Analytical methods for alkaloid components in Shenrong Gubenhuanshao Pills

Abstract

This application provides an analytical method for alkaloid 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 alkaloids in Shenrong Gubenhuanshao Pills and combining it with GNPS molecular network mass spectrometry processing, the alkaloid components in this preparation are identified, further determining the alkaloid components that enter 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 alkaloid components in the Shenrong Gubenhuanshao Pill. Background Technology

[0002] The Ginseng and Deer Antler Tonifying and Rejuvenating Pill originates from a prescription made by a renowned physician in Gansu during the mid-Qing Dynasty. It is prepared from 65 Chinese medicinal herbs using traditional processing techniques combined with modern pharmaceutical technology. It is believed to tonify the kidneys and strengthen yang, replenish qi and solidify the body, replenish essence and stop seminal emission, and strengthen muscles and bones. Of the 26 herbs in the Ginseng and Deer Antler Tonifying and Rejuvenating Pill, alkaloids are present. Their kidney-protective, anti-cancer, and neuroprotective activities are related to the overall efficacy of the formula, suggesting that alkaloids may be the key active ingredients. However, there is currently no research on the alkaloid components in the Ginseng and Deer Antler Tonifying and Rejuvenating Pill. Summary of the Invention

[0003] To address the aforementioned technical limitations, this application proposes an analytical method for alkaloid 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 alkaloid 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 and extraction, freeze dry, and centrifuge and filter after adjusting the volume 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 alkaloid 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:20ml, each time for 1h; after concentration under reduced pressure, it is extracted twice with chloroform (0.2% HCl solution to pH 2-3); the aqueous layer is taken and extracted three times with chloroform (pH adjusted to 10-11 with ammonia); the chloroform layers are combined and evaporated for concentration; after freeze-drying, it is diluted to volume with 70% methanol; centrifuged at 12,000 rpm for 10 min and filtered through a 0.22 μm filter membrane to obtain the Shenrong Gubenhuanshao Pill test solution.

[0006] Optionally, in the above analytical method, the chromatographic conditions in step S2 are: Infinity LabPoroshell 120 EC-C column. 18 The column has dimensions of 150 mm × 2.1 mm and a diameter of 1.9 μm; the column temperature is 30℃; the mobile phase is 0.1% formic acid water as mobile phase A and acetonitrile as mobile phase B; gradient elution is performed as follows: 0~3 min, 5~15% B; 3~20 min, 15~30% B; 20~30 min, 30~50% B; 30~50 min, 50~90% B; 50~55 min, 90~100% B; 55~60 min, 100~10% B; flow rate: 0.3 mL / min; injection volume: 3 μL.

[0007] Optionally, in the above analytical method, in step S2, the mass spectrometry conditions are as follows: ionization source is electrospray ionization source + 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 voltage is 3.5 kV; and the scanning mass range is... m / z 50~1700; collision energies in positive ion mode of secondary mass spectrometry (MS / MS) 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, construct a mass spectrometry database of alkaloids in Shenrong Gubenhuanshao Pills, 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, analyze and identify the alkaloid components in Shenrong Gubenhuanshao Pills.

[0009] Optionally, the above analytical method, specifically the method for constructing the alkaloid mass spectrometry database of Shenrong Gubenhuanshao Pill, involves: collecting and constructing an alkaloid mass spectrometry database (SRAMSD) containing 287 alkaloid components that may be contained in the formula of Shenrong Gubenhuanshao Pill by consulting literature from the TCMSP database, TCMID database, TCM database, HIT database, DrugBank database, Chemicalbook, Pubchem, Massbank, Chemspider, and Huayuan.com over the past 25 years.

[0010] Optionally, in the above analytical method, the mass spectrometry data processing strategy is selected as follows: total ion chromatogram combined with the SRAMSD alkaloid mass spectrometry database and the GNPS global natural product molecular network for the analysis and identification of alkaloid components.

[0011] 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 alkaloid components in traditional Chinese medicine compositions containing Shenrong Gubenhuanshao Pills.

