1 Introduction

Man-made materials and nanoparticles are being rapidly produced in large quantities throughout the world [1]. Many studies conducted in the past few decades suggest that nanomaterials have different toxicity profiles compared with larger particles because of their small size and high reactivity. In our daily life, more and more nanomaterials are introduced due their wide spread applications. But their adverse effects to the environmental and human health are a major concern [2]. Meanwhile, owing to their size and unusual properties, nanoparticles can enter the body and cross biological barriers relatively unimpeded. Several studies have reported that nanoparticles enter the body through the respiratory tract and can penetrate the pulmonary epithelium; they can reach the interstitium and be deposited in the peripheral lung tissue [3, 4]. Since insoluble metal oxide nanomaterials are expected to be more environmentally persistent, investigations on environmental perseverance, bioaccumulation and toxicity of these nanomaterials help to assess the potential risks and provide information to industry to develop safer nanomaterials [5, 6].

Owing to their specific functional characteristics and importance in a wide variety of technical applications, nickel oxide (NiO) is extensively investigated as electronic component, ceramic materials and catalysts. It is also known that nickel compounds (including NiO) have harmful effects. Nickel compounds are classified as class I carcinogenic material by the International Agency for Research on Cancer (IARC Monographs on the evaluation of Carcinogenic Risks to Human, Volume 49, 1990). In general, NiO is classified as having relatively lower toxicity than other nickel compounds [7, 8].

Recent studies indicate the great potential of NiO NPs in biomedical applications, including cancer diagnosis and therapy. Mariam et al. [9] reported that Ni and NiO NPs used to study the cytotoxic effect against for human colon adenocarcinoma (HT-29) cells. Adam et al. [10] have investigated the cytotoxicity and apoptotic effects of NiO NPs on human cervix epithelioid carcinoma (HeLa) cell line. Ahamed et al. [11] have been studied the NiO NPs exert cytotoxicity via oxidative stress and induce apoptotic response in human liver (HepG2) cancer cells. Duan et al. [12] reported that the NiO NPs induce apoptosis through repressing SIRT1 in human bronchial epithelial cancer cells. Ramasami et al. [13] investigated antimicrobial activity against the two bacterial (Bacillus cereus and Klebsiella aerogenes) strains and one fungal (Candida albicans) treated with NiO NPs. Talebian et al. [14] tested the antibacterial activity of NiO NPs against two common food borne pathogenic bacteria Stapylococcus aureus and Escherichia coli.

However, variations observed in the biological responses of metal oxide nanoparticles could be due to the differences in the ability to produce the reactive oxygen species (ROS) and their types. Apart from this, the physico-chemical properties (size, shape, surface area, surface coating, composition and dissolution) of metal oxide nanoparticles, method of synthesis, as well as impurities play a major role in their biological outcomes [15]. We have synthesised NiO nanoparticles in chemical and green synthesis method. Moreover, there was no literature found in the synthesis of NiO NPs using Gymnema sylvestre plant extracts.

The synthesised nanoparticles were characterized by X-ray diffraction studies (XRD), Fourier transform infra-red spectroscopy (FTIR), high resolution transmission electron microscopy (HRTEM) with selected area diffraction studies (SAED) and energy dispersive analysis (EDAX), UV–visible spectroscopy, photoluminescence (PL) studies, X-ray photoelectron spectroscopy (XPS), electron paramagnetic resonance (EPR) spectroscopy and zeta potential analysis. To the best of our knowledge, this is the first report for the comparative toxicity studies of chemical and green synthesised (G. sylvestre plant extract reduced) NiO NPs in bacterial and MCF 7 cancer cells.

2 Experimental methods

2.1 Synthesis of NiO NPs using G. sylvestre leaves extract

15 g of finely chopped G. Sylvestre leaves were weighed. Then, 150 ml of double distilled water was added and boiled at 60 °C for 15 min. The obtained extract was filtered using Whatman no. 1 filter paper and the filtrate was collected in a 250 ml Erlenmeyer flask. Thereafter, 0.1 M nickel nitrate hexahydrate (Ni(NO3)2·6H2O) solution was added into 150 ml of G. sylvestre leaves extract and it was stirred constantly at 80 °C for 6 h. A black colour precipitate was obtained. Further, the precipitate was dried at 120 °C for 6 h. The obtained NiO nanopowder was calcined at 600 °C for 5 h.

