Materials and Chemicals
Fresh matured leaves of Psidium guajava were collected from a local residence garden. To maintain the freshness of the leaves they were stored in a clear plastic bag in the lab refrigerator at 4oC. Silver nitrate (AgNO3, 99.8 - 100% pure), phosphate buffer, and acetylthiocholine iodide were purchased from Sigma – Aldrich (Merk, St. Louis, MO, USA). Thiobarbituric acid (TBA), Trichloroacetic acid, 5,5`dithiobis (2-nitrobenzoic acid), Sulphanilamide, and N – (1 – naphthyl) ethylenediamine were purchased from Biodiagnostic Co., Giza, Egypt.
Preparation of plant extract
Psidium guajava leaf extract (PGLE) was prepared as described by Bose and Chatterjee (2016) with slight modifications. The leaves were thoroughly washed under tap water and then soaked and washed with distilled water to remove any adsorbed dust and other particles. The leaves were then left to dry at room temperature on filter papers. 10 g of leaves were weighed and put in a chopper with 120 mL of distilled water and leaves were chopped until a green solution of fine pieces of leaves was produced. The produced mixture was then filtered using Whatman No.1 filter paper. The filtrate which was yellowish in color was then poured into test tubes and stored in the freezer as a stock solution for further use during the experiment.
Synthesis of silver nanoparticles
AgNPs were synthesized according to Bose and Chatterjee (2016) with some modifications as follows: 20 ml of a 5 mM AgNO3 solution was poured into a 50 ml Erlenmeyer flask and 0.2 ml of PGLE was added to the 20 ml of AgNO3 solution. The mixture was heated to 90 oC and stirred at 200 rpm for 20 minutes on a hotplate magnetic stirrer (MSH-20D, Daihan Scientific, Wonju, South Korea). The color of the stirred solution changed from colorless to dark orange or light brownish color, which confirmed the synthesis of the AgNPs and indicated the reduction of the AgNO3 solution by the PGLE to produce the AgNPs. The color of the produced silver nanoparticle aqueous solution was darkened with time, producing darker reddish-brown color indicating a further reduction of AgNO3 by the PGLE solution till its completion.
Size optimization of biosynthesized AgNPs
Optimization of AgNPs was accomplished via the change in the following parameters: molarity, pH, temperature, and volume of PGLE during the synthesis process. Different molarities of AgNO3 solution were investigated to optimize the particles size, these were: 1 mM, 3 mM, 5 mM, 10 mM, 13 mM, and 15 mM. Different pH values of the AgNO3 solution were investigated to optimize the particles size, these were pH: 6, 7, 9, 11, and 12. The pH was controlled by 0.1 N NaOH. The particles size was also optimized by synthesizing the AgNPs at different temperatures: 30 oC, 50 oC, 70 oC, and 90 oC while keeping other parameters constant. The PGLE volume was also changed through the synthesis trials to optimize the particle size. The extract volumes investigated were: 0.2, 0.4, 0.6, 0.8 and 1mL that were added to the 20 mL of AgNO3 solution. The different particle size optimization parameters are summarized in Table 1.
Characterization of Silver Nanoparticles
The size, shape, and surface charge of the AgNPs along with other factors play an important role in their effect on the biological system; therefore, the characterization of the synthesized nanoparticles is important to determine their shape, size, and surface area properties, and stability. The characterization of the synthesized and optimized AgNPs was done through the following techniques: UV-Vis spectrophotometry, Fourier transform infrared spectroscopy (FTIR), Transmission electron microscopy (TEM), and Dynamic light scattering (DLS).
UV- Vis Spectroscopy
AgNPs have a unique absorption peak known as the surface plasmon resonance (SPR) peak which usually occurs, for the PGLE biosynthesized particles, in the range of 419 - 460 nm (Bose and Chatterjee, 2016; Sharmila et al., 2018; Sougandhi and Ramanaiah, 2020). This peak is characteristic of the silver nanoparticles which could be used as a confirmation for the synthesis of silver nanoparticles; hence the absorption peak of the PGLE was also measured in the same range for further confirmation. The UV-Vis spectroscopy was carried out in the range of wavelengths of 350 – 650 nm on a UV-Vis Spectroscopy (Alpha – 1502, Shanghai Lab – spectrum, instruments Co, Ltd).
Fourier Transform Infrared Spectroscopy
The structures and compositions of the dried powder of the AgNPs, AgNO3, and PGLE were measured and analyzed using the FTIR technique in the range of 400 – 4000 cm-1 at a resolution of 4 cm-1 using an FTIR Spectrometer (FT/IR-4100 type A, JASCO, Japan).
Transmission Electron Microscopy
The size and morphology of the particles were determined by transmission electron microscope imaging using JEOL, JEM-1230, high performance, high contrast, 40 – 120 Kv, transmission electron microscope (TEM). The particles were placed on a carbon grid to be examined and captured. The size distribution of the captured particles was done by the image j software.
Dynamic Light scattering
The hydrodynamic size, size distribution, and zeta potential of the produced nanoparticles were measured by the dynamic light scattering device (Malvern Zetasizer, nano – Zs 90, Malvern Instruments Ltd., UK).
