Allium ampeloprasum var. Porrum (Alliaceae) Improves Metabolic and Reproductive Disorders Associated with Polycystic Ovary Syndrome in Wistar Rats

To provide scientific evidence of the efficacy of Allium ampeloprasum against female infertility, the effects of the aqueous extract of the said plant (AE) were evaluated in rats with letrozole-induced polycystic ovary syndrome (PCOS). AE was administered orally to PCOS rats at doses of 192, 384, and 768 mg/kg. The positive control was co-treated with clomiphene citrate (1 mg/kg) and metformin (200 mg/kg). Normal and negative controls received distilled water. The vaginal contents of rats were examined daily under a microscope before (7 days) and during treatment. Treatments were administered orally for 15 days, and then, 6 rats from each group were sacrificed for biochemical and histological analyses. The remaining rats were mated with males of proven fertility for 5 days. The daily examination of vaginal smears allowed the evaluation of fertility index. After parturition, additional fertility parameters were determined. Results showed that in PCOS rats, AE decreased body weight (p < 0.001), abdominal fat weight (p < 0.001), serum levels of LH (p < 0.001), testosterone (p < 0.001), total cholesterol (p < 0.05), and LDL cholesterol (p < 0.01). HDL cholesterol increased and atherogenic indices decreased (p < 0.001). The number of Graafian follicles and corpora lutea increased, while cystic (p < 0.001) and atretic (p < 0.05) follicles decreased. AE also decreased oxidative stress in the ovaries, restored the estrous cycle, induced uterine epithelial cell hypertrophy, and improved fertility. These effects were attributed to phenols, flavonoids, terpenoids, and anthocyanins present in AE. The overall results justify the traditional use of A. ampeloprasum against female infertility and suggest its potential use as a dietary supplement for PCOS patients.


Introduction
Infertility is considered a disease of the reproductive system characterized by the inability to achieve a clinical pregnancy after 12 months or more of regular unprotected sexual intercourse [1,2].10-15% of reproductive-aged couples are afected worldwide [3].Tis reproductive disease afects diferent areas of a couple's life as the ability to reproduce is closely linked to self-image, self-respect, and sexuality [4,5].Although male infertility contributes to about 50% of cases of global childlessness, infertility remains a woman's social burden [6].Te female reproductive function can be impaired by innate or acquired circumstances that afect the normal function of reproductive organs, illnesses, or psychological factors [4].One of the most common diseases afecting the function of female reproductive organs is polycystic ovary syndrome (PCOS).
PCOS is an endocrine and metabolic disorder afecting 5-20% of women of childbearing age [7].It is thought to be the most common cause of chronic hyperandrogenic anovulation and female infertility [8].Metabolic disorders associated with PCOS include obesity, insulin resistance, dyslipidemia, and type 2 diabetes mellitus [7,9].Hormonal changes occurring in PCOS women include hyperandrogenism, high levels of luteinizing hormone (LH), and hyperinsulinemia [9][10][11].Hyperandrogenism is an important criterion for the diagnosis of PCOS and plays a crucial role in the development and progression of PCOS [7].Chronic elevated levels of androgens induce the overproduction of gonadotropin-releasing hormone (GnRH) and LH by the hypothalamus and the anterior pituitary, respectively [12].In addition, hyperandrogenism stimulates pancreatic β-cells to release insulin [11].Te resulting hyperinsulinemia increases the bioavailability of androgens and insulin-like growth factor (IGF)-1 [13,14].IGF-1 and insulin potentiate the efect of LH on theca cells of the ovarian follicles to promote the production of androgens [15,16].High levels of insulin and IGF-1 also amplify the efect of LH on granulosa cells, causing premature diferentiation, follicle growth arrest, anovulation, and cyst formation [17,18].
Treatment for PCOS aims to relieve the clinical manifestations of PCOS and include antiandrogens, insulin sensitizers (e.g., metformin), or ovulation induction therapies (e.g., clomiphene citrate, gonadotropins, and laparoscopic ovarian drilling (LOD)) [19,20].Metformin and clomiphene citrate are often combined to increase ovulation and pregnancy rates [19,21].Gonadotropins are used when oral ovulation induction drugs fail to induce ovulation in patients with PCOS [19].LOD and medically assisted procreation (MAP) are generally performed in infertile PCOS patients who have not had a favorable outcome with drug treatments [19,22].However, current treatment plans still have many shortcomings [19,23] and some of them (gonadotropins and surgical interventions) are costly and time-consuming and their use requires intensive monitoring [19].It is therefore necessary to continue to develop more efective and accessible alternatives for better management of PCOS and its complications.
Previous studies suggest that medicinal plants could be potential alternatives in the management of PCOS [24,25].Several Cameroonian herbal medicines were found to correct PCOS-related symptoms in rats [26][27][28].Tese include Phyllanthus muellerianus [26], Myrianthus arboreus [27], and Milicia excelsa [28].Tese herbal essences have been shown to decrease serum testosterone and LH levels and to restore ovarian function and fertility in PCOS rats [26][27][28].To contribute to the popularization of the Cameroon pharmacopeia potentially efective against PCOS and associated infertility, we undertook to evaluate the efects of the aqueous extract of Allium ampeloprasum var.porrum (Alliaceae) in animals with PCOS.Tis plant, also called leek, is consumed in Cameroon as a vegetable or spice.It also has a medicinal virtue as it is traditionally recommended to cure female infertility [29].Previous work has reported the hypoglycemic, lipid-lowering, and antioxidant efects of this plant in diabetic rats [30], suggesting its ability to improve physiological disorders associated with PCOS.
In the present study, the aqueous extract of A. ampeloprasum was chemically screened for the detection of the classes of compounds that are present in it, in particular those endowed with antioxidant, estrogenic, hypoandrogenic, and hypolipidemic properties.Tese include phenols, favonoids, terpenoids, and anthocyanins.Indeed, the literature reports that phenolic compounds and terpenoids are endowed with anti-infammatory properties [31].Flavonoids are known to be estrogen-mimicking compounds [32,33].Polyphenols, such as favonoids and tannins, were found to have an antihyperglycemic efect [34].Soy isofavones were also found to improve endocrine (decrease in serum testosterone levels) and metabolic (decrease in serum lipid levels) status in women with PCOS [35,36].Finally, anthocyanins were found to have antioxidant activities [37].

