throughout the ages , plants have been the major source of medication for the treatment of diverse diseases and continue to be a major source of primary health care for today 's populations in most areas of the world . the medicinal value of plants has therefore been recognized by almost every society on this planet . in the developing countries in particular , for instance , the use of traditional herbal medicine is popular in central africa , particularly in cameroon , mainly because of economic constraints and the strength of traditional beliefs [ 4 , 5 ] . in our ongoing search for bioactive compounds from medicinal plants [ 610 ] , we have been studying the stem bark of millettia macrophylla , a cameroonian medicinal plant used in the treatment of menopausal symptoms . millettia macrophylla , also known as millettia aboensis , is used to alleviate sexual transmitted diseases , as well as some symptoms related to menopausal complaints , like other species of millettia genus such as m. conraui , m. drastica , m. griffoniana , m. zechiana , m. barteri , and m. dura [ 1117 ] . since there were no scientific data to support the above claims , we therefore evaluated the beneficial effects of m. macrophylla extracts on menopausal symptoms , in order to scientifically justify the claimed beneficial effects . using a model of rats exhibiting menopause - like symptoms in vivo and cells overexpressing estrogen receptors alpha or beta ( er or er ) in vitro , we previously showed that dcm and meoh extracts of m. macrophylla mimic estrogen - like effects on the genital tract and increase estrogen receptors transactivity , respectively . these results suggest that m. macrophylla may contain secondary metabolites that bind ers or alter coregulators of ers . however , the mechanism of actions that underpin the aforementioned effects is still unknown . therefore , we aimed to elucidate the underlying mechanisms by which m. macrophylla induces its estrogenic effects . to accomplish our goal , dcm and meoh soluble fractions of m. macrophylla were used with a pure and well - characterized antiestrogen ici 182,780 in a cotreatment regimen in vitro on cells transiently transfected with the estrogen  or  receptor expression plasmids and in vivo in ovariectomized rats . the pure antiestrogen ici 182,780 ( 7-[9-[4,4,5,5,5-pentafluoropentyl ) sulfinyl [ nonyl]-estra-1,3,5(10)-triene-3,17diol ) and 17-3-benzoate ( e2b ) ( estr-1 , 3,5 ( 10)-trien-3 , 16 , 17-triol ) were obtained from sigma - aldrich ( hamburg , germany ) . the hek293 t cells that contain the sv40 large t - antigen were purchased from atcc ( the global bioresource center , australia ) . the stem barks of millettia macrophylla were harvested at the former reserve of ediki ( kumba , southwest region of cameroon ) in january 2010 . our botanical sample was identified and authenticated by mr . victor nana , botanist at the national herbarium of cameroon ( hnc ) in yaounde ( voucher specimen number 49654/hnc ) . the well - dried and pulverized stem bark of m. macrophylla ( 2500  g ) was macerated in 10 liters of dichloromethane ( dcm ) for 72  h at room temperature , and 25  g of extract was obtained after filtration and evaporation of the solvent using a rotary evaporator in vacuum . the residue insoluble material was thereafter steeped in 10 liters of methanol for 72  h ( meoh ) , filtered through whatman paper n4 , and concentrated to give 53.4  g of methanol extract of m. macrophylla . for phytochemical analysis of these extracts , the lc analyses were performed on a thermo finnigan hplc system consisting of a vacuum degasser ( discovery supelco hs c-18 column ( 25  4.6  mm , 5  m particle size ) ) , a quaternary pump , an autosampler , and a dad ( diode array detector ) ( thermo finnigan , san jose , ca ) as previously reported . fifty female wistar rats ( rattus norvegicus ) aged 3 months ( 250  g ) were obtained from the breeding facility of the laboratory of animal physiology , university of yaounde 1 , cameroon . they had free access to a standard soy - free rat chow and water ab libitum . animal housing and experiments were carried out following the guidelines of the institutional ethics committee of the cameroon ministry of scientific research and technological innovation , which has adopted the guidelines established by the european union on animal care ( cee council 86/609 ) . in a previous study , we have found that m. macrophylla extracts are able to transactivate the estrogen receptors  and  , in human embryonic kidney 293 t cell line ( hek293 t ) . in the present study , we investigated whether this transactivation is lost in the presence of a pure antiestrogen ici 182,780 . to accomplish our goal , hek293 t were transiently transfected with the estrogen  receptor expression plasmid ( 200  ng  hek293t - er cells ) or the estrogen  receptor expression plasmid ( 200  ng  hek293t - er  cells ) , together