American Journal of Plant Sciences, 2012, 3, 1646-1653 Published Online November 2012 (
Aqueous Extracts of Dried Fruits of Zanthoxylum armatum
DC., (Rutaceae) Induce Cellular and Nuclear Damage
Coupled with Inhibition of Mitotic Activity in Vivo
Eros Vasil Kharshiing
Department of Botany, St. Edmund’s College, Shillong, India.
Received August 9th, 2012; revised September 12th, 2012; accepted October 15th, 2012
Bioactive compounds derived from plant natural compounds have proven to be valuable sources of metabolites which
can seldom be obtained from other sources. Plants belonging to the genus Zanthoxylum have been valued across various
cultures for their curative properties. Zanthoxylum armatum DC., belonging to the family Rutaceae is extensively used
in traditional practices in North-Eastern India and neighbouring regions including South-East Asia. However, the poten-
tial cytogenetic effects of Zanthoxylum armatum under in vivo conditions, and their causative mechanisms have not yet
been studied in detail. The current study was undertaken to evaluate the cytotoxic and genotoxic potential of aqueous
extracts of fruits of Z. armatum under in vivo conditions using the Allium test. Physiological and cellular data indicate
that the extracts induce clumped chromosomes at metaphase stage of cell division coupled with mitotic arrest. Electron
microscopy data reveal membrane damage of cellular organelles, chromatin condensation and chromatin marginalisa-
tion in cell of roots incubated in the extracts. The extracts also induce concentration dependent protein precipitation and
genomic DNA degradation.
Keywords: Clumped Chromosomes; Chromatin Condensation and Marginalisation; TEM; Toxicity; Zanthoxylum
1. Introduction
Plant derived natural compounds such as quinine, mor-
phine, digitoxin, artemisinin and various others have made
important contributions to modern civilisation. Advances
in natural products research have helped in developing
leads towards the identification and isolation of com-
pounds having significant biological activity [1]. Bioac-
tive secondary metabolites which are produced by plants
for their own defense have proven to be valuable sources
of new drugs some of which cannot be obtained from
other sources [2-4]. Research in plants therefore repre-
sents an invaluable source for discovering new sub-
stances, considering that most plants can contain a large
number of secondary metabolites. From approximately
more than 300,000 plant species reported, only a small
percentage has been the subject of phytochemical and
biological activity studies [5].
Plants belonging to the genus Zanthoxylum have been
reported to harbour medicinal properties which justifies
their use in traditional medicines worldwide [6,7]. Many
species have been used in different parts of the world
especially in Asia, Africa and America to treat a number
of diseases in humans and animals [8-11]. Extracts of Z.
nitidum exhibit antibacterial activity in vitro, as well as
antioxidative properties [12,13]. Aqueous extracts of Z.
macrophylla roots have been reported to show anti-sick-
ling effects in human erythrocytes [14]. Extracts of Z.
piperitum show scavenging activities towards active
oxygen species [15]. Crude extracts of Z. acanthopodium
and Z. alainthoides are reported to possess antioxidative
properties [16,17]. Z. armatum contains numerous che-
mical constituents reported to exhibit several biological
activities [18,19]. The genus Zan thoxylum is also re-
ported to harbour several metabolites that exhibit anti-
proliferative activity [20,21]. While the putative curative
properties of medicinal plants are widely reported, there
are also reports of the in vitro toxicity effects of plant
extracts utilised in traditional medicines [22-24]. In vitro
assays indicate that extracts of such plants induce sig-
nificant damage on cellular and nuclear material. Spiri-
donov et al., [25] reported acute in vitro cytotoxicity of
Potentilla erecta extracts, which is used for alleviating
symptoms of disease in cancer patients. The extracts of
aerial parts of Rumex acetosa used in traditional Korean
and Japanese medicine is also reported to show toxicity
Copyright © 2012 SciRes. AJPS
Aqueous Extracts of Dried Fruits of Zanthoxylum armatum DC., (Rutaceae) Induce Cellular and
Nuclear Damage Coupled with Inhibition of Mitotic Activity in Vivo
in vitro [26]. Similar reports are also available for Plan-
tago sp used in traditional Spanish medicine [27]. Plants
extracts used in herbal remedies are also reported to
show cytotoxic potential when used either singly or as
concoctions with extracts of other plants [22].
Zanthoxylum armatum DC. (syn. Z. alatum Roxb.)
belonging to the family Rutaceae is extensively used in
traditional practices of the Khasi tribe in North-Eastern
India and in neighbouring regions including South-East
Asia. According to reports of ethnobotanical properties
of the Zanthoxylum genus, the most commonly used ex-
traction methods use mainly water as a solvent. Herbal
formulations prepared from various parts of the plant are
reported to be anti-helminthic and hypoglycaemic, and
are used as treatment of cholera, tonic for fever, remedy
for skin diseases, diseases of the mouth and teeth and
others [28,29]. However based on currently available
data, the potential cytogenetic effects of Z. armatum un-
der in vivo conditions, and their causative mechanisms
have not yet been studied in detail. The current study was
undertaken to evaluate the cytotoxic and genotoxic po-
tential of aqueous extracts of Z. armatum DC in an in
vivo experimental system and gain some insight into the
mechanism(s) by which these extracts exert their toxic
2. Materials and Methods
2.1. Plant Material and Growth Conditions
The plant specimen used in this study was collected from
Shillong, Meghalaya and identified with the help of Dr. P.
