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Journal of Garmian University

https://doi.org/10.24271/garmian.15

Characterization of iron nanoparticles of Citrus sinensis peel extract and studying its antioxidant activity Sundus Hameed Ahmed Ministry of Science and Technology, Agriculture directorate, Biotechnology Department, Iraq. *Corresponding author. [email protected].

ABSTRACT Iron nano particles Fe3O4 were synthesized in water using Citrus cinensis peel extract as a reducing agent. A systematic characterization of iron nano particles Fe3O4 was performed using AFM, atomic fluorescence microscope;SEM, scanning electron microscopy; XRD, X-ray diffraction studies. The diameter of iron nanoparticles was predominantly within the range 22 to 100 nm. Nano particle was studied for its in vitro antioxidant activity using different models viz. DPPH radical scavenging, ABTS radical scavenging, iron chelating activity, lipid peroxidation assay. Its antioxidant activity was estimated by IC50 value and the values are 45 μg/mL (DPPH radical scavenging), 11.9 μg/mL (ABTS radical scavenging), 56 μg/mL (Iron chelating activity) and 20.5 μg/mL (lipid peroxidation). In all the testing, a significant correlation existed between concentrations of the nano particle and percentage inhibition of free radicals, metal chelation or inhibition of lipid peroxidation. These results clearly indicate that C.cinensis is effective against free radical mediated disease. Key words: Iron nano particle, UV, XRD, SEM DPPH, lipid peroxidation.

INTRODUCTION Nature has devised various processes for the synthesis of nano and micro-length scaled inorganic materials which have contributed to the development of relatively new and largely unexplored area of research based on the biosynthesis 164

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of nanomaterials. Synthesis using bio-organisms is compatible with the green chemistry principles. “Green synthesis” of nanoparticles makes use of environmental friendly, non-toxic and safe reagents (Qui et al., 2007; Sharma et al., 2009; Prathna et al., 2010). Green nanotechnology has attracted a lot of attention and includes a wide range of processes that reduce or eliminate toxic substances to restore the environment. The synthesis of metal nanoparticles using inactivated plant tissue (Padil et al., 2010), plant extracts (Shameli et al.,2012) , exudates (Lukman et al., 2011) and other parts of living plants (Wang et al., 2014) is a modern alter native for their production.

The

development in the field of green chemistry has delivered different nanomaterials as substitute antibacterial agents. In this present study, an effort is made to synthesize iron nanoparticles using leaves extract of Citrus cinensis peel extract as reducing agent. The characterization of green synthesized iron nanoparticles was characterized by Atomic Fluorescence Microscope (AFM), scanning electron microscopy (SEM), X-ray diffraction (XRD), and atomic force microscopy (AFM). Also antioxidant activity is detected by free radical scavenger (DPPH), Fé2+ Chelation assay, ABTS radical scavenging assay Lipid peroxidation and thiobarbituric acid reactions in Egg phosphatidylcholine.

MATERIALS AND METHODS Materials The C.cinensis peel extract fruit was collected from Baghdad/ Iraq market. Iron sulfate was purchased from Sigma–Aldrich and used as received. Preparation of C.cinensis peel extract C.cinensis peel extracts using as a reducing agent for preparing Iron nanoparticles. About 25 g of peel powder was taken in a 100 mL beaker containing 50 mL double distilled water and then the peel was boiled at 80°C for10 min and filtered through Whatman No. 1 filter paper twice. The resultant filtrate was stored at 4°C and used as reducing and stabilizing agent. 165

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Synthesis of Fe3O4nano particles Nanoparticles of Fe3O4 were synthesized according to literature reported protocol (Ponder and Darab, 2000; Daraio et al., 2006) by hydrolysis of aqueous solution containing iron salts and a base at room temperature in ambient atmosphere.FeCl3.6H2O (1.35 g) and FeCl2.4H2O (0.6 g) were dissolved in 50ml of distilled water at 30°C. To the mixture 50 mL of 0.5M NaOH was added rapidly while stirring vigorously under inert gas atmosphere (N 2). Fe3O4 sediment was collected using a permanent magnet. The black powder was rinsed with double distilled water followed by washing with ethanol for several times to remove all impurities. A black precipitate was obtained under N 2 atmosphere and stored until use. Iron monosulphide (FeS) (100 mesh, 99.9%) microparticles (MPs) was obtained from Aldrich. UV-Vis Spectra analysis Vis spectral analysis was done by using UV-Vis spectrophotometer at the range of 330 to 450 nm and observed the absorption peaks at 340 to 350 nm regions, which are identical to the characteristics UV-visible spectrum of metallic Iron and it was recorded. SEM analysis of silver nanoparticles Thin films of the sample were prepared on a carbon coated copper grid by just dropping a very small amount of the sample on the grid, extra solution was removed using a blotting paper and then the film on the SEM grid were allowed to dry for analysis. AFM analysis For AFM analysis, the nanoparticle suspension was diluted with distilled water 1:2500 and dropped onto a surface of unruffled mica. After air drying, probes were scanned with a Dual Scope C26 (DME, Herlev, Denmark).

