Fitoterapia 82 (2011) 939–949
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Fitoterapia j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e
A postmenopause-like model of ovariectomized Wistar rats to identify active principles of Erythrina lysistemon (Fabaceae) M.A. Mvondo a, D. Njamen a,⁎, S. Tanee Fomum b, J. Wandji b, Günter Vollmer c a b c
Department of Animal Biology and Physiology, Laboratory of Animal Physiology, University of Yaounde I, P.O. Box 812 Yaounde, Cameroon Department of Organic Chemistry, University of Yaounde I, P.O. Box 812 Yaounde, Cameroon Institut of Zoology, Chair of Molecular Cell Physiology and Endocrinology, University of Technology, Zellescher Weg 20b, 01217 Dresden, Germany
a r t i c l e
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Article history: Received 14 March 2011 Accepted in revised form 9 May 2011 Available online 23 May 2011 Keywords: Isoflavone Flavanone Estrogenicity Postmenopause Hot flushes Antiatherogenic
a b s t r a c t To determine whether the two major compounds of Erythrina lysistemon are active principles accounting for Erythrina estrogenic effects, we used a postmenopause-like model of ovariectomized Wistar rats to evaluate their effects on some menopausal problems. Ovariectomized rats were orally treated either with compound 1 or compound 2 at 1 and 10 mg/kg BW for 28 days. Estradiol valerate served as the reference substance. As results, compounds 1 and 2 displayed estrogen-like effects on the uterus and the vagina, and reduced atherogenic risks by decreasing the two assessed atherogenic parameters, the total cholesterol/HDL-cholesterol ratio and the atherogenic index of plasma. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Estrogens are classically known as steroid hormones with important functions for the regulation of specific sexual processes in the female organism. In addition, there is knowledge about the action of estrogens in other non-classical target tissues like the brain, the bone, the cardiovascular system, the kidney, the immune system and the liver [1]. Menopause marks the end of the reproductive life span of women and is characterized by a dramatic drop in circulating estrogen. Symptoms associated with estrogen deprivation include vasomotor instability (hot flushes), genitourinary atrophy, and depression [2]. In a long term, estrogen deficiency affects bone density [3,4] and the cardiovascular health [5,6]. Hormone replacement therapy (HRT) has been successfully used to treat the symptoms of menopause because estrogen has strong suppressive effects on climacteric complaints. Recent studies, however, have uncovered a greater understanding of
⁎ Corresponding author. Tel.: + 237 79 42 47 10. E-mail address:
[email protected] (D. Njamen). 0367-326X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2011.05.009
the hazards of HRT. The Women's Health Initiative Study was abandoned because several adverse effects including higher risk for breast cancer and coronary heart disease outweighed the benefits of hormone treatment for postmenopausal women [7]. Similar results were obtained from the more recent ‘Million Women Study’ [8]. These findings led to various attempts to search for alternative products to classical HRT among them, plant secondary metabolites with estrogenic activity, so-called “phytoestrogens”. Phytoestrogens are compounds found in plants and fungi that exhibit estrogenic activity both in vivo and in vitro. The scientific interest lies in the potential of phytoestrogens for medical use either as registered drug or mostly as dietary supplement. A decade earlier Kaufert et al. [9], reported that 80% of women aged 45–60 years were using non prescription therapies to manage menopausal symptoms. More recently, Ferrari [10] reported that in daily practice conditions, high doses of isoflavones, particularly genistein, can be used for the management of hot flushes in postmenopausal women not treated with HRT due to their early onset of action, efficacy and safety. Nevertheless, the effectiveness of phytoestrogens on breast cancer, cardiovascular diseases and bone health is still controversial [11,12]. Therefore, there are efforts to find new
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compounds that exert selective effects by acting as an estrogen receptor (ER) antagonist on neoplastic or normal breast and uterine tissues, and as an ER agonist on estrogen-responsive tissues like bones, liver and the central nervous system. In this search, the science of ethnobotany and pharmacognosy are being used as guide to lead scientists toward different sources and classes of compounds, and the tropical flora, by virtue of its diversity, continues to provide new leads. Erythrina is one of the worldwide represented genus which species are rich in isoflavonoids and widely used in many folkloric medicines [13–17]. Erythrina lysistemon in particular is a medicinal plant widely distributed in Africa and traditionally used as antibacterial, anti-inflammatory, analgesic medicine and as a palliative for women problems. It has been subject to a number of scientific investigations and some authors reported that it exhibited cyclo-oxygenase inhibition and anti-bacterial activities [18]. The alkaloids isolated from its seeds and flowers also exhibited DPPH radical scavenging activity [19,20]. More recently, the methanol extract of the stem bark of E. lysistemon was found to inhibit PTP1B activity by more than 80% at 30 μg/ml [21], revealing its capacity to treat Type 2 diabetes and obesity. In parallel the ethylacetate extract of the stem bark of E. lysistemon has been found to exert estrogen-like effects at 200 mg/kg BW both in vivo (on female rat sex organs) and in vitro (on yeast and Ishikawa cells) [22]. The authors concluded that this extract may contain compounds endowed with oestrogenic effects. From this extract we have isolated one isoflavone and one flavanone as its major constituents, namely alpinumisoflavone (compound 1) and abyssinone V-4′-methyl-ether (compound 2). Although they are not new compounds, their estrogen-like properties have not yet been reported. Our goal was to use a postmenopause-like model of ovariectomized Wistar rats to evaluate the effects of these compounds on some problems of menopause. The biological activity was primarily evaluated on female rat sex organs (uterus and vagina) since they are estrogen primary target organs. We also determined body weight gain, and analyzed serum lipid and gonadotropin concentrations to evaluate their effects on cardiovascular risk and hot flushes respectively. And finally, to determine whether these compounds bind estrogen receptors (ER), an in vitro study of ligand binding assay was carried out using fluorescence polarization on ERα and ERβ. 2. Materials and methods
eluted at ethylacetate/hexane (70 + 30) were subsequently subjected to repeated CC on silica gel to obtain alpinumisoflavone (48 mg) as yellow crystals. Fractions 273–344 (C5) eluted at ethylacetate/hexane (40 + 60) were further chromatographed to yield 4 compounds C5.1 (2 mg), abyssinone V-4′-methyl-ether (159 mg), oleanolic acid (10 mg) and warangalone (5 mg). Their structures have been successfully determined by their UV, MS, 1H- and 13C- NMR data, and also in comparison with the literature data.
