Green synthesis of iron oxide nanorods using Withania coagulans extract improved photocatalytic degradation and antimicrobial activity

Journal Pre-proof Green synthesis of iron oxide nanorods using Withania Coagulans extract improved photocatalytic degradation and antimicrobial activity

Shaheen Qasim, Ayesha Zafar, Muhammad Saqib Saif, Zeshan Ali, Maryem Nazar, Muhammad Waqas, Ain Ul Haq, Tuba Tariq, Shahbaz Gul Hassan, Faisal Iqbal, Xu-Gang Shu, Murtaza Hasan PII:

S1011-1344(19)31164-9

DOI:

https://doi.org/10.1016/j.jphotobiol.2020.111784

Reference:

JPB 111784

To appear in:

Journal of Photochemistry & Photobiology, B: Biology

Received date:

15 September 2019

Revised date:

6 December 2019

Accepted date:

9 January 2020

Please cite this article as: S. Qasim, A. Zafar, M.S. Saif, et al., Green synthesis of iron oxide nanorods using Withania Coagulans extract improved photocatalytic degradation and antimicrobial activity, Journal of Photochemistry & Photobiology, B: Biology(2020), https://doi.org/10.1016/j.jphotobiol.2020.111784

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© 2020 Published by Elsevier.

Journal Pre-proof

Green Synthesis of Iron Oxide Nanorods using Withania Coagulans extract improved Photocatalytic Degradation and Antimicrobial Activity Shaheen Qasima║, Ayesha Zafara║, Muhammad Saqib Saifa, Zeshan Alib, Maryem Nazara, Muhammad Waqasa, Ain Ul Haqa, Tuba Tariqa, Shahbaz Gul Hassand, Faisal Iqbalc, Xu-Gang Shue¥ and Murtaza Hasanae¥ Department of Biochemistry & Biotechnology (Baghdad-ul-Jadeed Campus), The Islamia

Department of Material Science and Engineering, College of Engineering, Peking University,

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b

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University of Bahawalpur, Bahawalpur 63100, Pakistan

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Beijing 100871, China

Department of Physics (Baghdad-ul-Jadeed Campus), The Islamia University of Bahawalpur,

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c

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a

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Bahawalpur 63100, Pakistan d

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Department of Chemical Engineering, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China

College of Chemistry and Chemical Engineering, Zhongkai University of Agriculture and

Engineering, Guangzhou 510225, China

Corresponding Authors

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Dr. Murtaza Hassan

([email protected])

Professor Xu-Gang Shu ([email protected])

║

These two authors have contributed equally in this work.

Journal Pre-proof Abstract: Present work compares the green synthesis of iron oxide nanorodes (NRs) using Withania coagulans and reduction precipitation based chemical method. UV/Vis confirmed the Sharp peak of Iron oxide NRs synthesized by biologically and chemically on 294 and 278 nm respectively. XRD and SEM showed highly crystalline nature of NRs with average size 16 ± 2 nm using

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Withania extract and less crystalline with amorphous Nanostructure of 18 ± 2 nm by chemical method. FTIR analysis revealed the involvement of active bioreducing and stabilizing

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biomolecules in Withania coagulans extract for synthesis of NRs. Moreover, EDX analysis

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indicates 34.91% of Iron oxide formation in biological synthesis whereas 25.8% of iron oxide

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synthesis in chemical method. The degradation of safranin dye in the presence of Withania

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Coagulans based NRs showed 30% more effectively than chemically synthesized Nanorods which were verified by the gradual decrease in the peak intensity at 553 nm and 550 nm

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respectively under solar irradiation. Furthermore, Withania Coagulans based NRs showed

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effective Antibacterial activity against S.aureus and P. aeuroginosa as compared to NRs by chemical method. Finally, we conclude that green synthesized NRs are more effective and

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functionally more efficient than chemically prepared NRs. Therefore, our work will help the researchers to boost the synthesis of nanoparticles via biological at commercial level. KEYWORDS: Iron oxide NRs, Withania coagulans, Antibacterial activity, Photocatalytic

Journal Pre-proof Introduction: Synthesis of functional nanomaterials is flourishing as the most favourable domain of researchers due to their immense applications like in bimolecular imaging, therapeutics, drugs delivery, biomedicines, cancer treatment, cosmetic surgery and molecular based detection etc. [1-6]. By considering these remarkable applications, scientists are attempting to synthesize functional

