Green synthesis of ZnO nanoparticles using Tecoma castanifolia leaf extract: Characterization and evaluation of its antioxidant, bactericidal and anticancer activities

Accepted Manuscript Green synthesis of ZnO nanoparticles using Tecoma castanifolia leaf extract: Characterization and evaluation of its antioxidant, bactericidal and anticancer activities

Govindasamy Sharmila, Marimuthu Chandrasekaran Muthukumaran

Thirumarimurugan,

PII: DOI: Reference:

S0026-265X(18)31256-6 https://doi.org/10.1016/j.microc.2018.11.022 MICROC 3467

To appear in:

Microchemical Journal

Received date: Revised date: Accepted date:

3 October 2018 12 November 2018 13 November 2018

Please cite this article as: Govindasamy Sharmila, Marimuthu Thirumarimurugan, Chandrasekaran Muthukumaran , Green synthesis of ZnO nanoparticles using Tecoma castanifolia leaf extract: Characterization and evaluation of its antioxidant, bactericidal and anticancer activities. Microc (2018), https://doi.org/10.1016/j.microc.2018.11.022

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ACCEPTED MANUSCRIPT Green synthesis of ZnO nanoparticles using Tecoma castanifolia leaf extract: Characterization and evaluation of its antioxidant, bactericidal and anticancer activities Govindasamy Sharmilaa*, Marimuthu Thirumarimuruganb, Chandrasekaran Muthukumarana Department of Industrial Biotechnology, Government College of Technology, Coimbatore -

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Department of Chemical Engineering, Coimbatore Institute of Technology, Coimbatore -

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b

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641013, Tamilnadu, India.

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641014, Tamilnadu, India.

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*Corresponding author Dr.G.Sharmila,

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Department of Industrial Biotechnology,

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Government College of Technology,

Coimbatore -641013, Tamilnadu, India.

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E-mail: [email protected]

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ACCEPTED MANUSCRIPT Abstract An efficient, facile and green route of zinc oxide nanoparticles was synthesized using Tecoma castanifolia leaf extract and characterized by UV–Vis spectroscopy, TEM, EDX, XRD and FTIR. Phytochemical constituents of T. castanifolia leaf extract were analyzed by GC-MS.

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UV–Vis absorption showed SPR band at 370- 400 nm which confirms the formation of ZnO NPs. TEM analysis exhibits spherical shape with size 70-75 nm and XRD results revealed the

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hexagonal phase of wurtzite structure. FTIR spectra confirmed the presence of O-H, C-H, amide-

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I, II groups, C-O bond and metal-oxygen groups. The presence of bioactive phytochemical constituents in the methanolic extracts of T. castanifolia was identified by GC-MS. An excellent

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antibacterial activity was observed for both Gram positive and Gram negative bacteria. Results of antioxidant activity showed that increase in concentration of ZnO nanoparticles increases the

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radical scavenging activity. Anticancer activity with IC50 value as 65 μg/mL which conferred

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better cytotoxic effects of ZnO NPs on proliferation of A549 cell line. The present study revealed

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that the pharmacologically active compounds present in the green synthesized ZnO nanoparticles

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pave the way to lead its effective application in biomedical and nano-drug delivery systems. Keywords: Anticancer; Antibacterial; Antioxidant; Green synthesis; Tecoma castanifolia; Zinc

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oxide nanoparticles.

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ACCEPTED MANUSCRIPT 1. Introduction Nanotechnology is an emerging field which focuses on the manipulation of matter at atomic and molecular level. It’s a multidisciplinary arena, provides a huge platform to create and engineer novel materials with unique properties. Exploration of nano sized particles between 1

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and 100 nm possess promising applications in various fields of science and create major impact in modern technology. Nanomaterials exhibits structures have distinct functionalities (large

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surface area, size-dependent properties) with enhanced catalytic, physical, chemical and also

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biological properties when reduced to nanosized level compared to bulk counterparts [1]. Recent years, metal nanoparticles attracted the research community due to its low melting point, large

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surface area, optical, catalytic and magnetic properties. These unique properties of nanoparticles

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made their exploitation in various industrial applications such as food, space, agriculture, cosmetics, chemical and medical field. This enables the consumers to turn for an eco-friendly,

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green approach towards the synthesis of nanoparticles [2]. ZnO NPs belongs to II–VI

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semiconductor type possess large band gap energy of 3.3 eV, and also high excitation energy of 60 eV enables it to withstand high temperature, large electric fields, under high power operations

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[3]. Owing to these properties, ZnO NPs are widely applicable in solar cells, chemical sensors

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and photocatalysis [4-6]. Basically, there are two approaches suggested for the synthesis of metal nanoparticles: (i) Top-down and (ii) Bottom-up approach. The top–down method relies on milling or break down of larger particle to nanoscale level whereas bottom-up involves the production of nanoparticles that starts from gaseous or liquid phase on the basis of atomic or molecular components [7]. Synthesis of nanoparticles is mediated by (i) physical, (ii) chemical and (iii) green or biogenic methods. Some physical methods such as high energy ball milling, physical vapor deposition, sputter deposition, laser ablation and chemical methods are (i) Liquid

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ACCEPTED MANUSCRIPT phase synthesis such as coprecipitation, colloidal methods, microemulsions, sol-gel processing, Solvothermal, hydrothermal, sonochemical and (ii) Vapor phase synthesis such as pyrolysis, inert gas condensation methods have been employed for the synthesis of metal nanoparticles [8]. But still the limitations of physiochemical methods are the need of high cost equipment, large

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surface area for equipment set up, high temperature and pressure, an additional use of capping

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agents and stabilizers, usage of toxic chemicals which are hazardous to the environment and

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living systems [9-10]. Thus to encounter these difficulties, green or biogenic methods are nowadays in practice for an eco-friendly, safe and cost-effective approach for the synthesis of

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metal nanoparticles. Green or biogenic route have the possibility of using least utilization of

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chemicals leads to less pollution, low cost and energy requirement [11]. Some of the natural moieties (plants, algae, fungi and bacteria) are used for the synthesis of ZnO NPs. Out of these

