IET Nanobio Published Paper

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Published in IET Nanobiotechnology

Received on 17th December 2012

Revised on 24th June 2013

Accepted on 9th July 2013

doi: 10.1049/iet-nbt.2012.0045

Special Issue: Recent Advances in Biosynthesis of

Nanoparticles and their Applications

ISSN 1751-8741

Evaluation of therapeutic potential of nanosilverparticles synthesised using aloin in experimentalmurine mastitis modelThota Venkata Chaitanya Kumar 1, Yegireddy Muralidhar 1, Pagadala Eswara Prasad 1,Tollamadugu Naga Venkata Krishna Vara Prasad 2 , Mekapogu Alpha Raj 3

1Department of Veterinary Biochemistry, College of Veterinary Science, Tirupati, AP, India 2

Nanotechnology Laboratory, Institute of Frontier Technology, Regional Agricultural Research Station, Acharya NG Ranga Agricultural University, Tirupati, AP, India 3 Department of Pharmacology and Toxicology, College of Veterinary Science, Proddatur, AP, India

E-mail: [email protected]

Abstract: Nanobiotechnology is an emerging biological branch of nanotechnology. Application of nanoparticles with specificsize and shape in biology has already shown unforeseen and interesting results. A study was conducted to evaluate thetherapeutic potential of phytogenically derived aloin mediated nanosilver particles (AAgNPs), prepared by reduction of silver nitrate with aloin, in Staphylococcus aureus induced murine mastitis. A total of 40 female mice were divided into five groupsof eight animals each. Group I served as lactating control, groups II-V were inoculated with 20 μl of 24 h broth culture of S.aureus containing 4.0 × 105 cfu/quarter under ketamine anaesthesia. After 6 h post inoculation, groups III and IV received 20μl of aloin nanosilver (AAgNPs) through intramammary and intraperitoneal routes, respectively. Group V received antibiotic cefepime at 1 mg/kg body weight through the intra-peritoneal route. After 18 h post-treatment, serum C reactive protein, weights of mammary glands, mammary gland bacterial load, thiobarbituric acid reactive substances content, reduced glutathione content, superoxide dismutase activity and catalase activity and histopathology were determined. The compound showed a minimum inhibitory concentration of 21.8 ng/ml against S. aureus. Significant reduction (98%) in poly-morphonuclear cell infiltration was observed with AAgNPs than antibiotic (50%).

1 Introduction

Biosynthesised metallic nanoparticles are gaining importancein the recent past because of their unusual and potentialapplications in biology and medicine in particular. Apart from the simple and cost-effective methods for production, bio-safety of biogenic metal nanoparticles has popularised them as ultimate tools for biological applications. Bovinemastitis is considered as the major constraint for the growthof dairy industry both in India and abroad [1] causingheavy losses in terms of reduced quality and quantity of milk besides incurring huge cost on treatment [2]. Theannual economic loss because of mastitis was estimated to be $35 billion worldwide and Rs.16 702 million in India[3]. The most common microorganisms responsible for intra-mammary infections include Staphylococcus,Streptococcus and Coliforms [4]. Staphylococcus aureuswas reported to be the leading cause of mastitis worldwide[2] and in India and also causes noscomial and community

acquired infections. The annual economic loss caused byStaphylococcal bovine mastitis globally was estimated to be$2 billion [5]. Furthermore, S. aureus was reported to bethe main reason for the use of antibiotics in dairy cows [ 6]

which also leads to the problem of antibiotic residues indairy products.

Silver nanoparticles owing to their characteristics likereduced size and increased surface area [7] were reported to provide enormous hope for use in medical field by virtue of broad spectrum antimicrobial activity [8, 9] which includes bactericidal action against both Gram positive and Gramnegative bacteria including (multi-drug resistant) MDR strains of bacteria [10–14]. Furthermore, the development of bacterial resistance has not been reported against silver nanoparticles [15, 16] making it a good choice for therapeutic consideration in mastitis. The additionaladvantage with silver nanoparticles resides in the fact that they are relatively non-toxic and safe antibacterial agents[16] requiring much smaller concentrations for antimicrobialeffect [12, 17]. Furthermore, the biological synthesis of nanoparticles using plant sources (phytogenic) isadvantageous over other methods of synthesis in being cost effective, practically non-toxic and eco friendly [18, 19].

