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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
b
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

a r t i c l e i n f o a b s t r a c t

Article history: Pineapple leaf was used in this study for the synthesis of silver nanoparticles based on the search for
Received 9 July 2013 sustainable synthetic means. Indeed, this offered an economical and sustainable synthetic route relative
Received in revised form to expensive and toxic chemical methods. The leaf extract was used and the corresponding nanoparti-
12 September 2013
cles obtained were subjected to UV–vis analysis at different times. The UV–vis was used to monitor the
Accepted 12 September 2013
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
Keywords:
460 nm. The crystals was equally characterized using FTIR, X-ray diffraction methods and TEM. The dif-
Pineapple leaf
Esherichia coli
ferent results obtained suggested the appearance of silver nanoparticles (SNPs) as determined by the
Gentamycin process parameters with a particle size of 12.4 nm. The sample was further screened against Staphylococ-
Antibacterial cus aureus, Streptococcus pneumoniae, Proteus mirabilis and Escherichia coli using Gentamicin as control.
Nanoparticles 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 range of materials can be prepared by a number of methods using

precursors from liquids, solid or gas phase (Mansoori, 2005).
The properties of matter at nanoscale level are significantly dif- It has been reported that silver nanoparticles (SNPs) are non
ferent from their macroscopic bulk properties. This makes the study toxic to humans but inhibits the growth of bacteria, virus and other
of nanotechnology interesting as it takes into consideration the eukaryotic micro organisms (Jeong et al., 2005).
designing, characterization, production and application of struc- Classically physical and chemical methods have been employed
tures, devices and systems by controlling shape and size at the in synthesizing nanoparticles and these methods are costly, toxic
nanometer scale (Mansoori, 2005). and non-eco friendly but in recent times scientists are looking
Nanoparticles are viewed as the fundamental building blocks forward to low cost, non-toxic and eco-friendly synthetic meth-
of nanotechnology (Mansoori, 2005; Mansoori et al., 2007). They ods. Most recently, biosynthesis of nanoparticles using bacteria
are the starting points for preparing many nanostructured mate- (Shiying et al., 2007; Husseiny et al., 2007), fungus and plants
rials and devices and their synthesis is an important component (Raut et al., 2009) have emerged as a simple and viable alterna-
of the rapidly growing research efforts in nanoscience and nano- tive to more complex physical and chemical synthetic procedures
engineering (Mansoori et al., 2007). The nanoparticles of a wide 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
∗ Corresponding author. Tel.: +234 8035642445; fax: +234 7063917311. agents etc. (Sharma et al., 2009). According to literature review,
∗∗ Corresponding author at: Department of Chemistry, Federal University of Agri- some studies have reported the synthesis of silver nanoparticles
culture Abeokuta, Nigeria. Tel.: +234 8035642445; fax: +234 7063917311. by plants such as Gliricidia sepium (Raut et al., 2009), Azadirachta
E-mail addresses: chemphilips@[Link] (E.E. Emeka), indica (Shankar et al., 2004), Capsicum annuum (Bar et al., 2009),
charlesbarken@[Link] (O.C. Ojiefoh), aleruchichuku@[Link] (C. Aleruchi),
Carica papaya (Jha and Prasad, 2010), Eucalyptus hybrida (Dubey
labulo@[Link] (L.A. Hassan), mfonbecca2000@[Link] (O.M. Christiana),
mfonbecca2000@[Link] (M. Rebecca), dare3160@[Link] (E.O. Dare), et al., 2009) and microorganisms such as Rhodopseudomonas cap-
turpsyelikay20@[Link] (A.E. Temitope). sulate (Shiying et al., 2007), Pseudomonas aeruginosa (Husseiny

0968-4328/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.
[Link]

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et al., 2007), Aspergillus fumigatus (Bhainsa and D’Souza, 2006), Cla- 1.2
dosporium cladosporioides (Balaji et al., 2009), Fusarium oxysporum
(Ahmad et al., 2003) and in our earlier research work, we reported
1
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 0.8 0mins

