International Journal of Clinical Medicine, 2011, 2, 185-195
doi:10.4236/ijcm.2011.23031 Published Online July 2011 (http://www.SciRP.org/journal/ijcm)
Copyright © 2011 SciRes. IJCM
185
Nephroprotective Effect of Nigella sativa and
Matricaria chamomilla in Cisplatin Induced Renal
Injury
Supportive Treatments in Cisplatin Nephrotoxicity
Ragaa Hamdy Mohamed Salama1*, Nahed Abdel-Mak sou d Abd -El -Hameed2, Sary
Khalil Abd-El-Ghaffar3, Zaghloul Thabet Mohammed2, Nagwa Mahmoud Aly Ghandour2
1Department of Medical Biochemistry, Assiut University, Assiut, Egypt; 2Forensic Medicine and Toxicology Faculty of Medicine,
Assiut, Egypt; 3 Pathology Department, Veterinary Medicine, Assiut University, Assiut, Egypt.
Email: *ragaa_2002@yahoo.com
Received August 15th, 2010; revised January 10th, 2011; accepted January 30th, 2011.
ABSTRACT
Nigella sativa and Matricaria chamomilla are extensively consumed as tea or tonic. Despite their widespread use as a
home remedy, relatively few trials evaluated their benefits in nephroprotection. Hence, this study evaluates the nephro-
protective effects of supportive treatments (N. sativa, M. chamomilla and vitamin E) in cisplatin nephrotoxicity rat
model. Eighty rats divided into 10 groups, of 8 animals each. The first group (G1) injected with saline intrapretoneal
(I.P). G2 injected with 5 mg/kg cisplatin I.P on zero day of experiment and repeated 4 times, with 5 days free interval.
G3 - G10 received daily supportive treatments, started 5 days before the experiment (–5 day). Concomitantly G4, G6,
G8 and G10 injected with 5 mg/kg cisplatin I.P like G2. On day sixteen, animal scarified, serum and/or kidney tissue
were used to determine kidney function tests (serum urea, creatinine, NAG, β-GAL), oxidative stress indices (NO, LPO),
antioxidant activities (SOD), sulphur compounds (GGT, GSH, total thiols), apoptotic indices (cathepsin D, DNA frag-
mentation), two minerals (Ca2+ and Zn2+). Cisplatin caused marked elevation in serum GGT that reduced significantly
in group received M. chamomilla with cisplatin (P < 0.001). There is a correlation between GGT and NAG in cisplatin
group (r = 0.731, P < 0.05) that may suggest one of possible mechanisms of renal injury by cisplatin. M. chamomilla
followed by N. sativa and vitamin E improved the biochemical and pathological renal injury, as determined by increas-
ing the body weight, normalizing the kidney functions, decreasing the oxidative stress markers, improving the apoptotic
markers, minimizing the pathological changes. Hence, N. sativa and M. chamomilla will be promising nephroprotective
agents for reducing cisplatin nephrotoxicity, most probably, by antioxidants effects and inhibition GGT production,
respectively.
Keywords: Cisplatin, Nephrotoxicity, Nephroprotection, Matricaria chamomilla, Nigella sativa, Vitamin E
1. Introduction
Cisplatin is a major antineoplastic drug for the treatment
of solid tumors, but it has dose-dependent renal toxicity.
It has multiple intracellular effects, causing direct cyto-
toxicity with reactive oxygen species as apoptosis, in-
flammation and fibrogenesis [1].
Despite all the marvelous advancements in modern
medicine, traditional herbal medicine has always been
practiced [2]. N. sativa is the most common herbal
medicine all over the world for the treatment and pre-
vention of a number of diseases. Their seeds/oil has
anti-inflammatory, analgesic, antipyretic, antimicrobial,
hypoglycemic and antineoplastic activity without sig-
nificant adverse effects. This may be related to their
cytoprotective and antioxidant actions [3]. M. chamo-
milla is a well-known medicinal plant as carminative,
analgesic, and anticonvulsant in traditional medicine [4].
It has moderate antioxidant, antimicrobial activities and
significant antiplatelet activity in vitro. Animal model
studies indicate potent anti-inflammatory action, some
antimutagenic and cholesterol-lowering activities, as
well as antispasmotic and anxiolytic effects [5]. Its
methanolic extract showed potent neuroprotective activ-
ity against global cerebral ischemia/reperfusion injury-
Nephroprotective Effect of Nigella sativa and Matricaria chamomilla in Cisplatin Induced Renal Injury
186
induced oxidative stress in rats [6]. There are numerous
varieties of Chamomile but the most two popular are
Roman Chamomile and German Chamomile. German
Chamomile which is called Matricaria chamomilla,
considered the more potent of the two and received more
scientific evaluation. The traditional drug of Matricaria
recutita is the dried flower heads [7].
