Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 112 (2013) 384387

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Spectrochimica Acta Part A: Molecular and

Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa

Bio-Fabrication of zinc oxide nanoparticles using leaf extract of Parthenium

hysterophorus L. and its size-dependent antifungal activity against plant fungal
pathogens
P. Rajiv a, Sivaraj Rajeshwari a,, Rajendran Venckatesh b
a
Department of Biotechnology, School of Life Sciences, Karpagam University, Eachanari Post, Coimbatore 641 021, Tamil Nadu, India
b
Department of Chemistry, Government Arts College, Udumalpet 642 126, Tamil Nadu, India

h i g h l i g h t s g r a p h i c a l a b s t r a c t

 This paper reports on the synthesis of

zinc oxide nanoparticles using weed
plant extract by novel method.
 Highly stable, spherical and
hexagonal zinc oxide nanoparticles
are synthesized.
 The particle sizes and shapes are
controlled by varying the Parthenium
leaf extract concentrations.
 Parthenium mediated zinc oxide
nanoparticles are proved to be good
antifungal agents.
 Green synthesized nanoparticles are
eco-friendly and have wide
applications in biomedical and
cosmetic industries.

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

Article history: The study reports the synthesis and characterization of zinc oxide nanoparticles from weed plant by a
Received 16 October 2012 novel method. The aim of this work is to synthesize zinc oxide nanoparticles from Parthenium hysteropho-
Received in revised form 10 April 2013 rus L. by inexpensive, ecofriendly and simple method. Highly stable, spherical and hexagonal zinc oxide
Accepted 15 April 2013
nanoparticles were synthesized by using different concentrations of 50% and 25% parthenium leaf
Available online 25 April 2013
extracts. Both the concentrations of the leaf extract act as reducing and capping agent for conversion of
nanoparticles. Formation of zinc oxide nanoparticles have been conrmed by UVVis absorption spectros-
Keywords:
copy, X-ray diffraction (XRD), Fourier trans-form infrared spectroscopy (FT-IR), scanning electron micros-
Parthenium
Antifungal activity
copy (SEM) and transmission electron microscopy (TEM) analysis with energy dispersive X-ray analysis
SEM and TEM (EDX). SEM, TEM and EDX analysis reveals that spherical and hexagonal zinc oxide nanoparticle sizes were
Zinc oxide nanoparticles 27 5 nm and 84 2 nm respectively and chemical composition of zinc oxide were present. We synthe-
Size-dependent sized different sized zinc oxide nanoparticles and explored the size-dependent antifungal activity against
plant fungal pathogens. Highest zone of inhibition was observed in 25 lg/ml of 27 5 nm size zinc oxide
nanoparticles against Aspergillus avus and Aspergillus niger. Parthenium mediated zinc oxide nanoparticles
were synthesized and proved to be good antifungal agents and environment friendly.
2013 Published by Elsevier B.V.

Introduction
Particles smaller than tens of nanometers in primary particle
diameter (nanoparticles) are of interest for the synthesis of new
Corresponding author. Tel./fax: +91 4222611146.
E-mail address: rajeshwarishivaraj@gmail.com (S. Rajeshwari).
materials because of their low melting point, special optical prop-
erties, high catalytic activity, and unusual mechanical properties
compared with their bulk material counterpart [1]. Zinc oxide is
attracting tremendous attention due to its interesting properties
like wide direct band gap of 3.3 eV at room temperature and high
exciton binding energy of 60 meV [2]. Zinc oxide is a unique

1386-1425/$ - see front matter 2013 Published by Elsevier B.V.

