Cytotoxicity and Antimicrobial efficiency of gold (Au) nanoparticles formulated by green approach using Andrographis paniculata leaf extract

2.1 Preparation of Andrographis paniculata leaf extract

Fresh Andrographis paniculata leaves were collected in the district of Virudhunagar, Tamil Nadu, India, washed thrice in de-ionised water, and then shade-dried for one week. Using a grinding machine, the dried leaves were crushed and sieved before being kept in a sealed container for subsequent analysis. Extract of leaves was made by heating 10 g of dried fine powdered leaves in 100 ml of Milli-Q water at 100 °C for 20 min. Using Whatman No. 1 filter paper, the resulting slurry was filtered and was refrigerated at 4 °C for further study.

2.2 Green synthesis of AuNPs

AuNPs were formed in a 100 ml beaker by heating 50 ml of 1 mM gold chloride solution to 80 °C for 10 min. The solution was then agitated for 15 min at 90 °C for 400 rpm. Then, 5 ml of 10% (w/v) Andrographis paniculata leaf extract was put in a dropwise manner. The reaction solution was held at ambient temperature for 20 min. to monitor for a colour change. Afterwards, the pale-yellow colored reaction mixture was changed to wine red indicated the development of gold nanoparticles. The green synthesized AuNPs was centrifugated at 8000 rpm for 20 min and the top most fluid was drained off. The obtained precipitate was washed multiple times with milli Q water and the content was dried at 50 °C in hot air oven (Hemmati et al., 2019).

2.3 Characterization of gold nanoparticles

The green generated gold nanoparticles' UV–Vis spectrum was measured at 1 nm resolution and de-ionized water as blank using a Shimadzu dual beam spectrophotometer (model UV – 1650 PC) with wavelengths spanning from 200 nm to 800 nm (Mani et al., 2021). FTIR analysis of AuNPs was performed using Fourier transform infra-red spectroscopy with a range of 4000–400 cm⁻¹ in Nicolet Impact 400 FT-IR spectrophotometer (Gandhi et al., 2021). The XRD analysis was determined in the scanning mode on an X’pert PROPAN analytical instrument. The resultant diffraction intensities were compared with the standard Joint Committee for Powder Diffraction Standard (JCPDS) files (Rathnakumar et al., 2019). The distribution of crystalline size of AuNPs was determined using a Tecnai F20 super-Twin FEI transmission electron microscope. DLS (Dynamic light scattering) was measured the average size and polydispersity index of green produced AuNPs. The zeta potential of the AP-AuNPs was measured using Zeta sizer-SZ-100 (Horiba, Japan). To obtain a valid result, the dilution of sample was made using milli Q water at 25 °C and injected into appropriate electrode using a 2 ml disposable syringe (Koperuncholan, 2015).

2.4 Anti-microbial activity

The anti-microbial property of AP-AuNPs was assessed against the human infectious microflora, including Escherichia coli (gram-negative), Staphylococcus aureus, and Enterococcus faecalis (gram-positive), as well as two fungus strains, Aspergillus niger and Candida albicans. The overnight grown pathogens were swabbed on Mueller Hinton Agar (MHA) plates. The AP-AuNPs with two different concentrations (50 and 100 µg/ml) were dispensed into the agar wells in the petri plates made using cork borer. Then the petri plates were kept overnight at 37 °C. The inhibitory zone was assessed and this study was done in five individual replicates. The minimum inhibitory concentration of AP-AuNPs were determined by broth dilution method using 96 well plate. The sample was diluted twofold at various concentrations and maintained overnight at ambient temperature. A reference was maintained with only organisms. Visual examination and ELISA plate reader (ERBA, LisaScan) measurements of optical density (OD) at 630 nm revealed growth. After obtaining a visual assessment, the OD was instantly quantified. The inhibition of growth in the sample loaded wells for each extract dilution was computed using the following formula; $$\text{Inhibition}\left(\%\right)=\frac{\mathit{OD}control-ODoftestsample}{\mathit{OD}ofcontrol}\times 100$$

2.5 Cell morphology study

The experimental cells (MCF7) were procured from the NCCS (National Centre for Cell Sciences) located in Pune, India. The cells were cultured in Dulbecco's modified Eagles medium for further study (DMEM, Sigma Aldrich, USA). MCF7 cells were cultured in 96 well plate and incubated at 37 °C for 24 h before each well was individually treated with two-fold diluted 5% DMEM (500 µl) at different concentrations of AuNPs (100, 50, 25, 12.5, 6.25 µg) and maintained in triplicates. Non treated wells are considered as control cells. The Olympus CKX41 with Optika Pro5 CCD camera phase contrast microscope was used to determine the cell morphology.

