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Summer Research Fellowship Programme of India's Science Academies

ANTIMICROBIAL COTTON FABRICS GRAFTED WITH ZINC NANOPARTICLES SYNTHESISED BY THE MANGROVE PLANT RHIZOPHORA ANNAMALAYANA KATHIR

Namitha Prathap

M.Sc Marine Biology, School of Marine Sciences, CUSAT, Kochi

Guided by:

Dr K Kathiresan

Center of Advanced Studies in Marine Biology, Annamalai University, Parangipettai 608502

Abstract

In a modified green protocol, leaf extract from mangroves Rhizophora Annamalayana was used to generate Zn nanoparticles. Characterization of Zn nanoparticles obtained thereof was achieved through Ultraviolet-Visible (UV-Vis) spectrophotometric analysis, Scanning Electron microscopy (SEM) analysis, FTIR analysis, and Dynamic Light Scattering (DLS) analysis. Crystalline nature of zinc nanoparticles was confirmed using XRD. The XRD pattern showed intense peaks in the whole spectrum of 2θ value ranging from 30 to 80 degrees. The shape and size of the zinc nanoparticles produced were studied by using Scherrer’s equation, Scanning Electron Microscopy and Atomic Force Microscopy. The particles were mostly spherical and cubical in nature with the size <35nm. Cotton fabric coated with Zn nanoparticles were tested for antimicrobial and antifungal activity using standard protocols. Nano Zn treated fabric surface exhibits swelling nature due to the cross-linking of zinc with fabrics.

Keywords: Zinc nanoparticles, zinc nanoparticle synthesis, plant extract, antimicrobial cotton fabrics.

INTRODUCTION

Nanoscience refers to the study, manipulation and engineering of matter, particles and structures on the nanometer scale (one millionth of a millimeter). Nanotechnology is the application of nanoscience leading to the use of nanomaterials and nanosize components in useful products. Nanomaterials are materials whose size range between 1 nm and 100 nm. As the particles get smaller, they tend to react differently with their environment than the larger particles. Their penetration power is also high, compared to larger particles. It includes nanocapsules, quantum dots, ultrafine aerosols, nanoparticulate materials, nanoparticles, nanotubes etc. Nanobiotechnology involves biological production and utilization of nano-materials using microorganisms as well as plant and animal-based products (Kathiresan 2007). Hence, both unicellular and multicellular organisms as well prokaryotes and eukaryotes are known to produce inorganic materials either intracellularly or extracellularly (Mann, 1996). In nature, numerous inorganic materials are synthesized by living organisms. These bioinorganic materials can be extremely complex both in structure and function. Inorganic materials in the form of hard tissues form an integral part of most multicellular biological systems. The interest in nanoparticle synthesis using plants has emerged quite recently. Biosynthesis of nanoparticles is accomplished by using plants through enzymes, metabolites, and reductants, generated by plant cell activities.

A recently devised wound dressing has become a major breakthrough in the management of wounds or infections. In order to prevent or reduce infection, a new generation of dressing, incorporating antimicrobial agents like silver was developed (Langford and Burrell, 1999). Most recently, the application of silver nanoparticles (AgNPs) to cotton fabrics received a great deal of attention particularly because of their high resistance to microbes (Vigneshwaran, et al., 2006). Nowadays, AgNPs based topical dressings are widely used as a treatment for infections in burns, open wounds, and chronic ulcers (Lansdown, 2002). Nanoparticles treated fabrics have some functional behaviours including antibacterial activity, UV-protection, self-cleaning etc. These types of textile materials, especially cotton fabrics, are needed in the increasingly demanding human society with environmental safety (Kitkulnumchai et al., 2008; Abidi et al., 2009; Sorapong et al., 2006). The applications of cotton fabrics increase day by day. The improved qualities and functionalities of cotton fabrics are necessary for children’s cloths, inner wear, medical bandage cloths, etc. (Teli and Shrish Kumar, 2007). The structural modification of a cellulose fabric is possible and this type of fabric has some interesting properties. A recent report deals with such a modification, that is, cyclodextrin modified cotton fabric with thymol for antibacterial activity (Rukmani and Sundrarajan, 2011) and PVP modification of cotton fabric and its improved antibacterial activity tested with ZnO nanoparticles coating (Selvam and Sundrarajan, 2012). The present work, therefore, was undertaken to find out antimicrobial efficiency of fabrics incorporated with different ratios of zinc nanoparticles.

AIM AND OBJECTIVES

Nanoparticles play a vital role in the field of biology and medicine. Among nanoparticles, zinc nanoparticles have important and diverse properties, as antioxidants, anticancer agents, nanofertilizers and antimicrobials. With the development of new chemical and physical methods for synthesis of zinc nanoparticles, the concern for environmental contaminations is also increasing. Hence, there is a growing need to develop a method for the synthesis of zinc nanoparticles, which should be environmentally benign, by not using toxic chemicals in the synthetic protocols and by not liberating the hazardous by-products. In this regard, nanoparticle synthesis by using biological systems such as microbes and plants are advantageous over chemical and physical methods as the biological method of synthesis is cost-effective and environment-friendly and does not require high pressure, energy, temperature and toxic chemicals (Kathiresan et al., 2009; Asmathunisha et al., 2010). The use of plant extracts for the synthesis of nanoparticles could be advantageous over other environmentally benign biological processes as they eliminate the elaborate process of maintaining microbial cell cultures. However, such attempts of using plant extracts are confined to a few terrestrial plants and not the mangrove plants. The present study attempts to test the potential of the mangrove plant in the synthesis of zinc nanoparticles and their antimicrobial activity for use in cotton napkins. The experiments are designed with the following objectives

1.      To synthesis zinc nanoparticles with the mangrove plant, Rhizophora annamalayana;

2.      To understand the biosynthesis as influenced by plant extraction, concentration of substrate and time of reaction;

3.      To characterize the zinc nanoparticles synthesized using UV spectra, SEM, FTIR, XRD, and DLS;

4.      To prepare antimicrobial cotton fabrics using zinc nanoparticles for testing their antimicrobial efficiency.

REVIEW OF LITERATURE

INTRODUCTION

Nano-biotechnology is the technology of the current century. Research and development in this field are growing rapidly throughout the world (Vidya et al., 2013). A major contribution of this field is the development of new materials in the nanometer scale (Sivakumar et al., 2011). These are usually particulate materials with at least one dimension of less than 100 nanometers (nm). The particles could even be zero dimension in the case of quantum dots (Vidya et al., 2013). Metal nanoparticles have been of great interest due to their distinctive features such as catalytic, optical, magnetic and electrical properties (Garima et al., 2011). Nanoparticles exhibit completely new or improved properties with the larger particles of the bulk materials, and these novel properties are derived due to the variation in specific characteristics such as size, distribution, and morphology of the particles (Ravindra et al., 2011). 

