Mussaenda frondosa L. mediated facile green synthesis of Copper oxide nanoparticles – Characterization, photocatalytic and their biological investigations

2.1 Preparation of leaf, stem and callus extract

1:10 ratio of powdered sample and distilled water was taken for the extraction of phytochemicals. The extraction was performed at 70–80 ⁰C with a reflux setup (8 h) and the extract was filtered through the filter paper (Whatmann No. 1); concentrated to 1/5^th of the volume in the vacuum concentrator and stored in the refrigerator until further use.

2.2 Synthesis of CuO-NPs

CuO-NPs were synthesized by solution combustion method by utilizing the dried aqueous extracts of leaf, stem and callus of M. frondosa according to the Suresh et al. (2015) method with slight modifications. Meant for NP synthesis the plant extract (2 g) was dissolved in sterile distilled water (100 ml) and stirred using magnetic stirrer (450 to 500 rpm) for about 15 min. Stoichiometric amount of Cupric nitrate trihydrate [Cu (NO₃)₂·3H₂O - Sigma Aldrich (AR)] was dissolved in the plant extract (5 ml) and was kept in a preheated muffle furnace at 400 ⁰C. Within 5 – 8 min, blackish amorphous powder of CuO-NPs was obtained which demonstrated the synthesis of NPs. The experiment was repeated with different concentrations (5, 10, 15, 20, 25 and 30 ml) of plant extracts. The synthesized nanoparticles were ground and stored in airtight containers for further use.

2.3 Morphological and structural characterization of CuO-NPs

Powder XRD (Shimadzu-7000) was carried out to study the crystalline structure and phase purity by using Cu K_α (1.541 Å) radiation operating at a voltage of 50 kV and a current of 30 mA (2θ ranging between 20^° to 80^°). The peak positions were compared with standard JCPDS files to identify the crystalline phase. UV–Vis spectra (Evolution-220, ThermoScientific) was recorded within the wavelength range of 280 – 800 nm to study the optical properties of the CuO-NPs. Surface morphology was analysed using FESEM (Carl Zeiss FESEM) with 10 kV acceleration voltages and TEM (JEOL JEM-1400). Elemental profile of CuO-NPs was carried out using EDS attached with SEM. FTIR spectra (Model AIM-8800) was obtained to study the chemical constituents present in the sample under the spectral range of 4000 – 400 cm⁻¹ with a resolution of 4 cm⁻¹. The size distribution, average size measurement, average zeta potential and stability of NPs in suspension was carried out by DLS (Microtrac USA) analysis. The surface area and pore size measurements of CuO NPs is carried out using BELSORP-mini II instrumentation. All the experiments were repeated thrice and the data was analysed using Origin 8 software.

3.1 Anti-oxidant activity: DPPH radical scavenging activity

Antioxidant activity of CuO-NPs was determined using DPPH as a free radical by 1, 1-Diphenyl-2- picrylhydrazyl (DPPH) assay (Zhishen et al., 1999). Varying concentrations of NPs (0 – 5000 µg/ml) was mixed with 0.3 ml of 0.5 mM DPPH in methanol. The mixture was vigorously shaken and kept in dark (30 min) at RT (Room Temperature) further, the decrease in absorbance was spectrophotometrically measured at λmax 520 nm against the blank (Absolute methanol). Ascorbic acid was used as a reference standard and the mixture which is devoid of test sample was considered as control. The ability to scavenge DPPH radical was evaluated using the equation: $$\mathrm{D}\mathrm{P}\mathrm{P}\mathrm{H}\mathrm{S}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}(\%)=\frac{\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}-\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}{\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}\times 100$$

The final result was expressed as 50% inhibitory concentration (IC₅₀) based on the percentage of DPPH radicals scavenged.

3.2 Anti bacterial activity

The antibacterial activity of CuO-NPs was evaluated by using the disc diffusion technique (Manasa et al., 2020). Two gram positive bacteria (Staphylococcus aureus and Bacillus subtilis) and three gram negative bacteria (Escherichia coli, Pseudomonas aeruginosa and Proteus vulgaris) were used as a test organisms to study the antibacterial efficacy of synthesized CuO-NPs. Bacterial test organisms were grown in nutrient broth (NB) for 24 h at 37 °C. Two hundred μl of overnight grown culture of each bacterial strain (1 × 10⁶cfu/ml) was inoculated into sterile NB and incubated for 4 – 5 h at 37 °C. Each individual culture (100 μl) was uniformly swabbed on the solidified Nutrient agar plates. The sterile antimicrobial susceptibility filter paper discs (5 mm - obtained from Himedia Lab.) was loaded with 20 μl of the CuO-NP and was placed on test organism swabbed plates. The activity was determined by measuring the zone of inhibition around the discs after 24 h of incubation at 37 °C.