Plant Collection and Authentication.
Te fresh samples of Allium ampeloprasum were purchased from the Dschang food market (West Region, Cameroon) in December 2020 and the plant was authenticated at the Cameroon National Herbarium under the number 67469/HNC.
Te aqueous extract of A. ampeloprasum was prepared following the method described by Edouard et al. [29].Briefy, 20 large fresh leeks (3 kg) were boiled in 5 L of distilled water for 20 minutes.After sieving and fltration on Whatman No. 4 flter paper, the fltrate obtained was freezedried (−45 °C) at the Institute for Medical Research and the Study of Medicinal Plant (Yaoundé, Cameroon), using a freeze dryer Christ Beta 1-8 LSCbasic (Osterode am Harz, Germany).Following this process, a total dry mass of 69.613 g of the aqueous extract of A. ampeloprasum was obtained and kept at 4 °C in an airtight container until use.

Chemical Screening of the Aqueous Extract of
A. ampeloprasum.To highlight the major classes of secondary metabolites of the aqueous extract of A. ampeloprasum, the analytical methods described by Harbone [38] were carried out.
Te presence of phenolic compounds in the aqueous extract of A. ampeloprasum was detected by suspending 0.01 g of extract in 3 ml of ethanol.Te mixture then received 3 drops of iron III chloride at 10% (V/V).At the end of this methodology, the appearance of a blue-violet or greenish color indicates the presence of phenols.
Te detection of favonoids was carried out using the Shinoda test.Briefy, 0.01 g of the extract was dissolved in 3 ml of methanol.Te mixture was treated with 0.05 g of magnesium chips and 3 drops of concentrated HCl.Te appearance of orange (favones), red (xanthones), and pink (favonols) colors indicate the presence of favonoids.For the detection of chalcones and aurones, 0.01 g of extract was mixed with 3 ml of concentrated sulfuric acid and then stirred for 5 minutes.Te presence of chalcones was characterized by the appearance of a red color, while aurones were marked by the appearance of a blue color.
Meyer test was used for the detection of alkaloids.Tus, 0.01 g of the extract was placed in a test tube in the presence of 3 ml of an aqueous solution of hydrochloric acid (50% V/ V).Te mixture was treated with 3 drops of "Meyer's reagent."Te formation of a white or yellowish color indicates the presence of alkaloids.
For the detection of saponins, 0.01 g of the extract was dissolved in 5 ml of distilled water and then boiled for 5 minutes.After cooling, each tube containing the dissolved extract was shaken vigorously for 30 seconds and then allowed to stand.Te appearance of persistent foam with a height of more than 1 cm characterizes the presence of saponins.

Biochemistry Research International
Te Liebermann-Burchard test was performed to detect the presence of triterpenoids and steroids.Tus, 0.01 g of the extract was dissolved in 3 ml of chloroform, and then, 3 ml of acetic anhydride acid was added and the mixture was cooled on ice for 3 minutes.Finally, a drop of concentrated sulfuric acid was added.Te presence of triterpenes was confrmed by the appearance of a purplish red color and that of steroids was confrmed by the appearance of a blue or green color.
Te presence of quinones was analyzed by dissolving 0.01 g of the extract in a mixture of 4 ml of ether-chloroform (1 : 1 V/V).Te resulting solution was treated with 4 ml of 10% (W/V) sodium hydroxide.Te appearance of a red color indicates the presence of quinones.
Tannins were analyzed by boiling 0.01 g of the extract in 5 ml of water for 5 minutes.After cooling, 5 ml of 2% NaCl (W/V) and 5 ml of 1% gelatin (W/V) were added to the preparation.Te appearance of a precipitate characterizes the presence of tannins.
Anthocyanins were detected by boiling 0.01 g of the extract in 5 ml of an aqueous solution of HCl (1% V/V).According to this protocol, the presence of anthocyanins is marked by the appearance of an orange coloration.
Te determination of the content of phenols, favonoids, and tannins in the aqueous extract of A. ampeloprasum was performed according to the methods described by Ramde-Tiendrebeogo et al. [39], Chang et al. [40], and Govindappa et al. [41], respectively.
Te total phenolic content of the aqueous extract of A. ampeloprasum was determined by the modifed Folin-Ciocalteu method as Ramde-Tiendrebeogo et al. [39] described.Tus, 100 μl of the Folin-Ciocalteu reagent dissolved in water 10 times and 80 μl of a 20% sodium carbonate reagent were added to 20 μl of the aqueous extract of A. ampeloprasum (2 mg/ml).Te mixture was shaken and incubated in a water bath at 20 °C for 30 minutes.Te absorbance was read at 765 nm.Te total phenolic content was expressed in milligrams of gallic acid equivalent/gram of extract (mgGAE/gE).
Te total favonoid content was determined using the aluminum chloride colorimetric method described by Chang et al. [40].Tus, 100 μl of the aqueous extract of A. ampeloprasum (2 mg/ml) was mixed with 50 μl of aluminum chloride (1.2%) and 50 μl of potassium acetate (120 mM).Te mixture was incubated at room temperature for 30 minutes and the absorbance was read at 415 nm.Total favonoid content was expressed in milligrams of quercetin equivalent/gram of extract (mgQE/ gE).
Te total tannin content was estimated using the Folin-Ciocalteu method described by Govindappa et al. [41].Tus, 100 μl of the aqueous extract of A. ampeloprasum (2 mg/ml) was mixed with 500 μl of the Folin-Ciocalteu reagent dissolved in water 10 times, 1000 μl of a 35% sodium carbonate reagent, and 8.4 ml of distilled water.Te mixture was shaken and incubated at room temperature for 30 minutes and the absorbance was read at 700 nm.Te total tannin content was expressed in milligrams of tannic acid equivalent/gram of the extract (mgTAE/gE).

