A characteristic spectrum of Wantong Yankang preparation and its construction method and application

Abstract

The present invention discloses a characteristic spectrum of a Wantong Yankang preparation, a construction method thereof, and an application thereof. (1) Sample preparation; (2) High-performance liquid chromatography analysis; (3) Construction of a characteristic spectrum. This method optimizes the characteristic spectrum detection conditions, and adopts a wavelength switching method in the high-performance liquid chromatography analysis to simultaneously determine the 11 components of the characteristic spectrum of Wantong Yankang, thereby realizing multi-component determination and analysis of the characteristic spectrum, greatly shortening the detection cycle, and improving the analysis efficiency. After a methodological investigation, the HPLC characteristic spectrum analysis method is finally determined. This method has the advantages of strong specificity, good precision, repeatability, and stability, and can be used to quantify 11 characteristic components at the same time. It is very suitable for controlling the overall quality of Wantong Yankang and ensuring the quality of the product, and has great application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine detection and quality control, and specifically relates to a method for constructing a characteristic spectrum of a compound Chinese patent medicine Wantong Yankang preparation and its standard characteristic spectrum, as well as the application of the characteristic spectrum in the quality standard and quality control of Wantong Yankang medicine. Background Art

[0002] Acute and chronic pharyngitis is a common disease, often caused by viral and / or bacterial infections. Worldwide, approximately 615 million cases occur annually across all age groups. To date, clinical treatment strategies for pharyngitis primarily include antibiotics, antiviral drugs, and glucocorticoids to alleviate pain, but these drugs are associated with toxicity and drug resistance.

[0003] Wantong Yankang is a Chinese patent medicine composed of Radix Scrophulariae and Radix Sarcosae. Its tablets are included in the Chinese Pharmacopoeia (2020 edition) Volume 1, and have the effects of dispelling wind and clearing heat, detoxifying and reducing swelling. It is used for pharyngeal swelling, gingival swelling, sore and furuncle swelling caused by exogenous wind-heat; acute and chronic pharyngitis, tonsillitis, gingivitis, sore and furuncle with the above symptoms. However, since Chinese patent medicines are made up of multiple medicinal materials, there are many deficiencies in the quality control of Wantong Yankang: (1) The Pharmacopoeia of the People's Republic of China only includes the method of determining the content of isoflurane by high performance liquid chromatography under the item [Content Assay] for Wantong Yankang Tablets. However, Wantong Yankang is made up of Radix Scrophulariae and Radix Sarcosae (1:1) It is not enough to evaluate the quality and efficacy of the drug based solely on the content of isoflurane in Radix Sarcosae. It is necessary to further improve the quality evaluation standards and establish more complete quality standards. Furthermore, the current standards for this drug issued by the State Food and Drug Administration also have deficiencies. A specific content determination method for rosmarinic acid, a chemical component of the prescription drug Radix Sarcosinae, has not been established, and a content determination method for the chemical components of the prescription drug Radix Scrophulariae is missing. The quality control level is low, which is not conducive to the monitoring of the production process and product quality. (2) There is a lack of characteristic fingerprint identification of the drug composition, and there is a lack of quality control and standards for its main active ingredients, which cannot meet the needs of the modernization of traditional Chinese medicine. It also fails to accurately reflect changes in production conditions and standard management. Therefore, it is urgent to construct a characteristic spectrum and quality control system for Wantong Yankang, comprehensively improve quality standards and quality control levels, and improve the monitoring of production processes and product quality. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for constructing a characteristic spectrum of Wantong Yankang preparation and its characteristic spectrum, which can effectively control the quality of the medicine and monitor the production process to solve the problems raised in the background technology.

[0005] The present invention is achieved through the following technical solutions:

[0006] A method for constructing a standard characteristic spectrum of a Wantong Yankang preparation, the method comprising the following steps:

[0007] S1: Preparation of the test sample Wantong Yankang preparation solution, the reference solution, and the negative control Scrophularia ningpoensis and Sarcandra scutellariae solutions;

[0008] S2: The test sample Wantong Yankang preparation solution, the reference substance solution and the negative control bitter Scrophularia and Sarcandra solution were respectively subjected to high performance liquid chromatography to obtain liquid chromatograms of the test sample Wantong Yankang preparation, the reference substance and the negative control bitter Scrophularia and Sarcandra;

[0009] The reference substances in the reference solution include: neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isofraquinone, verbascoside, isovarascoside, apigenin-7-O glucuronic acid, rosmarinic acid, scrophulariaceae IA and scrophulariaceae IA;

[0010] The high performance liquid chromatography is carried out using a high performance liquid chromatograph, C 18 Chromatographic column, mobile phase A is acetonitrile, mobile phase B is 0.1% phosphoric acid solution or 0.1% formic acid solution by volume, elution mode is gradient elution, and the gradient elution program is:

[0011] From 0 to 20 minutes, the volume fraction of the mobile phase A is increased from 15% to 20%;

[0012] 20-35 minutes, the volume fraction of the mobile phase A is increased from 20% to 30%;

[0013] 35-50 minutes, the volume fraction of the mobile phase A is increased from 30% to 60%;

[0014] The retention time of the liquid phase spectrum of the test sample Wantong Yankang preparation solution is compared with the retention time of the liquid phase spectrum of the reference solution to obtain the characteristic spectrum of the Wantong Yankang preparation.