Aloin, the major constituent of Aloe vera was reported to possess antimicrobial [20] and antiinflammatory properties[21, 22]. It is also reported to have anticancerous and antitumour activity [23]. Hence, for the present study,

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nanosilver particles derived phytogenically using aloin wereconsidered and also herewith, we report for the first time,the effect of aloin mediated nanosilver particles (AAgNPs)on Bovine mastitis using a murine mastitis model.

2 Material and methods

2.1 Grouping of albino mice

Adult female albino mice weighing 20–35 g and in 10–15days of lactation were housed in solid bottom poly propylene cages (five animals in each cage) at an ambient temperature of 24 ± 2°C and 45–55% relative humidity with12–12 h light and dark cycle. The mice were kept on ad libitum feed and water. Forty albino female mice between10 and 15 days of lactation were randomly divided into fivegroups (n = 8).

S. no. Group

1 normal lactating group2 mastitis group3 mastitis mice treated with aloin nanosilver i.m

(intra-mammary route)4 mastitis mice treated with aloin nanosilver i.p

(intra-peritoneal route)5 mastitis mice treated with cefepime i.p

(intra-peritoneal route)

2.2 Preparation and characterisation of AAgNPs

The AAgNPs were synthesised by the reduction of silver nitrate with aloin [24] at the Regional Agricultural ResearchStation, Tirupati and was used for the study. The prepared

AAgNPs were characterised by using scanning electronmicroscope (SEM), ultraviolet –vis spectra, DLS and Fourier transform infrared spectroscopy techniques. The toxicity of the compound was tested by in vitro dimethyl thiazolediphenyl tetrazolium bromide assay on mouse splenocytes[25] and acute oral toxicity in rats [26]. The minimum

inhibitory concentration (MIC) of the compound wasdetermined using antibiotic sensitivity test by tube dilutiontechnique [27].

2.3 Experimental induction of mastitis

Experimental induction of mastitis was carried out by

inoculating 20 µl of S. aureus (4.0 × 10

4

organisms)suspension through the teat orifice into L3 and L4mammary glands (Fig. 1) after inducing general anaesthesiausing ketamine (100 mg/kg; i.p). The guidelines of theCommittee for the Purpose of Control and SupervisionExperiments on Animals (CPCSEA) were followed duringthe procedure. Later, the mice were given butorphanol (3–5mg/kg) to prevent post-inoculation trauma [28]. After 6 h post inoculation, AAgNps (20 µl ) and cefepime (1 mg/kg)were injected in respective groups as per the experimental protocol. Whole blood was collected through tail vein puncture under light ether anaesthesia using heparinised capillary tubes. A volume of 0.4 ml whole blood wascollected in serum vacutainers and serum was separated

after allowing for clotting followed by centrifugation at 3000 RPM for 15 min. The serum was utilised for estimation of C reactive protein (CRP) [29]. After blood collection the animals were euthanised and mammaryglands were aseptically dissected and collected in pre-sterilised and pre-weighed Ependorf tubes. The weightsof the mammary glands were determined by the differenceof weights of Eppendorf tubes with and without mammaryglands. Initially, a one in ten homogenate (1 g in 10 ml) of each mammary gland was prepared in sterile phosphate buffer solution (PBS), which was further serially diluted (100-fold) in sterile PBS resulting in final dilutions of 10−3,10−5 and 10−7. A volume of 100 µl was plated on nutrient

agar plates in triplicate for each dilution. The plateswere incubated for 24 h at 37°C. The plates showing <300colonies were counted using colony counter. A 10%homogenate was prepared from the mammary gland using0.2 M Tris HCl buffer (pH 7.4). The homogenate was used for estimation of thiobarbituric acid reactive substances

