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ABSTRACT:To explore the metabolism of 1,5-dicaffeoylquinic acid (1,5-DCQA) in rats, liquid chromatography-mass spectrometry in parallel to diode-array detection was used for the rapid detection/characterization of the metabolites formed in bile, urine, and plasma of rats following oral administration of 1,5-DCQA (160 mg/kg). The methylation and glucuronidation of 1,5-DCQA occurring in vitro using rat liver and small intestinal microsomes and cytosols were studied in comparison with those occurring in vivo, and the enzymes involved were also determined. In addition, the anti-HIV (human immunodeficiency virus) activity of three important metabolites was preliminarily evaluated in MT-4 cells infected with HIV-1. A total of 22 metabolites in vivo and in vitro were identified, including four isomeric O-mono-methylated metabolites (M8-M11), nine isomeric O-di-methylated metabolites (M3, M6, M22, and M12-M17), four isomeric O-mono-methyl-glucuronidated metabolites (M2 and M19-M21), four isomeric O-di-methyl-glucuronidated metabolites (M1, M4, M5, and M7), and one glucuronidated metabolite (M18).The O-methylation positions of three important metabolites (M8, M9, and M12) were determined (3؆-, 3-, and 3,3؆-) by comparing with synthesized standards. The efficacy experiments showed that M8, M9, and M12 could inhibit HIV replication with IC 50 values of about 25, 25, and 46 M, respectively. These results suggest that O-methylation and glucuronidation are two important metabolic pathways of 1,5-DCQA, that both rat liver and small intestine can catalyze such reactions by catechol-O-methyltransferase and UDP-glucuronosyltransferases, and that the HIV-1 inhibitory activity of M8, M9, and M12 is comparable to or slightly weaker than that of 1,5-DCQA.Dicaffeoylquinic acids (DCQAs) are a class of natural polyphenolic compounds widely distributed in plants, such as fennel (Parejo et al., 2004), mugwort flowering tops (Fraisse et al., 2003), coffee (Moreira et al., 2001), sweetpotato leaf (Yoshimoto et al., 2002), Ilex brevicuspis Reisseck (Filip and Ferraro, 2003), Isertia pittieri (Um et al., 2002), and Eleutherococcus senticosus (Tolonen et al., 2002). Structurally, they are characterized by two caffeic acid molecules connected to one quinic acid molecule through ester bonds. In the past 10 years, DCQAs have been established as an important class of compounds with their potential effects of inhibiting HIV-1 integrase selectively and preventing HIV-1 replication in tissue culture at nontoxic concentrations (McDougall et al., 1998; Robinson et al., 1996a, b;King et al., 1999). HIV-1 integrase is an essential enzyme that mediates integration of the HIV genome into the host chromosomes. Zhu et al. (1999) reported that such inhibition of DCQAs on the HIV-1 integrase is irreversible toward its conserved amino acid residues in the central core domain during catalysis. The combinations of HIV integrase inhibitors with already existing inhibitors for HIV reverse transcriptase and protease have been suggested to be strongly synergetic (Ro...

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α-conotoxin GI, a short peptide toxin in the venom of Conus geographus, is composed of 13 amino acids and two disulfide bonds. It is the most toxic component of Conus geographus venom with estimated lethal doses of 0.029–0.038 mg/kg for humans. There is currently no reported analytical method for this toxin. In the present study, a sensitive detection method was developed to quantify GI in human plasma using a solid-phase extraction (SPE) column (polystyrene–divinyl benzene copolymer) combined with liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) in the multiple reaction monitoring (MRM) mode. The plasma samples were treated with a protein precipitating solvent (methanol: acetonitrile = 50:50, v/v). GI in the solvent was efficiently extracted with an SPE column and was further separated by a Grace Alltima HP C18 (50 × 2.1 mm, 5 μm) column at a flow rate of 0.4 mL/min. Water (with 2% methanol) acetonitrile (with 0.1% acetic acid) was selected as the mobile phase combination used in a linear gradient system. α-Conotoxin GI was analyzed by an API 4000 triple quadrupole mass spectrometer. In the method validation, the linear calibration curve in the range of 2.0 to 300.0 ng/mL had correlation coefficients (r) above 0.996. The recovery was 57.6–66.8% for GI and the internal standard. The lower limit of quantification (LLOQ) was 2 ng/mL. The intra- and inter-batch precisions were below 6.31% and 8.61%, respectively, and the accuracies were all within acceptance. GI was stable in a bench-top autosampler through long-term storage and freeze/thaw cycles. Therefore, this method is specific, sensitive and reliable for quantitative analysis of α-conotoxin GI in human plasma.

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