with the double estrogen response element ( ere ) fused to a luciferase reporter ( 250  ng(ere)2-tk - luc ) plasmid and -galactosidase reporter plasmid using lipofectamine reagent . they were then treated with different concentrations of m. macrophylla extracts alone or in combination with ici 182,780 ( 500  nm ) for 24  h. cells treated with e2 ( 10  nm ) served as positive control . luciferase reporter gene assays were performed using a commercial kit ( promega , australia ) according to the manufacturer 's instructions . luciferase activity was measured and normalised against -galactosidase activity determined by using the 2-nitrophenyl -d - galactopyranoside ( onpg ) method ( sigma - aldrich , sydney , australia ) . this experiment aimed to elucidate whether the estrogenic activity observed in vivo with m. macrophylla extracts is mediated through ers . all substances ( e2b , ici 182,780 , and m. macrophylla extracts ) used in this experiment were dissolved in olive oil . fourteen days after endogenous hormonal decline , animals were randomly distributed into 10 groups of 5 rats each . the negative control received the vehicle ( olive oil ) and the positive control was treated with estradiol benzoate ( e2b ) at the dose of 0.75  g / kg  bw / d . three groups received either dcm extract at the dose of 300  mg / kg or meoh extract at the doses of 100 and 300  mg / kg the last remaining five groups were treated with the same substances and at the same doses combined with ici 182,780 at the dose of 300  g / kg  bw / d . after the 3-day treatment , animals were sacrificed ; the uterine weight , total protein levels in uteri , uterine , and vaginal epithelial heights , and mammary gland were assessed . total uterine protein levels were determined in uteri using colorimetric methods described by gornall et al . . the histomorphological parameters of mammary gland , uteri , and vagina were assessed as described previously . one - way analysis of the variance ( anova ) followed by dunnett 's test for multiple comparisons and the student 's t - test was used for statistical comparison between different control and treated groups for in vivo and in vitro experiments , respectively . as expected , ici 182,780 significantly ( p < 0.001 ) blocked the transactivation induced by e2 alone in both hek - er and hek - er ( figure 1 ) . the results obtained after cotreatment of hek293 t cell lines transiently transfected with er and er showed a significant inhibition ( p < 0.001 ) with m. macrophylla extracts and ici 182,780 ( figure 1 ) . this result confirms that m. macrophylla extracts induced their in vitro activity through ers . regarding the uterine parameters , after the in vivo 3-day treatment , antiestrogen ici 182,780 ( faslodex : 300  g / kg ) totally abrogated ( p < 0.001 ) the increase of uterine wet weight induced by e2b ( figure 2(a ) ) , and it significantly ( p < 0.01 ) decreased uterine total protein level ( figure 2(b ) ) and uterine epithelial height ( figures 2(c ) and 2(d ) ) . compared with dcm treated group , the cotreatment of animal with dcm extract and ici 182,780 induced a significant ( p < 0.01 ) decrease in the uterine wet weight . however , it did significantly ( p < 0.01 ) increase total protein levels and uterine epithelial height . ici 182,780 significantly blocked the increase of these aforementioned parameters when cotreated with dcm and meoh extracts ( figures 2(b ) , 2(c ) , and 2(d ) ) . as shown in figure 3(a ) , e2b treatment significantly ( p < 0.001 ) increased the vaginal epithelial height by 266.71% as compared with control ( ovx ) . treatment with m. macrophylla extracts increased the vaginal epithelial height ( p < 0.01 ) at all tested doses . cotreatment of m. macrophylla extracts with ici 182,780 ( faslodex : 300  g / kg ) significantly ( p < 0.01 ) abrogated the increase of vaginal epithelial height induced by e2b or m. macrophylla extracts treatment alone ( figure 3(a ) ) . these effects were materialized in histological sections by the loss of the presence of stratification ( stratum granulosum or stratum corneum ) to a thin stratum germinativum consisting of few cell layers as observed in control group ( ovx ) ( figure 3(b ) ) . as shown in figure 4 , treatment with e2b induced the increment of the diameter and the lumen of alveoli as well as an abundant eosinophil secretion in the lumen of alveoli . the meoh extract increased the diameter of alveoli at all tested doses , however it induce eosinophil secretion only at the high dose . the results of the cotreatments with ici 182,780 and dcm or meoh m. macrophylla extracts showed that ici 182,780 inhibits the mammotrophic effects induce by this substance alone ; this was materialized by the loss of eosinophil secretion and the increment of acini height . m. macrophylla has demonstrated pharmacological effects on estrogen target organs of female , osteoporosis , and male