[0012] The third objective of this application is to provide a method for detecting the alkaloid components in the blood of Shenrong Gubenhuanshao Pills, including the following steps: T1. Rats were administered a water-soluble powder suspension of Ginseng and Deer Antler Pills 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 collected. The sample was filtered to remove impurities, dried with nitrogen, reconstituted with 50% methanol solution, and centrifuged again to collect the supernatant. The sample was then ready 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 C 18 Mobile phase: 2% acetonitrile-water containing 0.1% formic acid was mobile phase A, and acetonitrile containing 0.1% formic acid was mobile phase B. Gradient elution: 0–3.5 min, 2–2% 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, 95–95% B; 14.4–14.5 min, 95–2% B; 14.5–16 min, 2–2% B. Flow rate: 0.4 mL / min, injection: 3 μL, column temperature: 40℃. Mass spectrometry conditions: ESI ion source, scanning in positive ion mode; spray voltage: 3.5 kV, ion source temperature: 320°C; S-Lens RF Level: 40, scanning mass range: m / z. 70~1050; collision energies in positive ion mode of secondary mass spectrometry MS / MS are 20, 40, and 60 V; T3. Analyze the mass spectrometry data of the sample obtained in step T2 to achieve the analysis and identification of the alkaloid components of Shenrong Gubenhuanshao Pill.

[0013] Compared with the prior art, this application has the following advantages: The analytical methods for alkaloids in Shenrong Gubenhuanshao Pill and the detection methods for blood-entering alkaloids provided in this application, by constructing mass spectrometry data of alkaloids in Shenrong Gubenhuanshao Pill and combining it with GNPS molecular network mass spectrometry processing method, identify the alkaloids in this preparation, further determine the blood-entering alkaloids 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 image shown is a positive ion mode total ion chromatogram of the alkaloid fraction in the Shenrong Gubenhuanshao Pill, as described in one embodiment of this application.

[0015] Figure 2 The diagram shown illustrates the interaction between alkaloid components and pharmacological activity network during the construction of the alkaloid chemical component mass spectrometry database for the Shenrong Gubenhuanshao Pill, an embodiment of this application.

[0016] Figure 3 The image shown is a TIC plot of a mixed blood sample in positive ion mode, as illustrated in one embodiment of this application; wherein... Figure 3 A is the TIC plot of the positive ion pattern of mixed blood samples at various time points. Figure 3 B is the TIC plot of positive ion mode for mixed blood samples of various concentrations, S is the gavage solution, Blank is blank plasma, and Z is the mixed blood sample.

[0017] Figure 4 Shown as an embodiment of this application, six components ( Figure 3 The pharmacokinetic curves of peaks 1-1 (Betaine), 7-1 (Peimine), 8-1 (Peiminine), 10-13-18 (Benzoylmesaconine), and 18-19-10 (Benzoylaconine). Detailed Implementation

[0018] 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.

[0019] 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.

[0020] To further understand this application, the following detailed description is provided in conjunction with the preferred embodiments.

[0021] Example 1 Construction of a mass spectrometry database of alkaloid components in Ginseng and Deer Antler Strengthening Pill (SR): To identify the alkaloids in Shenrong Gubenhuanshao Pills, literature from the past 25 years was reviewed in databases including TCMSP, TCMID, TCM, HIT, DrugBank, Chemicalbook, PubChem, Massbank, Chemspider, and Huayuan.com. A SR Alkaloids Mass Spectrum Database (SRAMSD) was also collected, containing 287 alkaloids potentially present in the formula of Shenrong Gubenhuanshao Pills. A network correlation analysis was performed on the pharmacological effects of the alkaloids and the efficacy of Shenrong Gubenhuanshao Pills, revealing a significant correlation. The interaction diagram of the network relationship between some alkaloid components and pharmacological activities during the construction of the SR Alkaloids Mass Spectrum Database for Shenrong Gubenhuanshao Pills is shown below. Figure 2 As shown.

[0022] The selected databases and website information are shown in Table 1.

