The phytoestrogen ferutinin improves sexual behavior in ovariectomized rats

Abstract

The present study was designed to examine the effect of ferutinin chronic administration on sexual behavior of ovariectomized non-estrogen-primed rats. Starting from 3 weeks after ovariectomy, female rats were orally treated with ferutinin at the doses of 0.2 and 0.5 mg/kg, daily for 4 weeks. Ferutinin's effect was compared with that of estradiol benzoate, subcutaneously injected at the dose of 1.5 μg/rat twice a week. Animals were tested for sexual motivation, receptivity and proceptivity after 1, 2 and 3 weeks of treatment and for paced mating behavior after 4 weeks of treatment. Before each experimental test, they received progesterone injection (500 μg/rat).

Both dosages of ferutinin significantly increased the receptive behavior in a time-dependent manner, as well as estradiol benzoate did. Also proceptive behaviors increased in ferutinin-treated animals in comparison with control ones. During the partner preference test ferutinin was able to induce a significant preference for a sexually active male over a sexually receptive female. Moreover, ferutinin restored a normal paced mating behavior, which had been suppressed by ovariectomy. These results show that ferutinin exerts an estrogenic activity in ovariectomized non-estrogen-primed female rats.

Introduction

Ferutinin (jaeschkeanadiol p-hydroxybenzoate) is a daucane sesquiterpene ester identified in the plants belonging to the genus Ferula (family Umbelliferae), widely distributed throughout the Mediterranean area and Central Asia. It was identified as the main component of Ferula hermonis roots, while other daucane sesquiterpenes, such as teferdin (ferutinol benzoate) and teferin (ferutinol vanillate) were found in lower concentrations (Abourashed et al., 2001; Galal, 2000). Ferula hermonis Boiss, a small shrub with thin leaves and pale yellow flowers, is native to Lebanon but it also grows in other Middle-East regions, where it is traditionally used to treat erectile dysfunction in men and menopausal disturbances in women. Ferutinin was also isolated from the rhizomes of Ferula jaeschkeana Vatke, native to East Asia (Singh et al., 1988) as well from the roots of Ferula communis L. (Appendino et al., 2001) and Ferula arrigonii Bocchieri (Appendino et al., 1997), both growing in Sardinia island (Italy). The chemical structure of ferutinin (Fig. 1) is characterized by the presence of a daucane core which was reported to be of primary importance for the biological activity, whereas the parent polyol jaeschkeanadiol was inactive (Appendino et al., 2002). A structure–activity study performed by Appendino et al. (2004) showed the critical role of the double bond in the daucane core for the ferutinin activity. Due to the presence of an ester bond between the daucane core and the p-hydroxybenzoyl part, ferutinin could be substrate for esterases in plasma and other tissues. The stability of the substance in human plasma is strictly dependent on its binding to serum albumin, which seems to protect ferutinin from the chemical hydrolysis (Greige-Gerges et al., 2008).

From the biological point of view, earliest experiments performed in vivo showed that ferutinin was able to prevent pregnancy in adult female rats when administered orally on days 1–7 post-coitum and to increase uterine weight in ovariectomized immature female rats (Singh et al., 1988). The affinity of ferutinin for estrogen receptors was tested in different in vitro experiments (Appendino et al., 2002; Ikeda et al., 2002; Singh et al., 1988). In a yeast screen containing the human estrogen receptor alpha, ferutinin exhibited an estrogenic activity approximately 200-fold lower than that of estradiol (Appendino et al., 2002). In competitive binding assays ferutinin displayed a binding affinity for human estrogen receptors α (ERα) and β (ERβ) approximately 10% of that of estradiol on both receptors (Appendino et al., 2002). Differently from the most of phytoestrogens having a higher binding affinity for ERβ than ERα, ferutinin was found to be more active through ERα than ERβ, displaying IC₅₀ values of 33.1 nM for ERα and 180.5 nM for ERβ (Ikeda et al., 2002). Using a luciferase reporter assay, ferutinin was found to act as agonist for ERα and as agonist/antagonist for ERβ (Ikeda et al., 2002): therefore the compound could be considered a selective estrogen receptor modulator (SERM) (Appendino et al., 2002; Ikeda et al., 2002).