3.3 Minimum inhibitory concentration (MIC)

The macrobroth dilution method (CLSI, 2012) was used to determine the MIC of NPs at varying concentrations. The lowest concentration of the antimicrobial agent (NP) which did not allow the visible growth of test organisms after 24 h of incubation relying upon the turbidity of the culture is termed as MIC. Each bacterial culture (24 h old) were 100 folds diluted in Nutrient broth (NB) by adding bacterial inoculum (100 µl) into broth (10 ml) and dispensed into separate test tubes. Increasing concentrations of NPs were prepared from stock solutions (10 mg/ml in sterile distilled water) of NPs and added to the test tubes containing bacterial cultures. Test tube containing broth without NP is used as a negative control and streptomycin sulphate as a positive control. The inoculated tubes were incubated at 37 ⁰C for 24 h and observed for the visual turbidity before and after incubation.

3.4 Evaluation of anti-inflammatory activity

The anti-inflammatory activity of CuO-NPs was determined by human red blood cells membrane stabilization method (HRBCsMS) with slight modifications (Emamuzo et al. 2010). A blood sample were collected from healthy human volunteers and was mixed with an equal volume of Alsever's solution (2 % dextrose, 0.8 % sodium citrate, 0.5 % citric acid and 0.42 % sodium chloride). Subsequently, the mixture was centrifuged at 3, 000 rpm (5 min) and the packed cells were washed with isosaline (0.85%, pH 7.2) and the suspension was made to 10% v/v using isosaline. Varying concentrations of CuO-NP (50, 100, 150, 200, 250 µg/ml) was prepared with distilled water along with the Diclofenac Sodium as a standard. One ml of phosphate buffer (0.15 M, pH 7.4), 2 ml of hyposaline (0.36 %) and 0.5 ml of HRBC suspension were added to each concentration of test sample. The samples were incubated at 37 ⁰C for 30 min and centrifuged (3,000 rpm) for 3 min. The haemoglobin content of the supernatant was spectrophotometrically measured at 560 nm. The percentage inhibition of hemolysis was determined using the following equation, $$\%\mathrm{i}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{o}\mathrm{f}\mathrm{h}\mathrm{e}\mathrm{m}\mathrm{o}\mathrm{l}\mathrm{y}\mathrm{s}\mathrm{i}\mathrm{s}=\frac{\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}-\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}{\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}\times 100$$

4.1 α-amylase inhibition

α-amylase inhibitory activity of CuO-NP was analysed by Manasa et al. (2020) method with slight modifications. In brief, 20 μl of varying concentrations of CuO-NP (5–500 µg/ml) were allowed to react with 50 μl phosphate buffer (50 mM, pH = 5.8) and 50 μl α–amylase (16 U/mg) for 10 min at RT (27 ⁰C). To the above mixture 50 μl of 1 % soluble starch (in 50 mM Phosphate buffer at pH 5.8) was added and incubated at RT for 10 min. Same protocol was followed for the control where the enzyme was replaced with buffer and Acarbose was used as a standard. The activity was determined by recording the absorbance at 405 nm using a Micro plate reader (Synergy H1 BIOTEK) and the results were interpreted as IC ₅₀ values using percentage inhibitory activities. $$\text{Inhibitory activity}\left(\%\right)=\left(1-{\text{A}}_{\text{t405}}/{\text{A}}_{\text{c405}}\right)\times 100$$ where A_t is the absorbance of test sample and A_c is the absorbance of control.

