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Evaluation of antibacterial activities of silver nanoparticles green-synthesized using pineapple leaf (Ananas comosus) Elemike Elias Emeka a,∗ , Oseghale Charles Ojiefoh a , Chuku Aleruchi b , Labulo Ayomide Hassan a , Owoseni Mojisola Christiana b , Mfon Rebecca c , Enock Olugbenga Dare a,d,∗∗ , Adesuji Elijah Temitope a a
Department of Chemistry, Federal University Lafia, Nigeria Department of Microbiology, Federal University Lafia, Nigeria c Department of Physics, Federal University Lafia, Nigeria d Department of Chemistry, Federal University of Agriculture Abeokuta, Nigeria b
a r t i c l e
i n f o
Article history: Received 9 July 2013 Received in revised form 12 September 2013 Accepted 12 September 2013 Keywords: Pineapple leaf Esherichia coli Gentamycin Antibacterial Nanoparticles
a b s t r a c t Pineapple leaf was used in this study for the synthesis of silver nanoparticles based on the search for sustainable synthetic means. Indeed, this offered an economical and sustainable synthetic route relative to expensive and toxic chemical methods. The leaf extract was used and the corresponding nanoparticles obtained were subjected to UV–vis analysis at different times. The UV–vis was used to monitor the silver nanoparticle formation through sampling at time intervals. The formation of silver nanoparticles was apparently displayed within 2 min with evidence of surface plasmon bands (SPB) between 440 and 460 nm. The crystals was equally characterized using FTIR, X-ray diffraction methods and TEM. The different results obtained suggested the appearance of silver nanoparticles (SNPs) as determined by the process parameters with a particle size of 12.4 nm. The sample was further screened against Staphylococcus aureus, Streptococcus pneumoniae, Proteus mirabilis and Escherichia coli using Gentamicin as control. From the results, there is evidence of inhibition towards bacteria growth. It can now be inferred from the studies that biosynthesis of nanoparticles could be a gateway to our numerous health issues. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction The properties of matter at nanoscale level are significantly different from their macroscopic bulk properties. This makes the study of nanotechnology interesting as it takes into consideration the designing, characterization, production and application of structures, devices and systems by controlling shape and size at the nanometer scale (Mansoori, 2005). Nanoparticles are viewed as the fundamental building blocks of nanotechnology (Mansoori, 2005; Mansoori et al., 2007). They are the starting points for preparing many nanostructured materials and devices and their synthesis is an important component of the rapidly growing research efforts in nanoscience and nanoengineering (Mansoori et al., 2007). The nanoparticles of a wide
∗ Corresponding author. Tel.: +234 8035642445; fax: +234 7063917311. ∗∗ Corresponding author at: Department of Chemistry, Federal University of Agriculture Abeokuta, Nigeria. Tel.: +234 8035642445; fax: +234 7063917311. E-mail addresses: [email protected] (E.E. Emeka), [email protected] (O.C. Ojiefoh), [email protected] (C. Aleruchi), [email protected] (L.A. Hassan), [email protected] (O.M. Christiana), [email protected] (M. Rebecca), [email protected] (E.O. Dare), [email protected] (A.E. Temitope).
range of materials can be prepared by a number of methods using precursors from liquids, solid or gas phase (Mansoori, 2005). It has been reported that silver nanoparticles (SNPs) are non toxic to humans but inhibits the growth of bacteria, virus and other eukaryotic micro organisms (Jeong et al., 2005). Classically physical and chemical methods have been employed in synthesizing nanoparticles and these methods are costly, toxic and non-eco friendly but in recent times scientists are looking forward to low cost, non-toxic and eco-friendly synthetic methods. Most recently, biosynthesis of nanoparticles using bacteria (Shiying et al., 2007; Husseiny et al., 2007), fungus and plants (Raut et al., 2009) have emerged as a simple and viable alternative to more complex physical and chemical synthetic procedures to obtain nanomaterials. Silver nanoparticles are undoubtedly the most widely used nanomaterials and they have been used in textile industries, water treatment, sunscreen lotions, as anti-microbial agents etc. (Sharma et al., 2009). According to literature review, some studies have reported the synthesis of silver nanoparticles by plants such as Gliricidia sepium (Raut et al., 2009), Azadirachta indica (Shankar et al., 2004), Capsicum annuum (Bar et al., 2009), Carica papaya (Jha and Prasad, 2010), Eucalyptus hybrida (Dubey et al., 2009) and microorganisms such as Rhodopseudomonas capsulate (Shiying et al., 2007), Pseudomonas aeruginosa (Husseiny
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Please cite this article in press as: Emeka, E.E., et al., Evaluation of antibacterial activities of silver nanoparticles green-synthesized using pineapple leaf (Ananas comosus). Micron (2013), http://dx.doi.org/10.1016/j.micron.2013.09.003
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et al., 2007), Aspergillus fumigatus (Bhainsa and D’Souza, 2006), Cladosporium cladosporioides (Balaji et al., 2009), Fusarium oxysporum (Ahmad et al., 2003) and in our earlier research work, we reported the synthesis of silver nanoparticles using some selected alcoholic beverages in Nigeria (Adesuji et al., 2013). Pineapple (Ananas comosus L.), a leading edible member of the botanical family Bromeliaceae, is a perennial herb native to the American tropics (Bartholomew et al., 2003) which is well known for its freeness from harmful phytochemicals (Mateljan, 2007). Pineapples may be cultivated from a crown cutting of the fruit, possibly flowering in 20–24 months and fruiting in the following six months. Its leaves are narrow, fibrous and spiny and the fruit grows on a stalk in the center of the rosette of leaves. Beside fruits, pineapple fields yield large amounts of leaves that may be used for their high quality fibre or as feedstuff for ruminants (Ecocrop, 2011). Ananas comosus peels, core extracts and crown extract contain sugar especially fructose, sucrose and glucose (Nadzirah et al., 2013). The peak in sucrose concentration of matured fruit is attained at full-yellow stage and then declines. A wide range of volatiles (more than 280 compounds) have been identified, including esters, terpenes, lactones, aldehydes, ketones, alcohols, hydrocarbons and a group of miscellaneous compounds. The main volatile compounds found in pineapple pulp and cores are esters, followed by terpenes, ketones and aldehydes (ChangBin et al., 2011). Biomolecules with carbonyl, hydroxyl, and amine functional groups have the potential for metal ion reduction and capping the newly formed particles during their growth processes (Shiying et al., 2007). In view of this knowledge, a detailed approach was designed to explore the potential of pineapple leaf towards reduction, capping and stabilization of silver compounds. In this research work, a clear definition of the synthesis, characterization and antibacterial activities of the silver nanoparticles of pineapple leaf extract was fully investigated and reported. Our aim is to use materials, which are environmental wastes to reduce Ag+ to Ag0 . The synthesized silver nanoparticles was characterized using XRD, UV–vis spectrophotometer, FTIR, transmission electron microscope (TEM) and its application as antimicrobial agents was also investigated. 2. Methodology 2.1. Materials Pineapple leaf, AgNO3 . 2.2. Organisms Staphylococcus aureus, Streptococcus mirabilis and Escherichia coli.