Fig. 1 Gross pictures showing mastitis affected mammary gland in mice

a Mastitis affected mammary gland b Exposed mastitis affected mammary gland

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doi: 10.1049/iet-nbt.2012.0045



(TBARS) and glutathione (GSH). The homogenate was precipitated by ethanol and chloroform and the supernatant was utilised for estimation of superoxide dismutase (SOD)and catalase (CAT). Briefly, the precipitation procedureincluded addition of 0.5 ml of tissue homogenate with 0.25ml of ethanol and 0.15 ml of chloroform and centrifuged 3500 RPM for 15 min. The mammary glands dissected from each group were collected for histopathological

studies. The tissues were fixed in 10% neutral buffered formalin until further analysis. The samples were processed and sections of 4–7 µm were made and stained withhaematoxylin and eosin (H&E). The specimens wereexamined under a light microscope [30].

3 Results and discussion

The SEM image (Fig. 2) analysis of AAgNPs showed that the particles are cubical, rectangular, triangular and spherical inshape with uniform distribution. However, on most of theoccasions agglomeration of the particles was observed. Themeasured sizes of the agglomerated clusters of nanoparticles were in the range of 287.5–293.2 nm and theaverage size of an individual particle was ∼70 nm, whichwas in good agreement with the results of dynamic light scattering (DLS) which recorded the particle size as 68.7 nm.

The AAgNPs showed a MIC of 21.8 ng/ml against S. aureus which was isolated from a case of acute bovinemastitis. The compound showed an in vitro splenocyteviability of more than 90% at the highest concentration of 87.5 ppm per well proving that the compound wasnon-toxic. The compound was also found to be safe at thelimit dose in acute oral toxicity conducted in rats.

All the groups inoculated with S. aureus viz., groups II–Vshowed significantly higher mammary gland weights. The

bacterial load was significantly higher in mastitis group II,whereas all the treatments showed significant reduction in bacterial loads (Fig. 3) compared with mastitis control.Bacterial loads and mammary glands weights were presented in Table 1. The levels of CRP, which is anindicator of acute inflammation were found to besignificantly elevated in mastitis group II [31]. AAgNPstreatment groups III and IV significantly reduced theelevated CRP in comparison with antibiotic group V(Table 2). The TBARs values in mastitis group II and antibiotic group V were significantly elevated compared

Table 1 Mean bacterial loads and mammary gland weightspost treatment of all the experimental groups

Group Mean log10 cfu/g Mammary glandweight, g

I lactating – 0.27a ± 0.01II mastitis >9.48b* 0.45b ± 0.02III ANS i.m 8.44a ± 0.39 0.29b ± 0.02IV ANS i.p 8.52a ± 0.42 0.33b ± 0.01V antibiotic i.p 9.09a ± 0.27 0.39b ± 0.04

df (3,10) (5,18)F 548.56 4.81

Sig. 0.000 0.039

*Bacterial counts could not be performed in the mastitis groupbecause of formation of mat even at highest dilution of 10−7.Hence, the count is assumed to be >300× 108 cfu/g. ANS: aloinnanosilver; i.m = intra-mammary route; i.p = intra-peritoneal routeValues are mean± SDOne way ANOVA followed by Tukey’s post hoc test using SPSS17.0 VMeans with different superscript alphabets are significantlydifferent (P < 0.05)

Fig. 3 Bacterial load in different experimental groups

Group II showing mat formation, whereas treatment groups III–V showed significant reduced bacterial loads

Fig. 2 Scanning electron microscopic images of aloin nanosilver

particles showing cubical, rectangular, triangular and spherical

shape with uniform distribution

Table 2 Mean CRP values (mg/dl) in serum post treatment

Group Mean CRP, mg/dl

I lactating 0.023a ± 0.003II mastitis 0.061b ± 0.001III ANS i.m 0.013a ± 0.002IV ANS i.p 0.016a ± 0.003V antibiotic i.p 0.044ab ± 0.003

df (5,18)F 5.477

Sig. 0.006

Mean CRP value was significantly increased in the mastitiscontrol group. ANS administered by both routes significantlyreduced the CRP values in plasma. ANS = Aloin nanosilver; i.m =intra-mammary route; and i.p = intra-peritoneal route

