Estrogenic and Antioxidant Activities of Pterocarpus soyauxii (Fabaceae) Heartwood Aqueous Extract in Bilateral Oophorectomized Wistar Rat

Phytoestrogens are used to ease postmenopausal symptoms, a property probably due to estrogenic and antioxidant effects. Pterocarpus soyauxii (P. soyauxii) is empirically used in Cameroon to treat among others primary and secondary amenorrhea. The aim of this study is to evaluate estrogenic and antioxidant activities of P. soyauxii heartwood aqueous extract in bilateral oophorectomized Wistar rats. Firstly, a characterization of the extract was carried out. For that, flavonoids, phenols, and tannins levels in P. soyauxii extract were evaluated by colorimetric assays and UHPLC-MS analysis was realized. In vitro antioxidant analysis of P. soyauxii was conducted using DPPH, ABTS, and FRAP assays. Secondly, 2 sets of pharmacologic tests were carried out. The results revealed that P. soyauxii aqueous extract contains, respectively, 229.42 ± 3.62 mg EAG/g, 63.42 ± 2.16 mg EQ/g, and 27.88 ± 0.23 mg ETA/g of polyphenols, flavonoids, and tannins. UHPLC-MS enabled identifying seven components including mono(2-ethylhexyl) phthalate, cembrene, 3′,5′-dimethoxy-4-stilbenol, and linoleic acid. DPPH, ABTS, and FRAP assays revealed that P. soyauxii extract possessed a high antioxidant activity with IC50 value of 730.20 µg/mL, 892.90 µg/mL, and 765.75 mEAG/g of extract, respectively. In the uterotrophic assay, P. soyauxii extract induced significant increase of fresh uterine weight, uterine and vaginal epithelial size, and mammary glands differentiation compared to Ovx control. In the postmenopausal model, compared to the sham control, vagina and uterine dystrophies were observed in Ovx rats treated with distilled water. P. soyauxii aqueous extract expressed estrogenic-like effects on vagina and did not affect uterine epithelial height compared with vehicle groups. On the back of these vaginotrophic effects, the extract displayed antiatherogenic properties by reducing (p < 0.001) AI and LDL cholesterol level as compared to Ovx control group. The extract at 200 mg/kg significantly prevented the increase of MDA (p < 0.01) level and decreased nitrites (p < 0.001) and GSH (p < 0.01) levels compared to Ovx rats. These beneficial effects are related at least in part to the presence of compound such as mono(2-ethylhexyl) phthalate, 3′,5′-dimethoxy-4-stilbenol, and linoleic acid. Overall, P. soyauxii aqueous extract exhibits estrogenic and antioxidant effects which can inhibit postmenopausal symptoms by providing vaginal stratification, improving lipid profile and insulin sensitivity, and reducing oxidative stress without side effects on the endometrium and mammary gland in 84-day Ovx rats.


