[Comprehensive identification of metabolites and metabolic characteristics of luteolin and kaempferol in Simiao Yong'an Decoction in rats by UHPLC-LTQ-Orbitrap MS/MS].

Simiao Yong'an Decoction is a classic prescription for treating gangrene. Modern medical evidence has proven that Si-miao Yong'an Decoction has therapeutic effects on atherosclerosis(AS), vascular occlusion angeitides, and hypertension, while its pharmacodynamic mechanism remains unclear. The evidence of network pharmacology, molecular docking, literature review, and our previous study suggests that luteolin and kaempferol are two major flavonoids in Simiao Yong'an Decoction and can inhibit macrophage inflammation and exert anti-AS effects. However, due to lack of the metabolism studies in vivo, little is known about the metabolic characteristics of luteolin and kaempferol. This study employed ultra-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry(UHPLC-LTQ-Orbitrap MS/MS) and relevant software to identify the metabolites and metabolic pathways of luteolin and kaempferol in rat plasma, urine, and feces, after oral administration of luteolin and kaempferol, respectively. After the administration of luteolin, 10, 11, and 3 metabolites of luteolin were detected in the plasma, urine, and feces, respectively. After the administration of kaempferol, 9, 3, and 1 metabolites of kaempferol were detected in the plasma, urine, and feces, respectively. The metabolic pathways mainly involved methylation, glucuronidation, and sulfation. This study enriches the knowledge about the pharmacological mechanism of luteolin and kaempferol and supplies a reference for revealing the metabolic process of other flavonoids in Simiao Yong'an Decoction, which is of great significance for elucidating the pharmacological effects and effective substances of this decoction in vivo.

[Identification of chemical constituents in Simiao Yong'an Decoction based on UPLC-LTQ-Orbitrap-MS].

This study aims to identify the chemical constituents of Simiao Yong'an Decoction based on ultra-performance liquid chromatography coupled with linear quadrupole ion trap-orbitrap mass spectrometry(UPLC-LTQ-Orbitrap-MS). The elution was performed through a UPLC BEH C_(18) column(2.1 mm × 100 mm, 1.7 µm) with the mobile phase of water(containing 0.1% formic acid)-acetonitrile at a flow rate of 0.4 mL·min~(-1). LTQ-Orbitrap-MS with heat electrospray ion(HESI) source was employed to collect MS fragment information in the negative ion mode. A total of 72 compounds were identified based on reference substance comparison, fragmentation rules, accurate molecular weight, related reports and databases(MassBank and HMDB), including 30 iridoid glycosides, 9 organic acids, 15 flavonoids, 10 phenylpropanoids, 7 triterpenoids, and 1 saccharide. The method established in this study is comprehensive, rapid, and accurate, which can help summarize the fragmentation rules of constituents and provide reference for revealing the active constituents and pharmacodynamic mechanism of Simiao Yong'an Decoction.

Comparative Pharmacokinetics of Seven Major Compounds in Normal and Atherosclerosis Mice after Oral Administration of Simiao Yong'an Decoction.

Simiao Yong'an decoction (SMYAD), a classic traditional Chinese medicine formula, has been used to treat atherosclerosis (AS) in clinical in China, but its therapeutic mechanism and pharmacodynamic material basis are not clear. In this study, the AS model was caused by a high-fat diet and perivascular carotid collar placement (PCCP), and SMYAD was orally administered to the model and normal mice. A rapid, sensitive, selective, and reliable method using ultrahigh-performance liquid chromatography (UHPLC) system combined with a Q Exactive HF-X mass spectrometer (UHPLC-Q Exactive HF-X MS) was established and validated for the simultaneous determination of seven compounds, including harpagide, chlorogenic acid, swertiamarin, sweroside, angoroside C, liquiritin, and isoliquiritigenin in the plasma of normal and AS mice. The specificity, linearity, precision, accuracy, recovery, and stability of the method were all within the acceptable criteria. The results showed that some pharmacokinetic behaviors of harpagide, chlorogenic acid, and isoliquiritigenin were significantly different among the two groups of mice. The specific parameter changes were harpagide (AUC0-t and AUC0-∞ were 11075.09 ± 2132.38 and 16221.95 ± 5622.42 ng·mL-1·h, respectively; CLz/F was 2.45 ± 0.87 L/h/mg), chlorogenic acid (t 1/2 was 21.59 ± 9.16 h; AUC0-∞ was 2637.51 ± 322.54 ng·mL-1·h; CLz/F was 13.49 ± 1.81 L/h/mg) and isoliquiritigenin (AUC0-t and AUC0-∞ were 502.25 ± 165.65 and 653.68 ± 251.34 ng·mL-1·h, respectively; CLz/F was 62.16 ± 23.35 L/h/mg) were altered under the pathological status of AS. These differences might be partly ascribed to the changes in gastrointestinal microbiota, nonspecific drug transporters, and cytochrome P450 activity under the AS state, providing research ideas and experimental basis for pharmacological effects and pharmacodynamic material basis.

Mechanistic Insight into PPARγ and Tregs in Atherosclerotic Immune Inflammation.

Atherosclerosis (AS) is the main pathological cause of acute cardiovascular and cerebrovascular diseases, such as acute myocardial infarction and cerebral apoplexy. As an immune-mediated inflammatory disease, the pathogenesis of AS involves endothelial cell dysfunction, lipid accumulation, foam cell formation, vascular smooth muscle cell (VSMC) migration, and inflammatory factor infiltration. The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) plays an important role in lipid metabolism, inflammation, and apoptosis by antagonizing the Wnt/ß-catenin pathway and regulating cholesterol efflux and inflammatory factors. Importantly, PPARγ-dependant fatty acid uptake is critical for metabolic programming. Activated PPARγ can exert an anti-atherosclerotic effect by inhibiting the expression of various inflammatory factors, improving endothelial cell function, and restraining the proliferation and migration of VSMCs. Regulatory T cells (Tregs) are the only subset of T lymphocytes that have a completely negative regulatory effect on the autoimmune response. They play a critical role in suppressing excessive immune responses and inflammatory reactions and widely affect AS-associated foam cell formation, plaque rupture, and other processes. Recent studies have shown that PPARγ activation promotes the recruitment of Tregs to reduce inflammation, thereby exerting its anti-atherosclerotic effect. In this review, we provide an overview of the anti-AS roles of PPARγ and Tregs by discussing their pathological mechanisms from the perspective of AS and immune-mediated inflammation, with a focus on basic research and clinical trials of their efficacies alone or in combination in inhibiting atherosclerotic inflammation. Additionally, we explore new ideas for AS treatment and plaque stabilization and establish a foundation for the development of natural PPARγ agonists with Treg recruitment capability.