Analysis method of flavonoids in shensheng pill

Abstract

This application provides an analytical method for flavonoid components in Shenrong Gubenhuanshao Pills, employing ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS / MS) for detection, followed by analysis of the mass spectrometry data. Compared with existing technologies, this application has the following advantages: The analytical method for flavonoid components in Shenrong Gubenhuanshao Pills and the method for detecting flavonoid components entering the bloodstream from Shenrong Gubenhuanshao Pills provided in this application, by constructing flavonoid mass spectrometry data of Shenrong Gubenhuanshao Pills, combined with GNPS molecular network and MS-DIAL / MS-FINDER mass spectrometry processing methods, identifies the flavonoid components in this preparation, further determining the flavonoid components entering the bloodstream and their core targets. This provides a methodological basis for elucidating the quality evaluation of Shenrong Gubenhuanshao Pills and also provides a scientific basis for its pharmacodynamic material basis research.

Description

Technical Field

[0001] This application relates to the field of pharmaceutical component analysis technology, specifically to an analytical method for flavonoid components in Shenrong Gubenhuanshao Pill. Background Technology

[0002] Shenrong Guben Huanshao Pill (SRGBHSP) is a compound traditional Chinese medicine preparation refined using modern pharmaceutical technology from 65 kinds of Chinese herbs, including ginseng, deer antler, aconite, and cinnamon. It has the effects of tonifying the kidneys and strengthening yang, replenishing qi and solidifying the body, replenishing essence and stopping seminal emission, and strengthening muscles and bones. All 50 of the herbal ingredients contained in it contain flavonoids, and the pharmacological activities of these components, such as anti-cancer, kidney-protective, and anti-osteoporosis effects, are related to the efficacy of SRGBHSP. Therefore, flavonoids may be the main active ingredient in SRGBHSP. However, there is currently no research on the flavonoid components in SRGBHSP. Summary of the Invention

[0003] To address the aforementioned technical limitations, this application proposes an analytical method for flavonoid components in Shenrong Gubenhuanshao Pills, which overcomes the deficiencies and defects mentioned in the background art.

[0004] To achieve the above objectives, this application adopts the following technical solution: The inventive point of this application is to provide an analytical method for flavonoid components in the ginseng and deer antler tonifying pill, the analytical method comprising the following steps: S1. Accurately weigh the powder of Shenrong Gubenhuanshao Pill, perform ultrasonic extraction, filter the supernatant to obtain the total flavonoid extract, concentrate and freeze dry, centrifuge to obtain the supernatant after adjusting the volume, and filter through a filter membrane to obtain the test solution of Shenrong Gubenhuanshao Pill. S2. The test solution of Shenrong Gubenhuanshao Pill obtained in step S1 was detected by ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS / MS) to obtain high-resolution mass spectrometry data of the test solution. S3. Analyze the high-resolution mass spectrometry data of the test solution obtained in step S2 to achieve the analysis and identification of flavonoid components in Shenrong Gubenhuanshao Pill.

[0005] Optionally, in the above analytical method, in step S1, the powder of Shenrong Gubenhuanshao Pill is ultrasonically extracted twice with 80% ethanol at a ratio of 1g:25ml, the extraction temperature is 70℃, and the extraction time is 1h each time; after freeze-drying, it is diluted with 70% methanol; centrifuged at 12000 rpm for 10 min, and filtered through a 0.22 μm filter membrane to obtain the test solution of Shenrong Gubenhuanshao Pill.

[0006] Optionally, in the above analytical method, the chromatographic conditions in step S2 are as follows: Infinity LabPoroshell 120 EC-C18 column, 150 mm × 2.1 mm, 1.9 μm; column temperature 30℃; mobile phase: 0.1% formic acid water as mobile phase A, acetonitrile as mobile phase B; gradient elution 0~3 min, 10~20% B; 3~10 min, 20~25% B; 10~20 min, 25~30% B; 20~35 min, 30~58% B; 35~40 min, 58~85% B; 40~45 min, 85~10% B; flow rate: 0.3 mL / min, injection 3 μL.

[0007] Optionally, in the above analytical method, in step S2, the mass spectrometry conditions are as follows: the ionization source is an electrospray ionization source (ESI); the drying gas is N2; the drying gas temperature is 225℃; the drying gas flow rate is 10 L / min; the nebulizer gas pressure is 25 psi; the capillary voltages in positive ion mode and negative ion mode are 3500 V and 4000 V, respectively; and the scanning mass range is... m / z 20~1700, fragmentation voltage 80 V; secondary mass spectrometry MS 2 The collision energies in positive and negative ion modes are 10, 20, and 40 V.

[0008] Optionally, the analytical identification method in step S3 of the above-mentioned analytical method is as follows: first, a flavonoid mass spectrometry database of Shenrong Gubenhuanshao Pills is constructed, and then, based on the ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS / MS) technology in step S2, combined with various mass spectrometry data processing strategies, the flavonoid components in Shenrong Gubenhuanshao Pills are analyzed and identified.

[0009] Optionally, the above analytical method, specifically the method for constructing the flavonoid mass spectrometry database of Shenrong Gubenhuanshao Pill, involves: collecting and constructing the flavonoid mass spectrometry database SRFMSD containing 309 flavonoid compounds in the formula of Shenrong Gubenhuanshao Pill by consulting authoritative databases such as the Chinese Academy of Sciences Chemistry Database, CNKI, PubChem, SciFinder, Reaxys, Web of Science, Moxoc Chemistry, ZINC, ChemSpider, ChEMBL, and MassBank.

[0010] Optionally, in the above analytical method, the mass spectrometry data processing strategy is selected as follows: total ion chromatogram combined with the flavonoid mass spectrometry database SRFMSD, the Global Natural Product Molecular Network (GNPS), and MS-DIAL / MS-FINDER for the analysis and identification of flavonoid components.

[0011] The second inventive point of this application is to provide the use of the above-mentioned analytical method in the identification, quality identification, quality testing, quality evaluation, or quality control of flavonoid components in traditional Chinese medicine compositions containing Shenrong Gubenhuanshao Pills.

[0012] The third inventive point of this application is to provide a method for detecting the flavonoid components in the blood of Shenrong Gubenhuanshao Pills, including the following steps: T1. Ginseng and Deer Antler Strengthening Pills were suspended in water and administered to rats by gavage. Blood samples were collected at multiple time points after gavage. The blood was centrifuged and the supernatant was collected. Acetonitrile was added to the serum sample for protein precipitation. After vortexing and sonication, the sample was centrifuged and the supernatant was filtered to remove impurities. The sample was dried with nitrogen and then reconstituted with 50% methanol solution. The sample was centrifuged again and the supernatant was collected to obtain the sample for analysis. T2. The sample obtained in step T1 was analyzed using ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry (UHPLC-Q-Exactive Orbitrap MS). The chromatographic conditions were as follows: column: ACQUITY UPLC BEH C18; mobile phase: 2% acetonitrile-water containing 0.1% formic acid as mobile phase A, acetonitrile containing 0.1% formic acid as mobile phase B; gradient elution: 0–0.5 min, 2% B; 0.5–3.5 min, 2–25% B; 3.5–7.5 min, 25–35% B; 7.5–11 min, 35–50% B; 11–13 min, 50–95% B; 13–14.4 min, 95% B; 14.4–14.5 min, 95–2% B; 14.5–16 min, 2% B; flow rate: 0.2 mL / min; injection rate: 3 mL / min. μL; column temperature 40℃; mass spectrometry conditions: sample ionized by electrospray ionization, mass spectrometry signals acquired using positive and negative ion scanning modes respectively; scan range 70-1050 μL. m / z Sheath gas flow rate 50 arb, auxiliary gas flow rate 13 arb, heating temperature 450℃, capillary temperature 320℃, spray voltage in positive ion scanning mode 3500V, spray voltage in negative ion scanning mode -3000V, collision energy 20, 40, 60 V, Full MS resolution 70000, MS resolution 2 It is 17500; T3. Analyze the mass spectrometry data of the sample obtained in step T2 to achieve the analysis and identification of the flavonoid components in the blood of Shenrong Gubenhuanshao Pill.