4.2 α-glucosidase inhibition

The α-glucosidase inhibition was analysed according to Sanap et al. (2010) with slight modifications. A reaction mixture, contained 50 μl phosphate buffer (0.3 mM, pH = 6.8), 40 μl α-glucosidase (1 U/ml) and 20 μl of varying concentrations of NP (5–500 µg/ml). The mixture was pre-incubated at 37 ⁰C for 10 min. After incubation, 40 μl of 2 mM p - nitro phenyl-α-D-glucopyranoside (P-NPG) in 0.3 mM buffer was added and incubated further at 37 ⁰C for 10 min. The reaction was ceased by adding 70 μl of 0.2 M Na₂CO₃. The absorbance was recorded at 405 nm employing Multiplate reader (Synergy H1 BIOTEK) to examine the release of p-nitrophenol. The same mixture without CuO-NP served as the control and Acarbose served as the standard (Positive Control). The results were interpreted as IC₅₀ values using percentage inhibition activities, $$\text{Inhibitory activity}\left(\%\right)=\left(1-{\text{A}}_{\text{t 405}}/{\text{A}}_{\text{c405}}\right)\times 100$$ where A_t is the absorbance of test sample and A_c is the absorbance of control.

5.1 Cell culture

The A549 cells (Human lung adenocarcinoma cells) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM: Hi media, India) supplemented with foetal bovine serum (10 %) and 1 % penicillium/streptomycin (Hi media, India). The cells were incubated at 37 ⁰C under 5 % CO₂, subcultured upon attaining 70 % confluence and used for the experiments.

5.2 Cytotoxicity assessment using MTT assay

Cytotoxicity of the CuO-NPs was evaluated using MTT assay (Mosmann, 1983) against A549 cells. Approximately, 5000 cells were seeded to each well of 96 well microtiter plates and incubated for 24 h at 37 ⁰C under 5 % CO₂ in a humidified atmosphere of 95 % air. Varying concentrations of CuO-NPs (12.5, 25, 50, 100 and 200 µg/ml in DMSO) were added to A549 cells and incubated for 48 h. Further, 100 µl of MTT solution (1 mg/ml) was added to the wells and incubated for 4 h. DMSO was added to solubilize formazan crystals and the absorbance was read at 570 nm using multimode microplate reader (FluoSTAR Omega, BMG Labtech). Cisplatin was served as a positive control. Cell viability was determined by the ability of living cells to reduce the MTT dye (Yellow) to a formazan crystal (Blue) using the formula, $$\text{Percentage of cell viability}={\text{Abs}}_{\text{C570}}-{\text{Abs}}_{\text{T570}}/{\text{Abs}}_{\text{C570}})\times 100$$ where Abs_C570 is the absorbance of control and Abs_T570 is the absorbance of test at 570 nm.

The results were interpreted as IC₅₀ values.

5.3 Dual AO/EB fluorescent staining to detect apoptosis:

The influence of CuO-NPs to induce apoptosis in A549 cells was examined by acridine orange (AO) and ethidium bromide (EB) double staining (Azizi et al., 2017). AO and EB are DNA specific dyes which can distinguish dead cells from viable cells. Once the cell membrane is ruptured, AO penetrates into both live and dead cells whereas; EB can penetrate only into dead cells. Therefore, AO-stained live cells appear green in colour and cells appear greenish yellow during the early stages of apoptosis. EB stains dead cells which appear to be red in colour.

In brief, 5 × 10³A549 cells/well were cultured onto 6 well plates and incubated at 37 ⁰C under 5% CO₂ (24 h) for attachment. Subsequently after attachment the cells were treated with CuO-NPs. After 48 h of incubation, cells were fixed in chilled methanol for 20 min at RT. Further, the cells were stained with AO/EB (10 µl) for 15 min in dark (37 ⁰C) and washed with PBS to remove excess stain. Cells were overlaid with 1 ml PBS and images of cells were captured using fluorescent microscope (ZOE, Biorad) to examine the morphological and nuclear transformations.

5.4 Photocatalytic activity

The methylene dye (Hi-media, India) was used as a model dye to study the photocatalytic activity of CuO-NPs in a 150X75 mm batch reactor (Udayabhanu et al., 2016). A catalytic load of 20 mg of CuO-NPs in 100 ml of 5 ppm dye was prepared and the neutral pH was maintained throughout. The slurry was placed in the reactor and stirred magnetically with instantaneous UV exposure. Further, 4 ml slurry was drawn at the specific intervals (10 min), centrifuged to remove the interference of the catalyst and the absorbance was recorded using UV–Vis spectrophotometer at 660 nm to assess the rate of degradation (Begum et al., 2018). The photo-degradation efficiency was determined using the following equation. $$\%\text{Degradation}=\frac{{\text{C}}_{\text{i 660}}-{\text{C}}_{\text{f 660}}}{{\text{C}}_{\text{i 660}}}\times 100$$

• where C_{i 660} - The initial concentration of the methylene blue dye at 660 nm.

