An evaluation of the biological activity of zinc oxide nanoparticles fabricated from aqueous bark extracts of Acacia nilotica

2.5.1 UV–visible spectroscopy of AN-ZnO NPs

The colour conversion on blending thoroughly the bark extract of A.nilotica with zinc nitrate was assessed visually. Through periodic sampling of aliquots (2–3 mL), the biogenic reduction of zinc ions into AN-ZnO NPs in the aqueous reaction solution was checked. The maximum spectral absorption was executed from 200 to 800 nm in a UV–Visible spectrophotometer (JASCO V-670 PC UV–Visible spectrophotometer).

2.5.2 Fourier transform infrared (FTIR) spectral analysis of AN-ZnO NPs

The phytometabolites, their functional groups and chemical bonds existing in A.nilotica to reduce and stabilize the AN-ZnO NPs were verified in the unique spectra formed in the FTIR. Potassium bromide was mingled with AN-ZnO NPs to acquire the pellet under a hydraulic pellet machine, then placed within the sample holder and the scanning wavelength was ranged from 400 cm⁻¹ to 4000 cm⁻¹ at 4 cm⁻¹ (resolution) in the Nicolet iS50 (Thermo Fischer Scientific) FTIR.

2.5.3 Powdered X-ray diffraction (P-XRD) spectral assessment of AN-ZnO NPs

The crystallographic fingerprint and purity phase of AN-ZnO NPs were keenly assessed through powdered XRD. The X-ray diffraction was enforced adopting a Bruker D8 Advance X-ray diffractometer with CuK_α radiation, 2 theta range (20° − 80°) handled at 30 kV with 10 mA (0.5 s). The crystallite dimension of the fabricated AN-ZnO NPs was pointed out using Debye-Scherrer’s formula as mentioned, D = 0.89λ/βcosθ. Here, the crystallographic size (nm) of nanoparticles was denoted to be D, wavelength of the used X-ray as λ (lambda = 1.5406 Å), FWHM (full width at half maximum) as $\beta$ and Bragg’s angle of diffraction as θ.

2.5.4 SEM and EDX analysis of AN-ZnO NPs

The surface morphological features of engineered AN-ZnO NPs was assessed in JOEL 6390LA scanning electron microscope (SEM). On a copper grid coated with carbon, a thin layer of AN-ZnO NPs was placed, dried under a mercury lamp and the imaging was done with magnification. The purity of AN-ZnO NPs, qualitatively existed elements were resolved in Energy Dispersive X-ray analysis (EDX) integrated with the scanning electron microscope.

2.5.5 High-resolution transmission electron microscopy (HRTEM) and selected area electron diffraction (SAED) of AN-ZnO NPs

The nanostructure, geometry, and dimension of the AN-ZnO NPs were reported by the FEI-Tecnai G2 20 Twin high-resolution transmission electron (HRTEM) microscopic technique operated at a voltage of 80 kV. The ethanol sonicated suspension (4 µL) of AN-ZnO NPs was coated over the copper-carbon coated grid and scrutinized for surface morphology under the microscope. Image J Software (1.45 s) was used to evaluate the size of the nanocrystals. To further confirm the crystallinity of nanoparticles, the SAED (selected area electron diffraction) pattern was also recorded with HRTEM.

2.7.1 2, 2-diphenyl-1-picrylhydrazyl (DPPH) scavenging assay

The antiradical potential of phyo-inspired harnessed AN-ZnO NPs was assessed using the DPPH (2,2-diphenyl-1-picrylhydrazyl) method as previously documented with minor variations. At first, 1 mL of synthesized AN-ZnO NPs in a diverse range of concentrations (20 µg/mL to 60 µg/mL) were individually combined with the DPPH of 4 mL prepared as 0.004% solution in methanol and kept undisturbed at the ambient temperature in a dark condition for 30 min. The absorbance of the faded purple colour of DPPH to yellow colour was recorded spectrophotometrically at 517 nm. The AN-ZnO NPs reports were compared to the standard reference solution of ascorbic acid. As a blank, methanol was used. The radical quenching ability was calculated in percentage (%) as mentioned in the below-provided equation. $$\text{DPPH}\text{radical}\text{quenching}\text{ability}\left(\%\right)=\left[\left({\text{C}}_{(\text{Abs})}-{\text{S}}_{(\text{Abs})}\right)/{\text{C}}_{(\text{Abs})}\right]\times 100$$

Here, C_(Abs) - Control (DPPH in methanol) absorbance, S_(Abs) - AN-ZnO NPs/Ascorbic acid (along with DPPH) absorbance. The sample concentration offering 50% antiradical ability (IC₅₀) was also calculated.