http://dx.doi.org/10.1016/j.saa.2013.04.072
 P. Rajiv et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 112 (2013) 384387
material that exhibits semiconducting, piezoelectric, and pyroelec-
tric properties and has versatile applications in transparent elec-
tronics, ultraviolet (UV) light emitters, piezoelectric devices,
chemical sensors, spin electronics, personal care products, and
coating and paints [35]. In fact, zinc oxide is non-toxic and chem-
ically stable under exposure to both high temperatures and UV [6].
Plants and/or their extracts provide a biological synthesis route
of several metallic nanoparticles which are more eco-friendly and
allows a controlled synthesis with well-dened size and shape
[7]. Green synthesis of gold nanoparticles using plants such as
neem [8], lemon grass [9], alfalfa [10], Emblica ofcinalis [11], and
Cinnamomum camphora [12] have been reported. The use of envi-
ronmentally benign materials like plant leaf extract [13], bacteria
[14], fungi [15] and enzymes [16] for the synthesis of silver nano-
particles offers numerous benets of eco-friendliness and compat-
ibility for pharmaceutical and other biomedical applications as
they do not use toxic chemicals for the synthesis protocol. The en-
zymes [17], plant leaf extract [18] and bacteria [19] were vital role
in green synthesis of zinc oxide nanoparticles.
Plants are often affected by plant pathogens such as fungi, bac-
teria and viruses which results in great agricultural loss [20]. Sev-
eral conventional methods have been used for the control or
reduce of these pathogens and each of these methods has one or
more limitations (pesticides cause dangerous effect on the envi-
ronment and human health). Thus, use of nanoparticles has been
considered as an alternate and effective approach which is eco-
friendly and inexpensive for the control of pathogenic microbes
[21]. Nanoparticles have a vital role in the management of plant
diseases as compared to synthetic fungicides [22]. Zinc oxide nano-
particles have also been proposed as an anti-microbial preservative
for wood or food products [2325]. The size dependent activity
was studied in the range of 100 nm to 0.8 lm in Staphylococcus
aureus and Escherichia coli and 12, 45, and 200 nm range in
E. coli, however little is known about the activity of zinc oxide
nanoparticles in the 10300 nm range toward S. aureus [26,27].
Parthenium hysterophorus L. (family Asteraceae) is one of the 10
worst weeds in the world [28], which is a poisonous, pernicious
and aggressive weed and is reported to have pharmacological
properties against many diseases such as rheumatism, hepatic
amoebiasis and tumours [2931]. Parthenium is also known to
cause allergy and toxicity in animals [32]. In this study, we synthe-
sized two zinc oxide nanoparticles of varying sizes and tested their
antifungal activity using well diffusion method.
Materials and methods
Materials
P. hysterophorus L. plants were collected from follow lands in
and around Karpagam University, Eachanari, Coimbatore, India
(11160 N; 76580 E) on 2011 before owering stage. All the chemi-
cals were obtained from sigma-aldrich chemicals, India. Aspergillus
avus (MTCC-7589), Aspergillus niger (MTCC-2587), Aspergillus
fumigatus (MTCC-2550) Fusarium culmorum (MTCC-2090) and
Fusarium oxysporium (MTCC-3379) were obtained from the Depart-
ment of Microbiology, School of Life Sciences, Karpagam Univer-
sity, Coimbatore, India. The culture samples were maintained on
potato dextrose broth at room temperature (aerobically) at
200 rpm shaking for 3 days.
Synthesis of zinc oxide nanoparticles using Parthenium leaf extract
Zinc nitrate was used as a precursor material for nanoparticles
synthesis. The harvested plants were washed with tap water and
