Physicochemical Assessment of Zinc Oxide Nanoparticle and Moringa oleifera Supplementation on the Male Reproductive System of a Diabetic Rat Model

,


Introduction
Diabetes mellitus is a metabolic disorder associated with insufficient insulin secretion, insensitivity of the endogenous insulin to its receptors, and progressive failure of pancreatic β-cells resulting in hyperglycemia [1].It is a syndrome resulting from the interactions of environmental and hereditary factors that affect metabolism involving proteins, carbohydrates, and fats in addition to the risk of damaging the pancreas, liver, kidney, and testes [2,3].There is a crucial link between diabetes and compromised reproductive functioning in males.Diabetes mellitus elicits a decrease in testosterone levels along with a reduction in the functionality and concomitant weight of the testes, vas deferens, epididymis, prostate gland, and seminal vesicle.Moreover, testicular histology of chronic diabetic subjects reveals a decrease in testicular volume and weight along with lysis of seminiferous tubules, disorientation of spermatids, and reduction in Leydig cells [4].Diabetes mellitus is treated and managed by insulin and oral hypoglycemic drugs [5].However, effective therapy for diabetes mellitus is still not available in modern medicine, and oral synthetic hypoglycemic drugs are not completely successful in managing diabetes [6].
Zinc plays a crucial role in different metabolic pathways, including glucose metabolism [7].Zinc is also known to augment the functioning of insulin and plays a vital role in insulin biosynthesis, storage, and secretion [8].Bionanotechnology is the field of material science that arises at the intersection of biology and nanotechnology.Nanoparticles are the main products of this field.Nanoparticles are defined as ultra-small entities measuring 1-100 nm.They are being employed in fields as diverse as therapeutics, diagnostics, drug delivery, medical imaging, tumor suppression, etc.Within the context of DM, zinc oxide nanoparticles have been investigated to have promising potential and merit further investigations [9].
Zinc oxide nanoparticles (ZnONPs) are agents that have recently been utilized to deliver zinc and have implications in many disease therapies including diabetes mellitus [10].ZnONPs have been shown to effectively mitigate diabetesinduced pancreatic structural, ultrastructural, and functional injuries [11].Recent investigations have revealed that Zn and ZnONPs supplementation facilitates the normalization of blood glucose and serum insulin levels.It also restores the sensitivity of the insulin receptor to insulin [12].
Recent studies have demonstrated that supplementation with ZnONPs in rats exposed to nicotine significantly mitigates the adverse effects of such exposure by reducing oxidative stress and enhancing the expression of steroidogenic enzymes, thereby potentially enhancing male fertility [13].Thus, the development of a zinc-based agent would be promising in the treatment of diabetes and its associated complications as zinc supplements have shown ameliorating effects in various studies [10,14].In this regard, ZnONPs merit further investigation within the context of their antidiabetic potential and their effect on the male reproductive system as this aspect has not been fully explored previously.Herbs that contain various phytoconstituents, such as flavonoids, terpenoids, saponins, carotenoids, alkaloids, and glycosides, have been reported to possess antidiabetic properties.In developing countries, medicinal plants are used to treat diabetes mellitus to overcome the burden cost of conventional medicine [15].Moringa oleifera (MO), also known as the Drumstick tree, has been used to mitigate the effects of diabetes due to its antidiabetic properties.Leaves, fruits, and stem bark of this plant have been reported to have antidiabetic activity [16][17][18][19][20].A recent study in alloxan-induced diabetic rats found that M. oleifera leaves had a hypoglycemic effect and prevented body weight loss [21].However, the curative properties of this medical plant in alloxan-induced diabetes mellitus associated with the testicular gonadal axis and reproductive functionality are poorly understood and yet to be studied in detail.In the present study, we undertake such an endeavor.Hence, we aim to investigate the effects of ZnONPs singly and in combination with MO on alloxan-induced diabetic rats and the male rat gonadal tissue.