2.7.2 Nitric oxide (NO) scavenging assay

The Griess reaction was adopted to quantify the nitrite ions produced due to the interaction of an aqueous solution of sodium nitroprusside with oxygen, as earlier prescribed by Sylvie et al., (Sylvie et al., 2014) including a few alterations. Nitric oxide (NO) scavenging activity was screened for AN-ZnO NPs. About 20 µg/mL to 60 µg/mL concentrations of AN-ZnO NPs (each 1 mL) were well blended in separate test tubes with 2 mL of 10 mmol/L of sodium nitroprusside solution prepared using 50 mmol/L of phosphate buffer (pH 7.4). The reaction tubes were incubated for nearly 150 min at ambient temperature. Subsequently, added one mL of 0.33% of sulfanilic acid (diluted in 20% glacial acetic acid) to the reaction mixture (0.5 mL) and left for diazotization for 5 min. Before incubation for 30 min, 1 mL of 0.1 % napthylethylenediamine dihydrochloride was reacted to the test tube contents. Spectrophotometrically, the developed chromophore (pink colour) intensity was recorded at 540 nm. Ascorbic acid served as a positive reference. The blank solution was methanol. The below-provided formula was implemented to calculate the % (percentage) of reduction in nitric oxide radical by AN-ZnO NPs. $$\%\text{Radical}\left(\text{NO}\right)\text{reduction}=\left[\left({\text{C}}_{(\text{Abs})}-{\text{S}}_{(\text{Abs})}/{\text{C}}_{(\text{Abs})}\right)\right]\times 100$$

Here, C_(Abs) - Control (without AN-ZnO NPs) absorbance, S_(Abs) - AN-ZnO NPs /Ascorbic acid absorbance. IC₅₀ value was also computed.

2.7.3 Hydroxyl radical scavenging (OH^–) assay

The scavenging potentiality of AN-ZnO NPs on the hydroxyl radical (OH^–) was executed with the protocol implemented by Subramanian et al., (Gupta et al., 2015). Initially, 0.2 mL of AN-ZnO NPs of varied ranges (20 µg/mL to 60 µg/mL) were added into the independent test tubes along with 1 mL solution of EDTA (0.13 % of anhydrous ferrous ammonium sulphate with 0.26 % of EDTA diluted in 100 mL of distilled water) and completely blended with 0.85 % of DMSO (1 mL) in phosphate buffer of 0.1 M (pH 7.4) to trigger the reaction. Subsequently, 0.22 % of ascorbic acid (0.5 mL) was further added into the reacting mixture. The contents were boiled within the water bath for 15 min at 90 ℃. Termination in the reaction was done by pouring 17.5 % of ice-cold trichloroacetic acid (1 mL), 3 mL of Nash reagent (75 % of ammonium acetate, 2 mL of acetylacetone and 3 mL of glacial acetic acid were combined, and volume was brought to 1 L using distilled water) was poured into all reaction tubes, incubated at room condition for 15 min. The yellowish chromophore obtained was spectrophotometrically recorded at 412 nm. Ascorbic acid was utilized as positive reference control and without ascorbic acid, the reaction mixture served as a negative control. By implementing the below-provided formula, the hydroxyl radical scavenging capacity of AN-ZnO NPs was calculated. $$\text{Percentage}\text{of}{\text{OH}}^{-}\text{radical}\text{scavenged}=\left[\left({\text{C}}_{\text{(Abs)}}-{\text{S}}_{\text{(Abs)}}/{\text{C}}_{\text{(Abs)}}\right)\right]\times 100$$

Here, C_(Abs) – Control (without AN-ZnO NPs) absorbance, S_(Abs) – AN-ZnO NPs/ Ascorbic acid absorbance. IC₅₀ value was also computed.

2.7.4 ABTS (2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging assay

The protocol of the ABTS scavenging assay of AN-ZnO NPs was executed with slight variations as done by Kuppurangan et al., (Kuppurangan et al., 2016). By mingling an equal proportion of 7 mM solution of ABTS with 2.4 mM of potassium persulphate, a working solution was obtained and retained in a dark place for 12 h in room condition. Next, 1 mL of the resultant solution was thoroughly mingled with 1 mL of varying concentrations (20 µg/mL to 60 µg/mL) of AN-ZnO NPs. Six minutes later, the reacted mixture was read spectrophotometrically at 734 nm. The aptness of AN-ZnO NPs to scavenge ABTS was found out in percentage (%) by applying the below indicated formula: $$\text{Percentage}\text{of}\text{ABTS}\text{inhibition}=\left[\left({\text{C}}_{\text{(Abs)}}-{\text{S}}_{\text{(Abs)}}\right)/{\text{C}}_{\text{(Abs)}}\right]\times 100$$

Here, C_(Abs) - Control (without AN-ZnO NPs) absorbance, S_(Abs) - AN-ZnO NPs /Ascorbic acid absorbance. IC₅₀ value was also computed.

