Quercus Brantii-Extract-Mediated Silver Nanoparticles: a Local Solution for Qarasu River Problems

1. In Iran, oak bark (pericarp or fruit wall) was traditionally used to seal waterskins. Another traditional use of oak barks (especially testa or seed coat) is as agents for converting animal hides to leather (tanning), because of their ability to precipitate proteins. Oak bark heals effectively skin injuries and reduces inflammation and bleeding. Acorn seed and cupule were used for making flour and jam respectively. Tannins are somehow involved in all uses. The term “tannin” comes from the ancient celtic word for oak, a typical source for tannins for leather making. Bate-smith defined tannins as “water-soluble phenolic compounds having molecular weights between 500 and 3000… giving the usual phenolic reactions… and having special properties such as the ability to precipitate alkaloids, gelatin and other proteins”. Haslam has more recently substituted the term “polyphenol” for “tannin”, in an attempt to emphasize the multiplicity of phenolic groups characteristic of these compounds. He notes that molecular weights as high as 20000 have been reported, and that tannins complex not only with proteins and alkaloids but also with certain polysaccharides. Phenolics can affect the biological availability or activity of metal ions by chelating the metal. Chelation requires appropriate patterns of substitution and a pH above the pKa of the phenolic group. Bacterial siderophores with multiple phenolic groups and very high affinities for essential metals such as iron have been characterized. The similarity between siderophore orthodihydroxy substitution pattern and the substitution patterns on condensed and hydrolysable tannins suggests that tannins may also have very high affinities for metals. Metal ion chelation can alter the redox potential of the metal or prevent its participation in redox reaction. Through the studies on the substitution patterns at low pH, though Raymond et al. supposed that bidendate catecholate ligands in iron-enterobactin complexes were responsible for catecholate-type bond forming, but the experiment results showed that bond formation is favored in salicylate-type bonding, in which the bond is formed between the carbonyl oxygen atom and the deprotonated oxygen of phenolate at meta position. Hydrolysable tannins are derivatives of gallic acid (3,4,5-trihydroxy benzoic acid). Gallic acid is esterified to a core polyol, and the galloyl groups may be further esterified or oxidatively crosslinked to yield more complex hydrolysable tannins. The simplest hydrolysable tannins, the gallotannins, are simple polygalloyl esters of glucose. In seeking environmentally green route of nanoparticle preparation, herein, we report a method in which two precursors are required for nanoparticle grow: a silver salt and a reducing agent.10 Persian
oak acorns bark extract and silver nitrate (AgNO3) salt were used to acquire reducing agents and silver ions (Ag) respectively. In better compliance with the principles of green chemistry, synthesis was one-pot, single-step method, and was conducted at room temperature without using any external sources of energy. Due to the antibacterial action of the so-obtained synthesized silver nanoparticles against polluted water sample of Qarasu River and owing to in-situ remediation of the contaminated water, they could find an application as effective remediators; a probable local solution for Qarasu River problems.
2. Materials and methods Materials
Silver nitrate (AgNO3, PubChem CID: 24470), Folin-Ciocalteu reagent, tannic acid (PubChem CID: 16129778) and sodium carbonate (PubChem CID: 10340) were purchased from Merck (Germany). Ethanol 96˚ (PubChem CID: 702) was obtained from Kimia Alcohol Zanjan (Iran). All aqueous solutions were prepared by deionized water. Methods Quercus brantii, the Persian oak (covering more than 50% of the Zagros forest area) is the most important tree species of the Zagros region in Western Iran.20 The acorns barks, as residues of ripe acorns that fell off their parent trees, were collected (Chalabeh, Dorudfaraman Rural District, Kermanshah, Iran), dried at room temperature and ground with mortar and pestle. A stock solution of bark extract (15%, w/v) was prepared by macerating 7.5 g of oak bark powder in 50 ml of 70% aqueous ethanol (v/v) for one week in a capped reservoir flask. The eluate was filtered on paper and the filtrate was stored in refrigerator for further experiments. The following ratios were prepared: 1:1, 1:2, 1:3, 2:1 and 3:1, based on volume ratios of 50 mM aqueous silver nitrate (AgNO3, Merck) stock solution to bark extract stock solution (15%, w/v). The total volume of each sample was adjusted to 50 ml by adding deionized water. To know the amount of phenolic compounds in Quercus brantii acorns bark extract, the FolinCiocalteu method was carried out by adding of Folin-Ciocalteu reagent (1 N) to the acorns bark extract solution (0.01 ml, 1.5 % w/v) followed by the addition of sodium carbonate (20%, w/v), and the absorbance was measured after 40 min incubation. In parallel, a calibration standard curve using various known concentrations of tannic acid was used to quantify the extract (0.4-2.0 mg L-1 in water).21 The antimicrobial activity of extract and synthesized silver nanoparticles was determined against water sample collected from Qarasu River (Kohneh Bridge, Kermanshah, Iran), by spreading over the blood agar plates as bacterial culture medium. The river was contaminated with the bacterial effluent. Control samples were used to assess the antimicrobial activity of extract. The plates were incubated at 37 °C for 48 h for the bacterial and yeast cultures. The size and morphology of the silver nanoparticles were characterized by scanning electron microscopy (SEM, KYKY-EM3200, China) at 26 kV.
