Establishment of fingerprint spectrum of compound shouyangquan granules and determination of its component contents

Abstract

This invention discloses a method for establishing a fingerprint spectrum of Compound Shuyangquan Granules, comprising the following steps: detecting the test solution using high-performance liquid chromatography (HPLC) to obtain a chromatogram of Compound Shuyangquan Granules; analyzing the chromatogram of Compound Shuyangquan Granules using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system software to obtain a fingerprint spectrum of Compound Shuyangquan Granules. The method for establishing a fingerprint spectrum of Compound Shuyangquan Granules provided by this invention, using HPLC and with reasonable control of chromatographic conditions, allows for a more comprehensive quality evaluation of Compound Shuyangquan Granules and provides a basis for further research on the chemical composition and quality standard improvement of Compound Shuyangquan Granules.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine quality testing technology. Specifically, it relates to a method for establishing the fingerprint spectrum of Compound Shuyangquan Granules and a method for determining the content of its components. Background Technology

[0002] Chronic gastritis is caused by the repeated effects of Helicobacter pylori infection, bile reflux, chemical irritation, and psychological stress on the gastric mucosa over a long period. Its prevalence increases significantly with age. Current clinical treatments primarily focus on eradicating Helicobacter pylori, acid suppression, mucosal protection, and prokinetic drugs. However, relapse rates are high after drug discontinuation, and the reversal of glandular atrophy and intestinal metaplasia is limited. Recent studies have shown that traditional Chinese medicine compound formulas can promote gastric mucosal repair and reduce relapse rates through anti-inflammatory mechanisms, improved mucosal microcirculation, enhanced barrier function, and immune regulation, which is of great significance in controlling disease progression.

[0003] Compound Shuyangquan Granules are an in-house preparation of Shanghai Fourth People's Hospital, a classic formula modified by Professor Peng Peichu, a renowned traditional Chinese medicine doctor in Shanghai, based on decades of clinical experience. The formula consists of thirteen Chinese herbs: Shuyangquan, Scrophularia, Watercress, Prepared Rhubarb, Scutellaria barbata, Anemarrhena asphodeloides, Forsythia suspensa, Scutellaria baicalensis, Citrus medica, Prepared Magnolia officinalis, Honey-fried Citrus aurantium, Ziziphus jujuba, and Bupleurum chinense. Its treatment principle is to clear heat and detoxify, nourish yin and moisten dryness, and soothe the liver and stomach. It is mainly used to treat chronic gastritis and precancerous lesions of the stomach.

[0004] Compound Shuyangquan Granules are formulated with thirteen traditional Chinese medicines, resulting in a complex chemical composition. This composition includes the characteristic active ingredients of each individual herb, as well as interactions and transformations among multiple components. Current quality standards focus only on the qualitative identification of a few components, lacking both multi-index content determination and fingerprinting, making it difficult to evaluate batch consistency and stability. Therefore, there is an urgent need to establish a quality control system that combines multi-index quantitative analysis and characteristic fingerprinting to provide crucial technical support for uniform formulation quality and controllable efficacy. Summary of the Invention

[0005] The purpose of this invention is to provide a method for establishing the fingerprint spectrum of Compound Shuyangquan Granules.

[0006] Another objective of this invention is to provide a method for determining the content of components in Compound Shuyangquan Granules.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides a method for establishing the fingerprint spectrum of Compound Shuyangquan Granules, comprising the following steps:

[0009] Add the compound Shuyangquan granules to pure water or a methanol aqueous solution with a volume fraction of 8%~32% (preferably 10% or 30%), with a mass-to-volume ratio of compound Shuyangquan granules to pure water or methanol aqueous solution of 1:10~30 (preferably 1:20), shake well, extract by ultrasonication, cool to room temperature, make up the weight loss, shake well, centrifuge to remove the supernatant (centrifuge at 12000 r / min for 10 min), filter (0.22 µm filter membrane) to obtain the test solution;

[0010] The test solution was analyzed by high performance liquid chromatography to obtain the chromatogram of Compound Shuyangquan Granules;

[0011] High performance liquid chromatography (HPLC) conditions include:

[0012] Chromatographic column: octadecylsilane-bonded silica gel column; mobile phase: phase A is 0.05%~0.15% (v / v) formic acid aqueous solution, phase B is methanol; gradient elution is used with 20%~95% (v / v) phase A and 5%~80% (v / v) phase B; flow rate: 0.9~1.1 mL / min; column temperature: 38~48℃; injection volume: 8~12 µl; detection wavelength: 200~600 nm;

[0013] The chromatogram of the compound Shuyangquan granules was analyzed using the software of the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012) to obtain the fingerprint spectrum of the compound Shuyangquan granules.

[0014] The preparation method of the compound Shuyangquan granules includes the following steps:

[0015] Mix *Shuyangquan*, *Shuixiancao*, and *Banzhilian* in a mass ratio of 1:1:1, and add *Chaihu*, *Xuanshen*, *Foshou*, *Shudahuang*, *Qinglianqiao*, *Huangqin*, *Zhihoupo*, *Zhihou*, *Yuzhizi*, and *Houpu* (stir-fried with honey) in a mass ratio of 1:1:1:1:1:1:1:1:1. The mass ratio of *Shuyangquan* to *Chaihu* is 1~3:1 (preferably 1.7:1). Decoction is prepared by adding 8~10 times (preferably 8 times) of water and boiling for 1~2.5 hours (preferably 2 hours). Filter the decoction and retain the dregs. Add 6~8 times (preferably 6 times) of water and boil for 0.8~1.5 hours (preferably 1 hour). Filter the decoction and combine the two decoctions. Concentrate the decoction, spray dry it into powder, and granulate it to obtain compound *Shuyangquan* granules.

[0016] The ultrasonic extraction time is 20-50 min (preferably 30 min), the extraction power is 300-400 W (preferably 350 W), and the extraction frequency is 45-60 kHz (preferably 53 kHz).

[0017] The high-performance liquid chromatography conditions include:

[0018] The octadecylsilane-bonded silica gel column used was an Acclaim™ 120 C18, 4.6 mm × 250 mm, 5 µm.

[0019] Mobile phase A was a 0.1% (v / v) aqueous solution of formic acid, and mobile phase B was methanol. The gradient elution program was as follows: 0–5 min, 95% A; 5–15 min, 95% A–75% A; 15–45 min, 75% A–50% A; 45–55 min, 50% A–20% A; 55–60 min, 20% A; 60.1–65 min, 95% A.

[0020] The detection wavelength was 254 nm, the flow rate was 1 mL / min, the column temperature was 45 °C, and the injection volume was 10 µl.

[0021] The fingerprint spectrum of the compound Shuyangquan granules: the chromatographic peaks of the main components with good separation were selected as characteristic peaks, and a total of 26 common peaks were identified.

[0022] The attribution and identification of the common peaks were completed by comparing the chromatograms of the single-herb reference medicinal material solution, the taste-deficient negative sample solution, and the mixed reference solution of Compound Shuyangquan Granules.

[0023] The preparation method of the mixed reference solution includes the following steps: dissolving neomangiferin, mangiferin, forsythoside I, forsythoside A, baicalin, rutin, naringin, hesperidin, baicalin, wogonin, apigenin, and baicalein in a solvent to obtain the mixed reference solution; the solvent is a methanol solution containing 0%~10% DMSO by volume.

