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nanotechnology

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A PROJECTREPORT
ON
Eclipta alba leaf extract: A green factory for the synthesis of multi-
applicative zinc nanoparticles
S...
DECLARATION
I do hereby declare that project report entitled “ECLIPTA ALBA LEAF
EXTRACT: A GREEN FACTORY FOR THE SYNTHESIS...
AMITY UNIVERSITY
UTTAR PRADESH
LUCKNOW CAMPUS
CERTIFICATE
Certified that Priti Pal (enrolment no.A7100213018) has carried ...
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  1. 1. A PROJECTREPORT ON Eclipta alba leaf extract: A green factory for the synthesis of multi- applicative zinc nanoparticles SUBMITTED TO AMITY INSTITUTE OF BIOTECHNOLOGY AMITY UNIVERSITY UTTAR PRADESH LUCKNOW SUBMITTED BY PRITI PAL M.Sc. Biotechnology A7100213018 (IV Semester) UNDER THE SUPERVISION OF Dr. Akhilesh Kumar Singh and Dr. Satarudra PrakashSingh Assistant Professor OF AMITY INSTITUTE OF BIOTECHNOLOGY AMITY UNIVERSITY UTTAR PRADESH LUCKNOW
  2. 2. DECLARATION I do hereby declare that project report entitled “ECLIPTA ALBA LEAF EXTRACT: A GREEN FACTORY FOR THE SYNTHESIS OF MULTI –APPLICATIVE ZINC NANOPARTICLES” undertaken by me is an authentic record of my own work carried out at Amity Institute Of Biotechnology, Lucknow as a requirement of Project for the award of degree of M.Sc. in Biotechnology, Amity University, Lucknow, under the guidance of Dr. Akhilesh Kumar Singh and Dr. Satarudra Prakash Singh Assistant Professor of Amity Institute of Biotechnology during 10thFeb, 2015 to 08thMay, 2015. DATE: PLACE: Lucknow Priti Pal M.Sc. Biotechnology Amity University
  3. 3. AMITY UNIVERSITY UTTAR PRADESH LUCKNOW CAMPUS CERTIFICATE Certified that Priti Pal (enrolment no.A7100213018) has carried out the research work presented in this dissertation entitled “Eclipta alba leaf extract: A green factory for the synthesis of multi-applicative zinc nanoparticles”, for the award of Master Of Science in Biotechnology, from Amity Institute of Biotechnology, Amity University Uttar Pradesh, Lucknow, under our joint supervision. The dissertation embodies results of original work and studies carried out by the student herself and the contents of the dissertation do not form the basis for the award of any other degree to the candidate or to anybody else from this or any other University/Institution Dr. Akhilesh Kumar Singh Dr.Satarudra Prakash Singh Assistant Professor Assistant Professor Amity Institute of Biotechnology Amity Institute of Biotechnology AMITY University Uttar Pradesh AMITY University Uttar Pradesh Lucknow campus Lucknow campus DATE:
  4. 4. AMITY UNIVERSITY UTTAR PRADESH LUCKNOW CAMPUS PLAGIARISM FREE CERTIFICATION This is to certify that internship project detailed below has been evaluated by online anti-plagiarism software. The contents and material was found satisfactory and within the permissible limit of content copied. Name of the Student- Priti Pal Enrollment Number- A7100213018 Programand Batch-M.Sc. BiotechIVth Sem, 2013-2015 Title of the Project-“ECLIPTAALBA LEAF EXTRACT:A GREEN FACTORYFOR THE SYNTHESIS OF MULTI –APPLICATIVE ZINC NANOPARTICLES” Results- Student Internal Faculty Supervisor Date:
  5. 5. FACULTY GUIDE APPROVAL CERTIFICATE I, hereby declare that the project entitled entitled “ECLIPTA ALBA LEAF EXTRACT: A GREEN FACTORY FOR THE SYNTHESIS OF MULTI –APPLICATIVE ZINC NANOPARTICLES” is a record of an original work done by Ms Priti Pal from 10-02-15 to 08-05-15 at Amity Institute of Biotechnology Amity University, Lucknow. This work has been carried out under the guidance of me, Dr. Akhilesh Kumar Singh and Dr. Satarudra Prakash Singh, Assistant Professor of Amity Institute of Biotechnology. This project has not been submitted elsewhere for any other degree. Dr. Akhilesh Kumar Singh Dr. Satarudra PrakashSingh
  6. 6. ACKNOWLEDGEMENT The successful completion of any project would be incomplete without thanking the people who made it possible. With all my submissiveness I thank God for his constant presence throughout my work. It takes great pleasure in expressing a few words of gratitude and respect to all those who helped me in completing this project work. I am highly grateful to Dr. J.K. SRIVASTAVA HOI, Amity Institute of Biotechnology, Amity University Uttar Pradesh, Lucknow for accepting my candidature and permitting me to undertake the dissertation work. It is my proud privilege and pleasure to bring my deepest gratitude to my supervisor Dr. Akhilesh Kumar Singh and Dr. Satarudra Prakash Singh Assistant Professor of Amity Institute of Biotechnology Lucknow for the constant supervision, meticulous guidance, his ever encouragement, moral support, constructive criticism and above all most sympathetic attitude throughout the period of investigation. I feel great pleasure in expressing my earnest and deep sense of gratitude to Dr. Aditi Singh, Abhishek Kumar, AIB for their invariable guidance, invaluable healthy instructions, constant supervision and keen interest throughout the course. With profound happiness, I want to pay my heartfelt regards and feelings of indebtedness towards the eternal love and fondness showered on me by my family. Date: Priti Pal M.Sc. Biotechnology Amity University
  7. 7. TABLE OF CONTENTS PARTICULARS PAGE No. Introduction 01-09 1.1 Background 01 1.2 Nanoparticles 02 1.3 Unique properties of nanoparticles 02-03 1.4 Zn nanoparticles and its current status 03-04 1.4.1 Viable applications of Zn nanoparticles 04-05 1.5 Synthesis of nanoparticles 05-08 1.5.1. Physical method 06 1.5.2. Chemical method 07 1.5.3. Biological method 08 1.6. Advantages of Zn nanoparticles synthesis by plants over microbes 09 Objective 10 Review of literature 11-17 2.1 Plant sample Eclipta Alba (Bhringraj) 18 2.1.2 Viable application of Bhringraj 19 Materials and Methods 20 3.5. Preparation of zinc nanoparticles 21-24 3.12 Antimicrobial activity 25 Results and discussion 26-35 Conclusions 36 Future Prospect 37 References 38-40
  8. 8. LIST OF FIGURER Figures No. Title Page No. 1 Outline of the various approaches synthesis of nanoparticles 06 2 Synthesis of nanoparticles by physical (top down) and chemical method (bottom up) 08 3 Plant Eclipta alba showing leaves and flower 18 4 UV-Vis Spectrophotometer 24 5 UV Cuvette 24 6 UV–Vis. absorption spectra of ZnNPs at different concentrations of zinc acetate 26 7 UV–Vis. absorption spectra of ZnNPs at various amount Eclipta alba leaf extract. 27 8 Effect of temperature in the nanoparticles synthesis using leaf extract of Eclipta alba. 28 9 pH dependence UV–Vis. absorption spectra of ZnNPs. 29 10 Time dependant UV–Vis. absorption spectra of ZnNPs 31 11 Antimicrobial activity of ZnNPs against (a) E. Coli.,(b) Staphylococcus aureus 33 12 significant change in colour after reaction 35 13 Colour change in 24 hours. 35 14 Colour change is several days. 35
  9. 9. ABBREVIATIONS Zn - Zinc ZnO - Zinc oxide ZnNPs - Zinc nanoparticles UV - Ultra violet XRD - X-ray diffraction SEM - Scanning Electron Microscope FT-IR - Fourier Transform Infrared Spectroscopy TEM - Transmission electron microscopy ROS - Reactive oxygen species PL - Photoluminescence spectroscopy RPM - Revolutions per minute. gm - Gram °C - Degree Celsius µl - Micro liter ml - Milli liter nm - Nano metre
  10. 10. ABSTRACT This research attempts to describe the multiplicity of the field, starting with the past of nanotechnology, the physics of the nanoparticle and diverse strategies of synthesis, the assorted advantages and disadvantages of different methods, the probable mechanistic aspects of nanoparticle synthesis and ultimately ends with the potential applications and expectations perspectives. Although there are a small numbers of good reviews dealing with the synthesis and applications of nanoparticles, there appears to be very little information concerning the possible mechanistic aspects of nanoparticle synthesis. There are several interesting examples in nature where nanostructures are present and have important functions. In recent years, there is much growing concern towards the eco-friendly production of nanoparticles because of their novelty that make them feasible for various potential applications in different areas of science and technology. The present study exploit the formation of zinc nanoparticles from zinc acetate using leave extract of Eclipta alba. These leaves extract were prepared by boiling leaves at 1000C for 20 minutes followed by filtration and then centrifuge the sample. The freshly prepared zinc acetate solution was then treated with respective leave extracts. The resulting reaction mixture was placed under continuous stirring at 600C utilizing magnetic stirrer for regular intervals, where a colour change was observed from dark brown to yellow, thereby indicating the synthesis of zinc nanoparticles. Furthermore, the preliminary characterization of zinc nanoparticles was accomplish by using UV-Vis spectroscopy in the range of 250- 500nm. In this respect, biological methods involving plant extracts are more effective. Zinc nanoparticles are identified to be one of the multifunctional non-living nanoparticles with efficient antibacterial activity. This study aims to determine the antimicrobial efficacy of green synthesized Zn nanoparticle against Escherichia coli and Staphylococcus aureus. Results confirm that green Zn nanoparticles show more improved biocide activity against these bacteria. Hence Zn nanoparticle is accountable for significant higher antimicrobial activity. Since the results obtained it is recommended that green Zn NPs could be used efficiently in agricultural and food safety applications and also can deal with potential medical concerns. Keywords: Nanotechnology, Zinc nanoparticles, green synthesis, UV-Vis spectroscopy, Eclipta alba.
