(Fabaceae) Extracts on Estrogen Target Organs of Female Wistar Rat
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J Pharmacol Sci 123, 120 – 131 (2013) Journal of Pharmacological Sciences © The Japanese Pharmacological Society Full Paper Effects of Millettia macrophylla (Fabaceae) Extracts on Estrogen Target Organs of Female Wistar Rat Stéphane Zingue1,2,3, Dieudonné Njamen1,*, Job Tchoumtchoua1,3, Maria Halabalaki3, Evan Simpson4, Colin Clyne4, and Chantal Beatrice Magne Nde4 1Laboratory of Animal Physiology, Department of Animal Biology and Physiology, Faculty of Science, University of Yaounde I, P.O. Box 812 Yaounde, Cameroon 2Laboratory of Physiology, Department of Life and Earth Sciences, Higher Teachers’ Training College, University of Maroua, P.O. Box 55 Maroua, Cameroon 3Division of Pharmacognosy and Natural Products Chemistry, Faculty of Pharmacy, University of Athens, Panepistimioupoli Zografou 15771, Athens, Greece 4Prince Henry’s Institute of Medical Research, Monash Medical Centre, 246 Clayton Road, Level 4, VIC 3168, Melbourne, Australia Received May 30, 2013; Accepted July 14, 2013 Abstract. The present study aims to determine the estrogenicity of Millettia macrophylla, a Cameroonian medicinal plant, in ovariectomized rats and to investigate the underlying mecha- nisms, in order to justify scientifically its traditional use. To accomplish this objective, we used dichloromethane (DCM) and methanol (MeOH) extracts of the stem bark of M. macrophylla. In the cell culture based assay, the MeOH extract significantly transactivated estrogen receptor a (ERa) and estrogen receptor b (ERb); in addition, the estrogen-like effects of both, DCM and MeOH extracts, could be inhibited in vitro by the pure ER antagonist ICI 182,780, indicating that these effects were primarily mediated through ERs. In animal experiments, both DCM and MeOH extracts significantly increased the uterine and vaginal epithelial heights in the 3-day treatment assay, while only the MeOH extract exhibited such effects in the sub-chronic treatment regimen. Furthermore, the MeOH extract significantly decreased fasting serum triglycerides, total cholesterol levels and artherogenic risk in the sub-chronic treatment. These results indicate that M. macrophylla extracts have estrogen-like effects supporting their traditional use in Cameroon to alleviate some menopausal problems (See graphical abstract in Supplementary Fig. 1, available in the online version only). [Supplementary Figures: available only at http://dx.doi.org/10.1254/jphs.13094FP] Keywords: Millettia macrophylla, female reproductive health, estrogen-like effect, estrogen target organ, phytoestrogen Introduction women, low serum levels of 17b-estradiol often result in symptoms such as hot flashes, genital-urinary atrophy, Estrogens are steroid hormones integral to the human or degenerative processes such as osteoporosis (2). Over endocrine system produced principally by the ovaries decades, hormone replacement therapy (HRT) has been from puberty. They regulate the growth and development successfully used to alleviate these problems (3), of reproductive organs as well as the homeostasis of a although large clinical trials have mentioned serious variety of tissues (1). In climacteric and postmenopausal side effects (4, 5). This has resulted in a search for HRT alternatives, and plant-derived substances, so-called *Corresponding author. [email protected] phytoestrogens, are being considered (6). In most devel- Published online in J-STAGE on September 27, 2013 oping countries, 80% of the population still resort to doi: 10.1254/jphs.13094FP traditional medicine for their primary health care (7). For 120 Effects of Millettia macrophylla 121 instance, the use of traditional herbal medicine is popular HPLC-DAD analysis of Millettia macrophylla (Fabaceae) in Central Africa particularly in Cameroon mainly The LC analyses were performed on a Thermo because of economic constraints and the strength of Finnigan HPLC system consisting of a vacuum degasser, traditional beliefs (8, 9) The limited scientific evidence a quaternary pump, an autosampler, and a DAD, Diode- about traditional medicine / complementary and alterna- Array Detector (Thermo Finnigan, San Jose, CA, USA). tive medicine’s (TM/CAM’s) safety and efficacy as A Discovery SUPELCO HS C-18 column (25 × 4.6 mm, well as other considerations have led the Cameroon 5-mm particle size) was used for separations while the Government in collaboration with the World Health system was controlled by ChromQuestTM 4.1 software. Organisation (WHO) to put in place a strategic platform The binary mobile phase consisting of 1% acetic acid (A) for the practice and development of TM (10). M. Macro- and methanol (B) was used. A gradient program was phylla also known as Millettia aboensis is one of such applied as