Linga Rao et al: Green Synthesis of Silver Nanoparticles by A. cathartica L. Leaf Extract and Evaluation for Antimicrobial… 1  Research Paper

Green Synthesis of Silver Nanoparticles by Allamanda cathartica L. Leaf Extract and Evaluation for Antimicrobial Activity

M. Linga Rao, G. Bhumi and N. Savithramma Department of Botany, Sri Venkateswara University, Tirupati, Andhra Pradesh, India.

Received April 18, 2013; accepted June 21, 2013 ABSTRACT

Silver nanoparticles (SNPs) exhibit tremendous applications in medicine as antimicrobial agent. The use of different parts of plants for the synthesis of nanoparticles is considered as a green technology as it does not involve any harmful chemicals. In the present study, we report a rapid biosynthesis of silver nanoparticles from aqueous leaf extract of medicinal plant Allamanda cathartica. The active phytochemicals present in the plant were responsible for the quick reduction of silver ion to metallic silver nanoparticles. The reduced silver nanoparticles were characterized by using UV-Vis spectrophotometry, Scanning Electron Microscope (SEM), Energy Dispersive Analysis of X-ray (EDAX) and Atomic Force Microscopy (AFM). The spherical shaped silver nanoparticles were

observed and it was found to 19-40 nm range of size. These phytosynthesized SNPs were tested for their antimicrobial activity and it analyzed by measuring the inhibitory zone. A. cathartica aqueous leaf extract of SNPs showed highest toxicity to Pseudomonas followed by Klebsiella, Bacillus and E. coli and lowest toxicity towards Proteus. In fungal species, highest inhibition zone was noted against Rhizopus followed by Curvularia, Aspergillus flavus and Aspergillus niger and minimum inhibition zone was observed against Fusarium species. These results suggest a promising potential of Indian plant-based green chemistry for production of SNPs for biomedical and nanotechnology applications.

KEYWORDS: Allamanda cathartica; nanoparticles; antimicrobial activity; green chemistry.

Introduction Nanotechnology can be defined as the science and

engineering involved in the design, synthesis, characterization and application of materials and devices whose smallest functional organization in at least one dimension is on the nanometer scale or one billionth of a meter (Silva, 2004). It is the study of assembling, controlling and manipulating matter on molecular or atomic size, it has attracted a great interest in recent years due to its expected impact to many areas such as agricultural and food technology, energy, electronics and medicine. Nanoparticles synthesis and their assembly have exciting possibilities of development in the area of nanotechnology.

Nanoparticles have been gaining a booming scientific interest due to their unique electronic, optical, mechanical, magnetic and chemical properties that are significantly different from those of bulk materials (Yang et al., 2010). A bulk material has constant physical properties regardless of its size, but at the nanoscale they exhibit most interesting properties, due to the fact that nanoparticle possess a very high aspect ratio i.e. high surface to volume ratio (Thakkar et al., 2010). The formation of nanoparticles comprises mainly of three

types (i) Dry nanotechnology, derived from surface science, physical chemistry and mainly focuses on the fabrication of structures in carbon, silicion as well as other inorganic materials (ii) Wet nanotechnology, study of biological systems that exist primarily in water base system, such as enzymes, membranes and cellular components, (iii) Computational nanotechnology, modeling and stimulation of complex nanometer. Among metal, sulphide and magnetite nanomaterial metal nanoparticle have attended much interest due to their vast applications in diverse areas such as electronics, consmetics, coatings, packaging, and biotechnology (Jena et al., 2013).