Introduction
Menopause is a physiological state characterized by hypoestrogenism and lead to complications including hot flashes, tachycardia, vaginal dryness, urogenital atrophy, high visceral fat, increase of body weight, cardiovascular diseases, and disruption in sex steroid feedback on gonadotropin secretion [1]. e associated decline in estrogen-related antioxidant power results in the rapid development of menopausal symptoms such as cardiovascular diseases [2]. Oxidative stress is generated by an imbalance between the production of reactive oxygen species (ROS) and antioxidant defense system [3]. It is related to many risk factors of cardiovascular diseases such as obesity, atherosclerosis, insulin resistance, hypercholesterolemia, endothelial dysfunction, and vascular inflammation [4][5][6][7]. Hormone replacement therapy (HRT) is the main line for prevention and treatment of cardiovascular diseases in postmenopausal women. Despite its many benefits, HRT has adverse side effects such as breast and endometrial cancers [8]. People also use vitamins E and C to manage oxidative stress in postmenopausal symptoms [9]. Nevertheless, despite their protective effects, they lose their antioxidant power through oxidation [10]. In order to cope with these adverse side effects, research has focused on alternatives to HRT. e use of phytoestrogens attracted researchers over the last few years because of their antioxidant and preventive effects on such chronic diseases like cardiovascular diseases [11]. Indeed, they possess estrogenic-like activity and provide effective and secure alternative to HRT. Several medicinal plants are used empirically to cope with primary health problems and their estrogenic activity. Among them, Erythrina poeppigiana, Rheum rhaponticum, and Anthocleista schweinfurthii are reported to mimic estrogen activities in menopausal conditions [12][13][14]. Nevertheless, phytoestrogens are reported to induce gastrointestinal and endometrial hyperplasia side effects [15]; thus, additional preclinical and clinical studies are required to establish the safety profiles of plant extract.
Pterocarpus soyauxii Taub which is the subject of this work belongs to Fabaceae known as the African Padauk. Existent in Cameroon, Pterocarpus soyauxii (P. soyauxii) also returned to Nigeria, Gabon, and the Democratic Republic of Congo [16]. e wood and stem bark of P. soyauxii are empirically used to treat hypertension and diabetes. Ethnomedically, Pterocarpus species are used to treat urogenital pathologies and infertility. Moreover, pterostilbene identified in the extract of P. soyauxii has been reported to exhibit estrogenic activities in vitro [17,18]. Traditional healers of the Center Region of Cameroon assert that heartwood P. soyauxii maceration is used to treat primary and secondary amenorrhea and some menopausal symptoms. e main objective of the present study was to evaluate estrogenic and antioxidant activities of the aqueous extract of the heartwood of P. soyauxii in bilateral oophorectomized Wistar rats.

Plant Material.
e leaves and heartwood of Pterocarpus soyauxii Taub were harvested in Ngomedzap (Center Region, Cameroon) during the rainy season at 7 : 30 am on April 21, 2020. ese leaves were authenticated at the National Herbarium of Cameroon (HNC-IRA) by comparison with the specimen of omas O. W8175 deposited under the voucher number 56984HNC.

Extraction.
e collected Pterocarpus soyauxii heartwood pieces were air dried at room temperature for 30 days. 70 g of the powder obtained was macerated in 2 L of distilled water for 48 hours at room temperature. e macerate obtained was filtered using Whatman number 3 filter paper and the filtrate obtained was evaporated in a rotary oven at 45°C. is process made it possible to obtain 5.6 g of crude extract with a yield of 8%.

Determination of Study Doses.
e recommendations of the traditional healer allowed for obtaining from 400 mL of macerate 1.12 g of crude extract after drying. e dose used in rats was determined by multiplying the HED by 6.2 according to the method described by Nair [19], resulting in a dose of approximately 100 mg/kg. is dose was divided by 2 and then multiplied successively by 2 and 4 to obtain the doses of 50, 200, and 400 mg/kg. e dose of 300 mg/kg was obtained by averaging the doses of 200 and 400 mg/kg.

Phytochemical Analysis.
e analysis of the phytochemical composition of the aqueous extract of the heartwood of P. soyauxii was carried out by using quantitative assays. Indeed, the levels of flavonoids, polyphenols, and tannins were evaluated according to Broadhurst and Jones [20], Singleton and Rossi [21], and Zhishen et al. [22], respectively.