B. Gurung, Herbaria curator, Department of Botany,
North Eastern Hill University, Meghalaya. A voucher
specimen was deposited in the herbaria of the Depart-
ment of Botany, St. Edmund’s College and assigned a
voucher number SEC/BOT/EK/101 dated 01/07/2010.
For the Allium test, onion bulbs of approximately 45
cm in diameter were incubated in double distilled water
in cylindrical opaque plastic vials (25 mm × 50 mm) for
48 hr with water being changed every 24 hr. After 48 hr
the water was substituted with the appropriate solutions
and incubated for another 24 hr after which roots of
equal length were harvested for analyses. The light con-
ditions for growth were maintained at 14 hr light and 10
hrs dark cycles. Light (200 µmol·m2·s1) was supplied
via cool white fluorescent tubes. Temperature was main-
tained at 25˚C ± 1˚C respectively throughout the period
of incubation. For each experiment a minimum of 5
bulbs were used.
2.2. Preparation of Aqueous Extract
Fruits of locally growing Zanthoxylum armatum DC.,
were collected, washed and air dried in the shade until
constant weight was obtained. The dried fruits were then
ground to a coarse powder in a grinder at short bursts of
30 sec each. 5 gm of powdered material was soaked in
100 mL double distilled water and incubated in the dark
for 48 hr at 25˚C ± 1˚C with frequent agitation. After 48
hr the extract was filtered through Whatmann filter paper
No 1 and immediately used. Drying of the aqueous ex-
tract in a rotary vacuum evaporator at 40˚C resulted in
sticky brownish slurry, which could not be used further.
Therefore, the fresh aqueous extracts, hereon referred to
as Aqueous Extract of Dried Fruits (AEDF), were quan-
tified as total phenolic content [30] and used directly for
further experiments.
2.3. Determination of Total Phenolic Content
Total phenolic content of AEDF was estimated according
to Makkar et al., [30] with slight modifications. Fifty
micro litre (50 µl) of AEDF extract for each sample was
taken in a test tube and the volume was made to 1.0 ml
with double distilled water. Then, 0.5 ml of 1 N Folin
Ciocalteu reagent was added and mixed thoroughly after
which 2.5 ml of 0.7 M Na2CO3·H2O solution was added.
The resulting solution was mixed thoroughly and incu-
bated for 40 minutes at 25˚C ± 1˚C. Absorbance was
measured spectrophotometrically at 725 nm and total
phenolic content was estimated as tannic acid equivalent
and expressed as mg/ml. Tannic acid was used for gener-
ating the standard curve.
2.4. Root Growth Measurement and Mitotic
Squash Preparations
Root lengths were measured at 0 hr, 24 hr, 48 hr and 72
hr after incubation. The initial incubation in double dis-
tilled water was considered as 0 (zero) hr for all experi-
ments. Five of the longest roots per bulb were considered.
All measurements are represented as the mean ± S.D. of
5 roots per bulb with a minimum of 5 bulbs per experi-
Root squash preparations were prepared for analyses
of mitotic cells. At 72 hr of incubation, 5 - 10 of the
longest roots from each bulb were harvested at a distance
of 5 mm from the root tip. The roots were immediately
boiled in 2% (w/v) aceto-orcein. From each root a seg-
ment of 1 mm from the root tip was dissected out. This
was used for squash preparation for analyses of mitotic
cells. For each analysis a minimum of 1000 cells were
examined per root tip with a minimum of 5 roots per bulb.
Images of cells were taken with a microscope (Weswox
TRHL-66A) fitted with a digital camera (Sony, Japan).
2.5. Transmission Electron Microscope Studies
After incubation for 24 hr in AEDF, the roots were har-
Copyright © 2012 SciRes. AJPS
Aqueous Extracts of Dried Fruits of Zanthoxylum armatum DC., (Rutaceae) Induce Cellular and
Nuclear Damage Coupled with Inhibition of Mitotic Activity in Vivo
vested for TEM studies. Root tips of approximately 3
mm in length, measured from the tip of roots incubated
in AEDF and the corresponding regions of the control
roots were fixed in Karnovsky’s fixative for 1.5 hr. The
fixed roots were then washed thrice by immersion in 0.1
M cacodylate buffer for 15 min. The fixed roots were
further processed and examined using 100 CXII JEOL
transmission electron microscope at the Sophisticated
Analytical Instrumentation Facility, North-Eastern Hill
University, Shillong as described earlier by Choudhury
and Sharan [31]. A minimum of 3 roots each from 5 dif-
ferent bulbs were used. Bulbs grown only in double dis-
tilled water were taken as negative controls.