[0013] Compared with the prior art, this application has the following advantages: The analytical methods for flavonoid components in Shenrong Gubenhuanshao Pill and the detection methods for blood-entering flavonoid components provided in this application, by constructing mass spectrometry data of flavonoids in Shenrong Gubenhuanshao Pill and combining GNPS molecular network and MS-DIAL / MS-FINDER mass spectrometry processing methods, identify the flavonoid components in this preparation, further determine the blood-entering flavonoid components and their core targets, provide methodological basis for clarifying the quality evaluation of Shenrong Gubenhuanshao Pill, and also provide scientific basis for its pharmacodynamic material basis research. Attached Figure Description

[0014] Figure 1 The diagram shown illustrates the network relationship between some flavonoid compounds and efficacy in the flavonoid chemical composition mass spectrometry database of Shenrong Gubenhuanshao Pill, an embodiment of this application. Red represents Shenrong Gubenhuanshao Pill; green represents the name of the medicinal material; blue represents flavonoid compounds in the medicinal material; purple represents the pharmacological activity of the flavonoid compounds; and yellow represents the indications and functions of Shenrong Gubenhuanshao Pill.

[0015] Figure 2 The figure shown is a rutin standard curve established during the preparation of total flavonoids in the ginseng and deer antler tonifying pill, which is an example of the analytical method for flavonoid components in the ginseng and deer antler tonifying pill according to an embodiment of this application.

[0016] Figure 3 The diagram shown illustrates the single-factor experimental results for preparing total flavonoids in Shenrong Gubenhuanshao Pills, as described in an embodiment of this application. Figure 3 A represents the effect of the number of extractions on the flavonoid content. Figure 3 B represents the effect of ethanol volume fraction on flavonoid content. Figure 3 C represents the effect of the material-to-liquid ratio on the flavonoid content. Figure 3 D represents the effect of extraction time on flavonoid content. Figure 3 E represents the effect of extraction temperature on flavonoid content.

[0017] Figure 4 The image shown is an example of an analytical method for the flavonoid components of Shenrong Gubenhuanshao Pills, illustrating the total ion chromatograms (TICs) of the Shenrong Gubenhuanshao Pills test sample in both positive and negative ion modes.

[0018] Figure 5 The image shown is a total ion chromatogram of a rat serum sample from a Shenrong Gubenhuanshao Pill, as described in one embodiment of this application; wherein... Figure 5 A represents blank serum ES. + , Figure 5 B represents serum ES after drug administration. + , Figure 5 C represents blank serum ES. - , Figure 5D represents serum ES after drug administration.

[0019] Figure 6 The following is a pharmacokinetic curve of each compound after administration, as shown in one embodiment of this application: 1: baicalin; 2: hesperidin; 3: astragalin; 4: icariin; 5: naringenin; 6: apigenin-7-O-β-D-glucoside.

[0020] Figure 7 The diagram shows a network of 88 flavonoids and their corresponding targets in one embodiment of this application; orange diamond nodes represent the 88 flavonoids, and green circular nodes represent their corresponding targets. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this application clearer, a more detailed description is provided below. However, it should be understood that the description herein is merely for explaining this application and is not intended to limit its scope.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this application. All reagents and instruments used herein are commercially available, and the characterization methods involved can be found in relevant descriptions in the prior art, and will not be repeated here.

[0023] To further understand this application, the following detailed description is provided in conjunction with the preferred embodiments.

[0024] Example 1 Construction of a mass spectrometry database of flavonoid compounds in Ginseng and Deer Antler Strengthening Pill (SRGBHSP): To elucidate the structure of flavonoids in SRGBHSP, literature from the past 20 years was retrieved from 11 databases including SciFinder and PubChem. Research data on flavonoids from 50 herbal medicines in the SRGBHSP formulation were collected and standardized. A mass spectrometry database (SRGBHSPFlavonoids Mass Spectrum Database, SRFMSD) was successfully constructed, containing 309 flavonoids that may be present in SRGBHSP. This database includes 84 flavonoids, 67 isoflavones, 48 ​​flavonols, 37 dihydroflavonoids, 36 flavans, 25 chalcones, 5 high isoflavones, 4 anthocyanins, and 3 aurones. Further research based on network association analysis confirmed a high correlation between flavonoids and the efficacy of SRGBHSP, providing a basis for subsequent analysis of flavonoid components.

[0025] 1. Experimental Methods 1.1 Data Acquisition and Standardization Using "50 medicinal plant names (such as Scutellaria baicalensis)" Scutellaria baicalensis The search employed a combination of search terms, including "chemical composition," "LC-MS / MS," etc., to collect information on flavonoids reported over the past 20 years from authoritative databases such as the Chinese Academy of Sciences Chemistry Database, CNKI, PubChem, SciFinder, Reaxys, Web of Science, Moxochem, ZINC, ChemSpider, ChEMBL, and MassBank. This included molecular formulas, precise molecular weights, quasi-molecular ion peaks, and characteristic mass spectrometry fragments. Based on this, the compounds were integrated and screened. First, the collected flavonoids were normalized according to international compound identification standards to eliminate duplicate entries due to naming differences. Second, the compounds were matched to three-dimensional structures using SciFinder and PubChem structure search platforms to exclude isomers and compounds with unclear stereoconfigurations. Finally, the secondary fragments of the collected compounds were further examined using MassBank to ensure that the obtained mass spectrometry fragments conformed to the compound fragmentation patterns.

[0026] 1.2 Database Establishment A database of flavonoid compounds from the Shenrong Gubenhuanshao Pill was constructed using Excel software. First, a blank database was created based on a general template from the literature. The cleaned data was then systematically entered according to the template format. Finally, functions such as "find and replace" were used to correct the format and verify the content of compound names, molecular formulas, and mass spectrometry information in the database. Through this process, a high-precision mass spectrometry database of flavonoid compounds from the Shenrong Gubenhuanshao Pill was constructed.

[0027] 1.3 Network Association Analysis of Flavonoids and the Efficacy of Shenrong Gubenhuanshao Pill First, the indications and functions of Shenrong Gubenhuanshao Pills were scientifically classified through multi-source data integration; the sources and pharmacological activities of flavonoids were reviewed in statistical databases. Then, network association analysis was performed using Gephi 0.9.2 software: the classification system of Shenrong Gubenhuanshao Pills' indications and functions was matrixed with the pharmacological activity data of flavonoid components to construct an association network containing multiple layers of nodes: "Traditional Chinese Medicine - Flavonoids - Pharmacological Activity - Indications and Functions".