[0023] Table 1

[0024] Database construction and analysis: Based on research into relevant databases of Chinese herbal medicine components and literature, a database was constructed by collecting information on the chemical components of each single herb contained in Shenrong Gubenhuanshao Pill and its related plants. The database includes compound names, CAS numbers, molecular formulas, relative molecular masses, fragment ions, and mass-to-charge ratios.

[0025] The SRAMSD database was built using the Agilent MassHunter PCDL Manager software platform (v8.0). The specific implementation process includes the following key steps: First, a new database framework was created based on the system's preset CSV template architecture, and compound names, molecular formulas, structural formulas, precise mass numbers, and secondary fragment ion information were standardized and preprocessed. Then, the software's "Allow Editing" permission management mode was activated, and the "Import Compounds" function enabled automated batch data import. The final database covers structural types such as diterpenoids and isosteroid derivatives, and its data format strictly follows the Agilent PCDL database specifications (the first column is the compound's systematic name, and the second column is the molecular formula). It can seamlessly connect to MassHunter's spectral library search module, providing standardized data support for the subsequent efficient identification of alkaloid components in SRs.

[0026] Network association analysis of alkaloid components and the efficacy of Shenrong Gubenhuanshao Pills: The therapeutic indications of SR were classified using multi-source data, and the pharmacological activities of the alkaloid components of SRAMSD were queried. The classification relationship of SR therapeutic indications and the relationship of pharmacological activities of alkaloid components were processed to construct a correlation network of "traditional Chinese medicine - alkaloid components - structural type - pharmacological activity - therapeutic indications". The network relationship was visualized using Gephi 0.10.0 released.

[0027] Construction of a mass spectrometry database of alkaloid components: The SR consists of 65 medicinal herbs. Through searching and screening to remove chemical components with incomplete information, a total of 287 alkaloid components from 26 traditional Chinese medicines were obtained. The results are shown in Table 2.

[0028] Table 2

[0029] Relationship between alkaloid components and pharmacological effects: A review of the alkaloids with reported pharmacological effects in SR revealed that they are mainly classified into three structural types: diterpenoids, isosteroids, and isoquinolines. The alkaloids are predominantly found in Aconitum carmichaelii and Fritillaria thunbergii.

[0030] A systematic analysis method was used to classify the functions and indications of SR (Syndrome Resin) in multiple dimensions, categorizing its effects into eight categories: strengthening muscles and bones, enhancing immunity, treating neurasthenia, treating tinnitus and hearing loss, treating renal insufficiency, treating neurological diseases, lowering blood sugar, and anti-inflammation. The alkaloid components and pharmacological activities of SR were systematically analyzed, resulting in a network diagram showing the relationship between 54 alkaloid components and 48 pharmacological activities from 21 medicinal materials and the eight categories of SR effects, as shown below. Figure 2As shown in the figure. Analysis revealed that magnoflorine in Epimedium has anti-osteoporosis and osteocyte proliferation effects, which correspond precisely to the bone-strengthening effects of SR. Components such as benzoyl aconitine from Aconitum carmichaelii and fritillary alkaloids from Fritillaria thunbergii have anti-inflammatory, neuroprotective, and blood sugar-regulating effects. Betaine from Lycium barbarum, Astragalus membranaceus, Angelica sinensis, and Cistanche deserticola has anti-inflammatory, hypoglycemic, neuroprotective, and anti-aging effects. These pharmacological activities are highly consistent with the therapeutic effects of SR on neurasthenia, neurological diseases, hypoglycemia, and anti-inflammation. Therefore, the alkaloid components in SRAMSD are highly correlated with the overall effects of SR, such as tonifying the kidneys and strengthening yang, and strengthening bones and muscles, providing a basis for the selection of key components in the later stages.