Values are mean± SDOne way ANOVA followed by Tukey’s post hoc test using SPSS17.0 VMeans with different superscript alphabets are significantlydifferent (P < 0.05)

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with normal lactating group I indicating substantial damage tomembrane lipids in the mammary gland [32]. However,AAgNPs treatment groups III and IV showed noimprovement probably as the treatment was given 6 h post

infection, by which time the damage had already occured.The reduced GSH levels and SOD activities were not significantly affected either by the induction of mastitis or by different treatments whereas CAT activity was found to be significantly lowered in AAgNPs treatment groups IIIand IV compared with groups I and II (Table 3). Thehistopathological picture of mastitis group II showed heavyinfiltration of PMN cells within and in between acini [ 33,34] along with desquamation and vacuolation of the acinar epithelium. Both AAgNPs groups III and IV reduced theinfiltration of PMN to the extent of 99% with very mild

changes in the mammary tissue. Antibiotic group V showed a moderate reduction of infiltration of PMN to the extent of 40–50% (Fig. 4).

4 Conclusions

AAgNPs were found to possess novel antibacterial effect which was superior to cefepime. Furthermore, thecompound also exhibited profound anti-inflammatory effect in Staphylococcal mastitis, which was not found withantibiotic treatment. Hence, keeping in view the economicsof production, safety and ef ficacy of the compound, aloinderived AgNPs could provide a promising alternative to theuse of traditional antibiotics for treatment of bovine mastitis.

5 Acknowledgments

The authors are grateful to Acharya NG Ranga AgriculturalUniversity, Tirupati, India and Sri Venkateswara VeterinaryUniversity, Tirupati, India for providing research facilitiesto conduct this work.

6 References

1 Osteras, O., Solverad, L.: ‘ Norwegian mastitis control programme’, Ir.Vet. J., 2009, 62, pp. 26–33

2 Ruegg, P.L.: ‘Investigation of mastitis problems on farms’, Vet.Clin. N. Am., Food Anim. Pract., 2007, 19, pp. 47–74

3 Muhamed Mubarack, H., Doss, A., Dhanabalan, R., Venkataswamy, R.:‘Activity of some selected medicinal plan extracts against bovinemastitis pathogens’, J. Anim. Vet. Adv., 2011, 10, pp. 738–741

4 Bradley, A.J.: ‘Bovine mastitis: a evolving disease’, Vet. J., 2002, 164, pp. 116–128

5 Schmelcher, M., Powell, A.M., Stephen, C., Becker, Camp, M.J.,Donovan, D.M.: ‘Chimeric phage lysins act synergistically withlysostaphin to kill mastitis causing Staphylococcus aureus in murinemammary glands’, Appl. Environ. Microbiol., 2012, 78, pp. 2297–2305

6 Mitchell, J.M., Grif fiths, M.W., Mc Ewen, S.A., Mc Nab, W.B., Yee, A.J.: ‘Antimicrobial drug residues in milk and meat causes concerns, prevalence,regulations, tests, and test performance’, J. Food Prot.,1998, 61, pp. 742–756

7 Yeo, S.Y., Lee, H.J., Jeong, S.H.: ‘Preparation of nanocomposite fi bersfor permanent antibacterial effect ’, J. Mater. Sci., 2003, 38, pp. 2143–2147

8 Lok, C.N., Ho, C.M., Chen, R., et al.: ‘Proteomic analysis of the modeof antibacterial action of silver nanoparticles’, J. Proteome. Res., 2006,5, pp. 916–924