whipped with tissue paper. The plant leaf samples were weighed
385
carefully and nely cut. Samples were ground well by mortar
and pistle using de-ionized water. The mixture of plant extract
was heated with medium ame. After cooling this solution was l-
tered using lter paper (Whatman No. 42, Maidstone, England) and
store in refrigerator for further studies.
Zinc nitrate was dissolved in de-ionized water under constant
stirring. The 50% and 25% of plant extract was prepared with de-
ionized water and the volume was made up to 250 ml. In order
to, the zinc nitrate solution was added in plant extract under con-
stant stirring. This mixture of the solution was kept under vigorous
stirring at 90 C for 45 h. After this process a green color precipi-
tate was obtained. This mixture was centrifuged at 7000 rpm for
15 min and the green precipitate was discarded. The supernatant
was stirred again at 150 C for 1 h and a pale yellow color solid pre-
cipitate was obtained. The precipitate was washed with methanol
and air dried. This product was annealed at 400 C for 1 h. Finally,
white color powder was obtained. The whole experiment was re-
peated three times. The methodology is given in the Supplemen-
tary information A1.
Characterization of zinc oxide nanoparticles
The aqueous zinc oxide nanoparticles were characterized by UV
absorption spectra (UV-2450, shimadzu). The synthesized zinc
oxide nanoparticles purity and grain size were identied by X-
ray diffraction (PerkinElmer spectrum one instrument) Cu Ka
radiations (k = 0.15406 nm) in 2h range from 20 to 80. The pow-
der sample of zinc oxide nanoparticles functional groups were ana-
lyzed by using Fourier trans-form infrared spectroscopy (Perkin
Elmer 1725). The nanoparticles size and shape were character-
ized by using scanning electron microscopy (Model JSM 6390LV,
JOEL, USA). The nanoparticles average size and size distribution
were determined by transmission electron microscopy (JEOL
JEM-3100F). The samples analyzed for energy dispersive X-ray
analysis (RONTECs EDX system, Model QuanTax 200, Germany).
Determination of size-dependent antifungal activity of zinc oxide
nanoparticles by the well-diffusion method
Antifungal activities of the synthesized zinc oxide nanoparticles
of different sizes were determined using plant fungal pathogens by
a modied Kirby Bauer disc diffusion method [33]. The fungi were
cultured in potato dextrose broth at room temperature on an orbital
shaking incubator (Remi, India) at 200 rpm. A 100 lL of broth fun-
gal culture was prepared and spread on potato dextrose agar plates.
After that plates were allowed to stand for 10 min to allow for cul-
ture absorption. The 5 mm size wells were punched into the agar
with help of sterile gel puncher. A 100 lL (25 lg/ml) of the nano-
particles solution sample and 100 lL (10 lg/ml) of positive control
(amphotricin B) were poured into wells on all plates using micropi-
pette. After incubation at room temperature for 48 h, the size of the
zone of inhibition diameter in millimeter was measured. Each
screening treatment was conducted with three replicates and the
results are presented as mean SE (standard error of the mean).
Results and discussion
Characterization of zinc oxide nanoparticles
The UVVisible absorption spectra of the as-grown a new tech-
nique for the preparation of the monodispersed semiconductor
zinc oxide nanoparticles (50% and 25% plant extract) are shown
in Fig. 1a and b. The room temperature spectra exhibit strong exci-
tonic absorption peaks at 374.00 nm (3.32 eV) and 370.50 nm for
samples 50% and 25% plant extract, respectively, which is in good
agreement with the previous work [19,34,35].
386 P. Rajiv et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 112 (2013) 384387