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moieties, plant extract mediated synthesis have gained more attention for the synthesis of various

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metal nanoparticles based on its plenty availability, non-hazardous, cost-effective and simple methodology [12-13]. But still, its exploitation has to be further explored for the benefit of

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mankind in various fields. Plant extract act as bioreducers and stabilizers for the synthesis of

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ZnO NPs through in vitro approach using zinc salt. Biometabolites (phenolics, tannins, saponins, flavonoids, terpenoids, polypeptides, starches etc) present in the plant extracts act as a

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bioreducing and capping agent for a particular metal salts yields desired nanoparticles [14]. ZnO NPs have attracted most of the research community using various plant sources such as Calotropis gigantea [1], Borassus flabellifer [15], Parthenium hysterophorus [16], Tectona grandis [17], Passiflora caerulea [18] and Glycosmis pentaphylla [19]. Tecoma castanifolia belongs to Bignoniaceae family which is a common ornamental tree known by its general names such as yellow bells, yellow elder, yellow trumpet. The leaf of T.

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ACCEPTED MANUSCRIPT castanifolia contains flavonoids, terpenes, alkaloids and hydrocarbons act as bioreductant for the nanoparticle synthesis [20]. The leaves of T. castanifolia have anti-inflammatory and antinociceptive activity. Cancer is the most threatening disease leads to high mortality worldwide. Specifically, lung cancer affects most of the people ends with high mortality rate.

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The use of chemotherapy to treat the cancer patients has its limitation due to its low specificity

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and also restriction towards its dosage level. To overcome these limitations, an alternate way of

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therapies and drugs are essential to treat the cancer. Nowadays, eco-friendly based nanoparticles are attempted by the researchers as an alternate to control the cancerous cell growth [21-22].The nanoparticles aimed for safe and effective targeted drug delivery with

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phytosynthesized

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controlled dosage to treat the cancer patients. The present study reported on the green synthesis of ZnO NPs using leaves of T. castanifolia and screened for its antibacterial, antioxidant and

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anticancer activity. Further, the phytosynthesized nanoparticles are subjected to drug release

delivery.

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2. Materials and methods

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kinetics of 5 Fluorouracil (5-FU) using chitosan as a carrier for effective dose dependent

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2.1 Chemicals

Zinc sulphate (ZnSO4), nutrient agar, 1,1-Diphenyl-2-picryl-hydrazyl (DPPH),

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methylthiazolyldiphenyl- tetrazolium bromide (MTT), 5 Fluorouracil and Dimethyl sulfoxide (DMSO) were procured from HiMedia. Chemicals used in the present study were of analytical grade. 2.2 Plant material collection T. castanifolia leaves were obtained from GCT campus, Coimbatore, Tamilnadu, India. The fresh leaves of T. castanifolia were washed thoroughly with distilled water to remove the

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ACCEPTED MANUSCRIPT dirt particles and then shade dried for one week. After ensuring the complete dryness, the leaves were powdered using kitchen blender and stored in airtight container at room temperature for further usage. 2.3 Preparation of plant extract

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5 g of powdered T. castanifolia leaf sample was taken and added to 50 mL of double

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distilled water. It was kept in the water bath at 60 °C for 15 min. The mixture was allowed to

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cool and then mixed well with the aid of magnetic stirrer for 20-30 min to enhance better extraction. The extract was subjected to filtration using Whatman No.1 filter paper to remove the

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leaf debris. The clear extract filtrate obtained was stored at 4°C for further use.

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2.4 Preparation of salt solution

1 mM zinc sulphate solution was prepared for 100 mL and the prepared ZnSO4 solution

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2.5 Phytosynthesis of ZnO NPs

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was used for the phytosynthesis of ZnO NPs.

For ZnO nanoparticle synthesis, 10 mL of plant extract was taken and mixed well with 90

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mL of zinc sulphate solutions. The mixture was incubated at room temperature for 4 days and it

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was monitored at regular interval for the formation of nanoparticles by visually and also through UV-Vis spectroscopy analysis. After 4 days incubation, the mixture was allowed to centrifuge at

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5000 rpm for 15 min. The pellet was collected and it was resuspended in distilled water for further centrifugation. The collected pellet was processed repeatedly twice or thrice to remove the impurities present in it. Finally, the obtained pellet was dried in hot air oven till the moisture is completely removed. The obtained dried particles were collected and used for further characterization studies. 2.6 Characterization

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ACCEPTED MANUSCRIPT 2.6.1 UV-vis spectroscopy analysis The nanoparticles are formed by the bioreduction of metal ions in the salt solution was analyzed periodically at regular intervals of 24 h for 4 days. UV-Vis spectroscopy analysis was carried out as a function of time for the prepared ZnO NPs at room temperature using

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2.6.2 Transmission electron microscopy (TEM) analysis

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Analytikjena UV spectrophotometer.

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The synthesized ZnO nanoparticles were characterized for their shape and size distribution using Transmission electron microscope (TEM, TECNAI G2 F-30, FEI). ZnO NPs

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suspension was subjected to sonication using Citizen Digital Ultrasonic bath for 30 min before

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placing it on the carbon coated copper grid. 2.6.3 Energy dispersive x-ray (EDX) analysis

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The elemental presence of zinc was confirmed with EDX analysis. This was performed

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using EDX detector coupled with TECNAI Transmission electron microscope. 2.6.4 X-ray diffraction (XRD) analysis

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The crystalline nature of the synthesized ZnO NPs was characterized by X-ray

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diffractometer (X‟Pert power, Germany) instrument using Cu Kα radiation in the 2θ range of 10-

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2.6.5 FTIR analysis

The phytosynthesized ZnO NPs were analyzed to determine the functional groups present by FTIR spectroscopy. The analysis was done using Perkin-Elmer Spectrum Two model instrument by mixing the sample with KBr salt and further measurements were recorded at a scan range of 400- 4000 cm-1 with the resolution of 2 cm-1.