[0031] Through systematic literature analysis and integration with PCDL Manager, a high-resolution mass spectrometry (HMS) prediction database for SR alkaloids was successfully constructed, comprising 287 compounds. To improve the accuracy and reliability of the data, strategies such as synonym standardization, CAS number deduplication, structural formula verification, and cross-referencing of mass spectrometry data from PubChem and MassBank databases were employed. This database features multi-dimensional search capabilities based on compound name and molecular formula, enabling rapid screening of candidate compounds based on precise molecular weight and formula, and accurate identification using HMS data. This optimizes traditional component analysis workflows and improves screening efficiency. The establishment of this self-built database provides a basis for the accurate identification of SR alkaloids in the future.

[0032] Meanwhile, by establishing a network diagram of "traditional Chinese medicine - alkaloid components - structural type - pharmacological activity - indications," the pharmacodynamic role of alkaloid components in SR was systematically revealed. The results showed that the pharmacological activities of alkaloid components in SR, including anti-inflammatory, neuroprotective, glycemic, and anti-osteoporosis effects, were significantly correlated with the overall efficacy of the preparation, indicating that alkaloid components are key pharmacodynamic components in SR.

[0033] Example 2 Qualitative analysis method for alkaloid chemical components in Ginseng and Deer Antler Strengthening Pill: To identify the alkaloid chemical components in Shenrong Gubenhuanshao Pills, UHPLC-Q-TOF-MS / MS was used, employing an InfinityLab Poroshell 120 EC-C spectrometer. 18LC-MS was performed using a chromatographic column, a 0.1% formic acid-water-acetonitrile mobile phase, and an electrospray ionization source. Based on the total ion chromatogram, combined with SRAMSD (Alkaloid Mass Spectrometry Database) and the Global Natural Products Social Molecular Networking (GNPS), a total of 89 alkaloids were identified, with 1 unknown. The structural types were mainly divided into: 8 diester alkaloids; 21 amino alcohol alkaloids; 22 monoester alkaloids; 22 non-steroidal alkaloids; 1 organic amine alkaloid; 2 benzylisoquinoline alkaloids; 1 furoline alkaloid; 1 steroidal alkaloid; and 11 other types of alkaloids. The positive ion mode total ion chromatogram of the alkaloid fraction in the Shenrong Gubenhuanshao Pill is shown below. Figure 1 As shown.

[0034] Materials and Instruments: Ginseng and Deer Antler Tonifying Pills (Lanzhou Foci Pharmaceutical Co., Ltd., batch number: 22091; 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); acetonitrile, formic acid (Merck, Germany, mass spectrometry grade); water (Watsons Food & Beverage Co., Ltd., Guangzhou, purified water). The instruments and materials are shown in Table 3.

[0035] Table 3

[0036] Experimental methods 1. Chromatographic conditions: InfinityLab Poroshell 120 EC-C column 18 (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, 5~15% B; 3~20 min, 15~30% B; 20~30 min, 30~50% B; 30~50 min, 50~90% B; 50~55 min, 90~100% B; 55~60 min, 100~10% B; flow rate: 0.3 mL / min; injection 3 μL.

[0037] 2. Mass spectrometry conditions: The ionization source was an electrospray ionization source (+ESI); the drying gas was N2; the drying gas temperature was 225℃; the drying gas flow rate was 10 L / min; the nebulizer gas pressure was 25 psi; the capillary voltage was 3.5 kV; and the scanning quality range was... m / z 50~1700. Collision energies in positive ion mode of secondary mass spectrometry (MS / MS) are 10, 20, and 40 V.

[0038] 3. Preparation of test solution: Preparation of the test solution: Accurately weigh 10 g of SR powder, add 200 mL of 80% ethanol, and extract twice by sonication for 1 h each time. Concentrate under reduced pressure, and extract twice with chloroform (0.2% HCl solution to pH 2-3). Collect the aqueous layer and extract three times with chloroform (adjust pH to 10-11 with ammonia). Combine the chloroform layers, evaporate and concentrate, freeze-dry, and then bring the volume to 5 mL with 70% methanol. Centrifuge at 12,000 rpm for 10 min, and filter through a 0.22 μm filter membrane to obtain the test solution.