Conventional methods of synthesizing nanoparticles using chemicals are more expensive and toxic, involving hazardous chemicals responsible for various biological risks (Geoprincy et al., 2012). To avoid the use of toxic chemicals, scientists have developed better methods which can be approached in two ways. First one is the use of microorganisms such as bacteria, fungi, and yeast (Helan et al., 2013). Using microorganisms for the synthesis of nanoparticles were found to be more tedious and required more steps in maintaining cell culture, intracellular synthesis with more purification steps while the second one was with the use of plants known as Green synthesis or Biogenic synthesis.

This type of biosynthesis shows better advancement over chemical and physical methods as it is less toxic, cost effective, environmental friendly (Vidya et al., 2013), and also involves proteins as capping agents (Sangeetha, et al., 2011). Proteins give low toxic, degradable end products (Raja et al., 2014). Hence biogenic synthesis of nanoparticles was found to be more attractive for research as conventional methods were more expensive and non-ecofriendly (Raj and Jayalakshmy, 2015a). The nanoparticles synthesized using the plant system can be utilized for different commercial applications like pharmaceuticals, drugs, therapeutics, etc. (Mohanpuria et al., 2008 and Bhattacharya and Mukherjee, 2008). 

Zinc (Zn) is an essential nutrient required by all living organisms and represents the 23rd most abundant element on earth (Broadley et al., 2007) and the 2nd most abundant transition metal, subsequent to iron (Jain et al., 2010). It is required in six different classes of enzymes, which include oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases (Auld, 2001). Zinc has been considered as an essential micronutrient for metabolic activities in plants and animals including humans.

Zinc Nanoparticles are used in various agricultural experiments to understand its effect on growth, germination, and various other properties (Fageria et al., 2002). The crop yield and quality of products can be affected by the deficiency of Zn (Jamali et al., 2011). 

ZINC OXIDE NANOPARTICLES

ZnO is an n-type semiconducting metal oxide. Zinc oxide NP has drawn interest in the past tfew years due to its wide range of applicability in the field of electronics, optics, and biomedical systems (Anbuvannan et al., 2015; Sundrarajan, et al., 2015; Vanathi, et al., 2014; Jamdagni et al., 2016; Prasad and Jha, 2009; Patil and Taranath, 2016; Gunalan et al., 2012). Several types of inorganic metal oxides have been synthesized and remained in recent studies like TiO2, CuO, and ZnO. Of all these metal oxides, ZnO NPs is of maximum interest because they are inexpensive to produce, safe and can be prepared easily (Jayaseelan, et al., 2012). US FDA has enlisted ZnO as GRAS (generally recognized as safe) metal oxide (Pulit-prociak, et al., 2016). ZnO NPs exhibit tremendous semiconducting properties because of their large band gap (3.37 eV) and high exciton binding energy (60meV) like high catalytic activity, optic, UV filtering properties, anti-inflammatory, wound healing (Mirzaei and Darroudi, 2016; Patel et al., 2015; Stan, et al., 2015; Sherly et al., 2014; Sangeetha, et al., 2011; Elumalai, et al., 2015; Sheikhloo et al., 2015). Due to its UV filtering properties, it has been extensively used in cosmetics like sunscreen lotions (Wodka, et al., 2010). It has a wide range of biomedical applications like in drug delivery, anti- cancer, anti- diabetic, antibacterial, antifungal and agricultural applications (Sangani, et al., 2015; Hameed, et al., 2016; Movahedi, et al., 2014; Martínková, et al., 2009; Jain, et al., 2014). Although ZnO is used for targeted drug delivery, it still has the limitation of cytotoxicity which is yet to be resolved (Ma, et al., 2013). ZnO NPs have a very strong antibacterial effect at a very low concentration of gram negative and gram positive bacteria as confirmed by the studies. Their antibacterial effect is stronger than the ZnO NPs synthesized chemically (Vimala, et al., 2014; Venkatachalam, et al., 2016; Hazra, et al., 2016). They have also been employed for rubber manufacturing, paint, for removing sulfur and arsenic from water, protein adsorption properties, and dental applications. ZnO NPs have piezoelectric and pyroelectric properties (Jha, 2007; Nagajyothi, et al., 2014). They are used for disposal of aquatic weed which is resistant to all type of eradication techniques like physical, chemical and mechanical means (Rajeshkumar, 2016). ZnO NPs have been reported in different morphologies like nanoflake, nanoflower, nanobelt, nanorod and nanowire (Paulkumar, et al., 2013; Paulkumar, et al., 2014, Rajeshkumar, et al., 2014). 

Biosynthetic Zinc oxide nanoparticles (ZnO NPs) produced using a natural source are readily scalable and nontoxic compared with physical and chemical methods (Jayapaul et al., 2011). ZnO-NPs are used in the preparation of substances in medical and cosmetic processes. Due to its antibacterial properties, ZnO is applied against skin irritation, diaper rash, dry skin, and blisters. ZnO-NPs can also be used for selective destruction of tumor cells and has a great potential in drug delivery applications (Rasmussen et al., 2010). ZnO-NPs have also been shown to exhibit strong protein adsorption properties, which can be used to modulate cytotoxicity, metabolism or other cellular responses (Horie and Nishiok, 2009). Apart from these many applications, ZnO, due to its low toxicity is listed as “Generally Recognized as Safe” (GRAS) by the US Food and Drug Administration (21, CFR 182, 8991)..

Due to the increasing popularity of green methods, biosysnthesis of ZnO NPs using different sources like plants, bacteria, fungus, algae and others have been attampted. 

SYNTHESIS OF ZnO NANOPARTICLES FROM LEAF EXTRACTS

Coriandrum sativum leaf was used for the synthesis and characterization of ZnO nanoparticles prepared by green and chemical techniques. The characterization of ZnO-NPs was carried out by XRD, SEM, FTIR, and EDAX. The average size was found to be 66 nm in green synthesis method while the size was 81nm in the chemical method. The ZnO nanoparticles prepared from Coriandrum leaf extract were expected to have more extensive application in biotechnology, sensors, medical, catalysis, optical devices, DNA labeling, drug delivery and water remediation (Gnanasangeetha and Thambavani, 2013).

The synthesis of Zinc oxide nanoparticles was reported with the leaf extract of Ocimum tenuiflorum. Prepared ZnO nanoparticles were characterized by XRD, SEM and FTIR. The SEM image showed hexagonal shape and nanoparticle with diameter range of 11-25 nm (Raut et al., 2013).

Calotropis gigantea leaf extract was used for the synthesis of Zinc oxide nanoparticles. The synthesized Zinc nanoparticles were characterized using SEM and XRD. The synthesized nano crystallites of ZnO were in the range of 30-35 nm (Vidya et al., 2013), whereas the synthesis of leaf extract of Calotropis procera was reported by Poovizhi and Krishnaveni and FTIR and SEM characterized the synthesized ZnO-NPs. The size of the particles ranged from 100 to 200 nm. The antibacterial activity towards human bacterial and plant pathogens showed good sensitivity towards the green synthesized ZnO-NPs at all concentrations. The study indicated that the C. procera ZnO nanoparticles had strong antimicrobial activity against the tested human and plant bacterial pathogens along with the fungal pathogens (Poovizhi and Krishnaveni, 2015). Different parts of the Calotropis gigantea plant were used in traditional Indian medicine for the treatment of painful muscular spasm, dysentery, fever, rheumatism, asthma and as an expectorant and purgative (Ravindra et al., 2011).

