Laurus nobilis leaf extract mediated green synthesis of ZnO nanoparticles: Characterization and biomedical applications

Biomedicine & Pharmacotherapy 84 (2016) 1213–1222

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Laurus nobilis leaf extract mediated green synthesis of ZnO nanoparticles: Characterization and biomedical applications Sekar Vijayakumar, Baskaralingam Vaseeharan* , Balasubramanian Malaikozhundan, Malaikkarasu Shobiya Nanobiosciences and Nanopharmacology Division, Biomaterials and Biotechnology in Animal Health Lab, Department of Animal Health and Management, Alagappa University, Karaikudi-630004, Tamil Nadu, India

A R T I C L E I N F O

Article history: Received 28 July 2016 Received in revised form 5 October 2016 Accepted 13 October 2016 Keywords: Laurus nobilis ZnO NPs Flower structure Antibacterial Antibiofilm Anticancer activity

A B S T R A C T

The present study reports the green synthesis of zinc oxide nanoparticles using the aqueous leaf extract of Laurus nobilis (Ln-ZnO NPs) by co-precipitation method. The synthesized Ln-ZnO NPs were characterized by UV–Vis spectroscopy, FTIR, XRD, TEM, SEM and EDX. Ln-ZnO NPs were crystalline in nature, flower like and have hexagonal wurtzite structure with a mean particle size of 47.27 nm. The antibacterial activity of Ln-ZnO NPs was greater against Gram positive (Staphylococcus aureus) bacteria than Gram negative (Pseudomonas aeruginosa) bacteria. The zone of inhibition against S. aureus was 11.4, 12.6 and 14.2 mm at 25, 50 and 75 mg mL
1. The zone of inhibition against P. aeruginosa was 9.8, 10.2 and 11.3 mm at 25, 50 and 75 mg mL
1. The light and confocal laser scanning microscopic images evidenced that Ln-ZnO NPs effectively inhibited the biofilm growth of S. aureus and P. aeruginosa at 75 mg mL
1. The cytotoxicity studies revealed that Ln-ZnO NPs showed no effect on normal murine RAW264.7 macrophage cells. On the other hand, Ln-ZnO NPs were effective in inhibiting the viability of human A549 lung cancer cells at higher concentrations of 80 mg mL
1. The morphological changes in the Ln-ZnO NPs treated A549 lung cancer cells were observed under phase contrast microscope. ã 2016 Elsevier Masson SAS. All rights reserved.

1. Indroduction Nanoparticles exhibit a distinct property of larger surface-area to volume ratio, which makes them a better candidate than their bulk counterparts in the sense of their activity [1,2]. Nanoparticle synthesis is usually carried out by physical and chemical methods. Both these methods suffer from high energy demand or the use of toxic chemicals [3–5]. The biological method of synthesis involves the use of microorganisms [6], fungus [7], algae [8] and plants [9]. The development of biosynthesis of nanoparticles has received immense attention in the last few years in progressive manner due to the tremendous advantages it offers in terms of cost and ecofriendliness. It is evident from the earlier reports that plants are the better candidates for the synthesis of nanoparticles. The nanoparticles produced from plants parts are more stable and the rate of synthesis is rapid than in the case of microorganisms [9]. Now-a-days, researchers are focusing on the green synthesis of nanoparticles using noble metals such as zinc, gold, silver,

* Corresponding author. E-mail addresses: [email protected], [email protected] (B. Vaseeharan). http://dx.doi.org/10.1016/j.biopha.2016.10.038 0753-3322/ã 2016 Elsevier Masson SAS. All rights reserved.

platinum, and palladium because of their applications in medical and pharmaceutical products, besides their use in consumer goods such as shampoos, soaps, detergents, shoes, cosmetic products and toothpaste [10]. The green synthesis of ZnO NPs is highly advantageous because the biomolecules present in the plant extracts act as efficient capping agents thereby playing a pivotal and versatile role in NPs synthesis. The capping agents appear to stabilize NPs by different mechanisms that includes electrostatic stabilization, steric stabilization, stabilization by hydration forces, depletion stabilization and stabilization using van der Waals forces. The stabilization of NPs is important for their functions and different biological applications [11]. Zinc oxide nanoparticles (ZnO NPs) possess several interesting properties such as optical transparency, electric conductivity, piezoelectricity, non toxicity, wide availability, low cost and stability [12,13]. They find applications in various fields, including catalysis, gas sensors, solar cells, paints, varnishes, plastics, pharmaceuticals, laser and optoelectronic devices [14–17]. It is usually employed as a protective agent in cosmetics and sunscreen products because of its ability to filter UV irradiations [18]. ZnO NPs are well known for its antibacterial and antifungal activity [19,20]. For example ZnO NPs can interact with membrane lipids and

