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Colloids and Surfaces B: Biointerfaces 109 (2013) 20– 24

Contents lists available at SciVerse ScienceDirect

Colloids and Surfaces B: Biointerfaces

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ntimicrobial activity of Leucas aspera engineered silver nanoparticlesgainst Aeromonas hydrophila in infected Catla catla

acob Joe Antonya,1, Murugaiyan Nivedheethaa,1, Durairaj Sivaa,1,anesapandy Pradeephaa, Palanivel Kokilavania, Seenivasan Kalaiselvi a,runachalam Sankarganesha, Athmanathan Balasundaramb, Vivekanandan Masilamanib,hanmugam Achiramana,∗

Department of Environmental Biotechnology, Bharathidasan University, Tiruchirappalli-24, Tamil Nadu, IndiaDepartment of Zoology, Periyar E. V. R college, Tiruchirappalli-23, India

a r t i c l e i n f o

rticle history:eceived 30 November 2012eceived in revised form 6 March 2013ccepted 9 March 2013vailable online xxx

a b s t r a c t

This research describes green synthesis of silver nanoparticles (AgNPs) utilizing Leucas aspera. The syn-thesized nanoparticles were characterized by UV–visible spectroscopy (UV–vis), dynamic light scattering(DLS), transmission electron microscopy (TEM), Fourier transform infra-red spectroscopy (FTIR) andinductively coupled plasmon optical emission spectroscopy (ICP-OES). UV–vis analysis proved the wave-length of the sample to be 429 nm, resembling the surface resonance peak (SPR) specific for AgNPs. DLS

eywords:eucas asperaTIREMeromonas hydrophilaatla catla

analysis indicated particles with superior stability with an average diameter of 189.3 nm. TEM resultsshowed that the particles were in the size range of 29–45 nm. FTIR prediction indicated the presenceof possible polyphenol and protein encapsulates on the AgNPs. Antimicrobial activity of the AgNPs wastested against Aeromonas hydrophila. Catla catla, the model organism used for the experiment was dividedinto six groups with 15 animals in each group. In vivo analysis of biochemical parameters and histologicalarchitecture provided evidence for the antibacterial effect of AgNPs in the fish model.

. Introduction

Silver nanoparticles show diverse physical, chemical, and bio-ogical properties and therefore are a center of attraction forpplication in a wide range of fields [1]. Interactions betweenanoparticles and bacteria occur by various processes such asiosorption, particle aggregation and cellular uptake. This results

n membrane damage and toxicity in the target organism. The tar-et organism is less likely to develop resistance against AgNPsnd the probable mechanism of cell death is by inhibition of DNAeplication or protein inactivation through release of K+ ions. Sizend surface modification of the nanomaterial play a key role in itsntibacterial efficacy [2,3].

An increase in surface energy is created when specific areas of

anoparticles are increased [4]. They have the ability to encapsulate

drug and protect it from degradation [3]

∗ Corresponding author at: Department of Environmental Biotechnology,harathidasan University, Tiruchirappalli-24, Tamil Nadu,

ndia. Tel.: +91 431 2407088; fax: +91 431 2407045.E-mail address: achiramans@gmail.com (S. Achiraman).

1 These authors have contributed equally to this work.

927-7765/$ – see front matter © 2013 Published by Elsevier B.V.ttp://dx.doi.org/10.1016/j.colsurfb.2013.03.020

© 2013 Published by Elsevier B.V.

Hence, nanoparticles are a key tool in drug delivery. Silver isknown to possess antimicrobial activity from ancient times, whichhas formed a base for applying AgNPs as antimicrobial agents[5]. Aeromonas hydrophila, a water borne organism is responsiblefor diarrhea in immunocompromized patients and children [6]. Itis a common opportunistic pathogen that can cause septicemia,ulcer and red- sore disease in fish [7,8]. Previous reports indicatethat the chances of A. hydrophila becoming resistant to commonantibiotics are well-known [9]. Hence, an alternative drug thatpossesses minimal chances of providing the microbe to becomeresistant is required. Since Leucas aspera is known to possess anti-fungal, antioxidant, antimicrobial and cytotoxic activity, it waschosen to fabricate AgNPs, the antimicrobial agent of this study[10].