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nanomaterials by a variety of physical, chemical and biological methods [7-13]. Physical and Chemical method used for the production of Nanoparticles (NPs), with narrow range of size and

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morphology [14-16] but it is relatively expensive and involve the use of different chemicals

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which are potentially hazardous to environment and also responsible for various biological risks

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while green synthesis of NPs, which utilized different biological resources like plants, fungi and

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microorganism for production of NPs that are eco-friendly, prevent pollution and wastes production, efficient synthesis, time and energy saving and the most importantly its economical

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production of NPs. Recently there have been some reports of biological synthesis of Iron oxide

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nanoparticles by using extract of different plants like, Lagenaria siceraria, Camellia sinensis, Glycosmi mauritiana, Eichhornia crassipes, Fagonia cretica, Dracinea cochenchinesis and

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Artemisia vulgaris [17-21]. But it is revealed that the nanoparticles synthesized by chemical method are different in properties regarding practical application such as antimicrobial activity and photodegradation properties as compared to biologically synthesized nanoparticles. Multiple approaches are used to synthesized nanoparticles where they can make changes in the structure, shape, composition and surface chemistry to optimized the synthesis for further advance and vast application. The nanoparticles are modified by different methods so that effectiveness and effiecny is increased in the particular treated environment. There are many reports on synthesis of iron oxide nanoparticles However, there have been fewer studies on fabricating iron nanorods

Journal Pre-proof via green synthesis and their application for biological materials [22-24]. There is an urgent need to search out biological approach such as wathiana coagulans extract for FeO-NRs synthesis that proved to be a good, efficient, and promising antibacterial candidate due to its cost-effectiveness, non-toxicity, and facile synthesis procedures in therapeutic biomedical fields. Keeping in view the effectiveness of iron oxide NRs on commercial scale including the photodegradation of dye such as Safranin which is very important for biological stain in histology and cytology. It is

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used as a counter-stain in some staining protocols, coloring cell nuclei red. This is the

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classic counterstain in both Gram stains and endospore staining. Safranin can also be used for the

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detection of cartilage, mucin and mast cell granules[25]. Furthermore, iron oxide NRs contribute

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toward antibacterial activity towards common infections bacterial strains.

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In present work we have explored the efficiency of green synthesis and chemical precipitation method. In biological synthesis we have utilized a new plant named Withania coagulans as a

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reducing and capping agent for the synthesis of Iron oxide nanorodes that is not reported yet.

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Due to its high production rate, cost effectiveness as well as no interference of hazardous chemicals and any bacterial or fungal species which eradicate all associated clinical issues,

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hence, make Withania coagulans extract distinct bio reducing source to discover the new horizons of bio nanomaterials [26-28]. Where as in in chemical method we have employed FeCl3 as a precursor for the synthesis of Iron oxide nanorodes. Next the wide applicated phenomena as photocatalytic degradation of important dye and antibacterial activities against most common infectious strains had been tested to make this less pensive, abundantly found waithania at commercial scale. The biological approach was compared with chemical method to demonstrate and evaluate the difference among both possible outcomes and the reliabilities should be

Journal Pre-proof preferably used in various biomedical applications such as bio-nanotechnology, biomedicines and pharmaceuticals [29-32]. Experimental section Plant extract preparation:

Withania coagulans. Berries were taken from Agriculture Department of Islamia University of

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Bahawalpur, Pakistan. Wash these berries with distilled water and grind them. Finally, we get

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fine powder of berries of Withania Coagulans. Later on, 30 g powder of berries of Withania

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Coagulans was mixed in 250 ml of distilled water in 500 ml conical flask and placed it on shaker ◦

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for overnight at 37 C. After it, filter it by using filter paper and then filtrate was kept at 4 C for

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further use.

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Synthesis of Iron oxide NRs by biological method and chemical method In green synthesis Iron oxide NRs were prepared by mixing 1:2 molar FeCl3.6H2O and

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FeCl2.4H2O in 100 ml distilled water at 90 °C with mild stirring. After 30 minutes, 20ml of

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aqueous solution of Withania Coagulans plant extract was added to the mixture shown in Fig S1. The bio reduction of Fe ions in aqueous solution were observed visually by change in color from dark brown to black and monitored by repeated sampling of aliquots (150 µL) and UV-vis spectra of suspension was measured. To maintain pH of solution 20 ml of sodium hydroxide was added with ratios of 3 ml per minute. On completion of reaction, synthesized nanoparticles were collected at room temperature. In chemical precipitation reduction method, initially 30 ml of aqueous solution of 2 mol/dm3 FeCl3 was mixed with

20 ml of 1 mol/dm3 Na2SO3 solution. The indication of complex ions

Journal Pre-proof formation was visually observed by change in color from light yellow to red shown in Fig S2. Under vigorous stirring 50.8 ml of concentrated ammonia diluted to total volume of 800ml was added in mixture where the color changes again from red to black was observed. After 30 minutes’ suspension was placed on permanent magnet. On completion of reaction a black powder containing Iron oxide NRs were seen settling in the bottom of the beaker.