[0015] Preferably, the Wantong Yankang tablets or Wantong Yankang capsules.

[0016] Preferably, the HPLC conditions are: column temperature 30±5°C; flow rate 0.8-1.1 mL / min; detection wavelengths 264±2 nm and 324-343 nm.

[0017] The test sample Wantong Yankang preparation solution, the reference solution and the negative sample Scrophularia scabra and Sarcandra scutellariae solution

[0018] Preferably, the test sample, Wantong Yankang preparation solution, is obtained by removing the coating or capsule shell from 10-20 tablets / pills of Wantong Yankang preparation, grinding the mixture to a fine powder, accurately weighing 0.5-1.0 g, placing the mixture in a stoppered conical flask, and accurately adding 25-50 ml of 50-75% methanol, weighing the mixture. Ultrasonicate the mixture at a power of 450-500 W, a frequency of 35-40 kHz, and a time of 30-60 minutes. Allow to cool, weigh the mixture again, and make up the loss with 50-75% methanol. Shake the mixture well, filter, and collect the filtrate to obtain the solution.

[0019] The preparation process of the reference solution is as follows: accurately weigh appropriate amounts of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isofraquinone, verbascoside, isovarboside, apigenin-7-O-glucuronic acid, rosmarinic acid, scrophulariaceae IA, and scrophulariaceae IA reference substances, and add 50-75% methanol to prepare a mixed reference solution containing 20-50 μg of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, and caffeic acid, 50-100 μg of isofraquinone, 200-250 μg of verbascoside, rosmarinic acid, isovarboside, and apigenin-7-O-glucuronic acid, and 100-800 μg of scrophulariaceae IA and scrophulariaceae IA per 1 ml;

[0020] The preparation process of the negative Scrophularia ningpoensis and Radix Sarcosae solution is as follows: ① Preparation of Scrophularia ningpoensis solution: accurately weigh 0.8-1.0 g of Scrophularia ningpoensis medicinal material powder, accurately add 25 or 50 ml of 75-50% methanol solution, and weigh the weight; ultrasonic extraction for 30-60 min with a power of 450-500 W and a frequency of 35-40 kHz, let cool, make up the lost weight with 75-50% methanol, filter, and take the filtrate; Preparation of Radix Sarcosae solution: accurately weigh 0.8-1.0 g of Radix Sarcosae medicinal material powder, accurately add 25 or 50 ml of 75-50% methanol solution, and weigh the weight; ultrasonic extraction for 30-60 min with a power of 450-500 W and a frequency of 35-40 kHz, let cool, make up the lost weight with 75-50% methanol, filter, and take the filtrate.

[0021] Preferably, the characteristic spectrum of the Wantong Yankang preparation has a total of 11 chromatographic peaks, namely peak 1-neochlorogenic acid, peak 2-chlorogenic acid, peak 3-cryptochlorogenic acid, peak 4-caffeic acid, peak 5-isofraxinidin, peak 6-verascoside, peak 7-isoverascoside, peak 8-apigenin-7-O-glucuronic acid, peak 9-rosmarinic acid, peak 10-scrophulariaceae glycoside IB, and peak 11-scrophulariaceae glycoside IA.

[0022] Preferably, the identification and attribution of chromatographic peaks are mainly detected by UFLC-Q-TOF-MS / MS liquid chromatography-mass spectrometry, and the chromatographic conditions of liquid chromatography-mass spectrometry analysis are: the chromatographic column is C 18Chromatographic column; acetonitrile (A)-0.1% formic acid / acetic acid (B) as mobile phase, gradient elution; flow rate: 0.8~1.1ml / min; and use a diverter valve to adjust the flow rate entering the mass spectrometer to 0.2~0.3ml / min; column temperature 30℃, injection volume 5~10μl; mass spectrometry conditions ESI electrospray ion source, positive and negative ion modes, ion spray voltage: positive mode 5500V, negative mode -4500V; ion source parameters: spray gas 50psi; auxiliary heating gas 50psi; ion source temperature 500℃; curtain gas 35psi; collision gas pressure 10psi, primary mass spectrometry parent ion scan range: m / z 100~2000, collision activation scan is 15.0, and dynamic background subtraction is turned on.

[0023] The present invention also provides the application of the characteristic spectrum constructed above in the evaluation and control of the quality of Wantong Yankang preparation products.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. A method for determining the characteristic spectrum of Wantong Yankang preparations was established for the first time. Based on the structural characteristics and physicochemical properties of the active ingredients in Wantong Yankang preparations, analytical conditions such as sample preparation, mobile phase, detection wavelength, elution procedure, column temperature, and flow rate were screened and optimized, completing a systematic methodological validation. The resulting Wantong Yankang characteristic spectrum exhibited high resolution and excellent peak shape. Each characteristic chromatographic peak achieved good baseline separation, exhibited good stability, and exhibited a high number of characteristic peaks. This method provides a comprehensive, accurate, and intuitive assessment of the quality of Wantong Yankang preparations and is suitable for product quality control.

