Analysis method of triterpenoid components in shenshenghuangjiuwan pill

Abstract

This application provides an analytical method for triterpenoid 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: by constructing mass spectrometry data of triterpenoid compounds in Shenrong Gubenhuanshao Pills and combining it with GNPS molecular network mass spectrometry processing, the triterpenoid components in this preparation are identified, further determining the blood-entering triterpenoid components 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 triterpenoid components in the Shenrong Gubenhuanshao Pill. Background Technology

[0002] Shenrong Guben Huanshao Pill (SRGBHSP) is a traditional Chinese medicine preparation made from 65 kinds of Chinese herbs. It has the functions of tonifying the kidneys and strengthening yang, replenishing qi and consolidating essence, stopping seminal emission, and strengthening tendons and bones. The SRGBHSP formula contains 50 kinds of plant-based medicines, 10 kinds of animal-based medicines, and 5 kinds of mineral-based medicines. Among them, 20 kinds of plant-based medicines, such as ginseng, black beans, and licorice, are rich in triterpenoids, which have pharmacological effects such as anti-inflammatory, immunomodulatory, and bone metabolism regulation. These effects are strongly correlated with the efficacy of SRGBHSP, therefore, triterpenoids may be the key active ingredient of SRGBHSP. However, there is currently no research on the triterpenoids in SRGBHSP. Summary of the Invention

[0003] To address the aforementioned technical limitations, this application proposes an analytical method for triterpenoid 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:

[0005] The inventive point of this application is to provide an analytical method for triterpenoid components in the ginseng and deer antler tonifying pill, the analytical method comprising the following steps:

[0006] S1. Accurately weigh the powder of Shenrong Gubenhuanshao Pill, perform ultrasonic extraction, and vacuum dry to obtain the total triterpenoid extract of Shenrong Gubenhuanshao Pill;

[0007] S2. The total triterpenoid content of the ginseng and deer antler tonifying pill obtained in step S1 was first determined by the vanillin-perchloric acid colorimetric method, and then 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 total triterpenoid extract.

[0008] S3. Analyze the high-resolution mass spectrometry data of the test solution obtained in step S2 to achieve the analysis and identification of triterpenoid components in Shenrong Gubenhuanshao Pill.

[0009] Optionally, in the above analytical method, in step S1, the powder of Shenrong Gubenhuanshao Pill is ultrasonically extracted twice with 70% ethanol at a ratio of 1g:20mL, each time for 90 min; after vacuum concentration and vacuum drying, the total triterpenoid sample of Shenrong Gubenhuanshao Pill is obtained, which is then redissolved in 75% methanol and brought to a fixed volume; centrifuged at 12000 rpm for 10 min and filtered through a 0.22 μm filter membrane to obtain the total triterpenoid extract of Shenrong Gubenhuanshao Pill.

[0010] Optionally, in the above analytical method, in step S2, the vanillin-perchloric acid colorimetric method uses oleanolic acid as a reference standard for determination; the chromatographic conditions in UHPLC-Q-TOF-MS / MS technology are as follows: InfinityLabPoroshell 120 EC-C18 column (150 mm × 2.1 mm, 1.9 μm); column temperature 30℃; mobile phase A is 0.1% formic acid water, mobile phase B is acetonitrile; gradient elution 0~2 min, 10% B; 2~3 min, 10%~15% B; 3~25 min, 15%~35% B; 25~50 min, 35%~60% B; 50~75 min, 60%~95% B; flow rate: 0.3 mL / min, injection 3 μL.

[0011] Optionally, in the above analytical method, in step S2, the mass spectrometry conditions are as follows: electrospray ionization (ESI); drying gas is N2; drying gas temperature is 225℃; drying gas flow rate is 10 L / min; nebulizer gas pressure is 25 psi; 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 The range is 20~1700, and the breaking voltage is 80 V; the collision energies in the positive / negative ion modes of secondary mass spectrometry (MS / MS) are 10, 20, and 40 V.

[0012] Optionally, the analytical identification method in step S3 of the above-mentioned analytical method is as follows: first, a triterpenoid 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 Qualitative Workflows B.08.00 to analyze mass spectrometry information and retention time, the triterpenoid components in Shenrong Gubenhuanshao Pills are analyzed and identified.

[0013] Optionally, the above-mentioned analytical method, specifically the method for constructing the triterpenoid mass spectrometry database of Shenrong Gubenhuanshao Pill, is as follows: by consulting relevant data on the research of plant medicine triterpenoid components in the formulation of Shenrong Gubenhuanshao Pill over the past 25 years from CNKI, PubMed, Wanfang, PubChem, TCMIP, Web of Science, and MassBank, and after standardizing the collected data, a mass spectrometry database SRTMSD of 82 triterpenoid compounds that may be contained in Shenrong Gubenhuanshao Pill is constructed.

[0014] Optionally, the mass spectrometry data processing strategy described above is as follows: total ion chromatogram combined with the triterpenoid compound mass spectrometry database SRTMSD and the Global Natural Product Molecular Network (GNPS) for triterpenoid component analysis and identification.

[0015] The second objective 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 triterpenoid components in traditional Chinese medicine compositions containing Shenrong Gubenhuanshao Pills.

[0016] The third objective of this application is to provide a method for detecting the triterpenoid components in the blood of Shenrong Gubenhuanshao Pill, including the following steps:

[0017] T1. The powder suspension of Ginseng and Deer Antler Pills was administered to rats by gavage. Blood was 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, the supernatant was filtered to remove impurities, dried with nitrogen, and then dissolved in methanol solution by sonication to obtain the sample for analysis.