[0028] 2. Experimental Results and Discussion 2.1 Database Establishment By reviewing relevant literature on the chemical components of 50 herbal medicines in Shenrong Gubenhuanshao Pills, a relatively comprehensive collection of flavonoids with mass spectrometry information was obtained. Subsequent standardized procedures, including isomorphism standardization, chemical structure verification, and mass spectrometry data comparison, were implemented. A mass spectrometry prediction database of 309 flavonoids in Shenrong Gubenhuanshao Pills was successfully constructed using Excel software. This database covers 84 flavonoids, 67 isoflavones, 48 ​​flavonols, 37 dihydroflavonoids, 36 flavans, 25 chalcones, 5 high isoflavones, 4 anthocyanins, and 3 aurones, as shown in Table 2-1.

[0029] 2.2 Network Association Analysis of Flavonoids and the Efficacy of Shenrong Gubenhuanshao Pill A systematic analysis method was used to conduct a multidimensional analysis of the functions and indications of Shenrong Gubenhuanshao Pills, classifying its efficacy into ten categories: strengthening muscles and bones, anti-aging, enhancing immunity, treating impotence and premature ejaculation, treating infertility, treating tinnitus and deafness, treating chronic nephritis, treating neurasthenia, treating ulcerative colitis, and treating asthma. Subsequently, by searching databases such as CNKI and PubMed, the sources and pharmacological activities of the main flavonoids in the prescription were systematically analyzed, constructing a network relationship diagram containing 35 kinds of traditional Chinese medicine, 30 kinds of flavonoids, 25 kinds of pharmacological activities, and 10 kinds of efficacy of Shenrong Gubenhuanshao Pills. Figure 1 Analysis revealed that isorhamnetin, hyperoside, styracin, and icariin, derived from herbs such as Epimedium, Tribulus terrestris, and Lycium barbarum, possess nephroprotective effects, which are highly consistent with the efficacy of Shenrong Gubenhuanshao Pills in treating chronic nephritis, impotence, and premature ejaculation. Astragaloside, isorhamnetin, isoliquiritigenin, acaciain, icariin, verbascoside, and styracin can exert anti-osteoporosis effects, explaining the pharmacological effects of Shenrong Gubenhuanshao Pills in strengthening muscles and bones. Meanwhile, 10 flavonoids, including baicalin, alfalfa extract, and vitexin, have neuroprotective effects, providing a material basis for Shenrong Gubenhuanshao Pills to improve neurasthenia. Therefore, the flavonoids in the database are significantly correlated with the overall efficacy of Shenrong Gubenhuanshao Pills in tonifying the kidneys, strengthening yang, and replenishing essence.

[0030] 3. Summary By integrating 11 database systems with Excel, a mass spectrometry database of flavonoid components in Shenrong Gubenhuanshao Pills was successfully constructed. This database covers 309 flavonoid compounds across 9 categories, including flavonoids (84 types), isoflavones (67 types), and flavonols (48 types). The study employed data processing strategies such as synonym standardization, multi-source structural formula verification, and cross-validation using PubChem and MassBank mass spectrometry data, significantly improving the accuracy and reliability of the data. The database features multi-parameter search capabilities, including compound name and molecular formula. Precise molecular weight and molecular formula can quickly identify candidate compounds, which can then be verified using mass spectrometry data. This effectively achieves precise screening and identification of flavonoid components, optimizing the efficiency of traditional identification processes and enabling initial screening of flavonoid compounds without relying on reference standards, significantly reducing the cost of the identification process. The establishment of this database systematically reveals the chemical diversity of flavonoid components in Shenrong Gubenhuanshao Pills, providing crucial data support for component identification.

[0031] Subsequently, by establishing a network relationship between the pharmacological activities of flavonoids and the efficacy system of Shenrong Gubenhuanshao Pills, the core efficacy role of these components in the formulation was systematically revealed. Network correlation analysis showed that the antioxidant, renal protective, and neuroprotective biological characteristics of flavonoids in the 50 herbal ingredients of Shenrong Gubenhuanshao Pills were significantly correlated with the overall efficacy of the formulation, thus indicating that flavonoids are the core material basis for the clinical therapeutic value of Shenrong Gubenhuanshao Pills.

[0032] Example 2 Analysis of flavonoid components in Shenrong Gubenhuanshao Pills based on UHPLC-Q-TOF-MS / MS technology: To identify the flavonoids in sRGBHSP, a method for preparing the total flavonoid sample was first optimized using single-factor and orthogonal experiments. Specifically, an appropriate amount of sRGBHSP powder was accurately weighed, and extracted twice with ultrasonic extraction at 70℃ using 80% ethanol as solvent, a material-to-liquid ratio of 1:25, and a time of 60 min. The filtrates were combined, concentrated under reduced pressure, and then freeze-dried to obtain the total flavonoid extract. The total flavonoid content in the preparation was determined to be 14.99 mg / g using the sodium nitrite-aluminum nitrate colorimetric method. Next, UHPLC-Q-TOF-MS / MS was performed using an InfinityLab Poroshell 120EC-C18 column, a 0.1% formic acid-water-acetonitrile mobile phase, and an electrospray ionization source. Based on total ion chromatograms, and combined with three mass spectrometry data processing strategies—SRFMSD, Global Natural Products Social Molecular Network (GNPS), and MS-DIAL / MS-FINDER—131 flavonoids were successfully identified, including 58 flavonoids, 24 isoflavones, 21 flavonols, 16 dihydroflavonoids, 5 chalcones, 4 flavans, 2 anthocyanins, and 1 orange ketone.

[0033] 1. Instruments and Materials Instruments: Electronic analytical balance (FA2004, Shanghai Dudan Instrument Co., Ltd.); rotary evaporator (RE-5299, Shanghai Yarong Biochemical Instrument Factory); freeze dryer (CTFD-20S, Qingdao Yonghe Chuangxin Electronic Technology Co., Ltd.); ultraviolet spectrophotometer (UV-1700, SHIMADUZ Corporation, Japan); Agilent 1290 Infinity II UHPLC tandem Agilent 6560Q-TOF mass spectrometry system (Agilent Technologies, Santa Clara, CA, USA).

[0034] Materials: Ginseng and Deer Antler Tonic Pills (Lanzhou Foci Co., Ltd., batch number 220912; prepared according to the 11th volume of the "Pharmaceutical Standards of the Ministry of Health of the People's Republic of China for Traditional Chinese Medicine Compound Preparations" WS3-B-2179-96); anhydrous ethanol (analytical grade, Jinan Yuansu Chemical Co., Ltd.); NaOH (analytical grade, Tianjin Damao Chemical Reagent Factory); Al(NO3)3 (analytical grade, Tianjin Damao Chemical Reagent Factory); NaNO2 (analytical grade, Tianjin Baishi Chemical Co., Ltd.); rutin reference standard (purity ≥98%, batch number 153-18-4, Sichuan Weikeqi Biotechnology Co., Ltd.); formic acid (analytical grade, Jinan Yuansu Chemical Co., Ltd.); acetonitrile mass spectrometry grade (Beijing Bailingwei Technology Co., Ltd.); water was Watson's purified water.

