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Ameliorative effect of novel nanocomposite: basic curcumin nanoparticles
modified with basic nano black seeds (nigella sativa) and calcium ascorbate on
metabolic changes in experimentally induced tumor in female mice.
Omayma A. Ragab Abou Zaid, Abdel Fatah. M. Badwi, Nora Abd Elmohsen El Sayed.
Department of Biochemistry, Faculty of Veterinary Medicine, Benha University, Egypt.
A B S T R A C T
The ameliorative effect and antitumor activity of basic curcumin nanoparticles modified with basic nano
black seeds (nigella sativa) and calcium ascorbate were evaluated. eighty female mice were divided into
4 equal groups of 20 mice each. Group 1: non – tumor bearing mice (NTBM). Group 2: tumor bearing
mice(TBM). Group 3: NTBM + nanocomposite (4.750gm / kg. b.wt /day ) , orally for 6 weeks . Group
4: TBM + nanocomposite (4.750gm / kg. b.wt/day ), orally for 6 weeks . Blood samples for serum
separation and liver tissues specimens were collected from all animal groups three times after 2, 4 and
6 weeks from the onset of treatment. All serum was directly used for determination of ALT and AST
activities and urea and creatinine concentrations. Moreover, antioxidants enzymes (catalase, GPX,
SOD), GSH and MDA were detected in liver tissue. The obtained results showed significant decrease
in catalase, GPX and SOD activities and GSH concentration in liver tissue of tumor bearing mice.
Meanwhile, liver MDA, serum transferases(ALT and AST) activities, urea and creatinine concentrations
were significantly increased in tumor bearing mice . TBM treated with nanocomposite improve the
adverse biochemical changes induced by tumor. So, this nanocomposite has potential benefits in cancer
treatment.
Key words: nanocomposite, curcumin nanoparticles, black seed nanoparticles
(
http://www.bvmj.bu.edu.eg
) (BVMJ-29(2):
235-244,
2015)
1. INTRODUCTION
ancer is a group of diseases
involving abnormal cell growth
with the potential to invade or
spread to other parts of the body. It caused
about 8.2 million deaths or 14.6 % of all
human deaths. The risk of cancer increased
significantly with age and many cancers
occur more commonly in developed
countries. Rates are increasing as more
people live to an old age and as lifestyle
changes occur in the developing world
(Jemal, et al.,2012). Curcumin is one of the
most powerful and promising
chemopreventive and anticancer agent.
Curcumin act as a potent cancer preventing
agent by blocking the nuclear factor kappa
B (Sajithlal, et al.,1998). Also, Curcumin
loaded poly (D, L-lactic-co-glycolic acid)
PLGA nanoparticles has enhanced cellular
uptake, and increased bioactivity in vitro
and superior bioavailability in vivo over
curcumin (Anand, et al., 2010). Black seed
(nigella sativa) oil and its main constituents
are extensively described to protect several
organs against oxidative damage depending
on its high antioxidant activity (Mansour, et
al.,2002). The active principle fatty acids
derived from N. sativa, completely
inhibited the growth of Ehrlich ascites
carcinoma and Dalton’s lymphoma ascites
cells (Salomi, et al.,1992). Other important
and interesting study showed that nigella
sativa and its constituents Thymoquinine
(TQ) shows role in cancer prevention via
inactivation and activation of molecular
pathways (Rahmani, et al.,2014). Also,
C
BENHA
VETERINARY
MEDICAL
JOURNAL,
V
OL
.
29,
N
O
.
