Accepted Manuscript Antibacterial behaviour of Vitex negundo extract assisted ZnO nanoparticles against pathogenic bacteria S. Ambika, M. Sundrarajan PII: DOI: Reference:
S1011-1344(15)00065-2 http://dx.doi.org/10.1016/j.jphotobiol.2015.02.020 JPB 9957
To appear in:
Journal of Photochemistry and Photobiology B: Biology
Received Date: Revised Date: Accepted Date:
19 November 2014 27 January 2015 17 February 2015
Please cite this article as: S. Ambika, M. Sundrarajan, Antibacterial behaviour of Vitex negundo extract assisted ZnO nanoparticles against pathogenic bacteria, Journal of Photochemistry and Photobiology B: Biology (2015), doi: http://dx.doi.org/10.1016/j.jphotobiol.2015.02.020
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Antibacterial behaviour of Vitex negundo extract assisted ZnO nanoparticles against pathogenic bacteria S. Ambika and M. Sundrarajan* Advanced Green Organic Chemistry Lab, Department of Industrial Chemistry, School of Chemical Sciences,
Alagappa University, Karaikudi-630003, Tamil Nadu, India. *Corresponding author: Tel/Fax: + 91 94444 96151 /+91-04565-225202, E-mail: ([email protected]
). Abstract The biological routes are advantageous over the chemical and physical ones as unlike. These, the biological synthesis protocol occurs at ambient conditions, are cheap, non-toxic and eco-friendly. This research describes the synthesis of zinc oxide nanoparticles (ZnO NPs) using Vitex negundo plant extract with zinc nitrate hexahydrate as precursor. Biomolecules present in plant extract can be used to hydrolyze metal ions into metal oxide NPs in a single-step green synthesis. The hydrolyzing agents involved the various water soluble plant metabolites such as flavonoid, alkaloids, flavone, phenolic compounds, terpenoids and co-enzymes. Presence of isoorientin (flavone) in Vitex negundo plant extract is mainly responsible for the formation of ZnO NPs. The prepared ZnO NPs were calcinated at 450°C and were confirmed by XRD, FTIR, UV-visible, SEM with EDX and DLS analysis. The biological application of antibacterial activity was done by gram positive and gram negative bacteria. Key words: Green synthesis, Sol-gel, Vitex negundo, Nanoparticles, Antibacterial activity.
1. INTRODUCTION Green chemistry consists of chemicals and chemical processes designed to reduce or eliminate negative environmental impacts. The use and production of these chemicals may involve reduced waste products, non-toxic components and improved efficiency. Green chemistry is a highly effective approach to pollution prevention because of it is applies innovative scientific solutions to real-world environmental situations. Use feedstock derived from annually renewable resources or from abundant waste. Chemicals are less hazardous to human health and the environment are less toxic to organisms, ecosystems and inherently safer with respect to handling and use. Synthesis of inorganic metal oxide NPs using biological things has been great interest due to their unusual optical, chemical, photoelectrochemical and electronic properties . Research into one-dimensional nanostructured materials with the size of 1–100 nm have riveted significant interest in recent years owing to their fascinating properties diverse from the bulk phase arising from quantum size effect . Since, the properties of NPs are size and shape dependent, the synthesis process having better control on monodispersity, size and shape is an important area of research . ZnO NPs are of unique material that exhibits semiconducting, piezoelectric and pyroelectric properties, it has versatile applications in transparent electronics, ultraviolet (UV) light emitters, chemical sensors, spin electronics, personal care products, catalyst, coating and paints [4,5]. In fact, ZnO is non-toxic and chemically stable under exposure to both high temperatures and UV . Several physical and chemical processes have been used for the synthesis of large quantities of metal NPs in a relatively short period of time. Approaches such
solvothermal/hydrothermal, electrochemical and photochemical reduction techniques are most 2
widely used. Chemical method leads to the presence of some toxic chemicals adsorbed on the surface that may have adverse effects in medical applications and environment . In order to minimize the toxicity, we have a need for the development of simple and eco-friendly methods for the synthesis of NPs  Biological systems have well organized and controlled physiological processes, which their use in the NPs synthesis is rapidly gaining importance. Several strains of microorganisms are known for having metal resistance capability. They are endowed with various cellular mechanisms for metal detoxification. The synthesis of nanosized metal is one such strategy/adaptive feature [9, 10]. Plant based microbial has enormous therapeutics as they can serve the purpose without any side effects that are often associated with synthetic compounds . The use of highly structured