Paper 5 - Vernonia Nano

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Vernonia cinerea (L.) Less. silver nanocompositeand its antibacterial activity against a cotton pathogen

K. Sahayaraj M. Roobadevi S. Rajesh S. Azizi

Received: 4 February 2014 / Accepted: 15 April 2014 Springer Science+Business Media Dordrecht 2014

Abstract Noble-metal nanomaterials are of particular interest today because of their applications in many areas, including agriculture. The latter topic is one of themost active areas of research in metal nanomaterials. Metal nanoparticles are tra-ditionally synthesized by wet chemical techniques, in which the chemicals used areoften toxic and ammable. We report here biosynthesis of silver nanoparticles usingleaf extract of Vernonia cinerea (L.) Less. (Asteraceae). Treatment of aqueoussolution of AgNO 3 with V. cinerea leaf extract resulted in rapid formation of stablesilver nanoparticles. The growth of nanoparticles was monitored by UVVisiblespectrophotometry complemented by characterization using transmission electronmicroscopy (TEM), X-ray diffraction analysis, and Fourier-transform infraredspectroscopy. A feasible mechanism for the formation of nanomaterial and thedifference in the reduction time for silver nanoparticle synthesis is discussed. TEManalysis revealed the presence of polydisperse silver nanoparticles with average sizeof 550 nm. X-ray diffraction studies corroborated that the biosynthesized nano-particles were crystalline silver. Furthermore, this green biogenic approach is a

rapid and simple alternative to chemical synthesis. The biologically synthesizedsilver nanoparticles were found to be highly effective against Xanthomonascampestris pv. malvacearum (13.00 0.58 mm) with minimum inhibitory

K. Sahayaraj ( & ) S. RajeshCrop Protection Research Centre, Department of Advanced Zoology and Biotechnology,St. Xaviers College (Autonomous), Palayamkottai 627 002, Tamil Nadu, Indiae-mail: [email protected]

M. RoobadeviDepartment of Biotechnology, Nandha Arts and Science College, Erode 638052, Tamil Nadu, India

S. AziziDepartment of Chemistry, Faculty of Science, University Putra Malaysia, 43400 Serdang, Selangor,Malaysia

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concentration of 80 l g/mL. Hence, such biosynthesized silver nanoparticles can beused in control of cotton bacterial blight.

Keywords Vernonia cinerea Xanthomonas campestris pv. malvacearum

Silver nanoparticles Antibacterial MIC

Introduction

The future success of nanotechnology is akin to capturing a wild horse: powerfuland full of potential, but it must be tamed before it becomes useful. The taming of any beast requires deep understanding of the basic fundamental traits that govern itsbehavior, which for a nanomaterial is primarily a combination of its composition,

size, and shape. To advance nanotechnology for antimicrobial applications,bioengineered devices, and high-speed electronics, development of methods tounderstand and control the behavior of nanomaterials is needed. A nanomaterialmay be dened as any material (insulator, conductor or semiconductor), which hasbeen controllably synthesized in the size range of roughly 1100 nm. At this sizeand dimensional range, essentially any material will exhibit properties that aredifferent from those that it would show as an atomic cluster or as a larger, bulk material [ 1].

Production of nanoparticles can be achieved through different methods, among

which chemical approaches are the most popular. However, some chemical methodscannot avoid the use of toxic chemicals in the synthesis protocol. Sincenanoparticles of noble metals such as gold, silver, and platinum are widely appliedin contact with humans, there is a growing need to develop environmentally friendlyprocesses for nanoparticle synthesis that do not use toxic chemicals. Biologicalmethods of nanoparticle synthesis using microorganisms [ 24], enzymes [ 5], andplant or plant extracts [ 6] have been suggested as possible ecofriendly alternatives tochemical and physical methods of nanoparticle synthesis. Such methods can also besuitably scaled up for large-scale synthesis of nanoparticles [ 7]. However, the major

drawback of using microbes for bioreduction is the difculty in maintaining asepticconditions, which not only requires technical effort but also greatly increasesproduction costs to a high level at the industrial level.