pneumoniae,
Proteus
2.3. Preparation of extract Fresh pineapple was collected from Lafia market in Nasarawa state and the leaf was washed, chopped and blended. Afterwards, it was extracted with water (ratio 1:10 w/v) at 70 ◦ C and filtered with no. 1 Whatman filter paper. The filtrate was collected and kept at 4 ◦ C and was further used for synthesis of the nanoparticles (Raut et al., 2009). 2.4. Synthesis of silver nanoparticles using the pineapple leaf extract The filtrate (5.0 mL) of pineapple leaf extract was added to 20 mL of 1 mM aqueous AgNO3 solution. The resulting solution was heated at 70 ◦ C and colour change observed. Aliquot sample of the solution was taken at intervals (0–30 min) and rate of the reduction of Ag+
1.2 1
Absorbance
2
0mins
0.8
2mins 0.6
5mins 10mins
0.4
15mins 30mins
0.2 0
300 350 400 450 500 550 600 650 700 750 wavelength (nm)
Fig. 1. UV–vis absorption peaks of the synthesized silver nanoparticles using pineapple crown leaf extract.
ions to Ag0 monitored by measuring the absorbance or appearance of plasmon bands with T60 UV–vis spectrophotometer. 2.5. Characterization The bioreduction of the Ag+ to Ag0 was monitored using UV–vis spectrophotometer (T60 UV–vis spectrophotometer) at regular intervals with samples in Quartz cuvette operated at a resolution of 1 nm. The functional group responsible for the silver nanoparticles was analysed using FTIR (Perkin Elmer Spectrum, USA). The crystallinity of the silver nanoparticles was analysed using X-ray diffraction (XRD) methods while the size of synthesized nanoparticle was determined using scanning electron microscope (SEM) (JEOL Ltd, Tokyo, Japan). 2.6. Antibacterial analysis The antibacterial screening of the silver nanoparticles was carried out against S. aureus, S. pneumoniae, P. mirabilis and Esherichia coli using the Agar-well diffusion method. 0.5 mL each of the seeded broth containing 105 cfu/mL of the test organisms were incubated on solid nutrient agar plates and spread uniformly with a glass spreader. Three wells were cut out in the agar layer of the plate using an aluminium bore of 2 mm diameter (Prabakaran et al., 2012). Then, 0.2 mL each of the pineapple leaf extract, Gentamycin and normal saline were placed in the wells using a micropipette and further incubated at 37 ◦ C for 24 h. Gentamycin was used as the positive control while normal saline was used as the negative control (Fig. 1). The mean diameter of the zone of inhibition in mm obtained around the well was measured and the values shown in Table 1. 3. Results 3.1. UV–vis spectra analysis The UV–vis spectroscopy was used to examine the sizes and shapes of the nanoparticles in aqueous suspensions (Swarnalathan et al., 2012; Wiley et al., 2006) The synthesized pineapple leaf silver nanoparticle was reddish brown in aqueous solution due to excitation of electrons and changes in electronic energy levels reflecting the reduction of Ag+ into Ag0 (Dare et al., 2012). In general, morphology of the nanoparticles is greatly influenced by the surface plasmon resonance (SPR), since it is the basis for measuring adsorption of material onto the surface of metal nanoparticles. The formation of the nanoparticles was observed at intervals (0–30 min) with a characteristic plasmon band ranging between 440 and 460 nm appearing at about the second minute of the reaction. The intensity of the peaks continues to increase from 2 min up to the 30th min with little variation in the absorption maxima which
Please cite this article in press as: Emeka, E.E., et al., Evaluation of antibacterial activities of silver nanoparticles green-synthesized using pineapple leaf (Ananas comosus). Micron (2013), http://dx.doi.org/10.1016/j.micron.2013.09.003
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Table 1 The mean diameter of the zone of inhibition in mm. Diameter of zone of inhibition (mm) Test organisms
Gentamycin +ve control (mm)
Normal saline −ve control (mm)
Ag nanoparticles From pineapple leaf
Result
Staphylococcus aureus Streptococcus pneumoniae Proteus mirabilis Escherichia coli
27 13 15 25
0 0 0 0
15 5 0 20
+ + − +
+, susceptible; −, resistant.
spectra result shown in Fig. 3 represents the silver nanoparticles from the leaf extract while Fig. 4 describes the ordinary pineapple leaf extract. From Fig. 4, the broad peak at 3354 cm−1 could be assigned to O H stretch while the peaks at 2976 cm−1 , 2896 cm−1 , 2836 cm−1 could be attributed to the C H stretching vibrations of methyl, methylene, or methoxy groups. There appeared other sharper peaks located at 1654 cm−1 , 420 cm−1 which were assigned to the C O stretching in carbonyl group while the peak at 1062 cm−1 was assigned to the C O stretch of the alcoholic group. These biomolecules were identified as possible stabilizing groups that contributed to the nanostructuring of the silver ion and it was evident in the FTIR spectra in Fig. 3. Comparing the two spectra, there was no free C O found in the nanoparticle spectra (Fig. 3) as against the 1654 cm−1 in Fig. 4, suggesting that stabilization of the system may have resulted from binding of the carbonyl group of the reducing sugars to the silver (Venu et al., 2011).