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Ultra Violet- vis Spectrometery (UV)

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Analysis of Withania Coagulans extract and confirmation of synthesis of NRs was done by using

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ultra violet spectrometry. In this technique, bio-reduction of iron ions occurred which is monitored by using periodic sampling of aliquots and time by time spectra of the solution was

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measured. These spectra were recorded after every 30 minutes and UV spectrum of all aliquots

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were monitored as a function of time retortion on UV spectrophotometer operated at a resolution

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of 1nm.

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Scanning Electron Microscopy (SEM)

The morphology, texture and elemental composition of the biologically and chemically

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synthesized FeO NRs was studied using a Quanta Inspect of scanning electron microscope (SEM), operating at 25 kV in vacuum. For this purpose, iron oxide NRs were used in the powder form Ray Diffraction (XRD) The diffracted intensities of the air-dried samples of FeO NRs (150 mg) was recorded to determine the crystal structure and size of the nanoparticles using X-ray diffraction spectroscopy

Journal Pre-proof (XRD, Rigaku, Ultima IV, X-ray diffractometer system). By using CuKα radiatioltage, all diffracted intensities were recorded at 40 KV and 30 mA current at 20 to 80 scanning range. Fourier Transmission Infra-red Ray (FTIR) The Fourier-transform infra-red (FTIR) analysis was performed to detect the presence of functional groups and interaction of the biologically and chemically synthesized FeO NRs with

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capping agent. Air dried iron oxide NPs are converted into pellet and at the resolution of 4 cm− 1,

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Transmission Electron Microscopy (TEM)

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range of 50-3500 cm−1, FTIR analysis was carried out Thermo Scientific spectrophotometer.

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Samples for TEM analysis were prepared by drop coating the nanoparticle solutions on carbon-

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coated copper grids at room temperature. The excess NRs solution was removed with filter paper. The copper grid was finally dried at room temperature and was subjected to TEM analysis

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Photo-catalytic activity

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by the instrument Tecnai F20 model operated at an accelerating voltage of 200 kV.

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The Photocatalytic activity of synthesized Iron oxide NRs was investigated by degrading Safranin dye under natural sunlight. Intially,0.5mg of Iron oxide NPs powder was mixed in 100 ml dye solution containing 1mg of safranin in 100 ml. The suspension was placed in the dark for 30 minutes in order to achieve adsorption-desorption equilibrium and then, irradiated to solar light for time intervals of 30 minutes for 3 hours. Afterwards the sample were filtered and analyzed for the residual concentrations of dye by UV-vis spectrophotometer at 553nm.To observe the pure photolysis, a control experiment was also carried out under similar conditions. The percentage degradation of dye was evaluated by employing the equation. Dye degradation

Journal Pre-proof (%) = [C0 – Ct / C0] × 100 (C0 is the initial concentration of safranin solution and Ct is the final concentration of the dye after sun light exposure time “t”.) Antibacterial Activities Anti-bacterial activity of biologically synthesized and chemically synthesized iron oxide NRs was performed against gram positive and gram-negative bacterial strains of Pseudomonas

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aeroginosa and Staphylococcus Aureus.by disc diffusion method. Firstly, bacterial strains were

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isolated and grown on liquid broth media. Then homogenized solution was autoclaved to keep

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sterilized. After autoclaving, the media was gushed into medium sized test tubes. After pouring media, took 10 µl bacteria and added in media containing test tubes in it. Because the stock of

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bacteria was freeze therefore thaw it before adding. Then place pre thawed bacterial stock over ◦

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shaker at 37 C.After 24 hours the color of test tubes was observed. If test tubes turned to milky it

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was indication of bacterial growth. Then prepare the agar for plating of the bacteria to observe the bacterial growth against biologically synthesized and chemically synthesized NRs. When the

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agar plate become solidified then streaking of bacterial strain on plates was carried out. After

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streaking of bacteria, took some paper discs and dip them in solution of biologically synthesized and chemically prepared FeO NRs. Then took dipped paper discs and stuck them one by one on ◦

agar plates. After sticking of discs incubate all prepared plates in incubator for 12 hours at 37 C. After incubation period the bacterial growth was observed. Biologically synthesized and chemically synthesized FeO NRs were active in killing the bacterial strains. After accomplishing ◦

antibacterial activity, the stocks of all strains were prepared and stored at-30 C.

