Biogenic silver nanoparticles synthesised from ...

Int. J. Biomedical Nanoscience and Nanotechnology, Vol. 3, No. 3, 2014

Biogenic silver nanoparticles synthesised from Zingiber officinale and its antifungal properties Malini Soundararajan* Department of Biochemistry, Center for Post Graduate Studies, Jain University, 18/3, 9th Main Jayanagar, 3rd Block, Bangalore – 560 011, Karnataka, India E-mail: [email protected] *Corresponding author

Neha Deora ICMR, Biochemistry Lab, Desert Medicine Research Centre, New Pali Road, Jodhpur – 342 005, Rajasthan, India E-mail: [email protected]

Lynette Lincoln and Purandhi Roopmani C/o. Mrs. Malini Soundararajan, Department of Biochemistry, Center for Post Graduate Studies, Jain University, 18/3, 9th Main Jayanagar, 3rd Block, Bangalore – 560 011, Karnataka, India E-mail: [email protected] E-mail: [email protected]

Shikha Gupta C/o. Mr. Bharat Prasad, Ganapati Trading Company, #146A, Sheshapur A, Gorakhpur – 273 005, Uttar Pradesh, India E-mail: [email protected]

Copyright © 2014 Inderscience Enterprises Ltd.

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Rajatha Shambu C/o. Mrs. Malini Soundararajan, Department of Biochemistry, Center for Post Graduate Studies, Jain University, 18/3, 9th Main Jayanagar, 3rd Block, Bangalore – 560 011, Karnataka, India E-mail: [email protected] Abstract: A silver particle at the nanoscale level behaves as an effective antimicrobial agent and offers numerous applications in biosensing and medicine. The current study unveils the effect of rhizome of Zingiber officinale and its silver nanoparticles against the growth and hydrolytic enzyme of two lethal moulds, Alternaria alternata and Curvularia lunata. The rhizome of Zingiber officinale was extracted under aseptic conditions to get cold distilled water and silver nanoparticle extracts in order to check the effect on inhibition of cell mass formation and protease activity of Curvularia lunata and Alternaria alternata. The formation of silver nanoparticles was confirmed by UV-Vis absorption spectroscopy and scanning electron microscopy. The obtained results showed that the highest tested concentration (2%) of Zingiber officinale in cold distilled water and silver nanoparticle extracts strongly inhibited the cell mass formation as well as protease activity in test organisms. The silver nanoparticle extract showed potent antifungal activity when compared to cold distilled water extract. The study unravels the antifungal property of Zingiber officinale and its biogenically synthesised silver nanoparticles that can be exploited further for therapeutical and other industrial applications. Keywords: Alternaria alternate; antifungal; biogenic; Curvularia lunata; nanotechnology; silver nanoparticles; Ag-Np’s; Zingiber officinale; cold distilled water; CDW; silver nitrate; SN. Reference to this paper should be made as follows: Soundararajan, M., Deora, N., Lincoln, L., Roopmani, P., Gupta, S. and Shambu, R. (2014) ‘Biogenic silver nanoparticles synthesised from Zingiber officinale and its antifungal properties’, Int. J. Biomedical Nanoscience and Nanotechnology, Vol. 3, No. 3, pp.251–261. Biographical notes: Malini Soundararajan is an Assistant Professor in the Department of Biochemistry at the Jain University. Her interest in the field of biogenic nanoparticles has instigated her to mentor a young team of researchers who played a vital role in unravelling the antimicrobial potential of biogenic silver nanoparticles. She is currently working on plant metabolomics and antimicrobial effect of phytochemicals. She also has two international publications. Neha Deora is currently designated as a Supervisor at the Biochemistry Lab in Desert Medicine Research Center, Jodhpur, Rajasthan. Her contribution as a co-author has been enormous during the paper preparation. She has a thorough knowledge on cheminformatics and is also working towards pursuing PhD in Neurochemistry in the future. Lynette Lincoln is an MPhil Scholar in Biochemistry who has worked on microbial enzyme production and purification. As a co-author she has immensely dedicated her time for paper revision and has aided the

