Efficacy of Morus nigra L. on reproduction in female Wistar rats

Abstract

Morus nigra L. is a plant employed as a substitute for the conventional hormonal replacement therapy. This work analyzes the estrogenic effect of M. nigra on the reproductive system and embryonic development of Wistar rats. Female rats were orally treated with M. nigra hydroalcoholic extract (MnHE) at the dose levels of 25, 50, 75, 350 and 700 mg/kg of body weight over 15 days, and continued through mating until the 14th day of gestation. Vaginal smears were performed daily and the body weight of the females was recorded at 5 days intervals. On day 15 of gestation, the females were killed and their kidneys, liver, spleen and ovaries were removed and weighed. The number of implants, resorptions, and live and dead fetuses were evaluated. Histological sections of ovaries, measurement of the height of the uterine epithelium and vaginal smears were performed to assess the estrogenic activity. The results showed that the administration of MnHE did not significantly alter the analyzed variables. Therefore, considering the experimental model used in this study, the data obtained indicate that M. nigra did not exhibit any estrogenic activity nor did exert a toxic effect on the female reproductive system and on the embryonic development of rats.

Introduction

Morus nigra L. (Moraceae), known as mulberry (also as “amoreira”, “amora preta” or “negra”) (Ercisli and Orhan, 2007, Wasano et al., 2009), is a plant found throughout Brazil and is well known by the population for its medicinal properties. The fruits, leaves, barks and roots are used as laxative, sedative, expectorant, refresher, emollient, calmative, diuretic, hypoglycaemic agent, antiseptic, anti-inflammatory, antioxidant, and in the treatment of eczema, and oral inflammation (Oryan et al., 2003, Naderi et al., 2004, Ercisli and Orhan, 2007, Padilha et al., 2010). In China, the leaves of Morus sp. are used as an antioxidant, antimicrobial agent and anti-inflammatory (Song et al., 2009), however the leaves of Morus alba L. were reported to have cytotoxic activity in neoplastic cells (Dat et al., 2010).

The leaves of M. nigra are commonly used by women in menopause as a substitute for the conventional hormonal replacement therapy, with a similar effect to that obtained after estrogen use (Silva et al., 2003, Miranda et al., 2010). Moreover, the leaves are also used by younger women as a reliever of the symptoms of the premenstrual tension. The use of M. nigra usually involves the preparation of a “tea” (i.e. through decoction or infusion of the leaves) and is reported to ameliorate the symptoms of menopause (Miranda et al., 2010), particularly hot flashes (Castro, 2010), which are related to the sudden vasodilatation that causes the feeling of intense heat and redness of the skin, namely the face.

However, the experimental studies are controversial in regard to the tests employed to verify the estrogenic effect on oophorectomized and pubescent animal models (Vanoni, 2006, Bolzan, 2008, Castro, 2010). For instance, Silva et al. (2003) assessed the estrogenic activity of an infusion of Morus sp. leaves and did not find any estrogenic effect on oophorectomized rats.

Previous studies have shown the presence of various substances in different species of the genus Morus sp., such as fatty acids, vitamin C (originated from the plants primary metabolism), phenylflavonoid (morusine) (Petlevski et al., 2001); flavonoids (Naderi et al., 2004, Lin and Tang, 2007, Ercisli and Orhan, 2007, Pawlowska et al., 2008, Song et al., 2009), phenolic compounds (Ercisli and Orhan, 2007, Song et al., 2009, Kim et al., 2010), organic acids (Koyuncu, 2004), anthocyanins, karotenoids (Dugo et al., 2001, Hassimotto et al., 2007), alkaloids (Song et al., 2009), originating from the secondary metabolism of the plants. M. nigra fruit in particular has high concentrations of phenols and flavonoids (Ercisli and Orhan, 2007) in addition to four types of anthocyanins (Pawlowska et al., 2008), whereas terpenoids and steroids, such as β-sitosterol, have been found in the leaves of this species (Padilha et al., 2010).

Several pharmacological activities have already been described in relation to some of the components mentioned above, such as the ability to change gene expression and anticarcinogenic activity due to the presence of phenolic substances and β-sitosterol (Tapiero et al., 2002, Nakamura et al., 2003, Awad et al., 2005). Some flavonoids have been reported to have estrogenic activity, such as quercetin, with broad pharmacological application, such as anti-inflammatory, anticarcinogenic. The anthocyanins inhibit Cox-1 and Cox-2, enzymes that are involved in the synthesis of prostaglandins (Seeram et al., 2001).

Moreover, some studies have reported the possible estrogenic potential of M. nigra leaves in pubescent (Vanoni, 2006) and oophorectomized Wistar rats (Silva et al., 2003, Vanoni, 2006), and in oophorectomized rats submitted to chronic treatment (Castro, 2010). The pharmacologic activities previously mentioned and the phytoestrogens may have an adverse effect on the reproductive process by: interfering with the ovarian hormone synthesis and the vaginal cyclicity; the embryo cell proliferation (anticarcinogenic activity); the mechanisms involved in the gene expression during the complete development and differentiation of the embryo or the prostaglandin F2-α or E production, which are related to many reproductive mechanisms, such as ovulation (PGF2-α) and blastocyst implantation (PGE).

Most of the information available in the literature about the estrogenic effect of M. nigra was concerned with young females. Therefore, considering the use of this plant by women and the popular belief of its effects on the female organism, the present study was aimed at investigating the estrogenic activity of M. nigra leaves in adult females, its reproductive toxicity and the embryonic development.