follows: 2% – 98% B over 60 min, isocratic medicinal plants. It is popular in many Sub-Saharan 98% B for 5 min, 98% – 2% B over 1 min, and isocratic African countries including Cameroon, where different 2% B for 9 min before the next injection. The injection parts (stems, leaves, roots) are used as concoctions volume was set to 20 mL and the flow rate, to 1 mL/min. (generally prepared by mixing about 100 g of the plant in DAD conditions: 254, 280, and 365 nm and recording 1.5 liters of water for 15 min) or macerations (generally window from 200 to 700 nm. prepared by mixing about 1 kg of the plant in 5 liters of palm wine for 2 days) to treat various ailments including Cell culture, transfections, and luciferase assays respiratory difficulties, constipation, colds and headaches, In the first set of experiments, we tested the ability and jaundice (11, 12). M. Macrophylla is also used to of different extracts of M. macrophylla to activate ERa alleviate venereal diseases as well as some symptoms and ERb, in cell-based assays. Human Embryonic related to menopausal complaints like other species of Kidney 293T cell line (HEK293T) that contains the Millettia genus such as M. conraui, M. drastica, M. grif- SV40 Large T-antigen was purchased from ATCC foniana, M. zechiana, and M. barteri (11, 13 – 16). Since (The Global Bioresource Center, Sidney, Australia). there were no scientific data to support the above claims, HEK293T cells were transiently transfected with the we therefore aimed to evaluate the beneficial effects of estrogen a-receptor plasmid (200 ng HEK293T-ERa M. macrophylla extracts on female reproductive health, cells) or the estrogen b-receptor expression plasmid in particular menopausal problems, in order to justify (200 ng HEK293T-ERb cells), together with the double scientifically the claimed beneficial effects. To achieve estrogen response element (ERE) and a luciferase our goal, both non-polar (dichloromethane soluble) and reporter [250 ng (ERE)2-tk-Luc] plasmid kindly provided polar (methanol soluble) extracts of M. macrophylla by Dr. Simon Chu (Prince Henry’s Institute of Medical were produced and their ability to transactivate estrogen Research, Melbourne, Australia) and b-galactosidase receptor a (ERa) and estrogen receptor b (ERb) and to reporter plasmid using Lipofectamine Reagent (Invitro- mimic estrogenic activity in ovariectomized rats were gen, Mulgrave, Australia). They were then treated with evaluated. different concentrations of the MeOH or DCM extracts of M. macrophylla alone or in combination with E2 or Materials and Methods ICI 182,780 for 24 h. Cells treated with E2 alone served as positive control. Reporter gene assays in HEK293T- Plant material ERa cells and HEK293T-ERb cells were performed Stem barks of M. macrophylla were collected in using a commercial kit (Promega, Melbourne, Australia) Kumba (South-West region of Cameroon) in January according to the manufacurer’s instructions. Luciferase 2010 and identified at the Cameroon National Herbarium activity was measured and normalized against b-galacto- (HNC) (Voucher specimen No. 49654/HNC). The dried sidase activity determined by using the 2-nitrophenyl b- and ground stem bark of M. macrophylla (1000 g) was D-galactopyranoside (ONPG) method (Sigma-Aldrich, macerated in 5 liters of dichloromethane (DCM) for 72 h Sydney, Australia). Each experiment was performed in at room temperature, and 18 g of extract was obtained at least duplicate and repeated three times. after evaporation of the solvent using a rotary evaporator in vacuum. The residue powder was thereafter steeped Animals in 5 liters of methanol for 72 h (MeOH), filtered, Juvenile female Wistar rats aged 10 to 12 weeks and concentrated to give 47 g of methanol extract of (250 g) were obtained from the breeding facility of the M. macrophylla. laboratory of Animal Physiology, University of Yaounde I (Yaounde, Cameroon). They had free access to a stan- dard soy-free rat chow and water ad libitum. Animal 122 S Zingue et al housing and experiments were carried out following Histological analysis the guidelines of the Institutional Ethic Committee of the Using the complete Zeiss equipment consisting of a Cameroon Ministry of Scientific Research and Innova- microscope (Axioskop 40) connected to a computer tion, which has adopted the guidelines established by the where the image was transferred and analyzed with European Union on Animal Care (CEE Council 86/609). the MRGrab1.0 and Axio Vision 3.1 software, all pro- vided by Zeiss (Hallbermoos, Germany) (15, 17, 18); Study design the histomorphology of the mammary glands, as well as Estradiol valerate (Progynova®, 2 mg; Delpharm, the uterine and vaginal epithelial heights, were assessed Lille, France) and the extracts of M. macrophylla were from 5-mm sections of paraffin-embedded tissues follow-