2.2 Synthesis of NiO NPs by co-precipitation method

In this typical chemical synthesis of NiO NPs, the following high purity chemicals such as, Ni(NO3)2·6H2O and sodium hydroxide (NaOH) were used as the precursors without further purification. Required amount of Ni(NO3)2·6H2O (0.1 M) was completely dissolved in deionized water, aqueous NaOH (0.8 M) solution was added drop wise to the aqueous nickel nitrate and this solution was stirred at 80 °C for 6 h. A green colour precipitate was formed slowly, which was washed several times with double distilled water and ethanol. The precipitate was dried at 120 °C. The NiO NPs thus obtained were annealed at 600 °C for 5 h. Finally, black colour NiO NPs was collected and used for further studies.

2.3 Antimicrobial activity of NiO NPs

Antimicrobial activity of the green and chemically synthesized NiO NPs was carried out by agar well diffusion method against two pathogenic bacterial strains namely S. aureus (gram positive) and E. coli (gram negative) on Muller-Hinton agar, according to the guidelines of Clinical and Laboratory Standards Institute (CLSI) [16]. The media plates (MHA) were streaked with bacteria for 2–3 times by rotating the plate at 60° angle for each streak to ensure homogeneous distribution of the inoculums. Then the agar plates were swabbed with 100 ml each of overnight cultures of S. aureus and E. coli using a sterile L-shaped glass rod. Using a sterile cork-borer, wells (6 mm) were created in each petri plate. Varied concentrations of NiO NPs (1 µg/ml, 3 µg/ml and 5 µg/ml for both G+ and G− bacteria) were loaded onto the petri plates followed by incubation for 24 h at 37 °C for bacteria. After the incubation period, the diameter of the zone of inhibition (DZI) was recorded. Kanamycin (Hi-Media) was used as the positive control against gram negative and gram positive bacteria to compare the efficacy of the test samples. We have simultaneously studied whether the dimethyl sulfoxide (DMSO) without the NiO NPs plays any active role as a biocide or not. As there is no any disc zone of inhibition observed, it confirms that DMSO doesn’t show any biocidal property.

2.4 Anticancer activity

2.4.1 Cell culture

The MCF-7 breast adenocarcinoma cancer cells were cultured in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10% fetal bovine serum (Sigma, USA) and 10,000 IU penicillin and 100 μg/ml of streptomycin as antibiotics (Himedia, Mumbai, India), in 96 well culture plates, at 37 °C, in a humidified atmosphere of 5% CO2, in a CO2 incubator (Forma, Thermo Scientific, USA). All experiments were performed using cells from passage 15 or less.

2.4.2 MTT assay

The green and chemically synthesized NiO NPs were suspended in DMSO, diluted in culture medium and used to treat the chosen cell line (MCF-7) over a complex concentration range of 5–100 μg/ml for 24 h. DMSO solution was used as the solvent control. A miniaturized viability assay using 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was carried out [17]. To each well, 20 μl of 5 µg/ml MTT in phosphate buffer (PBS) was added. The plates were wrapped with aluminium foil and incubated for 4 h at 37 °C. The purple formazan product was dissolved by the addition of 100 μl of 100% DMSO to each well. The absorbance was monitored at 570 nm (measurement) and 630 nm (reference) using a 96 well plate reader (Biorad 680). The data were collected for four replicates. The percentage of inhibition was calculated from this data using the formula

$$\scriptsize \% {\text{ of inhibition }} = \frac{{{\text{mean OD of untreated cells }}\left( {\text{control}} \right) {-}{\text{mean OD of treated cells}}}}{{{\text{mean OD of untreated cells }}\left( {\text{control}} \right)}} \times 100$$

2.4.3 Acridine orange (AO) and ethidium bromide (EB) staining

AO/EB staining was performed as described by Spector et al. [18]. The cell suspension of each sample containing 30,000 cells/ml cells, was treated with 200 μl of AO/EB solution (100 μl/mg AO and 100 μl/mg EB in PBS) and examined by a Olympus inverted fluorescence microscope (Ti-Eclipse) using an UV filter (450–490 nm). One hundred cells per sample were counted in tetraplicate for each dose point. The cells were scored as viable, apoptotic or necrotic as judged by the fluorescence emittance, nuclear morphology and membrane integrity [18]. The morphological changes were also observed and photographed.