Combination of two or more herbs may be used with
chemotherapy to decrease toxic adverse effects, elevate
the immune function, and improve the quality of life in
the patients. However, this required further studies in
human.
The aim of the present study is to design cisplatin
nephrotoxicity rat model mimics human cycles of cis-
platin chemotherapy, then, evaluates the nephroprotec-
tive effects of N. sativa, M. chamomilla and vitamin E in
this model.
2. Materials and Methods
2.1. Preparation of Supportive Treatments and
Cisplatin
N. sativa (seeds) and M. chamomilla (dry leaves and
flowers) were selected with a fair degree of quality as-
surance from faculty of Pharmacy, Pharmacognosy de-
partment, Assiut University, Egypt. The identity of the
planets was verified by the Center of Medicinal, Aro-
matic and Poisonous Plants and a voucher specimen
were kept on record in the herbarium of the Faculty of
Pharmacy. The dry leaves crushed to powder and ex-
tracted according to method described by El-Daly [8].
Twenty five grams of either powder extracted by 95%
ethanol (500 ml) with continuous stirring at 4˚C, over-
night, this was repeated for three successive days. The
pooled extracts (1500 ml), evaporated under reduced
pressure using Duché rotary flash evaporator rendering
the product alcohol-free. Then, one gram of the product,
reconstituted with 20 ml saline so, final concentration
was 50 mg/ml and stored at 4˚C. N. sativa oil and Vita-
min E purchased from Pharco-Company, Egypt. Eight
grams of N. sativa oil (w/v) emulsified with 100 ml 2%
polyethylene glycol-400 w/v (final concentration was 80
mg/ml). Vitamin E (90 mg, w/v) dissolved in 30 ml corn
oil, so, final concentration was 3 mg/ml as described by
El-Daly [8]. Vial of 50 mg Cisplatin, Bristol-Myers
Squibb Company, USA, reconstituted in 20 ml saline
immediately before injection with final concentration
2.5 mg/ml.
2.2. Experimental Design
The study carried out on 80 healthy adult male Spra-
gue-Dawley rats, weighing 250 - 350 g. Their ages
ranged from 26 - 28 weeks. The animals were housed
conventionally in clean cages and fed with standard food
and water ad libitum. The animals were housed in
groups in 12-hour light/ dark cycle. The care and treat-
ments of the animals were approved and performed ac-
cording to the guidelines of Animal House and ethical
standards of Faculty of Medicine, Assiut University,
Egypt. The experiment carried out in 10 groups of 8
animals each (G1 to G10). The first group (G1) was the
healthy reference (H.R) group, injected with 1 ml saline
I.P in the same way as group 2. The second group (G2)
was cisplatin nephrotoxicity rat model, injected with 5
mg/kg cisplatin I.P on the zero day of experiment. The
injection repeated 4 times, with 5 days free interval in
between (on the day, zeroth, 5th, 10th, 15th) that mimics
human regimen. By the 5th day up to 43% of the admin-
istered cisplatin is recovered in the urine [9]. G3 and G4
received N. sativa extract (50 mg/kg I.P), G5 and G6
received N. sativa oil (400 mg/kg orally by tubing), G7
and G8 received M. chamomilla extract (50 mg/kg I.P),
G9 and G10 received vit. E (6 mg/kg I.P). The injection
of the three compounds started 5 days before the ex-
periment (–5 day) and continued daily till the end of the
experiment. In addition, G4, G6, G8 and G10 received
cisplatin injection one hour before supportive treatments
in the same way like group 2. On the day sixteenth, ani-
mals weighted, blood samples obtained, then animals
sacrificed and both kidneys were removed and weighted.
One kidney was homogenized in 3 ml ice cold saline
using a Glas-Col homogenizer fitted with teflon plunger
(Terre Haute, # 099C, USA). Then centrifuged at 4000 ×
g for 10 min. at 4˚C, supernatant was kept at –70˚C for
further biochemical measurements. The second kidney
prepared for histopathological examinations.
2.3. Biochemical Analysis
1) Serum samples used for determination of: a) Kidney
function tests: serum urea and creatinine using kits from
Stanbio, USA. b) Oxidative stress indices: nitric oxide
(NO) measured as total nitrite [10] and lipid peroxida-
tion (LPO) as Thio Barbituric Acid Reactive Substances
[11]. c) Antioxidants: Superoxide dismutase activity
(SOD) [12]. d) Sulphur compounds: Gamma glutamyl
transferase (GGT) using kit from QCA, Spain and total
thiols by chemical method [13]. e) Serum levels of re-
lated mineral as Ca2+ and Zn2+ by atomic absorption
spectrophotometer (Buck scientific, USA. Model 210
VGP) using nitrous oxide/acetone flame absorption at
wave length 422.7 nm and 213.9 nm, respectively. 2)
Tissue supernatants were used for determination of: a).