Fabrication of nanoparticles without toxicity is of paramountimportance and hence it has led to development of green methodsfor synthesis [11]. Though bacteria, actinomycetes, yeast and fungihave been exploited for biosynthesis, use of plants for biosynthe-sis is still largely an unexplored area and hence needs to be utilizedeffectively [12]. It includes various advantages compared to chemi-

cal means of synthesis with respect to cost effectiveness, large scalesynthesis and does not need high temperatures or energy [3]. Todate, there are a lot of reports on green synthesis of AgNPs whichcircles around these above mentioned advantages.

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nanofluid prepared at 95 ◦C for further analysis (Fig. 1). The surfaceplasmon resonance (SPR) of the synthesized AgNPs corresponds tonanomaterials of silver [18]. The sample prepared at 95 ◦C had theplasmon resonance peak in the SPR range of around 450 nm that

J.J. Antony et al. / Colloids and Sur

The novelty of this work is that this is the first ever report onntibacterial activity of AgNPs in fish model, in vivo. There are only

limited number of reports on antibacterial activities in systemsn vivo. AgNPs known to possess toxic effects on microbes are poorlynderstood with regard to its effects on vertebrate models [13].ence, this study would pave way for studies in animal models for

urther research in the near future.

. Material and methods

.1. Preparation of L. aspera bark extract

The plant material (L. aspera) was collected from Tiruchirap-alli, Tamil Nadu, India and confirmed by taxonomists. Barks wereashed thoroughly with distilled water to remove any possible

mpurities. It was shade dried to remove the moisture completely,hopped into small pieces and ground well to a fine powder thatas stored for further experiments.

.2. Preparation of aqueous extract

5 g of fine powder was dissolved in 100 ml of deionized waternd boiled for 10 min at 60 ◦C in a water bath. The mixture was fil-ered using Whatmann No.1 filter paper to get aqueous bark extractt desired concentration, and used for further work.

.3. Biosynthesis of AgNPs

Synthesis of nanoparticles was done as per the procedure fol-owed by Song and Kim, 2009 [3]. 1 mM of Silver nitrate (Qualigens

99.8%) and the filtrate at desired concentration were mixed inhe ratio of 95:5 and heated in water bath at various tempera-ures (60 ◦C, 90 ◦C, 95 ◦C). This heated mixture was centrifuged at0,000 rpm for 10 min. Supernatant was discarded and the pel-

et was resuspended in deionized water and centrifuged again toet rid of uncoordinated biomolecules. These processes were doneepeatedly for thrice to make sure that there is a better separationf the AgNPs.

.4. Characterization of AgNPs

Synthesis of AgNPs by reducing the respective metal ion solu-ion with bark extract was observed by UV–vis spectroscopy. Thebsorption spectra were measured using a Perkin-Elmer Lamda-45pectrophotometer in 200–1100 nm range. TEM analysis was doney using a Tecnai 10 Model instrument operated at an acceleratingoltage at 80 kV. DLS analysis was performed in a Malvern Zetasizeranoseries compact scattering spectrometer (Malvern Instrumentstd., Malvern, UK). FTIR measurements were carried out on a Spec-rum RX 1 model instrument in the diffuse reflectance mode at aesolution of 4 cm−1 with potassium bromide (KBr) pellets recordedn the 4000– 400 nm−1 range. The concentration of AgNPs was esti-

ated by Perkin Elmer Optima 5300 DV ICP-OES model.

.5. Experimental

Catla catla, were divided into 6 groups randomly and each groupontaining 15 fishes. Group I – intact; Group II – normal + AgNPs50 �l); Group III – normal + plant extract (50 �l); Group IV –eromonas induced; Group V – Aeromonas induced + AgNPs (50 �l);roup VI – Aeromonas induced + plant extract (50 �l). Treatmentas initiated three days after inducing the infection in fish with

eromonas and was continued for 15 days. The dose, 50 �l, was pre-icted by in vitro disc diffusion analysis (data not presented). A setf doses such as 25 �l, 50 �l and 75 �l were tested of which minimalnhibitory zone was observed at 50 �l dose and hence was selected

: Biointerfaces 109 (2013) 20– 24 21

for further analysis in vivo. Serum was collected after treatment andbiochemical parameters were performed along with hematologicalassays. Histological architecture was studied by observing sectionsof gills.

2.6. Biochemical parameters

Serum glutamate oxalate transaminase (SGOT), serum glu-tamate pyruvate transaminase (SGPT) and alkaline phosphatase(ALP) were studied for analyzing liver function. Lactate dehydro-genase (LDH) was studied for marking tissue injury. Hematologicalparameters were analyzed in cohesion.