physical and biosynthetic activities of microbial cells has recently emerged as a novel approach for the synthesis of nanosized materials. Increasing awareness towards green chemistry and other biological processes has led to a desire to develop an eco-friendly approach for the synthesis of NPs. The use of environmentally benign materials like plant extract , bacteria [13,14], fungi  and enzymes  have been used for the synthesis of metal oxide NPs for the benefits of eco-friendliness and compatibility for pharmaceutical and other biomedical applications as they do not use toxic chemicals. Biosynthesis of NPs using plants such as alfalfa  Cinnamomum camphora  neem , emblica officinalis  lemon grass  and tamarind  have been reported, the potential of plants as biological materials for the synthesis of NPs is yet to be fully explored. Vitex negundo Linn. (VN) (Family: Verbenaceae) is commonly known as five leaved chaste tree. It occurs wild in most parts of India near moist places. Although all parts VN are used as medicine in the indigenous system, the leaves are the most potent for medicinal use . 3
The whole plant is used in anticancer, inflammations, antiseptic, antipyretic, diuretic, antihistamine, antioxidant, antibacterial, antifungal, snake venom neutralization, mosquito repellant
hepatoprotective, antifertility, skin aging inhibitor and anti-dopaminergic effects . In this paper, we adopt a green chemistry approach for the synthesis of ZnO NPs using Vitex negundo herbal plant extract extract. This method does not need any special experimental conditions like special surfactant or temperature and pressure control. To the best of our knowledge, it is the first report on ZnO NPs synthesis using Vitex negundo extract. Synthesized ZnO NPs were characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (HR-SEM) with EDX analysis and their antibacterial activity against gram positive Staphylococcus aureus (S. aureus) and gram negative bacteria Escherichia coli (E. coli) was examined. 2. MATERIAL AND METHODS 2.1 Materials Vitex negundo (Nochi) is an herbal plant, leaves were collected from our local agricultural land of Karaikudi, Tamil Nadu, India. Zinc nitrate hexahydrate was purchased from Merck Chemicals Ltd, India and used as without any further purification. Deionized water was used through the complete experiment. 2.2 Preparation of plant extract The collected Vitex negundo leaves were gently washed with pure water and dried in a room temperature, dust free condition for about 10 days. Dried leaves were crushed into finest
powder using mortar and pestle. 5g of crushed powder was mixed with 50 ml of ethanol for 2 h at 60°C using soxhlet apparatus. The resulting extract was filtered using a pure cotton cloth followed by further filtration with Whatmann No.1 filter paper. The filtrate was collected in a closed vessel and stored in refrigerator for further use. This filtrate can be used as the stabilizer and a hydrolyzing agent in the synthesis of ZnO NPs. 2.3 Synthesis of ZnO NPs The ZnO NPs were prepared by using a stock solution of [Zn (NO3)2. 6H2O] mixed with 25ml of leaves extract was slowly added under vigorous stirring for about 5 h. The precipitation of Zinc hydroxide was obtained under the Erlenmeyer flask. Then the solutions settle for 24 h before centrifugation. The particles were separated by centrifugation (KUBOTA-6800 Centrifuge machine) at 6000 rpm for about 15 min for removing the unwanted organic matters and after they were filtered with deionized water and dried in an oven at 80°C to get the asprepared sample. The prepared sample was calcinated at 450°C. Finally ZnO NPs was obtained. The diagrammatic representation of ZnO NPs synthesis is as shown in Scheme 1. The utilized VN extracts are having rich sources such as
chrysophenol, luteolin, p-hydroxybenzoic acid and D-fructose . Among the functional active ingredients, polyphenolic compounds such as isoorientin and luteolin etc., are present as major component with high proportion [26, 27]. These natural polyphenolic compounds are responsible for the formation of ZnO NPs. In our synthesis, aromatic hydroxyl groups present in Vitex negundo extracts isoorientin with zinc ions to form the zinc-isoorientin complex with acid medium . These complexes undergo direct decomposition at 450°C and leads to ZnO NPs. The possible mechanism of ZnO NPs using Vitex negundo extract is shown in Scheme 2 5
2.4 Characterization techniques X-ray diffraction (XRD) analysis was carried out on PANanlytical X-Pert PRO. Diffractometer operating at 40 kV with a current of 30 mA using Cu-Kα for the determination of purity and crystalline size of the NPs. Size and surface morphology of the ZnO NPs was examined by JEOL JSM 6390 Scanning Electron Microscope (SEM) instrument operated at an accelerating voltage at 15kV. For Energy Dispersive X-ray (EDX) analysis, the particles were dried on a carbon coated copper grid and performed on SEM instrument with thermo EDX attachment.