  11. 11. INTRODUCTION 1.1 Background There are several interesting examples in nature where nanostructures are present and have important functions. In recent years, there is much growing concern towards the eco-friendly production of nanoparticles because of their novelty that make them feasible for various potential applications in different areas of science and technology (Borase et al. 2014). The outstanding progress of nanoscience and technology is the part and parcel of advancement in measurement systems, method and instruments. The recent trend of increased interest from bulk to nanotechnologies raises a number of new explicit problems due to the small dimensions and structures which needs to be addressed and explored in this area. Particularly, in nanotechnology the hypothesis applies: “If you can’t measure it accurately, you can’t construct it and reproduce it in number of times.” Nanotechnology may be the subsequently huge craze in science, and before that we will be possibly find ourselves immersed in it [1]. The idea of nanotechnology though appraise to be a recent science has its antiquity dating to as back as the 9th century. The artisans of Mesopotamia used gold and silver nanoparticles to produce an impressive effect to pots. The initial scientific explanation of the significance of nanoparticles was given in 1857 by Michael Faraday in his popular paper “Experimental relations of gold (other metals included) to light” (Faraday, 1857). In 1959, Richard Feynman a talked about explained molecular machines built with atomic accuracy. This was considered the initial talk on nanotechnology. This was entitled “There’s plenty of space at the bottom”. Nanotechnology can be termed as the synthesis, characterization, investigation and application of nanosized (1-100 nm) resources for the expansion of science [1, 2]. It deals with the resources whose structures reveal considerably new and enhanced physical, chemical, and biological properties, phenomena, and functionality appropriate to their nano scaled range. Because of their size, nanoparticles have a big surface part than macro-sized materials. The essential properties of metal nanoparticles are mostly determined by range, shape, composition, crystalline and morphology (Ranjan et al. 2009). Nanoparticles outing to their small size, have diverse properties compared to the mass form of the same substance, thus contribution many novel developments in the fields of biosensors, biomedicine, and bio
  12. 12. nanotechnology. Nanotechnology is furthermore human being utilized in medicine for diagnostic, therapeutic drug delivery and the improvement of treatments for many diseases and disorders. Nanotechnology is an a great deal potent technology, which holds an enormous promise for the design and development of many kinds of novel product with its prospective medical applications on untimely disease detection, treatment, and prevention. (Gopinath et al.2012)[2]. 1.2 Nanoparticles The word “nanoparticles” that is used to express a particle with extent in the range of 1nm-100nm, at slightest in one of the three possible dimensions. Inside this size range, the physical, chemical and biological properties of the nanoparticles changes in fundamentals conduct from the properties of both being atoms/molecules and of the equivalent bulk materials (Nour et al. 2010)[3]. Nanoparticles can be prepared of materials of various chemical nature, the largely frequent being metals, metal oxides, silicates, non-oxide ceramics, polymers, organics, carbon and biomolecules. Nanoparticles are present in numerous changed morphologies such as spheres, cylinders, platelets, tubes etc. Generally the nanoparticles are considered through surface modifications adapted to gather the needs of specific applications they are going to be used for. The enormous diversity of the nanoparticles arising from their wide chemical nature, shape and morphologies, the medium in which the particles are present, the state of dispersion of the particles and most importantly, the numerous possible surface modifications the nanoparticles can be subjected to make this an important active field of science now-a-days (Zamiri et al.2012)[3]. 1.3 Unique properties of nanoparticles A number of physical phenomena become more definite as the size of the system reduced. Some phenomena may not be significant as we move from macro (high) to micro (low) stage but may be notable at the nano scale. Another example of uniqueness of nanoparticle is that when we increase the surface area with respect to volume changes the catalytic, mechanical and thermal and properties of the material. Also it increases of the superiority in the behaviour of atoms on the surface of the particle over the atoms in the interior, thereby altering the properties. The properties (optical, electrical properties and chemical reactivity) of small clusters are different
  13. 13. from the properties of those components in bulk. Also there are size dependent properties of nanoparticles that are quantum incarceration in semiconductors, in metallic nanoparticles surface Plasmon resonance and paramagnetic in magnetic nanoparticles [4]. In resonance, the collective oscillations of the conduction of electrons with the light field are known as surface Plasmon resonance. This property emerges from the electron confinement in the nanoparticles. The frequency also depends on the shape and size of the nanoparticles,the dielectric properties of the medium surrounded and also the metal (Jain et al. 2007). For instance, the noble metals like gold and silver nanopartiles reveal unique and tuneable optical properties because of their surface Plasmon resonance. 1.4 Zn nanoparticles and its current status Zn appears as a white powder and is practically insoluble in water. Zn is one of the most significant semiconducting materials with exclusive properties and resourceful applications. Zn has a stable quartzite structure consisting of a number of planes composed of tetrahedral coordinated O2− and Zn2+ ions. Because of its no central symmetry, Zn is piezoelectric, which is a key property in building electromechanical coupled sensors and transducers (Albrecht et al.2006). Zinc nanoparticles can be present in ions only in the existence of strong oxidizing substances. Zn is an environmental friendly material and biocompatible which is desirable especially for biomedical applications. Zinc nanoparticles have received considerable attention due to their antimicrobial, UV blocking, high catalytic and photochemical activities. Zn is non-hazardous and is compatible with human skin building it a proper additive for textiles and surfaces that get nearer in contact with human body (Li et al. 2011). The increase in surface area of nanoscale Zn compared to immensity has the potential to develop the effectiveness of the material function. Zinc is a natural constituent and an intrinsic part of the environment. Exposure to natural environment levels of zinc in the biosphere is crucial for all living organisms, fulfilling important metabolic functions in humans, animals and plants. Although zinc oxide is generally recognized as safe, occupational, consumer, and environmental exposures are controllable and considered to pose little or no risk. Zinc occurs naturally in soil, sediment, water and air, from where it is taken up by organisms to fulfil essential functions in metabolism. As such, the distribution, transport and effects
  14. 14. (bioavailability) of zinc in the environment largely depend on the site‐specific physicochemical characteristics of the environment and an organism’s condition. Zn has received much attention from researchers and regulatory agencies due to its use in cosmetics and personal care products [5]. However, consumer and environmental safety interests have been demonstrated through various lines of evidence, including endorsed use of nano. Zn containing sunscreens by the U.S. FDA (Food and drug Administration) and Australian TGA (Therapeutic goods Administration) Following several assessments on potential occupational, consumer, and environmental exposure and effects, it has been demonstrated that the levels of nano Zn present in cosmetics, effluents, or environmental media do not pose health risks to humans. 1.4.1 Viable applications of Zn nanoparticles 1. It is used in paints, cosmetics, sunscreens, plastic and rubber manufacturing, electronics and pharmaceuticals products etc. 2. It is also potentially used to treat leukaemia and carcinoma cancer cell 3. It is also a physically powerful antibacterial agent. 4. It is also used as drug transporter. 5. Zn nanoparticles is also used in industrialized sectors together with environmental, synthetic textiles, food, packaging, medical care, healthcare, as well as construction an decoration. 6. Zinc Nanoparticles exhibited feasible applications such as therapeutics, drug delivery agents, biosensors, imaging contrast agents, transfixion vectors, and fluorescent labels. Plant mediated zinc nanoparticles showed best anticancer activity. 7. It has been used as a source of zinc which is an essential trace element in food industry. 8. It also widely applied to various cosmetic products like sunscreen products while it acts as an invisible obstacle which scatters UV radiation away from the skin relatively than permitting its destructive energy to be absorbed. But, absorption rate of zinc is low via oral ingestion and bulk-sized ZnO has poor dispersion property, resulting in low UV blocking capacity, low transparency on skin, and hard agglomeration compared to small-sized ZnO. Thus, nano-