[0026] 2. The present invention adopts HPLC wavelength switching method to simultaneously identify 11 components of the characteristic spectrum and determine their contents in Wantong Yankang preparation, thereby achieving comprehensive evaluation and control of the quality of Wantong Yankang preparation.

[0027] 3. The characteristic spectrum of the Wantong Yankang preparation established by the present invention overcomes the characteristics of the current standards with a single control index, low specificity, and low quality standard level. The characteristic spectrum of the Wantong Yankang preparation established has a total of 11 chromatographic peaks, namely Peak 1 (neochlorogenic acid), Peak 2 (chlorogenic acid), Peak 3 (cryptochlorogenic acid), Peak 4 (caffeic acid), Peak 5 (isofraxinidin), Peak 6 (verbascoside), Peak 7 (isoverbascoside), Peak 8 (apigenin-7-O-glucuronic acid), Peak 9 (rosmarinic acid), Peak 10 (scrophulariaceae IB), Peak 11 (scrophulariaceae IA). There are 6 characteristic components belonging to the medicinal material of Sarcandra scabra and 5 characteristic components belonging to the medicinal material of Scrophulariaceae. This can comprehensively evaluate the quality of the Wantong Yankang preparation and effectively ensure the quality of the finished product. At the same time, it can serve as an internal control standard for large-scale production enterprises and an evaluation basis for the consistency of the efficacy of Wantong Yankang tablets, providing a guarantee for the safe, effective, uniform and stable standardized production of the Wantong Yankang preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The UV absorption of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isofraquinone, verbascoside, isovarascoside, apigenin-7-O glucuronic acid, rosmarinic acid, scrophulariaceae IA and scrophulariaceae IA;

[0029] Figure 2 This is a comparison of the similarity of HPLC characteristic spectra of 10 batches of Wantong Yankang tablets. R: control characteristic spectrum; S1-S10: batch numbers 22052201, 22071901, 22101002, 22110902, 23010303, 23011802, 23020302, 22101802, 21102701, 22010701;

[0030] Figure 3 It is the common pattern of the characteristic spectrum of Wantong Yankang tablets (reference characteristic spectrum);

[0031] Figure 4 This is the HPLC chart of the reference medicinal material of Sarcandra scabra (Batch No. 230413);

[0032] Figure 5 This is the HPLC chart of the control medicinal material Scrophularia purpurogenum (Batch No. 220830);

[0033] Figure 6 This is the HPLC characteristic spectrum of Wantong Yankang Tablets;

[0034] Figure 7This is the common peak attribution diagram of the characteristic spectrum of Wantong Yankang tablets, A: mixed reference substance; B: Sarcandra scutellariae; C: Scrophulariaceae; D: Wantong Yankang tablets, Peak 1: neochlorogenic acid, Peak 2: chlorogenic acid, Peak 3: cryptochlorogenic acid, Peak 4: Caffeic acid, Peak 5: Isofraxin, Peak 6: Verbascoside, Peak 7: Isoverbascoside, Peak 8: Apigenin-7-O-glucuronic acid, Peak 9: Rosmarinic acid, Peak 10: Scrophulariaceae IB, Peak 11: Scrophulariaceae IA;

[0035] Figure 8 This is the characteristic spectrum of Wantong Yankang Capsules;

[0036] Figure 9 This is the total ion current diagram of Wantong Yankang tablets; DETAILED DESCRIPTION

[0037] The present invention is further described below by way of examples, but is not intended to limit the present invention.

[0038] 1. Experimental Preparation

[0039] 1. Instruments and test drugs

[0040] 1.1 Instrument

[0041] An electronic analytical balance (MS205DU, Mettler Toledo, Switzerland) with a 1 / 100,000 ppm ion flowmeter was used; an ultrapure water system (Simplicity, Millipore, USA) was used; a digital ultrasonic cleaner (KQ500DE, Kunshan Ultrasonic Instrument Co., Ltd.) was used; an Ultimate 3000 high-performance liquid chromatograph (Dionex, USA, equipped with an LPG-3400SD quaternary pump, an SRD-3600 degasser, a WPS-3000SL autosampler, a TCC3000-RS column oven, a DAD detector, and Chromeleon 7.0 data processing software; instrument number: 418-6) was used; and a UFLC-Q-TOF-MS / MS liquid chromatography-mass spectrometry system (UFLC XR ultra-high-performance liquid chromatograph, Shimadzu Corporation, Japan) was used; and a Triple TOF™ 5600+ liquid chromatography-mass spectrometry system (AB Sciex, USA) was used.

[0042] Chromatographic column: 1.C 18 Column - Manufacturer: Welch Specifications: UltimateXB-C18, 4.6*250, 5μm, SN: 60230803750

[0043] 2.C 18 Column - Manufacturer: ZORBAX Specification: Eclipse plus C18, 4.6*250, 5μm, SN: USUXA13448

[0044] 3.C 18Column - Manufacturer: Thermo Specifications: Hypersil gold, 4.6*250, 5μm, SN: 20260745

[0045] 1.2 Drug testing

[0046] Experimental reagents:

[0047] Methanol (Manufacturer: Fisher; Batch No.: F23N5N202; Purity: HPLC)

[0048] Liquid chromatography reagents:

[0049] Formic acid (manufacturer: Sigma; batch number: BCBM4407; purity: LC-MS, ≥99%);

[0050] Acetonitrile (Manufacturer: Fisher; Batch No.: F23N5N202; Purity: HPLC)

[0051] 1.3 Sample

[0052] Wantong Yankang tablets (capsules), Sarcandra scabra and Scrophularia ningpoensis were provided by Guangxi Wantong Pharmaceutical Co., Ltd., as shown in Table 1; the reference substances are shown in Table 2.