The synthesis of Zinc oxide nanoparticles was reported with the leaf extract of Olea europea. Prepared ZnO nanoparticles were characterized by UV-Vis Spectroscopy, FTIR, XRD and SEM. The average size of particles was found to be 500 nm and the thicknesses was about 20 nm by SEM studies. FT-IR analysis of aqueous Olea europea leaf extract indicated the presence of phytoconstituents such as amines, aldehydes, phenols and alcohols. These are surface active molecules that stabilize the Zinc oxide nanosheets (Awwad et al., 2014).

Chemotherapeutic agents from Catharanthus roseus are known for their pain relieving property in cancer treatment. It is cultivated mainly for its alkaloids, which have anticancer properties (Jaleel et al., 2009). The synthesized Zinc oxide nanoparticles were characterized using XRD, SEM, EDAX and FT-Raman Spectroscopy. The SEM results showed that the particles were spherical in shape with an average size of 23 to 57 nm. The synthesized ZnO-NPs were evaluated for antibacterial activity against Bacillus thuringiensis, Escherichia coli, Staphylococcus aureus and Pseudomonas aeuroginosa. The highest antimicrobial activity was observed against Pseudomonas aeuroginosa followed by Staphylococcus aureus (Bhumi and Savithramma, 2014b).

The synthesis of Zinc oxide nanoparticles was reported with the leaf extract of Adhatoda vasica. The Synthesized ZnO-NPs were characterized by UV-Vis Spectroscopy, SEM, EDAX, XRD, and FT- Raman Spectroscopy. The Synthesized ZnO-NPs were found to be discoid in shape with an average size of 19 - 60 nm. Phytochemicals present in the plant were responsible for the quick reduction of Zn+ ion to metallic Zinc Oxide nanoparticles. The synthesized ZnO-NPs had the potential to mitigate the bacterial cell proliferation particularly in Escherichia coli, Bacillus thuringiensis, Pseudomonas aeurogonisa and Staphylococcus aureus. Adhatoda vasica is a good source for rapid reduction of metallic Zinc oxide into nanoparticles with antibacterial activity. Adhatoda vasica has been used as the reducing material as well as a surface stabilizing agent for the synthesis of ZnO NPs (Bhumi et al., 2014a).

The leaf extract of Hibiscus rosa sinensis was used for the synthesis of Zinc oxide nanoparticles. The particle size and morphology of the synthesized nanoparticles were characterized by using SEM and XRD. The SEM image showed relatively spongy shape nanoparticles and size were found to be in the range of 30-35 nm. Bala et al. reported the synthesis of Zinc oxide nanoparticles from the leaf extract of Hibiscus subdariffa, and the formation of synthesized ZnO nanoparticles was confirmed by UV-Vis Spectroscopy, FTIR, XRD, HRTEM, EDX and FESEM. The synthesized ZnO nanoparticles had potential anti-bacterial properties which have been studied on Escherichia coli and Staphylococcus aureus. Another study has indicated that the small-sized ZnO NPs, stabilized by plant metabolites had better anti-diabetic effect on streptozotocin (STZ) induced diabetic mice than that of large-sized ZnO particles (Bala et al., 2015). The flowers of Hibiscus rosa sinensis are edible and are used in salads in the Pacific Islands. The flower is additionally used in hair care as a preparation (Devi and Gayathri, 2014).

The synthesis of Zinc oxide nanoparticles was reported with the leaf extracts of Azadirachta indica and Emblica officinalis. The formed nanoparticles were characterized by SEM, XRD, FTIR, and EDAX for their morphology, size, crystallinity and percentage composition. The synthesized nanoparticles were found to be in the range of 100-200 nm by SEM results. The qualitative examination of the aqueous extracts of the leaf samples of Azadirachta indica and Emblica officinalis showed the presence of phytochemical constituents such as Alkaloids, Carbohydrates, Glycosides, Steroids, Flavonoids, Terpenoids, Tannins, and Steroids (Gnanasangeetha and Thambavani, 2014).

Zinc oxide nanoparticles were synthesized from the leaf extract of Tanners cassia (Cassia auriculata). The synthesized Zinc oxide nanoparticles were confirmed by SEM, UV- Vis Spectrophotometer and FTIR. The SEM studies revealed that the synthesized ZnO-NPs were spherical in shape (Ramesh et al., 2014a).

Zinc oxide nanoparticles were synthesized with the leaf extract of green tea (Camellia sinensis). The synthesized Zinc oxide nanoparticles were characterized using XRD, UV–VIS spectrum and FTIR. The average size of the nanoparticles calculated using XRD data was 16 nm (Senthilkumar and Sivakumar, 2014) whereas Shah et al., reported on green tea and synthesized Zn nanoparticles were characterized by UV-Vis Spectroscopy, Particle size analyzer, and SEM. Particles size analyzer determined the size of the particles and was found to be 853 nm in diameter. The synthesized Zn nanoparticles showed significant antimicrobial activity against Gram positive and Gram negative bacteria as well as against a fungal strain. The study also suggested that the green synthesized Zn nanoparticles could be used as an alternative to existing antimicrobial agents (Shah et al., 2015). The synthesized ZnO NPs showed better and comparable antimicrobial activities compared to chemically synthesized drugs (Senthilkumar and Sivakumar, 2014).

The plant leaf extract of Brassica oleraceae was used for the synthesis of Zinc oxide nanoparticles. The characterization of the synthesized nanoparticles was examined by UV- Vis spectrum and SEM. The particles were spherical and sheet shape. The experimental results showed that the diameters of prepared nanoparticles in the solution had sizes between 1 and 100 nm. The whole plant body of Brassica oleraceae had possessed aphrodisiac activities, and it had a significant role in maintaining maleness. Siddha medicine explained it as rejuvenating herb, and it was known to possess coumarin, which was responsible for the hypolipidemic activity. The nanoparticles showed antibacterial activity against both Gram-positive and negative bacteria. The antibacterial activities increased as the concentration of Zinc oxide nanoparticles increased (Amrita et al., 2015).

The plant leaf extract of neem (Azadirachta indicia) was used for the synthesis of Zinc oxide nanoparticles. The synthesized ZnO nanoparticles were characterized using FTIR spectroscopy and SEM analysis. The SEM results revealed that the particles were spindle-shaped and their sizes were 50 μm (Noorjahan et al., 2015).

The leaf extract of Pyrus pyrifolia was used for the synthesis of Zinc oxide nanoparticles. The structural, morphological and optical properties of the synthesized nanoparticles have been characterized by using UV–Vis spectrophotometer, XRD, FTIR, AFM, and FE – SEM with EDX analysis. The synthesized ZnO NPS were found to be almost spherical in shape with a particle size of around 45 nm. The photocatalytic study concluded that the bio – ZnO NPs had the efficiency to dye degrade methylene blue under solar irradiation. Therefore, the study could find application in water treatment plants and textile industries (Parthiban and Sundaramurthy, 2015).