[0039] 4. Database construction and analysis: The mass information of compounds in SRAMSD was input into Mass Hunter 10.0 software. With a mass error of < ± 10 ppm as the standard, the alkaloid components in SR were compared with the accurate molecular mass and mass spectrometry fragmentation pattern of compounds in the PCDL self-built library, and the alkaloid components were finally determined.

[0040] 5. Construction and annotation of GNPS molecular networks: Raw UHPLC-Q-TOF-MS / MS data were imported into Proteo Wizard (v3.0.11781), converted to "mzXML" format, and uploaded to the GNPS online platform. A molecular network was constructed by setting parameters such as "Minimum cosine score" to 0.70 and "Minimum matched fragment ions" to 0.02. The "View Spectra Families" function was used to visualize the formed molecular network. Nodes represent compounds, and lines represent the correlations between compounds. The data was then imported into Cytoscape 3.10.0 for further visualization of the molecular network diagram. Nodes of the same color represent the same metabolite type, and node labels are the quasi-molecular ion peaks of the compounds. m / z value.

[0041] 6. Experimental Results: Chemical components were characterized in the alkaloid fraction of SR using mass spectrometry data obtained by UHPLC-Q-TOF-MS / MS, combined with SRAMSD search and GNPS molecular network strategy. The total ion chromatogram of the alkaloid fraction in SR in positive ion mode is shown below. Figure 1A total of 89 alkaloids were identified, with 1 unknown. The main structural types were: 8 diester alkaloids; 21 amino alcohol alkaloids; 22 monoester alkaloids; 22 non-steroidal alkaloids; 1 organic amine alkaloid; 2 benzylisoquinoline alkaloids; 1 furoline alkaloid; 1 steroidal alkaloid; and 11 other types of alkaloids.

[0042] The identification of alkaloid components in Shenrong Gubenhuanshao Pill using UHPLC-Q-TOF-MS / MS technology is shown in Table 4.

[0043] Table 4

[0044] In Table 4, a represents diester alkaloids; b represents amino alcohol alkaloids; c represents monoester alkaloids; d represents nonsteroidal alkaloids; e represents organic amine alkaloids; f represents benzylisoquinoline alkaloids; g represents furoline alkaloids; h represents other types of alkaloids; and i represents steroidal alkaloids.

[0045] Based on UHPLC-Q-TOF-MS / MS, combined with SRAMSD and GNPS molecular networks, 89 alkaloid components and 1 unknown component were identified. The structural types were mainly divided into: 8 diester alkaloids; 21 amino alcohol alkaloids; 22 monoester alkaloids; 22 nonsteroidal alkaloids; 1 organic amine alkaloid; 2 benzylisoquinoline alkaloids; 1 furoline alkaloid; 1 steroidal alkaloid; and 11 other types of alkaloids. The mass spectrometric fragmentation patterns of representative compounds of diester, amino alcohol, monoester, and nonsteroidal alkaloids were summarized. By comparing with SRAMSD, 60 compounds were identified; and through molecular network construction and annotation, 29 more alkaloid components were further identified. These results lay the foundation for serum medicinal chemistry research on SR.