Proximal convoluted tubules functional tests such as
beta-N-acetylglucosaminidase (NAG),
-Galactosidase
(
-GAL) [14]. b) Oxidative stress index: Lipid peroxi-
dation (LPO) [11]. c) Antioxidants: SOD [12], reduced
Copyright © 2011 SciRes. IJCM
Nephroprotective Effect of Nigella sativa and Matricaria chamomilla in Cisplatin Induced Renal Injury187
glutathione (GSH) determined by spectrophotometer
using chemical method [15]. d) Apoptotic indices:
Cathepsin D by chemical methods, as hemoglobin split-
ting activity of cathepsin [16], and DNA fragmentation
by colorimetric method using diphenylamine (98% v/v
glacial acetic acid; 1.5% v/v sulfuric acid; and 0.5% v/v
of 1.6% acetaldehyde) [17]. e) Total protein using kit
from Stanbio USA.
2.4. Histopathological Examinations
Kidney was divided into 2 parts, the 1st part fixed in
10% phosphate buffered formalin and stained by H&E
for light microscopic examination. The 2nd part fixed in
5% gluteraldhyde, then, prepared semithin sections
stained by toluden blue stain, and examined by light
microscope. Representative fields of the semithin sec-
tions were selected, then ultrathin sections (70 nm) were
cut with diamond knife using a Reichert OMVs ultra
microtom for electron microscopic studies using lead
citrate, uranyl acetate stain [18], and transmission elec-
tron microscope (Jo El JEM, Japan. Model 100 CxII) in
Electron Microscope Unit of Assiut University.
2.5. Statistical Analysis
Soft ware, SPSS version 16 was used for statistical
analysis. Values expressed as mean ± SE. Differences
between obtained values carried out by Mann-Whitney
U test using G1 as reference group. One way analysis of
variance (ANOVA) followed by post hoch test (Tukey
HSD) multiple comparison test using G2 as reference
group. A P < 0.05 is a criterion for significant.
3. Results
For clinical assessment, there was increase in body
weight in G1 (3.99%) and in groups received supportive
treatment only (G3, G5, G7, G9). Contrary, there was
reduction in body weight in all groups received cisplatin
plus supportive treatment. The maximum is in G2 that
received cisplatin only (7.59%), then G4 (4.55%), G6
(3.98%), G10 (3.75%) and the minimum is G8 that re-
ceived combination of cisplatin and M. chamomilla
(3.31%).
In this study, Cisplatin caused elevation in all kidney
function indices. Co-administration of M. chamomilla
extract with cisplatin provided the best protection for the
kidney by reducing the levels of urea, creatinine, NAG
and β-GAL followed by co-administration of vitamin E
then N. sativa orally and finally N. sativa extract as
shown in Table 1. Determination of oxidative stress and
antioxidant indices revealed that cisplatin caused sig-
nificant elevation in NO and LPO, significant decrease
in SOD and GSH as shown in Tables 2-4, respectively.
The best correction achieved in group received cisplatin
with vitamin E then groups treated with N.sativa (extract
or oil) and cisplatin. However, M. chamomilla extract
with cisplatin provided the lowest protection against
oxidative stress. Determination of sulpher compounds
revealed that cisplatin caused marked elevation in serum
GGT. The best reduction in the level of GGT achieved
in M. chamomilla with cisplatin treated group (P < 0.001)
as shown in Table 4. On the other hand, cisplatin caused
marked decrease in total thiols and GSH compared to
control (G1), but vit. E showed the best recovery for total
thiols and GSH due to its antioxidant action that reserve
GSH and total thiols. Co-administration of M. chamo-
milla with cisplatin approved better protection from
apoptosis, as shown in Figure 2(f), followed by co-ad-
ministration with N. sativa oil, vitamin E and finally N.
sativa extract as shown in Table 5. In this study, cis-
platin caused marked decrease in serum Ca2+ and Zn2+
levels. Co-administration of N. sativa oil with cisplatin
approved better protection from cisplatin- induced hypo-
calcaemia, followed by M. chamomilla, vitamin E and
finally N. sativa extract as shown in Table 6.
The histopathological changes demonstrated the pro-
tective effect of co-administration of supportive treat-
ments in different groups (Figure 2) in comparison to
new cisplatin rat model (Figure 1).The intensity of
histopathological lesions demonstrated the protective
effect of co-administration of supportive treatments in
different groups as shown in Table 7. The biochemical
and histological results of the rats’ kidney of groups 3, 5,
7, 9 that received supportive treatment only and served
as control groups for these compounds revealed no ab-
normal changes (data not shown).