Silver nanoparticles find use in many fields, and the major applications include their use as catalysts, as optical sensors of Zeptomole (10-21) concentration, in textile engineering, in electronics, in optics and most importantly in the medical field as a bactericidal and as a therapeutic agent. Silver ions are used in the formulation of dental resin composites, in coatings of medical devices as a bactericidal coating in water filters, as an antimicrobial agent in air sanitizer sprays, pillows, respirators, socks, wet wipes, detergents, soaps, shampoos, toothpastes, washing machines and many

 

International Journal of Pharmaceutical Sciences and Nanotechnology

Volume 6 •  Issue 4 • December 2013 (extra issue) IJPSN-4-18-13-LINGARAO

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Linga Rao et al: Green Synthesis of Silver Nanoparticles by A. cathartica L. Leaf Extract and Evaluation for Antimicrobial… 2261  wound dressing (Prabhu and Poulose, 2012). Silver has some medicinal uses going back for centuries. The Phoenicians are said to have stored water, wine and vinegar in silver bottles to prevent spoiling. In the early 1990’s silver gained regulatory approval as an anti-microbial agent (Kumar Reddy et al., 2012) and people would put silver coins in milk bottles to prolong the milks freshness (Nowack et al., 2011). Hippocrates, the father of medicine wrote that silver had beneficial healing and antimicrobial properties. In recent days, a number of living organisms are already well known to elaborate nanostructured composites such as cynobacteria, bacteria, fungi, actinomycetes, biomolecules and various plant materials such as Tritium vulgare (Armendariz et al., 2009), Svensonia hyderobadensis (Lingarao and Savithramma, 2011), Psidium guajava (Prasad et al., 2011), Boswellia ovalifoliolata and Shorea tumbuggaia (Savithramma et al., 2011), Ocimum tenuiflorum (Patil et al., 2012), Tribulus terrestris (Gopinatha et al., 2012), Artemisia nilagirica (Vijayakumar et al., 2013), Shorea tumbuggaia (Venkateswarlu et al., 2010) and Thespesia populnea (Bhumi et al., 2013).

In the present study we explored the biosynthesis of silver nanoparticles using leaves from Allamanda cathartica, a widely growing perennial ornamental shrub belonging to the family Apocynaceae and also commonly known as the Yellow Bell, Golden Trumpet or The Buttercup flower. It can grow up to a height of 15 feet tall or more is native to Brazil but widely cultivated throughout the world. The plant is used to relieve cough and made into decoctions for use as a purgative. This plant has anti-bacterial and anti-canerous properties, it was also widely used in the treatment of jaundice. The root and stem of this plant contain two rare lactones which are active against polio virus and pathogenic fungi. Root is also used in various formulations to treat malarial symptoms. The leaves, stem and branches of this plant are used against Snake bite (Gomes et al., 2010). Moreover, all the plant parts are reported to be poisonous and hence the plant has not been extensively used in medicine. Therefore, the present study was aimed to the synthesis of silver nanoparticles and tested their antimicrobial activity of A. cathartica.

Material and Methods

Plant Material and Preparation of the Extract Fresh and healthy leaves were collected from

Tirumala hills of Chittoor District, Andhra Pradesh, India during the year 2012. Primarily the leaves were washed, cleaned and pressed with blotted paper. Then the leaves were shade dried and ground to make a fine powder. 5 g of powder were taken into 250 ml conical flask and added 100 ml of sterile distilled water and boiled for 10 minutes at 1000C. Then the leaf extract was collected in separate conical flask by standard filtration method.

Synthesis of silver nanoparticles

1 mM AgNO3 solution was prepared and stored in amber color bottle. The leaf extract was added to 1 mM AgNO3 solution. The color change of the solution from yellow to brown indicated the silver nanoparticles were synthesized from the leaf for the characterization of silver nanoparticles and antimicrobial activity.

UV-Vis spectra analysis

The reduction of pure silver ions was monitored by measuring the UV-Vis spectrum of the reaction medium at 5 h after diluting a small aliquot of the sample into distilled water. UV-Vis spectral analysis was done by using UV-Vis spectrophotometer UV- 2450 (Shimadzu).

SEM analysis of silver nanoparticles

Scanning electron microscope (SEM) analysis was carried out by using Hitachi S-4500 SEM machine. Thin films of the sample were prepared on a carbon coated copper grid by just dropping a very small amount of the sample on the grid, extra solution was removed using a blotting paper and then the film on the SEM grid was allowed to dry.