Results and Discussion

Journal Pre-proof The synthesis procedure of NRs via biological method and chemical method involved the optimization of initial concentration of plant extract (wathiana coagulans), iron oxide, iron

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chloride, temperature and stirrer time point are crucial for formulation in as shown in Fig1.

Figure 1. Scheme illustrating biological and chemical method of NRs preparation and their application Iron oxide NRs synthesized by the bioreducing agents derived from the Withania coagulans showed characteristic absorption peak at 294 nm, whereas chemically prepared Iron oxide NRs showed peak at 278 nm due to plasmon vibration of surface molecules as shown in Fig 2(a). The reduction of Iron precursor by Withania coagulans extract and chemical method was clearly

Journal Pre-proof observed by contrasting the color from dark orange to light orange and then dark black showing the strong absorption of visible light due to the excitation of the NRs surface plasmons (Fig. S3, S4), which depends on the size, shape and concentration of the precursors [33,34]. Further to confirm the reduction process in both methods for Iron oxide NRs, UV-vis spectra was recorded at various time intervals of 5, 10, & 15 min as shown in Fig S5 where the maximum absorbance was observed at 294 nm for green synthesis and 278 nm for chemical synthesis respectively. The

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control sample does not show any peak due to absence of reducing agent (plant extract) results

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no formation of Iron Oxide NRs, while sample having Withania coagulans extract shows

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maximum intensity for 15 min convincing that plant extract plays a main role in reduction

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process. There is 18 nm change in shifting of peak from 278 nm to 294 nm of Iorn oxide NRS by chemical method to biologically method indicated most probably the coating of Withania

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coagulans extract and also predicts the involvement of bioactive reducing molecules. Most

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probably, plant extract has high level of hydroxyl and phenolic compound, are responsible for reduction and formation of NRs which helps in providing stability to the metal ions that

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gets unstablized thus synthesis is conducted using the reducing rich agents. Once the NRs are

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synthesized, they were used for antibacterial activity but it was kept a positive control so that it could be assured that plant extract is just providing stability for generating specific iron oxide NRs. These results are matching with previously reported by other researchers. [35-37]. There was big difference in the band gap energy of synthesized Iron oxide NRs evaluated by using plank’s Einstein equation E = hc/ λ (h=6.62607004 × 10-34 m2 kg / s planks constant, c=300,000 km/s speed of light and λ is wavelength of absorbed spectra). Using plank’s equation band gap energy of both biologically fabricated and chemically prepared Iron oxide NRs were calculated to be 4.20 eV and 4.48 eV respectively. Fourier transform infrared spectroscopy (FTIR) was

Journal Pre-proof carried out for further evaluation of various types of biologically active phytochemical components in the sample of Withania coagulans extract which caused reduction of iron ions and

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also responsible for capping and enveloping of NRs as shown in Fig 2(b).

Fig 2. Characterization of Iron oxide NRs Synthesized by Biological and Chemical method (a) Uv-Vis (b) FTIR analysis (c) XRD of Biological method (d) XRD of chemical method The FTIR spectrum of biologically fabricated Iron oxide NRs was compared with chemically synthesized NRs to evaluate the biomolecules. The characteristic peaks at 666cm−1 and 657 cm−1 for biologically and chemically prepared Iron oxide NRs corresponds to the metal oxygen bond vibration in Fe-O Fig 2(b). It is very engrossing to observe that there is a peak shift from

Journal Pre-proof 666 cm−1 to 657 cm−1 upon Withania extract coating of green synthesized Iron oxide NRs. In spectra of biologically synthesized Iron oxide NRs the absorption at 1044 cm− 1 refer to C=H bending,1593 cm− 1 corresponds to O-H bending mode,2351 cm− 1 refers to N-H bending and 2954 cm− 1 showing C-H adsorption. The functional group involve in the bio reduction of biologically synthesized Iron oxide NRs are lone pair containing hydroxyl (OH) group and NH2. The O-H bending at 1593 cm− 1, N-H bending at 2351 cm− 1 and C=H bending at 1593 cm− 1

