Green approach to synthesize nano zinc oxide via Moringa oleifera leaves for enhanced anti-oxidant, anti-acne and anti-bacterial properties for health & wellness applications

2.1 Materials

Zinc Acetate 99.5 % (C₄H₆Zn·2H₂O) from S D Fine-chem limited, Mumbai, India. Moringa oleifera leaves powder from Holy Natural, India. Extra pure 2,2-Diphenyl-1-Picrylhydrazyl (DPPH) from SRL Pvt. ltd. Distilled water having pH 5.5–7.0 & 1µS/cm conductivity. Bacterial cultures E. coli ATCC 10536, C. acne ATCC 11,827 and S. aureus ATCC 6538 were purchase through ATCC bank (American Type of Culture Collection) and standard drugs (Doxycycline, Azithromycin, Cefpodoxime) from local pharmacies.

2.2 Method of green synthesis ZnO nanoparticles

Prepare decoction of Moringa oleifera leaves powder with distilled water at 1:20 ratio under continuous stirring at 80–90 °C for 6 h. Filter the above mixture using Whatman filter paper-1 to get separated solid leaf pieces from the Moringa oleifera leaves extract. Add 0.272 mol of zinc acetate dihydrate (final concentration in mixture to be 0.0597 g/mL) to the filtered plant extract with continuously stirring for 2–3 h at 80–90 °C until precipitation is observed. Set the solution aside for 18 h in room temperature in order to facilitate the reaction completion and formation of ZnO nanoparticles. A dark green mixture having a pH of 7.45 and settled precipitates is observed. Centrifuge the solution at 3000 rpm to collect GsZnO-Nps and dry at 50 °C to make it powder for further evaluation Fig. 1. The possible mechanism of green synthesis of nano ZnO formation, using herbal extract is described as,

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Fig. 1

Systematic presentation of synthesis of GsZnO-Nps via Moringa oleifera leaves.

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The phytochemicals such as phenolic compounds and flavonoids (Ar-(OH) n) present in Moringa oleifera leaves extract are responsible for synthesis of GsZnO-Nps. (Ar-(OH) n) losses an electron.

for the reduction of Zn²⁺ to Zn^o and possible formation of Zn°-phenolate complex by chelation effect which helps in the synthesis of ZnO nanoparticle at 80–90 °C (Hasan et al., 2021).

2.3 Characterizations

UV–Visible spectrum was recorded by using UV–vis spectrophotometer Model-1800 from Shimadzu, Japan. The suspension of GsZnO-Nps and AR Grade ZnO was prepared in distilled water and sonicated for 10 min before measuring the UV–vis spectra. The absorbance range of ZnO was 200–700 nm. FT-IR spectrums were recorded by using FT-IR Spectrophotometer Model- IR Affinity-1S (Shimadzu) Japan. ATR (Attenuated Total Reflection) from SPECAC, U.K. in the wavelength of 4000–400 cm⁻¹. The crystallization properties, size, and phase analysis of GsZnO-Nps were examined by using an X-ray diffractometer (XRD Shimadzu-6100) having scanning range 2θ, degree 20°-70°, CuK1 radiation (λ = 1.54056 Å), voltage 40 kV and current 30 mA. The surface morphology of green synthesis ZnO nanoparticles (GsZnO-Nps) was explored by using SEM Model-JSM-6010 PLUS ILA. The range magnification was x50k to x100k, parameter 10 kV, WD 12 mm. The qualitative and quantitative elemental analysis was assessed by Energy Dispersive X-ray Spectroscopy (EDS).

2.4 Anti-oxidant efficacy

The anti-oxidant efficacy of GsZnO-Nps has been determined by using the DPPH assay. Methanolic solution of 1.52*10^-4 M of DPPH was used in 1:1 ratio with GsZnO-Nps at varying concentrations. anti-oxidant efficacy percentage was examined after incubating the mixture of GsZnO-Nps with DPPH for 30 min and recording the absorption at 517 nm. The half-maximal inhibitory concentration (IC₅₀) is measured to study the antioxidant potency of nanoparticles to reduce the DPPH. The control samples were prepared using DPPH solution and mixed only with respective solvents (aqua/methanol) at a 1:1 ratio and measured at 517 nm, the same method followed which was reported in the previously published literature (Bhalla et al., 2021).