[0024] Of the 26 common peaks, peak 10 (retention time 19.2 min) is neomangiferin, peak 11 (retention time 23.5 min) is mangiferin, peak 12 (retention time 26.6 min) is forsythoside I, peak 14 (retention time 31.6 min) is forsythoside A, peak 15 (retention time 32.2 min) is baicalin, peak 16 (retention time 33.3 min) is rutin, peak 17 (retention time 34.5 min) is naringin, peak 18 (retention time 36.0 min) is hesperidin, peak 19 (retention time 42.1 min) is baicalin, peak 24 (retention time 49.3 min) is wogonin, peak 25 (retention time 51.2 min) is apigenin, and peak 26 (retention time 53.0 min) is baicalein. The remaining common peaks were not identified.

[0025] A second aspect of the present invention provides a method for establishing a comparative fingerprint spectrum of Compound Shuyangquan Granules, comprising the following steps:

[0026] Take n batches of Compound Shuyangquan Granules and obtain the fingerprint chromatograms of each batch of Compound Shuyangquan Granules according to the method described above; analyze the fingerprint chromatograms of the n batches of Compound Shuyangquan Granules using the software of the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System to obtain the control fingerprint chromatogram of Compound Shuyangquan Granules; wherein, n≥10.

[0027] The comparative fingerprint spectrum of the compound Shuyangquan granules: the chromatographic peaks of the main components with good separation were selected as characteristic peaks, and a total of 26 common peaks were identified.

[0028] Of the 26 common peaks, peak 10 (retention time 19.2 min) is neomangiferin, peak 11 (retention time 23.5 min) is mangiferin, peak 12 (retention time 26.6 min) is forsythoside I, peak 14 (retention time 31.6 min) is forsythoside A, peak 15 (retention time 32.2 min) is baicalin, peak 16 (retention time 33.3 min) is rutin, peak 17 (retention time 34.5 min) is naringin, peak 18 (retention time 36.0 min) is hesperidin, peak 19 (retention time 42.1 min) is baicalin, peak 24 (retention time 49.3 min) is wogonin, peak 25 (retention time 51.2 min) is apigenin, and peak 26 (retention time 53.0 min) is baicalein. The remaining common peaks were not identified.

[0029] A third aspect of the present invention provides a reference fingerprint chromatogram of Compound Shuyangquan Granules obtained by the method described above. The reference fingerprint chromatogram of Compound Shuyangquan Granules, with mangiferin peak 11 as a reference peak, compares the relative retention times of each common peak with the corresponding peaks in the reference fingerprint chromatogram, with a deviation within ±5%. Within %; using peak 11 (mangiferin) as the reference peak, the relative retention times of each common peak should meet the following requirements: Peak 1 0.224~0.248, Peak 2 0.327~0.361, Peak 3 0.347~0.383, Peak 4 0.386~0.426, Peak 5 0.486~0.538, Peak 6 0.504~0.557, Peak 7 0.560~0.618, Peak 8 0.601~0.664, Peak 9 0.670~0.740, Peak 10 0.775~0.857, Peak 11 1.000, Peak 12 1.074~1.187, Peak 13 1. Peak 231~1.361, Peak 14 1.280~1.414, Peak 15 1.302~1.439, Peak 16 1.347~1.489, Peak 17 1.394~1.541, Peak 18 1.456~1.609, Peak 19 1.704~1.883, Peak 20 1.814~2.005, Peak 21 1.842~2.036, Peak 22 1.902~2.102, Peak 23 1.949~2.155, Peak 24 1.994~2.204, Peak 25 2.069~2.287, Peak 26 2.144~2.370.

[0030] A fourth aspect of the present invention provides a quality control method for Compound Shuyangquan Granules, comprising the following steps:

[0031] Take the Compound Shuyangquan Granules to be tested, and obtain the chromatogram of the Compound Shuyangquan Granules to be tested according to the method described above. Evaluate the similarity between the obtained chromatogram of the Compound Shuyangquan Granules to be tested and the reference fingerprint chromatogram of the Compound Shuyangquan Granules. If the similarity is ≥0.90, the quality of the Compound Shuyangquan Granules to be tested is determined to be qualified.

[0032] In a fifth aspect, the present invention provides a method for determining the content of chemical components in Compound Shuyangquan Granules, comprising the following steps:

[0033] The content of chemical components in Compound Shuyangquan Granules was determined by high performance liquid chromatography. The chemical components were selected from neomangiferin, mangiferin, forsythoside I, forsythoside A, baicalin, and wogonin.

[0034] The first step is to establish a standard curve for chemical composition:

[0035] New mangiferin, mangiferin, forsythoside I, forsythoside A, baicalin, and wogonin were dissolved in methanol solution containing 0%~10% DMSO to obtain a mixed reference solution. The solution was analyzed by high performance liquid chromatography. The chromatographic peaks of each chemical component were determined based on their retention times, and the peak areas of the corresponding chemical components were obtained. Standard curves for each chemical component were established, and the linear regression equations and correlation coefficients r (r≥0.999) for each chemical component were obtained.

[0036] In the mixed reference solution, the concentrations of neomangiferin, mangiferin, and forsythoside I were 5.07–101.4 µg / mL; the concentrations of forsythoside A, forsythoside A, baicalin, and forsythoside A were 100.6–2012 µg / mL; and the concentrations of wogonin were 20.16–403.2 µg / mL.

[0037] High performance liquid chromatography (HPLC) conditions include:

[0038] Chromatographic column: octadecylsilane-bonded silica gel column; mobile phase: phase A is 0.05%~0.15% (v / v) formic acid aqueous solution, phase B is methanol; gradient elution is used with 20%~95% (v / v) phase A and 5%~80% (v / v) phase B; flow rate: 0.9~1.1 mL / min; column temperature: 38~48℃; injection volume: 8~12 µl; detection wavelength: 200~600 nm;

[0039] The second step is to obtain the chromatographic peak areas of the chemical components in the compound Shuyangquan granules to be tested.

[0040] Add mass M of compound Shuyangquan granules to volume V of pure water or a volume fraction of 8%~32% methanol (preferably 10% or 30%) aqueous solution and shake well. The mass-to-volume ratio is 1:10~30 (preferably 1:20). Extract by ultrasonication, cool to room temperature, make up the weight loss, shake well, centrifuge and collect the supernatant (centrifuge at 12000 r / min for 10 min), filter (0.22 µm filter membrane) to obtain the test solution.

[0041] The test solution was detected under the same high-performance liquid chromatography conditions as in the first step. The chromatographic peaks of each chemical component were determined based on their retention times, and the peak areas of the corresponding chemical components were obtained.

[0042] The third step is to determine the content of chemical components in the compound Shuyangquan granules to be tested;

[0043] Based on the established standard curves for each chemical component, the chromatographic peak areas of the chemical components of the Compound Shuyangquan Granules to be tested are substituted into the corresponding linear regression equations. The concentration C of the corresponding component in the test solution is calculated using the external standard method. Then, the content of each chemical component in the Compound Shuyangquan Granules to be tested is calculated using the formula: Content = C × V / M, where V is the volume of the test solution and M is the sample weight.

[0044] The high-performance liquid chromatography conditions include:

[0045] The octadecylsilane-bonded silica gel column used was an Acclaim™ 120 C18, 4.6 mm × 250 mm, 5 µm.

