J Pharm Pharmaceut Sci (www.cspscanada.org) 8(3):387-393, 2005
Protective effect of aqueous saffron extract (Crocus sativus L.) and crocin, its active constituent, on renal ischemia-reperfusion-induced oxidative damage in rats
Hossein Hosseinzadeh1,
Hamid R. Sadeghnia2, Toktam Ziaee3 and Aghdas Danaee4
1-
2-
Department of Pharmacology, Faculty of
Medicine,
3-
Faculty of Pharmacy,
4-
Faculty of Veterinary,
Received March 13, 2005; Revised August 1, 2005; Accepted August 15, 2005, Published August 22, 2005
Corresponding author:
Hossein Hosseinzadeh,
Abstract
PURPOSE. The generation of reactive
oxygen species and lipid peroxidation are associated with tissue injury
following ischemic insult; therefore, the use of antioxidants appears rational
in the improvement of kidney diseases therapy. The aim of the present study was
to assess the effect of aqueous saffron extract (Crocus sativus L.)
and its active constituent, crocin, on oxidative stress following renal
ischemia-reperfusion injury (IRI) in rats. METHODS. The cellular redox
status (thiobarbituric acid reactive species (TBARS) and total thiol levels)
and antioxidant power (using ferric reducing/antioxidant power test) were
assessed in control and ischemic groups. The left kidney was exposed to warm
ischemia for 60 min followed by reperfusion for 90 min. The macerated aqueous
extract of saffron (with doses of 5, 20 and 80 mg/kg, i.p.) and crocin (with
doses of 50, 200 and 400 mg/kg, i.p.) were administrated prior to induction of
ischemia. Normal saline (10 ml/kg, i.p.) was injected to control group and a
sham group that did not have ischemia-reperfusion. RESULTS. Ischemia-reperfusion
(IR) caused a significant increase in TBARS levels (p<0.001) and decrement
in both antioxidant power (FRAP value) (p<0.05) and total thiol
concentration (p<0.001) in kidney homogenate samples. In crocin pretreated groups,
a reduction in TBARS levels (from 85.8 ± 5.4 to 20.9 ± 1.5 nmol/g tissue,
p<0.001; 400 mg/kg) and elevation in antioxidant power (FRAP value) (from
3.05 ± 0.16 to 4.15 ± 0.16 µmol/g tissue, p<0.001; 400 mg/kg) and total
thiol concentrations (from 0.38 ± 0.03 to 0.62 ± 0.03 mM, p<0.001; 200
mg/kg), as compared with control group, were observed. The aqueous extract also
reduced lipid peroxidation products (from 85.8 ± 5.4 to 15.9 ± 2.6 nmol/g
tissue, p<0.001; 80 mg/kg) and increased antioxidant power (from 2.98 ± 0.11
to 5.97 ± 0.56 µmol/g tissue, p<0.001; 80 mg/kg) in ischemia-reperfusion
injured rat kidneys. CONCLUSION. This study therefore suggests that the aqueous
saffron extract (Crocus sativus L.) and its active constituent,
crocin, may be useful agents for the prevention of renal ischemia-reperfusion
(IR)-induced oxidative injury in rats.
INTRODUCTION
The cellular depletion of ATP,
the initial pathophysiologic event and hallmark of ischemic injury, lead to a
series of morphologic, biochemical and physiologic derangements. Free oxygen
radical (ROS) generation is an important mechanism of cellular injury in
ischemic and reperfused tissues that causes oxidative damage to cellular
macromolecules including membrane lipids, proteins and nucleic acids [1, 2].
Renal ischemia is a
major cause of acute renal failure. Ischemic renal failure occurs following an
episode of severe hemorrhagic shock, endotoxin sepsis, thermal burns, or
transplantation surgery [3]. ROS per se have also been shown to
compromise renal function, depress glomerular filtration, impair glomerular
sieving function [4-6], and induce apoptosis in renal cells [7].
Cellular defense
against free radical injury is provided by enzymatic (catalase, superoxide
dismutases, and glutathione peroxidase) and nonenzymatic (GSH, a-tocopherol, vitamin C, and urate) free
radical scavenging systems, present in the cell [3]. Recent overwhelming
attention to plant products and alternative medicine has encouraged plant
chemists, pharmacologists, biochemists, and molecular biologists to combine
their efforts in a search for natural agents that can limit free
radical-mediated injuries during and following ischemia–reperfusion, for better
therapeutic management of IRI.
