1. INTRODUCTION:

The metal nanoparticles have been a great concern for researchers these days, because of their exclusive physicochemical potentials including the large surface area to volume ratio, and the phenomenon of surface plasmon resonance¹^,². The biological approaches used for the metal nanoparticle synthesis is an environment-friendly, cost- effective method which requires only a step in the reduction of silver nanoparticles thus saving the total time of reaction³. The surface functionalization of the metal surface enhances their stability, reproducibility, and functionality of the synthesized nanoparticles⁴. These metallic nanoparticles due to these properties have found its applications in the field of pharmaceuticals as drug delivery carrier, catalysis⁵, biological tagging, optoelectronics, food industries for its preservation and packaging⁶, and also photonics. The performance of these nanoparticles depends entirely on their shape, size, and constituents involved in materializing the nanoparticles⁷.

The Mucuna pruriens plant belongs to the Fabaceae family that originates in the tropical regions like India and West Africa, widely used against snake bites or as a medicinal agent for years.⁸ The plant and its different parts have been used as a tonic for curing any ailments in the nervous system. It is said to contain a high concentration of L-Dopa which has been exploited in the treatment of Parkinson’s disease⁹. It also possesses properties like antioxidant, itching¹⁰, antineoplastic, antimicrobial, anti -epileptic, anti- inflammatory, anti-cancer, analgesic and also anti-helminthic activities^11–13.

In this present study, the anticancer property of the silver nanoparticles has been exploited on lung cancer cell lines with the help of the seed extract which itself possess the same property. The synthesised Ag nanoparticles using Mucuna pruriens seed extract was characterised by checking the optical properties, analysed for its morphology using the microscopic techniques like SEM and TEM for its shape, size and the EDX spectroscopy used to check the presence of any elemental Ag ions, and FTIR analysis was used to check the functional groups of that compound responsible for the reduction of silver nanoparticles or its stabilization.

2. MATERIALS AND METHODS:

2.1 M. pruriens seed powder preparation:

The seeds were washed thoroughly in running tap water and were finally rinsed with Mili-Q water, and the seed was dried for 14 days at room temperature. The powder was made by blending it in the grinder and the powder was stored in air tight containers and was protected from sunlight till further use.

2.2 M. pruriens aqueous seed broth preparation and biosynthesis of silver nanoparticles:

The dried seeds were collected from online site Amazon, and the silver nitrate was purchased from Hi-media laboratories Pvt. Ltd., Mumbai, India. The seeds were dried and powdered in a grinder, and 1 g of that powder was used for the extract preparation. 100 mL of Mili-Q water was added to this powder and heated at 90℃ for 10 min for the phytochemicals to release from the seeds. Then, the filtrate was extracted by using the Whatman filter paper No.42. 10 mL of this filtrate was added to a 1 mM concentration of silver nitrate in 90 mL of Mili-Q water, maintaining a ratio of 1:9 of silver nitrate solution and seed extract. The concentration of the silver nitrate was optimized with (0.5, 1, 1.5, 2,4 mM), out of which it was found the maximum stability, sharp broad peak obtained, and optimized shape of the nanoparticles with 90 mL of the 2 mM concentration of silver nitrate. The filtrate was stored at low temperatures (-4℃) for further use.

2.3 Characterization of the silver nanoparticles:

The solution containing the seed extract and silver nitrate solution was put in a shaker, and the color change was observed and analyzed using the UV-Visible spectroscopy for checking the optical properties of the nanoparticles. The readings were taken at an interval of every 15 min for an hour. The solution was centrifuged at 10,000 rpm for 8 min and pellet was collected, freeze-dried in a lyophilizer, and the obtained particles were again re-dispersed in the Mili-Q water to check its stability. The silver nanoparticles were further subjected to SEM, EDX and FTIR analysis, to give an idea about the surface morphology, shape, the functional groups of the possible organic compounds which might be involved in the reduction or stabilization of the silver nanoparticles and the presence of elemental Ag particles present.