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disorganize the membrane structure, which leads to loss of membrane integrity, malfunction, and finally to bacterial death [21,22]. Lung cancer is the most common malignant tumor worldwide, and it is one of the leading causes of human cancer related deaths [23]. It is the leading cause of death worldwide, accounting for 29% of cancer deaths in men and 26% in women [24]. Non-small cell lung cancer (NSCLC) comprises more than 80% of lung cancers [25]. Cancer chemotherapy is limited by the development of drug resistance by cancer cells and the adverse effects of antitumor drugs. The search for novel anti-tumor agents that circumvent these limitations has turned to natural plants [26]. Bay leaf (Laurus nobilis) belongs to the family Lauraceae, is one of the most widely used culinary spices in all Western countries and Asian countries. Previous phytochemical investigations revealed that 1,8-cineole, linalool and a-terpinyl acetate were the basic componentsof the essential oil of bay leaves [27]. Epicate-chin, procyanidin dimer, procyanidin trimer, flavonol and flavonederivatives were the most prominent phenolic compounds found in bay leaves. Bay leaves has many volatile active components such as a-pinene, ß-pinene, myrcene, limonene, linalool, methyl chavicol, neral, a-terpineol, geranyl acetate, eugenol, and chavicol. These compounds are known to have been antiseptic, anti-oxidant, digestive, and thought to have anti-cancer properties [28]. Bay leaf traditionally has been used as herbal medicine to treat rheumatism, earaches, indigestion, sprains, and to promote perspiration [29]. Recent research revealed that it can be used in treating diabetes and preventing migraine [30]. Bay leaf were found to have different pharmacological properties including inhibitory effects on NO (Nitric oxide) production (anti inflammatory) [31], inhibitory effects on alcohol absorption [32], and enhancement of liver glutathione S-transferase (GST) activity [33]. As an alternative pharmaceutical, bay leaves were effective in reducing blood glucose and total cholesterol in people with type-2 diabetes and prevention of insulin resistance [34,35]. To the best of our knowledge, this is the first study to report on the green synthesis, characterization, antibacterial, and antibiofilm activity of ZnO nanoparticles synthesized from the leaf extract of L. nobilis (Ln-ZnO NPs). In addition, the anticancer activity of green synthesized zinc oxide nanoparticles was determined against human A-549 lung cancer cell lines. 2. Materials and methods 2.1. Chemicals used Zinc acetate dehydrate [Zn(CH3COO)2]2H2O, sodium hydroxide (NaOH), methanol (CH3OH), ethanol (C2H5OH), Whatman filter paper (WHA10348903), phosphate buffered saline (PBS) tablets (pH 7.4), Dulbecco modified eagle Medium (DMEM), Fetal bovine serum (FBS), Cell counting kit-8 (CCK-8), fluorescent dye propidium iodide (PI) (33342), acridine orange (235474) and crystal violet (c3886) were purchased from Sigma Aldrich, India. Nutrient broth (NB), Luria Bertani agar (LBA) and Luria Bertani broth (LBB) were purchased from HiMedia (India). Ultra-pure deionized water from PURITE (18 MV) system was used in all the experiments. All the chemicals used were of analytical grade. The glasswares used for experimental purposes were properly washed, sanitized and autoclaved.

