The Promising Anticancer Efficacy of Parsley Seeds Flavonoid (Apigenin) in Induced Mammary Adenocarcinoma (AMN3) Mice

Abstract

The cytotoxic effect of apigenin in mammary adenocarcinoma (AMN3) bearing mice was manifested through significant (P< 0.01) reduction in tumour volume and growth rate inhibition 90.8% after 24 days of oral administration.

Full text

Original Article
5
lavonoids are phenolic compounds which are
widely distributed in plants, and have been
reported to exert multiple biological effects, including
antioxidant, free radicals scavenging abilities anti-
inflammatory and anticarcinogenic activity.1,2,3 The
flavonoid apigenin is regarded as the active principle
of parsley and is known to possess an antioxidant
property and effectively militates against the pro-
oxidative activity of cadmium.4,5,6 In addition,
apigenin showed anticancer effect against lung
cancer7 as well as, growth inhibition of human colon
carcinoma cell lines.8 Human trials on the antioxidant
effects of beverages rich in polyphenolics, such as red
wine, fruit juice or tea and vegetables rich in
flavonoid like parsley, have been limited and results
are, at present, inconclusive, in part, to poor
methodologies available to measure oxidative
damage in vivo.9 Therefore, this study was performed
in normal and mammary adenocarcinoma (AMN3)
bearing mice to investigate 1) the effect of parsley
seeds apigenin on the growth of transplanting tumor
in mice, 2) lipid peroxidation (glutathione and malon-
dialdehyde concentration) and serum protein profile
in normal and treated animals, and 3) histopatho-
logical changes of liver, kidney and spleen as well as
tumor mass in normal and treated mice.
Materials and Methods
Flavonoid extraction and identification
Extraction of flavonoids from parsley seeds was
carried out according to Harbone method10 modified
by Al-Kawary, 2000.11 Acid hydrolysis was carried
out by 2N hydrochloric acid and, ethyl acetate and
petroleum ether as organic solvent.
Identification of flavonoids by thin layer
chromatography12 on silica gel plates was conducted
using, n- butanol : acetic acid : water (40:10:50) as a
mobile phase, UV detector was used to explore the
spot at 245 nm.
Immune-suppressed animals
Preparation of immune-suppressed mice was carried
out by dividing a group of 70 animals into two
groups:
In the first group , fifty mice were administered
orally 10 mg/kg body weight (BW) of levamisole in
3 days intervals, plus 0.5 % H2O2 in drinking water
for one month.13
The Promising Anticancer Efficacy of Parsley Seeds Flavonoid (Apigenin)
in Induced Mammary Adenocarcinoma (AMN3) Mice
Layla Hashim Alol, Kahtan Ahmed Al-Mzaien, Shalal Murad Hussein
Abstract
Extraction and identification of parsley (Petroselinum sativum) seeds flavonoids (apigenin), as well as
evaluation its anticancer efficacy was the main aim of the current study. Thin layer chromatography results
clarified that apigenin is the major flavonoid in parsley seeds. The cytotoxic effect of apigenin in mammary
adenocarcinoma (AMN3) bearing mice was manifested through significant (P ≤ 0.01) reduction in tumor
volume and growth rate inhibition (90.8 %) after 24 days of oral administration at a dose of 300 mg/kg body
weight. The volume of tumor in the treated group reached 1354.8 mm³ while the recorded size of the control
was 14758 mm³. Transplanted cancer mice showed a significant (P ≤ 0.01) elevation in concentration of liver,
heart, kidney and tumor mass tissue homogenate malondialdehyde as well as a significant depression in
glutathione concentration. Apigenin intubation caused a significant correction of the previous parameters
manifested by a significant elevation and depression in glutathione and malondialdehyde concentration
respectively. Agarose gel electrophoresis of blood serum of AMN3 transplanted mice, showed an increment in
globulin concentrations coincide with albumin concentration decrement, while apigenin administration
normalized to a great extent serum protein concentrations. Histopathological sections of liver, kidney, spleen as
well as tumor mass showed the significant role of apigenin in immune system stimulation manifested by
elevation of globulins, lymphocytes and macrophages and replacement of cancer tissues by connective tissues.
