Eco-Friendly Green Synthesis Of Zinc Oxide Nanoparticles (ZnONPs) Using Bio-Flavonoid Rutin

The present study reports an eco-friendly green synthesis of Zinc oxide nanoparticles (ZnONPs) using bio-flavonoid rutin. The synthesized ZnONPs were characterized by UV-Visible spectroscopy, XRD, FE-SEM, EDX, and DLS analyses. FE-SEM image showed that the synthesized ZnONPs were rod in shape. The surface charge of the synthesized ZnONPs was analyzed by Zeta sizer. The green synthesized ZnONPs exhibited significant antibacterial activity against both Gram-positive (S. aureus, B. subtilis, S. pneumoniae, and S. pyogenes) and Gram-negative (P. aeruginosa, A. hydrophila, P. vulgaris, K. pneumoniae, and E. coli) clinical bacterial pathogens by agar disk diffusion method.

Introduction

Multiple human diseases caused by Gram-positive and Gram-negative bacteria become resistant to commercial antibiotics and also natural traditional medicine. The development of bacterial resistance to antibiotics has grown to be a major issue to bio-pharma industries and human health. So, there is an urgent need for the development of new biocidal agents against multi-drug resistant bacterial pathogens. In recent times, zinc oxide nanoparticles (ZnONPs) show the impact of eradicating the bacterial pathogens with low cost and also eco-friendly manner.

Synthesis of ZnONPs has been carried out by different methods such as precipitation, sol-gel method, microwave assist method, ultrasonic synthesis, thermal decomposition, hydrothermal synthesis, and electrochemical method. All of these methods involve the use of toxic chemicals, which leads to harmful environmental problems. Recently, green chemistry method for the synthesis of ZnONPs, especially using plants has gained significant importance due to being simple, low cost and environmentally friendly. Synthesis of ZnONPs using medicinally valuable plants and single bio-compounds received importance due to their potential bioactive properties such as strong antibacterial and anti-proliferative agents.

Rutin is a natural bio-flavonoid and has potential bioactive properties such as strong antioxidant agents of the plant origin, cytotoxic and anti-proliferative agents and more. It prevents the proliferation of human lung, and colon carcinoma cells. However, the pathogen bacterial killing ability of rutin and synthesis of nanoparticles (NPs) using this commercially and biologically valuable flavonoid has gained less attention. Hence, the present study aimed to synthesis ZnONPs using pure bio-flavonoid rutin compound for the first time. Here, we demonstrated the synthesis and characterization of ZnONPs. In addition, antibacterial activity of synthesized ZnONPs was tested against both Gram-positive and Gram-negative clinical human bacterial pathogens.

Materials and methods

Chemicals and reagents

Zinc sulphate (ZnSO4.7H2O2), rutin (yellow powder), Muller Hinton Agar (MHA) were obtained from Sigma Aldrich. All other reagents and chemicals used in this study were of analytical grade.

Synthesis of Zinc Oxide nanoparticles

For the synthesis process, about 20 mL aqueous rutin (0.2mM) was added into 20 mL Zinc sulphate (o.5M) solution in a 100 mL conical flask and the mixture solution was subjected to continuous stirring with magnetic stirrer maintaining at 60 ºC for 10 min. The mixture solution pH was adjusted to 11 using NaOH (0.2M). The resultant suspension was washed with sterile distilled water and ethanol to remove loosely connected rutin molecules and then dried at 80 ºC for 24h.

Characterization techniques

The formation of ZnONPs was monitored by UV-Visible spectroscopy (JASCO-V-670). UV-Vis spectrum analysis was studied in the range from 300-800 nm. X-ray diffraction spectroscopy (XRD) analyses were carried out for the determination of the crystalline phase of synthesized ZnONPs (XPERT-PRO using 40 kV/40 mA current with Cu-Kα radiation). The surface morphology of the synthesized ZnONPs was observed by field emission scanning electron microscopy (FE-SEM, Sigma-Carl Zeiss). Presences of elements in the synthesized ZnONPs were identified and mapped using energy dispersive X-ray spectroscopy (EDX) attached with FE-SEM. The hydrodynamic size and surface charge of synthesized ZnONPs were determined by Dynamic light scattering spectroscopy (DLS-Malvern Instruments Ltd, Malvern, UK).

Antibacterial activity against clinical human pathogens

Clinical bacterial pathogen source

Most common clinical human pathogenic bacterial strains such as Gram-positive (Staphylococcus aureus, Bacillus subtilis, Streptococcus pneumoniae, and Streptococcus pyogenes) and Gram-negative pathogens (Pseudomonas aeruginosa, Aeromonas hydrophila, Proteus vulgaris, Klebsiella pneumoniae, and Escherichia coli) were obtained from PSGIMSR, Coimbatore and maintained in nutrient agar slants at 4˚C.

