There are different sources of plant parts which can be used to produce nanoparticles.

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1 LITERATURE REVIEW 2.1 INTRODUCTION The emergence of nanotechnology has provided a vast research area in recent years by intersecting with various other branches of science and forming impact on all forms of life. Nanotechnology is a field of science which deals with production, manipulation and use of materials ranging in nanometers. In nanotechnology nanoparticles research is an important aspect due to its innumerable applications. Nanoparticles have expressed significant advances owing to wide range of applications in the field of bio-medical, sensors, antimicrobials, catalysts, electronics, optical fibers, agricultural, bio-labeling and in other areas. As opposition with the conventional methods to produce nanoparticles, the term herbal nanotechnology has become of more interest as it makes use of nanoparticles which are made from herbal extracts and are less hazardous when interacted with human as it uses less toxic chemicals. The metallic nanoparticles produced by numerous herbal extracts are of more focus because they show extreme anti-microbial activity and the production of these are not time consuming and cost effective. The present work emphasizes reported plant resources for the synthesis of different nanoparticles. Plants contain various therapeutic compounds that have been exploited since the ancient times. These compounds are flavanoids, alkaloids, phenols etc. Recent studies of using plants as a sources for manufacturing nanoparticles is advantageous because it is easily available, easy to handle and non-hazardous. 2.2 SOURCES OF HERBAL EXTRACT NANOPARTICLES: There are different sources of plant parts which can be used to produce nanoparticles. In a study, the flowers of a plant Hibiscus rosa sinensis, which is known to have different medicinal properties and contains flavonoids, anthocyanine and polyphenols was used to

2 synthesis silver nanoparticles by its complete reduction in presence of AgNO 3. (Shabana et al, 2013) In a study carried out by Jain et al, (2009) papaya fruit extract was used t o synthesis silver nanoparticles. In a study, leaf extract of Eclipta alba was used to synthesis silver nanoparticles. (Saminathan, 2015) 2.3 SYNTHESIS OF NANOPARTICLES There are different methods for synthesizing nanoparticles. Each one will be discussed briefly below CHEMICAL METHODS: The most common approach for synthesis of silver nanoparticles is chemical reduction by organic and inorganic reducing agents. In a study, silver nanoparticles were synthesized using chemical reduction method. Silver nitrate was taken as the metal precursor and hydrazine hydrate as a reducing agent. The formation of the silver nanoparticles was monitored using UV-Vis absorption spectroscopy and the size of the colloidal silver nanoparticle was found to be 60nm. (Guzman et al, 2009) In a study, many silver nanoparticles were synthesized by solution phase route. Different shapes of silver nanoparticles were synthesized. Triangular prism nanoparticles were prepared by reducing silver nitrate at room temperature. Spherical silver nanoparticles were synthesized by using sodium citrate and sodium borohydride as reducing agents. (Dong et al, 2012) PHYSICAL METHODS: Evaporation-condensation and laser ablation are the most important physical approaches. The absence of solvent contamination in the prepared thin films and the uniformity of Nanoparticles distribution are the advantages of physical synthesis methods in comparison with chemical processes.

3 2.3.3 BIOLOGICAL METHODS: The biological method for synthesizing nanoparticles is making use of various microorganism and plants. In a study carried out by Daisy and Saipriya (2012), the bark of Cassia fistula was used to synthesize gold nanoparticles by phytochemical method. Finely coarse powder of C. fistula stem bark was used for phytochemical mediated synthesis of the gold nanoparticles, whereby bark powder was added distilled water and continuously stirred; aqueous HAuCl 4 was subsequently added to the mixture whilst stirring. There was an immediate change in color from brown to ruby red, indicating formation of green gold nanoparticles. The utilization of fungi to synthesis nanoparticles as a novel method has been described in many research papers. In a study, fungus Trichoderma Reesei is used to synthesize silver nanoparticles. In the process of biosynthesis the fungus mycelium is exposed to silver nitrate solution. As a result, the fungus produces enzymes and metabolites for its survival. These enzymes and metabolites convert the toxic silver nitrate into non-toxic silver nanoparticle. (Wahabi et al, 2011) In a study, different sizes of gold nanowires (10-20 nm) were synthesized by used cell-free extract of Rhodopseudomonas capsulata. The procedure used offered control in shapes when the concentration of HAuCl 4 was altered in the mixture resulting in reduction of gold ions in the solution and biosynthesis of morphologies of gold nanostructures. At lower concentration of gold ions in the aqueous solution the shape of the nanoparticle was spherical where as at higher concentration nanowires were produced. (He et al, 2008) In another study, a combination technique of microwave irradiation and bacteria was used to synthesize silver nanoparticles. It was concluded that by combining both these techniques, the production of nanoparticle was rapid. (Safiuddin et al, 2009) In another technique, extract of oven dried leaves of Pongamia pinnata (L) Pierre was used for the synthesis of silver nanoparticles. Stable and crystalline silver nanoparticles were formed by

