Antioxidant Effect of the Mill. Leaf Extract on Oxidative Stress Induced upon Human Spermatozoa.

This study was aimed at evaluating the effects of a 75% / ethanolic extract of leaves of Mill. (var. Bastarda Rossa, Mount Amiata, Tuscany, Italy) on ejaculated human sperm. Total polyphenols and total flavonoids contained in the extract were determined by a colorimetric assay and HPLC-DAD. The DPPH test and electrochemistry were utilized to study the antioxidant activity of the extract. Swim-up-selected sperm from 8 healthy men were treated with the leaf extract at different dilutions (1 : 100, 1 : 200, and 1 : 500), and sperm motility was assessed following the WHO guidelines. Swim-up-selected spermatozoa were incubated with 100 M HO to induce lipid peroxidation (LPO) and with HO and the leaf extract (1 : 100, 1 : 200, and 1 : 500) to test the antioxidant activity of the extract. The levels of LPO were determined by measuring malondialdehyde (MDA) concentrations. The treated samples were also analyzed by transmission electron microscopy (TEM) for ultrastructural evaluation. The chemical analysis showed that one-third of the polyphenols in the extract were made up of flavonoids, with hyperoside present in high concentration. A good antioxidant activity was demonstrated by both the DPPH test and electrochemical analysis. The leaf extract did not decrease sperm motility at all tested dilutions. Treatment with HO alone caused a significant increment in MDA levels ( = 0.006993), while the treatment with HO plus extract diluted to 1 : 100 and 1 : 200 significantly reduced MDA levels ( = 0.01476 and = 0.01571, respectively), with respect to HO alone. TEM analysis confirmed the protective effect of the extract on damage induced by LPO, in particular that occurring at the plasma membrane level. The leaf extract could be used in human and farm animal protocols for gamete handling, such as techniques of assisted reproduction and cryopreservation of semen, all conditions in which oxidative stress is exacerbated.