Anticancer and Antioxidant Activity of Morinda Citrifolia Leaf Mediated Selenium Nanoparticles

The goal of the present work is to fabricate selenium nanoparticles utilising Morinda citrifolia leaves extract via green approach. UV-Vis spectroscopy, FT-IR, and TEM were used to characterise the green nanoparticles. The size of generated SeNPs in Morinda citrifolia was anticipated to be 12-160 nm based on TEM images. The antioxidant activity of selenium NPs was discovered to be 66.7 to 83.7% of free radical inhibtion. When the concentration of nanoparticles rises, the viability of cancer cells decreases. It shows that biosynthesized selenium nanoparticles have anticancer properties that depend on the concentration. The brine shrimp lethality assay revealed that Morinda citrifolia mediated selenium nanoparticles have low cytotoxic e ﬀ ects.


Introduction
Nanotechnology addresses a progressive way for technological advancement that includes the administration of material at the nanometer scale (one billion times less than a meter) [1]. Nanotechnology implies any innovation on the nanoscale that has various applications in reality. Nanotechnology in a real sense incorporates the fabrication and utilization of chemical, physical, and biological systems at scales ranging from atoms to submicron measurements, and furthermore the combination of these subsequent nanomaterials into larger systems [2,3]. Selenium (Se) is a fundamental trace component present in our body. Selenium is found in proteins as selenocysteine (Sec), which is referred to as selenoproteins. The presence of selenium in enzymes, which have a critical function in shielding the organism from the effects of oxidative stress, explains the element's importance in human nutrition [4,5]. The presence of oxidoreductase in selenoproteins regulates redox equilibrium in the body. Because of the low toxicity of selenium nanoparticles (SeNPs), they have been investigated for variety of oxidative pressure as well as inflammatory diseases. Therefore, SeNPs pave a way to transmit various medications to the site of action (Amit [6]). The current study exposes the fabrication of SeNPs using Morinda citrifolia extract.
Fabrication of nanoparticles via green approach using medicianl plants extracts has recently gained popularity. Phytochemicals, in particular, constitute the backbone of plants, may readily produce nanoparticles with lower toxicity. As the benefits of Morinda citrifolia L. and its products become more widely known, researchers have begun to take an interest in them [7]. M. citrifolia L., belongs to the family Rubiaceae spread all over in tropical Asia and Polynesia. It appears to have been highly valued therapeutically in the Tropical regions, and the plant is normally produced for its root system, leaves, and organic matter. These plants roots are an excellent source of anthraquinones, which are typically found as aglycones and, to a lesser extent, as glycosides. All components of the tree have been widely used medicinal services for the relief of arthritic as well as other pains, and for their healing properties [8].

Materials and Methods
2.1. Preparation of Extract. Morinda citrifolia leaves were obtained from Thiruparuthikundaram, Kancheepuram. The collected leaves were thoroughly washed under the tap water. For seven days, the leaves were dried in the shade at room temperature. The leaves of Morinda citrifolia sepa-rately grounded using mixier grinder into fine powder. 1gm of powdered leaves of Morinda citrifolia was added to 100 mL of distilled water and heated at a temperature of 60-70°C using a heating mantle. Finally, using Whatmann No. 1 filter paper, the mixture was filtered and the extract was stored for further use (Figure 1).

Synthesis of Selenium
Nanoparticles. Sodium selenite (Na 2 SeO 3 ), 30 mM dissolved in 50 mL of distilled water. To that, 50 mL of Morinda citrifolia leaf extract was slowly added. Then the reaction mixture was kept on a magnetic stirrer at 650-700 rpm for 48-72 hours.

PURIFICATION AND CHARACTERIZATION of NPs
USING TEM &FT-IR. The collected NPs were kept for centrifugation at 8000 rpm for 10 min. The pellet obtained dried at 70°C in hot air oven for 12 h. The dried pellet was grinded   Journal of Nanomaterials using mortar and pestle and the powder was stored for further use. The TEM and FT-IR analysis was carried out using the powdered pellet.

Antioxidant
Assay. The DPPH assay of free radical scavenging using Morinda citrifolia leaves extract mediated SeNPs was carried out by the procedure reported in (Rajeshkumar et al. [9]). Various concentrations (10, 20, 30, 40 and 50 μg/mL) of Morinda citrifolia extract synthesized selenium nanoparticles was added to 1 mL of DPPH and 450 μl of TrisHCl buffer was added and kept in incubation for 30 mins. The free radical scavenging was analysed by measuring absorbance at 517 nm. As a control, BHT was used. Ascorbic acid was employed as a reference substance. The inhibition percentage was determined from the following equation, The solution was tested using the UV-visible spectroscopy and the optical density at 595 nm measured using DMSO as control. The percentage of cell viability was calculated using the below equation.

Cell viability
2.6. Cytotoxicity Activity. Weighing and dissolving 2 g of iodine-free salt in 200 mL of purified water. 10-12 ml saline water filled in 6 well ELISA plates. 10

Journal of Nanomaterials
After incubation, the plates were counted for the presence of live nauplii and the number was estimated using the following formula: number of dead nauplii/number of dead nauplii + number of live nauplii × 100 ð3Þ

Results and Discussion
3.1. Visual Observation. The formation of metal nanoparticles upon the addition the plant extract is accompanied by the colour change of the solution. As depicted in Figure 2 the Morinda citrifolia mediated selenium nanoparticles gradually changes its colour to light brown after 28 h and pale brown colour after 31 h and finally brownish red colour was obtained after 56 h.