UHPLC-MS Analysis of P. soyauxii Extract.
UHPLC-MS analysis was used to identify the phytochemical profile of P. soyauxii extract in an attempt of standardization. To obtain high-resolution mass spectra of extract, a spectrometer (QTOF Bruker, Germany) equipped with a Heated Electrospray Ionization (HESI) source was used. e spectrometer operates in positive mode (mass range: 100-1500, with a scan rate of 1.00 Hz) with automatic gain control to provide high-accuracy mass measurements within 0.40 ppm deviation using sodium formate as calibrant. Spray voltage of 4.5 kV and capillary temperature of 200°C were used for assays. Nitrogen was used as sheath gas (10 L/min). e spectrometer was attached to an Ultimate 3000 ( ermo Fisher, Germany) UHPLC system consisting of LC-pump, diode array detector (DAD) (λ: 190-600 nm), autosampler (injection volume 10 µL), and column oven (40°C). e separation was performed using a Synergi MAXRP 100A (50 × 2 mm, 2.5 µ particle size) with a H 2 O (+0.1% HCOOH) (A)/acetonitrile (+0.1% HCOOH) (B) gradient (flow rate 500 µL/min, injection volume 5 µL). e sample was analyzed using a gradient program as follows: 95% A isocratic for 1.5 min, linear gradient to 100% B over 6 min, after 100% B isocratic for 2 min, the system returned to its initial condition (90% A) within 1 min and was equilibrated for 1 min. Based on the mass of compounds previously identified in Pterocarpus genus, identification of compounds was performed.

Animal Material.
Healthy female albino Wistar rats (8-10 weeks old) weighing 120-130 g were supplied by the production facility of the Animal Physiology Laboratory, University of Yaoundé 1 (Cameroon). All rats were housed in clean plastic cages at the room temperature (natural cycle). ey had free access to tap water and soy-free rat chow. e composition of animal diet was corn (60%), bone flour (3%), peanuts (5%), wheat (10%), fish flour (20%), salt (1%), and vitamin complex (Olivitazol) (1%). All experiments were conducted in accordance with the principles and procedures of the European Union on Animal Care (CEE Council 86/609) guidelines adopted by the Cameroon Institutional National Ethic Committee, Ministry of Scientific Research and Technology Innovation (Reg. number FWA-IRD 0001954).

DPPH Radical Scavenging Assay.
e DPPH free radical scavenging assay was carried out for the evaluation of the antioxidant activity. is assay measures the free radical scavenging capacity of the investigated extract. DPPH is a molecule containing a stable free radical. In the presence of an antioxidant, which can donate an electron to DPPH, the purple color typical for free DPPH radical decays and the absorbance change is measured at 517 nm. e antiradical activity of the plant extract was examined based on the scavenging effect of the stable DPPH free radical activity [23]. Briefly, 2 mL of DPPH (0.1 mM prepared in methanol) was introduced into a test tube containing 0.5 mL of extract (0.1 to 1 mg/mL). en the mixture was stirred well for 5 min and incubated in the dark for 60 min at room temperature (20°C). For the control tube, methanol was used in place of the extract. e reference used was ascorbic acid at concentrations of 0.1 mg/mL to 1 mg/mL. A calibration curve was drawn from this reference. e antioxidant activity of the plant extract was expressed as a percentage inhibition following the relationship: e IC 50 value (µg/mL) is the effective concentration at which DPPH radicals were scavenged by 50% and the value was obtained by interpolation from linear regression analysis.

Determination of Ferric-Reducing Antioxidant Power (FRAP) Assay.
e assessment of ferric-reducing antioxidant power (FRAP) was performed based on the ability of the tested substance to reduce ferric tripyridyl triazine (Fe III TPTZ) complex to ferrous form (intense blue color) at low pH by using a modified method of Benzie and Strin [24]. e solution of TPTZ (2,4,6-tri (2-pyridyl)-s-triazine) was obtained by diluting TPTZ (10 mM) in 10 ml of HCl (400 mM diluted with distilled water) and 10 mL of 10 mM iron chloride (FeCl 3 ) solution was prepared in distilled water. FRAP reagent was obtained by mixing 100 mL of acetate buffer (pH 3.6) with 10 ml of TPTZ solution and 10 ml of iron chloride solution. In test tubes containing 2 mL of FRAP reagent, 75 μL of sample (extract/catechin) was added and the mixture was stirred and incubated for 15 minutes. Optical densities were read at the wavelength of 593 nm against white.