spectrophotometer. The powder sample of green synthesis ZnO NPs was analyzed by Fourier transform infrared spectroscopy (FT-IR) using a Shimadzu infrared spectrometer (Model 400) with KBr as background over the range of 400–4000 cm-1. 2.5 Antibacterial activity The antibacterial activity of ZnO NPs was determined by agar well diffusion methods (Kirby-Bauer) is a relatively swift and easily prompted the antibacterial activity. The ZnO NPs were tested against Staphylococcus aureus (gram positive) and Escherichia coli (gram negative) organisms. The suspension of bacteria was grown in nutrient broth medium. Test organisms were dispersed over the surface of agar plates. A small amount of sample is gently pushed over the nutrient agar plate inoculated with bacterial cells for intimate contact of the sample. The plates were incubated at 37°C for 18-24 h. The antibacterial activity of NPs was demonstrated by the diameter of the zone of inhibition developed around the sample. The zone of inhibition and minimum inhibitory concentration (MIC) were measured by subtracting the well diameter from the total inhibition zone diameter. A zone of inhibition is the area in which the bacterial growth
is stopped due to the bacteriostatic effect of the compound and it measures the inhibitory effect of compound towards a particular microorganism. 3. RESULTS AND DISCUSSION 3.1 XRD The purity, crystalinity, average particle size of ZnO NPs was confirmed by X-ray diffraction analysis technique. The XRD pattern of calcinated ZnO NPs is recorded in the range of 20-80°. The sharp peak indicates the purity and crystalinity of NPs in Fig. 1. The XRD peaks are consistent with the JCPDS data card 89-0510 hexagonal phase and primitive geometry of ZnO NPs. The detected peaks corresponded with those of hexagonal phase zincite were found at the lattice planes of (100), (002), (101), (102), (110), (103), (112) and (201) in the 2θ value: 31.68, 34.35, 36.17, 47.47, 56.52, 62.80, 67.88 and 69.01° respectively. The crystalline size of the most intense plane (101) was 38.17 nm, determined by employing Debye-Scherrer’s equation. Nanosized value of ZnO suggests that the Vitex negundo leaves extract can be employed as the hydrolytic and precipitating agent for the formation of ZnO NPs. 3.2 SEM with EDX analysis SEM image was used to study the surface morphology, size and shape of NPs. Fig. 2a and b shows the morphological SEM with EDX photograph of ZnO NPs. Image showed that the ZnO NPs has spherical in nature with the particle size approximately 75 to 80 nm. Fig. 2b exihibited the presence of zinc and oxygen. 3.3 FT-IR Spectroscopy FTIR analysis was performed to determine the functional groups in the synthesis of ZnO NPs. Fig.3 shows a broad prominent band at 3403.48 cm-1 are related to the –OH stretching 7
vibrations. The sharp peak 2357.70 indicates the C-H stretching mode. The peaks at 1595.42, 1424.02 and 1099 cm-1 represent the C=C and C-H symmetric vibration stretching vibration respectively. It is well known that the peak at 533 cm-1 is distinctive for Zn-O-Zn vibrations. It is apparent that the intensity of absorption in the region of 500 cm-1 characteristic of tetragonal ZnO vibration. This suggests that the biological molecules could possibly act as hydrolyzing agent for the metal oxide NPs. 3.4 UV–vis spectra of ZnO NPs In general, absorption edge characteristic of bulk ZnO particles is 400 nm with a band gap of 3.37 eV. Fig. 4 (a) and (b) shows the UV spectra Vitex negundo extract and ZnO NPs. Fig. 4(a) shows the peaks at 343 nm in Vitex negundo extract correspond to the ᴨ-ᴨ* transition of polyphenols in Vitex negundo extract. Fig. 4(b), the absorbance wavelength at 343 nm is shifted into 375 nm after reacting with zinc nitrate. The spectrum shows a typical excitation absorption band appeared at 375 nm that is blue shifted with respect to the bulk ZnO absorption edge which appears at 400 nm, this shift may be ascribed to the nanometric size effect of the synthesized ZnO and characteristic for the hexagonal ZnO NPs. The sharp and prominent absorption band may arise due to the transitions from valence band to conduction band and agrees with the reported literature for ZnO NPs . According to the literature, blue shift can be raised by the decrease of size and red shift by the increase of size. 3.5 DLS Spectra of ZnO NPs ZnO NPs were characterized using dynamic light scattering (DLS) analysis. The particle sizes starting from 10 to 100 nm with an average size 60 nm (Fig. 5). Relatively good symmetry of size distribution diagrams in DLS analysis illustrates uniformity of the formed NPs. The data 8
