Antibacterial activity of biochemically capped iron oxide nanoparticles: A view towards green chemistry

Accepted Manuscript Antibacterial activity of biochemically capped iron oxide nanoparticles: A view towards green chemistry

Rabia Irshad, Kamran Tahir, Baoshan Li, Aftab Ahmad, Azka R. Siddiqui, Sadia Nazir PII: DOI: Reference:

S1011-1344(16)31194-0 doi: 10.1016/j.jphotobiol.2017.04.020 JPB 10799

To appear in:

Journal of Photochemistry & Photobiology, B: Biology

Received date: Revised date: Accepted date:

29 December 2016 12 April 2017 17 April 2017

Please cite this article as: Rabia Irshad, Kamran Tahir, Baoshan Li, Aftab Ahmad, Azka R. Siddiqui, Sadia Nazir , Antibacterial activity of biochemically capped iron oxide nanoparticles: A view towards green chemistry. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Jpb(2017), doi: 10.1016/j.jphotobiol.2017.04.020

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ACCEPTED MANUSCRIPT Antibacterial activity of biochemically capped iron oxide nanoparticles: A view towards green chemistry Rabia Irshada, Kamran Tahira, b, Baoshan Li*a, Aftab Ahmada, Azka R Siddiquia, Sadia Nazirb

State Key Laboratory of Chemical Resource Engineering, School of Science, Beijing University

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Institute of Chemical Sciences, Gomal University, D. I. Khan, KP, Pakistan

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b

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of Chemical Technology, Beijing 100029, PR China.

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Corresponding Author = E-mail: [email protected]; Fax: +86 10 64445611; 5Tel: +86 10

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64445611.

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Abstract A green approach to fabricate nanoparticles has been evolved as a revolutionary discipline. Ecocompatible reaction set ups, use of non-toxic materials and production of highly active biological and photocatalytic products are few benefits of this greener approach. Here, we introduce a green

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method to synthesize Fe oxide NPs using Punica granatum peel extract. The formation of Fe

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oxide NPs was optimized using different concentrations of peel extract (20 mL, 40 mL and 60

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mL) to achieve small size and better morphology. The results indicate that the FeNPs, obtained using 40 mL concentration of peel extract possess the smallest size. The morphology, size and

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crystallinity of NPs was confirmed by implementing various techniques i.e. UV-Vis spectroscopy, X-ray diffraction, Scanning Electron Microscopy and Electron Diffraction Spectroscopy. The bio-chemicals responsible for reduction and stabilization of FeNPs were

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confirmed by FT-IR analysis. The biogenic FeNPs were tested for their size dependent antibacterial activity. The biogenic FeNPs prepared in 40 mL extract concentrations exhibited

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strongest antibacterial activity against Pseudomonas aeruginosa i.e. (22 (±0.5) mm than FeNPs

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with 20 mL and 60 mL extract concentrations i.e. 18 (±0.4) mm and 14 (±0.3) mm respectively. The optimized FeNPs with 40 mL peel extract are not only highly active for ROS generation but

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also show no hemolytic activity. Thus, FeNPs synthesized using the greener approach are found to have high antibacterial activity along with biocompatibility. This high antibacterial activity

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can be referred to small size and large surface area. Key words: Antibacterial activity, Green synthesis, iron oxide nanoparticles, Punica granatum.

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1. Introduction

Large scale urbanization and industrialization have contributed to today’s environmental calamities principally in aquatic domain. Nanoparticle synthesis is one of the most emerging processes to cope with various organic and inorganic toxic pollutants1-3. In recent years, iron nanoparticles due to their diversified applications are being actively looked into. Iron nanoparticles are characterized as active agents against many organic and inorganic pollutants. These iron-based nanoparticles have been reported in different states i.e. zero valent iron 3, Feball clay 4, iron oxide nanoparticles5. Minuscule size, large surface area and high degree of