ABTS Radical Scavenging Assay.
To determine ABTS radical scavenging assay, the method described by Re et al. [25] was used. Radical ABTS was obtained as follows: in an Erlenmeyer flask, 0.0384 g of ABTS and 0.00662 g of potassium persulfate (K 2 S 2 O 8 ) were weighed and then 10 mL of distilled water was added. e mixture was then solubilized for 5 min and incubated for 16 h at room temperature (20°C) in the dark before use. For the actual analysis, the ABTS solution was diluted with ethanol to an absorbance of 1.3 (±0.02) at 734 nm and stable at 30°C (initial OD). en in a test tube, 1.8 mL of this diluted ABTS solution and 0.2 mL of extract (1 mg/mL) were introduced and shaken well. e absorbance reading was taken at 734 nm and the values considered were those that remained stable at room temperature for approximately 1 minute. Ascorbic acid was used as the reference antioxidant at the same concentrations as extract. e results were expressed as a percentage of inhibition and calculated according to the following formula: is test was performed according to the protocol described by the OCDE [26]. Forty 8-to-10-week-old female Wistar rats were used. irty-five of these rats were ovariectomized using a dorsal approach and 5 more underwent simulation surgery and formed the sham-operated group. 14 days after ovariectomy, the ovariectomized animals (Ovx) were randomized and then divided into 7 groups of 5 animals each for oral Evidence-Based Complementary and Alternative Medicine administration of treatments for 3 days. e aqueous extract of Pterocarpus soyauxii heartwood was administered at doses of 50, 100, 200, 300, and 400 mg/kg and estradiol valerate (E 2 V) was administered at a dose of 1 mg/kg. A group of Ovx animals and the sham-operated group received distilled water at 10 mL/kg. At the end of the treatments, vaginal smears were carried out, and then the animals were sacrificed by decapitation under anaesthesia with diazepam and ketamine. e estrogen-dependent organs (uterus, vagina, and mammary gland) were removed and then fixed in 10% buffered formaldehyde solution for histological analysis. Before fixation, the uterus was weighed and part of it was homogenized in McEwen's solution for the evaluation of the level of uterine proteins. At last, the fresh uterine weight, uterine protein levels, and uterine and vaginal epithelial size as well as mammary gland alveolar and ductal differentiation were assessed.

Evaluation of Pterocarpus soyauxii Activities in a Postmenopausal Model.
irty-five rats were either shamoperated (sham) or bilaterally ovariectomized (Ovx) like in the first experiment. Eighty-four days later, animals were distributed in seven different groups (n � 5) and treated per os once daily for 28 consecutive days as follows: sham and an Ovx group received distilled water, the 4 others batches received, respectively, 1 mg/kg of E 2 V, and P. soyauxii aqueous extract at 100, 200, and 300 mg/kg. At day 21, insulin tolerance test was realized. Animals were weighted weekly. Twenty-four hours after the last administration (day 29) and following a 12 h of overnight fasting, animals were sacrificed under light anaesthesia. Blood samples were taken and centrifuged at 3500 g (15 min at 4°C) to obtain serum samples which were kept at −15°C for the determination of total cholesterol (TC), triglycerides (TG), and high-density cholesterol (HDL-C) levels. e concentration of lowdensity lipoproteins cholesterol (LDL-C) and very-lowdensity lipoproteins (VLDL) levels was calculated using the Friedewald equation [27]. Uterus, vagina, and aorta were dissected out and cleaned of all soft tissues. Prior to immersion-fixation of organs in the formaldehyde 10% buffered for histological analysis, they were weighted. A portion of aorta was homogenized in Mac even buffer to evaluate antioxidant parameters. Abdominal fat of each animal was weighted.

Vaginal Cellular Differentiation.
Vaginal smears were carried out at the end of the experiment using an eyedropper containing 10 µL of NaCl 0.9%, placed on ringed slides, fixed, and coloured with Papanicolaou method [28]. Cellular differentiation was observed under a light microscope at ×100 magnification.

