Anticolon Cancer Activity of Zinc Oxide Nanoparticles Using Fresh Leaf Extract Nyctanthes Arbor-tristis

The present study reports the green synthesis of Zinc Oxide nanoparticles using the aqueous leaf extract Nyctanthes arbor-tristisis by co-precipitation method. The synthesized zinc oxide nanoparticles are characterized by X-Ray diffraction (XRD), Fourier Transform Infrared Spectral Analysis (FTIR), UV-Visible spectroscopy (UV-Vis), Scanning Electron Microscopy (SEM) and Energy dispersive X-Ray Analysis (EDX). The Zinc Oxide nanoparticle are crystalline in nature and have hexagonal structure with the particle size of about 25 nm and is determined by XRD analysis. The capping agent and the functional groups of the Zinc Oxide nanoparticles are determined by FTIR spectral analysis. The band gap energy is about 3.18 eV for the synthesized ZnO nanoparticles which is calculated by UV-Visible spectroscopy. The surface morphological structure of ZnO nanoparticles is spherical in shape. The EDX spectroscopy is used to determine elemental composition of ZnO nanoparticles. The anticancer activity of Zinc Oxide nanoparticles have high toxicity against HT-29 cell line which is determined by MTT assay. The synthesized ZnO nanoparticles have decreased cell viability from 99% to 23% with increasing concentration from 10 to100 μg/mL .


Introduction
Nanotechnology is one of the most important research and promising new technologies of the 21 st century. A number of physical, chemical, biological and hybrid methods are employed to synthesize different types of nanoparticles [1]. The physical and chemical methods are mostly used to synthesize nanoparticles. The presence of toxic compounds in these methods limits their applications and also it costs high. Therefore to avoid the toxicity, the green methods are involved for synthesize the nanomaterials using plant materials [2][3][4]. The plant based materials seems to be the best candidates for large-scale biosynthesis of nanoparticles. The advantage of using green methods is safe, cost effective and eco-friendly when compared to conventional methods [5,6]. Therefore the use of non toxic leaf extracts as reducing and stabilizing agent is also an additional advantage for the green method. The presence of biomolecules in plant leaf extract, they can act as capping and stabilizing agent during synthesis process [7].
The Zinc is a mineral which is very essential for human health and the zinc oxide nanoparticle has good biocompatibility to human cells. Among metal oxides, a zinc oxide nanoparticle (ZnO NPs) is a bio-safe material and it has wide range of applications in medical, industrial, agricultural, and environmental fields [8,9]. The ZnO nanoparticle has more significant in controlling the growth of bacteria.
Cancer is characterized by invasive and uncontrolled cell division and the spread of abnormal cells [10]. Every year thousands of people worldwide die from different types of cancer.
Chemotherapy and radiotherapy are most common treatments of cancer but they evoke many serious side effects [11]. The cytotoxic agents or cancer drugs are very few and are not very efficient [12].
Alternative treatment options are very less. Medicinal plants have always been used as natural remedy for many ailments and they have been a boon for many diseases and disorders. Nanoparticles synthesizing using medicinal plants extracts further enhances their efficacy as natural drugs many folds [13][14][15]. In the present work, we report for the first time synthesis of ZnO NPs from leaves of Nyctanthes arbortristis.
Nyctanthes arbortristis is commonly known as night jasmine or Harshringar which is the most important medicinal plant mainly used in Ayurveda [16][17][18]. The leaf extract from the plant is used for various treatments of diseases such as sciatica, arthritis, fever, asthma, diabetes, cancer. The phytoconstituents of plant leaf contains flavanoid, glycoside, oleanic acid, essential oils, tannic acid, carotene, friedeline, lupeol, glucose, benzoic acid have been reported for significant hair tonic, hepatoprotective, anti-leishmaniasis, anti-viral, antifungal, anti-pyretic, anti-histaminic, anti-malarial, anti-bacterial, anti-inflammatory and anti-oxidant activities [19,20]. The biologically synthesize ZnO nanoparticle is investigated by various characterization techniques such as X-Ray diffraction, Fourier Transform-Infrared Spectroscopy (FTIR), UV-Visible spectroscopy, Scanning Electron Microscopy and Energy Dispersive X-Ray Analysis. The cytotoxic effect is evaluated by MTT assay against colon cancer cell line (HT-29 cells).