Crocus sativus L., commonly known as saffron, is
used in folk medicine as an antispasmodic, eupeptic, gingival sedative,
anticatarrhal, nerve sedative, carminative, diaphoteric, expectorant,
stimulant, stomachic, aphrodisiac and emmenagogue [8]. Furthermore, modern
pharmacological studies have demonstrated that saffron extract or its active
constituents have anticonvulsant [9], antidepressant [10], anti-inflammatory
[11] and antitumour effects, radical scavenger as well as learning and memory
improving properties [8, 12-15] and promote the diffusivity of oxygen in
different tissues [8]. Saffron extract also has chemopreventive and showed
protective effects on genotoxins-induced oxidative stress in Swiss albino mice [16-19].
The aim of present study was to assess the protective effects of aqueous saffron extract (Crocus sativus L.) and its active constituent, crocin, on renal IR-induced oxidative injury in rats.
MATERIALS AND METHODS
Animals
Adult male Wistar rats weighing
200-300 g were used throughout the study. All of them were kept in the same
room under a constant temperature (22 ± 2 °C) and illuminated 7:00
a.m. to 7:00 p.m., with food pellets and water available ad libitum.
The animals were divided into
eight groups, each of which contained 6-8 rats. Group 1 was the sham group in
which only surgery was done without induction of ischemia. Group 2 was the
control group in which saline solution (10 ml/kg) was given intraperitoneally.
In groups 3-8 aqueous saffron extract (5, 20 and 80 mg/kg, i.p) and crocin (50,
200 and 400 mg/kg, i.p.) were administrated prior to induction of ischemia.
Chemicals
DTNB (2, 2´-dinitro-5,
5´-dithiodibenzoic acid), TPTZ (2, 4, 6-tri (2-pyridyl)-1, 3, 5-triazine), TBA
(2-thiobarbituric acid), n-butanol, tris, Na2EDTA, sodium acetate,
glacial acetic acid, phosphoric acid, potassium chloride, tetramethoxypropane
(TMP), ferric chloride (FeCl3.6H2O), ferrous sulfate and
hydrochloric acid was obtained from Merck. Crocin was purchased from Fluka.
Preparation of aqueous saffron extract
Crocus sativus L. stigmas were collected from Ghaen
(Khorasan province, Northeast of Iran). In the maceration method, 3g of stigmas
were macerated in 400 ml distilled water for three days. The mixture was
subsequently filtered and concentrated under reduced pressure at 35ºC.
Induction of renal ischemia-reperfusion injury (IRI)
The animals were subjected to
left renal warm ischemia for 60 min, and reperfusion for 90 min. Briefly, under
intraperitoneal ketamine/xylazine anesthesia (60 mg/kg and 10 mg/kg,
respectively) and through a midline incision; the abdominal contents were
displaced to the right side. The left renal artery and vein were dissected and
the perirenal fat was preserved. The vascular pedicle was temporarily ligated
with 2–0 silk before the abdominal contents were replaced and the incision was
covered with a moistened pad. At the end of the ischemic period, the abdominal
cavity was reentered, the ligature was removed and reperfusion was supplied.
Throughout the experiments, body temperature was kept at 36-38 °C by placing
the rats under light source. At the 90th min of reperfusion, left kidney was
removed and maintained at -80 °C until analysis [20]. At the day of analysis,
the kidney tissues was homogenized in cold KCl solution (1.5%) to give a 10%
homogeny suspension and used for biochemical assays.
The aqueous saffron extract and
crocin were dissolved in physiologic saline and administrated prior to
induction of ischemia.
Tiobarbituric acid reactive species (TBARS) measurement
Malondialdehyde (MDA) levels,
as an index of lipid peroxidation, were measured. MDA reacts with
thiobarbituric acid (TBA) as a thiobarbituric acid reactive substance (TBARS)
to produce a red colored complex that has peak absorbance at 532 nm [21].
3 ml phosphoric acid (1%) and 1
ml TBA (0.6%) was added to 0.5 ml of homogenate in a centrifuge tube and the
mixture was heated for 45 min in a boiling water bath. After cooling, 4 ml of
n-butanol was added the mixture and vortex-mixed for 1 min followed by
centrifugation at 20000 rpm for 20 min. The organic layer was transferred to a
fresh tube and its absorbance was measured at 532 nm. The standard curve of MDA
was constructed over the concentration range of 0-40 µM [22].
