Epilepsy is a serious and common neurological disease. Nearly 70% of patients with epilepsy achieve seizure control with antiepileptic drugs. Pharmacological treatment of seizures has primarily involved modulation of voltage-gated ion channels, enhancement of GABAergic inhibition, and reduction of glutamatergic excitation.1 Phytochemical and pharmacological studies have been done on anticonvulsant plants, and an increasing number of patients in the United States and Europe use herbal medicines as a supplement to or substitute for prescription drugs. Such treatments are considered to be gentle and safe alternatives to synthetic drugs.2
Curcumin, the major component of the spice turmeric, can reduce oxidative and inflammatory damage.3 Recently, several studies have shown that curcumin has anticonvulsant effects against seizures induced by kainic acid (KA)4 and FeCl35 in rats. We have previously shown that high doses (100 and 300 mg/kg, i.p.), but not low doses (10 and 30 mg/kg), of curcumin inhibited amygdala-kindled seizures in rats.6 The aim of this study was to test the effects of curcumin on pilocarpine-induced seizures in rats, to confirm its anticonvulsant effects in an additional typical seizure model.
Seizures can induce the generation of epoxide and free radicals, and this has been suggested to contribute to the recurrence of seizures.7 Lipid peroxidation, resulting from an increase in free radicals, causes cellular membrane damage, which can also increase the recurrence of seizures.8 Therefore, we also investigated the generation of epoxide and free radicals in the hippocampus during pilocarpine-induced seizures and its modulation by curcumin treatment.
Male Sprague-Dawley (SD) rats (250–300 g) were used. All experiments were approved by the Fudan University Animal Care and Use Committee. All efforts were made to minimize the number of animals used and their suffering. Each rat was used only once, and each treatment group consisted of fifteen animals.
Drugs and dosing schedule
The following drugs were used: pilocarpine hydrochloride, curcumin, methyl scopolamine, and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; Sigma, USA). All injections were administered at a volume of 10 ml/kg. Appropriate vehicle controls were performed for each experiment.
Pilocarpine hydrochloride was dissolved in 0.9% saline and administered i.p. to induce seizures. Methyl scopolamine (1 mg/kg in 0.9% saline, i.p.) was administered 30 minutes before pilocarpine to minimize peripheral effects. Curcumin, suspended with dimethyl sulfoxide (DMSO) in double-distilled water, was administrated i.p. once daily for 5 days at the dose of 30, 100, or 300 mg∙kg-1∙d-1. The final dose of curcumin was administered 30 minutes before the pilocarpine injection. The dose of curcumin was chosen based on our previous experiments.
Behavioral seizures were classified as follows:9 class I, hypoactivity and mouth and facial automatisms; class II, head nodding and mastication; class III, forelimb clonus without rearing; class IV, bilateral forelimb clonus and rearing; class V, rearing and loss of posture. Rats were observed continuously for 120 minutes following pilocarpine administration for the occurrence of limbic seizures and status epilepticus (an uninterrupted class V seizure for 30 minutes). The latency to the first episode of convulsive behavior (forelimb clonus), and the latency to onset of status epilepticus was also recorded.
Rats were killed by decapitation 24 hours after pilocarpine administration. Brains were quickly removed and ice-cooled, and the hippocampus was isolated, weighed, and homogenized. Saline homogenate (10% wt/vol) was used for estimation of glutathione (GSH) content, malonaldehyde (MDA) content, lactate dehydrogenase (LDH) activity, and superoxide dismutase (SOD) activity. Nitric oxide synthase (NOS) activity was measured in the HEPES-buffered homogenate (20% wt/vol).
The HEPES homogenate was ultracentrifuged at 100 000 ×g for 1 hour at 4°C. One portion of the supernatant was kept on ice until the NOS assay, which was done within 4 hours of preparation.10 Nitric oxide (NO) synthesis was measured by spectrophotometric determination of the oxidation of oxyhemoglobin to methemoglobin by NO.11 The difference in absorption between 401 and 411 nm was monitored with a dual-wave length recording spectrophotometer at 37°C for 3 minutes. The second portion of the supernatant was diluted at 1:100. An aliquot of the saline homogenate was ultracentrifuged at 105 000 ×g for 45 minutes at 4°C. The cytosolic fraction was used to determine SOD and LDH activity. SOD (EC 126.96.36.199) activity was assayed at 420 nm according to the method of Marklund et al,12 and enzyme activity was expressed as U/mg protein. LDH (EC 188.8.131.52) activity was measured by monitoring the rate of increase in absorbance at 340 nm (3 minutes) resulting from reduction of nicotinamide adenine dinucleotide (NAD) into nicotinamide adenine dinucleotide-reduced (NADH), using a Stanbio reagent Kit (San Antonio, TX, USA). The total protein concentration was determined according to the method of Lowry et al.13 The other parts of the saline homogenate were mixed with medium appropriate for the parameter assayed.
