Optical and biomedical applications of eco-friendly biosynthesized silver nano spheres using zingiber officinale root extract

Eco-friendly bio-compatible silver nanoparticles (Ag NPs) were successfully synthesized using Zingiber officinale extract in a simple green route at room temperature. The phytoconstituents present in Zingiber officinale (Z. officinale) extract act as reducing and stabilizing agents. The size and crystallinity of spherical Ag NPs were confirmed through transmission electron microscopy (TEM) and X-ray diffraction (XRD) studies. The formation of silver nanoparticles was also confirmed from the UV–vis and FTIR spectra. Luminescence properties of europium (Eu) and samarium (Sm) complexes in the presence of silver were studied. The intensity of luminescence from Eu and Sm complexes were found to get enhanced or quenched with their concentrations in Ag NPs. Interesting nonlinear optical properties exhibited by Ag NPs were observed in the Z-scan experiment suggesting that they can be used as optical limiters for the picosecond (ps) time scale green laser. Silver nanoparticles were tested against colon cancer (HCT116) cells in vitro by MTT assay and they exhibited smaller IC50 values with better inhibition efficiency. Ag NPs induced apoptosis through the activation of Reactive oxygen species (ROS) and Caspase-3 pathways. Antibacterial activity of Ag NPs was analysed against Acinetobacter baumannii (A. baumannii) and Staphylococcus aureus (S. aureus) and they were found to be efficient in inhibiting the bacteria. The results indicate that the biosynthesized eco-friendly nanoparticles having high stability can lead to many applications such as good luminescence enhancement, optical limiting characteristics, anticancerous and antibacterial properties in optics and biomedicine.


