The green synthesis, characterization, and evaluation of the biological activities of silver nanoparticles synthesized from Leptadenia reticulata leaf extract
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Biosynthesis of silver nanoparticles (Ag Nps) was carried out using methanol leaves extract of L. reticulata. Ag Nps were characterized based on the observations of UV–visible spectroscopy, transmission electron microscopy, and X-ray diffraction (XRD) analysis. These Ag Nps were tested for antimicrobial activity by agar well diffusion method against different pathogenic microorganisms and antioxidant activity was performed using DPPH assay. Further, the in vitro cytotoxic effects of Ag Nps were screened against HCT15 cancer cell line and viability of tumor cells was confirmed using MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole)) assay. The nuclear condensation was studied using the propidium iodide-staining method. The color change from green to dark brown and the absorbance peak at about 420 nm indicated the formation of nanoparticles. XRD pattern showed characteristic peaks indexed to the crystalline planes (111), (200) and (220) of face-centered cubic silver. The nanoparticles were of spherical shape with varying sizes ranging from 50 to 70 nm. Biosynthesized Ag Nps showed potent antibacterial activity and effective radical scavenging activity. MTT assay revealed a dose-dependent decrease in cell viability. Microscopic observations showed distinct cellular morphological changes indicating unhealthy cells, whereas the control appeared normal. Increase in the number of propidium iodide positive cells were observed in maximum concentration. Methanolic leaf extract of L. reticulata acts as an excellent capping agent for the formation of silver nanoparticles and demonstrates immense biological activities. Hence, these Ag NPs can be used as antibacterial, antioxidant as well as cytotoxic agent in treating many medical complications.
KeywordsLeptadenia reticulata Nanoparticles Green synthesis TEM XRD Biological activities
In this era, nanotechnology is one of the most interesting areas which are used to describe the creation and utilization of materials with structural features between those of atoms and bulk materials with at least one dimension in the nano range. Nanoparticles are atomic or molecular aggregates with at least one dimension between 1 and 100 nm that can drastically modify their physico-chemical properties compared to the bulk material. It is worth noting that nanoparticles can be made from a full variety of bulk materials and that they can explicate their actions depending on both the chemical composition and on the size and/or shape of the particles (Brunner et al. 2006). Nanoparticles have been known to be used for numerous physical, biological, and pharmaceutical applications (Rai et al. 2009). Processes used for nanoparticles synthesis are chemical, physical, and a recently developed biological method. Chemical methods have various drawbacks including the use of toxic solvents, generation of hazardous by-products, and high energy consumption, which pose potential risks to human health and to the environment. Therefore, the biological method has an advantage over chemical and physical methods of nanoparticle synthesis, as it is cost-effective and environmentally friendly (Nabhikha et al. 2009). However, these methods also have the drawback of being rather slow (Balaji et al. 2009). The major biological systems involved in this are bacteria, fungi, and plant extracts (Prabhu and Poulose 2012). In recent years, the biosynthesis of nanoparticles using plant extracts has gained more importance. The synthesis and applications of silver nanoparticles from several plants have been studied by many researchers (Ankamwar et al. 2005; Kumar et al. 2010; Saxena et al. 2010; Ahmad et al. 2010). In most of the therapeutic applications, it is the antimicrobial property that is being majorly explored, though anti-inflammatory property, cytotoxicity and antioxidants have their fair share of applications.
Leptadenia reticulata (Retz) Wight & Arn. belonging to family Asclepiadaceae is an important endangered medicinal plant. In India it is found in Gujarat, Punjab, Himalayan ranges, Konkon, Nilgiris, and Southern part of India (Sudipta et al. 2011). The various reports on its multiple uses in curing several diseases such as hematopoiesis, emaciation, cough, dyspnoea, fever, burning sensation, and night blindness draw the attention for utilization of this plant as drugs. It is regarded as good cure for tuberculosis and effectively used for several ear and nose problems. 50 % methanolic extract of this plant is having antibacterial activity and is used for the treatment of skin infection and wounds (Sivarajan and Balachandran 1994). Anjaria in 1967 tried Leptaden tablets (a formulation from Leptadenia reticulata) on some clinical cases and reported its beneficial use as galactogogue for increasing milk (Anjaria and Gupta 1967). Hence in this study, for the first time we evaluated the synthesis and biological activities of Ag NPs using leaf extract of L. reticulata.
Materials and methods
The chemical silver nitrate (AgNO3), Mueller–Hinton agar (MHA), and Sabouraud dextrose agar (SDA) were purchased from Hi Media Laboratories Mumbai, India. Tissue culture plastic wares were obtained from Tarsons Products Pvt. Ltd., India. All organic solvents used were of HPLC grade. RPMI 1640, fetal bovine serum (FBS), and MTT (3-(4, 5-dimethyl-thiazol-2-yl)-2, 5-diphenyltetrazolium bromide) were purchased from Sigma Aldrich, India.
Collection of plant material
Healthy, disease-free leaves of Leptadenia reticulata were collected during the month of April in 2013 from in and around the Padmahree Campus, Kommagatta, Sulikeri post of Bangalore, India. The collected leaves were washed thoroughly in tap water and then in detergent water and were finally rinsed with distilled water until no foreign material remained. The freshly cleaned leaves were left to dry in sun light for approximately 10 days.
Preparation of the plant extract
The leaves (20 g) were washed twice in tap water and rinsed thrice in distilled water. Then they were surface sterilized by HgCl2 (0.1 %) for 1 min, cut into small pieces, dried in the micro-oven, and ground into powder using an electronic blender. About 100 g of leaf powder material was uniformly packed into a thimble and run in soxhlet extractor. It was extracted with methanol for the period of about 5–6 cycles. After that extracts were filtered with the help of Whatman No. 1 filter paper. The filtrates were then evaporated under reduced pressure and dried using a rotary evaporator at 55 °C. Then the extract was kept in refrigerator at 4 °C for future experiments.