Absorbance
the botanical family Bromeliaceae, is a perennial herb native to 2mins
the American tropics (Bartholomew et al., 2003) which is well
0.6 5mins
known for its freeness from harmful phytochemicals (Mateljan,
2007). Pineapples may be cultivated from a crown cutting of the 10mins
fruit, possibly flowering in 20–24 months and fruiting in the fol- 0.4
15mins
lowing six months. Its leaves are narrow, fibrous and spiny and
the fruit grows on a stalk in the center of the rosette of leaves. 30mins
0.2
Beside fruits, pineapple fields yield large amounts of leaves that
may be used for their high quality fibre or as feedstuff for rumi-
nants (Ecocrop, 2011). Ananas comosus peels, core extracts and 0
crown extract contain sugar especially fructose, sucrose and glu- 300 350 400 450 500 550 600 650 700 750
cose (Nadzirah et al., 2013). The peak in sucrose concentration of wavelength (nm)
matured fruit is attained at full-yellow stage and then declines.
A wide range of volatiles (more than 280 compounds) have been Fig. 1. UV–vis absorption peaks of the synthesized silver nanoparticles using
pineapple crown leaf extract.
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 ions to Ag0 monitored by measuring the absorbance or appearance
are esters, followed by terpenes, ketones and aldehydes (Chang- of plasmon bands with T60 UV–vis spectrophotometer.
Bin et al., 2011). Biomolecules with carbonyl, hydroxyl, and amine
functional groups have the potential for metal ion reduction and 2.5. Characterization
capping the newly formed particles during their growth processes
(Shiying et al., 2007). The bioreduction of the Ag+ to Ag0 was monitored using UV–vis
In view of this knowledge, a detailed approach was designed spectrophotometer (T60 UV–vis spectrophotometer) at regular
to explore the potential of pineapple leaf towards reduction, cap- intervals with samples in Quartz cuvette operated at a resolution
ping and stabilization of silver compounds. In this research work, of 1 nm. The functional group responsible for the silver nanopar-
a clear definition of the synthesis, characterization and antibacte- ticles was analysed using FTIR (Perkin Elmer Spectrum, USA). The
rial activities of the silver nanoparticles of pineapple leaf extract crystallinity of the silver nanoparticles was analysed using X-ray
was fully investigated and reported. Our aim is to use materials, diffraction (XRD) methods while the size of synthesized nanopar-
which are environmental wastes to reduce Ag+ to Ag0 . The syn- ticle was determined using scanning electron microscope (SEM)
thesized silver nanoparticles was characterized using XRD, UV–vis (JEOL Ltd, Tokyo, Japan).
spectrophotometer, FTIR, transmission electron microscope (TEM)
and its application as antimicrobial agents was also investigated. 2.6. Antibacterial analysis

2. Methodology The antibacterial screening of the silver nanoparticles was car-

ried out against S. aureus, S. pneumoniae, P. mirabilis and Esherichia
2.1. Materials coli using the Agar-well diffusion method.
0.5 mL each of the seeded broth containing 105 cfu/mL of the test
Pineapple leaf, AgNO3 . organisms were incubated on solid nutrient agar plates and spread
uniformly with a glass spreader. Three wells were cut out in the
2.2. Organisms 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
Staphylococcus aureus, Streptococcus pneumoniae, Proteus extract, Gentamycin and normal saline were placed in the wells
mirabilis and Escherichia coli. using a micropipette and further incubated at 37 ◦ C for 24 h. Gen-
tamycin was used as the positive control while normal saline was
2.3. Preparation of extract used as the negative control (Fig. 1).
The mean diameter of the zone of inhibition in mm obtained
Fresh pineapple was collected from Lafia market in Nasarawa around the well was measured and the values shown in Table 1.
state and the leaf was washed, chopped and blended. Afterwards, it
3. Results
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 3.1. UV–vis spectra analysis
4 ◦ C and was further used for synthesis of the nanoparticles (Raut
et al., 2009). 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 aque-
2.4. Synthesis of silver nanoparticles using the pineapple leaf ous solution due to excitation of electrons and changes in electronic energy levels
extract 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
The filtrate (5.0 mL) of pineapple leaf extract was added to 20 mL it is the basis for measuring adsorption of material onto the surface of metal nanopar-
ticles. The formation of the nanoparticles was observed at intervals (0–30 min) with
of 1 mM aqueous AgNO3 solution. The resulting solution was heated a characteristic plasmon band ranging between 440 and 460 nm appearing at about
at 70 ◦ C and colour change observed. Aliquot sample of the solution the second minute of the reaction. The intensity of the peaks continues to increase
was taken at intervals (0–30 min) and rate of the reduction of Ag+ 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 Normal saline Ag nanoparticles Result

+ve control (mm) −ve control (mm) From pineapple leaf

Staphylococcus aureus 27 0 15 +
Streptococcus pneumoniae 13 0 5 +
Proteus mirabilis 15 0 0 −
Escherichia coli 25 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