Thirty-four adult male Wistar rats were used in the present study. Their average weight was 152 ± 20 g. The animals were purchased from the national institute of cancer, Cairo, Egypt. On arrival, they were left for one week to acclimatize prior to the experimental procedures. They were maintained in standard environmental conditions of humidity, temperature, and 12 hours day and night cycles. The animals had access to food and water ad libitum.
The animals were divided into four groups, control (n = 10) received orally 0.9% saline solution, the second, third, and fourth groups (n = 8 / group) received orally AgNPs at 0.5, 5, 10 mg/kg, respectively. All rats in different groups were administered daily for 14 days. At the end of the experiment, the animals were sacrificed by sudden decapitation on the 14th day, one hour after the last administration. The brain of each rat was dissected and divided into two halves. The right half of the brain of 3 animals, from different groups, was separated for the ultrastructural transmission electron microscope (TEM) imaging. In the rest of the animals, the cortex and hippocampus brain regions were dissected. Each brain region was weighed and kept frozen at –30 oC until the biochemical analyses.
Ultrastructural examination of brain tissues by electron microscope
The ultrastructural examination was carried out to detect the presence of AgNPs in brain tissues by TEM. The samples were preserved in ice-cold glutaraldehyde solution as a first fixation step for examination. The tissues were then rinsed and fixed again with osmium tetroxide for better contrast of the image. Then they were dehydrated by different concentrations of ethanol and eventually embedded in epoxy resin to make them firm enough to handle the pressure of the cutting process. Ultra-thin sections, at approximately 75 – 90 μm in thickness, were obtained from the prepared resin by a Leica EM UC6 ultramicrotome (Leica Microsystems Co.) and examined by TEM, JOEL (JEM-1400 TEM) at direct magnification 2500x to 25000x at HV=80.0 KV with a scale bar of 500 nm - 2 mm.
Neurochemical analyses were carried out on the hippocampus and cortex areas of the rat’s brain. Each brain area was homogenized in ice-cold tris hydrochloric acid (tris HCL) buffer of pH 7.4. The homogenate was then centrifuged for 30 minutes at 4 oC at 5000 rpm in a high-speed cooling centrifuge (Type 3k-30, Sigma, Germany). Then the following parameters were measured.
Oxidative Stress parameters
Lipid peroxidation was measured according to the method of Ruiz-Larrea et al., (1994). This method depends on measuring malondialdehyde (MDA) as an indicator of lipid peroxidation. A 200 μL of the tissue homogenate was added to a 1000 μL of the thiobarbituric acid (TBA) (chromogen), and heated in a boiling water bath for 30 minutes, then left to cool. The absorbance of the resultant solution was measured at 534 nm.
Reduced glutathione (GSH) was measured on the homogenates of the hippocampus and cortex using the method of Beutler et al., (1963). A 250 μL of the tissue homogenate was added to a 250 μL of trichloroacetic acid, mixed well, allowed to stand for 5 minutes, and centrifuged at 3000 rpm for 15 min. 250 μL of the supernatant was then added to a 500 μL buffer solution with 250 μL of 5,5`dithiobis (2-nitrobenzoic acid). The absorbance of the resultant solution was measured at 405 nm.
The method used to measure nitric oxide (NO) uses Griess reagent based on the method of Montogomery and Dymock (1962). A 100 μL of tissue homogenate was added to a 1000 μL of sulphanilamide and left to stand for 5 minutes, then a 100 μL of N– (1– naphthyl) ethylenediamine was added to the mixture. The absorbance of the resultant solution was measured at 540 nm.
The activity of acetylcholinesterase (AchE) was determined according to the method of Gorun et al. (1978). A 10 μL of tissue sample was added to a 50 μL of the enzyme’s substrate (acetylcholine) and a 140 μL of 20 mM phosphate buffer (pH 7.6). The mixture was incubated at 38oC for 10 min. 1800 μL of 5,5`dithiobis (2-nitrobenzoic acid) was then added. The absorbance of the resultant solution was measured at 415 nm.
Measurements of monoamines neurotransmitters
The right half of the cortex was homogenized in 3 mL of an ice-cold solution of acidified n-butanol. The homogenates were centrifuged at 5000 rpm for 5 min. 2.5 mL of the supernatant was added to 1.6 mL of (0.2 N) acetic acid and 5 mL of heptane. The mixture was centrifuged again at 5000 rpm for 5 min to separate the aqueous layer from the alcoholic layer. The aqueous part was used for the estimation of dopamine (DA), norepinephrine (NE), and serotonin (5-hydroxytryptamine; 5H-T) according to the fluorometric method described by Ciarlone (1978). The fluorescence of DA, NE, and 5H-T was measured using a spectrofluorometer (model Jasco-FP-6500, Japan) with a source of xenon arc lamp 150 W at different excitation and emission wavelengths. DA was measured at 320 nm and 370 nm; NE was measured at 380 nm and 460 nm and 5H-T was measured at 355 and 470 nm.
The obtained data were analyzed statistically by one-way ANOVA followed by Duncan’s post hoc to compare different groups. The difference between groups was considered significant at P-value < 0.05. Data are presented as the mean ± SEM. To perform these analyses SPSS v26.0 was used.