Another portion of the homogenate was mixed with an equal amount of ice-cold 5% sulfosalicylic acid for determination of GSH using spectrophotometry at 412 nm.14 For determination of MDA content, another portion of the saline homogenate was mixed with ice-cold 2.3% KCl and centrifuged at 600 ×g for 15 minutes at 4°C. The level of thiobarbituric acid (TBA) reactive substance was determined as previously described15 and expressed in nmol MDA/g tissue.
Data were expressed as mean ± standard error (SE). for each experimental group. Analysis of the latency to seizures and status epilepticus was performed using one-way analysis of variance (ANOVA) with Dunnett’s post hoc test. Comparison of seizure severity scores was done with Kruskal-Wallis ANOVA and Dunnett’s post hoc test. Before using ANOVA, data were tested for normality (Shapiro-Wilk test) and homogeneity of variances (Levene’s test). Differences in MDA and GSH content and LDH, SOD, and NOS activity were tested using ANOVA followed by Duncan test. P <0.05 was considered to be statistically significant. SPSS 13.0 (SPSS Inc., USA) was used in this study.
All the rats in the control group (n=15) exhibited generalized limbic seizures after pilocarpine administration, at a latency of (9.53±0.25) minutes. Pretreatment with curcumin significantly delayed the onset of seizures at doses of 300 mg/kg ((20.00±1.14) minutes, n=15) and 100 mg/kg ((16.47±0.79) minutes, n=15) compared with control (F=37.01, P <0.01). The 30 mg/kg dose of curcumin had no significant effect ((10.33±0.68) minutes, P=0.50, n=15) (Figure 1A). Similarly, the mean seizure severity score was significantly reduced (F=25.41, P <0.01) compared with control (4.80±0.32, n=15) at curcumin doses of 300 mg/kg (3.37±0.19, n=15) and 100 mg/kg (3.40±0.71, n=15), but not at 30 mg/kg (4.60±0.47, P=0.36, n=15) (Figure 1B). Pretreatment with curcumin at 300 and 100 mg/kg also decreased the occurrence of pilocarpine-induced status epilepticus (F=36.69, P <0.01). Several rats died before the onset of status epilepticus (2/15 rats treated with 300 mg/kg, 2/15 rats treated with 100 mg/kg, 4/15 rats treated with 30 mg/kg, and 4/15 control rats). In the remaining rats, curcumin significantly delayed the onset of status epilepticus at doses of 300 mg/kg ((66.38±2.31) minutes, n=13) and 100 mg/kg ((60.08±2.19) minutes, n=13) compared with control ((37.55±1.94) minutes, n=11) (Figure 1C). Curcumin at 30 mg/kg did not delay the onset of status epilepticus ((43.36±2.44) minutes, P=0.09, n=11) compared with control.
|view in a new window |Figure 1. Effects of different doses of curcumin (30, 100, 300 mg/kg) on the latency to seizure (A), seizure severity (B), and onset to status epilepticus (SE) (C) in rats treated with pilocarpine (300 mg/kg). Curcumin was administered 30 minutes before an i.p. injection of pilocarpine. *P <0.01, compared with pilocarpine (P) control group. Data are expressed as mean ± SE (n=11–15).|
Pilocarpine-induced seizures significantly increased MDA content, and treatment with curcumin did not normalize this effect (F
=5.50, Figure 2A). Animals treated with pilocarpine showed an elevation in LDH activity that was restored by curcumin at 100 or 300 mg/kg (F
=17.85, Figure 3A). Pilocarpine also caused an increase in NOS activity (F
=37.31, Figure 3C), and curcumin at 100 and 300 mg/kg prevented this effect (P
<0.01, Figure 3C). Both GSH (Figure 2B) and SOD (Figure 3B) levels were decreased in pilocarpine-treated rats. GSH content (F
=60.43, Figure 2B) was restored to near normal in animals treated with curcumin at 100 and 300 mg/kg
<0.01, Figure 2B). Curcumin at 30 mg/kg failed to restore SOD activity (F
=0.06, Figure 3B), but the 100 and 300 mg/kg doses significantly increased it towards normal levels (P
<0.01, Figure 3B).