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
Fig. 2. Comparison of UV–vis of pineapple leaf synthesized SNPs (a) and beverage which are indexed as (1 1 1), (2 0 0), (2 2 0) and (3 1 1) bands of face centred cubic
mediated SNPs (b). (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
shows particle size variation (Muhammad et al., 2012). As the reaction progresses of the X-ray, ˇ full width at half maximum and  the Bragg angle in radians, the
after the first two minutes, there was evidence of further decrease in sizes of the average crystallite size of silver nanoparticles was found to be about 12.4 nm.
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 alco- 3.4. TEM analysis
holic 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 The shape, size and morphology of the synthesized silver nanoparticles were
promising one because it saves time and exhibits better stabilizing phenomenon. elucidated with the help of transmission electron microscopy. The TEM images
confirmed the formation of silver nanoparticles (Fig. 5). The nanoparticles are homo-
3.2. FTIR spectroscopy geneous 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
The FTIR analysis was carried out between 4000 and 600 cm−1 to identify the calculated from XRD analysis. Fig. 5b shows the size distribution of the silver
functional groups responsible for capping and stabilizing the Ag nanoparticles. The nanoparticle (Figs. 6 and 7).

120

110

100

90 3662.51

80 2825.33
%T

1012.63
70
3696.18 3680.72 2921.95 1054.71
60 2980.72 2865.06 1032.68
2972.49
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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120
110
100
90
80
70
2836.23 1420.19
60
1654.06 1086.48 878.24
50
%T 40 1024.51
30 2896.50
20 1043.00
10 3354.04 2976.74
0
-10
-20
-30
4000 3500 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 pneu-
moniae.

4. Discussion

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
Fig. 5. XRD spectra pattern of synthesized silver nanoparticle using pineapple leaf
surface plasmon bands at 440–460 nm proved the existence of sil-
extract.
ver 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 nanopar-
3.5. Antibacterial assay ticles almost started from the second minute of the reaction and
continued to give sharper peaks at varying wavelengths showing
The susceptibility of S. aureus, S. pneumoniae and E. coli showed that the silver
nanoparticles from pineapple leaf extract contained antibacterial properties that that the nanoparticles formed are varying in sizes and also stable.
inhibited both Gram +ve cocci (S. aureus and S. pneumoniae) and Gram −ve bacilli The application of the synthesized nanoparticles as antimicrobial
(E. coli) thereby placing antimicrobial activity on a broad spectrum plane. agents has equally yielded positive result as it inhibits the growth
Due to the highly susceptibility of S. aureus and E. coli, the synthesized silver
of bacteria though it showed lower hindrance abilities against
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
the tested isolates compared to the control (Gentamycin). It is
of mankind in the health sector. believed that Ag nanoparticles penetrate into the cell walls of the
microbes, causing cellular damage by interacting with phospho-
rus 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

The field of nanotechnology became imperative in this present

Fig. 6. (a) TEM image of synthesized silver nanoparticle using pineapple crown leaf time as it seems to offer solutions to the imminent industrial,
extract. (b) Particle size distribution of the synthesized silver nanoparticles. health and environmental problems of the world. Future research

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is underway as regards to the potentialities of the silver nanopar- Ecocrop, 2011. Ecocrop Database. FAO, [Link]
ticles from the pineapple leaf extract, as it has proven to exhibit (retrieved 12.06.13).
Husseiny, M.I., El-Aziz, M.A., Badr, Y., Mahmoud, M.A., 2007. Biosynthesis of
antimicrobial properties as well as enhanced optical properties. gold nanoparticles using Pseudomonas aeruginosa. Spectrochimica Acta Part A:
Molecular and Biomolecular Spectroscopy 67, 1003–1006.
Conflict of interest 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,
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The authors report no conflict of interest in this work. Jha, A.K., Prasad, K., 2010. Green synthesis of silver nanoparticles using Cycas leaf.
International Journal of Green Nanotechnology: Physics and Chemistry 1 (2),
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Acknowledgements Mansoori, G.A., 2005. Principles of Nanotechnology – Molecular-Based Study of
Condensed Matter in Small Systems. World Scientific Pub. Co., Hackensack, NJ.
The authors wish to acknowledge the Vice Chancellor of Federal Mansoori, G.A., George, T.F., Zhang, G., Assoufid, L., 2007. Molecular Building Blocks
for Nanotechnology. Springer, New York.
University Lafia, Prof. Ekanem Ikpi Braide, Dean Faculty of Science, Mateljan, G., 2007. The Worlds Healthiest Foods, vols. 46/45. United Sates, Seattle
Prof. Martin Ogbe and the entire Staff of Chemistry, Microbiology WA, pp. 8699–8702.
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Osuagwu Stanislaus for their technical support. inhibitory activities against Helicobacter pylori. International Journal of Molec-
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