3 Characterization techniques

The NiO NPs were characterized by X-ray diffractometer (model: X’PERT PRO PAN analytical). The diffraction pattern was recorded in the range of 25°–80° for the NiO NPs, at the monochromatic wavelength of 1.54 Å. The morphology of the synthesized NiO NPs was examined using HRTEM. Sample for HRTEM analysis was prepared by drop coating the NPs solutions on carbon-coated copper grids at room temperature. The excess NPs solutions were removed with filter paper. The copper grid was finally dried at room temperature and was subjected to TEM analysis by the instrument Tecnai F20 model operated at an accelerating voltage of 200 kV. The NiO NPs were analyzed by EDAX (model: ULTRA 55). The FT-IR spectrum was recorded in the range of 400–4000 cm−1 by using Perkin-Elmer spectrometer. Ultraviolet–visible (UV–Vis) spectra of NiO nanoparticles were recorded on a Perkin-Elmer UV-Lambda 25 spectrophotometer (Perkin-Elmer, Norwalk, Connecticut). The PL emission study of the sample was carried out using Horiba Jobin–YVON spectrofluorometer (model: FLUOROMAX-4, 450 W high pressure xenon lamp as the excitation source, photomultiplier at a range 325–550 nm). The XPS measurements were performed with an XPS instrument (Carl Zeiss) under ultra-high vacuum with Al Kα excitation at 250 W. To obtain information on defects and vacancies, EPR was recorded using X-band JEOL JES-RE1X at room temperature. Zeta potential measurement was carried out by using Malvern Zetasizer instrument.

4 Results and discussion

4.1 X-ray diffraction studies

The X-ray diffraction patterns of green and chemically synthesised NiO NPs are shown in Fig. 1a. The XRD result shows that all the samples have cubic nickel oxide (ICDD:78-0643) belonging to the Fm3m space group. Figure 1b shows that enlarged peak position of green and chemically synthesized NiO NPs. As compared to the green synthesized NiO NPs, the intensity of chemically synthesized NiO NPs increase because the energy from the heat can enhance the vibration and diffusion of the lattice atoms for crystallization. The peaks are located at an angle of 2θ at 37.2685, 43.5040, 63.0885, 75.3950 and 79.5011 for green synthesised NiO NPs and 37.2826, 43.3037, 62.9345, 75.3463 and 79.4478 for chemically synthesised NiO NPs which correspond to the (111), (200), (220), (311) and (222) plane of the cubic structure for green synthesized NiO and chemically synthesized NiO NPs respectively.

Fig. 1
figure 1

a X-ray powder diffraction patterns of green and chemically synthesised NiO NPs, b an enlarged version of the XRD pattern of NiO NPs between 35.4° and 38.7°

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The lattice constants ‘a’ of the cubic structure of NiO can be calculated by using the relation

$$\frac{1}{{d^{2} }} = \left( {\frac{{h^{2} + k^{2} + l^{2} }}{{a^{2} }}} \right)$$
(1)

Or

$${\text{a}} = \surd d^{2} \left( {h^{2} + k^{2} + l^{2} } \right)$$
(2)

The unit cell volume is calculated by the relation V = a3. The calculated lattice constant value and unit cell volume are presented in Table 1.

Table 1 X-ray diffraction parameter values of the green and chemically synthesised NiO NPs
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The average crystallite size of the samples are calculated by Debye–Scherrer’s relation

$${\text{Average crystalline size D }} = \frac{{{\text{k}}\uplambda}}{{\upbeta_{{{\text{D cos}}\uptheta}} }}$$
(3)

where λ—the wavelength of the radiation (1.54056 Å for Cu Kα radiation), k—a constant which is equal to 0.94, β—the peak width at half-maximum intensity, θ—the peak position. The average crystallite size is found to be 23 nm and 32 nm for chemical and green synthesised NiO NPs respectively. The average crystallite size decreases in the Chemical synthesized NiO NPs. The reduction in the particle size is mainly due to the temperature effect. Further, micro strain (\(\varepsilon\)) was estimated by the following relations [18]

$${\varepsilon = } \frac{{\upbeta{ \cos }\uptheta}}{4}$$
(4)

The micro-strain increases in the case of chemically synthesized NiO NPs as compared to that of green synthesized NiO NPs. It is evident that the increase in strain causes the increase in the lattice constant values, reduction in the particles size, the broadening and small shifts in the XRD peaks (Table 2).