Only in recent years have biosynthetic methods employing plant extractsreceived some attention as a simple and viable alternative to chemical procedures orphysical methods for synthesizing metal nanoparticles. Gardea-Torresdey et al. [ 8]rst reported the formation of gold and silver nanoparticles by living plants.Extracellular nanoparticle synthesis using plant leaf extracts rather than wholeplants would be more economical owing to the easier downstream processing.Shankar et al. [ 6] reported synthesis of pure metallic silver and gold nanoparticlesby reduction of Ag ? and Au ? ions using neem ( Azadirachta indica ) leaf broth.There have been recent reports on photosynthesis of silver and gold nanoparticles byemploying lemon grass extract [ 6], Aloe vera plant extract [ 9], Murraya koenigii[10], green tea ( Camellia sinensis ) [11 ], coriander leaves [ 12], sun-dried

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Cinnamomum camphora leaves [ 13], Cinnamon zeylanicum [14], phyllanthinextract [ 15], bioactive principle of henna leaves (apiin) [ 16], Acalypha indica [17],Curcuma longa [18], Hibiscus rosa-sinensis [19], Panicum virgatum [20], and Rosarugosa [21]. Green synthesis is very efcient for production of silver nanoparticles

because of its simplicity, accuracy, and cost-effectiveness, in addition to its greenchemistry perspective and agricultural and biomedical applications.

The antimicrobial activity of silver and its compounds has been exploitedworldwide [ 2224]. In addition, silver is known to exhibit an oligodynamic effectbecause of its ability to exert bactericidal activity even at minimum concentrations[25]. Development of resistance to silver in microbes is improbable due to its actionon a broad spectrum of targets in the cell [ 26]. The advantage of silver nanoparticlesover bulk metals or salts is the slow and regulated release of silver fromnanoparticles, thereby providing long-lasting protection against bacteria [ 27]. There

are various reasons for considering silver nanoparticles (AgNPs) as a universalmicrobicidal agent [ 2830].

Vernonia cinerea (L.) Less. (Asteraceae) is a slender-stemmed plant withvariable leaf shape and pinkish-purple owers, distributed in grassy areas of Southeast Asia and Hawaii. It has been documented and recommended in Thaitraditional medicine, as in other countries, for smoking cessation and relief of asthma, cough, fever, malaria, urinary calculi, and arthritis [ 30]. Active compoundof V. cinerea showed anti-inammatory, analgesic, and antipyretic activities [ 31],and anti-oxidant and anti-inammatory activity [ 32]. Furthermore, the methanol

extract [ 33] and benzene extract fraction [ 34] of V. cinerea exhibited anti-inammatory and antibacterial activity, respectively.The genus Xanthomonas (Proteobacteria) is a diverse and economically

important group of Gram-negative bacterial pathogens. Bacterial blight caused by Xanthomonas campestris pv. malvacearum (Smith) Dye (synonym Xanthomonasaxonopodis pv. malvacearum ) [35, 36] has become an increasing problem in cottonproduction worldwide. The focus of the present study is the synthesis of silvernanoparticles using aqueous extract of V. cinerea for the rst time, and theircharacterization. In addition, we recorded the antibacterial activity of the preparednanoparticles against a Gram-negative bacterium, X. campestris , an importantpathogen of the cotton plant. To the best of our knowledge, this is the rst report onthe use of V. cinerea as a biological system for synthesis of silver nanoparticles forcotton pathogen management.

Materials and methods

Materials

Silver nitrate was obtained from Hi-Media, Mumbai. All glassware was washedwith distilled water and oven-dried before use. Fresh aerial part of V. cinerea wascollected from St. Xaviers College campus (8.7166 N, 77.7333 E), Palayamkottai,identied, and deposited in St. Xaviers College Herbarium (XCH no. 25483) forfuture reference.

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Synthesis of silver nanoparticles

Plant leaf broth solution was prepared by taking 5 g thoroughly washed and nelycut leaves in a 300-mL Erlenmeyer ask with 100 mL sterile distilled water, then

boiling the mixture for 20 min before nally decanting it. Typically, 0.1 mL lteredleaf broth was added to 100 mL 10 - 3 M aqueous AgNO 3 solution for reduction of Ag ? ions at room temperature. The color of the solution changed from light yellowto cream, indicating the formation of silver nanoparticles (AgNPs).