2.2. Collection and processing of plant samples Bay leaf (L. nobilis) was collected from the Forest area in Nagercoil, Tamil Nadu, South India. A voucher specimen was matched with the authentic specimen (herbarium no: 6666,

Regional Plant Resource Center, Puri, India) and was deposited in the herbarium. 2.3. Preparation of leaf extracts Fresh leaves of L. nobilis, without any infestation were collected and washed with tap water followed by distilled water to remove the dust particles. The leaves were allowed to dry at room temperature (37  C). Twenty grams of leaves were soaked in 100 mL of double distilled water and allowed to boil under magnetic stirrer hot plate (Tarson Digital Spinot, Model MC02) at 60  C for 20 min [36]. During boiling, a light yellow colored solution was formed and was cooled at room temperature. Then, the yellow colored extract was filtered through Whatman No. 1 filter paper. The final volume of the extract obtained was 20 mL and was stored at refrigerator for further use. 2.4. Synthesis of ZnO nanoparticles using L. nobilis extract (Ln-ZnO NPs) Zinc oxide nanoparticles were synthesized following the co-precipitation method described by Singh et al. [37]. Zinc acetate dehydrate [Zn(CH3COO)2]2H2O and sodium hydroxide (NaOH) were used as the starting material. Briefly, zinc acetate (2 M) was prepared in 50 mL of deionized water under constant stirring. After complete dissolution of the mixture, 5 mL of leaf extract and 50 mL of 2 M NaOH were added to the prepared solution of zinc acetate.The mixture was kept under magnetic stirrer for 2 h. The resultant white precipitate was filtered and washed repeatedly with distilled water followed by ethanol to remove the impurities. Finally, a solid white powder was obtained after overnight drying of the purified precipitate at 60  C in an oven. The powder was then subjected to calcination under muffle furnace at 350  C for 3 h. The mechanism of synthesis is that the biomolecules such as apinene, ß-pinene, myrcene, limonene, linalool, methyl chavicol, neral, a-terpineol, geranyl acetate, eugenol, and chavicol present in the leaf extract form complexing agents with the precursors (zinc acetate) which initially starts the process of nucleation forming reverse micelle and then further causing reduction and shaping of NPs [38,39]. When zinc acetate was dissolved in water, colourless solution was formed due to the presence of [Zn (CH3COO)2]2H2O ions. The addition of NaOH produces a white precipitate of ZnO NPs in the core of a micelle. The capping agent acts as a stabilizing agent by adhering onto the surface of NPs forming a protective layer and controlling the particle size [40]. 2.5. Characterization of Ln-ZnO NPs The characterization of Ln-ZnO NPs was carriedout using UV–Vis spectroscopy, XRD, FTIR, SEM, EDX and TEM. The optical properties of Ln-ZnO NPs were studied using UV–Vis absorption (UV-1700 Spectrometer of Shimadzu) spectrophotometer with samples in quartz cuvette. The absorption spectra of the synthesized Ln-ZnO NPs were monitored at different wavelengths ranged between 200 and 800 nm [41]. The crystalline phase and size of the Ln-ZnO NPs crystal were analyzed using powdered X-ray diffraction (X’PERT PROPANalytical, PHILIPS). The possible functional group of Ln-ZnO NPs was examined under Fourier transform infrared spectroscopy (SHIMADZU, India). The morphology, topography and the size of Ln-ZnO NPs were determined by scanning electron microscopy (SEM, JSM 6701F-6701, JPEG, India) and transmission electron microscopy (JOEL model instrument 1200 EX instrument). The particle-size distribution of Ln-ZnO NPs was determined by computerized analysis of SEM images. This was carried out using the JMicroVision code. The code can calculate the

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average diameter (d) of the particles in an image from any one of p their geometrical characteristics, namely area (d = 2 [s/p]), perimeter (d = p/p), or average of longest and shortest diameters (d = 0.5[d1 + d2]) in 2-D [42]. The elemental composition of Ln-ZnO NPs was analyzed by energy dispersive X-ray spectrum (BRUKER, India).

