Research On An Eco-Friendly Synthesis Of ZnO NPs Using Pure Bioflavonoid Rutin

Introduction

Nanobiotechnology is an emerging as a fast developing research area with various applications in biomedical and pharmaceutical industries. Recently, metal oxide nanoparticles (MO NPs) of zinc, copper, ion, and cerium oxide has been focus of interest due to their unique physical, chemical, and biological properties. Among these MO NPs, Zinc oxide NPs (ZnO NPs) has often preferred due to their nontoxic and are applied in various fields. Synthesis of ZnO NPs has been carried out by different methods such as precipitation, sol-gel method, microwave assist method, sonochemical synthesis, thermal decomposition, hydrothermal synthesis, and electrochemical method. All of these chemical and physical methods involve the use of toxic chemicals, which leads to harmful environmental problems. Recently, green chemistry method for the synthesis of ZnO NPs, especially using phyto compounds has been gained significant importance due to being simple, low cost and environmental friendly. Multiple human diseases caused by Gram-positive and Gram-negative bacteria become resistant to commercial antibiotics, phytocompounds and also natural traditional medicines. The development of bacterial resistance to commercial 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 (ZnO NPs) show the impact of eradicating the bacterial pathogens with low cost and also eco-friendly manner. In a recent study, Ali et al. , (2018) reported that the synthesis of ZnO NPs from phytocompounds of Conyza canadensis showed significant antibacterial activity against bacterial pathogens.

The bioflavonoid Rutin is a polyphenolic compounds found in many medical plants and vegetables. It has potential bioactive properties such as strong antioxidant agents of the plant origin, cytotoxic and anti-proliferative agents. It prevents the proliferation of human lung, and colon carcinoma cells. However, the bacterial pathogen killing ability of rutin and synthesis of MO 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 have demonstrated the synthesis, characterization and antibacterial activity of ZnONPs. In addition, antioxidant properties of the ZnONPs were assayed using DPPH and H2O2 methods.

Materials and methods

Chemicals and reagents

Zinc sulphate, rutin, Muller hinton agar (MHA), 2, 2-diphenyl-1-picrylhydrazyl (DPPH), 3-[4, 5-96 dimethylthiazole-2-yl]-2, 5-diphenyltetrazolium bromide (MTT), Dulbecco’s Modified Eagle Medium (DMEM) was obtained from Himedia Laboratories. The experiments in this study were done using sterile distilled water.

Synthesis of Zinc Oxide nanoparticles

About 20 mL of aqueous rutin (0. 2 mM) was added into 20 mL of zinc sulphate (o. 5 M) solution in a 100 mL conical flask and the reaction mixture was subjected to continuous stirring with magnetic stirrer maintaining at 60 ºC for 20 min. The solutions pH was adjusted to 12 using NaOH (2 M) solution. The resultant precipitate was purified by washing with distilled water three times to remove loosely connected rutin molecules from the ZnO surface and purified NPs were dried at 80 ºC for 24h.

Characterization techniques

The synthesis of ZnO NPs was monitored by UV-Visible spectroscopy (JASCO-V-670). UV-Vis spectral analysis was studied in the range from 300 to 800 nm. X-ray diffraction spectroscopic (XRD) analysis was carried out for the determination of the crystalline structure of the ZnO NPs (XPERT-PRO using 40 kV/40 mA current with Cu-Kα radiation). The morphology of the ZnO NPs was studied by electron microscopy (FE-SEM, Sigma-Carl Zeiss). The presences of elements in ZnO NPs were identified and mapped using energy dispersive X-ray spectroscopy (EDX) attached with FE-SEM. Fourier transform infrared spectra of the samples were recorded on FTIR spectrometer from 500 – 4000 cm-1 at a resolution of 4 cm-1. The surface charge on ZnO NPs was determined by zeta potential measurement using the zeta sizer (Malvern Instruments Ltd, Malvern, UK).

Antibacterial activity of ZnO NPs

Totally, four pathogenic bacterial strains namely Staphylococcus aureus (MTCC 3160), Proteus vulgaris (MTCC 1771), Klebsiella pneumoniae (MTCC 530), and Escherichia coli (443) were used and maintained in nutrient agar slants at 4 ºC. The antibacterial activity of the ZnONPs was tested against the selected bacterial strains through agar disc diffusion method described by Saravanakumar et al. , (2018). In brief, each bacterial strain was swabbed (105 CFU/mL) on the muller hinton agar petri plates using sterile cotton swabs. Sterile discs of 5 mm diameter were soaked with different concentration of ZnO NPs (10, 20 and 40 µg/mL), rutin (20 µg/mL) and control streptomycin sulphate (10 µg/mL). The discs were carefully placed on swabbed plates. After incubation for 24 h at 37 ˚C, the zone of inhibition was measured.

Invitro antioxidant activity

Antioxidant activity of the ZnO NPs and aqueous rutin was estimated by DPPH and hydrogen peroxide (Yadav et al. , 2015) methods. The DPPH and H2O2 activity of ZnO NPs was expressed as percentage of activity and the percentage of activity was calculated using the following equation: Antioxidant activity (%) = [(I0 – I1)/I0] × 100. Where I0 was the absorbance of the control, and I1 was the absorbance of the samples.

