Accepted Manuscript Antibacterial effects of biosynthesized MgO nanoparticles using ethanolic fruit extract of Emblica officinalis Kalimuthan Ramanujam, Mahalingam Sundrarajan PII: DOI: Reference:

S1011-1344(14)00289-9 http://dx.doi.org/10.1016/j.jphotobiol.2014.09.011 JPB 9841

To appear in:

Journal of Photochemistry and Photobiology B: Biology

Received Date: Revised Date: Accepted Date:

11 June 2014 2 September 2014 6 September 2014

Please cite this article as: K. Ramanujam, M. Sundrarajan, Antibacterial effects of biosynthesized MgO nanoparticles using ethanolic fruit extract of Emblica officinalis, Journal of Photochemistry and Photobiology B: Biology (2014), doi: http://dx.doi.org/10.1016/j.jphotobiol.2014.09.011

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Antibacterial effects of biosynthesized MgO nanoparticles using ethanolic fruit extract of Emblica officinalis Kalimuthan Ramanujam and Mahalingam Sundrarajan * Advanced Green Chemistry Lab, Department of Industrial Chemistry, School of Chemical Sciences, Alagappa University, Karaikudi -630 003, Tamil Nadu, India. *Corresponding author; Tel/Fax: +91 9444496151 / +91-04565-225202 E-mail:[email protected]

Abstract Magnesium oxides nanoparticles were successfully synthesized from Mg (NO3)2.6H2O through a simple greener route using fruit extract (Emblica officinalis). The synthesized samples were characterized by different techniques such as X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy and Scanning electron microscopy (SEM) with Energy dispersive X-ray spectroscopy (EDX) analysis. The XRD pattern shows the face centred cubic structure of MgO nanoparticles was confirmed by the Debye-Scherrer’s Formula. From the surface morphology the average nanoparticle size 27nm.

MgO nanoparticles treated cotton fabric

produced stronger antibacterial activity. These types of treated fabrics are used in medical application.

Keywords: Green synthesis, Magnesium oxide nanoparticles, Emblica officinalis, Sol-gel, Antibacterial activity

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1. Introduction Nano biotechnology is a growing, interdisciplinary field of research interlacing material science, bio nanoscience and technology. The advances made in the field of nano biotechnology to harness the benefit of life sciences, health care and industrial biotechnology are remarkable [13]. Nanoparticles exhibit completely new or improved properties with larger particles of the bulk materials and these novel properties are derived due to the variation in specific characteristics such as size and morphology of the particles [4]. Magnesium oxide (MgO) is an interesting basic metal oxide that has many applications. For example, MgO with ultrafine, nanoscale particles and high specific surface area have shown great promise as destructive adsorbent for toxic chemical agents. Nanoscale MgO exhibits unique optical, electronic, magnetic, thermal, mechanical and chemical properties due to its characteristic structures. Chemical synthesized prepared nanoparticles of various methods such as, sol-gel process, micelle, precipitation, hydrothermal and pyrolysis etc. In general, chemicals used for the nanoparticles synthesis and stabilization are toxic and led to non-ecofriendly byproducts are very expensive and use of hazardous chemicals mostly which cause danger to the environment and human beings[5]. To overcome these problems, the eco-friendly synthesis of nanoparticles using environmentally benign materials like Plants [6], Fungi [7], Seaweed [8], Bacteria [9] and Enzymes [10] were employed. In the above different parts were used for biosynthesis and the plants were ruled good results in nanoparticle synthesis.

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The various works on the medicinal plant (emblica officinalis) have the anti-bacterial [1113], anti-fungal [14], anti-oxidant [15], cardio-protective [16], anti-anthelmintic [17] and antiinflammatory properties [18]. These plants are useful in conjunctivitis, inflammation, dyspepsia, ulcerative stomatitis, gastrohelicosis, cough, diarrhoea, dysentery, diabetes, asthma, bronchitis, opthmopathy, colic, jaundice, emaciation, cardiac disorder, intermittent fever, hepatopathy, hemorrhages, menorrhagia and skin diseases [19- 20]. Emblica officinalis is a deciduous tree commonly known as “Indian gooseberry” or “Amla” and “Nelli” in Tamil. According to early Indian tradition, it is the first tree to be created in the world. It belongs to the family Euphorbiaceae as given below (Figure 1.1).

