Flaxseed cyclolinopeptide-mediated regulation of α-linolenic acid metabolism alleviates high-fat diet-induced vascular inflammation

Methionine-containing cyclolinopeptide ([1-9-NαC]-linusorb B2, CLB) and α-linolenic acid (ALA), two anti-inflammatory flaxseed constituents, were investigated for their combined vascular benefits. CLB-enriched orbitides were prepared for long-term high-fat diet (HFD)-fed mouse studies, while high-purity CLB monomer (>97%) was isolated <i>via</i> preparative chromatography for acute <i>in vivo</i> and cell-based assays. Structural confirmation and purity validation were achieved <i>via</i> mass spectrometry and liquid chromatography. CLB-enriched orbitides co-administered with ALA in HFD-fed mice reduced body weight gain, serum trimethylamine <i>N</i>-oxide (TMAO), dyslipidemia, and vascular inflammation, histologically confirmed by reduced pro-inflammatory mediators and endothelial repair. Purified CLB combined with ALA further demonstrated efficacy in alleviating TMAO-induced acute vascular inflammation in mice. Mechanistically, CLB enhanced ALA metabolism by upregulating key enzymes (lipoxygenases and cytochrome P450) and directly binding to their catalytic pockets. Combined effect loss upon enzyme inhibition confirmed ALA metabolic modulation as the key mechanism. These findings highlight CLB's role in amplifying ALA's efficacy to combat vascular inflammation.