Introduction
Nanotechnology has gained attention and shows rapid growth due to its capability to tune the properties of materials [1]. Nanoparticles (NPs) exhibit exclusive electronic, magnetic, chemical, mechanical, optical, medicinal and catalytic properties in comparison with bulk materials because of the quantum size effect and large surface to volume ratio [2]. Among all the metals, silver is fascinating owing to its ease of preparation, chemical and physical properties, disinfecting nature and medicinal value particularly acting as effective anticancer agent. Its various applications extend to biosensors, ultrasensitive detection, bio-imaging, oxidative catalysis, nano-electronics, surface enhanced Raman scattering, and antimicrobial activity [3][4][5]. A number of techniques are feasible for the preparation of Ag NPs, such as chemical, electrochemical, photochemical, irradiation methods, Langmuir-Blodgett and biological techniques [6]. Many of these techniques are hazardous, complicated and costly. They have drawbacks due to toxic solvents and by-products with high energy consumption. Consequently, there is a demand to establish green synthesis of NPs using micro-organisms and plant extract to make it environmental friendly and cost-effective. Organic synthesis of NPs by microorganisms, enzymes is complex and requires elaborate process by cell culture and environmental issues. Thus plant extracts are promoted for preparation of nanoparticles [4]. Several reports have appeared on synthesis of silver NPs by plant extracts. Some of them are using: Radish [7], Pinus desiflora (red pine) [8], ginko, persimmon, mokryeon, oriental plane, Cycas leaf [9], drumstick leaf [10], Shorea tumbuggaia stem bark [11], Cinnamon [12], papaya [13], biskhapra roots [14], Jatropha curcas latex [15], jack fruit leaf [16], big-sage [17], coriander leaf [18] and wild croton leaf [19] etc.
Ginger root belongs to the family Zingiberaceae, which has been consumed as a spice and also as medicine. 3% of essential oil contained in ginger causes fragrance of spice. Sesquiterpenoids with zingiberene are major constituents of ginger. Compounds like farnesene and β-sesquiphellandrene bisabolene belong to sesquiterpenoids (citral, cineol and β-sesquiphellandrene). Gingerol-related components possess high antifungal, antimicrobial, anticancerous, antioxidant and anti-inflammatory pharmaceutical properties [20][21][22]. Hence ginger has been serving as an oriental traditional medicine used in treating many diseases like cold, cough, nausea, rheumatism, cardiac disorders, inflammation and tumors.
Luminophors (III) draw good attention because of their potential applications in fluoroimmunoassay, light emitting diodes (LED) and optical signal amplification. Mostly, recent work is concentrated on synergy of luminophores with metallic nanostructures, quantum dots, organic molecules, dyes or polymers to achieve higher luminescence. An increase in the radiative decay rate of luminophores can be achieved by introducing a resonant Plasmon mode of a metal nanoparticle. The ability of the photoluminescence (PL) enhancement is based on: (i) the distance between metal nanoparticle and rare-earth ion, (ii) coordination around rare-earth ions and (iii) the excitation wavelength. This demonstrates that the enhancement owes to coupling of the dipoles of lanthanide ion transitions with Plasmon modes of metallic nanostructures, resulting in enhancement several orders of magnitude in radiative decay rates when the separation them is below 20 nm [23][24][25][26]. Other reason for the enhancement of luminescence from emitters is due to the surface-enhanced fluorescence (SEF), where the separation between metal surface and emitter is a crucial factor [27].
Ag nanoparticles also exhibit good third order nonlinear optical (NLO) properties and showed their application as optical limiter [28]. Bio synthesized Ag NPs show better optical limiting properties even though the bio molecules cap the Ag NPs [29][30][31][32].
Cancer has been a major uncontrolled degenerative health problem. Identification with least side effects and locating the new anti-cancer drug has become crucial aspect of current research in cancer therapy. With extraordinary antimicrobial and anticancerous activities, application of Ag NPs have become more extensive in medicine as they are playing an important role in medicinal devices, dressings, nano-lotions, gels etc, [33]. They exhibit antibacterial activities against gram-positive and gram-negative bacteria. Majority of bacteria have grown their own resistance to antibiotic drugs, thus forcing us to develop a replacement for antibiotics in future. Ag NPs are more attractive due to their non-toxic and antibacterial behaviour in broad range with no side effects to human body. Various studies reported that Ag NPs remarkably diminish the function of mitochondria with induced cell apoptosis or necrosis. Au NPs are simply bound with thiol and amine groups, which modifies the surfaces with DNA and amino acids which have good biocompatibility in clinical applications. The cytotoxicity of these NPs depends on the size, shape and surface modification [34][35][36][37][38].
The goal of the study is to improve the role of green chemistry both in optics and in biomedicine. Current work demonstrates the luminescence efficiency of eco-friendly Zingiber officinale based biosynthesized Ag NPs on lanthanide complexes and also optical limiting efficiency. The study validates the possible in vitro antiproliferative effects of bio synthesized silver nanoparticles against the HCT116 cell lines. The report presents the antibacterial activity of the NPs against (A. baumannii) and Staphylococcus aureus (S. aureus). Moreover, the report elucidates the efficiency of NPs in luminescence enhancement, optical limiting, anticancerous and antibacterial activities.

Reagents
Silver nitrate (AgNO 3 ) was obtained by Sigma Aldrich. Zingiber officinale roots were purchased from a local market. Human colon (HCT116) cell lines were provided by National Centre for Cell Science (NCCS), Pune, India.

Preparation of extract from Zingiber officinale root extract
First the fresh roots were thoroughly rinsed in distilled water many times and 10 grams of it were cut and added to 100 ml of distilled water and heated for 5 min. The mixture was cooled to room temperature and was filtered with a filter paper (Whatman no. 1). The solution was again filtered with 1 mm filter paper to avoid granules and stored in a refrigerator.

Synthesis of Silver nanoparticles
Briefly, AgNO 3 (1 mM) aqueous solution was prepared and 3 ml of root extract was added to 30 ml of 1 mM silver nitrate solution. Initially the solution was transparent. After few minutes the solution turns to dark ash colour indicating the formation of Ag NPs. The experiment was repeated thrice to confirm the reproducibility.