[0018] 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: ACQUITY UHPLC HSS T3 column, 2.1 mm × 100 mm, 1.8 µm; column temperature 35℃; mobile phase: 0.1% formic acid water as mobile phase A, 0.1% formic acid acetonitrile as mobile phase B; gradient elution 0–17 min, 5–98% B; 17–17.2 min, 98–5% B; 17.2–20 min, 5% B; flow rate: 0.3 mL / min; injection volume: 3 μL. The mass spectrometry conditions were: electrospray ionization (ESI) with both positive and negative ion modes for mass spectrometry acquisition. + The spray voltage is 3800 V, ESI -The spray voltage was 3500 V, the sheath gas flow rate was 40 L / min, the ion transfer tube temperature was 320℃, and the atomization temperature was 350℃; the detection method was full scan / data-dependent secondary scan Full-MS / dd-MS. 2 Pattern, top 10 MS 1 MS / MS spectra of ions were obtained, with collision energies (CEs) normalized to step levels 20, 40, and 60; the first-order mass-to-charge ratio scan range was 90–1300. m / z ;

[0019] T3. Analyze the mass spectrometry data of the sample obtained in step T2 to achieve the analysis and identification of the blood-injecting triterpenoid components of Shenrong Gubenhuanshao Pill.

[0020] Compared with the prior art, this application has the following advantages:

[0021] The analytical methods for triterpenoid components in Shenrong Gubenhuanshao Pill and the detection methods for blood-entering triterpenoid components provided in this application, by constructing a mass spectrometry database of triterpenoid compounds in Shenrong Gubenhuanshao Pill and combining it with GNPS molecular network mass spectrometry processing method, identify the triterpenoid components in this preparation, further determine the blood-entering triterpenoid components and their core targets, provide a methodological basis for clarifying the quality evaluation of Shenrong Gubenhuanshao Pill, and also provide a scientific basis for its pharmacodynamic material basis research. Attached Figure Description

[0022] Figure 1 The diagram shown illustrates the interaction between triterpenoid components and pharmacological activity networks during the construction of the mass spectrometry database for the Shenrong Gubenhuanshao Pill, an embodiment of this application.

[0023] Figure 2 The diagram shown is an analysis method for triterpenoid components of the Ginseng and Deer Antler Tonic Pill in an embodiment of this application, including the molecular network and triterpenoid compound classification diagram of the Ginseng and Deer Antler Tonic Pill sample in positive ion mode.

[0024] Figure 3 The diagram shown is an analysis method for triterpenoid components of the Ginseng and Deer Antler Tonic Pill in an embodiment of this application, including a molecular network and triterpenoid compound classification diagram of the Ginseng and Deer Antler Tonic Pill sample under negative ion mode.

[0025] Figure 4 The image shown is an example of an analytical method for triterpenoid components in Shenrong Gubenhuanshao Pills, which is an embodiment of this application. The image shows the total ion chromatograms (TIC) of the Shenrong Gubenhuanshao Pills test sample in positive and negative ion modes.

[0026] Figure 5 The image shown is a total ion chromatogram of the treated serum and blank serum, as illustrated in one embodiment of this application; wherein... Figure 5A represents the positive ion mode ion chromatogram of the drug-treated serum; Figure 5 B represents the positive ion mode ion chromatogram of blank serum. Figure 5 C represents the negative ion mode ion chromatogram of the drug-treated serum; Figure 5 D represents the negative ion mode ion chromatogram of blank serum.

[0027] Figure 6 As shown in one embodiment of this application, oleanolic acid (… Figure 6 A) Glycyrrhizic acid ( Figure 6 B), glycyrrhetinic acid ( Figure 6 C) is the drug-time curve.

[0028] Figure 7 The diagram shows a network of targets related to 11 triterpenoid compounds in one embodiment of this application; orange diamond nodes represent the 11 triterpenoid compounds, and green circular nodes represent targets related to the 11 triterpenoid compounds. Detailed Implementation

[0029] 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.

[0030] 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.

[0031] To further understand this application, the following detailed description is provided in conjunction with the preferred embodiments.

[0032] Example 1

[0033] Construction of a mass spectrometry database of triterpenoid compounds in Ginseng and Deer Antler Strengthening Pill (SR):

[0034] To elucidate the structure of triterpenoids in sRGBHSP, relevant research data on plant-based triterpenoids in sRGBHSP formulations over the past 25 years were reviewed from seven databases: CNKI, PubMed, Wanfang, PubChem, TCMIP, Web of Science, and MassBank. After data standardization, a mass spectrometry database (SRTMSD) of 82 potentially contained triterpenoids in sRGBHSP was successfully constructed, including 43 pentacyclic triterpenoids and 39 tetracyclic triterpenoids. Further research based on network association analysis confirmed a high correlation between triterpenoids and the efficacy of sRGBHSP, providing a basis for subsequent analysis of triterpenoid components.

[0035] Data acquisition and standardization:

[0036] Using search terms such as "20 medicinal plant names rich in triterpenoids (e.g., Panax ginseng CA Mey)," "chemical composition," and "LC-MS," relevant literature from the past 20 years was retrieved from platforms including CNKI, Wanfang Data, and PubMed. This yielded research on the components of medicinal materials and collected information on reported triterpenoid compounds, including molecular formula, precise molecular weight, quasi-molecular ion peak, and characteristic mass spectrometry fragments. Based on this, the data was deduplicated and integrated. Furthermore, the CID numbers were recorded in the PubChem database to exclude compounds with unclear structures. The secondary fragments of the collected compounds were further examined using the MassBank database to ensure that the obtained mass spectrometry fragments conformed to the compound fragmentation patterns.

[0037] Database construction:

[0038] First, a blank database was created based on a general template. The collected compound names, molecular formulas, mass spectrometry information and other data were standardized and integrated according to the system's preset CSV template format. Then, the chemical structures were drawn using ChemDraw 18.0, and finally, a database of triterpenoid chemical components of Shenrong Gubenhuanshao Pill was obtained, laying the foundation for subsequent molecular docking experiments.

