Preparation of Moringa Oliefera Extract:
The plant Moringa oliefera, locally known as the Horse Reddish tree, was collected from fields of the University of Agriculture Faisalabad (UAF) in the month of January. Plants were identified by the Department of Botany, GCUF. Identification number for Moringa 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 100C in the refrigerator separately.
Zinc Oxide Nanoparticles Preparation
Zinc oxide nanoparticles were prepared in the biochemistry laboratory, University of Agriculture Faisalabad (UAF). A one-step solvothermal method was utilized to synthesize the zinc oxide nanoparticles. Firstly, 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 OC. Nanoparticles started to precipitate and the solution became turbid. It was stirred rigorously for two hours after which it was allowed to sit for 1 hour. Excess liquid was removed and the precipitate was twice washed with 50 ml methanol and collected by 20-minute centrifugation at 103 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 (Singh et al., 2012; Zak et al., 2011). The nanoparticles were characterized via, Zetasizer, UV-vis spectroscopy, and scanning electron microscopy.
Experimental Animal Used
A total of fifty healthy male albino Wistar rats weighing 250 ± 10 g were used in the experimental research study. Rats were obtained from the experimental animal house, institute of pharmacy, physiology, and pharmacology, University of Agriculture Faisalabad (UAF). They were kept in well-aerated cages at 24 ± 2 ͦ C in a 12 h 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, University of Agriculture Faisalabad, Pakistan.
Induction Of Experimental 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 rats were kept for 12 hours in a fasting condition. After 72 hours of injection, fasting blood glucose was measured with the help of the blood glucose monitoring system (On-Call EZ II). Blood glucose Test Strips (On Call Plus ACON Laboratories, Inc. USA) and the rats with blood glucose levels over 200 mg/dl were considered diabetic and grouped in the diabetic group.
Experimental Design
Animals were randomly divided into six groups (n = 8). Group 1 of animals was considered a negative control and received normal saline (1ml/kg body wt.) along with a routine diet for the duration of the six-week trial. Group 2, the positive control, received a single intraperitoneal dose of Alloxan monohydrate (130 mg/kg). Group three received a standard commercially available antidiabetic medication glimepiride at a dose rate of 0.1mg/kg of body weight The remaining three treatment groups were treated with ZnONPs, MO, and a combination of the two.
Group | Group I.D | Administration Route | Treatment |
Negative Control | NC | Oral gavage | Routine diet + normal saline (1.0 ml/kg body of body weight) |
Diabetic untreated Positive control | PC | Oral gavage | Alloxan + Routine diet + normal saline (1.0 ml/kg of body weight) |
Standard treatment group | Std. C | Oral gavage | Glimepiride 0.1mg/kg of body weight |
The treatment group was given zinc oxide nanoparticles | ZnONP | Oral gavage | Zinc oxide nanoparticles dosage at 7.5mg/kg of body weight |
The treatment group was given Moringa oleifera | MO | Oral gavage | Moringa oleifera dosage at 250mg/kg of body weight |
The treatment group was given zinc oxide nanoparticles plus Moringa oleifera | ZnONP + MO | Oral gavage | zinc oxide nanoparticles dosage at (7.5mg/kg) of body weight plus Moringa oleifera dosage at 250mg/kg of body weight |
The optimum, safe, and effective dosages of the ZnO nanoparticles and Moringa oleifera were assessed from the already published wealth of literature (Ryu et al., 2014; Srivastav et al., 2016; Abbasalipourkabir et al., 2015; Stohs et al., 2015). ZnONPs were dissolved in water by constant magnetic stirring for 25 minutes 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.
Determination Of Body Weight, Serum Glucose, Serum Insulin, And Serum Glucagon
The body weight was evaluated at regular intervals of seven days. The estimation of the blood glucose was done via the established strip method (Accu-Check by Roche R). The routinely employed enzyme-linked im-immunosorbent assay (ELISA) was employed for the assessment of serum insulin and glucagon (Ray biotech; Cat. Number E-EL-R0425)
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 Number. SE120089; My BioSource R.
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 (1500ml), formaldehyde (500ml), and glacial acetic acid (100ml).
Statistical Analysis
Statistical analysis was conducted by one-way analysis of variance (ANOVA) followed by Duncan’s multiple range (DMR) test at 5% level significance (P < 0.05) (Duncan, 1995; Steel et al., 1996). Statistical Package for the Social Sciences (SPSS) version 16.0 and graph pad prism software version 8.00 were used for statistical analysis.