Curcumin improves spatial memory and decreases oxidative damage in aged female rats
Aging induced cognitive impairment has been well documented for many years and several antioxidant strategies have been developed against this impairment. Curcumin is the active component of curcuma longa and has shown antioxidant, antiinflamatory and neuroprotective properties. We hypothesized that curcumin would have an influence on cognitive functions in aged female rats. The purpose of the present study was to investigate the effects of curcumin supplementation on cognitive impairment evaluated by Morris water maze (MWM) as well as the oxidative stress induced by aging in female rats. Rats were randomly divided into either control or curcumin-supplemented groups. Curcumin or vehicle (corn oil) were given once daily for a period of 12 days, beginning 7 days prior to and 5 days during the behavioral tests. Behavioral assessment was performed in MWM. At the end of the behavioral test, blood samples and brain tissues were taken for the analysis of malondialdeyde (MDA), protein carbonyl and glutathione levels. During the training session, curcumin supplementation decreased latency to reach to the platform and the total distance traveled. During the probe trial, curcumin supplementation increased the number of platform crossings. In addition to the behavioral testing, biochemical results showed that MDA levels decreased in brain tissue by curcumin supplementation. It may be concluded that, curcumin supplementation improves cognitive functions by decreasing the lipid peroxidation in brain tissue of aged female rats.
KeywordsCurcumin Aging Spatial learning Memory Oxidative stress
Aging is a biological change characterized by a decrease in biochemical and physiological functions of the most organs (Rattan 2012a; Demirovic and Rattan 2013). It also adversely affects cognitive functions such as, decrease in locomotor activities and coordination and impairment of learning and memory (Van Groen et al. 2002). Several lines of evidence suggest that learning and memory gradually decline with advancing age and this age related impairment extends to spatial learning and memory tasks. Many studies (Gallagher et al. 1993; Geinisman et al. 1995; D’Hooge and De Deyn 2001; Van Groen et al. 2002) have indicated that the age-related decline in spatial learning abilities originated from the alterations in function and morphology of the hippocampal formation. Brain is sensitive to oxidative damage because of the relatively low levels of both enzymatic and non-enzymatic antioxidant systems and huge amount of free radical production due to the high oxygen demand (Halliwell and Gutteridge 1985; Asha Devi et al. 2011). Therefore, oxidative damage induced by free radicals also play a pivotal role in the aging process, including the cognitive decline, age-related impairment in spatial learning and memory (Markesbery 1997). Additionally in females, loss of ovarian hormones as a result of the aging process may aggravate neurodegeneration and subsequent cognitive decline (Bimonte-Nelson et al. 2004). It has been clearly demonstrated that cognitive functions of the aged female rats were significantly impaired compared to the young counterparts, when assessed in several maze models including Morris water maze (MWM) and radial arm maze (Bimonte-Nelson et al. 2003).
MWM was described by Morris (1981) to investigate the spatial learning and memory in laboratory rats. It has been clearly demonstrated that MWM performance declines with increasing age. This decline may be due to age-related changes in swimming abilities, locomotion and exploration. The observed changes in MWM performance seem to be the part of a larger configuration of age-related cognitive and behavioral alterations (D’Hooge and De Deyn 2001). Besides, some authors (Küçük et al. 2008; Bergado et al. 2011) have suggested that age associated decrease in various neuromotor functions may also contribute to the MWM performance defects.
Since oxidative damage is one of the factors underlying the aging process, short term and long term antioxidant treatment may alleviate age related MWM deficits in rats. Some dietary supplements were found to have beneficiary effects on MWM performance. Dietary supplementation of blueberry, spinach or strawberry extracts (Joseph et al. 1999), folic acid (Singh et al. 2011) and ginko biloba extract (Topic et al. 2002) prevented age-associated decline in MWM performance in rats, which was attributed to the antioxidant activity of these extracts.