J Physiol Biomed Sci. 2012; 25(1): 5-12
Keywords: parsley seeds flavonoid, apigenin, anticancer efficacy, mammary adenocarcinoma, blood serum
electrophoresis
From the Department of Physiology and Pharmacology, College of
Veterinary Medicine / Baghdad University (L.H.A. and K.A.A-M.),
and Iraqi Center for Cancer and Medical Genetics Research /Al-
Mustansiriya University (S.M.H.) Baghdad, Iraq.
Corresponding author:
Kahtan Ahmed Al-Mzaien
Department of Physiology and Pharmacology,
College of Veterinary Medicine / Baghdad University
Baghdad, Iraq
E-mail: almzaien@yahoo.com
© 2012 Journal of Physiological and Biomedical Sciences
Available online at www.j-pbs.org
F

J Physiol Biomed Sci. 2012; 25(1): 5-12 L. H. Alol, et al.
6
In the second group, twenty mice were
administered orally 50 mg/kg of cyclosporine daily
(Novartis Pharma., France) until the end of the
experiment. Drinking water and food pellets were
sterilized for 20 and 10 minutes respectively.14 The
results showed that, levamisole plus H2O2-treated
mice were found to be infected by mammary adeno-
carcinoma while cyclosporine-treated group was not.
Transplantation of tumor cells in the immune-
suppressed mice
Mammary adenocarcinoma bearing mouse, from
the Iraqi Center for Cancer and Medical Genetic
Research (ICCMGR), was used as a source for tumor
cells transplantation into immune-suppressed adult
females albino mice supplied from ICCMGR.
Anticancer efficacy of apigenin in vivo
Group 1: Five mice with documented mammary
adenocarcinoma were administered a daily dose of
300 mg/kg BW of apigenin up to the end of the
experimental period (24 days). Administered dose
was selected according to the result of the
cytotoxicity test in vitro.15
Group 2: Five mice carrying mammary adeno-
carcinoma received only distilled water and
considered positive control for the first group.
Group 3: A group of ten normal mice were
administered apigenin orally, a daily dose of 300 mg
/kg BW for 30 days.
Group 4: Ten normal mice received distilled
water for 30 days to serve as a control group for
Group 3.
Group 5: Forty mice represented a group which
resisted AMN3.
Measurement of tissue glutathione16
Homogenized tissues (liver, spleen, kidney, tumor
mass) were prepared in Tris buffer (50 mM Tris, 0.1
mM EDTA, pH 7.6), 25% trichloroacetic acid
solution, 0.15 M imidazole (pH 7.4), 3 mM DTNB
(5, 5-dithiobis 2-nitrobenzoic acid) freshly prepared
in imidazole buffer. The optical density of the sample
was measured spectrophotometrically at 412 nm and
glutathione concentration was expressed as µmol/g
wet tissue.
Measurement of tissue malondialdehyde17
Homogenated tissues (liver, spleen, kidney, tumor
mass) were prepared in sodium chloride (0.9 %
NaCl)-2 mM NaN3 (sodium azide), trichloroacetic
acid (TCA 28%)-0.1 M Na-arsenite, thiobarbituric
acid 1% in 0.05 M NaOH (freshly prepared; heating
required), Tris (50 mM)-0.1 mM EDTA pH 7.6.
Hydrogen peroxide 30 % (11 µl 30 % H2O2 + 10
ml saline azide) was freshly prepared. The
absorbance was measured at 532 nm and 453 nm
using 1.53 x 105 M-1 as molar extinction coefficient
and the values were expressed as nmol TBA RS/g
wet tissue.
Tissue MDA = ∆A / (1.53 x 105)
[∆A = (Abs. sample 532 - blank 532) - 20% (Abs.
sample 453 - blank453)]
Agarose gel electrophoresis
Electrophoretic separation of serum proteins of
different treated groups according to their relative
mobility on agarose gel was carried out using
Hellabio agarose gel kit (Greece). Serum protein
fractions were fixed and stained by amido black and
the percent protein fractions were estimated by
Hellabioscan or densitometer at 520 nm.
Measurement of Tumor volume18
Tumor volume, was measured in three days intervals
up to the end of experimental period (24 days) using
vernier calipers, volume of the tumor was calculated
as follows
Tumor volume (mm3) = a x b 2 /2;
where a = tumor length and b = tumor width.
