https://orcid.org/0000-0002-8112-0383
Figures
Abstract
Allium Cepa Linn. (Onions) has extensively been used in traditional medicine, is one of the important Allium species regularly used in our daily diet, and has been the source of robust phenolic compounds. The current study is intended to evaluate the fecundity-enhancing effect of A. Cepa on the reproductive performance of two successive generations of rats; F0 and F1. A. Cepa extract was initially tested for in vitro antioxidant assay via DPPH and ROS, followed by in vivo toxicity testing. In the fecundity assessment, eighteen pairs of male and female rats (n = 36, 1:1, F0 generation) were divided into three groups and dosed with 75mg/kg and 150 mg/kg daily of A. Cepa extract and saline respectively, up to pre-cohabitation, cohabitation, gestation and lactation period. The reproductive performance, including body weight, live birth index, fertility index, and litter size, was assessed. Various parameters like Hematological, Hormonal (FSH, LH, Testosterone, estradiol), antioxidant markers (SOD, Glutathione peroxidase) and lipid profile of F0 and F1 generations were assessed with evaluation of histopathology of male and female organs. Ethanolic extract of A. Cepa showed the greatest antioxidant potential in DPPH and ROS methods. The continued exposure of the F0 and F1 generations to A. Cepa extract did not affect body weight, fertility index, litter size, and survival index. However, semen pH, sperm motility, sperm count, sperm viability, and semen volume were significantly improved in both generations. We have found pronounced fecundity outcomes in both genders of F0 and F1 generations with A. Cepa 150mg/kg/day extract as compared to control. Results showed that A. Cepa significantly increased (P < 0.05) hemoglobin, follicular stimulating hormone (FSH), luteinizing hormone (LH), plasma testosterone and glutathione peroxidase activities, while total lipid, LDL, and cholesterol were significantly decreased (P < 0.05) in both generations. Histology of both generations of animals reveals enhanced spermatogenesis and enhanced folliculogenesis with improved architecture. Altogether, the present results suggest that A. Cepa extract improved fecundity in both male and female rats by improving hormonal activities and oxidative stress.
Citation: Suri S, Khan SS, Naeem S, Nisa ZU, Alam N, Majeed S, et al. (2024) The beneficial effect of Allium Cepa bulb extract on reproduction of rats; A two-generation study on fecundity and sex hormones. PLoS ONE 19(3): e0294999. https://doi.org/10.1371/journal.pone.0294999
Editor: Minhui Li, Baotou Medical College, MONGOLIA
Received: March 7, 2023; Accepted: November 8, 2023; Published: March 14, 2024
Copyright: © 2024 Suri et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: The author(s) received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Infertility can be defined as the failure to accomplish a pregnancy clinically after 12 or more months of regular unsafe sexual intercourse [1]. It has been estimated that around 15% of couples are infertile globally [2]. Within all infertility issues, human sterility attributed to male fertility issues reports for 45–50% [3], and female accounts for 46.6% of cases with polycystic ovarian syndrome (PCOS) constituting the principal cause [4]. To date, factors underlying the dysfunction of the reproductive system are incompletely understood. The use of recent drugs to improve fertility dysfunction is insignificant in achieving her pregnancy outcomes [5]. So, the future focus will be on formulating more viable treatment choices to prevent or treat reproductive system dysfunctions to improve fecundity.
For several years, locally grown plants in place of supplements have been used globally to vitalize, energize, and enhance male sexual functions. Several plants extract have now been identified for improving fecundity and other sexual functions, including Parkia Biglobosa [6], Eugenia Uniflora [7], Montanoa Tomentosa [8], Bryonia Laciniosa [9], Terminalia Catappa [10], Tribulus Terrestris [11] and Lepidium Meyenii [12]. These plants’ active constituents, including terpenoids, phenols, alkaloids, and saponins, may have a role in restoring fertility [13]. Polyphenols, especially flavonoids, are recognized to have estrogenic [14] or androgenic capabilities [15] that have a crucial role in maintaining fecundity. Such findings pointed out that flavonoids can be essential in recovering spermatozoa and spermatogenesis [5]. Sperm infertility may be caused by various factors among which ROS over production constitutes a major risk factor. Enhanced ROS production oxidatively spoil almost all biological molecules in cell involving DNA leading to enhanced sperm membrane damage, declined motility and disability of sperm to fertilize the oocyte [16].
Allium Cepa Linn. (Onion) is the well-known edible bulb of the Alliaceae family and is considered among the oldest cultivated species in the world [17, 18]. It contains various constituents [19] studied because of their different pharmacological properties [20]. A. Cepa has been a valuable medicinal plant for decades due to its robust free radical scavenging potential [21]. It contains antioxidants such as vitamins, selenium, glutathione, and flavonoids like isorhamnetin and quercetin [22, 23]. It has been documented that incorporating antioxidants in the diet protects sperm DNA against oxidative damage and improves sperm health [24]. Moreover, Ghasemzadeh et al., [25] reported that A. Cepa ethanolic extract administration significantly compensated the blood antioxidant level in the experimental polycystic ovary (PCO) model of Wistar female rats. Recent researchers claim that the presence of these anti-oxidants in A. Cepa may be beneficial and protective in reproductive functions [26–28].
Adeleye et al. [29] reported that A. Cepa has increased the serum levels of some hormones, luteinizing hormone (LH), and follicle-stimulating hormone (FSH); it has also improved sperm motility in birds. Another study elucidated the protective effect of A. Cepa juice against dexamethasone-induced oxidative stress during lactation in rats [30]. The cadmium-induced sperm toxicity and testicular oxidative stress were successfully attenuated after the administration of A. Cepa aqueous extract to rats [31]. The evidence shows that A. Cepa has a protective potential against testicular oxidative damage and reproductive dysfunction by stimulating the free radical scavenging system and decrementing lipid peroxidation.
A. Cepa extract presented its exclusive actions by augmenting fecundity power and diminishing fecundity-related problems. Assessment of Body weight and blood parameters provide information regarding the health of the rodents and reproductive effects of A. Cepa extract [32]. However, no research has been executed to explore the beneficial role of A. Cepa on reproduction in two generations. Therefore, a two-generational study was established to explore the beneficial and protective use of A. Cepa extract on fecundity and reproductive hormones in experimental animals.
Materials and methods
Extraction of plant material
A. Cepa was purchased from a local market in Karachi, Pakistan, and was distinguished at Herbarium and Botanic Garden, University of Karachi and has got an identification number; G.H. No. 956008 (Allium Cepa L. of Alliaceae family).
The A. Cepa bulbs were washed with distilled water. The outer layer of the bulbs was peeled off manually and rinsed with sterile distilled water, then air dried properly, cut into small pieces, and chopped. The pieces were soaked in 96% ethanol for five days (100 grams of the sample soaked in 200 ml ethanol) at room temperature (25°C to 30°C) with intermittent shaking. The ethanol extract was filtered and later evaporated to dryness at 20°C using a rotary evaporator (Buchi Rotavapor, Germany) and stored at 2°C– 8°C in a refrigerator [33].