2.5 Anti-acne efficacy

GsZnO-Nps were studied for the anti-acne efficacy against C. acne using disk diffusion technique commonly known as Kirby Baurer method. This is a qualitative technique to test the ability of a material to inhibit microbial growth by measuring ZOI. Overnight bacterial culture was grown at 37 °C and its cell density was adjusted to 10⁸ cfu/mL (i.e. Optical density of 0.1 at 600 nm). 0.1 mL of bacterial culture was spread onto sterile Muller Hinton agar plates. Plates were allowed to dried followed by placing 6 mm sterile disc soaked for 30 min in GsZnO-Nps and Zinc acetate concentration 0.0183 g/mL. Post incubation at 37 °C for 24 h, ZOI around the disc was measured.

2.6 Antibacterial activity

The same disk diffusion technique (section 2.5) was used for determination of the antibacterial efficacy against S. aureus and E. coli (both of 10⁸ cfu/mL). 6 mm sterile disc soaked for 30 min in different concentrations of GsZnO-Nps, AR Grade ZnO and standard drugs were used for study. Post incubation at 37 °C for 24 h, ZOI around the disc was measured.

Minimum inhibitory concentration of GsZnO-Nps against Gram-positive and Gram-negative pathogens were determined by agar dilution method. Stock solution of GsZnO- Nps was diluted using Muller Hinton agar to achieve a final concentration of 900, 700, 500, 300, 100 µg/mL. Mixture was vortex well and pour onto sterile petri plate and allowed to solidify. After solidification, 10⁸ cfu/mL of saline suspension of test organisms E. coli and S. aureus were streaked on every plate and incubated at 37 °C for 48 h. Inhibition of growth was judged by comparison with growth in control plates. The MIC was defined as the lowest concentration of particles that showed no visible growth after incubation (Siddiqi et al., 2018).

3.1 UV–vis spectrum

The UV–Visible absorption spectrum of GsZnO-Nps and AR grade ZnO was examined by a UV–vis spectrophotometer in the range of 200–700 nm. The AR Grade ZnO exhibits the absorption peak at 386 nm with an energy bandgap of 3.20 eV correspondingly GsZnO-Nps exhibits a typical absorption peak in the UV range at 368.5 nm having bandgap energy of 3.36 eV shown in Fig. 2 (A and B).

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Fig. 2

The UV–vis absorption spectrum and band gap energy of (A) GsZnO-Nps (B) UV–vis spectrum AR grade ZnO given for comparative study.

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Based on UV wavelength, the band gap (Eg) was calculated with formula (Bhalla et al., 2022):

The high band gap energy of GsZnO-Nps clearly indicate the successful synthesis of nanoparticles. A blue shift of absorption bands was observed in the GsZnO-Nps spectra unlike bulk AR grade ZnO. The blue shift could be because of several reasons, like the quantum confinement effect explaining the bandgap energy inversely proportional to particle size, and shows a blue shift as the particle size decreases. The particle size decreases and approaches the size of the electron-hole distance, particle energy levels become quantum confined and separate thus confining the motion of the electron (Debanath and Karmakar, 2013).