The synthesis of Zinc oxide nanoparticles was done by using biological and chemical reducing agents. The synthesized nanoparticles in the biological method used Pithecellobium dulce and Lagenaria siceraria leaf extracts and in the chemical method using sodium hydroxide as reducing agents. In traditional medicine, the leaves of P. dulce was used for the treatment off earache, leprosy, peptic ulcer, toothache, venereal diseases and also showed emollient, anodyne, larvicidic (Sugumaran et al., 2008) and abortifacient, and antidiabetic properties in folk medicine. Various parts of plant were used for different purposes like a leaf as astringent, seed oil as spermicidal, anti-inflammatory, anti-oedemia, fruit and seed as edible, bark for tannin (Mohammed et al., 2004). Lagenaria siceraria, which had diuretic and anti-swelling effects, was used as food (Wang and Ng, 2000). A decoction of Lagenaria siceraria was employed in the treatment of anasarca, ascites, and beriberi (Anandh et al., 2014). The biologically synthesized ZnO Nanoparticles showed better antimicrobial activities with respect to the activities of synthetic drugs (Prakash and Kalyanasundharam, 2015).

The synthesis of Zinc nanoparticles was reported with the leaf extract of Thevetia peruviana. The plant contained a poisonous toxin called thevetin, which was used as a heart stimulant (Singh et al., 2012). These plant toxins were also used as biological pest controls (Kareru et al., 2010). The synthesized Zinc oxide nanoparticles were characterized using UV-Visible Spectroscopy, FTIR, Particle size analyzer, XRD, SEM, TEM, Inductively Coupled Plasma-Optical Emission Spectrophotometer. The average particle size of synthesized Zinc nanoparticles was found to be 53 nm. SEM and TEM data revealed the presence of triangular shaped and poly-dispersed zinc nanoparticles with a grain size of 50 ± 5 nm. The synthesized Zinc nanoparticles were applied on Arachis hypogaea L (peanut) pot-culture to estimate soil microbial population, soil exo-enzyme activities and physiological growth parameters of the peanut plants. Zinc nanoparticles applied to the peanut pot-culture exhibited good soil microbial and enzyme activities by showing significant variations compared to the control and enhanced the physiological growth parameters of peanut plants (Sindhura et al., 2015).

The Zinc oxide nanoparticles were synthesized with the leaf extract of Aloe vera. XRD, SEM, UV-Vis Spectroscopy, PL, BET, and TGA were used for characterization of synthesized ZnO nanoparticles. The particles were a hexagonal shape with an average size of 22.18 nm. Photodegradation and antibacterial activity of the nanoparticles were studied. The antibacterial studies of synthesized nanoparticles showed sensitivity to both Gram positive and Gram negative bacteria (Varghese and George, 2015).

Corymbia citriodora leaf extract was used for the synthesis of Zinc oxide nanoparticles. SEM, EDX, XRD, UV–VIS spectroscopy, Raman spectroscopy and TGA had been used for characterizing the biosynthesized ZnO NPs. The synthesized nanoparticles exhibited polyhedron shape with a size range between 20 and 120 nm and showed excellent dispersibility with an average size of 64 nm. The photocatalytic activity of biosynthesized ZnO NPs has been evaluated by the photodegradation of methylene blue. The results showed that the biosynthesized ZnO NPs had higher photocatalytic performance than normal hydrothermally prepared ZnO NPs due to the smaller size (Zheng et al., 2015). 

GREEN SYNTHESIS OF ZNO NPS USING BACTERIA

Nanoparticles synthesis using bacteria is a green approach but it has several disadvantages like screening of microbes is a time-consuming process, careful monitoring of culture broth and the entire process is required to avoid the contamination, lack of control on NP size, shape and cost associated with the media used to grow bacteria is also very high.

ZnO nanoflowers were synthesized by B. licheniformis through an eco-friendly approach which showed photocatalytic activity, degraded Methylene blue dye. These nanoflowers showed enhanced photocatalytic activity as compared to already present photocatalytic substances and it has been presumed that larger oxygen vacancy in the synthesized nanoparticles imparts it the property of enhanced photocatalytic activity. Photocatalysis generates active species by absorption of light which degrades the organic waste material and thus can be used as an effective bioremediation tool. Nanoflowers synthesized using B. licheniformis were 40 nm in width and 400 nm in height (Raliya and Tarafdar, 2013). Rhodococcus is able to survive in adverse condition and it has the ability to metabolize hydrophobic compounds thus, can help in biodegradation (Otari, et al., 2012). Spherical shaped NPs had been synthesized using Rhodococcus pyridinivorans and Zinc Sulphate as a substrate which showed size range of 100–130 nm confirmed through FE-SEM and XRD Analysis. It also demonstrated the presence of Phosphorus compound, secondary sulphonamide, mono-substituted alkyne, β -lactone, amine salt, amide II stretching band, enol of 1-3-di ketone, hydroxy aryl ketone, amide I bending band, alkane, and mononuclear benzene band confirmed through FTIR analysis (Tripathi et al., 2014). ZnO was used as a substrate to synthesize ZnO NP through A. hydrophilla. NPs synthesized showed size range of 42–64 nm, confirmed through AFM and XRD analysis with varied shapes like oval and spherical (Mehta et al., 2009). Singh et al. compared the antioxidant activity of bare ZnO NP and Pseudomonas aeruginosa rhamnolipid stabilized NPs and it had been found that rhamnolipid stabilizes the ZnO NP because it is tough to form micelle aggregates on surface of carboxymethyl cellulose (Kundu, et al., 2014) and it acts as a better capping agent because of its long carbon chain (Singh et al., 2014). It showed the formation of spherical shaped NP with nano size of 27–81 nm confirmed through TEM, XRD, and DLS analysis (Singh et al., 2014).  

GREEN SYNTHESIS OF ZNO NPS USING MICROALGAE AND MACROALGAE 

Algae are a photosynthetic organism rannging from unicellular forms (ex. Chlorella) to multicellular ones (ex. Brown algae). Algae lack basic plant structure like roots and, leaves. Marine algae are categorized based on the pigment present in them like Rhodophyta having red pigment, Phaeophyta with brown pigment and chlorophytes with green pigment. Algae have been used extensively for the synthesis of Au and Ag nanoparticles but its application for the ZnO nanoparticle synthesis is limited and reported in very less number of papers (Thema, et al., 2015). Microalgae draw special attention because of its ability to degrade toxic metals and convert them to less toxic forms (Bird et al., 2015). Sargassum muticum and S. myriocystum be-longing to Sargassaceae family have been used for ZnO NP synthesis. Sargassum muticum studied size of NPs using XRD and FE-SEM which showed similar ranges and hexagonal wurtzite structure with the presence of hydroxyl group and sulfated polysaccharides. S. myriocystum compared size using DLS and AFM which showed different size ranges with the presence of hydroxyl and carbonyl stretching in NPs which vary greatly in shape (Rajiv et al., 2013).

