International Journal of Drug Development and Research

Keywords

Silver nanoparticles, Justicia adhatoda, Green synthesis, FTIR, SEM, TEM, Antibacterial activity, Cytotoxicity.

INTRODUCTION

Nanotechnology is a field of applied science and technology whose control of matter on atomic and molecular scale generally about 1 to 100 nm. Nan particles or nano crystals are made of either metals, semiconductors or oxides with different electrical, optical and chemical properties. They have been used as chemical catalysts and quantum dots. Nana particles are constituted by bounded atoms roughly in the range of 10 to 104. Hence, they cannot be simply treated as freeatoms or small gas molecules. On the other hand, they are much smaller than polymers. When the size is too small, particles gain wave character.

Hence, the sole particle behaviour, the energy response will be modified. The properties of nanoparticles changes drastically with size. Nanoparticles can be synthesized using physical, chemical and biological methods. .Synthesis using biological sources are rapidly gaining importance owing to their growing success and simplicity. Many biological organisms are known to produce nano materials either intra or extracellularly [1,2,3]. Synthesis of silver and gold and silver nanoparticles have been reported using the plant extracts of Aloevera [4],Tamarind, Emblica officinalis [5,6], Carica papaya [7] ,Azadirachta indica [8],Jatropha curcas [9],Cuminum cyminum , Stigmaphyllon littorale and ,Boswellia serrata 10,11,12] and, Securinega leucopyrus [13], etc. in the present study, we repot the synthesis of silver nanoparticles mediated by the leaves extracts of the medicinal plant Justicia adhatoda. Justicia adhatoda is a herbal plant, also called Vaidyamata singhee belongs to Acanthaceae family and grows wild in abundance all over India. The plant is a source of vitamin C and acts as anti plasmodia, anti diabetic, anti-jaundice, bronchodilator, anti inflammatory and cardio protective agent. The leaves are used for checking postpartum hemorrhage and urinary trouble. The antimicrobial properties of silver nanoparticles were known since ancient times and silver ions are widely used as bactericidal agent. The leaves with such medicinal properties are used for the synthesis of silver nanoparticles which would act as effective application in the field of medicine.

MATERIAL AND METHODS:

Biosynthesis of silver nanoparticles:

Fresh and mature leaves of the plant Justicia adathoda was collected from the Osmania University campus, Hyderabad. The leaves are thoroughly washed with distilled water and cut into small pieces. The plant leaf broth solution was prepared by using 5 gm of washed and cut leaves in a 250 ml Erlenmeyer flask with 50 ml of distilled water and then boiling the mixture for 5 min. the aqueous extract was then filtered by Whitman No 1 filter paper and stored at 40C. for the synthesis of silver nanoparticles the leaves extract and 1m M silver nitrate solution are added in the ratio 1:10 ratio and kept on a sand bath for 600C for 10mins. The color of the reaction mixture has changed from pale yellow to reddish brown after incubation.

CHARACTERIZATION STUDIES:

The synthesized silver nanoparticles from the d extract of the plant of Justicia adhatoda were characterized with the help of Elico SL-159 UV Spectrophotometer by continuous scanning from 300nm to 700nm and the extract was used as the reference for the baseline correction. The binding properties behind the formation of silver nanoparticles using extracts of the plant were analyzed by FT-IR. The spectra were recorded in the wave number range of 500-4000cm-1. The analysis was carried using Paragon 500, Perkin Elmer-RX1 spectrophotometer in the diffuse reflectance mode at a resolution of 4cm−1 in KBr pellets. Further, the formed silver nanoparticles were centrifuged at 12,000 rpm for 15min, followed by redispersion of the pellet of silver nanoparticles in 5ml of double distilled water. . The synthesis of silver Nanoparticles image size study by SEM–EDX. The synthesized Nanoparticles morphology were characterized by scanning electron microscope. The elemental analysis of the sample was done using EDX analysis. The size of the nanoparticles was determined using TEM analysis. A drop of the solution was subsequently deposited onto a lacey C film supported on a Cu grid and allowed to evaporate under ambience conditions .The antibacterial assays of silver nanoparticles were done on human pathogens Gram positive and gram negative by disc diffusion method. In this disc diffusion method L.B Agar medium and sterile paper disc containing silver nanoparticles along with standard disc were placed in the Petri plate and incubated at 37° C for 48 hrs and observation was recorded. Ampicilline was used as the positive control.

Hela cell line maintenance and growth

Cell line maintenance and growth conditions the cytotoxicity potential of silver nanoparticle was studied against HeLa cell lines (Human epitheloid cervix carcinoma). Cell lines were purchased from NCCS, Pune, India. Hela cell lines were sub cultured and were maintained at 370C at 5% CO2 in CO2 incubator. Cultures were continuously every 24hr observed under an inverted microscope and to confirm the absence of any bacterial and fungal contaminants.

