Green synthesis of silver nanoparticles using Magnolia alba leaf extracts and evaluating their antimicrobial, anticancer, antioxidant, and photocatalytic properties

Abstract

The biosynthesis of silver nanoparticles has recently emerged as a promising approach in nanomedicine, particularly for targeted therapeutic applications. Green synthesized (plant-based) nanoparticles have been shown to offer enhanced reduction efficiency, greater bioavailability, and improved stability compared to synthetic nanoparticles. Here, we report the green synthesis of silver nanoparticles (AgNPs) using Magnolia alba leaf extract (MLE). The formation of these Magnolia-derived silver nanoparticles (MAgNPs) was verified through UV-Vis spectroscopy with a surface plasmon resonance peak at 440 nm, and further characterized by scanning electron microscopy, which showed that the MAgNPs have a mean diameter of 40 nm and a spherical morphology. MAgNPs exhibited significant antibacterial activity against Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterococcus faecalis, methicillin-resistant and -sensitive Staphylococcus aureus, with a minimum inhibitory concentration of 0.00043 mg/mL and a minimum bactericidal concentration of 0.00043 mg/mL and 0.0017 mg/mL, respectively. Disc diffusion and plaque assays with MAgNPs demonstrated strong antifungal activity against Candida albicans, with a zone of inhibition of 14 mm, and antiviral activity against T7 bacteriophage (p = 0.0004). In vitro studies with HCT-116 human colon cancer cells, MAgNPs exhibited bi-phasic, dose-dependent inhibition of viability with a 20-40% reduction, surpassing the positive control Camptothecin. Antioxidant assays indicated that MAgNPs showed significantly higher antioxidant activity compared to MLE, with enhanced Total Flavonoid Content (p = 0.0066), Total Phenol Content (p = 0.0013), and Total Antioxidant Capacity (p = 0.0051). Additionally, MAgNPs showed efficient photocatalytic degradation of the azo bond in methyl orange within 30 min. To our knowledge, this is the first report on the biosynthesis of MAgNPs and their multifunctional properties, highlighting the promise of MAgNPs in biomedical and environmental fields. (Insert attached Graphical abstract).

Keywords: Antimicrobial; Cancer; Candida; HCT-116; MRSA; Pneumoniae; T7 Bacteriophage.

Fig. 1

Green synthesis and UV–Vis characterization of MAgNPs. (A) Color change observed before and after MAgNPs synthesis. (B) The spectroscopic analysis of the MAgNPs synthesized for different periods and at different temperatures. Noted a SPR peak at 440 nm of the samples that were kept at 95 °C for 30 min, which is indicative of the presence of silver nanoparticles. There was no further change in peak height at 45 or 60 min.

Fig. 2

Characterization of MAgNPs using scanning electron microscopy (SEM) at different magnifications. SEM revealed that Magnolia silver nanoparticles (MAgNPs) had a mean diameter of 40 nm and were spherical, as shown at 1 μm (A), 500 nm (B and C), and 300 nm (D). At higher concentrations, MAgNPs aggregate and appear as grape-like clusters (C).

Fig. 3

Well and disc diffusion assays for antibacterial and antifungal properties of MAgNPs against medically relevant pathogens. Kanamycin (Kan, 100 μg/mL), Gentamycin (Gen, 50 μg/mL), Vancomycin (Vanc, 50 μg/mL), Fluconazole (FLZ 50 mM) Magnolia Silver Nanoparticles (MAgNP, 0.07 mg/mL), Magnolia leaf extract (MLE), and Saline were exposed to a lawn of bacteria and grown at 37 °C for 24 h. (A) E. coli BL21, (B) Klebsiella pneumoniae, (C) Pseudomonas aeruginosa, (D) Enterococcus faecalis, (E) MSSA, (F) MRSA and (G and H) C. albicans. (G) 0.00086 mg/mL of MAgNPs and (H) 0.00043 mg/mL of MAgNPs.