reproductive system of wistar rats . we hypothesize that m. macrophylla might mediate some of its actions by binding to the estrogen receptor , as it shares many estrogen - like actions in various physiological systems . to accomplish our goal , we used ici 182,780 ( faslodex ) which is the most potent estrogen receptor antagonist of the class of 7-alkylamide that has been approved for treatment of postmenopausal breast cancer patients who fail to respond to tamoxifen therapy . it was reported that the ability of agonists of ers to activate the transactivation ligand - dependent or ligand - independent is completely inhibited by ici 182,780 . in our study , ici 182,780 significantly blocked m. macrophylla extracts - induced transactivation of both  and  estrogen receptor subtypes in vitro . these results suggest that the induction of transactivation by m. macrophylla extracts is due to the binding of its secondary metabolites to ers , thus corroborating the observation of many authors who also observed the inhibition of ers (  and  ) transactivation in cellular systems [ 2427 ] . based on the fact that any beneficial effect obtained in vitro is not transferable in vivo because of the complexity of this later system , we also antagonized the plant extracts with ici 182,780 in vivo in ovariectomized rats . the results showed that cotreatment of the extracts with ici 182,780 significantly abrogated the increase of uterine wet weight , uterine , and vaginal epithelial height and uterine total protein levels after a 3-day treatment . moreover , ici 182,780 inhibits the effect of different treatments ( e2b , dcm , and meoh extracts ) on mammary gland . in fact , ici 182,780 blocks endometrial proliferation and uterine growth in rats and inhibits growth of breast tumor cells both in cell culture and in vivo [ 25 , 28 ] . ici 182,780 is known not only for its inhibitory effects on the functional activity of estrogen receptors but also for its ability to reduce the cellular level of ers . therefore , m. macrophylla extracts while cotreated with ici lose their effects probably due to a low level of ers available to exhibit the estrogen - like effects obtained in the absence of ici . this confirms that the active principles of m. macrophylla bind directly to ers or at least are part of the regulatory complex at the promoter region of ers target genes . phytoestrogens have been found to bind the estrogen receptor with an affinity 10-fold to 1000-fold lower than estrogen . m. macrophylla has exhibited estrogen agonistic effects in the range of some well - characterized phytoestrogens ; and these estrogenic effects were lost in the presence of ici 182,780 . therefore , millettia macrophylla should be classified as source of phytoestrogen like a number of plants consumed by humans such as soy , flax , red - clover , hops , citrus , grape , block cohosh , dong quai , ginseng , licorice , and wild mexican yam . our results are interesting when considering the data from epidemiological surveys and nutritional intervention studies , indicating that some phytoestrogens ameliorate menopausal symptoms and are protective against a variety of disorders , including cardiovascular disease , cancer , hyperlipidemia , and osteoporosis [ 3134 ] . the estrogenic activities of m. macrophylla extracts may be due to one or more of its constituents that bind to ers to induce their biological effects . more in vitro and in vivo studies will be needed to further characterise the molecular mechanism of action that underpins m. macrophylla extracts estrogenic activities .
millettia macrophylla is used traditionally to treat menopause related symptoms . 
 this plant was shown to exhibit estrogenic effects in vitro on human embryonic kidney cells and in vivo on ovariectomized rats . 
 the present study aimed at elucidating underlying mechanisms by which m. macrophylla induced its estrogenic effects . to accomplish our goal , kidney hek293 t cells transiently transfected with estrogen alpha or beta receptor expression 
 plasmids were cotreated with a pure antiestrogen ici 182,780 and the dichloromethane or methanol soluble fractions of m. macrophylla . to follow up , we cotreated ovariectomized rats with both extracts and ici 182,780 for 3 days in the classical uterotrophic assay . 
 animals were then sacrificed and the uterine wet weight , total protein levels in uteri , uterine , and vaginal epithelial heights , and mammary gland were assessed . in vitro , the results suggested that the induction of the estrogenic activity by m. macrophylla is due to the binding of its secondary metabolites to er and er. in vivo , the cotreatment of extracts and ici 182,780 significantly abrogated the biological responses induced by the extracts alone . taken together , these results indicate that the active principles of m. macrophylla induce their beneficial effects on menopausal symptoms by activating the ers .