Areca catechu Linn. Derived Silver Nanoparticles: A Novel

13 Pages·2011·1.03 MB·English

This article was downloaded by: [shanmugam achiraman]
On: 27 July 2011, At: 10:57
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House,
37-41 Mortimer Street, London W1T 3JH, UK
International Journal of Green Nanotechnology
Publication details, including instructions for authors and subscription information:
http://www.tandfonline.com/loi/ugnj20
Areca catechu Linn.–Derived Silver Nanoparticles:
A Novel Antitumor Agent against Dalton's Ascites
Lymphoma
Raman Sukirtha a , Muthukalingan Krishnan a , Rajamanickam Ramachandran a ,
Soundararajan Kamalakkannan b , Palanivel Kokilavani a , Devaraj SankarGanesh a ,
Soundarapandian Kannan c & Shanmugam Achiraman a
a Department of Environmental Biotechnology, School of Environmental Sciences,
Bharathidasan University, Tiruchirappalli, Tamilnadu, India
b Department of Animal Science, Bharathidasan University, Tiruchirappalli, Tamilnadu, India
c Department of Zoology, Bharathiar University, Coimbatore, Tamilnadu, India
Available online: 27 Jul 2011
To cite this article: Raman Sukirtha, Muthukalingan Krishnan, Rajamanickam Ramachandran, Soundararajan Kamalakkannan,
Palanivel Kokilavani, Devaraj SankarGanesh, Soundarapandian Kannan & Shanmugam Achiraman (2011): Areca catechu
Linn.–Derived Silver Nanoparticles: A Novel Antitumor Agent against Dalton's Ascites Lymphoma, International Journal of
Green Nanotechnology, 3:1, 1-12
To link to this article: http://dx.doi.org/10.1080/19430892.2011.571626
PLEASE SCROLL DOWN FOR ARTICLE
Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions
This article may be used for research, teaching and private study purposes. Any substantial or systematic
reproduction, re-distribution, re-selling, loan, sub-licensing, systematic supply or distribution in any form to
anyone is expressly forbidden.
The publisher does not give any warranty express or implied or make any representation that the contents
will be complete or accurate or up to date. The accuracy of any instructions, formulae and drug doses should
be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims,
proceedings, demand or costs or damages whatsoever or howsoever caused arising directly or indirectly in
connection with or arising out of the use of this material.
InternationalJournalofGreenNanotechnology,3:1–12,2011
Copyright(cid:1)c Taylor&FrancisGroup,LLC
ISSN:1943-0892print/1943-0906online
DOI:10.1080/19430892.2011.571626
BIOMEDICINE
Areca catechu Linn.–Derived Silver Nanoparticles: A Novel
Antitumor Agent against Dalton’s Ascites Lymphoma
1 RamanSukirtha
1
0 Muthukalingan Krishnan
2
y RajamanickamRamachandran
ul
J
7 SoundararajanKamalakkannan
2
7 PalanivelKokilavani
5
0: DevarajSankarGanesh
1
at SoundarapandianKannan
]
n ShanmugamAchiraman
a
m
a
r
hi
c
a
m
a
g
u
m
n
a
h
s
[
y
b
d
e
d
a
o
nl
w Received30January2011andaccepted4February2011.
o
D WegratefullyacknowledgeUniversityGrantCommission,UniversityGrantCommission-SpecialAssis-
tance Programs, Department of Science and Technology Fast Track Scheme, Department of Science and
Technology-Nano Mission, Council for Scientific and Industrial Research, government of India for their
financialsupport.TheauthorsthankBharathidasanUniversity,Tiruchirappalli,Tamilnadu,fortheUniversity
Research Fellowship. The authors report no conflict of interest. The authors alone are responsible for the
contentandwritingofthearticle.
RamanSukirtha,MuthukalinganKrishnan,RajamanickamRamachandran,PalanivelKokilavani,Devaraj
SankarGanesh,andShanmugamAchiramanareaffiliatedwiththeDepartmentofEnvironmentalBiotechnol-
ogy,SchoolofEnvironmentalSciences,BharathidasanUniversity,Tiruchirappalli,Tamilnadu,India.
SoundararajanKamalakkannanisaffiliatedwiththeDepartmentofAnimalScience,BharathidasanUni-
versity,Tiruchirappalli,Tamilnadu,India.