Measurement of percentage of tumor inhibition19
Following tumor volume calculation, inhibition in the
rate of tumor growth (GI) was carried out according
to the equation:
GI% = 100 x (A – B) / A;
where GI = growth inhibition. A = volume of
untreated tumor, B = volume of treated tumor.
Histopathological test
For histological studies, organs in five treated
animals (liver, spleen, kidney and tumor mass) were
extracted and preserved in 10% formalin until the
preparation of histopathological sections.
Statistical analysis
Data were analyzed statistically by using one way
analysis of variance (ANOVA) for tissue glutathione
(GSH) and malondialdehyde (MDA) determination.20
Results
Extraction and identification of flavonoids from
Petroselineum sativum seeds
The result of this study revealed that out of each
kilogram of Petroselineum sativum dry seeds, ap-
proximately 2.69 g crude flavonoids were obtained.
Apigenin appeared as a dominant flavonoid on silica
gel plates with Rf value of 0.81 (Figure 1).
Treatment of tumor by using apigenin
Daily administration of 300 mg/kg BW of apigenin to
a group of mice with induced mammary adenocarci-
noma caused significant (P ≤ 0.01) percent inhibition
(90.81 %, Table 1) in tumor volume after 24 days of
treatment as compared to zero time. Figure 3A and
3B confirmed clearly the significant reduction in
tumor volume of treated (Figure 3B) and untreated
(Figure 3A) animals.
Malondialdehyde (MDA) and glutathione (GSH)
Malondialdehyde (nmol/g) and glutathione (μmol/g)
concentration in heart, liver , kidney and tumor mass
homogenates wet tissues of normal mice, mice with

A
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Figur
e
acid:
flavon
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Figur
e
mary
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receiv
AM
N
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show
0.01)
tion
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n
with
A
value
% tumor growth inhibition
Figur
e
group
days)
;
A
B
n
cer efficacy of
a
e
1 Chromatog
water as mob
i
o
id (apigenin) i
n
e
3 Comparing
adenocarcino
m
e
d water orally
ing 300mg/kg o
f
N
3 cancer an
d
nin, were sh
o
ed significan
t
in malondial
d
in mammar
y
n
s and tumor
m
c
tivel
y
, as co
n
istration of 3
A
MN3 cause
d
s (P ≤ 0.01).
50
60
70
80
90
100
3
%
tumor
growth
inhibition
e
2 Percentag
(mice receiv
e
;
n = 5; P ≤ 0.01
a
pigenin
ram TLC analy
s
i
le phase; thre
n
ethyl acetate a
the tumor size
m
a; A, untreate
d
for 24 d; and
f
apigenin orally
d
mice with
o
wn in Table
t
elevation a
n
d
ehyde and g
l
y
adenocarc
i
m
ass homoge
n
mpared to th
e
00 mg/kg B
W
d
significant n
o
6912
Days after
e of tumor gro
w
e
d 300 mg/kg
.
s
is using butan
o
e different dil
u
re shown.
in mouse beari
n
d
(Group 2),
r
B, treated (G
for 24 d.
AMN3 treat
e
2 and 3. The
n
d depressio
n
l
utathione co
n
i
noma-
b
earin
g
n
ates (Table 2
e
control, w
h
W
of apigenin
t
o
rmalization
o
15 18 21
treatment
apigenin
w
th inhibition in
apigenin orally
7
o
l: acetic
u
tions of
n
g mam-
r
eceiving
roup 1),
e
d with
results
n
(P ≤
n
centra-
g
mice
and 3),
h
ile oral
t
o mice
o
f those
Ta
b
rel
a
un
t
m
a
D
a
tr
e
Gr
o
m
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ad
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su
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m
Ta
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G
r
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Co
Tr
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Co
for
ad
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Gr
o
30
0
sig
n
Ta
b
Gr
o
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Co
Tr
e
Fi
g
8.
6
ca
r
3,
mi
c
we
or
a
re
c
A
g
C
h
se
r
gl
o
an
i
T
h
th
e
th
e
24
treated
for 24
b
le 1 Compari
a
tive size (in
t
reated (Group
a
mmary adenoc
a
a
ys of
e
atment U
n
(
G
3 1,5
8
6 4,28
4
9 6,92
7
12 9,18
7
15 9,64
3
18 12,7
2
21 13,
7
24 14,7
o
up 1, mice wit
h
/kg apigenin or
a
e
nocarcinoma
r
p
e
r
scripted alp
h
m
be
r
of mice =
5
b
le 2 Malondial
r
oups Hea
r
n (-ve) 0.49±0
n (+ve) 1.37±0
e
ated 0.60±0
n (-ve), or Gro
u
30 d; Con (+
v
e
nocarcinoma r
e
o
up 1, mice be
a
0
mg/kg apigeni
n
ificance at P ≤
b
le 3 Glutathio
n
o
ups Hea
r
n (-ve) 6.93±0
n (+ve) 5.11±
0
e
ated 7.71±0
.
