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PAPER Jason B. Benedict et al. The role of atropisomers on the photo-reactivity and fatigue of diarylethene-based metal–organic frameworks

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Synthesis, Anti-bacterial, Anti-arthritic, Anti-oxidant and In-vitro Cytotoxicity Activities of ZnO Nanoparticles Using Leaf Extract of Tectona Grandis(L.) Received 00th January 20xx, Accepted 00th January 20xx

N. Senthilkumara, E. NandhaKumarb, P. Priyac, D. Sonid, M. Vimalane, I. Vetha Pothehera*

DOI: 10.1039/x0xx00000x www.rsc.org/

Abstract The novelty of this present work is to investigate the anti-bacterial, anti-arthritic, anti-oxidant and in-vitro cytotoxicity activities of green synthesized Zinc Oxide Nanoparticles (ZnO NPs) using leaf aqueous extract of TectonaGrandis (L.). Zinc nitrate act as the precursor and leaf extract act as the reducing agent. The synthesized ZnO NPs are confirmed by the powder X-ray diffraction (PXRD) analysis and the crystalline size was calculated by the Scherer’s formula. The FTIR analysis confirms the presence of various functional groups in the leaf extract as well as in the NPs. The UV absorption wavelength observed at the peak of 360 nm and the calculated energy band gap is found to be 3.4eV. The Dynamic Light Scatteing (DLS) analysis shows the stability and particle size of the synthesized ZnO NPs. The crystalline size, shape and surface morphology of the ZnO NPs is determined by using Field Emission Scanning Electron Microscopy (FE-SEM). Green synthesized ZnO NPs exhibited interesting antibacterial activity against Grampositive and Gram-negative bacteria and exerted excellent DPPH (di(phenyl)-(2,4,6-trinitrophenyl) iminoazanium) free radical scavenging activity. Also shows the maximum stabilization of protein denaturation (90.46 ± 0.02) and proteinase inhibitory activity (87.68±0.03) at a dose of 200µg/ml respectively. The anticancer activity of ZnO NPs is also tested against normal and cancer osteoblast MC3t3-E1 cell line and found the reduction in size of the cancer cell. Key Words: Green synthesis, Antibacterial, Anticancer, Nanoparticles, TectonaGrandis (L.) 1. INTRODUCTION

a

Department of Physics, University College of Engineering, Bharathidasan Institute of Technology Campus, Anna University, Tiruchirappalli – 620 024. b Department of Mechanical Engineering, Sri Ramakrishna Engineering College, Coimbatore - 641022 c Department of Chemistry, Advanced Materials Research Laboratory, Periyar University, Salem – 636 011 d Department of Biotechnology, University College of Engineering, Bharathidasan Institute of Technology Campus, Anna University, Tiruchirappalli – 620 024. e Department of Physics, Thirumalai Engineering College, Kilambi, Kancheepuram– 631 551 This journal is © The Royal Society of Chemistry 20xx *Corresponding author: Email: [email protected], Mobile: +919942994274

Nanotechnology is one of the most dynamic fields in modern advanced materials science and engineering [1]. Recent reports show that nanotechnology deals with the distribution, morphology of the materials having size less than 100 nm and it depends upon the nature of the NPs that are atomic or molecular aggregates [2-4]. So far various semiconducting nanomaterials such as TiO2, SnO2, GaN, CuO, GaAs, Si and ZnO have been synthesized and reported by various researchers. Among them, Zinc oxide (ZnO) has become particularly interesting because of its wide band-gap (3.37 eV) with n-type semiconductor and having high