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of Ln-ZnO NPs. After treatment, the plates were incubated for 24 h. MTT was added at a concentration of 5 mg mL
1 to each well and incubated for 4 h. Purple color formazan crystals formed were then dissolved in 100 mL of dimethyl sulfoxide (DMSO). Optical density was read at 570 nm in a multi well ELISA plate reader. Optical density value was subjected to percentage of viability by using the following formula:-

2.6. Antibacterial activity of Ln-ZnO NPs

OD value of experimental samples OD value of experimental controls  100

Percentage of cell viability ¼ The antibacterial activity of Ln-ZnO NPs was tested against the growth of Gram positive [Staphylococcus aureus (HQ116443)] and Gram negative [Pseudomonas aeruginosa (HQ116441)] bacteria. The antibacterial activity of Ln-ZnO NPs was determined by agar well diffusion method [43]. The test organisms were inoculated in Luria Bertani broth (pH 7.4) for 8 h. The isolates were seeded on Luria Bertani agar plates using sterilized cotton swabs. Agar surface was bored using sterilized gel borer to make wells (7 mm diameter). Different concentrations ranging from 25 to 75 mg mL
1 of Ln-ZnO NPs and 100 mL of sterilized distilled water (negative control) were poured into separate wells. The plates were incubated at 37  C for 24 h. Triplicate plates were maintained for each organism. 2.7. Antibiofilm assay To analyze the ability of Ln-ZnO NPs to prevent the biofilm formation of Staphylococcus aureus (HQ116443) and Pseudomonas aeruginosa (HQ116441), bacterial colonies (1 106 cfu mL
1) were allowed to grow on glass pieces (diameter 1 1 cm) placed in 24well polystyrene plates with 1 mL of nutrient broth supplemented with different concentrations of Ln-ZnO NPs (25–75 mg mL
1) and incubated for 24 h at 37  C. Glass pieces were stained with 0.04% crystal violet and examined under a Nikon inverted research microscope (ECLIPSE Ti100) at 40 magnification. Another set of glass pieces with biofilms grown as above was washed with PBS, stained with acridine orange (0.1%) and the biofilm growth was quantified using a confocal laser scanning microscope (CLSM- Carl Zeiss LSM 710) using a 488 nm argon laser and band path 500–640 band pass emission filter and running Zen 2009 software (Carl Zeiss, Germany). The Z-stack analysis (surface topography and three-dimensional architecture) was done with the Zen 2009 software (Carl Zeiss, Germany). To measure the biofilm thickness, sections were scanned and Z-stacks were acquired at z step-size of 0.388 mm. Each field size was 455 mm by 455 mm at 20 magnification. Microscope images were acquired with the Zen 2009 image software. 2.8. Anticancer activity of Ln-ZnO NPs The inhibitory concentration (IC50) value was evaluated using an MTT [3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay. Briefly, human lung cancer cell line A549 was procured from National Centre for Cell Science (NCCS), Pune, India. They were grown in DMEM (Dulbecco's modified eagle medium) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/mL streptomycin and 10% FBS. The cells were cultured using 75 cm2 cell culture flasks at 37  C in CO2 incubator (95% air, 5% CO2 and 100% relative humidity). They were then seeded into 96 well plates (5000 cells in each well) and incubated for 24 h. A549 cells were treated with different concentrations of Ln-ZnO NPs (10, 20, 40 and 80 mg mL
1). A respective control samples (100 mg mL
1) were simultaneously prepared with DMEM medium, saline, leaf extract and bulk zinc for comparison of cytotoxic effects. Subsequently, the toxicity of Ln-ZnO NPs was also tested on normal murine macrophage RAW264.7 cells (obtained from National Centre for Cell Sciences (NCCS), Pune, India.) in comparison with leaf extract and bulk zinc to confirm the antilung cancer potential

A549 cells were plated into a six well chamber plate at 3  105 cells/well. At >90% confluence, the cells were treated with Ln-ZnO NPs (80 mg mL
1) for 24 h. The cells were washed with PBS fixed in methanol: acetic acid (3:1 v/v) for 10 min. The morphological variations were examined under phase contrast microscope (Olympus, Japan). 2.9. Statistical analysis Experiments were carried out in a randomized block design with three replications. The data were analyzed using one way analysis of variance (ANOVA) by software SPS version 16. 3. Results and discussion 3.1. Characterization of Ln-ZnO NPs 3.1.1. UV–Vis spectroscopic analysis UV–vis spectrophotometer (UV-1700 Spectrometer of Shimadzu) was performed at a resolution of 1 nm with a wavelength range of 200–800 nm. For the analysis, 0.2 mL of reaction sample was placed in the cuvette and diluted to 1 mL with deinonised water. The color of reaction mixture changed from greenish yellow to pale white colored precipitate, indicating the synthesis of ZnO NPs. Ln-ZnO NPs showed the strong absorbance peak at 338 nm due to surface plasmon resonance (Fig. 1A). Our results are well supported by Azizi et al. [44] who reported that UV–vis absorbance spectrum of pure ZnO NPs was 334 nm. Similar findings have been documented by Vimala et al. [45] who reported that the absorbance spectra of ZnO NPs were recorded between 330 and 370 nm.

