Onion (Allium cepa L.) peel extract (OPE) regulates human sperm motility via protein kinase C-mediated activation of the human voltage-gated proton channel.

Onion (Allium cepa L.) and quercetin protect against oxidative damage and have positive effects on multiple functional parameters of spermatozoa, including viability and motility. However, the associated underlying mechanisms of action have not yet been identified. The aim of this study was to investigate the effect of onion peel extract (OPE) on voltage-gated proton (Hv1) channels, which play a critical role in rapid proton extrusion. This process underlies a wide range of physiological processes, particularly male fertility. The whole-cell patch-clamp technique was used to record the changes in Hv1 currents in HEK293 cells transiently transfected with human Hv1 (HVCN1). The effects of OPE on human sperm motility were also analyzed. OPE significantly activated the outward-rectifying proton currents in a concentration-dependent manner, with an EC50 value of 30 µg/mL. This effect was largely reversible upon washout. Moreover, OPE induced an increase in the proton current amplitude and decreased the time constant of activation at 0 mV from 4.9 ± 1.7 to 0.6 ± 0.1 sec (n = 6). In the presence of OPE, the half-activation voltage (V1/2 ) shifted in the negative direction, from 20.1 ± 5.8 to 5.2 ± 8.7 mV (n = 6), but the slope was not significantly altered. The OPE-induced current was profoundly inhibited by 10 µm Zn2+ , the most potent Hv1 channel inhibitor, and was also inhibited by treatment with GF109203X, a specific protein kinase C (PKC) inhibitor. Furthermore, sperm motility was significantly increased in the OPE-treated groups. OPE exhibits protective effects on sperm motility, at least partially via regulation of the proton channel. Moreover, similar effects were exerted by quercetin, the major flavonoid in OPE. These results suggest OPE, which is rich in the potent Hv1 channel activator quercetin, as a possible new candidate treatment for human infertility.

Effects of eupatilin on the contractility of corpus cavernosal smooth muscle through nitric oxide-independent pathways.

Eupatilin (5,7-dihydroxy-3,4,6-trimethoxyflavone) is one of the main compounds present in Artemisia species. Eupatilin has both antioxidative and anti-inflammatory properties and a relaxation effect on vascular contraction regardless of endothelial function. We evaluated the relaxant effects of eupatilin on the corpus cavernosum (CC) of rabbits and the underlying mechanisms of its activity in human corpus cavernosum smooth muscle (CCSM) cells. Isolated rabbit CC strips were mounted in an organ bath system. A conventional whole-cell patch clamp technique was used to measure activation of calcium-sensitive K+ -channel currents in human CCSM cells. The relaxation effect of eupatilin was evaluated by cumulative addition (10-5  m ~ 3 × 10-4  m) to CC strips precontracted with 10-5  m phenylephrine. Western blotting analysis was performed to measure myosin phosphatase targeting subunit 1 (MYPT1) and protein kinase C-potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17-kDa (CPI-17) expression and to evaluate the effect of eupatilin on the RhoA/Rho-kinase pathway. Eupatilin effectively relaxed the phenylephrine-induced tone in the rabbit CC strips in a concentration-dependent manner with an estimated EC50 value of 1.2 ± 1.6 × 10-4  m (n = 8, p < 0.05). Iberiotoxin and tetraethylammonium significantly reduced the relaxation effect (n = 8, p < 0.001 and p = 0.003, respectively). Removal of the endothelium or the presence of L-NAME or indomethacin did not affect the relaxation effect of eupatilin. In CCSM cells, the extracellular application of eupatilin 10-4  m significantly increased the outward currents, and the eupatilin-stimulated currents were significantly attenuated by treatment with 10-7  m iberiotoxin (n = 13, p < 0.05). Eupatilin reduced the phosphorylation level of MYPT1 at Thr853 of MLCP and CPI-17 at Thr38. Eupatilin-induced relaxation of the CCSM cells via NO-independent pathways. The relaxation effects of eupatilin on CCSM cells were partially due to activation of BKCa channels and inhibition of RhoA/Rho-kinase.