Antifungal and Cytotoxic Activities of Biosynthesized Silver, Zinc and Gold Nanoparticles by Flower Extract of Rhanterium epapposum

This study was designed to prepare silver, zinc and gold nanoparticles (NPs), AgNPs, ZnNPs and AuNPs, by biosynthesis technique using methanolic extract of Rhanterium epapposum flowers using AgNO 3 , Zn (CH 3 CO 2 ) 2 and HAuCl 4 ·3H 2 O as starting materials. The physical properties of the formed NPs were characterized by ultraviolet spectroscopy (UV), X-ray diffraction (XRD), transmission electron microscopy and Fourier transformed infrared spectroscopy (FTIR). The results revealed that, AgNPs were homogeneous and spherical in shape, with average diameter 16.3 nm. While, ZnNPs were approximately triangle and hexagonal shaped, with average diameter 23.5 nm. Most of the synthesized AuNPs were spherical in shape with average diameter 17.9 nm. The antifungal activity of different concentrations of the formed AgNPs, ZnNPs and AuNPs was tested against two human pathogens: Candida albicans and Aspergillus melleus and one plant pathogenic fungus: Phoma exigua, using agar diffusion assay. The best results recorded by 120 µg/ml AgNPs against the human pathogen: C. albicans where the inhibition zone was 23.5 mm. Additionally, the cytotoxicity of the tested NPs was evaluated against Breast adenocarcinoma (MCF-7), Hepatocellular carcinoma (HepG-2) and colorectal carcinoma (HCT 116) human cell lines. The most toxic was AuNPs where the IC50 against MCF-7, HepG2 and HCT116 was 55.02, 66.44 and 169.87 µg/mL respectively.


Introduction
Nanoparticles are considered as the building blocks of nanotechnology that they were synthesized in the range of 1 -100 nm [1] [2]. The synthesis of NPs by biological methods is rapidly growing for the field of nanotechnology due to its low toxicity and relatively cheap. Thus, these methods are reported as an economical valuable and alternative source for the synthesis of metal NPs [3]. Among all noble metal NPs, silver, zinc and gold had an interest based on their distinctive properties using different experiments as antibacterial activity [2] [4] [5], antitumor [1] [3], catalytic activity [6] [7] and chemical stability [8] [9] [10].
Incidentally, the researchers concern was attracted by the production possibility of NPs by biological methods. This was performed by using bacteria, algae, fungi or plant extracts as an alternative to the chemical and physical methods for producing NPs [11] [12]. The biosynthesis of NPs, which mediated by microbes, is not industrially feasible, because it requires the preservation of highly aseptic conditions and expensive growth media to grow [13]. It was reported that plant extracts showed numerous advantages, such as availability, safety handling and viability of metabolites. Moreover, the reaction media for the synthesis of inorganic NPs is more interesting over other microorganisms as well as less hazard than other organisms [14].
Medicinal plants possess high safety profile as well as high effectiveness, so, they are useful for human beings in many applicatory uses [15]. They are considered as an important source of bioactive metabolites which can be used for treatment of many diseases. In the meantime, some of these plant metabolites play vital roles as reducing and capping agents in metals NPs biosynthesis [16].
The green method for creating metal NPs is aimed at striving against the safety of applications of the nanotechnology, especially in medical fields.
Recently, there are several publications interested in application of nanotechnology in drug discovery. These reports showed the effect of Musa acuminata [3], Echinochloa frumentacea [5], Clinacanthus nutans [9], that used for biological synthesis of silver, zinc and gold NPs.
As there is no available reviews or publications indicated the production of metals NPs using the flower of Rhanterium epapposum (family: Asteraceae) which is considered a rich source of volatile compounds including limonene, linalool, 4-terpineol and α-cadinol [17] [18]. So that, the present study is interested in production of silver, zinc and gold NPs by green synthesis method using R. epapposum flower extract.
The study was designed to investigate the ability of the methanolic extract of R. epapposum flowers to biosynthesized AgNPs, ZnNPs and AuNPs without using any harmful reducing or capping agents. Then, the features of the formed Open Journal of Applied Sciences NPs were characterized by UV-Vis spectroscopy, XRD analysis, FTIR analysis and TEM. Further, the antifungal activity of the formed NPs was tested against C. albicans, P. exigua and A. melleus. In addition, their cytotoxicity on three cancer cell lines: Breast adenocarcinoma (MCF-7), Hepatocellular carcinoma (HepG-2) and colorectal carcinoma (HCT 116) human cell lines was also investigated. After that, each metallic NPs were settled by centrifugation at 10,500 rpm for 15 min. The NPs were subjected to the washing process with distilled water three times before drying them at 90˚C for 24 h for obtaining powdered NPs.