Agar disk diffusion - antibacterial assay

The antibacterial activity of ZnONPs was carried out by agar disk diffusion method against human bacterial pathogens. Muller Hinton Agar plates were prepared, sterilized and solidified. Each bacterial strain of human pathogenic bacteria (103 CFU/mL) was swabbed uniformly on the solidified petri plates using cotton swabs. Different concentration of ZnO-NPs (10, 20 and 40 µg/mL), rutin (20 µg/mL) and control streptomycin sulphate (10 µg/mL) separately loaded disks were carefully placed on swabbed plates. After incubation for 24 h at 37 ˚C, the plates were examined for the zone of inhibition.

Results and discussion

Synthesis of ZnONPs

The synthesis of ZnONPs was confirmed by the color change from yellow to white color suspension. The change in color may due to the process surface plasmon resonance in ZnO reaction solution. Similar results were observed by Chaudhuri et al., (2017). Presence of functional group in bio-flavonoid rutin may responsible for the reduction, capping, and synthesis of ZnONPs.

Characterization techniques

Initially, the synthesis of ZnONPs was monitored using UV-vis spectroscopy and the highest shift in spectral peak was observed at 355 nm (Fig. 1). It was reported earlier that the spectral absorbance around 355 nm is a characteristic feature of ZnONPs and the obtained results were correlated with previous reports.

XRD analysis can provide information about the crystalline structure of the ZnONPs. Fig.2 shows the XRD pattern of the green synthesized ZnONPs and the obtained XRD planes [(100), (002), (101), (102), (110), (103), (200), (112), (201), (004) and (002)] were indexed to a hexagonal phase with wurtzite structure of ZnO, which also matched with JCPDS card no. 36-145. The mean average crystallite size of ZnONPs was calculated using Debye-Scherrer’s formula: D = 0.9λ / β cos θ, Where “λ” is the wavelength of X-ray, β is FWHM in radians and θ diffraction angles. The average mean crystallite size was calculated to be 28 nm from all the breath of the refraction.

The morphology of ZnONPs was determined by FE-SEM. The morphology of green synthesized ZnONPs was the hexagonal rod structure with the average size of 30-150 nm (Fig. 3a-b). Similar to our study, the morphology of biologically synthesized ZnONPs exhibited rod shape with the varied size range between 80-130 nm. EDX spectrum and elemental mapping analysis from Fig. 3(c-g) represent, that signal from Zn together with O, which clearly shows the formation of ZnONPs and ZnO is the main element in synthesized nanoparticles. The hydrodynamic histogram of DLS showed that Z average size diameter of 182 nm (Fig. 4a). This is quite larger than the size reported by SEM and the difference mainly due to the method involved in sample preparation for DLS studies. The surface charge of the synthesized ZnONPs was determined by zeta sizer and it shows a negative charge (-29.9 mV) of synthesized ZnONPs (Fig. 4b). The negative potential value from zeta sizer results supports the high stability and dispersity of ZnONPs.

Antibacterial activity

The synthesized ZnONPs showed significant activity against clinically isolated most common bacterial pathogens. The disk diffusion bacterial killing efficacy of different concentration of ZnONPs (10, 20 and 40 µg/mL) and rutin (20 µg/mL) and control (10 µg/mL) was shown in Fig. 5. The highest antibacterial zone of inhibition was recorded in A. hydrophilla followed by S. pneumoniae, P. aeruginosa, S.aureus, B. subtilis, P. vulgaris, K. pneumoniae, S. pyogenes and E.coli (Table 1). Moreover, compared with control and rutin, synthesized ZnONPs exhibited enhanced antibacterial activity. The results of the bactericidal activity would differ depending on the cell wall nature of Gram-negative and Gram-positive bacteria, the cell wall of Gram-positive bacteria is wider than the cell wall of Gram-negative bacteria.

Conclusions

In the present study, a bioactive nano-flavonoid approach was given. An eco-friendly synthesis of ZnONPs using bio-flavonoid rutin was demonstrated. Flavonoid rutin in this study played a significant role as capping and stabilizing agents. The synthesized ZnONPs showed significant antibacterial activity against selected nine human bacterial pathogens. Collectively, the obtained results suggest that the pathogen killing ability of pure bio-flavonoid rutin could be increased by converting them into metal oxide nano-flavonoids.