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excitonic binding energy (~60 meV) [5-7]. The ZnO NPs have many applications such as luminescent material, optoelectronics, solar cells, photocatalyst, varistors, photodetectors and various biological applications [5]. Several methods are available for the synthesis of ZnO NPs namely, wet chemical, biosynthesis, chemical micro emulsion, hydrothermal, sol-gel, vapor phase process, solvothermal, microwave-assisted combustion, chemical, direct precipitation and sono-chemical method [7-9]. Nowadays increasing awareness of green chemistry and other biological processes have led to the development of an eco-friendly approach of the synthesis of NPs [10]. The metal oxide nanoparticles synthesized by various biological systems such as plants [7], bacteria [11], fungus [12] and other similar organisms [13] have been reported earlier. The green synthesized ZnO NPs are nontoxic, biosafe and biocompatible. Also they can be used as drug carriers, cosmetics in various medical materials [14]. Previous literature implies that green synthesized ZnO NPs show more biocidal activity against various microorgnisms when compared to chemically synthesized ZnO NPs [15, 16]. The mechanism of green synthesis of NPs in plants may be associated with the phytoremediation concept [9]. Recently, Suresh et al., reported green synthesis of ZnO NPs using Cassia fistula plant extract by solution combustion method, where plant extract contain reducing components such as polyphenols (11%) and flavonoids (12.5%) [17]. Sangeetha et al., reported biosynthesized ZnO NPs with the size of 36 nm. The synthesis was achieved by seaweed Sargassum myriocystum (microalgae) obtained from the gulf of Mannar which shows there is no visible changes even after 6 months and confirms the stability of NPs formed. FTIR result shows that, the fucoidan soluble pigments secreted from microalgae were responsible for the reduction and stabilization of the NPs [18]. Quasi-spherical shaped ZnO NPs was synthesized and reported by Ramesh et al. The reported diameter from Solanum nigrum leaf extract was around 29.79 nm, where the carbonyl groups and proteins were acted as both reducing and capping agent [19]. Aloe Vera leaf extract was also used as reducing and capping agent for the synthesis of spherical shaped ZnO NPs. Here the material was harvested by the presence of free carboxylic and the amino group in the plant extract [20]. ZnO NPs synthesized from Trifolium pretense flower extract showed similar peaks in UV-Vis spectra after 24, 48, 72, 96 and 120 hours of NPs formation confirms the stability of synthesized NPs [21]. Nyctanthes arbor-tristis was used as the biological reduction agent by Jamdagni et al., [22]. Jafarirad et al., reported the fruit extract of Rosa canina acted as both reducing and stabilizing agent for synthesis of ZnO NPs. The reducing and stabilizing property was confirmed by phytochemical studies and correlated from FTIR analysis. Bio-capping was done by carboxylic and phenolic acid present in the fruit extract [23]. Jayaseelan et al., synthesized the ZnO NPs using reproducible bacteria, Aeromonas hydrophila, which is an eco-friendly reducing and capping agent. Characterizations indicate that the formed ZnO NPs were spherical and oval shape with an average size of 57.72nm [24]. Vanathi et al developed the ZnO NPs using an aquatic weed of Eichhornia

crassipes as reducing agent [25]. In this way, TectonaGrandis.Linn. (Common name – Teak; Family Lamiaceae) is one of the most famous timbers in the world commonly found in India and other South-East Asian countries [31]. Teak is also used for many folklore medicines, treating inflammatory swelling and extract from various parts of the teak shows the healing capacity of biliousness, bronchitis, hyperacidity, diabetes, leprosy, astringent and dysentery [32]. Balraj et al., reported that the biosynthesized platinum NPs having the ability to produce prominent anticancer agent against cancer cell lines [33]. Also Premanathan et al., reported that ZnO NPs induce cytotoxicity in a cancer cell by specific and proliferation dependent manner in rapidly dividing tumour cells [34]. ZnO NPs may induce ROS (Reactive Oxygen Species) generation and regulate the cellular movements [35]. From the literature, So far there is no scientific reports exists for the anti-oxidant, anti-arthritics, in-vitro cytotoxicity and anti-bacterial activities of ZnO NPs synthesized by using Tectona Grandis(L.) leaf extract. Hence in the present study, investigation on the antibacterial, in-vitro anti-oxidant, in-vitro cytotoxicity and antiarthiritic activities of ZnO NPs prepared by using Tectona Grandis(L.) leaf extract is made and succeeded. The antibacterial properties of synthesized ZnO NPs were investigated and developed as antibacterial agents against a wide range of Gram-positive and Gram-negative bacteria to control and prevent the spreading of bacterial infections. The intake of anti oxidants provides protection against damage caused by free radicals. Cell viability assay was performed to MC3t3-E1cell line and evaluate the biocompatibility of these ZnO nanopowders in vitro. Antiarthritic activity of prepared ZnO NPs tested protein denaturation and proteinase inhibitory activity. All the results confirm that the ZnO NPs synthesized in the present work is a potential candidate for various biological activities and hence it can be useful for the medical industry. 2.

MATERIALS AND METHODS

2.1 Materials Fresh leaves of TectonaGrandis(L.) were collected from southern region of Tamilnadu, INDIA. All the chemicals used in the experiment were of analytical grade and were purchased from Merck. 2.2 Phytochemical Screening Test The preliminary screening test was used to find the secondary components presence in the leaf extract according to the standard methods [36]. Test for saponins: Froth test: - 1ml extract was slowly added to 2-3 ml of double distilled water. Then the mixture was shaken vigorously. Finally the formation of foam confirms the presence of saponin in the leaf extract [36, 37].

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Test for Alkaloids:

(Salkowski test):

A 3 ml of concentrated extract was taken in a test tube and 1 ml of hydrochloric (HCl) acid was added to the extract. Then the mixture was heated gently for 20 min and cool down to room temperature. Finally, the obtained mixture was filtered in the watt man filter paper. The filtrate was used for the following test.

5 ml of extract was mixed with 2 ml of chloroform followed by careful addition of 3 ml of concentrated sulphuric (H2SO4) acid. A layer of reddish brown coloration indicates the positive result for the presence of Terpenoids [38].

Hager’s test: - Presence of alkaloids was confirmed by the obtained yellow coloured precipitate when 1ml of the extract was treated with Hager’s reagent [36, 38].

2 ml of leaf extract was taken in a test tube and dissolved with 10 ml of chloroform. Equal volume of concentrated H2SO4 acid was added to the mixture through the side wall of the test tube. Steroid was confirmed by the changes in the upper layer of the solution as red and H2SO4 acid layer as yellow with green fluorescence [37].