Chemicals
Ferric Chloride, Petroleum ether, Ethanol (96%), Formalin, Potassium hydroxide, Glacial acetic acid, Sulphuric acid (concentrated), Sodium Hydroxide, Mayer’s and Dragendoff’s reagents, HCl 1%, Cholesterol reagent, Conjugate solutions for testosterone and estradiol, Tetramethylbenzidine (TMB), Stop solution, HRP (Horseradish peroxidase) Conjugate reagent, FBS (Fetal bovine serum), PBS (Phosphate-buffered saline), Na2CO3 (30 g/L) were procured from Sigma Chemicals Company (St Louis, MO, USA). Etoposide, 0.1 mM DPPH, DMEM (Dulbecco’s Modified Eagle Medium) and Penicillin-streptomycin were procured from Sigma–Aldrich Co., USA. Gallic acid and Folin-Ciocalteu reagent were obtained from Merck (Darmstadt, Germany). Olive oil was purchased from Saeed Ghani Ltd., Pakistan. All chemicals used in this study were of analytic grades. Elisa Kits was used to assay the levels of FSH (serial number-54371), LH (serial number-52167), plasma testosterone (serial number-55378), SOD and Glutathione (Glory Science Co.2022#45).
Preliminary phytochemical screening
The phytochemical tests of ethanolic extract, including tannins, alkaloids, saponins, flavonoids, glycosides, and phlobatannins, were performed by methods mentioned already with little modifications [34–36].
Total phenolic content analysis
The method used for total phenolic content described previously by Lee et al. [37] utilizes Gallic acid as a standard to obtain a curve by plotting. With the aid of serial dilutions utilizing deionized water, different concentrations of dilutions; 50.0, 25.0, 12.5, 6.2, and 3.1 mg/ml, were formulated. Constituent volumes were added to a single vessel: reagent 100 μL: Folin-Ciocalteu; 20 μL: sample-blank (DI water)-standard gallic acid; 80 μL: Na2CO3 (30 g/L). The microplate was allowed to incubate for 15 minutes at room temperature, followed by taking an absorbance reading at 540 nm utilizing a UV-VIS spectrometer (Genesys™ 10S UV-Vis, Thermo Scientific, USA). Dilutions were executed on the extract as considered necessary for the values of absorbance to fit among the standard curve. The results were declared as mg/g of gallic acid equivalent.
Antioxidant assays
Antioxidant assay of A. Cepa by DPPH.
An upgraded DPPH assay (1,1-diphenyl-2-picrylhydrazyl) was selected to evaluate the tested compound. The DPPH radicals diminish at 517 nm of absorbance in the presence of antioxidant potential. The test compound A. Cepa at different concentrations (10, 20, 100, 300 μg/ml) was added to 0.1mM DPPH (3ml) and allowed to react. The mixture was swirled and incubated for 30 min at room temperature. A microplate reader (SkanIt Software 5.0 of RE, ver. 5.0.0.42) was utilized to measure the absorbance at 517 nm. The test compound reducing power was calculated with the absorbance of ascorbic acid, which served as control [38].
Radical Scavenging Activity was equated in percentage via equation.
Antioxidant assay of A. Cepa by ROS.
Primary cortical cells were used for the determination of Reactive Oxygen Species (ROS) generation. The method [39] was adopted with some modifications. The untreated cells were used as a control. Cortical cells were harvested from the brain of two-day-old rat pups. Cells were grown in DMEM affixed with 10% FBS and then 1% penicillin-streptomycin (100 units/mL-100 μg/mL) respectively. Adhesive cell monolayers were cultured into T25 tissue culture flasks to be used for further experiments. The cortical cells were incubated at 37°C in a humidified atmosphere having 5% carbon dioxide (CO2). ROS Intracellular levels of living cells were assessed quantitatively by employing a fluorescent probe, 2′,7′ dichlorodihydrofluorescein diacetate (DCF-DA). Primary cortical cells were subjected to various concentrations of extract for 12 h. Untreated or treated cultures were allowed to incubate in the dark with 10 μM DCF-DA at 37°C for 45 minutes, and later on, washed with PBS. Relevant ROS level changes intracellularly were observed by fluorometric detection of DCF by using a fluorescent Microplate Readers RE, ver. 5.0.0.42 at emission and excitation wavelengths of 530 nm and 485 nm, respectively. The fluorescence intensity level of DCF is proportional to the ROS level generated intracellularly. Percentage Cell Viability can be calculated by formula [40].
Experimental animals.
In the experimental fecundity study, we used 36 healthy animals comprising 18 males and 18 females Wistar rats. The animals were bought from the animal house, H.E.J, University of Karachi, and were kept in plastic cages having proper ventilation under standard temperature conditions (25°C to 30°C) with 12 h light and dark cycle. The animals were given pelleted rodent food. The weight of animals ranged between 170g to 190g. The animals were placed in an animal house at the Department of Pharmacology, University of Karachi.
The experimental protocol fulfills the guidelines regarding the appropriate use and care of laboratory animals [41], and the experimental protocol and design were permitted by the Board of Advanced Studies and Research (BASR), Resolution No. 10(P)07, ASRB/No./05071/Pharm., University of Karachi.
Animal ethical approval.
The study was performed under the approval of the Board for Advanced Studies and Research dated 28-03-2019 (reference No 05071/pharm), University of Karachi and Pharmacology Departmental Research Committee, for the use of animals according to the National Institute of Health guidelines (NIH guidelines Islamabad, Pakistan) [42].
Brine shrimp toxicity testing.
Toxicity of A. Cepa was evaluated to rule out % Mortality with the help of the Brine shrimp method (Artemia Saline). Etoposide served as a positive control [43].
Acute oral toxicity.
The study on acute oral toxicity was executed according to the OECD-404 guidelines [44]. Both genders’ rats were used and supplied with distilled water and fed with adlibitum. Twenty rats were randomly divided into four groups (n = 5). A single oral dose of ethanolic extract of Allium Cepa, 300 mg/kg, 600 mg/kg, and 1200 mg/kg were administered as per body weight respectively, to each group in comparison to the control. The rats were examined for toxicity signs like behavioral changes and mortality for 48 hours [45].
Fecundity assessment.
The study was conducted for a total duration of seven months. A modified method of Vohra et al. [46] was used. Eighteen pairs of rats (n = 36, F0 generation) were placed in separate cages.
In the fecundity study, healthy and sexually mature male rats aged 12 weeks (155.42 ± 1.89 g) and female rats aged 14 weeks (169.1 ± 2.16 g) were used. Rats were isolated and evaluated for any gross symptoms of injury or disease. After one week of acclimatization, six experimental groups were formed. Three groups were of male rats, each comprising of six rats (n = 6), while the rest of the three groups comprises of female rats only (n = 6).