3.2 Fourier-transform infrared spectroscopy (FT-IR)

IR spectrum of moringa extract and GsZnO-Nps exhibits the functional group in different regions as shown in Fig. 3. Spectrum (A, B, and C) shows the sharp broad peak in the single bond region with a shift from 3076.63 to 3302.13 cm⁻¹ suggests the existence of the hydroxyl (OH) group which can be attributed to the presence of H₂O molecules. Spectrum (A) shows the two peaks at 2918.29 cm⁻¹ and 2848.86 cm⁻¹ could be due to symmetrical and asymmetrical C—H stretching of phytochemicals (Joshi et al., 2022). Small peak at 2362.79 cm⁻¹ observed Spectrum (C) could be due to O⚌C = O stretching probably due to the impurity during synthesis or measuring condition (Bhalla et al., 2022). The peak at 1635.63 cm⁻¹ and 1639.49 cm⁻¹ in spectrum (A & C) could be attributes to the stretching of (C⚌C) group of phenolic compounds (Joshi et al., 2022). The peaks at 1550.76, 1552.69 and 1350.17 cm⁻¹ in spectrum (B & C) could be due to asymmetrical and symmetrical stretching bonds of COO^– (Hasan et al., 2021). Peak at 1408.03 to 1433.11 cm⁻¹ in spectrum (A, B & C) could be attributed to the –C—H bending vibration. The peaks 1244.08 cm⁻¹ could be C—O stretching vibration. While 1045.41 cm⁻¹ peak is the due to the C-OH bending of carbohydrates present in moringa extract. The peak 1018.41 cm⁻¹ in spectrum B could be due to C-CH₃ framework originated from zinc acetate (Phoohinkong et al., 2017). While the peak at 956.69 cm⁻¹ exhibit the existence of vinyl C—H out-of-plane bend. The peak at 686.65 and 611.43 cm⁻¹ could be due to out-of-plane bend of OH vibration. Spectrum (C and D) shows the peaks at 420.48 cm⁻¹ and 410.83 cm⁻¹ indicating the presence of ZnO similar to previously reported literatures. (Bechambi et al., 2015; Hasan et al., 2021).

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Fig. 3

FT-IR spectrum of (A) Moringa oleifera leaves powder (B) Zinc acetate (C) GsZnO-Nps (D) AR Grade Zinc oxide in the spectral range of 400–4000 cm^−1.

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3.3 X-ray diffraction analysis

The crystalline structure of GsZnO-Nps was analyzed by an X-ray diffractometer. The diffractogram confirms the hexagonal wurtzite structure of GsZnO-Nps (Lei et al., 2012). The diffraction peaks were found at 2θ values of 31.53°, 34.19°, 36.02°, 47.31°, 56.37°, 62.63°, 66.16°, 67.72°, and 68.86°. These peaks are in line with JCPDS standard card 36–1451 of ZnO shown in Fig. 4 (A, B and C) which is in line with previously published literatures [4, 5 and 17]. The average crystallites size of GsZnO-Nps is 13.82 nm (Table 1) which is significantly lower than AR Grade ZnO is 30.87 nm and other previous reported literature (Jayachandran et al., 2021). Debye-Scherrer equation was used for evaluation of crystallite size,

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Fig. 4

XRD spectrum of (A) AR Grade ZnO (B) GsZnO-Nps and (C) JCPDS Standard card no. 36–1451 given for reference.

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D- Crystallites size, λ- The wavelength of X-ray, K-Scherrer constant (0.9), θ-Braggs angle in radians and β-Full width at half maxima of the peak in radians.

3.4 Scanning electron microscope analysis (SEM)

The surface morphology of the GsZnO-Nps was examined by using a scanning electron microscope having x50k and x100 k magnification. Fig. 5 (A, B, and C) shows monodispersed spherical particles of GsZnO-Nps having approximately 50 nm particle size as deduced from the cited scale. Fig. 5 (D) shows the energy-dispersive X-ray spectroscopy (EDS) analysis which reveals the quantitative elemental composition of GsZnO-Nps. The elemental analysis of green synthesis nanoparticles shows a mass % of Zn-77.07 and O- 22.93.

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Fig. 5

(A, B and C) Scanning Electron Microscopic images reveal the morphology of GsZnO-Nps shows spherical nanoparticles, (D) EDS of GsZnO-Nps revealing the elemental composition in graphical and tabulated form.

