Development of Silver Nanoparticles Using Moringa Oliefera Fruit Extract

Introduction

Lately, nanotechnology has breathtaking development because of its different applications in material science, medication & biotechnology and so forth nanoparticles have high consideration in light of their extraordinary surface to volume proportion, and colossally little size, which prompts distinction in physical properties when contrasted with same constitution of bulk material.¹ A nanoparticle with controlled size has extraordinary principal and mechanical applications as it gives answers for innovative and natural difficulties in the space of medication, catalysis and water treatment.² Nanotechnology is an exceptional science for the production of particles in nanosize, which has a wide scope of application in diagnosis and clinical applications. Due to distinct structure & physical characteristics of nanoparticles it plays a unique role in delivery of drugs, controlling the phase of abnormal cell multiplication & preventing the growth of microorganisms³.

Silver has extended history of its utilization in various forms and for various purposes for quite a long time, the antibacterial properties of silver have been utilized to treat consumable water in silver containers.⁴ There is roundabout evidence for the use of nanosilver in old Egypt and Rome⁵ . The Macedonians utilized silver plates to improve wound mending and Hippocrates utilized silver for the treatment of ulcers. In 1520 Paracelsus used silver for treating wounds, till to date silver is used for treatment of various wounds.⁶ In 1614 Angelo Sala used silver nitrate as a counterirritant, as a laxative and for treating cerebrum diseases.⁵ C. S. F. Crede for the first time published the use of silver in the form of eye drops using 1% silver nitare for treating the infection of eye in new born.⁶

Silver nanoparticles (AgNP) can be synthesized by various physical methods by using laser ablation, tube furnace, arc discharge and Evaporation-condensation Chemical methods like chemical reduction, micro emulsion, UV photo reduction, electrochemical reduction & microwave irradiation.⁷ AgNP prepared from above methods provide high yield of nanoparticles with desired size & surface characteristics but they are toxic to human use & environment as the nanoparticles produced by these methods are used for human benefits.⁸ To overcome the above toxicity problems much attention has been given for green synthesis of nanoparticles, where microorganisms or extracts of plants are used to reduce silver ion to form AgNP which are echo friendly, cost effective & solves the problems associated with chemical method. In contrast to bacteria & fungi, plant extracts require less time for reduction of silver ion as the active constituents present in the plant can do it rather quickly and replaces extravagant and tedious methods. Hence we applied/adopted the biosynthesis of AgNP by plant extract using green synthesis method.

Green synthesis of AgNP have been previously explored by using plant extracts like Melia azedarach,⁹ Alternanthera dentate,¹⁰ Andrographis paniculata,¹¹ Tribulus terrestris,¹² Calliandra haematocephala,¹³ Citrullus colocynthis,¹⁴ These studies have demonstrated the biological active compounds present in the extracts of plants can effectively act as reducing agents to from AgNP which are indeed eco friendly & non toxic for various biomedical applications.

Moringa oleifera, an important folklore medicinal plant of Moringaceae family which is used as drug for cardiovascular diseases.¹⁵ Phytochemical literature revealed that, Moringa oleifera plant extract is rich in phenols, sitosterol, caffeoylquinic acid, quercetin, and kaempferol¹⁶ which has shown immune modulator activity, helps in regulating blood sugar¹⁷ and cholesterol level.¹⁸ Interestingly, extract of Moringa oleifera is also known to have fair antiproliferative activity on cancerous alveolar and pancreatic cells. These properties have leaded us to explore the antiprolferative activity of Moringa oleifera AgNP. The present investigation 46 deals with aqueous extract (AE) of Moringa oleifera fruit mediated synthesis, characterization of AgNPs and its anti cancer activity against A549 human lung cancer cell lines

Materials and Methods

Materials

Fresh fruits of Moringa oleifera were collected from vegetable market of Bagalkot. Silver nitrate of analytical grade was purchased from SDFCL, Mumbai. dimethylsulfoxide and 3-(4,5-dimethylthi-azol-2-yl)-2,5-diphenyltetrazoli um bromide (MTT) were obtained from Sigma-Aldrich (St Louis, MO, USA). Dulbecco`s Modified Eagle Media (DMEM) with low glucose, fetal bovine serum (FBS), Trypsin-EDTA from Sigma-Aldrich,. Double distilled water was used throughout the study. 

