A gamma-glutamyl peptide isolated from onion (Allium cepa L.) by bioassay-guided fractionation inhibits resorption activity of osteoclasts.

One gram of onion added to the food of rats inhibits significantly (p < 0.05) bone resorption as assessed by the urinary excretion of tritium released from bone of 9-week-old rats prelabeled with tritiated tetracycline from weeks 1 to 6. To isolate and identify the bone resorption inhibiting compound from onion, onion powder was extracted and the extract fractionated by column chromatography and medium-pressure liquid chromatography. A single active peak was finally obtained by semipreparative high-performance liquid chromatography. The biological activity of the various fractions was tested in vitro on the activity of osteoclasts to form resorption pits on a mineralized substrate. Medium, containing the various fractions or the pure compound, was added to osteoclasts of new-born rats settled on ivory slices. After 24 h of incubation, the tartrate-resistant acid phosphatase positive multinucleated cells, that is, osteoclasts, were counted. Subsequently, the number of resorption pits was determined. Activity was calculated as the ratio of resorption pits/osteoclasts and was compared to a negative control, that is, medium containing 10% fetal bovine serum only and to calcitonin (10(-12) M) as a positive control. Finally, a single peak inhibited osteoclast activity significantly (p < 0.05). The structure of this compound was elucidated with high-performance liquid chromatography-electrospray ionization-mass spectrometry, time-of-flight electrospray ionization mass spectrometry, and nuclear magnetic resonance spectroscopy. The single peak was identified as gamma-L-glutamyl-trans-S-1-propenyl-L-cysteine sulfoxide (GPCS). It has a molecular mass of 306 Da and inhibits dose-dependently the resorption activity of osteoclasts, the minimal effective dose being approximately 2 mM. As no other peak displayed inhibitory activity, it likely is responsible for the effect of onion on bone resorption.

HMBC-RELAY: a combined technique for the differentiation of simultaneously detected 2J(C,H) and (n)J(C,H) connectivities.

We present a new pulse sequence that detects simultaneously (n)J(C,H) and 2J(C,H) connectivities. The corresponding coherences are created along independent pathways and therefore can be separated into two different subspectra. One spectrum is to show all (n)J(C,H) connectivities and the other is to show exclusively 2J(C,H) connectivities. In contrast to the previously published 2J/(n)J experiment, this sequence detects the 2J(C,H) connectivities via a C,H,H-RELAY pathway leading to an intensification of the 2J(C,H) signals. Strictly, the 2J(C,H) spectrum does not show 2J(C,H) but 3J(H,H) coupling interactions within 13CH(k)-12CH(l) fragments. Therefore, 2J(C,H) signals can appear even if the corresponding 2J(C,H) coupling constant is zero.