4 the treatment of aqueous solution of AgNO 3 with dried leaf extract of Pongamia pinnata (L) Pierre. (Raut et al, 2010) In a study carried out by Saxena et al (2010), an onion ( Allium cepa) extract was used to synthesize silver nanoparticles at a rapid rate. In the research, silver nitrate in the aqueous solution was reduced by onion extract. It was concluded that onion extract increases the reaction rate and hence is a convenient method of synthesizing nanoparticles. In a study, silver nanoparticles were formed by extracting Eucalyptus hybrida leaf. Bioactive silver nanoparticle synthesis by reacting the methanolic biomass of Eucalyptus hybrida leaf with aqueous solutions of silver nitrate. (Dubey et al, 2009) In a research, the synthesis of highly dispersed silver nanoparticles using a dried stem bark of Boswellia ovalifoliolata(an endemic plant) extract as the reducing agent was studied. After exposing the silver ions to bark extract, rapid reduction of silver ions is observed leading to the formation of silver nanoparticles in solution. (Ankanna et al, 2010) In a study carried out by Elumalai et al (2010), leaves of Euphorbia hirta were used to form silver nanoparticles. In the process dried leaves of the plant were mixed with aqueous solution of silver nitrate and stored at a low temperate. After centrifugation the supernatant was exposed to very high temperature forming silver nanoparticles. Marine bacteria too have been exploited to synthesize gold nanoparticles. This field is still under research for production of nanoparticles. In a study carried out by Sharma et al (2012), marine bacteria Marinobacter Pelagius was used to synthesize gold nanoparticle. In was concluded that the bacteria produced stable, monodisperse gold nanoparticles. In another study, Selenium nanoparticles were synthesized using Klebsiella pneumoniae in selenium chloride solution. The broth culture of K. pneumoniae was sterilized with selenium nanoparticles. Released selenium nanoparticles showed no chemical changes. It was concluded that the wet heat sterilization process can be used to recover elemental selenium from bacterial cells. (Fesharaki et al, 2010)

5 In a study it was reported that previously unexploited fungus Hormoconis resinae was exploited for synthesize of silver nanoparticle. On treatment of aqueous solutions of silver with fungus, silver nanoparticles could be rapidly fabricated within an hour. These nanoparticles were characterized with UV-Vis spectroscopy, XRD, TEM and EDX analysis which revealed that the silver nanoparticles are polydisperseand of different morphologies ranging from 20 to 80 nm in size. (Varshney et al, 2009) In another study Cu 2 O nanoparticles were synthesized from Fehling s solution using Tridax procumbens leaf extract. Water soluble components present in the leaf extract were responsible for the conversion of copper ions to nano-sized copper particles. (Gopalakrishnan et al, 2012) In another study carried out by Shanmugam et al (2013), reduction of marine bacteria Vibrio alginolyticus was carried to synthesize extracellular and intracellular silver nanoparticle. It was concluded that it is a eco-friendly method for synthesizing nanoparticles. In a study, nanoparticles were synthesized by using aqueous extract of Moringa oleifera and metal ions (silver ions). M. oleifera leaf extract was selected as it is of high medicinal value and it does not require any sample preparation and hence is cost-effective. The fixed ratio of plant extract and silver ions were mixed and kept at room temperature for reduction. The color change from yellow to reddish brown confirmed the formation of nanoparticles. Further, the synthesized nanoparticles were characterized by UV, EPMA, XRD and FTIR data. (Mubayi et al, 2012) 2.4 CHARACTERIZATION OF NANOPARTICLES: The need to characterize nanoparticles in solution before assessing the in vitro toxicity is a high priority. Particle size, size distribution, particle morphology, particle composition, surface area, surface chemistry, and particle reactivity in solution are important factors which need to be defined to accurately assess nanoparticle toxicity. In a study, characterization of a wide range of nanomaterials was carried out using dynamic light scattering (DLS) and tr ansmission electron microscopy, including metals, metal oxides, and