EDAX measurements

In order to carry out EDAX analysis, the drop of leaf extract with reduced silver nanoparticles was dried on coated with carbon film and performed on Hitachi S-3400 N SEM instrument equipped with thermo EDAX attachments.

AFM measurements

The silver nanoparticles extracted through above protocol were visualized with an atomic force microscope. A thin film of the sample was prepared on a glass slide by dropping 100 micro liters of the sample on the slide were allowed to dry for 5 min, the slides were then scanned with the AFM (Nano Surf ® AG, Switzerland, Product: BTO2089, BRO).

Antimicrobial Activity Microorganisms

Pure culture of Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Proteus vulgaris and Klebsiella pneumoneae species of bacteria and Fusarium oxysporum, Curvularia lunata, Rhizopus arrhizus, Aspergillus niger and Aspergillus flavus species of fungi were procured from the Department of Microbiology of Sri Venkateswara Institute of Medical Science (SVIMS). The experiments of antimicrobial activity were carried out in the Department of Microbiology, Sri Venkateswara University, Tirupati, Andhra Pradesh, India.

Antibacterial activity

The antibacterial activity of SNPs was carried out by disc diffusion method (Bauer et al., 1996). Nutrient agar medium plates were prepared, sterilized and solidified. After solidification bacterial cultures were swabbed on these plates. The sterile discs were dipped in silver

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nanoparticles solution (10 μg/ml) and placed in the nutrient agar plate and kept for incubation at 370C for 24 h. Zones of inhibition for control, SNPs and silver nitrate were measured. The experiments were repeated thrice and mean values of zone diameter were presented.

Antifungal activity

Potato dextrose agar plates were prepared, sterilized and solidified, after solidification fungal cultures were swabbed on these plates. The sterile discs were dipped in silver nanoparticles solution (10μg/ml) and placed in the agar plate and kept for incubation for 7 days. After 7 days zone of inhibition was measured.

Results and Discussion In the present study, silver nanopaticles were

synthesized by using aqueous leaf extract of Allamanda cathartica within 10 to 20 min of incubation period yellowish brown colour was developed rapidly by addition of Ag (NO3)2 to the extract. Change of colour has been ranging from light yellow to thick brown within 10 to 15 min in Allamanda cathartica (Fig.1). The signatory brown colour was obtained which resulted due to the excitation of the Surface Plasmon Resonance vibrations of the silver nanoparticles formed, similar results were observed in various plants studied by Savithramma et al. (2011); Lingarao and Savithramma (2012); Ankanna et al. (2010). The time duration of change in colour and thickness of the colour varies from plant to plant. The reason could be that the quantitative variation in the formation of SNPs (or) availability of H+ ions to reduce the silver. Silver nitrate is used as reducing agent as silver has distinctive properties such as good conductivity, catalytic and chemical stability. The aqueous silver ions when exposed to herbal extracts were reduced in solution, there by leading to the formation of silver hydrosol. The biomolecules found in plants induce the reduction of Ag+ ions from silver nitrate to silver nanoparticles (SNPs). The process of reduction is extracellular and fast which may lead to the development of easy biosynthesis of silver nanoparticles. Plants produce large amount of H+ ions during glycolysis along with NAD which acts as a strong redoxing agent, this seems to be responsible for the formation of SNPs. Water soluble anti oxidative agents like ascorbic acid further seems to be responsible for the reduction of SNPs, biomolecules like proteins, phenols and flavonoids not only play in reducing the ions to the nano size, but also play an important role in the capping of the nanoparticles (Jagadeesh et al., 2004).