2:235-244,
DECEMBER,
Abou Zaid et al. (2015)
236
Ethanol extract of nigella sativa seeds can
generate antioxidants, possess antitumor
activity, and ameliorate and prolong the life
span of mice bearing Ehrlich ascitic tumor
(Musa, et al., 2004). Ascorbate (vitamin C)
is implicated in preventing cancer due to its
ability in scavenging free radicals and
carcinogens maintaining the integrity of
connective tissue and improving
maintaining the integrity of connective
tissue and improving immunocompetence
and resistance to cancer (Cameron, et
al.,1979). Adequate vitamin C intake is
important to prevent nutrient deficiencies
and maintain antioxidant levels so the anti-
tumor defense system keeps working
effectively (Kulcsar, 2003). If cancer
become established, evidence suggests that
vitamin C may selectively kill cancer cells
via production of hydrogen peroxide (Chen,
et al.,2005), encapsulate tumors with a
collagen wall to prevent metastasis
(Cameron, et al.,1979), promote
macrophage function and removal of cancer
cells (Fiumara, et al.,1997) and
(Kaminogawa., et al,2004). Accordingly,
the antitumor effect of novel
nanocomposite: basic curcumin
nanoparticles modified with basic nano
black seeds (nigella sativa) and calcium
ascorbate on metabolic changes in
experimentally induced tumor in female
mice were evaluated.
2. MATERIALS AND METHODS
2.1. Animals
Eighty Australian females albino mice, 12 –
14 weeks old and weighting 25 – 30 gm
were used in this study .Mice was obtained
from the research institutes of
ophthalmology , Giza , Egypt . The animals
were housed in separate metal cages, fresh
and clean water was supplied ad-libtium
through specific nipple. Mice were kept at a
constant environmental and nutritional
condition throughout the period of the
experiment in special animal room at
national cancer institute, Egypt.
2.2. Ehrlich Ascites carcinoma cells
A line of Ehrlich Ascites carcinoma (EAC)
cells was supplied from national cancer
institute, Cancer Biology Department,
Egypt.
2.3. Tumor induction
Solid tumors were induced by
intramuscular inoculation of each mice with
0.2 ml of EAC, which contained 2.5 x 106
viable EAC cells, in the right thigh of the
lower limb of each mouse. The tumor
developed and become palpable in all
injected animals at (7 – 10) days post tumor
inoculation.
2.4. Chemicals:
Basic nano curcumin and nano black seed
were purchased from sigma Aldrich (sigma,
USA) company. Vitamin C was purchased
from El – Gomhoria Chemicals Company,
Egypt.
Nanocomposite compound: The novel
nanocomposite was composed of Basic
nano curcumin 3.0 gm + nano black seeds
6.0 gm + calcium ascorbate 1.0 gm.
2.5. Experimental design
Eighty female mice were divided into 4
groups each one contains 20 mice placed in
individual cages and classified as follows:
G 1: NTBM. G 2: TBM. G 3: NTBM+
nanocomposite (4.750 g / kg
b.wt/day) ,orally for 6 weeks. G 4: TBM
+nanocomposite (4.750 g / kg b.wt/day),
orally for 6 weeks.
2.6. Sampling
Blood samples and liver tissues specimens
were collected from all animal groups by
decapitation at 2, 4 and 6 weeks from the
onset of treatment in dry and clean tubes
and serum was separated by centrifugation
at 3000 r.pm for 15 minutes .The clear
serum was aspirated by Pasteur pipette and
received in dry sterile sample tube ,
processed directly for (ALT and AST)
activities determination. , then kept in a
deep freeze at -20 °C until used for
subsequent biochemical analysis .All serum
were used for determination ALT , AST,
urea and creatinine . The liver tissues then
Ameliorative effect of novel nanocomposite
237
were obtained in dry and clean plates then
kept in a deep freeze -20 c until used for
analysis. All liver tissues used for
determination CAT, SOD, GPX, GSH and
MDA.
2.7. Biochemical analysis
Catalase activity was assayed following the
method of Luck (1974). SOD was assayed
according to the method of Kakkar et al.
(1984). GPX: Glutathione peroxidase was
assayed according to the method described
by Placer et al. (1966). GPX level was
assessed using the method of Paglia and
Valentine (1967). Reduced glutathione was
determined by method of Moron et al.
(1979). MDA: The extent of lipid
peroxidation was estimated according to the
method of Okhawa et al. (1979). Serum
ALT was determined according to the
method described by Murray, et al. (1984).
Serum AST was determined according to
the method described by Murray, et al.
(1984). Urea was determined by a
colorimetric method as described by Kaplan,
et al. (1984). Creatinine was determined
according to the method described by
Murray, et al. (1984).