4. Discussion
The public received information about complementary
and alternative medicines (CAMS) inaccurate or incom-
plete [19] that, encourage the practitioners and research-
ers to evaluate the actual benefits of these agents. N.
sativa oil has been shown to possess 67 constituents,
many of which are capable of inducing beneficial phar-
macological effects in human [20]. By HPLC analysis of
N. sativa oil, thymoquinone (TQ), dithymquinone (DTQ),
thymohydroquinone, and thymol are considered the main
active ingredients [21]. Bisabololoxide A is the principle
constituents of some bioactivities of German chamomile
such as anti-inflammatory, gastrointestinal and antipru-
ritic actions [22].
For clinical assessment of cisplatin nephrotoxicity rat
model, there was 7.59% decrease in body weight in cis-
platin treated group along the period of experiment (cis-
platin 5 mg/kg , 4 doses, time of experiment 21 days);
compared to 14% decrease in body weights (cisplatin
15mg/kg, time of experiment 5 days) [23]. In this study,
Copyright © 2011 SciRes. IJCM
Nephroprotective Effect of Nigella sativa and Matricaria chamomilla in Cisplatin Induced Renal Injury
Copyright © 2011 SciRes. IJCM
188
Table 1. Kidney functions in rats treated with cisplatin compared to those treated with cisplatin combinations.
G1 G2 G4 G6 G8 G10
Serum
Urea
(mg/dl)
25.3 ± 1.2
60.9 ± 1.6
51.92 ± 3.4
46.34 ± 5
N.S**
41.53 ± 4.1
N.S**
N.S***
45.53 ± 3.8
N.S**
N.S***
N.S****
ANOVA test 0.0001* 0.4* 0.01* 0.0001* 0.006*
Creatinine
(mg/dl)
0.37 ± 0.01
0.64 ± 0.01
0.57 ± 0.03
0.53 ± 0.03
N.S**
0.46 ± 0.04
P < 0.05**
N.S***
0.53 ± 0.03
N.S**
N.S***
N.S****
ANOVA test
0.0001* 0.4* 0.04* 0.0001* 0.03*
Tissue
NAG
(mu/mg protein)
5.07 ± 0.2
8.54 ± 0.4
6.89 ± 0.5
6.8 ± 0.5
N.S**
6.33 ± 0.3
N.S**
N.S***
6.84 ± 0.5
N.S**
N.S***
N.S****
ANOVA test
0.0001* 0.02* 0.01* 0.001* 0.02*
β-GAL
(mu/mg protein)
2.88 ± 0.2
7.93 ± 0.3
6.44 ± 0.6
5.86 ± 0.6
N.S**
5.32 ± 0.4
N.S**
N.S***
5.96 ± 0.5
N.S**
N.S***
N.S****
ANOVA test 0.0001* 0.1* 0.01* 0.0001* 0.02*
G1 is the healthy reference group; G2 is cisplatin treated group; G4 received Nigella sativa extract + cisplatin; G6 received Nigella sativa oil + cisplatin; G8
received Matricaria chamomilla extract + cisplatin; G10 received vit. E + cisplatin. Data of groups 3, 5, 7, 9 that received only supportive treatment not shown
as it is within normal range. N. B: * versus G2; ** versus G4; ***versus G6; **** versus G8. ANOVA followed by Tukey test as post ANOVA test was used
to compare between group 2 and different groups.
Table 2. Oxidative stress in rats treated with cisplatin compared to those treated with cisplatin combinations.
G1 G2 G4 G6 G8 G10
Serum
NO
(µmol/ml)
14.06 ± 0.3
25.52 ± 1.7
19.01 ± 1.8
19.58 ± 1.1
N.S**
22.2 ± 1.8
N.S**
N.S***
19.05 ± 1.4
N.S**
N.S***
N.S****
ANOVA test 0.0001* 0.007* 0.02* 0.6* 0.008*
LPO
(nmol/ml)
0.37 ± 0.004
1.01 ± 0.02
0.76 ± 0.09
0.68 ± 0.09
N.S**
0.85 ± 0.08
N.S**
N.S***
0.66 ± 0.1
N.S**
N.S***
N.S****
ANOVA test 0.0001* 0.09* 0.005* 0.6* 0.002*
Tissue
LPO
(nmol/mg protein)
0.24 ± 0.01
0.73 ± 0.02
0.51 ± 0.08
0.48 ± 0.07
N.S**
0.64 ± 0.07
N.S**
N.S***
0.46 ± 0.08
N.S**
N.S***
N.S****
ANOVA test 0.0001* 0.07* 0.01* 0.9* 0.01*
G1 is the healthy reference group; G2 is cisplatin treated group; G4 received Nigella sativa extract + cisplatin; G6 received Nigella sativa oil + cisplatin; G8
received Matricaria chamomilla extract + cisplatin; G10 received vit. E + cisplatin. Data of groups 3, 5, 7, 9 that received only supportive treatment not shown
as it is within normal range. N. B: * versus G2; ** versus G4; ***versus G6; ****versus G8. ANOVA followed by Tukey test as post ANOVA test was used to
compare between group 2 and different groups.
