HPLC determination of malondialdehyde in ECV304 cell culture medium for measuring the antioxidant effect of vitexin-4''-O-glucoside.
To investigate the antioxidant effect of vitexin-4''-O-glucoside, a flavone glycoside, isolated from the leaves of Crataegus pinnatifida Bge. var. major, we developed a simple and sensitive high-performance liquid chromatography (HPLC) method to determine levels of malondialdehyde (MDA) in ECV304 cell culture medium after induction by tert-butyl-hydroperoxide (TBHP). The preparation of analyzed samples involved a one-step derivatization with thiobarbituric acid (TBA). HPLC analysis was performed on a Synergi Hydro-RP, a polar end-capped C18 column (250 x 4.6 mm, 4 mum), using an acetonitrile-ammonium acetate aqueous solution (10 mM, pH 6.8) as the mobile phase under linear gradient conditions with UV detection at 532 nm. The calibration curve was linear over 0.0125-1.25 microM MDA (r = 0.9951). Relative standard deviations (RSDs) of intra-day and inter-day precision were less than 6.1% and 5.0%, respectively. The mean recovery was 96.9 +/- 1.6%. The lower limit of quantification (LLOQ) of MDA was 0.0125 microM. This chromatographic method was successfully applied to investigating the in vitro antioxidant effect of vitexin-4''-O-glucoside. Vitexin-4''-O-glucoside (120 M) protected ECV304 cells from peroxidation induced by TBHP.
Ying
Xi-xiang
XX
School of Pharmacy, Liaoning University of TCM, No. 79 Chongshan East Road, Shenyang, China.
Li
Hai-bo
HB
Chu
Zheng-yun
ZY
Zhai
Yan-jun
YJ
Leng
Ai-jing
AJ
Liu
Xun
X
Xin
Chun
C
Zhang
Wen-jie
WJ
Kang
Ting-guo
TG
D000975:Antioxidants / Q000494:pharmacology*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D027825:Crataegus / Q000737:chemistry; D003470:Culture Media / Q000032:analysis; D005960:Glucosides / Q000494:pharmacology*; D007202:Indicators and Reagents; D007529:Isoflavones / Q000494:pharmacology*; D009682:Magnetic Resonance Spectroscopy; D008315:Malondialdehyde / Q000032:analysis* / Q000378:metabolism*; D016877:Oxidants / Q000633:toxicity; D018515:Plant Leaves / Q000737:chemistry; D012015:Reference Standards; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization; D013056:Spectrophotometry, Ultraviolet; D020122:tert-Butylhydroperoxide / Q000633:toxicity
D000975:Antioxidants; D003470:Culture Media; D005960:Glucosides; D007202:Indicators and Reagents; D007529:Isoflavones; D016877:Oxidants; C477953:isovitexin-4'-O-glucoside; D008315:Malondialdehyde; D020122:tert-Butylhydroperoxide