高效液相色谱-四极杆/静电场轨道阱高分辨质谱法快速分析三子散的入血成分及代谢产物

Sanzi San, a Mongolian medicine, comprises three herbs: Terminalia chebula, Melia toosendan, and Gardenia jasminoides. Clinically, Sanzi San is administered orally and distributed via blood to the action site, which implies that the absorption, distribution, metabolism, and excretion (ADME) are closely related to the pharmacological action and curative effect. Therefore, possible explanations for the material basis of Sanzi San were explored in this study preliminarily. A strategy based on serum pharmacochemistry was first applied to explore the absorbed bioactive components and metabolites of Sanzi San. Wistar rats were randomly divided into normal and dosing groups, which were provided with the Sanzi San’s water extract for three days. Then, the rat’s blood samples were obtained from their abdomiral aorta using a sterile blood collection tube after administering the medicine. The blood samples were then centrifuged at 3500 r/min for 10 min to obtain the serum samples. A practical method based on high performance liquid chromatography coupled with quadrupole and electrostatic field orbitrap high resolution mass spectrometry (HPLC-Q/Orbitrap HRMS) was developed to screen and analyze numerous bioactive components and metabolites adsorbed in the serum of the dosing rats after oral administration of the Sanzi San’s water extract. Chromatographic separation was achieved on a SHIMADZU GIST C(18) chromatographic column (150 mm×4.6 mm, 5 μm). The temperature of the column was maintained at 30 ℃. The flow rate was 0.5 mL/min, and the injection volume was 10 μL. The mobile phase comprised an aqueous solution of 0.1% formic acid and methanol under gradient elution. A heated electrospray ion (HESI) source was used with positive and negative ion scanning modes. To rapidly screen out and identify the absorbed bioactive components and metabolites of Sanzi San in the rat serum samples, a simple three-step approach was developed. First, the known components in Sanzi San were listed systematically by exploring various databases, such as the Web of Science, PubMed, and Chinese National Knowledge Infrastructure. In addition, relevant information on drug biotransformation and the characteristic fragmentation patterns of parent compounds were summarized. Second, the absorbed components and metabolites were ascertained using the Xcalibur 3.0 software. Based on the information related to the parent compound’s structure, the software could be used to identify the unique peaks by comparing the chromatograms of the normal and dosing samples. Consequently, the total ion chromatograms of serum samples were established. Finally, the Compound Discover 3.0 software was used to predict the metabolic pathways and fragmentation of the absorbed compounds. Using this approach, 55 compounds were characterized, including 41 prototype components and 14 metabolites. The main prototype components in the serum sample were tannins, iridoids, and phenolic acids. The details of these compounds have been summarized and presented. Regarding the absorbed bioactive components and metabolites in the serum samples of rats administered with Sanzi San, phase Ⅰ and phase Ⅱ biochemical reactions were involved in the biotransformation pathways. The phase Ⅰ reaction modified the components and created sites for the phase Ⅱ reaction, involving reduction and hydrolysis. The phase Ⅱ reaction coupled groups to existing conjugation sites, including glucuronide to glucuronic acid, sulfate, and methyl. MS/MS spectra indicated that methylation, demethylation, and dehydroxylation are the metabolic pathways of procyanidins. Additionally, glucuronidation, deglucosidation, hydration, and demethylation are the metabolic pathways of iridoids in Sanzi San. This study comprehensively analyzed the components of the Sanzi San’s water extract absorbed in the rat’s serum. Our results revealed information regarding the pharmacodynamic substances and the major pathways involved in the ADME of Sanzi San. Further, potential medicinal ingredients for the pharmacological effects and clinical use of Sanzi San were explored at the serum pharmacochemistry level.