Synthesis of silver nanoparticles (Ag NPs)
For reduction of silver ions, 10 ml of collected filtrate was treated with 90 ml of silver nitrate aqueous solution (21.2 g of AgNO3 powder in 125 ml of Milli Q water) and incubated at room temperature for 10 min, resulting in the formation of yellowish to bright yellow and to dark brown color indicating the synthesis of silver nanoparticles (Parashar et al. 2009). After 8 h of incubation, the solution was centrifuged with 12,000 rpm for 20 min, and their pellets were redispersed in sterile distilled water. The centrifugation and redispersion were repeated three times to ensure the complete separation of nanoparticles. After drying, purified nanoparticles were resuspended in deionized water and stored in a freezer for further study.
Characterization of nanoparticles
About 1 ml (diluted with 1:20 V/V Milli Q water) of plant-part-synthesised silver nanoparticle solution was monitored in UV–Vis spectrophotometer (Elico, India) (between 300 and 700 nm) with different time intervals (15 and 30 min; 4, 6, and 8 h).
Transmission electron microscopy (TEM)
Transmission electron microscope (TEM) analysis was done using JEM-1200EX electron microscope (JEOL, Japan). Thin films of sample were prepared on a carbon-coated copper grid by just dropping a very small amount of sample on the grid, extra solution was removed using a blotting paper, and then the film on the TEM grid were allowed to dry by putting it under incubator. In this technique, whereby a beam of electronics is transmitted through an ultra-thin specimen, interacting with the specimen as it passes through. An image is formed from the interaction of the electrons transmitted through the specimen. The image is magnified and focused on to an imaging device.
X-ray diffraction analysis (XRD)
XRD measurements of the silver nanoparticle solution drop-coated on glass were done on a XRD-6000 X-ray diffractometer model (Shimadzu, Japan) with 40 kV, 30 mA with Cu kα radiation at 2θ angle.
Evaluation of antibacterial activity
The silver nanoparticles (Ag NPs) synthesized using L. reticulata leaves extract were tested for antimicrobial activity by agar well diffusion method against different pathogenic microorganisms Escherichia coli (E. coli), Klebsiella pneumoniae (K. pneumoniae) (Gram negative), Streptococcus pneumoniae (S. pneumoniae), Micrococcus luteus (M. luteus), and Bacillus subtilis (B. subtilis) (Gram positive). The pure cultures of bacteria were subcultured on MHA. Each strain was swabbed uniformly onto the individual plates using sterile cotton swabs. Wells of 8 mm diameter were made on nutrient agar plates using gel puncture. Using a micropipette, different concentrations (25, 50, 75, 100, 150 μg/ml) of nanoparticle solution was poured onto each well on all plates. After incubation at 37 °C for 24 h, the diameter of zone inhibition was measured in millimeter, and was recorded as mean ± SD of the triplicate experiments.
Studies on antioxidant activity
DPPH free radical scavenging assay
Cytotoxity test of biosynthesized Ag NPs on human colon cancer cell line HTC15
Cell viability assay
The cells were seeded at 1 × 105 cells/well into a six-well chamber plate and incubated overnight. Later, the medium was replaced with maintenance medium DMEM without FBS containing 20 g/ml Ag NPs incubated for 48 h. The cytomorphology was examined under Nikon inverted microscope.
Propidium iodide staining
HCT15 cells were plated at 5 × 104 cells/well into a six-well chamber plate. At >90 % confluence, the cells were treated with Ag NPs for 48 h. The cells were washed with PBS fixed in methanol: acetic acid (3:1, v/v) for 10 min and stained with 50 g/ml Propidium iodide for 20 min. Nuclear morphology of apoptotic cells with condensed/fragmented nuclei was examined under Confocal microscope.
The grouped data were statistically evaluated using Microsoft excel worksheet. Values are presented as the mean ± SD of the three replicates of each experiment.
Results and discussion
In vitro antibacterial potential of biosynthesized Ag NPs using L. reticulata leaf extract
Ag NPs Conc. (μg/ml)
Zone of inhibition (mm)
11.2 ± 0.5
9.6 ± 0.6
11.2 ± 1.2
8.7 ± 1.4
13.1 ± 0.8
14.0 ± 2.1
10.3 ± 2.1
13.3 ± 0.9
11.0 ± 1.6
19.2 ± 1.2
17.0 ± 0.9
12.4 ± 1.8
15.3 ± 0.6
14.0 ± 0.4
20.0 ± 1.8
21.0 ± 1.2
17.6 ± 1.1
18.1 ± 0.5
17.1 ± 0.5
22.2 ± 0.3
24.0 ± 0.4
20.7 ± 1.0
23.0 ± 1.0
17.0 ± 1.5
24.3 ± 1.3
In conclusion, the leaf extract of L. reticulata can be used efficiently to produce Ag NPs. This is a simple, eco-friendly process and has potent applications in biomedical and pharmaceutical applications. The bio-reduction observed may be due to the activities various plant metabolites present in the leaf extracts. XRD pattern thus clearly illustrates that the silver nanoparticles formed in this present synthesis are crystalline in nature. The nanoparticles were spherical in shape with varying sizes ranging from 50 to 70 nm. The biosynthesized Ag NPs demonstrated an excellent antibacterial activity, antioxidant activity, and cytotoxic activity. Hence, Ag NPs can be explored as a new source of alternative medicine for treating many human ailments.
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