Nephroprotective Effect of Nigella sativa and Matricaria chamomilla in Cisplatin Induced Renal Injury189
Table 3. Levels of SOD in rats treated with cisplatin compared to those treated with cisplatin combinations.
G1 G2 G4 G6 G8 G10
Serum
SOD
(U/ml)
6.215 ± 0.3
3.55 ± 0.1
4.78 ± 0.5
P < 0.05*
4.8 ± 0.5
P < 0.05*
N.S**
2.93 ± 0.3
N.S*
P < 0.01**
P < 0.01***
5.125 ± 0.6
P < 0.05*
N.S**
N.S***
P < 0.01****
ANOVA test 0.0001* 0.4* 0.4* 0.9* 0.1*
Tissue
SOD
(U/mg protein)
0.69 ± 0.05
0.175 ± 0.01
0.38 ± 0.09
0.45 ± 0.09
N.S**
0.244 ± 0.08
N.S**
P < 0.05***
0.48 ± 0.1
N.S**
N.S***
P < 0.05****
ANOVA test 0.0001* 0.5* 0.1* 0.9* 0.09*
G1 is the healthy reference group; G2 is cisplatin treated group; G4 received Nigella sativa extract + cisplatin; G6 received Nigella sativa oil + cisplatin; G8
received Matricaria chamomilla extract + cisplatin ; G10 received vit. E + cisplatin. Data of groups 3, 5, 7, 9 that received only supportive treatment not shown
as it is within normal range. N.B: * versus G2; ** versus G4; ***versus G6; **** versus G8. ANOVA followed by Tukey test as post ANOVA test was used to
compare between group 2 and different groups.
Table 4. Sulpher related compound s in rats treated with cisplatin compared to those treated with cisplatin combinations.
G1
(N = 20)
G2
(N = 12)
G4
(N = 14)
G6
(N = 14)
G8
(N = 18)
G10
(N = 14)
Serum
GGT
(U/l)
5.92 ± 0.45
13.9 ± 1.4
12.08 ± 1.4
N.S*
8.24 ± 1.1
P < 0.01*
P < 0.05**
7.15 ± 0.7
P < 0.001*
P < 0.01**
10.05 ± 1.07
P < 0.05*
P < 0.05****
ANOVA test 0.0001* 0.9* 0.001* 0.0001* 0.09*
Tissue
GSH
(μmol/g protein)
4.14 ± 0.07
2.45 ± 0.07
2.77 ± 0.1
N.S*
3.12 ± 0.3
P < 0.05*
N.S**
2.64 ± 0.06
N.S*
N.S**
N.S***
3.23 ± 0.2
P < 0.05*
N.S**
N.S***
N.S****
ANOVA test 0.0001* 0.8* 0.02* 0.9* 0.004*
Total Thiols
(nmol/ml)
10.13 ± 1
3.39 ± 0.3
6.21 ± 1.1
P < 0.01*
4.96 ± 0.5
P < 0.05*
N.S**
4.36 ± 0.6
N.S*
N.S**
N.S***
5.17 ± 0.5
P < 0.05
N.S**
N.S***
N.S****
ANOVA test 0.0001* 0.1* 0.8* 0.9* 0.7*
G1 is the healthy reference group; G2 is cisplatin treated group; G4 received Nigella sativa extract + cisplatin; G6 received Nigella sativa oil + cisplatin; G8
received Matricaria chamomilla extract + cisplatin; G10 received vit. E + cisplatin. Data of groups 3, 5, 7, 9 that received only supportive treatment not shown
as it is within normal range. N.B: * versus G2; ** versus G4; ***versus G6; **** versus G8. ANOVA followed by Tukey test as post ANOVA test was used to
compare between group 2 and different groups.
there was 1.4 times increase in the percentage of kidney
weight to body weight after cisplatin-treatment. This
could be explained by the marked decrease in the total
body weight in cisplatin treated group. This was in
agreement with another studies, where there were 1.7
and 2 times increase respectively, in percentage of kid-
ney weight to body weight in cisplatin-treated animals
[24,25]. The co-administration of supportive treatment
with cisplatin resulted in decrease in this percentage
when compared to cisplatin treated groups; this may be
due to the relatively less reduction in the total body
weights in these groups.