  15. 15. sized ZnO has recently attracted much attention in order to enhance the uptake of zinc and increase UV filtering efficiency with cosmetic clarity. 9. The similar kind of active ingredient used in the calamine lotion and diaper cream. 10. It is now generally accepted that nanoparticles efficiently penetrate the cell membrane compared to micro-sized particles. However, few researches were performed to demonstrate the effects of physicochemical parameters of ZnO nanoparticles on cellular uptake, which may provide critical information on their toxicity potential [6]. 11. Most activities to conflict and have centered on the nano coatings or nano composites using nanoparticulates of silver and zinc oxide. Their antimicrobial effects of natural biological compounds have also been demonstrated and confirmed by various researches .Zinc oxide show evidence of antibacterial activity which increases with decreasing the particle size. ZnO is nontoxic and it is a strong antibacterial agent. 12. ZnO nanoparticles have strong resistance to microorganisms and this property is due to the production of reactive oxygen species (ROS) on the surface of nanoparticles. 13. Zinc nanoparticles are used as preservative for various materials and products such as plastics, ceramics, glass, pigments, foods, etc. 1.5 Synthesis of nanoparticles Synthesis of dimensionally controlled particles enlarges quantities and studies their properties to investigate novel applications is a preferred activity for researchers in nanomaterials. There are several physical, chemical procedures for synthesis of Zn nanoparticles in large quantities in a short period of time. Among them simple- solution based methods, chemical precipitation, sol-gel or hydrothermal, electrochemical and photochemical reduction method are preferable methods (Seow et al.2011). Zn nanoparticles can be synthesized using green method that exploits various species of plant leaf extract, microorganisms like bacteria and fungi. Further the green synthesis of nanoparticles also governs by various enzymes. There are a lot of different methods of Zn nanostructures preparation like metal organic vapour phase epitaxial, evaporation of high temperature, gas spraying, pulsed laser deposition,
  16. 16. sputtering, sol-gel, wet chemical and electrochemical methods. Different applications of nanoparticles based on different protocols which have been designed for synthesis of nanoparticles. Figure 1 summarized the different methods used for synthesis of nanoparticles (physical, chemical and biological mathods). Fig.1.Outline of the various approaches such as physical, chemical and biological for the synthesis of nanoparticles 1.5.1. Physicalmethod Physical methods developed for synthesis of Zn nanoparticles often require special equipments or operational control. The physical method often called top-down approach which includes methods like diffusion, irradiation, thermal decomposition and arc discharge etc. The thermal decomposition method is one of the significant top-down approaches. It is used for the creation of monodisperse nanoparticles. These approaches use larger initial structures or macroscopic units which can be externally controlled in the processing of nanostructures. Etching through the mask; ball milling and application of severe plastic deformation are common examples of physical top- down method of synthesis of nanoparticles[7]. The top-down method of synthesis is a model which generates on a larger scale for further reduction of nano scale. Mostly the top-down methods are not cheap and quick to manufacture. This method is slow
  17. 17. and not suitable for large scale production. In this method of synthesis, fatty acids are allowed to dissolve in the hot NaOH solution and then mixed with the metal salt solution. After that, the solution used to forms the metal precipitates. Crystals and short wires of copper are allowed to be encircled in the glass ampoules by the diffusion method. Then, it is sealed at low pressure followed by annealing at 5000˚C for 24 hours. Subsequently the crystals are eradicated from the ampoules. At room temperature, the ampoules free crystals are chilled on a metallic plate. In UV irradiation method, polycarbonate films are placed on glass microscope slide .Then this slide is exposed to UV radiation. Figure 2 depicts the synthesis of nanoparticles by top down method. 1.5.2. Chemicalmethod Moderate reaction position and suitable artificial manipulation have rendered chemical methods a striking option for accessing Zn nanoparticles. Conventional chemical routes that are recurrently used in modern years include sol-gel approach, chemical precipitation and colloidal synthesis. Surfactants or polymer based preparative method for Zn nonmaterials are also quite accepted to avoid agglomeration and accomplish better manages of Zn nanoparticles. Removal of surfactants or polymers, however, poses serious limitations to such methods. Hence, delicate and precise control of experimental factors for the production of nanoparticles with desired morphologies is a cherished goal for future device applications [7]. Homogeneous chemical precipitation method is often considered economically viable for preparation of mono disperse metal oxide particles of different shapes and sizes. The method also provides better control of chemical and morphological characteristics. Chemical methods, including precipitation from inorganic or organic solutions and sol-gel techniques can conveniently provide control of nucleation, growth and ageing of particles in the solution. The methods rely on advanced solution and coordination chemistry theories; enabling the synthesis of their quirked precursor particles utilizing a variety of parameters to enable control of the solid formation process. A major contribution to the growth of particles is through Ostwald ripening, where small particles with lower solubility product dissolve and re- precipitate on the surface of larger particles in solution. Agglomeration takes place in solution as the particles clog together to minimize surface energy. Mostly the chemical methods of synthesis of nanoparticles are the bottom up approaches. These
  18. 18. approaches include the miniaturization of material components (up to atomic level) with further self-assembly processes leading to the formation of nanostructures. During self-assembly the physical forces operating at nanoscale are used to combine basic units into larger stable structures. Typical examples are quantum dot formation during epitaxial growth and formation of nanoparticles from colloidal dispersion [8]. In these methods different metal particles are reduced to form nanoparticles. Various types of metal particles used like Sodium monohydrate and Sodium citrate etc. Other chemical reagents used are N, N-dimethylformamide (DMF), Poly (N-vinyl pyrrolidine (PVP), ethyl alcohol, tetra-n-tetra-fluroborate (TFATEB), CTAB, etc. This method initiates with a pattern which is generate larger scale, then reduced to nanoscale[9]. This method of synthesis of nanoparticles is cheap and quick to manufacture. This method is slow and not suitable for the large scale production. (Figure 2 displays the chemical method for nanoparticles synthesis) Fig.2. Synthesis of nanoparticles by physical (top down) and chemical method (bottom up) 1.5.3. Biologicalmethod Biosynthesis of nanoparticles is a sort of bottom up (chemical method) approach where the main reaction taking place is reduction/oxidation. The requirement for biosynthesis of nanoparticles increases as the physical and chemical methods were expensive. Often, chemical synthesis method leads to formation of some of the toxic chemical engrossed on the surface that may have unfavourable effect in the medical applications. This is not a problem when it comes to
  19. 19. biosynthesized nanoparticles via green synthesis way. So, in the investigation of cheaper pathways for nanoparticles synthesis, scientist used microbial enzymes and plant extracts (phytochemicals) [14]. With their antioxidant or dropping properties they are regularly dependable for the decrease of metal compounds into their particular nanoparticles. Green synthesis provides improvement over chemical and physical method as it is price effective, environment friendly; easily scaled up for large scale synthesis and in this method there is no need to use high pressure, energy, temperature and toxic chemicals. Green synthesis technique is proved as favourable over other techniques (physical and chemical methods) which are implemented for the production of nanoparticles. Green synthesis exploitation of biological materials like plant leaf extract, bacteria, fungi and enzymes methods are eco-friendly, advance and compatible for pharmaceutical and other biomedical applications, as the toxic chemicals are not used in these methods. As well, this method does not require of high pressure and temperature. 1.6. Advantages of Zn nanoparticles synthesis by plants over microbes The major advantage of using plant extracts for zinc nanoparticle synthesis is that they are simply accessible, safe, and non-hazardous in generally cases, have a broad multiplicity of metabolites that can assist in the reduction of zinc ions, and are faster than microbes in the synthesis because it’s not easy to maintenance of microbial cell culture and contamination [12]. The main method considered for the procedure is plant-assisted reduction due towards phytochemicals. The major phytochemicals concerned are, ketones, aldehydes, terpenoids, carboxylic acid flavones and amides. Organic acids, Flavones and quinones are water-soluble (soluble in water) phytochemicals that are dependable for the instant reduction of the ions. Studies have exposed that xerophytes have emodin, an anthraquinone that undergoes tautomerization, most important to the formation of the zinc nanoparticles. Inside the case of mesophytes, it was founds that they hold three types of benzoquinones: cyperoquinone, dietchequinone, and remirin. It was optional to directly implicate the phytochemicals in the reduction of the ions and productionof zinc nanoparticles[16].