Experimental details
Silver solutions were coated on a silica substrate and dried at room temperature to record the XRD spectrum by Cu−K α X-radiation (λ=1.5406 Å) of Bruker D8 diffractometer operated at 30 mA and 40 kV power with scan rate of 0.05°/min over the 2θ range 30-80°. The FTIR spectra of Ag NPs solution was recorded using Thermo-Nicolet 6700 in the 400-4000 cm −1 region. The reduction of silver ions to silver NPs was confirmed by recording the absorption spectrum on UV-visible spectrometer JASCO in the 300-800 nm range with 1 nm resolution. TEM images and Energy dispersive X-ray analysis (EDX) were recorded on TECHNAI G2 S-Twin at 20 kV voltage to investigate the morphology, size and crystallinity of Ag NPs. Field emission scanning electron microscopy (FESEM) studies were accomplished on Carl ZEISS-Ultra 55 FE-SEM model. Photoluminescence (PL) emission spectra of liquid Ag NPs with rare-earth ions were measured on HORIBA YVON spectrometer over the range of 570-670 nm by exciting at 350 nm. Third order nonlinear optical studies were performed by Z-scan using a ps Nd:YAG laser (EKSPLA-2143A) operating at 532 nm, having a pulse duration of 30 ps and operating at 10 Hz pulses. Ag NPs dispersed in water were used in 1 mm quartz cuvette for recording the Z-scan data. The output light is received with photodiode and a reference photodiode monitoring the laser fluctuations.

Cytotoxicity (MTT assay)
The cytotoxic assay of prepared Ag NPs on HCT116 cancer cells was obtained by MTT (3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide) assay. The cell lines were grown in Dulbecco's modified Eagle's medium (DMEM), with 10% fetal bovine serum (FBS), penicillin (100 μg ml −1 ), streptomycin (100 U ml −1 ) at 37°C atmosphere of 5% CO 2 . The cell lines were seeded for 24 h before exposure to Ag NPs. The actively grown HCT116 cells were seeded in a 96-well plate at a density of 1×10 4 per well, incubated in DMEM/1% FBS. After that, media was replaced with Ag NPs of various concentrations at 25, 50, 75, 100 and 125 μM. The cells were incubated at 37°C for 1 h with 20 μl MTT solution. The MTT solution was removed and replaced by DMSO. Optical density (OD) was obtained at 550 nm by scanning ELISA plate reader. Cell culture medium with cells act as negative control. Cell viability measurements were calculated from the ratio of mean optical density to the negative control. The cytotoxicity measurements of Ag NPs are compared with Oxaliplatin (OXP) drug as positive control.

Apoptosis assay using ANNEXIN V
Combining of Annexin V-FITC/PI to cancer cells was obtained using Annexin V-FITC Apoptosis detection Kit I (BD Biosciences, New Jersey, and USA). 2 ml of HCT116 cells (3×10 5 /2 ml) were treated with Ag-NPs and camptothecin (15 μM) for 24 h and cleaned with PBS twice. These were suspended in buffer (100 μl) and changed to culture tube (5 ml). Then, 5 μl of FITC Annexin V combined with cells incubated at 25°C in dark for 15 min. The binding buffer (400 μl) with PI (5 μl) added to every tube. Then the cells were studied on flow cytometer (Becton Dickinson, FACS Calibur, USA).

Caspase-3
In vitro caspase activity in Ag-NPs, both camptothecin (15 μM) treated and untreated cells were studied using FITC rabbit anti-active caspase-3 antibody (BD Biosciences, New Jersey, USA). Cancer cells were cleaned in PBS and permeabilized using BD Cytofix/Cytoperm Kit at RT for 20 min, then pelleted and cleaned with BD buffer. Cancer cells were subsequently stained with FITC rabbit anti-active caspase-3 antibody (BD Biosciences, clone C92-605). Then, cells were cleaned and suspended in BD buffer to study by flow cytometry (Becton Dickinson, FACS Calibur, New Jersey, USA).

Antibacterial activity
Here, D is mean size of particles, K is shape factor, λ is wavelength, β is full width half maximum of the peak, θ is angle of diffraction from the XRD spectrum. From above equation, the diameter of nanoparticles is estimated as ∼6.1 nm (calculated from [111] plane diffraction peak). This is comparable with the TEM data.
The diffraction peaks at 27.1°, 32.3°, and 46.3°are due to constituents present in Zingiber officinale, which act as capping and stabilizing agents. Hence, these results affirmed that silver nanoparticles were formed by reduction of silver ions by Zingiber officinale extract [3,39].

