The synergy effect of daidzein and genistein isolated from Butea superba Roxb. on the reproductive system of male mice

Abstract Butea superba Roxb. (BS) has been used in Thai men as an aphrodisiac, and prevent erectile dysfunction. Nevertheless, the active ingredients, dosages, have not been cleared. Hence, this study was to investigate the effect of compounds from the BS on the reproductive parameters of male mice. The results revealed that BS was extracted to afford biochanin A and genistein, which were first reported on BS, and daidzein. The mice were treated by daidzein and genistein alone and in combination. The results showed that the sperm number and motility, cholesterol and testosterone level of all isoflavones-treated groups were significantly higher than controls (p < 0.01). Obviously, daidzein plus genistein exhibited a synergistic effect, which is also the first report, and resulted in significantly displayed higher levels of these parameters compared to others. So, the synergistic activity of these isoflavones may be useful in improving libido, erectile capacity and assist infertility of poor spermatozoa in men.


Introduction
The Butea superba Roxb. (BS) herb has been employed as traditional herbal medicines, for rejuvenation, improve sexual performance, or prevent erectile dysfunction function, in Southeast Asian Nations since ancient times. The tubers of BS extract that contained carbohydrates showed the highest immuno-stimulating activity in vitro (Burana-Osot et al. 2010). The results of long-term treatment in rats and mice with tuberous roots of BS extract exhibited that significantly increased the sperm concentration and delayed the decreased motility with time (Tocharus et al. 2005), although, the effect of the tuberous roots of the BS extract on orchidectomized mature albino rats showed no androgenic and aphrodisiac activities (Smitasiri et al. 1992 ). Alternatively, low serum total testosterone may be a co-morbid feature and could also exacerbate risk factor for dementia of the Alzheimer's Type in men (Hogervorst et al. 2004). Whereas, Wistar-Unilever male rats received the soy isoflavone mixture that contain 45% genistein, 23% daidzein and 4% glycitein exhibited no significant effects on sperm count sperm production, or sperm morphology in any group (Faqi et al. 2004). Although, many of the experiments were carried out on the tuberous roots of BS crude extract, however, the active ingredients, dosages, duration of these compounds have not been cleared . Moreover, up to date, the in vivo results are still ambiguous whether the effect of the tuberous roots of BS extract increases cholesterol, testosterone on male mice or not.

Sperm count and morphology
The results of sperm count demonstrated the synergistic activity of daidzein plus genistein at FIC index 0.4 showing significantly higher and the highest sperm number than others (p < 0.01) ( Figure S1). The morphology of these sperms was revealed to be normal ( Figure  S3). Obviuosly, these findings provide evidence that this combination clearly increases sperm number.

Sperm motility
The results exhibited that there were significant increases in sperm motility of all post-treated groups compared to pre-treatment except for control (p < 0.05 and p < 0.01). Apart from this, the highest motility level was found in the combination of daidzein and genistein-treated group compared to others (p < 0.01) as a result of synergy effect of these compounds with FIC index 0.4 ( Figure S2). Clearly, these results imply that the combination undoubtedly rises sperm motility.

Histology of seminiferous tubules
It can be seen from Figure S3 that the spermatogenesis of all treated groups had more maturation of spermatids than the control .

Testosterone and cholesterol level
The results of these tested agents on testosterone and cholesterol exhibited that there were significant increases in testosterone and cholesterol level of all post-treated groups compared to pre-treatment except for the control (p < 0.01). Also, the synergistic activity of daidzein plus genistein observed with FIC index at 0.4 resulted in a significant great increase in testosterone and cholesterol level compared to other groups (p < 0.01) (Figures S5 and S6). Our findings provide the first evidence that this combination clearly enhances testosterone and cholesterol level.

Selected reproductive organs and body weight
The relative testis and seminal vesicle weight of genistein alone and combined with daidzeintreated groups were significantly heavier than others (p < 0.01) (Table S2).

Conclusion
The present work demonstrated that the tuberous roots of BS were isolated to afford biochanin A, genistein and daidzein. Interestingly, biochanin A and genistein were first reported on BS.
Our results found that using daidzein and genistein alone or in combination caused a significantly higher sperm number and motility, testosterone and cholesterol level than the controls (p < 0.01). Also, these parameters of the combination-treated group were significantly highest compared to other groups due to the synergistic activity. These findings provide evidence that cholesterol increased in these treated groups. As a consequence, testosterone also increased. For this reason, the daidzein and genistein isolated from BS used either alone or combined provide evidence that these would be useful to increase testosterone level in Ad patients, improve libido and erectile function in hypogonadal males and assisted infertility technology to improve sperm quality of poor quality spermatozoa in men after safety level is investigated.