[0046] Mobile phase A was a 0.1% (v / v) aqueous solution of formic acid, and mobile phase B was methanol. The gradient elution program was as follows: 0–5 min, 95% A; 5–15 min, 95% A–75% A; 15–45 min, 75% A–50% A; 45–55 min, 50% A–20% A; 55–60 min, 20% A; 60.1–65 min, 95% A.

[0047] The detection wavelength was 254 nm, the flow rate was 1 mL / min, the column temperature was 45 °C, and the injection volume was 10 µl.

[0048] By adopting the above technical solution, the present invention has the following advantages and beneficial effects:

[0049] The fingerprinting method for Compound Shuyangquan Granules provided by this invention employs high-performance liquid chromatography (HPLC). Through reasonable control of chromatographic conditions, multiple chemical components in Compound Shuyangquan Granules are effectively separated. The obtained fingerprint spectrum identified 26 common characteristic peaks, and 12 components were identified, including neomangiferin, mangiferin, forsythoside I, forsythoside A, baicalin, rutin, naringin, hesperidin, baicalin, wogonin, apigenin, and baicalein. The method exhibits good precision, stability, and reproducibility, and can more comprehensively and objectively reflect the chemical composition characteristics of Compound Shuyangquan Granules, providing a reliable experimental basis for improving quality standards.

[0050] This invention also provides a method for establishing a reference fingerprint spectrum for Compound Shuyangquan Granules and its application in quality control. By collecting samples from 11 different production batches, and using the optimized fingerprint spectrum method described above, the reference fingerprint spectrum for Compound Shuyangquan Granules was obtained through analysis using a Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version). This reference fingerprint spectrum has good representativeness and universality. By evaluating the similarity between the sample fingerprint spectrum and the aforementioned reference fingerprint spectrum, quality control of Compound Shuyangquan Granules can be achieved. The overall quality consistency can be quickly determined through similarity comparison.

[0051] This invention further provides a method for determining the chemical components in Compound Shuyangquan Granules. Using the same chromatographic conditions as the fingerprinting method, the procedure is simplified, enabling the simultaneous quantification of six chemical components: neomangiferin, mangiferin, forsythoside I, forsythoside A, baicalin, and wogonin. The method exhibits high sensitivity, accuracy, and repeatability, scientifically and comprehensively reflecting product quality and providing reliable quantitative evidence and technical support for improving quality standards. Attached Figure Description

[0052] Figure 1 This is the HPLC chromatogram for investigating the mobile phase system.

[0053] Figure 2 This is the HPLC chromatogram for column temperature determination.

[0054] Figure 3 The HPLC chromatogram was used to investigate the detection wavelength.

[0055] Figure 4 The HPLC chromatograms of the test sample solution under different extraction solvents are shown.

[0056] Figure 5 This is a schematic diagram of the fingerprint spectrum of 11 batches of Compound Shuyangquan Granules.

[0057] Figure 6 Schematic HPLC chromatograms of Compound Shuyangquan Granules test solution, single herbal sample solution, and mixed reference solution.

[0058] Figure 7 This is a schematic diagram of the HPLC chromatograms of the test solution, various negative sample solutions, and the mixed reference solution of Compound Shuyangquan Granules.

[0059] Figure 8 The total ion chromatogram of the test solution of Compound Shuyangquan Granules.

[0060] Figure 9 This is a schematic diagram of the comparative fingerprint spectrum of Compound Shuyangquan Granules.

[0061] Figure 10 This is a schematic diagram of the specificity spectrum for the determination of the content of Compound Shuyangquan Granules. Detailed Implementation

[0062] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting the scope of protection of the present invention.

[0063] The relevant descriptions of the instruments, reagents, and materials used in this application are as follows:

[0064] Instruments: Ultimate 3000 high-performance liquid chromatograph (Thermofisher, USA); Agilent 6545Q-TOF LC / MS system (Agilent Technologies, USA); ME155DU 0.0001 g / mL balance (METTLERTOLEDO, Switzerland); BT 224 S 0.0001 g / mL balance (Sartorius, Germany); Legend Micro 21R high-speed refrigerated centrifuge (Thermofisher, USA); Master-DUV ultrapure water system (Shanghai Hetai Instrument Co., Ltd.); SK7200H ultrasonic cleaner (Shanghai Kedao Ultrasonic Instrument Co., Ltd.); fully automatic ceramic decoction pot (Wanchanglong Hardware Products Factory, Jinshi Town, Chaoan District, Chaozhou City); R502B rotary evaporator (Shanghai Shensheng Technology Co., Ltd.); SHB-3 vacuum water pump (Shanghai Shensheng Technology Co., Ltd.); SCILOGEX MX-S vortex apparatus (Sciencel, USA); Traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition): National Pharmacopoeia Commission.

[0065] Reagents: Chromatographic grade methanol and acetonitrile were purchased from CNW GmbH, Germany; formic acid was purchased from CNW GmbH, Germany; ammonium acetate was purchased from ACS GmbH, USA; DMSO was purchased from biofroxx GmbH, Germany; and the test water was Master-DUV ultrapure water.

[0066] Materials: Reference standards, including new mangiferin (batch number KB368284), mangiferin (batch number C21M9Y61363), forsythoside I (batch number JB245799), forsythoside A (batch number J29HB186611), baicalin (batch number J31GB156318), rutin (batch number T20N11Z131674), hesperidin (batch number S23IB224130), baicalin (batch number JB246114), wogonin (batch number J05IB216330), and baicalein (batch number A07IB222187), were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; naringin (batch number 20120221) and apigenin (batch number 20121101) were purchased from the National Institutes for Food and Drug Control. The following ingredients were supplied by Shanghai Wanshicheng Pharmaceutical Co., Ltd., and all comply with the provisions of Part I of the Chinese Pharmacopoeia (2025 edition). The 11 batches of Compound Shuyangquan Granules (batch numbers 202306010401, 202307010401, 202310010401, 202406010401, 202409010401, 202502010401, 20250401, 20250402, 20250403, 20250501, 202506010401) are numbered S1 to S11 respectively.

[0067] Example 1

[0068] Fingerprint chromatogram establishment method for Compound Shuyangquan Granules:

[0069] (1) Preparation of Compound Shuyangquan Granules

[0070] Take the prescribed medicinal slices, by weight, including: 5 parts each of *Shuyangquan*, *Shuixiancao*, and *Banzhilian* slices; and 3 parts each of *Chaihu*, *Xuanshen*, *Foshou*, *Shudahuang*, *Qinglianqiao*, *Huangqin*, *Zhihoupu*, *Zhihou*, *Yuzhizi*, and *Houpu* stir-fried with honey. Decoct in water. First, add 8 times the amount of water and boil for 2 hours. Filter the decoction and retain the dregs. Second, add 6 times the amount of water and boil for 1 hour. Filter the decoction and combine the two decoctions. Concentrate the decoction into a clear paste, spray dry it into a dry powder, and granulate it to obtain Compound Shuyangquan Granules.