3.1.2. XRD analysis X-ray powder diffraction (XRD) was used to analyze the structural properties of the ZnO NPs and to identify the phase and crystallinity of Ln-ZnO NPs. The diffraction peaks corresponding to synthesized ZnO NPs match well with the standard ZnO hexagonal wurtzite structure. No extra diffraction peaks corresponding to impurities were detected, indicating that relatively pure ZnO NPs was obtained. The powder XRD patterns of asprepared and calcined ZnO samples are shown in hexagonal wurtzite phase of ZnO (JCPDS file no. 89-7102). After calcinations at 350  C, diffraction peaks were observed at 2uE 31.71, 34.31, 36.31, 47.51, 56.51, 62.81, 66.31, 67.91, 69.01, 72.61 and 76.81. These peaks are indexed as (100), (002), (101), (102), (110), (103), (200), (112), (201), (004) and (202) diffraction lattice planes respectively (Fig. 1B), corresponding to hexagonal ZnO with lattice parameters of a1/40.3250 nm and c1/40.5207 nm (JCPDS file no.89-7102). These results shows that after calcinations Zn (OH)2 decomposes and high purity of hexagonal structure of ZnO is formed. The crystallite sizes of as-prepared and calcined Ln-ZnO NPs were calculated by means of an X-ray line-broadening method using the Debye– Scherrer formula [46]: D 1/4 0:89l = b cos u, where D is the crystallite size in nanometers, 0.89 represents a dimension

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Fig. 1. A. UV–vis absorption spectra showing the surface plamon resonance of Ln-ZnO NPs. B. XRD pattern showing the various Bragg’s lattice planes of Ln-ZnO NPs. C. FTIR spectra showing the capping agents of Ln-ZnO NPs. D. Scanning electron microscopic image showing the flower shape of Ln-ZnO NPs. E. EDAX showing the elemental composition of Ln-ZnO NPs. F. Transmission electron microscopic image showing the size and shape of Ln-ZnO NPs. Scale bar indicates the distance on the specimen from the bar (50 nm). Circle represents hexagonal and wurzite structure of Ln-ZnO NPs.

lessconstant k, l is the wavelength of Cu-Ka (0.15406 nm), b is the fullwidth at half maximum (FWHM, radian) of the diffraction peak and u is the Bragg diffraction angle (degree). The crystallite sizes of the as-preparedand calcined ZnO powder at 350  C were found to be 24 nm. These results were well supported by the XRD pattern of ZnO NPs reported earlier [47,48].

3.2. FTIR analysis FTIR analysis was carried out to identify the possible biomolecules involved in the biosynthesis procedure. FTIR spectra of Ln-ZnO NPs were in the range of 500–4000 cm
1 (Fig. 1C). The Table 2 Minimum inhibitory concentrations of bulk zinc acetate, leaf extracts and Ln-ZnO NPs against different bacteria.

Table 1 Average diameters of the nanoparticle as calculated by three geometrical characteristics in the digital processing of scanning electron microscopy images. Nanoparticle

Average diameter (nm) Area

Perimeter

Dimension

Ln-ZnO NPs

34.5

52.28

55.03

Mean (nm) 47.27

Bacteria

Minimum inhibitory concentrationa (MIC) (mg mL
1) Bulk Zinc acetate

Leaf extract

Ln-ZnO NPs

Staphylococcus aureus Pseudomonaus aeruginosa

2.274  0.2ab 2.324  0.5ab

2.014  0.4ab 2.120  0.6ab

1.775  0.3b 1.998  0.7ab

Different letters indicate significant differences among values (P < 0.05) (ANOVA followed by Tukey’s HSD test). a Values are mean  SE of three replicates.