GREEN SYNTHESIS OF ZNO NPS USING FUNGUS

 Extracellular synthesis of NPs from the fungus is highly useful because of large scale production, convenient downstream pro-cessing and economic viability (Azizi et al., 2014). Fungal strains are chosen over bacteria because of their better tolerance and metal bioaccumulation property (Pati et al., 2014). ZnO NPs were synthesized from mycelia of As-pergillus fumigatus. DLS analysis revealed the size range of NPs to be 1.2 to 6.8 with the average size of 3.8. AFM confirmed the average height of NP to be 8.56 nm. Particle size was >100 nm for 90 days but after 90 days they formed an agglomerate of average size 100 nm which suggested the stability of formed NPs for 90 days (Pavani et al., 2012). NPs synthesized from Aspergillus terreus belonging to Tri-chocomaceae family had a size range of 54.8–82.6 nm confirmed by SEM and the average size of 29 nm calculated using Debye-Sherrer equation through XRD analysis results. It confirmed the presence of primary alcohol, primary or secondary amine, amide, aromatic nitro compounds in the NPs formed confirmed through FTIR studies (Hoffmann et al., 1995). NPs synthesized using Candida albicans showed similar size range 15–25 nm confirmed by SEM, TEM and XRD Analysis (Shamsuzzaman et al., 2013). Aspergillus species have been widely employed for the synthesis of ZnO NPs and NPs synthesized from fungal strain were spherical shaped in most of the cases. Table 4 gives a brief account of commonly used fungus utilized for ZnO NP synthesis.

GREEN SYNTHESIS OF ZNO NPS USING OTHER GREEN SOURCES

Biocompatible chemicals are used as some other green sources for the synthesis of nanoparticles. It is a fast, economic process which eliminates the production of any kind of side product in the nucleation and synthesis reaction of nanoparticles. It leads to the formation of controlled shape and size nanoparticles with their well-dispersed nature (Nagarajan and Kuppusamy 2013). Nanoparticles synthesized through wet chemical method render them special properties like enhanced anti-bacterial efficiency up to 99.9% when coated on a cotton fabric (Anita et al., 2010) Table 5 illustrated a few other green sources that have been employed for the synthesis of ZnO NP (Nagarajan and Kuppusamy 2013; Anita et al., 2010; Gharagozlou, et al., 2015).

MATERIALS AND METHODS

DESCRIPTION OF STUDY AREA: VELLAR ESTUARY

VELLAR.png
    Vellar estuary

    The study area is of a mangrove forest, rose artificially in 15 hectare area on the northern bank of the Vellar estuary (Lat. 11° 29’ 19.1-25.2”N; Long. 79° 45’ 51.9-57.3”E), located along the Bay of Bengal on the south eastern coast of the state of Tamil Nadu, India. The mangrove forests are dominated by two plant species, Avicennia marina (Forsk.) Vierh. and Rhizophora mucronata Poir., R. annamalayana Kathir., is a rare species (Fig.1)

    2.jpg
      Rhizophora annamalayana Kathir.

      PLANT USED FOR NANOPARTICLE SYTHESIS

      In the present study, the leaves of mangrove plant, Rhizophora annamalayana Kathir. were collected from 3rd and 4th Position from the tip of shoot branch.was and tested for the synthesis of zinc nanoparticles. (Fig.2)

      PREPARATION OF LEAF EXTRACT

      Leaf sample was extracted using different methods namely (i) extraction in boiling water; (ii) extraction by grinding followed by filtration through Whatman no 1 filter paper (iii) extraction by grinding under ice cold condition followed by filtration through muslin cloth. For boiling extraction, the leaf weighing 20 g was thoroughly washed, finely cut, and boiled in a 500-mL Erlenmeyer flask with 100 ml of sterile distilled water for 1 hour before finally decanting it. In fresh leaf extraction method, the cut leaves were ground using a mortar and pestle, and then it was filtered using a filter paper. The other method of leaf extraction was ice cold extraction, in which the cut leaves were ground in ice cold condition which was then filtered using muslin cloth. These three extractions were stored in 4 ºC. Among the three methods boiled extraction method gave the best result (Fig. 3).

      3.png
        Different method of plant extraction (A) Boiled extraction (B) Fresh leaf extraction (C) Ice cold extraction (D) Prepared plant extract

        TESTING OF LEAF EXTRACT FOR ZINC NANOPARTICLES SYNTHESIS

        For reduction of zinc ions, 5 ml of leaf extract was added to 45 ml of 5 different concentrations (0.1, 0.25, 0.5, 0.75, 1 mM) (Fig. 4) of zinc oxide solution in 1:9 ratio. The reduction of pure Zn ions was monitored by measuring the absorbance of the solution at regular intervals. The absorption was measured at the range between 200-700 nm at a spectrophotometer (Elico, Chennai). Reduction process was very slow, and formation of nanoparticles was identified from the colour change (pale yellow to reddish brown) of the solution only after 72 hours. For quick synthesis, the molar concentration of the zinc oxide was altered by taking 0.01, 0.025, 0.05, 0.075 and 0.1 M that was then mixed with plant extract in the same 1:9 ratio. The colour changed to dark reddish colour within 1 hour, as confirmed with UV-spectrum. Among the five concentrations, the colour intensity was recorded the highest in 0.1 M substrate concentration. Then it was taken for bulk synthesis.

        To study the effect of light and dark conditions, two sets were prepared. One set was incubated under dark and another one under light exposed condition. Both samples i.e., kept in dark and light showed similar rate of Zn nanoparticle synthesis, which indicated that intensity of light was not a factor influencing the nanoparticle synthesis. The colour change was seen within 30 minutes of sample preparation. In order to confirm the optimal ratio, the samples were prepared in different ratios i.e., 1:99 (1:99 ml), 1:19 (5:95 ml) and 1:9 (10:90 ml) in 100 ml; out of which, 1:9 gave quick synthesis. Similarly samples were prepared by using three different leaf extraction methods with 0.1 M substrate, the colour intensity was the highest in the boiled extract. The suspended nanoparticles were separated by centrifugation with 8000 rpm for 15 min and followed by 3000 rpm for 5 min. After decanting the mother liquor, the zinc nanoparticles were thoroughly washed with distilled water and the pure particles were separated and dried in vacuum and stored in a dark bottle at 4 ºC for further characterization.

        4.png
          (A) 0.1mM-1mM zinc oxide (B) 0.1mM-1mM zinc oxide

          BIOSYNTHESIS OF ZINC NANOPARTICLES

          For synthesis of zinc nanoparticles, 90 ml of zinc oxide solution was mixed with 10 ml of leaf extract of Rhizophora annamalayana in a 250 ml Erlenmeyer flask and incubated at 25oC in dark. Control (without addition of plant extract, only zinc oxide solution) was also run along with the experimental flask. One ml of sample was withdrawn and the optical density was taken at a broad range of 200 to 700 and at a narrow range of 360 to 380 nm.

          CHARACTERIZATION OF NANOPARTICLES

          Ultraviolet-Visible (UV-Vis) specrophotometric analysis

          UV-Visible spectroscopy is a fundamental system to find out the synthesis of nanoparticles as well as to determine the morphology and stability of nanoparticles. The colour change was observed in the metal ion solution incubated with plant extracts. The bioreduction of metal ions in solution was monitored by measuring the UV-Vis spectra of the solution in 10 mm optical-path-length quartz cuvettes with UV-Vis spectrophotometer at a resolution of 1mm between 200 and 700 nm.

