title: Identification of the anti-oxidant components in a two-step solvent extract of bovine bile lipid: Application of reverse phase HPLC, mass spectrometry and fluorimetric assays.
abstract: An ether extract of nine different bacterial metabolites in combination with two solvent extract (ether followed by ethanol) of bile lipids from ox gall bladder is used as an immune stimulator drug. Over the years bile acids are discussed regarding their anti-oxidant and lipid peroxidation properties. Since some of the bile acids are known to be potent antioxidants, presence of similar activity in the solvent extract of ox bile lipid was investigated using TLC and reverse phase HPLC systems. Fractions from HPLC were analyzed with mass spectrometry using electrospray ionization. The presence of twelve different bile acids along with other substances in small proportions including fatty acids, sulfate conjugates and bile pigments were confirmed. The twelve separated peaks had similar retention times as those of tauroursodeoxycholic acid, glycoursodeoxycholic acid, taurocholic acid, glycocholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, taurodeoxycholic acid, cholic acid, ursodeoxycholic acid, chenodeoxycholic acid, deoxycholic acid, and lithocholic acid. Subsequently, all fractions were tested for their anti-oxidative property on HepG2 cells exposed to H2O2 that served as an oxidative injury model. Four fluorescent dyes H2DCF DA, MitoSOX red, Amplex red and DAF-2 DA were used for estimation of reactive radicals in the HepG2 cells. Among the separated bile acids, tauroursodeoxycholic acid, glycoursodeoxycholic acid and ursodeoxycholic acid prevented the HepG2 cells from H2O2-induced oxidative stress.
authors.affiliation: Division of Structural Biology and Bioinformatics, CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Jadavpur, Kolkata 700032, India. Electronic address: namratasingh819@gmail.com.
authors.forename: Namrata
authors.initials: N
authors.lastname: Singh
authors.affiliation: Division of Structural Biology and Bioinformatics, CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Jadavpur, Kolkata 700032, India. Electronic address: debasish@iicb.res.in.
authors.forename: Debasish
authors.initials: D
authors.lastname: Bhattacharyya
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D001646:Bile / Q000737:chemistry*; D002417:Cattle; D005591:Chemical Fractionation; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D056148:Chromatography, Reverse-Phase / Q000379:methods; D005470:Fluorometry / Q000379:methods; D005704:Gallbladder / Q000737:chemistry*; D056945:Hep G2 Cells; D006801:Humans; D008055:Lipids / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D013058:Mass Spectrometry / Q000379:methods; D018384:Oxidative Stress / Q000187:drug effects
chemical_list: D000975:Antioxidants; D008055:Lipids
keywords: Anti-oxidant assay by fluorescence probes; HepG2 cells; Mass spectrometry; Ox bile; Oxidative stress; Reverse-phase HPLC; Separation and identification of bile acids