2.7. Histopathology

Gills were excised and fixed with 10% formalin. Section of 5 �mthickness were prepared, stained with hematoxylin, eosin andobserved under microscope at 40× [14].

2.8. Statistics

Results were expressed as mean ± SD. One-way ANOVA was per-formed by SPSS version 17 (SPSS Inc., Chicago, IL, USA) and LSD wasused for individual comparisons [15]. Statistical significance wasset at p < 0.05.

3. Results and discussion

There are three possible routes of conversion of silver nitrateto AgNPs. The secondary metabolites present in the plant mayaccount for the primary cause. The second reason might be bythe energy released during glycolysis during conversion of NAD toNADH. The third route of reduction might be due to the formationof electrons during the formation of radicals from ascorbate[16]. Our previous report has suggested the first route to be theresponsible route for synthesis [17].

UV–vis analysis provided a reading of 422 nm at 60 ◦C and429 nm for both 90 ◦C and 95 ◦C incubated samples. We used

Fig. 1. UV–vis analysis for samples prepared at varying temperatures such as 60 ◦C,90 ◦C, and 95 ◦C.

22 J.J. Antony et al. / Colloids and Surfaces B: Biointerfaces 109 (2013) 20– 24

Fig. 2. (A) Zeta potential analysis for the sample prepared at 95 ◦C.

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Fig. 3. TEM analysis performed at 100 nm scale.

ave been attributed to particles in size range of 2 nm to 100 nmith spherical shape [19–22]. Particles in the range of longer wave-

ength possess increased size to those of shorter wavelengths [23].ence, the synthesized particles were tiny in size and spherical in

hape as predicted by UV–vis analysis.The zeta potential of the sample was −25.3, indicating highly

table AgNPs (Fig. 2 A). As the sample potential moves towardshe vicinity of zero, the particles tend to aggregate. Negativeharge of particles tends to give more stability preventing particles

rom agglomeration leading to stable nanoparticles [22]. Hence,he synthesized particles were highly stable. Average particle size

easured by DLS was 189.3 nm (Fig. 2B). DLS being a sensitiveethod for detection of protein bound nanoparticles can detect

Fig. 4. FTIR analysis for sam

(B) Particle size distribution for the sample prepared at 95 ◦C.

smaller amounts of large sized particles formed due to agglomer-ation or contamination causing ambiguities in particle sizes [24].Due to this issue, an alternate method such as TEM is suitableto confirm that the particle sizes are not due to agglomerates[25]. The TEM readings displayed particles with the size of 29 to45 nm with spherical shape (Fig. 3). This correlates with the UVpredictions. FTIR bands were studied at 3409 cm−1, 2072 cm−1,1637 cm−1, 1368 cm−1, 1234 cm−1 and 683 cm−1 (Fig. 4). The peaksat 3409 cm−1, 1368 cm−1 and 1234 cm−1 may correspond to asharp peak for alcohols and phenols, indicating possible polyphenolencapsulation [26]. The peak around 2072 cm−1 could be attributedto a peak for metal reduction [27]. The peaks at 1637 cm−1 and683 cm−1 may correspond to primary amines correlating to pro-teins [28]. This proves to an extent that polyphenols and proteinsof the plant might be encapsulated agents [17]. The concentrationof AgNPs was found to be 89 mg/L as predicted by ICP-OES.

Biochemical and haematological parameters analyzed providedevidence for the antibacterial activity of AgNPs. Toxicity analy-sis revealed limited toxicity in the fish model in majority of theanalyses. SGOT, SGPT, ALP values increased in the diseased ani-mal. This was an evidence for hepatic injury as the increase inlevel of these enzymes proves the injury that has occurred to liver[29,30]. This was revived efficiently in the AgNPs treated group.LDH, a marker studied for tissue injury showed augmented levelsin diseased group as that of liver enzymes denoting tissue injury(Table 1, [31]). This was revived in treatment groups efficiently.

Hematological profiles proved further evidence for the antimicro-bial activity. The WBC and RBC levels decreased significantly inthe diseased group, which was revived efficiently in the treatedgroups (Table 2). Histopathology of gills, the respiratory organ,

ple prepared at 95 ◦C.

J.J. Antony et al. / Colloids and Surfaces B: Biointerfaces 109 (2013) 20– 24 23

Table 1Biochemical parameters.