Ferric Reducing / Antioxidant Power (FRAP) assay
The FRAP assay measures the
change in absorbance at 593 nm owing to the formation of a blue colored FeII-tripyridyltriazine
compound from the colorless oxidized FeIII form by the action of
electron donating antioxidants [23].
The FRAP reagent consist of 300
mM acetate buffer (3.1 g sodium acetate + 16 ml glacial acetic acid, made up to
1 liter with distilled water; pH=3.6), 10 mM TPTZ in 40 mM HCl and 20 mM FeCl3.6H2O
in the ratio of 10:1:1.
Briefly, 50 μl
of kidney homogenate was added to 1.5 ml freshly prepared and prewarmed (37 ºC) FRAP reagent in a test tube and
incubated at 37 ºC for 10 min. The absorbance of the blue colored complex was
read against reagent blank (1.5 ml FRAP reagent + 50 μl distilled water)
at 593 nm. Standard solutions of Fe II in the range of 100 to 1000
mM were prepared from ferrous sulphate (FeSO4.7H2O) in
distilled water. The data was expressed as mmol ferric ions reduced to ferrous
form per liter (FRAP value) [24].
Total sulfhydryl (SH) groups assay
Total
SH groups were measured using DTNB (2, 2´-dinitro-5, 5´-dithiodibenzoic acid)
as the reagent. This reagent reacts with the SH groups to produce a yellow
colored complex which has a peak absorbance at 412 nm [25].
Briefly, 1 ml Tris-EDTA buffer (pH=8.6)
was added to 50 μl kidney homogenate in 2 ml cuvettes and sample
absorbance was read at 412 nm against Tris-EDTA buffer alone (A1).
Then 20 μl DTNB reagent (10 mM in methanol) was added to the mixture and
after 15 min (stored in laboratory temperature), the sample absorbance was read
again (A2). The absorbance of DTNB reagent was also read as a blank
(B). Total thiol concentration (mM) was calculated from the following equation:
Total
thiol concentration (mM) = (A2-A1-B) × 1.07/0.05 ×
13.6
Statistical analysis
Data are expressed as mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by Tukey-Kramer post-hoc test for multiple comparisons. The p-values less than 0.05 were considered statistically significant.
RESULTS
Modulation of MDA levels by crocin and saffron extract
There was an increase (50 %) in
the MDA levels following IRI as compared with sham-operated animals (85.8 ± 5.4
vs. 42.9 ± 4.3 nmol/g tissue, p<0.001) (Figure 1).
Crocin and the
saffron extract pretreatment resulted in a significant reduction in the free
radical-mediated lipid peroxidation as indicated by a decrease in the MDA
levels, at various dose levels (Figure 1, 2).
In
crocin-pretreated groups, a reduction in TBARS levels (from 85.8 ± 5.4 to 20.9
± 1.5 nmol/g tissue, p<0.001; 400 mg/kg) was observed. The aqueous saffron
extract also reduced lipid peroxidation products (from 85.8 ± 5.4 to 15.9 ± 2.6
nmol/g tissue, p<0.001; 80 mg/kg) in ischemia-reperfusion injured rat
kidneys.
Figure 1: Effect of aqueous saffron extract on lipid peroxidation following
renal IRI. MDA levels were measured in 10%
homogenates of kidney samples from rats subjected to 60 min of ischemia and 90
min of reperfusion. All drugs were administrated intraperitoneally prior to
induction of ischemia. Values are mean ± SEM (n=6). ***p<0.001 as compared
with vehicle (normal saline) treated animals (One-way ANOVA followed by
Tukey-Kramer test)
Figure 2: Effect of crocin on lipid peroxidation following renal IRI. MDA levels were measured in 10% homogenates of kidney samples from
rats subjected to 60 min of ischemia and 90 min of reperfusion. All drugs were
administrated intraperitoneally prior to induction of ischemia. Values are mean
± SEM (n=8). ***p<0.001 as compared with vehicle (normal saline) treated
animals (One-way ANOVA followed by Tukey-Kramer test)
Modulation of FRAP value by crocin
and saffron extract
IRI caused a significant reduction in FRAP value (30.2 %) of kidney homogenate samples as compared with sham-operated animals (4.37 ± 0.10 vs. 3.05 ± 0.16 µmol/g tissue, p<0.001) (Figure 3).