[0046] Example 3 Analysis and network pharmacology of alkaloid components in the blood of Shenrong Gubenhuanshao Pills: To clarify the active ingredients of Shenrong Gubenhuanshao Pill, based on the principle that components entering the bloodstream may be the effective components in "serum pharmacochemistry," ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry (UHPLC-Q-Exactive Orbitrap MS) was first used to detect the serum of rats in the blank group and the gavage sRGBHSP administration group. A total of 18 alkaloid components entering the bloodstream were identified in the mixed serum at various time points. Betaine, fritillary A, fritillary B, benzoyl aconitine, benzoyl hypoaconitine, and benzoyl neoaconitine showed a pharmacokinetic relationship (pharmaceutical time relationship). Figure 3 , Figure 4 Secondly, network pharmacology and reverse molecular docking were used to predict the targets of blood-entering components. SwissTargetPrediction identified 434 targets for 14 blood-entering components. A PPI network was then established to clarify the relationship between targets and proteins, identifying 134 key targets for blood-entering components. GO and KEGG enrichment analyses were performed, revealing enrichment primarily in lipid and atherosclerosis signaling pathways, non-alcoholic fatty liver disease signaling, and cancer signaling pathways. Heatmap analysis of blood-entering components and diseases revealed associations with lung tumors, brain injury, inflammation, osteoarthritis, liver injury, autism, obesity, diabetes, rheumatoid arthritis, nervous system diseases, colon cancer, and liver cancer. Further targeting of the top five targets with the highest degree values—TP53, AKT1, TNF, ALB, and IL-6—was coupled with 14 blood-entering components. The results showed that, except for betaine, the binding scores were all less than 5.0, confirming that the 14 blood-entering components have a strong binding affinity to their corresponding targets. This preliminarily confirms that these components may be the active ingredients in Shenrong Gubenhuanshao Pills.

[0047] Materials and Instruments: SR (Lanzhou Foci Pharmaceutical Co., Ltd.) (batch number: 22091); acetonitrile, formic acid (Thermo Fisher Scientific, USA, mass spectrometry grade); water (Watsons Food & Beverage Co., Ltd., Guangzhou, purified water). Instrument materials are shown in Table 5, and the database is shown in Table 6.

[0048] Table 5

[0049] Table 6

[0050] Experimental methods: 1. Blood component analysis: 1) Animal husbandry conditions and grouping: Twelve male SD rats (250-300 g) were purchased from the Experimental Animal Center of Lanzhou University (License No.: SYXK(Gan)2023-0004). Hulling conditions: temperature 20-25℃, humidity 55%-70%, 12-hour light and dark cycle. After 7 days of acclimatization in the laboratory, rats were randomly assigned to groups. They were fasted for 12 hours before the experiment but had free access to water. The groups consisted of a treatment group (n=9) and a control group (n=3).

[0051] 2) Collection and processing of blood samples: Preparation of the gavage solution: SR was ground into powder. The required total amount was taken and dissolved in distilled water for later use. The dosage of SR was 6-12 g per person (60 kg) twice daily. After a 4-fold conversion of the dosage, and following a 7-day acclimatization period, the rats in the treatment group were administered the drug for one week, twice daily via gavage, at a dose of 6.48 g / kg (rats' body weight). Rats were fasted for 12 hours before the last gavage on day 7. Blood samples were collected from the orbital venous plexus at 0.083 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, and 8 h after gavage. The blood samples collected at each time point were centrifuged at 4°C and 3000 rpm for 15 min, and the supernatant was collected for later use. Furthermore, to comprehensively detect the components in the drug-containing serum, a mixed sample of serum samples from each time point after drug administration was prepared.

[0052] Each serum sample was precipitated with acetonitrile at a 1:3 ratio. After vortexing and sonication for 30 min, the samples were centrifuged at 14,000 rpm and 4°C for 15 min. The supernatant was collected and filtered to remove impurities. Subsequently, the samples were dried under nitrogen and reconstituted with 200 μL of 50% methanol solution. Finally, the samples were centrifuged again and the supernatant was collected as the samples for analysis.

[0053] 3) Detection method: (1) Chromatographic conditions: The chromatographic column was an ACQUITY UPLC BEH C. 18(100 mm × 2.1 mm; id, 1.7 µm; Waters, Milford, USA); Mobile phase A was 2% acetonitrile-water (containing 0.1% formic acid), mobile phase B was acetonitrile (containing 0.1% formic acid), gradient elution 0–3.5 min, 2–2% 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, 95–95% B; 14.4–14.5 min, 95–2% B; 14.5–16 min, 2–2% B; flow rate: 0.4 mL / min, injection 3 μL, column temperature 40℃.