Test for Proteins: Xanthoproteic test: - 2 ml of extract was treated with few drops of concentrated Nitric (HNO3) acid changes the color of the solution into yellow indicates the presence of proteins [36]. Test for Flavonoids: a) Alkaline reagent test: - Formation of intense yellow colour when a 2 ml of extract was treated with 10% NaOH solution indicates the presence of Flavonoid. b) Zn test: - 2 ml of extract was treated with Zn dust and conc.HCl changes the solution as red color indicates the presence of Flavonoids. c) Lead acetate test: 2 ml of leaf extract was treated with few drops of lead acetate solution. Formation of yellow color precipitate indicates the presence of Flavonoids [36]. Test for Phenol: Ferric Chloride test: - 2ml of leaf extract was treated with 4 drops of alcoholic Ferric chloride (FeCl3) solution. Changes in the solution as bluish black confirms the presence of phenolic compound [36].

Test for steroids

2.2 Synthesis of ZnO NPs using TectonaGrandis(L.) leaf extract The TectonaGrandis(L.) leaves were collected and 20 gm of leaves were weighed and washed thoroughly 2 to 3 times in tap water. All the leaves were cut into fine pieces and taken into round bottom flask and then 100 ml of double distilled water was added. Then it was heated at 60 ⁰C for 1hr and then residues were filtered by Whatman NO.1 filter paper. The filtered extract was used as reducing and capping agent for the further process. 0.1M of zinc nitrate was taken in a beaker and dissolved in 50 ml of double distilled water under mild stirring for 10 minutes using magnetic stirrer. After being stirred, 2 ml of the reducing agent as well as capping agent of leaf extract was added in the precursor solution and heated at 60 ⁰C to 90 ⁰C. The colour of the solution turned from clear white to yellow colour paste confirms the formation of ZnO NPs. The paste was transferred into ceramic crucible and kept into muffle furnace heated at 400oC for 2hr. The resultant powder was used for further characterization. The schematic representation of green synthesis of ZnO NPs is shown in Fig. 1.

Test for Cardial Glycosides: Keller-Killani Test: - 2 ml of leaf extract was treated with 2 ml of glacial acetic acid containing a drop of FeCl3. A brown colored ring formation indicates the presence of Cardial Glycosides [36]. Test for Carbohydrate: 1 ml of leaf extract was dissolved gradually in 5ml of double distilled water and filtered. The filtrate was used for the following test. Benedict’s test: - 2 ml of filtrate was treated with Benedict’s reagent and heated gently. Formation of orange red precipitate indicates the presence of carbohydrate [36].

Fig.1. Schematic Representation of green synthesized ZnO NPs


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2.3 Analytical Methods The powder XRD pattern of ZnO NPs was carried out using Xpert Pro Penalty CAL with Cu-Kα radiation (1.5406 Å) and the maximum peaks were well matched with standard files. The FTIR spectra of synthesized ZnO NPs were analyzed by using Perkin Elmer Spectrometer with KBr pellet technique which is used to determine the functional groups of the synthesized ZnO NPs and leaf extracts. Raman spectra for the solid samples were recorded using a confocal Raman microscope (RENISHAW, United Kingdom) to confirm the materials having vibrations at lower frequencies. The synthesized ZnO NPs were subjected to UV-Vis analysis using UV-Visible (JASCO V650) spectrometer. The zeta potential experiment was carried out in a Zeta sizer nanoseries (Malvern) to find out the stability of the synthesized NPs. The Morphological and topography of the synthesized ZnO NPs were analyzed by using FE-SEM (Carl Zeiss microscopy Ltd, UK & SIGMA). 2.4 Screening of antibacterial activity

Test control solution (0.5ml) consists of 0.45ml of bovine serum albumin (5% w/v aqueous solution) and 0.05ml of distilled water. Product control (0.5ml) consists of 0.45ml of distilled water and 0.05 ml of test solution. Standard solution (0.5ml) consists of 0.45ml of Bovine serum albumin (5% w/v aqueous solution) and 0.05ml of Diclofenac sodium [31]. Various concentrations (100, 150, 200 µg/ml) of ZnO NPs (test solution) and diclofenac sodium (standard) were taken. All the above solutions were adjusted to pH 6.3 using 1N HCl. The samples were incubated at 37°C for 20 min and the temperature was increased to keep the samples at 57 °C for 3 min. After cooling, 2.5 ml of phosphate buffer was added to the above prepared solutions. The absorbance was measured using UV-Visible spectrophotometer at 416 nm [32, 33]. The control represents 100% protein denaturation. The results were compared with diclofenac sodium. The percentage inhibition of protein denaturation can be calculated as indicated below: % Inhibition = 100 −

optical density of test solution − optical density of product control × 100 optical density of test control