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Fig. 2. Antibacterial activity of Ln-ZnO NPs against bacteria at different concentrations. (A) S.aureus (B) P. aeruginosa. (C) control (T1) 25 mg mL
1 (T2) 50 mg mL
1 (T3) 75 mg mL
1.

corresponds to the standard peak of ZnO due to stretching of Zn

O bonds.This suggests that the biological molecules could possibly act as hydrolyzing agent for the metal oxide nanoparticles. The result of FTIR corroborates with the FTIR spectrum of ZnO NPs synthesized from various plant extracts [49–52]. 3.3. SEM and EDX analysis

Fig. 3. Zone of inhibition level of Ln-ZnO NPs against bacteria. Each bar indicated mean  standard deviations of three replications. (*values are significant at p < 0.05 using ANOVA).

broad peak at 3418 cm
1 indicate the OH stretching vibrations. The sharp and intense band at 1435 cm
1 depicts the presence of C

C stretching of aromatics groups. The strong peak at 865 cm
1 attributed to the presence of C

H bend in alkane group. It is apparent that the intensity of absorption in the region of 413 cm
1

The pure Ln-ZnO NPs formed were agglomerated with a hexagonal wurtzite structures. The particle size of Ln-ZnO NPs ranged from 34.5, 52.28 and 55.03 nm by area, perimeter and dimensions respectively with a mean size of 47.27 nm (Fig. 1D). The particle size distribution determined by the three geometrical characteristics in the digital processing of scanning electron microscopy images are shown in Table 1. This agglomeration is due to polarity and electrostatic attraction of ZnO nanoparticles [53]. The particle size of the synthesized Ln-ZnO NPs was in close agreement with the findings of Vijayakumar et al. [48] and Divya et al. [54] who reported that the size of ZnO NPs were in the range of 20–50 nm. Furthermore, in the present study, SEM clearly shows that the particles are agglomerated to form flower like structures. Ln-ZnO NPs appear like a single “flower” consists of needle-like crystals (“petals”) radiating from the center. Similar kind of observation have been documented by Madan et al. [55] who

Fig. 4. Light microscopic image showing the antibiofilm activity of Ln-ZnO NPs against bacteria at different concentrations. (A) S.aureus (B) P.aeruginosa.

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Fig. 5. 2D view of confocal laser scanning microscopic image showing the antibiofilm activity of Ln-ZnO NPs against bacteria at different concentrations. (A) S.aureus (B) P. aeruginosa.

reported that ZnO NPs synthesized using Azadhirachta indica showed mushroom like structure when the volume of extract added was 2 mL. Similarly, Kazeminezhad et al. [56] reported that ZnO NPs were arranged in the form of closed pine like structures. EDX analysis of Ln–ZnO NPs revealed that the elemental composition of zinc was 57.16% (Fig. 1E). 3.4. Transmission electron microscopic analysis Transmission electron microscopic image reveals that the LnZnO NPs obtained were agglomerated particles, spherical and hexagonal wurtzite with an average particle size ranging between 47.27 nm (Fig. 1F). These results are in accordance with

Vijayakumar et al. [48] who reported that the particle size of Pam-ZnO NPs were between 20 and 50 nm. 3.5. Antibacterial assay of Ln-ZnO NPs The minimum inhibitory concentrations of Ln-ZnO NPs was comparatively lower than that of L. nobilis leaf extract and bulk zinc acetate (Table 2). The antibacterial activity of Ln-ZnO NPs was investigated on both Gram positive (S. aureus) and Gram negative (P. aeruginosa) bacteria by agar well diffusion method and the results are shown in Fig. 2. In the present study, the size of inhibition zone was different among pathogens and the inhibition size increased when the concentration of Ln-ZnO NPs was

Fig. 6. 3D view of confocal laser scanning microscopic image showing the biofilm inhibition of Ln-ZnO NPs against bacteria after treatment with Ln-ZnO NPs at 75 mg mL
1 concentrations. (A) S.aureus (B) P.aeruginosa.

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Fig. 7. Reduction in biofilm thickness of bacteria after treatment with Ln-ZnO NPs at 75 mg mL
1. Each bar indicated mean  standard deviations of three replications. (*values are significant at p < 0.01 and **values are significant at p < 0.05 using ANOVA).

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Fig. 9. Cell viability of human A549 lung cancer cells treated with Ln-ZnO NPs at different concentrations. Each bar indicated mean  standard deviations of three replications. (*values are significant at p < 0.05 using ANOVA).