3.3. XRD analysis
Fig. 2. Comparison of UV–vis of pineapple leaf synthesized SNPs (a) and beverage mediated SNPs (b).
shows particle size variation (Muhammad et al., 2012). As the reaction progresses after the first two minutes, there was evidence of further decrease in sizes of the nanoparticles within 5–15 min. However, sampling within 10 and 15 min recorded the same sizes as shown in the overlapping SPB. Thereafter, at the 30th min, the size of silver nanoparticle picks up displaying SPB of higher intensity. Comparing our previous work (Adesuji et al., 2013), on synthesis of nanoparticles from alcoholic drinks, where in some of the samples, the plasmon bands did not appear until the 30th min (Fig. 2), this present work now shows that the substrate is a more promising one because it saves time and exhibits better stabilizing phenomenon. 3.2. FTIR spectroscopy The FTIR analysis was carried out between 4000 and 600 cm−1 to identify the functional groups responsible for capping and stabilizing the Ag nanoparticles. The
The X-ray diffraction pattern of the pineapple leaf silver nanoparticles is shown in Fig. 5. Four sharp peaks are observed at 2 values of 38.20, 44.400, 64.60 and 77.50 which are indexed as (1 1 1), (2 0 0), (2 2 0) and (3 1 1) bands of face centred cubic (fcc) structures of silver. Similar values of diffraction peaks for nano silver have been reported by others (Vimala et al., 2009). Using the Debye–Scherrer equation, K/ˇ cos , where K is the Scherrer constant with value from 0.9 to 1, the wavelength of the X-ray, ˇ full width at half maximum and the Bragg angle in radians, the average crystallite size of silver nanoparticles was found to be about 12.4 nm.
3.4. TEM analysis The shape, size and morphology of the synthesized silver nanoparticles were elucidated with the help of transmission electron microscopy. The TEM images confirmed the formation of silver nanoparticles (Fig. 5). The nanoparticles are homogeneous and spherical which conforms to the shape of SPR band in the UV–vis spectrum with an average diameter of 12.4 nm. This particle size agrees with that calculated from XRD analysis. Fig. 5b shows the size distribution of the silver nanoparticle (Figs. 6 and 7).
120 110 100
%T
90
3662.51 2825.33
80 70 60
1012.63 3696.18 3680.72 2921.95 2980.72 2865.06 2972.49
1054.71 1032.68
50 40 30 20 4000
3500
3000
2500
2000
1500
1000
Wavenumbers (cm-1) Fig. 3. FTIR analysis of synthesized silver nanoparticles using pineapple leaf extract.
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%T
4
120 110 100 90 80 70 60 50 40 30 20 10 0 -10 -20 -30 4000
2836.23
1420.19 1654.06 1086.48
878.24 1024.51
2896.50 3354.04
3500
1043.00
2976.74
3000
2500
2000
1500
1000
Wavenumbers (cm-1) Fig. 4. FTIR analysis of pineapple leaf extract.
Fig. 7. Antibacterial activities of (a) S. aureus, (b) E. coli and (c) Streptococcus pneumoniae.
4. Discussion
Fig. 5. XRD spectra pattern of synthesized silver nanoparticle using pineapple leaf extract.
3.5. Antibacterial assay The susceptibility of S. aureus, S. pneumoniae and E. coli showed that the silver nanoparticles from pineapple leaf extract contained antibacterial properties that inhibited both Gram +ve cocci (S. aureus and S. pneumoniae) and Gram −ve bacilli (E. coli) thereby placing antimicrobial activity on a broad spectrum plane. Due to the highly susceptibility of S. aureus and E. coli, the synthesized silver nanoparticles would be very valuable in the treatment of various infections caused by the above two organisms, thereby truly converting waste to wealth to the benefit of mankind in the health sector.
This study has shown that pineapple leaf otherwise considered as waste has transformed silver nitrate to nanoparticles with great stability. The appearance of brownish colouration confirmed by surface plasmon bands at 440–460 nm proved the existence of silver nanoparticles. The FTIR result suggested that sugars such as sucrose, glucose etc. could be responsible for the bioreduction of the AgNO3 . From the kinetics studies, the formation of the nanoparticles almost started from the second minute of the reaction and continued to give sharper peaks at varying wavelengths showing that the nanoparticles formed are varying in sizes and also stable. The application of the synthesized nanoparticles as antimicrobial agents has equally yielded positive result as it inhibits the growth of bacteria though it showed lower hindrance abilities against the tested isolates compared to the control (Gentamycin). It is believed that Ag nanoparticles penetrate into the cell walls of the microbes, causing cellular damage by interacting with phosphorus and sulphur containing compounds, such as DNA and protein present inside the cell. Therefore, the bacteriocidal properties of silver nanoparticles synthesized are due to the release of Ag which confers the antimicrobial activity (Amarendra and Krishna, 2010). From Fig. 2, it is interesting to know that the UV absorption arising from pineapple leaf extract SNPs displayed a shift towards higher UV region (bathocromic shift). This characteristic behaviour is an indication of enhanced properties towards optical materials. It is therefore evident that the application will potentially transcend antimicrobial activities. Future work on the further application is underway. 5. Conclusion
Fig. 6. (a) TEM image of synthesized silver nanoparticle using pineapple crown leaf extract. (b) Particle size distribution of the synthesized silver nanoparticles.