1
2
g
ure 4 Agaros
e
, size of sampl
e
r
cinoma receivi
n
normal mice r
e
c
e inoculated
w
re resistant; 5,
n
a
lly for 30 d;
7
c
eiving distilled
w
g
arose
g
el ele
c
h
anges in th
e
r
um protein (
a
o
bulin and
γ
i
mals were sh
o
h
e average al
b
e
mice (G2)
w
e
correspond
J Physiol B
son of tumor v
o
% reduction)
2 and Grou
p
ar
cinoma.
Tumor volume
(
n
treated
G
roup2)
8
7±1.25
h
4
4
.6±2.45
g
9
7
.5±4.10
f
1
7
.5±5.31
e
1
3
.5±1.22
d
1
2
8.8±2.9
c
1
7
12±3.3
b
1,
4
58±3.27
a
1,
3
h
mammary ade
a
lly for 24 d; Gr
o
r
eceived distill
e
h
abets denote
5
.
dehyde concen
t
r
t Liver
.02
c
0.69±0.0
3
.05
a
2.66±0.0
6
.02
b
1.57±0.0
6
u
p 3, normal mi
v
e), or Group
2
e
ceiving distille
d
a
ring mammary
n for 24 d; sup
e
0.01; number o
f
n
e concentratio
n
r
t Liver
.03
b
15.0±0.3
b
0
.06
c
12.6±0.2
3
.
02a 16.0±0.2
0
2
3 4
e
gel electropho
e
5 µl. 1 & 6,
m
n
g 300 mg/kg a
p
e
ceiving distille
d
w
ith mammary
n
ormal mice re
c
7
, mice with
m
w
ater orally for
2
c
trophoresis
e
concentrati
o
a
lbumin, α1-g
γ
-globulin) i
n
o
wn in Table
4
b
umin concen
t
w
as 38.84 % o
f
ing values
iomed Sci. 2012
;
o
lume (mean +
in apigenin-t
r
p
1) mice wi
t
(
mm
3
)
Treated %
(Group 1)
4
80.5±2.04
g
9
56.5±2.45
f
,364±2.44
d
,533±1.22
b
,290±4.08
e
,605±2.04
a
4
85.9±2.05
c
3
54.8±1.63
c
nocarcinoma re
o
up 2, mice wit
h
e
d wate
r
orally
significance at
t
ration (nmol/g
w
Kidney
3
c
0.33±.003
c
6
a
0.46±0.17
a
6
b
0.40±.002
b
ce receiving di
s
2
, mice bearin
g
d
water for 24 d;
adenocarcino
m
e
rscripted alpha
b
f
mice = 5
n
(µmol/g wet ti
s
Kidney
b
1.18±0.01
a
3
c
0.63±0.62
c
0
a
1.09±0.03
b
5 6 7
resis of blood s
m
ice with mam
m
p
igenin orally f
o
d
water orally f
o
adenocarcino
m
c
eiving 300 mg/
k
m
ammary aden
o
2
4 d.
o
n and
p
atter
n
lobulin, α2-g
l
n
normal a
n
4
and Figure
4
t
ration in the
f
the total pro
t
in the con
t
;
25(1): 5-12
SEM) and
r
eated and
t
h induced
reduction
69.72
d
77.68
c
80.30
b
83.31
b
8
6.62
a,b
87.39
a
89.16
a
90.81
a
ceiving 300
h
mammary
for 24 d;
P ≤ 0.01;
w
et tissue)
Tumor
-
2.16±0.17
a
1.08±0.13
b
s
tilled water
g
mammary
Treated, or
m
a receiving
b
ets denote
s
sue)
Tumor
-
0.75±0.02
b
2.40±0.01
a
e
rum at pH
m
ary adeno-
o
r 24 d; 2 &
o
r 30 d; 4,
m
a but they
k
g apigenin
o
carcinoma
n
s of the
l
obulin, β-
n
d treated
4
.
serum of
t
ein, while
t
rol were

J Physiol Biomed Sci. 2012; 25(1): 5-12 L. H. Alol, et al.