2.2. Animals Juvenile female Wistar rats aged 10 to 12 weeks old (150 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 diet (SSniff R10-Diet, SSniff GmbH, Soest, Germany) and water. Housing of animals and all in vivo experiments were carried out following the guidelines of the institutional Ethic Committee of the Cameroon Ministry of Scientific Research and Technology Innovation, which has adopted the guidelines established by the European Union on Animal Care (CEE Council 86/609).
2.3. Study design Animals were either intact or ovariectomized (ovx). Eighty four days (12 weeks) after endogenous hormonal decline and installation of human-like postmenopausal condition in the case of ovx animals, all experimental groups were treated with the respective test compounds. Thereby, animals were randomly distributed into 6 lots of 5 rats and were daily treated per os for 28 days. As ovx-control, the first lot received the vehicle only. Positive control animals were treated with the reference substance, estradiol valerate (E2V) at the dose of 1 mg/kg BW. Compounds 1 and 2 were separately administered to the 4 remaining lots at doses of 1 and 10 mg/kg BW. Animals were weighed once every two weeks throughout the experiment. After the 28-day treatment, animals were sacrificed by decapitation after 12 h of fasting. Blood was collected from each rat and serum was immediately separated by centrifugation at 3500 rpm for 15 min at 4 °C, for lipid and gonadotropin measurement. Uterus and vagina were taken out and fixed, after determination of uterine wet weight (UWW), in 4% formalin for histological analysis.
2.1. Plant material: Extraction and isolation E. lysistemon Hutch (Fabaceae) was collected in Zimbabwe in May 2008 and was identified at the Zimbabwe Herbarium in comparison with the reference voucher specimen number KPAL00000083. Its stem bark (5 kg) was then dried ground and was extracted with ethylacetate for 72 h at room temperature. After concentration using a rotary evaporator in vacuum, a total mass of 209 g of crude extract was obtained. This was flash chromatographed using a mixture of hexane – ethylacetate – methanol of increasing polarity, to yield 554 fractions collected and combined on the basis of TLC into 12 collective fractions (C1–C12). Fractions 397–430 (C7; 6g)
2.4. Histological analysis Uterine and vaginal epithelial heights were assessed from 5-μm sections of paraffin-embedded uterine and vaginal tissues. Following the hematoxylin–eosin staining, uterine and vaginal epithelial heights were assessed on microphotography using the complete Zeiss equipment consisting of a microscope Axioskop 40 linked to a computer where the image was transferred, and analyzed with the MRGrab1.0 and Axio Vision 3.1 software, all provided by Zeiss (Hallbermoos, Germany).