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ACCEPTED MANUSCRIPT 2.6.6 GC-MS analysis The phytochemicals present in T. castanifolia leaf extract was analyzed by GC-MS. The instrument used was Agilent Technologies 7890B (GC) equipped with 5977A Mass Selective Detector (MSD). 1 μL of sample was injected to HP-5MS (5% phenyl methyl siloxane) capillary

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column of dimensions 30m×250μm×0.25μm and helium was used as carrier gas. The

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temperatures of injector and detector were 250°C and 290°C respectively. The column

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temperature was initially programmed at 50°C for 5 min, followed by an increase of 5°C/min to 270°C and maintained isothermally for 10 min. The MS was operating at 70 eV with m/z scan

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spectral data using the standard NIST library 2011.

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range of 40-600 amu. The phytochemical composition identified was compared with their mass

2.7 Antibacterial activity of ZnO NPs

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The green synthesized ZnO NPs were tested for their antibacterial activity against Gram

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positive (Bacillus subtilis and Staphylococcus aureus) and Gram negative (Escherichia coli and Pseudomonas aeruginosa). The well-diffusion assay was performed. Nutrient agar medium of

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approximately 20 mL was poured to the petriplates and 5 mm wells were made using cork borer.

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The wells were loaded with 50 μL of ZnO NPs of different concentration (25, 50, 75,100 μg) in each plate to determine the antibacterial activity. Ceftazidime-clavulanic acid (30/10 mcg)

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antibiotic disc was used as control and after 24 hrs of incubation at 37°C, the zone of inhibition was measured.

2.8 Antioxidant activity 2.8.1 DPPH assay ZnO NPs of various concentrations (10-100 μg/mL) were added to 2 mL methanol and 1 mL methanolic solution containing DPPH (2,2-diphenyl-1-picryl hydrazyl) radicals (0.012 g/100

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ACCEPTED MANUSCRIPT mL). The solution was mixed well, incubated for 60 min at room temperature and the absorbance was read at 517 nm against the blank. Ascorbic acid was used as standard and the DPPH solution was used as control. The scavenging ability was calculated using the following equation: Scavenging activity (%) =

𝐴517 𝑜𝑓 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴517 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑥 100 𝐴517 𝑜𝑓 𝑐𝑜𝑛𝑡𝑟𝑜𝑙

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2.9 Anticancer activity (MTT assay)

Human lung carcinoma cells (A549) was obtained from National Centre for Cell Sciences, Pune,

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India, were seeded (1 x 105 cells/ 25 mm T Flask) and cultured in Dulbecco’s Modified Eagle

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Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% antibiotic solution at 37°C with 5% CO2. Cells were sub-cultured every third day by trypsinization with trypsin phosphate

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versus glucose solution (TPVG). A549 cells (2.5×105) were seeded in 96-well titre plate and

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allowed to grow overnight at 37°C. The nanoparticles concentration ranging from 20 (μg/mL) 100 (μg/mL) were treated with the cells for 24 hrs. MTT reagent [3-(4,5-Dimethylthiazol-2-yl)-

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2,5-diphenyltetrazolium bromide] was added to each well and incubated further for 4 hrs at

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37°C. Dimethylsulphoxide (DMSO) was added to each well and incubated for 1 hr and the absorbance was recorded at 570 nm. Sample without the addition of the nanoparticles act as

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control. The concentration of nanoparticles required to inhibit 50% of viability (IC 50) was

formula.

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determined graphically and the cell viability percentage was calculated using the following

𝐶𝑒𝑙𝑙 𝑣𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦(%) =

Absorbance of treated cells 𝑥 100 Absobance of control cells

The effect of cytotoxicity observed in untreated and treated A549 cell line with ZnO NPs was examined using phase contrast microscope. 2.10 Evaluation of ZnO NPs for in-vitro drug delivery

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ACCEPTED MANUSCRIPT The phytosynthesized ZnO NPs interact with chitosan for the formulation of polyelectroyte nanoparticulate for drug delivery using 5-Flurouracil (5-FU) as a model drug. 5 mL aqueous solution containing 10 mg ZnO NPs and 10 mg 5-FU added drop by drop to 0.02% chitosan prepared in 2% (v/v) acetic acid under the sonication. The suspension was further

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sonicated for 30 min to obtain ZnO-Chitosan-5FU nanosuspension. The entrapment efficiency

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(%) was calculated by centrifuging the sample at 10000 rpm for 15 min at 4˚C and the

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supernatant was analyzed for free 5-FU at 266 nm. Dialysis sac method was used for in-vitro drug release of ZnO-Chitosan-5FU nanosuspension. Dialysis sac containing 4 mL of

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nanosuspension formulation was tied with thread to be immersed in 100 ml of 1X PBS buffer

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(release media). 3 mL of sample aliquots were withdrawn and replaced by the equal volume of fresh PBS at periodic intervals. The 5-FU contents in the samples was determined by measuring

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the absorbance at 266 nm. The release kinetic rate to determine the rate constants and the

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mechanism of 5-FU release from nanosuspension, was fitted to four models zero-order, firstorder, Higuchi and Koresmeyer–Peppas model. The release rate constant (k) for the applied

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models was determined graphically.

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3. Results and discussion 3.1 Characterization

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3.1.1 UV-Vis characterization The formation of ZnO NPs was observed by gradual colour change of the solution attributed to surface resonance plasmon (SPR) of the synthesized NPs. The bioreduction of Zn2+ to Zn0 ions was mainly due to the phytochemicals present in T. castanifolia leaf extract and the formation of ZnO NPs was monitored for 4 days periodically by UV-vis analysis. Fig.1 represent the UV-vis spectrum analysis of the synthesized ZnO NPs. A progressive SPR band appearance at the

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ACCEPTED MANUSCRIPT wavelength range of 370- 400 nm which confirms the formation of ZnO NPs. The maximum absorbance peak was obtained at 380 nm. The obtained SPR result was well accordance with the previously reported literature on green synthesized ZnO NPs [23]. Similar results of absorption peak obtained for ZnO NPs at 370 nm was reported by Padalia and Chanda [24].The result

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confirmed that phytochemicals such as alkaloids, trepenoids, phenolics, flavonoids, polypeptides

