UV–Vis Spectrophotometric studies: Recording of localized surface plasmon resonance (LSPR)
Bioreduction of aqueous AuCl4− ions can easily be followed by UV–Vis spectrophotometer, and one of the most important features in optical absorbance spectra of metal nanoparticles is surface plasmon band, which is due to collective electron oscillation around the surface mode of the particles. Previous studies have shown that gold exhibits red wine color and silver exhibits yellowish-brown color due to the excitations of their surface plasmon response (SPR) (Mulvaney 1996), when dissolved in water. The change in color of the solution was observed (from colorless to dark red wine color) after keeping the solution at 50 °C for 25 min (Fig. 2).
In the case of gold, the reduction started within 5 min after the addition and completed in 30 min. The possible explanation of difference in the reduction time could be due to the difference in their reduction potential for both the metal ions. Metal nanoparticles such as gold have free electrons, which give rise to SPR absorption band (Noginov et al. 2007) at and around 555 nm (Fig. 3). The reduction and stabilization of Au+ ions could be done by combinations of biomolecules found in the extracts such as proteins, aminoacids, polysaccharides and vitamins which are evidenced in FT-IR studies (Thirumurugan et al. 2010).
Transmission electron microscopy (TEM) and energy dispersion spectroscopic (EDS) measurements
The EDS spectrum of AuNPs synthesized at 50 °C is shown in Fig. 4. Strong signals from the gold atoms in the nanoparticles were observed, and signals from Si, K and C atoms were also recorded. The presence of C and K, signals were likely due to X-ray emission from carbohydrates/proteins/enzymes present in the cell wall of the biomass. The presence of the elemental gold can be observed in the graph obtained from EDS analysis, which also supports the XRD results. The TEM images of AuNPs are shown in (Figs. 5, 6). The TEM images have shown that the formed AuNPs were polydispersed and were predominately spherical in nature. But it is evident from TEM micrographs that triangular, hexagonal, rod and irregular shaped nanoparticles were also formed.
X-Ray diffraction (XRD): study of crystalline structure of gold nanoparticles
The XRD pattern of the AuNPs is shown in (Fig. 7). Bragg reflections obtained in the micrograph clearly indicated the presence of (111) and (200) sets of lattice planes which is a consequence of crystalline nature of formed AuNPs and indexed as face-centered-cubic (FCC) structure of gold. In addition to the Bragg peaks representative of FCC AuNPs, additional as yet unassigned peaks are also observed suggesting that the crystallization of bio-organic phase occurs on the surface of the nanoparticles.
Fourier transform infrared (FT-IR) spectroscopic studies
FT-IR results reveal the absorption bands at 3,444, 1,634 and 694 cm−1 (rhizome extraction) (Fig. 8); 3,424, 1,620 and 673 cm−1 (AuNPs at 50 °C) (Fig. 9b), respectively. The vibrational bands corresponding to the bonds such as amines (N–H stretch), –C=C (alkane), C–Cl (Halogens) which was in the region range of 694–3,444 cm−1. The most wide spectrum absorption was observed at 3,424 and 3,444 cm−1 and it can be attributed to the stretching vibrations of amino (N–H) (Rajasekharreddy et al. 2011), absorption peaks centered at 1,620 and 1,634 cm−1 can be attributed to the stretching vibration of –C=C (alkane) (Zhu 2000). Amines are a particularly attractive class of reducing agents because of their structural or chemical properties (Newman and Blanchard 2006). Thus, the FT-IR micrograph reveals that amides that are present in the extract are responsible for the reduction and stabilization of the gold nanoparticles.
Dynamic light scattering (DLS) technique: particle size measurement of hydrosol
Particle size determination of the formulated AuNPs was shown under different categories like size distribution by volume and intensity (Fig. 9). The size distribution by volume gives a bell shaped (Fig. 9) pattern which indicates the wide range size distribution of nanoparticles in the sample formulation. The volume % of the samples were found to be in the range of 0.1–1 × 108(AuNPs) The formed AuNPs are well distributed with respect to volume and intensity, an indication of the formation of well built AuNPs and their mono and poly disparity, respectively.
Antibacterial and cytotoxicity activities of AuNPs
The growth curves of S. aureus (41.1, 58.8, 61.3 and 80.77 %) S. epidermidis (31.8, 52.88, 62.14 and 86.5 %) and E. coli (19.1, 62.43, 69.84 and 82.2 %) in MH broth medium in the presence of AuNPs at 1, 0.5, 0.25 and 0.125 mM concentrations are shown in Fig. 10. Zhang et al. (2008) used an amido-amine coated AuNPs and tested their toxicity on a large suite of gram-negative and positive bacteria. At 2.8 mg/L, AuNPs demonstrated up to a 98 % inhibition of bacterial growth. Gold nanoparticles possess well-developed surface chemistry, chemical stability and appropriate smaller size, which make them easier to interact with the microorganisms (Nirmala Grace et al. 2007). Also, the particles interact with the building elements of the outer membrane and might cause structural changes, degradation and finally cell death (Zawrah and Sherein 2011).
The incubation of B16/F10 melanoma cells with synthesized AuNPs significantly reduced the viability of these cells in a dose dependent manner and all concentrations (0.2–1 mM) were found toxic to the cells (Fig. 11). The maximum inhibition of proliferation was 52.98 % at higher concentration (1 mM); 21.51 % minimum cell inhibition observed at lower concentration (0.2 mM). From the results, it is evident that synthesized AuNPs have inhibitory effect on SGT oral cancer cells. It is also important to recognize that a vast majority of gold (I) and gold (III) compounds show varying degrees of cytotoxicity to a variety of cells (Basset et al. 2003). Further size and shape dependent uptake of gold nanoparticles into mammalian cells has been reported and points to the need of in depth study of size and shape dependent antimicrobial and cytotoxic effects of nanoparticles (Devika Chitrani et al. 2006).