Figure 3: Effect of aqueous saffron extract on antioxidant power of kidney
homogenate samples following renal IRI. FRAP values were measured in 10%
homogenate samples from rats subjected to 60 min of ischemia and 90 min of
reperfusion. All drugs were administrated intraperitoneally prior to induction
of ischemia. Values are mean ± SEM (n=6). *p<0.05, ***p<0.001 as compared
with vehicle (normal saline) treated animals (One-way ANOVA followed by
Tukey-Kramer test)
Crocin pretreatment increased antioxidant power (FRAP value) of kidney homogenate samples, dose dependently (from 2.98 ± 0.11 to 4.15 ± 0.16 µmol/g tissue, p<0.001; 400 mg/kg). The aqueous saffron extract also increased antioxidant power (from 2.98 ± 0.11 to 5.97 ± 0.56 µmol/g tissue, p<0.001; 80 mg/kg) in ischemia-reperfusion injured rat kidneys (Figure 3, 4).
Figure 4: Effect of crocin on antioxidant power of kidney homogenate
samples following renal IRI. FRAP values were measured in 10% homogenate
samples from rats subjected to 60 min of ischemia and 90 min of reperfusion.
All drugs were administrated intraperitoneally prior to induction of ischemia.
Values are mean ± SEM (n=8). ***p<0.001 as compared with vehicle (normal
saline) treated animals (One-way ANOVA followed by Tukey-Kramer test)
Effect of crocin and saffron extract
on total thiol concentration
Following ischemia-reperfusion injury a significant reduction (37.7 %) in total SH groups (0.61 ± 0.03 vs. 0.38 ± 0.03 mM, p<0.001) in kidney homogenate samples were observed (Figure 5).
Figure 5: Effect of aqueous saffron extract on total thiol concentrations
following renal IRI. Total sulfhydryl (SH) groups were measured in
10% kidney homogenate samples from rats subjected to 60 min of ischemia and 90
min of reperfusion. All drugs were administrated intraperitoneally prior to induction
of ischemia. Values are mean ± SEM (n=6). ***p<0.001 as compared with
vehicle (normal saline) treated animals (One-way ANOVA followed by Tukey-Kramer
test)
Crocin pretreatment caused a significant and dose dependently elevation in total thiol concentration, as compared with control group (from 0.38 ± 0.03 to 0.62 ± 0.03 mM, p<0.001; 200 mg/kg). In contrast, the saffron extract failed to increase total SH groups following ischemia-reperfusion injury (Figure 5, 6).
Figure 6: Effect of crocin on total thiol concentrations following renal
IRI. Total sulfhydryl (SH) groups were measured in 10% kidney
homogenate samples from rats subjected to 60 min of ischemia and 90 min of
reperfusion. All drugs were administrated intraperitoneally prior to induction
of ischemia. Values are mean ± SEM (n=6). ***p<0.001 as compared with
vehicle (normal saline) treated animals (One-way ANOVA followed by Tukey-Kramer
test).
DISCUSSION
A great deal of effort has been
directed toward searching for compounds that can be used for better management
of the clinical consequences arising from renal ischemia–reperfusion injuries,
without much success. The results obtained in the present investigation suggest
that the saffron extract and its active constituent, crocin, have an overall
protective effect against kidney ischemia/reperfusion injury in a rat model.
The observed protective effects can be attributed to the water soluble chemical
constituents of saffron which would include mainly constituents such as crocin
and picrocrocin [13].
A number of
processes have been implicated in the pathogenesis of oxygen
deprivation–induced cell injury. These include the disturbances of cell calcium
homeostasis, depletion of adenine nucleotides, activation of enzymes like
phospholipases with production of toxic lipid metabolites, proteases and
endonucleases and generation of free radicals (ROS) that can cause oxidative
damage to cellular macromolecules [3, 26]. ROS have been shown to play a major
role in IRI [27, 28] and collectively are instrumental in impairing overall
renal function [4-6]. ROS can induce damage to endothelial, glomerular
mesangial and tubular epithelial cells (especially S3 segment of proximal
tubule) [27, 28] and induce apoptosis in renal cells [7]. Cellular death
following renal ischemia-reperfusion injury is well associated with ROS
production and lipid peroxidation and antioxidant therapy has been well
documented to help in the improvement of organ functions [29].
