A green synthetic approach by using oak fruit hull (Jaft) extract for preparation of silver nanoparticles (AgNPs) was developed and optimized. Parameters affecting the synthesis of AgNPs, such as temperature, extract pH, and concentration of extract (ratio of plant sample to extraction solvent), were investigated and optimized. Optimum conditions for the synthesis of silver nanoparticles are as follows: Ag+ concentration, 1 mM; extract concentration, 40 g/L (4% w/v); pH = 9 and temperature, 45°C. Biosynthesized silver nanoparticles were characterized using UV-visible absorption spectroscopy (UV-Vis), Fourier-transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), dynamic light scattering (DLS), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). TEM and DLS analyses have shown the synthesized AgNPs were predominantly spherical in shape with an average size of 40 nm. The cytotoxic activity of the synthesized AgNPs and Jaft extract containing AgNPs against human breast cancer cell (MCF-7) was investigated and the half maximal inhibitory concentrations (IC50) were found to be 50 and 0.04
Green chemistry is the design of chemical products and processes that reduce or eliminate the use or generate hazardous substances for human health and environment. Therefore, green chemistry protects the environment, not by cleaning up, but by introducing new chemical processes that do not pollute the environment.
Nowadays, development of green synthesis of metallic nanoparticles and their applications is one of the most important areas of research. For synthesizing silver nanoparticles (AgNPs) several methods such as chemical synthesis [
In several reports it was demonstrated that the antimicrobial activities of AgNPs are dependent on the size, shape, and stabilizing agents of nanoparticles. The antibacterial activities increased with size reduction of AgNPs [
Despite the widespread use of AgNPs in medical fields, relatively few in vitro studies have been accomplished to determine the cytotoxicity effects of AgNPs on different cell lines including HeLa [
There are many reports on the therapeutic properties of fruit, leave, gall, and Jaft of oak due to presence of phenols, tannins, and proteins in this plant. Extract of
In this work, synthesis of AgNPs using aqueous Jaft extract of
Silver nitrate, sodium hydroxide, ammonia solution (25%), and orthophosphoric acid were purchased from Merck Chemical Co. (Darmstadt, Germany). 2-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich (USA). All solutions were prepared with distilled water.
UV-Vis spectra were recorded by using a UV-Vis spectrophotometer (Jenway, model 6505, UK). The existence of biomolecules in shell of synthesized AgNPs was investigated using FT-IR (BRUKER, model TENSOR 27, Germany) analysis. Biosynthesis of AgNPs was demonstrated by X-ray diffraction (X’Pert PRO, SciSpec Co. Thailand) analysis. The shape of the freeze dried AgNPs was analyzed by SEM and TEM (SEM, Hitachi model S-4160 and TEM, Philips model CM30). The particle size distribution and zeta potential analysis of biosynthesized AgNPs were evaluated via dynamic light scattering (DLS) and zeta potential analysis by using a Malvern Zetasizer Nano range instrument (Malvern Instruments Ltd., Malvern, UK).
Fruits of oak trees were collected from Khorramabad Mountains in the west of Iran. Oak fruit hull (Jaft) was isolated and dried in 25°C in shadow. The same sample was used in the whole optimization study.
In order for preparation of extract, 5.0 g of air-dried and pulverized Jaft was extracted by ultrasonic bath for 24 h with 50 mL distilled water and filtered by using Whatman filter paper. Finally, the filtrate was centrifuged for 10 min at 4000 rpm. The supernatant was used for the synthesis of AgNPs.
40 mL (10% w/v) of Jaft aqueous extract, 10 mL of ammonia solution (1 M), and 10 mL of silver nitrate solution (10 mM) were mixed. The solution pH adjusts to the desired value by using sodium hydroxide or phosphoric acid solution and then was diluted until 100 mL with distilled water. The mixture was stirred for 4 h at 45°C.
After centrifuging of AgNPs solution for 10 min at 10000 rpm, AgNPs were sedimented at the bottom of the conical tube. The supernatant phase was removed and AgNPs were washed with 10 mL water for three times. After the washing, the residue was transferred to freeze dryer. Finally, the obtained powder was subjected to XRD, FT-IR, SEM, and TEM analyses.