FTIR
The FT-IR spectrum of Zingiber officinale extract produced silver nanoparticles is seen in figure 2. Major bands noticed at 3317, 1638, 1381, 1082, 815, 721 and 611 cm −1 are interpreted below. The characteristic peak 3317 cm −1 is the stretching mode of N-H or C=O. 1638 cm −1 is attributed to N-H bonding vibration of amides indicating their existence in extract during the reduction of nanoparticles. The band at 1381 cm −1 is ascribed to -C=C alkane stretching vibration. The band at 1082 cm −1 is attributed to the stretching vibration of -C=O. The bands at 815, 721 and 611 cm −1 are the strong signals of heterocyclic compounds, the active components of Z. officinale such as alkaloids and flavonoids, which act as capping agents. These peaks reveal the presence of heterocyclic compounds in extract which are responsible for reduction of the silver ions [40,41].  and its correspnding planes are observed. These have been supported by the XRD. Spherical particles' mean diameter is around 16.8 nm with a range from 12 to 24 nm. Figure 3(e) shows the EDX pattern indicating the presence of chemical composition of Ag. The percentage of elements found to be Silver (Ag) 13%, Iron (Fe) 24%, and Copper (Cu) 77%. The other elements like Fe and Cu served as capping agents bound to the surface of the Ag NPs. Table 1 compares the d-spacing values obtained from TEM and XRD where both of them match closely [42].

FESEM
Bio synthesized silver nanoparticles are confirmed by the structural analysis with FESEM and the results are shown in figure 4. FESEM analysis revealed that the formation of Ag NPs capped with biomolecules had uniform distribution with spherical shape. The size of the nanoparticles varied from 10 to 20 nm in diameter.

UV-vis absorption
When Z. officinale root extract was added to aqueous AgNO 3 , the solution turned from transparent to dark ash colour owing to excitations of surface Plasmon of Ag nanoparticles. Appearance of surface Plasmon resonance (SPR) peak in UV-vis absorption spectra which is around 435 nm. Figure 5 (inset 5(ii)) clearly indicates the formation of silver NPs. Shape of the Ag NPs determines the number of SPR bands appearing in the absorption spectra, as predicted by the Mie theory [26]. Based on this, absorption spectra of spherical NPs' have only one SPR band while the other shapes lead to two or more Plasmon bands. Single SPR band shown in inset (ii) indicates that the biosynthesized Ag NPs are uniformly distributed as nanospheres, which also matches with the XRD and TEM analysis.

