The Development Of New Biocidal Agents: An Eco-Friendly Preparation Of Chi-ZnO NPs Using Bio-Flavonoid Rutin

Abstract

The chitosan coated zinc oxide nanoparticles (Chi-ZnO NPs) were prepared for the first time by green chemistry approach using bioflavonoid rutin. The prepared Chi-ZnO NPs were characterized by UV-Visible spectroscopy, XRD, FE-SEM, EDX, and DLS analyses. FE-SEM image showed that the prepared Chi-ZnO NPs were rod in shape. Zeta sizer confirmed the positive surface charge of the prepared Chi-ZnO NPs.

The prepared Chi-ZnO NPs were 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, assayed using the agar disk diffusion assay. Furthermore, the prepared nanocomposites act as a potential photocatalyst for the degradation of methylene blue under sunlight irradiation. Collectively, our findings suggest that the prepared nanocomposites can be used as strong antibacterial agents and also a green photocatalyst.

Introduction

Multiple human diseases caused by Gram-positive and Gram-negative bacteria become very resistant to commercial antibiotics and also natural traditional medicines. 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 pathogenic bacterial strains with low cost and also eco-friendly manner.

There is various method have been reported to synthesis ZnONPs such as precipitation, sol-gel method, microwave assist method, hydrothermal synthesis, and electrochemical method. Most of these methods involve the use of toxic chemicals, which leads to dangerous environmental problems and also involve long synthesis procedure. Recently, green chemistry method for the synthesis of ZnONPs, especially using plants as a green source 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 significant bioactive properties such as strong antibacterial and anti-proliferative agents. In addition, ZnONPs used as strong photocatalytic agents for the removal of toxic and pollutant dyes from commercial industries due to their wide band gap energy. 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 killing ability of rutin and synthesis of NPs using this biologically and commercially valuable flavonoid has gained less attention. Hence, the present study aimed to preparation of chitosan coated ZnO NPs using pure bio-flavonoid rutin for the first time. Here, we have demonstrated the preparation, characterization and antibacterial activity of Chi-ZnO NPs. In addition, photocatalytic property of Chi- ZnO NPs was tested.

Chemicals and reagents

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

Synthesis of Zinc

Oxide nanoparticles (ZnO NPs) 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) aqueous solution. The resultant suspension was washed with sterile distilled water and ethanol to remove loosely connected rutin molecules and then dried at 60 ºC for 24h.

Preparation of Chitosan coated Zinc oxide nanoparticles (Chi-ZnO NPs)

About 100 mg of synthesized ZnO NPs were dissolved with 20 mL of chitosan solution (20 mg chitosan in 20 mL of acetic acid [0.1M]) by constant stirring at 60 ºC for 1 h. The obtained composite solution was centrifuged at 6000 rpm for 10 min to remove excess chitosan solution and then dried at 50 ºC for 24 h to get final product [sen].

Characterization of Chi-ZnO NPs

Primarily, the formation of Chi-ZnO NPs 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) analysis was carried out for the determination of the crystalline phase of prepared Chi-ZnO NPs (XPERT-PRO using 40 kV/40 mA current with Cu-Kα radiation). The surface morphology of the prepared Chi-ZnO NPs was determined by field emission scanning electron microscopy (FE-SEM, Sigma-Carl Zeiss).

Presences of elements in the prepared Chi-ZnO NPs were identified and mapped using energy dispersive X-ray spectroscopy (EDX) attached with FE-SEM. The chitosan and flavonoid molecules which are responsible for the capping and formation of Chi-ZnO NPs were identified by FT-IR (Nicolet iS5-FTIR). The size and surface charge of Chi-ZnO NPs were determined by DLS (DLS-Malvern Instruments Ltd, Malvern, UK).

Antibacterial activity of Chi-ZnO NPs:

Bacterial sources

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 hydrophil, Proteus vulgaris, Klebsiella pneumoniae, and Escherichia coli) were obtained from PSGIMSR, Coimbatore and maintained in nutrient agar (Himedia, Mumbai) slants at 4˚C.

Disk diffusion – antibacterial assay

The antibacterial activity of Chi-ZnO NPs was carried out by agar disk diffusion method against human bacterial pathogens. MHA plates were prepared, sterilized and solidified. Each bacterial strain of human pathogenic bacteria (103 CFU/mL) was swabbed uniformly on the solidified plates using cotton swabs. Different concentration of Chi-ZnO NPs (10, 20 and 40 µg/mL), rutin (20 µg/mL) and streptomycin (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. The antibacterial assay was carried out in triplicate.

Photocatalytic activity

The photocatalytic activity of Chi-ZnO NPs was assayed by evaluating the degradation of MB under sunlight irradiation as previously reported by Arunachalam et al. About 25 mg of prepared Chi-ZnO NPs were added to 100 mL of MB solution. Control was maintained without adding ZnONPs to the dye. Before to irradiation, the mixture was stirred for 30 min in dark conditions. After that, the suspension was subjected to sunlight irradiations (April – May 2018). At given time intervals, 5 mL of suspension was taken and centrifuged at 8000 rpm for 10 min, and the absorbance spectrum was measured using a UV-Vis spectroscopy. The degradation percentage was calculated from the equation, E (%) = (C0-Ct / C0) × 100 Where E is the degradation percentage, C0 is the absorbance before irradiation, and Ct is the absorbance at the different time (t).