ACCEPTED MANUSCRIPT dispersion of nanoparticles make them unique for their catalytic activity. Owing to high magnetic susceptibility and biocompatibility, iron nanoparticles have been magnificently employed in various therapeutics for cancer treatment and radiation oncology6. Various distinctive methods have been in practice to manufacture nanoparticles. The methods used for their production fall under chemical, physical and biosynthetic domains. Some of these

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chemical processes include thermal decomposition7-8, co-precipitation9, sol-gel method10, polyol

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methods11 and hydrothermal method12. However, these conventional methods are not as enticing as they lead to degradation of the ecosystem, exhibit low dispersion rates, are expensive, exhibit

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low uniformity in dispersion and are inconvenient to work with in scaled-up applications. Moreover, these aforementioned processes tend to operate under prime critical conditions i.e.

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temperature and pH.

Contemporary to these methods, Green synthesis stands out showcasing the encouraging results

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and a wide range of flexible effects which include no demand of optimum operating conditions, stable economical perspectives and their hospitable approach to environment. Green synthetic

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process has already been used to fabricate various metal nanoparticles and nano composites such Cu/ZnO nanoparticles13-19.

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as silver, palladium, gold nanoparticles, Au/TiO2 nanocomposite, ZnO nanoparticles and

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In the present work, Punica granatum’s (generally called as pomegranate, family Punicaceae) peel extract is used to synthesize iron oxide nanoparticles. Punica granatum is a primeval fruit

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and is widely cultivated. Its peel extract has been found to have divergent applications in various medical fields i.e. drugs and medicine18 and exhibits a potential ability against certain bacteria21

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and other microbes. In comparison to the pulp pomegranate, peel contains thrice the amount of polyphenols22. Punica granatum peel may contains different phenolic compounds i.e. ellagic and ellagic acid derivatives like punicalagin23-24. These compounds contribute in the stability of nanoparticles and have a reducing nature. The present work holds dual profits i.e. an innovative and eco-friendly method to synthesize iron nanoparticles as well as a movement to decline the pitch of pollution by using the peels which are disposed of as waste material. The green synthetic iron oxide nanoparticles have been examined for their antibacterial activity. The result illustrated that the nanoparticles prepared at 40 mL extract concentration have high antibacterial activity as compared to nanoparticles prepared at 20 mL and 60 mL peel extract.

ACCEPTED MANUSCRIPT 2. Materials and methods 2.1. Preparation of Punica granatum peel Extract Punica granatum was collected from local market in Beijing. Peels were thoroughly washed several times with distilled water to remove the dust particles, dried in dark and ground in minute sized granules. 7 g of this sample was taken in 250 mL beaker and enough distilled water was

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added to make the total volume upto 150 mL. The resulting solution was heated initially at 80 ˚C

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for about 30 minutes and then further stirred 60 minutes at 1000 rpm for. The peel extract was

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filtered using Whatman filter paper no.3 and stored at 4 ˚C for further use. 2.2. Synthesis of Fe nanoparticles using Punica granatum peel extract

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Fe nanoparticles were synthesized by adding different concentrations of peel extract i.e. 20 mL, 40 mL and 60 mL in 150 mL of 0.15 M solution of ferric chloride hexahydrate (FeCl3.6H2O) in a

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500 mL beaker. The color of salt solution turned from brown to black immediately. Then it was autoclaved for 5 hours at a constant 200 ˚C. After the formation of Fe oxide nanoparticles, it was

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centrifuged at 10,000 rpm. Later, it was dried in 6ES freeze drier for 4 days.

2.3. Screening for antibacterial activity by agar well diffusion method

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Agar well protocol was applied to check the antibacterial activity of greener Fe oxide

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nanoparticles25-26. A bacterial culture was prepared in nutrient broth at 37˚C for 24 hours in an incubator. Inocula of underlined bacteria was marked on Muller Hinton agar plates, using sterile swab. It ensured an even dense lawn of culture following incubation. Wells of 6 mm diameter

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were made on nutrient agar plates, using sterile cork borer. A solution of 1 mg Fe oxide nanoparticles in 1 mL distilled water was prepared and 50 µL of this solution was poured into the wells formed on nutrient agar plates. The agar plates then left to stay for 1 hour at 25 ˚C. Finally, the plates were incubated for 24 hours at 37 ˚C. The resultant diameter of zone of inhibition was measured cautiously. Streptomycin was used as standard.