3. Formation of blue chromophores of tungsten and molybdenum complexes due to the reduction of Folin-Ciocalteu’s reagent (phosphomolybdate-phosphotungstate) in presence of phenolic compounds in alkaline medium, verifying by UV-Visible spectroscopy at 725 nm provides the basis for development of an assay to quantify these compounds. Oak barks depending on different plant parts contain different types of tannins, especially gallotannic and quercitannic acids, in different levels. In this study, the total phenolic content as tannic acid was estimated to 17.6% w/w. Silver nanoparticles were characterized with scanning electron microscopy. Figure 2 shows SEM images recorded from sputter-coated gold film of the silver nanoparticles synthesized by treating the silver nitrate solution with oak acorns bark extract. From the image corresponding to 1:2 volume ratio (metal salt to extract volume ratio), it can be inferred that about 65% of particles are mainly ranged from 45 to 75 nm in size. One hundred particles were counted in each micrograph by using Image J software. At the critical proportion of 1:2, tannic acid complexes will be saturated with 20 silver atoms (around the o-dihydroxyphenyl groups of tannic acid) enabling rapid nucleation that results in smaller particle size. For values below and above this critical proportion, the interaction of such unsaturated compounds in solution leads to slower nucleation rates resulting in larger particle sizes. On the other hand, growth occurs due to the collision of nuclei/particles with chelated silver atoms. At a critical proportion of 1:2, the incorporation efficiency of atoms onto nuclei will be higher per collision resulting in higher growth rates and in smaller particle sizes The Antibiogram patterns (Figure 3) were expressed by spread-plate method against control samples (patterns h and i). Water sample was pipetted onto the surface of blood agar plates (patterns a-g). The off-white stains indicate the presence of colonies. Silver nanoparticles were present in the micelles of the plant-based surfactants. The addition of a nanoparticle solution with a certain amount of plantbased surfactants to the bacterial suspension probably led to coagulation of the nanoparticles via a charge neutralization mechanism between oppositely charged ions of bacterial cell surface and ionic surfactants. The low colloidal stability of the nanoparticles resulted in a decrease of effective silver concentration in the broth medium. Coagulation via a charge neutralization mechanism is most effective at an optimum coagulant dose. Most probably in our study, the nanoparticle levels at volume ratio of 1:2 correspond to the optimum surfactant concentration for effective coagulation and a slower killing trend was observed (pattern b). At a level lower than optimum nanoparticle level (volume ratio of 1:3), complete charge neutralization does not happen, whereas at a higher level (volume ratios of 1:1, 2:1 and 3:1), due to the charge reversal the nanoparticles regain their colloidal stability, leading to poor or ineffective coagulation. Consequently, in those cases the killing is either much faster or immediate.
4. Conclusion
It can be inferred that atomic reorganization occurs within phenolic compounds result in different particle size and size distribution. Although growth on agar plates is a more ready means of determining antimicrobial properties of silver nanoparticles, in this study experiments showed that at a critical proportion of silver nitrate to acorns bark extract, the obtained smaller nanoparticles will be less efficient in bactericidal applications for a short period of time. The explored energy-conserving, eco-friendly, simple, inexpensive and high efficiency silver nanoparticles production prepared by using locally based plant-derived materials (i.e. Quercus brantii acorns bark extract) are expected to have more extensive applications, among which waste and groundwater treatment, environmental remediation and refining. Although oak acorns bark consumption or other aforementioned uses are less common nowadays, such emerged exploitation is executable. Hence, embedding in-situ green synthesized silver nanoparticles in porous templates is the targeted long-term strategy.