| Summary: | This study aimed to isolate, prepare and identify the main flavonoids from a standardized <i>Smilax glabra</i> flavonoids extract (SGF) using preparative HPLC, MS, <sup>1</sup>H NMR and <sup>13</sup>C NMR, determine the contents of these flavonoids using UPLC, then compare their pharmacological activities in vitro. We obtained six flavonoids from SGF: astilbin (18.10%), neoastilbin (11.04%), isoastilbin (5.03%), neoisoastilbin (4.09%), engeletin (2.58%) and (−)-epicatechin (1.77%). The antioxidant activity of six flavonoids were evaluated by determining the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and 2,2′-Azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS<sup>+</sup>) radical scavenging activity and ferric reducing antioxidant power (FRAP). In addition, the anti-inflammatory activity of six flavonoids were evaluated by determining the production of cytokines (IL-1β, IL-6), nitric oxide (NO) using enzyme linked immunosorbent assay and the NF-κB p65 expression using Western blotting in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. The results showed that (−)-epicatechin, astilbin, neoastilbin, isoastilbin and neoisoastilbin had strong antioxidant activities, not only in DPPH and ABTS<sup>+</sup> radicals scavenging capacities, but in FRAP system. Furthermore, all the six flavonoids could significantly inhibit the secretion of IL-1β, IL-6, NO (<i>p</i> < 0.01) and the protein expression of NF-κB p-p65 (<i>p</i> < 0.01) in LPS-stimulated RAW264.7 cells. This study preliminarily verified the antioxidant and anti-inflammatory activities of six flavonoids in <i>S. glabra</i>.
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