• C_{f 660} - The residual concentration of the methylene blue dye at 660 nm.

5.5 Statistical analysis

Experimental results were mean of three parallel measurements (n = 3). Statistical analysis was performed by SPSS statistical package software version 20 with Duncan’s multiple range test grouping. All the statistical analysis were conducted at 95% confidence intervals (p < 0.0.5).

6.1 Powder X-ray diffraction

X-ray diffraction patterns of the CuO-NPs synthesized from M. frondosa extracts were depicted in Fig. 1. The diffraction patterns were reliable with the CuO-NPs and sharp peaks of XRD indicated the crystalline structure. CuO-NPs data was well coordinated with standard JCPDS Card (Joint Committee on Powder Diffraction Standards Card No.89-1397). The diffraction study reveals the peaks of CuO-NPs have a strong intensity and narrow width that indicates the synthesized products were of highly crystalline. The planes present in XRD [(1 1 0), (1 1 1) and (2 0 2)] reveals the formation of pure monoclinic structure of CuO-NPs and no other extra peaks were observed in the plots (Navada et al., 2021). The average particle size of the synthesized CuO-NPs was calculated by using the Scherrer formula and the average crystallite size was found to be 2, 10 nm for C-CuO-NPs; 2, 3 nm for L-CuO-NPs and 2, 4 nm for S-CuO-NPs prepared using 20 and 30 ml of extracts respectively (Moradnia et al., 2020a, 2020b). Hence it could be suggested that the plant extract (Fuel) plays a significant role in controlling the particle size of the bioreduced CuO-NPs.

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Fig. 1

Diffraction pattern of CuO-NPs synthesized from different parts of M. frondosa. A) Leaf- CuO-NP, B) Stem-CuO-NP; C) Callus-CuO-NP.

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6.2 UV–Visible spectra

The CuO-NPs synthesized using M. frondosa extracts were subjected to UV–visible spectra at room temperature is represented in Fig. 2. For uniform dispersion, the CuO-NPs were mixed with water (0.1 wt%), sonicated and then analysed in UV–visible spectrophotometer. The absorption spectra of CuO-NPs revealed a characteristic absorption maximum at 382 nm (L-CuO-NPs), 377 (S-CuO-NPs) and 380 (C-CuO-NPs) at the band gap of 4.66, 3.88 and 4.96 eV respectively. The values are comparable with previous literature (Navada et al., 2020; Moradnia et al., 2020a, 2020b). The absorbance peak is due to the Surface Plasmon Resonance (SPR) of CuO-NPs. When the wavelength of incident light exceeds the particle size diameter causes the transition of electrons from valence band to the conduction band and gives the absorption maxima at ̴ 380 nm indicates the formation of CuO-NPs (Khorsand Zak et al., 2012; Naika et al., 2015).

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Fig. 2

UV– visible spectra of CuO-NPs with inset graphs corresponding to Band Gap of CuO NPs A) L-CuO-NP; B) S-CuO-NP; C) C-CuO-NP (with inset graphs corresponding to Band Gap of CuO NPs).

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6.3 SEM imaging and EDS analysis

The structure of C-CuO-NP and S-CuO-NP were spherical in shape (Fig. 3) which is complementary to the results of CuO-NPs synthesized from Tabernaemontana divaricate leaf extracts (Sivaraj et al., 2014). The morphology of L-CuO-NPs exhibited rod shaped structures which is in accordance with the results of Sankar et al. (2014) in the plant Carica papaya. The SEM images of the CuO-NPs comprised of abandoned patches and clearly visible pores caused due the excessive release of hot gases during solution combustion synthesis process (Udayabhanu et al., 2016).

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Fig. 3

SEM images of synthesized CuO-NPs at different magnifications from M. frondosa. L-CuO-NP (A and D); S-CuO-NP (B and E); C-CuO-NP (C and F).

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Fig. 4 depicts the EDS spectra conjectured the existence of Copper and Oxygen as the predominant elements in the bioreduced CuO-NPs. Peaks located at 0.94, 8.04 and 8.94 keV correspond to the binding energies of Cu, while the peak at 0.5 keV register the presence of oxygen. The elemental analysis results were comparable with the previous reports of Sivraj et al. (2014) and Sankar et al. (2014). However, the peak at 0.27 keV is attributed to the presence of carbon in the carbon tape used for sample mounting during the analysis.

