Cytotoxic effect of silver nanoparticles synthesized from ethanolic extract of Allium sativum on A549 lung cancer cell line

2.1 Materials for synthesis of Ag NPs

The plant was collected from Kodaikanal, Dindigul district of TamilNadu. The garlic bulbs were identified and authenticated by Prof. P. Jayaraman, Plant Anatomy Research Center, Tambaram, Chennai. The garlic bulbs were thoroughly cleaned and cut into small pieces, dried in open air atmosphere and crushed into fine powder. After that, obtained garlic powder was extracted using ethanol at boiling temperature for 48 h by Soxhlet extractor (Lin et al., 1999). Whatman No 1 filter paper was used to filter the extract and the solvent was removed using rotary vacuum evaporator and stored for further analysis.

2.2 Synthesis of Ag NPs via green aproach

Initailly, 1 mM of silver nitrate (AgNO₃) was added into 50 ml of double distilled (DD) water under constant stirring. After that, 5 ml of ethanol extract of fresh garlic powder was mixed with above mentioned solution. During the reaction process, the solution colour gradually changed light brown to dark brown at 3 hrs constant stirring which indicates the NPs formation. However, the polyphenols present in the garlic extracts act as reducing agent and cause the formation of Ag NPs. The formation of dark brown color (Fig. 1) demonstrates the synthesis of Ag₂O. The synthesized Ag₂O were centrifuged and the pellet was collected. The collected product was kept in a lyophilizer for 24 h, to obtain powdered form of a NPs. The obtained powder was stored at 4 °C for further characterization studies and cytotoxicity analysis (Selvan et al., 2018).

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Fig. 1

Biosynthesis of Ag NPs.

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2.3 Characterization

The structure-based properties of Ag NPs was analyzed by powder X-ray diffraction (PXRD) pattern carried out by X’ Pert Pro instrument with Cu Kα radiation. Molecular analysis of the Ag NPs was analysed by Fourier-Transform Infrared (Nicolet b6700) spectrometer using KBr pellet technique in the range form 4000 to 450 cm⁻¹. The characteristic absorption peak of Ag NPs was examined using UV- 2450 (Shimadzu) from 300 to 600 nm. By this measurement, reduction of AgNO₃ to Ag⁺ ions was monitored. Morphological analysis of nanoparticles was carried out by Hitachi S-4500 FESEM analyzer. A drop of the sample was coated on to carbon film and dried for the analysis. Energy-dispersive X-ray spectroscopy (EDAX) was used to examine the metals and materials present in the synthesized material using Hitachi S-4500 instrument equipped with a Thermo EDAX attachments. High Resolution Transmission Electron Microscopy (HRTEM) analysis was done in order to determine the process of formation of Ag NPs and to study their size and shape. The analysis was performed by Jeol/JEM 2100, operating at 200 Kv. Samples for HRTEM grid was arranged through coating a drop of Ag NPs solution on carbon-coated copper grids. Dynamic Light Scattering is a method is performed to measure the particle size distributions in the range of 2–500 nm using the instrument Nanophox, Germany. It measures the particle size using the scattered light, which is passed from a laser source and it depends on the Rayleigh scattering from the suspended NPs.

The human lung cancer (A549) cell line was achieved from the National Centre for Cell Science (NCCS), Pune and facilitated to grow in Eagles Minimum Essential Medium (EMEM) and 10% Fetal Bovine Serum (FBS). The obtained cells were sustained at 37 °C, 100% relative humidity, 5% CO₂ and 95% air. By the inverted microscope, cell culture was continuously viewed to determine the absence of bacterial and fungal contaminants as well as quantity of confluence.

2.4 In- vitro cytotoxic activity

The cell viability test was made with the 3-(4, 5-dimethylthiazol-2-yl) −2, 5-diphenyltetrazolium bromide (MTT) assay to demonstrate the cytotoxic effect of Ag NPs and garlic extract. MTT is a tetrazolium salt which was used to investigate mitochondrial function. The enzymatic reduction of MTT was catalyzed by the mitochondrial succinate dehydrogenase enzyme, which breaks the tetrazolium ring, transforming it to an purple formazan which is not soluble (Mosmann, 1983). Consequently, the quantity of formazan formed is proportionate to the quantity of viable cells. A549 cells were taken in a 96-well plate at the density of 5x 10³ cells/well. The cells were grown in a 96-well plate for 24 hrs, in 200 μl of EMEM with 10% FBS. After 24 hrs, cells were exposed to Ag NPs at a different dose (1–20 mg/ml) for a period of 48 hrs. Each well in the plate containing the cells were then subjected to 15 µl of MTT (5 mg/ml) and incubated for 4 hrs at 37 °C. EMEM was then removed, and 100 μl of DMSO was added to each well and then measured using the microplate reader at 570 nm. EMEM without samples was served as control. $$\%Cellviability=100-Abs\left(\mathit{sample}\right)/Abs\left(\mathit{control}\right)\times 100$$