Photoluminescence
The effect of the presence of silver on the luminescence emission from Eu (TTFA) 3 and Sm (TTFA) 3 are investigated with various Ag NPs concentration (λ exc =350 nm) and the results are shown in figures 6((a) and (b)). Three luminescence emission peaks centred at 614, 577 and 590 nm are assigned to 5 D 0 → 7 F 2 (electric dipole), 5 D 0 → 7 F 0 and 5 D 0 → 7 F 1 (magnetic dipole) transitions for Eu(TTFA) 3 respectively ( figure 6(a)). Similarly, figure 6(b), shows the luminescence spectra of Sm (TTFA) 3 with different concentrations of silver. The three bands at ( 4 G 5/2 → 6 H 5/2 ) 566 nm, ( 4 G 5/2 → 6 H 7/2 ) 602 nm (magnetic dipole) and ( 4 G 5/2 → 6 H 9/2 ) 645 nm (electric dipole) appear. Inset figures show the dependence of luminescence intensity on the concentration of Eu complex (20, 30, 40 μl) and Sm complex (200, 220, 240 μl) with varying Ag concentrations from 5 to 300 μl. In the case of Eu 3+ ions, enhancement factors (EF) for electric-dipole transition ( 5 D 0 → 7 F 2 ) is 8.7. For magnetic dipole transitions ( 5 D 0 → 7 F 0 and 5 D 0 → 7 F 1 ) EFs are 3.81 and 5.44 in the presence of Ag NPs. In case of Sm 3+ ions, EF's of electric dipole transition ( 4 G 5/2 → 6 H 9/2 ) is 3.6 and magnetic dipole transitions ( 4 G 5/2 → 6 H 5/2 and 4 G 5/2 → 6 H 7/2 ) are 1.81 and 3.04 respectively.      The normalised emission intensity of electric dipole transition increases up to a certain concentration of Ag and then decreases with further increase of Ag, which could be due to re-absorption of Plasmon resonance (SPR). From inset of figure 6(ii), the absorption of Ag NPs over the wavelength range ∼300-600 nm practically covers the emission of the rare-earth complex ( figure 6). From Forster mechanism [19,33], the absorption is a competition between SPR band of Ag NPs and rare-earth complex. However, the energy transfer from lanthanum ions to silver NPs i.e., the emitted energy from complex is absorbed by Ag NPs. Therefore the enhancement is due to the balance between SPR and SEF (surface enhanced fluorescence) and also it depends on size, concentration and the medium around of Ag NPs.
The intensity ratio, shown in table 2, between electric and magnetic dipole transitions of rare-earth complexes indicate the symmetry around rare-earth ions. In case of Eu complex, at maximum emission enhancement, intensity emission ratio first decreases and then increases. It means that (i) there is an energy transfer between lanthanum ions and silver NPs, (ii) induced surface Plasmon resonance of silver NPs influence the field around rare-earth ions. The emission from rare-earth ions depends on the size of Ag NPs as the SPR peak changes with size. In case of Sm complex, the intensity ratio did not alter significantly, which indicates that Ag NPs' SPR does not have resonant interaction with the lanthanum ions. Figure 7 shows the fluorescence decay curves of 5 D 0 and 4 G 5/2 levels of Eu 3+ and Sm 3+ ions with and without 70 μl of Ag NPs at 613 nm and 645 nm under 350 nm excitation respectively. The transitions from 5 D 0 → 7 F 0,1, 2 show longer lifetimes. The curves were fitted with single exponential decay. The lifetime of Eu complex is 97 μs and in the presence of silver NPs, the life time increases to 216 μs. We observe only a small change from 6.3 μs to 10.2 μs in Sm complex owing to the presence of silver NPs. At the off resonant excitation of Ag NPs with 350 nm excitation, there is an appreciable energy transfer from the Ag NPs to the lanthanum ions (both Eu and Sm). This energy transfer keeps increasing as we increase the concentration of the Ag NPs. However, at higher concentration, the Ag NPs are likely to cluster and their resultant absorption shifts towards the red region. At such high concentrations, we would expect a reverse energy transfer, that is, from the Eu and Sm to the Ag NPs, thereby reducing the luminescence from the rare-earth complexes. This means that a small variation in luminescence lifetime of rare earth ion affects the luminescence yield. This enhancement increases the decay rates, thus increase in quantum yields [21,44]. The population of 5 D 0 or 4 G 5/2 level therefore depends on the concentration of rare-earth ions [23], or the emission intensity ratio is proportional to rare-earth concentration. The quantum efficiencies are unaltered which means lifetime and intensity ratios are invariant as shown in table 2.