In male rat groups, Group, I served as control and administered distilled water daily; Group II animals were given low dose A. Cepa extract of 75 mg/Kg/day while Group III animals were given A. Cepa extract at the high dose of 150 mg/Kg/day [47, 48]. A similar protocol was followed for the female group as well. All rats were dosed for 30 days before mating (Pre- Cohabitation) by gastric gavage between 14:00 and 16:00 h. Female rats were subjected to the soiled bedding of an adult male rat to align their estrus cycle (female rats were fertile and receptive during this period) [49]. During mating each male rats were individually paired with female rats of each group in separate cages for seven days (1:1). Inseminated females were separated from males and placed in cages after conception. The doses of A. Cepa were given continuously to female rats during cohabitation (21 days), gestation (21 days), delivery and lactation till postnatal day of 22. When pups were delivered, all F0 animals (male and female) were deeply anesthetized with intraperitoneal Ketamine (10 mg/Kg) and Xylazine (5 mg/Kg) [50], to alleviate the suffering of rats. Blood samples were taken from the aorta and the animals were sent to necropsy. Subsequently, reproductive organs were taken for histopathological assessment.
Fertility indices (fertility index, the number of pups, survival index) were estimated. Offspring were counted and observed for any abnormality on postnatal days 0, 4, 7 14, and 21. Body weight was recorded weekly before, during mating, and in gestational periods. All F0 male/female parental animals were monitored twice daily for toxicity signs.
For male rats’ fecundity assessment, the procedure described by Quadri & Yakubu [51] was selected to prepare the semen from the epididymis. The Epididymis was carefully detached and lacerated with scissors from the testis. The semen was aspirated 10 μl from the caudal epididymis of every rat by using a capillary tube and transferred into Petri dishes already having 1 ml, 0.1 M phosphate buffer having 7.4 pH. The homogeneity was maintained by slightly swirling each dish. The sperm cells were permitted to separate in the solution at 37°C for 10 min. Afterward, the semen volume, sperm count, pH, motility, and viability were estimated using standard methods.
For F1 generation study, pups were grown up to 4 weeks then the same doses of the A. Cepa extract were given to 4 weeks old pups (according to their bodyweight) during their growth, adulthood, and up to breeding. After one month, 8 weeks old rats were permitted mating by making pairs on a 1:1 basis within same treatment group, preventing sibling-mating, for 7 consecutive days. The vaginal plug was noticed to confirm the pregnancy of each female rat. Pregnant female rats of the F1 generation were separated from male rats and placed individually. A. Cepa extract continuously given during their pre-cohabitation, cohabitation period, gestation, and lactation until the F2 generation pups were weaned. Gestated female rats were permitted to naturally deliver pups (F2 generation).
Afterward, all F1 generation animals were anesthetized and examined as described previously. The same protocol of the F0 generation was followed for both F1 and F2 generation till the birth of F2 pups. For all F1 generation female rats’ fecundity assessment, the above procedure for determining fertility indices in F0 generation animals were used as described by Vohra et al. [46]. Similarly, for all F1 generation male rats’ fecundity assessment, the procedure described by Quadri & Yakubu [51] was selected as discussed above in F0 generation animals.
Reproductive performance of F0 and F1 rats.
The reproductive parameters studied were mentioned in the form of ratios, weights, and indices that analyze all stages starting from conception till weaning [46]. These fertility parameters were computed as follows:
Fertility index (%) = (Females count giving birth/females count lived together) × 100
Litter size = count of pups/count of gestated females
Live birth index (%) = (count of pups alive at day 0/count of pups born) × 100
Survival index—4 days (%) = (count of pups alive on day 4/ day 0 alive pups count) × 100
Survival index- 21 days (%) = (count of alive pups on day 21/ day 4 alive pups count) × 100
Blood parameter assessment.
When pups were delivered, all F0 and F1 generation male and female animals were anesthetized with Ketamine and Xylazine. Blood samples were taken from the aorta for biochemical assessment and kept in B-Ject gel clot activator vacuum tubes under aseptic conditions. Samples were centrifuged in an Eppendorf machine at 4000 rpm for 10 min. The separated serum was kept at -80° C for hormonal and biochemical testing. All samples were analyzed for hematological (Sysmex KX-21 hematological analyzer) [52], Total lipid profile (LDL, VLDL, HDL, Triglycerides, Cholesterol), hormonal estimation (FSH, LH, testosterone and estradiol [53, 54] were assayed via ELISA Roche Diagnostics, Basil). SOD and Glutathione (GPx) were estimated by the kit acquired from Glory Science Co., Ltd. (Glory Science Co.2022#45).
Histopathological evaluation.
For histopathological evaluation, the ovaries and testis of the selected rats were removed and cleaned from adherent connective tissues, then fixed in 10% formalin. Fresh tissue samples were dehydrated by exposing ethanol and dimethyl benzene of different concentrations (twice) at room temperature. Then, sex organs were embedded with paraffin at 60° C and cut into sections. Blocks of paraffin were cut serially and longitudinally at 4 μm thickness, followed by Hematoxylin-Eosin staining. Lastly, the stained sections were dehydrated and sealed with coverslips and imaged under microscope (Bio Base XS-208, China). Stained slides were analyzed and compared in groups [55].
Statistical analysis
Statistical analysis was implemented using the SPSS software (version 20.0). Multiple groups were compared by one-way analysis of variance applying the Bonferroni post-hoc test. The results were considered statistically significant if the value lies between P < 0.05 and highly significant when P < 0.01.
Results
% Yield and total phenolic content of A. Cepa bulb
A. Cepa bulb (12 Kg) was extracted with 96% ethanol and the total yield percentage of A. Cepa extract was found to be 10%.
Total Phenolic Content was determined in the extract of A. Cepa bulb by the method of Folin-Ciocalteu (F–C) using gallic acid as the standard. Absorbance values were determined at various concentrations of gallic acid to construct the calibration curve. The total phenolic content of the extract was estimated from the regression equation of the calibration curve (Y = 1.012x; R2 = 0.996) designated as gallic acid equivalents (GAE) (63 mg/g GAE) (Table 1).
Acute oral toxicity.
Regarding the safety of A. Cepa L. plant, acute toxicity study disclosed no signs of toxicity with no mortality in rats. However, some dose-dependent behavioral changes were observed in high-dose (1200 mg/kg) treated groups, including depression, clustering, folding and sleeping, unsteady gait, and loss of appetite. The results regarding Acute toxicity (LD50) of A. Cepa, by using Karber Method, is mentioned below (Table 2). The arithmetic method of Karber was utilized for calculation.
Preliminary Phytochemical Screening
The presence of A. Cepa constituents was mentioned in Table 3. The qualitative phytochemical analysis reveals that Allium Cepa possesses tannins, flavonoids, terpenoids, glycosides, saponins, phenols, and phlobatannins.
Antioxidant assays
Antioxidant Assay of A. Cepa by DPPH.