X-ray diffraction was taken to further conrm zinc oxide phase

of the nanoparticles. The XRD patterns of the obtained nanoparti-
cles and was shown in Supplementary information A2. The XRD
peaks were identied as (1 0 0), (0 0 2), (1 0 1), (1 0 2), (1 1 0), (1 1 2)
and (2 0 2) reections, respectively. All of the diffraction peaks
can be indexed to the spherical and hexagonal zinc oxide phase
by comparison with the data from JCPDS card No. 89-7102. The
narrow and strong diffraction peaks indicate that the product has
well crystalline particles structure. The Scherrer formula was used
for calculate the particles sizes and found to be in the range of 22
35 nm and 7590 nm respectively [36].
The spectrum showed bands at 433, 457 and 470 cm1 corre-
sponding to metaloxygen (MO). The peak in the region between
400 and 600 cm1 is allotted to ZnO [37,38]. The bands at 1446 Fig. 2. FTIR spectrum of synthesized zinc oxide nanoparticles. (A) 25% Extract and
and 1531, 1517 cm1 correspond to NH bending mode and the (B) 50% Extract.

band at 3375 cm1 can be attributed to free NH stretching vibra-

tions [39]. The spectrum recorded from untreated zinc oxide nano-
Size-dependent antifungal activity of zinc oxide nanoparticles against
particles showed a single band at 460 cm1 attributed to MO
plant fungal pathogens
vibrational band [39]. The synthesized zinc oxide nanoparticles
has peaks at 3520, 3574 cm1 which indicates phosphorous com-
The results of size-dependent antifungal activity of zinc oxide
pounds, secondary sulfornamide; 2405 cm1 represents alkynes
nanoparticles against plant fungal pathogens are given in Supple-
(monosubstituted), amine salts; 2303, 2326 cm1 indicates enol
mentary information A5. Actually, the zone of inhibition was in-
of 1, 3 diketone, 1624 cm1 represents (medium charge) vinyl,
creased in 100 ll (50 lg/ml) concentration of zinc oxide
cis-tri substituted; 1382 cm1 represents monosubstitued alkynes.
(27 5 nm) nanoparticles. The highest zone of inhibition occurred
It shows the combination of zinc oxide and plant secondary com-
in A. avus with a zone diameter of 24.66 0.57 mm at a concen-
pounds (Fig. 2a and b),
tration of 50 lg/ml, it is more than positive control (i.e.
The morphology of the nanostructures obtained from different
19.66 0.57 mm zone of inhibition) and which is very similar to
concentrations of plant extracts was studied using SEM. The SEM
previous studies [maximum zone of inhibition was observed in
images are shown in Supplementary information A3, which exhibit
the zinc oxide nanoparticles (57.72 nm size and 25 lg/mL) against
varieties of typical shapes obtained in this method, such as, spher-
A. avus (19 1.0 mm)] [19]. At the same time the lowest zone of
ical and hexagonal nanoparticles, depending on the different con-
inhibition occurred in F. culmorum with a zone diameter of
centrations of plant extract. The SEM pictures show distinguished
14 0.57 mm at same concentration and same size of zinc oxide
spherical and hexagonal morphology and generally in random
nanoparticles. The moderate zones of inhibition were observed in
and not uniform, which is very similar to those described in the
100 ll (50 lg/ml) concentration of zinc oxide (84 2 nm) nanopar-
previous literature [40].
ticles. The size of 27 5 nm nanoparticles inhibited the growth of
The transmission electron micrograph of the zinc oxide samples
fungal pathogens as compared to positive control and zinc oxide
gures are shown in Supplementary information A4. These gures
(84 2 nm) nanoparticles. The previous studies show the growth
undoubtedly indicate the morphology of the particles to be spher-
inhibition of microorganisms by six metal oxides nanoparticles
ical and hexagonal. Some of the particles are agglomerates. TEM
(MgO, TiO2, CuO, CaO, CeO2, and ZnO). Among them, zinc oxide
image conrms the formation of zinc oxide nanoparticles and it
nanoparticles show that signicant growth inhibition in a size-
has an average size about 27 5 nm and 84 2 nm respectively.
dependent manner under normal ambient lighting conditions
This is very similar to, synthesis of zinc oxide nanoparticles (35
[42]. Raghupathi et al. explained that the antibacterial activity of
40 nm) from aloe barbadensis miller leaf [18]. The spherical nano-
the zinc oxide nanoparticles was inversely proportional to the size
particles from 1-octadecene with longer reaction time have a
diameter ranging from 12 to 14 nm [41]. Figs. 3a and 3b represents
the strong signals from the zinc atoms in the nanoparticles re-
corded, and other signals from C and O atoms were also observed,
which is very similar to Jayaseelan et al. [19].

