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Protective effects of oral crocin against intracerebroventricular streptozotocin-induced spatial memory deficit and oxidative stress in rats
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Protective effects of oral crocin against intracerebroventricular streptozotocin-induced spatial memory deficit and oxidative stress in rats B. Naghizadeh a,1 , M.T. Mansouri a,∗,1 , B. Ghorbanzadeh b , Y. Farbood c , A. Sarkaki d a
Department of Pharmacology, School of Medicine, Physiology Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran Department of Pharmacology and Toxicology, School of Pharmacy, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran Department of Physiology, School of Medicine, Physiology Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran d Physiology Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran b c
a r t i c l e Keywords: Crocin Streptozotocin (STZ) Spatial memory Oxidative stress
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a b s t r a c t Intracerebroventricular (ICV) streptozotocin (STZ) has been shown to cause cognitive impairment, associated with free radical generation. In this study, we evaluated the effects of crocin on cognitive performance in ICV STZ-lesioned rats (3 mg/kg bilaterally, on day 1 and 3). Crocin (100 mg/kg, p.o.) was administered for 21 consecutive days, starting 1 h prior to the first dose of STZ. Cognitive performance was assessed using Morris water maze task while the parameters of oxidative stress assessed, were malondialdehyde (MDA) and total thiol levels besides glutathione peroxidase (GPx) activity. STZ-lesioned rats showed a severe deficit in memory associated with elevated MDA levels, reduced GPx activity and total thiol content. Crocin treatment improved cognitive performance and resulted in a significant reduction in MDA levels and elevation in total thiol content and GPx activity. This study demonstrates that crocin may have beneficial effects in the treatment of neurodegenerative disorders such as Alzheimer’s disease. © 2013 Elsevier GmbH. All rights reserved.
Introduction Senile dementia of the Alzheimer type (SDAT) is a degenerative disorder, characterized by a progressive deterioration of intellectual and social functions, memory loss, personality changes, and inability for self care. The severity of cognitive deficits correlates best with synaptic loss and formation of neurofibrillary tangles, particularly in cholinergic neurons. The presence of neuritic plaques containing amyloid beta protein and neurofibrillary tangles are the diagnostic hallmarks of SDAT, which contribute to neurodegeneration and disease progression, but their formation is probably triggered by pathological processes, involving oxidative stress (Weinstock and Shoham 2004). Oxidative stress can affect all classes of macromolecules (sugar, lipids, proteins, and DNA), leading inevitably to neuronal dysfunction (Polidori and Mecocci 2002).
Abbreviations: Cro, crocin; STZ, streptozotocin; GPx, glutathione peroxidase; TBARS, thiobarbituric acid reactive substances; ROS, reactive oxygen species; LPO, lipid peroxidation; MDA, malondialdehyde; DTNB, 2,2 -dinitro-5,5 -dithiodibenzoic acid; H2 O2 , hydrogen peroxide; GSH, reduced glutathione; AD, Alzheimer’s disease. ∗ Corresponding author. Tel.: +98 9133178795; fax: +98 9133178795. E-mail addresses: smt [email protected], [email protected] (M.T. Mansouri). 1 Equal contribution to the work.
The most widely used treatment for Alzheimer’s disease at present are acetylcholinesterase inhibitors, which aim to prolong cognitive function through increased synaptic activity, without providing neuroprotection. This treatment is only symptomatic and provides modest outcomes for patients (Stuchbury and Mnch 2005). The recent elucidation of the oxidative stress pathways involved in Alzheimer’s disease however has opened vistas for better treatment and prevention by targeting the cause of the disease rather than the symptoms. Reports indicate that administration of antioxidants may be useful in prevention and treatment of Alzheimer’s disease. In this respect scavenging of free radicals by non-enzymatic/exogenous antioxidants seems to be the most practical approach. This is due to the fact that many different nonenzymatic/exogenous antioxidants are known and many (e.g. vitamins E and C, melatonin, flavanoids and carotenoids) have no major side effect (Christen 2000; Sharma et al. 2005). Intracerebroventricular (ICV) injection of streptozotocin (STZ), in a sub diabetogenic dose in rat has been likened to sporadic dementia of Alzheimer’s disease (Sharma and Gupta 2001a). It is characterized by cognitive impairment, impaired glucose metabolism (Lannert et al. 1998), oxidative stress (Sharma and Gupta 2001b) and a decrease in cholinergic markers in the brain (Sharma et al. 2005). Crocin, one of the active components of saffron, is a carotenoid pigment and has the structure of crocetin di-gentiobiose ester (Ochiai et al. 2004a; Ochiai et al. 2004b). Crocin exhibits a variety of
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Please cite this article in press as: Naghizadeh, B., et al., Protective effects of oral crocin against intracerebroventricular streptozotocin-induced spatial memory deficit and oxidative stress in rats. Phytomedicine (2013), http://dx.doi.org/10.1016/j.phymed.2012.12.019
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pharmacological effects including inhibition of skin tumor growth (Konoshima et al. 1998), improvement of learning behavior previously impaired by ethanol or scopolamine (Abe and Saito 2000; Pitsikas et al. 2007), prevention of long-term potentiation inhibition caused by ethanol (Sugiura et al. 1995), anti-hyperlipidemic effect (Lee et al. 2005), treatment of colon adenocarcinoma (GarciaOlmo et al. 1999), anti-atherosclerotic property (He et al. 2005), anti-oxidant effect in PC-12 cells by increasing GSH synthesis (Ochiai et al. 2004a; Ochiai et al. 2004b), protection against inflammation-induced neurotoxicity (Nam et al. 2010), inhibition of reperfusion-induced oxidative/nitrative injury in the ischemic brain (Zheng et al.2007) and protective effects against cisplatininduced oxidative stress and nephrotoxicity (Naghizadeh et al. 2010). Alzheimer’s disease patients show a deficit in spatial memory (Simone and Baylis 1997). The Morris water maze is one of the choice apparatus to investigate the spatial memory deficits in rodent models of AD (D’Hooge and De Deyn 2001). The aim of the present study was to investigate the protective effects of crocin against memory deficit and cerebral oxidative stress induced by ICV injection of STZ. Materials and methods Animals Adult male Wistar Albino rats weighing 250–300 g were used throughout the study. All animals were obtained from the Animal House of Ahvaz Jundishapur University of Medical Sciences (Ahvaz, Iran). Animals were put separately in the cages in an airconditioned unit and allowed free access to standard laboratory chow and water, ad libitum. A 12-h light/dark cycle at 22 ± 2 ◦ C and 50% humidity conditions was maintained. All animal experiments were carried out in accordance with Ahvaz University of Medical Sciences (Ahvaz, Iran), Ethical Committee Acts. Chemicals Streptozotocin, DTNB (5,5 -dithiobis-2-nitrobenzoic acid) and 1,1,3,3-tetramethoxypropane (purity: 99%) were obtained from Sigma (England). 2-Thiobarbituric acid (TBA), n-butanol, Tris–HCl, Na2 EDTA, phosphoric acid and potassium chloride were obtained from Merck (Germany). Crocin was purchased from Fluka (Japan). GPx kit was purchased from Randox Company (England). All other chemicals were of analytical grade and prepared from Merck Company (Germany). Drug administration Animals were randomly divided into four groups (ten each) and individually put in the cages. The treatment schedule and the intervals for estimation of various parameters have been presented in Fig. 1. In the first group (sham), the rats were treated with normal saline (2 ml/kg, p.o.). The second group (STZ-lesioned or lesion) were injected with ICV STZ (3 mg/kg bilaterally, on day 1 and 3) and treated with normal saline (2 ml/day, p.o.) respectively for 21 days.
