In vitro antioxidant, antimicrobial, cytotoxic potential of gold and silver nanoparticles prepared using Embelia ribes

Abstract In recent years, the green synthesis of gold (GNPs) and silver (SNPs) nanoparticles has gained great interest among chemists and researchers. The present study reports an eco-friendly, cost-effective, rapid and easy method for the synthesis of gold and silver nanoparticles using the seed extract of Embelia ribes (SEEr) as capping and reducing agent. The synthesised GNPs and SNPs were characterised using the following techniques: UV–vis spectroscopy, DLS, HR-TEM, FT-IR and XRD. The free radical scavenging potential of GNPs and SNPs was measured by DPPH assay and Phosphomolybdenum assay. Further, the antimicrobial activity against two micro-organisms were tested using disc diffusion method and cytotoxicity of GNPs and SNPs was determined against MCF-7 cell lines at different concentrations by MTT assay. Both the GNPs and SNPs prepared from E. ribes comparatively showed promising results thereby proving their clinical importance.


Introduction
The wide variety of possible biomedical applications of GNPs include drug and gene delivery (Kumar et al. 2013), proteins and pathogen detection, deoxyribonucleic acid labelling, ABSTRACT In recent years, the green synthesis of gold (GNPs) and silver (SNPs) nanoparticles has gained great interest among chemists and researchers. The present study reports an eco-friendly, cost-effective, rapid and easy method for the synthesis of gold and silver nanoparticles using the seed extract of Embelia ribes (SEEr) as capping and reducing agent. The synthesised GNPs and SNPs were characterised using the following techniques: UV-vis spectroscopy, DLS, HR-TEM, FT-IR and XRD. The free radical scavenging potential of GNPs and SNPs was measured by DPPH assay and Phosphomolybdenum assay. Further, the antimicrobial activity against two micro-organisms were tested using disc diffusion method and cytotoxicity of GNPs and SNPs was determined against MCF-7 cell lines at different concentrations by MTT assay. Both the GNPs and SNPs prepared from E. ribes comparatively showed promising results thereby proving their clinical importance. fluorescent labelling, tissue engineering, photothermal ablation and contrast agents for magnetic resonance imaging and other imaging methods. The plant-mediated nanoparticle synthesis is preferred because it is cost-effective, environment friendly, single step process and safe for human therapeutic purpose (Kumar & yadav 2009). So far, different parts of plant materials such as extracts from leaves (Rahimi-Nasrabadi et al. 2014;Murugan et al. 2015), fruits (Prathna et al. 2011), bark (Sathishkumar et al. 2009), fruit peels (Bankar et al. 2010), root (Ahmad et al. 2010), seeds (Anbu et al. 2015) and callus (Nabikhan et al. 2010) have been studied for the synthesis of gold, silver and other metals.
In this present study, we have demonstrated the synthetic route of GNPs and SNPs using the seed extract of Embelia ribes plant (SEEr). E. ribes Burm. commonly known as 'Vidanga, ' is a large woody climbing shrub belongs to the family Myrsinaceae, widely distributed in India, Sri Lanka, Malaysia and South China (Guhabakshi et al. 2001). The antioxidant, cytotoxic, antibacterial potential of SEEr-GNPs and SNPs were studied. Both SEEr-GNPs and -SNPs comparatively showed promising results thereby proving their clinical importance.

Results and discussion
The formation of gold and silver nanoparticles from 1mM solution of silver nitrate and chloroauric acid was confirmed using UV-vis spectrophotometer analysis. The gold solution has changed from pale yellow to wine red colour showing a characteristic peak in the range between 500 and 550 nm whereas the silver solution has changed from colourless to yellowish-brown colour showing peak between 400 and 440 nm (Figure S1a and S1b) due to the reduction of metals ions to neutral. The constituents of E. ribes seed extract including alkaloids, quinones, proteins, reducing sugars and saponins may be responsible for the reduction of metal ions. The size of SEEr-GNPs and -SNPs were in the range of 10-30 and 5-35 nm, respectively. Figure S1c and S1d shows that the TEM images of GNPs and SNPs prepared from the seed extract of E. ribes plant are spherical and polydisperse in nature. Figure S1e and S1f shows the hydrodynamic diameter of seed extract mediated synthesised gold nanoparticles which was found in the range between 6 and 68 nm, while that of silver nanoparticles was 5 to 122 nm).
The proton donating capacity of SEEr-GNPs was found to be higher than SEEr-SNPs. The absorbance decreases as a result of a colour change from purple to yellow as radical was scavenged by antioxidants through donation of hydrogen to form the stable DPPH radical ( Figure S3a). The IC 50 of SEEr-GNPs and SEEr-SNPs to scavenge free radicals was found at 20 and 100 μg/mL, respectively. The phosphomolybdenum assay includes the reduction of Mo (VI) to Mo (V). In the present study, the results show that the seed extract showed 40% of PRP potential at 100 μg concentration, whereas SEEr-GNPs showed IC 50 at 40 μg and SEEr-SNPs at 60 μg concentration ( Figure S3b). At 100 μg concentration, SEEr-GNPs showed 66% reduction and SEEr-SNPs showed 60% reduction of phosphomolybdenum. The antioxidant potential of nanoparticles reveal that they have been reduced with phenolics in the seed extract of E. ribes. Figure S4 and S5 demonstrate that GNPs showed ~30% decrease in the tumour cells at 10 μg/mL and SNPs showed ~20% decrease in the cells at 20 μg/mL concentration. Both GNPs and SNPs showed their IC 50 values at 100 μg/mL concentration ( Figure  S6). SEEr-GNPs showed strong antibacterial action against Escherichia coli compared to SEEr-SNPs with the increase in the inhibition zone. When tested on Staphylococcus aureus, both GNPs and SNPs showed good antibacterial action. The zone diameter range of SEEr-GNPs treated E. coli was 28-34 mm and S. aureus was 22-27 mm. The diameter range of inhibition zones obtained in SEEr-SNPs treated E. coli was 20-28 mm, while for S. aureus was 22-27 mm ( Figure S7 and S8). When treated with tetracycline at 15 mg/mL concentration, E. coli and S. aureus showed inhibition zone of 11 and 17 mm, respectively (Table S1). The zone diameter was found to be larger for Gram-negative bacteria (E. coli) than Gram-positive bacteria (S. aureus) which may be due to the variation in the cell wall components. The cell wall of Gram-positive bacteria consists of a thick peptidoglycan layer, linear polysaccharide chains cross linked by short peptides that make the cell wall rigid thereby leading to difficult penetration of GNPs and SNPs (Ibrahim 2015).

Conclusion
The biosynthesised gold and silver nanoparticles using the seed extract of E. ribes were characterised: they are crystalline, uniform and spherical with average particles size of about 20-30 nm. The SEEr-GNPs and SEEr-SNPs showed good antioxidant property that has reduced the free radicals in both DPPH and PMA assay. Both GNPs and SNPs were found to be good antibacterial agent that has inhibited the growth of Gram-negative (E. coli) and Gram-positive (S. aureus), among which greater activity was found against Gram-negative bacterium. This green synthesis approach appears to be a cost-effective, non-toxic, eco-friendly alternative to the conventional microbiological, physical and chemical methods.

Supplementary material
Experimental details relating to this paper are available online, alongside Figures S1-S8 with Table S1.

Disclosure statement
No potential conflict of interest was reported by the authors.