The % inhibition of A. Cepa extract at different concentrations is shown in Table 4. The extract was observed with radical scavenging potential by DPPH utilizing Ascorbic Acid as a reference standard. Among various concentrations, A. Cepa exhibited the highest activity at 100 μg/ml and 300 μg/ml, i.e., 84.92 ± 0.11% and 80.27 ± 1.20%, respectively, while Ascorbic acid exhibited the strongest scavenging activity of 88.14 ± 1.28% at 300 μg/ml.
Antioxidant Assay of A. Cepa by ROS.
The DCF-DA method was employed to determine the ROS production and % Viability of A. Cepa extract on primary cortical neuronal cells was found nontoxic and significantly prevented H2O2-induced oxidative damage. % Cell viability in neuronal cells after incubation using indicated concentrations of A. Cepa is shown in Table 5, Fig 1, which shows no cytotoxic effect in neuronal cells at concentrations up to 50 (μg/ml). However, at the dose of 100 (μg/ml), A. Cepa extract shows protective efficacy against H2O2-induced toxicity on neuronal cells.
Brine shrimp lethality testing.
The A. Cepa extract was assessed for brine shrimp lethality studies and the results were mentioned in Table 6. No significant toxicity to brine shrimp Artemia salina was found with A. Cepa extract at different concentrations. The A. Cepa extract shows significant results, which represent that the extract is biologically safe. The % mortality of A. Cepa at 1000 μg/ml concentration is 16.66% compared to standard Etoposide, which shows 100% mortality at 1000 μg/ml, indicating the extract is significant and safe for use.
Fecundity assessment.
The two-generation fecundity study was conducted for seven months that showed no congenital abnormalities in any of the pups of all the groups of F0 and F1 generations. The body weights of F0 and F1 animals are mentioned in Table 7, representing insignificant differences among generations and control.
The number of pups was counted in each generation and noticed for congenital abnormalities. In all groups, no congenital abnormality was observed. The number of pups increased significantly in the first and second generations compared to the control group (df 5, 30; F 5.51; P < 0.05).
Post Hoc analysis through the Bonferroni test revealed that high-dose administration of Allium Cepa produced statistically significant differences in the number of pups in both generations compared to low dose group and control group rats, as shown in Table 8.
Reproductive performance.
The reproductive performance was assessed by observing several parameters, including live birth index, fertility index, and litter size. Insignificant changes were found in all treated groups’ live birth, fertility, and survival indexes (P > 0.05). All these parameters were improved in all treated groups, just like the control group; however, a significant increase in litter size was noticed in the treated group in both generations (P < 0.05) when compared to the control group, as shown in Table 9.
Additionally, in male rats, parameters like semen pH, sperm motility, sperm count, sperm viability, and semen volume are crucial indices of male fecundity as they are key markers in epididymal maturation and testicular spermatogenesis. All these parameters were significantly improved (P < 0.05) in all treated groups when compared to the control group, as shown in Table 10.
Hematological parameters.
Hematological analysis showed the non-significant result in male and female rats, including Hb, RBCs and WBCs. However, on Platelets, highly significant results (P < 0.01) in male F0 generation rats (df 2,15; F 14.56; P < 0.01) were found as shown in Fig 2. Similarly, F1 generation male and female rats showed significant results (df 2,15; F 6.62; P < 0.05), (df 2,15; F 6.66; P < 0.05), respectively, on platelets.
(A) F0 Male (B) F0 Female (C) F1 Male (D) F1 Female. n = 6, Mean ± SEM; *P < 0.05 significant; ** P < 0.01 highly significant as compared to control.
Biochemical analysis.
Biochemical Analysis showed statistically highly significant reduction in Cholesterol (df 2,15; F 5.68, 7.70; P < 0.01), LDL (df 2,15; F 12.76, 12.48; P < 0.01), significant reduction in triglycerides (df 2,15; F 3.91, 2.51; P < 0.05) and raised HDL effects (df 2,15; F 4.85, 5.42; P < 0.05) of F0 and F1 generation male animals respectively as shown in Fig 3. Correspondingly female animals of F0 generation showed highly significant reduction in Triglycerides (df 2,15; F 15.10; P<0.01), LDL (df 2,15; F 14.24; P<0.01) and a raised HDL (df 2,15; F 6.35; P<0.01) effect in comparison to control. Similarly, F1 generation female animals showed significant reduction in Cholesterol (df 2,15; F 46.66; P<0.05), Triglycerides (df 2,15; F 50.55; P<0.05), LDL (df 2,15; F 185.8; P<0.05) and VLDL (df 2,15; 10.87; P<0.05) and a raised HDL effect (df 2,15; F 4.82; P<0.05) when compared to control group.
(A) F0 Male (B) F0 Female (C) F1 Male (D) F1 Female. n = 6. Mean ± SEM; *P < 0.05 significant; **P < 0.01 highly significant as compared to control.
Hormonal parameters.
FSH, LH, Estradiol and Testosterone levels were tested in the plasma of both control and treated male and female rats. In higher doses, statistically significant raised values of FSH, LH and testosterone were found in F0 generation male rats (df 2,15; F 1610; P < 0.05) (df 2,15; F 50.724; P < 0.05) (df 2,15; F 2138; P < 0.05) respectively, when compared to the control. Similarly, F1 generation rats also exhibited a significantly raised Testosterone effect (df 2,15; F 3193; P < 0.05). The insignificant result in estradiol was found in male rats (df 2,15; F 3.26, 3.39; P > 0.05) as well as in female rats (df 2,15; F 5.29, 19.26; P > 0.05) respectively, of both F0 and F1 generation, as shown in Fig 4. In F1 generation females, statistically significant raised effect in FSH (df 2,15; F 70.05; P < 0.05) was found.
(A) F0 Male (B) F0 Female (C) F1 Male (D) F1 Female. n = 6, Mean ± SEM; *P < 0.05 significant; **F < 0.01 highly significant as compared to control.
However, in F0 generation female rats, insignificant changes were seen in FSH (df 2,15; F 16.025; P >0.05), LH (df 2,15; F 0.20; P > 0.05) and testosterone (df 2,15; F 16.05; P > 0.05) when compared to control animals.
Oxidative parameters.
Glutathione (GPx) levels in treated rats were found to be statistically significant in both F0 and F1 generations male rats (df 2,15: F 14.20; P < 0.01); (df 2,15: F 19.01; P < 0.05) and female rats (df 2,15; F 14.73, 63.65; P < 0.05) in comparison to control animals. In contrast, insignificant results of SOD were found in F0 male rats (df 2,15; F 1.13; P > 0.05) and F0 and F1 female rats (df 2,15; F 0.461, 0.124; P > 0.05) while F1 generation male rats showed a significant raised effect on SOD (df 2,15; F 1.08; P < 0.05) in comparison to the control group as shown in Fig 5.
(A) F0 Male (B) F0 Female (C) F1 Male (D) F1 Female. n = 6, Mean ± SEN1; *P < 0.05 significant; ** P < 0.01 highly significant as compared to control.
Histopathological evaluation of ovaries.