8
Table 4 Agarose protein electrophoresis (protein fraction, %)
Group Albumin Globulins
α1 α2 β γ
1 57.74 11.85 6.33 15.07 8.99
2 38.84 10.98 2.92 42.8 4.44
3 33.87 20.83 5.23 24.0 16.05
4 59.95 7.14 1.61 15.89 15.39
5 47.67 9.71 15.67 16.40 10.53
Group 1, mice with mammary adenocarcinoma + 300 mg/kg
apigenin orally for 24 d; Group 2, mice with mammary adeno-
carcinoma + distilled water orally for 24 d; Group 3, normal
mice + 300 mg/ apigenin orally for 30 d, Group 4, normal
mice + distilled water orally for 30 d; Group 5, mammary
adenocarcinoma-resistant mice.
59.95 %. On the other hand a considerable elevation
in albumin concentration was recorded in apigenin-
treated group 57.74 % of the total protein as
compared to the control, while normal mice received
orally 300 mg/kg BW of flavonoid (apigenin) showed
the lowest values 33.87 %. Moreover, albumin
concentration in the group of the mice which showed
endogenous resistances (failed cancer) was about
47.67 %. An increase in the level of α1- and α2-
globulin was recorded in all treated groups as
compared to the control which showed the lowest
values 7.14 and 1.61 %, respectively. The corres-
ponding values in mice with AMN3 cancer and
apigenin-treated groups were 10.98 and 11.85 for α1-
globulin, and 2.92 and 6.32 for α2-globulin,
respectively. The highest concentration for β-globulin
was recorded in mice with AMN3 cancer (42.8 %)
and the lowest values in the control (15.89 %). Mice
with AMN3 cancer administered with flavonoid
(apigenin) showed a value of 15.07 %. Finally, γ-
globulin fraction was lowest in mice with AMN3
cancer 4.44 % as compared with control 15.39 % and
apigenin-treated group 8.99 %.
Histopathological study
Liver. Histopathological sections of liver in
cancer-inoculated mice clearly showed cancer cell
presence, as distinguished with hyperchromatin and
pleomorphic in central veins and degenerative
changes in liver cells with ruptured cell and
cytoplasm (Figure 5A).
Spleen. Histopathological section of spleen in
untreated cancer mice showed congestion of blood
vessels and cancer cells in the cavity which was
distinguished with hyperchromatin and pleomorphic,
precipitation of amyloid in red pulp and wide spread
of white pulp (Figure 6C), while histopathological
examination of spleen in cancer, treated mice showed
clarified precipitation of amyloid in spleen with
hyperplasia of white pulp of cancer cells (Figure 6B).
Figure 6 Spleen histopathology. A, Group 3 (normal, treat-
ed), 100x; showing hypertrophy of medial layer of central
artery with hyperplasia of periarterial sheet; B, Group 1
(cancer, treated), 100x; arrow showing hyperplasia of white
pulp with amyloid like substance deposition in the red pulp;
C, Group 2 (cancer, untreated), 400x; arrow showing
congested red pulp with macrophage-associated tumor cells
Figure 5 Liver histpathology. A, Group 2 (cancer, untreat-
ed), 400x; arrow indicates macrophage-associated tumor cell
in the sinusoid; B, Group 1 (cancer, treated), 400x; K,
Kupffer cell proliferation; V, congested central vein; L,
lymphocytic aggregation around central vein; C, Group 3
(normal, treated), 100x; showing the presence of kupffer
cells and aggregation of monocyte (M).