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3.5 Anti-oxidant efficacy

Anti-oxidant efficacy of GsZnO-Nps and AR Grade ZnO were evaluated at different concentrations (10 to 100 µg/mL) by using nitrogen-centered free radical i.e. DPPH. The anti-oxidant efficacy of GsZnO-Nps and AR Grade ZnO was shown in Fig. 6 (A and C) up to 60 min. The systematic pictorial representation of scavenging of free radical color change due to GsZnO-Nps and AR Grade ZnO shown Fig. 6 (B and D). The free radical scavenging potency “FRSP” of GsZnO-Nps at concentration 50, 75 and 100 µg/mL shows 91 % to 99.99 % after 30 min. However, FRSP of AR Grade ZnO was 14 % to 16 %. The IC₅₀ concentration of GsZnO-Nps was 21.72 µg/mL and AR Grade ZnO was 345.57 µg/mL which is shown in Fig. 7 (A and B). The free radicle scavenging potency of GsZnO-Nps is significantly higher than in reported literature (Tettey and Shin, 2019). The high FRSP could be because of several reasons, influence of phytochemicals of Moringa oleifera leaves extract and their bioactive attraction towards charged nanoparticles (ZnO = Zn²⁺ + O²⁻) which synergistically increase the bioactivity, formation of nanoscale particles, high specific surface area. The activity effects commonly depend on the attachment sites of the metals and their subsequent impact on the potency of the free radical scavenging (Kumar et al., 2014).

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Fig. 6

(A) anti-oxidant efficacy of GsZnO-Nps different concentration (10 to 100 µg/mL vs Time), (B) Pictorial representation anti-oxidant efficacy of GsZnO-Nps, (C) anti-oxidant efficacy of AR Grade ZnO, (D) Pictorial representation anti-oxidant efficacy of AR Grade ZnO given of comparison.

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Fig. 7

IC₅₀ concentration (A) GsZnO-Nps and (B) AR Grade ZnO which is given for comparison.

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3.6 Anti-acne efficacy

The anti-acne efficacy of 0.0183 g/mL GsZnO-Nps and zinc acetate dihydrate was evaluated by disk diffusion technique against C. acne bacteria which is mainly responsible for causing acne on skin. The study was carried out in four different sets to minimize the experimental error. The average inhibition zone of GsZnO-Nps against C. acne was 33 mm having a standard deviation of 1.155 mm and a standard error of 0.557 mm. ZOI of zinc acetate dihydrate was 10.7 mm having a standard deviation of 0.559 mm and the standard error was 0.279 mm which is shown in Fig. 8 (A and B) and Table 2. The robust anti-acne efficacy could be due to several reasons like the influence of active chemical constituents of Moringa oleifera, the particle size of metal oxide in nano regime, generation of reactive oxygen species, the release of Zn²⁺ ions which can rupture the micro-organism cell walls and potentially damage the internal organelle of the bacterial cell. The anti-acne efficacy is significantly higher than other reported literature (Bae and Park, 2016).

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Fig. 8

Representative images of anti-acne study against C. acne bacteria (A) ZOI of GsZnO-Nps (B) ZOI of Zinc acetate dihydrate salt.

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3.7 Anti-bacterial efficacy

Anti-bacterial properties of GsZnO-Nps, AR Grade ZnO and standard drug were investigated by disk diffusion method. Different concentration like 10 µg/mL, 50 µg/mL, 100 µg/mL, 200 µg/mL of GsZnO-Nps, AR Grade ZnO and same for standard drugs used for the ZOI study against both Gram-positive (S. aureus) and Gram-negative (E. coli) pathogens which is shown in Fig. 9 (A and B). There is no inhibition zone observed in AR Grade ZnO. However, the higher concentration of GsZnO-Nps (200 µg/mL) have shown the average ZOI 26.75 mm against (E. coli) and 30.00 mm against (S. aureus). Standard drug (antibiotics) Doxycycline show the inhibition zone 24.0 mm and 22.5 mm against (E. coli) and 30.00 mm against (S. aureus), Azithromycin shows the strong inhibition zone against both (E. coli) and (S. aureus) respectively 31.00 mm and 30.00 mm as well as Cefpodoxime shows the inhibition zone approximately 20.25 mm and 19.75 mm against both (E. coli) and (S. aureus). The inhibition zone of GsZnO-Nps is directionally higher than standard drugs Doxycycline, Cefpodoxime and at par to Azithromycin inhibition zone at 200 µg/mL which is tabulated in Table 3. The anti-bacterial efficacy of GsZnO-Nps is significantly higher that reported literatures possibly due to influence of phytochemicals, high active specific surface area available for the interaction with microorganisms per unit mass, release of zinc ions, interaction of particles to cell membrane and generation of reactive oxygen species (Gupta et al., 2018).

