Abstract
The green alga Klebsormidium flaccidum var. zivo is a rich source of proteins, polyphenols, and bioactive small-molecule compounds. An approach involving chromatographic fractionation, anti-inflammatory activity testing, ultrahigh performance liquid chromatography-mass spectrometry profiling, chemometric analysis, and subsequent MS-oriented isolation was employed to rapidly identify its small-molecule anti-inflammatory compounds including hydroxylated fatty acids, chlorophyll-derived pheophorbides, carotenoids, and glycoglycerolipids. Pheophorbide a, which decreased intracellular nitric oxide production by inhibiting inducible nitric oxide synthase, was the most potent compound identified with an IC50 value of 0.24 µM in lipopolysaccharides-induced macrophages. It also inhibited nuclear factor kappaB activation with an IC50 value of 32.1 µM in phorbol 12-myristate 13-acetate-induced chondrocytes. Compared to conventional bioassay-guided fractionation, this approach is more efficient for rapid identification of multiple chemical classes of bioactive compounds from a complex natural product mixture.