Dose Determination. Te doses of the aqueous extract of
A. ampeloprasum used in this work were extrapolated from the dosage of Edouard et al. [29] against female infertility.Tese authors recommend drinking 3 glasses of the decoction of A. ampeloprasum daily, during the menstrual period.Considering a glass with a capacity of 250 ml, 3 glasses of the decoction of A. ampeloprasum, equivalent to a volume of 750 ml of that decoction, are therefore to be taken daily.After freeze-drying 750 ml of the decoction of A. ampeloprasum, a mass of 3.716 g of the aqueous extract of A. ampeloprasum was obtained.Tis mass was divided by 60 (average weight of a woman in kg [42]) to obtain the daily dose, which in this case was 61.93 mg/kg.To obtain the equivalent dose in rats, the human dose (61.93 mg/kg) was multiplied by 6.2 following the recommendations of Nair and Jacob [42].Tis method gave an equivalent dose of 384 mg/kg.Tis dose was divided and multiplied by 2 to obtain the minimum and maximum doses of 192 mg/kg and 768 mg/kg, respectively.

Animals.
Healthy young female Wistar rats 10-12 weeks old, with an average body weight of 150 g before the experiment, were obtained from the breeding facility of the Research Unit of Animal Physiology and Phytopharmacology (University of Dschang, Cameroon).Tey were housed in clean plastic cages at room temperature and lit by natural light.All rats had free access to a diet (a standard soyfree rat diet to eliminate exposure to exogenous estrogenic compounds) and tap water ad libitum.2.6.Experimental Protocol 2.6.1.PCOS Induction.PCOS was induced with letrozole as we described previously [28].Briefy, 55 female rats were force-fed with letrozole (a reversible aromatase inhibitor) at a dose of 1 mg/kg for 21 consecutive days.Control animals (n � 11) received distilled water instead during this period.Te onset of PCOS was confrmed by being overweight and by the blockage of the estrous cycle in the diestrus phase, as we reported previously [27,28].1).

Estrous Cycle Monitoring.
Te estrous cycle was examined by daily observation of vaginal smears under the microscope, for 25 days before the induction of PCOS and then, 7 days before the treatments, and throughout the treatment period, as we reported previously [27,28].
2.6.4.Sacrifce and Collection of Blood and Tissues.At the end of the treatment period, 6 rats per group were sacrifced under anesthesia (diazepam (10 mg/kg) and ketamine (50 mg/kg) administered intraperitoneally).Blood, collected (in dry tubes) from each rat by catheterization of the abdominal artery, was centrifuged at 3000 rpm at room temperature for 15 minutes.Te serum obtained was stored at −20 °C for biochemical analyses.Abdominal fat, ovaries, and uterus were collected and weighed.Te left ovary of each rat was homogenated in 0.9% NaCl (10% W/V) and then centrifuged at 3000 rpm at room temperature for 15 minutes.Te supernatant was collected and stored at −20 °C for biochemical analyses.Te right ovary and uterus were fxed in 10% formalin for histological analysis.

Measurement of Serum Lipid Levels.
Serum levels of triglycerides (TG), total cholesterol (TC), and HDLcholesterol (HDL-C) were measured by a fully automated enzymatic method using reagent kits purchased from SIGMA Diagnostics (Budapest, Hungary).Atherogenic indexes were determined using the formulae described by Anthony et al. [43] (CT/HDL-C) and Dobiášová and Frohlich [44] (Log (TG)/HDL-C).

Measurement of Serum Hormone Levels.
Te hormonal profle was examined by measuring serum LH, testosterone, and estradiol levels.Serum levels of these hormones were assessed by ELISA tests using reagent kits purchased from Calbiotech (El Cajon, California, USA).Te absorbance of calibrators and the specimen was determined using the ELISA microplate reader, the Multiskan Ascent plate reader, purchased from MTX Lab Systems, Inc. (Bradenton, USA).Hormone concentrations were evaluated by using calibration curves established by the calibrators supplied with the kits.