Insulin Tolerance Test (ITT) in Ovariectomized Rats.
Prior to an ITT test, rats were fasted for 12 h. A single dose of insulin at a dose of 2 UI/kg was injected i.p. into each rat. Blood samples were taken from tail veins and the blood sugar levels were measured using the Accu-chek reactive strips at 0 (just before the insulin injection), 10, 20, 30, and 60 min after injection of insulin.

Determination of Relative Weight of Organs.
e relative fresh weight of uterus, abdominal fat, and aorta was calculated using the following formula according to [29]: 2.9. Assays for Lipid Profile. Serum total cholesterol (TC), triglycerides (TG), and HDL cholesterol (HDL-C) levels were assessed using commercial diagnostic kits Fortress, UK. e levels of LDL cholesterol (LDL-C) were assessed by using the following formula: LDL-Chol (mg/dL) � Chol -(TG/5) -HDL-Chol. VLDL cholesterol (VLDL-C) level was calculated with the following formula: TG/5 [27]. Atherogenic index (AI) was calculated as TC on HDL-C.

2.10.
Oxidative Stress Parameters Investigation. Malondialdehyde (MDA) and reduced glutathione (GSH) in aorta homogenate were determined using the methods described by Wilbur et al. [30] and Ellman [31], respectively, while the nitrites content was determined using the method described by Green et al. [32].

Histomorphometric Analysis of Uterine, Vaginal, and
Aorta Tissues. Aortic, uterine, vaginal, and mammary gland tissues after fixation (2 weeks) in formaldehyde 10% buffered were trimmed and dehydrated in alcohol of croissant gradient (70%, 80%, 90%, and 100% (3 baths)). After tissues were clarified in 2 baths of xylene (1h30 min per bath) and impregnated in liquid paraffin at 60°C (for 5 hours), uterine and vaginal epithelial sizes as well as intima and media heights were assessed from 5 μm sections of paraffin-embedded and haematoxylin-eosin stained uterine and vaginal tissues. Epithelial sizes were assessed on microphotographies obtained by using a light microscope (Leitz wetzlar Germany 513) connected with a digital camera celestron 44421 linked to a computer where images were transferred.

Statistical
Analysis. Data were expressed as mean-± standard error on mean. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by the Tukey post hoc test using GraphPad Prism 8.0.1. A value of p ˂ 0.05 was considered statistically significant.

In Vitro Antioxidant Activity.
According to the results obtained in Table 3, vitamin C exhibits greater anti-free radical activity than that of our extract. It had the smallest IC 50 against the DPPH and ABTS radicals. e inhibitory concentration 50 of vitamin C was 24.56 µg/mL while the aqueous extract of P. soyauxii was 730.20 µg/mL for the DPPH radical while it was 37.75 for vitamin C against 892.90 for the extract of PS for the radical ABTS. e concentration of FRAP was 765.75 mEAG/g.  Figure 2 represents effects of 3-day treatment with aqueous extract of P. soyauxii heartwood on the relative weight of the uterus (Figure 2(a)), the size of the uterine epithelium (Figure 2(b)), and uterine protein level (Figure 2(c)). e extract induced a significant increase in the relative weight of the uterus and the size of the uterine epithelium at the doses of 200 and 300 mg/kg with respective probabilities of p < 0.001 and p < 0.01 as compared to the ovariectomized animals treated with distilled water. Figure 2(c) also shows that the extract induced a significant increase in uterine protein levels (p < 0.05) at doses of 100 and 400 mg/kg. e doses of 200 and 300 mg/kg also significantly increased this uterine protein level (p < 0.001) in comparison to animals in the Ovx control.