Materials and methods
The Zinc Oxide (ZnO) nanoparticle was synthesized by Co-precipitation method using fresh leaf extract Nyctanthes arbor-tristisis. The leaves were collected around the areas of Dharmapuri. The fresh leaf extract of Nyctanthes arbor-tristisis was boiled for 60 min until the color of the aqueous solution changes from watery to light yellow. Then the extract was cooled to room temperature and filtered using Whatman filter paper No. 1. The 10 ml of leaf extract is mixed with 0.35M of Zinc acetate dihydrate (50ml) aqueous solution which was prepared using deionized water and allowed to stir for 2 Hrs. While stirring 2M of NaOH (50ml) was added inorder to maintain pH at 13 and was stirred for 4 Hrs. After complete stirring, the white precipitate was formed. Next, the white precipitate was rinsed repeatedly for three to four times using distilled water inorder to eliminate impurities. The precipitate was dried in an oven for 100˚C for 3 Hrs and the obtained particles were calcinated under a muffle furnace at 400˚C for 3 Hrs [21, 22]. Then the particle was grinded using mortar to get fine nanoparticle. The process synthesis method is shown in Figure 1.

Characterization Techniques
The crystalline nature of ZnO nanoparticle is studied using X-Ray diffraction (Shimadzu XRD 6000 X-Ray diffractometer). The capping molecules and the functional groups are tested in The Energy Dispersive X-Ray analysis is used to analyze elemental composition and stoichiometric mass percentage of ZnO nanoparticles (EV018, Carl Zeiss).

4.1.X-Ray diffraction
The XRD is used to determine the particle size or the grain size of the zinc oxide nanoparticle.
The ZnO nanoparticles are found to be hexagonal and matched with JCPDS card no.79-2205 [24]. From the Table 1, it shows that the values of particle size for the ZnO nanoparticles are calculated from the above equation using three strongest peaks. Therefore the average particle size is The particle size of the ZnO nanoparticle indicates more crystalline in nature which also confirmed theoretically using Scherrer's equation.

4.2.Fourier Transform Infrared Spectral Analysis (FTIR)
The Figure 3 shows the FTIR spectrum of zinc oxide nanoparticles using Nyctanthes arbortristis leaf extract and ranges from 4000 cm -1 -400 cm -1 . The FTIR spectrum is used to investigate the

4.3.UV-Visible spectroscopy
The UV-Visible spectroscopy is used to determine the optical properties and has wide range of applications in biomedical science [28]. The zinc oxide nanoparticles have attracted great attention towards the optical properties. The UV absorption spectrum of ZnO nanoparticle shows the strongest absorption peak at 391.31 nm and the binding gap energy is 3.18 eV. The color of the ZnO nanoparticles is white in color due to its surface plasmon resonance which confirms that the solution is free from impurities. The Figure 4 shows the absorption peak of ZnO nanoparticle using Nyctanthes arbor-tristis leaf extract.

4.4.Scanning Electron Microscopy
The scanning electron microscopy is used to determine the surface morphology and shape of the ZnO nanoparticles. The micrographs of ZnO nanoparticles using fresh leaf extract Nyctanthes arbor-tristis are shown in Figure 5. The precursor is used as zinc acetate then the zinc oxide molecules are grown slowly, forms spherical shape and also agglomeration in the nanoparticles. The SEM results that the ZnO nanoparticles are uniformly distributed and they are in spherical shape [29].

4.5.Energy Dispersive X-Ray analysis (EDX)
The elemental composition of the ZnO nanoparticles is determined by EDX analysis. The Figure 6 shows the single peak of zinc and oxygen is present between 0 and 2. The two peaks of zinc are present between 8 and 10 [30]. The presence of elements in the sample confirms that the nanoparticles are formed high purity without any external disturbance. The stoichiometric mass percentage of zinc and oxygen are 66% and 34% respectively and is shown in Table 2.

Anticancer activity of green synthesized nanoparticles
The

Conclusion
The simple and economical green methods are used to synthesize ZnO nanoparticles using fresh         Figure 1 The systematic representation of preparing ZnO nanoparticles The XRD analysis of ZnO nanoparticle using leaf extract Nyctanthes arbor-tristis The FTIR spectrum of ZnO nanoparticle using Nyctanthes arbor-tristis leaf extract The UV-Visible spectroscopy of ZnO nanoparticle using Nyctanthes arbor-tristis leaf extract The SEM micrographs of ZnO nanoparticles using Nyctanthes arbor-tristis leaf extract.

Figure 6
The EDX spectrum of ZnO nanoparticles using aqueous leaf extract Nyctanthes arbor-tristis.

Figure 7
The cell viability of synthesized ZnO nanoparticles using fresh leaf extract Nyctanthes arbor-tristis against HT-29 cell lines for different concentrations Figure 8 The microscopic images of green synthesis of ZnO nanoparticles using fresh leaf extract Nyctanthes arbor-tristis against HT-29 cell lines.