B
C
V
K
L
M
A
B
C
A

A
ntica
n
On t
h
300
m
of w
h
nor
m
main
Ki
histo
p
arou
n
tissu
e
of
k
apig
e
b
eca
u
p
hag
e
p
res
e
kidn
e
no p
a
T
u
mam
m
sho
w
the
pr
p
oly
m
nucl
e
struc
t
of tu
m
the
p
cells
)
cells
tion
o
(Fig
u
large
8B).
were
The
R
appr
o
1984
,
to p
a
clov
e
majo
r
p
lant
s
cont
a
A
l
cond
i
and
a
indic
a
canc
e
facto
r
inhib
i
b
y
a
Besi
d
in t
h
intra
c
decr
e
cyto
c
apop
t
attrib
facto
r
facto
r
n
cer efficacy of
a
h
e other han
d
m
g/kg apigen
i
h
ite pulp. Am
y
m
al mice rece
i
cause of amy
l
i
dney. Kidne
y
p
athologicall
y
n
d kidney gl
o
e
s (Figure 7A
)
k
idney of tr
e
e
nin) showe
d
u
se of aggre
g
e
in
p
arench
y
e
nce of cancer
e
y of normal
m
a
thological ch
a
u
mor mass.
H
m
ary adenoc
a
w
ed progressi
v
r
esence of ca
n
m
orphic in di
f
e
us and canc
t
ure (Figure 9
A
m
or mass in
m
p
resence of la
r
)
surrounded
w
like macroph
a
o
f connectiv
e
u
re 9B), whil
e
tumor mass
c
Results con
c
summarized
i
R
f value of a
p
o
ximately the
,
under simil
a
a
rsley seeds,
e
s, grapes, o
n
r
sources of
s
-derived be
v
a
ined consider
a
l
ong the expe
r
i
tion, active,
w
a
ll treated
m
a
ting the pro
t
e
r da
m
age. I
n
r
like activat
o
i
tion of trans
c
a
pigenin coul
d
d
es, flavonoi
d
h
e plasma
m
c
ellular Na+
e
ased K+ c
o
c
hrome c f
r
t
osis.24 Antic
a
uted to the hi
r
-1α (HIF-1α
)
r
(VEGF) ex
p
a
pigenin
d
, spleen of n
o
i
n for 30 day
s
y
loid precipit
a
i
ved apigenin
,
l
oid (Figure 6
A
y
of untreated
y
heavy gro
w
o
meruli whic
h
)
, while histo
p
e
ated cancer
d
congestion
g
ation of ly
m
y
ma of kidne
y
cells (Figure
m
ice treated
w
a
nges.
H
istopatholog
i
a
rcinoma ma
s
v
e stage of c
a
n
cer cells wit
h
f
ferent size, w
i
er cell aggr
e
A
), while hist
o
m
ice treated
w
r
ge necrotic
a
w
ith large am
o
a
ge and lymp
h
e
tissue that
r
e
Figure 8 A
c
ompare
d
to t
r
c
erning histo
p
i
n Table 5.
Discussio
n
p
igenin recor
d
same as that
r
a
r diagnostic
c
apple, bea
n
n
ions, barley,
food rich ap
i
v
erage, inclu
d
a
ble amount
o
r
imental perio
w
ith increasin
m
ice remain
a
t
ective effect
o
n
hibition of
a
o
r protein (A
P
c
ription and c
a
d
be anothe
r
d
s inhibit Na+
-
m
embrane l
e
and Ca2+
o
ncentration,
r
om mitoch
o
a
ncer activity
gh inhibition
)
and vascula
r
p
ression in t
h
o
rmal mice r
e
s
showed hyp
e
a
te is not rec
o
,
since cance
r
A
).
cancer mice
s
w
th of canc
h
lead to ch
a
p
athological
c
mice (300
of blood
m
phocyte an
d
y
and there
w
7B) while se
c
w
ith apigenin
s
i
cal section
o
s
s, in untreat
e
a
ncer, manife
s
h
hype
r
chrom
a
i
th of
p
rolifer
a
e
gation at gl
a
o
pathological
w
ith apigenin
s
a
rea (necrotic
o
unt of infla
m
h
ocyte with p
r
r
eplaced canc
e
showe
d
macr
o
r
eated mouse
p
athological
c
n
d
ed in this st
u
r
ecorded by H
c
ondition. In
a
n
s, celery, c
tomatoes, r
e
i
genin. Fu
r
th
e
d
ing tea an
d
o
f apigenin.21
d, mice were
i
g food consu
m
a
live after tr
e
o
f apigenin22
a
nother trans
c
P
) which lead
s
a
scade of apo
p
r
possible p
a
-
K+ ATPase
e
e
ading to in
c
concentration
with librat
i
o
ndria result
i
of apigenin
m
of hypoxia i
n
r
endothelial
h
e tumor tissu
e
9
e
ceived
e
rplasia
o
rded in
r
is the
s
howe
d
er cell
a
nge in
c
hanges
mg/kg
vessels
macro-
w
as no
c
tion of
s
howed
o
f skin
e
d mice
s
ted by
a
tin and
a
tion of
a
ndular
section
s
howed
cancer
m
matory
r
olifera-
e
r cells
o
scopic
(Figure
c
hanges
u
dy was
arbone,
a
ddition
herries,
e
presen
t
e
rmore,
d
wine
i
n good
m
ption,
e
atment
against
c
ription
s
to the
p
tosis23
a
thway.