Fig. 4

Micro dilution assay for minimal inhibitory concentration (MIC) determination. Methicillin Resistant Staphylococcus aureus (MRSA), Methicillin Sensitive Staphylococcus aureus (MSSA) was with MAgNPs (A and B), and MLE (C and D), before incubation (A and C), after 20 h of incubation (B and D). In each 96-well plate, column 11 contained bacteria only (positive control) and column 12 contained media only (negative control). (A) MRSA and MSSA treated with MAgNPs using serial dilution at concentrations of 0.441 mg/mL to 0.00043 mg/mL at the start of the experiment (0 h). (B) MRSA and MSSA treated with MAgNPs after 20 h of incubation at 37 °C. (C) MRSA and MSSA treated with MLE using serial dilution at concentrations of 32 mg/mL to 0.06 mg/mL at the start of the experiment (0 h). (D) MRSA and MSSA treated with MLE after 20 h of incubation at 37 °C.

Fig. 5

Bacteriophage plaque assay to examine the antiviral activity of Magnolia silver nanoparticles (MAgNPs). T7 coliphage (bacteriophage) exposed to different agents for 20 min and then incubated with E. coli BL21 strain for plaque formation. (A) No exposure, (B) Magnolia leaf extract (MLE), and (C) Magnolia silver nanoparticles (MAgNPs). (D) PFU/mL for each of the exposure groups after incubating at 37 °C overnight. This experiment was repeated at least 3 times, and a representative data set is shown. The error bars are the standard error of the mean (SE). P-values were obtained using a t-test to compare the means of the groups.

Fig. 6

Crystal violet assay for the effects of MAgNPs and MLE on HCT-116 cells. (A) MAgNPs concentration range, from 0.1 to 500 µM in tenfold serial dilutions. (B) Selected MLE concentration range, from 25 to 250 µM. The MAgNPs depict an inhibitory bi-phasic effect on HCT-116 colorectal cancer cells. The biphasic trend showed enhanced inhibition of cells at lower concentrations and then again at higher concentrations. This suggests more than one mechanism of action which could indicate distinct cellular pathways being activated at different concentrations usually seen with antiproliferative and pro-apoptotic mechanisms of natural plant-derived bioactive molecules, or PBAMS, also known as phytochemicals. Camptothecin was 10 µM. The error bars are the standard error of the mean (SE).

Fig. 7

Antioxidant Assays with MAgNP. (A) Total Flavonoid Content of leaf extract and MAgNPs expressed as Quercetin equivalents (QE). Magnolia silver nanoparticles showed significantly higher TFC values compared to the leaf extracts only. (B) Total Phenolic Content of leaf extract and MAgNPs expressed as Gallic acid equivalents (GAE). Magnolia silver nanoparticles showed significantly higher TPC values compared to the leaf extracts. (C) Total Antioxidant Content of leaf extract and MAgNPs expressed as Ascorbic Acid Equivalents (AAE). Magnolia silver nanoparticles showed significantly higher TAC values compared to the leaf extracts. (D) FRAP analysis of leaf extract and MAgNPs. The ferric ion reducing power was observed to be higher in MAgNPs compared to the leaf extracts. The results showed that MAgNPs exhibited greater free radical scavenging activity within 4 min and after 6 min, whereas the leaf extracts scavenging activity also increased but at a slower rate. (E) Percent DPPH values for leaf extracts and MAgNPs. The MAgNPs demonstrated superior free radical scavenging activity compared to the leaf extracts. The higher reducing power and electron-donating property of MAgNPs support the presence of a higher TFC demonstrated above. The error bars are the standard error of the mean (SE). P-values were obtained using a t-test to compare the means of the groups.

Fig. 8

Photocatalytic activity of MAgNP on methyl orange. Radicals generated by exposure to sunlight attack the azo bond in methyl orange leading to degradation of the dye and formation of intermediates, ultimately resulting in the mineralization of the dye to a colorless final product in 30 min.