2.4. Determining Minimum inhibitory concentration

ACCEPTED MANUSCRIPT Serial dilution method was adopted to determine MIC of Fe oxide nanoparticles. 1 mL of biogenic FeNPs, with different concentrations of peel extract i.e. 20 mL, 40 mL and 60 mL, were taken in sterilized test tubes containing 1 mL of bacterial (Pseudomonas aeruginosa) solution having turbidity of 0.5 Mcfarland turbidity standard. The test tubes were mixed and then kept in incubator at 37 ˚C for 24 hours. These test tubes containing culture were taken as control. The concentration range of FeNPs used was from 2 mg mL-1 to 0.031 mg mL-1. MIC can be

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considered as the minimum concentration of compound which inhibits the growth of

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2.5. Reactive oxygen species generation by FeNPs

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microorganism. The assay was carried out in triplicates.

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2, 7-dichlorodihydrofluorescein diacetate (DCFH-DA) kit, a method for oxidative stress assessment of FeNPs treated microbes, was employed to verify the intracellular generation of reactive oxygen species (ROS). This fluorogenic organic dye is quite advantageous to detect

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hydroxyl, per hydroxyl and other reactive oxygen species (ROS) within the cell. Fe nano particles with 40 mL Punica granatum peel extract concentrations were incubated for 4 hours at

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250 rpm along with the tested bacterial (Pseudomonas aeruginosa) strain. After incubation, the

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suspension of bacterial cells (Pseudomonas aeruginosa) was collected (8000 rpm, 5 min) and the obtained pellet was washed thrice with phosphate buffer saline (PBS). A suspension of pellet in 1

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mL of buffer solution (PBS) was subsequently treated with 1 mL of 20 mM 2, 7dichlorodihydrofluorescein diacetate reagent for 40 minutes. The DCFH-DA treated cells were

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washed thrice with PBS to get rid of the excess dye from outer surface of cells. The fluorescence image of the suspension was determined by a fluorescence microscope (Olympus 1×51) at two

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wavelengths i.e. excitation wavelength of 488 nm and the emission wavelength of 535 nm27. 2.6. Hemolytic activity assay To check the hemolytic property of green synthesized FeNPs, the amount of hemoglobin released from red blood cells (RBCs) on treatment with biogenic FeNPs was measured. The blood was obtained from a male albino rat and was taken in a sterile Lithium Heparin Vacutainer. The test tube was then centrifuged at 1500 rpm for 20 minutes. The supernatant was removed cautiously and the pellet was sterilized three times with phosphate buffered saline (PBS). The pH of PBS was maintained at 7.4. Different amounts of FeNPs synthesized at optimized condition i.e. using 40 mL peel extract (20, 40, 60, 80, 100 and 120 mg) taken in PBS

ACCEPTED MANUSCRIPT solution and cells (5% v/v) in PBS were added in each tube to make the total volume up to 1 mL. RBCs in PBS were taken as negative control whereas RBCs in 1% Triton X-100 solution were taken as positive control. A shaking incubator maintained at 37 ˚C was used to incubate the reaction mixtures for 1 hour with gentle shaking. The tubes were then centrifuged at 1500 rpm for 10 minutes and the supernatant was observed keenly at 540 nm against their blank28.