2.6. Protein Precipitation Studies
Bovine serum albumin (Fraction V) diluted in double
distilled water to a final concentration of 1 mg/mL was
taken in different tubes. Increasing amounts of AEDF
quantified as total phenolic content were added into each
tube, mixed thoroughly and centrifuged at 2000 g for 10
minutes at room temperature. From each tube equal
volumes of solution were taken for total proten quantifi-
cation and the remaining solutions were incubated for 24
hr at 25˚C ± 1˚C in the dark. After incubation, the tubes
were centrifuged at 2000 g for 10 minutes at room tem-
perature and from each tube equal volumes of solution
were taken for total protein quantification. Total protein
concentration was determined by the Bradford assay
2.7. Genotoxicity Studies
For in vivo studies, total genomic DNA was extracted
from roots of Allium bulbs incubated in increasing con-
centrations of AEDF for 24 hrs as described earlier. Ge-
nomic DNA extraction was modified from Križman et al.,
[33]. Briefly, fresh root tissue was homogenised with
CTAB buffer (20 mM EDTA, 100 mM Tris-HCl [pH
8.0], 1.4 M NaCl, 2% [w/v] N-Cetyl-N,N,N, Trimethyl-
ammonium bromide [CTAB] and 1% [w/v] Polyviny-
polypyrrolidone) in a ratio of 0.2 gm:1.5 ml (tissue:
buffer) alongwith β-Mercaptoethanol (50 µl/ml buffer)
and 0.5% (w/v) activated charcoal and incubated at 55˚C
for 30 min with constant agitation followed with cen-
trifugation at 12,500 g for 10 min at room temperature.
The resulting supernatant was mixed with equal volumes
of chloroform:isoamyl alcohol (24:1) solution and cen-
trifuged at 12,500 g for 10 min at room temperature. The
supernatant was treated with 5 µl RNaseA (1 mg/ml) for
30 min and the DNA was precipitated with cold isopro-
panol, washed twice with 70% cold ethanol and dis-
solved in 100 µl of TE buffer. Total genomic DNA was
quantified by measuring the absorbance at A260 and elec-
trophoresed in 1% (w/v) agarose.
For studying the effects of AEDF on genomic DNA,
total genomic DNA from roots of Allium bulbs incubated
only in distilled water was extracted as described above.
5 µg of DNA was incubated with increasing concentra-
tions of AEDF for 12 hrs and electrophoresed in 1% (w/v)
3. Results
3.1. Analysis of Mitotic Activity and Changes in
Phase Index of Allium Root Cells under
Incubation with AEDF
Incubation of 2-day-old onion roots in AEDF for 24 hr
inhibited root growth by approximately 83% in com-
parison with the negative controls (Figure 1(b)). The
inhibition in root growth was comparable to that induced
by the Fenton’s reagent [34] which is a commonly used
inducer of oxidative stress. A dose response analysis of
the effect of AEDF on root growth indicated that half
maximal effective concentration (EC50) for inhibiton of
root growth was observed at total phenolics concentra-
tion of 0.4 mg/ml (Figure 1(c)). Analysis of mitotic ac-
tivity indicated that incubation of roots in AEDF caused
changes in the percentage of the distribution of particular
phases’ in comparison to the roots in the control. While
both the Fenton’s reagent and AEDF inhibited root
growth along with alterations in the mitotic indices, the
characteristic effect caused by AEDF, was an extensive
increase in the metaphase index during the period of in-
cubation (Figure 1(d)). Furthermore, incubation in AEDF
induced clumping of chromosomes in all metaphase cells
examined while the cells at interphase showed extensive
vacuolisation of the cell nuclei (Figures 1(e)-(j)). The
meristematic cells containing clumped chromosomes,
accounted for 5% of the total cells examined which cor-
responded to the percentage of cells in either metaphase
or anaphase stage of mitotic division in the negative con-
3.2. Transmission Electron Microscope Studies
Roots of Allium bulbs incubated in AEDF for 24 hr
showed inhibition of growth which was dependent on the
concentration of AEDF used. In most of the roots, the
inhibition in root growth was accompanied by swelling
of the region near the root tips (Figures 2(a)-(g)). Trans-
mission electron microscope studies of the roots incu-
bated in AEDF for 24 hr showed extensive alterations in
the ultra-structure of the cellular organelles investigated
compared to the negative controls. The cell wall and cell
membrane of the controls was intact and continuous. In
contrast, in the experimental roots, while the integrity of
Copyright © 2012 SciRes. AJPS
Aqueous Extracts of Dried Fruits of Zanthoxylum armatum DC., (Rutaceae) Induce Cellular and
Nuclear Damage Coupled with Inhibition of Mitotic Activity in Vivo
Figure 1. (a) Voucher specimen of Zanthoxylum armatum
DC. Inset shows dried fruit; (b) Root growth in Allium
bulbs after 24 hr, 48 hr and 72 hr of incubation; (c) Dose
response analysis of root growth in varying concentrations
of AEDF; (d) Mitotic phase indices of Allium roots after 24
hr incubation in the respective solutions; (e)-(j): Root tip
cells of Allium after incubation in dd H2O (e), (f), Fenton’s
reagent (g), (h) and AEDF (1 mg/ml) (i), (j). Allium bulbs
were incubated in dd H2O for 48 hr and then transferred
into the respective solutions for another 24 hr. Images are
representative of cells observed after 24 hr incubation in the
respective solutions. Arrows indicate normal mitotic stages
with visibly distinct chromosomes (e), (f), cells with cellular
and nuclear damage (g), (h) cells with clumped chromo-
somes and nuclear vacuolisation (i), (j). In (b) and (c),
*denotes point of incubation in AEDF as described in in
text. Bars = 50 μm.
the cell wall appeared to be intact, the cell membrane was
ruptured and discontinuous at several places (Figures
2(h)-(j)). The cell membrane also appeared to be de-
tached from the cell wall. The root cells of the controls
had a large number of mitochondria of varying shapes
Figure 2. (a) Morphology of root tips after incubation in
different concentrations AEDF (as indicated) for 24 hr.