[0035] 2. Experimental Methods 2.1 Preparation of total flavonoids from Shenrong Gubenhuanshao Pills The ginseng and deer antler tonifying pills were pulverized, and 1 g of powder was weighed into an Erlenmeyer flask. Different material-to-liquid ratios and different concentrations of ethanol solution were used as extraction solvents in an ultrasonic cleaner. Different extraction times, temperatures, and extraction cycles were designed. After ultrasonic treatment, the supernatant was filtered to obtain the total flavonoid extract, which was then concentrated and freeze-dried to obtain the total flavonoid sample from the ginseng and deer antler tonifying pills.

[0036] 2.1.1 Determination of total flavonoid content The content of flavonoids in Shenrong Gubenhuanshao Pills was determined using the NaNO2-Al(NO3)3-NaOH colorimetric method. 5.11 mg of rutin standard was accurately weighed, dissolved in 70% ethanol, and diluted to 25 mL to prepare a 0.2044 mg / mL rutin stock solution. 0.0, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mL of the rutin stock solution were added to 10 mL volumetric flasks. For each concentration gradient, 0.4 mL of 5% sodium nitrite solution was added, shaken well, and allowed to stand for 6 min. Then, 0.4 mL of aluminum nitrate solution was added, shaken again, and allowed to stand for 6 min. Finally, 4 mL of 4% sodium hydroxide solution was added, and the solution was brought to the mark with 70% ethanol. After standing at room temperature for 15 min, the absorbance of the solution was measured three times at 510 nm. The formula for calculating the total flavonoid content is as follows:

[0037] W It is the total flavonoid content (mg / g); C It is the mass concentration of the test solution (μg / mL); N It is the dilution factor; V It is the volume of the liquid to be tested (mL); m The weight (g) of the powder in the Ginseng and Deer Antler Strengthening Pill is as follows.

[0038] 2.1.2 Single-factor experiment The extraction conditions of total flavonoids from Shenrong Gubenhuanshao Pill were optimized through single-factor experiments. This study selected the number of extractions, ethanol volume fraction, solid-liquid ratio, extraction time, and extraction temperature for single-factor experiments.

[0039] Extraction times: Under fixed conditions of 80% ethanol volume fraction, 1:20 g / mL material-to-liquid ratio, 60 min extraction time, and 60°C extraction temperature, the effects of 1, 2, and 3 extraction times on the total flavonoid content of Shenrong Gubenhuanshao Pills were investigated.

[0040] Ethanol volume fraction: Under the conditions of fixed extraction times (2 times), material-to-liquid ratio (1:20 g / mL), extraction time (60 min), and extraction temperature (60°C), the effects of ethanol volume fractions of 60%, 70%, 80%, and 90% on the total flavonoid content of Shenrong Gubenhuanshao Pill were investigated.

[0041] Material-liquid ratio: Under the conditions of fixed extraction times (2 times), ethanol volume fraction (80%), extraction time (60 min), and extraction temperature (60°C), the effects of material-liquid ratios of 1:10, 1:15, 1:20, 1:25, and 1:30 g / mL on the total flavonoid content of Shenrong Gubenhuanshao Pill were investigated.

[0042] Extraction time: Under the conditions of fixed extraction times of 2 times, ethanol volume fraction of 80%, material-to-liquid ratio of 1:20 g / mL, and extraction temperature of 60°C, the effects of extraction times of 30, 60, 90, and 120 min on the total flavonoid content of Shenrong Gubenhuanshao Pill were investigated.

[0043] Extraction temperature: Under the conditions of fixed extraction times of 2 times, ethanol volume fraction of 80%, material-to-liquid ratio of 1:20 g / mL, and extraction time of 60 min, the effects of extraction temperatures of 40, 50, 60, 70, and 80℃ on the total flavonoid content of Shenrong Gubenhuanshao Pill were investigated.

[0044] 2.1.3 Orthogonal Experiment The extraction process was optimized using orthogonal experiments based on the results of single-factor experiments. The orthogonal experiment adopted an L9 (32) quadrature design. 3 An orthogonal design was used to optimize the effects of ethanol volume fraction (A), extraction temperature (B), and material-liquid ratio (C) on the total flavonoid content in Shenrong Gubenhuanshao Pills. The factor levels are shown in Table 1.

[0045] Table 1

[0046] 2.1.4 Data Processing Data processing and plotting were performed using statistical software SPSS 25.0, Excel, Origin 2019, and GraphPad Prism 9.

[0047] 2.2 Chromatographic and Mass Spectrometry Conditions Chromatographic conditions: InfinityLab Poroshell 120 EC-C18 column (150 mm × 2.1 mm, 1.9 μm); column temperature 30℃; mobile phase: 0.1% formic acid water (A) - acetonitrile (B); gradient elution: 0–3 min, 10–20% B; 3–10 min, 20–25% B; 10–20 min, 25–30% B; 20–35 min, 30–58% B; 35–40 min, 58–85% B; 40–45 min, 85–10% B; flow rate: 0.3 mL / min; injection: 3 μL.

[0048] Mass spectrometry conditions: Ionization source: electrospray ionization (ESI); drying gas: N2; drying gas temperature: 225℃; drying gas flow rate: 10 L / min; nebulizer gas pressure: 25 psi; capillary voltages: 3500 V and 4000 V in positive and negative ion modes, respectively; scanning mass range: m / z 20~1700, fragmentation voltage 80 V. Secondary mass spectrometry (MS) 2 The collision energies in the positive and negative ion modes are 10, 20, and 40 V.

[0049] 2.3 Preparation of test solution Accurately weigh 100 mg of the prepared total flavonoid sample, dilute to 10 mg / mL with 70% acetonitrile, centrifuge at 12000 rpm for 10 min, and filter the supernatant through a 0.22 μm filter membrane for analysis.

[0050] 2.4 Annotations for the self-built flavonoid database Precise mass-to-charge ratio of chromatographic peaks obtained based on high-resolution mass spectrometry (HSMS) m / z The data were used to derive candidate molecular formulas through elemental composition calculations. Then, multi-level matching (mass deviation threshold ±10 ppm) was performed with the self-built mass spectrometry database of flavonoid components in Shenrong Gubenhuanshao Pills. Combined with retention time correlation analysis and fragment ion matching degree verification, the accurate identification of flavonoid components in Shenrong Gubenhuanshao Pills was achieved.

[0051] 2.5 Construction and Annotation of GNPS Molecular Networks The raw MS / MS data obtained from the experiments were converted into mzML format using ProteoWizard MSConvert software, and then efficiently transferred to the Global Network of Natural Product Molecular Data (GNPS) platform via the Secure Transfer Protocol (SFTP) client WinSCP. During the molecular network construction phase, the parameters "Precursor Ion Mass Tolerance" was set to 0.02, "Fragment Ion Mass Tolerance" to 0.02, and "Minimum cosine score" to 0.70 to establish the initial network structure. Furthermore, to improve network specificity, a ranking filtering strategy was implemented (retaining only the edges between the top 10 most similar nodes). Finally, the maximum size of the molecular network was set to 100, and the lowest-scoring edges were removed from the molecular network until the network size fell below this threshold.

[0052] Molecular network visualization exhibits the following characteristics: nodes represent compounds (with precise annotation). m / z (Values), blue nodes represent known compounds directly matched to the GNPS spectral library, and each edge reflects the MS / MS spectrum similarity index. Based on the principle of spectral similarity, the chemical structure of adjacent nodes can be deduced using the structure of known compounds and their corresponding mass spectra, thus completing the annotation of unknown substances. GNPS-based visualization tools are used to analyze the acquired MS... 2 After the data is visualized, the generated molecular network is exported from GNPS in ".graphml" format and imported into Cytoscape for visualization and analysis.