Plants fix CO2 and in the presence of sunlight carbohydrates are the first cellular constituents formed by the photosynthesizing organisms. These carbohydrates are utilized by the cell as glucose through glycolysis. The free energy released in this process is used to form the high energy compounds, ATP and NADPH. Large amount of H+ ions are produced along with ATP. NADP+ is a coenzyme found in all living cells acting as NADP strong reducing agent which involves in redox reactions, carrying electrons from one reaction to

another. NADP+ is an oxidizing agent it accepts electron from other molecules and becomes reduced, this reaction forms NADPH can donate electrons, these electron transfer reactions are the main function of NADP. NADP+ keeps on getting reoxidised and gets constantly regenerated due to redox reactions this might have led to transformations of Ag ions to Ago. Another mechanism for the reduction of Ag ions to silver could be due to the presence of water soluble anti-oxidative substances like ascorbate. This acid is present at high levels in all parts of plants. Ascorbic acid is a reducing agent and can reduce, thereby neutralize, reactive oxygen species leading to the formation of ascorbate radical and an electron. This free electron reduces the Ag+ ion to Ag0.

Fig. 1. Synthesis of SNPs (colour change) by using leaf of Allamanda cathartica. The synthesis (i) Control (leaf extract), (ii) Formation of SNPs (plant extract and AgNO3 solution)

The synthesis of SNPs had been confirmed by measuring the UV-Vis spectrum of the reaction media. The UV-Vis spectrum of colloidal solutions of SNPs synthesized from aqueous leaf extract of Allamanda cathartica has the characteristic absorbance peaks ranging from 320 to 400 (Fig.2). The broadening of peak indicated that the particles are poly-dispersed. The weak absorption peak at shorter wavelengths due to the presence of several organic compounds which are known to interact with silver ions; same results were observed in Boswellia ovalifoliolata (Ankanna et al., 2010; Savithramma et al., 2011). The reaction could easily be tracked by the change in color and reconfirmed by UV-VIS spectroscopy. An absorption band at 270 nm is attributed to the aromatic amino acids of proteins. It is well known that the absorption band at 270 nm arises due to electronic excitations in tryptophan and tyrosine residues in the proteins. This observation indicates the release of proteins into solution by Allamanda cathartica and suggests a possible mechanism for the reduction of the metal ions present in the solution.

Linga Rao et al: Green Synthesis of Silver Nanoparticles by A. cathartica L. Leaf Extract and Evaluation for Antimicrobial… 2263  

SEM images of SNPs derived from the aqueous leaf extracts of Allamanda cathartica showed the particles in spherical shape and size ranged from 19.00 nm to 35.4 nm in 500 nm image (Fig.3), in 300 nm image shows the size range from 20 to 40 nm (Fig.3). The morphology of the SNPs was predominantly spherical and they appear to be monodisperse. Further analysis of the silver particles by Energy Dispersive Spectroscopy confirmed

the presence of the signal characteristic of silver (Fig.4). All the peaks of Ag are observed and are assigned. Peaks of Ag are from the grid used and the peaks of S, P and N correspond to the protein capping over the AgNPs. EDAX information has given the various elements along with SNPs of leaf extracts of Allamanda cathartica was identified elements like C, O, Mg, Si, Ag and Al with different percentages (Table 1).

Fig. 2. UV-Visible spectra analysis of silver nanoparticles of Allamanda cathartica.

Fig. 3. SEM images of silver nanoparticles of Allamanda cathartica.

(a) SEM image at 500 nm and (b) SEM image at 300 nm

Fig. 4. EDAX images of silver nanoparticles of Allamanda cathartica.

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TABLE 1

Energy dispersive analysis of X-rays (EDAX) of synthesized elements of Allamanda cathartica

Plant name Allamanda cathartica

Element Weight Atomic (%)

CK 22.60 62.68

OK 06.75 14.07

MgK 01.54 02.12

SiK 00.19 00.23

AgL 66.22 20.45

AuL 02.70 00.46

AFM analysis the SNPs were clearly distinguishable owing to their size difference. AFM images has given average sizes of SNPs of Allamanda cathartica is 26.3 nm with three dimensional structures. SNPs attached with one another and looks like a cluster in an area of 15 μm with rod shape in 3D view (Fig.5). The physicochemical properties of nanoparticles differ dramatically from fine particles of the same composition (Geraci and Castranova, 2010).