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present in the T. castanifolia leaf extract act as bioreductant which plays a significant role in the

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ZnO NPs formation [25]. 3.1.2 TEM and EDX characterization

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The size and shape of the synthesized ZnO NPs were analyzed as dark spots of spherical shape

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with size 70-75 nm observed in TEM analysis (Fig.2a). Individually dispersed spherical shaped dark spots of ZnO NPs was seen and few ZnO NPs were agglomerated due to high surface

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energy of particles which may be attributed when the synthesis was performed in aqueous

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medium [26]. Also, the TEM image depicts that ZnO NPs were capped by phytochemicals present in the T. castanifolia leaf extract. The chemical composition of prepared ZnO NPs was

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identified by EDX analysis. EDX spectrum (Fig.2b) confirmed the presence of oxygen and zinc

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signals in the ZnO NPs prepared using T. castanifolia leaf extract. The other signals in the EDX spectrum were may be due to bioorganics present in the leaf extract.

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3.1.3 XRD analysis

The XRD pattern for the phytosynthesized ZnO NPs was shown in Fig.3a. The Bragg‟s reflection peaks at 2θ = 30.96°, 34.08°, 36.51°, 47.32°, were indexed to the (100), (002), (101), and (102) corresponds to the hexagonal phase of wurtzite structure [23-24]. The characteristic peaks obtained were in good correlation with the standard JCPDS no-36-1451 [27]. 3.1.4 FTIR analysis

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ACCEPTED MANUSCRIPT The FTIR spectrum reveals the presence of functional groups present in the phytosynthesized ZnO NPs were determined in the scan range of 4000– 400 cm-1 (Fig.3b). The characteristic peak obtained at 3291 cm-1belongs to O-H group. The peaks at 2930 cm-1 represent the C-H stretching vibration mode in alkanes. The peaks (1530-1663 cm-1) showed the presence of amide-I and II

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groups observed was mainly due the presence of proteins in the T. castanifolia leaf extract. The

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bands at 1239 cm-1 and 1075 cm-1corresponds to C-O bond attributed to the characteristic group

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of alcohols and carboxylic acids. The peaks observed in the range of (400-600 cm-1) are due to the metal-oxygen groups. A peak at 459 cm-1 confirms the ZnO bond at bending vibration [28].

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The observed functional groups in the FTIR analysis revealed that the phytochemicals such as

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proteins, alkaloids, flavanoids and phenolics were contributed for the formation and stability of ZnO NPs synthesized by the biogenic reduction [26].

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3.2 GC-MS analysis

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GC/MS analysis was performed to identify the phytochemical constituents present in the methanolic leaf extract of T. castanifolia. Totally, 54 compounds were identified by GC-MS and

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the detailed information about the phytochemicals were listed in Table. 1. Majorly, 8 compounds

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having the highest composition peaks out of 54 compounds. The maximum peak composition were shared by1,15-Pentadecanediol (26.95%), 1,10-Decanediol (18.76), n-Hexadecanoic acid

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(7.36), 9,12-Octadecadienoic acid (Z,Z) (7.11), 9,12,15-Octadecatrienoic acid, (Z,Z,Z) (5.49), Squalene (4.38), Octadecanoic acid (3.43) and dibutyl phthalate (2.35). Further GC-MS reveals the presence of bioactive phytochemical constituents such as sugars, amides, alcohols, aldehydes, ethers, ketones, carboxylic acids, amino acids, fatty acids, alkaloids, phenolic compounds, flavonoids, tannins and terpenoids were present in the methanolic extracts of T. castanifolia. These bioactive phytochemical constituents can act as a key factor for the reduction

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ACCEPTED MANUSCRIPT of Zn ions to ZnO NPs and also responsible for the potent (antibacterial, antioxidant and anticancer) bioactivities enhanced by ZnO NPs derived from T. castanifolia leaf extract. Similar GC-MS results were reported out for the leaf extracts such as Broussonetia luzonica [29], Schinus terebinthifolius [30] for the identification of phytochemical compounds.

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3.3 Antibacterial activity of ZnO NPs

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Antibacterial activity against Gram positive (Bacillus subtilis and Staphylococcus aureus) and

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Gram negative (E. coli and Pseudomonas aeruginosa) bacterial strains with ZnO NPs of T. castanifolia leaf extract was studied and zone of inhibition was measured. The zone of inhibition

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observed for both Gram negative and Gram positive bacteria (Fig.4).The excellent antibacterial

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activity was observed at the concentration of (75 and 100 μg) on both Gram positive and Gram negative bacteria. The maximum zone of inhibition obtained for E. coli (17 mm)and

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Pseudomonas aeruginosa (15 mm), Bacillus subtilis (15 mm) and Staphylococcus aureus (17

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mm) at 100 μg of ZnO NPs (Table.2).The control antibiotic disc shows no inhibition. The results confirmed that ZnO NPs synthesized by T. castanifolia possess better antibacterial activity

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against all the tested bacterial strains. The mechanism of antibacterial activity relies on the action

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of ZnO NPs with the bacterial cell wall leads to the bacterial cell wall destruction results in the liberation of ions and ROS formation [31-33]. The antibacterial activity of ZnO NPs proved to be

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better candidate for the implementation of synthesizing bioactive food packaging materials to prolong its shelf-life with microbial resistance [34]. 3.4 Antioxidant activity of ZnO NPs DPPH, a stable free radical acquires the characteristic absorption at 517 nm in the UV-Vis analysis. DPPH assay was performed to assess the free radical scavenging activity of the green synthesized ZnO NPs by the decrease in the absorbance of DPPH. T. castanifolia leaf extract

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ACCEPTED MANUSCRIPT mediated ZnO NPs were assessed for radical scavenging activity by DPPH assay. Various concentrations ranging from 10-100 μg/mL was used and the results were in the following order 100 μg/mL (67%) >90 μg/mL (63%) >80, 70 μg/mL (54%) >60 μg/mL (53%) > 50 μg/mL (40%)> 40 μg/mL (35%) > 30 μg/mL (28%) > 20 μg/mL (23%) > 10 μg/mL (13%) as

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represented in Fig.5. The increase in concentration of ZnO NPs increases the radical scavenging

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activity and maximum 67 % was observed at 100 μg/mL. . The major phytochemical constituents

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such as 1,15-Pentadecanediol and 1,10-Decanediol present in T. castanifolia leaf extract can be responsible for greater antioxidant activity. Increased antioxidant activity enhanced by ZnO NPs

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was due to the activation of reactive oxygen species (ROS) generation results to cell death.