[0053] 2.6 MS-DIAL / MS-FINDER Combined Analysis First, a standardized data processing workflow was established on the MS-DIAL 5.5.2 platform. Parameter settings included: ionization mode set to Soft ionization; separation mode set to Chromatography; collision mode set to CID / HCD; primary and secondary mass spectrometry data types selected as Profile data; target omics set to Metabolomics; primary mass tolerance set to 0.01 Da; secondary mass tolerance set to 0.025 Da; peak height threshold set to 1000; smoothing level set to 2 scans; minimum peak width set to 5 scans; and the selected addition ions in positive ion mode were [M+H]. + [M+NH4] + [M+Na] + and [2M+H] + The additive ions selected for the negative ion mode are [MH]. - [M+HCOO] - and [M-H2O-H]- Other settings are default. Additionally, when identifying metabolites using MS-DIAL, the corresponding database must be imported; MSP files can be downloaded directly from MS-DIAL and imported. After MS-DIAL analysis, compounds with secondary mass spectrometry validation (MS / MS) are prioritized for double validation, combining retention time and fragment ion matching (>80%).

[0054] A multi-database joint search strategy (covering 15 cross-species metabolomics databases such as MassBank and HMDB, as well as natural product-specific databases such as COCONUT and UNPD) was used to obtain potential candidate structures of compounds, which were then ranked according to spectral similarity scores. The results will display the top-ranked candidate structures; however, if the critical fragment ion breakage pathway cannot be reasonably explained by the candidate structures, or if multiple structures have the same or similar matching scores, manual screening combining chemical structures and literature data is required.

[0055] 3. Experimental Results and Discussion 3.1 Preparation of total flavonoids from Shenrong Gubenhuanshao Pills 3.1.1 Rutin Standard Curve The obtained rutin standard curve equation is Y = 0.0095X - 0.0082 (r = 0.9995), showing good linearity in the range of 10.22–61.32 μg / mL. Figure 2 As shown.

[0056] 3.1.2 Results and Analysis of Single-Factor Experiments (1) Effect of extraction times on flavonoid content Depend on Figure 3 As shown in A, after two extractions, the total flavonoid content of Shenrong Gubenhuanshao Pills increases slowly, but the effect on the total flavonoid content is not significant. This indicates that two extractions can extract most of the flavonoid compounds in the test solution. Therefore, the optimal number of extractions is two.

[0057] (2) Effect of ethanol volume fraction on flavonoid content Depend on Figure 3As shown in Figure B, the total flavonoid content initially increases and then decreases within the ethanol volume fraction range of 60%-90%. Flavonoids generally exist in the form of flavonoid glycosides and flavonoid aglycones, which have different polarities. Therefore, solvent polarity is a key parameter in the extraction process. By adjusting the ethanol volume fraction, flavonoids of different polarities can be extracted. When the ethanol volume fraction increases in the 60-80% range, the flavonoid yield shows a significant upward trend, reaching its maximum at 80%. This phenomenon is mainly attributed to the fact that as the ethanol volume fraction increases, the polarity of the solvent and flavonoids gradually converges, and the reduction in their polarity effectively enhances the dissolution efficiency of flavonoids, thereby transferring more flavonoids to the extract. When the ethanol volume fraction exceeds 80%, the flavonoid extraction rate decreases accordingly. This may be because at higher ethanol volume fractions, the polarity of the ethanol solution weakens, and more low-polarity components in the sample are over-dissolved, leading to a decrease in the flavonoid extraction rate. Therefore, an ethanol volume fraction of 80% is chosen for the extraction of total flavonoids.

[0058] (3) Effect of material-to-liquid ratio on flavonoid content Figure 3 C shows the effect of the solid-liquid ratio on the total flavonoid content. The flavonoid extraction rate from Shenrong Gubenhuanshao Pills was highest at a solid-liquid ratio of 1:25 g / mL, reaching 11.95 mg / g. Within the range of 1:10-1:25 g / mL, the solubility of flavonoids in the solution gradually increased with increasing solvent volume, leading to a gradual increase in flavonoid content. When the solid-liquid ratio exceeded 1:25 g / mL, the solvent volume increased, but at this point, the dissolution of flavonoids reached its maximum, while other components dissolved relatively more, resulting in a decrease in the content of flavonoids during extraction. Furthermore, an excessively high solid-liquid ratio may also increase mass transfer resistance, causing a decrease in flavonoid leaching efficiency and affecting the extraction effect. Therefore, a solid-liquid ratio of 1:25 g / mL was ultimately selected.

[0059] (4) Effect of extraction time on flavonoid content Depend on Figure 3 As shown in D, the total flavonoid yield significantly increased within the time range of 30 to 60 minutes, and then stabilized between 60 and 120 minutes. This trend is likely due to the fact that when the ultrasonic time is too short, less flavonoid dissolves, resulting in a low dissolution rate. However, as the sample and solvent come into sufficient contact and are extracted for a certain period, the cavitation and mechanical vibration effects of ultrasound enhance the random thermal motion of molecules, increasing the dissolution of total flavonoids. When the extraction time exceeds 60 minutes, the total flavonoid dissolution reaches its maximum, and the extraction rate reaches equilibrium. To avoid high-temperature damage to the flavonoid molecular structure caused by prolonged extraction and to reduce the precipitation of non-flavonoid substances, 60 minutes was ultimately chosen as the extraction time.

[0060] (5) Effect of extraction temperature on flavonoid content like Figure 3 E indicates that within the extraction temperature range of 40-80°C, flavonoid extraction initially increases and then decreases with increasing extraction temperature, reaching a high extraction rate of 14.98 mg / g at 70°C. In the initial stage of extraction, increasing temperature accelerates the reaction process, effectively enhancing the transfer of flavonoids from plant tissue to the solvent, thus achieving efficient extraction of the target components. Subsequently, as the temperature continues to rise, molecular motion accelerates, increasing mass transfer between the sample and the ethanol solution, further promoting the diffusion and solubility of flavonoids in the ethanol solution. However, when the extraction temperature exceeds 70°C, some heat-sensitive groups in flavonoids may be destroyed, or some active groups in flavonoids may become more reactive due to heat, reacting with other compounds and leading to a significant decrease in the flavonoid extraction rate. Furthermore, the extraction rates of other compounds may also increase rapidly, further reducing the extraction efficiency of flavonoids. Therefore, the results indicate that the optimal extraction temperature for flavonoids is 70°C.

[0061] 3.1.3 Orthogonal Experiment Results and Optimal Process Verification Based on the single-factor experiments, the extraction time was fixed at 60 min, and the extraction was performed twice. Three experimental factors were selected: ethanol volume fraction (A), extraction temperature (B), and solid-liquid ratio (C). The L9 (3) model was then implemented. 3 An orthogonal experiment was conducted to optimize the extraction process of total flavonoids from Shenrong Gubenhuanshao Pills. Table 2 shows the results of the orthogonal experiment on the total flavonoid extraction process, and Table 3 shows the analysis of variance. The importance of each factor, from highest to lowest, is A > B > C. That is, the ethanol volume fraction is the most important factor in the extraction process, followed by the extraction temperature, and finally the solid-liquid ratio. All three factors have a significant impact on the extraction yield of total flavonoids from Shenrong Gubenhuanshao Pills. P <0.05); the optimal process combination is A2B2C2, that is, ethanol volume fraction of 80%, extraction temperature of 70℃, material-liquid ratio of 1:25 g / mL. Under these conditions, the experiment was repeated three times, and the total flavonoid content of Shenrong Gubenhuanshao Pill reached 14.99 mg / g, with an RSD of 1.33%.