[0071] (2) Preparation of the test solution of compound Shuyangquan granules

[0072] Accurately weigh 1.0g of Compound Shuyangquan Granules, place them in an Erlenmeyer flask, add 20mL of pure water, weigh, shake well, and sonicate for 30min at an extraction power of 350W and an extraction frequency of 53kHz. Then cool to room temperature, make up the weight loss, shake well, centrifuge at 12000r / min for 10min, take the supernatant and filter it through a 0.22µm filter membrane to obtain the test solution, and store it in a refrigerator at 4℃ for later use.

[0073] (3) Preparation of single-herb control medicinal material decoction and taste-deficient negative sample solution of Compound Shuyangquan Granules

[0074] Preparation method of standard decoction of compound Shuyangquan granules: Weigh 15g each of Shuyangquan, Shuixiancao, and Banzhilian slices, and 9g each of Chaihu, Xuanshen, Foshou, prepared Dahuang, Qinglianqiao, Huangqin, prepared Houpo, Zhimu, Yuzhizi, and honey-fried Zhishi slices, and place them in a ceramic decoction pot. For the first decoction, add 8 times the amount of water (g of medicinal slices / ml of water = 1:8), soak for 30 minutes, then bring to a boil over high heat (500w), and simmer over low heat (250w) for 2 hours. Filter the decoction through a 200-mesh sieve. For the second decoction, add 6 times the amount of water (g of medicinal slices / ml of water = 1:6), bring to a boil over high heat (500w), and simmer over low heat (250w) for 1 hour. Filter the decoction through a 200-mesh sieve. Combine the two filtrates, transfer to a rotary evaporator, and concentrate under reduced pressure (temperature 60℃, rotation speed 40~50r / min, vacuum degree -0.08~-0.1MPa) to 500mL to obtain the standard decoction of Compound Shuyangquan Granules.

[0075] Following the preparation method of the above-mentioned Compound Shuyangquan Granules prescription standard decoction, single-herb reference medicinal material solutions were prepared. Specifically, the medicinal materials of each single-herb reference herb in the above prescription (Shuyangquan, Shuixiancao, Banzhilian, Chaihu, Xuanshen, Foshou, Shudahuang, Qinglianqiao, Huangqin, Zhihoupo, Zhimu, Yuzhizi, and honey-fried Zhishi) were weighed separately. The weighing amount of each single-herb reference medicinal material was consistent with the weighing amount of the corresponding medicinal material in the standard decoction. They were placed separately in ceramic decoction pots. Each reference herb was prepared separately according to the preparation process of the standard decoction to obtain the solutions of each single-herb reference medicinal material for later use.

[0076] Further following the preparation method of the above-mentioned Compound Shuyangquan Granules prescription standard decoction, prepare the missing ingredient negative sample solution, that is, prepare the negative sample solution for each missing ingredient in the prescription. Specifically, remove one target ingredient from the prescription in sequence (remove any one of the following: Shuyangquan, Shuixiancao, Banzhilian, Chaihu, Xuanshen, Foshou, Shudahuang, Qinglianqiao, Huangqin, Zhihoupo, Zhimu, Yuzhizi, and honey-fried Zhishi). The weighing amount of each remaining ingredient is consistent with the weighing amount of the corresponding decoction piece in the standard decoction. Mix the remaining ingredients and place them in a ceramic decoction pot. Follow the complete preparation process of the standard decoction to obtain the corresponding missing ingredient negative sample solution for each ingredient, and set it aside.

[0077] (4) Preparation of mixed reference solution

[0078] Take the following 12 components: mangiferin, mangiferin, forsythoside I, forsythoside A, baicalin, rutin, naringin, hesperidin, baicalin, wogonin, apigenin, and baicalein. Accurately weigh them, dissolve them separately in methanol solution containing 0%~10% DMSO, and then dilute with methanol to obtain a mixed reference solution of the above 12 components.

[0079] (5) Determination of chromatographic conditions

[0080] Determination of the mobile phase system: An Acclaim™ 120 C18 (4.6 mm × 250 mm, 5 µm) column was used. The detection wavelength was 254 nm, the column temperature was 35 ℃, the flow rate was 1 mL / min, and the injection volume was 10 µL. Mobile phase A was aqueous phase, and mobile phase B was organic phase. Gradient elution was used. The gradient elution program for mobile phase system 1 (aqueous phase A was 0.1% formic acid aqueous solution, organic phase B was acetonitrile) was investigated: 0–10 min, 98%–95% A; 10–20 min, 95%–85% A; 20–40 min, 85%–70% A; 40–45 min, 70%–0% A; 45–50 min, 0% A. The effects of mobile phase A (50.1–55 min, 98% A), mobile phase 2 (aqueous phase A: 0.1% formic acid aqueous solution, organic phase B: methanol, gradient elution program: 0–30 min, 95%–60% A; 30–45 min, 60%–50% A; 45–55 min, 50%–25% A; 55.1–60 min, 95% A) and mobile phase 3 (aqueous phase A: 20 mmol / L ammonium acetate and 2% acetic acid aqueous solution, organic phase B: methanol, gradient elution program the same as mobile phase 2) on the chromatographic behavior of the test solution are as follows: Figure 1 As shown, Figure 1 The image shows the HPLC chromatogram for the mobile phase system. The results show that when using mobile phase system 2, the peak shapes and resolutions of each component in the test solution are good.

[0081] The gradient elution program was further optimized, and the mobile phase A was determined to be 0.1% formic acid aqueous solution, and the organic phase B was methanol. The final gradient elution program was as follows: 0~5 min, 95% A; 5~15 min, 95%~75% A; 15~45 min, 75%~50% A; 45~55 min, 50%~20% A; 55~60 min, 20% A; 60.1~65 min, 95% A.

[0082] Column temperature determination: An Acclaim™ 120 C18 (4.6 mm × 250 mm, 5 µm) column was used. The detection wavelength was 254 nm, and the flow rate was 1 mL / min. Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was methanol. The gradient elution program was: 0–5 min, 95% A; 5–15 min, 95%–75% A; 15–45 min, 75%–50% A; 45–55 min, 50%–20% A; 55–60 min, 20% A; 60.1–65 min, 95% A. The injection volume was 10 µL. The effect of column temperatures of 35℃, 40℃, and 45℃ on the chromatographic behavior of the test solution was investigated. The results are shown in the figure. Figure 2 As shown, Figure 2 The image shows the HPLC chromatogram for column temperature testing. The results indicate that when the column temperature is 45℃, the peak shapes and resolutions of each component in the test solution are good.

[0083] Determination of detection wavelength: An Acclaim™ 120 C18 (4.6 mm × 250 mm, 5 µm) column was used at a flow rate of 1 mL / min. Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was methanol. The gradient elution program was as follows: 0–5 min, 95% A; 5–15 min, 95%–75% A; 15–45 min, 75%–50% A; 45–55 min, 50%–20% A; 55–60 min, 20% A; 60.1–65 min, 95% A. The injection volume was 10 µL. A high-performance liquid chromatography (HPLC) DAD detector was set to acquire chromatograms in the wavelength range of 200–600 nm. The effect of different detection wavelengths on the chromatographic behavior of the test solution was investigated. The results are shown in [Figure number missing]. Figure 3 As shown, Figure 3 The HPLC chromatograms were used to investigate the detection wavelength. The results showed that at a detection wavelength of 254 nm, the sample solution chromatogram showed the largest number of chromatographic peaks of the components simultaneously, with relatively uniform responses from each component, and good peak shape and resolution.