Effect on the Size of the Vagina Epithelium.
According to Figure 3

Effects on the Uterine Epithelium.
e 28-day treatment with the extract of P. soyauxii did not have any effect on uterine epithelium at all doses. As shown by Figure 6, the uterine epithelium is simple cubic in all rats treated with the extract of P. soyauxii contrary to the one treated with estradiol valerate which is stratified. Figure 7, P. soyauxii at 100 mg/kg exhibits a cell differentiation and production of eosinophilic secretions according to Figure 8. Nevertheless, this differentiation is weaker than E 2 V one.  Figure 8(a) shows a significant increase in weight gain on days 7 and 14 of treatment, respectively, of p < 0.01 and p < 0.05 in ovariectomized rats compared with sham-operated animals. Likewise, Figure 7(b) shows a significant increase (p < 0.001) in the relative weight of abdominal fat in ovariectomized rats treated with distilled water compared to sham-operated rats. e extract of P. soyauxii at the dose of 300 mg/kg induced a significant decrease (p < 0.05) in weight gain on 14 and 21 days of treatment and the same effect (p < 0.001) on relative weight abdominal fat compared to Ovx animals. Extract at the dose of 200 mg/kg decreased only relative weight abdominal fat (p < 0.001) and weight gain on 21 days of treatment compared to Ovx animals.

Effects on Serum Glucose Levels during Insulin Resistance
Test. Figure 9 below shows a significant increase in serum glucose levels of (p < 0.01) and (p < 0.001), respectively, at 20 and 30 minutes in ovariectomized rats compared to sham-operated rats. e extract of P. soyauxii at the dose of 200 mg/kg resulted in a significant decrease in serum

Effects on Relative Weight of the Aorta and on Aortic
Protein. Figure 10 shows a significant decrease (p < 0.05) in the aortic protein.
ere was also a significant (p < 0.05) increase in the relative weight of the aorta in Ovx rats as compared to sham-operated rats (Figure 10(a)). e extract of P. soyauxii at the dose of 300 mg/kg resulted in a significant increase (p < 0.001) in aortic protein levels and a significant decrease (p < 0.001) in relative weight of this organ. P. soyauxii extract at the doses of 100 and 200 mg/kg significantly increased protein levels (p < 0.001) but had no effect on relative aortic weight.

Effects of P. soyauxii on Serum Lipid Levels.
Ovariectomy caused a substantial increase in the TC, TG, LDL-C, and VLDL-C levels ( Table 4) and also caused substantial decrease in HDL-C levels and increase in atherogenic index as compared to sham-operated control group (p < 0.001). In Ovx rats, treatment with 100 mg/kg/day of P.

Evidence-Based Complementary and Alternative Medicine
soyauxii induced a significant decrease in TG, LDL-C, and VLDL-C levels and atherogenic index (p < 0.05, p < 0.001, p < 0.001, and p < 0.05) but did not affect HDL-C and TC levels compared with the Ovx-control group. P. soyauxii at the dose of 200 mg/kg/day has significantly reduced TC, TG, LDL-C, and VLDL-C levels and atherogenic index (p < 0.01, p < 0.001, p < 0.001, p < 0.001 and p < 0.001) and increased HDL-C levels (p < 0.05) as compared to Ovx-control group. At the dose of 300 mg/kg/day, P. soyauxii extract significantly decreased TG, LDL-C, and VLDL-C levels and atherogenic index (p < 0.001, p < 0.001, p < 0.001, and p < 0.001) and significantly increased HDL-C levels (p < 0.05) as compared to Ovx group.

Effects of P. soyauxii on Oxidative Stress Status.
After 84 days, ovariectomy resulted in a significant increase (p < 0.001) in MDA level and a significant decrease (p < 0.001) in GSH and nitrite levels in the aorta. e plant at the dose of 100 mg/kg significantly increases the levels of nitrites but had no effect on the level of GSH and MDA. P. soyauxii extract at the dose of 200 and 300 mg/kg significantly decreased (p < 0.001, p < 0.01, resp.) the MDA level and likewise increased (p < 0.001, p < 0.01, resp.) GSH and nitrites levels ( Figure 11). Figure 12, after 84 days, ovariectomy induced leukocyte infiltration in aorta compared to sham-operated rats. Leucocyte infiltration was associated with a high ratio intima/ media (p < 0.001) in Ovx rats. e extract at the dose of 100, 200, and 300 mg/kg prevented leukocyte infiltration on aorta sections. Furthermore, P. soyauxii extract significantly reduced at all doses the ratio intima/media in Ovx rats compared to the control group treated with distilled water.

