In the present study, mehendi extract ( Lawsonia inermis ) was used for phytosynthesis of ZnO nanoparticles using 0.1 M Zn(NO 3 ) 2 as precursor under alkaline condition using NaOH with vigorous stirring for 2 h. ZnO NPs obtained were characterized by UV-Vis spectroscopy, XRD, SEM and TEM that showed change in shape and size. Hexagonal particles were formed due to plant extract relative to the rod shaped particles in absence of plant extract. Further the antibacterial property of ZnO NP synthesized by green method was more effective than those synthesized in absence of plant extract. The antibacterial activity study of both the synthesized ZnO nanoparticles reveals that the nanoparticles synthesized using mehendi extract are more effective than the particle synthesized without mehendi extract. Thus , the use of leaf extract as capping agent would improve the antibacterial property of ZnO nanoparticle. However, bacteriocidal effect of these nanoparticles varies with respect to the organism tested .
Nanotechnology is the art and science of manipulating matter at the nanoscale to create new and unique materials and products with enormous potential to change society [
Green synthesis or phytosynthesis of nanoparticle is an eco-friendly approach which is in common practice [
In our experiment the leaves of Lawsonia inermis were collected from Karimganj, Assam, India washed properly and was allowed to dry at room temperature. The plant was authenticated by the subject expert Dr. Partha Sarathi Das, Department of Botany and Biotechnology, Karimganj College Karimganj, Assam, India and confirmed by comparing the same with existing herbarium of the Department of Botany and Biotechnology, Karimganj College Karimganj, Assam, India. The dry leaf powder was obtained using grinder and used to prepare the plant extract by mixing 1 g of the leaf powder in 10ml of distilled water and heated in a water bath at 100˚C for 10 min. The solution was then filtered using Whatman No.1 filter paper and the filtrate was stored at 4˚C and used for ZnO NP synthesis. Different concentrations of the resulting filtrate (0%, 2.5%, 5%, 10%, 15%, 20%) were used as a reducing agent for ZnO NP synthesis and 5% plant extract was selected based UV-Vis spectroscopic studies.
In this method we have dissolved 2.97 g (0.1 M) of Zn(NO3)2 salt in 90.7 ml of double distilled water which was then titrated drop wise along the wall of the conical flask with 9.3 ml of 2 M NaOH for 2 h under vigorous stirring at room temperature to obtain 100 ml final volume after titration. After 2 h of vigorous stirring the solution was filtered through a filter paper and the residue in the form of Zn(OH)2 obtained was washed properly using double distilled water. The residue was then dried at 70˚C in a hot air oven for 48 h to obtain fine ZnO NP powder. In the ZnO NP-II synthesis the above solution for ZnO NP-I was titrated with 5% plant extract along with NaOH drop wise and then filtered, washed and dried using the aforesaid method for ZnO NP-I.
The above synthesized ZnO NP was characterized using UV-Vis spectroscopy, SEM, TEM and XRD. The morphology was investigated using field emission scanning electron microscopy. For FESEM alcoholic dispersion of synthesized ZnO NP was put on a properly cleaned glass slide followed by spin coating. Uv-Vis spectroscopy of the sample was done using Biospectrometer (Eppendorf) in which 10 mg of ZnO NP was resuspended in 15 ml distilled water and sonicated for 10 min after which the sample was scanned from wavelength 300 - 700 nm.
X-Ray diffraction of the sample powder was carried out in a PANalytical X-PERT PRO applying a monochromator to select the Kα1 component of the employed copper radiation (Wavelength of 1.54056 Å). The data have been collected in the range 20 - 80 with a step size of 5˚. The XRD data has been depicted in
D h k l = K λ / β p cos θ
where Dhkl is the average grain size, K the shape factor (here, 0.9), λ is the x-ray wavelength, βp is the full width at half maximum (FWHM) intensity (here, 101 and 002 peak of the ZnO nanoparticle ZnO NP-I and ZnO NP-II spectrum fitted with Gaussian for precision measurement and θ is the Bragg’s angle.
For TEM analysis, powder samples were dispersed in triple distilled water using sonication, and a drop of the dispersion was placed onto a carbon coated copper grid and dried at room temperature. Further selected area electron diffraction (SAED) patterns were recorded to determine the growth orientation of the synthesized ZnO and was put into a uniform carbon coated copper TEM grid and dried in vacuum.
The antimicrobial activity of both the synthesized ZnO nanoparticles was performed and their antimicrobial efficacy was compared against four bacterial strains (Escherichia Coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis). The lyophilized cultured of the four bacterial strains were revived in conical flasks containing 20 ml of nutrient broth with 5 ml of inoculum and incubated at 37˚C. From this revived bacterial culture 100 ul of inoculum were added to 20 ml of LB broth media followed by the addition of synthesized ZnO nanoparticle dispersion of concentration of 0 µg/ml, 100 µg/ml, 200 µg/ml and 500 µg/ml with respect to the total volume of 20 ml (
culture were incubated at 37˚C with gentle stirring. The growth of bacterial strains was observed in liquid medium by measuring absorbance at 600 nm against control using UV-VIS spectrophotometer [
Each experiment was repeated thrice and data presented are mean ± SE (n = 3) and least significance difference (LSD) test was used for comparison between pairs of treatments. The data analysis was carried out using a statistical package, SPSS v. 10 (SPSS Inc., Chicago, IL, USA).