Concentrations were determined as total phenolic content
(mg/ml); (b)-(g) Representative images of roots showing
distinct swelling under increasing concentrations of AEDF.
Concentrations used were same as that of panel (a); (h)-(p)
TEM micrographs of roots incubated in AEDF (1mg/ml)
for 24 hr; (h)-(j) cell wall and cell membrane showing dis-
rupted continuity in the treated roots (i), (j); (k)-(m) mito-
chondria showing cristolysis and with ruptured or de-
stroyed outer membrane in the treated roots (l), (m); (n)-(p)
nuclei with condensed or marginalized chromatin in the
treated roots (o), (p); (h), (k), (n) = control roots incubated
in ddH2O. CW-cell wall, CM-cell membrane, IS-intercellu-
lar space, MT-mitochondria, MC-mitochondrial membrane;
RER-rough endoplasmic reticulum, NM-nuclear membrane,
NC-nucleoli, CR-chromatin. Bars = 500 µm (b)-(g), 1 µm
(h)-(j), 0.5 µm (k)-(m), 2 µm (n)-(p).
and sizes with an outer membrane and very well defined
cristae (Figure 2(k)). The mitochondria of the cells in
roots incubated in AEDF, however, exhibited extensive
cristolysis and disarrangement of the cristae (Figures 2(l)
and (m)). Furthermore, the integrity of outer membrane
of the mitochondria in the root cells exposed to AEDF
was also disrupted, while in some cases the entire outer
membrane was destroyed (Figure 2(m)) whilst main-
taining the structural identity of the cristae. In the control
roots, the nuclei of the cells at interphase were spherical
Copyright © 2012 SciRes. AJPS
Aqueous Extracts of Dried Fruits of Zanthoxylum armatum DC., (Rutaceae) Induce Cellular and
Nuclear Damage Coupled with Inhibition of Mitotic Activity in Vivo
in shape with an intact double-layered nuclear envelope
and well-defined nucleolus with distinctly visible chro-
matin (Figure 2(n)). As observed with the other cellular
organelles, the integrity of the nuclear envelope in the
cells of the roots incubated in AEDF was also disrupted.
The disruption of the nuclear envelope was accompanied
with marked chromatin condensation. In most of the cells,
the electron-dense nuclear material was aggregated pe-
ripherally under the nuclear membrane (chromatin mar-
ginalisation) while in others a uniformly dense nuclei
was prevalent (Figures 2(o) and (p)).
3.3. Effect of AEDF on Protein and DNA
24 hr of incubation of protein solution (BSA) with in-
creasing amounts of AEDF resulted in a gradual decrease
in the absorbance of the incubated solutions at 595 nm
(Figure 3(a)). Normalisation of the absorbance obtained
for the different solutions with the absorbance at the
same wavelength prior to incubation, showed protein
precipitation to be proportional to the concentration of
AEDF used (Figure 3(b)).
Incubation of roots of Allium bulbs in increasing con-
centrations of AEDF for 24 hr did not show any visible
damage to the integrity of genomic DNA (Figure 3(c)).
Interestingly incubation of fresh genomic DNA with
AEDF for 12 hr, showed increased degradation of DNA
with increasing AEDF concentration (Figure 3(d)).
4. Discussion and Conclusions
The current study demonstrates that the aqueous extracts
of dried fruits (AEDF) of Z. armatum DC., can induce
nuclear and cellular damage coupled with mitotic arrest
in Allium roots. AEDF induced clumping of chromo-
somes in the meristematic cells thereby possibly inhibit-
ing further mitotic activity. Such a condition would in-
hibit the progression of cell division beyond metaphase,
thereby leading to subsequent mitotic arrest [35]. In
murine cell lines, drugs that induce cytotoxic effects
concurrently induce clumped metaphase chromosomes,
mitotic arrest at metaphase and subsequent cell death
[35]. In cell lines, it has been suggested that clumped
chromosomes during cell division may arise due to inhi-
bition of telomerase resulting in end-to-end fusion of the
chromosomes [36]. Various plant extracts used in tradi-
tional medicinal practices have also been reported to in-
hibit telomerase activity [37-39]. It is interesting to note
that in plants, germinating seedlings and root tips are
sites of maximum telomerase activity [40]. It could
therefore be possible that AEDF alters or inhibits telom-
erase activity in the dividing cells resulting in clumped
chromosomes at metaphase. While it is still premature at
this stage to conclusively ascertain how AEDF induces
Figure 3. Effect of AEDF on protein and DNA (a)-(b):
AEDF induces concentration dependent precipitation of
BSA after 24 hr of incubation; (c)-(d): Effects of AEDF on
genomic DNA in intact Allium roots (c) and isolated total
genomic DNA (d). 1-5 denotes concentrations of AEDF used
corresponding to 0.2, 0.4, 0.6, 0.8 and 1.0 mg/ml of total
phenolic content respectively, C denotes controls, M denotes
1 kb DNA ladder.