9 Gogoi, S.K., Gopinath, P., Paul, A., Ramesh, A., Ghosh, S.S.,Chattopadhyay, A.: ‘Green fluorescent protein-expressing Escherichia

coli as a model system for investigating the antimicrobial activities of silver nanoparticles’, Langmuir , 2006, 22, pp. 9322–932810 Alt, V., Bechert, T., Steinrucke, P., et al.: ‘ Nanoparticulate silver. A new

antimicrobial substance for bone cement ’, Biomaterials, 2009, 25, pp. 4383–4391

Table 3 Mean values of different antioxidant parameters of the mammary tissue of the experimental groups

Group Mean, MDA µg/g Mean Glutathione level, mg/g Mean (SOD),U/mg protein

Mean CAT, mg/dl

I lactating 0.07a ± 0.11 0.12 ± 0.13 04.57 ± 0.32 40.00b ± 2.96II mastitis 0.47b ± 0.22 0.11 ± 0.13 13.10 ± 0.04 35.80b ± 2.78III ANS i.m 0.29ab ± 0.19 0.14 ± 0.18 08.34 ± 0.58 12.99a ± 1.04IV ANS i.p 0.33b ± 0.22 0.13 ± 0.02 08.63 ± 0.65 13.01a ± 1.18V antibiotic i.p 0.64b ± 0.27 0.09 ± 0.03 16.11 ± 0.97 22.60ab ± 1.62

df (5,18) (5,18) (5,18) (5,18)F 7.302 0.191 0.827 3.967Sig. 0.003 0.901 0.497 0.027

ANS = Aloin nanosilver; i.m = intra-mammary route; i.p = intra-peritoneal routeValues are mean± SDOne way ANOVA followed by Tukey’s post hoc test using SPSS 17.0 VMeans with different superscript alphabets are significantly different (P < 0.05)

Fig. 4 Histopathology of mammary glands of treatment groups

(H&E)

a Group II mastitis control showing extensive PMN infiltration (×70)b Group III aloin nanosilver i.m treated group showing absence of PMN cells(×280)c Group IV aloin nanosilver i.p treated group showing absence of PMN cellinfiltration (×280)

d Group V cefepime i.p treated group showing moderate PMN cell infi

ltration(×70)P = PMN cells; G = mammary alveolar cells; H&E = haematoxylin and eosinstaining; PMN = polymorphonuclear cells; i.m = intra-mammary route; and i.p = intra-peritoneal route

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11 Baker, C., Pradhan, A., Pakstis, L., Pochan, D.J., Shah, S.I.: ‘Synthesisand antibacterial properties of silver nanoparticles’, J. Nanosci. Nanotechnol., 2005, 5, pp. 244–249

12 Panacek, A., Kvitek, L., Prucek, R., et al.: ‘Silver colloid nanoparticles:synthesis, characterization, and their antibacterial activity’, J. Phys.Chem. B, 2006, 110, pp. 16248–16253

13 Morones, J.R., Elechiguerra, J.L., Camacho, A., et al.: ‘The bactericidaleffect of silver nanoparticles’, Nanotechnology, 2005, 16, pp. 2346–2353

14 Ip, M., Lui, S.L., Poon, V.K.M., Lung, I., Burd, A.: ‘Antimicrobial

activities of silver dressings: an in vitro comparison’, J. Med. Microbio., 2006, 55, pp. 59–63

15 Neu, H.C.: ‘The crisis in antibiotic resistance’, Science, 1992, 257, pp. 1064–1073

16 Stewart, P.S., Costerton, J.W.: ‘Antibiotic resistance of bacteria in biofilms’, Lancet , 2001, 358, pp. 135–138

17 Kvitek, L., Vanickova, M., Panacek, A., et al.: ‘Initial study on thetoxicity of silver nanoparticles (NPs) against Paramecium caudatum’, J. Phys. Chem. C , 2009, 113, pp. 4296–4300

18 Aymonier, C., Schlotterbeck, U., Antonietti, L., et al.: ‘Hybrids of silver nanoparticles with amphiphilic hyperbranched macromoleculesexhibiting antimicrobial properties’, Chem. Commun., 2002, 24, pp. 3018–3019

19 Sun, Y.G., Xia, Y.N.: ‘Shape-controlled synthesis of gold and silver nanoparticles’, Science, 2002, 298, pp. 2176–2179