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Related antioxidant studies were reported in literature for plant extract mediated ZnO NPs [35-

3.5 Anticancer activity of ZnO NPs:

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36].

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Anticancer activity was evaluated for T. castanifolia mediated ZnO NPs against A549 cell line with the various concentrations ranging from 20 μg/mL, 40 μg/mL, 60 μg/mL, 80 μg/mL and

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100 μg/mL and the cell viability test was determined after 48 hrs. Fig.6a showed the altered

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morphology of A549 cells after treated with ZnO NPs based on dosage. Treated ZnO NPs revealed that the increase in concentration from 20-100 μg/mL of the ZnO NPs decreases the

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proliferation of A549 cell line compared to the control (Fig.6b). Cell viability (%) was calculated using the formula and the respective graph was plotted as cell viability (%) at Y-axis and concentration of ZnO NPs in X axis. Based on the graphical results, the IC 50 value was determined as 65 μg/mL confers better cytotoxic effects in the proliferation of A549 cell line. The similar cytotoxicity studies for the green synthesis of ZnO NPs were reported earlier by Senthilkumar et al. [17] and Baskar et al. [37].

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ACCEPTED MANUSCRIPT 3.6 In-vitro drug release studies The nanosuspension of TZ-chitosan-5FU was prepared and the entrapment efficiency (%) of 5-FU drug with the nanoparticles- chitosan polymer complex was determined by measuring the absorbance of unbound 5-FU in the supernatant after centrifugation. The

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entrapment efficiency for TZ-chitosan-5FU was calculated as 90.4%. Fig.7 represented the in-

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vitro drug release profile of TZ-chitosan-5FU and chitosan-5FU nanosuspension without the

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nanoparticles is used as control for comparative analysis. From Fig.7, it was observed that 99% of the drug was released from TZ-chitosan-5FU nanosuspension at 40 min respectively whereas

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for control, maximum release was observed at 22 min. From this results, it was revealed that

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TZ-chitosan-5FU nanosuspension showed sustained drug release as compared to chitosan-5FU

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3.6.1 In-vitro drug release kinetics:

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nanosuspension.

The rate constants with respect to 5-FU release of ZnO NPs-chitosan-5-FU nanosuspension were

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derived from the kinetic plots shown in Fig.8a-d. Higuchi model of R2=0.944 explains the linear

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relationship between drug release rate and drug concentration than zero order (R2=0.915) and first order (R2=0.786) models. The estimated rate constants and coefficient of determination (R2)

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were summarized in Table.3. The drug mechanism was explained by the ‘n’ value of Koresmeyer–Peppas plot. Fickian diffusion, non- Fickian diffusion, case-II transport, super caseII transport mechanisms are explained by the ‘n’ value of 0.45 < n < 0.89, 0.89 and >0.89 respectively The results of the study revealed that non-Fickian diffusion mechanism was adopted for 5-FU release from the ZnO NPs-chitosan-5-FU nanosuspension with the release exponent (n) value of 0.791 from the Koresmeyer–Peppas plot. Similar drug release studies for curcumin with

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ACCEPTED MANUSCRIPT beta-cyclodextrin coated PEG linked ZnO nanocomposite as a drug delivery vehicle for cancer therapy [38]. 4. Conclusion The present study concluded that the phytochemical constituents present in the plant

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extracts plays a vital role in the biogenic formation of ZnO NPs and also for the antibacterial,

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antioxidant, anticancer activities. T. castanifolia leaf extract derived ZnO NPs showed good

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antibacterial, antioxidant and anticancer activities were due to the synergistic effect of bioactive phytochemical constituents. The sustained release of ZnO NPs has its effective application for

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the delivery of chemotherapeutic drugs in the body to the targeted site in nano-drug delivery

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systems.

commercial, or not-for-profit sectors.

[1]

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References:

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Funding: This research did not receive any specific grant from funding agencies in the public,

S.K. Chaudhuri, L. Malodia, Biosynthesis of zinc oxide nanoparticles using leaf extract of

PT

Calotropis gigantea: characterization and its evaluation on tree seedling growth in nursery

[2]

CE

stage, Appl. Nanosci. 7 (2017) 501-512. P. Basnet, T.I. Chanu, D. Samanta, S. Chatterjee, A review on bio-synthesized zinc oxide

AC

nanoparticles using plant extracts as reductants and stabilizing agents, J. Photochem. Photobiol. B. 183 (2018) 201-221. [3]

P. Sutradhar, M. Saha, Green synthesis of zinc oxide nanoparticles using tomato (Lycopersicon esculentum) extract and its photovoltaic application, J. Exp. Nanosci. 11 (2016) 314-327.

16

ACCEPTED MANUSCRIPT [4]

X. Bai, L. Li, H. Liu, L. Tan, T. Liu, X. Meng, Solvothermal synthesis of ZnO nanoparticles and anti-infection application in vivo, ACS Appl. Mater. Interface. 7 (2015) 1308-1317.

[5]

M. Moritz, M. Geszke-Moritz, The newest achievements in synthesis, immobilization and

A. Reinert, C. Payne, L. Wang, J. Ciston, Y. Zhu, P. G. Khalifah, Synthesis and

IP

[6]

T

practical applications of antibacterial nanoparticles, Chem. Eng. J. 228 (2013) 596-613.

CR

characterization of visible light absorbing (GaN) 1-x(ZnO)x semiconductor nanorods, Inorg. Chem. 52 (2013) 8389-8398.

