Efficacy of Morus nigra L. on reproduction in female Wistar rats
Graphical abstract
The highest doses (350 and 700 mg/kg) of the extract did not produce morphological changes nor did interfere with the vaginal cyclicity of the rats. In fact, the occurrence of ovulation was detected in the treated group 4, as indicated by the presence of two oocytes in the uterine horn.
Highlights
► The estrogenic activity of Morus nigra leaves was tested in adult females. ► The reproductive toxicity and the embryonic development were assessed. ► The data obtained indicate that M. nigra did not exhibit any estrogenic activity.
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.
Section snippets
Materials and methods
The experimental protocol was approved by the Ethical Committee of the Centro de Biologia da Reprodução at the Federal University of Juiz de Fora (UFJF – Juiz de Fora, Minas Gerais State, Brazil) (Protocol No. 048/2009-CEEA), which follows the international principles in ethics for animal experimentation.
Phytochemistry
The phytochemical evaluation of MnHE M. nigra showed the presence of flavonoids, tannins, cumarins, and triterpenes/steroids. Flavonoids and triterpenes, are known to possess estrogen-like effects (Ibarreta et al., 2001, Jefferson, 2003).
Reproductive toxicology and embryonic development
During the experimental procedure, no deaths or any clinical signs of toxicity were observed. Food consumption (Fig. 2) and body weight (Fig. 3) gain were similar between control and treated animals. Death and clinical signs of toxicity as well as increase or
Discussion
Owing to the popular use of M. nigra leaves as a reliever of hot flashes and the symptoms of premenstrual tension (Miranda et al., 2010), it would be possible to suppose that substances capable of exerting some estrogenic or progesteronic effect would be present among the constituents of this plant’s leaves. The phytochemical analysis showed the presence of flavonoids, tannins, cumarins, polyphenols, triterpenes/steroids, of which two of these compounds, flavonoids and triterpenes, are known to
Conflict of Interest
The authors declare that there are no conflicts of interest.
Acknowledgments
The authors are grateful to Prof. Luis Cláudio Ribeiro (Statistics Department/UFJF) for doing the statistical analysis and “Rede Mineira de Bioterismo (172/08), “Rede Mineira de Toxicologia e Farmacologia de Produtos terapéuticos” (173/08), FAPEMIG, Brazil, for financial support and Roy Robinson for reviewing the english version of the manuscript.
References (45)
- et al.
Effect of resveratrol and betasitosterol in combination on reactive oxygen species and prostaglandin release by PC-3 cells
Prostaglandins Leukot. Essent. Fatty Acids
(2005) - et al.
Correlation between maternal toxicity and embryo/fetal effects
Reprod. Toxicol.
(1999) - et al.
The relationship of maternal and fetal toxicity in developmental toxicology bioassays with notes on the biological significance of the “no observed adverse effect level”
Reprod. Toxicol.
(2008) - et al.
Deglycosylation of flavonoid and isoflavonoid glycosides by human small intestine and liver β-glucosidase activity
FEBS Lett.
(1998) - et al.
Cytotoxic prenylated flavonoids from Morus alba
Fitoterapia.
(2010) - et al.
Chemical composition of white (Morus alba), red (Morus rubra) and black (Morus nigra) mulberry fruits
Food Chem.
(2007) - et al.
Quercetin glucosides interact with the intestinal glucose transport pathway
Free Radic. Biol. Med.
(1998) The biochemistry and medical significance of the flavonoids
Pharmacol. Ther. Oxford
(2002)- et al.
Pregnancy interceptive activity of Melia azedarach Linn In adult female Sprague–Dawley rats
Contraception
(2003) - et al.
Determination of total phenolic and flavonoid contents in selected fruits and vegetables, as well as their stimulatory effects on mouse splenocyte proliferation
Food Chem.
(2007)