Characterization

UVVisible spectral analysis

Bioreduction of AgNO 3 in aqueous solution was monitored using UVVisiblespectrophotometry at regular intervals. UVVisible spectra were recorded fromsamples in quartz cuvettes at resolution of 1 nm as a function of reaction time usinga Shimadzu UV-1601 spectrophotometer.

Transmission electron microscopy (TEM) and scanning electron microscopy (SEM)analyses

The size and shape of the biosynthesized nanoparticles were observed by TEM and

SEM measurements, respectively. Samples of silver nanoparticles synthesized usingV. cinerea leaf broth for TEM analysis were prepared by placing a drop of nanoparticle solution on a carbon-coated copper grid and allowing water toevaporate. TEM measurements were performed using a JEOL model 3010instrument operated at accelerating voltage of 120 kV. The morphology of thenanoparticles was observed using a JSM-6390 SEM. Samples for SEM analysiswere prepared by drop-coating the Ag nanoparticle solution onto a carbon-coatedcopper grid. The lms on the grids were allowed to dry prior to SEM measurement.

Powder X-ray diffraction

The X-ray diffraction (XRD) pattern of dry nanoparticle powder was obtained usingCu K a radiation (1.5406 A ; 45 kV, 30 mA). The XRD pattern was analyzed todetermine peak intensity, position, and width. The particle size was calculated usingthe following formula:

d 0:9k=b cos h;

where d is the mean diameter of the nanoparticles, k is the wavelength of the X-ray

radiation source, and b is the angular full-width at half-maximum (FWHM) of theXRD peak at diffraction angle h [37]. Powder samples for analysis were prepared bycentrifugation at 13,000 rpm for 15 min followed by redispersion in sterile distilledwater to remove any uncoordinated biological molecules. Centrifugation and redi-spersion were repeated thrice for better separation.

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Fourier-transform infrared (FTIR) spectroscopy analysis

After complete reduction of Ag ? ions by V. cinerea extract, 10 mL solution of silver nanoparticles was centrifuged at 4,000 rpm for 10 min, and the resulting

suspension was redispersed in 20 mL Millipore water; the process of centrifugationand redispersion was repeated three times so that the nanoparticles were free fromproteins or other bioorganic compounds present in the solution. Thereafter, thepuried suspension was completely dried and analyzed by FTIR spectrometer in therange of 4,000 to 400 cm - 1 at resolution of 4 cm - 1 . The FTIR spectra of leaf extract taken before and after synthesis of silver nanoparticles were analyzed andinvestigated for possible functional groups involved in the formation of silvernanoparticles.

Antimicrobial assays

Diffusion method

Xanthomonas campestris pv. malvacearum was isolated from infected cotton plantsand used for the experiment. The pathogen was isolated, subcultured on nutrientagar (HiMedia, India), and identied using standard protocol [ 38]. Antimicrobialactivity was assayed using the agar well diffusion method [ 39]. Petri plates (9 cm)were prepared with 20 mL sterile Mu llerHinton agar (HiMedia, India). Wells were

made using a sterile cork borer under aseptic conditions. V. cinerea leaf extractand synthesized nanoparticles with various concentrations (10, 20, 40, 80, and160 l g/mL) was added to the respective wells. Chloramphenicol (0.1 %; HiMedia,Mumbai, India) and deionized water were used as positive and negative control,respectively. After incubation for 24 h at 37 C, a clear zone around the well wasevidence of antibacterial activity. The diameter of the inhibition zone was measuredin millimeters using a HiMedia ruler. Each test was performed in triplicate.