3.6. Antibiofilm assay increased. This was in agreement with Sangeetha et al. [57] who stated that increasing the concentration of ZnO nanoparticles in wells and discs, the growth inhibition has also been increased consistently because of proper diffusion of nanoparticles in the agar medium. In the present study, no inhibition zone was observed in control. The antibacterial activity of Ln-ZnO NPs was greater against Gram positive S. aureus than Gram negative P. aeruginosa. The zone of inhibition against S. aureus was 11.4, 12.6 and 14.2 mm at 25, 50 and 75 mg mL
1 respectively (Fig. 3). On the other hand, the zone of inhibition against P.aeruginosa was 9.8, 10.2 and 11.3 mm at 25, 50 and 75 mg mL
1 respectively. These results are supported by Sangeetha et al. [57] who reported that nano ZnO showed maximum activity (26/23 mm) against S. aureus followed by P. mirabilis (27/24 mm), S. marcescens (24/21 mm) and C. freundii (19/16 mm). Premanathan et al. [58] reported that Gram-positive bacterium such as S. aureus is more susceptible to ZnO NP toxicity than Gram-negative bacteria such as E. coli and Pseudomonas aeruginosa, which may be attributed due to the differences in bacteria’s cell membrane structure that controls the toxicant’s access to sites of action.

In the present study, the light (Fig. 4) and confocal laser scanning microscopic 2D and 3D view (Figs. 5 and 6) showed well developed biofilm formation of S. aureus and P. aeruginosa in control, whereas treatment with Ln-ZnO NPs (25–75 mg mL
1) inhibited biofilm formation in a dose dependent manner. At higher concentration of Ln-ZnO NPs (75 mg mL
1), both S. aureus and P. aeruginosa showed disintegrated and recalcitrant biofilm architecture. The thickness of biofilm was also decreased after treatment with 75 mg mL
1 of Ln-ZnO NPs such that 7 mm and 9 mm in S. aureus and P. aeruginosa respectively as compared to control (16 and 30 mm respectively) (Fig. 7). The biofilm inhibitory activity of Ln-ZnO NPs against bacteria may be due to the rupture of bacterial cell membrane and surface activity of NPs in contact with bacterial surface. Stoimenov et al. [59] reported that the contact between nanoparticles and bacterial cell was initiated by the surface charges on the particle and the electrostatic interaction between bacterial surface and nanoparticle. After contact with bacterial membrane, high rate of reactive oxygen species generated from ZnO nanoparticles leads to the death of bacteria by chemical

Fig. 8. Phase contrast microscopic images showing the normal murine RAW264.7 macrophage cells treated with Ln-ZnO NPs in comparison with saline, DMSO medium, leaf extract and bare zinc acetate at 80 mg mL
1. Arrow indicates no morphological changes in the murine RAW264.7 macrphage cells.

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Fig. 10. Phase contrast microscopic images showing the morphological changes in human A549 lung cancer cells exposed to Ln-ZnO NPs in comparison with saline, DMSO medium, leaf extract and bare zinc acetate at 80 mg mL
1. Arrow indicates morphological changes such as cell shrinkage, clumping and loss of stability in the human A549 lung cancer cells.