Figure 1.1: Emblica officinalis

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In these fruits are present in chemical composition of quercetin, phyllemblin compounds, gallic acid, tannins, flavonoids, pectin, vitamin C and polyphenolic compounds [21]. It is also a wide range of components including terpenoids, alkaloids, flavonoids, and tannins. Since the plant has versatile properties suitable for potential application in various fields. Novelty of work is green synthesized MgO nanoparticles used fruit (emblica officinalis) extracts. This is the first report for the new, rapid, room temperature and size controlled MgO nanoparticles procedure using a green reagent and their low cost, nontoxic, time consuming, pollution free, economic viability and antimicrobial agent. In this study it is possible to use these types of cotton fibres used in wounds stressing, surgical clothes and cotton bandages and other textiles. The present study describes antibacterial effects of biosynthesized MgO nanoparticles using an ethanolic fruit extract of Emblica officinalis. 2. Materials and Methods 2.1. Sample Collection The sample of Emblica officinalis fruit was collected from Karaikudi, Sivagangai District, Tamil Nadu, India. The bleached fabric was purchased from the textiles industry in Tirupur. Magnesium nitrate hexahydrate was purchased from Merck chemicals in India. Double distilled water was used throughout the experimental work. 2.2. Preparation of fruit extract (Emblica officinalis) The collected fresh, healthy fruits were washed thoroughly with double distilled water. About 100 grams of the collected fruits were finely cut into small pieces and dried at room temperature for 10 days under dust free condition. The dried fruits were crushed into the powder. The 2gram of powder were extracted with 50ml ethanol under reflux condition at boiling for about 1 hour. After the extraction, it was filtered through Whattman No.1. filter paper. The 4

filtration of the extract was collected in a closed container and stored in refrigerators for further use. 2.3. Green Synthesis of MgO nanoparticles using fruit extract ( Emblica officinalis) Green synthesized MgO nanoparticles were prepared by sol-gel method [22]. In a typical experiment, 0.2M solution of Magnesium nitrate hexahydrate (Mg (NO3) 2.6H2O) was prepared with 50ml of fruit extracts (Emblica officinalis). The reaction mixtures were stirred for 4 hours at room temperature.

After that, the stirred process was complete process in

precipitation of magnesium hydroxide was recognized by the brown colour colloidal particles at the bottom of the flask. It is believed that the colour change indicates the development of the hydrolysis of Mg (OH)2. After the completion of the reaction it is formed brown coloured precipitate was allowed to settle for one day. The precipitate was separated from this solution by centrifugation at 1000 rpm for 10 minutes and washed with water repeatedly and to remove the impurities then dried in hot air oven 80°C. The dried Mg (OH)2 sample were grinding and crushing by mortar and pestle. These samples were calcination in muffle furnace at 450°C for 3 hours. After the dehydrated process by calcination white coloured MgO nanoparticles were obtained. 2.4. Coated cotton fabric with green synthesis of MgO nanoparticles Green synthesis MgO nanoparticles using fruit extracts were applied on cotton fabric. Coated cotton fabric with metal oxide nanoparticles were carried out by the pad-dry-cure method. The cotton fabric specimen of dimension 12cm×12cm was immersed in the solution containing 3% of citric acid as a crosslinking agent and 3% of biosynthesized metal oxide nanoparticles for about 3 hours. Then these fabric samples were padded continuously for 15 min 5

in a two bowl padding mangle. Finally the padded fabric was subjected to air drying and curing at 150ºC for 3 minutes [23]. 2.5 Antibacterial evaluations of MgO nanoparticles Bacterial resistance of green synthesized MgO was measured by Agar diffusion (Kirby Bauer) method. The microorganisms were used for testing against S.aureus (gram positive bacteria) and E.coli (gram negative bacteria). The growth media, nutrient agar was inoculated with the test organism. The sample was placed on it in intimate contact followed by incubation at 37 °C for 24 h or until visible growth was established. The diameter of the zone of inhibition was then examined directly underneath and around the sample. The inhibition cleared zone in and around the sample determined the efficiency of the antibacterial agent to inhibit the growth of bacteria. 2.6. Characterization X-ray diffraction (XRD) analysis of MgO powder samples of glass substrates was carried out on the JEOL LDX 8030 instrument operating at 40kV with a current of 30mA using Cu K α radiation λ=1.5405Å over a wide range of Bragg angles(10°≤ 2θ ≤ 75°). The surface morphology of MgO nanoparticle was measured using a JEOL JSM 6390 model. An elemental analysis of the sample was studied by energy dispersive analysis of X-rays. Fourier transform infrared spectroscopy (FTIR) analysis of the samples was performed by Perkin Elmer make Model Spectrum RX1 (Range 4000 cm-1- 400 cm-1). The sample was dried at room temperature and then KBr pellets were prepared. The transmittance of the samples was measured.