SoudarapandianKannanisaffiliatedwiththeDepartmentofZoology,BharathiarUniversity,Coimbatore,
Tamilnadu,India.
Address correspondence to Dr. S. Achiraman, Info Chemicals and Nano Oncology Lab, Department of
EnvironmentalBiotechnology,SchoolofEnvironmentalSciences,BharathidasanUniversity,Tiruchirappalli
620024,Tamilnadu,India.E-mail:[email protected]
1
2 ArecacatechuLinn.derivedSilverNanoparticles
ABSTRACT.Thepresentinvestigationemphasizesbiomimeticsynthesisofsilvernanoparticles(Ag-
NPs)usinganaqueousextractofArecacatechuanditsimpactonaDalton’sasciteslymphoma(DAL)
micemodel.Theultraviolet(UV)spectrumofAgNPsat428nmconfirmedthesphericalshapeofthe
particles and average size of 80 nm was determined using electron microscopic analysis. Elemental
silver and adhered biomolecules conferred a synergetic antitumor activity with a significant increase
in life span of tumor-induced mice with decreased body weight and tumor volume. Acridine Orange
stainingandDNAfragmentationstudiesofharvestedtumorcellsshowedhigherlevelofcytotoxicity
byAgNPswhencomparedtoaqueousextractofArecacatechu.
KEYWORDS.silvernanoparticles,Arecacatechu,Dalton’sasciteslymphoma
INTRODUCTION has been a popular masticatory throughout the
world.[13] The nuts, husks, young shoots, buds,
In outward appearance, colloidal nanoparti- and leaves have been used in various medici-
cles (NPs) prevail over the conventional anti- nal preparations and the nut possesses curative
1
1 tumor drugs that conferred resistance in tumor abilities against many diseases such as obesity,
0
y 2 cells.ColloidalNPsalsoreducesitstoxicityto- leprosy, anemia, and leukoderma.[14] The pres-
ul wardnormalcellsbyincreasingtheirselectivity enceoftotalphenolicsandtanninsintheAreca
J
27 toward cancer cells. In this regard, a potential nutiswelldocumentedandreflectstheirpoten-
57 entrantissilvernanoparticles(AgNPs)withthe tialantioxidantproperties.[15] Todatenoreports
0: possibilityforuseasatherapeuticagentincan- have been documented for A. catechu–derived
1
at cer therapy.[1] In biomimetics research, biolog- AgNPssynthesisandthestudyofantitumorac-
]
n ical sources are used for eco-friendly, reliable tivity of biosynthesized AgNPs is in evolution-
a
m metal nanoparticles synthesis.[2] Over the past ary phase. Hence, the present study highlights
a
r
hi decade, the use of biological systems such as thesynthesisofAgNPsfromA.catechunutand
c
a yeast, fungi, bacteria, and plants has been re- antitumor activity against Dalton’s ascites lym-
m
a portedfortheirnanoparticlessynthesis.Someof phoma(DAL)-inducedmicemodel.
g
u the well-known examples are extracellular syn-
m
n thesisofAgNPsbyasilver-tolerantyeaststrain
a
sh MKY3[3] and biosynthesis of silver-based crys-
[ RESULTSANDDISCUSSION
y tallinenanoparticlesfromPseudomonasstutzery
b
d AG259 isolated from silver mines.[4] Eukary-
e Environmentally friendly methodologies
d
a otic organisms such as fungi have also been
o have been gradually implemented due to their
wnl used to grow nanoparticles.[5] Though the syn- feasibilityinthesynthesisofnanostructures.[16]
o thesis of metal nanoparticles from microorgan-
D WefocusedonthebiosynthesisofAgNPsusing
isms continues to be investigated, the use of
A. catechu aqueous extract and its antitumor
plants is an exciting possibility that is unex-
activity was studied against a DAL-induced
plored and underexploited. Biocompatible syn-
mice model. Applying the principle of green
thesis of NPs by plants would be advantageous
chemistry,thebioreductionofsilvernitratewith
over other processes by eliminating the elabo-
aqueousextractofA.catechuatvarioustemper-
ratecellcultures.[6]Theeraofgreen-synthesized ◦
atures(30,60,90,95 C)foranincubationtime
NPssuchasgoldandsilverfromplantswasfirst
of10minresultedintheformationofAgNPsat
reported by Gardea-Torresdey et al.[7] Conse- ◦
anambienttemperatureof95 C.