the observed clumped chromosmes at metaphase, the
results clearly suggest that chromosome clumping is one
the mechanisms by which AEDF induces genotoxicity in
Assessing cell membrane integrity is one of the most
common ways to measure cell viability and cytotoxic
effects. Compounds that exhibit cytotoxic effects often
compromise cell membrane integrity. The results from
the current study conclusively show that AEDF can
cause loss of structural membrane integrity in cellular
organelles such as mitochondria, nuclei and cell mem-
branes, all of which are critical for proper functioning of
the cell. Lipid peroxidation is known to trigger the loss of
membrane integrity, structural damage to DNA, and cell
death. Since AEDF induced disruption of cell and organ-
elle membrane coupled with chromatin condensation and
marginalisation, it was of interest to investigate whether
the observed cellular damages could be attributed to lipid
peroxidation effected by AEDF. The formation of sub-
stances, which react with thiobarbituric acid (TBARS), is
characteristic of the terminal stage of lipid peroxidation
and indicates the breakdown of peroxidised lipids.
Analysis of roots of Allium bulbs incubated in varying
concentrations of AEDF exhibited a concentration de-
pendent inhibition of a yellow coloured complex, previ-
ously reported to be an aldehyde-TBA complex [41,42]
(result not shown). This is in agreement with previously
published reports of the inhibition of lipid peroxidation
by Zanthoxylum extracts [43,44] suggesting that the cel-
Copyright © 2012 SciRes. AJPS
Aqueous Extracts of Dried Fruits of Zanthoxylum armatum DC., (Rutaceae) Induce Cellular and
Nuclear Damage Coupled with Inhibition of Mitotic Activity in Vivo
lular damages induced by AEDF possibly does not in-
volve induction of lipid peroxidation and might occur via
an alternative mechanism(s).
Chromatin condensation and marginalisation are nu-
clear events that have been associated with the disruption
of cell division eventually resulting in cell death [45,46].
Chromatin condensation and nuclear DNA fragmentation
occur in plant cells during senescence [47] and/or in the
presence of cytotoxic agents [48] and prolonged expo-
sure to higher concentrations of cytotoxic agents can
induce loss of membrane integrity. Electron microscopy
analyses indicate that AEDF drastically alters the mor-
phology of the cellular organelles investigated. TEM
results reveal ultrastructural morphological characteris-
tics such as 1) electron-dense nuclei (marginalisation in
the early phase); 2) disorganized cytoplasmic organelles;
and 3) disrupted organelle membrane which indicate that
the cytotoxic potential of AEDF could be attributed to its
ability to possibly induce necrosis since the ultrastruc-
tural alterations induced by AEDF bear striking resem-
blances to typical necrotic hallmarks.
The results presented in this study indicates that AEDF
does not seem to induce any visible damage to the integ-
rity of nuclear DNA under in vivo conditions which is in
contrast to that reported for apoptotic cells or cells un-
dergoing programmed cell death, but can induce degra-
dation of isolated genomic DNA. The reason for such
variation could be that under in vivo conditions, the pro-
portion of DNA degraded by AEDF is much lesser com-
pared to the intact genomic DNA, thereby masking the
gentoxic potential of AEDF. This could be partially due
to the reason that not all the cells are as easily accessible
to AEDF as the meristematic cells present at the root tips.
Since the meristematic cells constitute only a small per-
centage of the total tissue from which the genomic DNA
was isolated, therefore, the integrity of the nuclear DNA
seemed unaffected by AEDF. However, under in vitro
conditions when all the nuclear DNA present was acces-
sible to AEDF, it induced extensive DNA degradation.
This implies that AEDF does have the potential to induce
acute gentoxic effects. In conclusion, the current study
clearly demonstrates that the aqueous extracts of dried
fruits (AEDF) of Zanthoxyllum armatum DC., which is
used extensively in traditional herbal remedies exert its
toxic potential in vivo by inducing membrane damage of
cellular organelles, chromatin condensation, chromatin
marginalisation, chromosome clumping and nuclear
DNA damage resulting in subsequent mitotic arrest.
5. Acknowledgements
The work was supported by the University Grants Com-
mission, India research grant (Ref. No. F-5-5/2010-11/
[1] D. J. Newman and G. M. Cragg, “Natural Products as
Sources of New Drugs over the last 25 Years,” Journal of
Natural Products, Vol. 70, No. 3, 2007, pp. 461-477.
[2] G. M. Cragg, D. G. I. Kingston and D. J. Newman,
“Anticancer Agents from Natural Products,” In: D. G. I.
Kingston, G. M. Cragg and D. J. Newman, Eds., CRC
Taylor & Francis, New York, 2005, pp. 1-3.
[3] P. B. Kaufman, A. Kirakosyan, M. McKenzie, P.
Dayanandan, J. E. Hoyt and C. Li, “The Uses of Plant
Natural Products by Humans and Risks Associated with
Their Use,” In: L. J. Cseke, A. Kirakosyan, P. B. Kauf-
man, S. Warber, J. A. Duke and H. L. Brielmann, Eds.,
Natural Products from Plants, CRC Taylor & Francis,
New York, 2006, pp. 442-468.
[4] S. M. Colegate and R. J. Molyneux, “An Introduction and
Overview,” In: S. M. Colegate and R. J. Molyneux, Eds.,
Bioactive Natural Products: Detection, Isolation, and
Structural Determination, 2nd Edition, CRC Press, New
York, 2007, pp. 1-3.
