2.1. Plant Sample
Chenopodium album (CA) were collected from the campus area of Iğdır University, Turkey, for thir research. Viscum album (VA) leaf was collected in Erzurum region, Turkey. PdCI2 was supplied at Sigma Aldrich company. The chemicals were used directly without any purification.
2.2. Preparation and characterization of VA-Pd/AC NPs nanoparticles
a. Preparation of activated carbon
Chenopodium album (CA) is known as a highly abundant invasive plant that grows in and around Igdir University. It was evaluated as the pre-eminent material for the activated carbon source. After the CA was washed, it was preserved in an oven for 24 hours at 80 ° C. Then, it was ground in intervals of 150-250 micrometers. It was kept for 30 minutes (in nitrogen environment) at a constant speed of 5oC/minute at 600 °C, for carbonization. The sample was cooled to room temperature. It was left in 0.1 M HCl solution throughout the day. Then, it was filtered and washed with distilled water until pH 7. The sample was left to be preserved at 105 °C for 24 hours.
b. Viscum album extracts preparation
In this research, Viscum album (VA) extract was used as the reducing agent. The (VA) leaf was thoroughly washed three times with distilled water to remove impurities and allowed to dry for 24 hours at 105 ° C. The dried VA samples were ground into very small pieces. A solution, containing 100 mL of deionized water and 10 g of VA was prepared, for VA extracts. The resulting mixture of VA was boiled for 2 h, and cooled down to room temperature. Then it was made ready to work by being filtered under vacuum.
c. Synthesis of VA-Pd/AC NPs
In green synthesis of VA-Pd/AC NPs, a solution containing 0.02 g of PdCI2, 0.02 g of CA in 40 mL of deionized water and 10 mL of VA extract were prepared in a glass vial and vigorously mixed at room temperature. The progress of the reaction was controlled by the color change that shows nanoparticle forming (Leal-Duaso et al., 2021). Thereafter, this solution was centrifuged at 12000 rpm for 10 minutes. The obtained sample was washed thoroughly and kept in the oven at 75 ° C for 24 hours (Fig. 1).
X-ray diffraction (XRD) analyzes of VA-Pd/AC NPs were performed with Panalytical EMPYREAN device (Malvern Panalytical Ltd, Malvern, United Kingdom) operating at 45 kV and capable of CuKα radiation. XRD analyzes were done to examine the XRD peaks and crystal size of VA-Pd/AC NPs. In addition, to reveal the distribution of Pd metal nanoparticles on AC surface and the average particle size, transmission electron microscopy (TEM) analyzes were performed with the Hitachi HT7800 (Hitachi Ltd, Tokyo, Japan) device operating at 200 kV. The average particle size was calculated using Imagej and origin package programs by counting spherical particles in TEM images (Calimli, 2020).
2.3. In vivo study
As living material of the research, adult wild-type zebrafish were maintained from Izmir Biomedicine and Genome Center. The used zebrafish larvae were younger than 5-days-old. So no need to require any license (Directive 86/609/EEC and EU Directive, 2010/63/EU). They were kept in 28°C with a 14-h light/10-h dark cycle, according to standard procedures (Westerfield, 1995). Fish were fed twice a day. In the first meal at 9 a.m. flake food (TetraMin Tropical Flakes 48% protein, 8% fat, and 2% fiber) were used, and in the second one, at 3 p.m. feeding was done with artemia.
b. Zebrafish embryo Exposition to VA-Pd/AC NPs
The study was planned based as a semi-static test with 4 replications. For preparing the different trial concentrations (10, 50, 100 and 500 mg/L); VA-Pd/AC NPs (2000 mg/L) stock solution had sonicated (50/60 kHz, Huber Minichiller Diagenode, Germany) in ultra-pure water with E3 (0.17 mM KCl, 0.33 mM MgSO4, 5 mM NaCl, and 0.33 mM CaCl2) for 3 h. These solutions were renewed daily and before solution regeneration, sonications were done for 30 min (Federici et al., 2007; Rocco et al., 2015). This procedure is known to be an effective method for dispersing and preventing agglomeration of nanoparticles of water suspensions (Sato et al., 2008).
The trial was organised of five groups. The first one was as control group (the embryo was preserved in E3 medium). For each group, 30 embryos were used and embryos were preserved in E3 medium. In the experiment, embryos were added to 5 ml test solution (VA-Pd/AC NPs) in different 5 plates each one including 30 embryos for 96 hpf. Each trial medium was renewed daily and all embryo groups were exposed to nanoparticle solution at the same conditions (28°C).
c. Determination of survival rate, hatching rate, and embryo morphological alterations
The larvae and embryos of all groups were visualized by a stereomicroscope (SZX16 Olympus microscope with SC50 Olympus camera) for evaluating the alterations after the application time (24-96 h). Not observe heartbeat in larvae at 24 hpf was accepted as not living.
2.4. Histopatological examination
The zebrafish larvae samples were detected in 4% paraformaldehyde solution for 48 hours. Following the routine tissue procedure, the larvae were embedded in paraffin blocks and 4 µm thick sections were taken from the blocks. Histopathological sections were stained with the hematoxylin-eosin (HE) staining procedure and examined with light microscopy (Olympus BX 51, Germany). Findings were evaluated as none (-), mild (+), moderate (++) and severe (+++) according to immune positivity.
2.5. Immunfloresans examination
After deparaffinization and dehydration processes, the sections (taken on poly-l-lysine slides) were kept in distilled water. It was boiled 2 times for 5 minutes to mask the antigens in the cells and allowed to cool at room temperature. Protein block was dropped onto the sections, washed with PBS for 10 minutes. Primary antibodies (caspase-3, Cat No: sc-56053 Diluent Ratio: 1/100, Santa Cruz, nNOS Cat No: ab16650 Diluent Ratio: 1/100, Abcam) were prepared and applied according to the usage conditions. After washing with PBS, an immunofluorescence antibody was used as a secondary marker (FITC Cat No: ab6717 Diluent Ratio: 1/1000, Abcam) and was washed in distilled water after being kept in the dark for 45 minutes. Then, DAPI with mounting medium (Cat no, D1306 diluent ratio: 1/200, Thermo) was dropped and kept in the dark for 5 minutes, and the tissues were washed with distilled water, covered with a coverslip. The sections were examined under a fluorescence attachment microscope (Zeiss Leica DM 1000 Germany). The findings were evaluated as none (-), mild (+), moderate (++) and severe (+++) according to immune positivity.
2.6. Statistical analyses
Statistical analysis of all data was performed by the SPSS program (SPSS for Windows, version 20.0). All data are given with statistical results, standard error, and mean values. The difference between the groups was determined by one-way (ANOVA) analysis of variance and then evaluated by Tukey's test (p < 0.05). The Kruskal-Wallis test (one of the nonparametric tests) was used for the analysis of the differences among the groups data (obtained semi-quantitatively) in histopathological examination, and the Mann Whitney U test was used for the comparison of paired groups.