Determination of minimum inhibitory concentration (MIC)

The broth microdilution assay was used to determine the MIC [ 40]. Test samples(75 l L) of various concentrations (10320 l g/mL) were added to sterile microtiterplates. Bacterial cell suspension (75 l L) corresponding to 1 9 10

8 colony-formingunits (CFU)/mL was added in all wells except control. Control well contained steriledistilled water and broth to check sterility, while the negative control wells werelled with nutrient broth and bacterial suspension to check for adequacy of the brothto support bacterial growth. The plates were placed in a sterile Petri plate andincubated at 37 C for 24 h. To indicate bacterial growth, 40 l L 0.2 mg/mL p-iodonitrotetrazolium chloride (INT, HiMedia) was added to each well, followed byincubation for another 30 min [ 40]. The colorless tetrazolium salt acts as an electronacceptor and is reduced to a red-colored formazan product by biologically activeorganisms. Inhibition of bacterial growth was indicated by a clear colorless well,and the presence of growth was detected by the presence of pinkred color. Thelowest concentration showing no color change was considered as the MIC.


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Results and discussion

We present a detailed study on extracellular synthesis of silver nanoparticles byV. cinerea aqueous extract and the antibacterial effect on a cotton-infecting

phytopathogen. It is generally recognized that UVVisible spectroscopy can be usedto examine size- and shape-controlled nanoparticles in aqueous suspension [ 41].Figure 1 shows the ltrate of V. cinerea biomass with Ag ? ions at the initial timepoint and at the end point after 1 h of reaction. After addition of the biomass, thesilver nitrate solution changed from colorless to pale yellow in about 30 min, thenal color deepening to cream or yellowish-brown within 1 h. Silver nanoparticles(AgNPs) have free electrons, which give rise to a surface plasmon resonance (SPR)absorption band [ 42], due to the combined vibration of electrons of the silvernanoparticles in resonance with the light wave [ 43, 44].

The synthesized silver nanoparticles exhibited an absorption maximum at430 nm in the visible region (Fig. 2) with yellowish-brown or cream color [ 4548].Due to the excitation of the plasmon resonances of interband transitions, somemetallic nanoparticle dispersions exhibit unique bands/peaks [ 49]. The broadness of the peak is a good indicator of the nanoparticle size. As the particle size increases,the peak becomes narrower with decreased bandwidth and increased band intensity[45, 47]. Moreover, the possible explanation for the difference in the reduction timecould be due to the difference in the reduction potential for both metal ions. Silvernanoparticles synthesized using V. cinerea were stable for more than 6 months

when stored at room temperature (2930

C).SEM analysis of the biologically synthesized silver nanoparticles clearly showeda particle size ranging from 5 to 50 nm (Fig. 3) and that the silver nanoparticleswere deposited on the surface. High-resolution transmission electron microscopy(HR-TEM) provided further insight into the morphology and size details of thesilver nanoparticles. HR-TEM micrographs were recorded from drop-coated lmsof silver nanoparticles synthesized by treating silver nitrate solution with plantextract for 1 h. Though shape variation was noticed, the majority of thenanoparticles were found to be spherical and polydisperse under HR-TEM. HR-TEM also conrmed the size of the nanoparticles to be in the range of 550 nm(Fig. 4). Similarly, silver nanoparticles synthesized using neem [ 6] and black tealeaf extract [ 50] were spherical and polydisperse, as observed for V. cinerea .

The XRD spectrum of the silver nanoparticles is shown in Fig. 5 and conrmsthe formation of metallic silver. The AgNPs showed diffraction peaks characteristicof metallic face-centered cubic silver phase at 2 h values of 38.08 (111), 44.17(200), 64.41 (220), and 77.34 (311). The average crystal size of the AgNPs wascalculated by applying the mentioned equation [ 37] and was found to be 33 nm.Hence, from the XRD pattern it was clear that the silver nanoparticles formed usingV. cinerea broth were essentially crystalline in nature.