[5] C. Tringali, “Bioactive Compounds from Natural Sources:
Isolation, Characterization and Biological Properties,”
CRC Taylor & Francis, New York, 2001, pp. 9-10.
[6] J. S. Negi, V. K. Bisht, A. K. Bhandari, P. Singh and R. C.
Sundriyal, “Chemical Constituents and Biological Activi-
ties of the Genus Zanthoxylum: A Review,” African
Journal of Pure and Applied Chemistry, Vol. 5, No. 12,
2011, pp. 412-416.
[7] L. O. J. Patiño, R. J. A. Prieto and S. L. E. Cuca, “Bioac-
tive Compounds in Phytomedicine: Zanthoxylum Genus
as Potential Source of Bioactive Compounds,” In: I. Ra-
sooli, Ed., InTech Europe, Rijeka, 2012, pp. 185-218.
[8] R. Diéguez, G. Garrido, S. Prieto, Y. Iznaga, L. González,
J. Molina, M. Curini , F. Epifano and M. C. Marcotullio,
“Antifungal Activity of Some Cuban Zanthoxylum Spe-
cies,” Fitoterapia, Vol. 74, No. 4, 2003, pp. 384-386.
[9] S. K. Adesina, “The Nigerian Zanthoxylum: Chemical and
Biological Values,” African Journal of Traditional, Com-
plimentary and Alternative Medicines, Vol. 2, No. 3,
2005, pp. 282-301.
[10] S. Rochfort, J. Anthony, J. A. Parker and F. R. Dunshea,
“Plant Bioactives for Ruminant Health and Productivity,”
Phytochemistry, Vol. 69, No. 2, 2008, pp. 299-322.
[11] S. S. Pereira, L. S. Lopes, R. B. Marques, K. A. Figueiredo,
D. A. Costa, M. H. Chaves and F. R. C. Almeida, “Anti-
nociceptive Effect of Zanthoxylum rhoifolium Lam. (Ru-
taceae) in Models Acute Pain in Rodents,” Journal of
Ethnopharmacology, Vol. 129, 2010, pp. 227-231.
[12] S. Bhattacharya, M. K. Zaman and P. K. Haldar, “Anti-
Copyright © 2012 SciRes. AJPS
Aqueous Extracts of Dried Fruits of Zanthoxylum armatum DC., (Rutaceae) Induce Cellular and
Nuclear Damage Coupled with Inhibition of Mitotic Activity in Vivo
bacterial Activity of Stem Bark and Root of Indian
Zanthoxylum nitidum,” Asian Journal of Pharmaceutical
and Clinical Research, Vol. 2, No. 1, 2009, pp. 30-24.
[13] L.-F. Shyur, J.-H. Tsung, J.-H. Chen, C.-Y. Chiu and C.-P.
Lo, “Antioxidant Properties of Extracts from Medicinal
Plants Popularly Used in Taiwan,” International Journal
of Appllied Science and Engineering, Vol. 3, No. 3, 2005,
pp. 195-202.
[14] I. Elekwa, M. O. Monanu and E. O. Anosike, “Effects of
Aqueous Extracts of Zanthoxylum macrophylla Roots on
Membrane Stability of Human Erythrocytes of Different
Genotypes,” Biokemistri, Vol. 17, No. 1, 2005, pp. 7-12.
[15] E. Yamazaki, “Extraction of Antioxidants from Zanthoxy-
lum piperitum DC. Fruit-Inhibition of Glycation with
Polyphenols from Zanthoxylum piperitum DC. Fruit,”
Reports of the Mie Prefectural Science and Technology,
Promotion Center Industrial Research Division, Vol. 26,
2002, pp. 1-5.
[16] A. H. Cahyana and L. Mardiana “The Use of Chemical
Components Isolated from Andaliman (Zanthoxylum
acanthopodium DC) Essential Oil as a Source of Natural
Antioxidant,” Journal of Food Science and Technology,
Vol. 1, No. 1, 2003, pp. 106-111.
[17] Y.-C. Chung, C.-T. Chien, K.-Y. Teng and S.-T. Chou,
“Antioxidative and Mutagenic Properties of Zanthoxylum
ailanthoides Sieb & Zucc.,” Food Chemistry, Vol. 97,
2006, pp. 418-425. doi:10.1016/j.foodchem.2005.05.019
[18] T. P. Singh and O. M. Singh, “Phytochemical and Phar-
macological Profile of Zanthoxylum armatum DC.—An
Overview,” Indian Journal of Natural Products and Re-
sources, Vol. 2, No. 3, 2011, pp. 275-285.
[19] Barkatullah, M. Ibrar, N. Muhammad and L. Tahir, “An-
timicrobial Evaluation, Determination of Total Phenolic
and Flavoniod Contents in Zanthoxylum armatum DC,”
Journal of Medicinal Plants Research, Vol. 6, No. 11,
2012, pp. 2105-2110.
[20] G. Pachon, H. Rasoanaivo, A. Azqueta, J. C. Rakotozafy,
A. Raharisololalao, A. L. De Cerain, J. De Lapuente, M.
Borràs, S. Moukha, J. J. Centelles, E. E. Creppy and M.
Cascante, “Anticancer Effect of a New Benzophenanth-
ridine Isolated from Zanthoxylum madagascariense (Ru-
taceline),” In Vivo, Vol. 21, No. 2, 2007, pp. 417-422.