|view in a new window |
Figure 2. Effect of different doses of curcumin (30, 100, 300 mg/kg, i.p.) on hippocampal concentrations of MDA (A) and GSH (B) after pilocarpine-induced seizures. Curcumin was administered 30 minutes before an i.p. injection of pilocarpine. *P <0.01 compared with vehicle control group. †P <0.01 compared with pilocarpine (P) control group. Data are expressed as mean ± SE (n=11–15).
|view in a new window |
Figure 3. Effect of different doses of curcumin (30, 100, 300 mg/kg, i.p.) on the activity of hippocampal LDH (A), SOD (B), and NOS (C) after pilocarpine-induced seizures. Curcumin was administered 30 minutes before an i.p. injection of pilocarpine. *P <0.01 compared with vehicle control group. †P <0.01 compared with pilocarpine (P) control group. Data are expressed as mean ± SE (n=11–15).
We have demonstrated that pretreatment with curcumin at doses of 100 and 300 mg/kg significantly delayed the onset of pilocarpine-induced limbic seizures and status epilepticus. These doses of curcumin also counteracted pilocarpine-induced changes in hippocampal NOS, SOD, and LDH activity and GSH content. Taken together, these results indicate that the anticonvulsant properties of curcumin may at least in part be mediated by the central nitric oxide system and free radical production.
Recent research has shown that curcumin exerted anticonvulsant effect against acute generalized seizures induced by maximal electroshock,16 KA,4 and FeCl3,5 and delayed the development of amygdala kindling.17 Taken together with the current study, these findings suggest that curcumin has anticonvulsant activity in a range of models.
The mechanisms underlying the potential anticonvulsant effects of curcumin are not yet fully understood. Previous research has indicated that curcumin has antioxidant and anti-inflammatory activity. Epidemiological studies showed that curcumin also had antioxidant effects after the injury of central nervous system.18 Curcumin was reported to be several times more potent than vitamin E as a free radical scavenger,19 and effective against nitric oxide-based radicals.20 Oxidative stress is known to play a role in epileptogenesis. Free radicals such as oxygen, superoxide, and nitrite, are generated during epileptogenesis.21 In lead-induced neurotoxicity in rats, curcumin (100 mg/kg, p.o.) has been shown to significantly decrease lipid peroxidation, and increase the levels of reduced glutathione and activity of superoxide dismutase and catalase.22 Thus, curcumin is an effective antioxidant, and this action may be responsible for its anticonvulsive activity.
An increase in MDA has been observed in the brain of rats with seizures induced by FeCl323 and N-methyl-D-aspartate (NMDA).24 In our experiment, MDA level in the hippocampus was also significantly increased after pilocarpine-induced seizures. However, administration of curcumin could not reverse this effect.
Recent research has shown that curcumin may trigger intracellular signaling responsible for modulation of NMDA receptor function25 and expression of brain-derived neurotrophic factor.26 Our previous research has indicated that curcumin modulates the influx of calcium mediated by 2-amino-3-(5-methyl-3-oxo-1,2- oxazol-4-yl) propanoic acid (AMPA) and kainate in cultured hippocampal neurons from rats.19 Thus, it is likely that the suppression of pilocarpine-induced seizures and status epilepticus is associated with modulation of neuronal excitability.
In conclusion, curcumin has anticonvulsant potential in the pilocarpine model of temporal lobe seizures. Curcumin is abundant, inexpensive, and relatively safe in humans. It can cross the blood-brain barrier and directly exert its effect on the brain.27 Curcumin may thus be a good supplement to or substitute for antiepileptic drugs. Further studies focusing on the mechanism of its antiepileptic effects should be conducted.
1. Rogawski MA, Löscher W. The neurobiology of antiepileptic drugs. Nat Rev Neurosci 2004; 5: 553-564.
2. Kaiboriboon K, Guevara M, Alldredge BK. Understanding herb and dietary supplement use in patients with epilepsy. Epilepsia 2009; 50: 1927-1932.
3. Menon VP, Sudheer AR. Antioxidant and anti-inflammatory properties of curcumin. Adv Exp Med Biol 2007; 595: 105-125.
4. Gupta YK, Briyal S, Sharma M. Protective effect of curcumin against kainic acid induced seizures and oxidative stress in rats. Indian J Physiol Pharmacol 2009; 53: 39-46.