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2.5. Serum lipids analysis Serum total cholesterol (TC), HDL-cholesterol (HDL-C), and triglyceride (TG) were measured in each animal by fully automated (Cobas Mira S auto analyzer) enzymatic method. Atherogenic index was calculated as total cholesterol on HDL cholesterol. In addition, atherogenic index of plasma (AIP) related to the particle size of lipoproteins was calculated as the logarithm of triglycerides/HDL cholesterol ratio [23]. The reagent kits used were purchased from Biolabo (France). 2.6. Serum gonadotropin analysis FSH and LH were determined by ELISA test using reagent kits purchased from HUMAN (Wiesbaden, Germany). The absorbance of calibrators and specimen was determined by using ELISA microplate readers or automated ELISA systems (like HUMAN's HUMAREADER or ELYSIS line). The concentration (IU/l) was evaluated by means of a calibration curve which is established from the calibrators supplied with the kit. 2.7. Ligand binding ability Ligand binding activity was examined directly by an Estrogen Receptor Competitor Assay based on fluorescence polarization [24] according to manufacturers (InVitrogen, Darmstadt, Germany) instructions. In brief, ER was added to a fluorescent estrogen ligand to form an ER/Fluormone Red complex. This complex was then added to individual test compounds in multiwell plates at different concentrations. If
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the test compound does not compete with Fluormone, then the ER/Fluormone Red complex will remain intact. In case of competitive replacement, Fluormone Red degrades rapidly resulting in a low polarization value. The change in polarization value in the presence of a test compound was used to determine relative affinity of that test compound for ER. The test was run in triplicates and repeated 3 times. 2.8. Statistical analysis The values in the figures of each experimental group are presented as the mean ± SEM. The non-parametric Mann– Whitney U test was used for statistical comparison. The ovxcontrol group was compared to the intact group while treated groups were compared to the ovx-control, and the significance of the difference was noted at P b 0.05. 3. Results 3.1. Phytochemical analysis The structures of all the compounds (alpinumisoflavone, warangalone, abyssinone V-4′-methyl-ether, oleanolic acid) were successfully determined by analyzing their UV, MS, 1Hand 13C- NMR data, and also in comparison with the literature data [25–29]. The two major compounds, alpinumisoflavone and abyssinone V-4′-methyl-ether, referred to as compounds 1 and 2 characteristics are summarized in Table 1. They were all known compounds extracted from other plant species. Since no work has been reported on these compounds with regard to their estrogen-like activity and as a follow up of our
Table 1 Structures and molecular weights of compounds 1 and 2. Compound Compound 1 alpinumisoflavone 5-hydroxy-7-(p-hydroxyphenyl)-2, 2-dimethyl-2H-6H-benzo-[1,2-b: 5,4-b]dipyran-6-one
Crystal color
Molecular weight structure
Yellow
O
O
OH
O
OH
Molecular Weight = 336.35 Molecular Formula = C20H16O5
Compound 2 abyssinone V-4′-methyl-ether 4H-1-benzopyran-4-one, 2,3-dihydro-5,7-dihydroxy-2-[4-methoxy-3,5-bis(3-methyl-2-buten-1-yl)phenyl]
White
OMe O
HO
OH
O
Molecular Weight = 422.53 Molecular Formula = C26H30O5
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previous finding with the crude stem bark extract, we looked at their potential biological activity using a postmenopauselike model of ovariectomized rats. Only compounds 1 and 2 were tested, because the other compounds isolated were in quantities so small as to only allow structures determination. 3.2. Uterine wet weight (UWW) The lost of ovarian estrogens led to a significant decrease (77.76%) of the UWW (Fig. 1). Following E2V administration, the UWW was increased by 278.9% compared to the ovxcontrol. In contrast, compound 1 decreased the UWW at 1 mg/kg BW (30.82%). Compound 2 acted in a dosedependent manner and decreased the UWW by 62.79% at 1 mg/kg BW and 40% at 10 mg/kg BW. 3.3. Uterine epithelial height
Fig. 2. Uterine epithelial height after a 28-day treatment. Intact = non-ovx animals, Control = ovx animals treated with the vehicle, E2V = ovx animals treated with estradiol valerate, [C1] = ovx animals treated with compound 1, [C2]= ovx animals treated with compound 2. Data are shown as mean ±S.E.M., n= 5, *Pb 0.05 and **P b 0.01 versus Control, ##Pb 0.01 versus Intact.
Gonadectomy decreased the uterine epithelial height to 65.17% (Fig. 2). Following E2V treatment, the uterine epithelial height increased by 217.43%. Compound 1 induced E2Vlike effect at the two tested doses and increased the uterine epithelial height by 65.95% at 1 mg/kg BW and 89.53% at 10 mg/kg BW. Compound 2 showed no significant effect whatever the tested dose.
Treatment with E2V led to a significant loss (258.49%) of body weight compared to that of the ovx-control group (P b 0.01). Compound 1 also induced weight loss at the two tested doses. Compound 2 significantly increased weight gain (180.4%) at 1 mg/kg BW and induced weight loss (164.6%) at 10 mg/kg BW.
3.4. Vaginal epithelial height
3.6.1. Serum triglyceride (TG) levels Following ovariectomy TG levels were increased by 43.26% (Fig. 5A). E2V failed to reverse ovariectomy-increased TG serum levels but showed a tendency to elevate it, an effect that did not turn to be significant compared to controls. In contrast, compound 1 decreased TG concentrations by 61.72% at 1 mg/kg BW and 69.64% at 10 mg/kg BW. Such a decrease was also obtained with compound 2 at 1 mg/kg BW (63.37%) whereas no effect was observed at 10 mg/kg BW compared to controls.
Following ovariectomy, the vaginal epithelial height decreased by 77% compared to intact animals (Fig. 3). E2V increased vaginal epithelial height by 384.56% (from 5.78 ± 0.46 μm to 28.04 ± 1.48 μm) compared to the ovx-control. Both compounds induced E2V-like effects at the dose of 1 mg/kg BW and they increased vaginal epithelial height by 87.83% and 143.09% respectively.