[0054] (2) Mass spectrometry conditions: The ion source was an ESI source, scanning in positive ion mode. The spray voltage was 3.5 kV, and the ion source temperature was 320°C; the S-Lens RF Level was 40, and the scan quality range was [not specified]. m / z 70~1050. Collision energies in positive ion mode of secondary mass spectrometry (MS / MS) are 20, 40, and 60 V.

[0055] 4) Data Analysis: Data analysis was performed using Xcalibur and Metworks software. Based on the database, targeted ion extraction was performed on the data. The possible elemental composition was calculated based on m / z within a specified error range (< ± 10 ppm). The molecular structure of the compounds was preliminarily inferred from the multi-stage mass spectrometry fragment data. The secondary mass spectrometry fragments of each component peak were compared with SRAMSD data to identify unknown compounds.

[0056] Target prediction: 1. Reverse target finding: (1) Target prediction of blood-entering components: First, the molecular structures of SR-infused components were queried one by one from the PubChem database to obtain SMILES numbers and target prediction was performed using Swiss Target Prediction. Then, the prediction results were visualized using Cytoscape 3.10.0 software.

[0057] (2) Constructing a protein-protein interaction network: String is commonly used to analyze protein-protein interactions. To explore the interactions between targets related to SR (serotonin) components entering the bloodstream, the obtained targets were imported into the String platform for online analysis. The study species was limited to humans, and isolated nodes were removed. To make the results more intuitive, the data was exported in TSV format, and a PPI (protein-protein interaction) network of SR components entering the bloodstream was constructed using Cytoscape 3.10.0. The CytoNCA plugin was then used for in-depth analysis of the targets, excluding targets with a degree value less than 25, and PPI maps were plotted for the remaining targets.

[0058] (3) Biological processes related to potential targets and the KEGG pathways involved: DAVID is primarily used for the functional and pathway enrichment analysis of differentially expressed genes. Potential targets are input into the DAVID database for online analysis, yielding data on the biological processes, cellular composition, molecular function (GO), and signaling pathways involved (KEGG). The results are then visualized using bubble charts generated by Microbioinformatics.

[0059] (4) Compound disease analysis: The Comparative Toxicology Genomics Database (CTD) is a public website research tool that uses scientific data to describe the relationships between chemicals and drugs. All blood-entering components are searched in the CTD, duplicates are removed, and a heatmap analysis of the compounds and diseases is performed.

[0060] 2. Molecular docking verification: By analyzing the results of blood-entry component-target network, protein-protein interaction network, and GO and KEGG enrichment analyses, the top 5 target proteins with the highest common degree were selected for molecular docking verification with the blood-entry components. PDB files for these 5 targets were downloaded from the PDB database. Pymol 2.6 was used to perform operations such as dehydration and hydrogenation, ligand removal, and determination of torsional forces on the proteins. 3D SDF structure files of the blood-entry components were downloaded from the PubChem database and converted to corresponding PDB files using OpenBabel. AutoDock-Tools 1.5.7 was used to obtain PDBQT files for the receptor and ligand, and active pocket data was obtained. Finally, AutoDock-vina was run for molecular docking, and Pymol 2.6 was used for visualization analysis.

[0061] 3. Experimental Results: Blood component identification: The identification conditions for the original components in SR-treated serum samples were consistent with those for in vitro component identification. A total of 18 alkaloid components were found in the mixed serum samples. The retention time of the total sample was used as the standard, with other times indicated as: Into (detectable) and None (not detected). Thirteen of these components were detectable at 0.083 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, and 8 h, namely peak 1 (Betaine), peak 2 (Isotalatizidine), peak 4 (Magnoflorine), peak 5 (Talatisamine), peak 7 (Peimine), peak 8 (Peiminine), and peak 9 (16-). O The in vitro biomarkers included: -Demethylbenzoylaconitine, peak 10 Benzoylmesaconine, peak 12 Solasodine, peak 13 Benzoylaconine, peak 14 14-Acetyltalatizamine, peak 16 Lauryl diethanolamine, and peak 18 Benzoylhypaconine. Peak 1 is an indicator component of Lycium barbarum; peaks 7 and 8 are indicator components of Fritillaria thunbergii; and peaks 10, 13, and 18 are monoester-type alkaloids, which are indicator components of Aconitum carmichaelii. Except for peak 4, the remaining 12 peaks were detectable in vitro. Peak 3, Bullatine B, was detected at all times except at 0.083 h. Furthermore, most of the components detected entering the bloodstream belong to Aconitum carmichaelii, indirectly indicating that the alkaloids in the principal herb Aconitum carmichaelii may play an important pharmacological role. Specific in vivo data are shown in Table 7, and the TIC plot is shown below. Figure 3 As shown.