[0039] Correlation analysis of triterpenoids and their pharmacological activities with the efficacy of Shenrong Gubenhuanshao Pill

[0040] First, by reviewing the sources and pharmacological activities of triterpenoids in the literature and statistical database, Gephi-0.10.1 software was used to conduct network association analysis on triterpenoid components, pharmacological activities of triterpenoid components, and the efficacy of Shenrong Gubenhuanshao Pills, and a multi-layered relationship network including "traditional Chinese medicine - triterpenoids - pharmacological activity - SRGBHSP indications" was constructed.

[0041] Experimental results:

[0042] 1) Database construction:

[0043] Literature analysis and database search were conducted on the analysis of triterpenoid components of 50 kinds of herbal medicines in Shenrong Gubenhuanshao Pills. A total of 926 articles were retrieved. After screening and removing irrelevant articles and duplicate information, more than 315 triterpenoid compounds were summarized. Further deduplication of compound information and screening of compounds with clear structures and secondary fragments resulted in 82 effective triterpenoid mass spectrometry information.

[0044] 2) Correlation analysis between triterpenoids and their pharmacological activities and the efficacy of Shenrong Gubenhuanshao Pills:

[0045] A multidimensional analysis of the functions and indications of Shenrong Gubenhuanshao Pills was conducted using systematic analysis methods, classifying its efficacy into 10 categories: strengthening muscles and bones, anti-aging, enhancing immunity, treating chronic nephritis, treating neurasthenia, treating ulcerative colitis, treating asthma, treating tinnitus and deafness, treating impotence and premature ejaculation, and treating infertility. Further literature review clarified the sources and pharmacological activities of triterpenoid compounds, and the constructed network diagram contained 79 nodes and 193 edges. Shenrong Gubenhuanshao Pills occupied 1 node, traditional Chinese medicine 26 nodes, triterpenoid compounds 20 nodes, the pharmacological activity of triterpenoid compounds 22 nodes, and the efficacy of Shenrong Gubenhuanshao Pills 10 nodes. Analysis revealed that the nephroprotective and neuroprotective effects of ginsenoside RG1 in ginseng and astragaloside A in astragalus are highly consistent with the efficacy of Shenrong Gubenhuanshao Wan in treating chronic nephritis, impotence, premature ejaculation, infertility, and neurasthenia. The immunomodulatory and anti-atherosclerotic effects of daidzein Bb in black beans and oleanolic acid in herbs such as Achyranthes bidentata and Chaenomeles speciosa are related to the immune-enhancing effects of Shenrong Gubenhuanshao Wan. Figure 1 The results showed that the triterpenoids in the database were significantly associated with the kidney-tonifying, yang-boosting, qi-tonifying, and solidifying effects of Shenrong Gubenhuanshao Pill.

[0046] Example 2

[0047] Analysis of triterpenoid components in Shenrong Gubenhuanshao Pills based on UHPLC-Q-TOF-MS / MS:

[0048] To identify the triterpenoid components of sRGBHSP, the preparation method for total triterpenoid analysis of sRGBHSP was first optimized through single-factor and orthogonal experiments. Specifically, an appropriate amount of sRGBHSP powder was accurately weighed, and extraction was performed twice at 60℃ using 70% ethanol as solvent at a material-to-liquid ratio of 1 g:20 mL for 90 min. The extraction was conducted twice using an ultrasonic-assisted method. The filtrates were combined, concentrated under reduced pressure, and vacuum dried to obtain the total triterpenoid extract of sRGBHSP. The total triterpenoid content in the preparation was determined to be 36.1106 mg / g by the vanillin-perchloric acid colorimetric method. Further, UHPLC-Q-TOF-MS / MS technology, combined with SRTMSD and GNPS molecular networks, was used for systematic structural analysis, successfully identifying 45 triterpenoid components, including 7 dammarane-type, 3 cycloatunane-type, 2 lanolinane-type, 29 oleanane-type, and 4 ursane-type.

[0049] 1. Materials and Instruments:

[0050] Experimental materials: Shenrong Gubenhuanshao Pills (Lanzhou Foci Pharmaceutical 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); reference standard oleanolic acid (508-02-1, purity ≥98%, Chengdu Aiboke Biotechnology Co., Ltd.); vanillin (Solarbio, catalog number V8080); perchloric acid; glacial acetic acid (Nanshi, C0680670277); anhydrous ethanol (analytical grade, Jinan Yuansu Chemical 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.

[0051] Instrumentation: Agilent 1290 UHPLC system in tandem with an Agilent 6560 ion mobility quadrupole time-of-flight LC-MS system; powder grinder (Joyoung JYS-M01); electronic analytical balance (FA2004, Shanghai Dudan Instrument Co., Ltd.); UV-1700 UV spectrophotometer (Shimadzu); ultrasonic cleaner (Ningbo Xinzhi Biotechnology Co., Ltd.); rotary evaporator (Zhengzhou Changcheng Science & Industry Trade Co., Ltd.); freeze dryer (CTFD-20S, Qingdao Yonghe Chuangxin Electronic Technology Co., Ltd.); OSB-2100 water bath (Haierang Instrument Co., Ltd.).

[0052] 2. Experimental Methods

[0053] 2.1 Study on the preparation method of triterpenoid analysis samples for Shenrong Gubenhuanshao Pills

[0054] 2.1.1 Determination of total triterpenoid content

[0055] The yield of total triterpenes was determined by the vanillin-perchloric acid colorimetric method using oleanolic acid as a reference standard. The principle is that perchloric acid oxidizes the phenolic hydroxyl groups of triterpenes to carboxyl groups, adding one double bond structure. Then, through double bond shift and addition with vanillin, a conjugated double bond system is formed, which forms a carbocation salt under the action of acid and is then colored.