Antimicrobial activity

In the present study the antimicrobial activity of silver nanoparticles was carried out against various pathogenic microbes such as gram negative and gram positive bacteria of E. coli, K. pneumoniae, P. vulgaris, P. aeruginosa and Bacillus subtilis, fungal species of Aspergillus niger, Aspergillus flavus, Fusarium, Curvularia and Rhizopus by using disc diffusion method. The extraction without silver nanoparticles served as control. Leaf aqueous extract of Allmanda cathartica showed broad spectrum of antimicrobial activity. The diameter of inhibition zone around each disc with SNPs is represented and each disc contains of 20 μl of SNPs solution (Table 2). The SNPs of leaf extract of Allmanda chatartica showed highest antibacterial activity against Pseudomonas followed by Klebsiella, Bacillus, E. coli and lowest activity towards Proteus and maximum inhibition zone was observed against fungal species of Rhizopus followed by Curvularia, Aspergillus flavus, and Aspergillus niger and minimum inhibition zone was observed against Fusarium (Fig.6 and Fig.7).

Fig. 5. AFM image of silver nanoparticles of Allamanda cathartica.

Linga Rao et al: Green Synthesis of Silver Nanoparticles by A. cathartica L. Leaf Extract and Evaluation for Antimicrobial… 2265  TABLE 2

Antimicrobial activity of leaf aqueous extract and silver nanoparticles of Allmanda cathartica

S. No. Bacterial species Standard

(Streptomycin/ Nystatin) Allmanda cathartica

Control SNPs AgNO3 1. Bacillus 20.0±1.0 6.33±0.57 8.33±0.57 13.53±0.57 2. E.coli 25.33±0.57 7.33±1.15 7.30±2.51 12.86±0.57 3. Klebsiella 21.56±1.15 4.50±1.0 9.56±3.51 16.66±0.57 4. Proteus 30.33±2.88 6.80±0.57 6.00±2.0 17.72±0.57 5. Pseudomonas 30.25±4.0 6.80±1.15 16.0±2.0 18.43±0.57

Fungal species 6. Aspergillus flavus 11.20±1.15 5.89±1.15 11.52±1.52 12.23±1.0 7. Aspergillus niger 25.30±1.15 5.68±1.15 10.08±2.0 6.35±1.15 8. Curvularia 14.60±0.57 6.00±1.15 12.23±2.0 7.42±1.15 9. Fusarium 16.22±0.57 5.28±0.57 6.78±2.30 7.12±1.15

10. Rhizopus 30.33±1.15 4.78±1.15 13.22±3.05 6.39±1.0

Note: ‘±’ indicates standard deviation

Fig. 6. Antimicrobial activity of Allamanda cathartica.

1. Plant aqueous extrat, 2. Silver nano-particles, 3. Silver nitrate, 4. Streptomycin / Nystatin

a. Proteus, b. E. coli, c. Bacillus, d. Klebsiella, e. Pseudomonas, f. Aspergillus flavus, g. Aspergillus niger, h. Fusarium, i. Curvularia and j. Rhizopus

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Fig. 7. Antimicrobial activity of Allamanda cathartica.

At low concentrations SNPs could prolonged the lag phase unit the concentration of SNPs was up to 40 μg/ml (Rajesh et al., 2012). The inhibitory effect of silver is probably the sum of distinct mechanisms of action. Some studies reported that silver ions react with SH groups of proteins (Liau et al., 1997; Feng et al., 2000) and play an essential role in bacterial inactivation (Morones et al., 2005). It is also reported to uncouple respiratory electron transport from oxidative phosphorylation which inhibits respiratory chain enzymes or interferes with membrane permeability to protons and phosphate (Feng et al., 2000). The presence of silver ions and sulphur in the electron dense granules observed after silver ion treatment in the cytoplasm of bacterial cells suggests an interaction with nucleic acids that probably results in the impairment of DNA replication (Feng et al., 2000). Thus, it is reasonable to that the biosynthesized nanosilver can be used to manage the disease caused by X. canpestris pv. malvacearum in cotton plant (Rajesh et al., 2012). Li et al. (2010) reported that the antibacterial mechanism of SNPs towards E. coli as a model organism and the leakage of proteins and reducing sugars at 2 h of exposure to 100 μg/ml of SNPs.