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Fig. 4

EDS Spectra of CuO-NPs from M. frondosa A) L-CuO-NP; B) S-CuO-NP; C) C-CuO-NP.

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6.4 TEM analysis

TEM analysis can be used to apprehend the crystalline faces and size of the bifabricated NPs. Fig. 5 (A and B) affirms the agglomerated spherical shaped CuO NPs which is also in accordance with the result acquired from SEM analysis (Andualem et al., 2020). CuO-NPs appear quite uniform in size with an average particle size of 7.625 nm which is in agreement with XRD results (Chand Mali et al. 2019). On a whole, the results of FESEM and TEM authenticate the biofabricated CuO NPs are agglomerated and spherical in shape.

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Fig. 5

TEM image (A and B) of biosynthesized CuO-NPs under different magnifications.

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6.5 DLS analysis

The average size (Table 1) and size distribution pattern of green synthesized CuO-NPs were shown in Fig. 6. The histograms revealed that the CuO-NPs were monodispersed. Zeta potential values are used to examine the surface charges acquired by CuO-NPs which gives an idea on the stability of the synthesized colloidal NPs. In the current study, the zeta potential analysis of CuO-NPs was found to be within +30 mV and −30 mV confirming the stability of the nanoparticles (Yedurkar et al., 2017). Absolutely high zeta potential values determine the strong repulsive force between the NPs preventing their aggregation (Sankar et al., 2014). PDI measurements of the CuO-NPs were > 0.7 confirming the monodispersion and homogeneity of the colloidal suspension (Table 1).

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Fig. 6

DLS analysis of CuO-NPs from M. frondosa. A) L-CuO-NP; B) S-CuO-NP; C) C-CuO-NP.

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6.6 FT-IR spectroscopy

FTIR spectra of aqueous plant extract (Leaf, stem and callus) and green synthesized CuO-NPs are shown in Fig 7 and 8. The spectra of CuO-NPs reveal the bands at 2352, 1690, 1529 and 1120 cm⁻¹ showed the presence of alkynes (—C≡C), primary amines (—C⚌O), amides in proteins (—N—H) and aliphatic and aromatic amines (—C—N) respectively. All the CuO-NP synthesized from leaf, stem and callus extracts exhibited the same pattern of bands in the spectra. Thus, the consequences confirmed the existence of phenols, flavonoids, terpenoids and some proteins that played an upper hand in capping, preventing the agglomeration of nanoparticles and aids in stabilizing the CuO-NPs. Similar trend was observed by Yugandhar et al. (2017), Padil and Černík (2013), Sivaraj et al. (2014) and Gunalan et al. (2012) in Syzygium alternifolium, Gum karaya, Acalypha indica and Aloe barbadensis respectively. The spectra showed the bands at 525 cm⁻¹ is due to the vibrations of Cu-O (Metal-oxygen) which corresponds to the B_2u mode (Padil and Černík, 2013; Taghavi Fardood et al., 2018). The bands observed at 2922, 3350 cm⁻¹ in the plant extracts are due to the symmetric and asymmetric stretching of aliphatic hydrocarbons (–CH₂) and OH stretching of intramolecular hydrogen bonding. The disappearance of 3350 cm⁻¹ band in the CuO-NP spectra indicates that the synthesized nanoparticles are free from moisture.

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Fig. 7

FTIR Spectra of (A) aqueous extracts of various parts of M. frondosa (B) FTIR Spectra of CuO-NPs of M. frondosa.

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Fig. 8

(A): BET plot of CuO NPs (B) nitrogen desorption/adsorption isotherms (C) BJH plot of CuO.

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6.7 Surface area studies

Fig. 8(A) represents BET surface area plot with regression coefficient of R² = 0.99882 Fig. 9(B) and (C) depicts nitrogen desorption/adsorption isotherms and Barret–Joyner–Halenda (BJH) pore size distributions corresponding to CuO NPs. The type IV nitrogen isotherm with a hysteresis loop is observed which represents the mesoporous nature of the CuO nanoparticles (Zedan et al., 2018). The pore width distribution by the BJH plot indicates the mixture of meso as well as macropores. However, irregular porosity is also observed in FESEM images of the CuO NPs. The BET and BJH measurement values are given in Table 2. The figures obtained indicates very large surface area of the nanoparticles prepared with increased porosity. The results are comparable with previous reports on green synthesized CuO nanoparticles (Navada et al., 2020). The increased surface area of the CuO can be due to “Quantum confinement effect”. The smaller size of the NPs results in a larger surface area. Decreased dimensions of NPs are concerned with the surface to volume ratio, hence enhancement in the specific surface area of the NPs (D'Souza et al., 2021).