quently, the synthesis of AgNPs from natural
products such as black tea,[8] Aloe vera plant
ConfirmationofAgNPsSynthesis
extract,[9] lemon grass,[10] neem leaf extract,[11]
greentea,[12] etc.,iswellestablished. Formationofyellowishbrowncoloratanam-
Since the fourth century, Areca catechu bient temperature of 95◦C confirmed the syn-
(Areca nut), commonly known as betel nut, thesis of AgNPs (Figure 1A). A color change
R.Sukirthaetal. 3
FIGURE 1. (a) Color intensity of A.catechu–derived colloidal AgNPs. (b) UV-Vis spectrum of
biosynthesizedAgNPswithaspecificplasmonicresonanceat428nm95◦C.(Colorfigureavailable
online.)
1
1
0
2
y
ul
J
7
2
7
5
0:
1
at
]
n
a
m
a
r
hi
c
a
m
a
g
u
m
n
a
h
s
[
y
b
d
e
d
a
o
nl
w
o
D
4 ArecacatechuLinn.derivedSilverNanoparticles
arose due to the excitation of surface plas- distribution of particles ranging from 20 to
mon vibration in the synthesized nanoparticles. 150 nm, with an average around 80 nm (Figure
With this color intensity evident, it was con- 2B).TEMresultsconfirmedtheaverageparticle
firmedthatmorethan90%formationofAgNPs sizeandtheseobtainedresultsshowedasimilar
was achieved at 95◦C compared to the other outcome as that in Mukherjee et al.[22] A sim-
temperatures. A notorious color change indi- ilar trend was also noticed in the TEM images
◦
cated the synthesis of AgNPs at 95 C and sim- ofgoldnanoparticlesobtainedfrom5%persim-
ilar results were observed by Shankar et al.[17] mon leaf broth and 1 mM HAuCl solution at
4
inaqueousneemleafbroth.Thebiosynthesized varioustemperatures.[19] Therefore,thetemper-
AgNPs were confirmed by their plasmonic res- ature was found to be the baseline to determine
onance peak at 428 nm in ultraviolet-visible thesynthesisrateandsizeoftheAgNPs.
(UV-Vis)spectroscopicanalysis(Figure1B).In
UV-Vis analysis, a maximum peak at 428 nm
CharacterizationofAssociatedMolecules
revealed a positive correlation between temper-
atureandAgNPssynthesis.Inaddition,AgNPs Energy-dispersive X-ray (EDX) analysis re-
1 synthesis is directly proportionate to reaction vealed strong signal in the silver region 3 keV
01 temperature.[18,19] Further,thesizeandshapeof confirmingthepresenceofelementalsilver(Fig-
2
y nanoparticles was studied by UV-Vis spectro- ure3A).Thiscompositionalanalysisofbiosyn-
ul
J scopic analysis. In the present study, spherical- thesizedAgNPsshowedthepresenceofcarbon,
7
2 shaped AgNPs were obtained at ambient tem- oxygen,andhydrogencountsalongwithAgNPs.
