Synergic effect of aqueous extracts of Ocimum sanctum and Trigonella foenum-graecum L on the in situ green synthesis of silver nanoparticles and as a preventative agent against antibiotic-resistant food spoiling organisms

The combination of Ocimum sanctum and Trigonella foenum-graecum L leaf water extract synergistically acts as a reducing and capping agent for the synthesis of narrow polydisperse silver nanoparticles (Ag NPs) with controlled sizes depending on the precursor (AgNO3) concentration in the plant extract. The toxicity of 40 nm-sized green synthesized Ag NPs is less than that of 10 nm-sized NPs. The Ag NP solution in Ocimum sanctum and Trigonella foenum-graecum L leaf water extract shows synergic antibacterial effect on Gram-negative bacteria by effecting the ester group of the lipids (hydrolysis) and also breaking the amide bonds of the bacterial chemical constituents, which leads to their rapid death.

Synthesis of Silver Nanoparticles. Silver nanoparticles were prepared in aqueous solution by reducing silver ions with a mixture of Ocimum sanctum and Trigonella foenum-graecum L leaf extract (1:1, v/v). The detailed synthetic procedure of silver nanoparticles, 1  Ocimum sanctum and Trigonella foenum-graecum L leaf extract, respectively. Synthesized Ag NPs were purified by centrifuged method at 6000 rpm to remove biproducts for AgNO 3 reduction.

Synthesis of Citrate capped Silver Nanoparticles
In 50 mL AgNO 3 (0.01 M) aqueous solution, tri-sodium citrate was added in molar ratio of AgNO 3 : tri-sodium citrate of 1:0.05 and stirring for 2h and then 0.2 ml of freshly prepared aqueous NaBH 4 (0.01 mM) solution was added for reduction. Synthesized Ag NPs were purified by centrifuged method at 6000 rpm to remove biproducts for AgNO 3 reduction. Samples were prepared by placing small drops of dispersed silver nanoparticles (lyophilized powder) in water on carbon coated copper grids and allowing the solvent to slowly evaporate at room temperature. Samples were analyzed without staining.
MTT Assay. Macrophages were collected by peritoneal lavage from sodium thioglycolatestimulated mice. The peritoneal cells were collected in RPMI-1640 medium (incomplete), centrifuged at 1400 x g for 10 min at 4 °C, washed twice and finally re-suspended in complete medium. Macrophages at a density of 1 x 10 6 cells mL ─1 were seeded in 96-well tissue culture plates (0.1 mL per well) and exposed to various size of Ag NPs (10 nm, 20 nm, 30 nm and 40 nm spherical shape at a concentration of 1, 2, 5, 10, 15, 20, 50 and 30 µg/mL for 12 h in a CO 2 incubator (5% CO 2 , 37° C) to calculate the LD 50 . Cell viability was evaluated using a modified MTT assay, where the conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to formazan by mitochondrial enzymes served as an indicator of cell viability and amount of dye produced is directly proportional to the number of metabolically active cells.
Accordingly, absorbance at 492 nm represented the number of live cells. LD 50 value was calculated from the dose-response curve and used as measure for drug resistance and the mean percentage viability was calculated as follow 3 Mean specific absorbance of treated Macrophages X 100 Mean specific absorbance of untreated Macrophages.
Hemolysis Assay. 5 mL blood was collected from an anonymous human donor, drawn directly into K 2 -EDTA-coated Vacutainer tubes to prevent coagulation and centrifuged at 3000 rpm for 10 mins and the supernatant was discarded. RBC were washed thrice with 1X PBS, PH 7.4 for 5 min.
washed erythrocytes were finally suspended in 1X PBS (1:9 dilution). To study Hemolysis, 950 µl of the above suspension was mixed with 50 µL solution of same concentration (15 µg/mL) of various sizes of silver nanoparticles (10, 20, 30 and 40 nm, respectively to obtain RBC suspension) and also 1:9 diluted RBC suspension was mixed with 1 %Triton X-100 solution for complete lysis of the erytrocytes. RBC suspended in 1mL PBS was used as negative control to assess background lysis, if any. Each suspension was incubated at 37 0 C for 15 min, 30 min, 60 min, 120 min and 180 min. After the predetermined time of incubation, the suspension was centrifuged and the supernatant was analyzed using spectrophotometer at 540 nm. The percentage of hemolysis was         Table S1 (data is an average zone of inhibition).
With increasing the concentration of Ag NPs and leaf extracts, zone of inhibition increases for the Pseudomonas fragi, Pseudomonas fluorescens and Salmonella. However, maximum antibacterial activity of green synthesized Ag NPs was observed against the Pseudomonas fluorescens. In comparison to the citrate capped Ag NPs, Ocimum sanctum and Trigonella foenum-graecum L water Ag NPs (0.6 mg/ml) shows statistically significant antibacterial properties against the all three bacteria. The green synthesized silver nanoparticles showing efficient for antibacterial application due to enhanced bacterial cell-wall permeability, cellular respiration of the bacteria, and interacting with phosphorus, nitrogen or sulfur containing group in DNA and protein of the bacteria to penetrate inside. 7