[0056] (1) Solution preparation

[0057] Preparation of 5% vanillin-glacial acetic acid solution: Weigh 5 g of vanillic acid solid into a beaker, dissolve it in a small amount of glacial acetic acid, and then bring the volume to 100 mL.

[0058] Preparation of standard stock solution: Accurately weigh 1.00 mg of oleanolic acid standard, dissolve it in methanol and dilute to a volumetric flask of 10 mL to obtain a 0.1 mg / mL oleanolic acid standard stock solution.

[0059] (2) Determination of the maximum absorption wavelength

[0060] Take 200 μL of the reference solution and place it in a test tube. After evaporating the solvent, add 0.2 mL of 5% vanillin-glacial acetic acid solution (freshly prepared), add 0.8 mL of perchloric acid, shake well, heat in a water bath at 60℃ for 15 min, place in an ice bath for 5 min, add 5 mL of glacial acetic acid, shake well, let stand for 15 min, scan at a wavelength of 300–800 nm, measure the absorbance value, and determine the maximum absorption wavelength.

[0061] (3) Drawing the standard curve

[0062] Take 0.6, 0.8, 1, 1.2, 1.4, 1.6 and 1.8 mL of oleanolic acid standard solution respectively, and measure its absorbance value at the maximum absorption wavelength according to method (2).

[0063] (4) Measurement of sample absorbance

[0064] Take 200 μL of the test solution and perform the determination according to the experimental procedure in (3).

[0065] (5) Formula for total triterpenoid yield

[0066]

[0067] In the formula, C is the concentration of the test solution (mg / mL); V is the volume of the test solution (mL); N is the dilution factor; and M is the mass of the Shenrong Gubenhuanshao Pill (g).

[0068] 2.1.2 Optimization of the extraction method of total triterpenoids in Shenrong Gubenhuanshao Pills

[0069] (1) Single-factor experimental design:

[0070] The extraction conditions were set as follows: ultrasonic temperature 60℃, ethanol concentration 80%, ultrasonic time 60 min, and material-liquid ratio 1:20 (g / mL). The effects of extraction times (1, 2, 3) on the yield of total triterpenes in Shenrong Gubenhuanshao Pill were investigated.

[0071] The extraction conditions were set as follows: ultrasonic temperature 60℃, extraction time 60 min, material-to-liquid ratio 1:20 (g / mL), and extraction times 2. The effect of ethanol concentration (60%, 70%, 80%, 90%) on the yield of total triterpenes in Shenrong Gubenhuanshao Pill was investigated.

[0072] The extraction conditions were set as follows: ultrasonic temperature 60℃, ethanol concentration 80%, extraction times 2, and material-to-liquid ratio 1:20 (g / mL). The effects of extraction time (0.5, 1.0, 1.5, 2.0) on the yield of total triterpenes in Shenrong Gubenhuanshao Pill were investigated.

[0073] The effects of material-to-liquid ratio (1:10, 1:15, 1:20, 1:25, 1:30, g:mL) on the yield of total triterpenes in Shenrong Gubenhuanshao Pill were investigated under the following extraction conditions: ultrasonic temperature 60℃, ethanol concentration 80%, extraction times 2, ultrasonic time 60 min.

[0074] Each factor level was repeated 3 times.

[0075] (2) Orthogonal experimental design:

[0076] To better investigate the effects of ultrasonic extraction parameters on the extraction of total triterpenoids from Shenrong Gubenhuanshao Pills, based on the results of single-factor experiments, extraction ethanol concentration, extraction time, and solid-liquid ratio were selected as the factors of investigation. The extraction rate of total triterpenoids from Shenrong Gubenhuanshao Pills was used as the index, and an L9(3)24-fold ultrasonic extraction method was designed. 3 Orthogonal experiments were used to determine the optimal extraction method for total triterpenoid components in Shenrong Gubenhuanshao Pills. The orthogonal experimental factor level table is shown in Table 1.

[0077] Table 1

[0078]

[0079] (3) Data processing

[0080] Data processing and plotting were performed using statistical software SPSS 25.0, Excel, Origin 2019, and GraphPad Prism 9.

[0081] 3. Analysis of triterpenoid components in Shenrong Gubenhuanshao Pills based on UHPLC-Q-TOF-MS / MS technology:

[0082] 3.1 Preparation of the test sample

[0083] To prepare the total triterpenoid sample of Shenrong Gubenhuanshao Pill, weigh 200 mg and place it in a 10 mL volumetric flask. Redissolve the triterpenoid in 75% methanol and bring the volume to a final volume. Mix thoroughly. Centrifuge at 12000 rpm for 10 min, filter through a 0.22 μm microporous membrane, and proceed with analysis.

[0084] 3.2 Chromatographic and Mass Spectrometry Conditions

[0085] 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: 0~2 min, 10% B; 2~3 min, 10%~15% B; 3~25 min, 15%~35% B; 25~50 min, 35%~60% B; 50~75 min, 60%~95% B; flow rate: 0.3 mL / min; injection: 3 μL.

[0086] Mass spectrometry conditions: Electrospray ionization (ESI); drying gas: N2; drying gas temperature: 225℃; drying gas flow rate: 10 L / min; nebulizer gas pressure: 25 psi; capillary voltages in positive and negative ion modes: 3500 V and 4000 V, respectively; scan mass range m / z The range is 20~1700, and the fragmentation voltage is 80 V. The collision energies in the positive / negative ion modes of secondary mass spectrometry (MS / MS) are 10, 20, and 40 V.