[0053] Table 1 Wantong Yankang sample situation

[0054]

[0055] 1.4 Reference substances

[0056] Table 2 Reference substance information

[0057]

[0058] Example 1

[0059] 1. Construction of the characteristic spectrum of Wantong Yankang tablets

[0060] 1.1 Preparation of test sample, reference sample and reference medicinal material solutions

[0061] Preparation of reference solution: Accurately weigh appropriate amounts of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isofraquinone, verbascoside, isovarboside, apigenin-7-O-glucuronic acid, rosmarinic acid, scroscoside IA, and scroscoside IB reference substances, and add 50% methanol to prepare a mixed reference solution containing 20 μg of neochlorogenic acid, 20 μg of chlorogenic acid, 20 μg of cryptochlorogenic acid, 20 μg of caffeic acid, 50 μg of isofraquinone, 100 μg of verbascoside, 200 μg of isovarboside, 200 μg of apigenin-7-O-glucuronic acid, 100 μg of rosmarinic acid, 800 μg of scroscoside IA, and 500 μg of scroscoside IB per 1 ml.

[0062] Preparation of test solution: Take 20 tablets of this product, remove the coating, accurately weigh, grind, take 0.6g (film-coated tablet), accurately weigh, place in a stoppered conical flask, accurately add 25ml of 50% methanol, weigh the weight, ultrasonically treat (power 500W 45kHz) for 30 minutes, let cool, weigh again, make up the lost weight with 50% methanol, shake well, filter, and take the filtrate.

[0063] Preparation of control medicinal material solutions: ① Preparation of the test solution of Scrophularia purpurogenum: Accurately weigh 0.8 g of the Scrophularia purpurogenum powder listed in the table above, accurately add 25 mL of 60% methanol solution, and weigh the weight; ultrasonically extract for 1 hour (power 500W 45kHz), cool, make up the loss with 60% methanol, filter, and obtain the filtrate. ② Preparation of the test solution of Sarcandra glabra: Accurately weigh 1 g of the Sarcandra glabra powder listed in the table above, accurately add 25 mL of 75% methanol solution, and weigh the weight; ultrasonically extract for 1 hour (power 500W 45kHz), cool, make up the loss with 75% methanol, filter, and obtain the filtrate.

[0064] Preparation of medicinal materials and test solution: Take 10 batches of Wantong Yankang tablets and reference substance, Sarcandra medicinal materials, and Scrophularia ningpoensis reference medicinal materials and prepare them according to the above-mentioned test solution preparation method to obtain the corresponding test solution.

[0065] 1.2 Optimization and selection of chromatographic methods

[0066] Mobile Phase Selection: The 2020 edition of the Chinese Pharmacopoeia (Volume 1) specifies that the HPLC method for determining the isofraxidin content in Sarcandra serrata (Symphora serrata) and Sarcandra serrata tablets uses acetonitrile-0.1% phosphoric acid (20:80 v / v) as the mobile phase. The HPLC method for determining the isofraxidin content in Sarcandra serrata extract uses acetonitrile-0.1% formic acid (20:80 v / v) as the mobile phase. Different mobile phases and elution gradient conditions were compared and screened using acetonitrile-0.1% phosphoric acid and acetonitrile-0.1% formic acid under the same elution gradient (see Table 3 below).

[0067] Table 3 Optimization of mobile phase elution gradient conditions

[0068]

[0069] After repeated tests and comparisons, and considering that phosphoric acid solution is not suitable for full-component detection of this product using UFLC-Q-TOF-MS / MS, acetonitrile-0.1% formic acid was finally selected as the mobile phase. The optimized chromatographic conditions for the determination of Wantong Yankang tablets are determined as follows:

[0070] Table 4 Mobile phase elution gradient (acetonitrile-0.1% formic acid)

[0071]

[0072] Detection wavelength: Use the above elution procedure to detect the Wantong Yankang tablets sample, record the chromatogram, and observe the absorption of the chromatogram at the full UV wavelength: the maximum absorption wavelengths of 9 substances, including neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isofraquinone, verbascoside, isoverascoside, apigenin-7-O glucuronic acid and rosmarinic acid, are concentrated around 340nm, while the maximum absorption wavelength of scrophulariaceae IA and scrophulariaceae glycoside IB is 264nm. It can be seen that at 264nm and 340nm, as much component information as possible (such as Figure 1 As shown), through analytical screening, 340 nm and 264 nm were selected as the detection wavelengths of the characteristic spectrum method.

