Chemometrics-Assisted Identification of Anti-Inflammatory Compounds from the Green Alga Klebsormidium flaccidum var. zivo.

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.