e
nzyme
c
reased
s, and
i
on of
i
ng in
m
ay be
n
ducible
growth
e
with
Fi
g
tre
a
Fi
g
un
t
Fi
g
ed
)
arr
o
A
g
ure 7 Kidney
a
ted); B, Group
g
ure 8 Tumor
t
reated), compa
r
g
ure 9 Tumor h
i
)
, showing she
e
o
w indicates ne
c
A
B
A
B
A
B
J Physiol B
histopathology
;
1 (cancer, treat
e
mass was lar
g
r
ed with B, Gro
u
i
stopathology;
A
e
t of pleomorph
i
c
rosis of tumor
c
iomed Sci. 2012
;
;
A, Group 2 (
c
e
d).
g
e in A, Group
u
p 1 (cancer, tr
e
A
, Group 2 (can
c
i
c hyperchroma
c
ells.
;
25(1): 5-12
c
ancer, un-
2 (cancer,
ated).
c
er, untreat-
tic cells; B,

J Physiol Biomed Sci. 2012; 25(1): 5-12 L. H. Alol, et al.
10
subsequent suppression of angiogenesis.7,25 Some
flavonoids with a 5-OH exhibited lower cytotoxicity
than their non-hydroxylated counterparts; therefore,
results indicated that 3,6 dihydroxyflavone showed
the most potent cytotoxic effect on cancer cells
(breast cancer, prostate cancer, and colorectal
carcinoma cells). It has been reported that flavonoids
in brownish scale of onion effect growth inhibition of
tumor by reducing the fluidity of tumor cell
membranes,26 while quercetin inhibited the express-
ion of specific oncogenes and genes controlling cell
cycle.27 Moon and his colleagues28 revealed that
cancer protection by flavonoids may include
alteration in detoxification enzyme (phase I and phase
II) which are known for their role in the metabolism
of foreign compounds, and many carcinogens are
metabolized by these enzymes system to biologically
inactive metabolites. One possible mechanism
suggested that apigenin may exhibit its cytotoxic
effect on tumor cell by its antioxidant and free radical
scavenging activities.29 As all flavonoids compounds,
apigenin may play a part in indirect inhibition of NF-
KB factor, in addition to inhibition of its binding to
DNA strand and transcription30 and or activation of
P38 MAPK.31
Malondialdehyde (MDA) and glutathione (GSH)
Increment in tissues MDA concentration and forma-
tion of MDA as a by-product of lipid peroxidation
(LPO) has been considered a simple and useful
diagnostic tool for the measurement of LPO.32
However, the result of heart, liver, kidney and
tumor mass of (AMN3) homogenates of mice after
24 days, of apigenin treatment showed a significant
decline (Table 2) in malondialdehyde concentration
as compared to the control and other treated groups.