Group 3 (sham + Cro) were injected ICV on day 1 and 3 with artificial CSF and treated with crocin (100 mg/kg, p.o.) for 21 days. In the next group (lesion + Cro), the rats were injected with ICV STZ (3 mg/kg bilaterally, on day 1 and 3) and treated with crocin (100 mg/kg, p.o.) for 21 days. Crocin was dissolved and administered as a solution in normal saline (2 ml/kg) orally for 21 consecutive days. On the day of ICV injections (day 1 and 3), crocin or normal saline was administered one hour prior to ICV injection. During the behavioral test, crocin was administered 60 min before the water maze training. The dose of crocin (100 mg/kg), used in this study, has been obtained from previous experiments in our lab (Naghizadeh et al. 2010). Intracerebroventricular administration of streptozotocin Rats were anesthetized with combination of ketamine/xylazine (60/6 mg/kg, i.p.). The head was positioned in a stereotactic frame and a midline saggital incision was made in the scalp. Burr holes were drilled in the skull on both sides over the lateral ventricles using the following coordinates: 0.8 mm posterior to bregma, 1.5 mm lateral to saggital suture, and 3.6 mm beneath the surface of brain. STZ (3 mg/kg) was injected ICV bilaterally on day 1 and 3 of the experiment (Sharma and Gupta 2001b). In the sham group, artificial CSF: 147 mM NaCl, 2.9 mM KCl, 1.6 mM MgCl2 , 1.7 mM CaCl2 and 2.2 mM dextrose was injected (20 l on each site) on the same days as STZ group. STZ was dissolved in artificial CSF. All microinjections were performed by delivering drug or vehicle solution slowly over a 1-min period and the needle remained in position for a further 5 min to prevent reflux along the injection tract. The progress of the injection was continuously monitored by following the movement of an air bubble in the tubing. Assessment of spatial memory via Morris water maze test The water maze used was a black circular tank (136 cm in diameter and 60 cm in height) that was filled with water (20 ± 1 ◦ C) to a depth of 25 cm. The maze was located in a room containing extra-maze cues (posters). The pool divided geographically into four quadrants [northeast (NE), northwest (NW), southeast (SE), southwest (SW)] and starting positions [north (N), south (S), east (E), west (W)] that were equally spaced around the perimeter of the pool. A hidden circular platform (diameter: 13 cm) was located 2 cm below the surface of the water on a fixed location in one of the four quadrants of the pool. A video camera was mounted directly above the water maze to record the rats’ swim paths. An automated tracking system (Radiab ver. 2, Tehran, Iran) was used to measure the escape latency, swimming speed of each rat and also percentage of the time in the target quadrant. On day 17 after the first dose of streptozotocin, rats were given four training trials each day for 4 consecutive days. For each training trial, the rats were placed in the water facing the pool wall at one of the four starting positions in a different order each day and allowed to swim until they reached the platform. The latency to reach the platform was recorded for up to 60 s. They remained on the platform for 30 s before being removed. If rat failed to reach the escape platform within the maximum allowed time of 60 s, it was gently placed on the platform
Fig. 1. The design of the treatment schedule and intervals for estimation of various parameters. ICV STZ = intracerebroventricular streptozotocin; MWM = Morris water maze; SAC = sacrificed.
Please cite this article in press as: Naghizadeh, B., et al., Protective effects of oral crocin against intracerebroventricular streptozotocin-induced spatial memory deficit and oxidative stress in rats. Phytomedicine (2013), http://dx.doi.org/10.1016/j.phymed.2012.12.019
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and allowed to remain there for the same interval of time. One day after the last training, a probe trial was conducted by removing the platform. Rats were allowed to swim freely into the pool for 60 s. The time spent in the target quadrant, which had previously contained the hidden platform, was recorded. The time spent in the target quadrant indicated the degree of memory consolidation that has taken place after learning. After the trials, the rats were dried with a towel and placed in a holding cage under a heating lamp before returning to the home cage (Morris 1984). Estimation of oxidative stress parameters At the end of the experiment, the animals (n = 5) were sacrificed by decapitation. The brain was removed and the hippocampus and cerebral cortex were dissected for the biochemical studies. Samples were kept at −80 ◦ C until used. The tissues were homogenized in cold KCl solution (1.5%) to give a 10% homogenate suspension used for measuring malondialdehyde (MDA) and total thiol contents and glutathione peroxidase (GPx) activity. Thiobarbituric acid reactive substances (TBARS) measurement Malondialdehyde (MDA) levels, an index of lipid peroxidation, produced by free radicals were measured. MDA reacts with thiobarbituric acid to produce a red colored complex that has peak absorbance at 532 nm. Briefly, 3 ml phosphoric acid (1%) and 1 ml TBA (0.6%) were 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 n-butanol was added to the mixture and vortex-mixed for 1 min followed by centrifugation at 2000 rpm for 20 min. The colored layer was transferred to a fresh tube and its absorbance was measured at 532 nm. MDA levels were determined using 1,1,3,3tetramethoxypropane as standard. The standard curve of MDA was constructed over the concentration range of 0–20 M (Ohkawa et al. 1979). Total thiol ( SH) groups assay Total SH groups were measured using DTNB (5,5 -dithiobis-2nitrobenzoic acid) as the reagent (Ellman 1959). This reagent reacts with the SH groups to produce a yellow colored complex which has a peak absorbance at 412 nm. Briefly, 1 ml Tris–EDTA buffer (pH 8.6) was added to 50 l brain homogenate in 2 ml cuvettes and absorbance was read at 412 nm against Tris–EDTA buffer alone (A1 ). Then, 20 l DTNB reagents (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:
Fig. 2. Effect of crocin on escape latency to find the hidden platform in ICV streptozotocin-lesioned rats. Data is expressed as mean ± S.E.M. (n = 10). a p < 0.05 and aa p < 0.01, STZ-lesioned compared to sham group from second day of the training sessions; b p < 0.05, STZ-lesioned compared to lesion + Cro group from third day of the training sessions (two-way ANOVA followed by Tukey’s test).
Results Effects of crocin on STZ-induced learning and memory impairment in Morris water maze task Fig. 2 shows the reduction of latency time to find the hidden platform in all groups during the four-day training trial. Group comparisons revealed that STZ-lesioned animals presented a higher latency time than sham group (p < 0.01), showing a poorer learning performance due to ICV-STZ infusion. Treatment with crocin significantly improved the learning performance in lesion + Cro group as compared to STZ-lesioned group (p < 0.05). According to Fig. 3, on the probe trial day with the platform removed, STZ-lesioned animals failed to remember the location of platform, spending less time in target quadrant than sham group (p < 0.05). However, the time spending in target quadrant was significantly increased via the administration of crocin as compared to STZ-lesioned group (p < 0.05). Furthermore, there was no significant difference among the swim speeds of all four groups (Fig. 4, p > 0.05).
Total thiol concentration(mM) = (A − A1 − B) × 1.07/0.05 × 13.6.
Glutathion peroxidase (GPx) assay GPx activity was measured with GPx kit (Randox Company, England). Data analysis Results were presented as mean ± SEM. Statistical differences were analyzed using ANOVA followed by Tukey’s test. The pvalue < 0.05 was considered statistically significant.