[0087] 3.3 Data Processing

[0088] (1) Identification of the components of Shenrong Gubenhuanshao Pill based on the self-built triterpenoid database: The mass spectrometry information and retention time of the triterpenoid components in the sample were analyzed by Qualitative Workflows B.08.00. Combined with the self-built database, the triterpenoid components in Shenrong Gubenhuanshao Pill were analyzed and identified.

[0089] (2) Identification of triterpenoids in Shenrong Gubenhuanshao Pills based on GNPS molecular network: The raw UHPLC-MS / MS data were converted from "wiff" format to "mzXML" format using MSConvertGUI and WinSCP software, uploaded to the GNPS online platform, and a molecular network was built using the platform's default parameters. The molecular network was then imported into Cytoscape 3.9.1 for visualization analysis using "download graphml for Cytoscape". The compounds were annotated based on the molecular network, and the mass spectra were further annotated using mass spectrometry fragmentation feature analysis to find and identify more triterpenoids.

[0090] (3) Summarize the mass spectrometry rules of known components: Confirm the preliminary analysis results obtained above, and summarize the mass spectrometry fragmentation rules of the two major categories of chemical structures.

[0091] Orthogonal experimental results of total triterpenoid extraction from Shenrong Gubenhuanshao Pill:

[0092] The results of the orthogonal experiment are shown in Table 2. Range analysis of the orthogonal experiment results showed that the influence of the three factors on the total triterpenoid content, from largest to smallest, was ethanol concentration, solid-liquid ratio, and extraction time, i.e., C>B>A. Based on the K values ​​of each factor, the optimal extraction combination for total triterpenoids was A2B2C2, which means extraction with 70% ethanol as the extraction solvent at 60℃, a solid-liquid ratio of 1:20 (g / mL), for 90 min, and repeated twice.

[0093] Verification experiment: The optimal conditions of the orthogonal experiment were verified using the total triterpenoid yield as an indicator. The experiment was conducted in three parallel groups. The total triterpenoid content of Shenrong Gubenhuanshao Pill was measured to be 36.1106 mg / g, with an RSD of 1.06%, proving that the method is stable and reliable.

[0094] Table 2

[0095]

[0096] Identification of triterpenoid components in Shenrong Gubenhuanshao Pills based on GNPS molecular network:

[0097] Seven molecular clusters of triterpenoids were obtained through the GNPS molecular network. In cluster I, two compounds matched the GNPS database (diamond nodes), and the quasi-molecular ion peaks of the nodes were as follows. m / z 974.531, and m / z 812.479, namely ginger-like notoginsenoside and ginsenoside Ro, are both oleanolic acid-type pentacyclic triterpenoids, possessing characteristic... m / z 439 and m / z Fragments of 191.

[0098] Mass spectra of clusters II, III, and VII were matched using the GNPS database, identifying three compounds (diamond-shaped nodes). The quasi-molecular ion peaks of these nodes were as follows: m / z 821.394 m / z 839.405 m / z 823 and 412 are glycyrrhizic acid, glycyrrhizin G2, and ural glycyrrhizin C, respectively, all of which are oleanane-type pentacyclic triterpenes with glycyrrhetinic acid as the aglycone. In positive ion mode, m / z 807 m / z 879、 m / z 805 and m / z 647 all have m / z The characteristic fragments of 471 indicate that the compound is based on glycyrrhetinic acid as an aglycone. Referring to the structure of glycyrrhetinic acid, it is inferred that they are deoxyglycyrrhizic acid, 22-acetoxyglycyrrhizic acid glycoside, glycyrrhizin C2, and glycyrrhetinic acid monoglucuronide. (Under negative ion mode) m / z 895 has m / z The characteristic ion peak at 645, based on the PubChem database, suggests that it is 2''-O-acetylgia saponin C.

[0099] One node in cluster IV was identified as calendula saponin E, which is an oleanane-type pentacyclic triterpenoid. m / z 647 is glycyrrhetinic acid monoglucuronide in positive ion mode, possessing... m / z Characteristic ions of 471.

[0100] Cluster V has two compounds that match the GNPS database (diamond nodes). The quasi-molecular ion peaks of the nodes in positive ion mode are as follows: m / z 437.342, and m / z 439 and 357 are oleanolic acid and 3-hydroxy-11-ursene-28,13-lactone, respectively, both of which are oleanolic acid-type pentacyclic triterpenes and have characteristic... m / z Fragments of 437.

[0101] Cluster VI has two compounds that match the GNPS database (diamond node). In negative ion mode, the quasi-molecular ion peaks of the node are respectively... m / z 845.487 and m / z 765 and 476 are ginsenoside RG1 and ginsenoside Rk1, respectively, both of which are tetracyclic triterpenoid compounds. (The text abruptly ends here, likely due to an incomplete sentence or missing information.) m / z Comparative analysis of mass spectrometry fragments of 633 with ginsenoside Rk1 revealed that its molecular formula is C633. 46 H 72 O 17 It is speculated that it is momordicin L.

[0102] In summary, based on the GNPS molecular network strategy, 10 triterpenoid compounds were further identified from Shenrong Gubenhuanshao Pill, including 8 oleanane-type, 1 ursane-type pentacyclic triterpenoid, and 1 dammarane-type tetracyclic triterpenoid.

[0103] In the analytical methods for triterpenoid components of Shenrong Gubenhuanshao Pills, the molecular network and triterpenoid compound classification of Shenrong Gubenhuanshao Pill samples under positive / negative ion modes are as follows: Figure 2 , Figure 3 As shown.

[0104] The total ion chromatograms (TICs) of the Ginseng and Deer Antler Strengthening Pill test sample in positive and negative ion modes are as follows: Figure 4 As shown.