2.5.2 Proteinase Inhibitory Activity

2.4.1 Preparation of inoculums °

Stock cultures were maintained at 4 C on slopes of nutrient agar. Active cultures of experiment were prepared by transferring a loop full of cells from the stock cultures to test tube of Muller-Hinton broth (MHB) for bacteria that were incubated without agitation for 24 hrs at 37°C and 25°C respectively. The cultures were diluted with fresh MullerHinton broth to achieve optical densities corresponding to 2.0X 106 colony forming units (CFU/ml) for bacteria. 2.4.2 Antimicrobial susceptibility test The disc diffusion method was used to screen the antimicrobial activity. In vitro antimicrobial activity was screened by using Muller Hinton Agar (MHA) obtained from Hi-media (Mumbai). The various concentrations of ZnO NPs were performed against bacterial strains viz. Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Salmonella paratyphi using disc diffusion assay. The MHA plates were prepared by pouring 15 ml of molten media into sterile petriplates. The plates were allowed to solidify for 5 minutes and 0.1% inoculums suspension was swabbed uniformly and the inoculums were allowed to dry for 5 minutes. The various concentrations of ZnO NPs were loaded on 6 mm sterile disc. The loaded disc was placed on the surface of medium and the extract was allowed to diffuse for 5 minutes and the plates were kept for incubation at 37oC for 24 hrs. Streptomycin was used as a control packed along with the solvents. At the end of incubation, inhibition zones formed around the disc were measured with transparent ruler in millimeter.

The test was performed according to the modified method of Oyedepo et al. [34]. The reaction mixture (2 ml) containing 0.06 mg trypsin, 1 ml 20 mM tris HCl buffer (pH 7.4) and 1 ml test sample of different concentrations. The mixture was incubated at 37 °C for 5 min and then 1 ml of 0.8% (w/v) casein was added. The mixture was incubated for an additional 20 min. 2 ml of 70% perchloric acid was added to terminate the reaction. Cloudy suspension was centrifuged and the absorbance of the supernatant was read at 210 nm against buffer as blank. The experiment was performed in triplicate to confirm the reproductivity of the result. The percentage inhibition of proteinase inhibitory activity was calculated as follows. % inhibition = 100 −

optical density of test solution × 100 optical density of control

2.6 Antioxidant activity The free Radical Scavenging Activity (RSA) of ZnO NPs was measured using a DPPH method, as previously described in literature [35]. Briefly, 50 µl of ZnO NPs (1, 0.75, 0.5, 0.25 or 0.125 mg/mL) was added to 100 µl DPPH solution in 96 well plates. The mixture was shaken vigorously and allowed to stand for 1 h in the dark to protect from the light radiation. After 1 h, the reduction of the DPPH radical was determined by measuring the absorbance at 517 nm. Ascorbic acid was used as a reference material to calibrate the resultant activity. The RSA was calculated by using the equation: %RSA = [(ADPPH ‒AS)/ADPPH]

100

2.5 Antiarthritic activity of ZnO NPs 2.5.1 Protein Denaturation

Where, ADPPH is the absorbance of the DPPH solution and AS is the absorbance of the solution when the sample is added.

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2.7 In vitro cytotoxicity The cells were cultured in a petri dish comprising DMEM medium containing 10 % fetal bovine serum and 1 % antibiotic kept in a 5 % CO2 incubator. The percentage of viable cells after exposure to samples was estimated by MTT assay. For this, MC3t3-E1 cells were obtained from the South Indian Textile Research Association, Coimbatore and seeded in a 96 well plates at a density of 1 × 104 cells per well in 200 µL medium. After 24 hr, the culture medium was replaced with a serial concentration of samples and with control 5Fluorouracil. The cells were grown for another 48 h. Then, 20 µl of MTT assay stock solution in PBS positive control was added to each well and kept at 37 °C for 4 h in dark for formation of formazon crystals. After 4 h, the medium containing MTT was removed and the formed purple formazon crystals were dissolved in 300 µl of DMSO. Absorbance of purple formazon product was measured at 570 nm using a microplate reader and IC50 values were obtained by plotting optical density versus concentration. The percentage of cell viability was expressed by the equation as follows !"# $%&' "( &"%)#"* &'**!

Cell viability % =

!"# $%&' "( )#'$)'+ &'**!

× 100

Fig.2. Formation mechanism of ZnO NPs

Saponins (Froth test)

positive

Alkaloids (Hager’s test)

The cells were stained with PI and calcein and imaged using the live/dead confocal microscope to evaluate the cellular viability qualitatively [39]. 3.

phenol and flavonoid compounds can act as a capping agent [45]. Phenols and flavonoids are secondary metabolites that are almost present in all medicinal plants have been reported to serve as a bio-reductants of metallic ions in aqueous medium and exhibit a wide range of biological activities including antioxidant and anti-carcinogenic activity [46]. Also the functional groups responsible for the reducing, capping and stabilizing agents were confirmed by the observed vibrational bands in FTIR spectrum (Fig. 4).