Curcumin, a yellow pigment which is extracted from the rhizome of the plant Curcuma longa has been used in Asian medicine as a traditional therapeutic agent to treat various disorders (Jovanovic et al. 2001; Aggarwal and Harikumar 2009). In some previous studies (Rajakumar and Rao 1994; Xu et al. 2005; Bala et al. 2006; Jagetia and Aggarwal 2007; Zhao et al. 2008), it has been demonstrated that curcumin has antioxidant, antiinflammatory, antiapoptotic, antiproliferative, anticancer, antidepressant, immunomodulatory and neuroprotective effects on humans and laboratory animals. Curcumin shows antioxidant activity equivalent to vitamins C and E (Zhao and Yang, 1986). Epidemiologic data have shown that regular curcumin intake improves cognitive function in healthy elderly humans, whereas in rat and mice models curcumin supplementation appears to reverse various forms of cognitive impairment (Ng et al. 2006; Reeta et al. 2009; Tian et al. 2012). In recent studies (Surh et al. 2008; Rattan et al. 2009; Lima et al. 2011) it has been reported that curcumin has hormetic effects via stabilizing nuclear factor-erythroid-2-related factor 2 (Nrf2) and increasing heme oxygenease-1 (HO-1) expression. Therefore curcumin is proposed as hormetin (Lima et al. 2011; Rattan 2012b). Antioxidant effects of curcumin on brain tissue have been clearly demonstrated in many studies (Bala et al. 2006; Ataie et al. 2010; Khurana et al. 2012). Curcumin shows its antioxidant effect by inhibiting the iron induced lipid peroxidation in the presence of chain breaking or H donating phenolic groups in its molecular structure (Bala et al. 2006). Additionally, dietary curcumin supplementation decreases biomarkers of inflammation and oxidative damage and also decreases beta amyloid induced memory deficits in animal models of Alzheimer’s disease (Ahmed and Gilani 2009). Frautschy et al. (2001) have shown that curcumin has ability to protect against the amyloid beta-protein induced brain damage in 22 months in old rats and thus they suggested its clinical application in prevention of Alzheimer’s disease.
Since curcumin has a strong antioxidant potential on brain tissue, in the present study, we hypothesized that short term curcumin supplementation may attenuate aging induced cognitive impairment in aged female rats by activating the antioxidant system and decreasing the oxidative stress in brain tissue. To our knowledge, no study to date has been investigated the effects of curcumin treatment on spatial learning and memory in aged female rats and its relation with oxidative stress. Therefore, the present study was carried out to evaluate the effect of curcumin supplementation on cognitive impairment as well as oxidative stress induced by aging in female rats using behavioral tests, as well as biochemical analysis in brain tissue and blood samples.
Material and methods
The study protocol was approved by the Ethics Committee of the Selcuk University Experimental Medicine Research and Application Center. National Institutes of Health Guide for Care and Use of Laboratory Animals (publication no. 85-23, revised 1985), the UK Animals Scientific Procedures Act 1986 or the European Communities Council Directive of 24 November 1986 (86/609/EEC) was followed in all the experiments.
Twenty aged (20 months of age) female Wistar rats weighing between 300 and 400 g were used in the present study. The percent lifespan of 20 months female Wistar rats are almost 75 % in our lab. Rats were obtained from the Experimental Medicine Research and Application Center of Selçuk University where the study was conducted. All animals were housed in polycarbonate cages (five rats/cage) with a 12-h light–dark cycle and environmentally controlled rooms (temperature: 21 ± 2 °C, relative humidity: 50 %). Rats were fed by a standard rat chow and tap water ad libitum.
Rats were randomly assigned to either control (CON) or curcumin-supplemented (CUR) groups (ten animals each). Curcumin was given once daily for a period of 12 days, beginning 7 days prior to and 5 days during the behavioral tests. Curcumin (C1386; Sigma Chemical, St. Louis, MO) dissolved in corn oil (C8267; Sigma Chemical, St. Louis, MO) was given in CUR group of rats via oral gavage, in a dose of 300 mg/kg. This dosage was chosen because previous studies (Thiyagarajan and Sharma 2004; Zhao et al. 2008; Reeta et al. 2009) have demonstrated antioxidant and neuroprotective effects of this dosage in rats. Control animals received an appropriate volume of corn oil as a vehicle alone. Rats were weighed every day before administration of the curcumin or vehicle.
Spatial memory function was measured with MWM. The MWM is a circular, galvanized steel tank with 150 cm diameter and 60 cm in depth, filled with water and was made opaque by nontoxic black paint. The temperature of water was maintained at 25 ± 2 °C. The surface area of the tank was imaginarily divided into four quadrants such as southeast (SE), southwest (SW), northeast (NE) and northwest (NW). A hidden square escape platform was placed 2 cm below the water surface. It was located into the SW region and fixed during the training period. Several identical cues were placed on the surrounding walls of MWM for the spatial orientation of rats.