3.6. Serum lipids analysis
3.5. Body weight From day-0 to day-84, animals' body weight gradually increased. The lost of endogenous estrogens due to ovariectomy induced a significant weight gain at days 42 (12.51%), 56 (9.42%), 70 (20.3%), and 84 (19.29%) compared to intact controls (Fig. 4A). At the end of treatments, the weight gain of the control was similar to that of the intact group (Fig. 4B).
3.6.2. Serum total cholesterol (TC) levels Eighty four days after ovariectomy, serum cholesterol levels increased by 32.97% although this effect was not statistically significant compared to intact animals (Fig. 5B). E2V significantly decreased TC by 42.74% (from 0.78 ± 0.05 g/l to 0.45 ± 0.07 g/l, P b 0.01). A comparable decrease in TC was also obtained with compound 1 (22.39% at 1 mg/kg BW and
Fig. 1. Uterine wet weight after a 28-day treatment. Intact = non-ovx animals, Control = ovx animals treated with the vehicle, E2V = ovx animals treated with estradiol valerate, [C1] = ovx animals treated with compound 1, [C2] = ovx animals treated with compound 2. Data are shown as mean ± S.E.M., n = 5, *P b 0.05 and **P b 0.01 versus Control, ##P b 0.01 versus Intact.
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3.6.4. TC/HDL-C ratio TC/HDL-C ratio was not significantly affected after ovariectomy (Fig. 5D). At 10 mg/kg BW, compound 1 decreased the ratio by 47.55% (from 1.51 ± 0.16 to 0.79 ± 0.1, P b 0.01) while compound 2 induced a decrease at the two tested doses (36.58% at 1 mg/kg BW and 60.37% at 10 mg/kg BW, P b 0.01).
Fig. 3. Vaginal epithelial height after a 28-day treatment. Intact = non-ovx animals, Control = ovx animals treated with the vehicle, E2V = ovx animals treated with estradiol valerate, [C1] = ovx animals treated with compound 1, [C2]= ovx animals treated with compound 2. Data are shown as mean ±S.E.M., n= 5, **Pb 0.01 versus Control, ##P b 0.01 versus Intact.
32.31% at 10 mg/kg BW, P b 0.05) and compound 2 (36.39% at 1 mg/kg BW and 42.87% 10 mg/kg BW, P b 0.01). 3.6.3. Serum HDL-cholesterol (HDL-C) levels Ovariectomy induced a slight and non significant increase is HDL-C (Fig. 5C). Compared to the control group, E2V decreased HDL-C by 31.17% (from 0.52 ± 0.02 g/l to 0.36 ± 0.03 g/l, P b 0.01). In contrast, compounds 1 and 2 increased, at 10 mg/kg BW, HDL-C concentrations by 26.62% and 43.35% respectively.
3.6.5. Atherogenic index of plasma (AIP) Following ovariectomy, AIP (Fig. 5E) was not significantly affected. E2V enhanced ovariectomy-increased AIP and increased it by 48.38%. Compounds 1 and 2 in contrast, significantly decreased AIP values at all tested doses. Results show that the AIP decreased by 88.41 and 136.92% following treatment with compound 1 at the doses of 1 and 10 mg/kg BW respectively. Compound 2 lowered AIP values by 95.52 and 35.9% at the doses of 1 and 10 mg/kg BW respectively, as compared to the ovx-control. 3.7. Serum gonadotropin analysis 3.7.1. Serum FSH levels FSH levels remained unaffected 84 days after ovariectomy (Fig. 6A). E2V, as expected, significantly decreased FSH levels by 29.38% (from 1.14 ± 0.018 IU/l to 0.805 ± 0.02 IU/l, P b 0.01) compared to the control group. A similar decrease was observed only with compound 1 at the tested doses (32.1% at 1 mg/kg BW and 31.75% at 10 mg/kg BW). Compound 2 showed no effect whatever the tested dose. 3.7.2. Serum LH levels Eighty four days of estrogen deprivation led to a 351.44% increase in LH levels (P b 0.01). E2V significantly decreased LH levels by 79.27% compared to the control group. In a dosedependent manner, compounds 1 and 2 also induced a significant decrease in LH serum levels (Fig. 6B). 3.7.3. FSH/LH ratio The ratio of FSH to LH was strongly decreased after ovariectomy (68.82%; P b 0.01). Compared to the ovx-control, E2V shifted the ratio toward FSH dominance and increased it by 147.52% (P b 0.01). A significant increase was also obtained with compounds 1 (95.69%) and 2 (67.86%) at 1 mg/kg BW (Fig. 6C). 3.7.4. ER binding assay Our results showed that compounds 1 and 2, as compared to estradiol (E2), bind to ERα with a relative binding affinity (RBA) of 0.150 and 0.151% respectively. Using regression analysis their IC50 values are respectively 4.49 and 4.46 μM. The RBA of both compounds for ERβ was 0.041 and 0.131% respectively; and their IC50 values were 1.49 and 4.69 μM respectively. The ratio of the RBA of compound 1 for ERα on its RBA for ERβ (equal to 3.66) was obviously shifted toward ERα preference. Compound 2 however, seems to bind ERα and ERβ in the same pattern since the ratio of its RBA for ERα on its RBA for ERβ was equal to 1.15.
