Bioassay-guided fractionation to isolate compounds of onion (Allium cepa L.) affecting bone resorption

In this work a hydrophilic ethanolic extract of onion (Allium cepa L.) devoid of flavonoids was found to inhibit significantly bone resorption in vitro and in vivo, whereas the flavonoids, claimed to posses phytoestrogen-like properties, were devoid of activity in vivo and toxic in vitro. Thus, in order to isolate the bone resorption inhibitory constituent(s) of onion, the hydrophilic ethanolic onion extract was subjected to an in vitro bioassay-guided fractionation using (semi)-preparative chromatographic techniques. Biological activity, i.e. bone resorption inhibitory activity, was determined in vitro using the osteoclast resorption pit assay: Medium, containing the fraction under investigation, was added to osteoclasts settled on ivory slices. After a 24-hour incubation period osteoclasts were counted and the number of resorption pits was determined. Activity was calculated as the ratio of resorption pits per osteoclasts and was compared to a negative control, i.e. medium only, and to calcitonin (10-12 M) as positive control. In this way, from the starting fraction which inhibited significantly (p < 0.05) the osteoclast activity at a dose of 30.0 mg / ml, a compound inhibiting significantly the osteoclast activity (0.53 mg / ml; p < 0.05) could be isolated. Structural analysis performed as well by nuclear magnetic resonance (NMR) as by electrospray-ionization mass-spectroscopy identified unambiguously the compound as γ-Lglutamyl-trans-S-1-propenyl-L-cysteine sulphoxide (γ-GPeCSO). Consequently, an adapted, scaled-up isolation by means of ion exchange - column chromatography was performed in order to isolate γ-GPeCSO in large amounts. Thus, sufficient amounts of γ-GPeCSO could be isolated to develop a high performance liquid chromatography method to quantify γ-GPeCSO in the fractions of the bioassay-guided fractionation. NMR experiments performed for structural confirmation of the isolated compound, revealed the presence of% acetic acid (weight/weight) in the sample which originated from solvents used in the ion exchange procedures. However, the acetic acid could be removed by solid phase extraction chromatography. Quantification of γ-GPeCSO in the fractions of the bioassay guided fractionation showed a significant (p < 0.05) correlation between the amounts of γ-GPeCSO therein and the osteoclast activity inhibition, thus indicating that γ-GPeCSO inhibited osteoclast activity in vitro.