          X-Ray Diffraction Pattern

          The bio-reduced metal ion solutions were drop-coated on to glass substrate and the powder X-ray diffraction measurement was carried out with an X-Ray diffractrometer (XPERT-PRO). The pattern was recorded by Zn Kα 1 radiation with λ of 1.5406 Ǻ and nickel monochromator filtering the wave tube voltage at 40 kV and tube current of 30 mA. The scanning was done in the region of 2θ from 300 to 800 at a speed of 0.020/min and the time constant was 2s. The size of the nanoparticles was calculated through the Debye-Scherrer’s formula.

          Scanning Electron microscopy (SEM)

          Scanning Electron Microscopic (SEM) analysis of nanoparticles synthesized by Rhizophora annamalayana, was carried out at the Department of Manufacture Engineering, Annamalai University. The ultra-thin sections were connected on copper grid stained with uranyl acetate and lead citrate and observed under JEOL JEM 100SX Scanning Electron Microscope at 80 KV.

          FTIR analysis

          For FTIR analysis, 100 ml of nanoparticle solution was centrifuged at 8000 rpm for 15 min. The supernatant was again centrifuged at 3000 rpm for 5 min and the pellet was obtained. This was followed by re-dispersion of the pellet of nanoparticles into 1 ml of deionized water. Thereafter, the purified suspension was freeze-dried to obtain dried powder. Finally, the dried nanoparticles were ground with KBr pellets and analyzed by FTIR instrument in the diffuse transmittance mode, operating at a resolution of 4 cm-1 over 4000-500 cm-1.

          Dynamic Light Scattering (DLS) Analysis

          Dynamic light-scattering measurement was performed for analyzing size groups of nanoparticles using a Nano ZS apparatus at 25ºC and started 2 min after the cuvette was placed in the DLS apparatus to allow the temperature to equilibrate. Measurement was carried out on 24 h after the preparation of the suspension.

          EFFECT OF ZINC NANOPARTICLES ON ANTIBACTERIAL ACTIVITY OF COTTON FABRICS

          Fabrication of nanoparticles

          Cotton fabrics used for the experiments were made by 100% cotton yarns. The fabrics were thoroughly cleaned and dried. The fabrications were performed on cotton materials with maximum dimension of 15 cm × 15 cm. Cotton fabrics were padded with different ratio (90:10, 95:5, 99:1) of zinc nanoparticle solutions at concentrations of 100 ppm; the concentrations were achieved through diluting the original solution of nanoparticles with distilled water. For complete dispersion of colloidal nanoparticles on fabrics, the padded fabrics were kept under UV light for 4 hours. All samples were immersed in the colloidal nanoparticles for 4 hour and then subjected to dryness under cold conditions (Fig. 5). Then the fabrics incorporated with nanoparticles were subjected to antibacterial assay.

          Picture1.png
            Cotton fabric treated with three different ratio (90:10, 95:5, 99:1) of zinc nanoparticles (A) Before treatment (B) After treatment

            SEM analysis

            Scanning Electron Microscopic (SEM) analysis of nanoparticles incorporated fabrics was carried out at the Department of Manufacture Engineering, Annamalai University. The ultrathin sections were connected on copper grid stained with uranyl acetate and lead citrate and observed under JEOL JEM 100SX Scanning Electron Microscope at 80 KV.

            Antimicrobial activity

            The antimicrobial activity of fabrics was evaluated against 3 bacterial pathogens and 2 fungal pathogens, Escherichia coli, Klebsiella oxytoca, Vibrio parahaemolyticus, penicillim sp., Candida sp.. In order to study the antibacterial activity of the fabrics, squares of 1 cm of each fabric was prepared in aseptic manner. Each square was placed in Muller Hinton Agar medium swabbed with bacterial pathogens and PDA medium swabbed with fungal pathogens. Control squares with or without addition of nanoparticles were also placed. The plates were kept under incubation at 37° C. After 24 hours, the inhibition zone diameter was measured in mm for antimicrobial activity. 

            RESULTS

            TESTING OF MANGROVE LEAF EXTRACT FOR THE SYNTHESIS OF NANOPARTICLES

            Rhizophora annamalayana extract was tested for the synthesis of nanoparticles. The plant extract was treated with zinc oxide in different concentrations for different durations of incubation up to 24 hours (Figs. 6 - 9). The colour change was observed visually as well as by using a spectrophotometer.

            Picture2.png
              Zinc nanoparticles synthesis using mM concentration of zinc oxide Initial
              Picture3.png
                Zinc nanoparticles synthesis using mM concentration of zinc oxide After
                6.png
                  Zinc nanoparticles synthesis using M concentration of zinc oxide Initial
                  6_1.png
                    Zinc nanoparticles synthesis using M concentration of zinc oxide After

                    VISUAL OBSERVATION OF COLOUR CHANGE

                    The change in colour of the reaction mixture was noted by visual observation. The plant extract incubated with zinc oxide, at the beginning of the reaction showed pale yellow colour, and gradually increased in colour intensity to dark orange, with the increasing period of incubation. The colour of the reaction mixture changed pale yellow to dark orange after one hour of incubation. Control without plant extract did not exhibit any colour change (Fig. 10).

                    10.jpg
                      Bulk synthesis of Zinc nanoparticles
                      11.jpg
                        Light and dark effect of Zinc nanoparticles (A) Initial (B) Light (C) Dark

                        SPECTRAL OBSERVATION OF COLOUR CHANGE

                        The surface plasmon resonance of zinc nanoparticles synthesized by Rhizophora annamalayana is depicted in Fig. 12. The peak of colour intensity was observed at 1st hour of incubation. There was no significant change of colour intensity beyond 1st hour of incubation. The highest colour intensity was recorded in 0.1 M concentration of zinc oxide. The optical density and peaks of zinc nanoparticles of Rhizophora annamalayana are depicted in Fig. 12.

                        12.png
                          UV absorbance spectrum of zinc nanoparticles

                          X-RAY DIFFRACTION PATTERN

                          The crystalline nature zinc nanoparticles produced by Rhizophora annamalayana was confirmed using XRD. X-ray diffraction pattern is shown in Fig 13. The XRD pattern showed intense peaks in the whole spectrum of 2Ɵ value ranging from 15 to 35.

                          13.png
                            . X-ray diffraction pattern of zinc nanoparticles

                            DYNAMIC LIGHT SCATTERING OF ZINC NANOPARTICLES

                            Dynamic light scattering (DLS) has become a popular method of measuring the size of colloidal nanoparticles. Fig. 14 represents the size distribution of zinc particles as determined by the DLS technique. The calculated particle size by volume was in the range of 15 – 30 nm for zinc nanoparticles.