Cell viability was calculated according to the method developed by Denizot and Lang [
All measurements were performed in triplicate. The one-way analysis of variance (ANOVA) was done for expressing experimental significance of results. In cell viability test the
The main parameters affecting the formation of nanoparticles, including pH of extract, temperature of synthesis process, and extract concentration, were investigated and optimized.
In green synthesis of AgNPs using plant extracts, various constituents may contribute in reduction process of silver ions. Therefore, changing the chemical state (e.g., ionization) of these constituents can be affected on performance and rate of reduction process. For this reason, the effect of extract pH on the synthesis of AgNPs in the range of 2–11 was investigated by using UV-Vis spectrophotometer. The results (Figure
Effect of extract pH on AgNPs synthesis. Synthesis conditions: silver nitrate concentration, 1 mM; extract concentration; 40 g/L, temperature, 45°C.
Synthesis of silver nanoparticles was performed at different temperatures in the range of 4 to 60°C. The results in Figure
Effect of temperature on AgNPs synthesis. Synthesis conditions: silver nitrate concentration, 1 mM; extract concentration; 40 g/L, extract pH, 9.
In order to complete reduction of silver ions to silver nanoparticles, different concentrations of extract (Jaft extract) were mixed with a constant volume of silver nitrate (1 mM). The results in Figure
Effect of extract concentration on AgNPs synthesis. Synthesis conditions: silver nitrate concentration, 1 mM; temperature, 45°C, extract pH, 9.
The XRD patterns of synthesized AgNPs are shown in Figure
XRD pattern of biosynthesized AgNPs (blue) and reference XRD pattern of cubic silver (red). Synthesis conditions: silver nitrate concentration, 1 mM; temperature, 45°C, extract pH, 9; extract concentration, 40 g/L.
The SEM and TEM images show that the synthesized AgNPs are in spherical structures (Figure
SEM (a) and TEM (b) images of biosynthesized AgNPs. Synthesis conditions: silver nitrate concentration, 1 mM; temperature, 45°C, extract pH, 9; extract concentration, 40 g/L.
DLS analysis (a) and zeta potential (b) measurement of biosynthesized AgNPs. Synthesis conditions: silver nitrate concentration, 1 mM; temperature, 45°C, extract pH, 9; extract concentration, 40 g/L.
Figure
FT-IR spectra of Jaft extract (a) and biosynthesized AgNPs (b). Synthesis conditions: silver nitrate concentration, 1 mM; temperature, 45°C, extract pH, 9; extract concentration, 40 g/L.
In previous reports [
As observed from Figure
MTT assay results. (a) Cytotoxic effects of dispersed AgNPs in the Jaft extract and AgNPs on cancer cell line (MCF-7) and (b) cytotoxic effects of dispersed AgNPs in the Jaft extract and AgNPs on normal cell line (human blood mononuclear cells). Data are calculated as mean ± SD of three experiments. AgNPs: dispersed AgNPs in purified water, Ex & AgNPs: dispersed AgNPs in the Jaft extract, control: untreated cell line. Percentage of cytotoxicity is expressed relative to untreated controls (
For the first time biosynthesis of AgNPs by using aqueous oak fruit hull (Jaft) extract was developed and optimized. TEM and DLS analyses have shown the biosynthesized AgNPs were spherical in shape with an average size of 40 nm. The results indicated cytotoxic activity of dispersed AgNPs in the Jaft extract was higher than AgNPs on MCF-7 breast cancer cell line. Therefore, the Jaft extract containing silver nanoparticles might be a potential alternative agent for human breast cancer therapy. The results of zeta potential show that the biosynthesized AgNPs have a long-term stability. Nanoparticles in extract solution were stable for one month.
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors gratefully acknowledge the support of Razi Herbal Medicines Research Center, Lorestan University of Medical Sciences and Islamic Azad University, Khorramabad Branch.