negative

Protein (Xanthoproteic test)

negative

Flavanoids

RESULTS AND DISSCUSSION

(Alkaline reagent test)

positive

3.1 Phytochemical Screening Analysis

(Zn test)

negative

The phytochemical screening test was carried out by using aqueous leaf extract of TectonaGrandis(L.). Generally plant extract is used as a potential substitute for the stabilizing and reducing agent due to the combination of its bio-components such as terpenoids, alkaloids, phenolics, tannins, proteins, amino acids, polysaccharides, enzymes, vitamins and saponins [40]. As shown in Table 1, phenols and flavonoids are the major chemical constituents of the essential extracts obtained from TectonaGrandis leaf extracts. Many reports have specified that phenols and flavonoids are involved in the bio-reduction, formation and stabilization of metal AND metal oxide NPs [41-43]. Presence of enormous OH groups in phenol and flavonoids are the responsible for reducing zinc nitrate into ZnO NPs. Previously researchers reported that C=O, C=O–C and C=C groups of heterocyclic compounds may act as a stabilizer [44]. The formation mechanism of the ZnO NPs was shown in the Fig. 2. In the present study, phenols and flavonoids in the aqueous leaf extract bind the surface of zinc in zinc nitrate to activate the formation of ZnO NPs and also control the size. The –OH groups from the

(Lead acetate test)

negative

Cardial Glygosidase (Keller-Killani test)

negative

Carbohydrate (Benedict’s test)

negative

Terpenoids (Salkowski test)

negative

Phenol (Ferric chloride test)

positive

Steroids

negative

Table 1. Preliminary qualitative screening analysis


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52]. The stabilization and capping agent of synthesized ZnO NPs may be due to the coordination of ZnO NPs with –OH Fig.3. shows the PXRD pattern of green synthesized and C=O groups. It may also conclude that the presence of ZnO NPs and it confirms the hexagonal wurtzite structure. phenolic and flavonoid group molecules is responsible for the From the PXRD pattern the noticeable reflection planes are reduction process [23, 47]. 110, 002, 101, 102, 110, 103, 200, 112, 201, 202 and corresponding diffraction angles are 31.64º, 34.29º, 36.27º, -1 O-H stretch (or) Nitrate ions (NO3 ) 47.48º, 56.47º, 62.80º, 66.35º, 67.71º, 69.10º, 76.83º. The Hydrogen bonded groups obtained peaks matches with the JCPDS NO: 36B) 1451confirms the ZnO hexagonal phase (wurtzite structure) 2115 without addition of any impurities [22]. The sharp and narrow diffraction peaks are indicates the pure crystalline nature of 1635 the material. The diffraction peak maximum observed at the M-O plane (101) and the crystalline size was calculated by using Scherrer’s formula, 3337 A)

2106 1634

Where, D is the crystalline size, λ is the X-ray wavelength, β is the full width half maximum of the peak. The average crystalline size of the ZnO NPs was found to be 59 nm.

-C=C stretch 3330 of alkynes 4000

3500

3000

2500

C=O Hydroxyl (or) Carboxyl groups 2000

1500

1000

500

-1

Wavenumber (cm )

700 31.64 (100) 34.29 (002) 36.27 (101)

ZnO (NP)

200 100 0 20

30

40

50

60

70

80

2θ (deg)

Fig.3. Powder XRD pattern of ZnO NPs 3.3 FTIR Analysis The FTIR spectrum was recorded for the determination of possible functional groups which leads to formation of ZnO NPs. Fig.4 (A & B) shows the FTIR spectrum of TectonaGrandis (L.) leaf extract and synthesized ZnO NPs. The broad stretch at 3330 cm-1 and 3337 cm-1 shows the presence of O–H stretch and hydrogen bonded groups in alcohol or phenolic or water molecules in the extract [47]. The peaks arises at 2106 cm-1 and 2115 cm-1 were associated with –C=C stretching vibration of alkynes [22]. The strong absorption peaks at 1635cm-1and 1634 cm-1 indicates the stretching vibration of C=O hydroxyl (or) carboxyl groups on the surface of the sample [48]. The peak around 1380 cm-1 indicates the asymmetric stretching vibration of nitrate ions (NO3-1) [49, 50]. The stretching vibration of ZnO NPs peak was observed at 513 cm-1 [51]. The vibrational peaks between 400 cm-1 to 600 cm-1 region denoted metal oxides [M–O] [20,

The most ZnO wurtzite crystal structure materials have hexagonal system with C6v4 (P63mc) and two formula units per primitive cell where all atoms occupy C3v sites [53]. According to that theory Raman spectroscopy has two photon modes i.e. active and inactive photon modes. In ZnO, the active photon modes are A1, E1and 2E2 type and inactive photon mode is B1 type [6]. Fig.5 shows the FT-Raman spectrum of green synthesized ZnO NPs. The peak at 437 cm-1 is due to E2 (high) mode of wurtzite ZnO [54]. The second order structure of 2E2 (M) generally associated to the mode at 332 cm-1 [6]. The disorder–related A1 longitudinal optical, A1 (LO) mode has the small peak observed at the peak around 580 cm-1, which is generally related to the structural defects induced oxygen vacancies and zinc interstitials in ZnO [55].

ZnO (NP)

Raman intensity (a.u.)