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DPPH radical scavenging assay To the methanolic solution of DPPH (1mM) an equal volume of the nano particle extract dissolved in water was added at various concentrations from 2 to 1000 μg/mL in a final volume of 1.0 ml. An equal amount of alcohol was added to the control. After 20 min, absorbance was recorded at 517 nm. Experiment was performed in triplicate (Sreejayan and Rao, 1996). ABTS radical scavenging assay To the reaction mixture containing 0.3 ml of ABTS radical, 1.7 mL phosphate buffer and 0.5 mL nano particle was added at various concentrations from 2 to 500 μg/mL. Blank was carried out without drug. Absorbance was recorded at 734 nm. Experiment was performed in triplicate (Sreejayan and Rao, 1996). Iron chelating activity assay The reaction mixture containing 1 mL O-Phenanthroline, 2 mL Ferric chloride, and 2 mL extract at various concentrations ranging from 2 to 1000 μg/mL in a final volume of 5 mL was incubated for 10 minutes at ambient temperature. The absorbance at 510 nm was recorded. Ascorbic acid was added instead of nano particle and absorbance obtained was taken as equivalent to 100% reduction of all ferric ions. Blank was carried out without drug. Experiment was performed in triplicate (Barnes et al., 2002). Lipid peroxidation assay The mixture (Egg phosphatidylcholine in 5 mL saline) was sonicated to get a homogeneous suspension of liposome. Lipid peroxidation was initiated by adding 0.05 mM ascorbic acid to a mixture containing liposome (0.1 mL). The pink chromogen was extracted with a constant volume of n-butanol and absorbance of the upper organic layer was measured at 532 nm (Govindarajan et al., 2003).

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Statistical analysis All results are expressed as mean ± S.E.M. Linear regression analysis (Origin 6.0 version) was used to calculate the IC50 values. RESULTS AND DISCUSSION The iron oxide nanoparticles (Fe3O4) synthesized by co-precipitation of ferric and ferrous chloride was validated by UV-Visible spectroscopic analysis and their scanning absorbance vs wave length (λ) has been established (Figure 1). The characteristics peaks of IO nanoparticles were observed at 370 nm, which is due to charge transfer spectra. Further analysis of the SEM image of synthesized iron oxide nanoparticles, showed a clear image of highly dense IO nanoparticles which are almost spherical in size (Figures1and2) showed biosynthesis of nanoparticle. Further analysis of the SEM image of synthesized iron oxide nanoparticles, showed a clear image of highly dense IO nanoparticles which are almost spherical in size(Barnes et al., 2002)(Figure 3). To investigate the morphology and dispersity of the synthesized nanoparticles, the AFM study has been performed and the resulting micrographs are presented in Figure4.It can be seen that the particles have irregular distribution and the particle size is about 35 nm. As evidenced by the, AFM and SEM studies, the particles are in cubic shape and average particle size is about 35 nm. Several concentrations ranging from 2 to 1,000 μg/mL of nano particle were tested for their antioxidant activity in different in vitro models. It was observed that free radicals were scavenged by the test compounds in a concentration dependent manner up to the given concentration in all the models. The percentage scavenging and IC50 values were calculated for all models given in Table 1. The antioxidative mechanism of antioxidants can result from metal chelation, free radical scavenging (hdrogen-donatation free radical quenching, or co-operative effects of these properties (Barnes et al., 2002). The ability of test sample to donate hydrogen was checked using stable free radical DPPH, is 168

