It is well known that the green synthesis of nanoparticles materials has attracted the attention of many studies due to their advancement over other methods as single step process, cost effective and emerged as ecofriendly environment [4].This report demonstrated for the first time the biosynthesis of AgNPs using aqueous extract from the equal mixture of Olea europaea subsp. europaea var. sylvestris and Pistacia lentiscus leaves. Here we proved that firstly, two Olea europaea subsp. europaea var. sylvestris and Pistacia lentiscus natural associated species can be used as natural sources to synthesize silver nanoparticles by enhancing their antimicrobial potential compared to other AgNPs synthesized from individually plant as reported previously in literature. For example as reported by the P lentiscus leaf extracts which possess moderate antimicrobial activities and scare works focused on the biosynthesis of silver nanomaterial from this specie [32].
Secondly, here the AgNPs synthesis were completely free from any chemical solvents and hazardous reagents similar to the green synthesis described previously [33], based on the use of two cultivars (Leccino and Carolea) of Olea europaea growing in the same pedoclimatic conditions for green biosynthesis of silver nanoparticles with new properties as antibacterial potential against, and were able to induce toxicity in breast cancer cell lines. In adition, here the mixed leaves extract enhance the biosynthesis of AgNPs, on one hand, the size of silver nanoparticles of 23nm, were smaller than those obtained only by Pistachio leaves extract as reported by [34]. show that the Ag NPs from Pistachia ethanol extract ranged from 24 to 26nm. On the other hand, the mixed leaves from both plant species enhance the antibacterial potential as compared to those obtained by silver nanoparticles from Pistachio which not exceed 13mm of zone inhibition agaisnt Escherchia coli and Staphylococcus aureus [34].
Face to the emergence of multidrug resistant bacteria and biofilms producer’s strains to currently used antibiotics, AgNPs may play a crucial role compared to the conventional antibiotics by action of multiple antagonism mechanism. In this work, we revealed a significant antibacterial and antifungal efficacy of AgNPs against all tested clinical strains. This finding may be attributed to its richness with secondary metabolites from both natural associated plant species. On the other hand, Olive leaves are polyphenol rich compounds that are known to have antioxidant, antimicrobial, and anti-inflammatory activities. In literature the anti-inflammatory and antibacterial effect against gram positive bacteria is due to the Olive component Oleuropein [35]. Furthermore, here the enhancement of AgNPs from the mixed leaves extract observed by the superior antibacterial activity by exhibiting maximum antibacterial effect against both gram negative and gram positive bacteria.
In addition, the results mentioned that the susceptibility of Gram positive and Gram negative bacteria to biosynthesized AgNPs was found to vary from study to other, related to the pathogen strains tested [36] and the concentration of the inoculum or solvent used [37]. For example, here the zone inhibition given by the AgNPs against Staphylococcus aureus and Escherchia coli were 18.66mm and15.33 mm respectively, these diameters were superior to those obtained by the AgNPs from Pistachia lenticus with 13mm and 13mm respectively against the same bacteria strains, these data could be due to the maximum richness of the mixed metabolites from both plant leaves [38]. Few works aimed the antifungal potential of AgNPs, here we successfully describe the high antifungal potential of our AgNPs against Candida species and Penicillium. The observed results on filamentous fungi confirmed that the silver nanoparticles enhanced the antifungal behavior against mycelium and spores [39]. Numerous works, described the antifungal potential of Olive plant against filamentous fungi like Rhizopus, Fusarium and Alternaria as reported by [40]. These funding mentioned that the antifungal behavior of the biosynthesized silver nanoparticles from mixed extracts could be due to the olive composition, which was able to affect the spores and the mycelium fungi growth. In literature, the major active components in Olive leaf are known to be Oleuropein and its derivatives as owing superior antifungal potential against fungi [41]. It is essential to note that, the new AgNPs exhibited novel bactericidal and fungicidal potential which may be highly relevant in infections caused by filamentous fungi and MDR bacteria strains. Consequently, the broad spectrum killing caused by AgNPs, have encouraged their use as antimicrobial drugs including multidrug strains MDR [2, 42].
Numerous studies have shown that nanoparticles generally improved the pharmaceutical characteristics of antifungals, as lower toxicity and enhancing antifungal potential, and the possibility of prolonged action [40]. The reported data show that the presence of AgNPs in the Candida growth can limit the virulence factor as enzyme production and biofilm formation.
In literature, several mechanisms have been reported for antimicrobial activity of AgNPs such as disruption of the bacterial cell membrane, interference in the respiratory electron transport chain formation of reactive oxygen species (ROS) [43, 44]. Nanoparticles exhibited new or improved properties depending upon their size and morphology. The pathogenicity of Candida species was attributed to the factor virulence such as enzyme hydrolases to invade host cells and biofilm formation to adhere to solid surface. The present work was the first to highlight the effect of silver nanoparticles from mixed leaves from Olive and Pistachia on Candida key virulence factors by means of enzyme production (proteinase, phospholipase) and morphogenesis reduction. Our results illustrated that the addition of silver nanoparticles can reduce the enzyme production and the germ tube and filamentous hypheae. Recently scare works, reported the reduction of enzyme and biofilm by Candida albicans by the addition of green silver nanoparticles. However, the exact mechanism of action on biofilm by silver nanoparticles is not known. In addition, the inhibition of yeast morphogenesis; like germ tube and filamentous hyphae lead to the suppression of biofilm formation in Candida strains [45, 46].
Furthermore, a combination of conventional antifungals with natural compounds can also minimize the toxicity of these drugs by reducing the dose request. Therefore, the focus of this study was to explore the new AgNPs by enhancing the antibacterial potential of conventional antibiotic drugs tested on clinical strains. Here the association of silver nanoparticles ameliorate the action of conventional antibiotic or make resistant bacteria more sensible against it. After demonstrating the simple method of synthesis, the antioxidant potential of AgNPs by testing DPPH radical scavenging, Ferric Antioxidant Reducing Power (FRAP) as well as the total antioxidant activity was determined, thus the combination of the antioxidant effects and the antibacterial and antifungal activities encourage the use of the green biosynthesized AgNPs in pharmaceutical field.
In conclusion to our knowledge, this is the first study evaluating the antioxidant and antimicrobial effects of silver nanoparticles biosynthesized from Olea europaea subsp. europaea var. sylvestris and Pistacia lentiscus leaves. The synthetized AgNPs is rich in secondary metabolites and has an antioxidant activity. The reported AgNPs exhibited markedly bactericidal and fungicidal effects against clinical pathogen strains. The synergistic interaction with the conventional antibiotic as well as the effect on bacteria biofilm and the spores of filamentous encouraged their formulation in pharmaceutical and medical purposes.