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corresponding author as well. Her passion for research has led her to pursue PhD in Biochemistry at the National Institute of Animal and Nutrition Physiology, Bangalore, India. Purandhi Roopmani is a PhD Scholar at the Sastra University, Tamil Nadu currently working on ‘Development of dual design multi-drug eluting stents for cardiovascular obstructive disorders’. She is trained in analytical instrumentation and also holds an honour’s programme on basic analytic techniques in chemistry. Shikha Gupta is a post-graduate student in biochemistry. Her passion for the subject had driven her to contribute maximum during the bench work of the project. She is currently a business analyst at renowned firm, Tata Consultancy Services, Ltd. at Bangalore. Rajatha Shambu is also a Post-Graduate Researcher in Biochemistry. She holds great interest on the microbial modes of action of nanoparticles as it is an emerging field. Her enthusiasm during the research project has kept the team going and perform excellently.

1

Introduction

New developments in nanoscience and nanotechnology have made a profound impact in the past decade, worldwide. The nanoparticles have enormous applications in biosensing (Jianrong et al., 2004) bioremediation of radioactive wastes (Marcato et al., 2007) functional electrical coating (Singh et al., 2006)and in medicine such as delivery of antigen for vaccination (Sung et al., 2007), topical aids for wound repair (Dowsett, 2004) and many more applications that have inspired researchers to develop novel methods for the synthesis of nanosized materials in an ecofriendly manner so as to avoid use of tedious, traditional synthetic procedures which are hazardous to health. This has stimulated our interest in developing biological methods to prepare Ag-Np’s through a green nano route using plant extract mediation and thereby reduce the dependence on chemical Ag-Np’s. The preparation method also provides an added advantage of being cost effective and less time consuming. Silver in its pure form was known by ancient Greeks as a great material to keep microbes at bay. If silver is transformed into a nanoparticle, its antimicrobial property is intensified, making it useful in effectively eliminating fungus, bacteria and viruses. Silver and its nanoparticles inhibit the growth of microorganisms by disrupting the biological processes (Jo et al., 2009). The biological pathways of these microorganisms are targeted by manifold modes of action thereby providing a crucial advantage by avoiding the development of resistance to a greater extent. The plant mediated biological synthesis of Ag-Np’s is known to have toxic effect against Escherichia coli (Singh et al., 2011b). Antifungal activity of Ag-Np’s was also demonstrated on Candida albicans and Saccahromyces cerevisiae (Nasrollahi et al., 2011). Streptomyces aureus and Bacillus cereus was also found to be inhibited by Ag-Np’s synthesised by biogenic method (Elumalai et al., 2010).Biosynthesis of Ag-Np’s by various plants such as Alfalfa (Torresdey et al., 2002), Aloe vera (Chandran et al., 2006), Carica papaya (Jain et al., 2009), Partheniumhy sterophorus (Ankamwar et al., 2005), Azadirachta indica (Shankar