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formed from the scavenger and the reaction is monitored by the decrease of the absorbance at 518 nm (Uirsini et al., 1994; Ganapaty et al., 2007). DPPH + antioxidant (purple color) ---------- DPPH-H+ antioxidant (yellow color) Oxidative stress has been implicated in the pathology of many diseases and conditions including diabetes, cardiovascular diseases, inflammatory conditions, cancer and ageing (Ganapaty et al., 2007). The reduction capability of DPPH radicals was determined by the decrease in its absorbance at 517 nm (RiceEvans and Miller, 1997). The antioxidant activity of the extract by this assay implies that action may be by either inhibiting or scavenging the ABTS radicals since both inhibition and scavenging properties of antioxidants towards this radical have been reported in earlier studies (Mahakunakorn et al., 2004). REFERENCES 1-Qui L, Shen Y, Xie A, Yu X, Zhang L and Zhang Q (2007). Green synthesis of silver nanoparticles using Capsicum annum L. extract, Green Chem.9:852-85. 2-Sharma VK, Yngard RA and Lin Y (2009).Silver nanoparticles Green synthesis and their antimicrobial activities, Adv. Collo.Interf. Sci. 145: 83–96. 3- Prathna TC, Mathew L, Chandrasekaran N, RaichurAM, Mukherjee A, (2010). Biomimetic Synthesis ofNanoparticles: Science, Technology and Applicability, Edited A. Mukherjee, InTech Publishers, Croatia, 1-20. 4- Padil, V.V.; Cerník, M. Green synthesis of copper oxide nanoparticles using gum karaya as abiotemplate and their antibacterial application. Int. J. Nanomedicine 2013, 8, 889–898. 5-. Shameli, K.; Ahmad, M.B.; Zamanian, A.; Sangpour, P.; Shabanzadeh, P.; Abdollahi. Y.; Zargar, M.Green biosynthesis of silver nanoparticles using Curcuma longa tuber powder. Int. J. Nanomedicine 2012, 7, 5603–5610.

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6- Lukman, A.I.; Gong, B.; Marjo, C.E.; Roessner, U.; Harris, A.T. Facile synthesis,

stabilization,and

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nanoparticles using Medicago sativa seed exudates.J. Colloid Interface Sci. 2011, 353, 433–44. 7- Ponder JG, Darab TE (2000). EnvironmentalScience& Technology,34:2564. 8 -Daraio XR, Wang CC, Talbot B(2006). “Room Temperature Solvent-Free Synthesis of Mono- disperse Magnetite Nanocrystals,” Journal of Nanoscience and Nanotechnology, Vol. 6, Num 3: 852-856. 9- Sreejayan N, Rao MNA(1996). Free radical scavenging activity of curcuminoids, Drug Res. 46:169. 10- Barnes J, Anderson LA, Phillipson JD(2002). A guide for health-care professionals. In:Herbal Medicines, Edited by: Barnes J, Anderson LA, Phillipson JD. London: Pharmaceutical Press, pp. 38. 11- Govindarajan R, VijayaM,

Kumar AKS,RawatS( 2003). Free radical

scavenging potential of PicrrorhizakurroaRoyle ex Benth., Indian J. Exptl. Biol. 41: 875. 12- UirsiniF, Maiorino M,Morazzoni K, Roveri A and PifferiG(1994) A novel antioxidant (IdB 1031) affecting molecular mechanisms of cellular . Free Radic.Bio.Med.16:547-553. 13- Barnes J, Anderson LA, Phillipson JD( 2002). A guide for health-care professionals. In: Herbal Medicines, Edited by: Barnes J, Anderson LA, Phillipson JD. London:Pharmaceutical Press, pp. 38. 14-Ganapaty

VM,Chandrashekhar

HR,Chitme

NM(2007).

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scavenging activity of gossypin and nevadensin: An in vitro relation, Indian Journal of Pharmacology,39: 281-283. 15- Rice-Evans C, Miller NJ(1997).Factors affecting the antioxidant activity determined by theABTS radical cation assay, Free Radic. Res. 195: 26-27. 16- Mahakunakorn M,Tohda Y, Murakami K, Matsumoto H, Watanabe (2004).Antioxidant and free radical scavenging activity of Choto-san and its related constituents, Biol. Pharm. Bull.27: 38-46 170

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2 1.5 1

0.5 0 1

2

3

4

5

6

7

8

9

Figure 1.The UV-VIS spectrum of Fe3O4nanoparticles.

Figure 2.Biosynthesis of nanoparticle.

Figure 3. SEM image of FeNPs synthesized using citrus peel extract.

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Figure 5. AFM photographs of synthesized magnetite nanoparticles.

Table 1.Comparison of IC50 values of Iron nanoparticle in compaction with standard.

No 1 2 3 4

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Test DPPH radical scavenging activity ABTS radical scavenging activity Iron chelating method Lipid peroxidation method

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IC50 µg/mL 45 11.9 56 20.5

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