[0084] Therefore, the optimal chromatographic conditions were determined as follows: Acclaim™ 120 C18 (4.6 mm × 250 mm, 5 µm) column; mobile phase A: 0.1% formic acid aqueous solution; mobile phase B: methanol; gradient elution program: 0–5 min, 95% A; 5–15 min, 95%–75% A; 15–45 min, 75%–50% A; 45–55 min, 50%–20% A; 55–60 min, 20% A; 60.1–65 min, 95% A. Flow rate: 1 mL / min; column temperature: 45 °C; detection wavelength: 254 nm; injection volume: 10 µL.

[0085] (6) Selection of extraction solvent for test sample

[0086] To investigate the effect of extraction solvent on sample analysis, pure water and methanol solutions with volume fractions of 10%, 30%, 50%, 70%, 90%, and 100% were used as extraction solvents. The sample solutions were prepared according to the method described in step (1) above, and analyzed using the optimal chromatographic conditions established in step (4). The results are shown in [Figure 1]. Figure 4 As shown, Figure 4 The HPLC chromatograms of the test solution under different extraction solvents are shown. The results show that as the proportion of methanol in the extraction solvent increases, the peak shape and resolution deteriorate. Pure water, 10% methanol, and 30% methanol solutions showed better extraction effects, while pure water extraction showed the best effect. Therefore, pure water was selected for extraction of the test solution.

[0087] (7) Establishment and component attribution of fingerprint spectrum

[0088] Eleven batches of Compound Shuyangquan Granules were tested using the optimal chromatographic conditions determined in step (4). The chromatograms of each batch of Compound Shuyangquan Granules were imported into the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012) software for analysis to obtain the fingerprint chromatograms of Compound Shuyangquan Granules. (See attached image). Figure 5 As shown, Figure 5 This is a schematic diagram of the fingerprint chromatograms of 11 batches of Compound Shuyangquan Granules. Chromatographic peaks of the main components with good separation were selected as characteristic peaks, and a total of 26 common peaks were identified.

[0089] The same chromatographic conditions were used to analyze the test solution of Compound Shuyangquan Granules, solutions of individual medicinal materials, negative sample solution, and mixed reference solution, supplemented by mass spectrometry (MS / MS) detection to identify common peaks. MS / MS conditions included: ESI (+ / -), capillary voltage 3500 V, sheath gas temperature 350 °C, sheath gas flow rate 11 L / min, drying gas flow rate 8 L / min, MS1 and MS2 scan range 100–1700 Da, and fixed collision energy 10 / 30 / 60 V. The retention times and MS / MS information of each component in the mixed reference solution were used as the basis for peak identification, and common peaks were preliminarily assigned. The results are shown in [Figure number missing]. Figures 6-8 As shown.

[0090] Figure 6 This is a schematic HPLC chromatogram of the test solution, single herbal sample solution, and mixed reference solution of Compound Shuyangquan Granules. Peak 10 is neomangiferin, peak 11 is mangiferin, peak 12 is forsythoside I, peak 14 is forsythoside A, peak 15 is baicalin, peak 16 is rutin, peak 17 is naringin, peak 18 is hesperidin, peak 19 is baicalin, peak 24 is wogonin, peak 25 is apigenin, and peak 26 is baicalin.

[0091] Figure 7This is a schematic diagram of the HPLC chromatograms of the test solution, negative sample solutions, and mixed reference solution of Compound Shuyangquan Granules. Peak 10 is neomangiferin, peak 11 is mangiferin, peak 12 is forsythoside I, peak 14 is forsythoside A, peak 15 is baicalin, peak 16 is rutin, peak 17 is naringin, peak 18 is hesperidin, peak 19 is baicalin, peak 24 is wogonin, peak 25 is apigenin, and peak 26 is baicalin.

[0092] Figure 8 The mass spectrometry total ion chromatograms of the test solution of Compound Shuyangquan Granules are shown. Among them, spectrum 1 is the total ion chromatogram in positive ion scanning mode and spectrum 2 is the total ion chromatogram in negative ion scanning mode.

[0093] The above analysis shows that Compound Shuyangquan Granules are made from 13 medicinal herbs: Shuyangquan (Shuyangquan), Scrophularia, Waterweed, Prepared Rhubarb, Scutellaria barbata, Anemarrhena asphodeloides, Forsythia suspensa, Scutellaria baicalensis, Citrus medica, Magnolia officinalis, Fried Citrus aurantium (with honey), Citrus aurantium (with honey), and Bupleurum chinense. The components of Compound Shuyangquan Granules include neomangiferin, mangiferin, forsythoside I, forsythoside A, baicalin, rutin, naringin, hesperidin, baicalin, wogonin, apigenin, and baicalein. The 26 common peaks were attributed to the following: among the identified common peaks, peak 10 (neomangiferin with a retention time of 19.2 min) and peak 11 (mangiferin with a retention time of 23.5 min) originated from Anemarrhena asphodeloides; peak 12 (forsythoside I with a retention time of 26.6 min) and peak 31.6 min... Peak 14 (retention time 32.2 min) of forsythoside A is derived from Forsythia suspensa; peak 15 (retention time 32.2 min) of baicalin is derived from Scutellaria barbata; peak 16 (retention time 33.3 min) of rutin is derived from Forsythia suspensa; peak 17 (retention time 34.5 min) of naringin and peak 18 (retention time 36.0 min) of hesperidin are derived from stir-fried Citrus aurantium and Citrus medica; peak 19 (retention time 42.1 min) of baicalin, peak 24 (retention time 49.3 min) of wogonin and peak 26 (retention time 53.0 min) of baicalein are derived from Scutellaria baicalensis; and peak 25 (retention time 51.2 min) of apigenin is derived from processed rhubarb. Among the unidentified common peaks, peak 1 with a retention time of 5.6 min may originate from *Hydrocotyle vulgaris*, *Sophora flavescens*, *Bupleurum chinense*, *Citrus aurantium*, *Scrophularia ningpoensis*, *Rheum palmatum* (processed), *Citrus medica*, *Scutellaria barbata*, and *Citrus aurantium* (stir-fried with honey bran); peaks 2 (8.1 min), 5 (12.0 min), 8 (14.8 min), and 13 (30.4 min) may originate from *Rheum palmatum*; peak 3 (8.6 min) may originate from *Hydrocotyle vulgaris*, *Sophora flavescens*, *Bupleurum chinense*, *Scrophularia ningpoensis*, *Scutellaria barbata*, *Citrus medica*, and *Citrus aurantium* (stir-fried with honey bran); peaks 4 (9.5 min) and 9 (16.6 min) may originate from *Forsythia suspensa*; peak 6 (12.5 min) may originate from *Hydrocotyle vulgaris*; peak 7 (13.8 min) may originate from *Scutellaria barbata*; and peak 44.9 min... Peak 20, with a retention time of 45.5 min, may originate from processed rhubarb and Scrophularia ningpoensis. Peaks 21 (45.5 min), 22 (47.0 min), and 23 (48.2 min) may originate from Scutellaria baicalensis. Based on the above results, this embodiment successfully established the fingerprint spectrum of Compound Shuyangquan Granules.