The field of nanotechnology became imperative in this present time as it seems to offer solutions to the imminent industrial, health and environmental problems of the world. Future research
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is underway as regards to the potentialities of the silver nanoparticles from the pineapple leaf extract, as it has proven to exhibit antimicrobial properties as well as enhanced optical properties. Conflict of interest The authors report no conflict of interest in this work. Acknowledgements The authors wish to acknowledge the Vice Chancellor of Federal University Lafia, Prof. Ekanem Ikpi Braide, Dean Faculty of Science, Prof. Martin Ogbe and the entire Staff of Chemistry, Microbiology and Physics Department for their support during the course of this research work. Special thanks also go to Mr. Danjuma Saidu and Mr Osuagwu Stanislaus for their technical support. References Adesuji, E.T., Elemike, E.E., Chuku, A., Labulo, A.H., Owoseni, M.C., Oseghale, C.O., Mfon, R., Dare, O.E., 2013. Synthesis of silver nanoparticles using some alcoholic beverages from Nigeria market. International Journal of Nano and Material Sciences 2 (1), 25–35. Ahmad, A., Mukherjee, P., Senapati, S., Mandal, D., Khan, M.I., Kumar, R., Sastry, M., 2003. Extracellular biosynthesis of silver nanoparticles using the fungus Fusarium oxysporum. Colloids and Surfaces B 28, 313–318. Amarendra, D.D., Krishna, G., 2010. Biosynthesis of silver and gold nanoparticles using chenopodium album leaf extract. Colloids and Surfaces A 369 (3), 27–33. Balaji, D.S., Basavaraja, S., Deshpande, R., Mahesh, D.B., Prabhakar, B.K., Venkataraman, A., 2009. Extracellular biosynthesis of functionalized silver nanoparticles by strains of Cladosporium cladosporioides fungus. Colloids and Surfaces B: Biointerfaces 68 (1), 88–92. Bar, H., Bhui, D.K., Sahoo, G.P., Sarkar, P., De, S.P., Misra, A., 2009. Green synthesis of silver nanoparticles using latex of Jatropha curcas. Colloids and Surfaces A: Physicochemical and Engineering Aspects 339, 134–139. Bartholomew, D., Paul, R., Rohrbach, K., 2003. The Pineapple: Botany, Production and Uses. CABI Publishing, Wallingford, UK, pp. 1–301. Bhainsa, K.C., D’Souza, S.F., 2006. Extracellular biosynthesis of silver nanoparticles using the fungus Aspergillus fumigates. Colloids and Surfaces B: Biointerfaces 47 (2), 160–164. Chang-Bin, W., Sheng-Hui, L., Yu-Ge, L., Ling-Ling, L., Wen-Xiu, Y., Guang-Ming, S., 2011. Characteristic aroma compounds from different pineapple parts. Molecules 16, 5104–5112. Dare, E.O., Makinde, O.W., Ogundele, K.T., Osinkolu, G.A., Fasasi, Y.A., Sonde, I., Bamgbose, J.T., Maaza, M., Sithole, J., Ezema, F., Adewoye, O.O., 2012. Zinc mediated synthesis,growth kinectic, and shaped evolution of silver nanoparticles. International Scholarly Research Network Nanomaterials, 1–8. Dubey, M., Bhadauria, S., Kushwah, B.S., 2009. Green synthesis of nanosilver particles from extract of Eucalyptus hybrida (Safeda) leaf. Digest Journal of Nanomaterials and Biostructures 4, 537–543.
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Ecocrop, 2011. Ecocrop Database. FAO, http://www.feedipedia.org/node/5368 (retrieved 12.06.13). Husseiny, M.I., El-Aziz, M.A., Badr, Y., Mahmoud, M.A., 2007. Biosynthesis of gold nanoparticles using Pseudomonas aeruginosa. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 67, 1003–1006. Jeong, S.H., Yeo, S.Y., Yi, S.C., 2005. The effect of filler particle size on the antibacterial properties of compounded polymer/silver fibres. Journal of Materials Science 40, 5407–5411. Jha, A.K., Prasad, K., 2010. Green synthesis of silver nanoparticles using Cycas leaf. International Journal of Green Nanotechnology: Physics and Chemistry 1 (2), 110–117. Mansoori, G.A., 2005. Principles of Nanotechnology – Molecular-Based Study of Condensed Matter in Small Systems. World Scientific Pub. Co., Hackensack, NJ. Mansoori, G.A., George, T.F., Zhang, G., Assoufid, L., 2007. Molecular Building Blocks for Nanotechnology. Springer, New York. Mateljan, G., 2007. The Worlds Healthiest Foods, vols. 46/45. United Sates, Seattle WA, pp. 8699–8702. Muhammad, A., Farooq, A., Muhammad, R., Saeed, A.J., Muhammad, A.I., Umer, R., 2012. Green synthesis of silver nanoparticles through reduction with Solanum xanthocarpum L. berry extract: characterization, antimicrobial and urease inhibitory activities against Helicobacter pylori. International Journal of Molecular Sciences 13, 9923–9941. Nadzirah, K.Z., Zainal, S., Noriham, A., Normah, I., Siti Roha, A.M., Nadya, H., 2013. Physico-chemical properties of pineapple variety N36 harvested and stored at different maturity stages. International Food Research Journal 20 (1), 225–231. Prabakaran, M., Subha, K., Thennarasu, V., Merinal, S., 2012. Biosynthesis of silver nanoparticles using sphaerulina albispiculata and evaluation of antibacterial activity. European Journal of Experimental Biology 2 (1), 297–303. Raut, R.W., Lakkakula, J.R., Kolekar, N.S., Mendhulkar, V.D., Kashid, S.B., 2009. Phytosynthesis of silver nanoparticle