Fig. 4. Body weight profile during 84 days of estrogen deprivation (A) and following treatment (B). Intact = non-ovx animals, ovx = ovariectomized animals, Control = ovx animals treated with the vehicle, E2V = ovx animals treated with estradiol valerate, [C1] = ovx animals treated with compound 1, [C2]= ovx animals treated with compound 2. Data are shown as mean ±S.E.M., n= 5, **Pb 0.01 versus Control and ###P b 0.001 versus Intact.
4. Discussion The aim of this study was to use a postmenopause-like model of ovariectomized Wistar rats to evaluate the estrogen-
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Fig. 5. Serum levels of TG (A), TC (B), HDL-C (C), TC/HDL-C ratio (D), and log(TG/HDL-C) (E), after a 28-day treatment. Intact = non-ovx animals, Control = ovx animals treated with the vehicle, E2V = ovx animals treated with estradiol valerate, [C1] = ovx animals treated with compound 1, [C2] = ovx animals treated with compound 2. Data are shown as mean ± S.E.M., n = 5, *P b 0.05, **P b 0.01 versus Control and ##P b 0.01 versus Intact.
like effects of an isoflavone and a flavanone derived from E. lysistemon and thereby, find out whether these major constituents are active principles accounting for the effects of E. lysistemon. Estrogen-like effects was primarily evaluated on female rat sex organs (uterus and vagina). We also determined body weight gain, and analyzed serum lipids and gonadotropins to evaluate their effects on cardiovascular risk and hot flushes respectively. And to determine whether these compounds bind estrogen receptors (ER), an in vitro study of ligand binding assay was carried out using fluorescence polarization on ERα and ERβ. This assay showed that both compounds bind ERα and ERβ with a higher affinity for ERα, especially compound 1, unlike most phytoestrogens whose
preference is for ERβ [12]. According to Casson et al. [30], compounds with relative binding affinity (RBA) ≥ 0.1, at a concentration of 1 μM can displace more than 90% of 17βestradiol bind to ER at the postmenopausal level of hormone (0.1 nM). And assuming that isoflavones administered to volunteers participating in a clinical trial can reach circulating concentrations of 2–5 μM [31], compounds 1 and 2 with RBA ≥ 0.1 can then be considered as potentially active. In vivo study shows that, the loss of ovarian estrogens following surgical menopause (ovariectomy) resulted in marked vaginal epithelium thinness, which, according to Westwood [32], is reduced to one cell layer, the stratum germinativum. The uterine wet weight as well as the
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Fig. 6. Serum levels of FSH (A) and LH (B), and FSH/LH ratio (C) after a 28-day treatment. Intact = non-ovx animals, Control = ovx animals treated with the vehicle, E2V = ovx animals treated with estradiol valerate, [C1] = ovx animals treated with compound 1, [C2] = ovx animals treated with compound 2. Data are shown as mean ± S.E.M., n = 5, *P b 0.05, **P b 0.01 versus Control and ##P b 0.01 versus Intact.
epithelial height decreased dramatically. After a 28-day treatment, E2V induced a significant increase of the vaginal epithelium. This result is in accordance with the observations of Buchanan et al. [33] who reported that estrogens consistently stimulate proliferation of the vaginal epithelium leading to the formation of a highly stratified epithelium. This effect is reported to be mediated through the ERα [33,34] as demonstrated by Couse et al. [35] who reported that E2 failed to induce vaginal epithelial proliferation and stratification in ERα knockout (αERKO) mice. Compounds 1 and 2 displayed
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E2V-like effects at 1 mg/kg BW as they raised the vaginal epithelial height. Since these vaginal events have been reported to be mediated through the ERα [33,34], and according to the significant relative binding affinity of both compounds for the ERα which was 0.15 and 0.151 respectively, we can hypothesized that at 1 mg/kg BW these compounds might stimulate ERα to raise vaginal epithelial height. On the contrary, at the dose of 10 mg/kg BW, both compounds would have down-regulated ERα expression and therefore decreased the sensitivity of vaginal epithelial cells to these compounds. As far as the uterus is concerned, E2V induced, as expected, a significant increase in uterine wet weight. These estrogen effects have long been attributed first to water imbibition of tissue and subsequently to proliferation of endo- and myometrial cells [36,37]. Results on the uterine epithelial height following treatment with E2V were in agreement with the hypothesis of estrogen-stimulation of the endometrial cell proliferation given rise to a tall columnar epithelium as reported Westwood [32], and which results in a marked increase in the uterine epithelial height. These uterine events induced by E2V are reported to be mediated via ERα as demonstrated by the lack of uterine stimulation and mitotic growth responses in αERKO mice [38]. Compound 1 decreased the uterine wet weight at the low dose (1 mg/kg BW) and showed no effect at 10 mg/kg BW. These results suggest that at the dose of 1 mg/kg BW, compound 1 might either antagonized the uterine ERα or agonized ERβ to decrease uterine wet weight, since ERβ is claimed to mediate ERα-antagonistic effects [39]. But at 10 mg/kg BW, the activated receptor