Table 2 FTIR peaks assignments of green and chemically synthesised NiO NPs [19, 20]
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4.2 Functional group analysis

The FTIR spectra of green and chemically synthesized NiO NPs are shown in Fig. 2. The broad absorption band located at 3441 and 3323 cm−1 are attributed the O–H stretching vibration of interlayer water molecules and H-bound to OH group for both NiO NPs respectively. The peaks at 2915 cm−1 are due to asymmetric stretching (C–H) mode of the CH2 groups for green synthesized NiO NPs [19]. The band at 2123 cm−1 is attributed to the C–H stretching vibrations are NiO NPs. The absorption peaks at 1643 cm−1 and 1656 cm−1 are due to the bending mode (H–O–H) of the water molecule of green synthesized NiO and chemically synthesized NiO NPs respectively. C–H bending vibrations are located at 1026 cm−1 and 1118 cm−1 for both NiO NPs respectively. The strong band at 423 and 436 cm−1 correspond to the Ni–O starching for green synthesized NiO and chemically synthesized NiO NPs [20].

Fig. 2
figure 2

FTIR spectra of the green and chemically synthesised NiO NPs

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4.3 Morphological analysis

In order to predict the morphology and size of the green and chemically synthesised NiO NPs, HRTEM images have been recorded as shown in Fig. 3a, b. It can be clearly observed that the synthesised product consist of nearly cube shaped particles with size around 10–20 nm. It is in good agreement with an average crystallite size obtained from Scherrer’s formula. Selected area diffraction pattern (SAED) originated from the NiO nanoparticles are shown in the Fig. 3c, d. The appearance of strong diffraction spots rather than diffraction rings confirmed the formation of single crystalline cubic nickel oxide.

Fig. 3
figure 3

a, b HRTEM image of green and chemically synthesised NiO NPs, c, d SAED pattern of green and chemically synthesised NiO NPs

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4.4 Elemental analysis

The quantitative chemical compositional analyses of green and chemically  synthesised NiO samples were carried out using EDAX. The typical EDAX spectra of the synthesized NiO NPs are shown in Fig. 4a, b. It revealed that the presence of Ni and O in chemically synthesized NPs is found to be 43.58 and 56.42% respectively. Likewise, the composition of Ni and O in the green synthesized sample is found to be 43.55% and 56.45% respectively. Moreover, no additional peaks corresponding to any other elements except Ni and O were observed.

Fig. 4
figure 4

EDAX spectra of a green synthesized NiO NPs and b chemically  synthesised NiO NPs

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4.5 Optical studies

UV–visible optical absorption spectra of green and chemically synthesised NiO NPs were recorded at room temperature in the wavelength range 350–800 nm as shown in Fig. 5. The samples were uniformly dispersed in distilled water, followed by ultra sonification for 15–20 min before recording UV–Vis spectra. Blue shift in the absorption edge is observed in the chemically synthesised NPs (243 nm) compared with green synthesised NiO NPs (259 nm). This is assigned to the intrinsic band gap absorption. These peaks are due to the electronic transition occurring between the valence band and the conduction band [21].