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shows presence of reducing agent (plant extract) on the surface of nanoparticles which is the

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basic reason of reduction of Iron oxide NRs. The increased intensities of N-H vibration for for

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biologically fabricated Iron oxide NRs confirmed the surface coating of Withania extract over

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them, and indicating the strong interaction between the amino group and Iron oxide NRs. FTIR of analysis of Withania extract was carried out as in Fig S6 which showing the presence of

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biologically active functional molecules which acts as reducing agents. The most probable

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reason for the deviation of peaks in UV and FTIR for biological preparation of nanorods as compared to chemical preparation is interaction of biomolecules present on the surface of

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nanoparticles synthesis by biological method. This is confirmed by spectroscopy which depends

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on the interference of electromagnetic radiation and provides the information of the different functional groups present on the surface of nanomaterials. Biological extracts have rich bio molecules with high reducing potential not only increase the rate of synthesis and efficiency but their presence on surface of nanorodes is most probable reason for the deviation of peaks in uv and FTIR. Furthermore, the complex chemistry of biological source as a plant extract used here as main cause of deviation so surface modified chemistry of biologically prepared NRs and chemical prepared NRs is quite different [38]. While in case of chemical synthesis there are pure reducing agent for synthesis of nanoparticles which give higher rate of absorption while in

Journal Pre-proof Biological method, there is a mixture of bio molecules which acts as reducing agent and in a complex form that suppress the intensity of absorption due to sharing of complex biomolecules residing in a extract [39]. The surface chemistry of nanoparticles synthesis by green method have diverse range of biomolecule such as phenols, flavonoids, tannins, proteins and glycosides that are the basic building and supporting units of the NRs. The surface plasmon response of the particles being synthesized tends to gain the reducing biomolecules of the extract and attract

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them, giving a different surface chemistry that is purely being supported by reducing molecules

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of the plants. Hence, making surface modification by better application solely depends on the

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naturally occurring entities. While in chemical methods , most nano materials are designed using

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common protecting agents including citrate and thiols which have proven to be sub optimal under physiological conditions as in here the pure chemical agents are reducing and supporting

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agent other than biological agents that give different chemical based surface chemistry which is

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pure and selectiveness in its nature[40-42]. Furthermore, Iron salt is made mixed with the plant extract, that is rich in the biomolecules that includes polyphenols, flavonoids and other

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molecules. Which is the reducing agent that reduces the FeCl3 and FeCl2 and oxidized itself. As

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the describe polyphenol gets oxidized and forms derivatives, as withanone converts into withaferin A after donating OH- and then this converts to withaferin D after giving an OH-. The donated OH- cause the Fe+3 to reduce to Fe+2 and then t Fe0 and hence proceeding towards the stability of the Iron and generating Iron Oxide rods. The complexed biomolecules get oxidized and form derivative and the concentration of specific biomolecule reduces by stabilizing the ion. Same is the case with Flavonoids, tannins, and other biomolecules that reduces the metal ion and oxidized itself. The XRD pattern of fabricated Iron oxide NRs is shown in Fig 2(c, d). The characteristic spectrum of x- ray diffraction for biologically synthesized Iron oxide NRs are well

Journal Pre-proof indexed at 2 theta values of 47° (311),54° (400), 60° (422),73° (440), 68° (511) and 77° (553nm) planes of crystal lattice. [43]. The intense narrow peaks and broad peaks are observed in spectra. Horde of narrow peaks indicates more crystalline structure of biologically fabricated particles and broad peaks show their reduction in size showing that they are more functional and remarkably more efficient as a result they are dominating in various biological fields and other diverse fields. The similar results were reported by using sea weeds mediated iron oxide

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nanoparticles synthesis [44-47]. On the other hand chemically manufactured Iron oxide NRs

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showed diffraction peaks only on 47° (311), 54° (400), 73° (440), 68° (511) and 77°(533) planes

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of crystal lattice. Less number of peaks were investigated in chemically synthesized Iron oxide

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NRs showing that they are less crystalline and more amorphous in structure [45]. The observed peaks were corresponding to the standard face centered cubic crystalline system of iron oxide

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nanoparticles (JCPDS software pdf ref. 01-088-0315). The Debye–Scherrer's equation (D=kλ/β

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cosθ) is used for the estimation of average crystal size and width of the Bragg's reflection of nanoparticles. The average crystallite size of was determined by using Scherrer’s formula 𝐷 =𝛽

𝐾𝜆

ℎ𝑘𝑙 𝑐𝑜𝑠𝜃

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[tauseef]:

Where, D = size of crystalline domains, λ = X-ray wavelength, k = 0.94 (shape factor), βhkl = full width of angular peak at half maximum and θ = measured Bragg’s angle. Biological = 26 nm Chemical = 34 nm The reduction in the crystallite size is occurred due to enveloping

of NRs with plant extract.