[21] G. Cebrián-Torrejón, S. A. Kahn, N. Lagarde, F. Castel-
lano, K. Leblanc, J. Rodrigo, V. Molinier-Frenkel, A.
Rojas de Arias, M. E. Ferreira, C. Thirant, A. Fournet, B.
Figadère, H. Chneiweiss and E. Poupon, “Antiprolifera-
tive Activity of Trans-Avicennol from Zanthoxylum chi-
loperone var. Angustifolium against Human Cancer Stem
Cells,” Journal of Natural Products, Vol. 75, No. 2, 2012,
pp. 257-261. doi:10.1021/np2004165
[22] J. Y. Seo, M. Y. Park, T. Y. Jung, H. Y. Choi, D. Kim, H.
S. Lee and S. K. Ku, “Genotoxicity Testing of Aqueous
Extracts of Mahwangyounpae-Tang, a Polyherbal For-
mula,” Food and Chemical Toxicology, Vol. 46, 2008, pp.
3827-3831. doi:10.1016/j.fct.2008.10.005
[23] C. J. van den Bout-van den Beukel, O. J. Hamza, M. J.
Moshi, M. I. Matee, F. Mikx, D. M. Burger, P. P. Koop-
mans, P. E. Verweij, W. G. Schoonen and A. J. van der
Ven, “Evaluation of Cytotoxic, Genotoxic and CYP450
Enzymatic Competition Effects of Tanzanian Plant Ex-
tracts Traditionally Used for Treatment of Fungal Infec-
tions,” Basic & Clinical Pharmacology & Toxicology,
Vol. 102, No. 6, 2008, pp. 515-526.
[24] J. Demma, E. Engidawork and B. Hellman, “Potential
Genotoxicity of Plant Extracts Used in Ethiopian Tradi-
tional Medicine,” Journal of Ethnopharmacology, Vol.
122, No. 1, 2009, pp. 136-142.
[25] N. A. Spiridonov, D. A. Konovalov and V. V. Arkhipov,
“Cytotoxicity of Some Russian Ethnomedicinal Plants
and Plant Compounds,” Phytotherapy Research, Vol. 19,
No. 5, 2005, pp. 428-432. doi:10.1002/ptr.1616
[26] N. J. Lee, J. H. Choi, B. S. Koo, S. Y. Ryu, Y. H. Han, S.
I. Lee and D. U. Lee, “ Antimutagenicity and Cytotoxic-
ity of the Constituents from the Aerial Parts of Rumex
acetosa,” Biological and Pharmaceutical Bulletin, Vol.
28, No. 11, 2005, pp. 2158-2161. doi:10.1248/bpb.28.2158
[27] M. Gálvez, C. Martín-Cordero, M. López-Lázaro, F.
Cortés and M. J. Ayuso, “Cytotoxic Effect of Plantago
spp. on Cancer Cell Lines,” Journal of Ethnopharmacol-
ogy, Vol. 88, No. 2-3, 2003, pp. 125-130.
[28] S. R. Hynniewta and Y. Kumar, “Herbal Remedies
among the Khasi Traditional Healers and Village Folk in
Meghalaya,” Indian Journal of Traditional Knowledge,
Vol. 7, No. 4, 2008, pp. 581-586.
[29] V. Jaiswal, “Culture and Ethnobotany of Jaintia Tribal
Community of Meghalaya, Northeast India—A Review,”
Indian Journal of Traditional Knowledge, Vol. 9, No. 1,
2010, pp. 38-44.
[30] H. P. S. Makkar, M. Bluemmel, N. K. Borowy and K.
Becker, “Gravimetric Determination of Tannins and Their
Correlations with Chemical and Protein Precipitation Me-
thods,” Journal of the Science of Food and Agriculture,
Vol. 61, No. 2, 1993, pp. 161-165.
[31] Y. Choudhury and R. N. Sharan, “Ultrastructural Altera-
tions in Liver of Mice Exposed Chronically and Trans-
generationally to Aqueous Extract of Betel Nut: Implica-
tions in Betel Nut-Induced Carcinogenesis,” Microscopy
Research and Techique, Vol. 73, No. 5, 2010, pp. 530-
[32] M. Bradford, “A Rapid and Sensitive Method for the
Quantitation of Microgram Quantities of Protein Utilizing
the Principle of Protein-Dye Binding,” Analalytical Bio-
chemistry, Vol. 72, 1976, pp. 248-254.
[33] M. Križman, J. Jakše, D. Baričevič, B. Javornik and M.
Prošek, “Robust CTAB-Activated Charcoal Protocol for
Plant DNA Extraction,” Acta Agriculturae Slovenica, Vol.
87, No. 2, 2006, pp. 427-433.
[34] F. Haber and J. Weiss, “On the Catalysis of Hydroperox-
ide,” Naturwissenschaften, Vol. 20, 1932, pp. 948-950.
[35] A. S. Multani, C. Li, M. Ozen, M. Yadav, D. F. Yu, S.