7
5 perature.Wileyetal.[20]reportedthatthepeakat An optical absorption band peak was observed
0:
1 ∼430 nm could be assigned to the plane dipole in the range of 3–4 keV, which is typical for
at
] resonanceofAgNPs,indicatingthepresenceof the absorption of silver nanocrystallites. Simi-
n
a sphericalparticleswithsmalldiameters.Theop- larly, a sharp band at 3 KeV observed in this
m
a tical absorption spectrum of AgNPs was domi- study confirmed the presence of elemental sil-
r
chi nated by the surface plasmon resonance (SPR) ver. In contrast, the weak signals present along
a
m band, which exhibits a shift toward the red or with the silver revealed the presence of carbon,
a
g blueenddependingupontheparticlesize,shape, oxygen, and hydrogen elements, which reflect
u
m state of aggregation, and surrounding dielec- the intensity of active biomolecules along with
n
a tric medium.[21] Thus, in our study the synthe- the biosynthesized AgNPs. It has also been re-
h
s
[ sisofspherical-shapedAgNPswithablueshift ported that biosynthesized nanoparticles using
y
b wasinitiallyconfirmedwiththeUV-Visspectral plant extracts are bounded by a thin layer of
d
de results. some capping organic material obtained from
oa theplantleafbroth.[23,24]
nl
w Further,thebioactivecompoundsinaqueous
CharacterizationofBiosynthesizedAgNPs
o
D extract of A. catechu responsible for bioreduc-
According to the results observed in UV-Vis tionofsilvernitrateintoAgNPswereconfirmed
spectroscopy, the temperature at which max- with their by their Fourier transform infrared
imum synthesis was obtained was passable (FTIR) band peaks. The presence of polyphe-
for further structural characterization and par- nolics such as terpenoids, flavonoids, and tan-
ticle size determination using scanning elec- nicacidwasconfirmedwiththeircorresponding
tron microscopy (SEM) and transmission elec- band peaks at 3580 and 3314 cm−1.The pres-
tron microscopy (TEM) analysis, respectively. ence of proteins was confirmed with the amide
The nanoparticles were uniformly distributed bondstretchofC-OandC-Ngroupswithpeaks
and most of the particles were spherical at 1634 and 1352 cm−1 (Figure 3B). It was ev-
in shape (Figure 2A). In the present study, idenced that biomolecules such as flavans and
SEM observation showed polydispersed Ag- tannins were present in a remarkable content in
NPs with a spherical shape, which was highly aqueous extract of A. catechu.[25] In addition,
similar to the topological reports of Song the presence of polyphenolics was confirmed
et al.[19] The images showed a broad size by band peaks at 3580 and 3314 cm−1, which
R.Sukirthaetal. 5
FIGURE 2. (a) Topographical appearance of biosynthesized AgNPs in SEM analysis. (b) TEM
imageofbiosynthesizedAgNPsshowingaverageparticlesizeof80nm.
1
1
0
2
y
ul
J
7
2
7
5
0:
1
at
]
n
a
m
a
r
hi
c
a
m
a
g
u
m
n
a
h
s
[
y
b
d
e
d
a
o
nl
w
o
D
6 ArecacatechuLinn.derivedSilverNanoparticles
FIGURE 3. (a) Strong signal of elemental silver at 3 KeV in EDX analysis. (b) FTIR spectra of
phytochemicals-coatedcolloidalAgNPs.(Colorfigureavailableonline.)
1
1
0
2
y
ul
J
7
2
7
5
0:
1
at
]
n
a
m
a
r
hi
c
a
m
a
g
u
m
n
a
h
s
[
y
b
d
e
d
a
o
nl
w
o
D
R.Sukirthaetal. 7
arose due to the sharp and free O-H bond. The The obtained results showed the potential of
biomoleculesresponsibleforthereductionofsil- these biomolecules as dependable for efficient
ver ions may be the presence of flavanoids and stabilizationofAgNPssynthesizedusingA.cat-
terpenoids.[17]Thebandpeaksat1634and1352 echu.Thecytotoxicandantitumorpropertiesof
cm−1developedforC-CandC-Nstretching,re- the biosynthesized AgNPs might have a syner-
spectively, reflect the presence of proteins. In getic effect along with the bound compounds
the current study the presence of C=O stretch- such as flavans and proteins, which possess an-
ing mode indicated the presence of a –COOH titumoractivity.[27]
group in the material bound to AgNPs. Similar
findings the presence of proteins as ligands for InVivoAntiTumorActivityof
AgNPs and a factor for increasing the stability
BiosynthesizedAgNPs
ofAgNPs.[26]Thus,theFTIRbandsat1634and
1352 cm−1 indicated the possibility of AgNPs In vivo study of AgNPs against DAL tu-
bound to proteins through free amine groups. mor cells resulted in a significant decrease in
1
1
0
y 2 FIGURE 4. (a) Histogram of significantly decreased tumor volume in AgNPs treated groups com-
ul paredtotumorgroup.Resultsarerepresentedasameanwithbarsshowingthestandarddeviation.