[0094] Example 2

[0095] Establishment of a comparative fingerprint spectrum for Compound Shuyangquan Granules:

[0096] The fingerprint chromatograms of 11 batches of Compound Shuyangquan Granules from Example 1 were imported into the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012) for analysis. The fingerprint chromatogram of Compound Shuyangquan Granules, numbered S1, was set as the reference chromatogram. The time window width was set to 0.2 min. The mean method was used for multi-point correction and automatic peak matching to obtain the control chromatogram of Compound Shuyangquan Granules. The results are shown in […]. Figure 9 As shown, Figure 9 This is a schematic diagram of the comparative fingerprint spectrum of Compound Shuyangquan Granules.

[0097] The obtained fingerprint spectrum of Compound Shuyangquan Granules: with mangiferin at peak 11 as the reference peak, the relative retention times of each common peak were compared with the corresponding peaks in the reference fingerprint spectrum, and the deviation was within ±5%. Specifically, using peak 11 (mangiferin) as the reference peak, the relative retention times of each common peak should conform to the following: Peak 1 0.224~0.248, Peak 2 0.327~0.361, Peak 3 0.347~0.383, Peak 4 0.386~0.426, Peak 5 0.486~0.538, Peak 6 0.504~0.557, Peak 7 0.560~0.618, Peak 8 0.601~0.664, Peak 9 0.670~0.740, Peak 10 0.775~0.857, Peak 11 1.000, Peak 12 1.074~1.187, Peak 13 1. Peak 231~1.361, Peak 14 1.280~1.414, Peak 15 1.302~1.439, Peak 16 1.347~1.489, Peak 17 1.394~1.541, Peak 18 1.456~1.609, Peak 19 1.704~1.883, Peak 20 1.814~2.005, Peak 21 1.842~2.036, Peak 22 1.902~2.102, Peak 23 1.949~2.155, Peak 24 1.994~2.204, Peak 25 2.069~2.287, Peak 26 2.144~2.370.

[0098] Example 3

[0099] Similarity evaluation and methodological investigation of fingerprint spectra of Compound Shuyangquan Granules:

[0100] (1) Similarity evaluation

[0101] The similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine (2012) was used to evaluate the similarity between the fingerprint chromatograms of 11 batches of Compound Shuyangquan Granules in Example 1 and the control chromatogram of Compound Shuyangquan Granules in Example 2. The results are shown in Table 1. The similarity between the fingerprint chromatograms of 11 batches of Compound Shuyangquan Granules and the control chromatogram of Compound Shuyangquan Granules was greater than 0.9. Using peak 11 (mangiferin), which showed a high response, moderate retention time, and good separation among the common peaks, as the reference peak, and the nominal value of the relative retention time of each common peak in the control chromatogram as the benchmark, the relative retention time (RRT) limit range of each common peak was calculated with an allowable relative deviation of ±5%. The results are shown in Table 2. The relative retention times of the common peaks in the fingerprint chromatogram of Compound Shuyangquan Granules should conform to the limit range in Table 2.

[0102] Table 1

[0103]

[0104] Table 2

[0105]

[0106] (2) Methodological investigation

[0107] Precision test: The test solution of Compound Shuyangquan Granules from batch S1 was injected and determined 6 times consecutively under the fingerprint chromatographic conditions determined above. Using peak 11 as the reference peak, the RSD of the relative retention time and relative peak area of ​​the common peaks were all less than 1.90%, and the similarity was 0.995. The results are shown in Tables 3 to 5, indicating that the method has good precision.

[0108] Table 3. Precision of the fingerprinting method for Compound Shuyangquan Granules (relative retention time)

[0109]

[0110] Table 4. Precision of the fingerprint spectral method for Compound Shuyangquan Granules (relative peak area)

[0111]

[0112] Table 5. Similarity of Precision Tests

[0113]

[0114] Repeatability test: Six portions of sample from batch S1 were accurately weighed and six test solutions were prepared. Each solution was injected for analysis. Using peak 11 as the reference peak, the RSD of the relative retention time and relative peak area of ​​the common peaks were both less than 2.32%, and the measured similarity was greater than 0.99. The results are shown in Tables 6 to 8, indicating that the repeatability of each component is good.

[0115] Table 6. Repeatability test of the fingerprint spectral method for Compound Shuyangquan Granules (relative retention time)

[0116]

[0117] Table 7. Repeatability test of the fingerprint spectral method for Compound Shuyangquan Granules (relative peak area)

[0118]

[0119] Table 8 Similarity of Repeatability Tests

[0120]

[0121] Stability test: Sample solutions from batch S6 were injected and analyzed at 0h, 2h, 4h, 6h, 8h, 12h, and 24h. Using peak 11 as the reference peak, the RSDs of the relative retention time and relative peak area of ​​the common peaks were all less than 3%, and the measured similarity was greater than 0.99. The results are shown in Tables 9-11, indicating that the sample solutions were stable within 24h.

[0122] Table 9. Stability study of the fingerprinting method for Compound Shuyangquan Granules (relative retention time)

[0123]

[0124] Table 10. Stability study of the fingerprinting method for Compound Shuyangquan Granules (relative peak area)

[0125]

[0126] Table 11 Similarity of stability tests

[0127]

[0128] Example 4

[0129] Methods for determining the content of six chemical components in Compound Shuyangquan Granules:

[0130] (1) Preparation of mixed reference solution

[0131] Accurately weigh neomangiferin, mangiferin, forsythoside I, forsythoside A, baicalin, and wogonin, dissolve them in methanol solution containing 10% DMSO, and then dilute with methanol to prepare stock solutions of each reference standard. Accurately measure appropriate amounts of each stock solution and prepare a mixed stock solution with methanol, wherein the concentrations of neomangiferin are 101.4 µg / mL, mangiferin is 102.2 µg / mL, forsythoside I is 406.4 µg / mL, forsythoside A is 401.6 µg / mL, baicalin is 2012 µg / mL, and wogonin is 403.2 µg / mL. Dilute the mixed stock solution sequentially with methanol to obtain six mixed reference solutions containing different known concentrations of each chemical component, including the mixed stock solution.

[0132] (2) Preparation of the test solution

[0133] Accurately weigh 1.0g of Compound Shuyangquan Granules, place them in an Erlenmeyer flask, add 20mL of pure water, weigh again, shake well, and extract by ultrasonication for 30min at an extraction power of 350W and an extraction frequency of 53kHz. Then cool to room temperature, make up the weight loss, shake well, centrifuge at 12000r / min for 10min, and filter the supernatant through a 0.22µm filter membrane as the test solution. Store at 4℃ for later use.

[0134] (3) Establishment of the standard curve

[0135] Six mixed reference solutions containing different known concentrations of each chemical component, prepared in step (1) above, were analyzed by liquid chromatography under the chromatographic conditions established in Example 1. The peak areas of each component were recorded. Standard curves for each chemical component were established with the peak area as the ordinate (y) and the corresponding concentration as the abscissa (x). The linear regression equations and correlation coefficients r of each chemical component were obtained. The sixth mixed reference solution with the lowest concentration was gradually diluted. The limit of quantitation (LOQ) was set at a signal-to-noise ratio (S / N) of 10:1, and the limit of detection (LOD) was set at an S / N of 3:1. The standard curves, LOQ, and LOD results for the six chemical components are shown in Table 12. The results show that the six chemical components all have a good linear relationship with their peak areas within their respective linear ranges.