We assessed the
effect of crocin and the aqueous saffron extract by studying their effects on
lipid peroxidation, which was measured in terms of MDA, a stable metabolite of
the free radical-mediated lipid peroxidation cascade. MDA levels increased
significantly following renal IRI. Crocin and the saffron extract reversed the
increase of MDA levels to a considerable extent, thereby confirming its
antioxidant role in IRI.
Sulfhydryl (SH)
groups known to be sensitive to oxidative damage and depleted following
ischemic insult [30], therefore we studied the effect
of these agents on total thiol concentration during IRI. Similarly, in our studies,
total sulfhydryl groups were decreased following ischemic-reperfusion injury.
Crocin pretreated rats exhibited higher SH contents than their respective
controls in the dose related pattern, indicating that crocin helped in
replenishing the total thiol pool. However saffron-mediated SH replenishment
was not as impressive as expected. Saffron pretreatment slightly increased
total thiol concentration following ischemic insult, but this elevation was not
significant as compared with control group. Premkumar et al showed oral
pretreatment with the saffron aqueous extract (40 and 80 mg/kg) for five
consecutive days inhibits genotoxins-induced oxidative stress in mice liver. In
this study, an increase in the levels of glutathione (GSH) concentration as
well as the activities of glutathione S-transferase (GST), glutathione
peroxidase (GPx), catalase and superoxide dismutase (SOD) were observed,
however, normal levels of GSH could not be attained [19]. In contrast, in the
study conducted by El Daly oral pretreatment with saffron extract (50 mg/kg 30
min prior to cisplatin administration for five alternative days, ip) had no
significant effect on the activities of enzymes such as alkaline phosphatase,
glutation reductase, isocitrate dehydrogenase, malate dehydraganse,
glucose-6-phosphate dehydro-geanse, etc. in the kidney of male albino rats as
compared with cisplatin treated animals [31]. It has been postulated that the
nephrotoxic mode of action of drug cisplatin is similar to that of the other
heavy metals, and is related to the decrease in the intracellular
concentrations of glutathione and protein-bound SH groups, which are required
for normal cellular, function [32]. The possibility that cisplatin itself has
the inhibitory effect on the enzyme activities was excluded by the fact that in
vitro addition of the drug to the reaction media did not affect the reaction
rates [33].
Under acute and
chronic pathologic conditions such as ischemia, the balance between oxidant and
antioxidant systems has been interrupted [2, 34]. Therefore we evaluate the
antioxidant or reducing potential of kidney homogenate samples following IRI,
using FRAP assay. As expected following IRI, a significant reduction in
antioxidant power, as indicated by FRAP value, was observed. Instead, crocin
and the saffron extract increased the antioxidant power of kidney homogenate
samples.
In rats, crocin
dyes are known to exert protective effects against acute hepatic damage induced
by aflatoxin B1 and dimethylnitrosamine [35]. Saffron has
chemopreventive effects and its extract inhibits tumor growth in vivo and in
vitro [12, 16, 17]. Escribano et al showed that the saffron extract and
its constituents, crocin, safranal and picrocrocin inhibit the growth of human
cancer cells (Hella cells) in vitro [13]. The saffron extract also has radical
scavenger properties [8] and protects from genotoxicity as well as
genotoxins-induced oxidative stress in mice [18, 19]. These observations
indicate that more than one mechanism of protection is operating. It is well
known that many naturally occurring compounds exhibit discrete mechanisms of
protection. Therefore, further extensive work is needed to identify the
mechanisms of action of saffron and its constituents.
In this study, the
saffron extract was more potent than crocin. This may be due to the fact that the aqueous saffron extract
consists of many constituents such as crocins (water soluble carotenoids which
are glycosyl esters of crocetin), crocetin, dimethyl crocetin and flovonoids
which quenching of free radicals and antioxidant effects and of these compounds
have established and may have role in protective effect of saffron on IRI. In addition,
saffron contains proteins, sugars, vitamins (especially riboflavin), amino
acids, minerals and gums [36].
In conclusion, the present study showed saffron extract and its constituent, crocin, have protective effect on IRI-induced oxidative stress in rats kidney that at least partly due to antioxidant properties of saffron.
ACKNOWLEDGEMENTS
The authors are thankful to the Vice Chancellor of Research, Mashhad University of Medical Sciences for financial support.
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Published by the Canadian Society for Pharmaceutical Sciences.
Copyright © 1998 by the Canadian Society for Pharmaceutical Sciences.
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