The rats were trained for four consecutive days with four trials per day and probe trial was performed 24 h after the last training session. During the training period, four starting points were randomized in each day to avoid the horizontal navigation to the platform. Each trial had a different starting position. Trials were terminated in 90s if the rats were unable to locate platform in this time interval. Then rats were placed on the platform for 30s. During training periods, following parameters were recorded: (a) latency to reach platform, (b) total distance traveled and (c) average swimming speed. The data of four consecutive trials for each of the groups obtained were averaged and presented as a block. Blocks in graphs were numbered consecutively for the day.
Twenty four hours after the last training session, a probe trial was performed to assess memory. During the probe trial, the hidden platform was removed from the tank and the rats were allowed to swim freely for 90s. The following variables were recorded: (a) total distance traveled, (b) average swimming speed, (c) time spent in each quadrant and (d) number of platform crossings.
All tests were recorded online using a video camera placed vertically above the test arenas. The videos of the behavioral tests were analyzed offline using an analytical software (Noldus Information Tech. Ethovision XT 8.0, Wageningen, The Netherlands).
Blood and tissue sampling
Twenty four hours after the last behavioral test, blood samples were taken by cardiac puncture under the ketamine hydrochloride (50 mg/kg body weight) and xylazine (10 mg/kg body weight) anesthesia. Within 1 min, blood samples were transferred into EDTA-coated tubes and plasma was separated by centrifugation at 1,750×g for 10 min at +4 °C and stored at −80 °C until to the time of analysis. Rats were sacrificed by cervical dislocation and tissue samples were immediately obtained from the brain. The tissue samples were washed with ice-cold saline and then frozen in liquid nitrogen and they were also stored at −80 °C until to the time of analysis. Malondialdehyde (MDA), protein carbonyl (PC) and glutathione (GSH) levels were analyzed both in the plasma and in the tissue samples.
After cutting the organs into small pieces, tissue samples were homogenized in ten volumes of ice-cold phosphate buffer (50 mmol/L, pH 7.4) using a homogenizer (Wise Mix HG-15; Daihan Scientific, Seoul, Korea). MDA, PC and GSH levels were measured in this homogenate. Also some part of the homogenate was centrifuged and its supernatant was separated. The supernatant solution was extracted with an equal volume of ethanol/chloroform mixture [5/3, volume per volume (v/v)]. After centrifugation at 5,000×g for 30 min, the upper layer (the ethanol phase) was used in the protein assays.
The lipid peroxidation product MDA was measured by using a commercially available kit (Bioxytech MDA-586 Assay Kit, Oxis Research, Poland) according to manufacturer’s instructions. The MDA assay is based on the reaction of chromogenic reagent N-methyl-2-phenylindole with MDA. In brief, 200 μL of sample was added to 650 μL of N-methyl-2-phenylindole in acetonitrile. Next, 150 μL of concentrated HCl was added and samples were incubated at 45 °C for 60 min. After the incubation time, samples were centrifuged at 10,000×g for 10 min and the clear supernatant was transferred to plate. MDA concentration was determined with an ELISA spectrophotometer (Power Wave XS, Biotek, Winooski, VT, USA) by measuring the absorbance at 586 nm and was calculated by using the standard curves. The MDA values were expressed as μmol/g wet tissue in tissues and as μmol/L in plasma.
The concentration of proteins containing carbonyl groups was determined spectrophotometrically according to the instructions of a commercially available kit (Cat. #10005020, Cayman Chemical, Ann Arbor, MI). Protein carbonyl levels were expressed as nmol/mg protein in tissues and nmol/mL in plasma.
GSH levels were determined by a Cayman’s GSH assay kit (Cat. #703002, Cayman Chemical, Ann Arbor, MI) using an enzymatic recycling method. Briefly, GSH reacts with 5,5-dithio-bis-2- nitrobenzoic acid (Ellman’s reagent) using the sulfhydryl group and produces a yellow-colored 5-thio-2-nitrobenzoic acid (TNB). The mixed disulfide, GSTNB (between GSH and TNB), which is concomitantly produced, is reduced by glutathione reductase to recycle the GSH and produce more TNB. The rate of TNB production is directly proportional to this recycling reaction, which in turn is directly proportional to the concentration of GSH in the sample. GSH levels expressed as μmol/mg protein and μmol/mL in tissues and plasma, respectively.
The protein contents of the tissues were measured by the method of Lowry et al. (1951).