Discussion
e aim of the present study was to assess estrogenic and antioxidant activities of aqueous extract of P. soyauxii heartwood in a model of oophorectomy in Wistar rats. For this, a 3-day uterotrophic assay according to the protocol of the Organization for Economic Cooperation and development [26] was used to verify estrogenic potential of the extract in ovariectomized rats. In vitro antioxidant capacity of P. soyauxii heartwood was evaluated using protocols described by numerous authors [23][24][25]. en, an 84-day postoophorectomy model was used as described in numerous studies [13,14] to assess properties of the plant extract on postmenopausal cardiovascular disorders and aortic oxidative stress. Compared to Ovx control, results of the 3-day uterotrophic assay showed that aqueous extract of P. soyauxii heartwood at 200 and 300 mg/kg exhibited uterotrophic activities characterized by an increase of relative weight, epithelial height, and protein level of the uterus. Plant extract also leaded to vagina cornification and stratification correlated with an increase of vaginal epithelium height and the density of foliaceous cells. e uterotrophic assay also showed an increased eosinophilic secretion in lumen of mammary acini at 200 and 300 mg/kg. Indeed, it is histologically known that oophorectomy leads to atrophy of estrogen-dependent tissues like a decrease of vaginal and uterine epithelia heights; it also reduced eosinophilic secretions in the lumen of mammary acini [33][34][35]. Estrogenic activities of P. soyauxii extract could be due to flavonoids found in the plant extract (63.42 ± 2.16 quercetin equivalent). As a matter of fact, flavonoids represent the most potent phytoestrogen family [36]. e results of in vitro antioxidant assay showed that P. soyauxii extract has induced inhibition of the DPPH and ABTS free radical with IC 50 of 730.20 and 892.90 µg/mL, respectively. is antioxidant activity could be linked to the richness of the plant extract in phenolic compounds and linoleic acid according to LC-MS analysis. Indeed, linoleic acid can prevent the production of ROS by protecting membranes composed of 1-palmitoyl 2-linoleoyl phosphatidylcholine [37]. Besides, purslane leaves like P. soyauxii aqueous extract exhibit antioxidant-like properties due to phenols and linoleic acid [38]. ese estrogenic and antioxidant activities of P. soyauxii extract can be beneficial for deep hypoestrogenism observed in postmenopausal women. Indeed, oophorectomy leads to a reduction in circulating estrogen levels [39]. In rats, longterm oophorectomy is an appropriate experimental research model to study postmenopausal disorders [40] such as atrophy of estrogen-dependent tissues, cardiovascular risk factors primarily obesity, and dyslipidaemia [41,42]. In this study, compared to sham-operated group, 84-days oophorectomy resulted in an atrophy of estrogen-     : Effects of a 21-day treatment with P. soyauxii on serum glucose levels during insulin resistance test. 1 p < 0.05; 2 p < 0.01; 3 p < 0.001, significant difference compared to sham-operated control; a p < 0.05; b p < 0.01; c p < 0.001, significant difference compared to Ovx control; PS � P. soyauxii.  Figure 10: Effects of a 28-day treatment with P. soyauxii on fresh aorta weight (a) and aorta total protein levels (b). 1p < 0.05; 2 p < 0.01; 3 p < 0.001, significant difference compared to sham-operated control; b p < 0.01; c p < 0.001, significant difference compared to Ovx control.
vagina after 28-days treatment could be due to linoleic acid, known as promoting vaginal cornification [44]. is lack of long-term action of the plant extract on mammary gland and uterus could reflect a potential selective effect, useful for preventing estrogen-dependent cancers. In addition, LC-MS analysis revealed the presence in the plant extract of mono(2-ethylhexyl) phthalate (MEHP) and 3′,5′-dimethoxy-4-Stilbenol or pterostilbene which are known as selective estrogen receptor modulators (SERM). is differing agonist or antagonist effects at the estrogen receptor in different tissues of pterostilbene contained in the plant extract can prevent hormone responsive cancers [45,46].