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3. Results and discussion

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3.1. Characterizations

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3 .1.1. UV spectroscopic analysis

UV-spectrophotometer (Shimadzo UV-2400) was used to verify the formation of Fe oxide

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nanoparticles. The UV spectrum obtained is shown in (Fig.1). The spectrum obtained noticeably specified the formation of Fe nanoparticles during the course of synthesis. Biogenic FeNPs

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showed maximum absorbance at wavelength of 300 nm that is in complete harmony with UV

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spectral analysis of metallic iron. Such UV spectral results have been already reported29. 3.1.2. Fourier Transform Infrared spectroscopic (FT-IR) analysis

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The presence of phytochemicals in peel extract of Punica granatum was revealed by FT-IR. IR spectrum was obtained using an ABB MB 3000 spectrophotometer. These phytochemicals play

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significant role as stabilizing and reducing agents. (Fig.2) represents the results of FT-IR spectrum of green Fe nanoparticles. The spectrum obtained specifies some prominent peaks at

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3426 cm-1, 2924 cm-1, 2848 cm-1, 1719 cm-1, 1616 cm-1, 1327 cm-1 and 1228 cm-1 respectively. Hydroxyl group (OH) is usually characterized by the presence of a broad peak i.e. at 3426 cm-1.

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The two other distinctive peaks obtained at 2924 cm-1 and 2848 cm-1 represent the C-H stretching frequencies. The peak at 1719 cm-1 indicates the presence of carbonyl group present in the organic moiety. The peak at 1616 cm-1 confirms the presence of C=C stretching frequency. The peaks at 1327 cm-1 and 1228 cm-1 indicate C-N stretching frequencies. However, a trend in inclination of peak intensities of functional groups was observed that can be accredited to the interaction of functional groups with the stabilizing agents of peel extract. Result obtained is in complete agreement with the previously reported work30-31. 3.1.3. X-ray Diffraction (XRD) analysis.

ACCEPTED MANUSCRIPT XRD analysis of green synthesized iron nanoparticles is shown in the (Fig.3). X-ray diffraction (XRD) measurements were carried out on a Rigaku D/Max 2500 VBZ+/PC diffractometer. The angle range θ was taken from 20˚-90˚.The analysis showed that no defined peak was observed by spectra. It gathers that the nanoparticles were amorphous instead of crystalline nature. Similar diffraction patterns for iron nanoparticles have also been reported previously32.Various studies have exposed that amorphous nature of iron nanoparticles supports catalytic activity in various

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reactions and dye degradation rate compared to crystalline iron nanoparticles.

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3.1.4. Scanning Electron Microscopy (SEM)

Biogenic iron nanoparticles were examined through SEM analysis to evaluate their morphology

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and their degree of dispersion. SEM spectrum were obtained using a Hitachi S-4700 scanning electron microscope. The SEM analysis of FeNPs, prepared at different extract concentrations i.e. 20 mL, 40 mL and 60 mL was studied (Fig.4). The result illustrated that FeNPs prepared at

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40 mL extract concentrations have small size and good dispersion rates. Some particles are slightly aggregated but not truly. The FeNPs prepared at 20 mL extract concentration have high

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degree of aggregation which may be due to the lesser amount of organic moiety. Similarly, the

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FeNPs prepared at 60 mL extract concentration also show large size and high aggregation. It is due to the fact that at high concentrations of the plant extract, the additional interactions occur

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between the surface stabilizing molecules and the molecules in the solution which as a result reduce the stabilizing and reducing efficacy of active phytochemicals.

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3.1.5. Electron Diffraction Spectroscopy (EDS) The elemental composition analysis of green synthesized iron nanoparticles was determined by

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EDS. EDS spectrum was obtained using a Hitachi S-4700 scanning electron microscope. The analysis depicted the presence of iron oxide nanoparticles by iron and oxygen peaks (Fig.4). Carbon peak was also observed approving the hypothesis that organic moiety played an effective role of capping agent. Similar confirmation has already been presented under different examinations33-34. 3.2 Application 3.2.1. Antibacterial activity

ACCEPTED MANUSCRIPT The rise in infectious epidemics and the incompetency of available drugs to counter them could lead to catastrophic results. To inhibit the actions of such microbial pathogens, new therapeutic agents must be utilized. Punica granatum is widely used as medicinal plant and its peel extract has been found to show a potential activity against different pathogens and infectious bacteria35, 21.