Results and discussion

Synthesis of ZnO NPs

The synthesis of ZnO NPs was carried out using pure bioflavonoid rutin. The synthesis of ZnO NPs was confirmed by the color change from light yellow to white color precipitate. In agreement with our results, Plectranthus amboinicus, Ruta graveolens and Jacaranda mimosifolia plant extract assisted synthesized ZnO NPs formed white color precipitate. The change in color may due to the process surface plasmon resonance (SPR) in the reaction samples. The functional groups and molecules presence in bioflavonoid rutin may responsible for the reduction of zinc salt to ZnO NPs. This was further confirmed by FTIR studies.

Characterization techniques

UV-Vis spectroscopy

Initially, the synthesis of ZnO NPs was monitored using UV-Vis spectrophotometer. The synthesized ZnO NPs showed the highest spectral absorbance peak at 355 nm. It was reported earlier that the spectral absorbance around 355 nm is a characteristic feature of ZnO NPs. Similar findings have been reported by Lingaraju et al. , (2016) who documented that the SPR absorbance spectrum of ZnO NPs was around 355 nm.

XRD analysis

XRD analysis can provide information about the crystalline structure of the ZnO NPs. The crystallite size of 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 calculated to be 38 nm from all the breath of the refraction. The strong and highest peak obtained at (101) suggested that the polycrystalline structure of synthesized NPs.

Electron microscopy and EDX mapping analysis

The morphology of the ZnO NPs was determined by FE-SEM analysis and has shown in Fig. 3 (a-d). ZnO NPs were found to have rod shape and an average size range of 20-130 nm. Similar to our study, morphology of the biologically synthesized ZnO NPs were exhibited rod shape with varied size range between 80-130 nm.

FTIR analysis

In ZnO NPs FTIR spectrum, the highest FT-IR peaks at 3290 cm-1 attributed to O–H stretch and H bond vibrations of alcohols, and phenols. FTIR spectral bands at 2891, 1632, and 1114 cm-1 correspond to C-O stretch, –C=C– stretch, and C – N stretch possible of alkenes, amines, and phenols. The medium spectral peak at 951 cm-1 assigned to the presence of aliphatic amines and phenols. The other short peaks from 500 to 800 cm-1 were assigned to the presence of metal-oxygen. This spectroscopic study was confirmed the presence of large amount of phenols, amines and alkenes compounds. FTIR spectrum supports the presence of above mentioned compounds were derived from rutin bioflavonoid. Rutin is a polyphenolic compound containing large amount of phenolic acids. Presence of these rutin derived molecules may responsible for the reduction, capping, stabilization ZnO NPs.

Zeta potential measurement

The surface charge on ZnO NPs was determined by zeta sizer. Fig. 6 shows the negative charge value (-29. 9 mV) on the synthesized ZnO NPs. The negative potential value from zeta sizer supports the high stability and dispersity of ZnO NPs.

Antibacterial activity

The synthesized ZnO NPs showed significant activity against common bacterial pathogens. The highest antibacterial zone of inhibition was recorded in E. coli (24. 5±0. 30 mm) followed by S. aureus (23±0. 40), P. vulgaris (22. 5±0. 20), and K. pneumoniae (20±0. 30).

Rutin bioflavonoid does not exhibit any zone of inhibition in tested pathogens. Moreover, compared to control and rutin, synthesized ZnO NPs showed higher antibacterial activity. Similarly, ZnO NPs synthesized using Laurus nobilis leaf extract showed significant antibacterial activity against P. aeruginosa and S. aureus. The results of the antibacterial properties of NPs would differ based on the cell wall nature of Gram-negative and Gram-positive bacteria. In the present study, synthesized ZnO NPs showed higher bactericidal activity against Gram-negative bacteria (E. coli) compared to Gram-positive bacteria (S. aureus, P. vulgaris, and K. pneumoniae). Similar findings were reported by Bhuyan et al. , (2015). This is due to the permeable capability of ZnO NPs into the cell wall of Gram-positive and negative bacteria.

Invitro antioxidant activity

DPPH scavenging activity

The scavenging ability of ZnO NPs was determined using DPPH and H2O2 assay. In DPPH assay, it was observed that the radical scavenging ability of ZnO NPs and rutin was increased in a concentration dependent manner. The percentage of DPPH activity was increase when increasing the concentration of samples. Similar to our report, ZnO NPs synthesized from stem barks of Ruta graveolens exhibited concentration depended manner of DPPH antioxidant activity. In the present study, the antioxidant capability of synthesized ZnO NPs showed potentially similar activity to bioflavonoid rutin. The free radical scavenging ability of ZnO NPs might be due to the presence of phenols that have the ability to donate the H in their OH groups.

Hydrogen peroxide scavenging activity

The H2O2 antioxidant assay was performed to evaluate the free radical scavenging ability of the synthesized ZnO NPs. The H2O2 inhibition percentage of ZnO NPs was increased with increasing concentration of ZnO NPs. Polyphenols, alkanes and tannins present in the ZnO NPs may contribute to the scavenging effect.

Conclusion

The present study was an eco-friendly synthesis of ZnO NPs using pure bioflavonoid rutin. Our synthesis protocol follows a facile green chemistry method. To our knowledge, this is the first report on synthesis of ZnO NPs using pure single bioflavonoid rutin compound. Synthesized nanoparticles were exhibited rod shape with varied size of 20-130 nm. The bioflavonoid rutin assisted synthesized ZnO NPs displayed significant antibacterial activity against both Gram-positive and Gram-negative bacteria. In addition, synthesized NPs were showed potential antioxidant activity which was confirmed by DPPH and H2O2 assay. Thus, we believe that the synthesized ZnO NPs in this study might be used as strong antibacterial and potential antioxidant agents in biomedical and pharmaceutical industries.