[0105] Retention time (RT) and mass-to-charge ratio of each triterpenoid compound m / z Table 3 shows the measured values, errors, and MS / MS fragmentation information of high-resolution mass spectrometry (UHPLC-Q-TOF-MS / MS identification of triterpenoids in Shenrong Gubenhuanshao Pill).

[0106] Table 3

[0107]

[0108]

[0109]

[0110]

[0111]

[0112] In Table 3, A represents compounds identified by the self-built database; B represents compounds identified by the GNPS molecular network.

[0113] Example 3

[0114] Analysis and target prediction of blood-entering triterpenoid components in Shenrong Gubenhuanshao Pills:

[0115] To clarify the active components of sRGBHSP, based on the serum pharmacochemistry theory that "components entering the bloodstream may be the active ingredients," we first used ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry (UHPLC-Q-ExactiveOrbitrap MS) to detect the serum of rats in the blank group and the sRGBHSP-administered group by gavage. A total of 11 triterpenoid components entering the bloodstream were identified, and compounds such as oleanolic acid, glycyrrhizic acid, and glycyrrhetinic acid were found to have potential pharmacokinetic relationships. Secondly, reverse molecular docking technology was used to predict the target sites of the bloodstream components. Using the PharmMapper database, 278 protein targets were predicted, and 100 key targets were screened using a protein-protein interaction network. Analysis revealed significant correlations with gastrointestinal diseases, lung diseases, and nervous system diseases. GO and KEGG analyses showed that the pathways were mainly enriched in cancer pathways and neurotrophic factor signaling pathways, validating the association between key targets and diseases. Further screening identified the top 15 core targets with the highest degree values, such as PIK3R1, PTPN11, and GRB2, and performed molecular docking with 11 blood-entering triterpenoid components. The results showed that 33.9% of the "compound-target" docking scores were less than -7, and 98% of the "compound-target" docking scores were less than -5, demonstrating that the 11 blood-entering components have a strong binding ability with their corresponding targets, preliminarily confirming that these components may be the active ingredients of sRGBHSP.

[0116] Identification and analysis of blood-entering triterpenoid components in Shenrong Gubenhuanshao Pills based on UHPLC-Q-Exactive Orbitrap MS technology:

[0117] 1. Materials and Instruments

[0118] Experimental materials: Ginseng and Deer Antler Rejuvenating Pills (Lanzhou Foci Pharmaceutical Co., Ltd., batch number 220912); anhydrous ethanol (analytical grade, Jinan Yuansu Chemical Co., Ltd.); mass spectrometry grade acetonitrile and methanol (Beijing Bailingwei Technology Co., Ltd.); mass spectrometry grade formic acid (analytical grade, Jinan Yuansu Chemical Co., Ltd.); Watson's purified water.

[0119] Instruments and equipment: UHPLC-Q-Exactive Orbitrap MS liquid chromatography-mass spectrometry system; low-temperature ultracentrifuge; electronic analytical balance (CPA225D, Sartorious Scientific Instruments, China; FA2004, Shanghai Dudan Instrument Co., Ltd.); ultrasonic cleaner (Ningbo Xinzhi Biotechnology Co., Ltd.); rotary evaporator (Zhengzhou Great Wall Science & Industry Trade Co., Ltd.); OSB-2100 water bath (Haierang Instrument Co., Ltd.).

[0120] 2. Laboratory animals

[0121] Twelve male SPF-grade SD rats, weighing 200±20 g, were purchased from Lanzhou University (Experimental Animal Center), license number: SYXK(Gan)2023-0004.

[0122] 3. Experimental Methods

[0123] 3.1 Animal grouping and administration

[0124] Weigh 20 g of Shenrong Gubenhuanshao Pill powder, add 50 mL of water, and mix well to prepare a gavage solution. Twelve male SD rats were acclimatized for 7 days in an SPF-grade animal facility (temperature 23±2℃, humidity 55±10%, 12-hour diurnal rhythm). They were then randomly divided into a Shenrong Gubenhuanshao Pill administration group (n=6) and a control group (n=6). Initial rat weights were recorded. The gavage dose was calculated to be 6.48 g / kg based on four times the adult dosage. The administration group and the control group were given the same volume of Shenrong Gubenhuanshao Pill aqueous solution and Wahaha water, respectively. All rats were administered the medication twice daily for three consecutive days.

[0125] 3.2 Collection and processing of serum samples

[0126] Before the last administration, the rats were fasted but allowed free access to water for 12 hours. At 5 min, 15 min, 30 min, 1 h, 2 h, 4 h and 8 h after the last administration, blood was collected from the orbital venous plexus of all rats. The blood was placed in a dry centrifuge tube, allowed to stand for 30-60 min, and then centrifuged (4℃, 3000 rpm, 15 min). The supernatant was collected in a centrifuge tube and stored at -80℃.

[0127] Take 300 μL of serum from each time period, mix them, vortex for 3 min to mix, add 6 mL of acetonitrile for protein precipitation, vortex for 30 min, and then sonicate for 15 min. Centrifuge at 13000 rpm for 15 min at 4℃, take the supernatant, filter it through a 0.22 μm filter membrane, blow dry with nitrogen, and sonicate to dissolve it with 0.2 mL of methanol-water solution (1:1) to obtain the sample.

[0128] 3.3 Chromatographic and Mass Spectrometry Conditions

[0129] Chromatographic conditions: Column: ACQUITY UHPLC HSS T3 (2.1 mm × 100 mm, 1.8 µm); Column temperature: 35℃; Mobile phase: 0.1% formic acid water (A) - 0.1% formic acid acetonitrile (B); Gradient elution: 0~17 min, 5~98%B; 17~17.2 min, 98~5%B; 17.2~20 min, 5%B; Flow rate: 0.3 mL / min; Injection volume: 3 μL.

