Investigation of Antimicrobial Activity and Biocompatibility of Biogenic Silver Nanoparticles Synthesized using Syzigyum cymosum Extract

Abstract

Nanotherapeutics has emerged as the most sought after approach to tackle the menace of drug-resistant pathogenic bacteria. Among others, biogenic silver nanoparticles (bAgNPs) synthesized using medicinal plant extracts demonstrate promising antibacterial propensity with excellent biocompatibility. Herein, bAgNPs were synthesized through the green chemistry approach using Syzygium cymosum leaf extract as a reducing agent at different pH values (i.e., 5, 7, 8, and 10). The average size of bAgNPs synthesized at pH 5, 7, 8, and 10 was 23.3, 21.3, 17.2, and 35.3 nm, respectively, and all the nanoparticles were negatively charged. Their antibacterial potential was investigated against Bacillus subtilis, Escherichia coli DH5α, E. coli K12, enteropathogenic E. coli, and Salmonella typhi. The highest antibacterial activity was exhibited by bAgNPs synthesized at pH 8 against all the tested bacterial strains, which can be attributed to their small size and greater surface area to volume ratio. The bAgNPs demonstrated the highest zone of inhibition (29.5 ± 0.8 mm) against B. subtilis through oxidation of membrane fatty acids that resulted in the formation of the malondialdehyde-thiobarbituric acid (MDA-TBA) adduct. However, bAgNPs demonstrated excellent hemocompatibility with rat and human red blood cells. Biogenic AgNPs synthesized at pH 8 also exhibited biocompatibility in terms of liver and kidney function biomarkers. Furthermore, hematoxylin and eosin staining of the tissue sections of vital organs (i.e., liver, kidneys, lungs, heart, spleen, and brain) also confirmed the biocompatibility of bAgNPs.

Scheme 1. Outline of the Synthesis of bAgNPs Using S. cymosum Leaf Extract

Figure 1

(a) CellTox Green uptake assay. The fluorescence intensity of bacteria treated with bAgNPs was measured at 490 nm using a spectrofluorometer. The values presented are mean ± SE of multiple samples (N = 3). The fluorescence intensity of bAgNP-treated bacterial strains was significantly higher than that of untreated bacteria and ***P < 0.001. (b) Both EPEC (Gram-negative) and B. subtilis RBW (Gram-positive) were treated with bAgNPs synthesized at pH 5 (i,v), 7 (ii,vi), 8 (iii,vii), and 10 (iv,viii). The treated bacteria were then incubated with CellTox green to stain the DNA of the cell wall-compromised bacteria, and green fluorescence was observed under a fluorescence microscope. Scale bar: 20 μm.

Figure 2

Lipid peroxidation assay. The cell membrane fatty acid oxidation potential of bAgNPs synthesized at pH 8 was measured through malondialdehyde–thiobarbituric acid (MDA–TBA) adduct assay. The absorbance of the MDA–TBA pink adduct was measured at 532 nm. The values presented are mean ± SE of multiple samples (N = 3). Data were analyzed using one-way ANOVA followed by Tukey’s multiple comparison test. The absorbance of the MDA–TBA adduct of bAgNP-treated bacteria were significantly higher than that of the untreated bacteria (i.e., control) and ***P < 0.01.

Figure 3

Hemocompatibility assay of bAgNPs synthesized at different pH values against (a) rat RBCs and (b) human RBCs. The values presented are mean ± SE and data and were analyzed using GraphPad Prism 5.0 (GraphPad software) using ANOVA followed by Tukey’s multiple comparison test. No statistical significant difference was observed among the different bAgNPs and p > 0.05.

Figure 4

In vivo cytotoxicity assay. The effect of bAgNPs synthesized at pH 8 on the liver was investigated by measuring the level of (a)ALT, (b)ALP, (c)AST, (d)γ-GT, and (e)albumin. Moreover, the effect of bAgNPs on kidney function was also investigated by measuring the level of (f) creatinine and (g) Uric acid. The values presented are mean ± SE, and four animals were taken per group. Data were analyzed using GraphPad Prism 5.0 (GraphPad software) using ANOVA. Dunnett test was used for posthoc comparison. No significant difference was observed when treatment groups were compared with the control and P > 0.05.

Figure 5

Representative images of histologic changes in liver tissues. All samples prepared from rats were stained with H&E and observed under a light microscope under 20× magnification. (a) bAgNPs at 5 mg/kg B.W at day 1, (b) bAgNPs at 10 mg/kg B.W at day 1, (c) bAgNPs at 50 mg/kg B.W at day 1, (d) bAgNPs at 5 mg/kg B.W at day 7, (e) bAgNPs at 10 mg/kg B.W at day 7, (f) bAgNPs at 50 mg/kg B.W at day 7, (g) bAgNPs at 5 mg/kg B.W at day 28, (h) bAgNPs at 10 mg/kg B.W at day 28, (i) 50 mg/kg B.W at day 28, (j) control group of rats at day 1, (k) control group of rats at day 7, and (l) control group of rats at day 28.

Figure 6

Representative images of histologic changes in kidney tissues. All samples prepared from rats were stained with H&E and observed under a light microscope under 20× magnification. (a) bAgNPs at 5 mg/kg B.W at day 1, (b) bAgNPs at 10 mg/kg B.W at day 1, (c) bAgNPs at 50 mg/kg B.W at day 1, (d) bAgNPs at 5 mg/kg B.W at day 7, (e) bAgNPs at 10 mg/kg B.W at day 7, (f) bAgNPs at 50 mg/kg B.W at day 7, (g) bAgNPs at 5 mg/kg B.W at day 28, (h) bAgNPs at 10 mg/kg B.W at day 28, (i) bAgNPs at 50 mg/kg B.W at day 28, (j) control group of rats at day 1, (k) control group of rats at day 7, and (l) control group of rats at day 28.

Figure 7

Representative images of histologic changes in lung tissues. All samples prepared from rats were stained with H&E and observed under a light microscope under 20× magnification. (a) bAgNPs at 5 mg/kg B.W at day 1, (b) bAgNPs at 10 mg/kg B.W at day 1, (c) bAgNPs at 50 mg/kg B.W at day 1, (d) bAgNPs at 5 mg/kg B.W at day 7, (e) bAgNPs at 10 mg/kg B.W at day 7, (f) bAgNPs at 50 mg/kg B.W at day 7, (g) bAgNPs at 5 mg/kg B.W at day 28, (h) bAgNPs at 10 mg/kg B.W at day 28, (i) bAgNPs at 50 mg/kg B.W at day 28, (j) control group of rats at day 1, (k) control group of rats at day 7, and (l) control group of rats at day 28.