FTIR measurements were carried out to identify the possible biomoleculesresponsible for the reduction of the Ag ? ions and capping of the bioreduced AgNPssynthesized by V. cinerea . The FTIR spectra of untreated and treated plant extractsamples containing AgNPs are depicted in Fig. 6a and b, respectively. The observedpeaks are more characteristic of tannins, which are abundant in V. cinerea extracts

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[33]. Generally, at alkaline pH, tannins are hydrolyzed to glucose and gallic acidunits [ 51]. At alkaline pH, gallic acid rapidly reduces silver nitrate to silvernanoparticles at room temperature, but the particles form aggregates in solution, asgallic acid is a poor stabilizing agent. The FTIR characterization after the reaction

of V. cinerea with Ag?

indicated that glucose might be responsible for thestabilization of the silver nanoparticles. Glucose is a weak reducing agent at roomtemperature, but a good stabilizing agent at alkaline pH [ 51]. In the FTIR spectrum,several absorption peaks were found centered at 666, 1,352, 1,633, 2,078, 2,432, and3,431 cm - 1 , lying in the range 6003,500 cm - 1 (Fig. 6b). The widest spectral

Fig. 1 Synthesis of silver nanoparticles using aqueous extract of Vernonia cinerea : a 10- 3 M AgNO 3 ,b initial point of time, c nal point of time

Fig. 2 UVvisible absorption spectra recorded as a function of time for silver nanoparticles obtainedusing aqueous extract of Vernonia cinerea aqueous extract: a 30 min, b 1 h, c 24 h


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absorption was observed at 1,633 (amide I, C=O groups) and 3,431 cm - 1 (OHstretching), whereas the absorption peaks centered at 1,515 and 1,540 cm - 1 can beattributed to stretching vibration of C=C (aromatic ring). A weak OH in-planebend of phenol (1,380 cm - 1 ) was lost due to capping of the plant extracts. Thesignal at 1,380 cm - 1 is characteristic of phenol functional group, as reported byPoljans ek and Matjaz Krajnc [52]. The various functional groups referred to aboveare mainly derived from heterocyclic compounds, which are water-solublecomponents of V. cinerea . So, it can be assumed that different water-solubleheterocyclic compounds such as tannins worked as the capping ligand during the

synthesis of silver nanoparticles and the presence of oxygen atoms helped in thestabilization of nanoparticles by facilitating absorption of heterocyclic compoundson the nanoparticles.

The antibacterial activity results for V. cinerea (VC) and V. cinerea biosynthesizedsilver nanoparticles (VCBSNs) against X. campestris are presented in Table 1. The

Fig. 3 Representative scanningelectron micrography imageillustrating silver nanoparticlesbiologically synthesized byreduction of Ag ? ions using

aqueous extract of Vernoniacinerea aqueous extract

Fig. 4 Representative TEM images illustrating silver nanoparticles biologically synthesized by reductionof Ag ? ions using aqueous extract of Vernonia cinerea aqueous extract: a 10 nm, b 20 nm

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crude extract showed no activity against the tested pathogen, whereas the VCBSNssignicantly ( P \ 0.05) inhibited growth of the pathogen (13.00 0.58 mm). Theminimum inhibitory concentration (MIC) was found to be 80 l g/mL. The mechanismof the bactericidal action of silver nanoparticles remains under debate and is not wellunderstood, but is closely associated with the formation of pits in the bacterial cellwall, leading to increased membrane permeability and resulting in cell death [ 53]. Thebactericidal action of silver ions results primarily from their interaction with the

Fig. 5 X-ray diffraction (XRD) spectrum of silver nanoparticles synthesized by reduction of Ag ? ionsusing aqueous extract of Vernonia cinerea aqueous extract

Fig. 6 FTIR spectra of a Vernonia cinerea aqueous extract and b silver nanoparticles synthesized byusing V. cinerea extract