Fig. 3. Effect of crocin on percentage of time spent in target quadrant during the probe trials. Values are expressed as mean ± S.E.M. (n = 10). *p < 0.05, as compared to STZ-lesioned group (one-way ANOVA followed by Tukey’s test).
Please cite this article in press as: Naghizadeh, B., et al., Protective effects of oral crocin against intracerebroventricular streptozotocin-induced spatial memory deficit and oxidative stress in rats. Phytomedicine (2013), http://dx.doi.org/10.1016/j.phymed.2012.12.019
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Fig. 4. Effect of crocin on swim speed. Data represent mean ± S.E.M., expressed in centimeters per second (n = 10). There were no significant differences between groups (p > 0.05, one-way ANOVA).
Effect of Crocin on MDA levels in the hippocampus and cerebral cortex The degree of free radical damage following STZ injection was assessed using lipid peroxidation (LPO), which was measured as MDA levels. According to Fig. 5, there was an increase in MDA levels of STZ-lesioned group (p < 0.05) as compared to sham-operated rats in the hippocampus and cerebral cortex. Oral administration of crocin resulted in a significant reduction of MDA levels, in the brain tissues of lesion + Cro animals as compared to STZ-lesioned group (p < 0.05).
Effect of Crocin on total thiol levels in the hippocampus and cerebral cortex The total thiol concentration (mM) was measured to evaluate the non-enzymatic defense potential of the cell against the oxidative stress. According to Fig. 6, total thiol levels in STZ-lesioned animals were found to be significantly depleted as compared to sham group animals in the hippocampus and cerebral cortex (p < 0.05). Chronic treatment with crocin in lesion + Cro group was able to raise total thiol levels significantly as compared to STZlesioned animals (p < 0.05).
Fig. 5. Effect of crocin on concentration of MDA in the hippocampus and cerebral cortex of STZ-lesioned rats. Values are expressed as mean ± S.E.M. (n = 5). *p < 0.05 as compared to STZ-lesioned group (one-way ANOVA followed by Tukey’s test).
Fig. 6. Effect of crocin on concentration of total thiol in the hippocampus and cerebral cortex of STZ-lesioned rats. Values are expressed as mean ± S.E.M. (n = 5). *p < 0.05 as compared to STZ-lesioned group (one-way ANOVA followed by Tukey’s test).
Effect of Crocin on GPx activity in the hippocampus and cerebral cortex GPx activity (u/l) was measured to evaluate the enzymatic defense potential of the cells against the oxidative stress. According to Fig. 7, the GPx activity was significantly (p < 0.01) decreased in STZ-lesioned group as compared to sham-operated group in both hippocampus and cerebral cortex. However, the decrease of the GPx activity was significantly restored by crocin treatment in the hippocampus and cerebral cortex of lesion + Cro group (p < 0.01). Discussion The present study shows that oral administration of crocin effectively attenuated spatial memory deficit and oxidative stress caused by intracerebroventricular streptozotocin in rats. In our previous studies, we have shown the protective effects of crocin against cisplatin-induced acute renal failure and relative oxidative stress (Naghizadeh et al. 2008). We found it worthwhile to investigate whether crocin has a protective role against ICV STZinduced cognitive dysfunction and oxidative stress in rats. The ICV STZ model in rat has been described as an appropriate animal model for sporadic Alzheimer type dementia. It is characterized by cognitive deficit, impaired glucose metabolism, oxidative stress and a decrease in cholinergic markers in the brain (Sharma et al. 2005). Free radical-induced oxidative stress has been expressed as an important factor mediating the behavioral impairments and memory deficits in age related neurodegenerative disorders such as AD (Sharma and Gupta 2002).
Fig. 7. Effect of crocin on Gpx activity in the hippocampus and cerebral cortex of STZlesioned rats. Values are expressed as mean ± S.E.M. (n = 5). *p < 0.05 and **p < 0.01 as compared to STZ-lesioned group (one-way ANOVA followed by Tukey’s test).
Please cite this article in press as: Naghizadeh, B., et al., Protective effects of oral crocin against intracerebroventricular streptozotocin-induced spatial memory deficit and oxidative stress in rats. Phytomedicine (2013), http://dx.doi.org/10.1016/j.phymed.2012.12.019
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Lipids and proteins are the major targets of oxidative modification by free radicals. Lipid peroxidation (LPO) and protein oxidation lead to loss of membrane integrity, an important factor in acceleration of aging and age-related neurodegenerative disorders. MDA is an end product of LPO; a measure of free radical generation. The neuronal defense against hydrogen peroxide (H2 O2 ), which is the most toxic molecule to the brain, is mediated primarily by the glutathione (GSH) system (Ishrat et al. 2006). GSH is an essential tripeptide, an antioxidant found in all animal cells which reacts with the free radicals and can protect cells from singlet oxygen, hydroxyl and superoxide radical damages (Sharma and Gupta 2001a). The selenium containing enzyme, glutathione peroxidase (GPx), protects cells against reactive oxygen substances via scavenging hydroperoxides and lipid peroxides (Naghizadeh et al. 2010). In this study, cerebral cortex and hippocampus were used for the biochemical estimations because of their involvement in behavioral and cognitive tasks. The results from the biochemical experiments showed a significant increase in MDA levels and a simultaneous decrease in total thiol contents and GPx activity in STZ-lesioned rats indicating neuronal damage caused by oxidative stress. While, crocin restored the oxidative damages via decreasing in MDA levels and increasing in total thiol contents and GPx activity. The reduction of MDA level in the brain with crocin, indicates attenuation of LPO. Lipid peroxidation may be enhanced by depletion of GSH content in the brain, which is often considered as the first line of defense as an endogenous antioxidant against oxidative stress. The antioxidant system uses reduced glutathione (GSH), the most abundant non-protein thiol, which buffers free radicals in brain tissue. It eliminates H2 O2 and organic peroxides by GPx. During detoxification, oxyradicals are reduced by GPx at the expense of GSH to form glutathione disulfide (GSSG). Oxidant radicals are known to inactivate GPx. However, chronic treatment with crocin significantly restored total thiol contents and GPx activity (Deshmukh et al. 2009). Crocin has been demonstrated to have antioxidant potential and reported to scavenge free radicals (Naghizadeh et al. 2010; Naghizadeh et al. 2008) which suggests that its ameliorating effects on memory dysfunction induced by STZ may be associated with this antioxidant effect. The Morris water maze test is used to test spatial memory by observing the latency to reach a hidden platform. A decrease in latency time in repeated trials demonstrates intact learning and memory function. STZ-lesioned rats did not show this decline in the latency time, while treatment with crocin in lesion + Cro animals decreased the time to reach the hidden platform. In probe trial, the time spent in target quadrant was significantly decreased in STZlesioned rats indicating poorer consolidation of memory. However, crocin treatment in lesion + Cro group, improved memory consolidation significantly as measured by increased time spent in target quadrant. Carotenoids are well known as highly efficient scavengers of oxygen radicals and other excited species (Stahl and Sies 2003). Thus, crocin may protect against oxidation of lipids, proteins and DNA. Our results suggest that beneficial effects of crocin on memory processing may be attributed to its favorable antioxidant effect. However further studies are required to demonstrate the potential use of crocin in AD and other agerelated neurodegenerative disorders, where oxidative stress is involved.
Conflict of interest The authors declare that there is no conflict of interest.