[0062] Table 2

[0063] Table 3

[0064] 3.2 Identification of flavonoids based on UHPLC-Q-TOF-MS / MS Three analytical methods—a self-built database, GNPS molecular network, and MS-DIAL / MS-FINDER—were used to rapidly and accurately identify flavonoids in Shenrong Gubenhuanshao Pills. A total of 131 flavonoids were identified, including 58 flavones, 24 isoflavones, 21 flavonols, 16 dihydroflavones, 5 chalcones, 4 flavans, 2 anthocyanins, and 1 orange ketone. The total ion chromatograms (TICs) of the ethanol extract of Shenrong Gubenhuanshao Pills in positive and negative ion modes are shown below. Figure 4 Retention time (t) of each chemical component R ), mass-to-charge ratio ( m / z The measured values, errors, and MS / MS fragmentation information of high-resolution mass spectrometry are shown in Table 4.

[0065] Table 4

[0066]

[0067]

[0068]

[0069]

[0070]

[0071] Example 3 Identification and target prediction of flavonoids entering the bloodstream in Shenrong Gubenhuanshao Pills: To clarify the active ingredients of SRGBHSP, based on the serum pharmacochemistry theory that the ingredients entering the blood may be the active ingredients, the serum of rats in the blank group and the SRGBHSP intragastric administration group was first detected by ultra-high performance liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry (UHPLC-Q-Exactive Orbitrap MS) technology. A total of 88 flavonoid ingredients entering the blood were identified, including 33 flavones, 24 flavonols, 15 dihydroflavones, 12 isoflavones, 2 chalcones, and 2 flavans. It was also found that compounds such as baicalin, hesperetin, astragalin, icariin, naringenin, and apigenin-7-O-β-D-glucoside had a relationship with drug time. Secondly, the reverse molecular docking technology was used to predict the action targets of the ingredients entering the blood. The PharmMapper database predicted 789 protein targets related to the ingredients entering the blood. After screening through the protein interaction network, 100 key targets were selected. Analysis showed that they were significantly related to a total of 10 types of diseases, including cancer, liver diseases, diabetes, kidney diseases, gastrointestinal diseases, neurodegenerative diseases, osteoporosis, prostate diseases, uterine diseases, and rheumatoid arthritis. GO analysis and KEGG analysis showed that the biological processes were mainly enriched in the positive regulation of gene expression, DNA transcription activation, and apoptosis inhibition, and the pathways were mainly enriched in cancer, hepatitis B, prostate cancer, AGE-RAGE signaling pathway in diabetic complications, etc. The results verified the correlation between the key targets and the 10 types of diseases. Further, 20 core targets such as TP53 and IL6 were screened for molecular docking with 88 flavonoid ingredients. The results showed that 17% of the "compound-target" scores were less than -7.0, and 82% of the "compound-target" scores were between -5.0 and -7.0, proving that the 88 ingredients entering the blood had a strong binding ability with the corresponding targets, and preliminarily confirming that these ingredients might be the efficacy ingredients of SRGBHSP.

[0072] 1. Instruments and Materials Instruments: VanquishTM UHPLC ultra-high performance liquid chromatography system; Q-Exactive TM Combined quadrupole Orbitrap TM Mass spectrometer (Thermo Scientific); low-temperature high-speed centrifuge (D1524R, DLAB); MX-S vortex mixer (Scilogex, USA).

[0073] Materials: SPF-grade male SD rats, weighing 180 - 200 g, purchased from the Experimental Animal Center of Lanzhou University, license number: SYXK(Gan)2023 - 0004. Shenrong Guben Huanshao Pills (Lanzhou Foci Co., Ltd., batch number 220912); formic acid (analytical pure, Jinan Yuansu Chemical Co., Ltd.); acetonitrile (mass spectrometry grade, Beijing百灵威科技有限公司); water was Watsons pure water.

[0074] 2. Experimental Methods 2.1 Analysis of Flavonoids in Shenrong Guben Huanshao Pills Entering the Blood 2.1.1 Preparation of Test Samples Solution Weigh 20 g of Shenrong Guben Huanshao Pills powder, add 50 mL of water, and stir with a glass rod until the solution becomes a homogeneous suspension state.

[0075] 2.1.2 Collection and Treatment of Serum Samples Take 12 male SD rats. After adapting to the SPF-level animal room for 7 days (temperature 23±2°C, humidity 55±10%, 12 h day-night rhythm), randomly divide them into a drug administration group and a blank group, with 6 rats in each group. The clinical dose of Shenrong Guben Huanshao Pills for adults is about 18 g / kg per day. After converting the dose by 4 times, the dosing dose for rats is about 6.48 g / kg. Rats in the drug administration group are gavaged 2 times a day for 3 consecutive days. Rats in the blank group are gavaged with an equal volume of pure water. Food is withheld 12 h before the last dose, but water is not restricted. Whole blood is collected from the marginal vein of the rat's eye at 5, 15, 30, 60, 120, 240, and 480 min after the last dose. Approximately 0.5 mL of blood is taken at each point and placed in a centrifuge tube. After the blood samples at each time point are left standing at room temperature for 30 min, they are centrifuged at 3000 rpm at 4°C for 15 min, and the supernatant is taken to obtain serum samples, which are stored in a -80°C refrigerator for future testing.

[0076] Take equal-volume serum samples (300 μL) at each time point and combine them into a mixed serum. Add 3 times the volume of pre-cooled acetonitrile for protein precipitation. After vortexing for 30 min, continue to sonicate for 30 min. The uniformly mixed samples are centrifuged at 13000 rpm at 4°C for 15 min. The supernatant is filtered through a 0.22 μm microporous filter membrane. The filtrate is concentrated to dryness under nitrogen. The residue is re-dissolved in 200 μL of 50% methanol solution, and centrifuged again for 10 min (13000 rpm, 4°C). The supernatant is taken for analysis.

[0077] 2.1.3 Chromatography and Mass Spectrometry Conditions Chromatographic conditions: Column: ACQUITY UPLC BEH C18 (100 mm × 2.1 mm id, 1.7 μm; Waters, Milford, USA); Mobile phase A: 2% acetonitrile-water (containing 0.1% formic acid); Mobile phase B: acetonitrile (containing 0.1% formic acid); Gradient elution: 0–0.5 min, 2% B; 0.5–3.5 min, 2–25% B; 3.5–7.5 min, 25–35% B; 7.5–11 min, 35–50% B; 11–13 min, 50–95% B; 13–14.4 min, 95% B; 14.4–14.5 min, 95–2% B; 14.5–16 min, 2% B; Flow rate: 0.2 mL / min; Injection: 3 μL; Column temperature: 40℃.