using Gliricidia sepium (Jacq.). Current Nanoscience 5, 117–122. Shankar, S.S., Rai, A., Ankamwar, B., Singh, A., Ahmad, A., Sastry, M., 2004. Biological synthesis of triangular gold nanoprisms, US National Library of Medicine National Institutes of Health. Nature Materials 3, 482–488. Sharma, V.K., Ria, A.Y., Lin, Y., 2009. Silver nanoparticles: green synthesis and their antimicrobial activities. Advances in Colloid and Interface Science 145, 83–96. Shiying, H., Zhirui, G., Yu, Z., Song, Z., Jing, W., Ning, G., 2007. Biosynthesis of gold nanoparticles using the bacteria Rhodopseudomonas capsulata. Materials Letters 61, 3984–3987. Swarnalathan, R., Christina, R., Shruti, B., Payas, 2012. Evaluation of in vitro antidiabetic activity of sphaeranthus amaranthoides silver nanoparticles. International Journal of Nanomaterials and Biostructures 2 (3), 25–29. Venu, R., Ramilu, T.S., Anandakumar, S., Rani, V.S., Kim, C.G., 2011. Bio-directed synthesis of platinum nanoparticles using aqeous honey solutions and their catalytic applications. Colloids and Surfaces A: Physicochemical and Engineering Aspects 384, 733–738. Vimala, K., Sivudu, K.S., Mohan, Y.M., Sreedhar, B., Raju, K.M., 2009. Permeation of sodium decyl sulfate through polyaniline-modified cellulose acetate membranes. Carbohydrate Polymers 75, 463–471. Wiley, B.J., Im, S.H., McLellan, J., Siekkinen, A., Xia, Y., 2006. Maneuvering the surface plasmon resonance of silver nanostructures through shape-controlled synthesis. The Journal of Physical Chemistry B 110, 15666–15675.
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ARTICLE IN PRESS Micron xxx (2013) xxx–xxx
Contents lists available at ScienceDirect
Micron journal homepage: www.elsevier.com/locate/micron
Evaluation of antibacterial activities of silver nanoparticles green-synthesized using pineapple leaf (Ananas comosus) Elemike Elias Emeka a,∗ , Oseghale Charles Ojiefoh a , Chuku Aleruchi b , Labulo Ayomide Hassan a , Owoseni Mojisola Christiana b , Mfon Rebecca c , Enock Olugbenga Dare a,d,∗∗ , Adesuji Elijah Temitope a a
Department of Chemistry, Federal University Lafia, Nigeria Department of Microbiology, Federal University Lafia, Nigeria c Department of Physics, Federal University Lafia, Nigeria d Department of Chemistry, Federal University of Agriculture Abeokuta, Nigeria b
a r t i c l e
i n f o
Article history: Received 9 July 2013 Received in revised form 12 September 2013 Accepted 12 September 2013 Keywords: Pineapple leaf Esherichia coli Gentamycin Antibacterial Nanoparticles
a b s t r a c t Pineapple leaf was used in this study for the synthesis of silver nanoparticles based on the search for sustainable synthetic means. Indeed, this offered an economical and sustainable synthetic route relative to expensive and toxic chemical methods. The leaf extract was used and the corresponding nanoparticles obtained were subjected to UV–vis analysis at different times. The UV–vis was used to monitor the silver nanoparticle formation through sampling at time intervals. The formation of silver nanoparticles was apparently displayed within 2 min with evidence of surface plasmon bands (SPB) between 440 and 460 nm. The crystals was equally characterized using FTIR, X-ray diffraction methods and TEM. The different results obtained suggested the appearance of silver nanoparticles (SNPs) as determined by the process parameters with a particle size of 12.4 nm. The sample was further screened against Staphylococcus aureus, Streptococcus pneumoniae, Proteus mirabilis and Escherichia coli using Gentamicin as control. From the results, there is evidence of inhibition towards bacteria growth. It can now be inferred from the studies that biosynthesis of nanoparticles could be a gateway to our numerous health issues. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction The properties of matter at nanoscale level are significantly different from their macroscopic bulk properties. This makes the study of nanotechnology interesting as it takes into consideration the designing, characterization, production and application of structures, devices and systems by controlling shape and size at the nanometer scale (Mansoori, 2005). Nanoparticles are viewed as the fundamental building blocks of nanotechnology (Mansoori, 2005; Mansoori et al., 2007). They are the starting points for preparing many nanostructured materials and devices and their synthesis is an important component of the rapidly growing research efforts in nanoscience and nanoengineering (Mansoori et al., 2007). The nanoparticles of a wide
∗ Corresponding author. Tel.: +234 8035642445; fax: +234 7063917311. ∗∗ Corresponding author at: Department of Chemistry, Federal University of Agriculture Abeokuta, Nigeria. Tel.: +234 8035642445; fax: +234 7063917311. E-mail addresses: [email protected] (E.E. Emeka), [email protected] (O.C. Ojiefoh), [email protected] (C. Aleruchi), [email protected] (L.A. Hassan), [email protected] (O.M. Christiana), [email protected] (M. Rebecca), [email protected] (E.O. Dare), [email protected] (A.E. Temitope).