2.6 Cytotoxicity assay

Fifteen milligram (15 mg) of MTT (Sigma, M−5655) was taken in 3 ml of Phosphate Buffered Saline until complete solubilization and filtered through a sterile filter paper and incubated for 24 h. After that, 30 µl of the diluted MTT solution was poured to all wells (both test and control), now the content was agitated gently and incubated in a humidified chamber with 5% CO₂ for 4 h at 37 °C. The top most layer was removed, then 100 µl of MTT solubilization solution was added and mixed well. Then, the absorbance was recorded at 540 nm in ELISA reader as described by Talarico et al. (2004).

The viability of cell in percentage was calculated by means of the following formula: $$\text{Viability of cell}=\left(\%\right)=\frac{\mathit{OD}valueofsample}{\mathit{OD}valueofcontrol}\times 100$$

2.7 Staining by AO/EtBr

Acridine Orange (AO) and Ethidium Bromide (EtBr) are DNA binding dyes. These two dyes were used for the detection of apoptotic and necrotic cell’s morphology. Acridine orange dye is received by viable and non-viable cells and produces green colour fluorescence if it was intercalated into double stranded DNA. The ethidium bromide dye is grasped by non-viable cells and releases red fluorescence due to the intercalation of DNA. The cells were treated with AP-AuNPs at a concentration of 90.62 µg/ml for 24 h, then washed with cold PBS and stained using AO/EtBr (100 µg/ml, w/v) at ambient temperature for 10 min. After staining, the cells were thoroughly washed two times using 1X phosphate buffered saline and observed under fluorescent microscope.

2.8 DNA fragmentation

The AP-AuNPs at 90.62 µg/ml concentration treated MCF7 cells were trypsinized and pelletized by centrifugation at 5000 rpm for 5 min at 4 °C. The harvested DNA was appended in Tris-EDTA buffer and the DNA fragmentation study was determined by agarose gel electrophoresis (AGE). After staining, the gel was envisioned under UV light in the gel documentation instrument.

2.9 Data analysis

Five individual replicates were carried out independently (n = 5) in each experiment and the standard error of mean (Mean ± SEM) for each group was computed. The post hoc tukey test followed by one-way ANOVA was performed to assess the significant difference of mean values and expressed as p < 0.05 and p < 0.001. IC₅₀ values were determined by dose–response curve generated using ED50 PLUS V1.0 software.

3.1 Characterization of AuNPs

Fig. 1 a shows the colour change of green synthesized gold nanoparticle from yellow to wine red colour. Fig. 1 b illustrates the UV–Visible spectrum of AP-AuNPs and its intense absorption peak was recorded at 533 nm. Fig. 1c depicts FTIR spectrum obtained from AP-AuNPs revealed that the stretching bands ranged from 3000 to 3800 cm⁻¹ which implies the occurrence of O–H vibration denotes the existence of phenolic compounds, flavonoid contents, anthocyanins and benzophenones in the leaf extract. A diminutive peak shift recommends that the C = O group in the leaf extract acts as capping and stabilizing agents for AP-AuNPs. The stretching vibration at 1510.26 cm⁻¹ indicates the presence of C = C aromatic groups and the region of 1249.87 cm⁻¹ indicates the amine group (C-N). The C-O stretching vibration band appeared at 1082.07 and 1035.77 cm⁻¹ in AP-AuNPs. The stretching occurs at 898.83, and 769.60 cm^-1which indicates the C–H bending of alkane groups and the peak at 599.86 and 424.12 cm⁻¹.

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Fig. 1

Characterization of AP-AuNPs: (a) colour change (b) UV (c) FTIR (d) XRD (e) TEM (f) particle size (g) zeta potential.

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XRD pattern of the A. paniculata mediated gold nanoparticles, which was crystalline in nature and an intense peak proved the structure of AP-AuNPs (Fig. 1d) to be face centered cubic (fcc) nature including 37.07◦, 44.25◦, 64.97◦, and 77.80◦ (111, 200, 220 and 311 respectively) and the obtained data was compared with JCPDS file (No. 00–004-0784). The TEM image of AP-AuNPs synthesized is represented in Fig. 1e. It revealed that the AuNPs is well dispersed with the leaf extract which was acting as the crowning agent to discrete the AuNPs from accumulation. The size of the AuNPs was ranged from 10 to 50 nm and most of them were sphere shaped, few were hexangular and trilateral in shape.

Dynamic light scattering is a standardized particle size analyzer detects an average size of AP-AuNPs was 40 nm by a standard deviation of 18.6 nm (Fig. 1f). The z- average of gold nanoparticle was 31.3 nm in the synthesis process with a poly dispersity index of 0.250. The zeta potential of the AP-AuNPs was −51.1 mV (Fig. 1g) with a tremendous stability.

