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Document heading
doi: 10.1016/S1995-7645(14)60258-3
Phytochemical
Molecular Biology and Biotechnology Department, Pan African University, Institute for Basic Sciences, Technology and Innovation
(PAUISTI JKUAT), Nairobi, Kenya
2
Biochemistry Department, Jomo Kenyatta University of Agriculture and Technology (JKUAT), Nairobi, Kenya
Faculty of Agriculture Research Park (FARP) and Biochemistry Department, Faculty of Agriculture, Cairo University, 12613 Giza, Egypt
ABSTRA
CT
ARTICLE INFO
Objective:
Article history:
Received 8 Jul 2014
To
determine
the
phytochem
ical
compositio
n,
antioxidant
and
anticancer
activities of
ethanolic
and water
leaves
extracts of
Annona
muricata
(A.
muricata)
from the
Eastern
Uganda.
Methods: P
hytochemi
cal
screening
was
conducted
using
standard
qualitative
methods
Keywords:
Annona muricata
Anticancer
1. Introduction
Cancer is the major cause of mortality
and
incidence
mor
bidit
World
Organization[1,2], annualauthor:
Health
*Corresponding
Hany A
El
-Shemy, Faculty of
Agriculture
Research
Park
(FARP)
and
Biochemistry
D
epartment, Faculty of
Agriculture, Cairo
U
niversity, 12613 Giza,
Egypt.
E-mail: helshemy@yahoo.com
Foundation Project: S upported by the Pan African
,4].
fri
ca
Lot 2013/2014 at the Institute for Basic Sciences,
is
Technology and Innovation in collaboration with Cairo
the Graduate Scholarship Student Research Funding
University to Y Gavamukulya.
551
200
with
a
mor
talit
y of
421
000[3
cancer
deathssynthetic
nonoccurred in low- andessential dietary
middle-income
agents to interrupt
countries[3,4].
Molecular
targeted
the
process
of
carcinogenesis
agents are currently
and to prevent or
being studied in all
delay
tumor
treatment
settings
growth[5,6].
The
including
that
of
available
chemoprevention,
treatment
which is defined as the
methods include
surgery,
chemotherapy,
and
radiation[7].
The
current
available methods
of
treatment
all
induce significant
side effects and
therefore the need
for
alternate
adjuvant therapies
Yahaya Gavamukulya et al./Asian Pac J Trop Med 2014; 7(Suppl 1): S355-S363
S356
has arisen[8]. Natural products are extremely an of ethanolic and water leaves
important source of medicinal agents. Althoughextracts of A. muricata from
there are some new approaches to drug discovery,Eastern Uganda, as an alternative
such as combinatorial chemistry and computer
medicine in the prevention and
based molecular modeling design, none of them
treatment of cancer and other
can replace the importance of natural products in
drug discovery and development[9,10]. Manyoxidative stress related diseases.
synthetic drugs cause severe side effects that are
not acceptable except as treatments of last resort2. Materials and methods
for terminal diseases such as cancer and the
metabolites discovered in medicinal plants may2.1. Sample collection and
authentication
avoid the side effect of synthetic drugs[11].
Antioxidants are a group of [11-13]
substances that are
useful for fighting cancer and other processes that
potentially
lead
to
diseases
such
as
Eastern
Kib
Northern
KALIR
UGANDA
Uganda
Kah
UgandaEastern
Nakibungulia
Buya IGANG
Ancient
therefore
aimed
to
determine
the
were
amounts
leaves
350
were
powdered.
Equal
g) of powdered
extracted
using
extracts
were
using
then
concentrated
rotary
certified suppliers
and
were
of
the
highest
(EACC)
cell lines
MDA
and
SKBR3
standard
procedures
as
previously
Yahaya Gavamukulya et al./Asian Pac J Trop Med 2014; 7(Suppl 1): S355-S363
S357
screened
for
the
following
phyto the absorbance was measured at
constituents:
alkaloids,
saponins, 700 nm. A standard curve for gallic
acid was generated and the linear
terpenoids, flavonoids, coumarins and equation was used to calculate the
lactones, anthraquinones, tannins, cardiac reducing power of the extracts.
glycosides, phenols and phytosterols.
2.8.