would have been down-regulated leading to a decrease in the uterine sensitivity to compound 1. Results on uterine epithelial heights showed that compound 1 increased uterine epithelial height at the two tested doses. These later results contradict the hypothesis that compound 1 might antagonize ERα or agonize ERβ since the increase in uterine epithelial height is reported to be an ERα-mediated event [38], and according to the in vitro binding affinity results, compound 1 seems to have a four-fold more preference for ERα than ERβ. These results suggest on one hand that compound 1 activated the uterine ERα in a way that could not stimulate water imbibition and the proliferation of the whole uterine tissue to increase the uterine wet weight; the decrease in uterine weight observed at the dose of 1 mg/kg BW being the result of the variation of animals' uterine weights in this group. On the other hand, these results suggest that the proliferation of endometrial cells responsible for the increase in uterine epithelial height is not enough to result in the augmentation of the uterine wet weight. This ability to stimulate epithelial cell proliferation is however reported to endanger the endometrium since endometrial hyperplasia has been considered as an indication of cancer endangerment [40], and call into question the long-term safety of compound 1 with regard to the endometrium. Compound 2, however, decreased the uterine wet weight and showed no significant effect on the epithelium. This compound might either antagonize ERα or agonize ERβ, or activate both ER subtypes favoring therefore the formation of ERα/ERβ heterodimers which are reported to attenuate ERαmediated events [41]. Consistently with this hypothesis, the relative binding affinity assay showed that the binding affinities of compound 2 for both ERα and ERβ were very
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close (0.151 vs 0.131) suggesting that this compound binds uterine ERα and ERβ in a same pattern. Therefore the tridimensional conformation of the complex compound 2ERα/ERβ might be in favor of the inhibition of water imbibition and proliferation of the uterus. Finally, all these results show that both compounds are ER ligands; whereas compound 1 seems to be an ERα agonist in both tissues (uterus and vagina), compound 2 might exert selective effects by acting as an ERα agonist on the vaginal epithelium and as an agonist of both ER subtypes on the uterine epithelium. In our previous work, we [22] reported that the crude extract of E. lysistemon increased vaginal epithelial height and showed a tendency to raise the uterine epithelial height. In this study, similar effects were observed only with compound 1 which rose vaginal and uterine epithelial heights. Compound 2 increased only vaginal epithelial height and showed no effect on the uterine epithelium. These results suggest that the effects reported with the crude extract result from the combined effects of compounds 1 and 2. Compound 2 would have inhibited, at least on the uterus, the effect of compound 1 and this inhibition might be related to their proportion in the crude extract. Phytochemical analysis revealed that the isolated amount of compound 2 (159 mg) represented 3.18% of the crude extract and was 3 times more abundant than compound 1 (48 mg). This abundance would have favored compound 2 for the competition of both compounds for ERs to produce the effects reported earlier with the crude extract. As far as body weight is concerned, our results showed that ovariectomized animals' body weight increased more rapidly than that of the intact control probably due to an increase in adipose deposition as suggested by Naaz et al. [42]. Furthermore, adipose tissue has been reported to be one of the most important extragonadal sources of steroids and particularly estrogens, due to the specific expression of steroidogenic enzymes, such as aromatase in this tissue [43]. This might be a natural process to fight weight gain in postmenopausal condition. E2V induced a significant body weight loss. This result is in accordance with the observations of Naaz et al. [42] who reported that estrogen reverses ovariectomy-induced body weight gain. This effect of E2V might be mediated via the ERα signaling pathway as shown in studies of ERα (αERKO), ERβ (BERKO) and double (DERKO) knockout mice models demonstrating that ERα mediates lipolytic or antilipogenic effects, while ERβ exerts lipogenic actions [44–46]. Compound 1 displayed E2V-like effect by inducing a significant body weight loss at the two tested doses, suggesting the effectiveness of this compound on ERα. At its lower dose (1 mg/kg BW), compound 2 failed to reverse ovariectomy-induced weight gain, whereas the higher dose (10 mg/kg BW) induced a significant weight loss. These results suggest that compound 2 acted either as an ERα antagonist or as an ERβ agonist to induce weight gain at 1 mg/kg BW. But at 10 mg/kg BW, the activated receptor would have been desensibilized favoring thereby the lipolytic pathway. These results suggest that both compounds might use two different signaling pathways in the adipose tissue to induce their effects. Lipid profile constitutes an important cardiovascular risk factor [47]. Estrogen is well known to modulate lipid metabolism and the loss of this hormone following menopause is largely recognized as the principal cause of the