Preparation of M.oleifera extract

The M. oleifera fruits were washed with 2% potassium permanganate solution followed by double distilled water for 2-3 times and dried at room temperature in shade and then fruits were chopped into small pieces and again dried at room temperature in shade then they were pulverized & powder was passed in sieve no 60. 10g dried powder of M. oleifera fruit was weighed and in a 250 ml closed Erlenmeyer flask and warmed with 100ml of double distilled water at 60 C for 10 min. After warming the extract was filtered using musclin cloth, then filtered by Whatmann filter paper no.1 and was centrifuged at 5000 rpm for 5 min and kept in refrigerator for further use. 

Phytochemical screening of AE of M.oleifera

The phytochemical screening for AE of M. oleifera was done according to the methods described by Khandelwal et al.¹⁹

Green synthesis of silver nanoparticles using AE of M.oleifera

Reduction of silver ion was done using, 1ml(F1), 2ml(F2), 3ml(F3) & 4ml(F4) of bioreductant AE of M. oleifera was mixed with 99ml, 98ml, 97ml & 96ml of 2mM silver nitrate solution. For the reduction of silver ions, AE of M.oleifera was used for reducing silver nitrate by adding extract to silver nitrate and heating at 60-800C for 20min, to prevent photo activation of silver nitrate, reaction mixture were incubated at dark place until change of color was observed from pale whites to orange.

Formation of M. oleifera silver nanoparticles (MO-AgNP) was confirmed by UV-Visible spectroscopy. To obtain the dry powder of green synthesized silver nanoparticles, dispersion of AgNP was centrifuged at 10,000 RPM for 15 min. After centrifuge obtained pellet was redisperesed in double distilled water and centrifugation process was repeated thrice to get rid of any un-reacted biological materials. The purified pellets were then dried in hot air oven at 60ºC to get silver nanoparticles; these nanoparticles were further used for characterization studies.

Characterization of green synthesized MO-AgNP

Green synthesized MO-AgNP were subjected to various characterization studies, formation of silver nanoparticles were confirmed by UV-Visible spectroscopy by scanning the samples from 200-800nmn with a resolution of 1nm (Shimadzu UV-1800, Japan). The Fourier transform infrared spectroscopy (FTIR) studies were done using Shimadzu IRAffinity-1S for AE of M.oleifera & MO-AgNPs using potassium bromide pellet in the wave number 2000-800 cm^-1. For determining average size of AgNP & zeta potential of MO-AgNPs solution was diluted ten folds using double distilled water & analyzed by Horiba Scientific instrument corp SZ-100. Surface morphology study of AgNP was done using atomic force microscopy (AFM) Bruker’s Dimension icon, for AFM studies a thin film was prepared on silica glass plate by placing few drops of nanoparticles & dried in nitrogen flow and deposited film was scanned using silicon probe cantilever in tapping mode at a resonance frequency 209-286 kHz. The crystallite of nanoparticles was done by X-ray diffraction (XRD) analysis using BYE FIEX 2002 model, Seifert, Germany by applying a voltage of 40 kV and current of 30 mA, with Cu Kα radiation in 2θ configurations.