Optical limiting studies
The nonlinear optical absorption measurements of Zingiber officinale synthesized silver NPs were carried through open aperture Z-Scan technique (figure 8) with Nd:YAG laser (EKSPLA-2143A) at 532 nm, 30 ps and 10 Hz. In short, in normal Z-scan experimental setup, a Gaussian profile beam is focused by a lens. 1 mm thick sample cuvette is moved along the focused beam. At focus, the sample sees a maximum intensity and it continuously reduces from focus in two directions. An f/40 configuration was used here. The width of the sample should be less than the Rayleigh range, which is ∼3 mm for the lens used in the present set up. Neutral density filters and apertures are used to alter the intensity of laser and for beam shaping, respectively. By moving the sample across the focus, the experimental data is measured through boxcar averager (model SRS 250) with the use of analog-to-digital (ADC) card to obtain a good averaging of the pulses and the output is given to a computer. The nonlinear absorption coefficient (a 2 ) was measured by fitting the transmittance equation   sample of length-L, a 0 is linear absorption coefficient. Figure 8 depicts the Z-scan of open aperture data with different intensities of green synthesized Ag NPs. Symbols represent the experimental data and solid lines are theoretical fit using equation (1). The experimental data shows a reverse saturable absorption (RSA) behaviour due to the excitations of SPR band to free carrier band of nanoparticles and also a two-photon absorption (TPA) from ground state. The theoretical fits obtained lead to the absorption coefficient α 2 =10.2×10 −9 − 15.2×10 −9 cm 2 W −1 at various intensities in the range of 0.78 GW cm −2 to 3.9 GW cm −2 [18]. Figure 8(b) displays the optical threshold limiting data of Ag NPs at 532 nm with ps laser. The measured optical limiting threshold value is 54 mJ cm −2 .

Cytotoxic assay (MTT assay)
The Zingiber officinale extract mediated Ag NPs were tested for cytotoxic effect using MTT on HCT116 cell lines as shown in figure 9. Cell lines were tested after 24 h incubation at 37°C in 5% CO 2 by varying the concentration of biosynthesized Ag NPs (25, 50, 75, 100, and 125 μM) in Zingiber officinale extract shows zero inhibition, indicating that the extract does not play any role in toxicity. The synthesized Ag NPs significantly increased the cell death in the treated HCT116 cell lines as shown in figure 9 and table 3 compares with commercially available drug Oxaliplatin (OXP). A significant cytotoxic effect of half maximal inhibitory concentration value (IC 50 ) of Ag NPs is 49.97 μg ml −1 . Ag NPs inhibited the proliferation of HCT116 cells dose dependently which demonstrates that the Ag NPs prepared using Zingiber officinale have great promise as an anticancer agent [46].   clusters and restricted cell spreading patterns as compared to control. This might be because of structural and functional changes in mitochondria. Bright spots on the cell surface might be due to adsorption of Ag NPs on the surface of the cells.

Apoptosis assay
Apoptosis is a programmed cell death process, in which, asymmetry of plasma membrane, blebs formation and condensation of nucleus will occur. The loss of plasma membrane asymmetry is early characteristic of apoptosis. In apoptotic bodies, phosphatidylserine (PS) is altered from inner leaflet to the outer leaflet of membrane [19]. Annexin V is a binding protein which binds to PS and propidium iodide (PI) is a dye used for labelling the early or late apoptotic bodies [20]. Live cells with undamaged membranes are not stained with PI and dead cells are stained with PI. Therefore, cells are viable means Annexin V and PI are negative (−); cells in early apoptosis means Annexin V is positive (+) and PI is negative (−); cells in late apoptosis or dead means Annexin V and PI are both positive (+).
In flow cytometry figure 11, HCT116-untreated and camptothecin-treated cells were stained with Annexin V and PI kit. In untreated, 93.3% cells were live and were non-apoptotic (Annexin V and PI are negative). In case of camptothecin-treated cells, 46.5% were live cells and increase in early-apoptotic (Annexin V(+) and PI(−)) cells were observed from 3% to 34.6% from untreated to treated cells. In case of Ag NPs treated, slight increase in late apoptotic (Annexin V(+) and PI(+)) cells is observed. Thus, Ag NPs treated cells were showing late apoptotic pathway. In FSC-H plots, the increment in the apoptotic cells is also reflected by altering the scattered light. In apoptosis, cells will shrink, which means decrement in the scattered light. As compared with untreated and camptothecin-treated cells, Ag NPs treated cells showed decrease in the scattered light. Percentage of apoptotic cells when untreated and when treated with Campthothecin and Ag NPs can be seen from the bar graph. The results indicates that Ag NPs treated cells are apoptotic bodies.