2.8. Statistical analysis
The statistical analysis was carried out
using ANOVA with two factors under
significance level of 0.05 for the whole
results using SPSS (ver. 22). Data were
treated as complete randomization design
according to (Steel, et al., 1997). multiple
comparison was carried out applying LSD.
3. RESULTS
The obtained results presented in tables (1,2
and 3) showed a significant decrease in liver
catalase, SOD, GPX activities and GSH
concentration in TBM group compared to
the control group. However, significant
increase in serum ALT and AST activities
and kidney functions (urea and creatinine),
in addition the liver MDA concentration in
TBM compared to NTBM. In TBM treated
with nanocomposite group, a significant
increase in the activity of liver antioxidants
(catalase, SOD, GPX) and GSH level and
decrease in liver MDA concentration, in
addition the serum ALT, AST, urea and
creatinine were observed when compared
with TBM non treated groups.
Table 1: Effect of two weeks oral administration of nanocomposite (4.750 g /kg. b.wt) on liver
CAT,SOD,GPX and MDA and serum ALT, AST, urea and creatinine in eexperimentally
induced tumor female mice
A, B & C: There is no significant difference (P>0.05) between any two means for the same attribute, within the
same row have the same superscript letter. Data are presented as (mean ± S.E) S.E= standard error.
Animal Groups/parameters G1 G2 G3 G4
CAT Mmol/g. tissue 78.83±5.27dB 78.83±5.27
dB 53.28±3.60cB 38.92±2.76
b
B
SOD U/g. tissue 25.56±3.72cB 4.77±0.69aA 8.40±0.39
b
A 9.55±0.87
b
A
GPX ng/g. tissue 30.99±2.46cB 16.15±1.34
aC 17.90±1.01aA 23.46±2.13
b
A
GSH ng/g. tissue 12.50±1.07dB 2.44±0.69aA 5.28±0.58
b
A 6.15±0.41cA
MDA Mmol/g. tissue 52.86±7.55aA 163.1±9.29dC 116.22±5.49cC 75.46±4.67
b
B
ALT U/L 46.84±3.03aA 97.21±6.04
cA 70.09±4.79
b
B 70.50±4.81
b
A
AST U/L 55.70±4.01aA 98.61±9.32
cA 79.43±4.90
b
A 87.14±5.72
b
A
Urea mg/dl 27.09±1.42
b
A 32.39±2.10
cA 26.09±1.52abA 21.77±1.90aA
Creatinine mg/dl 0.36±0.03aA 0.74±0.03cA 0.52±0.02
b
A 0.59±0.03
b
B
Abou Zaid et al. (2015)
238
Table 2: Effect of four weeks oral administration of nanocomposite (4.750 g /kg. b.wt) on liver
CAT,SOD,GPX and MDA and serum ALT, AST, urea and creatinine in eexperimentally
induced tumor female mice
G4 G3 G2 G1 Animal groups / parameters
34.32±2.74
b
A
44.90±2.35
cA
16.01±1.85
aA
58.35±3.24
dA
CAT Mmol / g. tissue
15.96±1.28
cB
10.78±1.17
b
B
5.35±0.46
aAB
22.23±0.94
dA
SOD U / g. tissue
26.81±1.88
cB
20.02±2.15
b
B
10.65±1.10
aA
30.08±1.73
dB
GPX ng / g. tissue
6.28±0.76
cA
4.74±0.65
b
A
2.23±0.50
aA
7.46±0.69
dA
GSH ng / g. tissue
59.30±5.88
aA
80.78±3.00
cB
114.45±8.62
dB
69.91±5.30
b
B
MDA Mmol / g. tissue
92.78±4.13
b
B
123.18±5.40
cC
142.64±4.99
dC
60.03±6.39
aB
ALT U/L
118.92±3.21
cB
102.56±3.75
b
B
165.84±7.19
dC
80.27±4.53
aB
AST U/L
24.44±2.67
aA
31.03±3.64
b
B
61.04±4.38
dB
40.18±3.17
cB
Urea mg/dL
0.51±0.03
aA
0.62±0.05
b
B
1.15±0.10
dB
0.85±0.04
cB
Creatinine mg/dL
a, b & c: There is no significant difference (P>0.05) between any two means, within the same column have the
same su perscript letter. A, B & C: There is no significant difference (P>0.05) between any two means for the same
attribute, within the same row have the same superscript letter. Data are presented as (mean ± S.E) S.E= standard
error.