These results confirmed the antioxidant role of
apigenin, and their free radical scavenging
properties.33 Therefore, apigenin administration was
accompanied with significant increment in anti-
oxidant capability and peroxidation inhibition which
was reflected by a significant decrease in MDA
concentration. Higher MDA and lower GSH
concentration confirmed the oxidizing role of cancer
in lipid peroxidation,34,35 reflecting glutathione
turnover for preventing oxidative damage in the
mice. Similar reports of lowered glutathione
concentration in cancers have been reported
earlier.33,36 In addition, several studies35,37,38 reported
similar findings in patients with malignant breast
tumor, and with colorectal cancer tissue. While
glutathione detoxification system is one of the
defense system against free radicals and carcinogen,
lower levels of glutathione may favor an over
production of free radicals and lipid peroxides, which
in turn may induce damage to the cell membrane and
cellular molecules (i.e. DNA, RNA) leading to
neoplasia. Similar lower erythrocytes glutathione
levels in cervical cancer patient were reported by.39
They have hypothesized that there may be an
impairment of the glutathione scavenger system due
to the carcinogenic process.40
Agarose Gel Electrophoresis
The changes in plasma protein concentration in a
cancer-bearing state may not be a specific tumor-
induced stimulus, but rather a generalized response to
an inflammatory state.41,42 Mice with AMN3 were
characterized by a considerable alteration in the
serum protein as compared to control or apigenin-
administered group. However, in this study serum
albumin decreased in cancer-bearing mice similar to
an acute inflammatory stress,41 dehydration and
malnutrition,43 and albumin decrement coincided
with α1-, α2-, β- and γ-globulin decrement as a result
of tissue damage and autoimmune disorders. In
addition, albumin decrement may be attributed to the
high level of catabolism and, as a consequence of
cancer, there was a possibility of free radical
increment which play a major role in LDL oxidation,
accompanied with elevation of β-globulin LDL-
vehicle.42
The higher permeability of cell membrane in
cancer-bearing mice may be considered another
factor for albumin decrement. Elevation in α1-
globulin in cancer group may be due to increased
Table 5 Key histopathological findings.
Group No. of
animals Liver Spleen Kidney Tumor
1 5 Kupffer cell proliferation;
congested central vein;
lymphocyte aggregation
around central vein
(Fig. 5 B)
White pulp
hyperplasia
with amyloid-like subs-
tance deposition in red
pulp (Fig. 6 B)
Congested blood ves-
sels (Fig. 7 B) Necrosis of tumor cells
(Fig. 9 A)
2 5 Presence of tumor-as-
sociated macrophages
(TAM) (Fig. 5 A)
"Metastatic tumor"
Congestion of red pulp
and tumor-associated
macrophage present in
congested blood vessel
(Fig. 6 C)
"Metastatic tumor"
Heavy growth of tumor
cells around glomeruli
(disapperance of tissue
features) (Fig. 7 A)
"Metastatic tumor"
Sheet of pleomorphic
hyperchromatic cells
(Fig. 9 B)
3 5 Presence of kupffer cells
and monocyte aggrega-
tion (Fig. 5 C)
Hyperatrophy of medial
layer of central artery with
hyperplasia of periarterial
sheet (Fig. 6 A)
No changes _

Anticancer efficacy of apigenin J Physiol Biomed Sci. 2012; 25(1): 5-12
11
trypsin enzyme in the lung that breaks down protein42
as a result of inflammation, and tissue damage.
Concerning α1- and α2-globulin concentration in
apigenin-treated group, the documented result
revealed the effective role of apigenin in the
improvement of the level of those fractions through
elevation of lymphocyte stimulation index. Therefore
apigenin has a certain immune co-stimulation
effect.45
The radical scavenging capability of the apigenin
may reduce LDL-C-oxidation and β-globulin frac-
tions required for the transportation of LDL-C.43 A
considerable elevation in γ-globulin was observed in
cancer bearing mice treated with apigenin, and this
may be attributed to immune system stimulation.
Liver and spleen recorded prevascular coughing of
lymphocytes which indicated immunity stimulation.46
Low values of gamma globulines was also recorded
in leukemia, cancer, starvation and sever dietary
deficiency.43,47
Histopathological study
Liver. Lesions occurred as a result of oxidative
damage by cancer, while apigenin treated mice
showed an increment in kupffer cells and lymphocyte
cells aggregation around central veins and there was
cancer cells disappearance (Figure 5B) as compared
with untreated group. Such results confirmed the
hepatoprotective role of apigenin44 while the liver of
normal mice treated with apigenin showed kupffer
cells and lymphocytes aggregation of and
macrophages in parenchyma and around central vein
(Figure 5C) and this may be attributed to the anti-
inflammatory action of apigenin.48
Spleen. Deposition of amyloidosis (diffuse) in
spleen may occur because of high level of immuno-
globulins and proteinaceous substances in the blood
serum. Amyloid precipitation is reversible patho-
logical changes and fetal in human and animals.49
These results explained that apigenin may
stimulate tumor necrotic factor (TNF), a group of
cytokines with important functions in immunity,
inflammation, differentiation, control of cell
proliferation and apoptosis. These cytokines induce
cell death through sequential recruitment by death
receptors and rapid activation of cascade of
caspases.50
Conflict of interest
None to declare.
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