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Fig. 9

Antioxidant activity of CuO-NPs. Results are expressed as mean ± SD (n = 3). Different letters indicates the significant difference at p < 0.05.

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7.1 Antioxidant activity

Numerous potential bioactive components viz., alkaloids, flavonoids, steroids, phenolic acids, glycosides have been reported from the plant M. frondosa (Manasa et al., 2017). These active components are very much familiar for their therapeutic effect like antioxidant activity. Antioxidants have a great impact on the metabolic functioning of the organisms. Free radicals and reactive oxygen species (ROS) were generated during routine physiological conditions. Being hazardous to the cell these free radicals should be eliminated from the system or else they tend to oxidise biomolecules subsequently causing oxidative stress resulting in tissue damage, cell death leading to the progression of injurious human health disorders like cancer, ageing, arthritis, cardiovascular diseases, neural diseases and so on (Gülçin et al., 2005). Anti oxidants are the molecules which combat such evil consequences of free radicals and ROS and aid in managing the redox homeostasis of the cell. In the present study green synthesized CuO-NPs possessed significant DPPH radical scavenging activity. The L-CuO-NP (IC ₅₀ value of 1570 μg/ml) and S- CuO-NP (IC ₅₀ value of 1536 μg/ml) exhibited on par antioxidant activity followed by C-CuO-NP (IC ₅₀ value of 2044 μg/ml) (Fig. 9). Lower IC ₅₀ value indicates the more reactive antioxidant molecule. Highest antioxidant efficiency of CuO-NPs against DPPH^. radical is probably due to the electrostatic attraction between charged bioactive molecules and CuO-NPs (Neutral or positively charged) (Kumar et al., 2017).

7.2 Anti microbial activity

Antimicrobial activity of green synthesized CuO-NPs has been revealed a remarkable growth inhibitory outcome against the tested bacterial strains. In this procedure, CuO-NPs (Test sample), aqueous plant extract, bulk CuO, streptomycin (Standard drug- Positive control), water (Negative control) were tested against the bacterial strains. Fig. 10 illustrates the statistical conclusions of the antimicrobial property of CuO-NPs. Varied degrees of inhibition was exhibited by CuO-NPs against P. aeruginosa. On par growth inhibition (p < 0.05) was observed by C-CuO-NP (20.13 mm) and standard streptomycin (20.31 mm) against P. vulgaris. S. aureus was highly susceptible to C-CuO-NP (22.54 mm) which was on par with streptomycin (22.56 mm) at p < 0.05. CuO-NP showed diverse range of growth inhibition against E.coli and was in the order of S-CuO-NP (20.15 mm), C-CuO-NP (18.21 mm) and L-CuO-NP (13.05 mm). S-CuO-NP (21.64 mm), C-CuO-NP (25.61 mm) and streptomycin (25.31 mm) showed on par antimicrobial activity against B. subtilis. Interestingly, the antimicrobial efficacy of CuO-NPs was virtually analogous to the standard drug against P. vulgaris, S. aureus and B. subtilis. Jayandran et al. (2015) reported the green synthesized CuO-NPs exhibited similar or higher antimicrobial activity than the standard drug against S. aureus, E. coli and B. subtilis. In the present study the CuO-NPs showed strong inhibition against both gram positive and gram-negative bacterial strains which is complementary to the report of Sharmila et al. (2019) in the plant Tecoma castanifolia.

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Fig. 10

Antimicrobial activity of CuO-NPs. Results are expressed as mean ± SD (n = 3). Different letters indicates the significant difference at p < 0.05.

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CuO-NPs showed conspicuous affinity towards the amines and carboxyl functional groups located on the outer surface of the bacterial cell wall. The liberated cupric ions subsequently bind to the DNA molecule altering the base pairs in the nucleic acid strands consequently altering the biochemical activities of the cell leading to the cell death (Abboud et al., 2014).