Statistical analysis was performed by using the SPSS statistical software (version 15.0.; SPSS, Inc., Chicago, IL, USA). The Shapiro-Wilks test was used to find the normal distribution of variables. The normally distributed parameters were analyzed with independent t test to compare the difference between two groups (CON vs. CUR). Data were not normally distributed, as revealed by the Shapiro–Wilks test, the Kruskal–Wallis test was applied for further statistical comparisons. Repeated measures analysis of variance (ANOVA) (group × time) followed paired t test was used to analyze the data collected during the training session of MWM. The level of significance for all the tests was set at 0.05.
Effects of curcumin supplementation on oxidative stress and antioxidant defense markers in brain and blood
μmol/g wet tissue
4.32 ± 2.03
2.45 ± 0.89*
6.5 ± 4.8
9.9 ± 10.5
19.2 ± 3.8
18.0 ± 6.7
0.22 ± 0.16
0.35 ± 0.28
186.1 ± 168.1
108.3 ± 78.1
11.7 ± 4.3
11.7 ± 2.6
The main finding of the present study is that improvement of cognitive function in aged female rats is accompanied by a decrease of lipid peroxidation in brain tissue when rats were treated with curcumin. In the previous studies (Conboy et al. 2009; Dong et al. 2012; Khurana et al. 2012) neuroprotective effects of curcumin during the post-treatment period has been investigated however, to our knowledge no study to date has been investigated antioxidant and neuroprotective effects of curcumin only for the period during which curcumin was given in aged female rats.
The MWM test has become one of the most frequently used research tools in behavioral neuroscience (D’Hooge and De Deyn 2001). It has been well documented that MWM performance declines with aging (D’Hooge and De Deyn 2001; Küçük et al. 2008; Bergado et al. 2011; Belviranli et al. 2012). Multiple factors lead to cognitive impairment in aging. These factors are age-related changes in transmitter systems (Gottfries 1990; Küçük et al. 2008) and increased oxidative stress in aging (Socci et al. 1995).
Investigations have been focused on the relationship between progression of aging and increase in oxidative stress biomarkers. Therefore, antioxidant supplementation may be beneficial for the treatment of aging induced cognitive impairment. Especially curcumin treatment may be beneficial, since curcumin has a strong antioxidant effect and crosses the blood–brain barrier and also induces a neuroprotection directly (Yang et al. 2005). Healthy aging may also be achieved by hormesis through mild and periodic, but not severe or chronic, physical and mental challenges, and by the use of nutritional hormesis incorporating mild stress-inducing molecules called hormetins (Rattan, 2008, 2012a). In recent studies (Surh et al. 2008; Rattan et al. 2009; Lima et al. 2011) curcumin was also proposed as hormetic compound via stabilizing Nrf2 and increasing HO-1 expression.
The antioxidant property of curcumin may be assigned to its nitric oxide scavenging ability, presence of two electrophilic α, β-unsaturated carbonyl groups which react with nucleophiles, metal-chelating property, ability to inhibit various oxidases like xanthine oxidase, lipooxygenases and phospholipase D and free radical scavenging property as well as inhibition on the upregulation of cyclooxygenase 2 and inducible NO synthase expression (Khurana et al. 2012). In the present study curcumin supplementation caused a decrease in MDA levels as a marker of lipid peroxidation in brain tissue. MDA is an end product of lipid peroxidation and its concentration is proportional to the magnitude of the reactive oxygen radicals induced membrane damage (Guzel et al. 2012). Therefore, decreased MDA levels indicated a reduced free radical production. In some previous studies (Reeta et al. 2009; Ataie et al. 2010; Dong et al. 2012) it has been stated that curcumin treatment prevents oxidative damage in brain tissue in a dose dependent manner. Our findings are consistent with these reports.