These capping agents restrict the nanoparticles to smaller size to attain proper stability. The XRD spectra obtained is more relevant to UV vis absorption observed. Size and shape of NRs is an important factor as it strongly effects the rate of diffusion through the cellular membranes,

Journal Pre-proof diffraction of light and their interaction with biological media. In previous research it has been reported that small size nanoparticles have more penetration power as compared larger size NPs, but too small size brings toxicity toward cell, so an appropriate size is desirable for innovative biological applications [46-49] Therefore, transmission electron microscopy (TEM) and Scanning electron microscopy (SEM) were used to evaluate the morphology and size distribution

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of prepared NRs via biological and chemical method prior to their applications.

Fig 3. TEM and SEM images of NRs Prepared by Biological method (a,c) Chemical Method (b,d)

Journal Pre-proof TEM images exhibited that biologically fabricated Iron oxide NRs with narrow size distribution of 33.3%, 15.3% ,32.4% and 10.8% in 0-50 nm ,60-100 nm,110-150 nm and 210-250 nm average length

with average diameter of 15-20 nm shown in Fig 3(a) and chemically

synthesized showed spindle shaped nanorays with size distribution of 36.4% ,29.4.% ,13.4%,9.6% ,7.2% and 3.8% in 60-100 nm, 160-200 nm, 0-50 nm, 110-150 nm, 210-250 nm and 260-300 nm average length with diameter of 10-15 nm in Fig 3(b). TEM result, showed that

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nanorods synthesized by biologically and chemically are different in size and morphology

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because of the precursor added in the reaction. Furthermore, it is interestingly found that the

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prepared iron oxide NRs via biologically methods are well organized as compared to NRs

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prepared by chemical method just like similar reported nanorods previously. [50, 51] SEM is instrument which gives detail informative about morphology and surface of prepared

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sample via biologically synthesized Iron oxide NRs which exhibited agglomeration of irregular

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clusters as shown in Fig 3(c) [52, 53]. The big adhesive clusters are formed due to accumulation of tiny building blocks of various bioactive reducing agents of plant extract, predominantly when

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chemical reaction undergoes between plant extract and chemical precursors nanorods Fig 3(d).

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Fig 4. EDX analysis of NRs Prepared by Biological method (a) Chemical Method (b) The results of present research are more relevant to previously reported results of green synthesized nanorods of silver While SEM of chemically manufactured showed NRs shaped iron oxide NRs which are arranged in the form of network of nano arrays [54-56]. Energy dispersive x-ray spectroscopy (EDX) is carried out to determine the elemental analysis of complex composition of Withania coagulans based iron oxide NRs and chemically synthesized iron oxide NRs. EDX technique is also performed to evaluate the purity level of synthesized NRs. The biologically synthesized nanoparticles have peaks around 6.4,2.4 and 0.7 keV are related to the

Journal Pre-proof binding energies of Fe with total percentage of 34.91%, along with the peak of oxygen 7.1 and 8.0 keV for oxygen with total 24.86. % Fig 4(a). Furthermore, there are some peaks for by products in the form of impurities like chlorine have peaks on 2.5 and 17.5 keV with total 40.2% while chemically prepared iron oxide NRs have 25.87% of iron,62.67% of oxygen,8.76% of nitrogen and 2.71% of chloride ion as shown in Fig 4(b). The presence of chlorine in the energy dispersive X-ray spectroscopy is commonly found in the synthesis of metal or metal oxide

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nanoparticles using the Withania coagulans extract. It has been observed that the impurities of

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elements can be further reduced by washing the precipitate with ethanol. These results are alike

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with previous results reported.[57]

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Photocatalytic activities.

