In Vitro and In Vivo Evaluation of Silver Nanoparticles Phytosynthesized Using Raphanus sativus L. Waste Extracts

The aim of the current paper is the development of phytosynthesized silver nanoparticles mediated by Raphanus sativus L. extracts obtained through two extraction methods (temperature and microwave) and to test their potential application for controlling apple crops pathogens. The phytosynthesized materials were characterized by X-ray diffraction, scanning electron microscopy, and transmission electron microscopy. All the materials were evaluated in terms of antioxidant and in vitro antimicrobial activity (against bacteria, molds, and yeast: Escherichia coli ATCC 8738, Staphylococcus aureus ATTC 25923, Pseudomonas aeruginosa ATCC 9027, Salmonella typhimurium ATCC 14028, Candida albicans ATCC 10231, Venturia inaequalis, Podosphaera leucotricha, Fusarium oxysporum ATCC 48112, Penicillium hirsutum ATCC 52323, and Aspergillus niger ATCC 15475). Considering the results obtained in the in vitro assays, formulations based on nanoparticles phytosynthesized using Raphanus sativus L. waste extracts (RS1N) were evaluated as potential antifungal agents for horticultural crops protection, against Venturia inaequalis and Podosphaera leucotricha through in vivo assays. For the DPPH assay, the inhibition (%) varied between 37.06% (for RS1N at 0.8 mg/mL concentration) and 83.72% (for RS1N at 7.2 mg/mL concentration) compared to 19.97% (for RS2N at 0.8 mg/mL) and only 28.91% (for RS2N at 7.2 mg/mL). Similar results were obtained for RS3N (85.42% inhibition at 7.2 mg/mL) compared with RS4N (21.76% inhibition at 7.2 mg/mL). Regarding the ABTS assay, the highest scavenger activity values were recorded for samples RS1N (91.43% at 1.6 mg/mL) and RS3N (96.62% at 1.6 mg/mL).


Evaluation of chemical composition of radish leaf extracts
Evaluation of total phenolic content of the extracts For the quantification of the total phenolic content, 150 μL of 10% diluted Folin-Ciocalteu reagent and 120 μL of 0.7 M Na2CO3 were added to 30 μL of the extracts dissolved in their solvents (240 μg/mL) and the mixtures were incubated for 30 min with shaking at room temperature. Absorbance was spectrophotometrically measured at 765 nm and the results expressed as milligrams of gallic acid equivalents (GAE) per gram of dry weight using Equation S1: where: Ctp-total phenolic content (mg/g) in GAE (gallic acid) equivalent, C-Concentration of gallic acid obtained from calibration curve in mg/mL, V-volume of extract in mL, m-mass of extract in grams. The calibration curves were constructed using analytic standards gallic acid (Sigma-Aldrich, Germany). The experiments were carried out in triplicate.

Evaluation of biological properties
1.2.1. DPPH assay DPPH is a stable free radical, at ambient temperature, presenting strong absorbance at 517 nm; in the presence of an antioxidant, it is reduced, the solution becomes yellow to colorless and the absorbance decreases. For the assay, 0.5 mL of different dilutions from analysed extracts or silver nanoparticles (concentration range 0.8-7.2 mg/mL) were mixed  with 3 mL of 0.1 mM DPPH ethanolic solution. The mixture was kept in the dark at room temperature and the absorbance of the DPPH solution was measured at λ = 517 nm before (Astart) and 30 min. after adding the test solutions (Aend). Ethanol was used as blank. The ability to scavenge the DPPH free radical was calculated using Equation S2: The extracts concentration that inhibited 50% of the DPPH free radical (EC50, mg/mL) was determined graphically from the linear regression curve plotted between percent (%) of inhibition and solutions concentration (mg/mL). All measurements were performed in triplicate.

ABTS • + radical cation scavenging assay
The ABTS radical cation was generated by incubation of ABTS diammonium salt (7 mM) with potassium persulfate (2.45 mM) in the dark, at room temperature for 16 h. The absorbance of the ABTS radical solution was equilibrated to a value of 0.700 ± 0.02 at λ = 734 nm after dilution with ethanol. 0.3 mL of several dilutions of analyzed extracts or silver nanoparticles (concentration range 0.004-0.6 mg/mL) were mixed with 3 mL reagent and the absorbance of the ABTS • + cation radical was measured at λ = 734 nm, before (Acontrol) and 6 min. after adding the test solutions (Asample). The scavenging activity was calculated using Equation S3: The extracts concentration that inhibited 50% of the ABTS • + free radical (EC50, mg/mL) was determined graphically from the linear regression curve plotted between percent (%) of inhibition and solutions concentration (mg/mL). All measurements were performed in triplicate.

Ferric reducing power assay
For the assay, 2.5 mL of several dilutions of analyzed extracts or silver nanoparticles (concentration range 0.8-7.2 mg/mL) were mixed with 2.5 mL of 0.2 M phosphate buffer (pH 6.6) and 2.5 mL of 1% potassium ferricyanide. Samples were kept at 50 °C in a water bath (Raypa, Spain) for 20 min. After, 2.5 mL of 10% trichloroacetic acid was added and the mixture was centrifuged at 2500 rpm for 5 min. (Universal 16 centrifuge). The upper layer (2.5 mL) was mixed with 2.5 mL water and 0.5 mL of a 0.1% ferric chloride solution. The absorbance was measured at λ = 700 nm, after 10 min., against a blank that contained all reagents except the tested solutions. A higher absorbance indicates a stronger reducing power.
The solutions concentration providing 0.5 of absorbance (EC50, mg/mL) was determined graphically from the linear regression curve plotted between absorbance and solutions concentrations (mg/mL). All measurements were performed in triplicate.

In vivo antifungal activity
Young seedlings, originated from "Idared" cvs. were inoculated in greenhouse with a suspension of 4.5 × 10 5 conidia/mL of V. inaequalis or P. leucotricha applied using a manual atomizer under controlled conditions (temperature 18-20 °C and humidity 80-100 %). The conidial suspension was provided from the naturally infected leaves of apples of three varieties: "Idared", "Starkrimson" and "Golden Delicious" cvs. from demonstrative apple plots of Research Institute for Fruit Growing Pitesti, Romania. The leaves were collected in 2018, from August to September, dried and kept in storage. The leaves were hydrated in distilled water and the inoculum of V. inaequalis or P. leucotricha was brought to 4.5 × 10 5 conidia/mL. After primary infection, the treatments with the developed materials were applied. The experimental variants were tested in triplicate.
Visual assessment regarding powdery mildew leaf area infection was carried out with the aid of the diseases rating scale, as presented in Table S1.
where: DD (%) = damage degree; F = attack frequency, %; I = attack intensity, estimated with notes between 1 and 6. The antifungal efficacy of the vegetal extracts on the diseases was evaluated after 14 days, as a percentage of inhibition calculated using Abbott Equation (S5): where: A and B represent the diseases area on the untreated and, respectively, treated plants.       Figure S4. Effect of vegetal extracts in powdery mildew control-Podosphaera leucotricha. Values without a common letter differ (p < 0.05) as analyzed by one-way ANOVA and the TUKEY test.