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Acknowledgment This research was supported by grants (PRC-69) from the Physiology Research Center, funded by the Vice Chancellor of Research, Ahvaz Jundishapur University of Medical Sciences (Iran). References Abe, K., Saito, H., 2000. Effects of saffron extract and its constituent crocin on learning behavior and long-term potentiation. Phytotherapy Research 14, 149–152. Christen, Y., 2000. Oxidative stress and Alzheimer disease. The American Journal of Clinical Nutrition 71, 621S–629S. Deshmukh, R., Sharma, V., Mehan, S., Sharma, N., Bedi, K.L., 2009. Amelioration of intracerebroventricular streptozotocin induced cognitive dysfunction and oxidative stress by vinpocetine – a PDE1 inhibitor. European Journal of Pharmacology 620, 49–56. D’Hooge, R., De Deyn, P.P., 2001. Applications of the Morris water maze in the study of learning and memory. Brain Research Reviews 36, 60–90. Ellman, G., 1959. Tissue sulfhydryl groups. Archives of biochemistry and biophysics 82, 70–77. Garcia-Olmo, D.C., Riese, H.H., Escribano, J., Ontanon, J., Fernandez, J.A., Atienzar, M., Garcia-Olmo, D., 1999. Effects of long-term treatment of colon adenocarcinoma with crocin, a carotenoid from saffron (Crocus sativus L.): an experimental study in the rat. Nutrition and Cancer 35, 120–126. He, S.H.Y, Qian, Z.Y., Tang, F.T., Wen, N., Xu, G.L., Sheng, L., 2005. Effects of crocin on experimental atherosclerosis in quails and its mechanisms. Life Science 77, 907–921. Ishrat, T., Badruzzaman Khan, M., Nasrul Hoda, M., Yousuf, S., Ahmad, M., Ansari, M.A., Ahmad, A.S., Islam, F., 2006. Coenzyme Q10 modulates cognitive impairment against intracerebroventricular injection of streptozotocin in rats. Behavioural Brain Research 171, 9–16. Konoshima, T., Takasaki, M., Tokuda, H., Morimoto, S., Tanaka, H., Kawata, E., Xuan, L.J., Saito, H., Sugiura, M., Molnar, J., Shoyama, Y., 1998. Crocin and crocetin derivatives inhibit skin tumour promotion in mice. Phytotherapy Research 12, 400–404. Lannert, H., Wirtz, P., Schuhmann, V., Galmbacher, R., 1998. Effects of estradiol (−17 beta) on learning, memory and cerebral energy metabolism in male rats after intracerebroventricular administration of streptozotocin. Journal of Neural Transmission 105, 1045–1063. Lee, I.A.H, Lee, J.H., Baek, N.I., Kim, D.H., 2005. Antihyperlipidemic effect of crocin isolated from the fractus of Gardenia jasminoides and its metabolite crocetin. Biological and Pharmaceutical Bulletin 28, 2106–2110. Morris, R., 1984. Development of a water-maze procedure for studying spatial learning in the rat. Journal of Neuroscience Methods 11, 47–60. Naghizadeh, B., Boroushaki, M.T., Vahdati Mashhadian, N., Mansouri, M.T., 2008. Protective effects of crocin against cisplatin-induced acute renal failure and oxidative stress in rats. Iranian Biomedical Journal 12, 93–100. Naghizadeh, B., Mansouri, M.T., Vahdati Mashhadian, N., 2010. Crocin attenuates cisplatin-induced renal oxidative stress in rats. Food and Chemical Toxicology 48, 2650–2655. Nam, K.N., Park, Y.M., Jung, H.J., Lee, J.Y., Min, B.D., Park, S.U., Jung, W.S., Cho, K.H., Park, J.H., Kang, I., Hong, J.W., Lee, E.H., 2010. Anti-inflammatory effects of crocin and crocetin in rat brain microglial cells. European Journal of Pharmacology 648, 110–116. Ochiai, T., Ohno, S., Soeda, S., Tanaka, H., Shoyama, Y., Shimeno, H., 2004a. Crocin prevents the death of rat pheochromyctoma (PC-12) cells by its antioxidant effects stronger than those of a-tocopherol. Neuroscience Letters 362, 61–64. Ochiai, T., Soeda, S.H., Ohno, S., Tanaka, H., Shoyama, Y., Shimeno, H., 2004b. Crocin prevents the death of PC-12 cells through sphingomyelinase-ceramide signaling by increasing glutathione synthesis. Neurochemistry International 44, 321–330. Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical Biochemistry 95, 351–358. Pitsikas, N., Zisopoulou, S., Tarantilis, P.A., Kanakis, C.D., Polissiou, M.G., Sakellaridis, N., 2007. Effects of the active constituents of Crocus sativus L., crocins on recognition and spatial rats’ memory. Behavioural Brain Research 183, 141–146. Polidori, M.C., Mecocci, P., 2002. Plasma susceptibility to free radical-induced antioxidant consumption and lipid peroxidation is increased in very old subjects wth Alzheimer disease. Journal of Alzheimer’s Disease 4, 517–522. Sharma, M., Briyal, S., Gupta, Y.K., 2005. Effect of alpha lipoic acid, melatonin and trans resveratrol on intracerebroventricular streptozotocin induced spatial memory deficit in rats. Indian Journal of Physiology and Pharmacology 49, 395–402. Sharma, M., Gupta, Y.K., 2002. Chronic treatment with trans resveratrol prevents intracerebroventricular streptozotocin induced cognitive impairment and oxidative stress in rats. Life Sciences 71, 2489–2498. Sharma, M., Gupta, Y.K., 2001a. Effect of chronic treatment of melatonin on learning, memory and oxidative deficiencies induced by intracerebroventricular streptozotocin in rats. Pharmacology Biochemistry and Behavior 70, 325–331. Sharma, M., Gupta, Y.K., 2001b. Intracerebroventricular injection of streptozotocin in rats produces both oxidative stress in the brain and cognitive impairment. Life Sciences 68, 1021–1029. Simone, P.M., Baylis, G.C., 1997. The role of attention in a spatial memory task in Alzheimer disease patients. Alzheimer Disease and Associated Disorders 11, 140–152.
Please cite this article in press as: Naghizadeh, B., et al., Protective effects of oral crocin against intracerebroventricular streptozotocin-induced spatial memory deficit and oxidative stress in rats. Phytomedicine (2013), http://dx.doi.org/10.1016/j.phymed.2012.12.019
G Model PHYMED-51365; No. of Pages 6 6
ARTICLE IN PRESS B. Naghizadeh et al. / Phytomedicine xxx (2013) xxx–xxx
Stahl, W., Sies, H., 2003. Antioxidant activity of carotenoids. Molecular Aspects of Medicine 24, 345–351. Stuchbury, G., Mnch, G., 2005. Alzheimer’s associated inflammation, potential drug targets and future therapies. Journal of Neural Transmission 112, 429–453. Sugiura, M., Shoyama, Y., Saito, H., Abe, K., 1995. The effects of ethanol and crocin on the induction of long-term potentiation in the CA1 region of rat hippocampal slices. Japanese journal of pharmacology 67, 395–397.
Weinstock, M., Shoham, S., 2004. Rat models of dementia based on reductions in regional glucose metabolism, cerebral blood flow and cytochrome oxidase activity. Journal of Neural Transmission 111, 347–366. Zheng, Y.Q., Liu, J.X., Wang, J.N., Xu, L., 2007. Effects of crocin on reperfusioninduced oxidative/nitrative injury to cerebral microvessels after global cerebral ischemia. Brain Research 1138, 86–94.