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et al., 2004) and Capsicum annuum (Bar et al., 2009) have also been reported. Many plant fungal diseases are curbed by the action of chemical management which is a matter of concern at present and it must be cut down by the use of biological Ag-Np’s. Zingiber officinale is valued worldwide as a delicacy, medicine and spice also possesses antifungal property. A protein with a novel N-terminal sequence isolated from rhizome with an apparent molecular mass of 32 kDa exerts antifungal activity against Botrytis cinerea, Fusarium oxysporum, Mycosphaerella arachidicola, and Physalosporapiricola (Wanga and Bun, 2005). Some of the essential oils like zingiberene (16.70%), (E, E)-_-farnesene (13.10%) and geranial (7.60%) in were Zingiber officinale has also found to have antimicrobial property (Sharma, 2006).This study reports the antifungal property of Zingiber officinale and its Ag-Np’s. Curvularia lunata and Alternaria alternata are two dematiaceous filamentous moulds characterised by melaninogenic pigmentation on their cell wall. They are mainly opportunistic pathogens with low pathogenicity that survives mostly as a saprophyte. They are known to cause variety of infections in humans and animals and have been shown to be a major disease causal agent in various cultivated crops posing serious threat to increase agricultural production. These pathogenic fungi possess a definite or specific pattern in order to infect the host by synthesis of extracellular enzymes. They can produce both constitutive as well as induced enzymes. Many phytopathogenic microorganisms are known to produce active proteases that along with other enzymes play an important role in pathogenesis; intrusion into the plant, irreversible inactivation of protected proteins, participation in transformation of pathogens own proteins (Valueva and Mosolov, 2004). Owing to the worldwide increase in the incidence of fungal infections, various attempts have been made to control the spread of fungal infections. These include improved cultural practices on the farm and chemical control using fungicides. But, most of these fungicides are toxic to humans and with the dwindling foreign exchange and prohibitive cost, most of the useful fungicides are usually out of reach of farmers. Moreover usage of chemical fungicide has become an important concern in disease management when consumers increase their attention on chemical free residue products as well as product quality. Also, the pathogen on its own builds up resistance to the fungicides used in medicinal practice therefore, new prototype antimicrobial agent or replacement of the chemical fungicides by biologically synthesised fungicides could be an appreciable alternative to address the situation. In the current study, the rhizome of Zingiber officinale has been used for the synthesis of Ag-Np’s.

2

Materials and methods

2.1 Test organisms C. lunata (2,098) and A. alternata (7,202) were obtained from Microbial Type Culture Collection (MTCC) and Gene bank. The cultures were grown on Waksman’s medium at 28°C before usage.


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2.2 Plant specimen Zingiber officinale was used as the plant source for production of Ag-Np’s using the extract of CDW and Ag-Np.

2.3 Preparation of plant extracts 2.3.1 Cold distilled water extraction Under aseptic conditions, the rhizome of Zingiber officinale was surface sterilised thrice with tap water and distilled water, further washed with mercuric chloride (1%, v/v) for 8min, followed by 70% ethanol for 30 sec and rinsed thrice with distilled water. After sterilisation, 20 g of rhizome was ground and then extracted by macerating in 100 ml of sterilised cold distilled water (CDW) for 24 h and the extract was filtered aseptically using Whatman filter paper. This served as the stock from which three different test concentrations were prepared (0.5%, 1% and 2%).

2.3.2 Synthesis of silver nanoparticles from CDW extract (Ag-Np extract) The Ag-Np extract was prepared by using the above CDW extract: 1 mM of SN was added and incubated for 1 h with continuous shaking in order to reduce SN into Ag-Np’s by phytochemicals present in the plant extract. The solution was analysed by using UV-Vis spectrophotometer and scanning electron microscopy (SEM).

2.4 UV-Vis spectra analysis After 1 h of incubation of Ag-Np extract, the preliminary detection of Ag-Np’s was performed by visual observation of colour change which was followed spectral analysis by using a UV-Vis spectrophotometer (ELICO SL 159) and the absorption spectra was scanned between 300 and 1,000 nm.

2.5 SEM measurements SEM analysis was performed using Quanta 200 ESEM to record the micrograph images of synthesised Ag-Np’s. 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 the film on the SEM grid was allowed to dry by placing it under a mercury lamp for 5 min.

2.6 Effect of extracts on the fungal biomass The measurement of fungal growth was made according to the method described by Kane and Mullins (1973). The Zingiber officinale extracts (CDW and Ag-Np) were mixed with 50 ml of Waksman’s medium at different concentrations after autoclaving. The medium without extract served as control. Mycelial discs were prepared using a cork borer (5 mm diameter) from the peripheral zones of seven days old culture. The flasks were incubated at 27°C for seven days after inoculation. Mycelia was separated using Whatman filter

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paper after the end of incubation period and the mats were washed carefully and dried up to constant dry weight at 70 to 80°C for 24 h. The experiments were performed in triplicates.