[0073] Selection of chromatographic column: With other chromatographic conditions unchanged, three chromatographic columns from different manufacturers, Welch Altimate XB-C18 (4.6×250mm, 5μm), Agilent ZORBAX Eclipse Plus C18 (4.6×250mm, 5μm), and ThermoHypersil gold (4.6×250mm, 5μm), were investigated. The peak shapes of each chromatographic peak were good, and basic separation could be achieved with a resolution of more than 1.2. Therefore, the C18 column was selected for characteristic spectrum detection.

[0074] Determination of characteristic chromatographic conditions: Octadecylsilane bonded silica gel was used as filler (Welch Ultimate XB-C 18 Column: 4.6 × 250 mm, 5 μm; mobile phase A: acetonitrile, mobile phase B: 0.1% formic acid solution, gradient elution according to Table 4; detection wavelengths: 340 nm and 264 nm; column temperature: 30°C; flow rate: 1 mL / min. Detection time: 50 minutes, with wavelength switching from 340 nm to 264 nm at 35 minutes; column temperature: 30°C; flow rate: 1 mL / min.

[0075] 1.3 Methodological Research

[0076] Methodological study on the optimized HPLC chromatographic conditions of Wantong Yankang tablets

[0077] ①Specificity test

[0078] The blank solvent was a 50% methanol solution, and a blank solvent chromatogram was obtained by injection. Wantong Yankang tablets (Batch No. 22101002) were analyzed according to the method described in Example 1 for the preparation of the test solution, using the same chromatographic conditions. The results showed that the identification of the 11 common chromatographic peaks was not affected by factors such as solvent and excipients, demonstrating good specificity.

[0079] ②Precision test

[0080] The same batch of Wantong Yankang tablets (batch number: 22101002) was accurately weighed and operated according to the method under the test solution preparation item in Example 1. Six consecutive injections were made on the high performance liquid chromatograph, and the chromatogram was recorded. Among them, because the IA peak of scrophulariaceae has good stability, high separation, and large peak area, and the transfer rate is high and the transferability is stable during the production process, scrophulariaceae IA was selected as the reference peak, and the relative retention time of the reference peak was compared with that of each peak. However, since the position of the IA peak is relatively late and belongs to scrophulariaceae, and the test sample is composed of scrophulariaceae and scrophulariaceae, isoflurane and rosmarinic acid belonging to scrophulariaceae were selected as reference peaks. In addition, the two compound reference substances are widely available, easy to obtain, stable, and have high separation. The results show that the RSD% of the relative retention values ​​of each characteristic peak is less than 1% (see Table 5), indicating that the method has good precision and meets the requirements of the characteristic spectrum.

[0081] Table 5 Relative retention value results of the precision of each characteristic peak in the characteristic spectrum

[0082]

[0083]

[0084] ③Stability test

[0085] The same batch of Wantong Yankang tablets (batch number: 22101002) was accurately weighed and operated according to the method under the test solution preparation item in Example 1. Samples were injected at 0, 12, 24, and 48 hours, the chromatograms were recorded, and the relative retention values ​​of each component in its characteristic spectrum were calculated (scrophulariaceae glycoside IA was the reference peak). The results are shown in the following table (Table 6): The RSD values ​​of the relative retention values ​​of each characteristic peak were all less than 3.00%, indicating that the Wantong Yankang tablets test solution and the mixed reference solution had good stability within 48 hours.

[0086] Table 6 Relative retention value results of the stability of each characteristic peak in the characteristic spectrum

[0087]

[0088] ④Repeatability test

[0089] The same batch of Wantong Yankang tablets (batch number: 22101002) was accurately weighed in 6 parallel portions and operated according to the method under the test solution preparation item in Example 1. The samples were injected according to the above chromatographic conditions, the chromatograms were recorded, and the relative retention values ​​of each component in the characteristic spectrum were calculated (scrophulariaceae glycoside IA was the reference peak). The results are shown in Table 7. The RSD values ​​of the relative retention values ​​of each characteristic peak were less than 1.00%, indicating that the method had good repeatability.

[0090] Table 7 Relative retention value results of each characteristic peak repeatability in the characteristic spectrum

[0091]

[0092] 1.4 Determination of common peaks

[0093] The 10 batches of Wantong Yankang tablets were subjected to HPLC analysis according to the chromatographic conditions under the "Preparation of Test Solution" and the HPLC chromatograms were recorded. The chromatograms of the 10 batches of Wantong Yankang tablets were analyzed, and 11 chromatographic peaks were stably reproduced. Combined with the mixed reference substance, Sarcandra scutellariae, Scrophularia ningpoensis single medicinal materials and literature reports, the common peaks of the characteristic spectra were identified and attributed. The characteristic peaks attributed to Sarcandra scutellariae are peaks 1, 2, 3, 4, 5, and 6 (such as Figure 4 The characteristic peaks attributed to Scrophularia odorata are peaks 7, 8, 9, 10, and 11 (as shown in Figure 5 shown).