Our previous studies investigated the influence of ferutinin and other daucane sesquiterpenes obtained from Ferula hermonis on male and female sexual behavior in rats. In males, ferutinin showed opposite effects on the copulatory activity, depending on the administration schedule. After acute administration ferutinin dosed at 2.5 mg/kg was able to improve sexual motivation and performance. On the contrary, when subchronically administered (0.25 mg/kg, daily for 10 consecutive days), it negatively affected the copulatory behavior (Zanoli et al., 2005a). These effects might be ascribed to testosterone serum levels which increased after the acute administration whereas decreased after the subchronic one (Zanoli et al., 2005a).

Recently, we demonstrated the crucial role exerted by the phytoestrogen ferutinin in the impairment of sexual function induced by Ferula hermonis in ovariectomized hormone-primed female rats (Zanoli et al., 2005b; Zavatti et al., 2006). The acute administration of ferutinin, dosed at 2.5 mg/kg, exerted an inhibitory effect on sexual receptivity, without affecting sexual motivation. The other tested components of Ferula extract (teferdin and teferin) did not show a significant activity on female sexual behavior (Zavatti et al., 2006). The negative effect of ferutinin on lordosis response was suggested to be due to the higher affinity of this compound for ERα rather than ERβ. On the other hand, the other compounds showed very low or no affinity for estrogen receptors in a protein binding assay (Singh et al., 1988). It must be underlined that ERα, but not ERβ, is primarily involved in the expression of sexual receptivity (Ogawa et al., 1998; Rissman et al., 1997; Walf and Frye 2005; Walf et al., 2008). Evidence obtained in vivo suggest that SERMs with greater activity at ERα might facilitate the lordosis response when administered in ovariectomized rats (Mazzucco et al., 2008; Walf and Frye, 2005). The above considerations prompted us to investigate the effect of ferutinin on female sexual behavior in ovariectomized non-estrogen-primed rats. The ovariectomized rat is widely used as a reliable animal model resembling the decline in estrogen levels in post-menopausal women. Therefore, this model allows the evaluation of phytoestrogen efficacy in estrogen-deficient diseases, even if higher doses are often needed in rats to produce effects comparable to those seen in humans (Whitten and Patisaul, 2001)

Sexual behavior of female rats is characterized by both appetitive and consummatory components, named proceptivity and receptivity (Beach, 1976). Receptive behavior consists in a reflexive posture, called lordosis, which represents the female readiness to allow copulation (Beach, 1976). In presence of a male partner, an estrous female exhibits a variety of soliciting behaviors, collectively termed proceptive behaviors, including hops, darts and ear-wigglings (Beach, 1976). These behaviors were suggested to represent the female's effort to arouse sexual interest of the male and to stimulate him to initiate mounting (Beach, 1976; Madlafousek and Hlinak, 1983). In addition, estrous female prefers to approach and remain in proximity to a sexually active male (sexual stimulus) rather than spending time with a female (social stimulus) during the partner preference test (Meyerson and Lindström, 1973; Vega Matuszczyk and Larsson, 1991). It was suggested that the preference for a sexually appropriate partner is an index of the female sexual motivation and it is dependent on distal clues rather than on coital stimulation (Clark et al., 2004; Erskine, 1989). It is well known that male rats are more active in seeking sexual contacts than females, therefore they usually control sexual interaction. Nevertheless, when given the opportunity in a suitable environment, an estrous female will approach and withdraw from the male, controlling hence the number and the timing of sexual contacts. This pattern is known as paced mating behavior (Zipse et al., 2000).

In the present study, we tested the effects of the chronic administration of ferutinin in ovariectomized non-estrogen-primed rats on the display of: (1) receptive and proceptive behaviors; (2) the preference for a sexual or a social stimulus; (3) the paced mating behavior.