  20. 20. OBJECTIVES A critical review of literature depicted that so far no report available on the green synthesis of Zn noparticles using Eclipta alba leaf extract as biological reducing agent .The synthesis of Zn nanoparticles is still in its infancy and need more research to further explore various other potential along with the mechanism of nanoparticle proceed which may lead to well tuning of the procedure eventually leading to the production of nanoparticles with a severe control in excess of the size and shape parameters. Taking all these facts into consideration the research was carried out with following objectives. 1. To produce Zinc nanoparticles from zinc acetate exploiting leaf extract of Eclipta alba. 2. To optimize the conditions for maximum production of zinc nanoparticles. 3. To check the feasibility of antimicrobial activity of Zn nanoparticles against microorganisms (Escherichia coli Staphylococcus aureus) 4. To characterization of Zn nanoparticles using UV-Vis spectrophotometer.
  21. 21. REVIEW OF LITERATURE The study about Nanoscience and nanotechnology provides the well developed application of exceptionally miniature things and be capable of the encroachment of all the fields of scientific research and development like Physics, Chemistry, Materials and Metallurgy engineering, Biology and also in Biotechnology. Bionanotechnology is the conjunction between biotechnology and nanotechnology for developing various biosynthetic and environmental eco-friendly technologies for the synthesis of various nanomaterials. Multiple of research on synthesis of nanoparticles has put forth great interest towards the emerging field of science due to their distinguishing physical and chemical properties than the macroscopic particles. The vast array of development of novel synthesis protocols and various characterization techniques are the evidences for the vast advancement for the nanotechnology. A critical review of literature depicted that currently, the nanobiotechnology is an interesting emerging and innovative field of research with possibility to explore various potential application. Taking this facts into consideration, now a days there is intensifying research towards the same area. Further the scientific/researcher commitments actively engage in the same field to drive and bring this area of research from infancy to maturity level. Jalal et al (2010) studied on “Zn nanofluids: Green synthesis, characterization, and antibacterial activity”. In this paper using a green solvent and ionic liquid, 1-butyl-3-methylimidazolium bis (trifluoromethylsulfonyl) imide, [bmim] [NTf2], Zinc oxide nanoparticles have been formed by microwave degeneration of zinc acetate precursor. Using transmission electron microscopy and X-ray diffraction, the structure and morphology of Zn nanoparticles have been characterized. The Zn nanofluids have been formulated by diffusing Zn nanoparticles in glycerol as a base fluid using a dispersant as ammonium citrate. The antimicrobial activity of diffusion of Zn nanofluids against (E. coli) has been assessed by roughly calculating the reducing percentage of the bacteria tested with Zn. Survival ratio of bacteria decreases with the increase in the concentration of Zn nanofluids and also with time. The final results show that an enhancement in the concentrations of Zn nanofluids produces strong antimicrobial activity regarding E. coli.
  22. 22. Sangeetha et al (2011) conducted investigation on “Green synthesis of zinc oxide nanoparticles by aloe barbadensis miller leaf extract: Structure and optical properties” Biological methods for nanoparticle have been recommended as possible eco-friendly alternatives to chemical and physical approach. In this paper, they have reported about the production of nanostructured zinc oxide particles by two approaches (chemical and biological method). Vastly constant and spherical zinc oxide nanoparticles are formed by using zinc nitrate and Aloe vera leaf extract. The particles were mostly spherical and size could be inhibited by changing the concentrations of leaf extract solution. The zinc oxide nanoparticles from Aloe vera leaf are ordinary to have applications in biomedical and industries. Espitia et al (2012) conducted studies “Zinc Oxide Nanoparticles: Synthesis, Antimicrobial Activity and Food Packaging Applications.”In this study Zinc oxide (ZnO) is an inorganic compound broadly used in daily applications. ZnO is presently listed as a usually recognized as safe material by the Food and Drug management and is used as food preservative. The arrival of nanotechnology has led the expansion of materials with novel properties for use as antimicrobial agents. Thus, Zn in nanoscale has shown antimicrobial properties and prospective applications in food conservation. Zn nanoparticles have been included in polymeric matrix in order to provide antibacterial activity to the binding material and get better packaging properties. This review presents the main production technique of ZnO nanoparticles, major description and mechanisms of antibacterial activity as well as the impact of their amalgamation in polymeric matrices. Security issues such as exposed routes and passage studies are also discussed. Thambavani et al (2013) search on “One Pot Synthesis of Zinc Oxide Nanoparticles via Chemical and Green Method.” currently the enhancement of green chemistry in the synthesis of nanoparticles with the use of plants has immersed a great consideration. This study information the exploit of aqueous leaf extract of Corriandrum sativum as an eco-friendly mediator for the pattern of Zinc Oxide nanoparticle using zinc acetate and sodium hydroxide as a substitute for Chemical process. The present examination describes the synthesis and characterization of ZnO nanoparticles performed by green and chemical technique (XRD, SEM, FTIR and EDAX). ZnO nanoparticles have found tremendous application in bimolecular detection, diagnostics, micro electronics and water remediation. Although chemical
  23. 23. and green methods are trendier for nanoparticles production, the biogenic green manufacture is a better option due to eco-friendliness. Ramesh et al (2014) work on this topic “Green Synthesis of Zinc Oxide Nanoparticles using flower Extract Cassia Auriculata”. Nanoparticles performed by plants are more established, and the rapidity of synthesis is more rapid with respect to the case of other organisms. Furthermore, the nanoparticles are a variety of in shape and size in evaluation with those formed by other organisms. The present enquiry was accepted to green production of zinc oxide nanoparticles with the help of medicinal plant Cassia auriculata (Tanners cassia). It was synthesized by insertion of 1mM aqueous solution of zinc acetate with aqueous extract of Cassia auriculata flower. The production of nanoparticles was evaluated by visualizing colour changes and it was investigate by Fourier Transform Infra-Red (FT-IR), electron microscope (SEM), UV-Vis spectrophotometer and spectroscopy. The results of different techniques confirmed the incidence Zinc oxide nanoparticles. Senthilkumar et al (2014) studied on “Green tea (camellia sinensis) mediated synthesis of zinc oxide (Zno) nanoparticles and studies on their antimicrobial activities.” In this study Green synthesis of Zinc oxide nanoparticles (ZnO Nps) was taken using the aqueous extract of green tea (Camellia sinensis) leaves. The UV-Vis Spectrum was observed to monitor the production of the nanoparticles, which shows a blue shifted absorption peak at 325 nm. The pattern XRD revealed well-defined peaks appearing at 2θ positions corresponding to the hexagonal quartzite structure of ZnO nanoparticles. The average size of the nanoparticles calculated using XRD data was 16 nm. FT-IR spectra were recorded for the green tea extract and for the ZnO nanoparticles to identify the biomolecules involved in the synthesis process. The higher percentage of phenolic compounds, with antioxidant potential, provided the reducing action on the metal oxides and significantly present amino acid, protein and lipids helped to stabilize the growth of the nanoparticles. Agar well -diffusion method was used to study the antibacterial and antifungal activities on selected pathogenic species. The synthesized ZnO Nps showed better and comparable antimicrobial activities with respect to the activities of synthetic drugs. Rajeshwari et al (2014) Investigate on this topic “Biogenic Zinc Oxide Nanoparticles Synthesis via Tabernaemontana Divaricate Leaf Extract and Its Anticancer Activity against MCF-7 Breast Cancer Cell Lines” This examination explains the green synthesis and description of zinc oxide nanoparticles from an
  24. 24. Indian medicinal plant by an environment friendly technique. The major intention of this study is to synthesize zinc oxide nanoparticles from Tabernaemontana divaricate leaves by a green chemistry procedure. Very stable, spherical zinc oxide nanoparticles were synthesized by using 50% volume of Tabernaemontana leaf extract. Production of zinc oxide nanoparticles have been characterized by X-ray diffraction (XRD), Raman spectroscopy and analysis through transmission electron microscopy (TEM). All the analysis reveals that zinc oxide nanoparticles were 36 ± 5 nm in dimension. Functional groups and chemical composition of zinc oxide were also confirmed. Tabernaemontana mediated zinc oxide nanoparticles showed effective cytotoxic effect against MCF-7 breast cancer cell lines with an IC50 value of 30.65 μg/ml/24 h by the MTT assay. These results clearly support the benefits of using biological method for synthesizing zinc oxide nanoparticles with anticancer activities. Singh et al (2014) studied on Biosynthesis of Stable Antioxidant ZnO Nanoparticles by Pseudomonas aeruginosa Rhamnolipids.” for the duration of the last several years, different chemical technique has been used for production of a diversity of metal nanoparticles. Most of these techniques pose severe ecological issues and biological risks; consequently the current study reports a biological way for formation of zinc oxide nanoparticles by means of Pseudomonas aeruginosa rhamnolipids and their antioxidant property. Formation of established ZnO nanoparticles gives mainly spherical particles with the particle size ranging between 35 to 80 nm. The ZnO nanoparticles were analyzed by UV-visible (UV–vis) spectroscopy. Gnanasangeetha et al (2014) Described on the “Biogenic Production of Zinc Oxide Nanoparticles Using Acalypha Indica” Zinc oxide nanoparticles have fascinated meticulous research interest because of its significant applications in the field of medicine, pigment electronics, spintronics and piezoelectricity. The biogenic invention of zinc oxide nanoparticle is a better option due to ecofriendliness. Aqueous leaf extract of Acalypha indica were used to synthesis zinc oxide nanoparticles not only in the laboratory scale, but also in their natural environs. This green synthesis approach shows that the environmentally benign and renewable aqueous leaf extract of Acalypha indica can be used as a stabilizing mediator for the synthesis of zinc oxide nanoparticles. The fashioned nanoparticles ranged in dimension of about 100- 200nm. Scanning Electron Microscope (SEM), Energy Dispersive X-Ray analysis (EDX), X-Ray Diffraction (XRD) and Fourier Transform Infrared Spectroscopy (FT- IR) well-established that the formed nanoparticles are zinc oxide nano cubes.