Measurement of Oxidative Stress-Related Parameters.
Te levels of malondialdehyde (MDA) in the ovary homogenates were assessed using the method of Wilbur et al. [45].Te principle of this assay stipulates that MDA or MDA-like substances and thiobarbituric acid react at 90-100 °C with the production of a pink pigment having an absorption maximum at 532 nm.Te ovarian levels of MDA were determined using the following formula: where [MDA] � concentration of MDA (nM/mg of tissue); DO � absorbance of the sample-absorbance of the reaction blank; ε � molar extinction coefcient (1.56 × 10 5 M −1 cm −1 ); l � path length (1 cm); and m � mass of the ovary used for the preparation of homogenates (mg).
Te levels of proteins in the ovary homogenates were assessed using a reagent kit purchased from Randox (London, UK) and following the manufacturer's instructions.Tis parameter was used to assess ovary levels of antioxidant enzymes (superoxide dismutase, catalase, and total peroxidases) as the amount of each of these enzymes in the ovary homogenates was assessed relative to the total protein content in this tissue.
Te measurement of superoxide dismutase (SOD) activity is based on the ability of SOD to inhibit the autooxidation of adrenaline to adrenochrome in an alkaline medium.SOD activity in the ovaries was assessed according to the method described by Habbu et al. [46] and the percentage of adrenaline inhibition was calculated as follows: where %I � percentage inhibition of adrenaline oxidation; DO sample : average between the absorbance at 30 seconds and the absorbance at 90 seconds of the sample; and DO blank : absorbance of the reaction blank.
Considering that 50% inhibition corresponds to one unit, the activity of SOD was expressed in units per amount of proteins as follows: where A � activity of SOD (in unit/mg of total proteins) and p � ovarian total protein levels (mg/dl).Catalase activity in the ovaries was estimated by the method described by Habbu et al. [46].Te measurement of the activity of this enzyme is based on the decomposition of H 2 O 2 into water by catalase (present in the sample).Te concentration of undecomposed H 2 O 2 was evaluated using a calibration curve established from a standard solution (50 mM H 2 O 2 ).Ovary catalase activity was determined as follows: where A � catalase activity (mole of H 2 O 2 /min/mg of total proteins); DO � absorbance of the sample-absorbance of the reagent blank; a � slope of the calibration curve; t � reaction time (1 min); and p � ovarian total protein level (mg/dl).Measurement of ovarian levels of total peroxidases was carried out following the method described by Habbu et al. [46].Briefy, 1 ml of KI solution (10 mM) and 1 ml of sodium 4 Biochemistry Research International acetate (40 mM) were added to 0.5 ml of the ovarian homogenates.After mixing, the absorbance of potassium iodide was read at 353 nm.Ten, 20 μl of H 2 O 2 (15 mM) was added and the change in the absorbance in 5 min was recorded.Te amount of total peroxidases in the ovaries was deduced by the law of Beer-Lambert [47] as follows: where C � concentration of ovarian total peroxidases (mM/ mg of total proteins); DO � optical density; ε � molar extinction coefcient (11.3 M −1 cm −1 ); l � path length (1 cm); and p � ovarian total protein level (mg/dl).

Fertility Test.
After sacrifce, the fve remaining rats in each group were mated with males of proven fertility for 5 consecutive days, the average duration of an estrous cycle [27,28].Te daily (7:30 a.m. to 8:30 a.m.) examination of vaginal smears allowed for determining the fertility index and the frst day of gestation.Te latter was fxed on the day the spermatozoa were observed in the vaginal smears (positive vaginal smears) as we reported previously [27,28].Rats were then followed up until parturition.After parturition, additional parameters of fertility were determined using the formulae we described previously [27].
(i) Fertility index: 100 × (number of vaginal smearpositive rats/number of mated rats) Biochemistry Research International (ii) Quantum gestation: 100 × (number of gestational rats/number of vaginal smear-positive rats) (iii) Gestation index: 100 × (number of rats with viable fetuses at birth/total number of gestational rats) (iv) Average litter size: total number of pups/number of gestational rats 2.9.Histological Analyses.Te histological analyses of the ovaries and uterus were performed on 5 μm sections of parafn-embedded tissues.Tese sections have been stained with hematoxylin and eosin.Histomorphological changes in these tissues were assessed on photomicrographs using a Scientico STM-50 microscope.Te latter was equipped with a Celestron MA411101 camera connected to a computer where the image was transferred and analyzed with Image J1.3 software.

Statistical Analysis.
Data are presented as mean-± standard error of the mean (SEM), except for the estrous cycle data which instead show the most represented stage of the estrous cycle in each group as we reported previously [27,28].GraphPad Prism 5.03 software was used to perform data analysis.Data from the control and treated groups were compared using a one-way analysis of variance (ANOVA) followed by Tukey's post-test for multiple comparisons.Diferences were considered signifcant at p < 0.05.

Efects of the Aqueous Extract of A. ampeloprasum on the
Body Weight.At the beginning of the experiment (week 1), the body weight of animals to be used as control and that of animals intended to receive letrozole (1 mg/kg) were comparable: 132.54 ± 4.88 g and 133.02 ± 3.21 g, respectively.During the experiment, a gradual increase in the body weight was observed in both groups.In the control group, the body weight was 132.54 ± 4.88 g at week 1.Tis parameter increased to 139.33 ± 2.89 g at week 2 (5% increase), 140.2 ± 3.22 g (6% increase) at week 3 and 147.30 ± 4.06 g (11% increase; p < 0.05) at week 4.In animals receiving letrozole, the body weight increased from 133.02 ± 3.21 g at week 1 to 145.22 ± 1.63 g (9% increase; p < 0.001) at week 2, 152 .77± 1.72 g (15% increase; p < 0.001) at week 3, and 160.25 ± 1.66 g (21% increase; p < 0.001) at week 4.In comparison with the control group, the body weight of animals receiving letrozole increased by 4% (p < 0.05) after one week of letrozole administration and 9% after two (p < 0.01) and three (p < 0.05) weeks of letrozole administration (Figure 2(a)).
During the 15 days of treatment, the body weight of the negative control remained higher than that of the normal control.Clomiphene citrate and metformin reduced animal body weight by 6%, 12% (p < 0.001), and 11% (p < 0.01) after 5, 10, and 15 days of treatment, respectively, compared to the negative control.Te same observation was made in animals treated with the aqueous extract of A. ampeloprasum at the doses tested, in comparison with the negative control (Figure 2 Figure 2(c) shows that the relative weight of abdominal fat increased by 145% (p < 0.01) in the negative control group where the abdominal fat weighed 1.035 ± 0.214 g/ 100 g BW, in comparison with the normal control where this parameter was on average 0.423 ± 0.031 g/100 g BW.Te combined administration of clomiphene citrate and metformin reduced the relative weight of abdominal fat by 65% (p < 0.001) as compared to the negative control.A similar efect was observed with the aqueous extract of A. ampeloprasum which reduced the relative weight of abdominal fat by 77% (p < 0.001) at the dose of 192 mg/kg, 30% at the dose of 384 mg/kg, and 19% at the dose of 768 mg/ kg.