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Protective effects of oral crocin against intracerebroventricular streptozotocin-induced spatial memory deficit and oxidative stress in rats B. Naghizadeh a,1 , M.T. Mansouri a,∗,1 , B. Ghorbanzadeh b , Y. Farbood c , A. Sarkaki d a
Department of Pharmacology, School of Medicine, Physiology Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran Department of Pharmacology and Toxicology, School of Pharmacy, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran Department of Physiology, School of Medicine, Physiology Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran d Physiology Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran b c
a r t i c l e Keywords: Crocin Streptozotocin (STZ) Spatial memory Oxidative stress
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a b s t r a c t Intracerebroventricular (ICV) streptozotocin (STZ) has been shown to cause cognitive impairment, associated with free radical generation. In this study, we evaluated the effects of crocin on cognitive performance in ICV STZ-lesioned rats (3 mg/kg bilaterally, on day 1 and 3). Crocin (100 mg/kg, p.o.) was administered for 21 consecutive days, starting 1 h prior to the first dose of STZ. Cognitive performance was assessed using Morris water maze task while the parameters of oxidative stress assessed, were malondialdehyde (MDA) and total thiol levels besides glutathione peroxidase (GPx) activity. STZ-lesioned rats showed a severe deficit in memory associated with elevated MDA levels, reduced GPx activity and total thiol content. Crocin treatment improved cognitive performance and resulted in a significant reduction in MDA levels and elevation in total thiol content and GPx activity. This study demonstrates that crocin may have beneficial effects in the treatment of neurodegenerative disorders such as Alzheimer’s disease. © 2013 Elsevier GmbH. All rights reserved.
Introduction Senile dementia of the Alzheimer type (SDAT) is a degenerative disorder, characterized by a progressive deterioration of intellectual and social functions, memory loss, personality changes, and inability for self care. The severity of cognitive deficits correlates best with synaptic loss and formation of neurofibrillary tangles, particularly in cholinergic neurons. The presence of neuritic plaques containing amyloid beta protein and neurofibrillary tangles are the diagnostic hallmarks of SDAT, which contribute to neurodegeneration and disease progression, but their formation is probably triggered by pathological processes, involving oxidative stress (Weinstock and Shoham 2004). Oxidative stress can affect all classes of macromolecules (sugar, lipids, proteins, and DNA), leading inevitably to neuronal dysfunction (Polidori and Mecocci 2002).
Abbreviations: Cro, crocin; STZ, streptozotocin; GPx, glutathione peroxidase; TBARS, thiobarbituric acid reactive substances; ROS, reactive oxygen species; LPO, lipid peroxidation; MDA, malondialdehyde; DTNB, 2,2 -dinitro-5,5 -dithiodibenzoic acid; H2 O2 , hydrogen peroxide; GSH, reduced glutathione; AD, Alzheimer’s disease. ∗ Corresponding author. Tel.: +98 9133178795; fax: +98 9133178795. E-mail addresses: smt [email protected], [email protected] (M.T. Mansouri). 1 Equal contribution to the work.
The most widely used treatment for Alzheimer’s disease at present are acetylcholinesterase inhibitors, which aim to prolong cognitive function through increased synaptic activity, without providing neuroprotection. This treatment is only symptomatic and provides modest outcomes for patients (Stuchbury and Mnch 2005). The recent elucidation of the oxidative stress pathways involved in Alzheimer’s disease however has opened vistas for better treatment and prevention by targeting the cause of the disease rather than the symptoms. Reports indicate that administration of antioxidants may be useful in prevention and treatment of Alzheimer’s disease. In this respect scavenging of free radicals by non-enzymatic/exogenous antioxidants seems to be the most practical approach. This is due to the fact that many different nonenzymatic/exogenous antioxidants are known and many (e.g. vitamins E and C, melatonin, flavanoids and carotenoids) have no major side effect (Christen 2000; Sharma et al. 2005). Intracerebroventricular (ICV) injection of streptozotocin (STZ), in a sub diabetogenic dose in rat has been likened to sporadic dementia of Alzheimer’s disease (Sharma and Gupta 2001a). It is characterized by cognitive impairment, impaired glucose metabolism (Lannert et al. 1998), oxidative stress (Sharma and Gupta 2001b) and a decrease in cholinergic markers in the brain (Sharma et al. 2005). Crocin, one of the active components of saffron, is a carotenoid pigment and has the structure of crocetin di-gentiobiose ester (Ochiai et al. 2004a; Ochiai et al. 2004b). Crocin exhibits a variety of
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Please cite this article in press as: Naghizadeh, B., et al., Protective effects of oral crocin against intracerebroventricular streptozotocin-induced spatial memory deficit and oxidative stress in rats. Phytomedicine (2013), http://dx.doi.org/10.1016/j.phymed.2012.12.019
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pharmacological effects including inhibition of skin tumor growth (Konoshima et al. 1998), improvement of learning behavior previously impaired by ethanol or scopolamine (Abe and Saito 2000; Pitsikas et al. 2007), prevention of long-term potentiation inhibition caused by ethanol (Sugiura et al. 1995), anti-hyperlipidemic effect (Lee et al. 2005), treatment of colon adenocarcinoma (GarciaOlmo et al. 1999), anti-atherosclerotic property (He et al. 2005), anti-oxidant effect in PC-12 cells by increasing GSH synthesis (Ochiai et al. 2004a; Ochiai et al. 2004b), protection against inflammation-induced neurotoxicity (Nam et al. 2010), inhibition of reperfusion-induced oxidative/nitrative injury in the ischemic brain (Zheng et al.2007) and protective effects against cisplatininduced oxidative stress and nephrotoxicity (Naghizadeh et al. 2010). Alzheimer’s disease patients show a deficit in spatial memory (Simone and Baylis 1997). The Morris water maze is one of the choice apparatus to investigate the spatial memory deficits in rodent models of AD (D’Hooge and De Deyn 2001). The aim of the present study was to investigate the protective effects of crocin against memory deficit and cerebral oxidative stress induced by ICV injection of STZ. Materials and methods Animals Adult male Wistar Albino rats weighing 250–300 g were used throughout the study. All animals were obtained from the Animal House of Ahvaz Jundishapur University of Medical Sciences (Ahvaz, Iran). Animals were put separately in the cages in an airconditioned unit and allowed free access to standard laboratory chow and water, ad libitum. A 12-h light/dark cycle at 22 ± 2 ◦ C and 50% humidity conditions was maintained. All animal experiments were carried out in accordance with Ahvaz University of Medical Sciences (Ahvaz, Iran), Ethical Committee Acts. Chemicals Streptozotocin, DTNB (5,5 -dithiobis-2-nitrobenzoic acid) and 1,1,3,3-tetramethoxypropane (purity: 99%) were obtained from Sigma (England). 2-Thiobarbituric acid (TBA), n-butanol, Tris–HCl, Na2 EDTA, phosphoric acid and potassium chloride were obtained from Merck (Germany). Crocin was purchased from Fluka (Japan). GPx kit was purchased from Randox Company (England). All other chemicals were of analytical grade and prepared from Merck Company (Germany). Drug administration Animals were randomly divided into four groups (ten each) and individually put in the cages. The treatment schedule and the intervals for estimation of various parameters have been presented in Fig. 1. In the first group (sham), the rats were treated with normal saline (2 ml/kg, p.o.). The second group (STZ-lesioned or lesion) were injected with ICV STZ (3 mg/kg bilaterally, on day 1 and 3) and treated with normal saline (2 ml/day, p.o.) respectively for 21 days.