I. Hussain, N.B. Singh, A. Singh, H. Singh, S.C. Singh, Green synthesis of nanoparticles

US

[7]

[8]

AN

and its potential application, Biotechnol. Lett. 38 (2016) 545-560. A.Naveed Ul Haq, A. Nadhman, I.Ullah, G.Mustafa, M.Yasinzai, I. Khan, Synthesis

M

approaches of zinc oxide nanoparticles: The dilemma of ecotoxicity, J. Nanomater. (2017)

[9]

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14 pages, doi:10.1155/2017/8510342.

R. Chandrasekaran, S. Gnanasekar, P. Seetharaman, R. Keppanan, W. Arockiaswamy, S.

PT

Sivaperumal, Formulation of Carica papaya latex-functionalized silver nanoparticles for

CE

its improved antibacterial and anticancer applications, J. Mol. Liq. 219 (2016) 232-238. [10] P. Dhandapani, A.S. Siddarth, S. Kamalasekaran, S. Maruthamuthu, G. Rajagopal, Bio-

AC

approach: Ureolytic bacteria mediated synthesis of ZnO nanocrystals on cotton fabric and evaluation of their antibacterial properties, Carbohydr. Polym. 103 (2014) 448-455. [11] G. Sharmila, S. Haries, M.F. Fathima, S. Geetha, N.M. Kumar, C. Muthukumaran, Enhanced catalytic and antibacterial activities of phytosynthesized palladium nanoparticles using Santalum album leaf extract, Powd. Technol. 320 (2017) 22-26.

17

ACCEPTED MANUSCRIPT [12] S.A. Khan, F. Noreen, S. Kanwal, A. Iqbal, G. Hussain, Green synthesis of ZnO and Cudoped ZnO nanoparticles from leaf extracts of Abutilon indicum, Clerodendrum infortunatum, Clerodendrum inerme and investigation of their biological and photocatalytic activities, Mater. Sci. Eng. C. 82 (2018) 46-59.

T

[13] J. Jeevanandam, Y.S. Chan, M.K. Danquah, Biosynthesis of metal and metal oxide

IP

nanoparticles, ChemBioEng Rev. 3 (2016) 55-67.

CR

[14] P. Singh, Y.J. Kim, D. Zhang, D.C. Yang, Biological synthesis of nanoparticles from plants and microorganisms, Trend. Biotechnol. 34 (2016) 588-599.

US

[15] K. Vimala, S. Sundarraj, M. Paulpandi, S. Vengatesan, S. Kannan, Green synthesized

AN

doxorubicin loaded zinc oxide nanoparticles regulates the Bax and Bcl-2 expression in breast and colon carcinoma. Process Biochem. 49 (2014) 160-172.

M

[16] A.Datta, C. Patra, H. Bharadwaj, S. Kaur, N. Dimri, R. Khajuria, Green synthesis of zinc

ED

oxide nanoparticles using Parthenium hysterophorus leaf extract and evaluation of their antibacterial properties, J. Biotechnol. Biomater. 7 (2017) 271.

PT

[17] N. Senthilkumar, E. Nandhakumar, P. Priya, D. Soni, M. Vimalan, I.V. Potheher, Synthesis

CE

of ZnO nanoparticles using leaf extract of Tectona grandis (L.) and their anti-bacterial, anti-arthritic, anti-oxidant and in vitro cytotoxicity activities, New J. Chem. 41 (2017)

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10347-10356.

[18] J. Santhosh kumar, S.V. Kumar, S. Rajeshkumar, Synthesis of zinc oxide nanoparticles using plant leaf extract against urinary tract infection pathogen, Resour. Effi. Technol. 3 (2017) 459-465.

18

ACCEPTED MANUSCRIPT [19] S. Vijayakumar, P. Krishnakumar, P. Arulmozhi, S. Mahadevan, N. Parameaswari, Biosynthesis, characterization and antimicrobial activities of zinc oxide nanoparticles from leaf extract of Glycosmis pentaphylla (Retz.) DC. Microb. Pathogenesis. 116 (2018) 44-48. [20] G. Sharmila, M. Thirumarimurugan, V.M. Sivakumar, Optical, catalytic and antibacterial

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properties of phytofabricated CuO nanoparticles using Tecoma castanifolia leaf

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extract, Optik. 127 (2016) 7822-7828.

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[21] A.B. Moghaddam, M. Moniri, S. Azizi, R. Abdul Rahim, A. Bin Ariff, M. Navaderi, R. Mohamad, Eco-friendly formulated zinc oxide nanoparticles: Induction of cell cycle arrest

US

and apoptosis in the MCF-7 cancer cell line, Genes. 8 (2017) 281.

AN

[22] D. Rehana, D. Mahendiran, R.S. Kumar, A.K. Rahiman, Evaluation of antioxidant and anticancer activity of copper oxide nanoparticles synthesized using medicinally important

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plant extracts, Biomed. Pharmacother. 89 (2017)1067-1077.

ED

[23] N. Bala, S. Saha, M. Chakraborty, M. Maiti, S. Das, R. Basu, P. Nandy, Green synthesis of zinc oxide nanoparticles using Hibiscus subdariffa leaf extract: Effect of temperature on

PT

synthesis, anti-bacterial activity and anti-diabetic activity, RSC Adv. 5 (2015) 4993-5003.

CE

[24] H. Padalia, S. Chanda, Characterization, antifungal and cytotoxic evaluation of green synthesized zinc oxide nanoparticles using Ziziphus nummularia leaf extract, Artif. Cell.

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Nanomed. Biotechnol. 45 (2017) 1751-1761. [25] A.K. Mittal, Y. Chisti, U.C. Banerjee, Synthesis of metallic nanoparticles using plant extracts, Biotechnol. Adv. 31 (2013) 346-356. [26] G. Rajakumar, M. Thiruvengadam, G. Mydhili, T. Gomathi, I.M. Chung, Green approach for synthesis of zinc oxide nanoparticles from Andrographis paniculata leaf extract and

19

ACCEPTED MANUSCRIPT evaluation of their antioxidant, anti-diabetic, and anti-inflammatory activities, Bioprocess Biosys. Eng. 41(2018) 21-30. [27] N. Matinise, X.G. Fuku, K. Kaviyarasu, N. Mayedwa, M. Maaza, ZnO nanoparticles via Moringa oleifera green synthesis: Physical properties & mechanism of formation, Appl.