[0130] Mass spectrometry conditions: Mass spectrometry data were acquired using both positive and negative electrospray ionization (ESI) modes. Spray voltage: 3800 V (ESI). + ) / 3500V (ESI) - The sheath gas flow rate was 40 L / min, the ion transfer tube temperature was 320℃, and the nebulization temperature was 350℃; the detection method was full scan / data-dependent two-stage scan (Full-MS / dd-MS). 2 ) mode, top 10 MS 1 MS / MS spectra of ions were obtained, with collision energies (CEs) normalized to step levels of 20, 40, and 60; the first-order mass-to-charge ratio scan range was 90–1300. m / z .

[0131] 4. Data Processing

[0132] UHPLC-Q-Exactive Orbitrap MS technology was used to compare and analyze blank serum and drug-containing serum. The raw mass spectrometry analysis data were imported into Xcalibur software, and the blood-entered components were identified by matching the raw data with the existing database.

[0133] 5. Experimental Results

[0134] Using Xcalibur software, the blood components of rats in the blank group and the drug-treated group were analyzed in positive and negative ion modes to obtain the total ion chromatogram. Figure 5 ).

[0135] Eleven blood-entering triterpenoids were identified. The results showed that the blood-entering triterpenoids of Shenrong Gubenhuanshao Pill were mainly pentacyclic triterpenoids. Specific data are shown in Table 4.

[0136] Table 4

[0137]

[0138] The pharmacodynamic curves for oleanolic acid, glycyrrhizic acid, and glycyrrhetinic acid are as follows: Figure 6 As shown.

[0139] 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 oleanolic acid, glycyrrhizic acid, and glycyrrhetinic acid as examples. Figure 6 As shown, oleanolic acid reached a peak blood concentration at 15 minutes, indicating that it undergoes absorption, distribution, and metabolism in vivo. The peak area of ​​oleanolic acid showed a continuous upward trend during the last 8 hours of testing, suggesting that other oleanolic acid-type pentacyclic triterpenes are metabolized into oleanolic acid, thus increasing its blood concentration. Similarly, glycyrrhizic acid also reached a peak blood concentration at 15 minutes, indicating that it undergoes absorption, distribution, and metabolism in vivo. Its peak area also showed a continuous upward trend during the last 8 hours of testing, suggesting secondary distribution. Glycyrrhetinic acid showed a decreasing trend at 5 min and an increasing trend from 4 h to 8 h. It is speculated that triterpenoid saponins with glycyrrhetinic acid as the aglycone begin to be metabolized into glycyrrhetinic acid at 5 min, thus increasing its blood concentration. Literature review found that the blood concentration of glycyrrhetinic acid reaches its peak at 12 h. This experiment only tested serum samples at 8 h, but the trend of the drug-time curve is somewhat similar to that of the literature, indicating that it undergoes absorption, distribution and metabolism in vivo.

[0140] Reverse molecular docking technology predicts potential active targets:

[0141] 1. Materials and Instruments

[0142] The main analysis software and data platforms used are shown in Table 5.

[0143] Table 5

[0144]

[0145] 2. Experimental Methods

[0146] 2.1 Acquisition and processing of molecular structures of triterpenoids entering the bloodstream

[0147] The 3D or 2D structures of 11 blood-inducing triterpenoid components of Shenrong Gubenhuanshao Pill, identified by UHPLC-Q-Exactive Orbitrap MS, were saved in SDF format from the PubChem database. The molecular structures underwent protonation state correction using MOE 2019.0102 software, followed by desalting and hydrogenation using the "PartialCharges" module to optimize charge distribution and eliminate potential bond length and angle anomalies in the original structures. Energy minimization was then performed on the small molecules, with a gradient of 0.1 RMS kcal / (A²·mol). The atomic coordinates were progressively adjusted using an algorithm to minimize the total molecular potential. The 11 optimized molecular structures were stored in the MOE molecular database in ".mdb" format.

[0148] 2.2 Collection of potential targets for triterpenoid components in Shenrong Gubenhuanshao Pills

[0149] The triterpenoid components that enter the bloodstream from Shenrong Gubenhuanshao Pills were submitted as SDF files to PharmMapperserver (http: / / 59.78.96.61 / pharmmapper / help.php) for optimization. Multiple conformations were generated. A human protein target library was selected, with other parameters set to default. PharmMapperserver automatically matched ligand small molecules with pharmacophores in its database and sorted them according to their z'-scores. All targets with a z'-score > 0.9 were analyzed. After removing common targets among compounds, the remaining targets constituted the target set of the compound.

[0150] 2.3 PPI Network Construction and Core Target Screening

[0151] The target set obtained in section 2.2 was uploaded to the STRING database (https: / / www.string-db.org / ), with "Homo sapiens" selected as the species. Target association confidence was ≥0.70, isolated nodes were removed, and a PPI network diagram was obtained. The resulting PPI diagram was imported as a TSV file into Cytoscape_v3.9.1 for visualization analysis. The DEGREE algorithm of the CytoHubba plugin in Cytoscape_v3.9.1 was used for analysis. The top 100 targets were selected as core targets for constructing a "compound-target-disease" network. Furthermore, the top 15 targets were selected for molecular docking with 11 blood-derived triterpenoid components.

[0152] 2.4 Construction of the triterpenoid-target-disease network diagram

[0153] Using DISEASES (https: / / diseases.jensenlab.org / Search),

[0154] The top 100 targets selected were analyzed using the 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 efficacy. A "compound-target-disease" network was constructed using Cytoscape_v3.9.1 software to explore potential targets for the treatment of diseases by Shenrong Gubenhuanshao Pill.