6 carbon-based materials, in water and cell culture media, with and without serum. Cell viability and cell morphology studies were conducted in conjunction with DLS experiments to evaluate toxicological effects from observed agglomeration changes in the presence or absence of serum in cell culture media. Observations of material-specific surface properties were also recorded. It was also necessary to characterize the impact of sonication, which is implemented to aid in particle dispersion and solution mixture. Additionally, a stock solution of nanomaterials used for toxicology studies was analyzed for changes in agglomeration and zeta potential of the material over time. Corresponding toxicity data revealed that the addition of serum to cell culture media can, in some cases, have a significant effect on particle toxicity possibly due to changes in agglomeration or surface chemistry. It was also observed that sonication slightly reduces agglomeration and has minimal effect on particle surface charge. Finally, the stock solution experienced significant changes in particle agglomeration and surface charge over time. (Murdock et al, 2007) In a study, the synthesis of antibacterial silver nanoparticles using leaf broth of medicinal herb, Ocimum sanctum (Tulsi). The synthesized Silver nanoparticles were characterized by UV-Vis spectroscopy, transmission electron microscopy (TEM), and X -ray diffractometry. The size of which was found to be 18 nm. The qualitative assessment of reducing potential of leaf extract was also carried out which indicated presence of significant amount of reducing molecules. FTIR analysis revealed that the silver nanoparticles were stabilized by eugenols, terpenes, and other aromatic compounds present in the extract. (Ramteke et al, 2013) In another study carried out by Mishra et al (2010), gold nanoparticles of various shapes like hexgon, triangles etc were synthesized within 20 minutes by reducing gold chloride in the presence of dry leaf powder of Stevia rebaudiana. This study was the first report of using this plant for nanomaterial synthesis. The characterization of which was carried out by ultravioletvisible (UV -Vis) spectrophotometry, high-resolution transmission electron microscopy (HR - TEM), energy-dispersive Xray (EDX) spectroscopy, and X-ray diffraction (XRD). The UV-Vis spectrum of the aqueous medium containing gold nanostructures showed a peak at 539 nm corresponding to the surface plasmon resonance band (SPR) of gold na noparticles. HR-TEM micrograph showed the formation of well-dispersed octahedral gold nanoparticles of size 8 20 nm. XRD spectrum of the gold nanoparticles exhibited Bragg s pattern of reflection.

7 2.5 TYPES OF METALLIC NANOPARTICLES: ZINC NANOPARTICLES: In a study carried out by Singh and Gopal (2007), zinc colloidal nanoparticles were synthesized using laser ablation technique. Zince nanoparticles were produced by pulsed laser ablation in an aqueous solution of SDS using distilled water as a solvent. Centrifugation was done to separate SDS and larger particles. TEM confirmed the presence of zinc nanoaparticle PLATINUM NANOPARTICLES: A method for synthesize of colloidal platinum nanoparticles was reported by Ahmedi et al (2004), with controlled shapes. In this method, the ratio of the concentration of the capping material to that of Pb 2+ was changed at room temperature. Therefore, changing its shape SILVER NANOPARTICLES: In a study, the synthesis of antibacterial silver nanoparticles using leaf broth of medicinal herb, Ocimum sanctum (Tulsi). The synthesized Silver nanoparticles were characterized by UV-Vis spectroscopy, transmission electron microscopy (TEM), and X -ray diffractometry. The size of which was found to be 18 nm. The qualitative assessment of reducing potential of leaf extract was also carried out which indicated presence of significant amount of reducing molecules. FTIR analysis revealed that the silver nanoparticles were stabilized by eugenols, terpenes, and other aromatic compounds present in the extract. (Ramteke et al, 2013) GOLD NANOPARTICLES: In another study carried out by Mishra et al (2010), gold nanoparticles of various shapes like hexgon, triangles etc were synthesized within 20 minutes by reducing gold chloride in the presence of dry leaf powder of Stevia rebaudiana. This study was the first report of using this plant for nanomaterial synthesis. The characterization of which was carried out by ultravioletvisible (UV -Vis) spectrophotometry, high-resolution transmission electron microscopy (HR - TEM), energy-dispersive Xray (EDX) spectroscopy, and X-ray diffraction (XRD). The UV-Vis spectrum of the aqueous medium containing gold nanostructures showed a peak at 539 nm