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3. Results and Discussion 3.1. XRD Pattern of MgO nanoparticles Figure 1, shows the XRD pattern of the green synthesized MgO nanoparticles positions and intensities of all relative peaks of MgO nanoparticles matched with the Joint Committee on Powder Diffraction Standards (JCPDS) file no. (39-7746). All the recorded peak intensity profile was characteristic of the nanoparticles cubic structure. The mean particles size of the nanoparticles were estimate from the (FWHM) used in Scherer’s formula, D = Kλ/βcosθ Where, K is a constant equal to 0.94, β is the full width half maximum height of the diffraction peak at an angle θ and λ is the wavelength. These sample showed the presence of peaks 2θ = 36.92°, 42.92°, 62.27°, 74.66°, and 78.61° corresponding to (111), (200), (220), (311) and (222) planes respectively. From the table 1, the crystalline size determined for the mean particle size of the samples is around 27nm, which is well matched with the measured crystal diameter obtained from SEM images. 3.2. FTIR analysis of MgO nanoparticles FT-IR measurements were established to identify the biomolecules for capping and stabilization of the metal oxide nanoparticles synthesized by fruit extract. Figure 2, shows the green synthesized MgO using plant (emblica officinalis) fruit extracts when the absorption peaks were found at 436 and 541cm-1Mg-O-Mg [24]. The probable reason for the formation of MgO nanoparticles of the Emblica officinalis extracts played an effective role as stabilizing agent for the formation of MgO. The magnesium hydroxide on calcination at 450°C the bio-molecules 7

gets removed by decomposition due to their low thermal stability behavior whereas the inorganic stable products of the material were formed by their stable crystalline nature. The pattern of absorptions at 664 cm-1 matches up aromatic C-H. The absorption peak at 875cm-1 point out the aromatic stretching (out of plane bending). Primary amine (R-NH2) shows two N-H stretching bands in the range 3433 cm-1. The absorption peak at 1015 cm-1 corresponds to C-O stretching of saturated primary alcohol. At the peak at 2916 cm-1 is assigned to aromatic symmetric CH3 stretching band. Band absorption near 2373 cm-1 corresponds to acid because of the overlapping of C-H. In the peak values of the 1714cm-1carbonyl group (C=O) stretching. The above extra peaks appeared due to biological molecules may be present in synthesized metal oxide nanoparticles. 3.3. Surface morphology of MgO nanoparticles Scanning electron microscopy (SEM) analysis to study the surface morphology of the MgO nanoparticles was observed in spherical structure. The SEM image of the green synthesized of nanoparticles was carried out at the different magnification such as X1600 and X 8,000 are shown in the Figure 3. The particle size of the MgO is 27nm. The synthesized nanoparticles showed some agglomeration due to the polarity and electrostatic attraction of MgO nanoparticles. 3.4. EDX analysis of MgO nanoparticles Energy dispersive X-ray spectroscopy (EDAX) was employed to establish the element identity of the observed particles in Figure 4. In the analysis by energy dispersive X-ray