2.8.1 Culture

The MCF-7 (Breast cancer cell line) was procured from NCCS (National Centre for Cell Science, Pune, India) and cultured in DMEM medium acquired from (Sigma-Aldrich, India), also boosted with 10% FBS (fetal bovine serum), 20 mL of antibiotic penicillin (1% w/v). The cells were sustained in an atmosphere of CO₂ (5%) with humidification (95%) at 37 ℃.

2.8.2 MTT -mediated cytotoxic evaluation of AN-ZnO NPs

MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide) assay was executed for AN-ZnO NPs on MCF-7 (Breast cancer cell line) cell line as previously performed by Menon et al., (Menon and Shanmugam, 2020). Exponentially proliferating MCF-7 cells (10⁵ cells/well) were aseptically placed into the 96-well cell-culture plate and left undisturbed for 24 h in 5% CO₂ at 37 ℃ for 1 day to acquire confluency. Diverged concentrations of prepared AN-ZnO NPs (10 µg/mL to 90 µg/mL) were exposed to the MCF-7 cells, re-incubated for 24 h at 37 ℃ respectively. Next, 100 µL of 0.5% MTT (1 mg/mL) diluted in PBS was included to all the wells and re-incubated for 4 h at ambient condition. The supernatant MTT solution used was discarded. Using PBS, cells were washed. The finally procured purplish coloured crystals of formazan were diluted in dimethyl sulfoxide (100 µL). The value of absorbance was monitored at 570 nm with the aid of ELISA plate reader for each concentration. The inhibition of cell viability percentage (%) was computed using the below-mentioned formula: $$\text{Cell}\text{viability}\left(\%\right)=[({\text{A}}_{570}\text{value}\text{of}\text{tested}\text{cells}/{\text{A}}_{570}\text{value}\text{of}\text{control}\text{cells})\times 100]$$

2.8.3 Determination of ROS using DCFH-DA (Dichloro-dihydrofluorescein diacetate method)

The in vivo ROS emergence in the MCF-7 cells on treatment with AN-ZnO NPs was assessed the using DCFH-DA staining protocol. The non-fluorescent DCFH-DA dye on interaction with the ROS generates a fluorescent compound DCF (2′,7′-dichlorofluorescein). In brief, 1 × 10⁵ MCF-7 cells/well were carefully laid into a microplate with 6-well. The MCF-7 cells were reacted with IC₅₀ concentration of AN-ZnO NPs for 24 h and 48 h within the CO₂ incubator. DCFH-DA stain (10 µM) was exposed to the cultured cells and further incubated for 30 min at room temperature (37 ℃). Consequently, the reacted cells were double washed with cold phosphate buffer saline (PBS) and resuspended in PBS. Analysis of cells (reacted and unreacted) was performed beneath the fluorescence microscopic technique (40 X objective). Doxorubicin (500 µg/mL) was utilized as a positive control. The fluorescence intensity was read with a fluorescence microplate reader at excitation (485 nm) and emission (550 nm) respectively.

2.8.4 Analysis of mitochondrial membrane potential (MMP) using Rhodamine 123 staining

In brief, MCF-7 cells (1 × 10⁵ cells/well) were placed into the 6-well microplate. AN-ZnO NPs at their respective IC₅₀ concentration was reacted to the MCF-7 cells and left undisturbed. After 24 h and 48 h, the reacted cells were carefully harvested and using PBS rinsed twice. Then the cells were exposed to Rhodamine-123 dye (Rh-123; 10 µg/mL) for 30 min at 37 ℃ in a dark condition inside the CO₂ incubator and re-rinsed using PBS. At last, the intensity of fluorescence given by the Rh-123 stain was visualized with a fluorescent microscope and photographs of the treated cells as well as untreated cells (negative control) along with doxorubicin (500 µg/mL) positive control were captured. The pattern of depolarization in the mitochondrial membrane was seen. The fluorescence intensity was read with a fluorescence microplate reader at excitation (485 nm) and emission (550 nm), respectively.

