Green synthesis of silver nanoparticles using Portulacaria characterization and evaluation of its antibacterial, anticancer activities

Applications of nanotechnology in different areas of research have expanded over the last years. Silver nanoparticles (AgNPs) have beneficial effects as antimicrobials, antioxidants and/or anticancer. Yet, one of the major limitations of their use was employing toxic chemicals as reducing agents. Biosynthesis was advantageous over the physical and chemical synthesis. The obtained nanoparticles were characterized using the High-Resolution Transmission Electron Microscope. Disc diffusion method was used to evaluate the antibacterial activity of AgNPs against Bacillus subtilis , Escherichia coli , Neisseria gonorrhoeae , Pseudomonas aeruginosa , Staphylococcus aureus , and Streptococcus faecalis . Cytotoxic activity of biosynthesized AgNPs was tested against humane breast cancer cell line (MCF-7). Results of characterization showed that AgNPs were irregular spherical in shape, with average diameter of 27.41nm, and width of 4.36nm. The antibacterial assay showed that Portulacaria afra extract had no inhibitory potential against the tested bacteria. However, both AgNO 3 and AgNPs exhibited recognized inhibitory potency against all tested bacteria. AgNPs exhibited wider inhibition zones than AgNO 3 . Cytotoxicity test revealed that green synthesized AgNPs had inhibitory activity against cancer cell line (MCF-7) which was concentration dependent, with IC 50 of 75.40 µmole. The aims of the present work were to study the possible green synthesis of AgNPs using P. afra aqueous leaf extract as a reducing agent; to characterize them, to investigate the antibacterial potency and cytotoxic potential of these biosynthesized AgNPs.


Introduction
Applications of nanotechnology in different areas of research have expanded over the last years, and scientists are continuously exploring all news about this technology. Nanomaterials have been reported to have unique characteristics including; their size, shape distribution, and morphology, which allowed them to have several applications. Among different types of nanomaterials; silver nanoparticles (AgNPs) were interestingly studied for their beneficial effects when used as antimicrobials, antioxidants and/or anticancer compounds. In addition to their medical uses, AgNPs were also used in clothing, food industry, paints, electronics and other fields (Abdel-Aziz et al., 2014).
AgNPs can be synthesized by several physical and chemical methods (Roopan et al., 2013) such as; UV or microwave irradiation, chemical reduction, photochemical method, electron irradiation and sonoelectrochemical method (Suman et al., 2013). However, the major limitations of these methods include high cost, employ of toxic chemicals as reducing and stabilizing agents, use of organic solvents or non-biodegradable agents (Zhang et al., 2013). According to Jagtap and Bapat, (2013), biosynthesis of nanoparticles was advantageous over the physical and chemical methods; as it was clean, non-toxic, involve the use of an ecofriendly renewable high energy efficient materials, benign reaction media, non-hazardous as well as non-toxic solvents. In last few years, the potential use of various plants extracts such as; banana, hibiscus, geranium leaves, Cinnamomum, Aloe vera, basil tansy fruits, and sweet pepper for the green synthesis of AgNPs were investigated (Malabadi et al., 2012). Remya et al., (2015) pointed out that plants serve as readily available sources of bioactive compounds including; alkaloids, amino acids, flavonoids, terpenoids and other phenolic intermediates; that could act as effective reducing agents for the bio-reduction of metals into nanoparticles (NP), which in turn have a wide range of biological applications. P. afra L. plant is a succulent member of the Didereaceae (formerly of the Portulacaceae), which was native to South Africa and commonly found in semi-arid areas. P. afra called elephant's food; has small succulent leaves. This plant can grow up to 2-5m in height as a large woody shrub, or small tree (Guralnick and Gladsky, 2017).

Collection of P. afra leaf samples and chemicals
Fresh leaves of P. afra (Fig. 1) were collected from local garden in Giza governorate, Egypt. The leaves were washed with deionized water, and then allowed to dry at room temperature. In order to prepare the extract, fresh leaves were cut into small pieces. All chemicals used were of analytical grade purchased from Sigma-Aldrich (USA).

Preparation of plant leaf extract
The extract was prepared by weighing 2.5 g of fresh leaves in 250 ml of deionized water, and then boiling them for 30 min. on a hot plate with magnetic stirrer. The resulting solution was cooled and filtered using a filter paper Whatman No. 1. The extract was colorless and clear, with pH 4.5. The extract solution was stored at 4°C until further use as a reducing agent (Suman et al., 2013).

Biosynthesis of AgNPs
The biosynthesis of AgNPs was performed by adding 1ml of P. afra extract to 30 ml of AgNO 3 (1mM) solution according to the method described by El-Chaghaby and Ahmad, (2011) with some modifications. This mixture was boiled with continuous stirring for 5 min. Formation of AgNPs was monitored using a UV-vis spectrophotometer (SpecorD 250 plus-Analytik Jena) in the wavelength range of 200-800 nm.
Novel Research in Microbiology Journal, 2019

Characterization of biosynthesized AgNPs
AgNPs biosynthesized using P. afra extract was characterized using High-Resolution Transmission Electron Microscope (HR-TEM, Tecnia G20, FEI, Netherlands), and particles size distribution was determined by laser diffractometer using Zeta Sizer nano-series (Nano ZS).