Table 3: Effect of six weeks oral administration of nanocomposite (4.750 g /kg. b.wt) on liver
CAT,SOD,GPX and MDA and serum ALT, AST, urea and creatinine in eexperimentally
induced tumor female mice :
G4 G3 G2 G1 Animal groups / parameters
39.66±2.17
b
B
55.19±2.40
cB
30.18±1.11
aB
74.98±2.56
dB
CAT Mmol / g . tissue
15.84±1.14
b
B
24.97±1.48
cC
7.25±1.11
aB
27.74±2.57
dB
SOD U / g . tissue
33.50±1.51
cC
27.16±1.60
b
C
13.62±1.07
aB
25.02±1.91
b
A
GPX ng / g . tissue
9.93±0.53
b
B
10.41±0.36
b
B
7.10±0.56
aB
12.16±0.57
cB
GSH ng / g . tissue
55.51±3.50
aA
55.83±6.99
aA
89.41±5.76
b
A
54.89±5.93
aA
MDA Mmol / g . tissue
106.39±3.66
cC
62.12±5.76
aA
122.82±3.62
dB
96.15±3.17
b
C
ALT U/L
119.7±2.28
cB
73.00±2.79
aA
139.22±5.25
dB
81.47±3.21
b
B
AST U/L
32.31±2.96
aB
62.60±3.54
b
C
61.57±4.11
b
B
58.28±1.64
b
C
Urea mg/dL
0.69±0.04
aC
0.99±0.05
b
C
1.31±0.06
cC
0.99±0.05
b
C
Creatinine mg/dL
a, b & c: There is no significant difference (P>0.05) between any two means, within the same column have the
sam e superscript lette r. A, B & C: There is no significant difference (P>0.05) between any two means for the same
attribute, within the same row have the same superscript letter. Data are presented as (mean ± S.E) S.E= standard
error.
4. DISCUSSION
The obtained results revealed that, a
significant decrease in liver SOD, GPX,
CAT activities and GSH concentration with
marked significant increase in MDA level
were observed in TBM group when
compared to control group .These findings
were in agreement with (Kumaraguruparan
et al., 2002) who found that, the presence
of tumor caused disequilibria of the
antioxidant defense system. Also, Hayat,
(2001) demonstrated that, lipid
peroxidation level was significantly
increased in blood, liver and tumor tissues
of EAC mice when compared with control
group. Moreover, Saygili et al., (2003)
demonstrated that a decrease in erythrocyte
GSH level has been reported in several
diseases including malignancies.
Ameliorative effect of novel nanocomposite
239
Additionally, a decline in SOD activity was
observed in mice bearing Ehrlich carcinoma
by (Sahu et al. 1977) who postulated that,
the loss of Mn-SOD activity could be due to
loss of mitochondria which leads to a
decrease in total SOD activity in different
tissues of the tumor host. It seems that
oxidative damage caused by decreased
capacity for H2O2 elimination is related to
suppressed activity of CAT, as well as to
suppressed direct antioxidant action of GSH.
This was in agreement with the previous
findings that CAT has a higher significant
role than GPX in protecting erythrocytes
against oxidative stress (Gaetani, et al.,1996)
and (Muller, et al.,1997). The present data
showed a marked depletion in GSH content
in liver of Ehrlich solid tumor (EST)
bearing mice accompanied by significant
reduction in SOD and catalase activities.
There is a close correlation between
depletion of GSH and antioxidant enzymes
and the increase in lipid peroxidation (Devi
and Ganasoundari, 1999). GSH plays an
important role as an endogenous
antioxidant system that is found particularly
in high concentration in liver and is known
to have key function in protecting cells by
scavenging ROS (Sinclair. et al.,
1990) and (Mates, et al., 1999), modulating
cellular redox status and acting as a cofactor
for antioxidant enzymes (Niki, 2008), (Sen,
2000) and (Agrawal, et al., 2011). On the
other hand, the free radical scavenging
system, CAT and SOD are to provide a
guard against the potentially injurious
reactivity of superoxide and hydrogen
peroxide (Rushmore and Picket, 1993),
(Rajkapoor, et al., 2007) and ( Kathiriya, et
al ., 2010 ). Level of GSH and antioxidant
status during tumor growth was previously
investigated by (Navarro, et al., 1999).