Efects of the Aqueous Extract of A. ampeloprasum on
Lipidemia.Serum triglyceride (TG) levels increased by 106% (p < 0.001) in the negative control as compared to those of the normal control.Te combined administration of clomiphene citrate and metformin decreased this parameter by 26% (p < 0.001) in comparison with the negative control.3(a)).Figure 3(b) shows that serum total cholesterol levels increased by 63% (p < 0.001) in the negative control relative to those of the normal control.Te co-treatment with clomiphene citrate and metformin reduced this parameter by 20% (p < 0.01) as compared to the negative control. A. ampeloprasum induced a similar efect by decreasing serum total cholesterol levels at the doses of 192 mg/kg (27% decrease; p < 0.001) and 768 mg/kg (15% decrease; p < 0.05), in comparison with the negative control.
Serum HDL cholesterol levels decreased by 77% (p < 0.001) in the negative control relative to those of the normal control.Te co-treatment with clomiphene citrate and metformin increased this parameter by 90% (p < 0.05)  Biochemistry Research International as compared to the negative control.A similar increase in serum HDL-cholesterol levels was observed with A. ampeloprasum at the doses tested: a 50% increase at 192 mg/kg, 56% increase at 384 mg/kg, and an 11% increase at 768 mg/kg, in comparison with the negative control (Figure 3(c)).Te atherogenic index of plasma (AIP) was 0.086 ± 0.021 in the normal control versus 1.020 ± 0.027 in the negative control, an increase of 1092% (p < 0.001).After co-treatment with clomiphene citrate and metformin, the AIP value decreased by 40% (p < 0.001) in comparison with the negative control.Te aqueous extract of A. ampeloprasum induced a similar efect (decrease in the AIP value) at the doses tested: 27% decrease at 192 mg/kg (p < 0.001), 49% decrease at 384 mg/kg (p < 0.001), and 11% decrease at 768 mg/kg, in comparison with the negative control (Figure 4(a)).
Te total-cholesterol/HDL-cholesterol (TC/HDL-C) ratio went from 1.262 ± 0.093 in the normal control to 8.726 ± 0.453 in the negative control, an increase of 592% (p < 0.001).Te combined administration of clomiphene citrate and metformin decreased this ratio by 57% (p < 0.001), in comparison with the negative control. A. ampeloprasum also decreased the TC/HDL-C ratio by 52% (p < 0.001) at the dose of 192 mg/kg, 38% (p < 0.001) at the dose of 384 mg/kg, and 24% (p < 0.001) at the dose of 768 mg/kg, in comparison with the negative control (Figure 4(b)).

Efects of the Aqueous Extract of A. ampeloprasum on the
Course of the Estrous Cycle.Te average duration of the estrous cycle in the normal control was 5 days and was made up of four stages including proestrus, estrus, metestrus, and diestrus.While each of the frst three phases of the estrous cycle varied over 24 hours, the last phase (the diestrus) spanned two days (Figure 5(a)).In the negative control, the estrous cycle was blocked in the diestrus phase throughout the experiment (Figure 5(b)).In animals co-treated with clomiphene citrate and metformin, the cyclic occurrence of the diferent phases of the estrous cycle from the proestrus phase, resumed after two days of treatment (Figure 5(c)).With A. ampeloprasum, the proestrus appeared after 14 days

Efects of the Aqueous Extract of A. ampeloprasum on the
Hormonal Profle.Serum LH levels went from 21.474 ± 1.741 mIU/ml in the normal control to 120.522 ± 6.087 mIU/ml in the negative control, an increase of 461% (p < 0.001).Tis parameter decreased by 61% (p < 0.001) after the combined administration of clomiphene citrate and metformin, in comparison with the negative control.A similar decrease in serum LH levels was observed with the aqueous extract of A. ampeloprasum at the doses tested: 74% decrease at the dose of 192 mg/kg (p < 0.001), 76% decrease at the dose of 384 mg/kg (p < 0.001), and 67% decrease at the dose of 768 mg/kg (p < 0.001), in comparison with the negative control (Figure 6(a)).Serum testosterone levels went from 56.256 ± 4.926 ng/ ml in the normal control to 140.225 ± 5.012 ng/ml in the negative control, an increase of 149% (p < 0.001).Clomiphene citrate and metformin lowered the mean value of serum testosterone levels by 69% (p < 0.001) in comparison with the negative control.A similar efect (decrease in serum testosterone levels) was induced by the aqueous extract of A. ampeloprasum at the doses tested: 30% decrease at the dose of 192 mg/kg (p < 0.001), 52% decrease at the dose of 384 mg/kg (p < 0.001), and 43% decrease at the dose of 768 mg/kg (p < 0.001), in comparison with the negative control (Figure 6(b)).
Serum estradiol levels decreased from 395.452 ± 25.498 pg/ ml in the normal control to 75.030 ± 6.927 pg/ml in the negative control, a decrease of 81% (p < 0.001).Tis parameter increased by 74% (p < 0.05) after the combined administration of clomiphene citrate and metformin, in comparison with the negative control.Following treatment with A. ampeloprasum, serum estradiol levels did not change statistically compared to the negative control, although a slight increase in this parameter was observed at the doses tested: an increase of 12% at 192 mg/kg, 13% increase at 384 mg/kg, and 62% increase at 768 mg/kg (Figure 6(c)).