[0078] Mass spectrometry conditions: The sample was electrospray ionized, and mass spectrometry signals were acquired using both positive and negative ion scanning modes. Scan range: 70-1050 Ω·cm m / z Sheath flow rate 50 arb, auxiliary flow rate 13 arb, heating temperature 450℃, capillary temperature 320℃, spray voltage (positive mode) 3500 V, spray voltage (negative mode) -3000 V, impact energy 20, 40, 60 V, resolution (Full MS) 70000, resolution (MS) 2 17500.

[0079] 2.1.4 Data Processing Based on the in vitro flavonoid composition analysis results of Shenrong Gubenhuanshao Pills, serum samples were systematically analyzed using Xcalibur 4.3 software. Based on primary and secondary mass spectrometry data, the flavonoid components of Shenrong Gubenhuanshao Pills that entered the bloodstream were analyzed and identified.

[0080] 2.2 Prediction Study of Flavonoid Components in Shenrong Gubenhuanshao Pills 2.2.1 Acquisition and processing of small molecule structures Based on UHPLC-Q-Exactive Orbitrap MS, this study systematically identified 88 flavonoids in the serum of the Shenrong Gubenhuanshao Pill administration group, successfully constructing the first database of flavonoid compounds in the serum of Shenrong Gubenhuanshao Pill. All small molecule compounds were able to download their SDF format 3D conformations from PubChem or generate initial 3D configurations in ChemDraw. All structures were imported into MOE 2019.0102 software, where protonation state correction was first performed, followed by desalting and hydrogenation using the "Partial Charges" module, and charge distribution optimization to eliminate potential bond length and bond angle anomalies in the original structures. Then, energy minimization was performed on the small molecules, with a gradient set to 0.1 RMS kcal / (A).2 The algorithm gradually adjusts the atomic coordinates (in mol) to minimize the total molecular potential energy. The resulting molecule is stored in the MOE database in ".mdb" format.

[0081] 2.2.2 Construction of Compound-Target Sets Eighty-eight target compounds were uploaded to the PharmMapper server (http: / / www.lilab-ecust.cn / pharmmapper / ) in SDF file format. Small molecules were optimized using "Conformation Generation" to generate multiple conformations. The "Human Protein Targets" database was then selected, with other parameters left at their default settings. PharmMapper identifies potential drug targets by matching compounds to its internal pharmacophore model database using inverse pharmacophore mapping and calculates a standardized matching score (z'-score). Targets with a z'-score greater than 0.5 were selected for analysis. After removing common targets among the 88 flavonoids, a protein target set for all compounds was constructed.

[0082] 2.2.3 Constructing a protein-protein interaction network Based on the potential targets identified in Section 2.2.2, a protein-protein interaction (PPI) network was constructed using the STRING database, with the species designated as "Homo sapiens." Isolated nodes were removed, and a confidence score ≥ 0.9 was used. For ease of analysis, the generated TSV format network data was imported into Cytoscape 3.9.1 for visualization. To identify key targets, the CytoHubba plugin was used, and the MCC algorithm was selected for deep mining. The top 100 core targets were ranked based on their MCC scores for constructing networks linking these targets to related diseases. Subsequently, the top 20 targets were selected and molecular docking was performed with 88 compounds.

[0083] 2.2.4 Construction of the target-disease network The top 100 protein targets screened were analyzed using the DISEASES (https: / / diseases.jensenlab.org / Search), TTD (http: / / bidd.nus.edu.sg / group / ttd / ttd.asp), and ETCM (http: / / www.tcmip.cn / ETCM / ) databases. The disease categories of the targets were defined by their confirmed or clinically investigated pharmacological effects. A "target-disease" network was constructed using Cytoscape software to explore potential targets for the treatment of diseases by Shenrong Gubenhuanshao Pill.

[0084] 2.2.5 GO functional enrichment and KEGG pathway enrichment analysis The GO functional enrichment and KEGG pathway enrichment analyses were performed on the top 100 protein targets using the David 2021 database to elucidate the biological effects of potential targets and their roles in signaling pathways.

[0085] 2.2.6 Molecular docking verification of key components and core targets Gene information for the top 20 protein targets was retrieved from the HUGO Gene Nomenclature Committee database (HGNC, https: / / www.genenames.org / ). After obtaining the corresponding UniProt IDs, the optimal protein crystal structure was screened using the UniProt database (https: / / www.uniprot.org / ). The PDB file of the selected structure was then downloaded from the Protein Database (PDB, https: / / www.rcsb.org / ). The obtained protein receptor was imported into the MOE software, and structure optimization was performed using the "Quickprep" module. Missing atomic structures were added, hydrogen atoms were added, and three-dimensional protonation was performed to construct a complete receptor molecular model. A semi-flexible docking mode was used, with the following parameters set in the "Dock" module: All-Atom docking was selected for the docking site; the ligand library file ".mdb" was selected; 30 docking postures were set, and the top 5 conformations were selected based on energy scores. The S-score (E_score) of the docking experiment was recorded. This index reflects the binding free energy of the ligand-acceptor complex through negative logarithmic transformation; a larger negative value indicates a stronger intermolecular interaction. The docking results were comprehensively evaluated using a built-in algorithm in the software, taking into account key parameters such as van der Waals forces, electrostatic interactions, and hydrogen bond formation.

[0086] 3. Experimental Results and Discussion 3.1 Analysis of Blood Flavonoid Components in Shenrong Gubenhuanshao Pills The serum of rats in the blank group and the drug-treated group was detected using UHPLC-Q-Exactive Orbitrap MS technology. The results are as follows: Figure 5 As shown. The blood-entry data were analyzed and processed using Xcalibur 4.3 software workstation. Combined with the in vitro flavonoid component analysis results of Shenrong Gubenhuanshao Pill, 88 flavonoid components of Shenrong Gubenhuanshao Pill were finally identified in the blood, including 33 flavonoids, 24 flavonols, 15 dihydroflavonoids, 12 isoflavones, 2 chalcones, and 2 flavans.

[0087] Drug-time curves reflect the dynamic processes of drug absorption, distribution, metabolism, and excretion in the body, providing crucial pharmacokinetic evidence for assessing whether a compound can produce a therapeutic effect. Therefore, to preliminarily explore drug metabolism trends, equal volumes of serum from six rats at each time point were mixed after drug administration, and changes in the peak area of ​​the compounds were detected. Drug-time curves were plotted using baicalin, hesperidin, astragalin, icariin, naringenin, and apigenin-7-O-β-D-glucoside as examples. Figure 6 As shown, the maximum peak area of ​​each compound in each chromatogram is taken as 100%, and the peak area percentages at each time point are expressed as relative peak areas. The results showed that the peak time of baicalin was 120 min, hesperidin was 30 min, astragaloside was 5 min, and icariin, naringenin, and apigenin-7-O-β-D-glucoside were all 15 min. Naringenin was nearly completely metabolized in vivo. The blood concentrations of astragaloside and icariin initially decreased before reaching a steady state, indicating a stable absorption mechanism in the gastrointestinal tract that effectively maintains the dynamic balance of their blood concentrations.