Groups SGOT SGPT ALP LDH

Normal 25.43 ± 0.011 35.47 ± 0.015 54.07 ± 0.940 1991 ± 1.154Normal + SNP 16.24 ± 0.010 38.44 ± 0.017 60.08 ± 0.026 2104 ± 0.577Normal + PE 18.27 ± 0.010 30.22 ± 0.023 56.58 ± 0.015 1867 ± 0.577Aeromonas induced 43.21 ± 0.011 79.14 ± 0.015 61.65 ± 0.026 2242 ± 1.527Aeromonas induced + SNP 24.35 ± 0.010 36.29 ± 0.010 55.07 ± 0.015 1962 ± 1.154Aeromonas induced + PE 32.93 ± 0.034 45.54 ± 0.01 56.86 ± 2.300 2148 ± 0.577

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Fig. 5. Histopathological profile of gills of infected and trea

howed supportive evidence for other experiments. Hyperemia andaemorrhagic lesions were observed in the control group. Normalistoarchitecture was at most observed in treatment groups (Fig. 5).

There are only limited reports that are being made on toxicffects of AgNPs on the ecosystem, particularly on aquatic ani-als. Toxicity tests performed by various groups prove that AgNPs

able 2ematological profile.

Groups WBC RBC

Normal 7500 ± 100.0 1.46 ± 0.05Normal + SNP 7866 ± 57.73 1.33 ± 0.57Normal + PE 8133 ± 57.73 1.73 ± 0.05Aeromonas induced 6766 ± 57.73 1.13 ± 0.05Aeromonas induced + SNP 7033 ± 57.73 1.36 ± 0.57Aeromonas induced + PE 7833 ± 57.73 1.20 ± 0.03

h. Group I, Group II, Group III, Group IV, Group V, Group VI.

were toxic to different varieties of fishes. Toxicity was dose depen-dant and embryos were more affected by AgNPs [32–34]. Smallsized AgNPs were found to be more toxic compared to large sizedparticles [35]. Reports made on toxic effects on fish also indicatethat toxicity was dependant on whether the fish is juvenile or anadult [13]. Early stage embryos were affected more compared todeveloped embryos [36]. But our results indicate that the particleswere of limited toxicity in the prescribed dose used for the experi-ment. Hence, this is a versatile report on the basis of its novelty andimportance.

4. Conclusion

We conclude that the AgNPs derived from plants can be usedeffectively as antimicrobial agents. This is a preliminary report andtherefore further in depth research can prove efficient in presenting

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onclusive evidence for decreasing the susceptibility of fish to bac-erial infections by applying AgNPs.

cknowledgements

The grants of University Grant Commission (UGC), Council forcientific and Industrial Research (CSIR), Department of Science andechnology (DST), DST-FIST, DST-PURSE, UGC-Innovative and UGC-on SAP, New Delhi, Government of India, were of great aid for

he completion of the research and the author acknowledges thegencies for their support.

eferences

[1] E.J. Park, S.W. Lee, I.C. Bang, H.W. Park, Nanoscale Res. Lett. 6 (2011) 223.[2] Y. Zhou, Y. Kong, S. Kundu, J.D. Cirillo, H. Liang, J. Nanobiotechnol. 10 (2012) 19.[3] R.B. Malabadi, G.S. Mulgund, N.T. Meti, K. Nataraja, S. Vijaya Kumar, Res. Pharm.

2 (2012) 10.[4] J.Y. Song, B.S. Kim, Bioprocess Biosyst. Eng. 32 (2009) 79.[5] K. Kim, W.S. Sung, S. Moon, J. Choi, J.G. Kim, D.G. Lee, J. Microbiol. Biotechnol.

18 (2008) 1482.[6] A.J.D. Britto, D.H.S. Gracelin, S.R. Sebastian, J. Biopest. 4 (2011) 57.[7] B.K. Das, S.K. Samal, B.R. Samantaray, S. Sethi, P. Pattnaik, B.K. Mishra, Aquacul-

ture 253 (2006) 17.[8] N. Nithilulworawong, Walailak J. Sci. Tech. 9 (2012) 195.[9] R. Son, G. Rusul, A.M. Sahilah, A. Zainuri, A.R. Raha, Salmah. Lett. Appl. Microbiol.

24 (1996) 479.10] M.S. Prajapati, J.B. Patel, K. Modi, M.B. Shah, Pharmacogn. Rev. 4 (2010) 85.11] A.M. Fayaz, K. Balaji, M. Girilal, R. Yadav, P.T. Kalaichelvan, R. Venketesan,

Nanomed.: Nanotechnol. Biol. Med. 6 (2010) 103.