[0136] Table 12 Standard curves and LOQ and LOD results for six chemical components

[0137]

[0138] (4) Specificity examination

[0139] The fourth mixed reference solution within the linear range and the test solution of Compound Shuyangquan Granules (S1) were analyzed, and the chromatogram was obtained as shown below. Figure 10 As shown. Figure 10 This is a schematic diagram of the specificity chromatogram for the determination of the content of Compound Shuyangquan Granules. Peak 1 is neomangiferin, peak 2 is mangiferin, peak 3 is forsythoside I, peak 4 is forsythoside A, peak 5 is baicalin, and peak 6 is wogonin. As can be seen from the figure, the test sample solution has chromatographic peaks with the same retention time at the corresponding positions of the reference standards for neomangiferin, mangiferin, forsythoside I, forsythoside A, baicalin, and wogonin, and the separation of each chemical component is good, indicating that this method has good specificity.

[0140] (5) Precision test

[0141] Quality control solutions were prepared with concentrations of 25.35 µg / mL for mangiferin, 25.55 µg / mL for mangiferin, 101.6 µg / mL forsythoside I, 100.4 µg / mL forsythoside A, 503 µg / mL for baicalin, and 100.8 µg / mL for wogonin. These solutions were injected six times consecutively over three days to determine intra-day and inter-day precision. The results are expressed as the RSD of each component concentration, as shown in Table 13. The table shows that the RSD values ​​for intra-day precision of mangiferin, mangiferin, forsythoside I, forsythoside A, baicalin, and wogonin were between 1.81% and 1.91%, and the RSD values ​​for inter-day precision were between 2.34% and 2.76%, indicating good instrument precision.

[0142] Table 13 Intra-day and inter-day precision of the six chemical components

[0143]

[0144] (6) Stability test

[0145] A test solution was prepared from Compound Shuyangquan Granules (S11). The solution was injected at 0h, 2h, 4h, 6h, 8h, 12h, and 24h according to the chromatographic conditions for content determination. The contents of neomangiferin, mangiferin, forsythoside I, forsythoside A, baicalin, and wogonin were measured at each time point. The RSD values ​​of each chemical component were calculated to be 2.65%, 1.27%, 1.49%, 2.30%, 1.63%, and 1.65%, respectively. The results indicate that the test solution has good stability after being placed in the injector for 24h.

[0146] (7) Repeatability test

[0147] Six parallel test solutions were prepared from Compound Shuyangquan Granules (S11). Each solution was analyzed under the specified chromatographic conditions, and the chromatograms were recorded. The contents of neomangiferin, mangiferin, forsythoside I, forsythoside A, baicalin, and wogonin were determined, and the RSD values ​​for each chemical component were calculated to be 1.37%, 1.90%, 1.91%, 2.22%, 1.85%, and 1.90%, respectively. The results indicate that the method has good repeatability.

[0148] (8) Recovery rate

[0149] Nine portions of Compound Shuyangquan Granules (S11) with known contents of six chemical components were accurately weighed and prepared into nine test solutions. Appropriate amounts of each test solution were precisely added to mixed reference solutions equivalent to 80%, 100%, and 120% of the contents of the six components in the test solution. Three samples were prepared for each level, and each sample was analyzed under the chromatographic conditions for content determination. The content of each chemical component was calculated. The recovery rate of each chemical component was calculated using the formula: Recovery rate (%) = (Measured amount - Original amount) / Added amount × 100%. The average recovery rate and RSD value were also calculated. The results are shown in Table 14. The average recoveries of neomangiferin, mangiferin, forsythoside I, forsythoside A, baicalin, and wogonin were 95.18%–101.54%, with RSD values ​​of 0.71%–2.66%. The results indicate that the method has good accuracy.

[0150] Table 14 Recovery rates of six chemical components

[0151]

[0152] (9) Content determination

[0153] The test solutions prepared from 11 batches of Compound Shuyangquan Granules in Example 1 were injected into a high-performance liquid chromatograph (HPLC) to obtain chromatograms of the Compound Shuyangquan Granules. The chromatographic peaks of the corresponding chemical components in the test solutions were determined based on their retention times, and the peak areas were obtained. According to the established standard curves for each chemical component, the concentration C of each chemical component was obtained from the peak areas of the 11 batches of Compound Shuyangquan Granules. The content of each chemical component in the 11 batches of Compound Shuyangquan Granules was calculated using the formula: content = C × V / M. The results are shown in Table 15. Based on 1 µg / mg = 1 mg / g, the content of each chemical component in Compound Shuyangquan Granules is expressed in mg / g. Among them, baicalin had the highest content.

[0154] Table 15 shows the content of each chemical component in Compound Shuyangquan Granules (numbers S1-S11) (mean ± standard deviation, mg / g, n=3).

[0155]

[0156] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for establishing the fingerprint spectrum of Compound Shuyangquan Granules, characterized in that, Includes the following steps: Add the compound Shuyangquan granules to pure water or a methanol aqueous solution with a volume fraction of 8%~32%, with a mass-volume ratio of 1:10~30 between the compound Shuyangquan granules and pure water or methanol aqueous solution. Shake well, extract by ultrasonication, cool to room temperature, make up the weight loss, shake well, centrifuge to remove the supernatant, filter, and obtain the test solution. The test solution was analyzed by high performance liquid chromatography to obtain the chromatogram of Compound Shuyangquan Granules; High performance liquid chromatography (HPLC) conditions include: Chromatographic column: Octadecylsilane-bonded silica gel column; Mobile phase: Phase A is an aqueous solution of formic acid with a volume fraction of 0.05%~0.15%, and Phase B is methanol; gradient elution is used with a volume fraction of 20%~95% Phase A and 5%~80% Phase B; flow rate is 0.9~1.1 mL / min; column temperature is 38~48℃; injection volume is 8~12 µl; detection wavelength is 200~600 nm; The chromatogram of the Compound Shuyangquan Granules was analyzed using the software of the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System to obtain the fingerprint spectrum of Compound Shuyangquan Granules.

2. The method for establishing the fingerprint spectrum of Compound Shuyangquan Granules according to claim 1, characterized in that, The preparation method of the compound Shuyangquan granules includes the following steps: Mix *Shuyangquan*, *Shuixiancao*, and *Banzhilian* in a mass ratio of 1:1:1, and add *Chaihu*, *Xuanshen*, *Foshou*, *Shudahuang*, *Qinglianqiao*, *Huangqin*, *Zhihoupu*, *Zhihou*, *Yuzhizi*, and *Houpu* (stir-fried with honey) in a mass ratio of 1:1:1:1:1:1:1:1:

1. The mass ratio of *Shuyangquan* to *Chaihu* is 1~3:

1. Decoction is prepared by adding 8~10 times the amount of water and boiling for 1~2.5 hours. Filter the decoction and retain the dregs. Add 6~8 times the amount of water and boil for 0.8~1.5 hours. Filter the decoction and combine the two decoctions. Concentrate the decoction, spray dry it into powder, and granulate it to obtain compound *Shuyangquan* granules.