5. Jyoti A, Sethi P, Sharma D. Curcumin protects against electrobehavioral progression of seizures in the iron-induced experimental model of epileptogenesis. Epilepsy Behav 2009; 14: 300-308.
6. Du P, Li X, Lin HJ, Peng WF, Liu JY, Ma Y, et al. Curcumin inhibits amygdaloid kindled seizures in rats. Chin Med J 2009; 122: 1435-1438.
7. Meador KJ. Neurodevelopmental effects of antiepileptic drugs. Curr Neurol Neurosci Rep 2002; 2: 373-378.
8. Adibhatla RM, Hatcher JF. Altered lipid metabolism in brain injury and disorders. Subcell Biochem 2008; 49: 241-268.
9. Racine RJ. Modification of seizure activity by electrical stimulation. I. After-discharge threshold. Electroencephalogr Clin Neurophysiol 1972; 32: 269-279.
10. Knowels RG, Merrett M, Salter M, Moncada S. Differential induction of brain, lung and liver nitric oxide synthase by endotoxin in the rat. Biochem J 1990; 270: 833-836.
11. Feelisch M, Noack EA. Correlation between nitric oxide formation during degradation of organic nitrates and activation of guanylate cyclase. Eur J Pharmacol 1987; 139: 19-30.
12. Marklund S, Marklund G. Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem 1974; 47: 469-474.
13. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin-phenol reagent J Biol Chem 1951; 193: 265-275.
14. Ahmed AE, Hussein GI, Loh J, Abdel-Rahman SZ. Studies on the mechanism of haloacetonitrile-induced gastrointestinal toxicity: interaction of dibromo-acetonitrile with glutathione and glutathione-Stransferase in rats. J Biochem Toxico 1991; 6: 115-121.
15. Mihara M, Uchiyama M. Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Anal Biochem 1978; 86: 271-278.
16. Bharal N, Sahaya K, Jain S, Mediratta PK, Sharma KK. Curcumin has anticonvulsant activity on increasing current electroshock seizures in mice. Phytother Res 2008; 22: 1660-1664.
17. Du P, Guo X, Peng WF, Ma Y, Fan W, Lin HJ, et al. The effect of curcumin on the AMPA/KA induced calcium influx in hippocampal neurons of rats. Chin J Neurol (Chin) 2009; 8: 258-261.
18. Das KC, Das CK. Curcumin (diferuloylmethane), a singlet oxygen ((1)O(2)) quencher. Biochem Biophys Res Commun 2002; 295: 62-66.
19. Zhao BL, Li XJ, He RG, Cheng SJ, Xin WJ. Scavenging effect of extracts of green tea and natural antioxidants on active oxygen radicals. Cell Biophys 1989; 14: 175-185.
20. Sreejayan N, Rao MN. Free radical scavenging activity of curcuminoids. Arzneimittelforschung 1996; 46: 169-171.
21. Maertens P, Dyken P, Graf W, Pippenger C, Chronister R, Shah A. Free radicals, anticonvulsants, and the neuronal ceroid-lipofuscinoses. Am J Med Genet 1995; 57: 225-228.
22. Shukla PK, Khanna VK, Khan MY, Srimal RC. Protective effect of curcumin against lead neurotoxicity in rat. Hum Exp Toxicol 2003; 22: 653-658.
23. Gupta YK, Chaudhary G, Sinha K, Srivastava AK. Protective effect of resveratrol against intracortical FeCl3-induced model of posttraumatic seizures in rats. Methods Find Exp Clin Pharmacol 2001; 23: 241-244.
24. Cicek E, Sutcu R, Gokalp O, Yilmaz HR, Ozer MK, Uz E, et al. The effects of isoniazid on hippocampal NMDA receptors: protective role of erdosteine. Mol Cell Biochem 2005; 277: 131-135.
25. Matteucci A, Cammarota R, Paradisi S, Varano M, Balduzzi M, Leo L, et al. Curcumin protects against NMDA-induced toxicity: a possible role for NR2A subunit. Invest Ophthalmol Vis Sci 2011; 52: 1070-1077.
26. Wang R, Li YB, Li YH, Xu Y, Wu HL, Li XJ. Curcumin protects against glutamate excitotoxicity in rat cerebral cortical neurons by increasing brain-derived neurotrophic factor level and activating TrkB. Brain Res 2008; 1210: 84-91.
27. Kaur C, Ling EA. Blood brain barrier in hypoxic-ischemic conditions. Curr Neurovasc Res 2008; 5: 71-81.