Control ovaries of F0 and F1 generation rats showed the external cortex surrounded with intact capsule having profuse stroma. A lot of blood vessels parted by unattached connective tissues in the medulla were also observed. The histological ovarian architecture of F0 and F1 generation treated with low-dose A. Cepa, animals was similar to controls. However, some primary follicles surrounded beneath the tunica albuginea of an intact primary oocyte can be seen in low-dose treated ovaries, and degenerative changes were also detected in follicles.
F0 and F1 generation’s animals of high-dose onion treatment showed Graafian follicle and zona pellucida along with the Nucleus of oocytes. Preantral, small antral, and large antral follicles and corpus luteum of different stages were observed. The number of follicle counts was significantly raised in the F1 high-dose treated animals as compared to other groups. Folliculogenesis was seen in all samples as shown in Fig 6.
(a) Micrograph, rat ovary F0 generation control (10x) (b) Micrograph, rat ovary F0 generation low dose A. Cepa treated (20x) (c) Micrograph, rat ovary F0 generation, high dose A. Cepa treated (20x) (d) Micrograph, rat ovary F1 generation control (10x) (e) Micrograph, rat ovary F1 generation, A. Cepa low dose treated (40x) (f) Micrograph, rat ovary F1 generation, A. Cepa high dose treated (10x).
Histopathological evaluation of testes.
Microscopic examination of control group testes of F0 and F1 both generation figures showed an architecture of tubular seminiferous epithelia present normally with occasional Sertoli cells and spermatogenic cells at the interstitial area and base of the tubule having negligible connective tissues at the tubular surroundings.
Microscopic examination of low-dose treated testes of F0 and F1 generation showed the normal seminiferous tubules having the normal arrangement of Sertoli and spermatogonial cells resting over intact basement membrane. Similarly, in F0 and F1 generation’s testes, high dose A. Cepa extract showed normal architecture of seminiferous epithelia of tubules along with Sertoli cells and spermatogenic cells having minimal connective tissues at the tubular surrounding. However, nearer to the tubule’s central part, primary and secondary spermatids at the center of the seminiferous tubule lumen were observed. Increased production of spermatids and spermatozoa was found as compared to the control and low-dose A. Cepa group as shown in Fig 7.
(a) Micrograph, rat testis of F0 generation control (20x) (b) Micrograph, rat testis F0 generation low dose A. Cepa treated (40x) (c) Micrograph, rat testis F0 generation, high dose A. Cepa treated (40x) (d) Micrograph, rat testis F1 generation control (40x) (e) Micrograph, rat testis F1 generation, A. Cepa low dose treated (20x) (f) Micrograph, rat testis F1 generation, A. Cepa high dose treated (40x).
Discussion
The therapeutic benefits of A. Cepa have been acknowledged since ancient civilizations. Important benefits under investigation nowadays include its effect on fecundity and reproduction [23]. Countless experimental researches were conducted to explore the reproductive potential of different herbal extracts to overcome infertility and related problems [7, 51]. A. Cepa is one of the herbs that have not been investigated widely for its fertility potential, only a few articles are being reported [26, 56]. This two-generation study was established to evaluate the role of A. Cepa extract in fecundity and the reproductive system.
In the current study, ethanolic extract of A. Cepa exhibited the presence of different phytochemicals, including tannins, flavonoids, saponins, terpenoids, and polyphenols as well as quantitative data suggest the presence of 63mg/g GAE total polyphenolic content. These findings are in accordance with Prakash et al., 2007 [57], who reported the presence of a rich amount of polyphenols in A. Cepa extract with promising antioxidant activity. Antioxidant activity of A. Cepa extract is evaluated by DPPH that showed the highest % inhibition at 100 μg/ml (84.92%). Similarly, in ROS method A. Cepa extract showed protective efficacy against H2O2-induced toxicity on neuronal cells. These results suggest dose-dependent radical scavenging activity against DPPH and superoxide anion radicals, which agrees with an earlier study [58].
In screening natural products for pharmacological activity, evaluating the toxic characteristics of medicinal extracts is usually an initial step. During such evaluations, the therapeutic dose determination and mortality rate is the initial step to be conducted [59]. In the Brine shrimp lethality test, the % mortality of A. Cepa was found at 16.66% at a maximum concentration, which shows that it is safe and significant to use. Similarly, the result of the oral acute toxicity test of A. Cepa ethanolic extract in-vivo showed that it has a very high safety range when administered orally, even at a maximum dose of 1200 mg/kg. According to Pitchaiah et al. [60], there were no mortality and gross behavioral changes in mice fed with A. Cepa extract, which suggested its safety in the acute study.
Body weight assessment provides information regarding the health of rodents [32]. The observations showed an insignificant decrease in body weights of F0 and F1 females following the treatment with lower (T1) and higher (T2) doses of A. Cepa. The reduction in body weight may be because A. Cepa has anti-obesity properties, thus being involved in reducing body weight and food intake in rats [61, 62]. But after week III, the body weights of females of both generations gradually increased during the gestation period [46]. This finding is in accordance with Mirabeau and Samson [63], who reported immune boosting and weight-improving capabilities of Allium Cepa.
Our data demonstrated increased pups in both F1 and F2 generation during the treatment with both doses of onion extract. This indicated that A. Cepa presented its beneficial actions by augmenting the fertility power of the female rats [26–28]. There was an increase in the litter size of treatment groups in both generations when compared to the control group. Khaki et al. [64] also reported increased reproductive performance after administering A. Cepa juice. In F1 off-springs, A. Cepa treatment caused a slight decrease in the live birth index of the high dose group when compared to control. No effects were observed on mating indices, viability index, and fertility indices on days 4 and 21. However, the live birth index was increased in the F2 generation. The outcomes of our study are endorsed further by the investigation of Ara et al. [65], who demonstrated that A. Cepa extract administration prevented tartrazine-induced noxious infertility in male and female mice. That could be due to polyphenolic compounds present in A. Cepa, especially quercetin that can repair oxidative damage and hormonal irregularities [66].
Furthermore, in the current study, we have found increased sperm count, volume, viability, and sperm motility by administration of A. Cepa. This improved result could be due to the antioxidant potential of A. Cepa, that is a rich source of endogenous and exogenous antioxidants like isorhamnetin and glutathione. It also holds trace minerals and different vitamins; A, B, and C that can assist in preventing damage by reactive oxygen species and protect the tissue from oxidative damage. All these effects may lead to enhanced fertility in males [65].
In our bi-generational experiment (F0, F1), blood parameters were normal besides platelet count, which increased in both treatment groups. Therefore, A. Cepa demonstrated potential benefits in maintaining the animal’s good health [67]. A study also reported that onion extract has blood lipid levels reducing potential [68]. Thus, we can conclude that Allium Cepa can efficiently decrease the health risk of cardiovascular events in both genders as it is rich in fiber and flavonoids [69, 70].