Experiments
2.1.Materials and Methods 2.1.1.Preparation of M. Oliefera Extract.The plant M. oliefera, oliefera, locally known as the Horse Reddish tree, was collected from fields of the University of Agriculture Faisalabad (UAF) in January.Plants were identified by the Department of Botany, GCUF.Identification number for M. oliefera was 284-bot-22.Plant leaves were separated and washed gently with water, cut into pieces, and kept for drying under shade.The leaves were dried completely after 25 days and then subjected to crushing and grinding to obtain a coarse powder.It was subjected to maceration.After complete maceration, the powder was filtered by using the Whatman number 2 filter paper twice.The filtrate obtained was evaporated using a rotary evaporator under reduced pressure until all the solvent evaporated leaving behind thick plant residue.A concentrated paste was obtained by drying in Petri plates.The leaf extracts were stored in bottles below 10°C in the refrigerator separately.
2.1.2.Zinc Oxide Nanoparticles Preparation.Zinc oxide nanoparticles were prepared in the Biochemistry Laboratory, UAF.A one-step solvothermal method was utilized to synthesize the zinc oxide nanoparticles.First, the dissolution of zinc acetate dihydrate (13.2 mmol) was undertaken in alcohols.A potassium hydroxide solution (28.4 mmol) was dropwise subjected to addition to the zinc acetate solution at 52°C.Nanoparticles started to precipitate and the solution became turbid.It was stirred rigorously for 2 hr after which it was allowed to sit for 1 hr.Excess liquid was removed and the precipitate was twice washed with 50 ml methanol and collected by 20 min centrifugation at 10 3 RPM.It was allowed to dry at room temperature for 1 day.The obtained solid was crushed to obtain white powder.It was subsequently dispersed in PVA with an ultrasonicator.The nanoparticles were stable and equally dispersed [22,23].The nanoparticles were characterized via, Zetasizer, UV-vis spectroscopy, and scanning electron microscopy.

Experimental Animal Used.
A total of 50 healthy male albino Wistar rats weighing 250 AE 10 g were used in the experimental research study.Rats were obtained from the experimental animal house at the Institute of Pharmacy, Physiology, and Pharmacology, UAF.They were kept in well-aerated cages at 24 AE 2°C in a 12 hr light and dark cycle, with surrounding humidity (40%-60%).For 6 weeks, the animals were provided ad libitum standard diet and water.Cleaning and changing water and food were done for all animals twice a day.Ethical rules on care and utilization of animal models for human disease were taken from the institutional bioethics committee (Ref no.145), University of Agriculture Faisalabad, Pakistan.).The trial was run in triplicate.Group 1 of animals was considered a negative control (NC) and received normal saline (1 ml/kg body wt.) along with a routine diet for the duration of the 6-week trial.Group 2, the positive control (PC), received a single intraperitoneal dose of Alloxan monohydrate (130 mg/kg).Group 3 received a standard commercially available antidiabetic medication glimepiride at a dose rate of 0.1 mg/kg of body weight.The remaining three treatment groups were treated with ZnONPs, MO, and a combination of the two (Table 1).The optimum, safe, and effective dosages of the ZnO nanoparticles and M. oleifera were assessed from the already published wealth of literature [24][25][26][27].ZnONPs were dissolved in water by constant magnetic stirring for 25 min at 37°C.The amount of ZnONPs and MO leaf extract for each adult rat was calculated based on their weight.The suspension was administered orally to each adult albino rat via oral gavage.

Induction of Experimental
2.1.6.Determination of Body Weight, Serum Glucose, Serum Insulin, and Serum Glucagon.The body weight was evaluated at regular intervals of 7 days.The estimation of the blood glucose was done via the established strip method (Accu-Check by Roche ® ).The routinely employed enzyme-linked im-immunosorbent assay (ELISA) was employed for the assessment of serum insulin and glucagon (Ray biotech; Catalog no.: E-EL-R0425).
2.1.7.Determination of Reproductive Hormones.For quantitative determination of follicle-stimulating hormone (FSH), standard ELISA was employed (Rat FSH ELISA Kit Catalog no.: MBS2502190; My BioSource R).Luteinizing hormone (LH) was assessed by Rat LH ELISA Kit; Catalog no.: MBS764675; My BioSource R. Rat testosterone was evaluated via the rat testosterone ELISA kit; Catalog no.: SE120089; My BioSource R.
2.1.8.Histopathological Examination of Testes.At the end of the trial, rat testes were isolated, and preserved in Bouin's solution for histopathological examination.Then, tissues were embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E).The composition of Bouin's solution is saturated picric acid (1,500 ml), formaldehyde (500 ml), and glacial acetic acid (100 ml).
2.1.9.Statistical Analysis.Statistical analysis was conducted by one-way analysis of variance (ANOVA) followed by Duncan's multiple ranges tests at 5% level significance (P <0:05) [28].Statistical Package for the Social Sciences (SPSS, version 16.0 and GraphPad Prism software version 8.00) was used for statistical analysis.