Caspase 3 expression
Caspases are important moderators of apoptosis, which normally play an important role in activating death protease and cleavage of cell proteins. Activation of caspase-3 proteolytically cleaves and activates other caspases, leading to apoptosis. Apopain, Rabbit IgG antibody can identify the active form of caspase-3. It has been used to visualize the existence of active form of caspase-3 in HCT116 cells.
From SSC-H plots in figure 12, as compared with untreated and Camptothecin-treated cells, Ag NPs treated cells showed decrease in the scattered light. The results from Counts and bar graphs also show that the untreated cells in M 1 region were negative for active caspase-3, whereas 66.49% treated cells were in M 2 region, i.e., positive for active caspase-3 staining. This result is in agreement with the cells treated with Camptothecin (48.26%), which is known to induce apoptosis, thus indicating Ag-NPs induced caspase-3 dependent apoptotic cell death in HCT116 cells [47].

Intracellular ROS assay by H 2 DCFDA
During apoptosis process, there is alteration of mitochondrial function, which results in overproduction of (reactive oxygen species) ROS. ROS contain hydroxyl radicals or peroxides with unpaired electrons. Excess ROS can damage the DNA, proteins, and lipids leading to cell death. H 2 DCFDA (2′, 7′-dichlorodihydrofluorescein diacetate) is a ROS indicator and non-fluorescent fluorescein, which is oxidized and converted into fluorescent 2′, 7′-dichlorofluorescein (DCF) by intracellular ROS. To investigate whether Ag-NPs induced ROS generation in HCT116 cells, we measured cellular ROS by H 2 DCFDA (Life Technologies, Invitrogen, India) from figure 13. We found that there was an increase in the percentage of ROS (43.15%) compared to the untreated control. This result is comparable with the cells treated with the Camptothecin (38.7%), which is known to induce ROS generation by apoptosis. The results indicate that the Ag-NPs induced excessive generation of ROS in HCT116 cells [48].

Antibacterial activity
The antibacterial studies of bio-synthesized silver nanoparticles were obtained against Acinetobacter baumannii (A. baumannii) and Staphylococcus aureus (S. aureus) ( figure 14). The antibacterial activities (inhibition zones) of   The Zingiber officinale synthesized silver nanoparticles against bacteria displayed unique zones of inhibitions in well diffusion method. In literature, it is reported that silver nanoparticles exhibited antibacterial activities against different bacteria, although the mechanism of silver nanoparticles affecting the system is not well understood. The bactericidal activity depends on the size, shape and synthesis methods of silver nanoparticles [49]. But among all the shapes and methods, spherical nanoparticles with smaller sizes (large surface area) and biologically synthesized have excellent antibacterial activities [50][51][52][53]. The results show that spherical biosynthesized silver nanoparticles are more effective against A. baumannii than S. aureus bacteria with increase in concentration.

Conclusions
Simple green synthesis technique is used to prepare eco-friendly biocompatible Ag NPs with no surfactants and chemicals, as the Zingiber officinale extract itself acted as reducing and stabilizing agent with its natural bioreduction potential. The synthesised nanoparticles were spherical in shape having small size around 20 nm and were highly stable for six months at ambient conditions indicating that the adapted synthesis technique is straight forward, economical, non-toxic and biodegradable. The presence of Ag NPs at particular concentrations exhibited appreciable luminescence enhancements with Eu 3+ and Sm 3+ ion complexes. The enhancement was found to be higher for europium complex due to an efficient energy transfer from NPs. This was also confirmed from the lifetime studies of Eu 3+ and Sm 3+ ion complexes, where Eu 3+ ion has shown a significant increase in the emitter life time. Ag NPs also exhibited enhanced optical limiting behaviour in ps regime, which is due to two photon absorption. In vitro cytotoxicity studies revealed that the Ag NPs exhibited excellent toxic effect against HCT116, colon cancer cell lines. Ag NPs exhibit greater abilities to inhibit HCT116 cells proliferation and enhanced cellular uptake. The studies showed that apoptosis was the dominant mode of cell death and Ag Nps promote Caspase-3 apoptotic pathway through ROS generation. Moreover, the NPs also