2.7 Determination of enzyme production 2.7.1 Plate test for protease production C. lunata and A. alternate were inoculated on casein media and plates were incubated at 27°C for seven days. The plates were then checked for the zone of clearance surrounding the fungal growth.

2.7.2 Estimation of protease activity Protease activity was determined according to Kunitz (1947) using casein (1%, w/v) as substrate. The seven days old culture filtrate was obtained by inoculating fungal sp. on growth medium containing casein and separating the mycelia from medium using filter paper. The obtained filtrate served as crude enzyme source. The reaction mixture containing 1 ml of crude enzyme and 1 ml of 1% (w/v) casein in 0.05 M citrate phosphate buffer, pH 6.0 was incubated at 30°C for 20 min. The reaction was stopped with 3 ml of 10% (v/v) trichloroacetic acid (TCA) and the mixture was centrifuged at 5,000 rpm for 10 min. The optical density of the supernatant was measured at 280 nm. The experiments were performed in triplicates. One unit of protease was defined as the activity that produced an increase in optical density of 1.0 in 20 min at 280 nm.

3

Results and discussion

3.1 Characterisation of Ag-Np’s Figure 1 represents the change in the colour of the sample solution from yellowish to reddish brown with time, which indicates the reduction process of Ag+ ions into Ag-Np’s. This is in agreement with Singh et al. (2011b) where they state that ascorbic acid and/or oxalic acid are/is the main organic components in the extract which play(s) the role of reducing silver ions into Ag-Np’s. It appears that these acids present in Zingiber officinale are/is chemically reduces Ag+ ions to Ag-Np’s. The possible stages of chemical reaction in the formation of Ag-Np’s from Zingiber officinale extract are nucleation, condensation, surface reduction and stabilisation. In addition to the above reducing acids there are other components like zingerones (C6H8O6) and phenylpropanoids in the phytochemicals of the Zingiber officinale extract of which zingerones (C6H8O6) act as a protecting material for stabilisation of Ag-Np’s. Like other nanomaterials the Ag-Np’s exhibit remarkably unusual physicochemical properties and biological activities by virtue of extremely small size. These distinctive properties extend its application in antibacterial, antifungal, anti-viral and anti-inflammatory therapy (Vaidyanatahan et al., 2009).

Biogenic silver nanoparticles synthesised Figure 1

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(a) Zingiber officinale extract (b) Colour change of leaf extract containing silver nitrate (see online version for colours)

(a)

(b)

3.2 UV-Vis spectroscopy studies Ag-Np’s are known to exhibit characterised absorption peak in the UV-Vis spectrum around 400 nm which can be observed in Figure 2. The phenomenon behind this is surface plasmon excitation, which is due to collective electron oscillation around the surface mode of the particles when excited by light at specific wavelength. Absorption and scattering of light depends upon size and shape of particles thus UV-Vis spectroscopy provides a mechanism to monitor how nanoparticles change over time (Singh et al., 2011b, 2011a). UV-Vis spectrograph of the colloidal solution of Ag-Np’s synthesised by reduction of Ag+ions using Zingiber officinale recorded at different nm (see online version for colours)

UV Spectra Analysis 3.5 3 A b s o rb an c e (A )

Figure 2

2.5 2 1.5 1 0.5 0 300

400

600 Wavelength (nm)

800

1000

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3.3 SEM analysis SEM analysis was carried out to understand the topology and size of Ag-Np’s which showed the synthesis of poly dispersed spherical nanoparticles in the range 70 to 80 nm with an average size of 75.3 nm (Figure 3). Figure 3

SEM micrograph of Ag-Np’s synthesised using the extract of Zingiber officinale (see online version for colours)