Results and Discussion
3.1.Nanoparticle Characterization: Zetasizer Analysis, UV-Vis Analysis, and Scanning Electron Microscopy Analysis.The spectroscopic analysis (V-730 UV-Visible Spectrophotometer, Jasco R) of ZnO nanoparticles was undertaken and showed a distinct peak at 370 nm, and it was found to agree with various research [23,29] (Figure 1(a)).The nanoparticle size was corroborated by evaluating the nanoparticles with a zeta sizer (Malvern Zetasizer; Nano-ZS90).The size of the ZnO nanoparticles was noted to be in the range of 25-35 nm, as shown in Figure 1(b).Scanning electron micrography (JSM-IT200 InTouch Scope™; Jeol R) revealed the formation of spherical and rhomboidal nanoparticles within the specified range of 25-35 nm, as shown in Figure 1(c).

Assessment of Zinc Oxide Nanoparticle and Moringa oliefera and a Combination Thereof on the Concentration of Serum Glucose, Serum Insulin, and Glucagon and Mean
Body Weight.The mean body weight was significantly normalized in the group ZnONP + MO, i.e., 230 AE 6 g as compared with the PC, i.e., 162 AE 4 g; P ≤ 0:05.This effect was markedly better than the MO treatment alone revealing a mean body weight of 188 AE 3 g; P ≤ 0:05 (Figure 2).The ZnONP + MO combination exerted a normoglycemic effect in the treatment group, i.e., 154.4 AE 4.5 mg/dl in contrast with the PC, i.e., 315.7 AE 3 mg/dl.This effect was followed by the effect of the M. oliefera treatment group, i.e., 212.6 AE 7 g.P ≤ 0:05 (Figure 2(a)).The NC revealed 16.4 UI/ml of insulin    Diabetes, now a day, has become one of the leading metabolic disorders in the world.It is on the rise, and its prevalence is increasing with each passing day, especially in the middle-income countries of the world.The available therapies have quite a few adverse effects in the present study, diabetes was induced in Wistar albino rats by alloxan monohydrate injected intraperitoneally at a dose of 130 mg/kg of body weight.Alloxan is a glucose analog having a toxic effect on pancreatic β-cells; it readily destroys them when administered to rodents.It was quickly and selectively taken ken up by β-cells followed by the production of reactive oxygen species ROS, superoxide radicals, and hydrogen peroxide by the redox cycle [30].Consequently, the antihyperglycemic and gonad protective effect of M. oliefera (MO) and zinc oxide nanoparticles (ZnONPs) alone and in combination Journal of Nanomaterials thereof was assessed.The one-step solvothermal method utilized in the present study was found to be a highly effective, cost-effective, and efficient method for nanoparticle synthesis.The nanoparticles synthesized were generally of uniform shape and within the size range of 25-35 nm.We report that one-step solvothermal process for zinc oxide nanoparticle synthesis can be upscaled for the bulk amount of nanoparticle production as well.The current study was designed to investigate the antidiabetic activity of the plant M. oleifera in Alloxan-induced diabetic rats.This plant was selected because it is taken as food and is also used as a medicinal plant for the treatment of the respiratory system, digestive system, ulcers, urinary system, and for inflammation.It has been reported to be used traditionally as an antidiabetic agent.The treated diabetic rats with a high dose of M. oleifera extract recorded a significant decrease in blood glucose level and a significant increase in insulin and this was in agreement with a previous study [31].Concerning the antidiabetic effects of MO, it is plausible that its active constituents are responsible for it.These include the antidiabetic constituents (glucoside, quercetin, chlorogenic acid, and moringinine).The therapeutic property of this plant may be due to its regenerative potential affecting pancreatic β-cell, increasing insulin secretion and uptake in skeletal muscles, and decreasing insulin resistance.However, further studies are needed to evaluate the precise mechanism of action for this beneficial herbal plant [32].Oral administration of zinc oxide nanoparticles resulted in significant antidiabetic effects, that is, improved glucose tolerance, higher serum insulin, and reduced blood glucose.Nanoparticles were systemically absorbed resulting in elevated zinc levels.Nanoparticles were safe up to 7.5 mg/kg body weight and previous studies have shown that they are 6 Journal of Nanomaterials safe up to 300 mg/kg dose in rats [11].In the present study, the ZnONP + MO combination exerted a normoglycemic effect in the treatment group F, i.e., 154.4 AE 4.5 mg/dl as in contrast with the PC, i.e., 315.7 AE 3 mg/dl.This effect was followed by the effect of M. oliefera treatment group, i.e., 212.6 AE 7 g.The main causal rationale behind these observed effects probably is because zinc has been shown to have an antihyperglycemic effect and it lowers blood glucose by increasing the uptake of glucose from the blood into the cells.Our observation agrees with earlier studies [33,34].Our work reveals that ZnONP + MO had a protective effect on the overall parenchymatous structure of the male reproductive tract including the seminiferous tubule shape, spermatogenesis activity, and presence or absence of Leydig cell.Within this context, research has already shown that the bulk form of zinc had a protective effect on the rat male reproductive system [35].
The present study supports their finding and we report that the combination of ZnONP + MO can plausibly be a suitable alternative to bulk supplementation by zinc.However, further work is still required to elucidate whether the protective effect of ZnONP + MO is localized or global within the context of the entire biochemical machinery of the organism.