ZnO nanoparticles were synthesized by using leaf extract of Lawsonia inermis extract as detailed above. The size and morphology of the nanoparticles was characterized by using various techniques. The UV-Vis spectra for ZnO NP-I and ZnO NP-II are shown in
The SEM morphology images of the ZnO NPs as synthesized without plant extract (NP-I) and with plant extract (NP-II) are shown in
The TEM micro graphs also confirm the synthesized particles to be in the same range as predicted XRD and SEM. It also shows formation of hexagonal structures with average particle size decreased from 100 nm for ZnO NP I to 75 nm in ZnO NP II. The results are in good agreement with that of XRD and SEM analysis.
Structural analysis of the sample was made by X-ray Diffraction studies with Cu Kα radiation (λ = 1.5418 Å) as X-ray source at 40 kV and 30 mA in the scanning angle (2θ) from 20˚ to 80˚. The resulted XRD pattern was analysed using
X’Pert High Score software by search match tool, it was found to match with the ICDD Reference Pattern of Zinc Oxide, 01-070-8072.
As depicted in
In case of E. coli the growth of bacterial populations has decreased significantly at 100 µg/ml and 200 µg/ml ZnO nanoparticle relative to the control. Absorbency
readings taken on 1st, 2nd, 3rd, 4th, 5th, 6th and 24th hours suggest that 200 µg/ml of both ZnO NP-I and ZnO NP-II nanoparticles show optimum antibacterial activity against E. coli. However when the antibacterial efficacy of ZnO NP-I and ZnO NP-II is compared readings suggest that ZnO NP-II has better antibacterial activity than ZnO NP-I compared to the control (
ingredients that ligated to ZnO nanoparticles (Zno NP-II). Guo et al. [
The antibacterial activity of ZnO NP-I and ZnO NP-II against P. aeruginosa is appreciable at 100 µg/ml, 200 µg/ml and 500 µg/ml concentration compared to control with 100 µg/ml and 200 µg/ml showing very good antibacterial effect relative to control on 1st, 2nd, 3rd, 4th, 5th and 6th hour (
inhibition which supports the present findings. At 24th hour the high absorbancy of the bacterial culture may be due to the formation of cell debris and toxic substances that increased the turbidity of the bacterial culture media. ZnO NP-II synthesized using Lawsonia inermis extract has enhanced antibacterial efficiency than ZnO NP-I which is due to the synergistic antibacterial effect of both heaxagonal wurtzite structure ZnO NP-II nanoparticles (as suggested by TEM images) that resulted in enhanced surface bioactivity and Lawsonia inermis derived bioactive compounds that ligated to the ZnO NP-II nanoparticles. Similar to our findings Guo et al., [
The present study of antibacterial activity of Zno NP-I and ZnO NP-II against Staphylococcus aureus suggest that both ZnO NP-I and ZnO NP-II have a little retarded antibacterial effect against S. aureus compared to other three bacteria in the present study (
Our study suggests that both ZnO NP-I and ZnO NP-II showed significant anti-Bacillus subtilis effect with 100 µg/ml and 200 µg/ml showing optimum antibacterial activity on 1st, 2nd, 3rd, 4th, 5th and 6th hour (
et al., [
ZnO NPs have potential antibacterial property that may be attributed to its various structural and functional properties. The antibacterial activity study of both the synthesized ZnO nanoparticles reveals that the nanoparticles synthesized using mehendi extractare are more effective than the particle synthesized without mehendi extract. Thus, the use of leaf extract as capping agent would improve the antibacterial property of ZnO nanoparticle. However, bacteriocidal effect of these nanoparticles varies with respect to the organism tested. Detail understanding of the molecular mechanism of the synthesized nanoparticles may help to modulate their antibacterial potential for the benefit of mankind.
The authors thankfully acknowledged the financial support from the DBT, Govt of India, under RGYI, Scheme (SAN No. 102/IFD/SAN/1716/2013-2014) and for the supply of rice seeds by Regional Agricultural Research Station (RARS), Akbarpur, Karimganj throughout the experiment. The authors are thankful to the Director, Institute of Advanced Study in Science and Technology (IASST), Guwahati 781035, Assam, India for allowing SEM and X-ray difraction analysis of ZnO NP at central instrumentation facility (CIF) of IASST.
Upadhyaya, H., Shome, S., Sarma, R., Tewari, S., Bhattacharya, M.K. and Panda, S.K. (2018) Green Synthesis, Characterization and Antibacterial Activity of ZnO Nanoparticles. American Journal of Plant Sciences, 9, 1279-1291. https://doi.org/10.4236/ajps.2018.96094