In the present study, neuroprotective effects of curcumin was observed more obviously during the probe trial, by demonstration of the significantly high number of platform crossings in the curcumin supplemented group. During a probe trial of MWM, the spatial accuracy of the animal was determined, represented by the time it spends looking for the platform in the quadrant where the platform used to be (target quadrant) or by the number of times it crosses the former platform area (D’Hooge and De Deyn 2001). There are some studies demonstrating the curcumin improved aging induced cognitive impairment (Conboy et al. 2009; Dong et al. 2012). Khurana et al. (2012) reported that curcumin supplementation improves spatial navigation task against colchicine-induced cognitive dysfunction in MWM. In consistent to the above mentioned reports, behavioral tests of the present study showed that short term of curcumin supplementation improves aging induced cognitive impairment in female rats. In the present study, especially female rats were chosen because all animals in previous studies were male rats and to our knowledge no study to date has been investigated the effects of curcumin supplementation on aging induced cognitive impairment in female rats. Therefore, this is probably the first study demonstrating the neuroprotective effect of curcumin on MWM performance in aged female rats. Performances of rats during the training session of MWM showed that although there was no significant difference between the CON and CUR groups, latency to reach platform and total distance traveled had a tendency to decrease with curcumin supplementation. These results partially explain the neuroprotective effects of short term curcumin treatment.
The oxidative stress (MDA, PC) and antioxidant defense (GSH) markers in brain and blood samples also investigated to observe the relationship between the spatial memory and oxidative stress and possible effect of curcumin on this relation. It has been clearly demonstrated that oxidative stress plays a pivotal role in the age associated cognitive decline (Ataie et al. 2010). Many studies (Kiray et al. 2004; Reeta et al. 2010; Asha Devi et al. 2011; Singh et al. 2011; Tian et al. 2012) have revealed that oxidative stress markers such as MDA increased in brain tissue of both aged male and female rats compared to their young counterparts. Age related memory impairment has been also linked to a decrease in brain and plasma antioxidants (Reeta et al. 2009). Curcumin supplementation caused a decrease in MDA levels as a marker of lipid peroxidation in brain tissue.
PC levels were also measured and remained unchanged in brain tissue and plasma with curcumin supplementation. PC is produced as a result of the oxidative modifications of proteins by free radicals (Dkhar and Sharma 2010). In a recent study (Dkhar and Sharma 2010) it has been shown that PC levels were elevated in brain tissue with aging and this increase depends on the production and accumulation of reactive oxygen species. Curcumin supplementation (90 mg/kg for 3 days) attenuated PC levels. However, in the present study, no difference was observed between the two groups. This may depend on the route of administration of curcumin. In a previous study (Dkhar and Sharma 2010), curcumin was given via intraperitoneal injection, while in the present study it was given via oral gavage. Although no difference has been reported (Shehzad et al. 2010) this may limit its bioavailability.
Glutathione is a non-enzymatic antioxidant which reacts with the free radicals and protects cells from singlet oxygen, hydroxyl radicals and superoxide radical damage (Reeta et al. 2009). It is observed that GSH levels remained unchanged in brain tissue and plasma. It has been clearly demonstrated that both enzymatic systems, including superoxide dismutase, glutathione peroxidase, and glutathione-S-transferase and non-enzymatic systems, such as glutathione were impaired with aging. However, curcumin supplementation ameliorates activities and levels of these antioxidant systems in a dose dependent manner in different brain regions such as cortex, hippocampus, cerebellum and medulla (Bala et al. 2006; Reeta et al. 2009; Khurana et al. 2012). In the present study no difference was observed between the two groups in brain tissue. This may depend on the dosage of curcumin, duration of treatment, age and/or gender of rats. Unchanged GSH levels may also depend on the biochemical analysis methods.
In conclusion, short term curcumin supplementation improves cognitive function by decreasing the lipid peroxidation in brain tissue of aged female rats. However, more detailed researches are needed in this subject especially in female rats to clarify the relationship among the ovarian hormones, oxidative stress, curcumin and cognitive functions.
The authors are grateful to Prof. Said Bodur for his assistance in the statistical work.
Conflict of interest
There is no conflict of interest.
- Guzel A, Kanter M, Guzel A, Yucel AF, Erboga M (2012) Protective effect of curcumin on acute lung injury induced by intestinal ischemia/reperfusion. Toxicol Ind Health [Epub ahead of print] Google Scholar
- Joseph JA, Shukitt-Hale B, Denisova NA, Bielinski D, Martin A, McEwen JJ, Bickford PC (1999) Reversals of age-related declines in neuronal signal transduction, cognitive, and motor behavioral deficits with blueberry, spinach, or strawberry dietary supplementation. J Neurosci 19:8114–8121PubMedGoogle Scholar
- Tian M, Wang L, Yu G, Liu B, Li Y (2012) Curcumin preserves cognitive function and improve serum HDL in chronic cerebral hypoperfusion aging-rats. Mol Neurodegener 7:S3Google Scholar