[0155] 2.5 GO functional enrichment and KEGG pathway enrichment analysis

[0156] The TOP 100 protein targets identified by PPI screening were enriched using GO function and KEGG pathway using the David 2021 database. Visualization analysis was then performed using the MicroBio platform to elucidate the biological effects of potential targets and their roles in signaling pathways.

[0157] 2.6 Molecular docking of key components and core targets

[0158] The key components of the selected Ginseng and Deer Antler Tonic Pill were input into PubChem, and their structures were exported as SDF files. Open Babel 3.1.1 was then used to convert them to MOL2 files for docking with bioactive small molecules. The optimal protein crystal structure of the core target protein was screened using the UniProt database, with the species source set to human only, selecting protein crystal structures with a resolution lower than 1.7 Å. The PDB files of the selected structures were then downloaded from the Protein Database (PDB, https: / / www.rcsb.org / ). The obtained protein structures were preprocessed using the "Quickprep" function in MOE software. Molecular docking was performed using the "DOCK" function in MOE software, with the following parameters set: All Atoms docking; Ligand selection: ligand library file ".mdb"; 30 docking positions were set, and the top 5 conformations were selected based on energy scores. Molecular docking was then performed on the key active components and the core target. According to the molecular docking score, a lower score reflects a lower binding energy between the molecule and the protein, indicating a stronger intermolecular interaction.

[0159] 3. Experimental Results

[0160] 3.1 Collection of potential targets for triterpenoid components in Shenrong Gubenhuanshao Pills

[0161] Based on 11 types of triterpenoid components from the blood-penetrating Ginseng and Deer Antler Tonic Pill, 817 targets were screened from the PharmMapper database. After deduplication and cleaning, 278 target proteins were obtained. The STRING analysis platform was used to convert the target protein names into their corresponding gene names. The data was imported into Cytoscape 3.9.1 software to construct an active ingredient-target map, resulting in a network diagram with 289 nodes and 792 interaction associations (e.g., ...). Figure 7 The orange diamond-shaped nodes in the middle represent 11 triterpenoid compounds, and the surrounding green circular nodes represent the target sites corresponding to these 11 triterpenoid compounds. Table 6 shows the target site information for the 11 blood-entering triterpenoid components in Shenrong Gubenhuanshao Pill.

[0162] Table 6

[0163]

[0164] 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 triterpenoid 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, and vacuum dry to obtain the total triterpenoid extract of Shenrong Gubenhuanshao Pill; The powder of Shenrong Gubenhuanshao Pill was ultrasonically extracted twice with 70% ethanol at a ratio of 1 g: 20 mL, each time for 90 min; after vacuum concentration and vacuum drying, the total triterpenoid sample of Shenrong Gubenhuanshao Pill was obtained, which was then reconstituted with 75% methanol and brought to a fixed volume; after centrifugation at 12000 rpm for 10 min and filtering through a 0.22 μm filter membrane, the total triterpenoid extract of Shenrong Gubenhuanshao Pill was obtained. S2. The total triterpenoid content of the total triterpenoid extract of the ginseng and deer antler tonifying pill obtained in step S1 was first determined by the vanillin-perchloric acid colorimetric method, and then 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 total triterpenoid extract. The vanillin-perchloric acid colorimetric method used oleanolic acid as a reference standard for determination. The chromatographic conditions for UHPLC-Q-TOF-MS / MS were as follows: InfinityLab Poroshell 120 EC-C18 column (150 mm × 2.1 mm, 1.9 μm); column temperature 30℃; mobile phase A was 0.1% formic acid in water, and mobile phase B was acetonitrile; gradient elution: 0–2 min, 10% B; 2–3 min, 10%–15% B; 3–25 min, 15%–35% B; 25–50 min, 35%–60% B; 50–75 min, 60%–95% B; flow rate: 0.3 mL / min; injection depth: 3 μL. Mass spectrometry conditions were as follows: electrospray ionization (ESI); drying gas was N2; drying gas temperature was 225℃; drying gas flow rate was 10 L / min; nebulizer gas pressure was 25 psi; capillary voltages in positive and negative ion modes were 3500 V and 4000 V, respectively; and the scanning mass range was... m / z The range is 20~1700, with a fragmentation voltage of 80 V; the collision energies in positive / negative ion modes of secondary mass spectrometry (MS / MS) are 10, 20, and 40 V; S3. Analyze the high-resolution mass spectrometry data of the total triterpenoid extract obtained in step S2 to achieve the analysis and identification of triterpenoid components in the Shenrong Gubenhuanshao pill.

2. The analysis method according to claim 1, characterized in that, The analytical identification method in step S3 is as follows: First, a mass spectrometry database of triterpenoids in 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) technique used in step S2, and combined with Qualitative Workflows B.08.00 software, the mass spectrometry information and retention time are analyzed to identify the triterpenoid components in Shenrong Gubenhuanshao Pills.

3. The analysis method according to claim 2, characterized in that, The method for constructing the mass spectrometry database of triterpenoids in Shenrong Gubenhuanshao Pills is as follows: By consulting relevant data on the study of plant-derived triterpenoid components in the formulation of Shenrong Gubenhuanshao Pills from CNKI, PubMed, Wanfang, PubChem, TCMIP, Web of Science, and MassBank over the past 25 years, and after standardizing the collected data, a mass spectrometry database of 82 triterpenoids that may be contained in Shenrong Gubenhuanshao Pills, SRTMSD, was constructed.

4. The analysis method according to claim 2, characterized in that, The mass spectrometry data processing strategy selected is as follows: total ion chromatogram combined with the triterpenoid compound mass spectrometry database SRTMSD and the Global Natural Product Molecular Network (GNPS) for triterpenoid component analysis and identification.

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 triterpenoid components in a traditional Chinese medicine composition containing Shenrong Gubenhuanshao Pill.

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