The cut-off value for the normal range of blood urea
nitrogen is (40 mg/dl) and serum creatinine is (0.2
mg/dl) based on the values obtained from normal un-
treated mice [23]. In this study, urea and creatinine levels
were 2.4 and 1.7 times increase in cisplatin-treated group
when compared to control goup. The increase in urea r
Copyright © 2011 SciRes. IJCM
Nephroprotective Effect of Nigella sativa and Matricaria chamomilla in Cisplatin Induced Renal Injury
190
Table 5. Apoptotic markers in rats treated with cisplatin compared to those treated with cisplatin combinations.
G1 is the healthy reference group; G2 is cisplatin treated group; G4 received Nigella sativa extract + cisplatin; G6 received Nigella sativa oil + cisplatin; G8
received Matricaria chamomilla extract + cisplatin ; G10 received vit. E + cisplatin. Data of groups 3, 5, 7, 9 that received only supportive treatment not shown
as it is within normal range. N.B: * versus G2; ** versus G4; ***versus G6; **** versus G8. ANOVA followed by Tukey test as post ANOVA test was used to
compare between group (2) and different groups.
Table 6. Serum calcium and zinc in rats treated with cisplatin compared to those treated with cisplatin combinations.
G1
(N = 20)
G2
(N = 12)
G4
(N = 14)
G6
(N = 14)
G8
(N = 18)
G10
(N = 14)
Ca+2
(ppm/ml)
35.4 ± 0.37
13.5 ± 0.36
19 ± 0.37
P < 0.01*
22.8 ± 0.37
P < 0.001*
P < 0.01**
22.2 ± 0.37
P < 0.001*
P < 0.01**
N.S***
22.06 ± 0.36
P < 0.001*
P < 0.01**
N.S***
N.S****
Zn+2
(ppm/ml)
0.68 ± 0.03
0.49 ± 0.03
0.41 ± 0.037
N.S*
0.5 ± 0.036
N.S*
N.S**
0.5 ± 0.036
N.S*
N.S**
N.S***
0.5 ± 0.037
N.S*
N.S**
N.S***
N.S****
G1 is the healthy reference group; G2 is cisplatin treated group; G4 received Nigella sativa extract + cisplatin; G6 received Nigella sativa oil + cisplatin; G8
received Matricaria chamomilla extract + cisplatin; G10 received vit. E + cisplatin. Data of groups 3, 5, 7, 9 that received only supportive treatment not shown
as it is within normal range. N.B: * versus G2; ** versus G4; ***versus G6; **** versus G8. ANOVA followed by Tukey test as post ANOVA test was used to
compare between group (2) and different groups.
level by 3.7 and 2 times and creatinine level by 2.3 and
4.5 times was reported [24,25]. NAG and β-GAL levels
are markers of early-impaired renal function and renal
tubular damage [26]. Cisplatin group, in this study, had
significant elevation of NAG and β-GAL levels when
compared to control group (Table 1) due to dysfunction
of tubular epithelial cells induced by increased traffic of
proteins in the tubular lumen [27,28]. This was con-
firmed by histopathological examinations (Figure 1(a)
and (c)). These pathological changes were minimized in
groups received cisplatin with supportive treatments es-
pecially in group treated with N. sativa oil (G6) or M.
chamomilla (G8) (Table 1, Figure 2(c), (d), (f) and (h)).
Oxidative stress was implicated in the pathogenesis of
cisplatin-induced nephrotoxicity. Oxidative stress, asso-
ciated with increased generation of reactive oxygen me-
tabolites (ROM) caused lipid peroxidation in kidney,
decreased levels of antioxidants and antioxidant enzymes
[29]. In this study, oxidative stress induced by cisplatin
was manifested by elevation in nitric oxide (NO) and
lipid peroxide levels. There was significant elevation in
lipid peroxide [25,30], decrease in nitric oxide level [24],
decrease in antioxidant as GSH and SOD activities was
reported in cisplatin-induced nephrotoxicity [31].
The expression of Cathepsin D, the lysosomal prote-
ases, had been shown to increase with protein degradation
occurred during apoptosis [32]. In this study, cisplatin
induced apoptosis in the form of significant increase in
cathepsin D level and DNA fragmentation that is docu-
mented by light and electron microscopy in Figures 1(b)
and (f). M. chamomilla extract provided significant re-
duction in these parameters followed by N. sativa oil and
vitamin E then, N. sativa extract as shown in Table 5.