2.4 Cytotoxicity (MTT) Assay:

The assay was performed on these human lung cancer cells to check the viability. The cell lines A549 were seeds in 96 well plates measured at a density of 4x10³and were incubated for 24h at 37℃ under the conditions of 5% CO₂in a humidified atmosphere,respectively. These cell lines were exposed to fresh medium containing the Ag nanoparticles synthesized from the seed extracts, at different concentrations from 5 to 100 µg/mL. The MTT solution of 10 µL from a stock solution of 5 mg/mL was added to individual wells and incubated at 37℃ for 4 h. The medium containing MTT solution was discarded and 100 µL DMSO solution was added to each well for formazan crystals formed to be dissolved, and mildly agitate it for 20 min. Now, the absorbance was measured at 570 nm and the viability was measured as:

Men of Sample OD

% Viabilty =--------------------------------------------------------x100

Men of Sample Control OD

% Cytotoxicity = 100 - (% Viability)

The results for the MTT reduction was expressed as percentage¹⁴^,¹⁵.

3. RESULTS AND DISCUSSION:

3.1 Visual identification and UV-Visible analysis:

The silver nanoparticles were produced by using optimized concentration of silver nitrate solution and the seed extract at room temperature. The different concentrations of silver nitrate solutions taken were (0.5, 1.0, 1.5, 2.0 and 4.0 mM), the optimized concentration was 2mM and it gave a maximum synthesis of silver nanoparticles. The plasmon peak was observed at this concentration to be maximum at 460 nm, as shown in the fig.2. The visual identification in fig.1 (A) shows the silver nitrate solution at 2 mM concentration (B) shows the color change when the seed extract was added initially and (C) shows the brown color change proving the silver nanoparticles have been produced with the mixing of plant extract. The brown color appearance is due to the absorption of the particular wavelength from the visible spectrum which explains the principle of surface plasmon resonance, was observed within 15min of addition of extract and the precursor solution. But, the maximum peak of 460 nm was observed after 1 h, and the peak remained stable for nearly 72 h. Initially, the band was observed first at 380 nm and then the peak was observed in the range of 400-460 nm, which is due to the red shift of wavelength with the increasing time. The stability was observed for weeks, and peak remained at constant level, proving that the nanoparticles were in stabilized form. And the broadening of the absorbance peak is maybe due to the rod-shaped nanoparticles which was proved with the SEM analysis. Similar absorbance of silver nanoparticles was also observed by N. Krithiga et al¹⁶ and also by Z. Abidin Ali et al¹⁷.

Fig. 1: A) silver nitrate solution B) the seed extract C) brown color change when silver nitrate solution was mixed with seed extract

Fig. 2: The maximum observance peak of silver nanoparticles at an optimized concentration

3.2 FTIR analysis:

The broad spectrum observed (A) in the range of 3305.99 nm proved the presence of phenols and alcohols (Hydrogen-bonded O-H Stretch), carboxylic acid, amine secondary (N-H stretch) and amides. The peak at 1635.64 corresponds to the functional group amides (C=O stretch and N-H bend), esters, aldehydes, ketones, carboxylic acids (C=O stretch) and alkenes (C-C=C Symmetric Stretch), peak at 503.42 and 472.56 nm. The intensity broad spectrum observed in the range of 3300 nm was drastically reduced to 3271.27 nm with same functional groups present in the seed extract which may be due to the adsorption of the organic compound on the surface of the silver nanoparticles. The FTIR analysis proves the presence of alkaloids, steroids, tannins, amino acids and etc. when compared with the phytochemicals present in the seed extract ¹⁸.

3.3 SEM Analysis and EDX analysis:

The SEM analysis shows the long rod-shaped of size nearly 20-60 nm nanoparticles produced with the optimized concentration of the seed extract as shown in the Fig. 4. The EDX analysis proves the presence of the elemental Ag with the highest peak and other compounds shows the presence of elemental phytochemical groups adsorbed on the surface of nanoparticle which might be responsible in the reduction of the silver nanoparticles. Similar results were also observed M. Vijayakumar et al¹⁹ proving that Ag elements are present on the nanoparticles. The Energy Dispersive X-ray spectroscopy analysis confirms the presence of elemental silver nanoparticles as given in the Fig.5. It showed characteristic absorption peaks at 3.4 keV which is maximum peak which indicates the presence of Ag, 0.3 keV, 0.6 keV, 2.1 keV indicates the presence of the elements like C, O, P and etc. The elements like carbon or oxygen present in the sample proves that might be involved in the stabilization of the nanoparticles. The lack of other elements explains the purity of the nanoparticles.