  25. 25. Salem et al (2015) Examine on the “Antibacterial activity of silver and zinc nanoparticles against Vibrio cholerae and enterotoxic Escherichia coli” Vibrio cholerae and enterotoxic Escherichia coli (ETEC) last two dominant bacterial causes of severe secretary diarrhea and still a crucial cause of death, mainly in developing countries. In order to explore new efficient and inexpensive therapeutic approaches, With the use of corresponding salt (silver or zinc nitrate) we analyzed nanoparticles synthesized with green aqueous extract of Caltropis procera fruit or leaves. They characterized the amount and quality of nanoparticles by UV–observable wavelength scans and nanoparticle tracking inspection. Synthesis of nanoparticles in the regeneration yield is around108 particles/ml with mode particles sizes of approx. 90– 100 nm. Antibacterial action against two pathogens was examining by minimal inhibitory concentration assays and endures curves. These pathogens show similar resistance characterized with minimal inhibitory concentrations ranging between 5 × 105 and 107 particles/ml. , Wonderfully, zinc nanoparticles exhibits moderately a better efficacy, but sub lethal concentrations give the opposite impact and ensue in the increased biofilm formation of V. cholerae. With the use of various utterance of the external membrane porin OmpT a signal for cAMP levels, it is stated that our various consequences is that zinc nanoparticles reduce adenylyl cyclase activity. The level of second messenger is been decreased by various segments and is basically known as the, inhibitor of biofilm formation. At the end, they express a single oral management of silver nanoparticles to infant mice colonized with V. cholerae or ETEC considerably inhibits the immigration rates of the pathogens by 75- or 100-fold, corresponding Elumala et al (2015) studied on “Green synthesis of zinc oxide nanoparticles using Moringa oleifera leaf extract and evaluation of its antimicrobial activity” The enhancement of the semiconductor materials made a significant development of catalytic methodology. In this case an easy and eco-friendly chemical route for the production of zinc oxide nanoparticles (ZnO NPs) with the use of leaf extract Moringa oleifera. The formed ZnO NPs were characterized and has distinct technology such as UV–Vis absorption spectroscopy, X-ray diffraction (XRD), field emission scanning electron microscopy (FE-SEM), energy dispersive X-ray analysis (EDX), Fourier transform infrared spectroscopy (FT-IR) and photoluminescence spectroscopy (PL). XRD investigation exposed the wurtzite hexagonal structure of ZnO NPs. FT-IR assured the occurrence of functional groups of these leaf extract and
  26. 26. ZnO NPs. The particles size, morphology and topography resolved from FE-SEM. The strong and narrow width of zinc and oxygen have better purity and crystalline were recognized using EDX. UV–Vis absorption it exhibits the characteristic absorption peak of ZnO NPs. As a result, the antimicrobial action exposed that maximum zones of inhibition was observed Gram (+ve) positive bacteria and followed by the Gram (−ve) negative bacterial and fungal at the volume of 200 μg/mL of ZnO NP s. Vijayakumar et al (2015) conducted investigation on “Plectranthus amboinicus leaf extract mediated synthesis of zinc oxide nanoparticles and its control of methicillin resistant Staphylococcus aureus biofilm and blood sucking mosquito larvae” In this review, with the use of the leaf extract of Plectranthus amboinicus (Pam-ZnO NPs) zinc oxide nanoparticles were biologically formed. The formation of Pam-ZnO NPs were categorized by UV–Vis spectrophotometer, FTIR, TEM and XRD examine. TEM investigation of Pam-ZnO NPs performed the standard size of about 20–50 nm. Pam-ZnO NPs enhance the development of methicillin- resistant Staphylococcus aureusbiofilms (MRSA ATCC 33591) at the quality of 8– 10 μg/ml. Confocal laser scanning microscope (CLSM) pictures exposed that Pam- ZnO NPs powerfully inhibited the biofilm production capacity of S. aureus. With the quantity of 8 and 10 μg/ml, Pam-ZnO NPs performed 100% mortality of fourth instars mosquito larvae of Anopheles stephensi, adding together Culex quinquefasciatus and Culex tritaeniorhynchus. The history of pathological studies showing the occurrence of damaged cells and tissues in the mid-gut, experienced by A. stephensi and C. quinquefasciatus larvae with the Pam-ZnO NPs. Foremost changes contain rupture and degeneration of epithelial layer and cellular vacuolization due to the trouble of injure tissue. It is stated with the current research that Pam-ZnO NPs exposed the valuable control of S. aureus biofilms and mosquito larvae by destructive the mid gut cells. Bala et al (2015) studied on this topic Green synthesis of zinc oxide nanoparticles using Hibiscus subdariffa leaf extract: effect of temperature on synthesis, anti-bacterial activity and anti-diabetic activity”. Leaf extract of Hibiscus subdariffa formed the Zinc oxide (ZnO) nanoparticles (NPs) . This study explains formation of particle and its dependency on temperature. Production of NPs was examined by UV-visible (UV-VIS) spectroscopy, X-ray diffraction (XRD) and Fourier transforms infrared (FTIR) spectroscopy. To investigate the morphology and
  27. 27. size distribution of the synthesized particles is done with the help of Electron microscopy. The produced ZnO nanoparticles as probable anti-microbial agents has explored on Escherichia coli and Staphylococcus aureus. Distint research has stated that the undersized ZnO NPs, stabilized by plant metabolites has superior anti- diabetic consequence on streptozotocin (STZ) initiate diabetic mice than that of big sized ZnO particles. It is also been noted by the enzyme linked immune sorbent assay (ELISA) and real time polymerase chain reaction (RT-PCR) that ZnO can give confidence the purpose of Th1, Th2 cells and expressions of insulin receptors and another genes of the pancreas related with diabetes. 2.1 Plant sample 2.1.1 Eclipta Alba(Bhringraj) Eclipta alba (L.) is small branched yearly botanical plant with a extensive history of traditional medicines uses in numerous countries specially in tropical and subtropical regions. This plant belongs to the family of Asteraceae. Root is greyish in colour, cylindrical in shape and is well developed. This plant is known as weed all over the world and is germinate usually in moist areas. It is broadly scattered all over India, China, Thailand, and Brazil. This herb is been recognized for its therapeutic properties and it is been exploit as antihyperglycemic, antioxidant, immunomodulatory, antimytotoxic, analgesic, antibacterial, antihepatotoxic, antihaemorrhagic, properties and it is measured as a fine rejuvenator too. In the current research it is been highlighted that anti-venom significance & corrosion pickling inhibitor activity on mild steel in hydrochloric acid. Chemical compound involves coumestans, alkaloids, thiopenes, flavonoids, polyacetylenes, triterpenes and has a very broad range and their glycosides has been secluded from species. Plant extracts and their metabolites are identified to acquire pharmacological properties. This involvement furnishes an inclusive check on ethno medicinal work, chemical composition, and the pharmacological background as medicinal plant. Focused treatment is provided to antihepatotoxic, analgesic, antioxidant, antihyperglycemic, antiaggresive, injure curative properties and insecticidal impact presented in the study, the possible use of the plant while in pharmaceutics or as an agricultural resource could be easily analysed. Similar with athlete foot, eczema and dermatitis, Eclipta alba even has a conventional exterior uses, For the treatment of scorpion
  28. 28. stings this leaves is been used and for hair loss it is been addressed on the scalp. In China and Brazil this plant is also been used as an anti-venom against snakebite. This plant (Bhringraj) also helps to recover hair growth and colour. Its leaves are also used for making food especially in India and it is grown up by the side of rice fields. Bhringraj’s juice which is basically oil made with mixture aid to hair growth and help in prevention of premature greying. Fig.3. Plant Eclipta alba showing leaves and flower 2.1.2 Viable applicationof Bhringraj i. Healthy hair: This supernatural herb is old in Ayurveda as a hair boost, for renaissance of hair and avoidance of hair drop, bluntness, dandruff and early brownish grey of hair. It is additional to shampoos for rebirth of hair and scalp. Correspondingly, bhringraj oil is of vast use to encourage hair health. It may be able to be used as a glue (of leaves) or fine particles useful to the scalp. Bhringraj is identified for its washing out and detoxifying property, which has a helpful pressure on hair and skin health. ii. Liver health: The crushed root of bhringraj has been conventionally used for liver disorders for example cirrhosis, hepatitis, jaundice, anaemia and enlarged spleen. It also provides assistance from piles, urinary tract infections and renal disorders. Bhringraj is too identified to eliminate impurities from blood and detoxification of liver. iii. Valuable for nervous system: establish to be valuable for insomnia and promoting noise sleep, the Ayurvedic doctor also suggested Bhringraj extract
  29. 29. as an successful solution for enhancing memory and sanity such as visualization and hearing. It is known that it cause a rejuvenating and anti- aging effect on the human body. Bhringraj oil is exposed to be helpful for alleviating trouble such as stress, anxiety, tension, headaches and migraine. iv. Healthy skin: The detoxification properties of bhringraj make it advantageous for healthy and shining skin and for treating skin exertion such as eczema, athlete’s foot, cracked heels, insect bites and stings. It can be useful to the skin or taken mouthy to encourage healthy skin. v. Cholesterol and blood sugar: Investigation have revealed bhringraj to be helpful for lowering LDL (bad) cholesterol and triglyceride levels as well as increasing HDL (good) cholesterol levels. It has also been exposed to assist decrease blood glucose level and blood pressure. vi. Anti-inflammatory: The anti-inflammatory properties of bhringraj make it an effectual medicine for situation such as joint pains and arthritis. It can also be helpful for treating cough, asthma and acidity. A mixture with honey, its leaf extract is used to treat worm infestation in infants. vii. Gynaecological problems: This herb has been established helpful for treating gynecological trouble such as miscarriage and abortion. Bhringraj leaves have been known to alleviate uterine flow of blood and post-delivery pain.