The intensity of these apoptotic changes was markedly
G1
(N = 20)
G2
(N = 12)
G4
(N = 14)
G6
(N = 14)
G8
(N = 18)
G10
(N = 12)
Catepsin D
UEA
0.024 ± 0.005
0.15 ± 0.02
0.102 ± 0.02
N.S*
0.052 ± 0.02
P < 0.01*
P < 0.05**
0.046 ± 0.02
P < 0.001*
P < 0.05**
N.S***
0.061 ± 0.02
P < 0.01*
P < 0.05**
N.S***
N.S****
ANOVA test 0.0001* 0.4* 0.0001* 0.0001* 0.002*
% of DNA
Fragmentation
27.2 ± 0.3
40.9 ± 0.3
36.6 ± 1.8
P < 0.05*
33.5 ± 2.2
P < 0.01*
N.S**
31.7 ± 2.4
P < 0.001*
N.S**
N.S***
34.5 ± 2.2
P < 0.05*
N.S**
N.S***
N.S****
ANOVA test 0.0001* 0.4* 0.01* 0.01* 0.05*
Copyright © 2011 SciRes. IJCM
Nephroprotective Effect of Nigella sativa and Matricaria chamomilla in Cisplatin Induced Renal Injury191
(a) (b)
(c) (d)
(e) (f)
Figure 1. Histopathological changes of new cisplatin nephrotoxicity rat model. The following changes were demonstrated: (a)
cystic dilatation of the renal tubules and vacuolar degeneration in the tunica media (arrows) of the blood vessels as well as
perivascular edema (double arrows) H&E 10 × 25. (b) evidences of apoptotic renal tubular epithelium cells (arrows ),
semithin section with toludin blue stain, 10 × 100. (c) necrosis of renal tubular epithelial cell and the mitochondrial
vacuolation (mv) with destruction of their cisternae, electron photomicrograph Lead citrate, uranyl acetate stain, ×10,000. (d)
acidophilic tubular casts (arrows) in medullary renal tubules. H&E 10 × 10. (e) karyorrhexis (arrows) and loss of cellular
membrane of the renal tubular epithelium, semithin section with toludin blue stain, 10 × 100. (f) sloughed apoptotic renal
tubular epithelium cell, conde nsed nucle ar chromatin (N) and c y toplasmic or ganelle s (C) with the prese nce of detached apop-
totic bodies (A), electron photomicrograph; lead citrate, uranyl acetate stain, ×14,000.
Copyright © 2011 SciRes. IJCM
Nephroprotective Effect of Nigella sativa and Matricaria chamomilla in Cisplatin Induced Renal Injury
192
(a) (b)
(c) (d)
(e) (f)
(g) (h)
Figure 2. Histopathological changes in kidney’s rat received cisplatin with supportive treatment. (a)(b) show (G4) that re-
ceived cisplatin with N. sativa extract (50 mg/kg I.P). (a) demonstrates necrosis of the renal tubular epithelium (N) in semithin
section stained with toludin blue 10 × 100. (b) illusterates cytoplasmolysis of renal tubular cell organelles (C) with marked
destruction of microvilli (arrows), Lead citrate, uranyl acetate stain, ×8000 by electron photomicrograph. (c)(d) show (G6)
that received cisplatin with N. sativa oil (400 mg/kg orally). (c) detects some intact tubules (T) and the others has necrotic
changes (N), semithin section stained by toludin blue , 10 × 100. (d) shows moderate destruction in the cytoplasmic organelles
in some cells (C), Lead citrate, uranyl acetate stain, electron photomicrograph ×5000. (e) vitamin E with cisplatin treated
group showing moderate degr ee of cystic dilatation of renal tubules (C) as well as interstitial ly mphoid cell reaction (arrows)
H&E 10 × 25. (f)-(h) evidences of apoptosis in few cells (arrows) of the renal cortex in G8 that received cisplatin with M.
chamomilla, semithin section with of toludin blue stain,10 × 100. (g) shows only increases in the mesengial matrix (M),
semithin section of renal glomeruli toludin blue stain, 10 × 100. (h) electron photomicrograph of renal tubular epithelial cells
in G8 with intact mitochondria (M) and rough endoplasmic reticulum (R), only destruction of some free ribosomes, Lead
citrate, uranyl acetate stain, ×6700.
Copyright © 2011 SciRes. IJCM
Nephroprotective Effect of Nigella sativa and Matricaria chamomilla in Cisplatin Induced Renal Injury
Copyright © 2011 SciRes. IJCM
193
Table 7. Intensity of the histopathological lesions in kidneys of rats of different cisplatin treated groups.