8 corresponding to the surface plasmon resonance band (SPR) of gold nanoparticles. HR -TEM micrograph showed the formation of well-dispersed octahedral gold nanoparticles of size 8 20 nm. XRD spectrum of the gold nanoparticles exhibited Bragg s pattern of reflection COPPER NANOPARTICLES: In a study carried out by Theivasanti and Alagar (2010), copper nanoparticles were synthesized by dissolving copper sulphate salt in distilled water and electrolysed. The copper nanoparticles were formed at the cathode and were carefully removed. Antibacterial activity of copper nanoparticles synthesized by electrolysis was evaluated by using standard Zone of Inhibition (ZOI) microbiology assay. The sample copper nanoparticles prepared in electrolysis method showed diameter of inhibition zone against E.Coli was 15 mm and B.megaterium was 5 mm. Other metallic nanoparticles like zinc, tin lead and iron have also being researched on. 2.6 ANTIMICROBIAL PROPERTIES OF NANOMATERIALS: The antimicrobial activity of many types of nanoparticles is certainly a function of their size but other features are important such as high surface area, unusual crystal morphologies (edges and corners) and reactive sites. It is recognized that the main mechanism or the pools of mechanisms behind the antimicrobial activity of these nanostructures are not fully elucidated. Hence, several studies focusing on the antimicrobial activity of different metals and metallic nanoparticles against many species of bacteria and fungi have to be carried out for a clear picture on this matter. A study was carried out to evaluate the potential of nano silver to remove bacterial contaminants. Experiment involved Nano silver with five rates (8, 10, 20, 50, 80 mg/l) in MS medium. Explants were cultured on MS medium and evaluate at four times (1, 2, 3, 4 weeks). Adding nano silver (50 mg L - 1) to media and evaluate at second week was fully effective to control the microorganism infection. This research shows that nano silver had a good potential for removing of the bacterial contaminants in plant tissue culture procedures. (Safavi et al, 2011)

9 In a study, a cost effective and environment friendly technique for green synthesis of silver nanoparticles from silver nitrate through the extract of papaya fruit as reducing as well as capping agent was studied. Nanoparticles were characterized using UV Vis absorption spectroscopy, FTIR, XRD and SEM. X-ray diffraction and SEM analysis showed the average particle size of 15 nm as well as revealed their cubic structure. The antimicrobial activity assay was done on human pathogen, Escherichia coli and Pseudomonas aeruginosa by standard disc diffusion method. The Silver nanoparticles synthesized via green route are highly toxic to multidrug resistant bacteria hence has a great potential in biomedical applications. (Jain et al,2009) In a study, silver nanoparticles were synthesized using chemical reduction method. Silver nitrate was taken as the metal precursor and hydrazine hydrate as a reducing agent. The formation of the silver nanoparticles was monitored using UV-Vis absorption spectroscopy and the size of the colloidal silver nanoparticle was found to be 60nm. The antibacterial activity of the nanopartículas dispersion was measured by Kirby-Bauer method. The nanoparticles of silver showed high antimicrobial and bactericidal activity against gram positive bacteria such as Escherichia Coli, Pseudimonas aureginosa and Staphylococcus aureus which is a highly methicillin resistant strain. (Guzman et al, 2009) In a study, many silver nanoparticles were synthesized by solution phase route. Different shapes of silver nanoparticles were synthesized. Triangular prism nanoparticles were prepared by reducing silver nitrate at room temperature. Spherical silver nanoparticles were synthesized by using sodium citrate and sodium borohydride as reducing agents. The antibacterial susceptibility of silver nanoparticles was evaluated using the zone inhibition method. A sample of bacterial suspension cultured in LB was spread on a nutrient agar plate. The plates were then holed and supplemented with spherical silver nanoparticles and triangular silver nanoprisms to determine the different antibacterial properties depending on size and shapes; the plates were then incubated further at 37 C. The zones of inhibition were calculated after 24 h of incubation. The result revealed that the triangular nanoprisms with sharp edges and vertexes pose very high antibacterial properties, compared with spherical-shaped silver nanoparticles. (Dong et al, 2012)