interactions between hydrogen peroxide and membrane proteins [60]. 3.7. Anticancer activity of Ln-ZnO NPs The in vitro cytotoxic activity of Ln-ZnO NPs was evaluated against human lung cancer A549 cells at different concentrations (10, 20, 40 and 80 mg mL
1). The reason to choose this cell line is that it is a typical cancer line that has widely been used for in vitro cytotoxicity study [62]. Previously, the cytotoxicity of ZnO and TiO2 nanoparticles against human A549 lung cancer epithelial cells have been reported [61]. Premanathan et al. [58] reported that zinc oxide nanoparticles have anticancer activity against human myeloblastic leukemia cells. In the present study, Ln-ZnO NPs were observed to be non-toxic to normal murine macrophage RAW264.7 cells. No changes in the viability (Data not shown) and morphology of RAW264.7 cells were noticed (Fig. 8). This was in agreement with the observations of Nagajyothi et al. [63] who stated that RAW264.7 macrophage cells showed excellent viability in the presence of up to 1 mg/mL of green synthesized ZnO NPs using Coptidis rhizome and Polygala tenuifolia indicating that the ZnO NPs had no toxic effect. On the other hand, the viability of A549 cells was decreased when the concentration of Ln-ZnO NPs was increased to 80 mg mL
1 (Fig. 9). This attributed that Ln-ZnO NPs are effective in inhibiting the viability of A549 lung cancer cells. To further confirm the antilung cancer effects of Ln-ZnO NPs on the apoptotic cell morphology, propidium iodide stained cells were visualized under phase contrast microscope (Olympus, Japan). When compared to L. nobilis leaf extract (80 mg mL
1), A549 cells treated with Ln-ZnO NPs showed nuclear morphological changes such as cell clumping and loss of membrane stability at 80 mg mL
1 after 24 h (Fig. 10). The cytotoxicity studies revealed that Ln-ZnO NPs were more effective in controlling the growth of A549 lung cancer cells compared to L. nobilis leaf extract. Generally, NPs have higher physical and chemical activities than their bulk counterparts because of their extremely high surface area to volume ratio [64]. Owing to their small size and large specific surface area, ZnO NPs have induced the elevation of intracellular

Zn2þ concentration, leading to the over generation of intracellular ROS, leakage of plasma membrane, dysfunction of mitochondria and cell death [65,66]. Previous studies by Hsiao and Huang [61] have shown that abnormally spherical shaped A549 cells were observed after exposure to ZnO nanorods, revealing that the cells were either damaged or dead. Vinardell and Mitjans [67] reported that zinc oxide NPs were used at a very low concentration and were found to exhibit activity against HepG2 (liver cancer) and MCF-7 (breast cancer) cancer cells in a dose-dependent manner. At a very low concentration, such as 25 mg/mL, the cell viability was less than 10% in the case of HepG2 cells. The present study suggests that the green synthesized Ln-ZnO NPs has the potential to control the human lung cancer cells compared to L. nobilis leaf extract and bulk ZnO. 4. Conclusion The present study concludes that Ln-ZnO NPs can be rapidly green synthesized using L. nobilis leaf extract and are inexpensive, non-toxic, ecofriendly with average size of 47.27 nm exhibiting flower like structures. The green synthesized Ln-ZnO NPs has greater antibacterial and antibiofilm activity against Gram positive (S.aureus) and Gram negative (P. aeruginosa) bacteria. Ln-ZnO NPs have shown antilung cancer activity against human A549 lung cancer cells. The outcomes of this study illustrate a broad range of notable applications of Ln-ZnO NPs in pharmaceutical and biomedical fields. Declaration of conflict of interest The authors report no conflicts of interest. Acknowledgement The first author S. Vijayakumar thanks the DST, New Delhi, India for financial support under INSPIRE programme (INSPIRE FellowIF140145). Dr.B.Vaseeharan thanks the Department of Biotechnology (DBT), New Delhi, India, for financial assistance under the

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Project grants code: BT/PR7903/AAQ/3/638/2013. Dr.B.Vaseeharan also thanks Dr.P.Ekambaram, Assistant Professor, Molecular Toxicology Laboratory, Department of Biotechnology, Bharathiar University, Coimbatore and Dr.Avinash Sonawane, School of Biotechnology, KIIT University, Bhubaneswar, Orissa, India for their help in cytotoxicity assay. References [1] F. Raimondi, G.G. Scherer, R. Kotz, Nanoparticles in energy technology: examples from electrochemistry and catalysis, Angew. Chem. Int. Ed. 44 (2005) 2190–2209. [2] A. Annamalai, T.B. Sarah, A.J. Niji, D. Sudha, V.L. Christina, Biosynthesis and characterization of silver and gold nanoparticles using aqueous leaf extraction of Phyllanthus amarus Schum & Thonn, World App. Sci. J. 13 (8) (2011) 1833–1840. [3] P. Raveendran, J. Fu, S.L. Wallen, Completely ‘green’ synthesis and stabilization of metal nanoparticles, J. Am. Chem. Soc. 125 (2003) 13940–13941. [4] N. Vigneshwaran, R.P. Nachane, R.H. Balasubramanya, P.V.A. 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