range of materials can be prepared by a number of methods using precursors from liquids, solid or gas phase (Mansoori, 2005). It has been reported that silver nanoparticles (SNPs) are non toxic to humans but inhibits the growth of bacteria, virus and other eukaryotic micro organisms (Jeong et al., 2005). Classically physical and chemical methods have been employed in synthesizing nanoparticles and these methods are costly, toxic and non-eco friendly but in recent times scientists are looking forward to low cost, non-toxic and eco-friendly synthetic methods. Most recently, biosynthesis of nanoparticles using bacteria (Shiying et al., 2007; Husseiny et al., 2007), fungus and plants (Raut et al., 2009) have emerged as a simple and viable alternative to more complex physical and chemical synthetic procedures to obtain nanomaterials. Silver nanoparticles are undoubtedly the most widely used nanomaterials and they have been used in textile industries, water treatment, sunscreen lotions, as anti-microbial agents etc. (Sharma et al., 2009). According to literature review, some studies have reported the synthesis of silver nanoparticles by plants such as Gliricidia sepium (Raut et al., 2009), Azadirachta indica (Shankar et al., 2004), Capsicum annuum (Bar et al., 2009), Carica papaya (Jha and Prasad, 2010), Eucalyptus hybrida (Dubey et al., 2009) and microorganisms such as Rhodopseudomonas capsulate (Shiying et al., 2007), Pseudomonas aeruginosa (Husseiny
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Please cite this article in press as: Emeka, E.E., et al., Evaluation of antibacterial activities of silver nanoparticles green-synthesized using pineapple leaf (Ananas comosus). Micron (2013), http://dx.doi.org/10.1016/j.micron.2013.09.003
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et al., 2007), Aspergillus fumigatus (Bhainsa and D’Souza, 2006), Cladosporium cladosporioides (Balaji et al., 2009), Fusarium oxysporum (Ahmad et al., 2003) and in our earlier research work, we reported the synthesis of silver nanoparticles using some selected alcoholic beverages in Nigeria (Adesuji et al., 2013). Pineapple (Ananas comosus L.), a leading edible member of the botanical family Bromeliaceae, is a perennial herb native to the American tropics (Bartholomew et al., 2003) which is well known for its freeness from harmful phytochemicals (Mateljan, 2007). Pineapples may be cultivated from a crown cutting of the fruit, possibly flowering in 20–24 months and fruiting in the following six months. Its leaves are narrow, fibrous and spiny and the fruit grows on a stalk in the center of the rosette of leaves. Beside fruits, pineapple fields yield large amounts of leaves that may be used for their high quality fibre or as feedstuff for ruminants (Ecocrop, 2011). Ananas comosus peels, core extracts and crown extract contain sugar especially fructose, sucrose and glucose (Nadzirah et al., 2013). The peak in sucrose concentration of matured fruit is attained at full-yellow stage and then declines. A wide range of volatiles (more than 280 compounds) have been identified, including esters, terpenes, lactones, aldehydes, ketones, alcohols, hydrocarbons and a group of miscellaneous compounds. The main volatile compounds found in pineapple pulp and cores are esters, followed by terpenes, ketones and aldehydes (ChangBin et al., 2011). Biomolecules with carbonyl, hydroxyl, and amine functional groups have the potential for metal ion reduction and capping the newly formed particles during their growth processes (Shiying et al., 2007). In view of this knowledge, a detailed approach was designed to explore the potential of pineapple leaf towards reduction, capping and stabilization of silver compounds. In this research work, a clear definition of the synthesis, characterization and antibacterial activities of the silver nanoparticles of pineapple leaf extract was fully investigated and reported. Our aim is to use materials, which are environmental wastes to reduce Ag+ to Ag0 . The synthesized silver nanoparticles was characterized using XRD, UV–vis spectrophotometer, FTIR, transmission electron microscope (TEM) and its application as antimicrobial agents was also investigated. 2. Methodology 2.1. Materials Pineapple leaf, AgNO3 . 2.2. Organisms Staphylococcus aureus, Streptococcus mirabilis and Escherichia coli.
pneumoniae,
Proteus
2.3. Preparation of extract Fresh pineapple was collected from Lafia market in Nasarawa state and the leaf was washed, chopped and blended. Afterwards, it was extracted with water (ratio 1:10 w/v) at 70 ◦ C and filtered with no. 1 Whatman filter paper. The filtrate was collected and kept at 4 ◦ C and was further used for synthesis of the nanoparticles (Raut et al., 2009). 2.4. Synthesis of silver nanoparticles using the pineapple leaf extract The filtrate (5.0 mL) of pineapple leaf extract was added to 20 mL of 1 mM aqueous AgNO3 solution. The resulting solution was heated at 70 ◦ C and colour change observed. Aliquot sample of the solution was taken at intervals (0–30 min) and rate of the reduction of Ag+
1.2 1
Absorbance
2
0mins
0.8
2mins 0.6
5mins 10mins
0.4
15mins 30mins
0.2 0
300 350 400 450 500 550 600 650 700 750 wavelength (nm)
Fig. 1. UV–vis absorption peaks of the synthesized silver nanoparticles using pineapple crown leaf extract.