  30. 30. MATERIALS AND METHODS MATERIALS: The following materials were exploited during course of research: 3.1. Glassware andApparatus All glass wares such as measuring cylinders, beakers, conical flasks, funnel, petriplates, filter paper (Whatman paper -1), eppendorf tubes, and microfuge tips, cuvettes, etc. were from Amity biotechnology lab. 3.2. Chemicals For all experimental studies pure and analytical grade chemicals were used. For synthesis of ZnO nanoparticles Zinc acetate dehydrate was (QUALIGENS) taken from amity lab. Nutrient Agar and Nutrient Broth media were from HIMEDIA. 3.3 Microorganisms andplant Escherichia coli strain B (KT45/45A) Lot number: #25029– Collected from Amity institute of biotechnology. Staphylococcus aureus (KT68) Lot number: 11118 - Collected from Amity institute of biotechnology. Eclipta alba plant leaves– Collected from Amity university. 3.4 Other apparatus and materials 1. Weight Machine 2. Micropipette 3. Centrifuge machine 4. Hot plates 5. Magnetic Stirrer 6. Autoclave 7. ph meter 8. Laminar 9. Refrigerator 10. Streaking needle 11. Double beam spectrophotometer 12. De-ionized water-collected from DI plant
  31. 31. METHODS 3.5. PREPARATION OF ZINC NANOPARTICLES The preparation of Zn nanoparticles involves following steps: 1. Preparation of leaf extract of bhringraj (Eclipta alba) 2. Preparation of Zinc acetate solutions of 1mM concentration. 3. Preparation of zinc nanoparticles by adding Eclipta albal leaf extract to zinc acetate solutions. 4. Incubation at room temperature to allow nanoparticles formation. 3.5.1 Preparationofplant extract Eclipta Alba (Bhringraj) plant leaves were collected from the amity university campus. To remove the dust particles the leaves are supposed to wash numerous times with water. Extract was prepared simply by boiling 10 gm of fresh leaves were boiled in 100 ml distilled water at 1000C for 20 min. The extract was chilled to room temperature and filtered by filter paper. Then the solution was incubated for 30 min. and then subjected to centrifuge for 10 min. at room temperature with 10000 rpm. The supernatant was separated and filtered with filter paper (Whatman filter paper-1). Then the solution was used for the reduction of Zn2+ ions to Zinc nanoparticles (Zn0). 3.5.2 PreparationofZinc acetate solutions of different concentrations Zinc Acetate Solutions are moderate to highly concentrated liquid solutions of Zinc Acetate. They are an excellent source of Zinc Acetate for applications requiring solubilised materials. Acetates are excellent precursors for production of ultra high purity compounds and certain catalyst and nanoscale (nanoparticles and nanopowders) materials. Weighed amount zinc acetate of was carefully transferred in a 50-ml volumetric flask following by addition of drop-wise de-ionized water while swirling to dissolve the salt up to the mark and solution was diluted as required and all the solutions were kept away from light and kept in dark.
  32. 32. 3.5.3 Preparation of zinc nanoparticles by adding Eclipta alba leaf extract to zinc acetate solutions For the synthesis of the Zinc nanoparticles, a certain volume of the Eclipta alba leaf extract 5 ml was added to the zinc acetate solution and the volume was adjusted to 10 ml with de-ionized water. The final concentration of zn+ was 1 × 10−3 M. The solution was stirred on magnetic stirrer at 600C for 10 min. The reduction process zn+ to zn0 nanoparticles was followed by the colour change of the solution from brownish-yellow to yellow to deep brown depending on parameters studied. The significant change in colour of solution indicating the synthesis of zinc nanoparticles, which was further, reconfirmed by the preliminary characterization using UV-Vis spectroscopy in the range of 200-500 nm at different time intervals. 3.5.4 Incubation at room temperature to allow nanoparticles formation In order to observe the formation and stability of zinc nanoparticles, it is required to incubate in room temperature and then regularly examine the formation of zinc nanoparticle and their stability. 3.6 Impact of concentrationof on zinc acetate synthesis ofZnNPs Concentration of Zinc acetate has significant impact on synthesis of Zn nanoparticles. Therefore, the absorption of Zn acetate mixture was optimized by changing the concentration of Zinc acetate 1, 3 and 5mM. 3.7 Effectof quantity of leaf extracton ZnNPs synthesis Testing for the effect of quantity of leaf extract on Zn nanoparticles synthesis, In sequence to optimize the amount of leaf extract for the synthesis of Zn nanoparticles the leaf extract was varied from 1 8mL in 5mL of zinc acetate solution. The formation of Zn nanoparticles were analysed by using UV–Vis spectrophotometer.
  33. 33. 3.8 Effectof temperature on synthesis of ZnNPs Temperature is one of the key influence factors in chemical reactions. In order to investigate the influence of temperature in the synthesis of zinc nanoparticles, the solution was heated from room temperature to 200C, 400C, 600C, 800C and 100°C, respectively. 3.9 Effectof pH on synthesis of ZnNPs pH play an significant task in the nanoparticles production, this factor enhance the reactivity of leaf extract with zinc ions. The impact of pH in the nanoparticles was evaluated below changed pH of the reaction mixture with the leaf extract. The effect of pH on synthesis of ZnNPs, pH is important feature for the biosynthesis of nanoparticles. The consequence of pH on the synthesis of ZnNPs was studied in the range of 4–9. To manage the pH, with the help of Hcl and NaOH (0.01 M) solutions. The other factors such as quantity of leaf extract, concentration of Zinc acetate and temperature were to keep it stable. The effect of pH on the production of ZnO NPs was monitored by UV–Vis spectroscopy. 3.10 Time dependant synthesis of ZnNPs With the time-dependent formation of Zn nanoparticles. As the time period increased, the nanoparticle synthesis also improved. Nanoparticle configuration was initiated within 5 min. The performance of nanoparticle synthesis was accomplishment after 1 hour as recognized in Figure. Due to the unsteadiness of the Zn nanoparticles the precipitation of nanoparticles occurred after 1 hour. Accumulation of nanoparticles exhibits the larger size of nanoparticles. So the best possible time duration for the synthesis of nanoparticles was 1 hours. Previous studies explain it was a long-duration procedure when using several sources such as plant which take 96 h in period for synthesizing the Zn nanoparticles, but in this research, it is a less time-consuming procedure. Some of the plants sources also took much longer time period for Zinc ion reduction process.