                            Picture4.png
                              DLS pattern of zinc nanoparticles

                              FTIR SPECTRUM OF ZINC NANOPARTICLES

                              The bands at 3838.34 cm-1, 3797.84 cm-1, 3749.62 cm-1 corresponded to hydroxyl group. The bands at 2360.87 cm-1, 2341.58 cm-1 corresponded to aromatic group. The bands 1770.65 cm-1, 1714.72 cm-1 corresponded to acid halide and open chain anhydride. The bands 1558.48 cm-1, 1541.12 cm-1, 1506.41 cm-1 corresponded to aliphatic nitro compounds. The bands at 1489.05 cm-1, 1473.62 cm-1 , 1456.26 cm-1 corresponded to methyl C-H bond. The band at 1338.60 cm-1 corresponded to methylene C-H bond. The bands at 507.28 cm-1, 493.78 cm-1, 486.06 cm-1, 480.28 cm-1, 470.63 cm-1 corresponded to polysulfides (s-s). The bands at 462.92 cm-1, 453.27 cm-1, 443.63 cm-1, 432.05 cm-1 corresponded to aryl sulfides(s–s). (Fig.15)

                              15.png
                                FTIR spectrum of zinc nanoparticles

                                Scanning Electron Microscopy (SEM) of zinc nanoparticles

                                The SEM micrograph of Zinc nanoparticles is shown in Fig. 16 & 17. The scanning electron microscopy showed the formation of nano-sized zinc particles with same shape and different size with scattered distribution. In general, all the nanoparticles were in spherical shape with the size less than 35 nm.

                                16.jpg
                                  SEM micrographs of zinc nanoparticles
                                  17.jpg
                                    SEM micrographs of zinc nanoparticles
                                    18.png
                                      EDS pattern zinc nanoparticles

                                      Fabrication of nanoparticles

                                      In SEM studies, the untreated cotton fabric surface had a clean, ribbon like structure and no deposition on its surface. In zinc treated fabric surface exhibited swelling nature and it is due to the cross-linking of zinc with fabrics, and the spherical shaped zinc nanoparticles are clearly seen in Fig. 19. The deposition of zinc nanoparticles is viewed at higher magnification (Fig. 19).

                                      19.jpg
                                        Microscopic view of cotton fabrics treated with zinc nanoparticles
                                        20.jpg
                                          Microscopic view of cotton fabrics treated with zinc nanoparticles
                                          21.png
                                            EDS profile of cotton fabrics treated with nanoparticles

                                             Antimicrobial activity of nanoparticles treated fabrics against clinical pathogens

                                            Antimicrobial activity of fabrics coated with zinc nanoparticles synthesized by Rhizophora annamalayana is shown in Figs. 18-21. The nanoparticles incorporated fabrics showed higher antimicrobial activity against all the test microbes. The highest inhibition zone of 42 mm diameter was formed against Vibrio parahemolyticus by the zinc nanoparticle (90:10) treated cotton fabrics, and the lowest of 13 mm was produced against Penicillium sp., by the zinc nanoparticle coated cotton fabric (Fig. 23). From the graph, all the ratio of zinc nanoparticles exhibited good antimicrobial activity. All the test pathogens were effectively inhibited by zinc nanoparticles (90:10) treated cotton fabrics, as compared with 95:5 and 99:10. Among the test microbes, Escherichia coli was most inhibited by all the three ratios of zinc nanoparticles incorporated fabrics.

                                            Picture5.png
                                              Disc diffusion method of different ratio of zincnanoparticles (1) 90:10 (2) 95:5 (3) 99:1 (C) Control
                                              Picture6.png
                                                Diameter of inhibition zone of microbial pathogens treated with zinc nanoparticles
                                                Picture7.png
                                                  Cotton fabrics treated with different ratio of zinc nanoparticles (1) 90:10 (2) 95:5 (3) 99:1 (C) Control
                                                  Picture8.png
                                                    Diameter of inhibition zone of microbial pathogens treated with 90:10 ratio of zinc nanoparticles

                                                    DISCUSSION

                                                    In the present investigation, zinc nanoparticles were synthesised by using the mangrove plant Rhizophora annamalayana. The nanoparticles were characterized by using ultra violet visible spectrophotometry, powder X-ray diffractometer (XRD) analysis, Fourier transform infrared spectroscopy (FTIR), Scanning electron microscopy (SEM), Transmission electron microscopy, and Dynamic light scattering.

                                                    UV-Vis spectroscopy is an effective technique to ascertain the formation and stability of nanoparticles in aqueous solution. Colloidal particles have an absorption band in visible region and the λmax is dependent on their size and the shape (Creighton and Eadon, 1991). According to the Mie theory, the isotropic spherical nanoparticles exhibit only one (out of plane) SPR absorption band, whereas anisotropic nanoparticles show two or three (in plane) SPR bands (Van de Hulst, 1957; Kerker, 1969). The formation of unusually shaped nanoparticles can be determined through plasmon absorption spectroscopy, because their optical properties of aqueous suspension intimately associate with the shapes (Shankar et al., 2005). The formation of in plane SPR band indicates the formation of nanoparticles of varied size and shape nanoparticles. Decreasing symmetry means more peaks of various size and shape in the localized SPR. Zinc nanoparticles were confirmed by techniques like UV-Vis spectroscopy, which showed the formation of broad resonance spectral band indicating an aggregated structure (Fig. 10) (Bali et al., 2006; Rajendran et al., 2010).

                                                    X-ray diffraction (XRD) pattern gives information about the crystal structure of the material. XRD of zinc nanoparticles revealed the formation of face centered cubic (fcc) and spherical structured nanocrystals. The mean size of zinc (Fig. 12) nanoparticles were calculated using the Debeye-Scherrer’s equation by determining the width of the (111) Bragg’s reflection (Borchert et al., 2005). All the nanoparticles exhibited the Bragg’s reflection of (200), (220) and (311), which were considerably weak and broadened relative to the intense (111) reflection. Moreover, the ratio between the intensity of the (200) and (111) diffraction peaks is lower than the conventional bulk intensity ratio suggesting that the (111) plane is predominantly oriented. XRD analysis of thin film of Zinc nanoparticles demonstrated that as the peak height decreased with corresponding increase in width, and there was a decrease in the crystallite size of nanoparticles (Fig. 12). The results of zinc are in good agreement with previous works (Clifford et al., 2007; Rajendran et al., 2010; Thi et al., 2012).

                                                    The applications of nanoparticles are dependent on their size and shape (Sankar Kalidas et al., 2009). The shape and size of zinc nanoparticles produced by Rhizophora annamalayana were mostly spherical in nature with the size of >35 nm (ranging from 15 -35 nm) as evident by SEM and also revealed by DLS (Figs. 12 and 14). Thus, sometimes, different techniques give different size averages. This difference in the particle sizes obtained using the microscopic techniques is perhaps due to differences in sample preparation and dispersity of the particles (Prathna et al., 2011). Such variation in the size of nanoparticles synthesized by biological systems is common. The zinc nanoparticles produced from wet chemical method is 60-75 nm (Rajendran et al., 2010). The leaf extract of Hibiscus rosa -sinensis used for the synthesis of Zinc oxide nanoparticles exhibits a relatively spongy shape and the size is found to be in the range of 30-35 nm.