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cytoplasm in the interior of the cell. Silver ions appear to penetrate through ionchannels without causing damage to the cell membrane; they then denature theribosome and suppress the expression of enzymes and proteins essential to productionof adenosine triphosphate (ATP), resulting in cell disruption [ 18, 54]. In the cell,silver ions may deactivate cellular enzymes and deoxyribonucleic acid (DNA) byreacting with electron-donating groups such as thiol (SH) groups and generatereactive oxygen species (ROS) [ 55, 56]. It has also been hypothesized that Ag ? ionsprimarily affect the function of membrane-bound enzymes, which play a vital role in

the respiratory chain [ 57]. Silver has a greater afnity to react with sulfur- orphosphorus-containing biomolecules in the cell. Thus, sulphur-containing proteins inthe membrane or inside the cells and phosphorus-containing elements such as DNAare likely to be preferential sites for silver nanoparticle binding [ 58, 59]. When silvernanoparticles enter the bacterial cell, they form a low-molecular-weight region in thecenter of the bacteria to which the bacteria thus conglomerates, protecting the DNAfrom the silver ions. The nanoparticles preferably attack the respiratory chain and celldivision, nally leading to cell death [ 53, 6062]. Thus, it is reasonable to infer thatbiologically synthesized AgNPs could be used to manage the disease caused by

X. campestris in cotton plant; there is a high possibility of generating a newantimicrobial agent.

The mean size of the silver nanoparticles interacting with bacteria was 5 2 nm.Particles were found inside the bacterial cell membrane. Furthermore, the particlepenetration was size dependent. Particles with size between 1 and 10 nm onlyinteract preferentially with bacteria. Such particle penetration ability was veriedearlier [ 53]. The inuence of size on antimicrobial activity was investigated byBaker et al. [ 63]. They recorded that the antibacterial properties were related to thetotal surface area of the nanoparticles. Smaller particles with larger surface-to-

volume ratios have greater antibacterial activity [ 64]. The size of VCBSNs rangedfrom 5 nm to 50 nm, thus this smaller size enabled higher activity against X. campestris . The surface plasmon resonance plays a major role in the opticalabsorption spectra of silver nanoparticles, shifting to longer wavelength withincreasing particle size. The small size of nanoparticles implies that they have a

Table 1 Antibacterial activity of aqueous silver nanoparticles biologically synthesized by V. cinereaagainst Xanthomonas campestris pv. malvacearum

Concentration ( l g/mL) VC VCBSNs

10 0.00 0.0020 0.00 0.00

40 0.00 7.67 0.34a

80 0.00 9.67 0.34a

160 0.00 13.00 0.58a

Positive control 22.34 0.89

Positive control 0.1 % chloramphenicol, VC V. cinerea aqueous extract, VCBSNs V. cinerea biosyn-thesized silver nanoparticlesa Signicant at 5 % level using Tukeys test

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large surface area to come into contact with the bacterial cells and hence a higherpercentage of interaction than for larger particles [ 61, 6569]. The VCBSNs werespherical and polydisperse. It required 80 l g/mL of nanoparticles to inhibit X. campestris . Thus, silver nanoparticles with different shapes have different effects

on bacterial cells.

Conclusions

Nanotechnology is emerging as a eld of applied science and technology. Synthesisof nanoparticles can be achieved by various physical and chemical methods, butbiological systems are gaining attention as an ecofriendly technique. Thebiosynthetic method employing plant parts has proved to be a simple and cost-

effective method for synthesis of nanoparticles. We demonstrated that use of anatural, low-cost biological reducing agent, namely V. cinerea aqueous extract, canproduce silver nanostructures through an efcient green nanochemistry methodol-ogy, avoiding the use of hazardous and toxic solvents and other synthetic chemicals.The nanoparticles were characterized using UVVisible spectrophotometry, XRD,FTIR, SEM, and TEM analyses. The biologically synthesized silver nanoparticlesshowed promising antimicrobial activity against X. campestris , an economicallyimportant pathogen of cotton plant causing severe yield loss worldwide. Theaforementioned advantages of the aqueous plant-based silver NPs make them ideal

for use in green industrial, medicinal, microbiological, agricultural, and otherapplications. The present biological synthesis is a simple, green, rapid, and low-costapproach for producing silver nanoparticles in the laboratory.

Acknowledgments Senior author K.S. acknowledges nancial support from the Ministry of EarthScience, New Delhi (ref. no. MRDF/01/33/P/07) provided for this work. We would like to thank the DSTunit of Nanoscience IIT, Madras for TEM analysis and Karunya University, Coimbatore for XRD andSEM analyses.

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Paper 5 - Vernonia Nano