Their study showed a reduction in blood
glutathione redox (GSH/GSSG) in Ehrlich
ascites carcinoma –bearing mice. They
attributed this result to the oxidative stress
that caused an elevation in peroxide
formation by cancer cells. GSH oxidation
occurred in the red blood cells leads to the
release of GSSG from the different tissues
to the blood stream ( Al Abdan, 2012). A
significant reduction in SOD and catalase
activities was observed in liver tissue of
tumor-bearing mice. Similar results of a
marked decrease in liver SOD activity was
observed in plasma, lung and liver of
Ehrlich carcinoma-bearing animals (Abu-
Zeid et al., 2000) . It was reported that,
Tumor development may lead to the
degradation of antioxidant enzymes such as
SOD and catalase as a result of uncontrolled
oxidative damage (Hasegawa, et al., 1992).
Malondialdehyde (MDA) the end product
of lipid peroxidation acts as a marker of
oxidative stress (Choi, et al .,
2012) and ( Niki , 2008). In the current
study, EST bearing mice displayed a
significant increase liver MDA
concentration. Previous studies
demonstrated that tumor growth disrupts
the antioxidant system and increases LPx in
tumor host vital organs (Gonenc, et al .,
2001), (Badr El-Din , 2004) and (Noaman,
et al ., 2008 ). The generation of lipid
peroxide and its increase in the mouse liver
could result from a chain reaction or could
be initiated by indirect mechanisms that
have escaped the antioxidant capacity of the
liver of EST bearing mice. In the present
study, untreated tumor bearing mice
showed significant elevation in serum ALT
and AST activities and urea and creatinine
concentrations as compared with control
group. The obtained results indicate that,
the development of tumor in the animal
body can affect many functions of vital
organs such as liver function. Similarly,
Gupta et al., (2004) showed elevation of
liver transaminases in EAC bearing mice
indicating liver dysfunction. Also, (Pal, et
al.,2005) demonstrated that, the activity of
the liver enzymes was increased in serum of
EAC-bearing mice indicating general
toxicity that occurred due to cancer
development. Also, liver and kidney
toxicity induced during tumor growth may
be due to the excessive production of ROS
that leads to oxidative damage (Borges, et
al., 2006). It was previously observed that
oxidative damage which appeared as
Abou Zaid et al. (2015)
240
increased lipid peroxidation and inhibition
of GSH content, catalase and SOD activity,
led to liver and kidney dysfunctions ( El –
Nahal , 2010) . The recorded increase in
plasma ALT and AST activities in tumor
bearing mice of the present study might be
due to generalized destruction of liver cells
and release of AST into plasma after tumor
induction. On the other hand, a significant
increase in serum urea concentration in
TBM was confirmed by the results
observed by (Hussein and Azab,1997) who
observed that, there was a highly significant
increase in plasma urea concentration in
tumor-bearing mice. The author attributed
such increase in blood urea concentration to
the increase in urea production as a result of
catabolic effect of tumor. The obtained
results showed a very highly significant
increase in serum creatinine concentration
in tumor bearing mice. These results were
similar to (Hussein, 2003) who observed
that, serum creatinine level showed a
significant increase in mice-bearing Ehrlich
ascites carcinoma due to muscle necrosis.
As confirmed by (Kawaguchi et al., 1991)
who suggested that, creatinine was
increased in tumor-bearing rats as the
glomerular lesions progressed, associated
with a rise in serum creatinine level.
Moreover, a highly significant increases
showed all over the experimental period in
(AST) activity (U/ml) as compared to
(NTBM) administrated nanocompsite ,
these results were similar results reported
by (Mokhtar et al., 2008) who showed that,
curcumin decreased the induction of (AST
and ALT) activity of rats treated with
Sodium arsenite. these results were
confirmed by (Tirkey et al., 2005) who
showed in studies with cyclosporine that,
treatment with curcumin was significantly
decreased the level of urea and creatinine
because of its role as potent antioxidant.