7.3 Minimum inhibitory concentration (MIC)

MIC experiment was performed to determine the lowest concentration of the CuO-NPs that inhibits the growth of microbes. Results of MIC values were found to be in the range of 96–593 µg/ml (Fig. 11). Lowest MIC values was observed against S. aureus (96 µg/ml) followed by B. subtilis (112 µg/ml), P. vulgaris (138 µg/ml), P. aeruginosa (154 µg/ml) and E. coli (305 µg/ml). Biosynthesized CuO-NPs possessed MIC value in the range of 12–50 µg/ml against Staphylococcus sp (Taran et al., 2017). Ahamed et al., 2014 reported the antimicrobial efficacy of CuO-NPs and found the MIC values of about 103 µg/ml and 120 µg/ml against E. coli and S. aureus respectively. Streptomycin exhibited highly significant MIC values than the CuO-NPs.

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Fig. 11

MIC values of CuO-NPs. Results are expressed as mean ± SD (n = 3). Different letters indicates the significant difference at p < 0.05.

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7.4 Anti inflammatory activity

Biofabricated CuO-NPs were evaluated for their anti inflammatory activity by Human red blood cells membrane stabilization method and the results were shown in the Fig. 12. L-CuO-NP and C-CuO-NP exhibited significantly (p < 0.05) on par HRBC membrane stabilizing activity of about 58 ± 0.65% at 100 µg/ml. Our results were compared with the standard drug Diclofenac sodium (78 ± 1.32%). Inflammation is a first line of body’s defense against any infection leading to the denaturation of protein which influences the membrane stabilization. CuO-NPs hampered the release of RBC lysosomal enzymes to certain levels and theirby effectively involved in membrane stabilization (Govindappa et al., 2018). Ananthi and Kala 2017 reported 41.31 ± 1.5% membrane protection employing CuO-NPs synthesized using Triumfetta rotundifolia. Higher membrane stabilizing activity was reported by silver nanoparticles synthesized from Calophyllum tomentosum (84 ± 1.4%) and mushrooms (84 ± 0.25%) (Govindappa et al., 2018; Sriramulu and Sumathi, 2017). Heretofore limited reports are available on the anti inflammatory activity of CuO-NPs and may provide as an excellent source for the development of green nanomedicine for the treatment of inflammation.

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Fig. 12

Anti inflammatory activity of CuO-NPs. Results are expressed as mean ± SD (n = 3). Different letters indicates the significant difference at p < 0.05.

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7.5 Anti diabetic activity

Diabetes is characterized by hyperglycaemia results from deficiency of insulin secretion and/or defects in the action of insulin with refashioning the carbohydrate, protein and fat metabolism. Masella et al. (2012) reported that hyperglycaemia is associated with chronic diabetes accomplished by oxidative stress caused due to the generation of ROS which plays a pivotal role in lipid peroxidation and membrane destruction. Intestinal α-glucosidases and pancreatic α-amylase are the strong inhibitors used for the management of diabetes, which are most significant in the carbohydrate metabolism (Rehana et al., 2017). Prevention of oxidative stress by ROS and inhibiting α-glucosidases and α-amylase activities plays a significant role in the treatment of Diabetes and associated pathophysiological conditions. Green synthesized CuO-NPs exhibited a potent antioxidant activity and hence CuO-NPs were evaluated to screen their efficiency for inhibiting the activity of these carbohydrate digestive enzymes.

In vitro α-amylase inhibitory studies of green synthesized CuO-NPs showed that S-CuO-NP (IC ₅₀ at 57.65 μg/ml) and C-CuO-NP (IC₅₀ at 59.05 μg/ml) possessed significantly similar activity and higher than that of L-CuO-NP (IC₅₀ at 68. 23 μg/ml) at p < 0.05 (Fig. 13). L-CuO-NP (22.31 μg/ml)(Ghosh et al., 2015) possessed significantly (P < 0.05) higher α–glucosidase inhibitory activity followed by S-CuO-NP and C-CuO-NPs (Fig. 14). Our results are in accordance with Ghosh et al., 2015 who reported CuO-NPs synthesized from Dioscorea bulbifera exhibited 99.09% inhibition of α–glucosidase activity. Saratale et al., 2018 reported IC ₅₀ value of 55.5 μg/ml and 51.7 μg/ml of α-amylase and α–glucosidase inhibitory activity respectively from the Argyreia nervosa leaf extract mediated silver nanoparticles. The CuO-NPs synthesized from leaf, stem and callus samples exhibited comparatively lower IC₅₀ values in terms of inhibition of α–glucosidase activity than the α-amylase inhibitory activity which is in accordance with Thatoi et al., 2016.

