Materials Research Bulletin 48 (2013) 3323–3327Contents lists available at SciVerse ScienceDirect Materials Research Bulletin journal homepage: www.elsevier.com/locate/matresbu Enhanced antibacterial activity of iron oxide magnetic nanoparticles treated with Argemone mexicana L. leaf extract: An in vitro study S. Arokiyaraj a, M. Saravanan b,*, N.K. Udaya Prakash a, M. Valan Arasu c, B. Vijayakumar a, S. Vincent d a R&D, Vel Tech High Tech Dr.Rangarajan Dr.Sakunthala Engineering College, Avadi, Chennai 600062, India Department of Biotechnology, SRM University, Kattankulathur, Chennai 603203, India c Department of Bio-Environmental Chemistry, Chungnam National University, Daejeon 305 764, South Korea d Department of Advanced Zoology and Biotechnology, Loyola College, Chennai 600034, India b A R T I C L E I N F O A B S T R A C T Article history: Received 8 November 2012 Received in revised form 30 April 2013 Accepted 7 May 2013 Available online 24 May 2013 The present study intended for the chemical synthesis of iron oxide nanoparticles (IO-NPs) followed by characterization and evaluation of antibacterial activity after treating with Argemone mexicana L. leaf extract. The formation of IO-NPs was confirmed by the colour change and further examined by UV–vis spectroscopy. The morphology was characterized by using SEM and TEM, which showed spherical particles of uniform size ranged between 10 and 30 nm and the crystallites were determined through XRD. The peaks in XRD pattern are in good agreement with that of face-centered cubic form of iron oxide nanoparticles. FT-IR spectroscopy confirmed the attachment of bioactive molecules of plant on the IONPs surfaces. Furthermore, the antibacterial efficacy of IO-NPs, plant extract and IO-NPs treated with plant extract was screened against Escherichia coli MTCC 443, Proteus mirabilis MTCC 425 and Bacillus subtilis MTCC 441. The results showed a noteworthy inhibition on P. mirabilis and E. coli with IO-NPs treated plant extract. This outcome may pave a way for using the magnetic nanoparticles as a drug carrier system to cure bacterial diseases. ß 2013 Elsevier Ltd. All rights reserved. Keywords: A. Magnetic materials B. Chemical synthesis C. Electron microscopy C. X-ray diffraction 1. Introduction Antibiotic resistance, a well-known phenomenon in nature assumes significant public health importance when it gets amplified many folds due to human misuse and neglect [1]. It has become a serious public health concern with economic and social implications throughout the world [2]. To overcome this, a newer area of research, i.e. nanoparticles in controlling bacterial growth is carried out by many authors. The antimicrobial activities of aluminum oxide, silver nanoparticles, gold nanoparticles and iron oxide nanoparticles have been previously reported [3–5]. Due to their antibacterial activities, metallic nanoparticles represent an effective solution for overcoming bacterial resistance [6]. Magnetic nanoparticles have applications in biomedicine due to their controllable size of less than 100 nm which give them the ability to attach with microbial cells. Magnetic biomaterials provide the ability to be directed and concentrated within the target tissue by means of external magnetic field and be removed once therapy was completed [7]. The drug loaded magnetic nanoparticles helps in the controlled release of drug which reduce side effects due to their lower dosage and minimize or prevent * Corresponding author. Tel.: +91 9443077097; fax: +91 044 27453903. E-mail address:
[email protected] (M. Saravanan). 0025-5408/$ – see front matter ß 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.materresbull.2013.05.059 drug degradation by using pathways other than gastrointestinal and have several advantages such as, small particle size, large surface area, magnetic response, bio-compatibility and nontoxicity [8]. The synergistic impact of nanoparticles as antimicrobials is already studied and showed enhanced result. The synergistic impact of silver nanoparticles with b-lactam antibiotics as an antibacterial agent [9], silver nanoparticles and antibiotics [10] as an effective antibacterial agent, antibacterial effect of iron oxide and titania [11], magnetic nanoparticle and microwave exposure [12], magnetic nanoparticle with glycol chitosan [13] and magnetic nanoparticles and cephalosporins [14] were reported. Similarly, the antiseptic qualities of medicinal plants have been long recognized. A major part of total population in developing countries still uses folklore medicines obtained from plant resources [15]. Biologically active compounds which are present in plants have always