Fig. 1. UVvis spectroscopy of synthesized zinc oxide nanoparticles. (A) 25% Extract
and (B) 50% extract. Fig. 3a. Analysis of chemical composition of zinc oxide nanoparticles (25% extract).
 P. Rajiv et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 112 (2013) 384387
Fig. 3b. Analysis of chemical composition of zinc oxide nanoparticles (50% extract).
of the nanoparticles in S. aureus [43]. Azam et al. were found the
antibacterial activity of CuO nanoparticles to be size-dependent.
In addition, the highly stable minimum-sized monodispersed cop-
per oxide nanoparticles synthesized and demonstrated a signi-
cant increase in antibacterial activities against both gram-
positive and negative bacterial strains [44]. The results conrm
that smaller sized zinc oxide nanoparticles indeed have higher
antifungal activity than larger sized nanoparticles under normal
room temperature.
Conclusion
The present work of biosynthesis method is a green approach,
inexpensive, pollution free and eco-friendly approach. The synthe-
sis of zinc oxide nanoparticles might have been obtained due to
some difference in the level of pH, which activates the pH sensitive
oxidoreductases enzymes. Antifungal activity experiments per-
formed on various fungal pathogens clearly conrmed the higher
effectiveness of zinc oxide nanoparticles against fungal growth
due to smaller particle size of this sample compared to other sam-
ples and control. It may selectively inhibit unnecessary plants
(such as weeds), kill harmful fungi and bacteria in agricultural
elds.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.saa.2013.04.072.
References
[1] R.W. Siegel, Annu. Rev. Mater. Sci. 21 (1991) 559578.
387
[2] S.S. Ashtaputre, A. Deshpande, S. Marathe, M.E. Wankhede, J. Chimanpure, R.
Pasricha, J. Urban, S.K. Haram, S.W. Gosavi, S.K. Kulkarni, Pramana J. Phys. 65
(2005) 615620.
[3] Z.L. Wang, Mater. Today 7 (2004) 2633.
[4] R. Wahab, Y.S. Kim, D.S. Lee, J.M. Seo, H.S. Shin, Sci. Adv. Mater. 2 (2010)
3542.
[5] M.S. Akhtar, S. Ameen, S.A. Ansari, O. Yang, J. Nanoeng. Nanomanuf. 1 (2011)
7176.
[6] A. Yadav, V. Prasad, A.A. Kathe, S. Raj, D. Yadav, C. Sundaramoorthy, N.
Vigneshwaran, Bull. Mater. Sci. 29 (2006) 641645.
[7] H. Bar, D.K. Bhui, G.P. Sahoo, P. Sarkar, S. Pyne, A. Misra, Colloids Surf.:
Physiochem. Eng. Aspects 348 (2009) 212216.
[8] M. Sastry, S.S. Shankar, A. Rai, A. Ahmad, J. Colloid Interface Sci. 275 (2004)
496502.
[9] S.S. Shankar, A. Rai, B. Ankamwar, A. Ahmad, M. Sastry, Nat. Mater. 3 (2004)
482488.
[10] J.L. Gardea-Torresdey, J.G. Parsons, E. Gornez, J. Videa, H.E. Troiani, P. Santiagol,
Nano Lett. 2 (2002) 397401.
[11] B. Ankamwar, D. Chinmay, A. Absar, S. Murali, J. Nanosci. Nanotechnol. 10
(2005) 16651671.