Group 3 (sham + Cro) were injected ICV on day 1 and 3 with artificial CSF and treated with crocin (100 mg/kg, p.o.) for 21 days. In the next group (lesion + Cro), the rats were injected with ICV STZ (3 mg/kg bilaterally, on day 1 and 3) and treated with crocin (100 mg/kg, p.o.) for 21 days. Crocin was dissolved and administered as a solution in normal saline (2 ml/kg) orally for 21 consecutive days. On the day of ICV injections (day 1 and 3), crocin or normal saline was administered one hour prior to ICV injection. During the behavioral test, crocin was administered 60 min before the water maze training. The dose of crocin (100 mg/kg), used in this study, has been obtained from previous experiments in our lab (Naghizadeh et al. 2010). Intracerebroventricular administration of streptozotocin Rats were anesthetized with combination of ketamine/xylazine (60/6 mg/kg, i.p.). The head was positioned in a stereotactic frame and a midline saggital incision was made in the scalp. Burr holes were drilled in the skull on both sides over the lateral ventricles using the following coordinates: 0.8 mm posterior to bregma, 1.5 mm lateral to saggital suture, and 3.6 mm beneath the surface of brain. STZ (3 mg/kg) was injected ICV bilaterally on day 1 and 3 of the experiment (Sharma and Gupta 2001b). In the sham group, artificial CSF: 147 mM NaCl, 2.9 mM KCl, 1.6 mM MgCl2 , 1.7 mM CaCl2 and 2.2 mM dextrose was injected (20 l on each site) on the same days as STZ group. STZ was dissolved in artificial CSF. All microinjections were performed by delivering drug or vehicle solution slowly over a 1-min period and the needle remained in position for a further 5 min to prevent reflux along the injection tract. The progress of the injection was continuously monitored by following the movement of an air bubble in the tubing. Assessment of spatial memory via Morris water maze test The water maze used was a black circular tank (136 cm in diameter and 60 cm in height) that was filled with water (20 ± 1 ◦ C) to a depth of 25 cm. The maze was located in a room containing extra-maze cues (posters). The pool divided geographically into four quadrants [northeast (NE), northwest (NW), southeast (SE), southwest (SW)] and starting positions [north (N), south (S), east (E), west (W)] that were equally spaced around the perimeter of the pool. A hidden circular platform (diameter: 13 cm) was located 2 cm below the surface of the water on a fixed location in one of the four quadrants of the pool. A video camera was mounted directly above the water maze to record the rats’ swim paths. An automated tracking system (Radiab ver. 2, Tehran, Iran) was used to measure the escape latency, swimming speed of each rat and also percentage of the time in the target quadrant. On day 17 after the first dose of streptozotocin, rats were given four training trials each day for 4 consecutive days. For each training trial, the rats were placed in the water facing the pool wall at one of the four starting positions in a different order each day and allowed to swim until they reached the platform. The latency to reach the platform was recorded for up to 60 s. They remained on the platform for 30 s before being removed. If rat failed to reach the escape platform within the maximum allowed time of 60 s, it was gently placed on the platform
Fig. 1. The design of the treatment schedule and intervals for estimation of various parameters. ICV STZ = intracerebroventricular streptozotocin; MWM = Morris water maze; SAC = sacrificed.
Please cite this article in press as: Naghizadeh, B., et al., Protective effects of oral crocin against intracerebroventricular streptozotocin-induced spatial memory deficit and oxidative stress in rats. Phytomedicine (2013), http://dx.doi.org/10.1016/j.phymed.2012.12.019
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and allowed to remain there for the same interval of time. One day after the last training, a probe trial was conducted by removing the platform. Rats were allowed to swim freely into the pool for 60 s. The time spent in the target quadrant, which had previously contained the hidden platform, was recorded. The time spent in the target quadrant indicated the degree of memory consolidation that has taken place after learning. After the trials, the rats were dried with a towel and placed in a holding cage under a heating lamp before returning to the home cage (Morris 1984). Estimation of oxidative stress parameters At the end of the experiment, the animals (n = 5) were sacrificed by decapitation. The brain was removed and the hippocampus and cerebral cortex were dissected for the biochemical studies. Samples were kept at −80 ◦ C until used. The tissues were homogenized in cold KCl solution (1.5%) to give a 10% homogenate suspension used for measuring malondialdehyde (MDA) and total thiol contents and glutathione peroxidase (GPx) activity. Thiobarbituric acid reactive substances (TBARS) measurement Malondialdehyde (MDA) levels, an index of lipid peroxidation, produced by free radicals were measured. MDA reacts with thiobarbituric acid to produce a red colored complex that has peak absorbance at 532 nm. Briefly, 3 ml phosphoric acid (1%) and 1 ml TBA (0.6%) were 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 n-butanol was added to the mixture and vortex-mixed for 1 min followed by centrifugation at 2000 rpm for 20 min. The colored layer was transferred to a fresh tube and its absorbance was measured at 532 nm. MDA levels were determined using 1,1,3,3tetramethoxypropane as standard. The standard curve of MDA was constructed over the concentration range of 0–20 M (Ohkawa et al. 1979). Total thiol ( SH) groups assay Total SH groups were measured using DTNB (5,5 -dithiobis-2nitrobenzoic acid) as the reagent (Ellman 1959). This reagent reacts with the SH groups to produce a yellow colored complex which has a peak absorbance at 412 nm. Briefly, 1 ml Tris–EDTA buffer (pH 8.6) was added to 50 l brain homogenate in 2 ml cuvettes and absorbance was read at 412 nm against Tris–EDTA buffer alone (A1 ). Then, 20 l DTNB reagents (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:
Fig. 2. Effect of crocin on escape latency to find the hidden platform in ICV streptozotocin-lesioned rats. Data is expressed as mean ± S.E.M. (n = 10). a p < 0.05 and aa p < 0.01, STZ-lesioned compared to sham group from second day of the training sessions; b p < 0.05, STZ-lesioned compared to lesion + Cro group from third day of the training sessions (two-way ANOVA followed by Tukey’s test).
Results Effects of crocin on STZ-induced learning and memory impairment in Morris water maze task Fig. 2 shows the reduction of latency time to find the hidden platform in all groups during the four-day training trial. Group comparisons revealed that STZ-lesioned animals presented a higher latency time than sham group (p < 0.01), showing a poorer learning performance due to ICV-STZ infusion. Treatment with crocin significantly improved the learning performance in lesion + Cro group as compared to STZ-lesioned group (p < 0.05). According to Fig. 3, on the probe trial day with the platform removed, STZ-lesioned animals failed to remember the location of platform, spending less time in target quadrant than sham group (p < 0.05). However, the time spending in target quadrant was significantly increased via the administration of crocin as compared to STZ-lesioned group (p < 0.05). Furthermore, there was no significant difference among the swim speeds of all four groups (Fig. 4, p > 0.05).
Total thiol concentration(mM) = (A − A1 − B) × 1.07/0.05 × 13.6.
Glutathion peroxidase (GPx) assay GPx activity was measured with GPx kit (Randox Company, England). Data analysis Results were presented as mean ± SEM. Statistical differences were analyzed using ANOVA followed by Tukey’s test. The pvalue < 0.05 was considered statistically significant.
Fig. 3. Effect of crocin on percentage of time spent in target quadrant during the probe trials. Values are expressed as mean ± S.E.M. (n = 10). *p < 0.05, as compared to STZ-lesioned group (one-way ANOVA followed by Tukey’s test).