20 Coopoosamy, R.M., Magwa, M.L.: ‘Antibacterial activity of aloe

emodin and aloin a isolated from Aloe excels‘

, Afr. J. Biotechnol.,2006, 5, pp. 1092–109421 Park, M.-Y., Kwon, H.-J., Sung, M.-K.: ‘Dietary aloin, aloesin, or

aloe-gel exerts anti-inflammatory activity in a rat colitis model’, LifeSci., 2011, 88, pp. 486–492

22 Park, M.Y., Kwon, H.J., Sung, M.K. : ‘Evaluation of aloin and aloe-emodin as anti-inflammatory agents in aloe by using murinemacrophages’, Bisci. Biotechnol. Biochem., 2009, 23, pp. 828–832

23 Matharasi, D.S.P., Abu-Yousef, I.A., Gopi, S., Kumar, S., Vijayakumar,B., Kanan, S.: ‘Synthesis and antibacterial activity of aloin Schiff ’s bases’, Eur. J. Sci. Res., 2010, 433, pp. 297–306

24 Prasad, T.N.V.K.V., Elumalai, E.K., Khateeja, S.: ‘Evaluation of theantimicrobial ef ficacy of phytogenic silver nanoparticles’, Asia-Pac. J. Trop. Biomed., 2011, 1, pp. 82–85

25 Mosmann, T.: ‘Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays’, J. Immunol. Methods, 1983, 65, pp. 55–63

26 OECD/OCDE: ‘OECD Guideline for testing of chemicals 420. Acuteoral toxicity – fixed dose procedure’. Available at http://www.iccvam.niehs.nih.gov/SuppDocs/FedDocs/OECD/OECD_GL420.pdf, 2001.

27 Jorgensen, J.H., Turnidge, J.D.: ‘Antibacterial susceptibility tests:

dilution and disk diffusion methods’, in Murray, P.R., Baron, E.J.,Jorgensen, J.H., Landry, M.L., Pfaller, M.A. (Eds.): Manual of clinical microbiology, (American Society for Microbiology,Washington, DC, 2007, 9th edn.), pp. 1152–1172

28 Gomez, M.I., Garcia, V.E., Gherardi, M.M., Cerquetti, M.C., Sordelli,D.O.: ‘Intramammary immunization with live-attenuated Staphylococcus aureus protects mice from experimental mastitis’, FEMS Immunol. Med. Microbiol., 1998, 20, pp. 21–27

29 Singh, U.B., Sulochana, S.: ‘Handbook of histological and histochemical techniques’, (Premier Publishing House, Hyderabad,1997), pp. 1–63

30 Marrack, J.R., Richards, C.B.: ‘Light-scattering studies of the formationof aggregates in mixtures of antigen and antibody’, Immunology, 1971,20, pp. 1019–1040

31 Lee, W.C., Hsiao, H.C., Chu, R.M.: ‘Serum C-reactive protein in dairyherds’, Can. J. Vet. Med., 2003, 67, pp. 102–107

32 Spengler, M.I., Svetaz, M.J., Leroux, M.B., Bertoluzzo, S.M., Parente,F.M., Bosch, P.: ‘Lipid peroxidation affects red blood cells membrane properties in patients with systemic lupus erythematosus’, Clin. Hemorheol. Microcirc., 2013, doi: 10.3233/CH-131716

33 Miao, J., Zheng, L., Zhang, J., Ma, Z., Zhu, W., Zou, S.: ‘The effect of taurine on the toll-like receptors/nuclear factor kappa B (TLRs/NF-κB)signaling pathway in Streptococcus uberis-induced mastitis in rats’, Int. Immunopharmacol., 2011, 11, pp. 1740–1746

34 Miao, J., Zhang, J., Zheng, L., Yu, X., Zhu, W., Zou, S.: ‘Taurineattenuates Streptococcus uberis-induced mastitis in rats by increasingT regulatory cells’, Amino Acids, 2012, 42, pp. 2417–2428

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