Fig. 5
figure 5

UV–visible spectra of green and chemically synthesised NiO NPs

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The direct band gap energy was calculated by using Tauc relation as given below.

$$\upalpha{\text{h}}\upupsilon = {\text{A}}\left( {{\text{h}}\upupsilon - {\text{E}}_{\text{g}} } \right)^{\text{n}}$$
(5)

where Eg is the optical band gap, α is the absorption co-efficient, A is a constant and the exponent n depends on the transition. Considering direct band transition in NiO NPs, a plot between (αhυ)2 versus photon energy (hυ) is drawn for green and chemically synthesized NiO NPs as shown in Fig. 6. The intercept of the tangent to the plot on the X-axis gives the direct band gap of NiO NPs. The optical band gap values obtained from Fig. 6 are 5.1 and 4.78 eV respectively for the chemical and green synthesised NiO NPs samples. The optical band gap of NiO in the present study is higher than the bulk value (3.65 eV). It is due to the quantum confinement effect. This effect is due the chemical defects or vacancies present in the intergranular regions generating new energy level to increase the band gap energy [22, 23].

Fig. 6
figure 6

Band gap spectra of green and chemically synthesised NiO NPs

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4.6 Photoluminescence (PL) studies

PL study is one of the important method to investigate the structural defects such as oxygen vacancies and quantum size effects. Figure 7 depicts the PL spectra of green and chemically synthesised NiO NPs annealed at 600 °C. The UV emission peak (K1) observed at 395, 396 (see Table 3) which correspond to a near band edge emission in all the samples due to direct recombination of the excitations through an exciton–exciton scattering. It can be attributed to the intrinsic transitions of exciton from the conduction band to the valance band, i.e., electronic transition involving 3d8 electrons of the Ni2+ ions [24]. The violet emission peaks K2 and K3 appear at 418, 424, 438 and 440 nm for green and chemically synthesised NiO NPs. The analysis of the emission peaks (K5 and K6) appear at 449, 456 and 468 nm which correspond to the blue region with major irregularities. It is due to the oxygen vacancies; these vacancies are responsible for the increment in defect levels in the crystals and thus enhance the visible emission spectra [25]. The green emission peaks (K7) appear at 491 and 505 nm for green and chemically synthesised NiO NPs. Besides, it should be noted that the PL spectra in Fig. 7 do not show any yellow-green visible luminescence from the green and chemically synthesised NiO NPs. It confirmed that there is no Ni vacancies present in both green and chemically synthesised NiO NPs.

Fig. 7
figure 7

Photoluminescence emission spectra of green and chemically synthesised NiO NPs

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Table 3 Comparison of the PL emission values of the green and chemically synthesised NiO NPs
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4.7 X-ray photoelectron spectroscopy (XPS) studies

The electronic structure and composition of the green and chemically synthesized NiO NPs are shown in Fig. 8a–c. Fig. 8a shows the XPS survey scan of green and chemically synthesised NiO NPs, which confirms the presence of Ni, O and C in the synthesized samples.

Fig. 8
figure 8

XPS spectra of the green and chemically synthesised NiO NPs. a Survey scan, b O 1s spectrum, c Ni 2p spectrum

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The asymmetric O (1s) signals are divided into three symmetrical signals (Fig. 8b), namely V1, V2 and V3 in the Gaussian fitting. The lower binding energy peak V1 at 529.17, 529.65 eV is from Ni2+ associated with the Ni–O octahedral bonding of cubic rock salt NiO [26,27,28]. The higher binding energy peak (V2) at 530.65 eV are attributed to Ni–O–Ni, whereas the peak (V3) at 532.40 eV is attributed to Ni–O–H at the surface of the NiO nanoparticles [29, 30], or it is related to a transition from O 1s states to the empty O-2p state hybridized with Ni-3d states.

The other peak noted for the binding energy at 535.39 eV is related to the empty O 2p states hybridized with Ni 4sp states [31, 32]. Chemical and green synthesized NiO nanoparticles samples show the positional shift in O (1s) spectra and lower binding energy values are given in Table 4.

Table 4 Comparison of the XPS binding energy values of the green and chemically synthesised NiO NPs
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Figure 8c shows the Cu (2p) core level binding energy spectrum of the green and chemically synthesized NiO nanoparticles. The two sharp peaks obtained at binding energies of 872.70 eV and 855.42 eV correspond to Ni 2p1/2 and Ni 2p3/2 while their satellite peaks due to shake-up process appear at about 879.32 eV and 861.57, 867.16 eV respectively [33]. The spin–orbit splitting between the Ni (2p3/2) and Ni (2p1/2) core level of NiO NPs is 17.28 eV and which is also agree with an earlier reports [34, 35]. The atomic percentage of Ni and O are calculated as 43.58% and 56.42% which reveals the presence of oxygen in excess of Ni. It shows that the particle is non-stoichiometric NiO which confirms the formation of Ni2+ vacancies.