Efects of the Aqueous Extract of A. ampeloprasum on the Relative Weight of the Ovaries and Uterus and the Uterine
Epithelial Height.Figure 7(a) shows that the relative weight of the ovaries increased by 59% (p < 0.001) in the negative control, relative to the normal control.After co-treatment with clomiphene citrate and metformin, the relative weight of the ovaries decreased by 38% (p < 0.001), in comparison with the negative control.A similar efect was induced by the aqueous extract of A. ampeloprasum as it decreased the relative weight of the ovaries by 21% (p < 0.05) at the dose of 192 mg/kg, 39% (p < 0.001) at the dose of 384 mg/kg, and 35% (p < 0.001) at the dose of 768 mg/kg, as compared to the negative control.
Te graphic representation of the relative weight of the uterus shows that this parameter decreased by 32% (p < 0.05) in the negative control, as compared to the normal control.After co-treatment with clomiphene citrate and metformin, the relative weight of the uterus increased by 75% (p < 0.001), in comparison with the negative control.A similar increase in the relative uterine weight was observed with the aqueous extract of A. ampeloprasum at the doses of 192 mg/kg (30% increase) and 384 mg/kg (32% increase), in comparison with the negative control (Figure 7(b)).
Te uterine epithelial height decreased by 54% (p < 0.001) in the negative control, as compared to the normal control.Te combined administration of clomiphene citrate and metformin increased this parameter by 323% (p < 0.001), in comparison with the negative control.Te aqueous extract of A. ampeloprasum induced a similar efect (increase in the uterine epithelial height) at the doses tested: 60% increase at the dose of 192 mg/kg, 82% increase Biochemistry Research International at the dose of 384 mg/kg (p < 0.05), and 28% increase at the dose of 768 mg/kg, in comparison with the negative control (Figure 7(c)).

Efects of the Aqueous Extract of A. ampeloprasum on the
Ovarian Follicle Growth.Te growth of ovarian follicles was analyzed on photomicrographs of ovarian sections (Figure 8).Te analysis was performed by counting the diferent types of ovarian follicles.Tese include primary, secondary, tertiary, and Graafan follicles, corpora lutea, and cystic and atretic follicles.In the negative control, the number of primary, secondary, tertiary and Graafan follicles and corpora lutea decreased by at least 39%, as compared to the normal control.In contrast, the number of cystic and atretic follicles increased by at least 80% in the negative control, relative to the normal control.Clomiphene citrate and metformin reversed the efects observed in the negative control as they increased (at least 53% increase) the number of primary, secondary, tertiary, and Graafan follicles and corpora lutea.Cystic and atretic follicles decreased by 73% (p < 0.001), as compared to the negative control.
A. ampeloprasum induced efects similar to those induced by clomiphene citrate and metformin at the doses tested (Table 3).Biochemistry Research International 895.299 ± 67.034 nM/mg of tissues in the negative control, an increase of 68% (p < 0.001).Te co-treatment with clomiphene citrate and metformin decreased ovarian MDA levels by 42% (p < 0.001), as compared to the negative control.A similar decrease in this parameter was observed with the aqueous extract of A. ampeloprasum at the doses tested: 21% decrease at the dose of 192 mg/kg, 28% decrease at the dose of 384 mg/kg (p < 0.05), and 38% decrease at the dose of 768 mg/kg (p < 0.01), in comparison with the negative control (Figure 9(a)).

Efects of the Aqueous Extract of A. ampeloprasum on
Concerning the ovarian superoxide dismutase (SOD) level, results show that this parameter did not vary significantly between the normal control (117.670± 3.445 U/mg of proteins) and the negative control (118.164± 1.421 U/mg of proteins).Te diferent treatments administered (clomiphene citrate and metformin, and the aqueous extract of A. ampeloprasum) did not signifcantly afect ovarian SOD level which remained close to that of the negative control (Figure 9(b)).
Results on the ovarian catalase level show that this parameter did not vary signifcantly between the normal control (4.020 ± 0.137 mM/mg of proteins) and the negative control (3.818 ± 0.126 mM/mg of proteins).Clomiphene citrate and metformin, as well as the aqueous extract of A. ampeloprasum, did not signifcantly afect the ovarian catalase level which remained close to that of the negative control (Figure 9(c)).
Ovarian total peroxidase levels were 28.925 ± 1.195 mM/ mg of proteins in the normal control versus 31.638± 2.434 mM/mg of proteins in the negative control, an increase of 9%.Te combined administration of clomiphene citrate and metformin reduced this parameter by 15%, in comparison with the negative control.Te aqueous extract of A. ampeloprasum induced a similar efect by decreasing ovarian total peroxidase levels by 33% (p < 0.01) at the dose of 192 mg/kg, 20% at the dose of 384 mg/kg, and 36% (p < 0.001) at the dose of 768 mg/kg, as compared to the negative control (Figure 9(d)).Biochemistry Research International