Characterization of AgNPs, ZnNPs and AuNPs:
The absorbance of the resulted NPs was measured at UV range of 300 -800 nm by UV-Visible spectrophotometer (Cary 8454 UV-Vis Diode Array System, United State). Also, the XRD of the biosynthesized NPs were analyzed on glass Open Journal of Applied Sciences substrate by using X-ray diffractometer Shimadzu X-ray (Cu kα) Diffract meter XRD-6000 (λ = 1.5406 Å) operated at 30 kV and 25 with a step of 0.02˚ in the Bragg angles 2θ at a scanning rate of 20 min −1 at 30˚ to 80˚. The functional groups of the samples were investigated in the 400 -4000 cm −1 range with a Thermo Nicolet 6700 Fourier transform infrared (FTIR) spectrometer (Thermo Scientific, Waltham, MA, USA). While the particle size and morphology of the The antifungal properties of the tested NPs were screened using the agar well diffusion method [4] [20]. For that purpose, 100 µL from each of the previously  and cultured in a 96 well tissue culture plate (3000 cells/well) for 24 h before treatment with the tested NPs. Cells exposed to six different concentrations of each NPs (0.01, 0.1, 1, 10, and 1000 µg) while the untreated cells served as control. The cells were incubated with the concentrations for 72 h and subsequently fixed with TCA (10% w/v) for 1 h at 4 ˚C. After several washings, cells were stained by 0.4% w/v SRB solution for 10 min in dark place. Excess stain was washed with 1% v/v glacial acetic acid. After drying overnight, the SRB-stained cells were dissolved with Tris-HCl and the color intensity was measured in microplate reader at 540 nm. The relation between viability percentage of each tumor cell line and NPs concentrations was analyzed to get the IC 50 (dose of the drug which reduces survival to 50%) using Sigma Plot 12.0 software. All the steps were repeated using the antitumor drug; Doxorubicin as control.
Statistical analysis: Statistical analysis of the present study was conducted, using the mean, standard deviation (SD) and analysis of variance (ANOVA) using Microsoft Excel 2016 program and the online free source; Free Statistics Calculators version 4.0. Statistical significance was acceptable to a level of p < 0.05. Graphs were plotted using GraphPad Prism software, version 6.00 (GraphPad Software, La Jolla, CA).

Results and Discussion
Biosynthesis of AgNPs, ZnNPs and AuNPs: The biosynthesis reaction started within a few minutes and the color reaction was observed in which clear AgNO 3 , Zn (CH 3 CO 2 ) 2 and HAuCl 4 ·3H 2 O solutions were changed into brown red color, yellowish milky color and dark pink respectively. The new colors indicated the formation of the corresponding NPs and confirm the ability of the methanolic extract of R. epapposum flowers to synthesize the NPs (Photo 1).

Characterization of AgNPs, ZnNPs and AuNPs
The UV-Vis spectra of the formed AgNPs, ZnNPs and AuNPs are shown in Figure 1. The distinct peaks observed at 423, 375 and 525 nm are distinguished to surface plasmon resonance of AgNPs, ZnNPs and AuNPs respectively. These results are compatible to results of other researches [1] [9]. The obtained nanoparticles showed high stability at the recorded peaks.   To study the morphology of the biosynthesized AgNPs, ZnNPs and AuNPs, the TEM analysis was carried out. The micrographs revealed that AgNPs were homogeneous and spherical in shape, with a size range from 3 -17 nm with average diameter 16.3 ± 3 nm. Shape of ZnNPs was approximately triangle and hexagonal, in which their size ranged from 9 -30.3 nm and the average diameter was 23.5 ± 10 nm (Photo 2). Most of the AuNPs were spherical in shape and their size ranged from 3 -19 nm with average diameter 17.9 ± 3 nm.
The FT-IR spectra of AgNPs, ZnNPs and AuNPs are shown in Figure 3

Conclusion
The silver, zinc and gold NPs were successfully prepared biologically using the alcoholic extract of R. epapposum flowers and the starting materials: AgNO 3 , Zn (CH 3 CO 2 ) 2 and HAuCl 4 ·3H 2 O. Then, the morphology and particle size of the formed NPs were established by UV spectroscopy, XRD, TEM and FT-IR. The tested NPs represented an antifungal activity with different degrees against C. albicans, P. exigua and A. melleus. Moreover, the formed NPs showed cytotoxic effects against three cancer cell lines (MCF-7, HepG-2 and HCT 116) where AgNPs exhibited the best results. According to these results, it was concluded that, the tested AgNPs, ZnNPs and AuNPs might be used as antitumor materials, but this still needs further studies.