been of great interest. Recently there has been a revival of interest in herbal medications due to a perception that there are lower incidences of adverse reactions to plant preparations compared to synthetic pharmaceuticals [16]. Argemone mexicana L. (Papaveraceae), commonly known as Mexican prickly poppy, a native of Tropical America is introduced and naturalized in India and generally occurs as wasteland weed in almost every part of the country. It is a prickly, glabrous, branching herb with yellow juice and showy yellow flowers. The plant mexicana and IO-NPS. 2.0125 M and 0. The cell suspensions were diluted with sterile MHB to provide an initial cell count of about 106 CFU/ml. Proteus mirabilis and Bacillus subtilis. skin-diseases. Pvt. mexicana and IO-NPs treated with A. mexicana L. Characterization methods The synthesized nanoparticles were analyzed using ultra violet–visible spectroscopy (UV–vis) (Perkin-Elmer Lambda 2 Spectrophotometer) in 300–800 nm wave length range. SEM. subtilis MTCC 441 consigned from Institute of Microbial Technology (IMTECH). The absorption spectrum of . Antibacterial assay of IO-NPs with ethanolic extract of A.6. Department of Biotechnology.5% of ammonium hydroxide was added at a rate of 0. Plant material collection and preparation of the extract Three bacterial strains chosen for studies of antibacterial assay were two Gram positive bacteria such as E. leaf extract. 2. The disc were gently pressed and incubated in inverted position for 24 h at 37 8C. The stored product was reconstituted again by using the same solvent for required concentration. The black colored nanoparticles were separated by magnetic decantation to form magnetite. The bacterial suspension (106 CFU/ml) swabbed on the Muller Hinton Agar (MHA) plates using sterile cotton swab. The XRD patterns of the synthesized magnetite nanoparticles were recorded by X-ray diffractometer (Bruker D8 Advance.025 M in deionized water were stirred and 29. The objective of this study is to synthesize iron oxide magnetic nanoparticles (magnetite) by chemical co-precipitation method and to characterize using UV–visible spectrophotometer. Fourier transform infrared (FT-IR) spectrometer was used to test the physicochemical interaction between leaf extract of A. protopine. Materials and methods 2. In the present study a new approach has been taken to study the antibacterial property of A. chelerytherine and the seed oil contains myristic. FT-IR spectra were recorded in the range 4000–400 cm 1 with Thermo Nicolet Avatar 370 FT-IR spectrophotometer. palmitic. 2. Approximately 150 mL of ethanol was used for making extract. Udaya Prakash. The discs with streptomycin (Himedia. mexicana ethanolic extracts treated with IO-NPs by disc diffusion method against Escherichia coli.e. The morphology of the synthesized IO-NPs was determined by scanning electron microscope (JSM-6390LV. mexicana and synthesized particles (IO-NPs) were taken in the ratio of 1:1 and dissolved in 1 mL of ethanol. were used in Soxhlet apparatus to form extract. Thus the selected leaves were washed in running tap water for removing the surface pollutants. inflammations and bilious fevers [17]. In Ayurveda. Ltd. The magnetites formed were washed 3 times with deionized water and 2 times with ethanol. the plant is used as a diuretic and purgative. XRD. Mumbai. and linoleic acids. India and it was acknowledged by one of the authors (N. Bruker AXS. the synergistic impact of magnetic nanoparticles of iron oxide treated with any of the plant extract as an antibacterial agent is not yet reported so far in the earlier literature and hence this study is conducted to find the antibacterial activity of magnetic nanoparticles. / Materials Research Bulletin 48 (2013) 3323–3327 contains alkaloids as berberine. mexicana was collected from Chengelpet district belonging to the state of Tamil Nadu. thus formed was filtered and concentrated using rotary evaporator. India. After the incubation period. After incubation this mixture was used for antibacterial assay to find the bactericidal activity. mexicana The antibacterial efficacy was assayed by the standard Kirby– Bauer disc diffusion method against the bacterial pathogen [19]. 