.

[0076] Figure 6 is the logarithmic comparison of the relative content of each compound in samples from different batches. Figure 5 and Figure 6 It can be seen that the content of each compound in the medicinal materials (Isatis Radix and Isatis Indigotica) varies greatly. It can be seen that the content differences of various compounds between batches gradually decrease during the process from Isatis Radix and Isatis Indigotica to water-extract paste, liquid paste, and granules. Isatis Radix, as the raw material for Isatis Radix extracts and compound preparations, has large quality differences between batches due to the influence of various factors such as growth environment, soil properties, cultivation technology, and production processing. The extraction process of water alcohol precipitation can effectively reduce batch differences and stabilize its quality.

[0077] These results suggest that the compounds uridine, adenosine, and guanosine, which exhibit transport properties, can be used as quality control compounds for Isatis granules due to their high concentrations (approximately 10-40 μg / g in the granules). Future HPLC-UV-based analytical methods should be developed for their content determination. However, the remaining compounds, isatin, isovitexin, tryptophan, and (R,S)-gaoitin, are either present at low concentrations or exhibit significant polarity differences from the three nucleoside components mentioned above. Therefore, alternative methods such as TLC or modified pretreatment methods to establish HPLC-UV-based assays are recommended for content determination.

[0078] 3 Summary

[0079] The chemical components of each Chinese herbal medicine slice in the medicinal materials, intermediates and finished granules of compound isatis root granules were confirmed by UHPLC-DAD-Q-TOF / MS, and the correlation of chemical components between the two Chinese herbal medicine slices (Isatis root and Isatis indigotica) in the prescription and the water extract paste, flow paste and granules was established, providing a basis for the quality control of Chinese herbal medicine slices-extract-granules in the industrialization research of compound isatis root granules.

[0080] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments. Those skilled in the art may make various equivalent modifications or substitutions without departing from the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. A quality inspection method for compound isatis root granules, wherein the compound isatis root granules are made from isatis root and isatis indigotica, characterized in that: The quality inspection method specifically uses UHPLC-DAD-Q-TOF / MS technology to identify the chemical components of the medicinal material slices, intermediates, and finished granules in the compound isatis root granules to determine the quality control compounds of the compound isatis root granules; and comprises the following steps: (A) Preparation of test solution of medicinal material slices: Randomly take Radix Isatidis / Leucophyllae from each batch and grind them into powder. Take 5 g of powder, weigh it accurately, and place it in a 50 ml conical flask with a stopper. Accurately add 20 ml of methanol solution, 20% methanol for Leucophyllae and 40% methanol for Radix Isatidis, respectively. Ultrasonicate for 30 min at 500 W power and 40 kHz frequency. Let it cool, shake well, and spin the extract at 3000 r·min. -1 Centrifuge for 10 min, take the upper layer solution into a 1.5 ml centrifuge tube, and centrifuge at 15000 r·min -1 Centrifuge for 10 min, let it stand for 10 min, and then take the supernatant again at 15000 r·min -1 Centrifuge for 10 min to obtain the product. (B) Preparation of test solutions of intermediate water extract paste and flow paste: Randomly take water extract paste and flow paste from each batch, accurately weigh them, and place them in a 50 ml stoppered conical flask. Add solvents in a ratio of 1:4 by volume, water extract paste: 20% methanol, flow paste: water, respectively. Ultrasonicate for 30 min at a power of 500 W and a frequency of 40 kHz. Cool, shake well, and spin the extract at 3000 r·min. -1 Centrifuge for 10 min, take the upper layer solution into a 1.5 ml centrifuge tube, and centrifuge at 15000 r·min -1 Centrifuge for 10 min, let it stand for 10 min, and then take the supernatant again at 15000 r·min -1 Centrifuge for 10 min to obtain the product. (C) Preparation of granule test solution: 5 g of each batch of granules was accurately weighed and placed in a 50 ml stoppered conical flask. 20 ml of water was accurately added to each batch and extracted at a ratio of 1:

4. Ultrasonic treatment was performed for 30 min at a power of 500 W and a frequency of 40 kHz. The solution was cooled and shaken. The extract was then stirred at 3000 r·min. -1 Centrifuge for 10 min, take the upper layer solution into a 1.5 ml centrifuge tube, and centrifuge at 15000 r·min -1 Centrifuge for 10 min, let it stand for 10 min, and then take the supernatant again at 15000 r·min -1 Centrifuge for 10 min to obtain the product. (D) Preparation of reference solution: Accurately weigh appropriate amounts of indigo carmine and indigo blue reference, add chloroform to make a solution containing 1 mg per 1 ml, sonicate and vortex to mix, and use as stock solutions 1 and 2; Accurately weigh appropriate amounts of leucine, arginine, valine, and indigo red reference, add 70% ethanol to make a solution containing 1 mg per 1 ml, sonicate and vortex to mix, and use as stock solutions 3-6; Accurately weigh RS-gaoichun and isovitexin, add methanol to make a solution containing 1 mg per 1 ml, sonicate and vortex to mix, and use as stock solutions 7-8; Accurately weigh appropriate amounts of L-tryptophan reference, add water to make a solution containing 1 mg per 1 ml, sonicate and vortex to mix, and use as stock solution 9; Accurately weigh appropriate amounts of adenosine and uridine reference, add water to make a solution containing 1 mg per 1 ml, sonicate and vortex to mix, and use as stock solutions 10 and 11; Accurately weigh appropriate amounts of guanosine reference, add water to make a solution containing 1 mg per 1 ml A solution containing 100 μg of the reference substance was dissolved by ultrasonication and vortexed to mix, which was used as the stock solution 12; Accurately pipette 200 μL each of stock solutions 1, 2, 4, and 6-9, place them in a 2 ml volumetric flask, mix them, and add methanol to prepare a mixed standard solution A with a concentration of 100 μg / mL for each standard. Accurately pipette 200 μL each of stock solutions 3, 5, 7, and 10-12, and 20 μL of stock solution 4, place them in a 2 ml volumetric flask, mix, and add methanol to prepare a mixed standard solution B with a concentration of 100 μg / mL for each standard and 10 μg / mL for arginine and guanosine; (E) Preparation of quality control samples: 10 μL of each test solution was accurately pipetted into a 2 mL centrifuge tube, vortexed to mix, and centrifuged at 15000 r·min. -1 Centrifuge for 10 min, let it stand for 10 min, and take the supernatant. (F) Compound analysis library establishment and data analysis: A prescription compound database was established based on Agilent's own defult file; data analysis was performed using Agilent MassHunter Qualitative Analysis 10.0 software; The chromatographic conditions were as follows: Agilent Poroshell 120, SB-C18 column, 4.6 mm × 150 mm, 2.7 μm, with a 0.1% formic acid aqueous solution (A)-methanol (B) as the mobile phase for gradient elution: 0-5 min, 2-7% B; 5-10 min, 7%-10% B; 10-20 min, 10-25% B; 20-45 min, 25-50% B; 45-50 min, 50-95% B; 50-55 min, 95% B; column temperature 35°C, flow rate 0.4 mL / min, injection volume 5 μL, DAD detection wavelengths set at 210, 230, and 260 nm; The mass spectrometry conditions were as follows: ESI ion source, data acquisition in positive ion mode; data acquisition range m / z 100–1700, ion source temperature 350°C, capillary voltage 3.5 kV positive ion, 4.0 kV negative ion, nebulizer gas pressure 45 Psi, drying gas flow rate 11 L / min, sheath gas flow rate 11 L / min, sheath gas temperature 350°C, and fragmentor voltage 140 V.

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