Efects of the Aqueous Extract of A. ampeloprasum on the
Fertility of Rats with PCOS.Te fertility index was 80% in the normal control versus 0% in the negative control.After the combined administration of clomiphene citrate and metformin, this index increased to 100%.Te aqueous extract of A. ameploprasum induced a similar efect (increase in the fertility index) at the doses tested: 60% increase at the dose of 192 mg/kg, and 80% increase at the doses of 384 and 768 mg/ kg (Table 4).Te quantum gestation as well as the gestation index was 100% in the normal control versus 0% in the negative control.Te co-treatment with clomiphene citrate and metformin, as well as the aqueous extract of A. ampeloprasum, increased each of these parameters to 100% (Table 4).
Te total number of pups per group was 0 in the negative control versus 35 in the normal control.After the cotreatment with clomiphene citrate and metformin, the total number of pups was 34.Te aqueous extract of A. ampeloprasum also increased this parameter at the doses tested: 18 pups at the dose of 192 mg/kg, 30 pups at the dose of 384 mg/kg, and 25 pups at the dose of 768 mg/kg (Table 4).
Te average litter size was 8.75 pups per female in the normal control versus 0 pups per female in the negative control.Te co-treatment with clomiphene citrate and metformin raised this parameter to a value of 6.8 pups per female.A similar observation was made with the aqueous extract of A. ampeloprasum where the average litter size was 6 pups per female at the dose of 192 mg/kg, 7.5 pups per female at the dose of 384 mg/kg, and 6.25 pups per female at the dose of 768 mg/kg (Table 4).