MTT assay of MO-AgNPs

Cells cultured in T-25 flasks were trypsinized and aspirated into a 5mL centrifuge tube. Cell pellet was obtained by centrifugation at 300 x g. The cell count was adjusted, using DMEM HG (Dulbecco’s Modified Eagle Medium with High Glucose) medium, such that 200μl of suspension contained approximately 10,000 cells. 200μl of the cell suspension was added in each well of 96 well microtitrate plates and the plates were incubated at 37℃ and 5% CO₂ atmosphere for 24 hr. After 24 hr, the spent medium was aspirated. 200μl of different test concentrations of MO-AgNPs formulation (25, 50, 100, 200 and 400 μg/mL serially diluted from stock) and standard drug Cisplatin (50 μg/mL diluted from stock) were added to the respective wells. The plates were then incubated at 37℃ and 5% CO₂ atmosphere for 24 hr. The plate was removed from the incubator and the silver nanoparticles containing media was aspirated. 200μl of medium containing 10% MTT reagent was then added to each well to get a final concentration of 0.5mg/mL and the plates were incubated at 37℃ and 5% CO₂ atmosphere for 3 hr. The culture medium was removed completely without disturbing the crystals formed. Then 100μl of solubilisation solution (DMSO) was added and the plate was gently shaken in a gyratory shaker to dissolve the formazan & absorbance’s was noted at 570 nm and also at 630 nm using microplate’s reader. The percentage growth inhibition was calculated, after subtracting the background and the blank, and concentration of test drug needed to inhibit cell growth by 50% (IC₅₀) was generated from the dose response curve for the cell line^20-21.

Results and Discussion

Phytochemical screening of AE of M.oleifera & UV-Visible spectroscopy

Phytochemical investigation of AE of M. oleifera was found to contain carbohydrates, proteins, saponins, tannins, steroids, flavonoids & phenolic phyto-constituents. The UV-Vis spectroscopy is universally accepted technique for structural characterization of silver nanoparticles.²² Synthesized silver nanoparticles exhibit strong absorption of electromagnetic waves in the visible range due to their optical resonant to its collective oscillation of conduction electrons Fig.1.a shows the UV-Visible absorption spectra of the aqueous extract & Fig 1.b shows after bioreduction AgNPs from Morniga oleifera. The peak of the above spectra was due to Surface Plasmon Resonance (SPR) property of silver nanoparticles. Surface Plasmon resonance (SPR) is defined by Link and Elligh²³ wave which induces a polarization of the electrons with respect to the much heavier ionic core of a nanoparticle. SPR is easily and rapidly measured by UV absorbance and peak widths and can indicate particle size and distribution from band widths and the λ peak maxima absorbance. This phenomenon complies with Mie’s theory for spherical particles. This method, however, has a narrow working size range of 2-20 nm, although the upper limit could be as large as 50-80 nm. The spectra recorded before addition of silver nitrate & after bioreduction observed the increased intensity in absorption spectra, indicating the formation of more number of AgNP in the solution. The prepared AgNPs showed absorption peaks at 426.5, 422 and 409 nm which is typical absorption band of nanoparticles due to their surface Plasmon thus biosynthesized AgNPs may be polydispersed.

FT-IR analysis of silver nanoparticle

FTIR analysis is used for confirming the reduction of silver ion to from AgNPs for the AE of M. oleifera and the resulting nanoparticles. Spectra of aqueous extract before & after bioreduction of silver ions are shown in Fig 2a. Absorbance bands were noted in the region of 500–4000 cm^-1 are 3418.97, 2926.14, 1623.17, 1401.34, 1236.42 cm^-1 these wave numbers can be correlated with the stretching vibrations of –C C–C O, –C C– [(in-ring) aromatic], –C–C– [(in-ring) aromatic], C–O (ethers, esters) and C–O (polyols), respectivel. In particular, the 1236.42 cm^-1 band arises likely from the C–O group of polyols like hydroxyflavones.²⁴ Absence of this wave number after the bioreduction Fig 2b was may be due to involvement of polyols for the bioreduction of silver ions, where polyols were oxidized themselves to unsaturated carbonyl groups leading to a broad peak at (1642.46 cm-1).²⁵