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Increasing the population leads to industrial growth which effluents contain toxic chemicals and organic dyes becoming the part of water reservoir with directly or indirectly influenced the

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human health. Degradation of organic industrial wastes like Safranin to test the efficiency of iron

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oxide NRs fabricated by biological and chemically method. Comparative quantification of the

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dye degraded with prepared iron oxide NRs with biological and chemical method was performed Fig (S5, S6). Solar irradiation activates the production of hydroxyl group which trigger the iorn oxide NRs that activates the degradation of dye illustrated in Fig. 5b. The results showed gradual degradation of dye from 0 to 180 min of incubation under sunlight. Visible color change of the dye from dark pink to colorless solution was observed. Optical density (OD) of dye was recorded on 0 min, 30 min, 60 min, 90 min, 120 min, 150 min, 180, with absorption starts from 2.2, 2.1, 1.4,1.1, 0.9, 0.8, 0.7 gradually reduced respectively (Fig. 5a). The iron oxide NRs prepared chemically was also tested with same time point 0 min, 30 min, 60 min, 90 min, 120 min, 150 min, 180, and the optical density was calculated 1.45, 1.35, 1.25,1.15, 1.1, 1.08, 1.09 which

Journal Pre-proof gradually reduced respectively as well (Fig. 5b). Present study showed that the photocatalytic activity of iron oxide NRs prepared by green synthesis showed more degradation as compared to iron oxide NRs prepared by chemical method. This is because of biological molecules involves in the formation of nanorods which generated the surface Plasmon on NRs for excitation. It means these biomolecules which acts as reducing agent acts like catalyst to boost the photocatalytic activity as previously reported [58-61]. There may be chances for the area of NRs,

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as the area of NRs synthesized by biological method is more due to enhancing the hydroxyl

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radical formation leading to the degradation of safranin as shown in (Fig. 6). Furthermore, both

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biological and chemical method of NRs shown the photocatalytic activity in mechanism via Fig.

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Fig 5. Photocatalytic activity of NRs on degradation of safranin prepared by Biological method (a) Chemical Method (b)

The dye Degradation begins when added with Nano-rods in the presence of sunlight that excite the electron and make them reach the conducting state. The water gets dissociated into ions and starts a cascade of reactions involving hydroxyl and hydrogen that first causes the oxidation and

Journal Pre-proof reduction of the of the dye with minimal rate. Later DH3that has the maximum reduction state of safranin Dye D that gets oxidized to DH2 and then DH giving rise to H+ ions that unite with water to give oxygen reactive species and OH- and H2O2 that help in degradation of the dye. Whereas DO3 has the maximum oxidation state of the safranin Dye D that gets reduced to DO2 and then DO give rise to O- that gives oxygen reactive species and H2O2 that degrades the dye. This is how photo degradation of dye takes place. But the particle that were green synthesized

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had different surface chemistry that different biomolecules attached on the surface which causes

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more reduction and oxidation steps, with increased amount of reactive oxygen species that

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degrade the dye more efficiently. That Phyto-chemical decorated particle tends to increase the

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reduction and oxidation potential that further increases the Redox by-products of the dye leading to higher rate of photocatalytic degradation of safranin. As Fig 6 (a) shows the mechanism of

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extract and the FeCl3, where the Phyto-chemicals get derived into their oxidized form by

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donating the OH- that reduces the Fe3+ to Fe0. Later the oxidized derivatives are formed and get attached to the surface. This surface decoration by the derived phytochemicals provide a rich

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source of OH- that attack on the N- group and generating amines and also replacing the CH- bond

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that form new bonds and break the previous one. In this manner more oxygen rich species are generated that degrade the dye by breaking and replacing the bond via highly reactive oxygen species. The reaction generates OH-, OOH-, H2O2 and degradation agents but the surface chemistry hand increased the degradation as it gives higher oxygen rich species then the chemical. Furthermore, the dissociation of H+ and O- from the dye give rise to dye-Oxidation and dye-reduction products. As shown in Fig 6 (b)

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Fig 6. (a) Illustrates the interacting mechanism of iron slats with wathiana coagulans extract (b) Photocatalytic mechanism of degradation of safranin by NRs Synthesis Biological method and Chemical Method.

Journal Pre-proof The antibacterial activity of biologically synthesized and chemically prepared Iron oxide NRs is performed along with standard ciprofloxacin against gram positive cocci (S. aureus) and gramnegative rods (P. aeruginosa) by using disc method. The size of inhibition zone varies from one species of bacteria to another species. Our results evaluated that Iron oxide NRs synthesized using extract of Withania coagulans exhibited more antibacterial activity than chemically prepared Iron oxide NRs Fig 7 (a). The antibacterial activity varies with concentration, surface

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area, morphology and crystalline structure of NRs.