Reproductive hormones are essential in the development and regular functions of the reproductive system. FSH and Testosterone are important for fulfilling reproductive abilities in males [71]. Studies showed that sex hormones like FSH, LH, and testosterone levels are associated with spermatogenesis [64]. LH activates Leydig cells to produce testosterone while FSH encourages Sertoli cells proliferation. Both these hormones affect testosterone levels that is necessary for sustaining the maturing sperm cells. Moreover, FSH and testosterone impacts the release and growth of spermatids. Thus, an appropriate concentration of FSH, LH and Testosterone is desired for spermatogenesis [72].
FSH, LH, testosterone, and Estradiol levels were estimated in both F0 and F1 generation animals (male and female rats). In male rats, significantly increased effects were observed by A. Cepa (150 mg/Kg) on FSH, LH, and Testosterone levels in both generations, depicting its potential as a fertility-enhancing agent. This result is in accordance with the study of Banihani, who reported that A. Cepa extract consumption enhances testosterone production in males [73]. Testosterone is vital for the maturation and maintenance of the structure and function of the male accessory sex glands. Moreover, insufficiency of this hormone hinders spermatogenic function [74]. Further, Banihani et al. suggested the mechanisms by which A. Cepa enhances testosterone production in males are mainly by enhancing the antioxidant defense mechanism (e.g., antioxidant enzymes, glutathione) in the testis, ameliorating insulin resistance and promoting nitric oxide production in Leydig cells [73].
Increased effects were observed on FSH levels in female rats (F1 generation), while in F0 generation, FSH levels were insignificant. Additionally, estradiol levels were insignificant and maintained as in the control group. Moreover, LH levels were maintained normal, indicating LH potentiates follicle-stimulating hormone (FSH), stimulates follicle growth, and improves female fertility [75]. Since FSH/LH surge is required for ovulation that is triggered by LH releasing hormone, dependent on estradiol levels [76]. Our results provide the rationale that A. Cepa in higher doses significantly improved FSH levels by balancing the estradiol and improving fecundity, probably due to the increased pituitary sensitivity to gonadotropin-releasing hormone [77]. These findings are in accordance with Mansouri et al., who explained that A. Cepa extract significantly improves sex hormone production and fertility in electromagnetic field-exposed mice [78]. The changing patterns of sex hormones in the present study are in line with the findings of earlier studies, as Adelakun et al. [79], reported that natural extracts have flavonoid contents that decrease oxidative stress and have pro-fertility properties, which are beneficial for fertility.
Recent studies have reported the anti-oxidant properties of A. Cepa are due to flavonoid quercetin [80]. Quercetin-induced upregulation of glutathione levels may elucidate the antioxidant effects of Allium Cepa [81]. A. Cepa extract increased the level of antioxidants in the body, such as glutathione [82]. Our study also confirmed the anti-oxidant potential of A. Cepa extract by increasing the glutathione (GPx) levels of both male and female Wistar rats. Present findings for glutathione (GPx) depicted the highest correlation between antioxidant potential (DPPH, ROS) and total phenolic contents in A. Cepa extract.
In Histopathological examination, female rats exhibited an improved number of follicles count as well as Folliculogenesis in the F1 high-dose treated animals. In addition, the male testis showed improved architecture of seminiferous tubular epithelia along with Sertoli cells and increased spermatogenic cells. These all above results are in line of agreement with Ara et al. [65], who reported that onion extract significantly ameliorated tartrazine-induced histopathological changes with improved architecture, granulosa cells, and corpus luteum. This could be due to the antioxidant activity of A. Cepa extract against reproductive deteriorations.
Conclusion
In conclusion, this study revealed that administration of A. Cepa ethanolic extract for seven months had beneficial effects on both male and female fecundity processes by enhancing ovulation and spermatogenesis with no toxic effect in both sexes. This could be related to its lipid-lowering action as manifested by the modulation of serum total cholesterol as well as its antioxidant and androgenic effects. Therefore, it is suggested that daily consumption of A. Cepa may provide an effective strategy to improve fertility. However, further infertility induced model studies are needed to investigate the effects of its active constituents and pure compounds in clinical trials.
Acknowledgments
The authors are grateful to the Department of Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, University of Karachi, for providing essential facilities for this research work.
References
- 1. Szamatowicz M. and Szamatowicz J.J.A.i.m.s, Proven and unproven methods for diagnosis and treatment of infertility. 2020. 65(1): p. 93–96.
- 2. Sun H., et al., Global, regional, and national prevalence and disability-adjusted life-years for infertility in 195 countries and territories, 1990–2017: results from a global burden of disease study, 2017. 2019. 11(23): p. 10952.
- 3. Kumar N. and Singh A.K.J.J.o.h.r.s, Trends of male factor infertility, an important cause of infertility: A review of literature. 2015. 8(4): p. 191.
- 4. Deshpande P.S. and Gupta A.S.J.J.o.h.r.s, Causes and prevalence of factors causing infertility in a public health facility. 2019. 12(4): p. 287.
- 5. Ye R.-J., et al., Interplay between male reproductive system dysfunction and the therapeutic effect of flavonoids. 2020. 147: p. 104756.
- 6. Obeten K.E., et al., Histomorphology and sperm profile of animal models administered with aqueous seed extract of Parkia biglobosa. 2022. 15(1): p. 1–8.
- 7. Nkpurukwe C.I., et al., Improvement in some Reproductive Parameters of Male Wistar Rats Administered with Leaf Extract of Eugenia uniflora. 2022. 5: p. 793–797.
- 8. Carro-Juárez M., et al., Aphrodisiac properties of Montanoa tomentosa aqueous crude extract in male rats. 2004. 78(1): p. 129–134.
- 9. Sud K. and Sud S.J.E.J.o.B, A Scientific Review on Shivlingi Beej (Bryonia Laciniosa): A Mystical Ethno-Medicine for Infertility. 2017. 4(8): p. 1098–102.
- 10. Oyeniran O.H., Ademiluyi A.O., and Oboh G.J.J.o.F.B, Comparative study of the phenolic profile, antioxidant properties, and inhibitory effects of Moringa (Moringa oleifera Lam.) and Almond (Terminalia catappa Linn.) leaves on acetylcholinesterase and monoamine oxidase activities in the head region of Fruitfly (Drosophila melanogaster Meigen) in vitro. 2021. 45(3): p. e13401.
- 11. Zheleva-Dimitrova D., Obreshkova D., and Nedialkov P.J.I.J.P.P.S., Antioxidant activity of tribulus terrestris—a natural product in infertility therapy. 2012. 4(4): p. 508–11.
- 12. Tafuri S., et al., Lepidium meyenii (Maca) in male reproduction. 2021. 35(22): p. 4550–4559.
- 13. Ogidi O.I., George D.G., and Esie N.G.J.J.o.M.P, Ethnopharmacological properties of Vernonia amygdalina (Bitter Leave) medicinal plant. 2019. 7(2): p. 175–181.
- 14. Akbaribazm M., Goodarzi N., Rahimi M.J.F.s, and nutrition, Female infertility and herbal medicine: An overview of the new findings. 2021. 9(10): p. 5869–5882.