4.8 Electron paramagnetic resonance studies

EPR spectroscopy is an useful technique for studying the materials with unpaired electrons and defects. In order to understand the effect of nanostructures, EPR measurement was carried out on the NiO NPs synthesised by chemical and green synthesis method. Figure 9 displays room temperature EPR spectra for two different synthesised NiO NPs. The calculated ‘g’ values of the samples are 2.250 and 2.214 for green and chemically synthesised NPs respectively. In addition, the Ni2+ ion in NiO has a g-value ~ 2.0. [36]. The relative intensity and line width of these absorption lines exhibit a strong dependence on different synthesis methods and solvents. The peak intensity increases significantly and the broad shift towards lower fields indicate a clear ferromagnetic signature of the both NiO materials. [37]. Thus, the enhanced intensity of EPR spectra and the larger g-value for the chemically synthesised NiO NPs may indicate the increasing amount of nickel vacancies.

Fig. 9
figure 9

EPR spectra of green and chemically synthesised NiO NPs

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4.9 Zeta potential distribution studies

Figure 10 depicts the zeta potential measurement of green and chemically synthesised NiO NPs. The zeta potential is a parameter widely used to predict colloidal suspension stability of synthesised NiO nanoparticles. It provides the degree of repulsion between similarly charged particles in dispersion medium. The greater stability of the nanoparticles is associated with its negative charge (i.e.) nanoparticles with more negative charge (< − 30) showed a greater stability [38]. In the current study, the zeta potential values of green and chemically synthesised NiO NPs are − 6.05 and − 17.9 mV respectively. This result reveals that the chemically synthesised NiO NPs is moderately stable than that of the green synthesised NiO NPs.

Fig. 10
figure 10

Zeta potential measurement of green and chemically synthesised NiO NPs

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5 Microbial toxicity studies

Figure 11 exhibits the zone of inhibition of green and chemically synthesised NiO nanoparticles at different concentrations (1, 3, 5 mg/ml) against S. aureus (gram positive) and E. coli (gram negative) bacterial pathogens. Figure 11a–d shows the size of the zone of inhibition and antibacterial action of both NiO NPs loaded with test samples. It is clear from Table 5, that the chemically synthesised NiO nanoparticles have shown effective antibacterial activities against all the pathogens. It is also observed that the green and chemically synthesized NiO nanoparticle was effective over Gram positive S. aureus showing a large value of zone of inhibition than the gram negative bacterial pathogens. This observation reveals the greater resistance or tolerance of gram negative pathogens over gram positive bacterial strains. The reactivity of the nanoparticles with bacterial cell depends not only on the metal oxide employed but also on the bacterial species tested [39].

Fig. 11
figure 11

Size of the zone of inhibition formed around each disc, loaded with test samples, indicating the antibacterial activity. a, b Green synthesised NiO NPs, c, d chemically synthesised NiO NPs against G+ and G− bacterial strains

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Table 5 Zone of inhibition value of green and chemically synthesised NiO NPs treated with micro organism  [46–48]
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Antibacterial activity of the nanoparticles depends on the particle size, stability and the concentration of the growth medium. As the outer cellular membrane of the bacterial strains is of nanoscale pores, there lies a better reactivity of the nanoparticle with that of the pathogens. The green and chemically synthesised NiO nanoparticles have a particle size of 13 nm, 27 nm and it is better proven through the obtained XRD and HRTEM results. Particle size has a significant role over the antibacterial activity and most of the studies have proven that the antibacterial activity increases with the decreasing particle size [40,41,42]. As both the green and chemically synthesized NiO nanoparticle has less particle size, it gains the tendency to attach to the cell membrane by electrostatic interaction [43] and trigger the initiation of the oxidative stress, thus resulting in free radical formation ROS, which disrupts the bacterial cell membrane as shown in Fig. 11. Production of ROS depends on the electronic structure of the metal oxides and the redox potentials of ROS generation reactions [44, 45]. The high antibacterial activity of the nanoparticles may be attributed to their definite chemical composition, particle size, tendency to release the metal ions and morphology, thereby inducing the trans membrane electron transfer, penetration and oxidization of cell components and production of secondary products (ROS), which results in cell damage. The précised toxicity mechanism of the metal oxide nanoparticle against various bacteria is not clearly understood. The governing mechanism for the antibacterial activity of the synthesized NiO nanoparticle maybe due to the electrostatic interaction between the positively charged nickel ions and the negatively charged bacterial cell membranes, which is in agreement with the studies of Basak et al. [46]. The release of nickel ion (Ni2+), from the NiO nanoparticles penetrate into the cell wall causing damage in the DNA, protein, mitochondria and also disturb in the electron transport resulting in cell death. Diffusion and accumulation of NiO NPs in the cell membrane and changes the membrane permeability with subsequent protein leakage [47, 48]. Another possible mechanism is represented below.