increased risk for atherosclerosis [48]. In this study, ovariectomy induced a significant increase in serum levels of triglycerides (TGs). Treatment with E2V did not attenuate the ovariectomy-increased TG levels. This observation is in accordance with the results from human studies that consistently demonstrated an increase in TG levels following estrogen treatment [49,50]. In an experimental animal study, Böttner et al. [51] also reported an increase in TG levels following treatment with estradiol benzoate (E2B). This effect might be due to the actions of E2 on hepatic lipase since it has been demonstrated that ethinylestradiol administration down-regulates the activity of this enzyme by decreasing its mRNA levels [52]. In agreement with this finding, further studies reported the inhibition of hepatic lipase by E2 [53,54]. A prominent decrease of TGs was observed following treatment with compound 1 at the two tested doses. Compound 2 induced a similar effect at its low tested dose (1 mg/kg BW). Our results also showed that ovariectomy induced nonsignificant variation of serum total cholesterol levels. Following treatment, E2V decreased the serum total cholesterol levels compared to the ovx-control. In their study, Moorthy et al. [55] reported that E2 inhibits the first enzyme in cholesterol biosynthesis known as hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in de novo cholesterol synthesis [56,57], thus reducing the synthesis of cholesterol. Furthermore, the effect on cholesterol metabolism might be mediated by ERα since αERKO mice have been reported to display elevated levels of total cholesterol [44]. Compounds 1 and 2 induced E2V-like effects by decreasing, probably via ERα, serum total cholesterol levels at all tested doses. To address the question as to whether compounds 1 and 2 might be efficient in the prevention of cardiovascular risks associated with estrogen deficiency, serum levels of HDL-cholesterol (HDL-C) were assessed to evaluate the atherogenic index calculated as total cholesterol (TC) on HDL cholesterol (HDL-C). In addition, atherogenic index of plasma (AIP) related to the particle size of lipoproteins was calculated as the logarithm of TG/HDL-C ratio [23]. Remarkably, we observed a decrease of HDL-C levels in our E2V-treated animals. This finding is in accordance with previous reports that demonstrated, in ovariectomized rats, reduced HDL-C levels following a 3-month treatment with subcutaneously applied E2 [58] or following a 5-day treatment with E2V administered per os [59]. In contrast, compounds 1 and 2 increased HDL-C concentrations at their high doses (10 mg/kg BW) and showed no effect at 1 mg/kg BW. This increase in HDL-C serum concentrations is in accordance with the results from human studies that consistently demonstrated an increase in HDL-C levels following estrogen treatment [60–62]. Results on atherogenic risks showed that, whereas AIP was significantly increased and TC/HDL-C ratio not significantly affected by E2V, compounds 1 and 2 significantly decreased both TC/HDL-C ratio and AIP. Compounds 1 and 2 then shifted the TC/HCL-C ratio toward HDL-C dominance. Moreover, by decreasing AIP, these compounds elevated HDL subpopulations. In fact, AIP has been reported to reflect the size of LDL and HDL subpopulations [63]. In the same study, the authors demonstrated that an increased concentration of medium and large VLDL and small LDL particles resulted in higher AIP while the
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values of this parameter decreased with increasing concentration of large LDL and large HDL subpopulations. Our results suggest that compounds 1 and 2 not only decreased the concentrations of TG (VLDL and LDL) and increased HDL-C, but also changed favorably the distribution of HDL subpopulations by increasing the proportion of large HDL. These changes might then result in markedly decreased values of AIP. Furthermore, AIP has been found to be highly correlated with the cholesterol esterification rate expressed as FERHDL [62] and both were found to be inversely correlated with the size of HDL particles. In small HDLs the esterification rate is high but large particles reduce it [64,65] and serve as the most effective vehicle for delivery of cholesteryl esters via scavenger receptor class B type 1 (SR-B1) to catabolic sites in liver and steroidogenic tissues [66]. Thereby, compounds 1 and 2 displayed anti-atherogenic effects by increasing the proportion of large HDL particles. FSH and LH secretion are known to be modulated by ovarian steroids. E2 exerts a negative effect on basal gonadotropin secretion [67–69]. But following menopause, serum concentrations of ovarian hormones become very low while those of FSH and LH increase markedly [70]. In this study, only serum concentrations of LH increased markedly after 84 days of estrogen deprivation while FSH levels remained unaffected thereafter and were similar to those of intact controls. These results suggest the existence of independent regulatory system driving FSH and not LH, as previously suggested by Genazzani et al. [71]. Moreover, we can hypothesize that the factor regulating FSH secretion, lost after ovariectomy, would have been restored 84 days later to normalize serum levels of FSH which are supposed to increase markedly following estrogen deprivation [70]. As