ions to Ag0 monitored by measuring the absorbance or appearance of plasmon bands with T60 UV–vis spectrophotometer. 2.5. Characterization The bioreduction of the Ag+ to Ag0 was monitored using UV–vis spectrophotometer (T60 UV–vis spectrophotometer) at regular intervals with samples in Quartz cuvette operated at a resolution of 1 nm. The functional group responsible for the silver nanoparticles was analysed using FTIR (Perkin Elmer Spectrum, USA). The crystallinity of the silver nanoparticles was analysed using X-ray diffraction (XRD) methods while the size of synthesized nanoparticle was determined using scanning electron microscope (SEM) (JEOL Ltd, Tokyo, Japan). 2.6. Antibacterial analysis The antibacterial screening of the silver nanoparticles was carried out against S. aureus, S. pneumoniae, P. mirabilis and Esherichia coli using the Agar-well diffusion method. 0.5 mL each of the seeded broth containing 105 cfu/mL of the test organisms were incubated on solid nutrient agar plates and spread uniformly with a glass spreader. Three wells were cut out in the agar layer of the plate using an aluminium bore of 2 mm diameter (Prabakaran et al., 2012). Then, 0.2 mL each of the pineapple leaf extract, Gentamycin and normal saline were placed in the wells using a micropipette and further incubated at 37 ◦ C for 24 h. Gentamycin was used as the positive control while normal saline was used as the negative control (Fig. 1). The mean diameter of the zone of inhibition in mm obtained around the well was measured and the values shown in Table 1. 3. Results 3.1. UV–vis spectra analysis The UV–vis spectroscopy was used to examine the sizes and shapes of the nanoparticles in aqueous suspensions (Swarnalathan et al., 2012; Wiley et al., 2006) The synthesized pineapple leaf silver nanoparticle was reddish brown in aqueous solution due to excitation of electrons and changes in electronic energy levels reflecting the reduction of Ag+ into Ag0 (Dare et al., 2012). In general, morphology of the nanoparticles is greatly influenced by the surface plasmon resonance (SPR), since it is the basis for measuring adsorption of material onto the surface of metal nanoparticles. The formation of the nanoparticles was observed at intervals (0–30 min) with a characteristic plasmon band ranging between 440 and 460 nm appearing at about the second minute of the reaction. The intensity of the peaks continues to increase from 2 min up to the 30th min with little variation in the absorption maxima which
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Table 1 The mean diameter of the zone of inhibition in mm. Diameter of zone of inhibition (mm) Test organisms
Gentamycin +ve control (mm)
Normal saline −ve control (mm)
Ag nanoparticles From pineapple leaf
Result
Staphylococcus aureus Streptococcus pneumoniae Proteus mirabilis Escherichia coli
27 13 15 25
0 0 0 0
15 5 0 20
+ + − +
+, susceptible; −, resistant.
spectra result shown in Fig. 3 represents the silver nanoparticles from the leaf extract while Fig. 4 describes the ordinary pineapple leaf extract. From Fig. 4, the broad peak at 3354 cm−1 could be assigned to O H stretch while the peaks at 2976 cm−1 , 2896 cm−1 , 2836 cm−1 could be attributed to the C H stretching vibrations of methyl, methylene, or methoxy groups. There appeared other sharper peaks located at 1654 cm−1 , 420 cm−1 which were assigned to the C O stretching in carbonyl group while the peak at 1062 cm−1 was assigned to the C O stretch of the alcoholic group. These biomolecules were identified as possible stabilizing groups that contributed to the nanostructuring of the silver ion and it was evident in the FTIR spectra in Fig. 3. Comparing the two spectra, there was no free C O found in the nanoparticle spectra (Fig. 3) as against the 1654 cm−1 in Fig. 4, suggesting that stabilization of the system may have resulted from binding of the carbonyl group of the reducing sugars to the silver (Venu et al., 2011).
3.3. XRD analysis
Fig. 2. Comparison of UV–vis of pineapple leaf synthesized SNPs (a) and beverage mediated SNPs (b).
shows particle size variation (Muhammad et al., 2012). As the reaction progresses after the first two minutes, there was evidence of further decrease in sizes of the nanoparticles within 5–15 min. However, sampling within 10 and 15 min recorded the same sizes as shown in the overlapping SPB. Thereafter, at the 30th min, the size of silver nanoparticle picks up displaying SPB of higher intensity. Comparing our previous work (Adesuji et al., 2013), on synthesis of nanoparticles from alcoholic drinks, where in some of the samples, the plasmon bands did not appear until the 30th min (Fig. 2), this present work now shows that the substrate is a more promising one because it saves time and exhibits better stabilizing phenomenon. 3.2. FTIR spectroscopy The FTIR analysis was carried out between 4000 and 600 cm−1 to identify the functional groups responsible for capping and stabilizing the Ag nanoparticles. The
The X-ray diffraction pattern of the pineapple leaf silver nanoparticles is shown in Fig. 5. Four sharp peaks are observed at 2 values of 38.20, 44.400, 64.60 and 77.50 which are indexed as (1 1 1), (2 0 0), (2 2 0) and (3 1 1) bands of face centred cubic (fcc) structures of silver. Similar values of diffraction peaks for nano silver have been reported by others (Vimala et al., 2009). Using the Debye–Scherrer equation, K/ˇ cos , where K is the Scherrer constant with value from 0.9 to 1, the wavelength of the X-ray, ˇ full width at half maximum and the Bragg angle in radians, the average crystallite size of silver nanoparticles was found to be about 12.4 nm.
3.4. TEM analysis The shape, size and morphology of the synthesized silver nanoparticles were elucidated with the help of transmission electron microscopy. The TEM images confirmed the formation of silver nanoparticles (Fig. 5). The nanoparticles are homogeneous and spherical which conforms to the shape of SPR band in the UV–vis spectrum with an average diameter of 12.4 nm. This particle size agrees with that calculated from XRD analysis. Fig. 5b shows the size distribution of the silver nanoparticle (Figs. 6 and 7).
120 110 100
%T
90
3662.51 2825.33
80 70 60
1012.63 3696.18 3680.72 2921.95 2980.72 2865.06 2972.49
1054.71 1032.68
50 40 30 20 4000
3500
3000
2500
2000
1500
1000
Wavenumbers (cm-1) Fig. 3. FTIR analysis of synthesized silver nanoparticles using pineapple leaf extract.
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%T
4
120 110 100 90 80 70 60 50 40 30 20 10 0 -10 -20 -30 4000
2836.23
1420.19 1654.06 1086.48
878.24 1024.51
2896.50 3354.04
3500
1043.00
2976.74
3000
2500
2000
1500
1000
Wavenumbers (cm-1) Fig. 4. FTIR analysis of pineapple leaf extract.