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Fig. 13

α-amylase inhibition of CuO-NPs. Results are expressed as mean ± SD (n = 3). Different letters indicates the significant difference at p < 0.05.

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Fig. 14

α-glucosidase inhibition of CuO-NPs. Results are expressed as mean ± SD (n = 3). Different letters indicates the significant difference at p < 0.05.

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7.6 Cytotoxicity studies

Cytotoxic potential of synthesized CuO-NPs was evaluated against A549 cell line (Human lung cancer cell lines) at different concentrations (5–100 µg/ml). A decreased cell viability with the increasing concentration of CuO-NPs was observed in the current study, suggesting the dose dependent activity (Fig. 15). Appreciable anticancer activity was noticed by CuO-NPs against A549 cells (Fig. 16). Biofabricated S-CuO-NP (85.66 μg/ml) exhibited potent anticancer activity when compared to L-CuO-NP (218.48 μg/ml) and C-CuO-NP (458.35 μg/ml) at p < 0.05 (Fig. 15). Sivaraj et al. (2014) reported an IC₅₀ value of 56.16 μg/ml from the biosynthesized CuO-NPs using Acalypha indica leaf extracts. Likewise, Ficus religiosa leaf extract mediated CuO-NPs induce cytotoxicity effect against A549 cells by the inhibition of histone deacetylase (Kalaiarasi et al., 2018). However, in the present study S-CuO-NP exhibited highest anticancer activity and further selected to study the impact of NP on morphological alterations against A549 cell lines by AO-EB attaining. S-CuO-NPs induced various morphological changes against A549 cells after 48 h of incubation (Fig. 17A). Diverse morphological changes characteristic to apoptosis were induced including chromatin condensation, structure alteration, chromatin fragmentation and cell clumping (Kosmider et al., 2004; Kouser et al., 2020). S-CuO-NP treated cells showed late apoptotic cells which were stained red. However, such changes were not found in untreated control cells which were healthy with intact nuclei and appeared green (Fig. 17B). Our results are in accordance with Sankar et al. (2014) who observed similar morphological changes from biosynthesized Cuo-NPs. From the results we suggest that the biosynthesized CuO-NPs could be effectively used as a chemotherapeutic drug for the treatment of lung cancer.

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Fig. 15

In vitro Cytotoxicity of green synthesized CuO-NPs by MTT assay against A549 cells. Results are expressed as mean ± SD (n = 3). Different letters indicates the significant difference at p < 0.05.

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Fig. 16

Anticancer activity (MTT assay) of CuO-NPs. Results are expressed as mean ± SD (n = 3). Different letters indicates the significant difference at p < 0.05.

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Fig. 17

Morphological observations of AO/EB stained A549 cells. S-CuO-NP; B) Negative control; C) Standard (Cisplatin).

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7.7 Photocatalytic activity

The CuO-NPs synthesized by green synthesis were used as a photocatalyst for the degradation of carcinogen organic dyes. As a result of these, CuO-NPs prepared from different parts of the plant extracts (callus, leaf, and stem) were subjected to photocatalytic activity, because of their better bulkiness, purity and high yield. The photocatalytic study was performed for the degradation of methylene blue (MB) in presence of UV light and the rate of degradation was observed in terms of decrease in intensity at absorption maxima of methylene blue dye at 663 nm. MB dye when incorporated into drinking water pose serious problem to the flora and fauna including human. Advanced activity of CuO NPs involve degradation of such harmful dyes (Begum et al. 2020). The results were presented in the Fig. 18(A, B, C). C-CuO-NP, L-CuO-NP and S-CuO-NPs degrades about 91.86%, 97.36% and 88.0% of the methylene blue dye in a time span of about 100, 120 and 140 min respectively in the presence of UV light. Semiconductors absorb the photon of energy under the irradiation of light which is higher than the band gap energy of semiconductors, creating a hole and a free electron in the valance and conduction band respectively. If the charge carriers do not recombine, they migrate on to the surface where the free electrons from the reduction of oxygen would lead to the formation of peroxides and superoxides and created holes oxidizes the water to form a highly reactive and unstable OH. The formed OH radicle ultimately leads to the degradation of organic dyes (Shahid et al., 2020a,b; (R and K M, 2021).

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Fig. 18

Methylene blue dye elimination of CuO-NPs under UV-light from A) L-CuO-NP; B) S-CuO-NP; C) C- CuO-NP.

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