[0062] Table 7

[0063]

[0064] Figure 3 middle, Figure 3 A is the TIC plot of the positive ion pattern of mixed blood samples at various time points. Figure 3 B is the TIC plot of positive ion mode for mixed blood samples of various concentrations, S is the gavage solution, Blank is blank plasma, and Z is the mixed blood sample.

[0065] in accordance with Figure 3As a result, for peaks 1 (Betaine), 7 (Peimine), 8 (Peiminine), 10 (Benzoylmesaconine), 13 (Benzoylaconine), and 18 (Benzoylhypaconine), pharmacokinetic curves (time on the x-axis and relative peak area on the y-axis) were plotted using Graphpad Prism 9.5 software. (See...) Figure 4 .

[0066] 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 alkaloid components in Shenrong Gubenhuanshao Pills, characterized in that, The analytical method includes the following steps: Accurately weigh the powder of Shenrong Gubenhuanshao Pill, perform ultrasonic extraction and extraction, freeze dry, and centrifuge and filter after adjusting the volume to obtain the test solution of Shenrong Gubenhuanshao Pill; The powder of Shenrong Gubenhuanshao Pills was ultrasonically extracted twice with 80% ethanol at a ratio of 1 g: 20 ml, each time for 1 h. After concentration under reduced pressure, it was extracted twice with 0.2% HCl solution and chloroform solution with pH 2-3. The aqueous layer was taken and extracted three times with chloroform solution adjusted to pH 10-11 with ammonia. The chloroform layers were combined, evaporated and concentrated, freeze-dried and then diluted to volume with 70% methanol. The solution was centrifuged at 12,000 rpm for 10 min and filtered through a 0.22 μm filter membrane to obtain the test solution of Shenrong Gubenhuanshao Pills. 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-C column. 18 The column dimensions are 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, 5~15% B; 3~20 min, 15~30% B; 20~30 min, 30~50% B; 30~50 min, 50~90% B; 50~55 min, 90~100% B; 55~60 min, 100~10% 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 voltage: 3.5 kV; scanning mass range: m / z 50~1700; collision energies in positive ion mode of secondary mass spectrometry MS / MS are 10, 20, and 40 V; The high-resolution mass spectrometry data of the test solution obtained in step S2 were analyzed to identify the alkaloid components in the Shenrong Gubenhuanshao Pill. The identification results are shown in the table below: 。 2. The analytical method according to claim 1, characterized in that, The analytical identification method in step S3 is as follows: First, a mass spectrometry database of alkaloids 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) technology in step S2, combined with various mass spectrometry data processing strategies, the alkaloid components in Shenrong Gubenhuanshao Pills are analyzed and identified.

3. The analytical method according to claim 2, characterized in that, The method for constructing the alkaloid mass spectrometry database of Shenrong Gubenhuanshao Pill is as follows: by consulting the TCMSP database, TCMID database, TCM database, HIT database, DrugBank database, Chemicalbook, Pubchem, Massbank, Chemspider, and Huayuan.com for nearly 25 years of literature, an alkaloid mass spectrometry database SRAMSD was constructed, which contains 287 alkaloid components that may be contained in the formula of Shenrong Gubenhuanshao Pill.

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 SRAMSD alkaloid mass spectrometry database and the GNPS global natural product molecular network for the analysis and identification of alkaloid 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 alkaloid components in a traditional Chinese medicine composition containing Shenrong Gubenhuanshao Pill.

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