[0088] 3.2 Prediction Study of Flavonoid Components in Shenrong Gubenhuanshao Pills 3.2.1 Construction of Compound-Target Sets Analysis of the blood-injecting components of Shenrong Gubenhuanshao Pills identified 88 bioactive flavonoids. Based on the principle of reverse pharmacophore matching, the PharmMapper database was used to screen these components for protein targets. With a z'-score greater than 0.5, 3585 potential targets were initially identified. After standardization and duplication removal using the UniProt database, 789 relevant protein targets were finally determined, involving multiple key target families such as nuclear receptors, kinases, and proteases. To systematically analyze the interaction network characteristics of "active ingredient-target," a visual network diagram containing 880 nodes and 3438 interaction associations was constructed using the Cytoscape 3.9.1 software platform. Figure 7 The top 10 compounds in terms of the compound-target network intensity are chopogonin B, isoxafen, baicalin, genistein, apigenin-7-O-β-D-glucoside, naringenin, baicalin, hesperidin, 7,8-dihydroxyflavone, and luteolin-7-glucuronide. These compounds, due to their multiple target sites, may become effective components of Shenrong Gubenhuanshao Pill.

[0089] 3.2.2 Constructing a protein-protein interaction network The protein-protein interaction network (PPI) visualization map constructed based on the STRING database includes 769 nodes, 3179 edges, an average degree of freedom of 8.27, and PPI enrichment. PValues ​​less than 1.0 e-16 indicate that the larger the degree value of the protein target, the larger the node, the darker the color, and the closer it is to the center. The MCC algorithm in the CytoHubba plugin was used to mine the core targets of the PPI network. The top 100 targets were selected and sorted from largest to smallest by the degree value of the core target group (see Table 5). It is speculated that these targets may be key targets for the efficacy of Shenrong Gubenhuanshao Pill. Therefore, a target-disease network will be constructed based on this.

[0090] Table 5

[0091] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for analyzing flavonoid components in Shenrong Gubenhuanshao Pills, characterized in that, The analytical method includes the following steps: S1. Accurately weigh the powder of Shenrong Gubenhuanshao Pill, perform ultrasonic extraction, filter the supernatant to obtain the total flavonoid extract, concentrate and freeze dry, centrifuge to obtain the supernatant after adjusting the volume, and filter through a filter membrane to obtain the test solution of Shenrong Gubenhuanshao Pill. The powder of Shenrong Gubenhuanshao Pill was extracted twice with 80% ethanol at a ratio of 1 g: 25 ml using ultrasonic extraction at 70℃ for 1 h each time. After freeze-drying, it was diluted to volume with 70% methanol. After centrifugation at 12000 rpm for 10 min and filtering through a 0.22 μm filter membrane, the test solution of Shenrong Gubenhuanshao Pill was obtained. S2. The test solution of Shenrong Gubenhuanshao Pill obtained in step S1 was detected by ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS / MS) to obtain high-resolution mass spectrometry data of the test solution. The chromatographic conditions were as follows: InfinityLab Poroshell 120 EC-C18 column, 150 mm × 2.1 mm, 1.9 μm; column temperature 30℃; mobile phase: 0.1% formic acid water as mobile phase A, acetonitrile as mobile phase B; gradient elution: 0–3 min, 10–20% B; 3–10 min, 20–25% B; 10–20 min, 25–30% B; 20–35 min, 30–58% B; 35–40 min, 58–85% B; 40–45 min, 85–10% B; flow rate: 0.3 mL / min, injection: 3 μL. Mass spectrometry conditions were as follows: ionization source: electrospray ionization (ESI); drying gas: N2; drying gas temperature: 225℃; drying gas flow rate: 10 L / min; nebulizer gas pressure: 25 psi; capillary voltages: 3500 V and 4000 V in positive and negative ion modes, respectively; scanning mass range: m / z 20–1700; fragmentation voltage: 80 V; secondary mass spectrometry (MS). 2 The collision energies in positive and negative ion modes are 10, 20, and 40 V; S3. Analyze the high-resolution mass spectrometry data of the test solution obtained in step S2 to achieve the analysis and identification of flavonoid components in Shenrong Gubenhuanshao Pill.

2. The analytical method according to claim 1, characterized in that, The analytical identification method in step S3 is as follows: First, a flavonoid mass spectrometry database of Shenrong Gubenhuanshao Pills is constructed. Then, based on the ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS / MS) technology in step S2, combined with various mass spectrometry data processing strategies, the flavonoid components in Shenrong Gubenhuanshao Pills are analyzed and identified.

3. The analytical method according to claim 2, characterized in that, The specific method for constructing the flavonoid mass spectrometry database of Shenrong Gubenhuanshao Pill is as follows: by consulting authoritative databases such as the Chinese Academy of Sciences Chemistry Database, CNKI, PubChem, SciFinder, Reaxys, Web of Science, Moxoc Chemistry, ZINC, ChemSpider, ChEMBL, and MassBank, a flavonoid mass spectrometry database SRFMSD containing 309 flavonoid compounds in the formula of Shenrong Gubenhuanshao Pill was collected and constructed.

4. The analytical method according to claim 2, characterized in that, The mass spectrometry data processing strategy selected is as follows: total ion chromatogram combined with the flavonoid mass spectrometry database SRFMSD, the Global Natural Product Molecular Network (GNPS), and MS-DIAL / MS-FINDER for the analysis and identification of flavonoid components.

5. The use of the analytical method according to any one of claims 1-4 in the identification, quality identification, quality testing, quality evaluation, or quality control of flavonoid components in a traditional Chinese medicine composition containing Shenrong Gubenhuanshao Pill.

6. A method for detecting flavonoid components in the blood of Shenrong Gubenhuanshao Pills, characterized in that, Includes the following steps: T1. Ginseng and Deer Antler Strengthening Pills were suspended in water and administered to rats by gavage. Blood samples were collected at multiple time points after gavage. The blood was centrifuged and the supernatant was collected. Acetonitrile was added to the serum sample for protein precipitation. After vortexing and sonication, the sample was centrifuged and the supernatant was filtered to remove impurities. The sample was dried with nitrogen and then reconstituted with 50% methanol solution. The sample was centrifuged again and the supernatant was collected to obtain the sample for analysis. T2. The sample obtained in step T1 was analyzed using ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry (UHPLC-Q-ExactiveOrbitrap MS). The chromatographic conditions were as follows: column: ACQUITY UPLC BEH C18; mobile phase: 2% acetonitrile-water containing 0.1% formic acid as mobile phase A, acetonitrile containing 0.1% formic acid as mobile phase B; gradient elution: 0–0.5 min, 2% B; 0.5–3.5 min, 2–25% B; 3.5–7.5 min, 25–35% B; 7.5–11 min, 35–50% B; 11–13 min, 50–95% B; 13–14.4 min, 95% B; 14.4–14.5 min, 95–2% B; 14.5–16 min, 2% B; flow rate: 0.2 mL / min; injection rate: 3 mL / min. μL; column temperature 40℃; mass spectrometry conditions: sample ionized by electrospray ionization, mass spectrometry signals acquired using positive and negative ion scanning modes respectively; scan range 70-1050 m / z, sheath gas flow rate 50 arb, auxiliary gas flow rate 13 arb, heating temperature 450℃, capillary temperature 320℃, spray voltage for positive ion scanning mode 3500 V, spray voltage for negative ion scanning mode -3000 V, collision energy 20, 40, 60 V, resolution Full MS 70000, resolution MS 2 It is 17500; T3. Analyze the mass spectrometry data of the sample obtained in step T2 to achieve the analysis and identification of the flavonoid components in the blood of Shenrong Gubenhuanshao Pill.

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