47.48 (102)

300

76.83 (202)

400

3.4 Raman Spectroscopy

62.80 (103) 66.35 (200) 67.71 (112) 69.10 (201)

500

Fig.4. FTIR spectrum of (A) leaf extract and (B) ZnO NPs

JCPDS NO:36-1451

56.47 (110)

600

-1

580.8 cm A1(LO)

E2H - E2L -1

332.9 cm

-1

437.9 cm E2(H)

200

250

300

350

400

450

500

550

600

-1

Raman shift (cm )

Fig.5. FT-Raman spectrum of ZnO NPs

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% Transmittance

D=0.94λ/β cos θ

Intensity (a.u.)


3.2 PXRD Analysis


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The optical property of synthesized ZnO NPs was studied by UV-Visible spectroscopy and shown in Fig.6. The absorption peak observed at 370 nm confirms the formation of ZnO NPs by the biosynthesis route [56]. The peak at 280 nm corresponds to the extract of TectonaGrandis (L.) leaf. In addition, the strong absorption band identified at 370 nm which can be assigned to the intrinsic band-gap absorption of ZnO due to the electron transitions from the valence band to the conduction band (O2p–Zn3d) [57, 58]. The energy band gap of ZnO NPs were calculated by using the formula E = hc/λ --------- (1) Where, h= (6.626x10-34Js) is planks constant, c= (3 x 108 m/s) is the velocity of light and λ (370nm) is the wavelength. The band gap energy of ZnO was found to be 3.35 eV. The strong absorption of the ZnO NPs in the UV region proves the applicability of this product in various medical applications such as sun-screen protectors or as antiseptic in ointments [17].

agglomerates or aggregates; agglomerate is a group of primary particles gathered by weak Vander Waals forces, and aggregate is referred to as a lump of primary particles held by the strong chemical bonds [60]. The high negative and larger hydrodynamic value of zeta potential confirms that the repulsion among the NPs and thereby increase in stability of the formulation which indicates the ZnO NPs are agglomerates not an aggregates in aqueous condition [60, 61]. The negative charge potential value of the synthesized ZnO NPs may be due to the reducing agents flavonoid and phenolic constituents present in the leaf extract. Also it confirms the presence of gross electro-static forces with the synthesized material [62]. So, that the Zeta potential value of -25.8 mV clearly indicates that the synthesized NPs have good stability. The particle size of the ZnO NPs was shown in the Fig.7 (B). The size of the ZnO NPs was found to be 124.6 nm.

2.0 370 nm Absorbance (a.u.)


3.5 UV-Vis Spectroscopy

Fig.7. (A) Zeta Potential and (B) Particle size analysis of ZnO NPs

1.5

1.0

3.7 Field (FESEM)

B)

280 nm

0.5 A) 0.0 200

300

400

500

600

700

Wavelength (nm)

Emission

Scanning

Electron

Microscopy

Fig.8 (A & B) shows different magnification of the surface morphology and topography of the synthesized ZnO NPs. Generally the synthesized ZnO NPs were reported as homogenous, agglomerated and without the presence of the other dominating phases [7, 15]. In the present study, FESEM image shows uniform distribution and partially spherical in shape of the ZnO NPs. The size of the particle was found to be 54 nm.

Fig.6. UV-Vis spectrum of (A) leaf extract and (B) ZnO NPs 3.6 Dynamic Light Scattering analysis (DLS) DLS used to measure the macromolecules and small particles in dilute suspension by using coherent light sources. The laser light and inducing Brownian movement works in DLS analysis. The measurement was done in liquid condition of ZnO NPs with pH of 6.4. Fig.7 (A) shows the stability of the ZnO NPs. In this respect, Zeta potential is the electric potential in the interfacial Double Layer (DL) at the location of the slipping plane versus a point in the bulk fluid away from the interface [59]. The magnitude of the zeta potential (30 mV to +30 mV) gives indication of the potential stability of the colloidal solution [9]. Therefore, zeta potential indicates the degree of repulsion between adjacent, similarly charged particles in dispersion [59]. The synthesized ZnO NPs in liquid dispersion of dry powder can form

Fig.8. (A & B) FE-SEM image of ZnO NPs 3.8 Transmission Electron Microscopy (TEM) TEM analysis can be used to understand the crystalline characteristics and size of the synthesized NPs. The analysis was carried out using Tecnai G2 20 with an accelerating voltage of 200 KV and the images with different magnification are shown in Fig. 9 (A&B). From the Figure, it


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is observed that, the average particle size was found to be 50150 nm. Also it is confirmed that, the particles are almost spherical with slight variation in thickness, which supports the SEM results. The particle size determined from TEM analysis is almost close to that of the size obtained from XRD, DLS and FESEM analysis.

[67]. Therefore existing excitons will lead to increase in the concentration of intracellular singlet oxygen 1O2, through so called oxidative stress. Excited oxygen molecules are mostly reactive due to the higher internal energy and subsequently enhance the antimicrobial activity [68]. The antibacterial activities of ZnO NPs depend on morphology, particle size, powder concentration, specific surface area, etc. Previous literature implies that the Sangeetha et al. reported green synthesized ZnO NPs showed more biocidal activity against various microorgnisms when compared to chemically synthesized ZnO NPs [15]. Susheela et al. reported green synthesized nanoparticles showed more effective antimicrobial activity than that of the plant extracts [16].