$$\begin{aligned} & \text{NiO} \, + {\text{ h}}\upupsilon \to \text{e}^{ - } + \, \text{h}^{ + } \\ & \text{h}^{ + } + \, \text{H}_{\text{2}} \text{O} \to {}^{ \cdot }\text{OH} \, + \, \text{H}^{ + } \\ & \text{e}^{ - } + \, \text{O}_{\text{2}} \to {}^{ \cdot }\mathop {\text{O}_{\text{2}} }\nolimits^{ - } \\ & {}^{ \cdot }\mathop {\text{O}_{\text{2}} }\nolimits^{ - } + \, \text{H}^{ + } \to \mathop {\text{HO}_{\text{2}} }\nolimits^{ \cdot } \\ & \mathop {\text{HO}_{\text{2}} }\nolimits^{ \cdot } + \, \text{H}^{ + } \to \text{H}_{\text{2}} \text{O}_{\text{2}} \\ \end{aligned}$$

When the band gap Eg is less than that of the incident photon energy, photo excitation takes place with the generation of holes (h+) in the valence band and electrons (e−) in the conduction band. These photo excited holes and electrons having high oxidizing and reducing properties, further react with donor (water and hydroxyl ions) and acceptor (molecular oxygen) and results in the production of different reactive oxygen species (ROS). The excited electron traps the molecular oxygen to generate superoxide anion (O2·−), which further reacts with free water molecules to form hydrogen peroxide (H2O ·2 ), a powerful oxidizing agent. The holes absorb electrons from water and hydroxyl ions to form hydroxyl radicals (·OH), and these reactive oxygen species generated are responsible for the damage of cell membrane, DNA and protein [49,50,51].

In the present study, XRD and HRTEM results reveals that there is a decrease in the particle size for chemically synthesised NiO NPs when compared to the green synthesised NiO NPs. EPR, PL and XPS studies confirmed that highest oxygen and Ni vacancies are present in the chemically synthesised NiO NPs and it produced more ROS in cell membrane. In this aforementioned mechanism and studies reveal that the chemically synthesised NiO NPs possess more microbial toxicity than that of the green synthesised NiO NPs.

6 In vitro cytotoxicity studies on MCF-7 cells

Because nanoparticles of different origin have diverse physicochemical characteristics, their biological responses and subsequent mechanisms will produce dissimilar cytotoxic effects. A thorough characterisation and identification of the important physicochemical properties on the different mechanisms of cytotoxicity require a broad range of cytotoxicity assays. This thesis will present two different cytotoxicity assays; MTT and acridine orange/ethidium bromide staining.

6.1 MTT assay

The cytotoxic effect of the green and chemically synthesized NiO NPs are examined on cultured MCF-7 human breast cancer cells by exposing cells for 24 h to medium containing the NiO NPs at 5–100 μg/ml concentrations (Fig. 12). In the present studies, the results reveal that MCF 7 cells respond to NiO NPs with dose dependent concentrations with increased cytotoxicity. The green synthesised NiO NPs at lower concentration (5 µg/ml) showed 98% cell viability and 92% of cell viability is shown in chemically synthesised NiO NPs respectively. The cell viability was decreased up to 22% for green synthesised NiO NPs and 15% for chemically synthesised NiO NPs at higher concentration (100 µg/ml). The half maximum inhibitory concentration (IC50) of green and chemically synthesised NiO NPs against MCF-7 cells was found to be 55 and 45 µg/ml respectively. IC50 value of the green and chemically synthesised NiO NPs are presented in Fig. 12. From these results, it is confirmed that the chemically synthesised NiO NPs possess highest cytotoxicity effects than that of the green synthesised NiO NPs.