These suggestion was confirmed by
(Farombi and Ekor, 2006) who found that,
the preventive effect of curcumin on the
gentamicin-induced decrease in the activity
of glutathione peroxidase (GSHPx) and
CAT could contribute to the restoration of
markers of renal tubular injury. It seems
reasonable to assume that curcumin is able
to suppress nephrotoxicity in kidney, as it
was demonstrated in studies with
adriamycin (Venkatesan, et al., 2000).
Kanter et al., (2005) demonstrates that, Vit
C treatment decreases serum urea and
creatinine concentrations and increase
antioxidant enzyme activities, and also
prevents renal damage in experimentally-
induced endotoxemic rats. An important
study in rats showed that TQ prevented the
ischemia/reperfusion induced alterations in
gastric mucosal glutathione (GSH) and
superoxide dismutase (SOD) (El-Abhar, et
al., 2003 ). Also, pre-treatment with TQ
protected organs against oxidative damage
induced by carbon tetrachloride (Nagi, et al.,
1999 ) and doxorubicin (al-Shabanah , et
al ., 1998 ).Moreever, another study showed
that, TQ has role in the protection of organs
against oxidative damage induced by a free
radical generating agents (Baynes , 1991 ).
Additionally, in vitro study proved that TQ
and a synthetic structurally-related TBHQ,
strongly inhibited iron-dependent
microsomal lipid peroxidation in a
concentration-dependent manner (Badary,
et al., 2003). CUR significantly and dose-
dependently improved urea and creatinine,
and decreasing the elevated levels of serum
urea and creatinine provides convincing
evidence for participation of reactive
oxygen species (ROS) in ciplastin (cis-
diammine dichloroplatinum) CDDP
induced renal dysfunction. So that, CUR
due to its potential antioxidants properties,
improves renal function via attenuating the
oxidative stress (Sumanont, et al., 2003)
and ( Vajragupta , et al ., 2003) . They
explained it on the antioxidant actions of
Niegla Sativa (NS) which prevented the
renal damage produced by reactive oxygen
species (Yaman, et al., 2010). Similar
findings had been reported earlier by Ali
(2004) who observed that, treatment of rats
with NS oil showed significant
alleviation in the biochemical parameters of
gentamicin nephrotoxicity, implying a rise
in the scavenger de-fense system and the
Ameliorative effect of novel nanocomposite
241
total anti-oxidant condition in kidney
parenchyma. Studies performed on NS in
various models of oxidative stress showed
that the majority of its pharmacological
effects are owing to its antioxidant potential
which is chiefly a result of its capability to
hunt reactive oxygen radicals and to slow
down the process of lipid peroxidation
(Gupta, et al., 2004). In the present study,
presumably the antioxidant action of the
Nigella sativa prevented the oxidative
damage of renal tissue with subsequent
reduction in serum levels of urea and
creatinine. Studies have shown that NS seed
extracts and its active
constituents possess
beneficial effects in kidney and liver
damage caused as a result of exposure to
various pharmacological agents (Ali, et al.,
2003).
Nigella sativa oil inhibited lipid
peroxidation in liposomes and act as a
scavenger of free radicals (Houghton, et al.,
1995).
In view of the action of Nigella
sativa on lipid peroxidation and antioxidant
defense system, it was also reported that
administration of Nigella sativa oil in
carbon tetrachloride induced toxicity in rats
resulted in an improvement of the
antioxidant defense system (Kanter, et al .,
2005 ).
5. CONCLUSION &
RECOMMENDATION
From the obtained results, it could be
concluded that curcumin nanoparticles
modified with basic nano black seeds
(nigella sativa) and calcium ascorbate have
higher antioxidant activity and protect liver
and kidney from the harmful catabolic
effect and oxidative damage of tumor. So
we recommended by using this novel
nanocomposite as prophylactic and
preventive for many diseases and as
adjuvant therapy in cancer treatment.
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