Mechanistic insights into the anti-oxidative and anti-inflammatory functions of covalent-reactive cinnamyl compounds within Cinnamomum cassia

Background: Cinnamomum cassia Presl (Lauraceae) is widely used as a medicinal plant in the folk medicine and pharmaceutic industry, for its promising anti-inflammatory, anti-oxidative, and anti-bacterial function. However, the major bioactive components were still in debate, and their underlying molecular mechanism was not yet fully understood.

Purpose: This study aimed to identify the bioactive ingredients of C. cassia and investigate the molecular mechanism using in vitro and in silico methods.

Methods: UPLC-QTOF/MS/MS analysis was used to characterize the chemical constituents of alcoholic extract from C. cassia. Reduced glutathione was employed to deplete covalent active cinnamyl compounds. Subsequently, the anti-inflammatory and antioxidant effects of covalent reactive and non-covalent reactive ingredients from C. cassia extract were compared. Their molecular mechanisms were investigated using untargeted metabolomics, in vitro assays, surface plasmon resonance (SPR), and molecular modeling.

Results: Chemical analysis and in vitro assays confirmed the covalent reactive cinnamyl compounds, such as cinnamaldehyde and 2-methoxycinnamaldehyde, exhibited anti-inflammatory and anti-oxidative activity on LPS-stimulated macrophages. Untargeted metabolomics revealed that cinnamaldehyde, one of the covalent reactive cinnamyl compounds, primarily affected amino acid metabolism, and glucose metabolism, promoted glutathione synthesis within LPS-stimulated macrophages, and affected the metabolic profile of M1 macrophages. Consistent with these findings, cinnamaldehyde significantly increased glutathione synthesis and induced glutathione efflux from murine macrophages. In contrast to the literature data, we observed that cinnamaldehyde did not cause GSH depletion, nor elevate the expression of glutamate-cysteine ligase (GCL) in proinflammatory macrophages at low concentrations. The SPR experiment and molecular modeling demonstrated that GCLC was the potential target of cinnamaldehyde.

Conclusions: Our study not only demonstrated the reactive cinnamyl species as the principal antioxidative component of C. cassia but also unveiled a novel molecular mechanism whereby covalent reactive compounds exert their antioxidative effects through covalent modification of GCLC at its active center.