Fig.9. (A & B) TEM image of ZnO NPs 3.9 Antibacterial activity The Fig. 10 (A, B, C & D) shows the antibacterial activity of ZnO NP’s was investigated by both Gram positive (Staphylococcus aureus, Bacillus subtilis) and Gram negative (Salmonella paratyphi, Escherichia coli) bacteria by zone inhibition methods. The diameter of inhibition zones in millimetre. In the agar well diffusion method, the ZnO NPs showed a highly significant antibacterial activity on all the four bacterial strains with distinct differences in the susceptibility to ZnO NPs in a dose dependent manner. A study by Kuhn et al. (2003) using the light and scanning electron microscopes reported that microbial destruction occurs through the direct damage of cell walls by the OH [63]. However, other studies suggest that H2O2 configuration is the primary effect contributing to the antibacterial activity taking place via penetration of H2O2 through the cell walls (Jalal et al., 2010; Sawai et al., 1998) [64, 65]. The generated H2O2 molecules can penetrate into the bacterial cell membrane inducing structural changes to membranes hence disturbing nutrient/protein transport and causing bacteria death [66]. The results indicated that the inhibition zone increased with increasing the concentration of ZnO NPs (20, 40, 60 and 100 µg/mL) which may be due to the increase of H2O2 concentration from the surface of ZnO NPs. The zone of inhibition was found to be highest zone for Escherichia coli (32 mm) when compared to other bacteria’s like Staphylococcus aureus (28 mm), Bacillus subtilis (30 mm) and Salmonella paratyphi (29 mm). The minimum inhibition concentration (MIC value in 20 µg/ml) of Staphylococcus aureus (15 mm) was potent than Bacillus subtilis (14 mm), Escherichia coli (13 mm) and Salmonella paratyphi (11 mm) which is shown in Table 1. Generally metal oxide NP’s with smaller sizes are correlated with a larger band gap and consequently unfavorable conditions for recombination of excitons. However, microorganisms carry a negative charge while metal oxides carry a positive charge, thus this interaction creates an “electromagnetic” attraction between the microorganism and treated surface (Xing et al., 2012)

Fig.10. (A, B, C & D) Zones of inhibition (mm) of various human pathogenic microorganism effects of various antimicrobial agents

Micro Organism a

Zone of inhibition (diameter in mm) at various concentrations 20 40 60 100 Contr (µg/ (µg/ (µg/ (µg/ ol ml) ml) ml) ml)

Staphylococcusa ureus

a

Bacillussubtilis

29

15

18

24

28

28

14

16

21

30

20

13

16

27

32

27

11

14

18

29

b

Escherichia coli

b

Salmonellaparat yphi

Control Streptomycin a Gram +ve bacteria b Gram -ve bacteria Table 2. Antibacterial activity of ZnO NPs synthesized from Tectona Grandis against Gram-positive and Gramnegative bacteria.

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Page 9 of 12

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DOI: 10.1039/C7NJ02664A

3.10 Anti-arthritic activity of Green synthesized ZnO NPs

3.11 Antioxidant activity

Inflammation is a common phenomenon occurs during arthritis. It is a reaction of living tissues towards injury. Denaturation of protein is the principle reason of inflammation. Steroids are capable of decreasing of inflammation and reduce the activity of immune response [35]. The present investigation was found that the protein denaturation (90.46±0.02) and proteinase inhibitory activity (87.68±0.03) was comparable with a powerful non steroidal anti-inflammatory standard drug (Diclofenac sodium) shown in Fig.11. The percentage of inhibition activity of protein denaturation and proteinase inhibitory activity is shown in Table 2. which clearly shows that ZnO NPs are better than the reference drug diclofenac sodium. With the increasing concentrations of ZnO the protein denaturation and proteinase inhibitory activity were found to be decreased and enhanced protection. Thus, the results reveal that ZnO NPs were capable of inhibiting denaturation of protein and proteinase inhibitory activity. Hence anti-arthritic activities of ZnO NPs were concentration dependant which confirms the antiarthritic activity of the extract against the denaturation of protein. It may be well recognized that free radical are critically involved in various pathological conditions like cancer, arthritis, inflammation and liver diseases.