T

Surf. Sci. 406 (2017) 339-347.

IP

[28] A.C. Dhanemozhi, V. Rajeswari, S. Sathyajothi, Green synthesis of zinc oxide nanoparticle

CR

using green tea leaf extract for supercapacitor application, Mater. Today: Proceed. 4 (2017) 660-667.

in

different

extracts

of

an

endemic

plant

Broussonetia

luzonica

AN

present

US

[29] F.P. Casuga, A.L. Castillo, M.J.A.T. Corpuz, GC–MS analysis of bioactive compounds

(Blanco)(Moraceae) leaves, Asian Pac. J. Trop. Biomed. 6 (2016) 957-961.

M

[30] F.B. Carneiro, P.Q. Lopes, R.C. Ramalho, M.T. Scotti, S.G. Santos, L.A. Soares,

ED

Characterization of leaf extracts of Schinus terebinthifolius raddi by GC-MS and chemometric analysis, Pharmacogn. Mag. 13 (2017) S672.

PT

[31] U. Kadiyala, E.S. Turali-Emre, J.H. Bahng, N.A. Kotov, J.S. VanEpps, Unexpected

CE

insights into antibacterial activity of zinc oxide nanoparticles against methicillin resistant Staphylococcus aureus (MRSA), Nanoscale. 10 (2018) 4927-4939.

AC

[32] S. Ahmed, S.A. Chaudhry, S. Ikram, A review on biogenic synthesis of ZnO nanoparticles using plant extracts and microbes: A prospect towards green chemistry, J. Photochem. Photobiol. B, 166 (2017) 272-284. [33] J. Karbowniczek, L. Cordero-Arias, S. Virtanen, S.K. Misra, E. Valsami-Jones, L. Tuchscherr, B. Rutkowski, K. Górecki, P. Bała, A. Czyrska-Filemonowicz, A.R. Boccaccini, Electrophoretic deposition of organic/inorganic composite coatings containing

20

ACCEPTED MANUSCRIPT ZnO nanoparticles exhibiting antibacterial properties, Mater. Sci. Eng. C. 77 (2017) 780789. [34] M. Hoseinnejad, S.M. Jafari, I. Katouzian, Inorganic and metal nanoparticles and their antimicrobial activity in food packaging applications, Crit. Rev. Microb. 44 (2018) 161-

T

181.

IP

[35] D. Rehana, D. Mahendiran, R. Senthil Kumar, A. Kalilur Rahiman, In vitro antioxidant and

CR

antidiabetic activities of zinc oxide nanoparticles synthesized using different plant extracts, Bioprocess Biosys. Eng. 40 (2017) 943-957.

US

[36] Siripireddy, B.K. Mandal, Facile green synthesis of zinc oxide nanoparticles by Eucalyptus

AN

globulus and their photocatalytic and antioxidant activity, Adv. Powd. Technol. 28 (2017) 785-797.

M

[37] G. Baskar, A. Gurugulladevi, T. Nishanthini, B.G. Garrick, R. Aiswarya, M. Gopinath,

ED

Synthesis of phytonanocomposite of zinc oxide by Ixora coccinea Linn for cancer treatment, J. Inorg. Organomet. Polym. Mater. 26 (2016) 876-880. Sawanta,

S.R.

Bamane,

PT

[38] V.J.

PEG-beta-cyclodextrin

functionalized

zinc

oxide

CE

nanoparticles show cell imaging with high drug payload and sustained pH responsive

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delivery of curcumin in to MCF-7 cells, J. Drug Deliv. Sci. Technol. 43 (2018) 397-408.

21

ACCEPTED MANUSCRIPT Table.1 GC–MS analysis report for the methanolic extract of T. castanifolia leaves extract.

Peak No

Retentio n time (min)

Molecular weight

Peak area (%)

1

2.819

2

3.463

1-Di(tert-butyl)silyloxy-2phenylethane Urea

264

0.49

CH4N2O

60

0.35

3

5.09

Furfural

C5H4O2

96

0.88

4

5.665

2-Furanmethanol

C5H6O2

98

0.18

5

7.11

1,2-Cyclopentanedione

C5H6O2

98

0.27

6

7.904

2-Furancarboxaldehyde, 5-methyl

C6H6O2

110

0.26

7

8.336

1-Octen-3-ol

C8H16O

128

0.95

8

10.056

Propionaldehyde, diethylhydrazone

C7H16N2

128

0.26

9

10.888

2-Octenoic acid, 4,5,7-trhydroxy

C8H14O5

190

0.28

10

11.658

C10H18O

154

0.27

11

13.103

144

0.41

12

14.341

1,6-Octadien-3-ol, 3,7-dimethyl 4H-Pyran-4-one, 2,3-dihydro-3,5dihydroxy-6-methyl Methyl salicylate

C8H8O3

152

1.08

13

14.698

Propanal, 2-methyl-3-phenyl

C10H12O

148

0.22

14

15.255

8,10-Dodecadien-1-ol, (E,E)

C12H22O

182

0.25

15

15.348

Benzofuran, 2,3-dihydro

C8H8O

120

0.50

16

15.661

C10H14O

150

0.48

17

17.694

150

0.37

18

18.839

Cyclohexanone, 2-(2-butynyl) Ethanone, 1-(2-hydroxy-5methylphenyl) Eugenol

C10H12O2

164

0.65

19

20.353

C11H14O

162

0.23

20

21.235

224

0.37

21

21.41

288

0.60

22

21.529

338

0.26

23

174

18.76

Molecular formula

Compound name

CE

PT

ED

M

AN

US

CR

IP

T

C16H28OSi

AC

Anisole, o-(1-ethylvinyl) Acetic acid, 7-hydroxy-1,3,4,5,6,7hexahydro-2H-naphthalen-4aylmethyl ester