Preparation of Chi-ZnO NPs

Primarily, the formation of Chi-ZnO NPs was confirmed by the color change from yellow to white color suspension. The change in color may due to the process of SPR (surface plasmon resonance) in ZnO and chitosan reaction medium. Similar to our study, Chitosan coated Zinc oxide nanoparticles capped with Poly vinyl alcohol (PVA) exhibited milky white color suspension.

Characterization techniques

The formation of Chi-ZnO NPs was monitored using UV-Vis spectroscopy and the highest shift in spectral peak was observed at 355 nm. It was reported earlier that the spectral absorbance around 355 nm is a characteristic feature of ZnO NPs and correlated with previous reports. AbdElhady et al.

XRD analysis of Chi-ZnO NPs

XRD analysis can provide information about the crystalline structure of the Chi-ZnO NPs. It shows the XRD pattern of the prepared Chi-ZnO NPs 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. 36-1451. Et al. reported that, XRD pattern of chitosan coated ZnO NPs exhibited wurtzite crystalline structure. The mean average crystallite size of Chi-ZnO NPs 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 estimated to be 32 nm from the breath of the refraction.

FE-SEM and EDX mapping analysis

The morphology of Chi-ZnO NPs was determined by FE-SEM. The morphology of the prepared Chi-ZnO NPs was rod shape with the average size of 30-150 nm. Similar to our study, the morphology of biologically prepared chitosan-neem seed coated ZnO NPs exhibited rod shape with the varied size range between 20 - 80 nm [revathi and thambi]. EDX spectrum and elemental mapping analysis was shown in. The EDX spectrum analysis confirmed the presence of Zn and O elements in the prepared Chi-ZnO NPs.

Fourier Transform Infrared spectroscopy analysis

FT-IR spectrum in Fig.5 indicates the presence of rutin and chitosan derived compounds which may responsible for the formation of Chi- ZnO NPs. The highest FT-IR peaks at 3389 and 2889 cm-1 attributed to O–H stretch, C–H stretch and H–bond vibrations of alkanes and phenols. FT-IR spectral bands at 1791, 1645, 1608, 1377 and 1301 cm-1 correspond to C=O stretch, –C=C– stretch, N–H bend, O-H stretch and C – H stretch possible of carboxylic acids, alkenes, amines, and alkanes vasa.

The sharp peak at 1014 cm-1 was assigned to the presence of aliphatic amines (C–N stretch) and the short peaks from 500 to 800 cm-1 were assigned to the presence of metal-oxygen (ZnO). These spectroscopic studies confirmed the presence of alkanes, phenols, carboxylic acids, amines and aliphatic amine compounds. Presence of these chitosan and rutin derived bio-compounds may responsible for the capping, reduction and formation of Chi-ZnO NPs.

DLS and Zeta potential analysis

The hydrodynamic histogram of DLS showed that Z average size diameter of 172 nm. This is moderately larger than the size observed by SEM and the difference mainly due to the process involved in NPs preparation for DLS studies. The surface charge of the prepared Chi-ZnO NPs was determined by zeta sizer and it shows a positive surface charge (+ 3.7 mV) of the prepared Chi-ZnO NPs. The positive values from zeta sizer results support the coating of chitosan on ZnO NPs. Wu et al reported that, Chi-ZnO NPs showed the positive zeta potential surface charge.

Antibacterial activity

The prepared Chi-ZnO NPs showed significant activity against clinically isolated most common bacterial pathogens. The disk diffusion antibacterial activity of different concentration of Chi-ZnO NPs (10, 20 and 40 µg/mL) and rutin (20 µg/mL) and control (10 µg/mL). 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. Moreover, compared with control and rutin, prepared Chi-ZnO NPs exhibited significantly enhanced antibacterial activity. The results of the bactericidal activity would differ depending on the cell wall nature of Gram-negative and positive bacteria, the cell wall of Gram-positive bacteria is wider than the cell wall of Gram-negative bacteria.

Photocatalytic activity of Chi-ZnO NPs

The catalytic activity of Chi-ZnO NPs was assayed for MB under sunlight irradiation. The absorbance spectra of dye were tested at the different time interval. The absorbance peaks decrease with the extension of the exposure of time. The prepared Chi-ZnO NPs exhibited significant catalytic activity against MB under sunlight irradiation. The Chi-ZnONPs catalysed 87% of dye degradation activity for MB cationic dye. Similar to our study, Chi-ZnO NPs showed time-dependent photocatalytic performance.

Conclusion

An eco-friendly preparation of Chi-ZnO NPs using bio-flavonoid rutin was demonstrated. Prepared Chi-ZnO NPs were exhibited rod-like shape with the size of 30-150 nm, determined by FE-SEM. The prepared Chi-ZnO NPs showed significant antibacterial activity against clinical pathogens. Furthermore, ZnONPs exhibited potential catalytic activity for methylene blue cationic dye. Collectively, obtained results suggest that synthesized ZnONPs can be used as a strong antibacterial agent and also a photocatalyst.