Three Polymethoxyflavones Purified from Ougan ( Citrus reticulata Cv. Suavissima ) Inhibited LPS-Induced NO Elevation in the Neuroglia BV-2 Cell Line via the JAK2/STAT3 Pathway.

In order to establish an efficient method for separation of polymethoxyflavones (PMFs) and explore the anti-inflammatory mechanism of PMF monomers, a citrus variety rich in PMFs, Ougan ( Citrus reticulata cv. Suavissima ), was selected, and three monomers, including nobiletin, tangeretin, and 5-demethylnobiletin, were purified by ultrasonic-assisted extraction, solid phase extraction, and high-speed countercurrent chromatography separation. UPLC-MS was used to identify the three monomers. UPLC determined purities of 99.87% to nobiletin, 99.76% to tangeretin, and 98.75% to 5-demethylnobiletin with the standard curve method. A lipopolysaccharide (LPS)-induced NO releasing model was performed in the mouse microglia BV-2 cell line. Results illustrated that PMF monomers inhibited the NO release and the inflammation-related cytokines, including IL-1β, IL-6, and TNFα elevation. QRT-PCR revealed that PMFs alleviated LPS-induced upregulation of iNOS , IL-6 , JAK2 , TNF α, IL-1β , and NF- κ B and LPS-induced downregulation of I κ B α, while they did not affect TLR1 , TLR2 , TLR4 , and TLR6 . STAT3 expression was repressed by tangeretin and 5-demethylnobiletin, but not by nobiletin. Western blot assay also showed a suppression of expression and phosphorylation of JAK2 by all three PMF monomers, while STAT3 phosphorylation was restrained by tangeretin and 5-demethylnobiletin. The mechanism was primarily verified by the JAK2 inhibitor Ruxolitinib and the STAT3 inhibitor Stattic.