[12] J. Huang, Q. Li, D. Sun, Y. Lu, Y. Su, X. Yang, H. Wang, Y. Wang, W. Shao, N. He, J.
Hong, C. Chen, Nanotechnology 18 (2007) 105104105115.
[13] V. Parashar, R. Parashar, A.C. Sharma, A.C. Pandey, Dig. J. Nanomat. Bios. 4 (1)
(2009) 4550.
[14] N. Saifuddin, C.W. Wong, A.A.N. Yasumira, E. J. Chem. 6 (1) (2009) 6170.
[15] K.C. Bhainsa, S.F.D. Sauza, Colloids Surf. B 47 (2006) 160164.
[16] B. Willner, B. Basnar, FEBS J. 274 (2007) 302309.
[17] K. Prasad, A.K. Jha, Nat. Sci. 1 (2009) 129135.
[18] G. Sangeetha, S. Rajeshwari, R. Venckatesh, Mater. Res. Bull. 12 (2011) 2560
2566.
[19] C. Jayaseelan, A. Abdul Rahuman, A. Vishnu Kirthi, S. Marimuthu, T.
Santhoshkumar, A. Bagavan, K. Gaurav, L. Karthik, K.V. Bhaskara Rao,
Spectrochimica Acta Part A 90 (2012) 7884.
[20] M.N. Esfahani, Indian Phytopathol. 59 (2006) 142147.
[21] V. Kumar, S.K. Yadav, J. Chem. Technol. Biotechnol. 84 (2009) 151157.
[22] H.J. Park, S.H. Kim, H.J. Kim, S.H. Choi, Plant Pathol. J. 22 (2006) 2534.
[23] V. Aruoja, H. Dubourguier, C. Kasamet, K.A. Kahru, Pseudokirchneriella
Subcapitata. Sci. Total Environ. 407 (2009) 14611468.
[24] L. Huang, L. Dian-Qing, W. Yan-Jun, G. Min David, E.D. Xue, J. Inorg. Biochem.
99 (2005) 986993.
[25] V.K. Sharma, R.A. Yngard, Y. Lin, Adv. Colloid Interface Sci. 145 (2009)
8396.
[26] N. Padmavathy, R. Vijayaraghavan, Sci. Technol. Adv. Mater. 9 (2008). Article
No. 035004.
[27] O. Yamamoto, Int. J. Inorg. Mater. 3 (2001) 643646.
[28] R.S. Rao, J. Bom. Nat. Hist. Soc. 54 (1956) 218220.
[29] S. Kumar, A. Pratap Singh, G. Nair, S. Batra, A. Seth, N. Wahab, R. Warikoo,
Parasitol. Res. 108 (2011) 853859.
[30] D. Mew, F. Balza, G.H.N. Towers, I.G. Levy, Planta Med. 45 (1982) 2327.
[31] G.L. Sharma, K.K. Bhutani, Planta Med. 54 (1988) 2022.
[32] H.C. Evans, Biocontrol News Inf. 18 (1997) 8998.
[33] A.W. Bauer, W.M. Kirby, J.C. Sherris, M. Turck, Am. J. Clin. Pathol. 45 (1966)
493496.
[34] Y.H. Ni, X.W. Wei, J.M. Hong, Y. Ye, Mater. Sci. Eng. B. 121 (2005) 4247.
[35] H.R. Nawaz, B.A. Solangi, B. Zehra, U. Nadeem, J. Sci. Ind. Res. 2 (2011) 164
170.
[36] H. Borchert, E.V. Shevchenko, A. Robert, I. Mekis, A. Kornowski, G. Grabel, H.
Weller, Langmuir 21 (2005) 19311936.
[37] A.C. Tas, P.J. Majewski, F. Aldinger, J. Am. Ceram. Soc. 85 (2002) 14211429.
[38] S.J. Jun, S. Kim, J.H. Han, J. Kor. Ceram. Soc. 35 (1998) 209.
[39] K. Nakamoto, Wiley, Chichester, 1978.
[40] M.A. Shah, African Phys. Rev. 2 (2008) 0011.
[41] T. Andelman, Y. Gong, M. Polking, M. Yin, I. Kuskovsky, G. Neumark, S. OBrien,
J. Phys. Chem. B 109 (2005) 14314.
[42] N. Jones, B. Ray, R.T. Koodali, A.C. Manna, FEMS Microbiol. Lett. 279 (2008) 71
76.
[43] K.R. Raghupathi, R.T. Koodali, A.C. Manna, Langmuir 27 (2011) 40204028.
[44] A. Azam, A.S. Ahmed, M. Oves, M.S. Khan, A. Memic, Int. J. Nanomed. 7 (2012)
35273535.