                                                     The synthesis of Zinc oxide nanoparticles is reported with the leaf extracts of Azadirachta indica and Emblica officinalis. The synthesized nanoparticles are found to be in the range of 100-200 nm by SEM results. The qualitative examination of the aqueous extracts of the leaf samples of Azadirachta indica and Emblica officinalis shows the presence of phytochemical constituents such as Alkaloids, Carbohydrates, Glycosides, Steroids, Flavonoids, Terpenoids, Tannins, and Steroids (Gnanasangeetha and Thambavani, 2014).

                                                     The Zinc oxide nanoparticles synthesized from the leaf extract of Tanners cassia (Cassia auriculata) reveal that the -NPs are spherical in shape (Ramesh et al., 2014a).

                                                     The Zinc oxide nanoparticles are synthesized with the leaf extract of green tea (Camellia sinensis) exhibit 16 nm as the average size of the nanoparticles calculated using XRD data (Senthilkumar and Sivakumar, 2014) whereas Shah et al (2015) have reported the synthesis of Zn nanoparticles by using green tea and characterized by UV-Vis Spectroscopy, Particle size analyzer, and SEM. Particles size analyzer has determined the size of the particles as 853 nm in diameter. In the present study, EDS profile showed zinc signals along with copper peak, which originated from the biomolecules that were bound to the surface of the zinc nanoparticles, suggesting that zinc nanoparticles were successfully synthesized using R. annamalayana leaf extract (Fig. 15).

                                                     The average particle size was found to decrease with increasing concentrations of substrates. The reason for the decrease in particle size with substrate concentration was not clear at this point. . It is considered that particle size and shape are dependent on various conditions such as plant type, nanoparticle types, reaction temperature and composition of reaction mixture and so on.

                                                    FTIR measurements were carried out to predict the potential biomolecules involved in reduction and the capping of bio reduced zinc nanoparticles. The IR bands of the zinc nanoparticles shows the presence of -OH hydroxyl groups revealed the nature of aromatic phenol, responsible for the reduction of zinc nanoparticles (Asmathunisha and Kathiresan, 2013). Presence of -NH stretch vibrations in the amide linkages of the proteins (Solumun et al., 2004; Philip 2009a; Philip 2009b ; Basavaraja et al., 2008), symmetric stretching of carboxyl (-COOH) side groups in the amino acid residues of the protein molecules (Huang et al., 2007; Shankar et al., 2004b; Ankamwar et al., 2005b; Shankar et al., 2003), and C-N stretching from amines (Inbakandan et al., 2010) are involved in capping and bioreduction of zinc and copper nanoparticles. Flavonones and terpenoids are likely responsible for the stabilization of the nanoparticles synthesised by mangrove extracts (Asmathunisha et al., 2012) (Fig.13).

                                                    In SEM studies, the untreated cotton fabric surface had a clean, ribbon like structure and no deposition on its surface (Fig. 16). In zinc treated fabric, the surface exhibited swelling (Fig. 16) and it was due to the cross-linking of zinc with fabrics, and the spherical shaped zinc nanoparticles are clearly seen in Fig. 16. The deposition of zinc nanoparticles can be viewed at higher magnification (Fig. 16). EDS profile also confirmed the presence of Zinc nanoparticles along with cotton fabrics (Fig. 17).

                                                    The nanoparticles incorporated fabrics showed higher antibacterial activity against all the test microbes. The highest inhibition zone of 42 mm diameter was formed against Vibrio parahemolyticus by the zinc nanoparticle (90:10) treated cotton fabrics, and the lowest of 13 mm was produced against Penicillium sp. by the zinc nanoparticles coated cotton fabric (Figs.18-21). From the graph, all the ratio of zinc nanoparticles exhibited antibacterial activity. All the test pathogens were effectively inhibited by zinc nanoparticles (90:10) treated cotton fabrics compared with 95:5 and 99:10. Among the test microbes, Escherichia coli was the most inhibited by all the ratios of zinc nanoparticles incorporated fabrics.

                                                    In general the antibacterial activity was high against Klebseilla and V. parahaemolytics in all the Zn nanoparticles treated fabrics. A similar report is available with Zhang et al., (2007). It seems that active oxygen species generated by ZnO particles can be a mechanism although there is no direct evidence from the results of the present study. . It has already been proved that both nano-sized and micron-sized ZnO suspensions are active in inhibiting the bacteria growth; the nano-sized ZnO suspension clearly has a much higher activity than the micro-sized ZnO suspension (Zhang et al., 2009). These results are in accordance with the present study.

                                                    SUMMARY

                                                    Of 10 different molar concentrations tested, the synthesis of zinc nanoparticles was maximum in 0.1M concentration as evident by the optical density of the reaction mixture after 1 hour of incubation.

                                                    The peak of colour intensity was observed at 1 hour of incubation. There was no significant change of colour intensity beyond 24 hours of incubation.

                                                    Leaf sample was extracted using different methods namely (i) extraction in boiling water; (ii) extraction by grinding followed by filtration through Whatman no 1 filter paper (iii) extraction by grinding under ice cold conditions followed by filtration through muslin cloth. Among the three methods boiled extraction method gave the best result.

                                                    Light was not a factor influencing zinc nanoparticle synthesis by Rhizophora annamalayana.

                                                    The crystalline nature of zinc nanoparticles produced by R. annamalayana was confirmed by using XRD. The XRD pattern showed intense peaks in the whole spectrum of 2θ value ranging from 15 to 35 nm.

                                                    The FTIR bands at 3800-3700 cm-1 corresponded to hydroxyl group. The bands at 2360.87 cm-1, 2341.58 cm-1 corresponded to aromatic group. The bands 1770.65 cm-1, 1714.72 cm-1 corresponded to acid halide and open chain anhydride. The bands 1500 cm-1 corresponded to aliphatic nitro compounds. The bands at 1480-1450 cm-1 corresponded to methyl C-H bond. The band at 1338.60 cm-1 corresponded to methylene C-H bond. The bands at 500-470 cm-1 corresponded to polysulfides (s-s). The bands at 460-430 cm-1 corresponded to aryl sulfides(s–s).

                                                    The shape and size of zinc nanoparticles produced by R. annamalayana were studied by using Scherrer’s equation, Scanning Electron Microscopy and Atomic Force Microscopy. The particles were mostly spherical and cubical in nature with the size <35 nm.

                                                    ·         The zinc treated fabric surface exhibited swelling nature, and it was due to the cross-linking of zinc with fabrics, and the spherical shaped zinc nanoparticles were clearly seen in the fabrics.

                                                    ·         The nanoparticles incorporated fabrics showed higher antimicrobial activity against all the test microbes. The highest inhibition zone of 42 mm diameter was formed against Vibrio parahemolyticus by the zinc nanoparticle (90:10) treated cotton fabrics, and the lowest of 13 mm was produced against Penicillium sp. by the zinc nanoparticle coated cotton fabric. From the graph, all the ratio of zinc nanoparticles exhibited good antimicrobial activity. All the test pathogens were effectively inhibited by zinc nanoparticles (90:10) treated cotton fabrics as compared with 95:5 and 99:10. Among the test microbes, Escherichia coli was the most inhibited by all the three ratio of zinc nanoparticles incorporated fabric.

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