3.1 PXRD analysis

Fig. 2a shows the PXRD pattern of Ag NPs synthesized from ethanol extract of garlic powder. From the Figure, the primary diffraction peaks at 2θ values of 28.05°, 32.41°, 38.31°, 46.38°, 55.01°, 57.62°, 67.68° and 77.61° respectively for (1 0 0), (1 1 0), (1 1 1), (2 0 0), (2 1 1), (2 1 1), (2 2 0) and (3 1 1) planes belonging to AgNP, which are well matched with standard JCPDS: 76–1393.The average size of the Ag NPs was determined from the PXRD pattern, according to the peak and line width of the plane, using Scherer's equation (Parikh et al., 2011). $$D=K\lambda /\beta cos\left(\theta \right)$$

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Fig. 2

(a) XRD pattern, (b) FTIR spectrum and (c)Uv–Visible spectrum of AgNPs.

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Where, ‘D’ denotes diameter, ‘K’represents the shape factor (0.94), ‘λ’denotes the wavelength of the X-ray, ‘β’ represents angular width at FWHM of the X-ray diffraction peak at Bragg’s diffraction angle ‘θ’. The results revealed that the average size of the Ag NPs was found to be 12 nm. Table 1 shows the PXRD parameters along with the particle size of each 2θ values. The Table revealed that, the obtained particle size in the present work is better than Ag NPs synthesized from aqueous Garlic extract (Theivasanthi and Alagar, 2012). The defects that occurred in the nanoparticles was calculated by the dislocation density (δ), as follows (Logeswari et al., 2015). $$\delta =1/D^2$$

Where D = crystallite size.

The dislocation density (δ) of Ag NPs was calculated as 6.94 × 10⁻³ (nm)⁻².Table 2 shows the comparative dislocation density (δ) of Ag NPs prepared from aqueous Garlic extract. From the Table, the dislocation density of Ag NPs synthesized from Allium sativum has lesser defects owing to its better Surface Plasmon Resonance (SPR) properties and larger surface area compared to previous reports.

3.2 FT-IR spectroscopy

The FT-IR spectrum for Ag NPs synthesized using garlic extract, showed a variation in the intensity of bands in different regions (Fig. 2b).The peak at 3434 cm⁻¹ was assigned to the –O–H stretching vibrations indicating the presence of hydroxyl groups. The peaks at 2925 cm⁻¹ and 2108 cm⁻¹ were due to the strong intensity of alkyl (–CH₂-) group. The peak at 1637 cm⁻¹ and 1560 cm⁻¹ was assigned to aryl aromatic ring. The O–H band corresponds to the peak at 1414 cm⁻¹. The peaks arise at 1385, 1189, 1125 and 1079 cm⁻¹ indicate the C-N stretch, which contributes to the presence of amine group in the synthesized material. The presence of sulfur compounds was primarily confirmed by the bands formed at 849, 779 and 638 cm⁻¹. The analysis confirmed the available functional groups may act as reducing and stabilizing agents for Ag NPs, either by carbonyl groups or sulfur containing groups (Saha et al., 2017).

3.3 UV–Vis spectral analysis

UV–Visible absrorption spectroscopy is an important tool to indentifing the formation and stability of metal NPs in an aqueous solution. The extracellular synthesis of Ag NPs using A. sativum extract involved in the reduction was identified by the UV–Visible spectroscopy and shown in Fig. 2c. The dark brown colour was observed from the synthesized Ag NPs which indicates the reduction of pure Ag⁺ ions due to its SPR properties (Varshney et al., 2009). The surface plasmon absorption band is observed at 452 nm in UV–Visible spectrum for A.sativum extract due to electronic transitions from the valence band to the conduction band, which indicates the formation of Ag NPs (Paul et al., 2015).

3.4 FESEM and EDAX

Fig. 3 depicts the typical FESEM image of green synthesized Ag NPs. The image depicts that the particles are spherical and rod shaped with an average size around 70 nm. EDAX spectrum of Ag NPs synthesized from ethanol extract of garlic is presented in Fig. 4. Figure shows the signal at 3 keV in the EDAX spectrum revealed that the existence of silver, which is a unique feature for the absorption of metallic silver nanocrystallites owing to their SPR property (Mahendran and RanjithaKumari, 2016). Also, the spectrum exhibits Ag, C and O elements only and no other impurities were presencein the prepared Ag NPs. Also, Figure shows the weight percentage of elemental constituents such as silver 58.34%, carbon 12.79% and oxygen 28.87%. According to the findings of Otunola et al., 2017 the weight percentage of Ag NPs synthesized from aqueous extract of garlic was 57.16% which showed that better yield was obtained in the present investigation.