Fig. 4: The SEM analysis of rod shaped silver nanoparticles

Fig. 5: The EDX analysis of the synthesized silver nanoparticles

3.4 Evaluation of cell viability using Ag nanoparticles synthesized from M. pruriens:

The MTT assay is known to check the cytotoxicity of the cancer cells or the reducing potential of the cancer cells by the anticancer drug or substance. Here, the lung cancer cell lines were subjected to silver nanoparticles at different concentrations and the reduction was evaluated. The cytotoxic effect is due to the interaction of the silver ions with the various intracellular proteins and also the nitrogenous bases or phosphate groups of the DNA²⁰.Different concentrations of Ag nanoparticles ranging from 5 µg to 100 µg was taken with an exposure of 24 h. Both the drug and the nanoparticle were successful in suppressing the growth of the cancer cell lines. But, the Ag nanoparticles proved to show better cytotoxicity due to sustained release in a dose-dependent manner. The cell death was seen even at lower concentrations, i.e. the IC₅₀value was noticed at 50 µg, while the drug cyclophosphamide showed very low cytotoxicity at the same concentration. Therefore, lower concentrations of the Ag nanoparticles can be an effective anticancer agent for the treatment of lung cancer. When compared with the ethyl acetate extract or the seed extract of different Mucuna species, showed an IC₅₀value in the concentration of 200-1000 µg/mL cytotoxicity on the cell lines²¹, while the bio-reduced silver nanoparticles showed an IC₅₀value in the concentration of 12- 30 µg. Thus, proving that low concentrations of silver nanoparticles are more effective against the cancer cell lines.

As reported in a study, the mechanism of the anticancer activity for the silver nanoparticles is maybe due to the permeability of the outer membrane, which results in leaking of cellular materials, also there is possibility that Ag nanoparticles are entering the inner membrane and preventing the activation of respiratory chain dehydrogenase, which directly inhibits the growth and respiration of these cells. Also, they are affecting the proteins or phosphate lipids, inducing a collapse of membrane and finally leads to apoptosis of these cancer cells eventually²².

Similar IC₅₀values were observed with silver nanoparticles reduced from P. brevicompactum (MTCC-1999) when they were treated against breast cancer cell lines²³ and also with A549 lung cancer cell lines, when silver nanoparticles reduced from Tetragonula iridipennis²⁴ or when Enterococcus sp. was used in extracellular synthesis of silver nanoparticles. According to S. Rajeshkumar et al 2016, there is an assumption that Ag nanoparticles may enter the ion channels into the cellular membrane and supressing the metabolic pathway when they are interacting with the functional groups of enzymes or intracellular proteins and also the nitrogenous bases of the DNA. It also has the ability of blocking the signalling pathways which leads to apoptosis²⁵.

Fig. 6: The cell viability was evaluated using Ag nanoparticle at different concentrations and was compared with drug cyclophosphamide

4. CONCLUSION:

The green synthesis of silver nanoparticles at optimized concentration using Mucuna prureins seed extract was succesfully performed and was characterized to check the optical properties of silver nanoparticles with the help of UV-Visible spectroscopy by checking the maximum absorption, the size and shape was analyzed with the help of microscopic techniques like SEM, TEM etc. and the elemental presence of silver ions was checked with the EDX analysis. The characterized silver nanoparticles were used to check the cytotoxicty on lung cancer cell lines, which can be considered as a potential agent for further biomedicinal applications.The mechanism on how the nanoparticles act on cancer cell lines should be exploited.

5. ACKNOWLEDGEMENT:

I would like to thank VIT University for providing lab facility to carry out the work.

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Received on 28.06.2017 Modified on 29.12.2017

Research J. Pharm. and Tech 2018; 11(9): 3887-3891.

DOI: 10.5958/0974-360X.2018.00712.6