Evaluation of cytotoxicity activity of the silver nanoparticles by MTT Assay

In present paper in-vitro study of cytotoxicity effect of silver nanoparticle was assessed by MTT (3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay. The process of assay is a colorimetric, non-radioactive assay for measuring cell viability through increased metabolization of tetrazolium salt [14]. HelaCell lines were subcultured and 100μl of media (containing 5000cells) were transferred into 96 well plates and incubated for 24 hr. The media was removed and added fresh media 100μl. Synthesized silver nanoparticle was added at different concentration (10-80 μg) and then final volume was made to 100μl with the media and incubated for 4 hr. After incubation media containing drug was removed. 10μl of MTT reagent (5mg/ml in PBS) was added to each well containing media and incubated for 2 hr at 37 °C under an atmosphere of 5% CO2 in incubator until a purple precipitate was observed. Media was removed (Don t disturb cells). 100μl DMSO (MTT solvent) was added to dissolve the purple precipitate [15].Absorbance was read at 562 nm. Percentage cytotoxicity was calculated as for protocol [16] and used for finding the IC50 value of the concentration required for 50% cell death by synthesis justicia adhatoda leaves silver nanoparticle.

RESULTS AND DISCUSSION:

The silver nanoparticles exhibit yellow brownish color in aqueous solution due to excitation of surface Plasmon vibrations in silver nanoparticles [17]. Upon incubation of mixture of the leaves extract with 1mM AgNO3 solution, the color of the solution changes from pale yellow to dark brown color (Fig. 1). In the FTIR study (Fig 2.), the peaks are more characteristic of eugenols, linalools, and terpenes that are abundant in the leaves of Justicia adhatoda plant leaves. The peaks found at 1635cm−1can be attributed to the C–C in alkene rings and C=C stretch of aromatic rings, respectively, whereas peaks at 1076 cm−1 can be attributed to the ether linkages and C–N stretching vibration of amine. 2344cm-1 cm-1 corresponds to -NH group of amines. 3403 cm-1 shows characteristics of O–H stretching of secondary alcohols. Depending on above observation, it can be assumed that the stabilization is achieved by the phenolic as well as aromatic compounds present in the extract. Studies have confirmed the fact that the carbonyl group form amino acid residues and proteins has the stronger ability to bind metal indicating that the proteins could possibly form a layer covering the metal nanoparticles.(Figure. 3) shows SEM images recorded from drop-coated films of the silver nanoparticles synthesized by treating seed powder extract with silver nitrate solution. The formed silver nanoparticles were predominantly spherical in shape. The shape of the nanoparticles plays a key role in optical and electrical properties. With Energy Dispersive Spectroscopy (EDS) analysis, the presence of elemental silver signal was confirmed in the sample (Figure 4and 5) shows TEM images of AgNPs at different magnifications. Images reveal that the formed AgNPs are predominantly spherical in shape with a smooth surface morphology. The figure also shows the histogram of particles size versus number of particles observed on TEM grid. It is clear from the histogram that more number of nanoparticles has the diameter within the range of 11-20 nm and the mean particle size of Ag-NPs is 18 nm. The synthesized silver nanoparticles have shown fairly effective action against different pathogenic bacteria. (Fig: 6, 7) and (table 1). In this study, we have employed a dose dependent approach to evaluate the cytotoxicity of the nanoparticles on human In-vitro cytotoxicity effect of silver nanoaprticle was studied against HeLa cell lines at different concentration (10, 20, 30, 40, 50, 60, 70, 80μg) (Figure .8). The in-vitro screening of the AgNPs showed potential cytotoxicity activity against the Hela cancer cell lines. The results were shown in Table 2. Complete mortality rate that is 82.15% cell death was observed in 80μg/μl concentration of Ag-NPs. Hence, the inhibitory concentration at 50% (IC50) was fixed at 55μg/μl of Ag-NPs for HeLa cells. At a concentration 50 μg/μl, 59.12% of cytotoxicity and at a concentration 60μg/μl, 41.19% cytotoxicity was recorded and the standard anticancer drug cisplatin (45μg/ml) was also used in this study to confirm and correlate the anticancer activity of Ag-NPs.

Acknowledgements:

The authors are gratefully acknowledged to the University Grants Commission, New Delhi for their financial support in the form of RFMS and Junior Research Fellowship. DBT-OU-ISLARIE –Coordinator and CFRD-Director, Osmania University,India for providing all support during the study period.

Authors’ contributions:

All authors read and approved the final manuscript. Madhukar Rao Kudle and Prashanthi .Y. Developed the concept and designed experiments. Research guide of this experimental study. Karunakar Rao and Manish R.Donda Performed plant collection, nanoparticles synthesis, and characterization and cell linef studies. Instrumental studies and advised on experimental part.

Tables at a glance

Table 1 Table 2

Figures at a glance

Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8

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