3.2 Anti-microbial activity

The antibacterial and antifungal efficacy of green synthesized AP-AuNPs was analysed against selected pathogenic microbial strains by agar well diffusion method. The gram positive bacterial strains selected in the study were S.aureus and E.faecalis, gram negative bacterium E.coli and fungal strains such as A.niger and C.albicans. The observed inhibitory zone is given in Fig. 2. It shows the higher concentration (100 µg/ml) gave better performance than the lower concentration (50 µg/ml). In this lower concentration the fungal strain does not exhibit any zone in the plate. But the standard antibiotic Clotrimazole exhibited better antifungal effect (34 and 26 mm) to A.niger and C.albicans respectively than the AP-AuNPs (14 and 15 mm) because of the chemical compound exclusively gave increased zone of inhibition. Meanwhile there is no zone of inhibition in the plant extract alone loaded well. The obtained result revealed that the increasing AuNPs concentration gives diminishing growth of microbial cells. The one-way ANOVA test between the low and high concentrations of AP-AuNPs and standard antibiotics such as streptomycin for bacterial strains and clotrimazole for fungal strains showed significant difference (p < 0.05) followed by the post hoc tukey test (p < 0.001).

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Fig. 2

Antimicrobial activity (inhibition zone in mm) of AP-AuNPs: (a) E. coli (b) E. faecalis (c) S.aureus (d) A.niger (e) C. albicans.

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Based on these information, the minimum inhibitory concentration (MIC) of the anti-microbial component required to prevent the growth of microflora was found to be 100 µg/ml against A.niger and E.faecalis (92.13 and 88.74 %) with the respective IC50 values of 15.98 and 15.49 µg/ml. Followed by this, E. coli strain was found to be good inhibitory effect (88.18%) at 100 µg/ml with the IC50 value (14.57 µg/ml). But more or less similar IC50 values were obtained for S. aureus and Candida albicans (24.126 and 24.425 µg/ml) respectively (Fig. 3).

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Fig. 3

MIC of AP-AuNPs against bacterial and fungal pathogens (a) E. coli, (b) S. aureus, (c) E. faecalis (d) A. niger (e) C. albicans.

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3.3 Anticancer study

MTT assay for cytotoxicity efficacy in MCF7 cells was used to determine the effect of AP-AuNPs on breast cancer cell lines. The AP-AuNPs meritoriously decreased MCF7 cell proliferative efficiency from 6.25 µg/ml in a dosage reliant manner (p < 0.001) in association through the unprocessed cells. It also explains the normal cell L929 was also used to analyze the proliferative activity using AuNPs for comparative study. It revealed that the greater impact of AuNPs on MCF7 than L929 cells (Fig. 4).

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Fig. 4

Cytotoxicity of different concentration of AuNPs (6.25 to 100 µg/ml) from Andrographis paniculata against MCF7 and L929 cells.

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The sigmoidal curve plotted for this dose dependent study of AuNPs on both cells revealed the IC50 value (half maximal) as 90.62 and 159.16 µg/ml. The one-way ANOVA and tukey post hoc test concluded that the proliferative inhibitory action was dose dependent way and each dose differences significantly (p < 0.001).

The cell morphology was critically analyzed by means of AP-AuNPs in different dosages. AuNPs treated cancer cells shows significant mortality at increased concentration. The damaged cells were shedding and renovating into an orbicular form. This kind of alteration does not materialize in L929 cell which indicates the experimental AP-AuNPs can inhibit cancer cells via hunting of reactive oxygen species and diminishing oxidative stress and also recommending that it could be expended as curative to skirmish carcinoma cells for anticancer drug development (Fig. 5).

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Fig. 5

Image of L929 (normal) and MCF7 (cancer cells) of untreated control and treated with AP-AuNPs.

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3.4 Apotosis and DNA fragmentation

The apoptosis study using AO/EtBr staining protocol to discriminate the viable and non– viable cells based on the emittance of fluorescence if it intercalated into double stranded DNA. The viable cell emits green and the non– viable cell emits red fluorescence. The present apoptotic result is predicted in Fig. 6. The observed data revealed the living cells are having typical green coloured nucleus, while in the apoptotic cells of early stage form an intense green coloured nucleus with compressed chromatin, the later stage apoptotic cell shows orange coloured stain with fragmentation and damaged (necrotic) cells are homogenously stained as orange red.

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Fig. 6

Apoptosis and DNA fragmentation of MCF7 cells control and treated with AuNPs.

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AP leaf extract mediated Gold (Au) nanoparticles stimulated programmed cell death which was reliable with the obtained result exhibiting that DNA disintegration take place as seen in Fig. 6. Strength of gold nanoparticles escorted a prolonged tail (DNA damage) supplementary sustaining the event that gold nanoparticles engendered caspase mediated cell death in MCF7 and the recorded information in Fig. 6 shows agarose gel electrophoresis of DNA fragmentation which was improved with intensifying acquaintance to AP-AuNPs. Green synthesized gold nanoparticles demonstrated cellular apoptosis over a diminished cell proliferative action and an improvement in DNA breakup.