  34. 34. 3.11 Characterizationof ZnNPs The formation of ZnNPs was monitored by UV–Vis. UV-Vis spectroscopy (SHIMADZU) Model: UV-1800 240 V (JAPAN) analysis describes the crystalline and an average size of synthesized material. The zn nanoparticles were characterized in a Shimadzu UV-VIS Spectrophotometer. The scanning range for the samples was 200-500 nm. The double distilled water and Zinc acetate used as a blank reference. Absorbance spectroscopy is used to determine the optical properties of a solution. A Light is send through the sample solution and the amount of absorbed light is measured. When the wavelength is varied and the absorbance is measured at each wavelength. The absorbance can be used to measure the concentration of a solution by using Beer-Lamberts Law. The examination of nanoparticles, the optical properties are much more complicated. For instance, the measured absorbance spectrum does not necessarily show the actual absorbance but the extinction of the light is both the absorbed and the scattered light from the particles. These wave lengths arise due to the surface Plasmon resonance of the particle. Fig 4.UV-Vis Spectrophotometer Fig 5.UV Cuvette
  35. 35. 3.12 Antimicrobial activity The antimicrobial activities of zinc nanoparticles. Where tested using likes Staphylococcus aureus (KT68) Lot number: 11118 and Escherichia coli strain B (KT45/45A) Lot number: #25029 by well diffusion method. Before experiment, bacterial strain was cultured on to nutrient broth prepare by Add 1.3 g of nutrient broth powder to 100 ml of distilled water and mix well on the other hand. Then a nutrient- broth was prepared; the pH was adjusted to 7.0-7.5 and autoclaved. [17] It was incubated overnight at 370C. The nutrient broth was poured into Petri dish and then master culture was sub cultured onto nutrient agar Petri dish and it was incubated overnight at 370 C and each strain was inoculated separately into culture broth. The culture Petri dish was incubated overnight at 37°C in shaker-incubator. Afterwards nutrient agar plate was arranged and punctured for wells and detect for 200uL of ZnNPs. The labelled well was loaded with samples of Zn naoparticles. This step was carried out for the Staphylococcus aureus, Escherichia coli bacterial strains for 6 replicates. The plates were incubated for 24h in incubator at 37°C before the results were observed.
  36. 36. RESULTS AND DISCUSSION 4.1 Effectof concentrationof Zinc acetate on synthesis of ZnNPs The impact of concentrations of zinc acetate on the formation of ZnNPs was monitored by using UV–Vis. spectroscopy. It is seen from the figure.6, at 1 mM zinc acetate larger SPR band eventuate at 324 nm that is a distinguishing band of ZnNPs with large size. At 3mM, zinc acetate the SPR small band at 324 nm designates minor size of ZnNPs. For 5mM zinc acetate lager SPR band acquired at 346 nm which is blue shift and that indicates large size distribution of ZnNPs. From the above conversation it is understandable that in the present investigation, a most favourable concentration of precursor for the production of ZnNPs by using Eclipta alba leaf extract (5 mL) is found to be 5mM. Zinc acetate related explanation was also reported by Kumar et al.[18] Fig.6. UV–Vis. absorption spectra of ZnNPs at different concentrations of zinc acetate nm. 200.00 300.00 400.00 500.00 Abs. 3.595 3.000 2.000 1.000 0.000 -0.307 Zincacetate solution 5mM 1mM
  37. 37. 4.2 Effectof quantity of leaf extracton ZnNPs synthesis Effect of amount of leaf extract on the formation of ZnNPs was estimated by UV–Vis. spectroscopy. As the amount of leaf extract expands the strength of the band (324 nm) of ZnNPs also modify which is shown in the Figure 8. From the picture it is understandable that for 6.0 and 7.0 mL of leaf extract, the absorption bands were broad which show larger particle size, at 7.0 mL of leaf extract absorbance band was equipped to be small that indicates narrow size allocation. Furthermore at 6.0 and 8.0 mL of leaf extract absorbance bands were blue shifted. From above conversation, it is understandable that optimized quantity of leaf extract for the preparation of ZnNPs was found to be 7.0 mL for 5.0mL zice acetate of (5mM). Fig.7. UV–Vis. absorption spectra of ZnNPs at various amount Eclipta alba leaf extract. nm. 200.00 300.00 400.00 500.00 Abs. 4.388 4.000 3.000 2.000 1.000 0.000 -0.270 1ml 8ml
  38. 38. 4.3 Effectof temperature on synthesis of ZnNPs Temperature plays one of the most significant physical parameter on the production of Zinc nanoparticles. Figure 8 indicate the effect of temperature in the nanoparticles production. The synthesis of nanoparticles was expanding while increasing the reaction temperature by using the Eclipta alba leaf extract. The absorbance band was intensify and placed at 320nm and 345 nm at the temperature of 20 °C and 60 °C, correspondingly. The elevated rate of reduction was arising at higher temperature due to the expenditure of zinc ions in the configuration of nuclei whereas the secondary reduction was stopped up on the surface preformed nuclei.[19] The increase peak was procured at low temperature indicate the development of large sized nanoparticles and the narrow peak was procured at high temperature, which performed the synthesis of nanoparticles are small in dimension and the higher speed of reduction of zinc ions was occurred in the 60°C. At the end, it was summarized that high temperature was most favourable for nanoparticles synthesis. Fig.8. Effectof temperature inthe nanoparticlessynthesisusingleaf extractof Eclipta alba. nm. 200.00 300.00 400.00 500.00 Abs. 4.267 4.000 3.000 2.000 1.000 0.000 -0.226 800 C 200 C1000 C
  39. 39. 4.4 Effectof pH on synthesis of ZnNPs pH has an inevitable role in the nanoparticles synthesis, this factor uplift the reactivity of leaf extract with zinc ions. The impact of pH in the nanoparticles was influence under various pH of the reaction mixture by the leaf extract. In low pH, small with broadening SPR band was produced indicates synthesis of large size of nanoparticles. The effect of pH on the production of ZnNPs was estimated by using UV–Vis. spectroscopic studies. Formation of ZnNPs mostly determined on the pH of the reaction medium and the results are shown in Figure 10. From figure it is understandable that as the pH increases from 4 to 9, the absorbance significance increases gradually which indicates the rate of production of ZnNPs enhanced from acidic to basic medium. At acidic condition pH 4 to 6, bands were wider and present red shift due to increase in particle size. In basic condition at pH 8 and 9, bands were narrow and exhibit blue shift due to decrease in particle size. The configuration of ZnNPs occurs quickly, in neutral and basic pH this could be due to the ionization of the alkaloid groups present in the leaf extract. The slow rate of configuration and aggregation of ZnNPs in acidic pH can be interrelated to electrostatic repulsion of anions here in the solution. At basic pH there is a prospect of ZnOH precipitation. On the basis of this reaction, it may be finished that the optimum state for the preparation of ZnNPs using Eclipta alba leaf extract was neutral medium. Fig.9. pH dependence UV–Vis. absorption spectra of ZnNPs. nm. 200.00 300.00 400.00 500.00 Abs. 4.390 4.000 3.000 2.000 1.000 0.000 -0.399 4pH 8pH
  40. 40. 4.5 Time dependant synthesis of ZnNPs The previous works also recommended that contact or incubation period also influence the synthesis of nanoparticles. This is the time interval required for accomplishment of all steps of the reaction. Dwivedi and Gopal described the enhancement in the sharpness of UV absorption spectrum peaks with an amplify in contact time while functioning with plant leaf extract [20]. They revealed that nanoparticles visible within 15 min of the reaction and increased up to 2 h, but subsequent to that only slight variation occurred. In a distinct study, Dubey et al. discover that in Tansy fruit-mediated synthesis the development of zinc and silver nano- particles started contained by 10 min of the reaction. In insertion, they found that the enhancement in contact time is dependable for the sharpening of the peaks in equally zinc and silver nanoparticles. They also declare an important lower contact time necessity in comparison to previous reports from Fayaz et al. and Shaligram et al. In addition, newly Vermin et al. known that due to the instability of nanoparticles produced an optimum period is necessary for full nucleation and consequent stability of nanoparticles. This investigate group experiential that the optimum time essential for the finishing point of the reaction for silver nanoparticle production was 60 min during their testing.[21] Correspondingly, Ghoreishi et al. also showed the condition of an optimum reaction period for the permanence of synthesized silver and gold nanoparticles by Rosa damascene. The impact of reaction period is shown in Fig.10.From figure and optical observations it is understandable that the colour of solution changes after insertion of extract into the zinc acetate solution and the aqueous solution of zinc acetate turned colourless to brown within 5 min. SPR strength of ZnNPs enhances with instant time and saturates within 75 min, this effect recommend the development of anisotropic molecules that are stabilized afterwards in the medium. Numerous researchers have described on the synthesis of ZnNPs by using different plant extract and time interval from some days to hours, normally time duration was 7 days, 4 and 2 hours correspondingly. In the present investigation the rate of synthesis of ZnNPs by using Eclpita alba a leaf extract was establish to be 5 min which is very high-speed as compared to above described plant extract, it may be due to the existence of alkaloid, terpenoid and poly phenolic compounds such as ruin and apigenin-7-glucoside in Eclipta alba leaf extract which acts as well-built reducing agent.