12] K.N. Thakkar, S.S. Mhatre, R.Y. Parikh, Nanomed.: Nanotechnol. Biol. Med. 6

(2010) 257.13] J.P. Wise, B.C. Goodale, S.S. Wise, G.A. Craig, A.F. Pongan, R.B. Walter, W.D.

Thompson, A. Ng, A. Aboueissa, H. Mitani, M.J. Spalding, M.D. Mason, Aquat.Toxicol. 97 (2010) 34.

[[

B: Biointerfaces 109 (2013) 20– 24

14] X. Ye, Y. Feng, Y. Tong, K.M. Ng, S. Tsao, G.K. Lau, C. Sze, Y. Zhang, J. Tang, J. Shen,S. Kobayashi, J. Ethnopharmacol. 124 (2009) 130.

15] J.H. Zar, Biostastical Analysis, second ed., Prentice Hall, Englewood Cliffs, NJ,1984.

16] M.L. Rao, K. Rukmini, P.S. Devi, Int. J. ChemTech Res. 3 (2011) 1394.17] J.J. Antony, P. Sivalingam, D. Siva, S. Kamalakkannan, K. Anbarasu, R. Sukirtha,

M. Krishnan, S. Achiraman, Colloids Surf. B 88 (2011) 134.18] P. Sivalingam, J.J. Antony, D. Siva, S. Achiraman, K. Anbarasu, Colloids Surf. B 98

(2012) 12.19] K. Vijayaraghavan, S.P. Kamala Nalini, N. Udaya Prakash, D. Madhankumar,

Colloids Surf. B 94 (2012) 114.20] J.J. Mock, M. Barbic, D.R. Smith, D.A. Schultz, S. Schultz, J. Chem. Phys. 116 (2002)

6755.21] Z. Zaheer, Rafiuddin, Colloids Surf. B 90 (2012) 48.22] U. Suriyakalaa, J.J. Antony, S. Suganya, D. Siva, R. Sukirtha, S. Kamalakkannan,

T. Pichiah, S. Achiraman, Colloids Surf. B 102 (2013) 189.23] N. Kannan, K.S. Mukunthan, S. Balaji, Colloids Surf. B 86 (2011) 378.24] S.A. Cumberland, J.R. Lead, J. Chromatogr. A 1216 (2009) 9099.25] M.A. Dobrovolskaia, A.K. Patri, J. Zheng, J.D. Clogston, N. Ayub, P. Aggarwal,

B.W. Neun, J.B. Hall, S.E. McNeil, Nanomed.: Nanotechnol. Biol. Med. 5 (2009)106.

26] K. Jilie, Y.U. Shaoning, Acta Biochim. Biophys. Sin. 39 (2007) 549.27] M. Rivallan, S. Thomas, M. Lepage, N. Takagi, H. Hirata, F. Thibault-Starzyk,

ChemCatChem 2 (2010) 1599.28] I.D.G. Macdonald, W.E. Smith, Langmuir 12 (1996) 706.29] M. Vuda, R. D’Souza, S. Upadhya, V. Kumar, N. Rao, V. Kumar, C. Boillat, P. Mungli,

Exp. Toxicol. Pathol. (2011), http://dx.doi.org/10.1016/j.etp.2011.03.00630] H. Upur, N. Amat, B. Blazekovic, A. Talip, Food Chem. Toxicol. 47 (2009)

2022.31] A. Koul, G. Binepal, S.C. Ganger, Indian J. Exp. Biol. 45 (2007) 359.32] K.W.H. Kwok, M. Auffan, A.R. Badireddy, C.M. Nelson, M.R. Wiesner, A. Chilkoti,

J. Liu, S.M. Marinakos, D.E. Hinton, Aquat. Toxicol. 120–121 (2012) 59.33] K. Park, G. Tuttle, F. Sinche, S.L. Harper, Arch Pharm. Res. 36 (2013) 125.34] T.M. Scown, E.M. Santos, B.D. Johnston, B. Gaiser, M. Baalousha, S. Mitov, J.R.

Lead, V. Stone, T.F. Fernandes, M. Jepson, R.V. Aerle, C.R. Tyler, Toxicol. Sci. 115(2010) 521.

35] A. Jahanbakhshi, F. Shaluei, A. Hedayati, World J. Zool. 7 (2012) 126.36] P.V. Asharani, Y.L. Wu, Z. Gong, S. Valiyaveettil, Nanotechnology 19 (2008)

255102.