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Fig. 9

Anti-bacterial efficacy of AR Grade ZnO, GsZnO-Nps, vs standard drugs antibiotics (Doxycycline, Azithromycin and Cefpodoxime) against both Gram-positive and Gram-negative organism (A) S. aureus and (B) E. coli.

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3.8 Minimum inhibitory concentration

The MIC of GsZnO-Nps and AR Grade ZnO was determined against E. coli and S. aureus bacteria. MIC of GsZnO-Nps was found to be 500 µg/mL which is significantly lesser as to AR Grade ZnO (>900 µg/mL) shown in Table 4 which is in line with previous published literature (Ansari et al., 2012). Both, the inhibition zone and the MIC allow a prediction of therapeutic success. Concentration of GsZnO-Nps can be vary in both ZOI & MIC because while the MIC is a direct measurement of a concentration needed at the site of growth for bactericidal effect and the inhibition zone is that a given sample inhibits in whatsoever way the growth of the target bacterium and express bacteriostatic property (Ansari et al., 2012). Number of factors are responsible for anti-bacterial activity like reactive oxygen species (ROS), release of Zn²⁺ and direct contact of ZnO nanoparticles.

3.9 Possible mechanisms of anti-bacterial efficacy

Possible anti-bacterial mechanisms have been reported in the previous literatures such as: direct interaction of zinc oxide molecule with the bacterial cell wall and subsequently destructing the integrity of the cell (Brayner et al., 2006), release of Zn²⁺ ions (Cao et al., 2020), and generation of reactive oxygen species (ROS) (Lipovsky et al., 2011).

ROS are released due to photo-generated electrons and holes migrate to the active sites present at the surface of the nanomaterial. The holes being highly oxidizing will either oxidize the cell membrane directly or by forming strong hydroxyl radicals OH∙ is an extremely powerful oxidizing agent that causes a partial or complete breakage of cell walls (Slavin et al., 2017).

It has also been reported that nanoparticle can release Zn²⁺ ions which potentially play a role in their anti-bacterial activities. Consequently, ion pathways and the dissociation of the plasma membrane allow ions to enter microbial cells (Song et al., 2010). Anaerobic metabolism and charge transfer are inhibited, electrical activity and permeation are disturbed, and cell damage occurs as a result of Zn²⁺ ions reacting with thiol compounds of metabolic and transport proteins and lipids in the membrane. The Zn²⁺ shows the momentous effect in enzyme disruption, active transport inhibition, and amino acid metabolism (Aydin Sevinç and Hanley, 2010).

Direct interaction of ZnO due to the surface defect phenomenon and surface charge of ZnO nanoparticles play vital role for anti-bacterial mechanism. ZnO nanoparticles electrostatically interact and causing severe damage to the microbial cell wall. The surface morphology having rough and sharp topography leads to cell membrane damage (Schmidt-Mende and MacManus-Driscoll, 2007; Padmavathy and Vijayaraghavan, 2008). Furthermore, phytochemicals around GsZnO-Nps may also playing role to special features that impact bacterial cell walls and disrupt the structure of the cell membranes, resulting in lowered membrane potential and adenosine triphosphate generation. Phytochemicals produce changes in barrier function, metal ion chelate, and interruption in the function of membrane bound proteins, all of which alter the bacterial biological response and lead to death (AlSheikh et al., 2020). Phenolic compounds have been shown to alter membrane structure and trigger the release of cell organelles, resulting in cell triphosphate deficiency (Gill and Holley, 2004).