Please cite this article in press as: Naghizadeh, B., et al., Protective effects of oral crocin against intracerebroventricular streptozotocin-induced spatial memory deficit and oxidative stress in rats. Phytomedicine (2013), http://dx.doi.org/10.1016/j.phymed.2012.12.019
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Fig. 4. Effect of crocin on swim speed. Data represent mean ± S.E.M., expressed in centimeters per second (n = 10). There were no significant differences between groups (p > 0.05, one-way ANOVA).
Effect of Crocin on MDA levels in the hippocampus and cerebral cortex The degree of free radical damage following STZ injection was assessed using lipid peroxidation (LPO), which was measured as MDA levels. According to Fig. 5, there was an increase in MDA levels of STZ-lesioned group (p < 0.05) as compared to sham-operated rats in the hippocampus and cerebral cortex. Oral administration of crocin resulted in a significant reduction of MDA levels, in the brain tissues of lesion + Cro animals as compared to STZ-lesioned group (p < 0.05).
Effect of Crocin on total thiol levels in the hippocampus and cerebral cortex The total thiol concentration (mM) was measured to evaluate the non-enzymatic defense potential of the cell against the oxidative stress. According to Fig. 6, total thiol levels in STZ-lesioned animals were found to be significantly depleted as compared to sham group animals in the hippocampus and cerebral cortex (p < 0.05). Chronic treatment with crocin in lesion + Cro group was able to raise total thiol levels significantly as compared to STZlesioned animals (p < 0.05).
Fig. 5. Effect of crocin on concentration of MDA in the hippocampus and cerebral cortex of STZ-lesioned rats. Values are expressed as mean ± S.E.M. (n = 5). *p < 0.05 as compared to STZ-lesioned group (one-way ANOVA followed by Tukey’s test).
Fig. 6. Effect of crocin on concentration of total thiol in the hippocampus and cerebral cortex of STZ-lesioned rats. Values are expressed as mean ± S.E.M. (n = 5). *p < 0.05 as compared to STZ-lesioned group (one-way ANOVA followed by Tukey’s test).
Effect of Crocin on GPx activity in the hippocampus and cerebral cortex GPx activity (u/l) was measured to evaluate the enzymatic defense potential of the cells against the oxidative stress. According to Fig. 7, the GPx activity was significantly (p < 0.01) decreased in STZ-lesioned group as compared to sham-operated group in both hippocampus and cerebral cortex. However, the decrease of the GPx activity was significantly restored by crocin treatment in the hippocampus and cerebral cortex of lesion + Cro group (p < 0.01). Discussion The present study shows that oral administration of crocin effectively attenuated spatial memory deficit and oxidative stress caused by intracerebroventricular streptozotocin in rats. In our previous studies, we have shown the protective effects of crocin against cisplatin-induced acute renal failure and relative oxidative stress (Naghizadeh et al. 2008). We found it worthwhile to investigate whether crocin has a protective role against ICV STZinduced cognitive dysfunction and oxidative stress in rats. The ICV STZ model in rat has been described as an appropriate animal model for sporadic Alzheimer type dementia. It is characterized by cognitive deficit, impaired glucose metabolism, oxidative stress and a decrease in cholinergic markers in the brain (Sharma et al. 2005). Free radical-induced oxidative stress has been expressed as an important factor mediating the behavioral impairments and memory deficits in age related neurodegenerative disorders such as AD (Sharma and Gupta 2002).
Fig. 7. Effect of crocin on Gpx activity in the hippocampus and cerebral cortex of STZlesioned rats. Values are expressed as mean ± S.E.M. (n = 5). *p < 0.05 and **p < 0.01 as compared to STZ-lesioned group (one-way ANOVA followed by Tukey’s test).
Please cite this article in press as: Naghizadeh, B., et al., Protective effects of oral crocin against intracerebroventricular streptozotocin-induced spatial memory deficit and oxidative stress in rats. Phytomedicine (2013), http://dx.doi.org/10.1016/j.phymed.2012.12.019
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Lipids and proteins are the major targets of oxidative modification by free radicals. Lipid peroxidation (LPO) and protein oxidation lead to loss of membrane integrity, an important factor in acceleration of aging and age-related neurodegenerative disorders. MDA is an end product of LPO; a measure of free radical generation. The neuronal defense against hydrogen peroxide (H2 O2 ), which is the most toxic molecule to the brain, is mediated primarily by the glutathione (GSH) system (Ishrat et al. 2006). GSH is an essential tripeptide, an antioxidant found in all animal cells which reacts with the free radicals and can protect cells from singlet oxygen, hydroxyl and superoxide radical damages (Sharma and Gupta 2001a). The selenium containing enzyme, glutathione peroxidase (GPx), protects cells against reactive oxygen substances via scavenging hydroperoxides and lipid peroxides (Naghizadeh et al. 2010). In this study, cerebral cortex and hippocampus were used for the biochemical estimations because of their involvement in behavioral and cognitive tasks. The results from the biochemical experiments showed a significant increase in MDA levels and a simultaneous decrease in total thiol contents and GPx activity in STZ-lesioned rats indicating neuronal damage caused by oxidative stress. While, crocin restored the oxidative damages via decreasing in MDA levels and increasing in total thiol contents and GPx activity. The reduction of MDA level in the brain with crocin, indicates attenuation of LPO. Lipid peroxidation may be enhanced by depletion of GSH content in the brain, which is often considered as the first line of defense as an endogenous antioxidant against oxidative stress. The antioxidant system uses reduced glutathione (GSH), the most abundant non-protein thiol, which buffers free radicals in brain tissue. It eliminates H2 O2 and organic peroxides by GPx. During detoxification, oxyradicals are reduced by GPx at the expense of GSH to form glutathione disulfide (GSSG). Oxidant radicals are known to inactivate GPx. However, chronic treatment with crocin significantly restored total thiol contents and GPx activity (Deshmukh et al. 2009). Crocin has been demonstrated to have antioxidant potential and reported to scavenge free radicals (Naghizadeh et al. 2010; Naghizadeh et al. 2008) which suggests that its ameliorating effects on memory dysfunction induced by STZ may be associated with this antioxidant effect. The Morris water maze test is used to test spatial memory by observing the latency to reach a hidden platform. A decrease in latency time in repeated trials demonstrates intact learning and memory function. STZ-lesioned rats did not show this decline in the latency time, while treatment with crocin in lesion + Cro animals decreased the time to reach the hidden platform. In probe trial, the time spent in target quadrant was significantly decreased in STZlesioned rats indicating poorer consolidation of memory. However, crocin treatment in lesion + Cro group, improved memory consolidation significantly as measured by increased time spent in target quadrant. Carotenoids are well known as highly efficient scavengers of oxygen radicals and other excited species (Stahl and Sies 2003). Thus, crocin may protect against oxidation of lipids, proteins and DNA. Our results suggest that beneficial effects of crocin on memory processing may be attributed to its favorable antioxidant effect. However further studies are required to demonstrate the potential use of crocin in AD and other agerelated neurodegenerative disorders, where oxidative stress is involved.
Conflict of interest The authors declare that there is no conflict of interest.