[0094] 1.5 Mass Spectrometry Confirmation of the Common Peak Molecular Structure of the Characteristic Spectrum of Wantong Yankang Tablets

[0095] The UFLC-Q-TOF-MS / MS technology was used to detect the mixed reference solution, Wantong Yankang tablet test solution, and the medicinal materials of Sarcandra suspensa and Scrophularia ningpoensis. Combined with the molecular ion peaks and pyrolysis fragment information of mass spectrometry, the components corresponding to the chromatographic peaks were confirmed to be: peak 1 (neochlorogenic acid), peak 2 (chlorogenic acid), peak 3 (cryptochlorogenic acid), peak 4 (caffeic acid), peak 5 (isofraxinidin), peak 6 (verbascoside), peak 7 (isoverbascoside), peak 8 (apigenin-7-O-glucuronic acid), peak 9 (scrophulariaceae IB), and peak 11 (scrophulariaceae IA).

[0096] 1.5 Construction of feature maps

[0097] During the experiment, 10 batches of Wantong Yankang tablets were tested. Their batch numbers were S1-S10: 22052201, 22071901, 22101002, 22110902, 23010303, 23011802, 23020302, 22101802, 21102701, and 22010701. Ten test solutions were prepared according to the method described in "Preparation of Test Solution." Chromatograms for these 10 batches of Wantong Yankang tablets were obtained. The similarity evaluation results are shown in Table 8. The similarity coefficients for all 10 batches of Wantong Yankang tablets were greater than 0.970, indicating good correlation between the 10 batches and demonstrating a stable production process and good product uniformity.

[0098] Table 8 Similarity evaluation results of 10 batches of Wantong Yankang tablets

[0099]

[0100] The 2012 version of the "Chinese Herbal Medicine Chromatographic Fingerprint Similarity Evaluation System" was used to perform pattern recognition on 10 batches of Wantong Yankang tablets. The common pattern of the HPLC characteristic spectrum of Wantong Yankang tablets was obtained by the average method ( Figure 3 ), establish a control feature map (such as Figure 3 The result is as shown in Figure 2 As shown, from left to right, they are characteristic peaks 1 to 11; R is the control characteristic spectrum; S1 to S10 are the characteristic spectra of batch numbers 22052201, 22071901, 22101002, 22110902, 23010303, 23011802, 23020302, 22101802, 21102701, and 22010701, respectively. The results demonstrate that the production process of Wantong Yankang is stable and the product uniformity is good.

[0101] Example 2

[0102] Wantong Yankang Capsules are another dosage form of Wantong Yankang preparations. There is no difference between Wantong Yankang Capsules and Wantong Yankang Tablets in terms of prescription composition, preparation process, etc. Both are composed of Scrophularia ningpoensis and Sarcandra chinensis, so the characteristic spectrum of Wantong Yankang Tablets can also be applied to Wantong Yankang Capsules.

[0103] 1Construction of characteristic spectrum of Wantong Yankang Capsules

[0104] 1.1 Preparation of reference solution: Accurately weigh appropriate amounts of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isofraquinone, verbascoside, isovarbascoside, apigenin-7-O-glucuronic acid, rosmarinic acid, scrophulariaceae IA, and scrophulariaceae IA reference substances, and add 50% methanol to prepare a mixed reference solution containing 20 μg of neochlorogenic acid, 20 μg of chlorogenic acid, 20 μg of cryptochlorogenic acid, 20 μg of caffeic acid, 50 μg of isofraquinone, 100 μg of verbascoside, 200 μg of isovarbascoside, 200 μg of apigenin-7-O-glucuronic acid, 100 μg of rosmarinic acid, 800 μg of scrophulariaceae IA, and 500 μg of scrophulariaceae IA per ml.

[0105] 1.2 Preparation of test solution: Take the contents of Wantong Yankang Capsules, about 0.6 g, accurately weigh, place in a stoppered conical flask, accurately add 10 ml of methanol, weigh the weight, ultrasonically treat (power 500w, frequency 40kHz) for 30 minutes, let cool, weigh again, make up the lost weight with methanol, shake well, filter through a 0.45μm filter membrane, and take the filtrate.

[0106] 1.3 Preparation of control medicinal material solutions: ① Preparation of the test solution of Radix Scrophulariae: Accurately weigh 0.8 g of Radix Scrophulariae powder, accurately add 25 mL of 60% methanol solution, and weigh the weight; ultrasonically extract for 1 hour (power 500W 45kHz), let cool, make up the lost weight with 60% methanol, filter, and take the filtrate. ② Preparation of the test solution of Radix Sarcandrae: Accurately weigh 1 g of Radix Sarcandrae powder, accurately add 25 mL of 75% methanol solution, and weigh the weight; ultrasonically extract for 1 hour (power 500W 45kHz), let cool, make up the lost weight with 75% methanol, filter, and take the filtrate.

[0107] 1.4 Preparation of medicinal materials and test solution: Take 10 batches of Wantong Yankang capsule finished product and reference substance, Sarcandra scutellariae medicinal material, and Scrophularia ningpoensis reference medicinal material and prepare them according to the above-mentioned test solution preparation method to obtain the corresponding test solution.

[0108] 1.5 High performance liquid chromatography analysis: Chromatographic conditions: Welch UltimateXB-C column 18 Column size: 4.6 mm × 250 mm × 5 μm; mobile phase A: acetonitrile, mobile phase B: 0.1% formic acid solution, gradient elution according to Table 1; detection wavelengths: 340 nm and 264 nm; column temperature: 30°C; flow rate: 1 mL / min. Detection time: 50 minutes, with wavelength switching from 340 nm to 264 nm at 35 minutes; column temperature: 30°C; flow rate: 1 mL / min.