Quantification
of
2.5. Determination of relative abundance of antioxidant activity using the
the phytochemicals
1, 1-diphenyl-2-picrylhydrazyl
critical=2
7055.
of Folin reagent
(1
concentrations of the
extracts (0, 250, 500, 750, 1 000, and 1
250 g/mL) were used. A volume of
%
2.5
100
muricata
The ethanolic leaves
extracts of A. muricata
fractionated using thin
layer chromatography
technique. The extract was applied
on silica gel 60 F254 TLC
aluminum sheets
Different
extremes
to
separate
the
petroleum
ether:
ethyl
(4:1:1).
and named as
EEAM1b-EEAM11.
2.10.
In
vitro
anti-cancer
000,
and 1 250 g/mL) were added. The tubes were 2.11. MTT assay for breast cancer
%
incubated at 37 C in the presence of 5 (v/v) CO2 cell lines MDA and SKBR3
for 2 h[31]. For each examined material (and control),
a new clean, dry small test tube was used and 10 L The culture medium was
of cell suspension, 80 L saline and 10 L trypanprepared using modified Earles
%
with 1.2 g/L sodium
blue (0.4 ) were added and mixed. Then thesalt
and
L-glutamine
number of living cells (non-stained) was calculatedcarbonate
%
using a hemocytometer slide by microscope (Nikon,(Gibco, Grand I sland, USA), 10
%
TMS). The extracts concentration providing 50 inactivated fetal bovine serum
inhibition (IC50) was calculated from the graph(Gibco) , and 100 units/m L
plotting inhibition percentages against logarithm ofpenicillin and 100 mg/ mL
concentration after transforming the concentrations.
Yahaya Gavamukulya et al./Asian Pac J Trop Med 2014; 7(Suppl 1): S355-S363
S358
Alkaloids; HIGH
Phytosterols; HIGH
Phenols; HIGH
Tannins; HIGH
and EEAM11
was evident that the TLC
EEAM4 had the highest
Compted Chi-square
Criticalfraction
value
reducing
power
whereas
%
graphically.
Cell death was calculated using
Figure 4. Phytochemicals present in water
fraction EEAM 11 registered no
the following formula:
leaf extracts of A. muricata with
reducing
power
activity.
% Cell death=(Control OD - Sample OD)/Control relative abundance computed from the
Although some fractions had a
OD 100
2 test.
reducing power of more than 15
3.2. Total phenolic
g/ml GAE,
most of them
2.12. Cytotoxicity effect of ethanolic leaves compounds
registered a very low value of
extracts of A. muricata on normal spleen(y=0.0026x+0.0044)
less than 10 g/mL GAE.
cells
Saponins; HIGH
(0.00). It
Spleen cells were isolated from normal healthyextract were computed to be (683
mice[33]. Spleen cells viability after and before .69 0.09) g/mL gallic acid
incubation with different concentrations of ethanolequivalents (GAE) while it was
CO2 for 2 h was(372.92 0.15) g/mL GAE in the
calculated using trypan blue technique[31]. Allethanolic extract. These values
at
37 C
in the presence of
(v/v)
3. Results
3.1. Phytochemical analysis of ethanolic
Terpenoids; HIGH
3.4.
Qua
ntifi
cati
on of antioxidant
activity using
the DPPH
Phenols; HIGH
method
Figure
Tannins; HIGH
S
shows
decrease
in
the
concentration
of
due
scavenging
n
s
;
DPPH
radical
to
the
ability of the
soluble
constituents in
L
O
W
the
ethanolic
and
water
leaves extracts
C
o
m
of A. muricata.
There
was a
direct positive
relationship
between
antioxidant
activity
increasing
concentration
of the extracts.
s
q
and
The
relationship
was
pronounced in
the
more
water
extract than in
the
extract. There
was ultimately
i
c
a
l
v
a
l
u
e
Figure 3. Phytochemicals
present in ethanolic leaves
extracts of A. muricata
with relative abundance
computed from the 2 test.
ethanolic
Yahaya Gavamukulya et al./Asian Pac J Trop Med 2014; 7(Suppl 1): S355-S363
the
ethanolic
extracts
as
represented
by
%RSA
on DPPH
the
100
200
400
600
800
1000
1200
Concentration ( g/mL)
% ell
and
EEAM11
showed
(15.85
relatively
%).
low
The
40
deathC
80
70
60
40
antioxidant
fractions.