Fig. 7. Antibacterial activities of (a) S. aureus, (b) E. coli and (c) Streptococcus pneumoniae.
4. Discussion
Fig. 5. XRD spectra pattern of synthesized silver nanoparticle using pineapple leaf extract.
3.5. Antibacterial assay The susceptibility of S. aureus, S. pneumoniae and E. coli showed that the silver nanoparticles from pineapple leaf extract contained antibacterial properties that inhibited both Gram +ve cocci (S. aureus and S. pneumoniae) and Gram −ve bacilli (E. coli) thereby placing antimicrobial activity on a broad spectrum plane. Due to the highly susceptibility of S. aureus and E. coli, the synthesized silver nanoparticles would be very valuable in the treatment of various infections caused by the above two organisms, thereby truly converting waste to wealth to the benefit of mankind in the health sector.
This study has shown that pineapple leaf otherwise considered as waste has transformed silver nitrate to nanoparticles with great stability. The appearance of brownish colouration confirmed by surface plasmon bands at 440–460 nm proved the existence of silver nanoparticles. The FTIR result suggested that sugars such as sucrose, glucose etc. could be responsible for the bioreduction of the AgNO3 . From the kinetics studies, the formation of the nanoparticles almost started from the second minute of the reaction and continued to give sharper peaks at varying wavelengths showing that the nanoparticles formed are varying in sizes and also stable. The application of the synthesized nanoparticles as antimicrobial agents has equally yielded positive result as it inhibits the growth of bacteria though it showed lower hindrance abilities against the tested isolates compared to the control (Gentamycin). It is believed that Ag nanoparticles penetrate into the cell walls of the microbes, causing cellular damage by interacting with phosphorus and sulphur containing compounds, such as DNA and protein present inside the cell. Therefore, the bacteriocidal properties of silver nanoparticles synthesized are due to the release of Ag which confers the antimicrobial activity (Amarendra and Krishna, 2010). From Fig. 2, it is interesting to know that the UV absorption arising from pineapple leaf extract SNPs displayed a shift towards higher UV region (bathocromic shift). This characteristic behaviour is an indication of enhanced properties towards optical materials. It is therefore evident that the application will potentially transcend antimicrobial activities. Future work on the further application is underway. 5. Conclusion
Fig. 6. (a) TEM image of synthesized silver nanoparticle using pineapple crown leaf extract. (b) Particle size distribution of the synthesized silver nanoparticles.
The field of nanotechnology became imperative in this present time as it seems to offer solutions to the imminent industrial, health and environmental problems of the world. Future research
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is underway as regards to the potentialities of the silver nanoparticles from the pineapple leaf extract, as it has proven to exhibit antimicrobial properties as well as enhanced optical properties. Conflict of interest The authors report no conflict of interest in this work. Acknowledgements The authors wish to acknowledge the Vice Chancellor of Federal University Lafia, Prof. Ekanem Ikpi Braide, Dean Faculty of Science, Prof. Martin Ogbe and the entire Staff of Chemistry, Microbiology and Physics Department for their support during the course of this research work. Special thanks also go to Mr. Danjuma Saidu and Mr Osuagwu Stanislaus for their technical support. References Adesuji, E.T., Elemike, E.E., Chuku, A., Labulo, A.H., Owoseni, M.C., Oseghale, C.O., Mfon, R., Dare, O.E., 2013. Synthesis of silver nanoparticles using some alcoholic beverages from Nigeria market. International Journal of Nano and Material Sciences 2 (1), 25–35. Ahmad, A., Mukherjee, P., Senapati, S., Mandal, D., Khan, M.I., Kumar, R., Sastry, M., 2003. Extracellular biosynthesis of silver nanoparticles using the fungus Fusarium oxysporum. Colloids and Surfaces B 28, 313–318. Amarendra, D.D., Krishna, G., 2010. Biosynthesis of silver and gold nanoparticles using chenopodium album leaf extract. Colloids and Surfaces A 369 (3), 27–33. Balaji, D.S., Basavaraja, S., Deshpande, R., Mahesh, D.B., Prabhakar, B.K., Venkataraman, A., 2009. Extracellular biosynthesis of functionalized silver nanoparticles by strains of Cladosporium cladosporioides fungus. Colloids and Surfaces B: Biointerfaces 68 (1), 88–92. Bar, H., Bhui, D.K., Sahoo, G.P., Sarkar, P., De, S.P., Misra, A., 2009. Green synthesis of silver nanoparticles using latex of Jatropha curcas. Colloids and Surfaces A: Physicochemical and Engineering Aspects 339, 134–139. Bartholomew, D., Paul, R., Rohrbach, K., 2003. The Pineapple: Botany, Production and Uses. CABI Publishing, Wallingford, UK, pp. 1–301. Bhainsa, K.C., D’Souza, S.F., 2006. Extracellular biosynthesis of silver nanoparticles using the fungus Aspergillus fumigates. Colloids and Surfaces B: Biointerfaces 47 (2), 160–164. Chang-Bin, W., Sheng-Hui, L., Yu-Ge, L., Ling-Ling, L., Wen-Xiu, Y., Guang-Ming, S., 2011. Characteristic aroma compounds from different pineapple parts. Molecules 16, 5104–5112. Dare, E.O., Makinde, O.W., Ogundele, K.T., Osinkolu, G.A., Fasasi, Y.A., Sonde, I., Bamgbose, J.T., Maaza, M., Sithole, J., Ezema, F., Adewoye, O.O., 2012. Zinc mediated synthesis,growth kinectic, and shaped evolution of silver nanoparticles. International Scholarly Research Network Nanomaterials, 1–8. Dubey, M., Bhadauria, S., Kushwah, B.S., 2009. Green synthesis of nanosilver particles from extract of Eucalyptus hybrida (Safeda) leaf. Digest Journal of Nanomaterials and Biostructures 4, 537–543.
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