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Fig. 3

FESEM of Ag NPs.

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Fig. 4

EDAX spectrum of Ag NPs.

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3.5 HRTEM analysis

A typical HRTEM image of biologically synthesized Ag NPs suggests that the particles were well segregated and distributed without any agglomeration. The stabilization of the category of Ag NPs is due to the domination of carbonyl and hydroxyl groups which give weak electrostatic interactions between Ag NPs and functional groups from garlic extract. Also, most of the particles were spherical shaped with varying size of 10–40 nm which is shown in Fig. 5a. The crystalline nature of the synthesized NPs was characterized by Selected Area Electron Diffraction (SAED) pattern and shown in Fig. 5b. The SAED pattern clearly shows the characteristic polycrystalline ring pattern for a FCC structure which exactly coincide with results of PXRD pattern (Mehmood et al., 2014). The sizes of the Ag NPs have been determined and the distribution of Ag NPs is shown in Fig. 5c. It indicated that the distribution with an average mean size of 30 nm. Green synthesized biocompatible nanoparticles synthesized using citrate and stabilized with garlic extract showed increase in size and polydispersity in TEM analysis (White et al., 2012). In another study, TEM micrograph of Ag NPs synthesized using A. sativum extract showed quasi spherical particles with an average size of 170 nm (Mohamed et al., 2022).

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Fig. 5

(a) HRTEM and (b) SAED pattern of Ag NPs.

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3.6 Dynamic light scattering (DLS)

Fig. 6 shows the DLS analysis which depict the Ag NPs particle size. From the analysis, the mean particle size of Ag NPs was observed between 3 nm and 10 nm. The comparision table of previously reported distribution of Ag NPs based on size is shown in Table 3. From the Table, the size of the synthesized NPs are very less when compared to previously reported size values, which confirms the prepared Ag NPs from garlic extract has hydrodymamic size and polydispersity in nature.

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Fig. 6

DLS spectrum of Ag NPs.

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3.7 In-vitro cytotoxicity

Assessment of cytotoxicity plays a major role for evaluation in toxicology when assessing its effectiveness as a drug that elucidate the cellular response to a toxicant. In the present study, the cytotoxicity impact on cell inhibition was examined by a dose-dependent approach at different concentration. Figs. 7a and 7b respectively shows the efficiency of synthesized Ag NPs and A. sativum extract alone in inhibiting cancer cells. From the Figures, it is evident that the percentage of cell viability was reduced by increasing concentration of Ag NPs and extract. Also, it is confirmed that, the required concentration of Ag NPs required was minimum when compared to the concentration of the extract (A. Sativum). This increasing cell inhibition activity with a higher concentration of Ag NPs indicated that more number of Ag NPs can penetrate into the cancer cells and finally leading to cell death. For the comparison, a plot has been drawn between concentration and the percentage of cell inhibition and shown in Figure 9a (g) and Figure 9b (g). From the Figures, it is observed that, 50% of cell inhibiton was observed by adding 22 μg/ml of Ag NPs fabricated from ethanol extract of A. sativum. At the same time, the same cell inhibition was achieved by adding 177 μg/ml of pure A. Sativum extract,which shows the better cytotoxicity effect attained from the biologically synthesized Ag NPs due to ROS promting and leads to cellular impairment and cell death (Jacob et al., 2012; Sankar et al., 2013). The cytotoxic effects of Ag NPs are higher in cancer cells when compared to normal cells. The small size and the large surface to volume ratio help in penetrating the cells easily. The major mechanism of cancer cell inhibition by metallic nanoparticles includes Reactive oxygen species generation, Caspase-3 activation, modification of mitochondrial membrane potential and DNA damage (Andleeb et al., 2021). Hence, from the observations, it was confirmed that the material proposed in the present work shows good inhibition activity against A549 cancer cell line.

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Fig. 7a

In-vitro cytotoxic activity of the biosynthesized Ag NPs against A549 cell line. (a) Untreated A549 cell line, (b) Ag NPs − 1.88 μg/ml, (c) Ag NPs − 3.75 μg/ml, (d) AgNPs − 7.5 μg/ml (e) Ag NPs − 15 μg/ml, (f) Ag NPs − 30 μg/ml (g) Percentage of cell inhibition of Ag NPs against A549 cell line.

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Fig. 7b

In-vitro cytotoxic activity of the ethanol garlic extract against A549 cell line. (a) Untreated A549 cell line (b) 18.75 μg/ml (c) 37.5 μg/ml (d) 75 μg/ml (e) 150 μg/ml (f) 300 μg/ml (g) Percentage of cell inhibition of A. sativum extract against A549 cell line.

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