30 mg/disc) was placed on the MHA plates maintained as control.5.1. The extract was prepared using Soxhlet apparatus with ethanol as a solvent. These experiments were performed in triplicates for achieving the optimum results.3324 S. mexicana in three different concentrations i. Arokiyaraj et al. Botanist) and deposited in the herbarium collections. Treatment of IO-NPs with ethanolic extract of A.. mirabilis MTCC 425 and one Gram negative bacteria B. Synthesis of iron oxide nanoparticles The iron oxide nanoparticles (magnetite) were synthesized by using co-precipitation method in alkaline media.2. TEM and evaluation of antibacterial activity after treating with A. The 2.2 mL/min to adjust the pH at 10 in a non-oxidizing environment. the susceptibility of the test organisms was determined by measuring the diameter of the zone of inhibition using Himedia zone scale and the obtained results were tabulated for evaluation.54056 A˚) radiation with scan range of 10–70 A˚ at 40 kV and 40 mA. India) and maintained for 24 h at 37 8C. Chandigarh. A. seeds and yellow juice are used to cure leprosy. 1 paper disc of 6 mm dimension was impregnated with the three solution components such as IO-NPs alone. Results and discussion 3. thus obtained was washed thrice with deionized water and twice with ethanol. The washed leaves were dried at room temperature for 2–3 days under dark condition.SR Engineering College. The thin film on the SEM grid was allowed to stand for 5 min to dry prior to measurement. To the best of our knowledge. coli MTCC 443. JEOL.5 mg/disc. mexicana Plant extract of A. Roots. Synthesis and characterization of IO-NPs UV–vis absorption spectrum measures the wavelength of the light that the nanoparticles absorb. Germany) equipped with Ni filter and CuKa (l = 1. using KBr pellet method. leaves. sarguinarine. Chennai. Japan) resolution at 300 nm at an accelerating voltage of 15 kV. The black precipitate.4.1. The fine powder of IO-NPs has been dispersed in ethanol on carbon coated copper grid and the size and shapes of the IO-NPs were obtained with TEM (TEM-JEOL 1200EX. P. After drying the leaves were powdered using electric blender.K. particle size of IO-NPs was calculated using full width at half maximum of face-centered cubic (3 1 1) using Debye–Scherrer equation (D = Kl/b cos u) from the highest intensity of XRD pattern [18]. India. 25 mg/ disc and 50 mg/disc respectively. 15 g of powder was made into three bags containing 5 g each. FTIR. The leaves that are collected were investigated for pathogenic infections and healthy leaves were selected after the examination. The concentrated extract was stored in refrigerator for further use. 3. Solutions of Fe2+ and Fe3+ with the molar ratio of 0. Microorganisms 2. Bacterial cultures were prepared in Mueller Hinton Broth (Hi-Media. oleic.3. ethanolic extract of A. The sterile Whatman No. optisine. The treated mixture was incubated in BOD incubator shaker at 45 8C for 12 h. and dried at room temperature. Japan) at an accelerating voltage of 120 kV. A thin film was prepared by drop coating synthesized IO-NPs on to carbon coated copper SEM grids and then the extra sample solution was removed using a blotting paper. 12. Veltech High Tech Dr.RR Dr. The plant extract (PE). 2. the morphology of the particles was Fig.818). Similar results were obtained by Gerko [23]. XRD pattern of iron oxide magnetic nanoparticles. 2. SEM image of synthesized IO-NPs. the synthesized IO-NPs recorded the peak at 315 nm (Fig. (a) FT-IR spectroscopy analysis of IO-NPs and Argemone mexicana leaf extract and (b) FT-IR spectroscopy analysis of IO-NPs treated with Argemone mexicana.308. (3 1 1). (4 0 0). The FT-IR spectrum of the magnetite showed three distinct bands at 566 cm 1.718. From the FT-IR data.318. 1). / Materials Research Bulletin 48 (2013) 3323–3327 3325 Fig. From the Scherrer’s formula the average crystallite size of the nanoparticles was found to be 11 nm. 4. 3. 35. FT-IR spectrum of plant extract shows H–C–H stretching and bending vibrations at 2917– 2849 cm 1 and 1466–1400 cm 1 respectively (Fig. The Fe–O stretching was established by vibration at 566 cm 1. and (4 4 0)] are quite identical to the peaks of Fe3O4 crystal with the cubic spinal structure (Fig. 4 shows the micrographs of scanning electron microscope of the synthesized magnetite. 2a) due to nalkane [22]. . 3). Arokiyaraj et al. Parallel results were observed by Cornell and Schwertmann. mexicana interacted with the synthesized Fe3O4 nanoparticles. The XRD pattern of the synthesized magnetite displayed six characteristic peaks (2u = 30. 2a). 53. The FT-IR spectrum of Fe3O4 nanoparticles after treatment with the plant extract also showed H–C–H stretching vibrations at 2972 and 2882 cm 1 and H–C–H bending vibrations at 1450 cm 1 (Fig. 2b). 1635 cm 1 and 3426 cm 1 (Fig. it is clear that the bioactive molecules present in the leaves extract of A. (5 1 1). O–H stretching vibration at 3426 cm 1 and O–H distorted vibration at 1635 cm 1 are due to surface adsorbed water.318. Fig. UV–visible spectral analysis of synthesized IO-NPs and the peak noted around 315 nm. (4 2 2). Similar results were reported by Yang et al. Fig. [21].S. marked by their indices [(2 2 0). 