Protective effect of a freshwater alga, <Emphasis Type="Italic">Spirogyra</Emphasis> sp., against lipid peroxidation in vivo zebrafish and purification of antioxidative compounds using preparative centrifugal partition chromatography

A freshwater alga, Spirogyra sp., collected in shallow ponds in South Korea, was evaluated for its antilipid peroxidative effect against 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH)-induced in vivo zebrafish, and antioxidative compounds from the alga were efficiently identified using 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS⁺) online high-performance liquid chromatography (HPLC) and preparative centrifugal partition chromatography. The ethyl acetate fraction of Spirogyra sp. (SPE) in each fraction showed the strongest 2,2-diphenyl-1-picrylhydrazyl scavenging activity and significantly scavenged 2′,7′-dichlorodihydrofluorescein diacetate and diphenyl-1-pyrenylphosphine fluorescence, respectively, in AAPH-induced zebrafish embryo without any cytotoxicity in the concentrations between 25 and 50 μg mL^?1. The two main antioxidative compounds in SPE were confirmed by ABTS⁺ online HPLC and were identified as gallic acid and methyl gallate, respectively, by HPLC–diode array detection (DAD)–electrospray ionization (ESI)/mass spectrometry (MS), ¹H- and ¹³C-NMR. We conclude therefore that Spirogyra sp. is rich in gallic acid and methyl gallate, and it might be useful as a strong antilipid peroxidation material.