Discussion
Te present study aimed to evaluate the efects of the aqueous extract of A. ampeloprasum on some metabolic and reproductive disorders associated with PCOS in female Wistar rats.PCOS was induced with a reversible aromatase inhibitor (letrozole) as we reported previously [28].Consistent with our previous results [27,28], the daily administration of the dose 12 Biochemistry Research International of 1 mg/kg of letrozole for 21 consecutive days induced a PCOS rat model possessing similar metabolic (overweight and dyslipidemia) and reproductive (hyperandrogenism, high LH levels, ovarian cysts, and infertility) disorders as seen in PCOS women.To induce this phenotype, letrozole acts by preventing the conversion of androgen to estrogens [48].
A. ampeloprasum reversed letrozole-induced efects as it decreased animal body weight and abdominal fat accumulation.Tis efect was associated with decreased serum testosterone levels.Indeed, androgen is known to induce visceral obesity by stimulating the diferentiation of pre-adipocytes into adipocytes preferentially in the abdomen [49].Terefore, by decreasing testosterone levels, A. ampeloprasum inhibited lipogenesis signaling pathways (inhibition of AMPK and diferentiation of pre-adipocytes into adipocytes), thereby reducing fat accumulation and consequently body weight.Te signifcant diference between control animals and animals treated with A. ampeloprasum in terms of body weight may be the result of an inhibition of weight gain, while animals in the control groups continued to grow.Also, since hyperandrogenism indirectly inhibits AMPK activity by stimulating hyperinsulinemia [11,50], the efects induced by A. ampeloprasum suggest an enhanced AMPK activity, hence the reduction of serum triglyceride levels in A. ampeloprasum-treated animals, as triglycerides are synthesized by the esterifcation of fatty acids to glycerol.Results on the other lipid parameters show that A. ampeloprasum decreased serum total cholesterol levels and increased serum HDL-cholesterol levels.Te mechanism through which A. ampeloprasum induced  these efects may include (i) the reduction of the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and/or cholesterol breakdown and (ii) the increase in the activity of lecithin: cholesterol acyl transferase (LCAT) and/or the inhibition of the hepatic HDLcholesterol uptake.HMG-CoA reductase is the ratelimiting enzyme involved in the biosynthesis of cholesterol in the liver [52].Its activity is inactivated by AMPK as cholesterol synthesis was found to be signifcantly suppressed in response to AMPK activators [52].Tis at least partly supports the hypothesis that A. ampeloprasum stimulates AMPK activity, thereby suppressing cholesterol synthesis.Cholesterol breakdown could be attributed to favonoids present in A. ampeloprasum, as this class of compounds has been shown to increase hepatic expression of the cytochrome P450 gene 7A1 [53] which codes for cholesterol-7α-hydroxylase, an enzyme that stimulates the conversion of cholesterol into bile acids in the liver [54].
LCAT is a plasma enzyme that esterifes peripheral tissue cholesterol onto nascent pre-β HDL (small complexes formed by the association of apolipoprotein A-1 (apoA1) (produced by the liver) with hepatic phospholipids and cholesterol via interaction with the ATP-binding cassette transporter A1 (ABCA1)), thus forming mature (larger) α-HDL, the major HDL species found in plasma [55][56][57].LCAT defciency prevents the formation of mature HDL, leading to an overall decrease in HDL levels [55,57].Terefore, the increase in serum HDL-cholesterol levels in rats treated with A. ampeloprasum indicates that this vegetable (or leek) would have promoted the hepatic production of apoA1 and increased the activities of ABCA1 and LCAT.Tis efect could be attributed to the favonoids present in the aqueous extract of A. ampeloprasum, because in our previous work, it was shown that isofavones promote the formation of HDL as they leaned the Apoa1/Scarb1 balance in favor of Apoa1 [53].Apoa1 and Sacrb1 are estrogensensitive genes associated with HDL synthesis (Apoa1; a gene coding for apolipoprotein A1) and clearance (Scarb1; a gene coding for SRB1, a receptor promoting the rapid clearance of HDL-cholesterol and its transport into bile) [53,58].Te improvement in lipid metabolism induced by the aqueous extract of A. ampeloprasum is consistent with the observations of Rahimi-Madiseh et al. [30] who reported the ability of the hydroalcoholic extract of Iranian leek to correct dyslipidemia in diabetic rats.Tis improvement in lipid metabolism contributed to reducing the atherogenic risk.
Regarding reproductive disorders associated with PCOS (hyperandrogenism, high serum LH levels, blockage of the estrous cycle in the diestrus phase, anovulation, ovarian cysts, and infertility), our results showed that the aqueous extract of A. ampeloprasum stimulated the resumption of the estrous cycle, improved ovarian dynamics, decreased serum LH and testosterone levels, and slightly increased serum estradiol levels.Te decrease in serum testosterone levels suggests that A. ampeloprasum reversed the inhibitory efect of letrozole on aromatase activity.However, the nonsignifcant change in estradiol levels somewhat refutes this hypothesis and suggests that A. ampeloprasum decreased LH and testosterone levels through a diferent pathway than aromatase activation.Te following hypothesis could be put forward to try to explain the decrease in LH and testosterone levels: Indeed, by inhibiting aromatase activity, letrozole causes an accumulation of androgens leading to hyperandrogenism.Te latter causes hyperinsulinemia [11] which in turn increases glutamate levels in the brain [12].High levels of this excitatory neurotransmitter overstimulate hypothalamic GnRH neurons.Te resulting excess release of GnRH causes the pituitary gland to release LH accordingly [12].A. ampeloprasum could have acted at the central level by promoting glutamate reuptake and therefore reducing its excitatory efects on hypothalamic GnRH neurons, thereby reducing pituitary release of LH and consequently the ovarian production of testosterone.Tis hypothesis paves the way for further research that will elucidate the mechanism by which A. ampeloprasum decreases serum LH and testosterone levels in PCOS rats.
Te reduction of serum testosterone levels induced by A. ampeloprasum contributed to the restoration of ovarian dynamics characterized by an increase in the number of tertiary and Graafan follicles (an indicator of follicle development and maturation) and corpora lutea (an indicator of ovulation), and a decrease in the number of cystic and atretic follicles.Literature reports that granulosa cell apoptosis is responsible for the increased number of atretic follicles in PCOS rats.Granulosa cells undergo apoptosis due to low estradiol levels associated with letrozole-induced aromatase inhibition [59].Tus, A. ampeloprasum would have induced an estrogenic-like efect by preventing granulosa cells from atresia, hence the increase in tertiary and Graafan follicles.Tis hypothesis of the estrogenic potential of A. ampeloprasum is confrmed by the hypertrophy of the uterus of treated animals.It is known that the hypertrophy of this main target of estrogens is mediated by the estrogen receptor alpha [60].Te favonoids present in this plant could be responsible for its estrogenic efect, as isofavones are known to be estrogen receptor ligands and therefore estrogen-mimicking compounds [32,33,53].
Additionally, cellular apoptosis is known to be initiated by reactive oxygen species (ROS) whose elevated levels indicate oxidative stress [61].Oxidative stress markers were found to be elevated in the serum and ovaries of animals with PCOS [27,59,62,63].In agreement with these reports, our results showed elevated levels of ovarian malondialdehyde (MDA, indicator of lipid peroxidation) associated with a slight increase in total peroxidases in the negative control.Te latter would have increased to reduce the damage caused by oxidative stress on granulosa cells.Tus, low estradiol levels increased oxidative stress in the ovaries of animals with PCOS.Tis could be the origin of the increase in the process of follicle atresia observed in the negative control.Te aqueous extract of A. ampeloprasum reversed this efect by decreasing ovarian MDA levels and increasing the levels of total peroxidases.Indeed, substances with antioxidant properties have been reported to decrease ROS production [62], lipid peroxidation [59,62,63], and follicle atresia [59,63].Tese data support the antioxidant properties of A. ampeloprasum reported by Rahimi-Madiseh et al. [30] in diabetic rats and could be attributed to anthocyanins found in A. ampeloprasum.
Tese benefcial efects of A. ampeloprasum could be attributed to its chemical composition which revealed the presence of phenols, favonoids, terpenoids, and anthocyanins.Tese classes of compounds were found to have antioxidant, hypolipidemic, hypoandrogenic, and estrogenic properties [31][32][33][35][36][37].Additionally, the corrective effects of A. ampeloprasum on hormonal profle and ovaries created a favorable physiological environment for fertility.Indeed, our results showed an increase in gestation index, quantum gestation, fertility index, and average litter size in animals treated with A. ampeloprasum.Tis confrms at least in part the traditional use of this plant against female infertility [29].
In conclusion, the aqueous extract of A. ampeloprasum improved PCOS-impaired physiological parameters in rats through its hypoandrogenic, hypolipidemic, estrogenic, and Biochemistry Research International antioxidant abilities.Te corrective efects of A. ampeloprasum on PCOS-related infertility justify the traditional use of this plant for the treatment of female infertility.Also, the overall results indicate that doses ranging from 192 mg/kg to 768 mg/kg are within the therapeutic range of the aqueous extract of A. ampeloprasum.Traditional practitioners could therefore prescribe a dose twice less than that currently prescribed to treat female infertility.Finally, in line with the suggestion made by Rahimi-Madiseh et al. [30] who proposed the use of A. ampeloprasum as a dietary supplement in diabetic patients, the present work suggests that supplementing PCOS patients with this vegetable may also be helpful as they experience similar metabolic disorders seen in diabetic patients.

2. 5 .
Ethical Statement.Animal handling and all experiments were carried out after approval of the research proposal by the Scientifc Committee of the Department of Animal Biology of the University of Dschang (Cameroon) on March 01, 2021, in conformity with the EU Directive 2010/63/EU for animal experiments.

Table 1 :
Classes of compounds detected in the aqueous extract of A. ampeloprasum.

Table 2 :
Phenol, favonoid, and tannin contents in the aqueous extract of A. ampeloprasum.

Table 4 :
Efects of aqueous extract of A. ampeloprasum on some fertility parameters in rats with PCOS.