Antioxidant is compound that protects cells against the damaging effect of reactive oxygen species. Recently natural antioxidants are in high demand because of their potential in disease prevention and health promotion. It is well known that antioxidant properties of the plant extracts can be evaluated by different methods due to the complex nature of phytochemicals [Singleton VL 1999] [69]. DPPH provides an easy and rapid method for estimating the free radical scavenging activity of green synthesized ZnO NP’s. The color changes from purple to yellow after reduction by ZnO NP’s, which can also confirmed by the decrease in absorbance at 517 nm [70]. Antioxidant activity of ZnO NP’s was found (Fig.12) to increase with increase in concentration from 12.5 µg/ml to 1000 µg/ml. IC50 of ZnO NPs is 39.67 µg/ml and IC50 value of standard ascorbic acid is 31.14 µg/ml. Compared to ascorbic acid, ZnO NPs shows a significant antioxidant activity. 100 ZnO NPs Ascorbic acid 80

60

40

20

0 12.5

25

50 100 250 Concentration (µg/mL)

500

1000

Fig.12. DPPH radical scavenging activity of ZnO NPs using ascorbic acid as a standard 3.12 Invitro cytotoxicity Fig.11. Anti-arthritic activity of ZnO NPs Conc. (µg/m)

% of Proteinase Denaturation

% of Proteinase Inhibitory Activity DS ZnO NPs 34.61± 70.88 0.97 ±0.03

DS

ZnO NPs

100

48.36±0.86

74.44±0.03

150

55.28±0.74

82.76±0.04

37.59± 1.06

78.93 ±0.04

200

58.42±0.93

90.46±0.02

58.42± 0.93

87.68 ±0.03

(Data is mentioned as Mean ± SD, DS- Diclofenac sodium) Table 3. Anti-arthritic activity of ZnO NPs

Cell viability assays are basic criteria in toxicology that elucidate the cellular response to a toxicant. The invitro cytotoxicity of the ZnO NPs was analyzed on MC3t3-E1 cell line treated with six different concentrations (25, 50, 100, 150, 200 and 250 µg/ml) for 48 h in comparison with positive control 5-Fluorouracil by MTT assay. As represented in Fig.13 (E), there were sharp percentage reductions in cell viability after ZnO NP’s exposure at concentrations ranging from 25-250 µg/ml. The concentration required for causing 50 % cell death (IC50) was 30.90 µg/ml for 5-Florouracil and 41.29 µg/ml in the MC3t3-E1 cell line treated with ZnO NPs. ZnO NPs and 5-Florouracil untreated cells exhibited negligible cytotoxicity upto 1mg/ml, which signified the cytotoxic effect of ZnO NPs is sufficient. The anticancer effect of ZnO NPs was further investigated by qualitative analysis using live/dead cell assay by confocal microscope (Fig.13 (A-D)) using two concentration of sample (100 and 250 µg/ml). Active esterase present in live cells with intact membranes modifies calcein AM to a green fluorescent


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% DPPH Radical Scavening Activity


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DOI: 10.1039/C7NJ02664A

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An efficient bio active ZnO NPs were synthesized using TectonaGrandis(L.) leaf extract and reported for the first time. The crystalline nature and the hexagonal structure of the synthesized ZnO NPs were confirmed by powder X-ray diffraction analysis. Various vibrational bands were identified the presence of components in the respective extracts as well as the ZnO NPs. Stretching of vibrational, rotational and low frequency transitions in molecules of ZnO NPs are found by FT-Raman spectroscopy. The formation of ZnO NPs was indicated by the observation of colour change and also it was revealed by a peak observed at 370 nm in UV–Vis spectroscopy. DLS studies confirm the particle size and potential stability of the colloidal solution of ZnO NPs. The spherical shape ZnO NPs with a group of agglomerated particles was found from FE–SEM and TEM analysis. The samples exhibited excellent antibacterial activity against both gram positive and gram negative bacteria and considerable antioxidant activity. The MTT assay results suggest that, when the concentration of green synthesized ZnO nano powders was 250µg/mL, the viability of cancer MC3t3E1osteoblast cells dropped to around 13 % with a IC50 valueof 41.29 µg/ml and no cytotoxicity was observed against normal sample untreated MC3t3-E1 cells upto 1mg/ml. Hence it is concluded that the ZnO NPs synthesized in the present work can be a potential candidate for various biological and medicinal related applications.

80 ZnO NPs Control

70 60

E

The author (Mr. N. Senthil Kumar) sincerely thanks to TEQIP – II for the financial support to carry out the work. The corresponding author Dr. I. Vetha Potheher acknowledges the TEQIP– II for providing the seed money to carry out the research work. Reference

50 40 30 20 10 0 25

50

100 150 200 Concentration (µg/ml)

250

Fig.13. Confocal microscopic live/dead cell images of MC3t3-E1 cell line after treatment with control (5Fluorouracil) (A) 100 µg/ml (B) 250 µg/ml and ZnO NPs (C) 100 µg/ml (D) 250 µg/ml; (E) The percentage of cell viability of control and ZnO NPs by MTT assay at various concentration from 50-250 µg/ml. The data are expressed as mean ± SD (n= 3)

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Conclusion

Acknowledgement

Cell viability (%)


compound (calcein), serving as a marker for viable cells. PI (Propidium iodide), a red fluorescent nucleic acid stain cannot penetrate normal unharmed cells and hence only damaged cells uptake the dye and stain positive [71]. As expected, the high density of dead cells (red) with slight morphological changes was observed after treating MC3t3-E1 cell line with ZnO NPs which indicated the enhanced cytotoxic effect of ZnO NPs in significant with the control.

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