clearly show broadening of size distribution and increase of average particle size distribution [30, 31]. 3.6 Antibacterial activity Fig. 6 reveals the antibacterial activity of ZnO NPs against S.aureus (ATCC 11632) and E.coli (ATCC 10536) by agar well diffusion method. ZnO NPs suspension (100µl) was prepared and resuspended in sterile distilled water and briefly sonicated for uniform dispersion to form a colloidal suspension. A zone of inhibition values is expressed millimetre (mm) in diameter for the control and ZnO NPs. The significant growth inhibitory effect against S. aureus and E. Coli bacteria are due to their large surface area of ZnO NPs. There are several possible mechanisms for the antibacterial activity of ZnO NPs. Reactive oxygen species (ROS) generated from the ZnO NPs actively inhibits the growth of S.aureus and E.coli cells by accumulation or deposition on the surface of both cells, ZnO NPs bind with cellular surface and kill the bacteria through electrostatic forces  and zinc ion bind to the membranes of microorganisms, may distort and damage bacterial cell, resulting in a leakage of intracellular contents and death of bacterial cells . From the result it can be calculated the antibacterial activity may be due to the presence of ZnO NPs. Table 1 shows the antibacterial activity of ZnO NPs and control. Based on the results observed from the test and control groups, the prepared ZnO NPs using Vitex negundo by green synthetic method could achieve the same antibacterial activity for environment friendly way. The microbicidal activity of ZnO NPs was checked by the MIC (minimum inhibitory concentration) method. The inhibitory effect of ZnO NPs (100 µl) was examined through UV-vis spectrophotometer by taking optical density (OD) values. The OD at 600 nm is due to the scattering of light by the bacterial cells. It is a function of bacterial cell density and thus 9
correlates with the growth of the colonies. Fig. 7 shows, Inhibit on the growth of gram-positive as well as gram-negative bacteria. The bacterial growth reduction value is decreased for both gram-positive and gram-negative bacteria after the addition of ZnO NPs suspension (Table 2). The growth rate of biomass was compared with addition of ZnO NPs with biomass. The growth rate of E. coli and S.aureus was decreased at the adding of ZnO NPs . The results of this study may be applicable to medical devices that are coated with NPs against microbes like fungal and bacterial pathogen. 4. Conclusion A simple, green and inexpensive technique has been established to prepare nanocrystalline ZnO NPs using Vitex negundo leaves extract at room temperature. Here, we demonstrated the antibacterial activities of ZnO NPs against S. aureus and E. coli by determining the growth graph of ZnO NPs treated and untreated bacterial cell. Isoorientin is a type of flavone, it’s responsible for the ZnO NPs formation. In conclusion, this study showed that ZnO NPs have potent antibacterial activities against S. aureus and E. coli bacterial cells. The growth rate ZnO NPs treated bacterial cells were quickly inhibited. To the best of our knowledge, this is the first report about the green synthesis ZnO NPs using Vitex negundo leaves extract. Acknowledgements The authors greatly acknowledge the Department of Physics, Alagappa University for providing XRD (DST-FIST) facilities and Department of Industrial Chemistry, Alagappa University for providing SEM facilities. References  Perumal Dhandapani, Sundram Maruthamuthu, Gopalakrishnan Rajagopal, Bio-mediated
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Fig. 1. XRD patterns of ZnO NPs. Fig. 2. SEM image of (a) ZnO NPs (b) EDX spectrum of ZnO NPs. Fig. 3. FTIR spectra of ZnO NPs. Fig. 4. UV-visible spectra of (a) VN extract, (b) ZnO NPs. Fig. 5. DLS analysis of ZnO NPs. Fig. 6. Antibacterial activity of ZnO NPs against (a) S. aureus and (b) E. coli. Fig.7. Graphs showing change in absorbance over time (2 h) for nutrient broth shake flask test. Table Captions Table. 1. Antibacterial assessment by agar well diffusion method Table. 2. Result of nutrient broth shake flask test in terms of growth reduction
Scheme 1 Schematic representation of ZnO NPs synthesis and its antibacterial Application
Scheme 2 Possible mechanism of ZnO NPs formation using Vitex negundo extracts
100 002 110 103 102
Fig. 1. XRD pattern of ZnO NPs.
Fig. 2. SEM image of (a) ZnO NPs (b) EDX spectrum of ZnO NPs.
3780.78 1595.42 8863.6
1424.02 1099.69 2357.70 533.78
Fig. 3. FTIR spectra of ZnO NPs.
a 0.0 300
Fig. 4. UV-visible spectra of (a) VN extract, (b) ZnO NPs.
Table. 1. Antibacterial assessment by agar well diffusion method Sample
Zone of inhibition (mm)
Control without ZnO NPs
Table. 2. Result of nutrient broth shake flask test in terms of growth reduction Growth OD @ 600nm for NPs E.Coli Control
S.aureus ZnO treated
Zinc oxide nanoparticles originally synthesized by using Vitex negundo plant extract.
This kind of economically feasible and environmentally benign greener method, remarkably shortens time and avoids the complicated synthetic procedures.
Polyphenolic compounds of Vitex negundo extract has played a major role for the hydrolysis of metal salt precursors.
Antibacterial efficiency of prepared Zinc oxide nanoparticles was significant against test pathogens.