UV-Visible Spectroscopy
Analysis. The formation of SeNPs by using Morinda citrifolia leaf extract was preliminarily confirmed by UV-visible spectroscopy (Figure 3). The readings were recorded at specific time intervals such as 6, 12, 24, 48 and 72 h. SeNPs exhibited an absorption peak at 390 nm in its UV spectrum.

Transmission Electron
Microscope. The morphological characteristics of biosynthesized SeNPs observed through    3.5. Antioxidant Activity. Antioxidants acts as a defense mechanism to prevent the body from experiencing alleviating chronic diseases by reducing the cellular oxidative damage caused by the free radicals [10]. The antioxidant 5 Journal of Nanomaterials activity of Morinda citrifolia mediated selenium nanoparticles was analysed by adopting DPPH method. As shown in Figure 6, the selenium nanoparticles showed significant free radical inhibition higher at 50 μL concentration in dose dependent manner. At 10 μL concentration the selenium nanoparticles showed free radical inhibition around 66.7% and 83.7% was obtained for 50 μL concentration.
3.6. Anticancer Activity. Figure 7 shows the Screening of biosynthesized SeNPs results in potential anticancer activity against HepG2. Different concentrations of selenium nanoparticles (5, 10, 20, 40, 80 μL) was used in this study. The cytotoxicity analysis of the SeNPs shown a concentration dependent response and higher cytotoxicity increament observed at higher concentration (80 μL).
3.7. Cytotoxic Effect of Brine Shrimp Lethality Assay. The cytotoxicity of green synthesized selenium nanoparticles was tested by adopting brine shrimp lethality assay. At day 2, the percentage of live nauplii at lower concentration (5 μl and 10 μl) was found to be 70% & 80%. As shown in Figure 8, only 30% of nauplii was alive at higher concentration (25 μL). Therefore, the cytotoxic results predicted low toxicity of selenium nanoparticles.

Discussion
The microbial synthesis of SeNPs using non-pathogenic bacterium Zooglea ramigera obtained trigonal selenium nanorods [11]. In previous research work, broccoli mediated selenium nanoparticles, FT-IR analysis showed peaks at 3235, 1595, 1407, and 1099 cm-1 indicates the presence of O-H stretch, N-H bend, C-F stretch and C-O stretch of aliphatic ether, respectively. SeNPs (64 g/mL) exhibited complete inhibitory action against Streptococcus agalactiae, Escherichia fergusonii, and Pseudomonas aeruginosa and could be used in replacement of antibiotics for treating cutaneous infections caused by bacteria [12]. Biosynthesis of SeNPs syntheisized using Clausea dentata exhibited strong larvicidal activity with increased concentration against the fourth instar larvae stage of Culex quinquefasciatus, Aedes Aegypti, and Anopheles stephensi [13]. SeNPs with doxorubicin combination exhibited excellent anti-cancer effect and it was found that SeNPs induced MCF-7cell death through apoptosis [14]. The microbial synthesis of SeNPs by using Bacillus species against Candida albicans and Aspergillus fumigatus exhibited good antifungal activity (Mojtaba Shakibaie et al., 2015). SeNPS were biosynthesized using Azoarcus sp. which transformed the selenite to Se and generated spherical SeNPs. Dried Vitis vinifera (raisin) extract synthesized uniformly shaped and biopolymer (lignin)-capped selenium nanoballs [15]. The biogenic fabrication of SeNPs via the flower extract of Bougainvillea spectabilis resulted in stable hollow SeNPs with an avergae size of 24.24 ± 2.95 nm [16]. The cytotoxic behaviour of SeNPs in combination with X-ray was demonstrated in treating lung cancer cell lines. The participation of SeNPs in caspase-3 activation and its downstream target showed that lung cancer cells undergo apoptosis [17][18][19][20][21][22]).

Conclusion
Using the leaf extract of Morinda citrifolia, a simple approach was explored to develop a green, eco-friendly manner to synthesize SeNPs. The production of SeNPs was validated by the brownish red color obtained after adding sodium selenite to Morinda citrifolia leaf extract. UV-Vis spectroscopy, FT-IR, and TEM were also used to confirm the SeNPs, which were found to be 120-160 nm in size in Morinda citrifolia. In Morinda citrifolia, selenium NPs was shown to have an antioxidant activity of 66.7 percent to 83.7 percent of free radicals. The viability of tumour cells lowered, while increasing the concentration of nanoparticles. It proves that selenium nanoparticles mediated Morinda citrifolia have anticancer properties. In the brine shrimp lethality assay, selenium nanoparticles produce less harmful findings. The findings of this study imply that selenium nanoparticles mediated by Morinda citrifolia can be employed in biomedical applications in the future.

Data Availability
The data used in this study is done by the authors.

Conflicts of Interest
The authors declare, there is no conflict of interest.