J
7 (b) Histogram representation of significant increase in life span of experimental groups compared
2
7 to tumor group. Results are represented as a mean with bars showing the standard deviation. (c)
5
0: Histogram representation of revival of normal body weight in the experimental groups compared
1
at tothetumorgroup.Resultsarerepresented asameanwithbarsshowing thestandard deviation.
n] (Colorfigureavailableonline.)
a
m
a
r
hi
c
a
m
a
g
u
m
n
a
h
s
[
y
b
d
e
d
a
o
nl
w
o
D
8 ArecacatechuLinn.derivedSilverNanoparticles
tumorvolume;however,theimpactwasgreater toredbodyweightasapreliminaryindicatorfor
in the AgNPs-treated group compared to the allexperimentalgroupsthroughouttheentirepe-
aqueous extract–treated and untreated tumor riod of treatment. In our results, AgNPs treated
groups (Figure 4A). Supporting the above re- DALbearingmiceexhibitedasignificantreduc-
sults, Badami et al.[28] stated that DAL tumor tioninbodyweightthanthecontrolandaqueous-
mice had increased ascites tumor volume. Re- treated groups (Figure 4C). These results were
ductionintumor volume reflectsthe direct effi- consistent with previous reports of Chanda et
cacyofchemotherapyandimmunotherapywith al.[29] Thenanoparticlesarepotentialindicators
the indications of natural immune response.[29] for antitumor activity and could act specifically
A significant increase in the life span of DAL onthebiomarkerspresentinthecancercells.[30]
model mice confirmed the antitumor activity of BiosynthesizedAgNPsusingaqueousextractof
biosynthesizedAgNPs(Figure4B).Toconclude A. catechu resulted in an average particle size
the overall therapeutic response we also moni- of ∼80 nm. There are reports suggesting that
1 FIGURE5. (a)ViableDALcellsexhibitinguniformgreenfluorescence.(b)EarlyapoptosisinDAL
01 cells treated withA.catechuextract exhibiting orange fluorescence. (c)–(e) Prominent membrane
2
y blebbing and nuclear condensation with apoptotic bodies exhibiting orange to red fluorescence in
ul AgNPs-treatedDALcells.(Colorfigureavailableonline.)
J
7
2
7
5
0:
1
at
]
n
a
m
a
r
hi
c
a
m
a
g
u
m
n
a
h
s
[
y
b
d
e
d
a
o
nl
w
o
D
R.Sukirthaetal. 9
FIGURE 6. DNA laddering assay. L1: marker; L2: DAL-induced tumor cells DNA; L3: tumor cells
DNAtreatedwith600µgAgNPs;L4:tumorcellstreatedwith800µgAgNPs;L5:tumorcellstreated
with1000µgAgNPs;L6:tumorcellstreatedwithaqueousextractofA.catechu.
1
1
0
2
y
ul
J
7
2
7
5
0:
1
at
]
n
a
m
a
r
hi
c
a
m
a
g
u
m
n
a
h particle size of 85 nm is sufficient to invade tu- sationwasobservedintheAgNPs-treatedDAL
s
y [ mor cells.[29] Consequently, the particle size of cells exhibiting a reddish orange fluorescence.
d b ∼85nmissufficienttoinvadethetumorcells.[29] OurresultsareinrelationtothefindingsofYen
e
d The therapeutic efficacy of biosynthesized Ag- etal.[31]whopredictedthatAgNPsarecytotoxic
a
nlo NPswassignificantlydifferent(p <0.05)com- in murine macrophages and fibroblasts.[32] In
w
pared to the aqueous extract–treated group and the present study, AgNPs provoked apoptotic
o
D
controlgroup. body formation in DAL tumor cells, which is
an indispensable indicator of the induction cell
death. In addition, DNA fragmentation was
Cytotoxicity ofBiosynthesizedAgNPs
observed in AgNPs treated DAL tumor cells
In acridine orange/ethidium bromide in a dose-dependent manner (Figure 6) than
(AO/EB) staining, untreated DAL cells exhib- the control (lane 2) and aqueous-treated group
ited green fluorescence, which reflected their (lane 6). In the present study, DNA fragments
percentage of viability (Figure 5A). A visible that formed around 100–150 bp revealed
nuclear condensation with yellowish orange that the biosynthesized AgNPs stimulated
fluorescence was noticed due to the formation mitochondrial-mediated apoptosis in DAL cell
of more apoptotic bodies in the AgNPs-treated lines. The previous findings of Yen et al.[31]
DALcells(Figures5C,5D,and5E)thaninthe and Kalishwaralal et al.[33] added sustaining
aqueousextract–treatedDALcells(Figure5B). evidence that the formation of a DNA ladder
The formation of blebbing and nuclear conden- wasduetothepositiveimpactofAgNPs.