Journal Pre-proof Fig 7. Antibacterial activity of Iron oxide NRs prepared by Biological and chemical methods (a) The zone inhibition for bacterial strains by Biological method (a) The zone inhibition for bacterial strains by chemical method It has observed that by increasing the surface area and concentration of

NRs antibacterial

activity increases. Different concentrations of 5,10 and 20 µg/ml of both biologically and chemically prepared Iron oxide NRs were used. It is observed that the gowth of S. aureus and P.

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aeruginosa were suppressed more when treated with biologically synthesized Iron oxide NRs

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than treated with chemically prepared NRs shown in Fig 7 (b). Moreover, biologically synthesized Iron oxide NRs showed maximum antibacterial activity for S. aureus while

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minimum in P. aeruginosa for all concentrations of (5,10,20 µg/ml). Similar results were

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evaluated for chemically prepared Iron oxide NRs but with lower activity as shown in Fig 7(c).

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Ciprofloxacin also shows better results for S.aureus than P.aeruginosa based on different concentrations of (5,10,20 µg/ml). NRs synthesized by the green method showed 30% higher

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activity because the surface chemistry of green synthesized NRs has naturally present complex

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biomolecules on the surface that shows higher activity. As the biomolecules existing in the

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complex form are the strong candidates for exhibiting reducing and stabilizing potential prior in synthesis and further acts as increasing the antibacterial activity. These biomolecules present in the plants provide the defense flora against various microbes without any external source. Thus, it has naturally existing antimicrobial property too. Hence combination of these biological entities with the metallic source raise the antibacterial potential as compared to chemically synthesized NRs, where there is only define set of purely formed compounds. Ruther than the naturally occurring mixture of compounds. Conclusively, the naturally occurring surface chemistry of green NRs synthesis gives 30% more activity than the NRs consisting pre define pure chemical surface chemistry in the chemical method. However, biologically synthesized Iron

Journal Pre-proof oxide NRs shows much better antibacterial activity for all concentrations of S. aureus and P. aeruginosa as compared to standard ciprofloxacin and chemically prepared NRs that shows that green biologically synthesized Iron oxide NRs are more effective and efficient in killing bacteria than commercially available Iron oxide NRs [62-66]. Biological extracts only play important role in synthesis of iron oxide nanorods while we have only wanted to investigate weather antibacterial activity is because of NPs or biological extract (positive control). In our previous

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reports, it has been clearly observed that antimicrobial activity is only because of nanoparticles

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not because of plant extract [67-69]. Here in we had used two most popular and significant strain

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of those bacteria that are being the main cause of infectious diseases. The selectivity is done by

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ourselves where we choose those bacteria that are most common. Although the bacteria can be killed by any materials but NRs shows the more effectiveness of in current studies. The treatment

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is 30% more effective for the most infectious strains that has the ability to cure efficiently and to

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Conclusion

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clear off the infection in an effective and efficient way than other materials [70-72].

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In closure, we have fruitfully synthesized highly crystalline agglomerated clusters of nano rods and nanorays of Iron oxide NRs with an average size of 16 ± 2 nm and 18 ± 2 nm by using aqueous reducing extract of Withania Coagulans and chemically by the reduction precipitation method respectively. The structural analysis indicated that biologically synthesized Iron oxide NRs are highly stable, more crystalline, less toxic and more compatible in nature than chemically prepared NRs. Furthermore, when photocatalytic activity and antibacterial behavior of synthesized Iron oxide NRs was employed then biologically synthesized NRs showed more effective and efficient results than that of chemically prepared particles. Finally, we conclude

Journal Pre-proof that, herbally synthesized Iron oxide NRs are cost effective, economical, energy efficient and biologically more functional. Acknowledgement The authors would like to thank, The Islamia University Bahawalpur, Pakistan, National Research Program for University (NRPU) for Higher Education Commission (9458), School of

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Engineering, Peking University, Beijing china, for providing TEM and EDX facilities.

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Author statement All authors do not have any financial and personal relationships with other people or

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organizations that could inappropriately influence (bias) their work or state.

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Conflict of interest

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There is no conflict of interest among authors for publication of this work

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Research Highlights Synthesis of Iron Oxide NRs through chemical & Biological Method

Characterizations of Iron Oxide NRs

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Mechanistic investigation of Degradation

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Degradation of Safranine dye under solar radiation

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Antimicrobial activities of Iron Oxide NRs by chemical & Biological Method

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