[0109] 1.6 Determination of common peaks:

[0110] The test solutions of 10 batches of Wantong Yankang Capsules were subjected to HPLC analysis according to the chromatographic conditions under "Preparation of test solution", and the HPLC chromatograms were recorded. The chromatograms of the 10 batches of Wantong Yankang Capsules were analyzed, and 11 chromatographic peaks were stably reproduced.

[0111] The mixed reference solution, Wantong Yankang capsule test solution, and Radix Sarcosae Radix and Radix Scrophulariae Radix solutions were tested. The common peaks of the characteristic spectra were identified and attributed by combining the analysis of the common peaks with the reference substance, reference medicinal material, UV spectrum comparison and literature search. Finally, the characteristic spectrum of Wantong Yankang capsule was determined as follows: Figure 8 shown.

[0112] Example 3

[0113] Determination of content in Wantong Yankang tablets (capsules)

[0114] This study investigated the characteristic spectrum methodology of the Wantong Yankang preparation. The method demonstrated excellent stability, high precision, and reproducibility, enabling comprehensive and objective evaluation of the quality of Wantong Yankang. The aforementioned chromatographic conditions were used for content determination. Ten batches of Wantong Yankang tablets and four batches of Wantong Yankang capsules produced by Guangxi Wantong Pharmaceutical Co., Ltd. were prepared according to the method described under "Preparation of Test Solution," and a reference sample was prepared according to the method described under "Preparation of Reference Solution." The aforementioned chromatographic conditions were used for content determination, and the contents of 11 characteristic peaks were calculated for the 14 batches of Wantong Yankang tablets and capsules. The results, shown in Tables 9 and 10, demonstrate that the characteristic spectrum of Wantong Yankang tablets can be used for content determination of characteristic peaks.

[0115] Table 9 Determination results of the content of each characteristic component in 10 batches of Wantong Yankang tablets

[0116]

[0117]

[0118] Table 10 Determination results of the content of each characteristic component in 4 batches of Wantong Yankang Capsules

[0119]

[0120]

[0121] Example 4

[0122] Confirmation of Mass Spectrum Peaks of Wantong Yankang Tablets (Capsules)

[0123] In the above investigation of the characteristic spectrum method of Wantong Yankang preparation, the common peaks of the characteristic spectrum were identified and attributed (such as Figure 6 and 7 The test sample was prepared according to the method under "Preparation of test solution", and the reference sample was prepared according to the method under "Preparation of reference solution". The following mass spectrometry conditions were used for LC-MS detection and analysis. The results are shown in the figure. Figure 9 shown.

[0124] LC-MS analysis: ① Chromatographic conditions: Welch UltimateXB-C column 18 , 4.6mm×250mm×5μm; acetonitrile (A)-0.1% formic acid water (B) as the mobile phase, gradient elution (as shown in the table below); flow rate: 1ml / min; and use a diverter valve to adjust the flow rate into the mass spectrometer to 0.3ml / min; column temperature 30℃, injection volume 10μl; elution program is as follows: mobile phase elution gradient

[0125]

[0126] ②Mass spectrometry conditions

[0127] ESI electrospray ionization source, positive and negative ion modes, ion spray voltage: 5500 V in positive mode, -4500 V in negative mode; ion source parameters: nebulizer gas 50 psi; auxiliary heater gas 50 psi; ion source temperature 500°C; curtain gas 35 psi; collision gas pressure 10 psi; primary mass spectrometry precursor ion scan range: m / z 100–2000. Collision activation scan was 15.0, and dynamic background subtraction was enabled.

[0128] The above experimental results show that the characteristic spectrum detection method provided by the present invention has good stability, high precision, good repeatability, and can comprehensively and objectively evaluate the quality of Wantong Yankang, which is of great significance for ensuring clinical efficacy.

[0129] The above embodiments are merely exemplary embodiments of the present invention and are not intended to limit the scope of the present invention. The scope of protection of the present invention is defined by the claims. Those skilled in the art may make various modifications or equivalent substitutions to the present invention within the spirit and scope of protection of the present invention, and such modifications or equivalent substitutions shall also be deemed to fall within the scope of protection of the present invention.