Antibacterial potential of AgNPs
Disc diffusion method was adopted to evaluate the antibacterial activity of biosynthesized AgNPs. The assay was done following the procedure described by Malabadi et al., (2012). Six bacterial species were tested including B. subtilis, E. coli, N. gonorrhoeae, P. aeruginosa, S. aureus and Strept. faecalis, obtained from Microbiology unit at Microanalytical center, Cairo University. In this experiment, filter paper discs (1 cm) were soaked separately in one of the following solutions: 1-P. afra extract (10 mg/ ml), 2-AgNO 3 (1mM), and 3-AgNPs (1 mM). 0.1 ml culture suspension of each bacterium adjusted to 10 8 cells/ ml, was seeded separately into Muller-Hinton agar medium, and then poured into Petri plates. One filter paper disc from each treatment was placed aseptically at the center of each of these plates. Plates were incubated at 37°C for 24 h. Three replicate plates were used for each treatment. Zones of inhibition of bacterial growth were measured around each disc and recorded in mm. (El-Chaghaby et al., 2014).

Statistical analysis
The data were statistically analyzed using one way ANOVA and the means were compared by Duncan's test at (p < 0.05).

Biosynthesis of AgNPs
Reduction of AgNO 3 solution after 5 min. from adding P. afra extract was visually noticed; where the colorless solution turned to yellowish brown in color which indicated extracellular synthesis of NP in accordance with Husseiny et al., (2015). To confirm the formation of AgNPs; UV-visible absorption pattern (Fig. 2) showed the characteristic surface plasma resonance of colloidal AgNPs which ranged between 300-500 nm, and exact peak was observed at 420 nm. AgNPs exhibited interesting optical properties directly related to their Localized surface plasma resonance (LSPR); which was highly dependent on the morphology of these NP. In a similar earlier study of El-Chaghaby and Ahmad, (2011), a typical plasma resonance band of AgNPs was confirmed by an absorption band in the range of 400-450 nm.

Characterization of biosynthesized AgNPs
HR-TEM image of the biosynthesized AgNPs were shown in Fig. (3a). This technique was employed to visualize the morphology and shape of the synthesized AgNPs. The AgNPs were irregular, spherical in shape and with different sizes.
Novel Research in Microbiology Journal, 2019 The AgNPs size distribution was shown in Fig. (3b); where, 99.8% of these NP had average diameters of 27.41 nm, and width of 4.36 nm.

Antibacterial potency of AgNPs
Results of detecting the antibacterial potential of AgNPs were shown in Table (1). The P. afra leaf extract had no inhibitory effect against the six studied bacterial spp. On the other hand, AgNO 3 and AgNPs exhibited significant inhibition against all tested bacteria. The inhibition zones observed due to activity of AgNPs were found to be wider than those caused by AgNO 3 . In reference to Zhang et al., (2013), this was attributed to the fact that the surface of NP could form a layer of water, Novel Research in Microbiology Journal, 2019 Table 1: Antibacterial potential of plant extract, AgNO 3 and AgNPs against some bacterial spp.

Inhibition zones (mm)
P. afra extract (10 mg/ ml) thus Ag ions could easily be released into the water. The bacterial cell-membrane was composed mainly of phospholipid bilayers and protein molecules; therefore, the whole cell membrane owned negative charge due to the presence of this phosphate group. The Ag ions had positive charge, thus were able to bind to the bacterial cell membrane quickly, causing deformation of this bacterial structural membrane. Moreover, Ag ions could also be strongly attracted to the negatively charged sulfhydryl group (SH) of the bacterial enzymes, causing their deactivation and subsequent inhibition of cell growth.
It could be also observed that the inhibitory effect of AgNPs was significantly (p<0.05) higher against some Gram negative compared to Gram positive bacteria. This might be because Gram positive bacterial cell wall was thicker; composed of thick peptidoglycan layer to provide structural strength to the cell, thus become able to counteract the cytoplasmic osmotic pressure (Zafar et al., 2016).

Cytotoxic activity of AgNPs against MCF-7 cell line
% Cell inhibition assay was an important test to measure the cellular response to drugs/toxicant, and also to provide information about cell survival, death and metabolic activities (Chandrasekaran et al., 2016). In the current work, it was noticed that AgNPs showed no cytotoxic effect against MCF-7 cell line at low concentrations up to 40 µmole. This was attributed to the green synthetic approach of AgNPs followed in this work; which might result in reducing its toxicity especially at low concentration, compared to AgNPs synthesized chemically. Above 40 µmole, the cytotoxicity of AgNPs against MCF-7 cell line was found to be concentration dependent, as the percentage of cell viability was decreased with increasing corresponding concentrations of the AgNPs ( Table 2).
The IC 50 is the dose required to kill 50% of the cultured breast cancer cells population, and it was estimated from the dose response curve plotted (Fig.  4). Currently, the IC 50 of AgNPs was recorded to be 75.40 µmole. Previous study of Valodkar et al., (2011) reported that NP induced cytotoxicity in a variety of mammalian cell lines (hepatocytes and fibroblasts), due to oxidative damage to the cell membrane and mitochondrial dysfunction. NP induced toxicity was exerted in the form of transferring electrons from molecular oxygen or by blockade of electron transport chain.

Conclusion
It could be concluded from the present findings that, AgNPs biosynthesized using P. afra leaf extract demonstrated potential antibacterial potency against both Gram positive and Gram negative bacteria. Moreover, it exhibited anticancer activity against breast cancer cell line (MCF 7). Therefore, these AgNPs have several advantages such as; cost effectiveness and eco-friendly technique for their green synthesis, which will help in their wide scale commercial production.