2.8.5 Morphological assessment of nucleus using DAPI

The condensation of chromosomes in the apoptotic cells was identified by DAPI (4,6-diamidino-2-phenylindole), a fluorescent probe specific to the nuclei. The MCF-7 cells treated with AN-ZnO NPs (IC₅₀ concentration) were incubated for 24 h and 48 h. Then, using PBS (phosphate buffer solution) the cells were repeatedly washed and fixed with formaldehyde (4%). Using DAPI solution (1 µg/mL) the cells were stained and left at 37 ℃ for 5 min. MCF-7 cells without AN-ZnO NPs exposure were utilized as a negative control whereas doxorubicin (500 µg/mL) exposed cells as a positive control. The cells were finally washed using PBS and the morphology of the nucleus was visualized under a fluorescence microscope with a blue filter (420 nm).

2.8.6 Western blotting technique

To detect the apoptotic (Bax, Caspase-3 and Caspase-9) and antiapoptotic expression of proteins (Bcl-2) along with proliferative protein marker expression (Cyclin D1 and PCNA) in the MCF-7 cells (1 x10⁵ density) exposed to AN-ZnO NPs were taken for western blot technique. MCF-7 cells cultured in 6-well tissue culture plates were reacted to AN-ZnO NPs (IC₅₀ concentration) for 24 h and 48 h. In this blot assay, β-actin served as a standard loading control. At the end of incubation time, harvesting of cells was done utilizing RIPA lysis buffer solution and then quantified protein concentration spectrophotometrically. Electrophoresis of protein was executed from the collected sample in 12% SDS-PAGE (sodium dodecyl sulphate -polyacrylamide gel electrophoresis). Transfer of proteins from the gel onto nitrocellulose membrane was carried out and the membrane was blocked with 5% BSA (bovine serum albumin) for 1 h to hinder non-specific binding. The membrane was exposed to primary mAb (monoclonal antibodies) and left for 24 h at 4 ℃. A gentle washing of the membrane using TBST buffer was done, then secondary antibodies were reacted to the membrane and re-incubated for 1 h at ambient condition. At last, the membrane was thoroughly washed in TBST buffer and the levels of expression of proteins were detected with the aid of a chemiluminescent detection system (Biorad, USA).

2.9.1 Larval whole-body homogenate preparation and acetylcholinesterase assay

The departed larvae of Anopheles stephensi (3rd instar) exposed to AN-ZnO NPs for 24 h were retrieved, washed with distilled water to remove adherents, and blotted with tissue paper to withdraw moisture content. Then homogenized with ice-cold sodium phosphate buffer solution (20 mM, pH 7.4), centrifuged at 8000 rpm for 15 min and finally the supernatant was taken for enzyme assay.

2.9.2 Acetylcholinesterase (AChE) inhibition by AN-ZnO NPs

The protocol of Ellman et al., (Ellman et al., 1961) with certain alterations was applied to assess the AChE inhibition activity of AN-ZnO NPs. Shortly, the solution of AChE (10 µL), AN-ZnO NPs (20 µL) of diverged concentrations and cold-phosphate buffer (150 µL) were placed in the 96-well microtiter plate followed by refrigeration (1–4 ℃). Then inhibitor of varying concentrations diluted in DMSO (0.1%), 0.4 mM acetylthiocholine iodide (20 µL) and DTNB (Ellman’s reagent) was poured into each well. The reacting mixture was incubated at 37 ℃ for 30 min and the colour alteration (yellowish/ colourless) of the solution was recorded at 412 nm by a microplate reader. The inhibition % of the acetylcholinesterase enzyme by AN-ZnO NPs was calculated by applying the below-provided equation: $$\text{Inhibition}\text{in}\%=\left[\left({\text{C}}_{(\text{Abs})}-{\text{S}}_{(\text{Abs})}\right)\right]/{\text{C}}_{(\text{A})}]\times 100$$

Here, C_(Abs) = Control absorbance, S_(Abs) = Sample absorbance.

2.9.3 Histological assessment of Anopheles stephensi

Histological analysis was executed to notify the alteration in the tissues of larvae. Both control larvae along with the AN-ZnO NPs exposed larvae (treated) of Anopheles stephensi mosquito were immersed in the fixative solution (10% formaldehyde), dehumidified with ethanol, cleansed with xylene solution, embedded in paraffin, and sectioned to get 5 µm thick tissue slices in a rotary ultramicrotome instrument. Paraffin was eradicated from the tissues and stained using Haematoxylin and Eosin stain and abnormalities were examined under an inverted microscope. The photographic images were captured with a digital camera.