Claims

1. A method for constructing a characteristic spectrum of a Wantong Yankang preparation, characterized in that: The following steps are involved: S1: Preparation of the test sample Wantong Yankang preparation solution, the reference solution, and the negative control Scrophularia ningpoensis and Sarcandra scutellariae solutions; S2: The test sample Wantong Yankang preparation solution, the reference solution and the negative bitter Scrophularia and Sarcandra solution were respectively subjected to high performance liquid chromatography to obtain liquid chromatography of the test sample Wantong Yankang preparation, the reference solution and the negative bitter Scrophularia and Sarcandra; The reference substances in the reference solution include: neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isoflurane, verbascoside, iso-verascoside, apigenin-7- O glucuronic acid, rosmarinic acid, scrophulariaceae IA and scrophulariaceae IB; The high performance liquid chromatography is carried out by using a high performance liquid chromatograph, C 18 Chromatographic column, mobile phase A is acetonitrile, mobile phase B is 0.1% by volume phosphoric acid solution or 0.1% by volume formic acid solution, elution mode is gradient elution, and the gradient elution program is: From 0 to 20 minutes, the volume fraction of the mobile phase A increased from 15% to 20%; From 20 to 35 minutes, the volume fraction of the mobile phase A is increased from 20% to 30%; 35-50 minutes, the volume fraction of the mobile phase A is increased from 30% to 60%; Comparing the retention time of the liquid phase spectrum of the test sample Wantong Yankang preparation solution with the liquid phase spectrum of the reference solution to obtain the characteristic spectrum of the Wantong Yankang preparation; The HPLC conditions are as follows: column temperature 30±5°C; flow rate 0.8~1.1 mL / min; detection wavelengths 264±2 nm and 324~343 nm; The preparation methods of the test sample Wantong Yankang preparation solution, the reference sample solution, and the negative sample solutions of Scrophularia ningpoensis and Sarcandra scutellariae are as follows: The process for obtaining the test sample Wantong Yankang preparation solution is as follows: take 10-20 tablets / capsules of Wantong Yankang preparation, remove the coating or capsule shell, grind finely, accurately weigh 0.5-1.0g, place in a stoppered conical flask, accurately add 25-50ml of 50-75% methanol, weigh the solution, sonicate at a power of 450-500W, a frequency of 35-40kHz, and a time of 30-60 minutes, let cool, weigh the solution again, make up the lost weight with 50-75% methanol, shake well, filter, and collect the filtrate to obtain the solution; The preparation process of the reference solution is as follows: accurately weigh neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isoflurane, verbascoside, iso-verascoside, apigenin-7- O - Add appropriate amounts of glucuronic acid, rosmarinic acid, scrophulariaceae IA, and scrophulariaceae IB reference substances to 50-75% methanol to prepare a mixed reference solution containing 20-50 μg of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, and caffeic acid, 50-100 μg of isoflurane, 200-250 μg of verbascoside, rosmarinic acid, isoverascoside, and apigenin-7-O glucuronic acid, and 100-800 μg of scrophulariaceae IA and scrophulariaceae IB per 1 ml; The preparation process of negative control Scrophularia ningpoensis and Radix Sarcosae is as follows: ① Preparation of Scrophularia ningpoensis solution: accurately weigh 0.8~1.0g of Scrophularia ningpoensis medicinal material powder, accurately add 25 or 50ml of 75~50% methanol solution, and weigh the weight; ultrasonic extraction for 30~60min with a power of 450~500W and a frequency of 35~40kHz, let cool, make up the lost weight with 75~50% methanol, filter, and take the filtrate; Preparation of Radix Sarcosae solution: accurately weigh 0.8~1.0g of Radix Sarcosae medicinal material powder, accurately add 25 or 50ml of 75~50% methanol solution, and weigh the weight; ultrasonic extraction for 30~60min with a power of 450~500W and a frequency of 35~40kHz, let cool, make up the lost weight with 75~50% methanol, filter, and take the filtrate.

2. The construction method according to claim 1, characterized in that The Wantong Yankang preparation is Wantong Yankang tablets or Wantong Yankang capsules.

3. The construction method according to claim 1, characterized in that The characteristic spectrum of the Wantong Yankang preparation has a total of 11 chromatographic peaks, namely peak 1-neochlorogenic acid, peak 2-chlorogenic acid, peak 3-cryptochlorogenic acid, peak 4-caffeic acid, peak 5-isofraxin, peak 6-verascoside, peak 7-isoverascoside, peak 8-apigenin-7-O-glucuronic acid, peak 9-rosmarinic acid, peak 10-scrophulariaceae glycoside IB, and peak 11-scrophulariaceae glycoside IA.

4. The construction method according to claim 1, wherein The test solution and the reference solution were analyzed by high performance liquid chromatography to obtain the high performance liquid chromatography fingerprint of Wantong Yankang; the main chromatographic peaks were attributed by UFLC-Q-TOF-MS / MS liquid chromatography-mass spectrometry technology. The chromatographic conditions of the liquid chromatography-mass spectrometry analysis were: the chromatographic column was C 18 Chromatographic column; acetonitrile A-0.1% formic acid / acetic acid B as mobile phase, gradient elution; flow rate: 0.8-1.1 ml / min; and a diverter valve was used to adjust the flow rate into the mass spectrometer to 0.2-0.3 ml / min; column temperature was 30°C, injection volume was 5-10 μl; mass spectrometry conditions: ESI electrospray ionization source, positive and negative ion modes, ion spray voltage: positive mode 5500 V, negative mode -4500 V; ion source parameters: spray gas 50 psi; auxiliary heating gas 50 psi; ion source temperature 500°C; curtain gas 35 psi; Collision gas pressure 10 psi, primary mass spectrometry precursor ion scan range: m / z 100-2000, collision activation scan was 15.0, and dynamic background subtraction was turned on.

5. Application of a characteristic spectrum obtained according to any one of the preparation methods of claims 1 to 4 in the evaluation and control of the quality of Wantong Yankang preparation products.

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