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Acknowledgment This research was supported by grants (PRC-69) from the Physiology Research Center, funded by the Vice Chancellor of Research, Ahvaz Jundishapur University of Medical Sciences (Iran). References Abe, K., Saito, H., 2000. Effects of saffron extract and its constituent crocin on learning behavior and long-term potentiation. Phytotherapy Research 14, 149–152. Christen, Y., 2000. Oxidative stress and Alzheimer disease. The American Journal of Clinical Nutrition 71, 621S–629S. Deshmukh, R., Sharma, V., Mehan, S., Sharma, N., Bedi, K.L., 2009. Amelioration of intracerebroventricular streptozotocin induced cognitive dysfunction and oxidative stress by vinpocetine – a PDE1 inhibitor. European Journal of Pharmacology 620, 49–56. D’Hooge, R., De Deyn, P.P., 2001. Applications of the Morris water maze in the study of learning and memory. Brain Research Reviews 36, 60–90. Ellman, G., 1959. Tissue sulfhydryl groups. Archives of biochemistry and biophysics 82, 70–77. Garcia-Olmo, D.C., Riese, H.H., Escribano, J., Ontanon, J., Fernandez, J.A., Atienzar, M., Garcia-Olmo, D., 1999. Effects of long-term treatment of colon adenocarcinoma with crocin, a carotenoid from saffron (Crocus sativus L.): an experimental study in the rat. Nutrition and Cancer 35, 120–126. He, S.H.Y, Qian, Z.Y., Tang, F.T., Wen, N., Xu, G.L., Sheng, L., 2005. Effects of crocin on experimental atherosclerosis in quails and its mechanisms. Life Science 77, 907–921. Ishrat, T., Badruzzaman Khan, M., Nasrul Hoda, M., Yousuf, S., Ahmad, M., Ansari, M.A., Ahmad, A.S., Islam, F., 2006. Coenzyme Q10 modulates cognitive impairment against intracerebroventricular injection of streptozotocin in rats. Behavioural Brain Research 171, 9–16. Konoshima, T., Takasaki, M., Tokuda, H., Morimoto, S., Tanaka, H., Kawata, E., Xuan, L.J., Saito, H., Sugiura, M., Molnar, J., Shoyama, Y., 1998. Crocin and crocetin derivatives inhibit skin tumour promotion in mice. Phytotherapy Research 12, 400–404. Lannert, H., Wirtz, P., Schuhmann, V., Galmbacher, R., 1998. Effects of estradiol (−17 beta) on learning, memory and cerebral energy metabolism in male rats after intracerebroventricular administration of streptozotocin. Journal of Neural Transmission 105, 1045–1063. Lee, I.A.H, Lee, J.H., Baek, N.I., Kim, D.H., 2005. Antihyperlipidemic effect of crocin isolated from the fractus of Gardenia jasminoides and its metabolite crocetin. Biological and Pharmaceutical Bulletin 28, 2106–2110. Morris, R., 1984. Development of a water-maze procedure for studying spatial learning in the rat. Journal of Neuroscience Methods 11, 47–60. Naghizadeh, B., Boroushaki, M.T., Vahdati Mashhadian, N., Mansouri, M.T., 2008. Protective effects of crocin against cisplatin-induced acute renal failure and oxidative stress in rats. Iranian Biomedical Journal 12, 93–100. Naghizadeh, B., Mansouri, M.T., Vahdati Mashhadian, N., 2010. Crocin attenuates cisplatin-induced renal oxidative stress in rats. Food and Chemical Toxicology 48, 2650–2655. Nam, K.N., Park, Y.M., Jung, H.J., Lee, J.Y., Min, B.D., Park, S.U., Jung, W.S., Cho, K.H., Park, J.H., Kang, I., Hong, J.W., Lee, E.H., 2010. Anti-inflammatory effects of crocin and crocetin in rat brain microglial cells. European Journal of Pharmacology 648, 110–116. Ochiai, T., Ohno, S., Soeda, S., Tanaka, H., Shoyama, Y., Shimeno, H., 2004a. Crocin prevents the death of rat pheochromyctoma (PC-12) cells by its antioxidant effects stronger than those of a-tocopherol. Neuroscience Letters 362, 61–64. Ochiai, T., Soeda, S.H., Ohno, S., Tanaka, H., Shoyama, Y., Shimeno, H., 2004b. Crocin prevents the death of PC-12 cells through sphingomyelinase-ceramide signaling by increasing glutathione synthesis. Neurochemistry International 44, 321–330. Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical Biochemistry 95, 351–358. Pitsikas, N., Zisopoulou, S., Tarantilis, P.A., Kanakis, C.D., Polissiou, M.G., Sakellaridis, N., 2007. Effects of the active constituents of Crocus sativus L., crocins on recognition and spatial rats’ memory. Behavioural Brain Research 183, 141–146. Polidori, M.C., Mecocci, P., 2002. Plasma susceptibility to free radical-induced antioxidant consumption and lipid peroxidation is increased in very old subjects wth Alzheimer disease. Journal of Alzheimer’s Disease 4, 517–522. Sharma, M., Briyal, S., Gupta, Y.K., 2005. Effect of alpha lipoic acid, melatonin and trans resveratrol on intracerebroventricular streptozotocin induced spatial memory deficit in rats. Indian Journal of Physiology and Pharmacology 49, 395–402. Sharma, M., Gupta, Y.K., 2002. Chronic treatment with trans resveratrol prevents intracerebroventricular streptozotocin induced cognitive impairment and oxidative stress in rats. Life Sciences 71, 2489–2498. Sharma, M., Gupta, Y.K., 2001a. Effect of chronic treatment of melatonin on learning, memory and oxidative deficiencies induced by intracerebroventricular streptozotocin in rats. Pharmacology Biochemistry and Behavior 70, 325–331. Sharma, M., Gupta, Y.K., 2001b. Intracerebroventricular injection of streptozotocin in rats produces both oxidative stress in the brain and cognitive impairment. Life Sciences 68, 1021–1029. Simone, P.M., Baylis, G.C., 1997. The role of attention in a spatial memory task in Alzheimer disease patients. Alzheimer Disease and Associated Disorders 11, 140–152.
Please cite this article in press as: Naghizadeh, B., et al., Protective effects of oral crocin against intracerebroventricular streptozotocin-induced spatial memory deficit and oxidative stress in rats. Phytomedicine (2013), http://dx.doi.org/10.1016/j.phymed.2012.12.019
G Model PHYMED-51365; No. of Pages 6 6
ARTICLE IN PRESS B. Naghizadeh et al. / Phytomedicine xxx (2013) xxx–xxx
Stahl, W., Sies, H., 2003. Antioxidant activity of carotenoids. Molecular Aspects of Medicine 24, 345–351. Stuchbury, G., Mnch, G., 2005. Alzheimer’s associated inflammation, potential drug targets and future therapies. Journal of Neural Transmission 112, 429–453. Sugiura, M., Shoyama, Y., Saito, H., Abe, K., 1995. The effects of ethanol and crocin on the induction of long-term potentiation in the CA1 region of rat hippocampal slices. Japanese journal of pharmacology 67, 395–397.
Weinstock, M., Shoham, S., 2004. Rat models of dementia based on reductions in regional glucose metabolism, cerebral blood flow and cytochrome oxidase activity. Journal of Neural Transmission 111, 347–366. Zheng, Y.Q., Liu, J.X., Wang, J.N., Xu, L., 2007. Effects of crocin on reperfusioninduced oxidative/nitrative injury to cerebral microvessels after global cerebral ischemia. Brain Research 1138, 86–94.
Please cite this article in press as: Naghizadeh, B., et al., Protective effects of oral crocin against intracerebroventricular streptozotocin-induced spatial memory deficit and oxidative stress in rats. Phytomedicine (2013), http://dx.doi.org/10.1016/j.phymed.2012.12.019