  41. 41. Fig.10.Time dependant UV–Vis. absorption spectra of ZnNPs 4.6 Antimicrobial activity The ZnNPs shows exceptional antimicrobial action against E. coli. Clear area of inhibition of bacterial expansion on nutrient agar dishes approximately the holes impregnated with analysis ZnNPs are shown in Fig.12. The radial distance of inhibition zone is 10 mm at the similar point the controlled cavities containing Eclipta alba leaf extract does not exposed any inhibition zone. The inhibitory action of Zinc compounds and Zinc ions had been traditionally documented and applied as a helpful beneficial agent for preventing injury infections. The inhibitory activity of zinc on bacterial cells is linked to the strong connections of zinc with phenol groups present in key respiratory enzymes in microorganisms. Whereas Nano crystalline Zinc shows the most efficient inhibitory activity with a fast inhibition rate [22]. In the present study Eclipta alba was taken for synthesis of ZnNPs because of its therapeutic values. Different studies have been done by various researchers which verify that Eclipta alba was found to be good quality antimicrobial nm. 200.00 300.00 400.00 500.00 Abs. 4.389 4.000 3.000 2.000 1.000 0.000 -0.415 0 min 75 min
  42. 42. agent adjacent to pathogenic and non-pathogenic organisms. The antimicrobial property of the bio-synthesized Zinc nanoparticles from Eclipta alba were also effectively investigated. Also there is a variety of information which has been provided that the evidences that Zinc nanoparticles were used as influential device in opposition to multidrug-resistant bacteria. Kora and Arunachalam conceded that zinc nanoparticles synthesized by UV photo-reduction technique are performance capable antibacterial activity on P. aeruginosa at a great deal poor concentrations. As these above information suggests that together Zinc nanoparticles and Eclipta alba plant extract have exposed good quantity of antibacterial activity. Durairaj et al. premeditated the antibacterial activity of purchased ZnNPs in (size 20-30nm) adjacent to 10 separates of P. aeruginosa incorporating of 5 MDR strains with an inhibition region of 11 mm experiential through µg quantity of the nanoparticles. The nanoparticles demonstrated MIC of 50µg/ml when added at the lag stage and the sub inhibitory concentration was measured as 100 µg/ ml.[23] In our testing, when we compared the antibacterial action of ZnNPs and plant extract, it was found that zinc ZnNPs have exposed additional antibacterial activity than plant extracts. Our results evidently shows that the conventional plant extract performance some antibacterial activity but not a large amount activity as ZnNPs does beside bacterial strains yet taken in amount 10 times more than ZnNPs . It clearly indicates that these green ZnNPs have shown significant quantity of action than that of plant extract. Antibacterial movement of ZnNPs considerably increases by more than 12-15% at very minor concentration i.e. approximately 2 out of 4 strains have showing region of inhibition comes at concentration of 4 mg/ml in example of plant extract while in case of ZnNPs 200 µg/ml is the maximum concentration we have used[24]. Consequently we further takings only with the results of antibacterial motion of ZnNPs. Also if we use synthetic or purchased ZnNPs then there may be a difficulty due to chemical mediator used. Therefore we synthesized ZnNPs by plant extract of Eclipta alba which potentially eliminate the difficulty of chemical mediator that could occur if we used any artificial or chemically synthesized ZnNPs, thus production nanoparticles biocompatible with the eco-friendly move towards. The antibacterial effectiveness of synthesized ZnNPs enhances because the use of Zinc and Eclipta alba as zinc inhibited in nano structure which increases its surface area, thus make ZnNPs more reactive and Eclipta alba enhances the medicinal value of ZnNPs due to its good antibacterial efficacy. As a result, in our study, ZnNPs prepared by Eclipta alba were
  43. 43. used for the advancement of antibacterial agents against Human. We have checked the antibacterial activity of ZnNPs by agar well diffusion method against pathogenic strains of bacteria. In our experiment, biosynthesized ZnNPs showed excellent antibacterial activity against pathogenic strains of Staphylococcus aureus, Escherichia coli. Our results showed that ZnNPs synthesized from Eclipta alba possess discrete antibacterial activity at concentrations of 200 µg/mL . The zone of inhibition ranges was clear. (a) (b) Fig.11. Antimicrobial activity of ZnNPs against (a) E. Coli. (b) Staphylococcus aureus  Red circle showed zn nanoparticles.  Green circle showed Eclipta alba leaf extract.  Blue circle showed zinc acetate.
  44. 44. 4.7 UV-Vis SPECTROPHOTOMETER ANALYSIS: Reduction of zinc ions into zinc nanoparticles during exposure to plant extracts was observed as an outcome of the colour modify. The colour modify is due to the Surface Plasmon Resonance phenomenon. The metal nanoparticles have free electrons, which give the SPR absorption band, due to the combined vibration of electrons of metal nanoparticles in resonance with light wave. The sharp bands of zinc nanoparticles were observed around 324 nm in case of Eclipta alba Figure: 7, Figure:8, Figure:9, & Figure:10) From different literatures it was found that the zinc nanoparticles show SPR peak at around 320-380 nm. From our studies we found the SPR peak Eclipta alba for at 324 nm .So we confirmed that Eclipta alba leaf extract has more potential to reduce zinc ions into zinc nanoparticles, which lead us for further research on synthesis of zinc nanoparticles from Eclipta alba leaf extracts. The intensity of absorption peak increases with increasing time period. This characteristic colour variation is due to the excitation of the SPR in the metal nanoparticles the insets to Figure: 7, Figure: 8, Figure: 9 & Figure: 10 represent the plots of absorbance at λmax (i.e., at 320 nm) versus time of reaction. The reduction of the metal ions occurs fairly rapidly; more than 90% of reduction of Zn+ ions is complete within 2 Hrs. after addition of the metal ions to the plant extract. The metal particles were observed to be stable in solution even 2 weeks after their synthesis. By stability, we mean that there was no observable variation in the optical properties of the nanoparticle solutions with time. Fig.12. Shown significant change in colour after reaction
  45. 45. Fig.13. showed the colour change in 24 hours. Fig.14.showen the colour change is several days.
  46. 46. CONCLUSIONS In summary, a green novel way for the rapid, eco-friendly and non-hazardous production of ZnNPs at room temperature using easily available Eclipta alba leaf extract which act as a green reducing furthermore stabilizing mediator. In the present study, Zn nanoparticles have been synthesized with the use of surfactant which was isolated from the leaves of Eclipta alba. Green synthesis of Zinc nanoparticles were successfuly carried out from Zinc acetate using bio components of leaf extract of Eclipta alba. The synthesized Zn nanoparticles have been characterized using UVss- Vis spectrometer. Zinc oxide nanoparticles arise out as one of the most adaptable materials, due to their various properties, functionalities, and applications. ZnNPs have fabulous optical and physical properties. They also acquire antimicrobial activities against some bacteria. Zinc nanoparticles were characterized by considering the absorption spectra confirmation from the Ultraviolet–visible spectral analysis for the samples. Development of zinc nanoparticles in aqueous colloidal solution were recorded using Ultraviolet–visible spectral investigation. Zinc nanoparticles usually give an idea about a broad peak in the UV–vis spectrum in the range of 315–345 nm. The results confirm assure for the improvement of a ‘‘green’’ process for nanoparticle synthesis. Bio molecules found in plants induce the reduction of Zn2+ions into Zn nanoparticles. The procedure of reduction is extracellular and quick leading to the enlargement of easy biosynthesis of zinc nanoparticles. In this study the UV-Vis absorbance spectrum for synthesised zinc nanoparticles is observed around 325 nm. Moreover the synthesized nanoparticles have shown antibacterial potential against pathogenic bacteria. Zn nanoparticles were found to inhibit bacterial growth in comparison to the plant extract. Inhibition of bacterial growth by Zn nanoparticle can be attributed to damage of the bacterial cell membrane and extrusion of the cytoplasmic contents there by resulting in the death of the bacterium. Since concentrations of Zn nanoparticles have impact on the antimicrobial activity, therefore concentration dependent studies of nano Zn structure synthesized under different reaction condition can be of great significance from technology point of view. The explored eco-friendly, high efficient Zn nanoparticles prepared from Eclipta alba leaves are expected to have more extensive application in biomedical fields and in cosmetic industries.
  47. 47. THE CURRENT RESEARCH WORK LEAVES THE SUBSEQUENT FUTURE PROSPECT  Zn nanoparticles produced through the Eclipta alba leaf extract may be investigated in details of the Anti-fungal and anti-cancerous activity.  To check the feasibility of large scale production of Zn nanoparticles using Eclipta alba leaf extract.  Elaborate mechanistic study of antimicrobial property of Zn nanoparticles fromed by Eclipta alba leaf extract.
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