Considering such active nature of this plant, peel extract of Punica granatum was used to

synthesize Fe oxide nanoparticles. The antibacterial activity against Pseudomonas aeruginosa

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was determined. The result obtained (Fig.5) evidently depends on the morphology of the

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nanoparticles. Nanoparticles synthesized using 40 mL peel extract and 0.15 M ferric chloride

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solution were found to have enticing antibacterial activity among the others (Table:1). The diameter zone of inhibition in case of optimized condition in case of 40 mL peel extract (22

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±0.5) was found to be highest when compared to other concentrations and the peel itself. This can be regarded as a result of small size and high dispersion of optimized FeNPs. Several studies

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have described the effect of metal nanoparticles interacting with certain bacteria36. Fig.6 (A) describes the SEM image of bacterial cells (Pseudomonas aeruginosa) before treatment with

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green synthesized iron oxide nanoparticles at optimized conditions i.e. 40 mL plant extract, (B) shows the morphology of bacterial cells after treatment with green synthesized iron oxide

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nanoparticles at optimized conditions i.e 40 mL plant extract whereas, (C) shows the condition of bacterial cells after treatment with Punica granatum peel extract only. It is unambiguously

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verified that bacterial cell undergoes denaturation and shrinkage on treatment with FeNPs with 40 mL plant extract concentration as observed in Fig.6 (B) whereas, comparatively less

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denaturation was observed on treatment with Punica granatum peel extract only. 3.2.1.1 Minimum inhibitory concentration

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Minimum inhibitory concentration is an appreciable method to quantitatively determine antibacterial activity. MIC refers to minimum concentration of the antibacterial agent that resists the growth of pathogen in artificial media after incubation period. In the present work, MIC was applied to check the antibacterial activity of greener FeNPs37. A dilute suspension of FeNPs was incubated along with Pseudomonas aeruginosa and bacterial growth was examined. After 24 hours of incubation the MIC found to be 0.062 mg /mL (Table: 2) 3.2.1.2 Reactive oxygen species generation by FeNPs

ACCEPTED MANUSCRIPT The production of intracellular reactive oxygen species (ROS) can be ascribed to the antimicrobial effect of iron oxide nanoparticles in the microbial cell. The reactive oxygen species such as superoxide free radical (O2·), hydrogen peroxide (H2O2) and hydroxyl free radical (OH·) are generated as a result of excited electrons of FeNPs. These excited electrons promote the production of ROS in the microbial cell. Such reactive species are not only responsible for the induction of oxidative stress but also tend to damage the biomolecules like Protein and DNA.

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The oxidation of 2, 7-dichlorofluorescin-diacetate into dichlorofluoroscein has been reported in

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the presence of reactive species. The emission of green fluorescence is also observed upon

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excitation at 488 nm. (Fig.7) clearly illustrates the green fluorescence when the samples undergoes a treatment with bio-directed iron nanoparticles. However, an increase in the

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intracellular fluorescence intensity of the sample was observed when it underwent exposure to greener iron nanoparticles. It is also in accordance with the enhanced antibacterial activity of

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iron oxide nanoparticles by producing reactive oxygen species. These active oxygen species interact and damage different cellular components like DNA, cell membrane and other vital

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enzymes eventually causing cell death. Hence, the results obtained suggest that biogenic FeNPs involve the interaction with the bacterial cell surface and induce the generation of intracellular

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reactive oxygen species and leakage of cytoplasmic materials as already represented in (Fig.)38.