3.4 Inhibition of cell mass formation by CDW and Ag-Np extracts The extracts were separately incorporated into the growth medium of the C.lunata and A.alternata test organisms to determine the best extract, appropriate concentration for application and its effectiveness on the pathogen. After the incubation period, it was observed that the mycelial growth of the test organisms had sequentially reduced by the extracts at different concentrations from 0.5 to 1% (Table 1) when compared to the control which was not poisoned with these extracts. The cell mass inhibition by CDW and Ag-Np extract allied with the results obtained by Fawzi et al. (2009) and Masuduzzaman et al. (2008), respectively. A similar observation was reported by Singh et al. (2011) on Escherchia coli. It was also observed that, Ag-Np extract showed higher inhibition when compared to CDW extract and the rate of inhibition increased with increase in concentration (2%) of Ag-Np’s. Table 1

Effect of botanical extracts of Zingiber officinale on inhibition of cell mass formation evaluated at different concentrations (0.5%, 1% and 2%) on the two test organisms CDW extract

Control

Ag-Np extract

C. lunata

A. alternata

C. lunata

A. alternata

0%

0%

0%

0%

0.5%

0.07 ± 0.01

0.08 ± 0.01

0.008 ± 0.00

0.03 ± 0.01

1%

0.03 ± 0.01

0.07 ± 0.005

0.006 ± 0.00

0.03 ± 0.01

2%

0.02 ± 0.01

0.06 ± 0.005

0.004 ± 0.00

0.02 ± 0.00

Note: The obtained data was analysed statistically by SPSS package and all values obtained have (p ≤ 0.05) and they were statistically significant.


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3.5 Plate test for protease production Protease production by C. lunata and A. alternata was confirmed by zone of clearance which was due to utilisation of casein provided in the growth medium.

3.6 Protease activity Concerning the effect of the extracts (CDW and Ag-Np) of Zingiber officinale on the activity of hydrolytic enzymes from C. lunata and A. alternata, protease activity was inhibited gradually by CDW extract as shown in Figure 4. These results are in confirmatory with the study made by Fawzi et al. (2009) where Zingiber officinale was shown to inhibit the activity of proteases produced by A. alternata and Fusarium oxysporum. Also, Masuduzzaman et al. (2008) stated Allamanda leaf extract showed to possess inhibitory effects against hydrolytic enzymes. Ag-Np extract inhibited the activity of enzyme strongly than CDW extract and exhibited maximum inhibition at the highest tested concentration (2%) as seen in Figure 5. Fungi possess a definite or specific infection pattern in order to infect the host plant. It is usually performed by the synthesis of a characteristic set of polymer-degrading enzymes to establish successful host-pathogen relationship. The present study demonstrated the higher effect of Ag-Np’s of Zingiber officinale over the grievous fungal enzyme having hazardous life threatening effect on cultivated crops. To best of our knowledge this is the first report of Ag-Np’s prepared from Zingiber officinale root extract to exhibit inhibitory effect against fungal proteases. Figure 4

The effect of CDW botanical extract with three different concentrations on the activity of protease from test moulds

Note: The obtained data was analysed statistically by SPSS package and all values obtained (p ≤ 0.05) were statistically significant.

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Figure 5

The effect of Ag-Np extract tested at three different concentrations (0.5%, 1% and 2%) on the protease activity of test moulds

Note: The obtained data was analysed statistically by SPSS package and all values obtained (p ≤ 0.05) were statistically significant.

4

Conclusions

CDW and Ag-Np extracts of Zingiber officinale were found to be effective in inhibiting the cell mass as well as proteases produced by pathogenic moulds of C. lunata and A. alternata. The biological method suggests being an economical, ecofriendly alternative to synthetic procedures and reduces the dependence on artificial fungicides. Thus, the Ag-Np extract of Zingiber officinale could be regarded as an effective antifungal agent and a promising material for alternative antimicrobial preparations to be incorporated in pharmaceutical formulations to treat various infections that affect the cultivated crops.

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