10 In a study carried out by Saxena et al (2010), an onion (Allium cepa) extract was used to synthesize silver nanoparticles at a rapid rate. The antimicrobial activity was studied by the growth curve of E.coli and Salmonella typhimurium. Fresh cultures were inoculated in LB broth. Silver nanoparticles were added at different concentrations (10-50 ug/ml). The nanoparticles at concentration 50ug/ml were showed complete antibacterial activity against E.coli and Salmonella typhimurium. In a study, the antibacterial properties of differently shaped silver nanoparticles against the gramnegative bacterium Escherichia coli, both in liquid systems and on agar plates. Energy-filtering transmission electron microscopy images revealed considerable changes in the cell membranes upon treatment, resulting in cell death. Truncated triangular silver nanoplates with a {111} lattice plane as the basal plane displayed the strongest biocidal action, compared with spherical and rodshaped nanoparticles and with Ag (in the form of AgNO 3 ). It is proposed that nanoscale size and the presence of a {111} plane combine promote this biocidal property. This study was the first comparative study on the bactericidal properties of silver nanoparticles of different shapes, and our results demonstrate that silver nanoparticles undergo a shape-dependent interaction with the gram-negative organism E. coli. (Pal et al, 2007) In a study carried out by Theivasanti and Alagar (2010), copper nanoparticles were synthesized by dissolving copper sulphate salt in distilled water and electrolysed. The copper nanoparticles were formed at the cathode and were carefully removed. Antibacterial activity of copper nanoparticles synthesized by electrolysis was evaluated by using standard Zone of Inhibition (ZOI) microbiology assay. The sample copper nanoparticles prepared in electrolysis method showed diameter of inhibition zone against E.Coli was 15 mm and B.megaterium was 5 mm. In a study, biological synthesis of silver nanoparticles was carried out by using leaf extract of Eclipta alba. The antibacterial activity was carried out using five different strains. Zone of inhibition in the plate showed that silver nanoparticles synthesized using aqueous leaf extract of Ecliptalva alba have the antibacterial activity against test pathogens namely Staphylococcus aureus, Escherichia coli, Pseudomonous sp, Proteus vulgaris, and Salmonella typhi through the inhibition of zone formation. Zone of inhibition was measured and compared with control silver nitrate solution. On comparison with the silver nitrate

11 and plant extracts silver nanoparticles outperformed in the bactericidal effect. (Saminathan, 2015) In a study, silver nanoparticles were green synthesized using leaf extract of Euphorbia hirta. Further, the silver nanoparticle revealed to possess an effective antibacterial property against B.cereus and S.aureus. The present study emphasizes the use of plants medicinal for the syntheis of Ag nanoparticles with potent antibacterial effect. (Elumalai et al, 2010) In another study Cu 2 O nanoparticles were synthesized from Fehling s solution using Tridax procumbens leaf extract. Water soluble components present in the leaf extract were responsible for the conversion of copper ions to nano-sized copper particles. The antimicrobial activity was also studied. By taking Escherichia coli as a model for Gram-negative bacteria, which always causes a variety of suppurative infections and toxinoses in humans, as a model bioparticle, the negative bioeffect of nano-cu2o on E.coli cells was evaluated by disc diffusion method. Concentrations of 10, 20 and 50 ug/ml of CuO 2 were used. The result showed that the growth of bacteria was inhibited by 65% at a concentration of 20 ug/ml. (Gopalakrishnan et al, 2012) In a study, emphasizes on screening of herb with potent antimicrobial activity, synthesis of silver nanoparticles using herb extracts, treatment of 100% cotton woven & Spunbonded/Melt blown/spunbonded (SM S) nonwoven fabrics with silver nanoparticles, assessment of their antimicrobial efficacy and finally assessment of wash durability was done. Based on the antimicrobial activity and availability Tulsi (Ocimum sanctum) was selected. Results of qualitative and quantitative analysis confirmed that silver nanoparticles synthesized from Tulsi leaf extract treated fabrics has shown maximum antimicrobial activity against Pseudomonas aeruginosa and multi drug resistant strain of Pseudomonas aeruginosa in both case (woven and nonwoven) among all bacterial species under study. The comparative studies reported that the treatment with silver nanoparticles from Tulsi leaf extract has maximum antimicrobial activity while comparing with QAC and silver nitrate for both sensitive and resistant strains of Pseudomonas aeruginosa. (Patel and Desai,2014) In another study, silver nanoaprticles were synthesized by using bark of a herbal plants Boswellia ovalifoliolata and Shorea tumbuggaia. These were also tested for their antimicrobial efficacy. The test cultures included Proteus,Klebsiella, Psuedomonas, Bacillus and E.coli. The inhibitory

12 zone was measured to detect the antimicrobial activity. The silver nanoparticles of Boswellia ovalifoliolata were more toxic to Proteus sp. and moderately toxic to E.coli. The silver nanoparticles of Shorea tumbuggaia were more toxic to Klebsiella sp. and moderately toxic to E.coli. (Savithramma et al, 2011)