Recently, proteomics analysis revealed that even a short exposure of E. coli to nanosilver resulted in alterations in the expression of panel of envelope and Heat shock proteins (HSP) (Lok, 2006). Therefore, these particles can penetrate and can disrupt the membranes of bacteria. A massive loss of intracellular potassium was induced by nanosilver. Furthermore, the molecular targets for the nanosilver could be protein thiol groups (respiratory enzymes). The phospholipid portion of the bacterial membrane may also be the site of action for the nanosilver (Rajesh et al., 2012).

The SNPs synthesized via green route are highly toxic towards bacterial strains when compared to fungal strains. Silver ions have been demonstrated to interact with the protein and possibly phospholipids associated with the proton pump of bacterial membranes (Savithramma et al., 2012). These results in a collapse of membrane proton gradient causing a disruption of many of the mechanisms of cellular metabolisms and hence cell death (Dibrov et al., 2002). Silver ions interact with a wide range of molecular processes within a microorganisms resulting in a range of effects from inhibition of growth and loss of ineffectiveness. The mechanism depends on both the concentration of silver ions present and the sensitivity of the microbial species to silver. The spectrum of activity is very wide and the development of resistance relatively low (Cooper, 2004). The use of plant extracts is an effective against various microorganisms including plant pathogens (Mishra et al., 2007).

The growth of microorganisms was inhibited by the green synthesized SNPs may be due to the presence of peptidoglycon, which is a complex structure and after contains teichoic acids or lipoteichoic acids which have a strong negative charge. This change may contribute to the sequestration of free silver ions (Savithramma et al., 2012).

The findings of Sreemaspum et al. (2008) suggested that the inhibition of oxidation based on biological process by penetration of metallic nanosized particles across the microsomal membrane. SNPs would interfere with the bacterial growth signaling pathway by modulating tyrosine phosphorylation of putative peptide substrates critical for cell viability and division (Srivastava et al., 2007). When compared with AgNO3

Linga Rao et al: Green Synthesis of Silver Nanoparticles by A. cathartica L. Leaf Extract and Evaluation for Antimicrobial… 2267  the SNPs have more toxicity towards selected microorganisms. The reason could be that the silver has more microbial efficacy and more effective in the presence of proteinaceous material.

World Health Organization (WHO) banned many agriculturally important pesticides due to wide range of toxicity against human (Barnard et al., 1997). Thus, there is an urgent need to search for other native methods for prevention of bio-determination without any toxicity to the consumer. Eco-friendly fungicides and bactericides can prepare through nano-biotechnology. Biologically synthesized silver nanoparticles are more toxic to pathogens. The findings of the present study are an important step towards a proof of validation of Allamanda cathartica as its nanoparticles are showed antibacterial and antifungal activity.

In the present scenario the bacterial and fungal strains are getting resistance to traditional and standardized drugs. It is inevitable for finding new drugs for curing ailments caused by microorganisms. The silver nanoparticles of Allamanda cathartica appear to serve as effective compounds for resistant microbial strains.

Conclusions

In conclusion, we found that Allamanda cathartica is good source for synthesis of silver nanoparticles. Applications of eco-friendly nanoparticles as bactericidal, fungicidal, wound healing and other medical and electronic applications. Toxicity studies of silver nanoparticles on human pathogen opens a door for a new range of antibacterial agent. The reduction of Ag+ ions and stabilization of silver nanoparticles are thought to occur through participation of plant proteins and metabolites. The reaction is simple and convenient to handle, and it is believed that it has advantages over other biological synthesis.

Acknowledgments

Authors are thankful to Dr. Ch. Paramageethama, Department of Microbiology, S.V. University, Tirupati and SAIF, IIT Madras for their help in experiments.

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Address correspondence to: M. Linga Rao, Research Scholar, Department of Botany, Sri Venkateswara University, Tirupati – 517 502, Andhra Pradesh, India. Mob: 09963214820; E-mail: matti2010rao@gmail.com