Conclusion
Diabetes adversely affects the rat's testes.There is increased blood glucose level, decreased body weight, decreased testicular weight, poor oral glucose tolerance, and unusual hematological parameters.Histopathology showed the disrupted epithelium of seminiferous tubules, the empty lumen of the tubules indicated no spermatogenic activity, and the destruction of Leydig cells.Diabetes-induced (morphological and histopathological) changes are recovered with zinc oxide nanoparticles and M. oleifera having a synergistic antidiabetic effect and protective effect on the rat reproductive system and LH, FSH, and testosterone levels.Histopathology of treated groups showed intact epithelium and sperms in the lumen of seminiferous tubules.

3. 4 .
Effect of Glimepiride, Zinc Oxide Nanoparticle, and Moringa oliefera on Histopathological Changes Induced by Alloxan in Testes of Rats.Histopathological changes in the testes of rats treated with alloxan alone showed the distortion in the epithelium of seminiferous tubules (Figure4(a); 40x; H&E stain).In the PC, the lumen of tubules was empty, which indicates no spermatogenic activity (Figure4(b); 40x; H&E stain).Immature spermatids were seen and tubules were reshaped, which ultimately reduced the testes' weight in the standard control (Figure4(c); 40x; H&E stain).Distorted seminiferous epithelium with an empty lumen was seen when treated with ZnO nanoparticles alone (Figure4(d); 40x; H&E stain).Destruction of the Leydig cells was noted which results in decreased testosterone production (Figure4(e); 40x; H&E stain).However, the histopathology of testes isolated from rats treated with the zinc oxide nanoparticle and M. oliefera combination showed almost normal spermatogenic activity, the lumen of seminiferous tubules contained sperms, normal spermatids, outer epithelium layer of seminiferous tubules was intact (Figure4(f); 40x; H&E stain).
Diabetes.Experimental diabetes mellitus was induced by a single intraperitoneal injection of alloxan monohydrate (Sigma Chemical Co., Poole, Dorst, UK) having a dose rate of 130 mg/kg.Before induction 2.1.5.Experimental Design.Animals were randomly divided into six groups (n = 8