G2 G4 G6 G8 G10
Apoptosis ++++ +++ ++ + ++
Necrobiosis of tubular epithelial cell ++++ +++ ++ + ++
Cystic dilatation ++++ +++ ++ -ve ++
Necrobiosis of glomerular tuft ++++ +++ ++ + ++
Renal Cortex
Interstitial lymphoid cell reaction +++ ++ ++ + ++
Tubular casts ++++ + + -ve +
Renal Medulla Angiopathic changes (bl.v.) +++ -ve -ve -ve -ve
(++++): Severe and involved all animals. (+++): Severe and involved most of animals. (++): Moderate and involved some animals. (+): Weak and involved few
animals. (-ve ): Negative. G1 is the healthy reference group; G2 is cisplatin treated group; G4 received Nigella sativa extract + cisplatin; G6 received Nigella
sativa oil + cisplatin ;G8 received Matricaria chamomilla extract + cisplatin; G10 received vit. E + cisplatin. Data of groups 3, 5, 7, 9 that received only sup-
portive treatment not shown as it is within normal range.
decreased in group treated with cisplatin and M. chamo-
milla as shown in Table 7 and Figure 2(f ) .
GGT, a key enzyme of GSH metabolism, can modu-
late crucial redox-sensitive functions, such as antioxi-
dant/antitoxic defenses and cellular proliferative/ apop-
totic balance. The mechanisms of GGT involvement in
various pathological processes suggest its potential role
as therapeutic target and diagnostic/prognostic marker
[33]. The highest level of activity is on the luminal sur-
face of the proximal tubule cells in the kidney. Its most
common physiological substrates are glutathione and
glutathione conjugates [34]. The nephrotoxicity of cis-
platin was the result of the binding of cisplatin to glu-
tathione and the subsequent metabolism of the cis-
platin-glutathione complex via a γ-glutamyl transpepti-
dase (GGT)-dependent pathway in the proximal tubules
[23]. GGT cleaved the gamma-glutamyl group of the
glutathione-conjugate, and aminopeptidase cleaved the
cysteinyl-glycine bond, resulting in platinum-cysteine-
conjugate. Finally the cysteine conjugate was metabo-
lized by cysteine-s-conjugate beta-lyase to reactive thiol
[35]. Also, they found that cisplatin was nephrotoxic in
wild-type mice but not in GGT-deficient mice and the
toxicity was specific to the proximal tubule cells. Acivi-
cin, an inhibitor of GGT, blocks the nephrotoxicity of
cisplatin in rats [36]. In this study, the highest level of
GGT occurred in cisplatin group and the lowest level in
(G8) that received combination of cisplatin and M.
chamomilla as shown in Table 4. There is a correlation
between GGT and NAG in cisplatin group (r = 0.731, P
< 0.05). Moreover, M. chamomilla doesn’t contain glu-
tamic or methionin [37] to help in regeneration of glu-
tathione. Also, Chamazulene (one of constituents of M.
chamomilla) affected free radical processes and inhibited
lipid peroxidation in a concentration and time-dependent
manner [38]. On the other hand, N. sativa seeds contain
glutamic acid and methionine [39]; this may explain its
antioxidant effect. The beneficial effects of the use of the
N. sativa seeds and thymoquinone (one of its constituent)
might be related to their cytoprotective and antioxidant
actions, and to their effect on some mediators of inflam-
mation [3].
Cisplatin administration caused hypocalcaemia [24,
40]. Calcium released from intracellular calcium storage
in the early phase of nephrotoxicity caused oxidative
stress in renal tubular epithelial cells [41]. The N. sativa
seeds are a source of calcium, iron, and potassium [42].
This can explain the improvement in cisplatin-induced
hypocalcaemia in groups received N. sativa oil with cis-
platin. Zinc pre-treatment caused significant protection
against cisplatin enhanced mortality in rats, and reduc-
tion in lipid peroxidation and NO [43]. However, zinc
content had an inverse correlation with platinum incor-
poration owing to a positive linkage with glutathione
(GSH), a zinc-dependent detoxification factor. The com-
bined treatment with cisplatin and Zn(II)-chelator in-
creased platinum uptake with a concomitant reduction of
intracellular GSH [44]. Zinc was effective factor for pro-
tection of weight loss and increased MDA levels in cis-
platin hepatotoxicity [45]. N. sativa and M. chamomilla
has been used as tea from long time indicate their safety
and minor side effects. In addition, their nephroprotective
effect may encourage physicians to prescribe them after
and inbetween chemotherapy.
5. Acknowledgements
The authors thank Professor Hanaa M. Sayed, Pharma-
cognosy Department, Faculty of Pharmacy, Assiut Uni-
versity, Egypt for her advice and help during extractions.
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