reported above, 84 days after ovariectomy, animals were heavier than intact controls. This increase in ovx animals' body weight has been associated with fat accumulation in adipose tissue [42] which is able to produce estrogens thanks to its aromatization capacity [43]. These estrogens could be involved in the normalization of serum levels of FSH but were not enough to modulate serum levels of LH. Finally, the system regulating FSH seems to be very sensitive to low concentrations of estrogen whereas the one driving LH appears to be less sensitive, needing normal estrogen concentrations. Following treatments, E2V, as expected, decreased serum levels of FSH and LH through a negative feedback effect on pituitary gonadotrophs, as reported in many studies [67–69], and shifted the FSH/LH ratio toward FSH dominance. Shupnik [72] reported that chronic treatment of rats with estrogens resulted in the suppression of gonadotropin gene transcription. This effect of E2V might be mediated via ERα as reported by Cosma et al. [46]. Compound 1 induced E2V-like effects by decreasing serum levels of FSH and LH at the two tested doses and shifted the FSH/LH ratio toward the dominance of FSH. These results suggest that compound 1 exerted an estrogenlike effect on the pituitary secretion of gonadotropins. Compound 2, at the two tested doses, decreased serum levels of LH while FSH levels remained unaffected, almost normal, and the FSH/LH ratio was obviously shifted toward the dominance of FSH. These findings are in accordance with the hypothesis according to which there is an independent regulatory system driving FSH and not LH [71]. Compound 2 seems to be more effective on the system regulating the
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pituitary secretion of LH. Since LH pulses have been involved in postmenopausal hot flushing [73,74], compounds 1 and 2 seem to be efficient to ameliorate hot flushes by favoring the dominance of FSH thanks to their negative feedback action on LH secretion. As Seidlova-Wuttke et al. [75] suggested the negative feedback action of E2 or substances with estrogenic activity on LH secretion can be used as an indirect measure of the potency to ameliorate hot flush activity. Taken altogether, the two tested compounds derived from E. lysistemon are ER ligands and exhibited an overall estrogenlike effects and effects contrary to those of estrogen on the assessed parameters. As estrogen-like effects, compound 1 increased uterine epithelial height, both compounds increased vaginal epithelial height, induced body weight loss, and reduced hot flushes by increasing the ratio of FSH to LH. As effects contrary to those of estrogen, these compounds decreased uterine wet weight and reduced atherogenic risks by decreasing the two assessed atherogenic markers (TC/ HDL-C, and AIP). Compounds 1 and 2 were thereby able to ameliorate some symptoms related to a postmenopause-like condition in female Wistar rats and might be active principles responsible for the estrogenic effects of the crude extract of Erythrina lysistemon on the uterus and the vagina. Acknowledgments Dieudonne Njamen is thankful to the Alexander von Humboldt Foundation for a fellowship at the University of Technology in Dresden, where part of this work was carried out. Special thanks are also extended to the German Academic Exchange Service (DAAD) for material support. References [1] Diel P, Schmidt S, Vollmer G. In vivo test systems for the quantitative and qualitative analysis of the biological activity of phytoestrogens. J Chromatogr B Analyt Technol Biomed Life Sci 2002;777(1–2): 191–202. [2] Pinkerton JV, Stovall DW, Kighlinger RS. Advances in the treatment of menopausal symptoms. Womens Health 2009;5:361–84. [3] McClung MR. The menopause and HRT. Prevention and management of osteoporosis. Best Pract Res Clin Endocrinol Metab 2003;17:53–71. [4] Turner RT, Riggs BL, Spelsberg TC. Skeletal effects of estrogen. Endocr Rev 1994;15:275–300. [5] Yang XP, Reckelhoff JF. Estrogen, hormonal replacement therapy and cardiovascular disease. Curr Opin Nephrol Hypertens 2011;20:133–8. [6] Mikkola TS, Clarkson TB. Estrogen replacement therapy, atherosclerosis, and vascular function. Cardiovasc Res 2002;53:605. [7] Derry PS. Hormones, menopause, and heart disease: making sense of the Women's Health Initiative. Womens Health Issues 2004;14: 212–9. [8] Beral V. Breast cancer and hormone-replacement therapy in the Million Women Study. Lancet 2003;362:419–27. [9] P. Kaufert, P. Boggs, B. Ettinger, N.F. Woods, W.H. Utian, Women and menopause: beliefs, attitudes and behaviors. The North American Menopause Society 1997 Menopause Survey, Menopause. 5 (1998) 197–202. [10] Ferrari A. Soy extract phytoestrogens with high dose of isoflavones for menopausal symptoms. J Obstet Gynaecol Res 2009;35:1083–90. [11] Eisenberg DM, Davis RB, Ettner SL, Appel S, Wilkey S, Van Rompay M. Trends in alternative medicine use in the United States, 1990–1997: results of a follow-up national survey. J Am Med Assoc 1998;280: 1569–75. [12] Wuttke W, Jarry H, Seidlová-Wuttke D. Isoflavones—safe food additives or dangerous drugs? Ageing Research Rev 2007;6:150–88. [13] F.A. Bisby, J. Buckingham, J.B. Harborne, Phytochemical Dictionary of the Leguminosae, Vol. 1, Plants and their Constituents, Chapman & Hall, London, 1994.
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