Fig. 12
figure 12

In vitro cytotoxicity of green and chemically synthesised NiO NPs against MCF-7 cells

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6.2 Fluorescence microscopic analysis of apoptotic cell death (AO and EB staining)

AO/EB staining adopting fluorescence microscopy also revealed apoptosis from the perspective of fluorescence. The MCF-7 cancer cells were exposed to the concentrations of green and chemically synthesised NiO NPs for 24 h. In this study, we used acridine orange/ethidium bromide (AO/EB) double staining assay [52]. Acridine orange is taken up by both viable and nonviable cells and emits green fluorescence if interrelated into double stranded nucleic acid (DNA). An Ethidium bromide is taken up only by the nonviable cells and emits red fluorescence by intercalation into DNA.

Apoptotic cells have orange to red nuclei with condensed or fragmented chromatin. Necrotic cells have an uniformly orange to red nuclei with condensed structure. As shown in Fig. 13a–c, the nuclei of untreated normal cells are stained green and showed normal structure, while NiO NPs treated cells are stained red and orange, representing the hallmark of apoptosis. Figure 13b, c exhibited the morphological alteration of MCF-7 cells treated with green and chemically synthesised NiO NPs with IC50 concentrations after staining with AO/EB.

Fig. 13
figure 13

Cells were treated NiO with IC50 concentrations and the morphological changes were observed using a fluorescent microscope after staining with AO/EB for a control, b chemically synthesized NiO NPs and c green synthesized NiO NPs

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Our results confirmed that NiO NPs induced apoptosis at the concentrations evaluated, in agreement with the cytotoxic results. The chemically synthesised NiO NPs possess highest apoptosis morphology as compared to green synthesised NiO NPs. This is due to their smaller size and large surface area which is generally causes the production of ROS.

7 Conclusion

NiO NPs were successfully synthesized by green and co-precipitation method. The G. sylvestre plant extract has been used as a potential reducing agent for the synthesis of NiO NPs which was confirmed by XRD studies. The cubic structure was identified for green and chemically synthesised NiO NPs through XRD analysis. The presence of all functional groups was ascertained from FTIR analysis. Cube shaped morphologies were found in both NiO NPs observed from HRTEM analysis. EDAX analysis has confirmed the presence of Ni and O in NiO NPs synthesised through both the methods. The XPS studies showed that from the indexed peaks corresponding to O (1s), Ni (2p), the respective binding energies of the elements were estimated. The PL emission studies showed that the chemically synthesised NiO NPs altered the band emission, which is due to oxygen vacancies and surface defects. An EPR study has confirmed the ferro magnetic in nature and also Ni vacancies present in the samples. Moderate zeta potential values were observed in green and chemically synthesised NiO NPs.

The potential microbial and MCF-7 cancer cell line toxicity were evaluated for green and chemically synthesised NiO NPs. Chemically synthesised NiO NPs have highest toxicity than green synthesised NiO NPs. With reference to cell death, a minimum concentration of 45 μg/ml for chemically synthesised NiO NPs was well enough to induce 50% cell mortality as compared to green synthesised NiO NPs (55 μg/ml). In the chemically synthesised NiO NPs, the wavelength of the blue green was greater than the green synthesised NiO NPs. This showed an increased number of oxygen vacancies and interstitial oxygen vacancies in the chemically synthesised NiO NPs, leading to a higher number of ROS as compared to that of the green synthesised NiO NPs. The mode of cell death was assessed through adopting an AO and EB staining. The chemically synthesised NiO NPs induced higher percentage of apoptosis than necrosis as compared to green synthesised NiO NPs. The green synthesised NiO NPs exhibit lowest toxicity and environmental friendly when compared chemically synthesised NiO NPs.