- 15. Abaho I., Masembe C., Akoll P., and Jones , The use of plant extracts to control tilapia reproduction: Current status and future perspectives. 2022. 53(3): p. 593–619.
- 16. Raeeszadeh M., Shokrollahi B., and Akbari A., Superior effect of broccoli methanolic extract on control of oxidative damage of sperm cryopreservation and reproductive performance in rats: A comparison with vitamin C and E antioxidant. Theriogenology, 2022. 181: p. 50–58. pmid:35063921
- 17. Sima N., Mahdieh A., Narges K.J.A.j.o.p, and pharmacology, Evaluation of analgesic and anti-inflammatory effects of fresh onion juice in experimental animals. 2012. 6(23): p. 1679–1684.
- 18. Akash M.S.H., Rehman K., and Chen S.J.N., Spice plant Allium cepa: Dietary supplement for treatment of type 2 diabetes mellitus. 2014. 30(10): p. 1128–1137. pmid:25194613
- 19. Benmalek Y., Yahia O.A., Belkebir A., and Fardeau M.-L.J.B., Anti-microbial and anti-oxidant activities of Illicium verum, Crataegus oxyacantha ssp monogyna and Allium cepa red and white varieties. 2013. 4(4): p. 244–248.
- 20. Marefati N., et al., A review of anti-inflammatory, antioxidant, and immunomodulatory effects of Allium cepa and its main constituents. 2021. 59(1): p. 285–300. pmid:33645419
- 21. Vidyavati H., et al., Hypolipidemic and antioxidant efficacy of dehydrated onion in experimental rats. 2010. 47(1): p. 55–60. pmid:23572601
- 22. Khaki A., Farnam A., Badie A.D., and Nikniaz H.J.B.m.j., Treatment effects of onion (Allium cepa) and ginger (Zingiber officinale) on sexual behavior of rat after inducing an antiepileptic drug (lamotrigine). 2012. 2012(3): p. 236–242.
- 23. Griffiths G., et al., Onions—a global benefit to health. 2002. 16(7): p. 603–615.
- 24. Martínez-Soto J.C., et al., Dietary supplementation with docosahexaenoic acid (DHA) improves seminal antioxidant status and decreases sperm DNA fragmentation. 2016. 62(6): p. 387–395.
- 25. Ghasemzadeh A., Farzadi L., Khaki A., and Ahmadi S.K.J.L.S.J., Effect of Allium cepa seeds ethanolic extract on experimental polycystic ovary syndrome (PCOS) apoptosis induced by estradiol-valerate. 2013. 10(4): p. 170–175.
- 26. KHAKI A., NOURI M., Fathi A.F., and KHAKI A.A., Evaluation of zingiber officinalis and allium cepa on spermatogenesis in rat. 2008.
- 27. Salehi P., et al., Effect of antioxidant therapy on the sperm DNA integrity improvement; a longitudinal cohort study. 2019. 17(2): p. 99.
- 28. Sejian V., et al., Effect of mineral mixture and antioxidant supplementation on growth, reproductive performance and adaptive capability of m alpura ewes subjected to heat stress. 2014. 98(1): p. 72–83.
- 29. Adeleye O., Adeleye A., Adetomiwa A., and Ariyo O.J.S.J.o.V.S, Effects of Allium cepa L. peels extract on gonadotropins, testosterone and sperm variables in Oba Marshal broiler cocks. 2020. 18(3): p. 137–143.
- 30. Jeje S., et al., Allium cepa Linn juice protect against alterations in reproductive functions induced by maternal dexamethsone treatment during lactation in male offspring of Wistar rats. 2020. 6(5): p. e03872. pmid:32395653
- 31. Ola-Mudathir K.F., et al., Protective roles of onion and garlic extracts on cadmium-induced changes in sperm characteristics and testicular oxidative damage in rats. 2008. 46(12): p. 3604–3611.
- 32. Aly H.A., Domènech Ò., Abdel-Naim A.B.J.F., and c. toxicology, Aroclor 1254 impairs spermatogenesis and induces oxidative stress in rat testicular mitochondria. 2009. 47(8): p. 1733–1738.
- 33. Ikegbunam M., Ukamaka M., and Emmanuel O.J.A.J.o.P.S, Evaluation of the antifungal activity of aqueous and alcoholic extracts of six spices. 2016. 7(1): p. 118–125.
- 34. Abdillah S., et al., Phytochemical screening and antimalarial activity of some plants traditionally used in Indonesia. 2015. 5(6): p. 454–457.
- 35. Alshammaa D.J.I.J.C.M.A.S., Preliminary Screening and Phytochemical Profile of Mangifera indica Leave’s Extracts, Cultivated in Iraq. 2016. 5: p. 163–173.
- 36. Esienanwan E.E., et al., Comparative qualitative phytochemical analysis of oil, juice and dry forms of garlic (Allium sativum) and different varieties of onions (Allium cepa) consumed in Makurdi metropolis. 2020. 12(1): p. 9–16.
- 37. Lee Y.H., et al., An appraisal of eighteen commonly consumed edible plants as functional food based on their antioxidant and starch hydrolase inhibitory activities. 2015. 95(14): p. 2956–2964.
- 38. Arif A., et al., Evaluation of Anti-inflammatory, Antioxidant and Xanthine Oxidase Inhibitory Potential of N-(2-hydroxy phenyl) Acetamide. 2022. 5(2): p. 309–318.
- 39. Vargas F.d.S, et al., Protective effect of alpha-tocopherol isomer from vitamin E against the H2O2 induced toxicity on dental pulp cells. 2014. 2014.
- 40. Kamiloglu S., Sari G., Ozdal T., and Capanoglu E.J.F.F., Guidelines for cell viability assays. 2020. 1(3): p. 332–349.
- 41. Council N.R., Guide for the care and use of laboratory animals. 2010.
- 42. Garber J.C., et al., Guide for the care and use of laboratory animals. 2011. 8: p. 220.
- 43. Meyer B., et al., Brine shrimp: a convenient general bioassay for active plant constituents. 1982. 45(05): p. 31–34.
- 44.
Co-operation, O.f.E. and Development, OECD Guideline for the testing of chemicals: Acute oral toxicity. 1987, OECD Paris.
- 45. Oyewusi J., Saba A., and Olukunle J.J.V.J.o.V.S, Phytochemical, elemental and acute toxic effects of methanol extract of onion (Allium cepa) bulbs in Wistar albino rats experimentaly pre-exposed and rested for a week. 2015. 10: p. 65–74.
- 46. Vohra P., Khera K.S.J.J.o.V.S, and Technology, Effect of imidacloprid on reproduction of female albino rats in three generation study. 2016. 7(4).
- 47. Amiri M., Jelodar G., Erjaee H., and Nazifi S., The effects of different doses of onion (Allium cepa. L) extract on leptin, ghrelin, total antioxidant capacity, and performance of suckling lambs. Comparative Clinical Pathology, 2019. 28: p. 391–396.