90
50
results
100
% ellC
death
SKBR
Figure 8.
l
i
ellC death
150
y=139.45xR=0.997
100
50
0 2 .3
2.4
2 .5
2.6
Log concentration (
Ethanolic extract activity
Linear (Ethanolic extract activity)
2.7
g/m L)
1400
Yahaya Gavamukulya et al./Asian Pac J Trop Med 2014; 7(Suppl 1): S355-S363
S360
high
anticancer
activity
had
high
DPPH
activity
in
this
already
outlined
independent
showed
that
species
F
% Cell
death
4. Discussion
Phytochemical screening conducted on leaves
extracts of
1. muricata
as
by
other
studies
that
among
ercentageP
alkaloid
the
presence
of
alkaloids, flavonoids, terpenoids,
coumarins
and
lactones,
anthraquinones, tannins, cardiac
glycosides, phenols, phytosterols,
and saponins confirms that A.
muricata leaves extracts contain
molecules known for extensive
use in the medical field both
traditionally and pharmaceutically.
This would be an indication for its
potential use in anti-inflammatory,
anti-allergic, antibacterial, and
antiviral, heart failure, antioxidant
and anticancer activity among
others. These findings emphasize
the value of traditional knowledge
in the use of plants for medicinal
use as well as pharmaceutical
development. The use of A.
muricata in traditional medicine is
validated by presence of these
phytochemicals of known health
benefits and thus further studies
on this species are needed.
The phenolic content of the A.
muricata was determined and all
results were expressed as GAE.
Typical phenolics that possess
antioxidant activity have been
characterized as phenolic acids
and flavonoids[36,37]. Phenols are
among
the
non-enzymatic
compounds obtained from natural
sources, which have received high
attention due to their proven
antioxidant capabilities. Although
phenolic compounds have been
related to antioxidant activity,
some studies have emphasized
specific classes such as flavonoids
and tannins[12] . Our results
revealed that the water leaves
extract had higher total phenolic
content as compared to the
ethanolic leaves extract of A.
muricata. The higher phenolic
BHA
and
BHT
were used as
Yahaya Gavamukulya et al./Asian Pac J Trop Med 2014; 7(Suppl 1): S355-S363
S361
extracts was far lower than the standard positive anticancer activity on three cell
controls, implying that the extracts, at similar lines of EACC, MDA and SKBR3 with
concentrations may not be competitively strongIC50 values which are low and very
antioxidants. It is however likely that the leavesclose to each other, despite the
extracts antioxidant activity of A. muricata may be difference in the method used and
as strong as standard BHA and BHT, given that thesource of the cells.
samples assayed in this study were crude extracts, An integrated part of cancer cell
while the standard controls are usually very purifieddevelopment is the resistance to
compounds.
programmed cell death (apoptosis)
It is not surprising that the water leaves extracts
of A. muricata had a stronger antioxidant activity asand therefore re -establishment of
compared to the ethanolic leaves extract. Thisapoptosis in cancer cells is a
revealed that the total phenolics were two fold
target mechanism for anticancer
higher in the water leaves extracts than the
ethanolic leaves extracts, and phenolics had longagents[39]. Some plant-derived
been associated with antioxidant activity. Similarly,products are known to selectively
the water leaves extracts had reducing powerinduce apoptosis
almost two times higher than that of the ethanolic
leaves extracts. In general however, the relatively
strong antioxidant activity makes this plant efficient
in managing oxidative stress related diseases; this
could be the reason why it is used in traditional
medicine to manage such diseases where the
water extracts are mostly applied.
Earlier
anticancer activity of the water leaves extract have high antioxidant activity while
despite its having a high antioxidant activity and the opposite trend is not[13].
reducing power compared to the ethanolic extract In this case, we propose that the
may elicit a number of theories pertaining theanticancer agents present in the
mechanism of action of the anticancer agents inethanolic leaves extracts may be
this plant which may be different from theacting in a very different mechanism
commonly generalized idea that anticancer activity
from that of the antioxidant
is directly related to antioxidant activity. Our results
mechanism.
These
compounds
are in line with earlier preliminary studies which
related to the anticancer activity may
showed a good relationship between antioxidant
efficacy of plant extracts and anticancer potency. Allalso be absent from the water
of the extracts which gave high anticancer potencyextract and not easily detected by
the
common
phytochemical
active
compounds
and
known
as
are
no
readily
available
Yahaya Gavamukulya et al./Asian Pac J Trop Med 2014; 7(Suppl 1): S355-S363
S362
TLC
the
African
Union
Commission
research
2013/2014.
funding
lot
Government
of
Kenya,
Jomo
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