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spectroscopy (EDX) of the MgO nanoparticles, the presence of elemental metal signal (magnesium and oxygen) was confirmed. 3.5. Antibacterial properties of MgO treated and untreated fabrics An agar diffusion method was determined the antibacterial activity of green synthesized MgO nanoparticles treated and untreated cotton fabric against S. aureus (gram positive) and E. coli (gram negative) of the two bacterial pathogens. The antibacterial performance was done by using the agar diffusion method using amikacin as standard antibiotic. Hence the differential sensitivity of S. aureus and E. coli towards MgO depends upon particle size, temperature of synthesis, bacterial cell wall structure, and the degree of contact with organisms with nanoparticles. E. coli contains many layers composed of lipids, proteins and lipopolysaccharides in its membrane, thereby preventing against MgO whereas no such layers in S. aureus bacterial membrane except one thick peptidoglycan layer which contains mixture of sugars and amino acids. Hence the interaction between S. aureus and MgO are stronger than with E. coli. The antibacterial activity of MgO coated fabrics were tested against both gram positive and gram negative. Figure 5(a & b) shows the zone of inhibition of untreated and treated cotton fabrics. The nanoparticles coated cotton, stronger antibacterial activity with S.aureus than E.coli. The main reason of the bactericidal effect of MgO generally has been attributed to the decay of bacterial outer membranes by (ROS), primarily °OH, which leads to phospholipid peroxidation and ultimately cell death. It was proposed that nanomaterials that can physically attach to a cell membrane can be bactericidal if they come into contact with this bacterial cell [25]. If the membrane of a bacterium is conceded, the cell could repair itself or, if the cut is severe, the cell component may release and finally the cell will die. 9

4. Conclusion A green method of synthesized magnesium oxide nanoparticles (MgO) used fruit (Emblica officinalis) extracts. We have utilized the natural, renewable sources for the synthesis of nanoparticles. The XRD patterns of the sample average particle size are around in 27nm. The MgO nanoparticles were confirmed by SEM with EDX. These nanoparticles coated cotton fabric showed good antibacterial activity. The used fruit extract in the treatment could be the safer, potent and cost effective way to treat infectious diseases for human beings. These kinds of the fabric used in surgical clothes, wound stressing, bandages bed lining, active cotton bandages, as well as for medical and food applications. Acknowledgements The authors express their sincere thanks to Professor and Head, Dept. of Industrial Chemistry and Dept. of Physics, Alagappa University, Karaikudi, Tamil Nadu, India, for their encouragement and providing excellent facilities XRD and SEM analysis for the above work. And also records their thanks to UGC-BSR for providing research fund to pursue this work. References 1. J.L. Gardea Torresdey, G.L. Parsons, Formation and growth of Au nanoparticles inside live Alfalfa, Plant. Nano Lett. 2(4) (2002)397-401. 2. J.R. Stephen and S.J. Macnaughton, Developments in terrestrial bacterial remediation of metals, Curr. Opin. Biotechnol. 10(3) (1999)230-233. 3. H.J. Lee, S.J. Yeo and S.H. Jeong, Antibacterial effect of nanosized silver colloidal solution on textile fabrics, J Mater Sci. 38(2003)2199-2204.

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Figure Caption: Figure 1: XRD Pattern of MgO nanoparticles Figure 2: FTIR analysis of MgO nanoparticles Figure 3: Surface morphology of MgO nanoparticles at different magnifications X1600 and X8,000

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Figure 4: EDX images of MgO nanoparticles Figure 5: Antibacterial Activity of MgO nanoparticles (a) untreated cotton fabric (b), treated cotton fabric S.aureus and E.coli.

Table: Table 1: Crystallinity parameter of MgO nanoparticles Table 2: Zone of inhibition of MgO nanoparticles untreated and treated cotton against bacterial pathogens

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Fig. 1

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Fig. 2

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Fig. 3

17

Fig. 4

18

Fig. 5

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Table 1: Crystallinity parameter of MgO nanoparticles

Pos. [°2Th.]

FWHM[°2Th.]

d-spacing[Å]

Rel. Int. [%]

36.92

0.38

2.43307

6.15

42.911

0.317

2.10668

100.00

62.27

0.30

1.48963

52.69

74.66

0.32

1.27018

6.86

78.61

0.48

1.21637

8.60

20

Mean Lattice particle Plane size 111

21.93

200

27.44

220

31.15

311

31.14

222

21.36

Table 2: Zone of inhibition of MgO nanoparticles untreated and treated cotton against bacterial pathogens

S.No.

1. 2.

Test sample

Zone of inhibition(mm) S.aureus

E.coli

0

0

30

27

Untreated fabric MgO nanoparticles treated fabric

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Highlights: A novel approach of synthesis of magnesium oxide nanoparticles using Emblica officinalis fruit. ► The fruit extract acts as bio-reducing and capping agents. ► These nanoparticles formed are spherical in shape 42nm of size synthesized at 25 °C and 60 °C, respectively. ► Act as a good antibacterial agent against Gram-negative and Gram-positive bacteria.

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