γ-glutamyl-propenyl-cysteine sulfoxide (GPCS), a gamma-glutamyl peptide from onion (Allium cepa L.). phytochemistry and pharmacology

摘要

Gamma-glutamyl peptides are widely found in plants. γ-Glutamyl-propenyl-cysteine sulfoxide (GPCS), a glutamyl peptide present in onion (Allium cepa L.), was identified by a bioassayguided fractionation in vitro to be the compound responsible for the inhibition of bone resorption by onion in rats. Therefore, GPCS was selected for an extended investigation in our studies. The aim of the first part of this work was to examine the presence of GPCS in various plants, which were previously shown to inhibit bone resorption in rats. A fast and reliable method to determine GPCS in plant material was developed and validated. GPCS was found in onion at various concentrations, but no GPCS was detected in the material from other plants. In the second part of the present work, the aim was to provide GPCS for further in vitro and in vivo studies. Small quantities (milligram-amounts) were obtained by isolation of GPCS from onion. As large-scale isolation (gram amounts) is not feasible under non-industrial conditions, and GPCS is not commercially available, an approach for synthesis was performed. The intermediate products S-(1-propenyl)-L-cysteine sulfoxide and Boc-Lglutamyl-α-t-butyl-N-oxo-succinimide ester were synthesised, but unfortunately, coupling of these two compounds failed and the final product could not be obtained. The aim of the third part of this work was to examine pharmacodynamic and pharmacokinetic parameters of GPCS. In vitro experiments on the effects of GPCS on the activity, activation and recruitment of osteoclasts (OCs) were performed. The effect of GPCS on the activity of OCs was tested in the pit assay. GPCS, at 8 mM, significantly reduced the number of pits per OCs. The effect of GPCS on the activation of OCs was examined by determination of the OCs with actin ring(s). After incubation of the cells with 2 and 8 mM of GPCS, the percentage of OCs with actin rings did not change. The formation of OCs was inhibited at GPCS concentrations of 1 mM and higher in our experiments with CSF-1/RANKL primed bone marrow cells. A structure-function relationship was assessed by testing GPCS structurally related compounds in the same assay, namely γ-glutamyl-cysteine-ethylester, glycylcysteine, allyl-cysteine, cysteine, glutamyl-glycine and glycyl-valine. All test substances which contained a cysteine moiety inhibited osteoclastogenesis while the others did not show an effect. The pharmacokinetic properties of GPCS were studied in vitro and in vivo. Within 24 hours no change in the concentration of GPCS was observed when GPCS was incubated with simulated gastric acid. 24-hour incubation of GPCS with simulated intestinal fluid caused a 90 % decrease of the GPCS concentration, while GPCS concentration in onion incubated with simulated intestinal fluid decreased only by 30 %. In a pilot study with rats on the kinetics of GPCS in vivo, the bioavailability of orally administered GPCS was determined to be about 1 %. Finally, onion without GPCS and onion containing GPCS, as well as GPCS-structure related compounds were tested in a rat model in vivo to analyse their effect on bone resorption. Allyl-cysteine and γ-glutamyl-cysteine-ethylester did not inhibit bone resorption while 3 g of onion without GPCS and 3 g of onion containing GPCS significantly inhibited bone resorption. The in vitro results indicate that the effects of GPCS might be mediated by the cysteine moiety of the molecule. An in vivo oral application of pure GPCS in an aqueous solution is not suitable due to the low bioavailability of GPCS, however, application of GPCS embedded in onion might prevent cleavage of the compound and therefore increase the bioavailability. Because both onion without GPCS and onion containing GPCS inhibited bone resorption in vivo, we can postulate that the inhibition of bone resorption by onion is not exclusively mediated by GPCS. Future experiments using natural GPCS-free onions spiked with synthetic GPCS should clarify, to which extent GPCS contributes to the inhibition of bone resorption.ud