43. Fig. 1. 2003 [20].618 and 62. The results are in agreement with the standard XRD pattern of Fe3O4 (JCPDS 750033). 57. [26] reported that the inactivation of E.2. The recorded activity is higher than that of the standard streptomycin (14 mm). subtilis the activity was found to be less and maximum inhibition was observed at 50 mg/disc. Lee et al.28]. Similar antibacterial effect of iron oxide nanoparticles was reported by Tran et al. Fig. The results are in agreement with observation from SEM images [24]. mexicana showed moderate activity. mexicana and combination effect of IO-NPs with A. The ethanolic extract of A. [32] from silver metals. The bactericidal effect of IO-NPs may be due to their smaller size. subtilis IO-NPs (mg/disc) Plant extract (mg/disc) IO-NPs + plant extract (mg/disc) Streptomycin (mg/disc) 12. coli and P. TEM image of iron oxide nanoparticles. 5). Reactive oxygen species (ROS) can cause damage to proteins and DNA in bacteria [31]. The present study proved that the immobilized nanomaterials of magnetite can effectively improve the drug loading and the antibacterial efficiency against the microbial pathogens. Acknowledgement Fig. coli by zero-valent iron nanoparticles could be because of the penetration of the small particles (sizes ranging from 10 to 80 nm) into E. observed to be spherical. leading to oxidative stress and causes disruption of the cell membrane. leaf extract material is the most rapid and ecofriendly method and it has a wide scope in opting as an excellent drug delivery system. [25]. Based on the observations. coli P. India for the characterization study. coli membranes. So. 6. It is conclude that further studies on this field are of enormous importance in developing eco-friendly bionanomaterial and is highly recommended for biomedicines. plant extract and IO-NPs treated with Argemone mexicana. Conclusion The synthesized IO-NPs treated with leaf extract of A. This phytochemical is known to be a DNA intercalator and an inhibitor of DNA synthesis through topoisomerase inhibition [27. (–) no zone of inhibition. proved to have outstanding antimicrobial efficacy against the bacterial pathogens. (b) ethanolic extract of Argemone mexicana and (c) combination effect of IO-NPs with Argemone mexicana against the bacterial pathogens. Taylor et al.5 25 50 12. . Antibacterial activity The antibacterial assay of IO-NPs. mexicana L. Arokiyaraj et al. The biological approach on IO-NPs treated with A. mexicana showed higher activity against the E. Chennai 600 036. Conflict of interest statement The authors have declared no conflict of interest. The size and morphology of the synthesized magnetite were also characterized by transmission electron microscopy (Fig. mirabilis B. In the case of B. 5. mexicana L. mexicana could be due to the synergistic effect of the phytochemicals and generation of ROS by IO-NPs. This bactericidal property has been evidenced by Park et al. 4.S. The mean zone diameter of different concentration level of IO-NPs was also determined. the inhibitory property of IO-NPs treated with ethanolic extract of A. 6). Indian Institute of Technology. and this inhibitory activity is may be due to the presence of phytochemical alkaloids. The antibacterial assay of (a) IO-NPs alone. The authors express their gratitude to Sophisticated Analytical Instrumentation facility. Bacteria E. it can be perceived that very lesser amount of activity against the tested bacterial strains was reported. [29] have also reported similar concentration dependent bacteria inhibition. Whereas the IO-NPs treated with ethnaolic extract of A. 3. mexicana against the bacterial pathogens was identified (Table 1 and Fig. ethanolic extract of A. mirablis which is reflected in the mean zone values of 13 mm and 18 mm respectively. / Materials Research Bulletin 48 (2013) 3323–3327 3326 Table 1 Antibacterial activity of IO-NPS.5 25 50 30 7 7 7 7 7 7 8 8 8 – 9 – 8 10 8 10 12 9 11 16 7 12 17 8 13 18 10 14 14 9 Values are expressed in mean of three independent experiments. Mechanism: the antibacterial drugs and antibiotics develop oxidative stress by generating reactive oxygen species [30].5 25 50 12. The magnetites were found to have smooth surface with well dispersed particles and the size ranged between 10 and 30 nm. Y.Y. Nayar. Andreescu. L. Health Prospect. Phelps. S. A. Park. J. Modi. [13] B. [12] P. Van Staden. QuetinLeclercq.M.C.C. O. [32] H. A.J. Ju-Yu. Nanomed. H. S. Li. Tran. Lee. Wei-Jen.L. Mihai. Wei-Jen. C. J. Inbaraj. Webster. Gerko.P. U. Guang Diau. Balaure. 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