- 48. Elberry A.A., et al., Immunomodulatory effect of red onion (Allium cepa Linn) scale extract on experimentally induced atypical prostatic hyperplasia in Wistar rats. Mediators of Inflammation, 2014. 2014. pmid:24829522
- 49. Khan R.A., Mirza T., Fayyaz T.B.J.E.-B.C., and Medicine A., The Effects of Ficus carica on Male and Female Reproductive Capabilities in Rats. 2022. 2022.
- 50. Joksimovic Jovic J., et al., Cardiovascular properties of the androgen-induced PCOS model in rats: the role of oxidative stress. Oxidative Medicine and Cellular Longevity, 2021. 2021. pmid:34512871
- 51. Quadri A. and Yakubu M.J.A., Fertility enhancing activity and toxicity profile of aqueous extract of Chasmanthera dependens roots in male rats. 2017. 49(10): p. e12775.
- 52. Fares A.K.J.S.J.I., Performance evaluation of two haematology analysers: the Sysmex KX-21 and the Beckman Coulter AC. T diff. 2001. 11(1).
- 53. Alam J.M., Anti-Müllerian Hormone (AMH): Predictor of Follicular Function, Fertility Status and Onset of Menopause.
- 54. Sultana I., Alam J.M., Ali H., and Noureen S., Age Groups of Men.
- 55. Ellenburg J.L., et al., Formalin versus bouin solution for testis biopsies: Which is the better fixative? 2020. 13: p. 2632010X19897262.
- 56. Shokoohi M., et al., Investigating the effects of onion juice on male fertility factors and pregnancy rate after testicular torsion/detorsion by intrauterine insemination method. 2018. 6(4): p. 499–505.
- 57. Prakash D., Singh B.N., and Upadhyay G.J.F.c, Antioxidant and free radical scavenging activities of phenols from onion (Allium cepa). 2007. 102(4): p. 1389–1393.
- 58. Chernukha I., et al., Antioxidant effect of ethanolic onion (Allium cepa) husk extract in ageing rats. 2021. 28(5): p. 2877–2885.
- 59. Porwal M., Khan N.A., and Maheshwari K.K.J.S.P., Evaluation of acute and subacute oral toxicity induced by ethanolic extract of Marsdenia tenacissima leaves in experimental rats. 2017. 85(3): p. 29.
- 60. Pitchaiah G., et al., Anxiolytic and anticonvulsant activity of methanolic extract of allium cepa Linn (Onion) bulbs in Swiss albino mice. 2015. 4: p. 131–5.
- 61. Kemp C.J., et al., Voluntary consumption of ethyl oleate reduces food intake and body weight in rats. 2008. 93(4–5): p. 912–918.
- 62. Momoh B.J., Okere S.O., Anyanwu G.O.J.C.C.M., and Pharmacology, The Anti-obesity Effect of Allium cepa L. Leaves on High Fat Diet Induced Obesity in Male Wistar Rats. 2022: p. 100035.
- 63. Mirabeau T.Y., Samson E.S.J.A.J.o.T, Complementary, and A. Medicines, Effect of Allium cepa and Allium sativum on some immunological cells in rats. 2012. 9(3): p. 374–379.
- 64. Khaki A., et al., Evaluation of androgenic activity of allium cepa on spermatogenesis in the rat. 2009. 68(1): p. 45–51.
- 65. Ara C., et al., Protective Potential of Aqueous Extract of Allium cepa against Tartrazine Induced Reproductive Toxicity. 2022. 42(3).
- 66. Zhao X.-X., et al., Recent advances in bioactive compounds, health functions, and safety concerns of onion (Allium cepa L.). 2021. 8.
- 67. Meraiyebu A., et al., Effects of Aqueous Extract of Onion (Allium cepa) on Blood Parameters in Adult Wistar Rats (Rattus novergicus). 2013.
- 68. Huang W., et al., Effect of onion on blood lipid profile: A meta‐analysis of randomized controlled trials. 2021. 9(7): p. 3563–3572.
- 69. Mattiuzzi C., Sanchis-Gomar F., Lippi G.J.N., Metabolism, and C. Diseases, Worldwide burden of LDL cholesterol: Implications in cardiovascular disease. 2020. 30(2): p. 241–244.
- 70. Rader D.J. and Hovingh G.K.J.T.L., HDL and cardiovascular disease. 2014. 384(9943): p. 618–625.
- 71. Walker W.H. and Cheng J.J.R., FSH and testosterone signaling in Sertoli cells. 2005. 130(1): p. 15–28.
- 72. Ijaz M.U., et al., Pinostrobin alleviates testicular and spermatological damage induced by polystyrene microplastics in adult albino rats. Biomedicine & Pharmacotherapy, 2023. 162: p. 114686. pmid:37044025
- 73. Banihani S.A., Testosterone in males as enhanced by onion (Allium Cepa L.). Biomolecules, 2019. 9(2): p. 75. pmid:30795630
- 74. Shulman L.M. and Spritzer M.D., Changes in the sexual behavior and testosterone levels of male rats in response to daily interactions with estrus females. Physiology & behavior, 2014. 133: p. 8–13. pmid:24813700
- 75. Alrezaki A., et al., Consumption of sage (Salvia officinalis) promotes ovarian function by stimulating estradiol hormone release and controlling folliculogenesis, steroidogenesis, and autophagy. 2021. 33(2): p. 101319.
- 76. Jeje S., et al., Protective role of Allium cepa Linn (onion) juice on maternal dexamethasone induced alterations in reproductive functions of female offspring of Wistar rats. 2021. 4: p. 145–154.
- 77. Lienou L.L., et al., Effect of the aqueous extract of Senecio biafrae (Oliv. & Hiern) J. Moore on sexual maturation of immature female rat. 2012. 12(1): p. 1–9.
- 78. Mansouri E., Keshtkar A., Khaki A.A., and Khaki A.J.I.J.W.H.R.S., Antioxidant effects of allium cepa and cinnamon on sex hormones and serum antioxidant capacity in female rats exposed to power frequency electric and magnetic fields. 2016. 4: p. 141–145.
- 79. Adelakun S.A., Akintunde O.W., Akingbade G.T., and Adedotun O.A.J.P.P., Bioactive component of aqueous extract of Solanum melongena ameliorate estradiol valerate induced ovarian-pituitary dysfunctions in female Sprague–Dawley rats: Histomorphological and biochemical evidence. 2022. 2(1): p. 100175.
- 80. Helen A., Krishnakumar K., Vijayammal P., and Augusti K.J.T.l, Antioxidant effect of onion oil (Allium cepa. Linn) on the damages induced by nicotine in rats as compared to alpha-tocopherol. 2000. 116(1–2): p. 61–68.
- 81. Lee B.K., Jung Y.-S.J.O.M., and Longevity C., Allium cepa extract and quercetin protect neuronal cells from oxidative stress via PKC-ε inactivation/ERK1/2 activation. 2016. 2016.
- 82. Mantawy M., et al., Antioxidant and schistosomicidal effect of Allium sativum and Allium cepa against Schistosoma mansoni different stages. 2012. 16: p. 69–80.