In addition to genital atrophy, oophorectomy resulted in an increase of relative weight of abdominal fat, weight gain, and insulin resistance correlated with dyslipidaemia and an increased atherogenic index, similar to studies carried by Somayeh et al. [47] and Dzeufiet et al. [11]. Indeed, estrogen deficiency increases lipoprotein lipase activity, leading to blood fatty acids accumulation, and thus dyslipidaemia [48]. According to Wade [49], estrogen negatively regulates food intake and weight gain. ereby, increased body weight and abdominal fat in ovariectomized rats in this study could be due to high food intake and fat accumulation in adipose tissue [50]. It has also been established that free radical   Figure 11: Effects of a 28-day treatment with P. soyauxii on MDA (a), GSH (b), and nitrites (c) aorta levels. 1 p < 0.05; 2 p < 0.01; 3 p < 0.001, significant difference compared to sham-operated control; a p < 0.05; b p < 0.01; c p < 0.001, significant difference compared to Ovx control; PS � P. soyauxii; MDA � Malondialdehyde; GSH � reduced glutathione. oxidation of LDL cholesterol leads to atherosclerosis [11]. In the present study, the increase of atherogenic index is associated with oxidative stress in ovariectomized rats. Indeed, in addition to an increased LDL cholesterol, ovariectomized rats exhibited a high lipid peroxidation via an increase of MDA level. ey showed also a decrease of GSH concentration which characterized an oxidative stress. Compared to Ovx control, P. soyauxii extract significantly reduced abdominal fat weight and body weights, insulin resistance, and dyslipidaemia. ese effects could be due to the 3′,5′dimethoxy-4-stilbenol also called pterostilbene which was revealed by LC-MS analysis. Indeed, pterostilbene is known for its antiadipogenic and hypotriglyceridemic properties through inhibition of proliferation and differentiation of 3T3-L1 cells into adipocytes, fatty acids accumulation, and expression of Diacylglycerol O-acyltransferase 1 (DGAT1)   which supply triglycerides synthesis. In addition, pterostilbene is known to reduce expression of Peroxisome Proliferator Activated Receptors c (PPARc) involved in the starting of insulin resistance and dyslipidaemia [51,52]. Oophorectomy is associated with a high activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, leading to the formation of free radicals in the mitochondria. e high production of free radicals is a major contributor in the pathogenesis of atherosclerosis through LDL cholesterol oxidation [53], starting process of endothelial dysfunction leading to a decreased nitrogen monoxide levels in aorta. is atherogenic process constitutes an inflammatory state materialized in this work by leukocyte infiltration on aortic sections in Ovx control. e treatment with plant extract improved the oxidative status as well as leukocyte infiltration in the aorta at 100, 200, and 300 mg/kg. Indeed, pterostilbene is known to exert antioxidant and antiinflammatory activities.
ey decreased expression of NADPH oxidase and inactivation of NF-κB via a downregulation of Toll-like 5 receptors [54].

Conclusions
Sixteen-week bilateral oophorectomy induced postmenopausal symptoms in rats like vagina atrophy, dyslipidaemia, insulin resistance, weight gain, and oxidative stress. P. soyauxii aqueous extract contains mono(2-ethylhexyl) phthalate, cembrene, 3′,5′-dimethoxy-4-stilbenol, and linoleic acid. Furthermore, this extract is able to exhibit estrogenic and antioxidant activities which are probably responsible of the prevention of postmenopausal symptoms.
ere is a need to evaluate different pathways involved by secondary metabolites of this extract on postmenopausal symptoms.