Copyright © 2012 SciRes. AJPS
Aqueous Extracts of Dried Fruits of Zanthoxylum armatum DC., (Rutaceae) Induce Cellular and
Nuclear Damage Coupled with Inhibition of Mitotic Activity in Vivo
Copyright © 2012 SciRes. AJPS
Wallace and S. Pathak, “Paclitaxel and Water-Soluble
Poly(L-Glutamic Acid)-Paclitaxel, Induce Direct Chro-
mosomal Abnormalities and Cell Death in a Murine Me-
tastatic Melanoma Cell Line,” Anticancer Research, Vol.
17, No. 6D, 1997, pp. 4269-4274.
[36] W. C. Chou, A. L. Hawkins, J. F. Barrett, C. A. Griffin
and C. V. Dang, “Arsenic Inhibition of Telomerase Tran-
scription Leads to Genetic Instability,” Journal of Clini-
cal Investigation, Vol. 108, No. 10, 2001, pp. 1541-1547
[37] S. Y. Lyu, S. H. Choi and W. B. Par, “Korean Mistletoe
Lectin-Induced Apoptosis in Hepatocarcinoma Cells Is
Associated with Inhibition of Telomerase via Mitochon-
drial Controlled Pathway Independent of p53,” Archives
of Pharmacal Research, Vol. 25, No. 1, 2002, pp. 93-101.
[38] S. H. Choi, S. Y. Lyu and W. B. Park, “Mistletoe Lectin
Induces Apoptosis and Telomerase Inhibition in Human
A253 Cancer Cells through Dephosphorylation of Akt.,”
Archives of Pharmacal Research, Vol. 27, No. 1, 2004,
pp. 68-76. doi:10.1007/BF02980049
[39] M. Pourhassan, N. Zarghami, M. Rahmati, A. Alibakhshi
and J. Ranjbari, “The Inhibitory Effect of Curcuma longa
Extract on Telomerase Activity in A549 Lung Cancer
Cell Line,” African Journal of Biotechnology, Vol. 9, No.
6, 2010, pp. 912-919.
[40] K. Riha, J. Fajkus, J. Siroky and B. Vyskot, “Develop-
mental Control of Telomere Lengths and Telomerase Ac-
tivity in Plants,” The Plant Cell, Vol. 10, No. 10, 1998,
pp. 1691-1698.
[41] H. Kosugi, T. Kato and K. Kikugawa, “Formation of
Yellow, Orange, and Red Pigments in the Reaction of
Alk-2-Enals with 2-Thiobarbituric Acid,” Analytical Bio-
chemistry, Vol. 165, No. 2, 1987, pp. 456-464.
[42] Q. Sun, C. Faustman, A. Senecal, A. L. Wilkinson and H.
Furr, “Aldehyde Reactivity with 2-Thiobarbituric Acid
and TBARS in Freeze-Dried Beef during Accelerated
Storage,” Meat Science, Vol. 57, No. 1, 2001, pp. 55-60.
[43] E. J. Cho, T. Yokozawa, D. Y. Rhyu, H. Y. Kim, N. Shi-
bahara and J. C. Park, “The Inhibitory Effects of 12 Me-
dicinal Plants and Their Component Compounds on Lipid
Peroxidation,” The American Journal of Chinese Medi-
cine, Vol. 31, No. 6, 2003, pp. 907-917.
[44] J. M. Hur, J. G. Park, K. H. Yang, J. C. Park, J. R. Park, S.
S. Chun, J. S. Choi and J. W. Choi, “Effect of Methanol
Extract of Zanthoxylum piperitum Leaves and of Its Com-
pound, Protocatechuic Acid, on Hepatic Drug Metaboliz-
ing Enzymes and Lipid Peroxidation in Rats,” Bioscience
Biotechnology Biochemistry, Vol. 67, No. 5, 2003, pp.
945-950. doi:10.1271/bbb.67.945
[45] N. Yao, Y. Tada, P. Park, H. Nakayashiki, Y. Tosa and S.
Mayama, “Novel Evidence for Apoptotic Cell Response
and Differential Signals in Chromatin Condensation and
DNA Cleavage in Victorin-Treated Oats,” Plant Journal,
Vol. 28, No. 1, 2001, pp. 13-26.
[46] K. Zheng, J.-W. Pan, L. Ye, Y. Fu, H.-Z. Peng, B.-Y.
Wan, Q. Gu, H.-W. Bian, N. Han, J.-H. Wang, B. Kang,
J.-H. Pan, H.-H. Shao, W.-Z. Wang and M.-Y. Zhu, “Pro-
grammed Cell Death-Involved Aluminum Toxicity in
Yeast Alleviated by Antiapoptotic Members with De-
creased Calcium Signals,” Plant Physiology, Vol. 143,
No. 1, 2007, pp. 38-49. doi:10.1104/pp.106.082495
[47] E. O’Brien, B. G. Murray, B. C. Baguley, B. A. Morris
and I. B. Ferguson, “Major Changes in Chromatin Con-
densation Suggest the Presence of an Apoptotic Pathway
in Plant Cells,” Experimental Cell Research, Vol. 241,
No. 1, 1998, pp. 46-54. doi:10.1006/excr.1998.4036
[48] S. Chakraborty, M. Roy, A. K. Taraphdar and R. K. Bhat-
tacharya, “Cytotoxic Effect of Root Extract of Tiliacora
racemosa and Oil of Semecarpus anacardium Nut in
Human Tumour Cells,” Phytotherapy Research, Vol. 18,
No. 8, 2004, pp. 595-600. doi:10.1002/ptr.1501.