3. The method for establishing the fingerprint spectrum of Compound Shuyangquan Granules according to claim 1, characterized in that, The ultrasonic extraction time is 20-50 min, the extraction power is 300-400 W, and the extraction frequency is 45-60 kHz; The high-performance liquid chromatography conditions include: The octadecylsilane-bonded silica gel column used was an Acclaim™ 120 C18, 4.6 mm × 250 mm, 5 µm. Mobile phase A was a 0.1% (v / v) aqueous solution of formic acid, and mobile phase B was methanol. The gradient elution program was as follows: 0–5 min, 95% A; 5–15 min, 95% A–75% A; 15–45 min, 75% A–50% A; 45–55 min, 50% A–20% A; 55–60 min, 20% A; 60.1–65 min, 95% A. The detection wavelength was 254 nm, the flow rate was 1 mL / min, the column temperature was 45 °C, and the injection volume was 10 µl.

4. The method for establishing the fingerprint spectrum of Compound Shuyangquan Granules according to claim 1, characterized in that, The fingerprint spectrum of the compound Shuyangquan granules: the chromatographic peaks of the main components with good separation were selected as characteristic peaks, and a total of 26 common peaks were identified.

5. The method for establishing the fingerprint spectrum of Compound Shuyangquan Granules according to claim 4, characterized in that, The attribution and identification of the common peaks were completed by comparing the chromatograms of the single-herb reference medicinal material solution, the taste-deficient negative sample solution, and the mixed reference solution of Compound Shuyangquan Granules.

6. The method for establishing the fingerprint spectrum of Compound Shuyangquan Granules according to claim 5, characterized in that, The preparation method of the mixed reference solution includes the following steps: dissolving neomangiferin, mangiferin, forsythoside I, forsythoside A, baicalin, rutin, naringin, hesperidin, baicalin, wogonin, apigenin, and baicalein in a solvent to obtain the mixed reference solution; the solvent is a methanol solution containing 0%~10% DMSO by volume. Of the 26 common peaks, peak 10 (retention time 19.2 min) is neomangiferin, peak 11 (retention time 23.5 min) is mangiferin, peak 12 (retention time 26.6 min) is forsythoside I, peak 14 (retention time 31.6 min) is forsythoside A, peak 15 (retention time 32.2 min) is baicalin, peak 16 (retention time 33.3 min) is rutin, peak 17 (retention time 34.5 min) is naringin, peak 18 (retention time 36.0 min) is hesperidin, peak 19 (retention time 42.1 min) is baicalin, peak 24 (retention time 49.3 min) is wogonin, peak 25 (retention time 51.2 min) is apigenin, and peak 26 (retention time 53.0 min) is baicalein. The remaining common peaks were not identified.

7. A method for establishing a comparative fingerprint spectrum of Compound Shuyangquan Granules, characterized in that, Includes the following steps: Take n batches of Compound Shuyangquan Granules and obtain the fingerprint chromatograms of each batch of Compound Shuyangquan Granules according to the method described above; analyze the fingerprint chromatograms of the n batches of Compound Shuyangquan Granules using the software of the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System to obtain the control fingerprint chromatogram of Compound Shuyangquan Granules; wherein, n≥10.

8. A comparative fingerprint spectrum of Compound Shuyangquan Granules obtained by the method of claim 7, characterized in that, The comparative fingerprint spectrum of the Compound Shuyangquan Granules, using mangiferin (peak 11) as the reference peak, shows the following relative retention times for each common peak: Peak 1: 0.224–0.248, Peak 2: 0.327–0.361, Peak 3: 0.347–0.383, Peak 4: 0.386–0.426, Peak 5: 0.486–0.538, Peak 6: 0.504–0.557, Peak 7: 0.560–0.618, Peak 8: 0.601–0.664, Peak 9: 0.670–0.740, Peak 10: 0.775–0.857, Peak 11: 1.000, Peak 12: 1.074–1.

187. Peak 13: 1.231-1.361; Peak 14: 1.280-1.414; Peak 15: 1.302-1.439; Peak 16: 1.347-1.489; Peak 17: 1.394-1.541; Peak 18: 1.456-1.609; Peak 19: 1.704-1.883; Peak 20: 1.814-2.005; Peak 21: 1.842-2.036; Peak 22: 1.902-2.102; Peak 23: 1.949-2.155; Peak 24: 1.994-2.204; Peak 25: 2.069-2.287; Peak 26: 2.144-2.

370.

9. A quality control method for Compound Shuyangquan Granules, characterized in that, Includes the following steps: Take the Compound Shuyangquan Granules to be tested, and obtain the chromatogram of the Compound Shuyangquan Granules to be tested according to the method described in claim 1. Evaluate the similarity between the obtained chromatogram of the Compound Shuyangquan Granules to be tested and the reference fingerprint chromatogram of the Compound Shuyangquan Granules. If the similarity is ≥0.90, the quality of the Compound Shuyangquan Granules to be tested is determined to be qualified.

10. A method for determining the content of chemical components in Compound Shuyangquan Granules, characterized in that, Includes the following steps: The content of chemical components in Compound Shuyangquan Granules was determined by high performance liquid chromatography. The chemical components were selected from neomangiferin, mangiferin, forsythoside I, forsythoside A, baicalin, and wogonin. The first step is to establish a standard curve for chemical composition: New mangiferin, mangiferin, forsythoside I, forsythoside A, baicalin, and wogonin were dissolved in methanol solution containing 0%~10% DMSO to obtain a mixed reference solution. The solution was analyzed by high performance liquid chromatography. The chromatographic peaks of each chemical component were determined based on their retention times, and the peak areas of the corresponding chemical components were obtained. Standard curves for each chemical component were established, and the linear regression equations and correlation coefficients r of each chemical component were obtained. In the mixed reference solution, the concentrations of neomangiferin, mangiferin, and forsythoside I were 5.07–101.4 µg / mL; the concentrations of forsythoside A and forsythoside A were 20.08–401.6 µg / mL; the concentrations of baicalin and forsythoside A were 100.6–2012 µg / mL; and the concentrations of wogonin and forsythoside B were 20.16–403.2 µg / mL. High performance liquid chromatography (HPLC) conditions include: Chromatographic column: Octadecylsilane-bonded silica gel column; Mobile phase: Phase A is an aqueous solution of formic acid with a volume fraction of 0.05%~0.15%, and Phase B is methanol; gradient elution is used with a volume fraction of 20%~95% Phase A and 5%~80% Phase B; flow rate is 0.9~1.1 mL / min; column temperature is 38~48℃; injection volume is 8~12 µl; detection wavelength is 200~600 nm; The second step is to obtain the chromatographic peak areas of the chemical components in the compound Shuyangquan granules to be tested. Add mass M of compound Shuyangquan granules to volume V of pure water or methanol aqueous solution with a volume fraction of 8%~32%, shake well, with a mass-to-volume ratio of 1:10~30, extract by ultrasonication, cool to room temperature, make up the weight loss, shake well, centrifuge to collect the supernatant, filter, and obtain the test solution. The test solution was detected under the same high-performance liquid chromatography conditions as in the first step. The chromatographic peaks of each chemical component were determined based on their retention times, and the peak areas of the corresponding chemical components were obtained. The third step is to determine the content of chemical components in the compound Shuyangquan granules to be tested; Based on the established standard curves for each chemical component, the chromatographic peak areas of the chemical components of the Compound Shuyangquan Granules to be tested are substituted into the corresponding linear regression equations. The concentration C of the corresponding component in the test solution is calculated using the external standard method. Then, the content of each chemical component in the Compound Shuyangquan Granules to be tested is calculated using the formula: Content = C × V / M, where V is the volume of the test solution and M is the sample weight.

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