C6H8O4

C9H10O2

C13H20O3

21.91

10-Chloro-1-decanol, pentafluoropropionate 1,10-Decanediol

C12H20ClF3 O2 C13H20ClF5 O2 C10H22O2

24

22.179

1,15-Pentadecanediol

C15H32O2

244

26.95

25

22.567

Isopulegol

C10H18O

154

1.46

26

22.742

1,7-Octadien-3-ol, 2,6-dimethyl

C10H18O

154

0.52

10-Chloro-1-decanol, trifluoroacetate

22

ACCEPTED MANUSCRIPT C16H32

224

0.67

C15H32O2

244

0.66

180

0.42

190

0.51

179

0.48

190

0.28

190

1.36

228

0.42

238

0.41

296

1.31

268

0.20

296

0.35

296

0.52

C17H34O2

270

0.24

C16H22O4

278

2.35

27

22.899

1-Nonylcycloheptane

28

22.98

29

23.286

30

24.406

31

24.744

32

25.201

1,15-Pentadecanediol 2(4H)-Benzofuranone, 5,6,7,7atetrahydro-4,4,7a-trimethyl-, (R) Megastigmatrienone 1,2,3,4,7,7a-Hexahydro-2,4,7trimethyl-6H-2-pyrindin-6-one Megastigmatrienone

33

25.488

Megastigmatrienone

C13H18O

34

28.547

C14H28O2

C11H16O2 C13H18O C11H17NO

35

28.779

36

30.011

37

30.142

38

30.518

39

30.881

40

31.769

Tetradecanoic acid Acetic acid, 2-(2,2,6-trimethyl-7-oxabicyclo[4.1.0]hept-1-yl)-propenyl ester 3,7,11,15-Tetramethyl-2-hexadecen1-ol 2-Pentadecanone, 6,10,14-trimethyl 3,7,11,15-Tetramethyl-2-hexadecen1-ol 3,7,11,15-Tetramethyl-2-hexadecen1-ol Hexadecanoic acid, methyl ester

41

32.538

Dibutyl phthalate

42

32.763

n-Hexadecanoic acid

C16H32O2

256

7.36

43

34.459

Heptadecanoic acid

C17H34O2

270

0.27

44

35.109

6-Octadecenoic acid, methyl ester

C19H36O2

296

0.23

45

35.353

Phytol

C20H40O

296

1.80

46

35.935

PT

IP

T

C13H18O

C18H32O2

280

7.11

47

36.085

278

5.49

48

36.404

9,12-Octadecadienoic acid (Z,Z) 9,12,15-Octadecatrienoic acid, (Z,Z,Z) Octadecanoic acid

C18H36O2

284

3.43

49

36.711

9,12-Octadecadienoic acid (Z,Z)

C18H32O2

280

0.44

50

37.411

C18H32O2

280

0.48

51

39.038

310

0.40

52

39.175

9,12-Octadecadienoic acid (Z,Z) Ethanol, 2-(9,12-octadecadienyloxy)-, (Z,Z) Ricinoleic acid

C18H34O3

298

0.69

53

39.738

Eicosanoic acid

C20H40O2

312

0.45

54

46.725

Squalene

C30H50

410

4.38

CR

US

AN

M

ED

CE

AC

C14H22O3 C20H40O C18H36O C20H40O C20H40O

C18H30O2

C20H38O2

23

ACCEPTED MANUSCRIPT Table.2 Antibacterial activity of T. castanifolia derived ZnO NPs

Zone of inhibition diameter (mm)

Test

50 µg

75 µg

100 µg

Control

E.coli

-

-

11

17

-

P.aeruginosa

-

-

11

15

-

S. auerus

-

-

11

17

-

B. subtilis

-

7

9

15

-

AC

CE

PT

ED

M

AN

US

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25 µg

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organisms

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ACCEPTED MANUSCRIPT Table.3 Estimated rate constants from kinetic models for in-vitro 5-FU release by ZnOchitosan nanosuspension.

Kinetic models Nanoparticle Higuchi

k0

R2

k1

R2

kH

R2

2.674

0.915

0.084

0.786

14.13

0.944

kKP

n

R2

6.22

0.791

0.938

AC

CE

PT

ED

M

AN

US

CR

ZnO

Koresmeyer-Peppas

T

First order

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Zero order

25

ACCEPTED MANUSCRIPT Figure captions: Fig.1 UV –Vis spectra showing the formation of ZnO NPs by bioreduction activity of T. castanifolia leaf extract. Fig.2 (a) TEM image and (b) EDX spectrum of biosynthesized ZnO NPs using T.

T

castanifolia leaf extract.

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Fig.3 (a) XRD pattern and (b) FTIR spectrum of biosynthesized ZnO NPs using T.

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castanifolia leaf extract.

Fig.4 Antibacterial activity of T. castanifolia leaf extract derived ZnO NPs on (a) E.coli,

US

(b) Pseudomonas aeruginosa, (c) Staphylococcus aureus and (d) Bacillus subtilis

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Fig.5 Antioxidant scavenging activity of ZnO NPs synthesized using T. castanifolia leaf extract

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Fig.6 (a) Phase contrast microscopy image showing the morphology of untreated and

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ZnO NPs treated A549 cells and (b) Anticancer activity of ZnO NPs synthesized using T. castanifolia leaf extract against A549 cell lines.

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Fig.7 In-vitro 5FU drug release profile of TZ-chitosan-5FU. The chitosan-5FU

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nanosuspension without nanoparticles is act as control. Fig.8 Kinetic models (a) zero-order, (b) first order, (c) Higuchi and (d) Koresmeyer–

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Peppas model for 5-FU drug release from T. castanifolia leaf extract derived ZnO NPs-chitosan-5-FU nanosuspension.

26

ACCEPTED MANUSCRIPT Highlights

First report on biosynthesis of ZnO NPs using T. castanifolia leaf extract



ZnO NPs characterized by Uv-Vis, TEM, XRD, FTIR



ZnO NPs showed bactericidal activity against Gram positive and negative bacteria.



ZnO NPs possess anticancer activity against A549 cell lines

AC

CE

PT

ED

M

AN

US

CR

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

27

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8