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3.2.2 Hemolytic activity

In order to determine the biocompatibility with normal cells, especially RBCs, hemolytic activity

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of greener FeNPs was carried out. The hemolytic analysis was done consuming different concentrations of FeNPs containing 40 mL peel extract (20, 40, 60, 80, 100 and 120 mg) on RBCs. It was noticed that FeNPs have no hemolytic activity against RBCs for different

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concentrations and it indicated almost equal hemolytic activity to that of the negative control (Table 3). This can possibly be due to the presence of capping agents containing different phenolic compounds of peel extract of Punica granatum. The existence of active organic moiety enhanced the plasma antiradical efficacy and induced a reduction in the prevention of erythrocyte membranes to oxidation and thus the resistance to hemolysis was substantially heightened. 4. Conclusion

ACCEPTED MANUSCRIPT Green-Nano technology has been proved to be one of the blooming fields that provide a nontoxic, eco-friendly, and an effective way to manufacture new materials. Such an approach has been used presently where Punica granatum peel extract has been consumed to fabricate Fe oxide nanoparticles. The peel extract played the role of capping and stabilizing agent, hereby increasing the efficiency of FeNPs. The FeNPs were optimized using different concentration of peel extract i.e. 20 mL, 40 mL and 60 mL in order to get better size, shape and dispersion of

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nanoparticles. The biogenic FeNPs were tested for their antibacterial activity. The results

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obtained clearly favor the statement that green synthesized FeNPs are highly active against

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Pseudomonas aeruginosa as well as show no hemolytic activity. This high efficiency of FeNPs can be referred to their small size and high dispersion. The fabricated FeNPs may require to

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check for their further applications. Acknowledgements

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The authors are obliged to China scholarship council (No.2016GXYS96) and the National Natural Science Foundation of China (grant No. 21271017), the National Science and

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Technology Supporting Plan of the Twelfth Five-year (No. 2014BAE12B0101) and the

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Fundamental Research Funds for the Central Universities (No. YS1406).

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ACCEPTED MANUSCRIPT (pomegranate) gel against S. mutans, S. mitis and C. albicans, Braz. Dent. J 17 (2006) 223–227. 38. A. Ahmad, Y. Wei, F. Syed, K. Tahir, A. U. Rehman, A. U. Khan, S. Ullah, Q. Yuan, The effects of bacteria-nanoparticles interface on the antibacterial activity of green

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synthesized silver nanoparticles, Microbial Pathogenesis 102 (2017) 133–142.

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Figure.1 UV spectroscopic analysis of biogenic Fe nanoparticles at 40 mL peel extract and 0.15

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Figure.2 FT-IR analysis of green synthesized iron oxide nanoparticles (using 40 mL peel extract)

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Figure.3 XRD analysis of green synthesized iron oxide nanoparticles (using 40 mL peel extract

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Figure.4 SEM analysis of Fe oxide nanoparticles (A) with 20 mL peel extract (B) with 40 mL

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Figure.5 Antibacterial activity of greener FeNPs with different concentrations of Punica granatum peel extract i.e. (A) 20 mL (B) 60 mL and (C) represents FeNPs with 40 mL peel

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Figure.6 SEM analysis of bacteria (Pseudomonas aeruginosa) (A) before treatment with

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biogenic FeNPs synthesized at optimized condition i.e 40 mL peel extract and (B) after treatment with biogenic FeNPs synthesized at optimized condition i.e 40 mL peel extract whereas, (C) on

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treatment with Punica granatum peel extract only.

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Figure.7 ROS generation (A) in the absence and (B) in the presence of FeNPs synthesized at

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optimized conditions i.e. 40 mL peel extract.

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FeNPs (40 mL

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Control 1% Triton X-100 Punica granatum extract(25µL) Fe-NPs (20 µg) Fe-NPs (40 µg) Fe-NPs (60 µg) Fe-NPs (80µg) Fe-NPs (100 µg) Fe-NPs (120 µg)

Hemolytic activity (%) (OD 540 nm) 1.18±0.11 99.9±0.03 1.22±0.11 1.46±0.12 1.51 ±0.15 1.54±0.11 1.57±0.13 1.60±0.12 1.63±0.11

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Experiments are in triplicates and the results are presented as a mean ± standard deviation. OD540 nm is optical density at 540 nm.*p< 0.05

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Graphical abstract

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Green synthesis of Iron oxide nanoparticles using punica granautm peel extract. The synthesis of nanoparticles were controlled by using various plant peel extract concentrations. The prepared nanoparticles were screened for their antibacterial activity.

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