Dynamic light Scattering and Zeta potential

The DLS size distribution of silver nanoparticles from F1, F2, F3 & F4. F1 showed average particle diameter 708.9nm with zeta potential -38.60 mV, F2 showed particle diameter was 439.4nm with zeta potential -38.22 mV, and F3 showed average particle diameter of 305.3nm with zeta potential -38.60 mV, F4 formulation showed the average particle diameter of 134.6nm with zeta potential -38.5mV.Above all the formulation. F4 was having least average particle diameter. From observed particle size distribution it was confirmed nanoparticle were having a wide size distribution of particles. This indicated synthesized nanoparticles were poly dispersed with negative charge in the concentration of aqueous extract from F1 to F4. It was absorbed that as the concentration of aqueous extract was increased the AgNPs size were decreased these values were in good concurrence with the reported literature.²⁶ The high negative zeta potential of the silver nanoparticles conform the repulsion among the particles and proves that they are very stable. As F4 formulation was having least average particle diameter so it was further used for evaluation.

X-Ray Diffraction

The nanoparticles synthesised in this method were characterized by using powder to confirm the particles as silver and to know the structural information fig 3 shows the XRD pattern of silver nanoparticles. The pattern clearly shows the main peaks at (2θ) 27.751, 32.174, 46.129, 54.75 and 57.467. By comparing, the typical pattern of green-synthesized AgNPs was found to possess a face centered cubic (fcc) structure. Unassigned peaks were also observed these peaks were weaker than those of silver. This was presumed due to bioorganic compounds occurring on the surface of the AgNPs. Unpredicted crystalline structures (76.66º and 85.44º) were also present due to organic compounds in the fruit extract the identified crystalline peaks (27.751, 32.174, 46.129, 54.75 and 57.467) which were according to the reported XRD patterns of silver nanoparticles ^27-29. 

Atomic force microscope (AFM)

AFM studies revealed that the AgNPs were similar in their shape and size, ranges between 53-70 nm. The three-dimensional views of the AgNPs are shown in Fig 4. AFM analysis was performed to analyze the size and shape of the AgNPs. From the AFM images, we realized that biosynthesized AgNPs were mostly FCC in shape with narrow size distribution as shown in Fig 5. Most of the AgNPs are sized between 53 nm and 70 nm and were aggregated. A similar result was reported by R. Sahana et al. ³⁰ DLS data revealed that average particle size of the biosynthesized AgNPs were 134.66 nm, where the DLS measures the hydrodynamic radii of the NPs and hence the particle size value was found to be high.

MTT Assay:

The in vitro anti-cancer activity of aqueous extract of Moringa oleifera and F4 formulation was tested on A549 cancer cell lines. A decrease in the viability of A549 cell lines were observed in a dose dependent manner on treatment with aqueous extract and AgNps the half maximum inhibitory concentration (IC50) for aqueous extract was 132.04 μg/ml were as for synthesized AgNps the IC50 was 110.18 μg/ml. There was significant change in cell viability of A549 cells treated with aqueous extract, 44.07% mortality rate was noted at 125 μg\ml concentration for AE of M.oleifera there was 51.77% mortality rate at 125 μg\ml concentration for synthesized AgNPs (F4). There was increased mortality A549 cells was absorbed for synthesized AgNps as compared to aqueous extract. This was may be due to poly phenols present in M.oleifera such as hydroxy flavones, which caped silver ions.

Conclusion

The present work was chosen to develop and eco-friendly AgNPs of M.oleifera fruit extract. AgNPs were biosynthesized by green synthesis which will not lead to hazards byproducts as in physical & chemical methods. Thus phytoconstituents of the plants are of choice for synthesis of silver nanoparticles, which may lead to the biosynthesis of eco-friendly and non-toxic green synthesized AgNPs for medical and biological applications. 

Conflict of interest

The authors declare no conflicts of interest.